anti-T Cell Research Articles

Haploidentical hematopoietic stem cell transplantation for hematologic malignancies: a novel conditioning regimen with anti-T lymphocyte immunoglobulin instead of anti-thymocyte globulin for in vivo T cell depletion.

We evaluated the safety and efficacy of a novel protocol for haploidentical stem cell transplantation (haplo-SCT) in 312 patients with hematologic malignancies. The protocol evolved from the Beijing platform replacing ATG with ATLG; adding Fludarabine and removing cytarabine and Simustine. GVHD prophylaxis combined Basiliximab and low-dose cyclophosphamide post-transplant; overall, the conditioning duration was shortened. Median times to neutrophil and platelet recovery were both 11 days. Graft rejection occurred in 0.96% of patients. Cumulative incidences of grades II-IV and III-IV acute GVHD by day 200 were 35.3% and 8.9%, respectively. Probabilities of total and extensive chronic GVHD at 2 years were 40.7% and 14.7%. CMV viremia was observed in 35.6% of patients, with a 1.9% 100-day CMV pneumonia incidence and no CMV-related mortality. Cumulative incidences of non-relapse mortality at 100 days, 1 year, and 2 years were 2.9, 4.4, and 6.6%. The 4-year OS, RFS, and GRFS rates were 78.9, 70.7, and 47.3%. Older recipient age was associated with higher NRM, while positive pre-transplant MRD predicted worse OS, RFS, and higher relapse incidence. Our novel protocol for haplo-SCT is associated with low infection rates and acceptable risks of graft failure, severe GVHD, and mortality, representing a safe and effective haploidentical transplantation strategy.

Sequential Conditioning With FLAMSA Does Not Improve Outcomes of Allogeneic Stem Cell Transplantation in Chronic Myelomonocytic Leukemia Patients

As with myelodysplastic syndromes (MDS), effective treatment options for Chronic myelomonocytic leukemia (CMML) are limited, and the optimal treatment approach remains undefined. Allogeneic stem cell transplantation (allo-SCT) is potentially curative therapy for patients with CMML. Sequential conditioning with FLAMSA was initially developed for refractory acute myeloid leukemia (AML) and has since been applied in the treatment of MDS and CMML. Data on optimal allo-SCT conditioning in CMML Patients is scarce. This retrospective study from the Department of Stem Cell Transplantation at the University Medical Center Hamburg, Germany, compared allo-SCT outcomes in CMML patients across three conditioning regimes: Thiotepa-Busulfan (TB), Sequential FLAMSA-Busulfan Fludarabine (FLAMSA-FB), and Treosulfan-Fludarabine (Treo-Flu). Sixty-nine consecutive patients with CMML who underwent allo-SCT between the years 2006-2022 were included in the study. Twenty-two received TB, 27 received FLAMSA-FB, and 20 received Treo-Flu conditioning. Transplant sources included matched related donors (MRD, 8 patients), mismatched related donors (MMRD, 8 all in the TB group), matched unrelated donors (MUD, 31), and mismatched unrelated donors (MMUD, 22) with significant group variations (p<0.001). Most patients received anti-T lymphocyte Globulin (ATLG) for graft versus host disease (GVHD) prophylaxis (TB 68%, FLAMSA-FB 93%, Treo-Flu 85%, p=0.08). CPSS-Molecular score was comparable between the groups. One TB patient experienced primary graft failure, but engraftment times were comparable across the groups. Although not statistically significant, the TB group showed a trend towards improved 3-year OS rates (80%) compared to FLAMSA-FB (37%) and Treo-Flu (55%) (p=0.05). The TB group also displayed significantly higher 3-year PFS rates (80%) compared to FLAMSA-FB (33%) and Treo-Flu (both 39%), (p=0.02). No significant differences were observed in 3-year non-relapse mortality (NRM) across the TB (20%), FLAMSA-FB (30%), and Treo-Flu (26%) groups (p=0.8). Interestingly, no TB patients relapsed at 3 years, contrasting with the FLAMSA-FB (41%) and Treo-Flu groups (30%, p=0.02). Lastly, cumulative incidences of acute and chronic GVHD were similar across groups. Our analysis suggests FLAMSA-FB does not improve transplant outcomes, however TB represents the preferred conditioning regimen for CMML patients undergoing allo-SCT. It demonstrates notable advantages in relapse prevention and leads to improved OS and PFS compared to FLAMSA-FB and Treo-Flu protocols.

P.490: Low-dose anti-T lymphocyte globulin (Grafalon) for kidney transplant induction immunosuppression. A pediatric cohort study.

P.490: Low-dose anti-T lymphocyte globulin (Grafalon) for kidney transplant induction immunosuppression. A pediatric cohort study.

P.536: The efficacy of anti T-lymphocyte globulin as induction therapy in kidney transplantation: A single-center retrospective analysis.

P.536: The efficacy of anti T-lymphocyte globulin as induction therapy in kidney transplantation: A single-center retrospective analysis.

686TiP Preliminary phase I results from a first-in-human study of ZG006, a trispecific anti-T cell engager targeting CD3/DLL3/DLL3, as monotherapy in patients with advanced small cell lung cancer or neuroendocrine carcinoma

686TiP Preliminary phase I results from a first-in-human study of ZG006, a trispecific anti-T cell engager targeting CD3/DLL3/DLL3, as monotherapy in patients with advanced small cell lung cancer or neuroendocrine carcinoma

VELOCITY-Lung substudy-03: A phase 2 study of neoadjuvant domvanalimab (dom)+zimberelimab (zim)+chemotherapy (chemo) or zim+chemo followed by adjuvant dom+zim or zim in patients with resectable stage II-III non-small cell lung cancer (NSCLC).

TPS8121 Background: Management of resectable NSCLC involves surgery with perioperative systemic therapy. Immune checkpoint inhibitors (CPIs) and targeted therapies are now used in the perioperative setting, leading to improved survival outcomes. The combination of dom (anti–T-cell immunoglobulin and ITIM domain [TIGIT]) and zim (anti–programmed death protein 1 [PD-1]) has shown promising antitumor activity with a manageable safety profile in metastatic NSCLC and may provide an opportunity to improve clinical outcomes in the perioperative setting in early-stage, resectable NSCLC. Substudy-03 of the VELOCITY-Lung platform study is evaluating novel perioperative treatment combinations in patients with newly diagnosed, resectable, stage II-III NSCLC. Methods: VELOCITY-Lung (NCT05633667) is an open-label, multicenter, phase 2 platform study with 3 ongoing substudies targeting the different NSCLC patient populations. Key eligibility criteria for Substudy-03 include age ≥ 18 years, histologically/cytologically confirmed stage II, IIIA, IIIB (T[3-4]N2) (per AJCC 8th ed.) squamous or nonsquamous NSCLC considered resectable with curative intent, no prior systemic or CPI therapy or radiotherapy, ECOG PS 0-1, any PD-1 ligand (PD-L1) status, and no actionable EGFR or ALK genomic alterations in patients with nonsquamous NSCLC. Randomization in Substudy-03 will be stratified by disease stage (II vs III) and baseline PD-L1 expression status (≥ 50% vs &lt; 50%). Current treatment groups in the neoadjuvant phase of the preliminary stage are: (A) dom+zim+platinum (Pt)-based chemo or (B) zim+Pt-based chemo for a maximum of 3 cycles; this will be followed by definitive surgery within 6 weeks of completing neoadjuvant treatment. Adjuvant treatment will include: (A) dom+zim or (B) zim and will begin within 12 weeks of surgery. Patients will continue to receive adjuvant treatment until disease recurrence, death, or unacceptable toxicity or to a maximum of 14 cycles. Dosing will be as follows: zim 360 mg intravenously (IV) once every 3 weeks (Q3W) and dom 1200 mg IV once Q3W (up to 3 cycles in the neoadjuvant phase and up to 14 cycles in the adjuvant phase). Pt-based chemo will be based on patient tumor histology (squamous vs nonsquamous). The primary endpoint is pathological complete response rate, defined as the percentage of patients with no residual invasive cancer in resected lung specimens and lymph nodes, assessed by local pathology review. Secondary endpoints include major pathological response rate (percentage of patients with ≤ 10% residual tumor in lung and lymph nodes at surgery), event-free survival, overall survival, and safety. Approximately 31 patients will be enrolled into each treatment group of the preliminary phase. The study is currently recruiting. Clinical trial information: NCT05633667 .

Anti-T-lymphocyte globulin exposure is associated with acute graft-versus-host disease and relapse in pediatric acute lymphoblastic leukemia patients undergoing hematopoietic stem cell transplantation: a multinational prospective study.

Anti-T-lymphocyte globulin (ATLG) is used in hematopoietic stem cell transplantation (HSCT) to prevent graft-versus-host disease (GVHD) and graft failure. To date, insight in ATLG pharmacokinetics and -dynamics (PK/PD) is limited, and population PK (POPPK) models are lacking. In this prospective study, we describe ATLG POPPK using NONMEM® and the impact of ATLG exposure on clinical outcome and immune reconstitution in a homogeneous cohort of pediatric acute lymphoblastic leukemia (ALL) patients transplanted with a matched unrelated donor and receiving uniform ATLG dosing. Based on 121 patients and 812 samples for POPPK analysis, a two-compartmental model with parallel linear and non-linear clearance and bodyweight as covariate, best described the ATLG concentration-time data. The level of ATLG exposure (day active ATLG <1 AU/mL, median 16 days post-HSCT) was strongly associated with aGVHD grade II-IV, with a lower incidence in patients with prolonged active ATLG exposure (≤day 16 50% vs. >day 16 8.2%; P<0.001). When stratified for remission state, patients transplanted in complete remission (CR) 2 or 3 with prolonged ATLG exposure had a higher relapse risk, while this effect was not seen in CR1 patients (P=0.010). High level ATLG exposure was associated with delayed CD4 T-cell recovery at 4 and 8 weeks post-HSCT, but not at 12 weeks, and overall and relapse-free survival were not influenced by CD4 recovery at 12 weeks post-HSCT. This study underlines the importance of individualized ATLG exposure with the use of model-informed precision dosing in order to optimize the HSCT outcome in pediatric ALL.

Immune reconstitution in children after haploidentical haematopoietic stem cell transplantation.

Immune reconstitution (IR) kinetics of paediatric patients underwent haploidentical haematopoietic stem cell transplantation (HSCT) with post-transplant cyclophosphamide (PTCy) have not been extensively studied. We compared IR patterns of children receiving HSCT from haploidentical (n = 92) and HLA-matched donors (n = 36), and analysed risk factors for viral infection in these patients. We prospectively measured lymphocyte subset numbers before HSCT and at 1, 3, 6 and 12 months after HSCT. Blood cytomegalovirus (CMV), Epstein-Barr virus, adenovirus, BK virus (BKV) and urine adenovirus and BKV viral loads were measured at designated time points. The median numbers of total T and T helper cells at 1 month were significantly lower in the haploidentical group compared with the HLA-matched group. Haploidentical HSCT recipients had significantly lower median numbers of several T cell subsets and B cells for 1 year after HSCT. The median NK cell count of the haploidentical group was lower at 1 month. BKV haemorrhagic cystitis, blood CMV and urine adenovirus reactivation were more frequently found in the haploidentical group. Post-haploidentical HSCT patients receiving anti-T lymphocyte globulin (ATG) had significantly lower median numbers of total T cells (at 1 month) and T helper cells (at 6 and 12 months) and higher rate of blood BKV reactivation compared with those without ATG. Paediatric patients who undergo haploidentical HSCT with PTCy are likely to have delayed IR and an increased risk of viral reactivation/infection compared with HLA-matched HSCT. The addition of ATG to PTCy delayed T cell recovery and increased risk of BKV reactivation.

Current Induction Therapy Strategies and Anti-T Lymphocyte Globulin Usage in Kidney Transplantation: Consensus-Based Recommendations by a Turkish Expert Panel

Current Induction Therapy Strategies and Anti-T Lymphocyte Globulin Usage in Kidney Transplantation: Consensus-Based Recommendations by a Turkish Expert Panel

Allogeneic Hematopoietic Cell Transplantation in the Elderly Results in Similar Survival with Matched Related or Unrelated and Haploidentical Donors: A Report from the Société Francophone De Greffe De Moelle Et De Thérapie Cellulaire (SFGM-TC)

Introduction Higher patient age is no longer considered to be a barrier for allogeneic hematopoietic cell transplantation (HCT). We sought to study the impact of different types of donors on the outcomes of HCT in subjects aged 65 years (y) or more, and to evaluate the determinants of these outcomes. Methods SFGM-TC registry data on first HCTs performed between January 2013 and July 2021 in patients aged 65 y or more, with myeloablative or reduced-intensity conditioning, and a 10/10 human leukocyte antigen (HLA) matched sibling donor (MSD), matched unrelated donor (MUD) or haploidentical donor (HAP) were analyzed. Main exclusion criteria were: MSD/MUD-HCT performed without anti-T lymphocyte globulin (ATG), and HAP-HCT performed without post-transplant cyclophosphamide (PTCY). Univariate analyses according to donor type, as well as multivariable analyses were performed. Results A total of 1176 patients met the eligibility criteria (215 MSD, 721 MUD, 240 HAP). Median follow-up was 2.8 y. In univariate analysis, there was no difference between donor types in terms of 3-y overall survival (OS: MSD 50%, MUD 49%, HAP 49%, p=0.52), progression-free survival (PFS: MSD 41%, MUD 45%, HAP 44%, p=0.51) and graft-versus-host disease-free, relapse-free survival (GRFS: MSD 34%, MUD 32%, HAP 35%, p=0.67). With MSD, the cumulative incidence of progression at 3 y was significantly higher at 47% (MUD 29%, HAP 29%, p&amp;lt;0.001), and non-relapse mortality (NRM) significantly lower at 12% (MUD 26%, HAP 27%, p&amp;lt;0.001). These MSD results were confirmed in multivariable analysis. Incidence of acute graft-versus-host disease (GVHD) at 180 days was lower with MSD: grades II-IV 21% (MUD 35%, HAP 38%, p&amp;lt;0.001) and grade III-IV 8% (MUD 13%, HAP 15%, p=0.05). Incidence of chronic GVHD at 2 y was comparable between MSD, MUD and HAP transplants: moderate to severe 14%, 18% and 15%, respectively, and severe 5%, 6% and 5%, respectively. Conclusions In patients aged 65 y or more, MSD/MUD HCT performed with ATG and HAP HCT performed with PTCY yielded similar survival outcomes. MSD HCT is associated with a lower acute GVHD and NRM but a higher relapse risk. Reducing the toxicity of MUD and HAP HCT may yield better results than with MSD HCT in this population. Acknowledgements: Nicole Raus, Data manager coordinator, SFGM-TC. Viviane Fossat, Secretary, SFGM-TC.

Single-dose antithymocyte globulin in standard immunological risk kidney transplant recipients: efficacy and kinetics of peripheral blood CD3+ T lymphocyte modulation.

Polyclonal anti-T cell antibodies (ATG or thymoglobulin®) are used as induction therapy in kidney transplant recipients. This study evaluates the safety, efficacy, and CD3+T lymphocyte modulation of two ATG regimens. The trial included two cohorts of kidney transplant recipients that were followed for one year. The study group, including standard immunological risk recipients, received one 3mg/kg dose of ATG. The comparator group, including standard and high immunological risk kidney transplant recipients, received a fractionated dose regimen (up to four 1.5mg/kg doses). Patient and graft outcomes and the kinetics of CD3+T lymphocyte modulation in the peripheral blood were evaluated. One hundred kidney transplant recipients were included in each group. The one-year incidence of treated acute rejection, and patient and graft survival did not differ between groups. Bacterial infections were significantly more frequent in fractionated-dose group patients (66%versus 5%; P = 0.0001). At one-year follow-up, there was no difference in the incidence of cytomegalovirus infection (P = 0.152) or malignancies (P = 0.312). CD3+T lymphocyte immunomodulation in the single-dose group was more effective in the first two days after transplantation. After the third post-transplant day, CD3+T lymphocyte modulation was more efficient in the fractionated dose group. Both regimens resulted in low rejection rates and equivalent survival. The single and reduced dose regimen protects from the occurrence of bacterial infections. CD3+T lymphocyte modulation occurred with different kinetics, although it did not result in distinct outcomes.

Impact of Allograft T-Cell (CD3) Dose on Outcomes When CD34 Cell Dose Is Capped: Analysis of 811 Consecutive Allogeneic PBSC Transplants from a Single Center

G-CSF mobilized PBSC allografts contain higher T-cell doses than bone marrow grafts and have been associated with a significantly higher risk of chronic GVHD. Higher CD34+ cell content in PBSC grafts has also been shown to predict higher rates of chronic GVHD and associated mortality 1,2 To mitigate this risk our center adopted a policy of capping CD34+ cell dose at 5 x 10e6/kg recipient weight for unmanipulated PBSC grafts. For this study, we assessed the effect of T-cell (CD3) dose administered under this capped CD34 strategy, on GVHD and other transplant outcomes. Consecutive first PBSC allografts for hematologic malignancy performed using the CD34 cap strategy between 2010 and 2022 (n=811) were included in this analysis. GVHD prophylaxis was tacrolimus/methotrexate for MRD and MUD and post-transplant cyclophosphamide based for Haplo. Patients receiving anti-T cell serotherapy or ex-vivo T-depletion were excluded. GVHD was uniformly assessed and graded by a dedicated practitioner for all cases. Patient characteristics were: median age 55 (18-80); 57% male; race -white 74%, black 21%, Asian 5%; diagnosis AML 38%, ALL 18%, MDS 18%, NHL 10%; donor type haplo 42%, MRD 32%, MUD 26%, %; high/v. high 25%, low/intermediate 73%; HCT-CI (0-2) 43%, (&amp;gt;3)57%, regimen - myeloablative 48%, RIC 31%, NMA 21%). Median follow-up for survivors was 64 (16-151) months. Median CD34 and CD3 doses infused were 5.0 x 10e6/kg (min 1.59) and 17.3 x 10e7/kg (range 1.2-83.3) respectively. Median CD3 dose infused were higher with female donors (20.5 vs 15.5 x 10e7/kg, p&amp;lt;0.001) and older donors (18.6 vs 15.9 x 10e7 for donors aged &amp;gt;38 vs &amp;lt;38, p&amp;lt;0.001). The effect of Infused CD3 dose was assessed in terciles (ranges 1.2-13.5, 13.6-22, 22.1-83.3 x 10e7/kg). Median peripheral blood donor CD3 chimerism was 100% for all CD3 dose terciles at d 30, through 180 post-transplant. On univariate analysis - estimated 3-year survival, DFS, NRM, relapse and acute GVHD incidence were not significantly different between the low, middle and high CD3 terciles. In contrast, the cumulative incidence (CI) of moderate-to-severe chronic GVHD at 3 years was significantly higher in the highest vs lowest tercile (36.8% vs 28.6%, p=0.042) (Fig 1), while severe chronic GVHD (14.5% vs 13%) was not significantly different. On stratified Cox multivariable analysis comparing the highest to the lowest CD3 dose terciles - survival, DFS, NRM, relapse and acute GVHD were not significantly different. Moderate-to-severe chronic GVHD was significantly higher in the highest versus lowest tercile (HR 1.53, p=0.005). In order to address specific effects of CD3 dose for particular donor groups, we analyzed incidence of chronic GVHD by CD3 dose terciles separately for the three donor types (MRD n=258, MUD n=210 and Haplo n=343). Whereas the CI of moderate-to-severe chronic GVHD was not significantly different for lowest versus highest CD3 tercile in MRD and Haplo transplant recipients, it was significantly higher for MUD recipients (50% vs 26%, p=0.008, GRFS 29% vs 9%, p=0.005). On multivariable analysis, risk of moderate-to-severe chronic GVHD was significantly higher for the highest versus lowest CD3 dose terciles (HR 2.62, p=0.002) and for the middle vs lowest terciles (HR1.77, p=0.043) in MUD patients (Fig 1). In contrast, CD3 dose had no significant impact on chronic GVHD for MRD and Haplo recipients. This large retrospective analysis of PBSC allografts using a CD34 cap (&amp;lt;5 x 10e6/kg) shows that this approach produced infused CD3 doses ranging between 1.2 and 83.3 x 10e6 /kg. The median and upper limit of infused CD3 dose was lower than previously reported by studies with no CD34 cap 2,3. CD3 dose had a significant impact on the rate of moderate-to-severe chronic GVHD in MUD recipients. A prospective comparison of capped versus uncapped CD34 dosing may help determine the value of this strategy in mitigating GVHD risk of PBSC grafts. These data suggest the need for more aggressive GVHD prophylaxis in patients infused with higher CD3 doses in MUD patients. 1. Mohty M, Bilger K, Jourdan E, et al. Leukemia. 2003;17(5):869-875. 2. Zaucha JM, Gooley T, Bensinger WI, et al. Blood. 2001;98(12):3221-3227. 3. Saad A, Lamb L, Wang T, et al. Biol Blood Marrow Transplant. 2019;25(9):1875-1883.

Genetic code expansion in E. coli enables production of a functional ‘ready-to-click’ T cell receptor-specific scFv

Antibody-based cancer therapies have been evolving at a rapid pace in the pharmaceutical market. Bispecific antibody-drug conjugates that engage immune cells to target and kill cancer cells with precision have inspired the development of immunotherapy. Miniaturized antibody fragments such as diabodies, nanobodies, or single-chain variable fragments (scFvs) hold great promise as antibody-drug conjugates as they specifically target tumor tissue and can penetrate it. Here, we optimized the soluble periplasmic expression of the scFv OKT3 comprising the variable VH and VL domains of the mouse anti-human CD3 antibody muromonab-CD3 (trade name Orthoclone OKT3) in E. coli. By an expansion of the genetic code, we site-specifically incorporated the reactive non-canonical amino acid Nε-((2-azidoethoxy)carbonyl)-L-lysine (AzK) into scFv OKT3 using an orthogonal pyrrolysyl-tRNA synthetase/tRNACUA pair. To confirm the AzK incorporation and to demonstrate the accessibility of the reactive azide group, we conjugated a fluorophore to scFv OKT3 AzK variants by copper-free strain-promoted alkyne-azide cycloaddition (‘click chemistry’). The scFv OKT3 wild type and the AzK variants bound T cells at nanomolar concentrations. In this study, a ‘ready-to-click’ scFv OKT3 was successfully developed for future applications, e.g. as controlled anti-T cell antibody-drug conjugate or bispecific T cell engager and for imaging immune T cell migration in cancers.

Selective targeting of auto reactive B cells using bispecific molecules

Abstract Type 1 diabetes occurs when autoreactive T cells attack insulin-producing cells in pancreatic islets. B cells promote disease by acting as essential antigen presenting cells. Targeted immune therapy for treatment and prevention of autoimmune diseases is preferable to drugs that cause global immunosuppression. We therefore investigated the efficacy of three constructs designed to specifically target and neutralize anti-insulin B cells. Construct one has a 4-arm PEG backbone with insulin attached to 2 of the arms (insulin-PEG). Construct two has insulin attached to 2 of the PEG arms and a trisaccharide molecule that acts as a ligand for inhibitory receptor CD22 on the remaining 2 PEG arms (insulin-PEG-CD22L). Construct three consists of insulin directly conjugated to CD22L (insulin-CD22L). In vitro experiments using transgenic anti-insulin B cells demonstrate that insulin-PEG unexpectedly promotes excessive proliferation and upregulation of CD86 in anti-CD40 treated anti-insulin B cells. Addition of CD22L (insulin-PEG-CD22L) restores anti-insulin B cell responses to baseline but has no overall benefit. Conversely, insulin-CD22L blunts the effects of anti-CD40 stimulation, reducing proliferation by 15% and IgM surface expression by 50% . CD22L alone has no effect on stimulated anti-insulin B cells. Preliminary in vivo data using 2-photon imaging shows that insulin-CD22L binds anti-insulin B cells in spleen, pancreatic lymph nodes and pancreatic islets within 30 minutes of injection. Thus, this novel insulin-CD22L construct directs the dampening effect of CD22 specifically to anti-insulin B cells that promote T1D, making it a promising therapeutic intervention that may also be useful when paired with anti-T cell treatments. P20GM1133117 T32GM008545 5T32AI7163-43

Abstract 4150: Novel preclinical model for the evaluation of cytokine release syndrome in response to CD19xCD3 bispecific antibody in humanized mice

Abstract Cytokine Release Syndrome (CRS) is a systemic inflammatory response that is caused by the large, rapid release of cytokines in the blood by immune cells affected by infection or immunotherapy and remains a prominent challenge for T cell-based therapeutics. Bispecific T cell engagers (BiTEs) are fusion proteins that are generated by linking the targeting region of two antibodies: one arm binds to the tumor-associated antigen, and the other arm binds to T cells to promote T cell-mediated cytotoxicity in an MHC-independent manner. A CD19xCD3 BiTE has been approved by the FDA for the treatment of CD19-positive B-cell lymphoma. Despite having great clinical efficacy, CRS remains a major concern for BiTE therapy. This necessitates the development of a translational model that could predict CRS toxicity more accurately. Various in-vitro models proposed to assess BiTE-related CRS have several shortcomings as they do not provide details about the systemic immune response, off-target effects, neurotoxicity, tissue damage, and organ failure. Here we propose an in-vivo approach to concomitantly assess the efficacy and CRS toxicity of CD19xCD3 BiTE either alone or in combination with Rituximab (monoclonal antibody targeting CD20) in the Raji luciferase xenograft model of Burkitt’s lymphoma in PBMC humanized NSG-MHC Class I/II knockout mice. Five days post human PBMC engraftment (Study Day- SD 5), the mice were injected with Raji-luciferase cells. On SD 6, the mice were treated with BiTE, Rituximab, or the combination of BiTE and Rituximab. Additional control groups included OKT3 and Anti-CD28, the anti-T cell antibodies that failed clinically due to severe CRS. Efficacy was assessed through in-vivo imaging on SD5, SD8, and SD11. We observed tumor growth inhibition with BiTE, Rituximab, and the combination treatment. Clinical observations were recorded daily post treatment. Following cytokines were measured 6 hours after dosing and at study termination - IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ. Elevated cytokine levels were observed in the BiTE/combination group 6 hours post dosing but not in the Rituximab alone group. Together, the data suggest that the model could be used to assess the preclinical efficacy and CRS toxicity of BiTE. Citation Format: Deep S. Shah, Catalina Simion, Sang hu, Rhonda Chronis, Tre McClellan, Jae Hwang, Ryan Yang, Heather Baker, Danying Cai, James Keck, Lindsay Shopland. Novel preclinical model for the evaluation of cytokine release syndrome in response to CD19xCD3 bispecific antibody in humanized mice. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4150.

Comparison of Benefits and Risks Associated with Anti-T-Lymphocyte Globulin (ATLG) Serotherapy in Methotrexate (MTX)- versus Mycophenolate Mofetil (MMF)-Based Hematopoietic Stem Cell Transplantation

Background: Serotherapy with anti-T lymphocyte globulin (ATLG, Grafalon, formerly ATG-Fresenius) is established for the prevention of severe graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation (HSCT). The evidence from prospective studies is predominantly derived from a setting where methotrexate (MTX) and a calcineurin inhibitor (CNI) are used as the backbone of GVHD prophylaxis. The efficacy of ATLG in combination with CNI and mycophenolate mofetil (MMF) has not been investigated as much, particularly in terms of a direct comparison with its effects when combined with CNI/MTX. A total of 401 HSCTs from two Austrian transplant centers were retrospectively evaluated. We included peripheral blood transplants from early- or intermediate-stage (excluding advanced/refractory) hematological diseases from matched siblings or 10/10 or 9/10 matched unrelated donors with CNI/MTX or CNI/MMF prophylaxis, either without (n = 219) or with ATLG (n = 182). Overall, ATLG significantly reduced the risk for all-cause mortality by multivariate Cox analysis (HR 0.53; p = 0.002). Stratification by postgrafting prophylaxis type revealed a significant survival advantage for ATLG in the CNI/MMF cohort (HR 0.49; p = 0.001; n = 193), while its effect on survival in the CNI/MTX cohort was not significant (HR 0.87; p = 0.56; n = 208). In unrelated HSCT with CNI/MMF prophylaxis, ATLG exhibited its greatest survival benefit (HR 0.34; p = 0.001; n = 104). In the context of CNI/MMF, ATLG may provide even greater benefits than in the setting of CNI/MTX for post-grafting immunosuppression. Future prospective studies on ATLG should therefore focus on CNI/MMF-based transplants, which are widely performed in elderly or comorbid patients not expected to tolerate a standard course of MTX.

Pre-transplant CD69+ extracellular vesicles are negatively correlated with active ATLG serum levels and associate with the onset of GVHD in allogeneic HSCT patients.

Graft versus host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (HSCT). Rabbit anti-T lymphocyte globulin (ATLG) in addition to calcineurin inhibitors and antimetabolites is a suitable strategy to prevent GVHD in several transplant settings. Randomized studies already demonstrated its efficacy in terms of GVHD prevention, although the effect on relapse remains the major concern for a wider use. Tailoring of ATLG dose on host characteristics is expected to minimize its side effects (immunological reconstitution, relapse, and infections). Here, day -6 to day +15 pharmacokinetics of active ATLG serum level was first assayed in an explorative cohort of 23 patients by testing the ability of the polyclonal serum to bind antigens on human leukocytes. Significantly lower levels of serum active ATLG were found in the patients who developed GVHD (ATLG_AUCCD45: 241.52 ± 152.16 vs. 766.63 +/- 283.52 (μg*day)/ml, p = 1.46e-5). Consistent results were obtained when the ATLG binding capacity was assessed on CD3+ and CD3+/CD4+ T lymphocytes (ATLG_AUCCD3: 335.83 ± 208.15 vs. 903.54 ± 378.78 (μg*day)/ml, p = 1.92e-4; ATLG_AUCCD4: 317.75 ± 170.70 vs. 910.54 ± 353.35 (μg*day)/ml, p = 3.78e-5. Concomitantly, at pre-infusion time points, increased concentrations of CD69+ extracellular vesicles (EVs) were found in patients who developed GVHD (mean fold 9.01 ± 1.33; p = 2.12e-5). Consistent results were obtained in a validation cohort of 12 additional ATLG-treated HSCT patients. Serum CD69+ EVs were mainly represented in the nano (i.e. 100 nm in diameter) EV compartment and expressed the leukocyte marker CD45, the EV markers CD9 and CD63, and CD103, a marker of tissue-resident memory T cells. The latter are expected to set up a host pro-inflammatory cell compartment that can survive in the recipient for years after conditioning regimen and contribute to GVHD pathogenesis. In summary, high levels of CD69+ EVs are significantly correlated with an increased risk of GVHD, and they may be proposed as a tool to tailor ATLG dose for personalized GVHD prevention.

S-40-3: MEMORY GAMMA DELTA T CELLS PLAY A ROLE IN HYPERTENSION

Objectives: A subset of T cells, γδ T cells, participate in the pathogenesis of hypertension. It has been shown that memory T cells develop after a hypertensive insult and sensitize mice to develop hypertension to subsequent mild hypertensive stimuli. However, whether memory γδ T cells develop and play a role in hypertension is unknown. We hypothesize that memory γδ T cells form after a hypertensive challenge, and that they sensitize mice to develop hypertension to mild hypertensive stimuli. Methods: Ten-12-week-old C57BL/6J mice were exposed or not to a pressor dose of angiotensin II (490 ng/kg/min, SC) for two weeks, followed by a two-week washout period, and then infused with a subpressor dose of angiotensin II (140 ng/kg/min, SC) for two weeks. Blood pressure (BP) was measured by telemetry and memory γδ T cells profiled by flow cytometry. A subset of mice was injected IP with 400 μg of anti-T cell receptor γδ-depleting or isotype control antibodies 1 day before and 6 days after the initiation of second hypertensive challenge. A final subset was injected IV with 2.5x105 γδ T cells isolated from hypertensive or normotensive mice, and then exposed to a subpressor dose of angiotensin II as above. Results: Repeated hypertensive challenges yielded a higher systolic BP than one mild hypertensive stimulus (154 ± 4 vs 120 ± 3 mm Hg, P &lt; 0.01). Two-week pressor dose angiotensin II increased effector memory γδ T cells in mesenteric artery perivascular adipose tissue (1.25 ± 0.37% vs. 0.24 ± 0.12%, P &lt; 0.05) and mesenteric lymph nodes (1.49 ± 0.03% vs 0.82 ± 0.15%, P &lt; 0.05). γδ T cell depletion reduced systolic BP elevation from day 8 (135 ± 5 vs 150 ± 5 mm Hg, P &lt; 0.05) to day 13 of the second hypertensive challenge (150 ± 5 vs 163 ± 2 mm Hg, P &lt; 0.05). Adoptive transfer of γδ T cells isolated from hypertensive compared to normotensive mice caused a greater systolic BP elevation in response to a subpressor dose of angiotensin II (159 ± 3 vs 142 ± 6 mm Hg, P &lt; 0.01). Conclusion: Memory γδ T cells develop after a hypertensive challenge and sensitize mice to mild hypertensive stimuli. Memory γδ T cells may represent a novel therapeutic target to treat hypertensive humans.

Blocking Tim-3 enhances the anti-tumor immunity of STING agonist ADU-S100 by unleashing CD4+ T cells through regulating type 2 conventional dendritic cells.

Rationale: An immunosuppressive tumor microenvironment (TME) is a major obstacle in tumor immunotherapy. Stimulator of interferon genes (STING) agonists trigger an inflammatory innate immune response to potentially overcome tumor immunosuppression. While STING agonists may hold promise as potential cancer therapy agents, tumor resistance to STING monotherapy has emerged in clinical trials, and the mechanisms remain unclear. Methods: The in vivo anti-tumor immunity of STING agonist ADU-S100 (S100), plus anti-T cell immunoglobulin and mucin-domain containing-3 antibody (αTim-3) were measured using murine tumor models. Tumor-specific T cell activation and alterations in the TME were detected using flow cytometry. The maturation and function of dendritic cells (DC) were also measured using flow cytometry, and the importance of CD4+ T cells in combination therapy was measured by blocking antibodies. Additionally, the effect of S100 on CD4+ T was verified via in vitro assays. Lastly, the impact of conventional dendritic cells (cDC) 2 with a high expression of Tim-3 on survival or therapeutic outcomes was further evaluated in human tumor samples. Results: S100 boosted CD8+ T by activating cDC1 but failed to initiate cDC2. Mechanistically, the administration of S100 results in an upregulation of Tim-3 expressed in cDC2 (Tim-3+cDC2) in both mice and humans, which is immunosuppressive. Tim-3+cDC2 restrained CD4+ T and attenuated the CD4+ T-driven anti-tumor response. Combining S100 with αTim-3 effectively promoted cDC2 maturation and antigen presentation, releasing CD4+ T cells, thus reducing tumor burden while prolonging survival. Furthermore, high percentages of Tim-3+cDC2 in the human TME predicted poor prognosis, whereas the abundance of Tim-3+cDC2 may act as a biomarker for CD4+ T quality and a contributing indicator for responsiveness to immunotherapy. Conclusion: This research demonstrated that blocking Tim-3 could enhance the anti-tumor immunity of STING agonist ADU-S100 by releasing CD4+ T cells through regulating cDC2. It also revealed an intrinsic barrier to ADU-S100 monotherapy, besides providing a combinatorial strategy for overcoming immunosuppression in tumors.

A Phase 1/1b Multicenter Ascending Dose Study to Evaluate the Safety of HA-1 Minor Histocompatibility Antigen-Reactive TCR-Modified T Cells (BSB-1001) in Patients Undergoing HLA-Matched Allogenic Hematopoietic Stem Cell Transplant (alloSCT) for MRD+ AML or ALL or Poor/Very Poor Risk MDS

Background: AlloSCT can cure patients with acute leukemia and high-risk myelodysplastic syndromes (MDS). However, post-transplant relapse remains the greatest single cause of post-transplant death, occurring in up to 80% of high-risk patients. In HLA-matched alloSCT, alloreactive donor T cells target minor histocompatibility antigens (miHAs), which are peptide products of coding polymorphisms that distinguish recipients from donors. These alloreactive T cells can target leukemic cells, mediating the graft-vs-leukemia (GVL) effect; unfortunately, they also can cause graft-vs-host disease (GVHD). Pre-clinical mouse models (Blood, 2011) demonstrate that CD8+ T cells that target miHAs with expression limited to hematopoietic cells can mediate GVL without causing GVHD. To translate this concept for how to safely augment GVL to the clinic, BlueSphere Bio (BSB) used its high throughput T cell receptor (TCR) discovery and characterization platforms to clone a panel of TCRs reactive against the HLA-A*02-restricted miHA, HA-1, which is expressed mostly in hematopoietic cells. These receptors were cloned from a multiparous A*02:01 HA-1- woman who was immunized to HA-1 through pregnancy. A high affinity TCR specific for HA-1 and not reactive against allogeneic HLA molecules was chosen to create a cellular therapeutic designed to mediate GVL and enhance donor engraftment with a lower risk for GVHD, relative to polyclonal donor T cells. The anti-HA-1 T cell product (BSB-1001) is composed of donor CD8+ T cells transduced with a lentivirus vector encoding the anti-HA-1 TCR and the RQR8 tag, which expresses an epitope from CD20, thereby enabling the in vivo killing of BSB-1001 cells with rituximab. CRISPR-cas9 editing of the CD8+ T cells deletes the endogenous TCRα and β chains with >95% efficiency. This increases expression of the TCR and reduces risk of GVHD caused by native TCRs, as well as possible off-target reactivities generated by pairing of endogenous and introduced TCR chains. BSB-1001 cells specifically kill HLA-A*02:01, HA-1+ targets, with no activity against cells that are HLA-A*02:01+ but HA-1-, or cells that are HA-1+ but are HLA-A*02:01-. BSB-1001 is also not reactive against a panel of B lymphoblastic cell lines (LCL) expressing allogeneic HLA molecules. Importantly, efficient target killing is seen at effector:target ratios as low as 0.1, and maintained after several rounds of ex-vivo expansion following target cell challenge, a likely predictor of potency and persistence in vivo. Study Design: The first in human Phase 1/1b study is a multicenter, open-label, single dose study with a 3+3 ascending design with the option to expand accrual at a promising dose. Subjects must have ALL, AML or high-risk MDS with evidence of disease after the last therapy, be HLA-A*02:01 HA-1+ and have a 10/10 HLA-matched HA-1- related or unrelated donor. GCSF-mobilized donors will undergo leukapheresis. From this product, a small fraction of cells will be removed for BSB-1001 manufacturing; the remainder will undergo CD34-selection and cryopreservation until SCT. After BSB-1001 manufacturing is complete, patients will undergo ablative conditioning (choice of three regimens), followed by infusion of the CD34-selected cells and then BSB-1001. Patients will not receive anti-T cell antibodies and will receive no prophylactic pharmacologic immunosuppression. Subjects must otherwise be suitable for ablative conditioning using standard criteria. The primary objective is to establish a recommended dose of BSB-1001 based on its dose-limiting toxicities and the magnitude of post-infusion BSB-1001 expansion. The primary endpoint is determining the incidence of treatment-emergent adverse events, including dose-limiting toxicities. Additional endpoints include rates of engraftment, incidences of cytokine release syndrome (CRS) and acute and chronic GVHD, quantitation of malignant hematopoiesis and lineage-specific donor chimerism. CRS and GVHD, should they occur, will be treated per institutional standards. Rituximab can be administered to kill BSB-1001 cells in case of refractory CRS or GVHD. Enrollment is anticipated to begin in 1Q2023. BSB is also developing panels of TCRs targeting additional relatively hematopoietically-restricted miHAs with prioritization based on the frequencies of the restricting HLAs and the immunogenic/nonimmunogenic miHA alleles.