Antibody function part 1

Question 1

Describe four different kinds of anti TNF blockers ibased on antibodies

Answer 1

1) infliximab which is chimeric (mouse Fab region +human Fab region).
2) adalimumab fully humanised anti TNFa mAb
3) Fuse TNF receptro with Fc portion.
4) Use Fab portion of anti TNF ab with pegylated molecule.

Question 2

What is broad antibody strucutre?

Answer 2

Two heavy chains with two light chains. Each heavy and light chain have a variable (Vh and VL) region and a constant region.

Question 3

What are the two light chain constant options?

Answer 3

kappa or lambda constant regions.

Question 4

What are the hypervaribale regions of the heavy and light chain? Which is most variable?

Answer 4

These are the complementarity determining regions (CDR) 1..2 and 3. CDR3 is most variable.

Question 5

What recombination do you have on the light chain? And what stage in B cell development does this occur?

Answer 5

You have V-J recombination. Occurs during pre B cell stage.

Question 6

What recombinatino do you have on the heavy chain and when do they occur in B cell development?

Answer 6

D-J recombination occurs during early pro B cell.
V-DJ occurs during late pro B cell.
VDJ heavy chain expressed and with surogate light chain in early pre B cell (pre B cell receptor).

Question 7

What three points do you generate diveristy in antibody recombination?

Answer 7

V(D)J recombination (e.g. 40 x (25) x 6).
Junctinoal diversity due to inaccurate and random joining)
Combinatino of heavy and light chains.

Question 8

What chains does TCR have? and What recombinatino do they undergo?

Answer 8

You have alpha and Beta chains.
a chain undergoes V-J recombination
B chain undergoes VDJ recombination.

Question 9

What istoypes of Ab are good for complement activation?

Answer 9

IgM, IgG3 (and IgG1).

Question 10

What isotypes are good at neutralisation

Answer 10

All IgG subtypes and IgA.

Question 11

what isotpyes are good for opsonisation and sensitizing NK and mast cells?

Answer 11

IgG1 and IgG3 for opsonisation.

IgG1 and IgG3 for NK and mast cells and especially IgE fo mast cells!!

Question 12

Limitations of IgM

Answer 12

Good for early reponses and activating complement, but low affinity and can’t access many areas due to size.

Question 13

What two things make secondary Ab responses more effective?

Answer 13

SHM and class swtiched antibodies.

Question 14

What enzyme is involved in class switching? And what regions does it target?

Answer 14

AID, will target switch regions within the constant regions. This enables structures to nick these switch sites and loop out and excise the DNA.

Question 15

Where might you find IgG4?

Answer 15

Very good at transporting across the placenta and after an allergic reaction. However, generally found at very low levels.

Question 16

Ab for transport across placenta?

Answer 16

IgG4 and IgG1 and 3.

Question 17

In HUMANS what isotype do type 2 responses generate?

Answer 17

IgG2a and IgE.

Question 18

what isotype responses for type 1?

Answer 18

IgG1 and IgG3.

Question 19

What isotpye responses for type 3?

Answer 19

IgG2b and IgA.

Question 20

How long do IgG antibodies have a half life for? What IgG antibody subtype has shortest half life and why?

Answer 20

3 weeks. IgG3 has lowest half life as doesn’t bind to the FcnR

Question 21

Why doIgG antibodies have the longest half life?

Answer 21

Because when they are endocytosed, at low pH they can bind FcnReceptors in the endosome. This enables them to be recycled and released and exocytosed at higher pH.
Other proteins endocytosed are degraded.

Question 22

How can Ab be used therapeutically?

Answer 22

Activate NK cells and complement. Block receptors or act as agonists. Checkpoint therapy and block ligands. Deliver drugs specifically. CAR T cell therapy.
Bring two targets into contact (bivalent antibodies)

Question 23

how to make hybridomas?

Answer 23

immunise mice, take B cells and fuse with melanoma cells using PEG. Transfer to selection media and select those that produce antibody to X. Grow in single cell wells for monoclonal antibodies.

Question 24

Problems with the OKT3 antibodies that target CD3?

Answer 24

depleted T cells, but were ineffective upon administration, causes allergic reactions and had strong side effects, were rapidly cleared.

Question 25

What are the characteristics of chimeric antibodies? and what ending do they have?

Answer 25

They have Fab of mice, but human constant region.

xi-mab ending.

Question 26

what are the characteristics of humanised antibodies and what is the ending?

Answer 26

They have CDR3 (or 1 and 2) and rest is humanised.

zu-mab

Question 27

how do you get humanised antibody? Whats the ending?

Answer 27

Use transgenic mice or phage display and ending is u-mab.

Question 28

What are the steps for chimerisation?

Answer 28

Take hybridoma culture and prepare mRNA and cDNA of VL and VH chain regions. Once you have cDNA, you can use PCR and transfer hem into vectors.
Vectors can have a human constant region.
then transform bacteria to produce mab.

Question 29

How does bacteriophage display work for humanised ab?

Answer 29

Take human B cells, and isolate mRNA and create cDNA, clone fragments into phage display vector transform phage with vector.
Phage displays the man and you select the phage which binds the desired epitope.
Then you can clone into cells that make large amounts of mab.

Question 30

What are single domain antibodies and what are the benefits/

Answer 30

Isolated the variable domains. these are smaller and can penetrate smaller areas, may have a higher affinity as easy to manipulate.