T-cell Delivery Of Interleukin-12 To The Triggers Potent Endogenous Anti-tumor Responses

Sid Kerkar, M.D. IL-12 Gene for Adoptiv e Cell Transfer Outline

• Background/Objective

• Adoptive cell transfer experiments with murine tumor- specific pmel-1 CD8+ T cells engineered to secrete IL-12

• The importance of receptor specificity for delivery of IL-12 directly to the tumor

• Mechanisms for enhanced tumor treatments IL-12 for Adoptiv e Cell Transfer Outline

• Background/Objective

• Adoptive cell transfer experiments with murine tumor- specific pmel-1 CD8+ T cells engineered to secrete IL-12

• The importance of T cell receptor specificity for delivery of IL-12 directly to the tumor

• Mechanisms for enhanced tumor treatments IL-12 Gene Therapy for Adoptiv e Cell Transfer Adoptive Cell Transfer

Rosenberg SA et al. Nat Rev . 2008; 8:299-308 IL-12 Gene Therapy for Adoptiv e Cell Transfer Immunosuppressive Tumor Microenvironment - +

Zou W. Nat Rev Cancer. 2005 Apr;5(4):263-74. IL-12 Gene Therapy for Adoptiv e Cell Transfer Mouse B16/ Model of Adoptive Cell Transfer

Cell Transfer C57/BL/6 CD8+ T cells Pmel-1 CD8+ T cells or C57BL/6 transduced with Pmel TCR or other hosts Lymphodepleting Irradiation

Blindly assess tumor Tumor Vaccine + IL-2 growth and analyze implantation immune response

A tripartite regimen is effective: i) 1 million Pmel-1 CD8+ cells ii) 2 x 107 plaque-forming units of rVV encoding hgp100 iii) administration of high-dose IL-2: 600,000 IU BID for 3 days

Overwijk WW et al. J Exp Med. 2003 Aug 18;198(4):569-80 IL-12 Gene Therapy for Adoptiv e Cell Transfer Outline

• Background/Objective

• Adoptive cell transfer experiments with murine tumor- specific pmel-1 CD8+ T cells engineered to secrete IL-12

• The importance of T cell receptor specificity for delivery of IL-12 directly to the tumor

• Mechanisms for enhanced tumor treatments IL-12 Gene Therapy for Adoptiv e Cell Transfer Tumor Specific Pmel-1 CD8+ T cells Engineered to Secrete IL-12

• We chose to genetically engineer tumor-antigen specific CD8+ T cells to secrete the heterodimeric IL-12 because of its capacity to augment the functionality of multiple end effectors in adaptive immunity

40 IL-12 MockMock IL-12 Mock 30 1 23 20

IL-12 (ng/ml) 10

CD8+ CD8+ 33 00 1010 1 0 IL-12IL-12 6 7 8 9 101112 NP

-log[hgp10025-33] IL-12 Gene Therapy for Adoptiv e Cell Transfer Tiny Numbers of pmel-1IL-12TD cells cause Regression of Large Established B16

140 140 300 150 )

2 No treatment Mock (1e6) 200 100 IL-12 (1e5) 120 120 IL-12 (5e4)

100 Weight Body% 50

IL-12 (1e4) Body % Weight Tumor Area (mm Area Tumor % Body Weight

100 0 1000 0 10 20 30 00 1010 2020 3030 0 510 15 20 25 30 Time (d) after transfer Time (d) after transfer TimeTime (d)(d) after after transfer transfer IL-12 Gene Therapy for Adoptiv e Cell Transfer Tiny Numbers of pmel-1IL-12TD cells are safe to adoptively transfer in mice

Liver Kidney Lung

NTr y

IL-12 IL-12 Gene Therapy for Adoptiv e Cell Transfer An Example of a Complete Response No Treatment

B16 Melanoma

Day 0 Day 14 Regressing Lesion PmelIL-12TD

B16 Melanoma Complete Response

Day 0 Day 9 Day 14 Day 21 IL-12 Gene Therapy for Adoptiv e Cell Transfer Pmel-1IL-12TD Cells Secrete High Levels Of IL-12 Directly Into The Tumor Microenvironment

d DayDay 77 postpost transfer/Tumortransfer/Tumor MockMock IL-12 00 2323

Thy1.1 Thy1.1 4949 00 5252 0

IL-12IL-12 IL-12 Gene Therapy for Adoptiv e Cell Transfer Outline

• Background/Objective

• Adoptive cell transfer experiments with murine tumor- specific pmel-1 CD8+ T cells engineered to secrete IL-12

• The importance of T cell receptor specificity for delivery of IL-12 directly to the tumor

• Mechanisms for enhanced tumor treatments IL-12 Gene Therapy for Adoptiv e Cell Transfer Generation of CD8+ T cells expressing a) the pmel-1 TCR alone; b) IL-12 alone, or c) both the pmel-1 TCR and IL-12

Pmel TCR Mock Pmel TCR IL-12 + IL-12 4 0 53 0 3224 24 13

β 0 0 40 12 V IL-12 IL-12 Gene Therapy for Adoptiv e Cell Transfer Only CD8+ T cells Expressing both IL-12 and the Pmel-1 TCR Destroy Tumor

300 ) 2 No treatment 200 TCR (1e6) IL-12 (1e6) IL-12(1e6) + TCR (1e6) 100 Double TD: TCR/IL-12 (1e6) Tumor Area (mm Area Tumor

0 0 510 15 20 25 30 35 Time (d) after transfer IL-12 Gene Therapy for Adoptiv e Cell Transfer TCR specificity enhances delivery of IL-12 directly to the tumor site

1000005 Tumor 10000006 Spleen 5 Lymph Nodes 10 10 10100000

100004 * 1000005 4 10 10 1010000

10003 4 3 gram tumor 10000 1000 gram tumor gram 10 10 gram tumor 10 CD8+ Thy1.1+ cells/ cells/ Thy1.1+ CD8+ CD8+ Thy1.1+ cells/ CD8+ Thy1.1+ cells/

2 3 2 10100 101000 10100 1234k 2 123k 2 4 123k 2 4 c R 1 R c R 1 R c R 1 R o C - C o C - C o C - C L L L M T I T 2 M T I T 2 M T I T 2 l l 1 l l 1 l l 1 e e - e e - e e - L L L m m I m m I m m I P P + P P + P P +

* p = 0.037 IL-12 Gene Therapy for Adoptiv e Cell Transfer Exogenous IL-12 is less effective than T cell produced IL-12 on anti-tumor activities of pmel-1 CD8+ T cells

300 No treatment )

2 x Mock (1e6)

200 Mock (1e6) + 0.25µg IL-12/day Mock (1e6) + 1µg IL-12/day Mock (1e6) + 2µg IL-12/day 100 Tc1-Mock (1e5) + Mock (9e5)

Tumor Area (mm IL-12 (1e5) + Mock (9e5)

0 0 510 15 20 25 30 Time (d) after transfer IL-12 Gene Therapy for Adoptiv e Cell Transfer

Outline

• Background/Objective

• Adoptive cell transfer experiments with murine tumor- specific pmel-1 CD8+ T cells engineered to secrete IL-12

• The importance of T cell receptor specificity for delivery of IL-12 directly to the tumor

• Mechanisms for enhanced tumor treatments IL-12 Gene Therapy for Adoptiv e Cell Transfer IL-12 Transduced Pmel-1 CD8+ T cells Express a Distinct Phenotype

We hypothesized that the phenotype and function of tumor-specific T cells transduced with IL-12 were altered in ways that increased their anti-tumor activities

Isotype Mock IL-12 Events CD44CD62L IL-7Rα IL-2Rα Sca-1 IL-12 Gene Therapy for Adoptiv e Cell Transfer IL-12 Engineered Pmel-1 Cells Secrete More IFN- and TNF-α, but Less IL-2

IFN- TNF-α IL-2 a 400 b 15 400 15 15 IL-12 IL-12 IL-12 IL-12 300 Mock Mock 300 Mock Mock 10 10

(ng/ml) 10

200 (ng/ml)  200 α IL-2 (ng/ml) IFN- 5 5 100100 TNF- 5

0 00 0 0 6 7 8 9 10 11 12 NP 6 7 8 9 101112 NP -log[hgp100 ] -log[hgp10025-33] 25-33 -log[hgp100 25-33 ] c IL-12 Gene Therapy for Adoptiv e Cell Transfer IL-12 Engineered Pmel-1 Cells have Increased Expression of T-bet and Down Regulation of Eomesodermin

Mock IL-12

8 1

4 0.5

Relative mRNA Relative 0 0 1 1 T-bet Eomes IL-12 Gene Therapy for Adoptiv e Cell Transfer PmelIL-12TD Cells are Less Lytic Against Peptide Pulsed Targets In Vitro

100 IL-12 Mock 50 IL-12 (NP) Mock (NP) Percent (%) Lysis (%) Percent 0 90 30 10 3 E:T Ratio IL-12 Gene Therapy for Adoptiv e Cell Transfer IL-12rβ2-/- Mice Were Used to Examine the Functional Importance of Cell-Intrinsic Characteristics

Pmel C57BL/6: Il12rb2-/-

X

Pmel-1 Il12rb2-/-

• Confirmed no differences in CD3+, CD4+, and CD8+ T cells in the spleen IL-12 Gene Therapy for Adoptiv e Cell Transfer Il12rβ2-/- Pmel-1IL-12TD Cells do not Display the Distinct Phenotype and Function of Wild Type (WT) Pmel-1IL-12TD Cells aa -/- Il12rb2IL-12R-/-IL-12IL-12 IL-12R-/-cells cells IL-12WTIL-12 WT cellscells Events Events

CD62L IL-2Rαα IL-7RIL-7Rαα Sca-1Sca-1 IL-12 IL-12IL-12 -/- b IL-12R-/-Il12rb2IL-12R-/-IL-12R-/- cells WTIL-12IL-12WT cells 50 9494

CD8+ CD8+ 33 0 0 1 IFN-IFN- IL-12 Gene Therapy for Adoptiv e Cell Transfer Il12rβ2-/- Pmel-1IL-12TD can Treat Tumors Similar to Wild Type (WT) Pmel-1IL-12TD Cells

300 ) 2 No treatment No treatment IL-12 200 IL-12RIl12rb2β-/-2-/-cells cells (1e5) (1e IL-12R-/- cells (1e5) WTWT cellscells (1e5)(1e5)(1e5)

100 Tumor Area (mm Area Tumor

0 0 510 15202530 Time (d) after transfer IL-12 Gene Therapy for Adoptiv e Cell Transfer Pmel-1IL-12TD are Less Effective in Il12rβ2-/- Mice Bearing Tumors

300 ) 2 IL-12RIl12rb2IL-12R-/-β2-/--/- Hosthost Host 200 No treatmenttreatment IL-12IL-12 (1e5)(1e5) WTWT HostHost 100 No treatmenttreatment Tumor Area (mm Area Tumor IL-12IL-12 (1e5)(1e5)

0 0 510 15 20 25 30 Time (d) after transfer IL-12 Gene Therapy for Adoptiv e Cell Transfer Pmel-1IL-12TD are Less Effective in IFN-R-/- Mice Bearing Tumors

300 ) 2 IFN-IfnrR-/--/- Host Host 200 No treatment IL-12 (1e5) WT Host 100 No treatment Tumor Area (mm Area Tumor IL-12 (1e5) * 0 0 510 15 20 25 30 35 Time (d) after transfer

 p = 0.014; Wilcoxon rank sum test IL-12 Gene Therapy for Adoptiv e Cell Transfer Pmel-1IL-12TD are more effective following lymphodepletion

300 ) 2 No Treatment 200 No treatment (5 Gy) IL-12 (1e5) IL-12 (1e5) (5 Gy) 100 Tumor Area (mm Area Tumor

0 0 510 15 20 25 30 Time (d) after transfer IL-12 Gene Therapy for Adoptiv e Cell Transfer Decreased Numbers of Intra-tumoral CD4+ Foxp3+ cells following 5 Gy Irradiation

Tumor Tumor 15 150 0 * Thy1.1- CD4+ cells / ) 2 0 GyGy 10 100 0 55 Gy Gy cells (10 + CD4

- 5500 gram tumor Thy1.1 Events 0 0 12 y y G Foxp3 G 0 5 IL-12 Gene Therapy for Adoptiv e Cell Transfer Conclusion

• Tiny numbers of tumor-specific CD8+ T cells engineered to produce IL-12 triggered the destruction of large vascularized tumors

• The expression of a tumor antigen-specific T cell receptor allowed for preferential delivery of IL-12 directly to the tumor microenvironment and was required for successful tumor treatments

• Host anti-tumor immunity triggered by IL-12 at the tumor site were critical for treatment responses IL-12 Gene Therapy for Adoptiv e Cell Transfer Clinical Applicability

• Gene Engineering with IL-12 may dramatically improve cellular against metastatic cancer

• Decreasing the number of cells required for adoptive transfer may help facilitate the widespread acceptance of a ACT

• These findings may open possibilities for delivering other molecules to the tumor microenvironment designed specifically to elicit endogenous anti-tumor immunity Acknowledgments

Pawel Muranski Paul Robbins Andrea Boni Ling Zhang Zhiya Yu Richard Morgan Luis Sanchez-Perez Lydie Cassard James Yang Andrew Kaiser Udai Kammula Luca Gattinoni Richard Sherry Doug Palmer Rob Reger Yun Ji Chris Klebanoff Zach Borman Madhu Sukumar

GIORGIO TRINCHIERI NICK P. RESTIFO STEVEN A. ROSENBERG