Structural & functional characterisation of antibody therapeutics

DigitalisDigitalis

Roy JefferisSchool of Immunity & Infection, University of Birmingham UK

CASSS – Practical Applications of Mass Spectrometry. Marina del Rey, September 2010

Market size: $22 billion in 2007

Projected for 2010: $30 billion

28 licensed; ~ 500 in development

80 in clinical trials; 200 companies

Challenge: To reduce:

“Cost of Goods” (CoG)

“Cost of Treatment” (CoT)

Natural & recombinant antibodies protect due to:

Neutralisation/blocking of infectious agents and their products:e.g. virus, toxins, venoms cytokines, e.g. TNFα

Infliximab, Adalimumab, Golimumab, Cimzia(Fab-Peg)

Formation of immune complexes and activation of killing mechanisms e.g. bacteria cancer cells

Rituximab, Herceptin, Cetuximab

Agonist or antagonist activity (autoimmunity!) Panitumumab, Cetuximab

Structural & functional characterisation of antibody therapeutics

Antibody classes, isotypes and allotypes

Antibody effector activities

IgG-Fc glycoforms

IgG-Fab glycoforms

Biosimilars & Antibody-drug conjugates

Human immunoglobulin classes & subclassesThe result of gene duplication, mutation and selection

IgM IgG IgD IgE IgA

IgG1, IgG2, IgG3, IgG4; IgA1, IgA2

The humoral immune response is “orchestrated” to provide optimal protection to a given “insult”

Four chain structure of the antibody molecule

VL

VH CH1 C

CL

H2 CH3

Hinge region, susceptible to enzymatic cleavage

Fab: antigen binding Fc: effector activation

OligosaccharideInterchain disulphide bridges

Domain structure of IgG

Antigen

binding

FcEffector functions

Ludger

FabFab

Antigen

binding

oligosaccharide

IgG2IgG2~ 25 %

IgG1IgG1~ 60 %

SpA/SpG bindingSpA/SpG binding

IgG4IgG4~ 5 %

IgG3IgG3~ 10 %

SpA/SpG bindingSpG binding

IgG subclass of licensed antibody therapeutics

Rituxan IgG1κ

Zenopax IgG1κ

Herceptin IgG1κ

Remicade IgG1κ

Simulect IgG1κ

Synagis IgG1κ

Campath IgG1κ

Humira IgG1κ

Tocilizumab IgG1κ

Golimumab IgG1κ

Xolair IgG1κ

Raptiva IgG1κ

Erbitux IgG1κ

Avastin IgG1κ

Mylotarg IgG4κ

Tysabri IgG4κ

Mylotarg IgG4κ

Vectibix IgG2κ

Biacore binding signals (RU) & dissociation constants (Kdiss) for anti-IgG-Fc: R10Z8E9 with human & non-human primate sera

Sample (serum) RU KDiss (M)

Human 1274 1.77 ×10−10

Cynomolgus 3 No binding

Baboon 0 No binding

Marmoset 5 No binding

Rhesus macaque −3 No binding

Chimpanzee 1077 2.21 ×10−10

Stubenrauch K, et al., J Pharm Biomed Anal. 49:1003-1008 (2009)

Human IgG gene polymorphism (allotypes)

Heavy chain Light chainIsotype IgG1 IgG2 IgG3 IgG4 κ

Allotype G1m G2m G3m G4m Km 1 23 21,28 4a 12 11, 5 4b 23 13,14 3

17 10,1516, 624,26

27

Jefferis, R. & Lefranc, M-P. mAbs 1:1-7 (2009)

Allele frequency varies between populations

Europeans (%) Orientals (%)

G1m(3) 80 0

G1m(17,1) 20 85

G1m(1,3) 0 15

Jefferis, R. & Lefranc, M-P. mAbs 1:1-7 (2009)

Magdelaine-Beuzelin, C., Jefferis R et al., Pharmacogen Genomics. 19:383 (2009).

Allotypes of licensed antibody therapeutics

Rituxan G1m(17,1) Km 3

Zenopax G1m(17,1) Km 3

Remicade G1m(17,1) Km 3

Campath G1m(17,1) Km 3

Humira G1m(17,1) Km 3

Herceptin G1m(17) Km 3

Xolair G1m(17) Km 3

Simulect G1m(3) Km 3

Synagis G1m(3) Km 3

Erbitux G1m(3) Km 3Carter, P. et al. Proc Natl Acad Sci. 89:4285-9 (1992)

Jefferis, R. and Lefranc, M-P. mAbs 1, 332-38 (2009)

Sequence correlates for IgG1 heavy chain allotypes

G1m(1) G1m(2)

G1m(17) K 214

E356-E- M358; Ala431

D356-E-L358; G431

G1m(3) R 214

G1m(17) IgG-Fc engineered to remove G1m(1) allotope

Carter, P. et al. Proc Natl Acad Sci. 89:4285-9 (1992)

Jefferis, R. and Lefranc, M-P. mAbs 1, 332-38 (2009)

Structural & functional characterisation of antibody therapeutics

Antibody classes, isotypes and allotypes

Antibody effector activities

IgG-Fc glycoforms

IgG-Fab glycoforms

Biosimilars & Antibody-drug conjugates

Fc receptors (FcγR) expressed on leucocytes:Mediators of inflammatory cascades : antibody dependent cellular

cytotoxicity, phagocytosis, super oxide, enzyme release etc

FcγRI FcγRIIa* FcγRIIb FcγRIIIa* FcγRIIIb(IgG1,3,4) (IgG1,2,3) (IgG1,3) (IgG 1,2,3,4*) (IgG 1,2,3,4*)

* dependent on FcγR polymorphisms

* dependent on IgG & FcγRIIIa glycoform

B

FcRn:

Neonatal Fc receptor

Catabolism (half-life):

IgG1, IgG2, IgG4 > 21 days; IgG3 ~ 7 days

Placental transport:

Passive transport of all IgG subclasses, providing immune protection for the newborn

Complement activation and complement receptors

Classical – C1 (IgG1,3) Lectin - MBL (IgG1,2,3,4)

Complement receptors on leucocytes

CR1, CR2, CR3, CR4

Membrane attack complex

Activities of aglycosylated IgG

FcγRI activation reduced x 10-2

FcγRII “ “ abolished

FcγRIII “ “ abolished

C1 “ “ abolished

FcRn half-life unaffected

Jefferis, R. WO2008030564. Expert Opin. Ther. Pat. 19:101-105 (2009).

Activities of aglycosylated IgG

FcRn catabolism unaffected

placental transport ??

Neutralisation unaffected

Apoptosis (caspase 3 dependent) unaffected

Agonist/antagonist unaffected

“Take home message ”

CQA defined as:

A physical, chemical, biological or microbiological property or characteristic that should be within an appropriate limit, range or distribution to ensure the desired product quality (ICH Q8 R1).

Requirement:

100 % occupancy & fidelity or 0 % occupancy

Glycosylation is a Critical Quality Attribute (CQA) for monoclonal antibodies therapeutics

Structural & functional characterisation of antibody therapeutics

Antibody classes, isotypes and allotypes

Antibody effector activities

IgG-Fc glycoforms

IgG-Fab glycoforms

Biosimilars & Antibody-drug conjugates

The glycoform profile may be species, tissue, site and/or gender specific

High mannose Complex diantennary

Glycosylation contributes to:

Protein folding

Quality control

Trafficking

Secretion

Solubility

Stability

Function

Immunogenicity

Oxford Glycobiology Institute

GPI

CD59

GPI: Glycosylphosphatidyl inositol

Co-translational glycosylation

Post-translational oligosaccharide processing

Plasma

membrane

Glycosylation of protein therapeutics:Human genome sequence shows that ~50 % of proteins express N-X-S/T glycosylation sequon(s) and may bear N-linked oligosaccharides; the possible presence of O -linked oligosaccharides cannot be predicted

Endoplasmic reticulum

Oligosaccharides released from polyclonal human IgG-Fc

-Gln-Tyr-Asn297-Ser-Thr-Tyr-Arg-

GlcNAc Fuc

GlcNAc

Manα(1- 6) α(1- 3)

Man ManGlcNAc

GlcNAc GlcNAc

GalGal

Neu5Ac Neu5Ac

G0bF

G1bF

G2bF

G0; G0F

G1; G1F

G2; G2F

HPLC of 2-aminopyridine derivativesOligosaccharides released from polyclonal human IgG-Fc

G0F

G0bFG0

Jefferis, R. Takahashi N., et al. Biochem. J. 268: 529-537 (1990)

Oligosaccharides released from monoclonal IgG1 & IgG4

IgG1-Cri

IgG-Wid IgG4-As

IgG4-Rea

G0F

G2FNon fucosylated

Jefferis, R. Takahashi N., et al. Biochem. J. 268: 529-537 (1990)

Myeloma protein Wid is glycosylated in the Fc & Fab SDS-PAGE; reducing conditions

Wid PNGase F Endo F

Heavy chain

Light chain

Mimura Y., Jefferis R. J Immunol Methods. 326:116-26 (2007)

MALDI-TOF MS of IgG-Fc: protein Wid

Native

Abu

ndan

ce

Deglycosylated

Mass (kDa)Mass (kDa)

Mimura, Y., Jefferis, R. et al., Molec Immunol 37:697-706 (2000)

ESI-MS analysis shows symmetric & asymmetric pairing

Glycoform Molecular mass (kDa) Observed Calculated

Degly 49871 49866

G0 + NM3N2 52263 52260

G1 + NM3N2 52425 52422

(G0)2 52467 52463

G0 + G1 52630 52625

G0 + G2/ (G1)2 52791 52788

G1 + G2 52951 52950

Hypogalactosylation of IgG-Fc in patients with Wegener’s Granulomatosis

Autoimmune vasculitis with antibodies to proteinase 3 (PR3)

G0

G0F

Normal Wegener’s

Mimura, Y., Jefferis R. et al. J Immunol Methods. 326:116-26 (2007)Holland, M., Jefferis, R. et al. Clin.Exp.Immunol. 129:183-190 (2002)

Oligosaccharides released from polyclonal human IgG-FcNP-HPLC analysis of 2-AB labelled glycans

G1(1,6)F

G0

G0F

G1(1,6)

G1(1,3)

G1(1,3)F

G1(1,6)FB

G2

G2F

G2FB

G2S1

G2FS1

G2FS2G0B

G0FB

G1(1,3)FB

Mimura, Y., Jefferis, R., Mimura-Kimura, Y., Abrahams, J., Rudd, P. M.: Therapeutic antibodies: From theory to practice, (Ed) An, Z., Wiley, 2009.

Adalimumab: CHO cells

Infliximab: NS0 cells

G0FG2FS1

G2FG1F

HPLC

Polyclonal IgGG0

Infliximab/Remicade (Sp2/0)

45000 46000 47000 48000 49000 50000 51000 520000

100

%

( ) [ ] ( ) ( ) (49520

49391

49550

51000

5112450962

50834

00

51155

C-terminal –Ser-Pro-Gly

C-terminal –Ser-Pro-Gly-Lys

G0FG

G0FK

G1FK

G1FG

Deglycosylated heavy chains

Herceptin/Trastuzumab (CHO)

G0F

45000 46000 47000 48000 49000 50000 51000 520000

100

%

49152

49182

49497

0 51000

); ( ,50596

50758

G1F

Deglycosylated heavy chain

C-terminal –Ser-Pro-Gly

Herceptin/Trastuzumab (CHO)

7200 147400 147600 147800 148000 148200 148400 148600 148800 149000 1492

148153147995

147830

148314

148468

100(G0F)2 G0F,G1F

(G1F)2

%

50

Prepn of homogeneous glycoforms of IgG & IgG-Fc

sialidase hexosaminidase

Manβ1 - 4GlcNAcβ1 - 4GlcNAcβ1-Asn297

S.A.α2 - 6Galβ1 - 4GlcNAcβ1 - 2Manα1

66

33

Fuc

66

α1

Endo Fβ-galactosidase α-mannosidase

M3N2(F)

MN2(F)

S.A.α2 - 6Galβ1 - 4GlcNAcβ1 - 2Manα1

Endo E

NGA2(F) or G0(F)

Preparation of homogeneous glycoforms of IgG-Fc (Wid)

Native

(NGA2)2

(M3N2)2

52,470

51,657

(MN2)250,999

Deglycosylated49,871

Mimura, Y., Jefferis, R. et al., Molec Immunol 37:697-706 (2000)

G0/G0F glycoforms may activate pattern recognition receptors

Mannan binding lectin (MBL); Mannose receptor (MR)

N

G2/G2F glycoforms may exhibit enhanced activity

C1 complement activation; FcRn placental passage

N

Jefferis, R.. Nature Reviews: Drug Discovery. 8, 226-234 (2009)

Presence or absence of fucose/bisecting N-acetylglucosamine is a CQA

N

Igeneon AG

Enhanced FcγRIIIa mediated ADCC

N?

Enhanced FcγRIIIa mediated ADCC

Sialylated glycoforms of IgG have a reduced inflammatory activity

N

Anthony RM, et al., Proc Natl Acad Sci U S A. 105:19571-8 (2008)

Glycoprotein production vehicles:

Mammalian: CHO, Sp2/0; NSO; Per.C6; HEK 293 etc

Transgenics: goat; sheep; cows; rabbits; pigs etc

Aves: chickens (eggs)

Yeasts: Pichia pastoris; Saccharomyces cerevisiae

Insect cells: Sf9 (baculovirus infected)

Plants: tobacco; corn; tomato; potato; moss

Bacteria: Escherichia coli; Bacillus subtilis

Glycoform analysis of specific antibody populations: IgG subclass tryptic glycopeptide

Capture Peptide Calculated Observed

IgG1 Protein A E293EQYNSTYR301 595.75 595.78

IgG2 Protein A E293EQFNSTFR301 579.76 579.79

IgG3 Protein G E293EQFNSTFR301 579.76 579.78

IgG4 Protein A E293EQFNSTYR301 587.75 587.79

Wuhrer M et al., Proteomics. 7:4070-4081 (2007)

Glycoform analysis of IgG1 & IgG2 subclass antibodies

G0F G1F G2F

IgG1

IgG2

Wuhrer M et al., Proteomics. 7:4070-4081 (2007)

Oligosaccharides released from anti-platelet antibodiesR

elat

ive

abun

danc

e

0.2

0.1

Wuhrer M. et al., J Proteome Res. 8 :450-6 (2009)

Glycoform profile of anti-PR3 autoantibodies from patients with Wegner’s Granulomatosis

Capture anti-PR3 ANCAPR3 Elisa plate

elute

Capture IgG1,2,4/3SpA/SpG plate

elute

Digest with trypsinnano-HPLC-ESI-MS

Wuhrer M et al., Proteomics. 7:4070-4081 (2007)

IgG glycoforms & structure/function stability

sialidase hexosaminidase

Manβ1 - 4GlcNAcβ1 - 4GlcNAcβ1-Asn297

S.A.α2 - 6Galβ1 - 4GlcNAcβ1 - 2Manα1

66

33

Fuc

66

α1

Endo Fβ-galactosidase α-mannosidase

M3N2(F)

MN2(F)

S.A.α2 - 6Galβ1 - 4GlcNAcβ1 - 2Manα1

Endo E

NGA2(F) or G0(F)

Structures: Krapp, S., Jefferis R et al. J.Mol.Biol. 325:979-989 (2003)

IC: Mimura, Y., Jefferis, R. et al. J.Biol.Chem. 276:45539-45547 (2001).

-0.2

0.0

0.2

0 10 20 30 40 50 60 70

0.0 0.5 1.0 1.5 2.0

-4

-2

0

2

-0.2

0.0

0.2

0 10 20 30 40 50 60 70

0.0 0.5 1.0 1.5 2.0 2.5 3.0

-6

-4

-2

04

2

0

2

0.0 0.5 1.0 1.5 2.0 2.5 3.0

6

4

2

0

-0.2

0.0

0.2

0.0 0.5 1.0 1.5 2.0 2.5 3.0

-6

-4

-2

0

DSC: Mimura Y., Jefferis R. et al., Molec Immunol. 37:697-706 (2000)

lated

G0F G1FG0

sialylated glycans

IgG3 WT

IgG3 F243A

G0

G0FG1F

G2F

F/A243 mutation results in high galactosylation & sialylation in CHO cells

G2FS1 G2FS2

Lund J., Jefferis R. et al. J Immunol. 157:4963-9 (1996) Mimura Y., Jefferis R. unpublished

Aglycosylated (Asn/Ala297) mIgG2b is sulphated at Tyr296

e lat

ive

inte

nsity

R

Matsuda K., Jefferis, R. et al., FEBS Lett. 584(15):3474-3479 2010

“Take home message ”

IgG-Fc glycoform profile is a CQA

Effector functions vary between IgG-Fc glycoforms

Non-fucosylated IgG binds FcγRIIIa with high affinity

FcRn, SpA & SpG binding are not dependent on glycosylation!!

IgG-Fc glycoform profiles can vary in health & disease

!! Gaza-Bulseco G, J Chromatogr A. 1216(12):2382-7 (2009)

Structural & functional characterisation of antibody therapeutics

Antibody classes, isotypes and allotypes

Antibody effector activities

IgG-Fc glycoforms

IgG-Fab glycoforms

Biosimilars & Antibody-drug conjugates

~ 30 - 40 % of normal polyclonal IgG is N-glycosylated in VH or VL

Mimura, Y., Jefferis R. et al. J Immunol Methods. 326:116-26 (2007)

Jefferis, R. Nature Reviews:Drug Discovery. 8, 226-234 (2009)

Asn297

|GlcNAc GlcNAc

Man

Man

Man

GlcNAc

GlcNAcGal

Neu5Ac

Neu5Ac

_

_

_

__

_

_

_

_

Fuc|\

\

GlcNAc _

IgG-Fc sugars

|

Gal

Asn297

|GlcNAc GlcNAc

Man

Man

Man

GlcNAc

GlcNAcGal

Neu5Ac

Neu5Ac

_

_

_

__

_

_

_

_

Fuc|\

\

GlcNAc _

IgG-Fab sugars

|

Gal

Glycan profile of cetuximab IgG1-Fc & IgG1-Fab

Produced in Sp2/0 cells

G3F

G3FSG4F

G2FS2

Fab N88Fc N297 G1F

G0F

G2F

a glycosylation sequon N31GS in VL is not occupied

Qian, J. et al., Anal. Biochem. 364,8–18 (2007)

Hypersensitivity reactions on exposure to Erbitux a therapeutic licensed for colorectal, head & neck cancer.

25/76 patients had a hyper -sensitivity reaction to the drug.

IgE antibodies were shown to be specific for galactose α(1-3) galactose present on the VH

Qian, J. et al., Anal. Biochem. 364:8–18 (2007)

Mendelsohn J. Semin Oncol. 33:369-85 (2006)

Chung, C.H. et al., N Engl J Med. 358:1109-1117 (2008)

“Take home message ”

IgG - Fab glycosylation may be beneficial e.g. for solubility and formulation at high concentrations

CHO cells may deliver IgG-Fab glycosylation fidelity

Sp2/0 & NS0 cells are not suitable for the production of IgG-Fab glycoforms – may extend to other complex glycoproteins

Wu SJ., Gilliland G et al., Protein Eng Des Sel. 23:643-51 (2010)

Structural & functional characterisation of antibody therapeutics

Antibody classes, isotypes and allotypes

Antibody effector activities

IgG-Fc glycoforms

IgG-Fab glycoforms

Biosimilars & Antibody-drug conjugates

Biosimilar recombinant proteins: What is the structure of the innovator product?

Gene sequence only provides sequence of neo-protein

Post translational modifications: glycosylation, γ -carboxylation, acetylation, proline isomerisation etc

Production/isolation/purification may result in chemical modifications: atypical conformation; aggregates; fragmentation; oxidation; deamidation; deimination; iso-aspartyl residues; glycation

Structural characterisation of the innovator product obtained from the pharmacy may vary depending on age & history.

Rapid comparison of a candidate biosimilar to an innovator monoclonal antibody with advanced liquid chromatography

and mass spectrometry techniques

Conclusion:

This “biosimilar” cannot be approved due to sequence differences between it and the innovator product

Xei H et al., mAbs 2:1-16. (2010)

Comparison of whole IgG masses

Xei H et al., mAbs 2:1-16. (2010)

Comparison of oligosaccharides

G0F

G1F

G1F

G2F

G0

G1

G1

Man5

Xei H et al., mAbs 2:1-16. (2010)

Comparison of light chain masses

Comparison of heavy chain masses

Comparison of deglycosylated heavy chain tryptic peptide masses

EPQVYTLPPSREEMTK

EPQVYTLPPSRDELTK

Xei H et al., mAbs 2:1-16. (2010)

Genentech sequence for Herceptin CH3 domain

GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

Harris R. et al., Journal of Chromatography B, 752:233–245 (2001)

Drug bank sequence for Herceptin CH3 domain

GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

http://www.drugbank.ca

Sequence correlates for IgG1 heavy chain allotypes

E356-E- M358; Ala431

D356-E-L358; G431

G1m(17) IgG-Fc engineered to remove G1m(1) allotope

Carter, P. et al. Proc Natl Acad Sci. 89:4285-9 (1992) Jefferis, R. and Lefranc, M-P. mAbs 1, 332-38 (2009)

Note Added to Proof

It has been brought to our attention that the level of deamidation reported for many of the peptides in the “biosimilar” antibody are much higher than previously reported for the innovator antibody.

We acknowledge that the digestion protocol was not optimized to minimize sample preparation-induced deamidation and believe that the high levels reported for certain peptides may not be reflective of the true level of deamidation in the sample prior to digestion.

Harris RJ et al., J Chromatogr B Biomed Sci Appl 752:233-45 (2001)

Present & future challenges:

Production vehicle specific host contaminants

Antibody/drug conjugates

Antibody fragments

Alternative scaffolds

Structural basis for adverse reactions:

immunogenicity; cross reactivity/non-specific binding to non-target molecules

Proceed on a “case by case” basis & expect the unexpected

Noriko TakahashiJohn Lund Margaret Goodall

Peter SondermannYusuke MimuraMark Holland

Press

Jefferis R. The antibody paradigm: present & future development as a scaffold for biopharmaceuticals. Biotech Gen Eng Rev 26:1-42. (2010).

Jefferis, R. & Lefranc, M-P. Human Immunoglobulin Allotypes: Possible implications for immunogenicity. mAbs 1:1-7 (2009)

Jefferis, R. Glycosylation as a strategy to improve antibody-based therapeutics. Nature reviews: Drug Discovery. 8: 226-234 (2009)

Jefferis, R. Recombinant antibody therapeutics: Impact of glycosylation on mechanisms of action. TIPS 30:356-62 (2009)