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

Steven A. Feldman, Ph.D.S B hSurgery Branch

National Cancer Institute

NCT ConferenceHeidelberg, GermanySeptember 24 2013September 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 i3. Passively transfer activated immune cells with anti-tumor activity • Adoptive cell transfer• Adoptive cell transfer

Advantages of Cell Transfer TherapyAdvantages 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 igrown in vitro.

4 The host can be manipulated to provide a favorable tumor4. 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 theof 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 normal2. 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 2IL-2Cytokine

IL 12IL-12

murine ( 100 NY ESO 1 MAGE A3)murine (gp100, NY-ESO-1, MAGE-A3)

TCRhuman (DMF5 NY ESO 1)human (DMF5, NY-ESO-1)

2nd gen 28Z (CD19 Meso)2nd gen-28Z (CD19, Meso)

CAR3rd gen 28BBZ (EGFRvIII VEGFR2)3rd gen-28BBZ (EGFRvIII, VEGFR2)

Interleukin 12

• IL-12 is a heterodimeric cytokine composed of a heavy chainIL 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 weeksStimulated with OKT-3, transduced and expanded, pInfuse after Cy/flu preparative regimenNo IL-2 administered

Cohort Number Result(# cells x 10-9) of patients

___________________________________________________________________________

0.001 1 1NR0.003 1 1NR0.01 7 7NR0 03 5 1CR (24+ mos); 4NR0.03 5 1CR (24+ mos); 4NR0.1 3 3NR0.3 3 3PR (4, 6, 12+)1.0 4 1PR (12+); 3NR3 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 gserum. 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 2IL-2Cytokine

IL 12IL-12

murine ( 100 NY ESO 1 MAGE A3)murine (gp100, NY-ESO-1, MAGE-A3)

TCRhuman (DMF5 NY ESO 1)human (DMF5, NY-ESO-1)

2nd gen 28Z (CD19 Meso)2nd gen-28Z (CD19, Meso)

CAR3rd gen 28BBZ (EGFRvIII VEGFR2)3rd gen-28BBZ (EGFRvIII, VEGFR2)

T-cell Receptor (TCR) Gene Therapy

TCR Cloning

IVSImmunize mice

TCR Vector (eg, MART1, NY‐ESO)

SD SA

TCR receptor

α β

TCRβΨ

TCRα 2A LTRLTR

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 TypesTypes

80R

50

60

70

r by

 RT‐PC

R

MAGE‐A3

30

40

50

tive tu

mor MAGE‐A1

NY‐ESO‐1

0

10

20

r C a n r a a

% of p

osit

Blad

der

NSCLC

Melan

oma

Ovarian

patocellu

lar

Myeloma

ll carcinom

a

He

quam

ous cel

Tumor Type

Sq

Tumor Type

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

LNZAT3WT4MSGINAPB, 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

0 100 200 300 400 500 600

624.38mel

IFN pg/ml

+ + Melanoma

IFN-, pg/ml

Phase II Study of Metastatic Cancer thatPhase 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 NYESO-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 ytoxicities.

Total PR CR ORTotal 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+)

*t t d t i*treated 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 TCRsp

2. Inability to target lipid / carbohydrate moleculesy g p y

3. Potential tumor “escape” via MHC loss / alterations in antigen3. Potential tumor escape via MHC loss / alterations in antigen processing

Surgery Branch Gene Therapy Products(by class)(by class)

IL 2IL-2Cytokine

IL 12IL-12

murine ( 100 NY ESO 1 MAGE A3)murine (gp100, NY-ESO-1, MAGE-A3)

TCRhuman (DMF5 NY ESO 1)human (DMF5, NY-ESO-1)

2nd gen 28Z (CD19 Meso)2nd gen-28Z (CD19, Meso)

CAR3rd gen 28BBZ (EGFRvIII VEGFR2)3rd gen-28BBZ (EGFRvIII, VEGFR2)

Chimeric Antigen Receptors (CARs)

Step 2Step 1 Step 3

Ig ScFv

Linker/TM

T cell signalingAntibody Producing Hybridoma Ig Genes

Chimeric Antigen Receptor (CAR)

CAR receptor

sd sa

CAR (CD19, Meso, EGFRvIII, VEGFR2

VL

VH

VL

VHscFvCD28 CD3 zetaAnti-tumor Ag-scFvLTR LTR

sd sa

CD28

CD3ζCD28 CD3 zetaLTR LTRsd sa

CD8 4-1BBAnti-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 precursorsCD19 is expressed by mature B cells, B cell precursors and plasma cells but not any other normal tissues.

Anti‐CD19 CAR

3’ LTRCD28                 FMC63 scFv CD3‐zeta5’ 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 CD20Kochenderfer 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 LymphomaZone 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 bTotal cyclo- Number of Response

Patient Age/Gender MalignancyNumberof prior

therapies

yphosphamide

dose(mg/kg)

CAR+ T cellsinfused

(X106/kg)

p(time after cellinfusion inmonths)

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 NE4 30/F PMBCL 3 120 2.5 NE5 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 conclusionsT 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 obtundation. The duration of these toxicities was short.

• Toxicity correlated with serum levels of inflammatory y ycytokines.

A Critical Challenge Confronting the Development of Human Cancer Immunotherapy is theof 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 normal2. 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 Cancersy

Receptor Type Cancers StatusIL‐12 Cytokine Adjuvant for all receptors Accruing

MART‐1 TCR Melanoma Closed

gp100 TCR Melanoma Closed

CEA TCR Colorectal ClosedCEA 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 developmentSSX 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 Accruingg

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 vivoantigen 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 o pa e s co oepithelial cancers.

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

Acknowledgments:

Surgery Branch, NCI:Steven A. Rosenberg, Chief

Lab of Molecular Biology, NCIIra Pastang,

Rick MorganMark DudleyJohn WunderlichPaul Robbins

Tapan Bera

Hematology Branch, NHLBIPaul RobbinsJames YangMaria ParkhurstNick Restifo

Dhana Chinnasamy

ETIB, NCIJim KochenderferSBVPF

TIL LABFACS LABClinical Staff

Jim Kochenderfer

Pediatric Oncology Branch, NCILing ZhangClinical Staff Ling Zhang

NYU Langone Medical CenterHoward Fine