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Immunotherapeutics Examples Master’s Programme: Engineering antibody molecules - 2 Nottingham University 9 th February 2009. by Mike Clark, PhD Department of Pathology Division of Immunology Cambridge University UK www.path.cam.ac.uk/~mrc7/.

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slide1

ImmunotherapeuticsExamplesMaster’s Programme: Engineering antibody molecules - 2 Nottingham University 9th February 2009

by

Mike Clark, PhD

Department of Pathology

Division of Immunology

Cambridge University

UK

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

slide2
Breakdown of Protein Therapeutics Estimated Market based on the sales of the top-selling biologics drugs.*

Slide courtesy of Bill Strohl, Centocor, September 2008

slide3

Examples of Therapeutic Antibody-relatedProducts on the Market

Slide courtesy of Bill Strohl, Centocor, September 2008

slide4

Sales of Existing Commercial Monoclonal Antibodies through 2006

Bioloogic Year

OKT3 1986

--- ///////

ReoPro® 1994

--- 1995

--- 1996

Rituxan® 1997

Zenapax® 1997

Remicade® 1998

Enbrel® 1998

Herceptin® 1998

Simulect® 1998

--- 1999

Mylotarg® 2000

Campath® 2001

Zevalin® 2002

Xolair® 2003

Raptiva® 2003

Amevive® 2003

Bexxar® 2003

Humira® 2003

Erbitux® 2003

Avastin® 2004

Tysabri® 2004

Actemra® 2005

Orencia® 2005

Lucentis® 2006

Vectibix® 2006

*

$271K

$3788K

*

$3768K

$4442K

$3020K

*

$115K

*

*

$533K

$90K

*

*

$2020K

$1069K

$2372K

*

*

$107K

$380K

*

* >$80M in 2006

$0.5B

$1.0B

$1.5B

$2.0B

$2.5B

$3.0B

$3.5B

$4.0B

2006 Sales

Slide courtesy of Bill Strohl, Centocor, September 2008

slide5

Fc fusion protein

Human MAb

Humanized MAb

Chimeric MAb

Murine MAb

Current and Projected Number of Marketed

Monoclonal Antibodies and Fusion Proteins

If all (unlikely) current Phase III candidates were

to be approved in next 3-4 years (100% POS)

60

60

75% POS (industry average) would yield approx

ca. 55 marketed Mabs and Fc fusion proteins

50

50

If ~50% of current Phase III candidates are

approved in next 3-4 years (50% POS)

40

40

If all current BLAs are approved

and result in marketed biologics

Cumulative number of MAbs and Fc fusion proteins approved

30

30

Current status as of Sept 2008; N=26

20

20

10

10

1995

2000

2005

2008

2010

Year

(Current status)

Slide courtesy of Bill Strohl, Centocor, September 2008

slide6

Form and Source of Existing Commercial and Phase III Monoclonal Antibodies

Monoclonal

Antibody Format

Murine

Chimeric

Humanized

Human

Monoclonal

Antibody Source

Murine hybridoma

Humanized mouse

hybridoma

Phage displayed

human antibody

library

Total Marketed Mabs = 22

Total Phase III Mabs = 30

3

3

6

2

11

11

2

14

20

16

1

8

1

6

2

4

6

8

10

12

14

16

18

20

Number of Monoclonal Antibodies

Slide courtesy of Bill Strohl, Centocor, September 2008

slide7

Time Required for Maturity of Technologies

11 years

Murine hybridoma

Kohler and

Milstein, 1975

Muronomab-OKT3®, 1986

10 years

Chimeric antibodies

Morrison et

al., 1984

ReoPro®, 1994

11 years

HumanizedCDR-

grafted antibodies

Jones et

al., 1986

Zenapax®, 1997

9 years

Fc fusion protein

Capon et al., 1989;

First Fc fusion

Enbrel®, 1998

12 years

Human antibodies from

phage display libraries

McCafferty

et al., 1990

Humira®, 2002

10 years

Human antibodies from

transgenic humanized mice

Lonberg et al., 1994;

Green et al., 1994

Vectibix®, 2004

Antibodies with modified,

muted Fc function

13 years

Alegre et al., 1994;

(first OKT3 ala-ala)

Soliris®, 2007

1980

1985

1990

1995

2000

2005

2010

1975

Slide courtesy of Bill Strohl, Centocor, September 2008

Year

slide8

Spectrum of IgG Activities

Substantially Moderate High

muted activity ADCC

  • FcgRI,II,III-
  • very active
  • Complement activity
  • FcgRI,II,III-
  • silent
  • No comple- ment activity
  • FcgRI,II,III-
  • silent
  • Complement
  • activity
  • FcgRI,III-silent
  • FcgRIIa-active
  • Little Comple-
  • ment activity
  • FcgRI,II,III-
  • active
  • Complement activity

IgG2m4

IgG2-4

IgG4ala-ala

Aglycosylated

IgG1

Engineered

IgG1

Standard IgG2

Standard IgG1

Non-Oncology, Cytokines, Oncology

Non-Infectious other soluble receptor

Diseases cell targets targets; ID

surface target

Slide courtesy of Bill Strohl, Centocor, September 2008

slide9

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

Slide courtesy of Bill Strohl, Centocor, September 2008

otelixizumab
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
novel antibodies to treat feto maternal alloimmune thrombocytopenia

NOVEL ANTIBODIES TO TREATFETO-MATERNAL ALLOIMMUNE THROMBOCYTOPENIA?

Lorna M Williamson, Kathryn Armour, Mike Clark

Departments of Haematology & Pathology,

University of Cambridge/National Blood Service

slide28

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
feto maternal alloimmune thrombocytopenia

Alloantigen positive

fetal platelets

(ab)

Alloantigen negative

Mother

(bb)

Fetal platelet material

Feto-maternal Alloimmune Thrombocytopenia
slide30

Feto-maternal Alloimmune Thrombocytopenia

Platelet destruction by maternal HPA alloantibodies

Maternal platelet antibodies

Anti-a

slide33

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
slide34

Ideal properties of an antibody for FMAIT therapy

  • HPA-1a specificity (B2 variable regions)
  • able to cross the placenta
  • inactive in FcgR-mediated cell destruction
  • unable to activate complement
proof of concept application alloimmune diseases as a target
Proof of concept applicationAlloimmune diseases as a target
  • We have been developing blocking antibodies for use in alloimmune disorders such as Feto-maternal Alloimmune Thrombocytopenia and Haemolytic Disease of the Newborn.
  • The desire is to have antibodies that block killing and are also not cytotoxic in their own right.
  • The properties demanded for such antibodies are applicable to other blocking or inhibitory therapies making use of antibodies or antibody Fc regions.
slide36

Summary of antibody activities

Mutants able to block function of active antibody

Used in HuVAP

& by Rinat/Pfizer

Tested in volunteers

slide37

RhD

HPA-1a

slide38

Chemiluminescent response of human monocytes to sensitised RBC

Fog-1

140

antibodies

120

G1

G1D a

100

G1D b

80

G1D c

% chemiluminescence

60

G1D ab

40

G1D ac

G2

20

G2D a

0

G4

-20

G4D b

0

5000

10000

15000

20000

25000

30000

G4D c

antibody molecules/cell

slide39

100

90

G1D b

80

G1D c

70

G1D ab

60

G1D ac

50

G2

% chemiluminescence

40

G2D a

30

G4D b

G4D c

20

10

0

0.1

1

10

100

1000

inhibitorconcentration,

m

g/ml

Inhibition of chemiluminescent response due to 2 mg/ml Fog-1 G1 by other Fog-1 antibodies

slide40

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

120

100

80

G1D ab

G2

G2D a

60

% RBC lysis

G4

G4D b

40

20

0

0.1

1

10

100

1000

10000

inhibitor antibody concentration, ng/ml

red cell survival study in normal volunteers
Red cell survival study in normal volunteers

Compare intravascular survival of RBC coated with Fog-1 G1 and Fog-1 G1Dnab in human volunteers.

99mTc G1

Label and coat autologous RBC

51Cr G1Dnab

Design 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
plasma counts

7

7

G1

G1Dnab

6

6

5

5

4

4

plasma count, % injected dose

3

3

2

2

1

1

0

0

-1

-1

1

10

100

1000

10000

1

10

100

1000

10000

time after injection, min

Plasma counts

subject 2

subject 4

subject 1

subject 3

subject 5

slide45
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

G1Dnab-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

next step

Next Step?

Completed in-vitro safety testing of anti-platelet antibody with similar Fc region to anti-RhD

Completed testing in an in-vivo mouse model of platelent survival

About to start a new volunteer study using anti-platelet antibody

slide47

Developing recombinant HPA-1a–specific antibodies with abrogated Fcγ receptor binding for the treatment of fetomaternal alloimmune thrombocytopenia J. Clin. Invest. 118(8): 2929-2938 (2008) Cedric Ghevaert et al

  • B2G1Δnab saturated HPA-1a+ platelets and substantially inhibited binding of clinical HPA-1a–specific sera to HPA-1a+ platelets.
  • The response of monocytes to B2G1Δnab-sensitized platelets was substantially less than their response to unmodified B2G1, as measured by chemiluminescence.
  • In addition, B2G1Δnab inhibited chemiluminescence induced by B2G1 and HPA-1a–specific sera.
  • In a chimeric mouse model, B2G1 and polyclonal Ig preparations from clinical HPA-1a–specific sera reduced circulating HPA-1a+ platelets, concomitant with transient thrombocytopenia
  • F(ab′)2 B2G1 was used as a proof of principle blocking antibody and prevented the in vivo platelet destruction seen with B2G1 and polyclonal HPA-1a–specific antibodies
slide48

Developing recombinant HPA-1a–specific antibodies with abrogated Fcγ receptor binding for the treatment of fetomaternal alloimmune thrombocytopeniaJ. Clin. Invest. Cedric Ghevaert, et al. 118:2929 doi:10.1172/JCI34708

summary
Summary
  • Fc regions with reduced functions
    • Based on existing human subclass sequences
    • Tested in humans
      • Suitable for use in inhibiting killing, cellular adhesion or blocking cytokine receptors.
  • Fc regions with enhanced activity
    • Single amino acid change over wild-type sequence (H268E)
      • Potential for applications where enhancement in cytotoxicity and cellular activation is desirable
slide50

Acknowledgements

Kathryn Armour

Chris Kirton

Cheryl Smith

University of Cambridge

Richard Farndale

Mike Peters

David Parry-Jones

NBS Cambridge/Division of Transfusion Medicine

Lorna Williamson

Cedric Ghaevert

Lotta Joutsi-Kornhonen

Simon Bowden

Sandy Preston

Steve Garner

Nick Watkins

Peter Smethurst

Willem Ouwehand

NBS, Bristol

Andrew Hadley

Belinda Kumpel

Marion Scott

Craig Turner

Keith Williams

NBS FOR FUNDING