Protein Structure and Function Lecture 7 Notes: Glycosylation and Therapeutic Antibodies Part 2 Therapeutic Antibodies - Therapeutic antibodies are monoclonal antibodies (mAbs) or antibody-drug conjugates which bind microscopically to certain cells/proteins. This treatment will stimulate the patient's immune system to attack those cells, deliver a radioactive or lethal drug dose (attached) localised to a target cell line, or bind to molecules involved in metabolic (e.g. T- cell) regulation. All licensed mAbs are IgGs. - In the preparation of these antibodies, it is important that they have consistent, defined structures (i.e. monoclonal) because specific targeting is required, and natural antibodies are 'fuzzy' in structure. Therapeutic protein characteristics such as safety, efficacy, complement activation, receptor affinity and serum half-life can be affected by differences in glycosylation pattern (can be variable at particular amino acid sites). Therefore, analysis of these patterns is an important part of characterisation of therapeutic antibodies (particularly of mAbs). This has stimulated the development of methods of analysis by instrument, software and reagent manufacturers; this is presently and active area of research (fastest-growing sector of pharmaceuticals). - Of over 80 mAbs with regulatory approval, over 20 have 'blockbuster' status (i.e. generates sales of over $1 billion a year). Currently 5 antibody-drug conjugates are marketed and over 30 are in clinical trials. - Designation of therapeutic antibodies is according to the source and method of production as indicated by the letters before 'mab' in the name. For example: (a) Human 'u' (e.g. adalimumab from transgenic mice then from e.g. CHO cells) (b) Humanised 'zu' (e.g. trastuzumab/Herceptin from e.g. CHO cells) (c) Chimeric 'xi' (e.g. infliximab/Remicade from fusing Fab and Fc regions from 2 species) (d) Mouse 'o' (e.g. edrecolomab/Panorex) - Therapeutic antibodies are produced via hybridoma technology or phage displays. For example, the production of mAbs from mice begins with immunisation of the mouse with targets leading to the production of mice antibodies. The humanised mAb will have human regions, either in the variable region or end-terminus of the variable region, thus they are chimeric antibodies. Phage displays or transgenic mice can produce human monoclonal Abs. (A) Mouse hybridoma Immunization with targets (B) Phage display soFv or Fab (C) Transgenic mouse (D) Single B cell Human Immunization with targets Harvest splenocytes, generate hybridomas Screening Phage-displayed Ab libraries Harvest splenocytes, generate hybridomas Biopanning with targets (3-5 cycles) Screening Screening PBMC 1 Sort B cells with labeled antigens Chimerization Chimeric mAb CDR graft Mouse mAb Construction of Human lgG 1 V.,DJ.C.1 V.J. C. Humanized mAb Human mAb PCR, construct Vy and VL
- Hybridoma technology is based on the ability of B cells to produce antibodies of single specificity (i.e. mAbs). Myeloma/tumour cells are capable of continuous divisions forming large numbers of cells. Hybrid cells will be having the property of B cells antibody production and the tumour cell's ability of continuous division. Hybrid cells ensures antibody production continuously in dividing cells. Monoclonal antibodies contain a glycosylation site on IgG heavy chains at Asn 297. The nature of glycosylation can vary, depending