Adoptively transferred tumor-specific T cells expanded ex vivo using HSV amplicons encoding 4-1BBL persist in the host and

demonstrate anti-tumor activity in vivo Kyung H. Yi1,4, Hovav Nechushtan5, William J. Bowers2,3, Dien G. Pham5, Yu Zhang5, Khaled A. Tolba5, Eckhard R. Podack4, Howard J. Federoff1,2,3, and Joseph D. Rosenblatt4,5,6,7 Departments of Microbiology and Immunology1, Neurology2, and the Center for Aging and Developmental Biology3, University of Rochester School of Medicine, Rochester, NY 14620, Department of Microbiology and Immunology4, University of Miami School of Medicine, Miami, FL 33136, and University of Miami Sylvester Comprehensive Cancer Center5, Miami, FL 33136.

Address correspondence to: Joseph D. Rosenblatt, University of Miami Sylvester Comprehensive Cancer Center, Division of Hematology/Oncology, 1475 NW 12th Avenue (D84), Suite 3300, Miami, FL 33136. Phone: 305-243-4860; Fax: 305-243-9161; email: jrosenblatt@med.miami.edu.

Running title: 4-1BBL in adoptive immunotherapy

Key words: Costimulation, T lymphocytes, adoptive immunotherapy, HSV amplicons, 4-1BBL

Footnotes
6

This work was supported by National Institutes of Health Grant RO1 CA87978 and RO1 CA74273, the Leukemia and Lymphoma Society of America, and the Rochester Nathan Shock Center.
7

Address correspondence and reprint requests to Dr. Joseph Rosenblatt, University of Miami Sylvester Comprehensive Cancer Center, Division of Hematology/Oncology, 1475 NW 12th Avenue (D8-4), Suite 3300, Miami, FL 33136. Email address: jrosenblatt@med.miami.edu
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Abbreviations used in this paper: 4-1BBL, 4-1BB ligand; LLC, Lewis lung carcinoma; HSV, herpes simplex virus I; LacZ, -galactosidase; MOI, multiplicity of infection.

Abstract 4-1BB is a T cell costimulatory receptor which binds its ligand 4-1BBL, resulting in prolonged T cell survival. We studied the anti-tumor effects of adoptively transferred tumorspecific T cells expanded ex vivo using tumors transduced with herpes simplex virus (HSV) amplicons expressing 4-1BBL as a direct source of antigen and costimulation. We constructed HSV amplicons encoding either 4-1BBL (HSV.4-1BBL) or B7.1 (HSV.B7.1) costimulatory ligands. Lewis lung carcinoma cells expressing ovalbumin (LLC/OVA) were transduced with HSV.4-1BBL, HSV.B7.1, or control HSV and used to stimulate GFP+ OVA-specific CD8+ T cells (OT-1/GFP). Nave or ex vivo-stimulated OT-1/GFP were then adoptively transferred into LLC/OVA tumor-bearing mice. Mice receiving OT-1/GFP stimulated with HSV.4-1BBL had significantly decreased tumor volumes compared to untreated mice (P<0.0001) or mice into which nave OT-1/GFP were transferred (P=0.0003). In contrast to HSV.4-1BBL-expanded OT1/GFP, transfer of HSV.B7.1-stimulated OT-1/GFP did not protect mice from tumor. Higher percentages of OT-1/GFP cells were seen in the peripheral blood for the HSV.4-1BBLstimulated OT-1/GFP group compared to other experimental groups. Splenocytes from mice that received HSV.4-1BBL-stimulated OT-1/GFP exhibited increased cytolytic activity against LLC/OVA and higher percentages of OT-1/GFP cells, which expressed CD44 and the memory marker Ly-6C, compared to controls. Increased tumor-free survival was observed 45 days following adoptive transfer of HSV.4-1BBL-stimulated OT-1 compared to transfer of nave OT1 into tumor-bearing mice. Tumor-specific T cells stimulated ex vivo using tumor transduced with HSV.4-1BBL expand in vivo following adoptive transfer, resulting in tumor eradication and the generation of tumor-specific T cells of memory phenotype.

Introduction Adoptive transfer of autologous tumor-specific T cells is a promising procedure for cancer immunotherapy. T cells isolated from tumor-infiltrating lymphocytes, tumor-draining lymph nodes, or peripheral blood contain tumor-specific cells, which can be expanded ex vivo and transferred back into the host to treat established disease. Several types of cancers including metastatic melanoma (1), renal cell carcinoma (2), and glioma (3), show encouraging results when treated with ex vivo-expanded cytotoxic T lymphocytes. Clinical trials have indicated that immune ablation is an effective preconditioning procedure that can increase T cell responses after adoptive transfer (1). Additional strategies are needed to improve generation of more potent tumor-reactive T cells for adoptive transfer. Areas of development include optimization of in vitro T cell expansion and culture, characterization of potent T cells for transfer, and preservation of function and survival of transferred cytotoxic T cells. Under favorable circumstances, adoptive transfer has the potential to induce long-lasting effects via the establishment of immunologic memory. 4-1BB (CD137, ILA, TNFRSF9) is a 30-kDa type I transmembrane glycoprotein belonging to the TNF receptor superfamily (4, 5). Expression of the transmembrane form of 41BB has been observed in a broad range of both myeloid and lymphoid cells, including CD4+ and CD8+ T cells, intraepithelial lymphocytes, natural killer cells, monocytes, and dendritic cells (5-7). 4-1BB is usually induced on cell surfaces following activation, but dendritic cells express 4-1BB constitutively (7). This is in contrast to CD28, which is expressed on the nave T cell. The temporal expression of 4-1BB suggested that CD28 might relay an initial costimulatory signal followed by 4-1BB signaling to further shape the T cell response. Ligation of 4-1BB induces cytokine secretion, especially IFN-, and significant cell proliferation and survival of T cells in

vitro and in vivo (5, 8-13). In vivo studies show 4-1BB costimulation plays a crucial role in the generation of effector and memory cytotoxic T lymphocytes (CTLs) by increasing the overall number of CTL produced (9, 14). Manipulation of 4-1BB responses has been suggested for purposes of tumor immunotherapy. Administration of agonistic anti-4-1BB monoclonal antibody (mAb) greatly enhanced tumor-specific CTL responses in mice and eradicated established tumor even in tumor models considered to be poorly immunogenic (15). In addition, antibodies against 4-1BB remarkably improved the anti-tumor effects seen with adoptive transfer of CD8+ T cells specific for tumor antigens (16-18). 4-1BB-mediated anti-tumor effects have been ascribed to the prevention of programmed cell death in T cells, thereby promoting accumulation of anti-tumor effector populations (16, 19). Previous experiments have demonstrated effectiveness of 4-1BBL8 gene transduction for anti-tumor immunity (20-23). Melero et al. first reported using a retroviral vector to stably transduce 4-1BBL into P815 mastocytoma and AG104A sarcoma cell lines (22). Mice inoculated with P815 tumor cells expressing 4-1BBL developed a strong CTL response and long-term immunity against wild-type tumor. In subsequent trials using B cell lymphoma (20) and squamous cell carcinoma (23) cell lines, gene transfer of 4-1BBL by retrovirus also induced CTL and reduced tumor growth. Other studies using replication-defective adenovirus for gene delivery showed enhanced survival and systemic protection against hepatic metastases induced by colon carcinoma with a combination of 4-1BBL and IL-12 gene transfer (21). We used herpes simplex virus I (HSV) amplicons as a vehicle for gene transfer of 41BBL. HSV amplicons were used to transduce costimulatory molecules into tumor cells because of their broad cellular tropism, large transgene capacity, and very high levels of gene expression.

Because of the ability of HSV amplicons to readily transduce primary tumor cells, we reasoned that HSV amplicons encoding 4-1BBL could be used to facilitate direct antigen presentation by tumor cells and to expand tumor-specific effector populations for adoptive transfer. We have noted activation of several toll-like receptors (TLRs) including TLR2 and TLR9 by administration of HSV amplicons in macrophage cells lines as well as human chronic lymphocytic leukemia (CLL) (Tolba K et al. unpublished data). HSV transduction can lead to cytokine induction and NKG2DL expression of transduced tumor cells as well as activation of anti-tumor CD8+ T cells (unpublished data). By triggering the innate immune response, HSV amplicons may serve to facilitate a more specific adaptive response. We also hypothesized that 4-1BBL-expanded CD8+ T cells would show desirable effector properties, including in vivo expansion and therapeutic efficacy in an immune replete host, as well as potentially confer a memory response. We used HSV.4-1BBL amplicons to transduce tumor for purposes of activating and expanding tumor-specific CD8+ OT-1 cells in vitro, and studied the behavior of adoptively transferred ex vivo expanded cells in LLC/OVA tumor-bearing mice. Our studies demonstrate that 4-1BBL expressed from HSV amplicons (HSV.4-1BBL) has the potential to induce significant expansion of cytolytic T cells in vitro and in vivo and that the adoptive transfer of expanded T cells may result in reduction of tumor growth in vivo as well persistence of CD44hiLy-6Chi tumor-specific memory T cells.

Materials and Methods

Animals and cells C57BL/6 mice were obtained from Jackson Laboratories. OT-1 TCR transgenic mice, a gift from Dr. M. Bevan, were bred to wild-type C57BL/6 mice to generate mice heterozygous for the OT-1 TCR transgene. OT-1 mice express a transgenic TCR that is specific for OVA (257264) (SIINFEKL) peptide bound to H-2Kb. GFP+ mice were obtained by permission of the producers (24); they were bred with OT-1 mice to generate OT-1/GFP T cells expressing green fluorescent protein. Mice were maintained in pathogen-free animal facilities at the University of Miami, and all procedures were performed in agreement with the Institutional Animal Care and Use Committee, according to the National Institutes of Health guidelines for animal use. Lewis lung carcinoma (LLC) stably transfected with ovalbumin (LLC/OVA) in pACNeo-ova was (25) and grown in IMDM plus 10% FBS, penicillin (50 units/ml), streptomycin (50 g/ml), 20 M 2-ME (I-10), and 1 mg/ml Geneticin.

Antibodies Directly conjugated monoclonal antibodies, including PE-conjugated anti-mouse CD3, CyChrome- (PE-Cy5-) or PE-conjugated anti-mouse CD8a, FITC-conjugated anti-mouse CD4, FITC-conjugated anti-mouse V2, and PE-conjugated anti-mouse V5.1,5.2 (BD PharMingen, San Diego, CA), were used to stain for T cells. Prior to staining, spleen cells were treated with purified anti-mouse CD16/CD32 (Fc-III/II receptor, BD PharMingen) to block Fc-mediated binding. Antibodies used to assess activation and/or differentiation states of T cells include: antiCD44-PE (eBiocscience), anti-human Granzyme B-PE (Caltag, Burlingame, CA), anti-mouse CD25-biotin (7D4) (BD PharMingen) or anti-mouse-Ly-6C-biotin (BD PharMingen) followed

by streptavidin-PE (Sigma, St Louis, MO), and purified anti-CD107a (1D4B) (BD PharMingen) followed by anti-rat IgG (H+L)-PE (Caltag). Tumor transduction was verified by staining for human B7.1 with anti-human CD80-FITC (BD PharMingen) and for murine 4-1BBL with anti4-1BBL (BD PharMingen), anti-rat IgG (H+L)-biotin (Caltag), and streptavidin-PE (Sigma).

Purification of CD8+ OT-1 cells Spleen cells from OT-1 TCR transgenic mice were purified by positive column selection using MACS anti-CD8a (Ly-2) MicroBeads (Miltenyi Biotec, Auburn, CA). Splenocytes were resuspended in 90 l of buffer (PBS containing 0.5% BSA) and 10 l of Ly-2 microbeads per 107 cells. After 15 min incubation at 4C, cells were washed and used for magnetic separation on LS columns. Purified cells were >95% CD8+ as judged by FACS analysis using antibodies (BD PharMingen) to CD8, variable (V) region (V2), and V (V5.1,5.2) chains.

HSV-amplicon-vector construction and helper virus-free packaging The cDNA of murine 4-1BBL with a KpnI restriction enzyme site 5 and NheI site 3 was amplified by RT-PCR from RNA of C57BL/6 spleen stimulated with LPS (15 g/ml) for 24 hours using the 5 primer: 5-GGTACCGCCATGGACCAGCACACACTTG-3 and the 3 primer: 5-GCTAGCTTCCCATGGGTTGTCGGGTTTCAC-3 based on its published

nucleotide sequence (26). The cDNA was inserted into pCR-Script Amp SK(+) (Stratagene, La Jolla, CA) (415.pCRScript) and again amplified by PCR, but with BamHI at its 5 and the stop codon plus EcoRI at its 3 using and the the 5 3 primer: primer: 55-

TCGGATCCGTAATGGACCAGCACACACTTG-3

GAGAATTCTCATTCCCATGGGTTGTCGGGTTTCAC-3.

The complete murine 4-1BBL

cDNA was then cloned into the polylinker region of the HSV-1 amplicon vector pHSVPrPUC (27) using the BamHI and EcoRI restriction enzyme sites and sequence verified. The cDNA of human B7.1 was cloned into HSVPrPUC as previously described (28). Packaging of virus vectors into helper-virus-free replication-defective HSV-1 viral amplicons was performed as described previously (29, 30). Purified and concentrated viral pellets were resuspended in PBS and stored at -80oC until use. Vector titers were determined as described previously by using expression and transduction titering methods (31). Amplicons containing the gene for Escherichia coli -galactosidase (LacZ) were prepared using the same vector system described above as a control for HSV transduction.

OT-1/GFP in vitro expansion for in vivo administration LLC/OVA tumor cells were treated with 2.5mM EDTA, washed in I-10, and resuspended at 106 cells/100 l in polypropylene tubes. LLC/OVA cells were transduced with either helper virusfree HSV.4-1BBL (MOI=1) or HSV.B7.1 (MOI=1) and incubated at 37oC for 1 h before being transferred to a six-well plate. One day later, transduced LLC/OVA were resuspended at 5 x 106 cells/ml, treated with 0.4 mg/ml mitomycin C for 20 min at 37oC in PBS, and washed three times in RPMI 1640 plus 10% FBS, penicillin (50 units/ml), streptomycin (50 units/ml), and 50 M 2ME (R-10). Freshly isolated OT-1/GFP cells were then plated with mitomycin C-treated LLC/OVA at an E:T ratio of 3:2 for three days in 24-well plates. Each well was plated with 2.4 x 106 OT-1/GFP cells plus 1.6 x 106 tumor cells in 2 ml, for a total of 4 x 106 cells/2ml. Supplemented R-10 media (0.5 ml) was added to each well after 2 days, and cells were harvested for in vivo adoptive transfer on the third day of co-culture.

Intracellular staining and flow cytometry Cells were washed, blocked for FcIII/II receptors using anti-CD16/32 (BD PharMingen), and stained on the surface with fluorochrome-conjugated anti-CD4 and anti-CD8a antibodies (BD PharMingen) in PBS at 4oC for 20 min. Cells were then washed with PBS and fixed using Cytofix/Cytoperm buffer (BD PharMingen) for 20 min at 4oC. 0.1% saponin/1%FBS in PBS was used to wash, stain with fluorochrome-conjugated antibodies, and wash again. Cells were analyzed using an LSR flow cytometer (BD Biosciences, San Jose, CA) and CellQuest software (BD Biosciences).

Blood collection and preparation for flow cytometry Sodium heparin from 10 ml Vacutainer blood collection tubes (Becton Dickinson, Franklin Lakes, NJ) was resuspended in PBS (5 ml) and aliquoted into microfuge tubes (100 l/tube). Mice were gently warmed with heat lamp, and blood (approximately 5 drops) was collected from the tail into microfuge tubes. Blood was transferred into flow tubes containing 3 ml of ACK lysing buffer. Samples were kept at room temperature for 5 min and centrifuged at 1400 rpm. Treatment with ACK lysing buffer was conducted twice more, and the cells were resuspended in PBS for antibody staining.

Detection of GFP+ cells in frozen tissue sections Spleens were frozen in O.C.T. compound (Sakura Finetek, Torrance, CA) with dry ice and stored at -80oC until sectioning. Tissues were cut 6 m thick using an electronic cryotome (Shandon, Pittsburgh, PA) and adhered onto Superfrost plus glass slides (VWR, West Chester, PA). Slides were kept cold to prevent diffusion of GFP and exposed in a closed-lid container to vapor from a

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napkin soaked with 37% formaldehyde (Sigma) at -20oC for 24 h, as first described by Jockusch et al. (32). Tissues were then outlined with ImmEdge pen (Vector Laboratories, Burlingame, CA), washed with PBS, and counterstained with 1 g/ml Hoechst 33342 (Sigma) for 15 min at 37oC. After washing with PBS, slides were mounted with Prolong Gold antifade reagent (Molecular Probes/Invitrogen, Eugene, OR). Sections were viewed using a Leica DMIRB Inverted Microscope (Bannockburn, IL) and captured with MetaMorph Imaging System (Molecular Devices Corporation, Downingtown, PA).

Tumor measurements and statistics Tumors were measured at two different diameters (D1 and D2) using a caliper. Tumor volume was calculated with the following formula: Tumor volume = 4/3((D1+D2)/4)3. For calculations of statistical significance, commercial software (StatView; SAS Institute, Cary NC) was used. Regression analysis was used for computing P values.

Cytotoxic T lymphocyte (CTL) assay Splenocytes were harvested and passed through a nylon mesh to make a single-cell suspension. Following red blood cell lysis with ACK buffer, splenocytes were enumerated and incubated with mitomycin C-treated LLC/OVA at a ratio of 10:1 for 5-6 days with recombinant murine IL2 (10-20 U/ml) in R-10 media. LLC/OVA and LLC targets (2 x 106 each) were labeled with 300 l
51

Cr (150 l/106 cells) for 5 h and washed in R-10 media. Splenocytes were collected,

counted, spun down to add fresh R-10 media, and plated in 96-well round-bottom plates according to the indicated E:T (effector:target) ratios in 100 l. Each sample dilution was plated in triplicate. Targets were then washed three times to remove excess
51

Cr and plated at 5 x 104

11

cells/100 l/well. Plates were briefly spun at 1400 rpm to increase contact between effectors and targets and incubated at 37oC for 8 h. After plates were spun at 1400 rpm for 5 min, supernatant (100 l) was collected from each well and added to 2ml of Ready Safe Liquid scintillation cocktail for aqueous samples (Beckman Coulter, Fullerton, CA). Samples were counted on a LS 6500 multi-purpose scintillation counter (Beckman Coulter). Percent lysis = (Sample counts Spontaneous counts)/(Maximum counts) * 100. Maximum counts were assessed from target lysis with Triton X-100.

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Results Tumors transduced by HSV.4-1BBL express murine 4-1BBL and can induce proliferation of CD8+ OT-1 T cells. Murine 4-1BBL cDNA was cloned into the herpes simplex virus I (HSV) amplicon vector pHSVPrPUC and packaged into replication defective HSV-1 viral particles, or amplicons (HSV.4-1BBL) using a helper virus-free methodology (Fig. 1A) (29, 30). The helper virus-free system employs a bacterial artificial chromosome (BAC) that encodes the entire HSV genome minus its cognate cleavage/packaging signals. This method leads to the production of virions that contain only amplicon genomes, without the propagation of cytotoxic helper virus particles (Fig. 1A). Human B7.1 was also packaged into HSV amplicons (HSV.B7.1) as previously described (28) and used in the following experiments to compare 4-1BB costimulation with stimulation through the CD28 receptor. Both mouse and human B7.1 can stimulate T cell CD28 receptors of either species (24). Once the HSV.4-1BBL and HSV.B7.1 plasmids were each packaged into amplicons, we tested the ability of the amplicons to transduce Lewis lung carcinoma stably expressing ovalbumin (LLC/OVA) and the parental LLC tumor cell line. As a control for effects of HSV amplicon transduction on endogenous costimulatory ligand expression, HSV.LacZ, which encodes bacterial -galatosidase, was used. Both LLC and LLC/OVA cells transduced with HSV.B7.1 or HSV.4-1BBL showed high levels of expression of B7.1 or 4-1BBL, respectively, by day 2 as shown by flow cytometric analysis (Fig. 1B). Several other murine tumor cell lines, including T cell lymphoma EL4, B cell lymphoma A20, melanoma B16-F10, and mammary adenocarcinoma EMT6, were also readily transduced by both amplicons (data not shown). HSV.LacZ did not induce either B7.1 or 4-1BBL on transduced LLC/OVA (Fig.1B). In addition,

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HSV.B7.1 did not induce 4-1BBL and vice versa (data not shown). Therefore, HSV amplicons can be used to efficiently express B7.1 and 4-1BBL on tumor cells. In order to more accurately follow the effects of 4-1BBL on T cells that are specific for tumor, we utilized the adoptive transfer of CD8+ OT-1 T cells, which express a transgenic TCR specific for OVA257-264 (SIINFEKL) peptide bound to H-2Kb, in our in vivo mouse experiments. Adoptive transfer of T cells from TCR transgenic mice into normal recipients provided a means of monitoring antigen-specific T cells during a response (33, 34). We used OT-1/GFP cells that were derived from OT-1 transgenic mice backcrossed into GFP+ mice to facilitate monitoring. Following adoptive transfer into normal C57BL/6J mice, OT-1/GFP cells were easily detectable by GFP+ fluorescence. Functionality of B7.1 or 4-1BBL on transduced LLC/OVA tumor cells was subsequently assessed by using the tumor cells to induce proliferation of CD8+ OT-1 T cells (Fig. 1C). LLC/OVA cells, which are recognized by OT-1, were transduced with HSV.B7.1, HSV.4-1BBL, or HSV.LacZ. One day post-transduction LLC/OVA cells were treated with mitomycin C to prevent proliferation and used to stimulate CD8+ OT-1 cells for 3 or 5 days. At day 3, robust proliferation was seen for OT-1 cells stimulated with tumor transduced with either HSV.B7.1 or HSV.4-1BBL (7-fold and 6-fold, respectively, compared to non-transduced tumor) (Fig. 1C). LLC/OVA cells transduced with HSV.LacZ did not augment proliferation of OT-1 in vitro. OT-1 stimulated with HSV.4-1BBL-transduced LLC/OVA continued to proliferate vigorously at day 5 (4-fold greater than with non-transduced tumor), although OT-1 stimulated with HSV.B7.1-transduced LLC/OVA proliferated at levels similar to non-transduced tumor (Fig. 1C). This suggested that initial proliferation could be prolonged in the presence of 4-1BB costimulation relative to that seen with costimulation through CD28. OT-1 cells which were

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cultured with parental LLC tumor with or without costimulatory ligands did not induce proliferation, signifying an absolute requirement for signal one in T cell activation (data not shown).

OT-1 cells costimulated ex vivo with 4-1BBL display an activated effector phenotype. The phenotype of expanded OT-1/GFP cells was characterized after three days of coincubation with tumor cells (Fig. 1D). CD44 is expressed on activated T cells and functions in lymphocyte homing and adhesion. CD25 (IL-2R) is a component of the high-affinity IL-2 receptor and is also upregulated on activated T cells. Granzyme B is a serine protease stored in the granules of cytotoxic T cells along with perforin (35). CD107a (LAMP-1) is a widely expressed intracellular antigen that appears on the T cell surface following activation-induced degranulation and has been used to enumerate activated antigen-specific CD8+ cytotoxic T cells (36). OT-1/GFP stimulated ex vivo with either HSV.4-1BBL- or HSV.B7.1-transduced LLC/OVA expressed high levels of CD44, intracellular granzyme B, and CD107a and modestly increased levels of CD25, indicating that they were activated and capable of cytotoxic activity (Fig. 1D). HSV.LacZ-stimulated OT-1/GFP also expressed CD44 and CD107a, but at lower levels than were observed for those stimulated with HSV.4-1BBL or HSV.B7.1. Nave OT1/GFP cells were used as a negative control for activation and did not express any of the aforementioned activation markers. Ly-6C is a marker for previously activated T cells and memory CD8+ T cells (37). Expression of Ly-6C was the highest in HSV.4-1BBL-stimulated OT-1/GFP, compared to HSV.B7.1- and HSV.LacZ-stimulated OT-1/GFP and nave OT-1/GFP (Fig. 1D).

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T cells costimulated with 4-1BBL ex vivo readily expand in vivo in response to tumor. HSV.4-1BBL amplicons were used as a means for activating and expanding tumorspecific cytotoxic CD8+ OT-1/GFP T cells in vitro. We then studied the extent of expansion of adoptively transferred T cells in vivo, as well as the effects of adoptive transfer of the ex vivoexpanded T cells on tumor growth. Ex vivo-stimulated OT-1/GFP were adoptively transferred into LLC/OVA tumor-bearing mice, and anti-tumor response was assayed. A schematic of the experimental procedures is given in Figure 2. Mice were bled at several time points following adoptive transfer of OT-1/GFP in order to detect expansion of OT-1/GFP in the peripheral blood (Fig. 3A). Six days following adoptive transfer, the number of OT-1/GFP was significantly greater in the LLC/OVA tumor-bearing group given OT-1/GFP stimulated with 4-1BBL compared to tumor-bearing groups given nave or B7.1-stimulated OT-1/GFP. Non-specific expansion of stimulated OT-1/GFP was determined by transferring OT-1/GFP into non-tumorbearing mice. Greater numbers of OT-1/GFP were seen in the peripheral blood following transfer of 4-1BBL-stimulated OT-1/GFP into non-tumor-bearing mice than nave or B7.1-stimulated OT-1/GFP, indicating that expansion of 4-1BBL-stimulated OT-1/GFP had occurred in vivo in the absence of viable tumor. The number of 4-1BBL-stimulated OT-1/GFP were significantly greater in the tumor-bearing mice compared to non-tumor-bearing mice, suggesting that 41BBL-stimulated OT-1/GFP cells were able to expand in vivo in a tumor-specific manner (Fig. 3A). The total numbers and percentages of tumor-specific OT-1/GFP cells in relation to the CD8+ population were determined in the spleen of LLC/OVA tumor-bearing mice 17 days postadoptive transfer. The percentage of GFP+ cells in the spleen was the greatest in the tumorbearing group given OT-1/GFP stimulated with HSV.4-1BBL-transduced tumor (19.8% of

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CD8+ cells) vs. HSV.B7.1-stimulated tumor (0.1%) or no tumor (1.1%) (Fig. 3B). Substantial numbers of OT-1/GFP were also detected in the spleen of non-tumor-bearing mice 17 days after transfer of 4-1BBL-stimulated OT-1/GFP (7% of CD8+ cells), suggesting that tumor-specific 41BBL-stimulated T cells can also persist in vivo in the absence of antigen (Fig. 3B). When the total number of OT-1/GFP in the spleen was determined 17 days following adoptive transfer, there were greater overall numbers of OT-1/GFP present in the spleen when stimulated with 4-1BBL compared to B7.1 or to no stimulation, as seen in the peripheral blood (Fig. 3C). Spleens from mice in each of the groups were sectioned to observe the presence and localization of GFP+ cells 32 days following adoptive transfer of OT-1/GFP. The spleens from mice given HSV.4-1BBL-stimulated OT-1/GFP showed the greatest degree of infiltration with GFP+ cells (Fig. 3D). These results indicate that tumor-specific T cells stimulated ex vivo with HSV.4-1BBL-transduced tumor can expand and persist in vivo.

CD8+ T cells expanded in vitro with 4-1BBL decrease tumor growth and increase survival. We tested for growth of LLC/OVA tumor following adoptive transfer of HSV ampliconstimulated T cells or controls. LLC/OVA tumor-bearing mice treated with HSV.4-1BBLactivated OT-1/GFP had statistically significant lower tumor burden than untreated mice (P < 0.0001) or mice treated with an identical number of HSV.B7.1-stimulated OT-1/GFP (P < 0.0001) or nave OT-1/GFP (P = 0.0003) (Fig 4A). Nave OT-1/GFP treatment did not reduce tumor burden significantly when compared to the untreated group (P = 0.06). Administration of HSV.B7.1-activated OT-1/GFP did not have major inhibitory effects on tumor size when compared to no treatment or to nave OT-1/GFP transfer (P > 0.05) (Fig. 4A).

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OT-1, either nave or stimulated with HSV.4-1BBL-tansduced tumor, was administered into LLC/OVA tumor bearing mice in order to determine effects on overall survival (Fig. 4B). The group treated with nave OT-1 had significantly better survival than those untreated (p=0.001, two-sided log-rank test). Four (80%) of 5 mice treated with 4-1BBL-stimulated OT-1 survived 45 days compared with 2 (20%) of 10 mice given nave OT-1, and is significant (p=0.04, one-sided log-rank test). Those mice that survived to day 45 were completely tumorfree.

4-1BBL-stimulated CD8+ OT-1 cells retain CTL activity and possess a memory phenotype. We measured CTL activity in mice that had received adoptively transferred OT-1/GFP cells. Splenocytes were harvested and cultured with LLC/OVA and recombinant IL-2 for six days. Effectors from the HSV.4-1BBL-activated OT-1/GFP group had substantially higher levels of CTL activity against LLC/OVA when compared to splenocytes from the HSV.B7.1stimulated OT-1/GFP, nave OT-1/GFP, and untreated groups (Fig. 5). When restimulated in vitro for 6 days, splenocytes from the HSV.B7.1 group showed lower CTL activity (14% lysis at E:T = 50:1) against LLC/OVA compared to the HSV.4-1BBL group (41% lysis at E:T = 50:1) and was similar to that of the nave OT-1/GFP group (17% lysis at E:T = 50:1) (Fig. 5). These results indicate that HSV.4-1BBL can be used for in vitro expansion of tumor-specific T cells for the purpose of adoptively transferring them into tumor-bearing host so as to augment anti-tumor responses. They also suggest that T cells expanded in this fashion may have more favorable effector characteristics than those obtained through HSV.B7.1-mediated stimulation. The phenotype of OT-1/GFP cells in each of the groups was characterized in freshly isolated splenocytes 17 days post-adoptive transfer of OT-1/GFP (Fig. 6). GFP+ cells from both

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tumor-bearing and non-tumor-bearing mice which had received HSV.4-1BBL-stimulated OT1/GFP or the control OT-1/GFP indicated were studied for levels of CD44 and Ly-6C, a CD8+ memory cell marker (Fig. 6) (37). Significantly higher levels of CD44+ OT-1/GFP were seen in mice which had received HSV.4-1BBL-stimulated OT-1/GFP (12.7% in tumor-bearing mice, 3.8% in non-tumor-bearing mice) than were seen for either HSV.B7.1-stimulated (0.1% in tumor-bearing and non-tumor-bearing mice) or nave (0.4% in tumor-bearing mice, 0.1% in nontumor-bearing mice) OT-1/GFP transfer (Fig. 6A). Similarly, HSV.4-1BBL-stimulated OT1/GFP also demonstrated higher levels of Ly-6C expression in comparison to the other groups, suggesting differentiation into memory cells was more pronounced in the HSV.4-1BBL-treated group (Fig. 6B). HSV.B7.1-stimulated OT-1/GFP cells had considerably lower levels of Ly-6C expression compared to HSV.4-1BBL-stimulated OT-1/GFP, although costimulation had occurred in vitro (Fig. 6B). This indicates that upon adoptive transfer of 4-1BBL-stimulated T cells, the T cells are maintained at high levels and exhibit phenotypic attributes of memory cells.

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Discussion The overall purpose of these studies was to understand the potential utility of the TNFR family member 4-1BB as a means of expanding and activating tumor-specific CD8+ T cells for adoptive immunotherapy. We were particularly interested in the purported ability of 4-1BB to sustain nascent responses and prevent activation-induced cell death in T cells (38). This effect on T cell survival is dependent on TRAF 2 signaling and NF-B activation (39, 40), which in turn induces Bcl-xL and Bfl-1, two pro-survival members of the Bcl-2 family. We reasoned that cells expanded using 4-1BB costimulation might have favorable effector characteristics and persist in vivo potentially leading to tumor eradication as well as improved survival. We investigated effects of 4-1BB engagement on anti-tumor immunity by using HSV amplicons to express high levels of 4-1BBL on tumor cells. HSV.4-1BBL was able to efficiently transduce a variety of tumor cell lines, including LLC/OVA. LLC/OVA transduced with HSV.41BBL were used to stimulate CD8+ TCR-transgenic OT-1 T cells. OT-1 cells responded in vitro by proliferating and expressing activation markers, namely CD44, CD25, Ly-6C, CD107a, and intracellular granzyme B, indicating priming of cytolytic activity and differentiation into cytolytic effectors. HSV.4-1BBL amplicons were used to transduce LLC/OVA tumors in vitro for purposes of ex-vivo expansion of tumor-specific OT-1/GFP T cells. Adoptive transfer of OT-1/GFP T cells that had been expanded ex vivo using HSV.4-1BBL-transduced LLC/OVA appeared to confer greater protection against LLC/OVA growth compared to nave OT-1/GFP, or OT-1/GFP cells incubated with HSV.B7.1-transduced tumor. Compared to splenocytes from the other groups, splenocytes from mice treated with HSV.4-1BBL-expanded OT-1/GFP showed higher percentages of tumor-specific OT-1/GFP+ cells, exhibited greater CTL activity, and expressed

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higher levels of CD44 and the CD8+ memory marker Ly-6C. Therefore, T cell costimulation with 4-1BBL expressed on tumor cells may be useful in facilitating expansion of tumor-specific T cells in vivo as well as ex vivo. We utilized the adoptive transfer of TCR-transgenic CD8+ OT-1/GFP T cells in order to more accurately follow the effects of 4-1BBL on T cells that were specific for defined tumorrelated antigens in our in vivo mouse experiments. This overcame a major difficulty in isolating, monitoring, and characterizing effective CD8+ CTL responses since antigen-specific CD8+ T cells are present in very low numbers in the animal (33, 34). Special consideration needs to taken, however, in order to apply information gained from this model to the human setting. Instead of expanding a population of nave TCR-transgenic T cells all of which are specific for tumor as in our experiments, in humans T cell expansion starts from a heterogeneous population in which relatively rare T cells can recognize tumor. Current cell culture techniques require several months to produce sufficient numbers of cells from single clones (41, 42). Other groups have examined the possibility of utilizing 4-1BB costimulation for the generation of tumor-reactive T cells for adoptive immunotherapy and have determined that addition of an agonistic anti-4-1BB antibody to in vitro culture enhances expansion and survival of T cells. Murine fibrosarcoma MCA 205 tumor-draining lymph node (TDLN) cells were expanded in vitro for 5 days with IL-2 and antibodies to CD3, CD28, and 4-1BB and found to produce greater numbers of T cells in vitro than those without anti-4-1BB, due to fewer apoptotic and necrotic cells (43). Anti-CD3/anti-CD28/anti-4-1BB-costimulated TDLN also produced more of the type 1 cytokine IFN- and granulocyte macrophage colony-stimulating factor (GMCSF) and less of the type 2 cytokine IL-10 compared to those stimulated without anti-4-1BB (43). When anti-CD3/anti-CD28/anti-4-1BB-expanded TDLN cells were adoptively transferred

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into MCA 205 tumor-bearing mice, significantly fewer metastatic lesions in the lungs were seen, and survival was prolonged compared to those mice treated with TDLN cells stimulated without anti-4-1BB (43). IFN- secretion was shown to be important in the 4-1BB-costimulated-TDLN effects using neutralizing antibody (43). IL-10 neutralization, on the other hand, resulted in significantly enhanced tumor regression (43). It would, therefore, be interesting to further study effects of HSV.4-1BBL-transduced tumor on IFN--/- T cells in order to assess the importance of IFN- in generating adept effector cells in our system. Strome et al. also observed that the combined use of anti-CD3/anti-CD28/anti-4-1BB in activating T cells for adoptive immunotherapy resulted in the generation of T cells that were more effective than those activated by anti-CD3 alone or anti-CD3/anti-CD28 in mediating antitumor reactivity (17). Intravenous adoptive transfer of dual CD28/4-1BB-costimulated T cells into mice bearing disseminated micrometastasis of a poorly immunogenic A9P melanoma resulted in a 60% cure rate. Unlike the previous experiments, we used ligands to CD28 and 4-1BB and employed them separately to differentiate the effects of CD28 and 4-1BB costimulation. When other groups used anti-CD3 plus agonistic anti-4-1BB antibody, without anti-CD28 antibody, in culture to expand a polyclonal T cell population for adoptive transfer, they were not successful in generating tumor-reactive T cells (17, 18). Kim et al. observed that TDLN cells activated in vitro with anti-CD3/anti-4-1BB were less potent than anti-CD3-activated cells in mediating tumor regression in vivo (18). In their study, the proliferation of TDLN cells in IL-2 following antiCD3/anti-4-1BB activation was significantly enhanced compared to anti-CD3 alone. However, the increased proliferation resulted in the expansion of non-tumor-reactive T cells. Based on our experiments, we can potentially overcome the difficulty of generating non-tumor-reactive clones

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by using HSV.4-1BBL amplicons to transduce tumor cells for specific stimulation of tumorreactive T cells. It is notable that Kim et al. added anti-4-1BB into culture in soluble form, taking into consideration crosslinking due to APCs (18), and Strome et al. used plate-fixed anti-4-1BB antibodies in their in vitro T cell expansion assays (17); however, both groups did not generate potent in vivo effectors through 4-1BB costimulation using solely anti-CD3 and anti-4-1BB antibodies. Some suggest that the lack of CD28 costimulation during in vitro culture in their studies diminished activation of tumor-primed reactive T cells. In contrast, our studies have shown that triggering the 4-1BB pathway using 4-1BBL, instead of anti-4-1BB antibody, independent of CD28 costimulation, can generate potent tumor-reactive T cells that are efficient in tumor eradication and can persist in vivo. Maus et al. have studied ex vivo expansion of human polyclonal and MHC tetramersorted antigen-specific cytotoxic T cells using K562 erythromyeloid cell lines stably transfected to express 4-1BBL and the Fc receptor CD32 to bind anti-CD3 and anti-CD28 antibodies on the surface (12). They demonstrated that these artificial APCs activate and rapidly expand polyclonal or sorted antigen-specific CD8+ T cells with substantial increases in expansion and recovery of T cells. In addition, apoptosis of cultured CD8+ T cells was decreased using 41BBL-expressing anti-CD3/anti-CD28-coated K562 cells versus anti-CD3/anti-CD28-coated K562. Although CD8+ T cells initially stimulated with anti-CD3/CD28-coated beads proliferate and produce IL-2, they can become unresponsive to re-stimulation in vitro (44). Maus et al. determined that the addition of 4-1BB costimulation facilitated the proliferation of T cells following re-stimulation with anti-CD3 and anti-CD28. Real-time quantitative RT-PCR showed that the addition of 4-1BB costimulation increased mRNA expression levels of IL-2 and Bcl-xL,

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two genes involved in T-cell survival and proliferation, respectively, in polyclonal CD8+ T cell cultures. Therefore, 4-1BBL in combination with CD28 costimulation was found to expand and increase survival in cytotoxic T cells ex vivo. Our studies show that 4-1BBL ligand stimulation alone, without prior CD28 costimulation, is also able to expand tumor-specific T cells, which were observed to elicit anti-tumor responses in vivo. In contrast to the work by Maus et al. in which 4-1BBL is stably transduced to a generic artificial APC, an advantage to using the highly efficient HSV amplicon system to express 4-1BBL is that it provides a theoretical means by which to expand tumor-specific effector populations using autologous tumor from patients without first pre-sorting tumor-reactive T cells. Using this system, T cells targeting unidentified tumor antigens are able to be expanded specifically. Another advantage to using HSV amplicons is their capacity to impart a strong innate stimulus to transduced cells, including macrophage cell lines and human chronic lymphocytic leukemia cells, resulting in cytokine secretion and NKG2DL expression by the transduced cells (45). HSV possesses at least three molecular components capable of activating the innate immune system: 1) dsRNA generated through self-hybridization of viral genes transcribed from complementary DNA strands (46) 2) envelope glycoproteins recognized by toll-like receptor (TLR) 2 (47), and 3) unmethylated CpG motifs encoded in the viral genome that activate TLR9 (48). Due to the fact that HSV amplicon DNA is concatamerized, the CpG effects on TLR9 could be quite potent. The enhanced capacity of transduced tumors to alert the innate immune response may serve to facilitate a more specific adaptive response. In certain settings, such as for human hematopoetic tumors, tissue may be available for and amenable to HSV transduction (e.g. chronic lymphocytic leukemia cells) (45). Since HSV vectors are theoretically safe and highly efficient means of gene transfer, the laboratory is

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pursuing pre-clinical development of these vectors for potential human use in chronic lymphocytic leukemia. Adoptive transfer of T cells permits screening of desirable functional and phenotypic qualities of T cells for transfer. Recently, Gattinoni et al. provided a new criterion for the generation and screening of optimal lymphocyte populations for adoptive immunotherapy (49). Despite enhanced in vitro anti-tumor properties (IFN- secretion and CTL activity), more differentiated effector T cells were less effective for in vivo tumor treatment than early effector cells. Several reasons why late effectors may be less potent in vivo include: 1) downregulation of homing molecules such as CD62L and/or costimulatory molecules, 2) inability to produce IL-2 and limited access to homeostatic cytokines, and 3) entry into a pro-apoptotic and replicative senescent state. Previous studies using agonistic anti-4-1BB antibody and 4-1BBL-expressing artificial APCs address the third point and indicate that use of 4-1BB signaling in combination with CD28 costimulation in in vitro expansion may prevent apoptosis in culture-expanded clones (9, 12, 17, 18, 43). We note that although in vitro 4-1BB-stimulated OT-1/GFP cells were highly positive for CD44, they had higher levels of CD62L-expressing cells than OT-1/GFP cells expanded using HSV.B7.1 (data not shown). Higher levels of CD62L may partially account for the enhanced function of HSV.4-1BBL-stimulated OT-1/GFP following adoptive transfer and may suggest that a higher proportion of therapeutically desirable early effectors arise as a result of 4-1BB-mediated expansion. Since we have not yet tested a combination of HSV.4-1BBL and HSV.B7.1, it is unknown whether HSV.B7.1 will further augment effects seen with HSV.4-1BBL-stimulation alone. HSV.B7.1-stimulated OT-1 failed to expand in vivo and inhibit LLC/OVA tumor growth. In some cases, mice in this group appeared to have subtly larger tumor burden compared to the

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group given nave OT-1 (not statistically significant). Since B7.1 serves as a ligand for both CD28 and CTLA-4, B7.1 may have bound to CTLA-4 expressed on activated OT-1 cells, inhibiting their clonal expansion and cytolytic activity. In addition, complete physical separation of OT-1/GFP and transduced LLC/OVA following in vitro activation was difficult due to cell debris. It is possible that the tumor-expressed B7.1 bound to CTLA-4 present on activated T cells in vivo. If this is the case, use of anti-CD28 antibodies for in vitro expansion may be preferred to use of the cognate B7.1 ligand. In summary, our studies suggest that costimulation with 4-1BB may be employed to enhance expansion and cytolytic activity of tumor-specific CD8+ T cells for the generation of long-term tumor-specific immunity. Although the OT-1 model was useful in demonstrating expansion of a tumor-specific response, results obtained with OT-1 may differ as compared to what would be observed using a polyclonal T cell population as a source of expanded T cells. Such a population might contain relatively fewer precursor T cells with anti-tumor activity, and expansion of relevant effectors might prove more difficult. Adoptive transfer experiments using polyclonal T cells from tumor-bearing mice are currently being performed in an effort to ascertain utility of this approach in a non-transgenic setting. Nevertheless, the vigorous cytolytic effector function as well as the increased expansion and persistence seen using HSV amplicontransduced tumor suggest this method should be explored further in the human setting.

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Figure Legends Figure 1. HSV amplicon vector encoding 4-1BBL and proliferation and phenotype of CD8+ OT-1 T cells in response to HSV amplicon-transduced tumor. A) The HSV.4-1BBL amplicon plasmid DNA is packaged into helper virus-free viral stocks using an HSV genomecontaining bacterial artificial chromosome (BAC), which has its cognate HSV packaging elements (pac sequences) deleted. Vhs, or the viral host shutdown gene, is expressed from a separate plasmid (pBS-vhs) in order to enhance viral titers. Baby hamster kidney (BHK) cell monolayers are transfected with all three vectors, and the HSV amplicon viral particles are later harvested, concentrated, and titered for use. B) LLC/OVA and LLC tumors were transduced with HSV amplicons encoding either human B7.1 (HSV.B7.1) or murine 4-1BBL (HSV.4-1BBL) as T cell costimulatory ligands or -galactosidase (HSV.LacZ) as an HSV control at MOI=1. After 2 days, the tumor cells were treated with 0.4 mg/ml mitomycin C and stained for B7.1 and 41BBL expression. Solid line indicates staining of untransduced tumor, and dashed line indicated staining of HSV.LacZ-transduced tumor. C) Purified OT-1 T cells (105) were added to mitomycin-treated tumor cells (2x104) and incubated at 37oC. Thymidine was added 20 h before the plate was harvested following 3 days and 5 days of co-culture. Thymidine uptake of tumor cells alone was minimal. SD of triplicates is shown. Data represents four separate experiments. D) CD8+ OT-1/GFP T cells were added to mitomycin C-treated LLC/OVA tumor cells. Three days later CD8+ OT-1/GFP T cells were stained for surface activation markers. CD44, CD25, and Ly-6C were measured by flow cytometry. CD107a and intracellular granzyme B were used as indicators of cytotoxic differentiation of OT-1/GFP. Red histogram = OT-1/GFP + LLC/OVA + HSV.4-1BBL; Pink line = OT-1/GFP + LLC/OVA + HSV.B7.1; Green line = OT-1/GFP + LLC/OVA + HSV.LacZ; Blue line = Nave OT-1/GFP

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Figure 2. Overview of adoptive transfer experiment. LLC/OVA tumor cells were transduced with HSV.4-1BBL or HSV.B7.1 at an MOI of 1 and cultured for one day. Tumor cells were then washed and treated with mitomycin C. CD8+ OT-1/GFP cells were purified from spleen and cocultured with transduced tumor for three days. Stimulated OT-1/GFP were separated from tumor cells using anti-CD8 antibody-magnetic beads. OT-1/GFP cells were administered i.v. into C57BL/6 mice bearing LLC/OVA tumor. LLC/OVA tumor was palpable following 3-4 days of s.c. injection. Peripheral blood and spleen were observed for the number of OT-1/GFP present. Spleen was also analyzed for cytolytic activity and the phenotype of OT-1/GFP cells.

Figure 3. Presence of OT-1/GFP cells in peripheral blood and spleen following adoptive transfer of expanded OT-1/GFP. A) Blood was collected 6 days post transfer of expanded OT1/GFP. Percentage of OT-1/GFP in blood was assessed using flow cytometry and used to calculate cell number from the volume of blood collected. N=5, 6, and 6 for the tumor bearing group. N=3 for each of the non-tumor bearing groups. Numbers are statistical results from a Students t-Test. B and C) Splenocytes from mice were collected on day 17 post OT-1/GFP adoptive transfer. They were stained with anti-CD8-Cy-Chrome and analyzed by flow cytometry. B) Blue numbers indicate percentage of GFP+ cells in the CD8+ gate. C) Total number of splenocytes x percentage of GFP+ cells in spleen = Number of GFP+ cells per spleen. Number of GFP+ cells per spleen on day 17 is shown. D) Frozen spleen harvested 32 days following adoptive transfer of OT-1/GFP cells were sectioned, fixed by formaldehyde vapor, and counterstained with Hoechst 33342 to show nuclei. GFP+ areas indicate infiltration by adoptively transferred CD8+ OT-1/GFP cells. Images were taken at 20x magnification. Upper

28

left: GFP+ spleen (positive control); upper right: transfer of nave OT-1/GFP; lower left: transfer of HSV.4-1BBL-expanded OT-1/GFP; lower right: transfer of HSV.B7.1-expanded OT-1/GFP.

Figure 4. Tumor volumes and survival in mice following adoptive transfer of OT-1 T cells. CD8+ OT-1 T cells were activated in vitro with transduced LLC/OVA at an E:T ratio of 3:2 for 3 days. A) Activated OT-1/GFP (O/G) or nave O/G (2 x 106) were administered into the tail vein of mice injected with tumor s.c. 4 days previously. Tumors were measured at the time points indicated. Error bars indicate SEM. P values were determined from regression analysis. O/G HSV.4-1BBL= 10 mice; O/G HSV.B7.1 = 5 mice; nave O/G = 5 mice; No O/G = 3 mice. B) Activated OT-1 or nave OT-1 (106) were administered into the tail vein of mice injected with LLC/OVA tumor (1.5 x 106) s.c. 3 days previously. Mice were sacrificed when tumor diameter reached 15 mm. Mice surviving by day 45 are tumor-free.

Figure 5. Tumor-specific cytolytic activity of splenocytes following administration of OT-1 T cells expanded ex vivo. At 22 days following tumor injection, splenocytes were harvested and cultured with recombinant human IL-2 (10 U/ml) and mitomycin C-treated LLC/OVA at an E:T ratio of 10:1 for 6 days. LLC/OVA cells were used as targets in an 8-h Percent lysis with standard deviation is shown.
51

Cr release assay.

Figure 6. CD44 and Ly-6C expression of OT-1/GFP in splenocytes. Splenocytes from mice were collected on day 17 following OT-1 adoptive transfer. They were stained with anti-CD8Cy-Chrome and A) anti-CD44-PE or B) biotinylated-anti-Ly-6C followed by streptavidin-PE and analyzed by flow cytometry. Numbers indicate percentages in the CD8+ gate.

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33

A
HSV pac site Ampr HSV Oris HSV IE 4/5 Promoter parC parB parA repE OriS HSV-BAC
A

B
SV40 poly A HSV Amplicon Murine 4-1BBL

LLC HSV.B7.1

LLC/OVA HSV.B7.1

pBS-vhs

100

101

102 FITC

103

104

100

101

102 FITC

103

104

US

A A A

B7.1
LLC HSV.4-1BBL

/ LLC/OVA / HSV.4-1BBL

UL

Transfection

Harvest

100

101

102 PE

103

104

100

101

102 PE

103

104

4-1BBL

C
Day 3
Thymidine Incorporation (cpm x 10-3)
80 70 60 50 40 30 20 10 0

D
CD44
10 0

10 2 10 1 ANTI-CD44-PE

10 3

10 4

CD25
10 0

10 2 10 1 ANTI-CD25-PE

10 3

10 4

T cells only

T + LLC/OVA T + LLC/OVA T + LLC/OVA T + LLC/OVA (-) HSV.B7.1 HSV.4-1BBL HSV.LacZ

CD107a
10 0
10 1 10 2 10 3 ANTI-CD107A-PE
10 4

Day 5
25

Thymidine Incorporation -3 (cpm x 10 )

20

Granzyme B, intracellular
10 0
10 1 10 2 10 3 ANTI-GRANZYME B-PE,I
10 4

15

10

Ly-6C
10 0
10 1 10 2 ANTI-LY6C-PE
10 3
10 4

T cells only T + LLC/OVA T + LLC/OVA T + LLC/OVA T + LLC/OVA (-) HSV.B7.1 HSV.4-1BBL HSV.LacZ

Yi--Figure 1

LLC/OVA (106) s.c. HSV.4-1BBL or HSV.B7.1 LLC/OVA 1 day Add to OT-1/ GFP 4-8 days

3 days Stimulated OT-1/GFP (1-2 x 106) i.v.

Bleed

Spleen: #/% OT-1/GFP CTL assay CD44/Ly-6C Granzyme B

Yi--Figure 2

A
1200 1000
GFP+ cells/ul blood

0.007

0.017

B
Nave OT-1/GFP

LLC/OVA-tumor bearing mice

non-tumor bearing mice

1.1
M1

0.4
M1

800

600

100
400

101

102 GFP

103

104

100

101

102 GFP

103

104

200

OT-1/GFP + L/O HSV.4-1BBL

19.8
M1

7.0
M1

T1/G 1B FP BL OT -1/ -stim HS GF u la t V. ed B7 P .1 OT st -1/ imu G F la P ted Na ve HS OT V. -1/ 4-1 GF BB P O T L -s -1/ tim HS GF u la t V. B7 P ed .1 OT st -1/ imu G F la P ted

100

101

Na ve O

102 GFP

103

104

100

101

102 GFP

103

104

OT-1/GFP + L/O HSV.B7.1

HS V

0.1
M1

0.3
M1

tumor bearing mice

.4 -

non-tumor bearing mice

100

101

102 GFP

103

104

100

101

102 GFP

103

104

C
1.4 1.2
C e ll N u m b e r (M illio n s )

D
GFP+ Spleen Nave OT-1/GFP Spleen

1.0 0.8 0.6 0.4 0.2 0.0

FP OT L/O -1/ G H S FP V. + 41B BL OT L/O -1/ G H S FP V. + B7 .1 No OT -1 /G FP
1 2 3 4 5 6 7

HSV.4-1BBL-stimulated OT-1/GFP Spleen

HSV.B7.1-stimulated OT-1/GFP Spleen

Na ve O

T1/G

Yi--Figure 3

A
1600 1400 Tumor volume (mm^3) 1200 1000 800 600 400 200 0 5 7 9 11 13 15 Days following tumor injection
O/G HSV.4-1BBL O/G HSV.B7.1 nave OT-1 No O/G

B
100 90 80 70 60 50 40 30 20 10 0 0

% Survival

L/O only (n=9) L/O + nave OT-1 (n=10) L/O + OT-1/HSV.4-1BBL (n=5)

10

20

30

40

50

Days post OT-1 transfer

Yi--Figure 4

P = <0.0001

P = 0.0003

45 40 35 30 % Lysis 25 20 15 10 5 0 50:1 10:1 E:T ratio 2:1 0.4:1 L/O + O/G HSV.4-1BBL L/O + O/G HSV.B7.1

L/O + nave O/G L/O only

Yi--Figure 5

tumor bearing mice

non-tumor bearing mice

A
Naive OT-1/GFP

0.4

0.1

0.8
10 0

0.2
10 0
10 1 10 2 GFP 10 3
10 4

10 1

10 2 GFP

10 3

10 4

OT-1/GFP + L/O HSV.4-1BBL

12.7

3.8

7.1
10 0

3.0
10 0 10 1
10 2 GFP 10 3 10 4

10 1

10 2 GFP

10 3

10 4

OT-1/GFP + L/O HSV.B7.1

0.1

0.1

0.2
10 0 10 1

0.2
10 0

10 2 GFP

10 3

10 4

10 1

10 2 GFP

10 3

10 4

B
Naive OT-1/GFP

0.7 0.4
10 0
10 1
10 2 GFP
10 3
10 4
10 0
10 1
10 2 GFP
10 3

0.1 0.1
10 4

OT-1/GFP + L/O HSV.4-1BBL

9.7 0.2
10 0
10 1
10 2 GFP
10 3
10 4
10 0
10 1
10 2 GFP
10 3

3.8 0.1
10 4

0.0
OT-1/GFP + L/O HSV.B7.1

0.1 0.0
10 0
10 1
10 2 GFP
10 3
10 4

0.1
10 0
10 1
10 2 GFP
10 3
10 4

Yi--Figure 6