Nucleic acid drugs have increasingly demonstrated their potential in tumor therapy and vaccine development, particularly exemplified by the recent advancements in mRNA-based cancer vaccines. Although mRNA cancer vaccines have shown promising clinical effects in cancer treatment, the inherent susceptibility of mRNA to degradation and its transient expression profile pose significant challenges in achieving long-term prevention of postoperative recurrence and metastasis. Herein, we propose the utilization of circular single-stranded DNA (Css DNA) as a novel gene expression vector to achieve robust expression for at least 315 days, which offers distinct advantages over conventional plasmids. It lacks bacterial-derived redundant sequences that are prone to clearance while exhibiting remarkably low immunogenicity due to its single-stranded conformation, which minimally activates the cGAS-STING pathway. With ovalbumin (OVA) as a model antigen, OVA-encoding Css DNA as a long-acting prophylactic vaccine has been demonstrated to significantly inhibit tumor growth and recurrence, thereby prolonging the survival time. Further combination with IL-12-encoding Css DNA, it elicited up to 7-fold cytotoxic CD8+ T cells and recruited innate immune cells in tumors. Our findings establish Css DNA as a versatile platform for cancer immunotherapy, combining durable antigen expression with potent immune activation to overcome current limitations of nucleic acid vaccines.

Chimeric antigen receptor (CAR)-T cell therapy is largely ineffective in most solid tumors, partially because of inadequate intratumoral trafficking. Bridging therapy, administered between leukapheresis and CAR-T cell infusion, offers a distinct opportunity to control the disease and precondition the tumor microenvironment without directly exposing CAR-T cells to concomitant drugs. Here, a poly (ADP-ribose) polymerase (PARP) inhibitor combined with anlotinib is evaluated as bridging therapy for ovarian cancer. In immunocompetent mice bearing ovarian tumors, this regimen induces durable intratumoral T cell accumulation associated with activation of the cGAS-STING pathway, vascular normalization, and reduced fibrillar collagen deposition. Furthermore, TGFβ-resistant, dual-target mesothelin (MSLN)/CD19 CAR-T cells are engineered using a bait-and-switch strategy. Niraparib plus anlotinib administered as bridging therapy before CAR-T cell infusion enhances CAR-T cell infiltration and antitumor activity across multiple preclinical ovarian cancer models. These findings inform an ongoing phase I clinical trial (NCT05141253) evaluating the safety of these engineered CAR-T cells following bridging therapy in patients with refractory MSLN-positive solid tumors. Collectively, our findings support a clinically actionable bridging therapy with translational potential for potentiating CAR-T cell therapy in ovarian cancer and highlight bridging therapy as a translational approach to improve CAR-T cell access and efficacy in solid tumors.

The universal chimeric antigen receptor T cell (UCAR-T) immunotherapy derived from healthy donors holds great promise in pan-cancer treatment. However, UCAR-T cell therapy faces a challenge in the rapid elimination of allogeneic cells by the host immune system. To address this, we introduced a T316I mutation in the leukocyte-specific protein tyrosine kinase (LCK) locus in CAR-T cells using the cytosine base editor (CBE) system. Concurrently, we disrupted endogenous T cell receptor alpha chain (TRAC) and beta-2 microglobulin (B2M) with the CRISPR-Cas9 system, along with dasatinib to overcome host immune rejection, an Src family kinase (SFK) inhibitor. The resulting LCK mutated UCAR-T (KM UCAR-T) cells exhibited normal phenotypes in activation, proliferation, differentiation, and tumor cytotoxicity in vitro. Moreover, KM UCAR-T cells demonstrated sustained expansion in mixed lymphocyte reactions (MLR) when incubated with T cells or peripheral blood mononuclear cells (PBMCs) from HLA-mismatched donors upon dasatinib treatment. Additionally, we illustrated that KM UCAR-T cells displayed antitumor activity in a xenograft murine model and verified the expansion and cytotoxicity of KM UCAR-T over traditional UCAR-T in the presence of allogeneic PBMCs when treated with dasatinib in vivo. These find-ings offer a novel strategy for UCAR-T cells to resist host immune rejection and achieve sustained expansion.

Poly(ADP-ribose) polymerase inhibitors (PARPis) are a class of agents targeting DNA damage repair that have become standard therapy for epithelial ovarian cancer (EOC) and multiple other solid tumors. In addition to targeting DNA damage repair, PARPis actively modulate antitumor immune responses, with efficacy being partially dependent on T cell activity. Here, we found that patient T cells sustain DNA damage during PARPi treatment, which reduces treatment efficacy. Leveraging paired pre- and posttreatment tumor samples from a clinical trial of patients with EOC treated with neoadjuvant niraparib as monotherapy, we showed that the PARPi caused DNA damage, slowed proliferation, and increased apoptosis in T cells, which we validated both in vitro and in mouse models. A genome-wide CRISPR (clustered regularly interspaced short palindromic repeats) knockout screen in primary human T cells identified PARP1 as the principal mediator of PARPi-induced T cell death. T cell-specific deletion of PARP1 or mutating Parp1 at its binding sites in transgenic mice led to reduced T cell DNA damage during PARPi treatment, resulting in improved efficacy of PARPis, alone or in combination with immune checkpoint inhibition. We then engineered PARPi-tolerant CAR T cells using cytosine base editing, which decreased PARPi-induced PARP1 trapping and led to reduced PARPi-induced DNA damage, resulting in superior antitumor efficacy in xenograft models compared with parental CAR T cells. This study highlights the relevance of PARPi-induced DNA damage to T cells and suggests opportunities to improve the efficacy of PARPis as monotherapy or in combination with immunotherapy.

Background: Multiple myeloma (MM) is a major hematologic malignancy driven by clonal plasma cells in the bone marrow. BCMA-directed CAR T cells and BCMA × CD3 TCEs have yielded promising responses in heavily pretreated patients, yet their optimal sequencing remains unclear. Deeper insight into how these therapies differentially reshape the immune microenvironment is essential for identifying response biomarkers and advancing MM immunotherapy. Results： Comprehensive single-cell atlas of 102 patients and 178 longitudinal samples We profiled 934 k bone-marrow cells (scRNA + TCR + BCR) spanning pre-therapy, early (1 mo), mid (2–5 mo), late (≥6 mo), and post-relapse timepoints after BCMA-CAR-T (n = 59) or BCMA-TCE (n = 43) treatment, creating the largest immune atlas to date in MM T-cell–redirecting therapy. TCE efficacy is driven by expansion of endogenous CD8⁺ effector T cells, whereas CAR-T globally reprograms the T-cell pool TCE responders showed a stepwise rise in CX3CR1⁺ CD8_Tef cells; CAR-T instead converted endogenous CD8 compartments toward Tem, γδ T, and naive states, suggesting broader lineage diversification and potential durability. CAR-CD8_Tef_CX3CR1 emerges as the dominant responder subset after infusion CAR sequence–based sorting revealed that this subset accounts for most proliferative CAR-T clones in responders. STARTRAC analysis shows therapy-specific state transitions CAR-T promoted extensive clonal sharing between CD8_Tem, CD8_Proliferating, CD8_Tex, γδ T, and CD8_Naive, whereas TCE selectively expanded CD8_Tef without inducing further differentiation. Distinct TCR-clonal dynamics underline divergent immune activation Most clones were therapy-specific (Exclusive_Pre/Exclusive_Post). TCE yielded more Expanding CD8_Tef clones; CAR-T produced more Expanding CD8_Tem, CD8_Tex, and γδ T clones. Persisting clones were virtually absent in CD4 subsets. Tumor-reactive versus by-stander effector T cells defined by clonal size and expansion Tumor-reactive Tef (Ttr) exhibited heightened cytotoxic signatures. Endogenous Ttr preferentially expanded under TCE, whereas CAR-T responders were enriched for CAR-Ttr clones. Coordinated TME cellular programs exhibiting varied associations with responsiveness An NMF-based analysis revealed three efficacy-linked bone-marrow programs. The Cytotoxic-Program (TIME-Tef) comprises CD8_Tef_CX3CR1 and γδ T cells and correlates strongly with favorable responses. In parallel, an Immune-Response Program (TIME-Mono)—dominated by NK cells and CD14⁺ monocytes—also associates with clinical outcome but displays a distinct, therapy-dependent pattern Temporal kinetics reveal modality-specific biomarkers of response In TCE therapy, a monotonic rise of CD8_Tef_CX3CR1 from early to late timepoints stratified responders, with clonal expansion peaking in mid/late phases. After CAR-T, Tex, proliferating, and γδ T fractions surged Pre → Early → Mid, while CD8_Tef declined and CD4 memory/naive rebounded at Mid, mirroring tumor-burden reduction. After CAR-T, Tex, proliferating, and γδ T fractions surged Pre → Early → Mid, while CD8_Tef declined and CD4 memory/naive rebounded at Mid, mirroring tumor-burden reduction. Pretreatment exhausted-like Tex predicts TCE failure but not CAR-T outcome A TCE-specific Predictive Index that integrates CD8_Tex_TOX, CD4_Treg_FOXP3, and MALT_SLC4A10 predicts both inferior M-protein reduction (AUC = 0.82) and significantly shorter progression-free survival (P < 0.01). Infusion-product (IP) features forecast CAR-T durability High CAR-Treg abundance and excessive G2M/S-phase CAR-T cells predicted inferior responses, whereas a naïve/memory-biased IP signature marked long-acting CAR-T products and was detectable in patients ≥12 mo post-infusion. Relapse landscape highlights antigen escape and a stem-like myeloma clone In six paired BCMA-CAR-T relapses, expression of BCMA, GPRC5D, and SDC1 uniformly declined. BCR-sharing exposed a stem-like malignant cluster whose gene signature, selected by Cox + Lasso, predicted post–CAR-T relapse in COMPASS and multiple GEO cohorts. Conclusions： These findings delineate modality-specific immune-remodeling trajectories, uncover dynamic and baseline predictors of efficacy, and nominate a relapse-linked stem-like myeloma program, collectively guiding optimal sequencing and biomarker-driven personalization of BCMA-targeted immunotherapy in MM.

Expanding the repertoire of CAR therapies to include intracellular antigens holds promise for treating a broad spectrum of malignancies. TCR-like T cells, capable of recognizing intracellular antigen-derived peptides in complex with HLA molecules (pHLA), represent a promising strategy in the field of engineered cellular therapy. This study introduced antibody-like TCR (abTCR) T cells that specifically targeted HLA-A*02:01-restricted LMP2426 peptides, a typical Epstein-Barr virus (EBV) latency II protein, for the treatment of EBV-associated lymphoproliferative diseases (EBV-LPDs). Compared with classic CAR T cells targeting the same epitope, abTCR T cells demonstrated superior efficiency, including increased CD107A expression, enhanced cytotoxicity, and elevated IFN-γ secretion, even when engaging with target cells that naturally present antigens. Moreover, a costimulatory signal-armed abTCR (Co-abTCR), which integrated a costimulatory structure with the abTCR, further enhanced the proliferation and in vivo tumoricidal efficacy of transfected T cells. Collectively, our study developed a potentially novel TCR-like T cell therapy that targets HLA-A*02/LMP2426 for the treatment of EBV-LPDs, providing a potential therapeutic solution for targeting of intracellular antigens in cancer immunotherapy.

Background: The persistence of CAR T cells is a critical factor in their therapeutic efficacy, particularly in the treatment of hematologic malignancies such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and large B-cell lymphoma (LBCL). Notably, in multiple myeloma (MM) therapies targeting B-cell maturation antigen (BCMA), prolonged CAR T-cell persistence has been linked to better clinical outcomes. In the FUMANBA-1 study, we previously confirmed a positive correlation between Equecabtagene Autoleucel (Eque-cel) persistence and the maintenance of MRD negativity, which was reported at the 2023 ASH conference (Wang D et al. 2023 ASH oral 4584). Objective: This study aims to analyze the relationship between CAR T-cell persistence and progression-free survival (PFS) as well as time to progression (TTP). Additionally, the study examines how the baseline level of soluble B-cell maturation antigen (sBCMA) influences these outcomes. Methods: A retrospective analysis was performed to analyze the population receiving Eque-cel treatment in the FUMANBA-1 study. CAR T-cell persistence was monitored via digital droplet polymerase chain reaction (ddPCR). The sBCMA levels were measured using enzyme-linked immunosorbent assay (ELISA). Patients were categorized based on the efficacy to target ratio (VCN duration/baseline sBCMA), with further analysis on PFS and TTP differences between the groups. Univariate and multivariate analyses were conducted to identify factors influencing CAR T-cell persistence. Results: During a median follow-up of 24.67 months, 107 patients in 14 centers were treated with Eque-cel. The median efficacy-to-target ratio, measured by the vector copy number (VCN) duration to baseline soluble B-cell maturation antigen (sBCMA), was 1.05 (Days*mL/ng). Patients were categorized into two groups: a high efficacy-to-target ratio group and a low ratio group . Those with a lower ratio had a significantly higher risk of disease progression, with a hazard ratio (HR) for PFS of 2.3 (95% CI: 1.27-4.14, p = 0.0045) and for TTP of 3.07 (95% CI: 1.51-6.24, p = 0.0011), both indicating a strong correlation between the efficacy-to-target ratio and disease progression risk. At the data cutoff, the ongoing remission group showed a longer median VCN duration than those with progression or relapse (12.52 vs. 9.03 months, p = 0.71), though this was not statistically significant. Among baseline characteristics, including ECOG score, R-ISS and ISS disease stages, tumor BCMA expression, tumor burden, baseline sBCMA level, previous autologous stem cell transplantation (ASCT), triple-class exposure, bridging therapy, prior CAR-T treatment, and lymphodepleting conditioning, only previous ASCT (n = 30) was significantly associated with Eque-cel persistence (HR = 0.35, p = 0.004). Post-infusion, patients with anti-drug antibodies (ADA) had a significantly higher risk of shorter VCN duration (HR = 5.79, p < 0.0001). Notably, Cmax levels, whether above or below the median, did not significantly impact VCN duration (HR = 0.93, p = 0.78). Among the 107 patients, only 14 exhibited an aplastic neutrophil recovery phenotype, characterized by persistent neutrophil counts below 500/μl for 14 days or more. This phenotype did not significantly affect VCN duration compared to patients without this condition. Conclusion: The efficacy-to-target ratio is a crucial determinant of clinical outcomes in patients treated with Eque-cel, underscoring the importance of CAR T-cell persistence. This ratio may serve as a biomarker for future treatment planning, highlighting the need for extended CAR T-cell persistence to achieve optimal disease control. While baseline characteristics like previous ASCT influence persistence, baseline sBCMA levels do not adversely affect the treatment efficacy of Eque-cel.

Refractory or relapsed T cell acute lymphoblastic leukemia (r/r T-ALL) patients have poor prognoses, due to the lack of effective salvage therapies. Recently, CD7-targeting chimeric antigen receptor (CAR)-T therapies show efficacy in patients with r/r T-ALL, but relapse with CD7 loss is common. This study evaluates a CD5-gene-edited CAR-T cell therapy targeting CD5 in 19 r/r T-ALL patients, most of whom had previously failed CD7 CAR-T interventions. CAR-T products were derived from previous transplant donors (Cohort A) or newly matched donors (Cohort B). Primary endpoints were dose-limiting toxicity at 21 days and adverse events within 30 days. Secondary endpoints were responses, pharmacokinetics and severe adverse events after 30 days. A total of 16 received infusions, 10 at target dose of 1 × 106 kg−1. All encountered grade 3–4 cytopenias and one had a grade 3 infection within 30 days. All patients (100 NCT05032599 . In a phase 1 trial, pediatric or adult patients with T-ALL who received CD5-targeted CAR-T cell therapy using cells from previous or newly matched donors showed an encouraging clinical response rate, with some severe adverse events that required attention and effective management.

The advent of multiple anti-BCMA CAR-T cell therapies represents a paradigm shift in the treatment of hematological malignancies, particularly multiple myeloma. This groundbreaking study on CT103A, a commercially available anti-BCMA CAR-T cell product, offers a multifaceted exploration of the intricate factors governing CAR-T cell functionality, encompassing CAR structure, antigen epitope interactions, and microenvironmental influences. In a comparative analysis with peer products featuring distinct CAR architectures, CT103A exhibited remarkable resilience against soluble BCMA (sBCMA) interference in the tumor microenvironment. This finding underscores the critical importance of CAR design in overcoming potential obstacles to efficacy. The study's utilization of crystallographic analysis to elucidate the CT103A-BCMA complex structure provides unprecedented insights into the molecular basis of their high-affinity interaction, contributing valuable structural biology data to the field of immunotherapy. The employment of alanine scanning mutagenesis on CT103A's complementarity-determining regions (CDRs) and saturation mutagenesis screening of BCMA represents a sophisticated approach to delineating the specific amino acid residues crucial for antigen recognition and binding. This methodological rigor not only enhances our understanding of the CT103A-BCMA interaction but also establishes a blueprint for future CAR optimization strategies. The study's integration of clinical cohort data to assess the impact of baseline sBCMA levels on CAR-T cell proliferation and clinical outcomes addresses a critical question in the field. The finding that sBCMA levels do not significantly influence these parameters challenges prevailing assumptions and may have important implications for patient selection and treatment strategies. This comprehensive investigation illuminates the multifaceted determinants of CT103A's functional characteristics, offering critical insights that extend beyond a single CAR-T cell product. The study's findings have broad implications for the rational design of CAR-T cell therapies, potentially informing strategies to modulate affinity, identify suitable patient populations, and monitor for high-risk antigen mutations that could lead to treatment resistance. Moreover, this research exemplifies the power of integrating structural biology, protein engineering, and clinical data to elucidate the complex interplay between CAR-T cell design, target antigen properties, and the tumor microenvironment. Such a holistic approach is essential for advancing our understanding of CAR-T cell biology and optimizing therapeutic outcomes. The insights gained from this study may catalyze the development of next-generation CAR-T cell therapies with enhanced efficacy, reduced toxicity, and broader applicability across various cancer types. Furthermore, the methodologies employed here could serve as a template for investigating other CAR-T cell products and target antigens, potentially accelerating progress in the field of cellular immunotherapy. In conclusion, this refined and comprehensive study not only advances our understanding of CT103A but also contributes significantly to the broader field of CAR-T cell therapy. By elucidating the intricate relationships between CAR structure, antigen binding, and microenvironmental factors, this research paves the way for more effective, personalized, and rationally designed cellular immunotherapies, holding promise for improved outcomes in patients with hematological malignancies and potentially other cancer types.

CT103A is a fully human chimeric antigen receptor T cell (CAR-T) product for targeting B cell maturation antigen. This study presents the updated safety and efficacy profiles of CT103A in patients with relapsed/refractory multiple myeloma (RRMM) after long-term follow-up. As of July 31, 2023, the median follow-up time after CAR-T cell infusion was 45.0 months (range, 0.7-58.3 months). During long-term follow-up, the incidence of adverse events gradually decreased over time. One patient had a maximum duration of response of nearly 5 years. All 18 patients (100%) achieved partial remission or better; 77.8% (14 of 18) of patients eventually exhibited complete response or stringent complete response (sCR), with response increasing over time. At the time of data cutoff, nine patients were still alive and seven patients had an sCR status with negative minimal residual disease. The median progression-free survival was 22.6 months, and the median overall survival was 50.2 months for all 18 patients. The median CAR transgene persistence was 14.0 months (range, 0.7-57.3 months). Long-term follow-up demonstrated that CT103A confers durable clinical benefit for RRMM patients based on the sustained presence of fully human CAR-T cells.

Extramedullary disease usually implies a dismal outcome in relapsed/refractory multiple myeloma patients, and requires novel treatment approaches. We designed a trial using Selinexor, a nuclear export protein 1 inhibitor, together with anti-B cell maturation antigen (BCMA) chimeric antigen receptor (CAR)-T cell product CT103A to treat these patients, and describe the first two cases in this report. Selinexor was administered with a novel two-step schedule in bridging therapy and in maintenance. The clinical responses and adverse events were recorded after CAR-T infusion and Selinexor administration. In vitro analysis of the influence of Selinexor on CAR-T cell function was performed using myeloma cell lines. After infusion, both patients achieved stringent complete remission (sCR), and were maintained in sCR at data-cutoff, with survival over 13 and 10 months, respectively. Neither immune effector cell-associated neurotoxicity syndrome nor over grade 2 cytokine release syndrome was observed. Meanwhile, the patients showed good tolerance to the combination. In addition, we demonstrated that low dose of Selinexor could upregulate the expression of BCMA on plasma cell lines and subsequently enhance the function of CAR-T cell in vitro. The combination of Selinexor and CT103A exerts preliminary synergistic effect, and can be developed as a promising strategy for relapsed/refractory extramedullary myeloma.

Background: Equecabtagene autoleucel (eque-cel, CT103A), which is designed with a fully human BCMA-specific CAR structure, was recently granted NMPA approval for the treatment of adult patients (pts) with relapsed/refractory multiple myeloma (RRMM) after ≥3 prior lines of therapy (LOT). The pivotal phase 2 FUMANBA-1 study (NCT05066646) demonstrated deep and durable responses with eque-cel in heavily pretreated patients with RRMM. Recent studies have demonstrated the utility of minimal residual disease (MRD) as a prognostic biomarker for long-term outcomes of RRMM, wherein achievement of sustained MRD-negative status was associated with an improvement in PFS of pts treated by eque-cel in FUMANBA-1 study. We sought to characterize the baseline and disease characteristics of pts with sustained MRD negativity (pts who continued to remain MRD negative ≥6 mo and ≥12 mo). Methods: FUMANBA-1 study enrolled RRMM pts who received ≥ 3 lines of prior therapies containing at least a proteasome inhibitor and an immunomodulatory agent and were refractory to their last line of treatment. All pts received a single infusion of eque-cel at the dose of 1.0 x 10 6 CAR-T cells/Kg. Pts who had progressed on previous BCMA CAR-T cell therapy were not included in this analysis. The primary endpoint was ORR. Additional endpoints included ≥CR rate (key secondary), DOR, PFS, and safety. ORR and CR were assessed per IMWG criteria; DOR and PFS were analyzed via Kaplan-Meier methods. MRD negativity was a secondary objective and was assessed on bone marrow samples at baseline; day 14, 28; and 2, 3, 6, 9, 12, 15, 18, 21 and 24 mo using EuroFlow next-generation flow, regardless of disease status. Evaluable samples passed calibration and quality control and included sufficient cells for evaluation at 10 -5 testing threshold. Characteristics were analyzed in pts who had MRD negativity <6 mo, or sustained ≥6 mo and ≥12 mo. Pts who did not achieve MRD negativity at any time point were considered to be MRD positive. Results: Of the 88 pts who achieved MRD negativity in FUMANBA-1, 74 had at least 6 mo follow up without progression after initial MRD-negativity and 43 had at least 12 mo follow up without progression after initial MRD-negativity. MRD negativity was sustained for ≥6 mo in 78.4% (58 of 74 with at least 6 mo follow-up without progression after initial MRD-negativity) and ≥12 mo in 74.4% (32 of 43 with at least 12 mo follow-up without progression after initial MRD-negativity; 7 pts evaluable for sustained MRD at ≥6 mo but not ≥12 mo had PD). Pts with sustained MRD negativity (≥6 mo, and ≥12 mo) had longer progression free survival (PFS; Figure 1A) compared with pts who did not (MRD negative <6 mo). Key pt and disease characteristics in these different groups (Figure 1B), and potential associations with sustained MRD negativity were analyzed descriptively. Baseline R-ISS disease stage III was more common in pts with MRD sustained for ≥6 mo (8.6%) and ≥12 mo (9.4%) compared with pts who had MRD negativity <6 mo (3.3%). Baseline high tumor burden diseases were less common in pts with MRD sustained for ≥6 mo (70.7%) and ≥12 mo (65.6%) compared with pts who had MRD negativity <6 mo (90%). Triple-class exposure was also less common in pts with MRD sustained for ≥6 mo (12.1%) and ≥12 mo (12.5%) compared with pts who had MRD negativity <6 mo (30%). Pts with sustained MRD negativity had trends toward shorter median time since diagnosis (38.6 mo and 40.2 mo for pts with MRD negativity sustained ≥6 and ≥12 mo, respectively versus 43.5 mo for those with MRD negativity <6 mo). Other baseline characteristics, including presence of high-risk cytogenetics, ECOG performance status, extramedullary disease and number of prior LOT did not differ across MRD subgroups, and were similar to the overall FUMANBA-1 population. Conclusions: Based on our descriptive analysis, pts receiving eque-cel achieved MRD negativity irrespective of their high-risk cytogenetics, extramedullary disease, number of prior LOT and performance status. Presence of high tumor burden at baseline, and prior triple-class exposure might be factors that impact achievement of sustained MRD negativity. These data suggest that while eque-cel is effective for a broad range of pts, specific pt and disease characteristics may be associated with sustained MRD negativity and better long-term outcomes. Sustained MRD negativity might be a promising prognostic indicator for RRMM pts treated by eque-cel.

BackgroundAcute myeloid leukemia (AML) treatment remains challenging. CD70 was reported as a promising AML-specific antigen. Preclinically, CAR T-cell with single-chain-variable fragment (scFv) or truncated CD27 targeting CD70 has been reported to treat AML. However, various disadvantages including spontaneous exhaustion, proteinase-mediated loss of functional receptors, and high immunogenicity, limited its further application to clinical settings. Alternatively, the single-variable domain on heavy chain (VHH), also known as nanobodies, with comparable binding ability and specificity, provides an optional solution.MethodWe generated CD70 knocked-out novel nanobody-based anti-CD70-CAR T-cells (nb70CAR-T) with two different VHHs for antigen detection. Next, we detected the CD70 expression on primary AML blasts by flow cytometry and associated the efficacy of nb70CAR-T with the target antigen density. Finally, epigenetic modulators were investigated to regulate the CD70 expression on AML cells to promote the functionality of nb70CAR-T.ResultsOur nb70CAR-T exhibited expected tumoricidal functionality against CD70-expressed cell lines and primary AML blasts. However, CD70 expression in primary AML blasts was not consistently high and nb70CAR-T potently respond to an estimated 40.4% of AML patients when the CD70 expression level was over a threshold of 1.6 (MFI ratio). Epigenetic modulators, Decitabine and Chidamide can up-regulate CD70 expression on AML cells, enhancing the treatment efficacy of nb70CAR-T.ConclusionCD70 expression in AML blasts was not fully supportive of its role in AML targeted therapy as reported. The combinational use of Chidamide and Decitabine with nb70CAR-T could provide a new potential for the treatment of AML.