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
What is the therapeutic application of Anti-CD20 mAb, rituximab?
- rheumatoid arthritis and many types of B lymphoma
- CD20: a surface protein with no known ligand. Function unclear - may act as a Ca+ channel
- all mature B cells express CD20 (not plasma cells)
- Rituximab depletes all CD20+ B cells (transiently)
- First trials (‘97, ‘98) on non-responding B lymphoma patients: 50% partial or complete remission
- because it doesn’t kill plasma cells you are left with all the antibody you had prior to the treatment, for most adults that is sufficient to keep us healthy and safe from most of the things that we encounter
- in Foll NHL and DLBCL about a doubling of survival rate
- doesn’t works so well in CLL - easier for these cells to produce CD20 escape variants
- turns out to be helpful in anti-TNF-resistant RA
What are T cell activating mAbs?
- used to “rev-up” T cells for immune and anti-tumour therapies
- first therapeutic mAb, anti-CD3
- Later, the super-agonist anti-CD28
- Early trials with anti-CD28 caused life-threatening complications in trial volunteers
- Global T cell activation lead to a “cytokine storm” causing severe tissue damage, clotting and organ failure
- error in dosing rate
What is Ipilimumab?
- Ipilimumab (anti-CTLA4) blocks a negative receptor on T cells, promoting activation efficacy in metastatic melanoma
- T cell activation requires 2 signals (TCR and CD28)
- After activation, CTLA4 is expressed on surface to inhibit further activation
- Ipilimumab binds to CTLA4 and blocks its negative role, enhancing T cell activation
- promotes overall survival for patients with severe metastatic melanoma
- 1 year survival is 46% vs 25%
- 2 year is 24% vs 14%
- costs $120,000 for a single course
What are the limitations of mAbs?
- some adverse reactions have been reported
- unpredictable effects: the Anti-CD28 disaster
- Cardiotoxicity
- Infections, when target = immune cells/molecules
- acute anaphylaxis
- generation of anti-mAb antibodies, so loss of efficacy
- modifying both the mAbs and the protocols are aimed at minimising these problems
What are chimeric antigen receptors (CARs)?
- grafting antibody specificity onto T cell activation molecules
- CARs include a T cell activation domain and an antigen recognition domain (Vh/Vl)
- fuses the antigen recognition specificity and affinity of antibody with cytotoxicity of T cells
- 28 patients treated in total with either CLL or follicular lymphoma (FL)
- Acute toxicities associated with elevated serum levels of inflammatory cytokines were noted in trials
How was Anti-CD19 CAR T cell therapy carried out?
- collect white blood cells for T cell activation and introduction of CAR gene vector
- Deplete patients lymphocytes with chemotherapy
- Re-infuse modified patient T cells
What have been some of the results regarding eradication of B cell cancers by CAR T-cell therapy?
- FACS of bone marrow before treatment, all CD19+ cells are CD5+, CLL phenotype
- Blood B cells, most are CLL, eliminated after treatment, assay at 14 months
A) Follicular lymphoma in bone marrow (CD19+)
B) 14 weeks after CAR T cell, no CD19+ cells in marrow, tumour or normal
What are orphan drugs e.g. Anti-IL1 beta?
- IL-1beta is a pro-inflammatory cytokine that drives inflammation and causes systemic signs of inflammation, such as fever, pain and in chronic inflammation, tissue damage
- monoclonal anti-IL1beta acts by ligand binding and blockade (like anti-TNG and anti-VEGFA)
- Currently licensed for a rare disorder: cryopyrin-associated periodic syndromes (CAPS) - a group of rare inherited auto-inflammatory conditions
- -> uncontrolled inflammation in multiple parts of the body starting in the newborn period
- recurrent rash/blistering, fever/chills, joint pain, fatigue, and eye pain/redness
- deafness, systemic amyloidosis (protein accumulation in tissues and organs, such as the kidneys), central nervous system disabilities (mental retardation and vision loss), and substantial joint and bone deformities
severe diseases have lower thresholds for using new drugs
can then be used in off label trials
also in trials for other inflammatory diseases:
- juvenile idiopathic arthritis (JIA), the most common and severe form of persistent arthritis in children
- type 2 (late onset) diabetes
- gout - VERY painful conditions of uric acid crystal accumulation in joints, characterised by recurrent attacks of acute inflammatory arthritis
- other chronic inflammatory conditions
-
lecture 131
-
lecture 229
-
lecture 335
-
Lecture 437
-
lecture 529
-
lecture 623
-
lecture 725
-
lecture 820
-
lecture 917
-
lecture 1026
-
lecture 1119
-
lecture 1224
-
lecture 1339
-
lecture 1433
-
lecture 1524
-
lecture 1621
-
lecture 1719
-
lecture 1830
-
lecture 1918
-
lecture 2038
-
lecture 2131
-
lecture 2216
-
lecture 2333
-
lecture 2418
-
lecture 2536
-
lecture 2645
-
lecture 2730
-
lecture 2824
-
lecture 2926
-
lecture 3030
-
lecture 3129
-
lecture 3225
-
lecture 3326
-
lecture 3434
-
lecture 3534
-
lecture 3627
