Antibody drug conjugates

Question 1

Describe the structure of antibodies.

Answer 1

Antibodies are a Y shaped protein that consists of constant regions (stalk of the Y, also known as the Fc region) and the variable regions (v of the Y, known as the Fab region). At the tip of the Fab region is the hypervariable regions that bind to the antigen.

Question 2

What are the differing functions between the Fc and Fab regions?

Answer 2

The Fc region is responsible for interacting with the Fc receptor on innate immune cells or with C1q, the recognition molecule of the complement system and hence initiates downstream signalling pathways.
Fab region is responsible for antigen binding.

Question 3

What are the four types of antibody that can be genetically modified?

Answer 3

Mouse
Chimeric
Humanised
Fully human antibody

Question 4

Describe which genetic modifications a chimeric antibody has undergone.

Answer 4

A chimeric antibody are molecules made up of two different domains. In application to the human mouse antibody, the Fc portion of the human antibody is combined with the hypervariable (antigen binding portion) region of mouse antibodies.

Question 5

What are the purpose of chimeric antibodies?

Answer 5

There were complications with the use of mouse antibody treatment as when used in humans, they would recognise it as foreign material and immune complex would form. This is known as the human anti-mouse antibody response. By switching the non-antigen binding region to human it reduces this adverse effect without alteration to the binding affinity of the antibody.

Question 6

Describe the structure of humanised antibodies.

Answer 6

Humanised antibodies are human antibodies with the exception of key murine sequences (complementary determining regions) in the hypervariable regions derived from mouse antibodies which are responsible for the antigen binding.

Question 7

What is the suffix at the end of drugs that are monoclonal antibodies?

Answer 7

-mab

Question 8

If there is a ‘u’ before the suffix -mab in the drug name, what type of monoclonal antibody is it?

Answer 8

Human

Question 9

If there is a ‘o’ before the suffix -mab in the drug name, what type of monoclonal antibody is it?

Answer 9

Mouse

Question 10

If there is a ‘xi’ before the suffix -mab in the drug name, what type of monoclonal antibody is it?

Answer 10

Chimeric

Question 11

If there is a ‘zu’ before the suffix -mab in the drug name, what type of monoclonal antibody is it?

Answer 11

Humanised

Question 12

If there is a ‘xi-zu’ before the suffix -mab in the drug name, what type of monoclonal antibody is it?

Answer 12

Hybrid of chimeric and humanised

Question 13

Describe the difference between classical and modern chemotherapeutics.

Answer 13

Classical chemotherapies such as anthracyclines, alkylating agents target cells that have increased rate of proliferation, often affecting the cell cycle. Quite simply cells entering the cell cycle more frequently and therefore going to be more affected by a drug preventing proliferation.
However among cancer cells, other more rapidly proliferating cells such as hair follicles, mucosal cells and immune cells are also affected, which can also be targeted by chemotherapies.
Modern chemotherapies however such as tyrosine kinase inhibitors and PARP inhibitors recognise and bind to unique proteins expressed on cancer cells, which distinguishes specifically between cancerous and healthy cells. These chemotherapies additionally identify mutations and over-expressing of proteins also.

Question 14

What are the two types of antibodies that can be used therapeutically?

Answer 14

-Polyclonal antibodies
-Monoclonal antibodies

Question 15

Compare polyclonal vs monoclonal antibodies.

Answer 15

Polyclonal antibodies are a heterogenous mixture of antibodies derived from the immune response of different plasma cells. Whilst the antibodies are not identical they each target the same antigen, but do not target the same epitope, resulting in an introduction of batch to batch variability.

Monoclonal antibodies are produced from clones of a single B-cell parent and therefore identical targeting the same antigen and the same epitope on the antigen, resulting in a decrease in cross-reactivity compared to polyclonal antibodies. These antibodies are produced by B-cell immortalisation by fusion with hybridoma cells, allowing for long-term generation of identical monoclonal antibodies.

Question 16

What is an epitope?

Answer 16

A protein sequence typically 4-6 amino acids in length.

Question 17

For which purposes are you more likely to use polyclonal or monoclonal antibodies?

Answer 17

Polyclonal antibodies are more useful for research applications due to their high affinity to the target antigen, are more sensitive compared to monoclonal antibodies due to their biophysical diversity. Furthermore they have relative tolerance to minor changes exhibited by the target antigen due to targeting multiple epitopes.

Monoclonal antibodies due to targeting a specific epitope are used more widely therapeutically due to no batch-batch variability as seen with polyclonal antibodies and has much more limited cross reactivity.

Question 18

Describe the advantages of polyclonal antibodies.

Answer 18

Inexpensive and manufacturing process is rapid (less than 3 months)
Higher overall antibody affinity against the antigen due to the recognition of multiple epitopes.
Have a high sensitivity for detecting low-quantity proteins.
High ability to capture the target protein (recommended as the capture antibody in a sandwich ELISA).
Antibody affinity results in quicker binding to the target antigen (recommended for assays that require quick capture of the protein; e.g., IP or ChIP).
Superior for use in detecting a native protein.
Easy to couple with antibody labels and rather unlikely to affect binding capability

Question 19

Describe the disadvantages of polyclonal antibodies.

Answer 19

Batch-to-batch variability as produced in different animals at different times.
High chance of cross-reactivity due to a recognition of multiple epitopes (affinity purified antibodies display a minimum cross-reactivity).

Question 20

Describe the advantages of monoclonal antibodies.

Answer 20

Batch-to-batch reproducibility (high homogeneity).
Possibility to produce large quantities of identical antibody (an advantage for diagnostic manufacturing and therapeutic drug development).
High specificity to a single epitope reflected in low cross-reactivity.
More sensitive in assays requiring quantification of the protein levels.
Low background noise

Question 21

Describe the disadvantages of monoclonal antibodies.

Answer 21

More expensive to produce due to necessity to produce a pool of several monoclonal antibodies.
Requires significantly more time to produce and develop the hybridized clone (+/- 6 months).
More susceptible to binding changes when labeled (e.g. with a chromogen or a fluorophore).

Question 22

State some monoclonal antibodies on the market and their indication.

Answer 22

• Adalimumab, brand name Humira(Anti-TNF) used for Rheumatoid arthritis
• Bevacizumab, brand name Avastin (Anti-VEGF) used for Colorectal cancer
  -Infliximab, brand name Remicade (Anti-TNF) used for Crohns disease
• Pembrolizumab, brand name Keytruda (Anti-PD-1) used for Metastatic melanoma
• Rituximab, brand name Rituxan (Anti-CD20) used for Non-Hodgkins Lymphona

Question 23

Why can’t antibodies be used for all tumours?

Answer 23

Whilst initially using antibodies appeared a promising strategy for identifying and target cancer cells, it can’t be used in the treatment of all cancers. Ideally cancers should be treated through identifying and targeting specific mutations that the tumours express, however whilst antibodies are effective as a steric block if the mutated protein is expressed extracellularly. However if the mutation is intracellularly antibodies are unable to target these mutations due to rapid degradation.

Question 24

What is the biological target of Trastuzumab?

Answer 24

Human epidermal growth factor receptor 2; the protein is found to be over-expressed in 20% of all breast cancers.

Question 25

Explain the mechanism of action of Trastuzumab.

Answer 25

Trastuzumab binds to an extracellular domain of the human epidermal growth factor receptor 2, a tyrosine kinase receptor. As a photo-oncogene gene amplification can result in over-expression of this protein, resulting in sustained proliferation.

The HER2 receptor is activated by the formation of homodimers or heterodimers with other EGFR proteins, leading to dimerization and autophosphorylation and/or transphosphorylation of specific tyrosine residues in EGFR intracellular domains. This then activates signalling cascades including Ras/Raf/mitogen-activated protein kinase (MAPK), the phosphoinositide 3-kinase/Akt, and the phospholipase Cγ (PLCγ)/protein kinase C (PKC) pathways promoting cell cycle progression. Trastuzumab works by inhibiting dimerization, down-modulating the signalling cascade leading to cell cycle arrest, additionally also recruits immune cells such as NK cells to the tumour with cells over-expressing HER2.

Question 26

Describe the screening process required before Trastuzumab can be prescribed.

Answer 26

Usually a immunohistochemistry test is completed, which involves comparison of tumour cells alongside healthy tissue. The technique is able to recognise antibody (often polyclonal antibodies) and antigen binding to recognise presence of a certain antigen - in this case HER2. 0 or 1 is HER2 negative, 2 is classified as borderline and 3 or more is considered positive.

If borderline by immunohistochemistry test, a fluorescence in situ hybridization) test checks the DNA of your cancer cells for extra copies of the HER2 gene by utilisation of a fluorescent probe.

Chromogenic in situ hybridization, is a process in which a labeled complementary DNA or RNA strand is used to localize a specific DNA or RNA sequence in a tissue specimen. CISH methodology may be used to evaluate gene amplification, gene deletion, chromosome translocation, and chromosome number, therefore detecting HER2 gene amplification.

Question 27

Describe cancer characteristics associated with HER2 (+) breast cancer.

Answer 27

HER2 (+) cancer cells have:
Increased risk of metastasis
Increased resistance to chemotherapy
Lower levels of estrogen and progesterone
Reduced prognosis as there is:
68% chance of five year survival compared with 74-88% chance depending on stage

Question 28

What is the rationale of antibody drug conjugates?

Answer 28

Antibodies, specifically monoclonal antibodies which are most commonly used therapeutically, have high specificity to a particular expressed epitope on an antigen. This can be an antigen on a tumour cell. This can be exploited as a method of drug delivery to ensure more targeted cytotoxic deposition at the tumour site, avoiding deposition into healthy tissue.

Question 29

What was the name of the first antibody drug conjugate? What was it licensed for?

Answer 29

Gemtuzumab - Ozogamicin, also known under the brand name Myotarg.
It is licensed to treat acute myeloid leukemia.

Question 30

Describe the four key aspects of X (Antibody, Linker-Antibody attachment, Linker-drug attachment, Drug)