scholarly journals Cytokine Release Syndrome and Tumor Responses in a First-in-Man Trial of a Novel Affinity-Enhanced TCR-Gene Transduced T Cell Transfer Targeting NY-ESO-1 Antigen

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 841-841
Author(s):  
Shinichi Kageyama ◽  
Mikiya Ishihara ◽  
Shigehisa Kitano ◽  
Yoshihiro Miyahara ◽  
Hidefumi Kato ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Cell Kinetics ◽  
Cell Transfer ◽  
T Cell Therapy ◽  
Car T Cell ◽  
Car T ◽  
T Cell Transfer

Abstract Adoptive cell transfers of receptor gene-engineered T cells include chimeric antigen receptor-gene transduced T (CAR-T) cell therapy and TCR-gene transduced T (TCR-T) cell therapy. In CD19-CAR-T cell therapy, high incidence of cytokine release syndrome (CRS) is associated with in vivo CAR-T cell proliferation and its clinical efficacy. In human TCR-T cell therapies, there have not been well known about CRS and its association with in vivo T cell kinetics or tumor responses. We have been developing a novel-type affinity-enhanced NY-ESO-1-specific TCR, and an original retrovirus vector that encodes siRNA to silence endogenous TCR creation. The NY-ESO-1 TCR is mutated for high affinity with replacements of G50A and A51E in CDR2 region, which is restricted with HLA-A*02:01 and A*02:06. We extensively examined potential cross-reactivities to different antigen-peptides in preclinical studies, and the high-affinity NY-ESO-1 TCR did not recognize analogous peptides. The new generation retroviral TCR-vector provides enhanced expression of transduced tumor-specific TCRs and an inhibition effect of formations of self-reactive TCRs. This is a first-in-man clinical trial of the novel NY-ESO-1-specfic TCR-T cell transfer to evaluate the safety, in vivo cell kinetics and clinical responses. It is designed as a cell-dose escalation from 5 x108 to 5 x109 cells. NY-ESO-1-expressing refractory cancer patients were enrolled, with 3+3 cohort design. Cyclophosphamide with/without fludarabine were administered prior to the TCR-T cell transfer as pre-conditioning. Six patients were treated with the NY-ESO-1 TCR-T cell transfer, and evaluated for the safety and in vivo cell kinetics. The TCR-T cells appeared in peripheral blood with a dose-dependent manner, associated with in vivo proliferation in an early phase. In three patients given 5x108 cells, no toxicities were seen. Two patients receiving 5x109 cells developed early-phase CRS (G2), with elevations of serum IL-6 and IFN-gamma. They were managed the treatment of anti-IL-6 receptor monoclonal antibody, tocilizumab. In a patient who developed CRS, an event of lung injury (G3) occurred, which was associated with marked infiltration of the NY-ESO-1 TCR-T cells. It was successfully treated with steroid. Two synovial sarcoma patients exhibited tumor responses of PRs. In one patient, progression-free survival lasted more than 8 months. In summary, the affinity-enhanced NY-ESO-1 TCR-T cell transfer exhibited CRSs in association with in vivo cell proliferation and sequential tumor responses. Disclosures No relevant conflicts of interest to declare.

scholarly journals IL-15 Serum Concentrations and CD19 CAR T-Cell Therapy: Impact on Clinical Outcomes and In Vivo CAR T Cell Kinetics

2021 ◽  
Vol 27 (3) ◽  
pp. S196-S197
Author(s):  
Jordan Gauthier ◽  
Cassie Chou ◽  
Alexandre V. Hirayama ◽  
Salvatore Fiorenza ◽  
Erik Kimble ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Therapy ◽  
Clinical Outcomes ◽  
Cell Kinetics ◽  
Serum Concentrations ◽  
T Cell Therapy ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

scholarly journals 124 Functionalizing CAR T cells for selective proliferation and dual-targeting using the meditope technology

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A133-A133
Author(s):  
Cheng-Fu Kuo ◽  
Yi-Chiu Kuo ◽  
Miso Park ◽  
Zhen Tong ◽  
Brenda Aguilar ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

BackgroundMeditope is a small cyclic peptide that was identified to bind to cetuximab within the Fab region. The meditope binding site can be grafted onto any Fab framework, creating a platform to uniquely and specifically target monoclonal antibodies. Here we demonstrate that the meditope binding site can be grafted onto chimeric antigen receptors (CARs) and utilized to regulate and extend CAR T cell function. We demonstrate that the platform can be used to overcome key barriers to CAR T cell therapy, including T cell exhaustion and antigen escape.MethodsMeditope-enabled CARs (meCARs) were generated by amino acid substitutions to create binding sites for meditope peptide (meP) within the Fab tumor targeting domain of the CAR. meCAR expression was validated by anti-Fc FITC or meP-Alexa 647 probes. In vitro and in vivo assays were performed and compared to standard scFv CAR T cells. For meCAR T cell proliferation and dual-targeting assays, the meditope peptide (meP) was conjugated to recombinant human IL15 fused to the CD215 sushi domain (meP-IL15:sushi) and anti-CD20 monoclonal antibody rituximab (meP-rituximab).ResultsWe generated meCAR T cells targeting HER2, CD19 and HER1/3 and demonstrate the selective specific binding of the meditope peptide along with potent meCAR T cell effector function. We next demonstrated the utility of a meP-IL15:sushi for enhancing meCAR T cell proliferation in vitro and in vivo. Proliferation and persistence of meCAR T cells was dose dependent, establishing the ability to regulate CAR T cell expansion using the meditope platform. We also demonstrate the ability to redirect meCAR T cells tumor killing using meP-antibody adaptors. As proof-of-concept, meHER2-CAR T cells were redirected to target CD20+ Raji tumors, establishing the potential of the meditope platform to alter the CAR specificity and overcome tumor heterogeneity.ConclusionsOur studies show the utility of the meCAR platform for overcoming key challenges for CAR T cell therapy by specifically regulating CAR T cell functionality. Specifically, the meP-IL15:sushi enhanced meCAR T cell persistence and proliferation following adoptive transfer in vivo and protects against T cell exhaustion. Further, meP-ritiuximab can redirect meCAR T cells to target CD20-tumors, showing the versatility of this platform to address the tumor antigen escape variants. Future studies are focused on conferring additional ‘add-on’ functionalities to meCAR T cells to potentiate the therapeutic effectiveness of CAR T cell therapy.

scholarly journals The roles of T cell competition and stochastic extinction events in chimeric antigen receptor T cell therapy

2021 ◽  
Vol 288 (1947) ◽  
Author(s):  
Gregory J. Kimmel ◽  
Frederick L. Locke ◽  
Philipp M. Altrock
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Chimeric antigen receptor (CAR) T cell therapy is a remarkably effective immunotherapy that relies on in vivo expansion of engineered CAR T cells, after lymphodepletion (LD) by chemotherapy. The quantitative laws underlying this expansion and subsequent tumour eradication remain unknown. We develop a mathematical model of T cell–tumour cell interactions and demonstrate that expansion can be explained by immune reconstitution dynamics after LD and competition among T cells. CAR T cells rapidly grow and engage tumour cells but experience an emerging growth rate disadvantage compared to normal T cells. Since tumour eradication is deterministically unstable in our model, we define cure as a stochastic event, which, even when likely, can occur at variable times. However, we show that variability in timing is largely determined by patient variability. While cure events impacted by these fluctuations occur early and are narrowly distributed, progression events occur late and are more widely distributed in time. We parameterized our model using population-level CAR T cell and tumour data over time and compare our predictions with progression-free survival rates. We find that therapy could be improved by optimizing the tumour-killing rate and the CAR T cells' ability to adapt, as quantified by their carrying capacity. Our tumour extinction model can be leveraged to examine why therapy works in some patients but not others, and to better understand the interplay of deterministic and stochastic effects on outcomes. For example, our model implies that LD before a second CAR T injection is necessary.

scholarly journals Efficient elimination of primary B-ALL cells in vitro and in vivo using a novel 4-1BB-based CAR targeting a membrane-distal CD22 epitope

2020 ◽  
Vol 8 (2) ◽  
pp. e000896
Author(s):  
Talia Velasco-Hernandez ◽  
Samanta Romina Zanetti ◽  
Heleia Roca-Ho ◽  
Francisco Gutierrez-Aguera ◽  
Paolo Petazzi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
T Cell Therapy ◽  
High Affinity ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

BackgroundThere are few therapeutic options available for patients with B-cell acute lymphoblastic leukemia (B-ALL) relapsing as CD19– either after chemotherapy or CD19-targeted immunotherapies. CD22-chimeric antigen receptor (CAR) T cells represent an attractive addition to CD19-CAR T cell therapy because they will target both CD22+CD19– B-ALL relapses and CD19– preleukemic cells. However, the immune escape mechanisms from CD22-CAR T cells, and the potential contribution of the epitope binding of the anti-CD22 single-chain variable fragment (scFv) remain understudied.MethodsHere, we have developed and comprehensively characterized a novel CD22-CAR (clone hCD22.7) targeting a membrane-distal CD22 epitope and tested its cytotoxic effects against B-ALL cells both in in vitro and in vivo assays.ResultsConformational epitope mapping, cross-blocking, and molecular docking assays revealed that the hCD22.7 scFv is a high-affinity binding antibody which specifically binds to the ESTKDGKVP sequence, located in the Ig-like V-type domain, the most distal domain of CD22. We observed efficient killing of B-ALL cells in vitro, although the kinetics were dependent on the level of CD22 expression. Importantly, we show an efficient in vivo control of patients with B-ALL derived xenografts with diverse aggressiveness, coupled to long-term hCD22.7-CAR T cell persistence. Remaining leukemic cells at sacrifice maintained full expression of CD22, ruling out CAR pressure-mediated antigen loss. Finally, the immunogenicity capacity of this hCD22.7-scFv was very similar to that of other CD22 scFv previously used in adoptive T cell therapy.ConclusionsWe report a novel, high-affinity hCD22.7 scFv which targets a membrane-distal epitope of CD22. 4-1BB-based hCD22.7-CAR T cells efficiently eliminate clinically relevant B- CD22high and CD22low ALL primary samples in vitro and in vivo. Our study supports the clinical translation of this hCD22.7-CAR as either single or tandem CD22–CD19-CAR for both naive and anti-CD19-resistant patients with B-ALL.

Quality Assessment of Pre-Clinical Studies of Chimeric Antigen Receptor T-Cell Therapy Products: A Point of Focus On Safety

2021 ◽  
Vol 16 ◽  
Author(s):  
Vikas Maharshi ◽  
Diksha Diksha ◽  
Pooja Gupta
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Chimeric Antigen Receptor ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

Background: Serious adverse reactions have been reported with the use of chimeric antigen receptor (CAR) T-cell therapy in clinical setting despite the success of these products in pre-clinical stages of development. Objective: We evaluated the quality of available pre-clinical safety data of CAR T-cell therapy products. Methods: A 21 items safety-checklist was designed specifically for CAR T-cell. Literature was searched using search/MeSH terms in PubMed (October 2019 – February 2020). Studies were screened from title and abstract. Original pre-clinical researches related to CAR T-cell anti-cancer therapy were included. Results: Of the search results, 152 studies (3 in vivo, 39 in vitro, and 110 combined) were included. Only 7.9% studies were specifically designed to evaluate/ improve product safety. Eleven studies included target antigen(s) and no study included co-stimulatory molecule(s) expressed exclusively by tumor tissue and/or CAR T-cells. One study used CRISPR-Cas9 for CAR gene insertion. The use of switch-off mechanism and purity assessment of CAR T-cell products were reported in 13.2% and 8.6% studies respectively. Of the 149 studies with in vivo component, immuno-competent animal models were used in 24.8%. Measurement of blood pressure, temperature, body weight and serum cytokines were reported in 0, 2.7, 29.2 and 27.4% studies respectively. The tissue distribution and CAR T-cells persistence were reported in 26.5% studies. Conclusion: Majority of the checklist parameters were not reported in the pre-clinical publications to be adequately predictive of the safety of CAR T-cells in a clinical setting.

scholarly journals PTK7-Targeting CAR T-Cells for the Treatment of Lung Cancer and Other Malignancies

2021 ◽  
Vol 12 ◽  
Author(s):  
Yamin Jie ◽  
Guijun Liu ◽  
Lina Feng ◽  
Ying Li ◽  
Mingyan E ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
T Cell Therapy ◽  
Lung Cancers ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

In spite of impressive success in treating hematologic malignancies, adoptive therapy with chimeric antigen receptor modified T cells (CAR T) has not yet been effective in solid tumors, where identification of suitable tumor-specific antigens remains a major obstacle for CAR T-cell therapy due to the “on target off tumor” toxicity. Protein tyrosine kinase 7 (PTK7) is a member of the Wnt-related pseudokinases and identified as a highly expressed antigen enriched in cancer stem cells (CSCs) from multiple solid tumors, including but not limited to triple-negative breast cancer, non-small-cell lung cancer, and ovarian cancer, suggesting it may serve as a promising tumor-specific target for CAR T-cell therapy. In this study, we constructed three different PTK7-specific CAR (PTK7-CAR1/2/3), each comprising a humanized PTK7-specific single-chain variable fragment (scFv), hinge and transmembrane (TM) regions of the human CD8α molecule, 4-1BB intracellular co-stimulatory domain (BB-ICD), and CD3ζ intracellular domain (CD3ζ-ICD) sequence, and then prepared the CAR T cells by lentivirus-mediated transduction of human activated T cells accordingly, and we sequentially evaluated their antigen-specific recognition and killing activity in vitro and in vivo. T cells transduced with all three PTK7-CAR candidates exhibited antigen-specific cytokine production and potent cytotoxicity against naturally expressing PTK7-positive tumor cells of multiple cancer types without mediating cytotoxicity of a panel of normal primary human cells; meanwhile, in vitro recursive cytotoxicity assays demonstrated that only PTK7-CAR2 modified T cells retained effective through multiple rounds of tumor challenge. Using in vivo xenograft models of lung cancers with different expression levels of PTK7, systemic delivery of PTK7-CAR2 modified T cells significantly prevented tumor growth and prolonged overall survival of mice. Altogether, our results support PTK7 as a therapeutic target suitable for CAR T-cell therapy that could be applied for lung cancers and many other solid cancers with PTK7 overexpression.

PTK7-Targeting CAR T-cells for the Treatment of Lung Cancer and other Malignancies

2020 ◽  
Author(s):  
Yamin Jie ◽  
Guijun Liu ◽  
Lina Feng ◽  
Ying Li ◽  
Mingyan E ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
T Cell Therapy ◽  
Lung Cancers ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

Abstract Background: In spite of impressive success in treating hematologic malignancies, adoptive therapy with chimeric antigen receptor modified T cells (CAR T) has not yet been effective in solid tumors, where identification of suitable tumor-specific antigens remains a major obstacle for CAR T-cell therapy due to the “on target off tumor” toxicity. Protein tyrosine kinase 7 (PTK7) is a member of the Wnt-related pseudokinases and identified as a highly expressed antigen enriched in cancer stem cells (CSCs) from multiple solid tumors, including but not limited to triple-negative breast cancer, non-small cell lung cancer, and ovarian cancer, suggesting it may serve as a promising tumor-specific target for CAR T-cell therapy. Methods: In this study, we constructed 3 different PTK7-specific CAR (PTK7-CAR1/2/3) each comprising a humanized PTK7-specific single chain variable fragment (scFv), hinge and transmembrane (TM) regions of the human CD8α molecule, 4-1BB intracellular co-stimulatory domain (BB-ICD), and CD3ζ intracellular domain (CD3ζ-ICD) sequence, and then prepared the CAR T cells by lentivirus mediated transduction of human activated T cells accordingly, and sequentially evaluated their antigen-specific recognition and killing activity in vitro and in vivo.Results: T cells transduced with all 3 PTK7-CAR candidates exhibited antigen-specific cytokine production and potent cytotoxicity against naturally expressing PTK7-positive tumor cells of multiple cancer types without mediating cytotoxicity of a panel of normal primary human cells; meanwhile, in vitro recursive cytotoxicity assays demonstrated that only PTK7-CAR2 modified T cells retained effective through multiple rounds of tumor challenge. Using in vivo xenograft models of lung cancers with different expression level of PTK7, systemic delivery of PTK7-CAR2 modified T cells significantly prevented tumor growth and prolonged overall survival of mice. Conclusion: Altogether, our results support PTK7 as a therapeutic target suitable for CAR T-cell therapy that could be applied for lung cancers and many other solid cancers with PTK7 overexpression.

scholarly journals Development of Target Modules for Early and Late Stage Cancer Treatment Using Switchable Unicar T Cell Therapy

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5613-5613
Author(s):  
Liliana Raquel Loureiro ◽  
Anja Feldmann ◽  
Ralf Bergmann ◽  
Nicole Berndt ◽  
Stefanie Koristka ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Side Effects ◽  
Cell Therapy ◽  
Tumor Burden ◽  
Tumor Site ◽  
T Cell Therapy ◽  
Car T Cell ◽  
Car T

The clinical efficacy of CAR T cell therapies has been widely recognized, particularly in the treatment of hematologic malignancies. Nevertheless, CAR T cells also have the capability to elicit undesired effects such as on-target/off-tumor recognition and cytokine release syndrome. To increase clinical safety of CAR T cell therapy, a novel modular universal CAR platform termed UniCAR was developed by our group. In the UniCAR system, antigen-binding specificity and signaling features are two distinct moieties, in which the antigen specificity is provided by targeting modules (TMs) to redirect UniCAR T cells in an individualized time- and target-dependent manner. In this way, UniCAR T-cells acquire killing potential only in the presence of a tumour-specific TM. Given the reduced size of such molecules, they are rapidly eliminated and therefore, need to be continuously infused. Thus, possible side effects and activation of UniCAR T cells can be easily monitored and controlled by TM dosing. During the onset of therapy, tumor burden and the risk for severe side effects are high and regulation of CAR T cell activity is particularly important at this stage. For this reason, TMs with extended half-life may play an important role by improving eradication of residual tumor cells in late phases of treatment and further expedite clinical application. In this line of thought, a set of novel short-lived and longer-lasting TMs directed against several tumor-associated antigens was developed. Short-lived TMs are composed of a tumor-specific binding moiety fused to the La peptide epitope (E5B9) which is recognized by UniCAR T cells. In order to generate extended half-life TMs, these two components are fused via an Fc domain derived from the human IgG4 molecule. In vitro and in vivo assays have shown that both short-lived and longer-lasting TMs efficiently redirect UniCAR T cells to cancer cells in a highly target-specific manner, thereby promoting the secretion of pro-inflammatory cytokines and tumor cell lysis. Further assays using PET-imaging, demonstrated that all TM formats specifically enriched at the tumor site presenting either short or prolonged serum half-lives. From a clinical point of view, after the initial reduction of tumor burden promoted by the small TMs, IgG4-based TMs could be subsequently administrated allowing a more convenient and personalized treatment of the patients avoiding the continuous infusion of the short-lived TMs. Furthermore, the specific accumulation of such IgG4-based TMs at the tumor site sets these molecules as attractive candidates for in vivo imaging and endoradiotherapy. Taken together, combination of switchable UniCAR T cells and TMs with different sizes, specificities and half-lives represent a flexible and individualized approach at different stages of cancer treatment. Disclosures Koristka: Intellia Therapeutics: Employment. Bachmann:GEMoaB Monoclonals: Equity Ownership, Patents & Royalties.

scholarly journals Targeting hypoxia downstream signaling protein, CAIX, for CAR T-cell therapy against glioblastoma

2019 ◽  
Vol 21 (11) ◽  
pp. 1436-1446 ◽  
Author(s):  
Jing Cui ◽  
Qi Zhang ◽  
Qi Song ◽  
Herui Wang ◽  
Pauline Dmitriev ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Intratumoral Injection ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

Abstract Background Glioblastoma survival remains unchanged despite continuing therapeutic innovation. Herein, we aim to (i) develop chimeric antigen receptor (CAR) T cells with a specificity to a unique antigen, carbonic anhydrase IX (CAIX), which is expressed in the hypoxic microenvironment characteristic of glioblastoma, and (ii) demonstrate its efficacy with limited off-target effects. Methods First we demonstrated expression of CAIX in patient-derived glioblastoma samples and available databases. CAR T cells were generated against CAIX and efficacy was assessed in 4 glioblastoma cell lines and 2 glioblastoma stem cell lines. Cytotoxicity of anti-CAIX CAR T cells was assessed via interferon gamma, tumor necrosis factor alpha, and interleukin-2 levels when co-cultured with tumor cells. Finally, we assessed efficacy of direct intratumoral injection of the anti-CAIX CAR T cells on an in vivo xenograft mouse model using the U251 luciferase cell line. Tumor infiltrating lymphocyte analyses were performed. Results We confirm that CAIX is highly expressed in glioblastoma from patients. We demonstrate that CAIX is a suitable target for CAR T-cell therapy using anti-CAIX CAR T cells against glioblastoma in vitro and in vivo. In our mouse model, a 20% cure rate was observed without detectable systemic effects. Conclusions By establishing the specificity of CAIX under hypoxic conditions in glioblastoma and highlighting its efficacy as a target for CAR T-cell therapy, our data suggest that anti-CAIX CAR T may be a promising strategy to treat glioblastoma. Direct intratumoral injection increases anti-CAIX CAR T-cell potency while limiting its off-target effects.

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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