Engineering Antibodies (1) MSc Programme University of Nottingham 14 th February 2005 by Mike Clark, PhD Department of Pathology Division of Immunology.

Engineering Antibodies (1)

MSc Programme University of Nottingham14th February 2005

by

Mike Clark, PhDDepartment of Pathology

Division of Immunology

Cambridge University

UK

www.path.cam.ac.uk/~mrc7/

Antibody based immunotherapeutics

IgG is the preferred class

Schematic view of IgG domains

Antibody fragments can also be used

Antibodies can be derived from immunised animals

The antibody immune response in-vivo can be T-cell dependent or independent

Antibody fragments can also be selected from in-vitro systems such as phage expression

Cycles of selection and mutation can give an artificial in-vitro immune response based

simply on binding affinity

The Selection of IgG Fc Regions for appropriate effector functions: The role of isotypes and polymorphisms

Effector functions of human IgG

IgG1 IgG2 IgG3 IgG4Complement activation

Classical pathway +++ + +++ Alternative pathway +

Fc receptor recognitionFcRI +++ +++ ++

FcRIIa, 131R/R ++ ++ FcRIIa, 131H/H + + ++ FcRIIb ++ ++ +

FcRIII + +/ + +/

Unlike mouse the human IgG subclasses are very similar in sequence but they still

have different properties

The IgG receptor FcRn

Interaction with FcRn and with Protein A through similar region

FcRn is important for IgG half-life and transport

For FcRI binding:

231 238IgG1, IgG3 A P E L L G G PIgG2 . . P V A - . .IgG4 . . . F . . . .sequence introduced into IgG1, IgG4

. . P V A - . .or . . P V A G . .

For C1q binding:

318 327 331IgG1, IgG3 E Y K C K V S N K A L P A PIgG2 . . . . . . . . . G . . . .IgG4 . . . . . . . . . G . . S Ssequence introduced into IgG1, IgG2

. . . . . . . . . G . . S S

Transferring motifs between human subclasses

b mutationc mutation

a mutation

Residues at key positions in mutatedconstant regions

Antibody 233 234 235 236 327 330 331

G1 E L L G A A PG1a E L L G G S SG1b P V A - A A PG1c P V A G A A PG1ab P V A - G S SG1ac P V A G G S SG2 P V A - G A PG2a P V A - G S SG4 E F L G G S SG4b P V A - G S SG4c P V A G G S S

Summary of terminology

Mutant residues

a Residues 327, 330, 331 of IgG4

b Lower hinge of IgG2; omitting Gly236

c Lower hinge of IgG2; including Gly236

Armour et al. Eur J Imm 1999; 29: 2613

Test systems: antibodies with CD52 and -RhD specificities

• Short, GPI-anchored glycoprotein

• Found on T cells and some B cells,

granulocytes and eosinophils

• About 45 x 104 molecules/cell

• Good target for CDC and ADCC

• Humanised variable domains of CAMPATH-1

antibody used

• Range of antibodies with same variable

domains already existed

CD52

Test systems: antibodies with CD52 and -RhD specificities

• Protein complex on erythrocyte membrane

• 1 - 3 x 104 molecules/cell

• Provides opportunities for use of agglutination

and rosetting assays

• Target for ADCC

• Used variable domains of Fog-1, a human IgG

isolated from hyperimmunised, RhD- blood

donor

-RhD

-5

0

5

10

15

20

25

30

35

40

45

0.1 1 10 100antibody, g/ml

% s

peci

fic

Cr

rele

ase

G1

G1 a

G1 b

G1 c

G1 ab

G1 ac

G2

G2 a

G4

G4 b

G4 c

CAMPATH-1 antibodies

Complement-mediated lysis of mononuclear cells

-5

0

5

10

15

20

25

1 10 100 1000

G2a concentration,g/ml

% s

peci

fic

Cr

rele

ase

Complement-mediated lysis withCAMPATH-1 G1(6.3 g/ml),

inhibited by CAMPATH-1 G2a

Binding to the FcRI-bearing cell line, B2KA, measured by fluorescence staining

G2

G1bG1c

G4

G1a

G1

CAMPATH-1H antibodies at 100 g/ml

0

20

40

60

80

100

120

140

160

0.001 0.01 0.1 1 10 100

antibody, g/ml

mea

n fl

uore

scen

ce

G1

G1a

G1 b

G1 c

G1 ab

G1 ac

G2

G2 a

G4

G4 b

G4 c

CAMPATH-1 antibodies

Binding to the FcRIa-bearing cell line, B2KA, measured by fluorescent staining

Chemiluminescent response of human monocytes to sensitised RBC

-20

0

20

40

60

80

100

120

140

0 5000 10000 15000 20000 25000 30000

antibody molecules/cell

% c

hem

ilum

ines

cenc

e

G1

G1a

G1b

G1c

G1ab

G1ac

G2

G2a

G4

G4b

G4c

Fog-1 antibodies

Inhibition of chemiluminescent response to clinical sera by Fog-1 G2a

280

0

40

80

120

160

200

240

0 10 100 1000

G2a concentration, g/ml

% c

hem

ilum

ines

cenc

e G1anti-D serum Aanti-D serum Banti-D serum Canti-D serum Danti-D serum Eanti-C+D serumanti-K serum

Binding to the cell line 3T6 + FcRIIa 131R, measured by flow cytometry

10

20

30

40

50

60

70

80

90

100

0.1 1 10 100

antibody concentration, g/ml

mea

n fl

uore

scen

ce

G1

G1a

G1b

G1c

G1ab

G1ac

G2

G2a

G4

G4b

G4c

G1g

IgA1,

Fog-1 antibodies

Binding to the cell line 3T6 + FcRIIa 131H, measured by flow cytometry

10

20

30

40

50

60

70

80

90

0.1 1 10 100

antibody concentration, g/ml

mea

n f

luor

esce

nce

G1

G1a

G1b

G1c

G1ab

G1ac

G2

G2a

G4

G4b

G4c

G1g

IgA1,

Fog-1 antibodies

Binding to the cell line 3T6 + FcRIIb1*, measured by flow cytometry

10

60

110

160

210

260

0.1 1 10 100

antibody concentration, g/ml

mea

n fl

uore

scen

ce

G1

G1a

G1b

G1c

G1ab

G1ac

G2

G2a

G4

G4b

G4c

G1g

IgA1,

Fog-1 antibodies

Binding to different forms of FcRII

antibody constant region

perc

enta

ge o

f G

1 bi

ndin

g

0% = binding of IgA1,

0

20

40

60

80

100

120G

1

G1

a

G1

b

G1

c

G1

ab

G1

ac G2

G2

a

G4

G4

b

G4

c

G1

g

FcRIIa 131R

FcRIIa 131H

FcRIIb1*

Activity of Fog-1 antibodies in ADCC

-20

0

20

40

60

80

100

120

0.1 1 10 100 1000 10000

antibody concentration, ng/ml

% R

BC

lysi

s

G1G1abG2G2aG4G4b

Inhibition by Fog-1 antibodies of ADCC due to Fog-1 IgG1 (at 2ng/ml)

inhibitor antibody concentration, ng/ml

% R

BC

lysi

s

0

5

10

15

20

25

30

35

40

45

0.0001 0.001 0.01 0.1 1 10 100 1000 10000

G1 a

G1 c

G2 a

G2

G1b, G1ab, G1ac, G4, G4b, G4c

{

Summary of antibody activities

Mutants of : G1 G2 G4wt a b c ab ac wt a wt b c

Binding to:FcRIFcRIIa R/RFcRIIa H/HFcRIIb1*FcRIIIb NA1FcRIIIb NA2

Monocyte actnComplement lysisADCC

Effect of mutations cannot always be predicted from wildtype antibody activities

Complement lysis: IgG2 activity is only ~3-fold lower than that of IgG1

but placing IgG2 residues in IgG1 (b, c) eliminates lysis.

FcRIIa 131H binding: IgG1 and IgG2 show equal binding

but G1b and G1c activities are 30-fold lower.

IgG1 binding may depend heavily on the mutated regions. Other subclasses may have additional sites of interaction with the effector molecules.

b and c mutants

The 3 pairs of b and c mutants show reduced activity in

all functions assayed but the residual levels of activity differ:

b slightly more active in FcRIIa 131H and 131R binding

c more active in FcRI binding, monocyte activation FcRIIIb NA1and NA2 binding and ADCC

These mutants differ only by -/+ G236.

This must affect the ability of the FcR to accommodate the

IgG2 lower hinge.

What of the immunogenicity of therapeutic antibodies?

Bad News

• Universal tolerance to all self-antigens does not exist.

• Auto and allo-immunity are common observations

• Human proteins can be immunogenic in humans. (e.g. recombinant insulin, EPO and Factor VIII)

• Human antibodies can be immunogenic in humans (anti-idiotype and anti-allotype) and this applies to chimeric, humanised and fully human antibodies.

Good News

• Auto and allo-immunity are common observations but these immune reponses can be modified and regulated.

• Human antibodies can be immunogenic in humans but this immunogenicity varies from antibody to antibody for complex reasons, and is probably more dependent on the mode of action, and not just the way they were made (i.e. chimeric, humanised or fully human).

Antigenicity and Immunogenicity

• Antigenicity is simply an ability of a molecule to be recognised by a pre-existing T-cell receptor (TCR) or a B-cell receptor (antibody)

• But once an antigen is recognised by a receptor it can either be immunogenic or tolerogenic.

• The same antigen can sometimes induce tolerance and sometimes provoke an immune response depending upon factors such as mode of administration and uptake by and co-stimulation of antigen presenting cells (APCs).

Immunogenicity

• Immunogenicity of T-cell dependent antigens relies on presentation by professional APCs (e.g. Dendritic cells).

• Dendritic cells (and other APCs) acquire antigen through use of innate receptors including complement receptors and Fc receptors, thus allowing recognition and uptake of immune complexes.

Benjamin,R.J., Cobbold,S.P., Clark,M.R., & Waldmann,H. (1986) J. Exp. Med. 163, 1539-1552. Tolerance to rat monoclonal antibodies: implications for serotherapy

Observation• Relatively easy to tolerise mice, with de-aggregated human immunoglobulin or with rat immunoglobulin, despite large differences in the constant region sequences between mouse, human and rat.

• However in mice which are tolerant of soluble rat IgG2b, administration of antibodies which bind to mouse cell surface antigens provokes a strong anti-idiotype response.

Explanation• Is this a function of the inherent immunogenicity of immune complexes?

•Aggregated antibody is more likely to activate complement and to bind to low affinity Fc receptors.

Induction of tolerance to therapeutic antibodies

Antibody selection and design

The choice of antibody constant region is largely dictated by functional requirements of the antibody.

But what about the V-regions ?

The V-region Mythology

Chimaeric65% Human ?

Humanised95% Human ?

• This commercial marketing mythology is based on an assumption that mouse and human antibody sequences are unique.

• However a study of the Kabat database shows that there is high sequence homology for antibodies from different species.

Kabat database variability of VH sequences

Human VH Mouse VH

Are chimaeric, humanised and fully human antibodies so very different in sequence?

Possible to select alternative V genes for humanisation

Gorman,S.D., Clark,M.R., Routledge,E.G., Cobbold,S.P. & Waldmann,H. P.N.A.S. 88, 4181-4185 (1991) Reshaping a therapeutic CD4 antibody.

Routledge,E., Gorman,S., & Clark,M. in Protein engineering of antibody molecules for prophylactic and therapeutic applications in man. (Ed. Clark,M. )

Pub. Academic Titles, UK (1993) pp. 13-44 Reshaping of antibodies for therapy.

• Gorman et al recognised that homology also extended through the CDR regions not just the framework regions • Homology to Kol was increased from 69% to 89% by the humanisation process.

The same strategy can be applied to almost any V-region

Antibody comparisons FR CDR Whole V

murine versus human germline

Campath-1G (68/87) 78% (14/34) 41% (82/121) 68%

Anti-Tac (67/87) 77% (14/29) 48% (81/116) 70%

OKT3 (67/87) 77% (12/32) 38% (81/119) 68%

humanised versus human germline

Campath-1H versus germline (78/87) 90% (8/34) 24% (86/121) 71%

Anti-Tac versus germline (77/87) 89% (14/29) 48% (91/116) 78%

OKT3 versus germline (76/87) 87% (6/32) 19% (82/119) 69%

human versus human germline

Fog-1 RhD versus germline (77/87) 89% (23/37) 62% (100/124) 81%

Sequence homologies of some rodent, humanised and human sequences

Antibody Specificity V-regionHomologous

VHJH Length Matches Homology

FOG-1 RhD Human V4-34 JH6A 124 100 0.807

anti-Tac CD25 Humanised HV1F10T JH6a 116 91 0.784

anti-Tac CD25 Mouse HV1F10T JH4D 116 84 0.724

anti-TNFa TNF-alpha Mouse VI-4-IB JH3B 117 84 0.718

Campath-1H CD52 Humanised DP-71_3D197D- JH4D 121 86 0.710

Campath-1G CD52 Rat DP-34_DA-10 JH4D 121 85 0.702

OKT3 CD3 Humanised b25 JH6a 119 82 0.689

OKT3 CD3 Mouse DP-7_21-2-.. JH6a 119 81 0.681

HD37 CD19 Mouse 6M27 JH4D 124 83 0.669

anti-CD20 CD20 Mouse DP-7_21-2- JH2 121 81 0.669

Homologies for antibody heavy chain V regionscompared with human germline sequences

Sorted by homology

What of the Emperor’s new clothes?

Appropriate selection of sequences of antibody Constant and Variable regions is likely to be only one factor controlling the immunogenicity of therapeutic antibodies.

However it is the final sequence of the antibodies which matters and not the route by which they were made. For example it is possible to come up with alternative humanised sequences for the same antibody. Similar sequences can often be found for mouse, rat and human variable regions within the databases.

Even fully human antibodies may contain unusual motifs or structures as a result of the somatic recombination and junctional diversity combined with somatic hypermutation.

What determines immunogenicity?

• Classical Self vs non-Self (Peter Medawar) Aquired neonatal tolerance to antigens.

• Danger Hypothesis (Polly Matzinger) Cell killing (inappropriate, non-apoptotic)

Inflammation (cytokine release)

• Pattern recognition (Charles Janeway) Innate receptors for infectious pathogens

Complement activation and fixation of C3 (Fearon)

( Fc receptors for immune complexes)

The effect of aglycosylation on the immunogenicity of a humanised therapeutic CD3 monoclonal antibody Routledge et al

1995 Transplantation 60, 847-853

Normal Mouse

(no antigen)

CD3 Transgenic

(cell surface)

Human IgG1 - +++

Aglycosyl IgG1 - +

The effect of aglycosylation on the immunogenicity of a humanised therapeutic CD3 monoclonal antibody Routledge et al

1995 Transplantation 60, 847-853

The human IgG1in the CD3 transgenic mice was able to kill target cells, to activate complement, to bind to FcR and to cause cytokine release. Whereas the aglycosylated antibody was poor in these functions and produced only a weak immune response.

Is this a special case or can it be generalised to other antibodies?

Is it consistent with the Matzinger “Danger Hypothesis” as applied to therapeutic administration of recombinant antibodies?

Elimination of the immunogenicity of therapeutic antibodies. Gilliland et al 1999 J.Immunol 162, 3663-3671

• Took CAMPATH antibody and mutated a key residue in the CDR region so as to prevent cell binding to CD52.

• This variant could be used to tolerise CD52 transgenic mice so that they no longer mounted an immune response to the wild type CAMPATH

Murine constant regionsV-region sequencesHuman Ig allotypes

Unusual glycosylation

Method of administrationFrequency of administration

Dosage of antibody

Patients' disease statusPatients' immune status

Patients' MHC haplotype

Specificity of antibodyCell surface or soluble antigen?

Formation of immune complexes with antigen

Complement activation by antibodyFc receptor binding by antibody

Inflammation and cytokine release

Factors likely to influence immunogenicity of therapeutic antibodies

Will the idiotype always be immunogenic?

The idiotype will obviously always be unique and thus antigenic.

However it may be possible through mode of use to influence whether this antigenic idiotype is immunogenic or tolerogenic!

Take home message to remember

In immunological terms antigenicity is certainly not the same as immunogenicity!