Potent anti-leukemia activities of humanized CD19-targeted Chimeric antigen receptor T (CAR-T) cells in patients with relapsed/refractory acute lymphoblastic leukemia

Introduction: Adoptive infusion of CD19-targeted chimeric antigen receptor T (CAR-T) cells has showed promising treatment effects for relapsed/refractory (R/R) acute lymphoblastic leukemia (ALL). While immune response induced by murine single-chain variable fragment (scFv) of the CAR leads to its premature elimination, and thus increases the risk of leukemia relapse. Humanized scFv is expected to reduce the immunogenicity of CAR, thereby promoting the survival time as well as improving the therapeutic efficacy of CAR-T treatment. We developed a humanized anti-CD19 scFv domain and now report on treatment with humanized CD19 CAR-T cells (hCART19s). Methods: A pilot phase 1 study of CAR-modified T cells containing a humanized anti-CD19 scFv domain (hCART19s) was performed with recruitment of children and adults with R/R B-ALL with or without prior exposure to a murine CD19 CAR-T cell product. Patient-derived T cells were transduced ex vivo with a lentiviral vector encoding a CAR composed of humanized anti-CD19 scFv, the human CD8 transmembrane, CD8 hinge, 4-1BB costimulatory domain, CD3ζ intracellular regions and T2A-EGFRt sequence. After lymphodepletion chemotherapy with cyclophosphamide (750 mg/m2, day -5) and fludarabine (30 mg/m2/day, days -5 to -2), patients received a single dose of autologous hCART19s infusion at a dose of 1×106 cells/kg (body weight) on day 0. The post-infusion responses, toxicities, expansion and persistence of hCART19s in patients were observed and monitored. Results: 51 R/R B-ALL patients aged 3-69 yr were treated with hCART19s. 5 patients was diagnosed as Ph+ B-ALL. 2 patients had received prior allogeneic stem cell transplant (SCT). A total of 8 patients had central nervous system leukemia (CNSL), and 1 had testicular leukemia (TL). Among 46 patients without previous CAR-T therapy, 38 (82.6%) achieved complete remission (CR) or CR with incomplete count recovery (CRi) on day 30, while 2 of 5 patients, who relapsed after murine CAR-T infusion, achieved CR after hCART19s infusion. The rates of event-free survival and overall survival were 63% (95% confidence interval [CI], 46 to 75) and 79% (95% CI, 64 to 88), respectively, at 6 months and 44% (95% CI, 27 to 59) and 67% (95% CI, 51 to 79) at 12 months. Among the 40 patients with CR or CRi, 17 had a relapse before receiving additional anticancer therapy. 12 patients underwent allogeneic hematopoietic stem-cell transplantation (HSCT) while in remission, 10 were alive without relapse and 2 had a relapse after HSCT. During treatment, 37 (72.5%) patients developed grade 1-2 cytokine release syndrome (CRS), and 11 (21.6%) patients developed grade 3-5 CRS. Neurologic events occurred in 4 (7.8%) patients within 8 weeks after infusion. 2 (3.9%) patients had grade 3 neurologic events; no grade 4 events or cerebral edema were reported. Conclusions: This study demonstrated that hCART19s have high therapeutic efficacy and safety in children and adults with R/R B-ALL. More importantly, hCART19s was confirmed to exert anti-leukemic activities in patients who relapsed after murine CAR-T infusion. HSCT is a potential approach to reduce leukemia relapse in patients who achieved CR after CAR-T therapy. Disclosures No relevant conflicts of interest to declare.

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Targeting Leukemias by CD123 Specific Chimeric Antigen Receptor

Blood ◽

10.1182/blood.v118.21.1908.1908 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1908-1908

Author(s):

Radhika Thokala ◽

Harjeet Singh ◽

Simon Olivares ◽

Richard Champlin ◽

Laurence J N Cooper

Keyword(s):

T Cells ◽

Chimeric Antigen Receptor ◽

Lymphoblastic Leukemia ◽

Antigen Receptor ◽

Leukemic Cell ◽

Sleeping Beauty ◽

Myelogenous Leukemia ◽

Hematopoietic Stem ◽

Car T Cells ◽

Car T

Abstract Abstract 1908 Chimeric antigen receptors (CARs) are employed to genetically modify T cells to redirect their specificity to target antigens on tumor cells. Typically a second generation CAR is derived by fusing an extracellular domain derived from the scFv of monoclonal antibody (CAR) specific to targeted antigen with CD3 zeta, and CD28 endodomains. CD123 (IL3RA) is expressed on 45% to 95%of acute myelogenous leukemia (AML) and B-cell lineage acute lymphoblastic leukemia (B-ALL). Expression of CD123 is high in the leukemic stem cell (LSC) population, but not in normal hematopoietic stem cells. Thus, CD123 appears to be potential target for immunotherapy in leukemias through chimeric antigen receptor (CAR). We hypothesized that the generation of CD123 specific CAR can redirect the specificity of T cells to CD123 and this was tested by cloning the scFv of CD123 mAb in our CAR construct. The sleeping beauty system was used to express the CAR and DNA plasmids were electroporated into peripheral blood mononuclear cells and cells were numerically expanded on artificial antigen presenting cells genetically modified to express co stimulatory molecules CD86, 4-1BBL, membrane-bound IL-15, and CD123 antigen in presence of IL-21 and 1L–2. CAR+ T numerically expanded to clinically relevant numbers and showed antigen specific cytotoxicity in leukemic celllines. CAR+ T cells expressed both effector and memory markers showing the potential for in vivo persistence after T cell infusion. The bonemarrow homing receptor CXCR4 was expressed by CAR T cells shows the potential to target LSC that reside in BM niches. The preliminary data suggests that mirroring an approach we are using to manufacture clinical grade CD19 specific CAR+ T cells.Figure 1:(A) CAR expression on day 35. (B) Cytotoxicity of CD123CAR in leukemic cell lines.Figure 1:. (A) CAR expression on day 35. (B) Cytotoxicity of CD123CAR in leukemic cell lines.CD3CD3 Disclosures: No relevant conflicts of interest to declare.

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The Impact of the Immunophenotyping Characteristics of Patients' Peripheral Blood on the Manufacturing and Clinical Outcome of CD7-Targeted Chimeric Antigen Receptor T Cells

Blood ◽

10.1182/blood-2021-147632 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3830-3830

Author(s):

Mingzhi Zhang ◽

Xiaorui Fu ◽

Huimin Meng ◽

Min Wang ◽

Yu Wang ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Chimeric Antigen Receptor ◽

Peripheral Blood ◽

Lymphoblastic Leukemia ◽

Antigen Receptor ◽

Car T Cells ◽

Significant Difference ◽

Car T ◽

The Impact

Abstract Background T cell acute lymphoblastic leukemia and T cell acute lymphoblastic lymphoma (T-ALL/LBL) is a highly aggressive hematological tumor characterized by immature lymphoblasts invading the bone marrow. Treatment options for patients with T-ALL/ LBL are currently limited. Chimeric antigen receptor (CAR)- T cell therapy has opened an era in the treatment of B-cell malignancies. However, the development of CAR-T therapy for T-ALL/LBL faces many challenges. One of them is that therapeutic targets are usually expressed on both tumor and normal T cells, which causes the potential risk of "cell fratricide". Therefore, the difficulty of manufacturing CAR T cells for T-ALL/LBL is dramatically increased. CD7, is 40kD membrane-bound glycoprotein majorly expressed on peripheral T-cells and NK cells and their precursors. CD7 is highly expressed in almost all T-ALL/LBL and considered to be one of the most promising targets for T-ALL/LBL treatment. Patients and Methods This study is based on a phase I clinical trial (NCT04004637) for patients with relapse/refractory CD7 + NK/T cell lymphoma and T-ALL/LBL. To manufacture CAR-T cells, the peripheral blood mononuclear cells (PBMC) were collected from the patients who met the enrollment criteria. The proportion of viability and the ratio of the T cell markers were analyzed. Subsequently, the isolated T cells were co-transduced with CD7 protein expression blocker (PEBL) and CD7-CAR lentiviruses to obtain CD7-CAR-T cells, which can avoid the fratricide of CD7-CAR-T cells. Before the infusion, the phenotypic characteristics and cytotoxicity of CD7-CAR-T products were analyzed. Then peripheral blood (PB) of patients was collected regularly after receiving treatment to analyze the immunophenotyping of T cells. Results From August 2019 to June 2021, 24 leukopaks from patients with CD7-positive T-ALL/LBL were collected, and a total of 32 batches of CD7-CAR T cells were manufactured, with a 78.13% (25/32) successful rate. Among the 7 batches of failure cases, one patient had undergone blood collection twice and CAR-T preparation for three times, but all of three attempts failed (brown icon). Another four patients failed to prepare once. Eight patents were recruited for CD7-CAR-T treatment and 87.5% of complete remission (CR) rate was achieved (7/8), of which a patient (P4, blue icon) has been maintaining CR for more than 15 months. Two other patients, P7 (red icon) and P8 (light red icon), had CD7 - relapse at the time of 6th month and 3rd month after CR, respectively. We divided all samples into successful preparation group (GS), infusion group (GI) and preparation failure group (GF). As shown in Fig. 1A, all three groups exhibited good viability of PBMC. There was no significant difference between GS and GF, but GI was higher than that of GF. The proportion of CD3 + cells in PBMC of GS was significantly lower than that of GF, and GI also showed this feature. Meanwhile, GS and GI both have a higher CD4 +/CD8 + ratio compared with GF. The immunophenotyping results showed CD7-CAR-T products had a majority of the central memory subsets (T CM; 69.41 ± 10.71%) and effect memory subsets (T EM; 28.56 ± 10.19%), with limited number of effector T cell (T E) and naive T cells (T N) (Fig. 1B). The percentage of CAR +CD8 +CD27 + and CD4 +CD25 +CD127 - subsets associated with T cells activation and proliferation, as well as CD223 + and CD279 + subsets related to T cells suppression and exhaustion were lower, except for CD366 + subgroup that also indicated depletion signal (Fig. 1B). In addition, CD7-CAR-T cells showed strong cytotoxicity against CEM (CD7 +) tumor cells accompanied by the release of cytokines, in which the level of IL-2 is extremely low (Fig. 1C). Subsequently, we performed statistics on the proportion of CD3 + and CD4 +/CD8 + cells in the PB of patients after infusion. The proportion of CD3 + cells in the PB of the P4 has been maintained at a high level, and the ratio of CD4 +/CD8 + keeps low (Fig. 1D). P7 showed a significant decrease in the amount of T cells on the 60th day after CAR T infusion, while the ratio of CD4 +/CD8 + showed an upward trend. Conclusion The results indicate that the success rate of CD7-CAR-T manufacturing is positively correlated with higher viability, lower CD3 + and higher CD4 + of PBMC. There was no significant difference among P4 (CR more than 15 months), P7 (CD7 - relapse at 6 th month after CR) and P8 (CD7 - relapse at 3rd month after CR). Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Anti-CD37 chimeric antigen receptor T cells are active against B- and T-cell lymphomas

Blood ◽

10.1182/blood-2018-04-842708 ◽

2018 ◽

Vol 132 (14) ◽

pp. 1495-1506 ◽

Cited By ~ 35

Author(s):

Irene Scarfò ◽

Maria Ormhøj ◽

Matthew J. Frigault ◽

Ana P. Castano ◽

Selena Lorrey ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Chimeric Antigen Receptor ◽

Lymphoblastic Leukemia ◽

Antigen Receptor ◽

B Cell Lymphoma ◽

Car T Cells ◽

T Cell Lymphomas ◽

Car T

Abstract Chimeric antigen receptor (CAR) T cells have emerged as a novel form of treatment of patients with B-cell malignancies. In particular, anti-CD19 CAR T-cell therapy has effected impressive clinical responses in B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma. However, not all patients respond, and relapse with antigen loss has been observed in all patient subsets. Here, we report on the design and optimization of a novel CAR directed to the surface antigen CD37, which is expressed in B-cell non-Hodgkin lymphomas, in chronic lymphocytic leukemia, and in some cases of cutaneous and peripheral T-cell lymphomas. We found that CAR-37 T cells demonstrated antigen-specific activation, cytokine production, and cytotoxic activity in models of B- and T-cell lymphomas in vitro and in vivo, including patient-derived xenografts. Taken together, these results are the first showing that T cells expressing anti-CD37 CAR have substantial activity against 2 different lymphoid lineages, without evidence of significant T-cell fratricide. Furthermore, anti-CD37 CARs were readily combined with anti-CD19 CARs to generate dual-specific CAR T cells capable of recognizing CD19 and CD37 alone or in combination. Our findings indicate that CD37-CAR T cells represent a novel therapeutic agent for the treatment of patients with CD37-expressing lymphoid malignancies.

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A Phase I Study of CD19 Chimeric Antigen Receptor-T Cells Generated By the PiggyBac Transposon Vector for Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood-2021-150469 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3831-3831

Author(s):

Nobuhiro Nishio ◽

Ryo Hanajiri ◽

Yuichi Ishikawa ◽

Makoto Murata ◽

Rieko Taniguchi ◽

...

Keyword(s):

T Cells ◽

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Phase I ◽

Chimeric Antigen Receptor ◽

Lymphoblastic Leukemia ◽

Piggybac Transposon ◽

Car T Cells ◽

Car T ◽

Grade 3

Abstract Introduction: Chimeric antigen receptor-modified T cells targeting CD19 (CD19.CAR-T cells) have shown clinical success in patients with hematological malignancies. Despite the encouraging results obtained with this novel therapy, a major concern to its global spread, particularly in developing countries, is its high cost. We developed a method of non-viral gene transfer using piggyBac transposon to reduce the cost of CAR-T therapy. In preclinical study, the median number and transduction efficiency of CAR-T cells obtained from 2x10 7 PBMC in 9 donors were 1.0x10 8 (range, 0.58-1.8x10 8) and 51% (range, 29-73%), respectively. The major subset of CAR-T cells was phenotypically CD8+CD45RA+CCR7+, closely related T-memory stem cells. Ex vivo, CD19.CAR-T cells showed cytotoxic effect on CD19 positive tumor cell lines. In NSG mice model, CD19.CAR-T cells successfully inhibit tumor growth. CAR gene integration sites were determined by inverse polymerase chain reaction and subsequent next-generation sequencing using MiSeq and equally distributed throughout the genome without preference for specific sites. The pre-clinical testing in mouse demonstrated safe toxicity profile at the 50 times dose of CD19.CAR-T cells. We started a human clinical trial to define feasibility, toxicity, maximum tolerated dose and clinical response of CD19.CAR-T cells (jRCTa040190099). Methods: We report the results of cohort 1 of the study in which the safety and efficacy of autologous CD19.CAR-T in patients with relapsed or refractory B-precursor acute lymphoblastic leukemia were evaluated. We engineered autologous T cells via the piggyBac transposon system with CD19.CAR-expression transposon vector and piggyBac transposase-expression vector to express CD19.CAR incorporating CD28 costimulatory domain. We designed this phase I trial using a modified 3 + 3 design to enroll 3-12 patients with relapsed or refractory acute lymphoblastic leukemia in both children and adults. In this study, patients in cohorts 1 (16-60 years old) and 2 (1-15 years old) receive 1 × 10 5 CAR-transduced T cells per kg. Patients in cohorts 3 and 4 (both 1-60 years old) receive 3 × 10 5 and 1 × 10 6 CAR-transduced T cells per kg, respectively. All patients receive 25mg/m 2/d of fludarabine and 250mg/m 2/d of cyclophosphamide for 3 days followed by a single infusion of CAR-T cells. Results: Three patients were enrolled in cohort 1 and infused with 1 × 10 5 CAR-transduced T cells per kg. All patients had previously undergone allogeneic hematopoietic stem cell transplantation. All patients had achieved a hematological complete response with salvage treatment before CAR-T therapy. None of the patients had dose-limiting toxicities (DLT) defined as nonhematological toxicities above grade 4 or cytokine release syndrome (CRS) above grade 4 or graft versus host disease (GVHD) above 4, or grade 3 nonhematological toxicities and GVHD not improved to grade 2 within 4 weeks after CAR-T infusion. There was no occurrence of non-hematological adverse events above grade 3. CRS was observed in one patient (grade 1) who also developed headache due to infiltration of CAR-T cells into the spinal fluid. In two patients, B cell aplasia lasted 2 and 9 months, respectively. Elevation of serum cytokine levels was observed in all patients and the peak time point was 7-21 days after CAR-T cell infusion. Conclusions: CD19.CAR-T cell infusion produced by the piggyBac transposon gene engineering system was safe in cohort 1 of our study. As no patients had DLT in cohort 1, we are enrolling the patients in further cohorts. Disclosures Murata: MSD: Honoraria; Kyowa Kirin: Honoraria; Sumitomo Dainippon Pharma: Honoraria; FUJIFILM: Honoraria; Toyama Chemical: Honoraria; Novartis: Honoraria; JCR Pharmaceutical: Honoraria; Astellas: Honoraria; Miyarisan Pharmaceutical: Honoraria; Asahi Kasei: Honoraria; GlaxoSmithKline: Honoraria; Celgene: Honoraria; Otsuka Pharmaceutical: Honoraria.

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Anti-CD19 CAR-T Cells for Relapsed or Refractory Acute Lymphoblastic Leukemia and Lymphomas

Case Medical Research ◽

10.31525/ct1-nct04196205 ◽

2019 ◽

Author(s):

Keyword(s):

T Cells ◽

Acute Lymphoblastic Leukemia ◽

Lymphoblastic Leukemia ◽

Car T Cells ◽

Car T

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Study of CD 19 Specific Chimeric Antigen Receptor (CAR-T) Cells in ALL and NHL

Case Medical Research ◽

10.31525/ct1-nct04206943 ◽

2019 ◽

Author(s):

Keyword(s):

T Cells ◽

Chimeric Antigen Receptor ◽

Antigen Receptor ◽

Car T Cells ◽

Car T

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112 Rational design of chimeric antigen receptor T cells against glypican 3 decouples toxicity from therapeutic efficacy

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0112 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A124-A124

Author(s):

Letizia Giardino ◽

Ryan Gilbreth ◽

Cui Chen ◽

Erin Sult ◽

Noel Monks ◽

...

Keyword(s):

T Cells ◽

Chimeric Antigen Receptor ◽

Antigen Receptor ◽

Single Chain ◽

High Affinity ◽

Car T Cells ◽

Car T ◽

Animal Care ◽

Cytotoxic Function

BackgroundChimeric antigen receptor (CAR)-T therapy has yielded impressive clinical results in hematological malignancies and it is a promising approach for solid tumor treatment. However, toxicity, including on-target off-tumor antigen binding, is a concern hampering its broader use.MethodsIn selecting a lead CAR-T candidate against the oncofetal antigen glypican 3 (GPC3), we compared CAR bearing a low and high affinity single-chain variable fragment (scFv,) binding to the same epitope and cross-reactive with murine GPC3. We characterized low and high affinity CAR-T cells immunophenotype and effector function in vitro, followed by in vivo efficacy and safety studies in hepatocellular carcinoma (HCC) xenograft models.ResultsCompared to the high-affinity construct, the low-affinity CAR maintained cytotoxic function but did not show in vivo toxicity. High-affinity CAR-induced toxicity was caused by on-target off-tumor binding, based on the evidence that high-affinity but not low-affinity CAR, were toxic in non-tumor bearing mice and accumulated in organs with low expression of GPC3. To add another layer of safety, we developed a mean to target and eliminate CAR-T cells using anti-TNFα antibody therapy post-CAR-T infusion. This antibody functioned by eliminating early antigen-activated CAR-T cells, but not all CAR-T cells, allowing a margin where the toxic response could be effectively decoupled from anti-tumor efficacy.ConclusionsSelecting a domain with higher off-rate improved the quality of the CAR-T cells by maintaining cytotoxic function while reducing cytokine production and activation upon antigen engagement. By exploring additional traits of the CAR-T cells post-activation, we further identified a mechanism whereby we could use approved therapeutics and apply them as an exogenous kill switch that would eliminate early activated CAR-T following antigen engagement in vivo. By combining the reduced affinity CAR with this exogenous control mechanism, we provide evidence that we can modulate and control CAR-mediated toxicity.Ethics ApprovalAll animal experiments were conducted in a facility accredited by the Association for Assessment of Laboratory Animal Care (AALAC) under Institutional Animal Care and Use Committee (IACUC) guidelines and appropriate animal research approval.

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