Poster CC16 - Reprogramming an ancient kinase network motif for reversible and humanized control of T cell receptor signaling

Introduction: The diametrically opposing cytoplasmic tyrosine kinases CSK (C-terminal Src kinase) and SFKs (src family kinases) form the core of an archetypal tyrosine kinase regulatory network that predates the evolution of multicellularity. CSK is unique amongst tyrosine kinases because its kinase activity inhibits the downstream activation of kinase signaling cascades. Perhaps due to its peculiarities, no tools exist to turn on CSK activity in an experimentally controlled manner. We reasoned that a novel CSK switch that we term CSKdim might be created through chemically forced dimerization and SRC like membrane localization, that resembles the ligand induced dimerization of receptor tyrosine kinases and the forced dimerization of oncogenic kinase fusions. The potential activation of CSK through forced dimerization could be useful for studying and controlling immune cell dynamics in a variety of cell types given its role in downregulating SFK activity. Relatedly, the LCK and FYN kinases signaling are critical cytoplasmic nodes for both CART signalling and TCR signaling. Engineered cell therapies that utilize CARTs or transgenic TCRs require LCK and FYN signaling to function. Therefore, beyond a research tool, the effective modulation of CSK might be used to create a reversible switch for the control of immune cell therapies. Our research aims to reprogram an ancient regulatory motif to develop a universal, reversible, non-viral, and immune-compatible switch that regulates TCR and CAR-T cell responses.

Materials and

Methods: a reversible switch would hold numerous advantages in the mitigation of adverse effects such as cytokine release syndrome (CRS) and tumor lysis syndrome (TLS) in therapeutic settings. These advantages include 1) the reversibility of inactivation 2) the complete absence of microbial/viral sequences (i.e. fully humanized), and 3) the regulation would be independent of the CART generation/design and transferable to transgenic TCRs (enabling it to be “bolted on” to approved therapies).
We engineered a construct combining the SRC myristoylation site (for membrane targeting), FKBP12 F36V (DmrB) homodimerizer for reversible regulation, a portion of the TEL protein as an internal linker, and the CSK kinase domain. This construct was transduced into native T cells and first to third generations of FMC63 CAR-T cells targeting CD19. We assessed our CSK switch's activity by detecting phosphorylation states of LCK at the inhibitory Y505 residue and the activating Y394 residue. We measured IL-2 and IFN-γ secretion via ELISA in both T cells and CAR-T cells with the CSK switch active and inactive. Additionally, we conducted cell viability assays using blinatumomab, a bispecific T-cell engager, against BV173 and Raji CD19+ cells, and further evaluated the CSK switch in anti-CD19 CAR-T cells against CD19+ cells (BV173, Raji, K562).

Results, Conclusions, and Discussions: Chemically regulated dimerization of CSK reversibly controlled LCK activity by phosphorylating the tyrosine residue at position 505 (Y505) on LCK and decreased phosphorylation at Y394. Activation of the CSK switch significantly and reversibly reduced both intra- and extracellular cytokine levels, including IL-2, TNF-α, and IFN-γ. CSKdim did this in the context of multiple CART receptor designs and an endogenous TCR. In cytotoxicity assays, the presence of the dimerizer substantially reduced T cell-mediated cytotoxicity against BV173 and Raji cells. Similarly, CAR-T cell killing of cancer cells was hindered with the CSK switch active, without affecting T cell viability. These results suggest that activation of the CSK switch can reversibly and universally regulate T cell therapies, potentially creating a novel and improved handle to control the safety of these important therapies with a design that is 100% human sequence.
Despite being fundamentally inactive and opposite in function to better studied kinases, we were able to reprogram CSK to inducibly inhibit SFK function as a chemically dimerizable integral membrane fusion protein. This suggests a deeper conservation of the function of well studied kinase regulatory mechanisms than was previously known. Our reversible CSK-based switch also modulates TCR and CAR-T cell activity. Beyond a new tool, this switch represents a novel strategy for reversibly controlling any engineered T- cell’s function in the clinic with a fully humanized design. Universal, reversible and humanized designs create stronger translational potential because 1) they can be introduced alongside any CAR-T or Transgenic TCR. 2) Instead of a kill switch that eliminates the cell therapy, reversible designs temporarily inhibit an expensive cell therapy that is essentially working too well. Finally, as opposed to designs that utilize viral sequences, a fully human switch is less likely to generate an immune response.

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