The efficacy of chimeric antigen receptor (CAR) T cell therapy against poorly responding tumors can be enhanced by administering the cells in combination with immune checkpoint blockade inhibitors. Alternatively, the CAR construct has been engineered to coexpress factors that boost CAR-T cell function in the tumor microenvironment. We modified CAR-T cells to secrete PD-1-blocking single-chain variable fragments (scFv). These scFv-secreting CAR-T cells acted in both a paracrine and autocrine manner to improve the anti-tumor activity of CAR-T cells and bystander tumor-specific T cells in clinically relevant syngeneic and xenogeneic mouse models of PD-L1 hematologic and solid tumors. The efficacy was similar to or better than that achieved by combination therapy with CAR-T cells and a checkpoint inhibitor. This approach may improve safety, as the secreted scFvs remained localized to the tumor, protecting CAR-T cells from PD-1 inhibition, which could potentially avoid toxicities associated with systemic checkpoint inhibition.

A novel set of GAC (kidney glutaminase isoform C) inhibitors able to inhibit the enzymatic activity of GAC and the growth of the triple negative MDA-MB-231 breast cancer cells with low nanomolar potency is described. Compounds in this series have a reduced number of rotatable bonds, improved ClogPs, microsomal stability and ligand efficiency when compared to the leading GAC inhibitors BPTES and CB-839. Property improvements were achieved by the replacement of the flexible n-diethylthio or the n-butyl moiety present in the leading inhibitors by heteroatom substituted heterocycloalkanes.

T-cell receptor (TCR)-modified T-cell gene therapy can target a variety of extracellular and intracellular tumor associated antigens, yet has had little clinical success. A potential explanation for limited antitumor efficacy is a lack of T-cell activation in vivo. We postulated that expression of pro-inflammatory cytokines in TCR-modified T cells would activate T cells and enhance antitumor efficacy. We demonstrate that expression of interleukin 18 (IL18) in tumor-directed TCR-modified T cells provides a superior proinflammatory signal than expression of interleukin 12 (IL12). Tumor-targeted T cells secreting IL18 promote persistent and functional effector T cells and a pro-inflammatory tumor microenvironment. Together, these effects augmented overall survival of mice in the pmel-1 syngeneic tumor model. When combined with sublethal irradiation, IL18secreting pmel-1 T cells were able to eradicate tumors, whereas IL12-secreting pmel-1 T cells caused toxicity in mice through excessive cytokine secretion. In another xenograft tumor model, IL18 secretion enhanced the persistence and antitumor efficacy of NY-ESO-1-reactive TCR-modified human T cells as well as overall survival of tumorbearing mice. These results demonstrate a rationale for optimizing the efficacy of TCRmodified T-cell cancer therapy through expression of IL18.

While T cell receptor (TCR)-modified T cell therapies have shown promise against solid tumors, overall therapeutic benefits in clinical practice have been modest due in part to suboptimal T cell persistence and activation in vivo, alongside the possibility of tumor antigen escape. In this study, we demonstrate an approach to enhance the in vivo persistence and activation of TCR-T cells through combination with Amphiphile (AMP)-vaccination including cognate TCR-T peptides. AMP-modification improves lymph node targeting of conjugated tumor immunogens and adjuvants, thereby coordinating a robust T cell-priming endogenous immune response. Vaccine combination with TCR-T cell therapy provided simultaneous in vivo invigoration of adoptively transferred TCR-T cells and in situ priming of the endogenous anti-tumor T cell repertoire. The resulting induction of an adoptive and endogenous anti-tumor effect led to durable responses in established murine solid tumors refractory to TCR-T cell monotherapy. Protection against recurrence was associated with antigen spreading to additional tumor-associated antigens not targeted by vaccination. Enhanced anti-tumor efficacy was further correlated with pro-inflammatory lymph node transcriptional reprogramming and increased antigen presenting cell maturation, resulting in TCR-T cell expansion and functional enhancement in lymph nodes and solid tumor parenchyma without lymphodepletion. In vitro evaluation of AMP-peptides with matched human TCR-T cells targeting NY-ESO-1, mutant KRAS, and HPV16 E7 illustrated the clinical potential of AMP-vaccination to enhance human TCR-T cell proliferation, activation, and anti-tumor activity. Taken together, these studies provide rationale and evidence to support clinical evaluation of the combination of AMP-vaccination with TCR-T cell therapies to augment anti-tumor activity.

BackgroundClinical results from TCR-T Cell therapies demonstrate anti-tumor efficacy, although therapeutic benefits remain transient due to suboptimal T Cell functional persistence and tumor infiltration alongside antigen escape mechanisms.1 2 3 4 5 Amphiphile (AMP) vaccines improve lymph node targeting of cancer immunogens, stimulating an enhanced endogenous anti-tumor response.6 7 We describe an approach to generate robust and durable anti-tumor responses by combining AMP lymphatic targeting with TCR-T Cell therapy. AMP cognate peptides traffic to lymph nodes and improve TCR-T Cell activation, persistence, and function compared to soluble (SOL) peptide vaccination or TCR-T Cells alone, inducing a superior anti-tumor effect.MethodsC57BL/6J mice were subcutaneously implanted with B16F10 10 days prior to transduced pmel-1 T cell transfer or 75 days after T cell treatment for rechallenge experiments. Tumor-bearing mice received 5 doses, 2x/week of AMP-GP100/AMP-CpG, SOL-GP100/SOL-CpG, or PBS by tail-base vaccination. Caliper measurements determined tumor progression and overall survival. TCR-T Cell persistence was assessed bi-weekly through retro-orbital bleeds. Tumors and lymph nodes from treated mice were excised and analyzed by Nanostring for differential gene expression and flow cytometry for TCR-T Cell functional persistence and T cell epitope spread. Human T Cells (HTCs) and Dendritic Cells (DCs) were isolated from autologous PBMCs, transduced with KRAS-specific TCRs, and cultured with AMP-KRAS-peptide pulsed DCs before assaying T Cell boosting.ResultsWe demonstrate that AMP vaccination expands tumor specific TCR-T Cells in vivo up to 46-fold while enhancing the activation, cytokine secretion, and pro-inflammatory gene expression of tumor-infiltrating TCR-T Cells. Endogenous tumor-infiltrating T cells from AMP vaccinated mice produced up to 17-fold greater cytokine secretion following re-stimulation with non-targeted tumor epitopes. These results correspond to the eradication of established B16F10 tumors and a resistance to secondary tumor challenge in cured mice. Providing clinical relevance, HTCs transduced with KRAS-specific TCRs and boosted with AMP-KRAS-peptide pulsed DCs exhibited enhanced T cell activation, Th1 cytokine secretion, and cytolytic capacity compared to HTCs exposed to unlabeled DCs.ConclusionsAMP vaccination delivers cognate peptides to lymph nodes providing in vivo activation of tumor-specific TCR-T Cells which amplifies anti-tumor potency of such adoptively transferred cells. AMP vaccination significantly enhanced TCR-T Cell anti-tumor response and led to durable cures of solid tumors in an established, syngeneic tumor model. Additionally, AMP-peptide pulsed autologous DCs enhanced the function of clinically relevant KRAS-specific TCR-T cells in vitro. Taken together, these studies provide direct rationale and evidence for the combination of AMP vaccination with TCR-T Cell therapies to augment clinical responses.ReferencesRobbins PF, Morgan RA, Feldman SA, Yang JC, Sherry RM, Dudley ME, Wunderlich JR, Nahvi AV, Helman LJ, Mackall CL, et al. Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1. J Clin Oncol 2011;29:917–924. doi: 10.1200/JCO.2010.32.2537.Rapoport AP, Stadtmauer EA, Binder-Scholl GK, Goloubeva O, Vogl DT, Lacey SF, Badros AZ, Garfall A, Weiss B, Finklestein J, et al. NY-ESO-1-specific TCR-engineered T cells mediate sustained antigen-specific antitumor effects in myeloma. Nat Med 2015;21:914–921. doi: 10.1038/nm.3910.Doran SL, Stevanovic S, Adhikary S, Gartner JJ, Jia L, Kwong MLM, Faquin WC, Hewitt SM, Sherry RM, Yang JC, et al. T-Cell Receptor Gene Therapy for Human Papillomavirus-Associated Epithelial Cancers: A First-in-Human, Phase I/II Study. J Clin Oncol 2019;37:2759–2768. doi: 10.1200/JCO.18.02424.Chandran SS, Klebanoff CA. T cell receptor-based cancer immunotherapy: Emerging efficacy and pathways of resistance. Immunol Rev 2019;290:127–147. doi: 10.1111/imr.12772.D’Angelo SP, Melchiori L, Merchant MS, Bernstein D, Glod J, Kaplan R, Grupp S, Tap WD, Chagin K, Binder GK, et al. Antitumor Activity Associated with Prolonged Persistence of Adoptively Transferred NY-ESO-1 (c259)T Cells in Synovial Sarcoma. Cancer Discov 2018;8:944–957. doi: 10.1158/2159-8290.CD-17-1417.H Liu, KD Moynihan, Y Zheng, GL Szeto, AV Li, B Huang, DS Van Egeren, C Park, DJ Irvine. Structure-based programming of lymph-node targeting in molecular vaccines. Nature 2014;507: 519–522. doi: 10.1038/nature12978.KD Moynihan, CF Opel, GL Szeto, A Tzeng, EF Zhu, JM Engreitz, RT Williams, K Rakhra, MH Zhang, AM Rothschilds, S Kumari, RL Kelly, BH Kwan, W Abraham, K Hu, NK Mehta, MJ Kauke, H Suh, JR Cochran, DA Lauffenburger, KD Wittrup, DJ Irvine. Eradication of large established tumors in mice by combination immunotherapy that engages innate and adaptive immune responses. Nat Med 2016; 22: 1402–1410. doi: 10.1038/nm.4200.

<div>Abstract<p>T-cell receptor (TCR)–modified T-cell gene therapy can target a variety of extracellular and intracellular tumor-associated antigens, yet has had little clinical success. A potential explanation for limited antitumor efficacy is a lack of T-cell activation <i>in vivo</i>. We postulated that expression of proinflammatory cytokines in TCR-modified T cells would activate T cells and enhance antitumor efficacy. We demonstrate that expression of interleukin 18 (IL18) in tumor-directed TCR-modified T cells provides a superior proinflammatory signal than expression of interleukin 12 (IL12). Tumor-targeted T cells secreting IL18 promote persistent and functional effector T cells and a proinflammatory tumor microenvironment. Together, these effects augmented overall survival of mice in the pmel-1 syngeneic tumor model. When combined with sublethal irradiation, IL18-secreting pmel-1 T cells were able to eradicate tumors, whereas IL12-secreting pmel-1 T cells caused toxicity in mice through excessive cytokine secretion. In another xenograft tumor model, IL18 secretion enhanced the persistence and antitumor efficacy of NY-ESO-1–reactive TCR-modified human T cells as well as overall survival of tumor-bearing mice. These results demonstrate a rationale for optimizing the efficacy of TCR-modified T-cell cancer therapy through expression of IL18.</p><p><i>See related commentary by Wijewarnasuriya et al., p. 732</i></p></div>

Clinical trials utilizing T cells genetically engineered to express a tumor-targeted T-cell receptor (TCR) illustrate the promise of this therapy in treating cancer patients. However, there are obstacles that must be overcome to improve the efficacy of TCR gene therapy. Two separate signals are needed to completely activate T cells and drive T-cell proliferation and differentiation. Co-stimulatory molecules present on the surface of cancer cells can become downregulated as an escape mechanism, thus depriving the T cells of complete activation and leading to T-cell apoptosis or anergy. This project aims to armor tumor-associated antigen-specific T cells with proinflammatory cytokines, specifically IL-12 and IL-18. We hypothesize that this genetic modification will provide an alternate form of co-stimulation that will augment antitumor efficacy. Tumor-associated antigen specific pmel-1 T cells transduced with retroviral constructs were shown to be more functional through increased secretion of IFNγ when co-cultured with antigen-positive tumor cells. Additionally, the armored pmel T cells were able to significantly enhance lysis of the B16F10 melanoma cell line in vitro. Subsequently, the in vivo efficacy of the armored T cells was assessed in a syngeneic, immune-competent B16F10 melanoma mouse model. The armored pmel T cells were confirmed to significantly delay the B16F10 tumor progression and enhance the survival of these mice in comparison to control pmel T cells. These results provide future insight into applying the concept of armored TCR T cells to a therapeutically relevant human tumor target to enhance TCR-modified T-cell antitumor responses. Citation Format: Dylan J. Drakes, Sarwish Rafiq, Terence Purdon, Renier Brentjens. Engineering armored TCR-modified T cells to enhance anti-tumor efficacy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A37.

<p>Supplementary Figures 1-8</p>