1. NY-ESO-1 Specific T Cell Therapy for Patients with Metastatic Sarcoma
Seth M. Pollack, MD
Associate in Clinical Research,
Fred Hutchinson Cancer Research Center
Acting Instructor, University of Washington

2. Tumor specific proteins are composed of peptides
Cancer immunology review:
Sarcoma Cell
CD8+ T cell
Tumor specific T cells recognize peptides displayed in the MHC complex.
MHC complex .
Tumor specific proteins are composed of peptides
Different HLA types:

3. Re-directed specifity
EFFECTOR CELLS
Endogenous
Non-specific
LAK
TIL
Specific
CD8
CD4
Genetically modified
Re-directed specifity
TCR
CAR
Enhanced Function
Pollack SM and Yee C Innate Immune Regulation and Cancer Immunotherapy Springer 2012

4. Re-directed specifity
EFFECTOR CELLS
Endogenous
Non-specific
LAK
TIL
Specific
CD8
CD4
Genetically modified
Re-directed specifity
TCR
CAR
Robbins et al., JCO 2011
Enhanced Function
Pollack SM and Yee C Innate Immune Regulation and Cancer Immunotherapy Springer 2012

5. Cancer Testis Antigens are Promising Targets
In adults, only expressed in testis (some are also expressed in placenta)
Expressed in many cancers
Epigenetically regulated***
Unknown biologic function
Includes NY-ESO-1, LAGE-1, PRAME, MAGE-A3
A: MAGE-A4 in tests
B: MAGE-C1 in fetal ovary
C: MAGE-A in trophoblastic placenta
D: NY-ESO-1 in bladder cancer
Simpson AJ Nat Rev Cancer, 2005

6. NY-ESO-1 Is a Cancer Testis Antigen Frequently Expressed Target In Synovial Sarcoma
NY-ESO stained 20/25 patients.
Staining homogonous in 14/20 patients
4 of the 5 patients without NY-ESO expression were biphasic phenotype (usually ssx-1)
Targeted in the NCI Surgery Branch Study
Jungbluth AA et al., International Journal of Cancer, 2001

7. Computed tomography scans demonstrating tumor regression.
Computed tomography scans demonstrating tumor regression. Radiologic studies were obtained before therapy and after adoptive transfer of NY-ESO-1 T-cell receptor (TCR) –transduced T cells. Tumors indicated by arrows. (A) Regression of multiple recurrent axillary lymph nodes from metastatic melanoma in patient 5 with a complete response now ongoing at 20 months. (B) Regression of a perihepatic chest wall lesion of synovial cell sarcoma in patient 16, who demonstrated a partial response lasting 8 months. (C) Regression of multiple lung metastases of synovial cell sarcoma in patient 13, who received two treatments with NY-ESO-1 TCR-transduced T cells and demonstrated a partial response lasting 18 months. No toxicities attributable to the administered cells were observed in these patients.
Robbins P F et al. JCO 2011;29:
©2011 by American Society of Clinical Oncology

8. Question: Are there other sarcoma subtypes that frequently express NY-ESO-1?

9. Myxoid/ Round Cell Liposarcomas Always Express NY-ESO-1, Usually Homogenously
We examined 25 Myxoid Liposarcoma tumors, all expressed NY-ESO-1 (100%)
>70% of cases, homogenous
In all but 2 case, patients would qualify for trials of NY-ESO-1 directed therapy based on staining.
MRCL cell lines can be lysed using NY-ESO-1 specific effectors
No other disease (including synovial sarcoma) expresses NY-ESO- 1 with this frequency
Pollack et al., Cancer 2012

10. Question: If Cancer Testis Antigens are epigenetically regulated, can sarcomas be induced to express them?

11. Decitabine induces NY-ESO-1 and MAGE-A3 in tumors from lung cancer patients
Patients on a phase I trial of decitabine for lung cancer
Serial biopsies performed
Analyzed retrospectively for CT Antigen Expression
Increased expression following treatment
This increase was sustained months after treatment
Analysis of NY-ESO-1, MAGE-3, and p16 expression in tumor biopsies before and after DAC treatment. A, representative immunohistochemical analysis of NY-ESO-1, MAGE-3, and p16 expression in non–small cell lung cancer (NSCLC) and small-cell lung cancer (SCLC) biopsies before and after DAC treatment. CTA induction was more uniform in small-cell lung cancer in all likelihood due to higher basal levels of NY-ESO-1 and MAGE-3 before treatment. The results are consistent with our observations concerning induction of NY-ESO-1, MAGE-3, and p16 in cultured cancer lines. B, quantitative RT-PCR analysis of NY-ESO-1, MAGE-3, and p16 expression in lung cancer tissues from a patient exhibiting prolonged stabilization of disease following DAC treatment. A progressive increase in NY-ESO-1 and MAGE-3 mRNA copy numbers was observed; in contrast, p16 mRNA copy numbers remained relatively constant. Immunohistochemistry and serum ELISA done in a blinded manner revealed NY-ESO-1 protein expression and serum antibodies to NY-ESO-1 at the 6 months time point and thereafter.
Schrump D S et al. Clin Cancer Res 2006;12:

12. A minority of chondrosarcoma tumors express NY-ESO-1/LAGE-1.
36% of tumors expressed either NY-ESO-1 or LAGE-1 at a level that might be targeted – could this be improved?
Pollack et al. Plos One, 2012

13. Decitabine (5-Aza-dC) Treatment Increases mRNA Expression of
NY-ESO-1, LAGE-1s and PRAME by qPCR.
A. NY-ESO-1 Expression in Chondrosarcoma Cell Lines Relative to Mel375
B. PRAME Expression in Chondrosarcoma Cell Lines Relative to Mel375
C. LAGE-1s Expression in Chondrosarcoma Cell Lines Relative to JJ
Untreated
5-Aza-dC
Pollack et al. Plos One, 2012

14. Lysis of the FS and JJ Cell Lines With and Without Decitabine (5-Aza-dC) Using NY-ESO-1/LAGE-1s and PRAME Specific Effector T Cells
Pollack et al. Plos One, 2012

15. Question: Can we isolate and expand NY-ESO-1 specific T cells from sarcoma patients with NY-ESO-1 expressing tumors?

16. Generation of Autologous NY-ESO-1 Specific T Cells
CD25 depletion (to reduce regulatory T cells)
Step 1: Leukapharesis
Step 2:
Generation of dendritic cells
Step 3:
Stimulation of T cells using peptide pulsed dendritic cells
Step 4: Clinical grade sorting
Rapid Expansion

17. Examples of Clinical Grade Products
tetramer
Sort and Expand
NY-ESO-1 Tetramer
CD8
+ 3 additional wells <1% tetramer +
Patient 2537-S02
Patient 2537-S04
+ 1 additional well <1% tetramer +

18. Peptide Titration With NY-ESO-1 Specific Effectors:
Pros and cons of autologous vs. virally transfected NY-ESO-1 specificity?

19. FHCRC Protocol #2537 Autologous NY-ESO-1 specific T cell therapy
Metastatic or unresectable Synovial Sarcoma and Myxoid/round cell liposarcoma
Patient must have HLA type A*0201
2 patients have been treated
4 additional patients have T cell products frozen
All finalized products kill tumor in vitro.
Lymphodelpetion with Cyclophosphamide as is considered standard

20. Adoptively transferred NY-ESO-1 Specific T cells Persist Following Infusion (from first patient treated)
% Tetramer + (% of CD8+ cells)
Day (infusion-day 0)
6 wks
8 wks
10 wks

21. Elimination of some small nodules
Prior to infusion
4 weeks post- infusion

22. Post-treatment (10 weeks)
Response in Large Right Upper Lobe Tumor
Pre-treatment
Post-treatment (10 weeks)
But the response was mixed …

23. NY-ESO-1 Remains Post-Tx (20x)
4/9/2012
7/2/2012
By IHC, few CD3+ cells could be seen in the tumor.
We expanded these few cells and a very small percentage were tetramer positive (a lower percentage than was in the peripheral blood).
These few cells could be sorted and expanded and remained functional.
NY-ESO-1 Tetramer

24. Conclusion
NY-ESO-1 is generally homogenously expressed in Myxoid/ Round Cell Liposarcoma tumors
NY-ESO-1/LAGE-1s and PRAME can be up-regulated in Chondrosarcoma cell lines
NY-ESO-1 specific T cells can be isolated and expanded from patients with NY-ESO-1 expressing sarcomas

25. Future Directions
Expand to different HLA types, cancer testis antigens and sarcoma subtypes.
Explore mechanisms of immune evasion in sarcoma tumors.

26. THANK YOU, SARC!!!

27. Robin Jones Acknowledgements: Mentors: Cassian Yee, Stan Riddell
FHCRC Immunology Program: Eric Farrar Ivy Lai Dawn Stief Heather Sloan
The SCCA Sarcoma Group
Chappie Conrad
Janet Eary
Doug Hawkins
Darin Davidson
Elizabeth Loggers
Gabrielle Kane
Edward Kim
Benjamin Hoch
Gary Mann
Venu Pillarisetty
Jennifer Hammilton
Funding:
SARC Career Development Award
Doug and Maggie Walker Foundation
Gilman Sarcoma Foundation
MSKCC/Ludwig
Achim Jungbluth
Sacha Gnjatic