Therapeutic Humanised Monoclonal Antibodies

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THERAPEUTIC HUMANISED MONOCLONAL ANTIBODIES Syed M Shoaib & Sibu

Transcript of Therapeutic Humanised Monoclonal Antibodies

THERAPEUTIC HUMANISED

MONOCLONAL ANTIBODIES

Syed M Shoaib & Sibu

Objectives

Introduction

What are humanised antibodies?

How they are developed

Mechanisms of action

Examples of humanised mAbs

Future work

Summary

References

Introduction• In 1975 Kohler and Milstein used hybridoma technology to

produce Monoclonal Antibodies (mAbs) in therapy.• These mAbs were derived from mice• Humans who were treated with these mAbs developed

immunogenicity and human anti-mouse antibody (HAMA) responses.

• Chimeric mAbs were developed to overcome the potential of patients developing immunogenicity and triggering an anti-globulin or HAMA response to the rodent derived mAbs.

• Although chimeric mAbs decreased the probability of triggering HAMA responses and reduced immunogenicity in patients, the problems were not overcome. Thus the requirement to develop more ‘humanized’ mAbs increased.

Introduction contd.• Humanised mAbs consist of mouse derived complementary-

determining regions (CDR) which are bound to a human IgG framework.

• CDRs have specificity and affinity to human tumour cells.• Humanised mAbs contain 5-10% of mouse proteins unlike chimeric

mAbs which contains approximately 34% murine protein.• Humanized mAbs decrease the immunogenicity and anti-globulin

responses even when administered repeatedly and during long term usage in therapy.

• Humanized mAbs are ideally employed as part of a therapeutic regime against diseases such as Cancers e.g. Non-Hodgkin’s Lymphoma (NHL), breast cancer and renal cell carcinoma.

Fig.1: Schematic diagram of different structures of antibodies

Development of hmAbCloning and sequencing of VH & VL of murine mAbCloning and sequencing of VH & VL of murine mAb

Gene synthesis of humanized V fragments.Gene synthesis of humanized V fragments.

Insertion of humanized V genes in staging vectorsInsertion of humanized V genes in staging vectors

Construction of expression vector for hmAbsConstruction of expression vector for hmAbs

hmAbs in Sp2/0 CellshmAbs in Sp2/0 Cells

Evaluation of specificity and avidityEvaluation of specificity and avidity

Ocrelizumab• On trial for patients with relapsing-remitting Multiple sclerosis

(MS).• Focal inflammatory demyelinating lesions cause relapse.• The targets antigen is CD20 on B Cells.• Non human primate – passive transfer of monoclonal anti Myelin

oligodendrocyte glycoprotein (MOG) led to demyelination in Experimental autoimmune encephalomyelitis (EAE).

• MOG is located on outer surface of myelin sheath.• Potential mechanisms of action not understood.• The rationale for B cell therapy.• Induces a ADCC response for B cell clearance, unlike rituximab • It has a lower safety profile than rituximab.

Ocrelizumab

• In a study carried out by, use of this monoclonal antibody saw a decrease of 89% - 96% of new gadolinium enhancing lesions and 80% reduction in relapse patients.

• Adverse – one patient on the 600 mg group died during trial. Discontinued for RA trial.

• Advantage over INF-β, Natilizumab and Alemzuzumab.

• Improved efficacy profile when compared to Rituximab.

• Ideal for monotherapy based on trial results.

Ocrelizumab

Fig.2: Dose dependent Ocrelizumab against interferon beta-1a over 24 weeks (Barkhof et al., 2011)

Ocrelizumab

Fig.3: Annualised relapse rate at trial’s end.

Bevacizumab: Anti-VEGF monoclonal antibody

• Vascular endothelial growth factor (VEGF) is an endothelial angiogenic inducer.

• It is expressed in many human tumours as lung, breast, GI tract and ovarian carcinomas.

• First anti-angiogenic approved by FDA.• Inactivates all active isoforms of VEGF to inhibit angeogenesis.• Effective against solid malignancies in metastatic colorectal cancer,

non-small-cell lung cancer & renal cell carcinoma.• VEGF is released because of hypoxia and genetic mutations in

oncogenes and tumour supressor genes.• Decreases tumour perfusion vascular volume, microvascular

density and circulating endothelial cells.(Adams & Weiner, 2005; Valabrega et al., 2007; Presta et al., 1997)

• Important Trial:

Miller et al. in 2007 carried out a trial on 722 patients with metastatic breast cancer. Patients were randomly assigned to receive paclitaxel either alone or with bevacizumab. The combination arm significantly prolonged progression-free survival as compared with paclitaxel alone.

Bevacizumab: Anti-VEGF monoclonal antibody

Fig.4: Signalling pathway induced by recognition of VEGF by VEGFR-1 resulting in angiogenesis promotion

Conjugated Monoclonal antibodies

Antibody selection• Selective binding to the tumour with little cross reactivity to other

cells.• High avidity binding to tumour cells.

Drug selection • Must show high potency to tumour cell lines.• Must contain a suitable functional group for antibody linkage.• Reasonable solubility in aqueous solution.

Linker selection• Allow stability in circulation. • Allow rapid release of fully active drug in tumour.

Conjugated Monoclonal antibodies

Fig.5: Selected conjugated Monoclonal antibody

Trastuzumab • Treats breast cancer by targeting the extracellular domain of the HER-

2 receptor. Mode of action not fully understood. Suggestions are:• Inhibition of P13K/AKT & MAPK signalling pathways.• Inhibition of angiogenesis. • Induction of antibody dependent cellular cytotoxicity. • Induction of apoptosis.• Resistance through Activation of the P13K/AKT signalling pathway &

signalling through the 95KD intracellular pathway Multiple mechanism are not fully understood.

• Resistance avoided by addition of Lapatanib • Cardio toxic.

Trastuzumab monotherapy • It is a safe first line treatment for monotherapy.

• However a small subset of patients do not respond.

• Majority of patients develop disease progression.

• Not suitable for a rapid response.

• Great selectivity, however, Binding doesn’t always lead to cytotoxicity.

• Transtuzumab can be used in conjunction with chemotherapy & endocrine therapy.

Trastuzumab with endocrine therapy

Table. : Breast cancer drugs used as monotherapy and with Trastuzumab

Anastrozole Letrozole Anastrozole + Mab

Letrozole + Mab

TTP (Time to progression)

2.4 months 3.3 months 4.8 months 14.1 months

OS (Overall survival)

23.9 months NR 28.5 months NR

Trastuzumab with chemotherapy

Docetaxel Trastuzumab/Docetaxel

Overall response rate 34% 61%

Time to disease progression

6.1 months 11.7months

Overall survival 22.7 months 31.2 months

Table 2: Docetaxel with and without trastuzumab.

Trastuzumab Emtansine

• Antibody drug conjugate against HER-2.

• Mertansine linked to the Fc portion and is non toxic in conjugated form. Inhibits microtubule assembly.

• Improves therapeutic index by limited exposure to normal tissues.

• Currently in phase 3 trial.

Transtuzumab Emtansine mechanism

Fig.6: The internalization of T-DM1 & the HER-2 receptor

Adapted from Burris et al., 2011

Trastuzumab Emtansine

Fig.7 : The response of resistant HER-2 over-expressing breast carcinoma cell lines to Trastuzumab & T-DM1. Burris lll et al., 2011

Tocilizumab • Inhibits soluble and membrane bound IL-6 receptors.• Approved in 90 countries to treat Rheumatoid arthritis & a candidate

for 17 other diseases.• In animal models blockade of IL6 receptors showed reduction and

severity of arthritis.• 50% of patients in the RADIATE trial improved after discontinuing

anti TNF treatment.• Activation of the SAA in AA amyloidis depends on the IL-6. • 3 case studies showed AA fibril deposits completely disappear after

3 injections.• Maintains the balance between TH17 & T regulatory cells.• No interaction with other drugs .• Affective for other diseases such as Haemophilia A.

Tocilizumab graph against DMARDS

Fig.8 : The effect of DMARD & Tocilizumab on Rheumatoid arthritis Hashimoto et al., 2007

Tocilizumab

Fig.9: Inhibition of IL-6 Tanaka et al., 2011

Tocilizumab

Fig.10: Regulation between Th17 and Tregs

Future Work• Identification of new epitopes on existing targets.• Radio isotopes and toxins are added to antibodies to induce cell

death.• Data on predicting response to therapies.• Markers that can predict patient response to mAb therapy.• Combination of chimeric-humanised antibody therapy rather then

humanised-chemotherapic treatment.• Systemic clearance of non targeted antibodies.• Multifunctional mAb binding to pair of antigens on tumour cells.• By recognizing specific epitopes adverse reaction could be avoided.• Radio isotopes and toxins could directly kill tumour cell.• Systemic clearance so that mAbs do not attach to other normal

antigens causing adverse reaction.• Multifunctional mAbs for more specificity

• Adams, G.P. And Weiner, L.M. (2005) Monoclonal Antibody Therapy of Cancer, Nature Biotechnology , 23,1147-1157.

• Albanell, J., Baselga, J., Clinton, G.M., García-Conde, J., García-Conde, J., Keenan, E.J., Lluch, A., Molina, M.A., Ramsey, E.E., Rojo, F. & Sáez, R. 2006. p95HER-2 predicts worse outcome in patients with HER-2-positive breast cancer. Clinical Cancer Research, 12(2), p.424-31.

• Antón, A., Chan, S., Conte, P., Extra, J.M., Green, M., Grimes, D., Kennedy, J., Lluch, A., Maraninchi, D., Marty, M., Mauriac, L., Mayne, K., O'Byrne, K., Tubiana-Hulin, M. & Ward, C. 2005. Randomized phase II trial of the efficacy and safety of Trastuzumab combined with Docetaxel in patients with human epidermal growth factor receptor 2-positive metastatic breast cancer administered as first-line treatment: the M77001 study group. Journal of clinical oncology, 23(19), p.4265-74

• Barkhof, F., Bar-Or, A., Calabresi, P.A., Glanzman, R., Hauser, S.L., Kappos, L., Li, D., O’Connor, P., Tinbergen, J., Yin, M. 2011. Ocrelizumab in relapsing-remitting multiple sclerosis: a phase 2, randomised, placebo-controlled, multicentre trial. Lancet, 378, p.1779-87

• Beck, A., Cochet, O. & Wurch, T. 2010. GlycoFi’s technology to control the glycosylation of recombinant therapeutic proteins. Expert opinion on drug discovery, 5 (4), p95-111.

• Burris III, H.A., Holden, S.N., Lewis-Phillips, G.D., Sliwkowski, M.X. & Tibbitts, J. 2011. Trastuzumab Emtansine (T-DM1): A Novel Agent for Targeting HER2+ Breast Cancer. Clinical Breast Cancer, 11(5), p.275-82

References

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• Ferrara, N., Hillan, K.J., Novotny, W. (2005) Bevacizumab (Avastin), a Humanized Anti-VEGF monoclonal Antibody for Cancer Therapy, Biochemical and Biophysical Research Communication, 333, 328-335.

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• Hashimoto, J., Kawai, S., Kishimoto, T., Miyasaka, N., Murata, N., Nishimoto, N., Takeuchi, T. & van der Heijde, D. 2007. Study of active controlled monotherapy used for rheumatoid arthritis, an IL-6 inhibitor (SAMURAI): evidence of clinical and radiographic benefit from an x ray reader-blinded randomised controlled trial of Tocilizumab. The annals of Rheumatic diseases, 66(9), p.1162-67

 

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• Presta, L.G., Chen, H., O’Connor, S. ., Chisholm, V., Meng, Y. ., Krummen, L., Winkler, M. & Ferrara N., (1997) Humanization of an Anti-Vascular Endothelial Growth Factor Monoclonal Antibody for the Therapy of Solid Tumors and Other Disorders, Cancer Research, 57, 4593-4599.