Single-domain antibody fragments derived from heavy-chain …vri.cz/docs/vetmed/57-9-439.pdf ·...

Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article

439

Single-domain antibody fragments derived from heavy-chain antibodies: a review

L. Eyer, K. Hruska

Veterinary Research Institute, Brno, Czech Republic

ABSTRACT: Single-domain antibody (sdAb) fragments derived from heavy-chain antibodies of camelids and cartilagi-nous fish represent a new generation of therapeutic agents and immunoreagents. Due to their unique characteristics, such as low molecular weight, high physical-chemical stability, good water solubility, and the ability to bind antigens inaccessible to conventional antibodies, they could potentially act as a substitute for conventional therapeutic drugs in the treatment of serious human diseases, and, moreover, could be broadly used in analyses and diagnostics. In this review article, an analysis of 826 publications oriented to heavy-chain antibodies and their sdAb fragments indexed in the Web of Science® database since 1993 has been carried out. Attention has predominantly been paid to papers published from 2010 to June 2012. Key publications are presented in tables and are characterised by descriptive words, abstracts and references. The presented publications have been sorted according to seven basic criteria: review articles and monographs, heavy-chain antibodies of camelids and sharks, production of sdAb fragments using recombinant technology, characteristic properties of sdAb fragments, application of sdAb fragments in therapy, application of sdAb fragments in diagnostic and immunoanalytical methods and other prospective uses of sdAb fragments. This review article should highlight the typical properties of heavy-chain antibodies and sdAb fragments which differentiate them from conventional antibodies and other available recombinant fragments, and also emphasize their extremely broad application potential, mainly in human disease therapy. At the same time it allows an easy and rapid orientation in numerous publications written on this subject, and facilitates the search for the required data.

Keywords: single-domain antibody fragment; heavy-chain antibody; antigen-binding site; camelid; shark; therapy; recombinant technology

Supported by the Ministry of Education, Youth and Sports, Czech Republic (AdmireVet; Grant No. CZ 1.05/2.1.00/01.0006-ED 0006/01/01) and the Ministry of Agriculture of the Czech Republic (Grant No. MZE 0002716202).

Contents

1. Introduction2. Review

2.1. Methods of searching and publication analysis2.2. Review articles and monographs2.3. Heavy-chain antibodies of camelids and sharks2.4. Production of sdAb fragments using recom-

binant technology2.5. Characteristic properties of sdAb fragments2.6. Application of sdAb fragments in therapy2.7. Application of sdAb fragments in diagnostic

and immunoanalytical methods2.8. Other prospective uses of sdAb fragments

3. References

Tables and figures

Table 1. Authors, countries, institutions and jour-nals (Top 5) showing the highest publication activities concerning heavy-chain antibodies and single-domain antibody fragments (Web of Science®, 826 papers from 1993 to June 2012)

Table 2. Important review articles and monographs concerning heavy-chain antibodies and single-domain antibody fragments

Table 3. Camelid and shark heavy-chain antibodiesTable 4. Production of single-domain antibody

fragments using recombinant technologyTable 5. Characteristic properties of single-domain antibody fragments

Review Article Veterinarni Medicina, 57, 2012 (9): 439–513

440

1. Introduction

A surprising discovery was made in the 1990s: in addition to conventional antibodies, mammals of Camelidae family and also some cartilaginous fish have evolved a distinctive type of antibody molecule composed entirely of two identical heavy chains (Hamerscasterman et al. 1993; Greenberg et al. 1995). Because these antibodies are devoid of light chains, they are called heavy-chain antibodies. The antigen-binding site of heavy-chain antibodies is confined to one single domain referred to as the VHH (Variable domain of the Heavy chain of the Heavy-chain antibody) in Camelidae and VNAR (Variable domain of the shark New Antigen Receptor) in car-tilaginous fish. These variable domains can be eas-ily expressed in bacteria, yeasts or in other hosts as recombinant single-domain antibodies (sdAb), which are the smallest available intact antigen-bind-ing fragments. SdAb fragments derived from llama and camel heavy-chain antibodies are often referred to as nanoantibodies or nanobodies®. Due to their biophysical and pharmaceutical properties, which are conferred by their single-domain nature and small molecule size, sdAb fragments offer a broad application potential, especially as new immuno-therapeutic drugs and also as efficient reagents in immunoanalytical and diagnostic methods. The aim of this review is to highlight the unique features of heavy-chain antibodies and sdAb fragments and

show their numerous novel feasible applications in research and in clinical development. Publications, which contain key information concerning heavy-chain antibodies and their sdAb fragments, are in this new designed review presented in tables and are characterized with a few descriptive words, full or shortened abstracts and source references. The text in the tables contains several format imperfec-tions, which exist in the Web of Science® database and are caused by transmission and copying of data between various information sources. The missing format of lower and upper indexes (e.g. Ig(2) instead of Ig2 or 10(–6) instead of 10–6) can be given as a typical example. Special characters, such as α, Å or °C were transcribed as alpha, angstrom or degree C, respectively. The attention is predominantly focused on papers published in the last three years, although some older key papers had been also included. The presented publications have been classified ac-cording to seven basic criteria: review articles and monographs, heavy-chain antibodies of camelids and sharks, production of sdAb fragments using recombinant technology, characteristic properties of sdAb fragments, application of sdAb fragments in therapy, application of sdAb fragments in diagnos-tic and immunoanalytical methods and other pro-spective uses of sdAb fragments. This review article should facilitate orientation in the above mentioned subjects and enable rapid and easy searching for particular data and information.

Table 6A. Application of single-domain antibody fragments in therapy: inhibition of enzymes, toxins and other soluble proteins

Table 6B. Application of single-domain antibody fragments in therapy: activity modulation of cell surface proteins

Table 6C. Application of single-domain antibody fragments in therapy: pathogen neutralisation

Table 6D. Application of single-domain antibody fragments in therapy: intracellular expression of single-domain antibody fragments

Table 6E. Application of single-domain antibody fragments in therapy: oral administration of single-domain antibody fragments

Table 6F. Application of single-domain antibody fragments in therapy: prevention of amyloid plaque formation and protein aggregation

Table 6G. Application of single-domain antibody fragments in therapy: multispecific and multi-functional constructs

Table 6H. Application of single-domain antibody fragments in therapy: humanised single-domain antibody fragments

Table 7. Application of single-domain antibody frag-ments in diagnostic and immunoanalytic methods

Table 8. Other prospective uses of single-domain antibody fragments

Figure 1. The number of publications on heavy-chain antibodies and single-domain antibody frag-ments during the period from 1993 to June 2012

Figure 2. Structure of antibodies. (a) Molecule of con-ventional antibody (down), recombinant fragments (up); (b) molecule of camelid heavy-chain antibody (down), single-domain antibody fragment (up); (c) molecule of shark heavy-chain antibody (down), single-domain antibody fragment (up)


441

2. Review

2.1. Methods of searching and publication analysis

The publications were retrieved from the Web of Science® database using the following search profile: Topic = (nanobod* OR nanoantibod* OR “single-domain antibod*” OR “heavy-chain anti-bod*” OR “heavy chain-only antibod*” OR VHH OR IgNAR OR “llama* antibod*” OR “camel* an-tibod*” OR “new antigen receptor”), Timespan = all years or 2010–2012, Citation databases = SCI-EXPANDED, SSCI, A&HCI, CPCI-S, CPCI-SSH, BKCI-S and BKCI-SSH, Chemical Databases = CCR-EXPANDED and IC.

Using the above mentioned search profile, a total of 826 publications were retrieved. The first paper describing the occurrence of heavy-chain antibod-ies was published in 1993 by a Belgium research group. Since 2003, publication activity on the topic has sharply increased (Figure 1). In the last three years, 297 papers were published. In 2012, sixty-four papers have already been published at the time of our manuscript preparation. The older publications are predominantly oriented to the elucidation of the structure and characteristics of heavy-chain anti-bodies and their antigen-binding sites. The latest papers describe the possibilities of practical use of sdAb fragments, especially in medicine and therapy.

The Web of Science® utilities have been em-ployed for search results analysis. Of all the pub-lished papers, original research articles prevail

(78.2%). Our analysis shows that 262 institutions from 47 countries are concerned with the subject of sdAb fragments derived from heavy-chain antibod-ies. Authors, institutions, countries and scientific journals which show the highest publication activi-ties (Top 5) are listed in Table 1.

2.2. Review articles and monographs

Eighteen review articles are listed in Table 2 and characterized by key words (left column), abstracts (in the middle), and references (right column). These papers offer an overall review regarding all important topics, especially about the structure, properties, generation and evolution of heavy-chain antibodies in camelids and sharks (Conrath et al. 2003; Tillib 2011), engineering of sdAb fragments (Deffar et al. 2009), their production, structure and properties (Muyldermans et al. 2001; Harmsen and de Haard 2007), use of sdAb fragments as building blocks for the construction of multivalent and mul-tispecific conjugates (Saerens et al. 2008), applica-tion of sdAb fragments in therapy (Van Bockstaele et al. 2009; Wesolowski et al. 2009) and in im-munoanalytical and diagnostic methods (Huang et al. 2010; de Marco 2011). All these topics are discussed in detail also in original papers, which are presented in Tables 3 to 8. Recently, the new monograph “Single Domain Antibodies” (Saerens and Muyldermans 2012) has been launched with a complete methodology and key protocols for the construction of sdAb libraries and for the selec-

0

20

40

60

80

100

120

140

160

1993 1995 1997 1999 2001 2003 2005 2007 2009 2011

Figure 1. The number of publications on heavy-chain antibodies and single-domain antibody fragments during the period from 1993 to June 2012


442

tion and expression of sdAb fragments and their advanced derivatives. This publication hihlights the broad application potential of sdAb fragments and can be used as a practical guide to sdAb recombi-nant technologies.

2.3. Heavy-chain antibodies of camelids and sharks

Heavy-chain antibodies are homodimers of di-sulphide-linked heavy chains (Figure 2) that occur naturally in the serum of camelids and cartilaginous fish. The total amount of heavy-chain antibodies of all serum immunoglobulins has been determined to be about 45% in llama species, while in camels it is about 75% (Hamerscasterman et al. 1993). The function of heavy-chain antibodies in the immune system has not yet been completely explained. Due to the lack of light chains, the antigen-binding site of heavy-chain antibodies is formed by only three complementary determining regions (CDRs), com-pared to six CDRs in conventional antibodies. The CDR3 region of the heavy-chain antibody, which usually plays a crucial role in an antibody-antigen interaction, can be formed by a long finger-like polypeptide loop. Therefore, the antigen-binding site of the heavy-chain antibodies often exhibits a convex shape and differs sharply from the con-cave, groove-shaped antigen-binding site of con-ventional antibodies. With regard to the shape of the antigen-binding site, heavy-chain antibodies recognise preferably epitopes buried in clefts on protein surfaces, e.g. in enzyme active sites, which are usually less antigenic to conventional antibod-ies (Lauwereys et al. 1998; Decanniere et al. 1999; De Genst et al. 2006). The convex shaped antigen-binding site of heavy-chain antibodies is not very suitable for the binding of small molecules (hap-tens). Therefore, only a few papers focused on anti-hapten heavy-chain antibodies have been published to this date (Spinelli et al. 2001; Ladenson et al. 2006; Alvarez-Rueda et al. 2007). Other structural features of heavy-chain antibodies, which differen-tiate them from conventional antibodies, include a higher proportion of hydrophilic amino acids in variable domains (VHH/VNAR), the presence of disulfide bonds stabilizing the antigen-binding site, and an unusually high number of mutational hot spots responsible for structural variability of heavy-chain antibodies (Harmsen and de Haard 2007). In addition to structural studies, recent research

has been focused also on the evolution of heavy-chain antibodies (Nguyen et al. 2002; Flajnik et al. 2011), their possible function in the immune system (Ferrari et al. 2007; Saccodossi et al. 2012), and molecular mechanisms of germline gene segment rearrangement (Nguyen et al. 2000). Some investi-gators have also used heavy-chain antibodies as a suitable alternative to conventional polyclonal an-tibodies, mainly for diagnostic purposes (Anderson and Goldman 2008; Torigoe et al. 2012). Twenty-three papers (eleven of them published from 2010 to 2012) are presented in Table 3.

2.4. Production of sdAb fragments using recombinant technology

Due to their simple modular structure and a single-gene nature, sdAb fragments are easily produced in vitro as recombinant proteins. The production of sdAb fragments is based on the cloning of VHH or VNAR gene segments into phage display vectors, construction of large phage libraries and selection of high-affinity binders us-ing a biopanning process. SdAb fragments are ef-fectively expressed in different microbial hosts, of which Escherichia coli, Saccharomyces cerevisiae and Pichia pastoris are the most commonly used (Makvandi-Nejad et al. 2011; Ezzine et al. 2012; Gorlani et al. 2012). Bacterial species enable high-yield expression of sdAb fragments in the cytosol or in periplasm, whereas yeast cells utilize secre-tory pathways resulting in efficient disulfide bond formation, N-glycosylation and secretion of the recombinant product into the growth medium. Recent papers also describe several alternative strategies for sdAb fragment engineering and pro-duction, such as yeast or ribosome display systems (Yau et al. 2003; Ryckaert et al. 2010), expression of recombinant fragments in filamentous fungi (Joosten et al. 2005), in mammalian cells (Bazl et al. 2007) and in transgenic mice (Zou et al. 2005). Mutagenesis and recombination of CDR regions to improve the affinity and specificity of sdAb fragments have also been discussed (Swain et al. 2010; Fanning and Horn 2011). Current studies are focused on the development of new cloning and expression strategies to produce humanised fragments, multivalent constructs and fusions of antibody fragments to other proteins (Saerens et al. 2005; Vincke et al. 2009; Bell et al. 2010; Pollithy et al. 2011). Selected articles are presented in Table 4.


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2.5. Characteristic properties of sdAb fragments

Recombinant sdAb fragments are formed from only one single polypeptide chain containing about 120 amino acids. Due to their size (4 × 2.5 nm) and molecular weight (12 kDa to 15 kDa), sdAb fragments are the smallest available recombinant antibodies. Their small molecular size enables sdAb to penetrate easily into tissues and inter-cellular spaces, but is, however, the cause of their rapid clearance from the blood by renal filtration (Cortez-Retamozo et al. 2002). The rapid clear-ance of sdAb fragments from the blood by kid-

neys can negatively affect their therapeutic activity. Therefore, several strategies to prolong the half-life of sdAb fragments in serum have been described, e.g. conjugation of sdAb to another recombinant fragment binding specifically to serum albumins or immunoglobulins (Harmsen et al. 2005). SdAb frag-ments that bind to and are internalised by cerebro-microvascular endothelial cells are able to cross the blood-brain barrier and transmigrate into tissues of the central nerve system (Abulrob et al. 2005). Due to their simple monomeric structure, sdAb fragments are conformationally very stable (Dumoulin et al. 2002) and refold easily after heating to achieve their original native structure (Dolk et al. 2005b; Walper et

Figure 2. Structure of antibodies. (a) Molecule of conventional antibody (down), recombinant fragments (up); (b) molecule of camelid heavy-chain antibody (down), single-domain antibody fragment (up); (c) molecule of shark heavy-chain antibody (down), single-domain antibody fragment (up)


444

al. 2012). They show an increased resistance to high pressure and to low pH (Dumoulin et al. 2002) and maintain their biological activity even in an environ-ment with a high concentration of proteases and detergents (Dolk et al. 2005a; Hussack et al. 2011a). Owing to a higher content of hydrophilic amino acids, sdAb fragments are characterised by good solubility in water and limited agglutination ability (Conrath et al. 2005). Another characteristic feature particularly concerns the affinity of sdAb fragments, which remains usually equivalent to that of the native heavy-chain antibodies. This is a substantial differ-ence from other recombinant fragments, that show often lower affinity compared with their polyclonal or monoclonal counterparts. Eight papers published from 2010 to 2012 describing typical properties of sdAb fragments are presented in Table 5. Eight older articles should also be considered.

2.6. Application of sdAb fragments in therapy

The unique physicochemical and pharmacological properties of camelid and shark sdAb fragments give them a prospective use as new-generation thera-peutic agents for the treatment of serious human diseases. At present, some sdAb-based drugs are in the stage of preclinical testing on animal models or in vitro. Some other drugs have advanced to phase I or II of clinical testing in volunteers. The therapeutic effect of sdAb fragments is particularly based on the following principles: Firstly, a remarkable prefer-ence of sdAb fragments for binding into clefts and cavities on protein surfaces offers the possibility to develop selective therapeutics for efficient inhibition of enzymes (Paalanen et al. 2011), neutralization of proteolytic toxins (Hussack et al. 2011b) and activity modulation of cell surface proteins, such as recep-tors, ion channels and leukocyte ecto-enzymes (Wei et al. 2011; Altintas et al. 2012) involved in cancer and inflammatory diseases. Moreover, sdAb frag-ments recognize also cryptic epitopes hidden deeply in the clefts of virus capsids and in surface envelopes of parasites (Stijlemans et al. 2004; Henderson et al. 2007; Forsman et al. 2008). Secondly, intracellular expression of sdAb fragments as “intrabodies” is a potential strategy to target intracellular antigens, e.g. anti-apoptotic proteins, oncogenes or several viral proteins (Vercruysse et al. 2010). Thirdly, the high structural stability of sdAb fragments in harsh conditions makes them ideally suited for the immu-

notherapy of gastrointestinal disorders using oral ad-ministration (Harmsen et al. 2006; Vandenbroucke et al. 2010). Fourthly, the ability of sdAb fragments to transmigrate across the blood-brain barrier and to prevent amyloid plaque formation could be utilized in the diagnostics and therapy of neurodegenerative diseases (De Genst et al. 2010; Rutgers et al. 2011). Fifthly, a modular nature of sdAb fragments allows an easy engineering of multivalent or multispecific formats that show an increased therapeutic potency compared with monovalent sdAb fragments. Recent research is focused also on the generation of bifunc-tional constructs by coupling of sdAb fragments with enzymes and toxic substances for specific drug de-livery to bacterial or tumour cells (Zhang et al. 2004; Stone et al. 2007). Finally, the non-human origin of camelid and shark sdAb fragments could elicit their neutralisation by the human immune response and a decrease in their therapeutic effect. Therefore, a general strategy to produce humanised sdAb frag-ments has been described as a promising way for lowering the potential risk of immunogenicity of therapeutic sdAb fragments (Vincke et al. 2009). More than half of the 826 analysed papers discuss possible therapeutic applications of sdAb fragments. Key publications are presented in Tables 6A to H.

2.7. Application of sdAb fragments in diagnostic and immunoanalytical methods

Their effective penetration into tissues makes sdAb fragments good candidates for the construction of imaging probes used for in vivo monitoring of tu-mours, metastatic lesions, amyloid fibrils, etc. Such immuno-imaging probes are prepared by the labelling of sdAb fragments with short-lived isotopes, mainly with 99mTc. In contrast to radiolabelled monoclonal antibodies, small sdAb fragments show rapid antigen targeting and fast clearance from blood resulting in a contrast-enhanced imaging signal, reduced accumula-tion of labelled fragments in liver and lower radiation burden. The main limitation of sdAb-based imaging probes, however, is their high non-specific uptake in kidney and bladder (Vaneycken et al. 2010, 2011b). An innovative approach based on sdAb fragments fused to fluorescent proteins and expressed in living cells as “chromobodies” offers the possibility to trace intra-cellular antigens and to modulate protein function in living cells (Schmidthals et al. 2010). Nanoantibodies can also be used in immunoassays and in biosensors


445

for the quantitative analysis of contaminants and tox-ins in food and environmental samples (Conway et al. 2010; Dona et al. 2010; Kim et al. 2012) and also for diagnostics of serious human diseases (De Marni et al. 2012). Their small size and lower aggregation propensity enable immobilisation of extremely high numbers of nanoantibody molecules on the surface of the well of microtitre plate or biosensor transducer, which leads to a substantial increase in the sensitivity of the immunodetection system. Moreover, highly stable sdAb fragments are resistant to denaturing con-ditions used during biochip regeneration. For details of new publications see Table 7.

2.8. Other prospective uses of sdAb fragments

Besides possible therapeutic, diagnostic and immunoanalytic applications, sdAb fragments

derived from camelid and shark heavy-chain an-tibodies can also be employed as crystallography chaperones to stabilise the conformation of pro-teins during crystallisation trials (Abskharon et al. 2011). Immunoaffinity chromatography represents another prospective field for sdAb fragment ap-plication. SdAb fragments are highly resistant to the effects of organic solvents used for the elution of the captured ligands and for regeneration of im-munoaffinity columns (Franco et al. 2010). Recent applications, such as the development of transport systems across epithelia (Iqbal et al. 2011), inacti-vation of phages infecting dairy bacterial cultures (Hultberg et al. 2007) and mimotope selection strat-egies (Simmons et al. 2008) show the extremely broad application potential of sdAb fragments. Ten recent publications discussing some alternative ap-plications of sdAb fragments are listed in Table 8. This table also includes two older papers which make the review complete.

Table 1. Authors, countries, institutions and journals (Top 5) showing the highest publication activities con-cerning heavy-chain antibodies and single-domain antibody fragments (Web of Science®, 826 papers from 1993 to June 2012)

Item Number of publicationsAuthors (685 in total)Muyldermans S 96Wyns L 32Rahbarizadeh F 28Conrath K 25Tanha J 23Verrips CT 23Institutions (262 in total)Free University of Brussels/Flanders Institute for Biotechnology 188Utrecht University 61National Research Council Canada 47Ablynx NV 32Ghent University 23Countries (47 in total)USA 200Belgium 193Netherlands 115 England 78 France 63Journals (140 in total)Journal of Biological Chemistry 30Journal of Molecular Biology 25Molecular Immunology 24Journal of Immunological Methods 22PLoS ONE 19


446

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ecie

s, th

ese

spec

ial a

ntib

odie

s are

dev

oid

of li

ght c

hain

s and

are

com

pose

d of

a h

eavy

-cha

in h

omod

imer

.Thes

e so

-cal

led

heav

y-ch

ain

antib

odie

s (H

CA

bs) a

re e

xpre

ssed

aft

er a

V-D

-J re

arra

ngem

ent a

nd re

quir

e de

dica

ted

cons

tant

gam

ma-

gene

s. A

n im

mun

e re

spon

se is

rais

ed in

thes

e so

-cal

led

heav

y-ch

ain

antib

odie

s fol

low

ing

clas

sica

l im

mun

izat

ion

prot

ocol

s. Th

ese

HC

Abs

are

eas

ily

puri

fied

from

seru

m, a

nd th

e an

tigen

-bin

ding

frag

men

t int

erac

ts w

ith p

arts

of t

he ta

rget

that

are

less

ant

igen

ic to

con

vent

iona

l an

tibod

ies.

Sin

ce th

e an

tigen

-bin

ding

site

of t

he d

rom

edar

y H

CA

b is

com

pris

ed in

one

sing

le d

omai

n re

ferr

ed to

as v

aria

ble

dom

ain

of h

eavy

cha

in o

f HC

Ab

(VH

H) o

r nan

obod

y (N

b), w

e de

sign

ed a

stra

tegy

to c

lone

the

Nb

repe

rtoi

re o

f an

imm

uniz

ed

drom

edar

y an

d to

sele

ct th

e N

bs w

ith sp

ecifi

city

for o

ur ta

rget

ant

igen

s. Th

e m

onoc

lona

l Nbs

are

wel

l pro

duce

d in

bac

teri

a, a

re

very

stab

le a

nd h

ighl

y so

lubl

e, a

nd b

ind

thei

r cog

nate

ant

igen

with

hig

h affi

nity

and

spec

ifici

ty. W

e ha

ve su

cces

sful

ly d

evel

oped

re

com

bina

nt N

bs fo

r res

earc

h pu

rpos

es, a

s pro

be in

bio

sens

ors,

to d

iagn

ose

infe

ctio

ns, a

nd to

trea

t dis

ease

s lik

e ca

ncer

or t

rypa

-no

som

osis

.

Muy

lder

-m

ans e

t al.

2009


447

Nan

obod

y te

chno

logy

This

shor

t rev

iew

pro

vide

s an

intr

oduc

tion

to th

e ra

pidl

y de

velo

ping

fiel

d of

gen

erat

ion

and

utili

zatio

n of

“cam

el n

anoa

ntib

odie

s”

(or “

nano

bodi

es”)

. The

term

“nan

oant

ibod

y”or

“nan

obod

y” w

as g

iven

to si

ngle

-dom

ain

vari

able

frag

men

ts o

f spe

cial

type

of a

nti-

bodi

es th

at n

atur

ally

exi

st (i

n ad

ditio

n to

cla

ssic

al ty

pes o

f ant

ibod

ies)

in b

lood

of C

amel

idae

fam

ily a

nim

als a

nd in

som

e ch

on-

dric

hthy

an fi

shes

. The

exis

tenc

e of

ver

y effi

cien

t tec

hnol

ogy

of n

anob

ody

gene

ratio

n an

d so

me

very

use

ful c

hara

cter

istic

feat

ures

pr

omis

e a

big

pote

ntia

l for

thei

r use

in im

mun

obio

tech

nolo

gy a

nd m

edic

ine.

Tilli

b 20

11

Nan

obod

ies

Prop

etie

sPr

oduc

tion

App

licat

ions

Nan

obod

ies a

re a

ntib

ody-

deriv

ed th

erap

eutic

pro

tein

s tha

t con

tain

the

uniq

ue st

ruct

ural

and

func

tiona

l pro

pert

ies o

f nat

ural

ly o

ccur

-rin

g he

avy-

chai

n an

tibod

ies.

The

Nan

obod

y te

chno

logy

was

orig

inal

ly d

evel

oped

follo

win

g th

e di

scov

ery

that

cam

elid

ae (c

amel

s and

lla

mas

) pos

sess

fully

func

tiona

l ant

ibod

ies t

hat l

ack

light

cha

ins.

Thes

e he

avy-

chai

n an

tibod

ies c

onta

in a

sing

le v

aria

ble

dom

ain

(VH

H)

and

two

cons

tant

dom

ains

(CH

(2) a

nd C

H(3

)). Im

port

antly

, the

clo

ned

and

isola

ted

VH

H d

omai

n is

a pe

rfec

tly st

able

pol

ypep

tide

har-

borin

g th

e fu

ll an

tigen

-bin

ding

cap

acity

of t

he o

rigin

al h

eavy

-cha

in a

ntib

ody.

Thes

e ne

wly

disc

over

ed V

HH

dom

ains

with

thei

r uni

que

stru

ctur

al a

nd fu

nctio

nal p

rope

rtie

s for

m th

e ba

sis o

f a n

ew g

ener

atio

n of

ther

apeu

tic a

ntib

odie

s whi

ch w

ere

nam

ed N

anob

odie

s. Th

e ai

m o

f thi

s pap

er is

to sh

ow th

e pr

oper

ties o

f Nan

obod

ies,

thei

r pro

duct

ion

and

expr

essio

n, a

pplic

atio

ns a

nd th

eir c

linic

al st

atus

.

Deff

ar e

t al.

2009

Bio

tech

nolo

gica

l ap

plic

atio

nsSe

lect

ion

syst

ems

Cam

elid

s pro

duce

func

tiona

l ant

ibod

ies d

evoi

d of

ligh

t cha

ins o

f whi

ch th

e si

ngle

N-t

erm

inal

dom

ain

is fu

lly c

apab

le o

f ant

igen

bi

ndin

g. Th

ese

sing

le-d

omai

n an

tibod

y fr

agm

ents

(VH

Hs o

r Nan

obod

ies)

hav

e se

vera

l adv

anta

ges f

or b

iote

chno

logi

cal a

pplic

a-tio

ns. Th

ey a

re w

ell e

xpre

ssed

in m

icro

orga

nism

s and

hav

e a

high

stab

ility

and

solu

bilit

y. Fu

rthe

rmor

e, th

ey a

re w

ell s

uite

d fo

r co

nstr

uctio

n of

larg

er m

olec

ules

and

sele

ctio

n sy

stem

s suc

h as

pha

ge, y

east

, or r

ibos

ome

disp

lay.

This

min

irev

iew

offe

rs a

n ov

er-

view

of t

heir

pro

pert

ies a

s com

pare

d to

con

vent

iona

l ant

ibod

ies,

thei

r pro

duct

ion

in m

icro

orga

nism

s, w

ith a

focu

s on

yeas

ts, a

nd

thei

r the

rape

utic

app

licat

ions

.

Har

mse

n an

d de

H

aard

200

7

Ant

igen

-bin

ding

sit

eM

utat

ions

VH

-VL

pair

Nov

el e

pito

pes

The

antig

en-b

indi

ng si

te o

f ant

ibod

ies f

rom

ver

tebr

ates

is fo

rmed

by

com

bini

ng th

e va

riab

le d

omai

ns o

f a h

eavy

cha

in (V

H) a

nd

a lig

ht c

hain

(VL)

. How

ever

, ant

ibod

ies f

rom

cam

els a

nd Il

amas

are

an

impo

rtan

t exc

eptio

n to

this

in th

at th

eir s

era

cont

ain,

in

addi

tion,

a u

niqu

e ki

nd o

f ant

ibod

y th

at is

form

ed b

y he

avy

chai

ns o

nly

The

antig

en-b

indi

ng si

te o

f the

se a

ntib

odie

s con

sist

s of

one

sing

le d

omai

n, re

ferr

ed to

as V

HH

. This

art

icle

revi

ews t

he m

utat

ions

and

stru

ctur

al a

dapt

atio

ns th

at h

ave

ta k

en p

lace

to

resh

ape

a V

H o

f a V

H-V

L pa

ir in

to a

sing

le-d

omai

n V

HH

with

rete

ntio

n of

a su

ffici

ent v

aria

bilit

y, Th

e V

HH

has

a p

oten

t ant

igen

-bi

ndin

g ca

paci

ty a

nd p

rovi

des t

he a

dvan

tage

of i

nter

actin

g w

ith n

ovel

epi

tope

s tha

t are

inac

cess

ible

to c

onve

ntio

nal V

H-V

L pa

irs.

Muy

lder

-m

ans e

t al.

2001

Can

cer

ther

apeu

tics

Ove

r the

yea

rs, m

any

antib

odie

s hav

e be

en su

cces

sful

ly g

ener

ated

to tr

eat p

atie

nts w

ith li

fe-t

hrea

teni

ng d

isea

ses,

mos

t not

ably

ca

ncer

. Whi

le th

e fir

st g

ener

atio

n of

ant

ibod

ies,

orig

inat

ing

from

mic

e, c

ause

d se

vere

side

effe

cts a

nd w

ere

rela

tivel

y in

effici

ent,

tech

nolo

gica

l adv

ance

s hav

e m

ade

it po

ssib

le to

obt

ain

fully

hum

an a

ntib

odie

s for

ther

apeu

tic u

se. ‘

Hea

vy-c

hain

onl

y’ a

ntib

odie

s ha

ve re

cent

ly b

een

disc

over

ed in

the

bloo

d of

cam

elid

s. Be

caus

e of

thei

r siz

e, th

e an

tigen

-bin

ding

uni

ts o

f the

se a

ntib

odie

s com

pris-

ing

only

a si

ngle

Ig fo

ld a

re c

alle

d N

anob

odie

s. Th

ese

antib

ody

frag

men

ts h

ave

seve

ral r

emar

kabl

e fe

atur

es th

at m

ake

them

idea

l ca

ndid

ates

as n

ext-

gene

ratio

n ca

ncer

ther

apeu

tics.

Part

icul

arly

app

ealin

g is

thei

r abi

lity

to si

mul

tane

ously

inhi

bit v

ario

us c

ruci

al

grow

th fa

ctor

rece

ptor

s or t

heir

ligan

ds w

ith a

sing

le m

olec

ule.

In a

dditi

on, t

hey

are

easy

to c

lone

and

exp

ress

on

the

tip o

f fila

men

-to

us p

hage

, whi

ch o

pens

the

poss

ibili

ty to

sele

ct fo

r Nan

obod

ies i

nduc

ing

part

icul

ar b

iolo

gica

l effe

cts.

Nan

obod

ies h

ave

pote

ntia

l to

beco

me

impo

rtan

t can

cer t

hera

peut

ics i

n th

e ne

ar fu

ture

, dis

play

ing

uneq

ualle

d an

d un

prec

eden

ted

effica

cies

in tr

eatm

ent.

Roov

ers e

t al

. 200

7

Stru

ctur

al p

rope

rtie

sTh

erap

euti

c ap

plic

atio

nC

linic

al tr

ials

ALX

-008

1

Nan

obod

ies a

re th

erap

eutic

pro

tein

s der

ived

from

the

heav

y-ch

ain

vari

able

(V(H

)H) d

omai

ns th

at o

ccur

nat

ural

ly in

hea

vy-c

hain

-on

ly Ig

mol

ecul

es in

cam

elid

ae. Th

ese

V(H

)H d

omai

ns a

re th

e sm

alle

st k

now

n an

tigen

-bin

ding

ant

ibod

y fr

agm

ents

. Nan

obod

ies

can

be e

asily

pro

duce

d in

pro

kary

otic

or e

ukar

yotic

hos

t org

anis

ms,

and

thei

r uni

que

biop

hysi

cal a

nd p

harm

acol

ogic

al c

hara

cter

-is

tics r

ende

r the

se m

olec

ules

idea

l can

dida

tes f

or d

rug

deve

lopm

ent.

This

revi

ew d

escr

ibes

the

stru

ctur

al p

rope

rtie

s of n

anob

odie

s an

d fo

cuse

s on

thei

r uni

que

feat

ures

, whi

ch d

istin

guis

hes t

hese

mol

ecul

es fr

om o

ther

ant

ibod

y fo

rmat

s and

smal

l-mol

ecul

e dr

ugs.

Poss

ible

ther

apeu

tic a

pplic

atio

ns o

f nan

obod

ies a

re d

iscu

ssed

and

dat

a fr

om p

hase

I cl

inic

al tr

ials

of t

heno

vel ‘

first

-in-

clas

s’ an

ti-th

rom

botic

age

nt A

LX-0

081

(Abl

ynx

NV

) are

pre

sent

ed.

Van

Bock

-st

aele

et a

l. 20

09


448

Ther

apeu

tic

appl

icat

ions

Viru

ses

In 1

989,

a n

ew ty

pe o

f ant

ibod

y w

as id

entifi

ed, fi

rst i

n th

e se

ra o

f dro

med

arie

s and

late

r als

o in

all

othe

r spe

cies

of t

he C

amel

idae

fa

mily

. Thes

e an

tibod

ies d

o no

t con

tain

a li

ght c

hain

and

als

o la

ck th

e fir

st c

onst

ant h

eavy

dom

ain.

Tod

ay it

is st

ill u

ncle

ar w

hat

the

evol

utio

nary

adv

anta

ge o

f suc

h he

avy

chai

n-on

ly a

ntib

odie

s cou

ld b

e. In

shar

p co

ntra

st, t

he b

road

app

licab

ility

of t

he is

olat

ed

vari

able

ant

igen

-bin

ding

dom

ains

(VH

H) w

as ra

pidl

y re

cogn

ized

, esp

ecia

lly fo

r the

dev

elop

men

t of t

hera

peut

ic p

rote

ins,

cal

led

Nan

obod

ies.

Her

e w

e su

mm

ariz

e fir

st so

me

of th

e un

ique

cha

ract

eris

tics a

nd fe

atur

es o

f VH

Hs.

Thes

e w

ill n

ext b

e de

scri

bed

in

the

cont

ext o

f diff

eren

t exp

erim

enta

l the

rape

utic

app

licat

ions

of N

anob

odie

s aga

inst

diff

eren

t vir

uses

: HIV

, Hep

atiti

s B v

irus

, in

fluen

za v

irus

, Res

pira

tory

Syn

cytia

l vir

us, R

abie

s vir

us, F

MD

V, P

olio

viru

s, R

otav

irus

, and

PER

Vs. N

ext,

the

diag

nost

ic a

pplic

a-tio

n of

VH

Hs (

Vacc

inia

vir

us, M

arbu

rg v

irus

and

pla

nt T

ulip

vir

us X

), as

wel

l as a

n in

dust

rial

app

licat

ion

(lytic

lact

ococ

cal 9

36

phag

e) w

ill b

e de

scri

bed.

In a

dditi

on, t

he d

escr

ibed

dat

a sh

ow th

at m

onov

alen

t Nan

obod

ies c

an p

osse

ss u

niqu

e ch

arac

teri

stic

s not

ob

serv

ed w

ith c

onve

ntio

nal a

ntib

odie

s. Th

e st

raig

htfo

rwar

d fo

rmat

ting

into

biv

alen

t, m

ultiv

alen

t, an

d/or

mul

tispe

cific

Nan

obod

-ie

s allo

wed

tailo

ring

mol

ecul

es fo

r pot

ency

and

cro

ss-r

eact

ivity

aga

inst

vir

al ta

rget

s with

hig

h se

quen

ce d

iver

sity

.

Vanl

and-

scho

ot e

t al

. 201

1

VH

HV

NA

RIm

mun

e fu

ncti

on

mod

ulat

ing

Toxi

n/m

icro

be ta

rget

ing

Ant

ibod

ies a

re im

port

ant t

ools

for e

xper

imen

tal r

esea

rch

and

med

ical

app

licat

ions

. Mos

t ant

ibod

ies a

re c

ompo

sed

of tw

o he

avy

and

two

light

cha

ins.

Bot

h ch

ains

con

trib

ute

to th

e an

tigen

-bin

ding

site

whi

ch is

usu

ally

flat

or c

onca

ve. I

n ad

ditio

n to

thes

e co

nven

tiona

l ant

ibod

ies,

llam

as, o

ther

cam

elid

s, a

nd sh

arks

als

o pr

oduc

e an

tibod

ies c

ompo

sed

only

of h

eavy

cha

ins.

The

antig

en-

bind

ing

site

of t

hese

unu

sual

hea

vy c

hain

ant

ibod

ies (

hcA

bs) i

s for

med

onl

y by

a si

ngle

dom

ain,

des

igna

ted

VH

H in

cam

elid

hcA

bs

and

VN

AR

in sh

ark

hcA

bs. V

HH

and

VN

AR

are

easi

ly p

rodu

ced

as re

com

bina

nt p

rote

ins,

des

igna

ted

sing

le d

omai

n an

tibod

ies

(sdA

bs) o

r nan

obod

ies.

The

CD

R3 re

gion

of t

hese

sdA

bs p

osse

sses

the

extr

aord

inar

y ca

paci

ty to

form

long

fing

erlik

e ex

tens

ions

th

at c

an e

xten

d in

to c

aviti

es o

n an

tigen

s, e

.g.,

the

activ

e si

te c

revi

ce o

f enz

ymes

. Oth

er a

dvan

tage

ous f

eatu

res o

f nan

obod

ies

incl

ude

thei

r sm

all s

ize,

hig

h so

lubi

lity,

ther

mal

stab

ility

, ref

oldi

ng c

apac

ity, a

nd g

ood

tissu

e pe

netr

atio

n in

viv

o. H

ere

we

revi

ew

the

resu

lts o

f sev

eral

rece

nt p

roof

-of-

prin

cipl

e st

udie

s tha

t ope

n th

e ex

citin

g pe

rspe

ctiv

e of

usi

ng sd

Abs

for m

odul

atin

g im

mun

e fu

nctio

ns a

nd fo

r tar

getin

g to

xins

and

mic

robe

s.

Wes

o-lo

wsk

i et a

l. 20

09

Imm

unos

enso

rIm

mun

oaffi

nity

ch

rom

atog

raph

yIm

mun

o-im

agin

g

With

the

adve

nt o

f new

ant

ibod

y en

gine

erin

g te

chno

logi

es, c

onve

ntio

nal a

ntib

odie

s hav

e be

en m

inim

ized

into

smal

ler a

ntib

ody

form

ats.

Smal

l siz

e is

an im

port

ant a

dvan

tage

for c

urre

nt a

nd fu

ture

dia

gnos

tic d

evel

opm

ent.

Nan

obod

ies (

Abl

ynx)

are

am

ong

the

smal

lest

kn

own

antig

en-b

indi

ng a

ntib

ody

frag

men

ts, a

nd a

re d

eriv

ed fr

om th

e he

avy-

chai

n on

ly a

ntib

odie

s tha

t occ

ur n

atur

ally

in th

e se

rum

of

Cam

elid

ae. E

ndow

ed b

y na

tura

l evo

lutio

n, th

ese

Nan

obod

ies i

nher

ently

exh

ibit

uniq

ue b

ioph

ysic

al, b

ioch

emic

al a

nd p

harm

acol

ogic

al

char

acte

ristic

s. In

add

ition

to th

eir e

xcel

lent

pot

entia

l as m

olec

ules

in d

rug

deve

lopm

ent,

Nan

obod

ies p

osse

ss v

ery

attr

activ

e fu

nctio

nal

prop

ertie

s tha

t aid

in th

eir d

evel

opm

ent f

or d

iagn

ostic

tool

s. H

ere

we

pres

ent s

ever

al e

xam

ples

of c

urre

ntly

ava

ilabl

e ap

plic

atio

ns o

f N

anob

odie

s to

the

field

of i

mm

unos

enso

r for

can

cer,

imm

unoa

ffini

ty c

hrom

atog

raph

y, in

viv

o an

d in

trac

ellu

lar i

mag

ing.

Hua

ng e

t al

. 201

0

Bio

tech

nolo

gica

l ap

plic

atio

nsTh

e di

scov

ery

of th

e si

ngle

-dom

ain

antib

ody’s

pot

entia

ls h

as st

imul

ated

thei

r use

in a

n in

crea

sing

var

iety

of fi

elds

. The

rapi

d ac

cum

ulat

ion

of a

rtic

les d

escr

ibin

g ne

w a

pplic

atio

ns a

nd fu

rthe

r dev

elop

men

ts o

f est

ablis

hed

appr

oach

es h

as m

ade

it, th

eref

ore,

ne

cess

ary

to u

pdat

e th

e pr

evio

us re

view

s with

a n

ew a

nd m

ore

com

plet

e su

mm

ary

of th

e to

pic.

Con

clus

ions

: Bes

ide

the

nece

ssar

y ta

sk o

f upd

atin

g, th

is w

ork

anal

yses

in d

etai

l som

e ap

plic

ativ

e as

pect

s of t

he si

ngle

-dom

ain

antib

odie

s tha

t hav

e be

en o

vers

een

in th

e pa

st, s

uch

as th

eir e

ffica

cy in

affi

nity

chr

omat

ogra

phy,

as c

o-cr

ysta

lliza

tion

chap

eron

es, p

rote

in a

ggre

gatio

n co

ntro

llers

, en

zym

e ac

tivity

tune

rs, a

nd th

e sp

ecifi

citie

s of t

he u

ncon

vent

iona

l sin

gle-

dom

ain

frag

men

ts.

de M

arco

20

11


449

Imm

uno-

imag

ing

Imm

uno-

imag

ing

is a

dev

elop

ing

tech

nolo

gy th

at a

ims a

t stu

dyin

g di

seas

e in

pat

ient

s usi

ng im

agin

g te

chni

ques

such

as p

ositr

on

emis

sion

tom

ogra

phy

in c

ombi

natio

n w

ith ra

diol

abel

ed im

mun

oglo

bulin

der

ived

targ

etin

g pr

obes

. Nan

obod

ies a

re th

e sm

alle

st

antig

en-b

indi

ng a

ntib

ody-

frag

men

ts a

nd sh

ow fa

st a

nd sp

ecifi

c ta

rget

ing

in v

ivo.

Thes

e pr

obes

are

cur

rent

ly u

nder

inve

stig

atio

n as

th

erap

eutic

s but

pre

clin

ical

stud

ies i

ndic

ate

that

nan

obod

ies c

ould

als

o be

com

e th

e ne

xt g

ener

atio

n of

mag

ic b

ulle

ts fo

r im

mun

o-im

agin

g. In

itial

dat

a sh

ow th

at im

agin

g ca

n be

per

form

ed a

s ear

ly a

s one

hou

r pos

t-in

ject

ion

enab

ling

the

use

of sh

ort-

lived

radi

o-is

otop

es. Th

ese

uniq

ue p

rope

rtie

s sho

uld

enab

le p

atie

nt fr

iend

ly a

nd sa

fe im

agin

g pr

otoc

ols.

This

revi

ew fo

cuse

s on

the

curr

ent

stat

us o

f rad

iola

bele

d na

nobo

dies

as t

arge

ting

prob

es fo

r im

mun

o-im

agin

g.

Vane

ycke

n et

al.

2011

a

Man

-mad

e co

njug

ate

Ant

ibod

ies a

re la

rge

and

com

plex

mol

ecul

es, w

ith tw

o id

entic

al p

arts

that

bin

d in

depe

nden

tly o

f eac

h ot

her o

nto

the

antig

en a

nd th

e th

ird p

art o

f the

mol

ecul

e th

at d

icta

tes t

he e

ffect

or fu

nctio

n(s)

. To

impr

ove

the

ther

apeu

tic v

alue

of a

ntib

odie

s, pr

otei

n-en

gine

erin

g en

deav

ors r

educ

ed th

e si

ze o

f the

ant

igen

-bin

ding

moi

ety

to a

sing

le-d

omai

n un

it. O

ccas

iona

lly, i

t was

dem

onst

rate

d th

at th

e si

ngle

-do

mai

n an

tigen

-bin

ding

der

ivat

ives

of a

ntib

odie

s can

hav

e –

on th

eir o

wn

– an

ago

nist

ic (o

r ant

agon

istic

) effe

ct o

n th

eir t

arge

t. Th

e sm

all s

ize

and

stri

ct m

onom

eric

beh

avio

r, in

com

bina

tion

with

oth

er b

ioch

emic

al p

rope

rtie

s suc

h as

hig

h so

lubi

lity

and

high

spec

ific-

ity a

nd a

ffini

ty fo

r the

cog

nate

ant

igen

, mak

e si

ngle

-dom

ain

antib

odie

s ide

al to

des

ign

nove

l man

-mad

e co

njug

ates

har

ness

ed w

ith

inno

vativ

e eff

ecto

r fun

ctio

ns o

utsi

de th

e re

ach

of c

lass

ical

ant

ibod

ies.

Saer

ens e

t al

. 200

8

Smal

ler

reco

mbi

nant

an

tibo

dy fr

agm

ents

Mul

tiva

lent

/mul

tisp

ecifi

c re

agen

tSi

ngle

ant

ibod

y do

mai

nsEn

hanc

ed th

erap

euti

c effi

cacy

With

18

mon

oclo

nal a

ntib

ody

(mA

b) p

rodu

cts c

urre

ntly

on

the

mar

ket a

nd m

ore

than

100

in c

linic

al tr

ials

, it i

s cle

ar th

at e

ngi-

neer

ed a

ntib

odie

s hav

e co

me

of a

ge a

s bio

phar

mac

eutic

als.

In fa

ct, b

y 20

08, e

ngin

eere

d an

tibod

ies a

re p

redi

cted

to a

ccou

nt fo

r >

30%

of a

ll re

venu

es in

the

biot

echn

olog

y m

arke

t. Sm

alle

r rec

ombi

nant

ant

ibod

y fr

agm

ents

(for

exa

mpl

e, c

lass

ic m

onov

alen

t an

tibod

y fr

agm

ents

(Fab

, scF

v)) a

nd e

ngin

eere

d va

rian

ts (d

iabo

dies

, tri

abod

ies,

min

ibod

ies a

nd si

ngle

-dom

ain

antib

odie

s) a

re

now

em

ergi

ng a

s cre

dibl

e al

tern

ativ

es. Th

ese

frag

men

ts re

tain

the

targ

etin

g sp

ecifi

city

of w

hole

mA

bs b

ut c

an b

e pr

oduc

ed m

ore

econ

omic

ally

and

pos

sess

oth

er u

niqu

e an

d su

peri

or p

rope

rtie

s for

a ra

nge

of d

iagn

ostic

and

ther

apeu

tic a

pplic

atio

ns. A

ntib

ody

frag

men

ts h

ave

been

forg

ed in

to m

ultiv

alen

t and

mul

tispe

cific

reag

ents

, lin

ked

to th

erap

eutic

pay

load

s (su

ch a

s rad

ionu

clid

es,

toxi

ns, e

nzym

es, l

ipos

omes

and

vir

uses

) and

eng

inee

red

for e

nhan

ced

ther

apeu

tic e

ffica

cy. R

ecen

tly, s

ingl

e an

tibod

y do

mai

ns h

ave

been

eng

inee

red

and

sele

cted

as t

arge

ting

reag

ents

aga

inst

hith

erto

imm

unos

ilent

cav

ities

in e

nzym

es, r

ecep

tors

and

infe

ctio

us

agen

ts. S

ingl

e-do

mai

n an

tibod

ies a

re a

ntic

ipat

ed to

sign

ifica

ntly

exp

and

the

repe

rtoi

re o

f ant

ibod

y-ba

sed

reag

ents

aga

inst

the

vast

ra

nge

of n

ovel

bio

mar

kers

bei

ng d

isco

vere

d th

roug

h pr

oteo

mic

s. A

s thi

s rev

iew

aim

s to

show

, the

re is

trem

endo

us p

oten

tial f

or a

ll an

tibod

y fr

agm

ents

eith

er a

s rob

ust d

iagn

ostic

reag

ents

(for

exa

mpl

e in

bio

sens

ors)

, or a

s non

imm

unog

enic

in v

ivo

biop

harm

a-ce

utic

als w

ith su

peri

or b

iodi

stri

butio

n an

d bl

ood

clea

ranc

e pr

oper

ties.

Hol

liger

an

d H

udso

n 20

05

Mol

ecul

ar im

agin

gTa

rget

-ori

ente

d th

erap

ies

Ant

ibod

y de

riva

tive

Alt

erna

tive

pro

tein

The

rapi

d an

d on

goin

g di

scov

ery

of n

ew d

isea

se re

late

d bi

omar

kers

lead

s to

a dr

amat

ic p

arad

igm

cha

nge

in h

uman

hea

lthca

re a

nd

cons

titut

es th

e ba

sis f

or a

trul

y pe

rson

aliz

ed m

edic

ine.

Mol

ecul

ar im

agin

g en

able

s ear

ly d

etec

tion

and

clas

sific

atio

n of

hum

an d

isea

ses

and

prov

ides

val

uabl

e da

ta fo

r opt

imiz

ed, t

arge

t-or

ient

ed th

erap

ies.

By n

ow, t

he b

ioch

emic

al a

nd p

hysi

olog

ical

pro

pert

ies o

f ant

ibod

y de

riva

tives

or a

ltern

ativ

e pr

otei

n sc

affol

ds c

an b

e en

gine

ered

for t

he d

etec

tion

of a

wid

e ra

nge

of ta

rget

stru

ctur

es. Th

e su

cces

sful

ap

plic

atio

n of

thes

e re

agen

ts in

ani

mal

s, xe

nogr

aft m

odel

s and

cel

ls in

pre

clin

ical

rese

arch

cle

arly

dem

onst

rate

thei

r util

ity fo

r mol

ecu-

lar i

mag

ing.

Des

pite

thes

e pr

omis

ing

pers

pect

ives

, onl

y a

few

ant

ibod

ies a

nd re

com

bina

nt p

rote

ins a

re u

sed

yet f

or m

olec

ular

imag

-in

g in

hum

an m

edic

ine.

Esp

ecia

lly th

e hi

gh sa

fety

dem

ands

and

the

need

to e

limin

ate

off ta

rget

effe

cts i

n hu

man

s req

uire

ext

ensi

ve

rese

arch

and

dev

elop

men

t effo

rts.

Rom

er e

t al

. 201

1


450

X-r

ay c

ryst

allo

grap

hyC

ryst

alliz

atio

n

chap

eron

eC

onfo

rmat

iona

l he

tero

gene

ity

The

prep

arat

ion

of d

iffra

ctio

n qu

ality

cry

stal

s rem

ains

the

maj

or b

ottle

neck

in m

acro

mol

ecul

ar X

-ray

cry

stal

logr

aphy

. A c

ryst

alliz

a-tio

n ch

aper

one

is a

n au

xilia

ry p

rote

in, s

uch

as fr

agm

ents

of m

onoc

lona

l ant

ibod

ies,

that

bin

ds to

and

incr

ease

s the

cry

stal

lizat

ion

prob

abili

ty o

f a ta

rget

mol

ecul

e of

inte

rest

. Suc

h ch

aper

ones

redu

ce c

onfo

rmat

iona

l het

erog

enei

ty, m

ask

coun

terp

rodu

ctiv

e su

rfac

es

whi

le e

xten

ding

surf

aces

pre

disp

osed

to fo

rmin

g cr

ysta

l con

tact

s, a

nd p

rovi

de p

hasi

ng in

form

atio

n. C

ryst

alliz

atio

n ch

aper

ones

gen

-er

ated

usi

ng re

com

bina

nt te

chno

logi

es h

ave

emer

ged

as su

peri

or a

ltern

ativ

es th

at in

crea

se th

e th

roug

hput

and

elim

inat

e in

here

nt

limita

tions

ass

ocia

ted

with

ant

ibod

y pr

oduc

tion

by a

nim

al im

mun

izat

ion

and

the

hybr

idom

a te

chno

logy

.

Koi

de 2

009

Rec

entl

y pu

blis

hed

m

onog

raph

Sing

le D

omai

n A

ntib

odie

s

http

://w

ww.

spri

nger

.com

/bio

med

/imm

unol

ogy/

book

/978

-1-6

1779

-967

-9Th

e de

velo

pmen

t of t

he h

ybri

dom

a te

chno

logy

cre

ated

the

poss

ibili

ty to

obt

ain

unlim

ited

amou

nts o

f mon

oclo

nal a

ntib

odie

s (m

Ab)

w

ith h

igh

spec

ifici

ty a

nd a

ffini

ty fo

r any

targ

et a

nd to

intr

oduc

e m

Abs

in a

wid

e ra

nge

of a

pplic

atio

ns. E

xam

ples

of a

ntib

ody-

base

d dr

ugs i

n th

erap

eutic

sett

ings

and

ant

ibod

y-ba

sed

prob

es in

dia

gnos

tics a

re in

finite

. How

ever

, the

bul

ky si

ze o

f mA

bs, c

ostly

pro

duc-

tion,

and

cum

bers

ome

engi

neer

ing

reta

rded

or h

ampe

red

regu

larly

thei

r str

eam

lined

dev

elop

men

t in

som

e ap

plic

atio

ns. C

onse

quen

tly,

mA

bs b

ecam

e th

e fo

cus o

f man

y at

tem

pts t

o m

inim

ize

the

size

and

com

plex

ity o

f the

ir an

tigen

-bin

ding

frag

men

ts. E

vent

ually

, the

se

effor

ts le

d to

the

reco

mbi

nant

pro

duct

ion

of sm

alle

r ant

igen

bind

ing

frag

men

ts su

ch a

s Fab

or s

cFv

(whe

re a

synt

hetic

link

er c

onne

cts

the

vari

able

dom

ains

of h

eavy

and

ligh

t cha

in, i

.e.,

VH

and

VL)

, and

eve

n sd

Abs

(sin

gle

dom

ain

antib

odie

s der

ived

mos

tly o

f the

VH

). A

lthou

gh th

e fir

st se

t of s

dAbs

offe

red

sign

ifica

nt a

dvan

tage

s, th

ey a

lso

suffe

red

from

mul

tiple

shor

tcom

ings

, all

of w

hich

hav

e be

en

rem

edia

ted

by e

lega

nt e

ngin

eeri

ng. I

nter

estin

gly,

whi

le sc

ient

ists

wer

e de

sign

ing,

eng

inee

ring

, and

shap

ing

the

idea

l sdA

b, a

sere

ndip

i-to

us d

isco

very

show

ed th

at a

sim

ilar e

ngin

eeri

ng o

ccur

red

alre

ady

in n

atur

e in

the

cam

elid

s, an

d la

ter o

n, it

was

foun

d th

at c

artil

agi-

nous

fish

ant

ibod

ies p

erfo

rmed

the

exer

cise

eve

n ea

rlier

on

in e

volu

tion.

Thes

e an

imal

s hav

e in

thei

r blo

od fu

nctio

nal a

ntib

ody

isot

ype

com

pose

d of

hea

vy c

hain

s – o

nly

that

lack

ligh

t cha

ins,

in a

dditi

on to

the

clas

sica

l ant

ibod

ies c

onta

inin

g tw

o he

avy

and

two

light

ch

ains

. Thes

e he

avy-

chai

n an

tibod

ies (

HC

Abs

) rec

ogni

ze th

e an

tigen

via

a si

ngle

var

iabl

e do

mai

n, re

ferr

ed to

as V

HH

or V

-NA

R. Th

e V

HH

or V

-NA

R is

the

smal

lest

inta

ct a

ntig

en-b

indi

ng fr

agm

ent t

hat c

an b

e pr

oduc

ed re

com

bina

ntly

at l

ow c

ost.

The

valu

able

pro

per-

ties o

f man

-mad

e sd

Abs

, VH

Hs,

and

V-N

ARs

incl

udin

g so

lubi

lity

and

stab

ility

, hig

h affi

nity

and

spec

ifici

ty fo

r the

ir co

gnat

e an

tigen

, sm

all s

ize

and

stri

ct m

onom

eric

beh

avio

r offe

r man

y op

port

uniti

es. A

s a re

sult,

seve

ral s

pin-

off c

ompa

nies

hav

e be

en fo

unde

d in

Aus

-tr

alia

, Bel

gium

, Eng

land

, Ger

man

y, N

ethe

rland

s, an

d Sc

otla

nd th

at in

trod

uced

thes

e pr

otei

ns su

cces

sful

ly in

a w

ide

rang

e of

app

lica-

tions

to c

over

a sp

ecia

l nee

d in

rese

arch

or e

ven

to p

rodu

ce n

ext-

gene

ratio

n th

erap

eutic

s in

the

clin

ic (P

refa

ce b

y th

e ed

itors

).

Title

s of t

he c

ontr

ibut

ions

:PA

RT I

OV

ERV

IEW

OF

SIN

GLE

DO

MA

IN A

NT

IBO

DIE

SFr

om W

hole

Mon

oclo

nal A

ntib

odie

s to

Sing

le D

omai

n A

ntib

odie

s: Th

ink

Smal

lIn

trod

uctio

n to

Hea

vy C

hain

Ant

ibod

ies a

nd D

eriv

ed N

anob

odie

s O

verv

iew

and

Dis

cove

ry o

f IgN

ARs

and

Gen

erat

ion

of V

NA

RsPA

RT II

SIN

GLE

DO

MA

IN A

NT

IBO

DY

LIBR

ARY

CO

NST

RUC

TIO

NC

reat

ion

of th

e La

rge

and

Hig

hly

Func

tiona

l Syn

thet

ic R

eper

toire

of H

uman

VH

and

Vκ

Dom

ain

Ant

ibod

ies

Prep

arat

ion

of a

Naï

ve L

ibra

ry o

f Cam

elid

Sin

gle

Dom

ain

Ant

ibod

ies

PART

III S

ELEC

TIO

N O

F SI

NG

LE D

OM

AIN

AN

TIB

OD

IES

Sele

ctio

n by

Pha

ge D

ispl

ay o

f Sin

gle

Dom

ain

Ant

ibod

ies S

peci

fic to

Ant

igen

s in

Thei

r Nat

ive

Con

form

atio

nSe

mia

utom

ated

Pan

ning

of N

aive

Cam

elid

ae L

ibra

ries

and

Sel

ectio

n of

Sin

gle-

Dom

ain

Ant

ibod

ies A

gain

st P

eptid

e A

ntig

ens

Pich

ia S

urfa

ce D

ispl

ay: A

Too

l for

Scr

eeni

ng S

ingl

e D

omai

n A

ntib

odie

sBa

cter

ial T

wo

Hyb

rid:

A V

ersa

tile

One

-Ste

p In

trac

ellu

lar S

elec

tion

Met

hod

Intr

acel

lula

r Ant

ibod

y C

aptu

re (I

AC

) Met

hods

or S

ingl

e D

omai

n A

ntib

odie

sSe

lect

ion

of F

unct

iona

l Sin

gle

Dom

ain

Ant

ibod

y Fr

agm

ents

for I

nter

feri

ng w

ith P

rote

in–P

rote

in In

tera

ctio

ns In

side

Cel

ls: A

“One

Pl

asm

id” M

amm

alia

n Tw

o-H

ybri

d Sy

stem

Saer

ens

and

Muy

l-de

rman

s 20

12


451

Cel

l-Fre

e Se

lect

ion

of D

omai

n A

ntib

odie

s by

In V

itro

Com

part

men

taliz

atio

n Se

lect

ion

of V

HH

s Und

er A

pplic

atio

n C

ondi

tions

Isol

atio

n an

d C

hara

cter

izat

ion

of C

lost

ridi

um d

iffici

le T

oxin

-Spe

cific

Sin

gle-

Dom

ain

Ant

ibod

ies

Sele

ctio

n of

VH

H A

ntib

ody

Frag

men

ts Th

at R

ecog

nize

Diff

eren

t Aβ

Dep

ositi

ons U

sing

Com

plex

Imm

une

Libr

arie

sPA

RT IV

EX

PRES

SIO

N O

F SI

NG

LE D

OM

AIN

AN

TIB

OD

IES

AN

D D

ERIV

ATIV

ESEx

pres

sion

of S

ingl

e-D

omai

n A

ntib

odie

s in

Bact

eria

l Sys

tem

sEx

pres

sion

of V

HH

s in

Sacc

haro

myc

es c

erev

isia

eSt

able

Exp

ress

ion

of C

him

eric

Hea

vy C

hain

Ant

ibod

ies i

n C

HO

Cel

lsPr

oduc

tion

of C

amel

-Lik

e A

ntib

odie

s in

Plan

tsPA

RT V

IMPR

OV

EMEN

T A

ND

APP

LIC

ATIO

NS

OF

SIN

GLE

DO

MA

IN A

NT

IBO

DIE

SSe

lect

ing

and

Puri

fyin

g A

uton

omou

s Hum

an V

aria

ble

Hea

vy (V

H) D

omai

nsSo

lubi

lity

and

Stab

ility

Eng

inee

ring

of H

uman

VH

Dom

ains

Impr

ovem

ent o

f Pro

teol

ytic

Sta

bilit

y Th

roug

h In

Sili

co E

ngin

eeri

ngSe

lect

ion

of H

uman

VH

Sin

gle

Dom

ains

with

Impr

oved

Bio

phys

ical

Pro

pert

ies b

y Ph

age

Dis

play

Im

prov

emen

t of S

ingl

e D

omai

n A

ntib

ody

Stab

ility

by

Dis

ulfid

e Bo

nd In

trod

uctio

nC

hara

cter

izat

ion

of S

ingl

e-D

omai

n A

ntib

odie

s with

an

Engi

neer

ed D

isul

fide

Bond

Affi

nity

Mat

urat

ion

of S

ingl

e-D

omai

n A

ntib

odie

s by

Yeas

t Sur

face

Dis

play

Mul

tival

ent D

ispl

ay o

f Sin

gle-

Dom

ain

Ant

ibod

ies

Met

hods

for D

eter

min

ing

the

PK P

aram

eter

s of A

lbud

Abs

and

of L

ong

Seru

m H

alf-

Life

Dru

gs M

ade

Usi

ng th

e A

lbud

Ab

Tech

nolo

gyFl

uore

scen

t Pro

tein

Spe

cific

Nan

otra

ps to

Stu

dy P

rote

in–P

rote

in In

tera

ctio

ns a

nd H

isto

ne-T

ail P

eptid

e Bi

ndin

gSi

te-S

peci

fic L

abel

ing

of H

is-T

agge

d N

anob

odie

s with

99m

Tc: A

Pra

ctic

al G

uide

N

anob

ody-

Base

d C

hrom

atin

Imm

unop

reci

pita

tion

Use

r-Fr

iend

ly E

xpre

ssio

n Pl

asm

ids E

nabl

e th

e Fu

sion

of V

HH

s to

App

licat

ion-

Spec

ific

Tags

App

licat

ion

of S

ingl

e-D

omai

n A

ntib

odie

s in

Tum

or H

isto

chem

istr

yPA

RT V

I CA

SE S

TU

DIE

SN

anob

odie

s as S

truc

tura

l Pro

bes o

f Pro

tein

Mis

fold

ing

and

Fibr

il Fo

rmat

ion

Mol

ecul

ar Im

agin

g U

sing

Nan

obod

ies:

A C

ase

Stud

yC

ase

Stud

y on

Liv

e C

ell A

popt

osis

-Ass

ay U

sing

Lam

in-C

hrom

obod

y C

ell-L

ines

for H

igh-

Con

tent

Ana

lysi

s


452

Tabl

e 3.

Cam

elid

and

sha

rk h

eavy

-cha

in a

ntib

odie

s

Hea

vy-c

hain

dim

erC

amel

us d

rom

edar

ius

Ant

igen

-bin

ding

re

pert

oire

CH

1 do

mai

nA

ntib

ody

engi

neer

ing

Ran

dom

ass

ocia

tion

of V

L an

d V

H r

eper

toir

es c

ontr

ibut

es c

onsi

dera

bly

to a

ntib

ody

dive

rsit

y. T

he d

iver

sity

and

the

affin

ity

are

then

incr

ease

d by

hyp

erm

utat

ion

in B

cel

ls lo

cate

d in

ger

min

al c

entr

es. E

xcep

t in

the

case

of ‘

heav

y ch

ain’

dis

ease

, nat

u-ra

lly o

ccur

ring

hea

vy-c

hain

ant

ibod

ies

have

not

bee

n de

scri

bed,

alth

ough

ant

igen

bin

ding

has

bee

n de

mon

stra

ted

for

sepa

-ra

ted

heav

y ch

ains

or

clon

ed V

H d

omai

ns. H

ere

we

inve

stig

ate

the

pres

ence

of c

onsi

dera

ble

amou

nts

of Ig

G-l

ike

mat

eria

l of

M(r

) 100

K in

the

seru

m o

f the

cam

el (C

amel

us d

rom

edar

ius)

. The

se m

olec

ules

are

com

pose

d of

hea

vy-c

hain

dim

ers

and

are

devo

id o

f lig

ht c

hain

s, b

ut n

ever

thel

ess

have

an

exte

nsiv

e an

tige

n-bi

ndin

g re

pert

oire

, a fi

ndin

g th

at c

alls

into

que

stio

n th

e ro

le o

f lig

ht c

hain

s in

the

cam

el. C

amel

hea

vy-c

hain

IgG

s la

ck C

H1,

whi

ch in

one

IgG

cla

ss m

ight

be

stru

ctur

ally

rep

lace

d by

an

ext

ende

d hi

nge.

Hea

vy-c

hain

IgG

s ar

e a

feat

ure

of a

ll ca

mel

ids.

The

se fi

ndin

gs o

pen

new

per

spec

tive

s in

the

engi

neer

ing

of a

ntib

odie

s.

Ham

er-

scas

ter-

man

et

al. 1

993

Nur

se sh

ark

New

ant

igen

rec

epto

rSe

quen

ce a

naly

sis

Rea

rran

gem

ent

Som

atic

div

ersi

ficat

ion

Imm

unog

lobu

lin a

nd T

-cel

l rec

epto

r (T

CR)

mol

ecul

es a

re c

entr

al to

the

adap

tive

imm

une

syst

em, S

eque

nce

cons

erva

tion

, si

mila

riti

es in

dom

ain

stru

ctur

e, a

nd u

sage

of s

imila

r re

com

bina

tion

sig

nal s

eque

nces

and

rec

ombi

nati

on m

achi

nery

indi

cate

th

at th

ere

was

pro

babl

y a

tim

e du

ring

evo

luti

on w

hen

an a

nces

tral

rec

epto

r di

verg

ed to

the

mod

ern-

day

imm

unog

lobu

lin

and

TC

R(1–

3). O

ther

mol

ecul

es th

at u

nder

go r

earr

ange

men

t hav

e no

t bee

n de

scri

bed

in v

erte

brat

es, n

or h

ave

inte

rmed

iate

s be

en id

enti

fied

that

hav

e fe

atur

es o

f bot

h th

ese

gene

fam

ilies

, We

repo

rt h

ere

the

isol

atio

n of

a n

ew m

embe

r of

the

imm

uno-

glob

ulin

sup

erfa

mily

from

the

nurs

e sh

ark,

Gin

glym

osto

ma

cirr

atum

, whi

ch c

onta

ins

one

vari

able

and

five

con

stan

t dom

ains

an

d is

foun

d as

a d

imer

in s

erum

. Ana

lyse

s of

com

plem

enta

ry D

NA

clo

nes

show

ext

ensi

ve s

eque

nce

dive

rsit

y w

ithin

var

iabl

e do

mai

ns, w

hich

is g

ener

ated

by

both

rea

rran

gem

ent a

nd s

omat

ic d

iver

sific

atio

n m

echa

nism

s. O

ur r

esul

ts s

ugge

st th

at r

ear-

rang

ing

loci

dis

tinc

t fro

m im

mun

oglo

bulin

and

TC

R ha

ve a

rise

n du

ring

evo

luti

on.

Gre

en-

berg

et

al. 1

995

Am

ino

acid

sub

stit

utio

nD

isul

phid

e bo

ndC

yste

in

The

ant

igen

-bin

ding

frag

men

t of f

unct

iona

l hea

vy c

hain

ant

ibod

ies

(HC

Abs

) in

cam

elid

s co

mpr

ises

a s

ingl

e do

mai

n, n

amed

th

e va

riab

le d

omai

n of

hea

vy c

hain

of H

CA

bs (V

HH

). T

he V

HH

har

bors

rem

arka

ble

amin

o ac

id s

ubst

ituti

ons

in th

e fr

ame-

wor

k re

gion

-2 to

gen

erat

e an

ant

igen

-bin

ding

dom

ain

that

func

tion

s in

the

abse

nce

of a

ligh

t cha

in p

artn

er. T

he s

ubst

ituti

ons

prov

ide

a m

ore

hydr

ophi

lic, h

ence

mor

e so

lubl

e, c

hara

cter

to th

e V

HH

but

dec

reas

e th

e in

trin

sic

stab

ility

of t

he d

omai

n.

Her

e w

e in

vest

igat

e th

e fu

ncti

onal

rol

e of

an

addi

tion

al h

allm

ark

of d

rom

edar

y V

HH

s, i.

e. th

e ex

tra

disu

lfide

bon

d be

twee

n th

e fir

st a

nd th

ird

anti

gen-

bind

ing

loop

s. A

fter

sub

stitu

ting

the

cyst

eine

s fo

rmin

g th

is in

terl

oop

cyst

ine

by a

ll 20

am

ino

acid

s, w

e se

lect

ed a

nd c

hara

cter

ized

sev

eral

VH

Hs

that

ret

ain

anti

gen

bind

ing

capa

city

. Alth

ough

VH

H d

omai

ns c

an fu

nc-

tion

in th

e ab

senc

e of

an

inte

rloo

p di

sulfi

de b

ond,

we

dem

onst

rate

that

its

pres

ence

con

stitu

tes

a ne

t adv

anta

ge. F

irst

, the

di

sulfi

de b

ond

stab

ilize

s th

e do

mai

n an

d co

unte

ract

s th

e de

stab

iliza

tion

by

the

fram

ewor

k re

gion

-2 h

allm

ark

amin

o ac

ids.

Se

cond

, the

dis

ulfid

e bo

nd r

igid

ifies

the

long

thir

d an

tige

n-bi

ndin

g lo

op, l

eadi

ng to

a s

tron

ger

anti

gen

inte

ract

ion.

Thi

s du

al

bene

ficia

l eff

ect e

xpla

ins

the

in v

ivo

anti

body

mat

urat

ion

proc

ess

favo

ring

VH

H d

omai

ns w

ith a

n in

terl

oop

disu

lfide

bon

d.

Gov

aert

et

al.

2012


453

Am

ino

acid

alig

nmen

tC

DR

iden

tific

atio

nC

onfo

rmat

ion

stud

y

Cam

elid

s hav

e a

spec

ial t

ype

of A

b, k

now

n as

hea

vy c

hain

Abs

, whi

ch a

re d

evoi

d of

cla

ssic

al A

b lig

ht c

hain

s. R

elat

ive

to c

lass

ical

A

bs, c

amel

id h

eavy

cha

in A

bs (c

Abs

) hav

e co

mpa

rabl

e im

mun

ogen

icity

, Ag

reco

gniti

on d

iver

sity

and

bin

ding

affi

nitie

s, h

ighe

r sta

-bi

lity

and

solu

bilit

y, an

d be

tter

man

ufac

tura

bilit

y, m

akin

g th

em p

rom

isin

g ca

ndid

ates

for a

ltern

ate

ther

apeu

tic sc

affol

ds. R

atio

nal

engi

neer

ing

of c

Abs

to im

prov

e th

erap

eutic

func

tion

requ

ires

kno

wle

dgeo

f the

diff

eren

ces o

f seq

uenc

e an

d st

ruct

ural

feat

ures

be

twee

n cA

bs a

nd c

lass

ical

Abs

. In

this

stud

y, am

ino

acid

sequ

ence

s of 2

7 cA

b va

riab

le re

gion

s (V

(H)H

) wer

e al

igne

d w

ith th

e re

spec

tive

regi

ons o

f 54

clas

sica

l Abs

to d

etec

t am

ino

acid

diff

eren

ces,

ena

blin

g au

tom

atic

iden

tifica

tion

of c

Ab

V(H

)H C

DRs

. CD

R an

alys

is re

veal

ed th

at th

e H

1 of

ten

(and

som

etim

es th

e H

2) a

dopt

s div

erse

con

form

atio

ns n

ot c

lass

ifiab

le b

y es

tabl

ishe

d ca

noni

cal

rule

s. A

lso,

alth

ough

the

cAb

H3

is m

uch

long

er th

an c

lass

ical

H3

loop

s, it

oft

en c

onta

ins c

omm

on st

ruct

ural

mot

ifs a

nd so

me-

times

a d

isul

fide

bond

to th

e H

1. L

ever

agin

g th

ese

obse

rvat

ions

, we

crea

ted

a M

onte

Car

lo-b

ased

cA

b V

(H)H

stru

ctur

al m

odel

ing

tool

, whe

re th

e C

DR

H1

and

H2

loop

s exh

ibite

d a

med

ian

root

-mea

n-sq

uare

dev

iatio

n to

nat

ives

of 3

.1 a

nd 1

.5 a

ngst

rom

, res

pec-

tivel

y. Th

e pr

otoc

ol g

ener

ated

8–1

2, 1

4–16

, and

16–

24 re

sidu

e H

3 lo

ops w

ith a

med

ian

root

-mea

n-sq

uare

dev

iatio

n to

nat

ives

of

5.7,

4.5

, and

6.8

ang

stro

m, r

espe

ctiv

ely.

The

larg

e de

viat

ion

of th

e pr

edic

ted

loop

s und

ersc

ores

the

chal

leng

e in

mod

elin

g su

ch lo

ng

loop

s. c

Ab

V(H

)H h

omol

ogy

mod

els c

an p

rovi

de st

ruct

ural

insi

ghts

into

inte

ract

ion

mec

hani

sms t

o en

able

dev

elop

men

t of n

ovel

A

bs fo

r the

rape

utic

and

bio

tech

nolo

gica

l use

.

Sirc

ar e

t al

. 201

1

Prim

ary

stru

ctur

eM

ALD

I-T

OF/

TO

FLC

-MS/

MS

Sequ

enci

ng

The

prim

ary

stru

ctur

e of

a 1

3.6

kDa

sing

le h

eavy

cha

in c

amel

id a

ntib

ody

(V(H

)H) w

as d

eter

min

ed b

y m

atri

x-as

sist

ed la

ser d

esor

p-tio

n io

niza

tion-

time-

of-fl

ight

/tim

e-of

-flig

ht (M

ALD

I-TO

F/TO

F) to

p-do

wn

sequ

ence

ana

lysi

s. Th

e m

ajor

ity o

f the

sequ

ence

was

ob

tain

ed b

y m

ass s

pect

rom

etri

c de

nov

o se

quen

cing

, with

the

N-t

erm

inal

14

amin

o ac

id re

sidu

es b

eing

det

erm

ined

usi

ng T

(3)-

sequ

enci

ng a

nd d

atab

ase

inte

rrog

atio

n. Th

e de

term

ined

sequ

ence

was

con

firm

ed b

y liq

uid

chro

mat

ogra

phy

tand

em m

ass s

pec-

trom

etry

(LC

-MS/

MS)

ana

lysi

s of a

tryp

tic d

iges

t, w

hich

als

o pr

ovid

ed h

igh-

ener

gy c

ollis

iona

lly in

duce

d di

ssoc

iatio

n (C

ID) d

ata

perm

ittin

g th

e cl

ear a

ssig

nmen

t of t

hree

of t

he 1

4 is

obar

ic L

eu/I

le re

sidu

es. F

ive

of th

e 11

Leu

/Ile

am

bigu

ities

cou

ld b

e re

solv

ed b

y ho

mol

ogy

com

pari

sons

with

kno

wn

V(H

)H se

quen

ces.

The

mon

oiso

topi

c m

olec

ular

wei

ght o

f the

was

det

erm

ined

by

ultr

ahig

h-re

solu

tion

orth

ogon

al e

lect

rosp

ray

(ESI

)-TO

F an

alys

is a

nd fo

und

to b

e 13

610

.606

6 D

a, in

exc

elle

nt a

gree

men

t with

the

esta

blis

hed

sequ

ence

. To

our k

now

ledg

e, th

is is

the

first

tim

e th

at th

e en

tire

prim

ary

stru

ctur

e of

a p

rote

in w

ith a

mol

ecul

ar w

eigh

t > 1

3 kD

a ha

s bee

n es

tabl

ishe

d by

mas

s spe

ctro

met

ric

top-

dow

n se

quen

cing

.

Rese

-m

ann

et

al. 2

010

VH

H/h

apte

n

inte

ract

ions

Azo

-dye

X-r

ay s

truc

ture

Cam

elid

s, c

amel

s an

d lla

mas

, hav

e a

uniq

ue im

mun

e sy

stem

abl

e to

pro

duce

hea

vy-c

hain

onl

y an

tibo

dies

. The

ir V

H d

omai

ns

(VH

Hs)

are

the

smal

lest

bin

ding

uni

ts p

rodu

ced

by im

mun

e sy

stem

s, a

nd th

eref

ore

suit

able

for

biot

echn

olog

ical

app

licat

ions

th

roug

h he

tero

logo

us e

xpre

ssio

n. T

he r

ecog

niti

on o

f pro

tein

ant

igen

s by

thes

e V

HH

s is

rat

her

wel

l doc

umen

ted,

whi

le le

ss

is k

now

n ab

out t

he V

HH

/hap

ten

inte

ract

ions

. The

rec

ently

rep

orte

d X

-ray

str

uctu

re o

f a V

HH

in c

ompl

ex w

ith a

cop

per-

cont

aini

ng a

zo-d

ye s

ettle

d th

e ab

ility

of V

HH

to r

ecog

nize

hap

tens

by

form

ing

a ca

vity

bet

wee

n th

e th

ree

com

plem

enta

rity

-de

term

inin

g re

gion

s (C

DR)

. Her

e w

e re

port

the

stru

ctur

es o

f a V

HH

(VH

H A

52) f

ree

or c

ompl

exed

with

an

azo-

dye,

RR1

, w

ithou

t met

al io

n. T

he s

truc

ture

of t

he c

ompl

ex il

lust

rate

s th

e in

volv

emen

t of C

DR2

, CD

R3 a

nd a

fram

ewor

k re

sidu

e in

a

late

ral i

nter

acti

on w

ith th

e ha

pten

. Suc

h a

late

ral c

ombi

ning

site

is c

ompa

rabl

e to

that

foun

d in

cla

ssic

al a

ntib

odie

s, a

lthou

gh

in th

e ab

senc

e of

the

VL.

Spin

elli

et a

l. 20

01


454

Caff

eine

Met

hylx

anth

ines

Hea

t-st

abili

tyEL

ISA

We

have

isol

ated

and

cha

ract

eriz

ed a

caff

eine

-spe

cific

, hea

vy-c

hain

-onl

y an

tibod

y fr

agm

ent (

V-H

H) f

rom

llam

a th

at is

cap

able

of

bein

g ut

ilize

d to

ana

lyze

caff

eine

in h

ot a

nd c

old

beve

rage

s. C

amel

id sp

ecie

s (lla

ma

and

cam

el) w

ere

sele

cted

for i

mm

uniz

atio

n be

caus

e of

thei

r pot

entia

l to

mak

e he

at-s

tabl

e, h

eavy

-cha

in-o

nly

antib

odie

s. L

lam

as a

nd c

amel

s wer

e im

mun

ized

with

caff

eine

co

vale

ntly

link

ed to

key

hole

lim

pet h

emoc

yani

n, a

nd re

com

bina

nt a

ntib

ody

tech

niqu

es w

ere

used

to c

reat

e ph

age

disp

laye

d lib

rar-

ies o

f var

iabl

e re

gion

frag

men

ts o

f the

hea

vy-c

hain

ant

ibod

ies.

Caff

eine

-spe

cific

VH

H fr

agm

ents

wer

e se

lect

ed b

y th

eir a

bilit

y to

bi

nd to

caff

eine

/bov

ine

seru

m a

lbum

in (B

SA) a

nd c

onfir

med

by

a po

sitiv

e re

actio

n in

a c

affei

ne e

nzym

e-lin

ked

imm

unos

orbe

nt

assa

y (c

affei

ne E

LISA

). O

ne o

f the

se V

HH

frag

men

ts (V

SA2)

was

exp

ress

ed a

s a so

lubl

e pr

otei

n an

d sh

own

to re

cove

r its

reac

tiv-

ity a

fter

exp

osur

e to

tem

pera

ture

s up

to 9

0 de

gree

s C. I

n ad

ditio

n, V

SA2

was

abl

e to

bin

d ca

ffein

e at

70

degr

ees C

. A c

ompe

titio

n ca

ffein

e EL

ISA

was

dev

elop

ed fo

r the

mea

sure

men

t of c

affei

ne in

bev

erag

es, a

nd c

once

ntra

tions

of c

affei

ne o

btai

ned

for c

offee

, C

oca-

Col

a C

lass

ic, a

nd D

iet C

oke

agre

ed w

ell w

ith h

igh

perf

orm

ance

liqu

id c

hrom

atog

raph

y (H

PLC

) det

erm

inat

ion

and

liter

atur

e va

lues

. VSA

2 sh

owed

min

imal

cro

ss re

activ

ity w

ith st

ruct

ural

ly re

late

d m

ethy

lxan

thin

es.

Lade

n-so

n et

al.

2006

Met

hotr

exat

eIm

mun

e-lla

ma

libra

ryA

nti-

MT

X V

HH

gen

esH

ydro

solu

ble

hapt

en

Sing

le-d

omai

n an

tibod

ies s

peci

fic to

met

hotr

exat

e (M

TX

) wer

e ob

tain

ed a

fter

imm

uniz

atio

n of

one

llam

a (L

lam

a gl

ama)

. Spe

cific

V

HH

dom

ains

(V-D

-J-r

egio

n) w

ere

sele

cted

by

pann

ing

from

an

imm

une-

llam

a lib

rary

usi

ng p

hage

dis

play

tech

nolo

gy. Th

e an

ti-bo

dy fr

agm

ents

spec

ific

to M

TX

wer

e pu

rifie

d fr

om E

sche

rich

ia c

oli (

C41

stra

in) p

erip

lasm

by

imm

obili

zed

met

al a

ffini

ty c

hrom

a-to

grap

hy w

ith a

n ex

pres

sion

leve

l of a

roun

d 10

mg/

l. A

sing

le b

and

arou

nd 1

6 00

0 D

a co

rres

pond

ing

to V

HH

frag

men

ts w

as fo

und

afte

r ana

lysi

s by

SDS-

PAG

E an

d W

este

rn b

lott

ing,

whi

le c

ompe

titio

n EL

ISA

dem

onst

rate

d se

lect

ive

bind

ing

to so

lubl

e M

TX

. Su

rfac

e pl

asm

on re

sona

nce

(SPR

) ana

lysi

s sho

wed

that

ant

i-MT

X V

HH

dom

ains

had

affi

nitie

s in

the

nano

mol

ar ra

nge

(29–

515

nM) t

o M

TX

-ser

um a

lbum

in c

onju

gate

s. Th

e ge

nes e

ncod

ing

anti-

MT

X V

HH

wer

e fo

und

by IM

GT

/V-Q

UES

T to

be

sim

ilar t

o th

e pr

evio

usly

repo

rted

llam

a an

d hu

man

IGH

V g

erm

line

gene

s. Th

e V

-D a

nd D

-J ju

nctio

n re

arra

ngem

ents

in th

e se

ven

anti-

MT

X

CD

R3 se

quen

ces i

ndic

ate

that

they

wer

e or

igin

ated

from

thre

e di

stin

ct p

roge

nito

r B c

ells

. Our

resu

lts d

emon

stra

te th

at c

amel

id

sing

le-d

omai

n an

tibod

ies a

re c

apab

le o

f hig

h affi

nity

bin

ding

to lo

w m

olec

ular

wei

ght h

ydro

solu

ble

hapt

ens.

Fur

ther

mor

e, th

ese

anti-

MT

X V

HH

giv

e ne

w in

sigh

ts o

n ho

w th

e an

tigen

bin

ding

repe

rtoi

re o

f lla

ma

sing

le-d

omai

n an

tibod

y ca

n pr

ovid

e co

mbi

ning

si

tes t

o ha

pten

s in

the

abse

nce

of a

VL.

This

type

of s

ingl

e-do

mai

n an

tibod

ies o

ffers

adv

anta

ges c

ompa

red

to m

urin

e re

com

bina

nt

antib

odie

s in

term

s of p

rodu

ctio

n ra

te a

nd se

quen

ce si

mila

rity

to th

e hu

man

IGH

V3

subg

roup

gen

es.

Alv

arez

-Ru

eda

et

al. 2

007

C(H

)1 d

omai

n ab

senc

eSe

quen

ce a

naly

sis

Splic

ing

The

mol

ecul

ar b

asis

for t

he a

bsen

ce o

f the

C(H

)1 d

omai

n in

nat

ural

ly o

ccur

ring

hea

vy-c

hain

ant

ibod

ies o

f the

cam

elid

s was

ass

esse

d by

det

erm

inin

g th

e en

tire

Cam

elus

dro

med

ariu

s gam

ma

2a h

eavy

-cha

in c

onst

ant g

ene.

The

orga

niza

tion

of th

e ca

mel

gam

ma

2a c

on-

stan

t hea

vy-c

hain

gen

e ob

tain

ed fr

om a

live

r gen

omic

libr

ary

appe

ars t

o be

typi

cal o

f all

othe

r mam

mal

ian

gam

ma

gene

s seq

uenc

ed to

da

te. I

t con

tain

s the

switc

h, C

(H)1

, hin

ge, C

(H)2

, C(H

)3, M

1 an

d M

2 ex

ons.

In c

ontr

ast t

o th

e ca

se in

mou

se a

nd h

uman

hea

vy c

hain

di

seas

es, t

he c

amel

gam

ma

2a g

ene

show

s no

maj

or st

ruct

ural

def

ect,

and

its e

quiv

alen

t C(H

)1 e

xon

is in

tact

. How

ever

, seq

uenc

e an

alys

is h

as re

veal

ed th

at th

e sp

licin

g si

te, i

mm

edia

tely

afte

r the

C(H

)1 e

xon,

is d

efec

tive

due

to p

oint

mut

atio

ns, e

spec

ially

the

G(+

1) to

A(+

1) tr

ansv

ersi

on se

ems t

o be

det

rim

enta

l. It

is c

oncl

uded

that

the

loss

of t

he sp

lice

cons

ensu

s sig

nal i

s res

pons

ible

for t

he

rem

oval

of t

he e

ntire

C(H

)1 d

omai

n in

cam

el g

amm

a 2a

hea

vy-c

hain

imm

unog

lobu

lins.

Add

ition

ally

, a c

lose

r ana

lysi

s of t

he h

inge

ex

on su

gges

ts th

e po

ssib

le in

volv

emen

t of t

rans

poso

ns in

the

gene

tic v

aria

tion

of m

amm

alia

n C

gam

ma

hing

es.

Ngu

yen

et a

l. 19

99


455

Inhi

bito

ry a

ntib

odie

sEn

zym

e’s

acti

ve s

ite

Car

boni

c an

hydr

ase

Alp

ha-a

myl

ase

Rec

ombi

nant

pro

tein

Evid

ence

is p

rovi

ded

that

dro

med

ary

heav

y-ch

ain

antib

odie

s, in

viv

o-m

atur

ed in

the

abse

nce

of li

ght c

hain

s, a

re a

uni

que

sour

ce o

f inh

ibito

ry a

ntib

odie

s. A

fter

imm

uniz

atio

n of

a d

rom

edar

y w

ith b

ovin

e er

ythr

ocyt

e ca

rbon

ic a

nhyd

rase

and

por

cine

pa

ncre

atic

alp

ha-a

myl

ase,

it w

as d

emon

stra

ted

that

a c

onsi

dera

ble

amou

nt o

f hea

vy-c

hain

ant

ibod

ies,

act

ing

as tr

ue c

ompe

titiv

e in

hibi

tors

, cir

cula

te in

the

bloo

dstr

eam

. In

cont

rast

, the

con

vent

iona

l ant

ibod

ies

appa

rent

ly d

o no

t int

erac

t with

the

enzy

me’s

ac

tive

site

. Nex

t we

illus

trat

ed th

at p

erip

hera

l blo

od ly

mph

ocyt

es a

re s

uita

ble

for

one-

step

clo

ning

of t

he v

aria

ble

dom

ain

frag

-m

ents

in a

pha

ge-d

ispl

ay v

ecto

r. By

bio

-pan

ning

, sev

eral

ant

igen

-spe

cific

sin

gle-

dom

ain

frag

men

ts a

re re

adily

isol

ated

for

both

en

zym

es. I

n ad

ditio

n w

e sh

ow th

at a

mon

g th

ose

isol

ated

frag

men

ts a

ctiv

e si

te b

inde

rs a

re w

ell r

epre

sent

ed. W

hen

prod

uced

as

reco

mbi

nant

pro

tein

in E

sche

rich

ia c

oli,

thes

e ac

tive

site

bin

ders

app

ear

to b

e po

tent

enz

yme

inhi

bito

rs w

hen

test

ed in

chr

o-m

ogen

ic a

ssay

s. T

he lo

w c

ompl

exity

of t

he a

ntig

en-b

indi

ng s

ite o

f the

se s

ingl

e-do

mai

n an

tibod

ies

com

pose

d of

onl

y th

ree

loop

s co

uld

be v

alua

ble

for

desi

gnin

g sm

alle

r sy

nthe

tic in

hibi

tors

.

Lauw

-er

eys e

t al

. 199

8

VH

HA

ntig

en-b

indi

ng lo

ops

RN

ase

AEn

zym

e in

hibi

tor

Can

onic

al s

truc

ture

s

Back

grou

nd: C

amel

id s

erum

con

tain

s a

larg

e fr

actio

n of

func

tiona

l hea

vy c

hain

ant

ibod

ies

– ho

mod

imer

s of

hea

vy c

hain

s w

ithou

t lig

ht c

hain

s. T

he v

aria

ble

dom

ains

of t

hese

hea

vy-c

hain

ant

ibod

ies

(VH

H) h

ave

a lo

ng c

ompl

emen

tari

ty d

eter

min

ing

regi

on 3

(CD

R3) l

oop

that

com

pens

ates

for

the

abse

nce

of th

e an

tigen

-bin

ding

loop

s of

the

vari

able

ligh

t cha

ins

(VL)

. In

the

case

of t

he V

HH

frag

men

t cA

b-Ly

s3, p

art o

f the

24

amin

o ac

id lo

ng C

DR3

loop

pro

trud

es fr

om th

e an

tigen

-bin

ding

sur

face

an

d in

sert

s in

to th

e ac

tive-

site

cle

ft o

f its

ant

igen

, ren

deri

ng c

Ab-

Lys3

a c

ompe

titiv

e en

zym

e in

hibi

tor.

Resu

lts: A

dro

med

ary

VH

H w

ith s

peci

ficity

for

bovi

ne R

Nas

e A

, cA

b-RN

05, h

as a

sho

rt C

DR3

loop

of 1

2 am

ino

acid

s an

d is

not

a c

ompe

titiv

e en

zym

e in

hibi

tor.

The

str

uctu

re o

f the

cA

b-RN

05-R

Nas

e A

com

plex

has

bee

n so

lved

at 2

.8 a

ngst

rom

. The

VH

H s

caffo

ld a

rchi

tect

ure

is c

lose

to th

at o

f a h

uman

VH

(var

iabl

e he

avy

chai

n). T

he s

truc

ture

of t

he a

ntig

en-b

indi

ng h

yper

vari

able

1 lo

op (H

1) o

f bot

h cA

b-RN

05 a

nd c

Ab-

Lys3

diff

er fr

om th

e kn

own

cano

nica

l str

uctu

res;

in a

dditi

on th

ese

H1

loop

s re

sem

ble

each

oth

er. T

he

CD

R3 p

rovi

des

an a

ntig

en-b

indi

ng s

urfa

ce a

nd s

hiel

ds th

e fa

ce o

f the

dom

ain

that

inte

ract

s w

ith V

L in

con

vent

iona

l ant

ibod

ies.

C

oncl

usio

ns: V

HH

s ad

opt t

he c

omm

on im

mun

oglo

bulin

fold

of v

aria

ble

dom

ains

, but

the

antig

en-b

indi

ng lo

ops

devi

ate

from

th

e pr

edic

ted

cano

nica

l str

uctu

re. W

e de

fine

a ne

w c

anon

ical

str

uctu

re fo

r th

e H

1 lo

op o

f im

mun

oglo

bulin

s, w

ith c

Ab-

RN05

an

d cA

b-Ly

s3 a

s re

fere

nce

stru

ctur

es. T

his

new

loop

str

uctu

re m

ight

als

o oc

cur

in h

uman

or

mou

se V

H d

omai

ns. S

urpr

isin

gly,

only

two

loop

s ar

e in

volv

ed in

ant

igen

reco

gniti

on; t

he C

DR2

doe

s no

t par

ticip

ate.

Nev

erth

eles

s, th

e an

tigen

bin

ding

occ

urs

with

nan

omol

ar a

ffin

ities

bec

ause

of a

pre

fere

ntia

l usa

ge o

f mai

ncha

in a

tom

s fo

r an

tigen

inte

ract

ion.

Dec

an-

nier

e et

al

. 199

9

Cle

fts

on p

rote

in

surf

aces

C

onve

x pa

rato

peA

ntig

en-c

ombi

ning

sit

eLy

sozy

me

inhi

bito

rC

ryst

al s

truc

ture

Cle

fts

on p

rote

in s

urfa

ces

are

avoi

ded

by a

ntig

en-c

ombi

ning

site

s of

con

vent

iona

l ant

ibod

ies,

in c

ontr

ast t

o he

avy-

chai

n an

tibod

ies

(HC

Abs

) of c

amel

ids

that

see

m to

be

attr

acte

d by

enz

ymes

’ sub

stra

te p

ocke

ts. T

he e

xpla

natio

n fo

r th

is p

rono

unce

d pr

efer

ence

of H

CA

bs w

as in

vest

igat

ed. E

ight

sin

gle

dom

ain

antig

en-b

indi

ng fr

agm

ents

of H

CA

bs (V

HH

) with

nan

omol

ar a

ffin

i-tie

s fo

r ly

sozy

me

wer

e is

olat

ed fr

om th

ree

imm

uniz

ed d

rom

edar

ies.

Six

of e

ight

VH

Hs

com

pete

with

sm

all l

ysoz

yme

inhi

bito

rs.

Thi

s ra

tio o

f act

ive

site

bin

ders

is a

lso

foun

d w

ithin

the

VH

H p

ool d

eriv

ed fr

om p

olyc

lona

l HC

Abs

pur

ified

from

the

seru

m o

f th

e im

mun

ized

dro

med

ary.

The

cry

stal

str

uctu

res

of s

ix V

HH

s in

com

plex

with

lyso

zym

e an

d th

eir

inte

ract

ion

surf

aces

wer

e co

mpa

red

to th

ose

of c

onve

ntio

nal a

ntib

odie

s w

ith th

e sa

me

antig

en. T

he in

terf

ace

size

s of

VH

H a

nd c

onve

ntio

nal a

ntib

odie

s to

lyso

zym

e ar

e ve

ry s

imila

r as

wel

l as

the

num

ber

and

chem

ical

nat

ure

of th

e co

ntac

ts. T

he m

ain

diffe

renc

e co

mes

from

the

com

pact

pro

late

sha

pe o

f VH

H th

at p

rese

nts

a la

rge

conv

ex p

arat

ope,

pre

dom

inan

tly fo

rmed

by

the

H3

loop

and

inte

ract

ing,

al

thou

gh w

ith d

iffer

ent s

truc

ture

s, in

to th

e co

ncav

e ly

sozy

me

subs

trat

e-bi

ndin

g po

cket

. The

refo

re, a

sin

gle

dom

ain

antig

en-

com

bini

ng s

ite h

as a

cle

ar s

truc

tura

l adv

anta

ge o

ver

a co

nven

tiona

l dim

eric

form

at fo

r ta

rget

ing

clef

ts o

n an

tigen

ic s

urfa

ces.

De

Gen

st

et a

l. 20

06


456

Car

tila

gino

us fi

shcD

NA

seq

uenc

e an

alys

isIg

NA

RIg

W

The

cart

ilagi

nous

fish

(chi

mer

as, s

hark

s, sk

ates

and

rays

) are

the

olde

st g

roup

rela

tive

to m

amm

als i

n w

hich

an

adap

tive

imm

une

syst

em fo

unde

d up

on im

mun

oglo

bulin

s has

bee

n fo

und.

In th

is m

anus

crip

t we

char

acte

rize

the

imm

unog

lobu

lins o

f the

spin

y do

gfish

(S

qual

us a

cant

hias

) at b

oth

the

mol

ecul

ar a

nd e

xpre

ssed

pro

tein

leve

ls. D

espi

te th

e pr

esen

ce o

f hun

dred

s of I

gM c

lust

ers i

n th

is

spec

ies t

he se

rum

leve

ls of

this

isot

ype

are

com

para

tivel

y lo

w. H

owev

er, a

naly

sis o

f cD

NA

sequ

ence

s and

seru

m p

rote

in su

gges

ts

mic

rohe

tero

gene

ity in

the

IgM

hea

vy c

hain

s and

supp

orts

the

prop

osal

that

diff

eren

t clu

ster

s are

pre

fere

ntia

lly u

sed

in th

e tw

o fo

rms

(mon

omer

or p

enta

mer

) of t

his i

soty

pe. W

e al

so fo

und

that

the

IgN

AR

isot

ype

in th

is sp

ecie

s exi

sts i

n a

prev

ious

ly u

nkno

wn

mul

ti-m

eric

form

at in

seru

m. F

inal

ly, w

e id

entifi

ed a

new

form

of t

he Ig

W is

otyp

e (th

e sh

ark

IgD

ort

holo

gue)

, in

whi

ch th

e le

ader

is sp

liced

di

rect

ly to

the

first

con

stan

t dom

ain,

resu

lting

in a

mol

ecul

e la

ckin

g an

ant

igen

-bin

ding

dom

ain.

Smith

et

al. 2

012

V(N

AR

) dom

ain

Rib

osom

e di

spla

yEr

ror-

pron

e

mut

agen

esis

Mut

atio

nal p

last

icit

ySt

ruct

ural

mod

ellin

g

The

shar

k an

tigen

-bin

ding

V(N

AR)

dom

ain

has t

he p

oten

tial t

o pr

ovid

e an

att

ract

ive

alte

rnat

ive

to tr

aditi

onal

bio

ther

apeu

tics

base

d on

its s

mal

l siz

e, a

dvan

tage

ous p

hysi

oche

mic

al p

rope

rtie

s, a

nd u

nusu

al a

bilit

y to

targ

et c

left

s in

enzy

mes

or c

ell s

urfa

ce

mol

ecul

es. Th

e V

(NA

R) sh

ares

man

y of

the

prop

ertie

s of t

he w

ell-c

hara

cter

ised

sing

le-d

omai

n ca

mel

id V

(H)H

but

is m

uch

less

un

ders

tood

at t

he m

olec

ular

leve

l. W

e ch

ose

the

hen-

egg-

lyso

zym

e-sp

ecifi

c ar

chet

ypal

Typ

e I V

(NA

R) 5

A7

and

used

ribo

som

e di

spla

y in

com

bina

tion

with

err

or-p

rone

mut

agen

esis

to in

terr

ogat

e th

e en

tire

sequ

ence

spac

e. W

e fo

und

a hi

gh le

vel o

f mut

atio

nal

plas

ticity

acr

oss t

he V

(NA

R) d

omai

n, p

artic

ular

ly w

ithin

the

fram

ewor

k 2

and

hype

rvar

iabl

e re

gion

2 re

gion

s. A

num

ber o

f res

i-du

es im

port

ant f

or a

ffini

ty w

ere

iden

tified

, and

a tr

iple

mut

ant c

ombi

ning

AID

, S61

R, a

nd G

62R

resu

lted

in a

K(D

) of 4

60 p

M fo

r he

n eg

g ly

sozy

me,

a 2

0-fo

ld im

prov

emen

t ove

r wild

-typ

e 5A

7, a

nd th

e hi

ghes

t K(D

) yet

repo

rted

for V

(NA

R)-a

ntig

en in

tera

ctio

ns.

Thes

e fin

ding

s wer

e ra

tiona

lised

usi

ng st

ruct

ural

mod

ellin

g an

d in

dica

te th

e im

port

ance

of r

esid

ues o

utsi

de th

e cl

assi

cal c

ompl

e-m

enta

rity

det

erm

inin

g re

gion

s in

mak

ing

nove

l ant

igen

con

tact

s tha

t mod

ulat

e affi

nity

. We

also

loca

ted

two

solv

ent-

expo

sed

resi

-du

es (G

15 a

nd G

42),

dist

ant f

rom

the

V(N

AR)

par

atop

e, w

hich

reta

in fu

nctio

n up

on m

utat

ion

to c

yste

ine

and

have

the

pote

ntia

l to

be e

xplo

ited

as si

tes f

or ta

rget

ed c

oval

ent m

odifi

catio

n. O

ur fi

ndin

gs w

ith 5

A7

wer

e ex

tend

ed to

all

know

n N

AR

stru

ctur

es u

sing

an

in-d

epth

bio

info

rmat

ic a

naly

sis o

f seq

uenc

e da

ta a

vaila

ble

in th

e lit

erat

ure

and

a ne

wly

gen

erat

ed V

(NA

R) d

atab

ase.

This

stud

y al

low

ed u

s to

iden

tify,

for t

he fi

rst t

ime,

bot

h V

(NA

R)-s

peci

fic a

nd V

(NA

R)/I

g V

(L)/

TCR

V(a

lpha

) ove

rlap

ping

hal

lmar

k re

sidu

es,

whi

ch a

re c

ritic

al fo

r the

stru

ctur

al a

nd fu

nctio

nal i

nteg

rity

of t

he si

ngle

dom

ain.

Intr

igui

ngly

, eac

h of

our

des

igna

ted

V(N

AR)

-sp

ecifi

c ha

llmar

ks a

lign

prec

isel

y w

ith p

revi

ousl

y de

fined

mut

atio

nal ‘

cold

spot

s’ in

nat

ural

nur

se sh

ark

cDN

A se

quen

ces.

Thes

e fin

ding

s will

aid

futu

re V

(NA

R) e

ngin

eeri

ng a

nd o

ptim

isat

ion

stud

ies t

owar

ds th

e de

velo

pmen

t of V

(NA

R) si

ngle

-dom

ain

prot

eins

as

via

ble

biot

hera

peut

ics.

Fenn

ell

et a

l. 20

10

IgM

IgN

AR

IgW

Hou

ndsh

ark

In th

is st

udy,

cDN

As e

ncod

ing

the

secr

eted

form

s of t

he im

mun

oglo

bulin

(Ig)

hea

vy c

hain

s of I

gM, I

gNA

R, a

nd Ig

W w

ere

clon

ed

from

the

band

ed h

ound

shar

k Tr

iaki

s scy

llium

. Tw

o cl

ones

for t

he Ig

M h

eavy

cha

ins e

ncod

ed 5

69 a

nd 5

70 a

min

o ac

ids,

who

se c

on-

serv

ed (C

) reg

ion

show

ed 4

7–70

% a

min

o ac

id id

entit

ies t

o th

ose

repo

rted

in o

ther

car

tilag

inou

s fish

. Fou

r clo

nes f

or th

e Ig

NA

R en

code

d 67

3–67

0 am

ino

acid

s with

con

serv

ed Ig

-sup

erfa

mily

dom

ains

. The

IgN

AR

C re

gion

show

ed 5

6–69

% a

min

o ac

id id

enti-

ties t

o th

ose

so fa

r rep

orte

d. H

igh-

thro

ughp

ut se

quen

cing

reve

aled

that

in m

ost o

f the

IgN

AR

sequ

ence

s, th

e tw

o va

riab

le re

gion

s (C

DR1

and

CD

R3) e

ach

poss

ess a

cys

tein

e re

sidu

e. Th

ree

type

s of I

gW w

ere

iden

tified

; one

con

tain

ed Ig

-sup

erfa

mily

dom

ains

th

at a

re in

oth

er c

artil

agin

ous fi

sh, o

ne la

cks t

he 3

rd d

omai

n in

the

cons

tant

regi

on, a

nd o

ne la

cks t

he 3

rd to

5th

dom

ains

. Des

pite

th

ese

diffe

renc

es, t

he Ig

W is

ofor

ms c

lust

ered

with

IgW

s of o

ther

car

tilag

inou

s fish

es a

nd th

e C

regi

ons s

how

ed 4

7–89

% a

min

o ac

id

iden

titie

s. m

RNA

s for

IgM

and

IgN

AR

wer

e de

tect

ed in

var

ious

tiss

ues,

whi

le Ig

W m

RNA

was

mai

nly

dete

cted

in p

ancr

eas.

The

band

ed h

ound

ed sh

ark

also

has

IgM

, IgW

and

IgN

AR

as w

ell a

s the

oth

er c

artil

agin

ous fi

sh w

ith u

niqu

e Ig

W is

ofor

m.

Hon

da e

t al

. 201

0


457

New

ant

igen

rec

epto

rD

isul

phid

e bo

ndLo

op s

tabi

lizat

ion

Cry

stal

logr

aphi

c st

udie

s

The

new

ant

igen

rece

ptor

(IgN

AR)

ant

ibod

ies f

rom

shar

ks a

re d

isul

phid

e bo

nded

dim

ers o

f tw

o pr

otei

n ch

ains

, eac

h co

ntai

ning

on

e va

riab

le a

nd fi

ve c

onst

ant d

omai

ns. Th

ree

type

s of I

gNA

R va

riab

le d

omai

ns h

ave

been

dis

cove

red,

with

Typ

e 3

appe

arin

g ea

rly

in sh

ark

deve

lopm

ent a

nd b

eing

ove

rtak

en b

y th

e an

tigen

-dri

ven

affini

ty-m

atur

ed T

ype

1 an

d 2

resp

onse

. Her

e, w

e ha

ve d

eter

-m

ined

the

first

stru

ctur

e of

a n

atur

ally

occ

urri

ng T

ype

2 Ig

NA

R va

riab

le d

omai

n, a

nd id

entifi

ed th

e di

sulp

hide

bon

d th

at li

nks a

nd

stab

ilize

s the

CD

R1 a

nd C

DR3

loop

s. Th

is d

isul

phid

e br

idge

lock

s the

CD

R3 lo

op in

an

“upr

ight

” con

form

atio

n in

con

tras

t to

othe

r sh

ark

antib

ody

stru

ctur

es, w

here

a m

ore

late

ral c

onfig

urat

ion

is o

bser

ved.

Fur

ther

, we

soug

ht to

mod

el th

e Ty

pe 3

isot

ype

base

d on

th

e cr

ysta

llogr

aphi

c st

ruct

ure

repo

rted

her

e. Th

is m

odel

incr

indi

cate

s tha

t int

erna

l Typ

e 3-

spec

ific

resi

dues

com

bine

to p

ack

into

a

com

pact

imm

unog

lobu

lin c

ore

that

supp

orts

the

CD

R lo

op re

gion

s, a

nd th

at d

espi

te a

ppar

ent l

ow-s

eque

nce

vari

abili

ty, t

here

Is

suffi

cien

t pla

stic

ity in

the

CD

R3 lo

op to

form

a c

onfo

rmat

iona

lly d

iver

se a

ntig

en-b

indi

ng su

rfac

e.

Stre

ltsov

et

al.

2005

Ant

ibod

y ev

olut

ion

Phyl

ogen

etic

and

phe

no-

typi

c re

lati

onsh

ips

Cam

elid

aeSh

arks

The

emer

genc

e in

Cam

elid

ae sp

ecie

s of f

unct

iona

l ant

ibod

ies d

evoi

d of

ligh

t cha

ins (

refe

rred

to a

s hea

vy-c

hain

ant

ibod

ies o

r H

CA

bs) i

s an

intr

igui

ng e

volu

tiona

ry e

vent

. Hom

odim

eric

HC

Abs

hav

e al

so b

een

docu

men

ted

in sp

otte

d ra

tfish

(Cos

5-A

bs) a

nd

nurs

e sh

ark

(NA

R). T

o re

veal

the

evol

utio

nary

his

tory

of H

CA

bs, w

e ev

alua

ted

the

phyl

ogen

etic

and

phe

noty

pic

rela

tions

hips

am

ong

HC

Abs

and

con

vent

iona

l ant

ibod

ies a

cros

s tax

a an

d co

nfirm

ed th

e cu

rren

t vie

wpo

int t

hat d

iffer

ent g

roup

s of H

CA

bs h

ave

evol

ved

inde

pend

ently

in th

e th

ree

linea

ges.

At l

east

, in

the

cam

elid

s, H

CA

bs a

re n

ot th

e re

sult

of re

susc

itatio

n of

dor

man

t gen

es.

They

are

der

ived

from

the

conv

entio

nal a

ntib

odie

s with

in th

e C

amel

idae

line

age,

and

are

app

aren

tly th

e ou

tcom

e of

mor

e re

cent

ad

aptiv

e ch

ange

s occ

urri

ng in

the

com

part

men

t of h

eter

omer

ic a

ntib

odie

s. Th

e sh

ared

stru

ctur

al p

rope

rtie

s of H

CA

bs a

cros

s tax

a ar

e th

eref

ore

expl

aine

d by

con

verg

ent e

volu

tion

due

to si

mila

r con

stra

ints

rela

ted

to th

e ab

senc

e of

pai

ring

to th

e lig

ht c

hain

. It

appe

ars t

hat i

nnov

ativ

e ev

olut

iona

ry c

hang

es in

Cam

elid

ae h

ave

led

to a

new

leve

l of a

ntig

en b

indi

ng re

pert

oire

div

ersi

ficat

ion

and

have

allo

wed

acq

uisi

tion

of n

ovel

ant

igen

-rec

epto

r pro

pert

ies.

Ngu

yen

et a

l. 20

02

Ada

ptiv

e im

mun

e re

spon

seM

icro

bial

enz

yme

in

hibi

tion

P-la

ctam

ase

In 1

993,

a fr

actio

n of

ant

ibod

ies (

Abs

) dev

oid

of L

cha

in w

as fo

und

natu

rally

occ

urri

ng in

the

Cam

elid

ae. Th

ey w

ere

foun

d to

lack

L

chai

ns, a

s wel

l as t

he fi

rst c

onst

ant h

eavy

-cha

in d

omai

n (C

HI)

and

ther

efor

e th

ey w

ere

nam

ed “h

eavy

-cha

in A

bs” (

HC

Abs

). Su

bseq

uent

stud

ies f

ocus

ed o

n th

e fu

nctio

nal,

stru

ctur

al a

nd b

ioch

emic

al p

rope

rtie

s of r

ecom

bina

nt v

aria

ble

frag

men

ts (r

VH

Hs)

of

HC

Abs

. It w

as st

ated

that

rVH

Hs h

ave

an a

ugm

ente

d ca

paci

ty to

inte

ract

with

“par

tially

hid

den”

epi

tope

s, li

ke e

nzym

es a

ctiv

e si

tes,

and

hav

e an

incr

ease

d st

abili

ty to

ther

mal

and

che

mic

al a

ggre

ssio

n. It

has

bee

n su

gges

ted

that

thes

e un

conv

entio

nal A

bs

coul

d re

pres

ent a

n ev

olut

iona

ry a

dvan

tage

, bei

ng m

ore

effici

ent t

han

conv

entio

nal A

bs to

inhi

bit m

icro

bial

enz

ymes

, and

thus

ex

ertin

g a

mor

e pr

otec

tive

imm

une

resp

onse

aga

inst

pat

hoge

ns. Th

e pr

esen

t wor

k fo

cuse

s on

the

imm

unob

iolo

gica

l rol

e of

H

CA

bs, i

n th

eir c

apac

ity to

inhi

bit m

icro

bial

enz

ymes

. Tw

o an

imal

mod

els w

ere

sele

cted

, com

pris

ing

a m

odel

for c

omm

on v

er-

tebr

ates

with

out H

CA

bs (r

abbi

ts),

and

a m

odel

for v

erte

brat

es w

ith b

oth

conv

entio

nal a

nd u

ncon

vent

iona

l Abs

(Lam

a gl

ama)

. A

reco

mbi

nant

bac

teri

al P

-lact

amas

e (C

TX

-M-2

) was

sele

cted

as t

he m

icro

bial

enz

ymat

ic a

ntig

en. A

fter

con

vent

iona

l im

mun

izat

ion

sche

dule

s, n

eith

er se

rum

tite

rs n

or se

rum

inhi

bito

ry c

apac

ity sh

owed

sign

ifica

nt d

iffer

ence

s whe

n ra

bbits

and

llam

as w

ere

com

-pa

red.

Thes

e re

sults

indi

cate

that

the

a pr

iori

ass

umpt

ion

that

the

adap

tive

imm

une

syst

em o

f cam

elid

s cou

ld b

e be

tter

“pre

pare

d”

to re

spon

d to

bac

teri

al e

nzym

es b

ecau

se o

f the

pre

senc

e of

HC

Abs

, is n

ot a

lway

s acc

urat

e. F

urth

erm

ore,

whe

n th

e di

ffere

nt ll

ama

antib

ody

isot

ypes

and

subc

lass

es w

ere

puri

fied,

it w

as d

emon

stra

ted

that

the

inhi

bito

ry c

apac

ity o

f tot

al se

rum

was

due

exc

lusi

vely

to

IgG

I. H

CA

bs n

ot o

nly

faile

d to

inhi

bit C

TX

-M-2

, but

inst

ead

they

act

ivat

ed it

s enz

ymat

ic a

ctiv

ity. A

ltoge

ther

, the

se re

sults

in

dica

te th

at th

e hy

poth

eses

ext

rapo

late

d fr

om th

e rV

HH

s pro

pert

ies n

eed

to b

e re

vise

d; th

e re

al ro

le o

f HC

Abs

in v

ivo

rem

ains

un

know

n, a

s wel

l as t

heir

evo

lutio

nary

cau

se.

Ferr

ari e

t al

. 200

7


458

Imm

unol

ogic

al

prop

erti

esC

ompl

emen

t act

ivat

ing

acti

vity

Ant

icom

plem

enta

ry

acti

vity

Hem

aggl

utin

atio

n

Hea

vy c

hain

ant

ibod

ies (

HC

Abs

), de

void

of t

he li

ght c

hain

s and

the

CH

1 do

mai

n, a

re p

rese

nt in

the

seru

m o

f cam

elid

s. Ig

G(2

) an

d Ig

G(3

) are

HC

Abs

; whe

reas

IgG

(1) h

as th

e co

nven

tiona

l str

uctu

re. I

n or

der t

o st

udy

the

imm

unol

ogic

al p

rope

rtie

s of l

lam

a H

CA

bs, f

rom

whi

ch to

dat

e lit

tle is

kno

wn,

llam

as (L

ama

glam

a) H

CA

bs c

DN

A w

ere

clon

ed, s

eque

nced

and

com

pare

d w

ith o

ther

m

amm

alia

n Ig

s. Th

e se

quen

ce a

naly

sis s

how

ed th

at ll

ama

HC

Abs

cD

NA

org

aniz

atio

n is

sim

ilar t

o ot

her m

amm

alia

n Ig

s and

the

pres

ence

of c

onse

rved

bin

ding

mot

ifs to

Pro

tein

A, P

rote

in G

, Fc

gam

ma

RI, F

c ga

mm

a RI

II a

nd C

1q in

HC

Abs

wer

e ob

serv

ed. I

n a

prev

ious

wor

k, d

iffer

ent I

gG is

otyp

es p

urifi

ed b

y Pr

otei

n A

and

Pro

tein

G c

hrom

atog

raph

y w

ere

assa

yed

for t

heir

abi

lity

to fi

x co

mpl

emen

t. Bo

th Ig

G(1

) and

the

tota

l ser

um w

ere

able

to fi

x co

mpl

emen

t, w

here

as Ig

G(2

) and

IgG

(3) fi

xed

com

plem

ent e

ven

in

the

abse

nce

of a

ntig

en (a

ntic

ompl

emen

tary

act

ivity

). Th

eref

ore,

in th

is w

ork

we

perf

orm

ed th

e co

mpl

emen

t act

ivat

ing

activ

ity o

f th

e di

ffere

nt Ig

G is

otyp

es p

urifi

ed u

nder

phy

siol

ogic

al c

ondi

tions

usi

ng S

epha

dex

G-1

50 a

nd th

eir a

bilit

y to

indu

ce h

emag

glut

ina-

tion.

Lla

mas

wer

e im

mun

ized

with

shee

p re

d bl

ood

cells

(RBC

) str

oma

and

the

diffe

rent

isot

ypes

wer

e pu

rifie

d fr

om se

ra. W

hole

se

rum

and

IgG

(1) c

ould

act

ivat

e co

mpl

emen

t; ho

wev

er, H

CA

bs (I

gG(2

) + Ig

G(3

)) c

ould

not

, des

pite

the

pres

ence

of t

he C

1q b

ind-

ing

mot

if in

thei

r pri

mar

y se

quen

ce. U

nlik

e Ig

G(1

), th

e fr

actio

n co

rres

pond

ing

to Ig

G(2

) + Ig

G(3

) did

not

dis

play

hem

aggl

utin

atin

g ac

tivity

. Our

find

ings

sugg

est t

hat H

CA

bs c

anno

t cro

sslin

k effi

cien

tly w

ith d

iffer

ent a

ntig

ens a

nd th

at th

e C

1q b

indi

ng si

te m

ight

be

hin

dere

d by

the

prox

imity

of t

he v

aria

ble

dom

ains

.

Sacc

o-do

ssi e

t al

. 201

2

Ant

igen

-bin

ding

re

pert

oire

Ger

mlin

e da

taba

seSe

quen

ce c

ompa

riso

nEv

olut

ion

Som

atic

mut

atio

n

The

antig

en-b

indi

ng si

te o

f the

cam

el h

eavy

-cha

in a

ntib

odie

s dev

oid

of li

ght c

hain

con

sist

s of a

sing

le v

aria

ble

dom

ain

(VH

H)

that

obv

ious

ly la

cks t

he V

-H-V

-L c

ombi

nato

rial

div

ersi

ty, T

o ev

alua

te th

e ex

tent

of t

he V

HH

ant

igen

-bin

ding

repe

rtoi

re, a

ger

-m

line

data

base

was

con

stru

cted

from

PC

R-am

plifi

ed T

HH

/V-H

segm

ents

of a

sing

le sp

ecim

en o

f Cam

elus

dro

med

ariu

s. A

tota

l of

33

VH

H a

nd 3

9 V

H u

niqu

e se

quen

ces w

ere

iden

tified

, enc

oded

by

42 a

nd 5

0 di

ffere

nt g

enes

, res

pect

ivel

y. Se

quen

ce c

ompa

ri-

son

indi

cate

s tha

t the

V(H

)Hs e

volv

ed w

ithin

the

VH

subg

roup

III,

Nev

erth

eles

s, th

e V

HH

ger

mlin

e se

gmen

ts a

re h

ighl

y di

vers

e,

lead

ing

to a

bro

ad st

ruct

ural

repe

rtoi

re o

f the

ant

igen

-bin

ding

loop

s, S

even

VH

H su

bfam

ilies

wer

e re

cogn

ized

, of w

hich

five

wer

e co

nfirm

ed to

be

expr

esse

d lit

viv

o. C

ompa

riso

n of

ger

mlin

e an

d cD

NA

sequ

ence

s dem

onst

rate

s tha

t the

rear

rang

ed V

(H)H

s are

ex

tens

ivel

y di

vers

ified

by

som

atic

mut

atio

n pr

oces

ses,

lead

ing

to a

n ad

ditio

nal h

yper

vari

able

regi

on a

nd a

hig

h in

cide

nce

of n

ucle

-ot

ide

inse

rtio

ns o

r del

etio

ns, Th

ese

dive

rsifi

catio

n pr

oces

ses a

re d

rive

n by

hyp

erm

utat

ion

and

reco

mbi

natio

n ho

tspo

ts e

mbe

dded

in

the

VH

H g

erm

line

gene

s at t

he re

gion

s affe

ctin

g th

e st

ruct

ure

of th

e an

tigen

-bin

ding

loop

s.

Ngu

yen

et a

l. 20

00

App

licat

ion

Hum

an le

ukoc

yte

an

tige

nC

ance

r

A n

ovel

mon

oclo

nal a

nti-

pan

hum

an le

ukoc

yte

antig

en (H

LA) c

lass

I he

avy

chai

n an

tibod

y, EM

R8-5

, was

est

ablis

hed.

It c

ould

de

tect

HLA

-A, -

B, a

nd -C

ant

igen

s in

form

alin

-fixe

d pa

raffi

n em

bedd

ed ti

ssue

s. B

y im

mun

ohis

toch

emic

al st

aini

ng u

sing

the

EMR8

-5 a

ntib

ody,

vari

ous c

ance

r tis

sues

from

246

cas

es w

ere

exam

ined

for H

LA c

lass

I ex

pres

sion

. It w

as fo

und

that

HLA

cla

ss I

expr

essi

on w

as d

ecre

ased

in 2

0% to

42%

of t

he c

ases

of l

ung

canc

er, h

epat

ocel

lula

r car

cino

ma,

col

on c

ance

r, re

nal c

ell c

arci

nom

a,

and

urot

helia

l car

cino

ma.

In c

ontr

ast,

85%

of b

reas

t can

cer c

ases

had

loss

of o

r dec

reas

ed H

LA c

lass

I ex

pres

sion

. Of t

he 3

5 br

east

ca

ncer

cas

es th

at h

ad d

ecre

ased

HLA

cla

ss I

heav

y ch

ain

expr

essi

on, 3

3 (9

4%) a

lso

had

decr

ease

d be

ta2-

mic

rogl

obul

in e

xpre

ssio

n de

tect

ed b

y im

mun

ohis

toch

emic

al st

aini

ng. I

t was

sugg

este

d th

at H

LA c

lass

I do

wn-

regu

latio

n m

ight

be

a co

mm

on c

hara

cter

istic

of

bre

ast c

ance

r mos

tly c

ause

d by

the

dow

n-re

gula

tion

of b

eta2

-mic

rogl

obul

in e

xpre

ssio

n.

Tori

goe

et a

l. 20

12


459

App

licat

ion

Prio

n im

mun

othe

rapy

Scra

pie

PrP

conf

orm

atio

n

Alth

ough

ther

e is

cur

rent

ly n

o eff

ectiv

e tr

eatm

ent f

or p

rion

dis

ease

s, si

gnifi

cant

adv

ance

s hav

e be

en m

ade

in su

ppre

ssin

g its

pr

ogre

ss, u

sing

ant

ibod

ies t

hat b

lock

the

conv

ersi

on o

f PrP

(C) i

nto

PrP(

Sc).

In o

rder

to b

e eff

ectiv

e in

trea

ting

indi

vidu

als t

hat

have

pri

on d

isea

ses,

ant

ibod

ies m

ust b

e ca

pabl

e of

arr

estin

g di

seas

e in

its l

ate

stag

es. Th

is re

quir

es th

e de

velo

pmen

t of a

ntib

od-

ies w

ith h

ighe

r affi

nity

for P

rP(S

c) a

nd sy

stem

s for

effe

ctiv

e tr

ansl

ocat

ion

of a

ntib

odie

s acr

oss t

he b

lood

bra

in b

arri

er in

ord

er to

ac

hiev

e hi

gh c

once

ntra

tions

of i

nhib

itor a

t the

site

of p

rote

in re

plic

atio

n. A

n ad

ditio

nal a

dvan

tage

is th

e ab

ility

of t

hese

ant

ibod

ies

to a

cces

s the

cyt

osol

of a

ffect

ed c

ells

. To

this

end

, we

have

gen

erat

ed P

rP-s

peci

fic a

ntib

odie

s (kn

own

as P

rioV

) by

imm

uniz

atio

n of

cam

els w

ith m

urin

e sc

rapi

e m

ater

ial a

dsor

bed

to im

mun

omag

netic

bea

ds. Th

e Pr

ioV

ant

ibod

ies d

ispl

ay a

rang

e of

spec

ifici

ties

with

som

e re

cogn

izin

g th

e Pr

P (2

7–30

) pro

tein

ase

K-re

sist

ant f

ragm

ent,

othe

rs sp

ecifi

c fo

r PrP

(C) a

nd a

num

ber w

ith d

ual b

ind-

ing

spec

ifici

ty. I

ndep

ende

nt o

f the

ir P

rP c

onfo

rmat

ion

spec

ifici

ty, o

ne o

f the

Pri

oV a

ntib

odie

s (Pr

ioV

3) w

as sh

own

to b

ind

PrP(

C)

in th

e cy

toso

l of n

euro

blas

tom

a ce

lls. I

n m

arke

d co

ntra

st, c

onve

ntio

nal a

nti-P

rP a

ntib

odie

s pro

duce

d in

mou

se a

gain

st si

mila

r ta

rget

ant

igen

wer

e un

able

to c

ross

the

neur

onal

pla

sma

mem

bran

e an

d in

stea

d fo

rmed

a ri

ng a

roun

d th

e ce

lls. Th

e Pr

ioV

ant

i-Pr

P an

tibod

ies c

ould

pro

ve to

be

a va

luab

le to

ol fo

r the

neu

tral

izat

ion/

clea

ranc

e of

PrP

(Sc)

in in

trac

ellu

lar c

ompa

rtm

ents

of a

ffect

ed

neur

ons a

nd c

ould

pot

entia

lly h

ave

wid

er a

pplic

abili

ty fo

r the

trea

tmen

t of s

o-ca

lled

prot

ein-

mis

fold

ing

dise

ases

.

Taye

bi e

t al

. 201

0

App

licat

ion

Com

peti

tive

flui

d ar

ray

imm

unoa

ssay

Trin

itro

tolu

ene

Llam

as p

osse

ss u

niqu

e su

bcla

sses

of a

ntib

odie

s tha

t lac

k lig

ht c

hain

s, a

nd th

us a

re m

ade

by th

e pa

irin

g of

two

heav

y ch

ains

. IgG

w

as p

urifi

ed fr

om tw

o lla

mas

whi

ch h

ad b

een

imm

uniz

ed w

ith tr

initr

oben

zene

-key

hole

lim

pet h

emoc

yani

n. C

onve

ntio

nal I

gG1

and

heav

y ch

ain

IgG

2 an

d Ig

G3

subc

lass

es w

ere

frac

tiona

ted

usin

g affi

nity

chr

omat

ogra

phy.

The

effec

tiven

ess o

f hea

vy c

hain

an

tibod

ies f

or th

e de

tect

ion

of tr

initr

otol

uene

(TN

T) u

sing

a c

ompe

titiv

e flu

id a

rray

imm

unoa

ssay

was

eva

luat

ed a

nd c

ompa

red

to

both

the

llam

a Ig

G1

as w

ell a

s a m

urin

e m

onoc

lona

l ant

i-TN

T a

ntib

ody.

It w

as fo

und

that

hea

vy c

hain

ant

ibod

y bo

und

TN

T w

ith

sele

ctiv

ity si

mila

r to

conv

entio

nal a

ntib

odie

s, y

et th

e he

avy

chai

n an

tibod

ies p

osse

ssed

gre

ater

ther

mal

stab

ility

. The

titer

of t

he

heav

y ch

ain

antib

odie

s how

ever

was

foun

d to

be

10-f

old

low

er th

an th

e Ig

Gl1

; thu

s ana

lytic

al a

ssay

s wer

e be

st d

emon

stra

ted

usin

g th

e lla

ma

IgG

1 co

nven

tiona

l ant

ibod

y. Th

e T

NT

com

petit

ive

imm

unoa

ssay

on

the

Lum

inex

flui

d an

alyz

er h

ad a

dyn

amic

rang

e fr

om si

mila

r to

100

ng/m

l to

10 µ

g/m

l. U

tiliz

ing

the

sam

e tw

o-st

ep c

ompe

titiv

e as

say

form

at th

e dy

nam

ic ra

nge

of th

e m

onoc

lona

l an

tibod

y w

as fo

und

to h

ave

a br

oad

rang

e (1

ng/

ml t

o 1

µg/m

l). Th

is m

etho

d w

as d

emon

stra

ted

on T

NT

con

tam

inat

ed so

il ex

trac

ts

usin

g bo

th th

e lla

ma

IgG

1 an

d th

e m

ouse

mon

oclo

nal v

alid

atin

g th

e ut

ility

of m

etho

d fo

r ana

lysi

s of h

eld

sam

ples

.

And

er-

son

and

Gol

dman

20

08


460

Tabl

e 4.

Pro

duct

ion

of s

ingl

e-do

mai

n an

tibo

dy fr

agm

ents

usi

ng r

ecom

bina

nt te

chno

logy

E. c

oli

Azo

xyst

robi

nM

etal

affi

nity

chr

oma-

togr

aphy

ELIS

A

Thr

ee V

(H)H

s ag

ains

t the

mod

el h

apte

n, a

zoxy

stro

bin

(MW

403

), w

ere

isol

ated

from

a h

yper

-im

mun

ized

pha

ge-d

ispl

ayed

V

(H)H

libr

ary.

Thi

s lib

rary

was

con

stru

cted

by

isol

atin

g th

e V

(H)H

-cod

ing

gene

s fr

om th

e ly

mph

ocyt

es c

olle

cted

from

a

Lam

a gl

ama

that

was

imm

uniz

ed w

ith a

zoxy

stro

bin

conj

ugat

ed to

bov

ine

seru

m a

lbum

in (B

SA).

Six

roun

ds o

f pan

ning

wer

e pe

rfor

med

aga

inst

azo

xyst

robi

n co

njug

ated

to e

ither

ova

lbum

in (O

VA) o

r ra

bbit

ser

um a

lbum

in (R

SA) t

o en

rich

clo

nes

cont

aini

ng V

(H)H

s sp

ecifi

c to

the

hapt

en. A

fter

scr

eeni

ng 9

5 cl

ones

, thr

ee V

(H)H

s (A

27, A

72, a

nd A

85) w

ith d

iffer

ent a

min

o ac

id s

eque

nces

wer

e id

enti

fied,

exp

ress

ed in

sol

uble

form

at in

Esc

heri

chia

col

i HB2

151,

and

pur

ified

usi

ng n

icke

l-im

mo-

biliz

ed m

etal

aff

init

y ch

rom

atog

raph

y. C

ompe

titi

ve in

hibi

tion

enz

yme-

linke

d im

mun

osor

bent

ass

ay (C

I-EL

ISA

) sho

wed

th

at A

27 a

nd A

85 w

ere

spec

ific

to a

zoxy

stro

bin

whi

le A

72 w

as n

ot. T

he IC

(50)

val

ues

of A

27 a

nd A

85 V

(H)H

s w

ere

7.2

and

2.0

µm, r

espe

ctiv

ely.

To

our

know

ledg

e A

85 is

one

of t

he h

ighe

st a

ffin

ity

V(H

)Hs

that

has

yet

bee

n is

olat

ed a

gain

st a

hyd

ro-

phob

ic h

apte

n su

ch a

s az

oxys

trob

in.

Mak

-va

ndi-

Nej

ad e

t al

. 201

1

Pich

ia p

asto

ris

Aah

I’ sc

orpi

on to

xin

Alc

ohol

oxi

dase

pr

omot

erA

-fac

tor

secr

etio

n si

gnal

Usi

ng e

ukar

yoti

c ho

sts,

ant

ibod

y fr

agm

ents

are

gen

eral

ly e

xpre

ssed

by

targ

etin

g to

the

secr

etor

y pa

thw

ay. T

his

enab

les

not

only

eff

icie

nt d

isul

fide

bond

form

atio

n bu

t als

o se

cret

ion

of s

olub

le a

nd c

orre

ctly

fold

ed p

rodu

ct. F

or th

is g

oal,

a re

com

bi-

nant

vec

tor

was

con

stru

cted

to p

rodu

ce a

sin

gle-

dom

ain

anti

body

(NbA

ahI’2

2) d

irec

ted

agai

nst A

ahI’s

corp

ion

toxi

n us

ing

the

met

hylo

trop

hic

yeas

t. T

he c

orre

spon

ding

com

plem

enta

ry D

NA

was

clo

ned

unde

r co

ntro

l of t

he a

lcoh

ol o

xida

se p

ro-

mot

er in

fram

e w

ith th

e Sa

ccha

rom

yces

α-f

acto

r se

cret

ion

sign

al a

nd th

en tr

ansf

erre

d to

P. p

asto

ris

cell

stra

in X

-33.

Usi

ng

Wes

tern

blo

t, w

e de

tect

ed th

e ex

pres

sion

of t

he r

ecom

bina

nt N

bAah

I’22

excl

usiv

ely

in th

e cu

lture

med

ium

. Tar

geti

ng to

th

e hi

stid

ine

labe

l, th

e se

cret

ed n

anob

ody

was

eas

ily p

urifi

ed o

n ni

ckel

nitr

ilotr

iace

tic

acid

res

in a

nd th

en te

sted

in e

nzym

e-lin

ked

imm

unos

orbe

nt a

ssay

. Int

eres

ting

ly, t

he p

rodu

ctio

n le

vel o

f the

NbA

ahI’2

2 in

its

new

gly

cosy

late

d fo

rm r

each

ed m

ore

than

six

fold

that

obt

aine

d in

E. c

oli.

The

se fi

ndin

gs g

ive

mor

e ev

iden

ce fo

r th

e ut

iliza

tion

of P

. pas

tori

s as

a h

eter

olog

ous

expr

essi

on s

yste

m.

Ezzi

ne

et a

l. 20

12

Sacc

haro

myc

es

cere

visi

ae

Secr

etio

n effi

cien

cyJ s

egm

ent

Vari

able

dom

ains

of l

lam

a he

avy-

chai

n an

tibo

dies

(VH

H) a

re b

ecom

ing

a po

tent

tool

for

a w

ide

rang

e of

bio

tech

nolo

gica

l an

d m

edic

al a

pplic

atio

ns. B

ecau

se o

f str

uctu

ral f

eatu

res

typi

cal o

f the

ir s

ingl

e-do

mai

n na

ture

, the

y ar

e re

lati

vely

eas

y to

pro

-du

ce in

low

er e

ukar

yote

s, b

ut it

is n

ot u

ncom

mon

that

som

e m

olec

ules

hav

e po

or s

ecre

tion

eff

icie

ncy.

We

ther

efor

e se

t out

to

stu

dy th

e pr

oduc

tion

yie

ld o

f VH

H. W

e co

mpu

tati

onal

ly id

enti

fied

five

key

resi

dues

that

are

cru

cial

for

fold

ing

and

secr

e-ti

on, a

nd w

e va

lidat

ed th

eir

impo

rtan

ce w

ith s

yste

mat

ic s

ite-d

irec

ted

mut

atio

ns. T

he o

bser

vati

on th

at a

ll ke

y re

sidu

es w

ere

loca

lised

in th

e V

seg

men

t, in

pro

xim

ity

of th

e J s

egm

ent o

f VH

H, l

ed u

s to

stu

dy th

e im

port

ance

of J

seg

men

t in

secr

etio

n ef

ficie

ncy.

Intr

igui

ngly

, we

foun

d th

at th

e us

e of

spe

cific

J se

gmen

ts in

VH

H c

ould

str

ongl

y in

fluen

ce th

e pr

oduc

tion

yie

ld.

Sequ

ence

ana

lysi

s an

d ex

pres

sion

exp

erim

ents

str

ongl

y su

gges

ted

that

inte

ract

ions

with

cha

pero

nes,

esp

ecia

lly w

ith th

e J

segm

ent,

are

a cr

ucia

l asp

ect o

f the

pro

duct

ion

yiel

d of

VH

H.

Gor

lani

et

al.

2012


461

Mag

neto

spir

illum

gr

yphi

swal

dens

e M

agne

tic

nano

part

icle

Mag

neto

som

eR

ed fl

uore

scei

n pr

otei

n

Num

erou

s ap

plic

atio

ns o

f con

vent

iona

l and

bio

geni

c m

agne

tic n

anop

artic

les

(MN

Ps),

such

as

in d

iagn

ostic

s, im

mun

omag

netic

se

para

tions

, and

mag

netic

cel

l lab

elin

g, re

quir

e th

e im

mob

iliza

tion

of a

ntib

odie

s. T

his

is u

sual

ly a

ccom

plis

hed

by c

hem

ical

co

njug

atio

n, w

hich

, how

ever

, has

sev

eral

dis

adva

ntag

es, s

uch

as p

oor

effic

ienc

y an

d th

e ne

ed fo

r co

uplin

g ch

emis

try.

Her

e,

we

desc

ribe

a n

ovel

str

ateg

y to

dis

play

a fu

nctio

nal c

amel

id a

ntib

ody

frag

men

t (na

nobo

dy) f

rom

an

alpa

ca (L

ama

paco

s) o

n th

e su

rfac

e of

bac

teri

al b

ioge

nic

mag

netic

nan

opar

ticle

s (m

agne

toso

mes

). M

agne

toso

me-

spec

ific

expr

essi

on o

f a re

d flu

ores

-ce

nt p

rote

in (R

FP)-

bind

ing

nano

body

(RBP

) in

vivo

was

acc

ompl

ishe

d by

gen

etic

fusi

on o

f RBP

to th

e m

agne

toso

me

prot

ein

Mam

C in

the

mag

netit

e-sy

nthe

sizi

ng b

acte

rium

Mag

neto

spir

illum

gry

phis

wal

dens

e. W

e de

mon

stra

te th

at is

olat

ed m

agne

to-

som

es e

xpre

ssin

g M

amC

-RBP

eff

icie

ntly

reco

gniz

e an

d bi

nd th

eir

antig

en in

vitr

o an

d ca

n be

use

d fo

r im

mun

opre

cipi

tatio

n of

RFP

-tag

ged

prot

eins

and

thei

r in

tera

ctio

n pa

rtne

rs fr

om c

ell e

xtra

cts.

In a

dditi

on, w

e sh

ow th

at c

oexp

ress

ion

of m

onom

eric

RF

P (m

RFP

or it

s va

rian

t mC

herr

y) a

nd M

amC

-RBP

resu

lts in

intr

acel

lula

r re

cogn

ition

and

mag

neto

som

e re

crui

tmen

t of R

FP

with

in li

ving

bac

teri

a. T

he in

trac

ellu

lar

expr

essi

on o

f a fu

nctio

nal n

anob

ody

targ

eted

to a

spe

cific

bac

teri

al c

ompa

rtm

ent o

pens

ne

w p

ossi

bilit

ies

for

in v

ivo

synt

hesi

s of

MN

P-im

mob

ilize

d na

nobo

dies

. Mor

eove

r, in

trac

ellu

lar

nano

trap

s ca

n be

gen

erat

ed to

m

anip

ulat

e ba

cter

ial s

truc

ture

s in

live

cel

ls.

Polli

thy

et a

l. 20

11

Toba

cco

expr

essi

on

syst

emA

grob

acte

rium

Seve

ral p

lant

s are

the

faci

le a

nd e

cono

mic

bio

reac

tor f

or la

rge-

scal

e pr

oduc

tion

of in

dust

rial

and

pha

rmac

eutic

al a

gent

s lik

e pr

o-te

ins a

nd a

ntib

odie

s. H

ere,

we

have

sele

cted

toba

cco

as th

e ho

st p

lant

bec

ause

of l

arge

scal

e pr

oduc

tion

capa

bilit

y an

d m

any

othe

r ad

vant

ages

such

as g

reat

er sa

fety

and

low

er p

rodu

ctio

n co

sts w

hen

com

pare

d to

ani

mal

-bas

ed sy

stem

s. In

this

stud

y, w

e ha

ve su

b-cl

oned

VH

H g

ene

into

pBI

121

usin

g ph

asm

id p

CA

NTA

B5E.

The

new

con

stru

ct w

as u

sed

to tr

ansf

orm

the

Agr

obac

teri

um st

rain

s C

58G

V31

01 a

nd L

BA44

04. A

grob

acte

rium

stra

in C

58G

V31

01 sh

owed

a h

ighe

r vir

ulen

ce o

n le

af d

isks

of N

icot

iana

taba

cum

(N

C25

). Tr

ansg

enic

toba

cco

plan

ts w

ere

then

dev

elop

ed b

y in

trod

ucin

g V

HH

gen

e un

der t

he c

ontr

ol o

f CaM

V 3

5S p

rom

oter

. The

pres

ence

of t

he V

HH

ant

ibod

y ge

ne in

the

plan

t gen

ome

was

ver

ified

by

PCR

anal

ysis

and

Sou

ther

n hy

brid

izat

ion

expe

rim

ents

. N

orth

ern

blot

ana

lysi

s sho

wed

that

the

gene

s cod

ing

for t

he V

HH

cou

ld b

e ex

pres

sed

in to

bacc

o pl

ants

. Thre

e lin

es o

f tra

nsge

nic

plan

t tha

t exp

ress

es h

igh

leve

ls o

f mRN

A w

ere

scre

ened

in a

furt

her a

naly

sis.

The

expr

essi

on o

f VH

H w

as th

en o

bser

ved

in tr

ans-

geni

c pl

ants

by

ELIS

A u

sing

the

spec

ific

antib

ody

deve

lope

d, th

e re

sults

show

ed th

ree

to fi

ve fo

lds h

ighe

r tha

n no

n-tr

ansg

enic

to

bacc

os.

Kor

ou-

zhde

hy

et a

l. 20

11

Tum

our

necr

osis

fact

orFu

sion

Elas

tin-

like

poly

pept

ide

Toba

cco

Tum

our n

ecro

sis f

acto

r (T

NF)

is a

maj

or p

ro-in

flam

mat

ory

cyto

kine

invo

lved

in m

ultip

le in

flam

mat

ory

dise

ases

. The

detr

imen

tal

activ

ity o

f TN

F ca

n be

blo

cked

by

vari

ous a

ntag

onis

ts, a

nd c

omm

erci

al th

erap

eutic

s bas

ed u

pon

this

pri

ncip

le h

ave

been

app

rove

d fo

r tre

atm

ent o

f dis

ease

s inc

ludi

ng rh

eum

atoi

d ar

thri

tis, C

rohn

’s di

seas

e an

d ps

oria

sis.

In a

sear

ch fo

r new

, im

prov

ed a

nti-

infla

mm

ator

y th

erap

eutic

s we

have

des

igne

d a

sing

le-d

omai

n m

onoc

lona

l ant

ibod

y (V

(H)H

), w

hich

reco

gniz

es T

NF.

The

antib

ody

com

pone

nt (T

NF-

V(H

)H) i

s bas

ed u

pon

an a

nti-h

uman

TN

F C

amel

idae

hea

vy-c

hain

mon

oclo

nal a

ntib

ody,

whi

ch w

as li

nked

to a

n el

astin

-like

pol

ypep

tide

(ELP

). W

e de

mon

stra

te th

at E

LP fu

sion

to th

e T

NF-

V(H

)H e

nhan

ces a

ccum

ulat

ion

of th

e fu

sion

pro

tein

du

ring

bio

man

ufac

turi

ng in

tran

sgen

ic to

bacc

o pl

ants

. With

this

stud

y, w

e sh

ow fo

r the

firs

t tim

e th

at th

is p

lant

-der

ived

ant

i-hu

man

TN

F-V

(H)H

ant

ibod

y w

as b

iolo

gica

lly a

ctiv

e in

viv

o. Th

eref

ore,

ther

apeu

tic a

pplic

atio

n of

TN

F-V

(H)H

-ELP

fusi

on p

rote

in

was

test

ed in

hum

aniz

ed T

NF

mic

e an

d w

as sh

own

to b

e eff

ectiv

e in

pre

vent

ing

deat

h ca

used

by

sept

ic sh

ock.

The

in v

ivo

pers

is-

tenc

e of

the

ELPy

late

d an

tibod

y w

as si

mila

r to

24 fo

ld lo

nger

than

that

of n

on-E

LPyl

ated

TN

F-V

(H)H

.

Con

rad

et a

l. 20

11


462

Pann

ing

met

hods

Bot

ulin

um n

euro

toxi

nB

indi

ng k

inet

ics

Sing

le-d

omai

n an

tibo

dies

(sdA

b) s

peci

fic fo

r bo

tulin

um n

euro

toxi

n se

roty

pe A

(BoN

T A

) wer

e se

lect

ed fr

om a

n im

mun

e lla

ma

phag

e di

spla

y lib

rary

der

ived

from

a ll

ama

that

was

imm

uniz

ed w

ith B

oNT

A to

xoid

. The

con

stru

cted

pha

ge li

brar

y w

as p

anne

d us

ing

two

met

hods

: pan

ning

on

plat

es c

oate

d w

ith B

oNT

A to

xoid

(BoN

T A

Td) a

nd B

oNT

A c

ompl

ex to

xoid

(B

oNT

Ac

Td) a

nd p

anni

ng o

n m

icro

sphe

res

coup

led

to B

oNT

A T

d an

d Bo

NT

A to

xin

(BoN

T A

Tx)

. Bot

h pa

nnin

g m

etho

ds

sele

cted

for

bind

ers

that

had

iden

tica

l seq

uenc

es, s

ugge

stin

g th

at p

anni

ng o

n to

xoid

ed m

ater

ial m

ay b

e as

eff

ecti

ve a

s pa

n-ni

ng o

n be

ad-i

mm

obili

zed

toxi

n fo

r is

olat

ing

spec

ific

bind

ers.

All

of th

e is

olat

ed b

inde

rs te

sted

wer

e ob

serv

ed to

rec

ogni

ze

bead

-im

mob

ilize

d Bo

NT

A T

x in

dir

ect b

indi

ng a

ssay

s, a

nd s

how

ed v

ery

little

cro

ss-r

eact

ivit

y to

war

ds o

ther

BoN

T s

ero-

type

s an

d un

rela

ted

prot

ein.

San

dwic

h as

says

that

inco

rpor

ated

sel

ecte

d sd

Ab

as c

aptu

re a

nd tr

acer

ele

men

ts d

emon

stra

ted

that

all

of th

e sd

Ab

wer

e ab

le to

rec

ogni

ze s

olub

le (“

live”

) BoN

T A

Tx

and

BoN

T A

c Tx

with

vir

tual

ly n

o cr

oss-

reac

tivi

ty

with

oth

er B

oNT

ser

otyp

es. T

he is

olat

ed s

dAb

did

not e

xhib

it th

e hi

gh d

egre

e of

ther

mal

sta

bilit

y of

ten

asso

ciat

ed w

ith

thes

e re

agen

ts; a

fter

the

first

hea

ting

cyc

le m

ost o

f the

bin

ding

act

ivit

y w

as lo

st, b

ut th

e po

rtio

n of

the

prot

ein

that

did

ref

old

and

reco

ver

anti

gen-

bind

ing

acti

vity

sho

wed

onl

y m

inim

al lo

ss o

n su

bseq

uent

hea

ting

and

coo

ling

cycl

es. T

he b

indi

ng k

inet

-ic

s of

sel

ecte

d bi

nder

s, a

sses

sed

by b

oth

an e

quili

briu

m fl

uid

arra

y as

say

as w

ell a

s su

rfac

e pl

asm

on r

eson

ance

(SPR

) usi

ng

toxo

ided

mat

eria

l, ga

ve d

isso

ciat

ion

cons

tant

s (K

(D))

in th

e ra

nge

2.2

× 10

(–11

) to

1.6

× 10

(–10

)M. T

hese

hig

h-af

finit

y bi

nd-

ers

may

pro

ve b

enef

icia

l to

the

deve

lopm

ent o

f rec

ombi

nant

rea

gent

s fo

r th

e ra

pid

dete

ctio

n of

BoN

T A

, par

ticu

larl

y in

fiel

d sc

reen

ing

and

mon

itori

ng a

pplic

atio

ns.

Swai

n et

al

. 201

0

Picl

oram

Rib

osom

e di

spla

yIn

vit

ro tr

ansc

ript

ion/

tr

ansl

atio

nPo

int m

utat

ion

Bin

ding

kin

etic

sM

olec

ular

evo

luti

on

Picl

oram

-spe

cific

var

iabl

e fr

agm

ents

(V(H

H)s

) of h

eavy

cha

in a

ntib

odie

s (H

CA

bs) w

ere

sele

cted

from

a n

aive

-llam

a lib

rary

usi

ng

ribo

som

e di

spla

y te

chno

logy

. A c

DN

A li

brar

y of

V(H

H)s

was

con

stru

cted

from

lym

phoc

ytes

of a

non

-im

mun

ized

llam

a an

d en

gi-

neer

ed to

allo

w in

vitr

o tr

ansc

ript

ion

and

tran

slat

ion.

With

no

stop

cod

ons p

rese

nt o

n th

e tr

ansc

ript

s, tr

imer

ic c

ompl

exes

of r

ibo-

som

es, m

RNA

s and

nas

cent

pep

tides

wer

e pr

oduc

ed fo

r affi

nity

sele

ctio

n, i.

e. p

anni

ng. A

fter

thre

e cy

cles

of p

anni

ng, s

even

diff

er-

ent V

(HH

)s a

ll be

long

ing

to th

e V

-HH

subf

amily

1 w

ere

isol

ated

. Fol

low

ing

anot

her t

hree

cyc

les o

f sel

ectio

n, o

nly

two

of th

e se

ven

V(H

H)s

per

sist

ed. A

com

pari

son

of th

ese

two

sequ

ence

s with

kno

wn

sequ

ence

s in

the

liter

atur

e su

gges

ts th

at p

oint

mut

atio

ns

may

hav

e be

en in

trod

uced

into

the

DN

A p

ool d

urin

g PC

R am

plifi

catio

n st

eps o

f lib

rary

con

stru

ctio

n, p

anni

ng a

nd/o

r clo

ning

. Th

ree

sepa

rate

poi

nt m

utat

ions

cau

sing

thre

e in

depe

nden

t am

ino

acid

cha

nges

(non

syno

nom

ous m

utat

ions

) acc

umul

ated

in th

e sa

me

sequ

ence

and

enr

iche

d th

roug

hout

the

sele

ctio

n pr

otoc

ol, s

ugge

stin

g th

at th

ese

chan

ges c

onfe

r bin

ding

adv

anta

ges.

Sur

face

pl

asm

on re

sona

nce

(SPR

) ana

lysi

s was

use

d to

det

erm

ine

bind

ing

kine

tics o

f the

two

clon

es (3

-1D

2 an

d 3-

1F6)

repr

esen

ting

the

two

diffe

rent

sets

of i

sola

ted

com

plem

enta

rity

det

erm

inin

g re

gion

(CD

R)3s

. Mea

sure

d K

(D)s

wer

e 3

and

254

µM, r

espe

ctiv

ely.

The

resu

lts in

dica

te th

at ri

boso

me

disp

lay

tech

nolo

gy c

an b

e us

ed to

effi

cien

tly is

olat

e ha

pten

-spe

cific

ant

ibod

y (A

b) fr

agm

ents

from

a

naiv

e lib

rary

and

con

curr

ently

intr

oduc

e di

vers

ity to

the

sele

cted

poo

l the

reby

faci

litat

ing

mol

ecul

ar e

volu

tion.

Rib

osom

e di

spla

y te

chno

logy

can

com

pens

ate

for t

he li

mite

d di

vers

ity o

f a V

-HH

nai

ve li

brar

y an

d pr

ovid

e an

unl

imite

d so

urce

of a

ffini

ty-m

atur

ed

imm

unoa

ctiv

e re

agen

ts in

vitr

o.

Yau

et

al. 2

003


463

Rib

osom

e di

spla

yEr

ror-

pron

e

mut

agen

esis

Lyso

zym

eSo

lven

t-ex

pose

d

resi

dues

We

chos

e th

e he

n-eg

g-ly

sozy

me-

spec

ific

arch

etyp

al T

ype

I V(N

AR)

5A

7 an

d us

ed ri

boso

me

disp

lay

in c

ombi

natio

n w

ith e

rror

-pr

one

mut

agen

esis

to in

terr

ogat

e th

e en

tire

sequ

ence

spac

e. W

e fo

und

a hi

gh le

vel o

f mut

atio

nal p

last

icity

acr

oss t

he V

(NA

R)

dom

ain,

par

ticul

arly

with

in th

e fr

amew

ork

2 an

d hy

perv

aria

ble

regi

on 2

regi

ons.

A n

umbe

r of r

esid

ues i

mpo

rtan

t for

affi

n-ity

wer

e id

entifi

ed, a

nd a

trip

le m

utan

t com

bini

ng A

ID, S

61R

, and

G62

R re

sulte

d in

a K

(D) o

f 460

pM

for h

en e

gg ly

sozy

me,

a

20-f

old

impr

ovem

ent o

ver w

ild-t

ype

5A7,

and

the

high

est K

(D) y

et re

port

ed fo

r V(N

AR)

-ant

igen

inte

ract

ions

. Thes

e fin

ding

s wer

e ra

tiona

lised

usi

ng st

ruct

ural

mod

ellin

g an

d in

dica

te th

e im

port

ance

of r

esid

ues o

utsi

de th

e cl

assi

cal c

ompl

emen

tari

ty d

eter

min

-in

g re

gion

s in

mak

ing

nove

l ant

igen

con

tact

s tha

t mod

ulat

e affi

nity

. We

also

loca

ted

two

solv

ent-

expo

sed

resi

dues

(G15

and

G42

), di

stan

t fro

m th

e V

(NA

R) p

arat

ope,

whi

ch re

tain

func

tion

upon

mut

atio

n to

cys

tein

e an

d ha

ve th

e po

tent

ial t

o be

exp

loite

d as

si

tes f

or ta

rget

ed c

oval

ent m

odifi

catio

n. O

ur fi

ndin

gs w

ith 5

A7

wer

e ex

tend

ed to

all

know

n N

AR

stru

ctur

es u

sing

an

in-d

epth

bi

oinf

orm

atic

ana

lysi

s of s

eque

nce

data

ava

ilabl

e in

the

liter

atur

e an

d a

new

ly g

ener

ated

V(N

AR)

dat

abas

e. Th

is st

udy

allo

wed

us

to id

entif

y, fo

r the

firs

t tim

e, b

oth

V(N

AR)

-spe

cific

and

V(N

AR)

/Ig

V(L

)/TC

R V

(alp

ha) o

verl

appi

ng h

allm

ark

resi

dues

, whi

ch

are

criti

cal f

or th

e st

ruct

ural

and

func

tiona

l int

egri

ty o

f the

sing

le d

omai

n. In

trig

uing

ly, e

ach

of o

ur d

esig

nate

d V

(NA

R)-s

peci

fic

hallm

arks

alig

n pr

ecis

ely

with

pre

viou

sly

defin

ed m

utat

iona

l ‘co

ld sp

ots’

in n

atur

al n

urse

shar

k cD

NA

sequ

ence

s. Th

ese

findi

ngs

will

aid

futu

re V

(NA

R) e

ngin

eeri

ng a

nd o

ptim

isat

ion

stud

ies t

owar

ds th

e de

velo

pmen

t of V

(NA

R) si

ngle

-dom

ain

prot

eins

as v

iabl

e bi

othe

rape

utic

s.

Fenn

ell

et a

l. 20

10

Yeas

t sur

face

dis

play

Pich

ia p

asto

ris

Gre

en fl

uore

scen

t pr

otei

n

Yeas

t sur

face

dis

play

is a

n effi

cien

t too

l for

isol

atin

g an

d en

gine

erin

g an

tibod

y fr

agm

ents

, bot

h sc

Fv a

nd F

ab. W

e de

scri

be th

e us

e of

pro

tein

dis

play

on

Pich

ia p

asto

ris f

or th

e ra

pid

sele

ctio

n of

cam

elid

ant

ibod

ies c

ompo

sed

only

of h

eavy

cha

ins (

nano

bodi

es)

from

a li

brar

y de

rive

d fr

om a

Ilam

a im

mun

ized

with

Gre

en F

luor

esce

nt P

rote

in. Th

e lib

rary

of n

anob

ody-

codi

ng se

quen

ces w

as

fuse

d to

the

C-t

erm

inal

par

t of t

he S

acch

arom

yces

cer

evis

iae

alph

a-ag

glut

inin

gen

e (S

AG

1) a

nd e

xpre

ssed

in g

lyco

engi

neer

ed

P. p

asto

ris.

A h

igh

effici

ency

tran

sfor

mat

ion

prot

ocol

yie

lded

a li

brar

y of

5 ×

10(

7) c

lone

s. A

bout

80%

of t

he c

lone

s str

ongl

y ex

pres

sed

the

nano

body

fusi

on. N

anob

ody-

disp

layi

ng c

lone

s wer

e ra

pidl

y en

rich

ed b

y flu

ores

cenc

e ac

tivat

ed c

ell s

ortin

g (F

AC

S),

and

GFP

-spe

cific

nan

obod

y-di

spla

ying

clo

nes w

ere

isol

ated

and

equ

ilibr

ium

dis

soci

atio

n co

nsta

nts (

K(d

)) d

eter

min

ed. Th

is

tech

nolo

gy fo

r dis

play

ing

prot

ein

libra

ries

on

P. p

asto

ris e

nabl

es th

e is

olat

ion

and

engi

neer

ing

of a

ntib

ody-

deri

ved

mol

ecul

es in

a

robu

st e

ukar

yotic

exp

ress

ion

host

.

Ryck

aert

et

al.

2010

Surf

ace

disp

lay

Expr

essi

on c

asse

ttes

Lact

obac

illus

par

acas

eiR

otav

irus

A se

ries o

f exp

ress

ion

cass

ette

s whi

ch m

edia

te se

cret

ion

or su

rfac

e di

spla

y of

ant

ibod

y fr

agm

ents

was

stab

ly in

tegr

ated

in th

e ch

rom

o-so

me

of L

acto

baci

llus p

arac

asei

. L. p

arac

asei

pro

duci

ng su

rfac

e-an

chor

ed v

aria

ble

dom

ain

of ll

ama

heav

y ch

ain

(VH

H) (

ARP

1) d

irect

ed

agai

nst r

otav

irus s

how

ed e

ffici

ent b

indi

ng to

rota

viru

s and

pro

tect

ion

in th

e m

ouse

mod

el o

f rot

aviru

s inf

ectio

n.

Mar

tin

et a

l. 20

11

Fung

al e

xpre

ssio

n sy

stem

Asp

ergi

llus

awam

ori

We

repo

rt th

e ex

pres

sion

and

pro

duct

ion

of ll

ama

vari

able

hea

vy-c

hain

ant

ibod

y fr

agm

ents

(V(H

H)s

) by

Asp

ergi

llus a

wam

ori.

Frag

men

ts e

ncod

ing

V(H

H)s

wer

e cl

oned

in a

suita

ble

Asp

ergi

llus e

xpre

ssio

n ve

ctor

and

tran

sfor

man

ts se

cret

ing

V-H

H fr

agm

ents

w

ere

anal

ysed

for i

nteg

rate

d ge

ne c

opy-

num

bers

, mRN

A le

vels

and

pro

tein

pro

duct

ion.

Fun

ctio

nal V

(HH

)s w

ere

dete

cted

in th

e cu

lture

med

ium

, ind

icat

ing

the

feas

ibili

ty o

f pro

duci

ng th

is ty

pe o

f pro

tein

in a

fung

al e

xpre

ssio

n sy

stem

. Sec

rete

d V

(HH

)s w

ere

subj

ecte

d to

(ext

race

llula

r) d

egra

datio

n, w

hich

cou

ld b

e pa

rtia

lly p

reve

nted

by

the

addi

tion

of B

SA to

the

cultu

re m

ediu

m.

Joos

ten

et a

l. 20

05


464

Chi

nese

ham

ster

ova

ry

cells

Expr

essi

on v

ecto

rsR

epor

ter

gene

GFP

-fus

ion

prot

ein

Ther

e is

an in

crea

sing

inte

rest

in th

e ap

plic

atio

n of

nan

obod

ies s

uch

as V

HH

in th

e fie

ld o

f the

rapy

and

imag

ing.

In th

e pr

esen

t stu

dy a

st

able

gen

etic

ally

eng

inee

red

cell

line

of C

hine

se h

amst

er o

vary

(CH

O) o

rigin

tran

sfec

ted

usin

g tw

o se

ts o

f exp

ress

ion

vect

ors w

as c

on-

stru

cted

in o

rder

to p

erm

it th

e cy

topl

asm

ic a

nd e

xtra

cellu

lar e

xpre

ssio

n of

sing

le d

omai

n an

tibod

y al

ong

with

gre

en fl

uore

scen

t pro

tein

(G

FP) a

s rep

orte

r gen

e. Th

e qu

ality

of t

he c

onst

ruct

s wer

e ex

amin

ed b

oth

by th

e re

stric

tion

map

as w

ell a

s seq

uenc

e an

alys

is. Th

e ge

ne

tran

sfec

tion

and

prot

ein

expr

essio

n w

as fu

rthe

r exa

min

ed b

y re

vers

e tr

ansc

riptio

n-po

lym

eras

e ch

ain

reac

tion

(RT-

PCR)

. The

tran

sfec

ted

cells

wer

e gr

own

in 2

00 µ

g/m

l hyg

rom

ycin

con

tain

ing

med

ia a

nd th

e st

able

cel

l lin

e ob

tain

ed sh

owed

fluo

resc

ent a

ctiv

ity fo

r mor

e th

an

a pe

riod

of 1

80 d

ays.

The

prod

uctio

n of

fusio

n pr

otei

n w

as a

lso d

etec

ted

by fl

uore

scen

t mic

rosc

opy,

fluor

esce

nt sp

ectr

osco

py a

s wel

l as

by e

nzym

e-lin

ked

imm

unos

orbe

nt a

ssay

(ELI

SA) a

naly

sis. Th

is st

rate

gy a

llow

s a ra

pid

prod

uctio

n of

reco

mbi

nant

fluo

bodi

es in

volv

ing

VH

H, w

hich

can

be

used

in v

ario

us e

xper

imen

ts su

ch a

s im

agin

g an

d de

tect

ion

in w

hich

a p

rimar

y la

bele

d an

tibod

y is

requ

ired.

Bazl

et

al. 2

007

Expr

essi

on in

mou

se

germ

line

Dro

med

ary

tran

sgen

eG

amm

a 2a

H c

hain

In m

atur

e B

cells

of m

ice

and

mos

t mam

mal

s, c

ellu

lar r

elea

se o

f sin

gle

H c

hain

Abs

with

out L

cha

ins i

s pre

vent

ed b

y H

cha

in a

sso-

ciat

ion

with

Ig-s

peci

fic c

hape

rons

in th

e en

dopl

asm

ic re

ticiu

lum

. In

prec

urso

r B c

ells

, how

ever

, sur

face

exp

ress

ion

of µ

-H c

hain

in

the

abse

nce

of su

rrog

ate

and

conv

entio

nal L

cha

in h

as b

een

iden

tified

. Des

pite

this

, Ag-

spec

ific

sing

le H

cha

in Ig

repe

rtoi

res,

usi

ng

µ-, g

amm

a-, e

psilo

n-, o

r alp

ha-H

cha

ins f

ound

in c

onve

ntio

nal A

bs, a

re n

ot p

rodu

ced.

Mor

eove

r, re

mov

al o

f H c

hain

or,

sepa

rate

ly,

L ch

ain

(kap

pa/la

mbd

a) lo

cus c

ore

sequ

ence

s by

gene

targ

etin

g ha

s pre

vent

ed B

cel

l dev

elop

men

t. In

con

tras

t, H

cha

in-o

nly

Abs

ar

e pr

oduc

ed a

bund

antly

in C

amel

idae

as H

2 Ig

G w

ithou

t the

C(H

)l do

mai

n. T

o te

st w

heth

er H

cha

in A

bs c

an b

e pr

oduc

ed in

m

ice,

and

to in

vest

igat

e ho

w th

eir e

xpre

ssio

n aff

ects

B c

ell d

evel

opm

ent,

we

intr

oduc

ed a

rear

rang

ed d

rom

edar

y ga

mm

a 2a

H

chai

n in

to th

e m

ouse

ger

mlin

e. Th

e dr

omed

ary

tran

sgen

e w

as e

xpre

ssed

as a

nat

ural

ly o

ccur

ring

Ag-

spec

ific

disu

lphi

de-li

nked

ho

mod

imer

, whi

ch sh

owed

that

B c

ell d

evel

opm

ent c

an b

e in

stig

ated

by

expr

essi

on o

f sin

gle

H c

hain

s with

out L

cha

ins.

Lym

pho-

cyte

dev

elop

men

t and

B c

ell p

rolif

erat

ion

was

acc

ompl

ishe

d de

spite

the

abse

nce

of L

cha

in fr

om th

e BC

R co

mpl

ex. E

ndog

enou

s Ig

coul

d no

t be

dete

cted

, alth

ough

V(D

)J re

com

bina

tion

and

IgH

/L tr

ansc

ript

ion

was

una

ltere

d. F

urth

erm

ore,

cro

ssin

g th

e dr

om-

edar

y H

cha

in m

ice

with

mic

e de

void

of a

ll C

gen

es d

emon

stra

ted

with

out a

dou

bt th

at a

H c

hain

-onl

y A

b ca

n fa

cilit

ate

B ce

ll de

velo

pmen

t ind

epen

dent

of e

ndog

enou

s Ig

expr

essi

on, s

uch

as µ

- or d

elta

-H c

hain

, at e

arly

dev

elop

men

tal s

tage

s.

Zou

et

al. 2

005

Uni

vers

al h

uman

ized

na

nobo

dy s

caffo

ldN

anob

odie

s, si

ngle

-dom

ain

antig

en-b

indi

ng fr

agm

ents

of c

amel

id-s

peci

fic h

eavy

-cha

in o

nly

antib

odie

s offe

r spe

cial

adv

anta

ges

in th

erap

y ov

er c

lass

ic a

ntib

ody

frag

men

ts b

ecau

se o

f the

ir sm

alle

r siz

e, ro

bust

ness

, and

pre

fere

nce

to ta

rget

uni

que

epito

pes.

A

Nan

obod

y di

ffers

from

a h

uman

hea

vy c

hain

var

iabl

e do

mai

n in

abo

ut te

n am

ino

acid

s spr

ead

all o

ver i

ts su

rfac

e, fo

ur h

allm

ark

Nan

obod

y-sp

ecifi

c am

ino

acid

s in

the

fram

ewor

k-2

regi

on (p

ositi

ons 4

2, 4

9, 5

0, a

nd 5

2), a

nd a

long

er th

ird

antig

en-b

indi

ng lo

op

(H3)

fold

ing

over

this

are

a. F

or th

erap

eutic

app

licat

ions

the

cam

elid

-spe

cific

am

ino

acid

sequ

ence

s in

the

fram

ewor

k ha

ve to

be

mut

ated

to th

eir h

uman

hea

vy c

hain

var

iabl

e do

mai

n eq

uiva

lent

, i.e

. hum

aniz

ed. W

e pe

rfor

med

this

hum

aniz

atio

n ex

erci

se w

ith

Nan

obod

ies o

f the

subf

amily

that

repr

esen

ts c

lose

to 8

0% o

f all

drom

edar

y-de

rive

d N

anob

odie

s and

inve

stig

ated

the

effec

ts o

n an

tigen

affi

nity

, sol

ubili

ty, e

xpre

ssio

n yi

eld,

and

stab

ility

. It i

s dem

onst

rate

d th

at th

e hu

man

izat

ion

of N

anob

ody-

spec

ific

resi

dues

ou

tsid

e fr

amew

ork-

2 ar

e ne

utra

l to

the

Nan

obod

y pr

oper

ties.

Sur

pris

ingl

y, th

e G

lu-4

9 ->

Gly

and

Arg

-50

-> L

eu h

uman

izat

ion

of

hallm

ark

amin

o ac

ids g

ener

ates

a si

ngle

dom

ain

that

is m

ore

stab

le th

ough

pro

babl

y le

ss so

lubl

e. Th

e ot

her f

ram

ewor

k-2

subs

titu-

tions

, Phe

-42

-> V

al a

nd G

ly/A

la-5

2 ->

Trp

, are

det

rim

enta

l for

ant

igen

affi

nity

, due

to a

repo

sitio

ning

of t

he H

3 lo

op a

s sho

wn

by

thei

r cry

stal

stru

ctur

es. Th

ese

insi

ghts

wer

e us

ed to

iden

tify

a so

lubl

e, st

able

, wel

l exp

ress

ed u

nive

rsal

hum

aniz

ed N

anob

ody

scaf

-fo

ld th

at a

llow

s gra

fts o

f ant

igen

-bin

ding

loop

s fro

m o

ther

Nan

obod

ies w

ith tr

ansf

er o

f the

ant

igen

spec

ifici

ty a

nd a

ffini

ty.

Vin

cke

et a

l. 20

09


465

Hum

aniz

ed n

anob

odie

sLo

op-g

raft

ing

In v

ivo

imag

ing

Scaff

old

Car

cino

embr

yoni

c an

tige

n

Nan

obod

ies

are

a no

vel t

ype

of im

mun

oglo

bulin

like,

ant

igen

-bin

ding

pro

tein

with

ben

efic

ial p

harm

acol

ogic

and

pha

rmac

oki-

netic

pro

pert

ies

that

are

idea

lly s

uite

d to

targ

etin

g ce

llula

r an

tigen

s fo

r m

olec

ular

imag

ing

or th

erap

eutic

pur

pose

s. H

owev

er,

beca

use

of th

eir

cam

elid

, non

hum

an o

rigi

n, th

e po

ssib

le im

mun

ogen

icity

of N

anob

odie

s w

hen

used

in th

e cl

inic

is a

con

cern

. H

ere

we

pres

ent a

new

str

ateg

y to

qui

ckly

gen

erat

e hu

man

ized

Nan

obod

ies

for

mol

ecul

ar im

agin

g pu

rpos

es. M

etho

ds: W

e ge

netic

ally

gra

fted

the

antig

en-b

indi

ng lo

ops

of N

bCEA

5, a

Nan

obod

y w

ith s

peci

ficity

for

the

colo

n ca

rcin

oma

mar

ker

carc

i-no

embr

yoni

c an

tigen

(CEA

), on

to th

e fr

amew

ork

of a

hum

aniz

ed N

anob

ody

scaf

fold

. Thi

s sc

affo

ld h

as b

een

prev

ious

ly c

hara

c-te

rize

d in

our

labo

rato

ry a

s a

stab

le N

anob

ody

that

can

ser

ve a

s a

univ

ersa

l loo

p ac

cept

or fo

r an

tigen

-bin

ding

loop

s fr

om d

onor

N

anob

odie

s an

d ha

s be

en a

dditi

onal

ly m

utat

ed a

t abo

ut 1

0 cr

ucia

l sur

face

-exp

osed

site

s to

rese

mbl

e th

e se

quen

ce o

f hum

an

vari

able

imm

unog

lobu

lin d

omai

ns. T

he 3

reco

mbi

nant

Nan

obod

ies

(NbC

EA5,

hum

aniz

ed s

caffo

ld, a

nd h

uman

ized

CEA

5 gr

aft)

w

ere

prod

uced

in b

acte

ria

and

puri

fied.

Unl

abel

ed a

nd (9

9m)T

c-la

bele

d N

anob

odie

s w

ere

bioc

hem

ical

ly c

hara

cter

ized

in v

itro

and

test

ed a

s pr

obes

for

SPEC

T/C

T o

f xen

ogra

fted

tum

ors.

Res

ults

: The

suc

cess

of l

oop-

graf

ting

was

con

firm

ed b

y co

mpa

ring

th

ese

Nan

obod

ies

for

thei

r ca

paci

ty to

reco

gniz

e so

lubl

e C

EA p

rote

in in

enz

yme-

linke

d im

mun

osor

bent

ass

ay a

nd b

y su

rfac

e pl

asm

on re

sona

nce

and

to b

ind

to C

EA-p

ositi

ve L

S174

T c

olon

car

cino

ma

cells

and

CEA

-tra

nsfe

cted

but

not

unt

rans

fect

ed C

hi-

nese

ham

ster

ova

ry c

ells

in fl

ow c

ytom

etry

. Spe

cific

ity o

f bin

ding

was

con

firm

ed b

y co

mpe

titio

n st

udie

s. A

ll N

anob

odie

s w

ere

heat

-sta

ble,

cou

ld b

e ef

ficie

ntly

labe

led

with

(99m

)Tc,

and

reco

gniz

ed b

oth

solu

ble

and

mem

bran

e-bo

und

CEA

pro

tein

in b

ind-

ing

stud

ies.

Fin

ally

, bio

dist

ribu

tion

expe

rim

ents

wer

e pe

rfor

med

with

intr

aven

ousl

y in

ject

ed (9

9m)T

c-la

bele

d N

anob

odie

s in

LS

174T

tum

or-b

eari

ng m

ice

usin

g pi

nhol

e SP

ECT

/mic

ro-C

T. T

hese

in v

ivo

expe

rim

ents

reve

aled

spe

cific

ity o

f tum

or ta

rget

ing

and

rapi

d re

nal c

lear

ance

for

all N

anob

odie

s, w

ith lo

w s

igna

ls in

all

orga

ns b

esid

es th

e ki

dney

s. C

oncl

usio

n: T

his

stud

y sh

ows

the

pote

ncy

of a

ntig

en-b

indi

ng lo

op-g

raft

ing

to e

ffic

ient

ly g

ener

ate

hum

aniz

ed N

anob

odie

s th

at re

tain

thei

r ta

rget

ing

capa

ci-

ties

for

noni

nvas

ive

in v

ivo

imag

ing

of tu

mor

s.

Vane

y-ck

en e

t al

. 201

0

Gra

ftin

gH

apte

nM

etho

trex

ate

Cry

stal

str

uctu

re

anal

ysis

Whi

le s

ever

al a

nti-

hapt

en V

HH

s ha

ve b

een

gene

rate

d, li

ttle

is k

now

n re

gard

ing

the

unde

rlyi

ng s

truc

tura

l and

ther

mod

y-na

mic

bas

is fo

r ha

pten

reco

gniti

on. H

ere,

an

anti-

met

hotr

exat

e V

HH

(ant

i-M

TX

VH

H) w

as g

ener

ated

usi

ng g

raft

ing

met

hods

w

here

by th

e th

ree

com

plem

enta

rity

det

erm

inin

g re

gion

s (C

DR

s) w

ere

inse

rted

ont

o an

exi

stin

g V

HH

fram

ewor

k. T

herm

ody-

nam

ic a

naly

sis

of th

e an

ti-M

TX

VH

H C

DR1

–3 G

raft

reve

aled

a m

icro

mol

ar b

indi

ng a

ffin

ity, w

hile

the

crys

tal s

truc

ture

of t

he

com

plex

reve

aled

a s

omew

hat s

urpr

isin

g no

ncan

onic

al b

indi

ng s

ite w

hich

invo

lved

MT

X tu

nnel

ing

unde

r th

e C

DR1

loop

. Due

to

the

clos

e pr

oxim

ity o

f MT

X to

CD

R4, a

non

hype

rvar

iabl

e lo

op, t

he C

DR4

loop

seq

uenc

e w

as s

ubse

quen

tly in

trod

uced

into

th

e C

DR1

–3 g

raft

, whi

ch re

sulte

d in

a d

ram

atic

100

0-fo

ld in

crea

se in

the

bind

ing

affin

ity. C

ryst

al s

truc

ture

ana

lysi

s of

bot

h th

e fr

ee a

nd c

ompl

ex a

nti-

MT

X C

DR1

–4 g

raft

reve

aled

CD

R4 p

lays

a s

igni

fican

t rol

e in

bot

h in

term

olec

ular

con

tact

s an

d bi

ndin

g si

te c

onfo

rmat

ion

that

app

ear

to c

ontr

ibut

e to

war

d hi

gh a

ffin

ity b

indi

ng. A

dditi

onal

ly, t

he a

nti-

MT

X V

HH

pos

sess

ed re

lativ

ely

high

spe

cific

ity fo

r M

TX

ove

r cl

osel

y re

late

d co

mpo

unds

am

inop

teri

n an

d fo

late

, dem

onst

ratin

g th

at V

HH

dom

ains

are

cap

able

of

bin

ding

low

-mol

ecul

ar w

eigh

t lig

ands

with

hig

h af

finity

and

spe

cific

ity, d

espi

te th

eir

redu

ced

inte

rfac

e.

Fann

ing

and

Hor

n 20

11


466

Gra

ftin

gTo

bacc

oLy

sozy

me

‘Uni

vers

al’ n

anob

ody

fram

ewor

k

Her

e w

e de

scri

be h

igh-

leve

l exp

ress

ion,

in N

icot

iana

ben

tham

iana

, of t

hree

ver

sion

s of a

n an

ti-he

n eg

g w

hite

lyso

zym

e (H

EWL)

na

nobo

dy w

hich

incl

ude

the

orig

inal

VH

H fr

om a

n im

mun

ized

libr

ary

(cA

bLys

3), a

cod

on-o

ptim

ized

der

ivat

ive,

and

a c

odon

-op

timiz

ed h

ybri

d na

nobo

dy c

ompr

isin

g th

e C

DRs

of c

AbL

ys3

graf

ted

onto

an

alte

rnat

ive

‘uni

vers

al’ n

anob

ody

fram

ewor

k. H

is6-

an

d St

repI

I-ta

gged

der

ivat

ives

of e

ach

nano

body

wer

e ta

rget

ed fo

r acc

umul

atio

n in

the

cyto

plas

m, c

hlor

opla

st a

nd a

popl

ast u

sing

di

ffere

nt p

re-s

eque

nces

. Whe

n ta

rget

ed to

the

apop

last

, int

act f

unct

iona

l nan

obod

ies a

ccum

ulat

ed a

t an

exce

ptio

nally

hig

h le

vel

(up

to 3

0% to

tal l

eaf p

rote

in),

dem

onst

ratin

g th

e gr

eat p

oten

tial o

f pla

nts a

s a n

anob

ody

prod

uctio

n sy

stem

.

Teh

and

Kav

a-

nagh

20

10

Epid

erm

al g

row

th

fact

or r

ecep

tor

Pent

abod

yLo

ng s

erum

hal

f lif

eFc

frag

men

t

In th

e se

arch

for b

ette

r ant

ibod

y fo

rmat

s for

in v

ivo

imag

ing

and/

or th

erap

y of

can

cer,

thre

e ty

pes o

f sdA

b-ba

sed

mol

ecul

es

dire

cted

aga

inst

epi

derm

al g

row

th fa

ctor

rece

ptor

(EG

ER) w

ere

cons

truc

ted,

cha

ract

eriz

ed a

nd te

sted

. Ele

ven

sdA

bs w

ere

isol

ated

fr

om a

pha

ge d

ispl

ay li

brar

y co

nstr

ucte

d fr

om th

e sd

Ab

repe

rtoi

re o

f a ll

ama

imm

uniz

ed w

ith a

var

iant

of E

GFR

. A p

enta

mer

ic

sdA

b, o

r pen

tabo

dy, V

2C-E

G2

was

con

stru

cted

by

fusi

ng o

ne o

f the

sdA

bs, E

G2,

to a

pen

tam

eriz

atio

n pr

otei

n do

mai

n. A

chi

mer

ic

HC

Ab

(cH

CA

b), E

G2-

hFc,

was

con

stru

cted

by

fusi

ng E

G2

to th

e fr

agm

ent c

ryst

alliz

able

(Fc)

of h

uman

IgG

1. W

here

as E

G2

and

V2C

-EG

2 lo

caliz

ed m

ainl

y in

the

kidn

eys a

fter

i.v.

inje

ctio

n, E

G2-

hFc

exhi

bite

d ex

celle

nt tu

mor

acc

umul

atio

n, a

nd th

is w

as la

rgel

y at

trib

uted

to it

s lon

g se

rum

hal

f life

, whi

ch is

com

para

ble

to th

at o

f IgG

s. Th

e m

oder

ate

size

(sim

ilar t

o 80

kD

a) a

nd in

tact

hum

an

Fc m

ake

HC

Abs

a u

niqu

e an

tibod

y fo

rmat

whi

ch m

ay o

utpe

rfor

m w

hole

IgG

s as i

mag

ing

and

ther

apeu

tic re

agen

ts.

Bell

et

al. 2

010

Uni

vers

al fr

amew

ork

Gra

ftin

gC

him

era

Cam

el si

ngle

-dom

ain

antib

ody

frag

men

ts (V

HH

s) a

re p

rom

isin

g to

ols i

n nu

mer

ous b

iote

chno

logi

cal a

nd m

edic

al a

pplic

atio

ns.

How

ever

, som

e co

nditi

ons u

nder

whi

ch a

ntib

odie

s are

use

d ar

e so

dem

andi

ng th

at th

ey c

an b

e m

et b

y on

ly th

e m

ost r

obus

t VH

Hs.

A u

nive

rsal

fram

ewor

k off

erin

g th

e re

quire

d pr

oper

ties f

or u

se in

var

ious

app

licat

ions

(e.g

. as i

ntra

body

, as p

robe

in b

iose

nsor

s or o

n m

icro

-arr

ays)

is h

ighl

y va

luab

le a

nd m

ight

be

furt

her i

mpl

emen

ted

whe

n em

ploy

men

t of V

HH

s in

hum

an th

erap

y is

env

isag

ed. W

e id

entifi

ed th

e V

HH

fram

ewor

k of

cA

bBC

II10

as a

pot

entia

l can

dida

te, u

sefu

l for

the

exch

ange

of a

ntig

en sp

ecifi

citie

s by

com

plem

en-

tari

ty d

eter

min

ing

regi

on (C

DR)

gra

fting

. Due

to th

e la

rge

num

ber o

f CD

RH lo

op st

ruct

ures

pre

sent

on

VH

Hs,

this

gra

fting

tech

-ni

que

was

exp

ecte

d to

be

rath

er u

npre

dict

able

. Non

ethe

less

, the

pla

stic

ity o

f the

cA

bBC

II10

fram

ewor

k al

low

s suc

cess

ful t

rans

fer o

f an

tigen

spec

ifici

ty fr

om d

onor

VH

Hs o

nto

its sc

affol

d. Th

e cA

bBC

II10

was

cho

sen

esse

ntia

lly fo

r its

hig

h le

vel o

f sta

bilit

y (4

7 kJ

/mol

, go

od e

xpre

ssio

n le

vel (

5 m

g/l i

n E.

col

i) an

d its

abi

lity

to b

e fu

nctio

nal i

n th

e ab

senc

e of

the

cons

erve

d di

sulfi

de b

ond.

All

five

chim

e-ra

s gen

erat

ed b

y gr

aftin

g C

DR-

Hs,

from

don

or V

HH

s bel

ongi

ng to

subf

amily

2 th

at e

ncom

pass

75%

of a

ll an

tigen

-spe

cific

VH

Hs,

on

the

fram

ewor

k of

cA

bBC

II10

wer

e fu

nctio

nal a

nd g

ener

ally

had

an

incr

ease

d th

erm

odyn

amic

stab

ility

. The

graf

ting

of C

DR-

H lo

ops

from

VH

Hs b

elon

ging

to o

ther

subf

amili

es re

sulte

d in

chi

mer

as o

f red

uced

ant

igen

-bin

ding

cap

acity

.

Saer

ens

et a

l. 20

05

VH

H-S

NA

P co

nstr

uct

Erv1

p su

lfhy

dryl

ox

idas

e

Cam

elid

ae si

ngle

dom

ain

antib

odie

s (V

HH

s) h

ave

stru

ctur

al a

nd b

indi

ng fe

atur

es th

at re

nder

them

suita

ble

alte

rnat

ives

to c

on-

vent

iona

l IgG

ant

ibod

ies.

VH

Hs a

re u

sual

ly e

asie

r to

prod

uce

as re

com

bina

nt p

rote

ins t

han

othe

r ant

ibod

y fr

agm

ents

. How

ever

, fo

r som

e of

the

biot

echn

olog

ical

app

licat

ions

for w

hich

they

hav

e be

en p

ropo

sed,

such

as i

mm

unoc

hrom

atog

raph

y an

d as

sist

ed-

crys

tallo

grap

hy, l

arge

am

ount

s of p

urifi

ed a

ntib

odie

s are

nec

essa

ry, w

here

as so

me

VH

H-f

usio

ns w

ith c

omm

on ta

gs su

ch a

s GFP

an

d SN

AP

are

poor

ly e

xpre

ssed

in th

e ba

cter

ial p

erip

lasm

. Her

e w

e ha

ve sh

own

that

the

co-e

xpre

ssio

n of

Erv

1p su

lfhyd

ryl o

xida

se

resu

lted

in a

n as

toni

shin

g yi

eld

incr

ease

of V

HH

-SN

AP

cons

truc

ts e

xpre

ssed

in th

e ba

cter

ial c

ytop

lasm

. The

resu

lting

reco

m-

bina

nt a

ntib

odie

s wer

e al

so m

ore

stab

le th

an th

e an

tibod

ies p

rodu

ced

usin

g th

e sa

me

plas

mid

, but

in w

ild-t

ype

bact

eria

. Usi

ng

this

app

roac

h, it

was

pos

sibl

e to

obt

ain

tens

of m

illig

ram

s of p

urifi

ed fu

sion

ant

ibod

ies u

sing

a b

asic

flas

k fe

rmen

tatio

n pr

otoc

ol.

Ther

efor

e, th

e de

scri

bed

met

hod

repr

esen

ts a

val

id so

lutio

n to

pro

duce

inex

pens

ivel

y la

rge

amou

nts o

f sin

gle

dom

ain

antib

odie

s fo

r in

vitr

o ap

plic

atio

ns a

nd w

e ex

pect

it w

ill b

e su

itabl

e fo

r the

pro

duct

ion

of o

ther

ant

ibod

y fr

agm

ents

.

Vegg

iani

an

d de

M

arco

20

11


467

Tabl

e 5.

Cha

ract

eris

tic

prop

erti

es o

f sin

gle-

dom

ain

anti

body

frag

men

ts

Blo

od-b

rain

bar

rier

pe

rmea

bilit

yTr

ypan

osom

a br

ucei

Surf

ace

glyc

opro

tein

V

SG

Expe

rim

enta

l app

roac

h: W

e ha

ve a

sses

sed

the

bloo

d-br

ain

barr

ier p

erm

eabi

lity

of N

b_A

n33,

a n

anob

ody

agai

nst t

he T

rypa

noso

ma

bruc

ei b

ruce

i var

iant

-spe

cific

surf

ace

glyc

opro

tein

(VSG

). Th

is a

naly

sis w

as p

erfo

rmed

in h

ealth

y ra

ts a

nd in

rats

that

wer

e in

the

ence

phal

itic

stag

e of

Afr

ican

tryp

anos

omia

sis u

sing

intr

acer

ebra

l mic

rodi

alys

is, s

ingl

e ph

oton

em

issi

on c

ompu

ted

tom

ogra

phy

(SPE

CT

) or a

com

bina

tion

of b

oth

met

hodo

logi

es. Th

is e

nabl

ed th

e qu

antifi

catio

n of

unl

abel

led

and

Tc-9

9m-la

belle

d na

nobo

dies

us

ing,

resp

ectiv

ely,

a se

nsiti

ve V

SG-b

ased

nan

obod

y-de

tect

ion

ELIS

A, r

adio

activ

ity m

easu

rem

ent i

n co

llect

ed m

icro

dial

ysat

es a

nd

SPEC

T im

age

anal

ysis

. Key

resu

lts: Th

e co

mbi

ned

read

-out

met

hodo

logi

es sh

owed

that

Nb_

An3

3 w

as d

etec

ted

in th

e br

ain

of h

ealth

y ra

ts fo

llow

ing

i.v. i

njec

tion,

infla

mm

atio

n-in

duce

d da

mag

e to

the

bloo

d-br

ain

barr

ier,

as in

the

late

enc

epha

litic

stag

e of

tryp

anos

o-m

iasi

s, si

gnifi

cant

ly in

crea

sed

the

effici

ency

of p

assa

ge o

f the

nan

obod

y th

roug

h th

is b

arri

er. C

ompl

emen

ting

SPEC

T a

naly

ses w

ith

intr

acer

ebra

l mic

rodi

alys

is im

prov

ed a

naly

sis o

f bra

in d

ispo

sitio

n. Th

ere

is c

lear

val

ue in

ass

essi

ng p

enet

ratio

n of

the

bloo

d-br

ain

bar-

rier

by

mon

oval

ent n

anob

odie

s in

mod

els o

f CN

S in

flam

mat

ion.

Our

dat

a al

so su

gges

t tha

t rap

id c

lear

ance

from

blo

od m

ight

ham

per

effici

ent t

arge

ting

of sp

ecifi

c na

nobo

dies

to th

e C

NS.

Con

clus

ions

and

impl

icat

ions

: Nan

obod

ies c

an e

nter

the

brai

n pa

renc

hym

a fr

om

the

syst

emic

circ

ulat

ion,

esp

ecia

lly in

pat

holo

gica

l con

ditio

ns w

here

the

bloo

d-br

ain

barr

ier i

nteg

rity

is c

ompr

omis

ed.

Cal

jon

et

al. 2

012

Bet

a-am

yloi

dTr

ansm

igra

tion

effi

cien

cyA

lzhe

imer

’s di

seas

eC

ereb

ral a

myl

oid

angi

-op

athy

Prev

ious

ly se

lect

ed a

myl

oid

beta

reco

gniz

ing

heav

y ch

ain

antib

ody

frag

men

ts (V

HH

) affi

nity

bin

ders

der

ived

from

the

Cam

elid

he

avy

chai

n an

tibod

y re

pert

oire

wer

e te

sted

for t

heir

pro

pens

ity to

cro

ss th

e bl

ood-

brai

n ba

rrie

r (BB

B) u

sing

an

esta

blis

hed

in v

itro

BBB

co-c

ultu

re sy

stem

. Of a

ll te

sted

VH

H, n

i3A

show

ed h

ighe

st tr

ansm

igra

tion

effici

ency

whi

ch is

, in

part

, fac

ilita

ted

by a

thre

e am

ino

acid

subs

titut

ions

in it

s N-t

erm

inal

dom

ain.

Add

ition

al st

udie

s ind

icat

ed th

at th

e m

echa

nism

of t

rans

cellu

lar p

assa

ge o

f ni

3A is

by

activ

e tr

ansp

ort.

As V

HH

ni3

A c

ombi

nes t

he a

bilit

y to

reco

gniz

e am

yloi

d be

ta a

nd to

cro

ss th

e BB

B, it

has

pot

entia

l as

a to

ol fo

r non

-inva

sive

in v

ivo

imag

ing

and

as e

ffici

ent l

ocal

dru

g ta

rget

ing

moi

ety

in p

atie

nts s

uffer

ing

from

cer

ebra

l am

yloi

dosi

s su

ch a

s Alz

heim

er’s

dise

ase

(AD

) and

cer

ebra

l am

yloi

d an

giop

athy

(CA

A).

Rutg

ers

et a

l. 20

11

Blo

od-b

rain

bar

rier

Tran

scyt

osis

Endo

cyto

sis

Cer

ebra

l end

othe

lial

cells

Puta

tive

rec

epto

r

Ant

ibod

ies a

gain

st re

cept

ors t

hat u

nder

go tr

ansc

ytos

is a

cros

s the

blo

od-b

rain

bar

rier

(BBB

) hav

e be

en u

sed

as v

ecto

rs to

targ

et

drug

s or t

hera

peut

ic p

eptid

es in

to th

e br

ain.

We

have

rece

ntly

dis

cove

red

a no

vel s

ingl

e do

mai

n an

tibod

y, FC

5, w

hich

tran

smi-

grat

es a

cros

s hum

an c

ereb

ral e

ndot

helia

l cel

ls in

vitr

o an

d th

e BB

B in

viv

o. Th

e pu

rpos

e of

this

stud

y w

as to

cha

ract

eriz

e m

echa

-ni

sms o

f FC

5 en

docy

tosi

s and

tran

scyt

osis

acr

oss t

he B

BB a

nd it

s put

ativ

e re

cept

or o

n hu

man

bra

in e

ndot

helia

l cel

ls. Th

e tr

ans-

port

of F

C5

acro

ss h

uman

bra

in e

ndot

helia

l cel

ls w

as p

olar

ized

, cha

rge

inde

pend

ent a

nd te

mpe

ratu

re d

epen

dent

, sug

gest

ing

a re

cept

or-m

edia

ted

proc

ess.

FC

5 ta

ken

up b

y hu

man

bra

in e

ndot

helia

l cel

ls c

o-lo

caliz

ed w

ith c

lath

rin

but n

ot w

ith c

aveo

lin-1

by

imm

unoc

hem

istr

y an

d w

as d

etec

ted

in c

lath

rin-

enri

ched

subc

ellu

lar f

ract

ions

by

wes

tern

blo

t. Th

e tr

anse

ndot

helia

l mig

ratio

n of

FC

5 w

as re

duce

d by

inhi

bito

rs o

f cla

thri

n-m

edia

ted

endo

cyto

sis,

K+

depl

etio

n an

d ch

lorp

rom

azin

e, b

ut w

as in

sens

itive

to c

aveo

lae

inhi

bito

rs, fi

lipin

, nys

tatin

or m

ethy

l-bet

a-cy

clod

extr

in. F

ollo

win

g in

tern

aliz

atio

n, F

C5

was

targ

eted

to e

arly

end

osom

es, b

ypas

sed

late

end

osom

es/ly

soso

mes

and

rem

aine

d in

tact

aft

er tr

ansc

ytos

is. Th

e tr

ansc

ytos

is p

roce

ss w

as in

hibi

ted

by a

gent

s tha

t affe

ct a

ctin

cy

tosk

elet

on o

r int

race

llula

r sig

nalin

g th

roug

h PI

3-ki

nase

. Pre

trea

tmen

t of h

uman

bra

in e

ndot

helia

l cel

ls w

ith w

heat

germ

agg

lu-

tinin

, sia

lic a

cid,

alp

ha(2

,3)-

neur

amin

idas

e or

Maa

ckia

am

uren

sis a

gglu

tinin

that

reco

gniz

es a

lpha

(2,3

)-, b

ut n

ot w

ith S

ambu

cus

nigr

a ag

glut

inin

that

reco

gniz

es a

lpha

(2,6

)-si

alyl

gala

ctos

yl re

sidu

es, s

igni

fican

tly re

duce

d FC

5 tr

ansc

ytos

is. F

C5

faile

d to

reco

gniz

e br

ain

endo

thel

ial c

ells

-der

ived

lipi

ds, s

ugge

stin

g th

at it

bin

ds lu

min

al a

lpha

(2,3

)-si

alog

lyco

prot

ein

rece

ptor

whi

ch tr

igge

rs c

lath

rin-

med

iate

d en

docy

tosi

s. Th

is p

utat

ive

rece

ptor

may

be

a ne

w ta

rget

for d

evel

opin

g br

ain-

targ

etin

g dr

ug d

eliv

ery

vect

ors.

Abu

lrob

et

al.

2005


468

Ther

mal

sta

bilit

yR

esis

tanc

e to

tryp

sin

and

extr

eme

pHC

lost

ridi

um d

iffici

leTo

xin

AO

ral a

dmin

istr

atio

n

The

extr

eme

pH a

nd p

rote

ase-

rich

env

iron

men

t of t

he u

pper

gas

troi

ntes

tinal

trac

t is a

maj

or o

bsta

cle

faci

ng o

rally

-adm

inis

tere

d pr

otei

n th

erap

eutic

s, in

clud

ing

antib

odie

s. Th

roug

h pr

otei

n en

gine

erin

g, se

vera

l Clo

stri

dium

diffi

cile

toxi

n A

-spe

cific

hea

vy c

hain

an

tibod

y va

riab

le d

omai

ns (V

(H)H

s) w

ere

expr

esse

d w

ith a

n ad

ditio

nal d

isul

fide

bond

by

intr

oduc

ing

Ala

/Gly

54C

ys a

nd Il

e78C

ys

mut

atio

ns. M

utan

t ant

ibod

ies w

ere

com

pare

d to

thei

r wild

-typ

e co

unte

rpar

ts w

ith re

spec

t to

expr

essi

on y

ield

, non

-agg

rega

tion

stat

us, a

ffini

ty fo

r tox

in A

, cir

cula

r dic

hroi

sm (C

D) s

truc

tura

l sig

natu

res,

ther

mal

stab

ility

, pro

teas

e re

sist

ance

, and

toxi

n A

-neu

-tr

aliz

ing

capa

city

. The

mut

ant V

(H)H

s wer

e fo

und

to b

e w

ell e

xpre

ssed

, alth

ough

with

low

er y

ield

s com

pare

d to

wild

-typ

e co

un-

terp

arts

, wer

e no

n-ag

greg

atin

g m

onom

ers,

reta

ined

low

nM

affi

nity

for t

oxin

A, a

lbei

t the

maj

ority

show

ed so

mew

hat r

educ

ed

affini

ty c

ompa

red

to w

ild-t

ype

coun

terp

arts

, and

wer

e ca

pabl

e of

in v

itro

toxi

n A

neu

tral

izat

ion

in c

ell-b

ased

ass

ays.

Far

-UV

and

ne

ar-U

V C

D sp

ectr

osco

py c

onsi

sten

tly sh

owed

shift

s in

peak

inte

nsity

and

sele

ctiv

e pe

ak m

inim

a fo

r wild

-typ

e an

d m

utan

t V(H

)H

pair

s; ho

wev

er, t

he o

vera

ll C

D p

rofil

e re

mai

ned

very

sim

ilar.

A si

gnifi

cant

incr

ease

in th

e th

erm

al u

nfol

ding

mid

poin

t tem

pera

ture

w

as o

bser

ved

for a

ll m

utan

ts a

t bot

h ne

utra

l and

aci

dic

pH. D

iges

tion

of th

e V

(H)H

s with

the

maj

or g

astr

oint

estin

al p

rote

ases

, at

biol

ogic

ally

rele

vant

con

cent

ratio

ns, r

evea

led

a si

gnifi

cant

incr

ease

in p

epsi

n re

sist

ance

for a

ll m

utan

ts a

nd a

n in

crea

se in

chy

mot

-ry

psin

resi

stan

ce fo

r the

maj

ority

of m

utan

ts. M

utan

t V(H

)H tr

ypsi

n re

sist

ance

was

sim

ilar t

o th

at o

f wild

-typ

e V

(H)H

s, a

lthou

gh

the

tryp

sin

resi

stan

ce o

f one

V(H

)H m

utan

t was

sign

ifica

ntly

redu

ced.

Ther

efor

e, th

e in

trod

uctio

n of

a se

cond

dis

ulfid

e bo

nd in

th

e hy

drop

hobi

c co

re n

ot o

nly

incr

ease

s V(H

)H th

erm

al st

abili

ty a

t neu

tral

pH

, as p

revi

ousl

y sh

own,

but

als

o re

pres

ents

a g

ener

ic

stra

tegy

to in

crea

se V

(H)H

stab

ility

at l

ow p

H a

nd im

part

pro

teas

e re

sist

ance

, with

onl

y m

inor

per

turb

atio

ns in

targ

et b

indi

ng

affini

ties.

Thes

e ar

e al

l des

irab

le c

hara

cter

istic

s for

the

desi

gn o

f pro

tein

-bas

ed o

ral t

hera

peut

ics.

Hus

sack

et

al.

2011

a

Hea

t den

atur

atio

nEp

ider

mal

gro

wth

fact

or

rece

ptor

Pich

ia p

asto

ris

Mal

igna

ncy

It h

as b

een

show

n th

at, i

n co

ntra

st w

ith c

onve

ntio

nal a

ntib

ody

frag

men

ts, t

he v

aria

ble

dom

ains

of t

hese

hea

vy-c

hain

ant

ibod

ies

are

func

tiona

l at o

r aft

er e

xpos

ure

to h

igh

tem

pera

ture

s. In

the

pres

ent s

tudy

, the

VH

H (v

aria

ble

dom

ain

of h

eavy

-cha

in a

ntib

ody)

ca

mel

ant

ibod

y w

as su

bclo

ned

into

vec

tor P

picz

c an

d ex

pres

sed

in P

ichi

a pa

stor

is. O

RBI-

83 V

HH

ant

ibod

y re

cogn

izes

the

exte

rnal

do

mai

n of

the

mut

ant E

GFR

[EG

F (e

pide

rmal

gro

wth

fact

or) r

ecep

tor]

, EG

FR V

III.

This

tum

our-

spec

ific

antig

en is

liga

nd-i

nde-

pend

ent,

cont

ains

a c

onst

itutiv

ely

activ

e ty

rosi

ne k

inas

e do

mai

n an

d ha

s bee

n sh

own

to b

e pr

esen

t in

a nu

mbe

r of h

uman

mal

ig-

nanc

ies.

We

repo

rt h

ere

that

, alth

ough

exp

ress

ion

from

P. p

asto

ris r

esul

ted

in a

sign

ifica

ntly

incr

ease

d le

vel o

f exp

ress

ion

of th

e an

ti-EG

FR V

III V

HH

ant

ibod

ies c

ompa

red

with

Esc

heri

chia

, thi

s ant

ibod

y se

lect

ivel

y bo

und

to th

e EG

FR V

III p

eptid

e an

d re

acte

d sp

ecifi

cally

with

the

imm

unoa

ffini

ty-p

urifi

ed a

ntig

en fr

om n

on-s

mal

l-cel

l lun

g ca

ncer

. Fur

ther

mor

e, th

erm

al d

enat

urat

ion

stab

il-ity

and

CD

spec

tra

anal

ysis

of t

he C

amel

us b

actr

ianu

s (Ba

ctri

an c

amel

) VH

H a

nd h

eavy

-cha

in a

ntib

odie

s at d

iffer

ent t

empe

ratu

re

prov

ed re

vers

ibili

ty a

nd b

indi

ng a

ctiv

ity a

fter

hea

t den

atur

atio

n. O

ur re

sults

indi

cate

that

the

P. p

asto

ris e

xpre

ssio

n sy

stem

may

be

usef

ul fo

r the

exp

ress

ion

of c

amel

sing

le d

omai

n an

tibod

y an

d th

e ab

ility

of t

he e

xpre

ssed

pro

tein

to re

vers

ibly

mel

t with

out a

ggre

-ga

tion,

allo

win

g it

to re

gain

bin

ding

act

ivity

aft

er h

eat d

enat

urat

ion.

Om

idfa

r et

al.

2007


469

Den

atur

ing

agen

tsC

elia

c pa

tien

tsPr

olam

inG

liadi

nEL

ISA

Food

inte

nded

for c

elia

c pa

tient

s’ co

nsum

ptio

n m

ust b

e an

alyz

ed fo

r the

pre

senc

e of

toxi

c pr

olam

ins u

sing

hig

h de

lect

abili

ty te

sts.

Thou

gh 6

0% e

than

ol is

the

mos

t com

mon

ly u

sed

solv

ent f

or p

rola

min

s ext

ract

ion,

2-m

erca

ptoe

than

ol (2

-ME)

and

gua

nidi

nium

chl

o-ri

de (G

uHC

l) ca

n be

add

ed to

incr

ease

pro

tein

reco

very

. How

ever

, eth

anol

and

den

atur

ing

agen

ts in

terf

ere

with

ant

igen

reco

gniti

on

whe

n co

nven

tiona

l ant

ibod

ies a

re u

sed.

In th

e pr

esen

t wor

k, a

new

met

hod

for g

liadi

ns q

uant

ifica

tion

is sh

own.

The

met

hod

is b

ased

on

the

sele

ctio

n of

llam

a si

ngle

dom

ain

antib

ody

frag

men

ts a

ble

to o

pera

te u

nder

den

atur

ing

cond

ition

s. Si

x ou

t of 2

8 V

HH

-pha

ges

obta

ined

reta

ined

thei

r bin

ding

cap

acity

in 1

5% e

than

ol. S

elec

ted

clon

es p

rese

nted

a lo

ng C

DR3

regi

on c

onta

inin

g tw

o ad

ditio

nal

cyst

eine

s tha

t cou

ld b

e re

spon

sibl

e fo

r the

hig

her s

tabi

lity.

One

of t

he c

lone

s (na

med

VH

H26

) was

fully

ope

rativ

e in

the

pres

ence

of

15%

eth

anol

, 0.5

% 2

-ME,

and

0.5

M G

uHC

l. C

aptu

re E

LISA

usi

ng V

HH

26 w

as a

ble

to d

etec

t glia

dins

in sa

mpl

es sh

own

as n

egat

ives

by

con

vent

iona

l ELI

SA. Th

eref

ore,

this

new

stra

tegy

app

ears

as a

n ex

celle

nt p

latfo

rm fo

r qua

ntita

tive

dete

rmin

atio

n of

pro

tein

s or a

ny

othe

r im

mun

ogen

ic c

ompo

und,

in th

e pr

esen

ce o

f den

atur

ing

agen

ts, w

hen

spec

ific

reco

gniti

on u

nits

with

hig

h st

abili

ty a

re re

quire

d.

Don

a et

al

. 201

0

Ente

roto

xin

BD

enat

ured

pro

tein

re

fold

ing

Dia

gnos

tic

assa

y

Back

grou

nd: C

amel

ids a

nd sh

arks

pos

sess

a u

niqu

e su

bcla

ss o

f ant

ibod

ies c

ompr

ised

of o

nly

heav

y ch

ains

. The

antig

en b

ind-

ing

frag

men

ts o

f the

se u

niqu

e an

tibod

ies c

an b

e cl

oned

and

exp

ress

ed a

s sin

gle

dom

ain

antib

odie

s (sd

Abs

). Th

e ab

ility

of t

hese

sm

all a

ntig

en-b

indi

ng m

olec

ules

to re

fold

aft

er h

eatin

g to

ach

ieve

thei

r ori

gina

l str

uctu

re, a

s wel

l as t

heir

dim

inut

ive

size

, mak

es

them

att

ract

ive

cand

idat

es fo

r dia

gnos

tic a

ssay

s. R

esul

ts: H

ere

we

desc

ribe

the

isol

atio

n of

an

sdA

b ag

ains

t Sta

phyl

occo

cus a

ureu

s en

tero

toxi

n B

(SEB

). Th

e cl

one,

A3,

was

foun

d to

hav

e hi

gh a

ffini

ty (K

d =

75 p

M) a

nd g

ood

spec

ifici

ty fo

r SEB

, sho

win

g no

cro

ss

reac

tivity

to re

late

d m

olec

ules

such

as S

taph

yloc

occa

l ent

erot

oxin

A (S

EA),

Stap

hylo

cocc

al e

nter

otox

in D

(SED

), an

d Sh

iga

toxi

n.

Mos

t rem

arka

bly,

this

ant

i-SEB

sdA

b ha

d an

ext

rem

ely

high

Tm

of 8

5 de

gree

s C a

nd a

n ab

ility

to re

fold

aft

er h

eatin

g to

95

degr

ees

C. Th

e sh

arp

Tm d

eter

min

ed b

y ci

rcul

ar d

ichr

oism

, was

foun

d to

con

tras

t with

the

grad

ual d

ecre

ase

obse

rved

in in

trin

sic

fluor

es-

cenc

e. W

e de

mon

stra

ted

the

utili

ty o

f thi

s sdA

b as

a c

aptu

re a

nd d

etec

tor m

olec

ule

in L

umin

ex b

ased

ass

ays p

rovi

ding

lim

its o

f de

tect

ion

(LO

Ds)

of a

t lea

st 6

4 pg

/ml.

Con

clus

ion:

The

anti-

SEB

sdA

b A

3 w

as fo

und

to h

ave

a hi

gh a

ffini

ty a

nd a

n ex

trao

rdin

arily

hi

gh T

m a

nd c

ould

still

refo

ld to

reco

ver a

ctiv

ity a

fter

hea

t den

atur

atio

n. Th

is c

ombi

natio

n of

hea

t res

ilien

ce a

nd st

rong

, spe

cific

bi

ndin

g m

ake

this

sdA

b a

good

can

dida

te fo

r use

in a

ntib

ody-

base

d to

xin

dete

ctio

n te

chno

logi

es.

Gra

ef e

t al

. 201

1

Den

atur

ing

cond

itio

nsR

efol

ding

Ther

mod

ynam

ic st

abili

ty

A v

arie

ty o

f tec

hniq

ues,

incl

udin

g hi

gh-p

ress

ure

unfo

ldin

g m

onito

red

by F

ouri

er tr

ansf

orm

infr

ared

spec

tros

copy

, fluo

resc

ence

, ci

rcul

ar d

ichr

oism

, and

surf

ace

plas

mon

reso

nanc

e sp

ectr

osco

py, h

ave

been

use

d to

inve

stig

ate

the

equi

libri

um fo

ldin

g pr

oper

ties o

f si

x si

ngle

-dom

ain

antig

en b

inde

rs d

eriv

ed fr

om c

amel

id h

eavy

-cha

in a

ntib

odie

s with

spec

ifici

ties f

or ly

sozy

mes

, bet

a-la

ctam

ases

, and

a

dye

(RR6

). Va

riou

s den

atur

ing

cond

ition

s (gu

anid

iniu

m c

hlor

ide,

ure

a, te

mpe

ratu

re, a

nd p

ress

ure)

pro

vide

d co

mpl

emen

tary

and

in

depe

nden

t met

hods

for c

hara

cter

izin

g th

e st

abili

ty a

nd u

nfol

ding

pro

pert

ies o

f the

ant

ibod

y fr

agm

ents

. With

all

bind

ers,

com

plet

e re

cove

ry o

f the

bio

logi

cal a

ctiv

ity a

fter r

enat

urat

ion

dem

onst

rate

s tha

t che

mic

al- i

nduc

ed u

nfol

ding

is fu

lly re

vers

ible

. Fur

ther

mor

e,

dena

tura

tion

expe

rim

ents

follo

wed

by

optic

al sp

ectr

osco

pic

met

hods

and

affi

nity

mea

sure

men

ts in

dica

te th

at th

e an

tibod

y fr

agm

ents

ar

e un

fold

ed c

oope

rativ

ely

in a

sing

le tr

ansi

tion.

Thus

, unf

oldi

ng/r

efol

ding

equ

ilibr

ium

pro

ceed

s via

a si

mpl

e tw

o-st

ate

mec

hani

sm

(Nre

vers

ible

arr

ow U

), w

here

onl

y th

e na

tive

and

the

dena

ture

d st

ates

are

sign

ifica

ntly

pop

ulat

ed. Th

erm

ally

- ind

uced

den

atur

atio

n,

how

ever

, is n

ot c

ompl

etel

y re

vers

ible

, and

the

part

ial l

oss o

f bin

ding

cap

acity

mig

ht b

e du

e, a

t lea

st in

par

t, to

inco

rrec

t ref

oldi

ng

of th

e lo

ng lo

ops (

CD

Rs),

whi

ch a

re re

spon

sibl

e fo

r ant

igen

reco

gniti

on. M

ost i

nter

estin

gly,

all t

he fr

agm

ents

are

rath

er re

sist

ant t

o he

at-in

duce

d de

natu

ratio

n (a

ppar

ent T

-m =

60–

80 d

egre

es-C

), an

d di

spla

y hi

gh c

onfo

rmat

iona

l sta

bilit

ies (

Del

taG

(H2O

) = 3

0–60

kJ/

mol

). Su

ch h

igh

ther

mod

ynam

ic st

abili

ty h

as n

ever

bee

n re

port

ed fo

r any

func

tiona

l con

vent

iona

l ant

ibod

y fr

agm

ent,

even

whe

n en

gi-

neer

ed a

ntig

en b

inde

rs a

re c

onsi

dere

d. H

ence

, the

redu

ced

size

, im

prov

ed so

lubi

lity,

and

high

er st

abili

ty o

f the

cam

elid

hea

vy-c

hain

an

tibod

y fr

agm

ents

are

of s

peci

al in

tere

st fo

r bio

tech

nolo

gica

l and

med

ical

app

licat

ions

.

Dum

ou-

lin e

t al.

2002


470

Hea

t sho

ckC

onfo

rmat

ion

stud

ies

Den

atur

atio

nR

efol

ding

In a

pre

viou

s stu

dy w

e ha

ve sh

own

that

llam

a V

HH

ant

ibod

y fr

agm

ents

are

abl

e to

bin

d th

eir a

ntig

en a

fter

a h

eat s

hock

of 9

0 de

gree

s C, i

n co

ntra

st to

the

mur

ine

mon

oclo

nal a

ntib

odie

s. H

owev

er, t

he m

olec

ular

mec

hani

sm b

y w

hich

ant

ibod

y: a

ntig

en

inte

ract

ion

occu

rs u

nder

thes

e ex

trem

e co

nditi

ons r

emai

ns u

ncle

ar. T

o ex

amin

e in

mor

e de

tail

the

stru

ctur

al a

nd th

erm

odyn

amic

as

pect

s of t

he b

indi

ng m

echa

nism

, an

exte

nsiv

e C

D, I

TC, a

nd N

MR

stud

y w

as in

itiat

ed. I

n th

is st

udy

the

inte

ract

ion

betw

een

the

llam

a V

HH

-R2

frag

men

t and

its a

ntig

en, t

he d

ye R

eact

ive

Red-

6 (R

R6) h

as b

een

expl

ored

. The

data

show

cle

arly

that

mos

t of t

he

VH

H-R

2 po

pula

tion

at 8

0 de

gree

s C is

in a

n un

fold

ed c

onfo

rmat

ion.

In c

ontr

ast,

CD

spec

tra

repr

esen

ting

the

com

plex

bet

wee

n V

HH

-R2

and

the

dye

rem

aine

d th

e sa

me

up to

80

degr

ees C

. Int

eres

tingl

y, ad

ditio

n of

the

dye

to th

e de

natu

red

VH

H-R

2 at

80

degr

ees C

yie

lded

the

spec

trum

of t

he n

ativ

e co

mpl

ex. Th

ese

resu

lts su

gges

t an

indu

ced

refo

ldin

g of

den

atur

ed V

HH

-R2

by it

s an

tigen

und

er th

ese

extr

eme

cond

ition

s. Th

is in

duce

d re

fold

ing

show

ed so

me

sim

ilari

ties w

ith th

e w

ell e

stab

lishe

d “in

duce

d fit

” m

echa

nism

of a

ntib

ody-

antig

en in

tera

ctio

ns a

t am

bien

t tem

pera

ture

. How

ever

, the

mai

n di

ffere

nce

with

the

“indu

ced

fit01

48”

mec

hani

sm is

that

at t

he st

art o

f the

add

ition

of t

he a

ntig

en m

ost o

f the

VH

H m

olec

ules

are

in a

n un

fold

ed c

onfo

rmat

ion.

The

refo

ldin

g ca

pabi

lity

unde

r the

se e

xtre

me

cond

ition

s and

the

stab

le c

ompl

ex fo

rmat

ion

mak

e V

HH

s use

ful i

n a

wid

e va

riet

y of

app

li-ca

tions

.

Dol

k et

al

. 200

5b

Bac

illus

ant

hrac

isTh

erm

al s

tabi

lity

Den

atur

atio

nR

efol

ding

Sign

ifica

nt e

ffort

s to

deve

lop

both

labo

rato

ry a

nd fi

eld-

base

d de

tect

ion

assa

ys fo

r an

arra

y of

pot

entia

l bio

logi

cal t

hrea

ts st

arte

d w

ell b

efor

e th

e an

thra

x at

tack

s of 2

001

and

have

con

tinue

d w

ith re

new

ed u

rgen

cy fo

llow

ing.

Whi

le n

umer

ous a

ssay

s and

met

h-od

s hav

e be

en e

xplo

red

that

are

suita

ble

for l

abor

ator

y ut

iliza

tion,

det

ectio

n in

the

field

is o

ften

com

plic

ated

by

requ

irem

ents

for

func

tiona

lity

in a

uste

re e

nvir

onm

ents

, whe

re li

mite

d co

ld-c

hain

faci

litie

s exi

st. I

n an

effo

rt to

ove

rcom

e th

ese

assa

y lim

itatio

ns fo

r Ba

cillu

s ant

hrac

is, o

ne o

f the

mos

t rec

ogni

zabl

e th

reat

s, a

seri

es o

f sin

gle

dom

ain

antib

odie

s (sd

Abs

) wer

e is

olat

ed fr

om a

pha

ge

disp

lay

libra

ry p

repa

red

from

imm

uniz

ed ll

amas

. Cha

ract

eriz

atio

n of

targ

et sp

ecifi

city

, affi

nity

, and

ther

mal

stab

ility

was

con

duct

ed

for s

ix sd

Ab

fam

ilies

isol

ated

from

roun

ds o

f sel

ectio

n ag

ains

t the

bac

teri

al sp

ore.

The

prot

ein

targ

et fo

r all

six

sdA

b fa

mili

es w

as

dete

rmin

ed to

be

the

S-la

yer p

rote

in E

A1,

whi

ch is

pre

sent

in b

oth

vege

tativ

e ce

lls a

nd b

acte

rial

spor

es. A

ll of

the

sdA

bs e

xam

ined

ex

hibi

ted

a hi

gh d

egre

e of

spec

ifici

ty fo

r the

targ

et b

acte

rium

and

its s

pore

, with

affi

nitie

s in

the

nano

mol

ar ra

nge,

and

the

abili

ty

to re

fold

into

func

tiona

l ant

igen

-bin

ding

mol

ecul

es fo

llow

ing

seve

ral r

ound

s of t

herm

al d

enat

urat

ion

and

refo

ldin

g. Th

is re

sear

ch

dem

onst

rate

s the

cap

abili

ties o

f the

se sd

Abs

and

thei

r pot

entia

l for

inte

grat

ion

into

cur

rent

and

dev

elop

ing

assa

ys a

nd b

iose

nsor

s.

Wal

per

et a

l. 20

12

Dan

druff

Mal

asse

zia

furf

urSh

ampo

oD

enat

urin

g co

ndit

ions

Am

ino

acid

rep

lace

men

t

As p

art o

f res

earc

h ex

plor

ing

the

feas

ibili

ty o

f usi

ng a

ntib

ody

frag

men

ts to

inhi

bit t

he g

row

th o

f org

anis

ms i

mpl

icat

ed in

dan

druff

, w

e is

olat

ed a

ntib

ody

frag

men

ts th

at b

ind

to a

cel

l sur

face

pro

tein

of M

alas

sezi

a fu

rfur

in th

e pr

esen

ce o

f sha

mpo

o. W

e fo

und

that

ph

age

disp

lay

of ll

ama

sing

le-d

omai

n an

tibod

y fr

agm

ents

(VH

Hs)

can

be

exte

nded

to v

ery

hars

h co

nditi

ons,

such

as t

he p

rese

nce

of sh

ampo

o co

ntai

ning

non

ioni

c an

d an

ioni

c su

rfac

tant

s. W

e se

lect

ed se

vera

l VH

Hs t

hat b

ind

to th

e ce

ll w

all p

rote

in M

alf1

of M

. fu

rfur

, a fu

ngus

impl

icat

ed in

cau

sing

dan

druff

. In

addi

tion

to h

igh

stab

ility

in th

e pr

esen

ce o

f sha

mpo

o, th

ese

VH

Hs a

re a

lso

stab

le

unde

r oth

er d

enat

urin

g co

nditi

ons,

such

as h

igh

urea

con

cent

ratio

ns. M

any

of th

e st

able

VH

Hs w

ere

foun

d to

con

tain

arg

inin

e at

po

sitio

n 44

. Rep

lace

men

t of t

he n

ativ

e am

ino

acid

at p

ositi

on 4

4 w

ith a

rgin

ine

in th

e m

ost s

tabl

e V

HH

that

lack

ed th

is a

rgin

ine

resu

lted

in a

dra

mat

ic fu

rthe

r inc

reas

e in

the

stab

ility

. The

com

bina

tion

of th

e un

ique

pro

pert

ies o

f VH

Hs t

oget

her w

ith a

pplie

d ph

age

disp

lay

and

prot

ein

engi

neer

ing

is a

pow

erfu

l met

hod

for o

btai

ning

hig

hly

stab

le V

HH

s tha

t can

be

used

in a

wid

e ra

nge

of

appl

icat

ions

.

Dol

k et

al

. 200

5a


471

Solu

bilit

yC

DR

3H

ydro

phili

c am

ino

acid

sTr

ypan

osom

e su

rfac

e gl

ycop

rote

in

Hea

vy c

hain

onl

y an

tibod

ies o

f cam

elid

s bin

d th

eir a

ntig

ens w

ith a

sing

le d

omai

n, th

e V

HH

, whi

ch a

cqui

red

adap

tatio

ns re

lativ

e to

cl

assi

cal V

Hs t

o fu

nctio

n in

the

abse

nce

of a

VL

part

ner.

Add

ition

al C

DR

loop

con

form

atio

ns, o

utsi

de th

e ca

noni

cal l

oop

stru

ctur

es

of V

Hs,

bro

aden

the

repe

rtoi

re o

f the

ant

igen

-bin

ding

site

. The

com

bine

d eff

ects

of p

art o

f the

CD

R3 th

at fo

lds o

ver t

he “f

orm

er”

VL

bind

ing

site

and

fram

ewor

k-2

mut

atio

ns to

mor

e hy

drop

hilic

am

ino

acid

s, e

nhan

ce th

e so

lubi

lity

of V

HH

dom

ains

and

pre

vent

V

L pa

irin

g. c

AbA

n33,

a V

HH

dom

ain

spec

ific

for t

he c

arbo

hydr

ate

moi

ety

of th

e va

rian

t sur

face

gly

copr

otei

n of

tryp

anos

omes

, ha

s a sh

ort C

DR3

loop

that

doe

s not

cov

er th

e fo

rmer

VL

bind

ing

site

as w

ell a

s a V

H-s

peci

fic T

rp47

inst

ead

of th

e V

HH

-spe

cific

G

ly47

. Res

urfa

cing

its f

ram

ewor

k-2

regi

on (m

utat

ions

Tyr

37Va

l, G

lu44

Gly

and

Arg

45Le

u) to

mim

ic th

at o

f a h

uman

VH

rest

ores

th

e V

L bi

ndin

g ca

paci

ty. I

n so

lutio

n, th

e hu

man

ised

VH

H b

ehav

es a

s a so

lubl

e, m

onom

eric

ent

ity, a

lbei

t with

redu

ced

ther

mod

y-na

mic

stab

ility

and

affi

nity

for i

ts a

ntig

en. C

ompa

riso

n of

the

crys

tal s

truc

ture

s of c

AbA

n33

and

its h

uman

ised

der

ivat

ive

reve

als

ster

ic h

indr

ance

exe

rted

by

VH

H-s

peci

fic re

sidu

es T

yr37

and

Arg

45 th

at p

reve

nt th

e V

L do

mai

n pa

irin

g, w

here

as G

lu44

and

Arg

45

are

key

elem

ents

to a

void

inso

lubi

lity

of th

e do

mai

n.

Con

rath

et

al.

2005

Seru

m h

alf-

live

Porc

ine

im

mun

oglo

bulin

GE.

col

i F4

fimbr

iae

The

ther

apeu

tic p

aren

tera

l app

licat

ion

of ll

ama

sing

le-d

omai

n an

tibod

y fr

agm

ents

(VH

Hs)

is h

ampe

red

by th

eir

smal

l siz

e,

resu

lting

in a

fast

elim

inat

ion

from

the

body

. Her

e w

e de

scri

be a

met

hod

to in

crea

se th

e se

rum

hal

f-lif

e of

VH

Hs

in p

igs

by

fusi

on to

ano

ther

VH

H b

indi

ng to

por

cine

imm

unog

lobu

lin G

(pIg

G).

We

isol

ated

19

pIgG

-bin

ding

VH

Hs

from

an

imm

uniz

ed

llam

a us

ing

phag

e di

spla

y. S

ix V

HH

s w

ere

gene

tical

ly fu

sed

to m

odel

VH

H K

609

that

bin

ds to

Esc

heri

chia

col

i F4

fimbr

iae.

All

six

yeas

t-pr

oduc

ed g

enet

ic fu

sion

s of

two

VH

H d

omai

ns (V

HH

2s) w

ere

func

tiona

l in

ELIS

A a

nd b

ound

to p

IgG

with

hig

h af

fin-

ity (1

–33

nM).

Four

pIg

G-b

indi

ng V

HH

2s w

ere

adm

inis

tere

d to

pig

s an

d sh

owed

a 1

00-f

old

exte

nded

in v

ivo

resi

denc

e tim

es a

s co

mpa

red

to a

con

trol

VH

H2

that

doe

s no

t bin

d to

pIg

G. T

his

coul

d pr

ovid

e th

e ba

sis

for

ther

apeu

tic a

pplic

atio

n of

VH

Hs

in

pigs

.

Har

mse

n et

al.

2005

Bio

dist

ribu

tion

stu

dies

Blo

od c

ircu

lati

onTu

mou

r re

tent

ion

Tum

our-

targ

etin

g ve

hicl

e

Two

cam

el si

ngle

-dom

ain

frag

men

ts, c

Ab-

Lys2

and

cA

b-Ly

s3, r

ecog

nizi

ng a

n ov

erla

ppin

g ep

itope

of l

ysoz

yme

with

a d

isso

ciat

ion

cons

tant

of 2

nM

and

6S

nM, r

espe

ctiv

ely,

and

a bi

vale

nt c

Ab-

Lys3

wer

e in

vest

igat

ed fo

r the

ir a

bilit

y to

targ

et tr

ansg

enic

tum

ors

expr

essi

ng ly

sozy

me

on th

eir m

embr

ane.

Bio

dist

ribu

tion

stud

ies r

evea

led

that

thes

e no

n-im

mun

ogen

ic m

onom

eric

and

biv

alen

t ca

mel

sing

le-d

omai

n an

tigen

bin

ders

spec

ifica

lly ta

rget

lyso

zym

e-ex

pres

sing

tum

ors a

nd m

etas

tatic

lesi

ons.

The

exce

ss o

f ant

i-bo

dy is

rapi

dly

elim

inat

ed fr

om th

e bl

ood

circ

ulat

ion

and

no c

Ab

rete

ntio

n w

as o

bser

ved

in n

orm

al o

rgan

s. Th

e tu

mor

to o

rgan

cA

b-ra

tios a

t 2 a

nd 8

h w

ere

in th

e (2

.1–1

0.8)

: 1

and

(6.2

–23.

7) :

1 ra

nge,

resp

ectiv

ely.

The

degr

ee a

nd sp

ecifi

city

of t

umor

rete

n-tio

n is

inde

pend

ent o

f the

affi

nity

of t

he re

com

bina

nt c

amel

sing

le-d

omai

n fr

agm

ents

for t

heir

ant

igen

and

from

thei

r uni

vale

nt

mon

omer

ic (1

5 kD

a) o

r biv

alen

t for

mat

(33

kDa)

. This

stud

y de

mon

stra

tes t

he su

cces

sful

and

spec

ific

in v

ivo

targ

etin

g of

tum

ors

by c

amel

sing

le-d

omai

n fr

agm

ents

. It m

ay o

pen

pers

pect

ives

for t

heir

futu

re u

se a

s tum

or-t

arge

ting

vehi

cle,

due

to th

eir s

mal

l si

ze, s

olub

le b

ehav

iour

and

bec

ause

they

are

non

-imm

unog

enic

and

inte

ract

with

epi

tope

s tha

t are

less

ant

igen

ic fo

r con

vent

iona

l an

tibod

ies.

Cor

tez-

Reta

-m

ozo

et

al. 2

002


472

Bin

ding

kin

etic

sR

icin

toxo

idEL

ISA

Surf

ace

plas

mon

res

o-na

nce

Sing

le d

omai

n an

tibod

ies a

re th

e re

com

bina

ntly

exp

ress

ed b

indi

ng fr

agm

ents

der

ived

from

hea

vy c

hain

ant

ibod

ies f

ound

in c

amel

s an

d lla

mas

. Thes

e un

ique

bin

ding

ele

men

ts o

ffer m

any

desi

rabl

e pr

oper

ties s

uch

as th

eir s

mal

l siz

e (s

imila

r to

15 k

Da)

and

ther

-m

al st

abili

ty, w

hich

mak

es th

em a

ttra

ctiv

e al

tern

ativ

es to

con

vent

iona

l mon

oclo

nal a

ntib

odie

s. W

e cr

eate

d a

phag

e di

spla

y lib

rary

fr

om ll

amas

imm

uniz

ed w

ith ri

cin

toxo

id a

nd se

lect

ed a

num

ber o

f sin

gle

dom

ain

antib

odie

s. P

hage

sele

cted

on

rici

n w

ere

foun

d to

bin

d to

eith

er ri

cin

A c

hain

or t

he in

tact

mol

ecul

e; n

o ri

cin

B ch

ain

bind

ers w

ere

iden

tified

. By

pann

ing

on B

cha

in, w

e id

entifi

ed

bind

ers a

nd h

ave

char

acte

rize

d th

eir b

indi

ng to

the

rici

n B

chai

n. W

hile

they

hav

e a

poor

er a

ffini

ty th

an th

e pr

evio

usly

des

crib

ed A

ch

ain

bind

ers,

it w

as fo

und

that

they

per

form

ed d

ram

atic

ally

bet

ter a

s cap

ture

reag

ents

for t

he d

etec

tion

of ri

cin,

pro

vidi

ng a

lim

it of

det

ectio

n in

enz

yme

linke

d im

mun

osor

bent

ass

ay (E

LISA

) bel

ow 1

00 p

g/m

L an

d ex

celle

nt sp

ecifi

city

for r

icin

ver

sus t

he h

ighl

y re

late

d RC

A 1

20 (1

to 1

0 00

0). W

e al

so re

eval

uate

d th

e pr

evio

usly

isol

ated

ant

iric

in si

ngle

dom

ain

antib

ody

bind

ing

kine

tics u

sing

su

rfac

e pl

asm

on re

sona

nce

and

foun

d th

eir K

(d)s

mat

ched

clo

sely

to th

ose

prev

ious

ly o

btai

ned

unde

r equ

ilibr

ium

bin

ding

con

di-

tions

mea

sure

d us

ing

the

Lum

inex

flow

cyt

omet

er.

And

er-

son

et a

l. 20

10

Nan

obod

y pr

oper

ties

Epid

erm

al g

row

th fa

ctor

re

cept

orD

rug

targ

etin

g

The

dis

cove

ry o

f nat

ural

ly o

ccur

ring

hea

vy c

hain

onl

y an

tibo

dies

and

thei

r fu

rthe

r de

velo

pmen

t int

o sm

all r

ecom

bina

nt

‘nan

obod

ies’

offe

rs a

ttra

ctiv

e ap

plic

atio

ns in

dru

g ta

rget

ing.

Her

e, w

e de

scri

be th

e pr

oper

ties

of n

anob

odie

s th

at h

ave

been

de

velo

ped

to ta

rget

the

epid

erm

al g

row

th fa

ctor

rec

epto

r (E

GFR

) and

con

tras

t the

se to

the

char

acte

rist

ics

of h

eavy

cha

in o

nly

anti

bodi

es a

nd c

onve

ntio

nal a

ntib

odie

s. E

GFR

is o

vere

xpre

ssed

in m

any

tum

ors

and

is a

n at

trac

tive

targ

et fo

r tu

mor

-dir

ecte

d dr

ug ta

rget

ing.

Alti

ntas

et

al.

2012


473

Tabl

e 6A

. App

licat

ion

of s

ingl

e-do

mai

n an

tibo

dy fr

agm

ents

in th

erap

y: in

hibi

tion

of e

nzym

es, t

oxin

s an

d ot

her

solu

ble

prot

eins

Prot

ein

kina

se C

Enzy

me

mod

ulat

orTh

e 10

isoz

ymes

of t

he p

rote

in k

inas

e C

(PK

C) f

amily

can

hav

e di

ffere

nt ro

les o

n th

e sa

me

biol

ogic

al p

roce

ss, m

akin

g is

ozym

e sp

ecifi

c an

alys

is o

f fun

ctio

n cr

ucia

l. C

urre

ntly

, onl

y fe

w p

harm

acol

ogic

al c

ompo

unds

with

mod

erat

e is

ozym

e sp

ecifi

c eff

ects

exi

st

thus

ham

peri

ng re

sear

ch in

to in

divi

dual

PK

C is

ozym

es. Th

e an

tigen

bin

ding

regi

ons o

f cam

elid

sing

le c

hain

ant

ibod

ies (

VH

Hs)

co

uld

prov

ide

a so

lutio

n fo

r obt

aini

ng P

KC

isoz

yme

spec

ific

mod

ulat

ors.

In th

e pr

esen

t stu

dy, w

e ha

ve su

cces

sful

ly se

lect

ed a

nd

char

acte

rize

d PK

C e

psilo

n sp

ecifi

c V

HH

ant

ibod

ies f

rom

two

imm

une

VH

H li

brar

ies u

sing

pha

ge d

ispl

ay. Th

e V

HH

s wer

e sh

own

to e

xclu

sive

ly b

ind

to P

KC

eps

ilon

in E

LISA

and

imm

unop

reci

pita

tion

stud

ies.

Str

ikin

gly,

five

of th

e V

HH

s had

an

effec

t on

PKC

ep

silo

n ki

nase

act

ivity

in v

itro.

VH

Hs A

10, C

1 an

d D

1 in

crea

sed

PKC

eps

ilon

kina

se a

ctiv

ity in

a c

once

ntra

tion-

depe

nden

t man

ner

(EC

(50)

val

ues:

212–

310

nM),

whe

reas

E6

and

G8

inhi

bite

d PK

C e

psilo

n ac

tivity

(IC

(50)

val

ues:

103–

233

nM).

Non

e of

thes

e V

HH

s had

an

effec

t on

the

activ

ity o

f the

oth

er n

ovel

PK

C is

ozym

es P

KC

del

ta a

nd P

KC

thet

a. T

o ou

r kno

wle

dge,

thes

e an

tibod

ies

are

the

first

des

crib

ed V

HH

act

ivat

ors a

nd in

hibi

tors

for a

pro

tein

kin

ase.

Fur

ther

mor

e, th

e de

velo

pmen

t of P

KC

eps

ilon

spec

ific

mod

ulat

ors i

s an

impo

rtan

t con

trib

utio

n to

PK

C re

sear

ch.

Paal

anen

et

al.

2011

Ant

i-id

ioty

pic

Alli

inas

eM

olec

ular

mim

icry

Abz

yme

Scre

enin

g of

inhi

bito

ry A

b1 a

ntib

odie

s is

a c

ritic

al s

tep

for

prod

ucin

g ca

taly

tic a

ntib

odie

s in

the

anti-

idio

typi

c ap

proa

ch. H

ow-

ever

, the

inco

mpa

tible

sur

face

of t

he a

ctiv

e si

te o

f the

enz

yme

and

the

antig

en-b

indi

ng s

ite o

f het

erot

etra

mer

ic c

onve

ntio

nal

antib

odie

s be

com

e th

e lim

iting

ste

p. B

ecau

se c

amel

id-d

eriv

ed n

anob

odie

s po

sses

s th

e po

tent

ial t

o pr

efer

entia

lly b

ind

to th

e ac

tive

site

of e

nzym

es d

ue to

thei

r sm

all s

ize

and

long

CD

R3, w

e ha

ve d

evel

oped

a n

ovel

app

roac

h to

pro

duce

ant

ibod

ies

with

al

liina

se a

ctiv

ities

by

expl

oitin

g th

e m

olec

ular

mim

icry

of c

amel

nan

obod

ies.

By

scre

enin

g th

e ca

mel

id-d

eriv

ed v

aria

ble

regi

on

of th

e he

avy

chai

n cD

NA

pha

ge d

ispl

ay li

brar

y w

ith a

lliin

ase,

we

obta

ined

an

inhi

bito

ry n

anob

ody

VH

HA

4 th

at re

cogn

izes

the

activ

e si

te. F

urth

er s

cree

ning

with

VH

HA

4 fr

om th

e sa

me

vari

able

dom

ain

of th

e he

avy

chai

n of

a h

eavy

-cha

in a

ntib

ody

libra

ry

led

to a

hig

her

inci

denc

e of

ant

i-id

ioty

pic

Ab2

abz

ymes

with

alli

inas

e ac

tiviti

es. O

ne o

f the

abz

ymes

, VH

HC

10, s

how

ed th

e hi

ghes

t act

ivity

that

can

be

inhi

bite

d by

Ab1

VH

HA

4 an

d al

liina

se c

ompe

titiv

e in

hibi

tor

peni

cilla

min

e an

d si

gnifi

cant

ly s

up-

pres

sed

the

B16

tum

or c

ell g

row

th in

the

pres

ence

of a

lliin

in v

itro.

The

resu

lts h

ighl

ight

the

feas

ibili

ty o

f pro

duci

ng a

bzym

es

via

anti-

idio

typi

c na

nobo

dy a

ppro

ach.

Li e

t al.

2012

Allo

ster

ic e

ffect

orD

ihyd

rofo

late

red

ucta

seIn

hibi

tion

kin

etic

sC

ryst

al s

truc

ture

Alth

ough

it h

as b

een

know

n fo

r man

y ye

ars t

hat a

ntib

odie

s dis

play

pro

pert

ies c

hara

cter

istic

of a

llost

eric

effe

ctor

s, th

e m

olec

ular

m

echa

nism

s res

pons

ible

for t

hese

effe

cts r

emai

n po

orly

und

erst

ood.

Her

e, w

e de

scri

be a

sing

le-d

omai

n an

tibod

y fr

agm

ent (

nano

-bo

dy) t

hat m

odul

ates

pro

tein

func

tion

by c

onst

rain

ing

conf

orm

atio

nal c

hang

e in

the

enzy

me

dihy

drof

olat

e re

duct

ase

(DH

FR).

Nan

obod

y 21

6 (N

b216

) beh

aves

as a

pot

ent a

llost

eric

inhi

bito

r of D

HFR

, giv

ing

rise

to m

ixed

hyp

erbo

lic in

hibi

tion

kine

tics.

The

crys

tal s

truc

ture

of N

b216

in c

ompl

ex w

ith D

HFR

reve

als t

hat t

he n

anob

ody

bind

s adj

acen

t to

the

activ

e si

te. H

alf o

f the

epi

tope

co

nsis

ts o

f res

idue

s fro

m th

e fle

xibl

e M

et20

loop

. This

loop

, whi

ch o

rdin

arily

osc

illat

es b

etw

een

occl

uded

and

clo

sed

conf

orm

a-tio

ns d

urin

g ca

taly

sis,

ass

umes

the

occl

uded

con

form

atio

n in

the

Nb2

16-b

ound

stat

e. U

sing

stop

ped

flow

, we

show

that

Nb2

16

inhi

bits

DH

FR b

y st

abili

sing

the

occl

uded

Met

20 lo

op c

onfo

rmat

ion.

Sur

pris

ingl

y, ki

netic

dat

a in

dica

te th

at th

e M

et20

loop

reta

ins

suffi

cien

t con

form

atio

nal fl

exib

ility

in th

e N

b216

-bou

nd st

ate

to a

llow

slow

subs

trat

e tu

rnov

er to

occ

ur.

Oye

n et

al

. 201

1


474

Sial

idas

eTr

ypan

osom

a cr

uzi

The

sia

lic a

cid

pres

ent i

n th

e pr

otec

tive

surf

ace

muc

in c

oat o

f Try

pano

som

a cr

uzi i

s ad

ded

by a

mem

bran

e an

chor

ed tr

ans-

sial

idas

e (T

cTS)

, a m

odifi

ed s

ialid

ase

that

is e

xpre

ssed

from

a la

rge

gene

fam

ily. I

n th

is w

ork,

we

anal

yzed

sin

gle

dom

ain

cam

elid

an

tibod

ies

prod

uced

aga

inst

tran

s-si

alid

ase.

Lla

mas

wer

e im

mun

ized

with

a re

com

bina

nt tr

ans-

sial

idas

e an

d in

hibi

tory

sin

gle-

dom

ain

antib

ody

frag

men

ts w

ere

obta

ined

by

phag

e di

spla

y se

lect

ion,

taki

ng a

dvan

tage

of a

scr

eeni

ng s

trat

egy

usin

g an

inhi

bi-

tion

test

inst

ead

of th

e cl

assi

c bi

ndin

g as

say.

Fou

r si

ngle

dom

ain

antib

odie

s di

spla

ying

str

ong

tran

s-si

alid

ase

inhi

bitio

n ac

tivity

ag

ains

t the

reco

mbi

nant

enz

yme

wer

e id

entif

ied.

The

y sh

are

the

sam

e co

mpl

emen

tari

ty-d

eter

min

ing

regi

on 3

leng

th (1

7 re

si-

dues

) and

hav

e ve

ry s

imila

r se

quen

ces.

Thi

s re

sult

indi

cate

s th

at th

ey li

kely

der

ived

from

a u

niqu

e cl

one.

Pro

babl

y th

ere

is o

nly

one

stru

ctur

al s

olut

ion

for

tight

bin

ding

inhi

bito

ry a

ntib

odie

s ag

ains

t the

TcT

S us

ed fo

r im

mun

izat

ion.

To

our

surp

rise

, thi

s si

ngle

dom

ain

antib

ody

that

inhi

bits

the

reco

mbi

nant

TcT

S, fa

iled

to in

hibi

t the

enz

ymat

ic a

ctiv

ity p

rese

nt in

par

asite

ext

ract

s.

Ana

lysi

s of

indi

vidu

al re

com

bina

nt tr

ans-

sial

idas

es s

how

ed th

at e

nzym

es e

xpre

ssed

from

diff

eren

t gen

es w

ere

inhi

bite

d to

dif-

fere

nt e

xten

ts (f

rom

8 to

98%

) by

the

llam

a an

tibod

ies.

Am

ino

acid

cha

nges

at k

ey p

ositi

ons

are

likel

y to

be

resp

onsi

ble

for

the

diffe

renc

es in

inhi

bitio

n fo

und

amon

g th

e re

com

bina

nt e

nzym

es. T

hese

resu

lts s

ugge

st th

at th

e pr

esen

ce o

f a la

rge

and

dive

rse

tran

s-si

alid

ase

fam

ily m

ight

be

requ

ired

to p

reve

nt th

e in

hibi

tory

resp

onse

aga

inst

this

ess

entia

l enz

yme

and

mig

ht th

us c

onst

i-tu

te a

nov

el s

trat

egy

of T

. cru

zi to

eva

de th

e ho

st im

mun

e sy

stem

.

Ratie

r et

al. 2

008

Clo

stri

dium

diffi

cile

Nos

ocom

ial i

nfec

tion

Nor

th A

mer

ica

Exot

oxin

A/B

Gas

troi

ntes

tina

l tra

ct

Clo

stri

dium

diff

icile

is a

lead

ing

caus

e of

nos

ocom

ial i

nfec

tion

in N

orth

Am

eric

a an

d a

cons

ider

able

cha

lleng

e to

hea

lthca

re

prof

essi

onal

s in

hos

pita

ls a

nd n

ursi

ng h

omes

. The

Gra

m-p

ositi

ve b

acte

rium

pro

duce

s tw

o hi

gh m

olec

ular

wei

ght e

xoto

xins

, to

xin

A (T

cdA

) and

toxi

n B

(Tcd

B), w

hich

are

the

maj

or v

irul

ence

fact

ors

resp

onsi

ble

for

C. d

iffic

ile-a

ssoc

iate

d di

seas

e an

d ar

e ta

rget

s fo

r C

. diff

icile

-ass

ocia

ted

dise

ase

ther

apy.

Her

e, re

com

bina

nt s

ingl

e-do

mai

n an

tibod

y fr

agm

ents

(V(H

)Hs)

, whi

ch

spec

ifica

lly ta

rget

the

cell

rece

ptor

bin

ding

dom

ains

of T

cdA

or

TcdB

, wer

e is

olat

ed fr

om a

n im

mun

e lla

ma

phag

e di

spla

y lib

rary

an

d ch

arac

teri

zed.

Fou

r V

(H)H

s (A

4.2,

A5.

1, A

20.1

, and

A26

.8),

all s

how

n to

reco

gniz

e co

nfor

mat

iona

l epi

tope

s, w

ere

pote

nt

neut

raliz

ers

of th

e cy

topa

thic

effe

cts

of to

xin

A o

n fib

robl

ast c

ells

in a

n in

vitr

o as

say.

The

neu

tral

izin

g po

tenc

y w

as fu

rthe

r en

hanc

ed w

hen

V(H

)Hs

wer

e ad

min

iste

red

in p

aire

d or

trip

let c

ombi

natio

ns a

t the

sam

e ov

eral

l V(H

)H c

once

ntra

tion,

sug

gest

-in

g re

cogn

ition

of n

onov

erla

ppin

g Tc

dA e

pito

pes.

Bia

core

epi

tope

map

ping

exp

erim

ents

reve

aled

that

som

e sy

nerg

istic

com

-bi

natio

ns c

onsi

sted

of V

(H)H

s re

cogn

izin

g ov

erla

ppin

g ep

itope

s, a

n in

dica

tion

that

fact

ors

othe

r th

an m

ere

epito

pe b

lock

ing

are

resp

onsi

ble

for

the

incr

ease

d ne

utra

lizat

ion.

Fur

ther

bin

ding

ass

ays

reve

aled

Tcd

A-s

peci

fic V

(H)H

s ne

utra

lized

toxi

n A

by

bind

ing

to s

ites

othe

r th

an th

e ca

rboh

ydra

te b

indi

ng p

ocke

t of t

he to

xin.

With

favo

rabl

e ch

arac

teri

stic

s su

ch a

s hi

gh p

rodu

ctio

n yi

eld,

pot

ent t

oxin

neu

tral

izat

ion,

and

intr

insi

c st

abili

ty, t

hese

V(H

)Hs

are

attr

activ

e sy

stem

ic th

erap

eutic

s bu

t are

mor

e so

as

oral

ther

apeu

tics

in th

e de

stab

ilizi

ng e

nvir

onm

ent o

f the

gas

troi

ntes

tinal

trac

t.

Hus

sack

et

al.

2011

b


475

Stap

hylo

cocc

us a

ureu

sH

emol

ysin

‘Hot

-col

d’ h

emol

ytic

ac

tivi

ty

Obj

ectiv

e: T

o is

olat

e an

d ch

arac

teri

ze S

taph

yloc

occu

s aur

eus (

S. a

ureu

s) h

emol

ysin

neu

tral

izin

g dA

bs fr

ont p

hage

dis

play

libr

ary

of In

dian

des

ert c

amel

. Met

hods

: Pha

ge d

ispl

ay li

brar

y of

5 ×

107 d

Ab

clon

es o

f LPS

-im

mun

ized

Indi

an d

eser

t cam

el c

onst

ruct

ed

in o

ur la

bora

tory

was

use

d fo

r sel

ectio

n of

S. a

ureu

s exo

toxi

n sp

ecifi

c cl

ones

by

pann

ing

tech

niqu

e. E

nric

hmen

t of A

g-sp

ecifi

c cl

ones

in su

cces

sive

roun

ds o

f pan

ning

was

ass

esse

d by

pha

ge-E

LISA

and

pha

ge ti

trat

ion.

Diff

eren

t dA

b cl

ones

bin

ding

to S

. aur

eus

exot

oxin

Ags

wer

e ex

pres

sed

with

C-t

erm

inal

6 X

His

tag

in E

. col

i and

pur

ified

by

Ni-

chel

ate

chro

mat

ogra

phy.

The

expr

essi

on

was

ver

ified

by

SDS-

PAG

E an

d w

este

rn b

lott

ing.

The

puri

fied

clon

es w

ere

test

ed fo

r inh

ibiti

on o

f ‘ho

t-co

ld’ h

emol

ytic

act

ivity

in

vitr

o. R

esis

tanc

e to

ther

mal

inac

tivat

ion

of th

e dA

b cl

ones

was

stud

ied

by o

bser

ving

the

effec

t of h

eat t

reat

men

t fro

m 5

0 de

gree

s C

to 9

9 de

gree

s C fo

r 30

min

on

the

‘hot

-col

d’ h

emol

ytic

act

ivity

in v

itro.

Res

ults

: Sev

eral

dA

b cl

ones

bin

ding

to S

. aur

eus e

xoto

xins

w

ere

isol

ated

and

enr

iche

d by

thre

e ro

unds

of p

anni

ng. Th

e so

lubl

e dA

b cl

ones

wer

e ap

prox

imat

ely

sim

ilar t

o 16

kD

a in

size

and

re

acte

d w

ith 6

X H

is ta

g sp

ecifi

c m

urin

e m

onoc

lona

l ant

ibod

y in

wes

tern

blo

t. O

ne o

f the

Ni-

chel

ate

affini

ty p

urifi

ed d

Ab.

6 X

His

cl

ones

, inh

ibite

d S.

aur

eus b

eta-

hem

olys

in a

ctiv

ity in

vitr

o an

d re

sist

ed th

erm

al in

activ

atio

n up

to 9

9 de

gree

s C. C

oncl

usio

ns: A

n S.

au

reus

bet

a-he

mol

ysin

neu

tral

izin

g dA

b cl

one

of p

ossi

ble

ther

apeu

tic p

oten

tial h

as b

een

isol

ated

.

Pooj

a an

d A

jit

2010

Thro

mbi

n ac

tiva

tabl

e fib

rino

lysi

s in

hibi

tor

Clo

t lys

is

Back

grou

nd: B

ecau

se a

ctiv

ated

thro

mbi

n ac

tivat

able

fibr

inol

ysis

inhi

bito

r (TA

FIa)

has

ver

y po

wer

ful a

ntifi

brin

olyt

ic p

rope

rtie

s, co

-ad

min

istra

tion

of t-

PA a

nd a

TA

FIa

inhi

bito

r enh

ance

s t-P

A tr

eatm

ent.

Obj

ectiv

e: W

e ai

med

to g

ener

ate

nano

bodi

es sp

ecifi

cally

inhi

bit-

ing

the

TAFI

a ac

tivity

and

to te

st th

eir e

ffect

on

t-PA

indu

ced

clot

lysis

. Res

ults

: Fiv

e na

nobo

dies

, rai

sed

tow

ards

an

activ

ated

mor

e st

able

TA

FIa

mut

ant (

TAFI

a A

(147

)-C

(305

)-I(3

25)-

I(329

)-Y(3

33)-

Q(3

35)),

are

des

crib

ed. Th

ese

nano

bodi

es in

hibi

t spe

cific

ally

TA

FIa

activ

ity,

resu

lting

in a

n in

hibi

tion

of u

p to

99%

at a

16-

fold

mol

ar e

xces

s of n

anob

ody

over

TA

FIa,

IC(5

0)’s

rang

e be

twee

n 0.

38- a

nd >

16-

fold

m

olar

exc

ess.

In v

itro

clot

lysis

exp

erim

ents

in th

e ab

senc

e of

thro

mbo

mod

ulin

(TM

) dem

onst

rate

that

the

nano

bodi

es e

xhib

it pr

ofi-

brin

olyt

ic e

ffect

s. H

owev

er, i

n th

e pr

esen

ce o

f TM

, one

nan

obod

y ex

hibi

ts a

n an

tifibr

inol

ytic

effe

ct w

here

as th

e ot

her n

anob

odie

s sho

w a

sli

ght a

ntifi

brin

olyt

ic e

ffect

at l

ow c

once

ntra

tions

and

a p

rono

unce

d pr

ofibr

inol

ytic

effe

ct a

t hig

her c

once

ntra

tions

. This

biph

asic

pat

tern

w

as h

ighl

y de

pend

ent o

n TM

and

t-PA

con

cent

ratio

n. Th

e na

nobo

dies

wer

e fo

und

to b

ind

in th

e ac

tive-

site

regi

on o

f TA

FIa

and

thei

r tim

e-de

pend

ent d

iffer

entia

l bin

ding

beh

avio

r dur

ing

TAFI

a in

activ

atio

n re

veal

ed th

e oc

curr

ence

of a

yet

unk

now

n in

term

edia

te c

onfo

r-m

atio

nal t

rans

ition

. Con

clus

ion:

Thes

e na

nobo

dies

are

ver

y po

tent

TA

FIa

inhi

bito

rs a

nd c

onst

itute

use

ful t

ools

to a

ccel

erat

e fib

rinol

ysis

. O

ur d

ata

also

dem

onst

rate

that

the

profi

brin

olyt

ic e

ffect

of T

AFI

a in

hibi

tion

may

be

reve

rsed

by

the

pres

ence

of T

M. Th

e id

entifi

catio

n of

a

new

con

form

atio

nal t

rans

ition

pro

vide

s new

insig

hts i

nto

the

conf

orm

atio

nal i

nact

ivat

ion

of th

e un

stab

le T

AFI

a.

Hen

-dr

ickx

et

al. 2

011

Tum

our

necr

osis

fact

orFu

sion

Elas

tin-

like

poly

pept

ide

Toba

cco

Tum

our n

ecro

sis f

acto

r (T

NF)

is a

maj

or p

ro-in

flam

mat

ory

cyto

kine

invo

lved

in m

ultip

le in

flam

mat

ory

dise

ases

. The

detr

imen

tal

activ

ity o

f TN

F ca

n be

blo

cked

by

vari

ous a

ntag

onis

ts, a

nd c

omm

erci

al th

erap

eutic

s bas

ed u

pon

this

pri

ncip

le h

ave

been

app

rove

d fo

r tre

atm

ent o

f dis

ease

s inc

ludi

ng rh

eum

atoi

d ar

thri

tis, C

rohn

’s di

seas

e an

d ps

oria

sis.

In a

sear

ch fo

r new

, im

prov

ed a

nti-

infla

mm

ator

y th

erap

eutic

s we

have

des

igne

d a

sing

le-d

omai

n m

onoc

lona

l ant

ibod

y (V

(H)H

), w

hich

reco

gniz

es T

NF.

The

antib

ody

com

pone

nt (T

NF-

V(H

)H) i

s bas

ed u

pon

an a

nti-h

uman

TN

F C

amel

idae

hea

vy-c

hain

mon

oclo

nal a

ntib

ody,

whi

ch w

as li

nked

to a

n el

astin

-like

pol

ypep

tide

(ELP

). W

e de

mon

stra

te th

at E

LP fu

sion

to th

e T

NF-

V(H

)H e

nhan

ces a

ccum

ulat

ion

of th

e fu

sion

pro

tein

du

ring

bio

man

ufac

turi

ng in

tran

sgen

ic to

bacc

o pl

ants

. With

this

stud

y, w

e sh

ow fo

r the

firs

t tim

e th

at th

is p

lant

-der

ived

ant

i-hu

man

TN

F-V

(H)H

ant

ibod

y w

as b

iolo

gica

lly a

ctiv

e in

viv

o. Th

eref

ore,

ther

apeu

tic a

pplic

atio

n of

TN

F-V

(H)H

-ELP

fusi

on p

rote

in

was

test

ed in

hum

aniz

ed T

NF

mic

e an

d w

as sh

own

to b

e eff

ectiv

e in

pre

vent

ing

deat

h ca

used

by

sept

ic sh

ock.

The

in v

ivo

pers

is-

tenc

e of

the

ELPy

late

d an

tibod

y w

as si

mila

r to

24 fo

ld lo

nger

than

that

of n

on-E

LPyl

ated

TN

F-V

(H)H

.

Con

rad

et a

l. 20

11


476

L-pl

asti

nM

alig

nanc

yF-

acti

n bu

ndlin

gFi

lopo

dia

L-pl

astin

, a c

onse

rved

mod

ular

F-a

ctin

bun

dlin

g pr

otei

n, is

ect

opic

ally

exp

ress

ed in

tum

or c

ells

and

con

trib

utes

to c

ell m

alig

nanc

y an

d in

vasi

on. Th

e un

derl

ying

mol

ecul

ar m

echa

nism

s inv

olve

d re

mai

n un

clea

r, in

par

t, be

caus

e sp

ecifi

c in

hibi

tors

of L

-pla

stin

are

la

ckin

g. W

e us

ed re

com

bina

nt a

lpac

a-de

rive

d L-

plas

tin si

ngle

-dom

ain

antib

odie

s (na

nobo

dies

) as e

ffect

or o

f L-p

last

in fu

nctio

n in

cel

ls. K

ey fi

ndin

gs w

ere

com

pare

d w

ith L

-pla

stin

dow

n-re

gula

tion

by R

NA

i. W

e sh

ow th

at n

anob

odie

s str

ongl

y in

tera

ct w

ith

L-pl

astin

by

targ

etin

g di

scre

te c

onfo

rmat

iona

l epi

tope

s with

nan

omol

ar a

ffini

ty. A

nan

obod

y th

at se

lect

ivel

y in

tera

cts w

ith th

e ta

ndem

ABD

s in

L-pl

astin

com

plet

ely

inhi

bits

F-a

ctin

bun

dlin

g at

equ

imol

ar ra

tios,

in c

ontr

ast t

o a

cont

rol g

reen

fluo

resc

ent p

ro-

tein

(GFP

) nan

obod

y. Th

is “k

nock

out”

nan

obod

y in

hibi

ts fi

lopo

dia

form

atio

n, m

otili

ty, a

nd in

vasi

on w

hen

expr

esse

d in

PC

-3 c

ells

. L-

plas

tin R

NA

inte

rfer

ence

show

ed n

o si

gnifi

cant

effe

ct o

n fil

opod

ial i

nteg

rity

and

onl

y m

argi

nally

rest

rain

ed th

e m

otile

pro

pert

ies

of c

ells

. L-p

last

in n

anob

odie

s uni

quel

y ex

pose

a fu

ndam

enta

l rol

e fo

r thi

s pro

tein

in fi

lopo

dia

form

atio

n an

d ce

ll m

igra

tion.

Ther

e-fo

re, t

hese

mol

ecul

es re

pres

ent a

pot

ent i

nstr

umen

t to

abla

te fu

nctio

ns o

f str

uctu

ral p

rote

ins w

ithou

t man

ipul

atin

g ge

ne e

xpre

s-si

on. I

n ad

ditio

n, w

e sh

ow th

at th

ey c

an b

e in

stru

men

tal i

n un

cove

ring

new

func

tions

of p

rote

ins t

hat r

emai

n ob

scur

ed b

y RN

Ai.

Del

anot

e et

al.

2010

Tabl

e 6B

. App

licat

ion

of s

ingl

e-do

mai

n an

tibo

dy fr

agm

ents

in th

erap

y: a

ctiv

ity

mod

ulat

ion

of c

ell s

urfa

ce p

rote

ins

Nan

obod

y pr

oper

ties

Epid

erm

al g

row

th fa

ctor

re

cept

orD

rug

targ

etin

g

The

disc

over

y of

nat

ural

ly o

ccur

ring

hea

vy c

hain

onl

y an

tibod

ies a

nd th

eir f

urth

er d

evel

opm

ent i

nto

smal

l rec

ombi

nant

‘nan

obod

-ie

s’ off

ers a

ttra

ctiv

e ap

plic

atio

ns in

dru

g ta

rget

ing.

Her

e, w

e de

scri

be th

e pr

oper

ties o

f nan

obod

ies t

hat h

ave

been

dev

elop

ed to

ta

rget

the

epid

erm

al g

row

th fa

ctor

rece

ptor

(EG

FR) a

nd c

ontr

ast t

hese

to th

e ch

arac

teri

stic

s of h

eavy

cha

in o

nly

antib

odie

s and

co

nven

tiona

l ant

ibod

ies.

EG

FR is

ove

rexp

ress

ed in

man

y tu

mor

s and

is a

n at

trac

tive

targ

et fo

r tum

or-d

irec

ted

drug

targ

etin

g.

Alti

ntas

et

al.

2012

Vasc

ular

end

othe

lial

grow

th fa

ctor

re

cept

or-2

Neo

vasc

ular

izat

ion

Met

asta

sis

Vasc

ular

end

othe

lial g

row

th fa

ctor

rece

ptor

-2 (V

EGFR

2) is

an

impo

rtan

t tum

or-a

ssoc

iate

d re

cept

or a

nd b

lock

ade

of th

e V

EGF

rece

p-to

r sig

nalin

g ca

n le

ad to

the

inhi

bitio

n of

neo

vasc

ular

izat

ion

and

tum

or m

etas

tasis

. Nan

obod

ies a

re th

e sm

alle

st in

tact

ant

igen

bin

ding

fr

agm

ents

der

ived

from

hea

vy c

hain

-onl

y an

tibod

ies o

ccur

ring

in c

amel

ids.

Her

e, w

e de

scrib

e th

e id

entifi

catio

n of

a V

EGFR

2-sp

ecifi

c N

anob

ody,

nam

ed 3

VG

R19,

from

dro

med

arie

s im

mun

ized

with

a c

ell l

ine

expr

essin

g hi

gh le

vels

of V

EGFR

2. W

e de

mon

stra

te b

y FA

CS,

th

at 3

VG

R19

Nan

obod

y sp

ecifi

cally

bin

ds V

EGFR

2 on

the

surf

ace

of 2

93K

DR

and

HU

VEC

s cel

ls. F

urth

erm

ore,

the

3VG

R19

Nan

obod

y po

tent

ly in

hibi

ts fo

rmat

ion

of c

apill

ary-

like

stru

ctur

es. Th

ese

data

show

the

pote

ntia

l of N

anob

odie

s for

the

bloc

kade

of V

EGFR

2 sig

nal-

ing

and

prov

ide

a ba

sis fo

r the

dev

elop

men

t of n

ovel

can

cer t

hera

peut

ics.

Behd

ani

et a

l. 20

12

Lept

in r

ecep

tor

Subd

omai

nN

euro

pept

ide

YB

lood

-bra

in b

arri

er

The

adip

ocyt

e-de

rived

cyt

okin

e le

ptin

act

s as a

met

abol

ic sw

itch,

con

nect

ing

the

body

’s m

etab

olism

to h

igh-

ener

gy c

onsu

min

g pr

oces

ses

such

as r

epro

duct

ion

and

imm

une

resp

onse

s. A

ccum

ulat

ing

evid

ence

sugg

ests

that

lept

in p

lays

a ro

le in

hum

an p

atho

logi

es, s

uch

as

auto

imm

une

dise

ases

and

can

cer,

thus

pro

vidi

ng a

ratio

nale

for t

he d

evel

opm

ent o

f lep

tin a

ntag

onist

s. In

the

pres

ent s

tudy

, we

gene

rate

d an

d ev

alua

ted

a pa

nel o

f neu

tral

izin

g na

nobo

dies

targ

etin

g th

e LR

(lep

tin re

cept

or).

A n

anob

ody

com

prise

s the

var

iabl

e do

mai

n of

the

natu

rally

occ

urrin

g sin

gle-

chai

n an

tibod

ies f

ound

in m

embe

rs o

f the

Cam

elid

ae fa

mily

. We

iden

tified

thre

e cl

asse

s of n

eutr

aliz

ing

nano

-bo

dies

targ

etin

g di

ffere

nt L

R su

bdom

ains

: i.e

. the

CRH

2 (c

ytok

ine

rece

ptor

hom

olog

y 2)

, Ig-

like

and

FNII

I (fib

rone

ctin

type

III)

dom

ains

. O

nly

nano

bodi

es d

irect

ed a

gain

st th

e C

RH2

dom

ain

inhi

bite

d le

ptin

bin

ding

. We

coul

d sh

ow th

at a

nan

obod

y th

at ta

rget

s the

Ig-li

ke

dom

ain

pote

ntly

inte

rfer

ed w

ith le

ptin

-dep

ende

nt re

gula

tion

of h

ypot

hala

mic

NPY

(neu

rope

ptid

e Y)

exp

ress

ion.

As a

con

sequ

ence

, dai

ly

intr

aper

itone

al in

ject

ion

incr

ease

d bo

dy w

eigh

t, bo

dy fa

t con

tent

, foo

d in

take

, liv

er si

ze a

nd se

rum

insu

lin le

vels

. All

of th

ese

char

acte

ris-

tics r

esem

ble

the

phen

otyp

e of

lept

in a

nd L

R-de

ficie

nt a

nim

als.

The

resu

lts o

f the

pre

sent

stud

y su

ppor

t pro

pose

d m

odel

s of t

he a

ctiv

ated

LR

com

plex

, and

dem

onst

rate

that

it is

pos

sible

to b

lock

LR

signa

lling

with

out a

ffect

ing

ligan

d bi

ndin

g. Th

ese

nano

bodi

es fo

rm n

ew to

ols

to st

udy

the

mec

hani

sms o

f BBB

(blo

od-b

rain

bar

rier)

lept

in tr

ansp

ort a

nd th

e eff

ect o

f LR

inhi

bitio

n in

dise

ase

mod

els.

Zab

eau

et a

l. 20

12


477

Car

cino

embr

yoni

c an

tige

n re

late

d ce

ll ad

hesi

on m

olec

ule

Can

cer

Car

cino

embr

yoni

c an

tigen

rela

ted

cell

adhe

sion

mol

ecul

e (C

EAC

AM

) 6 is

ove

r-ex

pres

sed

in d

iffer

ent t

ypes

of c

ance

r cel

ls. I

n ad

di-

tion,

it h

as a

lso

been

impl

icat

ed in

som

e in

fect

ious

dis

ease

s. Ta

rget

ing

this

mol

ecul

e by

an

antib

ody

mig

ht h

ave

appl

icat

ions

in d

iver

se

tum

or m

odel

s. Si

ngle

dom

ain

antib

ody

(sdA

b) is

bec

omin

g ve

ry u

sefu

l for

mat

in a

ntib

ody

engi

neer

ing

as p

oten

tial t

ools

for t

reat

-in

g ac

ute

and

chro

nic

dise

ase

cond

ition

s suc

h as

can

cer f

or b

oth

diag

nost

ic a

s wel

l as t

hera

peut

ic a

pplic

atio

n. G

ener

ally

, sdA

bs w

ith

good

affi

nity

are

isol

ated

from

an

imm

une

libra

ry. D

isco

very

of a

new

targ

et a

ntig

en w

ould

requ

ire a

new

imm

uniz

atio

n w

ith p

urifi

ed

antig

en w

hich

is n

ot a

lway

s eas

y. In

this

stud

y, w

e ha

ve is

olat

ed, b

y ph

age

disp

lay,

an sd

Ab

agai

nst C

EAC

AM

6 w

ith a

n affi

nity

of 5

nM

fr

om a

llam

a im

mun

ized

with

can

cer c

ells

. The

antib

ody

has g

ood

biop

hysi

cal p

rope

rtie

s, an

d it

bind

s to

the

cells

exp

ress

ing

the

targ

et

antig

en. F

urth

erm

ore,

it re

duce

s can

cer c

ells

prol

ifera

tion

in v

itro

and

show

s an

exce

llent

tum

or ta

rget

ing

in v

ivo.

This

sdA

b co

uld

be

usef

ul in

dia

gnos

is a

s wel

l as t

hera

py o

f CEA

CA

M6

expr

essi

ng tu

mor

s. Fi

nally

, we

envi

sage

it w

ould

be

feas

ible

to is

olat

e go

od sd

Abs

ag

ains

t oth

er in

tere

stin

g tu

mor

ass

ocia

ted

antig

ens f

rom

this

libr

ary.

Ther

efor

e, th

is im

mun

izat

ion

met

hod

coul

d be

a g

ener

al st

rate

gy

for i

sola

ting

sdA

bs a

gain

st a

ny su

rfac

e an

tigen

with

out i

mm

uniz

ing

the

anim

al w

ith th

e an

tigen

of i

nter

est e

ach

time.

Bara

l et

al. 2

011

12 n

anob

ody

clon

esO

ligoc

lona

l nan

obod

ies

Bre

ast c

ance

r ce

llsH

ER2

Mod

ern

anti-

HER

2 an

tibod

y th

erap

y te

nds t

o ex

ploi

t a p

anel

of d

iffer

ent a

ntib

odie

s aga

inst

diff

eren

t epi

tope

s on

the

antig

en. F

or

this

aim

, nan

obod

ies a

re v

ery

stri

king

targ

etin

g ag

ents

and

can

be

easi

ly p

rodu

ced

agai

nst a

ny c

ell-s

peci

fic m

embr

ane

antig

en. Th

e ol

igoc

lona

l nan

obod

ies c

an b

e us

ed to

blo

ck m

ore

than

one

func

tiona

l epi

tope

on

a ta

rget

ant

igen

and

inhi

bit t

he g

ener

atio

n of

esc

ape

vari

ants

ass

ocia

ted

with

can

cer t

hera

py. I

n th

is st

udy,

12 n

anob

ody

clon

es se

lect

ed fr

om a

n im

mun

e ca

mel

libr

ary

wer

e ex

amin

ed fo

r th

eir a

bilit

y to

diff

er b

etw

een

tum

or m

arke

rs. Th

ese

olig

oclo

nal n

anob

odie

s tar

gete

d br

east

can

cer c

ells

bette

r tha

n ea

ch in

divi

dual

na

nobo

dy. I

n ep

itope

map

ping

, sev

eral

nan

obod

ies o

verla

pped

in th

e ep

itope

reco

gniz

ed b

y tr

astu

zum

ab a

nd so

me

of th

e no

n-ov

er-

lapp

ing

nano

bodi

es c

ould

affe

ct th

e bi

ndin

g of

tras

tuzu

mab

to H

ER2.

This

stud

y de

mon

stra

tes t

hat t

he o

ligoc

lona

l nan

obod

ies a

re

pote

ntia

l the

rape

utic

tool

s tha

t can

be

used

inst

ead

of, o

r in

com

bina

tion

with

tras

tuzu

mab

to a

sses

s tum

or v

iabi

lity

duri

ng tr

eatm

ent.

Jam

nani

et

al.

2012

Ion

chan

nel M

2In

fluen

za A

Influ

enza

A v

irus

pos

es se

riou

s hea

lth th

reat

to h

uman

s. N

eutr

aliz

ing

antib

odie

s aga

inst

the

high

ly c

onse

rved

M2

ion

chan

nel i

s th

ough

t to

offer

bro

ad p

rote

ctio

n ag

ains

t infl

uenz

a A

vir

uses

. Her

e, w

e sc

reen

ed sy

nthe

tic C

amel

sing

le-d

omai

n an

tibod

y (V

HH

) lib

rari

es a

gain

st n

ativ

e M

2 io

n ch

anne

l pro

tein

. One

of t

he is

olat

ed V

HH

s, M

2-7A

, spe

cific

ally

bou

nd to

M2-

expr

esse

d ce

ll m

em-

bran

e as

wel

l as i

nflue

nza

A v

irio

n, in

hibi

ted

repl

icat

ion

of b

oth

aman

tadi

ne-s

ensi

tive

and

resi

stan

t infl

uenz

a A

vir

uses

in v

itro,

and

pr

otec

ted

mic

e fr

om a

leth

al in

fluen

za v

irus

cha

lleng

e. M

oreo

ver,

M2-

7A sh

owed

blo

ckin

g ac

tivity

for p

roto

n in

flux

thro

ugh

M2

ion

chan

nel.

Thes

e pi

eces

of e

vide

nce

colle

ctiv

ely

dem

onst

rate

for t

he fi

rst t

ime

that

a n

eutr

aliz

ing

antib

ody

agai

nst M

2 w

ith b

road

sp

ecifi

city

is a

chie

vabl

e, a

nd M

2-7A

may

hav

e po

tent

ial f

or c

ross

pro

tect

ion

agai

nst a

num

ber o

f var

iant

s and

subt

ypes

of i

nflue

nza

A

viru

ses.

Wei

et a

l. 20

11


478

Tabl

e 6C

. App

licat

ion

of s

ingl

e-do

mai

n an

tibo

dy fr

agm

ents

in th

erap

y: p

atho

gen

neut

rali

sati

on

Biv

alen

t nan

obod

yIn

fluen

za v

irus

In

tran

asal

ad

min

istr

atio

n

Influ

enza

A v

irus

infe

ctio

ns im

pose

a re

curr

ent a

nd g

loba

l dis

ease

bur

den.

Cur

rent

ant

ivir

als a

gain

st in

fluen

za a

re n

ot a

lway

s eff

ectiv

e. W

e as

sess

ed th

e pr

otec

tive

pote

ntia

l of m

onov

alen

t and

biv

alen

t Nan

obod

ies (

Abl

ynx)

aga

inst

cha

lleng

e w

ith th

is v

irus

. Th

ese

Nan

obod

ies w

ere

deri

ved

from

llam

as a

nd ta

rget

H5N

1 he

mag

glut

inin

. Int

rana

sal a

dmin

istr

atio

n of

Nan

obod

ies e

ffect

ivel

y co

ntro

lled

hom

olog

ous i

nflue

nza

A v

irus

repl

icat

ion.

Adm

inis

trat

ion

of N

anob

odie

s bef

ore

chal

leng

e st

rong

ly re

duce

d H

5N1

viru

s re

plic

atio

n in

the

lung

s and

pro

tect

ed m

ice

from

mor

bidi

ty a

nd m

orta

lity

afte

r a le

thal

cha

lleng

e w

ith H

5N1

viru

s. Th

e bi

vale

nt

Nan

obod

y w

as a

t lea

st 6

0-fo

ld m

ore

effec

tive

than

the

mon

oval

ent N

anob

ody

in c

ontr

ollin

g vi

rus r

eplic

atio

n. In

add

ition

, Nan

o-bo

dy th

erap

y af

ter c

halle

nge

stro

ngly

redu

ced

vira

l rep

licat

ion

and

sign

ifica

ntly

del

ayed

tim

e to

dea

th. E

pito

pe m

appi

ng re

veal

ed

that

the

VH

H N

anob

ody

bind

s to

antig

enic

site

B in

H5

hem

aggl

utin

in. B

ecau

se N

anob

odie

s are

smal

l, st

able

, and

sim

ple

to p

ro-

duce

, the

y ar

e a

prom

isin

g, n

ovel

ther

apeu

tic a

gent

aga

inst

influ

enza

.

Iban

ez e

t al

. 201

1

Mul

tim

eric

/bis

peci

fic

cons

truc

tsIn

fluen

za

For e

ffici

ent p

reve

ntio

n of

vir

al in

fect

ions

and

cro

ss p

rote

ctio

n, si

mul

tane

ous t

arge

ting

of m

ultip

le v

iral

epi

tope

s is a

pow

er-

ful s

trat

egy.

Llam

a he

avy

chai

n an

tibod

y fr

agm

ents

(VH

H) a

gain

st th

e tr

imer

ic e

nvel

ope

prot

eins

of R

espi

rato

ry S

yncy

tial V

irus

(F

usio

n pr

otei

n), R

abie

s vir

us (G

lyco

prot

ein)

and

H5N

1 In

fluen

za (H

emag

glut

inin

5) w

ere

sele

cted

from

llam

a de

rive

d im

mun

e lib

rari

es b

y ph

age

disp

lay.

Neu

tral

izin

g V

HH

reco

gniz

ing

diffe

rent

epi

tope

s in

the

rece

ptor

bin

ding

site

s on

the

spik

es w

ith a

ffini

-tie

s in

the

low

nan

omol

ar ra

nge

wer

e id

entifi

ed fo

r all

the

thre

e vi

ruse

s by

vira

l neu

tral

izat

ion

assa

ys. B

y fu

sion

of V

HH

with

var

i-ab

le li

nker

leng

ths,

mul

timer

ic c

onst

ruct

s wer

e m

ade

that

impr

oved

neu

tral

izat

ion

pote

ncie

s up

to 4

000-

fold

for R

SV, 1

500-

fold

fo

r Rab

ies v

irus

and

75-

fold

for I

nflue

nza

H5N

1. Th

e po

tenc

ies o

f the

VH

H c

onst

ruct

s wer

e si

mila

r or b

ette

r tha

n be

st p

erfo

rmin

g m

onoc

lona

l ant

ibod

ies.

The

cros

s pro

tect

ion

capa

city

aga

inst

diff

eren

t vir

al st

rain

s was

als

o im

prov

ed fo

r all

thre

e vi

ruse

s, b

oth

by

mul

tival

ent (

two

or th

ree

iden

tical

VH

H) a

nd b

ipar

atop

ic (t

wo

diffe

rent

VH

H) c

onst

ruct

s. B

y co

mbi

ning

a V

HH

neu

tral

izin

g RS

V

subt

ype

A, b

ut n

ot su

btyp

e B

with

a p

oorl

y ne

utra

lizin

g V

HH

with

hig

h affi

nity

for s

ubty

pe B

, a b

ipar

atop

ic c

onst

ruct

was

mad

e w

ith lo

w n

anom

olar

neu

tral

izin

g po

tenc

y ag

ains

t bot

h su

btyp

es. T

riva

lent

ant

i-H5N

1 V

HH

neu

tral

ized

bot

h In

fluen

za H

5N1

clad

e1 a

nd 2

in a

pse

udot

ype

assa

y an

d w

as v

ery

pote

nt in

neu

tral

izin

g th

e N

IBRG

-14

Influ

enza

H5N

1 st

rain

with

IC(5

0) o

f nin

e pi

com

olar

. Biv

alen

t and

bip

arat

opic

con

stru

cts a

gain

st R

abie

s vir

us c

ross

neu

tral

ized

bot

h 10

diff

eren

t Gen

otyp

e 1

stra

ins a

nd

Gen

otyp

e 5.

The

resu

lts sh

ow th

at m

ultim

eriz

atio

n of

VH

H fr

agm

ents

targ

etin

g m

ultip

le e

pito

pes o

n a

vira

l tri

mer

ic sp

ike

prot

ein

is a

pow

erfu

l too

l for

ant

i-vi

ral t

hera

py to

ach

ieve

“bes

t-in

-cla

ss” a

nd b

road

er n

eutr

aliz

atio

n ca

paci

ty.

Hul

tber

g et

al.

2011

HIV

vir

usC

XC

R4

Epit

ope

map

ping

The

impo

rtan

t fam

ily o

f G p

rote

in-c

oupl

ed re

cept

ors h

as so

far n

ot b

een

targ

eted

ver

y su

cces

sful

ly w

ith c

onve

ntio

nal m

onoc

lona

l an

tibod

ies.

Her

e w

e re

port

the

isol

atio

n an

d ch

arac

teri

zatio

n of

func

tiona

l VH

H-b

ased

imm

unog

lobu

lin si

ngle

var

iabl

e do

mai

ns

(or n

anob

odie

s) a

gain

st th

e ch

emok

ine

rece

ptor

CX

CR4

. Tw

o hi

ghly

sele

ctiv

e m

onov

alen

t nan

obod

ies,

238

D2

and

238D

4, w

ere

obta

ined

usi

ng a

tim

e-effi

cien

t who

le c

ell i

mm

uniz

atio

n, p

hage

dis

play

, and

cou

nter

sele

ctio

n m

etho

d. Th

e hi

ghly

sele

ctiv

e V

HH

-ba

sed

imm

unog

lobu

lin si

ngle

var

iabl

e do

mai

ns c

ompe

titiv

ely

inhi

bite

d th

e C

XC

R4-m

edia

ted

sign

alin

g an

d an

tago

nize

d th

e ch

emoa

ttra

ctan

t effe

ct o

f the

CX

CR4

liga

nd C

XC

L12.

Epi

tope

map

ping

show

ed th

at th

e tw

o na

nobo

dies

bin

d to

dis

tinct

but

pa

rtia

lly o

verl

appi

ng si

tes i

n th

e ex

trac

ellu

lar l

oops

. Sho

rt p

eptid

e lin

kage

of 2

38D

2 w

ith 2

38D

4 re

sulte

d in

sign

ifica

ntly

incr

ease

d affi

nity

for C

XC

R4 a

nd p

icom

olar

act

ivity

in a

ntic

hem

otac

tic a

ssay

s. In

tere

stin

gly,

the

mon

oval

ent n

anob

odie

s beh

aved

as n

eutr

al

anta

goni

sts,

whe

reas

the

bipa

rato

pic

nano

bodi

es a

cted

as i

nver

se a

goni

sts a

t the

con

stitu

tivel

y ac

tive

CX

CR4

-N3.

35A

. The

CX

CR4

na

nobo

dies

dis

play

ed st

rong

ant

iret

rovi

ral a

ctiv

ity a

gain

st T

cel

l-tr

opic

and

dua

l-tr

opic

HIV

-1 st

rain

s. M

oreo

ver,

the

bipa

rato

pic

nano

body

effe

ctiv

ely

mob

ilize

d C

D34

-pos

itive

stem

cel

ls in

cyn

omol

gus m

onke

ys. Th

us, t

he n

anob

ody

plat

form

may

be

high

ly

effec

tive

at g

ener

atin

g ex

trem

ely

pote

nt a

nd se

lect

ive

G p

rote

in-c

oupl

ed re

cept

or m

odul

ator

s.

Jah-

nich

en e

t al

. 201

0


479

HIV

vir

usEn

velo

pe p

rote

inC

D4

rece

ptor

Man

y of

the

neut

ralis

ing

antib

odie

s, is

olat

ed to

dat

e, d

ispl

ay li

mite

d ac

tiviti

es a

gain

st th

e gl

obal

ly m

ost p

reva

lent

HIV

-1 su

btyp

es

A a

nd C

. Ther

efor

e, th

ose

subt

ypes

are

con

side

red

to b

e an

impo

rtan

t tar

get f

or a

ntib

ody-

base

d th

erap

y. Va

riab

le d

omai

ns o

f lla

ma

heav

y ch

ain

antib

odie

s (V

HH

) hav

e so

me

supe

rior

pro

pert

ies c

ompa

red

with

cla

ssic

al a

ntib

odie

s. Th

eref

ore

we

desc

ribe

the

appl

icat

ion

of tr

imer

ic fo

rms o

f env

elop

e pr

otei

ns (E

nv),

deri

ved

from

HIV

-1 o

f sub

type

A a

nd B

/C, f

or a

pro

long

ed im

mun

izat

ion

of tw

o lla

mas

. A p

anel

of V

HH

, whi

ch in

terf

ere

with

CD

4 bi

ndin

g to

HIV

-1 E

nv w

ere

sele

cted

with

use

of p

anni

ng. Th

e re

sults

of

bind

ing

and

com

petit

ion

assa

ys to

var

ious

Env

, inc

ludi

ng a

var

iant

with

a st

abili

zed

CD

4-bi

ndin

g st

ate

(gp1

20(D

s2))

, cro

ss-c

ompe

-tit

ion

expe

rim

ents

, mat

urat

ion

anal

ysis

and

neu

tral

isat

ion

assa

ys, e

nabl

ed u

s to

clas

sify

the

sele

cted

VH

H in

to th

ree

grou

ps. Th

e V

HH

of g

roup

I w

ere

effici

ent m

ainl

y ag

ains

t vir

uses

of s

ubty

pe A

, C a

nd B

’/C. Th

e V

HH

of g

roup

II re

sem

ble

the

broa

dly

neu-

tral

isin

g an

tibod

y (b

nmA

b) b

12, n

eutr

aliz

ing

mai

nly

subt

ype

B an

d C

vir

uses

, how

ever

som

e ha

d a

broa

der n

eutr

alis

atio

n pr

ofile

. A

repr

esen

tativ

e of

the

thir

d gr

oup,

2E7

, had

an

even

hig

her n

eutr

aliz

atio

n br

eadt

h, n

eutr

aliz

ing

21 o

ut o

f the

26

test

ed st

rain

s be

long

ing

to th

e A

, A/G

, B, B

/C a

nd C

subt

ypes

. To

eval

uate

the

cont

ribu

tion

of c

erta

in a

min

o ac

ids t

o th

e po

tenc

y of

the

VH

H

a sm

all s

et o

f the

mut

ants

wer

e co

nstr

ucte

d. S

urpr

isin

gly

this

yie

lded

one

mut

ant w

ith sl

ight

ly im

prov

ed n

eutr

alis

atio

n po

tenc

y ag

ains

t 92U

G37

.A9

(sub

type

A) a

nd 9

6ZM

651.

02 (s

ubty

pe C

). Th

ese

findi

ngs a

nd th

e w

ell-k

now

n st

abili

ty o

f VH

H in

dica

te th

e po

tent

ial a

pplic

atio

n of

thes

e V

HH

as a

nti-H

IV-1

mic

robi

cide

s.

Stro

kap-

pe

et a

l. 20

12

Rot

avir

usG

astr

oent

erit

isIm

mun

ocap

ture

ele

ctro

n m

icro

scop

y

Rota

viru

s is t

he m

ain

caus

e of

vir

al g

astr

oent

eriti

s in

youn

g ch

ildre

n. Th

eref

ore,

the

deve

lopm

ent o

f ine

xpen

sive

ant

ivir

al p

rod-

ucts

for t

he p

reve

ntio

n an

d/or

trea

tmen

t of r

otav

irus

dis

ease

rem

ains

a p

rior

ity. P

revi

ousl

y w

e ha

ve sh

own

that

a re

com

bina

nt

mon

oval

ent a

ntib

ody

frag

men

t (re

ferr

ed to

as A

nti-R

otav

irus

Pro

tein

s or A

RP1)

der

ived

from

a h

eavy

cha

in a

ntib

ody

of a

llam

a im

mun

ised

with

rota

viru

s was

abl

e to

neu

tral

ise

rota

viru

s inf

ectio

n in

a m

ouse

mod

el sy

stem

. In

the

pres

ent w

ork

we

inve

stig

ated

th

e sp

ecifi

city

and

neu

tral

isin

g ac

tivity

of t

wo

llam

a an

tibod

y fr

agm

ents

, ARP

1 an

d A

RP3,

aga

inst

13

cell

cultu

re a

dapt

ed ro

tavi

-ru

s str

ains

of d

iver

se g

enot

ypes

. In

addi

tion,

imm

unoc

aptu

re e

lect

ron

mic

rosc

opy

(IEM

) was

per

form

ed to

det

erm

ine

bind

ing

of

ARP

1 to

clin

ical

isol

ates

and

cel

l cul

ture

ada

pted

stra

ins.

ARP

1 an

d A

RP3

wer

e ab

le to

neu

tral

ise

a br

oad

vari

ety

of ro

tavi

rus s

ero-

type

s/ge

noty

pes i

n vi

tro,

and

in a

dditi

on, I

EM sh

owed

spec

ific

bind

ing

to a

var

iety

of c

ell a

dapt

ed st

rain

s as w

ell a

s str

ains

from

cl

inic

al sp

ecim

ens.

Thes

e re

sults

indi

cate

d th

at th

ese

mol

ecul

es c

ould

pot

entia

lly b

e us

ed a

s im

mun

opro

phyl

actic

and

/or i

mm

uno-

ther

apeu

tic p

rodu

cts f

or th

e pr

even

tion

and/

or t

reat

men

t of i

nfec

tion

of a

bro

ad ra

nge

of c

linic

ally

rele

vant

rota

viru

s str

ains

.

Ala

din

et

al. 2

012

Plas

mod

ium

viv

axFy

blo

od g

roup

Duff

y an

tige

n re

cept

or

for

chem

okin

es

Fy b

lood

gro

up a

ntig

ens a

re c

arri

ed b

y th

e D

uffy

antig

en re

cept

or fo

r che

mok

ines

(DA

RC),

a re

d ce

lls re

cept

or fo

r Pla

smod

ium

vi

vax

broa

dly

impl

icat

ed in

hum

an h

ealth

and

dis

ease

s. R

ecom

bina

nt V

HH

s, o

r nan

obod

ies,

the

smal

lest

inta

ct a

ntig

en b

ind-

ing

frag

men

t der

ivat

ive

from

the

heav

y ch

ain-

only

ant

ibod

ies p

rese

nt in

cam

elid

s, w

ere

prep

ared

from

a d

rom

edar

y im

mun

ized

ag

ains

t DA

RC N

-ter

min

al e

xtra

cellu

lar d

omai

n an

d se

lect

ed fo

r DA

RC b

indi

ng. A

des

crib

ed V

HH

, CA

52, d

oes r

ecog

nize

nat

ive

DA

RC o

n ce

lls. I

t inh

ibits

P. v

ivax

inva

sion

of e

ryth

rocy

tes a

nd d

ispl

aces

inte

rleu

kin-

8 bo

und

to D

ARC

. The

targ

eted

epi

tope

ov

erla

ps th

e w

ell-d

efine

d D

ARC

Fy6

epi

tope

. K(D

) of C

A52

-DA

RC e

quili

briu

m is

sub-

nano

mol

ar, h

ence

idea

l to

deve

lop

diag

nos-

tic o

r the

rape

utic

com

poun

ds. I

mm

unoc

aptu

re b

y im

mob

ilize

d C

A52

yie

lded

hig

hly

puri

fied

DA

RC fr

om e

ngin

eere

d K

562

cells

. Th

is fi

rst r

epor

t on

a V

HH

with

spec

ifici

ty fo

r a re

d bl

ood

cell

prot

ein

exem

plifi

es V

HH

s’ po

tent

ialit

ies t

o ta

rget

, to

puri

fy, a

nd to

m

odul

ate

the

func

tion

of c

ellu

lar m

arke

rs.

Smol

arek

et

al.

2010


480

Tryp

anos

oma

bruc

eiVa

rian

t-sp

ecifi

c su

rfac

e gl

ycop

rote

inN

anob

ody-

med

iate

d ly

sis

The

Afr

ican

tryp

anos

ome

Tryp

anos

oma

bruc

ei, w

hich

per

sist

s with

in th

e bl

oods

trea

m o

f the

mam

mal

ian

host

, has

evo

lved

pot

ent

mec

hani

sms f

or im

mun

e ev

asio

n. S

peci

fical

ly, a

ntig

enic

var

iatio

n of

the

vari

ant-

spec

ific

surf

ace

glyc

opro

tein

(VSG

) and

a h

ighl

y ac

tive

endo

cyto

sis a

nd re

cycl

ing

of th

e su

rfac

e co

at e

ffici

ently

del

ay k

illin

g m

edia

ted

by a

nti-V

SG a

ntib

odie

s. C

onse

quen

tly, c

on-

vent

iona

l VSG

-spe

cific

inta

ct im

mun

oglo

bulin

s are

non

-try

pano

cida

l in

the

abse

nce

of c

ompl

emen

t. In

shar

p co

ntra

st, m

onov

a-le

nt a

ntig

en-b

indi

ng fr

agm

ents

, inc

ludi

ng 1

5 kD

a na

nobo

dies

(Nb)

der

ived

from

cam

elid

hea

vy-c

hain

ant

ibod

ies (

HC

Abs

) rec

og-

nizi

ng v

aria

nt-s

peci

fic V

SG e

pito

pes,

effi

cien

tly ly

se tr

ypan

osom

es b

oth

in v

itro

and

in v

ivo.

This

Nb-

med

iate

d ly

sis i

s pre

cede

d by

ver

y ra

pid

imm

obili

satio

n of

the

para

site

s, m

assi

ve e

nlar

gem

ent o

f the

flag

ella

r poc

ket a

nd m

ajor

blo

ckad

e of

end

ocyt

osis

. This

is

acc

ompa

nied

by

seve

re m

etab

olic

per

turb

atio

ns re

flect

ed b

y re

duce

d in

trac

ellu

lar A

TP-

leve

ls a

nd lo

ss o

f mito

chon

dria

l mem

-br

ane

pote

ntia

l, cu

lmin

atin

g in

cel

l dea

th. M

odifi

catio

n of

ant

i-VSG

Nbs

thro

ugh

site

-dir

ecte

d m

utag

enes

is a

nd b

y re

cons

titut

ion

into

HC

Abs

, com

bine

d w

ith u

nvei

ling

of tr

ypan

olyt

ic a

ctiv

ity fr

om in

tact

imm

unog

lobu

lins b

y pa

pain

pro

teol

ysis

, dem

onst

rate

s th

at th

e tr

ypan

olyt

ic a

ctiv

ity o

f Nbs

and

Fab

s req

uire

s low

mol

ecul

ar w

eigh

t, m

onov

alen

cy a

nd h

igh

affini

ty. W

e pr

opos

e th

at th

e ge

nera

tion

of lo

w m

olec

ular

wei

ght V

SG-s

peci

fic tr

ypan

olyt

ic n

anob

odie

s tha

t im

pede

end

ocyt

osis

offe

rs a

new

opp

ortu

nity

for

deve

lopi

ng n

ovel

tryp

anos

omia

sis t

hera

peut

ics.

In a

dditi

on, t

hese

dat

a su

gges

t tha

t the

ant

igen

-bin

ding

dom

ain

of a

n an

ti-m

icro

-bi

al a

ntib

ody

harb

ours

bio

logi

cal f

unct

iona

lity

that

is la

tent

in th

e in

tact

imm

unog

lobu

lin a

nd is

reve

aled

onl

y up

on re

leas

e of

the

antig

en-b

indi

ng fr

agm

ent.

Stijl

e-m

ans e

t al

. 201

1

Sial

idas

eTr

ypan

osom

a cr

uzi

The

sial

ic a

cid

pres

ent i

n th

e pr

otec

tive

surf

ace

muc

in c

oat o

f Try

pano

som

a cr

uzi i

s add

ed b

y a

mem

bran

e an

chor

ed tr

ans-

sial

idas

e (T

cTS)

, a m

odifi

ed si

alid

ase

that

is e

xpre

ssed

from

a la

rge

gene

fam

ily. I

n th

is w

ork,

we

anal

yzed

sing

le d

omai

n ca

mel

id

antib

odie

s pro

duce

d ag

ains

t tra

ns-s

ialid

ase.

Lla

mas

wer

e im

mun

ized

with

a re

com

bina

nt tr

ans-

sial

idas

e an

d in

hibi

tory

sing

le-

dom

ain

antib

ody

frag

men

ts w

ere

obta

ined

by

phag

e di

spla

y se

lect

ion,

taki

ng a

dvan

tage

of a

scre

enin

g st

rate

gy u

sing

an

inhi

bitio

n te

st in

stea

d of

the

clas

sic

bind

ing

assa

y. Fo

ur si

ngle

dom

ain

antib

odie

s dis

play

ing

stro

ng tr

ans-

sial

idas

e in

hibi

tion

activ

ity a

gain

st

the

reco

mbi

nant

enz

yme

wer

e id

entifi

ed. Th

ey sh

are

the

sam

e co

mpl

emen

tari

ty-d

eter

min

ing

regi

on 3

leng

th (1

7 re

sidu

es) a

nd

have

ver

y si

mila

r seq

uenc

es. Th

is re

sult

indi

cate

s tha

t the

y lik

ely

deri

ved

from

a u

niqu

e cl

one.

Pro

babl

y th

ere

is o

nly

one

stru

ctur

al

solu

tion

for t

ight

bin

ding

inhi

bito

ry a

ntib

odie

s aga

inst

the

TcT

S us

ed fo

r im

mun

izat

ion.

To

our s

urpr

ise,

this

sing

le d

omai

n an

ti-bo

dy th

at in

hibi

ts th

e re

com

bina

nt T

cTS,

faile

d to

inhi

bit t

he e

nzym

atic

act

ivity

pre

sent

in p

aras

ite e

xtra

cts.

Ana

lysi

s of i

ndiv

idua

l re

com

bina

nt tr

ans-

sial

idas

es sh

owed

that

enz

ymes

exp

ress

ed fr

om d

iffer

ent g

enes

wer

e in

hibi

ted

to d

iffer

ent e

xten

ts (f

rom

8 to

98

%) b

y th

e lla

ma

antib

odie

s. A

min

o ac

id c

hang

es a

t key

pos

ition

s are

like

ly to

be

resp

onsi

ble

for t

he d

iffer

ence

s in

inhi

bitio

n fo

und

amon

g th

e re

com

bina

nt e

nzym

es. Th

ese

resu

lts su

gges

t tha

t the

pre

senc

e of

a la

rge

and

dive

rse

tran

s-si

alid

ase

fam

ily m

ight

be

requ

ired

to p

reve

nt th

e in

hibi

tory

resp

onse

aga

inst

this

ess

entia

l enz

yme

and

mig

ht th

us c

onst

itute

a n

ovel

stra

tegy

of T

. cru

zi

to e

vade

the

host

imm

une

syst

em.

Ratie

r et

al. 2

008


481

Ant

igen

var

iati

onVa

rian

t sur

face

gl

ycop

rote

ins

Cry

ptic

/con

serv

ed

epit

opes

Tryp

anos

omos

is th

erap

yA

ntib

ody:

bet

a-la

ctam

ase

fusi

on

Ant

igen

var

iatio

n is

a su

cces

sful

def

ense

syst

em a

dopt

ed b

y se

vera

l inf

ectio

us a

gent

s to

evad

e th

e ho

st im

mun

e re

spon

se. Th

e pr

inci

ple

of th

is d

efen

se st

rate

gy in

the

Afr

ican

tryp

anos

ome

para

digm

invo

lves

a d

ense

pac

king

of v

aria

nt su

rfac

e gl

ycop

rote

ins

(VSG

) exp

osin

g on

ly h

ighl

y va

riab

le a

nd im

mun

o-do

min

ant e

pito

pes t

o th

e im

mun

e sy

stem

, whe

reas

con

serv

ed e

pito

pes b

ecom

e in

acce

ssib

le fo

r lar

ge m

olec

ules

. Red

ucin

g th

e si

ze o

f bin

ders

that

targ

et th

e co

nser

ved,

less

-im

mun

ogen

ic, c

rypt

ic V

SG e

pito

pes

form

s an

obvi

ous s

olut

ion

to c

omba

t the

se p

aras

ites.

This

goa

l was

ach

ieve

d by

intr

oduc

ing

drom

edar

y H

eavy

-cha

in a

ntib

odie

s. W

e fo

und

that

onl

y th

ese

uniq

ue a

ntib

odie

s rec

ogni

ze e

pito

pes c

omm

on to

mul

tiple

VSG

cla

sses

. Aft

er p

hage

dis

play

of t

heir

an

tigen

-bin

ding

repe

rtoi

re, w

e is

olat

ed a

sing

le d

omai

n an

tibod

y fr

agm

ent w

ith h

igh

spec

ifici

ty fo

r the

con

serv

ed A

sn-li

nked

ca

rboh

ydra

te o

f VSG

. In

shar

p co

ntra

st to

labe

led

conc

anav

alin

-A th

at st

ains

onl

y th

e fla

gella

r poc

ket w

here

car

bohy

drat

es a

re

acce

ssib

le b

ecau

se o

f les

s den

se V

SG p

acki

ng, t

he si

ngle

dom

ain

bind

er st

ains

the

entir

e su

rfac

e of

via

ble

para

site

s, ir

resp

ectiv

e of

th

e V

SG ty

pe e

xpre

ssed

. This

cor

robo

rate

s the

idea

that

smal

l ant

ibod

y fr

agm

ents

, but

not

larg

er le

ctin

s or c

onve

ntio

nal a

ntib

ody

frag

men

ts, a

re a

ble

to p

enet

rate

the

dens

e V

SG c

oat t

o ta

rget

thei

r epi

tope

. The

diag

nost

ic p

oten

tial o

f thi

s fluo

resc

ently

labe

led

bind

er w

as p

rove

n by

the

dire

ct, s

elec

tive,

and

sens

itive

det

ectio

n of

par

asite

s in

bloo

d sm

ears

. The

empl

oym

ent o

f thi

s bin

der a

s a

mol

ecul

ar re

cogn

ition

uni

t in

imm

unot

oxin

s des

igne

d fo

r try

pano

som

osis

ther

apy

beco

mes

feas

ible

as w

ell.

This

was

illu

stra

ted

by

the

spec

ific

tryp

anol

ysis

indu

ced

by a

n an

tibod

y: b

eta-

lact

amas

e fu

sion

act

ivat

ing

a pr

odru

g.

Stijl

e-m

ans e

t al

. 200

4

Cry

ptic

epi

tope

sA

pica

l mem

bran

e

anti

gen

1Pl

asm

odiu

m fa

lcip

arur

mEx

tens

ive

poly

mor

phis

mIg

NA

R

Api

cal m

embr

ane

antig

en 1

(AM

A1)

is e

ssen

tial f

or in

vasi

on o

f ery

thro

cyte

s and

hep

atoc

ytes

by

Plas

mod

ium

par

asite

s and

is

a le

adin

g m

alar

ial v

acci

ne c

andi

date

. Alth

ough

con

vent

iona

l ant

ibod

ies t

o A

MA

1 ca

n pr

even

t suc

h in

vasi

on, e

xten

sive

pol

y-m

orph

ism

s with

in su

rfac

e-ex

pose

d lo

ops m

ay li

mit

the

abili

ty o

f the

se A

MA

1 in

duce

d an

tibod

ies t

o pr

otec

t aga

inst

all

para

site

ge

noty

pes.

Usi

ng a

n A

MA

1-sp

ecifi

c Ig

NA

R si

ngle

-var

iabl

e-do

mai

n an

tibod

y, w

e pe

rfor

med

targ

eted

mut

agen

esis

and

sele

ctio

n ag

ains

t AM

A1

from

thre

e P.

falc

ipar

urm

stra

ins.

We

pres

ent c

ocry

stal

stru

ctur

es o

f tw

o an

tibod

y-A

MA

1 co

mpl

exes

whi

ch re

veal

ex

tend

ed Ig

NA

R C

DR3

loop

s pen

etra

ting

deep

into

a h

ydro

phob

ic c

left

on

the

antig

en su

rfac

e an

d co

ntac

ting

resi

dues

con

serv

ed

acro

ss p

aras

ite sp

ecie

s. C

ompa

riso

n of

a se

ries

of a

ffini

ty-e

nhan

cing

mut

atio

ns a

llow

ed d

isse

ctio

n of

thei

r rel

ativ

e co

ntri

butio

ns

to b

indi

ng k

inet

ics a

nd c

orre

latio

n w

ith in

hibi

tion

of e

ryth

rocy

te in

vasi

on. Th

ese

findi

ngs p

rovi

de in

sigh

ts in

to m

echa

nism

s of

sing

le-d

omai

n an

tibod

y bi

ndin

g, a

nd m

ay e

nabl

e de

sign

of r

eage

nts t

arge

ting

othe

rwis

e cr

yptic

epi

tope

s in

path

ogen

ant

igen

s.

Hen

der-

son

et a

l. 20

07


482

Tabl

e 6D

. App

licat

ion

of s

ingl

e-do

mai

n an

tibo

dy fr

agm

ents

in th

erap

y: in

trac

ellu

lar

expr

essi

on o

f sin

gle-

dom

ain

anti

body

frag

men

ts

Apo

ptos

isC

aspa

se 3

Neu

rode

gene

rati

ve

dise

ases

Can

cer

Apo

ptos

is, o

r pro

gram

med

cel

l dea

th, i

s an

esse

ntia

l pro

cess

affe

ctin

g ho

meo

stas

is o

f cel

l gro

wth

, dev

elop

men

t, an

d th

e el

imin

a-tio

n of

dam

aged

or d

ange

rous

cel

ls. I

napp

ropr

iate

cel

l dea

th c

ause

d by

oxi

dativ

e st

ress

has

bee

n im

plic

ated

in th

e de

velo

pmen

t of

neu

rode

gene

rativ

e di

seas

es su

ch a

s Alz

heim

er’s,

Par

kins

on’s,

and

stro

ke. O

n th

e ot

her h

and,

a d

efec

t in

the

cell

deat

h pr

oces

s le

ads t

o th

e de

velo

pmen

t of c

ance

r. Fo

r exa

mpl

e, th

e m

ain

play

er o

f apo

ptos

is, p

53, i

s def

ectiv

e in

man

y of

the

hum

an c

ance

rs.

Apo

ptos

is is

regu

late

d by

the

inte

rpla

y of

pro

-apo

ptot

ic a

nd a

nti-a

popt

otic

pro

tein

s fro

m th

e Bc

l-2 fa

mily

and

cas

pase

s. In

par

-tic

ular

, spe

cific

mod

ulat

ors o

f the

act

ivity

of C

aspa

se 3

cou

ld b

e ve

ry im

port

ant f

or th

e de

velo

pmen

t of t

hera

pies

for d

isea

ses s

uch

as n

euro

dege

nera

tion

and

canc

er. I

n th

is st

udy,

two

V(H

)Hs s

peci

fic to

Cas

pase

3 (V

hhC

asp3

1 an

d V

hhC

asp3

2) w

ere

isol

ated

fr

om a

hea

vy c

hain

ant

ibod

y va

riab

le d

omai

n (V

(H)H

) pha

ge d

ispl

ay li

brar

y an

d te

sted

for t

heir

apo

ptos

is-m

odul

atin

g eff

ects

. W

hile

Vhh

Cas

p31

was

foun

d to

be

anta

goni

stic

tow

ards

Cas

pase

3, V

hhC

asp3

2 w

as a

goni

stic

. Fur

ther

mor

e, w

hen

expr

esse

d as

in

trab

odie

s in

SHSY

-5Y

neur

obla

stom

a ce

lls, V

hhC

asp3

1 re

nder

ed c

ells

resi

stan

t to

oxid

ativ

e-st

ress

-ind

uced

apo

ptos

is, w

here

as

Vhh

Cas

p32

resu

lted

in a

popt

osis

. Thes

e V

(H)H

ant

agon

ist a

nd a

goni

st o

f apo

ptos

is c

ould

hav

e po

tent

ial f

or th

e de

velo

pmen

t of

ther

apeu

tics f

or n

euro

dege

nera

tive

dise

ases

and

can

cer,

resp

ectiv

ely.

McG

oni-

gal e

t al.

2009

Prot

ein

kina

se C

Enzy

me

mod

ulat

orTh

e 10

isoz

ymes

of t

he p

rote

in k

inas

e C

(PK

C) f

amily

can

hav

e di

ffere

nt ro

les o

n th

e sa

me

biol

ogic

al p

roce

ss, m

akin

g is

ozym

e sp

ecifi

c an

alys

is o

f fun

ctio

n cr

ucia

l. C

urre

ntly

, onl

y fe

w p

harm

acol

ogic

al c

ompo

unds

with

mod

erat

e is

ozym

e sp

ecifi

c eff

ects

exi

st

thus

ham

peri

ng re

sear

ch in

to in

divi

dual

PK

C is

ozym

es. Th

e an

tigen

bin

ding

regi

ons o

f cam

elid

sing

le c

hain

ant

ibod

ies (

VH

Hs)

co

uld

prov

ide

a so

lutio

n fo

r obt

aini

ng P

KC

isoz

yme

spec

ific

mod

ulat

ors.

In th

e pr

esen

t stu

dy, w

e ha

ve su

cces

sful

ly se

lect

ed a

nd

char

acte

rize

d PK

C e

psilo

n sp

ecifi

c V

HH

ant

ibod

ies f

rom

two

imm

une

VH

H li

brar

ies u

sing

pha

ge d

ispl

ay. Th

e V

HH

s wer

e sh

own

to e

xclu

sive

ly b

ind

to P

KC

eps

ilon

in E

LISA

and

imm

unop

reci

pita

tion

stud

ies.

Str

ikin

gly,

five

of th

e V

HH

s had

an

effec

t on

PKC

eps

ilon

kina

se a

ctiv

ity in

vitr

o. V

HH

s A10

, C1

and

D1

incr

ease

d PK

C e

psilo

n ki

nase

act

ivity

in a

con

cent

ratio

n-de

pend

ent

man

ner (

EC(5

0) v

alue

s: 21

2–31

0 nM

), w

here

as E

6 an

d G

8 in

hibi

ted

PKC

eps

ilon

activ

ity (I

C(5

0) v

alue

s: 10

3–23

3 nM

). N

one

of

thes

e V

HH

s had

an

effec

t on

the

activ

ity o

f the

oth

er n

ovel

PK

C is

ozym

es P

KC

del

ta a

nd P

KC

thet

a. T

o ou

r kno

wle

dge,

thes

e an

tibod

ies a

re th

e fir

st d

escr

ibed

VH

H a

ctiv

ator

s and

inhi

bito

rs fo

r a p

rote

in k

inas

e. F

urth

erm

ore,

the

deve

lopm

ent o

f PK

C e

psi-

lon

spec

ific

mod

ulat

ors i

s an

impo

rtan

t con

trib

utio

n to

PK

C re

sear

ch.

Paal

anen

et

al.

2011

IRF-

1Tu

mor

sup

pres

sor

Mf1

dom

ain

Tran

scri

ptio

nal a

ctiv

ity

IRF-

1 is

a tu

mor

supp

ress

or p

rote

in th

at a

ctiv

ates

gen

e ex

pres

sion

from

a ra

nge

of p

rom

oter

s in

resp

onse

to st

imul

i spa

nnin

g vi

ral

infe

ctio

n to

DN

A d

amag

e. S

tudi

es o

n th

e po

st-t

rans

latio

nal r

egul

atio

n of

IRF-

1 ha

ve b

een

ham

pere

d by

a la

ck o

f sui

tabl

e bi

o-ch

emic

al to

ols c

apab

le o

f tar

getin

g th

e en

doge

nous

pro

tein

. In

this

stud

y, ph

age

disp

lay

tech

nolo

gy w

as u

sed

to d

evel

op a

mon

o-cl

onal

nan

obod

y ta

rget

ing

the

C-t

erm

inal

Mf1

dom

ain

(res

idue

s 301

–325

) of I

RF-1

. Int

race

llula

r exp

ress

ion

of th

e na

nobo

dy

dem

onst

rate

d th

at th

e tr

ansc

ript

iona

l act

ivity

of I

RF-1

is c

onst

rain

ed b

y th

e M

f1 d

omai

n as

nan

obod

y bi

ndin

g ga

ve a

n in

crea

se in

ex

pres

sion

from

IRF-

1-re

spon

sive

pro

mot

ers o

f up

to 8

-fol

d. F

urth

erm

ore,

Mf1

-dir

ecte

d na

nobo

dies

hav

e re

veal

ed a

n un

expe

cted

fu

nctio

n fo

r thi

s dom

ain

in li

miti

ng th

e ra

te a

t whi

ch th

e IR

F-1

prot

ein

is d

egra

ded.

Thus

, the

incr

ease

in IR

F-1

tran

scri

ptio

nal

activ

ity o

bser

ved

on n

anob

ody

bind

ing

is a

ccom

pani

ed b

y a

sign

ifica

nt re

duct

ion

in th

e ha

lf-lif

e of

the

prot

ein.

In su

ppor

t of t

he

data

obt

aine

d us

ing

nano

bodi

es, a

sing

le p

oint

mut

atio

n (P

325A

) inv

olvi

ng th

e C

-ter

min

al re

sidu

e of

IRF-

1 ha

s bee

n id

entifi

ed,

whi

ch re

sults

in g

reat

er tr

ansc

ript

iona

l act

ivity

and

a si

gnifi

cant

incr

ease

in th

e ra

te o

f deg

rada

tion.

The

resu

lts p

rese

nted

her

e su

ppor

t a ro

le fo

r the

Mf1

dom

ain

in li

miti

ng b

oth

IRF-

1-de

pend

ent t

rans

crip

tion

and

the

rate

of I

RF-1

turn

over

. In

addi

tion,

the

data

hig

hlig

ht a

rout

e fo

r act

ivat

ion

of d

owns

trea

m g

enes

in th

e IR

F-1

tum

or su

ppre

ssor

pat

hway

usi

ng b

iolo

gics

.

Mol

ler e

t al

. 201

0


483

Hep

atit

is B

HB

eAg

IgN

AR

Intr

acel

lula

r pr

ecor

e pr

otei

n

The

Hep

atiti

s B v

irus

pre

core

pro

tein

is p

roce

ssed

in th

e en

dopl

asm

ic re

ticul

um (E

R) in

to se

cret

ed h

epat

itis B

e a

ntig

en (H

BeA

g),

whi

ch a

cts a

s an

imm

une

tole

roge

n to

est

ablis

h ch

roni

c in

fect

ion.

Dow

nreg

ulat

ion

of se

cret

ed H

BeA

g sh

ould

impr

ove

clin

ical

ou

tcom

e, a

s pat

ient

s who

effe

ctiv

ely

resp

ond

to c

urre

nt tr

eatm

ents

(IFN

-alp

ha) h

ave

sign

ifica

ntly

low

er se

rum

HBe

Ag

leve

ls.

Her

e, w

e de

scri

be a

nov

el re

agen

t, a

sing

le v

aria

ble

dom

ain

(V(N

AR)

) of t

he sh

ark

imm

unog

lobu

lin n

ew a

ntig

en re

cept

or (I

gNA

R)

antib

odie

s. V

(NA

RS) p

osse

ss a

dvan

tage

s in

stab

ility

, siz

e (s

imila

r to

14 k

Da)

and

cry

ptic

epi

tope

reco

gniti

on c

ompa

red

to c

onve

n-tio

nal a

ntib

odie

s. Th

e V

(NA

R) d

omai

n di

spla

yed

biol

ogic

ally

use

ful a

ffini

ty fo

r rec

ombi

nant

and

nat

ive

HBe

Ag,

and

reco

gnis

ed

a un

ique

con

form

atio

nal e

pito

pe. T

o as

sess

ther

apeu

tic p

oten

tial i

n ta

rget

ing

intr

acel

lula

r pre

core

pro

tein

to re

duce

secr

eted

H

BeA

g, th

e V

(NA

R) w

as e

ngin

eere

d fo

r ER-

targ

eted

in v

itro

deliv

ery

to fu

nctio

n as

an

intr

acel

lula

r ant

ibod

y (in

trab

ody)

. In

vitr

o da

ta fr

om H

BV/p

reco

re h

epat

ocyt

e ce

ll lin

es d

emon

stra

ted

effec

tive

intr

abod

y re

gula

tion

of p

reco

re/H

BeA

g.

Wal

sh e

t al

. 201

1

Hep

atit

is B

vir

usH

BcA

gH

BeA

g

Six

sing

le-d

omai

n an

tibod

ies (

VH

Hs)

targ

etin

g th

e co

re a

ntig

en o

f HBV

(HBc

Ag)

hav

e be

en g

ener

ated

and

thre

e of

thes

e bo

und

stro

ngly

to H

BcA

g of

bot

h su

btyp

e ay

w a

nd a

dw. Th

ese

thre

e V

HH

s wer

e st

udie

d as

intr

abod

ies d

irec

ted

tow

ards

the

nucl

eus

or th

e cy

topl

asm

of a

hep

atom

a ce

ll lin

e th

at w

as c

o-tr

ansf

ecte

d w

ith H

BV. A

spec

kled

stai

ning

of H

BcA

g w

as o

bser

ved

in th

e cy

topl

asm

of c

ells

tran

sfec

ted

with

nuc

leot

ropi

c V

HH

intr

abod

ies.

Mor

eove

r, an

incr

ease

d in

trac

ellu

lar a

ccum

ulat

ion

of h

epat

itis

B e

antig

en (H

BeA

g) a

nd a

com

plet

e di

sapp

eara

nce

of in

trac

ellu

lar H

BcA

g si

gnal

wer

e ob

serv

ed w

ith n

ucle

ar ta

rget

ed H

BcA

g-sp

ecifi

c V

HH

s. Th

ese

resu

lts su

gges

t tha

t HBc

Ag-

spec

ific

VH

Hs t

arge

ted

to th

e nu

cleu

s affe

ct H

BcA

g an

d H

BeA

g ex

pres

sion

and

tr

affick

ing

in H

BV-t

rans

fect

ed h

epat

ocyt

es.

Serr

uys e

t al

. 201

0

HIV

-1 v

irus

Rev

pro

tein

Rep

licat

ion

Mul

tim

eriz

atio

n do

mai

n

The

hum

an im

mun

odefi

cien

cy v

irus

, typ

e 1

(HIV

-1)-

enco

ded

Rev

prot

ein

is e

ssen

tial f

or th

e ex

pres

sion

of l

ate

vira

l mRN

As.

Rev

fo

rms a

larg

e or

gani

zed

mul

timer

ic p

rote

in-p

rote

in c

ompl

ex o

n th

e Re

v re

spon

se e

lem

ent o

f the

se v

iral

mRN

A sp

ecie

s and

tran

s-po

rts t

hem

from

the

nucl

eus t

o th

e cy

topl

asm

, exp

loiti

ng th

e C

RM1-

med

iate

d ce

llula

r mac

hine

ry. H

ere

we

repo

rt th

e se

lect

ion

of

a na

nobo

dy, d

eriv

ed fr

om a

llam

a he

avy-

chai

n on

ly a

ntib

ody,

that

effi

cien

tly b

lock

s the

ass

embl

y of

Rev

mul

timer

s. Th

e na

nobo

dy

inhi

bits

HIV

-1 re

plic

atio

n in

cel

ls a

nd sp

ecifi

cally

supp

ress

es th

e Re

v-de

pend

ent e

xpre

ssio

n of

par

tially

splic

ed a

nd u

nspl

iced

H

IV-1

RN

A. I

n H

IV-s

usce

ptib

le c

ells

, thi

s nan

obod

y th

us h

as p

oten

tial a

s an

effec

tive

anti-

HIV

age

nt u

sing

gen

etic

imm

uniz

atio

n st

rate

gies

. Its

bin

ding

site

was

map

ped

to R

ev re

sidu

es L

ys-2

0 an

d Ty

r-23

loca

ted

in th

e N

-ter

min

al a

lpha

-hel

ical

mul

timer

izat

ion

dom

ain.

In th

e pr

esen

ce o

f thi

s nan

obod

y, w

e ob

serv

ed a

n ac

cum

ulat

ion

of d

imer

ic R

ev sp

ecie

s, su

ppor

ting

a he

ad-t

o-he

ad/t

ail-

to-t

ail m

olec

ular

mod

el fo

r Rev

ass

embl

y. Th

e re

sults

indi

cate

that

the

olig

omer

ic a

ssem

bly

of R

ev fo

llow

s an

orde

red

step

wis

e pr

oces

s and

iden

tify

a ne

w e

pito

pe w

ithin

Rev

that

cou

ld g

uide

stra

tegi

es fo

r the

dev

elop

men

t of n

ovel

HIV

inhi

bito

rs.

Ver-

cruy

sse

et

al. 2

010

AD

P-ri

bosy

lati

onSa

lmon

ella

SpvB

toxi

n

AD

P-ri

bosy

latio

n of

hos

t cel

l pro

tein

s is a

com

mon

mod

e of

cel

l int

oxic

atio

n by

pat

hoge

nic

bact

eria

l tox

ins.

Ant

ibod

ies i

nduc

ed

by im

mun

izat

ion

with

inac

tivat

ed A

DP-

ribo

syla

ting

toxi

ns p

rovi

de e

ffici

ent p

rote

ctio

n in

cas

e of

som

e se

cret

ed to

xins

, e.g

., di

ph-

ther

ia a

nd p

ertu

ssis

toxi

ns. H

owev

er, o

ther

AD

P-ri

bosy

latin

g to

xins

, suc

h as

Sal

mon

ella

Spv

B to

xin,

are

secr

eted

dir

ectly

from

the

Salm

onel

la-c

onta

inin

g va

cuol

e in

to th

e cy

toso

l of t

arge

t cel

ls v

ia th

e SP

I-2

enco

ded

bact

eria

l typ

e II

I sec

retio

n sy

stem

, and

thus

ar

e in

acce

ssib

le to

con

vent

iona

l ant

ibod

ies.

Sm

all-m

olec

ule

AD

P-ri

bosy

latio

n in

hibi

tors

are

frau

ght w

ith p

oten

tial s

ide

effec

ts

caus

ed b

y in

hibi

tion

of e

ndog

enou

s AD

P-ri

bosy

ltran

sfer

ases

. Her

e, w

e re

port

the

deve

lopm

ent o

f a si

ngle

-dom

ain

antib

ody

from

an

imm

uniz

ed ll

ama

that

blo

cks t

he c

apac

ity o

f Spv

B to

AD

P-ri

bosy

late

act

in a

t a m

olar

ratio

of 1

: 1.

The

sing

le-d

omai

n an

tibod

y, w

hen

expr

esse

d as

an

intr

abod

y, eff

ectiv

ely

prot

ecte

d ce

lls fr

om th

e cy

toto

xic

activ

ity o

f a tr

ansl

ocat

ion-

com

pete

nt c

him

eric

C

2IN

-C/S

pvB

toxi

n. T

rans

fect

ed c

ells

wer

e al

so p

rote

cted

aga

inst

cyt

oske

leta

l alte

ratio

ns in

duce

d by

wild

-typ

e Sp

vB-e

xpre

ssin

g st

rain

s of S

alm

onel

la. Th

is p

roof

of p

rinc

iple

pav

es th

e w

ay fo

r dev

elop

ing

new

ant

idot

es a

gain

st in

trac

ellu

lar t

oxin

s.

Alz

ogar

ay

et a

l. 20

11


484

Bot

ulin

um n

euro

toxi

nsPr

otea

se in

hibi

tor

Botu

linum

neu

roto

xins

(BoN

Ts) f

unct

ion

by d

eliv

erin

g a

prot

ease

to n

euro

nal c

ells

that

cle

ave

SNA

RE p

rote

ins a

nd in

activ

ate

neur

otra

nsm

itter

exo

cyto

sis.

Sm

all (

14 k

Da)

bin

ding

dom

ains

spec

ific

for t

he p

rote

ase

of B

oNT

sero

type

s A o

r B w

ere

sele

cted

fr

om li

brar

ies o

f hea

vy c

hain

onl

y an

tibod

y do

mai

ns (V

HH

s or n

anob

odie

s) c

lone

d fr

om im

mun

ized

alp

acas

. Sev

eral

VH

Hs b

ind

the

BoN

T p

rote

ases

with

hig

h affi

nity

(K(D

) nea

r 1 n

M) a

nd in

clud

e po

tent

inhi

bito

rs o

f BoN

T/A

pro

teas

e ac

tivity

(K(i)

nea

r 1

nM).

The

VH

Hs r

etai

n th

eir b

indi

ng sp

ecifi

city

and

inhi

bito

ry fu

nctio

ns w

hen

expr

esse

d w

ithin

mam

mal

ian

neur

onal

cel

ls a

s in

trab

odie

s. A

VH

H in

hibi

tor o

f BoN

T/A

pro

teas

e w

as a

ble

to p

rote

ct n

euro

nal c

ell S

NA

P25

prot

ein

from

cle

avag

e fo

llow

ing

into

xica

tion

with

BoN

T/A

hol

otox

in. Th

ese

resu

lts d

emon

stra

te th

at V

HH

dom

ains

hav

e po

tent

ial a

s com

pone

nts o

f the

rape

utic

ag

ents

for r

ever

sal o

f bot

ulis

m in

toxi

catio

n.

Trem

blay

et

al.

2010

Intr

acel

lula

r pr

otei

nsE.

col

iIn

trac

ellu

lar

deliv

ery

Mol

ecul

ar s

yrin

ge

Intr

acel

lula

r pro

tein

s hav

e a

grea

t pot

entia

l as t

arge

ts fo

r the

rape

utic

ant

ibod

ies (

Abs

) but

the

plas

ma

mem

bran

e pr

even

ts-

acce

ss to

thes

e an

tigen

s. A

b fr

agm

ents

and

IgG

s are

sele

cted

and

eng

inee

red

in E

. col

i and

this

mic

roor

gani

sm m

ay b

e al

so a

n id

eal v

ecto

r for

thei

r int

race

llula

r del

iver

y. In

this

wor

k w

e de

mon

stra

te th

at si

ngle

-dom

ain

Ab

(sdA

bs) c

an b

e en

gine

ered

to b

e in

ject

ed in

to h

uman

cel

ls b

y E.

col

i bac

teri

a ca

rryi

ng m

olec

ular

syri

nges

ass

embl

ed b

y a

type

III p

rote

in se

cret

ion

syst

em (T

3SS)

. Th

e in

ject

ed sd

Abs

acc

umul

ate

in th

e cy

topl

asm

of H

eLa

cells

at l

evel

s ca.

10(

5)–1

0(6)

mol

ecul

es p

er c

ell a

nd th

eir f

unct

iona

l-ity

is sh

own

by th

e is

olat

ion

of sd

Ab-

antig

en c

ompl

exes

. Inj

ectio

n of

sdA

bs d

oes n

ot re

quir

e ba

cter

ial i

nvas

ion

or th

e tr

ansf

er

of g

enet

ic m

ater

ial.

Thes

e re

sults

are

pro

of-o

f-pr

inci

ple

for t

he c

apac

ity o

f E. c

oli b

acte

ria

to d

irec

tly d

eliv

er in

trac

ellu

lar s

dAbs

(in

trab

odie

s) in

to h

uman

cel

ls fo

r ana

lytic

al a

nd th

erap

eutic

pur

pose

s.

Blan

co-

Tori

bio

et

al. 2

010

Tabl

e 6E

. App

licat

ion

of s

ingl

e-do

mai

n an

tibo

dy fr

agm

ents

in th

erap

y: o

ral a

dmin

istr

atio

n of

sin

gle-

dom

ain

anti

body

frag

men

ts

Ther

mal

sta

bilit

yTr

ypsi

nEx

trem

e pH

Clo

stri

dium

diffi

cile

Toxi

n A

The

extr

eme

pH a

nd p

rote

ase-

rich

env

iron

men

t of t

he u

pper

gas

troi

ntes

tinal

trac

t is a

maj

or o

bsta

cle

faci

ng o

rally

-adm

inis

tere

d pr

otei

n th

erap

eutic

s, in

clud

ing

antib

odie

s. Th

roug

h pr

otei

n en

gine

erin

g, se

vera

l Clo

stri

dium

diffi

cile

toxi

n A

-spe

cific

hea

vy c

hain

an

tibod

y va

riab

le d

omai

ns (V

(H)H

s) w

ere

expr

esse

d w

ith a

n ad

ditio

nal d

isul

fide

bond

by

intr

oduc

ing

Ala

/Gly

54C

ys a

nd Il

e78C

ys

mut

atio

ns. M

utan

t ant

ibod

ies w

ere

com

pare

d to

thei

r wild

-typ

e co

unte

rpar

ts w

ith re

spec

t to

expr

essi

on y

ield

, non

-agg

rega

tion

stat

us, a

ffini

ty fo

r tox

in A

, cir

cula

r dic

hroi

sm (C

D) s

truc

tura

l sig

natu

res,

ther

mal

stab

ility

, pro

teas

e re

sist

ance

, and

toxi

n A

-neu

-tr

aliz

ing

capa

city

. The

mut

ant V

(H)H

s wer

e fo

und

to b

e w

ell e

xpre

ssed

, alth

ough

with

low

er y

ield

s com

pare

d to

wild

-typ

e co

un-

terp

arts

, wer

e no

n-ag

greg

atin

g m

onom

ers,

reta

ined

low

nM

affi

nity

for t

oxin

A, a

lbei

t the

maj

ority

show

ed so

mew

hat r

educ

ed

affini

ty c

ompa

red

to w

ild-t

ype

coun

terp

arts

, and

wer

e ca

pabl

e of

in v

itro

toxi

n A

neu

tral

izat

ion

in c

ell-b

ased

ass

ays.

Far

-UV

and

ne

ar-U

V C

D sp

ectr

osco

py c

onsi

sten

tly sh

owed

shift

s in

peak

inte

nsity

and

sele

ctiv

e pe

ak m

inim

a fo

r wild

-typ

e an

d m

utan

t V(H

)H

pair

s; ho

wev

er, t

he o

vera

ll C

D p

rofil

e re

mai

ned

very

sim

ilar.

A si

gnifi

cant

incr

ease

in th

e th

erm

al u

nfol

ding

mid

poin

t tem

pera

ture

w

as o

bser

ved

for a

ll m

utan

ts a

t bot

h ne

utra

l and

aci

dic

pH. D

iges

tion

of th

e V

(H)H

s with

the

maj

or g

astr

oint

estin

al p

rote

ases

, at

biol

ogic

ally

rele

vant

con

cent

ratio

ns, r

evea

led

a si

gnifi

cant

incr

ease

in p

epsi

n re

sist

ance

for a

ll m

utan

ts a

nd a

n in

crea

se in

chy

mot

-ry

psin

resi

stan

ce fo

r the

maj

ority

of m

utan

ts. M

utan

t V(H

)H tr

ypsi

n re

sist

ance

was

sim

ilar t

o th

at o

f wild

-typ

e V

(H)H

s, a

lthou

gh

the

tryp

sin

resi

stan

ce o

f one

V(H

)H m

utan

t was

sign

ifica

ntly

redu

ced.

Ther

efor

e, th

e in

trod

uctio

n of

a se

cond

dis

ulfid

e bo

nd in

th

e hy

drop

hobi

c co

re n

ot o

nly

incr

ease

s V(H

)H th

erm

al st

abili

ty a

t neu

tral

pH

, as p

revi

ousl

y sh

own,

but

als

o re

pres

ents

a g

ener

ic

stra

tegy

to in

crea

se V

(H)H

stab

ility

at l

ow p

H a

nd im

part

pro

teas

e re

sist

ance

, with

onl

y m

inor

per

turb

atio

ns in

targ

et b

indi

ng

affini

ties.

Thes

e ar

e al

l des

irab

le c

hara

cter

istic

s for

the

desi

gn o

f pro

tein

-bas

ed o

ral t

hera

peut

ics.

Hus

sack

et

al.

2011

a


485

Clo

stri

dium

diffi

cile

Nos

ocom

ial i

nfec

tion

Nor

th A

mer

ica

Exot

oxin

A/B

Cel

l rec

epto

r bi

ndin

g do

mai

n

Clo

stri

dium

diffi

cile

is a

lead

ing

caus

e of

nos

ocom

ial i

nfec

tion

in N

orth

Am

eric

a an

d a

cons

ider

able

cha

lleng

e to

hea

lthca

re p

rofe

s-si

onal

s in

hosp

itals

and

nurs

ing

hom

es. Th

e G

ram

-pos

itive

bac

teri

um p

rodu

ces t

wo

high

mol

ecul

ar w

eigh

t exo

toxi

ns, t

oxin

A (T

cdA

) an

d to

xin

B (T

cdB)

, whi

ch a

re th

e m

ajor

vir

ulen

ce fa

ctor

s res

pons

ible

for C

. diffi

cile

-ass

ocia

ted

dise

ase

and

are

targ

ets f

or C

. diffi

cile

-as

soci

ated

dis

ease

ther

apy.

Her

e, re

com

bina

nt si

ngle

-dom

ain

antib

ody

frag

men

ts (V

(H)H

s), w

hich

spec

ifica

lly ta

rget

the

cell

rece

ptor

bi

ndin

g do

mai

ns o

f Tcd

A o

r Tcd

B, w

ere

isol

ated

from

an

imm

une

llam

a ph

age

disp

lay

libra

ry a

nd c

hara

cter

ized

. Fou

r V(H

)Hs (

A4.

2,

A5.

1, A

20.1

, and

A26

.8),

all s

how

n to

reco

gniz

e co

nfor

mat

iona

l epi

tope

s, w

ere

pote

nt n

eutr

aliz

ers o

f the

cyt

opat

hic

effec

ts o

f tox

in

A o

n fib

robl

ast c

ells

in a

n in

vitr

o as

say.

The

neut

raliz

ing

pote

ncy

was

furt

her e

nhan

ced

whe

n V

(H)H

s wer

e ad

min

iste

red

in p

aire

d or

trip

let c

ombi

natio

ns a

t the

sam

e ov

eral

l V(H

)H c

once

ntra

tion,

sugg

estin

g re

cogn

ition

of n

onov

erla

ppin

g Tc

dA e

pito

pes.

Biac

ore

epito

pe m

appi

ng e

xper

imen

ts re

veal

ed th

at so

me

syne

rgis

tic c

ombi

natio

ns c

onsi

sted

of V

(H)H

s rec

ogni

zing

ove

rlapp

ing

epito

pes,

an in

dica

tion

that

fact

ors o

ther

than

mer

e ep

itope

blo

ckin

g ar

e re

spon

sibl

e fo

r the

incr

ease

d ne

utra

lizat

ion.

Fur

ther

bin

ding

ass

ays

reve

aled

Tcd

A-s

peci

fic V

(H)H

s neu

tral

ized

toxi

n A

by

bind

ing

to si

tes o

ther

than

the

carb

ohyd

rate

bin

ding

poc

ket o

f the

toxi

n. W

ith

favo

rabl

e ch

arac

teri

stic

s suc

h as

hig

h pr

oduc

tion

yiel

d, p

oten

t tox

in n

eutr

aliz

atio

n, a

nd in

trin

sic

stab

ility

, the

se V

(H)H

s are

att

ract

ive

syst

emic

ther

apeu

tics b

ut a

re m

ore

so a

s ora

l the

rape

utic

s in

the

dest

abili

zing

env

ironm

ent o

f the

gas

troi

ntes

tinal

trac

t.

Hus

sack

et

al.

2011

b

Infla

mm

ator

y bo

wel

di

seas

eTu

mor

-nec

rosi

s fa

ctor

Lact

ococ

cus

lact

isSe

cret

ion

Infla

mm

ator

y bo

wel

dis

ease

(IBD

) is a

chr

onic

infla

mm

ator

y ga

stro

inte

stin

al d

isor

der.

Syst

emic

trea

tmen

t of I

BD p

atie

nts w

ith

anti-

tum

or n

ecro

sis f

acto

r (T

NF)

-alp

ha a

ntib

odie

s has

pro

ven

to b

e a

high

ly p

rom

isin

g ap

proa

ch, b

ut se

vera

l dra

wba

cks r

emai

n,

incl

udin

g si

de e

ffect

s rel

ated

to sy

stem

ic a

dmin

istr

atio

n an

d hi

gh c

ost o

f tre

atm

ent.

Lact

ococ

cus l

actis

was

eng

inee

red

to se

cret

e m

onov

alen

t and

biv

alen

t mur

ine

(m) T

NF-

neut

raliz

ing

Nan

obod

ies a

s the

rape

utic

pro

tein

s. Th

ese

ther

apeu

tic p

rote

ins a

re d

eriv

ed

from

frag

men

ts o

f hea

vy-c

hain

cam

elid

ant

ibod

ies a

nd a

re m

ore

stab

le th

an c

onve

ntio

nal a

ntib

odie

s. L

. lac

tis-s

ecre

ted

anti-

mT

NF

Nan

obod

ies n

eutr

aliz

ed m

TN

F in

vitr

o. D

aily

ora

l adm

inis

trat

ion

of N

anob

ody-

secr

etin

g L.

lact

is re

sulte

d in

loca

l del

iver

y of

ant

i-m

TN

F N

anob

odie

s at t

he c

olon

and

sign

ifica

ntly

redu

ced

infla

mm

atio

n in

mic

e w

ith d

extr

an su

lfate

sodi

um (D

SS)-

indu

ced

chro

nic

colit

is. I

n ad

ditio

n, th

is a

ppro

ach

was

als

o su

cces

sful

in im

prov

ing

esta

blis

hed

ente

roco

litis

in in

terl

euki

n 10

(IL1

0)(–

/–) m

ice.

Fi

nally

, L. l

actis

-sec

rete

d an

ti-m

TN

F N

anob

odie

s did

not

inte

rfer

e w

ith sy

stem

ic S

alm

onel

la in

fect

ion

in c

oliti

c IL

10(–

/–) m

ice.

In

conc

lusi

on, t

his r

epor

t det

ails

a n

ew th

erap

eutic

app

roac

h fo

r tre

atm

ent o

f chr

onic

col

itis,

invo

lvin

g in

situ

secr

etio

n of

ant

i-m

TN

F N

anob

odie

s by

oral

ly a

dmin

iste

red

L. la

ctis

bac

teri

a. Th

erap

eutic

app

licat

ion

of th

ese

engi

neer

ed b

acte

ria

coul

d ev

entu

ally

lead

to

mor

e eff

ectiv

e an

d sa

fer m

anag

emen

t of I

BD in

hum

ans.

Vand

en-

brou

cke

et a

l. 20

10

F4 fi

mbr

iae

E. c

oli

Dia

rrho

eaPr

oteo

lyti

c st

abili

tyG

astr

ic fl

uid

We

prev

ious

ly d

emon

stra

ted

that

ora

l app

licat

ion

of th

e re

com

bina

nt si

ngle

-dom

ain

antib

ody

frag

men

t (V

HH

) clo

ne K

609,

di

rect

ed a

gain

st E

sche

rich

ia c

oli F

4 fim

bria

e, re

duce

d E.

col

i-ind

uced

dia

rrho

ea in

pig

lets

, but

onl

y at

hig

h V

HH

dos

es. W

e ha

ve

now

show

n th

at a

larg

e po

rtio

n of

the

oral

ly a

pplie

d K

609

VH

H is

pro

teol

ytic

ally

deg

rade

d in

the

stom

ach.

Str

inge

nt se

lect

ion

for p

rote

olyt

ic st

abili

ty id

entifi

ed se

ven

VH

Hs w

ith 7

- to

138-

fold

incr

ease

d st

abili

ty a

fter

in v

itro

incu

batio

n in

gas

tric

flui

d. B

y D

NA

shuffl

ing

we

obta

ined

four

clo

nes w

ith a

furt

her 1

.5- t

o 3-

fold

incr

ease

d in

vitr

o st

abili

ty. Th

ese

VH

Hs d

iffer

ed b

y at

mos

t te

n am

ino

acid

resi

dues

from

eac

h ot

her a

nd K

609

that

wer

e sc

atte

red

over

the

VH

H se

quen

ce a

nd d

id n

ot o

verl

ap w

ith p

redi

cted

pr

otea

se c

leav

age

site

s. Th

e m

ost s

tabl

e cl

one,

K92

2, re

tain

ed 4

1% a

ctiv

ity a

fter

incu

batio

n in

gas

tric

flui

d an

d 90

% in

jeju

nal fl

uid.

O

ral a

pplic

atio

n of

K92

2 to

pig

lets

con

firm

ed it

s im

prov

ed p

rote

olyt

ic st

abili

ty. I

n ad

ditio

n, K

922

boun

d to

F4

fimbr

iae

with

hig

her

affini

ty a

nd in

hibi

ted

fimbr

ial a

dhes

ion

at lo

wer

VH

H c

once

ntra

tions

. K92

2 is

thus

a p

rom

isin

g ca

ndid

ate

for p

reve

ntio

n of

pig

let

diar

rhoe

a. F

urth

erm

ore,

our

find

ings

cou

ld g

uide

sele

ctio

n an

d im

prov

emen

t by

gene

tic e

ngin

eeri

ng o

f oth

er re

com

bina

nt a

nti-

body

frag

men

ts fo

r ora

l use

.

Har

m-

sen

et a

l. 20

06


486

Rot

avir

usD

iarr

hoea

Dev

elop

ing

coun

trie

sPa

ssiv

e im

mun

othe

rapy

Tran

sfor

med

lact

obac

illi

Back

grou

nd. R

otav

irus

-indu

ced

diar

rhea

pos

es a

wor

ldw

ide

med

ical

pro

blem

in c

ausi

ng su

bsta

ntia

l mor

bidi

ty a

nd m

orta

lity

amon

g ch

ildre

n in

dev

elop

ing

coun

trie

s. W

e th

eref

ore

deve

lope

d a

syst

em fo

r pas

sive

imm

unot

hera

py in

whi

ch re

com

bina

nt

lact

obac

illi c

onst

itutiv

ely

expr

ess n

eutr

aliz

ing

vari

able

dom

ain

of ll

ama

heav

y-ch

ain

(VH

H) a

ntib

ody

frag

men

ts a

gain

st ro

tavi

rus.

Met

hods

. VH

H w

ere

expr

esse

d in

Lac

toba

cillu

s par

acas

ei, i

n bo

th se

cret

ed a

nd c

ell s

urfa

ce-a

ncho

red

form

s. E

lect

ron

mic

rosc

opy

was

use

d to

inve

stig

ate

the

bind

ing

effica

cy o

f VH

H-e

xpre

ssin

g la

ctob

acill

i. To

inve

stig

ate

the

in v

ivo

func

tion

of V

HH

-exp

ress

ing

lact

obac

illi,

a m

ouse

pup

mod

el o

f rot

avir

us in

fect

ion

was

use

d. R

esul

ts. E

ffici

ent b

indi

ng o

f the

VH

H a

ntib

ody

frag

men

ts to

ro

tavi

rus w

as sh

own

by e

nzym

e-lin

ked

imm

unos

orbe

nt a

ssay

and

scan

ning

ele

ctro

n m

icro

scop

y. V

HH

frag

men

ts e

xpre

ssed

by

lact

obac

illi c

onfe

rred

a si

gnifi

cant

redu

ctio

n in

infe

ctio

n in

cel

l cul

ture

s. W

hen

adm

inis

tere

d or

ally

, lac

toba

cilli

-pro

duci

ng su

rfac

e-ex

pres

sed

VH

H m

arke

dly

shor

tene

d di

seas

e du

ratio

n, se

veri

ty, a

nd v

iral

load

in a

mou

se m

odel

of r

otav

irus

-ind

uced

dia

rrhe

a w

hen

adm

inis

tere

d bo

th fr

esh

and

in a

free

ze-d

ried

form

. Con

clus

ions

. Tra

nsfo

rmed

lact

obac

illi m

ay fo

rm th

e ba

sis o

f a n

ovel

form

of

prop

hyla

ctic

trea

tmen

t aga

inst

rota

viru

s inf

ectio

ns a

nd o

ther

dia

rrhe

al d

isea

ses.

Pant

et

al. 2

006

Tabl

e 6F

. App

licat

ion

of s

ingl

e-do

mai

n an

tibo

dy fr

agm

ents

in th

erap

y: p

reve

ntio

n of

am

yloi

d pl

aque

form

atio

n an

d pr

otei

n ag

greg

atio

n

Bet

a-am

yloi

d tr

ansm

i-gr

atio

n effi

cien

cyA

lzhe

imer

’s di

seas

eC

ereb

ral a

myl

oid

an

giop

athy

Prev

ious

ly se

lect

ed a

myl

oid

beta

reco

gniz

ing

heav

y ch

ain

antib

ody

frag

men

ts (V

HH

) affi

nity

bin

ders

der

ived

from

the

Cam

e lid

hea

vy

chai

n an

tibod

y re

pert

oire

wer

e te

sted

for t

heir

prop

ensi

ty to

cro

ss th

e bl

ood-

brai

n ba

rrie

r (BB

B) u

sing

an

esta

blis

hed

in v

itro

BBB

co-

cultu

re sy

stem

. Of a

ll te

sted

VH

H, n

i3A

show

ed h

ighe

st tr

ansm

igra

tion

effici

ency

whi

ch is

, in

part

, fac

ilita

ted

by a

thre

e am

ino

acid

su

bstit

utio

ns in

its N

-ter

min

al d

omai

n. A

dditi

onal

stud

ies i

ndic

ated

that

the

mec

hani

sm o

f tra

nsce

llula

r pas

sage

of n

i3A

is b

y ac

tive

tran

spor

t. A

s VH

H n

i3A

com

bine

s the

abi

lity

to re

cogn

ize

amyl

oid

beta

and

to c

ross

the

BBB,

it h

as p

oten

tial a

s a to

ol fo

r non

-inva

-si

ve in

viv

o im

agin

g an

d as

effi

cien

t loc

al d

rug

targ

etin

g m

oiet

y in

pat

ient

s suff

erin

g fr

om c

ereb

ral a

myl

oido

sis s

uch

as A

lzhe

imer

’s di

seas

e (A

D) a

nd c

ereb

ral a

myl

oid

angi

opat

hy (C

AA

).

Rutg

ers

et a

l. 20

11

Bet

a-am

yloi

dA

lzhe

imer

’s di

seas

ePr

oteo

lyti

c na

nobo

dyA

ggre

gati

on

Dep

ositi

on o

f bet

a-am

yloi

d (A

bet

a) is

con

side

red

an im

port

ant e

arly

eve

nt in

the

path

ogen

esis

of A

lzhe

imer

’s di

seas

e (A

D),

and

redu

ctio

n of

A b

eta

leve

ls in

the

brai

n co

uld

be a

via

ble

ther

apeu

tic a

ppro

ach.

A p

oten

tially

non

infla

mm

ator

y ro

ute

to fa

cilit

ate

clea

r-an

ce a

nd re

duce

toxi

city

of A

bet

a is

to d

egra

de th

e pe

ptid

e us

ing

prot

eoly

tic n

anob

odie

s. H

ere

we

show

that

a p

rote

olyt

ic n

anob

ody

engi

neer

ed to

cle

ave

A b

eta

at it

s alp

ha-s

ecre

tase

site

has

pot

entia

l the

rape

utic

val

ue. Th

e A

sec-

1A p

rote

olyt

ic n

anob

ody,

deri

ved

from

a p

aren

t cat

alyt

ic li

ght c

hain

ant

ibod

y, pr

even

ts a

ggre

gatio

n of

mon

omer

ic A

bet

a, in

hibi

ts fu

rthe

r agg

rega

tion

of p

refo

rmed

A

beta

agg

rega

tes,

and

redu

ces A

bet

a-in

duce

d cy

toto

xici

ty to

war

d a

hum

an n

euro

blas

tom

a ce

ll lin

e. Th

e na

nobo

dy a

lso

redu

ces t

oxic

-ity

indu

ced

by o

vere

xpre

ssio

n of

the

hum

an a

myl

oid

prec

urso

r pro

tein

(APP

) in

a C

hine

se h

amst

er o

vary

(CH

O) c

ell l

ine

by c

leav

ing

APP

at t

he a

lpha

-sec

reta

se si

te w

hich

pre

clud

es fo

rmat

ion

of A

bet

a. T

arge

ted

prot

eoly

sis o

f APP

and

A b

eta

with

cat

alyt

ic n

anob

od-

ies r

epre

sent

s a n

ovel

ther

apeu

tic a

ppro

ach

for t

reat

ing

AD

whe

re p

oten

tially

har

mfu

l sid

e eff

ects

can

he

min

imiz

ed.

Kas

-tu

rira

n-ga

n et

al.

2010

Am

yloi

doge

nic

lyso

zym

eA

lpha

-syn

ucle

inPa

rkin

son’

s di

seas

eSe

lf-a

ssoc

iati

on

Nan

obod

ies a

re si

ngle

cha

in a

ntib

odie

s tha

t are

uni

quel

y pr

oduc

ed in

Cam

elid

ae, e

.g. c

amel

s and

llam

as. Th

ey h

ave

the

desi

r-ab

le fe

atur

es o

f sm

all s

izes

(Mw

< 1

4 kD

a) a

nd h

igh

affini

ties a

gain

st a

ntig

ens (

Kd

sim

ilar t

o nM

), m

akin

g th

em id

eal a

s str

uctu

ral

prob

es fo

r bio

med

ical

ly re

leva

nt m

otifs

bot

h in

vitr

o an

d in

viv

o. W

e ha

ve p

revi

ousl

y sh

own

that

nan

obod

y bi

ndin

g to

am

yloi

do-

geni

c hu

man

lyso

zym

e va

rian

ts c

an e

ffect

ivel

y in

hibi

t the

ir a

ggre

gatio

n, th

e pr

oces

s tha

t is a

t the

ori

gin

of sy

stem

ic a

myl

oid

dis-

ease

. Her

e w

e re

port

the

NM

R as

sign

men

ts o

f a n

ew n

anob

ody,

term

ed N

bSyn

2, w

hich

reco

gnis

es th

e C

-ter

min

us o

f the

intr

insi

-ca

lly d

isor

dere

d pr

otei

n, h

uman

alp

ha-s

ynuc

lein

(aS)

, who

se a

berr

ant s

elf-

asso

ciat

ion

is im

plic

ated

in P

arki

nson

’s di

seas

e.

Vuch

elen

et

al.

2009


487

Rot

avir

usD

iarr

hoea

Dev

elop

ing

coun

trie

sPa

ssiv

e im

mun

othe

rapy

Tran

sfor

med

lact

obac

illi

Back

grou

nd. R

otav

irus

-indu

ced

diar

rhea

pos

es a

wor

ldw

ide

med

ical

pro

blem

in c

ausi

ng su

bsta

ntia

l mor

bidi

ty a

nd m

orta

lity

amon

g ch

ildre

n in

dev

elop

ing

coun

trie

s. W

e th

eref

ore

deve

lope

d a

syst

em fo

r pas

sive

imm

unot

hera

py in

whi

ch re

com

bina

nt

lact

obac

illi c

onst

itutiv

ely

expr

ess n

eutr

aliz

ing

vari

able

dom

ain

of ll

ama

heav

y-ch

ain

(VH

H) a

ntib

ody

frag

men

ts a

gain

st ro

tavi

rus.

Met

hods

. VH

H w

ere

expr

esse

d in

Lac

toba

cillu

s par

acas

ei, i

n bo

th se

cret

ed a

nd c

ell s

urfa

ce-a

ncho

red

form

s. E

lect

ron

mic

rosc

opy

was

use

d to

inve

stig

ate

the

bind

ing

effica

cy o

f VH

H-e

xpre

ssin

g la

ctob

acill

i. To

inve

stig

ate

the

in v

ivo

func

tion

of V

HH

-exp

ress

ing

lact

obac

illi,

a m

ouse

pup

mod

el o

f rot

avir

us in

fect

ion

was

use

d. R

esul

ts. E

ffici

ent b

indi

ng o

f the

VH

H a

ntib

ody

frag

men

ts to

ro

tavi

rus w

as sh

own

by e

nzym

e-lin

ked

imm

unos

orbe

nt a

ssay

and

scan

ning

ele

ctro

n m

icro

scop

y. V

HH

frag

men

ts e

xpre

ssed

by

lact

obac

illi c

onfe

rred

a si

gnifi

cant

redu

ctio

n in

infe

ctio

n in

cel

l cul

ture

s. W

hen

adm

inis

tere

d or

ally

, lac

toba

cilli

-pro

duci

ng su

rfac

e-ex

pres

sed

VH

H m

arke

dly

shor

tene

d di

seas

e du

ratio

n, se

veri

ty, a

nd v

iral

load

in a

mou

se m

odel

of r

otav

irus

-ind

uced

dia

rrhe

a w

hen

adm

inis

tere

d bo

th fr

esh

and

in a

free

ze-d

ried

form

. Con

clus

ions

. Tra

nsfo

rmed

lact

obac

illi m

ay fo

rm th

e ba

sis o

f a n

ovel

form

of

prop

hyla

ctic

trea

tmen

t aga

inst

rota

viru

s inf

ectio

ns a

nd o

ther

dia

rrhe

al d

isea

ses.

Pant

et

al. 2

006

Tabl

e 6F

. App

licat

ion

of s

ingl

e-do

mai

n an

tibo

dy fr

agm

ents

in th

erap

y: p

reve

ntio

n of

am

yloi

d pl

aque

form

atio

n an

d pr

otei

n ag

greg

atio

n

Bet

a-am

yloi

d tr

ansm

i-gr

atio

n effi

cien

cyA

lzhe

imer

’s di

seas

eC

ereb

ral a

myl

oid

an

giop

athy

Prev

ious

ly se

lect

ed a

myl

oid

beta

reco

gniz

ing

heav

y ch

ain

antib

ody

frag

men

ts (V

HH

) affi

nity

bin

ders

der

ived

from

the

Cam

e lid

hea

vy

chai

n an

tibod

y re

pert

oire

wer

e te

sted

for t

heir

prop

ensi

ty to

cro

ss th

e bl

ood-

brai

n ba

rrie

r (BB

B) u

sing

an

esta

blis

hed

in v

itro

BBB

co-

cultu

re sy

stem

. Of a

ll te

sted

VH

H, n

i3A

show

ed h

ighe

st tr

ansm

igra

tion

effici

ency

whi

ch is

, in

part

, fac

ilita

ted

by a

thre

e am

ino

acid

su

bstit

utio

ns in

its N

-ter

min

al d

omai

n. A

dditi

onal

stud

ies i

ndic

ated

that

the

mec

hani

sm o

f tra

nsce

llula

r pas

sage

of n

i3A

is b

y ac

tive

tran

spor

t. A

s VH

H n

i3A

com

bine

s the

abi

lity

to re

cogn

ize

amyl

oid

beta

and

to c

ross

the

BBB,

it h

as p

oten

tial a

s a to

ol fo

r non

-inva

-si

ve in

viv

o im

agin

g an

d as

effi

cien

t loc

al d

rug

targ

etin

g m

oiet

y in

pat

ient

s suff

erin

g fr

om c

ereb

ral a

myl

oido

sis s

uch

as A

lzhe

imer

’s di

seas

e (A

D) a

nd c

ereb

ral a

myl

oid

angi

opat

hy (C

AA

).

Rutg

ers

et a

l. 20

11

Bet

a-am

yloi

dA

lzhe

imer

’s di

seas

ePr

oteo

lyti

c na

nobo

dyA

ggre

gati

on

Dep

ositi

on o

f bet

a-am

yloi

d (A

bet

a) is

con

side

red

an im

port

ant e

arly

eve

nt in

the

path

ogen

esis

of A

lzhe

imer

’s di

seas

e (A

D),

and

redu

ctio

n of

A b

eta

leve

ls in

the

brai

n co

uld

be a

via

ble

ther

apeu

tic a

ppro

ach.

A p

oten

tially

non

infla

mm

ator

y ro

ute

to fa

cilit

ate

clea

r-an

ce a

nd re

duce

toxi

city

of A

bet

a is

to d

egra

de th

e pe

ptid

e us

ing

prot

eoly

tic n

anob

odie

s. H

ere

we

show

that

a p

rote

olyt

ic n

anob

ody

engi

neer

ed to

cle

ave

A b

eta

at it

s alp

ha-s

ecre

tase

site

has

pot

entia

l the

rape

utic

val

ue. Th

e A

sec-

1A p

rote

olyt

ic n

anob

ody,

deri

ved

from

a p

aren

t cat

alyt

ic li

ght c

hain

ant

ibod

y, pr

even

ts a

ggre

gatio

n of

mon

omer

ic A

bet

a, in

hibi

ts fu

rthe

r agg

rega

tion

of p

refo

rmed

A

beta

agg

rega

tes,

and

redu

ces A

bet

a-in

duce

d cy

toto

xici

ty to

war

d a

hum

an n

euro

blas

tom

a ce

ll lin

e. Th

e na

nobo

dy a

lso

redu

ces t

oxic

-ity

indu

ced

by o

vere

xpre

ssio

n of

the

hum

an a

myl

oid

prec

urso

r pro

tein

(APP

) in

a C

hine

se h

amst

er o

vary

(CH

O) c

ell l

ine

by c

leav

ing

APP

at t

he a

lpha

-sec

reta

se si

te w

hich

pre

clud

es fo

rmat

ion

of A

bet

a. T

arge

ted

prot

eoly

sis o

f APP

and

A b

eta

with

cat

alyt

ic n

anob

od-

ies r

epre

sent

s a n

ovel

ther

apeu

tic a

ppro

ach

for t

reat

ing

AD

whe

re p

oten

tially

har

mfu

l sid

e eff

ects

can

he

min

imiz

ed.

Kas

-tu

rira

n-ga

n et

al.

2010

Am

yloi

doge

nic

lyso

zym

eA

lpha

-syn

ucle

inPa

rkin

son’

s di

seas

eSe

lf-a

ssoc

iati

on

Nan

obod

ies a

re si

ngle

cha

in a

ntib

odie

s tha

t are

uni

quel

y pr

oduc

ed in

Cam

elid

ae, e

.g. c

amel

s and

llam

as. Th

ey h

ave

the

desi

r-ab

le fe

atur

es o

f sm

all s

izes

(Mw

< 1

4 kD

a) a

nd h

igh

affini

ties a

gain

st a

ntig

ens (

Kd

sim

ilar t

o nM

), m

akin

g th

em id

eal a

s str

uctu

ral

prob

es fo

r bio

med

ical

ly re

leva

nt m

otifs

bot

h in

vitr

o an

d in

viv

o. W

e ha

ve p

revi

ousl

y sh

own

that

nan

obod

y bi

ndin

g to

am

yloi

do-

geni

c hu

man

lyso

zym

e va

rian

ts c

an e

ffect

ivel

y in

hibi

t the

ir a

ggre

gatio

n, th

e pr

oces

s tha

t is a

t the

ori

gin

of sy

stem

ic a

myl

oid

dis-

ease

. Her

e w

e re

port

the

NM

R as

sign

men

ts o

f a n

ew n

anob

ody,

term

ed N

bSyn

2, w

hich

reco

gnis

es th

e C

-ter

min

us o

f the

intr

insi

-ca

lly d

isor

dere

d pr

otei

n, h

uman

alp

ha-s

ynuc

lein

(aS)

, who

se a

berr

ant s

elf-

asso

ciat

ion

is im

plic

ated

in P

arki

nson

’s di

seas

e.

Vuch

elen

et

al.

2009

Agg

rega

tion

Alp

ha-s

ynuc

lein

Fibr

illar

am

yloi

d

stru

ctur

esPa

rkin

son’

s di

seas

e

The

aggr

egat

ion

of th

e in

trin

sica

lly d

isor

dere

d pr

otei

n al

pha-

synu

clei

n to

form

fibr

illar

am

yloi

d st

ruct

ures

is in

timat

ely

asso

ciat

ed

with

a v

arie

ty o

f neu

rolo

gica

l dis

orde

rs, m

ost n

otab

ly P

arki

nson

’s di

seas

e. Th

e m

olec

ular

mec

hani

sm o

f alp

ha-s

ynuc

lein

agg

rega

-tio

n an

d to

xici

ty is

not

yet

und

erst

ood

in a

ny d

etai

l, no

t lea

st b

ecau

se o

f the

pau

city

of s

truc

tura

l pro

bes t

hrou

gh w

hich

to st

udy

the

beha

vior

of s

uch

a di

sord

ered

syst

em. H

ere,

we

desc

ribe

an

inve

stig

atio

n in

volv

ing

a si

ngle

-dom

ain

cam

elid

ant

ibod

y, N

bSyn

2,

sele

cted

by

phag

e di

spla

y te

chni

ques

to b

ind

to a

lpha

-syn

ucle

in, i

nclu

ding

the

expl

orat

ion

of it

s effe

cts o

n th

e in

vitr

o ag

greg

atio

n of

th

e pr

otei

n un

der a

var

iety

of c

ondi

tions

. We

show

usi

ng is

othe

rmal

cal

orim

etri

c m

etho

ds th

at N

bSyn

2 bi

nds s

peci

fical

ly to

mon

o-m

eric

alp

ha-s

ynuc

lein

with

nan

omol

ar a

ffini

ty a

nd b

y m

eans

of N

MR

spec

tros

copy

that

it in

tera

cts w

ith th

e fo

ur C

-ter

min

al re

sidu

es

of th

e pr

otei

n. Th

is la

tter fi

ndin

g is

con

firm

ed b

y th

e de

term

inat

ion

of a

cry

stal

stru

ctur

e of

NbS

yn2

boun

d to

a p

eptid

e en

com

pass

ing

the

nine

C-t

erm

inal

resi

dues

of a

lpha

-syn

ucle

in. Th

e N

bSyn

2 : a

lpha

-syn

ucle

in in

tera

ctio

n is

med

iate

d m

ainl

y by

side

-cha

in in

tera

c-tio

ns w

hile

wat

er m

olec

ules

cro

ss-li

nk th

e m

ain-

chai

n at

oms o

f alp

ha-s

ynuc

lein

to a

tom

s of N

bSyn

2, a

feat

ure

we

belie

ve c

ould

be

impo

rtan

t in

intr

insi

cally

dis

orde

red

prot

ein

inte

ract

ions

mor

e ge

nera

lly. Th

e ag

greg

atio

n be

havi

or o

f alp

ha-s

ynuc

lein

at p

hysi

olog

ical

pH

, inc

ludi

ng th

e m

orph

olog

y of

the

resu

lting

fibr

illar

stru

ctur

es, i

s rem

arka

bly

unaff

ecte

d by

the

pres

ence

of N

bSyn

2 an

d in

deed

we

show

that

NbS

yn2

bind

s str

ongl

y to

the

aggr

egat

ed a

s wel

l as t

o th

e so

lubl

e fo

rms o

f alp

ha-s

ynuc

lein

. Thes

e re

sults

giv

e st

rong

sup-

port

to th

e co

njec

ture

that

the

C-t

erm

inal

regi

on o

f the

pro

tein

is n

ot d

irect

ly in

volv

ed in

the

mec

hani

sm o

f agg

rega

tion

and

sugg

est

that

bin

ding

of N

bSyn

2 co

uld

be a

use

ful p

robe

for t

he id

entifi

catio

n of

alp

ha-s

ynuc

lein

agg

rega

tion

in v

itro

and

poss

ibly

in v

ivo.

De

Gen

st

et a

l. 20

10

Agg

rega

tion

Ocu

loph

aryn

geal

m

uscu

lar

dyst

roph

yA

lani

ne e

xpan

sion

PAB

PN1

Ocu

loph

aryn

geal

mus

cula

r dys

trop

hy is

cau

sed

by sm

all a

lani

ne e

xpan

sion

s in

poly

aden

ylat

e bi

ndin

g pr

otei

n nu

clea

r 1 (P

ABP

N1)

pr

otei

n re

sulti

ng in

its i

ntra

nucl

ear a

ccum

ulat

ion

in sk

elet

al m

uscl

e. 3

F5 ll

ama

antib

ody

spec

ifica

lly in

terf

eres

with

the

PABP

N1

aggr

egat

ion

proc

ess i

n vi

tro

and

in v

ivo.

To

unde

rsta

nd th

e st

ruct

ural

bas

is fo

r its

epi

tope

reco

gniti

on w

e m

appe

d th

e bi

ndin

g in

terf

ace

of 3

F5 w

ith P

ABP

N1

and

prov

ide

a st

ruct

ural

mod

el o

f the

3F5

-PA

BPN

1 co

mpl

ex. W

e sh

ow th

at 3

F5 c

ompl

emen

tari

ty

dete

rmin

ing

regi

ons c

reat

e a

cavi

ty in

whi

ch P

ABP

N1

alph

a-he

lix d

omai

n re

side

s by

invo

lvin

g cr

itica

l res

idue

s pre

viou

sly

impl

i-ca

ted

in th

e ag

greg

atio

n pr

oces

s. Th

ese

resu

lts m

ay in

crea

se o

ur u

nder

stan

ding

of t

he P

ABP

N1

aggr

egat

ion

mec

hani

sm a

nd th

e th

erap

eutic

pot

entia

l of 3

F5.

Impa

gli-

azzo

et

al. 2

010


488

Tabl

e 6G

. App

licat

ion

of s

ingl

e-do

mai

n an

tibo

dy fr

agm

ents

in th

erap

y: m

ulti

spec

ific

and

mul

tifu

ncti

onal

con

stru

cts

Pent

abod

y Ve

roto

xin

B-s

ubun

itE.

col

iTh

erm

osta

bilit

yPr

otea

se r

esis

tanc

e

We

desc

ribe

a n

ovel

type

of m

olec

ule

in w

hich

sing

le-d

omai

n an

tibod

ies (

sdA

bs) i

sola

ted

from

a n

aive

llam

a si

ngle

dom

ain

anti-

body

libr

ary

are

linke

d to

an

olig

omer

izat

ion

dom

ain

to g

ener

ate

high

-avi

dity

ant

igen

-bin

ding

reag

ents

. An

sdA

b is

fuse

d to

the

B-su

buni

t of E

sche

rich

ia c

oli v

erot

oxin

, or s

higa

-like

toxi

n, w

hich

self-

asse

mbl

es to

form

a h

omop

enta

mer

and

resu

lts in

sim

ul-

tane

ous s

dAb

pent

amer

izat

ion

and

intr

oduc

tion

of a

vidi

ty. M

olec

ular

mod

elin

g in

dica

ted

that

this

fusi

on p

rote

in (P

DB:

10J

F),

term

ed p

enta

body

, has

stru

ctur

al fl

exib

ility

for b

indi

ng to

surf

ace-

pres

ente

d an

tigen

. In

the

inst

ance

of a

n sd

Ab

spec

ific

for a

pe

ptid

e an

tigen

, pen

tam

eriz

atio

n re

sulte

d in

a d

ram

atic

incr

ease

in fu

nctio

nal a

ffini

ty fo

r im

mob

ilize

d an

tigen

. The

pent

abod

y w

as

expr

esse

d in

hig

h yi

eld

in E

. col

i in

a no

n-ag

greg

ated

stat

e, a

nd e

xhib

ited

exce

llent

ther

mos

tabi

lity

and

prot

ease

resi

stan

ce. Th

is

tech

nolo

gy p

rovi

des a

rela

tivel

y ra

pid

mea

ns o

f gen

erat

ing

nove

l ant

igen

-bin

ding

mol

ecul

es th

at b

ind

stro

ngly

to im

mob

ilize

d an

ti-ge

n. It

is e

xpec

ted

that

pen

tava

lent

sdA

bs w

ill h

ave

gene

ral a

pplic

abili

ty in

pro

teom

ics,

imm

unoc

hem

ical

stai

ning

, can

cer d

iagn

osis

an

d ot

her a

pplic

atio

ns in

whi

ch a

ntig

ens a

re p

rese

nted

mul

tival

ently

.

Zha

ng e

t al

. 200

4

Bip

arat

opic

nan

obod

yEp

ider

mal

gro

wth

fact

or

rece

ptor

Ant

ican

cer

ther

apeu

tics

The

epid

erm

al g

row

th fa

ctor

rece

ptor

(EG

FR) h

as b

een

show

n to

be

a va

lid c

ance

r tar

get f

or a

ntib

ody-

base

d th

erap

y. A

t pre

sent

, se

vera

l ant

i-EG

FR m

onoc

lona

l ant

ibod

ies h

ave

been

succ

essf

ully

use

d, su

ch a

s cet

uxim

ab a

nd m

atuz

umab

. X-r

ay c

ryst

allo

grap

hy

data

show

that

thes

e an

tibod

ies b

ind

to d

iffer

ent e

pito

pes o

n th

e ec

to-d

omai

n of

EG

FR, p

rovi

ding

a ra

tiona

le fo

r the

com

bine

d us

e of

thes

e tw

o an

tibod

y sp

ecifi

citie

s. W

e ha

ve p

revi

ousl

y re

port

ed o

n th

e su

cces

sful

isol

atio

n of

ant

agon

istic

ant

i-EG

FR n

anob

od-

ies.

In o

ur st

udy,

we

aim

ed to

impr

ove

the

effica

cy o

f the

se m

olec

ules

by

com

bini

ng n

anob

odie

s with

spec

ifici

ties s

imila

r to

both

ce

tuxi

mab

and

mat

uzum

ab in

to a

sing

le b

ipar

atop

ic m

olec

ule.

Car

eful

ly d

esig

ned

phag

e na

nobo

dy se

lect

ions

resu

lted

in tw

o se

ts

of n

anob

odie

s tha

t spe

cific

ally

blo

cked

the

bind

ing

of e

ither

mat

uzum

ab o

r cet

uxim

ab to

EG

FR a

nd th

at d

id n

ot c

ompe

te fo

r eac

h ot

hers

’ bin

ding

. A c

ombi

natio

n of

nan

obod

ies f

rom

bot

h ep

itope

gro

ups i

nto

the

bipa

rato

pic

nano

body

CO

NA

N-1

was

show

n to

bl

ock

EGFR

act

ivat

ion

mor

e effi

cien

tly th

an m

onov

alen

t or b

ival

ent (

mon

ospe

cific

) nan

obod

ies.

In a

dditi

on, t

his b

ipar

atop

ic n

ano-

body

pot

ently

inhi

bite

d EG

F-de

pend

ent c

ell p

rolif

erat

ion.

Impo

rtan

tly, i

n an

in v

ivo

mod

el o

f ath

ymic

mic

e be

arin

g A

431

xeno

-gr

afts

, CO

NA

N-1

inhi

bite

d tu

mou

r out

grow

th w

ith a

n al

mos

t sim

ilar p

oten

cy a

s the

who

le m

Ab

cetu

xim

ab, d

espi

te th

e fa

ct th

at

CO

NA

N-1

is d

evoi

d of

an

Fc p

ortio

n th

at c

ould

med

iate

imm

une

effec

tor f

unct

ions

. Com

pare

d to

ther

apy

usin

g bi

vale

nt, m

ono-

spec

ific

nano

bodi

es, C

ON

AN

-1 w

as c

lear

ly m

ore

pote

nt in

tum

our g

row

th in

hibi

tion.

Thes

e re

sults

show

that

the

ratio

nal d

esig

n of

bip

arat

opic

nan

obod

y-ba

sed

antic

ance

r the

rape

utic

s may

yie

ld p

oten

t lea

d m

olec

ules

for f

urth

er d

evel

opm

ent.

Roov

ers

et a

l. 20

11

Biv

alen

t nan

obod

yIn

fluen

za v

irus

Intr

anas

al a

dmin

istr

a-ti

on

Influ

enza

A v

irus

infe

ctio

ns im

pose

a re

curr

ent a

nd g

loba

l dis

ease

bur

den.

Cur

rent

ant

ivir

als a

gain

st in

fluen

za a

re n

ot a

lway

s eff

ectiv

e. W

e as

sess

ed th

e pr

otec

tive

pote

ntia

l of m

onov

alen

t and

biv

alen

t Nan

obod

ies (

Abl

ynx)

aga

inst

cha

lleng

e w

ith th

is v

irus

. Th

ese

Nan

obod

ies w

ere

deri

ved

from

llam

as a

nd ta

rget

H5N

1 he

mag

glut

inin

. Int

rana

sal a

dmin

istr

atio

n of

Nan

obod

ies e

ffect

ivel

y co

ntro

lled

hom

olog

ous i

nflue

nza

A v

irus

repl

icat

ion.

Adm

inis

trat

ion

of N

anob

odie

s bef

ore

chal

leng

e st

rong

ly re

duce

d H

5N1

viru

s re

plic

atio

n in

the

lung

s and

pro

tect

ed m

ice

from

mor

bidi

ty a

nd m

orta

lity

afte

r a le

thal

cha

lleng

e w

ith H

5N1

viru

s. Th

e bi

vale

nt

Nan

obod

y w

as a

t lea

st 6

0-fo

ld m

ore

effec

tive

than

the

mon

oval

ent N

anob

ody

in c

ontr

ollin

g vi

rus r

eplic

atio

n. In

add

ition

, Nan

o-bo

dy th

erap

y af

ter c

halle

nge

stro

ngly

redu

ced

vira

l rep

licat

ion

and

sign

ifica

ntly

del

ayed

tim

e to

dea

th. E

pito

pe m

appi

ng re

veal

ed

that

the

VH

H N

anob

ody

bind

s to

antig

enic

site

B in

H5

hem

aggl

utin

in. B

ecau

se N

anob

odie

s are

smal

l, st

able

, and

sim

ple

to p

ro-

duce

, the

y ar

e a

prom

isin

g, n

ovel

ther

apeu

tic a

gent

aga

inst

influ

enza

.

Iban

ez e

t al

. 201

1


489

Mul

tim

eric

and

bi

spec

ific

cons

truc

tsIn

fluen

za

For e

ffici

ent p

reve

ntio

n of

vir

al in

fect

ions

and

cro

ss p

rote

ctio

n, si

mul

tane

ous t

arge

ting

of m

ultip

le v

iral

epi

tope

s is a

pow

er-

ful s

trat

egy.

Llam

a he

avy

chai

n an

tibod

y fr

agm

ents

(VH

H) a

gain

st th

e tr

imer

ic e

nvel

ope

prot

eins

of R

espi

rato

ry S

yncy

tial V

irus

(F

usio

n pr

otei

n), R

abie

s vir

us (G

lyco

prot

ein)

and

H5N

1 In

fluen

za (H

emag

glut

inin

5) w

ere

sele

cted

from

llam

a de

rive

d im

mun

e lib

rari

es b

y ph

age

disp

lay.

Neu

tral

izin

g V

HH

reco

gniz

ing

diffe

rent

epi

tope

s in

the

rece

ptor

bin

ding

site

s on

the

spik

es w

ith a

ffini

-tie

s in

the

low

nan

omol

ar ra

nge

wer

e id

entifi

ed fo

r all

the

thre

e vi

ruse

s by

vira

l neu

tral

izat

ion

assa

ys. B

y fu

sion

of V

HH

with

var

i-ab

le li

nker

leng

ths,

mul

timer

ic c

onst

ruct

s wer

e m

ade

that

impr

oved

neu

tral

izat

ion

pote

ncie

s up

to 4

000-

fold

for R

SV, 1

500-

fold

fo

r Rab

ies v

irus

and

75-

fold

for I

nflue

nza

H5N

1. Th

e po

tenc

ies o

f the

VH

H c

onst

ruct

s wer

e si

mila

r or b

ette

r tha

n be

st p

erfo

rmin

g m

onoc

lona

l ant

ibod

ies.

The

cros

s pro

tect

ion

capa

city

aga

inst

diff

eren

t vir

al st

rain

s was

als

o im

prov

ed fo

r all

thre

e vi

ruse

s, b

oth

by m

ultiv

alen

t (tw

o or

thre

e id

entic

al V

HH

) and

bip

arat

opic

(tw

o di

ffere

nt V

HH

) con

stru

cts.

By

com

bini

ng a

VH

H n

eutr

aliz

ing

RSV

subt

ype

A, b

ut n

ot su

btyp

e B

with

a p

oorl

y ne

utra

lizin

g V

HH

with

hig

h affi

nity

for s

ubty

pe B

, a b

ipar

atop

ic c

onst

ruct

was

m

ade

with

low

nan

omol

ar n

eutr

aliz

ing

pote

ncy

agai

nst b

oth

subt

ypes

. Tri

vale

nt a

nti-

H5N

1 V

HH

neu

tral

ized

bot

h In

fluen

za

H5N

1 cl

ade1

and

2 in

a p

seud

otyp

e as

say

and

was

ver

y po

tent

in n

eutr

aliz

ing

the

NIB

RG-1

4 In

fluen

za H

5N1

stra

in w

ith IC

(50)

of

9 pi

com

olar

. Biv

alen

t and

bip

arat

opic

con

stru

cts a

gain

st R

abie

s vir

us c

ross

neu

tral

ized

bot

h 10

diff

eren

t Gen

otyp

e 1

stra

ins a

nd

Gen

otyp

e 5.

The

resu

lts sh

ow th

at m

ultim

eriz

atio

n of

VH

H fr

agm

ents

targ

etin

g m

ultip

le e

pito

pes o

n a

vira

l tri

mer

ic sp

ike

prot

ein

is a

pow

erfu

l too

l for

ant

i-vi

ral t

hera

py to

ach

ieve

“bes

t-in

-cla

ss” a

nd b

road

er n

eutr

aliz

atio

n ca

paci

ty.

Hul

tber

g et

al.

2011

Bio

sens

ors

Dec

abod

yC

ellu

lose

filt

erS.

aur

eus

Ant

ibod

y en

gine

erin

g ha

s allo

wed

for t

he ra

pid

gene

ratio

n of

bin

ding

age

nts a

gain

st v

irtu

ally

any

ant

igen

of i

nter

est,

pred

omi-

nant

ly fo

r the

rape

utic

app

licat

ions

. Con

side

rabl

y le

ss a

tten

tion

has b

een

give

n to

the

deve

lopm

ent o

f dia

gnos

tic re

agen

ts a

nd b

io-

sens

ors u

sing

eng

inee

red

antib

odie

s. R

ecen

tly, w

e pr

oduc

ed a

nov

el p

enta

vale

nt b

ispe

cific

ant

ibod

y (i.

e., d

ecab

ody)

by

pent

amer

iz-

ing

two

sing

le-d

omai

n an

tibod

ies (

sdA

bs) t

hrou

gh th

e ve

roto

xin

B su

buni

t (V

TB)

and

foun

d bo

th fu

sion

par

tner

s to

be fu

nctio

nal.

Usi

ng a

sim

ilar a

ppro

ach,

we

have

eng

inee

red

a bi

spec

ific

pent

amer

ic fu

sion

pro

tein

con

sist

ing

of fi

ve sd

Abs

and

five

cel

lulo

se-

bind

ing

mod

ules

(CBM

s) li

nked

via

VT

B. T

o fin

d an

opt

imal

des

ign

form

at, w

e co

nstr

ucte

d si

x bi

spec

ific

pent

amer

s con

sist

ing

of

thre

e di

ffere

nt C

BMs,

fuse

d to

the

Stap

hylo

cocc

us a

ureu

s-sp

ecifi

c hu

man

sdA

b H

VH

P428

, in

both

ori

enta

tions

. One

bis

peci

fic

pent

amer

, con

tain

ing

an N

-ter

min

al C

BM9

and

C-t

erm

inal

HV

HP4

28, w

as so

lubl

e, n

on-a

ggre

gatin

g, a

nd d

id n

ot d

egra

de u

pon

stor

age

at 4

deg

rees

C fo

r ove

r six

mon

ths.

This

mol

ecul

e w

as d

ually

func

tiona

l as i

t bou

nd to

cel

lulo

se-b

ased

filte

rs a

s wel

l as S

. au

reus

cel

ls. W

hen

impr

egna

ted

in c

ellu

lose

filte

rs, t

he b

ispe

cific

pen

tam

er re

cogn

ized

S. a

ureu

s cel

ls in

a fl

ow-t

hrou

gh d

etec

tion

assa

y. Th

e ab

ility

of p

enta

mer

ized

CBM

s to

bind

cel

lulo

se m

ay fo

rm th

e ba

sis o

f an

imm

obili

zatio

n pl

atfo

rm fo

r mul

tival

ent d

ispl

ay

of h

igh-

avid

ity b

indi

ng re

agen

ts o

n ce

llulo

sic

filte

rs fo

r sen

sing

of p

atho

gens

, bio

mar

kers

and

env

iron

men

tal p

ollu

tant

s.

Hus

sack

et

al.

2009


490

Chi

mer

ic a

ntig

en

rece

ptor

Ant

i-M

UC

1 na

nobo

dyC

ance

r im

mun

othe

rapy

The

cruc

ial r

ole

of T

lym

phoc

ytes

in a

nti-

tum

or im

mun

ity h

as le

d to

the

deve

lopm

ent o

f nov

el st

rate

gies

that

can

targ

et a

nd a

cti-

vate

T c

ells

aga

inst

tum

or c

ells

. Rec

ombi

nant

DN

A te

chno

logy

has

bee

n us

ed to

gen

erat

e no

n-M

HC

-res

tric

ted

chim

eric

ant

igen

re

cept

ors (

CA

Rs).

Her

e, w

e co

nstr

ucte

d a

pane

l of r

ecom

bina

nt C

AR

that

har

bors

the

anti-

MU

C1

nano

body

and

the

sign

alin

g an

d co

-sig

nalin

g m

oiet

ies (

CD

3 ze

ta/C

D28

) with

diff

eren

t spa

cer r

egio

ns d

eriv

ed fr

om h

uman

IgG

3 w

ith o

ne o

r tw

o re

peat

s of t

he

hing

e se

quen

ce o

r the

hin

ge re

gion

of F

c ga

mm

a RI

I. Th

e Ph

iC31

inte

gras

e sy

stem

was

em

ploy

ed to

inve

stig

ate

if th

e re

com

bina

-tio

n effi

cien

cy c

ould

be

recr

uite

d fo

r hig

h an

d st

able

exp

ress

ion

of T

cel

l chi

mer

ic re

cept

or g

enes

. The

effec

t of n

ucle

ar lo

caliz

atio

n si

gnal

(NLS

) and

two

diffe

rent

pro

mot

ers (

CM

V a

nd C

AG

) on

effica

cy o

f Phi

C31

inte

gras

e in

hum

an T

cel

l lin

es w

as e

valu

ated

. The

pres

ence

of i

nteg

rase

in c

ombi

natio

n w

ith N

LS, m

edia

ted

up to

7.6

and

8.5

fold

incr

ease

s in

CA

R ex

pres

sion

in Z

CH

N-a

ttB

and

ZCH

HN

-att

B ca

sset

te in

tegr

ated

T c

ells

, res

pect

ivel

y. O

ur re

sults

show

ed th

at h

ighl

y effi

cien

t and

stab

le tr

ansd

uctio

n of

the

Jurk

at

cell

line

by P

hiC

31 in

tegr

ase

is a

feas

ible

mod

ality

for g

ener

atin

g an

ti-ca

ncer

chi

mer

ic T

cel

ls fo

r use

in c

ance

r im

mun

othe

rapy

.

Iri-S

ofla

et a

l. 20

11

Nan

obod

y-m

icel

le

conj

ugat

eEp

ider

mal

gro

wth

fact

or

rece

ptor

Lyso

zym

eD

rug

targ

etin

g

The

aim

of t

his s

tudy

was

to d

evel

op p

oly(

ethy

lene

gly

col)-

b-po

ly[N

-(2-

hydr

oxyp

ropy

l) m

etha

cryl

amid

e-la

ctat

e] (m

PEG

-b-

p(H

PMA

m-L

ac(n

)) c

ore-

cros

slin

ked

ther

mos

ensi

tive

biod

egra

dabl

e po

lym

eric

mic

elle

s sui

tabl

e fo

r act

ive

tum

or ta

rget

ing,

by

coup

ling

the

anti-

EGFR

(epi

derm

al g

row

th fa

ctor

rece

ptor

) EG

a1 n

anob

ody

to th

eir s

urfa

ce. T

o th

is e

nd. P

EG w

as fu

nctio

naliz

ed

with

N-s

ucci

nim

idyl

3-(

2-py

ridy

ldith

io)-

prop

iona

te (S

PDP)

to y

ield

a P

DP-

PEG

-b-p

(HPM

Am

-Lac

(n))

blo

ck c

opol

ymer

. Mic

elle

s co

mpo

sed

of 8

0% m

PEG

-b-p

(HPM

Am

-Lac

(n))

and

20%

PD

P-PE

G-b

-p(H

PMA

m-L

ac(n

)) w

ere

prep

ared

and

lyso

zym

e (a

s a m

odel

pr

otei

n) w

as m

odifi

ed w

ith N

-suc

cini

mid

yl-S

-ace

tylth

ioac

etat

e, d

epro

tect

ed w

ith h

ydro

xyla

min

e hy

droc

hlor

ide

and

subs

eque

ntly

co

uple

d to

the

mic

ella

r sur

face

. The

mic

ella

r con

juga

tes w

ere

char

acte

rize

d us

ing

SDS-

PAG

E an

d ge

l per

mea

tion

chro

mat

ogra

phy

(GPC

). U

sing

the

know

ledg

e ob

tain

ed w

ith ly

sozy

me

conj

ugat

ion,

the

EGa1

nan

obod

y w

as c

oupl

ed to

mPE

G/P

DP-

PEG

mic

elle

s an

d th

e co

njug

atio

n w

as su

cces

sful

as d

emon

stra

ted

by w

este

rn b

lot a

nd d

ot b

lot a

naly

sis.

Rho

dam

ine

labe

led

EGa1

-mic

elle

s sh

owed

subs

tant

ially

hig

her b

indi

ng a

s wel

l as u

ptak

e by

EG

FR o

ver-

expr

essi

ng c

ance

r cel

ls (A

431

and

UM

-SC

C-1

4C) t

han

unta

r-ge

ted

rhod

arni

ne la

bele

d m

icel

les.

Inte

rest

ingl

y, no

bin

ding

of t

he n

anob

ody

mic

elle

s was

obs

erve

d to

EG

FR n

egat

ive

cells

(3T

3)

as w

ell a

s to1

4C c

ells

in th

e pr

esen

ce o

f an

exce

ss o

f fre

e na

nobo

dy. Th

is d

emon

stra

tes t

hat t

he b

indi

ng o

f the

nan

obod

y m

icel

les i

s in

deed

by

inte

ract

ion

with

the

EGF

rece

ptor

. In

conc

lusi

on, E

Ga1

dec

orat

ed (m

PEG

/PD

P-PE

G)-

b-(p

HPM

Am

-Lac

(n))

pol

ymer

ic

mic

elle

s are

hig

hly

prom

isin

g sy

stem

s for

act

ive

drug

targ

etin

g.

Tale

lli e

t al

. 201

1

Nan

obod

y-go

ld n

ano-

part

icle

con

juga

tes

HER

2Ph

otot

herm

al th

erap

y

Bran

ched

gol

d na

nopa

rtic

les a

re p

oten

tial p

hoto

ther

mal

ther

apy

agen

ts b

ecau

se o

f the

ir la

rge

abso

rptio

n cr

oss s

ectio

n in

the

near

-in

frar

ed w

indo

w. U

pon

lase

r irr

adia

tion

they

pro

duce

eno

ugh

heat

to d

estr

oy tu

mor

cel

ls. I

n th

is w

ork,

bra

nche

d go

ld n

anop

artic

les a

re

blof

unct

iona

lized

with

nan

obod

ies,

the

smal

lest

fully

func

tiona

l ant

igen

-bin

ding

frag

men

ts e

volv

ed fr

om th

e va

riabl

e do

mai

n, th

e V

HH

, of

a c

amel

hea

vy c

hain

-onl

y an

tibod

y. Th

ese

nano

bodi

es b

ind

to th

e H

ER2

antig

en w

hich

is h

ighl

y ex

pres

sed

on b

reas

t and

ova

rian

canc

er

cells

. Flo

w c

ytom

etric

ana

lysis

and

dar

k fie

ld Im

ages

of H

ER2

posit

ive

SKO

V3

cells

incu

bate

d w

ith a

nti-H

ER2

conj

ugat

ed b

ranc

hed

gold

na

nopa

rtic

les s

how

spec

ific

cell

targ

etin

g. L

aser

Irra

diat

ion

stud

ies r

evea

l tha

t HER

2 po

sitiv

e SK

OV

3 ce

lls e

xpos

ed to

the

anti-

HER

2 ta

r-ge

ted

bran

ched

gol

d na

nopa

rtic

les a

re d

estr

oyed

afte

r five

min

utes

of l

aser

trea

tmen

t at 3

8 W

/cm

2 usin

g a

690

nm c

ontin

uous

wav

e la

ser.

Star

ting

from

a n

anop

artic

le o

ptic

al d

ensit

y of

4, c

ell d

eath

is o

bser

ved,

whe

reas

the

cont

rol s

ampl

es, n

anop

artic

les w

ith a

nti-P

SA n

ano-

bodi

es, n

anop

artic

les o

nly,

and

lase

r onl

y, do

not

show

any

cel

l dea

th. Th

ese

resu

lts su

gges

t tha

t thi

s new

type

of b

ioco

njug

ated

bra

nche

d go

ld n

anop

artic

les a

re e

ffect

ive

antig

en-t

arge

ted

phot

othe

rmal

ther

apeu

tic a

gent

s for

can

cer t

reat

men

t.

Van

de

Broe

k et

al

. 201

1


491

Nan

obod

y-lip

osom

e co

njug

ate

Epid

erm

al g

row

th fa

ctor

re

cept

orR

ecep

tor

do

wnr

egul

atio

n

The

epid

erm

al g

row

th fa

ctor

rece

ptor

(EG

FR) i

s a re

cogn

ized

targ

et fo

r tum

or th

erap

y an

d m

onoc

lona

l ant

ibod

ies (

mA

bs, e

.g.

cetu

xim

ab) h

ave

been

dev

elop

ed to

inhi

bit r

ecep

tor a

ctiv

atio

n. B

esid

es b

lock

ing

ligan

d (e

.g. E

GF)

bin

ding

to th

e re

cept

or, r

epor

ts

have

show

n th

at m

Abs

pro

mot

e sl

ow re

cept

or in

tern

aliz

atio

n an

d de

grad

atio

n in

lyso

som

es, i

.e. d

ownr

egul

atio

n. Th

e effi

cacy

of

rece

ptor

dow

nreg

ulat

ion

was

rece

ntly

show

n to

dep

end

on th

e si

ze o

f rec

epto

r clu

ster

s for

med

at t

he c

ell s

urfa

ce. I

n th

is st

udy,

a m

ultiv

alen

t pla

tform

is p

rese

nted

, con

sist

ing

of n

anob

odie

s rec

ogni

zing

the

ecto

dom

ain

of E

GFR

(EG

a1) c

oupl

ed to

PEG

-lip

osom

es, a

nd th

e in

vitr

o an

d in

viv

o eff

ects

of t

his s

yste

m o

n EG

FR in

tern

aliz

atio

n an

d do

wnr

egul

atio

n w

ere

inve

stig

ated

. Nan

o-bo

dies

are

the

smal

lest

func

tiona

l ant

igen

-bin

ding

imm

unog

lobu

lin fr

agm

ents

and

the

EGa1

nan

obod

y ha

s bee

n de

scri

bed

as a

n EG

FR-a

ntag

onis

t. EG

a1-li

poso

mes

(EG

a1-L

) ind

uced

a m

ore

than

90%

rem

oval

of E

GFR

from

the

cell

surf

ace,

as a

resu

lt of

rece

p-to

r int

erna

lizat

ion.

Fur

ther

mor

e, th

is m

assi

ve se

ques

trat

ion

of E

GFR

med

iate

d by

EG

a1-L

lead

to re

cept

or d

egra

datio

n, w

hile

no

degr

adat

ion

was

det

ecte

d w

ith th

e m

onov

alen

t nan

obod

y. Th

e do

wnr

egul

ator

y ca

paci

ty h

ere

repo

rted

was

foun

d to

be

inde

pend

-en

t of t

he e

pito

pe o

n EG

FR re

cogn

ized

by

the

graf

ted

nano

body

, and

exc

lusi

ve to

the

nano

body

-lipo

som

es, a

s ant

i-EG

FR si

ngle

ch

ain

vari

able

frag

men

ts (s

cFv)

cou

pled

to li

poso

mes

wer

e un

able

to in

duce

this

effe

ct. I

mpo

rtan

tly, E

Ga1

-L in

duce

d a

sign

ifica

nt

inhi

bitio

n of

tum

or c

ell p

rolif

erat

ion,

in v

itro,

an

effec

t lik

ely

med

iate

d by

the

com

bina

tion

of re

cept

or d

ownr

egul

atio

n an

d re

cep-

tor a

ntag

onis

m. A

lso

in v

ivo,

EG

FR d

ownr

egul

atio

n w

as o

bser

ved

in tu

mor

s of m

ice

intr

aven

ousl

y in

ject

ed w

ith E

Ga1

-L, i

ndic

atin

g th

at th

is m

ultiv

alen

t pla

tform

blo

cks l

igan

d bi

ndin

g to

the

rece

ptor

and

sim

ulta

neou

sly

indu

ces t

he d

ownr

egul

atio

n of

EG

FR.

Oliv

eira

et

al.

2010

Nan

obod

y-β-

lact

amas

e co

njug

ate

Car

cino

embr

yoni

c an

tige

nC

ance

r th

erap

y

We

iden

tified

a n

anob

ody

with

subn

anom

olar

affi

nity

for t

he h

uman

tum

or-a

ssoc

iate

d ca

rcin

oem

bryo

nic

antig

en. Th

is n

anob

ody

was

con

juga

ted

to E

nter

obac

ter c

loac

ae b

eta-

lact

amas

e, a

nd it

s site

-sel

ectiv

e an

tican

cer p

rodr

ug a

ctiv

atio

n ca

paci

ty w

as e

valu

-at

ed. Th

e co

njug

ate

was

read

ily p

urifi

ed in

hig

h yi

elds

with

out a

ggre

gatio

n or

loss

of f

unct

iona

lity

of th

e co

nstit

uent

s. In

vitr

o ex

peri

men

ts sh

owed

that

the

nano

body

-enz

yme

conj

ugat

e eff

ectiv

ely

activ

ated

the

rele

ase

of p

heny

lene

diam

ine

mus

tard

from

the

ceph

alos

pori

n ni

trog

en m

usta

rd p

rodr

ug 7

-(4-

carb

oxyb

utan

amid

o) c

epha

losp

orin

mus

tard

at t

he su

rfac

e of

car

cino

embr

yoni

c an

tigen

-exp

ress

ing

LS17

4T c

ance

r cel

ls. I

n vi

vo st

udie

s dem

onst

rate

d th

at th

e co

njug

ate

had

an e

xcel

lent

bio

dist

ribu

tion

profi

le

and

indu

ced

regr

essi

ons a

nd c

ures

of e

stab

lishe

d tu

mor

xen

ogra

fts.

The

easy

gen

erat

ion

and

man

ufac

turi

ng y

ield

of n

anob

ody-

base

d co

njug

ates

toge

ther

with

thei

r pot

ent a

ntitu

mor

act

ivity

mak

e na

nobo

dies

pro

mis

ing

vehi

cles

for n

ew g

ener

atio

n ca

ncer

th

erap

eutic

s.

Cor

tez-

Reta

-m

ozo

et

al. 2

004

Nan

obod

y-ap

olip

opro

tein

L-

I con

juga

teTr

ypan

osom

iasi

s th

erap

yIm

mun

otox

in

Hig

h sy

stem

ic d

rug

toxi

city

and

incr

easi

ng p

reva

lenc

e of

dru

g re

sist

ance

ham

pers

effi

cien

t tre

atm

ent o

f hum

an A

fric

an tr

ypan

oso-

mia

sis (

HAT

). H

ence

, dev

elop

men

t of n

ew h

ighl

y sp

ecifi

c tr

ypan

ocid

al d

rugs

is n

eces

sary

. Nor

mal

hum

an se

rum

(NH

S) c

onta

ins

apol

ipop

rote

in L

-I (a

poL-

I), w

hich

lyse

s Afr

ican

tryp

anos

omes

exc

ept r

esis

tant

form

s suc

h as

Try

pano

som

a br

ucei

rhod

esie

nse.

T.

b. rh

odes

iens

e ex

pres

ses t

he a

poL-

I neu

tral

izin

g se

rum

resi

stan

ce-a

ssoc

iate

d (S

RA

) pro

tein

, end

owin

g th

is p

aras

ite w

ith th

e ab

ility

to

infe

ct h

uman

s and

cau

se H

AT. A

trun

cate

d ap

oL-I

(Tr-

apoL

-I) h

as b

een

engi

neer

ed b

y de

letin

g its

SR

A-i

nter

actin

g do

mai

n,

whi

ch m

akes

it ly

tic fo

r T. b

. rho

desi

ense

. Her

e, w

e co

njug

ated

Tr-

apoL

-I w

ith a

sing

le-d

omai

n an

tibod

y (n

anob

ody)

that

effi

cien

tly

targ

ets c

onse

rved

cry

ptic

epi

tope

s of t

he v

aria

nt su

rfac

e gl

ycop

rote

in (V

SG) o

f try

pano

som

es to

gen

erat

e a

new

man

mad

e ty

pe o

f im

mun

otox

in w

ith p

oten

tial f

or tr

ypan

osom

iasi

s the

rapy

. Tre

atm

ent w

ith th

is e

ngin

eere

d co

njug

ate

resu

lted

in c

lear

cur

ativ

e an

d al

levi

atin

g eff

ects

on

acut

e an

d ch

roni

c in

fect

ions

of m

ice

with

bot

h N

HS-

resi

stan

t and

NH

S-se

nsiti

ve tr

ypan

osom

es.

Bara

l et

al. 2

006


492

Nan

obod

y-gl

ucos

e

oxid

ase

conj

ugat

eSt

rept

ococ

cus

mut

ans

Ant

imic

robi

al a

gent

s

Enzy

mes

such

as l

acto

pero

xida

se a

nd g

luco

se o

xida

se (G

Ox)

are

use

d as

ant

imic

robi

al a

gent

s in

oral

car

e pr

oduc

ts. Th

eir l

ow

spec

ifici

ties a

nd su

bsta

ntiv

enes

s can

be

redu

ced

by c

oval

ent c

oupl

ing

of a

ntim

icro

bial

mol

ecul

es to

ant

ibod

ies.

Var

iabl

e do

mai

ns

(V-H

H) d

eriv

ed fr

om ll

ama

heav

y-ch

ain

antib

odie

s are

par

ticul

arly

suite

d fo

r suc

h an

app

roac

h. Th

e an

tibod

ies a

re c

ompo

sed

sole

ly o

f hea

vy-c

hain

dim

ers;

ther

efor

e, p

rodu

ctio

n of

act

ive

fusi

on p

rote

ins b

y us

ing

mol

ecul

ar b

iolo

gy-b

ased

tech

niqu

es is

less

co

mpl

icat

ed th

an p

rodu

ctio

n by

use

of c

onve

ntio

nal a

ntib

odie

s. In

this

stud

y, a

fusi

on p

rote

in c

onsi

stin

g of

V-H

H a

nd G

Ox

was

co

nstr

ucte

d an

d ex

pres

sed

by S

acch

arom

yces

cer

evis

iae.

A ll

ama

was

imm

uniz

ed w

ith S

trep

toco

ccus

mut

ans s

trai

n H

G98

2. S

ubse

-qu

ently

, B ly

mph

ocyt

es w

ere

isol

ated

and

cD

NA

frag

men

ts e

ncod

ing

the

V-H

H fr

agm

ents

wer

e ob

tain

ed b

y re

vers

e tr

ansc

ript

ion-

PCR

. Aft

er c

onst

ruct

ion

of a

V-H

H li

brar

y in

Esc

heri

chia

col

i and

scre

enin

g of

the

libra

ry a

gain

st m

utan

s gro

up st

rept

ococ

ci a

nd

Stre

ptoc

occu

s san

guin

is st

rain

s, w

e fo

und

two

V-H

H fr

agm

ents

with

hig

h sp

ecifi

citie

s for

S. m

utan

s str

ains

. A G

Ox

gene

was

lin

ked

to th

e tw

o V

-HH

gen

es a

nd c

lone

d in

to S

. cer

evis

iae

yeas

ts. Th

e ye

asts

exp

ress

ed a

nd se

cret

ed th

e re

com

bina

nt p

rote

ins i

nto

the

grow

th m

ediu

m. Th

e te

st o

f bin

ding

of f

usio

n pr

otei

ns to

ora

l bac

teri

a th

roug

h th

eir V

-HH

frag

men

ts sh

owed

that

S. m

utan

s ha

d be

en sp

ecifi

cally

targ

eted

by

GO

x-S1

20, o

ne o

f the

fusi

on p

rote

in c

onst

ruct

s. A

low

con

cent

ratio

n of

the

fusi

on p

rote

in w

as

also

abl

e to

sele

ctiv

ely

kill

S. m

utan

s with

in 2

0 m

in in

the

pres

ence

of l

acto

pero

xida

se a

nd p

otas

sium

iodi

de. Th

ese

findi

ngs d

emon

-st

rate

that

the

fusi

on p

rote

in G

Ox-

V-H

H is

pot

entia

lly v

alua

ble

in th

e se

lect

ive

killi

ng o

f tar

get b

acte

ria

such

as S

. mut

ans.

Szyn

ol e

t al

. 200

4

Bis

peci

fic a

ntib

ody

Dec

abod

yLi

nker

seq

uenc

eVe

roto

xin

B s

ubun

it

Bisp

ecifi

c an

tibod

ies p

rese

nt u

niqu

e op

port

uniti

es in

term

s of n

ew a

pplic

atio

ns fo

r eng

inee

red

antib

odie

s. H

owev

er, d

esig

ning

id

eal b

ispe

cific

ant

ibod

ies r

emai

ns a

cha

lleng

e. H

ere

we

desc

ribe

a n

ovel

bis

peci

fic a

ntib

ody

mod

el in

whi

ch fi

ve si

ngle

dom

ain

antib

odie

s (sd

Abs

) are

fuse

d vi

a a

linke

r seq

uenc

e to

the

N-t

erm

inus

of t

he v

erot

oxin

B (V

TB)

subu

nit,

a pe

ntam

eriz

atio

n do

mai

n,

and

five

sdA

bs a

re fu

sed

via

a lin

ker s

eque

nce

to th

e V

TB

C-t

erm

inus

. Fift

een

such

dec

aval

ent b

ispe

cific

mol

ecul

es, t

erm

ed

deca

bodi

es, w

ere

cons

truc

ted

and

char

acte

rize

d fo

r the

pur

pose

of i

dent

ifyin

g an

opt

imal

dec

abod

y de

sign

. One

of t

he fi

ftee

n m

olec

ules

exi

sted

in a

non

-agg

rega

ted

deca

vale

nt fo

rm. I

n co

njun

ctio

n w

ith th

e is

olat

ion

of sd

Abs

with

the

desi

red

spec

ifici

ties

from

non

-imm

une

phag

e di

spla

y lib

rari

es, t

he d

ecab

ody

stra

tegy

pro

vide

s a m

eans

of g

ener

atin

g hi

gh a

vidi

ty b

ispe

cific

ant

ibod

y re

agen

ts, w

ith g

ood

phys

ical

pro

pert

ies,

rela

tivel

y qu

ickl

y.

Ston

e et

al

. 200

7

Tabl

e 6H

. App

licat

ion

of s

ingl

e-do

mai

n an

tibo

dy fr

agm

ents

in th

erap

y: h

uman

ised

sin

gle-

dom

ain

anti

body

frag

men

ts

VH

HC

DR

3H

uman

sca

ffold

pro

tein

Gra

ftin

gU

biqu

itin

VH

H is

the

bind

ing

dom

ain

of th

e Ig

G h

eavy

cha

in. S

ome

VH

Hs h

ave

an e

xtre

mel

y lo

ng C

DR3

that

con

trib

utes

to a

ntig

en b

indi

ng.

We

stud

ied

the

antig

en b

indi

ng a

bilit

y of

CD

R3 b

y gr

aftin

g a

CD

R3 fr

om a

n an

tigen

-bin

ding

VH

H o

nto

a no

nbin

ding

VH

H.

cAb-

CA

05-(

1RI8

), th

e C

DR3

-gra

fted

VH

H, h

ad a

n an

tigen

-bin

ding

abi

lity.

To fi

nd a

hum

an sc

affol

d pr

otei

n ac

cept

able

for V

HH

C

DR3

gra

ftin

g, w

e fo

cuse

d on

the

cons

erve

d st

ruct

ure

of V

HH

, esp

ecia

lly th

e N

-ter

min

al a

nd C

-ter

min

al a

min

o ac

id re

sidu

es o

f th

e C

DR3

loop

and

the

Cys

resi

due

of C

DR1

. Hum

an o

rigi

n pr

otei

n st

ruct

ures

with

the

sam

e or

ient

atio

n w

ere

sear

ched

in P

DB

and

ubiq

uitin

was

sele

cted

. Ubi

-(1R

I8),

the

CD

R3-g

raft

ed u

biqu

itin,

had

ant

igen

-bin

ding

abi

lity,

thou

gh th

e affi

nity

was

rela

tivel

y lo

w c

ompa

red

to c

Ab-

CA

05-(

1RI8

). Th

e th

erm

odyn

amic

par

amet

ers o

f Ubi

-(1R

I8) b

indi

ng to

HEW

L w

ere

diffe

rent

from

cA

b-C

A05

-(1R

I8).

Hyd

roge

n-de

uter

ium

exc

hang

e ex

peri

men

ts sh

owed

dec

reas

ed st

abili

ty a

roun

d th

e C

DR3

gra

ftin

g re

gion

of

Ubi

-(1R

I8),

whi

ch m

ight

exp

lain

the

decr

ease

d an

tigen

-bin

ding

abi

lity

and

the

diffe

renc

es in

ther

mod

ynam

ic p

rope

rtie

s. W

e co

n-cl

uded

that

the

orie

ntat

ion

of th

e C

DR3

sequ

ence

of U

bi-(

1RI8

) cou

ld n

ot b

e re

cons

truc

ted

corr

ectly

.

Inou

e et

al

. 201

1


493

Aah

II s

corp

ion

toxi

nH

uman

ized

nan

obod

yRe

cent

ly, a

pot

ent A

ahII

scor

pion

toxi

n-ne

utra

lizin

g na

nobo

dy w

as id

entifi

ed. H

owev

er, t

his N

bAah

II10

con

tain

s a si

ngle

Cys

in

its fi

rst a

ntig

en-b

indi

ng lo

op, l

eadi

ng to

Nb

dim

eriz

atio

n up

on p

rolo

nged

stor

age.

In th

is w

ork,

we

first

inve

stig

ate

the

effica

cy o

f N

bAah

II10

var

iant

s in

whi

ch th

is C

ys w

as su

bstit

uted

by

Ala

, Ser

or Th

r. Se

cond

, the

NbA

ahII

10 C

ys/S

er m

utan

t dis

play

ing

the

best

func

tiona

l pro

pert

ies i

s sub

sequ

ently

hum

aniz

ed. I

t is d

emon

stra

ted

that

the

max

imal

ly h

uman

ized

ver

sion

of N

bAah

II10

C

ys/S

er m

aint

ains

its h

igh

affini

ty fo

r the

ant

igen

with

out c

once

ding

muc

h on

exp

ress

ion

yiel

d an

d st

abili

ty. M

ore

impo

rtan

tly, i

ts

neut

raliz

ing

capa

city

is p

rese

rved

as a

ll m

ice

surv

ive

inje

ctio

ns o

f sev

en L

D(5

0) a

nd 5

0 of

mic

e su

rviv

ed n

ine

LD(5

0) o

f the

scor

-pi

on to

xin.

Thus

, thi

s hum

aniz

ed N

b is

the

best

can

dida

te to

dev

elop

a th

erap

y in

hum

an a

gain

st th

e m

ost t

oxic

ven

om c

ompo

und

of o

ne o

f the

mos

t dan

gero

us sc

orpi

ons.

Ben

Abd

er-

raze

k et

al

. 201

1

Hum

aniz

ed n

anob

odie

sLo

op-g

raft

ing

In v

ivo

imag

ing

Scaff

old

Car

cino

embr

yoni

c an

tige

n

Nan

obod

ies a

re a

nov

el ty

pe o

f im

mun

oglo

bulin

like,

ant

igen

-bin

ding

pro

tein

with

ben

efici

al p

harm

acol

ogic

and

pha

rmac

okin

etic

pr

oper

ties t

hat a

re id

eally

suite

d to

targ

etin

g ce

llula

r ant

igen

s for

mol

ecul

ar im

agin

g or

ther

apeu

tic p

urpo

ses.

How

ever

, bec

ause

of

thei

r cam

elid

, non

hum

an o

rigi

n, th

e po

ssib

le im

mun

ogen

icity

of N

anob

odie

s whe

n us

ed in

the

clin

ic is

a c

once

rn. H

ere

we

pres

ent

a ne

w st

rate

gy to

qui

ckly

gen

erat

e hu

man

ized

Nan

obod

ies f

or m

olec

ular

imag

ing

purp

oses

. Met

hods

: We

gene

tical

ly g

raft

ed th

e an

tigen

-bin

ding

loop

s of N

bCEA

5, a

Nan

obod

y w

ith sp

ecifi

city

for t

he c

olon

car

cino

ma

mar

ker c

arci

noem

bryo

nic

antig

en (C

EA),

onto

the

fram

ewor

k of

a h

uman

ized

Nan

obod

y sc

affol

d. Th

is sc

affol

d ha

s bee

n pr

evio

usly

cha

ract

eriz

ed in

our

labo

rato

ry a

s a

stab

le N

anob

ody

that

can

serv

e as

a u

nive

rsal

loop

acc

epto

r for

ant

igen

-bin

ding

loop

s fro

m d

onor

Nan

obod

ies a

nd h

as b

een

addi

-tio

nally

mut

ated

at a

bout

10

cruc

ial s

urfa

ce-e

xpos

ed si

tes t

o re

sem

ble

the

sequ

ence

of h

uman

var

iabl

e im

mun

oglo

bulin

dom

ains

. Th

e 3

reco

mbi

nant

Nan

obod

ies (

NbC

EA5,

hum

aniz

ed sc

affol

d, a

nd h

uman

ized

CEA

5 gr

aft)

wer

e pr

oduc

ed in

bac

teri

a an

d pu

ri-

fied.

Unl

abel

ed a

nd (9

9m)T

c-la

bele

d N

anob

odie

s wer

e bi

oche

mic

ally

cha

ract

eriz

ed in

vitr

o an

d te

sted

as p

robe

s for

SPE

CT

/CT

of

xen

ogra

fted

tum

ors.

Res

ults

: The

succ

ess o

f loo

p-gr

aftin

g w

as c

onfir

med

by

com

pari

ng th

ese

Nan

obod

ies f

or th

eir c

apac

ity to

re

cogn

ize

solu

ble

CEA

pro

tein

in e

nzym

e-lin

ked

imm

unos

orbe

nt a

ssay

and

by

surf

ace

plas

mon

reso

nanc

e an

d to

bin

d to

CEA

-po

sitiv

e LS

174T

col

on c

arci

nom

a ce

lls a

nd C

EA-t

rans

fect

ed b

ut n

ot u

ntra

nsfe

cted

Chi

nese

ham

ster

ova

ry c

ells

in fl

ow c

ytom

etry

. Sp

ecifi

city

of b

indi

ng w

as c

onfir

med

by

com

petit

ion

stud

ies.

All

Nan

obod

ies w

ere

heat

-sta

ble,

cou

ld b

e effi

cien

tly la

bele

d w

ith

(99m

)Tc,

and

reco

gniz

ed b

oth

solu

ble

and

mem

bran

e-bo

und

CEA

pro

tein

in b

indi

ng st

udie

s. F

inal

ly, b

iodi

stri

butio

n ex

peri

men

ts

wer

e pe

rfor

med

with

intr

aven

ousl

y in

ject

ed (9

9m)T

c-la

bele

d N

anob

odie

s in

LS17

4T tu

mor

-bea

ring

mic

e us

ing

pinh

ole

SPEC

T/

mic

ro-C

T. Th

ese

in v

ivo

expe

rim

ents

reve

aled

spec

ifici

ty o

f tum

or ta

rget

ing

and

rapi

d re

nal c

lear

ance

for a

ll N

anob

odie

s, w

ith lo

w

sign

als i

n al

l org

ans b

esid

es th

e ki

dney

s. C

oncl

usio

n: Th

is st

udy

show

s the

pot

ency

of a

ntig

en-b

indi

ng lo

op-g

raft

ing

to e

ffici

ently

ge

nera

te h

uman

ized

Nan

obod

ies t

hat r

etai

n th

eir t

arge

ting

capa

citie

s for

non

inva

sive

in v

ivo

imag

ing

of tu

mor

s.

Vane

y-ck

en e

t al

. 201

0

Uni

vers

al h

uman

ized

na

nobo

dy s

caffo

ldN

anob

odie

s, si

ngle

-dom

ain

antig

en-b

indi

ng fr

agm

ents

of c

amel

id-s

peci

fic h

eavy

-cha

in o

nly

antib

odie

s offe

r spe

cial

adv

anta

ges

in th

erap

y ov

er c

lass

ic a

ntib

ody

frag

men

ts b

ecau

se o

f the

ir sm

alle

r siz

e, ro

bust

ness

, and

pre

fere

nce

to ta

rget

uni

que

epito

pes.

A

Nan

obod

y di

ffers

from

a h

uman

hea

vy c

hain

var

iabl

e do

mai

n in

abo

ut te

n am

ino

acid

s spr

ead

all o

ver i

ts su

rfac

e, fo

ur h

allm

ark

Nan

obod

y-sp

ecifi

c am

ino

acid

s in

the

fram

ewor

k-2

regi

on (p

ositi

ons 4

2, 4

9, 5

0, a

nd 5

2), a

nd a

long

er th

ird

antig

en-b

indi

ng lo

op

(H3)

fold

ing

over

this

are

a. F

or th

erap

eutic

app

licat

ions

the

cam

elid

-spe

cific

am

ino

acid

sequ

ence

s in

the

fram

ewor

k ha

ve to

be

mut

ated

to th

eir h

uman

hea

vy c

hain

var

iabl

e do

mai

n eq

uiva

lent

, i.e

. hum

aniz

ed. W

e pe

rfor

med

this

hum

aniz

atio

n ex

erci

se w

ith

Nan

obod

ies o

f the

subf

amily

that

repr

esen

ts c

lose

to 8

0% o

f all

drom

edar

y-de

rive

d N

anob

odie

s and

inve

stig

ated

the

effec

ts o

n an

tigen

affi

nity

, sol

ubili

ty, e

xpre

ssio

n yi

eld,

and

stab

ility

. It i

s dem

onst

rate

d th

at th

e hu

man

izat

ion

of N

anob

ody-

spec

ific

resi

dues

ou

tsid

e fr

amew

ork-

2 ar

e ne

utra

l to

the

Nan

obod

y pr

oper

ties.

Sur

pris

ingl

y, th

e G

lu-4

9 ->

Gly

and

Arg

-50

-> L

eu h

uman

izat

ion

of

hallm

ark

amin

o ac

ids g

ener

ates

a si

ngle

dom

ain

that

is m

ore

stab

le th

ough

pro

babl

y le

ss so

lubl

e. Th

e ot

her f

ram

ewor

k-2

subs

titu-

tions

, Phe

-42

-> V

al a

nd G

ly/A

la-5

2 ->

Trp

, are

det

rim

enta

l for

ant

igen

affi

nity

, due

to a

repo

sitio

ning

of t

he H

3 lo

op a

s sho

wn

by

thei

r cry

stal

stru

ctur

es. Th

ese

insi

ghts

wer

e us

ed to

iden

tify

a so

lubl

e, st

able

, wel

l exp

ress

ed u

nive

rsal

hum

aniz

ed N

anob

ody

scaf

-fo

ld th

at a

llow

s gra

fts o

f ant

igen

-bin

ding

loop

s fro

m o

ther

Nan

obod

ies w

ith tr

ansf

er o

f the

ant

igen

spec

ifici

ty a

nd a

ffini

ty.

Vin

cke

et a

l. 20

09


494

Tabl

e 7.

App

licat

ion

of s

ingl

e-do

mai

n an

tibo

dy fr

agm

ents

in d

iagn

osti

c an

d im

mun

oana

lyti

c m

etho

ds

Non

inva

sive

det

ecti

onTe

chne

tium

-99m

Ath

eros

cler

otic

pla

ques

Sing

le-p

hoto

n em

issi

on

com

pute

d to

mog

raph

y

We

aim

ed to

gen

erat

e, ra

diol

abel

, and

eva

luat

e an

ti-V

CA

M1

nano

bodi

es fo

r non

inva

sive

det

ectio

n of

ath

eros

cler

otic

lesi

ons.

Met

hods

and

Res

ults

: Ten

ant

i-VC

AM

1 na

nobo

dies

wer

e ge

nera

ted,

radi

olab

eled

with

tech

netiu

m-9

9m, a

nd sc

reen

ed in

vitr

o on

mou

se a

nd h

uman

reco

mbi

nant

VC

AM

1 pr

otei

ns a

nd e

ndot

helia

l cel

ls a

nd in

viv

o in

apo

lipop

rote

in E

-defi

cien

t (A

poE(

–/–)

) m

ice.

Ano

ntar

getin

g co

ntro

l nan

obod

y w

as u

sed

in a

ll ex

peri

men

ts to

dem

onst

rate

spec

ifici

ty. A

ll na

nobo

dies

dis

play

ed n

anom

olar

affi

nitie

s for

mur

ine

VC

AM

1. F

low

cyt

omet

ry a

naly

ses u

sing

hum

an h

uman

um

bilic

al v

ein

endo

thel

ial c

ells

indi

cate

d m

urin

e an

d hu

man

VC

AM

1 cr

oss-

reac

tivity

for 6

of 1

0 na

nobo

dies

. The

lead

com

poun

d cA

bVC

AM

1-5

was

cro

ss-r

eact

ive

for h

uman

VC

AM

1 an

d ex

hibi

ted

high

lesi

on-t

o-co

ntro

l (4.

95 ±

0.8

5), l

esio

n-to

-hea

rt (8

.30

± 1.

11),

and

lesi

on-t

o-bl

ood

ratio

s (4.

32 ±

0.4

8) (P

< 0

.05

vers

us c

ontr

ol C

57Bl

/6J m

ice)

. Aor

tic a

rch

athe

rosc

lero

tic le

sion

s of A

poE(

–/–)

mic

e w

ere

succ

essf

ully

iden

tified

by

sing

le-p

hoto

n em

issi

on c

ompu

ted

tom

ogra

phy

imag

ing.

Tc-

99m

-cA

bVC

AM

1-5

bind

ing

spec

ifici

ty w

as d

emon

stra

ted

by in

viv

o co

mpe

titio

n ex

peri

men

ts. A

utor

adio

grap

hy a

nd im

mun

ohis

toch

emis

try

furt

her c

onfir

med

cA

bVC

AM

1-5

upta

ke in

VC

AM

1-po

sitiv

e le

sion

s. C

oncl

usio

ns: Th

e Tc

-99m

-labe

led,

ant

i-VC

AM

1 na

nobo

dy c

AbV

CA

M1-

5 al

low

ed n

onin

vasi

ve d

etec

tion

of V

CA

M1

expr

essi

on

and

disp

laye

d m

ouse

and

hum

an c

ross

-rea

ctiv

ity. Th

eref

ore,

this

stud

y de

mon

stra

tes t

he p

oten

tial o

f nan

obod

ies a

s a n

ew c

lass

of

radi

otra

cers

for c

ardi

ovas

cula

r app

licat

ions

.

Broi

sat e

t al

. 201

2

Tum

our

upta

keTu

mou

r bu

rden

Epid

erm

al g

row

th fa

ctor

re

cept

orB

iolu

min

esce

nce

im

agin

g

We

stud

ied

the

rela

tions

hip

betw

een

tum

or u

ptak

e of

the

epid

erm

al g

row

th fa

ctor

rece

ptor

(EG

FR)-

spec

ific

nano

body

(99m

)Tc

-7C

12 a

nd tu

mor

bur

den

and

eval

uate

d th

e po

ssib

ility

of u

sing

this

pro

be to

mon

itor t

umor

resp

onse

to e

rlot

inib

. The

spec

ific-

ity a

nd a

ffini

ty o

f (99

m)T

c-7C

12 w

as d

eter

min

ed o

n A

431

cells

. Cel

ls e

xpre

ssin

g fir

efly

luci

fera

se w

ere

used

to e

valu

ate

tum

or

burd

en u

sing

bio

lum

ines

cenc

e im

agin

g. W

e ev

alua

ted

the

effec

t of e

rlot

inib

on

tum

or b

urde

n an

d (9

9m)T

c-7C

12 u

ptak

e in

vitr

o as

wel

l as i

n vi

vo. I

n vi

vo b

iolu

min

esce

nce

imag

ing

was

per

form

ed fo

llow

ed b

y pi

nhol

e si

ngle

-pho

ton

emis

sion

com

pute

d to

mog

-ra

phy/

mic

ro-c

ompu

ted

tom

ogra

phy.

(99m

)Tc-

7C12

bin

ds sp

ecifi

cally

to th

e re

cept

or w

ith h

igh

affini

ty (3

.67

± 0.

59 n

M).

Erlo

tinib

re

duce

d tu

mor

upt

ake

and

cell

viab

ility

in a

con

cent

ratio

n-de

pend

ent m

anne

r. Tu

mor

upt

ake

of (9

9m)T

c-7C

12 sh

owed

goo

d co

r-re

latio

n w

ith tu

mor

bur

den.

Erl

otin

ib tr

eatm

ent r

esul

ted

in a

pro

gres

sive

redu

ctio

n of

tum

or b

urde

n an

d tu

mor

upt

ake

of (9

9m)

Tc-7

C12

. (99

m)T

c-7C

12 b

inds

to E

GFR

with

hig

h affi

nity

and

spec

ifici

ty. T

umor

upt

ake

is c

orre

late

d w

ith tu

mor

bur

den.

Qua

ntifi

-ca

tion

of (9

9m)T

c-7C

12 u

ptak

e is

pro

mis

ing

for m

onito

ring

ther

apy

resp

onse

of E

GFR

-exp

ress

ing

tum

ors.

Gai

nkam

et

al.

2011

Nan

opro

beQ

uant

um d

ots

Flow

cyt

omet

ryC

arci

noem

bryo

nic

anti

gen

Bio

psy

sam

ples

Com

mon

stra

tegy

for d

iagn

ostic

s with

qua

ntum

dot

s (Q

Ds)

util

izes

the

spec

ifici

ty o

f mon

oclo

nal a

ntib

odie

s (m

Abs

) for

targ

etin

g.

How

ever

QD

-mA

bs c

onju

gate

s are

not

alw

ays w

ell-

suite

d fo

r thi

s pur

pose

bec

ause

of t

heir

larg

e si

ze. H

ere,

we

engi

neer

ed u

ltr-

asm

all n

anop

robe

s thr

ough

ori

ente

d co

njug

atio

n of

QD

s with

13-

kDa

sing

le-d

omai

n an

tibod

ies (

sdA

bs) d

eriv

ed fr

om ll

ama

IgG

. M

onom

eric

sdA

bs a

re 1

2 tim

es sm

alle

r tha

n m

Abs

and

dem

onst

rate

exc

elle

nt c

apac

ity fo

r ref

oldi

ng. s

dAbs

wer

e ta

gged

with

QD

s th

roug

h an

add

ition

al c

yste

ine

resi

due

inte

grat

ed w

ithin

the

C te

rmin

al o

f the

sdA

b. Th

is a

ppro

ach

allo

wed

us t

o de

velo

p sd

Abs

-Q

D n

anop

robe

s com

pris

ing

four

cop

ies o

f sdA

bs c

oupl

ed w

ith a

QD

in a

hig

hly

orie

nted

man

ner.

sdA

bs-Q

D c

onju

gate

s spe

cific

to

carc

inoe

mbr

yoni

c an

tigen

(CEA

) dem

onst

rate

d ex

celle

nt sp

ecifi

city

of fl

ow c

ytom

etry

qua

ntita

tive

disc

rim

inat

ion

of C

EA-p

ositi

ve

and

CEA

-neg

ativ

e tu

mor

cel

ls. M

oreo

ver,

the

imm

unoh

isto

chem

ical

labe

ling

of b

iops

y sa

mpl

es w

as fo

und

to b

e co

mpa

rabl

e or

ev

en su

peri

or to

the

qual

ity o

btai

ned

with

gol

d st

anda

rd p

roto

cols

of a

nato

mop

atho

logy

pra

ctic

e. sd

Abs

-QD

-ori

ente

d co

njug

ates

as

dev

elop

ed re

pres

ent a

new

gen

erat

ion

of u

ltras

mal

l dia

gnos

tic p

robe

s for

app

licat

ions

in h

igh-

thro

ughp

ut d

iagn

ostic

pla

tform

s.

Sukh

-an

ova

et

al. 2

012


495

Chr

omob

ody

Post

tran

slat

iona

l mod

i-fic

atio

ns

The

unde

rsta

ndin

g of

cel

lula

r pro

cess

es a

nd th

eir p

atho

phys

iolo

gica

l alte

ratio

ns re

quir

es c

ompr

ehen

sive

dat

a on

the

abun

danc

e,

dist

ribu

tion,

mod

ifica

tion,

and

inte

ract

ion

of a

ll ce

llula

r com

pone

nts.

On

the

one

hand

, art

ifici

ally

intr

oduc

ed fl

uore

scen

t fus

ion

prot

eins

pro

vide

info

rmat

ion

abou

t the

ir d

istr

ibut

ion

and

dyna

mic

s in

livin

g ce

lls b

ut n

ot a

bout

end

ogen

ous f

acto

rs. O

n th

e ot

her

hand

, ant

ibod

ies c

an d

etec

t end

ogen

ous p

rote

ins,

pos

ttra

nsla

tiona

l mod

ifica

tions

, and

oth

er c

ellu

lar c

ompo

nent

s but

mos

tly in

fix

ed a

nd p

erm

eabi

lized

cel

ls. H

ere

we

high

light

a n

ew te

chno

logy

bas

ed o

n th

e an

tigen

-bin

ding

dom

ain

of h

eavy

-cha

in a

ntib

od-

ies (

VH

H) f

rom

Cam

elid

ae. Th

ese

extr

emel

y st

able

VH

H d

omai

ns c

an b

e pr

oduc

ed in

bac

teri

a, c

oupl

ed to

mat

rice

s, a

nd u

sed

for

affini

ty p

urifi

catio

n an

d pr

oteo

me

stud

ies.

Alte

rnat

ivel

y, th

ese

VH

H d

omai

ns c

an b

e fu

sed

with

fluo

resc

ent p

rote

ins a

nd e

xpre

ssed

in

livi

ng c

ells

. Thes

e flu

ores

cent

ant

igen

-bin

ding

pro

tein

s cal

led

“chr

omob

odie

s” c

an b

e us

ed to

det

ect a

nd tr

ace

prot

eins

and

oth

er

cellu

lar c

ompo

nent

s in

vivo

. Chr

omob

odie

s can

, in

prin

cipl

e, d

etec

t any

ant

igen

ic st

ruct

ure,

incl

udin

g po

sttr

ansl

atio

nal m

odifi

-ca

tions

, and

ther

eby

dram

atic

ally

exp

and

the

qual

ity a

nd q

uant

ity o

f inf

orm

atio

n th

at c

an b

e ga

ther

ed in

hig

h-co

nten

t ana

lysi

s. D

epen

ding

on

the

epito

pe c

hose

n, c

hrom

obod

ies c

an a

lso

be u

sed

to m

odul

ate

prot

ein

func

tion

in li

ving

cel

ls.

Schm

idt-

ha

ls et

al.

2010

deG

radF

PG

reen

fluo

resc

ent p

ro-

tein

Prot

ein

knoc

kout

The

use

of g

enet

ic m

utat

ions

to st

udy

prot

ein

func

tions

in v

ivo

is a

cen

tral

par

adig

m o

f mod

ern

biol

ogy.

Rece

nt a

dvan

ces i

n re

vers

e ge

netic

s suc

h as

RN

A in

terf

eren

ce a

nd m

orph

olin

os a

re w

idel

y us

ed to

furt

her a

pply

this

par

adig

m. N

ever

thel

ess,

such

syst

ems a

ct

upst

ream

of t

he p

rote

ic le

vel,

and

prot

ein

depl

etio

n de

pend

s on

the

turn

over

rate

of t

he e

xist

ing

targ

et p

rote

ins.

Her

e w

e pr

esen

t de

Gra

dFP,

a g

enet

ical

ly e

ncod

ed m

etho

d fo

r dir

ect a

nd fa

st d

eple

tion

of ta

rget

gre

en fl

uore

scen

t pro

tein

(GFP

) fus

ions

in a

ny

euka

ryot

ic g

enet

ic sy

stem

. This

met

hod

is u

nive

rsal

bec

ause

it re

lies o

n an

evo

lutio

nari

ly h

ighl

y co

nser

ved

euka

ryot

ic fu

nctio

n, th

e ub

iqui

tin p

athw

ay. I

t is t

race

able

, bec

ause

the

GFP

tag

can

be u

sed

to m

onito

r the

pro

tein

kno

ckou

t. In

man

y ca

ses,

it is

a re

ady-

to-

use

solu

tion,

as G

FP p

rote

in-t

rap

stoc

k co

llect

ions

are

bei

ng g

ener

ated

in D

roso

phila

mel

anog

aste

r and

in D

anio

reri

o.

Cau

ssi-

nus e

t al.

2012

Alz

heim

er’s

dise

ase

Pept

ide

conf

orm

atio

nA

myl

oid

beta

Neu

roto

xici

ty

Neu

roto

xic

olig

omer

s of a

myl

oid

beta

(A b

eta)

pep

tide

have

bee

n in

crim

inat

ed in

the

path

ogen

esis

of A

lzhe

imer

’s di

seas

e. F

urth

er

expl

orat

ion

of th

is is

sue

has b

een

ham

pere

d to

this

dat

e by

the

fact

that

all

prev

ious

ly d

escr

ibed

ant

i-A

bet

a an

tibod

ies a

re u

nabl

e to

dis

crim

inat

e be

twee

n th

e di

ffere

nt c

onfo

rmat

ions

of t

he p

eptid

e (o

ligom

ers,

pro

tofib

rils

and

fibr

ils).

Her

e w

e de

scri

be th

e ge

n-er

atio

n of

nov

el c

amel

id si

ngle

-cha

in b

indi

ng d

omai

ns (V

HH

s) th

at re

cogn

izes

spec

ifica

lly lo

w m

olec

ular

-wei

ght (

MW

) olig

omer

s. Th

ree

VH

H sp

ecifi

c fo

r A b

eta

wer

e ob

tain

ed fr

om a

n im

mun

ized

alp

aca

phag

e di

spla

y lib

rary

. Tw

o w

ere

able

to re

cogn

ize

sele

c-tiv

ely

intr

aneu

rona

l A b

eta

olig

omer

s: fu

rthe

rmor

e, o

ne o

f the

m, V

31-1

, pre

vent

ed A

bet

a-in

duce

d ne

urot

oxic

ity a

nd in

hibi

ted

fibri

l for

mat

ion.

This

stud

y co

nfirm

s tha

t VH

Hs m

ay re

cogn

ize

non-

conv

entio

nal e

pito

pes a

nd il

lust

rate

s the

ir p

oten

tial f

or th

e im

mun

odia

gnos

tic o

f dis

ease

s due

to p

rote

in a

ccum

ulat

ion.

Lafa

ye e

t al

. 200

9


496

Hum

aniz

ed n

anob

odie

sLo

op-g

raft

ing

In v

ivo

imag

ing

Scaff

old

Car

cino

embr

yoni

c an

tige

n

Nan

obod

ies a

re a

nov

el ty

pe o

f im

mun

oglo

bulin

like,

ant

igen

-bin

ding

pro

tein

with

ben

efici

al p

harm

acol

ogic

and

pha

rmac

okin

etic

pr

oper

ties t

hat a

re id

eally

suite

d to

targ

etin

g ce

llula

r ant

igen

s for

mol

ecul

ar im

agin

g or

ther

apeu

tic p

urpo

ses.

How

ever

, bec

ause

of

thei

r cam

elid

, non

hum

an o

rigi

n, th

e po

ssib

le im

mun

ogen

icity

of N

anob

odie

s whe

n us

ed in

the

clin

ic is

a c

once

rn. H

ere

we

pres

ent

a ne

w st

rate

gy to

qui

ckly

gen

erat

e hu

man

ized

Nan

obod

ies f

or m

olec

ular

imag

ing

purp

oses

. Met

hods

: We

gene

tical

ly g

raft

ed th

e an

tigen

-bin

ding

loop

s of N

bCEA

5, a

Nan

obod

y w

ith sp

ecifi

city

for t

he c

olon

car

cino

ma

mar

ker c

arci

noem

bryo

nic

antig

en (C

EA),

onto

the

fram

ewor

k of

a h

uman

ized

Nan

obod

y sc

affol

d. Th

is sc

affol

d ha

s bee

n pr

evio

usly

cha

ract

eriz

ed in

our

labo

rato

ry a

s a

stab

le N

anob

ody

that

can

serv

e as

a u

nive

rsal

loop

acc

epto

r for

ant

igen

-bin

ding

loop

s fro

m d

onor

Nan

obod

ies a

nd h

as b

een

addi

-tio

nally

mut

ated

at a

bout

10

cruc

ial s

urfa

ce-e

xpos

ed si

tes t

o re

sem

ble

the

sequ

ence

of h

uman

var

iabl

e im

mun

oglo

bulin

dom

ains

. Th

e 3

reco

mbi

nant

Nan

obod

ies (

NbC

EA5,

hum

aniz

ed sc

affol

d, a

nd h

uman

ized

CEA

5 gr

aft)

wer

e pr

oduc

ed in

bac

teri

a an

d pu

ri-

fied.

Unl

abel

ed a

nd (9

9m)T

c-la

bele

d N

anob

odie

s wer

e bi

oche

mic

ally

cha

ract

eriz

ed in

vitr

o an

d te

sted

as p

robe

s for

SPE

CT

/CT

of

xen

ogra

fted

tum

ors.

Res

ults

: The

succ

ess o

f loo

p-gr

aftin

g w

as c

onfir

med

by

com

pari

ng th

ese

Nan

obod

ies f

or th

eir c

apac

ity to

re

cogn

ize

solu

ble

CEA

pro

tein

in e

nzym

e-lin

ked

imm

unos

orbe

nt a

ssay

and

by

surf

ace

plas

mon

reso

nanc

e an

d to

bin

d to

CEA

-po

sitiv

e LS

174T

col

on c

arci

nom

a ce

lls a

nd C

EA-t

rans

fect

ed b

ut n

ot u

ntra

nsfe

cted

Chi

nese

ham

ster

ova

ry c

ells

in fl

ow c

ytom

etry

. Sp

ecifi

city

of b

indi

ng w

as c

onfir

med

by

com

petit

ion

stud

ies.

All

Nan

obod

ies w

ere

heat

-sta

ble,

cou

ld b

e effi

cien

tly la

bele

d w

ith

(99m

)Tc,

and

reco

gniz

ed b

oth

solu

ble

and

mem

bran

e-bo

und

CEA

pro

tein

in b

indi

ng st

udie

s. F

inal

ly, b

iodi

stri

butio

n ex

peri

men

ts

wer

e pe

rfor

med

with

intr

aven

ousl

y in

ject

ed (9

9m)T

c-la

bele

d N

anob

odie

s in

LS17

4T tu

mor

-bea

ring

mic

e us

ing

pinh

ole

SPEC

T/

mic

ro-C

T. Th

ese

in v

ivo

expe

rim

ents

reve

aled

spec

ifici

ty o

f tum

or ta

rget

ing

and

rapi

d re

nal c

lear

ance

for a

ll N

anob

odie

s, w

ith lo

w

sign

als i

n al

l org

ans b

esid

es th

e ki

dney

s. C

oncl

usio

n: Th

is st

udy

show

s the

pot

ency

of a

ntig

en-b

indi

ng lo

op-g

raft

ing

to e

ffici

ently

ge

nera

te h

uman

ized

Nan

obod

ies t

hat r

etai

n th

eir t

arge

ting

capa

citie

s for

non

inva

sive

in v

ivo

imag

ing

of tu

mor

s.

Vane

y-ck

en e

t al

. 201

0

HER

-2B

reas

t can

cer

Non

inva

sive

imag

ing

Acc

urat

e de

term

inat

ion

of tu

mor

hum

an e

pide

rmal

gro

wth

fact

or re

cept

or 2

(HER

2)-s

tatu

s in

brea

st c

ance

r pat

ient

s is p

ossi

ble

via

noni

nvas

ive

imag

ing,

pro

vide

d ad

equa

te tr

acer

s are

use

d. In

this

stud

y, w

e de

scri

be th

e ge

nera

tion

of a

pan

el o

f 38

nano

bodi

es,

smal

l HER

2-bi

ndin

g fr

agm

ents

that

are

der

ived

from

hea

vy-c

hain

-onl

y an

tibod

ies r

aise

d in

an

imm

uniz

ed d

rom

edar

y. In

sear

ch

of a

lead

com

poun

d, a

subs

et o

f nan

obod

ies w

as b

ioch

emic

ally

cha

ract

eriz

ed in

dep

th a

nd p

recl

inic

ally

test

ed fo

r use

as t

race

rs

for i

mag

ing

of x

enog

raft

ed tu

mor

s. Th

e se

lect

ed c

ompo

und,

2Rs

15d,

was

foun

d to

be

stab

le a

nd to

inte

ract

spec

ifica

lly w

ith H

ER2

reco

mbi

nant

pro

tein

and

HER

2-ex

pres

sing

cel

ls in

ELI

SA, s

urfa

ce p

lasm

on re

sona

nce,

flow

cyt

omet

ry, a

nd ra

diol

igan

d bi

ndin

g st

udie

s with

low

nan

omol

ar a

ffini

ties,

and

did

not

com

pete

with

ant

i-HER

2 th

erap

eutic

ant

ibod

ies t

rast

uzum

ab a

nd p

ertu

zum

ab.

Sing

le-p

hoto

n-em

issi

on c

ompu

ted

tom

ogra

phy

(SPE

CT

) im

agin

g qu

antifi

catio

n an

d bi

odis

trib

utio

n an

alys

es sh

owed

that

(99m

)Tc

-labe

led

2Rs1

5d h

as a

hig

h tu

mor

upt

ake

in 2

HER

2+ tum

or m

odel

s, fa

st b

lood

cle

aran

ce, l

ow a

ccum

ulat

ion

in n

onta

rget

org

ans

exce

pt k

idne

ys, a

nd h

igh

conc

omita

nt tu

mor

-to-

bloo

d an

d tu

mor

-to-

mus

cle

ratio

s at 1

h a

fter

intr

aven

ous i

njec

tion.

Thes

e va

lues

w

ere

dram

atic

ally

low

er fo

r an

irre

leva

nt c

ontr

ol (9

9m)T

c-na

nobo

dy a

nd fo

r (99

m)T

c-2R

s15d

targ

etin

g a

HER

2– tum

or.

Vane

y-ck

en e

t al

. 201

1b


497

VH

H-b

ased

imm

unoa

ssay

3-ph

enox

yben

zoic

aci

dP

yret

hroi

d in

sect

icid

es

We

expr

esse

d V

HH

s fro

m a

n im

mun

ized

alp

aca

and

deve

lope

d a

VH

H-b

ased

imm

unoa

ssay

usi

ng 3

-phe

noxy

benz

oic

acid

(3-P

BA),

a m

ajor

met

abol

ite o

f pyr

ethr

oid

inse

ctic

ides

as a

mod

el sy

stem

. A p

hage

VH

H li

brar

y w

as c

onst

ruct

ed, a

nd se

ven

VH

H c

lone

s w

ere

sele

cted

by

com

petit

ive

bind

ing

with

3-P

BA. Th

e be

st im

mun

oass

ay d

evel

oped

with

one

of t

hese

VH

Hs s

how

ed a

n IC

50 o

f 1.

4 ng

/ml (

limit

of d

etec

tion

(LO

D) =

0.1

ng/

ml).

Thes

e pa

ram

eter

s wer

e fu

rthe

r im

prov

ed b

y us

ing

the

phag

e bo

rne

VH

H, I

C50

=

0.1

ng/m

l and

LO

D =

0.0

1 ng

/ml.

Both

ass

ays s

how

ed a

sim

ilar t

oler

ance

to m

etha

nol a

nd d

imet

hyls

ulfo

xide

up

to 5

0% in

ass

ay

buffe

r. Th

e as

say

was

hig

hly

spec

ific

to 3

-PBA

and

its 4

-hyd

roxy

late

d de

riva

tive,

4-h

ydro

xy 3

-PBA

, (15

0% c

ross

reac

tivity

) with

neg

-lig

ible

cro

ss re

activ

ity w

ith o

ther

test

ed st

ruct

ural

ana

logu

es, a

nd th

e re

cove

ry fr

om sp

iked

uri

ne sa

mpl

e ra

nged

from

80

to 1

12%

. In

con

clus

ion,

a h

ighl

y sp

ecifi

c an

d se

nsiti

ve V

HH

for 3

-PBA

was

dev

elop

ed u

sing

sequ

ence

s fro

m im

mun

ized

alp

aca

and

phag

e di

spla

y te

chno

logy

for a

ntib

ody

sele

ctio

n.

Kim

et a

l. 20

12

Bot

ulin

um n

euro

toxi

nTo

xin

neut

raliz

ing

ca

pabi

lity

Hig

h te

mpe

ratu

res

Back

grou

nd: Th

ere

are

curr

ently

7 k

now

n se

roty

pes o

f bot

ulin

um n

euro

toxi

n (B

oNT

) cla

ssifi

ed u

pon

non-

cros

s rea

ctiv

ity o

f ne

utra

lizin

g im

mun

oglo

bulin

s. N

on-n

eutr

aliz

ing

imm

unog

lobu

lins,

how

ever

, can

exh

ibit

cros

s-re

activ

ities

bet

wee

n 2

or m

ore

sero

type

s, p

artic

ular

ly m

osai

c fo

rms,

whi

ch c

an h

ampe

r the

dev

elop

men

t of h

ighl

y sp

ecifi

c im

mun

oass

ays,

esp

ecia

lly if

bas

ed o

n po

lycl

onal

ant

iser

a. H

ere

we

empl

oy fa

cile

reco

mbi

nant

ant

ibod

y te

chno

logy

to su

btra

ctiv

ely

sele

ct li

gand

s to

each

of t

he 7

BoN

T

sero

type

s, re

sulti

ng in

pop

ulat

ions

with

ver

y hi

gh sp

ecifi

city

for t

heir

inte

nded

sero

type

. Met

hods

and

Fin

ding

s: A

sing

le ll

ama

was

imm

uniz

ed w

ith a

coc

ktai

l of 7

BoN

T to

xoid

s to

gene

rate

a p

hage

dis

play

libr

ary

of si

ngle

dom

ain

antib

odie

s (sd

Ab,

VH

H o

r na

nobo

dies

) whi

ch w

ere

sele

cted

on

live

toxi

ns. R

esul

ting

sdA

b w

ere

capa

ble

of d

etec

ting

both

toxi

n an

d to

xin

com

plex

with

the

best

com

bina

tions

abl

e to

det

ect 1

00s-

10s o

f pg

per 5

0 µl

sam

ple

in a

liqu

id b

ead

arra

y. Th

e m

ost s

ensi

tive

sdA

b w

ere

com

bine

d in

a

hept

aple

x as

say

to id

entif

y ea

ch o

f the

BoN

T se

roty

pes i

n bu

ffer a

nd m

ilk a

nd to

a le

sser

ext

ent i

n ca

rrot

juic

e, o

rang

e ju

ice

and

cola

. Sev

eral

ant

i-A(1

) sdA

b re

cogn

ized

A2

com

plex

, sho

win

g th

at su

btyp

e cr

oss-

reac

tivity

with

in a

sero

type

was

evi

dent

. Man

y of

our

sdA

b co

uld

act a

s bot

h ca

ptor

and

trac

er fo

r sev

eral

toxi

n an

d to

xin

com

plex

es su

gges

ting

sdA

b ca

n be

use

d as

arc

hite

c-tu

ral p

robe

s to

indi

cate

BoN

T o

ligom

eris

atio

n. S

ix o

f 14

anti-

A c

lone

s exh

ibite

d in

hibi

tion

of S

NA

P-25

cle

avag

e in

the

neur

o-2A

as

say

indi

catin

g so

me

sdA

b ha

d to

xin

neut

raliz

ing

capa

bilit

ies.

Man

y sd

Ab

wer

e al

so sh

own

to b

e re

fold

able

aft

er e

xpos

ure

to h

igh

tem

pera

ture

s in

cont

rast

to p

olyc

lona

l ant

iser

a, a

s mon

itore

d by

cir

cula

r dic

hroi

sm. C

oncl

usio

ns: O

ur p

anel

of m

olec

ular

ly fl

exib

le

antib

odie

s sho

uld

not o

nly

serv

e as

a g

ood

star

ting

poin

t for

rugg

ediz

ing

assa

ys a

nd in

hibi

tors

, but

ena

ble

the

intr

icat

e ar

chite

c-tu

res o

f BoN

T to

xins

and

com

plex

es to

be

prob

ed m

ore

exte

nsiv

ely.

Con

way

et

al.

2010

ELIS

ATo

xin

SdA

b-al

kalin

e

phos

phat

ase

fusi

on

Nai

ve li

brar

ies o

f sin

gle

dom

ain

antib

odie

s (sd

Abs

) ena

ble

rapi

d is

olat

ion

of b

inde

rs to

nea

rly

any

targ

et. Th

ese

bind

ers,

how

ever

, la

ck th

e be

nefit

s bes

tow

ed b

y in

viv

o affi

nity

mat

urat

ion

and

typi

cally

hav

e lo

w a

ffini

ty to

war

d th

eir t

arge

ts. W

e ex

pres

sed

five

low

-affi

nity

toxi

n bi

ndin

g sd

Abs

, pre

viou

sly

sele

cted

from

a n

aive

libr

ary

deri

ved

from

var

iabl

e re

gion

s of l

lam

a he

avy

chai

n-on

ly

antib

odie

s, a

s fus

ions

with

a h

yper

activ

e m

utan

t Esc

heri

chia

col

i alk

alin

e ph

osph

atas

e (A

P) a

nd e

xam

ined

the

impa

ct o

n ap

pare

nt

affini

ty a

nd u

tility

. AP

spon

tane

ousl

y di

mer

izes

in so

lutio

n, e

ffect

ivel

y di

mer

izin

g th

e fu

sed

sdA

bs, i

mpa

rtin

g av

idity

in p

lace

of t

he

low

er a

ffini

ty m

onom

eric

inte

ract

ions

. The

sdA

b-A

P fu

sion

als

o co

mbi

nes t

he ta

rget

reco

gniti

on d

omai

n w

ith a

sign

al tr

ansd

uctio

n do

mai

n, c

omm

only

use

d in

enz

yme-

linke

d im

mun

osor

bent

ass

ays (

ELIS

As)

. The

func

tiona

l affi

nity

of t

he sd

Ab-

AP

fusi

ons,

oft

en

incr

ease

d by

a fa

ctor

of 1

0 ov

er u

nfus

ed sd

Abs

, and

thei

r util

ity a

s tra

cer r

eage

nts i

n EL

ISA

s was

dra

mat

ical

ly im

prov

ed, g

ivin

g lim

its o

f det

ectio

n of

300

ng/

ml o

r les

s, w

here

as p

aren

tal s

dAbs

gav

e no

dis

cern

ible

sign

al a

t the

toxi

n co

ncen

trat

ions

test

ed. Th

e fu

sion

of s

dAbs

to A

P pr

esen

ts a

val

uabl

e ro

ute

to fa

cilit

ate

the

impl

emen

tatio

n of

sdA

b-ba

sed

imm

unor

eage

nts r

apid

ly se

lect

ed

from

exi

stin

g na

ive

libra

ries

tow

ard

new

or e

mer

ging

thre

ats.

Swai

n et

al

. 201

1


498

ELIS

AR

icin

Bot

ulin

um A

toxi

nC

y-3

fluor

esce

nt d

ye

Prev

ious

ly, w

e se

lect

ed sd

Ab

that

wer

e sp

ecifi

c fo

r bot

h ri

cin

and

botu

linum

A (B

oNT

A) t

oxin

com

plex

from

pha

ge d

ispl

ay li

brar

-ie

s of s

dAb

and

eval

uate

d th

e so

lubl

y ex

pres

sed

prot

ein.

Her

e, p

hage

-dis

play

ed sd

Ab

wer

e us

ed a

s rep

orte

r rea

gent

s and

com

pare

d to

solu

ble,

unf

used

sdA

b. W

e fo

und

that

usi

ng p

hage

-dis

play

ed sd

Ab

as re

port

er e

lem

ents

in im

mun

oass

ay fo

rmat

s gav

e im

prov

ed

dete

ctio

n ov

er u

sing

unf

used

, sol

uble

sdA

b re

port

ers.

In e

nzym

e-lin

ked

imm

unos

orbe

nt a

ssay

s (EL

ISA

s), t

he lo

wes

t lev

el o

f tox

in

dete

cted

for b

oth

rici

n an

d Bo

NT

A to

xoid

com

plex

was

dec

reas

ed b

y on

e to

two

orde

rs o

f mag

nitu

de u

sing

pha

ge-d

ispl

ayed

sdA

b as

repo

rter

reag

ents

. Use

of t

he p

hage

pre

serv

ed th

e ab

ility

to d

iscr

imin

ate

rici

n an

d RC

A12

0 by

at l

east

a fa

ctor

of 1

0 fo

ld. I

n an

eff

ort t

o re

duce

the

num

ber o

f ste

ps in

the

assa

ys, w

e di

rect

ly la

bele

d ph

age

disp

layi

ng sd

Ab

with

a C

y-3

fluor

esce

nt d

ye. S

igna

l w

as g

reat

ly d

ecre

ased

usi

ng th

e dy

e-la

bele

d ph

age

com

pare

d to

bio

tinyl

ated

pha

ge fo

llow

ed b

y st

rept

avid

in-p

hyco

eryt

hrin

. In

thes

e as

says

the

use

of p

hage

-dis

play

ed sd

Ab

give

s mor

e se

nsiti

ve d

etec

tion

than

solu

ble

sdA

b al

one,

how

ever

dir

ectly

dye

labe

ling

the

phag

e fa

iled

to p

rovi

de re

spon

ses o

f a si

mila

r mag

nitu

de.

Gol

dman

et

al.

2010

Ant

ibod

y m

icro

arra

ysSu

rfac

e ex

pres

sion

Fibr

obla

st g

row

th fa

ctor

re

cept

or 1

The

prep

arat

ion

of e

ffect

ive

conv

entio

nal a

ntib

ody

mic

roar

rays

dep

ends

on

the

avai

labi

lity

of h

igh

qual

ity m

ater

ial a

nd o

n th

e co

rrec

t acc

essi

bilit

y of

the

antib

ody

activ

e m

oiet

ies f

ollo

win

g th

eir i

mm

obili

zatio

n on

the

supp

ort s

lide.

We

show

that

spot

ting

bact

eria

that

exp

ose

reco

mbi

nant

ant

ibod

ies o

n th

eir e

xter

nal s

urfa

ce d

irec

tly o

n na

nost

ruct

ured

-TiO

(2) o

r epo

xy sl

ides

(pur

i-fic

atio

n-in

depe

nden

t mic

roar

ray

– PI

M) i

s a si

mpl

e an

d re

liabl

e al

tern

ativ

e fo

r pre

pari

ng se

nsiti

ve a

nd sp

ecifi

c m

icro

arra

ys fo

r an

tigen

det

ectio

n. V

aria

ble

dom

ains

of s

ingl

e he

avy-

chai

n an

tibod

ies (

VH

Hs)

aga

inst

fibr

obla

st g

row

th fa

ctor

rece

ptor

1 (F

GFR

1)

wer

e us

ed to

cap

ture

the

antig

en d

ilute

d in

seru

m o

r BSA

solu

tion.

The

FGFR

1 de

tect

ion

was

per

form

ed b

y ei

ther

dir

ect a

ntig

en

labe

ling

or u

sing

a sa

ndw

ich

syst

em in

whi

ch F

GFR

1 w

as fi

rst b

ound

to it

s ant

ibod

y an

d su

cces

sive

ly id

entifi

ed u

sing

a la

bele

d FG

F.

In b

oth

case

s the

sign

al d

istr

ibut

ion

with

in e

ach

spot

was

uni

form

and

spot

mor

phol

ogy

regu

lar.

The

sign

al-t

o-no

ise

ratio

of t

he

sign

al w

as e

xtre

mel

y el

evat

ed a

nd th

e sp

ecifi

city

of t

he sy

stem

was

pro

ved

stat

istic

ally

. The

LOD

of t

he sy

stem

for t

he a

ntig

en w

as

calc

ulat

ed b

eing

0.4

ng/

ml a

nd th

e dy

nam

ic ra

nge

betw

een

0.4

ng/m

l and

10

μg/m

l. Th

e m

icro

arra

ys p

repa

red

with

bac

teri

a ex

pos-

ing

antib

odie

s rem

ain

fully

func

tiona

l for

at l

east

31

clay

s aft

er sp

ottin

g. W

e fin

ally

dem

onst

rate

d th

at th

e m

etho

d is

suita

ble

for

othe

r ant

igen

-ant

ibod

y pa

irs a

nd e

xpec

t tha

t it c

ould

be

easi

ly a

dapt

ed to

furt

her a

pplic

atio

ns su

ch a

s the

dis

play

of s

cFv

and

IgG

an

tibod

ies o

r the

aut

oant

ibod

y de

tect

ion

usin

g pr

otei

n.

De

Mar

ni e

t al

. 201

2

Com

peti

tive

ass

ayTr

iclo

carb

anU

sing

tric

loca

rban

(TC

C) a

s a m

odel

hap

ten,

we

foun

d th

at c

onve

ntio

nal a

ntib

odie

s, Ig

G1

frac

tion,

reac

ted

with

free

TC

C w

ith a

hi

gher

rela

tive

affini

ty (I

C(5

0) 5

1.0

ng/m

l) th

an d

id th

e sd

Abs

(IgG

2 an

d Ig

G3,

497

and

370

ng/

ml,

resp

ectiv

ely)

. A V

HH

libr

ary

was

pre

pare

d, a

nd b

y el

utio

n of

pha

ge w

ith li

miti

ng c

once

ntra

tions

of T

CC

and

com

petit

ive

sele

ctio

n of

bin

ders

, we

wer

e ab

le to

is

olat

e hi

gh-a

ffini

ty c

lone

s, K

(D) 0

.98–

1.37

nM

(SPR

), w

hich

allo

wed

dev

elop

men

t of a

com

petit

ive

assa

y fo

r TC

C w

ith a

n IC

(50)

=

3.5

ng/m

l (11

nM

). Th

is re

pres

ents

a 1

00-f

old

impr

ovem

ent w

ith re

gard

to th

e pe

rfor

man

ce o

f the

sdA

b se

rum

frac

tion,

and

it

is 1

00-f

old

bett

er th

an th

e IC

(50)

att

aine

d w

ith o

ther

ant

ihap

ten

VH

Hs r

epor

ted

thus

far.

Des

pite

the

mod

est o

vera

ll an

tihap

ten

sdA

bs re

spon

se in

llam

as, a

smal

l sub

popu

latio

n of

hig

h-affi

nity

VH

Hs i

s gen

erat

ed th

at c

an b

e is

olat

ed b

y ca

refu

l des

ign

of th

e se

lect

ion

proc

ess.

Taba

res-

da R

osa

et a

l. 20

11


499

Den

atur

ing

agen

tsC

elia

c pa

tien

tsPr

olam

inG

liadi

nEL

ISA

Food

inte

nded

for c

elia

c pa

tient

s’ co

nsum

ptio

n m

ust b

e an

alyz

ed fo

r the

pre

senc

e of

toxi

c pr

olam

ins u

sing

hig

h de

lect

abili

ty te

sts.

Thou

gh 6

0% e

than

ol is

the

mos

t com

mon

ly u

sed

solv

ent f

or p

rola

min

s ext

ract

ion,

2-m

erca

ptoe

than

ol (2

-ME)

and

gua

nidi

nium

chl

o-ri

de (G

uHC

l) ca

n be

add

ed to

incr

ease

pro

tein

reco

very

. How

ever

, eth

anol

and

den

atur

ing

agen

ts in

terf

ere

with

ant

igen

reco

gniti

on

whe

n co

nven

tiona

l ant

ibod

ies a

re u

sed.

In th

e pr

esen

t wor

k, a

new

met

hod

for g

liadi

ns q

uant

ifica

tion

is sh

own.

The

met

hod

is b

ased

on

the

sele

ctio

n of

llam

a si

ngle

dom

ain

antib

ody

frag

men

ts a

ble

to o

pera

te u

nder

den

atur

ing

cond

ition

s. Si

x ou

t of 2

8 V

HH

-pha

ges

obta

ined

reta

ined

thei

r bin

ding

cap

acity

in 1

5% e

than

ol. S

elec

ted

clon

es p

rese

nted

a lo

ng C

DR3

regi

on c

onta

inin

g tw

o ad

ditio

nal

cyst

eine

s tha

t cou

ld b

e re

spon

sibl

e fo

r the

hig

her s

tabi

lity.

One

of t

he c

lone

s (na

med

VH

H26

) was

fully

ope

rativ

e in

the

pres

ence

of

15%

eth

anol

, 0.5

% 2

-ME,

and

0.5

M G

uHC

l. C

aptu

re E

LISA

usi

ng V

HH

26 w

as a

ble

to d

etec

t glia

dins

in sa

mpl

es sh

own

as n

egat

ives

by

con

vent

iona

l ELI

SA. Th

eref

ore,

this

new

stra

tegy

app

ears

as a

n ex

celle

nt p

latfo

rm fo

r qua

ntita

tive

dete

rmin

atio

n of

pro

tein

s or a

ny

othe

r im

mun

ogen

ic c

ompo

und,

in th

e pr

esen

ce o

f den

atur

ing

agen

ts, w

hen

spec

ific

reco

gniti

on u

nits

with

hig

h st

abili

ty a

re re

quire

d.

Don

a et

al

. 201

0

ELIS

ATa

enia

sol

ium

Cys

tice

rcos

is

Taen

ia so

lium

cys

ticer

cosi

s is a

maj

or h

elm

inth

zoo

nosi

s in

deve

lopi

ng c

ount

ries

. Pig

s are

the

inte

rmed

iate

hos

ts m

edia

ting

tran

s-m

issi

on o

f inf

ectio

n. S

peci

fic a

ssay

s to

diag

nose

livi

ng c

ysts

in p

igs a

re la

ckin

g. Th

e m

onod

onal

-bas

ed a

ntig

en d

etec

tion

ELIS

A is

ge

nus-

spec

ific

and

cros

s-re

actio

ns w

ith T

aeni

a hy

datig

ena

ham

per t

he u

se o

f thi

s tes

t to

scre

en p

igs.

We,

ther

efor

e, a

imed

to in

tro-

duce

nan

obod

ies,

cam

elid

-der

ived

sing

le-d

omai

n an

tibod

ies s

peci

fic fo

r T. s

oliu

m c

ystic

erco

sis,

to d

evel

op u

nam

bigu

ous t

ests

. N

anob

odie

s wer

e cl

oned

follo

win

g im

mun

izat

ion

of tw

o dr

omed

arie

s with

T. s

oliu

m a

ntig

en a

nd e

ight

T. s

oliu

m-s

peci

fic n

ano-

bodi

es w

ere

sele

cted

aft

er p

hage

dis

play

. Thei

r bin

ding

cha

ract

eris

tics a

nd p

oten

tial f

or th

e di

agno

sis o

f por

cine

cys

ticer

cosi

s wer

e in

vest

igat

ed. Th

e na

nobo

dies

do

not c

ross

-rea

ct w

ith T

hyd

atig

ena,

Tae

nia

sagi

nata

, Tae

nia

cras

sice

ps o

r Tri

chin

ella

spir

alis

and

w

ere

cate

gori

zed

into

four

epi

tope

-bin

ding

gro

ups.

The

targ

et p

rote

in w

as id

entifi

ed a

s 14

kDa

diag

nost

ic g

lyco

prot

ein

(Ts1

4), b

ut

the

nano

bodi

es a

lso

reac

ted

with

oth

er p

rote

ins o

f the

sam

e fa

mily

. Nan

obod

ies w

ere

test

ed in

a sa

ndw

ich

ELIS

A w

ith c

yst fl

uid,

an

d on

e pa

rtic

ular

nan

obod

y de

tect

ed it

s cog

nate

seru

m a

ntig

ens i

n a

spec

ies-

spec

ific

inhi

bitio

n EL

ISA

. Con

side

ring

thei

r ben

efi-

cial

pro

duct

ion

and

stab

ility

pro

pert

ies,

thes

e hi

ghly

spec

ific

nano

bodi

es c

onst

itute

a p

rom

isin

g to

ol to

dia

gnos

e cy

stic

erco

sis a

fter

fu

rthe

r im

prov

emen

t of t

he se

nsiti

vity

and

futu

re a

ssay

val

idat

ion.

Dec

k-er

s et a

l. 20

09

Bio

sens

orEb

olav

irus

Clin

ical

and

en

viro

nmen

tal

sam

ples

IgN

AR

Hae

mor

rhag

ic fe

ver

Mem

bers

of t

he g

enus

Ebo

lavi

rus c

ause

fulm

inat

ing

outb

reak

s of d

isea

se in

hum

an a

nd n

on-h

uman

pri

mat

e po

pula

tions

with

a

mor

talit

y ra

te u

p to

90%

. To

faci

litat

e ra

pid

dete

ctio

n of

thes

e pa

thog

ens i

n cl

inic

al a

nd e

nvir

onm

enta

l sam

ples

, rob

ust r

eage

nts

capa

ble

of p

rovi

ding

sens

itive

and

spec

ific

dete

ctio

n ar

e re

quir

ed. I

n th

is w

ork

reco

mbi

nant

ant

ibod

y lib

rari

es w

ere

gene

rate

d fr

om

mur

ine

(sin

gle

chai

n va

riab

le d

omai

n fr

agm

ent;

scFv

) and

nur

se sh

ark,

Gin

glym

osto

ma

cirr

atum

(IgN

AR

V) h

osts

imm

unis

ed

with

Zai

re e

bola

viru

s. Th

is p

rovi

des t

he fi

rst r

ecor

ded

IgN

AR

V re

spon

se a

gain

st a

par

ticul

ate

antig

en in

the

nurs

e sh

ark.

Bot

h m

urin

e sc

Fv a

nd sh

ark

IgN

AR

V li

brar

ies w

ere

pann

ed b

y ph

age

disp

lay

tech

nolo

gy to

iden

tify

usef

ul a

ntib

odie

s for

the

gene

ratio

n of

imm

unol

ogic

al d

etec

tion

reag

ents

. Tw

o m

urin

e sc

Fv w

ere

show

n to

hav

e sp

ecifi

city

to th

e Z

aire

ebo

lavi

rus v

iral

mat

rix

prot

ein

VP4

0. T

wo

isol

ated

IgN

AR

V w

ere

show

n to

bin

d to

the

vira

l nuc

leop

rote

in (N

P) a

nd to

cap

ture

via

ble

Zai

re e

bola

viru

s with

a h

igh

degr

ee o

f sen

sitiv

ity. A

ssay

s dev

elop

ed w

ith Ig

NA

R V

cro

ss-r

eact

ed to

Res

ton

ebol

avir

us, S

udan

ebo

lavi

rus a

nd B

undi

bugy

o eb

ola-

viru

s. D

espi

te th

is b

road

reac

tivity

, nei

ther

of I

gNA

R V

show

ed re

activ

ity to

Cot

e di

voir

e eb

olav

irus

. IgN

AR

V w

as su

bsta

ntia

lly

mor

e re

sist

ant t

o ir

reve

rsib

le th

erm

al d

enat

urat

ion

than

mur

ine

scFv

and

mon

oclo

nal I

gG in

a c

ompa

rativ

e te

st. Th

e de

mon

stra

ble

robu

stne

ss o

f the

IgN

AR

V d

omai

ns m

ay o

ffer e

nhan

ced

utili

ty a

s im

mun

olog

ical

det

ectio

n re

agen

ts in

fiel

dabl

e bi

osen

sor a

pplic

a-tio

ns fo

r use

in tr

opic

al o

r sub

trop

ical

cou

ntri

es w

here

out

brea

ks o

f Ebo

lavi

rus h

aem

orrh

agic

feve

r occ

ur.

Goo

d-ch

ild e

t al

. 201

1


500

Bio

sens

orD

ecab

ody

Cel

lulo

se fi

lter

sS.

aur

eus

Ant

ibod

y en

gine

erin

g ha

s allo

wed

for t

he ra

pid

gene

ratio

n of

bin

ding

age

nts a

gain

st v

irtu

ally

any

ant

igen

of i

nter

est,

pred

omi-

nant

ly fo

r the

rape

utic

app

licat

ions

. Con

side

rabl

y le

ss a

tten

tion

has b

een

give

n to

the

deve

lopm

ent o

f dia

gnos

tic re

agen

ts a

nd b

io-

sens

ors u

sing

eng

inee

red

antib

odie

s. R

ecen

tly, w

e pr

oduc

ed a

nov

el p

enta

vale

nt b

ispe

cific

ant

ibod

y (i.

e., d

ecab

ody)

by

pent

amer

iz-

ing

two

sing

le-d

omai

n an

tibod

ies (

sdA

bs) t

hrou

gh th

e ve

roto

xin

B su

buni

t (V

TB)

and

foun

d bo

th fu

sion

par

tner

s to

be fu

nctio

nal.

Usi

ng a

sim

ilar a

ppro

ach,

we

have

eng

inee

red

a bi

spec

ific

pent

amer

ic fu

sion

pro

tein

con

sist

ing

of fi

ve sd

Abs

and

five

cel

lulo

se-

bind

ing

mod

ules

(CBM

s) li

nked

via

VT

B. T

o fin

d an

opt

imal

des

ign

form

at, w

e co

nstr

ucte

d si

x bi

spec

ific

pent

amer

s con

sist

ing

of

thre

e di

ffere

nt C

BMs,

fuse

d to

the

Stap

hylo

cocc

us a

ureu

s-sp

ecifi

c hu

man

sdA

b H

VH

P428

, in

both

ori

enta

tions

. One

bis

peci

fic

pent

amer

, con

tain

ing

an N

-ter

min

al C

BM9

and

C-t

erm

inal

HV

HP4

28, w

as so

lubl

e, n

on-a

ggre

gatin

g, a

nd d

id n

ot d

egra

de u

pon

stor

age

at fo

ur d

egre

es C

for o

ver s

ix m

onth

s. Th

is m

olec

ule

was

dua

lly fu

nctio

nal a

s it b

ound

to c

ellu

lose

-bas

ed fi

lters

as w

ell a

s S.

aure

us c

ells

. Whe

n im

preg

nate

d in

cel

lulo

se fi

lters

, the

bis

peci

fic p

enta

mer

reco

gniz

ed S

. aur

eus c

ells

in a

flow

-thr

ough

det

ectio

n as

say.

The

abili

ty o

f pen

tam

eriz

ed C

BMs t

o bi

nd c

ellu

lose

may

form

the

basi

s of a

n im

mob

iliza

tion

plat

form

for m

ultiv

alen

t dis

play

of

hig

h-av

idity

bin

ding

reag

ents

on

cellu

losi

c fil

ters

for s

ensi

ng o

f pat

hoge

ns, b

iom

arke

rs a

nd e

nvir

onm

enta

l pol

luta

nts.

Hus

sack

et

al.

2009

SRP-

sens

orEL

ISA

M. t

uber

culo

sis

Phag

e di

spla

y se

lect

ion

stra

tegi

esH

eat s

hock

pro

tein

Back

grou

nd: R

ecom

bina

nt a

ntib

odie

s are

pow

erfu

l too

ls in

eng

inee

ring

of n

ovel

dia

gnos

tics.

Due

to th

e sm

all s

ize

and

stab

le

natu

re o

f lla

ma

antib

ody

dom

ains

sele

cted

ant

ibod

ies c

an se

rve

as a

det

ectio

n re

agen

t in

mul

tiple

xed

and

sens

itive

ass

ays f

or M

. tu

berc

ulos

is. M

etho

dolo

gy/P

rinc

ipal

Fin

ding

s: A

ntib

odie

s for

Myc

obac

teri

um tu

berc

ulos

is (M

. tb)

reco

gniti

on w

ere

rais

ed in

A

lpac

a, a

nd, b

y ph

age

disp

lay,

reco

mbi

nant

var

iabl

e do

mai

ns o

f hea

vy-c

hain

ant

ibod

ies (

VH

H) b

indi

ng to

M. t

uber

culo

sis a

ntig

ens

wer

e is

olat

ed. T

wo

phag

e di

spla

y se

lect

ion

stra

tegi

es w

ere

follo

wed

: one

dir

ect s

elec

tion

usin

g se

mi-

puri

fied

prot

ein

antig

en, a

nd a

de

plet

ion

stra

tegy

with

lysa

tes,

aim

ing

to a

void

cro

ss-r

eact

ion

to o

ther

myc

obac

teri

a. B

oth

pann

ing

met

hods

sele

cted

a se

t of b

ind-

ers w

ith w

idel

y di

fferi

ng c

ompl

emen

tari

ty d

eter

min

ing

regi

ons.

Sel

ecte

d re

com

bina

nt V

HH

s wer

e pr

oduc

ed in

E. c

oli a

nd sh

own

to b

ind

imm

obili

zed

lysa

te in

dir

ect E

nzym

elin

ked

Imm

unos

orbe

nt A

ssay

(ELI

SA) t

ests

and

solu

ble

antig

en b

y su

rfac

e pl

asm

on

reso

nanc

e (S

PR) a

naly

sis.

All

test

ed V

HH

s wer

e sp

ecifi

c fo

r tub

ercu

losi

s-ca

usin

g m

ycob

acte

ria

(M. t

uber

culo

sis,

M. b

ovis

) and

ex

clus

ivel

y re

cogn

ized

an

imm

unod

omin

ant 1

6 kD

a he

at sh

ock

prot

ein

(hsp

). Th

e hi

ghes

t affi

nity

VH

H h

ad a

dis

soci

atio

n co

nsta

nt

(KD

) of 4

× 1

0(–1

0) M

. Con

clus

ions

/Sig

nific

ance

: A b

road

set o

f diff

eren

t lla

ma

antib

odie

s spe

cific

for 1

6 kD

a he

at sh

ock

prot

ein

of M

. tub

ercu

losi

s is a

vaila

ble.

This

pro

tein

is h

ighl

y st

able

and

abu

ndan

t in

M. t

uber

culo

sis.

The

VH

H th

at d

etec

t thi

s pro

tein

are

ap

plie

d in

a ro

bust

SPR

sens

or fo

r ide

ntifi

catio

n of

tube

rcul

osis

-cau

sing

myc

obac

teri

a.

Trill

ing

et a

l. 20

11


501

Tabl

e 8.

Oth

er p

rosp

ecti

ve u

ses

of s

ingl

e-do

mai

n an

tibo

dy fr

agm

ents

Cry

stal

str

uctu

reEd

itos

ome

Tryp

anos

oma

bruc

ei

The

para

site

Try

pano

som

a br

ucei

, the

cau

sativ

e ag

ent o

f sle

epin

g si

ckne

ss a

cros

s sub

-Sah

aran

Afr

ica,

dep

ends

on

a re

mar

kabl

e U

-inse

rtio

n/de

letio

n RN

A e

ditin

g pr

oces

s in

its m

itoch

ondr

ion.

A a

ppro

xim

atel

y 20

S m

ulti-

prot

ein

com

plex

, cal

led

the

edito

-so

me,

is a

n es

sent

ial m

achi

nery

for e

ditin

g pr

e-m

RNA

mol

ecul

es e

ncod

ing

the

maj

ority

of m

itoch

ondr

ial p

rote

ins.

Edi

toso

mes

co

ntai

n a

com

mon

cor

e of

twel

ve p

rote

ins w

here

six

OB-

fold

inte

ract

ion

prot

eins

, cal

led

A1-

A6,

pla

y a

cruc

ial r

ole.

Her

e, w

e re

port

th

e st

ruct

ure

of tw

o si

ngle

-str

and

nucl

eic

acid

-bin

ding

OB-

fold

s fro

m in

tera

ctio

n pr

otei

ns A

3 an

d A

6 th

at su

rpri

sing

ly, f

orm

a h

et-

erod

imer

. Cry

stal

gro

wth

requ

ired

the

assi

stan

ce o

f an

anti-

A3

nano

body

as a

cry

stal

lizat

ion

chap

eron

e. U

nexp

ecte

dly,

this

ant

i-A

3 na

nobo

dy b

inds

to b

oth

A3(

OB)

and

A6,

des

pite

onl

y si

mila

r to

40%

am

ino

acid

sequ

ence

iden

tity

betw

een

the

OB-

fold

s of A

3 an

d A

6. Th

e A

3(O

B)-A

6 he

tero

dim

er b

urie

s 35%

mor

e su

rfac

e ar

ea th

an th

e A

6 ho

mod

imer

. This

is a

ttri

bute

d m

ainl

y to

the

pres

ence

of

a co

nser

ved

Pro-

rich

loop

in A

3(O

B). Th

e im

plic

atio

ns o

f the

A3(

OB)

-A6

hete

rodi

mer

, and

of a

dim

er o

f het

erod

imer

s obs

erve

d in

th

e cr

ysta

ls, f

or th

e ar

chite

ctur

e of

the

edito

som

e ar

e pr

ofou

nd, r

esul

ting

in a

pro

posa

l of a

‘five

OB-

fold

cen

ter’

in th

e co

re o

f the

ed

itoso

me.

Park

et

al. 2

012

X-r

ay c

ryst

allo

grap

hyPr

ion

In s

itu

prot

eoly

sis

Mic

rose

ed m

atri

x sc

reen

ing

Prio

n pr

otei

ns (P

rPs)

are

diffi

cult

to c

ryst

alliz

e, p

roba

bly

due

to th

eir i

nher

ent fl

exib

ility

. Sev

eral

PrP

s str

uctu

res h

ave

been

solv

ed

by n

ucle

ar m

agne

tic re

sona

nce

(NM

R) te

chni

ques

; how

ever

, onl

y th

ree

stru

ctur

es w

ere

solv

ed b

y X

-ray

cry

stal

logr

aphy

. Her

e w

e co

mbi

ned

in-s

itu p

rote

olys

is w

ith a

utom

ated

mic

rose

ed m

atri

x sc

reen

ing

(MM

S) to

cry

stal

lize

two

diffe

rent

PrP

(C)-

nano

body

(N

b) c

ompl

exes

. Nan

obod

ies a

re si

ngle

-dom

ain

antib

odie

s der

ived

from

hea

vy-c

hain

-onl

y an

tibod

ies o

f cam

elid

s. In

itial

cry

stal

-liz

atio

n sc

reen

ing

cond

ition

s usi

ng in

situ

pro

teol

ysis

of m

ouse

pri

on (2

3–23

0) in

com

plex

with

a n

anob

ody

(Nb_

PrP_

01) g

ave

thin

ne

edle

agg

rega

tes,

whi

ch w

ere

of p

oor d

iffra

ctio

n qu

ality

. Nex

t, w

e us

ed th

ese

mic

rocr

ysta

ls a

s nuc

lean

ts fo

r aut

omat

ed M

MS.

G

ood-

qual

ity c

ryst

als w

ere

obta

ined

from

mou

se P

rP (8

9–23

0)/N

b_Pr

P_01

, bel

onge

d to

the

mon

oclin

ic sp

ace

grou

p P

1 21

1, w

ith

unit-

cell

para

met

ers a

= 5

9.13

, b =

63.

80, c

= 6

9.79

ang

stro

m, β

= 1

01.9

6° a

nd d

iffra

cted

to 2

.1 a

ngst

rom

reso

lutio

n us

ing

sync

hro-

tron

radi

atio

n. H

uman

PrP

(90–

231)

/Nb_

PrP_

01 c

ryst

als b

elon

ged

to th

e m

onoc

linic

spac

e gr

oup

C2,

with

uni

t-ce

ll pa

ram

eter

s a

= 13

1.86

, b =

45.

78, c

= 4

5.09

ang

stro

m, β

= 9

6.23

° and

diff

ract

ed to

1.5

ang

stro

m re

solu

tion.

This

com

bine

d st

rate

gy b

enefi

ts

from

the

pow

er o

f the

MM

S te

chni

que

with

out s

uffer

ing

from

the

draw

back

s of t

he in

situ

pro

teol

ysis

. It p

rove

d to

be

a su

cces

sful

st

rate

gy to

cry

stal

lize

PrP-

nano

bodi

es c

ompl

exes

and

cou

ld b

e ex

ploi

ted

for t

he c

ryst

alliz

atio

n of

oth

er d

ifficu

lt an

tigen

antib

ody

com

plex

es.

Abs

kha-

ron

et a

l. 20

11

X-r

ay c

ryst

allo

grap

hyC

onfo

rmat

iona

l in

term

edia

tes

Am

yloi

doge

nesi

sB

eta

2-m

icro

glob

ulin

Ato

mic

-leve

l str

uctu

ral i

nves

tigat

ion

of th

e ke

y co

nfor

mat

iona

l int

erm

edia

tes o

f am

yloi

doge

nesi

s rem

ains

a c

halle

nge.

Her

e w

e de

mon

stra

te th

e ut

ility

of n

anob

odie

s to

trap

and

cha

ract

eriz

e in

term

edia

tes o

f bet

a 2-

mic

rogl

obul

in (b

eta

2m) a

myl

oido

gene

sis

by X

-ray

cry

stal

logr

aphy

. For

this

pur

pose

, we

sele

cted

five

sing

le d

omai

n an

tibod

ies t

hat b

lock

the

fibri

lloge

nesi

s of a

pro

teol

ytic

am

yloi

doge

nic

frag

men

t of b

eta

2m (D

elta

N6

beta

2m

). Th

e cr

ysta

l str

uctu

re o

f Del

ta N

6 be

ta 2

m in

com

plex

with

one

of t

hese

na

nobo

dies

(Nb2

4) id

entifi

es d

omai

n sw

appi

ng a

s a p

laus

ible

mec

hani

sm o

f sel

f-as

soci

atio

n of

this

am

yloi

doge

nic

prot

ein.

In

the

swap

ped

dim

er, t

wo

exte

nded

hin

ge lo

ops-

corr

espo

ndin

g to

the

hept

apet

ide

NH

VT

LSQ

that

form

s am

yloi

d in

isol

atio

n-ar

e un

mas

ked

and

fold

into

a n

ew tw

o-st

rand

ed a

ntip

aral

lel b

eta-

shee

t. Th

e be

ta-s

tran

ds o

f thi

s she

et a

re p

rone

to se

lf-as

soci

ate

and

stac

k pe

rpen

dicu

lar t

o th

e di

rect

ion

of th

e st

rand

s to

build

larg

e in

term

olec

ular

bet

a-sh

eets

that

run

para

llel t

o th

e ax

is o

f gro

win

g ol

igom

ers,

pro

vidi

ng a

n el

onga

tion

mec

hani

sm b

y se

lf-te

mpl

ated

gro

wth

.

Dom

an-

ska

et a

l. 20

11


502

Tran

spor

t sys

tem

sIm

mun

oglo

bulin

re

cept

orC

arri

er s

yste

mTr

ansc

ytos

isEp

ithe

lial m

onol

ayer

The

poly

mer

ic im

mun

oglo

bulin

rece

ptor

(pIg

R) e

nsur

es th

e tr

ansp

ort o

f dim

eric

imm

unog

lobu

lin A

(dIg

A) a

nd p

enta

mer

ic im

mu-

nogl

obul

in M

(pIg

M) a

cros

s epi

thel

ia to

the

muc

osal

laye

r of f

or e

xam

ple

the

inte

stin

es a

nd th

e lu

ngs v

ia tr

ansc

ytos

is. P

er d

ay th

e hu

man

pIg

R m

edia

tes t

he e

xcre

tion

of 2

to 5

gra

ms o

f dIg

A in

to th

e m

ucos

a of

lum

inal

org

ans.

This

syst

em c

ould

pro

ve u

sefu

l fo

r the

rapi

es a

imin

g at

exc

retio

n of

com

poun

ds in

to th

e m

ucos

a. H

ere

we

inve

stig

ated

the

use

of th

e va

riab

le d

omai

n of

cam

elid

de

rive

d he

avy

chai

n on

ly a

ntib

odie

s, a

lso

know

n as

VH

Hs o

r Nan

obod

ies,

targ

etin

g th

e hu

man

pIg

R, a

s a tr

ansp

ort s

yste

m a

cros

s ep

ithel

ial c

ells

. We

show

that

VH

Hs d

irec

ted

agai

nst t

he h

uman

pIg

R ar

e ab

le to

bin

d th

e re

cept

or w

ith h

igh

affini

ty (s

imila

r to

1 nM

) and

that

they

com

pete

with

the

natu

ral l

igan

d, d

IgA

. In

a tr

ansc

ytos

is a

ssay

bot

h na

tive

and

phag

e-bo

und

VH

H w

ere

only

abl

e to

get

acr

oss p

olar

ized

MD

CK

cel

ls th

at e

xpre

ss th

e hu

man

pIg

R ge

ne in

a b

asol

ater

al to

api

cal f

ashi

on. I

ndic

atin

g th

at th

e V

HH

s ar

e ab

le to

tran

sloc

ate

acro

ss e

pith

elia

and

to ta

ke a

long

larg

e pa

rtic

les o

f car

go. F

urth

erm

ore,

by

mak

ing

mul

tival

ent V

HH

s we

wer

e ab

le to

enh

ance

the

tran

spor

t of t

he c

ompo

unds

bot

h in

a M

DC

K-hp

IgR

and

Cac

o-2

cell

syst

em, p

roba

bly

by in

duci

ng re

cep-

tor c

lust

erin

g. Th

ese

resu

lts sh

ow th

at V

HH

s can

be

used

as a

car

rier

syst

em to

exp

loit

the

hum

an p

IgR

tran

scyt

otic

syst

em a

nd

that

mul

tival

ent c

ompo

unds

are

abl

e to

sign

ifica

ntly

enh

ance

the

tran

spor

t acr

oss e

pith

elia

l mon

olay

ers.

Emm

er-

son

et a

l. 20

11

Tran

spor

t sys

tem

sM

icel

leEG

FRD

oxor

ubic

inEn

caps

ulat

ion

effici

ency

Cor

e-cr

ossl

inke

d th

erm

osen

sitiv

e an

d bi

odeg

rada

ble

poly

mer

ic m

icel

les w

ere

activ

ely

targ

eted

to E

GFR

-ove

rexp

ress

ing

canc

er

cells

by

conj

ugat

ing

an a

nti-E

GFR

nan

obod

y on

the

surf

ace

of th

e m

icel

les.

A m

etha

cryl

ated

dox

orub

icin

der

ivat

ive

cont

aini

ng a

n ac

id se

nsiti

ve h

ydra

zone

spac

er w

as e

ncap

sula

ted

and

subs

eque

ntly

cov

alen

tly a

ttac

hed

duri

ng c

ore-

cros

slin

king

of t

he m

icel

les.

Enca

psul

atio

n effi

cien

cy w

as 6

0% a

nd d

oxor

ubic

in (D

OX

) was

com

plet

ely

rele

ased

aft

er 2

4 h

at p

H 5

, whi

le h

ardl

y an

y D

OX

was

re

leas

ed a

t pH

7.4

. DO

X-lo

aded

mic

elle

s sho

wed

toxi

city

sim

ilar t

o fr

ee d

oxor

ubic

in to

war

ds o

vari

an c

arci

nom

a ce

lls.

Tale

lli e

t al

. 201

0

Dru

g de

liver

y ve

hicl

eSo

dalis

glo

ssin

idiu

sPa

ratr

ansg

enes

isTr

ypan

osom

a br

ucei

Back

grou

nd: S

odal

is g

loss

inid

ius,

a g

ram

-neg

ativ

e ba

cter

ial e

ndos

ymbi

ont o

f the

tset

se fl

y, ha

s bee

n pr

opos

ed a

s a p

oten

tial i

n vi

vo

drug

del

iver

y ve

hicl

e to

con

trol

tryp

anos

ome

para

site

dev

elop

men

t in

the

fly, a

n ap

proa

ch k

now

n as

par

atra

nsge

nesi

s. D

espi

te th

is

inte

rest

of S

. glo

ssin

idiu

s as a

par

atra

nsge

nic

plat

form

org

anis

m in

tset

se fl

ies,

few

pot

entia

l effe

ctor

mol

ecul

es h

ave

been

iden

tified

so

far a

nd to

dat

e no

ne o

f the

se m

olec

ules

hav

e be

en su

cces

sful

ly e

xpre

ssed

in th

is b

acte

rium

. Res

ults

: In

this

stud

y, S.

glo

ssin

idiu

s w

as tr

ansf

orm

ed to

exp

ress

a si

ngle

dom

ain

antib

ody,

(Nan

obod

y) N

b_A

n33,

that

effi

cien

tly ta

rget

s con

serv

ed c

rypt

ic e

pito

pes

of th

e va

rian

t sur

face

gly

copr

otei

n (V

SG) o

f the

par

asite

Try

pano

som

a br

ucei

. Nex

t, w

e an

alyz

ed th

e ca

pabi

lity

of tw

o pr

edic

ted

secr

etio

n si

gnal

s to

dire

ct th

e ex

trac

ellu

lar d

eliv

ery

of si

gnifi

cant

leve

ls o

f act

ive

Nb_

An3

3. W

e sh

ow th

at th

e pe

lB le

ader

pep

tide

was

succ

essf

ul in

dir

ectin

g th

e ex

port

of f

ully

func

tiona

l Nb_

An3

3 to

the

peri

plas

m o

f S. g

loss

inid

ius r

esul

ting

in si

gnifi

cant

leve

ls

of e

xtra

cellu

lar r

elea

se. F

inal

ly, S

. glo

ssin

idiu

s exp

ress

ing

pelB

Nb_

An3

3 ex

hibi

ted

no si

gnifi

cant

redu

ctio

n in

term

s of fi

tnes

s, de

term

ined

by

in v

itro

grow

th k

inet

ics,

com

pare

d to

the

wild

-typ

e st

rain

. Con

clus

ions

: Thes

e da

ta a

re th

e fir

st d

emon

stra

tion

of

the

expr

essi

on a

nd e

xtra

cellu

lar r

elea

se o

f fun

ctio

nal t

rypa

noso

me-

inte

rfer

ing

Nan

obod

ies i

n S.

glo

ssin

idiu

s. F

urth

erm

ore,

Sod

alis

st

rain

s tha

t effi

cien

tly re

leas

ed th

e eff

ecto

r pro

tein

wer

e no

t affe

cted

in th

eir g

row

th, s

ugge

stin

g th

at th

ey m

ay b

e co

mpe

titiv

e w

ith

endo

geno

us m

icro

biot

a in

the

mid

gut e

nvir

onm

ent o

f the

tset

se fl

y. C

olle

ctiv

ely,

thes

e da

ta re

info

rce

the

notio

n fo

r the

pot

entia

l of

S. g

loss

inid

ius t

o be

dev

elop

ed in

to a

par

atra

nsge

nic

plat

form

org

anis

m.

De

Voog

ht

et a

l. 20

12


503

Blo

od-b

rain

bar

rier

Dru

g bi

odis

trib

utio

nIn

viv

o im

agin

gLi

poso

me

nano

carr

ier

The

deve

lopm

ent o

f im

agin

g an

d th

erap

eutic

age

nts a

gain

st n

euro

nal t

arge

ts is

ham

pere

d by

the

limite

d ac

cess

of p

robe

s int

o th

e ce

ntra

l ner

vous

syst

em a

cros

s the

blo

od b

rain

bar

rier

(BBB

). Th

e ev

alua

tion

of d

rug

pene

trat

ion

into

the

brai

n in

exp

erim

enta

l m

odel

s oft

en re

quir

es c

ompl

ex p

roce

dure

s, in

clud

ing

drug

radi

olab

elin

g, a

s wel

l as d

eter

min

atio

ns in

mul

tiple

ani

mal

s for

eac

h co

nditi

on o

r tim

e po

int.

Pros

pect

ive

in v

ivo

imag

ing

of d

rug

biod

istr

ibut

ion

may

pro

vide

an

alte

rnat

ive

to “c

lass

ical

” pha

rmac

oki-

netic

s and

bio

dist

ribu

tion

stud

ies i

n th

at a

con

tras

t-en

hanc

ed im

agin

g si

gnal

cou

ld se

rve

as a

surr

ogat

e fo

r the

am

ount

of d

rug

or

biol

ogic

del

iver

ed to

the

orga

n of

inte

rest

. For

the

brai

n-ta

rget

ing

appl

icat

ions

, it i

s nec

essa

ry to

dev

elop

form

ulat

ion

stra

tegi

es

that

ena

ble

a si

mul

tane

ous d

rug

and

cont

rast

age

nt d

eliv

ery

acro

ss th

e BB

B. In

this

cha

pter

, we

desc

ribe

met

hods

for e

ncap

sula

ting

drug

s int

o lip

osom

e na

noca

rrie

rs w

ith su

rfac

e di

spla

y of

bot

h th

e im

agin

g co

ntra

st a

gent

for o

ne o

r mul

tiple

imag

ing

mod

aliti

es

and

the

sing

le-d

omai

n an

tibod

y th

at u

nder

goes

rece

ptor

-med

iate

d tr

ansc

ytos

is a

cros

s the

BBB

. Con

tras

t-en

hanc

ed im

agin

g si

gnal

de

tect

ed in

the

brai

n af

ter i

ntra

veno

us in

ject

ion

of su

ch fo

rmul

atio

n(s)

is p

ropo

rtio

nal t

o th

e am

ount

of d

rug

deliv

ered

into

the

brai

n pa

renc

hym

a. Th

is m

etho

d al

low

s for

a p

rosp

ectiv

e, n

onin

vasi

ve e

stim

atio

n of

dru

g de

liver

y, ac

cum

ulat

ion,

and

elim

inat

ion

from

the

brai

n.

Iqba

l et

al. 2

011

Mim

otop

e se

lect

ion

IgN

AR

Plas

mod

ium

falc

ipar

um

Mim

otop

es m

imic

the

thre

e-di

men

sion

al to

polo

gy o

f an

antig

en e

pito

pe, a

nd a

re fr

eque

ntly

reco

gniz

ed b

y an

tibod

ies w

ith a

ffini

-tie

s com

para

ble

to th

ose

obta

ined

for t

he o

rigi

nal a

ntib

ody-

antig

en in

tera

ctio

n. P

eptid

es a

nd a

nti-

idio

typi

c an

tibod

ies a

re tw

o cl

asse

s of p

rote

in m

imot

opes

that

mim

ic th

e to

polo

gy (b

ut n

ot n

eces

sari

ly th

e se

quen

ce) o

f the

par

enta

l ant

igen

. In

this

stud

y, w

e co

mbi

ne th

ese

two

clas

ses b

y se

lect

ing

mim

otop

es b

ased

on

sing

le d

omai

n Ig

NA

R an

tibod

ies,

whi

ch d

ispl

ay e

xcep

tiona

lly lo

ng

CD

R3 lo

op re

gion

s (an

alog

ous t

o a

cons

trai

ned

pept

ide

libra

ry) p

rese

nted

in th

e co

ntex

t of a

n im

mun

oglo

bulin

fram

ewor

k w

ith

adja

cent

and

supp

ortin

g C

DR1

loop

s. B

y sc

reen

ing

an in

vitr

o ph

age-

disp

lay

libra

ry o

f IgN

AR

vari

able

dom

ains

(V(N

AR)

s) a

gain

st

the

targ

et a

ntig

en m

onoc

lona

l ant

ibod

y M

Ab5

G8,

we

obta

ined

four

pot

entia

l mim

otop

es. M

Ab5

G8

targ

ets a

line

ar tr

ipep

tide

epito

pe (A

YP) i

n th

e fle

xibl

e si

gnal

sequ

ence

of t

he P

lasm

odiu

m fa

lcip

arum

Api

cal M

embr

ane

Ant

igen

-1 (A

MA

1), a

nd th

is o

r si

mila

r mot

ifs w

ere

dete

cted

in th

e C

DR

loop

s of a

ll fo

ur V

(NA

R)s.

The

V(N

AR)

s, 1

-A-2

, -7,

-11,

and

-14,

wer

e de

mon

stra

ted

to

bind

spec

ifica

lly to

this

par

atop

e by

com

petit

ion

stud

ies w

ith a

n ar

tifici

al p

eptid

e an

d al

l sho

wed

enh

ance

d affi

nitie

s (3–

46 n

M)

com

pare

d to

the

pare

ntal

ant

igen

(175

nM

). C

ryst

allo

grap

hic

stud

ies o

f rec

ombi

nant

pro

tein

s 1-A

-7 a

nd 1

-A-1

1 sh

owed

that

the

SYP

mot

ifs o

n th

ese

V(N

AR)

s pre

sent

ed a

t the

tip

of th

e ex

pose

d C

DR3

loop

s, id

eally

pos

ition

ed w

ithin

bul

ge-li

ke st

ruct

ures

to

mak

e co

ntac

t with

the

MA

b5G

8 an

tibod

y. Th

ese

loop

s, in

par

ticul

ar in

1-A

-11,

wer

e fu

rthe

r sta

biliz

ed b

y in

ter-

and

intr

a- lo

op

disu

lphi

de b

ridg

es, h

ydro

gen

bond

s, e

lect

rost

atic

inte

ract

ions

, and

aro

mat

ic re

sidu

e pa

ckin

g. W

e ra

tiona

lize

the

high

er a

ffini

ty o

f th

e V

(NA

R)s c

ompa

red

to th

e pa

rent

al a

ntig

en b

y su

gges

ting

that

adj

acen

t CD

R1 a

nd fr

amew

ork

resi

dues

con

trib

ute

to b

indi

ng

affini

ty, t

hrou

gh in

tera

ctio

ns w

ith o

ther

CD

R re

gion

s on

the

antib

ody,

thou

gh o

f cou

rse

defin

itive

supp

ort o

f thi

s hyp

othe

sis w

ill

rely

on

co-c

ryst

allo

grap

hic

stud

ies.

Alte

rnat

ivel

y, th

e se

lect

ion

of m

imot

opes

from

a la

rge

(< 4

× 1

08 ) con

stra

ined

libr

ary

may

hav

e al

low

ed se

lect

ion

of v

aria

nts w

ith e

ven

mor

e fa

vora

ble

epita

pe to

polo

gies

than

pre

sent

in th

e or

igin

al a

ntig

enic

stru

ctur

e, il

lust

rat-

ing

the

pow

er o

f in

vivo

sele

ctio

n of

mim

otop

es fr

om p

hage

-dis

play

ed m

olec

ular

libr

arie

s.

Sim

mon

s et

al.

2008


504

Bac

teri

opha

ge p

2M

ajor

cap

sid

prot

ein

Rec

epto

r-bi

ndin

g

prot

eins

Neu

tral

isat

ion

assa

y

Back

grou

nd: B

acte

riop

hage

s inf

ectin

g la

ctic

aci

d ba

cter

ia (L

AB)

are

wid

ely

ackn

owle

dged

as t

he m

ain

caus

e of

milk

ferm

enta

tion

failu

res.

In th

is st

udy,

we

desc

ribe

the

surf

ace-

expr

essi

on a

s wel

l as t

he se

cret

ion

of tw

o fu

nctio

nal l

lam

a he

avy-

chai

n an

tibod

y fr

agm

ents

, one

bin

ding

to th

e m

ajor

cap

sid

prot

ein

(MC

P) a

nd th

e ot

her t

o th

e re

cept

or-b

indi

ng p

rote

ins (

RBP)

of t

he la

ctoc

occa

l ba

cter

ioph

age

p2, b

y la

ctob

acill

i in

orde

r to

neut

ralis

e la

ctoc

occa

l pha

ges.

Res

ults

: The

antib

ody

frag

men

t VH

H5

that

is d

irec

ted

agai

nst t

he R

BP, w

as fu

sed

to a

c-m

yc ta

g an

d ex

pres

sed

in a

secr

eted

form

by

a La

ctob

acill

us st

rain

. The

frag

men

t VH

H2

that

is

bin

ding

to th

e M

CP,

was

fuse

d to

an

E-ta

g an

d an

chor

ed o

n th

e su

rfac

e of

the

lact

obac

illi.

Surf

ace

expr

essi

on o

f VH

H2

was

co

nfirm

ed b

y flo

w c

ytom

etry

usi

ng a

n an

ti-E-

tag

antib

ody.

Effici

ent b

indi

ng o

f bot

h th

e V

HH

2 an

d th

e se

cret

ed V

HH

5 fr

agm

ent

to th

e ph

age

antig

ens w

as sh

own

in E

LISA

. Sca

nnin

g el

ectr

on m

icro

scop

y sh

owed

that

lact

obac

illi e

xpre

ssin

g V

HH

2 an

chor

ed a

t th

eir s

urfa

ce w

ere

able

to b

ind

lact

ococ

cal p

hage

s. A

neu

tral

isat

ion

assa

y al

so c

onfir

med

that

the

secr

eted

VH

H5

and

the

anch

ored

V

HH

2 fr

agm

ents

pre

vent

ed th

e ad

sorp

tion

of la

ctoc

occa

l pha

ges t

o th

eir h

ost c

ells

. Con

clus

ion:

Lac

toba

cilli

wer

e ab

le to

exp

ress

fu

nctio

nal V

HH

frag

men

ts in

bot

h a

secr

eted

and

a c

ell s

urfa

ce fo

rm a

nd re

duce

d ph

age

infe

ctio

n of

lact

ococ

cal c

ells

. Lac

toba

cilli

ex

pres

sing

llam

a he

avy-

chai

n an

tibod

y fr

agm

ents

repr

esen

t a n

ovel

way

to li

mit

phag

e in

fect

ion.

Hul

tber

g et

al.

2007

Prot

ein

swit

ches

Com

bina

tori

al h

isti

dine

lib

rary

pH-d

epen

dent

bin

ding

Ther

e is

gro

win

g in

tere

st in

the

deve

lopm

ent o

f pro

tein

switc

hes,

whi

ch a

re p

rote

ins w

hose

func

tion,

such

as b

indi

ng a

targ

et

mol

ecul

e, c

an b

e m

odul

ated

thro

ugh

envi

ronm

enta

l tri

gger

s. E

ffort

s to

engi

neer

hig

hly

pH se

nsiti

ve p

rote

in-p

rote

in in

tera

c-tio

ns ty

pica

lly re

ly o

n th

e ra

tiona

l int

rodu

ctio

n of

ioni

zabl

e gr

oups

in th

e pr

otei

n in

terf

ace.

Suc

h ex

peri

men

ts a

re ty

pica

lly ti

me

inte

nsiv

e an

d of

ten

sacr

ifice

the

prot

ein’

s affi

nity

at t

he p

erm

issi

ve p

H. Th

e un

derl

ying

ther

mod

ynam

ics o

f pro

ton-

linka

ge d

ic-

tate

that

the

pres

ence

of m

ultip

le io

niza

ble

grou

ps, w

hich

und

ergo

a p

K(a

) cha

nge

on p

rote

in b

indi

ng, a

re n

eces

sary

to re

sult

in

high

ly p

H-d

epen

dent

bin

ding

. To

test

this

hyp

othe

sis,

a n

ovel

com

bina

tori

al h

istid

ine

libra

ry w

as d

evel

oped

whe

re e

very

pos

sibl

e co

mbi

natio

n of

his

tidin

e an

d w

ild-t

ype

resi

due

is sa

mpl

ed th

roug

hout

the

inte

rfac

e of

a m

odel

ant

i-RN

ase

A si

ngle

dom

ain

VH

H

antib

ody.

Ant

ibod

ies w

ere

cose

lect

ed fo

r hig

h-affi

nity

bin

ding

and

pH

-sen

sitiv

ity u

sing

an

in v

itro,

dua

l-fun

ctio

n se

lect

ion

stra

tegy

. Th

e re

sulti

ng a

ntib

odie

s ret

aine

d ne

ar w

ild-t

ype

affini

ty y

et b

ecam

e hi

ghly

sens

itive

to sm

all d

ecre

ases

in p

H, d

rast

ical

ly d

ecre

as-

ing

thei

r bin

ding

affi

nity

, due

to th

e in

corp

orat

ion

of m

ultip

le h

istid

ine

grou

ps. S

ever

al tr

ends

wer

e ob

serv

ed, s

uch

as h

istid

ine

“hot

-spo

ts”,

whi

ch w

ill h

elp

enha

nce

the

deve

lopm

ent o

f pH

switc

h pr

otei

ns a

s wel

l as i

ncre

ase

our u

nder

stan

ding

of t

he ro

le o

f io

niza

ble

resi

dues

in p

rote

in in

terf

aces

. Ove

rall,

the

com

bina

tori

al a

ppro

ach

is ra

pid,

gen

eral

, and

robu

st a

nd sh

ould

be

capa

ble

of

prod

ucin

g hi

ghly

pH

-sen

sitiv

e pr

otei

n affi

nity

reag

ents

for a

num

ber o

f diff

eren

t app

licat

ions

.

Mur

-ta

ugh

et

al. 2

011

Imm

unoa

ffini

ty

chro

mat

ogra

phy

Caff

eine

Gra

ftin

g

This

wor

k de

mon

stra

tes t

he fe

asib

ility

of u

sing

a c

amel

id si

ngle

dom

ain

antib

ody

for i

mm

unoa

ffini

ty c

hrom

atog

raph

ic se

pera

tion

of sm

all m

olec

ules

. An

anti-

caffe

ine

VH

H a

ntib

ody

was

pro

duce

d by

gra

ftin

g th

e co

mpl

emen

tant

y de

term

inin

g se

quen

ces o

f a

prev

ious

ly g

ener

ated

ant

ibod

y on

to a

n an

ti-RN

ase

A a

ntib

ody

scaff

old

follo

wed

by

expr

essi

on in

E. c

oli.

Ana

lysi

s of t

he b

indi

ng

prop

ertie

s of t

he a

ntib

ody

by E

LISA

and

fluo

resc

ence

-bas

ed th

erm

al sh

ift a

ssay

s sho

wed

that

it re

cogn

izes

not

onl

y ca

ffein

e, b

ut

also

theo

phyl

line,

theo

biom

ine

and

para

xant

hine

, alb

eit w

ith lo

wer

affi

nity

. Fur

ther

inve

stig

atio

n of

the

effec

t of e

nvir

onm

enta

l co

nditi

ons,

i.e.

, tem

pera

ture

, pH

, and

ioni

c st

reng

th, o

n th

e an

tibod

y us

ing

thes

e m

etho

ds p

rovi

ded

usef

ul in

form

atio

n ab

out

pote

ntia

l ela

tion

cond

ition

s to

be u

sed

in c

hrom

atog

raph

ic a

pplic

atio

ns. I

mm

obili

zatio

n of

the

VH

H o

nto

a hi

gh fl

ow-t

hrou

gh

synt

hetic

supp

ort m

ater

ial r

esul

ted

In a

stat

iona

ry p

hase

cap

able

of s

epar

atin

g ca

ffein

e an

d its

met

abol

ites.

Fran

co e

t al

. 201

0


505

Prot

ein

conf

orm

atio

nSp

ectr

al p

rope

rtie

sG

reen

fluo

resc

ent

prot

ein

Prot

ein

tran

sloc

atio

n

Prot

ein

conf

orm

atio

n is

cri

tical

ly li

nked

to fu

nctio

n an

d of

ten

cont

rolle

d by

inte

ract

ions

with

regu

lato

ry fa

ctor

s. H

ere

we

repo

rt

the

sele

ctio

n of

cam

elid

-der

ived

sing

le-d

omai

n an

tibod

ies (

nano

bodi

es) t

hat m

odul

ate

the

conf

orm

atio

n an

d sp

ectr

al p

rope

rtie

s of

the

gree

n flu

ores

cent

pro

tein

(GFP

). O

ne n

anob

ody

coul

d re

vers

ibly

redu

ce G

FP fl

uore

scen

ce b

y a

fact

or o

f 5, w

here

as it

s dis

plac

e-m

ent b

y a

seco

nd n

anob

ody

caus

ed a

n in

crea

se b

y a

fact

or o

f 10.

Str

uctu

ral a

naly

sis o

f GFP

-nan

obod

y co

mpl

exes

reve

aled

that

the

two

nano

bodi

es in

duce

subt

le o

ppos

ing

chan

ges i

n th

e ch

rom

opho

re e

nvir

onm

ent,

lead

ing

to a

ltere

d ab

sorp

tion

prop

ertie

s. U

nlik

e co

nven

tiona

l ant

ibod

ies,

the

smal

l, st

able

nan

obod

ies a

re fu

nctio

nal i

n liv

ing

cells

. Nan

obod

y-in

duce

d ch

ange

s wer

e de

tect

ed b

y ra

tio im

agin

g an

d us

ed to

mon

itor e

vent

s suc

h as

the

tam

oxife

n-in

duce

d nu

clea

r loc

aliz

atio

n of

est

roge

n re

cept

or. Th

is w

ork

dem

-on

stra

tes t

hat p

rote

in c

onfo

rmat

ions

can

be

man

ipul

ated

and

stud

ied

with

nan

obod

ies i

n liv

ing

cells

.

Kir

ch-

hofe

r et

al. 2

010

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Received: 2012–08–30Accepted after corrections: 2012–09–25

Corresponding Author:

Mgr. Ludek Eyer, Ph.D., Veterinary Research Institute, Hudcova 70, 621 00 Brno, Czech RepublicTel. +420 533 331 911, E-mail: [email protected]

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