Antibodies Flashcards
What are the 7 steps to the role of antibodies in the immune system?
What are the 2 methods of blocking antigen effect?
How does clonal expansion allow for evolutionary pressure/selection?
- IgM/IgD antibody expressed on B cell & binds to antigen by chance
- B cell internalises antigen & cleaves it into peptides
- Peptides expressed on surface B cell at grooves called MHC
- MHC-peptide interacts with T-cell receptors
- T-cells release cytokines such that B-cells can clone itself in clonal expansion
- B-cells undergo class switching & release IgG into blood plasma
- IgG interact with antigens & block effect
Opsonisation - antigen coated in antibodies triggering macrophages to ingest
Trigger complement - triggering other sections of immune system like complement to kill cells expressing antigen
Mutations occur in antibody sequence - antibodies that outcompete eachother with better binding & B-cells are expanded selectively
How are polyclonal antibodies made? briefly
What is the problem?
How are monoclonal antibodies different?
Why are polyclonal antibodies not crystallisable?
Why are monoclonal antibodies not crystallisable?
Each B cell produces 1 type of antibody in large numbers
End up with a mixture of antibodies each in small volume
Each B cell produces the same type of antibody - higher volume
Mixture of antibodies - not pure
Light chains are flexible -
What are the fragments made of & where are they?
Fab
Fv
Fc
Hinge
Where are the disulphide bonds? (3)
VL & CL - light chains
VL & VH - variable region at end of both light & heavy - antigen binding site
CH2 & CH3 - constant region
CH1 & CH2 - gives flexibility
- between the hinge
- within each domain
- connecting C-terminus light chain with heavy chains
With re to the following loci, what do these regions encode?
Lambda
Kappa
Heavy
What gene segments do the heavy regions have that lambda & kappa don’t?
How do these introduce antibody random variability? 2 reasons
What is the need for class switching?
How is random variability introduced?
2x light, heavy
D regions - diversity
Frameshift - 6 reading frames
Imprecise recombination - extra bases added to end of D fragment
Optimise antibody binding
DNA splicing of gene segments
What kind of receptors bind antibodies?
What kind of molecule is C1q?
What are the 2 steps for the activation of C1q?
How does the concentration effect C1q activation?
Fc receptors
Effector
- C1q binds to 2+ Fc fragments (antibody receptors) & triggers compliment-dependency cytotoxicity
- Molecule is lysed inducing cascade to membrane-attack complex
Cluster of IgG on cell surface - C1q binds & triggers cascade more effectively (IgM pentamer)
How are IgM or IgD class switches produced?
IgE & IgA?
Why is DNA splicing unidirectional?
What is the immunoglobulin fold made up of?
What secondary structures are present at the variable antigen binding site (FV)?
Where are carbohydrates bound?
mRNA splicing
DNA splicing - IgA from spliced DNA from IgE
DNA been excised out - as a result cannot transcribe/translate
Beta sandwich - linked by disulphide bonds
Protein loops
2x bound to CH2
How do VH & VL interact?
How do the CH2 domains interact?
With what does the Fc fragment (CH2 & CH3) interact?
What was Wu & Kubat’s conclusion for antibody structure?
How was this determined?
How many loops are there?
What was not quite right about their conclusion?
5 stranded beta sheet in a barrel
Carbohydrates/sugars mediate interaction between 2 domains
Cell-surface receptors/Fc receptors & C1q
Hypervariable regions that form loops & come together to form Complimentary Domain Regions CDRs to form the binding site
Variability plots & aligning sequences
6 loops - 3 heavy 3 light
Hypervariable region describes regions where antigen can bind but loop structure is relatively constant unlike the variable protein (residues) sequence
Where are the CDR genes along the antibody loci?
Which genes are at the centre of the binding site (in their protein product)?
How have antibodies evolved?
What did Clothia in the 1980s observe?
What does antibody variability depend on?
How did Clothia discover this?
CDRH1/L1 in V, CDRH2/L2 in V, CDRH3 over V, D, J and CDR-L3 over V & J
CDRH3 & L3
Maximise diversity at centre of antibody
Conformations/structure conserved including when loop length varied
CDR length, key residues in & around CDR, and other residues have small conformational effect
Overlapped CDR loop structures & examined antibody structures
What 3 reasons are why antibodies make good drugs?
How do antibodies have a longer half life?
What is immunogenicity?
What can antibody-based drugs not access? (2)
Briefly describe the 4 modes of action for antibody based drugs.
- High affinity/specificity
- Small molecule drugs make few interactions due to size & has to bind to a pocket
- Antibodies are larger so can interact with more & different surfaces & doesn’t require pocket
Fc fragment has binding site for near-natal Fc receptor - allows for antibody recycling instead of degradation
Lead to an immune response to attack the antibody-drug
Blood-brain barrier, penetrate into cells/tumours
- Conventional drugs - binding to a substrate & blocking interactions
- Immune activation - inducing complement-dependent cytotoxicity with C1q activation
- Delivering other drugs - attaching payload to antibodies
- Cross-linking - Making bi-specific antibodies (antibody targets both T-cell & antigen on cell) to localise T-cells at cancer cells
How are bispecific antibodies engineered?
What is an advantage of bispecific antibodies?
How can you engineer the Fab fragment?
What 2 ways can you make an Fv fragment more stable?
What happens if the linker is short?
CH3 domain engineered - 1 side has large residues & other site has smaller residues to favour interactions between molecules
Can use certain fragments & not whole antibody
pegylation
- introduce disulfide bonds between VH & VL = dsFv
- introduce linker GGGG( x5) between C-terminus & N-terminus of VH & VL = scFv
Forms diabody
What 3 ways could you engineer the Fc fragment and for what reason?
What is the HAMA method for producing antibody based drugs?
Chimeric Human Antibodies?
What is the problem with this?
What is re-surfacing?
- Binding - disable interaction with C1q to increase FcRn binding & half life
- Immune activating - interactions with C1q or Fc gamma receptor
- Half life - decrease FcRn binding to decrease half life
Produce antibodies in mice & inject into humans so humans make antibodies against these
Add human Fc domain to mouse antibody to interact with human Fc receptors & C1q
Still lots of foreign protein - antigenic
Putting human-equivalent residues on mouse FV region
What is the combining site?
What did Riechmann do with humanistion by CDR grafting?
What happened?
How was this improved?
What is adair patent?
Where H & light chains combine (FAB & FV) with CDR of residues involved in interactions
Put Rat antibody CDRs onto human antibody
Not good binding & more residues needed for good interaction
Mutated key residues in CDR & residues that pack CDR
Position of amino acids to consider for certain antibodies such that they must match the donor for good binding
What is the single cell sequencing technique?
What is the basis of phage display?
What are the 7 steps of phage display?
What is the idea of transgenic mice?
In what expression system are antibodies normally done?
B-cell from recovering patient & sequence to find antibody sequences - use to produce large quantities
Phage DNA encodes antibody of interest & use it to isolate, sequence & improve/express antibody for binding
1. Modify bacteriophage DNA to produce single chain Fv (scFv) - antibodies/scFv attach to coat proteins presenting on the phage - produces library of many different scFv/antibodies 2. Immobilise antigen on plate & add phage with scFv 3. Wash phage off 4. Ones that stick remain on plate & elute these 5. Stock & titrate 6. Amplify - allowing phage grow in bacteria ○ Can use low stringency copying (non-optimal temperature) to allow errors in copying process which simulates sematic hypermutation allowing mutations to incorporate into antibodies to allow explore different sequences to enhance binding Enter another cycle - 3 cycles end up with antibodies that bind well to antigen
Engineered mouse to have human antibody gene segments to produce fully human antibodies
CHO - eukaryotic & PTM
