Belatacept: An Opportunity to Personalize Immunosuppression? Andrew Adams MD/PhD Emory Transplant Center

Disclosure Research Funding from BMS.

Learning Objectives -Define belatacept-resistant rejection - Identify pathways and cell subsets involved in belatacept-resistant rejection -Propose possible therapeutic interventions to prevent belatacept-resistant rejection

Current Immunosuppressive Strategies Basiliximab Thymoglobulin Campath Calcineurin Inhibitor Mycophenolate +/- Steroids Time

CD154 CD40 T Cell Cytokine IL-2, IL-15, IL-7 APC Steroids Signal 3 CNI X γ chain Signal 1 MMF TCR Signal 2 (+) CD28 p/mhc CD80/86

Targeted immunosuppression Neurotoxicity Hypertension Hyperlipidemia Near Ubiquitous Target Distribution CNI Cellcept Steroids Rapamycin Diabetes Immunosuppression Nephrotoxicity

Targeted immunosuppression Specifically Target T cell Immunosuppression

CD154 CD40 T Cell Cytokine IL-2, IL-15, IL-7 APC Signal 3 γ chain Signal 1 Signal 2 (+) (+) CD28 TCR p/mhc CD80/86

CTLA4 mutated human IgG1 CTLA-4 T cell IgG1 CTLA4-Ig Dimer Retains Fc binding Non-complement fixing Serum T1/2 ~2 wks Immunologic tool and therapeutic fusion protein

Belatacept (LEA29Y): Engineered for more potent co-stimulation blockade Extracellular portion of CTLA4 (CD152) A29 L104 Second generation CTLA4 derivative human IgG1 Fc fusion protein aa substitutions at position 29 and 106 2-fold higher avidity to B7-1 4-fold higher avidity to B7-2 than parent CTLA4-Ig molecule 10-fold higher biologic potency in vitro Larsen et al AJT 2005

7yr Patient/Graft Survival

BENEFIT-Trial: Improved Cardiovascular, Metabolic and Renal health NEJM 2016

Belatacept & Rejection Acute rejection rates

FDA Approval 2011

Current Immunosuppressive Strategies Basiliximab Belatacept X Calcineurin Inhibitor Mycophenolate Steroids Time

% rejection

Update Belatacept Protocol 0 4 8 12 16 Weeks Belatacept 10 mg/kg IV 5 mg/kg Q4wks IV Tacrolimus 5-8 ng/ml 3-5 ng/ml 3 mo 6 mo 9 mo 12 mo

Percent 100 Freedom from Rejection *p=0.91 80 60 40 20 0 6 12 18 24 0 180 360 540 720 Months

Percent 100 Patient Survival *p=0.86 80 60 40 Bela 2.1 (N=169) 20 0 6 12 18 24 0 180 360 540 720 Months

Percent 100 Graft Survival *p=0.63 80 60 40 Bela 2.1 (N=169) 20 0 6 12 18 24 0 180 360 540 720 Months

Renal Function On Treatment Analysis- Combined

Percent 100 Freedom From DSA *p=0.002 95 90 TAC: 85 Bela 1.0-2.1 80 75 0 365 1 730 2 1095 2 4 36 Months

Belatacept and Rejection Better long-term renal function post-transplant Higher acute rejection rate and more severe pathology Why does belatacept-resistant rejection occur? Acute rejection rates

Costimulation Blockade Resistant Rejection alternative costimulatory molecules provide escape signals certain T cell subsets are less dependent on traditional costimulatory signals Memory T cells acquired directly vs via heterologous response? Other cell subsets The Emory Transplant Center

Understanding Mechanisms of Costimulation Independence Belatacept + MMF + Steroids d0 Belatacept Resistance d140 Belatacept Susceptibility d306 Rejection Rejection

% Survival Understanding Mechanisms of Costimulation Independence Pre-Transplant Immune Phenotype? Belatacept + MMF + Steroids d0 Belatacept Resistance d140 Belatacept Susceptibility d306 Rejection Rejection Days Post-Transplant

% Survival Understanding Mechanisms of Costimulation Independence Days Post-Transplant

CD127 MFI 800 600 CD28hi TEMRA P<.0001 **** CD28hi TEMRA Cells Express High Levels of IL-7Rα IL7Rα CD16 IL6st CXCR3 CCR6 IL6Rα LFA-1 VLA-4 400 200 CD154 ICOS CD27 CD2 0 CD28hi TEMRA CD28null TEMRA TIM3 LAG3 PD1 Ox40 CD28hi TEMRA

The Role of IL-7Rα Signaling in Transplantation Lymphopoiesis γc IL-7Rα TCR CD28 Homeostatic Proliferation or Depletion TNF Signal 3 Cytokine? IFN GzmB Target Cell Death

CD28 %IFNg+TNF+ CD8 T cells Addition of IL-7 Augments Effector Function IL-7 Augments Effector Function 2.78 6.98 8 6 *** ** 4 2 1.61 13.9 IFNγ 0 Unstim P+I P+I+IL7 TNF IFN GzmB Target Cell Death

Mouse Allogeneic Skin Graft Experimental Design Balb/c Ear (R) Tail (L) C57BL/6 Donor Recipient Treatment administered via IP injection at day 0, 2, 4 and 6 anti-il7rα + Costimulation Blockade Costimulation Blockade Assess Skin Graft Survival

Percent survival Blocking IL-7Rα Prevents Costimulation Independent Rejection CoB vs. CoB + anti-cd127 100 P<.0001 **** 50 CoB+aIL7Ra CoB No Rx 0 0 50 Days Post-Transplant

Role of IL-7Rα in Costimulation Independent Rejection C57BL/6 Ova Expressing Graft CTLA4-Ig 24 hours CTLA4-Ig+ Anti-IL7Rα Adoptive Transfer of OT1 & OTII CD4 and CD8 Graft specific T cells Untreated Thy1.1+ Tracking Graft Specific T cells

% survival Addition of Anti-IL-7Rα Synergistically Prevents Costimulation Independent Rejection CTLA4-Ig vs. CTLA4-Ig+anti-CD127 100 P<.0001 **** 50 No Rx CTLA4-Ig CTLA4-Ig +ail7ra 0 0 10 20 30 40 50 60 70 80 90 Days Post-Transplant

Abs. # of OTII Cells Abs. # of OTI Cells Anti-IL-7Rα + CoB Results in Decreased Expansion of Graft Reactive T cells CD4 T Cells CD8 T cells 10 6 * * 10 6 * ** ** 10 5 10 5 10 4 10 4 10 3 10 3 10 2 No Rx ail7rα CTLA4-Ig+aIL7Rα 10 2 No Rx ail7rα CTLA4-Ig CTLA4-Ig+aIL7Rα

TNF Anti-IL-7Rα+CoB Decreased Effector Function of Graft Reactive T cells CD8 T cell Effector Function % IFNg+TNF+ of OT1 20 15 10 5 0 No Rx ail7rα No Rx *** CTLA4-Ig CTLA4-Ig+aIL7Rα ail7rα CTLA4-Ig TNF IFN GzmB Target Cell Death CTLA4-Ig+aIL7Rα IFNγ

CD4 + CD25 + CD127 lo/- cells express highest level of Foxp3 Strongest correlation with functional activity Used as phenotype in clinical trials

Anti-IL-7Rα + Costimulation Blockade Impact on Graft Specific CD4 T cells Treg Phenotype IL7Rα (CD127) Negative, CD25 (IL2Rα) Positive Immune Regulation IL-10 TGFβ CTLA4 Graft Protection

FoxP3 %CD25+FoxP3+ of OTII Anti-IL-7Rα + Costimulation Blockade increased Frequency of Tregs 20 15 * Immune Regulation 10 IL-10 5 0 No Rx No Rx ail7rα CTLA4-Ig CTLA4-Ig+aIL7Rα ail7rα CTLA4-Ig TGFβ CTLA4 Graft Protection CTLA4-Ig+aIL7Rα CD25

%CD25+FoxP3+ of OTII FoxP3 CD25 Anti-IL-7Rα + CoB increased Frequency of Tregs Sustained at Day 20 25 20 ** Immune Regulation 15 10 5 IL-10 CTLA4 Graft Protection 0 No Rx CTLA4-Ig CTLA4-Ig+aIL7Rα No Rx CTLA4-Ig TGFβ CTLA4-Ig+aIL7Rα

% survival %CD25+FoxP3+ of OTII Anti-IL-7Rα + CoB increased Frequency of Tregs Sustained at Day 30 Immune Regulation IL-10 30 20 * ** ** No Rx CTLA4-Ig CTLA4-Ig+aIL7Ra TGFβ CTLA4 Graft Protection 100 10 CTLA4-Ig vs. 0CTLA4-Ig+anti-CD127 0 10 20 30 40 Days Post-Transplant P<.0001 **** 50 No Rx CTLA4-Ig CTLA4-Ig +ail7ra 0 0 10 20 30 40 50 60 70 80 90 Days Post-Transplant

Costimulation Independent CD8 T cells Decrease CD28 Expression, Increase IL-7Rα CD28 CD8 OTI Thy1.1 No Rx % of Thy1.1 CD8 (OT1) 80 60 40 20 0 No Rx CTLA4-Ig CTLA-4 Ig CD127

TIGIT % OTI Cells Anti-IL-7Rα + CoB Induces an Exhaustion-like Phenotype in Graft Reactive T cells CD8 OTI Thy1.1 PD1+TIGIT+ OTI T cells (Day 20) No Rx CTLA-4 Ig CTLA4-Ig+aIL7Rα 60 **** 40 20 PD1 0 No Rx CTLA4-Ig TLA4-Ig+aIL7Rα

% Graft Survival Anti-IL7Ra+CoB Mediated Graft Survival is PD1-Dependent Dependent Survival Benefit 100 CTLA4-Ig CTLA4-Ig+aIL7Ra CTLA4-Ig+aIL7Ra+aPD1 P<.0001 **** 50 0 0 30 60 90 Days Post-Transplant

CTLA-4 Next Generation Costimulation Blockade T cell function is tightly regulated by a fine balance of costimulatory and coinhibitory signals CD28

Lulizumab- a domain antibody V PEG traditional antibody (IgG) - monovalent, pegylated - non-activating - no evidence of cytokine release syndrome

Selective blockade of CD28 is superior to CTLA4-Ig Liu et al., 2014 J Exp Med

2B4 Upregulation after CD28 dab The Emory Transplant Center

Transplant CD28 dab Renal allograft Protocol d 0 4 7 14 28 35 42 140 ultrasound biopsy CD28 dab mg/kg 3 3 3 3 3 3 3 Anti-IL-2R Solumedrol 20mg/day 2mg/day 1mg/day MMF 15 mg bid 15 mg qd No treatment of acute rejection episodes

Belatacept vs CD28-specific Rx 100 Belatacept 100 anti-cd28 dab 80 80 60 60 40 40 20 20 0 0 50 100 150 0 0 50 100 150 The Emory Transplant Center

Next Generation Costimulation Blockade CD28 selective therapy Primate studies supportive, superior to belatacept Mechanistic studies,? Increase in Treg activity Appears safe and efficacious No overt safety signal in clinical trial for SLE Next Step- Clinical Trial(s) in Renal Transplant recipients The Emory Transplant Center

Proposed Immunosuppression Protocol Thymoglobulin (total dose 6mg/kg) biopsy 3 mo 6 mo 9 mo 12 mo 18 mo MMF steroid 1000mg po BID 5mg CD28 dab (Lulizumab) dosing 12.5 mg weekly 12.5mg q 2wk vs Tacrolimus 8-12ng/ml 5-8ng/ml 3-5ng/ml

Conclusions Belatacept holds promise for improved outcomes Pre-transplant phenotyping may be able to identify patients at risk of rejection Adjunct therapies may improve outcomes blockade of IL-7 pathway, IL-15 CD28 selective therapy