Presentation is loading. Please wait.

Presentation is loading. Please wait.

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

Similar presentations


Presentation on theme: "Engineering Antibodies for therapeutic applications in man Part II Lecture 5 th March 2009 by Mike Clark, PhD Department of Pathology Division of Immunology."— Presentation transcript:

1 Engineering Antibodies for therapeutic applications in man Part II Lecture 5 th March 2009 by Mike Clark, PhD Department of Pathology Division of Immunology Cambridge University UK www.path.cam.ac.uk/~mrc7/

2 Breakdown of Protein Therapeutics Estimated Market based on the sales of the top-selling biologics drugs.* Slide courtesy of Bill Strohl, Centocor, September 2008

3 Product Name (Manufacturer) DateIndicationProtein Rituxan (Biogen/Idec) 1998NHL IgG1 , chimeric Herceptin (Genentech) 1998Breast cancer IgG1 , humanized Mylotarg (Wyeth) 2000Leukemia IgG4 , humanized; conj. NP Campath (Berlex) 2000Leukemia IgG1 , humanized Zevalin (Biogen/Idec) 2002NHL IgG1  murine, conjugate Bexxar (Corixa)2003NHLIgG2aλ - Iodine-131, conj. Erbitux (ImClone/BMS) 2004Colorectal cancer IgG1 , chimeric Avastin (Genentech) 2004Colorectal cancer IgG1, human Vectibix (Amgen) 2006Cancer IgG2  ; human Examples of Therapeutic Antibody-related Products on the Market Product Name (Manufacturer) DateIndi- cation Protein ReoPro (Lilly)1994CVFab, chimeric Simulect (Novartis)1998GVHD IgG1 , chimeric Remicade (J&J)1998RA IgG1 , chimeric Synagis (AZ)1998RSV IgG1 , chimeric Zenapax (Roche)1998GVHDIgG1, humanized Enbrel (Immunex)1998RATNFR-Fc fusion Xolair ( Genentech) 2003Asthma IgG1 , humanized Raptiva ( Genentech) 2003Psoriasis IgG1 , humanized Humira (Abbott)2003RA IgG1 , human Orencia (BMS)2005RACTLA4-Fc fusion; Modified Fc Tysabri (Biogen/Elan)2005MS IgG4  ; humanized Lucentis (Genentech)2006AMD Fab  ; humanized Soliris (Alexion)2007PNHIgG2/4 humanized; Modifed Fc Arcalyst (Regeneron)2008CAPSIL-1 – Fc fusion Cimzia (UCB)2008RAPEGylated Fab Nplate (Amgen)2008 TCP Peptide-Fc fusion; Aglycosylated Fc Slide courtesy of Bill Strohl, Centocor, September 2008

4 BioloogicYear OKT31986 ---/////// 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 $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 Slide courtesy of Bill Strohl, Centocor, September 2008

5 2000200520082010 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 MAb Fc fusion protein Humanized MAb Chimeric MAb Murine MAb Year Cumulative number of MAbs and Fc fusion proteins approved If all (unlikely) current Phase III candidates were to be approved in next 3-4 years (100% POS) Current and Projected Number of Marketed Monoclonal 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 Slide courtesy of Bill Strohl, Centocor, September 2008

6 Monoclonal Antibody Format Murine Chimeric Humanized Human Monoclonal Antibody Source Murine hybridoma Humanized mouse hybridoma Phage displayed human antibody library 2468101214161820 Number of Monoclonal Antibodies 3 3 6 2 11 2 14 20 16 1 1 6 8 Total Marketed Mabs = 22 Total Phase III Mabs = 30 Form and Source of Existing Commercial and Phase III Monoclonal Antibodies Slide courtesy of Bill Strohl, Centocor, September 2008

7 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

8 Time Required for Maturity of Technologies Year 1975 1980198519901995200020052010 Kohler and Milstein, 1975 Muronomab-OKT3®, 1986 11 years Morrison et al., 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 McCafferty et 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 from transgenic humanized mice Human antibodies from phage display libraries Fc fusion protein HumanizedCDR- grafted antibodies Chimeric antibodies Murine hybridoma Slide courtesy of Bill Strohl, Centocor, September 2008

9 Substantially Moderate High muted activity ADCC Fc  RI,II,III- silent No comple- ment activity Fc  RI,II,III- silent Complement activity Fc  RI,III-silent Fc  RIIa-active Little Comple- ment activity Fc  RI,II,III- active Complement activity Fc  RI,II,III- very active Complement activity IgG2m4 IgG2-4 IgG4ala-ala Aglycosylated IgG1 Standard IgG2Standard IgG1 Engineered IgG1 Non-Oncology, Cytokines, Oncology Non-Infectious other soluble receptor Diseases cell targets targets; ID surface target Spectrum of IgG Activities Slide courtesy of Bill Strohl, Centocor, September 2008

10 Summary: Examples of “fit-for-purpose” Mabs and Fc Fusions with Isotypes other than IgG1 Slide courtesy of Bill Strohl, Centocor, September 2008

11 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

12 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

13 Immunogenicity of Approved Antibody Drugs mAbTargetMoleculeImmuno- suppression Comed with immuno- suppressor Immunogenicity OKT3CD3Murine mAb, IgG2aYes 17-63% ReoProIIb/IIIaChimaeric FabNo 5.8% (1 st dose) RituxanCD20 Chimaeric IgG1,  YesNo1-10% ZenapaxCD25 Humanized IgG1,  Yes 14-34% Remicade TNF-  Chimaeric IgG1,  Yes 10-61%* HerceptinHER2 Humanized IgG1,  NoYes/No0-1% SynagisRSV Humanized IgG1,  No 0.7-1.8% SimulectCD25 Chimaeric IgG1,  Yes 1-20% MylotargCD33Humanized IgG4Yes 2 pts in Phase I CampathCD52 Humanized IgG1,  YesNo1-90% ZevalinCD20 Murine IgG1,  YesNo3.8% HUMIRA TNF-  Human IgG1,  YesYes/No1-12% All antibodies administered IV; data from product inserts; *Baert et al, N. Engl. J. Medicine 2003

14 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

15 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

16 Engineered antibody fragments Holliger and Hudson, Nat. Biotechnology (Sept 2005)

17 Antibody fragments Antibody fragments: size vs half life Holliger and Hudson, Nat. Biotechnology (Sept 2005)

18 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

19 Declaration of interests (rights as an inventor) CD52IlexOncology/Genzyme (Campath® humanisation) CD4TolerRx/Genentech (for induction of tolerance) CD4BTG (improved method of humanisation) CD3TolerRx/GSK (immunosuppression and tolerance) RhDNBS / University collaboration HPA-1aNBS / University collaboration

20 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

21

22

23

24 The antibody isotype is important

25

26

27

28

29

30 Chimeric and humanised

31

32 Rat IgG2b is effective in therapy

33 Human IgG1 also effective in therapy

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

35

36

37 NOVEL ANTIBODIES TO TREAT FETO-MATERNAL ALLOIMMUNE THROMBOCYTOPENIA? Lorna M Williamson, Kathryn Armour, Mike Clark Departments of Haematology & Pathology, University of Cambridge/National Blood Service

38 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

39 Alloantigen negative Mother (bb) Alloantigen positive fetal platelets (ab) Fetal platelet material Feto-maternal Alloimmune Thrombocytopenia

40 Maternal platelet antibodies Platelet destruction by maternal HPA alloantibodies Anti-a

41 FMAIT causes neonatal purpura

42 FMAIT causes intracranial haemorrhage in utero

43 An ideal blocking antibody for FMAIT 1. HPA-1a specificity & competes with FMAIT sera 2. Inert Fc without increased immunogenicity 3. Inactive in Fc  R binding & Fc  R-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

44

45 2. Inert Fc without increased immunogenicity

46

47 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

48 Ideal properties of an antibody for FMAIT therapy HPA-1a specificity (B2 variable regions) able to cross the placenta inactive in Fc  R-mediated cell destruction unable to activate complement

49 RhD HPA-1a

50 Chemiluminescent response of human monocytes to sensitised RBC -20 0 20 40 60 80 100 120 140 050001000015000200002500030000 antibody molecules/cell % chemiluminescence G1 G1  a G1  b G1  c G1  ab G1  ac G2 G2  a G4 G4  b G4  c Fog-1 antibodies

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

52 Inhibition by Fog-1 antibodies of ADCC due to clinically relevant polyclonal anti-RhD (at 3ng/ml) 0 20 40 60 80 100 120 0.1110100100010000 inhibitor antibody concentration, ng/ml % RBC lysis G1  ab G2 G2  a G4 G4  b

53 3. No triggering of cell destruction in vivo

54 Red cell survival study in normal volunteers Compare intravascular survival of RBC coated with Fog-1 G1 and Fog-1 G1  nab in human volunteers. 99m TcG1 Label and coat autologous RBC 51 CrG1  nab Design allows simultaneous comparison in same donor assessment of survival over several days ( 51 Cr) gamma camera imaging of sites of red cell accumulation ( 99m Tc) Armour et al, Blood 2006;107:2619-2626

55 RBC incubated with antibodies at 50  g/ml, giving 75% saturation of RhD sites. Subjects 123456 Age / Sex47 / F53 / M45 / M41 / M48 / M38 / M Predicted Rh genotypeR1R2R1R2 R2R2R2R2 R1R2R1R2 R1rR1rR1rR1rR1rR1r Antibody molecules/cell: G111700 (  2700) 19900 (  2300) 6800 (  500) 17400 (  3500) 13300 (  1300) G1Δnab11200 (  2100) 19900 (  2500) 6500 (  600) 15600 (  2600) 13700 (  2100) 13500 (  3000)

56 Plasma counts subject 1 subject 2 subject 3 subject 4 subject 5

57 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 G1  nab-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

58 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 ?

59 The V-region Mythology Chimaeric 65% Human ? Humanised 95% 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.

60

61 Kabat database variability of VH sequences Human VH Mouse VH

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

63

64 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.

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

66 Antibody comparisonsFRCDRWhole 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

67 AntibodySpecificityV-region Homologous VH JHLengthMatchesHomology FOG-1RhDHumanV4-34JH6A1241000.807 anti-TacCD25HumanisedHV1F10TJH6a116910.784 anti-TacCD25MouseHV1F10TJH4D116840.724 anti-TNFaTNF-alphaMouseVI-4-IBJH3B117840.718 Campath-1HCD52HumanisedDP-71_3D197D-JH4D121860.710 Campath-1GCD52RatDP-34_DA-10JH4D121850.702 OKT3CD3Humanisedb25JH6a119820.689 OKT3CD3MouseDP-7_21-2-..JH6a119810.681 HD37CD19Mouse6M27JH4D124830.669 anti-CD20CD20MouseDP-7_21-2-JH2121810.669 Homologies for antibody heavy chain V regions compared with human germline sequences Sorted by homology

68 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.


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

Similar presentations


Ads by Google