Humanisation of antibodies and Techniques

Humanisation of antibodies and Techniques such as CDR Grafting, Phage Display Technology, Transgenic animals.

1. HUMANISATION OF ANTIBODIES Presented by : megh Vithalkar Roll no. 07 m.Pharm semester-I Department of pharmacology Goa college of pharmacy

2. Introduction I n 1975 K ohler and M ilstein used H ybridoma technology to produce monoclonal antibodies ( mAbs ) in therapy. T hese mAbs were derived from mice. H umans who were treated with these mAbs developed immunogenicity and a peculiar response called humans anti-mouse antibody (HAMA) . A fter that incident 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 . A lthough 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.

3. ctd … H umanised mAbs consist of mouse derived ‘complementary determining regions’ (CDR) which are bound to a human IgG framework. H umanised mAbs contain 5-10% of mouse proteins unlike chimeric mAbs which contains approximately 34% murine protein. H umanized mAbs decrease the immunogenicity and anti-globulin responses even when administered repeatedly and during long term usage in therapy. H umanized 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.

4. Definition H umanization is a process by which xenogeneic antibody sequences are modified to reduce the immunogenicity. H ere, antibodies are from non-human species whose protein sequences have been modified to increase their similarity to antibody variants produced naturally in humans. T he process of "humanization" is usually applied to monoclonal antibodies developed for administration to humans (for example, antibodies developed as anti-cancer drugs). The international non proprietary names of humanized antibodies end in zumab , as in omalizumab .

5. Structure of antibody Fab region or Fc region or Antigen binding site

6. T he heavy and light chains that make up each arm of the antibody are composed of two regions, called constant (c) and variable (v) . T hese regions are distinguished on the basis of amino acid similarity—that is, constant regions have essentially the same amino acid sequence in all antibody molecules of the same class (IgG, I gM, I gA, I gD , or IgE ), but the amino acid sequences of the variable regions differ quite a lot from antibody to antibody. T his makes sense, because the variable regions determine the unique shape of the antibody-binding site. the tail of the molecule, which does not bind to antigens, is composed entirely of the constant regions of heavy chains.

7. Need for humanisation of antibodies

8. Techniques of humanizing m A b 1.CDR grafting. 2.Phage display technologies . 3.Transgenic animals.

9. CDR grafting T he complementarity determining regions (CDRs) are part of the variable ‘ends’ of an antibody which are responsible for that very antibody to bind to a specific antigen. CDR grafting is a humanization technique whereby humanized antibody sequences are generated by carefully selecting the CDRs of the parental antibody and grafting them into a human framework (typically murine/mice origin), but increasingly other species are being humanized, including rabbits). Complementarity-determining regions (CDRs) are part of the variable chains in I gS (antibodies) where these molecules bind to their specific antigen variable region.

10. REFERENCE: Robert R, Streltsov VA, Newman J, Pearce LA, Wark KL, Dolezal O. Germline humanization of a murine Abeta antibody and crystal structure of the humanized recombinant Fab fragment. Protein Sci. 2010: 19:299-308.

11. References https://www.cancer.net/navigating-cancer-care/how-cancer-treated/immunotherapy-and- vaccines/understanding-immunotherapy. https://www.fusionantibodies.com/blog/2016/january/ebr-antibody-humanization-article. https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4190434/. https://www.clinicaloptions.com/oncology/programs/immunotherapy-in-cancer/cco- slideset/ immunotherapyslides . http://www.scielo.br/scielo.php?script=sci_arttext&pid=s1415-47572005000100001. https://www.ncbi.nlm.nih.gov/pmc/articles/pmc3011222/. https://www.ncbi.nlm.nih.gov/pmc/articles/pmc5440098/.

12. Thank you To be continued…