Phage display and scFvs Flashcards
1
Q
what PTM is needed for the Fc region of the antibodies to interact with effector cells?
A
glycosylated
2
Q
what benefits are there of antibodies compared to chemicals?
A
- binds tight to target
- binds only to target
- long serum half life
- block protein protein interactions
- direct cell killing
- can target outside blood
3
Q
what properties do chemicals have that antibodies cant acheive yet?
A
- target pockets
- target inside cells
- oral route
- chemical synthesis
4
Q
what is the minimal structure that is needed to still get high specificty of targetting?
A
variable region - VH and Vl subunits
5
Q
what one thing can be changed about antibodies to solve a lot of the issues there are associatated with them for therapeutic use?
A
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
6
Q
how are the Vh and Vl subunit joined together in ScFv?
A
a linker connects the Nterminus of the variable light chain to the c terminus of the variable heavy chain
7
Q
what is the linker of the scFv composed of?
A
Linker is composed of 4 glycines followed by a serine and repeated 3 times
8
Q
why did gregory winter use glycine in the linker in ScFvs?
A
It tends not to form alpha helices – meaning its very flexible so its not going to constrain the chains from coming together
9
Q
why did gregory winter use serine in the linker of scFvs?
A
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)
10
Q
describe the structure of single chain variants ScFvs?
A
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
11
Q
what are the advantages of scFv?
A
- 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.
12
Q
what are two disadvantages of scFvs?
A
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)
13
Q
what do all scFv variations rely on?
A
all rely on generation of high affinity by phage display
14
Q
what things can scFvs do that monoclonal antibodies cant?
A
Target pockets
Target outside blood
Chemical synthesis
15
Q
what are the three broad stages of scFv production?
A
scFv library generation
phage display
panning for antigen specificity
16
Q
what phage is used in phage display for scFv production?
A
filamentous phage
17
Q
describe the steps of vh vl dna isolation
A
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
18
Q
describe the steps for preparing the gene construct
A
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
19
Q
what are the key features of the phagemid expression vector?
(9)
A
- 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
20
Q
what is special about the phagemid expression vector?
A
it can be expressed in ecoli as well as phages
21
Q
what are the three types of stop codon?
A
TAG - amber
TAA - ochre
TGA - opal or umber
22
Q
what happens when the same suppressor and stop codon are used?
eg when amber stop codon is put into an amber suppressor
A
it ignore it and continues through the sequence
23
Q
why is TG1 (an amber suppressor) used in the phage mid for creation of scFv?
A
- 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
24
Q
describe the steps for creation of transformed phage
A
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
25
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
26
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
27
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
28
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)
29
why is the g3p signal important?
targets scfv to the periplasmic space which is essential for secretion of scfv into the supernatant
30
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
31
whats the possible number of heavy- and light- chain combination in the human?
2.3million combinations
32
how many heavy chain V D and J segments are there?
V = 45
D = 23
J = 6
## Footnote
possible combination = 6210
33
how many kappa light chain V and J segments are there?
V = 41
J = 5
## Footnote
possible combinations = 205
34
how many lamda light chain V and J segments are there?
V = 33
J 5
## Footnote
possible combinations = 165
35
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.
36
where do we add diversity to scFv?
all variation and diversity needs to be put into the CDR loops
37
how can we introduce diversity into scFvs?
* Introducing mutation using Taq polymerase – Error prone PCR
* DNA shuffling
* Oligonucleotide directed mutagenesis
* CDR walking
38
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
*
39
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
40
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)
41
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.
42
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)
43
what is DNA shuffling?
a tehcnique used to introduce diversity into a sequence, used in scFv
mimics DNA recombination
44
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)
45
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)
46
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
47
what is oligonucleotide directed mutagenesis?
a non‐transgenic base pair‐specific precision gene editing platform
48
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
## Footnote
(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
49
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
50
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
51
naive human libraries can be made from what samples?
* Human libraries can be made using peripheral blood mononuclear cells (PBMCs) and bone marrow.
- These are Naïve Libraries as no antigen has been given
52
what does pooling people into a scfv library do?
everyone has a slightly different repertoire – to increase diversity
53
human libraries from immuniised repertoires have be made from what?
- PBMCs/ lymph/ bone marrow taken from patients with infections, auto-immune disease or tumours.
54
what ways can we create human scfv libraries?
- PBMCs/ lymph/ bone marrow taken from patients with infections, auto-immune disease or tumours.
- bone marrow from a healthy patient and isolate b cells
- DNA synthesising machine using computer design (synthetic)
- combination of real and synthetic scfb (semi synthetic)
55
what are naive libraries?
**constructed using B cells of unimmunized or healthy donors**, normally focusing on the IgM isotype. Naïve libraries are supposedly able to be used for the isolation of mAbs against any antigen
56
what are immune libraries?
generated from disease infected host or immunization against an infectious agent. Antibodies derived from immune libraries are distinct from those derived from naïve libraries as the host's in vivo immune mechanisms shape the antibody repertoire to yield high affinity antibodies
57
what are synthetic libraries?
constructed using designed synthetic DNA that facilitates the use of highly optimized human frameworks and enables the introduction of defined chemical diversity at positions that are most likely to contribute to antigen recognition.
58
what are semi-synthetic libraries?
constructed wherein diversity is controlled by the oligonucleotide synthesis. Semi-synthetic libraries are derived from unrearranged V-genes from pre-B cells or a single antibody framework with at least one complementary determining region (CDR) region genetically randomized.
59
what are chimeric antibodies?
molecules made up of domains from different species
the Fc region or all the constant regions of a mouse mAb may be replaced with those of a human or (any other species) antibody.
take variable regions from mouse and stitch onto human backbone/Fc region
60
what is a humanised antibody?
A type of antibody made in the laboratory by combining a human antibody with the CDR regions of a mouse/rat antibody.
The mouse or rat part of the antibody binds to the target antigen, and the human part makes it less likely to be destroyed by the body's immune system.
61
describe the background of humira development
- Mouse mAb (MAK195) generated against TNF-α to investigate potential use in treating inflammatory disease. – showed really high affinity and neutralising activity, bound to very specific epitope
- Mouse Abs are immunogenic and so can’t be used in the clinic.
- Used the mouse antibody as a template and created a fully human antibody
- Phage display of scFvs used to humanise mouse mAb, generated adalimumab (Humira).
- Used to treat many inflammatory diseases including rheumatoid arthritis
- TNF alpha a cytokine involved in systemic ifnlammation.
62
what is guided selection?
involves blocking steps or the use of two or more antigens, and is typically used for the isolation of epitope-specific antibodies or to find antibodies that recognize shared epitopes on different antigens
63
describe the process of guided selection
1. Clone appropriate segments of heavy (Hm) and light chains (Lm) into scFv.
2. Murine heavy chain (Hm) paired with library of naïve human light chain (Lh).
3. Murine Light chain (Lm) paired with library of naïve human Heavy chain (Hh).
Created library of one specific vh or vl combined with millions of different vl or vh regions
All mouse parts are identical but the human are all different
4. Combinations screened against human TNF- α binding. (panning stage) found the high affinity binder)
5. Successful Hh and Lh combined.
6. Stepwise mutagenesis of CDRs to maximise binding.
7. Transfer CDR sequences into the appropriate human IgG –adalimumab
End with fully human scfv identical to the specificity of the original mouse one
In this example they wanted the full antibody so they stitched it on to the base
64
describe how the gene construct is prepared for guided selection
a mouse (vh or vl) segment is combined with a human (vl or vh) and connected by a linker ((gly4ser)3)
add primers containing restriction sites (Sfi1 and Not1)
do 30 cycles of PCR
digest sfi1 and not1 to create sticky ends
65
all methods of display follow what same principle?
the genotype is connected to the phenotype
## Footnote
they all also isolate vh and vl chains from b cells and isolate the DNA to create a libray for transfection into something
66
what are the three major display systems?
virus surface display
cell surface display
cell free display
67
what are the two types of virus surface display?
phage display
eukaryotic virus display
68
what are the three forms of cell surface display?
bacterial display
yeast display
mammalian cell display
69
what are the four types of cell free display systems?
ribosome display
mRNA display
covalent DNA display
CIS display
70
what is yeast surface display?
a type of surface display that uses a yeast cell
a powerful technology used to isolate and engineer proteins to improve their activity, specificity, and stability. In this method, gene expression is regulated by promoters, and secretion efficiency is affected by secretion signals
71
in yeast surface display what factor links genotype to phenotype?
mating adhesion protein Aga2p
## Footnote
the Aga2p and Aga1 protein interaction on the cell surface: the genotype is on the inside of the cell, phenotype on the outside of the cell
72
what is the composition of the gene constrct needed for yeast surface display?
* Relying on mating adhesion protein Aga2- - links our genotype to the phenotype
- gene sequence containing vh linker and vl
* Gene sequence flanked on either side by HA and Cmyc tag (used to screen and pan later on )
* The construct will go into expression vector – yeast display plasmid
* Under control of galactose promoter – how we can turn it on and off
73
describe the process of yeast surface display
* Transfect yeast cell with expression vector
* When protein expressed it will be made in the ER then shuttle into golgi
* Once in the golgi the aga2p will interact with endogenous aga1 protein via its disulphide bridge
* Whole complex shuttled out onto cell membrane
* Isolate scfv of interest by panning
* Take further through directed mutagenesis (to increase affinity of scfv (diversification))
* The HA and c-myc epitope tags can be used for immuno-fluorescent labeling and detection
74
what components are used in ribosome display?
- mRNA for the scfv
- only necessary proteins purified from Ecoli cells (those involved in translation: 302 ribosome, translation factors, initiation factors, elongation factors – everything needed to translate mrna into protein)
75
what is the most succesful system for ribosome display?
Protein synthesis Using Recombinant Elements (PURE) system
76
what are the main advantages to ribosome display?
* contain little nucleases and protease so no degradation of mrna or protein so you can get a good yield
* cell free is you are not effected by transformation efficiency – its inefficient to get the dna into cells
77
how is genotype connected to phenotype in ribosome display?
## Footnote
what bond?
Linking genotype to phenotype by **the stable ribosome complex**
## Footnote
Normally once translated the protein will dissociate from ribosome – however conditions are such that it forms a stable complex between all three molecules: mRNA, ribosome and scfv, importantly this is a non covalent bond so conditions are critically controlled so to maintain that bond and the complex stays together
78
describe the process of ribosome display using the PURE system
* (i) Transcription of DNA library to mRNA library.
* (ii) Formation of the mRNA–ribosome–polypeptide ternary complex by translation from the mRNA library by the ribosome - Conditions tightly controlled to allow folding and stable complex to form between rRNA and scfv
* (iii) Selection of the ternary complex bound to an antigen by antigen panning
* (iv) Isolation of mRNA from the selected ternary complex.
* (v) Preparation of DNA library for the next round of selection by reverse transcription (RT)-PCR.
then diversification steps
79
what is ribosome display?
a cell free display mechanisms that uses translational proteins from ecoli to form a complex of mrna scfv and ribosome
a technique used to perform in vitro protein evolution to create proteins that can bind to a desired ligand
80
how is mRNA protein fusion similar and different to ribosome display?
Similar to ribosome display in that it is cell free
unlike ribosome display that uses Ecoli protein it uses enzymes isolated from rabbit reticular sites
and
the genotype is linked to the phenotype by a covalent puromysocin attachment (more stable) where as in ribosome display it is a noncovalent ribosome complex that links them
81
how is genotype connected to phenotype in mRNA protein fusion?
Linking genotype to phenotype via covalent puromyocin attachment
## Footnote
puromycin is attached to 3’ end of the mRNA - its structure is similar of tyrosol trna and so it serves as a small molecule mimic of trna
as ribosomes move down the mRNA during translation the puromycin brought in close proximity enough to enter the binding in site within the ribosome so mRNA gets covalently attached to the c terminal of the peptide being produced
82
describe the process of mRNA protein fusion
(ai) mRNA of interest is ligated to a synthetic oligonucleotide containing puromycin at its 3′ end.
(aii) The ribosome initiates synthesis of the template and reads in a 5′→3′ direction. tRNAs and amino acids are in the P- and A-sites of the ribosome.
translation continues and when it reaches the 3' end
(b) Puromycin enters the ribosome, covalently attaching the template mRNA to the C terminus of the nascent peptide.
(c) Reverse transcription generates cDNA that can be amplified by PCR and diversified
(d) selection can then take place
83
what is the typical max library size generated when phage display is used?
10^10 to 10^11
84
what is the typical max library size generated when yeast surface display is used?
10^7 to 10^9
85
what is the typical max library size generated when ribosome display or mRNA protein fusion is used?
10^12 to 10^13
86
what selection methods can be performed after phage display?
immobilised antigen, tissue sections, cells, subcellular fractions
## Footnote
versitile
87
what selection methods can be performed after yeast surface display?
cell sorting technique such as magnetic beads, Fluorescence-activated cell sorting (FACS)
88
what selection methods can be used adter ribosome display or mRNA protein fusion?
immobilised antigen
## Footnote
limited
89
what potential applications are there for phage display?
- Selection of Ab fragments from natural and synthetic libraries.
- Affinity maturation.
- Stability increase.
90
what potential application are there for yeast surface display?
- Selection of Ab fragments from synthetic libraries.
- Affinity maturation.
- Stability / expression increase.
91
what potential applications are there for ribosome binding and mrna protein fusion?
- Selection of Ab fragments from synthetic libraries.
- Affinity maturation.
- Stability / expression increase.
92
what are the main advantages of phage display?
- Large libraries widely available.
- Technically robust.
- Most established.
- Easy to use.
- Automated
93
what are the main disadvantages to phage display?
- Introduction of diversity by cloning is slow.
- Large libraries difficult to make,
- limited by transformation efficiency.
94
what are the main advantages of yeast surface display?
- Fast in combination with random mutagenesis.
- Direct screening for kinetic parameters (e.g. affinity constants) measured in cells.
- Multiplex screening.
- Post-translational modification.
95
what are the main disadvantages of yeast surface display?
- Sorting expertise and equipment needed.
- Sorting speed limited.
- Transformation efficiency
96
what are the main advantages of ribosome display and mrna protein fusion?
- Intrinsic mutagenesis.
- Fastest of all systems.
- Amenable to automation.
- Enables selection of otherwise toxic Ab fragments.
- No cell transformation.
97
what are the main disadvantages to ribosome display and mrna protein fusion?
- Small mAb panels.
- Limited selection scope.
- Technically sensitive.
- Selection in buffers different from intracellular milieu (folding issues).
98
what are four beenfits of scFvs?
* No requirement for immunisation.
* Can be produced in large amounts in inexpensive expression systems (E.coli, yeast).
* Small size (25kDa vs 150 kDa of full sized mAb) allows for better penetration of tissues (tumours).
* Can easily be tagged with toxins/radionuclides for therapy and diagnosis.
99
give 6 examples of phage display derived antibodies approved by the FDA or EMA
* adalimumab (TNFalpha)
* belimumab (b lymphocyte stimulator)
* necitumumab (EGFR)
* ramucirumab (VEGFR2)
* ranibizumab (VEGFA)
* rexibacumab (protective antigen for anthrax)
100
why is the serum half life of scfv so short?
Scfv is removed from the body much faster only lasting a couple of hours – because it has no fc region so cant interact with FcRn
they are also more suseptible to proteases
101
what benefits are there for imporving the serum half life of scFvs?
If in body longer – have greater time to exert therapeutic effects, patient doesnt need to visit hospital as regularly
102
how can we improve the serum half life of scFvs?
* Make them a little bit bigger. (not as big as mAb) – creating different antibody fragment formats – makes them more resistance to proteases in the blood and reduces their clearance in the kidneys
* Conjugate them to PEG and other polymers - PEGylation (polyethylene glycol), Polysialylation, HPMA (N-(2 Hydroxypropyl)methacrylamide), Dextran
* Conjugate them to albumin; utilises FcRn recycling pathway
103
what are the possible antibody fragment formats?
* Vl gomain
* Vh domain
* scFv
* triabody (trivalent)
* tetrabody (tetravalent)
* IgG
* minibody (bivalent)
* Fab (bispecific)
* Diabody (bispecific)
* Bis-scFv (bispecific)
104
what are the advantages to a short serum half life?
scFvs can easily be tagged with toxins/radionuclides for therapy and diagnosis.
these gamma or beta emitters can do their job and are quickly removed from the body thereby reducing the toxic efffects
105
what are the benefits for using a scFv with a short serum half life for detection?
:reduced background
(high tumour:blood uptake)
: reduced toxicity
: improved tissue penetration
106
what are the advantages of using a scFv with a short serum half life for therapy?
: reduced toxicity
: improved tissue penetration
107
give an example for when the short serum half life of a scFv is beneficial
for radioimmunotherapy to target the beta emitter drug to cancer cell, short half life means a reduced toxic effect because it will be removed form body quickly
108
give an example for when the short serum half life of a scFv is beneficial
for radioimmunotherapy to target the beta emitter drug to cancer cell, short half life means a reduced toxic effect because it will be removed form body quickly
109
give three examples of radionucleotides used for detection that cna be tagged onto scFvs?
| gamma emitters
99m Techntium
123Iodine
124Iodine (PET)
110
give two examples of radionucleotides used for radiotherapy that can be tagged onto scFvs?
| beta emitters
90Yttrium (liver cancer)
131Iodine (thyroid cancer)
111
what toxins/drugs can be tagged onto scFvs for cancer therapy?
* chemotherapy eg doxorubicin, taxol
* BL22 (anti-CD22 fused to a fragment of Pseudomonas aeruginosa exotoxin A)
112
what are peptide bicyles and how do they increase serum half life?
## Footnote
gregory winters work once again
* Utilises 2 CDR loops (hence bicycle) identified and optimised for specific antigen binding using phage display. Still binds very tightly with high specificity to target.
* These loops are stabilised by a chemical crosslinking compound tris bromomethyl benzene so the three bromines react with the three cysteines to create stable peptide structure (react in water)
* High plasma stability due to low protease interaction.
## Footnote
the two main CDR loops that ocntirbute to affinity
reaction is rapid
113
how can we restrore effector function in scFv
1. Fusion of scFv to an appropriate Fc-domain.
2. Fusion of scFv to cytokines.
| requires bivalent binding
## Footnote
to stimulate immune cell types such as monocytes, macrophages, natural killer cells, dendritic cells, T cells and B cells and restore some effector function
114
give an example of recovering effector function by Fusion of scFv to an appropriate Fc-domain?
scFv targeting FGFR1 fused to CH2 and CH3 of IgG1 for treatment of lung cancer
constant region of IgG antibody fused – now bivalent so it can now direct cell killing
now its 100kda instead of 25kda which is still less than a full antibody 150kDa so will help with tissues penetrance
115
give an example of recovering effector function by Fusion of scFv to cytokines?
Immunocytokines combine specific antigen binding with bioactivity of cytokines, demonstrate immunomodulatory function.
e.g. L19-IL2; Diabody targeting extra-domain of fibronectin fused to IL2. In phase III clinical trials for malignant melanoma
il2 important for t cell activation and proliferation
now scfv will bind to antigen on target and activate the t cells with the IL2
116
what are intrabodies?
an antibody that works within the cell to bind to an intracellular protein
* Full length antibodies cannot fold correctly or form stable structures within cells due to the highly reducing environment (no –S-S- bonds). (rely on cysteins too much for structure)
* They are also unable to penetrate the cell membrane hindering their ability to inhibit intracellular targets.
* Intrabodies designed to retain antigen affinity and are most commonly scFvs or single domain VH (-S-S- bonds appear not to be so important in stabilising structure).
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what role do the cysteines in antibody structure have in their ability to penertrate the cell membrane
* Full length antibodies cannot fold correctly or form stable structures within cells due to the highly reducing environment (no –S-S- bonds). (rely on cysteins too much for structure)
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how are intrabodies made?
Add coding sequence of scFv fragment which blocks target function into a vector either (if we want to target it to the nucleus we can add a nuclear localisation signal). Transfect this into a cell where it is transcribe and translated and then can go about its function
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how are intrabodies made?
Add coding sequence of scFv fragment which blocks target function into a vector either (if we want to target it to the nucleus we can add a nuclear localisation signal). Transfect this into a cell where it is transcribe and translated and then can go about its function
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what things can we use to get intrabodies into target cells?
viral delivery: adenovirus, adenoassociated virus, retrovirus/lentivirus
non viral delivery: antibody couple vector delivery, dendrimer, liposome, nanoparticles with polymers, protein transduction domain peptide
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give an example of an intrabody used for cancer
e.g. TAR1, a human anti-p53 scFv.
Specifically binds mutant p53 with high affinity and restores its wild-type conformation.
Induces transcriptional transactivation of p53 target genes and induced apoptosis.
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instead of using antibody scaffold what other protein scaffold can we engineer for binding?
A – Affibody
B – Knottins
C – Designed ankyrin repeat protein (DARPin)
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what are affibodies
Affibody: Three helix bundle that is a natural Ig-binding domain. (engineered antibody/ antibody mimetics - affibody molecules are more stable and have a lower molecular weight of ~7 kDa)
: Used for in vivo imaging.
For example used for her2 targeting, bind with high affinity which has been good for in vivo imaging
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what are knottins?
Knottins: Cystine knot peptide, contains a common folding motif in which a disulphide bond laces between a peptide loop formed by two others.
: Used as imaging agents.
Small and really stable
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what are DARPins?
Designed ankyrin repeat protein (DARPin):
are genetically engineered antibody mimetic proteins typically exhibiting highly specific and high-affinity target protein binding. They are derived from natural ankyrin repeat proteins, one of the most common classes of binding proteins in nature, which are responsible for diverse functions such as cell signaling, regulation and structural integrity of the cell. DARPins consist of at least three, repeat motifs or modules
Modular scaffolds that consist of tandem repeats of a 10–50 amino acid sequence.
: Potential for novel diagnostic and therapeutic applications
