CAR-T Cells In Patients Research Articles

Addition of Nivolumab Tailored by Expansion of CAR-T Cells in Patients with Stable/Progressive Large B Cell Lymphoma at Lymphodepletion—A Phase 2, Prospective Interventional Study

Patients with large B-cell lymphoma (LBCL) in stable or progressive disease (SD/PD) at lymphodepletion prior to chimeric antigen receptor T cell (CAR-T) therapy have an inferior outcome. we hypothesized that enhancing in-vivo expansion of CAR-T cells could overcome this grim prognosis leading to improved outcomes. We conducted a phase 2 prospective trial (NCT05385263) investigating the addition of nivolumab to enhance CAR-T cell expansion and response in patients with SD/PD-LBCL. Eligible patients received 1 dose of nivolumab between day +5 and +9 post CAR-T infusion. An additional dose of nivolumab was administered on day +19 only to patients whose CAR-T cell levels in peripheral blood were below 100 cells/µL at day +7. Twenty patients were enrolled and received anti-CD19 CAR-T (Axicabtagene ciloleucel, n = 12; tisagenlecleucel, n = 8). Eight were ineligible to receive nivolumab due to active CAR-T-associated toxicities. Overall, the protocol was safe. One-month PET-CT showed an 84% overall response rate (complete response, 53%). The cumulative incidence of progression-free survival at 6 and 12 months were 50% (95% CI 36%-64%) and 42% (95% CI 26%-58%), respectively. The cumulative incidence of overall survival at 6 and 12 months were 85% (95% CI 72%-98%) and 51% (95% CI 31%-71%), respectively. Nivolumab administration significantly reduced PD-1 expression on all immune cells. CAR-T cell expansion was similar between nivolumab-eligible and noneligible patients. Notably, there was a significant enrichment of CD45RO-CD27+ CD8+ cells and CD45RO-CD27+ CD8+ CAR-T cells in the nivolumab-eligible group compared to those ineligible, suggesting that specific cell enrichment could potentially contribute to an enhanced response rate. We conclude that the addition of nivolumab based on CAR-T cell expansion in patients with SD/PD-LBCL is safe and yields promising early response rates.

An exploratory investigator-initiated trial via intrathecal or intracerebroventricular delivery of B7H3-specific allogeneic universal CAR-T cells in patients with recurrent high-grade gliomas.

23 Background: The MT027, developed by T-Maximum, is a B7-H3 (CD276)-directed genetically modified allogeneic T cell, targeting CD276-overexpressed tumor cells. Here, we reported a first-in-human, single-center, open-label investigator-initiated trial (IIT) via intrathecal or Intracerebroventricular delivery targeting recurrent high-grade glioma (ChiCTR2100047968). Methods: As of March 7, 2024, a total of 50 adult patients with recurrent high-grade glioma, B7H3+ expression, and KPS ≥40 were enrolled and administered at least one dose of MT027 (FAS), of whom, 32 received at least 3 doses (PPS1), 15 subjects with ≥3 doses and at least 1 efficacy visit (PPS2). 4 dose levels of 1.0, 1.5, 2.0, 2.5×107 cells per injection were administered once every 4 weeks. Results: The overall results demonstrated the favorable tolerability, safety, PK/PD characteristics, and preliminary efficacy. In FAS, the most common AE were fever and headache, without any CRS, ICANS, GvHD, or drug-related death. There were 4 cases (8%) of grade 3 AE or greater and 3 cases (6%) of SAEs. The copies of CAR in all groups reached 10000-100000 copy/ug, and persisted for > 60 days after a single dose. After multiple doses, the MT027 CAR-T cell numbers maintained at a high level (median 8850 copies /μg (range: 200-385900)) and for a long time. The levels of IL6, TNF-α and IFN-γ in CSF increased significantly and maintained for a long time. The mOS was 13.54 m (95% CI: 7.45; 19.63) and 20.73 m (95% CI: 16.12; 25.34) in PPS1 and PPS2, respectively. The 12-month OS rate was 53.30% (95%CI: 37.5%-75.7%) and 84.60% (95%CI: 67.1%-100%) in PPS1 and PPS2, respectively (historical data: 14%). The objective response rate (ORR) was 33% and the disease control rate (DCR) was 80% in PPS2. Conclusions: In patients with relapsed high-grade glioma, the intrathecal or ICV administration of MT027 cell therapy was found to be safe and well-tolerated. The toxicity of grade 3 or above was 1/5 of similar products. The pharmacokinetics and pharmacodynamics of single-dose and multiple-dose treatments were stable. MT027 exhibited longer persistence in the body of the patients, and the cell numbers and cytokine responses induced in vivo were 1-2 orders of magnitude higher than those of similar products. MT027 cell therapy demonstrated the excellent efficacy in treating relapsed high-grade glioma, with significantly prolonged overall survival and higher objective response rate compared to the historical data and similar product data. Currently, we are preparing for the IND filing in US (expected in 2024) with the recurrent glioblastoma as the first indication. Clinical trial information: ChiCTR2100047968 .

Safety and biological outcomes following a phase 1 trial of GD2-specific CAR-T cells in patients with GD2-positive metastatic melanoma and other solid cancers

BackgroundChimeric antigen receptor (CAR) T cell therapies specific for the CD19 and B-cell maturation antigen have become an approved standard of care worldwide for relapsed and refractory B-cell malignancies. If...

Liquid biopsy approach to monitor the efficacy and response to CAR-T cell therapy

BackgroundChimeric antigen receptor (CAR)-T cells are approved for use in the treatment of hematological malignancies. Axicabtagene ciloleucel (YESCARTA) and brexucabtagene autoleucel (TECARTUS) genetically modified autologous T cells expressing an anti-CD19...

Single-cell analysis of refractory anti-SRP necrotizing myopathy treated with anti-BCMA CAR-T cell therapy

Immune-mediated necrotizing myopathy (IMNM) is an autoimmune disorder associated with the presence of autoantibodies, characterized by severe clinical presentation with rapidly progressive muscular weakness and elevated levels of creatine kinase, while traditional pharmacological approaches possess varying and often limited effects. Considering the pathogenic role of autoantibodies, chimeric antigen receptor (CAR)-T cells targeting B cell maturation antigen (BCMA) have emerged as a promising therapeutic strategy. We reported here a patient with anti-signal recognition particle IMNM refractory to multiple available therapies, who was treated with BCMA-targeting CAR-T cells, exhibited favorable safety profiles, sustained reduction in pathogenic autoantibodies, and persistent clinical improvements over 18 mo. Longitudinal single-cell RNA, B cell receptor, T cell receptor sequencing analysis presented the normalization of immune microenvironment after CAR-T cell infusion, including reconstitution of B cell lineages, replacement of T cell subclusters, and suppression of overactivated immune cells. Analysis on characteristics of CAR-T cells in IMNM demonstrated a more active expansion of CD8+ CAR-T cells, with a dynamic phenotype shifting pattern similar in CD4+ and CD8+ CAR-T cells. A comparison of CD8+ CAR-T cells in patients with IMNM and those with malignancies collected at different timepoints revealed a more NK-like phenotype with enhanced tendency of cell death and neuroinflammation and inhibited proliferating ability of CD8+ CAR-T cells in IMNM while neuroinflammation might be the distinct characteristics. Further studies are warranted to define the molecular features of CAR-T cells in autoimmunity and to seek higher efficiency and longer persistence of CAR-T cells in treating autoimmune disorders.

Overall review of curative impact and barriers of CAR-T cells in osteosarcoma.

Osteosarcoma (OS) is a rare form of cancer and primary bone malignancy in children and adolescents. Current therapies include surgery, chemotherapy, and amputation. Therefore, a new therapeutic strategy is needed to dramatically change cancer treatment. Recently, chimeric antigen receptor T cells (CAR-T cells) have been of considerable interest as it has provided auspicious results and patients suffering from low side effects after injection that resolve with current therapy. However, there are reports that cytokine release storm (CRS) can be observed in some patients. In addition, as researchers have faced problems that limit and suppress T cells, further studies are required to resolve these problems. In addition, to maximize the therapeutic benefit of CAR-T cell therapy, researchers have suggested that combination therapy could be better used to treat cancer by overcoming any problems and reducing side effects as much as possible. This review summarizes these problems, barriers, and the results of some studies on the evaluation of CAR-T cells in patients with osteosarcoma.

Chimeric Antigen Receptor-T Cell Therapy for Lymphoma: New Settings and Future Directions.

In the last decade, anti-CD19 CAR-T cell therapy has led to a treatment paradigm shift for B-cell non-Hodgkin lymphomas, first with the approval for relapsed/refractory (R/R) large B-cell lymphomas and subsequently for R/R mantle cell and follicular lymphoma. Many efforts are continuously being made to extend the therapeutic setting in the lymphoma field. Several reports are supporting the safety and efficacy of CAR-T cells in patients with central nervous system disease involvement. Anti-CD30 CAR-T cells for the treatment of Hodgkin lymphoma are in development and early studies looking for the optimal target for T-cell malignancies are ongoing. Anti-CD19/CD20 and CD19/CD22 dual targeting CAR-T cells are under investigation in order to increase anti-lymphoma activity and overcome tumor immune escape. Allogeneic CAR product engineering is on the way, representing a rapidly accessible 'off-the-shelf' and potentially more fit product. In the present manuscript, we will focus on recent advances in CAR-T cell therapy for lymphomas, including new settings and future perspectives in the field, reviewing data reported in literature in the last decade up to October 2023.

SOR-CART-MM-001 Study: A Phase I, Open-Label, Dose-Escalation, Pharmacokinetic, and Pharmacodynamic Study of Safety and Efficacy of CAR2 Anti-CD38 A2 CAR-T Cells in Patients with Relapsed or Refractory Multiple Myeloma (RRMM)

Background: B-cell maturation antigen (BCMA) directed chimeric antigen receptor (CAR-) T cells, show high response rates in patients with highly refractory and heavily treated myeloma, and have become a standard treatment in this setting. The durability of these responses, however, has been limited with few long term remissions. One approach to potentially improve CAR T outcome is to direct these cells to alternative targets such as CD38 almost universally expressed on malignant plasma cells. In this open-label phase I multi-institutional study we explored the efficacy and safety of CAR2 anti-CD38 A2 CAR-T cells in patients with RRMM. Methods: The SOR-CART-MM-001 study was a multicenter, open-label, “3+3” dose-escalation, phase 1b trial of a single administration of a fully human antibody with lambda light chain generated by transduction of activated T cells with an anti-CD38 A2 CAR expressing retroviral vector. A single dose of CAR2 Anti-CD38 CAR-T cells was administered 2 days post lymphodepletion with single-agent cyclophosphamide 1.5 g/m 2. The primary endpoint was determination of the maximum tolerated dose (MTD) assessed by the occurrence of treatment-emergent dose-limiting toxicities during the 28-day treatment period in the dose-escalation phase of the study. Dosing was divided into three cohorts ranging from 1 × 10 5 cells/kg (Cohort 1) to 1 × 10 7 cells/kg (Cohort 3). Circulating CAR-T cells were measured at different timepoints post infusion. Key eligibility criteria included ≥3 lines of therapy with prior proteasome inhibitor, immunomodulatory IMID drug or daratumumab exposure, measurable and progressive MM per International Myeloma Working Group criteria and ECOG ≤ 2. Results: Between March 2018 and March 2022, 9 patients were enrolled and assigned to each of the three Cohorts. Patient demographics and baseline characteristics are described in Table 1. Hematologic toxicity was the most common treatment emergent adverse event experienced (6/9, 66.7%), all Grade 3 or Grade 4. All cytopenias recovered in 21 days in subjects treated with no more than MTD. Two subjects within Cohort 3 exhibited mild cytokine release syndrome (CRS) (Grade 1 and 2) with one of these two subjects also developing mild neurotoxicity (Grade 2). No CRS or neurotoxicity was observed at either of the lower dose levels. Two serious adverse events (SAEs) were reported: Klebsiella sepsis 60 days after treatment that recovered in 4 days after antibiotics and muscle weakness at 24 days after treatment that received within 1 day without treatment and neither were deemed related to the study medication. The first 2 patients in Cohort 3 met dose limiting toxicity (DLT) criteria after treatment due to grade 3 hematological toxicity and the next lower dose 1 x 10 6 cells/kg (Cohort 2) became the MTD. Pharmacokinetic data showed the percentage of the detectable circulating CAR-T cells in any subject at any time point was low with many showing no detectable cells. 4 deaths were reported after the end of the study period, and none were considered related to the investigated CAR-T cells (3 due to progression and one due to COVID-19). Overall, a total of 3 patients had a partial response (PR) and 3 patients had stable disease (SD) across 3 dose levels. PR constituted the best response in the study and the durations of objective clinical responses were each less than 3 months. These treatment responses are summarized in Table 2. Conclusion: This phase I trial demonstrated that CAR2 anti-CD38 A2 CAR-T cells were generally well tolerated at the Cohort 2 (MTD) dose and demonstrated objective though short responses even in patients progressing after daratumumab and belantamab mafodotin. More clinical studies are needed to optimize the efficacy and tolerability of CAR-T cell therapies for patients with RRMM. This study was completed, and no more patients will be enrolled. DART-RRMM-101 is a phase 1b, open-label, dose-escalation study ongoing to assess allogeneic Anti-CD38 A2 Dimeric Antigen Receptor T Cells in RRMM (ClinicalTrials: NCT05007418).

Long-Term Follow-up of Humanized and Murine CD19 CAR-T Cell Therapy for B-Cell Malignancies

Aim: CD19 chimeric antigen receptor T cell (CAR-T) therapy is a revolutionary treatment for relapsed/refractory (R/R) B-cell hematologic malignancies. However, the safety and efficacy of murine CAR-T cells which have been widely used as commercialized products still need to be improved. Similarly, murine CAR-T encountered the problem that CAR-related immunogenicity existed after the infusion, which led to the failure of the re-infusion of CAR-T cells. As an improved generation of murine CAR-T cells, whether humanized CAR-T therapy can overcome the above shortcomings remains to be explored. Methods: In this study, the differences in safety, efficacy, and long-term follow-up results of murine and humanized CD19 CAR-T cells in patients with B-cell malignancies were collected and analyzed. A total of 130 patients were enrolled, including 35 patients with murine CAR-T and 95 with humanized CAR-T cells (NCT 02965092, NCT 04008251). Results： The proportion of patients with cytokine release syndrome (CRS) in the murine and humanized groups was 54.3%（19/35） and 61.1%（58/95）, respectively. A significantly higher proportion of patients suffered from severe CRS in the murine group than that in the humanized CAR-T group (17.14% vs. 7.4%, P=0.0233), and one patient in each group died of Grade 5 CRS. The incidence of grade 1-2 Immune effector cell-associated neurotoxicity syndrome (ICANS) was 11.4% (murine: 4/35) and 5.3% (humanized: 5/95), but high-grade ICANS was not observed. Among patients receiving murine CAR-T cells, 80% achieved objective response (OR), 71.43% achieved complete response (CR) and the CR rate of patients with leukemia was 74.19%. The OR rate and CR rate of patients in the humanized group were 75.79% and 63.16%, respectively, while the CR rate of patients with leukemia receiving humanized CAR-T cells was 89.58%. The median follow-up time of patients in the murine and humanized groups was 7 months (0.6-75 months) and 5.5 months (0.5-34 months), respectively. The median progression-free survival (PFS) of patients with murine CAR-T cells was 11 months and that of patients with humanized CAR-T products was 12 months. Both of the median overall survival (OS) were not reached. Among the 48 patients with a bone marrow burden over 20% at baseline, humanized CAR-T therapy was associated with a higher OS rate (84.64% vs. 59.26%, P=0.1479) and a significantly improved PFS (43.94% vs. 33.33%, P=0.0217, Figure 1). Bridging transplantation was an independent factor in prolonging OS (x 2=8.017, p=0.0046) and PFS (x 2=6.584, p=0.0103) of patients with leukemia. Neither species of chimeric antigen receptor nor common risk factors such as age, recurrence, bone marrow burden, BCR-ABL fusion gene had significant effect on patients' long-term follow-up outcomes (Table 1). Four patients received CAR-T therapies more than once. Three of them still achieved varying degrees of long-term remission after multiple humanized CAR-T infusions. However, one patient relapsed as soon as one month after his second infusion of murine CAR-T cells. Conclusion: Results indicate that humanized CAR-T therapy showed better safety and durable efficacy, especially in patients with higher tumor burden in bone marrow. Moreover, it could overcome immunogenicity-induced CAR-T resistance, providing treatment options for patients' failures of CAR-T therapies.

Real-Word Experience of CAR T-Cells in Patients with Relapsed/Refractory Follicular Lymphoma : A Descart Registry Analysis from the Lysa

Background: Anti-CD19 CAR T-cells have revolutionized the treatment of aggressive B-cell non-Hodgkin lymphomas (NHL) by demonstrating durable responses. Although follow-up remain short, axicabtagene ciloleucel (axi-cel, ZUMA-5) and tisagenlecleucel (tisa-cel, ELARA) demonstrated promising complete response rates (CRR) of 86% and 69%, respectively, in phase II trials including relapsed or refractory (R/R) follicular lymphoma (FL).. Tisa-cel and axi-cel are now approved after at least 2 lines of systemic therapy by the FDA. Few real-word evidence (RWE) data have been reported so far. Methods: Patients were eligible if aged >18 years, had FL histology (grade 1-3A) and had received at least 2 previous treatment lines for tisa-cel and 3 for axi-cel according to the French early access program label. From Dec 2021 to Jan 2023, a total of 112 patients were included in early access program by 21 French centers and in DESCAR-T registry (NCT04328298), among them 87 were infused with a CAR product. At the data cut-off on March 2023, 70 had at least 1 month of follow-up (FU) with PET-CT evaluation, and thus were considered for the safety and efficacy sets (62 tisa-cel and 8 axi-cel,). Results: Patient and disease characteristics were as follows: 46 males (65.7%), median age 62 years (range: 34-79), 3 median prior lines of therapy (range 2-9, including bi-specific antibody in 12.9% and autoSCT in 44.3%), FLIPI 0-2 in 40.5% and 3-5 in 49.5%, bulky disease (>5cm) in 22%, and LDH > N 52.2%. POD24 after 1 st systemic immunochemotherapy (IC) was reported in 44 patients (62.8%). Before CAR T-cell infusion, 58.6% of patients received a bridging therapy (20 (48.8%) chemotherapy, 25 (61%) monoclonal antibodies, 4 (9.8%) kinase inhibitors, 15 (36.6%) lenalidomide, and 3 (7.3%) radiotherapy). All patients but one received a fludarabine and cyclophosphamide-based lymphodepletion. Median time from order to infusion was 48 days (range 34-204), and 41 days (30-328) from leukapheresis to infusion. Median FU was 7.3 months [6.4; 8.2] from product order, and 5.4 months [3.4; 6] from CAR infusion. Best ORR and CRR were 97.5 and 87.5%, respectively with 72.5% of patients in CR at 1 month with projected DOR and DOCR of 72.4% [56.8; 83.1%], and 79.7% [62.7%; 89.6%], respectively. Projected 6-months PFS, OS, were 71.8% [95% CI, 56.6%; 82.4%], 97.4% [83.2%; 99.6%], respectively. Fifteen (21.4%) patients progressed (median time from infusion to first relapse: 3 months (range 1-10 mo). Only one patient died from lymphoma progression. Any-grade CRS and ICANS were reported for 74.3% and 27.1% of patients, but grade 3-4 CRS/ICANS were very unfrequent,1.4% and 4.3%, respectively. No grade 5 was reported. Persisting grade 3-4 hematologic toxicities at 1 and 3 months were: neutropenia (50% and 12.3%), thrombocytopenia (18.6% and 0%), and anemia (8.6% and 0%). Medically relevant bacterial and viral infections were reported in 20% and 14.3% of patients, respectively. Conclusions: Although longer FU is needed to assess disease control, RWE data from the DESCAR-T registry confirm the excellent response rates and safety profile of CAR T-cells in R/R FL after at least 2 lines of previous therapy.

High Efficacy and Excellent Safety Profile of Actalycabtagene Autoleucel, a Humanized CD19 CAR-T Product in r/r B-Cell Malignancies: A Phase II Pivotal Trial

Background: Commercially approved CD19 CAR-T cell therapies are effective in r/r B cell malignancies, but are associated with significant albeit manageable toxicities. These toxicities contribute to significant morbidity. We have developed a novel, humanized CD19 CAR-T cell therapy, Actalycabtagene autoleucel (Actaly-cel) and previously reported the safety in Phase I study (Dwivedi et al. Mol Cancer Ther.2021, Karulkar et al. ASH 2022). Here, we present the pooled results from Phase I and a planned interim analysis of the Phase II study evaluating Actaly-cel. Materials and methods: The Phase I study (n=10) was a single-center trial to test the safety of Actaly-cel at a dose of 1x10 7 to 5x10 9 CAR-T cells in patients with r/r DLBCL, tFL and PMBCL (CTRI/2021/04/032727). The Phase II study (n=50) was a single-arm, multi-center study conducted across 3 centers in India (CTRI/2022/12/048211). Patients with r/r B-cell malignancies (including aggressive and indolent B-cell lymphomas and acute lymphoblastic leukemia) aged ≥ 15 years, with normal organ function, ECOG PS 0-2 and measurable disease were eligible. Actaly-cel was manufactured using an integrated semi-automated system. Bridging therapy was allowed at the clinician's discretion. Patients received infusion of Actaly-cel two days after conditioning with fludarabine plus cyclophosphamide. The target dose was ≥5x10 6/kg CAR T-cells (efficacy-evaluable cohort), however doses of ≥0.5x 10 6/kg CAR-T cells were allowed. Patients who received Actaly-cel at any dose were considered in the safety analysis. The primary endpoint was objective response rate, with duration of response, adverse events, PFS and OS as the secondary endpoints. A Simon II-stage design was used to test the hypothesis that the ORR would increase from 25% (historical data) to 40% (⍺- 0.1 and β- 0.2), >15 responses were required out of 50 patients to reject the null hypothesis. The CRS and neurotoxicities (ICANS) were graded and treated as per ASTCT guidelines, other toxicities were graded as per CTCAE v5. Exploratory studies were performed to study the correlation between patient and product related characteristics with response. Results: A total of 59 patients with r/r DLBCL (41), ALL (16) and indolent lymphomas (2) were enrolled (Phase I; 06/2021 till 06/2022 and Phase II; 12/2022 till 07/2023), 56 underwent leukapheresis (ITT set) and 47 received Actaly-cel (safety set). 43 patients received the target dose (Figure 1A). The median age was 43 years (16-71) with 46 (65%) male patients. Patients had a median of 2 prior lines of therapy (1-6), 65% had refractory disease,25% of lymphoma patients had bulky disease, and the median bone marrow blast was 69% (6-98%) in the B-ALL cohort. The median vein-to-vein time was 17(7-132) days. Total 78%(33/43) patients reached the day 28 timepoint at the time of analysis (Figure 1B). The ORR was 70% (23/33), including 58% (19/33) with a CR. In the lymphoma cohort, the ORR was 71%(17/24) and CR was observed in 54%(13/24), while in the leukemia cohort, the CR rate was 66% (6/9, n=5 MRD negative). The median follow-up of evaluable patients was 57 days (21-453). All nine patients and 3 out of 4 patients maintained the response after 3 months and 12 months follow-up, respectively. None of the patients developed ICANS of any grade. CRS developed in 22/33(66%) cases (grade 1/2 in 61%, grade 3/4 in 6%). Of note, no grade >3 CRS was noticed in the lymphoma cohort. Grade 3/4 cytopenias developed in all cases. The median duration of neutropenia was 7 (4-32) days. On Day 28, grade 3/4 neutropenia was observed in 11/33(33%) cases, grade 3/4 thrombocytopenia was observed in 7/33 (21%). Only 1(3%) patient required ICU admission, 2(6%) required vasopressor support, tocilizumab was administered to 18 (55%) patients, median 1 dose (1-4) and steroids were used in 5 (15%) cases. Patients were hospitalized for a median of 8 days (7-19 ). There were no treatment-related deaths. On exploratory analysis, higher CAR-T dose, fewer prior lines of therapy and higher peak CAR-T copies/µg DNA correlated with response. Conclusions: Actaly-cel demonstrated efficacy in r/r B-cell malignancies with a very favorable safety profile. The absence of ICANS, shorter duration of cytopenias and a lower incidence of grade 3/4 CRS makes it one of the safest CD19 CAR-T cell therapy products. Actaly-cel can improve the ease of delivery of CAR T-cell therapy in a wide-range of settings.

Functional diversification and dynamics of CAR-T cells in patients with B-ALL

Understanding of cellular evolution and molecular programs of chimeric antigen receptor-engineered (CAR)-T cells post-infusion is pivotal for developing better treatment strategies. Here, we construct a longitudinal high-precision single-cell transcriptomic landscape of 7,578 CAR-T cells from 26 patients with B cell acute lymphoblastic leukemia (B-ALL) post-infusion. We molecularly identify eight CAR-T cell subtypes, including three cytotoxic subtypes with distinct kinetics and three dual-identity subtypes with non-T cell characteristics. Remarkably, long-term remission is coincident with the dominance of cytotoxic subtypes, while leukemia progression is correlated with the emergence of subtypes with B cell transcriptional profiles, which have dysfunctional features and might predict relapse. We further validate invitro that the generation of B-featured CAR-T cells is induced by excessive tumor antigen stimulation or suppressed TCR signaling, while it is relieved by exogenous IL-12. Moreover, we define transcriptional hallmarks of CAR-T cell subtypes and reveal their molecular changes along computationally inferred cellular evolution invivo. Collectively, these results decipher functional diversification and dynamics of peripheral CAR-T cells post-infusion.

Safety and efficacy of CRISPR-based non-viral PD1 locus specifically integrated anti-CD19 CAR-T cells in patients with relapsed or refractory Non-Hodgkin's lymphoma: a first-in-human phase I study.

Thus far, all approved chimeric antigen receptor (CAR)-T products are manufactured using modified viruses, which increases the risk of tumorigenesis, costs and production time. We aimed to evaluate the safety and efficacy of a kind of virus-free CAR-T cells (PD1-19bbz), in which an anti-CD19 CAR sequence is specifically integrated at the PD1 locus using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, in adults with relapsed/refractory (r/r) B cell non-Hodgkin's lymphoma (B-NHL). This single-arm phase I dose-escalation clinical trial evaluated PD1-19bbz in adult patients with r/r B-NHL from May 3rd 2020 to August 10th 2021. The patients were recruited and treated at the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China. Patients underwent leukapheresis and lymphodepleting chemotherapy before PD1-19bbz infusion. After the dose-escalation phase including three cohorts: 2×106/kg, 4×106/kg, 6×106/kg with three patients at each dose level, the optimal biological dose was determined to be 2×106/kg, which was then applied to an extended cohort of nine patients. The primary endpoint was the incidence of dose-limiting toxicities (DLT). The secondary endpoint was the response and survival. This trial was registered at www.clinicaltrials.gov as #NCT04213469. Twenty-one patients received PD1-19bbz infusion. Among all treated patients, 19 (90%) patients were diagnosed with stage III or IV disease. Meanwhile, 19 (90%) were stratified as intermediate risk or worse. Of note, four participants had >50% programmed death ligand-1 (PD-L1) expression in pre-treatment tumour sample, including two with extremely high levels (∼80%). There was no DLT identified. Fourteen patients had low-grade (1-2) cytokine release syndrome and two patients received tocilizumab. Four patients experienced immune effector cell-associated neurotoxicity syndrome of grade 1-2. The most common adverse events were hematologic toxicities, including anaemia (n=6), lymphocyte count decreased (n=19), neutrophil count decreased (n=17), white blood cell count decreased (n=10), and platelet count decreased (n=2). All patients had objective response and 18 patients reached complete response. At a median follow-up of 19.2 months, nine patients remained in remission, and the estimated median progression-free survival duration was 19.5 months (95% confidence interval: 9.9-infinity), with the median overall survival not reached. In this first-in-human study of non-viral specifically integrated CAR-T products, PD1-19bbz exhibited promising efficacy with a manageable toxicity profile. A phase I/II trial of PD1-19bbz in a larger patient cohort is underway. National Key R&D Program of China, National Natural Science Foundation of China, Key Project of Science and Technology Department of Zhejiang Province, Shanghai Zhangjiang National Independent Innovation Demonstration Area, Key Projects of Special Development Funds.

Abstract 1152: NKTR-255, a polymer-conjugated IL-15, dramatically improves ROR1 CAR-T cell persistence and anti-tumor efficacy in an autochthonous model of ROR1+ lung cancer

Abstract CAR-T cells have mediated remarkable clinical responses against hematological malignancies, but this success has yet to extend to solid tumors, where many factors in the tumor microenvironment (TME) inhibit their persistence and function. In a phase 1 trial at our Center, CAR-T cells targeting the tumor-associated antigen ROR1 in patients with non-small cell lung cancer (NSCLC) and triple negative breast cancer rapidly became dysfunctional and persisted poorly at tumor sites, ultimately failing to mediate objective responses. To evaluate strategies to improve ROR1 CAR-T efficacy, we adapted the KrasLSL-G12D/+;p53fl/fl (KP) autochthonous model of lung adenocarcinoma to express ROR1 (KPROR1). This aggressive model mimics the initiation, progression, and suppressive TME of human NSCLC. ROR1 CAR-T cells in KPROR1 mice rapidly declined in number, lost the ability to produce pro-inflammatory cytokines, and failed to significantly enhance tumor control, recapitulating the major barriers to CAR-T efficacy we observed in patients. NKTR-255 is a novel immunotherapeutic consisting of a polymer conjugated recombinant human interleukin-15 (rhIL-15) that could enhance the activity of CAR-T cells. NKTR-255 binds to the IL-15 receptor and IL-2/IL-15 receptor, thus maintaining the full potential spectrum of IL-15 biology while exerting a sustained effect on CD8+ memory T cell and NK cell expansion and persistence. We hypothesized that the combination of CAR-T cells and NKTR-255 would be feasible and safe and may extend the duration of efficacy of ROR1 CAR-T cells in ROR1+ lung tumors by improving their expansion and persistence. To test this, we treated tumor-bearing KPROR1 mice with control or ROR1 CAR-T cells with or without weekly injections of NKTR-255. NKTR-255 treatment resulted in a robust expansion of ROR1 CAR-T cells in the peripheral blood relative to vehicle-treated mice, which correlated with increased CAR-T cell infiltration into lung tumors. CAR-T cells in NKTR-255-treated mice also showed reduced signs of T cell exhaustion, including reduced expression of inhibitory receptors and enhanced ability to produce effector cytokines upon ex vivo re-stimulation. Importantly, combination therapy with both NKTR-255 and ROR1 CAR-T cells resulted in significantly improved tumor control relative to either treatment alone. Together, our work shows that NKTR-255 dramatically improves the persistence, function, and anti-tumor activity of ROR1 CAR-T cells in an aggressive autochthonous model of ROR1+ lung cancer. Our findings provide rationale to combine NKTR-255 with CAR-T cell therapy as a strategy to enhance the anti-tumor efficacy of CAR-T cells in patients with solid tumors. Citation Format: W. Sam Nutt, Takahiro Miyazaki, Mario Marcondes, Stanley R. Riddell, Shivani Srivastava. NKTR-255, a polymer-conjugated IL-15, dramatically improves ROR1 CAR-T cell persistence and anti-tumor efficacy in an autochthonous model of ROR1+ lung cancer [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 1152.

MRD Status By Clonoseq ® Is a Poor Predictor of Long-Term Outcomes after Bispecific LV20.19 CAR T-Cell Therapy for Relapsed, Refractory B-Cell NHL

INTRODUCTION: The clonoSEQ® Assay (Adaptive Biotechnologies Corporation, Seattle, USA) uses next-generation sequencing (NGS) based testing to identify disease-specific immunoglobulin sequences and detect evidence of minimal residual disease (MRD). MRD assessments have been shown to predict progression-free survival and overall survival in non-Hodgkin's lymphoma (NHL) patients. We report MRD results via the commercially available clonoSEQ® assay and its association with durability of response in patients treated with bispecific LV20.19 CAR in our phase 1/2 trial (NCT04186520). METHODS: We conducted a phase 1/2 single-center prospective trial (NCT04186520) to evaluate the efficacy and safety of LV20.19 CAR T-cells in patients with NHL at 2.5x106 cells/kg dose. LV20.19 CAR T-cells were manufactured onsite at varying lengths of manufacturing (8 vs 12 days) in the CliniMACS Prodigy device with IL7+15 for cell expansion, with the goal of fresh infusion. The commercial clonoSEQ® assay (by Adaptive Biotechnologies) was used to identify and track tumor clonotypes in responding patients at first assessment performed between Day 28-60. We evaluated associations between MRD status and long-term response post LV20.19 CAR-T cell therapy. Descriptive statistics were utilized for baseline characteristics. Survival analyses were calculated utilizing the Kaplan-Meier method and differences between groups were evaluated via the log-rank test. RESULTS: Among the 32 patients enrolled in the trial, there were 26 responders on Day 28. Of these responders, 23 patients had clonoSEQ® B-cell MRD assay done between Day 28-60 post CAR infusion (Table 1). MRD was performed in 11 patients with Diffuse Large B-cell Lymphoma (DLBCL), 3 patients with Follicular lymphoma (FL), and 9 patients with Mantle Cell Lymphoma (MCL). The median age was 64 years (45-74) and the median lines of prior therapy were 4 (2-11). Of the 23 patients with early MRD status available, the day 28 objective response rate (ORR) was 100% (complete response=57%, n=13 and partial response=43%, n=10). Eighteen patients (78%) were MRD negative at first assessment between days 28-60 and 5 (22%) were MRD positive. Of the 5 patients who were MRD positive, 3 became MRD negative at day 90, 1 patient was non-evaluable due to a non-relapse mortality event, and the remaining patient remains positive but with decreasing MRD status at Day 90. None of the patients with MRD positivity at day 28 have relapsed to date. Among patients who achieved early MRD negativity (n=18), 7 (39%) have relapsed, and all were patients with either DLBCL or FL. No MRD-negative MCL patient has relapsed to date. The duration of response in MRD-negative patients was significantly improved in MCL patients compared to DLBCL/FL patients (Figure 1), p=0.02. Among the 7 MRD negative patients who relapsed, only 1 patient was detected to have disease relapse by MRD positivity, 2 were detected simultaneously on MRD testing and imaging, while 3 were detected by physical exam or imaging findings and did not have MRD testing. Interestingly, 1 patient had a central nervous system relapse while being MRD negative. CONCLUSION: Relapse after CAR T-cell therapy remains a significant clinical challenge. New predictors are indicated to determine which responding patients after CAR-T cell therapy are more likely to relapse. Utilizing the commercially available Adaptive clonoSEQ® assay, early MRD assessment was not predictive of long-term clinical outcomes in DLBCL and FL. MRD positive patients maintained durable remission and converted to MRD negativity, while early MRD negative patients continued to relapse. In contrast, MRD negativity post-CAR-T therapy did appear to predict durable clinical remission in MCL patients. Newer assays, including cell-free technologies, may be more informative for non-MCL histologies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Improved anti-solid tumor response by humanized anti-podoplanin chimeric antigen receptor transduced human cytotoxic T cells in an animal model.

Recently, research has been conducted with chimeric antigen receptor (CAR)-T cells to improve efficacy against solid tumors. Humanized CAR improved the long-term survival of CAR-T cells in patients' peripheral blood, resulting in increased therapeutic efficacy. Therefore, the humanization of the CAR-gene sequence is considered an effective method. Podoplanin (PDPN) is a glycosylated transmembrane protein that is highly expressed in solid tumors and is associated with poor prognosis in patients with cancer. Therefore, PDPN is considered a biomarker and good target for cancer treatment with CAR-T cells. Previously, an anti-PDPN CAR was generated from a conventional nonhumanized antibody-NZ-1, the only anti-PDPN antibody for which a CAR was produced. In this study, we investigated other anti-PDPN CARs from the antibody NZ-27, or humanized NZ-1, to enhance the therapeutic potential of CAR-T cells. The CAR signal intensity was enhanced by the efficient expression of CAR proteins on the T-cell surface of NZ-27 CAR-T cells, which show tumor-specific cytotoxicity, proinflammatory cytokine production, and anti-tumor activity against PDPN-expressing tumor xenografts in mice that were significantly better than those in nonhumanized NZ-1 CAR-T cells.

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

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

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

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

Phase 1 study of P-PSMA-101 CAR-T cells in patients with metastatic castration-resistant prostate cancer (mCRPC).

98 Background: P-PSMA-101 is an autologous CAR-T therapy targeting PSMA, with a high percentage of stem cell memory T cells (TSCM) associated with efficacy, safety, and bone homing (particularly relevant to prostate cancer). It is manufactured using a novel non-viral transposon system (piggyBac) that creates high TSCM products. Genes are inserted encoding a PSMA-targeted Centyrin CAR, iCasp9-based safety switch, and DHFR to purify CAR-T cells. P-PSMA-101 completely eliminated tumors in intractable murine models of prostate cancer, providing rationale for this phase 1 trial (NCT04249947). Methods: Patients with mCRPC treated with or not eligible for a CYP17 inhibitor or second-generation antiandrogen, and a taxane were enrolled. P-PSMA-101 was manufactured from apheresed T cells and administered IV following a standard 3-day cy/flu lymphodepletion regimen. Dose escalation from 0.25-15 x 106 cells/kg is planned. Results: As of September 30, 2021, P-PSMA-101 had been administered to 10 heavily pretreated patients (median 7 prior regimens; range 3-15). Single infusions of 0.25 (n=5) to 0.75 (n=5) x 106 cells/kg have been assessed, with dose escalation continuing. P-PSMA-101 cells were shown to expand in blood via qPCR assay, peaking 2-3 weeks after infusion, consistent with the high percentage of TSCM. Significant antitumor responses were seen in this preliminary data set. Declines in PSA were seen in 7 patients (>50% in 3 and >99% in 1). Of 4 patients who had pre- and post-treatment FDG and PSMA-PET imaging, 3 demonstrated marked to complete resolution of abnormal uptake at known metastatic disease sites, with concordance in bone and CT scans, and/or circulating tumor cells (CTC). In 1 case, post-treatment tumor biopsy demonstrated infiltration by P-PSMA-101 CAR-T cells and elimination of tumor cells (pathologic complete response). Safety was consistent with expectations for a CAR-T product. CRS was seen in 60% (10% Gr ≥3) of patients. DLT was seen in 1 patient with macrophage activation syndrome/uveitis, and was the only Gr ≥3 CRS event. Immune effector cell-associated neurotoxicity syndrome (ICANS) has not occurred. CRS marker elevations were modest (max IL-6: 642.6 pg/mL). The most common AEs were cytopenias, infections, and constitutional symptoms (Gr ≥3 60%, 10%, and 0%), as expected with lymphodepletion. Treatable related ocular AEs were noted in 3 patients. Conclusions: These results parallel preclinical findings that P-PSMA-101 can produce marked efficacy in mCRPC, and very low doses are highly efficacious, consistent with unique product attributes such as the TSCM phenotype and bone tropism. This is the first report demonstrating profound antitumor effects of a novel PSMA-directed CAR-T-cell platform with concordant biochemical, radiographic, and pathologic parameters, demonstrating that therapeutic benefit of unarmored CAR-T cells in a major solid tumor is possible. Clinical trial information: NCT04249947.

A first-in-human clinical trial of piggyBac transposon-mediated GMR CAR-T cells against CD116-positive acute myeloid leukemia and juvenile myelomonocytic leukemia

Although several types of chimeric antigen receptor (CAR)-modified T cells (CAR-T cells) targeting myeloid antigens have been developed for acute myeloid leukemia (AML) globally, significant clinical benefits have not yet been reported. Furthermore, CAR-T cells targeting juvenile myelomonocytic leukemia (JMML) have not yet been developed. All JMML cells and 63-83% of AML cells express granulocyte macrophage-colony stimulating factor (GM-CSF) receptor (GMR, CD116/CD131 complex). Therefore, we created ligand-based CAR-T cells targeting GMR using the piggyBac transposon system. We further redesigned the CAR construct by optimizing the affinity of the antigen-binding region and length of the spacer region. The GMR CAR-T cells with a mutated GM-CSF at residue 21 (E21K) and a G4S spacer showed superior antitumor effects in the human AML-xenograft model. Safety tests revealed that the toxicity of GMR CAR-T cells was restricted to normal monocytes. Based on the promising results of the nonclinical study, we started a first-in-human clinical trial of GMR CAR-T cells in patients with CD116-positive AML and JMML in 2021.