Monoclonal antibodies

mab

1. Monoclonal antibodies Dr. Satyabrata Sahoo PDT Clinical & Experimental Pharmacology School of Tropical Medicine, Kolkata

2. Introduction An antibody is a protein used by the immune system to identify and neutralise foreign objects like bacteria and viruses. Each antibody recognises a specific antigen unique to its target. Polyclonal antibodies are antibodies that are derived from different cell lines. They differ in aminoacid sequence Immunoglobulins are structurally related glycoproteins that function as antibodies Monoclonal antibodies are antibodies that are identical because they were produced by one type of immune cell,all clones of a single parent cell

3. Cont.. Monoclonal antibodies: An antibody of unique specificity Derived from single B cell clone These can be made in large quantities in the labouratory Cornerstone of immunology These are also termed as magic bullets

4. Classification of antibodies Polyclonal antibodies Monoclonal antibodies Antibody fragments Chimeric antibodies Humanized antibodies Bispecific antibodies

5. Antibodies Polyclonal Monoclonal Cost Less expensive More expensive Yield Limited supply Infinite supply Ease Easily , rapidly produced Time consuming, more technical skill Potential for cross-reactivity High Low

6. Antibodies Polyclonal Monoclonal Produced by: Many B cell clones A single B cell clone Bind to: Multiple epitopes of all antigens used in the immunisation A single epitope of a single antigen Antibody class: A mixture of different antibody classes( isotypes ) All of a single antibody class Ag-binding site: Different antigen binding sites All antibodies have the same antigen binding site

7. History First monoclonal antibody was generated in 1975 by George kohler and Cesar Milstein. Both of them and Niels Kaj Jerne shared the nobel prize in physiology or medicine in 1984 for the discovery Fully licenced in 1986 The first licenced monoclonal antibody was Orthoclone OKT3(muromonab-CD3) approved in 1986 for use in preventing kidney transplant rejection It is a monoclonal mouse IgG2a antibody whose cognate antigen is CD3 Its use was limited to acute cases due to reported side effects(human anti-mouse antibody response) First fully human monoclonal antibody – Adalimumab (2003) Recently there are so many monoclonal antibodies generated and used for diagnostic and therapeutic purpose

8. Principle of Mabs

9. Conventional production of Mabs The hybridoma technology: Spleen cells from immunised mice are fused with the murine myeloma cells The several process had been developed at large scale. According to the different cell culture methods, it can classified into four fields 1. Robottle cell culture process 2. Membrane blinded cell culture process 3. Microcarrier cell culture process 4. Suspended cell culture process

10. Production in animals(In-vivo) Mouse ascites method: Hybridoma cells injected in mouse Produce ascites Fluid contains high concentration of antibodies No further concentration required Purification required Easy and inexensive Animal mortality

11. Production in cell culture(In-vitro) Batch tissue culture method: Grow hybridoma cells in batches Purify Mabs from the culture media Fetal bovine serum commonly used Low concentration Denature during concentration Semi permeable membrane based system: A barrier- hollow fibre or a membrane Larger compartment containing culture media Smaller chamber to isolate cells and Mabs High concentrations Method of choice for large scale production

12. Types of monoclonal antibody Naked monoclonal antibodies: those without any drug or radioactive material attached to them Mark the cells for the immune system Attach to receptors- block binding of growth factors E.g. 1. Transtuzumab - For advanced breast cancer(HER-2) 2.Cetuximab- For B cell non Hodgkin lymphoma(CD 20) 3.Bevacizumab- For metastatic colorectal cancer(VEGF) 4.Alemtuzumab- For B cell chronic lymphocytic leukemia (CD 52)

13. Types of Monoclonal antibodies Conjugated/loaded/ labeled Mabs : Coupled with drugs/toxins/radioactive atoms Chemo- labeled antibodies: Mabs conjugated with chemotherapeutic agents e.g. Brentuximab vedotin and ado- transtuzumab emtasine Brentuximab vedotin,attached to a chemo drug(MMAE) targets the CD 30 antigen(present on T and B- cells) in treatment of Hodgkin lymphoma and non-responding anaplastic large cell lymphoma Ado- transtuzumab emtansine , attached to DM1 chemo drug, targets the HER2 protein antigen used for curing advanced breast cancer patients

14. Types of Monoclonal antibodies Immune toxins: conjugated with toxins e.g.Denileukin diftitox Used to treat some cancers( cutaneous T- cell lymphoma and many others) Consists of IL2 protein attached to a toxin( derived from the germ causing diptheria ) IL-2 normally attaches to cells that express the CD 25 antigen and thus helps in delivering the toxin to these cells Radio-immune antibodies: e.g. Ibritumomab An Mab against the CD20 antigen on B cells (and lymphomas) Conjugated to either the radioactive isotope indium-111(111In) or Yttrium-90(90Y) for treatment of lymphoma patients

15. Nomenclature

16. Mechanism of action

17. Pharmacokinetics:Mabs Routes of administration: Subcutaneously( Rituximab , Transtuzumab,Adalimumab ) Intramuscularly( Palivizumab ) Intravenously( Infliximab ) Route for elimination of antibodies: Via uptake and catabolism by reticuloendothelial system and target tissue

18. Pharmacokinetics:Mabs Half life Chimeric : 4-15 days Humanised: 3-24 days Recombinant human: 11-24 days Human antimouse antibody(HAMA) response develops 7-10 days following exposure to murine antibody

19. Adverse effects of Mabs Naked Mabs : Mild, often allergic reaction on 1 st infusion Cytokine release syndrome Infusion toxicities, cytopenias Conjugated Mabs : More Adverse effects, depend on substances attached Antilymphocytes Mabs : Immunosuppression Increase risk of infection Cancer Anti TNF- α Mabs : Reactivation of tuberculosis lymphomas

20. Applications of Mabs Diagnostic applications Mab is used to detect pregnancy as early as weak or two after conception by reacting with human chorionic gonadotrophin HIV diagnostic kits Rapid diagnosis of hepatitis, inflenza , herpes, streptococcal,chlamydia and corona virus Identification and characterisation of tumor specific antigen: classification of cancer Imaging: localisation of cancer Diagnostic tools in research and labouratory Different technologies in which Mabs are used include: western blot, immunodot blot, ELISA, RIA,flow cytometry , immunohistochemistry , fluorescence microscopy, electron microscopy, confocal microscopy

21. Therapeutic uses Immunosuppression Autoimmune diseases Malignancies Antiplatelet therapy Infectious diseases Asthma Osteoporosis

22. Cancer Strategies for the Mabs in cancer: Immune reaction directed destruction of cancer cells Interference with the growth& differentiation of malignant cells Antigen epitope directed transport of anticancer agents to malignant cells Variety of agents conjugated to Mabs for selective delivery to cancer cells

23. Future applications Fight against bioterrorism Inhalational anthrax(potent biological terrorism) is caused by breathing the bacterial spores of Bacillus anthracis Raxibacumab injection is used to treat infectious inhalational anthrax when alternative therapies are failed

24. FDA approved Mabs

25. Ethical issues Freunds complete adjuvant(FCA)(to enhance the immune response): painful lesions at the injection site Pristane as a priming agent- granulomatous reactions Respiratory distress due to ascites The volume of FCA and pristane used should not exceed 0.1 ml and 0.2 ml respectively Individual mice should not be inoculated adjuvant more than 3 times Ascites fluid should only be harvested once at the time of euthanasia

26. Limitations of Mabs The typical doses of Mab drugs needed for treatment are significantly higher than those required for other drugs( or products) The huge demand to increase production of these drugs and the drive to lower the cost of these expensive medicines is a continuous challenge to the present industry Hybridoma technology is labourious and time consuming The presence of retroviruses as a part of the mammalian chromosomes is a common occurence This possess a great danger, since there is no guarentee that Mab production is totally virus-free, despite the purification. For this reason USFDA insists that Mab for human use should be totally free from all pathogenic organisms including viruses

27. Summary The monoclonal antibody production technology has revolutionized the world of biotechnology Advances in genetic engineering over the years have provided numerous ways to design Mabs that are more robust and efficacious compared with their original murine version Animals are utilise to produce Mabs but these antibodies are associated with immunogenicity and ethical problems Recombinant DNA technology, genetic engineering and transgenic animals are used to produce humanized Mabs or pure human Mabs with fewer ADRs Used for treatment of cancer, autoimmune disorders, graft rejections , infections, asthma, osteoporosis etc Somany newer Mabs are under trial phase now for future benefit

28. References 1.Goodman & Gilman’s the pharmacological basis of therapeutics 13 th edition 2. Leavy O. Therapeutic antibodies: past, present and future. Nat Rev Immunol 2010;10(5):297. 3. Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody at predefined specificity. Nature 1975;256: 495-7. 4. Robert A. Burger, M.D., Mark F. Brady, Ph.D., Michael A. Bookman, M.D.,Gini F. Fleming, M.D., Bradley J. Monk, M.D., Helen Huang, M.S., n engl j med 365;26 nejm.org december 29, 2011 5. CLINICAL MICROBIOLOGY REVIEWS, JUly 1988, p. 313-329 Vol. 1, No. 30893-8512/88/030313-17$02.00/0Copyright © 1988, American Society for Microbiology 6. L. Gandhi, D. Rodriguez‑Abreu , S. Gadgeel , E. Esteban, E. Felip , F. De Angelis,M . Domine , P. Clingan , M.J. Hochmair , S.F. Powell ,N Engl J Med 2018;378:2078-92.DOI: 10.1056/NEJMoa1801005 7. Patrick Mayrhofer and Renate Kunert,Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), 1190 Vienna, Austria, Human Antibodies 27 (2019) 37–51 37DOI 10.3233/HAB-180347IOS Press

29. References 8. Masami Suzuki1, Chie Kato1, and Atsuhiko Kato1, J Toxicol Pathol 2015; 28: 133–139 9. Patrick Chames1, Marc Van Regenmortel2, Etienne Weiss2 and Daniel Baty1, British Journal of Pharmacology (2009), 157, 220–233 Journal compilation © 2009 The British Pharmacological Society No claim to original French government works All rights reserved 0007-1188/09 10. Monoclonal Antibody Production http://www.nap.edu/catalog/9450.html 11. The CAMMS223 Trial Investigators*, n engl j med 359;17 www.nejm.org october 23, 2008 12. Maria Alice Vieira Willrich1*, 454 JALM | 454–457 | 02:03 | November 2017

30. Thank you