Foundations in Pharmacology PHRM2005 Antibodies Part B Lecture Notes PDF

Foundations in Pharmacology PHRM2005 Dr Ricky Lareu Antibodies Part B 1 Reading Immunology for Pharmacy, DK Flaherty, 2012 ▪ Chapters 13 Curtin library online recourse – chapters are also downloadable as PDF 2 Polyclonal Vs Monoclonal Antibodies POLYCLONAL Antibodies that are collected from sera of...

Foundations in Pharmacology PHRM2005 Dr Ricky Lareu Antibodies Part B 1 Reading Immunology for Pharmacy, DK Flaherty, 2012 ▪ Chapters 13 Curtin library online recourse – chapters are also downloadable as PDF 2 Polyclonal Vs Monoclonal Antibodies POLYCLONAL Antibodies that are collected from sera of immunized animals Recognize multiple antigenic sites of injected antigen Produced by many plasma cells MONOCLONAL Individual B lymphocyte hybridoma is cloned and cultured. Secreted antibodies are collected from culture media Recognize only one antigenic site of injected antigen Produced by one type of plasma cell 3 Polyclonal Antibodies Produced by immunising an animal with the appropriate antigen. The immunized animal’s serum is collected. Antibodies can then be purified from the serum. Since one antigen induces the production of many antibodies the result is a ‘polyclonal’ mixture of antibodies. Polyclonal antibodies are much less expensive to produce than monoclonal antibodies. https://www.goldbio.com/articles/article/an-overview-of-polyclonal-and-monoclonal-antibodies-theirdifferences-and-how-to-choose Monoclonal Antibodies Monoclonal Antibodies (mAb) - identical antibodies produced in large quantities by an immortalized hybridoma cell line. Benefits: identical antibodies available in unlimited quantity; easily purified Drawbacks: may not be useful for all techniques; a limited number of species (mouse, rat, hamster, rabbit) Much more complicated and expensive to produce than polyclonal antibodies. One polyclonal preparation Many monoclonal preparations 5 Monoclonal Antibody Production Process begins by immunizing an animal (commonly a mouse) with an antigen - minimum 2 injections. The animal’s spleen is removed. B-cells are fused with myeloma cells (B cell cancer) resulting in hybridomas. Note, selection media only allows hybridomas to grow. Hybridomas are serially-diluted and cultured. Hybridoma growing solution is screened to find those producing antibodies to the antigen with which they were immunized. Each hybridoma cell is derived from one B-cell so the antibodies that a clonal population of hybridoma cells produces are monoclonal antibodies. ▪ Monoclonal antibodies recognize one epitope only. 6 Monoclonal Antibody Production cont. Hybridoma: the B cell fusion with a myeloma cell ▪ Produced by using polyethylene glycol (PEG) to fuse cells o The myeloma cells provide: immortal growth properties o The B cells provide: the genetic information for the synthesis of specific antibody Selected by using HAT medium (hypoxanthine, aminopterin, and thymidine) o Myeloma cells are unable to grow (due to a mutation) o B cells are able to survive, but can not live for extended periods o Therefore, hybridoma cells can survive and grow and produce antibodies. 7 Humanised Monoclonal Antibodies Production of mAb in the lab resolved the problem of limited reagents. BUT as the monoclonal Ab is from mouse → foreign to the human immune system: ▪ repeated use will generate a response with neutralising Ab, reduce the effectiveness of treatment Chimeric mAb Humanised mAb ▪ risk of developing serum sickness 33% mouse 5% mouse ▪ The solution is to humanise the monoclonal Ab Chimeric and humanised mAb are generated: ▪ Hybridomas are produced as normal ▪ The mouse variable region genes/CRDs are cloned and combined with human genes to complete the Ab structure → Expressed in bacteria Human monoclonal Ab: start with a transgenic mouse with human genes for the IgG regions → hybridomas are produced as normal CRD, complementarity-determining regions 8 Monoclonal Antibodies for Therapy Monoclonals offer exquisite specificity and affinity for both secreted and cell-surface targets. Different formats of antibody can be used to mop up circulating proteins: Monoclonals used to block signalling pathways, drive the internalization and degradation of cell-surface receptors, deliver small-molecule payloads to specific cell types (immunotoxins, radioimmunotherapy), recruit immune cells to cancer cells, and more. Medicinal chemists can take years to identify small molecules with activity against a given target, antibody discovery can take a matter of months. Antibodies are twice as likely to succeed in trials as small molecules. ▪ Their approval rate is higher than non-monoclonal antibody drugs FDA https://www.lachmanconsultants.com/2019/07/approval-rate-fortherapeutic-monoclonal-antibodies-continues-to-grow/ 9 Immunoassays - COVID-19 Rapid Antigen Test (RAT) (a) RAT is a lateral flow immunoassay that qualitatively detects the presence of viral proteins. Uses monoclonal antibodies (mAb) to detect SARSCoV-2 nucleocapsid (N) in secretions. A nasal or nasopharyngeal sample is mixed with an extraction solution. (b) Drops are placed in the cassette well/inlet. (c) Capillary action moves solution and proteins through to the conjugate pad where the viral proteins interact with anti-N mAb and control (C) protein with anti-C mAb. Antibodies are labelled with a coloured bead (d) Ab/Ab-protein complexes move over test and control lines, where: Ab-N interacts with more anti-N mAb fixed to the material → forms coloured line. The same for the C-mAb complex - control confirms the test worked. False positives can occur with asymptomatic individuals. Immunotoxins Immunotoxins are monoclonal antibodies linked with a toxin Regular chemotherapy and radiation therapy that is delivered to all cells/generalised areas, and patients significant side effects eg hair loss, nausea, low energy levels. Immunotoxins are very specific and target a single cell type/tissues Commonly used toxins: From bacteria that prevent protein synthesis: Diphtheria toxin (DT), Pseudomonas exotoxin (PE) From plant that inactivate ribosomes → stop protein production: gelonin, pokeweed antiviral protein, dodecandron; a chain for ricin (RTA) For these toxins to act, the immunotoxin must be internalised therefore the linker is stable extracellularly but liable intracellularly for toxin to enter the cytosol. https://www.researchgate.net/figure/Immunotoxin-targeting-in-leukaemia-a-Immunotoxins-are-therapeutic-molecules-composed_fig1_26855626 11 Radioimmunotherapy Radioimmunotherapy (RIT) combines radiation therapy and immunotherapy. Monoclonal antibodies are conjugated with radioactive materials, called radiotracers. When injected into your bloodstream, they bind to cancer cells and deliver a high dose of radiation directly to the tumor Used to treat non-Hodgkin lymphoma and other types of cancer. 12 Examples of monoclonals used to treat disease. Infliximab: against TNF-a, used for various disorders including Rheumatoid arthritis Adalimumab: Also against TNF-a, similar uses to Infliximab + psoriasis Omalizumab: against IgE – used to combat severe allergies Rituximab: Against CD20 on b-lymphocytes – used for Chronic Lymphocytic Leukemia and non-Hodgkin Lymphoma. Basiliximab: Against IL-2 receptor a-chain on T-cells – used in immunosuppression Summary Polyclonal antibodies are a mixture of antibodies detecting many epitopes on an antigen/protein and monoclonal antibodies just detect one epitope on an antigen/ protein. Monoclonal antibodies are produced by hybridomas that are a fusion of a plasma cell (a B cell that produces antibody) and a myeloma cell (immortal cell). To reduce the immunogenicity of mouse monoclonals for human therapy, chimeric and humanised monoclonal antibodies have been developed. The development and approval of monoclonals for therapy is rapidly on the rise. Some applications of monoclonals include diagnostic tests, delivering toxins and radiotracers to target diseased cells. 14