Donor regulatory T-cell therapy to prevent graft-versus-host disease Free

Allogeneic hematopoietic cell transplantation (HCT) is a curative therapy for patients with hematological malignancies and bone marrow disorders. Graft-versus-host disease (GVHD) causes significant morbidity and mortality after myeloablative human leukocyte antigen (HLA)–matched HCT and limits the success of the therapy.¹ At the time that the trial was conducted, the standard immunosuppressive GVHD prophylaxis regimen of a calcineurin inhibitor and methotrexate resulted in incidences of grade 3 to 4 acute GVHD of 10% to 20% and moderate to severe chronic GVHD of 30% to 50%.^2-4 Both calcineurin-based and calcineurin-free approaches have not improved the composite end point of chronic GVHD-free or relapse-free survival, and new approaches, such as the use of posttransplant cyclophosphamide or engineering the cell composition of the donor graft, are being explored.⁵ 

Preclinical models support a nontoxic strategy to prevent GVHD through the therapeutic administration of purified donor regulatory T cells (Tregs).^6-8 These rare cells limit and help control immune responses. Tregs in the donor graft appear to reduce GVHD and improve immune reconstitution while retaining graft-versus-tumor (GVT) effects in animal and early clinical studies.^6,7,9-11 Donor Tregs were tested in haploidentical HCT and expanded third-party cord blood HCT with apparent reduction in GVHD.^12-16 GVHD prevention is enhanced when pure Tregs are infused 2 to 3 days before the infusion of conventional T cells (Tcons), which drive both GVHD and GVT.¹⁷ 

Our clinical strategy in HLA-matched or 7/8 HCT involves the isolation of highly purified Tregs from the granulocyte colony-stimulating factor (G-CSF) mobilized donor peripheral blood progenitor cells. In the high-precision Treg therapy, the Treg product (2 × 10⁶/kg to 3 × 10⁶/kg) is administered the same day as the hematopoietic stem and progenitor cell product, followed 2 to 3 days later by Tcon at a dose of 3 × 10⁶ CD3⁺ T cells/kg.¹⁸ Prior clinical studies have suggested that as few as 5 × 10⁴ CD3⁺ T cells/kg without GVHD prophylaxis and as few as 1 × 10⁶ CD3⁺ T cells/kg with single-agent GVHD prophylaxis result in significant GVHD.^19,20 Thus, dual-agent prophylaxis is normally required for 3 × 10⁶ CD3⁺ T cells/kg. However, earlier evaluations showed that the high-precision Treg therapy plus single-agent GVHD prophylaxis had a low incidence of GVHD.^18,21 

Here, we evaluate, in an open-label, single-center, 2-stage phase 2 study, whether the engineered Treg-based therapy plus single-agent prophylaxis could improve GVHD-free relapse-free survival (GRFS) at 1 year.

All trials were conducted according to the Declaration of Helsinki and Good Clinical Practice. All patients provided written informed consent.

This trial was designed and opened as a phase 1/2 trial with phase 1 completed between 16 August 2011 and 1 May 2017, and reported elsewhere.¹⁸ The phase 2 efficacy portion of the trial reported here was an investigator-initiated, open-label, single-center (Stanford University) trial (NCT01660607) approved by the institutional review board at Stanford University (IRB-21275). Unrelated donor participation was approved by the National Marrow Donor Program institutional review board (IRB-2020-0421). The first author (E.H.M.) served as US Food and Drug Administration sponsor and holds the Investigational New Drug (IND) at Stanford University (IND number 014686). Data were collected and trial procedures were overseen by the trial investigators. The phase 2 portion of the trial was designed as a 2-stage study, with the first stage randomizing patients to receive Treg therapy alone (n = 12) or Treg therapy plus single-agent GVHD prophylaxis (n = 12) and was completed between 12 November 2018 and 2 April 2019, and reported elsewhere.²¹ The phase 2 second stage was completed between 3 April 2019 and 20 January 2022.

Eligible patients were aged 13 to 72 years, although no patients aged <18 years were enrolled. This study included patients with acute leukemia in complete remission or active disease, chronic myelogenous leukemia (failed multiple tyrosine kinase inhibitors, history of accelerated or blast phase), myelodysplastic syndrome (intermediate 2 or high-risk, IPSS-R), myeloproliferative disorders, or non-Hodgkin lymphoma with poor risk features not suitable for autologous HCT. Patients were required to be candidates for myeloablative conditioning. Patients were eligible if they had a Karnofsky performance-status score ≥70%, a left ventricular ejection fraction of at least 45%, a diffusing capacity of the lung for carbon monoxide of at least 50%, a creatinine clearance of at least 60 mL/min, and serum levels of alanine aminotransferase and aspartate aminotransferase that were no more than 7.5 times the upper limit of the normal range.

Most patients had 8/8 HLA-matched (at HLA-A, HLA-B, HLA-C, or HLA-DRB1 by high-resolution typing) related (n = 21) or unrelated donors (n = 12), although single-allele mismatched (7/8 HLA-allele matched) unrelated donors were permitted (n = 11).

For purposes of general comparison, a single-center contemporaneous standard-of-care (SOC) cohort of HLA-matched patients with HCT (N = 129) were identified who would have been otherwise eligible for this trial and participated in our institutional biobanking study (Table 1). Patients in the SOC cohort received an unmanipulated, G-CSF mobilized donor peripheral blood product followed by dual-agent GVHD prophylaxis methotrexate and tacrolimus.

G-CSF mobilized products were obtained by institutional continuous-flow cell separator (Spectra Optia, Terumo BCT) from HLA-matched volunteer related and unrelated donors after 5 daily doses of 10 to 16 μg/kg G-CSF (Neupogen, Amgen). Apheresis collections occurred as a single-day large-volume collection (18-30 L) or 2 consecutive 15-L collections on days 4 and 5 of G-CSF that were pooled or as a single large volume collection on day 5.

Each Treg therapy is custom made for each individual patient and was produced at a centralized guanosine monophosphate OrcaBio manufacturing facility (Sacramento, CA). Hematopoietic stem progenitor cell (HSPC) and Treg drug products were shipped without cryopreservation to the transplant center for infusion. The Tcon drug product was shipped as a cryopreserved product, with infusion of the thawed Tcon drug product on day +2 to 3. Target Treg dose was 2 × 10⁶ to 3 × 10⁶ cells/kg, CD34 cell dose was >2 × 10⁶ cells/kg, and CD3⁺ target dose was 3 × 10⁶ cells/kg.

All patients received myeloablative conditioning regimens, either total body irradiation (TBI) or busulfan based (Table 2). Tacrolimus was started at 0.03 mg/kg per day 12 to 24 hours following Tcon infusion and targeted to serum trough levels of 6 to 8 ng/mL with taper starting at day +60 and target discontinuation by day +180. Six of 11 HLA 7/8 mismatch recipients had an additional mycophenolate mofetil starting at HSCT and taper off by day 30. Supportive care was provided according to institutional guidelines.¹⁸ 

The phase 2 primary end point was 1-year GRFS, which is a composite end point of no occurrence of acute GVHD grade ≥3 (per Mount Sinai Acute GVHD International Consortium [MAGIC] criteria²²), moderate to severe chronic GVHD (per 2015 National Institutes of Health Consensus Guidelines²³), relapse, or death.

Peripheral blood samples were collected at baseline and defined times after treatment with Treg therapy and HCT. Chimerism was measured by Clinical Laboratory Improvement Amendments–certified single tandem repeat and sequence-based method by the Stanford Clinical Laboratory. Serum interleukin-2 levels were measured at day +14 by high-sensitivity electrochemiluminescence assay (S-Plex, K151Z2S-1, MesoScale Discovery, Rockville, MD). Hematopoietic and peripheral blood immune cell populations were measured by clinical flow cytometry using a FACSAria (BD Biosciences, Milpitas, CA).

For time-to-event end points of acute GVHD, chronic GVHD, and infection, the nonparametric estimation of the cumulative incidence rate in the presence of the competing risk event of death at selected time points and the corresponding 95% confidence interval (CI) are provided. For the end points of relapse-free survival, GRFS, and overall survival, Kaplan-Meier method was used. A significance level of 0.05 was applied to phase 2 end point analysis of the observed vs expected incidence of GRFS. All time-to-event end points (overall survival, progression-free survival, GRFS, and nonrelapse mortality [NRM]) were computed using the R package survival (Therneau 2021, https://CRAN.R-project.org/package=survival). All binomial proportions and associated CIs were estimated using the method of Wilson and computed using the R package binom (Dorai-Raj 2014, https://CRAN.R-project.org/package=binom). Statistical analyses were performed with the use of SPSS software, version 22 (IBM), and R software, version 4.1.0 (R Core Team, 2021; https://www.R-project.org/). We computed the unadjusted GRFS point estimate using Kaplan-Meier and the associated 95% CI using the cumulative hazard. GraphPad Prism 10.0.3 was used to generate all graphs.

For the single-center phase 2 efficacy study, 44 patients (33 HLA matched and 11 single-allele HLA mismatched) were enrolled between January 2019 and January 2022. The last follow-up was 1 April 2024 (median, 34 months; range, 1-63 months). All patients met an end point or achieved at least 2 years of follow-up. Baseline patient characteristics are summarized in Table 1 and supplemental Table 1 (available on the Blood website). An SOC reference cohort was identified for the same period and included 129 patients (42 HLA-matched measurable residual disease, 76 HLA-matched unrelated donor (URD), and 11 single-allele HLA mismatched; Table 3).

For all Treg therapy recipients, products were manufactured, delivered, and infused without complication (schema in Figure 1A). Overall time from North American donor centers to central manufacturing to Stanford was under expiry of 72 hours for all products. No infusion reactions were attributed to the donor Treg therapy. The mean HSPC drug product cell dose target of >2.0 × 10⁶ cells/kg was achieved in all patients with a mean dose of 6.7 × 10⁶ per kg (2.1 × 10⁶ to 10 × 10⁶), with fewer than 1.0 × 10⁵ cells/kg Tcon in all products. The mean Treg therapy dose was 2.8 × 10⁶ per kg (1.2 × 10⁶ to 3 × 10⁶), with purity of 97% (range, 92%-99%). Tcon products were controlled to target dose of 3 × 10⁶ cells/kg with the option to match Tcon dose with Treg dose for those patients who had <2 × 10⁶ cells/kg at a ratio 1:1 (which is the case for 2 patients). The Treg therapy approach results in a precise donor cell infusion with low variability centered around a 1:1 ratio of Treg to Tcon (Figure 1B). On the basis of preclinical mouse studies, the Treg and Tcon products are delivered in a timed, sequential manner (Figure 1A). Because the Tregs are given 2 to 3 days before the Tcon, the Tregs are expected to expand in vivo between 4 to 9 cell cycles. Thus, the effective Tregs/Tcon ratio on the day of Tcon infusion is estimated to be between 15:1 and 500:1. In this strategy, highly purified Tregs are given a significant advantage over their naturally occurring ratio (≈1:20 Treg/Tcon; Figure 1B), which was hypothesized to shape immune reconstitution toward immune tolerance.

All patients engrafted with median times to neutrophil and platelet engraftment of 12 days (range, 10-16 days) and 15.5 days (range, 11-56 days), respectively. No primary graft failures were observed. The median time from transplant to discharge was 15 days (range, 12-29 days) vs 19 days (range, 14-33 days) for Stanford SOC patients. Full donor myeloid chimerism was achieved in 99% of patients at day +90 to 100 (Figure 1C). Over 80% of patients also achieved full donor T-cell chimerism at day +90 to 100 (Figure 1C). There was no relationship between achieving full or mixed CD3 T-cell chimerism by day 90 to 100 and disease relapse, although this trial is not powered to address this question.

For all patients receiving Treg therapy, 11 of 44 (25%; 95% CI, 13%-39%) had grade 2 to 4 and 3 had grade 3 to 4 acute GVHD (7%; 95% CI, 1.7%-17%; Figure 1D). The single-center SOC reference cohort had acute GVHD grade 2 to 4 in 49 of 129 patients (38%; 95% CI, 30%-46%) and grade 3 to 4 in 29 of 129 patients (22%; 95% CI, 16%-30%; Table 4).⁵ Additional details of GVHD manifestations are provided in Tables 5 and 6 and supplemental Tables 2 and 3.

The 1-year cumulative incidence of moderate to severe chronic GVHD was 5 of 44 patients (11%; 95% CI, 4.1%-23%) (Figure 1E). For context, the SOC reference cohort, 42 of 129 patients developed moderate to severe chronic GVHD (33%; 95% CI, 25%-41%).

The 1-year cumulative NRM was 3 of 44 (4.5%; 95% CI, 0.8%-14%) for trial participants and 18 of 129 (13%; 95% CI, 8%-20%) in the SOC reference cohort.

The phase 2 trial was powered for an estimated historical incidence of 40% GRFS at 1 year posttransplant and achieved statistical significance for the HLA matched alone at 67% (P = .002) and all the patients enrolled at 64% (P = .002). The SOC reference cohort had a 1-year GRFS of 36% (95% CI, 28%-45%; Figure 1F). The relapse-free survival for phase 2 trial participants at 1 year was 82% (95% CI, 71%-94%).

For the patients with grade 2 to 4 acute GVHD, 10 of 11 (91%) responded to front-line corticosteroid therapy, and the remaining patient did not respond to further therapy, limited by concomitant severe sinusoidal obstruction syndrome (SOS)/veno-occlusive disease (VOD) (Figure 2A). For the patients with grade 2 to 4 acute GVHD in the SOC cohort, 24 of 49 (50%) responded to corticosteroids, an additional 15 of 49 (28%) responded to further therapy, and the remainder died of GVHD (22%; Figure 2A).

Given the evidence for reduced GVHD and improved GVHD response, we wanted to assess the potential effect on later-term NRM by HLA match type with evidence that trial participants had reduced mortality compared with what might be expected (Figure 2B). The reference SOC had more URD transplants compared with the trial participants and likewise the trial participants included proportionately more major histocompatibility complex mismatch. With this mind and with the improved response to corticosteroids observed in trial participants, we assessed the 2-year cumulative incidence of the combined end point of RFS and survival off all immunosuppression by donor type showed among trial participants at 57% for HLA-matched sibling, 75% for HLA-matched unrelated donor, and 45% for HLA single-allele mismatch by donor type differed substantially from that seen in the SOC cohort at 39% for HLA-matched sibling, 28% for HLA-matched unrelated donor, and 0% for HLA single-allele mismatch (Figure 2C). An evaluation at 2 years was believed necessary as it better captures late-term effects of chronic GVHD and other complications.

The frequency of nonhematologic grade ≥3 severe adverse events within 90 days after donor Treg product infusion was 23% (n = 10 of 44). The incidence of grade ≥3 infections was 5% (by Bone Marrow Transplant Clinical Trials Network Manual of Procedure criteria). Cytomegalovirus status and reactivation are shown with no end-organ involvement noted (Table 3). Sinusoidal obstruction syndrome cases and other complications occurred at low frequency (Table 4). Further details of the results seen in HLA-mismatch transplant can be found in supplemental Tables 4 and 5.

Total and CD4 and CD8 subset immune reconstitution showed recovery of appropriate levels of cells within 6 months, and mean CD4 was >50 per μL at day 30 (Figure 2D). Serum interleukin-2 levels at day +14 were substantially higher in single-allele mismatch recipients (Figure 2E), which may explain why the CD4/Treg and CD8/Treg ratios were substantially higher in this group (Figure 2F).

GVHD and immune dysregulation remain significant causes of morbidity and mortality after allogeneic HCT. To improve HCT, new methods that facilitate better donor T-cell reconstitution and immune tolerance are needed. By investigating the mechanisms that control T-cell reconstitution, strategies were developed to use highly purified donor Treg product to reduce or replace immunosuppression. In this clinical report, the single-center phase 2 trial met its primary efficacy end point of improved GVHD relapse-free survival from expected, because of a low incidence of grade 3 to 4 acute GVHD, moderate to severe chronic GVHD, and NRM. The trial was reliant on donor blood collection centers across the continental United States and Hawaii, demonstrating the safety and feasibility of manufacturing and distributing a new category of precision-selected Treg therapy.

The donor Treg product had no associated infusion reactions or exceptional adverse events and showed evidence for earlier neutrophil engraftment compared with SOC. This could be because of the removal of methotrexate, which exacerbates mucositis and can delay engraftment, or it is possible the Tregs themselves may be facilitative. There were no exceptional infectious disease risks or complications, and patients seemed to recover quickly, especially given the low incidence of chronic GVHD, which profoundly limits quality of life and can drive long-term morbidity. Mortality was substantially reduced compared with the SOC cohort and historical expectations, and the sought after end point of no relapse and off immunosuppression at 2 years appears substantially greater in trial participants.

Because the donor Treg product and other graft constituents are more precisely determined than in a full apheresis product in which T cells can vary logs fold, there is the impression of a smoother and more reproducible immune recovery. In preclinical models, Treg improve immune reconstitution because of the prevention of GVHD, which damages lymphoid organs where immune recovery takes place.¹¹ It is likely that combined strategies of T-cell depletion and other agents that work synergistically with Tregs or improve Treg function, like genetic modification, could allow new opportunities for continued improvement.

Although the patients on trial had heterogeneous primary diseases, GVT effect in trial recipients appeared intact. A central trade-off in the HCT field is that increased chemotherapy intensification reduces relapse but at the cost of increased NRM due to GVHD, infectious complications, and organ toxicity.²⁴ The current results suggest the donor Treg therapy reduces immune dysfunction and may allow more intensive chemotherapy to reduce relapse without increased NRM. Furthermore, the avoidance of toxic posttransplant therapies, like methotrexate, could facilitate earlier treatment of malignancy, reduced infection risk, or better tolerability for older or sicker patients and for patients who have only 7/8 HLA-matched donors available.

A potential limitation of the study is that posttransplant cyclophosphamide has become more commonly used for HLA-matched and 7/8 myeloablative transplantation at many centers across the United States but, at the study site, the approach was used on clinical trials during the period of this study, and these patients are not included in our SOC cohort. Although posttransplant cyclophosphamide has decreased the incidence of GHVD, the overall severity and treatment response has not changed substantially, and some key complications observed with the use of posttransplant cyclophosphamide, such as engraftment syndrome, graft loss, and cardiac toxicity, were not observed in this trial.

The high-precision Treg therapy described herein has evolved into a therapy called “Orca-T.” On the basis of the results of these trials, a multicenter phase 1b trial of Orca-T was conducted and a randomized phase 3 trial comparing Orca-T with an unmanipulated allograft plus standard GVHD prophylaxis is underway (NCT05316701).

Funding was received from the National Institutes of Health (NIH), National Heart, Lung, and Blood Institute grants RO1 HL1145591 (R.S.N.) and K08HL119590 (E.H.M.), and NIH, National Cancer Institute grant P01 CA 49605 (R.S.N.); an ORCA Sponsored Research Agreement (E.H.M.); and a National Marrow Donor Program Amy Manesevit Award (E.H.M.).

Contribution: E.H.M. was responsible for overall trial conduct of the phase 2 trial and correlative science and was responsible for analysis and writing of the manuscript; A. Pavlova, K.S., C.B., A.V.-P., and P.K. contributed to trial conduct, analysis, and writing, along with R.S.N., S.S., and V.A. who served as site directors and/or contributed to patient care and accrual; S.B., S.D., M.F., S.A., L.J., D.M., A.R., P.S., J.S., W.-K.W., and R.L. contributed to patient care and trial conduct; N.F. and J.S.M. contributed to trial conduct and cell product manufacturing; A. Putnam contributed to cell product scaling and manufacturing; A. Pavlova, C.B., A.V.-P., B.X., and R.D.P. contributed to correlative science; R.L. provided independent GVHD assessment and review; J.T. and Y.L. contributed to statistical evaluations; and all authors had access to the data, confirm the completeness and accuracy of the data and the fidelity of the trial to the protocol, solely contributed to the writing and review of the manuscript, and had final responsibility for the manuscript content.

Conflict-of-interest disclosure: E.H.M. receives sponsored research support from Orca Biosciences for the conduct of this and other clinical trials and has equity in and consulted for GigaMune, Jura Bioscience, and TRACT Therapeutics. R.L. receives sponsored research support from Orca Biosciences for the conduct of this and other clinical trials. The remaining authors declare no competing financial interests.

Correspondence: Everett H. Meyer, Division of Bone Marrow Transplantation and Cellular Therapy, Department of Medicine, Stanford University, 269 West Campus Dr, CCSR 2245c, Stanford, CA 94305; email: evmeyer@stanford.edu.