Biologic Drug Development Flashcards
What is the relevance of antibodies in the pharmaceutical industry?
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
What is the central dogma of molecular biology?
DNA –(transcription)–> RNA –(post-transcription modification)–>mRNA–(translation via ribosome)–>Protein
What happens to hydrophobic or hydrophillic amino acids in a polypeptide chain?
Hydrophobic amino acids are internal, while hydrophillic amino acids are external
This type of organization results in globular structures
What are the four levels of protein structure?
- Primary structure
- Secondary structure
- Tertiary structure
- Quaternary structure
In general, how do antibodies work?
Antibodies can target individual proteins and modulate structure and function
Binding is mediated by CDR loops
What are the components of humoral immunity?
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)
What are the components of cell-mediated immunity?
T-cells bind to antigen-presenting cells and either release cytokines or kill infected cells
What is the basic structure of antibodies?
Heterotetramer with two identical light chains and heavy chains.
Fab portion is important for antigen binding
What are some effector functions of antibodies?
- Inactivates, prevents binding of pathogens and toxins to cells
- Induce phagocytosis or lysis
- NK cell-induced apoptosis (useful in cancer)
- Indice degranulation
What are the different types of anti-cancer antibodies?
- Tumour-specific IgG
- Angiogenesis inhibition
- Checkpoint blockade
- Radioimmunotherapy
- Antibody-drug conjugate therapy
- Bispecific antibody (BiTE therapy)
- CAR T-cells
review slide 12
Why are antibodies specific to a specific antigen?
Antigen and antibody binding sites need to be structurally and chemically compatible
CDR loop confirmations enables binding to diverse antigens
How is antibody diversity generated?
Exon mix and match in the IgH locus results in the diversity of CDR loop confirmations
see slide 15
In geneal, how are antibodies manufactured?
- 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
What is a major issue of murine (sourced from mice) antibodies?
They are immunogenic
Are humanized antibodies also immunogenic?
There will be a reduced immune attack and foriegn body detection responses with humanized aantibodies vs. murine antibodies
How does Xenomouse (Trangenic mice) technology work?
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
What is phage display?
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
How does a naive antibody library respond to a novel antigen?
Trial and error process of testing native antibodies against antigens
What are the four methods for making antibodies?
- Mouse hybridoma
- Phage display
- Transgenic mouse
- Single B cell
slide 22
What are the types of antibiotic fragments used in phage-display?
- Full IgG
- Fab
- scFv
- VH/VHH
slide 27
What is the favoured antibody type for solid tumour therapies?
scFV has improved penetration into solid tumours
Do antibody-based drugs have a lower success rate for FDA approval?
No, success rates are almost 3x compared to small molecules (23% vs. 7%)
What is the most common type of approved antibody?
Canonical antibody (full IgE molecule alone)
Are most antibodies designed to target unique targets?
No, 45% of antibodies are targeted against the top ten targets
PD1/PDL1, CD20, TNF, HER2 are some common targets for antibodies
What is an examples of a canonical antibody?
Bevacizumab
Humanized murine mAb targeting VEGF A (inhibition of angiogenesis)
How was bevacizumab humanized?
CDR regions from murine antibody were grafted onro a human Fab fragment sequence
What is the advantage of engineering Fc fragments for antibodies?
They help the antibody evade degradation
slide 35
What is the role of a linker in antibody drug conjugates?
They connect the antibody to the payload
Payload release is dependent on the linker and it can be triggered by tumour specific factors
What is the payload in antibody drug conjugates?
- Can be active drug or radioactive material
- Ensures low impact on non-targeted tissues (narrow window)
What are some functions of bispecific antibodies?
- Bridging cells (in-trans): T-cell and antigen brought into proximity
- Receptor inhibition (in cis): prevent HER2 dimerization
- Receptor activation (in cis): bring together two cell surface proteins and enhance activation
- Co-factor mimetic
- Piggybacking (use receptors for other drugs, to help import across BBB)
slide 39
What is a quadroma?
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)
What is knob-in-holes technology in the context of bispecific antibodies?
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
What is the use of Cross-Mab technology in the context of bispecific antibodies?
Light chain crossover is prevented by fusing the light chain with the heavy chain
