Immune recognition Flashcards
Innate immunity
broad range of recognition, DAMPS, PAMPS – fast response – action is short lived, no memory, recruits adaptive leukocytes
Adaptive immunity
humoral (B cell) cell mediated (T cell)?? Also get cytokines to stimulate both adaptive and innate, express extreme number of antigen receptors, specificity high, proliferation needed, slower sponse, but around for long and generate memory
PAMPs
recognised by innate immune system
expressed on a wide variety of different pathogens
Antigens
any structure that can illicit an adaptive immune response via antigen receptors
recognised by adaptive immune system, unique to a specific pathogen and recognised by a specific antigen receptor
Adaptive immune cells have to:
Recognise foreign antigen
Destroy or produce products that can destroy the pathogen
Develop immune memory
Disseminate immunity around the body
Lymphocytes
B cells - Recognise pathogens outside cells - produced in bone marrow
T cells - Recognise intracellular antigens that have invaded - produced in thymus
Compare BCRs to TCRs
Both have complementary determining regions
BCRs:
Identify a wide range of antigens
Binds native antigen, so doesn’t require antigen processing
co receptors aren’t needed
secreted (as antibodies)
not MHC restricted
TCRs:
Antigens of peptide or lipid
doesn’t bind native antigen, requires processing
Co-receptors needed
not secreted
MHC restricted
CD8+ cytotoxic T-cells
- recognize virus-infected or cancerous cells.
- induce cell lysis
- produce antiviral cytokines
CD4+ Helper T-cells
provide critical help to B cells and CD8+ T cells
3 signals required for T cell priming
1st TCR signalling via MHC moleciles
2nd - co-stimulatory e,g, B7
3rd - cytokines e.g IL2,IL12
Results in genetic and transcriptional changes that causes T cell activation and large pools of T cells that all target the particular antigen
CD8 vs CD4 MHC classes
CD8 = MHC I
CD4 = MHC II
what is MHC restriction
The requirement of the TCR to engage with both peptide and MHC concurrently
What does MHC stand for, and another name for them
Major Histocompatibility Complex
also called HLA
MHC I and II differences
Cell expression: I = All nucleated cells
II = Restricted (e.g. APCs)
Peptide origin: I = Cytosol, II = Endosomal
Co-receptor: I = CD8, II = CD4
extracellular domains of MHC I vs MHCII
Extracellular domains of both form an antigen binding cleft – generates a cradle for a short amino acid fragment – bound by anchor residues in. Binding pockets
MHC1 binding cleft has closed stucture so can only bind small peptides 8-10aa
MHC2 less closed, often overhang regions and can bind longer polypeptide chains
Structure of MHC I vs II
SEE PHOTO
HLA/MHC genes are polymorphic and polygenic, define both
Polymorphic: Multiple forms of each gene exist within the population (termed alleles)
Polygenic: Multiple genes with the same function but slightly different structures: broad range of peptide binding specificities (3 pairs of the gene which encodes a different HLA type (for HLA-1))
How are MHC alleles expressed
MHC are co-dominantly expressed
Set of linked alleles (haplotypes) inherited from parents (express everything that you inherit)
Designed to create a combination if genes that is almost unique to each individuals (this is what makes organ donation difficult)
Why do we have such diversity in HLA
Diversity allows wide variety of peptides to be presented
With three HLA class I genes and four HLA class II genes on each chromosome 6, a human typically expresses six different HLA class I molecules and eight different HLA class II molecules on his/her cells:
Total theoretical number of combinations ~ HLA-I(1.2 x 10^7 ) x HLA-II(1.8 x 10^10) = 2.25 x 10^17
Where do polymorphic residues i MHC tend to reside
Within the binding groove
Different types can differ from 30aa or just a few – but still enough to change specificity
MHC I and II presentation/expression
MHCI – expressed on surface of all nucleated cells within the body. Antigens generated – processed by proteasome and cut into peptides, moved to ER where bind to MHC, this is then transported to the surface to be recognized by cytotoxic T cells so that the cell gets destroyed
MHCII – present antigens that are derived from extracellular pathogens that have been engulfed (bacteria and parasites) - degrade into peptides – loaded onto MHC II – transported to cell surface where its presented to helper T cells
Structure of classical Class I MHC
- Highly polymorphic heavy chain around 44kDa:
3 extracellular domains, a1-3, then transmembrane segment followed by a cytoplasmic tail - Lighter chain (called beta-microglobulin) non-covalently attached to the heavy chain around 12kDa
- A3 and B2m each contain two cystine residues - form disulphide bonds - folded structure - closely resemble immunoglobulin domains - crucial for T cell induction through the interaction with co-receptors and co-stimulating molecules
- B2m interacts extensively with a1 and a2 domains - to enhance peptide binding in groove and to stabilise MHC heavy chain
- A3 has no impact on peptide binding but is still important for the overall integrity of MHC
Which region of MHC I is polymorphic
a1+a2 domains form a domain composed of two alpha helices sat onto of 8 beta strands
^forms cleft in which peptide antigens can bind (peptide binding cleft) - also plays a role in T cell receptor specific recognition
- Global polymorphism analysis of all available HLA sequences shows this (Abualrous 2021)
Antigen processing and presentation MHC I
- All nucleated cells express MHC I on surface
- MHC I present endogenous peptide antigens to CD8+ T cells
Presentation generated within cytoplasm
1. cut up by proteasome, then transported through the Transporter associated with antigen processing (Tap) complex
2. move to the ER where further trimmed by ER amino-peptidases, creating peptides of 8-10 AAs
3. Association of Heavy chain and B2m with these peptides is coordinated through larger complex called a peptide loading complex, helps with loading and then transport to the cell surface for CD* cytotoxic T cell recognition
Structure of the MHC I peptide binding site
- Peptide binding groove has closed ends - acting to restrict the size of the bound peptide to around 8-11AAs
- Each half of the a1/a2 complex contributes half the 8 beta stranded sheet, along with an alpha helix from each creating a “wall” - this creates the groove
- Most variable residues in groove either face into the groove or up from the top of Both piecesn?? - these act to confers unique peptides and for TCR binding
- Majority of variable resides are located in the central portion of the cleft, with clusters of highly conserved (often aromatic) resides that hold the peptide termini in place
Explain the experiment of polymorphism done by Abualrous et al (2021)
Polymorphisms only occur in restricted number of residues
calculated an entropy score (a measure of diversity) for each AA in the MHC I molecule (HLA A, B and C)
residues with high entropy discovered tended to line peptide groove
Calculated entropy score for each AA of MHC II molecule (HLA-DR, DQ, DP) - found same thing, and in this case were predominantly from the b-strand. Also HLA DQ and DP showing high entropy at b-86 (P1) residue but DR did not
Explain how the relative arrangment of the polymorphic residues that line the peptide binding cleft, creates pockets that accommodate the predominant AA side chains of the peptide antigen (therefore anchor peptide onto MHC)
- 6 major molecular pockets (A-F) - position of the pockets is the same in every binding cleft of an MHC molecule
- 2 deep pockets - called Primary anchor residues: - form H binds to bind that peptide to groove
B - accommodates 2nd residue (P2) from its N terminus
F - Side chain of the C-terminal AA can insert deeply - Therefore P2 and C terminus play significant roles in interaction between peptides ands MHC
- Can also get secondary anchor residues - located in middle, weakly bind - can enhance and further tune the affinity of a particular peptide
- Position of pockets = highly conserved
MHC molecules bind and present peptides with…
a combination of sequence-independent and -dependent features (allows binding and presentation of a wide range of peptides)
Sequence-independent binding of MHC I
- Size and shape of the peptide-binding groove - determines length of peptide that can bind
-Chemistry of the amino acid residues that line the groove - provides specificity
- residues that line the groove are primarily hydrophobic and anchor the peptide in place
Sequence-dependent binding of MHC I
- The specific amino acid residues that form hydrogen bonds with the peptide
- Primary and secondary anchor residues
- Wide tolerance for many side chains at the other positions.
-Overall charge distribution of the groove
Sequence independent binding provides a mechanism by which…
a single MHC I allele can bind a large variety of peptides - important for its antigen presentation function
Describe peptide binding in a sequence independant manner of class I MHC
Bjorkman 2016
Comparisons of multiple MHC I peptide complex structures determines through X-ray crystallography have shown that the termini are always mostly in same position
H bonds to conserved residues at each end of the groove:
- backbone atoms of N-terminal residues H bond with conserved tyrosines (within A pocket)
-backbone atoms of C-terminus H bond with conserved residues of pocket F
Explain the class I allele specific binding motifs
Allele specific sequence motifs - (defined by two primary anchor residues (at least)) - means that each allele will have a distinct peptide specificity
(Abualrous 2021) - pooled sequencing of eluted peptides from HLA molecules expressed at the cell surface, key residues identified using mass spec
e.g. HLA-A (02) allele - includes a hydrophobic residue within pocket B - this means that it will accomedate a peptide with a medium sized hydrophobic AA at the P2 position (e.g. Leu, Ile, Val, Met). also in P9 pocket will bind the same
HLA-B (27) allele - Pocket B made up of an acidic residue - and so will preferentially accomediate a peptide with an arganine (R) at position 2 (its basic). At position 9 will accomedate Leu, Phe, Arg, Lys.
(antigen) Peptide specificity is dependant on what?
The Particular polymorphism within the peptide binding groove pocket on the MHC molecule
What do the non-conserved residues of the class I MHC molecules allow for in antigen binding?
Non-conserved = NOT position 2 or C-terminal (often 9) residues
Allow accommodation of a wide variety of variants of peptide sequences
How do longer peptides fit into the small MHC I binding groove
Create a bulge (or peptide arch) in the centre (P3-P6), still anchored o the anchoring residues (2 and C-terminus)
What does the extent at which a peptide bulges from the binding groove help determine
The variable immunogenicity of different peptides
conformation of the bound peptide influences how the MHC interacts and presents the peptide to the appropriate TCR
Where are MHC II molecules expressed
A small subset of highly specialised immune cells called professional APCs - Macrophages, dendritic cells, neutrophils and B cells
Structure of MHC II (more detailed)
Hetrodimeric
Alpha chain (a1,a2) and beta chain (b1,b2) (the 2 subunits)
Each subunit contains:
- Half the extracellular peptide binding domain
- Immunoglobulin like domain (also extracellular) - stabalise peptide binding groove and provide intecation site for CD4 co-receptor
- Transmembrane alpha helix
- short cytoplasmic tale
Antigen processing and presentation of MHC II
Present peptides that have originated from exogenous/external proteins
- these proteins are first taken up by the APC by phagocytosis
- transferred to an early then late endosomal compartment
- Loaded onto MHC II protein
- shuttled up to surface for presentation
Structure of peptide binding groove of MHC II
8 stranded b sheets creating a platform, laterally enclosed by 2 alpha helices
binding groove open at both ends - peptides as long as 20 AAs can bind, creating overhang regions that protrude out the ends
each subunit provides 1 helical region and 4 beta strands (helices are roughly antiparallel)
Unlike MHCI, Symmetry is broken by short 3 (subsript10) helix of the alpha strand, with beta strand you get a prominent kink in the middle of the helical region - its thought that these features contribute to the canonical N to C orientation of the peptides that accommodate the groove
Two types of interactions in MHC II that hold peptide in place
- Conserved network of 12-15 H bonds between the peptide backbone and MHC II residues (regularly spread throughout length of binding site) - stops peptide folding - keeps it extended
- Interaction of Polymorphic side chains of peptide antigen and the specificity pocket that sits at the bottom of the binding groove - these pockets are termed P1,4,6,7,9 (named after the respective side chains of the bound peptide that accommodates them)
Where does the most allelic variation occur for MHC II
within the peptide binding region (same as MHC I)
BUT unlike MHC I - most polymorphism occurs within the beta chain, whereas alpha chain shows little polymorphism
The preferential binding of a specific peptide sequence (to MHC II), can be determined by…
The polymorphism present in the MHC II binding pockets
How have scientists gone about identifying different peptide repertoires that MHC molecules bind?
The technique of Immunopeptidomics
Explain how immunopeptidomics is used in terms of MHC molecules
MHC associated peptide purification, identification and validation by UPLC tandem mass spec:
1. MHC molecules purified (from biopsy, tissue cells ect) - Homogenise in a bead beater to break up tissue and cell membranes
2. Cell lysate now produced - this is purified to get MHC molecules by immunoprecipitaton
3. MHC molecules still attached to beads eluted from mixture by acid elution
4. now left with MHC molecule components along with the peptides they are bound to
5. High performance liquid chromatography (HPLC) to separate out MHC associated peptides
6. Peptide fraction collected analysed by liquid chromatography and tandem mass spec, sequences are identified using spectral interpretation
7. Use label free quantification and motif analysis - to quantify the numbers of the different peptide sequences
these allow for the lengths (MHC I shorter and MHC II longer) and specific residues of the peptides to be determined and gives us useful info
TCR recognition of peptide-MHC complex
CD8+ T cells recognise peptide bound to MHC I with their TCR as well as with co-receptor CD8
CD4+ T cells recognise peptide bound to MHC II with their TCR as well as co-receptor CD4
TCR structure
(alpha and beta chain) Transmembrane tether, Constant domain, Variable domain containing 6 CDR loops
TCR antigen binding domain structure
CDR loops: CDR2a, CDR1a, CDR3a, CDR3b, CDR1b, CDR2b
How does TCR diversity arise
- Permutations of V(D)J segments for a and beta chains
- Imprecise joining of segments in recombination
- Random addition of non-template nucleotides at junction sites
- combination of a and b chains
Explain briefly VDJ recombination and how it leads to diversity
- TRA gene and TRB gene (coding for alpha and beta chain)
- for TRA, V and J recombination, End up with germline encoded (CDR1a and 2a - made from V) and hyper-variable (CDR3a - made from J and C)
- for TRB, VDJ recombination, end up with germline encoded (CDR1b and 2b - made from V) and Hyper-variable (CDR3b - made from D, J and C)
- in both cases combination occurs from transcription, splicing, and translation
SEE PHOTO
What/where do mutations in VDJ recmobination occur
P/N-mutations
Occur in the hyper-variable regions
TRA - after V before J
TRB - after B, either side of D, before J
Where does most of the understanding of molecular details by which TCRs engage with peptide MHC come from?
Crystallographic studies
Explain TCR engagement with MHC peptide (for CD8 and CD4 cells)
TCR interacts with MHC molecule, interaction is assisted by CD8/4 molecule which binds directly to MHC
How does the TCR dock to the MHC peptide complex
Docks over the top of the MHC peptide complex, in a diagonal manner on top of the peptide binding groove (normally around 45^)
alpha chain positioned over the top of MHC alpha 2 helix, beta chain over alpha 1 MHC helix
CDR 1 and 2 loops interact with MHC iteslef, Hypervariable CRD3 loops that engage and interogate the peptide
Explain additional mechanisms requred as well as hypervariability of the CRD3 loop to allow TCR plasticity and therefore recognition of all possible foreign antigens
- Altered angle/ register
- Flexibility in the CDR loops
- Tolerance of amino acid substitutions
ExplainTCR plasticity by altered angle/register
Macrolevel changes
altered angle - angle at which the TCR is coming down on top of the binding groove
altered register - position of binding in reference to central point of peptide MHC complex
- both of these can change to allow for TCR adoption to recognise different peptides
Explain TCR plasticity by flexibility in CDR loops
micro level flexibility
Flexibility of CRD loops - allows them to accommodate different shapes of the peptide (think of CDR loops as fingers coming out)
Explain TCR plasticity by tolerance of amino acid substitutions
TCRs tend to focus their interaction on 2-4 upward facing AAs of the peptide (called hotspots)
This residue focused TCR engagement allows for tolerance of substitutions at other AA residues of the peptide
Positive implications of TCR plasticity
small NO. of TCRs (25mil) provide immune cover for greater theoretical No. of foreign peptides
T-cell cross-reactivity - fewer T cells needed to scan forign peptides before on interacts - ensuring rapid response and challenge for pathogens to escape recognition
allows heterologous immunity (single T cell can respond to several infections)
Negative implications of TCR plasticity
Autoimmune diseases - T cells activated by pathogens and then cross-reacting with a self ligand (molecular mimicry)
Acute rejection of HLA-mismatched grafted organs
What is needed other than binding of pMHC and TCR for T cell activation and why
affinity of binding between a given pMHC and a TCR is relatively low.
Sustained interaction between the naïve T cell and APC is needed to trigger T cell activation - done by co-stimulation and cytokine help (IL-2 binds to IL2R - Autocine for CD4, paracrine (from Th cells) for CD8)
Explain the conformational changes leading to T-cell activation
After peptide recognition with TCR, conformational changes occur
- Change in TCR to bring variable regions even closer to peptide to stabilise interaction - allowing TCR to make specific contacts with antigen and recognise with higher affinity
- signal transmission into cell - associated CD3 chains bound to membrane, contact with constant regions of the TCR, allows the intracellular CD3 chains to become phosphorylated in their ITAM regions by LCK
- This leads to the recruitment of another kinase called ZAP70, which then goes on to phosphorylate tyrosine residues of the LAT signalosome, leading to other various downstream signalling molecules to become active - eventual T cell activation
MHC and disease predisposition
Lack of immune response to infection or cancer - suffer from the disease
Inappropriate immune response - autoimmunity diseases or hypersensitivity occur
Give examples of autoimmune diseases and the particular association of MHC alleles linked to them
MS - DR2
Graves disease - DR3
RA - DR4
Explain how knowledge of how antigens interact with MHC molecules and TCRs - thus their immunogenicity, is useful
can be leveraged for vaccine and drug development.
requires an understanding of which peptides will have the greatest opportunity to bind to any given HLA allele
Vaccine design for infectious diseases, cancer and autoimmunity
Prediction of MHC class I-presented peptides is a critical tool
Computer-aided methods developed to identify candidate peptides e.g. deep-learning models: rank peptides’ binding affinities to MHC class I molecule(s)
These models are created with the goal of predicting which peptides will have the highest binding affinity for the HLA alleles
Explain augmenting the antigenicity of a cytotoxic T lymphocyte epitope according to the structure of MHC–peptide complex.
Normally involves replacement of amino acids within the sequence of a peptide epitope
Typically MHC anchor residues or to alter the TCR-interacting residues within antigen peptide.
increasing the stability of the MHC-peptide-TCR complex - augmented antigens can achieve more potent immune responses
Give two examples of augmenting MHC anchor residues
(Chen 2005)
SLLMWITQC -> SLLMWITQV
- HLA–A02 with tumor epitope
- induces greater numbers of cross-reactive cytotoxic T lymphocyte
(Ibarz 2006)
RMFPNAPYL -> YMFPNAPYL
- HLA -A02 with WT1 oncoprotein
- Improved T-cell responses
Example of augmenting antigen peptide sequence
Slansky (2000)
- used mouse model to see effects of immunisation with altered peptides
- non-mutated H-2Ld (mouse HLA) restricted peptide antigen derived from gp70 amino acids 423–431 (AH1; sequence SPSYVYHQF).
- gp70 expression is silent in most normal tissues but is active in many mouse tumours.
- AH1 is the dominant target for the CD8 T cell responses against the CT26 colorectal tumour
- AH1 has relatively high affinity for H-2Ld but provides relatively weak immunization against CT26 - challenge!!
- Alanine scanning mutagenesis showed that alanine substitution of valine at residue five in the AH1 peptide enhanced stability of the MHC-p-TCR complex
- SPR used to show the enhanced binding mediated by the AH1-A5 peptide relative to the AH1 peptide was not a result of an increased affinity for MHC BUT H-2Ld -AH1-A5 binds TCR with higher affinity than H-2Ld-AH1.
Increasing the stability of the MHC-peptide-TCR complex does what?
achieves more potent immune responses
Features of BCRs
- located on the surface of B cells
- composed of two identical heavy chains and two identical light chains that are held together by disulfide bonds
- variable regions of the heavy and light chains form the antigen-binding site allowing BCR to recognize specific antigens
- allows B cells to recognize a wide range of antigens and mount an effective immune response against them
What happens when an antigen binds to a BCR
triggers a series of intracellular signaling events that activate the B cell
proliferation of B cells, differentiation into antibody-secreting plasma cells, and the generation of memory B cells
differences in struture of the two forms of BCR
Membrane bound - extracellular part same as secreted form. 2 Transmembrane spacer with a hydrophobic segment, cytosolic segment
Secreted form - antibody - Hydrophillic segment on end
Structure of BCR
2 heavy chains and 2 light chains.
Each chain contains a constant region (CH or CL) and a variable region (VH or VL)
hinge region between Fc domain and Fab domains
SEE PHOTO FOR FULL STRUCTURE
Structure of BCR complex
BCR associated with Iga/Igb heterodimer
BCR signalling
BCR association with Iga/Igb heterodimer (e.g. CD79) causes phosphorylation of the ITAMS present on this by LYN
many different signalling cascades occur, resulting in translocation of FOXO, NFAT, ERK and NFkB to the nucleus to induce gene transcription leading to proliferation and differentiation of B cells
Immunoglobulin classes
IgG - bound and secreted
IgM - bound and secreted (as pentamer)
IgD - mainly bound
IgA - Bound and secreted (as dimer)
IgE - Bound and secreted
Compare membrane bound structure of immunoglobulins IgM-BCR and IgG-BCR
All have Vh, Vl and Cl
as well as CH1-3
IgM has extra CH4 region at the bottom of the receptor, this is what associates with Iga/Igb (compared to CH3 in IgG)
Explain the antigen binding domain of BCRs
made up of six complementarity-determining regions (CDRs) - protrude from the V domains
CDRs - most variable regions responsible for specificity of antigen binding
CDR1 and CDR2 are relatively short and located at the N-terminal end of the variable domains, while CDR3 is the longest and most diverse
CDRL1-3, CDRH3-1
Comparison of BCR and TCR structure
SEE PHOTO
BCR bigger
when it comes to the complementary determining regions (CDRs) main difference is in name
BCR - CDRH1-3, CDRL1-3
TCR - CDRb1-3, CDRa1-3
Antibody positions that contact the antigen
Averaged known crystal structures of antibody antigen binding partners
Seen that the majority of AAs making contact woth the antigen reside within the CDR regions
There are als structural positions (residues) within the CDR that are seen to never make contact with the antigen
also seen that some of the frame residues (not in the CDR) may also play a role in antigen binding - theres a commin farme position seen that is sometimes refered to as CDRH4
Molecular interactions between CDRs of BCRs and antigens
- Electrostatic interactions: attractive or repulsive depending on charges
- H bonds: relativly weak, but important
- Van der Waals interactions: important in creating close contact
- Hydrophobic interactions: Occur between nonpolar AAs
1-4: important in stabilising he interaction between CDRs and antigen
- Cation-pi interactions: Noncovalent interaction between cation and an electron cloud of a nearby aromatic group
- Shape complementarity: CDRs can adopt different conformations to fit the shape of the antigen, and the shape of the antigen can also be complementary to the shape of the CDRs
factors contributing to specificity of CDRs
AA sequence: generated through somatic recombination and somatic hypermutation - allows for a wide range of possible amino acid sequences and a corresponding diversity of potential antigen binding sites
Structural conformation - flexibility: CDRs are highly flexible and their specific conformation of the CDR loops contributes to the specificity of the BCR
Structural conformation - binding pockets: The CDR loops form a binding pocket or groove that is specific to each BCR
Affinity: The CDRs can impact the affinity of the receptor for its antigen by affecting the strength of the interactions between the receptor and the antigen
Amino acid sequence of CDRs
(North 2010)
compared 703 solved antibody structures, looked at CDR sequences and length
Sequence: fairly common consensus of AAs flanking the CDR regions
Length: In light chain - CDR1 10-17 AAs
CDR2 8-12
CDR3 1-13
In Heavy chain - CDR1 10-16
CDR2 8-15
CDR3 5-26
They showed that CDR H3 showed most variability in length and sequence
Explain how CDR diversity occurs
VDJ recombination (heavy)
VJ recombination (light)
can combine in many ways to create around 2.3 million potential different structures
also non-homologous recombination is very error prone - ups possible combinations to 4 million in the CDR3 loop
Why is the CDR3 region of BCRs most variable?
Maps to where VDJ come together in heavy chain and where V and J come together in light - error prone non-homologous recombination adds diversity
Where does the most change in BCRs occur in affinity maturation
Variable domain - creates changes from initial to high affinity antibody
Role of CDR 3 in antigen binding in BCR
CDR 3 located very centrally, main thing that binds to antigen
also shows the most conformational change between ligand bound and ligand unbound state
Structural conformation - flexibility of BCRs - explain
Adaptation to antigen shape: The flexibility of the BCR allows it to adapt to different shapes and sizes of antigens.
High specificity: The CDRs are able to adopt a wide range of conformations, which allows the BCR to bind to a diverse range of antigens with high specificity.
High affinity: The BCR can adjust its shape to make more contacts with the antigen, resulting in a stronger binding interaction.
Type of topography of the CDRs correlate to what?
The type of antigen it can bind
e.g.
pockets that can bind haptens
Grooves bind peptides
Plateau’s bind proteins (large)
can also have some elongated CDR3 loops that protrude into antigens e.g. an antibody that can bind a HIV antigen called gp120
Affinity and avidity
Affinity
– antigen binding by just one arm of an antibody
- Measured as the dissociation constant (Kd)
Avidity
– overall binding between multivalent antibody and the antigen.
- Influenced by both the valence of the antibody and the valence of the antigen.
- More than the sum of the individual affinities.
What can be used to measure the binding affinity of antibodies
SPR
detection of the changes of a refracted index of a thin piece of metal film
immobalise the antigen of interest - flow as solution containing antibody over
as it binds t the target, the index changes, this is detected as a shift in resonance angle by a detector
the rate and extent if shift is directly proportional to conc. of antibody being bound and its affinity to the antigen
can find dissociation constant (Koff) and equilibrium constant (Kd) and stoichiometry of binding (bi vs divalect ect.)
Methods to improve antibody affinity
Affinity maturation: selection and cloning of antibody variants with higher affinity for the target antigen - can be done by repeatedly exposing the antibody to the antigen and selecting the variants with the highest affinity
Site-directed mutagenesis: making specific changes to the antibody’s amino acid sequence to optimize its interaction with the target antigen
Protein display systems (Phage, ribosome, yeast): This involves displaying a library of antibody fragments on the surface of bacteriophage/ribosome/yeast and selecting the variants with the highest affinity for the target antigen
Protein engineering: This involves using computational methods to design new antibody variants with improved affinity for the target antigen. This can be done by predicting the effects of specific mutations on the antibody’s structure and function
Applications of structural biology in antigen recognition
Design of vaccines
Development of therapeutics
Understanding immune system function
Engineering improved antibodies
