Significance Patients with compromised T-cell function are at risk for opportunistic fungal infections. We have developed a novel approach to restore immunity by using a fungal pattern-recognition receptor Dectin-1 to redirect T-cell specificity to carbohydrate antigen in the fungal cell wall. We did so by genetically modifying T cells using the nonviral Sleeping Beauty gene-transfer system to enforce expression of a chimeric antigen receptor (CAR) that recapitulates the specificity of Dectin-1 (D-CAR). The D-CAR + T cells can be electroporated and propagated on artificial activating and propagating cells in a manner suitable for human application, enabling this immunology to be translated into immunotherapy. This approach has implications for genetically modifying T cells to express CARs with specificity for carbohydrate and thus broadening their application in the investigational treatment of pathogens and malignancies.

T cells expressing CD19-targeting chimeric antigen receptors (CARs) reveal high efficacy in the treatment of B cell malignancies. Here, we report that T cell receptor fusion constructs (TRuCs) comprising an antibody-based binding domain fused to T cell receptor (TCR) subunits can effectively reprogram an intact TCR complex to recognize tumor surface antigens. Unlike CARs, TRuCs become a functional component of the TCR complex. TRuC-T cells kill tumor cells as potently as second-generation CAR-T cells, but at significant lower cytokine release and despite the absence of an extra co-stimulatory domain. TRuC-T cells demonstrate potent anti-tumor activity in both liquid and solid tumor xenograft models. In several models, TRuC-T cells are more efficacious than respective CAR-T cells. TRuC-T cells are shown to engage the signaling capacity of the entire TCR complex in an HLA-independent manner.

Purpose The human endogenous retrovirus (HERV-K) envelope (env) protein is a tumor- associated antigen expressed on melanoma, but not normal cells. This study was designed to engineer a chimeric antigen receptor (CAR) on T cell surface, such that they target tumors in advanced stages of melanoma. Experimental Design Expression of HERV-K protein was analyzed in 220 melanoma samples (with various stages of disease) and 139 normal organ donor tissues using immuno-histochemical (IHC) analysis. HERV-K env-specific CAR derived from mouse monoclonal antibody was introduced into T cells using the transposon-based Sleeping Beauty (SB) system. HERV-K env-specific CAR+ T cells were expanded ex vivo on activating and propagating cells (AaPC), and characterized for CAR expression and specificity. This includes evaluating the HERV-K-specific CAR+ T cells for their ability to kill A375-SM metastasized tumors in a mouse xenograft model. Results We detected HERV-K env protein on melanoma, but not in normal tissues. After electroporation of T cells and selection on HERV-K+ AaPC, over 95% of genetically-modified T cells expressed the CAR with an effector memory phenotype and lysed HERV-K env+ tumor targets in an antigen specific manner. Even though there is apparent shedding of this TAA from tumor cells which can be recognized by HERV-K env-specific CAR+ T cells, we observed a significant anti-tumor effect. Conclusion Adoptive cellular immunotherapy with HERV-K env-specific CAR+ T cells represents a clinically-appealing treatment strategy for advanced-stage melanoma and provides an approach for targeting this TAA on other solid tumors.

We have previously reported that human endogenous retrovirus-K (HERV-K) envelope () protein is a tumor-associated antigen (TAA) for cancer vaccines, and that its antibodies (mAbs) possess antitumor activity against cancer. In this study, a chimeric antigen receptor (CAR) specific for HERV-K env protein (K-CAR) was generated using anti-HERV-K mAb. K-CAR T cells from peripheral blood mononuclear cells (PBMCs) of 9 breast cancer (BC) patients and 12 normal donors were able to inhibit growth of, and to exhibit significant cytotoxicity toward, BC cells but not MCF-10A normal breast cells. The antitumor effects in cancer cells were significantly reduced when control T cells were used, or the expression of HERV-K was knocked down by an shRNA. Secretion of multiple cytokines, including IFNγ, TNF-α, and IL-2, was significantly enhanced in culture media of BC cells treated with K-CARs. Significantly reduced tumor growth and tumor weight was observed in xenograft models bearing MDA-MB-231 or MDA-MB-435.eB1 BC cells. Importantly, the K-CAR prevented tumor metastasis to other organs. Furthermore, downregulation of HERV-K expression in tumors of mice treated with K-CAR correlated with upregulation of p53 and downregulation of MDM2 and p-ERK. Importantly, the expression of HERV-K env protein in metastatic tumor tissues treated with K-CAR T cells correlated with the expression of Ras. Our results indicate that HERV-K env protein is an oncoprotein and may play an important role in tumorigenesis related to p53 and Ras signaling pathways. Anti-HERV-K treatment, including K-CAR treatment, shows potential for immunotherapy of BC.

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CTL was characterized as to eps specificity and restricting HLA allele. The pool of CTL donors inherited HLA alleles in frequencies similar to those in caucasian and black populations, with certain alleles overrepresented. In 55% of the CTL lines, the ID T-cell response (TCr) was restricted (restr.) by 3 HLA alleles: A0201(25%), B0702(21%) and B 3501-11(9%); 45% of CTL lines were restr. by other class-I alleles, and 17/123(14%) were restr. by class-II alleles. Certain CMVpp65 15 mers contained overlapping eps presented by both class-I and class-II, which elicited more robust TCr. CTLs from all B0702 + donors (26/26) were restr. by B0702. In comparison, CTLs from 30/39(77%) A0201 + donors and 9/19 (47%) HLA B3501-11 + donors were restr. by A0201 and B3501-11 respectively. Donors coinheriting A0201 and B 0702 (9/9) universally demonstrated B0702 restr. ID TCr. In comparison, 11/12 (91.6%) donors coinheriting A0201 and B 4401-04 demonstrated HLA A0201 restr. CTLs. Strikingly, only 1/123 and 0/123 A1101 + and A0301 + donors respectively demonstrated CTLs restr. by these commonly inherited class eI alleles. This analysis thus indicates an immunodominance hierarchy for CMVpp65 eps and their presenting HLA alleles. In a series of 239 MUD, 137 MMUD, and 100 DUCB consecutive HSCT at MSKCC, we could identify an immediately available CMVpp65 specific CTL line matched with the patient at 2-3 alleles and restr. by a shared HLA allele in this GMP-CTL bank in 86%, 89% and 80% cases respectively. This CTL bank thus provides a clinical reagent for the treatment of CMV infections in HSCT recipients. The delineated immunodominance hierarchy for CMVpp65 may also facilitate selection of an appropriately restr. CTL line for treatment with predicted activity in the recipient. We can also successfully generate CMV-CTLs against subdominant CMVpp65 eps presented prevalent HLA alleles using a panel of artificial antigen presenting cells (AAPCs). Expansion of this bank with CTLs generated with AAPCs can broaden its applicability to almost all HSCT recipients.

Murine neurocysticercosis is a parasitic infection transmitted through the direct ingestion of Taenia solium eggs, which differentially disrupts the barriers that protect the microenvironment of the central nervous system. Among the host factors that are involved in this response, matrix metalloproteinases (MMPs) have been recently described as important players. Doxycycline is a commonly prescribed antimicrobial drug that acts as an anti-inflammatory agent with broad inhibitory properties against MMPs. In this study, we examined the effects of doxycycline treatment in a murine model of neurocysticercosis. Animals treated with doxycycline exhibited reduced morbidity and mortality throughout the course of infection. Although similar levels of leukocyte infiltration were observed with both treatment regimens, doxycycline appeared to provide improved conditions for host survival, as reduced levels of apoptosis were detected among infiltrates as well as in neurons.

BackgroundMesothelin (MSLN) is a glycosylphosphatidylinositol (GPI)-anchored membrane protein with high expression levels in an array of malignancies including mesothelioma, ovaria, non-small cell lung cancer, and pancreatic cancers and is an attractive target antigen for immune-based therapies. Early clinical evaluation of autologous MSLN-targeted chimeric antigen receptor (CAR)-T cell therapies for malignant pleural mesothelioma has shown promising acceptable safety1 and have recently evolved with incorporation of next-generation CAR co-stimulatory domains and armoring with intrinsic checkpoint inhibition via expression of a PD-1 dominant negative receptor (PD1DNR).2 Despite the promise that MSLN CAR-T therapies hold, manufacturing and commercial challenges using an autologous approach may prove difficult for widespread application. EBV T cells represent a unique, non-gene edited approach toward an off-the-shelf, allogeneic T cell platform. EBV-specific T cells are currently being evaluated in phase 3 trials [NCT03394365] and, to-date, have demonstrated a favorable safety profile including limited risks for GvHD and cytokine release syndrome.3 4 Clinical proof-of-principle studies for CAR transduced allogeneic EBV T cell therapies have also been associated with acceptable safety and durable response in association with CD19 targeting.5 Here we describe the first preclinical evaluation of ATA3271, a next-generation allogeneic CAR EBV T cell therapy targeting MSLN and incorporating PD1DNR, designed for the treatment of solid tumor indications.MethodsWe generated allogeneic MSLN CAR+ EBV T cells (ATA3271) using retroviral transduction of EBV T cells. ATA3271 includes a novel 1XX CAR signaling domain, previously associated with improved signaling and decreased CAR-mediated exhaustion. It is also armored with PD1DNR to provide intrinsic checkpoint blockade and is designed to retain functional persistence.ResultsIn this study, we characterized ATA3271 both in vitro and in vivo. ATA3271 show stable and proportional CAR and PD1DNR expression. Functional studies show potent antitumor activity of ATA3271 against MSLN-expressing cell lines, including PD-L1-high expressors. In an orthotopic mouse model of pleural mesothelioma, ATA3271 demonstrates potent antitumor activity and significant survival benefit (100% survival exceeding 50 days vs. 25 day median for control), without evident toxicities. ATA3271 maintains persistence and retains central memory phenotype in vivo through end-of-study. Additionally, ATA3271 retains endogenous EBV TCR function and reduced allotoxicity in the context of HLA mismatched targets. ConclusionsOverall, ATA3271 shows potent anti-tumor activity without evidence of allotoxicity, both in vitro and in vivo, suggesting that allogeneic MSLN-CAR-engineered EBV T cells are a promising approach for the treatment of MSLN-positive cancers and warrant further clinical investigation.ReferencesAdusumilli PS, Zauderer MG, Rusch VW, et al. Abstract CT036: A phase I clinical trial of malignant pleural disease treated with regionally delivered autologous mesothelin-targeted CAR T cells: Safety and efficacy. Cancer Research 2019;79:CT036-CT036.Kiesgen S, Linot C, Quach HT, et al. Abstract LB-378: Regional delivery of clinical-grade mesothelin-targeted CAR T cells with cell-intrinsic PD-1 checkpoint blockade: Translation to a phase I trial. Cancer Research 2020;80:LB-378-LB-378.Prockop S, Doubrovina E, Suser S, et al. Off-the-shelf EBV-specific T cell immunotherapy for rituximab-refractory EBV-associated lymphoma following transplantation. J Clin Invest 2020;130:733–747.Prockop S, Hiremath M, Ye W, et al. A Multicenter, Open Label, Phase 3 Study of Tabelecleucel for Solid Organ Transplant Subjects with Epstein-Barr Virus-Driven Post-Transplant Lymphoproliferative Disease (EBV+PTLD) after Failure of Rituximab or Rituximab and Chemotherapy. Blood 2019; 134: 5326–5326.Curran KJ, Sauter CS, Kernan NA, et al. Durable remission following ‘Off-the-Shelf’ chimeric antigen receptor (CAR) T-Cells in patients with relapse/refractory (R/R) B-Cell malignancies. Biology of Blood and Marrow Transplantation 2020;26:S89.