Antibodies and Small Molecule Drugs Flashcards
What are extra hypervariable loops known as? What is the rest of the structure called?
Complementarity determining regions, framework
How many CDRs are there on the heavy and light chains?
3 on each
Which CDR is the most variable?
CDR3 on heavy chain
What are constant domains recognised by?
FcR receptors
What is the Fc region?
CH2 and CH3 domain
Which receptor does IgG bind to?
Fc-gamma-R which prevents it being degraded
Which receptor does IgA bind to?
Fc-alpha-R - mucosal layer, gut lining, lactation
Which receptor does IgA/M bind to?
Fc-alpha/u-R
Which receptor does IgE bind to?
Fc-E-R
What recognises constant domains?
Complement receptors
WHat do complement receptors circulate as?
Inactive precursors
What does the flu immunity antibody do?
Blocks sialic acid binding site on haemaglutinin
What kind of binding is there between flu antibody and haemagglutinin?
Induced fit
What does C1q do?
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
What targets are antibodies restricted to?
Surface or soluble
What are polyclonal antibodies used for?
Research, cheap and easy to produce
What are monoclonal antibodies for?
Clinical use because they are pure
What is an epitope?
Site for the antibody to bind to
How do you get polyclonal antibodies?
Inject mouse with antigen, get antibodies from more than one B cell whihc will bind to different epitopes
How do you make monoclonal antibodies?
Fuse antibody producing cells with a myeloma (B cell cancer) > select so unfused cells die > use binding assay to select highest affinity antibodies
What is a mAb? What do they cause?
Mouse monoclonal antibody - cause human anti-mouse response (HAMA), Fc region interacts poorly with human Fc receptors so reduced half life
What are chimeric antibodies?
Use mouse variable regions on a human constant region to fix FcR problems
What is a humanised antibody?
Use CDR grafting - only need the three variable regions from the mouse and get the rest from a human
How do you make human antibodies from transgenic mice?
Inactivate mice Ig genes in stem cells, introduce human ones, breed them and some mice will only have human antibodies
What is an scFv fused by?
SHort protein linker
What does a FAB fragment do?
Binds to toxic/pathogenic region and blocks function
How do you make an scFv library?
Use PCR to get a VH and VL genes > put them in a plasmid > create population of bacteriophage > check they bind to antigen
How do you make scFv be expressed on the surface?
Fuse it with a phage coat protein
WHy do we use drugs at the top of cascades?
At the bottom you need more due to amplification
What binds to EGF receptor?
ErbB2
What does humira/adalimumab do?
Generated by phage display, prevent TNF binding to receptor, for RA and Crohn’s
How does humira/adalimumab work?
Reduces TNF-alpha activity > less cytokines etc. > less inflammatory infiltrate > less apoptosis and basement mebrane invasion > less inflammation
How does herceptin work?
Binds to ErbB2 but doesn’t affect dimerisation, causes receptor internalisation and controls cytotoxicity
What is between the lobes of a kinase?
ATP binding site
How does tarceva work?
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
What is immunoprecipitation?
Using an antibody to catch a protein of interest and any proteins that have associated with it (co-immunoprecipitation)
How do you identify proteins following immunoprecipitation?
Mass spec
What is an epitope tag?
Like immunoprecipitation but adding a short AA sequence to the protein you’re looking for so you can use higher affinity antibodies
What does an interactome show?
Which proteins interact with each other
How does the Ras signalling cascade work?
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
