Cytokines

Cytokines are signaling molecules involved in inflammation. They include Interferons, Interleukins, Chemokines, colony-stimulating factors, Tumor necrosis factors, and Transforming growth factors. At baseline their concentration in the blood is low. Overrelease can cause a cytokine storm, triggering massive inflammation.

Ref (1)

Functional categorizations

• Type I cytokines, mediate development of cellular response: IL-2, IL-12, IFN-g
• Type II cytokines, mediate development of humoral response: IL-4, IL-5, IL-10, IL-13
• Regulatory cytokines, which moderate inflammation and maintain homeostasis: IL-10, TGF-b

Barriers to therapeutic application

WT cytokines are, as of 2023: “relegated to second-line therapy”, and none have been approved since the mid 1990s:

1. Biological pleiotropism: context-dependent responses are variable and dynamic, and immunosuppressive and immunostimulatory effects must be decoupled.
2. Poor drug-like properties:
   • Short half life
   • Narrow therapeutic window
3. Acute toxicity and morbidity

Synthekines

Synthetic cytokines that dimerize novel receptor pairs. This has been achieved using IL-2, IL-4, and type I interferons. These can also include Nanobodies and Single chain variable fragments that mediate receptor dimerization.

Antibody-cytokine conjugates

• Fusing Fc-regions to cytokines reduces renal clearance (citation needed) and intracellular digestion via FcRn-mediated uptake. This approach has been tested with IL-2, IL-7, IL-15, IL-22. The major challenge is dealing with the fact that Fc regions are dimeric, and many cytokines are not.
• One example is a nanobody fused to IL-2 or IFN-g that targets PD-L1
• Another is the IL-10-Pavilizumab hybrid, where monomerized IL-10 was added to CDRL1.

Fusion to albumin

• This is of interest since Albumin is the most abundant protein in blood
• Fusion of IFN-g to Human serum albumin increased half-life from 4-8 hours to 144 hours
• This may cause more distribution in lymph nodes and spleen
• Can introduce steric hindrance due to 67 kDa size of HSA, reducing activity (this has been attributed to IFN-a conjugated to albumin, which retained 1.4% of in vitro activity)
• Fusing to Albumin-binding domains (which are much smaller) can counteract this

Masked cytokines

Masked IFNs get around ubiquitous expression of receptor by conjugating to soluble receptor via matrix metalloprotease-cleavable linker to conceal cytokine until it reaches tumor microenvironment, where high concentrations of the protease are present. Also done with IL-12, IL-2, IFN-beta

Miscellaneous

• Dimeric Cytokines have been monomerized using various approaches:
  • IL-10 via a GGGSGG linker
  • IL-12 to fuse with Fc domain, which increased half-life
  • IGN-g via a similar GS linker (details unclear)

This note cites liberally from (2)

1.

Pires IS, Hammond PT, Irvine DJ. Engineering Strategies for Immunomodulatory Cytokine Therapies: Challenges and Clinical Progress. Advanced Therapeutics. 2021;4(8). Available from: https://doi.org/10.1002/adtp.202100035

2.

Aung T, Grubbe WS, Nusbaum RJ, Mendoza JL. Recent and future perspectives on engineering interferons and other cytokines as therapeutics. Trends in Biochemical Sciences. 2023;48(3):259–73. Available from: https://doi.org/10.1016/j.tibs.2022.09.005