Allogeneic stem cell transplantation combined with conditioning regimen including donor-derived CAR-T cells for refractory/relapsed B-cell lymphoma

TO THE EDITOR: In recent years, immunochemotherapy and targeted therapies have significantly improved patient survival [1], whereas the proportion of patients with refractory/relapsed B-NHL (R/R BNHL) was found to be lower than 50%, with extremely poor survival [2–4]. In 2017, the United States Food and Drug Administration approved the administration of chimeric antigen receptor (CAR) T-cell therapy targeting CD19 antigen for the third or later line of systemic therapy for patients with R/R B-NHL [5], whereas ~20–30% of patients still failed to respond or had relapses. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been presented for multi-line therapy of R/R B-NHL patients [6], whereas the long-term survival rate in patients with high tumor burden (HTB) before transplantation was only ~4% [7, 8]. To explore appropriate therapeutic options for patients with R/R BNHL, CAR-T cells from the same donor (donorderived CAR-T) were involved in the conditioning of allo-HSCT, utilizing the immune effects to reduce the HTB before transplantation. This procedure was conducted to decrease the high relapse rate and to improve survival. From September 2020 to May 2021, 8 eligible adult patients with R/R B-NHL were enrolled, including high-grade B-cell lymphoma (n= 4), diffuse large B-cell lymphoma, NOS (n= 3), and Richter transformation (n= 1). Based on the 5th edition of the World Health Organization Classification of Haematolymphoid Tumors: Lymphoid Neoplasms. Patients aged 18–60 years, with Eastern Cooperative Oncology Group status 0–2 and no uncontrollable infections, intracranial hypertension, or organ failure (Table S1 and S2). Patients were required to give written informed consent. The protocol was approved by the Institutional Review Board of Beijing Boren Hospital (ChiCTR2000040665).To be eligible for participation in this study, an individual must meet the criteria described in the supplemental materials. Peripheral blood mononuclear cells were isolated from the haplotype donors for allo-HSCT, and CD3+ T lymphocytes were separated by using antigencoated immunomagnetic beads. CD19/ CD20/CD22 expression in tumor tissues was identifed by IHC and FCM, which was the basis for selecting targets for CAR T-cells. Patients received allo-HSCT in combination with donor-derived CAR T-cell therapy with the T/Bu/Flu (thiotepa 5mg/kg·d, -16d~15d;busulfan 3.2 mg/kg·d, -16d~-14d;fludarabine 30mg/m2·d, -16d~-12d)-based conditioning regimen. Te detailed dosages were adjusted according to the fundamental status and tolerance of the patients. Taking the date of hematopoietic stem cells transfusion as day 0, donor-derived CAR T-cell was transfused on day-9 and -8. Tacrolimus, antithymocyte globulin and a short course of MTX and mycophenolate mofetil were used to prevent graft-versus-host disease (GVHD) (Fig. S1). Multicolor fow cytometer and quantitative polymerase chain reaction (qPCR) were used to detect the CAR T-cell concentration in the blood. Enzyme-linked immunosorbent assay was used to dynamically monitor the peripheral serum cytokines. The criteria for cytokine release syndrome (CRS), neurotoxicity, GVHD, and tumor assessment are listed in the supplemental materials. Overall survival (OS) was defined as the time from the date of allo-HSCT to the censoring date of follow-up examination or death. Progression-free survival (PFS) was defined as the time from the date of allo-HSCT to disease relapse or the censoring date of the follow-up examination. The t-test or Wilcoxon test was implemented to compare the CAR-T cell expansion and percentage of different lymphocyte subtypes. The Kaplan–Meier method was employed to estimate the probability of OS and EFS. The results obtained in this study have been represented in Table 1.


TO THE EDITOR:
In recent years, immunochemotherapy and targeted therapies have significantly improved patient survival [1], whereas the proportion of patients with refractory/relapsed B-NHL (R/R B-NHL) was found to be lower than 50%, with extremely poor survival [2][3][4]. In 2017, the United States Food and Drug Administration approved the administration of chimeric antigen receptor (CAR) T-cell therapy targeting CD19 antigen for the third or later line of systemic therapy for patients with R/R B-NHL [5], whereas 20-30% of patients still failed to respond or had relapses. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been presented for multi-line therapy of R/R B-NHL patients [6], whereas the long-term survival rate in patients with high tumor burden (HTB) before transplantation was only~4% [7,8]. To explore appropriate therapeutic options for patients with R/R B-NHL, CAR-T cells from the same donor (donor-derived CAR-T) were involved in the conditioning of allo-HSCT, utilizing the immune effects to reduce the HTB before transplantation. This procedure was conducted to decrease the high relapse rate and to improve survival.
From September 2020 to May 2021, 8 eligible adult patients with R/R B-NHL were enrolled, including high-grade B-cell lymphoma (n = 4), diffuse large B-cell lymphoma, NOS (n = 3), and Richter transformation (n = 1). Based on the 5th edition of the World Health Organization Classification of Haematolymphoid Tumors: Lymphoid Neoplasms. Patients aged 18-60 years, with Eastern Cooperative Oncology Group status 0-2 and no uncontrollable infections, intracranial hypertension, or organ failure (Table S1 and S2). Patients were required to give written informed consent. The protocol was approved by the Institutional Review Board of Beijing Boren Hospital (ChiCTR2000040665).To be eligible for participation in this study, an individual must meet the criteria described in the supplemental materials.
Peripheral blood mononuclear cells were isolated from the haplotype donors for allo-HSCT, and CD3 + T lymphocytes were separated by using antigencoated immunomagnetic beads. CD19/ CD20/CD22 expression in tumor tissues was identifed by IHC and FCM, which was the basis for selecting targets for CAR T-cells. Patients received allo-HSCT in combination with donor-derived CAR T-cell therapy with the T/Bu/Flu (thiotepa 5 mg/kg·d, -16d~-15d;busulfan 3.2 mg/kg·d, -16d~-14d;fludarabine 30 mg/m2·d, -16d~-12d)-based conditioning regimen. Te detailed dosages were adjusted according to the fundamental status and tolerance of the patients. Taking the date of hematopoietic stem cells transfusion as day 0, donor-derived CAR T-cell was transfused on day-9 and -8. Tacrolimus, antithymocyte globulin and a short course of MTX and mycophenolate mofetil were used to prevent graft-versus-host disease (GVHD) (Fig. S1).
Multicolor fow cytometer and quantitative polymerase chain reaction (qPCR) were used to detect the CAR T-cell concentration in the blood. Enzyme-linked immunosorbent assay was used to dynamically monitor the peripheral serum cytokines. The criteria for cytokine release syndrome (CRS), neurotoxicity, GVHD, and tumor assessment are listed in the supplemental materials. Overall survival (OS) was defined as the time from the date of allo-HSCT to the censoring date of follow-up examination or death. Progression-free survival (PFS) was defined as the time from the date of allo-HSCT to disease relapse or the censoring date of the follow-up examination.
The t-test or Wilcoxon test was implemented to compare the CAR-T cell expansion and percentage of different lymphocyte subtypes. The Kaplan-Meier method was employed to estimate the probability of OS and EFS.
The results obtained in this study have been represented in Table 1.

DISEASE STATUS AFTER CYTOREDUCTION BEFORE TRANSPLANTATION
Eight eligible patients were enrolled, all of whom had failed autologous anti-CD19 CAR-T therapy (Table S1). After the bridging therapy (including chemotherapy and targeted therapy), 5, 1, and 2 patients had partial remission (PR), stable disease (SD), and PD, respectively.

DONOR-DERIVED CAR T-CELL EXPANSION AND PERSISTENCE
The median dose of donor-derived CAR-T infusion was 4 (range, 0.78-4.88) × 10 6 /kg. FCM detected the expansion of donor-derived CAR-T cell in the peripheral blood of all 8 patients.
Donor-derived CAR-T cells peaked at a median of 10 (range, 7-35) days with a median percentage of peak CAR vector transgene copies(CAR vector transgene copies/Human single copy gene from PB mononuclear cells) 8.0162% (range, 0.09-116.8%) by qPCR in blood. The results of statistical analysis showed that the dose of infusion of donor-derived CAR-T was not significantly correlated with the peak value of in vivo expansion of CAR-T cells (P = 0.09). The peak donor-derived CAR-T was higher in patients in the PR condition than in SD/PD before allo-HSCT (P < 0.0001) (Figs. S2 and S3).
Theoretically, donor-derived lymphocytes overcame the failure of autologous CAR-T cell treatment caused by the malfunction of autologous lymphocytes. However, the obstacles for unmodified allogeneic CAR-T cells are poor in vivo expansion and persistence due to alloreactivity, failing to achieve the goal of reducing tumor burden [9]. Therefore, the conditioning regimen by allo-HSCT proposed in this study could simultaneously achieve two goals: firstly, the intensity of conditioning disrupted the patients' immunity defense and eliminated the lymphocytes, ensuring the expansion of donor-derived CAR-T, and the immunological effects further reduced the tumor burden before transplantation; secondly, the consequent transfusion of hematopoietic stem cells allowed for hematopoiesis and immunological reconstruction, and the donor-derived CAR-T cells from the same immune platform survived and exerted oncological surveillance effects.
In general, we found that allo-HSCT combined with conditioning regimen including donor-derived CAR-T cells are safe. CRS was manageable, and no neurotoxicity occurred. Furthermore, the incidence of aGVHD did not increase post-transplantation, and transplant-related complications were also under control.
In addition, further analysis showed that PFS (6-month PFS 62.5%) was longer in our study than in previous studies [8] (1-year PFS 29-44%). We speculated that the observed differences might be due to the following: firstly donor-derived CAR T cells expanded successfully and persistence at low doses in the body possibly reduced tumor burden and led to remission in patients; However, testing efficacy after infusion of donor CAR-T cells was difficult because of the myelosuppressed state of the patient in the transplant ward and the high risk of infection. Therefore, it was not possible to accurately assess the contribution of donor CAR -T cells to reducing tumor burden. Secondly immunosuppression was not increased to prevent GVHD, according to previous reports, so the allogeneic graft-versus-lymphoma effect probably also played a role in reducing tumor burden [10]. However, patients with pre-transplant status SD or PD could not benefit from this treatment. Therefore, a long-term follow-up period is required. This study was registered in the Chinese Clinical Trial Registry/ International Clinical Trial Registry Platform of WHO (ClinicalTrials#: ChiCTR2000040665).