1. Syed M Shoaib & Sibu

2. Objectives
 Introduction
 What are humanised antibodies?
 How they are developed
 Mechanisms of action
 Examples of humanised mAbs
 Future work
 Summary
 References

3. 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.

4. 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.

5. Fig.1: Schematic diagram of different structures of antibodies

6. Development of hmAb
Cloning and sequencing of VH & VL of murine mAb
Gene synthesis of humanized V fragments.
Insertion of humanized V genes in staging vectors
Construction of expression vector for hmAbs
hmAbs in Sp2/0 Cells
Evaluation of specificity and avidity

7. 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.

8. 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.

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

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

11. 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)

12. • 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

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

14. 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.

15. Conjugated Monoclonal antibodies
Fig.5: Selected conjugated Monoclonal antibody

16. 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.

17. 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.

18. 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

19. 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.

20. 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.

21. Transtuzumab Emtansine mechanism
Fig.6: The internalization of T-DM1 & the HER-2 receptor
Adapted from Burris et al., 2011

22. 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

23. 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.

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

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

26. Tocilizumab
Fig.10: Regulation between Th17 and Tregs

27. 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

28. • 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
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