Proteins and Immunology Flashcards
Limits of Small drug molecules
1) Intractable molecular targets
2) Restrictable ‘classical’ drug action
3) Traditional drug discovery less successful
What is a biologic
Medicinal product whose synthesis, extraction or manufacture involves living sources (human, animal or microbiological)
Insulin structure
2 linked peptide chains synthesised by enzymatic cleavage from a single protein-precursor
Injection, formulation/stability, immunogenicity
1920’s - banting and best
Advantages of antibody technology
1) Tackle targets resistant to small molecule intervention
large complex binding sites, orphan diseases, unknown binding sites
2) Higher affinity and selectivity
closely related targets, mutant forms of target
3) Diverse mechanisms of action
Messenger molecule, immune-directed cytotoxicity
Risks of antibody technology
Lack of efficacy and pharmacokinetic challenges
administration/delivery to target
species variation
Two main parts of antibody structure
Fab- antigen binding fragment domain (Fv responsible for antigen recognition) 2 X fab domains - bivalent
Fc - constant domain, directs cellular interactions and immunogenic response
Fc and pharmacokinetics
Regulates Ig transport and extends plasma half-life of Ig molecules
IgG structure
2 X Heavy chains & 2 X Light chains (disulphide bonds, different genes)
VH/VL - complex gene organisation generates diverse aa sequences responsible for antigen recognition. 3 hyper variable regions (complementarity determining regions) between 4 more conserved framework regions.
Define polyclonal and monocolonal
P - many different IgG molecules with increased affinity for antigen purified from serum after immunisation
M - IgG producing B cells isolated from immunised mouse
How antibodies made
1) Immunisation of mouse (isolate immune cells)
2) antibody forming cells
3) Hybridomas (screened for production of antibody)
4) antibody producing hybridomas clones
5) monoclonal antibodies (clonal expansion)
Problems with monoclonal
Antimouse antibodies (HAMA's) Rapid degradation - short 1/2 life Immunogenicity Fc domains have lack of efficacy as chimeric/humanised required for IgG biologics
What is phage display
Virus coats engineered to express human IgG domains. Screening and selection for affinity, without immunisation.
Lack key post translational modifications - glycosylation
How can they work?
1) receptor antagonists/inhibitors - cetuximab, antagonist for ligand binding at EGF receptor, limits proliferation
2) antagonism of growth factor - Anti VEGF - bevacuzimab or anti TNFalpha - infliximab
3) Agonists - agonists for death receptors, TRIAL
4) Cell cytotoxicity - Fc domains recruit macrophages to IgG bound antigens or tumour cells
Describe Trastuzumab
Herceptin
Targets EGF related receptor HER2, direct binding/inhibition of dimers
endocytosis and degradation of receptors
Nanoparticle aims
1) Smaller antibody templates
2) Streamline synthesis and manufacture
3) Simplify engineering
4) Increase stability
5) Improve access to target tissues
Strategies to generate IgG fragments which retain antigen binding
1) Enzymatic digestion
Pepsin - cleaves Fc domain, retaining disulphide linked bivalent antigen binding domains f(ab)2
Papain - individual monomeric f(ab)’ domains
2) Recombinant technology
Single chain (sc)fv - variable domains are joined by peptide linker in one polypeptide encoded by 1 gene.
ScFc or f(ab)’ =
Reduction in size. 160-30
Simplified manufacture, single-gene encoding and lack of glycosylation.
Antibody production in bacteria
Problems with no Fc domain
May be important
Reduces structural stability -prone to aggregation
Short 1/2 life
Full length IgG’s
Predictable pharmacokinetic profile
Long held life(15-30 days)
No renal elimination
Reduced proteolytic degradation after phagocytosis. unbound recycled to plasma FCGAMMA R
Abciximab
Chimeric f(ab)’ binds platelet glycoprotein 11b/111 preventing fibrinogen cross-linking and aggregation.
Short 1/2 life - 10-30mins
Suitable for indication
high affinity means binding long lasting
Certolizumab pegol
Humanised f(ab)', pegylated - increases size, reducing renal elimination Anti TNFalpha f(ab)' solubility = improved PK
Nano bodies - sharks and camelids
Antigen-binding domain is fully encoded by single heavy chain small- 15-18KD monomeric - less risk of aggregation pH stable - oral delivery require humanisation
HVR of nanobodies
Longer CDR loops
Convex binding surface able to recognise binding sites
Caplacizumab
Thrombotic Thrombocytopenia Purpura
Deficient processing of vWF - aggregate platelets
Multivalent binding
Increased affinity for a single targets altered pharmacological properties
Conjugation can help cause
1) Optimisation of pharmacokinetic properties
2) CNS penetration
3) Delivery Vehicles
How can we achieve CNS penetration
Hijack receptors mediating transcytosis across BBB, antibody conjugation to fab’ domain binding transferring receptor which carries antibody across BBB via enosomal transcytosis pathway.
Pharmacopoeia standards
1) Chemical formula
2) Methods of identification
3) Formulation
4) Excipients
5) Disintegration tests/bioavailability
Why biosimilar
Organism from which biosimilar and reference drug are produced may not be identical and the production process expires. not exact duplicates.
Inherent variability, no batch is identical but shouldn’t affect clinical efficacy
