Biologic Drug Development - Antibodies and Recombinant Proteins Flashcards
The best selling type of pharmaceuticals are?
Antibodies
How are antibodies made and what do they do? (3)
- Antibodies are made by B-cells to recognize foreign substances
- Secreted antibodies neutralize threats
- Binding is mediated by CDR loops
What is the structure of antibodies? (3)
- Heterotetramer with two identical light chains and heavy chains
- Fab portion is important for antigen binding. Highly diverse to bind multiple antigens
- Fc portion binds to Fc receptors expressed on various cells for effector functions
What are some examples of antibody use in cancer therapy? (7)
- Tumor-specific IgG
- Angiogenesis inhibition
- Checkpoint blockade
- Radioimmunotherapy
- Antibody-drug conjugate therapy
- Bispecific antibody therapy
- CAR T cell
Describe molecular recognition by antibodies. (2)
- Structural and biochemical complementarity determines binding
- Diversity of CDR loop conformations enables binding to diverse antigens
How is antibody diversity generated?
Immunoglobulin genes and recombination
The total diversity of antibody genes in body is > ________
billions
How do we make antibodies to proteins of interest? (2)
- Hybridoma method
- Xenomouse technology
What is the hybridoma method of making antibodies? (2)
- Murine antibodies are immunogenic
- Humanized antibodies such as Trastuzumab made from hybridoma
What is xenomouse technology/how does it work? (2)
- Antibody genes in mice replaced with human genes
- Several similarities between human and mouse proteins cross-reactivity can limit antibody development
What is phage display and selection of antibodies? (4)
- M13 bacteriophage has been a workhorse for protein engineering
- Proteins are expressed on the phage surface as fusion to coat proteins (mostly p3 or p8)
- Phages are propagated at low MOI in E.coli to maintain genotype to phenotype linkage
- Libraries of ~ 1010 variants can be generated and screened
Go through the flowchart of mouse hybridoma (making antibodies) (5)
- Immunization with targets –>
- Harvest splenocytes, generate hybridomas –>
- Mouse mAb can go 2 ways –>
4a. Chimeric mAb
4b. Humanized mAb
Go through the flowchart of phage display (making antibodies) (4)
- Phage displayed Ab libraries –>
- Biopanning with targets (3-5 cycles) –> screening
- Construction of Human IgG –>
- Human mAb
Go through the flowchart of transgenic mouse (making antibodies) (3)
- Immunization with targets –>
- Harvest splenocytes, generate hybridomas –> screening
- Human mAb
Go through the flowchart of single B cell (making antibodies) (4)
- Human, PBMC –>
- Sort B cells with labeled antigens –>
- PCR, construct VH and VL –>
- Human mAb
Full-length IgG is not compatible with phage-display. What then, is done? (2)
- Several formats are used in antibody libraries
- Significant engineering necessary for smaller formats such as scFv and VH/VHH
What is the success rate from phase I study to FDA approval of antibody drugs compared to small molecule drugs?
23% as compared to 7% for small molecule drugs
~45% of antibodies are targeted against top 10 candidates. What are those candidates?
- PD1/PDL1
- CD3
- HER2
- CTLA4
- SARS-CoV-2
- 4-1BB
- LAG3
- EGFR
- CD20
- CD47
What is Bevacizumab?
Humanized murine monoclonal antibody targeting VEGFA which inhibits angiogenesis
What are the indications for Bevacizumab?
Age-related macular degeneration and several cancers
How was Bevacizumab humanized? (2)
- CDR regions were grafted into a human Fab fragment sequence
- Point mutations switching back to murine sequence allowed tight binding
What can mutations in Fc result in? (2)
- Can alter binding affinity to Fc gamma receptors
- Can increase or decrease antibody effector functions as desired
How can Fc fragments be engineered to increase half-life?
Increased affinity at low pH to FcRn receptor increases half-life by preventing degradation in lysosomes
What is the mechanism of bispecific antibodies? (2)
- Binding to two different antigens/epitopes enables new modalities of targeting
- Biparatopic binding increases crosslinking and internalization
What is required for a quadroma?
Need advanced protein purification methodologies and multi-species antibodies
What is knob-in-holes technology? (2)
- Mutations prevent/reduce homodimerization of heavy chain
- Light chain crossover can still happen
What does cross-mab technology allow for? (2)
- Enables purification of correctly paired antibodies.
- Light chain crossover is prevented
What are bispecific antibodies?
Allow engagement of multiple targets in one drug and allows bridging cancer cells with immune cells (CAR T-like approach)
What is best bispecific antibody success story as of right now? (Only 3 approved bispecifics so far)
Emicizumab for treating hemophilia
What are the advantages of antibodies? (3)
- High affinity and specificity
- Long half-life
- Good safety profile
What are the disadvantages of antibodies? (3)
- High cost of production
- Poor tissue penetration
- IP conflicts for developing new drugs
What are the advantages of alternative scaffolds? (3)
- Low cost of production
- Better tissue penetration
- Target epitopes not accessible to antibodies
What are the disadvantages of alternative scaffolds? (2)
- Immunogenicity
- Stability
What are protein domain-based affinity reagents? (2)
- A structural and biochemical match with an epitope on target protein, results in new protein interaction
- The engineered protein binds with 1nM affinity and disrupts VEGFA-VEGFR2 interaction
