scholarly journals 132 HLA-independent T cell receptors effectively target low abundance antigens

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A142-A142
Author(s):  
Jorge Mansilla-Soto ◽  
Justin Eyquem ◽  
Sascha Haubner ◽  
Mohamad Hamieh ◽  
Judith Feucht ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
High Sensitivity ◽  
Target Antigen ◽  
Dependent Manner ◽  
Cell Surface Antigens ◽  
Car T Cells ◽  
Car T ◽  
Increased Sensitivity

BackgroundChimeric antigen receptors (CARs) engage antigen independently of HLA and enable sustained T cell proliferation when they are endowed with both activating and costimulatory functions. While remission rates have been noticeably elevated in numerous clinical trials targeting CD19, CD22 or BCMA, relapses are common. One of the several underlying relapse mechanisms is antigen escape, which refers to a relapsing tumor that is either negative for the targeted antigen or expresses the latter at a low level. Failure to eliminate antigen-low tumors raises questions about the sensitivity of CARs and the minimum antigen density that is required for effective tumor eradication. Unlike CARs, TCRs engage antigen in an HLA-dependent manner, and they do so with high sensitivity. We hypothesized that a TCR/CD3 complex containing the same heavy and light immunoglobulin chains as a CAR will display increased sensitivity to the target antigen.MethodsWe edited the TRAC locus in human primary T cells to establish a novel antigen receptor structure, termed HLA-independent TCR or HIT receptor, by incorporating into the TCR/CD3 complex the same heavy and light chains as those of a corresponding CAR. We assessed their antigen sensitivity against a panel of cell lines expressing different antigen levels, analyzing their cytotoxicity, cytokine secretion, signaling response and degranulation activity. HIT and CAR T cells were further evaluated for their anti-tumor response using established ALL and AML mouse models.ResultsCD19-TRAC-HIT and CD19-TRAC-CAR T cells lysed wild-type NALM6 (~27,000 CD19 molecules) and NALM6 variants with 100-fold less CD19. As CD19 levels decreased further, CAR T cells no longer killed their target, in contrast to HIT T cells. HIT T cells showed increased expression of IFN-gamma, IL-2 and TNF-alpha upon exposure to NALM6 cells expressing ~20 CD19 molecules per cell, compared to CAR T cells. This increased sensitivity of HIT receptors correlated to their greater signaling response, upon exposure to the low-antigen-density NALM6. Phospho-proteomic analyses further confirmed this increased response of HIT T cells to low antigen levels. Altogether, these results confirm that HIT receptors endow T cells with greater antigen sensitivity than canonical CARs. We further showed that HIT T cells have higher in vivo anti-tumor activity compared to CAR T cells in mice bearing low-antigen-density ALL or AML.ConclusionsHIT receptors consistently afford high antigen sensitivity and mediate tumor recognition beyond what current CARs can provide. HIT receptors open new prospects for targeting cell surface antigens of low abundance.Ethics ApprovalEight- to 12-week-old NOD/SCID/IL-2Rgamma-null (NSG) male mice (Jackson Laboratory) were used under a protocol approved by the MSKCC Institutional Animal Care and Use Committee.

scholarly journals "IF-Better" Gating: Combinatorial Targeting and Synergistic Signaling for Enhanced CAR T Cell Efficacy

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2774-2774
Author(s):  
Sascha Haubner ◽  
Jorge Mansilla-Soto ◽  
Sarah Nataraj ◽  
Xingyue He ◽  
Jae H Park ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Target Antigen ◽  
Normal Cells ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Provision Of Services ◽  
Xenograft Models

Abstract CAR T cell therapy provides a potent therapeutic option in various B cell-related hematologic malignancies. One of the major efficacy challenges is escape of tumor cells with low antigen density, which has been clinically observed in several malignancies treated with CAR therapy. Novel concepts of CAR design are needed to address phenotypic heterogeneity including clonal variability of target antigen expression. In the study presented here, we focused on AML and selected ADGRE2 as CAR target due to its high rate of positivity on AML bulk and leukemic stem cells (LSC) in a molecularly heterogeneous AML patient population. We chose an ADGRE2-CAR with optimized scFv affinity and fine-tuned CD3zeta signaling to achieve an ideal killing threshold that would allow for sparing of ADGRE2-low normal cells. We hypothesized that co-targeting of a second AML-related antigen may mitigate potential CAR target antigen-low AML escape and we identified CLEC12A as preferential co-target due to its non-overlapping expression profiles in normal hematopoiesis and other vital tissues. We developed ADCLEC.syn1, a novel combinatorial CAR construct consisting of an ADGRE2-targeting 28z1XX-CAR and a CLEC12A-targeting chimeric costimulatory receptor (CCR). ADCLEC.syn1 operates based on what we describe as "IF-BETTER" gate: High CAR target expression alone triggers killing, whereas low CAR target expression does not, unless a CCR target is present. Additional CCR interaction lowers the threshold for CAR-mediated killing through increased avidity and costimulation, allowing for higher CAR sensitivity that is purposefully limited to target cells expressing both antigens. In the context of ADCLEC.syn1, ADGRE2-high/CLEC12A-negative AML cells can trigger cell lysis while ADGRE2-low/CLEC12A-negative normal cells are spared. Importantly, ADGRE2-low/CLEC12A-high AML cells are also potently eliminated, preventing ADGRE2-low AML escape. Using NSG in-vivo xenograft models of engineered MOLM13 AML cell line variants with low levels of ADGRE2 to model antigen escape, we found that ADCLEC.syn1 outperforms a single-ADGRE2-CAR lacking assistance via CLEC12A-CCR. Importantly, ADCLEC.syn1 also outperformed an otherwise identical alternative dual-CAR version (OR-gated ADGRE2-CAR+CLEC12A-CAR) in the setting of both ADGRE2-high and ADGRE2-low MOLM13, further underlining the importance of fine-tuned overall signaling. We confirmed high in-vivo potency against diverse AML cell lines with a wide range of ADGRE2 and CLEC12A levels reflecting population-wide AML heterogeneity. At clinically relevant CAR T cell doses, ADCLEC.syn1 induced complete and durable remissions in xenograft models of MOLM13 (ADGRE2-high/CLEC12A-low) and U937 (ADGRE2-low/CLEC12A-high). ADCLEC.syn1 CAR T cells were found to be functionally persistent for >70 days, with a single CAR T cell dose potently averting relapse modeled via AML re-challenges. In summary, we provide pre-clinical evidence that an "IF-BETTER"-gated CAR+CCR T cell (ADCLEC.syn1) can outperform a single-CAR T cell (ADGRE2-CAR) and a dual-CAR T cell (ADGRE2-CAR+CLEC12A-CAR). ADCLEC.syn1 enhances antileukemic efficacy and prevents antigen-low AML escape via detection of a rationally selected combinatorial target antigen signature that is commonly found in AML but limited in vital normal cells. Using phenotypically representative AML xenograft models and clinically relevant T cell doses, we demonstrate high therapeutic potential of ADCLEC.syn1 CAR T cells, further supporting clinical translation of an "IF-BETTER"-gated CAR concept into a phase 1 trial. Disclosures Haubner: Takeda Pharmaceuticals Company Ltd.: Patents & Royalties: Co-inventor of IP that MSK licensed to Takeda, Research Funding. Mansilla-Soto: Takeda Pharmaceuticals Company Ltd.: Patents & Royalties; Atara Biotherapeutics: Patents & Royalties; Fate Therapeutics: Patents & Royalties; Mnemo Therapeutics: Patents & Royalties. He: Takeda Pharmaceuticals Company Ltd.: Ended employment in the past 24 months, Patents & Royalties. Park: Curocel: Consultancy; BMS: Consultancy; Innate Pharma: Consultancy; Autolus: Consultancy; Servier: Consultancy; Kite Pharma: Consultancy; Affyimmune: Consultancy; Intellia: Consultancy; Minerva: Consultancy; PrecisionBio: Consultancy; Amgen: Consultancy; Kura Oncology: Consultancy; Artiva: Consultancy; Novartis: Consultancy. Rivière: Juno Therapeutics: Patents & Royalties; Fate Therapeutics: Other: Provision of Services, Patents & Royalties; Centre for Commercialization of Cancer Immunotherapy: Other: Provision of Services; The Georgia Tech Research Corporation (GTRC): Other: Provision of Services (uncompensated); FloDesign Sonics: Other: Provision of Services. Sadelain: NHLBI Gene Therapy Resource Program: Other: Provision of Services (uncompensated); St. Jude Children's Research Hospital: Other: Provision of Services; Minerva Biotechnologies: Patents & Royalties; Mnemo Therapeutics: Patents & Royalties; Juno Therapeutics: Patents & Royalties; Fate Therapeutics: Other: Provision of Services (uncompensated), Patents & Royalties; Ceramedix: Patents & Royalties; Takeda Pharmaceuticals: Other: Provision of Services, Patents & Royalties; Atara Biotherapeutics: Patents & Royalties.

scholarly journals Locally secreted BiTEs complement CAR T cells by enhancing solid tumor killing

2021 ◽  
Author(s):  
Yibo Yin ◽  
Jesse Rodriguez ◽  
Nannan Li ◽  
Radhika Thokala ◽  
MacLean P Nasrallah ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumor ◽  
Tumor Therapy ◽  
Target Antigen ◽  
Car T Cells ◽  
Car T ◽  
Anti Tumor Activity

Bispecific T-cell engagers (BiTEs) are bispecific antibodies that redirect T cells to target antigen-expressing tumors. BiTEs can be secreted by T cells through genetic engineering and perform anti-tumor activity. We hypothesized that BiTE-secreting T cells could be a valuable T cell-directed therapy in solid tumors, with distinct properties in mono- or multi-valent strategies incorporating chimeric antigen receptor (CAR) T cells. Glioblastomas represent a good model for solid tumor heterogeneity and represent a significant therapeutic challenge. We detected expression of tumor-associated epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), and interleukin-13 receptor alpha 2 (IL13Rα2) on glioma tissues and glioma cancer stem cells. These antigens formed the basis of a multivalent approach, using a conformation-specific tumor-related EGFR targeting antibody (806) and Hu08, an IL13Rα2-targeting antibody, as the scFvs to generate new BiTE molecules. Compared with 806CAR T cells and Hu08CAR T cells, BiTE T cells demonstrated prominent activation, cytokine production, and cytotoxicity in response to target-positive gliomas. Superior response activity was also demonstrated in BiTE secreting bivalent targeting T cells compared with bivalent targeting CAR T cells, which significantly delayed tumor growth in a glioma mouse model. In summary, BiTEs secreted by mono- or multi- valent targeting T cells have potent anti-tumor activity in vitro and in vivo with significant sensitivity and specificity, demonstrating a promising strategy in solid tumor therapy.

PL03.3.A Development and characterization of CD317-specific CAR T cells as an innovative immunotherapeutic strategy against glioblastoma

2021 ◽  
Vol 23 (Supplement_2) ◽  
pp. ii2-ii2
Author(s):  
L Hänsch ◽  
M Peipp ◽  
R Myburgh ◽  
M Silginer ◽  
T Weiss ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Glioma Cells ◽  
Target Antigen ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract BACKGROUND Due to the limited success of existing therapies for gliomas, innovative therapeutic options are urgently needed. Chimeric antigen receptor (CAR) T cell therapy has been successful in patients with hematological malignancies. However, using this treatment against solid tumors such as glioblastomas is more challenging. Here, we generated CAR T cells targeting the transmembrane protein CD317 (BST-2, HM1.24) which is overexpressed in glioma cells and may therefore serve as a novel target antigen for CAR T cell-based immunotherapy. MATERIAL AND METHODS CAR T cells targeting CD317 were generated by lentiviral transduction of human T cells from healthy donors. The anti-glioma activity of CD317-CAR T cells was determined in lysis assays using different glioma target cell lines with varying CD317 expression levels. The efficiency of CD317-CAR T cells to control tumor growth in vivo was evaluated in clinically relevant orthotopic xenograft glioma mouse models. RESULTS We created a second-generation CAR construct targeting CD317 and observed strong anti-glioma activity of CD317-CAR T cells in vitro. Glioma cells with a CRISPR/Cas9-mediated CD317 knockout were resistant to CD317-specific CAR T cells, demonstrating their target antigen-specificity. Since CD317 is also expressed by T cells, transduction with a CD317-directed CAR resulted in fratricide of the transduced T cells. Silencing of CD317 in CAR T cells by integrating a specific shRNA into the CAR vector significantly increased the viability, proliferation and cytotoxic function of the CAR T cells. Importantly, intratumoral treatment with CD317-CAR T cells prolonged the survival and cured a significant fraction of glioma-bearing nude mice. CONCLUSION We demonstrate strong CD317-specific anti-tumor activity of CD317-CAR T cells against various glioma cell lines in vitro and in xenograft glioma models in vivo. These data lay a scientific basis for the subsequent evaluation of this therapeutic strategy in clinical neuro-oncology.

scholarly journals Understanding the CD8+ CAR T Cell Response to Antigen-Low Leukemias

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 736-736
Author(s):  
Kole Degolier ◽  
Jennifer Cimons ◽  
Michael Yarnell ◽  
Mark Eric Kohler ◽  
Terry J. Fry
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Response ◽  
Target Antigen ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Tumor Clearance

Abstract Chimeric antigen receptor (CAR) T cell therapy has emerged as a highly efficacious treatment for B-lineage acute lymphoblastic leukemias (B-ALL). However, downregulation of the CAR-targeted antigen on leukemia cells, predicted to reduce cellular avidity, is associated with post-CAR T cell leukemic relapse following CD22 CAR treatment (Fry et al., Nat. Med., 2017). We have observed reduced function of human CAR T cells against low target antigen site density (Ag Lo) human leukemia in immunodeficient mouse models, relative to CARs responding to high-antigen expressing leukemia. Thus, a better understanding of CAR responses to Ag Lo leukemia could help to increase the durability of remissions. We set out to develop a model system in which we could further interrogate the consequences of low-avidity interactions on CAR immunobiology, generating variants of a murine B-ALL driven by the E2A-PBX fusion protein (E2A) with different levels of target antigen to use in an immunocompetent syngeneic mouse model. We observed impaired expansion (p<0.0001) and tumor clearance (p<0.001) of CAR T cells responding to low-antigen variants of E2A (E2A-Ag Lo) as compared to wildtype E2A expressing high levels of antigen (E2A-WT). While CD8+ CAR T cell (CAR8) transcription factor (TF) expression in response to E2A-Ag Lo versus E2A-WT was largely similar early after CAR infusion, by day 9 post-CAR, CAR8s responding to E2A-Ag Lo exhibited decreased expression of multiple TFs, with Eomes (p<0.01), Irf4 (p<0.001) and Blimp1 (p<0.01) showing the largest magnitude change relative to CAR8s responding to E2A-WT. Additionally, CAR8s from mice bearing E2A-Ag Lo became enriched for cells of a "terminally exhausted" phenotype (Eomes+/PD1 Hi/TOX Hi) by day 11 post-CAR, and negatively-enriched for the "progenitor exhausted" (Tcf1+/PD1 Int) phenotype which can be functionally rescued by anti-PD1 therapy (p<0.0001, p<0.01). These data suggest that continual stimulation by low density antigen leads to a gradual reduction in the ability of CAR8s to mount an effector response, and eventually to T cell states with sub-optimal anti-tumor efficacy. Following in vitro stimulation of human CD22 CARs across a range of leukemic antigen densities, we saw that the percentage of CAR+ cells capable of producing IFNγ and IL2 corresponded to target antigen density (p<0.01, p<0.001). As human CARs are commonly manufactured from heterogenous bulk donor T cells, we hypothesized that antigen sensitivity is impacted by the prior antigen-experience of a given T cell. We predicted that T cells which had encountered cognate antigen through their TCR prior to CAR manufacturing (CAR8 AgEx) would have enhanced capacity to respond to low-avidity stimulation compared to CARs manufactured from naïve CD8+ T cells (CAR8 Naïve). We used a well-characterized ovalbumin vaccination model with OT-I TCR-transgenic T cells, allowing defined control of T cell antigen experience, to generate CAR8 AgEx. We found that CAR8 AgEx were highly antigen-sensitive relative to CAR8 Naïve, showing almost no reduction in numbers of cells capable of producing IFNγ and TNFα in vitro against E2A-Ag Lo as compared to E2A-WT. In vivo, CAR8 AgEx showed near complete depletion of E2A-Ag Lo in bone marrow by day 11 post-CAR, while mice treated with CAR8 Naïve maintained a substantial tumor burden (p<0.01). To test our hypothesis in human cells, we manufactured CD22 CAR T cells from naïve (CD45RO-) versus non-naïve (CD45RO+) starting T cell populations, and again found that CAR AgEx outperformed CAR Naïve against Ag Lo leukemia in production of IFNγ and IL2 in vitro (p<0.001, p<0.01) and in early leukemic clearance in vivo (p<0.0001, day 13). In conclusion, we have established a model to study the immunobiology of the CAR T cell response to Ag Lo B-ALL in an intact host. Preliminary findings indicate impaired expansion and tumor clearance of Ag Lo leukemia, associated with altered CAR T cell transcriptional profiles and features of T cell exhaustion. Furthermore, T cell history prior to CAR manufacturing has a drastic impact on the capacity to respond to Ag Lo leukemia. Future studies with this model will expand our characterization of CAR T cells responding to Ag Lo leukemia, with the goal of optimizing antigen sensitivity. We expect that advancing our understanding on the interplay of antigen density and CAR differentiation status will prove useful in developing more effective iterations of this therapy. Disclosures Fry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company.

scholarly journals 120 Chemokine receptor engineering enhances trafficking and homing of primary and iPSC-derived CAR-T cells to solid tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A129-A129
Author(s):  
Martin Hosking ◽  
Soheila Shirinbak ◽  
Joy Grant ◽  
Yijia Pan ◽  
Angela Gentile ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Chemokine Receptor ◽  
Dependent Manner ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

BackgroundChimeric antigen receptor (CAR)-T cells for solid tumors have shown modest effectiveness as compared to hematologic malignancies, a consequence of antigen heterogeneity, the immuno-suppressive tumor microenvironment (TME), limited cell persistence, and perhaps most notably, the trafficking of the CAR-T cell to the tumor itself. Early detection of CAR-T cells within a solid tumor has been associated with better outcomes across several clinical trials in diverse tumor settings, suggesting that strategies focused on enhancing CAR-T cell homing to and infiltration into the tumor can yield therapeutic benefit.MethodsHere, we demonstrate that following irradiation or exposure to common chemotherapy drugs, selected tumor cell lines (breast, ovarian, and prostate) specifically upregulate several chemokines, notably the CXCR2 ligand, interleukin (IL)-8, up to 4-fold over baseline control (e.g. 24ng/ml increased to 79ng/ml for SKOV3; 2.9ng/ml increased to 12.5ng/ml for MDA-MB-231). To leverage the upregulation of IL-8 as a mechanism of directing CAR-T cells to the tumor site, we initially engineered primary CAR-T cells to express CXCR2 and demonstrated functional migration, in a dose-dependent manner, to recombinant IL-8 in an in vitro transwell chemotaxis assay; maximal migration of approximately 2-fold over baseline was observed with 10ng/ml of rhIL-8. Similarly, supernatant from pre-conditioned tumor lines also elicited functional enhancements in migration (up to 4-fold specific migration). In addition, ovarian tumors were sub-optimally treated with paclitaxel in vivo, which promoted infiltration of CXCR2+ CAR-T cells and demonstrated enhanced tumor control.ResultsWe then incorporated these findings into our off-the-shelf, iPSC-derived CAR-T cell product platform. Induced pluripotent stem cells (iPSCs) were precisely engineered to co-express CAR and CXCR2 and subsequently differentiated to T cells to generate iPSC-derived CAR-T cells (CAR-iT cells). Like their primary CAR-T cell counterparts, functional chemotaxis of CXCR2+ CAR-iT cells was also observed in response to recombinant IL-8 and preconditioned tumor media. Importantly, CXCR2 expression did not limit CAR-dependent cytolytic function and the specificity of CAR-iT cells, underscoring the compatibility of this approach. Further in vitro and in vivo studies are ongoing and will be presented.ConclusionsCollectively, these data demonstrate that rational engineering of unique chemokine receptors to deliver the ideal chemokine/chemokine receptor match between tumors and effector cells can be leveraged to enhance tumor targeting and trafficking of CAR-iT cells for more effective treatment of solid tumors.Ethics ApprovalThese studies were approved by Fate Therapeutics Institutional Animal Care and Use Committee and were carried out in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals.

scholarly journals Base-edited CAR T Cells for combinational therapy against T cell malignancies

2020 ◽  
Author(s):  
Christos Georgiadis ◽  
Jane Rasaiyaah ◽  
Soragia Athina Gkazi ◽  
Roland Preece ◽  
Aniekan Etuk ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Specific Binding ◽  
Haematopoietic Stem Cell ◽  
Target Antigen ◽  
Car T Cells ◽  
Car T ◽  
T Cell Malignancies

AbstractTargeting T cell malignancies using chimeric antigen receptor (CAR) T cells is hindered by ‘T v T’ fratricide against shared antigens such as CD3 and CD7. Genome-editing can overcome such hurdles through targeted disruption of problematic shared antigens. Base editing offers the possibility of seamless disruption of gene expression through the creation of stop codons or elimination of splice donor or acceptor sites. We describe the generation of fratricide resistant, T cells by orderly removal of shared antigens such as TCR/CD3 and CD7 ahead of lentiviral mediated expression of CARs specific for CD3 or CD7. Molecular interrogation of base edited cells confirmed virtual elimination of chromosomal translocation events detected in conventional Cas9 treated cells. Interestingly, co-culture of 3CAR and 7CAR cells resulted in ‘self-enrichment’ yielding populations that were 99.6% TCR-/CD3/-CD7-. 3CAR or 7CAR cells were able to exert specific cytotoxicity against their relevant target antigen in leukaemia lines with defined CD3 and/or CD7 expression as well as primary T-ALL cells. Co-cultured 3CAR/7CAR cells exhibited the highest level of cytotoxicity against T-ALL targets expressing both target in vitro and an in vivo human:murine chimeric model. While APOBEC editors can reportedly exhibit guide-independent deamination of both DNA and RNA, we found no evidence of promiscuous base conversion activity affecting CAR antigen specific binding regions which may otherwise redirect T cell specificity. Combinational infusion of fratricide resistant anti-T CAR T cells may enable enhanced molecular remission ahead of allogeneic haematopoietic stem cell transplantation for T cell malignancies.

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.

scholarly journals Base-edited CAR T cells for combinational therapy against T cell malignancies

Leukemia ◽  
2021 ◽  
Author(s):  
Christos Georgiadis ◽  
Jane Rasaiyaah ◽  
Soragia Athina Gkazi ◽  
Roland Preece ◽  
Aniekan Etuk ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Specific Binding ◽  
Base Editing ◽  
Car T Cells ◽  
Dna And Rna ◽  
Car T ◽  
T Cell Malignancies

AbstractTargeting T cell malignancies using chimeric antigen receptor (CAR) T cells is hindered by ‘T v T’ fratricide against shared antigens such as CD3 and CD7. Base editing offers the possibility of seamless disruption of gene expression of problematic antigens through creation of stop codons or elimination of splice sites. We describe the generation of fratricide-resistant T cells by orderly removal of TCR/CD3 and CD7 ahead of lentiviral-mediated expression of CARs specific for CD3 or CD7. Molecular interrogation of base-edited cells confirmed elimination of chromosomal translocations detected in conventional Cas9 treated cells. Interestingly, 3CAR/7CAR co-culture resulted in ‘self-enrichment’ yielding populations 99.6% TCR−/CD3−/CD7−. 3CAR or 7CAR cells were able to exert specific cytotoxicity against leukaemia lines with defined CD3 and/or CD7 expression as well as primary T-ALL cells. Co-cultured 3CAR/7CAR cells exhibited highest cytotoxicity against CD3 + CD7 + T-ALL targets in vitro and an in vivo human:murine chimeric model. While APOBEC editors can reportedly exhibit guide-independent deamination of both DNA and RNA, we found no problematic ‘off-target’ activity or promiscuous base conversion affecting CAR antigen-specific binding regions, which may otherwise redirect T cell specificity. Combinational infusion of fratricide-resistant anti-T CAR T cells may enable enhanced molecular remission ahead of allo-HSCT for T cell malignancies.

109 Dominant-negative TGFβ receptor 2 enhances GPC3-targeting CAR-T cell efficacy against hepatocellular carcinoma

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A121-A121
Author(s):  
Nina Chu ◽  
Michael Overstreet ◽  
Ryan Gilbreth ◽  
Lori Clarke ◽  
Christina Gesse ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Dominant Negative ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

BackgroundChimeric antigen receptors (CARs) are engineered synthetic receptors that reprogram T cell specificity and function against a given antigen. Autologous CAR-T cell therapy has demonstrated potent efficacy against various hematological malignancies, but has yielded limited success against solid cancers. MEDI7028 is a CAR that targets oncofetal antigen glypican-3 (GPC3), which is expressed in 70–90% of hepatocellular carcinoma (HCC), but not in normal liver tissue. Transforming growth factor β (TGFβ) secretion is increased in advanced HCC, which creates an immunosuppressive milieu and facilitates cancer progression and poor prognosis. We tested whether the anti-tumor efficacy of a GPC3 CAR-T can be enhanced with the co-expression of dominant-negative TGFβRII (TGFβRIIDN).MethodsPrimary human T cells were lentivirally transduced to express GPC3 CAR both with and without TGFβRIIDN. Western blot and flow cytometry were performed on purified CAR-T cells to assess modulation of pathways and immune phenotypes driven by TGFβ in vitro. A xenograft model of human HCC cell line overexpressing TGFβ in immunodeficient mice was used to investigate the in vivo efficacy of TGFβRIIDN armored and unarmored CAR-T. Tumor infiltrating lymphocyte populations were analyzed by flow cytometry while serum cytokine levels were quantified with ELISA.ResultsArmoring GPC3 CAR-T with TGFβRIIDN nearly abolished phospho-SMAD2/3 expression upon exposure to recombinant human TGFβ in vitro, indicating that the TGFβ signaling axis was successfully blocked by expression of the dominant-negative receptor. Additionally, expression of TGFβRIIDN suppressed TGFβ-driven CD103 upregulation, further demonstrating attenuation of the pathway by this armoring strategy. In vivo, the TGFβRIIDN armored CAR-T achieved superior tumor regression and delayed tumor regrowth compared to the unarmored CAR-T. The armored CAR-T cells infiltrated HCC tumors more abundantly than their unarmored counterparts, and were phenotypically less exhausted and less differentiated. In line with these observations, we detected significantly more interferon gamma (IFNγ) at peak response and decreased alpha-fetoprotein in the serum of mice treated with armored cells compared to mice receiving unarmored CAR-T, demonstrating in vivo functional superiority of TGFβRIIDN armored CAR-T therapy.ConclusionsArmoring GPC3 CAR-T with TGFβRIIDN abrogates the signaling of TGFβ in vitro and enhances the anti-tumor efficacy of GPC3 CAR-T against TGFβ-expressing HCC tumors in vivo, proving TGFβRIIDN to be an effective armoring strategy against TGFβ-expressing solid malignancies in preclinical models.Ethics ApprovalThe study was approved by AstraZeneca’s Ethics Board and Institutional Animal Care and Use Committee (IACUC).

Combinatorial Antigen Targeting Strategy for Acute Myeloid Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 22-23
Author(s):  
Pinar Ataca Atilla ◽  
Mary K McKenna ◽  
Norihiro Watanabe ◽  
Maksim Mamonkin ◽  
Malcolm K. Brenner ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Tumor Control ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Introduction: Efforts to safely and effectively treat acute myeloid leukemia (AML) by targeting a single leukemia associated antigen with chimeric antigen receptor T (CAR T) cells have had limited success. We determined whether combinatorial expression of chimeric antigen receptors directed to two different AML associated antigens would augment tumor eradication and prevent relapse in targets with heterogeneous expression of myeloid antigens. Methods: We generated CD123 and CD33 targeting CARs; each containing a 4-1BBz or CD28z endodomain. We analyzed the anti-tumor activity of T cells expressing each CAR alone or in co-transduction with a CLL-1 CAR with CD28z endodomain and CD8 hinge previously optimized for use in our open CAR-T cell trial for AML (NCT04219163). We analyzed CAR-T cell phenotype, expansion and transduction efficacy by flow cytometry and assessed function by in vitro and in vivo activity against AML cell lines expressing high, intermediate or low levels of the target antigens (Molm 13= CD123 high, CD33 high, CLL-1 intermediate, KG1a= CD123 low, CD33 low, CLL-1 low and HL60= CD123 low, CD33 intermediate, CLL-1 intermediate/high) For in vivo studies we used NOD.SCID IL-2Rg-/-3/GM/SF (NSGS) mice with established leukemia, determining antitumor activity by bioluminescence imaging. Results: We obtained high levels of gene transfer and expression with both single (CD33.4-1BBʓ, CD123.4-1BBʓ, CD33.CD28ʓ, CD123.CD28ʓ, CLL-1 CAR) and double transduction CD33/CD123.4-1BBʓ or CD33/CD123.CD28ʓ) although single-transductants had marginally higher total CAR expression of 70%-80% versus 60-70% after co-transduction. Constructs containing CD28 co-stimulatory domain exhibited rapid expansion with elevated peak levels compared to 41BB co-stim domain irrespective of the CAR specificity. (p<0.001) (Fig 1a). In 72h co-culture assays, we found consistently improved anti-tumor activity by CAR Ts expressing CLL-1 in combination either with CD33 or with CD123 compared to T cells expressing CLL-1 CAR alone. The benefit of dual expression was most evident when the target cell line expressed low levels of one or both target antigens (e.g. KG1a) (Fig 1b) (P<0.001). No antigen escape was detected in residual tumor. Mechanistically, dual expression was associated with higher pCD3ʓ levels compared to single CAR T cells on exposure to any given tumor (Fig 1c). Increased pCD3ʓ levels were in turn associated with augmented CAR-T degranulation (assessed by CD107a expression) in both CD4 and CD8 T cell populations and with increased TNFα and IFNɣ production (p<0.001 Fig 1d). In vivo, combinatorial targeting with CD123/CD33.CD28ʓ and CLL-1 CAR T cells improved tumor control and animal survival in lines (KG1a, MOLM13 and HL60) expressing diverse levels of the target antigens (Fig 2). Conclusion: Combinatorial targeting of T cells with CD33 or CD123.CD28z CARs and CLL-1-CAR improves CAR T cell activation associated with superior recruitment/phosphorylation of CD3ʓ, producing enhanced effector function and tumor control. The events that lead to increased pCD3ʓ after antigen engagement in the dual transduced cells may in part be due to an overall increase in CAR expression but may also reflect superior CAR recruitment after antigen engagement. We are now comparing the formation, structure, and stability of immune synapses in single and dual targeting CARs for AML. Disclosures Brenner: Walking Fish: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Tumstone: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Founder; Maker Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Memmgen: Membership on an entity's Board of Directors or advisory committees; Allogene: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Atilla:Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Tumstone: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: founder; Marker Therapeuticsa: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Other: Founder, Patents & Royalties; Allogene: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Walking Fish: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Memgen: Membership on an entity's Board of Directors or advisory committees; KUUR: Membership on an entity's Board of Directors or advisory committees.

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