Long-term safety and efficacy of the fully human CAR-T therapy CT103A in relapsed/refractory multiple myeloma.

Introduction

Patients with relapsed or refractory multiple myeloma (RRMM) have limited outcomes when treated with conventional chemotherapy, with a 5-year overall survival (OS) rate below 30%. Although novel treatments, such as proteasome inhibitors, immunomodulatory drugs, and anti-CD38 monoclonal antibodies, have been introduced, most patients with relapsed or refractory disease experience short remission periods or no remission at all, highlighting the need for therapies with new mechanisms of action. 1 , 2 , 3 , 4 , 5

B cell maturation antigen (BCMA)-targeting chimeric antigen receptor T cell (CAR-T) therapy has emerged as the most promising cellular immunotherapy for RRMM. 6 , 7 , 8 , 9 In 2021 and 2022, idecabtagene vicleucel (ide-cel) and ciltacabtagene autoleucel (cilta-cel) were approved for the clinical treatment of RRMM. 10 In June 2023, the National Medical Products Administration also approved equecabtagene autoleucel (eque-cel or CT103A), the product studied in this research, for the clinical treatment of RRMM. CT103A is a fully human-origin CAR-T cell product that has shown promising efficacy and safety in early clinical trials, with an overall response rate (ORR) of 100% and a complete response (CR) rate of 72.2% among 18 adult patients in a previous report. 11 Despite the high response rate, the long-term durability of CAR-T efficacy still needs further exploration. Recent studies with extended observation periods for other products have been published ( Table S1 ). 6 , 7 , 12 , 13 , 14 At a median 47.8 months of follow-up, LCAR-B38M had an ORR of 87.8%, median progression-free survival (PFS) was 18.0 months, and median OS was not reached. 13 Idecabtagene vicleucel had an ORR of 75.8%, median PFS was 8.8 months, and median OS was 34.2 months with a median 18.1 months of follow-up. 14 Ciltacabtagene autoleucel had an ORR of 97.9% and median PFS and median OS were not reached, with a median 27.7 months of follow-up. 7 However, long-term studies focusing on fully human-derived BCMA CAR-T cells remain limited.

Here, we report an updated, long-term follow-up analysis of CT103A therapy for RRMM based on a phase 1 trial.

Results

Patient characteristics and response

From September 7, 2018, to July 8, 2019, 24 patients were enrolled in the phase 1 clinical trial, six of whom withdrew before CAR-T cell infusion, three due to progression, one due to heart failure, one due to severe liver dysfunction, and one due to severe infection ( Figure 1 ). A total of 18 patients were treated with CT103A, the sex ratio (male/female) was 1.25, and the median age was 53.5 years (range, 38–66 years). Five patients had extramedullary disease (EMD), and patient 01-013 also had plasma cell leukemia before infusion. The median number of prior lines of therapy was four (range, 3–6 lines), and four patients (22.2%) had received prior anti-BCMA CAR-T cell treatment. Only one patient received daratumumab and dexamethasone as bridging therapy. The detailed patient characteristics were previously reported, 11 and updated characteristics are shown in Tables 1 and S2 . Figure 1 Flowchart of this trial ∗Three patients were excluded because they did not meet the inclusion criteria or because of rapid progression. † Three patients who received lymphodepletion were not diagnosed because of heart failure, severe liver function damage, or severe infection. ‡ Two patients died of infection. § One patient refused follow-up at the study site. One patient died of infection. Table 1 Baseline characteristics of patients Characteristic Patients ( N = 18) Age, median (range), years 53.5 (38–66) Male gender, no. (%) 10 (55.6) Monoclonal globulin, no. (%) IgG 8 (44.4) IgA 1 (5.6) IgM 0 (0) IgD 1 (5.6) Light chain 5 (27.8) Nonsecretory 3 (16.7) ISS stage, no. (%) Ⅰ 10 (55.6) Ⅱ 8 (44.4) High-risk cytogenetics a , no. (%) 13 (72.2) Double-hit, no. (%) 6 (33.3) EMD and/or PCL, no. (%) 5 (27.8) BCMA expression on plasma cells ≥50%, no. (%) 18 (100) Previous therapy lines, median (range) b 4 (3–6) Previous CAR-T infusion, no. (%) 4 (22.2) Previous proteasome inhibitor, no. (%) Bortezomib 18 (100) Ixazomib 3 (16.7) Carfilzomib 2 (11.1) Previous immunomodulatory drug, no. (%) Lenalidomide 18 (100) Pomalidomide 2 (11.1) Previous anti-CD38 monoclonal antibody, no. (%) Daratumumab 2 (11.1) Other drug exposure, no. (%) Cyclophosphamide 15 (83.3) Etoposide 6 (33.3) Doxorubicin 6 (33.3) Cisplatin 6 (33.3) Melphalan 1 (5.6) ECOG, Eastern Cooperative Oncology Group; ISS, International Staging System; EMD, extramedullary disease; PCL, plasma cell leukemia; BCMA, B cell maturation antigen; CAR-T, chimeric antigen receptor T cell. a The high risk of cytogenetics was defined as that at least one of t(4;14), t(14;16), t(14;20), Del 17p, and Gain 1q detected by fluorescence in situ hybridization was positive. b Two patients were proteasome inhibitor, immunomodulatory drug, and daratumumab triple class exposed.

Each patient’s response, bone marrow assessments with minimal residual disease (MRD), and survival profiles are visualized as a bar chart in Figure 2 A. All 18 patients assessed for efficacy exhibited a partial response (PR) or better. Patient 01-012 died 20 days post infusion, and the efficacy evaluation of day 14 did not include MRD assessment. Therefore, the rates of MRD negative complete response (CR) or stringent complete response (sCR) for evaluable and intent-to-treat population were 82.4% (14 of 17) and 77.8% (14 of 18), respectively. Patients’ accumulated best responses to CT103A were enhanced over time ( Figure 2 B). Of the nine patients still alive at the date cutoff, seven remained in ongoing sCR without disease progression ( Figure 2 A). The response rates did not appear to be influenced by key covariates such as age, sex, International Staging System (ISS) stage, high-risk cytogenetic abnormalities, prior CAR-T cell therapy, or EMD according to survival analysis ( Figure S1 ). Figure 2 Treatment response and outcome in 18 patients after CT103A infusion (A) Patient response, bone marrow assessments with MRD, and survival profiles. (B) Patients’ accumulated best responses to CT103A in the long-term follow-up. The red line indicates that the rate of CR or sCR increased over time. sCR, stringent complete response; CR, complete response; VGPR, very good partial response; PR, partial response; MR, minor response; SD, stable disease; PD, progressive disease; NA, not applicable; MRD, minimal residual disease.

Long-term survival

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). The median PFS was 22.6 months, and the median OS was 50.2 months ( Figure 3 ). For patients with CR or sCR, the median PFS was 44.5 months, while the median OS had not been reached. PFS and OS were significantly longer for patients who achieved CR compared to those who did not achieve CR ( p < 0.0001, p = 0.0014; Figure S2 ). The recurrence rates at 1, 2, 3, and 4 years post CT103A treatment were 28%, 7%, 9%, and 0%, respectively. Figure 3 Survival analysis of patients PFS (A) and OS (B) with 95% confidence intervals for all 18 patients. PFS, progression-free survival; OS, overall survival.

Subtype-specific prognostic factors were further analyzed. Remarkably, the PFS of patients without relapse/progressive disease (PD) was significantly higher than that of patients with relapse/PD ( p = 0.0023; Figure 4 A). The OS of patients without/with relapse/PD had the same trend as the PFS, but with no significant difference ( p = 0.1083; Figure 4 B). No significant survival differences were found based on sex, EMD, prior CAR-T cell therapy, double-hit or high-risk cytogenetic features ( Figure S3 ). 15 Figure 4 Subgroup analysis of PFS and OS in patients Subgroup analysis of PFS (A) and OS (B) in patients with or without relapse/progressive disease. The red lines represent patients who had relapse/progressive disease, and the blue lines represent patients who did not experience relapse/progressive disease.

Of the four patients who received prior anti-BCMA CAR-T cell treatment, two patients achieved sCR and remained in sCR state at the study cutoff. Patient 01-007 achieved sCR, but the disease progressed 20 months post infusion, although the patient remained alive at the cutoff date. Patient 01-013, who had EMD and secondary plasma cell leukemia, achieved the best response of very good partial response and survived for 7.4 months ( Table S3 ).

Among the eight patients with relapse/progressive disease, six received subsequent therapies and only two patients were still alive at the cutoff date. The details of post-relapse survival and subsequent lines of therapy are presented in Table S2 .

Adverse events

Long-term adverse events (AEs) were analyzed in multiple stages after CAR-T cell infusion, including within 8 weeks, from 8 weeks to 1 year, and from 1 year to the cutoff date. AEs within 8 weeks post infusion were reported in our previous study. 11 The most common late AE occurring beyond 8 weeks after CAR-T cell infusion was hematologic toxicity ( Table 2 ). Leukopenia, neutropenia, anemia, and thrombocytopenia were observed in 11 patients (61.1%), 11 patients (61.1%), 10 patients (55.6%), and 9 patients (50.0%), respectively. The median durations for these conditions were 76 days (range, 2–550 days), 78 days (range, 1–550 days), 184 days (range, 22–429 days), and 148 days (range, 17–632 days), respectively. All hematologic toxicities were reversible, and no patient received stem cell boost. We rescored the CAR-HEMATOTOX score to screen for patients who could develop immune effector cell-associated hematotoxicity (ICAHT). Consequently, seven patients’ CAR-HEMATOTOX scores were ≥2, four of whom had ICAHT after 1 year ( Table S4 ). No significant differences in survival were detected in patients with CAR-HEMATOTOX scores <2 or ≥2 ( p = 0.2899, p = 0.2899; Figure S4 ). Nine patients (50.0%) experienced hypoglobulinemia after 8 weeks. The globulin and percentage of CD19 + cells after CT103A infusion are shown in Figure S5 . Among the 18 patients, B cell recovery was observed in five patients, and globulin recovery was observed in 15 patients. However, of the seven living patients with sCR, only two had recovered B cells. Table 2 Adverse events that occurred in patients after CAR-T cell infusion Events Beyond 8 weeks From 8 weeks to 1 year From 1 year to cutoff date Grade ≥3 All grades Grade ≥3 All grades Grade ≥3 All grades Hematologic Leukopenia 2 (11.1%) 11 (61.1%) 2 (11.1%) 8 (44.4%) 0 5 (27.8%) White blood cell count increased 0 1 (5.6%) 0 1 (5.6%) 0 0 Neutropenia 8 (44.4%) 11 (61.1%) 7 (38.9%) 10 (55.6%) 1 (5.6%) 3 (16.7%) Neutrophil count increased 0 1 (5.6%) 0 1 (5.6%) 0 0 Lymphopenia 0 5 (27.8%) 0 4 (22.2%) 0 2 (11.1%) Lymphocyte count increased 0 1 (5.6%) 0 1 (5.6%) 0 1 (5.6%) Anemia 3 (16.7%) 10 (55.6%) 2 (11.1%) 6 (33.3%) 1 (5.6%) 4 (22.2%) Thrombocytopenia 2 (11.1%) 9 (50.0%) 0 7 (38.9%) 2 (11.1%) 6 (33.3%) Metabolic Elevated LDH 0 9 (50.0%) 0 7 (38.9%) 0 4 (22.2%) Elevated ALT 0 6 (33.3%) 0 6 (33.3%) 0 1 (5.6%) Elevated AST 0 6 (33.3%) 0 5 (27.8%) 0 1 (5.6%) Hypokalemia 0 5 (27.8%) 0 4 (22.2%) 0 2 (11.1%) Hypoglobulinemia 0 9 (50.0%) 0 9 (50.0%) 0 0 Infections a Pulmonary infection 3 (16.7%) 6 (33.3%) 2 (11.1%) 6 (33.3%) 2 (11.1%) 2 (11.1%) Urinary tract infection 0 5 (27.8%) 0 4 (22.2%) 0 2 (11.1%) Anal cryptitis 0 1 (5.6%) 0 1 (5.6%) 0 0 Herpes zoster 2 (11.1%) 3 (16.7%) 2 (11.1%) 3 (16.7%) 0 0 Herpes virus infection 0 1 (5.6%) 0 1 (5.6%) 0 0 Necrotic fasciitis 1 (5.6%) 1 (5.6%) 1 (5.6%) 1 (5.6%) 0 0 Cellulitis 1 (5.6%) 1 (5.6%) 1 (5.6%) 1 (5.6%) 0 0 Infectious enteritis 0 1 (5.6%) 0 1 (5.6%) 0 0 Appendicitis 1 (5.6%) 1 (5.6%) 1 (5.6%) 1 (5.6%) 0 0 Hepatitis B 0 1 (5.6%) 0 1 (5.6%) 0 0 Septic shock 1 (5.6%) 1 (5.6%) 0 0 1 (5.6%) 1 (5.6%) a Eight patients (44.4%) had repeated infections beyond 8 weeks after CAR-T cell infusion.

Given the deficiencies in leukocytes, lymphocytes, and globulin, infections were closely monitored. Eleven patients (61.1%) experienced 26 infections beyond 8 weeks, including ten patients (55.6%) who had 21 infections from 8 weeks to 1 year and five patients (27.8%) who had five infections after 1 year. Six patients (33.3%) had eight pulmonary infections. Five patients (27.8%) had seven urinary tract infections. Three patients (16.7%) had four herpes zoster infections. The main severe AEs occurring 8 weeks after CAR-T cell infusion were infections. Six patients (33.3%) had nine severe infections ( Table 3 ). All infections were controlled with proper and prompt treatment except for one patient with grade 5 septic shock. Table 3 Severe adverse events of infections from 8 weeks post infusion to cutoff date Patient no. Events Grade Days post infusion 01-001 pulmonary infection 3 101 01-001 herpes zoster 3 108 01-001 pulmonary infection 3 698 01-002 pulmonary infection 3 82 01-009 septic shock 5 361 01-016 herpes zoster 3 65 01-019 appendicitis 3 161 01-024 cellulitis 3 205 01-024 necrotic fasciitis 3 223

AEs that occurred from 1 year post infusion to the cutoff date were similar to those that occurred from 8 weeks post infusion to 1 year post infusion and included leukopenia, neutropenia, anemia, thrombocytopenia, elevated lactate dehydrogenase (LDH), proteinuria, pulmonary infection, and urinary tract infection. The frequency and types of AEs gradually decreased over time. During long-term follow-up, no patients experienced late neurotoxicity or secondary neoplasia.

Nine patients died before the cutoff date. Five patients died after disease progression and withdrawal from the clinical trial, one patient died of PD, and the condition of four patients was unknown. Four patients died of severe AEs related to infection, and two patients exhibited sCR at the time of death.

CAR-T cell pharmacokinetics and pharmacodynamics

CAR transgene copy numbers at serial points after infusion are shown in Figure 5 A. The duration of CAR transgene persistence varied among patients (median, 14.0 months; range, 0.7–57.3 months). CAR transgenes remained detectable in seven patients before the cutoff date, six of whom were at the sCR stage. Patients with relapse/PD had poorer expansion and persistence of CAR-T cells than patients without relapse/PD ( p = 0.0268, p = 0.0048) ( Figures S6 A and S6B). Patients with relapse/PD had a poorer AUC 0–∞ of CAR-T cells than patients without relapse/PD ( p = 0.0366) ( Figure S6 C). In addition, patients with EMD had poorer expansion of CAR-T cells than patients without EMD ( p = 0.0387) ( Figure S6 D). Similarly, patients with EMD had poorer persistence of CAR-T cells than patients without EMD, but the difference was not statistically significant ( p = 0.1756) ( Figure S6 E). Figure 5 Kinetics of CAR-T cells and serum BCMA levels after CT103A infusion (A) CAR-T cell expansion and persistence in patients are shown at serial time points post infusion. The red lines represent patients who experienced relapse/progressive disease. (B) Serum BCMA levels in patients are shown at serial time points post infusion. The data were obtained through the latest follow-up of each patient. The red lines represent patients who had relapse/progressive disease.

Serum BCMA (sBCMA) levels were measured at screening, before infusion, and then regularly after infusion ( Figure 5 B). Compared with the baseline, the sBCMA levels were significantly lower at 1 month post infusion. In our previous report, 11 either delayed clearance or an increase in sBCMA levels was observed for patients with relapse or PD. With the extension of follow-up time, of the eight evaluable patients with PD, the sBCMA levels rebounded much more than in the ten evaluable non-PD patients. These results indicate that sBCMA can be used as a biomarker for response monitoring.

We also analyzed the correlation between sBCMA and CAR-T cell pharmacokinetics in patients ( Figure S7 ). Patients with baseline sBCMA above upper limit of normal (ULN) had poorer expansion and persistence of CAR-T cells than patients with baseline sBCMA below ULN, but the difference was not statistically significant ( p = 0.9789, p = 0.0699).

Humoral immunogenicity

Anti-drug antibodies (ADAs) were tested in all patients before infusion and/or at serial follow-up visits after infusion if possible. As in our previous report, 11 only two samples from patient 01-016 were positive (at 90 and 120 days). The remaining samples were all negative for ADAs.

Discussion

ORRs from published studies on BCMA-based CAR-T cells generally exceed 70%, which seems to be much greater than those for other strategies for treating RRMM patients. 6 , 7 , 8 , 13 In addition to the high response rate, the durability of CAR-T cell efficacy is also a topic of interest for researchers, as longer survival provides patients more opportunities to receive future therapies. This study presents nearly 4 years of follow-up data.

With extended of follow-up, the CR rate further improved to 77.8% (14 of 18), compared to the 72.2% (13 of 18) reported in phase 1. This improvement was due to one patient (01-011) who achieved sCR in the second year post infusion. Some patients also achieved CR at approximately 3–6 months after CT103A infusion, indicating the sustained efficacy of CT103A. In terms of survival, the median PFS was 22.6 months, and the median OS was 50.2 months. Nine out of the 18 patients were still alive at the cutoff time, and seven currently exhibit sCR. In comparison, the pivotal trial of ide-cel showed an ORR of 73% and a median PFS of 8.8 months in patients with heavily pretreated RRMM. 14 In LEGEND-2, the ORR was 87.8%, and the median PFS was 18.0 months with the median follow-up 47.8 months. 13 In a study that combined anti-BCMA and anti-CD19 CAR-T cells with a median follow-up of 21.3 months, the ORR was 92% and the median PFS was 18.3 months. 12 The impressive efficacy of CT103A may result from its fully human CAR construct, which differs from scFv constructs used in other trials. This fully human scFv may exhibit slower binding, longer retention, and slower dissociation from BCMA. 11 Subtle differences in binder properties, in combination with other signals such as 4-1BB and CD3z incorporated in the CAR molecule, may lead to complex variations in antigenic signaling strength and duration. 11 In our study, none of the single high-risk factors, such as EMD, high-risk genetic mutation, or previous CAR-T cell therapy, had a negative impact on the length of remission or survival, indicating an ideal effect of CT103A for these specific patients. Nevertheless, the small sample size may have resulted in bias during the statistical analysis; therefore, the phase 1b/2 study will provide more convincing conclusions regarding these factors.

At the latest follow-up, CAR transgenes were still detectable in 77.8% of the surviving patients. Among the seven patients with persistent sCR, CAR-T cells were still detectable in six. Patients with relapse/PD had poorer persistence and expansion of CAR-T cells than patients without relapse/PD. A clear trend was also observed in the survival time and AUC 0-∞ of CAR-T cells between the two groups. These results demonstrated that the survival advantage is largely dependent on long-term exposure to CAR-T cells in vivo . Notably, while some patients maintained high CAR transgene levels, others with low copy numbers still benefited, indicating that, even at a low copy number, patients may still benefit from the sustained presence of CAR-T cells with continuous tumor killing/monitoring effects.

Several factors may influence CAR-T cell proliferation and persistence, including extrinsic and intrinsic factors. Extrinsically, Mu et al. from our center demonstrated that EMD can impair C max , which in turn affects long-term survival. 16 This adverse impact of EMD remains unchanged during long-term follow-up. For intrinsic factors, scientists have focused on modifying CAR structures, including the ectodomain, transmembrane domain, and endodomain, to achieve sustained CAR-T cell efficacy. 17 , 18 , 19 The prolonged persistence of CT103A may be attributed to its design, which shows greater specificity for membrane BCMA and less susceptibility to serum BCMA. Further investigation is needed to explore these structural advantages. While no evidence links ADAs to CAR-T cell kinetics, the lower incidence of ADAs in this study compared to others suggests that fully human structures may have reduced immunogenicity. 13 , 20

CT103A also demonstrated a manageable safety profile. Previous reports indicated low cytokine release syndrome (CRS) and absence of immune effector cell-associated neurotoxicity syndrome (ICANS) in early stages, along with AEs within 8 weeks post infusion. We are more concerned with the impact of AEs on patients during long-term follow-up, especially those that affect survival and quality of life. Late AEs were divided into two periods: from 8 weeks to 1 year and from 1 year to the cutoff date. Hematologic AEs, now termed immune effector cell-associated hematotoxicity (ICAHT) by the European Society for Blood and Marrow Transplantation and European Hematology Association, 20 , 21 were the most frequent from 8 weeks to 1 year. We applied the CAR-HEMATOTOX score to predict ICAHT and found that 71.4% of cases could be anticipated, with the highest scores among those with persistent ICAHT beyond 1 year. Low platelet count and compromised hematopoietic reserves were major predictors of ICAHT. 22 , 23

Another important AE was infection, which was the dominant AE after 8 weeks. Twenty-one infection events were reported in ten patients from 8 weeks to 1 year, and five infection events were reported in five patients after 1 year. One of the infections was fatal and occurred after 1 year without B cell recovery. The incidence was similar to that reported by the consensus form of the European Myeloma Network, which was 1.5 AEs per patient. 24 According to the consensus, the risk factors for infection after anti-BCMA treatment include patient-specific, disease-specific, and treatment-related factors. However, the occurrence of infections during long-term follow-up is mostly related to immune deficiency caused by B cell aplasia and hypogammaglobulinemia. Therefore, proper management of infection, including infection treatment and prevention, is necessary from 8 weeks to 1 year after CAR-T cell infusion. 25 , 26 , 27

During long-term follow-up, no patients who received CT103A experienced secondary neoplasia. However, secondary primary malignancies were reported in 16 patients (16/97, 16.5%) who underwent cilta-cel treatment. 7 Four patients (4/74, 5.4%) who underwent LCAR-B38M treatment developed second primary non-hematologic tumors. 13 Of the secondary neoplasia observed, none were classified as related to CAR-T therapy. Therefore, the occurrence of the secondary neoplasia needs further exploration.

The origins of ADA production remain unclear, involving factors such as antibody sources, patient immune functions, and acquired immunity. 28 , 29 , 30 , 31 Even though fully human CAR-T cells exhibit lower immunogenicity, they can still trigger immune responses due to their synthetic nature.

Lastly, our previous reports indicated that either delayed clearance or increased sBCMA levels were associated with relapse or PD. 11 With the extension of follow-up time, the sBCMA levels of patients with relapse or PD significantly rebounded compared with those of the non-PD/relapse patients. These results indicate that sBCMA can be used as a biomarker for response monitoring. 32 , 33

In summary, long-term follow-up data showed that CT103A induced a durable response in patients with RRMM with a manageable long-term safety profile. Future studies will be needed to explore the reasons for the strong therapeutic effect of CT103A on malignant plasma cells. As a single-arm trial, future randomized controlled trials will be conducted to provide a more rigorous comparison of CT103A’s efficacy.

Materials and methods

Study design and treatment

This was a phase 1, single-arm, open-label study conducted at Tongji Hospital in China and registered with the Chinese Clinical Trial Registry ( http://www.chictr.org.cn ; #ChiCTR1800018137). The cutoff date of this study is July 31, 2023. CT103A is an engineered autologous T cell product transduced with a CAR plasmid via a lentivirus transfer system. The functional elements of the CAR construct include two BCMA-targeting single-domain antibodies, a CD8a hinge and transmembrane domain, and a 4-1BB/CD3ζ intracellular signaling domain. Lymphodepletion was performed using fludarabine at 25 mg/m 2 plus cyclophosphamide at 20 mg/kg daily for 3 consecutive days (days −4 to −2) before CT103A infusion. CT103A was administered at 1 × 10 6 , 3 × 10 6 , and 6 × 10 6 CAR-positive T cells/kg in the dose-escalation phase and at 1 × 10 6 CAR-positive T cells/kg in the expansion phase. The details of this clinical trial, including the study design, CAR-T cell preparation, patients, and treatments, can be found in our previous report. 11

Long-term assessments

The primary endpoint was to evaluate the safety and tolerability. The secondary endpoints were efficacy and pharmacokinetic and pharmacodynamic data of CT103A. Efficacy was assessed by the criteria of response according to the International Myeloma Working Group consensus recommendations, including ORR, OS, and PFS. 34 The ORR was defined as the proportion of patients who achieved a PR or better. OS was calculated from the date of CAR-T cell infusion to the date of death or the cutoff date. PFS was defined as the duration from CT103A infusion to any of the following events: disease progression or any cause-related mortality. The CR evaluation in patients with EMD included bone marrow aspirate and biopsy, blood and urine M protein, and imaging. 35 MRD was assessed at visits when bone marrow aspiration was performed and was assessed by using multiparametric flow cytometry and next-generation sequencing if experimentally feasible. The ADA effect of CT103A was evaluated by using an electrochemiluminescence bridging assay on the MSD-ECL platform (Meso Scale Discovery, Gaithersburg, MD). 36 , 37 CAR transgene copies were assessed at each visit and were monitored by using digital droplet PCR. 38 For the determination of sBCMA levels, the Human BCMA/TNFRSF17 DuoSet ELISA Kit (DY193, R&D Systems) and DuoSet ELISA Ancillary Reagent Kit 2 (DY008, R&D Systems) were used to detect and quantify the sBCMA levels. The correlations of the pharmacokinetic profile (virus copy number), ADA, and sBCMA with clinical outcomes were also analyzed.

AEs were graded using the National Cancer Institute Common Terminology Criteria for Adverse Events version 5.0. The CAR-HEMATOTOX score was calculated prior to lymphodepletion using the online CAR-HEMATOTOX calculator from the German Lymphoma Alliance: https://www.german-lymphoma-alliance.de/Scores.html . 39 CRS and ICANS were assessed using the modified criteria proposed by the American Society for Transplantation and Cellular Therapy consensus. 40 This study analyzed long-term AEs in multiple stages after CAR-T cell infusion, including within 8 weeks, 8 weeks to 1 year, and 1 year to the cutoff date.

Statistical analysis

Analysis of categorical variables was performed using the χ 2 test or Fisher’s exact test, and analysis of continuous variables was performed using Student’s t test. PFS and OS and the corresponding 95% exact confidence intervals were calculated using the Kaplan-Meier method. Statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) 22.0 software (SPSS, Chicago, USA) and GraphPad Prism software (version 9.0). A two-tailed p value of <0.05 was considered to indicate statistical significance.

Data and code availability

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Acknowledgments

We thank all the patients who participated in this trial along with their families and caregivers; the physicians and nurses who cared for the patients and the staff members at the clinical sites, the clinical trial teams, and those involved in data collection, analyses, and medical writing. We also thank the late Jianfeng Zhou, MD of Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, for his contributions and instrumental guidance for the study concept, development of the protocol, and conduct of the trial. This study was conducted in compliance with the principles of the Declaration of Helsinki. Ethics approval from the Institutional Review Board of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology was obtained before the start of the study. Written informed consent was obtained from all participants. This trial was registered in the Chinese Clinical Trial Registry (ChiCTR, number ChiCTR1800018137). This work was supported by funding from the 10.13039/501100001809 National Natural Science Foundation of China ( 82170223 to Chunrui Li) and the Clinical Research Program of Huazhong University of Science and Technology affiliated with Tongji Hospital ( 2020003 to Chunrui Li).

Author contributions

Chunrui Li and A.D. designed and supervised the clinical study. Q.Y. and D.W. analyzed the data and wrote and revised the manuscript. Q.Y., Z.L., N.A., Chunhui Li, B.Y., X.W., X.L., and C.S. collected the clinical data. Q.Y., D.W., Z.L., and W.M. performed the statistical analyses. D.W., J.W., L.J., P.Z., and Chunrui Li enrolled and cared for patients.

Declaration of interests

W.W. holds interests in patent applications related to the CT103A. H.Y., H.G., S.C., G.H., and W.W. are employees of Nanjing IASO Medical Technology Co., Ltd. and hold interests in the company.

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