Monoclonal antibodies, Cancer immunotherapy and molecular imaging Flashcards
Monoclonal antibodies
Antibodies that are made by identical immune cells in culture. All monoclonal antibodies bind to the same unique epitope - can differentiate even if the antigen itself is conserved. Antibodies identified that bind to epitopes specific to individual antigens
Polyclonal Antibodies
Antigens carry many different epitopes so if you inject an animal with a single antigen, they’ll produce a mixture of antibodies made by a different clone of B cells
Hybridoma technology
George Kohler and Cesar Milstein at the MRC in Cambridge in 1975. Nobel Prize in Physiology or Medicine in 1984. Identification of an antibody with a pre-defined specificity. Fusion of a B cell (Ig+ with finite lifespan) and a myeloma cell (Ig- with inifinte lifespan). Create HYBRIDOMA = infinite lifespan producing monoclonal antibodies.
Hybridoma production
Challenge a mouse with an antigen of choice. Test it’s serum to see if it is producing antibodies. If yes, euthanise it and dissect it’s spleen out. Rupture spleen - many splenocytes producing antibodies specific to antigen. Fuse these splenocytes with myeloma cells and plate in 96-wells. Bathe in HAT medium to kill the unfused myeloma cells, the unfused B cells will die naturally, leaving isolated hybridomas. Dilute each well down to extinction i.e. only one hybridoma per well - divide within 12 hours - one clone of specific hybridoma producing a specific monoclonal antibody. Can store in liquid nitrogen - immortal.
HAT medium
Hypoxanthine, aminopterin, thymidine.
Aminopterin is a toxin and blocks the biosynthetic pathway of nucleic acid synthesis.
B cells have thymidine kinase which, with endogenous thymidine, allows the synthesis of DNA.
B cells have HGPRT (hypoxanthine:guanine phosphoribosyl transferase) which allows the production of RNA with endogenous hypoxanthine.
Myeloma cells do not have HGPRT and so aren’t able to use this salvage pathway to create DNA/RNA –> die.
Hybridoma has HGPRT from B cell so survives HAT with the immortality of myeloma cell.
B cells die naturally within a few days.
HGPRT
hypoxanthine:guanine phosphoribosyl transferase.
Problem with hybridoma technology
When you immunise an animal, they tend to elicit the immune response against the immunodominant epitope. You might want a B cell that doesn’t recognise this epitope.
Phage display
Peptides/proteins are expressed along the bacteriophage as a fusion with a coat protein.
Fused proteins are displayd on the surface of the virion whilst the DNA encoding fusion resides within the virion.
M13 phage
Filamentous phage. Multiple copies of diffreent proteins along it’s lengh or at different termini. of pVIII protein, has around 2700 copies. pIII has around 5 copies.
Antibody combinatorial libraries
Get a B cell source (either naive or immunised) and extract the mRNA the encodes antibody proteins. Using reverse transcriptase, create cDNA. Use PCR to amplify the V-gene families (Vh and Vl). Assemble these at random to create a range of fragments with varying specificities and affinities. Clone as scFv to pIII protein of phage.
Compare hybridoma and phage display technologies- end result
Hybridoma gives you a full length antibody with full Fc and Fab domains.
Phage display technology gives you fragments of an antibody that do the binding.
Uses of monoclonal antibodies
WIDESPREAD IN DIAGNOSTICS. Diagnostic pathology (in cytology and histology). In vitro diagnostics for pathogens and biomarkers of disease, based on ELISA, PLA and LFA. Affinity purification and characterisation of antigens.
Why do we need to humanidse mouse monoclonal antibodies?
- Immunogenicity - mouse origin so they are xenogeneic and will result in a HAMA response.
- Origin - mouse origin deminishes its ability to elicit mechanisms such as ADCC and complement.
HAMA reaction
Human anti-mouse antibody reaction
ADCC
antibody-dependent cell-mediated cytotoxicity
Humanisation of mABs
disguise as a human antibody as xenoantibodies are seen as foreign by the human immune system.
Human hybridomas
Production of human hybridomas using human B lymphocytes
Chimeric antibody (Fab - mouse, Fc -human)
replacement of constant region of mouse mAB with human antibody
CDR Grafting
Replacement of CDRs of a human Ab with those of a mouse
Transgenic mice
Produce fully humanised mAb in transgenic mice (genetically modified to only produce human immune cells)
how to produce CDR grafted antibody
Get a mouse mAb cell line and extract the mRNA that encodes for the variable domains. Clone within an expression vector (variable domains from mouse and constant regions from human antibody). Put into the cell of a chinese hamster ovary and culture. Will produce a humanised mAb.
Chimeric antibody example
Rituximab (“xi”). Non-hodkin lymphoma. Rituxan.
Humanised antibody example
Trastuzumab (“zu”), Breast cancer. Herceptin
Fully human antibody (phage display) example
Adalimumab (“mu”). Inhibits TNF-alpha signalling. Auto-immune disorders. Humira.
How does the immune system play a role in cancer.
Immunocompromised people are more likely to get cancer. IL-2 is a FDA approved cancer therapy. tumor microenvironment is rich in tumour infiltrating T lymphocytes (TIL)
How is an unfolded protein normally treated in the cell?
Unfolded protein is usually bound to a soluble protein (calreticulin) which blocks its transport from the ER to the Golgi body. As well as MHCI signalling, Calreticulin also moves to the plasma membrane and sends out an “eat me” signals which tells T cells to form killer t cells to eradicate the tumour.
