Proteins and Immunology

Question 1

Limits of Small drug molecules

Answer 1

1) Intractable molecular targets
2) Restrictable ‘classical’ drug action
3) Traditional drug discovery less successful

Question 2

What is a biologic

Answer 2

Medicinal product whose synthesis, extraction or manufacture involves living sources (human, animal or microbiological)

Question 3

Insulin structure

Answer 3

2 linked peptide chains synthesised by enzymatic cleavage from a single protein-precursor
Injection, formulation/stability, immunogenicity
1920’s - banting and best

Question 4

Advantages of antibody technology

Answer 4

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

Question 5

Risks of antibody technology

Answer 5

Lack of efficacy and pharmacokinetic challenges
administration/delivery to target
species variation

Question 6

Two main parts of antibody structure

Answer 6

Fab- antigen binding fragment domain (Fv responsible for antigen recognition) 2 X fab domains - bivalent
Fc - constant domain, directs cellular interactions and immunogenic response

Question 7

Fc and pharmacokinetics

Answer 7

Regulates Ig transport and extends plasma half-life of Ig molecules

Question 8

IgG structure

Answer 8

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.

Question 9

Define polyclonal and monocolonal

Answer 9

P - many different IgG molecules with increased affinity for antigen purified from serum after immunisation
M - IgG producing B cells isolated from immunised mouse

Question 10

How antibodies made

Answer 10

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)

Question 11

Problems with monoclonal

Answer 11

Antimouse antibodies (HAMA's)
Rapid degradation - short 1/2 life
Immunogenicity
Fc domains have lack of efficacy as chimeric/humanised required for IgG biologics

Question 12

What is phage display

Answer 12

Virus coats engineered to express human IgG domains. Screening and selection for affinity, without immunisation.
Lack key post translational modifications - glycosylation

Question 13

How can they work?

Answer 13

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

Question 14

Describe Trastuzumab

Answer 14

Herceptin
Targets EGF related receptor HER2, direct binding/inhibition of dimers
endocytosis and degradation of receptors

Question 15

Nanoparticle aims

Answer 15

1) Smaller antibody templates
2) Streamline synthesis and manufacture
3) Simplify engineering
4) Increase stability
5) Improve access to target tissues

Question 16

Strategies to generate IgG fragments which retain antigen binding

Answer 16

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.

Question 17

ScFc or f(ab)’ =

Answer 17

Reduction in size. 160-30
Simplified manufacture, single-gene encoding and lack of glycosylation.
Antibody production in bacteria

Question 18

Problems with no Fc domain

Answer 18

May be important
Reduces structural stability -prone to aggregation
Short 1/2 life

Question 19

Full length IgG’s

Answer 19

Predictable pharmacokinetic profile
Long held life(15-30 days)
No renal elimination
Reduced proteolytic degradation after phagocytosis. unbound recycled to plasma FCGAMMA R

Question 20

Abciximab

Answer 20

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

Question 21

Certolizumab pegol

Answer 21

Humanised f(ab)', pegylated - increases size, reducing renal elimination
Anti TNFalpha
f(ab)' solubility = improved PK

Question 22

Nano bodies - sharks and camelids

Answer 22

Antigen-binding domain is fully encoded by single heavy chain
small- 15-18KD
monomeric - less risk of aggregation
pH stable - oral delivery
require humanisation

Question 23

HVR of nanobodies

Answer 23

Longer CDR loops

Convex binding surface able to recognise binding sites

Question 24

Caplacizumab

Answer 24

Thrombotic Thrombocytopenia Purpura

Deficient processing of vWF - aggregate platelets

Question 25

Multivalent binding

Answer 25

Increased affinity for a single targets altered pharmacological properties

Question 26

Conjugation can help cause

Answer 26

1) Optimisation of pharmacokinetic properties
2) CNS penetration
3) Delivery Vehicles

Question 27

How can we achieve CNS penetration

Answer 27

Hijack receptors mediating transcytosis across BBB, antibody conjugation to fab’ domain binding transferring receptor which carries antibody across BBB via enosomal transcytosis pathway.

Question 28

Pharmacopoeia standards

Answer 28

1) Chemical formula
2) Methods of identification
3) Formulation
4) Excipients
5) Disintegration tests/bioavailability

Question 29

Why biosimilar

Answer 29

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