Efficacy and follow‐up of humanized anti‐BCMA CAR‐T cell therapy in relapsed/refractory multiple myeloma patients with extramedullary‐extraosseous, extramedullary‐bone related, and without extramedullary disease

Abstract The prognosis of patients with multiple myeloma (MM) with extramedullary disease (EMD) remains poor. A high overall response rate (ORR) has been reported following anti‐B‐cell maturation antigen (BCMA) chimeric antigen receptor (CAR)‐T cell therapy in relapsed/refractory (R/R) patients with MM; however, data on patients with EMD remain limited. Herein, we compared and analyzed the efficacy and long‐term follow‐up of anti‐BCMA CAR‐T cell therapy in R/R MM patients with extramedullary‐extraosseous (EM‐E), extramedullary‐bone related (EM‐B), and without extramedullary disease. No difference in the ORR was observed between the three groups. The long‐term efficacy of anti‐BCMA CAR‐T cell therapy in the EM‐E group was worse than that in patients without EMD and with EM‐B. In the EM‐E group, disease progression was the reappearance of extramedullary lesions without an increase in the MM cell percentage or M protein level. Although no difference in the proportion of CAR‐T cells was detected among the three groups, the EM‐E group might exhibit a relatively high grade of cytokine release syndrome following anti‐BCMA CAR‐T therapy. Interleukin‐6 levels in the without EMD group were lower than those in the EM‐E and EM‐B groups. However, given the small number of cases in the three groups, statistical analysis was not performed.(ChiCTR1800017051 and ChiCTR2000033925).


| NTRODUCTION
Multiple myeloma (MM) is a hematologic malignancy characterized by proliferative disorders of neoplastic plasma cells. Although proteasome inhibitors, monoclonal antibodies, immunomodulatory agents, and autologous hematopoietic stem cell transplantation (auto-HSCT) could significantly improve efficacy and survival, [1][2][3] some patients develop relapsed/refractory (R/R) MM with an extremely poor prognosis. [4][5][6] MM cells are often not confined to bone marrow, migrating from the bone marrow, infiltrating into various extramedullary organs throughout the body, and even infiltrating the peripheral blood. 7 This extramedullary MM (EMM) is a rare type of MM, with the lesion termed extramedullary disease (EMD). 8 It might develop at initial diagnosis, during relapse, or followup. Two types of EMDs are known to exist: extramedullaryextraosseous (EM-E), leading to soft tissue tumors at a site far from the bone, and extramedullary-bone related (EM-B), which invades the surrounding soft tissues. 9,10 Although several new EMD treatments are available, the prognosis of MM patients with EMD remains poor. [11][12][13] Anti-B cell maturation antigen (anti-BCMA) is a member of the tumor necrosis factor receptor superfamily, mainly expressed in MM cells of almost all patients. 14 Therefore, anti-BCMA is considered an ideal target for chimeric antigen receptor (CAR)-T cell therapy in patients with R/R MM. 15,16 A high overall response rate (ORR) has been reported following anti-BCMA CAR-T cell therapy in patients with R/R MM 17,18 ; however, data on MM patients with EMD are limited. Herein, we compared and analyzed the efficacy, safety, and long-term follow-up of humanized anti-BCMA CAR-T cell therapy in R/R MM patients with EM-E, EM-B, and without EMD.

| Patients enrolled in the study
Twenty-one patients were diagnosed with R/R MM and enrolled in clinical trials of humanized anti-BCMA CAR-T cell therapy (ChiCTR1800017051 and ChiCTR2000033925) between September 2018 and June 2020, including six patients with at least one EM-E, six patients with at least one EM-B, and nine patients without any assessable EMD. All patients had BCMA expression in MM cells at enrollment. The cutoff date was 31 July 2021. All patients were observed after anti-BCMA CAR-T cell therapy for more than 12 months unless death occurred due to R/R MM.

| Preparation of humanized anti-BCMA CAR-T cells and anti-BCMA CAR-T cell therapy
Peripheral blood mononuclear cells were collected from patients with R/R MM and isolated by Ficoll density gradient centrifugation. CD3+ T cells were selected using CD3 microbeads (Miltenyi Biotec, Inc.).  were also assessed.

| Adverse events following humanized anti-BCMA CAR-T cell therapy
We evaluated Adverse events (AEs) associated with humanized anti-BCMA CAR-T cell therapy. The grade of cytokine release syndrome (CRS) was determined according to the National Cancer Institute Common Terminology Criteria for Adverse Events v4.03. 20 Immune effector cell-associated neurotoxicity syndrome (ICANS) was used to assess neurotoxicity. 21 FCM was used to assess the proportion of anti-BCMA CAR-T cells in peripheral blood on days 0, 4, 7, 14, 28, and 60 after infusion; the proportion of these cells was also determined in pleural effusion. Cytokine levels, including interleukin-6 (IL-6), IL-2R, and tumor necrosis factor-α (TNF-α), were assessed on days 0, 7, 14, 28, and 60 using enzyme-linked immunosorbent assay.

| Statistical analysis
Data are expressed as the mean � standard error (SE). Probabilities of PFS and OS were estimated using the Kaplan-Meier method and compared using the log-rank test. All statistical analyses were performed using GraphPad Prism 7 and SPSS 17.0. Statistical significance was set at p < 0.05.

| Transduction and amplification efficiency, infusion dose of humanized anti-BCMA CAR-T cells
The mean humanized anti-BCMA CAR transduction efficiency for final products of the EM-E, EM-B, and without EMD group were 37.87 � 9.65%, 36.32 � 8.04%, and 39.57 � 9.08%, respectively. On  In EM and EB groups, assessable EMD of patients with R/R MM who reached ORR disappeared or shrunk on reaching optimal response. However, it failed to shrink in patients who did not achieve the ORR.   respectively. Given the small number of cases in the three groups, statistical analysis was not performed.

| Proportions of humanized anti-BCMA CAR-T cells in peripheral blood
The proportion of anti-BCMA CAR-T cells was detected at days 0, 4,   29,30 In particular, the median OS of patients with relapse after auto-HSCT was less than 1 year. 31 Some studies have suggested that following the use of proteasome inhibitors and/or immunomodulatory agents, the PFS and OS of patients with EMM were less pronounced than those in MM patients without EMD. 32,33 In general, the prognosis for EM-E is worse than EM-B. 34 Anti   Assessing a larger sample size could provide more definitive results.
In conclusion, the long-term efficacy of anti-BCMA CAR-T cell therapy in R/R MM patients with EM-E was considerably worse than in patients without EMD and those with EM-B. Therefore, further therapy after anti-BCMA CAR-T cell therapy to maintain the efficacy of CAR-T therapy for longer is necessary for this patient group, possibly prolonging survival. In addition, attention should be paid to potential side effects in R/R MM patients with EM-E.