Anti-inflammatory immunosuppressive drugs

Question 1

Abatacept

Answer 1

CTLA4 mimetic for treatment of RA
Recombinant fusion protein comprising of the extracellular domain of human CTLA4 and FC domajn of human IgG1 engineered to prevent complement fixation.
Binds to and inactivates B7, higher affinity for B7 than CD28
Blocks costimulatory binding on niave T cells preventing T cell activation and proliferation.
Effective in combination with disease-modifying anti-rheumatic drugs (DMARDs).

Question 2

Why develop a drug that can activate T cells in tbe abscence of a presented antigen?

Answer 2

For selective manipulation of supressive T cell populations, Tregs.
Treg activation is a potential treatment strategy for autoimmune disorders.
Treg inhibition is a potential strategy for enhancing cancer cell immunogenicity.

Question 3

CD28 superagonists

Answer 3

T cel activation achieved by bivalent binding of superagonistic antibodies to membrane-proximal epitope on CD28. I.e antigen independent T cell activation.
In vitro all T cels activated
In rodents and monkeys in vivo, preferal expansion of Tregs.
Phase 1 trials, all T cells activated, multiple organ failure, cytokine storm, fever, swelling, vascular collapse, white blood cell depletion.
High risk drug target.

Question 4

IL-2 receptor signalling on T cells

Answer 4

IL-2 increases B cell proliferation and antibody production, it also increases NK cell proliferation and cytolytic activity. IL-2 binding permits transphosphorylation on Tyr and activation of JAKs 1 and 3.
Active JAKs phosphorylate specific Tyr residues on the b chain cytoplasmic domain.
SH2 domains on STAT-5a and -5b bind to p-Tyrs on receptor where they are phosphorylated by the active JAKs. Phosphorylated STATs form dimers that translocate to the nucleas to initiate target gene transcription.

Question 5

JAKs as targets for immunosuppressive drugs

Answer 5

JAK3 expression is restricted to myeloid, NK, activated T and B cells. (other JAKs are widely expressed thus drugs have side effects.)
Some genetically inhertiable forms of XSCID are due to loss of function mutations in JAK3.
It is relatively straight forward to screen for JAK selective kinase inhibitors.

Question 6

JAKs as targets for anti-myeloproliferative drugs

Answer 6

Diseases - polycythemia Vera, myelofibrosis.
Hyperactivation and proliferation of haematopoietic cells leading to bone marrow failure.
This prompts the spleen and liver to take over blood cell production causing enlargement of the spleen and debilitating symptoms.
>90% of PV and >50% of myelofibrosis cases due to an activating mutation in JAK2.

Question 7

JAK inhibitor drugs

Answer 7

Ruxolitinib - oraly available JAK/JAK2 ATP binding site inhibitor. FDA approved for myelofibrosis and other myeloproliferative disorders. In phase 3 trials for PV.

Tofacitinib - orally available, moderately selective JAK3 inhibitor. Approved for RA patients who are intolerant or resistant to conventional RA treatments.

Question 8

Antibody based theraputics

Answer 8

Inhibitory mimetics - abatacept (CTLA4-IgG fusion)
Anti-cytokines - infliximab, adilimumab, anti-TNFa
Receptor decoys - Etanercept (TNF receptor Ig fusion)
Receptor inhibitors - trastuzimab (anti-HER2 receptor mAb)
Advantages - specificity of targetting.

Question 9

Making a monoclonal antibody

Answer 9

Proliferative myeloma cells, defective jn antibody production, are fused with non-proliferative spleen cells from a mouse immunised with the desired antigen. Cells are then cultured in a selective medium to isolate proliferative Ab secreting myeloma cells generated frkm the fusion “hybridomas”.

Question 10

Making human monoclonal antibodies

Answer 10

1. Transgenic Mice - gentically engineering mice in which human Ig genes have been knocked in to replace mouse Ig genes. This way all mAbs produced are 100% human.
   Example panitumumab an anti-HER1 Ab, binds extracellular domains to block EGF signalling. Used in colorectal cancer.
2. Phage display - human genes encoding Ab diversity are cloned into a bacteriophage library. The library is then screened for clones with high affinity for defined target. Hits are recovered for cloning into CDRs of a human IgG1 scaffold. Molecule is further optimised for desirable properties (ie long half-life, rapid elimination of unconjuagted target). Thus all mAbs produced are 100% human.
   Adalimumab is an example.

Possible to control antibodies capacity to trigger complement-directed-cytotoxicity and antibody-dependent cytotoxicity.