CAR-T Cell Expansion Research Articles

PB2137: INFLUENCE OF PREVIOUS RADIOTHERAPY ON THE EFFECT OF CART CELL THERAPY IN AGGRESSIVE B-CELL LYMPHOMA

Background: In previous studies, radiotherapy has an effect on the number and function of lymphocytes. In recent years, with the advent of CART cell therapy, the PFS and OS of relapsed and refractory highly aggressive B-cell lymphoma have been significantly improved. Whether previous radiotherapy is effective for CART cell therapy The impact is inconclusive. Aims: To investigate the influence of the radiotherapy history before CART cell therapy on the culture, expansion and treatment effect of CART cells in patients with aggressive B-cell lymphoma. Methods: A retrospective study from December 2018 to September 2021 in adult patients with aggressive B-cell lymphoma treated with CART cells at our center who underwent radiotherapy before lymphocyte collection. The patient’s previous radiotherapy dose, radiotherapy distance from lymphocyte collection time, peripheral blood lymphocyte count before lymphocyte collection, CART cell culture dose, reinfusion dose, and curative effect statistics of 3 months after CART cell reinfusion were counted. Statistical methods such as logistic regression and spearman correlation analysis were used for analysis. Results: 16 males and 18 females were eligible, with a median age of 45.5 years (18-86 years). These included 2 Burkitt lymphoma, 26 unspecified diffuse large B-cell lymphoma, 5 primary mediastinal B-cell lymphoma, and 1 primary central lymphoma. The median time from lymphocyte collection to previous radiotherapy was 151 (5-4772) days, and the median radiation dose was 46 (4-66) Gy. The absolute value of lymphocytes before the collection of lymphocytes was 0.79 (0.14-2.58)×10^9/L, which was lower than the normal reference value. The median culture dose of CART cells was 0.837(0.1-7.03)×10^6/kg, and the median infusion dose was 0.837(0.1-4.2)×10^6/kg. After CART cell infusion, the peak CART cell expansion was 18.6(0-919)×10^6/kg, 3-month CR: 20.59%, 3-month ORR: 44.12%. The time interval between previous radiotherapy and lymphocyte collection had a significant effect on 3-month CR, P=0.0417. The effect on 3-month ORR was not statistically significant, P=0.1388. There was no significant effect of previous radiotherapy dose on CR/ORR at 3 months, with P values ​​of 0.9188/0.5078, respectively. The effect of previous radiotherapy dose on the CART cell culture dose and the maximum value of CART cell expansion was not found to be statistically significant. CR and ORR at 3 months after reinfusion were significantly correlated with the reinfusion dose, with P values ​​of 0.0102 & 0.0121, respectively. There was no significant relationship between the absolute value of lymphocytes and the proportion of lymphocytes, the culture dose and the CR/ORR of the curative effect. Summary/Conclusion: Radiotherapy affects the number and cell quality of lymphocytes, the median culture dose is significantly lower than the 2×10^6/kg reported in the literature. In patients with aggressive B-cell lymphoma who had a history of radiotherapy, the CR/ORR at 3 months after CART cell therapy was significantly lower than that reported. This adverse effect was independent of radiation dose. The CR rate is affected by the time interval between radiotherapy and lymphocyte collection. The longer the interval, the greater the adverse effect.

P1446: PHASE I STUDY DATA UPDATE OF PHE885, A FULLY HUMAN BCMA-DIRECTED CAR-T CELL THERAPY MANUFACTURED USING THE T-CHARGETM PLATFORM FOR PATIENTS WITH RELAPSED/REFRACTORY (R/R) MULTIPLE MYELOMA (MM)

Background: Chimeric antigen receptor (CAR)-T cell therapy (tx) is approved for treatment of r/r MM. However, prolonged product manufacturing times and a need for longer duration of response are a major obstacle in this population, necessitating fast manufacture of a reliable and durable CAR-T cell product. Aims: Herein we report updated clinical data from a Phase I trial assessing the fully human B-cell maturation antigen (BCMA) CAR-T cell product PHE885 in adult patients (pts) with r/r MM. Methods: This multicenter, open-label study (NCT04318327) characterizes the safety and efficacy of PHE885 manufactured using the novel T-ChargeTM platform, which takes <2 d to manufacture the final product and relies on in vivo expansion after CAR-T cell infusion. Pts with MM r/r to ≥2 prior lines of tx, including an immunomodulatory drug, proteasome inhibitor, and anti-CD38 monoclonal antibody, were eligible. Pts received fludarabine and cyclophosphamide for lymphodepletion prior to a single PHE885 intravenous injection. Primary objectives were safety, including dose-limiting toxicities (DLTs) and adverse events (AEs). Secondary objectives included clinical response, T-ChargeTM process evaluation, and cellular kinetics. Results: At abstract submission, 22 pts were infused with PHE885 at the following fixed doses: 2.5×106 (n=4), 5×106 (n=12), 10×106 (n=5), and 14.3×106 (n=1) CAR-T cells; 15 pts (4 at 2.5×106, 10 at 5×106, and 1 at 14.3×106) were evaluable for safety and efficacy at the data cutoff for the abstract (October 13, 2021). Longer follow up of these 15 pts and data from additional pts treated at doses of 5×106 and 10×106 will be presented at the annual meeting. Of the 15 pts evaluable at data cutoff, DLTs were reported in 3 pts and included asymptomatic grade (gr) 3 transaminitis (n=1, 14.3×106), asymptomatic gr ≥3 elevated pancreatic enzymes (n=1, 5×106), and gr 4 neutropenia (n=1, 2.5×106). Most frequent tx-related gr ≥3 AEs included neutropenia (n=15, 100%), anemia (n=13, 87%), and thrombocytopenia (n=11, 73%). All pts experienced cytokine release syndrome (CRS) per Lee 2014 criteria; 2 had gr ≥3 CRS (n=1, 5×106; n=1, 14.3×106). Median times to CRS onset and resolution were 7 d (range, 4-12 d) and 11 d (range, 9-22 d), respectively. Tocilizumab (n=13, 87%), steroids (n=11, 73%), anakinra (n=4, 27%), and pressors (n=3, 20%) were used to manage CRS. Gr 2 neurotoxicity occurred in 4 pts (27%). Pts had an overall response rate of 93%, stringent complete response in 33%, very good partial response (VGPR) in 20%, and PR in 40%. Of evaluable pts at 3 mo post infusion, 43% (3/7) had minimal residual disease (MRD) at <10–5 sensitivity[JC(1], while 33% (2/6) were MRD negative at <10–6 by next-generation sequencing. Robust expansion of BCMA CAR-T cells was observed in all pts via qPCR and flow cytometry; maximum expansion (geometric mean Cmax) was 316,000 copies/μg by qPCR and 76% of circulating T cells by flow cytometry. Maximum expansion was reached at a median of 14.2 d by qPCR (13.9 d by flow cytometry). Naive-like T cells present in the leukapheresis were preserved during manufacturing of most PHE885 products as measured by flow cytometry, and an increase in naive/Tscm populations is observed in ≥VGPR pts post-infusion. Summary/Conclusion: Low doses of PHE885 manufactured by T-ChargeTM in <2 d have a manageable safety profile and encouraging clinical activity in pts with r/r MM. PHE885 expands rapidly in vivo, persists for prolonged periods, and demonstrates a relatively naive T-cell phenotype.

S263: PHASE I OPEN-LABEL SINGLE ARM STUDY OF GPRC5D CAR-T CELLS (ORICAR-017) IN PATIENTS WITH RELAPTSED/REFRACTORY MULTIPLE MYELOMA (POLARIS)

Background: G-protein–coupled receptor class 5-member D (GPRC5D), a type-C 7-pass transmembrane receptor protein, is predominantly expressed on malignant plasma cell phenotype, including most malignant plasma cells from pts with multiple myeloma (MM). The autologous GPRC5D-directed CAR-T cell (OriCAR-017), owning to an additional proprietary Ori element, is the standard second generation CAR-T cell with improvement in expansion and durability of CAR-T cells post-transfusion. Here we report initial results from the phase I open-label single arm study of GPRC5D CAR-T Cells (OriCAR-017) in pts with Relapsed/Refractory (R/R) MM (NCT05016778). Aims: The primary objective in the dose escalation phase was to evaluate safety and tolerability of OriCAR-017. Secondary endpoints included efficacy and pharmacokinetics (expansion and persistence of OriCAR-017. Methods: Key enrolment criteria included adults with measurable MM, R/R or intolerant to established MM therapies, prior BCMA-targeted therapy allowed. Patient received lymphodepleting chemotherapy with Fludarabine 30mg/m2 daily and cyclophosphamide 300mg/m2 daily for 3 days followed by a single infusion of OriCAR-017. The trial followed a standard 3 + 3 design with the following dose cohorts: 1×106/kg, 3×106/kg and 6×106/kg CAR+ T cells. Results: Eleven pts with R/R MM were enrolled and underwent apheresis during June 9, 2021 and February 25, 2022. Nine of them have completed OriCAR-017 infusion to date. 2 pts were suspended infusion due to rapid disease progression. Of the 9 pts infused, median age of 65 years (range: 41-71) and a median of 6 (range 3-17) prior lines of therapy; 4 (44.4%) prior BCMA CART therapy. 3 (33.3%) pts with extramedullary plasmacytoma ≥ 1 and 7 (77.8%) pts with bone marrow plasma cells ≥60% at baseline. Five out of 6 pts had high-risk cytogenetic profiles, including 2 pts with del(17p). No dose-limiting toxicities occurred. The most common treatment-related AEs were hematological toxicities. All pts experienced Cytokine Release Syndrome (CRS, with 8(88.9%) pts in G1, 1 (11.1%) patient in G2), no G3/4 CRS was observed. All CRS cases were rapidly relieved after conventional CRS intervention, including tocilizumab and steroids. No neurologic toxicities were reported to date. All 9pts median follow up time was 118 days (range33—220). A 100.0% ORR were observed with 3 (33.3%) pts achieved CR/sCR, 3 pts (33.3%) were VGPR, 3 pts were PR (33.3%). 4 pts relapsed from BCMA CAR-T therapy had responses with 1 sCR, 2 VGPR, 1 PR. All 9 pts were Minimal Residual Disease (MRD) negative in the bone marrow by flow cytometry (sensitivity: 10-5) at day 28 after infusion, and 6pts continued MRD negative at month 3 and 2 pts at month 6 after infusion. Robust OriCAR-017 expansion in peripheral blood by using qPCR in 3 dose cohorts, median peak was 349866.1copies/ml (range 60837.5 – 2240358.5), median time to peak expansion was 10 days (range 7-14). 8 out of 9 patients detected CAR + cell in bone marrow at D28 after infusion with median 498048.3copies/ml (range 495.4- 2358538.6). Image:Summary/Conclusion: In this phase I study, OriCAR-017 was safe and showed impressive efficacy among previously heavily treated R/RMM pts. Majority of AEs were transient, manageable, and reversible. 100% ORR and 100% MRD negative rate, along with favorable safety evidenced support OriCAR-017 could be a competitive therapy for pts with R/RMM. Furthermore, pts who had relapsed from BCMA CAR-T therapy may still benefit from OriCAR-017 treatment.

S185: CARTITUDE-2 COHORT B: UPDATED CLINICAL DATA AND BIOLOGICAL CORRELATIVE ANALYSES OF CILTACABTAGENE AUTOLEUCEL IN PATIENTS WITH MULTIPLE MYELOMA AND EARLY RELAPSE AFTER INITIAL THERAPY

Background: CARTITUDE-2 (NCT04133636) is a phase 2, multicohort study evaluating the efficacy and safety of the chimeric antigen receptor (CAR) T-cell therapy ciltacabtagene autoleucel (cilta-cel) in patients (pts) with multiple myeloma (MM). Cohort B enrolled pts who had early relapse after initial therapy. These pts have functionally high-risk disease, as early relapse following autologous stem cell transplantation (ASCT) is associated with poor prognosis, representing an unmet medical need. Aims: To present updated results from CARTITUDE-2 cohort B. Methods: All pts provided informed consent. Pts had MM, received 1 prior line of therapy (proteasome inhibitor and immunomodulatory drug required), had disease progression per International Myeloma Working Group criteria (either ≤12 months after ASCT or ≤12 months after initiation of anti-myeloma therapy for pts not treated with ASCT), and were naive to CAR-T or other anti-B-cell maturation antigen therapies. After lymphodepletion, a single cilta-cel infusion was administered at a target dose of 0.75×106 CAR+ viable T cells/kg. Efficacy and safety were evaluated. The primary endpoint was minimal residual disease (MRD) negativity at 10-5. Patient management strategies were used to minimize risk of movement and neurocognitive adverse events (MNTs). Pharmacokinetic (PK) analyses, including Cmax and Tmax of CAR+ T-cell transgene levels in blood are being performed. Additional assessments include levels of cytokine release syndrome (CRS)-related cytokines (eg, IL-6) over time, peak levels of cytokines by response and CRS, association of cytokine levels with immune effector cell-associated neurotoxicity syndrome (ICANS), and CAR+ T cell CD4/CD8 ratio by response, CRS, and ICANS. Results: 19 pts (median age: 58.0 years [range: 44-67]; 74% male) received cilta-cel as of January 2022; 79% received prior ASCT. Median follow-up was 13.4 months (range: 5.2-21.7). Overall response rate was 100.0%, with 90% of pts achieving ≥complete response, and 95% achieving ≥very good partial response. Median time to first response was 0.95 months (range: 0.9-9.7) and median time to best response was 5.1 months (range: 0.9-11.8). Among 15 MRD-evaluable pts, 14 (93%) achieved MRD 10-5 negativity. Median duration of response was not reached. The 12-month progression-free survival rate was 90%; 12-month event-free rate was 88.9%. CRS occurred in 16 (84.2%) pts (1 grade 4); median time to onset was 8 days (range: 5-11) and all events resolved. ICANS (grade 1) and MNT (grade 3, previously reported) occurred in 1 pt each. 1 pt died post cilta-cel (progressive disease, day 158). Preliminary PK data showed peak expansion of CAR-T cells on day 13.1 (range: 8.96-209.9); median persistence was 76.9 days (range: 40.99-221.8). Summary/Conclusion: This functionally high-risk pt population with early relapse after initial therapy experienced deep and durable responses with manageable safety following a single cilta-cel infusion. Cilta-cel led to responses in pts with ineffective or insufficient response to ASCT. Follow-up is ongoing and responses continue to deepen. We will present updated and detailed PK, cytokine, and CAR-T subset analyses as well as clinical correlation to provide insight into biological correlates of efficacy and safety in these pts.

Abstract 2838: Antigen-independent expansion enhances efficacy of CAR-T cells against solid tumor

Abstract One key hurdle for the CAR-T cell treatment of solid tumors is the limited accessibility of solid tumor antigens outside the tumor microenvironment, which prohibits the expansion of solid tumor-targeting CAR-T cells in patients. Here, we report the characterization of prostatic acid phosphatase (PAP) as a feasible CAR-T target for prostate cancer and a novel approach, named CoupledCAR, to expand solid tumor-targeting CAR-T cells lacking solid tumor antigens based on the observation of non-transduced T cells proliferating together with CD19 CAR-T cells during the treatment of acute lymphocyte leukemia. We demonstrated that CoupledCAR can significantly enhance the expansion and antitumor efficacy of PAP CAR-T cells both in vitro and in vivo. Furthermore, we showed that the expansion of solid tumor-targeting CAR-T cells does not depend on CAR/CD3ζ stimulation through direct antigen binding with CAR but enhances the memory status of CAR-T cells and causes little exhaustion. Since the CoupledCAR system does not rely on solid tumor antigens, we propose that it can be utilized in all CAR-T and T cell therapies for the treatment of solid tumors. Citation Format: Zhiyuan Cao, Chengfei Pu, Xianyang Jiang, Guiting Han, Yuzhe Peng, Wensheng Wang, Wei Ding, Xiaogang Shen, Dongqi chen, Beibei Jia, Xiaoqiang Xu, Zhipeng Huang, Xi Huang, Wenbi Liu, Ruihong Zhu, Lee Tian, Christopher Ballas, Victor.X Lu, Zhao Wu, Lei Xiao. Antigen-independent expansion enhances efficacy of CAR-T cells against solid tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2838.

Abstract 3603: Reverse fate mapping of CD19-targeted CAR T cells in patients with large B-cell lymphoma

Abstract Autologous T cells genetically engineered to express a chimeric antigen receptor targeting CD19 (CD19-CAR) have achieved high complete response rates in patients with hematologic malignancies, but &amp;gt;50% of patients progress following therapy. Here, we sought to understand key T-cell intrinsic factors impacting efficacy, namely CAR T-cell expansion, persistence, and homing to the tumor. Using an approach called reverse fate mapping, we followed individual T-cell clones at the single-cell level from pre-manufacture apheresis to the infusion product, tumor-involved lymph node, and blood at peak and late expansion in 12 adult patients with relapsed or refractory large B-cell lymphoma treated with axicabtagene ciloleucel, an FDA-approved CD19-CAR T-cell immunotherapy. The resulting CAR T-cell atlas comprises matched transcriptome (scRNA-seq) and surface protein expression (CITE-seq) for 322,028 cells from 44 samples, with 119,397 unique T-cell receptor (TCR) clonotypes identified. This atlas enabled us to ask questions like: “What were the phenotypes of the most successful CAR T-cell clones at the time of infusion or pre-manufacture apheresis?” We found that T-cell clonotypes with juvenile features at apheresis, including IL7R expression, were the most successful at expansion to higher frequencies in the infusion product, while clones with effector gene expression programs, such as those encoding perforin and granzymes, contracted between apheresis and product. Conversely, it was GZMK-expressing T cells in pre-manufacture apheresis that were dominant in the tumor early following CAR T-cell infusion. Further, T-cell clonotypes with active effector programs at infusion dominated at peak expansion. Finally, we defined active expression modules and pathways in the infusion product for CAR T-cell clones that homed to the tumor or became dominant at late expansion. These analyses pinpoint the molecular mechanisms that could be modulated to rationally steer CAR T-cell differentiation trajectories at the genetic or pharmacological level. This work was supported in part by the Parker Institute for Cancer Immunotherapy, California Institute for Regenerative Medicine, Kite Pharma, and Stanford Cancer Institute. Citation Format: Zinaida Good, Mark P. Hamilton, Jay Y. Spiegel, Sreevidya Kurra, Moksha Desai, Snehit Prabhu, Eric Yang, Michael G. Ozawa, Paul J. Hanson, Fang Wu, Matthew J. Frank, John H. Baird, Lori Muffly, Gursharan K. Claire, Juliana Craig, Katherine A. Kong, Dhananjay Wagh, John Coller, Sylvia K. Plevritis, Bita Sahaf, David B. Miklos, Crystal L. Mackall. Reverse fate mapping of CD19-targeted CAR T cells in patients with large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3603.

Abstract 561: CAR-targeted IL-2 drives selective CAR-T cell expansion and improves anti-tumor efficacy

Abstract Chimeric antigen receptor T cell (CAR-T) therapies have transformed the treatment of some hematological malignancies and are showing promising preliminary results in solid tumors. Recent studies have shown that in vivo expansion and persistence of CAR-Ts are correlated with improved therapeutic outcomes in patients. Administration of IL-2 enhances CAR-T engraftment, persistence, and functionality in preclinical models. However, the clinical potential of IL-2 stimulation with cell therapies is limited using current molecules due to severe toxicity of high-dose IL-2 and the inadequate selectivity of existing engineered IL-2 variants which expand multiple endogenous cells in addition to CAR-T cells. To address this challenge, we have applied our cis-targeting technology to develop CAR-T selective IL-2 fusion molecules that specifically activate CAR-Ts, while exhibiting minimal activity on CAR-negative cells. Cis-targeted IL-2 fusions are comprised of a fusion between a targeting antibody and an IL-2 mutein having attenuated binding to IL2R⍺ and IL2Rβ. The attenuated cytokine selectively binds and activates IL-2 receptors on CAR-Ts via the avidity provided by the targeting arm. We engineered two cis-targeted CAR-T selective IL-2 fusions, CAR-IL2 or EGFRt-IL2. CAR-IL2 targets the FMC63 CAR directly without blocking CD19 antigen recognition, enabling targeting of approved anti-CD19 CAR-T products, and EGFRt-IL2 targets the EGFRt tag co-expressed with the CAR. Molecules were characterized in vitro using primary human CAR-transduced T cells. The in vivo activity was examined using tumor bearing NSG mice infused with human CAR-Ts. The specificity of the CAR-IL2 or EGFRt-IL2 molecules was demonstrated by their ability to selectively induce pSTAT5 signaling resulting in &amp;gt;1000-fold preferential STAT5 activity in CAR-expressing cells compared to CAR-negative cells. The ability of the fusion proteins to selectively expand CAR-Ts in vivo was shown through a substantial and specific expansion of the infused CAR-Ts. The CAR-T fraction increased from approximately 50% of infused T cells, to over 93% of T cells, demonstrating a nearly 40-fold expansion in vivo. Re-expansion of CAR-Ts was demonstrated after allowing the CAR-Ts to rest in vivo &amp;gt;100 days; re-dosing resulted in significant and selective re-expansion of CAR-T cells. Furthermore, treatment with the CAR-IL2 molecule enhanced tumor regression of CD19+ lymphoma-bearing mice infused with suboptimal doses of CAR-Ts. Together, these data demonstrate that our cis-targeted IL-2 molecules selectively activate CAR-Ts in vitro and enhance CAR-T expansion and anti-tumor efficacy in vivo. Cis-targeted IL-2 fusion molecules directed by anti-CAR or anti-tag antibodies represent a promising approach to confer enhanced CAR-T activity in a specific and temporally controlled manner. Citation Format: Nathan D. Mathewson, Kelly D. Moynihan, Sara Sleiman, Wei Chen, Paul Bessette, Christopher Kimberlin, Eric Wigton, Danielle Pappas, Terrence Park, Ton N. Schumacher, Saar I. Gill, Yik Andy Yeung, Ivana Djuretic. CAR-targeted IL-2 drives selective CAR-T cell expansion and improves anti-tumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 561.

Abstract CT196: Early results of a safety and efficacy study of allogeneic TruUCAR™ GC502 in patients with relapsed/refractory B-cell acute lymphoblastic leukemia (r/r B-ALL)

Abstract Introduction: CD19 targeted autologous CAR-T therapies have been approved for the treatment of r/r B-ALL and greatly improved outcome. However, some patients may not be eligible to receive autologous CAR-T. TruUCAR™ GC502 is an allogeneic, universal CAR-T product with CD19/CD7 dual directed CAR. Preclinical data of GC502 were reported at ASH 2021 (Abstract 148500). Here, we report early clinical results from a phase I open-label, non-randomized, prospective investigator initiate trial (IIT) of GC502 in r/r B-ALL patients. Methods: GC502 is manufactured using leukopaks from HLA-unmatched healthy donors. It contains a 4-1BB based CD19/CD7 dual directing CAR, a T cell enhancer, and genetically disrupted TRAC and CD7 loci to avoid GvHD and fratricide. Patients (pts) with r/r B-ALL were enrolled and treated with one of two different formulations (A or B) in escalating dose levels ranging from 1.0x107 (DL1) to 1.5x107 (DL2) cells/kg. Prior to infusion of GC502, pts received a Flu/Cy based lymphodepletion regimen. Adverse events, disease response and expansion kinetics were evaluated in this study. Results: To date, 4 pts (15-34 yrs) were enrolled from Sep. 2021 to Jan. 2022. All patients were heavily pretreated, and had received either autologous or donor derived CD19 or CD19-CD22 targeted CAR-T therapy. Baseline marrow blast levels ranged from 19.5% to 92% (median 48.1%). 1 pt had extramedullary involvement. At data cut-off (Jan. 28, 2022), all patients had received a single dose of GC502: 1 pt at DL 1 1.0x107cells/kg and 3 patients at DL 2 1.5x107cells/kg. At day 28 post CAR-T infusion, 2 out of 3 response evaluable patients had achieved CR/CRi; 1 pt achieved PR at month 1 and subsequently received HSCT on day 39. TEAEs presented as Gr 3 febrile neutropenia (2/3), Gr 4 thrombocytopenia (1/3) and Gr 3 anemia (3/3). All TEAE resolved after treatment with SOC. Non-hematological TEAE presented as Gr≤3 γ-GT increase (3/3), Gr≤3 AST increase (2/3) and Gr≤3 ALT increase (3/3). CRS presented as Gr 2 in 2 patients with formulation B. 2 pts received formulation A and experienced Gr 3 CRS with a duration of 7 and 10 days respectively (CRS was graded according ASTCT Consensus Grading). CRS in all pts was manageable and resolved after treatment with Ruxolitinib, SOC and supportive care. No ICANS or aGvHD were observed. The pt treated in DL 1 did not show adequate GC502 cellular expansion. Peak expansions of GC502 in peripheral blood were observed between week 1-2 in DL 2. Median peak CAR copies were 149,945 copies/ug DNA (range 10,849-195,400). Conclusions: TruUCAR™ GC502 demonstrated promising early results with a manageable safety profile. Robust CAR-T cell expansion was observed in DL2 at 1.5x107cells/kg in heavily pretreated r/r B-ALL patients, including those previously treated with CD19 CAR-T therapies. The study is ongoing and continues accruing patients. Citation Format: Shiqi Li, Xinxin Wang, Zhongtao Yuan, Lin Liu, Yu Li, Jia Liu, Jiaping He, Zhimin Li, Wei Zhao, Jianning Ge, Yajin Ni, Lianjun Shen, Wei Cao, Xi Zhang, Martina Sersch, Sanbin Wang. Early results of a safety and efficacy study of allogeneic TruUCAR™ GC502 in patients with relapsed/refractory B-cell acute lymphoblastic leukemia (r/r B-ALL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT196.

Abstract 3575: Differential dynamics of response at single cell resolution between axi-cel and tisa-cel CAR-T therapy in refractory B-cell lymphomas

Abstract Chimeric Antigen Receptor (CAR)-T cell therapy has revolutionized the treatment of hematologic malignancies. Approximately half of patients with refractory large B-cell lymphomas achieve durable responses from CD19-targeting CAR-T treatment, across the commercially available CAR-T products with differing designs. Known failure mechanisms such as antigen loss account for only a fraction of cases without durable responses, and this knowledge gap has limited advances in CAR-T engineering and optimal targeting to patients. We hypothesized that characterization of the transcriptional programs and temporal evolution of CAR-T and host immune cell populations could provide novel insights into the basis of clinical response to CAR-T cell therapy for B cell lymphoma. We performed 10X single-cell RNA sequencing on serial samples collected from 32 individuals with high grade B cell lymphoma treated with the two first FDA-approved CD19 CAR-T products: axicabtagene ciloleucel (axi-cel, utilizing a CD28z costimulatory domain) and tisagenlecleucel (tisa-cel, with a 4-1BB domain). We analyzed 106 samples, including pre-infusion blood samples, infusion product, and post-infusion T cells sorted by flow cytometry into CAR+ and CAR- populations. Analyzing 602,577 single-cell transcriptomes, we discerned major differences in the dynamics of response of the two products. Tisa-cel responders showed dramatic expansion of CD8+ T cells at day 7 after infusion, which represented less than 10% of cells in the product. Conversely, CD8+ T cells in products of non-responders failed to expand to the same degree post-infusion and had a more effector- than memory-like T cell phenotype. In one tisa-cel–treated patient who had no CD8+ T cell expansion after initial infusion and relapsed at 6 months post-infusion, re-treatment with a second dose of the same product led to a durable response and was associated with greater CD8+ T cell expansion as well as a shift in CD4+ T phenotype from cytotoxic to helper. In contrast, axi-cel responders had pre-expanded effector populations distributed more heterogeneously among CD4+ and CD8+ T cells. Finally, we identified nominal elevations in CAR-T regulatory cells (CAR-Tregs) among both axi-cel and tisa-cel non-responders in our dataset, which we confirmed in an external dataset. These small increases in CAR-Tregs were sufficient to uniformly suppress conventional CAR-T cell expansion and drive late relapses in an in vivo mouse model of lymphoma after treatment with CARs with either CD28z or 4-1BB co-stimulatory domains. In summary, this represents the largest CAR-T scRNAseq cohort established thus far and provides important insights into (i) the temporal dynamics of a successful CAR-T response, (ii) the molecular phenotypes of CAR-T cells with different costimulatory domains, and (iii) the capacity for small increases in CAR-Tregs to drive relapse. Citation Format: Nicholas J. Haradhvala, Mark B. Leick, Katie Maurer, Satyen Gohil, Rebecca C. Larson, Estelle Yao, Matthew J. Frigault, Shuqiang Li, Kenneth J. Livak, Kahn Rhrissorrakrai, Filippo Utro, Chaya Levovitz, Raquel A. Jacobs, Kara Slowik, Brian P. Danysh, Laxmi Parida, Catherine J. Wu, Gad Getz, Marcela V. Maus. Differential dynamics of response at single cell resolution between axi-cel and tisa-cel CAR-T therapy in refractory B-cell lymphomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3575.

IMMU-01. Combining CD28 and 4-1BB costimulationin trans enhances the anti-glioma efficacy and persistence of B7-H3 CAR T cells in immune-competent brain tumor models

We and others have demonstrated that B7-H3 CAR T-cells have potent antitumor responses in xenograft models for brain tumors; however, these models do not recapitulate the immunosuppressive tumor microenvironment (TME) in patients with high-grade glioma. To evaluate the safety and efficacy of antigen-specific CAR T-cells, we adapted the immune-competent GL261 glioma model which recapitulates human disease and host immune barriers. We generated a library of B7-H3 CARs with different transmembrane (CD8, CD28), costimulatory (CD28, 4-1BB), and activation (ζ, mutζ) domains. We then compared their cytolytic activity, expansion, and anti-tumor activity. Results show that B7-H3 CARs with CD28 transmembrane and costimulatory domains have superior efficacy compared to CARs with CD8 and 4-1BB domains. Additionally, CARs with mutated ζ activation domain have better overall persistence. However, providing costimulation signals through CD28 or 4-1BB alone does not induce superior anti-glioma efficacy of B7-H3 CAR T-cells in vivo. Thus, we next investigated whether incorporating 4-1BB signaling into CD28-based CARs using in trans design enhances the therapeutic efficacy of B7-H3 CAR T-cells. We found that in repeat stimulation assays, surface expression of 4-1BBL enhanced expansion of B7H3 CAR T-cells at least 300-folds more than T-cells with CD28 or 4-1BB costimulatory domains alone. Additionally, 4-1BBL expression significantly enhanced the sequential killing capacity compared to CD28- or 41BB-based B7-H3 CAR T-cells. High dimensional flow cytometry analysis of GL261 tumors post CAR T-cell injection revealed unique immune clusters including dendritic cells and lymphoid predominant populations in mice treated with 4-1BBL expressing CARs. Thus, expression of 4-1BBL on CD28-based CARs reshaped the TME and enhanced persistence and anti-glioma efficacy of B7-H3 CAR T-cells. Studies examining transcriptional and epigenetic programs, and TME/CAR T-cell interactions are in progress. Results will define pathways that dictate CAR T-cell performance and will identify unique mechanisms for further improvements utilizing other members of TNF-superfamily.

STRIVE-02: A first-in-human phase 1 trial of systemic B7H3 CAR T cells for children and young adults with relapsed/refractory solid tumors.

10011 Background: B7H3 (CD276) has limited expression in normal tissues and high cell-surface expression in pediatric solid malignancies providing rationale for immunologic therapeutic targeting. We present a first-in-human experience of B7H3 chimeric antigen receptor T cells (CAR-T) for children and young adults (CYA) with relapsed or refractory solid tumors (R/RST). Methods: CYA patients with R/RST were enrolled onto a Phase 1 trial (NCT04483778) to examine the safety of autologous T cells genetically modified to express scFV-IgG4hinge-CD28tm-4-1BB-zeta B7H3-specific CAR with the methotrexate resistance/selection cassette DHFRdm and the tracking/suicide construct EGFRt. All patients received lymphodepleting fludarabine and cyclophosphamide prior to infusion of cryopreserved CAR-T at the prescribed dose level. The maximal tolerated dose or biologically effective dose (BED) was determined based upon observed toxicity through day 28 from initial CAR-T infusion and using a 3+3 statistical design. Results: Sixteen subjects (age 11-24, median 17 years) enrolled and received dose level (DL) 1 (0.5 x 106 CAR-T/kg, n = 3) or DL2 (1 x 106 CAR-T cells/kg, n = 6). No dose limiting toxicity was observed following first infusion, most common toxicities were fatigue and cytokine release syndrome (CRS) (n = 2, maximum CTCAE grade 2). Maximum circulating CAR-T expansion on first infusion was 4.98 cells/uL (range 0.23-4.98 cells/uL) with median persistence of 28 days (range 14-90). Best overall response of Stable Disease was observed in 3 of the 9 subjects infused. Given observed expansion and persistence, DL2 was determined to be the BED. A second infusion at DL2 in one subject demonstrated CAR T expansion to 1590 cells/uL (86% of circulating CD3 cells) with CTCAE grade 2 CRS and transient dose limiting CTCAE grade 4 liver enzyme elevation. A partial metabolic response on FDG-PET by PERCIST criteria was observed in this subject at Day 28. Conclusions: B7H3 CAR T cells are safe and demonstrate anti-tumor activity in CYA with R/RST. CAR-T cell expansion and persistence may be necessary to achieve objective responses. STRIvE-02 Arm B will explore dual expression of CD19 CAR with B7H3 CAR, using lymphocytic CD19 expression to drive CAR expansion and persistence. Clinical trial information: NCT04483778.

Biological correlative analyses and updated clinical data of ciltacabtagene autoleucel (cilta-cel), a BCMA-directed CAR-T cell therapy, in lenalidomide (len)-refractory patients (pts) with progressive multiple myeloma (MM) after 1–3 prior lines of therapy (LOT): CARTITUDE-2, cohort A.

8020 Background: Cohort A of the multicohort phase 2 CARTITUDE-2 (NCT04133636) study is evaluating cilta-cel safety and efficacy in pts with MM who received 1–3 prior LOT and were len-refractory – a difficult-to-treat population with poor prognosis. We present updated results. Methods: Pts had progressive MM after 1–3 prior LOT, including a PI and IMiD, were len-refractory, and had no prior exposure to BCMA-targeting agents. A single cilta-cel infusion (target dose 0.75×106 CAR+ viable T cells/kg) was given post lymphodepletion. Safety and efficacy were assessed, and the primary endpoint was MRD negativity at 10-5. Management strategies were implemented to minimize risk of movement/neurocognitive AEs (MNTs). Pharmacokinetic (PK) analyses (Cmax and Tmax of CAR+ T-cell transgene levels in blood) are being conducted, as well as analyses of levels of CRS-related cytokines (eg, IL-6) over time, peak levels of cytokines by response and CRS, association of cytokine levels with ICANS, and CAR+ T cell CD4/CD8 ratio by response, CRS, and ICANS. Results: As of January 2022 (median follow-up [MFU] 17.1 mo [range 3.3–23.1]), 20 pts (65% male; median age 60 y [range 38–75]) received cilta-cel. Pts received a median of 2 (range 1–3) prior LOT, and a median of 3.5 y (range 0.7–8.0) since MM diagnosis. 95% were refractory to last LOT, and 40% were triple-class refractory. ORR was 95%, 90% achieved CR or better, and 95% had ≥VGPR. Median times to first and best response were 1.0 mo (range 0.7–3.3) and 2.6 mo (range 0.9–13.6), respectively. 16 pts were MRD-evaluable, all of whom achieved MRD negativity at 10-5. Median DOR was not reached and 12-mo event-free rate was 79%. The 12-mo PFS rate was 75%. Median time to onset of CRS was 7 d (range 5–9) and occurred in 95% of pts (gr 3/4: 10%), with median duration of 3 d (range 2–12). Neurotoxicity occurred in 30% of pts (5 gr 1/2; 1 gr 3/4). 3 pts (15%) had ICANS (all gr 1/2); 1 pt had gr 2 facial paralysis. No MNTs were seen. 1 death occurred due to COVID-19 (assessed as tx-related by the investigator), 2 due to progressive disease, and 1 due to sepsis (not related to tx). Preliminary PK analyses indicate that peak expansion of CAR-T cells occurred at d 10.5 (range 8.7–42.9) and median persistence was 153.5 d (range 57.1–336.8). Conclusions: At a longer MFU of 17.1 mo, a single cilta-cel infusion led to deepening and durable responses in pts with MM who had 1–3 prior LOT and were len-refractory. Follow-up is ongoing. Updated and in-depth PK, cytokine, and CAR-T subset analyses and clinical correlation will be presented and provide novel insights into biological correlates of efficacy and safety in this pt population. This pt population is being further evaluated in the CARTITUDE-4 study (NCT04181827), which has concluded enrollment. Clinical trial information: NCT04133636.

Biological correlative analyses and updated clinical data of ciltacabtagene autoleucel (cilta-cel), a BCMA-directed CAR-T cell therapy, in patients with multiple myeloma (MM) and early relapse after initial therapy: CARTITUDE-2, cohort B.

8029 Background: In cohort B of the multicohort phase 2 CARTITUDE-2 (NCT04133636) study, the efficacy and safety of cilta-cel are being evaluated in patients (pts) with MM who had early relapse after initial therapy. These pts have functionally high-risk disease, with early relapse post autologous stem cell transplantation (ASCT) being a poor prognostic factor and representing an unmet medical need. We present updated results. Methods: Eligible pts had MM, received 1 prior LOT (PI and IMiD required), had disease progression per IMWG (either ≤12 mo after ASCT or ≤12 mo after start of anti-myeloma therapy for pts who did not undergo ASCT), and were tx-naive to CAR-T/anti-BCMA therapies. A single cilta-cel infusion (target dose 0.75×106 CAR+ viable T cells/kg) was given post lymphodepletion. Safety and efficacy were assessed, and the primary endpoint was MRD negativity at 10-5. Management strategies were implemented to minimize risk of movement/neurocognitive AEs (MNTs). Pharmacokinetic (PK) analyses (Cmax and Tmax of CAR+ T-cell transgene levels in blood) are being conducted, as well as analyses of levels of CRS-related cytokines (eg, IL-6) over time, peak levels of cytokines by response and CRS, association of cytokine levels with ICANS, and CAR+ T cell CD4/CD8 ratio by response, CRS, and ICANS. Results: As of January 2022, 19 pts (median age 58.0 y [range 44–67]; 74% male; median follow-up 13.4 mo [range 5.2–21.7]) received cilta-cel. 79% of pts received prior ASCT. ORR was 100.0%, 90% achieved CR or better, and 95% achieved ≥VGPR. Median time to first response and best response were 0.95 mo (range 0.9–9.7) and 5.1 mo (range 0.9–11.8), respectively. Of pts who were MRD-evaluable (n = 15), 14 (93%) achieved MRD 10-5 negativity during this study. Median DOR was not reached and 12-mo event-free rate was 88.9%. The 12-mo PFS rate was 90%. Median time to onset of CRS was 8 d (range 5–11) and occurred in 16 (84.2%) pts (1 gr 4). CRS resolved in all pts. ICANS (gr 1) occurred in 1 pt; MNT (gr 3) occurred in 1 pt, previously reported. 1 pt died post cilta-cel due to PD at d 158. Preliminary PK analyses indicate that peak expansion of CAR-T cells occurred on d 13.1 (range 8.96–209.9) and median persistence was 76.9 d (range 40.99–221.8). Conclusions: A single cilta-cel infusion led to deep and durable responses in a functionally high-risk pt population who experienced early clinical relapse/tx failure to initial therapy, with a manageable safety profile. In this pt population with ineffective or insufficient response to ASCT, cilta-cel led to responses. Responses continue to deepen, and follow-up is ongoing. Updated and in-depth PK, cytokine, and CAR-T subset analyses and clinical correlation will be presented and provide novel insights into biological correlates of efficacy and safety in this pt population. Clinical trial information: NCT04133636.

Immunogenicity of CAR-T Cell Therapeutics: Evidence, Mechanism and Mitigation.

Chimeric antigen receptor T cell (CAR-T) therapy demonstrated remarkable success in long-term remission of cancers and other autoimmune diseases. Currently, six products (Kymriah, Yescarta, Tecartus, Breyanzi, Abecma, and Carvykti) are approved by the US-FDA for treatment of a few hematological malignancies. All the six products are autologous CAR-T cell therapies, where delivery of CAR, which comprises of scFv (single-chain variable fragment) derived from monoclonal antibodies for tumor target antigen recognition is through a lentiviral vector. Although available CAR-T therapies yielded impressive response rates in a large number of patients in comparison to conventional treatment strategies, there are potential challenges in the field which limit their efficacy. One of the major challenges is the induction of humoral and/or cellular immune response in patients elicited due to scFv domain of CAR construct, which is of non-human origin in majority of the commercially available products. Generation of anti-CAR antibodies may lead to the clearance of the therapeutic CAR-T cells, increasing the likelihood of tumor relapse and lower the CAR-T cells efficacy upon reinfusion. These immune responses influence CAR-T cell expansion and persistence, that might affect the overall clinical response. In this review, we will discuss the impact of immunogenicity of the CAR transgene on treatment outcomes. Finally, this review will highlight the mitigation strategies to limit the immunogenic potential of CARs and improve the therapeutic outcome.

Sex differences in the pre-treatment cytokine signatures associated with response and survival in B cell lymphoma patients treated with anti-CD19 CAR T-cell therapy

Abstract Many autoimmune diseases, and in some instances, immune response to infections exhibit gender bias. However, it is not known if gender influences the response of lymphoma patients to anti-CD19 CAR T-cell therapy. To address this, we profiled 51 pre-treatment serum cytokines and chemokines to identify those that were differentially associated with response and survival in male vs. female relapsed/refractory large B-cell lymphoma (R/R LBCL) patients treated with Axicabtagene ciloleucel. Male non-responders (stable/progressive disease) had significantly higher baseline levels of IL-6, IL-8, IL-1RA, MIP-1α, GM-CSF and CRP compared to responders (complete/partial response). Higher levels of IL-6, IL-8, IL-27, and CRP were significantly associated with poor overall survival (OS), and IL-6, IL-8, MIP-1β, and CRP with poor progression-free survival (PFS) in male but not female patients. Baseline metabolic tumor volume (MTV) and lactate dehydrogenase (LDH) levels were also significantly higher in male patients with poor OS and PFS. Finally, baseline IL-8 and CRP were significantly correlated with baseline MTV and LDH levels in male but not female patients. Taken together, elevated baseline levels of IL-8, IL-6, CRP with high pretreatment tumor burden in male non-responder R/R LBCL patients point to an immunosuppressive tumor microenvironment that can potentially hinder anti-tumor activity prior to treatment and CAR T-cell expansion upon treatment in these patients, and result in poor outcomes.

Long-term Outcomes With Ibrutinib Treatment for Patients With Relapsed/Refractory Mantle Cell Lymphoma: A Pooled Analysis of 3 Clinical Trials With Nearly 10 Years of Follow-up.

Long-term Outcomes With Ibrutinib Treatment for Patients With Relapsed/Refractory Mantle Cell Lymphoma: A Pooled Analysis of 3 Clinical Trials With Nearly 10 Years of Follow-up.

Non-cleavable hinge enhances avidity and expansion of CAR-T cells for acute myeloid leukemia.

Chimeric antigen receptor (CAR) Tcell therapy is effective in lymphoid malignancies, but there has been limited data in myeloid cancers. Here, we start with a CD27-based CAR to target CD70 ("native") in acute myeloid leukemia (AML), and we find modest efficacy invivo, consistent with prior reports. We then use orthogonal approaches to increase binding on both the tumor and CAR-T cell sides of the immune synapse: a pharmacologic approach (azacitidine) to increase antigen density of CD70 in myeloid tumors, and an engineering approach to stabilize binding of the CAR to CD70. To accomplish the latter, we design a panel of hinge-modified regions to mitigate cleavage of the extracellular portion of CD27. Our CD8 hinge and transmembrane-modified CD70 CAR-T cells are less prone to cleavage, have enhanced binding avidity, and increased expansion, leading to more potent invivo activity. This enhanced CD70-targeted CAR is a promising candidate for further clinical development.

Clinical features of hemophagocytic syndrome following BCMA CAR-T cell therapy in patients with relapsed/refractory multiple myeloma.

To analyze the clinical features of hemophagocytic syndrome (HLH) following B cell maturation antigen (BCMA) chimeric antigen receptor (CAR) T cell therapy in patients with relapsed/refractory multiple myeloma. Ninety-nine patients with relapsed/refractory multiple myeloma (including 3 cases of plasma cell leukemia) undergoing BCMA CAR-T cell therapy (monocentric phaseⅠclinical trial, ChiCTR1800017404) in the First Affiliated Hospital, Zhejiang University School of Medicine from July 2018 to December 2021 were enrolled in the study. The baseline features, laboratory findings, treatment, and clinical response of these patients were analyzed. CAR-T cell associated HLH (carHLH) occurred in 20 patients (20.20%), and the median onset time was 7(0-19) d after cytokine release syndrome (CRS). Patients with carHLH were maily male patients, and manifested as high percentage of abnormal plasma cells, higher incidence of severe CRS (grade 3-4), and robust expansion of CAR-T cells in the peripheral blood (all P<0.05). The levels of interleukin (IL)-6, IL-10 and interferon (IFN)-γ, the peak value of international normalized ratio and D-dimer were elevated, and the valley value of fibrinogen was decreased in patients with carHLH (all P<0.01). All carHLH patients resolved with proper intervention (including 7 cases with tocilizumab, 5 with steroids, 6 with both). The objective response rate in carHLH patients was slightly higher than that in non-carHLH patients [100.0% (17/17) vs. 94.87% (74/78), P>0.05]. The incidence of carHLH is relatively high in BCMA CAR-T cell treated patients, which is closely related to pretreatment tumor cell percentage in bone marrow, expansion of CAR-T cells and the secretion of cytokines. Medication based on tocilizumab and steroids can achieve considerable therapeutic effects in patient with carHLH.

Superior antitumor immunotherapy efficacy of kynureninase modified CAR-T cells through targeting kynurenine metabolism

ABSTRACT Accumulated oncometabolites in the tumor microenvironment (TME) suppresses the metabolism, expansion, and function of T cells. Immunosuppressive TME also impeded Chimeric Antigen Receptor (CAR)-T cells mediated cytotoxicity since CAR-T cells had to adapt the in vivo metabolic characteristics with high levels of oncometabolites. We screened oncometabolites for the inhibition of glucose uptake in CD8 + T cells and found Kynurenine (Kyn) showed the strongest inhibiting effect on glucose uptake. In vitro experiments showed that 120 μM Kyn treatment in CD8 + T cells resulted in inhibiting the expansion of CD8 + T cells, decreasing the production of granzyme B and interferon-γ. CAR-T cells mediated cytotoxicity was also impaired by the high Kyn treatment from killing assay. We then explored the anti-tumor effect of Kynureninase (KYNU) modified CAR-T cells through catabolism o oncometabolites Kyn. KYNU over-expression (OE) CAR-T cells showed a superior killing effect against cancer cells even in the immunosuppressive TME with high Kyn levels. In vivo experiments confirmed KYNU-OE CAR-T cells showed an excellent anti-tumor effect in a TME with high Kyn levels since it improved the survival of mice bearing NALM6 cancer cells and NALM6-IDO1 cancer cells. The KYNU-modified CAR-T cells displayed distinct phenotypes related to the expansion, function, and memory differentiation status of CAR-T cells. This study explores an immunotherapy strategy for patients with alterations in Kyn metabolism. KYNU-OE CAR-T cells take advantage of Kyn catabolism to improve anti-tumor activity in the metabolic immunosuppressive TME with high Kyn.

PiggyBac Transposon-Mediated CD19 Chimeric Antigen Receptor-T Cells Derived From CD45RA-Positive Peripheral Blood Mononuclear Cells Possess Potent and Sustained Antileukemic Function.

The quality of chimeric antigen receptor (CAR)-T cell products, namely, memory and exhaustion markers, affects the long-term functionality of CAR-T cells. We previously reported that piggyBac (PB) transposon-mediated CD19 CAR-T cells exhibit a memory-rich phenotype that is characterized by the high proportion of CD45RA+/C-C chemokine receptor type 7 (CCR7)+ T-cell fraction. To further investigate the favorable phenotype of PB-CD19 CAR-T cells, we generated PB-CD19 CAR-T cells from CD45RA+ and CD45RA− peripheral blood mononuclear cells (PBMCs) (RA+ CAR and RA− CAR, respectively), and compared their phenotypes and antitumor activity. RA+ CAR-T cells showed better transient gene transfer efficiency 24 h after transduction and superior expansion capacity after 14 days of culture than those shown by RA− CAR-T cells. RA+ CAR-T cells exhibited dominant CD8 expression, decreased expression of the exhaustion marker programmed cell death protein-1 (PD-1) and T-cell senescence marker CD57, and enriched naïve/stem cell memory fraction, which are associated with the longevity of CAR-T cells. Transcriptome analysis showed that canonical exhaustion markers were downregulated in RA+ CAR-T, even after antigen stimulation. Although antigen stimulation could increase CAR expression, leading to tonic CAR signaling and exhaustion, the expression of CAR molecules on cell surface after antigen stimulation in RA+ CAR-T cells was controlled at a relatively lower level than that in RA− CAR-T cells. In the in vivo stress test, RA+ CAR-T cells achieved prolonged tumor control with expansion of CAR-T cells compared with RA− CAR-T cells. CAR-T cells were not detected in the control or RA− CAR-T cells but RA+ CAR-T cells were expanded even after 50 days of treatment, as confirmed by sequential bone marrow aspiration. Our results suggest that PB-mediated RA+ CAR-T cells exhibit a memory-rich phenotype and superior antitumor function, thus CD45RA+ PBMCs might be considered an efficient starting material for PB-CAR-T cell manufacturing. This novel approach will be beneficial for effective treatment of B cell malignancies.