Adoptive Cell Therapy: Treating Cancer with Genetically Engineered - - PowerPoint PPT Presentation

Adoptive Cell Therapy: Treating Cancer ith G ti ll E i d T C ll with Genetically Engineered T Cells

Steven A. Feldman, Ph.D. S B h Surgery Branch National Cancer Institute

NCT Conference Heidelberg, Germany September 24 2013 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)
• Inhibit regulatory factors (PD-1, CTLA-4)
• 2. Active immunizations to enhance anti-tumor reactions
• Cancer vaccines

3 P i l f i d i ll i h i

• 3. Passively transfer activated immune cells with anti-tumor

activity

• Adoptive cell transfer
• Adoptive cell transfer

Advantages of Cell Transfer Therapy 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. p g y

• 3. Large numbers of tumor-specific lymphocytes can be

i i grown in vitro. 4 The host can be manipulated to provide a favorable tumor

• 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

• f Human Cancer Immunotherapy is the
• f 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 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) (by class)

IL 2 IL-2 Cytokine IL 12 IL-12 murine (

100 NY ESO 1 MAGE A3)

murine (gp100, NY-ESO-1, MAGE-A3) TCR human (DMF5 NY ESO 1) human (DMF5, NY-ESO-1) 2nd gen 28Z (CD19 Meso) 2nd gen-28Z (CD19, Meso) CAR 3rd gen 28BBZ (EGFRvIII VEGFR2) 3rd gen-28BBZ (EGFRvIII, VEGFR2)

Interleukin 12

• IL-12 is a heterodimeric cytokine composed of a heavy chain

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

Production Only in the Tumor Microenviroment

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) Gene Encoding IL-12 (9/28, 32% OR)

TIL grown for 2-3 weeks Stimulated with OKT-3, transduced and expanded , p Infuse after Cy/flu preparative regimen No IL-2 administered Cohort Number Result (# cells x 10-9)

• f patients

___________________________________________________________________________ 0.001 1 1NR 0.003 1 1NR 0.01 7 7NR 0 03 5 1CR (24+ mos); 4NR 0.03 5 1CR (24+ mos); 4NR 0.1 3 3NR 0.3 3 3PR (4, 6, 12+) 1.0 4 1PR (12+); 3NR 3 0 4 1CR (5+) 3PR (9+ 7 5) 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

T i fil i l h d d i h NFAT IL12

• Tumor infiltrating lymphocytes transduced 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 g y g g

• 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) (by class)

IL 2 IL-2 Cytokine IL 12 IL-12 murine (

100 NY ESO 1 MAGE A3)

murine (gp100, NY-ESO-1, MAGE-A3) TCR human (DMF5 NY ESO 1) human (DMF5, NY-ESO-1) 2nd gen 28Z (CD19 Meso) 2nd gen-28Z (CD19, Meso) CAR 3rd gen 28BBZ (EGFRvIII VEGFR2) 3rd gen-28BBZ (EGFRvIII, VEGFR2)

T-cell Receptor (TCR) Gene Therapy

TCR Cloning IVS Immunize mice

TCR Vector (eg, MART1, NY‐ESO)

SD SA

TCR receptor

α β

TCRβ

Ψ

TCRα

2A

LTR LTR

CD3ζ,γ,ε,δ

Cancer/Testes Antigens - Shared Tumor Specific Antigens

Expressed during fetal development

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 Types

80

R

50 60 70

r by RT‐PCR

MAGE‐A3

30 40 50

tive tumor

MAGE‐A1 NY‐ESO‐1

10 20

r C a n r a a

% of posit

Bladder NSCLC Melanoma Ovarian patocellular Myeloma ll carcinoma He quamous cel

Tumor Type

Sq

Tumor Type

Recognition of Non-melanoma Tumors by NY- Recognition of Non melanoma Tumors by NY ESO-1 TCR Transduced PBL

LNZAT3WT4

MSGIN APB, NY-ESO-1 TCR ESO A2

• +

Glioblastoma NCI H526 LN-18 TC-71

+ + + + + +

SCLC Glioblastoma Ewing’s sarcoma MDA453s-A2 SKN-AS-A2 NCI H526

+ + + + + +

Breast cancer Neuroblastoma SCLC NCI H345 Saos-2

+ + + +

SCLC Osteosarcoma

100 200 300 400 500 600

624.38mel

IFN pg/ml + +

Melanoma

IFN-, pg/ml

Phase II Study of Metastatic Cancer that Phase II Study of Metastatic Cancer that Expresses NY-ESO-1 Using Lymphodepleting Conditioning Followed by Infusion of Anti-NY- 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 g y toxicities.

Total PR CR OR 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%) 10 (63%) Sarcoma (29+**,14*, 12**,10, 8, 6+, 5, 4, 3**,2+)

*t t d t i *treated twice **plus ALVAC vaccine (Robbins et al J Clin Oncol29:917-924, 2011)

Limitation of TCR gene transfer

• 1. HLA-restriction limits ability to treat patients / requires

multiple TCRs p 2. Inability to target lipid / carbohydrate molecules y g p y 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) (by class)

IL 2 IL-2 Cytokine IL 12 IL-12 murine (

100 NY ESO 1 MAGE A3)

murine (gp100, NY-ESO-1, MAGE-A3) TCR human (DMF5 NY ESO 1) human (DMF5, NY-ESO-1) 2nd gen 28Z (CD19 Meso) 2nd gen-28Z (CD19, Meso) CAR 3rd gen 28BBZ (EGFRvIII VEGFR2) 3rd gen-28BBZ (EGFRvIII, VEGFR2)

Chimeric Antigen Receptors (CARs)

Step 2 Step 1 Step 3 Ig ScFv Linker/TM T cell signaling Antibody Producing Hybridoma Ig Genes Chimeric Antigen Receptor (CAR)

CAR receptor



sd sa

CAR (CD19, Meso, EGFRvIII, VEGFR2

VL VH VL VH

scFv

CD28 CD3 zeta Anti-tumor Ag-scFv

LTR LTR



sd sa

CD28 CD3ζ

CD28 CD3 zeta

LTR LTR



sd sa CD8 4-1BB Anti-tumor Ag-scFv

B-Cell Malignancies

(A ti E d N E ti l N l Ti ) (Antigens Expressed on Non-Essential Normal Tissues)

Approximately 22 000 people die of B cell malignancies

• Approximately 22,000 people die of B-cell malignancies

annually in the U.S.

• CD19 is expressed by more than 90% of B cell
• CD19 is expressed by more than 90% of B-cell

malignancies.

• CD19 is expressed by mature B cells B-cell precursors

CD19 is expressed by mature B cells, B cell precursors and plasma cells but not any other normal tissues. Anti‐CD19 CAR

 3’ LTR CD28 FMC63 scFv CD3‐zeta 5’ LTR

Bone marrow biopsies showed extensive CLL before and nearly absent B-lineage cells after treatment

Before treatment 3 months after treatment

y g

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 0.3x107 PR (7) Lymphoma 1b 48/M Follicular 5 1.3x107 PR (40+) Lymphoma 2 48/M Follicular 5 0.3x107 NE Lymphoma (died of influenza) 3 61/M CLL 3 1.1x107 CR (24) 4 55/M Splenic Marginal 3 1.1x107 PR (12) Zone Lymphoma Zone Lymphoma 5 54/M CLL 4 0.3x107 SD (6) 6 57/M CLL 7 1.7x107 PR (7) ( ) 7 61/M CLL 4 2.8x107 CR (31+) 8 63/M Follicular 7 3.0x107 PR (11)*

Patient 1 was treated twice.

*Patient developed squamous cell carcinoma of the larynx.

Lymphoma

Response to Therapy with CD19 CAR and No IL-2 (11/14 78 5% OR) (11/14, 78.5% OR)

N b Total cyclo- Number of Response Patient Age/Gender Malignancy Number

• f prior

therapies y phosphamide dose (mg/kg) CAR+ T cells infused (X106/kg) p (time after cell infusion in 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 2 5 NE 4 30/F PMBCL 3 120 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 NHL 4 60 1 PR (4+) NHL 11 58/F DLBCL from CLL 13 60 1 PR (2) 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 T cell trial conclusions

• Biological activity of the infused cells was demonstrated
• 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
• btundation. The duration of these toxicities was short.
• Toxicity correlated with serum levels of inflammatory

y y cytokines.

A Critical Challenge Confronting the Development

• f Human Cancer Immunotherapy is the
• f 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 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 y

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 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 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 antigen receptors can mediate cancer regression in vivo.

• The ability to genetically modify human T cells opens
• The ability to genetically modify human T cells opens

possibilities to improve the effectiveness of cell transfer immunotherapy and extend it to patients with common u o e apy a d e e d

• pa e

s co

• epithelial cancers.

Personalized immunotherapy using anti-tumor receptor gene-modified lymphocytes receptor gene modified lymphocytes

Acknowledgments:

Surgery Branch, NCI:

Steven A. Rosenberg, Chief

Lab of Molecular Biology, NCI

Ira Pastan g, Rick Morgan Mark Dudley John Wunderlich Paul Robbins Tapan Bera

Hematology Branch, NHLBI

Paul Robbins James Yang Maria Parkhurst Nick Restifo Dhana Chinnasamy

ETIB, NCI

Jim Kochenderfer SBVPF TIL LAB FACS LAB Clinical Staff Jim Kochenderfer

Pediatric Oncology Branch, NCI

Ling Zhang Clinical Staff Ling Zhang

NYU Langone Medical Center

Howard Fine