Results of a Phase 1 Trial of Treg Adoptive Cell Transfer (TRACT) in De Novo Living Donor Kidney Transplant Recipients A Skaro , A LeFever, J Mathew, L Gallon, J Hie, C Hansen, D Stare, G Johnson, J Leventhal June 13, 2016 ATC Boston MA
As we understand immune regulation, immunosuppression is evolving Drug-free donor- Corticosteroids Induction Maintenance Rx Azathioprine Therapy specific (Lymphodepletion) CNIs tolerance CsA, FK MMF, mTOR Co-stim. blockade Therapeutic cell transfer PAST PRESENT FUTURE
Cell Therapies being considered for Tolerance Induction HSC to induce chimerism HSC to induce immunomodulation Regulatory T cells Dendritic cells (DC) Mesenchymal Stem Cells (MSC) Apoptotic Cell Delivery (ECDI, ECP) Future possibilities Combination of cell types (HSC + Treg) Single vs multiple infusions
Regulatory CD4 + CD25 + FoxP3 + T cells Derived from the thymus and/or peripheral tissues Demonstrated to broadly control T cell reactivity. Control immune responsiveness to alloantigens Contribute to operational tolerance in transplantation models Wood KJ and Sakaguchi S. Nat Rev Immunol. 2003 Mar;3(3):199-210.
Tregs in transplantation: clinical evidence Higher circulating numbers of Tregs in tolerant kidney transplant recipients Increased numbers of Tregs in tolerant liver transplant recipients Improved outcomes in stem cell transplant patients receiving infusion of expanded Tregs Sagoo P, Perucha E, Sawitzki B, et al. Development of a cross-platform biomarker signature to detect renal transplant tolerance in humans. J Clin Invest 2010; 120:1848 . Wood KJ, Regulatory T. cells in transplantation. Transplant Proc 2011; 43: 2135. Leventhal JR, Mathew JM, Salomon DR, et al. Am J Transplant. 2016 Jan;16(1):221-34 . Nonchimeric HLA-Identical Renal Transplant 5 Tolerance: Regulatory Immunophenotypic/Genomic Biomarkers Sawitzki B, Brunstein C, Meisel C, et al. Prevention of graft-versus-host disease by adoptive T regulatory therapy is associated with active repression of peripheral blood Toll-like receptor 5 mRNA expression. Biol Blood Marrow Transplant. 2014 Feb;20(2):173-82.
Lymphodepletion maximizes efficacy of Treg therapy 100 % Graft Survival 80 60 No nTreg 40 TGF-β/IL-2 nTreg 20 RA/TGF-β/IL-2 nTreg TSA/RA/TGF-β/IL-2 nTreg 0 0 14 28 42 56 70 84 98 Days Post-Transplantation Lymphodepletion to reduce numbers of circulating effector T cells may be an important adjunct to the use of Tregs as a cellular therapy in organ transplantation
A PHASE 1 TRIAL OF TREG ADOPTIVE CELL TRANSFER (TRACT) IN LIVING DONOR KIDNEY TRANSPLANT RECIPIENTS Design: Single center, open label, nonrandomized Objectives: Determine the safety of using expanded Treg Adoptive Cell Transfer (TRACT). Investigating whether TRACT leads to transplant rejection/allosensitization and/or nonspecific immunosuppression Performing limited immune monitoring . Methodology : dose escalation of expanded autologous nTregs in experimental arm (0.5, 1, 5 & x10 9 cells/subject , n=3 in each tier) Primary Outcome Measure: Patient and Graft survival @ 1year
Inclusion Criteria Patients males or females age 18-65 years . No prior organ transplant . Patients who are single-organ recipients ( kidney only ). Women of childbearing potential must have a negative serum pregnancy test and agree to use a medically acceptable method of contraception throughout the treatment period. Recipient is able to understand the consent form and give written informed consent.
Exclusion Criteria 1. Known sensitivity or contraindication to Sirolimus, tacrolimus or MMF. 2. Patient with significant or active infection. 3. Patients with a positive flow cytometric crossmatch using donor lymphocytes and recipient serum. 4. Patients with PRA >20%. 5. Patients with current or historic donor specific antibodies . 6. Body Mass Index (BMI) of < 18 or > 35. 7. Patients who are pregnant or nursing mothers. 8. Patients whose life expectancy is severely limited by diseases other than renal disease. 9. Ongoing active substance abuse, drug or alcohol. 10. Major ongoing psychiatric illness or recent history of noncompliance. 11. Significant cardiovascular disease (e.g.): • Significant non-correctable coronary artery disease; • Ejection fraction below 30%; • History of recent myocardial infarction. 12. Malignancy within 3 years, excluding non-melanoma skin cancers. 13. Serologic evidence of infection with HIV or HBVsAg positive. 14. Patients with a screening/baseline total white blood cell count < 4,000/mm3; platelet count < 100,000/mm3; triglyceride > 400 mg/dl; total cholesterol > 300 mg/dl. 15. Investigational drug within 30 days prior to transplant surgery. 14. Anti-T cell therapy within 30 days prior to transplant surgery.
Summary of Enrolled Subjects Subject Subject Gender Age at Race Cause of ESRD # Initials Transplant 1 ECW Male 52y 1m Black HTN/FSGS 2 L-N Female 28y 9m White FSGS 3 A-O Male 47y 8m Hispanic/Latino PCKD 4 J-R Male 30y 3m Hispanic/Latino Membranous Nephropathy 5 JTM Male 53y 0m White HTN 6 V-P Female 24y 6m Native Lupus Hawaiian / Pacific Islander 7 KMG Male 37y 2m White IgA Nephropathy 8 K-G Male 62y 3m White PCKD 9 B-G Female 57y 7m White PCKD PCKD – polycystic kidney disease HTN – hypertension FSGS – focal segmental glomerulosclerosis
Clinical Protocol Pretransplant Day 0: Living Donor Initiate isolation and collection of Kidney Transplant: expansion of recipient Alemtuzumab autologous Tregs; lymphocytes via Induction, Tacrolimus infusion of expanded leukopheresis; and Mycophenolate cells Day +60; cryopreservation based IS; conversion protocol biopsy at 3 of cells (1 week - from Tac to Sirolimus months and 1 year 1year Day +30 post-Tx Pretransplant)
Isolation and Manufacture of Autologous Polyclonal Tregs Leukopheresis of recipient pre-transplant Immunomagnetic selection of Tregs (CD8,CD19 negative depletion, CD25 positive selection) from cryopreserved “raw product” Ex vivo culture and expansion of Tregs In process testing of expanded cells for phenotype, function, and sterility
Release Criteria for expanded Tregs >70% cell viability CD4 + /CD25 + > 70%; CD8 + .CD19 + <10% endotoxin < 5 EU/kg gram stain negative; aerobic, anaerobic and fungal sterility mycoplasma negative residual bead count <3,000 beads per 10 8 cells >50% suppression at a 1:2 Treg:Teffector cell ratio in a mixed lymphocyte reaction
Inhibition of Recipient’s MLR by Expanded Tregs 100% Day 0 Day 14 Inhibition by Tregs 80% Day 21 60% 40% P < 0.05 20% 0% 1:2 1:4 1:8 1:16 1:32 1:64 Treg : Responder Ratio In Process Testing = Day 14
Kinetics of CD3 + CD4 + cells post Treg infusion 1200 Pt. # 1 0.5x10 9 Tregs CD3 + CD4 + per μ l blood Pt. # 2 1000 6.8x10 6 /KG Pt. # 3 Pt. # 4 1x10 9 Tregs 800 Pt. # 5 15x10 6 /KG Pt. # 6 Pt. # 7 600 5x10 9 Tregs Pt. # 8 50x10 6 /KG Pt. # 9 400 200 0 Pre 1m 3m 6m 9m 12m Months after Transplant
Increased circulating Tregs post Treg infusion 25 Pt. #1 0.5x10 9 Tregs Pt. #2 % of CD4 + CD127 - CD25 high FoxP3 + Cells 6.8x10 6 /KG 20 Pt. #3 Fold Change from Pre-Tx in TRACT Pt. #4 1x10 9 Tregs Pt. #5 15x10 6 /KG 15 Pt. #6 Pt. #7 5x10 9 Tregs Pt. #8 50x10 6 /KG 10 Pt. #9 5 0 Pre 3m 6m 9m 12m
Current Status of Enrolled Recipients Subject Initials Transplant Treg infusion Cell # 3m 3m 1yr 1yr Graft # Date Date Administered biopsy DSA biopsy DSA loss (10 9 ) 1 ECW 7/10/14 9/8/2014 0.5 NR - NR + NO 2 L-N 8/7/14 10/6/2014 0.5 NR - NR - NO 3 A-O 8/29/14 10/28/2014 0.5 NR - NR - NO 4 J-R 9/22/14 11/21/2014 1.0 NR - NR - NO 5 JTM 12/4/14 2/2/2015 1.0 NR - NR - NO 6 V-P 1/8/15 3/9/2015 1.0 NR - NR - NO 7 KMG 2/2/2015 4/3/2015 5.0 NR - NR - NO 8 K-G 3/27/2015 5/26/2015 5.0 NR - SCR + NO C4d + 9 B-G 4/23/2015 6/22/2015 5.0 NR - NR - NO *NR= no rejection on biopsy *SCR=subclinical rejection
Summary & Conclusions Efficient expansion of Tregs from cryopreserved leukopheresis product from all recipients met release criteria No infusion related serious adverse events (5 x 10 9 poly Tregs are safe) No evidence of over immunosuppression (opportunistic infections) Post-infusion protocol biopsies at 3 months have been normal Serial immunophenotypic analysis of subjects shows a sustained increase in circulating Tregs following Treg infusion Data support the design and initiation of a Phase 2 trial FDA approval for Phase 2 trial secured
Acknowledgements Woman’s Board of Northwestern Memorial Hospital John & Lillian Mathews Regenerative Medicine Endowment Miltenyi BioTec National Kidney Foundation of Illinois TRACT Therapeutics Inc. Jessica Heinrichs
Thank you
Questions? Results of Phase 1 Trial of Treg Adoptive Cell Transfer in Living Donor Kidney Transplant Recipients
FOXP3 Expression in Expanded Tregs CD127-PE SS FS CD4-ECD FOXP3 + Cells CD25-PC7 in ungated sample 71.1 ± 12.3% (n=7) FOXP3-PC5
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