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

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:

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

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

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

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

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;29:917-924 ©2011 by American Society of Clinical Oncology

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

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

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

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;12:5777-5785

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

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

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

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

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

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 +

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

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

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

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

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

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

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

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

THANK YOU, SARC!!!

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