Chimeric antigen receptor (CAR) T cell therapy has led to impressive clinical responses in patients with hematological malignancies; however, its effectiveness in patients with solid tumors has been limited. While CAR T cells for the treatment of advanced prostate and pancreas cancer, including those targeting prostate stem cell antigen (PSCA), are being clinically evaluated and are anticipated to show bioactivity, their safety and the impact of the immunosuppressive tumor microenvironment (TME) have not been faithfully explored preclinically. Using a novel human PSCA knockin (hPSCA-KI) immunocompetent mouse model, we evaluated the safety and therapeutic efficacy of PSCA-CAR T cells. We demonstrated that cyclophosphamide (Cy) pre-conditioning significantly modified the immunosuppressive TME and was required to uncover the efficacy of PSCA-CAR T cells in metastatic prostate and pancreas cancer models, with no observed toxicities in normal tissues with endogenous expression of PSCA. This combination dampened the immunosuppressive TME, generated pro-inflammatory myeloid and T cell signatures in tumors, and enhanced the recruitment of antigen-presenting cells, as well as endogenous and adoptively transferred T cells, resulting in long-term anti-tumor immunity.

BackgroundThe immune suppressive tumor microenvironment (TME) that inhibits T cell infiltration, survival, and antitumor activity has posed a major challenge for developing effective immunotherapies for solid tumors. Chimeric antigen receptor (CAR)-engineered T cell therapy has shown unprecedented clinical response in treating patients with hematological malignancies, and intense investigation is underway to achieve similar responses with solid tumors. Immunologically cold tumors, including prostate cancers, are often infiltrated with abundant tumor-associated macrophages (TAMs), and infiltration of CD163+ M2 macrophages correlates with tumor progression and poor responses to immunotherapy. However, the impact of TAMs on CAR T cell activity alone and in combination with TME immunomodulators is unclear.MethodsTo model this in vitro, we utilized a novel co-culture system with tumor cells, CAR T cells, and polarized M1 or M2 macrophages from CD14+ peripheral blood mononuclear cells collected from healthy human donors. Tumor cell killing, T cell activation and proliferation, and macrophage phenotypes were evaluated by flow cytometry, cytokine production, RNA sequencing, and functional blockade of signaling pathways using antibodies and small molecule inhibitors. We also evaluated the TME in humanized mice following CAR T cell therapy for validation of our in vitro findings.ResultsWe observed inhibition of CAR T cell activity with the presence of M2 macrophages, but not M1 macrophages, coinciding with a robust induction of programmed death ligand-1 (PD-L1) in M2 macrophages. We observed similar PD-L1 expression in TAMs following CAR T cell therapy in the TME of humanized mice. PD-L1, but not programmed cell death protein-1, blockade in combination with CAR T cell therapy altered phenotypes to more M1-like subsets and led to loss of CD163+ M2 macrophages via interferon-γ signaling, resulting in improved antitumor activity of CAR T cells.ConclusionThis study reveals an alternative mechanism by which the combination of CAR T cells and immune checkpoint blockade modulates the immune landscape of solid tumors to enhance therapeutic efficacy of CAR T cells.

Chimeric antigen receptor (CAR) T cell therapeutic responses are hampered by limited T cell trafficking, persistence, and durable anti-tumor activity in solid tumors. However, these challenges can be largely overcome by relatively unconstrained synthetic engineering strategies. Here, we describe CAR T cells targeting tumor-associated glycoprotein-72 (TAG72), utilizing the CD28 transmembrane domain upstream of the 4-1BB co-stimulatory domain as a driver of potent anti-tumor activity and IFNγ secretion. CAR T cell-mediated IFNγ production facilitated by IL-12 signaling is required for tumor cell killing, which is recapitulated by engineering an optimized membrane-bound IL-12 (mbIL12) molecule in CAR T cells. These T cells show improved antigen-dependent T cell proliferation and recursive tumor cell killing in vitro, with robust in vivo efficacy in human ovarian cancer xenograft models. Locoregional administration of mbIL12-engineered CAR T cells promotes durable anti-tumor responses against both regional and systemic disease in mice. Safety and efficacy of mbIL12-engineered CAR T cells is demonstrated using an immunocompetent mouse model, with beneficial effects on the immunosuppressive tumor microenvironment. Collectively, our study features a clinically-applicable strategy to improve the efficacy of locoregionally-delivered CAR T cells engineered with antigen-dependent immune-modulating cytokines in targeting regional and systemic disease.

BackgroundThe immune suppressive tumor microenvironment (TME) that inhibits T cell infiltration, survival, and anti-tumor activity has posed a major challenge for developing effective immunotherapies for solid tumors. Chimeric antigen receptor T cell therapy has shown unprecedented clinical response in treating patients with hematological malignancies, and intense investigation is underway to achieve similar responses with solid tumors. Immunologically cold tumors, including prostate cancers, are often infiltrated with abundant macrophages, and infiltration of M2 macrophages correlates with metastasis and poor prognosis.MethodsTo model this in vitro, we utilized a novel co-culture system with tumor cells, prostate stem cell antigen (PSCA)-directed CAR T cells, and polarized macrophages. To investigate the TME in vivo, we took advantage of ”humanized” MISTRG mice, which are immunocompromised mice with knocked-in human genes that support human hematopoiesis and efficient tumor-infiltration of myeloid cell populations. Humanized MISTRG mice were intratibially engrafted with LAPC9 tumor cells to model bone metastatic disease.ResultsWe observed significant hampering of PSCA-CAR T cell activity in vitro with the presence of M2 macrophages, but not M1 macrophages, coinciding with a robust induction of PD-L1 in both tumor cells and macrophages. We also observed PD-L1 expression in tumor-associated macrophages infiltrating tumors following PSCA-CAR T cell therapy in the humanized mice. Anti-PD-L1 monoclonal antibodies in combination with CAR-T cell therapy altered phenotype and survival of M2 macrophages, resulting in improved anti-tumor activity of PSCA-CAR T cells in the presence of M2 macrophages.ConclusionsRecently, immune checkpoint (IC) blockade (ICB) has been utilized in combination with chimeric antigen receptor (CAR) T cell therapy, with the notion that induction of immune responses with CAR T cells may instigate checkpoint pathways in immunologically cold tumors that would otherwise not respond to ICB. This study gives insights to a mechanism by which CAR T cells and ICB work in synergy to modulate immune landscape of immunologically cold tumors, and our ongoing studies will continue to elucidate the TME-mediated immunosuppression of CAR T cell therapy.

While chimeric antigen receptor (CAR) T cells have received FDA approvals in treating hematological malignancies, clinical responses of CAR T cells for solid tumors have been underwhelming. Intense investigation is underway to improve CAR T cells trafficking, persistence and efficacy. Interleukin-12 (IL-12) is a potent inflammatory cytokine that recruits and activates various endogenous and adoptively transferred immune cells, but development of its clinical applications is hindered by toxicity of systemic IL-12 signaling. In this study, we engineered and expressed membrane-bound IL-12 (mbIL12) in CAR T cells to overcome current limitations in CAR T cell and IL-12 therapies. We evaluated CAR T cell function using co-culture assays and preclinical murine models bearing human tumor xenografts. We modeled systemic metastasis of ovarian and breast cancer and assessed the impact of mbIL12 in CAR T cells on targeting regional and systemic diseases. Syngeneic mouse models were used to evaluate toxicity induced by mbIL12 in CAR T cells. We observed a greater number of CAR T cells in the peripheral blood of mice receiving locoregionally delivered CAR T cells engineered with mbIL12, suggesting that mbIL12 improves CAR T cell trafficking and persistence. Furthermore, mice bearing regional and systemic diseases experienced more potent and durable anti-tumor activity when treated with mbIL12 endowed CAR T cells. These results were mirrored in vitro where mbIL12 improved CAR T cells activation, proliferation and IFNg secretion when co-cultured with tumor cells, recapitulating our findings in vivo. Toxicities observed in immune-competent mice receiving systemic IL-12 delivery were not detected in those administered CAR T cells with mbIL12. We demonstrated that mbIL12 enhances anti-tumor function of CAR T cells for treatment of solid tumors. This study also showed that mbIL12 is safe, providing a translatable engineering strategy to improve CAR T cell therapy. IL-12 signaling is well known to modulate function of various immune cells, but the impact of CAR T cells with mbIL12 on immune tumor microenvironments remains to be elucidated. We are currently investigating changes in the immune landscape that CAR T cells with mbIL12 induce. Citation Format: Hee Jun Lee, Yuki Yamaguchi, Jackson Gibson, Cody Cullen, John P. Murad, Anthony K. Park, Isabel Monroy, Cari Young, Lea Christian, Lauren Adkins, Lawrence Stern, Wen-Chung Chang, Catalina Martinez, Stephen J. Forman, Saul J. Priceman. Regional administration of IL-12 endowed CAR T cells effectively targets systemic disease. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4090.