Monoclonal Antibodies
Biotech Drugs - Monoclonal Antibodies
Course Overview
Course Code: BIT
Title: Biotech Drugs - Monoclonal Antibodies
Lecturer: Dr. Kulwinder Kaur
Institution: RCSI Royal College of Surgeons in Ireland Coláiste Ríoga na Máinleá in Éirinn
Lecture Learning Outcomes
Recap on immune regulation and cytokines
Comprehend the role of cytokines as pharmaceutical targets
Describe the role of monoclonal antibodies (MAbs)
Discuss how therapeutic MAbs are produced
Define the different types of antibodies
Explain production methods of MAbs using examples
Immune Regulation and Cytokines
Definition of Cytokines:
Cytokines are chemical communicators in the immune system that regulate the functioning of cells. They trigger intracellular transduction events by binding to specific cell surface receptors.
Cytokines include various molecules such as interleukins and chemokines.
Role in Immune System:
Cytokines have a central role in regulating immune and inflammatory responses.
Primarily act on or are produced by leukocytes (white blood cells).
Receptors:
Each cytokine acts through its specific receptor on target cells.
Common receptor families include members of the immunoglobulin (Ig) superfamily and tumor necrosis factor (TNF) family.
Cells in the Immune System
Major cell types in the immune system:
Monocytes
Macrophages
Mast Cells
Dendritic Cells
Natural Killer (NK) Cells
Neutrophils
Eosinophils
Basophils
T Cells
B Cells
Monoclonal Antibodies
Definition:
Monoclonal antibodies are identical antibodies produced by a single clone of B-cells, designed to bind specifically to one particular antigen or epitope.
Comparison to Polyclonal Antibodies:
Polyclonal antibodies consist of a mix and bind to multiple epitopes, while monoclonal antibodies are uniform and highly specific.
History:
The first monoclonal antibody was generated in 1975, and the first fully licensed monoclonal antibody was approved in 1986.
Approximately 30 monoclonal antibodies are currently FDA approved for treating various diseases including cancer and chronic inflammatory diseases.
Clinical Applications:
Cancer
Autoimmune diseases
Infectious diseases
Imaging tumor markers
Protein detection
Kohler and Milstein's Contribution
B-cell myeloma cancer cells produce a single type of antibody.
Hybridoma Technology:
Kohler and Milstein fused immortal myeloma cells with antibody-producing B lymphocytes.
A proportion of these hybrid cells, stable and cancerous, produce antibodies, creating a source of monoclonal antibodies.
They were awarded the Nobel Prize in 1984 for this discovery.
Production of Monoclonal Antibodies - Hybridoma Technology
Immunization:
Immunize an animal (usually a mouse) subcutaneously with an appropriate antigen.
Multiple injections at various sites performed several times stimulate B-lymphocytes specific to the antigen.
Antibody concentrations in serum are monitored.
The spleen is aseptically removed to release B cells.
The lymphocytes are separated by density gradient centrifugation.
Cell Fusion:
Lymphocytes are fused with myeloma cells using polyethylene glycol (PEG).
This fusion creates hybridoma cells that inherit antibody-producing ability from B cells and the immortality of myeloma cells.
PEG is washed away and cells are kept in fresh medium, resulting in a mixture of hybridomas, free myeloma cells, and free lymphocytes.
Selection of Hybridomas:
Cells are cultured in HAT medium (Hypoxanthine-Aminopterin-Thymidine), where only hybridoma cells can grow while unfused B cells die off naturally within a few days.
This selection occurs typically within 7-10 days of culture.
Screening the Products:
Hybridomas are screened for the secretion of the desired antibody using Enzyme-Linked ImmunoSorbent Assay (ELISA) and Radioimmunoassay (RIA).
These assays involve the antibody binding to a specific antigen, allowing unbound components to be washed off.
Antibodies produced by hybridomas are classified as monoclonal antibodies.
Culture for Mass Production:
Two methods are utilized:
Ascites Production (in vivo):
Hybridoma cells are injected into the peritoneal cavity of mice, acting as fermentation chambers.
It can be costly and may involve contamination from mouse proteins.
Animal Cell Culture (in vitro):
Cells are extracted from the antibody-containing media and purified through chromatographic steps.
Therapeutic Monoclonal Antibodies
Evolution from fully mouse antibodies to fully human antibodies.
First Licensed Monoclonal Antibody
Orthoclone OKT3 (muromonab-CD3):
Approved in 1986 for preventing kidney transplant rejection.
It is a monoclonal mouse IgG2a antibody with limited use due to side effects such as infections from immune suppression.
Mechanism of Action:
OKT3 binds to CD3 receptors on T cells, activating and depleting them, thus modulating immune responses and preventing transplant rejection.
Monoclonal Antibodies Approved by the EMA and FDA
Example Products and Their Details
Tysabri® (Natalizumab):
Company: Biogen Idec and Elan
Target: VLA-4
Type: Humanized IgG4
EU Approval: 2006
FDA Approval: 2004
Rituxan® (Rituximab):
Company: Biogen Idec, Genentech (Roche)
Target: CD20
Type: Chimeric IgG1
EU Approval: 1998
FDA Approval: 1997
Other examples:
Remicade® (Infliximab): Target: TNFα.
Humira® (Adalimumab): Target: TNFα.
Simponi® (Golimumab): Target: TNFα.
Actemra® (Tocilizumab): Target: IL-6 receptor.
Cimzia® (Certolizumab pegol): Target: TNFα.
Zenapax® (Daclizumab): Target: CD25 (IL2 receptor).
Chimeric Antibodies
Definition:
Chimeric antibodies are genetically engineered monoclonal antibodies that contain regions from two different species.
An example includes a mouse-rabbit chimeric antibody with components from both species.
They are designed to reduce the immunogenicity of mouse antibodies while retaining antigen specificity.
Humanized Antibodies
Definition:
Humanized antibodies are monoclonal antibodies that are mostly human but have antigen-binding sites derived from a non-human species, usually a mouse.
They consist of approximately 95% human origin and result in lower binding affinity compared to the original murine antibodies.
Examples include natalizumab and daclizumab.
Case Studies: Infliximab
Definition:
Infliximab is a chimeric human-murine monoclonal antibody targeting TNFα.
It consists of approximately 30% murine variable region amino acid sequence responsible for binding to human TNFα, and 70% human IgG1 constant region.
Mechanism of Action:
Binds to and neutralizes both membrane-bound TNF-α and soluble TNF-α, destroying TNF-α producing cells, thus inhibiting inflammation.
Administration:
Marketed as Remicade, presented as a sterile lyophilized powder for injection.
Configuration:
Each vial contains 100 mg of infliximab.
Mixed with buffer substances with no preservatives.
Production - Animal Cell Culture
Fermentation and Harvesting:
Infliximab is produced from SP2/0 mouse myeloma cells through continuous perfusion cell culture.
Harvests undergo filtration for clarification followed by purification.
Purification Process:
Affinity and anion chromatography are employed.
Characterization:
High-quality characterization data for infliximab provided.
Problems with Infliximab
Reported side effects include fever, rashes, and bacterial infections.
Administration is via the intravenous route, which can be challenging.
Adalimumab (Humira) may be a more suitable choice for some patients due to its subcutaneous injection route.
Adalimumab
Definition:
TNF-inhibitor monoclonal antibody.
Binds to TNF-α to reduce the inflammatory response.
Produced through hybridoma technology with recombinant techniques using CHO cells.
Market and Sales:
Marketed as Humira, costing approximately $3,100 per month.
Achieved global sales of $13 billion in 2014.
The first adalimumab biosimilar launched at a price of $200 in 2014.
U.S. patent expired in 2016.
Summary
Overview of antibodies, their production, and case studies involving various monoclonal antibodies.
Emphasis on therapeutic applications and advances in biotechnology related to monoclonal antibodies.