504 PVRIG, a novel T cell checkpoint, is preferentially expressed in TLS on stem-like memory T cells, potentially inhibiting their expansion (2022)

<h3>Background</h3> Tertiary lymphoid structures (TLS) recently emerged as an intra-tumoral niches of immune-cell aggregates with a predictive value for cancer immunotherapy responses.<sup>1</sup> LAMP3<sup>+</sup>DCs in the TLS were shown to interact and support the differentiation of stem-like CD8 T-cells into effector-like cells, that then expand in the tumor micro-environment (TME) and may exert anti-tumor responses.<sup>2</sup> We investigated the expression of DNAM-1 axis genes: PVRIG, TIGIT, DNAM-1 and their ligands PVRL2 and PVR in the TME.<sup>3</sup> <h3>Methods</h3> MERFISH technology was employed to detect the expression of 350 distinct mRNA transcripts at sub-cellular resolution in CRC sections. Publicly available TME scRNA-seq datasets were analyzed for expression of PVRIG and PVRL2 across immune populations and validated by flow-cytometry. An extensive omics profiling was performed for patients with pre- and on-treatment biopsies from COM701 (anti-PVRIG antibody) and COM701+nivolumab Phase-1 study (NCT03667716). <h3>Results</h3> Spatial distribution of gene transcripts allowed identifying localization of stem-like T-cells in TLS regions of two CRC patients (figure 1, p&lt;0.001). While, CTLA-4, PD-1, and TIM3 were mainly expressed by tumor infiltrating T cells, PVRIG and other genes of DNAM-1 axis were also largely expressed in tumor bed, and even more intensely in TLS (p&lt;0.05, figure 2). Furthermore, high resolution unsupervised scRNA gene co-expression analysis in the TME further validated that while PD-1 is strongly correlated with TIM3, CTLA-4, and other markers of exhausted T-cells, PVRIG uniquely clusters with markers of stem-like T-cells. The PVRIG protein expression was increased on CD28+ stem-like T-cells across indications (figure 3). RNA and protein expression data identified PVRL2, PVRIG ligand, preferentially expressed across DC-subtypes compared to PD-L1 and PVR (figure 4). PVRIG blockade could therefore enhance memory T-cell activation by DCs, resulting in their increased expansion and differentiation. Accordingly, COM701 monotherapy induced CD8+ T-cell numbers and immune activation in the TME of ovarian cancer patient (figure 5). Moreover, MSS-CRC patient with partial response to COM701+nivolumab, demonstrated an increase in TCR numbers, clonality, T-cell infiltration and activation in the TME (figure 6). Finally, preliminary analysis of serum from two patients clinically responding to COM701+nivolumab (RECIST criteria), revealed induction of activated-DC markers, compared to non-responders (figure 7). <h3>Conclusions</h3> By leveraging spatial and scRNA transcriptomics, we identified PVRIG<sup>+</sup>CD8<sup>+</sup>T-cells predominantly localized within TLS, interacting with PVRL2<sup>+</sup>LAMP3<sup>+</sup>DCs. PVRIG blockade could therefore enhance the differentiation and expansion of stem-like CD8<sup>+</sup> T-cells into effector cells (figure 8). Accordingly, early clinical data shows increased T-cells infiltration and immune activation in patients treated with COM701 or COM701+nivolumab. <h3>References</h3> Schumacher TN, Thommen DS. Tertiary lymphoid structures in cancer. <i>Science</i>. 2022;<b>375</b>(6576):eabf9419 Magen A, Hamon P, Fiaschi N, Troncoso L, Humblin E, D’souza D, Dawson T, Park MD, Kim J, Hamel S, Buckup M. Intratumoral mregDC and CXCL13 T helper niches enable local differentiation of CD8 T cells following PD-1 blockade. <i>bioRxiv</i>. 2022 Alteber Z, Kotturi MF, Whelan S, Ganguly S, Weyl E, Pardoll DM, Hunter J, Ophir E. Therapeutic targeting of checkpoint receptors within the DNAM1 axis. <i>Cancer Discovery</i>. 2021;<b>11</b>(5):1040–51.