scholarly journals PS1210 DEVELOPMENT OF A NEXT GENERATION ALLOGENEIC CAR-T CELL PLATFORM WITHOUT GENE EDITING

HemaSphere ◽  
2019 ◽  
Vol 3 (S1) ◽  
pp. 551
Author(s):  
P.A. Sotiropoulou ◽  
A. Michaux ◽  
S. Raitano ◽  
S. Bornschein ◽  
J. Bolsée ◽  
...  
Keyword(s):  
T Cell ◽  
Gene Editing ◽  
Next Generation ◽  
Car T Cell ◽  
Car T

scholarly journals Next Generation NKG2D-based CAR T-cells (CYAD-02): Co-expression of a Single shRNA Targeting MICA and MICB Improves Cell Persistence and Anti-Tumor Efficacy in vivo

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3931-3931
Author(s):  
Martina Fontaine ◽  
Benjamin Demoulin ◽  
Simon Bornschein ◽  
Susanna Raitano ◽  
Steve Lenger ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Next Generation ◽  
Activated T Cells ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Anti Tumor Activity ◽  
Nkg2d Receptor

Background The Natural Killer Group 2D (NKG2D) receptor is a NK cell activating receptor that binds to eight different ligands (NKG2DL) commonly over-expressed in cancer, including MICA and MICB. The product candidate CYAD-01 are chimeric antigen receptor (CAR) T-cells encoding the full length human NKG2D fused to the intracellular domain of CD3ζ. Data from preclinical models have shown that CYAD-01 cells specifically target solid and hematological tumors. Encouraging preliminary results from the Phase I clinical trial THINK, assessing CYAD-01 safety, showed initial signals of objective clinical responses in patients with r/r AML and MDS. The clinical development of CAR T-cells has been limited by several challenges including achieving sufficient numbers of cells for clinical application. We have previously shown that NKG2D ligands are transiently expressed on activated T cells and that robust cell yields are generated through the addition of a blocking antibody and a PI3K inhibitor during cell manufacture. Here, we investigated the ability of an optimized short hairpin RNA (shRNA) technology to modulate NKG2DL expression on CYAD-01 cells and to determine if there is an increase in the anti-tumor activity of NKG2D-based CAR T-cells (termed CYAD-02). Methods Molecular and cellular analyses identified MICA and MICB as the key NKG2DL expressed on activated T-cells and highly likely to participate in driving fratricide. In silico analysis and in vitro screening allowed the identification of a single shRNA targeting the conserved regions of MICA and MICB, thus downregulating both MICA and MICB expression. The selected shRNA was incorporated in the NKG2D-based CAR vector, creating the next-generation NKG2D-based CAR T-cell candidate, CYAD-02. In addition, truncated versions of the NKG2D receptor were generated to explore the mechanisms of action of NKG2D receptor activity in vivo. The in vivo persistence and anti-tumor activity of CYAD-02 cells was evaluated in an aggressive preclinical model of AML. Results Injection of CAR T-cells bearing truncated forms of the NKG2D-CAR in immunosuppressed mice resulted in similar persistence to the control T-cells. In contrast, CYAD-01 cells had reduced persistence, suggesting that the recognition of the NKG2DL by the NKG2D receptor could contribute to this effect. Analysis of cell phenotype upon CAR T-cell activation showed that MICA and MICB were transiently expressed on T-cells during manufacturing. These results collectively suggested that downregulating MICA and MICB expression in CYAD-01 cells could be a mean to increase CAR T-cell persistence in vivo. Candidate shRNA were screened for efficient targeting of both MICA and MICB at the mRNA and protein level. T-cells transduced with a single vector encoding for the NKG2D-based CAR and the selected shRNA targeting MICA and MICB (CYAD-02) demonstrated 3-fold increased expansion during in vitro culture in the absence of the blocking antibody used to increase cell yield during manufacture. When injected into immunosuppressed mice, CYAD-02 cells generated with the Optimab process showed 10-fold higher engraftment one week after injection and potent anti-tumor activity resulting in 2.6-fold increase of mouse survival in an aggressive AML model. Conclusions By using a single vector encoding the NKG2D-based CAR next to a shRNA targeting MICA and MICB and combined with improved cell culture methods, CYAD-02, the next-generation of NKG2D-based CAR T-cells, demonstrated enhanced in vivo persistence and anti-tumor activity. Following FDA acceptance of the IND application, a Phase 1 dose-escalation trial evaluating the safety and clinical activity of CYAD-02 for the treatment of r/r AML and MDS is scheduled to start in early 2020. Disclosures Fontaine: Celyad: Employment. Demoulin:Celyad: Employment. Bornschein:Celyad: Employment. Raitano:Celyad: Employment. Machado:Horizon Discovery: Employment. Moore:Avvinity Therapeutics: Employment, Other: Relationship at the time the work was performed; Horizon Discovery: Employment, Equity Ownership, Other: Relationship at the time the work was performed; Centauri Therapeutics: Consultancy, Other: Current relationship. Sotiropoulou:Celyad: Employment. Gilham:Celyad: Employment.

scholarly journals Predictive Value of Next-Generation Sequencing-Based Minimal Residual Disease after CAR-T Cell Therapy

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2842-2842
Author(s):  
Yue Huang ◽  
Houli Zhao ◽  
Mi Shao ◽  
Linghui Zhou ◽  
Guoqing Wei ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
T Cell ◽  
Cell Therapy ◽  
Patient Specific ◽  
Next Generation ◽  
T Cell Therapy ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T ◽  
Generation Sequencing

Abstract Background Minimal residual disease (MRD) is closely associated with risk stratification of hematological malignancies. Monitoring the MRD levels of patients during treatment and at time points after remission is critical for prevention of relapse. Chimeric antigen receptor T (CAR-T) cell therapy redirects genetically modified immune cells to fight against hematologic malignancies. However, given the high relapse rate after CAR-T cell therapy, MRD monitoring by traditional techniques cannot accurately quantify the disease burden, nor can they perform high-sensitivity in-depth monitoring. Further treatment options are still controversial at such a time point when the flow cytometry (FC) results show MRD < 0.01%, while tumor clones remain. Next-generation sequencing (NGS) - MRD screens out patient-specific T/B cell receptors and accurately and quantitatively monitor patient-specific tumor cells (up to 10E-6) to reveal accurate and highly sensitive MRD levels, plus provide more timely intervention criteria. Method Between June 2016 and June 2020, we retrospectively enrolled 27 patients who achieved complete remission at the first 4-week evaluation after CAR-T cell therapy. BM samples were harvested and stored before CAR-T cell infusion and 10-154 days after infusion. We evaluated 63 specimens in total 27 patients, plus tracked immunoglobulin (IG) sequencing rearrangement by next-generation sequencing (NGS) in 20 patients. Next, we classified 17 patients into high-risk (HR, NGS-MRD positive) and low-risk (LR, NGS-MRD negative) groups according to the lower limit of NGS detection (10E-6), as well as performed leukemia-free survival (LFS) and overall survival (OS) analysis. Results At least one trackable IG clonal sequence was identified in the pre-CAR-T BM specimens from 20/27 of the cases analyzed. The two diagnostic samples with low DNA quantity, in addition to five samples lacking a dominant index sequence were excluded from further analysis. We measured the MRD in 63 samples, using a threshold of 0.01%, the determination of the presence (or absence) of leukemia was concordant in 46/63 (73%) of the samples. Discordance between NGS and FC was identified in 17/63 samples (27%). Of the 20 patients for whom trackable sequences were identified, 17 with detectable clonal index sequences on +30 days were included in our subsequent analysis cohort (Figure 1). The baseline characteristics of the patients are presented in Table 1. NGS identified 9 out of the 17 patients (52.9%) whose level of MRD was > 0.01%, but MRD negative as measured by FC. This controversial group (n=9) had inferior LFS than those whose MRD was less than 0.01% by NGS (median, 56 days vs. 219 days, p = 0.037) (Figure 2A). The OS rate was comparable between the two groups (p = 0.129) (Figure 2B). Patients with positive NGS-MRD at day 30 had comparable LFS compared with those with negative NGS-MRD (p = 0.103) (Figure 3A). For subgroup analysis, we further analyzed the influence of HSCT on prognosis of HR and LR patients. Of the total 11 patients in HR group, seven non-HSCT subjects all relapsed within three months, with a median LFS of 38 days. In contrast, the remaining four HSCT patients in HR subgroup had a significantly better LFS than the non-HSCT patients (median, 495 days vs. 38 days, p = 0.003) (Figure 3B). These four patients also exhibited better OS to that of the non-HSCT group (median, 768 days vs 409 days, p = 0.006) (Figure 3C). As for the LR cohort, no significant difference was found in LFS and OS between the HSCT and non-HSCT groups (LFS: p = 0.782, OS: p = 0.782) (Median LFS and OS data not shown). All of the evidence demonstrated that NGS-MRD early after CAR-T is an efficient prognostic factor, especially for early relapse prediction. Early post-CAR-T NGS-MRD status may be recommended for HSCT timing. As such, for HR patients, whose NGS-MRD results were positive, timely HSCT may significantly improves the prognosis and therefore is highly recommended at an early point after CAR-T cell therapy. Conclusion NGS-MRD elaborately demonstrated the tumor dynamics. NGS-MRD evaluated early after CAR-T cell therapy is an efficient prognostic factor, and timely HSCT is highly recommended for NGS-MRD positive patients at an early point after CAR-T cell therapy. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Clinical Development of a Non-Gene-Edited Allogeneic Bcma-Targeting CAR T-Cell Product in Relapsed or Refractory Multiple Myeloma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 27-28
Author(s):  
A. Samer Al-Homsi ◽  
Sebastien Anguille ◽  
Jason Brayer ◽  
Dries Deeren ◽  
Nathalie Meuleman ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Gene Editing ◽  
Cell Product ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Background Autologous CAR T-cell therapy targeting the B-cell maturation antigen (BCMA) has shown impressive objective response rates in patients with advanced multiple myeloma (MM). Clinical grade manufacturing of autologous CAR T-cells has limitations including vein-to-vein delivery time delay and potentially sub-optimal immunological capability of T-cells isolated from patients with advanced disease. Allogeneic CAR T-cell products, whereby cells from healthy third-party donors are used to generate an "off-the-shelf" CAR T-cell product, have the potential to overcome some of these issues. To circumvent the primary potential risk of graft-versus-host disease (GvHD) associated with the use of allogeneic T-cells, abrogation of the T-cell receptor (TCR) expression in the CAR T-cells, via gene editing, is being actively pursued. To avoid the potential safety risks and manufacturing challenges associated with gene editing, the allogeneic CYAD-211 CAR T-cell product exploits short hairpin RNA (shRNA) interference technology to down-regulate TCR expression thus avoiding the risk of life-threatening GvHD. Aim The aim is to generate a BCMA-specific allogeneic CAR T-cell product using a non-gene editing approach and study its activity both in vitro and in vivo. CYAD-211 combines a BCMA-specific CAR with a single optimized shRNA targeting the TCR CD3ζ subunit. Downregulation of CD3ζ impairs the TCR expression on the surface of the donor T-cells, preventing their reactivity with the normal host tissue cells and potential GvHD induction. Maintaining all the elements required for the therapy within a single vector (all-in-one vector) provides some significant manufacturing advantages, as a solitary selection step will isolate cells expressing all the desired traits. Results CYAD-211 cells produce high amounts of interferon-gamma (IFN-γ) during in vitro co-cultures with various BCMA-expressing MM cell lines (i.e., RPMI-8226, OPM-2, U266, and KMS-11). Cytotoxicity experiments confirmed that CYAD-211 efficiently kills MM cell lines in a BCMA-specific manner. The anti-tumor efficacy of CYAD-211 was further confirmed in vivo, in xenograft MM models using the RPMI-8226 and KMS-11 cell lines. Preclinical data also showed no demonstrable evidence of GvHD when CYAD-211 was infused in NSG mice confirming efficient inhibition of TCR-induced activation. Following FDA acceptance of the IND application, IMMUNICY-1, a first-in-human, open-label dose-escalation phase I clinical study evaluating the safety and clinical activity of CYAD-211 for the treatment of relapsed or refractory MM patients to at least two prior MM treatment regimens, is scheduled to begin recruitment. IMMUNICY-1 will evaluate three dose-levels of CYAD-211 (3x107, 1x108 and 3x108 cells/infusion) administered as a single infusion after a non-myeloablative conditioning (cyclophosphamide 300 mg/m²/day and fludarabine 30 mg/m²/day, daily for 3 days) according to a classical Fibonacci 3+3 design. Description of the study design and preliminary safety and clinical data from the first cohort will be presented at ASH 2020. Conclusion CYAD-211 is the first generation of non-gene edited allogeneic CAR T-cell product based on shRNA technology. The IMMUNICY-1 clinical study seeks to provide proof of principle that single shRNA-mediated knockdown can generate fully functional allogeneic CAR T-cells in humans without GvHD-inducing potential. We anticipate that subsequent generations of this technology will incorporate multiple shRNA hairpins within a single vector system. This will enable the production of allogeneic CAR T-cells in which multiple genes of interest are modulated simultaneously thereby providing a platform approach that can underpin the future of this therapeutic modality. Figure 1 Disclosures Al-Homsi: Celyad: Membership on an entity's Board of Directors or advisory committees. Brayer:Janssen: Consultancy; Bristol-Myers Squibb, WindMIL Therapeutics: Research Funding; Bristol-Myers Squibb, Janssen, Amgen: Speakers Bureau. Nishihori:Novartis: Other: Research support to institution; Karyopharm: Other: Research support to institution. Sotiropoulou:Celyad Oncology: Current Employment. Twyffels:Celyad Oncology: Current Employment. Bolsee:Celyad Oncology: Current Employment. Braun:Celyad Oncology: Current Employment. Lonez:Celyad Oncology: Current Employment. Gilham:Celyad Oncology: Current Employment. Flament:Celyad Oncology: Current Employment. Lehmann:Celyad Oncology: Current Employment.

Bypassing the Constraint for Chimeric Antigen Receptor (CAR) Development in T-Cells Expressing the Targeted Antigen: Improvement of Anti-CS1 CAR Activity in Allogenic TCRa/CS1 Double Knockout T-Cells for the Treatment of Multiple Myeloma (MM)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 116-116 ◽  
Author(s):  
Roman Galetto ◽  
Isabelle Chion-Sotinel ◽  
Agnès Gouble ◽  
Julianne Smith
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Mouse Model ◽  
B Cell ◽  
Cd8 T Cells ◽  
Gene Editing ◽  
Transcription Activator ◽  
Car T Cell ◽  
Car T

Abstract Adoptive immunotherapy with autologous T-cells expressing chimeric antigen receptors (CARs) targeting CD19 has achieved long-term remissions in patients with B cell leukemia, pointing out that CAR technology may become a new alternative in cancer treatment. In this work we assessed the feasibility of targeting the CS1 antigen (SLAMF7) for the treatment of Multiple Myeloma (MM). MM is a B-cell neoplasia characterized by clonal expansion of malignant plasma cells in the bone marrow. Even if currently available therapies can improve overall survival, MM still remains incurable in most patients. Immunotherapy against MM is therefore an area in which extensive research is being made, with novel antigenic targets being considered. Among these is the CS1 glycoprotein, which is highly expressed on tumor cells from most patients with MM. However, CS1 is also expressed on normal CD8+ T-cells, which may be problematic for a CAR-based approach as antigen-expressing T cells will be targeted, impacting both the number and the phenotype of the final CAR T cell population. To circumvent this issue we have used our highly-efficient transcription activator-like effector nuclease (TALEN) gene-editing technology to inactivate CS1 in T-cells prior to transduction with a viral vector encoding an anti-CS1 CAR. Our results demonstrate that while non-gene-edited T-cells expressing an anti-CS1 CAR display limited cytolytic activity against MM cell lines, and resulted in a progressive loss of CD8+ T-cells. CS1-gene-edited CAR cells display significantly increased cytotoxic activity, with the percentage of CD8+ T-cells remaining unaffected. In addition, experiments in an orthotopic MM mouse model showed that CS1 disrupted T-cells were able to mediate an in vivo anti-tumoral activity. Subsequently, we have utilized this strategy for CS1 in the context of our allogeneic "off-the-shelf" engineered CAR+ T-cell platform. This allogenic platform utilizes TALEN gene editing technology to inactivate the TCRα constant (TRAC) gene, eliminating their potential to mediate Graft versus Host Disease (GvHD). We have previously shown that editing of the TRAC gene can be achieved at high frequencies, allowing efficient production of TCR-deficient T-cells that no longer mediate alloreactivity in a xeno-GvHD mouse model. Our results also show that multiplex genome editing is possible and can lead to the production of double KO (TRAC and CS1) T-cells, allowing large scale manufacturing of allogeneic, non alloreactive CS1 specific T-cells with enhanced antitumor activity. Moreover, these allogenic T-cells could be easily available for administration to a large number of MM patients. Disclosures Galetto: Cellectis SA: Employment. Chion-Sotinel:Cellectis SA: Employment. Gouble:Cellectis SA: Employment. Smith:Cellectis: Employment, Patents & Royalties.

scholarly journals Genome Editing as a Vehicle to Drive Successful Chimeric Antigen Receptor T Cell Therapies to the Clinic

2021 ◽  
Author(s):  
Caitlin R Hopkins ◽  
Joseph A Fraietta
Keyword(s):  
T Cell ◽  
Genome Editing ◽  
Chimeric Antigen Receptor ◽  
Gene Editing ◽  
Antigen Receptor ◽  
Cell Therapies ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Specific Antigens

Chimeric antigen receptor (CAR) T cells have emerged as an effective therapy for patients with relapsed and refractory haematological malignancies. However, there are many challenges preventing clinical efficacy and thus broader translation of this approach. These hurdles include poor autologous T cell fitness, manufacturing issues and lack of conserved tumour-restricted antigens to target. Recent efforts have been directed toward incorporating genome editing technologies to address these challenges and develop potent CAR T cell therapies for a diverse array of haematopoietic cancers. In this review, the authors discuss gene editing strategies that have been employed to augment CAR T cell fitness, generate allogeneic ‘off-the-shelf’ CAR T cell products, and safely target elusive myeloid and T cell cancers that often lack appropriate tumour-specific antigens.

ATA3219: A Potent Next-Generation Allogeneic Off-the-Shelf CD19-CAR T Therapy without the Need for Gene-Editing

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 9-9
Author(s):  
C Pham ◽  
T Spindler ◽  
E Hwang ◽  
A Brito ◽  
Y Bulliard ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Gene Editing ◽  
Next Generation ◽  
Publicly Traded ◽  
Car T Cells ◽  
Car T ◽  
Signaling Domain

INTRODUCTION The development of allogeneic CD19 chimeric antigen receptor (CAR) T cells from healthy donors is a significant focus in cell therapy and is anticipated to overcome the technical and logistical challenges associated with autologous CAR-T cells. Unlike gene-edited approaches, which require inactivation of the endogenous αβ T cell receptor to reduce the risk of Graft-versus-Host Disease (GvHD), allogeneic Epstein-Barr Virus (EBV)-targeted T cells represent a clinically-advanced treatment modality that, to-date, has demonstrated a favorable safety profile with limited risks of GvHD or cytokine release syndrome [Prockop et al. JCI, 2020; Prockop et al. Blood, 2019] . As an allogeneic CAR T cell platform, EBV T cells represent a unique composition that retains critical transducibility and function, and minimizes risks for GvHD and other host interactions, without requiring complex gene editing or other cell engineering approaches to facilitate use in the allogeneic setting. Recent clinical experience with allogeneic CD19 CAR-modified EBV T cells have further supported safe and effective clinical experience in the context of B cell malignancies [Curran KJ et al. TCT 2020]. Recent advances in next-generation stimulatory domains also represent potential for improvement on current CAR-T therapies. Specifically, a modified CD3ζ domain retaining signaling capacity in 1 of 3 immune-receptor-tyrosine-based-activation-motif (ITAM) regions (referred to as 1XX) is designed to extend functional persistence without compromising potency via calibration of antigen induced CAR signaling intensity to more physiologic levels [Feucht et al. Science Trans Med 2018]. Here, we describe the first preclinical evaluation of ATA3219, a next-generation allogeneic CD19 CAR T cell therapy, combining a non-edited allogeneic EBV T cell approach with a CAR signaling domain designed to improve upon the currently clinically validated CD19 targeted CAR therapies. METHODS and RESULTS We generated EBV T cells engineered with a CD19-targeted CAR containing a modified CD3ζ signaling domain, 1XX (CD19-1XX CAR+ EBV T cells). CD19-1XX CAR+ EBV T cells demonstrate high CAR expression, polyfunctionality, expansion and in vitro potency through HLA-independent killing of CD19+ targets. Furthermore, CD19-1XX CAR+ EBV T cells demonstrate highly potent antitumor activity in an established disseminated tumor model of acute lymphoblastic leukemia and is associated with long-term persistence of the product. No treatment-related toxicities were observed in this animal model. CONCLUSIONS This preclinical dataset for CD19-1XX CAR+ EBV T cells demonstrate, persistence, polyfunctional phenotype and efficient targeting of CD19-expressing tumor cells, both in vitro and in vivo, with limited allocytoxicity against antigen-negative, HLA-mismatched targets. These findings support advancing ATA3219 to clinical evaluation. Disclosures Pham: Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Spindler:Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Hwang:Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Brito:Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Bulliard:Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Aftab:Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company.

scholarly journals 136 ATA3271: An armored, next-generation off-the-shelf, allogeneic, mesothelin-CAR T cell therapy for solid tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A145-A145
Author(s):  
Xianhui Chen ◽  
Jiangyue Liu ◽  
Shuai Yang ◽  
Amogh Oke ◽  
Sarah Davies ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Next Generation ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

BackgroundMesothelin (MSLN) is a GPI-anchored membrane protein with high expression levels in an array of malignancies including mesothelioma and is an attractive target antigen for tumor surface antigen-targeting therapies. Regional administration of autologous, 2nd generation MSLN-targeted CAR-T cells for malignant pleural mesothelioma has shown promise in early clinical evaluation.1 2 More recently, a next-generation MSLN-targeted, autologous CAR T therapy leveraging 1XX CAR signaling and PD1DNR is currently under investigation for advanced mesothelioma [NCT04577326]. Although autologous MSLN CAR-T holds promise, an allogeneic approach may have more widespread application. EBV T-cells represent a unique, non-gene edited approach for allogeneic T-cell therapy. EBV-specific T-cells are currently in a phase 3 trial for EBV-positive post-transplant lymphoproliferative disease [NCT03394365] and, to-date, have demonstrated a favorable safety profile with no evidence for GvHD and cytokine release syndrome attributable to EBV T-cells. Clinical proof-of-principle studies for CAR transduced CD19-targeted allogeneic EBV T-cell therapies have shown acceptable safety and durable response.3 The first preclinical evaluation of ATA3271 was reported last year.4 Here, we describe updated preclinical data for this potential off-the-shelf, allogeneic cell therapy.MethodsWe engineered MSLN CAR+ EBV T-cells (ATA3271) with a novel 1XX signaling domain that is associated with strong effector function and favorable persistence, as well as armored with PD1DNR to provide intrinsic checkpoint blockade.5 Anti-tumor effect of ATA3271 was assessed using a MSTO-211H-derived tumor cell line overexpressing MSLN and PDL1.ResultsUpon MSLN engagement, ATA3271 showed proliferation, efficient tumor cell lysis in the presence of high-level cell-surface PD-L1 expression and secretion of effector cytokines [IL-2, TNF-α, granzyme B]. In a 16-day serial stimulation assay, with PD-L1+ tumor cells added every 2–3 days, ATA3271 expanded 4 to 45-fold without the need for external cytokines, and retained comparable antitumor function as CD3/CD28-stimulated ‘autologous’ CAR-T cells. In an orthotopic mouse model of pleural mesothelioma, ATA3271 demonstrated anti-tumor efficacy without toxicities. Memory markers [CD62L, CCR7] play a key role for T-cell persistence in vivo. We identified donor-to-donor variability in memory marker expression on ATA3271 and optimized our process to maximize their expression. Memory marker expression impact on ATA3271 potency, both in vitro and in vivo, will be presented.ConclusionsOverall, these in vitro and in vivo data show potent anti-tumor activity without evidence of toxicity, suggesting that ATA3271 may be a promising approach for the treatment of MSLN-positive cancers that warrants further clinical investigation.ReferencesAdusumilli Prasad S, 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 Res 2019;79(13 Suppl):Abstract CT036.Adusumilli Prasad S, et al. A phase I trial of regional mesothelin-targeted CAR T-cell therapy in patients with malignant pleural disease, in combination with the anti-PD-1 agent pembrolizumab. Cancer Discov 2021.Curran Kevin J, et al. Durable remission following ‘off-the-shelf’ chimeric antigen receptor (CAR) T-cells in patients with relapse/refractory (R/R) B-cell malignancies. Biol Blood Marrow Transplant 2020;26.3: S89.Liu Jiangyue, et al. 98 ATA3271: an armored, next-generation off-the-shelf, allogeneic, mesothelin-CAR T cell therapy for solid tumors. JITC 2020;8.Feucht Judith, et al. Calibration of CAR activation potential directs alternative T cell fates and therapeutic potency. Nat Med 2019;25.1: 82–88.

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