Adoptive Cell Therapy: Treating Cancer

Adoptive Cell Therapy: Treating Cancer with Genetically Engineered T Cells Steven A. Feldman, Ph.D. Surgery Branch National Cancer Institute NCT Conference Heidelberg, Germany September 24, 2013

Three Main Approaches to Cancer Immunotherapy 1. Non-specific stimulation of immune reactions Stimulate effector cells (IL-2, IL-12) Inhibit regulatory factors (PD-1, CTLA-4) 2. Active immunizations to enhance anti-tumor reactions Cancer vaccines 3. Passively transfer activated immune cells with anti-tumor activity Adoptive cell transfer

Advantages of Cell Transfer Therapy 1. High avidity anti-tumor T cell receptors (TCR) can be identified and cloned using in vitro assays. 2. Peripheral blood lymphocytes can be genetically modified to express these high avidity TCRs. 3. Large numbers of tumor-specific lymphocytes can be grown in vitro. 4. The host can be manipulated to provide a favorable tumor microenvironment prior to administering the cells. 5. ACT can mediate tumor regressions.

Development of Adoptive Cell Transfer Therapy

A Critical Challenge Confronting the Development of Human Cancer Immunotherapy is the Identification of Antigens to Target 1. Differentiation antigens overexpressed on cancers compared to normal tissue (MART-1, gp100, CEA, Her-2, Mesothelin) 2. Antigens expressed on cancers and on non-essential normal tissues (CD19, thyroglobulin) 3. Shared antigens unique to cancer (cancer-testes antigens, NY- ESO-1, MAGE-A) 4. Mutations unique to each cancer (EGFRvIII) 5. Critical components of the tumor stroma (VEGFR2, FAP)

Surgery Branch Gene Therapy Products (by class) Cytokine TCR CAR IL-2 IL-12 murine (gp100, NY-ESO-1, MAGE-A3) A3) human (DMF5, NY-ESO-1) 2 nd gen-28z (CD19, Meso) 3 rd gen-28bbz (EGFRvIII, VEGFR2)

Interleukin 12 IL-12 is a heterodimeric cytokine, composed of a heavy chain (p40) and a light chain (p35), Coordinated production of the two chains lead to the secretion of the biologically active p70 IL-12 is produced by activated hematopoietic phagocytic cells (monocytes, macrophages, neutrophils) and dendritic cells (DC) Activates effector cells: CD4+, CD8+, and NK cells

Development of an Inducible Vector to Mediate IL-12 Production Only in the Tumor Microenviroment MSGV1.NFAT.IL12.PA2 In vitro In vivo No IL 2, no vaccine Zhang L, Kerkar SP et al, Molecular Therapy, 2011

Phase I Study of ACT Using TIL Transduced with Gene Encoding IL-12 (9/28, 32% OR) TIL grown for 2-3 weeks Stimulated with OKT-3, transduced and expanded Infuse after Cy/flu preparative regimen No IL-2 administered Cohort Number Result (# cells x 10-9 ) of patients 0.001 1 1NR 0.003 1 1NR 0.01 7 7NR 003 0.03 5 1CR (24+ mos); 4NR 0.1 3 3NR 0.3 3 3PR (4, 6, 12+) 1.0 4 1PR (12+); 3NR 30 3.0 4 1CR (5+) 3PR (9+, 7, 5) In first 5 cohorts 1 of 17 patients responded. At doses greater than 0.1X10-9, 8 of 11 patients responded.

IL-12 Gene Therapy (M.S. 3x107 IL12Td CD8+ TIL)

Summary TIL IL-12 Tumor infiltrating i lymphocytes transduced d with NFAT.IL12 vector secrete IL-12 upon stimulation. 28 patients with metastatic melanoma received the autologous TILs genetically modified by NFAT.IL12 vector. Following IL12 Td TILs infusion, 2 patients experienced dose limiting toxicity correlated with high levels of IFN-g and IL-12 in their serum. All patients recovered completely. 9 t f 28 ( 32%) ti t d d t IL 12 Td TIL t t t 9 out of 28 ( 32%) patients responded to IL-12 Td-TIL treatment based on RECIST. IL-2 not needed to achieve OR in this setting.

Surgery Branch Gene Therapy Products (by class) Cytokine TCR CAR IL-2 IL-12 murine (gp100, NY-ESO-1, MAGE-A3) A3) human (DMF5, NY-ESO-1) 2 nd gen-28z (CD19, Meso) 3 rd gen-28bbz (EGFRvIII, VEGFR2)

T-cell Receptor (TCR) Gene Therapy TCR Cloning IVS Immunize mice TCR Vector (eg, MART1, NY ESO) TCR receptor α β LTR SD Ψ SA TCRα 2A TCRβ LTR CD3ζ,γ,ε,δ

Cancer/Testes Antigens - Shared Tumor Specific Antigens Expressed during fetal development Restricted in their expression in adult normal tissues to germ cells Up-regulated in 10-80% of cancers from multiple tissues NY-ESO-1 Family Small family of X-linked genes that includes NY-ESO-1 and LAGE-1 MAGE Family Family of ~ 45 X-linked genes

Cancer/Testis Antigens Expressed in Multiple Tumor Types 80 70 60 50 40 30 20 10 0 MAGE A3 MAGE A1 NY ESO 1 He Squamous cel Bladder NSCLCC Melanomaa Ovarian patocellular Myeloma ll carcinomaa % of positive tumor by RT PCRR Tumor Type

Recognition of Non-melanoma Tumors by NY- ESO-1 TCR Transduced PBL ESO A2 - + Glioblastoma + + + + + + SCLC + + LNZAT3WT4 Ewing s sarcoma TC-71 Glioblastoma LN-18 SCLC NCI H526 Neuroblastoma SKN-AS-A2 MSGIN APB, NY-ESO-1 TCR + + Breast cancer MDA453s-A2 + + Osteosarcoma Saos-2 + + SCLC + + Melanoma NCI H345 624.38mel 0 100 200 300 400 500 600 IFN-, pg/ml

Phase II Study of Metastatic Cancer that Expresses NY-ESO-1 Using Lymphodepleting Conditioning Followed by Infusion of Anti-NY- ESO-1 TCR-Gene Engineered Lymphocytes J. Clinical Oncology, 29:917 924, 2011

DC (Melanoma) CR 30+

HK (Synovial Cell Sarcoma) PR (14*)

SUMMARY: NY-ESO-1 TCR Engineered T cells TCR gene therapy targeting CTA antigen NY-ESO-1 can lead to cancer regression in melanoma and synovial cell sarcoma without associated toxicities. Total PR CR OR number of patients (duration in months) Melanoma 18 5 (28%) 4 (22%) 9 (50%) (18+,10**, 8, 4, 3) (48+, 37+, 25, 21+**) Synovial Cell 16 10 (63%) 0 10 (63%) Sarcoma (29+**,14*, 12**,10, 8, 6+, 5, 4, 3**,2+) *treated t twice **plus ALVAC vaccine (Robbins et al J Clin Oncol 29:917-924, 2011)

Limitation of TCR gene transfer 1. HLA-restriction limits ability to treat patients / requires multiple TCRs 2. Inability to target lipid / carbohydrate molecules 3. Potential tumor escape via MHC loss / alterations in antigen 3. Potential tumor escape via MHC loss / alterations in antigen processing

Surgery Branch Gene Therapy Products (by class) Cytokine TCR CAR IL-2 IL-12 murine (gp100, NY-ESO-1, MAGE-A3) A3) human (DMF5, NY-ESO-1) 2 nd gen-28z (CD19, Meso) 3 rd gen-28bbz (EGFRvIII, VEGFR2)

Chimeric Antigen Receptors (CARs) Step 2 Step 1 Step 3 Ig ScFv Linker/TM Antibody Producing Hybridoma Ig Genes T cell signaling Chimeric Antigen Receptor (CAR) CAR (CD19, Meso, EGFRvIII, VEGFR2 CAR receptor LTR sd sa Anti-tumor Ag-scFv CD28 CD3 zeta LTR scfv V L V H V L V H LTR sd sa Anti-tumor Ag-scFv CD8 CD28 4-1BB CD3 zeta LTR CD28 CD3ζ

B-Cell Malignancies (Antigens Expressed on Non-Essential Normal Tissues) Approximately 22,000 people die of B-cell malignancies annually in the U.S. CD19 is expressed by more than 90% of B-cell malignancies. CD19 is expressed by mature B cells, B-cell precursors and plasma cells but not any other normal tissues. Anti CD19 CAR 5 LTR FMC63 scfv CD28 CD3 zeta 3 LTR

Bone marrow biopsies showed extensive CLL before and nearly absent B-lineage cells after treatment Before treatment 3 months after treatment CD19 CD19 CD20 CD20 Kochenderfer et al. Blood 2012

Tumor regression and elimination of normal B cells

Patient characteristics and response original patients treated with IL-2 (6/8, 75% OR) Response Number of Number of CAR+ (months since Patient Age/sex Disease prior therapies cells infused/kg infusion) 1a 47/M Follicular 4 0.3x10 7 PR (7) Lymphoma 1b 48/M Follicular 5 1.3x10 7 PR (40+) Lymphoma 2 48/M Follicular 5 0.3x10 7 NE Lymphoma (died of influenza) 3 61/M CLL 3 1.1x10 7 CR (24) 4 55/M Splenic Marginal 3 1.1x10 7 PR (12) Zone Lymphoma 5 54/M CLL 4 0.3x10 7 SD (6) 6 57/M CLL 7 1.7x10 7 PR (7) 7 61/M CLL 4 2.8x10 7 CR (31+) 8 63/M Follicular 7 3.0x10 7 PR (11)* Lymphoma Patient 1 was treated twice. *Patient developed squamous cell carcinoma of the larynx.

Response to Therapy with CD19 CAR and No IL-2 (11/14, 78.5% OR) Patient Age/Gender Malignancy Number of prior therapies Total cyclo- Number of Response phosphamide CAR+ T cells (time after cell dose infused infusion in (mg/kg) (X10 6 /kg) months) 1 56/M SMZL 4 120 5 PR (20+) 2 43/F PMBCL 4 60 5 CR (19+) 3 61/M CLL 2 60 4 CR (16+) 4 30/F PMBCL 3 120 25 2.5 NE 5 63/M CLL 4 120 2.5 CR (10+) 6 48/M CLL 1 60 2.5 CR (7+) 7 42/M DLBCL 5 60 2.5 CR (4+) 8 44/F PMBCL 10 60 2.5 PR (6+) 9 38/M PMBCL 3 120 2.5 SD (1) 10 57/F Low-grade 4 60 1 PR (4+) NHL 11 58/F DLBCL from 13 60 1 PR (2) CLL 12 60/F DLBCL 3 60 1 SD (1+) 13 68/M CLL 4 60 1 PR (2+) 14 43/M DLBCL 2 60 1 PR (1+)

Autologous anti-cd19 CAR-transduced T cell trial conclusions Biological activity of the infused cells was demonstrated by depletion of CD19+ cells 17/22 (77%) evaluable patients obtained remissions, but the contribution of CAR-transduced T cells to the remissions is unclear. Substantial toxicity occurred including hypotension and obtundation. The duration of these toxicities was short. Toxicity correlated with serum levels of inflammatory cytokines.

A Critical Challenge Confronting the Development of Human Cancer Immunotherapy is the Identification of Antigens to Target 1. Differentiation antigens overexpressed on cancers compared to normal tissue (MART-1, gp100, CEA, Her-2, Mesothelin) 2. Antigens expressed on cancers and on non-essential normal tissues (CD19, thyroglobulin) 3. Shared antigens unique to cancer (cancer-testes antigens, NY- ESO-1, MAGE-A) 4. Mutations unique to each cancer (EGFRvIII) 5. Critical components of the tumor stroma (VEGFR2, FAP)

Program for the Application of Cell Transfer Therapy to a Wide Variety of Human Cancers Receptor Type Cancers Status IL 12 Cytokine Adjuvant for all receptors Accruing MART 1 TCR Melanoma Closed gp100 TCR Melanoma Closed CEA TCR Colorectal Closed 2G1 TCR Renal Accruing Hu NY ESO 1 TCR Epithelial/Sarcoma Accruing Mu NY ESO 1 TCR Epithelial/Sarcoma In development MAGE A3 TCR Epithelial In development SSX 2 TCR Epithelial In development HPV E6/E7 TCR Cervical In development Thyroglobulin TCR Thyroid In development CD19 CAR Lymphomas Accruing VEGFR2 CAR All cancers Accruing EGFRvIII CAR Glioblastoma Accruing Mesothelin CAR Pancreatic/Mesothelioma/Ovarian Accruing CSPG4 CAR Melanoma/Pancreatic/Breast In development

Conclusions Autologous peripheral lymphocytes genetically modified to express anti-tumor T cell receptors and chimeric antigen receptors can mediate cancer regression in vivo. The ability to genetically modify human T cells opens possibilities to improve the effectiveness of cell transfer immunotherapy and extend e it to patients with common o epithelial cancers.

Personalized immunotherapy using anti-tumor receptor gene-modified lymphocytes

Acknowledgments: Surgery Branch, NCI: Steven A. Rosenberg, Chief Rick Morgan Mark Dudley John Wunderlich Paul Robbins James Yang Maria Parkhurst Nick Restifo SBVPF TIL LAB FACS LAB Clinical Staff Lab of Molecular Biology, NCI Ira Pastan Tapan Bera Hematology Branch, NHLBI Dhana Chinnasamy ETIB, NCI Jim Kochenderfer Pediatric Oncology Branch, NCI Ling Zhang NYU Langone Medical Center Howard Fine