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

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

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

scholarly journals Analysis of CAR-T and Immune Cells within the Tumor Micro-Environment of Diffuse Large B-Cell Lymphoma Post CAR-T Treatment By Multiplex Immunofluorescence

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 678-678 ◽  
Author(s):  
Pei-Hsuan Chen ◽  
Mikel Lipschitz ◽  
Kyle Wright ◽  
Philippe Armand ◽  
Caron A. Jacobson ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract BACKGROUND: Axicabtagene ciloleucel is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy that shows efficacy in patients with refractory diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma and transformed follicular lymphoma after failure of conventional therapy. However, the exact mechanism of anti-tumor immunity is poorly understood, in part due to the dearth of data on the events in the tumor micro-environment (TME) that occur upon exposure to CAR-T cells. We sought to quantify and characterize both CAR-T cells and non-CAR T cells within the TME of DLBCL using tissue biopsy samples collected in the ZUMA-1 multicenter trial of CAR-T cell therapy for patients with refractory DLBCL. METHODS: Tumor samples obtained from patients 5-30 days (median 10 days) after CAR-T infusion ("CAR-treated", n=14) and randomly-selected untreated ("untreated ", n=15) archival DLBCL tissue samples were analyzed by multiplex immunofluorescence using formalin-fixed, paraffin embedded tissue sections, with successive labeling by the primary antibodies KIP-1 and/or KIP-3 (recognizing separate CD19 CAR epitopes), PAX5, PD-1, CD4, and CD8, followed by secondary amplification and tyramide-conjugated fluorophores. For each case, at least 3 representative 20x fields of view were selected and imaged using a multispectral imaging platform. Two specific image analysis algorithms were designed to accurately identify CD4 and CD8 T cells and PAX5+ DLBCL cells simultaneously, then to threshold PD-1 and KIP-1/-3 by relative fluorescent units (RFU) in each phenotype. RESULTS: We identified CAR T-cells within the fixed biopsy samples of CAR-treated DLBCLs by immunostaining with CAR T-cell specific antibody KIP-1; at the timepoints analyzed, CAR T-cells comprised only a small minority of total T- cells (<2%) and included CD4+ and CD8+ T-cells. Immunostaining with a second antibody, KIP-3, validated the presence of CAR T-cells in these cases and confirmed the KIP-1 results. Expression of the T cell activation marker PD-1 was detected among majority of KIP-1+ cells. Further analysis that included KIP1-negative cells revealed that the percentage of CD8+ cells co-expressing PD-1 across all CD8+ cells was higher in the CAR-treated DLBCLs compared to the untreated DLBCLs (mean 50.1% vs 17.5%, p<0.0001 with unpaired t test ), indicating CD8 T cell activation within the tumor environment. In contrast, PD-1 positivity across CD4+ T cells were equivalent between the two groups (mean 21.8% vs 21.6%, ns with unpaired t test). The percentages of total, CD4+, and CD8+ T-cell populations in the TME were similar between the CAR-treated DLBCL and untreated biopsies. CONCLUSIONS: CD4+ and CD8+ CAR-T cells can be detected in CAR-treated DLBCL patient tissue biopsies by multiplex immunofluorescence. At the time points analyzed to date, CAR-T cells comprise only a small percentage of all T-cells (<2%) within the TME. However, the presence of gene marked T cells with downregulated CAR protein expression is also possible. The activation marker PD-1 is preferentially expressed by KIP-1-negative CD8+ T cells compared to CD4+ T cells in CAR-T treated DLBCLs relative to untreated DLBCLs. These data implicate preferential activation of CD8+ non-CAR "by-stander" T-cells in the post CAR-T TME, and the possible benefit of combining PD-1 blockade with CAR-T therapy in DLBCL. *PH.C and M.L share equal contribution. Disclosures Armand: Otsuka: Research Funding; Affimed: Consultancy, Research Funding; Pfizer: Consultancy; Infinity: Consultancy; Adaptive: Research Funding; Merck: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Roche: Research Funding; Tensha: Research Funding. Roberts:KITE: Employment. Rossi:KITE: Employment. Bot:KITE: Employment. Go:KITE: Employment. Rodig:Merck: Research Funding; Bristol Myers Squibb: Research Funding; Affimed: Research Funding; KITE: Research Funding.

scholarly journals Allogeneic Hematopoietic Stem Cell Transplantation with Conditioning Including Donor Humanized CAR-T Cells for Refractory/Relapsed B-Cell Non-Hodgkin Lymphoma and Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 557-557
Author(s):  
Fan Yang ◽  
Hui Shi ◽  
Yang Lei ◽  
Ruiting Li ◽  
Teng Xu ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Hodgkin Lymphoma ◽  
Tumor Burden ◽  
Car T Cells ◽  
Car T Cell ◽  
Non Hodgkin Lymphoma ◽  
Car T

Abstract Background: The prognosis of refractory/relapsed aggressive B-cell non-Hodgkin lymphoma (r/r B-NHL) and multiple myeloma (r/r MM) is extremely poor, especially for the patients who failed to CAR-T cells therapy and/or ASCT. Aims: Forr/r B-NHLand r/r MM, a clinical trial using Allo-HSCT with conditioning including donor humanized CAR-T cells from the same donor (allo-CAR-T) has been registered, and the safety and efficacy will be evaluated. Methods: From September 2020 to May 2021, 11 patients were enrolled.The median age was 41 (26-64) years old. The diagnosis included high grade B-cell lymphoma (n=9) and Multiple myeloma (n=2). Seven cases were with TP53 mutations.All patients was progressive disease (PD) who failed to multi-line therapies, including chemotherapy (n=11), ASCT (n=4), autologous CAR-T (n=11).In order to further reduce the tumor burden, all patients were treated with combination therapy before transplantation. Before the trial, the expression of CD19 and/or CD22 or CD20 antigen in tumor tissue of r/r B-NHL and BCMA antigen in r/r MM patients was positive confirmed by immunohistochemistry.There were matched sibling identical donor in 1 case,matched unrelated donor in 1 case and haploidentical donor in 9 cases；Conditioning with busulfan, fludarabine-based regimen combined with allo-CAR-T was applied. Tacrolimus, mycophenolate mofetil, a short-term methotrexate and antithymocyte globulin were used for GVHD prophylaxis. The kinetics and function of CAR-T cells was monitored by quantitative PCR and flow cytometry. The efficacy was evaluated by PET-CT in r/r NHL as well as bone marrow puncture and immunofixation electrophoresis in r/r MM every 2 month after CAR-T infusion. Results: The median allo-CAR-T cells infused were 4 (range,0.78-4.88)×10 6/kg. CRS occurred in all cases with 6 cases in grade I, 1 case in grade II and 4 cases in grade III.The peak of cytokine IFN-γ and IL-6 in grade III CRS were significantly higher than those with grade I-II.No ICANS was noted. Four cases with grade III CRS were relieved with methylprednisolone. G-CSF-mobilized PBSC were infused 7 days after allo-CAR-T with the median CD34 + cells 6 (range,3-8.19)×10 6/kg. The neutrophil and platelets engraftment was achieved in all cases on median days 13 (range,11-24) and 16 (range,14-85) respectively post-transplant .All cases were donor type by STR analysis.Three cases of grade II acute GVHD were seen. CMV viremia occurred in 7 cases.For allo-CAR-T cell expansion,the peak time in vivo was on median 14(range,7-28) days after infusion.The median peak lever was 221 (range,0.191-1502)×10 6/L, which positively correlated with the number of allo-CAR-T infused. The tumor burden before transplantation was not significantly associated with allo-CAR-T expansion.Levels of allo-CAR-T cells were very low after the first 2 months of HSCT which detected persistently in 9/11(81.8%) patients, and the longest lasting time was 239 days post-transplant so far. B-cell aplasia was documented in 8/9 cases of r/r B-NHL during the follow-up. With the median follow-up 171 (range,100-295) days, 7/11(63.6%) patients survived,five cases(5/11,45.5%) achieved CR,one cases(1/11,9.1%) obtained PR, and 1 case(1/11,9.1%) of MM achieved SD and survival with tumor .Three cases(3/11,27.3%) with DLBCL died of PD whose disease status before transplantation were SD or PD, one patient(1/11,9.1%) died of infection.Significantly lower levels of Cumulative CAR T cell levels (AUC) during the first 2 month post transplantation were observed in patients who relapsed compared with those who had durable responses (P=0.0001).aGVHD were not associated directly with in vivo CAR T-cell expansion(P=0.193). Conclusion: Our preliminary results have shown that CRS is manageable and has no influence on hematopoiesis reconstitution. Allo-CAR-T cells still exist persistently post-transplant in majority of patients, which may contribute a long-term anti-lymphoma effect.With current protocol, aGVHD and viral reactivation was mild. Allo-HSCT with conditioning including allo-CAR-T cells is a safe and effective strategy for r/r B-NHL and MM. The Poor clinical efficacy was associated with high tumor burden before transplantation. [Key words] refractory/relapsed B-cell non-Hodgkin lymphoma; refractory/relapsed multiple myeloma;allogeneic CAR-T cell; allogeneic hematopoietic stem cell transplantation Disclosures No relevant conflicts of interest to declare.

scholarly journals Systematically optimized BCMA/CS1 bispecific CAR-T cells robustly control heterogeneous multiple myeloma

2020 ◽  
Author(s):  
Eugenia Zah ◽  
Eunwoo Nam ◽  
Vinya Bhuvan ◽  
Uyen Tran ◽  
Brenda Y. Ji ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
B Cell ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

ABSTRACTChimeric antigen receptor (CAR)-T cell therapy has shown remarkable clinical efficacy against B-cell malignancies but also demonstrated marked vulnerability to antigen escape and tumor relapse. Here, we report the rational design and systematic optimization of bispecific CAR-T cells with robust activity against multiple myeloma (MM), including heterogeneous MM that is resistant to conventional CAR-T cell therapy targeting B-cell maturation antigen (BCMA). We demonstrate that BCMA/CS1 bispecific CAR-T cells exhibit significantly higher CAR expression levels and greater antigen-stimulated proliferation compared to T cells that co-express individual BCMA and CS1 CARs. Compared to single-input BCMA- or CS1-targeting CAR-T cells, BCMA/CS1 bispecific CAR-T cells significantly prolong the survival of animals bearing heterogeneous MM tumors. Combination therapy with anti–PD-1 antibody further accelerates the rate of initial tumor clearance in vivo, but CAR-T cell treatment alone was able to achieve durable tumor-free survival even upon tumor re-challenge. Taken together, the BCMA/CS1 bispecific CAR presents a promising treatment approach to prevent antigen escape in CAR-T cell therapy against MM, and the vertically integrated optimization process can be used to develop robust cell-based therapy against novel disease targets.

scholarly journals Adoptive Immunotherapy beyond CAR T-Cells

Cancers ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 743
Author(s):  
Aleksei Titov ◽  
Ekaterina Zmievskaya ◽  
Irina Ganeeva ◽  
Aygul Valiullina ◽  
Alexey Petukhov ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Solid Tumors ◽  
Adoptive Immunotherapy ◽  
Γδ T Cells ◽  
Car T Cells ◽  
Car T Cell ◽  
Cell Immunotherapy ◽  
Car T

Adoptive cell immunotherapy (ACT) is a vibrant field of cancer treatment that began progressive development in the 1980s. One of the most prominent and promising examples is chimeric antigen receptor (CAR) T-cell immunotherapy for the treatment of B-cell hematologic malignancies. Despite success in the treatment of B-cell lymphomas and leukemia, CAR T-cell therapy remains mostly ineffective for solid tumors. This is due to several reasons, such as the heterogeneity of the cellular composition in solid tumors, the need for directed migration and penetration of CAR T-cells against the pressure gradient in the tumor stroma, and the immunosuppressive microenvironment. To substantially improve the clinical efficacy of ACT against solid tumors, researchers might need to look closer into recent developments in the other branches of adoptive immunotherapy, both traditional and innovative. In this review, we describe the variety of adoptive cell therapies beyond CAR T-cell technology, i.e., exploitation of alternative cell sources with a high therapeutic potential against solid tumors (e.g., CAR M-cells) or aiming to be universal allogeneic (e.g., CAR NK-cells, γδ T-cells), tumor-infiltrating lymphocytes (TILs), and transgenic T-cell receptor (TCR) T-cell immunotherapies. In addition, we discuss the strategies for selection and validation of neoantigens to achieve efficiency and safety. We provide an overview of non-conventional TCRs and CARs, and address the problem of mispairing between the cognate and transgenic TCRs. Finally, we summarize existing and emerging approaches for manufacturing of the therapeutic cell products in traditional, semi-automated and fully automated Point-of-Care (PoC) systems.

scholarly journals 221 CRISPR screen identifies loss of IFNγR signaling and downstream adhesion as a resistance mechanism to CAR T-cell cytotoxicity in solid but not liquid tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A234-A234
Author(s):  
Rebecca Larson ◽  
Michael Kann ◽  
Stefanie Bailey ◽  
Nicholas Haradhvala ◽  
Kai Stewart ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Solid Tumors ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

BackgroundChimeric Antigen Receptor (CAR) therapy has had a transformative impact on the treatment of hematologic malignancies1–6 but success in solid tumors remains elusive. We hypothesized solid tumors have cell-intrinsic resistance mechanisms to CAR T-cell cytotoxicity.MethodsTo systematically identify resistance pathways, we conducted a genome-wide CRISPR knockout screen in glioblastoma cells, a disease where CAR T-cells have had limited efficacy.7 8 We utilized the glioblastoma cell line U87 and targeted endogenously expressed EGFR with CAR T-cells generated from 6 normal donors for the screen. We validated findings in vitro and in vivo across a variety of human tumors and CAR T-cell antigens.ResultsLoss of genes in the interferon gamma receptor (IFNγR) signaling pathway (IFNγR1, JAK1, JAK2) rendered U87 cells resistant to CAR T-cell killing in vitro. IFNγR1 knockout tumors also showed resistance to CAR T cell treatment in vivo in a second glioblastoma line U251 in an orthotopic model. This phenomenon was irrespective of CAR target as we also observed resistance with IL13Ralpha2 CAR T-cells. In addition, resistance to CAR T-cell cytotoxicity through loss of IFNγR1 applied more broadly to solid tumors as pancreatic cell lines targeted with either Mesothelin or EGFR CAR T-cells also showed resistance. However, loss of IFNγR signaling did not impact sensitivity of liquid tumor lines (leukemia, lymphoma or multiple myeloma) to CAR T-cells in vitro or in an orthotopic model of leukemia treated with CD19 CAR. We isolated the effects of decreased cytotoxicity of IFNγR1 knockout glioblastoma tumors to be cancer-cell intrinsic because CAR T-cells had no observable differences in proliferation, activation (CD69 and LFA-1), or degranulation (CD107a) when exposed to wildtype versus knockout tumors. Using transcriptional profiling, we determined that glioblastoma cells lacking IFNγR1 had lower upregulation of cell adhesion pathways compared to wildtype glioblastoma cells after exposure to CAR T-cells. We found that loss of IFNγR1 reduced CAR T-cell binding avidity to glioblastoma.ConclusionsThe critical role of IFNγR signaling for susceptibility of solid tumors to CAR T-cells is surprising given that CAR T-cells do not require traditional antigen-presentation pathways. Instead, in glioblastoma tumors, IFNγR signaling was required for sufficient adhesion of CAR T-cells to mediate productive cytotoxicity. Our work demonstrates that liquid and solid tumors differ in their interactions with CAR T-cells and suggests that enhancing T-cell/tumor interactions may yield improved responses in solid tumors.AcknowledgementsRCL was supported by T32 GM007306, T32 AI007529, and the Richard N. Cross Fund. ML was supported by T32 2T32CA071345-21A1. SRB was supported by T32CA009216-38. NJH was supported by the Landry Cancer Biology Fellowship. JJ is supported by a NIH F31 fellowship (1F31-MH117886). GG was partially funded by the Paul C. Zamecnik Chair in Oncology at the Massachusetts General Hospital Cancer Center and NIH R01CA 252940. MVM and this work is supported by the Damon Runyon Cancer Research Foundation, Stand Up to Cancer, NIH R01CA 252940, R01CA238268, and R01CA249062.ReferencesMaude SL, et al. Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. N Engl J Med 2018;378:439–448.Neelapu SS, et al. Axicabtagene ciloleucel CAR T-cell therapy in refractory large B-cell lymphoma. N Engl J Med 2017;377:2531–2544.Locke FL, et al. Long-term safety and activity of axicabtagene ciloleucel in refractory large B-cell lymphoma (ZUMA-1): a single-arm, multicentre, phase 1–2 trial. The Lancet Oncology 2019;20:31–42.Schuster SJ, et al. Chimeric antigen receptor T cells in refractory B-cell lymphomas. N Engl J Med 2017;377:2545–2554.Wang M, et al. KTE-X19 CAR T-cell therapy in relapsed or refractory mantle-cell lymphoma. N Engl J Med 2020;382:1331–1342.Cohen AD, et al. B cell maturation antigen-specific CAR T cells are clinically active in multiple myeloma. J Clin Invest 2019;129:2210–2221.Bagley SJ, et al. CAR T-cell therapy for glioblastoma: recent clinical advances and future challenges. Neuro-oncology 2018;20:1429–1438.Choi BD, et al. Engineering chimeric antigen receptor T cells to treat glioblastoma. J Target Ther Cancer 2017;6:22–25.Ethics ApprovalAll human samples were obtained with informed consent and following institutional guidelines under protocols approved by the Institutional Review Boards (IRBs) at the Massachusetts General Hospital (2016P001219). Animal work was performed according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) (2015N000218 and 2020N000114).

scholarly journals Absolute lymphocyte count proliferation kinetics after CAR T-cell infusion impact response and relapse

2021 ◽  
Vol 5 (8) ◽  
pp. 2128-2136
Author(s):  
Sophia Faude ◽  
Jane Wei ◽  
Kavitha Muralidharan ◽  
Xiaoming Xu ◽  
Gerald Wertheim ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Lymphocyte Count ◽  
Absolute Lymphocyte Count ◽  
Rapid Expansion ◽  
Proliferation Kinetics ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract CD19-directed chimeric antigen receptor (CAR) T cells show characteristic proliferation kinetics after infusion that correlate with response. Clearance of circulating disease, B-cell aplasia (BCA), and cytokine release syndrome (CRS) are used to observe CAR T-cell function, given the lack of commercial CAR T-cell measurement assays. We investigated the utility of common hematology laboratory parameters in 166 patients with B-cell acute lymphoblastic leukemia (B-ALL) who were treated with CAR T-cell therapy targeting CD19. CAR T-cell infusion was followed by disappearance of circulating blasts in 86% of patients at a median of 6 days. After a lag phase, there was a rapid expansion in absolute lymphocyte count (ALC) in the second week that coincided with the appearance of atypical lymphocytes. The expansion phase was followed by a contraction phase with a concomitant decrease in atypical lymphocytes. In vitro CAR T-cell studies showed similar kinetics and morphological changes. Peak ALC and overall expansion was greater in sustained responders compared with that in nonresponders. Patients with early loss of BCA and those with eventual CD19+ minimal residual disease/relapse showed lower overall lymphocyte expansion compared with the controls. Pleomorphic lymphocytosis was noted in the cerebrospinal fluid at post-CAR time points. We conclude that lymphocyte counts and differential can also be used to evaluate CAR T-cell expansion after infusion, along with BCA and CRS. This is the first report to characterize the morphology of CAR T cells and determine the utility of lymphocyte kinetics.

scholarly journals Early Response Observed in Pediatric Patients with Refractory/Relapsed B-Cell Non-Hodgkin Lymphoma Treated with Sequential Chimeric Antigen Receptor T Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1945-1945 ◽  
Author(s):  
Wenqun Zhang ◽  
Bo Hu ◽  
Ling Jing ◽  
Jing Yang ◽  
Shan Wang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Hodgkin Lymphoma ◽  
Pediatric Patients ◽  
Response Rate ◽  
Car T Cell ◽  
Non Hodgkin Lymphoma ◽  
Car T ◽  
To Receive

Background:Outcomes for pediatric patients with relapsed/refractory B-cell non-Hodgkin lymphoma (NHL) are poor despite use of high-intensity chemotherapy. CAR-T has shown efficacy in treating refractory/relapsed leukemia in pediatric patients and non-Hodgkin lymphoma in adult patients. Objectives:To assess the safety and efficacy of sequential CAR-T in the treatment of refractory/ relapsed B-NHL in pediatric patients. Design/Methods:In our ongoing clinical trial (ChiCTR1800014457), we enrolled and treated 17 pediatric patients with refractory/relapsed B-NHL. Following leukapheresis, T cells were activated with CD3 and CD28 antibodies for 24h, then transduced with lentivirus encoding anti-CD19-CD3zeta-4-1BB CAR and cultured for 5-6 days in serum-free media containing IL2, IL7, IL15, IL21. Meanwhile, all patients briefly received lympho-depleting chemotherapies consisting of fludarabine (30 mg/m2/day) and cyclophosphamide (250 mg/m2/day) on days −5, −4 and −3 according to tumor burden and patient state. On day 0, all patients received a single-dose infusion of CAR-T cells. CAR-T cell dose ranged from 0.5 to 3 million/kg. CAR-T cell numbers and cytokines were measured weekly. Tumor responses were evaluated at day 30 and day 60 post infusion and every two months thereafter. Adverse events were graded according to CTCAEv4 except cytokine release syndrome (CRS) was graded according to Lee et al. Results:Treated patients had relapsed/refractory Burkitt lymphoma (BL) (13/17), diffuse large B cell lymphoma (DLBCL) (2/17), B-lymphoblastic lymphoma (B-LBL) (2/17), and ranged from 4.5-18.0 years old. By St Jude's staging, 9 cases (46.7%) were in stage III, 8 cases (53.3%) were in stage IV. There were 3 cases with CNS involvement (17.6%) and 7 cases with bone marrow involvement (41.2%). They all failed at prior treatment including an average of 8.9 (6-15) courses of chemotherapy. They were then treated with sequential CAR-T cell therapy. A total of 26 courses of CAR-T cell infusion were administered. The overall complete response rate (CRR) was 41.7% (7/17) when first course of CAR-T therapy was conducted, which were all CD19 targeted. Among the 10 patients who did not achieve CR, 2 patients achieved PR with ongoing response, 1 patient died of severe CRS and progression at day 6 and another patient refused to continue the following therapy when tumor progressed at day 99, and he died 1 week later, the other 6 continued to receive second course of CAR-T therapy targeting CD20 or CD22, and 3 of them achieved CR. Thus the overall CRR increased to 58.8% (10/17). The 3 patients, who still did not achieve CR, continued to receive third course of CAR-T therapy targeting CD20 or CD22. Two of them finally achieved CR and the other failed to get CR and is now retreated with chemotherapy and oral Olaparib and Venclexta. Thus, with a median follow-up of 6.2 months (1-18 months), the overall response rate of sequential CAR-T therapy was 94.1% (16/17) and the overall CRR was 70.6% (12/17). Toxicity information through day 30 revealed the occurrence of mild CRS in 8 subjects (47.1%, grade I n=8, grade II n=0), severe CRS in 9 subjects (52.9%, grade III n=8, grade IV n=1). Neurotoxicity was observed in 7 cases (41.2%, seizure in 3 cases, tremor in 4 cases, headache in 1 cases). One case who died rapidly at day 6 of therapy suffered severe CRS (high fever, Capillary leak syndrome, severe pleural effusion, respiratory failure, shock, cardiopulmonary arrest) and neurotoxicity besides disease progression. Other patients with severe CRS and neurotoxicity recovered fully after glucocorticoid use and symptomatic treatment including anti-epilepsy, fluid, dehydrating agent. No case used tocilizumab. Response assessments were performed at day 15, 30, 45, 60. Updated enrollment, toxicity and response assessments will be presented. Conclusion: CD19/CD20/CD22-CAR-T therapy showed promising efficacy for pediatric patients with r/r B-NHL and the toxicities are tolerable with proper symptomatic and supportive treatment. Sequential CAR-T therapy can improve the efficacy compared with a single course of CAR-T infusion. Disclosures No relevant conflicts of interest to declare.

scholarly journals Efficacy and Safety of JWCAR029 in Adult Patients with Relapsed and Refractory B-Cell Non-Hodgkin Lymphoma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4187-4187 ◽  
Author(s):  
Zixun Yan ◽  
Wen Wang ◽  
Zhong Zheng ◽  
Ming Hao ◽  
Su Yang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Hodgkin Lymphoma ◽  
Dose Level ◽  
Car T Cells ◽  
Car T Cell ◽  
Non Hodgkin Lymphoma ◽  
Car T

Abstract Introduction JWCAR029 is a novel CD19-directed 4-1BB stimulated chimeric antigen receptor T (CAR-T) cell type, which is different from JWCAR017 with independent production of CD4 and CD8 T cells and transfusion in non-fixed ratio. We conducted a single arm, open-label, dose escalation Phase I trial of JWCAR029 in relapsed and refractory B-cell non-Hodgkin lymphoma (NCT03355859). Methods From January to July 2018, 10 patients have been enrolled in this trial, including eight diffused large B cell lymphoma (DLBCL) and two MALT lymphoma, with median age of 47 years (range 32 to 59 years). All the patients received immunochemotherapy as induction and more than two lines of salvage treatment. Two patients received bridging chemotherapy after T-cell collection due to rapid tumor progression, followed by re-evaluation before CAR-T cell infusion. Lymphodepletion preconditioning was accomplished by fludarabine 25mg/m2/d and cyclophosphamide 250mg/m2/d on Day-4 to D-2, followed by CAR-T cell infusion on Day0. JWCAR029 was administrated as a single infusion in escalation dose levels, from 2.5×107 CAR-T cells (dose level 1, DL1) to 5.0×107 CAR-T cells (dose level 2, DL2) and to 1.0×108 CAR-T cells (dose level 3, DL3) according to mTPI-2 algorithm. Circulating blood count, serum biochemistry, and coagulation status were follow-up after infusion. Cytokines were assessed on a Luminex platform. Tumor evaluation was performed on Day 29 by PET-CT. PK data were detected by flow cytometry and real-time quantitative polymerase chain reaction system. All the adverse events were recorded. The study was approved by the Shanghai Rui Jin Hospital Review Board with informed consent obtained in accordance with the Declaration of Helsinki. Results The demographic characteristics of the patients were demonstrated in Table 1. Among six evaluable patients (3 of DL1 and 3 of DL2), the ORR was 100% on Day 29, including four complete remission and 2 partial remission. Cytokine release syndrome (CRS) was 100% in Gr 1, with main symptoms as fever (<39.0 degrees), fatigue, and muscle soreness. No neurotoxicity was observed. Four of the six patients with fever >38.0 degrees used prophylactic IL-6 Inhibitor (8mg/kg, ACTEMRA, two patients administered twice). No patients received steroids. The CRS showed no difference between dose level groups (p>0.99). Adverse effects included leukopenia (Gr 3-4: 83.3%, Gr 1-2: 16.7%), hypofibrinogenemia (Gr 1: 16.7%, Gr 2-4: 0%), liver dysfunction (Gr 1: 33.3%, Gr 2-4: 0%), elevated CRP (Gr 1: 83.3%, Gr 2-4: 0%), ferritin (Gr 1-2: 83.3%, Gr 2-4: 0%), or IL-6 (Gr 1-2:100%, Gr 3-4: 0%, Table 2). Conclusion Although long-term follow-up was needed, the preliminary data of six patients in this trial have demonstrated high response rates and safety of JWCAR029 in treating relapsed and refractory B-cell non-Hodgkin lymphoma. Disclosures Hao: JW Therapeutics: Employment, Equity Ownership.

scholarly journals P01.22 Extending CAR T cell therapy applications via drug inducible control of transgene expression

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A18.2-A19
Author(s):  
B Kotter ◽  
N Werchau ◽  
W Krueger ◽  
A Roy ◽  
J Mittelstaet ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Transgene Expression ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Part Time ◽  
Anti Cd20

BackgroundAdoptive transfer of chimeric antigen receptor (CAR)-modified T cells has emerged as a promising treatment modality for a broad range of cancers highlighted by the approval of Kymriah™ and Yescarta™ for the treatment of B cell malignancies. However, lack of control of CAR T cell function and consequent excessive inflammation in patients can result in severe side effects especially when targeting tumor-associated rather than tumor-specific antigens. Thus, temporal and tunable control of CAR activity is of major importance for the clinical translation of innovative CAR designs. While the activation of suicide switches results in the apoptotic elimination of the transferred cells, other strategies, e.g. anti-tag CARs or small molecule-gated CARs, enable the reversible control of CAR-mediated function at the protein level but are restricted to a particular CAR design. Focusing on the control of expression rather than CAR signaling, transcriptional regulators represent a versatile tool facilitating a wide range of CAR T cell applications.Materials and MethodsTo maintain control over the infused CAR T cell product and mitigate risks for the patient, we describe here the development of an inducible switch system for the transcriptional regulation of transgene expression in primary, human T cells. Chemically regulated synthetic transcription factors composed of a zinc finger DNA-binding domain, an inducible control domain and a transcription activation domain were designed, screened for functionality, and evaluated in T cells regarding their potential to control CAR expression both in vitro and in vivo.ResultsBy screening, we identified a synthetic transcription factor, which shows high transcriptional output in T cells in the presence of a clinically relevant inducer drug and absence of background activity in the non-induced state. Using this system we were able to control the expression of a CAR recognizing the CD20 antigen present on B cells and B cell leukemic blasts. The addition of the inducer drug resulted in rapid expression of the anti-CD20 CAR on the T cell surface. Moreover, inducible anti-CD20 CAR T cells executed cytolytic activity against CD20 positive target cells and secreted cytokines upon stimulation in vitro. Effectivity in co-cultures was thereby comparable to T cells expressing the anti-CD20 CAR under a conventional constitutive promoter. Furthermore, we could fine-tune CAR activity by titrating the inducer concentration. By defining the time-point of induction, modulation of the onset of therapy was achieved. Upon inducer drug discontinuation, inducible CD20 CAR T cells lost CAR expression and concurrently all CAR-related functions, indicating that the ‘on’ and ‘off’ status can be tightly controlled by the administration of the drug. After pausing of CAR T cell-mediated activity, we could re-induce CAR expression suggesting complete reversibility of effector function. Finally, we were able to show that inducible CD20 CAR T cells mediate a significant, strictly inducer-dependent antitumor activity in a well-established mouse model of B cell lymphoma.ConclusionsThe zinc-finger-based transcriptional control system investigated in this study provides small molecule-inducible control over a therapeutically relevant anti-CD20 CAR in primary T cells in a time- and dose-dependent manner. The tight regulation of CAR expression will pave the way for safer cellular therapies.Disclosure InformationB. Kotter: A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. & Co. KG. N. Werchau: A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. & Co. KG. W. Krueger: A. Employment (full or part-time); Significant; Lentigen Technology Inc. A. Roy: A. Employment (full or part-time); Significant; Lentigen Technology Inc. J. Mittelstaet: A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. & Co. KG. A. Kaiser: A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. & Co. KG.

scholarly journals GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells

2019 ◽  
Vol 11 (485) ◽  
pp. eaau7746 ◽  
Author(s):  
Eric L. Smith ◽  
Kim Harrington ◽  
Mette Staehr ◽  
Reed Masakayan ◽  
Jon Jones ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
B Cell ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell Therapy ◽  
Car T

Early clinical results of chimeric antigen receptor (CAR) T cell therapy targeting B cell maturation antigen (BCMA) for multiple myeloma (MM) appear promising, but relapses associated with residual low-to-negative BCMA-expressing MM cells have been reported, necessitating identification of additional targets. The orphan G protein–coupled receptor, class C group 5 member D (GPRC5D), normally expressed only in the hair follicle, was previously identified as expressed by mRNA in marrow aspirates from patients with MM, but confirmation of protein expression remained elusive. Using quantitative immunofluorescence, we determined that GPRC5D protein is expressed on CD138+ MM cells from primary marrow samples with a distribution that was similar to, but independent of, BCMA. Panning a human B cell–derived phage display library identified seven GPRC5D-specific single-chain variable fragments (scFvs). Incorporation of these into multiple CAR formats yielded 42 different constructs, which were screened for antigen-specific and antigen-independent (tonic) signaling using a Nur77-based reporter system. Nur77 reporter screen results were confirmed in vivo using a marrow-tropic MM xenograft in mice. CAR T cells incorporating GPRC5D-targeted scFv clone 109 eradicated MM and enabled long-term survival, including in a BCMA antigen escape model. GPRC5D(109) is specific for GPRC5D and resulted in MM cell line and primary MM cytotoxicity, cytokine release, and in vivo activity comparable to anti-BCMA CAR T cells. Murine and cynomolgus cross-reactive CAR T cells did not cause alopecia or other signs of GPRC5D-mediated toxicity in these species. Thus, GPRC5D(109) CAR T cell therapy shows potential for the treatment of advanced MM irrespective of previous BCMA-targeted therapy.

Sign in / Sign up

Export Citation Format

Share Document