850 Dual blockade of the EP2 and EP4 PGE2 receptors with TPST-1495 is an optimal approach for drugging the prostaglandin pathway

BackgroundProstaglandin E2 (PGE2) is a bioactive lipid produced by tumor cells that drives disease progression through stimulating tumor proliferation, enhancing angiogenesis and suppressing immune function in the TME.1 PGE2 is also a mediator of adaptive resistance to immune checkpoint inhibitor therapy via the upregulation of cyclooxygenase-2 (COX-2). While the role of PGE2 signaling in cancer is clear, how best to inhibit PGE2 for cancer treatment remains under investigation. Inhibition of COX-1 and/or COX-2 has shown promising results in observational studies and meta-analyses, but inconsistent results in prospective studies. PGE2 signals through four receptors, EP1-4, that are variably expressed on tumor and immune cells and have distinct biological activities. The EP2/EP4 receptors signal through cAMP and drive pro-tumor activities, while EP1/EP3 receptors signal through calcium flux and IP3 and drive immune activation and inflammation. While COX-2 and single EP inhibitors continue to be developed, the nature of PGE2 signaling supports our rationale to inhibit PGE2 by dual antagonism of the pro-tumor EP2/EP4 receptors, while sparing the pro-immune EP1/EP3 receptors.MethodsWe utilized human and murine whole blood to perform in vitro characterization of PGE2/inhibitor activity. In vivo, CT26 tumors and APCmin/+ mice were used to model CRC and measure immune endpoints.ResultsIn mouse and human whole blood assays, dual blockade of EP2 and EP4 receptors with TPST-1495 reversed PGE2-mediated suppression of LPS induced TNF-α, while EP4 receptor antagonists were unable to block suppression at higher PGE2 concentrations. Similarly, in murine and human T cells in vitro, TPST-1495 inhibited PGE2-mediated suppression, resulting in a significant increase of IFN-γ production in response to stimulation with cognate peptide Ag. In vivo, TPST-1495 therapy alone also significantly reduced tumor outgrowth in CT26 tumor bearing mice, correlated with increased tumor infiltration by NK cells, CD8+ T cells, AH1-specific CD8+ T cells, and DCs. The induced NKp46+CD4-CD8- cell population appeared to have an important role in TPST-1495 efficacy, as significant anti-tumor activity was observed in murine models lacking T Cells, particularly CT26 tumor-bearing RAG2-/- mice. TPST-1495 monotherapy demonstrated a decrease of both the intestinal tumor size and number in Adenomatous Polyposis (APCmin/+) mice, as compared to a single EP4 antagonist.ConclusionsTPST-1495 is a potent inhibitor of PGE2 mediated immune suppression and is currently being evaluated in an ongoing Phase 1 first-in-human study (NCT04344795) to characterize PK, PD, safety, and to identify a recommended phase 2 dose for expansion cohorts in key indications and biomarker selected patients.ReferenceZelenay S, van der Veen AG, Böttcher JP, et al. Cyclooxygenase-dependent tumor growth through evasion of immunity. Cell 2015;162(6):1257–70. doi: 10.1016/j.cell.2015.08.015

848 Novel small molecule HPK1 inhibitor PCC-1 induces strong anti-tumor activity

BackgroundHematopoietic progenitor kinase 1 (HPK1, MAP4K1), is a negative regulator of T and B cell receptor signaling.1 2 3 A strong anti-tumor immunogenic response and tumor rejection was observed in mice with HPK1 gene knocked out.3 Treatment of HPK1 kinase dead mice with immune check-point blockers (ICBs) demonstrated enhanced tumor growth inhibition.3 Hence, HPK1 is an attractive therapeutic strategy for immuno-oncology based treatment in cancers. In comparison to our previous HPK1 small molecule inhibitor, PCC,4 we present here a differentiated novel HPK1 inhibitor, PCC-1 with good anti-T cell kinases selectivity and stronger anti-tumor efficacy in CT26 tumor model. In addition, using the syngeneic model of MC38 expressing human PD-L1, we present for the first time, the combination efficacy of a HPK1 inhibitor with the clinical ICB, Atezolizumab.MethodsIntuitive medicinal chemistry complemented by structure-based drug design was used to identify & develop potent inhibitors of HPK1 with optimal kinase selectivity, PK and in vivo efficacy profile. The SAR efforts were guided by biochemical assays, functional read-outs and primary human in vitro T-cell activation assays. In vivo target engagement and pharmacodynamic data was generated using CT26 and MC38-hPD-L1 tumor models.ResultsPCC-1 has sub-nanomolar HPK1 inhibition potency and strong target engagement resulting in pSLP76 inhibition, enhanced anti-tumor cytokine production of IL-2 and/or IFNgamma in Jurkat cells, human PBMCs and human whole blood. PCC-1 also demonstrated nanomolar potency in inducing a complete reversal of PGE2 or adenosine mediated immunosuppression. Oral dosing of PCC-1 as a single agent, induced strong tumor growth inhibition (TGI) in the syngeneic model of CT26 and MC38-hPD-L1 tumor models. Combination of PCC-1 with anti-CTLA4 in CT26 tumor model induced significantly greater TGI than anti-CTLA4 alone. Moreover, as a first, the combination of PCC-1 with clinical ICB, Atezolizumab in MC38-hPD-L1 induced enhanced rejection of tumors. These results strongly suggest PCC-1 as a promising candidate for HPK1 inhibition and as a combination partner with ICBs in clinic.ConclusionsPCC-1 is a novel, orally active HPK1 inhibitor that demonstrates excellent stand-alone efficacy and enhances current immunotherapy efficacy. Further evaluation of PCC-1 is ongoing to advance towards clinic.AcknowledgementsWe thank Dnyaneshwar Dahale, Sanjay Patale, Sandip Patil, Vidya Kattige, Jiju Mani, Namrata Singh, Ekta Kashyap, Sandeep Thorat, Pankaj Jain and Pramod Sagar for their contributions to the projectTrial RegistrationN/AReferencesKiefer F, et al. The EMBO Journal 1996.Hu, et al. Genes and Development 1996.Sawasdikosol, Burakoff. eLife 2020;9:e55122.Sachin S Chaudhari, et al. Poster#1709, AACR Annual Meeting April-May 2021.Ethics ApprovalThe studies involving animals have obtained ethics approval from Institutional Animal Ethics Committee (IAEC), The Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), New Delhi, India, GRC/IAEC/472/2020-1. Participants of the studies have given informed consent before taking part.

P08.03 Neoantigen cancer vaccine auguments anti CTLA-4 efficacy

BackgroundImmunotherapy based on anti CTLA-4 (αCTLA-4) and anti PD1 (αPD1) is being tested in combination with different therapeutic approaches including other immunotherapy approaches such as neoantigen cancer vaccines (NCV). Here we explored, in two cancer murine models, different therapeutic combinations of αCTLA-4 and/or αPD1 with a plasmid DNA vaccine expressing neoantigens and delivered by electroporation (EP).Materials and MethodsTo evaluate the impact of NCV in the MC38 and in the CT26 tumor model three plasmid vaccines were generated with or without CD4 epitopes. Therapeutic DNA vaccines were delivered by EP in different therapeutic protocols including large tumors. Flow cytometry was utilized to measure CD8, CD4, T-reg, and B cells as well as neoantigen-specific immune responses, which were also measured by IFN-γ ELIspot.ResultsImmune responses were augmented in combination with αCTLA4 but not with αPD1 in the MC38 tumor model with significantly impacting tumor growth. Similarly, neoantigen-specific T cell immune responses were observed in the CT26 tumor model where large tumors regressed in all mice treated with αCTLA-4 and NCV. In line with previous evidence, we observed an increased switched memory B cells in the spleen of mice treated with αCTLA-4 alone or in combination with NCV.ConclusionsThese results support the use of NCV delivered by DNA-EP with αCTLA-4 and suggest a new combined therapy for clinical testing.Disclosure InformationF. Palombo: A. Employment (full or part-time); Significant; Neomatrix biotech. E. Salvatori: A. Employment (full or part-time); Significant; Takis biotech. L. Lione: A. Employment (full or part-time); Significant; Takis biotech. M. Compagnone: A. Employment (full or part-time); Significant; Neomatrix biotech. A. Conforti: A. Employment (full or part-time); Significant; Evvivax. L. Aurisicchio: A. Employment (full or part-time); Significant; Neomatrix biotech, Takis biotech, Evvivax.

PET Imaging of CD8 via SMART for Monitoring the Immunotherapy Response

Imaging of CD8 receptors on T-cells by positron emission tomography (PET) has been considered a promising strategy for monitoring the treatment response to immunotherapy. In this study, a trial of imaging CD8 with our newly developed sequential multiple-agent receptor targeting (SMART) technology was conducted. Mice bearing a subcutaneous colorectal CT26 tumor received three times different immunotherapy treatments (PD1 or CTLA4 or combined). On either day 7 or day 14 after the first time treatment, the PET imaging study was performed with sequentially administered TCO-modified anti-CD8 antibody and 64Cu-labeled MeTz-NOTA-RGD. However, no positive response was detected, probably due to (1) inappropriate selection of biomarkers for the SMART strategy, (2) limited TCO modification on the anti-CD8 antibody, and (3) inadequate response of the CT26 tumor to the selected immunotherapies. Therefore, the potential of applying SMART in imaging CD8 was not demonstrated in this study, and further optimization will be necessary before it can be applied in imaging CD8.

253 Anti-TIGIT antibodies require enhanced FcγR co-engagement for optimal T and NK cell-dependent anti-tumor immunity

BackgroundT-cell immunoreceptor with Ig and ITIM domains (TIGIT) is an important negative regulator of the immune response to cancer that contributes to resistance/relapse to anti-PD-1 therapy.1 In clinical trials, anti-human (h) TIGIT antibodies have shown promising activity in combination with anti-PD-1/PD-L1 antibodies for the treatment of various solid tumors.2 However, the optimal format for anti-TIGIT antibodies remains controversial. Here we describe a novel Fcγ receptor (FcγR)-dependent mechanism of action that is critical for enhancing T and NK cell anti-tumor immunity, and, further informs on the optimal design of anti-TIGIT antibodies.MethodsWe investigated a panel of Fc-silent, Fc-competent, and Fc-engineered anti-mouse (m) TIGIT antibody variants in syngeneic murine CT26 tumor-bearing or B16F10 pseudo-metastases models. To further elucidate the relative contribution of T and NK cells in controlling tumor growth, we assessed the activity of Fc-engineered anti-TIGIT antibodies in NK cell-depleted or T cell-deficient (Nu-Foxn1nu) CT26 tumor-bearing mice. Immune-related pharmacodynamic changes in the tumor microenvironment were assessed by flow cytometry. We further validated these findings in primary human T and NK cell activation assays using Fc-engineered anti-human TIGIT antibodies.ResultsThe Fc-engineered anti-mTIGIT antibody, which demonstrates enhanced binding to mouse FcγRIV, was the only variant to deliver single agent anti-tumor activity. The Fc-enhanced variant outperformed the Fc-competent variant while the Fc-inert variant had no anti-tumor activity. Tumor control by anti-mTIGIT antibodies was not dependent on Treg depletion, but rather on increased frequency of CD8+ T cells and activated NK cells (Ki67, IFNγ, CD107a and TRAIL) in the tumor microenvironment. Concordant with observations in the mouse, Fc-engineered anti-hTIGIT antibodies with improved binding to FcγRIIIA demonstrate superior T and NK cell activation in PBMC-based assays compared to a standard hIgG1 variant. Notably, superior activity of the Fc-engineered anti-hTIGIT antibody was observed from PBMC donors that express either high or low affinity FcγRIIIA. Blockade of FcγRIIIA or depletion of CD14+ and CD56+ cells reduced the functional activity of the Fc-enhanced anti-TIGIT antibody, confirming the requirement for FcγR co-engagement to maximize T cell responses.ConclusionsOur data demonstrate the importance of FcγR co-engagement by anti-TIGIT antibodies to promote immune activation and tumor control. First generation anti-TIGIT antibodies are not optimally designed to co-engage all FcγRIIIA variants. However, Fc-enhanced anti-TIGIT antibodies unlock a novel FcγR-dependent mechanism of action to enhance T and NK cell-dependent anti-tumor immunity and further improve therapeutic outcomes.ReferencesJohnston RJ, et al., The immunoreceptor TIGIT regulates antitumor and antiviral CD8(+) T cell effector function. Cancer Cell 2014; 26:923–37.Rodriguez-Abreu D, et al., Primary analysis of a randomized, double-blind, phase II study of the anti-TIGIT antibody tiragolumab (tira) plus atezolizumab (atezo) versus placebo plus atezo as first-line (1L) treatment in patients with PD-L1-selected NSCLC (CITYSCAPE). Journal of Clinical Oncology 2020; 38:15_suppl, 9503–9503.

A recombinant oncolytic Newcastle virus expressing MIP-3α promotes systemic antitumor immunity

BackgroundThe oncolytic Newcastle disease virus (NDV) is inherently able to trigger the lysis of tumor cells and induce the immunogenic cell death (ICD) of tumor cells and is also an excellent gene-engineering vector. The macrophage inflammatory protein-3α (MIP-3α) is a specific chemokine for dendritic cells (DCs). Thus, we constructed a recombinant NDV expressing MIP-3α (NDV-MIP3α) as an in vivo DC vaccine for amplifying antitumor immunities.MethodsThe recombinant NDV-MIP3α was constructed by the insertion of MIP-3α cDNA between the P and M genes. Western blotting assay and ELISA were used to detect MIP-3α, HMGB1, IgG, and ATP in the supernatant and sera. The chemotaxis of DCs was examined by Transwell chambers. The phenotypes of the immune cells (eg, DCs) were analyzed by flow cytometry. The antitumor efficiency of NDV-MIP3α was observed in B16 and CT26 tumor-bearing mice. Immunofluorescence and immunohistochemistry were applied to observe the ecto-calreticulin (CRT) and intratumoral attraction of DCs. Adoptive transfer of splenocytes and antibodies and depletion of T-cell subsets were used to evaluate the relationship between antitumor immunities and the role of the T-cell subtype.ResultsThe findings show that NDV-MIP3α has almost the same capabilities of tumor lysis and induction of ICD as the wild-type NDV (NDV-WT). MIP-3α secreted by NDV-MIP3α could successfully attract DCs in vitro and in vivo. Both B16 and CT26 cells infected with NDV-MIP3α could strongly promote DC maturation and activation. Compared with NDV-WT, intratumoral injection of NDV-MIP3α and the adoptive transfer of T lymphocytes from mice injected with NDV-MIP3α resulted in a significant suppression of B16 and CT26 tumor growth. The NDV-MIP3α-induced production of tumor-specific cellular and humoral immune responses was dependent on CD8+ T cells and partially on CD4+ T cells. A significant reversion of tumor microenvironments was found in the mice injected with NDV-MIP3α.ConclusionsCompared with NDV-WT, the recombinant NDV-MIP3α as an in vivo DC vaccine demonstrates enhanced antitumor activities through the induction of stronger system immunities and modulation of the tumor microenvironment. This strategy may be a potential approach for the generation of an in vivo DC vaccine.