Cancer Gene Therapy One T Cell Kills Multiple Tumor Cells David L. Liu, MD, PhD Instructor in Medicine, Assistant in Biology Harvard Cancer Gene Therapy Center BIDMC and Harvard Medical School

Background Landmark of Adoptive Immunotherapy 1977, Rosenberg SA, et al. Passive Immunotherapy of cancer in animals and man 1985 Rosenberg-Lotz, LAK cells 1987 Spiess-Rosenberg, TILs 1987 Yamada, Human IL2 gene in murine T cells 1987 Kuwana, first reported IgTCR chimeric receptor 1995 Treisman-Hwu, IL2 transduced lymphocytes 2001 Maher-Sadelain, chimeric TCR/CD28 receptor 2002 R. Junghans, IgCD28TCR (Tandem) T cells 2003 Liu, IgTCR-GM-CSF transduced T cells

Classic Concept-1 Tumors are killed by LAK cells or TILs or IgTCR or Tandem-transduced T cells that is resulted from proliferation of injected T cells in tumor tissue. Dr. Rosenberg wrote: LAK cells or TILs traffic to and accumulate in tumor deposits. This finding led to attempts to genetically alter TILs to increase their anti-tumor activity at the tumor site.

Classic Concept-2 In the past, Dr. Richard Junghans also wrote in application of research proposal: … He has freshed his concept because several new findings have been observed in BDL Dr. James Mier also said Proliferation of injected T cells in tumor tissue is the mechanism killing tumor, everyone says so. In 2003, an Israel research group still reported “proliferation of intratumorally injected IgTCR-TD T cells in tumor (Cancer Res), this is absolutely untrue.

Classic Concept-3 In the textbook of Cellular and Molecular Immunology, Dr. Abul Abbas writes: 1. CTL killing is antigen specific. 2. CTL killing requires cell contact. 3. CTLs themselves are not injured during lysis of target cells. 4. Suppose each individual CTL is capable of sequentially killing multiple target cells, but so far no direct proof has been demonstrated.

Classic Concept-4  Dr. James Allison presented and demonstrated the modality how antigen presenting cells deliver a signal to T cells or to tumor cells. He was asked to answer how much long is needed for this process?  He didn’t have definite data, but we do have!

Classic Concept-5  Generally, it is easy to follow the hypothesis, assumption and theory that suggested by a great professor or big head.  Universally, it is hard to accept a new finding that conflicts with the traditional concept.

Hypothesis-1  Over the past 7 years, we examined >2000 tumor tissue slides in mice immunized with hybrid tumor vaccines, <5% of samples showed infiltration of T cells in tumor tissue.  During the past 14 months, we examined >100 tumor slides in nude mice treated with IgTCR or Tandem-TD T cells, no proliferation of IgTCR or Tandem-TD T cells was seen in tumor tissue.

Hypothesis-2  A tumor at size of 6x6mm in diameters (approximately 500x10 6 tumor cells) that could be cured with 50x10 6 transduced T cells in our in vivo therapy, how does one transduced T cell kill 5 to 10 tumor cells?

Hypothesis-3 If one T cell can really kill 5 to 10 tumor cells, what modality of killing process is it? Crazed T cells? The killing process needs direct contact of TD-T cells and tumor cells, what contact modality is it? Leech Mouth-Like T Cells? What morphology changes in T cells and tumor cells will happen when kiss-death of TD-T cells and tumor cells is initiated? Both cell types?

Materials and Methods  Human PBLs from blood donor  Transduced with IgTCR or IgTCR-CD28 viral sup.  High percentage of transduced T cells (35-76%)  50x10 6 CFSE labeled and WI2 stained T cells I.v.  Ex vivo killing tests: 5, 10, 15, 30, 60, 90, 120 min.  In vivo proliferation in regressing tumor tissue  Computed digital light microscope  Invital fluoresence microscopy  Confocal microscopy  Flow cytometry

Stains-Double Labeling Of Cells Labeling transduced T cells: CFSE, purified WI2 + GAM Rhodamine Labeling ECA-expressing tumor cells Purified MN14 ab + GAM Rhodamine or PE Anti-human CD3, CD8 T cell Immunochemical staining

IgTCR – chimeric immunoglobulin – T cell receptor Ig CEATCR Modified T Cell Advantage: Conferring the specificity of antibody to TCR and bypassing the MHC-TCR pathway for T cell cytotoxicity It is a prerequisite for a successful adoptive immunotherapy to have a large amount of T cells (10 11 ). Tumor Cell

Cloning of anti-GD3 IgTCR, 2 nd generation CD28 LTR gag scFV (anti-CEA) LTR CD3z CD8h  Nco I Blp 1 2nd generation Anti-CEA IgCD28Zeta LTR gag scFV (anti-GD3) LTR CD3z CD8h  Nco I Blp 1 1st generation Anti-GD3 IgTCR

Structure of the CEA-Ig Chimera Molecule

Constructs of IgTCR, Ig28TCR

Transgene Transfection Protein Viral particle Genome Packaging cell line Target cells Integration polgag env pol gag env Viral RNA DNA Production of Retrovirus Particles Producer cell line

Subpopulation of T cells during transfection CD4/CD8 ratio: on day 7 OKT OKT3+CD PHA 1.51

Positively Transduced IgTCR or IgTCR-CD28 T Cells

76% positively transduced IgTCR T Cells

EXPERIMENTAL GROUPS FIRST COURSE OF KILLING TEST T cells:Tumor cells = 3:1  Jurkat-ATCC (30x10 6 ) + MIP-CEA (10x10 6 )  Jurkat-IgTCR (30x10 6 ) + MIP-CEA (10x10 6 )  Jurkat-Tandem (30x10 6 ) + MIP-CEA (10x10 6 )  Activated T cells (30x10 6 ) + MIP-CEA (10x10 6 )  IgTCR-TD T cells (30x10 6 ) + MIP-CEA (10x10 6 )  Tandem-TD T cells (30x10 6 ) + MIP-CEA (10x10 6 )

FIRST COURSE OF KILLING TEST [2, 3, 4 and 5 days-AICD] T Cells : Tumor Cells at 3:1 J-ATCC J-IgTCR J-Tandem Act. T IgTCR Tandem No killing No killing No killing No Killing 40% 50% No killing No killing No killing No Killing 60% 75% No killing No killing No killing No Killing >90% 100% No killing No killing No killing AICD AICD AICD

RE-ACTIVATION OR RE-KILLING: GROUPING T cells:Tumor cells = 1:1 Activated T cells (30x10 6 ) + MIP-CEA (10x10 6 ) IgTCR-TD T cells (30x10 6 ) + MIP-CEA (10x10 6 ) Tandem-TD T cells (30x10 6 ) + MIP-CEA (10x10 6 ) T cells:Tumor cells = 3:1 Activated T cells (30x10 6 ) + MIP-CEA (10x10 6 ) IgTCR-TD T cells (30x10 6 ) + MIP-CEA (10x10 6 ) Tandem-TD T cells (30x10 6 ) + MIP-CEA (10x10 6 )

RE-KILLING TEST (Day 2, 3 (AICD), 8) T Cells : Tumor Cells at 1:1 T Cells : Tumor Cells at 3:1 Act. T IgTCR Tandem Act. T IgTCR Tandem No killing 5% 5% No Killing 40% 50% No killing 20% 25% No Killing 60% 80% No killing AICD AICD No Killing AICD AICD No killing 40% 10% 60% 25% 10% re-growing re-growing re-growing

CEA - Control CEA+ Regressed

Human CEA+ Cancer Regressed after Gene Therapy

One Gene Transfected T Cell Kills Multiple Tumor Cells

Apoptosis in Tumor Cell

Leech Mouth-Like T Cells

Injected TD-T cells in Spleen (2hs)

Homing or Proliferation of TD-T Cells in Spleen (Enlarged from the last slid)

Necessary Time For Change In T Cell Morphology 5 minno change in morphology 15 minno change in morphology 30 min5-10% T cells had change in morphology 45 min15% T cells had change in morphology 60 min20% T cells had change in morphology 2hs30% T cells had change in morphology

Blue Color Shows Perforin Produced by Gene Transfected T Cells

Blue Color Shows Perforins Produced by Gene Transfected T cells

Centralized Killing Modality of T Cells

Conclusion 1. IgTCR or IgTCR-CD28 gene transfected T cells induced massive necrosis of CEA-expressing human colon cancer from day 1 to day 3 after treatment. 2. A new finding, leech mouth-like T cells are plunged onto surface of CEA-expressing tumor cells, is for the first time described. 3. Direct evidences of one T cell kills >5 tumor cells in vivo have been confirmed by 3 types of specific staining, HE staining, anti-hCD3 immunochemical staining and CFSE labeling-TD T cells. 4. The modality of centralized T cells killing tumor cells in vivo has been consistently observed. 5. Injected TD-T cells in vivo have 3 fates: reactivation and killing tumor cells inside tumor; proliferation in spleen and re-circulation into tumor; and become apoptotic or activation induced cells death. 6. No evidence of proliferation of T cells in tumor was seen.