animal health trust antibodies as therapeutic entities duncan hannant animal health trust newmarket

Animal Health Trust

Antibodies as Therapeutic Entities

Antibodies as Therapeutic Entities

Duncan Hannant

Animal Health Trust Newmarket

Animal Health Trust

Summary of this SessionSummary 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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Monoclonal antibodies: SummaryMonoclonal antibodies: Summary

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)

Hybridoma selection based on drug resistance

HGPRT = hypoxanthine-guanine phosphoribosyltransferaseTK = thymidine kinase

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Monoclonal antibodies: SummaryMonoclonal antibodies: Summary

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).

Hybridoma selection based on drug resistance

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Biosynthetic mutations in mutant myeloma cellsBiosynthetic mutations in mutant myeloma cells

Enzyme Present Absent ResultHGPRT × 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 = hypoxanthineA = aminopterinT = thymidine

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Species-specific monoclonal antibodies: summarySpecies-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

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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)

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Historical: Antibodies used in passive therapyHistorical: 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)

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ImmunotherapyImmunotherapy 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.

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Non-specific immunotherapyNon-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.

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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.

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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.

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Factors affecting the use of MAbs in therapy: GeneralFactors 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.

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Factors affecting the use of MAbs in therapy: Mab efficacyFactors 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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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)

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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.

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MAbs in therapy: Overcoming some problemsMAbs 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.

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MAbs in therapy: Overcoming some problemsMAbs 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.

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Some current applications of MAbs in disease therapySome 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.Allergic lung disease therapy. Cancer 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.

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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.

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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)

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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).

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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.

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MAbs in disease therapyMAbs in disease therapy

In all these cases, a reduction in immunogenicity is seen with MAbs in this order:

Murine

Chimera

Humanised

Human

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Fender Telecaster Custom1973

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Humanised Mab Therapy: AsthmaHumanised 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.

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MAbs to IgE molecule. MAbs to IL-5

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Humanised Mab Therapy: AsthmaHumanised 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)

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Humanised Mab Therapy: AsthmaHumanised 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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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!

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Gibson SG Standard1973

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0.75

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MAbs in Cancer TherapyMAbs 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)

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Two examples described:

Lymphoma therapy (Lym-1 and Lym-2)

Cancer vaccine (anti-idiotype for colorectal cancer - Licenced from CRC as Onyvax-105)

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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.

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MAbs in Cancer TherapyMAbs in Cancer TherapyCancer 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.

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MAbs in Cancer TherapyMAbs in Cancer TherapyCancer 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)

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Gibson Chet Atkins Tennessean 1990

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Review and Discussion:

MAbs in therapy of human disease


MAbs in therapy of human disease

Drug deliveryDelivery of BRMs?Risks (human-derived genetic material)Other potential use (anti-idiotypes etc)

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New approaches under clinical and experimental investigation


New approaches under clinical and experimental investigation

Bispecific Abs in LymphomaDendritic cell vaccines (pulsed with Ag or mRNA)

DNA (or mRNA?) vaccinesAny other thoughts, possibilities?

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Monoclonal antibody index:Monoclonal antibody index:

http://www.gallartinternet.com/mai

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MONOCLONAL ANTIBODY INDEX. Cancer -- 6th edition.All rights reserved. Copyright by Gallart Biotech SL, 2003. E-mail: [email protected]

MOAB DATA TABLEOAB

NAMERELATEDMOAB

SPECIES

IGTYPE

MOAB/ CELLLINECHARACTERI STICS

TARGETCELL/TISSUE

TARGETCANCER

AG/ EPITOPENAME

AGMw/ kDa(ip)

AGEPITOPE

STUDYPHASE

PRODUCER/ SUPPLIER/ PATENT APPLI CANT(other names of themoab or itsderivatives)

ym-1 IL-2fusionproteinsand scFv

m G2a Ka= 4.02x10(8)M(-1) / bsc=1.1x10(6)

Blymphocytes

lymphoma(non-Hodgkin's),leukemia

- 31-35 light chainofpolymorphic HLA-DR10(class I I )protein

I I / I I I Southern California Univ./ Northwestern Univ. /Techniclone, US(Oncolym) / DamonBiotech, US / Schering,Germany / PeregrinePharm., US

BI BLI OGRAPHY

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

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

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MAbs used in human medicine To suppress the immune system: OKT3: Binds to molecule on surface of T cells – prevents rejection of transplants, also good in autoimmune diabetes. Daclizumab (Zenopax): Binds to part of IL-2 receptor on activated T cells, prevents acute transplant rejection. Infliximab (Remicade): Binds to TNF-alpha, good against RA, but has some adverse effects (can convert TB to active case) Omalizumab (Xolair): Binds to free IgE and stops it binding to mast cells, shows promise against allergic asthma. To kill malignant cells: Rituximab (Rituxan): Binds to CD20 on most B cells, treatment for lymphoma. Herceptin: Binds HER2 receptor for epidermal growth factor (EGF) found on some tumour cells (breast, lymphomas) Mylotarg: Conjugate of Mab to CD33 on AML cells and calicheamicin (breaks DS DNA) – first immunotoxin in cancer. LymphoCide: Binds to CD22 on B cell leukemias. Alemtuzumab (MabCampath): Binds to CD52 on WBC, has produced complete remission of chronic lymphocytic leukemia. Lym-1 (Oncolym): Binds to discontinuous epitope on HLA DR10 (MHC class II antigen). Cetuximab (Erbitux): Blocks HER1 – an EGF receptor. To stop angiogenesis: Vitaxin: Binds to a vascular integrin (alpha-v/beta-3) on tumour but not normal blood vessels – shrinks solid tumours. Bevacizumab (Avastin): Blocks vascular endothelial GF (VEGF) receptor – used for colorectal cancers (US FDA Feb 2004).

Others: Abciximab (ReoPro): Inhibits platelet clumping, prevents re-clogging in patients after angioplasty.

NB: List supplied as printed sheetPlease insert “Humanised” and “Chimera” identifiers

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animal health trust antibodies as therapeutic entities duncan hannant animal health trust newmarket

Documents

session n

n antitoxin

n intravenous injection

n strange things

balbc mice n

staphs n bcg

n coleys vaccine

n unfused myeloma cells