Biologic Drug Development

Question 1

What is the relevance of antibodies in the pharmaceutical industry?

Answer 1

Antibodies are versatile drugs and dominate the pharmaceutical market (by revenue)

They are expensive for consumers, but are easier for manufacturers to turn a profit

Question 2

What is the central dogma of molecular biology?

Answer 2

DNA –(transcription)–> RNA –(post-transcription modification)–>mRNA–(translation via ribosome)–>Protein

Question 3

What happens to hydrophobic or hydrophillic amino acids in a polypeptide chain?

Answer 3

Hydrophobic amino acids are internal, while hydrophillic amino acids are external

This type of organization results in globular structures

Question 4

What are the four levels of protein structure?

Answer 4

1. Primary structure
2. Secondary structure
3. Tertiary structure
4. Quaternary structure

Question 5

In general, how do antibodies work?

Answer 5

Antibodies can target individual proteins and modulate structure and function

Binding is mediated by CDR loops

Question 6

What are the components of humoral immunity?

Answer 6

B lymphocytes are the main cells involved in humoral immunity

B cell (with antibodies on cell surface and activated by helper T-cells)

Antibody-secreting B cell (release free antibodies)

Question 7

What are the components of cell-mediated immunity?

Answer 7

T-cells bind to antigen-presenting cells and either release cytokines or kill infected cells

Question 8

What is the basic structure of antibodies?

Answer 8

Heterotetramer with two identical light chains and heavy chains.

Fab portion is important for antigen binding

Question 9

What are some effector functions of antibodies?

Answer 9

• Inactivates, prevents binding of pathogens and toxins to cells
• Induce phagocytosis or lysis
• NK cell-induced apoptosis (useful in cancer)
• Indice degranulation

Question 10

What are the different types of anti-cancer antibodies?

Answer 10

1. Tumour-specific IgG
2. Angiogenesis inhibition
3. Checkpoint blockade
4. Radioimmunotherapy
5. Antibody-drug conjugate therapy
6. Bispecific antibody (BiTE therapy)
7. CAR T-cells

review slide 12

Question 11

Why are antibodies specific to a specific antigen?

Answer 11

Antigen and antibody binding sites need to be structurally and chemically compatible

CDR loop confirmations enables binding to diverse antigens

Question 12

How is antibody diversity generated?

Answer 12

Exon mix and match in the IgH locus results in the diversity of CDR loop confirmations

see slide 15

Question 13

In geneal, how are antibodies manufactured?

Answer 13

• An antigen is injected into mouse
• Mouse creates antibodies in B cells
• These antigen-producing B cells are fused with a tumour cell to generate a viable hybridoma
• These hybridomas will contnually generate monoclonal antibodies against the original antigen

Question 14

What is a major issue of murine (sourced from mice) antibodies?

Answer 14

They are immunogenic

Question 15

Are humanized antibodies also immunogenic?

Answer 15

There will be a reduced immune attack and foriegn body detection responses with humanized aantibodies vs. murine antibodies

Question 16

How does Xenomouse (Trangenic mice) technology work?

Answer 16

Antibody genes in mice are replaced with human genes

Subsequent human antibodies are inserted into B cells and fused with tumour cells to form hybridomas

The hybridomas produce fully human monoclonal antibodies

Question 17

What is phage display?

Answer 17

M13 bacteriophage (antibodies are expressed on the phage surface as fusion to coat proteins)

Direct site mutagenesis is used to make antibody variants

Libraries of 10^10 variants can be generated and screened

Question 18

How does a naive antibody library respond to a novel antigen?

Answer 18

Trial and error process of testing native antibodies against antigens

Question 19

What are the four methods for making antibodies?

Answer 19

1. Mouse hybridoma
2. Phage display
3. Transgenic mouse
4. Single B cell

slide 22

Question 20

What are the types of antibiotic fragments used in phage-display?

Answer 20

• Full IgG
• Fab
• scFv
• VH/VHH

slide 27

Question 21

What is the favoured antibody type for solid tumour therapies?

Answer 21

scFV has improved penetration into solid tumours

Question 22

Do antibody-based drugs have a lower success rate for FDA approval?

Answer 22

No, success rates are almost 3x compared to small molecules (23% vs. 7%)

Question 23

What is the most common type of approved antibody?

Answer 23

Canonical antibody (full IgE molecule alone)

Question 24

Are most antibodies designed to target unique targets?

Answer 24

No, 45% of antibodies are targeted against the top ten targets

PD1/PDL1, CD20, TNF, HER2 are some common targets for antibodies

Question 25

What is an examples of a canonical antibody?

Answer 25

Bevacizumab Humanized murine mAb targeting VEGF A (inhibition of angiogenesis)

Question 26

How was bevacizumab humanized?

Answer 26

CDR regions from murine antibody were grafted onro a human Fab fragment sequence

Question 27

What is the advantage of engineering Fc fragments for antibodies?

Answer 27

They help the antibody evade degradation slide 35

Question 28

What is the role of a linker in antibody drug conjugates?

Answer 28

They connect the antibody to the payload Payload release is dependent on the linker and it can be triggered by tumour specific factors

Question 29

What is the payload in antibody drug conjugates?

Answer 29

- Can be active drug or radioactive material - Ensures low impact on non-targeted tissues (narrow window)

Question 30

What are some functions of bispecific antibodies?

Answer 30

1. Bridging cells (in-trans): T-cell and antigen brought into proximity 2. Receptor inhibition (in cis): prevent HER2 dimerization 3. Receptor activation (in cis): bring together two cell surface proteins and enhance activation 4. Co-factor mimetic 5. Piggybacking (use receptors for other drugs, to help import across BBB) slide 39

Question 31

What is a quadroma?

Answer 31

It is the viable result of the fusion of two hybridomas From this process, a bispecific antibody can be generated (quadroma contains CDR regions for different antibodies)

Question 32

What is knob-in-holes technology in the context of bispecific antibodies?

Answer 32

These mutations prevent/reduce homodimerization of heavy chain due to steric hindrance (light chain cross over can still happen) This increases the proportion of desired bispecific antibodies

Question 33

What is the use of Cross-Mab technology in the context of bispecific antibodies?

Answer 33

Light chain crossover is prevented by fusing the light chain with the heavy chain