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Antibodies as Therapeutic Entities

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1 Antibodies as Therapeutic Entities
Duncan Hannant Animal Health Trust Newmarket

2 Summary of this Session
Part 1: Rapid review of mouse and species-specific MAbs. Part 2: Historical - antibodies in therapy e.g. “antiserum”, antitoxins, passive transfer. Part 3: Concepts of Immunotherapy - biological response modifiers, immune enhancers, immune suppressors, MAbs. Part 4: MAbs used in human medicine - general approaches, problems, applications and benefits. Part 5: MAbs in Allergic Human Disease. Part 6: MAbs in Cancer Therapy. Part 7: Student discussion on potential of MAbs for treatment of human diseases. Part 8: Resources

3 Summary of this Session
Part 1: Rapid review of mouse and species-specific MAbs. Part 2: Historical - antibodies in therapy e.g. “antiserum”, antitoxins, passive transfer. Part 3: Concepts of Immunotherapy - biological response modifiers, immune enhancers, immune suppressors, MAbs. Part 4: MAbs used in human medicine - general approaches, problems, applications and benefits. Part 5: MAbs in Allergic Human Disease. Part 6: MAbs in Cancer Therapy. Part 7: Student discussion on potential of MAbs for treatment of human diseases. Part 8: Resources

4 Monoclonal antibodies: Summary
Hybridoma selection based on drug resistance Identify immortalised mouse myeloma cell line (e.g. NS1 myeloma derived from BALB/c mice) Mutate to make deficient in enzymes necessary for DNA synthesis (HGPRT and TK) HGPRT = hypoxanthine-guanine phosphoribosyltransferase TK = thymidine kinase

5 Monoclonal antibodies: Summary
Hybridoma selection based on drug resistance Mutated myeloma cells will not grow in presence of aminopterin. Fuse myeloma cells to normal B cells from immunised mouse (PEG used routinely now) Unfused myeloma cells die (aminopterin) Unfused normal B cells die after 1-2 weeks anyway Only hybridomas survive (they can use hypoxanthine and thymidine).

6 Biosynthetic mutations in mutant myeloma cells
Enzyme Present Absent Result HGPRT × Can’t use hypoxanthine TK × Can’t use thymidine Rescue path  Can use Uridine BUT: Culture medium contains HAT which blocks the Rescue Pathway H = hypoxanthine A = aminopterin T = thymidine

7 Species-specific monoclonal antibodies: summary
Very few human myeloma cell lines Even less myeloma cell lines in other species Inter-species fusions are possible but give low hybridisation frequency and unstable hybrids Possible Solution: Use heterohybridoma cell lines as fusion partners

8 Species-specific Mabs: basic method
e.g. Consider the horse Antibodies produced from: (Horse x Mouse) x Horse Heterohybridomas Examples of equine antibodies to influenza virus (previous lecture)

9 Summary of this Session
Part 1: Rapid review of mouse and species-specific MAbs. Part 2: Historical - antibodies in therapy e.g. “antiserum”, antitoxins, passive transfer. Part 3: Concepts of Immunotherapy - biological response modifiers, immune enhancers, immune suppressors, MAbs. Part 4: MAbs used in human medicine - general approaches, problems, applications and benefits. Part 5: MAbs in Allergic Human Disease. Part 6: MAbs in Cancer Therapy. Part 7: Student discussion on potential of MAbs for treatment of human diseases. Part 8: Resources

10 Historical: Antibodies used in passive therapy
Anti-tetanus antibodies derived from horses (>100 years ago). Anti-toxin (e.g. snake venom etc). Intravenous injection of maternally derived antibodies into neonates (failure of colostral transfer - many animals). Infusion of immune serum into diseased animals (e.g. Rhodococcus equi in foals). Immunised goat serum components (HIV and MS)

11 Summary of this Session
Part 1: Rapid review of mouse and species-specific MAbs. Part 2: Historical - antibodies in therapy e.g. “antiserum”, antitoxins, passive transfer. Part 3: Concepts of Immunotherapy - biological response modifiers, immune enhancers, immune suppressors, MAbs. Part 4: MAbs used in human medicine - general approaches, problems, applications and benefits. Part 5: MAbs in Allergic Human Disease. Part 6: MAbs in Cancer Therapy. Part 7: Student discussion on potential of MAbs for treatment of human diseases. Part 8: Resources

12 Immunotherapy Based on the body’s natural defense system.
Enhance this response ~ cancer therapy. Reduce this response ~ asthma therapy. Cytokine and MAb therapy are commonplace in cancer treatment. Cytokine enhancement of anti-virus responses/vaccines are common in vet science.

13 Non-specific immunotherapy
Stimulators of immune responses (biological response modifiers). Coley’s vaccine (~1900, gruesome, Streps and Staphs) BCG ( equally so, granulomas etc) Strange things: goat serum, heterologous proteins, quartz and other particulates.

14 Pathogen related/specific immunotherapy (i)
Interferon alpha (IFN-alpha, effective in virus diseases and cancers such as multiple myeloma, chronic myeloid leukemia, malignant melanoma). Cytokines e.g. IL-2 (kidney cancer and melanoma) IL-2, IL-6 (adjuvants in virus vaccines, enhancing anti-tetanus responses in aged people) and TNF-alpha. Side effects can be significant. Cancer vaccines: cancer antigen(s) + other components - e.g. anti-idiotypic MAbs for colorectal cancer (QMC Nottingham - see later) Ref: Durrant LG and Spendlove I (2003) Cancer vaccines entering Phase III clinical trials. Expert Opin. Emerg. Drugs 8,

15 Pathogen related/specific immunotherapy (ii)
Monoclonal antibodies Becoming useful in allergic disease treatment Now part of standard cancer treatment Potential as “guided missile” delivery systems for drugs, toxins etc.

16 0.25

17 Summary of this Session
Part 1: Rapid review of mouse and species-specific MAbs. Part 2: Historical - antibodies in therapy e.g. “antiserum”, antitoxins, passive transfer. Part 3: Concepts of Immunotherapy - biological response modifiers, immune enhancers, immune suppressors, MAbs. Part 4: MAbs used in human medicine - general approaches, problems, applications and benefits. Part 5: MAbs in Allergic Human Disease. Part 6: MAbs in Cancer Therapy. Part 7: Student discussion on potential of MAbs for treatment of human diseases. Part 8: Resources

18 Factors affecting the use of MAbs in therapy: General
Extremely high target specificity but antibodies are large proteins c.f. drugs. Slow kinetics of distribution. Limited tissue-penetration. Dependent upon blood supply to locate to solid tumours. Dependent upon high level of target antigen expression (and its accessibility) in tumours. Dependent upon MAb isotype, avidity etc.

19 Factors affecting the use of MAbs in therapy: Mab efficacy
Often not completely known, many bystander effects. Blocking function of target antigen. Killing target cell (cytotoxicity) Complement activation (cytotoxicity) Recruitment of phagocytes/NK cells etc. Blocking/activating a receptor for intracellular signal transduction (e.g. NF-kappaB) Transport of toxins, drugs, radionuclides to diseased tissues. Recombinant peptide inclusion in MAb (e.g. TNFalpha antagonist, Etanercept).

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21 Factors affecting the use of MAbs in therapy: Problems (i)
Mouse Fc may not interact with human FcR: reduced effector function. Rapid clearance of foreign protein from bloodstream. Development of human anti-mouse antibodies (HAMA). Retreatment could result in anaphylaxis/allergy Retreatment could be less effective than previous treatments. NB: What about positive benefits of HAMA? De Nardo GL et al (2003) Human antiglobulin response to foreign antibodies: therapeutic benefit? Cancer Immunol. Immunother. 52, (Printed copy of abstract supplied)

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23 Factors affecting the use of MAbs in therapy: Problems (ii)
First injection induces multiple systemic symptoms (chills, headache, pyrexia, vomiting, diarrhoea, tachycardia, respiratory distress, hypotension, arthralgia) - common with OKT3 (anti CD3) and Campath-1-H (anti CD52), also seen with TNFalpha antagonists. Due to massive systemic cytokine release after T cell activation by MAbs. OKT3 also induces C’ activation. NB:these are reversible and not life-threatening.

24 MAbs in therapy: Overcoming some problems
Q: How can we reduce the problem of HAMA? A1: Chimeric MAbs (ag binding = mouse, effector function = human) A2: Humanised MAbs (replace resident hypervariable region with new amino acids) A3: Transgenic mice (human Ab gene loci inserted into body by embryonic stem cell transfer) A4: “Knock-out” mice with Ab producing genes deleted.

25 MAbs in therapy: Overcoming some problems
Potential of transgenic mouse approaches to reduce HAMA are: Mouse can be immunised with desired antigen. Mouse produces human antibodies. ASC can be fused with myeloma cells to produce all-human MAbs.

26 Some current applications of MAbs in disease therapy
Inhibition of alloimmune reactivity (graft rejection). Inhibition of autoimmune reactivity (RA, Crohn’s disease, SLE). Allergic lung disease therapy. Cancer therapy. Infectious disease therapy (RSV). Antiplatelet therapy (acute coronary syndromes) NB: 25% of all biotech drugs in R&D are MAbs - >30 in clinical evaluation now.

27 Some current applications of MAbs in disease therapy
Inhibition of alloimmune reactivity (graft rejection). Ortho Multicentre Transplant Study Group: A randomised clinical trial of OKT3 monoclonal antibody for acute rejection of cadaveric renal transplants. N. Eng. J. Med. 1985, 313, Kahan BD et al. Reduction in occurrence of acute cellular rejection among renal allograft recipients treated with basilimixab, a chimeric anti-interleukin-2 receptor monoclonal antibody. Transplantation 1999, 67, Vincenti F et al. Phase I trial of humanised anti-interleukin-2 receptor antibody in renal transplantation. Transplantation 1997, 63,

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29 Some current applications of MAbs in disease therapy
Inhibition of autoimmune reactivity (RA, Crohn’s disease, SLE). Maini RN et al. Infliximab (chimeric anti-TNFalpha monoclonal antibody) versus placebo in rheumatoid arthritis patients receiving concomitant methotrexate: a randomised phase III trial. Lancet 1999, 354, Breedveld FC. New TNFalpha biologic therapies for rheumatoid arthritis. Eur. Cytokine Netw. 1998, 9, (Etanercept, IgG1 fusion protein) Geletka RC, St Clair (2005) Infliximab for treatment of early rheumatoid arthritis Expert Opin. Biol. Ther. 5, (MAb against TNF)

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31 Some current applications of MAbs in disease therapy
Infectious disease therapy (RSV). Malley R et al. Reduction of respiratory syncytial virus (RSV) in tracheal aspirates in intubated infants by use of humanised monoclonal antibody to RSV F protein. J. Inf. Dis. 1998, 178, (Palivizumab - Synagis) Mejias A et al (2004) Asthma and RSV: new opportunities for therapeutic intervention. An. Pediatr. (Barc) 61, (use of MAB against RSV in patients).

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33 Some current applications of MAbs in disease therapy
Antiplatelet therapy (acute coronary syndromes). Hamm CW et al. Benefit of abciximab in patients with refractory unstable anginain relation to serum troponin T levels. c7E3 Fab Antiplatelet Therapy in Unstable Refractory Angina (CAPTURE) Study Investigators. N. Eng.J Med. 1999, 340, (based on MAb to platelet receptor, gpIIb/IIIa). Note: Two clinical trials with placebo stopped because of highly significant benefit.

34 MAbs in disease therapy
In all these cases, a reduction in immunogenicity is seen with MAbs in this order: Murine Chimera Humanised Human

35 Fender Telecaster Custom
1973

36 Summary of this Session
Part 1: Rapid review of mouse and species-specific MAbs. Part 2: Historical - antibodies in therapy e.g. “antiserum”, antitoxins, passive transfer. Part 3: Concepts of Immunotherapy - biological response modifiers, immune enhancers, immune suppressors, MAbs. Part 4: MAbs used in human medicine - general approaches, problems, applications and benefits. Part 5: MAbs in Allergic Human Disease. Part 6: MAbs in Cancer Therapy. Part 7: Student discussion on potential of MAbs for treatment of human diseases. Part 8: Resources

37 Humanised Mab Therapy: Asthma
Monoclonal antibodies made against human IgE. Antibodies selected that bound to FcεR1-binding region of free IgE. The MAbs formed complexes with free IgE: stopped attachment to mast cells and basophils. Problems with allergic responses and reduced therapeutic efficacy (cleared quickly) because MAbs were immunogenic.

38 Humanised Mab Therapy: Asthma
MAbs to IgE molecule. MAbs to IL-5

39 Humanised Mab Therapy: Asthma
Recombinant humanised MAb to IgE (Omalizumab) prepared which also cleared free IgE from circulation. Resulted in a dose-related and sustained fall in plasma IgE. Reduced early bronchoconstriction responses to inhaled allergen were seen but also the late responses (T cell and eosinophil mediated). Also, showed reduction in high affinity IgE receptors on circulating basophils (because levels of IgE in circulation regulate FcεR1 in vivo)

40 Humanised Mab Therapy: Asthma
Very low occurrence of adverse side reactions to Omalizumab (humanised). Immune complexes of MAb/IgE cleared by reticuloendothelial system (no kidney damage). Although rather expensive, only 5% allergic patients have severe asthma. Cost is acceptable c.f. alternatives (steroids, oral gold, other drugs - high frequency of toxicity) See Holgate et al (2005)paper: next slide

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42 Humanised Mab Therapy: Asthma
Mab to IL-5 IL-5 ~ eosinophilic inflammation of asthma. IL-5 gene deletion or anti-IL-5 MAbs markedly reduce eosinophilic inflammation and airway reactivity in mice. Anti-IL-5 MAb effects last for 3 months in primates (chronic asthma therapy possible?). Humanised anti-IL-5 MAb (Mepolizumab) reduces blood eosinophils by >95% but only 50% in lung. No significant clinical benefit. Needs more work!

43 Gibson SG Standard 1973

44 Summary of this Session
Part 1: Rapid review of mouse and species-specific MAbs. Part 2: Historical - antibodies in therapy e.g. “antiserum”, antitoxins, passive transfer. Part 3: Concepts of Immunotherapy - biological response modifiers, immune enhancers, immune suppressors, MAbs. Part 4: MAbs used in human medicine - general approaches, problems, applications and benefits. Part 5: MAbs in Allergic Human Disease. Part 6: MAbs in Cancer Therapy. Part 7: Student discussion on potential of MAbs for treatment of human diseases. Part 8: Resources 0.75

45 MAbs in Cancer Therapy Subject areas previously covered in lectures:
Design of MAbs MAb-cytotoxic agent conjugates Pharmakokinetics Immunogenicity. Engineering antibody molecules for therapy (Dr Clarke) Applications of engineered antibodies (Dr Clarke) Engineering antibodies for human therapy (Dr Athwal)

46 MAbs in Cancer Therapy Two examples described:
Lymphoma therapy (Lym-1 and Lym-2) Cancer vaccine (anti-idiotype for colorectal cancer - Licenced from CRC as Onyvax-105)

47 MAbs in Cancer Therapy Lymphoma therapy with MAbs Lym-1 and Lym-2.
Tumour cell nuclei used as immunogens. Mouse MAbs reactive with B cells (tumour and normal) but not myeloid cells, or T cells. Target antigen is HLA-DR (discontinuous epitope on beta chain of HLA-DR10, but also other DRs) Selectivity for lymphoma cells on basis of target antigen density and MAb avidity. Further details: see notes in “Resources” section.

48 MAbs in Cancer Therapy Cancer vaccine (anti-idiotype for colorectal cancer) Smart news from Nottingham! Human monoclonal anti-idiotypic antibody made which mimics a TAA (CD55) on colorectal cancer. Phase I trial: non-toxic, enhanced patient survival. Stimulates anti-tumour T cell responses (Th1 and increased IL-2 production). T and NK cell invasion of solid tumours seen (very valuable). Sequencing of the anti-idiotype and CD55 shows that patients with HLA-DR1, DR3 and DR7 should show enhanced T cell responses to tumour.

49 MAbs in Cancer Therapy Cancer vaccine (anti-idiotype for colorectal cancer) Continued: 83% patients carrying these HLA-DR haplotypes responded to this vaccine. 88% patients not expressing these haplotypes were non-responders. Onyvax started Phase II trials in 2000 Ref: Durrant LG et al (2000) A neoadjuvant clinical trial in colorectal cancer patients of the human anti-idiotypic antibody 105AD7, which mimics CD55. Clin. Cancer Res. 6, See also Pritchard-Jones et al (2005) use of 105AD7 in osteosarcoma (next slide)

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51 Gibson Chet Atkins Tennessean 1990

52 Summary of this Session
Part 1: Rapid review of mouse and species-specific MAbs. Part 2: Historical - antibodies in therapy e.g. “antiserum”, antitoxins, passive transfer. Part 3: Concepts of Immunotherapy - biological response modifiers, immune enhancers, immune suppressors, MAbs. Part 4: MAbs used in human medicine - general approaches, problems, applications and benefits. Part 5: MAbs in Allergic Human Disease. Part 6: MAbs in Cancer Therapy. Part 7: Student discussion on potential of MAbs for treatment of human diseases. Part 8: Resources

53 Review and Discussion: MAbs in therapy of human disease
Drug delivery Delivery of BRMs? Risks (human-derived genetic material) Other potential use (anti-idiotypes etc)

54 Review and Discussion: New approaches under clinical and experimental investigation
Bispecific Abs in Lymphoma Dendritic cell vaccines (pulsed with Ag or mRNA) DNA (or mRNA?) vaccines Any other thoughts, possibilities?

55 Summary of this Session
Part 1: Rapid review of mouse and species-specific MAbs. Part 2: Historical - antibodies in therapy e.g. “antiserum”, antitoxins, passive transfer. Part 3: Concepts of Immunotherapy - biological response modifiers, immune enhancers, immune suppressors, MAbs. Part 4: MAbs used in human medicine - general approaches, problems, applications and benefits. Part 5: MAbs in Allergic Human Disease. Part 6: MAbs in Cancer Therapy. Part 7: Student discussion on potential of MAbs for treatment of human diseases. Part 8: Resources

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57 Monoclonal antibody index:

58 Using Monoclonal Antibody Index:
e.g Lym-1 ( Oncolym) for Lymphoma

59 Using Monoclonal Antibody Index: e.g Lym-1 ( Oncolym) for Lymphoma

60 NB: List supplied as printed sheet
Please insert “Humanised” and “Chimera” identifiers

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