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Abstract 741 We have previously shown that a costimulation blockade-containing regimen could provide effective protection against acute GvHD (aGvHD) in a non-human primate model. We therefore designed a first-in-disease trial of in vivo CD28:CD80/86-directed costimulation blockade with CTLA4-Ig (abatacept) to prevent aGvHD after unrelated-donor HSCT for patients > 12y. (Clinical Trials.Org #NCT01012492). In this trial, 10mg/kg abatacept was administered IV on day −1, +5, +14, +28 in addition to standard prophylaxis with cyclosporine + MTX. Enrollment of all patients is complete, and the study is evaluable for feasibility, toxicity, engraftment, and the primary immunologic outcome: the incidence of Grade III-IV aGvHD by d+100. Patient Characteristics and Survival: 10 patients, with a median age of 44.5 y (17–74) were enrolled and treated. 6 patients received HLA-mismatched grafts (matched at 7/8 alleles) and 4 received 8/8 HLA-matched URD grafts. 8 received PBSCs and 2 received BM. All received high-intensity conditioning (Bu/Cy, TBI/Cy or Flu/Mel). With a median follow-up of 367 days (262–680), 7 patients are alive and in remission. 2 patients died of relapse (Day +121 and Day +147). One patient died, in remission, of multi-organ failure on day +453 post-transplant. Feasibility, Pharmacokinetics and Pharmacodynamics of Abatacept in HSCT: All 10 patients received all 4 scheduled abatacept doses, without infusion reactions. The average peak (230.9 +/− 7.4 mg/ml) and trough (45.9 +/− 2.8 mg/ml) abatacept levels, as well as the terminal T1/2 (19.6 +/− 1.9 days) were similar to that observed previously. Importantly, as has been previously established in vitro, patients receiving abatacept demonstrated significant inhibition of post-transplant CD4+ T cell proliferation (with >80% reduction in the accumulation of Ki-67+ proliferating CD4+ T cells at d+14 and +28 compared to a control cohort who received cyclosporine + methotrexate without abatacept, Figure 1). This data establishes, for the first time, the feasibility of giving abatacept to this new patient population, and that the PK and PD parameters in HSCT closely mirror those previously shown to effectively control immune-mediated diseases. Engraftment: All patients achieved neutrophil engraftment (median d+16.5) and donor engraftment (100% CD33 chimerism at d+30). Lymphocyte recovery was rapid: Day +100 mean peripheral blood counts showed ALC = 1053 +/− 259 cells/ml, total T cells = 741 +/− 208 cells/ml, and CD8+ T cells = 381 +/− 99 cells/ml. The Day +100 CD4+ T cells = 285 +/− 105 cells/ml, not significantly different from historical controls (n = 43) that received CNI/MTX aGvHD prophylaxis without abatacept (262 +/−26 cells/ml). GvHD: Patients receiving the abatacept-containing regimen had encouragingly low rates of early severe aGvHD: Two patients developed aGvHD before day +100, with one of these patients (Gr II) progressing to steroid-dependent cGvHD of the liver and one patient (Gr III) with complete resolution of aGvHD (and currently off steroids). The cumulative incidence of grade II-IV and III-IV aGvHD by day 100 was thus 20% and 10%, respectively (Figure 2). Importantly, there was no Gr IV aGvHD, no patient received salvage therapy for aGvHD, and there were no deaths from aGvHD. After day +100, one patient developed late acute GvHD (currently off all immunosuppression), and two patients developed overlap syndrome. Both had responsive disease and are currently either weaning or off corticosteroids. No other patient developed late aGvHD, and no other patient has developed moderate or severe cGvHD. Infection: No life-threatening infections occurred. 5 patients developed CMV reactivation, all responsive to antivirals. No patient developed CMV disease. No EBV viremia >1000 copies/ml occurred. One patient developed EBV+ plasmacytic hyperplasia of the tongue, which resolved without intervention. No other EBV-related disease occurred. Conclusions: This trial demonstrates, for the first time, the feasibility of adding in vivo T cell costimulation blockade with abatacept for aGvHD prevention. The decreased CD4+ T cell proliferation post-transplant and the encouragingly low rates of early, severe aGvHD suggest that costimulation blockade may be an effective agent for aGvHD prophylaxis and support the conduct of a larger, randomized phase 2 study. Disclosures: Off Label Use: Abatacept was used in this trial. It is a costimulation blockade agent which was tested for its ability to prevent aGvHD.

Abstract 2998 Introduction: GvHD remains the most deadly complication of HSCT despite current prevention strategies. To address the unmet need for better GvHD control, we have created a non-human primate (NHP) model with which to rigorously test mechanism and efficacy of novel therapeutics. In this study, we determined whether a novel combination of mTOR inhibition (with sirolimus) and CD28:CD80/86 costimulation blockade (with belatacept) could control GvHD. Here we show for the first time that these two agents combine synergistically to prevent both the clinical and immunologic manifestations of primate aGvHD. Methods: Rhesus macaque recipients were irradiated (9.6 Gy in 2 fractions at 7cGy/min), and then transplanted with G-CSF-mobilized PBSC from a haplo-identical donor (1–5×108 TNC/kg). Recipients were treated with either sirolimus alone (n = 4, troughs targeted at 5–10 ng/mL), belatacept alone (receiving weekly doses of 20 mg/kg), or combination therapy. Clinical GvHD was monitored using our previously described NHP grading scale (Miller et al., Blood 2010), and multiparameter flow cytometric analysis was performed. Results: Untreated controls (n = 5) developed rapid, severe histopathologically-proven aGvHD and succumbed rapidly (MST = 7 days). Recipients treated with either sirolimus or belatacept alone were partially protected from the clinical manifestations of GvHD. Sirolimus-treated recipients (n = 6) developed predominantly GI disease (with diarrhea but no elevation of bilirubin) and had an MST of 14 days (Figure 1). Recipients treated with belatacept alone (n = 3) developed primarily liver aGvHD (bilirubin rapidly rising to 6–30 × normal with histologically-confirmed lymphocytic infiltration) and an MST of 11 days. In striking contrast, recipients treated with combined sirolimus + belatacept (n = 5) demonstrated neither uncontrolled diarrhea nor hyperbilirubinemia at the timed terminal analysis (1 month post-transplant). We employed multiparameter flow cytometry to determine the immunologic consequences of sirolimus and belatacept on T cell proliferation (using Ki-67 expression) and cytotoxity (using granzyme B expression). We found that the clinical synergy observed with combined therapy was recapitulated immunologically. Thus, while untreated aGvHD was associated with rampant CD8+ proliferation (with 83 +/− 14% Ki-67+ CD8+ vs 4.7 +/− 0.6% pre-transplant), sirolimus or belatacept as monotherapy both partially controlled proliferation (35 +/− 3% and 65 +/− 23% Ki-67+ CD8+ with sirolimus or belatacept, respectively). Combined sirolimus + belatacept dramatically reduced proliferation (to 8 +/− 3%, favorably comparing with 13% Ki-67+ CD8+ T cells using standard Calcineurin Inhibitor/Methotrexate (CNI/MTX) prophylaxis). Sirolimus and belatacept both also partially controlled GvHD-related T cell cytotoxicity. Thus, while untreated aGvHD was associated with excessive granzyme B expression in CD8+ T cells (82 +/− 2% granzyme Bvery high CD8+ cells vs 0.3 +/− 0.2% pre-transplant) sirolimus or belatacept monotherapy also partially controlled cytotoxicity (8 +/− 1% and 35 +/− 1% granzyme Bvery high with sirolimus or belatacept, respectively). Combination therapy dramatically reduced the proportion of these cells, to 1.5 +/− 0.8 % granzyme Bvery high, favorably comparing with 4% granzyme Bvery high using CNI/MTX. The ability of sirolimus, belatacept, or the combination to control Ki-67 and Granzyme B expression closely correlated with survival (Figure 2A, B) supporting a pathogenic role for these highly proliferative and cytotoxic cells in aGvHD pathology. Moreover, significant co-expression of granzyme B in the Ki-67+ cells was observed (Figure 2C) suggesting that dual-positive Ki-67/Granzyme B cells may mark a pathogenic population, amenable to tracking in the peripheral blood. Implications: These results reveal a previously undiscovered synergy between sirolimus and belatacept in the control of primate aGvHD, and provide support for future clinical investigation of this novel prevention strategy. They also identify CD8+/Ki-67+/Granzyme Bvery high dual-positive T cells as a potentially sensitive biomarker of GvHD pathogenesis, amenable to monitoring in either the blood or in GvHD target organs. Disclosures: No relevant conflicts of interest to declare.

Abstract 4078 Background: Acute GvHD remains the major cause of complications and death following unrelated-donor HSCT. In a non-human primate model, we have previously shown that in vivo costimulatory blockade of donor T-cells could provide effective protection against GVHD. To begin to explore its clinical utility, we are conducting a trial (Clinical Trials.Org # NCT01012492) to determine the feasibility of combining abatacept (CTLA4-Ig) with cyclosporine and methotrexate as acute GVHD prophylaxis for patients undergoing unrelated marrow and peripheral blood stem cell transplants for hematologic malignancies. Methods: Patients older than 12 with advanced hematologic malignancies, conditioned with either TBI/Cytoxan, Busulfan/Cytoxan or Fludarabine/Melphalan are eligible. Abatacept is administered IV on days −1, +5, +14, and +28 at 10 mg/kg in addition to standard GvHD prophylaxis consisting of cyclosporine (day −2 to day 100), and methotrexate (15 mg/m2 on day +1 and 10 mg/m2 on days +3, 6 and 11). Patients are then followed for clinical outcomes and immunologic reconstitution through day +365. Results: 9 patients (planned enrollment = 11 patients) have thus far been enrolled on the study of which 5 are evaluable for engraftment, toxicity and acute GvHD. The other four patients consist of 2 who are currently receiving abatacept, 1 who was discovered to have an ongoing viral infection at the start of the first abatacept infusion so was removed from the treatment regimen, and 1 who is awaiting transplant. The median age for the 5 evaluable patients is 47 years (17–74 years). 3 patients had AML and 2 had ALL. Patients were conditioned with Bu/Cy (n=1), TBI/Cy (n=2) and Flu/Melphalan (n=2). 4 donor-recipient pairs were allele matched at 9 of 10 loci (A, B, C, DRB1 and DQB1), while 1 was fully matched. Four of the 5 patients are currently alive and in remission and 1 relapsed at day +98 (and died on day +121 with refractory AML). The four other patients are surviving without relapse with a follow-up of 155–313 days. All 5 patients received the 4 scheduled abatacept doses. No infusional side effects were noted. All patients achieved neutrophil engraftment (median day +20 (11–47). 4 of 5 patients have achieved platelet engraftment (median day +27 (14–35). Donor engraftment (100% CD33 and 99–100% CD3 at Day +30) occurred in all cases. All patients have demonstrated rapid lymphocyte engraftment, with the mean ALC reconstituting to >500 cells/μL by day +21 post-transplant. At day +100, the mean CD3+ count was 673 +/− 251 cells/μL. Both CD8+ and CD4+ T cells reconstituted by day 100, with the mean CD8+ count = 384 +/− 148 cells/μL and the mean CD4+ count = 229 +/− 119 cells/μL. T cell reconstitution was accompanied by a shift away from naïve (Tn, CCR7+/CD45RA+) toward a CCR7-/CD45RA- effector memory (Tem)-predominant phenotype. Thus, the average proportion of CD4+ Tem cells in the recipient increased from 22 +/− 6% pre-transplant to 46 +/− 7% at day +100 with a concomitant loss of CD4+ Tn cells. Likewise, the proportion of CD8+ Tem also significantly increased, from an average of 15 +/− 4% pre-transplant to 32 +/− 7% at day +100, also with a reciprocal decrease in CD8+ Tn cells. One patient developed steroid responsive grade 3 acute GVHD involving the skin and the liver, followed by steroid responsive liver chronic GvHD. This patient is currently weaning corticosteroids. Another patient developed steroid responsive late-onset (day +217) acute GVHD (liver and GI) during cyclosporine weaning, which was also steroid responsive, and is also currently weaning corticosteroids. No other systemic acute or chronic GvHD has occurred. No unexpected complications or life-threatening infections were observed. 3 patients have experienced 5 episodes of CMV reactivation, all responsive to antiviral therapy. One patient developed polyclonal EBV-related PTLD (plasmacytic hyperplasia) in the absence of EBV viremia, which regressed without intervention. No other EBV-related disease has occurred. Conclusions: These preliminary data suggest that abatacept can be safely added to cyclosporine and methotrexate for GVHD prophylaxis in recipients of hematopoietic grafts from unrelated donors, with encouraging rates of acute GVHD. As such, they support the conduct of a larger, randomized phase 2 study. Disclosures: Off Label Use: Abatacept: It is an immunosuppressive agent that targets the CD28/B7 T cell costimulation pathway. It is approved for use in Rheumatoid arthritis.