Hillyar Internalisation of radioimmunotherapeutics rev 2014

319ISSN 2041-599010.4155/TDE.14.6 © 2014 Future Science Ltd Ther. Deliv. (2014) 5(3), 319–335

Review

Radioimmunotherapy (RIT) holds enormous potential for treating localized and metastatic tumors. Radioimmunotherapeutic agents are commonly synthesized from tumor antigen-specific antibodies that are radiohalogenated at tyrosines with 123I, 125I or 77Br. This involves radiolabeling methods that use the Na-salt chol-ramine-T(N-chloro para-toluenesulfonylamide, sodium chloro([4-methyl phenylsulfonyl]aza-nide) or Iodogen (1,3,4,6-tetrachloro-3a,6a-diphenylglucoluril) to oxidize the radiohalogens, which, subsequently, rapidly undergo electro-philic substitution with activated aromatic rings of the tyrosine constituents of antibodies [1,2]. Dehalogenation of antibodies can be sig-nificantly reduced and their cellular retention increased through alternative radiohalogenation methods based on the Bolton-Hunter reagent N-succinimidyl 3-(4-hydroxy-3-iodophenyl)propionate, or the almost identical compound N-succinimidyl 3-iodobenzoate [3]. 211At can be attached to antibodies using the acetylation agent N-succinimidyl 3-[211At]astato-4-gua-nidinomethylbenzoate [4]. Radionuclides such as 90Y, 111In or 177Lu can be coupled to lysine residues in antibodies via the acyclic diethyl-enetriaminepentaacetic acid (DTPA) and the macrocyclic 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). The choice of labeling agent may influence the tumor and normal tissue distribution of RIT agents [5].

Strategic targeting of upregulated or aber-rantly expressed tumor-associated antigens aims

to accumulate RIT agents in tumor tissue with a high degree of selectivity. Some radionuclides emit cytotoxic ionizing radiation and their tumor targeting reduces irradiation of healthy tissues, which may occur with conventional external radiation therapy. Zevalin (90Y-labeled ibritu-momab tiuxetan [murine IgG1k]) and Bexxar (131I-labeled tositumomab [murine IgG2al]) are both noninternalizing anti-CD20 RIT agents. Zevalin and Bexxar were US FDA approved in 2002 and 2003, respectively, for the treatment of non-Hodgkin lymphomas [6]. These RIT agents can be administered as combination treatments with other therapeutics that enhance their effi-cacy and/or reduce toxicity. Clinical trials are actively investigating the efficacy of 90Y-labeled ibritumomab–tiuxetan in combination with high-dose melphalan and stem cell transplant, or with external beam radiotherapy and stem cell transplant, or with subsequent maintenance rituximab, while 131I-labeled tositumomab in combination with dose-intensive chemotherapy and immunotherapy, or with Fludarabine fol-lowed by stem cell transplantation, is also being investigated (Table 1). Zevalin and Bexxar have been used with great success to treat non-Hodg-kin’s lymphomas, an exquisitely radiosensitive group of tumors of the hematopoietic and lym-phoid tissues that include acute lymphocytic leu-kemia and lymphomas of the mucosa associated lymphoid tissue [27,28].

Recently, significant effort has been directed towards advancing RIT agents aimed at treating

Uptake, internalization and nuclear translocation of radioimmunotherapeutic agents

Radioimmunotherapy (RIT) agents that incorporate short-range particle-emitting radionuclides exploit the high linear energy transfer of a-particles and Auger electrons. Both are densely ionizing, generate complex DNA double-strand breaks and so are profoundly cytotoxic. Internalizing RIT agents enter tumor cells through receptor-mediated endocytosis and by incorporation of cell-penetrating peptides. Once internalized, some RIT agents mediate escape from endosomes and/or translocate to the nucleus. In the classical nuclear import pathway, a/b-importins recognize nuclear localization sequences in RIT agents. Translocation through nuclear pores enables RIT agents to bind to nuclear targets induced by, for example, cellular stress, growth factors or anticancer therapy, such as gH2AX or p27KIP-1. This review discusses RIT agents designed to exploit the mechanisms underlying these complex processes and compares them with noninternalizing RIT agents.

Christopher RT Hillyar, Bart Cornelissen & Katherine A Vallis*Cancer Research UK/Medical Research Council Gray Institute for Radiation Oncology & Biology, Department of Oncology, University of Oxford, OX3 7DQ, UK *Author for correspondence: Tel.: +44 1865 225850 [email protected]

Review | Hillyar, Cornelissen & Vallis

Ther. Deliv. (2014) 5(3)320 future science group

Tab

le 1

. Rad

ioim

mu

no

ther

apeu

tic

acti

vely

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ng

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die

d in

clin

ical

tri

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tig

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tle

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Ref

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ve c

hem

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rapy

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bin

ed w

ith

mon

ocl

onal

ant

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ther

apy

and

targ

eted

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mun

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rapy

for

unt

reat

ed p

atie

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wit

h hi

gh-r

isk

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ell n

on-

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dgk

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lym

phom

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nive

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CT0

0577

629

[7]

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2013

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osi

tum

omab

(B

exxa

r)A

clin

ical

tria

l eva

luat

ing

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mom

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20) w

ith

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latin

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llow

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tolo

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s or

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ed o

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frac

tory

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mph

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in p

atie

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year

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e an

d o

lder

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ton

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sort

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effi

cacy

of

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ter/

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of

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ton

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sort

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NC

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0739

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209

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or p

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ultip

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of R

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Uptake, internalization & nuclear translocation of radioimmunotherapeutic agents | Review

www.future-science.com 321future science group

Tab

le 1

. Rad

ioim

mu

no

ther

apeu

tic

acti

vely

bei

ng

stu

die

d in

clin

ical

tri

als

(co

nt.

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tSt

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tle

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(Zev

alin

)

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hase

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y of

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d ib

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mom

ab t

iuxe

tan

(Zev

alin

) for

eld

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pat

ient

s w

ith

prev

ious

ly u

ntre

ated

dif

fuse

larg

e B

-cel

l lym

phom

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emor

ial S

loan

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terin

g C

ance

r C

ente

rN

CT0

005

842

2[17]

B113

1 I-a

nti-

B1B

EAM

+ 13

1 I-a

nti-

B1 r

adio

imm

unot

hera

py a

nd a

uto

log

ous

hem

ato

po

ieti

c st

em c

ell

tran

spla

ntat

ion

for

the

trea

tmen

t of

rec

urre

nt n

on-H

od

gkin

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mph

oma

Uni

vers

ity

of N

ebra

ska

NC

T005

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09

[18]

CD

3717

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etal

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)A

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se I

/II s

tud

y of

177 L

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alut

in) r

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imm

unot

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py f

or t

reat

men

t of

re

laps

ed C

D37

+ n

on-H

od

gkin

’s ly

mph

oma

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dic

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ovec

tor

AS

NC

T017

961

71[19]

Co

lon

CEA

90 Y

-M5A

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e I t

rial o

f ra

dio

imm

unot

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py (

90 Y

-M5A

) in

com

bina

tion

wit

h FO

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I and

b

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izum

ab c

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rapy

for

met

asta

tic

colo

rect

al c

arci

nom

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ity

of H

op

e M

edic

al C

ente

rN

CT0

1205

022

[20]

CEA

90 Y

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hase

II t

rial o

f ra

dio

imm

unot

hera

py (

90 Y

-M5A

) fo

llow

ing

hepa

tic

rese

ctio

n an

d FO

LFIR

I or

FOLF

OX

che

mot

hera

py [+

/-b

evac

izum

ab],

or

XEL

OX

for

met

asta

tic

colo

rect

al c

arci

nom

a to

the

live

r

Cit

y of

Ho

pe

Med

ical

Cen

ter

NC

T013

206

83

[21]

CEA

90 Y

-cT8

4.6

6A

Pha

se I

/II t

rial o

f ra

dio

imm

unot

hera

py (

90 Y

-ct8

4.6

6),

gem

cita

bin

e an

d he

pati

c ar

teria

l inf

usio

n of

flo

xuri

din

e fo

r m

etas

tati

c co

lore

ctal

car

cino

ma

to t

he li

ver

Cit

y of

Ho

pe

Med

ical

Cen

ter

NC

T00

645

710

[22]

Lun

g

CEA

90 Y

-cT8

4.6

6A

Pha

se I

tria

l of

radi

oim

mun

othe

rapy

(9

0 Y-m

x-D

TPA

-ct8

4.6

6) a

fter

com

plet

ion

of

radi

atio

n th

erap

y al

one,

or

radi

atio

n th

erap

y pl

us s

yste

mic

the

rapy

in u

nres

ecta

ble

or m

edic

ally

ino

per

able

, non

met

asta

tic

CEA

-pro

duci

ng s

tag

e I–

IIIb

non

-sm

all-

cell

lung

can

cer

Cit

y of

Ho

pe

Med

ical

Cen

ter

NC

T007

38

452

[23]

Bra

in

GD

213

1 I-3

F8A

tria

l of

radi

oim

mun

othe

rapy

, red

uced

-do

se e

xter

nal b

eam

cra

nio

spin

al r

adia

tion

th

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y w

ith

inte

nsit

y-m

odu

late

d ra

diat

ion

ther

apy

bo

ost

, and

che

mot

hera

py f

or

pati

ents

wit

h st

anda

rd-r

isk

med

ullo

blas

tom

a

Mem

oria

l Slo

an-K

ette

ring

Can

cer

Cen

ter

NC

T00

058

370

[24]

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213

1 I-3

F8C

ombi

nati

on o

f ta

rget

ed 13

1 I–3

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edia

ted

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oim

mun

othe

rapy

and

bev

aciz

umab

in

pat

ient

s w

ith

rela

psed

or

refr

acto

ry n

euro

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tom

a: a

Pha

se I

stud

yM

emor

ial S

loan

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terin

g C

ance

r C

ente

rN

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045

082

7[25]

Fibr

onec

tin

extr

a d

omai

n B

131 I

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A p

rosp

ecti

ve n

onra

ndom

ized

stu

dy

of 13

1 I-L

19SI

P ra

dio

imm

unot

hera

py in

co

mbi

nati

on w

ith

who

le b

rain

-rad

iati

on t

hera

py in

pat

ient

s w

ith

mul

tiple

bra

in

met

asta

ses

from

so

lid t

umor

s

Philo

gen

SpA

NC

T011

250

85[26]

CEA

: Car

cino

mem

bry

oni

c an

tig

en; D

TPA

: Die

thyl

inet

riam

inep

enta

ceti

c ac

id; R

IT: R

adio

imm

unot

hera

py.



solid tumors [29], including those arising in brain, colon, liver, ovary and lung (Table 1). In the clinical setting, long-range (12 mm) b-emitting radioimmunotherapeutics, such as 90Y-M5A and 90Y-cT84.66 that target carcino-membryonic antigen (CEA) overexpressed in colorectal carcinoma and non-small-cell lung cancer, are being developed. In the preclinical setting, RIT agents that are internalized by can-cer cells, and subsequently traffic to the nucleus, are gaining much attention. Localization of low energy (<23 keV), very short-range (nm–µm) radioimmunotherapeutics, such as Auger electron emitters, in the nucleus optimizes the geometry of their dose-point kernel, defined as the limited space in which radially emitted particles slow down according to the stopping power function of the surrounding cells or tis-sue. Geometrically optimizing the delivery of a radioactive payload to the nucleus causes exten-sive DNA damage and, potentially, damage to other subnuclear protein structures within the local decay site for maximum therapeutic effect. The biological processes underpinning nuclear localization of RIT agents involves a complex sequence of distinct events, includ-ing internalization, endocytic vesicle escape, cytoplasmic trafficking towards the nuclear-pore complex, nuclear-pore navigation, and resistance to intracellular degradation.

The therapeutic eff icacy of RIT agents that incorporate long-range b-emitting radio-nuclides (90Y, 131I and 177Lu) depends upon the mean tumor radiation dose, which is lim-ited by the amount of off-target toxicity that builds up in normal tissue [30]. In contrast, short-range RIT agents that localize at specific critical targets inside individual cancer cells have a therapeutic ratio that depends upon how their localization relates geometrically to their critical targets, while their mean tumor radiation dose may be relatively low. Internal-ization of short-range RIT agents by tumor-specific mechanisms can avoid off-target toxic-ity to normal tissue, such as potentially fatal myelosuppression. Uptake, internalization and nuclear translocation of short-range RIT agents forms the basis for the following discus-sion, while other therapies that can be given in combination with RIT agents to reduce toxic-ity and/or improve efficacy fall outside of the scope of this review.

Bryan et al. have shown that internalizing 64Cu-labeled cBR96 antibodies and nonin-ternalizing 64Cu-labeled cT84.66 antibodies,

which both target Lewis Y antigen expressed by LS174T colon carcinoma cells, have differ-ent uptake and biodistribution characteristics in athymic mice bearing LS174T xenografts. Over 48 h, noninternalizing antibodies (cT84.66) increased the amount of radioactivity that accumulated in the spleen and liver of these mice compared with internalizing antibodies (cBR96) [31]. Optimizing the geometry of some RIT agents, such as very low-energy (<23 keV), very short-range Auger emitters or high-energy (2.28 MeV) and short-range a-emitters by tar-geting tumor specific antigens that either selec-tively translocate to, or are expressed inside the nucleus of tumor cells, may not only enhance their therapeutic efficacy, but also reduce their toxicity to normal tissue. This is an advantage over noninternalizing RIT agents, which may cause irreparable damage to cancer cells and to normal tissue, leading to normal tissue side effects. The emissions of long-range noninter-nalizing RIT agents are capable of inducing damage to critical targets inside the nucleus of normal tissue cells from relatively distal loca-tions in the extracellular microenvironment. Thus, it is hypothesized that short-range inter-nalizing RIT agents may be safer than long-range noninternalizing RIT agents, because short-range emissions are less likely to produce toxic off-target effects in normal cells, unless they are geometrically positioned in locations close to the critical targets.

RadionuclidesThe choice of radionuclide for RIT depends upon its physical properties, availability, label-ing methods, possibility of imaging and radia-tion safety. Historically, RIT agents have incor-porated b particle-emitting radionuclides with emission ranges of up to a few milimeters, such as 90Y, 131I, and 177Lu (see Table 2 for a list of radionuclides used in RIT and selected clinical experiences). These radionuclides emit b radia-tion that is high energy, but sparsely ionizing, that is, the linear energy transfer (LET; the amount of energy transferred to biological mate-rial per unit track length) of the b emissions is low (0.2 keV/µm). Cross-fire effects occur due to the traversal of b particles across neighbor-ing targeted, or indeed, nontargeted tumor cells (FiguRe 1). It is important to note that cross-fire effects are a double-edged sword that can increase damage to normal tissue. An exam-ple of a b-emitting radio immunotherapeutic under preclinical investigation is 90Y-labeled

Key Terms

Radioimmunotherapeutic agent: Radioactive antibody produced by linking a radioactive element to an antibody that is capable of delivering radioactivity to cancer cells, due to its ability to bind to an antigen that is predominantly expressed or overexpressed by cancer cells.

Range: Length of the path of a particle emitted by a decaying radionuclide, equal to the distance between the first and last of a sequence of ionizations produced in tissue by interactions between the emitted particle and the molecules of tissue.

Internalization: Biological process that can be exploited to localize very short range, high linear energy transfer radioimmunotherapeutic agents (e.g., Auger electron-emitting agents) present outside of a cancer cell to a position that is inside the target cell. This process commonly results in the internalization of a receptor protein that is present at the cell surface membrane of the target cell to which the radioimmunotherapeutic agent has become bound.

Nuclear translocation: Biological process that can be exploited for the relocation of very short range, high linear energy transfer radioimmunotherapeutic agents (e.g., Auger electron-emitting agents) present inside the cytoplasm of a cancer cell to a position that is inside the nucleus. This process usually involves a nuclear import pathway called the classical pathway.

Linear energy transfer: Number of ionizations or amount of energy that is deposited by a particle emitted by a decaying radionuclide over a given distance in tissue. This process breaks chemical bonds in molecules in the cells of tissue. Therefore, the higher the linear energy transfer, typically the greater the amount of damage caused to targeted cancer cells.



panitumumab (Vectibix), a fully humanized anti-EGF receptor (EGFR) antibody. 90Y-labeled panitumumab is taken up by, and causes growth delay of an EGFR-positive head and neck cancer (UM-SCC-22B) tumor model in vivo [37].

In contrast to b radiation, a-particles, which are Helium nuclei (4He), have high LET (80–100 keV/µm, increasing to 300 keV/µm at the Bragg peak). They are emitted by such radionuclides as 213Bi, 212Pb, 211At, 225Ac, 227Th and 223Ra. a-particle tracks are short-range and they traverse, at most, a few cells from the source of decay (FiguRe 1). This makes them ideal for treating small volume or disseminated disease. Also, RIT agents that incorporate low energy (<23 keV), very short-range (nm–µm) particle-emitting radionuclides (e.g., 111In) can be directed effectively and safely against small volume or microscopic disease, because once they are internalized by tumor cells they deposit high amounts of energy around the local decay site and produce minimal cross-fire effects, which limits normal tissue side effects. In some cases, the antibody moiety can be designed to simultaneously disrupt the biological function of specific proteins, augmenting radiosensitiv-ity. Auger electrons produced during decay of radionuclides such as 125I, 123I and 111In have high LET [38]. 111In decays by electron capture, emitting g-rays (171–245 keV) as well as internal

conversion (145–219 keV) and Auger (3–19 keV) electrons. The Auger electrons emitted by 111In, at the rate of 14.7 per decay, have a very short-path length of 0.25 nm to 13.6 µm in water. The localization of Auger electron-emitting RIT agents in the nucleus or perinuclear area pro-duces an optimal therapeutic effect. For Auger emitters such as 125I, it has been shown that a 30-fold higher radiation dose is imparted to the nucleus (and the DNA within) when they are localized in the nucleus compared with at the cell membrane (FiguRe 1) [38].

It is worth noting that there is evidence to sug-gest that antibodies that do not internalize after binding to a target antigen may also be effective delivery vehicles for Auger electron radionuclide therapy [39]. Both short- and long-range emitters, such as 125I- and 186Re-labeled RIT agents that target epithelial cellular adhesion molecule, can be rapidly internalized into colorectal carcinoma cells, leading to excellent intratumoral retention [40,41]. 111In-cycloheximide (CHX)-A”-DTPA-panitumumab is an effective Auger emitter, which is internalized after it binds to EGFR. Upon bind-ing EGFR, a RTK often overexpressed in breast cancer, 111In-CHX-A”-DTPA-panitumumab translocates to the nucleus [42]. Interestingly, the choice of chelate has been shown to influ-ence internalization and intracellular retention of the radionuclide 177Lu that was delivered by

Table 2. Radionuclides used in radioimmunotherapy and selected clinical experiences.

Radionuclide Half life Maximum particle energy

Maximum range in tissue

Clinical experience Ref.

b particles90Y 64.1 h 2.28 MeV 11.3 mm 90Y-ibritumomab tiuxetan (Zevalin) is US FDA approved

for CD20 positive non-Hodgkin’s lymphoma131I 8.0 days 606 keV 2.3 mm 131I-tositumomab (Bexxar) FDA approved for CD20

positive non-Hodgkin’s lymphoma177Lu 6.7 days 498 keV 1.8 mm 177Lu-labeled Rituximab for radioimmunotherapy of

31 patients with relapsing follicular, mantle cell, and other indolent B-cell lymphomas

[32]

a particles211At 7.2 h 5.99 MeV 60 µm 211At-labeled antitenascin monoclonal antibody 81C6

for regional RIT of six recurrent brain tumor patients

[33]

213Bi 45.6 min 1.43 MeV 84 µm 213Bi-labeled anti-CD33 antibody HuM195 for RIT of 31 patients with relapsed or refractory acute myeloid leukemia

[34]

Auger electrons125I 60.2 days 500eV nm–µm 125I-labeled anti-EGF receptor monoclonal antibody for

RIT of 192 patients with glioblastoma multiform

[35]

111In 2.8 days 19 keV nm–µm Mainly used clinically for imaging and dosimetry prior to therapeutic administration of Zevalin

RIT: Radioimmunotherapy. Adapted with permission from [36].

Key Term

Auger electron: High linear energy transfer, short range particle emitted in the form of an electron usually as a result of the decay of proton-rich-radioactive elements. Auger electrons travel very short distances, but collectively deposit very high energy in their intended target if the radioimmunotherapeutic is transported to distances very close to target.



anti-EGFR antibodies [43]. This may in part have been due to the varying stabilities of the metal ion-chelate complexes and/or changing affinity of the 177Lu-labeled anti-EGFR antibodies.

�� RIT agents & the tumor microenvironmentTo reach the cancer cells in a vascularized solid tumor, systemically administered RIT agents must extravasate across the tumor blood vessel wall into the tumor milieu, through which it must then migrate. The tumor milieu consists of a heterogeneous mixture of tumor and stromal cell types. Migration through the tumor milieu relies almost exclusively on diffusion from the tumor neovasculature. Tumor vessels are highly disorganized and much leakier than normal cap-illaries. The lack of lymphatic drainage results in increased interstitial pressure that results in the collapse of some of these vessels. Temporary ces-sation, or even reversal of plasma flow, limits the accumulation of RIT agents. Areas of low vessel density give rise to regions of hypoxia, making some tumor cells more resistant to the radio-biological effects of (low LET) ionizing radia-tion than others, although cell death induced by a-particle-emitting EGFR-targeted RIT agents has been shown to occur independently of hypoxia [44].

The enhanced perfusion and retention mech-anism, which nonspecifically concentrates large molecules in the tumor milieu, contributes to passive targeting of tumor tissue. The large size of antibodies (~155 kDa) enhances their reten-tion, but simultaneously limits the diffusion of RIT agents deep into the extravascular extra-ceullar space of the tumor. The same uptake limitations are true of antibody fragments, such as diabodies or minibodies, or affibodies, which do not possess improved tumor tissue uptake properties, but reduce normal tissue retention through enhanced clearance rates. Examples of RIT agents based on antibody fragments include, 177Lu-DOTA-ABY-027, a HER2/neu-targeted affibody-albumin-binding domain fusion protein radiolabeled with 177Lu via DOTA [45]; and 111In-CHX-A”-DTPA-panitumumab F(ab´)

2, an EGFR-targeted F(ab´)

2 fragment of

panitumumab [46].Strategies that manipulate the tumor micro-

environment can enhance the uptake of RIT agents. Pretreatment of cervical and head and neck squamous-cell carcinoma with the platinum chemotherapeutic agent, cisplatin, enhances the in vivo tumor uptake of 188Re-labeled antibodies directed against the human papilloma virus E6 oncoprotein. This effect was attributed to the induction of apoptosis of tumor cells in the vicinity of the tumor neovascula-ture, rather than internalization and nuclear transportation mechanisms [47]. Nevertheless, it is worth noting that 188Re-labeled anti-E6 antibodies significantly retard E6-transfected SiHa xenograft growth in athymic mice [48].Changes to the composition of the tumor milieu may occur due to the effects of the RIT agents. The same concentration of radioactivity split into several fractions reduced the toxicity, while maintaining therapeutic efficacy of 227Th-BnDOTA-trastuzumab, an a-particle-emitting HER2-targeted RIT agent. 227Th-BnDOTA-trastuzumab was found by dynamic contrast enhanced-MRI to increase the rate of diffusion in extravascular extraceullar space and the rate of clearance from plasma [49]. This suggests that fractionated RIT regimens cause the remodeling of the tumor microenvironment, significantly increasing tumor uptake over time, and there-fore, improve toxicity as fractionation progresses. Interestingly, pulsed high-frequency-focused ultrasound exposure also increased the uptake of 111In-labeled RIT agents directed against the LeV tumor antigen in A431 epidermal carcinoma xenograft-bearing athymic mice [50]. However,

Figure 1. Long- versus short-range radioimmunotherapy agents. (A) Off-target effects of long-range b-particle emitting radioimmunotherapy agents are a double-edge sword that causes more homogeneous irradiation of the tumor, but also causes the surrounding normal tissue to be irradiated, even though it does not express the target antigen. (B) The internalization and nuclear transport of short-range, high-linear energy transfer radioimmunotherapy agents can reduce cross-fire effects while increasing radiation dose to the nucleus.



uptake of 111In-labeled RIT agents in surround-ing normal tissue was also increased by the pulsed high frequency focused ultrasound.

Early examples of membrane-associated tumor antigens targeted by RIT agents include CEA, tumor-associated glycoprotein-72, MUC-1, L6, prostate-specific membrane antigen, placental-like alkaline phosphatase, and CD147(reviewed in [51]). The differential expression of such tumor antigens can result in wide heterogeneity in the local concentration of RIT agents even in efficiently targeted tumor regions. However, these heterogeneities can be overcome by cross-irradiating tumor cells that do not express the target antigen and that would otherwise escape being targeted by shorter-range RIT agents. RIT agents labeled with 90Y have a longer range (11.3 mm) compared with 131I and 177Lu. Although 131I-labeled RIT agents targeting a transmembrane glycoprotein of the immuno-globulin family, A33, are highly internalized by colorectal carcinoma cells [52,53], the shorter range of 131I (2.3 mm) and 177Lu (1.8 mm) com-pared with 90Y means that cross-irradiation of antigen negative tumour cells occurs less, but spares more normal tissue.

Tumor accumulation of noninternalizing, membrane-associated tumor antigen-targeted

diagnostic immuno-SPECT agents does not always correlate with tumor antigen density at the cell membrane [54]. Theranostic RIT agents, treatments that combine therapeutics with diag-nostics (i.e., immuno-SPECT), that internal-ize into tumor cells can be used to delineate antigen-positive tumor regions using SPECT. Although the SPECT signal may not directly correlate with the expression of antigen inside tumor cells, adequate contrast with normal tis-sue can be achieved following a wash-out period that clears excess radioimmunoconjugates from the tumor milieu. Thus, the SPECT signal can show how much theranostic was delivered to the tumor (whether correlated to antigen expression or not) (FiguRe 2).

A recent example of an internalizing immuno-SPECT agent is an111In-labeled antibody that targets the CDK inhibitor p27KIP-1, which is induced in the nucleus following treatment of tumor cells with the HER2-targeted immuno-therapy, trastuzumab (Herceptin). In athymic mice bearing MDA-MB-361 breast adenocarci-noma xenografts, the retention of 111In-DTPA-anti-p27KIP-1-Tat (which is capable of binding to p27KIP-1 in the nucleus after internalizing inside cancer cells due to the integration of the cell-penetrating peptide, Tat) was significantly

Clearance

γ

γ

γ

γ

Figure 2. Internalizing radioimmunotherapy agents for diagnostic SPECT-imaging. Following internalization, which protects g-ray-emitting theragnostic radioimmunotherapy agents from plasma clearance, a tumor SPECT signal is produced that contrasts with normal tissue and shows how much theranostic was delivered to the tumor.



higher in trastuzumab-treated compared with control xenografts in vivo [55]. Furthermore,111In-BnDTPA-anti-gH2AX-Tat (a RIT agent that targets phosphorylated H2AX, gH2AX, a marker of DNA DSB) tracks changes in DNA damage in vivo using mouse xenograft models of human cancer. 111In-BnDTPA-anti-gH2AX-Tat colocalized with gH2AX and produced SPECT signals proportional to the amount of DNA damage induced in tumors after local x-ray irradiation or bleomycin treatment [56]. Also, 111In-BnDTPA-anti-gH2AX-Tat and ion-izing radiation (10 Gy) increased the number of gH2AX foci compared with ionizing radia-tion alone. Similarly, coadministration reduced the surviving fraction of MDA-MB-231/H2N cells in vitro and tumor growth rate of MDA-MB-231/H2N xenografts in athymic mice in vivo [57]. Thus, measurements of the expres-sion of tumor-associated antigens can be made using internalizing RIT agents that are retained inside cancer cells, whilst RIT agents in extra-vascular extracellular space are washed out over time from the tumor milieu. Such internaliz-ing RIT agents are superior to noninternalizing counterparts, because such theranostic agents may also possess potent antitumor properties.

Internalization of RIT agents�� Receptor-mediated endocytosis

Many RIT agents target the RTKs, so the fol-lowing uses examples of RTKs to discuss issues of RIT internalization. RTKs, such as EGFR (ErbB1) and HER2 (ErbB2), are commonly over expressed in breast cancer and inappro-priately activate signal-transduction networks implicated in cancer development. Receptor-mediated endocytosis, which internalizes ligands bound to membrane-bound target receptors such as RTKs, may be exploited for the internaliza-tion of RIT agents. Receptor internalization and receptor recycling, the trafficking of internalized receptors back to the cell membrane, controls the amount of antigen available at the cell surface. EGFR undergoes rapid internalization via clath-rin-coated pits following capture of its ligand, EGF, and this process sustains EGFR signaling [58]. Clathrin-dependent endocytosis involves recruitment of the clathrin adaptor protein com-plex AP2. Endocytic vesicles are formed through the inwards invagination of clathrin-coated pits that pinch off from the plasma membrane, a process that requires the GTPase dynamin [59].

EGFR family receptors can shuttle to the nucleus. This was first observed for EGFR in

hepatocytes during liver regeneration [60] and EGF has also been found in the nucleus [61]. A constitutively active nuclear variant of EGFR called EGFRvIII was reported in hormone-refractory prostate cancer [62] and primary glioblastomas [63]. EGFRvIII can be targeted with internalizing 211At-labeled monoclonal antibodies (L8A4), which have higher levels of uptake into EGFR-transfected glioblastoma cells (U87MGDEGFR) than 131I-labeled iodo-gen-L84A [64]. Interestingly, phosphatidic acid generated at the plasma membrane by phos-pholipase D has been shown to induce type 4 phosphodiesterase-mediated ligand-indepen-dent internalization of EGFR. This leads to the accumulation of EGFR in juxtamembrane endosomes due to decreased receptor recycling [65]. Phospholipase D, an oncogene, which has been shown to play a role in cell invasion and metastasis in a murine model of breast can-cer [66], may therefore also cause resistance to EGFR-targeted RIT agents by downregulat-ing expression of EGFR at the cell membrane. HER2, which is targeted by 111In-labeled trastuzumab [67], full length HER3 (ErbB3) and the C-terminal fragment of HER4 (ErbB4) have all been found in the nucleus of cancer cells [68–71]. In contrast, CEA, a glycoprotein involved in cell adhesion, is noninternaliz-ing and thus has not been found expressed in the nucleus.

Boudousq et al. compared internalizing HER2-targeted RIT agents with noninter-nalizing CEA-targeted RIT agents [72]. They found that median survival of epidermal car-cinoma A431 xenograft-bearing athymic mice injected intraperitoneally with short-range a-particle-emitting 212Pb-labeled RIT agents was improved by the greatest extent by target-ing the internalizing HER2 receptor, compared with the noninternalizing antigen, CEA. This effect was observed even though the HER2-targeting RIT agents deposited a lower radia-tion dose (27.6 Gy) compared with the nonin-ternalizing CEA-targeted RIT agents (35.5 Gy) [72]. This evidence suggests that internalization of RIT agents enhances their relative biologi-cal effectiveness. However, noninternalizing CEA-targeted RIT agents labeled with the Auger electron emitter, 125I, have been shown to deliver a mean absorbed-radiation dose of 11.6–16.7 Gy to the tumor in athymic mice bearing A431 ovarian squamous carcinoma xenografts. This resulted in an increase in median survival from 32 days to 46–73 days,



depending on whether the RIT agents were administered as a single dose versus repeated intravenous infusions, respectively [73]. The therapeutic eff icacy of these noninternaliz-ing RIT agents may have been due to toxic interactions between Auger electrons (21 per decay and with energy of 2.3–22.9 keV) and the tumor cell membrane. The higher energy emissions of 125I, such as the g-rays (35 keV; 0.07/decay) and internal conversion electrons (27-32 keV; 0.93/decay), may have also con-tributed to the radiation dose, although their contribution would be negligible due to their low abundance per decay compared with Auger electrons. The cell membrane of A431 cells as well as another ovarian carcinoma cell line, SKOV3, has been found to be more radiosen-sitive to the effects of Auger electrons than the cytoplasm [74]. Subsequently, noninternalizing RIT agents have been found to be suitable for RIT of small-volume peritoneal carcinomatosis in a mouse model [39].

In contrast to noninternalizing RIT agents, internalizing RIT agents may be taken up into early endosomes positive for the GTPase Rab5 and can either traffic back to the cell membrane via a Rab4-regulated mechanism, or fuse with the lysosome under the regulation of Rab7 [75]. As the endosome traffics through the cyto-plasm it matures into an endolysosome, which is accompanied by a drop in the pH inside the endosome from 6.5 to 4.5 due to the influx of H+ ions via the endosomal V-ATPase, as it fuses with the dedicated lysosomes [75]. If internal-izing RIT agents are to be directed toward the nucleus, they must be capable of escape from the endosome before the leakage of metal ions from the chelate occurs at low pH, and they must also avoid being trafficked back to the cell membrane or undergoing proteolytic deg-radation in the lysosome through the action of acid-sensitive lysosomal hydrolases (FiguRe 3). Further modification of RIT agents may be necessary to promote their endosomal escape and this is an area that requires further atten-tion because better approaches for maximizing endosomal escape may lead to improvements in the efficacy and/or toxicity of internalizing high-LET RIT agents that have a very short–range (nm–µm), such as Auger emitters (125I, 123I and 111In).

Endosomal escape may not be as impor-tant to the therapeutic efficacy of RIT agents with emissions that traverse distances greater than those separating endosomes from the

critical targets in the nucleus. For example, 212Pb-DOTA-trastuzumab is internalized into endosomes, where 212Pb, leaking from DOTA due to the reduced pH of endosomes, likely becomes trapped [72]. a emissions originating from 212Pb at the cell membrane or trapped in endosomes, deliver a dose to the nucleus because their range is comparable to that of the diameter of the average cell (10–30 µm). b-emitting RIT agents, such as 177Lu-labeled anti-FAP antibod-ies, are rapidly internalized by FAP-expressing melanoma cells [76]. However, their hypotheti-cal translocation to the nucleus would not enhance the therapeutic efficacy of their long-range (µm–mm) b emissions. The anticancer efficacy of Auger electron-emitting RIT agents that internalize and transport to the nucleus can be measured by monitoring the formation of gH2AX foci. gH2AX foci form approximately ten nucleotides from the decay site of 125I and are detectable at sites of DNA double-stranded break damage [77].

�� Cell-penetrating peptidesCell-penetrating peptides (CPPs) mediate their own transduction into cells because they con-tain positively charged amino acids or alter-nating polar and nonpolar hydrophobic amino acid motifs that disrupt cell membranes. A list of CPPs is shown in Table 3 [75]. The mecha-nism of entry of CPPs into tumor cells is not fully understood. Proteoglycans expressed at the cell surface may act as low affinity recep-tors for some CPPs. Cell entry may involve a variety of clathrin-dependent and -indepen-dent pathways. Our poor understanding of the internalization mechanism is not aided by the fact that the precise endocytic mechanism may depend on the type of CPP, the type of antibody or antibody fragment, the mode of linkage of CPP to the RIT agent, the ratio of CPP to antibody and the lipid composition of the plasma membrane.

CPPs are capable of promoting escape from endocytic vesicles [85]. The same mechanism that disrupts cell membranes and promotes cell entry may also apply to endosomal mem-branes. Negatively charged phospholipid phos-phatidylserine, which has affinity for CPPs, is present in the opposite leaflet of the endosome to the leaflet that is adjacent to the endocytosed CPP-conjugated RIT agents. This arrangement may generate a membrane potential that cre-ates an electropore through which CPP-conju-gated RIT agents translocate [85]. Enrichment



of the inner leaflet of maturing late endosome bilayers with negatively charged phospho-lipid bis(monoacylglycero)phosphate has been hypothesized to promote Rab-dependent lipid mixing and membrane disruption properties of CPPs such as Tat at an optimal pH of 5.5 (FiguRe 3) [75].

Nuclear transportation of RIT agentsReceptors that are found in the nucleus, some of which have been targeted by RIT agents, include the EGFR family members, FGF receptor, c-Met, IGF-1 receptor, VEGF recep-tor, TrkA, interleukin receptors, IFN-g recep-tor and growth hormone receptor [86–89]. The

α/β-importin-mediated

nuclear transport

Receptor-mediatedendosomal

escape

Binding to nuclearantigen

Binding to nuclearantigen

Cell membraneantigen binding

Receptor-mediatedinternalization

Rab4+ Rab4+Rab7+ Rab7+

Rab5+Rab5+

CPP-mediated cellmembrane mixing and

internalization

CPP-mediatedendosomal escape via

PhtSer and BMP

pH 5.5

A B

Figure 3. Receptor-versus cell-penetrating peptide-mediated internalization and nuclear transport of radioimmunotherapy agents. (A) Membrane receptor-targeted internalizing short-range radioimmunotherapy (RIT) agents enter cells upon recognition of their target membrane receptor and, following receptor mediated endocytosis, are trafficked in the endosome, which pinches off from the cell membrane under Rab5 regulation. If the RIT agent is to be transported to the nucleus it must escape from the endosome, a process mediated by the target receptor, to which the RIT agent is bound, before it is either degraded in the lysosome or trafficked back to the cell membrane under Rab7 and Rab4 regulation, respectively. In the cytoplasm, the RIT agent–target receptor complex is recognized by the a /b-importin complex, which aids in the navigation through the nuclear pore complex. Subsequently, the RIT agent is released in the nucleus where it may be capable of binding to a nuclear target either through bi-specific targeting or by affinity of the target receptor for specific regulatory gene elements or proteins. The RIT agents’ short range high linear energy transfer emissions deposit a highly toxic radiation dose inside the nucleus. (B) CPP-functionalized internalizing short-range RIT agents mediate their own transduction into cells. This may involve a combination of endocytosis-dependent and -independent mechanisms. During endocytosis, CPP-functionalized RIT agents are trafficked in the endosome, which pinches off from the cell membrane under Rab5 regulation. The CPP aids endosomal escape via interactions with negatively charged phosphatidylserine and bis(monoacylglycero)phosphate before the RIT agent is either degraded in the lysosome or trafficked back to the cell membrane. In the cytoplasm, a nuclear localization sequence contained in the CPP allows the a /b-importin complex to recognize the RIT agent and navigation through the nuclear-pore complex ensues. Subsequently, the RIT agent is released in the nucleus, where it may be capable of binding to a nuclear target through specific targeting of nuclear proteins. There the CPP-functionalized RIT, which has short range, high linear energy transfer emissions, deposits a highly toxic radiation dose inside the nucleus or at specific subnuclear domains. CPP: Cell-penetrating peptide; BMP: bis(monoacylglycero)phosphate; PhtSer: Phosphatidylserine.



nuclear translocation of RIT agents occurs via positive diffusion, nonclassical mechanisms (i.e., CPPs), or through the classical nuclear-import pathway, which involves the concerted action of adaptor proteins called importins. Importin a binds directly to nuclear localization sequences (NLS) contained within motifs present in the cell-surface receptor or CPP. Importin a also interacts with a carrier protein called impor-tin b, a member of the b-karyopherin family. Importin b mediates interactions with the tri-meric nuclear-pore complex and is responsible for the navigation of carrier proteins through the nuclear pore, translocating the entire complex into the nucleus. Once in the nucleus, the GTP-bound form of a small Ras family GTPase called Ran (Ran-GTP) causes a conformational change in importin b. This dissociates the importin-a/b complex and releases its cargo into the nucleoplasm (FiguRe 3) [90].

The NLS of cell membrane tumor antigens and CPPs that interact with importin a contain posi-tively charged arginines and lysines. The EGFR family of receptors contain a tripartite NLS, highlighted in bold (RRRHIVRKRTLRR), while Tat contains a noninterrupted NLS com-prising six essential amino acids, highlighted in bold (GRKKRRQRRRPP). Such NLS can be used to potentiate the nuclear uptake of cell membrane tumor antigen-targeted RIT agents. 111In-DTPA-NLS-trastuzumab (111In-labeled trastuzumab incorporating the SV-40 large T antigen [CGYGPKKKRKVGG]) produces a greater retardation in the rate of growth of MDA-MB-361 breast adenocarci-noma xenografts in athymic mice compared with 111In-DTPA-trastuzumab, which lacks the SV-40 large T antigen [91]. This effect was attrib-uted to a greater deposition of radiation dose from Auger electrons to DNA. In this instance, the incorporation of the SV-40 large T antigen into HER2-targeted 111In-labeled RIT agents overcame resistance to trastuzumab treatment

[92]. Similarly, SV-40 large T antigen incorpo-ration into 111In-labeled anti-CD33 antibodies resulted in the effective killing of methotrexate-resistant acute-myeloid leukemia HL-60-MX-1 cells [93]. Furthermore, incorporation of SV-40 large T antigen is critical for nuclear uptake of 111In-labeled anti-CD123 antibodies, promising RIT agents directed against CD123+/CD131- leukemia stem cells [94].

Interestingly, intranuclear tumor antigens can become extracellular targets due to the high turnover of fast-growing tumor cells. Release of the intranuclear oncogene E6 into the tumor milieu from apoptotic or necrotic FaDu head and neck squamous-cell carcinoma cells per-mitted E6 to be targeted using an 188Re-labeled anti-E6 antibody injected intraperitoneally into E6-transfected FaDu xenograft-bearing athymic mice in vivo [95]. This was proof-of-principle that intranuclear tumor antigens may be targeted after they are released from inside the tumor cell into the tumor microenvironment.

Pathways that degrade RIT agentsUnder physiological conditions intracellular degradation pathways result in efflux of radio-nuclides from tumor cells (FiguRe 4). Levels of eff lux can be relatively high for radiometals with an oxidation state of +2, marginal for +3 radiometals, and as yet unknown for +4 radio-metals. Where RIT agents are concerned, these mechanisms may produce variable reductions in absorbed radiation dose. Where the final target is the nucleus, this may reduce damage caused by some Auger emitters to critical targets, for example, DNA. Proteolysis of the radiolabeled antibody may also abrogate any direct inhibi-tory properties. In size exclusion chromatogra-phy experiments, Auger electron-emitting RIT agents labeled with 111In obtained from cyto-plasmic extracts of BT-474 breast ductal car-cinoma cells were found to be present in high-molecular-weight forms (which represented

Table 3. Selected cell-penetrating peptides.

Type of CPP Name of CPP Sequence Ref.

Truncated version of the HIV-1 transactivation protein Tat peptide GRKKRRQRRR [78]

Derivative of Drosophila antennapedia homeoprotein Penetratin RQIKIWFQNRRMKWKK [79]

Herpes simplex virus VP22 protein VP22 peptide APPGANAVASGRPLAFS [80]

Chimeric peptide Transportan GWTLNSAGYLLGKINLKALAALAKKIL [81]

Amphipathic peptide MPG SVVDRVAEQDTQA [82–84]

Amphipathic peptide Pep-1 KETWWETWWTEWSQPKKKRKV [82–84]

Amphipathic peptide ppTG1 GLFLALLKLLKSLWKLLLKA [82–84]

CPP: Cell-penetrating peptide.



nondegraded constructs) to a greater extent compared with 123I-labeled RIT agents also obtained from cytoplasmic extracts of BT-474 cells. Subsequently, the 111In associated with 111In-labeled RIT agents was eff luxed from the cytoplasm to a lesser extent compared with

the 123I associated with 123I-labeled RIT agents [96]. 111In-labelling of RIT agents was achieved by conjugation of DTPA anhydride or pSCN-BnDTPA to lysines. In contrast, the radioha-lodination of tyrosines with 123I was achieved using the Iodogen method.

Efflux via amino acid transporter

Proteosomal degradation

TRIM21 recognition

and ubiquitylation

Rab5+

Low pH and lysosomal

hydrolase-mediated degradation

Rab5+

Rab7+

Rab7+

pH 4.5

Radiohalogenated RIT agent

DTPA-linked RIT agent

pH 4.5

Figure 4. Hypothesized mechanisms of degradation of radiohalogenated and diethylenetriaminepentaacetic acid-linked radioimmunotherapy agents. After receptor mediated endocytosis of radiohalogenated RIT agents under Rab5 regulation, acidification of the endo-lysosome and fusion with the lysosome produces a lysosomal pH of 4.5. Low pH coupled with the action of pH-dependent endopeptidases cause the digestion of the antibody component of RIT agents. It is hypothesized that radiohalogenated RIT agents that escape the endosome could be targeted by the intracellular antibody receptor, TRIM21 (although this is speculative at present). TRIM21 may mark the RIT agent with ubiquitin, leading to its degradation in the proteosome. Halogenated tyrosines are effluxed from the cytoplasm via amino acid transporters. Although DTPA-conjugated RIT agents may also be degraded in the lysosome, and may be targeted by TRIM21 in the cytoplasm, radiolabeled DTPA-conjugated lysines do not interact with amino acid transporters. Thus, high amounts of activity are retained within target cells when the RIT agent is radiolabeled via DTPA compared with when it is radiohalogenated. DTPA: Diethylenetriaminepentaacetic acid; RIT: Radioimmunotherapy.

Key Term

TRIM21: Protein that is capable of binding to antibodies that are internalized into cells and automatically marking the antibody for destruction inside the cytoplasm. TRIM21 has evolved as a component of the immune system that attaches small proteins called ubiquitins to the surface of antibodies inside the cytoplasm. Ubiquitin-coated antibodies are subsequently recognized by the proteosome, a large protein complex that breaks bonds between the amino acids comprising the antibody. This process completely destroys internalized antibodies. Therefore, it is speculated that TRIM21 is capable of also binding to radioimmunotherapeutic agents, because their structure is based upon the incorporation of antibodies, and mediating their destruction via the proteosome.



111In- and 123I-labeled RIT agents enter dif-ferent degradation pathways. 123I-labeled RIT agents are catabolized to iodotyrosine, which is eff luxed from cells and subsequently deio-dinated. However, 111In-labeled RIT agents linked to DTPA are degraded by proteolysis to 111In-DTPA-e-lysine, which is retained inside the cytoplasm due to its anionic charge and inability to interact with amino acid transport-ers. Both pathways lead to the destruction of the antibody component of the RIT agent. Radiohalogenation by direct tyrosine labeling is susceptible to radiohalogenases, leading to significant radioiodine uptake in the thyroid and stomach in vivo. In contrast, 111In-labelling via DTPA enhances the amount of radioactivity retained within cancer cells and therefore can increase the radiation dose or SPECT tumor-to-normal tissue signal ratio [96].

An emerging degradation mechanism, which mediates the proteosomal degradation of anti-bodies that become bound to virus particles, involves a recently discovered intracellular antibody receptor. As a result of this intracel-lular antibody receptor that targets infecting virus particles simply by recognizing the host antibodies that coat these virus particles, it is speculated that the same mechanisms may inadvertently target the antibody compo-nent of RIT agents inside tumor cells. The prototypic tripartite motif (TRIM) protein, TRIM21 (also known as Ro52), is the intracel-lular antibody receptor that is responsible for recognizing antibodies in the cytoplasm and mediating their destruction. Human, canine, guinea pig, monkey, mouse, and rat species of antibody become bound by TRIM21 with high affinity (Kd ≈ 10 µM). The conserved PRYSPRY (B30.2) domain of TRIM21 binds to the Fc region. The binding of TRIM21 to the antibody directs the antibody into a con-served degradation pathway. In the cytoplasm, the E3 ubiquitin ligase activity of the TRIM21 RING domain marks the antibody, and the virus to which it may be bound with ubiqui-tin. This results in the proteosomal degrada-tion of the antibody (and virus) within 2 h of internalization [97,98].

Consistent with a hypothesis that internal-ized antibodies might be targeted by TRIM21, the antibody Fc domain is not detectable at high levels in the cytoplasm of HeLa cells engi-neered to overexpress the antibody Fc domain, suggesting that ectopically overexpressed Fc is turned over efficiently, consistent with a

degradation mechanism [99]. However, high levels of Fc were found in this same model of ectopic Fc overexpression following treat-ment of the HeLa cells with shRNA specific for TRIM21 or 2 µM epoxomycin, a selective proteosome inhibitor. The use of an inhibitor of the E3 ligase activity or antibody binding properties of TRIM21 may lead to the stabi-lization of RIT agents inside tumor cells [100]. Mutation of the ‘HNH’ motif at positions 433–435 in the Fc region, which forms inter-actions with W381, W383, D355 and D452 of TRIM21, may abolish TRIM21 binding. Thus, antibody fragments, such as HER2-targeted 177Lu-DOTA-ABY-027 and EGFR-targeted 111In-CHX-A”-DTPA-panitumumab F(ab´)

2, may avoid TRIM21 binding [45,46]. An

interesting approach to enhancing the efficacy and/or toxicity of RIT may be the combination of proteosome inhibitors with internalizing RIT agents.

Future perspectiveRecent trends in developing RIT agents that incorporate short range particle-emitting radionuclides exploit the high LET properties of a-particle and Auger electron emissions to deposit a high radiation dose at the decay site. Internalization and nuclear transport of short-range RIT agents is dependent on internalizing cell-membrane antigens or the incorporation of a CPP. CPPs enhance endocytosis and nuclear transport of RIT agents that target cell-mem-brane antigens. Innovative combinations of CPPs with mono- or bi-specific antibodies will enhance the endocytosis and nuclear transport of RIT agents, causing greater cell killing by induc-ing higher numbers of cytotoxic DNA double-strand breaks, whilst minimizing cross-fire to normal tissue.

Internalization of theranostic RIT agents used for SPECT could be exploited for diag-nostic imaging of overexpressed or therapy- and stress-induced antigens inside cancer cells. This will produce a tumor SPECT signal that shows how much theranostic was delivered to the tumor (whether correlated to antigen expression or not) following a wash-out period that removes excess RIT agent. The tumor microenvironment presents a significant obstacle to the mobility of RIT agents and the accumulation of RIT agents may not correlate closely with the expression of the tumor tar-get. Thus, an important question to address is how fractionated RIT is capable of increasing



the diffusion within extravascular extracellu-lar space and plasma clearance of RIT agents. Greater understanding of these areas will increase uptake and selectivity of RIT agents.

The internalization of cell-membrane anti-gens is a well understood mechanism. Exactly how RIT agents, especially CPP-linked RIT agents, escape the endosome is poorly under-stood. It has been hypothesized that Tat inter-acts with negatively charged phosphatidylserine and bis(monoacylglycero)phosphate for escape into the cytoplasm [85]. RIT agents that contain an NLS, or are bound to a receptor containing an NLS, translocate to the nucleus through the nuclear-pore complex by forming an associa-tion with a/b-importin. This process can be exploited for the localization of RIT agents within specific subnuclear sites, such as DNA damage foci.

Degradation mechanisms cause the efflux of radiohalogenated RIT agents from tumor cells. Radiohalogenated tyrosines are eff luxed from tumor cells, while RIT agents integrating metal ion chelators such as DTPA are metabolized to radionuclide-DTPA-e-lysine complexes that are incapable of interacting with amino acid transporters. It is possible, although not yet tested that TRIM21, an intracellular antibody

receptor, may also mediate the degradation of RIT agents inside tumor cells. TRIM21 binds to the Fc region of antibodies and its E3 ubiq-uitin ligase marks material for proteosomal degradation. Consideration of these degrada-tion pathways may be key to RIT develop-ment. Future work is needed to focus on the interactions between RIT agents and TRIM21. Mutation of the ‘HNH’ motif – which forms interactions with TRIM21 – may abolish TRIM21 binding and use of antibody frag-ments or affibodies, which lack an Fc domain, will avoid TRIM21 binding. Furthermore, the combination of proteosome inhibitors with internalizing RIT agents may be an interest-ing strategy for enhancing the efficacy and/or toxicity of RIT.

Financial & competing interests disclosureThe authors wish to acknowledge support from Cancer Research UK and the Medical Research Council. The authors have no other relevant affiliations or financial involvement with any organization or entity with a finan-cial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Executive summary

Radionuclides

�� Radioimmunotherapy (RIT) agents that incorporate short range, particle-emitting radionuclides exploit the high linear energy transfer (LET) properties of a-particle and Auger electron emissions.

�� a-particle and Auger electron emissions cause cell killing by inducing complex cytotoxic DNA double-strand breaks, while minimizing cross-fire and toxicity to normal tissue, as compared with low-LET, longer range b-emitting radionuclide 90Y-labeled RIT agents.

RIT agents & the tumor microenvironment

�� Fractionated short-range RIT regimens may be capable of increasing the diffusion within extravascular extracellular space and plasma clearance of RIT agents.

Internalization of RIT agents

�� Short range, high LET RIT agents, especially Auger emitters, may be internalized and transported to the nucleus for optimal efficacy.

�� Internalization of short range, high LET RIT agents is dependent on internalizing cell membrane antigens or the incorporation of a cell-penetrating peptide (CPP) to target an intracellular antigen.

�� Internalization of theranostic RIT agents may be exploited for diagnostic SPECT/PET imaging of overexpressed or induced tumor antigens.

�� Internalization of cell membrane antigens occurs by receptor-mediated endocytosis, while for CPPs internalization may vary depending on cell- and CPP-type.

�� Endosomal escape of Auger-emitting RIT agents is important for therapeutic effect.Nuclear translocation of RIT agents

�� RIT agents that contain nuclear localization sequences translocate to the nucleus via the classical nuclear-import pathway.Pathways that degrade RIT agents

�� Degradation mechanisms cause the direct efflux of radiohalogenated RIT agents, while others, for example +3 radiometals such as diethylinetriaminepentacetic acid-linked 111In, are retained within tumor cells.

�� It is possible to hypothesize that TRIM21, a recently discovered intracellular antibody receptor, may mediate the ubiquitin-dependent proteosomal degradation of RIT agents, and interference with this mechanism with proteosome inhibitors may improve RIT.



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