antibodies

Question 1

what produces antibodies, what happens when antigen binding happens

Answer 1

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

Question 2

how are antibodies adapted

Answer 2

they have both high affinity and specificity for a huge range of anitbodies

they have conserved trigger functions (sameish effects)

Question 3

what is general structure of antibodies

Answer 3

made up of 4 chains;

2long/heavy chains which are roughly 440AAs each

2 short/light chains which are roughly 220AAs each

Question 4

what is difference between polyclonal and monoclonal antibodies

Answer 4

polyclonal antibodies are mixed antibodies binding the same antigen, monoclonal are identical antibodies

Question 5

do antibodies crystalise

Answer 5

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.

Question 6

what is action of enzymes which split antibodies

Answer 6

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

Question 7

what are domains of antibodies

Answer 7

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

Question 8

where are sugars/ disulphide bonds in antigen

Answer 8

sugars are attatched to CH2 domains

hinge has 2-4 disulphide bonds

Question 9

which antibodies have slightly different structure to normal

Answer 9

IgM and IgE have additional CH4 domain

Question 10

how many loci controls gene transcription for antibodies

Answer 10

three loci in the genome: lamda light chains, kappa light chains and heavy chains are at seperate loci

Question 11

describe structure of lamda light chain locus

Answer 11

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

Question 12

describe structure of kappa light chain locus

Answer 12

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

Question 13

describe structure of heavy chain locus

Answer 13

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.

Question 14

what are the anitbody classes, what are their functions, how do they vary

Answer 14

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

Question 15

how is switching between types of antibody enabled genetically

Answer 15

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.

Question 16

what are Fc receptors

Answer 16

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.

Question 17

which antibodies for multimers

Answer 17

IgM and IgD can form multimers (not together), J chains mediate this. IgM forms a pentamer, IgD forms a dimer

Question 18

what is C1q

Answer 18

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

Question 19

describe the domain structure of antibodies

Answer 19

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

Question 20

what is antigen binding site on antibodies

Answer 20

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

Question 21

what are interactions between antibody domains

Answer 21

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

Question 22

what are antigens

Answer 22

proteins, peptides, carbohydrates, lipids, small molecules

Question 23

what enables antigen binding

Answer 23

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)

Question 24

describe antibody structure at antigen binding sites

Answer 24

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)

Question 25

how large is antibody repertoire

Answer 25

over 10^15, since 1.3 billion variable domains from splicing undergo post translational mutations

Question 26

how were hypervariable loops first predicted

Answer 26

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

Question 27

which CDR is particularly variable

Answer 27

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

Question 28

how are CDR variability effects maximised

Answer 28

CDR H3 is site of maximum variability, is also in centre of CDR interface

Question 29

what is chothia’s analysis

Answer 29

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

Question 30

describe structure of CDR-L1

Answer 30

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)

Question 31

how is CDR-H2 structure predicted

Answer 31

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

Question 32

how did chonthia describe CDR conformations

Answer 32

he described the different conserved structures as cononicals, defined by key residues

Question 33

what are uses of antibodies in vitro

Answer 33

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

Question 34

what are uses of antibodies in vivo

Answer 34

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

Question 35

what advantages do antibodies have over small molecules in in vivo therapeutics

Answer 35

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

Question 36

what is a negative of antibody therapeutics

Answer 36

antibodies are expensive to produce and so are usually reserved for hard to treat diseases

Question 37

what is are examples of antibodies used therapeutically

Answer 37

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

Question 38

how do antibodies help administer drugs

Answer 38

mylotarg is anti-CD33 which is expressed on tumour cells, helps guide drug to target

Question 39

what is ADEPT

Answer 39

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

Question 40

how are catalytic antibodies used

Answer 40

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

Question 41

how are Fab fragments used

Answer 41

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

Question 42

how are Fv fragments used

Answer 42

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)

Question 43

how are antibodies produced for treatment

Answer 43

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

Question 44

what are problems with therapeutic monoclonal antibodies

Answer 44

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

Question 45

how are mouse antibodies humanised

Answer 45

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

Question 46

what are processes for producing full human antibodies

Answer 46

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)