therapeutic antibodies

Question 1

what are the advantages of therapeutic antibodies vs small molecules?

Answer 1

specific, shorter time to market

Question 2

what are the disadvantages of therapeutic antibodies vs small molecules?

Answer 2

given as injection, complex and expensive to manufacture

Question 3

describe the structure of IgG

Answer 3

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

Question 4

what is antigen binding driven by?

Answer 4

complementary determining regions supported by framework regions found at the tip of Fab

Question 5

what are the mechanisms by which antibodies can cause a response?

Answer 5

• antagonism
• interfering with signalling cascades
• complement dependent cytotoxicity
• antibody dependent cellular toxicity
• as a vehicle for something else (e.g. prodrug, fluorophores)

Question 6

what is immunogenicity?

Answer 6

the ability of a substance to evoke an immune response

Question 7

what problems can immunogenicity of an antibody cause?

Answer 7

• acute/delayed hypersensitivity
• pharmacokinetic changes
• reduced efficacy
• cross-reactivity with endogenous proteins

Question 8

by which two methods can antibodies be discovered?

Answer 8

• immunisation

- phage display

Question 9

how can antibodies be discovered by immunisation?

Answer 9

immunise rodent/rabbit with antigen -> isolate B cells and generate hybridomas with myeloma cells -> screen for specificity, identify Ab and engineer

Question 10

how can antibodies be discovered by phage display?

Answer 10

isolate antibody and add to phages from a phage library -> incubate -> wash -> elute

Question 11

list the solutions to immunogenicity

Answer 11

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

Question 12

what is a chimeric antibody?

Answer 12

fusion protein composed of regions of genes from two species; usually a combination of mouse variable regions with human constant regions

Question 13

what are the disadvantages of a chimeric antibody?

Answer 13

lack the human acceptor framework and can have reduced affinity

Question 14

what are humanised antibodies?

Answer 14

antibodies from non-human sources whose protein sequences have been modified to increase their similarity to human antibodies

Question 15

how can CDRs be identified

Answer 15

using bioinformatics to compare residue variation, it will be higher for CDRs compared to framework regions

Question 16

what must be considered when choosing a sequence for a CDR graft?

Answer 16

some framework residues are directly involved in antigen binding
framework residues can be responsible for holding CDR in correct conformation

Question 17

which factors affect immunogenicity?

Answer 17

• foreign sequence
• physical presentation
• genetic background

Question 18

how can physical presentation be engineered?

Answer 18

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

Question 19

do the following protein expression systems produce protein with disulphide bonds?
mammalian, bacterial, Yeast, Insect

Answer 19

mammalian- Yes
bacterial- No but possible in engineered cell lines
yeast - yes
insect - yes

Question 20

do the following protein expression systems produce proteins with glycosylation? mammalian, bacterial, yeast, insect

Answer 20

mammalian - yes
bacterial - no
yeast- yes
insect - yes

Question 21

give some examples of commercially driven changes to expression

Answer 21

• changing codon usage to accomodate microbial codon bias
• changing the mRNA structure to improve translation rates
• single amino acid changes

Question 22

how could you engineer a higher serum half-life?

Answer 22

• 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

Question 23

how does FcRn increase half-life?

Answer 23

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

Question 24

what does the efficacy of killing by CDC or ADCC rely on?

Answer 24

• affinity for the antigen
• antigen density
• affinity of antibody for FcYRIIIa