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BackgroundTargeting PD1 with monoclonal antibodies has yielded clinical success across a variety of tumor types, yet overcoming inhibitory receptor (IR)-mediated tolerance is essential to improve immunotherapeutic responses. LAG3 co-expresses with PD1 on CD8+ tumor-infiltrating T cells (TIL), signifying a highly exhausted phenotype, and dual PD1/LAG3 blockade in C57BL/6 mice enhances antitumor immunity. As CD8+ TIL is the dominant LAG3-expressing TIL population, it is hypothesized that PD1 and LAG3 synergizes to limit CD8+ TIL function controlling antitumor immunity.MethodsTo understand the cellular and mechanistic basis for PD1/LAG3 synergy, conditional knock-in mice ”surgically dissect” Pdcd1 and/or Lag3 floxed alleles restricted to CD8+ T cells expressing E8ICre.GFP. These mice were crossed with the Pmel transgene to assess PD1 and/or LAG3-sufficient or deficient gp100-specific CD8+ T cell populations. CD8+ Pmel cells were isolated and adoptively transferred into C57BL/6 mice harboring a B16-gp100-overexpressing tumor to observe therapeutic benefit, or to assess T cell functionality within the tumor.ResultsWhile little therapeutic benefit was observed with a prophylactic adoptive transfer of wild-type CD8+ Pmel cells into mice which then received B16-gp100 tumor, there was reduced tumor growth in mice receiving PD1-deficient CD8+ Pmel cells, which was further enhanced in mice receiving PD1/LAG3-deficient CD8+ Pmel cells with long-term tumor-free survival. Likewise, a therapeutic administration of PD1/LAG3-deficient CD8+ Pmel cells into mice once the tumor has been established showed an initial therapeutic benefit, with enhanced survival, that was not demonstrated with adoptive transfer of PD1 or LAG3-deficient, or wild-type, counterparts. Each PD1 and/or LAG3-sufficient or deficient CD8+ Pmel mice were differentially congenically marked to assess each of the four genotypes that can be adoptively transferred into the same host as a ”quad transfer” system. Recovery of these populations within the tumor show that the PD1/LAG3-deficient CD8+ Pmel cells out-compete PD1 or LAG3-deficient, or wild-type, counterparts due to enhanced proliferation (Ki67/BrdU). Furthermore, PD1/LAG3-deficient CD8+ T cells were more functional with increased IFNg and GzmB release observed by flow cytometry.ConclusionsOverall PD1 and LAG3 limit anti-tumor immune effects as removal of both IRs on a gp100 antigen-specific CD8+ T cell population results in reduced B16-gp100 tumor growth and enhanced survival in an adoptive transfer model, as a result of enhanced CD8+ TIL functionality and proliferation. These results provide striking evidence that the development of anti-LAG3 agents in the clinic would yield improved responses with anti-PD1.

Checkpoint blockade immunotherapy established a new paradigm in cancer treatment: for certain patients curative treatment requires immune reinvigoration. Despite this monumental advance, only 20%–30% of patients achieve an objective response to standard of care immunotherapy, necessitating the consideration of alternative targets. Optimal strategies will not only stimulate CD8+T cells, but concomitantly modulate immunosuppressive cells in the tumor microenvironment (TME), most notably regulatory T cells (Tregcells). In this context, the immunoregulatory receptor Neuropilin-1 (NRP1) is garnering renewed attention as it reinforces intratumoral Tregcell function amidst inflammation in the TME. Loss of NRP1 on Tregcells in mouse models restores antitumor immunity without sacrificing peripheral tolerance. Enrichment of NRP1+Tregcells is observed in patients across multiple malignancies with cancer, both intratumorally and in peripheral sites. Thus, targeting NRP1 may safely undermine intratumoral Tregcell fitness, permitting enhanced inflammatory responses with existing immunotherapies. Furthermore, NRP1 has been recently found to modulate tumor-specific CD8+T cell responses. Emerging data suggest that NRP1 restricts CD8+T cell reinvigoration in response to checkpoint inhibitors, and more importantly, acts as a barrier to the long-term durability of CD8+T cell-mediated tumor immunosurveillance. These novel and distinct regulatory mechanisms present an exciting therapeutic opportunity. This review will discuss the growing literature on NRP1-mediated immune modulation which provides a strong rationale for categorizing NRP1 as both a key checkpoint in the TME as well as an immunotherapeutic target with promise either alone or in combination with current standard of care therapeutic regimens.

Meeting abstracts Inhibitory receptors control immune responses preventing exacerbated T cell activation and the onset of autoimmunity; however, they also limit antitumor immunity. Enhanced co-expression of PD1 and LAG3 phenotypically mark functionally exhausted tumor-specific T cells, with dual

Meeting abstracts The tumor microenvironment is a complex system, which is composed of various types of non-tumor cells including stromal fibroblasts, the blood and lymphatic vascular networks, the extracellular matrix and notably, the infiltrating immune cells. Regulatory T cells (Treg cells) play