Antibody based

Question 1

Explain a Fab (Fragment antigen-binding) fragment.

Answer 1

Fab is a region on an antibody that binds to antigens. It is composed of one constant and one variable domain of each of the heavy and the light chain. It is the variable part of the antibody.

Question 2

What differs polyclonal from monoclonal antiboides?

Answer 2

Polyclonal:
- Origin in many different antibody-producing B cells (immune cells)
- Have the affinity for the same antigen but different epitopes (parts of the target antigen)
- Recognize the antigen with different binding strength (affinity)
- Can bind many different epitopes with different functionalities

Monoclonal:
- Origin in a single antibody-producing B cell
- Recognize one part (epitope) of the target antigen
- Antibodies recognize the antigen with a single, defined binding strength (affinity)

Question 3

What are the risks of polyclonal antibodies?

Answer 3

• There is a risk of viral contamination of human antibodies derived from human subjects.
• There is a risk of immune response against animal-derived antibodies.

Question 4

What are the risks of polyclonal and monoclonal antibodies?

Answer 4

Polyclonal:
- There is a risk of viral contamination of human antibodies derived from human subjects.
- There is a risk of immune response against animal-derived antibodies.

Monoclonal:
- May be of animal or human origin and the latter is likely to not induce substantial immune response against the antibody during treatment.

Question 5

What are antibody-druggable targets?

Answer 5

Antigens exposed to the extracellular environment:
- Membrane proteins/receptors
- Extracellular matrix
- Soluble mediators (e.g. cytokines, chemokines)
- Viral and bacterial surface proteins

Question 6

What are the steps to get 1-3 clones out of thousands?

Answer 6

1. Antibody/antibody fragment development
2. Primary screening
3. Sequencing
4. Affinity determination
5. Specificity screening assay
   …
6. Epitope mapping
   …
7. Animal model testing (in vivo)

Question 7

Methods in primary screening phase?

Answer 7

• Immunochemical assays (e.g. ELISA) on recombinant antigen
• High throughput flow cytometry screening assay
• Next Generation Sequencing (NGS)

Question 8

What is cross-reactivity testing and why is it required?

Answer 8

Cross-reactivity testing identifies clones with a problematic behavior and it is highly advantageous to do on tests in preclinical animal in vivo model. May be required for functional validation and assessment of side effects such as on-target and off-target toxicity.