Discovery of Novel Therapeutic Strategies for Cancer Immunotherapy

Project Summary The central hypothesis of my F99-phase proposal is that the lack of T-cell infiltration into human tumors may be caused by the presence of tumor-derived T-cell excluders (TCEs) in the TME, which are overexpressed in tumor tissues and impair the function of T cells including their ability to migrate into tumor tissues. This provides

a potential explanation why both spontaneous and therapeutic T cell-mediated anti-tumor immunity fail frequently in patients with immunologically “cold” tumors. By employing a secretome-wide in vitro T-cell transwell migration high-throughput screening (HTS) platform, we tested over a thousand soluble human proteins on the migration

of activated T cells towards chemokine signal. SLIT2 was identified as a candidate target and validated to directly inhibit T-cell chemotaxis towards CXCL11, a common chemokine found in human tumor tissues. The N-terminal fragment of SLIT2 was first confirmed to mediate T-cell chemotaxis inhibition. Detailed

functional domain mapping demonstrated that the first two leucine-rich repeat (LRR) domains, where the canonical receptor ROBO1 binds, are dispensable to SLIT2’s function in regulating T-cell chemotaxis. Meanwhile, ROBO1 expression could not be detected on T-cell surface with FACS staining, and soluble ROBO1 extracellular

fragment fusion protein failed to neutralize SLIT2’s inhibitory effect on T cells, collectively suggesting a novel T- cell specific SLIT2 signaling axis independent of ROBO1. In a syngeneic mouse pancreatic cancer model, dramatically elevated T-cell infiltrated was observed in SLIT2 knock-out Pan02 tumors. SLIT2-KO tumors were

rejected by the immune-competent C57BL/6 mice, while the SLIT2 wild-type tumors grew. Such difference diminished when the SLIT2-WT/KO tumors were inoculated into immune-compromised NSG mice. Anti-SLIT2 monoclonal antibody 11C8 neutralized the inhibitory effect of SLIT2 on T-cell chemotaxis in vitro, and its single-

agent treatment led to Pan02 tumor regression in vivo. The functional SLIT2 receptor expressed on T cells, the broader impact of tumoral SLIT2 to the immune contexture within the TME, and the potential synergistic effect of anti-SLIT2/anti-PD-1 combo are subject to further investigation during the F99 phase training.

In the K00 phase of this proposal, I would like to expand my study to understand dysfunction of T cells during tumor progression. These studies might include the discovery of additional TME-specific extracellular factors that drive dysfunctional anti-tumor immunity in T-cell inflamed tumors. I propose to establish in vitro HTS

platforms to identify candidate targets that (1) promote central memory T-cell phenotype that favors egression into secondary lymphoid organs, (2) reduce T-cell proliferative capacity and induce apoptotic signatures, (3) suppress T-cell effector functions and drive exhaustion phenotype. Multi-omics studies and inducible expression

animal models will be utilized in parallel for target identification and validation purposes. Collectively, this proposed research will extend our understanding of tumor immune evasion mechanisms, and lead to the discovery of novel immunotherapy strategies for cancer patients.