Analysis of Gene Expression in Adoptively Transferred TCR- Engineered T Cells

Analysis of gene expression in adoptively transferred TCR- engineered T cells

SITC Annual Meeting Nov 4th, 2011 Daniel Abate‐Daga Surgery Branch. NCI‐NIH. Presenter Disclosure Information

Daniel Abate-Daga, Ph.D.

The following relationships exist related to this presentation:

No Relationships to Disclose Gene-modified PBL are able to mediate objective tumor regression

Park et al, Trends in Biotechnology, 2011 Gene-modified PBL are able to mediate objective tumor regression

Response rate NY-ESO1 TCR: 45% for melanoma, 67 % for SCS

Robbins et al, J. Clin. Oncol., 2011 TCR-transduced T cells persist after transfer but TCR gene expression decreases due to a global down-regulation in gene expression.

Relative Endogenous TCR expression in Vector expressed TCR T cells Infusion

Burns et al, Blood, 2009

Highly activated Resting TCR+ TCR+ cells cells

TCR expression Reactivity

TCR expression Goal: Reactivity Analysis of gene expression: Sample processing

≈‐21 0 ≈30 Time (days) Transduction and REP IL‐2 Leukapheresis Infusion Leukapheresis

1 month post‐ Pre‐infusion Infusion infusion samples FACS SORT Post‐SORT analysis mTCRb CD3

CD3+ mTCR+ Thawed and Thawed Thawed and magnetically FACS‐sorted sorted for CD3+ CD3+ mTCR‐

Donor: patient from gp100 (154) trial Representative sorting results from patients included in analysis

Patient 1 Patient 2 Patient 3 35 days post‐infusion 25 days post‐infusion 31 days post‐infusion

43% 72% 11%

mTCRbeta CD3

‐Received 55.8e9 cells. ‐Received 23.5e9 cells. ‐Received 68e9 cells. ‐No response. ‐Partial response ‐Complete response. Screening for genes involved in down regulation of TCR expression by transcriptomic analysis

Pathway‐focused PCR array

Pros: Genes analyzed -Accurate quantification Lymphocyte Regulatory genes: BTLA, CBLB, CD27, CD28, FAS, FOXP3, IL15, IL2, IL2RA, IL4, LAT, TGFB1, TNFRSF14, TNFSF14, BTLA, CD27, CD40, CD40LG, FOXP1, FOXP3, HDAC9, IL4. -No technical validation required Cytokines, Receptors and their related Proteins: CCL3L1, CCR4, CD40LG, CD70, CSF1, CSF2, FASLG, IFNG, IL10, IL10RA, IL13, IL15, Cons: IL17A, IL1A, IL2, IL2RA, IL2RB, IL31, IL4, IL5, IL6, IL7R, LEP, LTA, PRF1, -Low throughput PTGER2, PTGS2, TGFB1, TNFSF10, TNFSF14, TNFSF8. TNF Superfamily Members and their Receptors: CD40LG, CD70, FAS, FASLG, LTA, TNFRSF10A, TNFRSF4, TNFRSF18, TNFRSF4, TNFRSF8, TNFRSF9, TNFSF10, TNFSF14, TNFSF8.

Transcriptional Regulators: CDK2, CDK4, EGR2, EGR3, EOMES, FOS, FOXP1, FOXP2, FOXP3, GATA3, HDAC9, IFNG, ING4, IRF4, JAK3, JUN, MEF2A, NFATC1, NFATC2, NFATC3, NFKB1, NHLH2, NOTCH1, STAT3, STAT6, TBX21, TGFB1.

Other Genes involved in T‐cell Anergy: CMA1, CTLA4, DGKA, DGKZ, GZMB, ICAM1, ICOS, ITCH, ITGA1, JAK1, LGALS3, PDCD1, PRKCG, RNF128, SELL. Post-infusion mTCRb+ vs Infusion Statistical significance

FC<-2 FC>2 (FC>2, <-2; p<0.05) Gene expression overview Pre Inf Post

Differentiation markers Cytokines

CD27 CD70 IL2 IL10 IL13 LTA

Pre Inf Post Pre Inf Post

CD28 IL2RA IL15 IL5

SELL IL17A CSF2 Relative expression

IL7R GZMB TGFB1 CSF1

Pre Inf Post Pre Inf Post Pre Inf Post Pre Inf Post Gene expression overview (cont.) Pre Inf Post

Co-stimulatory/co-inhibitory receptors Transcription factors PDCD1 LIGHT FOXP3 JUN STAT3

Pre Inf Post Pre Inf Post HVEM NFATC2 STAT6 Relative expression Relative

Pre Inf Post

FOXP1 FOS

Pre Inf Post Pre Inf Post Examples of regulated genes

1- FOXP1

2- PDCD1

3- HVEM / LTA 1) Forkhead box protein 1 (FOXP1)

Adapted from Brown et al, Blood, 2009

9 FOXP1 isoforms described

TCR signaling

FOXP1 Skon and Jameson, Nat Immunology 2011 p<0.05

p<0.001 p<0.001 FOXP1 protein expression

Pre Inf Post Pre Inf Post Pre Inf Post Pre Inf Post

Isoform 1/4 FoxP1 Isoform 3

Tubulin

1.00 0.28 0.14 1.00 0.05 0.39 1.00 0.16 0.56 1.00 0.26 0.52 Densitometry Patient 1 Patient 2 Patient 3 Patient 4 Pre Inf Post 2) PDCD1 (PD-1, CD279)

PDCD1 PD-1 Relative p<0.001 p<0.05 Isotype ctrl Max mTCR+

expression in of change in % gene post-Inf cells expression: Pre Inf Post PD-1

Functional RPMI 624 624-PDL1 effect:

TNFa

IFNg

IFNg (+) cells upon coculture

p=<0.05 1 month post-Inf cells are less reactive against PD-L1 targets: %Percentage of max %Percentage 3) HVEM and binding partners

(-) (-) (+) (+)

T cell

Adapted from Cai, G. and Freeman, G.J., Immunological Review, 2009 HVEM as a receptor in T cells

HVEM LTA p<0.01 p<0.05 p<0.01 p<0.001 Relative change in gene Fold change expression: Fold change Pre Inf Post Pre Inf Post

Functional Media 624 624-LTA effect: 0.76 0.02 17.7 3.7 18.4 8.5 CD107 CD8+ IFNg 98.2 1 76.3 2.3 68.4 4.7

p= 0.005 LTA increases reactivity of infusion TCR cells: Summary

-Of 84 genes analyzed, 38 were found to be differentially expressed in one month post-infusion engineered T cells as compared to matched infusion samples.

Examples:

Transcription factor FOXP1 was dramatically under-expressed in infusion samples compared to pre- infusion T cells and presented intermediate levels in post-infusion T cells.

-PDCD1 was overexpressed in post-infusion T cells, compared to infusion samples. Surface expression was confirmed by flow cytometry and sensitivity to PD-L1 was observed in coculture experiments.

-HVEM and the genes encoding for its binding partners, LTA and LIGHT, were found to be differentially expressed between post-infusion and infusion T cells. Overexpression of LTA resulted in increased production of IFN gamma by infusion T cells in coculture experiments. Potential genes for intervention

TCR expression Reactivity -Knock-down of FOXP1 -Knock-down of PD-1 -Overexpression of LTA

TCR expression Reactivity Acknowledgements

Morgan Lab:

Zhili Zheng,Ph.D. FACS laboratory Samy Chinnasamy, Ph.D. Arnold Mixon Ling Zhang, MD., Ph.D. Shawn Farid Richard A. Morgan, Ph.D. Shannon Rosati, M.D. Steven A. Rosenberg, M.D., Ph.D. Ken‐ichi Hanada, MD., Ph.D. TIL lab Clinical Staff Surgery Branch, NCI Research nurses