therapeutic antibodies Flashcards
what are the advantages of therapeutic antibodies vs small molecules?
specific, shorter time to market
what are the disadvantages of therapeutic antibodies vs small molecules?
given as injection, complex and expensive to manufacture
describe the structure of IgG
heterotetramer of 2 identical light chains, and 2 identical heavy chains. heavy and light chains folded into distinct domains. functionally, the antibody is separated into Fab domain (antigen binding) and Fc (fragment crystallisable) domain
what is antigen binding driven by?
complementary determining regions supported by framework regions found at the tip of Fab
what are the mechanisms by which antibodies can cause a response?
- antagonism
- interfering with signalling cascades
- complement dependent cytotoxicity
- antibody dependent cellular toxicity
- as a vehicle for something else (e.g. prodrug, fluorophores)
what is immunogenicity?
the ability of a substance to evoke an immune response
what problems can immunogenicity of an antibody cause?
- acute/delayed hypersensitivity
- pharmacokinetic changes
- reduced efficacy
- cross-reactivity with endogenous proteins
by which two methods can antibodies be discovered?
- immunisation
- phage display
how can antibodies be discovered by immunisation?
immunise rodent/rabbit with antigen -> isolate B cells and generate hybridomas with myeloma cells -> screen for specificity, identify Ab and engineer
how can antibodies be discovered by phage display?
isolate antibody and add to phages from a phage library -> incubate -> wash -> elute
list the solutions to immunogenicity
1) co-therapy with an immunosuppressant
2) sequential IgG type therapy regime
3) use of antibody fragments
4) use of human monoclonal antibodies
5) use of recombinant chimeric antibodies or humanised antibodies
what is a chimeric antibody?
fusion protein composed of regions of genes from two species; usually a combination of mouse variable regions with human constant regions
what are the disadvantages of a chimeric antibody?
lack the human acceptor framework and can have reduced affinity
what are humanised antibodies?
antibodies from non-human sources whose protein sequences have been modified to increase their similarity to human antibodies
how can CDRs be identified
using bioinformatics to compare residue variation, it will be higher for CDRs compared to framework regions
what must be considered when choosing a sequence for a CDR graft?
some framework residues are directly involved in antigen binding
framework residues can be responsible for holding CDR in correct conformation
which factors affect immunogenicity?
- foreign sequence
- physical presentation
- genetic background
how can physical presentation be engineered?
tendency to form aggregates is dependent on thermal stability, the CH2 domain on the Fc component has the lowest Tm, this can be engineered to be higher by altering the amino acid sequence
do the following protein expression systems produce protein with disulphide bonds?
mammalian, bacterial, Yeast, Insect
mammalian- Yes
bacterial- No but possible in engineered cell lines
yeast - yes
insect - yes
do the following protein expression systems produce proteins with glycosylation? mammalian, bacterial, yeast, insect
mammalian - yes
bacterial - no
yeast- yes
insect - yes
give some examples of commercially driven changes to expression
- changing codon usage to accomodate microbial codon bias
- changing the mRNA structure to improve translation rates
- single amino acid changes
how could you engineer a higher serum half-life?
- adding protein domains that have long serum half lives
- FcRn binding
- adding attachment sites for non-protein structures
- adding albumin binding structures to enable in vivo attachment to serum albumin
how does FcRn increase half-life?
IgG transported into the cell -> FcRn binds to IgG in acidified endosome -> non receptor bound proteins are degraded -> endosome recycling occurs, FcRn complex is removed from the cell -> pH alters and IgG released back into circulation
what does the efficacy of killing by CDC or ADCC rely on?
- affinity for the antigen
- antigen density
- affinity of antibody for FcYRIIIa
