1
Q
Fc effector region components
A
CH2 and 3
2
Q
Fab region components
A
CH1, CL, VH and VL
3
Q
How many total CDR loops per antibody
A
12 total. 3 on each variable heavy and 3 on each variable light chain. 2 Fabs so 2x6=12.
4
Q
Method of using mice to make mAbs
A
Inject antigen -> leave for a few weeks -> kill and mash spleen -> isolate B cells -> form hybridoma -> makes antibodies.
5
Q
Disadvantages of mAbs
A
Large so poor penetrance in tissues/into cells and can’t bind small pockets. Have to administer intravenously. Can’t chemically synthesise as need euk cell machinery (eg. glycosylation). Time consuming and expensive to make.
6
Q
Advantages of mAbs
A
Tight and specific binding. Long serum half life. Recruit effectors. Block protein-protein interactions. Direct cell killing.
7
Q
How Winter came up with scFv idea
A
Worked out only VL and VH minimum structure needed to maintain binding and specificity. But need linker to stop falling apart and maintain function.
8
Q
ScFvs
A
Single chain variable fragments. Use filamentous phages to display. VL and VH connected by linker.
9
Q
Linker sequence of scFvs
A
4Gly and 1Ser repeated
10
Q
Purpose of Gly in scFv linker
A
Tends not to form a helices so gives flexibility
11
Q
Purpose of Ser in scFv linker
A
Hydrophillic so keeps linker out of interface between VH and VL
12
Q
Advantages of scFvs
A
Small so good penetrance and can bind small pockets. Cheap to produce. Large libraries can be screened rapidly. No requirement for immunisation. Easily tagged with toxins/radionuclides for therapy and diagnosis. Efficient and highly adaptable. Don’t need any PTMs (don’t need euk cell machinery).
13
Q
Disadvantages of scFvs
A
No Fc so no effector function and can’t bind FcRn. Short 1/2 life. But both can be overcome with modifications.
14
Q
Bacteriophage
A
Virus that infects and replicates in bacteria
15
Q
Idea of phage display
A
Use to direct enzyme evolution to antibody evolution
16
Q
Filamentous phage components
A
ssDNA and coat proteins
17
Q
Ends of VH and VL
A
Highly conserved so can design PCR primers for the ends.
18
Q
Source of starting material for phage display
A
B cell DNA encoding VH and VL from hybridomas (so human)
19
Q
Preparation of gene construct for PCR
A
Linker ends extended with VH and VL conserved ends.
20
Q
Self annealing PCR purpose
A
Stick together VH, linker, and VL to form scFv construct
21
Q
Self annealing PCR steps
A
Denature 94C to single DNA strands. Reanneal 50-60C. Elongate with DNA pol 72C. Repeat again.
22
Q
Preparation of gene construct for insertion into expression vector
A
Design primers including restriction sites targeting the conserved ends. Amplify with PCR. Digest with enzymes so sticky ends. Insert into cloning site of phagemid expression vector.
23
Q
Example phagemid expression vector
A
pCANTAB 5 E
24
Q
Key features of phagemid expression vector
A
Cloning site. E-tag. Amber stop codon. Fd gene 3. M13 ori. Amp. ColE1 ori. Lac promoter. g3 signal.
25
Purpose of lac promoter in expression vector
Turn gene on/off
26
Purpose of g3 signal in expression vector
Target scFv to periplasmic space at the end of the process so secreted onto e.coli membrane.
27
Purpose of E tag in expression vector
For antibody detection
28
Purpose of amber stop codon in expression vector
Control whether the antibody/protein is fused to the phage coat protein or produced as a soluble protein
29
Purpose of Fd gene 3 in expression vector
Phage minor coat protein. Displays scFv on cell surface
30
Purpose of M13 ori in expression vector
Phage origin of replication site
31
Purpose of ColE1 ori in expression vector
E.coli origin of replication site
32
Purpose of Amp in expression vector
Ampicillin resistance. Select for cells that take up vector.
33
Example of first E.coli strain used in phage display method
TG1 E.coli. Amber suppressor strain.
34
Amber suppressor
Reads through the amber stop codon.
35
First transformation step in phage display
Tranform TG1 e.coli with phagemid. Done at concentration so that each e.coli only gets one phagemid vector so only encodes one scFv. Ignores amber stop codon so produces scFv linked to pIII. Phagemid replicates but can't form filamentous phage.
36
Helper phage
Eg. M13 KO7. Contains structural genes for viral assembly. Has defective origin of replication so itself can't replicate. Doesn't assemble very quickly so can't reform a helper phage inside e.coli.
37
Helper phage step in phage display
Infect e.coli with helper phage -> produces all structural proteins for forming filamentous phage -> millions of phage particles produced -> lysis of e.coli -> particles in supernatant
38
Phage display connection between phenotype and genotype
scFv on surface of filamentous phage with encoding DNA within.
39
Panning
Coat plate with antigen -> add solution with scFvs -> wash -> elute (reduce pH) off non-specific binding. Increase washes/ wash for longer to leave only higher affinity binding. Bulk identifies scFvs for good affinity, not individual clones.
40
Off rate
Amount of time it stays bound to its target
41
Avidity
Able to bind multiple antigens if high enough density - weak binding appears to bind strongly. Additive effect. Low density antigen means only affinity, not avidity, has effect.
42
Step after panning in phage display
Reinfect e.coli with selected phages. One phage per e.coli. Spread on agar plate with ampicillin and so that each singular e.coli forms a separate colony.
43
Step after reinfecting e.coli with selected phages and plating
Pick off individual colonies from agar plate and transfer into 96 well plate. Rescue with KO7 helper phage to reform phage particles -> scFvs released into supernatant. Each well contains many of one scFv variant. Additional panning to determine very high affinities.
44
Step after 96 well plate reinfection
ELISA assay. Antigens immobilised at bottom of well plate -> wash -> secondary antibody against filamentous phage coat protein linked to eg. HRP. Fluorescence shows bound scFvs (with good affinities). Identifies individual clones with good affinities.
45
HRP
Horse radish peroxidase. Catalyses a reaction the produces fluorescence
46
Step after ELISA assay in phage display
Reinfect e.coli strain that isn't an amber suppressor (eg. HB2151). Recognises amber stop codon so only produces scFv with E-tag. No longer linked targeted to membrane. Spin down cells and remove e.coli from solution, leaving scFvs. Antibody against E-tag used to check production and affinities etc. Can go on to use scFv DNA to make parge quantities/ graft into other constructs/ add stuff etc.
47
Phagemid library
Millions of phages encoding a unique scFv.
48
Diversification
Improves affinity of binding
49
Summarise steps of scFv phage display
Isolation of mRNA and amplification of VH and VL cDNA -> construction of scFv sequence and phagemid library -> transformation into TG1 e.coli -> rescue with KO7 helper phage -> panning and selection -> diversification -> transformation into HB2151 e.coli -> production and analysis of soluble scFv.
50
VDJ regions in light vs heavy chain
Heavy - VDJ. Light only V and J.
51
Why increase diversity
Number of potential structures is finite - rely only on B cell natural diversification of VDJ recombination. There may be blind spots - won't make self-antibodies or against toxins as not otherwise exposed. More likely to isolate high affinity antibodies.
52
Number of combinations only from B cell diversification
4 million
53
Number of combinations necessary to be certain the library contains a high binder for antigen
1 trillion (10^12)
54
Error prone PCR
Change conditions to force more errors. Change dNTP ratios. Increase Mg2+. Add Mn2+. Taq polymerase without proof reading.
55
Limitations of using error prone PCR
No control as to where the mutations go. Mutations in structural regions may give non-viable structures.
56
DNA shuffling
Mimics non-homologous recombination. Partially digest scFv DNA -> self priming PCR rejoin fragments -> shuffled DNA
57
Limitations of using DNA shuffling
Non-random cleavage of DNA - prefer to cleave sites adjacent to pyrimidines. Overlaps of fragments must be large enough for elongation - less diversity with larger fragments.
58
Oligonucleotide-directed mutagenesis
Control degree and position of mutation. Can target CDR loops and avoid structural regions. PCR using degenerate codons in primers. Read and replicate DNA. Random but controlled.
59
Degenerate codon examples
MNN. NNC. NWW. NNT. VVC. RST.
60
MNN codon
All 20 amino acids
61
NNC codon
15 amino acids
62
NWW codon
Charged/hyrdophobic amino acids but no cys (no S-S)
63
NNT codon
Mixed
64
VVC codon
Hydrophillic
65
RST
Small side chains
66
Number of variants generated using 1 or 5 MNN codons
1 -> 32 variants. 5 -> 34 million variants
67
CDR walking
Mutate one CDR at a time, find highest affinity scFv, then mutate other CDRs.
68
Naive libraries
No antigen given. B cells taken from human donor blood/bone marrow.
69
Immune library
Patients already exposed to antigen
70
Synthetic library
In silico design and DNA synthesis. Standard framework for conserved regions then design CDRs.
71
Semi-synthetic library
CDRs from donor as well as adding in silico designs
72
4 main ways to generate human scFv libraries
Naive. Immune. Synthetic. Semi-synthetic.
73
Examples where human scFvs have been used
anti-HIV mAbs. Treating systemic lupus erythematosus. CAR-T cell therapy. mAbs against SARs-CoV-2
74
Limitations of human scFv libraries
Somewhat limited library - no self antibodies and not exposed to antigen of interest.
75
MAK195
Mouse Ab targets TNF-a with high affinity
76
Increased TNFa implications
Alzheimers. Cancer. Inflammatory and autoimmune diseases.
77
Guided selection
Way to fully humanise mouse antibody (and increase affinity). Replace one mouse antibody chain (VH or VL) while keeping the other constant, then selecting new binding partners from naive human library. Panning steps. Isolate human chain and combine with other human chain. Can transfer human CDR seqs into human IgG.
78
3 main display systems
Virus-surface display (phages). Cell-surface display. Cell-free display.
79
Yeast surface display
Euk so can add PTMs. Gene of interest flanked by epitope markers. Aga2p gene adjacent to its secretion sequence so whole construct goes to surface. Fusion protein shuttled ER->Golgi-> aga2p forms 2S-S with aga1p which anchors it to membrane.
80
What links phenotype to genotype in yeast surface display
Aga2p protein
81
Epitope markers
Short protein seqs used for downstream detection using antibodies against them and readout of proper folding and display of scFv
82
Advantages of yeast display system
Fast. Direct screening for kinetic parameters. Can use flow cytometry and cell sorting eg for affinity sorting. PTMs.
83
PURE
Protein synthesis using recombinant elements
84
Ribosome display system
Generate cDNA scFv library then convert to mRNA (with ribosome binding site). Take necessary proteins from e.coli cell for translation. Conditions for correct folding and stable mRNA-ribosome complex. Little degradation as few proteases/nucleases. Panning steps then recover mRNA -> reveerse transcription -> cDNA -> diversification etc.
85
Necessary proteins for translation
TFs. Elongation factors. Ribosome subunits. Etc.
86
Advantages of cell free systems
Intrinsic mutagenesis. Fastest. Automated. Can select for otherwise toxic Abs. Not limited by transformation efficiency.
87
What links phenotype to genotype in ribosome display system
Stable ribosome complex
88
mRNA protein fusion system
All necessary proteins for translation (rabbit reticulocyte lysate). Covalently attach puromycin to 3' scFv mRNA. Analogue of aminoacyl-tRNA so enters ribosome binding site and covalently attaches to protein. Ribosome dissociates. Reverse transcription -> cDNA -> diversification etc.
89
What links phenotype to genotype in mRNA protein fusion system
Puromycin
90
Limitations of phage display
Transformation efficiency. Slow introduction of diversity.
91
Typical library size of phage/yeast/cell free systems
Phage- 10^11. Yeast- 10^8. Cell free- 10^13.
92
Selection/panning methods of phage/yeast/cell free systems
Phage- immobilised antigen/tissue sections. Yeast- immobilised antigen/cell sorting. Cell free- only immobilised antigen.
93
Advantages of phage display
Fairly large libraries. Robust. Most established. Easy. Automated.
94
Limitations of yeast surface display
Sorting expertise and equipment needed. Sorting speed limited. Transformation efficiency (worse than bacteria).
95
Limitations of cell free systems
Limited selection (only use immobilised antigens). Technically sensitive. Selection of buffers different from intracellular env. - might fold differently.
96
Improving serum half life of scFvs
Reduce sensitivity to proteases and liver clearance. Make bigger while maintaining good penetrance. Many Ab fragment formats (eg diabodies). Conjugate polymers like PEGylation (polyethylene glycol)/dextran/HPMA/polysialylation. Conjugate albumin - can also bind FcRn.
97
Advantages of short serum half life
Diagnosis. PET tracers. Vehicle for radionuclides to target tissues. Only needed for minutes/hours. Can penetrate solid tumours. Reduce exposure to healthy tissues.
98
Peptide bicycles
Only main two CDRs with 3 cys introduced -> react with tribromomethyl benzene to make stable. High plasma stability due to low protease interaction.
99
IL2
T cell activation and proliferation
100
What binds Fc for complement
C1q
101
Normal form of binding required for effector function
Bivalent binding. Eg. bring 2 antigens together.
102
Restoring effector function to scFv
Fuse to Fc domain or fuse to cytokine.
103
Intrabodies
scFv or single VH domain. Disulphide bonds not important for structure so remain correctly folded in cell. Intracellular targets.
104
How to introduce intrabodies to cells
Insert scFv gene in expression vector -> inside cell -> produce scFv.
105
Different vector types
Viral or non-viral (lipid nanoparticles).
106
Why can't mAbs target intracellularly
Unable to penetrate cell membrane. Can't fold correctly (no S-S) in reducing env so lose activity.
107
Example intrabody
TAR1. anti-p53 scFv. Binds mutant p53 and restores WT function -> apoptosis.
108
Alternative protein scaffold applications
Imaging. Clinical diagnostics. Biosecurity. Industrial biocatalysis. Faster, easier and cheaper than Abs.
109
Engineering alternative protein scaffolds
Same high throughput display technologies and diversification etc as scFvs as long as has specific binding. Assist directed evolution for increased affinity and specificity.
110
Affibody
3 helix bundle that is a natural Ig-binding domain.
111
Knottins
Cysteine knot peptide containing common folding motif with disulphide bond laces between peptide loop formed by two others.
112
DARPin
Designed ankyrin repeat protein. Modular scaffolds that consist of tandem repeats of a short seq. Takes advantage of mechanisms of repeating structural motifs mediating protein-protein interactions.
Biomedical Technology
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