5. Monoclonal antibodies

Question 1

What is a mAb?

Answer 1

Monoclonal antibody: a pure population of Abs with one target specificity made by a clonal population of Ab secreting plasma cells

Question 2

Why do mAbs have great therapeutic potential?

Answer 2

1. Extremely well tolerated: serum >10mg/ml Ig
   * stable, not seen as foreign
2. Long lived: normally half life of several weeks
3. Very specific and selective: binding constants ≥ 1/10^9 M for their targets

Question 3

What are the therapeutic applications of mAbs?

Answer 3

• 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)

Question 4

What are the “Big 5”?

Answer 4

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

Question 5

Idea behind mAb

Answer 5

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

Question 6

What is the “humanisation” of mAbs?

Answer 6

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.

Question 7

Generic recipe to make a monoclonal Ab

Answer 7

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)

Question 8

Advantages and disadvantages of immortalising human memory B cells

Answer 8

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

Question 9

Arming mAbs

Answer 9

1. Naked mAb: activate c’ dependent cytotoxicity
2. 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)
3. Multistep targeting: modified

Question 10

Mechanisms of action of mAbs

Answer 10

1. Ligand blockade
2. Receptor blockade or down-modulation
3. Target cell depletion
   - complement mediated lysis
   - ADCC: Ab-dependent, cell-mediated cytotoxicity
   - FcR-mediated phagocytosis
4. Target cell activation

Question 11

1. Ligand blockade

Answer 11

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”

Question 12

2. Receptor blockade or down-modulation

Answer 12

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

Question 13

3. Depletion I

Answer 13

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

Question 14

3. Depletion II

Answer 14

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.

Question 15

3. Depletion III

Answer 15

mark for phagocytosis!

Fc-dependent phagocytosis and lysosomal degradation of tumour cell

Question 16

Combination therapies

Answer 16

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!

Question 17

4. Target cell activation

Answer 17

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

Question 18

4. Target cell activation

Answer 18

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

Question 19

4. Target cell activation: regulation of T cell activation

Answer 19

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

Question 20

Limitations of mAbs

Answer 20

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

Question 21

Chimeric Antigen Receptors (CARs)

Answer 21

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

Question 22

Anti-CD19 CAR T cell therapy procedure

Answer 22

1. Collect WBCs for T cell activation and introduction of CAR gene vector by transduction with gammaretroviral vector encoding CAR gene
2. Deplete patients lymphocytes with chemotherapy whilst Ex vivo cell processing occurs with result of step 1
3. Re-infuse modified patient T cells (Anti-CD19 CAR cells returned to patient)

Question 23

What is IL1β?

Answer 23

A proinflammatory cytokine that drives inflammation and causes system signs of inflammation, such as fever, pain, and in chronic inflammation, tissue damage.

Question 24

What is the orphan drug, Anti-IL1β?

Answer 24

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)