5. Monoclonal antibodies Flashcards
Monoclonal Abs as therapeutics: -how they are made -mechanisms of action -therapeutic applications -limitations as therapeutics -experimental mAb-based therapies
What is a mAb?
Monoclonal antibody: a pure population of Abs with one target specificity made by a clonal population of Ab secreting plasma cells
Why do mAbs have great therapeutic potential?
- Extremely well tolerated: serum >10mg/ml Ig
* stable, not seen as foreign - Long lived: normally half life of several weeks
- Very specific and selective: binding constants ≥ 1/10^9 M for their targets
What are the therapeutic applications of mAbs?
- malignancies
- autoimmune conditions (these 2 make up 80% of market)
- transplant rejection (anti-T cell)
- infectious disease (respiratory syncytial virus)
- cardiovascular disease (glycoprotein IIb/IIIa)
- macular degeneration (VEGF-A)
- paroxysmal nocturnal hemoglobinuria (complement C5)
What are the “Big 5”?
Anti-TNF: infliximab (Remicade), adalimumab (Humira)
-rheumatoid arthritis, Crohn’s disease, psoriasis
Anti-HER2: trastuzumab (Herceptin)
-cancer, especially breast cancer
Anti-VEFGA: bevacizumab (Avastin)
-anti-angiogenesis, cancer therapy
Anti-CD20: rituximab (Rituxan)
-rheumatoid arthritis and non-Hodgkin’s lymphoma
Idea behind mAb
Fusion of B cell making specific Ab to a cell with continual growth, and therefore transferring properties of immortality to a specific B cell.
ie. B cell with myeloma cells to form hybridomas, which are cultured and harvested
What is the “humanisation” of mAbs?
Made with human sequences replacing mouse sequences where applicable in vitro to avoid anti-mAb immune reaction in the patient
Initial first generation mAbs lacked some important effector functions in humans because their constant region domains were mouse-derived
in vitro technology using molecular biology or can make mAbs in transgenic mice whose Ig genes (some or all: constant regions only or V & C) have been replaced with human Ig genes to make fully human Abs
Alternatively, can make mAbs from immune humans.
Generic recipe to make a monoclonal Ab
Binding specificity + function + immortality
Using human memory cells:
Memory B cells of humans who have survived infection are high affinity & switched to IgG
+ Infection with virus that immortalises B cells (EBV/glandular fever)
Advantages and disadvantages of immortalising human memory B cells
Adv: not rejected by patients, captures Ab specificities that were effective in protecting the donor from past infection
Disadv: limited to foreign specificities
*B cells that reflect the donor’s infection history
**used for prophylaxis
Arming mAbs
- Naked mAb: activate c’ dependent cytotoxicity
- Immunoconjugates: modified to improve therapeutic value, coupling with toxins, enzymes, liposome (with toxins inside) and targeted to specific parts of body, can have hybrid Ab with 2 specificities (use as bridge/linking agent)
- Multistep targeting: modified
Mechanisms of action of mAbs
- Ligand blockade
- Receptor blockade or down-modulation
- Target cell depletion
- complement mediated lysis
- ADCC: Ab-dependent, cell-mediated cytotoxicity
- FcR-mediated phagocytosis - Target cell activation
- Ligand blockade
Anti-TNFα
Acts on TNF, binding to it to prevent TNF binding to its receptor and triggering inflammation. Inflammation begets more inflammation, hence by short circuiting this process you neutralise the action of TNF and kill or render the cell inert.
Anti-VEGFA
blocks formation of new blood vessels so large tumour masses cannot grow at new sites
Anti-RANKL
prevent bone resorption in MM and menopause “Denosunab”
- Receptor blockade or down-modulation
Prevent receptor from binding ligand/prevents change of receptor to correct shape/conformation.
OR
Can mimic ligand, receptor taken into cell and destroyed without activating cell/signal
Anti-HER2
HER2 is a member of the epidermal growth factor receptor family (drives breast cancer prolif.), signals through cytoplasmic tyrosine kinase domain when it sees its ligand, neuregulin.
-internalised into cell, stops signaling
- Depletion I
complement mediated lysis
Abs binding activate c’ cascade which forms membrane attack complex (MAC) that triggers normal imm response to kill cell by drilling holes in the cell
ie. Anti-CD20, rituximab
- Depletion II
Antibody dependent cell mediated cytotoxicity (ADCC)
Antibody coated tumour cell
Coat so that specialised cells (ie. NK cells) recognise these Ab coated cells and release perforin and granzymes to kill cell.
- Depletion III
mark for phagocytosis!
Fc-dependent phagocytosis and lysosomal degradation of tumour cell
Combination therapies
Rituximab improves survival, beneficial, but rarely a cure. Least effective in CLL, easier for these to express CD20 variants –> no longer sensitive to Ab.
Rituxan also helpful in anti-TNF-resistant RA
(50% of RA patients respond to anti-TNF, and 50% of the non responsive are responsive to rituximab)
Rituximab now widely used!
- Target cell activation
Cross link T cell receptor and drive all T cells crazy to release lots of cytokines irrespective of specificity, therefore improving the activity of some tumour-specific T cells.
- used to “rev” up T cells for immune and anti-tumour therapies
- 1st therapeutic mAb, anti-CD3
- later super-agonist anti-CD28
- **error in dosing rate caused life-threatening complications in trial volunteers, global T cell activation lead to “cytokine storm” causing severe tissue damage, clotting, organ failure
- Target cell activation
Cross link T cell receptor and drive all T cells crazy to release lots of cytokines irrespective of specificity, therefore improving the activity of some tumour-specific T cells.
- used to “rev” up T cells for immune and anti-tumour therapies
- 1st therapeutic mAb, anti-CD3
- later super-agonist anti-CD28
- **error in dosing rate caused life-threatening complications in trial volunteers, global T cell activation lead to “cytokine storm” causing severe tissue damage, clotting, organ failure
- Target cell activation: regulation of T cell activation
T cell activation requires 2 signals (TCR & 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 activation efficacy in metastatic melanoma
and anti-CTLA4 promotes overall survival for these patients
Limitations of mAbs
Some adverse reactions have been reported
- unpredictable effects: the anti-CD28 disaster
- cardiotoxicity: given over extended time can lead to heart failure
- infections, when target = immune cells/molecules
- acute anaphylaxis: imm reaction to Abs, repeated reaction = anaphylaxis
- generation of anti-mAb Abs so loss of efficacy
Modifying both the mAbs and the protocols are aimed at minimising these problems
Chimeric Antigen Receptors (CARs)
CARs include a T cell activation domain and an Ag recognition domain (Vh/Vl)
Fusion of Ag recognition specificty and affinity of Ab with cytotoxicity of T cells
-currently 28 patients treated (CLL & FL) with acute toxicities associated with elevated serum levels of inflam cytokines noted in trials
Anti-CD19 CAR T cell therapy procedure
- Collect WBCs for T cell activation and introduction of CAR gene vector by transduction with gammaretroviral vector encoding CAR gene
- Deplete patients lymphocytes with chemotherapy whilst Ex vivo cell processing occurs with result of step 1
- Re-infuse modified patient T cells (Anti-CD19 CAR cells returned to patient)
What is IL1β?
A proinflammatory cytokine that drives inflammation and causes system signs of inflammation, such as fever, pain, and in chronic inflammation, tissue damage.
What is the orphan drug, Anti-IL1β?
Monoclonal anti-IL1β acts by ligand binding and blockade.
Currently licensed for a rare disorder: Cryopyrin-associated periodic syndromes (CAPS) - a rare group of inherited auto-inflam conditions
Uncontrolled inflammation occurs in multiple parts of body starting in newborn period. 2-3 cases worldwide
Produce name = Canakinumab
well tolerated, one shot, no side effects, long lasting
now in trial for other inflamm diseases (JIA, Type II diabetes, gout)
