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Antibodies as Therapeutic Entities. Duncan Hannant Animal Health Trust Newmarket. 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.

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antibodies as therapeutic entities

Antibodies as Therapeutic Entities

Duncan Hannant

Animal Health Trust Newmarket

summary of this session
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
summary of this session1
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
monoclonal antibodies summary
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

monoclonal antibodies summary1
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).
biosynthetic mutations in mutant myeloma cells
Biosynthetic mutations in mutant myeloma cells

EnzymePresentAbsentResult

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

species specific monoclonal antibodies summary
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

species specific mabs basic method
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)

summary of this session2
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
historical antibodies used in passive therapy
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)
summary of this session3
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
immunotherapy
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.
non specific immunotherapy
Non-specific immunotherapy
  • Stimulators of immune responses (biological response modifiers).
  • Coley’s vaccine (~1900, gruesome, Streps and Staphs)
  • BCG (1922 - equally so, granulomas etc)
  • Strange things: goat serum, heterologous proteins, quartz and other particulates.
pathogen related specific immunotherapy i
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, 489-500.

pathogen related specific immunotherapy ii
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.
summary of this session4
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
factors affecting the use of mabs in therapy general
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.
factors affecting the use of mabs in therapy mab efficacy
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).
factors affecting the use of mabs in therapy problems i
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, 309-316 (Printed copy of abstract supplied)
factors affecting the use of mabs in therapy problems ii
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.
mabs in therapy overcoming some problems
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.

mabs in therapy overcoming some problems1
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.
some current applications of mabs in disease therapy
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.
some current applications of mabs in disease therapy1
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, 337-342.
    • 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, 276-284.
    • Vincenti F et al. Phase I trial of humanised anti-interleukin-2 receptor antibody in renal transplantation. Transplantation 1997, 63, 33-38.
some current applications of mabs in disease therapy2
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, 1932-1939.
    • Breedveld FC. New TNFalpha biologic therapies for rheumatoid arthritis. Eur. Cytokine Netw. 1998, 9, 233-238. (Etanercept, IgG1 fusion protein)
    • Geletka RC, St Clair (2005) Infliximab for treatment of early rheumatoid arthritis Expert Opin. Biol. Ther. 5, 405-407. (MAb against TNF)
some current applications of mabs in disease therapy3
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, 1555-1561 (Palivizumab - Synagis)
    • Mejias A et al (2004) Asthma and RSV: new opportunities for therapeutic intervention. An. Pediatr. (Barc) 61, 252-60 (use of MAB against RSV in patients).
some current applications of mabs in disease therapy4
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, 1623-1629 (based on MAb to platelet receptor, gpIIb/IIIa).
      • Note: Two clinical trials with placebo stopped because of highly significant benefit.
mabs in disease therapy
MAbs in disease therapy
  • In all these cases, a reduction in immunogenicity is seen with MAbs in this order:

Murine

Chimera

Humanised

Human

summary of this session5
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
humanised mab therapy asthma
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.
humanised mab therapy asthma1
Humanised Mab Therapy: Asthma
  • MAbs to IgE molecule.
  • MAbs to IL-5
humanised mab therapy asthma2
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)
humanised mab therapy asthma3
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
humanised mab therapy asthma4
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!
summary of this session6
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

mabs in cancer therapy
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)
mabs in cancer therapy1
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)
mabs in cancer therapy2
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.
mabs in cancer therapy3
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.
mabs in cancer therapy4
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, 422-430.
  • See also Pritchard-Jones et al (2005) use of 105AD7 in osteosarcoma (next slide)
slide51

Gibson

Chet Atkins Tennessean

1990

summary of this session7
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
review and discussion mabs in therapy of human disease
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)

review and discussion new approaches under clinical and experimental investigation
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?

summary of this session8
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
monoclonal antibody index
Monoclonal antibody index:

http://www.gallartinternet.com/mai

slide58

Using Monoclonal Antibody Index:

e.g Lym-1 ( Oncolym) for Lymphoma

slide59

Using Monoclonal Antibody Index:

e.g Lym-1 ( Oncolym) for Lymphoma

slide60

NB: List supplied as printed sheet

Please insert “Humanised” and “Chimera” identifiers