Challenges and opportunities in targeting the costimulation pathway in solid organ transplantation

Seminars in Immunology 23 (2011) 157–164Contents lists available at ScienceDirectSeminars inImmunologyj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /y s m imReviewChallenges and opportunities in targeting the costimulation pathway in solid organ transplantationDavid Wojciechowski,Flavio Vincenti ∗University of California,San Francisco,Kidney Transplant Service,United Statesa r t i c l ei n f oKeywords:Kidney transplant Costimulation Belatacepta b s t r a c tSignaling through the costimulatory pathway is critical in the regulation of T cell activation.Abatacept,a selective costimulatory antagonist FDA approved for the treatment of moderate to severe rheumatoid arthritis,binds to CD80and CD86on antigen presenting cells,blocking the interaction with CD28on T cells.Belatacept,a second generation CTLA4-Ig with 2amino acid substitutions,has shown considerable promise in clinical transplantation as part of a maintenance immunosuppression regimen.This review will summarize the role of costimulation in T cell activation,detail the development of costimulation antagonists and highlight the pertinent clinical trials completed and ongoing utilizing belatacept as part of an immunosuppressive regimen in organ transplantation.© 2011 Elsevier Ltd. All rights reserved.1.IntroductionT cells are important mediators of acquired or adaptive immu-nity and play a crucial role in both appropriate and aberrant immune responses.Insights into the immunologic pathways involving antigen presentation and T cell responses have provided novel targets for therapeutic intervention.Advances in protein engineering have now enabled the creation of tailored therapeu-tics,maximizing target specificity with the hope of minimizing unwanted collateral toxicities.These two achievements have led to the creation of monoclonal antibodies (mAbs;murine,chimeric,and humanized)designed to interrupt these critical T cell activation pathways.This review will detail the development of therapeutic proteins that block the costimulation pathway,with an empha-sis on the therapeutic drug belatacept,which has demonstrated significant promise when utilized in organ transplant recipients.2.Costimulation and T cellsT cells play a central role in the initiation and regulation of the adaptive immune response to antigen,whether foreign or native.Naïve T cells require 2signals for activation [1].The first,Sig-nal 1,is an antigen-specific signal provided by the T cell receptor∗Corresponding author at:University of California,San Francisco,Kidney Trans-plant Service,505Parnassus Avenue,Room 884M,San Francisco,CA 94143-0780,United States.Tel.:+14153531322;fax:+14153538974.E-mail addresses:david.wojciechowski@ (D.Wojciechowski),Flavio.Vincenti@ ,vincentif@ ,david.wojciechowski@ (F.Vincenti).interacting with the MHC and antigenic peptide complex on the antigen-presenting cell (APC).Costimulation,which is Signal 2,is provided by the interactions between specific receptors on the T cell and their ligands on the APC.Following these two signals several pathways are acti-vated.They include the calcium–calcineurin pathway,the RAS-mitogen-activated protein kinase pathway,and the nuclear factor-␬B(NF ␬B)pathway with subsequent activation of several transcription factors for numerous effectors including the cytokine interleukin-2(IL-2).IL-2activates the downstream proliferation through the ␥-chain/JAK3pathway also referred to as Signal 3.The sum total of these events leads to T cell proliferation with the gen-eration of an effector CD4+T cell pool (T-helper)and the clonal expansion of activated CD8+or cytotoxic T cells [2].If the T cell does not receive a costimulation signal because of pathway blockade,it becomes anergic and undergoes apoptosis.Several costimulatory pathways are involved in T cell regulation which can either up regulate or down regulate T cell activa-tion [3].One of the most critical and best characterized of these costimulatory interactions is the one between CD28/CTLA4and CD80and CD86in the B7family (Fig.1)[3].Another important receptor-ligand pair for T cell activation is CD40and CD154.The CD40–CD154pathway was initially described as critical for B cell activation and differentiation,but it was subsequently reported to contribute to T cell activation by upregulating the B7family ligands CD80and CD86on APCs.CD154is expressed on vascular endothe-lial cells,smooth muscle cells,and macrophages,suggesting an expanded role for the CD40–CD154pathway during immunity [4].A nonhuman primate (NHP)model of kidney transplant treated with an anti-CD154mAb demonstrated prolonged allograft sur-vival [5].However,the initial trial in humans utilizing Hu5C8,1044-5323/$–see front matter © 2011 Elsevier Ltd. All rights reserved.doi:10.1016/j.smim.2011.07.002158 D.Wojciechowski,F.Vincenti /Seminars in Immunology 23 (2011) 157–164Fig.1.Costimulation pathways identified in the B7and TNF family.Numerous costimulation pathways have been identified;the best characterized of these are in the B7and TNF family.a humanized anti-CD154,demonstrated unacceptable thrombotic events leading to the discontinuation of the study as well as ces-sation of the clinical development of several anti-CD154mAbs in transplantation and autoimmune disease [6].Interest has now shifted to targeting CD40,which unlike CD154is not expressed on platelets and should not be associated with thrombotic events.In the interim blockade of the CD28costimulatory pathway has emerged as a leading therapeutic strategy for selectively modulat-ing full T cell activation.3.The CD28pathwayCD28signals promote T cell differentiation into TH1pheno-type cells and enhance both the production of antibodies by B cells and the proliferation of previously activated T cells [7].The two ligands for CD28,CD80and CD86,have distinct but overlapping functions.CD86is constitutively expressed and is rapidly upreg-ulated on APCs coincident with Signal 1.CD80expression levels are very low on the resting cell and higher expression is usually induced after more prolonged T cell stimulation [8,9].These dif-ferences suggest distinct roles for the two ligands.CD86may be important in mediating initial T cell activation and CD80may play a greater part in perpetuating the immune response.After T cell activation,a second receptor,CTLA4,is upregulated [7].CTLA4is structurally homologous to CD28but has higher avidity to the CD80and CD86ligands.However,unlike CD28,CTLA4is a negative reg-ulator of T cells.Consequently,the upregulated CTLA4successfully competes for the CD80/CD86ligands and turns off T cell activation.The receptor fusion protein CTLA4-Ig (consisting of the extracel-lular binding domain of CTLA4linked to a modified Fc domain of human IgG1)has been developed to inhibit costimulation and T cell activation by binding to CD80/CD86.A biologic that selectively targets CD28is a more desirable therapeutic target than CTLA4-Ig because it would allow the T cell to receive inhibitory signals through CTLA4when bound to CD80/CD86.It has been shown that direct blockade of CD28could in fact inhibit a mixed lymphocyte culture in vitro [10].A rat model of transplantation showed that anti-CD28therapy induced tolerance.However,several mAbs to CD28have been found to activate rather than just block the receptor [11].A superagonist anti-CD28mAb,TGN1412,which in preclinical studies produced marked expan-sion of T-regulatory cells but not inflammatory T cells,produced acytokine storm and multiorgan failure in 6volunteers [12].So while targeting of anti-CD28remains a focus for clinical development,the transition to the clinic will be slow and challenging.4.Clinical development of costimulation blockade:abatacept to belataceptThe dimeric fusion protein abatacept (CTLA4-Ig)was developed to block the interactions of CD28with CD80/CD86.Abatacept binds more avidly than CD28to CD80/CD86,as does native CTLA4.The addition of the IgG1domain solubilizes the CTLA4domain,creat-ing a soluble receptor for CD80/CD86[13].Numerous studies have shown that abatacept acts to inhibit immune responses both in vitro and in vivo .The in vitro binding of CTLA4-Ig to CD80and CD86down-regulates T cell proliferation and inhibits humoral immune responses [13–15].Therapeutic inhibition of the CD28pathway by using CTLA4-Ig was first established in the transplantation setting;however,early experiments were mixed.Prolongation of graft survival or induction of tolerance using co-stimulation blockade in rodent transplantation experiments could not be reproduced in non-human primates [16,17].CTLA4-Ig did not achieve as good affinity to CD86as compared with CD80and was the likely cause of failure in a more stringent animal model [16].The lower avidity of CTLA4-Ig to CD86compared with CD80resulted in a rational design concept to improve the binding characteristics of CTLA4-Ig to CD86specifically,due to the pre-sumed importance of CD86in the initiation of the alloimmune reaction [16].A mutagenesis and screening strategy was used to develop belatacept (Fig.2).Codon-based mutagenesis of CTLA4-Ig was performed on 24individual amino acids within the com-plementary determining region (CDR)and CDR3analogous loops (i.e.,not antibody-derived CDRs,but the analogous regions on the CTLA4portion of abatacept),and the region C-terminal to the CDR3-like loop,because these regions were previously iden-tified as critical for high-avidity receptor binding.A total of 2300mutants were screened by using a binding assay,and a variant with a slower off rate was identified.This variant,which had a leucine to glutamate switch at position 104,was then used in a second round of mutagenesis and screening.The second round yielded belatacept with a second mutation (alanine to tyrosine)at position 29.D.Wojciechowski,F.Vincenti/Seminars in Immunology23 (2011) 157–164159Fig.2.Rational design of LEA29Y.Mutagenesis procedure employed to modify CTLA4-Ig to develop LEA29Y for use in organ transplantation.Belatacept,a second-generation CTLA4-Ig,binds CD802-fold better and CD864-fold better than CTLA4-Ig and has a10-fold more potent inhibition of T-cell activation in vitro versus CTLA4-Ig[18]. Abatacept,the parent molecule,was shown to be effective in the treatment of autoimmune diseases and has been approved by the FDA for the treatment of moderate to severe rheumatoid arthritis [19,20].5.Kidney transplantation:an overviewRecipients of kidney transplants generally require lifelong immunosuppression to maintain a state of low immunoresponsive-ness to the allograft.Recent advances in therapies and treatment strategies that have resulted in a decrease in acute rejection have not been matched by similar improvements in long-term patient or graft survival[21].New small molecules and biologics were successfully intro-duced in the clinic during the1990s.Consequently,rejection rates which were in the40%range fell to10–15%[21].Since acute rejec-tion has been shown to be a risk for graft loss this decrease in acute rejection was anticipated to lead to a significant improvement in long-term graft survival.Thus far outcome data from the Scien-tific Registry of Transplant Recipients have not demonstrated an increase in long-term graft survival in the recipients of primary kid-ney transplants[21].The one year renal allograft survival rate is95% and89%for recipients of living and deceased donor kidneys,respec-tively[22].At5years post-transplantation renal allograft survival falls to a disappointing80%and67%for living and deceased donor kidney recipients,respectively[22].Importantly,the long-term survival of both the allografts and the recipients are affected by non-immune toxicities caused, in part,by the immunosuppressive therapies used to prevent graft rejections.Chronic allograft nephropathy(CAN),character-ized by interstitialfibrosis and tubular atrophy(IF/TA)on biopsy, remains the most common cause of late allograft loss[23]while cardiovascular disease remains the leading cause of death post-transplantation[24].Immunosuppression protocols which include a calcineurin inhibitor[11],currently the cornerstone therapy for chronic maintenance of immunosuppression,contribute to the pro-gression of CAN through several Is are clearly nephrotoxic and can cause IF/TA[25].However,CNIs also may not adequately suppress the chronic humoral response which has recently been reported to be an important cause of late allograft failure[26].Additionally,CNIs exert negative effects on the car-diovascular and metabolic risk profile by worsening hypertension, diabetes and dyslipidemia[27–29].6.Immunosuppression strategiesDuring the induction phase(early post-transplant period),when immune responses to the allograft are at their highest,regimens use more potent immunosuppression.Induction agents,which may be depleting or non-depleting,include protein therapeutics (either polyclonal antibodies or murine,chimeric,or humanized mAbs),which act mainly by either depleting lymphocytes or sup-pressing proliferation and blunting the effects of T cell activation. Protein therapeutics target specific cell surface glycoproteins or membrane-bound receptors.Depleting biologics such as polyclonal antithymocyte globu-lin are used to reduce early rejection by eliminating T cells at the time of antigen presentation and thus preventing activation of alloreactive T cell clones.They can be associated with cytokine release as well as the immunologic consequences of prolonged T cell depletion,such as infectious complications and posttransplant lymphoproliferative disease(PTLD).Daclizumab and basiliximab are nondepleting mAbs.These agents specifically target the␣-chain of the IL-2receptor(IL-2R), inhibiting the signal for proliferation resulting from initial T cell activation.Unlike the T cell depleting agents,these compounds are associated with a low incidence of toxicities and side effects[30]. However,despite their specificity,the anti-IL-2R mAbs have lim-ited efficacy because of the redundancy of the cytokine receptor system and are generally inappropriate for long-term use[31].After the induction phase,transplant patients transition to what is known as the maintenance phase of immunosuppres-sion[32].Effective maintenance immunosuppression is the key to preventing rejection throughout the life of the graft.Mod-ern maintenance therapies include a CNI backbone along with antiproliferative agents(mycophenolate mofetil[MMF]/enteric-coated mycophenolate acid or mTOR inhibitors such as sirolimus and everolimus)and I regimens require frequent ther-apeutic drug monitoring and dosage adjustment to remain within160 D.Wojciechowski,F.Vincenti /Seminars in Immunology 23 (2011) 157–164Fig.3.The treatment regimen for subjects enrolled in BENEFIT and BENEFIT-EXT.The belatacept regimen differed for belatacept MI compared to belatacept LI.Belatacept MI had more frequent dosing to Day 84and higher dosing (10mg/kg)to 6months.Belatacept LI had less frequent dosing to Day 84,and the maintenance phase (5mg/kg)started at approximately 4months,versus 6months for the MI regimen (MI:more intensive;LI:less intensive).the narrow therapeutic windows and are associated with multiple toxicities [32].These limitations have motivated a search for novel agents with greater specificity and an improved side effect profile.The focus of much clinical research over the last decade has turned toward costimulation blockade.The hope of costimulation blockade is to provide durable selective and specific immunosuppression without the renal and non-renal toxicities seen with current maintenance agents with the goal of ultimately improving long-term patient and allograft survival.7.Costimulation blockade in clinical transplantationThe first clinical trial with belatacept in renal transplantation was designed with a regimen that provided efficacy in a non-human primate model [16].In these experiments,a CNI-free regimen with belatacept and a combination of an anti-interleukin-2receptor antibody and maintenance therapy with MMF and steroids resulted in marked prolongation of the survival of renal allografts [16].These findings were used to design a phase II multicenter clin-ical trial comparing the safety and efficacy of 2dosing regimens (more or less intensive)of belatacept compared to cyclosporine [33].In this trial belatacept performed with equivalent efficacy to cyclosporine and was associated with better renal function and histology.Lipid levels and blood-pressure values were similar or slightly lower in the belatacept groups.Given the promising phase II trial results two phase III trials were undertaken.The 12month results of these phase III trials evaluating the effectiveness and side-effect profile of belatacept as part of a CNI free regimen have recently been published.Both trials have provided important data that will be used to help develop the appropriate role for belatacept in the clinical arena as part of a CNI free immunosuppression regimen.The first phase III trial,Belatacept Evaluation of Nephroprotec-tion and Efficacy as First-line Immunosuppression Trial (BENEFIT)[34]is a 3-year,randomized,active-controlled,parallel-group mul-ticenter phase III trial conducted at 100centers worldwide.Adult patients receiving a living donor or standard criteria deceased donor kidney were eligible.First-time patients with a panel reac-tive antibody ≥50%,re-transplants with a panel reactive antibody≥30%,recipients of prior or concurrent non-renal solid organ trans-plants,and recipients of extended criteria donor kidneys were excluded.Patients were randomized to one of three regimens for maintenance immunosuppression:a more intensive regimen (MI)of belatacept,a less intensive regimen (LI)of belatacept or cyclosporine.The treatment regimen for both phase III trials can be seen in Fig.3.Patients in all treatment arms received basiliximab induction and were maintained on mycophenolate mofetil and cor-ticosteroids.Lymphocyte depleting therapy was permitted only in the cyclosporine group for delayed graft function.The trial was designed with three co-primary outcomes:com-posite patient and graft survival;composite renal impairment endpoint;incidence of acute rejection.Protocol biopsies were performed at implantation and at week 52.Secondary outcomes included:mean measured glomerular filtration rate,mean cal-culated glomerular filtration rate using the modification of diet in renal disease (MDRD)equation [35],and the prevalence of chronic allograft nephropathy on protocol biopsies.Cardiovascu-lar and metabolic parameters measured at month 12included:mean systolic and diastolic blood pressure,the incidence of new-onset diabetes after transplant (NODAT),and mean change in serum lipids.A total of 527patients were randomized to three treatment groups,transplanted and completed the initial 12month treatment phase.Recipient demographics and baseline characteristics as well as donor characteristics were similar between the three groups.Both belatacept regimens were non-inferior to cyclosporine on the primary endpoint of patient and graft survival.At one year patients enrolled in the MI,LI and cyclosporine treatment groups had a 95%,97%,and 93%patient and graft survival,respectively.The mean measured GFR was 65mL/min/1.73m 2,63.4mL/min/1.73m 2,and 50.4mL/min/1.73m 2in the MI,LI,and cyclosporine treated patients,respectively (p <0.0001for both MI and LI versus cyclosporine).The prevalence of chronic allograft nephropathy on protocol biopsies was lower in belatacept treated patients compared to cyclosporine treated patients (18%MI;24%LI;32%cyclosporine).There was a higher incidence of acute rejection at 12months in the belatacept treated groups compared with the cyclosporine treated group (22%MI;17%LI;7%cyclosporine).The incidence of acute rejection met the non-inferiority cutoff for LI versus cyclosporine groups but not for MI versus cyclosporine groups.D.Wojciechowski,F.Vincenti/Seminars in Immunology23 (2011) 157–164161Almost100%of rejections occurred within thefirst6months post-transplantation.Belatacept treated patients had more type IIa and IIb rejections compared to cyclosporine treated patients but were not associated with an increase in donor specific antibody.The mean measured GFR at month12was higher in belatacept treated patients with acute rejection compared to cyclosporine treated patients without acute rejection.Belatacept treated patients had a significantly lower mean blood pressure(MI133/79mmHg;LI131/79mmHg)compared to cyclosporine treated patients(139/82mmHg)(p≤0.0273for MI or LI versus cyclosporine in all comparisons).The mean change in non-HDL cholesterol from baseline was significantly different in belatacept treated patients(MI8.1mg/dL;LI8.0mg/dL)compared to cyclosporine treated patients(18.3mg/dL)(p=0.0115for MI and p=0.0104for LI versus cyclosporine).The incidence of NODAT was not significantly different between the three groups:MI7%,LI4%, and cyclosporine10%(p=NS for MI or LI versus cyclosporine).By12months1,2,and1patient in the MI,LI,and cyclosporine groups developed PTLD,respectively.Additionally,after month 12two additional patients in the MI group developed cen-tral nervous system(CNS)PTLD.Four of the six patients who developed PTLD had known risk factors.One patient had Epstein-Barr virus(EBV)negative serology pre-transplant,one patient received lymphocyte depleting therapy as treatment for an acute rejection and two patients had both EBV negative serology and received lymphocyte depleting stly,two patients with EBV negative serology received transplants from EBV seropositive donors.The second phase III trial,Belatacept Evaluation of Nephro-protection and Efficacy as First-line Immunosuppression Trial-EXTended criteria donors(BENEFIT-EXT)[36]is a3-year,random-ized,multicenter phase III trial conducted at79centers worldwide in patients who received a kidney transplant from an extended cri-teria donor defined as:≥60years old;≥50years old and at least two other risk factors(cerebrovascular accident,hypertension,serum creatinine>1.5mg/dL);anticipated cold ischemia time of≥24h; or donation after cardiac death.Patients were treated with basil-iximab induction,MMF and steroids.Patients were randomized to receive either belatacept MI,belatacept LI or cyclosporine.Lympho-cyte depleting therapy was allowed for anticipated delayed graft function only in cyclosporine treated patients and for the treatment of rejection in all patient groups.The primary outcomes were the composite endpoint of patient and graft survival as well as the composite endpoint of renal impairment at12months.Secondary outcomes included measured GFR,calculated GFR using the MDRD equation,the prevalence of biopsy-proven CAN and the incidence and severity of biopsy proven acute rejection.Protocol biopsies were performed at implantation and at week52.Cardiovascular and metabolic endpoints included mean systolic and diastolic blood pressure,the incidence of NODAT and the mean change in serum lipids.A total of543patients were randomized and transplanted(n=184MI;n=175LI;n=184 cyclosporine).There were no differences in baseline characteristics between the three groups.Both belatacept regimens were non-inferior to cyclosporine on the primary endpoint of patient and graft survival.Graft loss or death occurred in14%,11%and15%of the patients treated with MI,LI,and cyclosporine,respectively.The prevalence of biopsy proven CAN was similar between the three groups(MI 45%;LI46%;cyclosporine52%).The mean measured GFR at 12months was52.1mL/min/1.73m2,49.5mL/min/1.73m2,and 45.2mL/min/1.73m2for the MI,LI and cyclosporine treated groups, respectively.The difference in measured GFR was significantly better in the MI treated patients versus the cyclosporine treated patients(p=0.0083)but was not significantly different for the LI group compared to cyclosporine(p=0.1039).There was not a significant difference in the incidence of acute rejection between the three groups(MI17.9%;LI17.7%; cyclosporine14.1%).However,more type IIB rejections occurred in belatacept treated patients compared to cyclosporine treated patients.Mean systolic and diastolic blood pressure was lower for both belatacept groups compared to the cyclosporine treated group (MI141/78mmHg;LI141/78mmHg;cyclosporine150/82mmHg). The incidence of NODAT was significantly lower in the MI group compared to the cyclosporine group(MI2%versus cyclosporine 9%;p=0.0308),however there was not a significant difference in NODAT in the LI group compared to cyclosporine(LI5%;p=0.2946). The mean change in non-HDL cholesterol from baseline was sig-nificantly different in the MI(12.6mg/dL)and LI(11.2mg/dL) groups compared to cyclosporine(29.3mg/dL)(p=0.0016MI versus cyclosporine;p=0.0006LI versus cyclosporine).One patient(0.5%)in the MI group and2patients(1%)in the LI group developed PTLD during the12month follow-up period.One additional patient in each of the belatacept groups developed PTLD after month12.No patients in the cyclosporine group developed PTLD.Three of thefive PTLD patients had negative EBV serology pre-transplant.None of the patients who developed PTLD were exposed to lymphocyte depleting therapy.8.Utilizing belatacept to improve long-term outcomesThe substitution of belatacept for a CNI resulted in a higher GFR, lower blood pressure and improved lipid profile.When both BENE-FIT and BENEFIT-EXT are analyzed together there is also a decreased incidence of NODAT in patients treated with belatacept versus a CNI (Fig.4).When one considers the impact of CAN on allograft loss and the effect of hypertension,diabetes and hyperlipidemia on prema-ture patient death with a functioning allograft due to cardiovascular disease it becomes clear that an immunosuppression regimen that minimizes these effects has the potential to prolong allograft and patient survival.The higher rate of CAN in cyclosporine treated patients in BENEFIT/BENEFIT-EXT as well as the significantly better measured GFR by a mean of at least12.9mL/min/1.73m2in both belatacept groups compared to the cyclosporine group in BENEFIT suggests a trend toward improved long-term renal function.The two year BENEFIT data,presented at the2010American Transplant Congress (ATC),demonstrates that this improvement in GFR persisted with a15–17mL/min/1.73m2higher mean measured GFR for the belat-acept groups compared to the cyclosporine group(p<0.0001MI or LI versus cyclosporine)[37].In BENEFIT-EXT the two year mean measured GFR was52mL/min/1.73m2,50mL/min/1.73m2,and 45mL/min/1.73m2for the MI,LI and cyclosporine groups,respec-tively(p=0.28MI vs.cyclosporine;p=0.108LI vs cyclosporine) [38].Typically GFR declines by approximately1–2mL/min/1.73m2 annually in renal allografts and ultimately leads to graft failure [39].The BENEFIT/BENEFIT-EXTfindings indicate that with a higher intercept GFR and a promising GFR stage at2years,if sustained, would result in better preservation of renal function and ultimately in prolonged graft survival.Blood pressure has emerged as an indicator of allograft and patient survival.Opelz et al.have demonstrated that for every 10mm Hg increase in systolic blood pressure above140mm Hg at one year post transplant there is an increased risk of graft failure during the following six years[40].Adjusting for renal func-tion there is a15%reduction in the rate of allograft survival for each10mm Hg increase in systolic blood pressure[41].For every 10mm Hg increase in blood pressure there is not only an increase risk of graft failure but also an increase risk of death[42].In BENE-FIT there was a significant improvement of6–9mm Hg in the mean162 D.Wojciechowski,F.Vincenti/Seminars in Immunology23 (2011) 157–164BENEFIT and BENEFIT-EXT: New-OnsetBENEFIT BENEFIT-EXT Pooled AnalysisDiabetes at Month 12Bela MIBela LICsA4682PercentofPatients(95%CI)inNtreatment with antiNODAT was de fined as-diabetic medication for a duration of ≥30 days or at least two fastingplasma glucose (FPG) tests indicating FPG ≥126 mg/dL (7.0 mmol/L); NOD AT was assessed only after Week 4;P=0.03 vs cyclosporine (CsA); *P<0.02 vs cyclosporineanalysis156168162133136118289304280Fig.4.BENEFIT,BENEFIT-EXT and pooled analysis of new onset diabetes at Month12.In the pooled data analysis there is a significantly lower rate of new onset diabetes at 12months in belatacept treated patients compared to cyclosporine treated patients.systolic blood pressure of belatacept treated patients compared tocyclosporine treated patients.In BENEFIT-EXT the mean systolicblood pressure was150mm Hg in cyclosporine treated patients,significantly higher than belatacept treated patients.The improve-ment in blood pressure control in belatacept treated patientsoccurred despite the fact that more patients in the two cyclosporinetreatment groups required≥3anti-hypertensive medications com-pared to the belatacept treated patients.Improved blood pressurecontrol in belatacept treated patients will likely translate intoimproved long-term graft and patient survival.Non-HDL cholesterol was significantly better in patients treatedwith belatacept compared to those who received cyclosporine.Dyslipidemia following renal transplant has been associated withgreater patient death post-transplant but not graft loss;however,a lower incidence of hyperlipidemia benefits mortality due to car-diovascular events[43].Improvements in this risk profile will likelytranslate into a lower rate of cardiac events and reduce the cardio-vascular mortality seen the renal transplant population.9.Costimulation and acute rejectionIn various studies certain features of acute rejection episodeshave been found to be poor prognostic factors for graft outcomes.These features include:high Banff grade,association with thedevelopment of donor specific antibodies(DSA),a late rejection,poor renal function after rejection,and recurrent rejection episodes[44–48].Belatacept was associated with more rejections that werehistologically more severe(higher Banff grade)than cyclosporinetreated patients,but lacked the other characteristics that are usu-ally associated with poor outcomes.The question remains:Why do patients on belatacept reject?Several factors are involved in analyzing this question.Some studieshave suggested that memory cells may be resistant to costimu-lation blockade[49,50].As previously discussed,T cell activationmay also occur through other costimulation pathways[51].Addi-tionally,negative signaling occurs through CTLA-4engagement,which is blocked with stly,there may be the deple-tion of important subsets of T-regulatory cells.Data has suggestedthat patients treated chronically with belatacept maintained nor-mal T regulatory cells;it remains unclear if a subset of adaptive Tregulatory cells may have been adversely affected[52].The major safety concern that has arisen is the development ofPTLD in belatacept treated patients.This risk can be minimized byusing the LI regimen and selecting for therapy only patients that areEBV-positive and avoiding the use of lymphocyte depleting agentsto treat acute rejection.10.Belatacept as part of steroid avoidancePatients and clinicians have advocated for the development ofsteroid sparing immunosuppression regimens due to the myriadof side effects seen with chronic corticosteroid use.Data from abelatacept-based corticosteroid avoiding regimen in de novo kid-ney transplant recipients has recently been published[53].In thisrandomized,phase II trial89patients were enrolled and receiveda kidney transplant.Patients received one of the following treat-ment regimens:belatacept with MMF,belatacept with sirolimus,or tacrolimus with MMF.Thymoglobulin was administered to alltreatment arms.Patients received an initial4day pulse of corti-costeroids which could be extended until day10if thymoglobulininfusions continued(then corticosteroid free).The primary effi-cacy endpoint was the incidence of acute rejection by month6.Secondary endpoints included the incidence and severity of acuterejection,patient and graft survival,and calculated GFR.Cardiovas-cular and metabolic parameters were also evaluated.Four patients in the belatacept-MMF group(12%),1in thebelatacept-sirolimus group(4%)and1in the tacrolimus-MMFgroup(3%)experienced acute rejection by month 6.By12months91%of the belatacept-MMF group,92%of the belatacept-sirolimus group,and100%of the tacrolimus-MMF group werealive with functioning grafts.At month12the mean cGFR was8–10mL/min/1.73m2higher in the belatacept groups than in thetacrolimus-MMF group.No significant changes in blood pressureoccurred.No clinically relevant differences in lipids or antihyper-lipidemic medication use were noted between the groups.Newonset diabetes occurred in a total of3patients(n=2belatacept-sirolimus and n=1tacrolimus-MMF).There were no cases of PTLDreported with either belatacept regimen.Overall the use of belatacept as part of a steroid sparing regi-men was well tolerated and was associated with a rejection ratecomparable or better to other studies utilizing a CNI or steroidfree regimen.The use of belatacept compared to tacrolimus was。