CD19-specific Chimeric Antigen Receptor Research Articles

CD19-CAR-DNT cells (RJMty19) in patients with relapsed or refractory large B-cell lymphoma: a phase 1, first-in-human study

Current approved chimeric antigen receptor (CAR) T-cell products are autologous cell therapies that are costly and poorly accessible to patients. We aimed to evaluate the safety and antitumor activity of a novel off-the-shelf anti-CD19 CAR-engineered allogeneic double-negative T cells (RJMty19) in patients with relapsed/refractory large B-cell lymphoma. We report the results from a first-in-human, open-label, single-dose, phase 1 study of allogeneic CD19-specific CAR double-negative T (CAR-DNT) cells. Eligibility criteria included the presence of measurable lesions, at least 2 lines of prior immunochemotherapy, and an ECOG score of 0-1. We evaluated four dose levels (DL) of RJMty19 in a 3+3 dose-escalation scheme: 1×106, 3×106, 9×106 and 2×107 CAR-DNT cells per kilogram of body weight. All patients received lymphodepleting chemotherapy with fludarabine and cyclophosphamide. The primary endpoints were dose-limiting toxicities (DLTs), incidence of adverse events (AEs), and clinically significant laboratory abnormalities. Secondary endpoints included evaluation of standard cellular pharmacokinetic parameters, immunogenicity, objective response rates (ORR), and disease control rate (DCR) per Lugano 2014 criteria. A total of 12 patients were enrolled between 22 July 2022 and 27 July 2023. Among these patients, 66% were classified as stage IV, 75% had an IPI score of 3 or higher, representing an intermediate risk or worse. The maximum tolerated dose was not reached because no DLT was observed. Four patient experienced grade 1 or 2 cytokine release syndrome and dizziness. The most common AEs were hematologic toxicities, including neutropenia (N=12, 100%), leukopenia (N=12, 100%), lymphopenia (N=10, 83%), thrombocytopenia (N=6, 50%), febrile neutropenia (N=3, 25%), and anemia (N=3, 25%). Seven subjects died till the cut-off date, five of them died of disease progression and two of them died of COVID 19. In all patients (N=12), the ORR was 25% and CRR was 8.3%. DL1 and DL2 patients benefited less from the therapy (ORR: 17%, N=1; DCR: 33%, N=2). However, all DL3 patients achieved disease control (N=3, 100%), and all DL4 patients achieved objective response (N=3, 100%). Our results demonstrate that CD19-CAR-DNT cells appear to be well tolerated with promising antitumor activity in LBCL patients. Further study of this product with a larger sample size is warranted. This phase 1 study is registered on clinicaltrials.gov (NCT05453669). Wyze Biotech. Co., Ltd.

Abstract 4014: Humanized mouse model to assess PBMC donor derived autologous CD19 CAR Ts for efficacy against Raji tumors and related cytokine release

Abstract Immuno-oncology cell therapies engage the immune system to treat cancer. In recent years, adoptive T-cell therapy with CD19-specific chimeric antigen receptors (CARs) has shown promise for treatment of various malignancies, including acute lymphoblastic leukemia. However, CAR-T-cell therapies have been hampered by various adverse events, including cytokine release syndrome (CRS). To predict and minimize CRS effects from CAR-T therapies, a human immune preclinical murine model that combines efficacy and toxicity readouts is essential. Here, we describe the development of PBMC humanized mouse model based on the NSG-MHC I/II-DKO strain to study autologous CD19 CAR-T treatment related tumor growth inhibition and CRS in vivo. Three different PBMC donors were used to generate CAR-T (CD28-Cd3z) directed against a CD19 epitome. Our preliminary data demonstrate that CD19 CAR-T treatment stimulated dynamic and PBMC donor dependent cytokine release with IFN-g, IL-10, IL-4, IL-2, IL-6 and TNF. Our model showed elevation of shared proinflammatory cytokines (IFN-g, IL-10, IL-2 and TNF) observed in humans after CD19 CAR-T treatment. Currently, we are evaluating our PBMC based cytokine release model in CD19 CAR-T cells tumor control study. Our results to date indicate that PBMC-engrafted NSG-MHC I/II-DKO mouse models are vital platforms to screen adoptive T-cell therapy for potential CRS. Citation Format: Senthil Ramalingam, Danying Cai, Lindsay Shopland, James G. Keck. Humanized mouse model to assess PBMC donor derived autologous CD19 CAR Ts for efficacy against Raji tumors and related cytokine release [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4014.

Modulating Cholesterol Metabolism via ACAT1 Knockdown Enhances Anti-B-Cell Lymphoma Activities of CD19-Specific Chimeric Antigen Receptor T Cells by Improving the Cell Activation and Proliferation.

CD19-specific CAR-T immunotherapy has been extensively studied for the treatment of B-cell lymphoma. Recently, cholesterol metabolism has emerged as a modulator of T lymphocyte function and can be exploited in immunotherapy to increase the efficacy of CAR-based systems. Acetyl-CoA acetyltransferase 1 (ACAT1) is the major cholesterol esterification enzyme. ACAT1 inhibitors previously shown to modulate cardiovascular diseases are now being implicated in immunotherapy. In the present study, we achieved knockdown of ACAT1 in T cells via RNA interference technology by inserting ACAT1-shRNA into anti-CD19-CAR-T cells. Knockdown of ACAT1 led to an increased cytotoxic capacity of the anti-CD19-CAR-T cells. In addition, more CD69, IFN-γ, and GzmB were expressed in the anti-CD19-CAR-T cells. Cell proliferation was also enhanced in both antigen-independent and antigen-dependent manners. Degranulation was also improved as evidenced by an increased level of CD107a. Moreover, the knockdown of ACAT1 led to better anti-tumor efficacy of anti-CD19 CAR-T cells in the B-cell lymphoma mice model. Our study demonstrates novel CAR-T cells containing ACAT1 shRNA with improved efficacy compared to conventional anti-CD19-CAR-T cells in vitro and in vivo.

Acute kidney injury following treatment with CD19-specific CAR T-cell therapy in children, adolescent, and young adult patients with B-cell acute lymphoblastic leukemia.

CD19-specific chimeric antigen receptor (CAR) T-cell therapy has shown promising disease responses in patients with high-risk B-cell malignancies. However, its use may be related to complications such as immune-mediated complications, infections, and end-organ dysfunction. The incidence of post-CAR T-cell therapy acute kidney injury (AKI) in the children, adolescent, and young adult (CAYA) patient population is largely unreported. The objectives of this study were to determine the incidence of AKI in CAYA patients with high-risk B-cell malignancies treated with CD19-CAR T-cell therapy, evaluate potential risk factors for developing AKI, and determine patterns of kidney function recovery. We conducted a retrospective analysis of 34 CAYA patients treated with CD19-CAR T-cell at a single institution. There was a cumulative incidence of any grade AKI by day 30 post-infusion of 20% (n = 7), with four cases being severe AKI (stages 2-3) and one patient requiring kidney replacement therapy. All episodes of AKI developed within the first 14days after receiving CAR T-cell therapy and 50% of patients with AKI recovered kidney function to baseline within 30days post-infusion. No evaluated pre-treatment risk factors were associated with the development of subsequent AKI; there was an association between AKI and cytokine release syndrome and neurotoxicity. We conclude that the risk of developing AKI following CD19-CAR T-cell therapy is highest early post-infusion, with most cases of AKI being severe. Frequent monitoring to facilitate early recognition and subsequent management of kidney complications after CD19-CAR T-cell therapy may reduce the severity of AKI in the CAYA patient population.

Decoding the mechanisms of chimeric antigen receptor (CAR) T cell-mediated killing of tumors: insights from granzyme and Fas inhibition

Chimeric antigen receptor (CAR) T cell show promise in cancer treatments, but their mechanism of action is not well understood. Decoding the mechanisms used by individual T cells can help improve the efficacy of T cells while also identifying mechanisms of T cell failure leading to tumor escape. Here, we used a suite of assays including dynamic single-cell imaging of cell-cell interactions, dynamic imaging of fluorescent reporters to directly track cytotoxin activity in tumor cells, and scRNA-seq on patient infusion products to investigate the cytotoxic mechanisms used by individual CAR T cells in killing tumor cells. We show that surprisingly, overexpression of the Granzyme B (GZMB) inhibitor, protease inhibitor-9 (PI9), does not alter the cytotoxicity mediated by CD19-specific CAR T cells against either the leukemic cell line, NALM6; or the ovarian cancer cell line, SkOV3-CD19. We designed and validated reporters to directly assay T cell delivered GZMB activity in tumor cells and confirmed that while PI9 overexpression inhibits GZMB activity at the molecular level, this is not sufficient to impact the kinetics or magnitude of killing mediated by the CAR T cells. Altering cytotoxicity mediated by CAR T cells required combined inhibition of multiple pathways that are tumor cell specific: (a) B-cell lines like NALM6, Raji and Daudi were sensitive to combined GZMB and granzyme A (GZMA) inhibition; whereas (b) solid tumor targets like SkOV3-CD19 and A375-CD19 (melanoma) were sensitive to combined GZMB and Fas ligand inhibition. We realized the translational relevance of these findings by examining the scRNA-seq profiles of Tisa-cel and Axi-cel infusion products and show a significant correlation between GZMB and GZMA expression at the single-cell level in a T cell subset-dependent manner. Our findings highlight the importance of the redundancy in killing mechanisms of CAR T cells and how this redundancy is important for efficacious T cells.

Safety and feasibility of anti-CD19 CAR T cells expressing inducible IL-7 and CCL19 in patients with relapsed or refractory large B-cell lymphoma

Although CD19-specific chimeric antigen receptor (CAR) T cells are curative for patients with relapsed or refractory large B-cell lymphoma (R/R LBCL), disease relapse with tumor antigen-positive remains a challenge. Cytokine/chemokine-expressing CAR-T cells could overcome a suppressive milieu, but the clinical safety and efficacy of this CAR-T therapy remain unclear. Here we report the preclinical development of CD19-specific CAR-T cells capable of expressing interleukin (IL)-7 and chemokine (C-C motif) ligand (CCL)-19 upon CD19 engagement (referred to as 7 × 19 CAR-T cells) and results from a phase 1 and expansion phase trial of 7 × 19 CAR-T cell therapy in patients with R/R LBCL (NCT03258047). In dose-escalation phase, there were no dose-limiting toxicities observed. 39 patients with R/R LBCL received 7 × 19 CAR-T with doses ranged from 0.5 × 106–4.0 × 106 cells per kg body weight. Grade 3 cytokine release syndrome occurred in 5 (12.8%) patients and ≥ grade 3 neurotoxicity in 4 (10.3%) patients. The overall response rate at 3 months post-single infusion was 79.5% (complete remission, 56.4%; partial response, 23.1%). With a median follow-up of 32 months, the median progression-free survival was 13 months, and median overall survival was not reached, with an estimated rate of 53.8% (95% CI, 40.3% to 72.0%) at two years. Together, these long-term follow-up data from the multicenter clinical study suggest that 7 × 19 CAR-T cells can induce durable responses with a median overall survival of greater than 2 years, and have a manageable safety profile in patients with R/R LBCL.

Cyclosporine A-resistant CAR-T cells mediate antitumour immunity in the presence of allogeneic cells

Chimeric antigen receptor (CAR)-T therapy requires autologous T lymphocytes from cancer patients, a process that is both costly and complex. Universal CAR-T cell treatment from allogeneic sources can overcome this limitation but is impeded by graft-versus-host disease (GvHD) and host versus-graft rejection (HvGR). Here, we introduce a mutated calcineurin subunit A (CNA) and a CD19-specific CAR into the T cell receptor α constant (TRAC) locus to generate cells that are resistant to the widely used immunosuppressant, cyclosporine A (CsA). These immunosuppressant-resistant universal (IRU) CAR-T cells display improved effector function in vitro and anti-tumour efficacy in a leukemia xenograft mouse model in the presence of CsA, compared with CAR-T cells carrying wild-type CNA. Moreover, IRU CAR-T cells retain effector function in vitro and in vivo in the presence of both allogeneic T cells and CsA. Lastly, CsA withdrawal restores HvGR, acting as a safety switch that can eliminate IRU CAR-T cells. These findings demonstrate the efficacy of CsA-resistant CAR-T cells as a universal, ‘off-the-shelf’ treatment option.

Allogeneic Veto-CAR CD8 T Cells Enabling Prolonged Graft Survival, Anti-Tumor Specificity, and Low Risk of Graft Versus Host Disease

¥ Equal contribution Studies in mice have demonstrated that CD8 T cells exhibit marked veto activity against host T cells with anti-graft specificity, enhancing engraftment after non-myeloablative T cell depleted mismatched bone marrow transplantation. To reduce the risk of GVHD associated with allogeneic CD8 veto T cells, these studies used naïve CD8 T cells stimulated against 3rd party stimulators under cytokine deprivation and subsequent expansion in the presence of IL-15. Recently, we showed that mouse CD8 veto T cells can be generated by stimulating CD8 memory T cells from ovalbumin-immunized mice under cytokine deprivation, using ovalbumin as a third-party antigen (Or-Geva et al., Leukemia, 2019). Based on these insights, human anti-viral CD8 central memory veto cells were successfully generated from CMV and EBV positive donors. Donor PBMC collected by leukapheresis were initially depleted of CD45RA+, CD4+ and CD56+ cells, and co-cultured with dendritic cells from the same donor, pulsed with a mixture of CMV, EBV, Adeno and BKV viral peptides. Within 12 days of culture, starting with 3 days of cytokine deprivation, more than 3x10 9 highly homogenous CD8 T cells, predominantly expressing a central memory phenotype could be routinely harvested. These cells exhibited marked veto activity in-vitro with more than a 3 log-depletion of alloreactivity as measured by limiting dilution analysis. Based on these results, a first in human phase 1-2 clinical trial is currently in progress, testing the safety of such veto cells in the context of non-myeloablative haploidentical T cell-depleted hematopoietic stem cell transplantation (HSCT). Preliminary results indicate that infusion of 5-10x10 6 veto cells / kg supports engraftment with a low risk of GVHD (Champlin et al. Blood, 2022). Most attempts to use allogenic chimeric antigen receptor (CAR) T cells involve gene editing, knocking down the TCR to avoid GVHD, and eliminating MHC expression to avoid graft rejection. Our veto cell preparation could potentially offer an attractive platform for attaining allogeneic CAR T cells capable of deleting host anti-donor T cell clones by virtue of their veto activity, with a very low risk for GVHD and without the need of gene editing. Using our current protocol for production of anti-viral central memory veto CD8 T cells, we first evaluated transduction efficacy at different time points during the veto production using a retroviral CAR directed against the Her-2 antigen (N29). The scFv N29 was attached to a costimulatory determinant of CD28 and a T cell activating domain followed by a GFP reporter gene. Our results suggested that optimal CAR transduction was attained on day 5 of culture. The veto cell product harvested on day 12 exhibited 92% CD8 T cells, of which 72% expressed the transduced N29 CAR. Notably, the transduced cells continued to exhibit veto activity and functional assays showed killing of target cells in a dose dependent manner. Next, we performed similar experiments transducing veto Tcm with a clinical grade gammaretroviral vector encoding a second-generation 4-1BB co-stimulated CD19-specific CAR. A typical experiment, shown in Fig.1, demonstrates a transduction level of 66.7 % (Fig. 1B,) and confirms the central memory phenotype of the transduced cells (Fig. 1C). Notably, upon infusion of 3x10 6 veto-CD19CAR into NSG mice implanted 3 days earlier with 0.25x10 6 Nalm-6-Luc+ tumor cells (CD19+), a marked eradication of tumor cells (Fig.1D) and survival benefit (Fig.1E) were found, while infusion of 3x10 6 non-transduced veto cells were ineffective. Based on these results, we plan to initiate clinical trials using haploidentical VETO-CD19CAR for the treatment of B-cell malignancies, in combination with hematopoietic transplantation and as off the shelf cellular therapy. .

Ablation of Cullin-5 in Primary Human T Cells Improves Tumor Killing and Persistence in BCMA-Targeting CAR-T Cells in a Multiple Myeloma Model

While adoptive T cell therapies have led to remarkable clinical responses in certain subsets of B cell leukemia and lymphoma, many challenges remain that are limiting the efficacy in other hematological malignancies. Compromised T cell function and exhaustion due to chronic antigen exposure can impair the persistence of chimeric antigen receptor (CAR)-T and T-cell-receptor (TCR)-T cells. Gene editing strategies have the potential to circumvent some of these challenges and enhance T cell function. To identify key genes that are involved in regulating T cell dysfunction, we performed a genome-wide CRISPR loss-of-function screen in primary human T cells in conditions that mimic chronic antigen exposure. Primary T cells from two donors were transduced with lentivirus containing a NY-ESO-1 TCR as well as a genome-wide sgRNA library and subsequently CRISPR edited using Cas9-RNP electroporation. These antigen specific TCR-T cells were then co-cultured with the NY-ESO-1+ melanoma cell line A375 at an effector-to-target (E:T) ratio of 1:1 for 48 hours and challenged under the pressure of chronic tumor stimulation for 9 rounds in total. The resulting next-generation sequencing data was then analyzed utilizing the MAGeCK pipeline to identify top scoring hits for validation regarding their role in T cell fitness and persistence. Among the hits with the highest positive fold changes, we found seven genes which are part of the Cullin-RING ligase 5 multi-protein complex (Fig. 1). These hits were then validated in a CAR-T cell context. We first edited primary human T cells by knocking out CUL5 as well as the endogenous TCR. The template for the B Cell Maturation Antigen (BCMA)-specific CAR bb2121 was then delivered to the TRAC locus via CRISPR/Cas9-RNP and adeno-associated virus (AAV). Here, we tested a panel with four CAR modifications, including two different co-stimulatory domains (CD28ζ, 4-1BB), a CAR with mutated immunoreceptor tyrosine-based activation motifs (ITAM) (1XX CAR), and a CAR containing an alternative BCMA-targeting single-chain variable fragment. This panel of CUL5 ablated CAR-T cells were then repeatedly co-cultured with BCMA-expressing tumor cells. After 6 rounds of tumor exposures, CUL5 ablated T cells showed a striking tumor killing advantage compared to control edited cells in a functional tumor killing assay (Fig. 2). Notably, we also observed vastly increased production of the cytotoxic cytokines INF-γand TNF-α in CUL5 ablated cells. Furthermore, the distinct tumor killing advantage of CUL5 KO cells in vitro was reproduced in the context of a CD19-specific CAR, which will be relevant in other clinical settings. Finally, we evaluated the striking tumor killing capacity of CUL5 ablated BCMA-specific CAR-T cells in vivo. For this, NOD.Cg- PrkdcscidIl2rgtm1Wjl/SzJ mice engrafted with luciferase-expressing BCMA+ multiple myeloma cell line OPM2 were treated with this panel of CUL5 ablated and control CAR-T cells and tumor burden was measured via Bioluminescent Imaging (BLI) over time. Mice treated with CUL5 KO BCMA-specific CAR-T cells showed lower median radiance measured by BLI, except for the 1XX CAR condition where both cohorts completely cleared the disease. In addition to the complete tumor clearance in both the control and CUL5 edited cohorts, we observed that the BLI levels in the 1XX CAR treated animals remained stable even after the mice were subjected to five tumor rechallenges. After 90 days, the mice were sacrificed and CAR+ human T cells were isolated from the bone marrow and spleens and phenotypically assessed. Analysis of these samples revealed a significantly higher frequency of CAR+ cells in the mouse bone marrow in the CUL5 KO group compared to control, while there were equivalent numbers in the spleens, suggesting an antigen-specific expansion advantage for the CUL5 KO CAR-T cells. We then co-cultured the isolated BCMA CAR-T cells from the spleens of these mice with OMP2 tumor cells in different E:T ratios and assessed tumor killing. We found a significant advantage in tumor killing of CUL5 ablated T cells compared to control cells in this ex vivo setting, suggesting an improved functional persistence. In summary, our findings identify the CUL5 complex as a novel potential therapeutic target to improve T cell persistence and counteract T cell exhaustion in order to improve the efficacy of adaptive cell immunotherapies for the treatment of BCMA+ hematological malignancies.

Phase I Open-Label Single-Arm Study of Sscart-19 in Adult Patients with Relapsed/Refractory B-Cell Acute Lymphoblastic Leukemia in China

Background: Although CAR-T therapy has shown amazing efficacy in the treatment of hematologic malignancies in recent years, it is also accompanied by high-frequency of cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), especially in acute lymphoblastic leukemia patients. Therefore, it is not recommended to enroll patients with active CNS involvement in CAR-T therapy. ssCART-19 is an autologous CD19-specific chimeric antigen receptor T-cell with short hairpin RNA-IL-6 gene silencing element. ssCART-19 can simultaneously express CAR structure and IL-6 silencing element. CAR structure can recognize and bind CD19 positive tumor cells, enhance T cell amplification, and finally exert T cell killing activity, such as releasing cytokines and perforin. Post-transcriptional expression of short hairpin RNA-IL-6 gene silencing element can recognize mRNA of IL-6, inhibit the expression of endogenous IL-6 in CAR-T cells through RNA interference mechanism, and then reduce the stimulation or activation intensity of downstream immune cells (mainly monocytes), with the control of the overall release intensity of cytokines, it reduces the incidence of severe CRS and ICANS, at the end reduces the clinical risk of CAR-T therapy. Methods: The trial of ssCART-19 in adult patients with R/R B-cell ALL (NCT04825496) is a single-arm, open-label Phase 1 study conducted in China. Patients underwent leukapheresis to obtain T cells for ssCART-19 manufacturing. Patients received fractionated infusions split over 3 days (10%; 30%; 60%) after lymphodepleting chemotherapy (3 days fludarabine and cyclophosphamide). Adverse events (AEs) were graded according to CTCAE, v5.0; CRS and ICANS were graded according to ASTCT Consensus Grading. The overall response rate(ORR) of complete response (CR) and CR with incomplete hematological recovery (CRi) within 3 months, duration of remission, relapse-free survival, and OS were assessed as secondary endpoints. Results : ssCART-19 was administered at 3 doses levels (DL) of 1×10 6/kg, 5×10 6/kg, or 10×10 6/kg. As of June 30th 2023 data cutoff (the median follow-up 15.9 months), 13 patients enrolled and underwent leukapheresis, 12 patients were received ssCART-19 treatment, 5 patients in DL1, 6 patients in DL2, and 1 patients in DL3. ssCART-19 was manufactured using serum-free culture system and no manufacture failure was reported. All 12 patients diagnosed with B-cell ALL were relapsed (75.0%) and refractory (25.0%) to multiple lines of prior therapy (100.0% patients had received 2 or more therapies). Five patients (41.7%) had more than 50% blasts in the bone marrow. All 12 patients did not have DLT events within 28 days. The most common ssCART-19-related AEs were transient neutropenia (100.0%), lymphopenia (100.0%), leukopenia (100.0%), anaemia (100.0%) and thrombocytopenia (83.3%). Adverse events of special interest (AESI) were CRS and ICANS. Among the 12 patients, 9 patients (75.0%) developed CRS, and 3 patients (25.0%) were grade 3 CRS. Notably, none of the 12 patients were occurred ICANS. No death cases were reported due to CRS or ICANS. A 83.3% overall response rate was achieved in efficacy-evaluable patients (n=12), with 7 complete response (CR, 58.3%), and 3 CR with incomplete hematological recovery (CRi,25.0%). The median duration of remission, relapse-free survival and OS have not been reached. Among them, 6 patients were in ongoing remission for over 12 months. Notably, one of the 12 patients (S012) had central nervous system infiltration with grade 2 before ssCART-19 infusion(Figure 1). After ssCART-19 therapy, the patient had no ICANS and got continuous remission. Robust CAR T-cell expansion occurred in all patients with a median time to T max of 12.5 days (range 9-14d), and peak copy number (C max) of 52495 (194-348000) copies/μg DNA. Conclusions ： ssCART-19 CAR-T cell therapy can effectively reduce the incidence of severe CRS and ICANS, especially control the occurrence of ICANS. And ssCART-19 achieved a high rate of remission in adult patients with R/R B-cell ALL. The ORR within 3 months were 83.3%. And, ssCART-19 shows good clinical efficacy in the treatment of leukemia patients with central nervous system infiltration. Thus, with the combination of short hairpin RNA-IL-6 gene silencing element, ssCART-19 can greatly improve the safety of CAR-T therapy, especially in central nervous system infiltration patients.

Nonactivated and IL-7 cultured CD19-specific CAR T cells are enriched in stem cell phenotypes and functionally superior

AbstractCD19-specific chimeric antigen receptor (CAR) T cells have demonstrated impressive responses in patients with relapsed and refractory B cell malignancies. However, many patients relapse or fail to respond to CD19 CAR T cells, demonstrating the need to improve its efficacy and durability. Current protocols for generating CAR T cells involve T cell activation through CD3 stimulation to facilitate efficient CAR transfer followed by ex vivo expansion with exogenous cytokines to obtain adequate cell numbers for treatment. Both T cell activation and expansion inevitably lead to terminal differentiation and replicative senescence, which are suboptimal for therapy. Interleukin-7 (IL-7) was previously shown to allow for lentiviral transduction of T cells in the absence of activation. In these studies, we used IL-7 to generate CD19 CAR T cells without stimulating CD3. Nonactivated and IL-7 cultured (NICE) CD19 CAR T cells were enriched with the T memory stem cell population, retained novel markers of stemness, had lower expression of exhaustion markers, and increased proliferative potential. Furthermore, our findings are consistent with engraftment of NICE CD19 CAR T cells and demonstrate a superior therapeutic response in both intraperitoneal and subcutaneous in vivo B cell lymphoma models. These results suggest that NICE CD19 CAR T cells may improve outcomes for B cell malignancies and warrant clinical evaluation.

How to integrate CD19 specific chimeric antigen receptor T cells with other CD19 targeting agents in diffuse large B-cell lymphoma?

About one third of patients with diffuse large B-cell lymphoma (DLBCL) have a relapsing/refractory (R/R) disease after first line chemo-immunotherapy, with particularly poor outcomes observed in patients with primary refractory disease and early relapse. CD19 specific chimeric antigen receptor (CAR) T cell therapy is a game changer that results in durable and complete response rates in almost half of the patients with R/R DLBCL. Other emerging CD19-targeting therapies include monoclonal antibodies, bispecific antibodies and targeting antibody-drug conjugates, which also show encouraging results. However, the timing and sequencing of different anti-CD19-targeting agents and how they might interfere with subsequent CAR T cell treatment is still unclear. In this review, we summarize the results of the pivotal clinical trials as well as evidence from real-world series of the use of different CD19-targeting approved agents. We discuss the effect of various therapies on CD19 expression and its implications for treatment sequencing.

Tumor-derived exosomes induce initial activation by exosomal CD19 antigen but impair the function of CD19-specific CAR T-cells via TGF-β signaling.

Tumor-derived exosomes (TEXs) enriched in immune suppressive molecules predominantly drive T-cell dysfunction and impair antitumor immunity. Chimeric antigen receptor (CAR) T-cell therapy has emerged as a promising treatment for refractory and relapsed hematological malignancies, but whether lymphoma TEXs have the same impact on CAR T-cell remains unclear. Here, we demonstrated that B-cell lymphoma-derived exosomes induce the initial activation of CD19-CAR T-cells upon stimulation with exosomal CD19. However, lymphoma TEXs might subsequently induce CAR T-cell apoptosis and impair the tumor cytotoxicity of the cells because of the upregulated expression of the inhibitory receptors PD-1, TIM3, and LAG3 upon prolonged exposure. Similar results were observed in the CAR T-cells exposed to plasma exosomes from patients with lymphoma. More importantly, single-cell RNA sequencing revealed that CAR T-cells typically showed differentiated phenotypes and regulatory T-cell (Treg) phenotype conversion. By blocking transforming growth factor β (TGF-β)-Smad3 signaling with TGF-β inhibitor LY2109761, the negative effects of TEXs on Treg conversion, terminal differentiation, and immune checkpoint expression were rescued. Collectively, although TEXs lead to the initial activation of CAR T-cells, the effect of TEXs suppressed CAR T-cells, which can be rescued by LY2109761. A treatment regimen combining CAR T-cell therapy and TGF-β inhibitors might be a novel therapeutic strategy for refractory and relapsed B-cell lymphoma.

Integrome signatures of lentiviral gene therapy for SCID-X1 patients.

Lentiviral vector (LV)-based gene therapy holds promise for a broad range of diseases. Analyzing more than 280,000 vector integration sites (VISs) in 273 samples from 10 patients with X-linked severe combined immunodeficiency (SCID-X1), we discovered shared LV integrome signatures in 9 of 10 patients in relation to the genomics, epigenomics, and 3D structure of the human genome. VISs were enriched in the nuclear subcompartment A1 and integrated into super-enhancers close to nuclear pore complexes. These signatures were validated in T cells transduced with an LV encoding a CD19-specific chimeric antigen receptor. Intriguingly, the one patient whose VISs deviated from the identified integrome signatures had a distinct clinical course. Comparison of LV and gamma retrovirus integromes regarding their 3D genome signatures identified differences that might explain the lower risk of insertional mutagenesis in LV-based gene therapy. Our findings suggest that LV integrome signatures, shaped by common features such as genome organization, may affect the efficacy of LV-based cellular therapies.

Sustained Remission and Decreased Severity of CAR T-Cell Related Adverse Events: A Pivotal Study Report of CNCT19 (inaticabtagene autoleucel) Treatment in Adult Patients with Relapsed/Refractory B-Cell Acute Lymphoblastic Leukemia (R/R B-Cell ALL) in China

CNCT19 (inaticabtagene autoleucel) is an autologous CD19-specific chimeric antigen receptor (CAR) T-cell product. The patent protected CAR structure of CNCT19 contains a unique CD19 scFv, HI19a, which is different from commonly used FMC63. Together with using 4-1BB co-stimulatory domain in the CAR structure, CNCT19 is expected to reduce the severity of treatment-associated cytokine release syndrome (CRS) and neurologic toxicities (NT) while maintaining a stronger and longer durable anti-tumor effect.CNCT19 has been granted Breakthrough Therapy Designation by China National Medical Products Administration and Orphan Drug Designation by the U.S. FDA for the treatment of ALL.The trial of CNCT19 in adult Chinese patients with R/R B-cell ALL (NCT04684147) is a single-arm, open-label pivotal study conducted at 10 centers in China. The primary endpoint was the overall complete response rate (OCR) of complete response (CR) and CR with incomplete hematological recovery (CRi) within 3 months and at the end of Month 3 after CNCT19 infusion by central assessment.All 39 patients diagnosed with B-cell ALL were refractory and relapsed to multiple lines of prior therapy. Among the 39 patients 32 (82.1%) had reached MRD-negative OCR within 3 months after CNCT19 infusion, The median duration of response and OS have not been reached. 25 patients (64.1%) remained on CR (51.3%) or CRi (12.8%). at the end of Month 3 after CNCT19 infusion These patients had sustained long-term remission regardless of whether subsequent allo-HSCT treatment was done or not. The most common CNCT19-related adverse events (AEs) were CRS and NT and there were 4 cases of Grade ≥ 3 CRS (n=4, 10.3%) and 3 cases of Grade ≥ 3 NT (n=3, 7.7%). Following CNCT19 infusion, all the patients recovered. No death cases were reported due to CRS or NT.CNCT19 CAR-T cell therapy achieved a high rate of MRD-negative complete remission in adult patients with R/R B-cell ALL. Thus, with its distinct CAR structure containing a unique CD19 scFv (HI19a), CNCT19 provides effective treatment with potential long-term clinical benefits for adult patients with R/R B-cell ALL.

Cord blood-derived CD19-specific chimeric antigen receptor T cells: an off-the-shelf promising therapeutic option for treatment of diffuse large B-cell lymphoma.

Autologous chimeric antigen receptor (CAR) T cell therapy is one of the most significant breakthroughs in hematological malignancies. However, a three-week manufacturing cycle and ineffective T cell dysfunction in some patients hinder the widespread application of auto-CAR T cell therapy. Studies suggest that cord blood (CB), with its unique biological properties, could be an optimal source for CAR T cells, providing a product with 'off-the-shelf' availability. Therefore, exploring the potential of CB as an immunotherapeutic agent is essential for understanding and promoting the further use of CAR T cell therapy. We used CB to generate CB-derived CD19-targeting CAR T (CB CD19-CAR T) cells. We assessed the anti-tumor capacity of CB CD19-CAR T cells to kill diffuse large B cell lymphoma (DLBCL) in vitro and in vivo. CB CD19-CAR T cells showed the target-specific killing of CD19+ T cell lymphoma cell line BV173 and CD19+ DLBCL cell line SUDHL-4, activated various effector functions, and inhibited tumor progression in a mouse (BALB/c nude) model. However, some exhaustion-associated genes were involved in off-tumor cytotoxicity towards activated lymphocytes. Gene expression profiles confirmed increased chemokines/chemokine receptors and exhaustion genes in CB CD19-CAR T cells upon tumor stimulation compared to CB T cells. They indicated inherent changes in the associated signaling pathways in the constructed CB CAR T cells and targeted tumor processes. CB CD19-CAR T cells represent a promising therapeutic strategy for treating DLBCL. The unique biological properties and high availability of CB CD19-CAR T cells make this approach feasible.

Immunogenetic Metabolomics Reveals Key Enzymes That Modulate CAR T-cell Metabolism and Function.

Immune evasion is a critical step of cancer progression that remains a major obstacle for current T cell-based immunotherapies. Hence, we investigated whether it is possible to genetically reprogram T cells to exploit a common tumor-intrinsic evasion mechanism whereby cancer cells suppress T-cell function by generating a metabolically unfavorable tumor microenvironment (TME). In an in silico screen, we identified ADA and PDK1 as metabolic regulators. We then showed that overexpression (OE) of these genes enhanced the cytolysis of CD19-specific chimeric antigen receptor (CAR) T cells against cognate leukemia cells, and conversely, ADA or PDK1 deficiency dampened this effect. ADA-OE in CAR T cells improved cancer cytolysis under high concentrations of adenosine, the ADA substrate, and an immunosuppressive metabolite in the TME. High-throughput transcriptomics and metabolomics analysis of these CAR T cells revealed alterations of global gene expression and metabolic signatures in both ADA- and PDK1-engineered CAR T cells. Functional and immunologic analyses demonstrated that ADA-OE increased proliferation and decreased exhaustion in CD19-specific and HER2-specific CAR T cells. ADA-OE improved tumor infiltration and clearance by HER2-specific CAR T cells in an in vivo colorectal cancer model. Collectively, these data unveil systematic knowledge of metabolic reprogramming directly in CAR T cells and reveal potential targets for improving CAR T-cell therapy.

Enhancing the effectiveness of γδ T cells by mRNA transfection of chimeric antigen receptors or bispecific T cell engagers

Adoptive cell therapy (ACT) utilizing γδ Tcells is becoming a promising option for the treatment of cancer, because it offers an off-the-shelf allogeneic product that is safe, potent, and clinically effective. Approaches to engineer or enhance immune-competent cells for ACT, like expression of chimeric antigen receptors (CARs) or combination treatments with bispecific Tcell engagers, have improved the specificity and cytotoxic potential of ACTs and have shown great promise in preclinical and clinical settings. Here, we test whether electroporation of γδ Tcells with CAR or secreted bispecific Tcell engager (sBite) mRNA is an effective approach to improve the cytotoxicity of γδ Tcells. Using a CD19-specific CAR, approximately 60% of γδ Tcells are modified after mRNA electroporation and these cells show potent anticancer activity invitro and invivo against two CD19-positive cancer cell lines. In addition, expression and secretion of a CD19 sBite enhances γδ Tcell cytotoxicity, both invitro and invivo, and promotes killing of target cells by modified and unmodified γδ Tcells. Taken together, we show that transient transfection of γδ Tcells with CAR or sBite mRNA by electroporation can be an effective treatment platform as a cancer therapeutic.

Development of a liquid tumor potency assay for evaluation of immune cell-mediated cytotoxicity in vitro

Abstract The development of immunotherapies relies on the use of in vitro potency assays—which are key for understanding complex interactions between immune (effector) cells and cancer (target) cells—to evaluate the function, specificity, and sensitivity of a product. A variety of in vitro assays are used to characterize the proliferation, cytokine release, and cell-mediated cytotoxicity of engineered immune cells, such as chimeric antigen receptor (CAR) T cells, with each representing an important aspect driving clinical efficacy of the cellular product. Previous work has shown that impedance-based potency assays provide a real-time, label-free measure of effector cell-mediated cytotoxicity for adherent target cells. Here, we describe an in vitro potency assay that quantifies effector cell-mediated cytotoxicity of liquid tumor target cells. First, the wells were coated with antibody to tether the liquid tumor target cells (Raji = anti-CD40, K562 = anti-CD71, Daudi = anti-CD40). Tethering the liquid tumor target cells significantly increased the impedance measurement, as compared to untethered controls. CD19-specific CAR T effector cells were added 24 hours after the target cells and co-cultured with the target cells across a range of effector to target ratios. Cytolysis of the target cells was calculated by comparing the impedance in treated wells to no treatment control wells. As expected, the CD19-specific CAR T cells produced significantly higher cytolysis in the Daudi (54.3%) and Raji (70.4%) cells, which express CD19, as compared to the K562 cells (30.5%). These results support a real-time, label-free potency assay with liquid tumor target cells.

Abstract 4105: Improvement of CD133-specific chimeric antigen receptor T cells by secreting anti-PD-L1 single-chain variable fragment against cholangiocarcinoma

Abstract Cholangiocarcinoma (CCA) is rare but hard-to-treat solid cancer affecting people worldwide. Cancer stem cells (CSCs) in CCA contribute to tumor heterogeneity and resistance to conventional therapies. Thus, CSCs are notable targets for cancer immunotherapy. CD133 is a potential CSC surface marker overexpressed in various cancers. T cell-based immunotherapy, namely chimeric antigen receptor (CAR) T-cell targeting CD133 has shown potential in eradication of some solid tumors including CCA. Nevertheless, anti-tumor function of CAR T cell could be decreased under immunosuppressive tumor microenvironment (TME). Thus, to improve its function under TME, the next-generation of CD133-specific CAR T-cell is required. We therefore constructed two lentiviral vectors containing: (i) anti-CD133 single-chain variable fragment (scFv), 4-1BB costimulatory molecule, and CD3ζ (133CAR), and (ii) anti-CD133 scFv, 4-1BB, and CD3ζ in tandem with anti-PD-L1 scFv (133CARsL). The lentiviruses were transduced to generate CD133-specific CAR T cells expressing the second-generation (133CART) or anti-CD133 CAR with self-secreting anti-PD-L1 scFv (133CARTsL). The latter was able to simultaneously target CD133 and PD-L1 molecules on CCA cells. CCA cells endogenously expressing CD133 and PD-L1 were cocultured with either 133CART or 133CARTsL. In short-term coculture, the antitumor effects of 133CARTsL were not different from that of 133CART. However, in long-term coculture and after tumor re-challenge, 133CARTsL showed lower expression of programmed cell death protein (PD-1) and better cytotoxic function against high CD133 and PD-L1 CCA cells. The higher potential of 133CARTsL with reduced exhaustion profile would offer great promise for application of CD133-targeting CAR T cells in solid tumors. Citation Format: Thanich Sangsuwannukul, Kamonlapat Supimon, Thaweesak Chieochansin, Kornkan Choomee, Jatuporn Sujjitjoon, Mutita Junking, Pa-thai Yenchitsomanus. Improvement of CD133-specific chimeric antigen receptor T cells by secreting anti-PD-L1 single-chain variable fragment against cholangiocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4105.