Identification of a clinically efficacious CAR T cell subset in diffuse large B cell lymphoma by dynamic multidimensional single-cell profiling.

Chimeric antigen receptor (CAR) T cells used for the treatment of B cell malignancies can identify T cell subsets with superior clinical activity. Here, using infusion products of individuals with large B cell lymphoma, we integrated functional profiling using timelapse imaging microscopy in nanowell grids with subcellular profiling and single-cell RNA sequencing to identify a signature of multifunctional CD8+ T cells (CD8-fit T cells). CD8-fit T cells are capable of migration and serial killing and harbor balanced mitochondrial and lysosomal volumes. Using independent datasets, we validate that CD8-fit T cells (1) are present premanufacture and are associated with clinical responses in individuals treated with axicabtagene ciloleucel, (2) longitudinally persist in individuals after treatment with CAR T cells and (3) are tumor migrating cytolytic cells capable of intratumoral expansion in solid tumors. Our results demonstrate the power of multimodal integration of single-cell functional assessments for the discovery and application of CD8-fit T cells as a T cell subset with optimal fitness in cell therapy.

Fulvestrant increases the susceptibility of enzalutamide-resistant prostate cancer cells to NK-mediated lysis.

BACKGROUND: Enzalutamide, a next-generation antiandrogen agent, is approved for the treatment of metastatic castration-resistant prostate cancer (CRPC). While enzalutamide has been shown to improve time to progression and extend overall survival in men with CRPC, the majority of patients ultimately develop resistance to treatment. Immunotherapy approaches have shown limited clinical benefit in this patient population; understanding resistance mechanisms could help develop novel and more effective treatments for CRPC. One of the mechanisms involved in tumor resistance to various therapeutics is tumor phenotypic plasticity, whereby carcinoma cells acquire mesenchymal features with or without the loss of classical epithelial characteristics. This work investigated a potential link between enzalutamide resistance, tumor phenotypic plasticity, and resistance to immune-mediated lysis in prostate cancer. METHODS: Models of prostate cancer resistant to enzalutamide were established by long-term exposure of human prostate cancer cell lines to the drug in culture. Tumor cells were evaluated for phenotypic features in vitro and in vivo, as well as for sensitivity to immune effector cell-mediated cytotoxicity. RESULTS: Resistance to enzalutamide was associated with gain of mesenchymal tumor features, upregulation of estrogen receptor expression, and significantly reduced tumor susceptibility to natural killer (NK)-mediated lysis, an effect that was associated with decreased tumor/NK cell conjugate formation with enzalutamide-resistant cells. Fulvestrant, a selective estrogen receptor degrader, restored the formation of target/NK cell conjugates and increased susceptibility to NK cell lysis in vitro. In vivo, fulvestrant demonstrated antitumor activity against enzalutamide-resistant cells, an effect that was associated with activation of NK cells. CONCLUSION: NK cells are emerging as a promising therapeutic approach in prostate cancer. Modifying tumor plasticity via blockade of estrogen receptor with fulvestrant may offer an opportunity for immune intervention via NK cell-based approaches in enzalutamide-resistant CRPC.

An Interleukin-15 Superagonist Enables Antitumor Efficacy of Natural Killer Cells Against All Molecular Variants of SCLC.

INTRODUCTION: SCLC is a highly aggressive tumor with a 5-year survival rate of less than 6%. A heterogeneous disease, SCLC is classified into four subtypes that include tumors with neuroendocrine and non-neuroendocrine features. Immune checkpoint blockade has been recently added for the frontline treatment of SCLC; however, this therapy has only led to modest clinical improvements. The lack of clinical benefit in a cancer type known to have a high tumor mutational burden has been attributed to poor T-cell infiltration and low expression of MHC-class I in most SCLC tumors. In an attempt to devise a more effective immunotherapeutic regimen, this study investigated an alternate approach on the basis of the use of the clinical-stage interleukin-15 superagonist, N-803. METHODS: Preclinical models of SCLC spanning all molecular subtypes were used to evaluate the susceptibility of SCLC to natural killer (NK)-mediated lysis in vitro, including NK cells activated by N-803. Antitumor activity of N-803 was evaluated in vivo with a xenograft model of SCLC. RESULTS: In vitro and in vivo data revealed differences in susceptibility of SCLC subtypes to lysis by NK cells and that NK cells activated by N-803 effectively lyse SCLC tumor cells across all variant subtypes, regardless of their expression of MHC-class I. CONCLUSIONS: These findings highlight the potential of a novel immune-based intervention using a cytokine-based therapeutic option for the treatment of SCLC. We hypothesize that N-803 may provide benefit to most patients with SCLC, including those with immunologically cold tumors lacking MHC expression.

Remodeling the tumor microenvironment via blockade of LAIR-1 and TGF-ß signaling enables PD-L1-mediated tumor eradication.

Collagens in the extracellular matrix (ECM) provide a physical barrier to tumor immune infiltration, while also acting as a ligand for immune inhibitory receptors. Transforming growth factor-ß (TGF-ß) is a key contributor to shaping the ECM by stimulating the production and remodeling of collagens. TGF-ß activation signatures and collagen-rich environments have both been associated with T cell exclusion and lack of responses to immunotherapy. Here, we describe the effect of targeting collagens that signal through the inhibitory leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) in combination with blockade of TGF-ß and programmed cell death ligand 1 (PD-L1). This approach remodeled the tumor collagenous matrix, enhanced tumor infiltration and activation of CD8+ T cells, and repolarized suppressive macrophage populations, resulting in high cure rates and long-term tumor-specific protection across murine models of colon and mammary carcinoma. The results highlight the advantage of direct targeting of ECM components in combination with immune checkpoint blockade therapy.

Vaccine Increases the Diversity and Activation of Intratumoral T Cells in the Context of Combination Immunotherapy.

Resistance to immune checkpoint blockade therapy has spurred the development of novel combinations of drugs tailored to specific cancer types, including non-inflamed tumors with low T-cell infiltration. Cancer vaccines can potentially be utilized as part of these combination immunotherapies to enhance antitumor efficacy through the expansion of tumor-reactive T cells. Utilizing murine models of colon and mammary carcinoma, here we investigated the effect of adding a recombinant adenovirus-based vaccine targeting tumor-associated antigens with an IL-15 super agonist adjuvant to a multimodal regimen consisting of a bifunctional anti-PD-L1/TGF-ßRII agent along with a CXCR1/2 inhibitor. We demonstrate that the addition of vaccine induced a greater tumor infiltration with T cells highly positive for markers of proliferation and cytotoxicity. In addition to this enhancement of cytotoxic T cells, combination therapy showed a restructured tumor microenvironment with reduced Tregs and CD11b+Ly6G+ myeloid cells. Tumor-infiltrating immune cells exhibited an upregulation of gene signatures characteristic of a Th1 response and presented with a more diverse T-cell receptor (TCR) repertoire. These results provide the rationale for the addition of vaccine-to-immune checkpoint blockade-based therapies being tested in the clinic.

CAR T-cells that target acute B-lineage leukemia irrespective of CD19 expression.

Chimeric antigen receptor (CAR) T-cells targeting CD19 demonstrate remarkable efficacy in treating B-lineage acute lymphoblastic leukemia (BL-ALL), yet up to 39% of treated patients relapse with CD19(-) disease. We report that CD19(-) escape is associated with downregulation, but preservation, of targetable expression of CD20 and CD22. Accordingly, we reasoned that broadening the spectrum of CD19CAR T-cells to include both CD20 and CD22 would enable them to target CD19(-) escape BL-ALL while preserving their upfront efficacy. We created a CD19/20/22-targeting CAR T-cell by coexpressing individual CAR molecules on a single T-cell using one tricistronic transgene. CD19/20/22CAR T-cells killed CD19(-) blasts from patients who relapsed after CD19CAR T-cell therapy and CRISPR/Cas9 CD19 knockout primary BL-ALL both in vitro and in an animal model, while CD19CAR T-cells were ineffective. At the subcellular level, CD19/20/22CAR T-cells formed dense immune synapses with target cells that mediated effective cytolytic complex formation, were efficient serial killers in single-cell tracking studies, and were as efficacious as CD19CAR T-cells against primary CD19(+) disease. In conclusion, independent of CD19 expression, CD19/20/22CAR T-cells could be used as salvage or front-line CAR therapy for patients with recalcitrant disease.

Interleukin-8: A chemokine at the intersection of cancer plasticity, angiogenesis, and immune suppression.

Tumor progression relies on the ability of cancer cells to effectively invade surrounding tissues and propagate. Among the many mechanisms that contribute to tumor progression is the epithelial-to-mesenchymal transition (EMT), a phenotypic plasticity phenomenon that increases the cancer cells' motility and invasiveness and influences their surrounding microenvironment by promoting the secretion of a variety of soluble factors. One such factor is IL-8, a chemokine with multiple pro-tumorigenic roles within the tumor microenvironment (TME), including stimulating proliferation or transformation of tumor cells into a migratory or mesenchymal phenotype. Further, IL-8 can increase tumor angiogenesis or recruit larger numbers of immunosuppressive cells to the tumor. Prognostically, observations in many tumor types show that patients with higher levels of IL-8 at baseline experience worse clinical outcomes. Additionally, studies have shown that the chemokine directly contributes to the development of resistance to both chemotherapy and molecularly targeted agents. More recently, clinical studies evaluating levels of IL-8 in patients receiving immune checkpoint inhibition (ICI) therapy deduced that myeloid tumor infiltration driven by IL-8 contributes to resistance to ICI agents and that peripheral IL-8 can predict outcomes to ICI therapy. Further, pre-clinical data demonstrate that targeting IL-8 or its receptors enables improved tumor killing by immune cells, and treatment strategies combining blockade of the IL-8/IL-8R axis with ICI ultimately improve anti-tumor efficacy. Based on these results and the prognostic capacity of IL-8, there are a number of ongoing clinical trials evaluating the addition of IL-8 targeting strategies to immune-based therapies.

Locoregional delivery of CAR T cells to the cerebrospinal fluid for treatment of metastatic medulloblastoma and ependymoma.

Recurrent medulloblastoma and ependymoma are universally lethal, with no approved targeted therapies and few candidates presently under clinical evaluation. Nearly all recurrent medulloblastomas and posterior fossa group A (PFA) ependymomas are located adjacent to and bathed by the cerebrospinal fluid, presenting an opportunity for locoregional therapy, bypassing the blood-brain barrier. We identify three cell-surface targets, EPHA2, HER2 and interleukin 13 receptor α2, expressed on medulloblastomas and ependymomas, but not expressed in the normal developing brain. We validate intrathecal delivery of EPHA2, HER2 and interleukin 13 receptor α2 chimeric antigen receptor T cells as an effective treatment for primary, metastatic and recurrent group 3 medulloblastoma and PFA ependymoma xenografts in mouse models. Finally, we demonstrate that administration of these chimeric antigen receptor T cells into the cerebrospinal fluid, alone or in combination with azacytidine, is a highly effective therapy for multiple metastatic mouse models of group 3 medulloblastoma and PFA ependymoma, thereby providing a rationale for clinical trials of these approaches in humans.

Tumor Plasticity and Resistance to Immunotherapy.

Tumor cell plasticity exhibited as an epithelial-mesenchymal transition (EMT) has been identified as a major obstacle for the effective treatment of many cancers. This process, which involves the dedifferentiation of epithelial tumor cells towards a motile, metastatic, and mesenchymal tumor phenotype, mediates resistance to conventional therapies and small-molecule targeted therapies. In this review, we highlight current research correlating the role of tumor plasticity with resistance to current immunotherapy approaches and discuss future and ongoing combination immunotherapy strategies to reduce tumor cell plasticity-driven resistance in cancer.

Simultaneous inhibition of CXCR1/2, TGF-ß, and PD-L1 remodels the tumor and its microenvironment to drive antitumor immunity.

BACKGROUND: Despite the success of immune checkpoint blockade therapy in the treatment of certain cancer types, only a small percentage of patients with solid malignancies achieve a durable response. Consequently, there is a need to develop novel approaches that could overcome mechanisms of tumor resistance to checkpoint inhibition. Emerging evidence has implicated the phenomenon of cancer plasticity or acquisition of mesenchymal features by epithelial tumor cells, as an immune resistance mechanism. METHODS: Two soluble factors that mediate tumor cell plasticity in the context of epithelial-mesenchymal transition are interleukin 8 (IL-8) and transforming growth factor beta (TGF-ß). In an attempt to overcome escape mechanisms mediated by these cytokines, here we investigated the use of a small molecule inhibitor of the IL-8 receptors CXCR1/2, and a bifunctional agent that simultaneously blocks programmed death ligand 1 (PD-L1) and traps soluble TGF-ß. RESULTS: We demonstrate that simultaneous inhibition of CXCR1/2, TGF-ß, and PD-L1 signaling synergizes to reduce mesenchymal tumor features in murine models of breast and lung cancer, and to markedly increase expression of tumor epithelial E-cadherin while reducing infiltration with suppressive granulocytic myeloid-derived suppressor cells, significantly enhancing T-cell infiltration and activation in tumors, and leading to improved antitumor activity. CONCLUSIONS: This study highlights the potential benefit of combined blockade of CXCR1/2 and TGF-ß signaling for modulation of tumor plasticity and potential enhancement of tumor responses to PD-L1 blockade. The data provide rationale for the evaluation of this novel approach in the clinic.

Retraction Note: A homing system targets therapeutic T cells to brain cancer.

This Article has been retracted; see accompanying Retraction.

A homing system targets therapeutic T cells to brain cancer.

Successful T cell immunotherapy for brain cancer requires that the T cells can access tumour tissues, but this has been difficult to achieve. Here we show that, in contrast to inflammatory brain diseases such as multiple sclerosis, where endothelial cells upregulate ICAM1 and VCAM1 to guide the extravasation of pro-inflammatory cells, cancer endothelium downregulates these molecules to evade immune recognition. By contrast, we found that cancer endothelium upregulates activated leukocyte cell adhesion molecule (ALCAM), which allowed us to overcome this immune-evasion mechanism by creating an ALCAM-restricted homing system (HS). We re-engineered the natural ligand of ALCAM, CD6, in a manner that triggers initial anchorage of T cells to ALCAM and conditionally mediates a secondary wave of adhesion by sensitizing T cells to low-level ICAM1 on the cancer endothelium, thereby creating the adhesion forces necessary to capture T cells from the bloodstream. Cytotoxic HS T cells robustly infiltrated brain cancers after intravenous injection and exhibited potent antitumour activity. We have therefore developed a molecule that targets the delivery of T cells to brain cancer.

TEM8/ANTXR1-Specific CAR T Cells as a Targeted Therapy for Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive disease lacking targeted therapy. In this study, we developed a CAR T cell-based immunotherapeutic strategy to target TEM8, a marker initially defined on endothelial cells in colon tumors that was discovered recently to be upregulated in TNBC. CAR T cells were developed that upon specific recognition of TEM8 secreted immunostimulatory cytokines and killed tumor endothelial cells as well as TEM8-positive TNBC cells. Notably, the TEM8 CAR T cells targeted breast cancer stem-like cells, offsetting the formation of mammospheres relative to nontransduced T cells. Adoptive transfer of TEM8 CAR T cells induced regression of established, localized patient-derived xenograft tumors, as well as lung metastatic TNBC cell line-derived xenograft tumors, by both killing TEM8+ TNBC tumor cells and targeting the tumor endothelium to block tumor neovascularization. Our findings offer a preclinical proof of concept for immunotherapeutic targeting of TEM8 as a strategy to treat TNBC.Significance: These findings offer a preclinical proof of concept for immunotherapeutic targeting of an endothelial antigen that is overexpressed in triple-negative breast cancer and the associated tumor vasculature. Cancer Res; 78(2); 489-500. ©2017 AACR.

Trivalent CAR T cells overcome interpatient antigenic variability in glioblastoma.

Background: Glioblastoma (GBM) is the most common primary malignant brain cancer, and is currently incurable. Chimeric antigen receptor (CAR) T cells have shown promise in GBM treatment. While we have shown that combinatorial targeting of 2 glioma antigens offsets antigen escape and enhances T-cell effector functions, the interpatient variability in surface antigen expression between patients hinders the clinical impact of targeting 2 antigen pairs. This study addresses targeting 3 antigens using a single CAR T-cell product for broader application. Methods: We analyzed the surface expression of 3 targetable glioma antigens (human epidermal growth factor receptor 2 [HER2], interleukin-13 receptor subunit alpha-2 [IL13Rα2], and ephrin-A2 [EphA2]) in 15 primary GBM samples. Accordingly, we created a trivalent T-cell product armed with 3 CAR molecules specific for these validated targets encoded by a single universal (U) tricistronic transgene (UCAR T cells). Results: Our data showed that co-targeting HER2, IL13Rα2, and EphA2 could overcome interpatient variability by a tendency to capture nearly 100% of tumor cells in most tumors tested in this cohort. UCAR T cells made from GBM patients' blood uniformly expressed all 3 CAR molecules with distinct antigen specificity. UCAR T cells mediated robust immune synapses with tumor targets forming more polarized microtubule organizing centers and exhibited improved cytotoxicity and cytokine release over best monospecific and bispecific CAR T cells per patient tumor profile. Lastly, low doses of UCAR T cells controlled established autologous GBM patient derived xenografts (PDXs) and improved survival of treated animals. Conclusion: UCAR T cells can overcome antigenic heterogeneity in GBM and lead to improved treatment outcomes.

Tandem CAR T cells targeting HER2 and IL13Rα2 mitigate tumor antigen escape.

In preclinical models of glioblastoma, antigen escape variants can lead to tumor recurrence after treatment with CAR T cells that are redirected to single tumor antigens. Given the heterogeneous expression of antigens on glioblastomas, we hypothesized that a bispecific CAR molecule would mitigate antigen escape and improve the antitumor activity of T cells. Here, we created a CAR that joins a HER2-binding scFv and an IL13Rα2-binding IL-13 mutein to make a tandem CAR exodomain (TanCAR) and a CD28.ζ endodomain. We determined that patient TanCAR T cells showed distinct binding to HER2 or IL13Rα2 and had the capability to lyse autologous glioblastoma. TanCAR T cells exhibited activation dynamics that were comparable to those of single CAR T cells upon encounter of HER2 or IL13Rα2. We observed that TanCARs engaged HER2 and IL13Rα2 simultaneously by inducing HER2-IL13Rα2 heterodimers, which promoted superadditive T cell activation when both antigens were encountered concurrently. TanCAR T cell activity was more sustained but not more exhaustible than that of T cells that coexpressed a HER2 CAR and an IL13Rα2 CAR, T cells with a unispecific CAR, or a pooled product. In a murine glioblastoma model, TanCAR T cells mitigated antigen escape, displayed enhanced antitumor efficacy, and improved animal survival. Thus, TanCAR T cells show therapeutic potential to improve glioblastoma control by coengaging HER2 and IL13Rα2 in an augmented, bivalent immune synapse that enhances T cell functionality and reduces antigen escape.

The Evolution of T-cell Therapies for Solid Malignancies.

Primary resistant, recurrent, and relapsed solid tumors are often nonresponsive to conventional antineoplastic therapies. Moreover, in responsive tumors, the therapeutic-to-toxic range of these interventions remains quite narrow, such that side effects of therapy are substantial. Targeted therapies, such as adoptive T-cell transfer, not only spare normal tissues but also use alternative killing mechanisms to which the tumor cells are usually not immune. Adoptive T-cell transfer for solid tumors faces unique challenges because of the inherent heterogeneity of tumor parenchyma, the complexity of the tumor microenvironment, and tumor occurrence in areas with limited therapeutic accessibility. In this review, we examine the recent evolution of various T-cell-based immunotherapeutics, the mechanisms of action behind their antitumor activity, their increasing complexity, and the prospect of building on previous successes in the treatment of solid tumors.