lecture 6 Flashcards
How do you do CDR grafting (humanisation I)?
- in vitro, using molecular biology
- graft variable region from the mouse onto constant region from human
- just the pieces of DNA that eventually form the antigen binding bits
- effectively grafted the specificity of the mouse bit onto a completely human structure
What is the second method of humanisation?
- making mAbs in transgenic mice whose Ig genes (some or all: C regions only, or V and C) have been replaced with human Ig genes
- so when you recover hybridomas from the mouse it will be making a fully humanised mAb even though from the mouse
How can we by-pass the mouse altogether in the making of mAbs?
- making mAbs from immune humans
- memory B cells of humans who have survived infection are high affinity (binding specificity) and switched to IgG (function) + infection of these cells with a virus that immortalises B cells (e.g. Epstein Barr Virus, EBV) (immortality)
What are the advantages and disadvantages of immortalising human memory B cells?
Advantages
- not rejected by patients
- captures antibody specificities that were EFFECTIVE in protecting the donor from a past infection
Disadvantages
- limited to foreign specificities
How do we use mAbs for passive immunisation of patients?
- give the patient purified, specific and high affinity antibody
- e.g. Sars, H5N1 influenza, HIV or in acute infections where there is not time or capacity to immunise
- theoretically, could produce a specific antibody for any infection experienced during one’s lifetime
How can we modify mAbs for better therapeutic “value”?
- improve potency
- improve functionality
- longer half-life
- tissue distribution
- solubility
- cost….
a lot of things done include coupling specific drugs with the Ab i.e. use it as a delivery system (Ab specific for the thing you are delivering the drug to)
- coupling toxins e.g. Biotinylated radioactive ligand
- coupled with liposomes filled with other things e.g. drugs etc
- enzyme to activate prodrug into active drug
- everything about antibodies is about specificity and targeting
- more adventurous –> Ab made with two different specificities - one for target and one for e.g. killer cell i.e. using it as a bridge/linking agent
What is the normal function of an antibody?
- to induce complement dependent cytotoxicity (i.e. activate the complement cascade and kill cells)
- to trigger other cells to kill the cell to which the antibody is bound - antibody dependent cytotoxicity
What are the mechanisms of action of mAbs?
- ligand blockade
- receptor blockade or down-modulation
- target cell depletion
- complement mediated lysis
- ADCC: antibody-dependent, cell-mediated cytotoxicity
- FcR-mediated phagocytosis - Target cell activation
What is the therapeutic application of Anti-TNF alpha drugs infliximab and adalimumab?
- rheumatoid arthritis, Crohn’s disease (inflammatory bowel disease), psoriasis
- a strong pro-inflammatory cytokine involved in joint destruction
- TNF mAbs bind to TNF and prevents it from bind to its receptor. It is the binding of TNF to its receptor that stimulates the inflammation that causes many autoimmune diseases. This process either kills the inflammatory cell, or renders it inert.
What is the therapeutic application of Anti-VEGFA drug, bevacizumab?
- metastatic colorectal, ling, breast, renal cancer
- anti-angiogenesis agent: blocks the formation of new blood vessels so large tumour masses cannot grow at new sites
- also binds the soluble ligand
- 1-year survival rate: 63% vs 74%
- 2 year survival: 30% vs 45%
What is the therapeutic function of Anti-RANKL mAbs e.g. denosunab?
- prevent bone resorption in MM and menopause
- RANKL, produced by MM cells, stimulate osteoclasts, which leads to the destruction of bone
- helps to treat one of the more debilitating side-effects of MM
- mAb binds RANKL and prevents from binding to receptor
What is the therapeutic application of Anti-HER2 mAb, trastuzumab (Herceptin)?
- cancer, especially breast
- HER2 is a member of the Epidermal Growth Factor Receptor family, signals through cytoplasmic tyrosine kinase domain when it sees ligand, neuregulin
- Anti-HER2 binds to receptor instead of ligand
- Used for Her2+ breast tumours
- slightly less than double time to progression in terms of months when using Herceptin and conventional therapy as opposed to just conventional therapy
What is an example of a mAb whose mechanism of action is depletion via complement mediated lysis?
- Rituximab binds to CD20 on B cells, targeting them for recognition by the IS
- initiates the complement cascade, which creates the membrane attack complex, which drills holes in the cells and kills them
- i.e. an antibody that triggers the normal functions of the immune system to kill the cells to which it binds
What is an example of a mAb whose mechanism of action is depletion via antibody-dependent cell-mediated cytotoxicity (ADCC)?
- coat the target cell (e.g. tumour cell) so that they can then be recognised by other killer cells e.g. NK cells
- NK have special receptors that recognise bound antibody
- they release toxic granules that kill the target cell
- i.e. taking another component of the immune system and giving it specificity and direction to kill the cell to which they are bound
What is the third example of mAbs whose mechanisms of action is depletion?
- Fc-dependent phagocytosis and lysosomal degradation of tumour cell
- targeting cell for phagocytosis of macrophage
