AbstractPancreatic adenocarcinoma is a devastating disease, characterized by resistance to current therapies and extremely poor prognosis. New, effective treatments are urgently needed. Cellular immune responses can be initiated via specialized antigen presenting cells, such as dendritic cells (DCs) that stimulate naive T cells in regional draining lymph nodes. DC and T cell trafficking is regulated by chemokines, such as CCL21. Our data indicate that intratumoral administration of CCL21, a chemoattractant for DCs, as well as for natural killer (NK) cells and T cells, slows the growth of pancreatic tumors. Furthermore, we have data that suggests intratumoral CCL21 injection can delay the growth of distant tumors. The number of DCs present in the blood and lymphoid organs is controlled by hematopoietic growth factors, for example, fms-like tyrosine kinase-3 ligand (Flt3L). In this study, we tested the hypothesis that sustained DC expansion by Flt3L and DC attraction by CCL21 will result in increased therapeutic activity against pancreatic cancer. We tested this hypothesis by examining the anti-tumor effect of simultaneous administration of Flt3L and CCL21, as well as both individually, against pancreatic adenocarcinomas established subcutaneously in C57BL/6 mice. Increasing DC numbers by intramuscular Flt3L treatment significantly delayed tumor growth. Similarly, treatment with intratumoral CCL21 reduced the rate of tumor growth. Surprisingly, the combination of both systemic Flt3L and intratumoral CCL21 was not more effective than either cytokine alone. To begin to determine whether manipulation of the timing of Flt3L and CCL21 treatments, relative to each other, would improve therapeutic efficacy, we analyzed the kinetics of Flt3L and CCL21 activity. In these experiments, adenoviral delivery of Flt3L and CCL21 was used, because it permits high transduction efficiency and transgene expression in vivo. We observed that subcutaneous injection of Adv-CCL21 was superior to recombinant CCL21 (rCCL21) not only in increasing DC, NK cell, and T cell populations in the local lymph node, but also in prolonging the duration of that increase (21 days in Adv-CCL21 injected mice versus 8 days in rCCL21 injected ones). Furthermore, Adv-Flt3L significantly expanded DC populations in the spleen with the peak of the expansion at 6-8 days post-injection. Overall, these observations suggest that 1) both CCL21 and Flt3L may be therapeutic for pancreatic cancer, and 2) peak activities for Adv-Flt3L and Adv-CCL21 are observed at 6-8 days and 7-9 days, respectively. Our subsequent study will be to inject Adv-CCL21 intratumorally 6-8 days after intravenous Adv-Flt3L.

Conclusions and Future Directions

CCL21 AND FLT3 LIGAND AS IMMUNOTHERAPEUTICS FOR PANCREATIC CANCERAbdelkader Ashour1,2, H. R. Turnquist1,3, A. J. Reber1, J. Kampf1, S. Kurz3, D. LaFace4, M. A. Hollingsworth1,3, R. K. Singh1,3, J. E. Talmadge1,3, and J. C. Solheim1,2,3.

Eppley Institute1, Department of Biochemistry and Molecular Biology2 and Department of Pathology and Microbiology3 University of Nebraska Medical Center, Omaha, NE; Canji Inc.4

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and immunity. Annu Rev Immunol 2001; 19:47-64.3. Albert ML, Sauter B, Bhardwaj N. Dendritic cells acquire antigen

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5. Willimann K, Legler DF, Loetscher M, Roos RS, Delgado MB, Clark-Lewis I, Baggiolini M, Moser B. The chemokine SLC is expressed in T cell areas of lymph nodes and mucosal lymphoid tissues and attracts activated T cells via CCR7. Eur J Immunol 1998; 28:2025-2034.

6. Sharma S, Stolina M, Luo J, Strieter RM, Burdick M, Zhu LX, Batra RK, Dubinett SM. Secondary lymphoid tissue chemokine mediates T cell-dependent antitumor responses in vivo. J Immunol 2000; 164:4558-4563.

7. Kirk CJ, Hartigan-O’Connor D, Nickoloff BJ, Chamberlain JS, Giedlin M, Aukerman L, Mule JJ. T cell-dependent antitumor immunity mediates by secondary lymphoid tissue chemokine: augmentation of dendritic cell-based immunotherapy. Cancer Res 2001; 61:2062-2070.

8. Robinson S, Mosley RL, Parajuli P, Pisarev V, Sublet J, Ulrich A, Talmadge J. Comparison of the hematopoietic activity of Flt-3 ligand and granulocyte-macrophage colony-stimulating factor acting alone or in combination. J Hematother Stem Cell Res 2000; 9:711-720.

9. McKenna HJ. Role of hematopoietic growth factors/flt3 ligand in expansion and regulation of dendritic cells. Curr Opin Hematol 2001; 8:149-154.

10. Pulendran B, Banchereau J, Burkeholder S, Kraus E, Guinet E, Chalouni C, Caron D, Maliszewski C, Davoust J, Fay J, Palucka K. Flt3-ligand and granulocyte colony-stimulating factor mobilize distinct human dendritic cell subsets in vivo. J Immunol 2000; 165:566-572.

11. Parajuli P, Mosley RL, Pisarev V, Sublet J, Ulrich A, Varney M, Singh RK, Talmadge JE. Flt3 ligand and granulocyte-macrophage colony-stimulation factor preferentially expand and stimulate different dendritic cell and T cell subsets. Exp Hematol 2001; 29:1185-1193.

12. Bjorkdahl, O., Barber, K.A., Brett, S.J., Daly, M.G., Plumpton, C., Elshourbagy, N.A., Tite, J.P., and Thomsen, L.L. Characterization of CC-chemokine receptor 7 expression on murine T cells in lymphoid tissues. Immunol 2003; 110: 170-179.

13. Dieu, M., Vanbervliet, B., Vicari, A., Bridon, J., Oldham, E., Aït-Yahia, S., Brière, Zlotnik, A., Lebecque, S., and Caux C. Selective recruitment of immature and mature dendritic cells by distinct chemokines expressed in differenct anatomical sites. J. Exp. Med. 1998; 188(2): 373-386.

14. Cambell, J.J., Hedrick, J., Zlotnik, A., Siani, M.A., Thompson, D.A., and Butcher, E.C. Chemokines adnd the arrest of lymphocytes rolling under flow conditions. Science, 279: 381-384, 1998.

15. Yanagawa Y, and Onoe K. CCR7 ligands induce rapid endocytosis in mature dendritic cells with concomitant up-regulation of Cdc42 and Rac activities. Blood 2003; 10: 4923-4929.

CCL21 and Flt3L Can Individually Delay Pancreatic Tumor Growth

Figure 3. Untreated tumors are inhibited in growth following intratumoral treatment of separate tumors with CCL21. Panc02 cells were inoculated into both flanks of C57BL/6 female mice, and upon development of palpable right and left flank tumors, mice were administered intratumoral CCL21 (n = 12) or PBS alone (n = 11) on days 1, 2, 3, 8, 9, and 10 in the right flank tumor, and the growth of both right and left flank tumors was monitored. (A) Change in CCL21-or PBS-treated right flank tumor volumes over time. (B) Change over time in untreated left flank tumor volumes in CCL21- or PBS-treated mice. The asterisks indicate statistical significance (p≤0.05).

Figure 4. Intratumoral administration of CCL21 slows tumor growth in mice that were injected with Panc02.Neo tumors. Differences in time until death from sacrifice upon reaching a 1.2 cm tumor size were assessed with log-rank statistical analysis (Graph Pad Prism) and are displayed on a Kaplan-Meier survival curve.

Introduction The disease of pancreatic adenocarcinoma is a highly progressive and lethal cancer, with only 4% of patients surviving five years after diagnosis. It is the fifth leading cause of cancer-related death in the U.S., and during the last decade its incidence has increased worldwide (1). Treatments lengthening the lives of those developing this disease are drastically needed. Given the resistance of pancreatic cancer to both chemo- and radiotherapy, we are focusing on the development of immunotherapy for this disease. The ability of the immune system to respond to malignancies relies on the presentation to T lymphocytes of peptides derived from new or over expressed protein sequences in tumor cells. DCs present peptide antigens derived from tumor cells on MHC class I molecules to naïve CD8+ T cells to activate them against the tumor (2). Therefore, to initiate a robust immune response, DCs need to gain access to the tumor, where they can obtain antigen from apoptotic cells (3). The DCs then traffic to the regional lymph nodes where they present the tumor antigens to T cells. The chemokine CCL21 (SLC) acts as a strong chemoattractant for both DCs and Th1 cells in vitro and in vivo (4, 5). In a murine model of lung cancer, intratumoral injection of SLC enhanced the antitumor effect of DCs (6). Similarly, in murine models of melanoma and mammary cancer, injection of recombinant SLC inhibited tumor growth (7). In the melanoma model, the inhibition was accompanied by infiltrating DCs, CD4+ T cells, and CD8+ T cells and was dependent on CD8+ T cells but not CD4+ T cells (7). Flt3L is a hematopoietic growth factor that can expand DCs in the blood and in lymphoid and parenchymal tissues (8-10). In ability to expand DCs, Flt3L is superior to granulocyte macrophage-colony stimulating factor (11). Expansion of DCs by Flt3L, alone or combined with attraction of DCs by intratumoral CCL21 administration, should be a viable approach to increasing the immune response against pancreatic tumors.

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Figure 2. Tumor growth is inhibited by intratumoral CCL21 treatment of Panc02 tumors in wild type mice, but not in mice lacking fully functional T, B, and NK lymphocytes. Tumor cells (1x106 Panc02 cells) were injected subcutaneously above the scapulae in female wild type C57BL/6 mice (A) or C57BL/6 RAG2-/-Pfp-/- mice (B). Following development of palpable tumors, mice were treated on days 1, 2, 3, 8, 9, and 10 with intratumoral injections of 1 µg recombinant murine CCL21 in PBS+0.05% NMS (n=8) or PBS+0.05% NMS alone (n=8). The graphs display the changes in tumor volumes over time, with the first day of treatment as day 1.

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AcknowledgmentsThis work was supported by an LB595/Cattlemen’s Ball Grant (to J.C.S.), by the Nebraska Research Initiative Program in Molecular Therapeutics (to J.E.T., J.C.S., R.K.S. and M.A.H.), and the NIH Specialized Program in Research Excellence Grant P50 CA72712 (to M.A.H. and J.C.S.). A.A. received support from a UNMC Graduate Fellowship. H.R.T. received support from the NIH Training Grant T32 CA09476, UNMC Graduate Fellowships, and a UNMC Presidential Fellowship.

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Combination CCL21/Flt3L ImmunotherapyCombination CCL21/Flt3L ImmunotherapyExperimental PlanExperimental PlanSurvival and tumor growth were examined in C57BL/6 mice challenged s.c. with 1 X 106 Panc02 cells Mice with tumors 1.5 - 3.5 mm were treated in groups: Flt3L (15 g/dose-Day 1 and 6) in Pro-Gelz™ i.m. + CCL21 in Pro-Gelz™ (3 g/dose- Day 1 and 8) i.t. CCL21 (3 g/dose-Day 1 and 8) in Pro-Gelz™ i.t. Flt-3L (15 g/dose-Day 1 and 6) in Pro-Gelz™ i.m. Pro-Gelz™ i.m. (Day 1 and 6) + Pro-Gelz™ (Day 1 and 8) i.t.

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Figure 6. Tumor growth following pCCL21/pFlt3L Figure 7. Survival following pCCL21 (SLC)/pFlt3L

Kinetics of CCL21 ActivityKinetics of CCL21 ActivityExperimental PlanExperimental PlanGroups of C57BL/6 mice were given 1X s.c. injections in the right flank with 50 l of: PBS, PBS containing 10 g recombinant CCL21 protein (rCCL21), 1X1011 adenoviral-CCL21 (Adv-CCL21), or 1X1011 adenoviral control (Ad-control) Lymph nodes (LNs) and spleens were harvested at different time points and examined by flow cytometry for DCs Injection sites were histologically examined

Conclusions

Treatment with intratumoral CCl21 (SLC) in Pro-Gelz™ is therapeutic for pancreatic cancer

Increasing DCs through systemic Flt-3L treatment significantly increased survival

Two effective immunotherapy modalities for pancreatic cancer

Recombinant CCL21 delivered intratumorally produces immune-mediated tumor growth inhibition

Intratumoral administration of CCL21 in a pancreatic tumor model causes significant immune cell infiltration of the tumor mass and delays growth of both directly treated and untreated tumors

Administration of Adv-Flt3L significantly expands both the DC1 and DC2 populations within the spleen

The expansion of DCs by Adv-Flt3L occurs by as much as 10 fold over the basal level with the peak of the expansion at 6-8 days post-injection.

Future Directions

Inject Adv-CCL21 intratumorally 6-8 days after intravenous Adv-Flt3L.

Examine the possible synergistic effect of agents that can stimulate maturation of DCs, such as CD40 ligand and CpG DNA on CCL21/Flt3L immunotherapy

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Kinetics of Flt3L ActivityKinetics of Flt3L ActivityExperimental PlanExperimental PlanBALB/c mice (4/group) were given 1X i.v. injection with 1x1011 Adv-Flt3L (shown) or with PBS (not shown) Mice were sacrificed, and spleen and peripheral blood were harvested at day 0, 2, 4, 6, 8, 10, or 12. DC populations (DC1 and DC2) were examined by flow cytometry.

Figure 1. CCL21 (secondary lymphoid tissue ckemokine, SLC) acts as a strong chemoattractant for both DCs and Th1 cells. Colocalization is possible due to expression of the shared G-protein coupled receptor, CCR7, on both cell types (12,13). This allows both cells to migrate on chemical gradients formed on the extracellular matrix (ECM) of endothelial cells of the high endothelial venules which constitutively secrete CCL21. Also, CCL21 binding to CCR7 results in intracellular signaling which initiates integrin-mediated firm adhesion that allows cellular migration and extravasation into the T cell zones of the lymphoid tissue (14). In addition to aiding cellular homing, CCL21 has been found to produce rapid endocytosis in mature DC (15).

Figure 1.

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Figure 15. Figure 16.

Figure 17.

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