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Refractory metastatic rhabdomyosarcoma is largely incurable. Here we analyze the response of a child with refractory bone marrow metastatic rhabdomyosarcoma to autologous HER2 CAR T cells. Three cycles of HER2 CAR T cells given after lymphodepleting chemotherapy induces remission which is consolidated with four more CAR T-cell infusions without lymphodepletion. Longitudinal immune-monitoring reveals remodeling of the T-cell receptor repertoire with immunodominant clones and serum autoantibodies reactive to oncogenic signaling pathway proteins. The disease relapses in the bone marrow at six months off-therapy. A second remission is achieved after one cycle of lymphodepletion and HER2 CAR T cells. Response consolidation with additional CAR T-cell infusions includes pembrolizumab to improve their efficacy. The patient described here is a participant in an ongoing phase I trial (NCT00902044; active, not recruiting), and is 20 months off T-cell infusions with no detectable disease at the time of this report., Recurrent metastatic rhabdomyosarcoma remains largely incurable. Here, the authors describe a child with metastatic rhabdomyosarcoma who has durable response to HER2-specific CAR T cells and shows endogenous immune reactivity. more...

In humans, the NK cell marker CD161 identifies several subsets of T cells, including a polyclonal CD8 αβ T cell receptor expressing subset with characteristic specificity for tissue-localized viruses. This subset has also been reported to display enhanced cytotoxic and memory phenotypes. Here, we characterized the biology and functional properties of this unique T cell subset and determined its potential suitability for use in chimeric antigen receptor (CAR) T cell therapy. In mice, gene expression profiling among the CD161 equivalent CD8(+) T cell populations (CD8(+)NK1.1(+)) revealed substantial upregulation of granzymes, perforin, killer lectin-like receptors and innate signaling molecules in comparison to CD8(+)NK1.1(−) T cells. Adoptive transfer of CD8(+)NK1.1(+) cells from previously exposed animals offered substantially enhanced protection and improved survival against melanoma tumors and influenza infection compared to CD8(+)NK1.1(−) cells. Freshly isolated human CD8(+)CD61(+) T cells exhibited heightened allogeneic killing activity in comparison to CD8(+)CD61(−) T cells or total peripheral blood mononuclear cells (PBMC). To determine if this subset might be utilized to improve the antitumor efficacy CAR T cell therapy in the solid tumor setting, we compared bulk PBMC, CD8(+)CD161(−), and CD8(+)CD161(+) T cells transduced with a HER2-specific CAR construct. In vitro, CD8(+)CD161(+) CAR-transduced T cells killed HER2(+) targets faster and with greater efficiency. Similarly, these cells mediated substantially enhanced in vivo anti-tumor efficacy and survival in xenograft models of HER2(+) pancreatic ductal adenocarcinoma, exhibiting elevated expression of granzymes and reduced expression of exhaustion markers. These data suggest that this unique T cell subset presents an opportunity to improve CAR T cell therapy for the treatment of solid tumors. more...

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. more...

Background & Aim Early clinical trials have demonstrated the safety of chimeric antigen receptor (CAR) T cells targeting glioblastoma (GBM), however, their efficacy remains limited by multiple obstacles including the highly immunosuppressive tumor microenvironment. These adoptively transferred CAR T cells are susceptible to inhibition via the engagement of inhibitory receptors on their surface including PD1, CTLA4, LAG3 and BTLA. The recruitment of Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2) to the immune synapse upon engagement of these co-inhibitory receptors may represent a common mechanism of T cell inhibition. Upon its recruitment and activation, SHP2 dephosphorylates key stimulatory signaling molecules, thus limiting T cell activation. We hypothesized that disrupting SHP2 expression will simultaneously offset multiple co-inhibitory pathways, thereby improving the anti-tumor activity of CAR T cells. Methods, Results & Conclusion CRISPR/Cas9 technology was used to knockout (KO) SHP2 from human T cells. Retroviral vector transduction was used to express a clinically-utilized second generation CAR (with a CD28 signaling endodomain) targeting HER2. The phenotype of wild-type and edited CAR T cells was investigated using mass cytometry and confirmed with flow cytometry. Their function was tested in vitro using xcelligence (an electrical impedance-based long-term cytotoxicity assay) and in vivo in an orthotopic xenograft mouse model of GBM. Efficient SHP2 KO in human T cells was verified using western blotting. The Inference of CRISPR Efficiency (ICE) Assay confirmed efficient editing of the PTPN11 gene encoding SHP2. An anti-HER2 CAR was efficiently expressed in the SHP2KO T cells. SHP2 deletion did not significantly affect T cell expansion, proliferation or phenotypic profile. However, SHP2KO CAR T cells eliminated LN229 cells (a GBM cell line) in vitro more efficiently compared to wild-type CAR T cells. In vivo, SHP2KO CAR T cells showed better control of engrafted LN229 tumor cells and improved the survival of treated mice in comparison to wild-type CAR T cells. In conclusion, these results suggest that SHP2 may limit the function of CAR T cells and that SHP2 deletion may improve their anti-GBM activity. more...

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. more...

Early clinical trials have demonstrated the therapeutic potential of chimeric antigen receptor (CAR) T cells against glioblastoma (GBM), but also highlighted the obstacles that need to be addressed to improve their anti-tumor activity. GBM is highly immunosuppressive and thus necessitates endowing adoptively transferred CAR T cells with resistance mechanisms to achieve sustained treatment responses. This is, in part, due to the effect of immune-inhibitory receptors, including PD1, CTLA4, LAG3 and BTLA, that antagonize the tumor-directed T cells. A common mechanism of T cell inhibition involves the recruitment of Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2) to the immune synapse upon engagement of multiple co-inhibitory receptors. SHP2, in turn, de-phosphorylates key signaling proteins involved in T cell activation. We hypothesized that disrupting SHP2 expression will offset multiple co-inhibitory pathways, thereby improving the anti-tumor activity of CAR T cells. Using CRISPR/Cas9 gene editing, we disrupted the SHP2-encoding gene locus, PTPN11, in primary human T cells by generating and testing a library of single-guide RNA (sgRNA). We selected the top candidate that reproducibly reduced the SHP2 protein expression to Citation Format: Khaled Sanber, Zeid Nawas, Vita Salsman, Ahmed Gad, Pretty Rose Matthew, Daniel Landi, Ciaran Lee, Amel Sengal, Rikhia Chakraborty, Sujith Joseph, Ahmed Nabil, Meenakshi Hegde. CRISPR-Cas9 gene editing of inhibitory receptor signaling pathways improves CAR T cell activity against glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2173. more...

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. more...

3031 Background: Early clinical trials have demonstrated the safety of chimeric antigen receptor (CAR) T cells targeting glioblastoma (GBM), however, their efficacy remains limited by multiple obstacles including the immunosuppressive tumor microenvironment. Adoptively transferred CAR T cells remain susceptible to inhibition via the engagement of co-inhibitory receptors on their surface such as PD1, BTLA, CTLA4 and LAG3. The subsequent recruitment of Src homology region 2 containing protein tyrosine phosphatase 2 (SHP2) by these receptors to the immune synapse may represent a common mechanism of T cell inhibition, as SHP2 can de-phosphorylate key signaling molecules that mediate T cell activation (including CD28 and CD3ζ). We hypothesized that SHP2 deletion will simultaneously offset the effects of multiple co-inhibitory receptors, thereby improving the anti-tumor activity of CAR T cells. Methods: Electroporation of sgRNA/Cas9 ribonucleoprotein complexes into human T cells was used to knockout (KO) SHP2. Retroviral vector transduction was used to express a clinically-utilized second generation CAR (with a CD28 endodomain) targeting HER2. The phenotype of wild-type (WT) and SHP2KO CAR T cells was evaluated with mass cytometry and flow cytometry. Their anti-tumor function was tested in vitro using the xCELLigence assay (an impedance-based cytotoxicity assay), and in vivo, in an orthotopic xenograft mouse model of GBM. Results: Efficient and reproducible depletion of the SHP2 protein in human T cells was verified using western blotting. The Inference of CRISPR Efficiency (ICE) Assay confirmed efficient editing of the PTPN11 gene encoding SHP2. An anti-HER2 CAR was efficiently expressed in the SHP2KO T cells. SHP2 deletion did not significantly affect CAR T cell expansion, proliferation or baseline phenotype. However, following co-culture with HER2+ LN229-GBM cells, the CD8+ central memory (CCR7+ CD45RA-) and effector memory (CCR7- CD45RA-) subsets were enriched to a greater extent in the SHP2KO CAR T cells. The pattern of cytokine co-expression varied between donors in a single-cell analysis comparing SHP2KO to WT CAR T cells after encountering LN229 cells. Functionally, SHP2KO CAR T cells derived from the majority of healthy donor and patient peripheral blood eliminated LN229 cells more rapidly in vitro. In an orthotopic mouse model of GBM, SHP2KO CAR T cells showed better early control of established LN229 xenografts and improved survival in comparison to WT CAR T cells. Conclusions: SHP2 deletion in CD28ζ.CAR T cells improves their anti-tumor activity. more...

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. more...

Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) can be modified to function as heterologous tumor directed effector cells that survive longer in vivo than tumor directed T cells without virus specificity, due to chronic stimulation by viral antigens expressed during persistent infection in seropositive individuals. We evaluated the nonviral piggyBac (PB) transposon system as a platform for modifying EBV-CTLs to express a functional human epidermal growth factor receptor 2-specific chimeric antigen receptor (HER2-CAR) thereby directing virus-specific, gene modified CTLs towards HER2-positive cancer cells. Peripheral blood mononuclear cells (PBMCs) were nucleofected with transposons encoding a HER2-CAR and a truncated CD19 molecule for selection followed by specific activation and expansion of EBV-CTLs. HER2-CAR was expressed in ~40% of T cells after CD19 selection with retention of immunophenotype, polyclonality, and function. HER2-CAR-modified EBV-CTLs (HER2-CTLs) killed HER2-positive brain tumor cell lines in vitro, exhibited transient and reversible increases in HER2-CAR expression following antigen-specific stimulation, and stably expressed HER2-CAR beyond 120 days. Adoptive transfer of PB-modified HER2-CTLs resulted in tumor regression in a murine xenograft model. Our results demonstrate that PB can be used to redirect virus-specific CTLs to tumor targets, which should prolong tumor-specific T cell survival in vivo producing more efficacious immunotherapy. more...

Purpose: Glioblastoma multiforme (GBM) is the most aggressive human primary brain tumor and is currently incurable. Immunotherapies have the potential to target GBM stem cells, which are resistant to conventional therapies. Human epidermal growth factor receptor 2 (HER2) is a validated immunotherapy target, and we determined if HER2-specific T cells can be generated from GBM patients that will target autologous HER2-positive GBMs and their CD133-positive stem cell compartment. Experimental Design: HER2-specific T cells from 10 consecutive GBM patients were generated by transduction with a retroviral vector encoding a HER2-specific chimeric antigen receptor. The effector function of HER2-specific T cells against autologous GBM cells, including CD133-positive stem cells, was evaluated in vitro and in an orthotopic murine xenograft model. Results: Stimulation of HER2-specific T cells with HER2-positive autologous GBM cells resulted in T-cell proliferation and secretion of IFN-γ and interleukin-2 in a HER2-dependent manner. Patients' HER2-specific T cells killed CD133-positive and CD133-negative cells derived from primary HER2-positive GBMs, whereas HER2-negative tumor cells were not killed. Injection of HER2-specific T cells induced sustained regression of autologous GBM xenografts established in the brain of severe combined immunodeficient mice. Conclusions: Gene transfer allows the reliable generation of HER2-specific T cells from GBM patients, which have potent antitumor activity against autologous HER2-positive tumors including their putative stem cells. Hence, the adoptive transfer of HER2-redirected T cells may be a promising immunotherapeutic approach for GBM. Clin Cancer Res; 16(2); 474–85 more...

Human epidermal growth factor receptor 2 (HER2) is expressed by the majority of human osteosarcomas and is a risk factor for poor outcome. Unlike breast cancer, osteosarcoma cells express HER2 at too low, a level for patients to benefit from HER2 monoclonal antibodies. We reasoned that this limitation might be overcome by genetically modifying T cells with HER2-specific chimeric antigen receptors (CARs), because even a low frequency of receptor engagement could be sufficient to induce effector cell killing of the tumor. HER2-specific T cells were generated by retroviral transduction with a HER2-specific CAR containing a CD28.zeta signaling domain. HER2-specific T cells recognized HER2-positive osteosarcoma cells as judged by their ability to proliferate, produce immunostimulatory T helper 1 cytokines, and kill HER2-positive osteosarcoma cell lines in vitro. The adoptive transfer of HER2-specific T cells caused regression of established osteosarcoma xenografts in locoregional as well as metastatic mouse models. In contrast, delivery of nontransduced (NT) T cells did not change the tumor growth pattern. Genetic modification of T cells with CARs specific for target antigens, expressed at too low a level to be effectively recognized by monoclonal antibodies, may allow immunotherapy to be more broadly applicable for human cancer therapy. more...

Cancer cells can live and grow if they succeed in creating a favorable niche that often includes elements from the immune system. While T lymphocytes play an important role in the host response to tumor growth, the mechanism of their trafficking to the tumor remains poorly understood. We show here that T lymphocytes consistently infiltrate the primary brain cancer, medulloblastoma. We demonstrate, both in vitro and in vivo, that these T lymphocytes are attracted to tumor deposits only after the tumor cells have interacted with tumor vascular endothelium. Macrophage Migration Inhibitory Factor (MIF)” is the key chemokine molecule secreted by tumor cells which induces the tumor vascular endothelial cells to secrete the potent T lymphocyte attractant “Regulated upon Activation, Normal T-cell Expressed, and Secreted (RANTES).” This in turn creates a chemotactic gradient for RANTES-receptor bearing T lymphocytes. Manipulation of this pathway could have important therapeutic implications. more...

Gliomas are invasive cancers that resist all forms of attempted therapy. Immunotherapy using Ag-pulsed dendritic cells has improved survival in some patients. We present evidence that another level of complexity may also contribute to lack of responses by the lymphocytes toward gliomas. Atomic force microscopy of four different glioma types—human U251 and rat T9 and F98 glioma cells, including freshly isolated human glioblastoma multiforme neurosphere cultures (containing “stem cell-like cells”)—revealed a complex surface topography with numerous microvilli and filopodia. These structures were not found on other cell types. Electron microscopy and immunofluorescence microscopy of glioma cells confirmed that microvilli are present. U251 cells with microvilli resisted the cytolytic actions of different human effector cells, (lymphokine-activated killer cells, γδ T cells, conventional CTLs, and chimeric Ag-receptor–redirected T cells) better than their nonmicrovilli-expressing counterparts. Killer lymphocytes released perforin, which was detected within the glioma’s microvilli/filopodia, indicating these structures can receive the cytolytic effector molecules, but cytotoxicity is suboptimal. Air-dried gliomas revealed nodes within the microvilli/filopodia. The microvilli that penetrated 0.4-μm transwell chamber’s pores resisted the actions of CTLs and physical damage. Those nodelike structures may represent a compartmentalization that resists physical damage. These microvilli may play multiple roles in glioma biology, such as invasion and resistance to lymphocyte-mediated killing. more...

BACKGROUND: We demonstrated that GBM patients’ T cells expressing HER2-specific chimeric antigen receptors (CAR) killed autologous HER2 + GBM cells and induced regression of autologous xenografts; however, tumors recurred in a number of treated animals, likely due to limited T-cell persistence and/or tumor antigen loss variants. Since the majority of GBMs express CMVpp65, we expressed HER2.CARs in CMV-specific cytotoxic T cells (CTLs) to enhance their persistence following native T-cell receptor (alphabetaTCR) engagement with CMV-antigens and improve their antitumor activity by simultaneously targeting CMV and HER2. METHODS: To study the feasibility of generating CMV CTLs from GBM patients, we determined the precursor frequency of CMV-specific T cells in 25 patients using IFN-gamma-ELIspot assays. To generate CMV CTL, blood mononuclear cells were transduced with an adenovirus vector, encoding the immunodominant CMV-pp65 antigen (Ad5f35pp65) and then stimulated with autologous EBV-transformed Ad5f35pp65-transduced irradiated lymphoblastic cell lines (EBV-LCL). CTLs were then transduced with a retroviral vector encoding a HER2.CAR. We detected the expression of the CAR using flow cytometry. To concomitantly identify the CMV-specific T cell population, we used pentamers for CMVpp65 and performed Cr-release assays to test their functionality. RESULTS: CMV-specific T cells were readily detectable in the blood of GBM patients, as judged by IFN-gamma secretion after stimulation with CMVpp65-PepMixes. We were able to generate CTLs that are specific for CMV through their endogenous alphabetaTCR and specific for HER2 through CAR. Approximately 50% of CMVpp65.CTLs expressed HER2.CARs on the cell surface. CMV.HER2.CAR CTLs recognized and killed the HER2+GBM cells as well as autologous T cells pulsed with CMVpp65-PepMixes in Cr-release assays. Neither the HER2 cell lines nor autologous T cells were killed. CONCLUSION: Our data demonstrate the feasibility of generating CAR-expressing CMVpp65– specific CTLs that are functional against HER2 and CMV expressed in GBM. more...