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PHAR 211 > Biologic Drug Development - Antibodies and Recombinant Proteins > Flashcards

Biologic Drug Development - Antibodies and Recombinant Proteins Flashcards

1
Q

The best selling type of pharmaceuticals are?

A

Antibodies

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2
Q

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

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

What is the structure of antibodies? (3)

A
  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
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4
Q

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

A
  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
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5
Q

Describe molecular recognition by antibodies. (2)

A
  1. Structural and biochemical complementarity determines binding
  2. Diversity of CDR loop conformations enables binding to diverse antigens
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6
Q

How is antibody diversity generated?

A

Immunoglobulin genes and recombination

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7
Q

The total diversity of antibody genes in body is > ________

A

billions

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8
Q

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

A
  1. Hybridoma method
  2. Xenomouse technology
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9
Q

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

A
  1. Murine antibodies are immunogenic
  2. Humanized antibodies such as Trastuzumab made from hybridoma
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10
Q

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

A
  1. Antibody genes in mice replaced with human genes
  2. Several similarities between human and mouse proteins cross-reactivity can limit antibody development
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11
Q

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

A
  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
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12
Q

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

A
  1. Immunization with targets –>
  2. Harvest splenocytes, generate hybridomas –>
  3. Mouse mAb can go 2 ways –>
    4a. Chimeric mAb
    4b. Humanized mAb
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13
Q

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

A
  1. Phage displayed Ab libraries –>
  2. Biopanning with targets (3-5 cycles) –> screening
  3. Construction of Human IgG –>
  4. Human mAb
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14
Q

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

A
  1. Immunization with targets –>
  2. Harvest splenocytes, generate hybridomas –> screening
  3. Human mAb
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15
Q

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

A
  1. Human, PBMC –>
  2. Sort B cells with labeled antigens –>
  3. PCR, construct VH and VL –>
  4. Human mAb
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16
Q

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

A
  1. Several formats are used in antibody libraries
  2. Significant engineering necessary for smaller formats such as scFv and VH/VHH
17
Q

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

A

23% as compared to 7% for small molecule drugs

18
Q

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

A
  1. PD1/PDL1
  2. CD3
  3. HER2
  4. CTLA4
  5. SARS-CoV-2
  6. 4-1BB
  7. LAG3
  8. EGFR
  9. CD20
  10. CD47
19
Q

What is Bevacizumab?

A

Humanized murine monoclonal antibody targeting VEGFA which inhibits angiogenesis

20
Q

What are the indications for Bevacizumab?

A

Age-related macular degeneration and several cancers

21
Q

How was Bevacizumab humanized? (2)

A
  1. CDR regions were grafted into a human Fab fragment sequence
  2. Point mutations switching back to murine sequence allowed tight binding
22
Q

What can mutations in Fc result in? (2)

A
  1. Can alter binding affinity to Fc gamma receptors
  2. Can increase or decrease antibody effector functions as desired
23
Q

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

A

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

24
Q

What is the mechanism of bispecific antibodies? (2)

A
  1. Binding to two different antigens/epitopes enables new modalities of targeting
  2. Biparatopic binding increases crosslinking and internalization
25
Q

What is required for a quadroma?

A

Need advanced protein purification methodologies and multi-species antibodies

26
Q

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

A
  1. Mutations prevent/reduce homodimerization of heavy chain
  2. Light chain crossover can still happen
27
Q

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

A
  1. Enables purification of correctly paired antibodies.
  2. Light chain crossover is prevented
28
Q

What are bispecific antibodies?

A

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

29
Q

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

A

Emicizumab for treating hemophilia

30
Q

What are the advantages of antibodies? (3)

A
  1. High affinity and specificity
  2. Long half-life
  3. Good safety profile
31
Q

What are the disadvantages of antibodies? (3)

A
  1. High cost of production
  2. Poor tissue penetration
  3. IP conflicts for developing new drugs
32
Q

What are the advantages of alternative scaffolds? (3)

A
  1. Low cost of production
  2. Better tissue penetration
  3. Target epitopes not accessible to antibodies
33
Q

What are the disadvantages of alternative scaffolds? (2)

A
  1. Immunogenicity
  2. Stability
34
Q

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

A
  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

PHAR 211 (10 decks)

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