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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

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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

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As we understand immune regulation, immunosuppression is evolving

Induction Therapy (Lymphodepletion)

Maintenance Rx CsA, FK MMF, mTOR Co-stim. blockade

PRESENT

Corticosteroids Azathioprine CNIs

Drug-free donor- specific tolerance

PAST FUTURE Therapeutic cell transfer

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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

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 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

Regulatory CD4+CD25+FoxP3+ T cells

Wood KJ and Sakaguchi S. Nat Rev Immunol. 2003 Mar;3(3):199-210.

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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 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.

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Lymphodepletion maximizes efficacy

f Treg therapy

20 40 60 80 100 14 28 42 56 70 84 98

Days Post-Transplantation

% Graft Survival

No nTreg TGF-β/IL-2 nTreg RA/TGF-β/IL-2 nTreg TSA/RA/TGF-β/IL-2 nTreg

 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

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 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 & x109 cells/subject , n=3 in each tier)  Primary Outcome Measure: Patient and Graft survival @ 1year

A PHASE 1 TRIAL OF TREG ADOPTIVE CELL TRANSFER (TRACT) IN LIVING DONOR KIDNEY TRANSPLANT RECIPIENTS

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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.

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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.

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Summary of Enrolled Subjects

PCKD – polycystic kidney disease HTN – hypertension FSGS – focal segmental glomerulosclerosis

Subject # Subject Initials Gender Age at Transplant Race Cause of ESRD 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 Hawaiian / Pacific Islander Lupus 7 KMG Male 37y 2m White IgA Nephropathy 8 K-G Male 62y 3m White PCKD 9 B-G Female 57y 7m White PCKD

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Pretransplant collection of recipient lymphocytes via leukopheresis; cryopreservation

f cells (1 week -

1year Pretransplant) Day 0: Living Donor Kidney Transplant: Alemtuzumab Induction, Tacrolimus and Mycophenolate based IS; conversion from Tac to Sirolimus Day +30 Initiate isolation and expansion of autologous Tregs; infusion of expanded cells Day +60; protocol biopsy at 3 months and 1 year post-Tx

Clinical Protocol

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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

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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 108 cells  >50% suppression at a 1:2 Treg:Teffector cell ratio in a mixed lymphocyte reaction

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0% 20% 40% 60% 80% 100% 1:2 1:4 1:8 1:16 1:32 1:64

Inhibition by Tregs Treg : Responder Ratio

Day 0 Day 14 Day 21

Inhibition of Recipient’s MLR by Expanded Tregs

In Process Testing = Day 14

P < 0.05

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Kinetics of CD3+CD4+ cells post Treg infusion

200 400 600 800 1000 1200

Pre 1m 3m 6m 9m 12m

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CD3+CD4+ per μl blood 0.5x109 Tregs 6.8x106/KG 1x109 Tregs 15x106/KG 5x109 Tregs 50x106/KG Months after Transplant

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5 10 15 20 25

Pre 3m 6m 9m 12m

Pt. #1
Pt. #2
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Fold Change from Pre-Tx in % of CD4+CD127-CD25highFoxP3+ Cells

TRACT

Increased circulating Tregs post Treg infusion

0.5x109 Tregs

6.8x106/KG

1x109 Tregs

15x106/KG

5x109 Tregs

50x106/KG

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Current Status of Enrolled Recipients

Subject # Initials Transplant Date Treg infusion Date Cell # Administered (109) 3m biopsy 3m DSA 1yr biopsy 1yr DSA Graft loss

1 ECW 7/10/14 9/8/2014 0.5 NR

+ NO 2 L-N 8/7/14 10/6/2014 0.5 NR

3 A-O 8/29/14 10/28/2014 0.5 NR

4 J-R 9/22/14 11/21/2014 1.0 NR

5 JTM 12/4/14 2/2/2015 1.0 NR

6 V-P 1/8/15 3/9/2015 1.0 NR

7 KMG 2/2/2015 4/3/2015 5.0 NR

8 K-G 3/27/2015 5/26/2015 5.0 NR

C4d+ + NO 9 B-G 4/23/2015 6/22/2015 5.0 NR

*NR= no rejection on biopsy *SCR=subclinical rejection

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Summary & Conclusions

Efficient expansion of Tregs from cryopreserved leukopheresis product from all recipients met release criteria No infusion related serious adverse events (5 x 109 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

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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

Acknowledgements

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Thank you

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Questions?

Results of Phase 1 Trial of Treg Adoptive Cell Transfer in Living Donor Kidney Transplant Recipients

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FOXP3 Expression in Expanded Tregs

SS FS CD127-PE CD4-ECD CD25-PC7 FOXP3-PC5