Antibodies and Small Molecule Drugs

Question 1

What are extra hypervariable loops known as? What is the rest of the structure called?

Answer 1

Complementarity determining regions, framework

Question 2

How many CDRs are there on the heavy and light chains?

Answer 2

3 on each

Question 3

Which CDR is the most variable?

Answer 3

CDR3 on heavy chain

Question 4

What are constant domains recognised by?

Answer 4

FcR receptors

Question 5

What is the Fc region?

Answer 5

CH2 and CH3 domain

Question 6

Which receptor does IgG bind to?

Answer 6

Fc-gamma-R which prevents it being degraded

Question 7

Which receptor does IgA bind to?

Answer 7

Fc-alpha-R - mucosal layer, gut lining, lactation

Question 8

Which receptor does IgA/M bind to?

Answer 8

Fc-alpha/u-R

Question 9

Which receptor does IgE bind to?

Answer 9

Fc-E-R

Question 10

What recognises constant domains?

Answer 10

Complement receptors

Question 11

WHat do complement receptors circulate as?

Answer 11

Inactive precursors

Question 12

What does the flu immunity antibody do?

Answer 12

Blocks sialic acid binding site on haemaglutinin

Question 13

What kind of binding is there between flu antibody and haemagglutinin?

Answer 13

Induced fit

Question 14

What does C1q do?

Answer 14

Recognises constant domain (binds to IgM or IgG antobodies) > conformational change activates C1r protease > leads to complement cascade and membrane attack complex activation > forms pores in cell membrane and leads to cell death and inflammation

Question 15

What targets are antibodies restricted to?

Answer 15

Surface or soluble

Question 16

What are polyclonal antibodies used for?

Answer 16

Research, cheap and easy to produce

Question 17

What are monoclonal antibodies for?

Answer 17

Clinical use because they are pure

Question 18

What is an epitope?

Answer 18

Site for the antibody to bind to

Question 19

How do you get polyclonal antibodies?

Answer 19

Inject mouse with antigen, get antibodies from more than one B cell whihc will bind to different epitopes

Question 20

How do you make monoclonal antibodies?

Answer 20

Fuse antibody producing cells with a myeloma (B cell cancer) > select so unfused cells die > use binding assay to select highest affinity antibodies

Question 21

What is a mAb? What do they cause?

Answer 21

Mouse monoclonal antibody - cause human anti-mouse response (HAMA), Fc region interacts poorly with human Fc receptors so reduced half life

Question 22

What are chimeric antibodies?

Answer 22

Use mouse variable regions on a human constant region to fix FcR problems

Question 23

What is a humanised antibody?

Answer 23

Use CDR grafting - only need the three variable regions from the mouse and get the rest from a human

Question 24

How do you make human antibodies from transgenic mice?

Answer 24

Inactivate mice Ig genes in stem cells, introduce human ones, breed them and some mice will only have human antibodies

Question 25

What is an scFv fused by?

Answer 25

SHort protein linker

Question 26

What does a FAB fragment do?

Answer 26

Binds to toxic/pathogenic region and blocks function

Question 27

How do you make an scFv library?

Answer 27

Use PCR to get a VH and VL genes > put them in a plasmid > create population of bacteriophage > check they bind to antigen

Question 28

How do you make scFv be expressed on the surface?

Answer 28

Fuse it with a phage coat protein

Question 29

WHy do we use drugs at the top of cascades?

Answer 29

At the bottom you need more due to amplification

Question 30

What binds to EGF receptor?

Answer 30

ErbB2

Question 31

What does humira/adalimumab do?

Answer 31

Generated by phage display, prevent TNF binding to receptor, for RA and Crohn's

Question 32

How does humira/adalimumab work?

Answer 32

Reduces TNF-alpha activity > less cytokines etc. > less inflammatory infiltrate > less apoptosis and basement mebrane invasion > less inflammation

Question 33

How does herceptin work?

Answer 33

Binds to ErbB2 but doesn't affect dimerisation, causes receptor internalisation and controls cytotoxicity

Question 34

What is between the lobes of a kinase?

Answer 34

ATP binding site

Question 35

How does tarceva work?

Answer 35

Competes with ATP for binding in EGF receptor kinase, kinase closes over it more than ATP, binds more tightly because froms hydrophobic not just hydrogen

Question 36

What is immunoprecipitation?

Answer 36

Using an antibody to catch a protein of interest and any proteins that have associated with it (co-immunoprecipitation)

Question 37

How do you identify proteins following immunoprecipitation?

Answer 37

Mass spec

Question 38

What is an epitope tag?

Answer 38

Like immunoprecipitation but adding a short AA sequence to the protein you're looking for so you can use higher affinity antibodies

Question 39

What does an interactome show?

Answer 39

Which proteins interact with each other

Question 40

How does the Ras signalling cascade work?

Answer 40

Growth factor binds to RTK > cytosolic domain autophosphorylated > Grb2 binds to phosphotyrosine peptide segment via its SH2 domain > also binds to Pro rich segments on Sos via its SH3 domains > Sos is activated and exchanged Ras's GDP to GTP > Ras binds to Raf > then MAP kinase cascade phosphorylates MEK > MEK phosphorylates MAPK > MAPK migrates to nucleus to phosphorylate Fos and Jun > Ras is inactivated by GTP hydrolysis by GTPase activating proteins > returns to resting state using protein phosphatases