Extended characterization of anti-CD19 CAR T cell products manufactured at the point of care using the CliniMACS Prodigy system: comparison of donor sources and process duration.

BACKGROUND AIMS: The CliniMACS Prodigy closed system is widely used for the manufacturing of chimeric antigen receptor T cells (CAR-T cells). Our study presents an extensive immunophenotypic and functional characterization and comparison of the properties of anti-CD19 CAR-T cell products obtained during long (11 days) and short (7 days) manufacturing cycles using the CliniMACS Prodigy system, as well as cell products manufactured from different donor sources of T lymphocytes: from patients, from patients who underwent HSCT, and from haploidentical donors. We also present the possibility of assessing the efficiency of transduction by an indirect method. METHODS: Seventy-six CD19 CAR-T cell products were manufactured using the CliniMACS Prodigy automated system. Immunophenotypic properties, markers of cell activation and exhaustion, antitumor, anti-CD19 specific activity in vitro of the manufactured cell products were evaluated. As an indirect method for assessing the efficiency of transduction, we used the method of functional assessment of cytokine secretion and expression of the CD107a marker after incubation of CAR-T cells with tumor targets. RESULTS: The CliniMACS Prodigy platform can produce a product of CD19 CAR-T cells with sufficient cell expansion (4.6 × 109 cells-median for long process [LP] and 1.6 × 109-for short process [SP]), transduction efficiency (43.5%-median for LP and 41.0%-for SP), represented mainly by T central memory cell population, with low expression of exhaustion markers, and with high specific antitumor activity in vitro. We did not find significant differences in the properties of the products obtained during the 7- and 11-day manufacturing cycles, which is in favor of reducing the duration of production to 7 days, which may accelerate CAR-T therapy. We have shown that donor sources for CAR-T manufacturing do not significantly affect the composition and functional properties of the cell product. CONCLUSIONS: This study demonstrates the possibility of using the CliniMACS Prodigy system with a shortened 7-day production cycle to produce sufficient amount of functional CAR-T cells. CAR transduction efficiency can be measured indirectly via functional assays.

Efficacy and safety of a reduced dose of plerixafor in combination with granulocyte colony-stimulating factor in healthy haploidentical stem cell donors.

BACKGROUND AND OBJECTIVES: Implementation of the technique of immunomagnetic selection requires the procurement of a large number of CD34+ cells from haploidentical donors within a single apheresis procedure. The release of stem cells with granulocyte colony-stimulating factor (G-CSF) alone is unsatisfactory in a number of donors, and plerixafor, a CXCR4 chemokine receptor antagonist, could be used as an additional mobilization agent. The aim of our study was to examine whether a lower dose of plerixafor (0.12 mg/kg) can provide sufficient increase in CD34+ cells in the peripheral blood of allogeneic healthy donors in comparison with a historical control group. In addition, we assessed the risk of inability to provide the recipient with a transplant containing the optimal dose of 8-10 × 106 CD34+ cells/kg body weight of the recipient. MATERIALS AND METHODS: In a prospective, single-arm study, we examined the results of 105 mobilizations in healthy adult haploidentical donors with G-CSF and plerixafor at a dose of 0.12 mg/kg. The historical control group consisted of 106 mobilizations with G-CSF and plerixafor at 0.24 mg/kg. RESULTS: The median increase in the number of CD34+ cells from day 4 to day 5 of mobilization was 69 cells/µl (range, 28-240) versus 77 cells/µl (24-217) in the groups of 0.12 and 0.24 mg/kg of plerixafor, respectively (p-value 0.255). The apheresis products contained a median of 14.4 × 106 /kg recipient body weight CD34+ cells versus 12.9 × 106 /kg in the groups that received 0.12 and 0.24 mg/kg of plerixafor, respectively (p-value 0.118). The obtained differences were not significant, which means the application of a decreased dose of plerixafor did not affect the results of mobilization. CONCLUSION: The obtained differences in collection were not significant, and thus the application of a decreased dose of plerixafor did not affect the results of mobilization.

Mismatched related vs matched unrelated donors in TCRαß/CD19-depleted HSCT for primary immunodeficiencies.

TCRαß+/CD19+ graft depletion effectively prevents graft-versus-host disease (GVHD). In the current study, we compared the outcomes of hematopoietic stem cell transplantation (HSCT) with TCRαß+/CD19+ depletion from matched unrelated donors (MUDs) and mismatched related donors (MMRDs) in patients with primary immunodeficiency (PID). A total of 98 pediatric patients with various PIDs underwent HSCT with TCRαß+/CD19+ graft depletion from MUDs (n = 75) and MMRDs (n = 23). All patients received a fludarabine-/treosulfan-based conditioning regimen, with 73 also receiving a second alkylating agent. For GVHD prophylaxis, all but 2 received serotherapy (antithymocyte globulin) before HSCT and a short course of posttransplant immunosuppression. Neutrophil and platelet engraftment in both the MUD and MMRD groups occurred on days 14 and 13, respectively. The incidence of secondary graft failure was 0.16 and 0.17 (P = .85), respectively. The cumulative incidence of acute GVHD grade 2 to 4 was 0.17 in the MUD group and 0.22 in the MMRD group (P = .7). The incidence of cytomegalovirus (CMV) viremia was 0.5 in the MUD group and 0.6 in the MMRD group (P = .35). The frequency of CMV disease was high (17%), and the most common manifestation was retinitis. The kinetics of immune recovery was similar in both groups. The overall survival was 0.86 in the MUD group and 0.87 in the MMRD group (P = .95). In our experience, there was no difference in the outcomes of HSCT performed from MUD and MMRD. Hence, given the immediate availability of donors, in the absence of HLA-identical siblings, HSCT with TCRαß+/CD19+ graft depletion from MMRDs can be considered as the first choice in patients with PID.

αß T Cell-Depleted Haploidentical Hematopoietic Stem Cell Transplantation without Antithymocyte Globulin in Children with Chemorefractory Acute Myelogenous Leukemia.

We evaluated the outcome of αß T cell-depleted haploidentical hematopoietic stem cell transplantation (HSCT) in a cohort of children with chemorefractory acute myelogenous leukemia (AML). Twenty-two patients with either primary refractory (n = 10) or relapsed refractory (n = 12) AML in active disease status received a transplant from haploidentical donors. The preparative regimen included cytoreduction with fludarabine and cytarabine and subsequent myeloablative conditioning with treosulfan and thiotepa. Antithymocyte globulin was substituted with tocilizumab in all patients and also with abatacept in 10 patients. Grafts were peripheral blood stem cells engineered by αß T cell and CD19 depletion. Post-transplantation prophylactic therapy included infusion of donor lymphocytes, composed of a CD45RA-depleted fraction with or without a hypomethylating agent. Complete remission was achieved in 21 patients (95%). The cumulative incidence of grade II-IV acute graft-versus-host disease (GVHD) was 18%, and the cumulative incidence of chronic GVHD was 23%. At 2 years, transplantation-related mortality was 9%, relapse rate was 42%, event-free survival was 49%, and overall survival was 53%. Our data suggest that αß T cell-depleted haploidentical HSCT provides a reasonable chance of long-term survival in a cohort of children with chemorefractory AML and creates a solid basis for further improvement.

Impact of Natural Killer Cell-Associated Factors on Acute Leukemia Outcomes after Haploidentical Hematopoietic Stem Cell Transplantation with αß T Cell Depletion in a Pediatric Cohort.

The technique of αß T cell depletion (αßTCD) is a well-established method of hematopoietic stem cell transplantation (HSCT) for children with acute leukemia owing to the low rates of graft-versus-host disease and nonrelapse mortality (NRM). The graft-versus-leukemia effect is generally ascribed to natural killer (NK) cells conserved within the graft. It is not known whether NK-related factors affect the outcome of αßTCD HSCT, however. The aim of this retrospective study was to explore the impact of NK alloreactivity (based on donor-recipient killer immunoglobulin-like receptor [KIR] mismatch), graft NK cell dose, and blood NK cell recovery on day +30 post-HSCT on the incidences of leukemia relapse and NRM. The pediatric acute leukemia cohort comprised 295 patients who underwent their first HSCT from a haploidentical donor in complete remission. During post hoc analysis, the total cohort was divided into subcohorts by diagnosis (acute lymphoblastic leukemia [ALL]/acute myeloid leukemia [AML]), NK alloreactivity prediction (KIR match/KIR mismatch), graft NK cell dose (less than versus greater than the median value), and blood NK cell recovery on day +30 post-HSCT (less than versus greater than the median value). We also investigated the influence of serotherapy (antithymocyte globulin [ATG] group) versus abatacept + tocilizumab combination [aba+toci] group) on relapse risk in the context of KIR mismatch. The risks of relapse and NRM were calculated by the cumulative risk method, and groups were compared using the Gray test. Multivariate analysis revealed no apparent impact of predicted NK alloreactivity or any other studied NK cell-related factors for the entire cohort. For patients with AML, a significantly higher relapse risk associated with high NK cell graft content on the background of no predicted KIR mismatch (P = .002) was shown. Multivariate analysis confirmed this finding (P = .018); on the other hand, for the KIR-mismatched patients, there was a trend toward a lower risk of relapse associated with high NK cell dose. The use of ATG was associated with a trend toward reduced relapse risk (P = .074) in the AML patients. There was no significant impact of NK-related factors in the ALL patients. Overall, the evaluated NK-related factors did not show a clear and straightforward correlation with the key outcomes of HSCT in our cohort of children with acute leukemia. In practice, the data support prioritization of KIR-mismatched donors for patients with AML. Importantly, a potential interaction of KIR ligand mismatch and NK cell content in the graft was identified. Indirect evidence suggests that additional cellular constituents of the graft could influence the function of NK cells after HSCT and affect their role as graft-versus-leukemia effectors.

Targeted Therapy With Venetoclax and Daratumumab as Part of HSCT Preparative Regimen in Children With Chemorefractory Acute Myeloid Leukemia.

The long-term outcome of allogeneic hematopoietic stem cell transplantation (HSCT) in chemorefractory acute myeloid leukemia (AML) remains suboptimal because of a high relapse rate. Enhancement of conditioning regimens by the incorporation of targeted anti-leukemia agents is a potential approach to improve the efficacy of HSCT. In a pilot trial and extended access cohort, we evaluated the safety and potential value of adding combinations of venetoclax and daratumumab to a preparative regimen among children with chemorefractory acute myeloid leukemia grafted with αß T-cell-depleted peripheral blood stem cells. All 20 patients had active disease status of AML at the time of transplantation. The preparative regimen included myeloablative conditioning based on either total body irradiation or treosulfan. A haploidentical related donor was used as a graft source for all patients. Engraftment was not compromised, and no excess toxicity was noted. Minimal residual disease-negative complete remission was achieved in 17 patients (85%). The cumulative incidence of grade II to IV acute graft-versus-host disease (GVHD) was 17%, and the cumulative incidence of chronic GVHD was 7%. At 2 years, nonrelapse mortality was 10%, relapse incidence was 46%, event-free survival was 44%, and overall survival was 65%. Our data show the possibility of safely adding targeted agents to conditioning regimens; however, no evidence of a significant improvement in long-term transplantation outcomes in this cohort of patients was observed.

Serotherapy-Free Regimen Improves Non-Relapse Mortality and Immune Recovery Among the Recipients of αß TCell-Depleted Haploidentical Grafts: Retrospective Study in Childhood Leukemia.

Depletion of αß T cells from the graft prevents graft-versus-host disease (GVHD) and improves the outcome of hematopoietic stem cell transplantation (HSCT) from haploidentical donors. Delayed recovery of adaptive immunity remains a problem, which can be approached by adoptive T-cell transfer. In a randomized trial, we have assessed the safety and efficacy of low-dose memory (CD45RA-depleted) donor lymphocytes (mDLI) after HSCT with αß T-cell depletion. Antithymocyte globulin (ATG) is viewed as an essential component of preparative regimen, critical for both prevention of graft failure and GVHD. Variable pharmacokinetics of ATG may significantly affect lymphocyte subpopulations after HSCT. To uncover the potential of mDLI, we replaced rabbit ATG with tocilizumab and abatacept. Here we compare post hoc the immune recovery and the key clinical outcomes, including nonrelapse mortality (NRM), overall- and event-free survival (OS and EFS), between the cohort enrolled in the prospective randomized trial and a historical cohort, comprised of patients grafted with a conventional ATG-based HSCT with αß T cell depletion. A cohort of 149 children was enrolled in the prospective trial and 108 patients were selected as historical controls from a prospectively populated database. Patient population was comprised of children with high-risk hematologic malignancies, with more than 90% represented by acute leukemia. Median age at enrollment was 8.8 years. In the prospective cohort 91% of the donors were haploidentical parents, whereas in the historical cohort 72% of the donors were haploidentical. Conditioning was based on either 12Gy total body irradiation or treosulfan. Thiotepa, fludarabine, bortezomib, and rituximab were used as additional agents. Patients in the historical cohort received rabbit ATG at 5 mg/kg total dose, while prospective cohort patients received tocilizumab at 8 mg /kg on day -1 and abatacept at 10 mg/kg on days 0, 7, 14, and 28. Patients in the prospective trial cohort were randomized 1:1 to receive mDLI starting on day 0, whereas 69% of historical cohort patients received mDLI after engraftment, as part of previous trials. Primary engraftment rate was 99% in the prospective cohort and 98% in the historical cohort. The incidence of grade II-IV aGVHD was 13% in the prospective cohort and 16 % in the control group. Chronic GVHD developed among 13% (historical) and 7% (prospective) cohorts (P = .07). The incidence of cytomegalovirus viremia was 51% in the prospective cohort arm and 54% in the historical control arm (p = ns). Overall, in the prospective cohort 2-year NRM was 2%, incidence of relapse was 25%, EFS was 71%, and OS was 80%, whereas in the historical cohort 2-year NRM was 13%, incidence of relapse was 19%, EFS was 67%, and OS was 76%, difference non-significant for relapse and survival. NRM was significantly improved in the ATG-free cohort (P = .002). Recovery of both αß- and γδ- T cells was significantly improved at days +30 and +60 after HSCT in recipients of ATG-free preparative regimens, as well as recovery of naïve T cells. Among the recipients of αß T-cell-depleted grafts, replacement of ATG with nonlymphodepleting abatacept and tocilizumab immunomodulation did not compromise engraftment and GVHD control and was associated with significantly lower NRM and better immune recovery early after HSCT.

Safety and efficacy of the low-dose memory (CD45RA-depleted) donor lymphocyte infusion in recipients of αß T cell-depleted haploidentical grafts: results of a prospective randomized trial in high-risk childhood leukemia.

Depletion of αß T cells from the graft prevents graft-vs.-host disease (GVHD) and improves outcome of HSCT from haploidentical donors. In a randomized trial, we aimed to evaluate the safety and efficacy of low-dose memory (CD45RA-depleted) donor lymphocytes (mDLI) after HSCT with αß T-cell depletion. A cohort of 149 children was enrolled, 76 were randomized to receive scheduled mDLI and 73 received standard care. Conditioning was based on either 12 Gy total body irradiation or treosulfan. Rabbit antithymocyte globulin was replaced by tocilizumab and abatacept. Primary end points were the incidence of acute GVHD grades II-IV and the incidence of cytomegalovirus (CMV) viremia. The incidence of grades II-IV aGVHD was 14% in the experimental arm and 12% in the control arm, p-0.8. The incidence of CMV viremia was 45% in the experimental arm and 55% in the control arm, p-0.4. Overall, in the total cohort 2-year NRM was 2%, cumulative incidence of relapse was 25%, event-free survival 71%, and overall survival 80%, without difference between the study arms. Memory DLI was associated with improved recovery of CMV-specific T-cell responses in a subcohort of CMV IgG seropositive recipients.

T-cell tracking, safety, and effect of low-dose donor memory T-cell infusions after αß T cell-depleted hematopoietic stem cell transplantation.

The delayed recovery of adaptive immunity underlies transplant-related mortality (TRM) after αß T cell-depleted hematopoietic stem cell transplantation (HSCT). We tested the use of low-dose memory donor lymphocyte infusions (mDLIs) after engraftment of αß T cell-depleted grafts.A cohort of 131 pediatric patients (median age 9 years) were grafted with αß T cell-depleted products from either haplo (n = 79) or unrelated donors (n = 52). After engraftment, patients received mDLIs prepared by CD45RA depletion. Cell dose was escalated monthly from 25 × 103 to 100 × 103/kg (haplo) and from 100 × 103 to 300 × 103 /kg (MUD). In a subcohort of 16 patients, T-cell receptor (TCR) repertoire profiling with deep sequencing was used to track T-cell clones and to evaluate the contribution of mDLI to the immune repertoire.In total, 343 mDLIs were administered. The cumulative incidence (CI) of grades II and III de novo acute graft-versus-host disease (aGVHD) was 5% and 2%, respectively, and the CI of chronic graft-versus-host disease was 7%. Half of the patients with undetectable CMV-specific T cells before mDLI recovered CMV-specific T cells. TCR repertoire profiling confirmed that mDLI-derived T cells significantly contribute to the TCR repertoire up to 1 year after HSCT and include persistent, CMV-specific T-cell clones.

Multiple site place-of-care manufactured anti-CD19 CAR-T cells induce high remission rates in B-cell malignancy patients.

Chimeric antigen receptor (CAR) T cells targeting the CD19 antigen are effective in treating adults and children with B-cell malignancies. Place-of-care manufacturing may improve performance and accessibility by obviating the need to cryopreserve and transport cells to centralized facilities. Here we develop an anti-CD19 CAR (CAR19) comprised of the 4-1BB co-stimulatory and TNFRSF19 transmembrane domains, showing anti-tumor efficacy in an in vivo xenograft lymphoma model. CAR19 T cells are manufactured under current good manufacturing practices (cGMP) at two disparate clinical sites, Moscow (Russia) and Cleveland (USA). The CAR19 T-cells is used to treat patients with relapsed/refractory pediatric B-cell Acute Lymphocytic Leukemia (ALL; n = 31) or adult B-cell Lymphoma (NHL; n = 23) in two independently conducted phase I clinical trials with safety as the primary outcome (NCT03467256 and NCT03434769, respectively). Probability of measurable residual disease-negative remission was also a primary outcome in the ALL study. Secondary outcomes include complete remission (CR) rates, overall survival and median duration of response. CR rates are 89% (ALL) and 73% (NHL). After a median follow-up of 17 months, one-year survival rate of ALL complete responders is 79.2% (95%CI 64.5‒97.2%) and median duration of response is 10.2 months. For NHL complete responders one-year survival is 92.9%, and median duration of response has not been reached. Place-of-care manufacturing produces consistent CAR-T cell products at multiple sites that are effective for the treatment of patients with B-cell malignancies.