Preclinical evaluation of anti-ROR1 CAR T cells employing a ROR1 binding SCFV derived from the clinical stage mab cirmtuzumab.

2020 ◽  
Vol 38 (5_suppl) ◽  
pp. 41-41
Author(s):  
Charles E. Prussak ◽  
Christopher Oh ◽  
Juliana Velez Lujan ◽  
Sharon Lam ◽  
Jieyu Zhang ◽  
...  
Keyword(s):  
T Cells ◽  
Cancer Cells ◽  
In Vivo Studies ◽  
Tumor Escape ◽  
Cancer Stemness ◽  
2Nd Generation ◽  
Car T Cells ◽  
Car T

41 Background: Chimeric antigen receptor (CAR)-modified T cells (CAR-T) were generated targeting cells expressing ROR1, which is present on many malignant cancers and has been associated with cancer stemness and chemo-resistance. The ROR1 CAR utilizes the humanized single-chain fragment variable (scFv) binding domain of UC-961 (cirmtuzumab), which exhibits high affinity and specificity for human ROR1 and has demonstrated an excellent safety profile in Phase 1 studies. Methods: CAR constructs with varying spacer regions and intracellular co-stimulatory domains, using the scFV of cirmtuzumab, were constructed and used to generate CAR-T cells from healthy donors. These ROR1 CAR-T cells were tested for cytotoxicity against lymphoid cancer cells in vitro and in vivo studies that employed immune-deficient mice engrafted with labeled human leukemia cells MEC1 or MEC1-ROR1, which had been transfected to stably express ROR1. Results: The 2nd generation and 3rd generation CAR-T-cells with analogous spacer regions were comparably potent and selectively cytotoxic for cells bearing the ROR1 target antigen. But the 2nd generation CARs demonstrated greater potency in vitro even at low effector to target ratios. For the in vivo studies, mice received a single injection of ROR1 CAR-T cells or activated T cells from the same donor as a control. The ROR1 CAR-T cells rapidly cleared the leukemic cells from the animals, whereas animals receiving control T cells or no therapy quickly succumbed to progressive disease within 3 weeks. The administered CAR-T products remained highly active following administration and could be detected for ≥ 3 months without evidence for T cell exhaustion. Conclusions: The generated CAR-T cells utilizing constructs with the Fv of cirmtuzumab, a humanized mAb highly specific for ROR1, onco-embryonic surface antigen, effectively and selectively killed neoplastic cells bearing ROR1 both in vitro and in vivo. As ROR1 expression and signaling has been associated with cancer stemness and chemo-resistance utilizing ROR1 CAR-T therapy to target cancer cells might mitigate tumor escape. These data strongly support the rationale for continued development of our ROR1 CAR-T.

scholarly journals 114 Preclinical development of a novel iPSC-derived CAR-MICA/B NK cell immunotherapy to overcome solid tumor escape from NKG2D-mediated mechanisms of recognition and killing

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A126-A126
Author(s):  
John Goulding ◽  
Mochtar Pribadi ◽  
Robert Blum ◽  
Wen-I Yeh ◽  
Yijia Pan ◽  
...  
Keyword(s):  
T Cells ◽  
Cancer Immunotherapy ◽  
Tumor Immunity ◽  
Immune Cell ◽  
Nk Cell ◽  
Tumor Escape ◽  
Car T Cells ◽  
Car T

BackgroundMHC class I related proteins A (MICA) and B (MICB) are induced by cellular stress and transformation, and their expression has been reported for many cancer types. NKG2D, an activating receptor expressed on natural killer (NK) and T cells, targets the membrane-distal domains of MICA/B, activating a potent cytotoxic response. However, advanced cancer cells frequently evade immune cell recognition by proteolytic shedding of the α1 and α2 domains of MICA/B, which can significantly reduce NKG2D function and the cytolytic activity.MethodsRecent publications have shown that therapeutic antibodies targeting the membrane-proximal α3 domain inhibited MICA/B shedding, resulting in a substantial increase in the cell surface density of MICA/B and restoration of immune cell-mediated tumor immunity.1 We have developed a novel chimeric antigen receptor (CAR) targeting the conserved α3 domain of MICA/B (CAR-MICA/B). Additionally, utilizing our proprietary induced pluripotent stem cell (iPSC) product platform, we have developed multiplexed engineered, iPSC-derived CAR-MICA/B NK (iNK) cells for off-the-shelf cancer immunotherapy.ResultsA screen of CAR spacer and ScFv orientations in primary T cells delineated MICA-specific in vitro activation and cytotoxicity as well as in vivo tumor control against MICA+ cancer cells. The novel CAR-MICA/B design was used to compare efficacy against NKG2D CAR T cells, an alternative MICA/B targeting strategy. CAR-MICA/B T cells showed superior cytotoxicity against melanoma, breast cancer, renal cell carcinoma, and lung cancer lines in vitro compared to primary NKG2D CAR T cells (p<0.01). Additionally, using an in vivo xenograft metastasis model, CAR-MICA/B T cells eliminated A2058 human melanoma metastases in the majority of the mice treated. In contrast, NKG2D CAR T cells were unable to control tumor growth or metastases. To translate CAR-MICA/B functionality into an off-the-shelf cancer immunotherapy, CAR-MICA/B was introduced into a clonal master engineered iPSC line to derive a multiplexed engineered, CAR-MICA/B iNK cell product candidate. Using a panel of tumor cell lines expressing MICA/B, CAR-MICA/B iNK cells displayed MICA specificity, resulting in enhanced cytokine production, degranulation, and cytotoxicity. Furthermore, in vivo NK cell cytotoxicity was evaluated using the B16-F10 melanoma cell line, engineered to express MICA. In this model, CAR-MICA/B iNK cells significantly reduced liver and lung metastases, compared to untreated controls, by 93% and 87% respectively.ConclusionsOngoing work is focused on extending these preclinical studies to further support the clinical translation of an off-the-shelf, CAR-MICA/B iNK cell cancer immunotherapy with the potential to overcome solid tumor escape from NKG2D-mediated mechanisms of recognition and killing.ReferenceFerrari de Andrade L, Tay RE, Pan D, Luoma AM, Ito Y, Badrinath S, Tsoucas D, Franz B, May KF Jr, Harvey CJ, Kobold S, Pyrdol JW, Yoon C, Yuan GC, Hodi FS, Dranoff G, Wucherpfennig KW. Antibody-mediated inhibition of MICA and MICB shedding promotes NK cell-driven tumor immunity. Science 2018 Mar 30;359(6383):1537–1542.

scholarly journals P06.09 Anti-hPSMA CAR engineered NK-92 cells: An off-the-shelf cellular therapeutic for targeted elimination of prostate cancer cells

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A45.1-A45
Author(s):  
G Zuccolotto ◽  
A Penna ◽  
IM Montagner ◽  
D Carpanese ◽  
A Rosato
Keyword(s):  
T Cells ◽  
Cell Therapy ◽  
Cancer Cells ◽  
Cell Line ◽  
Cell Population ◽  
Tumor Escape ◽  
Tumor Models ◽  
Car T

BackgroundAdoptive cell therapy of malignant diseases takes advantages of the cellular immune system to recognize and destroy cancer cells. Despite the remarkable success in B cell malignancies after adoptive transfer of CD19 CAR T cells, CAR T cell therapy in solid tumors has shown less encouraging clinical results, above all caused by tumor escape mechanisms.In order to overcome such limitations, NK-92, a permanent and IL-2-dependent cell line with a high cytotoxicity in vitro, has been engineered in preclinical models with CAR. In this project, we exploited a CAR directed against the human antigen hPSMA that is overexpressed in prostate tumors. This project aimed at transducing NK-92 cell line to obtain a hPSMA-specific CAR NK-92 cell population, to be thereafter characterized in vitro and in vivo for antigen-specific functional activity.Materials and MethodsNK-92 cell line was transduced with a lentiviral vector (LV) carrying a CAR anti-hPSMA. The cell population obtained was then sorted and analyzed for degranulation capacity, IFNγ production and lytic activity against hPSMA+ (PC3-hPSMA, LNCaP) or hPSMA-tumor cell lines. In vivo therapeutic efficacy of CAR-transduced NK-92 was evaluated initially using Winn-Assay and than in subcutaneous and orthotopic tumor models.ResultsCAR-expressing LV efficiently transduced NK-92 cells, which in turn produced cytokines, degranulated and exerted a relevant cytotoxic upon challenge with PSMA+ prostate tumor cells, irrespective of 10 Gy γ-irradiation. In all the in vivo, tumor models CAR-transduced NK-92 shown a statistically significant inhibition of tumor growth.ConclusionsChimeric antigen receptor-engineered NK-92 could offer a valid and cost-effective alternative to primary CAR NK or T cells, in particular in cases, where a suitable donor is not available or the sophisticated infrastructure needed for cell isolation, expansion and genetic modification is missing. This work demonstrates that CAR-engineered NK-92 cells display a high and specific recognition of hPSMA+ PC both in vitro as is in vivo, and could represent an efficient strategy as a new therapeutic intervention against prostate carcinoma, thus paving the way to an Off-The-Shelf cellular therapeutic for targeted elimination of cancer cells and induction of protective antitumor immunity.Disclosure InformationG. Zuccolotto: None. A. Penna: None. I.M. Montagner: None. D. Carpanese: None. A. Rosato: None.

scholarly journals Engineering light-controllable CAR T cells for cancer immunotherapy

2020 ◽  
Vol 6 (8) ◽  
pp. eaay9209 ◽  
Author(s):  
Ziliang Huang ◽  
Yiqian Wu ◽  
Molly E. Allen ◽  
Yijia Pan ◽  
Phillip Kyriakakis ◽  
...  
Keyword(s):  
T Cells ◽  
Cancer Cells ◽  
Cancer Immunotherapy ◽  
Tumor Antigens ◽  
Nuclear Translocation ◽  
Car T Cells ◽  
Car T ◽  
Target Cancer

T cells engineered to express chimeric antigen receptors (CARs) can recognize and engage with target cancer cells with redirected specificity for cancer immunotherapy. However, there is a lack of ideal CARs for solid tumor antigens, which may lead to severe adverse effects. Here, we developed a light-inducible nuclear translocation and dimerization (LINTAD) system for gene regulation to control CAR T activation. We first demonstrated light-controllable gene expression and functional modulation in human embryonic kidney 293T and Jurkat T cell lines. We then improved the LINTAD system to achieve optimal efficiency in primary human T cells. The results showed that pulsed light stimulations can activate LINTAD CAR T cells with strong cytotoxicity against target cancer cells, both in vitro and in vivo. Therefore, our LINTAD system can serve as an efficient tool to noninvasively control gene activation and activate inducible CAR T cells for precision cancer immunotherapy.

CAR-T cells to deliver engineered peptide antigens and reprogram antigen specific T cell responses against solid tumors.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 2530-2530
Author(s):  
Daniel Lee ◽  
Andy J Minn ◽  
Lexus R Johnson
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Tumor Cells ◽  
In Vivo Studies ◽  
Peptide Antigens ◽  
Car T Cells ◽  
Car T

2530 Background: Neoantigen depleted malignancies such as colorectal cancer demonstrate primary resistance to immune checkpoint blockade, and solid tumors in general have shown resistance to chimeric antigen receptor (CAR) T cell therapy. However, CAR-T cells have been shown to be capable of delivering various therapeutic molecules in a targeted fashion to the tumor microenvironment, in some cases through extracellular vesicles (EVs). In vivo studies have shown that the presentation of foreign viral peptides by solid tumors can reprogram bystander virus-specific cytotoxic T cells (CTLs) against tumor cells. In this study, we demonstrate that CAR-T cells can deliver engineered peptide antigens to solid tumors, leading to presentation on tumor cells and anti-tumor response. Methods: Second generation CAR-T cells (41BB endodomain) targeting human CD19 (19BBz) or human mesothelin (M5BBz) were generated via retroviral and lentiviral transduction respectively. CAR-T cells were engineered to co-express peptides such as SIINFEKL of ovalbumin and NLVPMVATV of CMV pp65 among others. Peptides were isolated from EVs via ultracentrifugation. For in vivo studies, C57BL/6 or NSG mice were injected on the flank with relevant tumors and treated with peptide-CAR-T cells. In vitro studies utilized flow cytometry and xCELLigence killing assays. Results: Murine 19BBz CAR-T cells expressing the SIINFEKL peptide of ovalbumin (ova-19BBz) were found to transfer SIINFEKL peptide to tumor cells via EVs in vitro and in vivo, leading to peptide presentation on MHC-I of tumor cells. This resulted in significantly delayed tumor growth in tumor bearing mice transfused with OT-I T cells to mimic an existing antigen specific T cell pool. We expanded on these findings by isolating EVs from human M5BBz CAR-T cells expressing CMV viral peptides. Peptide-CAR-T EVs were co-cultured with human ovarian cancer cells to assess presentation to Jurkat T cells. Finally, we utilized primary human T cells from CMV+ healthy donors to assess the clinical feasibility of our peptide delivery approach. Conclusions: CAR-T cells can be engineered to deliver peptides to tumor cells for presentation and subsequent targeting by antigen specific CTLs. This represents a novel strategy for the treatment of non-immunogenic tumors.

scholarly journals Effective and persistent antitumor activity of HER2-directed CAR-T cells against gastric cancer cells in vitro and xenotransplanted tumors in vivo

2017 ◽  
Vol 9 (10) ◽  
pp. 867-878 ◽  
Author(s):  
Yanjing Song ◽  
Chuan Tong ◽  
Yao Wang ◽  
Yunhe Gao ◽  
Hanren Dai ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
T Cells ◽  
Antitumor Activity ◽  
Cancer Cells ◽  
Gastric Cancer Cells ◽  
Car T Cells ◽  
Car T

scholarly journals CAR T Cells Equipped With a Fully Human scFv Targeting Trop2 Can Be Used to Treat Pancreatic Cancer

2021 ◽  
Author(s):  
Hong Jia Zhu ◽  
Yujie Jia ◽  
Jingwen Tan ◽  
Xiaoyan Fang ◽  
Jing Ye ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
T Cells ◽  
Cancer Cells ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Car T Cells ◽  
Pancreatic Cancer Cells ◽  
Car T

Abstract Purpose: Chimeric antigen receptor (CAR) T cell therapy has demonstrated clinical success in treating haematologic malignancies but has not been effective against solid tumours thus far. Trop2 is a tumour-related antigen broadly overexpressed on a variety of tumours and has been reported as a promising target for pancreatic cancers. Our study aimed to determine whether CAR T cells designed with a fully human Trop2-specific single-chain fragment variable (scFv) can be used in the treatment of Trop2-positive pancreatic tumours.Methods: We designed Trop2-targeted chimeric antigen receptor engineered T cells with a novel human anti-Trop2 scFv (2F11) and then investigated the cytotoxicity, degranulation, and cytokine secretion profiles of the anti-Trop2 CAR T cells when they were exposed to Trop2+ cancer cells in vitro. We also studied the antitumour efficacy and toxicity of Trop2-specific CAR T cells in vivo using a BxPC-3 pancreatic xenograft model.Results: Trop2-targeted CAR T cells designed with 2F11 effectively killed Trop2-positive pancreatic cancer cells and produced high levels of cytotoxic cytokines in vitro. In addition, Trop2-targeted CAR T cells, which persistently circulate in vivo and efficiently infiltrate into tumour tissues, significantly blocked and even eliminated BxPC-3 pancreatic xenograft tumour growth without obvious deleterious effects observed after intravenous injection into NSG mice. Moreover, disease-free survival was efficiently prolonged.Conclusion: These results show that Trop2-targeted CAR T cells equipped with a fully human anti-Trop2 scFv could be a potential treatment strategy for pancreatic cancer and could be useful for clinical evaluation.

767 Interferon gamma reduces CAR-T exhaustion and toxicity without compromising therapeutic efficacy in hematologic malignancies

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A815-A815
Author(s):  
Stefanie Bailey ◽  
Sonika Vatsa ◽  
Amanda Bouffard ◽  
Rebecca Larson ◽  
Irene Scarfo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Cancer Cells ◽  
Tumor Cells ◽  
Interferon Gamma ◽  
Hematologic Malignancies ◽  
Car T Cells ◽  
Car T

BackgroundChimeric antigen receptor (CAR) T cell therapy has shown remarkable efficacy in hematologic malignancies, ultimately leading to its FDA approval for relapsed/refractory acute lymphoblastic leukemia and large cell lymphomas in 2017. Despite the success of CAR T cells in the clinic, toxicities such as cytokine release syndrome (CRS) can be severe. Attempts to mitigate these effects have primarily focused on the blockade of macrophage-derived cytokines, such as IL-6 and IL-1B. Herein, we show that the pharmaceutical blockade or genetic deletion of interferon gamma (IFNg, a CAR-T-derived cytokine that strongly correlates with CRS in the clinic, appears to be a viable target for the reduction of CAR-T-associated toxicities.MethodsPharmacologic (blocking antibody) and genetic (CRISPR/Cas9) approaches were used to block IFNg signaling and/or production by CAR T cells. In vitro CAR-T function and cytotoxicity was tested using ELISA, flow cytometry and short-/long-term killing assays prior to their assessment in vivo. NSG mice were injected with Nalm6 or JeKo-1 cancer cells prior to treatment with IFNg-modified CAR-T and tumor size and IFNg production were measured. To determine how the loss of IFNg might affect innate immune cells, CAR-T, macrophages and tumor cells were co-cultured and assessed by flow cytometry, immunofluorescence, Luminex and RNA sequencing.ResultsIFNg could be blocked using an anti-IFNg antibody or CRISPR/Cas9 editing of the CAR T cells without affecting T cell activation, proliferation or cytokine production (IL-2, TNFa, GM-CSF). Successful blockade of the IFNg signaling pathway was confirmed by reduced phosphorylation of JAK1, JAK2 and STAT1, even in the presence of exogenous IFNg. Loss of IFNg did not reduce the cytotoxic potential or persistence of CAR-T against hematologic malignancies in vitro or in vivo. When cultured with macrophages and cancer cells, IFNg knockout (IFNgKO) CAR-T yielded decreased levels of IL-1B, IL-6, IL-13, MCP1 and CXCL10, indicating a reduction in macrophage activation induced by CAR-T in the absence of IFNg. Serum from tumor-bearing mice treated with IFNgKO CAR-T elicited lower activation of macrophages in vitro compared to those treated with IFNg-producing CAR-T cells. Furthermore, IFNgKO CAR T cells co-cultured with tumor cells and macrophages demonstrated less exhaustion as shown by reduced expression of PD1, Tim3 and Lag3 and increased IFNgKO CAR-T expansion.ConclusionsCollectively, these data suggest that IFNg is not required for the efficacy of CAR-T in hematologic malignancies and can potentially be targeted to reduce toxicity and enhance CAR-T efficacy and persistence in the clinic.

scholarly journals 765 Contextual secretion of nanoscale interleukin (IL)-12 by CAR T cells for the treatment of cancer

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A813-A813
Author(s):  
Zhifen Yang ◽  
Maggie Bobbins ◽  
Hana Choi ◽  
Ofir Stefanson ◽  
Jin Yang ◽  
...  
Keyword(s):  
T Cells ◽  
Cancer Cells ◽  
Systemic Toxicity ◽  
Car T Cells ◽  
Car T ◽  
Her 2 ◽  
Ifn Γ ◽  
Fadu Cells

BackgroundInterleukin(IL)-12 activates T cells and macrophages pivoting the switch that turns chronic into acute inflammation and results in cancer rejection. However, despite formidable antitumor effects in preclinical models, its clinical utilization is limited by severe systemic toxicity. Here, we present a conditional, antigen-dependent, non-editing CRISPR-activation (CRISPRa) circuit (RB-2-12) that purposefully induces minimally effective doses of IL-12 for autocrine activation of CAR-T.MethodsRB-2-12 is a CAR T cell engineered to express the IL-12 heterodimer via conditional transcription of its two endogenous subunits p35 and p40. The circuit includes a lentiviral constructs encoding an anti-HER2 (4D5) single chain variable fragment, with CD28 and CD3ζ co-stimulatory domains linked to a tobacco etch virus (TEV) protease and two single guide RNAs (sgRNA) targeting the promoter region for IL-12A orL-12B. A second constructs encodes linker for activation of T cells, complexed to nuclease-deactivated/dead Cas9 (dCas9)-VP64-p65-Rta transcriptional activator (VPR) via a TEV-cleavable linker (LdCV). Activation of CAR brings CAR-TEV in proximity to LdCV releasing dCas9 for nuclear localization to the regulatory regions and conditionally and reversibly induce nanoscale expression of the p70 heterodimer. RB-2-12 was compared in vitro to control (cRB-2-12, lacking the IL-12 sgRNAs).ResultsRB-2-12 induced autocrine production of low concentrations of IL-12 upon exposure to HER2+ FaDu cancer cells resulting in significantly enhanced production of interferon (IFN)-γ, cytotoxic activity and proliferation (figure 1a). These effects were comparable to co-culturing conventional HER2-specific CAR-T cells with a modified FaDu cell line expressing high doses of IL-12 (figure 1b).Abstract 765 Figure 1Conditional autocrine release of nanoscale-dose p70/IL-12 byConditional autocrine release of nanoscale-dose p70/IL-12 by RB-2-12 resulting in enhanced IFN-γ production, cytotoxicity and proliferation in vitro after three days of exposure to FaDu cells (figure 1a). Constitutive high dose release of p70-IL-12 by HER2+ Fadu cells engineered to constitutively express IL-12 (FaDu/IL-12) and its effect on IFN-γ secretion, cytotoxicity and proliferation of conventional HER-2-specific CAR T-cells three days after exposure to FaDu or FaDu/IL-12 cancer cells (figure 1b). N.T. = non-transduced T cells; NOsg = cRB-2-12 CAR-T cells missing the sgRNAs for the two IL-12 subunits; IL12sg = complete product incorporating the full CRISPRa functions.ConclusionsWe have previously shown that tandem suppression of PD-1 expression upon HER-2 CAR activation using CRISPR interference enhances anti-cancer properties of CAR-T cells in vivo against HER2-FaDu xenografts by promoting their persistence and long-term tumor colonization (companion abstract submitted to SITC annual meeting). We hypothesize that addition of a Th1 polarizing component such as IL-12 will exponentially increase the efficacy of reprogrammed CAR-T cells by combining enhancement of effector functions to cellular fitness. At the same time, the autocrine effects of nanoscale IL-12 production limit the risk of off-tumor leakage and systemic toxicity. Such cumulative synthetic biology approaches are currently investigated in vitro and in vivo model systems. Current work is testing the effectiveness of RB-2-12 in vivo against FaDu xenografts.AcknowledgementsNoneTrial RegistrationN.A.Ethics ApprovalNot ApplicableConsentNot ApplicableReferencesNone

scholarly journals Effective antitumor activity of 5T4‐specific CAR‐T cells against ovarian cancer cells in vitro and xenotransplanted tumors in vivo

MedComm ◽  
2020 ◽  
Vol 1 (3) ◽  
pp. 338-350
Author(s):  
Cuiyu Guo ◽  
E Dong ◽  
Qinhuai Lai ◽  
Shijie Zhou ◽  
Guangbing Zhang ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
T Cells ◽  
Antitumor Activity ◽  
Cancer Cells ◽  
Ovarian Cancer Cells ◽  
Car T Cells ◽  
Car T

98 ATA3271: an armored, next-generation off-the-shelf, allogeneic, mesothelin-CAR T cell therapy for solid tumors

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A109-A109
Author(s):  
Jiangyue Liu ◽  
Xianhui Chen ◽  
Jason Karlen ◽  
Alfonso Brito ◽  
Tiffany Jheng ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Next Generation ◽  
Phase 3 ◽  
T Cell Therapy ◽  
Cell Therapies ◽  
Car T Cells ◽  
Car T

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.

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