Immunoanalysis Lecture 4 PDF

Introduction to Immunoanalysis BIOT6002: Lecture 4 Lecturer: Caroline Browne Learning Objectives Describe the characteristics of monoclonal antibodies (mAbs) Understand the meaning of the mAb with the following suffixes Omab, Ximab, Zamab, and umab Define Naked mAb, Conjugated...

Introduction to Immunoanalysis BIOT6002: Lecture 4 Lecturer: Caroline Browne Learning Objectives Describe the characteristics of monoclonal antibodies (mAbs) Understand the meaning of the mAb with the following suffixes Omab, Ximab, Zamab, and umab Define Naked mAb, Conjugated mAb and Bispecific mAb Understand how mAbs are generated Compare and contrast the utility of mAbs and PAbs Monoclonal Antibodies (mAbs) Secreted by a plasma cell “clone” derived from a single B-lymphocyte precursor. Produced by plasma B cells grown in culture. These cell lines are hybridomas which secrete only one type of Ab. Homogeneous Ab directed against a single epitope on antigen molecule. mAb therapeutics – suffixes of relevance Omab - Murine: Rituximab Indication: Lymphoma & autoimmune These antibodies are derived from mouse proteins diseases MOA: Targets CD20, a surface antigen Ximab - Chimeric: on B cells Abs are combination of mouse and human proteins Zumab - Humanized: Abs consist of small portions of mouse proteins Trastuzumab Indication: Brest cancer fused with human proteins MAO: Targets HER2 (Receptor tyrosine- protein kinase erbB-2) Umab – Human Abs are entirely composed of human proteins Ofatumumab Indication: Multiple sclerosis MOA: Depletion of B cells mAbs for Oncology mAb therapeutics are available to treat non-small cell lung, brain tumors, ovarian, breast, gastric, melanoma, colorectal, Hodgkin’s lymphoma, and other malignancies. Naked mAbs standalone therapy – one drug only. Conjugated mAbs (tagged, loaded or labelled mAbs) Converted into radioactive particles, or Combined with drug (therapy) Bispecific mAbs Modified to target and attack two specific antigens simultaneously Monoclonal Antibody Production Step 1 - Immunization & isolation of splenocytes: Mice are immunized with an antigen. After a set period, their blood is screened for antibody production. The antibody-producing splenocytes are then isolated for in vitro hybridoma production. Step 2 - Preparation of myeloma cells: Myeloma cells are immortalized cells. Once fused with spleen cells, that can result in hybridoma capable of unlimited growth. Monoclonal Antibody Production Step 3 - Fusion: Myeloma cells and isolated splenocytes are fused together to form hybridomas in the presence of polyethylene glycol (PEG). PEG causes the cell membranes to fuse. Step 4 - Clone screening/selection: Clones are screened and selected based on antigen specificity and immunoglobulin class. Monoclonal Antibody Production Step 5 - Functional characterization: Confirm, validate and characterize (e.g. ELISA) each potentially high-producing colony Step 6 - Scale up and wean: Scale up clones producing desired antibodies Wean off selection agents used to generate mAbs Step 7 - Expansion: Expand clones producing desired antibodies (e.g. hollow fiber bioreactors or large flasks (spinner flasks). Monoclonal Antibody Production Advantages of mAb production Batch-to-batch reproducibility (high homogeneity). Production of large quantities of identical antibody is possible. This is an advantage for diagnostic manufacturing and therapeutic drug development. Low cross-reactivity - high specificity to a single epitope of Ag. Increased sensitive for quantification of protein levels. Low background noise. Limitations of mAb production More expensive to produce. necessary to produce a pool of several monoclonal antibodies. Requires significantly more time to produce and develop the hybridized clone (+/- 6 months). More susceptible to binding changes when labelled. Activity – Compare/contrast MAbs & PAbs Ab population Ag detection Specificity Cost of production Time required to produce Stability Utility The global market for mAb production According to Vision Research Reports, the monoclonal antibodies market is poised to grow at a CAGR of 11.07% from 2024 to 2033. The global monoclonal antibodies market size was estimated at 237.64 billion USD in 2023. It is expected to surpass around 679.03 billion USD by 2033. Future areas of growth Autoimmune disease Infectious disease Neurological disorders Learning Objectives Describe the characteristics of monoclonal antibodies (mAbs) Understand the meaning of the mAb with the following suffixes Omab, Ximab, Zamab, and umab Define Naked mAb, Conjugated mAb and Bispecific mAb Understand how Mabs are generated Compare and contrast the utility of mAbs and PAbs