Alloreactive Immune Responses Research Articles

Treatment of acute myeloid leukemia with Orca-T.

6552 Background: Allogeneic hematopoietic stem cell transplants (alloHSCT) offer a potential curative treatment for many hematological cancers, however, traditional alloHSCT is associated with high mortality from complications including infection, graft versus host disease (GvHD) and relapse. Orca-T is an investigational allogeneic T-cell immunotherapy that includes stem and immune cells, derived from allogeneic donors, that leverages highly purified, polyclonal donor regulatory T cells to control alloreactive immune responses. In this sub-group analysis, we evaluated the safety and efficacy of patients with acute myeloid leukemia who were treated with Orca-T. Methods: Data from 37 patients with the diagnosis of acute myeloid leukemia (AML) in CR/CRi who received myeloablative conditioning with busulfan, fludarabine, and thiotepa (BFT) followed by Orca-T by 6/30/22 as part of a multicenter phase 1b single-arm trial (NCT04013685) are reported here. Patients enrolled in the phase 1b trial who did not meet these criteria were not included in this analysis. Patients received BFT prior to Orca-T, followed by single-agent GvHD prophylaxis with tacrolimus, and had an 8/8 related or unrelated matched donor. Donors were matched via DNA-based high-resolution typing of HLA-A, -B, -C, and -DRB1. Results: Orca-T was successfully manufactured at a centralized GMP facility, distributed, and infused at study sites throughout the U.S. Vein-to-vein time (i.e. time between end of donor apheresis to start of recipient’s Orca-T infusion) was < 72 hours for all patients, with the majority < 60 hours. Median age was 51 years, 49% were female and 16% were of Hispanic or Latino ethnicity. Twenty-three (62%) had matched related donors and 14 (38%) had matched unrelated donors. Baseline HCT-CI score was 3 and 4 in 27% and 8% of the patients, respectively. The majority had intermediate DRI scores (89%) and 41% were MRD positive at baseline. The median duration of follow-up was 14 months. Relapse-free survival at 12 months was 81.4% (95% CI: 62.9, 91.2). Non-relapse mortality and overall survival were 0% and 100% at 12 months, respectively. Conclusions: These encouraging results suggest that investigational Orca-T could represent a reduced toxicity alternative to conventional alloHSCT. This combination of Orca-T with myeloablative BFT led to > 80% RFS without treatment related mortality, and 100% overall survival in this AML patient population. These outcomes were accomplished with consistent and reliable cell manufacturing and distribution of Orca-T at a national scale. A multi-center randomized controlled phase 3 trial comparing Orca-T to SOC, utilizing BFT or TBI-based conditioning, will complete enrollment during the first half of 2024. (NCT05316701). Clinical trial information: NCT04013685 .

Histocompatibility Testing: A Fundamental Aspect of Renal Transplant Workup

Histocompatibility testing is pivotal in any renal transplantation workup, aimed at enhancing prospective donor recipient compatibility and improving transplant outcomes. The evolution and advancement of histocompatibility testing, particularly HLA typing, have significantly improved its precision. This study outlines the historical progression from serologic to DNA-based HLA typing, emphasizing the role of HLA proteins in immune response. Anti-HLA antibodies, targeting HLA proteins, pose challenges in renal transplantation. Monitoring and managing these antibodies are critical for renal transplant success. Complement-dependent cytotoxicity crossmatch and flow cytometry crossmatch are essential techniques for assessing donor–recipient compatibility. Panel-reactive antibody assesses antibodies against a panel of donor antigens, often HLA. Higher PRA levels (percentage) complicate donor matching, requiring specialized protocols. Virtual crossmatch evaluates recipient anti-HLA antibodies against potential donors through synthetic beads. This approach predicts crossmatch outcomes by comparing antibody profiles, offering a valuable tool for the risk assessment of renal transplantation. Despite advancements, a comprehensive understanding of alloreactive immune responses requires a combination of assays, emphasizing the importance of a multifaceted approach in histocompatibility testing. This is an attempt to compile the relevant information, providing a basis for comparison in a clear and foundational format for histocompatibility testing laboratories.

Optimizing Outcomes with Myeloablative Conditioning in Older Patients: Efficacy and Safety of Precision Engineered Orca-T in Patients > 55 Years Old with Hematologic Malignancies

Background Allogeneic hematopoietic stem cell transplants (alloSCT) offer the only curative treatment for many hematological cancers but carry the risk of significant toxicity including graft versus host disease (GvHD). Additionally, while the use of a myeloablative conditioning (MAC) regimen optimizes disease control, it can increase the complications associated with transplant and non-relapse mortality. Therefore, older patients are often deemed unfit for MAC and relegated to treatment with reduced intensity conditioning (RIC) regimens, which result in a higher incidence of relapse. Orca-T is a high-precision cell therapy biologic that includes stem and immune cells, derived from allogeneic donors, that leverages highly purified, polyclonal donor regulatory T cells to control alloreactive immune responses. The specific subsets selected in the Orca-T manufacturing process retain cells with therapeutic benefit while removing those that pose potential risks, allowing for intensification of concomitant chemotherapy and resulting in overall positive outcomes to date in the phase 1b trial (NCT04013685). To assess the safety and efficacy in an older patient population, we compared outcomes in those at least 55 years old to younger patients treated with Orca-T, receiving chemotherapy-based MAC with tacrolimus as single-agent GvHD prophylaxis. Methods As of 6/02/23, 38 patients ≥18 to <55 (younger) and 25 patients ≥55 years of age (older) received Orca-T as part of a multicenter Phase 1b single-arm trial and had the following diagnosis: acute myeloid, lymphoid or mixed phenotype leukemia in CR/CRi, myelodysplastic syndrome or chronic myeloid leukemia that was in chronic phase. Patients with active disease at the time of transplantation and patients with myelofibrosis were not included in this analysis. Patients received a myeloablative regimen consisting of intravenous busulfan, fludarabine, and thiotepa (BFT) prior to Orca-T, followed by single-agent GvHD prophylaxis with tacrolimus, and had an 8/8 related or unrelated matched donor. Donors were matched via DNA-based high-resolution typing of HLA-A, -B, -C, and -DRB1. Results Orca-T was successfully manufactured at a centralized GMP facility, distributed and infused at study sites throughout the U.S. Vein-to-vein time (i.e. time between end of donor apheresis to start of recipient's Orca-T infusion) was < 72 hours for all patients, majority < 60 hours. Median age was 47 in the younger group and 59 in the older group. The younger group was 50% female, and the older group was 36% female. The percentage that were of Hispanic or Latino ethnicity were 11% in the younger group, and 12% in the older group. 8%, 74% and 11% vs 12%, 44% and 32% had ALL, AML and MDS in the younger and older groups, respectively. A greater proportion of patients in the younger group had a baseline HCT-CI score of 0 (53%), while more had a baseline HCT-CI score of 4 (12%) in the older group. The median duration of follow-up for both groups was 12 months (Table). Disease free survival at 1 year was similar in both groups at 89% in the younger patients and 86% in the older patients (Figure). Modified GRFS* rate at 12 months among patients in the younger group was 86% compared to 77% in the older group. Non-relapse mortality at 1 year was 0% in both of these groups who received BFT MAC. Overall survival was high at 100% and 96% at 12 months in the younger and older patients, respectively. There were two patients > 65 when they received Orca-T; both remain alive without evidence of grade 3-4 acute GvHD, moderate to severe cGvHD, or relapse to date. Only 2 patients experienced grade 3 mucositis (no grade 4-5) and 1 younger patient (2.6%) and 1 older patient (4.0%) required total parenteral nutrition. Conclusions Our analysis suggests that Orca-T cell therapy represents a reduced toxicity alternative to conventional transplant and is well-tolerated by patients 55 and older, with comparable clinical outcomes to those 18 - < 55. In both patient groups, relapse incidence was low, with high GRFS. Remarkably, non-relapse mortality was zero at 1 year with excellent overall survival , suggesting that this regimen could potentially improve outcomes for older patients. An ongoing Phase 3 trial evaluating Orca-T vs. standard of care is currently enrolling throughout the United States.

High Disease-Free Survival in Patients with High-Risk MDS Treated with Orca-T

BACKGROUND Allogeneic hematopoietic cell transplant (alloHCT) may be a curative treatment option for patients with myelodysplastic syndrome (MDS). The use of investigational therapies that potentially reduce transplant toxicity could improve outcomes in this patient population with difficult to treat disease. Per CIBMTR, survival after alloHCT for MDS using matched donors is approximately 65% at 1 year and relapse-free survival at 1 year was ~69% in MDS patients in the RICMAC trial who received myeloablative conditioning (MAC). Orca-T is a high-precision cell therapy biologic that includes stem and immune cells, derived from allogeneic donors, that leverages highly purified, polyclonal donor regulatory T cells to control alloreactive immune responses. In this phase 1b study, Orca-T was evaluated in MDS patients who were eligible for transplant per 2017 International Expert Panel recommendations. METHODS As of 03/31/23, 16 patients with intermediate to very high risk (HR) MDS with ≤10% blasts in the bone marrow at the time of transplant had received Orca-T in the phase 1b study. IPSS-M prognostic scores were Low (6%), Moderate Low (6%), Moderate High (19%), High (44%), and Very High (25%). Baseline HCT-CI scores were 0 (n=6), 1 (n=3), 2 (n=2), 3 (n=3), and 4 (n=2). The median f/u was 19.1 months (range 6.3 - 32.2). Patients were aged 43 - 65 (median 59) and 56% female. Donors were 8/8 HLA-matched related (n=7) or unrelated (n=9). Patients received a busulfan-based myeloablative chemotherapy regimen prior to Orca-T, followed by single-agent GvHD prophylaxis with tacrolimus. RESULTS Orca-T was successfully manufactured in a single, centralized GMP manufacturing facility for all patients enrolled, distributed, and infused throughout the U.S. Orca-T was delivered within < 72 hours vein-to-vein (i.e. time between end of donor apheresis to start of recipient's Orca-T infusion). Disease-free survival (RFS) at 12 months was 94% (Figure). At 1 year, no patients had experienced grade ≥ 3 acute GvHD and moderate to severe chronic GvHD had occurred in 2 patients. Modified GRFS (survival free of Grade 3-4 aGvHD, moderate-to-severe cGvHD and relapse) at 12 months was 75%. Non-relapse mortality (NRM) and overall survival (OS) were 0% and 94%, respectively, at 12 months. Additionally, one MDS patient with very poor prognosis, characterized by mutated TP53 and complex karyotype, remains in CR without active signs of acute or cGvHD at 500 days post-alloHCT. CONCLUSIONS Orca-T demonstrated promising results in this HR-MDS patient population, including high RFS, low incidence of GvHD, and very high OS at 1 year. A randomized controlled phase 3 multi-center trial comparing Orca-T to SOC in patients with MDS, AML, and ALL is currently enrolling across the US (NCT05316701).

Drug exporter expression correlates with group 3 innate lymphoid cell resistance to immunosuppressive agents

Allogeneic hematopoietic cell transplantation (HCT) is often complicated by graft versus host disease (GvHD), an alloreactive immune response triggered by tissue damage. Interleukin (IL)-22 producing type 3 innate lymphoid cells (ILC3) protect epithelial tissues against chemo(radio)therapy-induced damage, suppress alloreactive T cells and mitigate acute GvHD symptoms after allogeneic HCT. Relatively high numbers of ILC before and after allogeneic HCT has been associated with significantly reduced tissue damage and less acute GvHD. While most transplantation conditioning and GvHD prophylaxis regimens are aimed at eliminating host and alloreactive donor lymphocytes, the effect of these regimens on ILC remain elusive. Here, we studied the effect of conditioning chemotherapy and immunosuppressive agents on the survival, proliferation, activation and function of human ILC3 in vitro. Tonsil-derived ILC3 were activated and incubated with agents commonly used to prevent and treat GvHD. While fludarabine, rapamycin, mycophenolic acid and prednisolone suppressed ILC3 to a similar degree as T cells, the effect of other agents, including cyclosporine A, methotrexate, imatinib, ibrutinib and ruxolitinib, was milder on ILC3 than on T cells. ILC3 are less sensitive to immunosuppressants potentially because of their expression of functionally active ATP Binding Cassette Subfamily B Member 1 (ABCB1) drug exporter proteins. This suggests less intracellular accumulation of immunosuppressive agents, which renders ILC3 resistant to these compounds. The present findings may help to develop strategies to simultaneously maintain the tissue protective properties of ILC3 and at the same time suppress alloreactive lymphocytes, which is important in the prevention and treatment of acute GvHD.

The adaptation model of immunity: allograft rejection versus allograft adaptation

Abstract Rejection of allografts and acceptance of grafts between identical twins appear to be associated with allogeneity or nonself entity of allografts as suggested by the self-nonself (SNS) model of immunity. Nevertheless, such guilt by association does not necessarily follow a cause-effect direction. The adaptation model proposes that graft rejection or tolerance is determined by the expression of the tissue-specific adaptation receptors (AdRs) which are linked to anti-apoptotic pathways for tolerating the recipient’s alloreactive immune response induced by surgical procedure during organ transplantation. A better engraftment of less matched living-donor organs compared with matched cadaver-donor organs is because of a severely altered AdRs in the latter. To this end, tissue adaptation to an anti-graft immune response suggests that AdRs can be restored in allografts if treated properly. Re-transplantation studies demonstrated that second fresh skin allograft rejection while the first adapted allograft from the same donor remaining viable in the presence of all-reactive T cells. Also, constitutive expression of a bidirectional AdR, B7-H1 linked to Bcl-xL anti-apoptotic pathway, in the cornea and retina protects them from rejection while block-ade of B7-H1 accelerates allograft rejection. Also, severe GVHD following autologous stem cell transplantation could be due to chemotherapy-induced alterations in the AdRs in target tissues. This is supported by demonstrating that autologous T RMcells that are involved in local tissue homeostasis but not alloreactive T EMcells are involved in GVHD. Interestingly, restoration of the putative AdR, B7-H1, expression by parenchymal tissues could prevent or ameliorate GVHD in humans. This work was supported by the Office of the Assistant Secretary of Defense for Health Affairs through the Breast Cancer Research Program under Award No. W81XWH2210793. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Department of Defense. This work was also supported by multi-investigator pilot funding from the VCU Massey Cancer Centre, supported in part with funding from NIH/NCI Cancer Centre support grants P30 CA016059.

Increased frequency of CD39+CD73+ regulatory T cells and Deltex-1 gene expression level in kidney transplant recipients with excellent long-term graft function

BackgroundThe ability of regulatory T cells (Tregs) to limit inflammatory responses has been demonstrated. However, different subpopulations of this cell have varying abilities to suppress alloreactive immune responses. The primary goal of this study was to assess the frequency of CD4+FOXP3+CD39+CD73+ Tregs and Deltex-1 gene expression on long-term renal transplant function. MethodsA total of 49 subjects were divided into 3 groups: (i) the excellent long-term graft function (ELTGF) group, (ii) the chronic graft dysfunction (CGD) group, and (iii) the healthy control (HC) group. Following sample collection, peripheral blood mononuclear cells (PBMCs) were isolated, and the Deltex-1 gene expression level and the frequency of CD4+FOXP3+CD39+CD73+ Tregs were evaluated. ResultsThe ELTGF group had more CD4+FOXP3+ Tregs than the CGD group, but the difference was not statistically significant (P = 0.07). However, the frequency of CD4+FOXP3+CD39+CD73+ Tregs and the ratio of these cells to total CD4+ lymphocytes significantly increased in the ELTGF group than in the CGD group (P = 0.04 and P = 0.02 respectively). In addition, the expression level of the Deltex-1 gene was significantly lower in the CGD group than in the other 2 groups (P = 0.01 and P = 0.04 respectively). ConclusionsGiven the increased frequency of CD4+FOXP3+CD39+CD73+ Tregs and the expression level of the Deltex-1 gene in the ELTGF group, it appears that these factors probably improved function and long-term survival of the transplanted organ through the suppression of alloreactive responses and reduction of inflammation. In other words, one of the immunological mechanisms involved in the CGD group may be a deficiency in Tregs.

Precision-Engineered Cell Therapy Orca-T Demonstrates High Relapse-Free Survival at 1 Year While Reducing Graft-Versus-Host Disease and Toxicity

BACKGROUND Allogeneic hematopoietic stem cell transplants (alloHSCT) remain the only curative treatment for many advanced hematologic malignancies; however, despite improvements in donor matching and supportive care, these transplants are associated with significant toxicity and reduced quality of life. Strategies to maintain or enhance the graft-vs-tumor and graft-vs-infection effects while eliminating graft-vs-host disease (GVHD) have long been a key goal in the field. Orca-T is a high-precision, immunotherapy consisting of stem and immune cells, derived from allogeneic donors, that leverages highly purified, polyclonal donor regulatory T cells to control alloreactive immune responses. Orca-T has demonstrated a favorable safety profile and promising control of both relapse and GVHD. METHODS As of 15 July 2022, 180 patients have received Orca-T. Of those patients, 127 have ≥180 days of follow-up and a diagnosis of acute leukemia in CR [AML (n=62), ALL (n=42), mixed phenotype acute leukemia(n=4), CML with prior blast crisis (n=4)], or high risk MDS (n=15). Patients received Orca-T as part of a single-center Phase 2 study (n=32, NCT01660607) or a multicenter Phase 1b study (n=95, NCT04013685). Median follow-up was 387 days (range 41-2062); 72 and 36 patients have > 1 year and >18 months of follow-up, respectively. Patients were aged 19-69 (median 48) and 57% male. Donors were HLA-matched related (n=66) or unrelated (n=61). Patients received investigator's choice of myeloablative conditioning regimens (busulfan-based, n=98; TBI-based, n=29) prior to Orca-T, followed by single-agent GVHD prophylaxis with either tacrolimus (n=124) or sirolimus (n=3). For comparison purposes, an independent CIBMTR-based cohort was identified which consisted of patients with AML, ALL, or MDS who received myeloablative alloHSCT with a PBSC source between 2016-2018 followed by tac/methotrexate PPX. Matching for relapse risk was based on minimal residual disease (MRD) status in acute leukemia patients; development of an additional risk matched CIBMTR comparator based on DRI score and MRD status is ongoing. RESULTS Orca-T was successfully manufactured in a single, centralized GMP manufacturing facility, distributed throughout the U.S., and infused for all patients enrolled. The relapse-free survival was 81% at both 1 year and 18 months in Orca-T recipients. MRD status was determined for 77 patients with acute leukemia by multicolor flow cytometry; of these patients, 31% were MRD+ when they received Orca-T. Amongst MRD- patients (n=53), RFS with Orca-T was 90% at both 1 year as compared to 66% in the CIBMTR cohort (n=324 MRD- patients). Amongst MRD+ patients (n=24), RFS was 68% at one year with Orca-T as compared to 48% in the comparator cohort (n=104). Relapse prevention with Orca-T appeared to be enhanced further with a conditioning regimen consisting of busulfan, fludarabine, and thiotepa ("BFT", n=56 patients, median f/u 342 days); RFS was 90% at 12 months in this group. This included patients with MDS (n=6, 100% RFS at 1 yr), MRD+ acute leukemia (n=14, RFS 73% at 1 yr), MRD- acute leukemia (n=26, RFS 96% at 1 year), and acute leukemia with unknown MRD status (n=11, 91% RFS at 1 yr) (Figure 1). As with relapse, severe infections were low following Orca-T with 11% of patients developing Grade 3 infections per the BMT-CTN grading scale. Median times to neutrophil and platelet engraftment were rapid with Orca-T at 13 and 16 days, respectively; graft failure was rare at 1.6%. Grade ≥ 3 aGVHD and mod/severe cGVHD rates were low with Orca-T at 5% and 6%, respectively, through 1 year post-transplant. Non-relapse mortality was low at 5% at 1 year; NRM with BFT was 0%. Overall, Orca-T shows GRFS of 76% and 69% at 1 year and 18 months post-transplant, respectively; OS was 91% and 86% at these time points post-transplant. No formal statistical comparison to the CIBMTR cohort was performed (Table 1). CONCLUSIONS At more than 1 year of median follow-up, outcomes with Orca-T, a high-precision immunotherapy, demonstrate robust graft-vs-leukemia and graft-vs-infection effects while markedly reducing GVHD and NRM despite MAC. These outcomes were accomplished with consistent and reliable cell manufacturing and distribution of Orca-T at a national scale. A multi-center randomized controlled phase 3 trial comparing Orca-T to SOC, utilizing BFT or TBI-based conditioning, is currently enrolling across the US (NCT05316701). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CT-524 Orca-T, A Precision Engineered Allograft, Results in High GVHD-Free and Relapse-Free Survival Following Myeloablative Conditioning for Hematological Malignancies

Rates of graft-versus-host disease (GVHD) and non-relapse mortality (NRM) following myeloablative allogeneic hematopoietic stem cell transplant (MA-alloHSCT) remain unacceptably high. Orca-T is a high-precision, allogeneic investigational cell therapy product comprised of stem and immune cells that leverages highly purified, polyclonal donor regulatory T cells to control alloreactive immune responses, reducing the need for pharmacologic GVHD prophylaxis. Orca-T is produced in a central GMP facility and has been successfully scaled to clinical centers throughout the U.S. The aim of these studies was to evaluate the safety and efficacy of Orca-T in patients with hematologic malignancies. Data are reported for 138 patients with high-risk hematologic malignancies who received Orca-T in a single-center Phase 1/2 study (n=41) and a multicenter Phase 1b study (n=97) and had ≥ 100 days of follow-up as of 2/28/22. Orca-T was produced from G-CSF-mobilized peripheral blood (PB) from MRD (n=72), MUD (n=62), or MMUD (n=4). Median follow-up for recipients was 300 days (range: 27-1941). Median age was 49 years, and diagnoses included AML (43%), ALL (27%), MDS (10%), myelofibrosis (7%), and CML (6%). Patients received MAC (busulfan-based, n=109; TBI-based, n=27; BCNU, n=2) followed by GVHD prophylaxis with either single-agent tacrolimus (n=127), sirolimus (n=7), or tacrolimus plus mycophenolate (n=4, MMUD). A contemporaneous CIBMTR-based control arm was obtained that consisted of patients with similar diagnoses who received MA-alloHSCT from a PB source followed by tacrolimus/methotrexate prophylaxis. Orca-T was successfully manufactured, distributed, and infused for all patients. Overall time from donor centers to recipient centers was under 60 hours in all cases. Median time to neutrophil engraftment was 13 days. Rates of grade ≥ 3 acute GVHD in the first 180 days and moderate to severe chronic GVHD through 1 year were low at 4% and 5%, respectively. NRM was infrequent at 4% through 1 year. Orca-T exhibited GVHD-free relapse-free survival (GRFS) of 71% & OS of 90% at 1 year. No formal comparison to CIBMTR cohort was performed. Results from patients treated with Orca-T, a high-precision Treg-engineered donor product, suggest a reduction in cGVHD, improved GRFS, and low toxicity relative to historic data. A multicenter randomized-control phase 3 trial comparing Orca-T to SOC is enrolling.

S237: ORCA-T, AN ENGINEERED ALLOGRAFT, RESULTS IN HIGH GVHD-FREE AND RELAPSE-FREE SURVIVAL FOLLOWING MYELOABLATIVE CONDITIONING FOR HEMATOLOGICAL MALIGNANCIES

Background: Rates of graft versus host disease (GVHD) and non-relapse mortality (NRM) following myeloablative allogeneic hematopoietic stem cell transplant (MA-alloHSCT) remain unacceptably high. Strategies to reduce GVHD and NRM have been compromised by limited efficacy or increased risk of infection and relapse, emphasizing the need for new approaches that holistically improve outcomes. Orca-T is a high-precision, allogeneic investigational cell therapy product comprised of stem and immune cells that leverages highly purified, polyclonal donor regulatory T cells to control alloreactive immune responses, reducing the need for pharmacologic GVHD prophylaxis. Orca-T is produced in a central GMP facility and has been successfully scaled to clinical centers throughout the U.S. Aims: The aim of these studies was to evaluate the safety and efficacy of Orca-T in patients with hematologic malignancies. Methods: As of 28 February 2022, 138 patients with high-risk hematologic malignancies have received Orca-T in a single-center Phase 1-2 study (NCT01660607, n=41) and a multicenter Phase 1b study (NCT04013685, n=97) and have ≥ 100 days of follow-up. Informed consent was obtained from all transplant recipients and donors, and the studies received IRB approval from participating institutions. Orca-T was produced from G-CSF-mobilized peripheral blood (PB) from matched related donors (n=72), matched unrelated donors (n=62), or mismatched unrelated donors (MMUD, n=4). Median follow-up for recipients was 300 days (range: 27-1941). Median age was 49 years, and diagnoses included AML (43%), ALL (27%), MDS (10%), myelofibrosis (7%), and CML (6%). Patients received myeloablative conditioning (busulfan-based, n=109; TBI-based, n=27; BCNU, n=2) followed by GVHD prophylaxis with either single-agent tacrolimus (tac, n=127), sirolimus (n=7), or tac plus mycophenolate (n=4, MMUD). A contemporaneous CIBMTR-based control arm was obtained that consisted of patients with similar diagnoses who received myeloablative alloHSCT from a PB source followed by tac/methotrexate PPX. Results: Orca-T was successfully manufactured, distributed, and infused for all patients enrolled. Overall time from donor centers to recipient centers was under 60 hours in all cases. Median time to neutrophil engraftment was 13 days. The rates of grade ≥ 3 acute GVHD in the first 180 days and moderate to severe chronic GVHD through 1 year were low with Orca-T at 4% and 5%, respectively. NRM was infrequent at 4% through 1 year. Orca-T exhibited GRFS of 71% & OS of 90% at 1 year. No formal comparison to the CIBMTR cohort was performed, and a Phase 3 study has been initiated to confirm these findings. Longitudinal immune reconstitution data was collected and will be presented. Clinical data is summarized in Table 1. *MAGIC Criteria **NIH Consensus Grading Image:Summary/Conclusion: Results from patients treated with Orca-T, a high-precision Treg-engineered donor product, suggest a reduction in cGVHD, improved GRFS, and low toxicity relative to historic data. Orca-T manufacturing was accomplished with consistent and reliable cell manufacturing and distribution across a wide geographic area. A multicenter randomized-control trial phase 3 trial comparing Orca-T to SOC has been initiated.

Lung transplant recipients with idiopathic pulmonary fibrosis have impaired alloreactive immune responses.

Telomere dysfunction is associated with idiopathic pulmonary fibrosis (IPF) and worse outcomes following lung transplantation. Telomere dysfunction may impair immunity by upregulating p53 and arresting proliferation, but its influence on allograft-specific immune responses is unknown. We hypothesized that subjects undergoing lung transplantation for IPF would have impaired T cell proliferation to donor antigens. We analyzed peripheral blood mononuclear cells (PBMC) from 14 IPF lung transplant recipients and 12 age-matched non-IPF subjects, before and 2 years after transplantation, as well as PBMC from 9 non-transplant controls. We quantified T cell proliferation and cytokine secretion to donor antigens. Associations between PBMC telomere length, measured by quantitative PCR, and T cell proliferation to alloantigens were evaluated with generalized estimating equation models. IPF subjects demonstrated impaired CD8+ T cell proliferation to donor antigens pre-transplant (p < 0.05). IL-2, IL-7, and IL-15 cytokine stimulation restored T cell proliferation, while p53 upregulation blocked proliferation. IPF subjects had shorter PBMC telomere lengths than non-IPF subjects (p < 0.001), and short PBMC telomere length was associated with impaired CD8+ T cell proliferation to alloantigens (p=0.002). IPF as an indication for lung transplant is associated with short PBMC telomere length and impaired T cell responses to donor antigens. However, the rescue of proliferation following cytokine exposure suggests that alloimmune anergy could be overcome. Telomere length may inform immunosuppression strategies for IPF recipients.

Dysregulated regulatory T cell responses target fibroblasts and lead to chronic rejection after heart transplantation

Abstract Suppressive and reparative regulatory T cell (Treg) functions are described. However, in transplantation (Tx), recipient immune responses to persistent MHC differences will sustain injury and impact repair. The development of immunosuppressant-resistant graft vasculopathy and fibrosis, or chronic rejection (CR) remains a leading cause of death post-heart Tx (HTx) and may reflect failed tissue repair. We tested the hypothesis that Treg repair responses become dysregulated due to sustained alloreactive immune responses. Our studies utilized IL-33 as a model injury signal, or damage-associated molecular pattern (DAMP) and B6 (H-2b) CD45.1+ mice received heterotopic HTx from MHCII-mismatched Bm12 (H2-I-Abm12) donors that were IL-33-deficient or competent. On day (d)14, scRNAseq and CITE-seq were used to compare CD45.1+ cells from HTx digests (n=4). IL-33 modulated &amp;gt;800 genes in HTx-infiltrating Treg, including the growth factor amphiregulin (Areg; 2.25x; P=0.0241). Areg is implicated in tissue repair, yet when B6 Foxp3YFP-CrexAregfl/fl and Foxp3YFP-Cre mice receiving Bm12 HTx were assessed at d100 (n=6–10), the deletion of Areg in Treg protected against CR. Using IncuCyte Live-Cell imaging, it was clear that Areg from Treg stimulated fibroblast proliferation and migration. Tregs stimulated with IL-2 alone or with IL-33 were assessed at d14 or d21 for CpG methylation of Treg-associated genes. These data revealed that sustained exposure to IL-33 increased methylation of Foxp3, suggesting that sustained DAMP stimulation makes Treg unstable. In total, our studies identify how a sustained and dysregulated repair response involving interactions between fibroblasts and recipient Treg contributes to the progression of HTx CR. Supported by grants from NIH (F30AI147437, T32 CA082084) (GKD), NIH (R01HL122489, R01AR073527) (HRT)

Occurrence of De novo Donor-Specific Antibodies After COVID-19 in Kidney Transplant Recipients Is Low Despite Immunosuppression Modulation

IntroductionDecreased immunosuppression has been proposed for kidney transplant recipients infected with coronavirus disease 2019 (COVID-19), but the impact on the alloreactive immune response during and after infection has been poorly investigated. We evaluated the occurrence of antihuman leukocyte antigen (HLA) donor-specific antibodies (DSAs) (post–COVID-19) and rejection episodes after COVID-19 with particular focus on immunosuppression modulation.MethodsKidney transplant recipients from 2 French institutions had anti-HLA antibody screening before and after COVID-19. Management of immunosuppression, rejection episodes, COVID-19 severity, inflammatory markers, and antiviral therapies were recorded.ResultsFrom 251 recruited patients, 72 were excluded because of COVID-19–related death (n = 25) and incomplete immunologic follow-up (n = 47). Among the remaining 179 included patients, almost half were hospitalized (49.2%). Antimetabolites were interrupted in 47% of patients (82% in hospitalized, median time of resumption of 23 days and in 15% nonhospitalized, median time of resumption of 7 days). Calcineurin inhibitors were interrupted in 12% of patients (all hospitalized, median time of resumption of 11 days). The incidence of post–COVID-19 DSA was 4% (8% and 0% in hospitalized and nonhospitalized, respectively). Allograft rejection occurred in 3 patients (1.7%) and all were hospitalized. Younger age, transplantation <1 year, and preexisting DSA were more frequently observed in patients with post–COVID-19 DSA, whereas inflammatory markers, lymphopenia, and use of antiviral therapies were not.ConclusionThe incidence of post–COVID-19 DSA among COVID-19–positive kidney transplant recipients was low (4%) despite a significant decrease in immunosuppression and was mainly restricted to high-risk immunologic patient’s status. COVID-19 severity was not associated with post–COVID-19 DSA and/or rejection.

Outcomes for Myelofibrosis Patients Following Myeloablative Allogeneic Stem Cell Transplantation Using the Orca-T Graft from HLA-Matched Related and Unrelated Donors

Background: Allogeneic stem cell transplantation (allo SCT) is the only potentially curative therapy for patients with intermediate-2 and high-risk myelofibrosis (MF). Allo SCT for MF has historically been challenging as many patients are transplanted with active or advanced disease and immune reconstitution in MF patients is often hindered by inflammation and bone marrow fibrosis. When compared to non-SCT therapies, MF patients who receive allo SCT achieve long-term survival benefits. However, it comes with an upfront cost of an increased risk of non-relapse mortality in the first year after allo SCT (Gowin K et al, Blood Av 2020). Therefore, there is an unmet need to improve on the early outcomes of transplantation for MF.Methods: We reviewed MF patients who underwent myeloablative allo SCT with Orca-T derived from HLA matched donors on NCT01660607 and NCT04013685. Orca-T is an investigational cellular product comprising stem and immune cells that leverages highly purified donor regulatory T cells to control alloreactive immune responses. Single agent GVHD prophylaxis was administered as tacrolimus or sirolimus.Results: We present outcomes for 7 MF patients with DIPSS risk of int-1 (n=1), int-2 or higher risk MF (n=6) treated with the Orca-T graft and compared this to 6 MF patients who underwent standard of care (SOC) allo SCT at Stanford, between 2017 - 2021 (Table 1). All patients were high or very high-risk based on MIPSS70 plus score v, 2.0. All patients had 10/10 HLA-matched donors, except 1 of 7 Orca-T patients who had 9/10 HLA-matched donor. All but 1 patient in both, Orca-T and SOC, recipients had prior Jakafi exposure.All Orca-T recipients had splenomegaly with spleen size of 16.3 - 23 cm. Engraftment occurred at median of D+13 (range 10-29) and 6 of 7 patients had 100% CD3 donor chimerism at D+100. 1 patient was 90% CD3 donor chimerism at D+100 but achieved 100% CD3 donor chimerism at D+180. All patients had significant marrow fibrosis, MF grade 2 or 3, before allo SCT. Regression of marrow fibrosis to MF grade 0 or 1 was noted by D+ 100 in 7 of 7 Orca-T recipients but only in 1 of 6 SOC patients. Orca-T recipients did not have grade 2-4 acute GVHD and 1 of 7 recipients had extensive chronic GVHD. Tacrolimus was tapered off in 3 of 7 patients at D+180. SOC recipients had 2 of 6 recipients with grade 2-4 acute GVHD, 3 of 6 recipients with extensive chronic GVHD and only 1 recipient was taken off tacrolimus at D+180. There were no deaths in Orca-T recipients and 2 deaths in SOC recipients before D+180, cause of death was acute GVHD and relapse. Finally, when compared to standard allo SCT, Orca-T recipients may have a reduced frequency and severity of infections without significant consequences while the SOC patients had severe and often multiple infections.Conclusion: Our data indicates that Orca-T graft was well tolerated and potentially efficacious in MF patients undergoing allo SCT. We observed early regression of marrow fibrosis at D+100 in all Orca-T recipients. This limited data suggests that Orca-T may afford an early resolution of an inflammatory microenvironment that allows for robust immune reconstitution and potentially lower incidence of serious infections. Orca-T is under continued investigation to reduce early non-relapse mortality for MF. [Display omitted] DisclosuresGandhi: CareDx Inc: Honoraria; Gamida Cell: Consultancy, Membership on an entity's Board of Directors or advisory committees. Muffly: Pfizer, Amgen, Jazz, Medexus, Pfizer: Consultancy; Astellas, Jasper, Adaptive, Baxalta: Research Funding; Adaptive: Honoraria, Other: fees for non-CME/CE services: , Research Funding. Shiraz: Kite Pharma-Gilead: Research Funding. Fernhoff: Orca Bio: Current Employment. McClellan: Orca Bio: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Gotlib: Allakos: Consultancy; Cogent Biosciences: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Chair for the Eligibility and Central Response Review Committee, Research Funding; PharmaEssentia: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kartos: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Deciphera: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Blueprint Medicines: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Meyer: Triursus Therapeutics: Current holder of stock options in a privately-held company; GigaImmune: Current holder of stock options in a privately-held company; Orca Biosystems: Research Funding; Indee, Jura: Consultancy.

Orca-T Results in High Gvhd-Free and Relapse-Free Survival Following Myeloablative Conditioning for Hematological Malignancies: Results of a Single Center Phase 2 and a Multicenter Phase 1b Study

Orca-T Results in High Gvhd-Free and Relapse-Free Survival Following Myeloablative Conditioning for Hematological Malignancies: Results of a Single Center Phase 2 and a Multicenter Phase 1b Study

Alloreactive Immune Response Associated to Human Mesenchymal Stromal Cells Treatment: A Systematic Review.

The well-known immunomodulatory and regenerative properties of mesenchymal stromal cells (MSCs) are the reason why they are being used for the treatment of many diseases. Because they are considered hypoimmunogenic, MSCs treatments are performed without considering histocompatibility barriers and without anticipating possible immune rejections. However, recent preclinical studies describe the generation of alloantibodies and the immune rejection of MSCs. This has led to an increasing number of clinical trials evaluating the immunological profile of patients after treatment with MSCs. The objective of this systematic review was to evaluate the generation of donor specific antibodies (DSA) after allogeneic MSC (allo-MSC) therapy and the impact on safety or tolerability. Data from 555 patients were included in the systematic review, 356 were treated with allo-MSC and the rest were treated with placebo or control drugs. A mean of 11.51% of allo-MSC-treated patients developed DSA. Specifically, 14.95% of these patients developed DSA and 6.33% of them developed cPRA. Neither the production of DSA after treatment nor the presence of DSA at baseline (presensitization) were correlated with safety and/or tolerability of the treatment. The number of doses administrated and human leucocyte antigen (HLA) mismatches between donor and recipient did not affect the production of DSA. The safety of allo-MSC therapy has been proved in all the studies and the generation of alloantibodies might not have clinical relevance. However, there are very few studies in the area. More studies with adequate designs are needed to confirm these results.

NK Cells Produce High Levels of IL-10 Early after Allogeneic Stem Cell Transplantation and Suppress Development of Acute GVHD

Natural killer (NK) cells reconstitute rapidly following allogeneic stem cell transplantation (allo-SCT) at a time when alloreactive T cell immunity is being established. We investigated very early NK cell reconstitution in 82 patients following T cell-depleted allo-SCT. NK cell number increased rapidly, exceeding T cell reconstitution such that the NK:T cell ratio was over 40 by day 14. NK cells at day 14 (NK-14) were donor-derived, intensely proliferating and expressed chemokine receptors targeted to lymphoid and peripheral tissue. Spontaneous production of the immunoregulatory cytokine IL-10 was observed in over 70% of cells and transcription of cytokines and growth factors was augmented. NK-14 cell number was inversely correlated with the incidence of grade II-IV acute graft versus host disease (GVHD). These findings reveal that robust reconstitution of immunoregulatory NK cells by day 14 after allo-SCT is an important determinant of clinical outcome and suggest NK cells may suppress development of the T cell-mediated alloreactive immune response through production of IL-10. DisclosuresNo relevant conflicts of interest to declare.

Regulatory T-cell phenotypes in children with sickle cell disease.

Regulatory T cells (Tregs) are linked to a reduction in alloreactive immune responses, but few studies have investigated the impact of hydroxyurea (HU) therapy on Tregs in sickle cell disease (SCD). Our case-controlled study presented here included two groups, the first comprising 60 pediatric SCD patients, 30 of whom did not receive any treatment and 30 who received HU, and the second group consisting of 30 healthy controls. Flow cytometry was used to evaluate the percentage of CD4+CD25+highFoxp3+ Tregs present and their phenotypes. The percentage of CD4+CD25+high Tregs was significantly increased in untreated SCD patients in comparison to treated SCD patients and controls. Conversely, treated SCD children had a lower percentage of CD4+CD25+high Tregs than controls. In addition, a significant increase in the percentage of CD4+CD25+highFoxp3+ Tregs was found in untreated SCD patients, compared to in HU-treated patients and controls. The percentage of naive CD45RA+ Tregs was significantly decreased in untreated SCD patients when compared to other groups. Among children with SCD, HU treatment exhibited significant qualitative and quantitative effects on Tregs by decreasing their frequency, and increasing the proportion of naive CD45RA+ Tregs and reducing levels of the most suppressive Tregs: HLA-DR+, CD39+, and CD69+. Among children with, SCD, HU treatment exhibited significant qualitative and quantitative effects on Tregs. HU treatment in SCD decreases the frequency of Tregs, as well as the levels of the most suppressive Tregs: HLA-DR+, CD39+, and CD69+. At the same time, HU increases the proportion of naive CD45RA+ Tregs. Our study showed the impact of HU therapy on Tregs in children with SCD.

HLA-A gene knockout using CRISPR/Cas9 system toward overcoming transplantation concerns

BackgroundThe treatment of many cancers and genetic diseases relies on novel engraftment approaches such as cell therapy and hematopoietic stem cell transplantation (HSCT). However, these methods are hindered by the alloreactive immune responses triggered by incompatible human leukocyte antigen (HLA) molecules. A successful HSCT procedure requires the eradication of donor and recipient HLA alloimmunization. Eliminating HLA-A gene expression using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 nuclease (CRISPR/Cas9) could be a great approach to increase the possibility of a successful HSCT through extending pool of unrelated donors.ResultsOur dual gRNA approach introduced a large deletion in the HLA-A gene. Among 22 single-cloned cells, two clones (9.09%) and 11 clones (50%) received homozygous and heterozygous large deletions, respectively. Finally, the real-time PCR results also revealed that HLA-A gene expression was diminished significantly.ConclusionThe results suggested that CRISPR/Cas9 could be used as an efficient technique to introduce HLA-A gene knockout; thus, it can considerably lessen the burden of finding a fully matched donor by lowering the alleles required for a successful HSCT.

CD52/GPI− T-Cells Are Enriched for Alloreactive Specificity and Predict Acute Graft-Versus-Host-Disease After Stem Cell Transplantation

Alemtuzumab is a CD52-specific lympho-depleting antibody. CD52- T cells emerge under alemtuzumab selection pressure. We sought to investigate the phenotype and function of the CD52- T cell fraction and related their presence to clinical outcome. We obtained longitudinal peripheral blood samples from 67 consecutive patients undergoing allo-HSCT between 2013-2016. Forty-seven patients (70%) had a myeloid disease (acute myelogenous leukemia or myelodysplastic syndrome) whereas 20 patients had lymphoid disease. All patients received in vivo alemtuzumab (10 mg/d from day -5 for 5 days) as part of their conditioning protocol. Sixty-three (94%) received reduced-intensity conditioning chemotherapy, whereas 4 (6%) received a myeloablative regimen. All patients received post-transplantation cyclosporine A for graft-versus-host disease (GVHD) prophylaxis. Six (9%) also received methotrexate, whereas 2 (3%) patients also received mycophenolate mofetil. Overall survival at 2 years was 68%, and relapse-free survival was 48%. Twenty-none percent of patients experienced acute GVHD (grade 2 or above), and 15% developed chronic GVHD. CD52- T cells were detectable in 66 of 67 consecutive patients. CD52- T cells demonstrated low binding of fluorescent aerolysin, indicating downregulation of the glycophosphatidylinositol anchor, although we did not detect any mutations in the PIG-A gene as is typically seen in patients with paroxysmal nocturnal hemoglobinuria. CD52- T cells were almost exclusively CD4+ and exhibited a dominant memory phenotype with only small numbers of CD25+ CD127low Foxp3+ regulatory T cells. CD52- T cells exhibited alloreactive specificity in vitro and have a distinct TCR repertoire to CD52+ T cells. Early after allo-hematopoietic stem cell transplantation, the presence of a significant population of CD52- T cells (comprising >51% of the T cell fraction) was found to be an independent risk factor for acute GvHD. This was confirmed in a validation cohort of 28 patients obtained between 2017-2018. These data suggest that the CD52- T cell fraction may represent a residual "footprint" of an early CD4+ T cell alloreactive response and may have been rescued from alemtuzumab-mediated lysis by antigen engagement in vivo. These data help to delineate the nature of T cell escape from alemtuzumab surveillance and contribute to increasing interest in the importance of CD4+ T cells in alloreactive immune responses, which could help inform immunotherapy protocols.