Experimental And Clinical Studies Of Abatacept For Acute Graft-versus-host Disease Prophylaxis

BackgroundAllogeneic hematopoietic stem cell transplantation(allo-HSCT)is the only curative treatment option for most of malignant hematological diseases.Acute graft-versus-host disease(aGVHD)is a major complication after allo-HSCT,especially in haploidentical hematopoietic stem cell transplantation(haplo-HSCT),with an incidence of up to 50%.Although aGVHD management has evolved from classical pharmacological prophylaxis to innovative prevention and treatment approaches including gene and cellular therapies as experience has been accumulated in clinical practice,aGVHD remains a significant cause of death in patients after HSCT.aGVHD occurs when donor-derived T cells attack recipient target tissues while exerting anti-leukemia/tumor(GVL/GVT)effects,and the goal of its prevention and treatment is to attenuate deleterious damage by donor T cells while preserving and enhancing GVL/GVT effects.Abatacept is a soluble CTLA-4 fusion protein consisting of the extracellular structural domain of CTLA-4 and a fragment of the Fc structural domain of human immunoglobulin G1(hinge-CH2-CH3 structural domain).Abatacept can compete with CD28 to bind CD80/CD86 on the surface of antigen-presenting cells(APCs),thereby inhibiting T cell proliferation activation and inducing post-transplant immune tolerance.Since subcutaneous abatacept entered the Chinese market in 2020,we conducted experimental and clinical studies on the efficacy and safety of subcutaneous abatacept to prevent aGVHD.The studies were aiming to investigate the efficacy and safety of abatacept to control aGVHD after haplo-HSCT,and explore its immune mechanism which facilitates the new therapeutic strategies for the prevention of aGVHD after haplo-HSCT in Chinese population.MethodsIn the first part,SPF-grade wild-type C57BL/6(H-2b,female)mice were used as donor mice and SPF-grade BALB/c(H-2d,female)mice were used as recipient mice to construct a mouse aGVHD model with different abatacept concentrations for subcutaneous injection.The survival and aGVHD histopathological scores of recipient mice were recorded in order to preliminary determine whether abatacept could prevent aGVHD in mice,and to evaluate the most appropriate dosage of subcutaneous abatacept for mouse aGVHD prophylaxis.The most appropriate dose of abatacept was subsequently used for aGVHD prophylaxis in mice,and the survival,body weight,aGVHD clinical score of recipient mice were recorded.The number and proportion of immune cell subpopulations,as well as the expression of intracellular cytokines and immune checkpoint molecules were detected using flow cytometry.In order to investigate the effect of abatacept on the survival rate of humanized mice,HLA-A2(02:01)-negative healthy human donor was selected to collect peripheral blood mononuclear cell(PBMNC)for establishing a humanized aGVHD mouse model,with SPF B-NDG HLA-A2.1 mice as recipient mice.In the second part,we conducted a prospective clinical trial(number in clinincaltrials.gov:NCT04686929)to evaluate the safety,efficacy and immunological effects of abatacept in the prevention of aGVHD during haplo-HSCT.All the participants received abatacept 250 mg(d-1),125 mg(d+5,d+14,d+21,d+28,d+35,d+42,d+49,d+56)subcutaneously during HSCT in combination with cyclosporine,methotrexate and mycophenolate for aGVHD prophylaxis.The primary study endpoint was grade Ⅱ-Ⅳ aGVHD at day 100,and the secondary study endpoints were treatment-related mortality(TRM)and relapse.To explore the immunological effects of abatacept,we screened patients who underwent haplo-HSCT during the same period and did not use abatacept for aGVHD prevention as a control group.PB specimens were collected from patients in the abatacept and control groups after transplantation,and the number and proportion of immune cell subpopulations,intracellular cytokines,and cell surface molecules in clinical patients were examined by flow cytometry.The molecular mechanism of abatacept modulating aGVHD was investigated by transcriptome sequencing(RNA sequencing,RNA-seq)of CD3+T lymphocytes of PB.Results1.In the first part,we explored the regulatory effects and mechanisms of aGVHD in recipient mice using abatacept by constructing a mouse allogeneic transplantation aGVHD model.(1)The dose-escalating experiment revealed that different doses of abatacept(5 mg/kg,10 mg/kg,20 mg/kg,50 mg/kg)improved the survival of allo-HSCT recipient mice compared to the control group with subcutaneous NaCl injection.Pathological examination suggested that 5 mg/kg of abatacept subcutaneously had the most significant protective effect against histopathological damage in aGVHD.However,since both the smaller subcutaneous injection dosages of 5 mg/kg and 10 mg/kg significantly improved survival and attenuated aGVHD scores in allo-HSCT mice,we further compared the allogeneic transplant recipient mice with these two dosages of abatacept and found that the abatacept treatment dose of 5 mg/kg was more effective in protecting mice body weight and reduced the severity of aGVHD.Therefore,we used abatacept of 5mg/kg subcutaneously in all subsequent mice experiments.(2)The proportion of CD4+T cells was decreased and the proportion of CD8+T cells was increased in PB,lung and liver of recipient mice treated with abatacept 5 mg/kg subcutaneously compared with control mice.In addition,abatacept inhibited the activation of splenic CD4+ T cells and CD8+T cells and the secretion of TNF-α and IFN-y in recipient mice,but promoted the expression of PD-1 on CD4+T.Flow cytometry was also performed on myeloid cells from mice spleen,and the results suggested that abatacept did not affect the proliferation levels of myeloid cells such as dendritic cells(DCs),monocytes,macrophages and granulocytes.(3)In the humanized aGVHD mouse model,both abatacept of 5 mg/kg and 10 mg/kg significantly prolonged the survival of mice compared to the control group using NaCl injection.2.In the second part,a total of 25 cases were recruited into the abatacept trial from June 1st,2021 to February 28th,2023,and 22 cases who completed follow-up were enrolled into analyses,in comparisons with 16 controlled patients who underwent haplo-HSCT in the same timeframe.(1)The cumulative incidence of grade Ⅰ-ⅣaGVHD,Ⅱ-Ⅳ aGVHD and Ⅲ-Ⅳ aGVHD at 100 days in patients of abatacept trial was 27.3%±0.8%,13.6%±0.6%and 4.5%±0.2%,respectively.There were 5 patients developed cGVHD by the end of follow-up,and the cumulative incidence of cGVHD was 28.4%±1.2%.(2)By the end of follow-up,one patient in the abatacept group relapsed and three patients died,all of whom died of infections.The 1-year cumulative incidence of relapse(CIR)and TRM of patients in abatacept group were 10.3%±1.1%and 17.4%±1.0%.The 1-year overall survival(OS),leukemia-free survival(LFS),and Graft-versus-host disease-free and relapse-free survival(GRFS)were 82.6%±9.1%,72.3%±12.5%,and 75.6%±1.2%,respectively.(3)There were no statistical differences between the abatacept cohort and the control cohort in terms of age,gender,diagnosis,follow-up time,conditioning regimen,graft source,and median count of MNC in the grafts.The median CD34+cell count in grafts was higher in the control group than in abatacept group(P=0.026).(4)There was no significant difference in engraftment,neutrophil recovery and platelet recovery between the two cohorts.Neutrophil recovery rates at 28 days and platelet recovery rates at 100 days were similar between the two groups.(5)The rate of Epstein-Barr virus reactivation(9.1%vs.6.3%,P=0.999),cytomegalovirus reactivation(31.8%vs.25.0%,P=0.729),median time to cytomegalovirus reactivation(57.0d vs.54.5d),incidence of pulmonary infection(50%vs.18.8%),sepsis(22.7%vs.0%),and hemorrhagic cystitis(13.6%vs.18.8%)in patients in the abatacept cohort were not significantly different from those in the control group.(6)PB samples were collected from patients in the abatacept cohort at sequential time points.Results of flow cytometry showed that CD4+ T cells and CD8+T cells started to recovery at+8w and+4w post-transplantation,respectively.Comparison of PB specimens at+8w between abatacept cohort and control cohort by flow cytometry hinted that abatacept inhibited CD4+T cell proliferation,granzyme expression in NK cells and CD8+T cells,and perforin expression in CD8+T cells,while promoted CD8+T cell and regulatory T lymphocyte proliferation,PD-1 expression in CD4+T cells,and TIM-3 expression in CD8+T cells.(7)RNA-seq was performed on CD3+T-lymphocyte of PB samples from abatacept and control cohorts with PB specimens at 3 months post-transplantation.Compared to the control group,512 genes were upregulated and 493 genes were downregulated in the abatacept group.GO analysis and GSEA revealed functional alterations related to major histocompatibility complex Ⅱ molecule binding,T cell activation and differentiation,DCs development,stem cell proliferation,and TOR signaling in the subjects.Conclusion1.The optimal dose of subcutaneous abatacept for the prevention of aGVHD after allogeneic transplantation in mice was 5 mg/kg.In a mouse model of aGVHD after allo-HSCT at this dose,the subcutaneous abatacept did not affect the proliferation of APCs populations such as DCs.It could establish immune tolerance by inhibiting the proliferation of CD4+T lymphocytes and the secretion of pro-inflammatory factors in the early post-transplantation period as well as promoting the expression of CD4+T PD-1.Thereby the survival of aGVHD model mice was improved through protecting their body weight,and reducing the manifestation of aGVHD and pathological damage.2.In prospective clinical trial,abatacept was safe for preventing aGVHD after haplo-HSCT.Abatacept could affect immune cell activation and proliferation,thus reducing the incidence of acute and chronic GVHD and preserving the GVL effect,which is expected to improve OS,LFS and GRFS of haplo-HSCT patients.