T Cells Engineered With Chimeric Antigen Receptors Targeting NKG2D Ligands Display Lethal Toxicity in Mice.

Ligands for the NKG2D receptor are overexpressed on tumors, making them interesting immunotherapy targets. To assess the tumoricidal properties of T cells directed to attack NKG2D ligands, we engineered murine T cells with two distinct NKG2D-based chimeric antigen receptors (CARs): (i) a fusion between the NKG2D receptor and the CD3ζ chain and (ii) a conventional second-generation CAR, where the extracellular domain of NKG2D was fused to CD28 and CD3ζ. To enhance the CAR surface expression, we also engineered T cells to coexpress DAP10. In vitro functionality and surface expression levels of all three CARs was greater in BALB/c T cells than C57BL/6 T cells, indicating strain-specific differences. Upon adoptive transfer of NKG2D-CAR-T cells into syngeneic animals, we observed significant clinical toxicity resulting in morbidity and mortality. The severity of these toxicities varied between the CAR configurations and paralleled their in vitro NKG2D surface expression. BALB/c mice were more sensitive to these toxicities than C57BL/6 mice, consistent with the higher in vitro functionality of BALB/c T cells. Treatment with cyclophosphamide prior to adoptive transfer exacerbated the toxicity. We conclude that while NKG2D ligands may be useful targets for immunotherapy, the pursuit of NKG2D-based CAR-T cell therapies should be undertaken with caution.

TSTEM-like CAR-T cells exhibit improved persistence and tumor control compared with conventional CAR-T cells in preclinical models.

Patients who receive chimeric antigen receptor (CAR)-T cells that are enriched in memory T cells exhibit better disease control as a result of increased expansion and persistence of the CAR-T cells. Human memory T cells include stem-like CD8+ memory T cell progenitors that can become either functional stem-like T (TSTEM) cells or dysfunctional T progenitor exhausted (TPEX) cells. To that end, we demonstrated that TSTEM cells were less abundant in infused CAR-T cell products in a phase 1 clinical trial testing Lewis Y-CAR-T cells (NCT03851146), and the infused CAR-T cells displayed poor persistence in patients. To address this issue, we developed a production protocol to generate TSTEM-like CAR-T cells enriched for expression of genes in cell replication pathways. Compared with conventional CAR-T cells, TSTEM-like CAR-T cells had enhanced proliferative capacity and increased cytokine secretion after CAR stimulation, including after chronic CAR stimulation in vitro. These responses were dependent on the presence of CD4+ T cells during TSTEM-like CAR-T cell production. Adoptive transfer of TSTEM-like CAR-T cells induced better control of established tumors and resistance to tumor rechallenge in preclinical models. These more favorable outcomes were associated with increased persistence of TSTEM-like CAR-T cells and an increased memory T cell pool. Last, TSTEM-like CAR-T cells and anti-programmed cell death protein 1 (PD-1) treatment eradicated established tumors, and this was associated with increased tumor-infiltrating CD8+CAR+ T cells producing interferon-γ. In conclusion, our CAR-T cell protocol generated TSTEM-like CAR-T cells with enhanced therapeutic efficacy, resulting in increased proliferative capacity and persistence in vivo.

Tissue-resident memory T cells from a metastatic vaginal melanoma patient are tumor-responsive T cells and increase after anti-PD-1 treatment.

BACKGROUND: Vaginal melanoma (VM) is a rare cancer and has a poor response to immune checkpoint blockade (ICB). CD8+Tissue Resident Memory (TRM) T cells proliferate in response to ICB and correlate with longer survival in metastatic cutaneous melanoma. However, their capacity to respond to VM and their neoantigens is not known. METHODS: Using longitudinal samples, we explored the evolution of VM mutations by whole-exome sequencing and RNAseq, we also defined the immune context using multiplex immunohistochemistry and nanostring pan cancer immune profile. Then using fresh single cell suspensions of the metastatic samples, we explored VM T cells via mass cytometry and single cell RNAseq and T cell receptor sequencing (TCRseq). Finally, we investigated TRM, pre-TRM and exhausted T cell function against melanoma neo-antigens and melanoma differentiation antigens in vitro. RESULTS: Primary VM was non-inflamed and devoid of CD8+ TRM cells. In contrast, both metastases showed proliferating CD8+ TRM were clustered at the tumor margin, with increased numbers in the second ICB-refractory metastasis. The first metastasis showed dense infiltration of CD8+ T cells, the second showed immune exclusion with loss of melanoma cell Major histocompatibility complex (MHC)-I expression associated with downregulation of antigen presentation pathway gene expression. CD8+ TRM from both metastases responded to autologous melanoma cells more robustly than all other CD8+ T cell subsets. In addition, CD8+ TRM shared TCR clones across metastases, suggesting a response to common antigens, which was supported by recognition of the same neoantigen by expanded tumor infiltrating lymphocytes. CONCLUSIONS: In this study, we identified TRM clusters in VM metastases from a patient, but not primary disease. We showed TRM location at the tumor margin, and their superior functional response to autologous tumor cells, predicted neoantigens and melanoma differentiation antigens. These CD8+ TRM exhibited the highest tumor-responsive potential and shared their TCR with tumor-infiltrating effector memory T cells. This suggests VM metastases from this patient retain strong antitumor T cell functional responses; however, this response is suppressed in vivo. The loss of VG MHC-I expression is a common immune escape mechanism which was not addressed by anti-PD-1 monotherapy; rather an additional targeted approach to upregulate MHC-I expression is required.

Understanding T cell phenotype for the design of effective chimeric antigen receptor T cell therapies.

Rapid advances in immunotherapy have identified adoptive cell transfer as one of the most promising approaches for the treatment of cancers. Large numbers of cancer reactive T lymphocytes can be generated ex vivo from patient blood by genetic modification to express chimeric antigen receptors (CAR) specific for tumor-associated antigens. CAR T cells can respond strongly against cancer cells, and adoptive transferred CAR T cells can induce dramatic responses against certain types of cancers. The ability of T cells to respond against disease depends on their ability to localize to sites, persist and exert functions, often in an immunosuppressive microenvironment, and these abilities are reflected in their phenotypes. There is currently intense interest in generating CAR T cells possessing the ideal phenotypes to confer optimal antitumor activity. In this article, we review T cell phenotypes for trafficking, persistence and function, and discuss how culture conditions and genetic makeups can be manipulated to achieve the ideal phenotypes for antitumor activities.

Using Mass Cytometry to Analyze the Tumor-Infiltrating Lymphocytes in Human Melanoma.

Here we describe the application of mass cytometry to analyze tumor-infiltrating lymphocytes in human melanoma. Mass cytometry is the coupling of flow cytometry and mass spectrometry, which allows for the simultaneous measurement of 40+ cell parameters on a per cell basis. Heavy metal-labeled antibodies can bind to proteins (CD markers, transcription factors, cytokines) on the cell surface and in the cytoplasm/nucleus. As labeled cells pass through the CyTOF, the instrument detects the heavy metals. Combining these signals allows description of melanoma tumor-infiltrating lymphocytes at a greater depth than alternative phenotyping strategies and enables detailed analyses of a variety of cellular parameters, including immune cell lineage, activation status, and functional polarization.

A Histone Deacetylase Inhibitor, Panobinostat, Enhances Chimeric Antigen Receptor T-cell Antitumor Effect Against Pancreatic Cancer.

PURPOSE: In this article, we describe a combination chimeric antigen receptor (CAR) T-cell therapy that eradicated the majority of tumors in two immunocompetent murine pancreatic cancer models and a human pancreatic cancer xenograft model. EXPERIMENTAL DESIGN: We used a dual-specific murine CAR T cell that expresses a CAR against the Her2 tumor antigen, and a T-cell receptor (TCR) specific for gp100. As gp100 is also known as pMEL, the dual-specific CAR T cells are thus denoted as CARaMEL cells. A vaccine containing live vaccinia virus coding a gp100 minigene (VV-gp100) was administered to the recipient mice to stimulate CARaMEL cells. The treatment also included the histone deacetylase inhibitor panobinostat (Pano). RESULTS: The combination treatment enabled significant suppression of Her2+ pancreatic cancers leading to the eradication of the majority of the tumors. Besides inducing cancer cell apoptosis, Pano enhanced CAR T-cell gene accessibility and promoted CAR T-cell differentiation into central memory cells. To test the translational potential of this approach, we established a method to transduce human T cells with an anti-Her2 CAR and a gp100-TCR. The exposure of the human T cells to Pano promoted a T-cell central memory phenotype and the combination treatment of human CARaMEL cells and Pano eradicated human pancreatic cancer xenografts in mice. CONCLUSIONS: We propose that patients with pancreatic cancer could be treated using a scheme that contains dual-specific CAR T cells, a vaccine that activates the dual-specific CAR T cells through their TCR, and the administration of Pano.

Primary and metastatic breast tumors cross-talk to influence immunotherapy responses.

The presence of a tumor can alter host immunity systematically. The immune-tumor interaction in one site may impact the local immune microenvironment in distal tissues through the circulation, and therefore influence the efficacy of immunotherapies to distant metastases. Improved understanding of the immune-tumor interactions during immunotherapy treatment in a metastatic setting may enhance the efficacy of current immunotherapies. Here we investigate the response to αPD-1/αCTLA4 and trimAb (αDR5, α4-1BB, αCD40) of 67NR murine breast tumors grown simultaneously in the mammary fat pad (MFP) and lung, a common site of breast cancer metastasis, and compared to tumors grown in isolation. Lung tumors present in isolation were resistant to both therapies. However, in MFP and lung tumor-bearing mice, the presence of a MFP tumor could increase lung tumor response to immunotherapy and decrease the number of lung metastases, leading to complete eradication of lung tumors in a proportion of mice. The MFP tumor influence on lung metastases was mediated by CD8+ T cells, as CD8+ T cell depletion abolished the difference in lung metastases. Furthermore, mice with concomitant MFP and lung tumors had increased tumor specific, effector CD8+ T cells infiltration in the lungs. Thus, we propose a model where tumors in an immunogenic location can give rise to systemic anti-tumor CD8+ T cell responses that could be utilized to target metastatic tumors. These results highlight the requirement for clinical consideration of cross-talk between primary and metastatic tumors for effective immunotherapy for cancers otherwise resistant to immunotherapy.

High-dimensional analyses reveal a distinct role of T-cell subsets in the immune microenvironment of gastric cancer.

OBJECTIVES: To facilitate disease prognosis and improve precise immunotherapy of gastric cancer (GC) patients, a comprehensive study integrating immune cellular and molecular analyses on tumor tissues and peripheral blood was performed. METHODS: The association of GC patients' outcomes and the immune context of their tumors was explored using multiplex immunohistochemistry (mIHC) and transcriptome profiling. Potential immune dysfunction mechanism/s in the tumors on the systemic level was further examined using mass cytometry (CyTOF) in complementary peripheral blood from selected patients. GC cohorts with mIHC and gene expression profiling data were also used as validation cohorts. RESULTS: Increased CD4+FOXP3+ T-cell density in the GC tumor correlated with prolonged survival. Interestingly, CD4+FOXP3+ T cells had a close interaction with CD8+ T cells rather than tumor cells. High densities of CD4+FOXP3+ T cells and CD8+ T cells (High-High) independently predicted prolonged patient survival. Furthermore, the interferon-gamma (IFN-γ) gene signature and PDL1 expression were up-regulated in this group. Importantly, a subgroup of genomically stable (GS) tumors and tumors with chromosomal instability (CIN) within this High-High group also had excellent survival. The High-High GS/CIN tumors were coupled with increased frequencies of Tbet+CD4+ T cells and central memory CD4+ T cells in the peripheral blood. CONCLUSION: These novel findings identify the combination of CD8+ T cells and FOXP3+CD4+ T cells as a significant prognostic marker for GC patients, which also could potentially be targeted and applied in the combination therapy with immune checkpoint blockades in precision medicine.

Chimeric antigen receptor-engineered T cells as oncolytic virus carriers.

The use of engineered T cells in adoptive transfer therapies has shown significant promise in treating hematological cancers. However, successes treating solid tumors are much less prevalent. Oncolytic viruses (OVs) have the capacity to induce specific lysis of tumor cells and indirectly impact tumor growth via vascular shutdown. These viruses bear natural abilities to associate with lymphocytes upon systemic administration, but therapeutic doses must be very high in order to evade antibodies and other components of the immune system. As T cells readily circulate through the body, using these cells to deliver OVs directly to tumors may provide an ideal combination. Our studies demonstrate that loading chimeric antigen receptor-engineered T cells with low doses of virus does not impact receptor expression or function in either murine or human T cells. Engineered T cells can deposit virus onto a variety of tumor targets, which can enhance the tumoricidal activity of the combination treatment. This concept appears to be broadly applicable, as we observed similar results using murine or human T cells, loaded with either RNA or DNA viruses. Overall, loading of engineered T cells with OVs represents a novel combination therapy that may increase the efficacy of both treatments.

Designed ankyrin repeat proteins are effective targeting elements for chimeric antigen receptors.

BACKGROUND: Adoptive cell transfer of tumor-specific T lymphocytes (T cells) is proving to be an effective strategy for treating established tumors in cancer patients. One method of generating these cells is accomplished through engineering bulk T cell populations to express chimeric antigen receptors (CARs), which are specific for tumor antigens. Traditionally, these CARs are targeted against tumor antigens using single-chain antibodies (scFv). Here we describe the use of a designed ankyrin repeat protein (DARPin) as the tumor-antigen targeting domain. METHODS: We prepared second generation anti-HER2 CARs that were targeted to the tumor antigen by either a DARPin or scFv. The CARs were engineered into human and murine T cells. We then compared the ability of CARs to trigger cytokine production, degranulation and cytotoxicity. RESULTS: The DARPin CARs displayed reduced surface expression relative to scFv CARs in murine cells but both CARs were expressed equally well on human T cells, suggesting that there may be a processing issue with the murine variants. In both the murine and human systems, the DARPin CARs were found to be highly functional, triggering cytokine and cytotoxic responses that were similar to those triggered by the scFv CARs. CONCLUSIONS: These findings demonstrate the utility of DARPins as CAR-targeting agents and open up an avenue for the generation of CARs with novel antigen binding attributes.