Biologic Drug Development - Antibodies and Recombinant Proteins

Question 1

The best selling type of pharmaceuticals are?

Answer 1

Antibodies

Question 2

How are antibodies made and what do they do? (3)

Answer 2

1. Antibodies are made by B-cells to recognize foreign substances
2. Secreted antibodies neutralize threats
3. Binding is mediated by CDR loops

Question 3

What is the structure of antibodies? (3)

Answer 3

1. Heterotetramer with two identical light chains and heavy chains
2. Fab portion is important for antigen binding. Highly diverse to bind multiple antigens
3. Fc portion binds to Fc receptors expressed on various cells for effector functions

Question 4

What are some examples of antibody use in cancer therapy? (7)

Answer 4

1. Tumor-specific IgG
2. Angiogenesis inhibition
3. Checkpoint blockade
4. Radioimmunotherapy
5. Antibody-drug conjugate therapy
6. Bispecific antibody therapy
7. CAR T cell

Question 5

Describe molecular recognition by antibodies. (2)

Answer 5

1. Structural and biochemical complementarity determines binding
2. Diversity of CDR loop conformations enables binding to diverse antigens

Question 6

How is antibody diversity generated?

Answer 6

Immunoglobulin genes and recombination

Question 7

The total diversity of antibody genes in body is > ________

Answer 7

billions

Question 8

How do we make antibodies to proteins of interest? (2)

Answer 8

1. Hybridoma method
2. Xenomouse technology

Question 9

What is the hybridoma method of making antibodies? (2)

Answer 9

1. Murine antibodies are immunogenic
2. Humanized antibodies such as Trastuzumab made from hybridoma

Question 10

What is xenomouse technology/how does it work? (2)

Answer 10

1. Antibody genes in mice replaced with human genes
2. Several similarities between human and mouse proteins cross-reactivity can limit antibody development

Question 11

What is phage display and selection of antibodies? (4)

Answer 11

1. M13 bacteriophage has been a workhorse for protein engineering
2. Proteins are expressed on the phage surface as fusion to coat proteins (mostly p3 or p8)
3. Phages are propagated at low MOI in E.coli to maintain genotype to phenotype linkage
4. Libraries of ~ 1010 variants can be generated and screened

Question 12

Go through the flowchart of mouse hybridoma (making antibodies) (5)

Answer 12

1. Immunization with targets –>
2. Harvest splenocytes, generate hybridomas –>
3. Mouse mAb can go 2 ways –>
   4a. Chimeric mAb
   4b. Humanized mAb

Question 13

Go through the flowchart of phage display (making antibodies) (4)

Answer 13

1. Phage displayed Ab libraries –>
2. Biopanning with targets (3-5 cycles) –> screening
3. Construction of Human IgG –>
4. Human mAb

Question 14

Go through the flowchart of transgenic mouse (making antibodies) (3)

Answer 14

1. Immunization with targets –>
2. Harvest splenocytes, generate hybridomas –> screening
3. Human mAb

Question 15

Go through the flowchart of single B cell (making antibodies) (4)

Answer 15

1. Human, PBMC –>
2. Sort B cells with labeled antigens –>
3. PCR, construct VH and VL –>
4. Human mAb

Question 16

Full-length IgG is not compatible with phage-display. What then, is done? (2)

Answer 16

1. Several formats are used in antibody libraries
2. Significant engineering necessary for smaller formats such as scFv and VH/VHH

Question 17

What is the success rate from phase I study to FDA approval of antibody drugs compared to small molecule drugs?

Answer 17

23% as compared to 7% for small molecule drugs

Question 18

~45% of antibodies are targeted against top 10 candidates. What are those candidates?

Answer 18

1. PD1/PDL1
2. CD3
3. HER2
4. CTLA4
5. SARS-CoV-2
6. 4-1BB
7. LAG3
8. EGFR
9. CD20
10. CD47

Question 19

What is Bevacizumab?

Answer 19

Humanized murine monoclonal antibody targeting VEGFA which inhibits angiogenesis

Question 20

What are the indications for Bevacizumab?

Answer 20

Age-related macular degeneration and several cancers

Question 21

How was Bevacizumab humanized? (2)

Answer 21

1. CDR regions were grafted into a human Fab fragment sequence
2. Point mutations switching back to murine sequence allowed tight binding

Question 22

What can mutations in Fc result in? (2)

Answer 22

1. Can alter binding affinity to Fc gamma receptors
2. Can increase or decrease antibody effector functions as desired

Question 23

How can Fc fragments be engineered to increase half-life?

Answer 23

Increased affinity at low pH to FcRn receptor increases half-life by preventing degradation in lysosomes

Question 24

What is the mechanism of bispecific antibodies? (2)

Answer 24

1. Binding to two different antigens/epitopes enables new modalities of targeting
2. Biparatopic binding increases crosslinking and internalization

Question 25

What is required for a quadroma?

Answer 25

Need advanced protein purification methodologies and multi-species antibodies

Question 26

What is knob-in-holes technology? (2)

Answer 26

1. Mutations prevent/reduce homodimerization of heavy chain 2. Light chain crossover can still happen

Question 27

What does cross-mab technology allow for? (2)

Answer 27

1. Enables purification of correctly paired antibodies. 2. Light chain crossover is prevented

Question 28

What are bispecific antibodies?

Answer 28

Allow engagement of multiple targets in one drug and allows bridging cancer cells with immune cells (CAR T-like approach)

Question 29

What is best bispecific antibody success story as of right now? (Only 3 approved bispecifics so far)

Answer 29

Emicizumab for treating hemophilia

Question 30

What are the advantages of antibodies? (3)

Answer 30

1. High affinity and specificity 2. Long half-life 3. Good safety profile

Question 31

What are the disadvantages of antibodies? (3)

Answer 31

1. High cost of production 2. Poor tissue penetration 3. IP conflicts for developing new drugs

Question 32

What are the advantages of alternative scaffolds? (3)

Answer 32

1. Low cost of production 2. Better tissue penetration 3. Target epitopes not accessible to antibodies

Question 33

What are the disadvantages of alternative scaffolds? (2)

Answer 33

1. Immunogenicity 2. Stability

Question 34

What are protein domain-based affinity reagents? (2)

Answer 34

1. A structural and biochemical match with an epitope on target protein, results in new protein interaction 2. The engineered protein binds with 1nM affinity and disrupts VEGFA-VEGFR2 interaction