scholarly journals A Bcma and CD19 Bispecific CAR-T for Relapsed and Refractory Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3147-3147 ◽  
Author(s):  
Hua Zhang ◽  
Lei Gao ◽  
Li Liu ◽  
Jishi Wang ◽  
Sanbin Wang ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Multiple Myeloma ◽  
T Cells ◽  
Clinical Study ◽  
Cell Line ◽  
Target Cells ◽  
Car T Cells ◽  
Car T

Introduction Chimeric Antigen Receptor T cells (CAR-T) therapy, e.g. B Cell Maturation Antigen (BCMA)-directed CAR-T has provided an encouraging modality for relapsed and refractory management of multiple myeloma (MM). However, a significant portion of patients still relapse with progressive disease after monospecific anti-BCMA CAR-T treatment. It has been demonstrated that CD19-directed CAR-T was effective in certain MM patients, likely due to CD19 expression on subsets of MM cells, and/or undetectable level of CD19 on MM cells. In addition, it has been reported that CD19 could express on the myeloma progenitor cells. To further improve the efficacy and to reduce relapse, we have designed a bispecific CAR-T targeting both BCMA and CD19. In addition to the conventionally-manufactured BCMA-CD19 CAR-T, the bispecific CAR-T was also successfully manufactured in our newly developed FasT CAR-T platform, which shortened the production time to one day. Here we report the results from pre-clinical studies and early results from the first-in-human clinical study. Methods The BCMA-CD19 bispecific CAR was constructed by linking BCMA and CD19 scFv, joined by a CD8 hinge, transmembrane domain, co-stimulatory domain and CD3. CAR-T cells were produced using either the conventional process (GC012) or the FasT CAR-T platform (GC012F). Peripheral blood (PB) mononuclear cells were obtained by leukapheresis either from healthy donors for the pre-clinical study or from patients for the clinical trial. T cells were isolated and used for CAR-T manufacturing. A xenograft mouse model was used to determine the efficacy in vivo. From March 2019 to July 2019, 5 adult relapsed/refractory MM patients (Age 50-59), who had previously received multiple lines of therapies, were enrolled (Table). Among them, 2 had extramedullary diseases. One patient did not receive lymphodepletion, and all other 4 patients received i.v. fludarabine and cyclophosphamide for 3 days. All patients received a single infusion of CAR-T cells, either at dose 1x106/Kg (DL1) (2 patients) or at dose 2x106/Kg (DL2) (3 patients), and the dose escalation is still ongoing. The endpoints of the exploratory trial were to evaluate the safety, feasibility, PK, and clinical efficacy of BCMA-CD19 bispecific CAR-T. Results In pre-clinical study, BCMA-CD19 bispecific CAR-T were very effective in killing CD19+ and/or BCMA + target cells including MM cell lines RPMI8226 and MM.1s (Fig 1). Increased IFN production and CD107a up-regulation were also observed. We demonstrated that BCMA-CD19 CAR-T completely eliminated BCMA+ MM cell line RPMI8226, MM.1s, and CD19+ ALL cell line Nalm6 in in vivo xenograft models. Additionally BCMA-CD19 CAR-T cells were shown to be more cytotoxic than single CAR-T both in vitro and in vivo. BCMA-CD19 CAR-T manufactured in the FasT CAR-T platform was more effective in eliminating MM in a xenograft model (Fig. 1). In the clinical study, the median observation time was 68 days (27-144 days up to 2019/7/30). Five patients were evaluated between 15-59 days post CAR-T infusion. Despite the relatively short disease evaluation time, all 5 patients responded to the treatment: 1 patient achieved sCR, 3 achieved VGPR and 1 achieved PR. Notably, although patient KM001 did not receive any pre-conditioning, however, the patient achieved sCR status on Day 15 and has maintained sCR up to now (129 days). CAR-T PK in the PB was monitored by qPCR and flow cytometry. The CAR-T proliferation peak was reached on Day 10 (D7-D14), and the median peak copy number was 34,039 (12,897-128,775) copies /ug DNA (Fig. 2). Remarkably, despite the encouraging clinical response to the CAR-T treatment, no severe adverse events were encountered during the observation period. Three patients experienced only grade 1 cytokine release syndrome (CRS) and no subject suffered from neurotoxicity of any level (Table). Conclusion Pre-clinical data demonstrated BCMA-CD19 CAR-T cells are effective in eliminating MM tumor cells both in vitro and in vivo. The first-in-human clinical trial also showed extraordinary safety profile and efficacy of BCMA-CD19 bispecific CAR-T in treating R/R MM. The long-term benefit and effect on relapse are being further studied. Bispecific CAR-T manufacturing on the FasT CAR-T platform is successful and has been shown to be more potent. A clinical study to evaluate safety and efficacy of FasT BCMA-CD19 CAR-T is ongoing. Disclosures No relevant conflicts of interest to declare.

scholarly journals Chimeric Antigen Receptor T Cells Specific for CLL-1 for Treatment of Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2205-2205 ◽  
Author(s):  
Elisa De Togni ◽  
Miriam Y Kim ◽  
Matt L Cooper ◽  
Julie Ritchey ◽  
Julie O'Neal ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Line ◽  
Myeloid Leukemia ◽  
Cytotoxic Effects ◽  
Car T Cells ◽  
Human T Cells ◽  
Car T ◽  
Acute Myeloid

Abstract Chimeric antigen receptor (CAR) T cells are a novel therapeutic approach which have shown good clinical outcomes in patients receiving CD19 CAR T cells for B cell acute lymphoblastic leukemia. CAR T cells are made to express a CAR that recognizes a specific surface antigen on a cell upon which they can then exert cytotoxic effects. We aim to extend the success of this therapy to acute myeloid leukemia (AML), a disease with generally poor clinical outcomes. However, due to the genetic heterogeneity characteristic of AML and the limited number of distinctive tumor markers, it has been difficult to find effective targets for CAR T cells on AML. C-type lectin like molecule-1 (CLL-1), also known as CD371, is a transmembrane glycoprotein that is expressed on about 90% of AML patient samples. CLL-1 may function as an inhibitory signaling receptor, as it contains an intracellular immunoreceptor tyrosine based inhibitory motif (ITIM). CLL-1 is primarily expressed on myeloid lineage cells in the bone marrow and in peripheral blood. While CLL-1 has been shown to be expressed on some granulocytes in the spleen, it is not reported to be expressed in non-hematopoietic tissues or on hematopoietic stem cells, which make CLL-1 a potential therapeutic target for AML. We generated two types of CLL-1 CARs, termed A and B, by using two different single chain variable fragments (scFvs) recognizing CLL-1. We used second generation CARs containing the scFvs, CD8 hinge and transmembrane domain, 4-1BB co-stimulatory domain, and CD3 zeta signaling domains. Using a lentiviral vector, we transferred the CAR gene into healthy donor human T cells and detected CAR expression by flow cytometry. We then tested the specific cytotoxic effects of CLL-1 CART-A and B on a CLL-1-expressing AML cell line, U937, by conducting a 4-hour chromium release assay. We found that both CAR T cells exhibited a dose-dependent killing of U937 (CLL-1 positive), while the untransduced (UTD) T cells had no cytotoxic effect (Figure 1A). We also found that U937 induces degranulation of CLL-1 CAR T cells as measured by CD107a expression by flow cytometry, while Ramos, a CLL-1 negative cell line, does not (Figure 1B). We then proceeded to investigate the in vivo efficacy of the CAR T cells. We injected NOD/SCID/IL2RG-null (NSG) mice with 1 x 106 THP-1 cells, a CLL-1 positive cell line. We confirmed engraftment by bioluminescent imaging (BLI) after 7 days and then injected 4 x 106 UTD, CLL-1 CART-A or CLL-1 CART-B. Surprisingly, only one of the CAR constructs, CLL-1 CART-A, showed significant activity in vivo, although both CARs had shown comparable activity in vitro. CLL-1 CART-A treated mice had delayed tumor progression and significantly increased length of survival (85 days vs. 63 days, p = 0.0021) compared to mice injected with UTD (Figure 1C and D). While CLL-1 CART-B treated mice also exhibited slower tumor growth and a trend towards better survival (72 days vs. 63 days, p=0.0547) this was not statistically significant. Post-mortem analysis showed that human T cells that continued to express CAR were present in the tumor, bone marrow and spleen of mice treated with CLL-1 CART-A only, while the UTD and CLL-1 CART-B treated mice showed tumor in all organs and no T cells. In summary, we show that CLL-1 CAR T cells can selectively eliminate CLL-1 positive target cells in vitro and in vivo, albeit with different degrees of efficacy modulated by the scFv. Studies are ongoing to investigate the mechanism behind the differential activity of these CAR constructs and to increase the long-term antitumor efficacy. Our results demonstrate that targeting CLL-1 using CAR T cell therapy holds promise for the treatment of AML. Disclosures Cooper: WUGEN: Consultancy, Equity Ownership.

GRP78 Is Expressed on the Cell Surface of Pediatric AML Blasts and Can be Targeted with GRP78-CAR T Cells without Toxicity to Hematopoietic Progenitor Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 12-12 ◽  
Author(s):  
Nikhil Hebbar ◽  
Rebecca Epperly ◽  
Abishek Vaidya ◽  
Sujuan Huang ◽  
Cheng Cheng ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Surface ◽  
Cell Lines ◽  
Target Cells ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T ◽  
Pediatric Aml

Finding the ideal immunotherapy target for AML has proven challenging and is limited by overlapping expression of antigens on hematopoietic progenitor cells (HPCs) and AML blasts. Intracellular Glucose-regulated-protein 78 (GRP78) is a key UPR regulator, which normally resides in the endoplasmic reticulum (ER). GRP78 is overexpressed and translocated to the cell surface in a broad range of solid tumors and hematological malignancies in response to elevated ER stress, making it an attractive target for immune-based therapies with T cells expressing chimeric antigen receptors (CARs). The goal of this project was to determine the expression of GRP78 on pediatric AML samples, generate GRP78-CAR T cells, and evaluate their effector function against AML blasts in vitro and in vivo. To demonstrate overexpression of GRP78 in AML, we performed gene expression analysis by RNAseq on a cohort of cord blood CD34+ cell samples (N=5) and 74 primary AML samples. Primary AML samples included RUNX1-RUNX1T1 (N=7), CBFB-MYH11(N=17), KMT2A rearrangement (N=28) and NUP98 (N=22). Analysis showed increased GRP78 expression in AML samples, especially in KMT2A- and NUP98-rearranged AML. To demonstrate surface expression of GRP78, we performed flow cytometry of AML (Kg1a, MOLLM13, THP-1, MV4-11) cell lines as well as 11 primary AML samples and 5 PDX samples; non transduced (NT) T cells served as control. All AML samples, including cell lines, primary AML blasts, and PDX samples, showed increased expression of GRP78 on their cell surface in comparison to NT T cells We then designed a retroviral vector encoding a GRP78-CAR using a GRP78-specific peptide as an antigen recognition domain, and generated GRP78-CAR T cells by retroviral transduction of primary human T cells. Median transduction efficiency was 82% (± 5-8%, N=6), and immunophenotypic analysis showed a predominance of naïve and terminal effector memory subsets on day 7 after transduction (N=5). To determine the antigen specificity of GRP78-CAR T cells, we performed coculture assays in vitro with cell surface GRP78+ (AML cell lines: MOLM13, MV-4-11, and THP-1 and 3 AML PDX samples) or cell surface GRP78- (NT T cells) targets. T cells expressing CARs specific for HER2-, CD19-, or a non-functional GRP78 (DGRP78)-CAR served as negative controls. GRP78-CAR T cells secreted significant amounts of IFNg and IL-2 only in the presence of GRP78+ target cells (N=3, p<0.005); while control CAR T cells did not. GRP78-CAR T cells only killed GRP78+ target cells in standard cytotoxicity assays confirming specificity. To test the effects of GRP78-CAR T cells on normal bone marrow derived HPCs, we performed standard colony forming unit (CFU) assays post exposure to GRP78-CAR or NT T cells (effector to target (E:T) ratio 1:1 and 5:1) and determined the number of BFU-E, CFU-E, CFU-GM, and CFU-GEMM. No significant differences between GRP78-CAR and NT T cells were observed except for CFU-Es at an E:T ratio of 5:1 that was not confirmed for BFU-Es. Finally, we evaluated the antitumor activity of GRP78-CAR T cells in an in vivo xenograft AML model (MOLM13). Tumor growth was monitored by serial bioluminescence imaging. A single intravenous dose of GRP78-CAR T cells induced tumor regression, which resulted in a significant (p<0.001) survival advantage in comparison to mice that had received control CAR T cells. In conclusion, GRP78 is expressed on the cell surface of AML. GRP78-CAR T cells have potent anti-AML activity in vitro and in vivo and do not target normal HPCs. Thus, our cell therapy approach warrants further active exploration and has the potential to improve outcomes for patients with AML. Disclosures Hebbar: St. Jude: Patents & Royalties. Epperly:St. Jude: Patents & Royalties. Vaidya:St. Jude: Patents & Royalties. Gottschalk:TESSA Therapeutics: Other: research collaboration; Inmatics and Tidal: Membership on an entity's Board of Directors or advisory committees; Merck and ViraCyte: Consultancy; Patents and patent applications in the fields of T-cell & Gene therapy for cancer: Patents & Royalties. Velasquez:St. Jude: Patents & Royalties; Rally! Foundation: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Novel CS1 CAR-T Cells and Bispecific CS1-BCMA CAR-T Cells Effectively Target Multiple Myeloma

Biomedicines ◽  
2021 ◽  
Vol 9 (10) ◽  
pp. 1422
Author(s):  
Vita Golubovskaya ◽  
Hua Zhou ◽  
Feng Li ◽  
Robert Berahovich ◽  
Jinying Sun ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Plasma Cells ◽  
Target Cells ◽  
Ifn Gamma ◽  
Myeloma Cells ◽  
Car T Cells ◽  
Car T ◽  
First Time

Multiple myeloma (MM) is a hematological cancer caused by abnormal proliferation of plasma cells in the bone marrow, and novel types of treatment are needed for this deadly disease. In this study, we aimed to develop novel CS1 CAR-T cells and bispecific CS1-BCMA CAR-T cells to specifically target multiple myeloma. We generated a new CS1 (CD319, SLAM-7) antibody, clone (7A8D5), which specifically recognized the CS1 antigen, and we applied it for the generation of CS1-CAR. CS1-CAR-T cells caused specific killing of CHO-CS1 target cells with secretion of IFN-gamma and targeted multiple myeloma cells. In addition, bispecific CS1-BCMA-41BB-CD3 CAR-T cells effectively killed CHO-CS1 and CHO-BCMA target cells, killed CS1/BCMA-positive multiple myeloma cells, and secreted IFN-gamma. Moreover, CS1-CAR-T cells and bispecific CS1-BCMA CAR-T cells effectively blocked MM1S multiple myeloma tumor growth in vivo. These data for the first time demonstrate that novel CS1 and bispecific CS1-BCMA-CAR-T cells are effective in targeting MM cells and provide a basis for future clinical trials.

scholarly journals Engineered IL-7 Receptor Enhances the Therapeutic Effect of AXL-CAR-T Cells on Triple-Negative Breast Cancer

2020 ◽  
Vol 2020 ◽  
pp. 1-13 ◽  
Author(s):  
Zhenhui Zhao ◽  
Yan Li ◽  
Wei Liu ◽  
Xun Li
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
Xenograft Model ◽  
Target Cells ◽  
Car T Cells ◽  
Car T

Triple-negative breast cancer (TNBC) is a very aggressive malignant type of tumor that currently lacks effective targeted therapies. In hematological malignancies, chimeric antigen receptor T (CAR-T) cells have shown very significant antitumor ability; however, in solid tumors, the efficacy is poor. In order to apply CAR-T cells in the treatment of TNBC, in this study, constitutively activated IL-7 receptor (C7R) that has been reported is used to enhance the antitumor function of constructed CAR-T cells by ourselves. Using in vitro coincubation experiments with target cells and in vivo antitumor experiments in mice, we found that the coexpressed C7R can significantly improve the activation, cell proliferation, and cytotoxicity of CAR-T cells. In addition, the in vivo experiments suggested that the enhanced CAR-T cells displayed significant antitumor activity in a TNBC subcutaneous xenograft model, in which in vivo, the survival time of CAR-T cells was prolonged. Together, these results indicated that CAR-T cells that coexpress C7R may be a novel therapeutic strategy for TNBC.

scholarly journals 5-Aza-2'-Deoxycytidine Promotes Cytotoxic Effect of CART-Cells on Leukaemia Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5812-5812
Author(s):  
Alla Dolnikov ◽  
Swapna Rossi ◽  
Ning Xu ◽  
Guy Klamer ◽  
Sylvie Shen ◽  
...  
Keyword(s):  
T Cells ◽  
B Cell ◽  
Target Cells ◽  
Car T Cells ◽  
Anti Tumour Activity ◽  
Car T ◽  
Pre Treatment ◽  
Tumour Activity

Abstract T cells modified to express CD19-specific chimeric antigen receptors (CAR) have shown anti-tumour efficacy in early phase clinical trials in patients with relapsed and refractory B-cell malignancies. In addition to direct cytotoxicity, chemotherapeutic drugs can have an immunomodulatory effect both through enhancing the tumour-specific immune response and increasing the tumour’s susceptibility to immune mediated destruction. Hence, combining immunomodulatory chemotherapy and CAR T-cells is an attractive approach for eliminating tumours, particularly in advanced stages. 5-aza-2'-deoxycytidine (5-AZA) is a hypomethylating agent that induces terminal differentiation, senescence or apoptosis in haematological malignancies. Here, we have explored a CAR-based immunotherapy combined with 5-AZA to maximise the effect of the CAR T-cells against CD19+ B-cell leukaemia. A second generation CAR including CD3zeta and the CD28 co-stimulatory domain was cloned into the PiggyBac-transposon vector and was used to generate CAR19 -T cells. Cord blood -derived mononuclear cells (MNC) were transfected with CAR19-transposon/transposase plasmids and expanded with CD3/28 beads for 2 weeks in the presence of 20ng/ml IL2 and 10ng/ml IL7. CAR19 T-cells efficiently induced cytolysis of CD19+ leukaemia cells in vitro and exhibited anti-tumour activity in vivo in a xenograft mouse model of leukaemia. Pre-treatment with 5-AZA produced greater leukaemia cell cytolysis in vitro and maximised anti-tumour activity of CAR19 T-cells in vivo against xenograft primary leukaemia compared to 5-AZA or CAR19 T-cells alone. In vitro analysis revealed that pre-treatment with 5-AZA up-regulates CD19 expression in leukaemia cells and improves CAR19 T-cell recognition of target cells increasing the formation of effector/ target cell conjugates and target cell cytolysis. Therefore using 5-AZA pre-treatment can be particularly useful for B-cell leukaemias with reduced expression of CD19. We have also demonstrated that pre-treatment of target cells with 5-AZA potentiates the effect of CAR19 T-cells used at low dose or low effector:target (E:T) suggesting that even small numbers of CAR19 T-cells can mediate a potent antitumor effect when combined with 5-AZA pre-treatment of target cells. This is particularly important for patients receiving limited numbers of CAR T-cells or for patients with large leukaemic burden. In addition, we speculate that the enhanced cellular cytotoxicity produced by 5-AZA-conditioning may allow the infusion of decreased numbers of CAR19 T-cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals 105 4–1BB and optimized CD28 co-stimulation enhances function of human mono- and bi-specific third-generation CAR T cells

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A115-A116
Author(s):  
Emiliano Roselli ◽  
Justin Boucher ◽  
Gongbo Li ◽  
Hiroshi Kotani ◽  
Kristen Spitler ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
The Other ◽  
Target Cells ◽  
Third Generation ◽  
Car T Cells ◽  
Nsg Mice ◽  
Car T

BackgroundCo-stimulatory signals regulate the expansion, persistence, and function of chimeric antigen receptor (CAR) T cells. Most studies have focused on the co-stimulatory domains CD28 or 4-1BB. CAR T cell persistence is enhanced by 4-1BB co-stimulation leading to NF-κB signaling, while resistance to exhaustion is enhanced by mutations of the CD28 co-stimulatory domain.MethodsWe hypothesized that a third-generation CAR containing 4-1BB and CD28 with only PYAP signaling motif (mut06) would provide beneficial aspects of both. We designed CD19-specific CAR T cells with 4-1BB or mut06 together with the combination of both (BB06). We evaluated their immune-phenotype, cytokine secretion, real-time cytotoxic ability and polyfunctionality against CD19-expressing cells. We analyzed LCK recruitment by the different constructs by immunoblotting. We further determined their ability to control growth of Raji cells in NSG mice. Additionally, we engineered bi-specific CARs against CD20/CD19 combining 4-1BB and mut06 and performed repeated in vitro antigenic stimulation experiments to evaluate their expansion, memory phenotype and phenotypic (PD1+CD39+) and functional exhaustion. Bi-specific CAR T cells were transferred into Raji or Nalm6-bearing mice to study their ability to eradicate CD20/CD19-expressing tumors.ResultsCo-stimulatory domains combining 4-1BB and mut06 confers CAR T cells with an increased polyfunctionality and LCK recruitment to the CAR (figure 1A), after repeated-antigen stimulation these cells expanded significantly better than second-generation CAR T cells (figure 1B). A bi-specific CAR targeting CD20/CD19, incorporating 4-1BB and mut06 co-stimulation, showed enhanced antigen-dependent in vitro expansion with lower exhaustion-associated markers (figure 1C). Bi-specific CAR T cells exhibited improved in vivo anti-tumor activity with increased persistence and decreased exhaustion (figure 1D).Abstract 105 Figure 1A. pLCK is increased in h19BB06z CAR T cells and associated with the CAR. CAR T cells were stimulated with irradiated 3T3-hCD19 cells at a 10:1 E:T ratio for 24hr. Cells were lysed and CAR bound and unbound fractions were western blotted. A single-cell measure of polyfunctional strength index (PSI) of CAR T cells. B. h19BB06z CAR T cells have the highest proliferation after repeated antigen stimulations. 5x105 CAR T cells were stimulated with 1x105 irradiated 3T3-hCD19 cells. After 1 week, half of the cells were enumerated by flow cytometry and the other half was re-stimulated with 1x105 fresh irradiated 3T3-hCD19 cells. This was repeated for a total of 4 weeks. C. 5x105 CAR T cells were co-cultured with 5x105 target cells (Raji-CD19High). After 1 week half the cells were harvested enumerated and stained by flow cytometry while the other half was re-stimulated with 5x105 fresh target cells (indicated by arrows). This was repeated for a total of 4 weeks. Frequency of PD1+CD39+ cells within CD8+ CAR T cells. D. Raji-FFLuc-bearing NSG mice were treated with 1x106 CAR T cells 5 days after initial tumor cell injection. Tumor burden (average luminescence) of mice treated with bi-specific or monospecific CAR T cells, UT and tumor control. Each line represents an individual mouse. (n = 7 mice per group).ConclusionsThese results demonstrate that co-stimulation combining 4-1BB with an optimized form of CD28 is a valid approach to optimize CAR T cell function. Cells with both mono- and bi-specific versions of this design showed enhanced in vitro and in vivo features such as expansion, persistence and resistance to exhaustion. Our observations validate the approach and justify clinical studies to test the efficacy and safety of this CAR in patients.

scholarly journals Engineering an Optimized Trimeric APRIL-Based CAR to Broaden Targetability of Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2059-2059 ◽  
Author(s):  
Andrea Schmidts ◽  
Maria Ormhoj ◽  
Allison O. Taylor ◽  
Selena J. Lorrey ◽  
Irene Scarfò ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Research Funding ◽  
Antigen Expression ◽  
Tumor Burden ◽  
Monomeric Form ◽  
Car T Cells ◽  
Car T

Abstract Background: Targeting BCMA (B-cell maturation factor) with chimeric antigen receptor (CAR) T cells has shown great success in the treatment of multiple myeloma (MM), but is limited by heterogeneous antigen expression and imminent antigen escape of tumor cells. Combinatorial antigen targeting may help address these challenges. Taking the naturally occurring receptor-ligand pairs as a model, we designed monomeric and trimeric APRIL- (A proliferation-inducing ligand) based CARs targeting BCMA and TACI (transmembrane activator and CAML interactor) simultaneously. Methods: The following 2nd generation CARs were designed to target BCMA and TACI concurrently: membrane-tethered truncated APRIL monomer ("APRIL-CAR") and three truncated and fused APRIL monomers ("TriPRIL-CAR"). A single chain variable fragment-based anti(α)-BCMA CAR served as control. CAR multimerization and binding affinity to BCMA and TACI were characterized. In vitro effector function was compared by cytotoxic potency, activation (CD69), degranulation (CD107a), cytokine production and proliferation in response to target antigens. In vivo anti-tumor efficiency was assessed in a xenograft mouse model of MM. Results: CAR T cell manufacturing of all three constructs was accomplished successfully (transduction efficiency 46-78%) from three different donors. Western blot analysis of CARs showed multimerized forms of the TriPRIL and α-BCMA CAR, while only the monomeric form of the APRIL CAR was detected. Binding affinity to soluble BCMA and TACI was higher for the TriPRIL CAR compared to the APRIL CAR. Evaluating the cytotoxic potential, activation and degranulation kinetics as well as long-term proliferation against a panel of BCMA and/or TACI positive target cells, the TriPRIL CAR T cells outperformed the APRIL CAR T cells. All three CAR constructs demonstrated robust antigen-specific production of Th1-type cytokines, like Il-2, IFNƔ, GM-CSF and TNFα. Next, we performed an in vivo stress test, engrafting NSG mice with high tumor burden of MM.1s myeloma cells. The TriPRIL and α-BCMA CAR T cells were able to eradicate the tumors while the APRIL CAR T cells only led to a stabilization of tumor burden. In vivo studies with a mixed antigen population aiming at modeling heterogeneous antigen expression and antigen escape are ongoing. Conclusion: Our APRIL-based chimeric antigen receptors were able to redirect T cell cytotoxicity to both BCMA and TACI positive tumor cells. Since both these receptors are consistently up-regulated on malignant plasma cells this is an attractive method to target MM. Furthermore, we found that using a trimeric form of APRIL rather than monomeric form as the CAR binding domain increased recognition of MM antigens in vitro and in vivo. Disclosures Maus: crispr therapeutics: Consultancy, Research Funding; adaptimmune: Consultancy; novartis: Consultancy; kite therapeutics: Consultancy, Research Funding; windmil therapeutics: Consultancy; agentus: Consultancy, Research Funding.

scholarly journals Characterization of novel dual tandem CD19/BCMA chimeric antigen receptor T cells to potentially treat multiple myeloma

2020 ◽  
Author(s):  
Liqing Kang ◽  
Jian Zhang ◽  
Minghao Li ◽  
Nan Xu ◽  
Wei Qi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Tumor Cells ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Cytokine Release ◽  
Car T Cells ◽  
Car T

Abstract Background: Treatment with chimeric antigen receptor (CAR)-engineered T cells directed against the B-cell maturation antigen (BCMA) promoted transient recovery from multiple myeloma (MM). However, the absence of this antigen on immature plasma cells may limit the efficacy of this modality and facilitate relapse. The purpose of this study is to characterize a novel CAR that includes both a single-chain variable fragment (scFv)-BCMA and an scFv-CD19 in tandem orientation (tan-CAR) in an attempt to target both BCMA and CD19 expression on MM cells. Method: The scFv sequences from the anti-CD19 antibody FMC63 and the anti-BCMA antibody C11D5.3 were ligated in tandem with transmembrane and T-cell signaling domains to generate the tan-CAR construct. Specificity and efficacy of activated tan-CAR T cells were analyzed using in vitro proliferation, cytokine release, and cytolysis assays. We also evaluated the in vivo efficacy with a xenograft mouse model that included target tumor cells that expressed CD19 or BCMA and compared the results to those obtained with conventional CAR T cells. Results: The in vitro studies revealed specific activation of tan-CAR T cells by K562 cells that overexpressed CD19 and/or BCMA. Cell proliferation, cytokine release, and cytolytic activity were all comparable to the responses of single scFv CAR T cells. Importantly, in vivo studies of tan-CAR T cells revealed specific inhibition of tumor growth in the mouse xenograft model that included cells expressing both CD19 and BCMA. Systemic administration of tan-CAR T cells resulted in complete tumor remission, in contrast to the reduced efficacies of BCMA-CAR T and CD19-CAR T alone in this setting. Conclusion: We report the successful design and execution of novel tan-CAR T cells that promote significant anti-tumor efficacy against both CD19 and BCMA antigen-positive tumor cells in vitro and in vivo . The data from this study reveal a novel strategy that may help to reduce the rate of relapse in the treatment with single scFv-CAR T cells.

scholarly journals A Small Number of HER2 Redirected CAR T Cells Significantly Improves Immune Response of Adoptively Transferred Mouse Lymphocytes against Human Breast Cancer Xenografts

2020 ◽  
Vol 21 (3) ◽  
pp. 1039 ◽  
Author(s):  
Gábor Tóth ◽  
János Szöllősi ◽  
Hinrich Abken ◽  
György Vereb ◽  
Árpád Szöőr
Keyword(s):  
T Cells ◽  
Complete Remission ◽  
Cytolytic Activity ◽  
Scid Mice ◽  
Target Cells ◽  
Her2 Positive ◽  
Car T Cells ◽  
Car T

HER2 positive JIMT-1 breast tumors are resistant to trastuzumab treatment in vitro and develop resistance to trastuzumab in vivo in SCID mice. We explored whether these resistant tumors could still be eliminated by T cells redirected by a second-generation chimeric antigen receptor (CAR) containing a CD28 costimulatory domain and targeting HER2 with a trastuzumab-derived scFv. In vitro, T cells engineered with this HER2 specific CAR recognized HER2 positive target cells as judged by cytokine production and cytolytic activity. In vivo, the administration of trastuzumab twice weekly had no effect on the growth of JIMT-1 xenografts in SCID mice. At the same time, a single dose of 2.5 million T cells from congenic mice exhibited a moderate xenoimmune response and even stable disease in some cases. In contrast, when the same dose contained 7% (175,000) CAR T cells, complete remission was achieved in 57 days. Even a reduced dose of 250,000 T cells, including only 17,500 CAR T cells, yielded complete remission, although it needed nearly twice the time. We conclude that even a small number of CAR T lymphocytes can evoke a robust anti-tumor response against an antibody resistant xenograft by focusing the activity of xenogenic T cells. This observation may have significance for optimizing the dose of CAR T cells in the therapy of solid tumors.

scholarly journals Apheresis Products from Patients with Multiple Myeloma Treated with G-CSF Are a Suitable Source of T Cells for the Production of BCMA-Targeting CAR-T Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 480-480
Author(s):  
Anthony M Battram ◽  
Aina Oliver-Caldés ◽  
Miquel Bosch i Crespo ◽  
María Suárez-Lledó ◽  
Miquel Lozano ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Progenitor Cells ◽  
Research Funding ◽  
Car T Cells ◽  
Car T

Abstract Background: Autologous chimeric antigen receptor-T (CAR-T) cells that target BCMA (BCMA-CARs) have emerged as a promising treatment for multiple myeloma (MM). Current clinical protocols dictate that BCMA-CAR therapy is only used after patients have repeatedly relapsed. However, at this stage, the immunosuppressive nature of advanced MM and/or side-effects of the previous therapies cause T cell dysfunction and an unfavourable phenotype, such as exhaustion, senescence and loss of early memory cells. An alternative and convenient pool of 'fitter' T cells are apheresis products that are routinely collected to obtain progenitor cells for autologous stem cell transplantation (ASCT), an intervention that is often carried out early in MM treatment. However, to mobilise the progenitor cells, patients are treated with G-CSF, which could have negative effects on T cells such as reduce proliferation, impair CD8 + T cell function and induce regulatory T cell (Treg) expansion. Whether this has an effect on the BCMA-CARs generated from these T cells, however, is unknown. Therefore, we aimed to establish whether G-CSF treatment had detrimental effects on T cell phenotype, and moreover, to ascertain whether BCMA-CARs that are generated from these T cells were impaired compared to those produced from T cells prior to G-CSF infusion. Methods: T cells were isolated from the blood of 9 patients with MM before and after 4 days of subcutaneous G-CSF administration (PRE G-CSF and POST G-CSF, respectively) prior to peripheral blood CD34 + cell harvesting for an ASCT as consolidation after first-line induction treatment. Following stimulation with anti-CD3/anti-CD28 beads and IL-2, T cells were transduced with ARI2h, an anti-BCMA CAR produced at our institution that is currently being explored in a clinical trial for relapsed/refractory MM (NCT04309981). Freshly-isolated T cells or expanded ARI2h cells were analysed by flow cytometry for markers of cell identity, activation, dysfunction and memory, or alternatively, challenged with an MM cell line (ARP-1 or U266) and then tested for cytokine production and cytotoxic ability. In addition, PRE and POST G-CSF ARI2h CARs were injected into ARP-1 tumour-bearing mice to assess their in vivo function. Results: Firstly, the phenotype of PRE G-CSF and POST G-CSF T cells, before CAR production, was analysed to identify effects of G-CSF treatment. Interestingly, there were fewer POST G-CSF CD8 + T cells with a stem cell memory (CCR7 +CD45RA +CD95 +) phenotype, but the proportion of naïve (CCR7 +CD45RA +CD95 -) cells and other memory populations was not significantly different. Moreover, POST G-CSF T cells had a lower CD4:CD8 ratio, but did not contain more senescent-like cells or display evidence of pre-activation or increased expression of exhaustion markers. Due to the known effect of G-CSF on CD4 + Treg expansion, the percentage of Tregs was also compared between the PRE G-CSF and POST G-CSF samples, but no difference was observed. Following T-cell activation and CAR transduction, comparable transduction efficiencies and proliferation rates were obtained. Likewise, the in vitro function of PRE G-CSF and POST G-CSF ARI2h cells, as determined by assessing their cytotoxic response to MM cell lines and ability to produce effector molecules such as granzyme B, was similar. To test the in vivo function of ARI2h CAR-T cells expanded from PRE G-CSF and POST G-CSF samples, they were injected into a murine xenograft model of advanced MM. Mice administered with both PRE and POST G-CSF ARI2h cells survived longer than those given untransduced T cells (p=0.015 and p=0.039, respectively), but there was no difference in the longevity of mice between the PRE G-CSF and POST G-CSF groups (p=0.990) (Figure 1). The similarity of the in vitro and in vivo function of PRE and POST G-CSF ARI2h cells was reflected in the phenotype of the CAR-T cells after ex vivo expansion, with cells from both groups displaying equal levels of activation, exhaustion, and importantly for CAR-T cell activity, memory/effector phenotype. Conclusions: The in vitro and in vivo functions of ARI2h CAR-T cells when generated from either PRE G-CSF or POST G-CSF samples were comparable, despite G-CSF administration decreasing the CD8 + stem cell memory pool. Overall, we conclude that T cells from apheresis products, performed to collect G-CSF-mobilised peripheral blood progenitor cells for ASCT, are suitable for BCMA-CAR manufacture. Figure 1 Figure 1. Disclosures Lozano: Grifols: Honoraria; Terumo BCT: Honoraria, Research Funding; Macopharma: Research Funding. Fernandez de Larrea: BMS: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Takeda: Honoraria, Research Funding; GSK: Honoraria; Sanofi: Consultancy; Janssen: Consultancy, Honoraria, Research Funding.

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