BS2520 Protein Structure and Function Lecture 8 Glycosylation - This is an example of non-enzymatic glycosylation. - Advanced glycation end products are harmful compounds - example of AGEs is Amadori Products. - Effects of AGES: o Ageing, diabetes ffincluding vascular complications), atherosclerosis, chronic kidney disease, Alzheimer's disease, stroke, cataract development, asthma, arthritis. - A receptor for AGEs has been identified. Role of Amadori products in diabetes: Early glycated protein As a causative agent in diabetes mellitus Human serum albumin Type 1 and type 2 diabetes with nephropathy and retinopathy Collagen Diabetic retinopathy Immunoglobulins (lgG, IgA, IgM) Plasma proteins Type 1 and type 2 with nephropathy Type 2 diabetes Diabetic atherosclerosis Lipoproteins (LDL) Histone LDL: Low density lipoprotein Therapeutic Antibodies Diabetes - A form of therapy that uses monoclonal antibodies ffmAbs) or antibody-drug conjugates to bind mono-specifically to certain cells or proteins. - The objective is that this treatment will stimulate the patient's immune system to attack those cells or deliver a radioactive or lethal drug dose to a target cell line or bind to molecules involved in metabolic regulation ffeg. T cell). - So far, all monoclonal antibodies are IgGs. - Since specific targeting is required and natural antibodies are 'fuzzy' in structure, monoclonal antibodies are required. - However, therapeutic protein characteristics ffincluding safety, efficacy, complement activation, receptor affinity and serum half-life) can also be affected by differences in their glycosylation pattern - therefore, the analysis of these patterns is an important part of the characterization of therapeutic glycoproteins, particularly mAbs. - This has stimulated the development of methods of analysis by instrument and kit manufacturers. - Therapeutic antibodies are the fastest growing sector of the pharmaceutical industry. - Currently 2 antibody-drug conjugates are marketed and >30 are in clinical trials. Human vs. Humanised mAbs:
Fully Human Monoclonal Antibody Mouse IgG locus replaced with human DNA sequence Humanized Monoclonal Antibody Mouse lgG locus intact Mouse injected with human therapeutic target 2 Target-specific antibody DNA extracted 3 Antibody genes cloned, and mAb expressed from CHO cells Human recombinant antibody Mouse injected with human therapeutic target 2 Target-specific antibody DNA extracted 3 CDR engineered for high affinity and grafted onto human framework, cloned, and mAb expressed from CHO cells Humanized recombinant antibody COR=complementariny determining region; CHO= Chinese harester ovary: Igzimmunoglobulin; ILzimmerleskin; rultemonocional antibody Designations of therapeutic antibodies: Human u e.g. adalimumab Humanised zu Chimeric xi e.g. infliximab (Remicade) Mouse 0 e.g. edrecolomab (Panorex) originally via hybridoma technology Hierarchy of mAbs: - Binding region Light chain -Heavy chain- [ Variable Constant Domains Glicans Fully Human via transgenic mice then from e.g. CHO cells e.g. trastuzumab (Herceptin) from e.g. CHO cells, $6.6b sales in 2015 by fusing Fab and Fc regions from 2 species Humanized Low immunogenicity Importance of Glycosylation Chimeric Murine High immunogenicity - 70% of therapeutic proteins are glycosylated. - Structural diversity: Fuc, Gal, Neu5Ac, GIcNAc, Man units - their arrangement. - Glycosylation determines biological, including therapeutic activity, physicochemical properties, ADCC and CDC ffAntibody & Complement-Dependent Cytotoxicity's). - Variability depending on the