Engineering Antibodies for therapeutic applications in man Part II Lecture 5 th March 2009 by Mike Clark, PhD Department of Pathology Division of Immunology.

Engineering Antibodies for therapeutic applications in man

Part II Lecture

5th March 2009 by

Mike Clark, PhDDepartment of Pathology

Division of Immunology

Cambridge University

UK

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

Breakdown of Protein Therapeutics Estimated Market based on the sales of the top-selling biologics drugs.*

Category 2006 (in $B)

2010 (projected) (in $B)

Monoclonal antibodies and Fc fusions 23.1 41.2 Epoetins 12.0 12.8** Insulin-related 9.0 13.0 Interferons 6.8 7.7 Antifibrinolytics 4.5 6.3 Immunostimulatory (xCSF) 4.3 5.5 Growth hormones 2.4 2.5 Other (mixed mechanisms) 3.2 4.7 Totals 65.3 93.7 * Based on published 2005 and 2006 sales, and 2010 projected sales of the top 70 biologics currently on the market, excluding vaccines (multiple sources). ** Projections were made prior to published safety concerns in mid-2007, which have depressed overall sales of epoetins.

Slide courtesy of Bill Strohl, Centocor, September 2008

Product Name (Manufacturer)

Date Indication Protein

Rituxan (Biogen/Idec)

1998 NHL IgG1, chimeric

Herceptin (Genentech)

1998 Breast cancer

IgG1, humanized

Mylotarg (Wyeth)

2000 Leukemia IgG4, humanized; conj. NP

Campath (Berlex)

2000 Leukemia IgG1, humanized

Zevalin (Biogen/Idec)

2002 NHL IgG1 murine, conjugate

Bexxar (Corixa) 2003 NHL IgG2aλ - Iodine-131, conj.

Erbitux (ImClone/BMS)

2004 Colorectal cancer

IgG1, chimeric

Avastin (Genentech)

2004 Colorectal cancer

IgG1, human

Vectibix (Amgen)

2006 Cancer IgG2; human

Examples of Therapeutic Antibody-relatedProducts on the Market

Product Name (Manufacturer)

Date Indi-cation

Protein

ReoPro (Lilly) 1994 CV Fab, chimeric

Simulect (Novartis) 1998 GVHD IgG1, chimeric

Remicade (J&J) 1998 RA IgG1, chimeric

Synagis (AZ) 1998 RSV IgG1, chimeric

Zenapax (Roche) 1998 GVHD IgG1, humanized

Enbrel (Immunex) 1998 RA TNFR-Fc fusion

Xolair (Genentech) 2003 Asthma IgG1, humanized

Raptiva (Genentech) 2003 Psoriasis IgG1, humanized

Humira (Abbott) 2003 RA IgG1, human

Orencia (BMS) 2005 RA CTLA4-Fc fusion;

Modified Fc

Tysabri (Biogen/Elan) 2005 MS IgG4; humanized

Lucentis (Genentech) 2006 AMD Fab; humanized

Soliris (Alexion) 2007 PNH IgG2/4 humanized;

Modifed Fc

Arcalyst (Regeneron) 2008 CAPS IL-1 – Fc fusion

Cimzia (UCB) 2008 RA PEGylated Fab

Nplate (Amgen) 2008 TCP Peptide-Fc fusion;

Aglycosylated Fc


Bioloogic Year

OKT3 1986--- ///////ReoPro® 1994--- 1995--- 1996Rituxan® 1997Zenapax® 1997Remicade® 1998Enbrel® 1998Herceptin® 1998Simulect® 1998--- 1999Mylotarg® 2000Campath® 2001Zevalin® 2002Xolair® 2003Raptiva® 2003Amevive® 2003Bexxar® 2003Humira® 2003Erbitux® 2003Avastin® 2004Tysabri® 2004Actemra® 2005Orencia® 2005Lucentis® 2006Vectibix® 2006

$0.5B $1.0B $1.5B $2.0B $2.5B $3.0B $3.5B $4.0B

2006 Sales

$271K

$3788K

$3768K$4442K

$3020K

$533K

$2020K$1069K

$2372K

$380K

$90K

$115K

*

*

*

**

**

**

* >$80M in 2006

*

$107K

Sales of Existing Commercial Monoclonal Antibodies through 2006


2000 2005 2008 2010

10

20

30

40

50

60

Current status as of Sept 2008; N=26

If all current BLAs are approved and result in marketed biologics

If ~50% of current Phase III candidates are approved in next 3-4 years (50% POS)

Human MAbFc fusion protein

Humanized MAbChimeric MAbMurine MAb

Year

Cu

mu

lati

ve n

um

ber

of

MA

bs

and

Fc

fusi

on p

rote

ins

app

rove

d If all (unlikely) current Phase III candidates were to be approved in next 3-4 years (100% POS)

Current and Projected Number of MarketedMonoclonal Antibodies and Fusion Proteins

10

20

30

40

50

60

1995(Current status)

75% POS (industry average) would yield approx ca. 55 marketed Mabs and Fc fusion proteins


MonoclonalAntibody Format

Murine

Chimeric

Humanized

Human

Monoclonal Antibody Source

Murine hybridoma

Humanized mouse hybridoma

Phage displayed human antibody library

2 4 6 8 10 12 14 16 18 20Number of Monoclonal Antibodies

33

62

1111

214

2016

1

16

8

Total Marketed Mabs = 22Total Phase III Mabs = 30

Form and Source of Existing Commercial and Phase III Monoclonal Antibodies


Antibody Technologies and Products

75 80 85 90 95 00 05

Products/Drugs

Hybridoma

Humanised

antibodies

Technologies

OKT3 7/86

Panorex 1/95

Chimaeric

antibodies

Remicade 8/98

ReoPro 12/94

Rituxan 11/97

Simulect 5/98

Radiolabeled Abs Immunotoxins (Pro)drug Conjugates Enzyme/Cytokine Fusions Multispecific/Multivalent Engineered Fc Cocktails

Emerging Technologies

HUMIRA 12/02

Bexxar 06/03

Mylotarg 05/00

Zevalin 02/02

Phage antibodies

Human Ab mice

Zenapax 12/97

Herceptin 9/98

Synagis 6/98

Campath 05/01

Xolair 06/03

Raptiva 10/03

Avastin 02/04

Erbitux 02/04

Tysabri 03/06

Time Required for Maturity of Technologies

Year1975 1980 1985 1990 1995 2000 2005 2010

Kohler and Milstein, 1975

Muronomab-OKT3®, 1986

11 years

Morrison etal., 1984

ReoPro®, 1994

10 years

11 years

Jones et al., 1986

Zenapax®, 1997

9 years

Capon et al., 1989;First Fc fusion

Enbrel®, 1998

12 years

McCaffertyet al., 1990

Humira®, 2002

Lonberg et al., 1994;Green et al., 1994

Vectibix®, 2004

10 years

13 years

Alegre et al., 1994;(first OKT3 ala-ala) Soliris®, 2007

Antibodies with modified,muted Fc function

Human antibodies fromtransgenic humanized mice

Human antibodies from phage display libraries

Fc fusion protein

HumanizedCDR-grafted antibodies

Chimeric antibodies

Murine hybridoma


Substantially Moderate High muted activity ADCC

• FcRI,II,III- silent• No complement activity

• FcRI,II,III- silent• Complement activity

• FcRI,III-silent• FcRIIa-active• Little Comple- ment activity

• FcRI,II,III- active• Complement activity

• FcRI,II,III- very active• Complement activity

IgG2m4IgG2-4IgG4ala-ala

AglycosylatedIgG1

Standard IgG2 Standard IgG1EngineeredIgG1

Non-Oncology, Cytokines, OncologyNon-Infectious other soluble receptorDiseases cell targets targets; IDsurface target

Spectrum of IgG Activities


US Trade NamAe (generic name)

Company Approval Date (US)

Molecular target

Major Indication

Protein format

Orencia® (Abatacept)

BMS 12/23/ 05 CD80/CD86 (T-cell)

RA CTLA4-Fc fusion protein with modified IgG1 Fc

Soliris® (Ecolizumab)

Alexion Pharma

03/16/07 Complement C5

PNH(reduce hemolysis)

Humanized IgG2/4 hybrid

Nplate® (Romiplostim)

Amgen 08/22/08 TPO-R Thrombo-cytopenia

Fc-peptide fusion („peptibody“) with aglycosylated Fc

Teplizumab; HOKT31- (Ala-Ala)

Macrogenics / Eli Lilly

Phase III CD3 Diabetes Humanized Modified Fc

Otelixizumab (ChAglyCD3; TRX4)

GSK / Tolerx Phase III CD3 Type 1 Diabetes

Aglycosylated, humanized IgG

Removab® (Catumaxomab)

Fresenius / Trion

Phase II/III

EpCAM and CD3

Cancer Rat-murine hybrid

MEDI-563 (formerly BIW-8405 )

Astra-Zeneca Phase II IL-5R SLE Afucosylated IgG based on BioWa’s Potelligent technology

PRO-131921 Genentech Phase I/II CD20 (third generation)

Oncology IgG with modified Fc for increased ADCC

AME-133v (LY2469298)

Eli Lilly Phase I/II CD20 NHL IgG with modified Fc for increased ADCC

TRU-015 Wyeth Phase II CD20 RA Small modular immuno-pharmaceutical product (SMIP)

MEDI-557 Astra-Zeneca Phase I F-protein on RSV

RSV YTE mutant – longer half-life Mab

Summary: Examples of “fit-for-purpose” Mabs and Fc Fusions with Isotypes other than IgG1


Trends in engineering Antibody fragments

From : Baker, Nature Biotechnology 2005, Figure 1. Anatomy of an optimized antibody. Red indicates sites for potential engineering. From top to bottom: the variable region, binding sites for Fc Rs and complement, and binding site for FcRn. (Source: Xencor, Monrovia, CA, USA.)

Effector Engineering

Binding site engineering

Antibody fragments and other binding

proteins

Combining antibodies

Looking beyond the initial success: remaining issues for Antibody Therapeutics

• Limited efficacy

• Safety (side effects)

• Immunogenicity

• Complex, expensive drugs

• Manufacturing (COGS)

• Delivery only possible via injection

Immunogenicity of Approved Antibody Drugs

mAb Target Molecule Immuno-suppression

Comed with immuno-suppressor

Immunogenicity

OKT3 CD3 Murine mAb, IgG2a Yes Yes 17-63%

ReoPro IIb/IIIa Chimaeric Fab No No 5.8% (1st dose)

Rituxan CD20 Chimaeric IgG1, Yes No 1-10%

Zenapax CD25 Humanized IgG1, Yes Yes 14-34%

Remicade TNF- Chimaeric IgG1, Yes Yes 10-61%*

Herceptin HER2 Humanized IgG1, No Yes/No 0-1%

Synagis RSV Humanized IgG1, No No 0.7-1.8%

Simulect CD25 Chimaeric IgG1, Yes Yes 1-20%

Mylotarg CD33 Humanized IgG4 Yes Yes 2 pts in Phase I

Campath CD52 Humanized IgG1, Yes No 1-90%

Zevalin CD20 Murine IgG1, Yes No 3.8%

HUMIRA TNF- Human IgG1, Yes Yes/No 1-12%

All antibodies administered IV; data from product inserts; *Baert et al, N. Engl. J. Medicine 2003

Engineering opportunities

• Find better binding site

• Increase affinity / potency

• Modulate on/off-rate

• Broaden epitope reactivity

• Combine binding sites

• Engineer improved effectors

• Enhance pharmacokinetics

• Diminish immunogenicity

• Increase productivity

• Improve stability

• Reduce product heterogeneity

Three Major Techniques for Making Antibodies for Therapy

I. Humanization : Immunization, hybridoma, cloning, then chimerization or humanization via CDR grafting or deimmunization

II. Mice carring Human Ab genes : Engineering the mouse, immunization, hybridoma/B-cell (Ab-gene rescue)

III. Phage Display : Creating phage display library (non-immune/immune/synthetic); selection of Ab from library

Fab IgG

Engineered antibody fragments

Holliger and Hudson, Nat. Biotechnology (Sept 2005)

Antibody fragmentsAntibody fragments: size vs half life

Holliger and Hudson, Nat. Biotechnology (Sept 2005)

University Research Programmes

• Immunosuppression

CD4, CD3, monovalent CD3, CD52 (Campath)

• Tumour Therapy

CD52 (Campath), bispecific CD3

• Organ Transplantation

CD52, CD3, CD4, synergistic CD45 pair

• Allo and auto-immunity

RhD, HPA-1a

• Chronic Inflammation

CD18, VAP-1

Declaration of interests (rights as an inventor)

• CD52 IlexOncology/Genzyme (Campath® humanisation)

• CD4 TolerRx/Genentech (for induction of tolerance)

• CD4 BTG (improved method of humanisation)

• CD3 TolerRx/GSK (immunosuppression and tolerance)

• RhD NBS / University collaboration

• HPA-1a NBS / University collaboration

Otelixizumab • Started out as a depleting monovalent rat antibody for use in

immunosuppression of graft rejection. Clark,M., Bindon,C., Dyer,M., Friend,P., Hale,G., Cobbold,S., Calne,R., & Waldmann,H.

Eur. J. Immunol. 19, 381-388 (1989) The improved lytic function and in-vivo efficacy of monovalent monoclonal CD3 antibodies.

Abbs,I.C., Clark,M., Waldmann,H., Chatenoud,L., Koffman,C.G. & Sacks,S.H. Therapeutic Immunology 1, 325-331 (1994) Sparing of the first dose effect of a monovalent anti-CD3 antibody used in allograft rejection is ssociated with diminished release of pro-inflammatory cytokines.

• Humanised as a monovalent depleting antibody. Routledge, E.G., Lloyd, I., Gorman, S., Clark, M. & Waldmann, H. Eur. J. Immunol. 21,

2717-2725 (1991) A humanized monovalent CD3 antibody which can activate homologous complement.

• Converted to a non-depleting form by modification of the Fc region.

Bolt,S., Routledge,E., Lloyd,I., Chatenoud,L., Pope,H., Gorman,S.D., Clark,M. & Waldmann,H. Eur. J. Immunol. 23, 403-411 (1993) The generation of a humanised, non-mitogenic CD3 monoclonal antibody

which retains in vitro immunosuppressive properties

The antibody isotype is important

Chimeric and humanised

Rat IgG2b is effective in therapy

Human IgG1 also effective in therapy

Antibodies (eg CD52 Campath) can be effective in killing cancer cells (BCLL)

NOVEL ANTIBODIES TO TREATFETO-MATERNAL ALLOIMMUNE

THROMBOCYTOPENIA?

Lorna M Williamson, Kathryn Armour, Mike Clark

Departments of Haematology & Pathology,

University of Cambridge/National Blood Service

Fetomaternal alloimmune thrombocytopenia

• Maternal IgG raised against fetal platelet alloantigens can

cross the placenta and cause fetal platelet destruction

• If the fetal platelet count falls dangerously low, cerebral

hemorrage or death may result

• Current therapies are intrauterine platelet transfusion and

maternal therapy with high dose IVIG

Alloantigen negativeMother

(bb)

Alloantigen positivefetal platelets

(ab)

Fetal platelet material

Feto-maternal Alloimmune Thrombocytopenia

Feto-maternal Alloimmune Thrombocytopenia

Maternal platelet antibodies

Platelet destruction by maternal HPA

alloantibodies

Anti-a

FMAIT causes neonatal purpura

FMAIT causes intracranial haemorrhage in utero

An ideal blocking antibody for FMAIT

1. HPA-1a specificity & competes with FMAIT sera

2. Inert Fc without increased immunogenicity

3. Inactive in FcR binding & FcR-mediated cell

destruction

4. Blocks destruction of cells by active antibody

5. No triggering of cell destruction in vivo

6. No effects on platelet production or function

7. Normal half-life and placental transport

2. Inert Fc without increased immunogenicity

Can a protective antibody be developed?

• 90% severe cases FMAIT are due to antibodies

against the alloantigen HPA-1a on GPIIIa

• Single B cell epitope (Leu-33) could be blocked

to prevent the binding of harmful antibodies

• Outcome depends on antibody titre

Williamson et al. Blood 1998; 92: 2280 Jaegtvik et al. Br J Obs Gynae 2000; 107: 691

Ideal properties of an antibody for FMAIT therapy

• HPA-1a specificity (B2 variable regions)

• able to cross the placenta

• inactive in FcR-mediated cell destruction

• unable to activate complement

RhDHPA-1a

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 due to 2 g/ml Fog-1 G1 by other Fog-1 antibodies

0

10

20

30

40

50

60

70

80

90

100

0.1 1 10 100 1000

inhibitor concentration, g/ml

% c

hem

ilum

ines

cenc

e

G1 b

G1 c

G1ab

G1ac

G2

G2a

G4b

G4c

Inhibition by Fog-1 antibodies of ADCC due to clinically relevant polyclonal anti-RhD (at 3ng/ml)

0

20

40

60

80

100

120

0.1 1 10 100 1000 10000

inhibitor antibody concentration, ng/ml

% R

BC

lysi

s

G1 ab

G2

G2a

G4

G4 b

3. No triggering of cell destruction in vivo

Red cell survival study in normal volunteers

Compare intravascular survival of RBC coated with Fog-1 G1 and Fog-1 G1nab in human volunteers.

99mTc G1

Label and coat autologous RBC

51Cr G1nabDesign allows

• simultaneous comparison in same donor• assessment of survival over several days (51Cr)• gamma camera imaging of sites of red cell

accumulation (99mTc)

Armour et al, Blood 2006;107:2619-2626

RBC incubated with antibodies at 50 g/ml,giving 75% saturation of RhD sites.

Subjects

1 2 3 4 5 6

Age / Sex 47 / F 53 / M 45 / M 41 / M 48 / M 38 / M

Predicted Rh genotype R1R2 R2R2 R1R2 R1r R1r R1r

Antibody molecules/cell:

G1 11700 ( 2700)

19900 ( 2300)

6800 ( 500)

17400 ( 3500)

13300 ( 1300)

G1Δnab 11200 ( 2100)

19900 ( 2500)

6500 ( 600)

15600 ( 2600)

13700 ( 2100)

13500 ( 3000)

Plasma counts

-1

0

1

2

3

4

5

6

7

1 10 100 1000 10000

G1nab

-1

0

1

2

3

4

5

6

7

1 10 100 1000 10000

time after injection, min

plas

ma

coun

t, %

inje

cted

dos

e

G1

subject 1 subject 2 subject 3 subject 4 subject 5

G1-coated cells

• Complete, irreversible clearance by 200 min

• Appearance of plasma radiolabel

• Accumulation in spleen and, at high coating levels, in liver

Total cell clearance and destruction

G1nab-coated cells

• Clearance incomplete and transient

• No appearance of plasma radiolabel

• Accumulation in spleen but not in liver even at high coating levels

No destruction of red cells but sequestration in the spleen

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.

Engineering Antibodies for therapeutic applications in man Part II Lecture 5 th March 2009 by Mike Clark, PhD Department of Pathology Division of Immunology.

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