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

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

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

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.

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.

5

Lymphodepletion maximizes efficacy of Treg therapy

0

20

40

60

80

100

0 14 28 42 56 70 84 98

Days Post-Transplantation

% G

ra

ft S

urv

iva

l

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

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

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

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

Pretransplant collection of

recipient lymphocytes via leukopheresis;

cryopreservation of 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

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 108 cells

>50% suppression at a 1:2 Treg:Teffector cell

ratio in a mixed lymphocyte reaction

0%

20%

40%

60%

80%

100%

1:2 1:4 1:8 1:16 1:32 1:64

Inhib

itio

n b

y T

regs

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

Kinetics of CD3+CD4+ cells post Treg infusion

0

200

400

600

800

1000

1200

Pre 1m 3m 6m 9m 12m

Pt. # 1Pt. # 2Pt. # 3Pt. # 4Pt. # 5Pt. # 6Pt. # 7Pt. # 8Pt. # 9

CD

3+C

D4

+ p

er μ

l b

loo

d 0.5x109 Tregs

6.8x106/KG

1x109 Tregs

15x106/KG

5x109 Tregs

50x106/KG

Months after Transplant

0

5

10

15

20

25

Pre 3m 6m 9m 12m

Pt. #1

Pt. #2

Pt. #3

Pt. #4

Pt. #5

Pt. #6

Pt. #7

Pt. #8

Pt. #9

Fo

ld C

han

ge f

rom

Pre

-Tx i

n

% o

f C

D4

+C

D127

- CD

25

hig

hF

oxP

3+

Cell

s

TRACT

Increased circulating Tregs post Treg infusion

0.5x109 Tregs 6.8x106/KG

1x109 Tregs 15x106/KG

5x109 Tregs 50x106/KG

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 - 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

C4d+

+ NO

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 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

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

Thank you

Questions?

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

FOXP3 Expression in Expanded Tregs

SS

FS

CD

127-P

E

CD4-ECD

CD

25-P

C7

FOXP3-PC5

FOXP3+ Cells

in ungated sample

71.1 ± 12.3%

(n=7)