antibodies Flashcards
what produces antibodies, what happens when antigen binding happens
b cells produce antibodies on its surface, when antigen binds clonal expansion occurs, IgM and IgD class switch to IgG, IgG binds to antigen which causes opsonisation etc
how are antibodies adapted
they have both high affinity and specificity for a huge range of anitbodies
they have conserved trigger functions (sameish effects)
what is general structure of antibodies
made up of 4 chains;
2long/heavy chains which are roughly 440AAs each
2 short/light chains which are roughly 220AAs each
what is difference between polyclonal and monoclonal antibodies
polyclonal antibodies are mixed antibodies binding the same antigen, monoclonal are identical antibodies
do antibodies crystalise
antibodies are flexible and so do not crystallize, however papain cleaves antibodies into 2 types of fragment; Fab containing a light chain and heavy chain dimer and Fc which is a heavy chain homodimer. Fab is created in twice the quanitity that Fc is created. Fab can be crystallized in monoclonals.
what is action of enzymes which split antibodies
pepsin cleaves IgG antibodies into 2 fragments in a 1:1 ratio, one Fc fragment and one Fab’ fragment which is 2 Fab fragments bonded via a disulphide bond
what are domains of antibodies
light chains have 2 domains: VL and CL
heavy chains have 4 domains: 3 constant : CH1-3 and VH
CH1/CL is closest to antigen binding site, then CH2 then 3
antigen binding site is VH/VL
where are sugars/ disulphide bonds in antigen
sugars are attatched to CH2 domains
hinge has 2-4 disulphide bonds
which antibodies have slightly different structure to normal
IgM and IgE have additional CH4 domain
how many loci controls gene transcription for antibodies
three loci in the genome: lamda light chains, kappa light chains and heavy chains are at seperate loci
describe structure of lamda light chain locus
lamda light chain locus contains 4 different parts; L sections 1-30 which are paired with V sections 1-30 and J sections 1-4 which are paired with C sections 1-4, L and V can be ignored, J stands for joining. The V+J at DNA level creates the v at protein level in antibodies, since V and J combine 120 possible outcomes
describe structure of kappa light chain locus
in kappa light chain locus L and V are still paired however there are 40 pairs not 30, J is not paired with C, there are 5 J regions and only one C region, producing 200 possible outcomes
describe structure of heavy chain locus
at the heavy chain locus the Ls and Vs are still paired, there are 65 L-V pairs, there is an additional D section, of which there are 27 regions, and there are 8 J regions, J regions are not paired with C regions. D stands for diversity, D+V+J creates v at protein level, 65x27x8x300= 4,212,000. 300 comes about due to frameshift mutations and imprecise recombination in D sections, since D can be read in any of the 6 reading frames.
what are the anitbody classes, what are their functions, how do they vary
There are 5 antibody classes (isotypes): IgM, IgD, IgG, IgA, IgE
IgM is involved in early immune response, they are soluble and there are B-cell membrane bound versions . Can trigger immune response
IgD is involved in early immune response, B cell membrane bound versions
IgG is involved in late immune response, major antibody found in blood plasma, most common one,
IgA is found in secretions such as saliva, tears, mucus
IgE is involved in degranulation of mast cells and histamine release
these isotypes only vary in terms of heavy chain regions
how is switching between types of antibody enabled genetically
class switching :
heavy chain locus contains a series of alternative constant domains
switch sites occur between the series of constant domains, DNA between these sites may be spliced resulting in the lack of these constant domains. This is called class switching, class switching is uni directional, one the DNA has been spliced it cannot be undone.
what are Fc receptors
Fc receptors:
FcgR (binds IgG): located primarily on neutrophils, macrophages and monocytes. Phagocytose IgG coated pathogens
FceR(binds IgE): primarily on eosinophils, basophils and mast cells. Trigger histamine release from intracellular granules.
FcaR (binds IgA): located on neutrophils, monocytes, macrophages and eosinophils. Causes phagocytosis and antibody dependent cell mediated cytotoxicity (ADCC). Also causes release of superoxide and inflammatory mediators.
which antibodies for multimers
IgM and IgD can form multimers (not together), J chains mediate this. IgM forms a pentamer, IgD forms a dimer
what is C1q
C1q (part of complement system) binds to IgM and IgG, must bind to at least to 2 IgG to be active, since it is Fc’s are fairly flexible they are unlikely to lead to conformational change, mechanism is likely due to concentration effect due to multiple antibody activation
activates classical complement pathway, leads to MAC formation
describe the domain structure of antibodies
Domain structure of antibodies;
immunoglobulin fold, is a beta sandwich (2 beta sheets on top of each other), two sheets are inclined at 30 degrees, very stable structure. Containing disulphide bonds between each domain.
VH/VL domains can rotate with respect to eachother
what is antigen binding site on antibodies
on the end of VH and VL are complementary binding regions (CDRs) where antigens bind
there are 6 loops on these domains, 3 from heavy chain 3 from the light, there form a relatively continuous surface for antigen to interact with
loops are labelled L1-3 and H1-3, they are organised the same (they have rotational symetry)
L3 and H3 are in middle, L3 is sandwiched between L1 and H2, and is above H3 which is sandwiched between H1 and L2:
H2L3L1
H1H3L2
H3 and L3 are hypervariable loops or CDRs
what are interactions between antibody domains
there are interactions between VL, VH, CH1 to CL, CH3 to CH3 (CH1 and Cl are next to eachother on each branch, same as VH and VL, so are CH3 and CH3)
CH2 to CH2 interactions are mediated via carbohydrate groups attached to them
interactions (such as covalent/ionic bonds etc) are sparse allowign for flexibility
VL and VH interact via 5 strand sheets to form a barrel
what are antigens
proteins, peptides, carbohydrates, lipids, small molecules
what enables antigen binding
shape complementarity and chemical complementarity
mediated by overall combining site shape of antibody and specific residue sequence composition of antibody; bulk of sidechains influences shape, specific residues have different chemical interactions
(similar to enzyme substrate binding)
describe antibody structure at antigen binding sites
the protein may have a flat surface, it may have a narrow groove, or a normal groove or pocket…. lots of shapes
flat surfaces are used to bind to protein antigens
narow grooves are used to bind to small molecules such as DNA
grooves are used to bind to peptide antigens
pockets are used to bind to carbohydrate/hapten antibodies
(a hapten is a small organic molecule, cant illicit an immune response, however if bound to a carrier protein they may ellicit an immune response, however hapten alone binds)
how large is antibody repertoire
over 10^15, since 1.3 billion variable domains from splicing undergo post translational mutations
how were hypervariable loops first predicted
loop structure was predicted by Wu and Kabat using variability plots, they analysed VL and VH domains which contained 3 highly variable regions, position in backbone was compared to variability in antibodies, 3 regions peaked, they predicted these regions would be loops
which CDR is particularly variable
CDR-H3 (loop H3) is particularly variable
since in light chain there is only Vand J splicing to create variability
in heavy chain there is V, D and J splicing, D introduces extra variability
variability plots show much higher variability in 3rd loop compared to rest of domain, thus CRD-H3 is very variable, D segment in gene encodes hypervariable region
how are CDR variability effects maximised
CDR H3 is site of maximum variability, is also in centre of CDR interface
what is chothia’s analysis
CDRs other than CDR-H3 have relatively similar structures across diferently antibodies
although loops tend to differ in length the base structure is relatively well conserved
he proposed that variability depends on CDR length and certain “key” residues, using this information you can predict the loop structure
note this does not apply to CDR H3
describe structure of CDR-L1
10 residues in CDR-L1, key residues are alanine, valine and methionine which point inwards and have hydrophobic interactions with eachother which maintains structure, allowing for more variability in residues pointing outward
key residues are:
position 2 must be isoleucine
position 25 must be alanine
position 29 must be valine/leucine/isoleucine
position 33 must be leucine/methionine
position 71 must be tyrosine/phenylalanine
(these are positions in VL domain)
how is CDR-H2 structure predicted
2 possible conformations:
two possible conformations, each have 10 amino acids and associated key residues which are conserved through different antibodies
class 2 and class 3 are the conformations
how did chonthia describe CDR conformations
he described the different conserved structures as cononicals, defined by key residues
what are uses of antibodies in vitro
they are useful in lab experiments as well as diagnostic/therapeutic agents, this is due to high specificity and affinity of antibodies
uses in vitro: purification, labelling (western blots), for in vitro clinical diagnostics to detect presence of antigen (monoclonals and sheep antibodies used)
antibodies may be purchased off the shelf, some companies will make custom made antibodies
what are uses of antibodies in vivo
categories of use of in vivo antibodies:
imaging such as in gamma-imaging and for guiding surgery
therapeutic antibodies can cause immune activation and can also have direct effects, common use for treatments is cancer
used for targetting processes such as conjugation with drug
may also be used for catalysis
what advantages do antibodies have over small molecules in in vivo therapeutics
for high affinity to occur there must be lots of connections between drug and site of action
due to small size of small molecules for high affinity to occur at target there must be a deep pocket which is relatively specific for the drug, not many targets have this
lots of targets do not have this small grooves, antibodies may be designed for structures such as convex faces
what is a negative of antibody therapeutics
antibodies are expensive to produce and so are usually reserved for hard to treat diseases
what is are examples of antibodies used therapeutically
trastuzumab (herceptin) is anti-HER2, used to treat HER2 positive breast cancer
reopro prevents clumping and reclogging of coronary artieries after angioplasty (after patient is given a stent)
also used to modulate immune system
how do antibodies help administer drugs
mylotarg is anti-CD33 which is expressed on tumour cells, helps guide drug to target
what is ADEPT
an anti tumour antibody is coupled to an enzyme which activates a pro drug, allowing for specific area metabolism of pro drug
(antibody directed enzyme pro-drug therapy)
does not work: since concentration created of active drug is relatively small so very toxic drugs are required, and these would then circulate and have distal side effects,
enzyme may also cause autoimmune response (if enzyme is human)/immune response
how are catalytic antibodies used
antibody itself acts as an enzyme either via engineering in enzyme activity into antibody or via an antibody which stabilise and thus lower energy required for transition states of reactions
how are Fab fragments used
Fab fragments, since VH and VL are desired regions, however on their own they are unstable so CH1, CL stabilise them
however these structures still relatively unstable, things added to stabilise them such as PEG (polyethylene glycol) addition on to end of Fab fragment allows for increased stability
these are used for activity where binding is activity wanted as opposed to interaction with immune system which requires Fc fragment
how are Fv fragments used
ocassionally only variable regions are used (Fv fragment), dsFv is an artificial disulphide added to help stabilise this structure as well as scFV which is an artificial linker which effective makes the 2 variable domains a single chain, if this scFV is short it causes pairing of antibodies either resulting in a bivalent antibody ( 2 of same variable regions facing opposite ways) or a bispecific antibody ( 2 different variable regions facing opposite ways)
how are antibodies produced for treatment
human monoclonals cannot be produced by conventional routes
HAMA, human anti mouse antibody is used
protein of interest is purified and injected into mouse/rat/rabbit, antibodies are harvested through b cell harvest
b cells are fused with tumour cells which imortalises the cell
this process is not done with human antibodies since human b cells cannot be immortalised and injection of human protein into human is unlikely to produce and immune response and so antibodies
antibodies are then harvested from these immortalised b cells
what are problems with therapeutic monoclonal antibodies
human immune system recognises mouse antibodies as foreign and so produces immune response and antibodies against these, drugs are given to treat this are anti- antibodies
problems is that in best case scenario immune system wipes out drug first time round and produces antibodies, this allows drug to work first time round but will not work again, worst case scenario is it causes anaphylactic shock
this problem is tried to overcome by humanisation of mouse antibodies
how are mouse antibodies humanised
variable remains from mice can be joined onto human constant domains thus decreasing chance of immune response, or only mouse CDRs joined onto an otherwise full human antibody, thus using minimal mouse component
problem with mouse CDR only is it does not have strong binding with targets
one way of doing this is the changing of serine at H27 to phenylalanine, serine was found in humans, phenylalanine in mouse, simply one example
the adair patent is a patent on the technique
what are processes for producing full human antibodies
phage display (a type of display technique)
transgenics: transgenic mice, mouse antibody genes replaced with human antibody genes, these then produce human antibodies when antigen is given (lots of drugs produced this way)
key thing is that DNA is coupled to protein that produces it (display techniques)
