Phage display and scFvs Flashcards
what PTM is needed for the Fc region of the antibodies to interact with effector cells?
glycosylated
what benefits are there of antibodies compared to chemicals?
- binds tight to target
- binds only to target
- long serum half life
- block protein protein interactions
- direct cell killing
- can target outside blood
what properties do chemicals have that antibodies cant acheive yet?
- target pockets
- target inside cells
- oral route
- chemical synthesis
what is the minimal structure that is needed to still get high specificty of targetting?
variable region - VH and Vl subunits
what one thing can be changed about antibodies to solve a lot of the issues there are associatated with them for therapeutic use?
shrinking the antibody
Gregory winter tried to figure out how to utilise the benefits while bypass the negative factors – broke his leg in car accident so couldn’t work in the lab so sat himself in front of computer and stared at the structure – decided most problems could be gotten rid of if you shrink the structure
how are the Vh and Vl subunit joined together in ScFv?
a linker connects the Nterminus of the variable light chain to the c terminus of the variable heavy chain
what is the linker of the scFv composed of?
Linker is composed of 4 glycines followed by a serine and repeated 3 times
why did gregory winter use glycine in the linker in ScFvs?
It tends not to form alpha helices – meaning its very flexible so its not going to constrain the chains from coming together
why did gregory winter use serine in the linker of scFvs?
Its hydrophillic - meaning serine wants to stay on the outside of structure
(so it doesn’t get in the way of structure and keeps flexible linker on the outside of the structure so it doesn’t get in the way)
describe the structure of single chain variants ScFvs?
Variable heavy chain (with CDR H1-3) and variable light chain (with CDR L1-3) linked by a linker molecule made up of (Gly4Ser)3
what are the advantages of scFv?
- Small size (25kDa vs 150 kDa of full sized mAb) allows for better penetration of tissues (tumours) and protein pockets (ability to target outside of blood is better) (Potential to target inside cell aswell because its only one polypeptide so wont fall apart)
- Cheap to produce using bacterial expression systems. (synthesise chemically bcos no glycosylation is needed as no Fc region)
- Large libraries of scFv sequences can be screened rapidly.
- No requirement for immunisation. (less ethical questions)
- Can easily be tagged with toxins/radionuclides for therapy and diagnosis.
what are two disadvantages of scFvs?
Serum half life is lower (but there are things we can do to extend it)
No longer have fc region so cant interact with effector cells (however there are things we can do to add this back on)
what do all scFv variations rely on?
all rely on generation of high affinity by phage display
what things can scFvs do that monoclonal antibodies cant?
Target pockets
Target outside blood
Chemical synthesis
what are the three broad stages of scFv production?
scFv library generation
phage display
panning for antigen specificity
what phage is used in phage display for scFv production?
filamentous phage
describe the steps of vh vl dna isolation
purify the vh and vl mRNA from hybridoma or B cells
convert it to cDNA using reverse transcriptase - by first strand cDNA synthesis
amplify by PCR
do PCR with variable region specific primer
the end results is lots of vh and vl pieces of DNA - each is unique hence need for a screening step
describe the steps for preparing the gene construct
make the linker using DNA synthesiser
then use self annealing PCR to stick the pieces (vh vl and linker) together
denature DNA to ssDNA and reanneal by lowering the temperature to generate some variations of the pordcut we wanat
heat to 72degrees celcius for elongation by DNA polymerase
then denature again to convert into ssDNA
then reanneal to form some products
then heat to 72 degrees for elongation by DNA pol and create the final complete gene fragment
now add the primers to the ends and extend them to add restrictions sites Sfi1 and Not1
put through 30 cycles of PCR to amplify
add in restriction enzymes to create sticky ends
now ready for addition into expression vector
what are the key features of the phagemid expression vector?
(9)
- Cloning site – Sfi-I and Not I (drop our fragment into)
- E-tag – for Ab detection (used in screening)
- Amber stop codon (important)
- Fd gene 3 – Phage minor coat protein
- M13 ori
- Ampr – Ampacilin resistance
- ColE1 ori
- Plac – Promoter (can turn off and on)
- g3 signal
what is special about the phagemid expression vector?
it can be expressed in ecoli as well as phages
what are the three types of stop codon?
TAG - amber
TAA - ochre
TGA - opal or umber
what happens when the same suppressor and stop codon are used?
eg when amber stop codon is put into an amber suppressor
it ignore it and continues through the sequence
why is TG1 (an amber suppressor) used in the phage mid for creation of scFv?
- TG1 is an amber suppressor, meaning that it reads through the amber stop codon and expresses the scFv linked to the group 3 minor coat protein.
making screening much easier
describe the steps for creation of transformed phage
transform phagemid into some ecoli at such a concentration that only one phagemid enters one ecoli
(so each ecoli will produce a unique single variation (specificity)
the plasmid will replicate itself so that a high concnetration of scfv are produced
(only one coat protein is included - gene 3)
the helper phage is transformed into the cell aswell to provide the other coat proteins necessay for assembly
assembly of new phages containing phage mid
lyse the cell and release all scfv into the supernatant
now we havemillions of phage particles with scFvs on their surface AND the DNA that codes for it
what is critical to the product after the transfromed phage is created
linking your phenotype to the genotype
– so when you identify the phage that binds to your target to high effinity you can make loads quickly and easily
phage particles have scFv on thier surface AND the DNA that codes for it
what is the structure and purpose of the KO7 helper phage?
- contains structural genes for viral assembly
- defective origin of replication so it doesnt replicate its own DNA
- doesnt assemble very quickly
- provides the structures (coat proteins etc) need for the phage to assemble
describe the panning steps to scfv production?
-want to find those that are specific)
coat a solid phase (plate) with the antigen of interest and pour scfv containing supernatant over it
those that have the right specificity will bind, the rest that dont bind will be washed away
now elute them off the plate (use a weak acid to interfere with the binding interactions)
reneutralise pH
-now want to find highest affinity-
reinfect ecoli with eluted pahges (1:1)
streak and grow on agar plate (containing ampicillin - the selection marker)
only those that have the plasmid will grow (amp resistance gene)
pick individual colonies off of the plate and put into 96 well microtitre plate
make another load of phages - rescue stage with KO7
each well has supernatant and millions of phages - each well will have a unique type of phage that binds to yur target with diff affinity
do ELISA to find out which ones have a higher affinity
transfer supernatant to elisa plate (antigen immobilised on the bottom, phage binds to antigen by scFv)
add in an antibody targetted to one of the coat protein on phage conjugated to a hrp
so when you add a substrate, any well sthat contain phage binding an antigen will have hrp co.our change and be able to identify the well that contain phages that bind the antigen of interest
describe the steps to growing scfvs in bulk
take the colonies that bind our antigen of interest nad infect a new strain of ecoli - one that is not an amber suppressor so the sequence will stops when it gets to the amber stop codon (no longer produce co protein 3 )
streak and grow onto agar plate (with ampicillin)
growth of culture from colony
production of soluble single chain Fv - end result is a high affinity antibody fragment that binds specifically to you rantigen of interest (importantly you also have the DNA sequence coding for it so you can make more)
why is the g3p signal important?
targets scfv to the periplasmic space which is essential for secretion of scfv into the supernatant
the larger the diversity within the scFv library means…
…the greater the probability you will find a scFv that will bind to the target with high affinity
whats the possible number of heavy- and light- chain combination in the human?
2.3million combinations
how many heavy chain V D and J segments are there?
V = 45
D = 23
J = 6
possible combination = 6210
how many kappa light chain V and J segments are there?
V = 41
J = 5
possible combinations = 205
how many lamda light chain V and J segments are there?
V = 33
J 5
possible combinations = 165
why increase diversity of scFv libraries?
- The number of potential structures is finite (even though it is large).
- There maybe potential ‘blind spots’ in these libraries e.g. due to recognition of self. (no self antigens)
Or toxic antigens (wouldn’t have been exposed to that) - The larger the library, the greater the probability of isolating high affinity antibodies.
- You need ~ 1 trillion (1012) particles to be certain the library contains a high binder for your antigen of interest.
where do we add diversity to scFv?
all variation and diversity needs to be put into the CDR loops
how can we introduce diversity into scFvs?
- Introducing mutation using Taq polymerase – Error prone PCR
- DNA shuffling
- Oligonucleotide directed mutagenesis
- CDR walking
how do you introduce diversity using taq polymerase?
error prone PCR
Changing the conditions in the PCR tube so it makes more mistakes
* Add nucletdies in different ratios
* Increase conc of magnesium to make taq pol error prone
* Add manganese effect ability of taq pol to be accurate
* Use a taq polymerase that doesn’t have proof reading ability
*
how do you introduce diversity using taq polymerase?
error prone PCR
Changing the conditions in the PCR tube so it makes more mistakes
* Add nucletdies in different ratios
* Increase conc of magnesium to make taq pol error prone
* Add manganese effect ability of taq pol to be accurate
* Use a taq polymerase that doesn’t have proof reading ability
under optimum conditions what is Taq polymerases error rate?
- Under optimum conditions Taq polymerase’s error rate is ~1 in 104 nucleotides. (also has proof reading ability)
what can the error rate be increased to in error prone PCR?
- Error rate can be increased 7-fold by the addition of 0.5 mM Mn2+ and an imbalance in the concentration of dNTPs.
what is the major limitation of error prone PCR in increasing diversity?
no control over where these errors will occur
(we want it in cdr but we cant specify this, so really stringent screening process is needed)
what is DNA shuffling?
a tehcnique used to introduce diversity into a sequence, used in scFv
mimics DNA recombination
how can diversity be increased in scFvs using DNA shuffling?
treat target genes with DNase1 (it will randomly cut up the DNA)
rejoin the segements using self annealing PCR
this allows you to mis up the positions of the six CDR regions - results in shuffled genes
Need to make sure that where the cuts are there is enough over lap that you can put it back together (reshuffles cdr)
what is the major limitations of DNA shuffling for introducing diversity?
is not completely random, tends to cut sites adjacent to pyrimidines, so Cs and Ts.
And the fragments need not be so small that the PCR no longer works.
Cross over can preferentially occurs at locations of sequence homology (blocks of parental DNA being duplicated)
what is the major benefit of oligonucleotide directed mutagenesis for increaseing diversity?
that the position and degree of ranomisation can be really precisely controlled
we can add them purely into cdr regions
what is oligonucleotide directed mutagenesis?
a non‐transgenic base pair‐specific precision gene editing platform
how can oligonucleotide directed mutagenesis increase diversity in scFvs?
Makes use of degenerate codons
on a DNA synthesiiser add a bottle of a known combo of bases
Add N (or X letter) bottle, which contains have a mix of all the nucleotides, so when you put N in the sequence the machine will add one of the four into the sequence (or one of the (you don’t know which one so it will be random)
After one MNN codon you can generate 32 diff variants
After 5 repeats you create 34 million diff variants
Introducing thse just into the cdr region
You don’t really want to add a stop codon
an example of this is CDR walking
(N, K, S, M, W, R, V) each letter represents a combo of bases eg N=G/A/T/C or R=A/G or V = G/A/C
what is CDR walking?
how does it introduce diversity?
introduction of mutations in the CDR loops one at a time
uses degenerate primers with PCR to bind in front of the CDR region and introduce mutations into the code
generates three fragments
use self annealing PCR to stick them back together
then repeat 2-10 cyclines
amplify then clone
what is the problem with human scFv libraries?
Immunogenic responses in humans to antibodies originating in other species may give rise to potentially fatal hypersensitivity reactions (anaphylaxis).
: Can’t inoculate human subjects with desired antigen
