Judith A. Shizuru

CD19-directed chimeric antigen receptor T cell therapy (CAR19) improves survival in relapsed/refractory large B cell lymphoma compared with immunochemotherapy with intent for autologous hematopoietic cell transplantation (autoHCT). However, major toxicities include prolonged cytopenias, infection and secondary hematologic malignancies. Here, to investigate these complications, we studied patients with lymphoma receiving CAR19 and compared clinical outcomes to propensity-matched autoHCT-treated controls. CAR19-treated patients exhibited impaired immune reconstitution and increased infection. Bone marrow analysis showed that prolonged post-CAR cytopenias are associated with clonal cytopenias of undetermined significance and interferon-mediated inflammation. Clonal hematopoiesis (CH) commonly expanded following CAR19 infusion and was associated with impaired immune reconstitution and treatment-related myeloid malignancy (tMN). Post-CAR tMN was molecularly and clinically comparable to post-autoHCT tMN. Single-cell DNA analysis revealed mostly independent CH clones, while CAR integration into CH-mutant T cells occurred frequently and may drive expansion and persistence. Together, these findings implicate CH expansion and marrow inflammation in key CAR19-associated toxicities.

Mixed hematopoietic chimerism after allogeneic hematopoietic cell transplantation (HCT) promotes tolerance of transplanted donor-matched solid organs, corrects autoimmunity, and could transform therapeutic strategies for autoimmune type 1 diabetes (T1D). However, development of nontoxic bone marrow conditioning protocols is needed to expand clinical use. We developed a chemotherapy-free, nonmyeloablative (NMA) conditioning regimen that achieves mixed chimerism and allograft tolerance across MHC barriers in NOD mice. We obtained durable mixed hematopoietic chimerism in prediabetic NOD mice using anti–CD117 monoclonal antibody, T cell depleting antibodies, JAK1/2 inhibition, and low-dose total body irradiation prior to transplantation of MHC-mismatched B6 hematopoietic cells, preventing diabetes in 100% of chimeric NOD:B6 mice. In overtly diabetic NOD mice, NMA conditioning followed by combined B6 HCT and islet transplantation durably corrected diabetes in 100% of chimeric mice without chronic immunosuppression or graft-versus-host disease (GVHD). Chimeric mice remained immunocompetent, as assessed by blood count recovery and rejection of third-party allogeneic islets. Adoptive transfer studies and analysis of autoreactive T cells confirmed correction of autoimmunity. Analysis of chimeric NOD mice revealed central thymic deletion and peripheral tolerance mechanisms. Thus, with NMA conditioning and cell transplantation, we achieved durable hematopoietic chimerism without GVHD, promoted islet allograft tolerance, and reversed established T1D.

CD19-directed chimeric antigen receptor T-cell therapy (CAR19) improves survival in patients with relapsed/refractory large B-cell lymphoma (rrLBCL) compared to immunochemotherapy with intent for autologous hematopoietic cell transplantation (HCT). However, major toxicities of CAR19 therapy include prolonged cytopenias, infection, and secondary hematologic malignancies. To investigate the mechanisms underlying these toxicities we studied a cohort of lymphoma patients receiving CAR19. CAR19-treated patients exhibited impaired immune reconstitution and increased infection compared to propensity-matched HCT-treated controls. Bone marrow analysis revealed prolonged post-CAR cytopenias is associated with clonal cytopenias of undetermined significance (CCUS) and is characterized by interferon-mediated inflammation. Despite durable lymphoma remissions, clonal hematopoiesis (CH) commonly expanded following CAR19 infusion and was associated with impaired immune reconstitution and the development of treatment related myeloid malignancy (tMN). The molecular composition and clinical outcomes of post-CAR tMN were comparable to those of post-HCT tMN. Single-cell DNA analysis revealed that most post-CAR CH clones harbored a single independent mutation and that CAR integration into T cells with CH mutations may drive persistence. These findings broadly implicate CH mutation burden and CH expansion in the development of post-CAR cytopenias and malignancies as well as mechanistically suggest these expansions occur in a background of marrow inflammation. Together, our results provide insight into the origins of key CAR19-associated toxicities, including infection and tMN.

Current hematopoietic stem cell transplantation (HSCT) conditioning strategies cause widespread tissue damage and systemic toxicities, especially in patients with DNA-repair deficiencies such as Fanconi anemia (FA). We have developed an alternative conditioning approach that incorporates the anti-CD117 antibody, briquilimab, which targets host hematopoietic stem and progenitor cells in place of genotoxic irradiation- and busulfan-based chemotherapy. Here we report a phase 1b clinical trial in patients with FA and bone marrow failure, evaluating safety and efficacy of briquilimab-based conditioning in combination with rabbit anti-thymocyte globulin, cyclophosphamide, fludarabine and rituximab immunosuppression and T cell receptor (TCR)αβ⁺ T cell-depleted and CD19⁺ B cell-depleted haploidentical HSCT. Primary endpoints of the trial included safety and engraftment, and secondary endpoints included pharmacokinetic measures and hematological and immunological recovery. All three patients have each undergone 2 years of follow-up to complete the phase 1b analysis. No treatment-emergent adverse events or acute graft-versus-host disease was observed. Patients experienced minimal toxicities, with typical mucositis and no veno-occlusive disease. Median neutrophil engraftment was 11 days (range 11–13 days) with robust donor chimerism up to 2 years post-HSCT (99–100%), meeting the primary endpoints of the study. Briquilimab cleared in each patient before HSCT without the need for adjustment. Red blood cell, platelet and lymphocyte recovery was comparable to previous reports with TCRαβ⁺ T cell-depleted and CD19⁺ B cell-depleted grafts. All patients are alive and well with resolution of earlier chromosomal breakage abnormalities in peripheral blood lymphocytes post treatment. These data demonstrate the broad potential of this protocol in maintaining HSCT efficacy while reducing toxicity. The phase 2 trial is ongoing (ClinicalTrials.gov identifier: NCT04784052).

MGTA-145 or GROβT, a CXCR2 agonist, has shown promising activity for hematopoietic stem cell (HSC) mobilization with plerixafor in pre-clinical studies and healthy volunteers. Twenty-five patients with multiple myeloma enrolled in a phase 2 trial evaluating MGTA-145 and plerixafor for HSC mobilization (NCT04552743). Plerixafor was given subcutaneously followed 2 h later by MGTA-145 (0.03 mg/kg) intravenously with same day apheresis. Mobilization/apheresis could be repeated for a second day in patients who collected 6 CD34+ cells/kg. Lenalidomide and anti-CD38 antibody were part of induction therapy in 92% (n = 23) and 24% (n = 6) of patients, respectively. Median total HSC cell yield (CD34+ cells/kg × 10⁶) was 5.0 (range: 1.1–16.2) and day 1 yield was 3.4 (range: 0.3–16.2). 88% (n = 22) of patients met the primary endpoint of collecting 2 ×10⁶ CD34+ cells/kg in ≤ two days, 68% (n = 17) in one day. Secondary endpoints of collecting 4 and 6 × 10⁶ CD34+ cells/kg in ≤ two days were met in 68% (n = 17) and 40% (n = 10) patients. Grade 1 or 2 adverse events (AE) were seen in 60% of patients, the most common AE being grade 1 pain, usually self-limited. All 19 patients who underwent transplant with MGTA-145 and plerixafor mobilized HSCs engrafted successfully, with durable engraftment at day 100. 74% (17 of 23) of grafts with this regimen were minimal residual disease negative by next generation flow cytometry. Graft composition for HSCs and immune cells were similar to a contemporaneous cohort mobilized with G-CSF and plerixafor.

Chimeric antigen receptor (CAR) T cells targeting CD22 (CD22-CAR) provide a therapeutic option for patients with CD22⁺ malignancies with progression after CD19-directed therapies. Using on-site, automated, closed-loop manufacturing, we conducted parallel Phase 1b clinical trials investigating a humanized CD22-CAR with 41BB costimulatory domain in children and adults with heavily treated, relapsed/refractory (r/r) B-ALL. Of 19 patients enrolled, 18 had successful CD22-CAR manufacturing, and 16 patients were infused. High grade (3–4) cytokine release syndrome (CRS) and immune effector-cell-associated neurotoxicity syndrome (ICANS) each occurred in only one patient; however, three patients experienced immune-effector-cell-associated hemophagocytic lymphohistiocytosis-like syndrome (IEC-HS). Twelve of 16 patients (75%) achieved CR with an overall 56% MRD-negative CR rate. Duration of response was overall limited (median 77 days), and CD22 expression was downregulated in 4/12 (33%) available samples at relapse. In summary, we demonstrate that closed-loop manufacturing of CD22-CAR T cells is feasible and is associated with a favorable safety profile and high CR rates in pediatric and adult r/r B-ALL, a cohort with limited CD22-CAR reporting.

Allogeneic hematopoietic cell transplantation (HCT) is a curative therapy limited by graft-versus-host disease (GVHD). Donor regulatory T cells (Tregs) appear to prevent GVHD and promote healthy immunity. We have developed a high-precision Treg cell therapy manufactured from donor mobilized peripheral blood. We conducted an open-label, single-center, phase 2 efficacy study investigating if the use of a precision engineered donor Treg cell therapy improves one-year GVHD-free relapse free survival (GRFS) after myeloablative transplantation. Thirty-three patients with HLA-matched donors were enrolled. All donor Treg cell products were successfully manufactured and administered fresh within 72 hours. One-year incidence of acute grade III-IV GVHD of 6%, moderate to severe chronic GVHD of 7% and non-relapse mortality rate of 6%. The primary endpoint of significantly improved one-year GRFS was achieved at 67% for HLA-matched patients compared to a predicted 40% (p < 0.002). Trial participants had a reduced incidence of GVHD and improved GRFS, as compared to rates common to highly variable unmanipulated donor grafts and multi-agent immune suppression.

Depletion of hematopoietic stem cells (HSCs) is used therapeutically in many malignant and nonmalignant blood disorders in the setting of a hematopoietic cell transplantation (HCT) to eradicate diseased HSCs, thus allowing donor HSCs to engraft. Current treatments to eliminate HSCs rely on modalities that cause DNA strand breakage (ie, alkylators, radiation) resulting in multiple short-term and long-term toxicities and sometimes even death. These risks have severely limited the use of HCT to patients with few to no comorbidities and excluded many others with diseases that could be cured with an HCT. 5-Azacytidine (AZA) is a widely used hypomethylating agent that is thought to preferentially target leukemic cells in myeloid malignancies. Here, we reveal a previously unknown effect of AZA on HSCs. We show that AZA induces early HSC proliferation in vivo and exerts a direct cytotoxic effect on proliferating HSCs in vitro. When used to pretreat recipient mice for transplantation, AZA permitted low-level donor HSC engraftment. Moreover, by combining AZA with a monoclonal antibody (mAb) targeting CD117 (c-Kit) (a molecule expressed on HSCs), more robust HSC depletion and substantially higher levels of multilineage donor cell engraftment were achieved in immunocompetent mice. The enhanced effectiveness of this combined regimen correlated with increased apoptotic cell death in hematopoietic stem and progenitor cells. Together, these findings highlight a previously unknown therapeutic mechanism for AZA which may broaden its use in clinical practice. Moreover, the synergy we show between AZA and anti-CD117 mAb is a novel strategy to eradicate abnormal HSCs that can be rapidly tested in the clinical setting.

Despite impressive progress, more than 50% of patients treated with CD19-targeting chimeric antigen receptor T cells (CAR19) experience progressive disease. Ten of 16 patients with large B cell lymphoma (LBCL) with progressive disease after CAR19 treatment had absent or low CD19. Lower surface CD19 density pretreatment was associated with progressive disease. To prevent relapse with CD19⁻ or CD19^lo disease, we tested a bispecific CAR targeting CD19 and/or CD22 (CD19-22.BB.z-CAR) in a phase I clinical trial (NCT03233854) of adults with relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL) and LBCL. The primary end points were manufacturing feasibility and safety with a secondary efficacy end point. Primary end points were met; 97% of products met protocol-specified dose and no dose-limiting toxicities occurred during dose escalation. In B-ALL (n = 17), 100% of patients responded with 88% minimal residual disease-negative complete remission (CR); in LBCL (n = 21), 62% of patients responded with 29% CR. Relapses were CD19^−/lo in 50% (5 out of 10) of patients with B-ALL and 29% (4 out of 14) of patients with LBCL but were not associated with CD22^−/lo disease. CD19/22-CAR products demonstrated reduced cytokine production when stimulated with CD22 versus CD19. Our results further implicate antigen loss as a major cause of CAR T cell resistance, highlight the challenge of engineering multi-specific CAR T cells with equivalent potency across targets and identify cytokine production as an important quality indicator for CAR T cell potency.