Rebecca A. Evans

Induction of T cell immunity overcomes complete resistance to PD-1 and CTLA-4 blockade and improves survival in pancreatic carcinoma

Winograd and colleagues used an engineered KPC mouse model of pancreatic ductal adenocarcinoma (PDA) reflecting that of human PDA to show that baseline refractoriness to checkpoint inhibitors can be rescued by priming a T-cell response with αCD40 plus chemotherapy with gemcitabine and nab-paclitaxel. Disabling the function of immune checkpoint molecules can unlock T-cell immunity against cancer, yet despite remarkable clinical success with monoclonal antibodies (mAb) that block PD-1 or CTLA-4, resistance remains common and essentially unexplained. To date, pancreatic carcinoma is fully refractory to these antibodies. Here, using a genetically engineered mouse model of pancreatic ductal adenocarcinoma in which spontaneous immunity is minimal, we found that PD-L1 is prominent in the tumor microenvironment, a phenotype confirmed in patients; however, tumor PD-L1 was found to be independent of IFNγ in this model. Tumor T cells expressed PD-1 as prominently as T cells from chronically infected mice, but treatment with αPD-1 mAbs, with or without αCTLA-4 mAbs, failed in well-established tumors, recapitulating clinical results. Agonist αCD40 mAbs with chemotherapy induced T-cell immunity and reversed the complete resistance of pancreatic tumors to αPD-1 and αCTLA-4. The combination of αCD40/chemotherapy plus αPD-1 and/or αCTLA-4 induced regression of subcutaneous tumors, improved overall survival, and conferred curative protection from multiple tumor rechallenges, consistent with immune memory not otherwise achievable. Combinatorial treatment nearly doubled survival of mice with spontaneous pancreatic cancers, although no cures were observed. Our findings suggest that in pancreatic carcinoma, a nonimmunogenic tumor, baseline refractoriness to checkpoint inhibitors can be rescued by the priming of a T-cell response with αCD40/chemotherapy. Cancer Immunol Res; 3(4); 399–411. ©2015 AACR.

Tumor-promoting desmoplasia is disrupted by depleting FAP-expressing stromal cells

Malignant cells drive the generation of a desmoplastic and immunosuppressive tumor microenvironment. Cancer-associated stromal cells (CASC) are a heterogeneous population that provides both negative and positive signals for tumor cell growth and metastasis. Fibroblast activation protein (FAP) is a marker of a major subset of CASCs in virtually all carcinomas. Clinically, FAP expression serves as an independent negative prognostic factor for multiple types of human malignancies. Prior studies established that depletion of FAP(+) cells inhibits tumor growth by augmenting antitumor immunity. However, the potential for immune-independent effects on tumor growth have not been defined. Herein, we demonstrate that FAP(+) CASCs are required for maintenance of the provisional tumor stroma because depletion of these cells, by adoptive transfer of FAP-targeted chimeric antigen receptor (CAR) T cells, reduced extracellular matrix proteins and glycosaminoglycans. Adoptive transfer of FAP-CAR T cells also decreased tumor vascular density and restrained growth of desmoplastic human lung cancer xenografts and syngeneic murine pancreatic cancers in an immune-independent fashion. Adoptive transfer of FAP-CAR T cells also restrained autochthonous pancreatic cancer growth. These data distinguish the function of FAP(+) CASCs from other CASC subsets and provide support for further development of FAP(+) stromal cell-targeted therapies for the treatment of solid tumors.

Exclusion of T Cells From Pancreatic Carcinomas in Mice Is Regulated by Ly6Clow F4/80+ Extratumoral Macrophages

BACKGROUND & AIMS Immunotherapies that induce T-cell responses have shown efficacy against some solid malignancies in patients and mice, but these have little effect on pancreatic ductal adenocarcinoma (PDAC). We investigated whether the ability of PDAC to evade T-cell responses induced by immunotherapies results from the low level of immunogenicity of tumor cells, the tumors immunosuppressive mechanisms, or both. METHODS Kras(G12D/+);Trp53(R172H/+);Pdx-1-Cre (KPC) mice, which develop spontaneous PDAC, or their littermates (controls) were given subcutaneous injections of a syngeneic KPC-derived PDAC cell line. Mice were then given gemcitabine and an agonist of CD40 to induce tumor-specific immunity mediated by T cells. Some mice were also given clodronate-encapsulated liposomes to deplete macrophages. Tumor growth was monitored. Tumor and spleen tissues were collected and analyzed by histology, flow cytometry, and immunohistochemistry. RESULTS Gemcitabine in combination with a CD40 agonist induced T-cell-dependent regression of subcutaneous PDAC in KPC and control mice. In KPC mice given gemcitabine and a CD40 agonist, CD4(+) and CD8(+) T cells infiltrated subcutaneous tumors, but only CD4(+) T cells infiltrated spontaneous pancreatic tumors (not CD8(+) T cells). In mice depleted of Ly6C(low) F4/80(+) extratumoral macrophages, the combination of gemcitabine and a CD40 agonist stimulated infiltration of spontaneous tumors by CD8(+) T cells and induced tumor regression, mediated by CD8(+) T cells. CONCLUSIONS Ly6C(low) F4/80(+) macrophages that reside outside of the tumor microenvironment regulate infiltration of T cells into PDAC and establish a site of immune privilege. Strategies to reverse the immune privilege of PDAC, which is regulated by extratumoral macrophages, might increase the efficacy of T-cell immunotherapy for patients with PDAC.

CD40 immunotherapy for pancreatic cancer

Pancreatic ductal adenocarcinoma (PDA) is a highly aggressive and lethal cancer which is poorly responsive to standard therapies. Although the PDA tumor microenvironment is considered especially immunosuppressive, recent data mostly from genetically engineered and other mouse models of the disease suggest that novel immunotherapeutic approaches hold promise. Here, we describe both laboratory and clinical efforts to target the CD40 pathway for immunotherapy in PDA. Findings suggest that CD40 agonists can mediate both T-cell-dependent and T-cell-independent immune mechanisms of tumor regression in mice and patients. T-cell-independent mechanisms are associated with macrophage activation and the destruction of PDA tumor stroma, supporting the concept that immune modulation of the tumor microenvironment represents a useful approach in cancer immunotherapy.

CD4+ T lymphocyte ablation prevents pancreatic carcinogenesis in mice.

Zhang and colleagues show that Kras-expressing epithelial cells recruit CD4+ T cells that repress the activity of CD8+ T cells to establish the immunosuppressive microenvironment in the iKras* mouse model of pancreatic cancer; elimination of CD4+ T cells uncovers the antineoplastic function of CD8+ T cells. Pancreatic cancer, one of the deadliest human malignancies, is associated with oncogenic Kras and is most commonly preceded by precursor lesions known as pancreatic intraepithelial neoplasias (PanIN). PanIN formation is accompanied by the establishment of an immunotolerant microenvironment. However, the immune contribution to the initiation of pancreatic cancer is currently poorly understood. Here, we genetically eliminate CD4+ T cells in the iKras* mouse model of pancreatic cancer, in the context of pancreatitis, to determine the functional role of CD4+ T cells during mutant Kras-driven pancreatic carcinogenesis. We show that oncogenic Kras-expressing epithelial cells drive the establishment of an immunosuppressive microenvironment through the recruitment and activity of CD4+ T cells. Furthermore, we show that CD4+ T cells functionally repress the activity of CD8+ T cells. Elimination of CD4+ T cells uncovers the antineoplastic function of CD8+ T cells and blocks the onset of pancreatic carcinogenesis. Thus, our studies uncover essential and opposing roles of immune cells during PanIN formation and provide a rationale to explore immunomodulatory approaches in pancreatic cancer. Cancer Immunol Res; 2(5); 423–35. ©2014 AACR.

Mammary Carcinoma Cell Derived Cyclooxygenase 2 Suppresses Tumor Immune Surveillance by Enhancing Intratumoral Immune Checkpoint Activity

IntroductionSystemic inhibition of the inflammatory enzyme cyclooxygenase (COX) 2 decreasesthe risk of breast cancer and its recurrence. However, the biology of COX-2 in themulticellular tumor microenvironment is poorly defined.MethodsMammary tumor onset and multiplicity were examined in ErbB2 transgenic mice thatwere deficient in mammary epithelial cell COX-2 (COX-2MECKO) comparedto wild type (WT) mice.Tumors were analyzed, by real time PCR, immune-staining and flow cytometry, forproliferation, apoptosis, angiogenesis and immune microenvironment. LentiviralshRNA delivery was used to knock down (KD) COX-2 in ErbB2-transformed mouse breastcancer cells (COX-2KD), and growth as orthotopic tumors was examined in syngenicrecipient mice, with or without depletion of CD8+ immune cells.ResultsMammary tumor onset was delayed, and multiplicity halved, in COX-2MECKOmice compared to WT. COX-2MECKO tumors showed decreased expression ofKi67, a proliferation marker, as well as reduced VEGFA, its receptor VEGFR2,endothelial NOS and the vascular endothelial marker CD31, indicating reduced tumorvascularization. COX-2MECKO tumors contained more CD4+ Thelper (Th) cells and CD8+ cytotoxic immune cells (CTL)consistent with increased immune surveillance. The ratio of Th markersTbet (Th1) to GATA3 (Th2) was higher, and levels of Retnla,a M2 macrophage marker, lower, in COX-2MECKO tumor infiltratingleukocytes compared to WT, suggesting a prevalence of pro-immune Th1over immune suppressive Th2 lymphocytes, and reduced macrophagepolarization to the immune suppressive M2 phenotype. Enhanced immune surveillancein COX-2MECKO tumors was coincident with increased intratumoral CXCL9,a T cell chemoattractant, and decreased expression of T lymphocyte co-inhibitoryreceptors CTLA4 and PD-1, as well as PD-L1, the ligand for PD-1. PD-L1 was alsodecreased in IFNγ-treated COX-2KD mouse mammary cancer cells in vitro and, compared to control cells, growth of COX-2KD cells as orthotopic tumorsin immune competent mice was markedly suppressed. However, robust growth ofCOX-2KD tumor cells was evident when recipients were depleted of CD8+ cells.ConclusionsThe data strongly support that, in addition to its angiogenic function, tumor cellCOX-2 suppresses intratumoral cytotoxic CD8+ immune cell function,possibly through upregulation of immune checkpoints, thereby contributing to tumorimmune escape. COX-2 inhibition may be clinically useful to augment breast cancerimmunotherapy.

Abstract A48: CD4+ T lymphocyte ablation prevents pancreatic carcinogenesis in mice

This abstract is also being presented as a short talk in Session 6: Immunology. A full abstract is printed in the Proffered Abstracts section (PR11) of the Conference Proceedings. Citation Format: Yaqing Zhang, Wei Yan, Esha Mathew, Filip Bednar, Shanshan Wan, Meredith A. Collins, Rebecca A. Evans, Theodore H. Welling, Robert H. Vonderheide, Marina Pasca di Magliano. CD4+ T lymphocyte ablation prevents pancreatic carcinogenesis in mice. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A48.

Abstract B23: CD4+ T lymphocytes promote pancreatic cancer progression by suppressing anti-tumor immune responses

Pancreatic cancer, one of the deadliest human malignancies, is associated with oncogenic Kras and is most commonly preceded by precursor lesions known as Pancreatic Intraepithelial Neoplasias (PanINs). PanIN formation is accompanied by the establishment of an immune-tolerant microenvironment. However, the immune contribution to the initiation of pancreatic cancer is currently poorly understood. Here, we show that oncogenic Kras-expressing epithelial cells drive the establishment of an immunosuppressive microenvironment through the recruitment and activity of CD4+ T cells. We further show that CD4+ T cells functionally repress the activity of CD8+ T cells. Elimination of CD4+ T cells uncovers CD8+ T cells anti-neoplastic function, and blocks the onset of pancreatic carcinogenesis. Thus, our studies uncover essential and opposing roles of immune cells during PanIN formation, and provide rationale to explore immune-modulatory approaches in pancreatic cancer. Citation Format: Yaqing Zhang, Wei Yan, Wei Yan, Esha Mathew, Filip Bednar, Shanshan Wan, Meredith A. Collins, Rebecca A. Evans, Theodore H. Welling, Robert H. Vonderheide, Marina Pasca di Magliano, Marina Pasca di Magliano. CD4+ T lymphocytes promote pancreatic cancer progression by suppressing anti-tumor immune responses. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B23. doi: 10.1158/1557-3125.RASONC14-B23

Abstract IA12: Oncogenic KRAS and the inflammatory microenvironment in pancreatic cancer

Pancreatic cancer -one of the deadliest human malignancies- is preceded by precursor lesions, most commonly Pancreatic Intraepithelial Neoplasia (PanIN). PanINs are characterized by changes in the epithelial architecture, and by the accumulation of a fibroinflammatory stroma. Oncogenic mutation in Kras are almost invariably associated with invasive pancreatic cancer (1-3) and often detected in PanIN lesions (4). Expression of oncogenic Kras in the mouse pancreas recapitulates the step-wise progression of the human disease (5). We have previously published a genetically engineered mouse model that allows inducible, tissue specific and reversible expression of oncogenic Kras in the pancreas, the iKras* mouse (6). By taking advantage of the reversible nature of oncogenic Kras expression in this model, we were able to address the role of this oncogene in PanIN and cancer maintenance. First, we showed that inactivation of oncogenic Kras at the PanIN stage leads to regression of the lesions and recovery of the pancreatic parenchyma. Then, by crossing iKras* mice with a mouse conditionally expressing a mutant form of the tumor suppressor p53 (7), we generated a model of metastatic pancreatic cancer, the iKras*p53* mouse(8). Inactivation of oncogenic Kras* in iKras*p53* mice bearing invasive tumors and metastases led to tumor regression, albeit without complete eradication of the malignant cells. Taking advantage of the reversible nature of oncogenic Kras expression in our model, we have undertaken to investigate the effect of modulating epithelial Kras expression on the tumor stroma. We have previously shown that the extensive myofibroblast population surrounding PanINs and invasive cancer depends on epithelial Kras for its proliferation, active status and maintenance. Recently, we have started characterizing the changes in immune cells infiltrating the pancreas during the PanIN stage. The induction of acute pancreatitis leads to extensive immune infiltration both in wild-type and iKras* mice. However, in wild type animals the immune infiltration is transient, and coincides with the dynamics of tissue repair. In contrast, in iKras* mice the immune infiltration is sustained over time. Moreover, the nature of the infiltrating cells is different in iKras* mice, with increased CD4+ T cells, and immature myeloid cells and fewer CD8+ T cells. This infiltration of immune cells is dependent on oncogenic Kras over time, as inactivation of Kras expression leads to a reduction in the number of inflammatory cells over time. While the nature of infiltrating immune cells over pancreatic carcinogenesis has been characterized before (9), the function of each specific subset is not fully understood. CD4 T cells are prevalent within the Kras-mutant pancreas, and include immune-suppressive cells such as regulator T cells and Th17 cells. Thus, we hypothesized that CD4+ T cells are required in pancreatic cancer. By genetically ablating CD4 T cell in iKras* mice (thus generating iKras*;CD4-/- animals) we determined that CD4 T cells are required for PanIN formation. Furthermore, we showed that CD4+ T cells promote pancreatic carcinogenesis by inhibiting the anti-tumor activity of CD8 + T cells. Thus, our results indicate that a potential anti-cancer immune response is inhibited by CD4+ T cells in response to signals derived from Kras mutant epithelial cells. Given the potential of immune-modulation as part of pancreatic cancer therapy, we plan to follow up on our initial observations by identifying the epithelial signals that modulate the immune response in pancreatic cancer. References: 1. Hruban RH, Adsay NV, Albores-Saavedra J, Compton C, Garrett ES, Goodman SN, et al. Pancreatic intraepithelial neoplasia: a new nomenclature and classification system for pancreatic duct lesions. Am J Surg Pathol. 2001;25:579-86. 2. Jones S, Zhang X, Parsons DW, Lin JC, Leary RJ, Angenendt P, et al. Core signaling pathways in human pancreatic cancers revealed by global genomic analyses. Science. 2008;321:1801-6. 3. Biankin AV, Waddell N, Kassahn KS, Gingras MC, Muthuswamy LB, Johns AL, et al. Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes. Nature. 2012;491:399-405. 4. Kanda M, Matthaei H, Wu J, Hong SM, Yu J, Borges M, et al. Presence of Somatic Mutations in Most Early-Stage Pancreatic Intraepithelial Neoplasia. Gastroenterology. 2012. 5. Hingorani SR, Petricoin EF, Maitra A, Rajapakse V, King C, Jacobetz MA, et al. Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse. Cancer Cell. 2003;4:437-50. 6. Collins MA, Bednar F, Zhang Y, Brisset JC, Galban S, Galban CJ, et al. Oncogenic Kras is required for both the initiation and maintenance of pancreatic cancer in mice. J Clin Invest. 2012;122:639-53. 7. Olive KP, Tuveson DA, Ruhe ZC, Yin B, Willis NA, Bronson RT, et al. Mutant p53 gain of function in two mouse models of Li-Fraumeni syndrome. Cell. 2004;119:847-60. 8. Collins MA, Brisset JC, Zhang Y, Bednar F, Pierre J, Heist KA, et al. Metastatic pancreatic cancer is dependent on oncogenic Kras in mice. PLoS One. 2012;7:e49707. 9. Clark CE, Hingorani SR, Mick R, Combs C, Tuveson DA, Vonderheide RH. Dynamics of the immune reaction to pancreatic cancer from inception to invasion. Cancer Res. 2007;67:9518-27. Citation Format: Yaqing Zhang, Wei Yan, Filip Bednar, Shanshan Wan, Meredith A. Collins, Rebecca A. Evans, Esha Mathew, Theodore H. Welling, Robert H. Vonderheide, Marina Pasca di Magliano. Oncogenic KRAS and the inflammatory microenvironment in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr IA12. doi: 10.1158/1557-3125.RASONC14-IA12

Abstract PR4: Restoring T cell immuno-surveillance in pancreatic carcinoma using CD40 agonists.

A hallmark of the tumor microenvironment in pancreatic ductal adenocarcinoma (PDA) is a profound immune infiltrate dominated by myeloid cells with a scarcity of T cells. Immune suppression imposed by this early immune reaction to PDA represents a significant clinical challenge to T cell immunotherapy. To circumvent this immune suppression, we have studied CD40 agonists for their well-recognized ability to induce potent T cell dependent anti-tumor immunity. However, in our studies using the KPC mouse model of PDA (KrasG12D/+ Trp53R172H/+ Pdx-1 Cre), we have found that delivery of a CD40 agonist alone or in combination with chemotherapy is unable to induce productive T cell immunosurveillance. Here, we examine barriers to the development of anti-tumor T cell immunity in PDA. By transplanting PDA tumor cells or whole PDA tumor tissue obtained from tumor-bearing KPC mice into syngeneic littermates, we demonstrate that a CD40 agonist (FGK45) in combination with gemcitabine chemotherapy can synergize to induce productive anti-tumor immunity leading to complete and sustained regression of established subcutaneous tumors (IgG2a alone – 0%; gemcitabine alone – 0%; FGK45 alone – 38%; gemcitabine + FGK45 – 71%; p < 0.001). In contrast to our findings in the KPC model, depletion of either CD4 (GK1.5) or CD8 (2.43) T cells abolished this effect (IgG2a alone – 0%; gemcitabine + FGK45 – 75%; gemcitabine + FGK45 + GK1.5 – 0%; gemcitabine + FGK45 + 2.43 – 14%; p <0.001). This observation suggested that PDA cells retain their immunogenicity and are capable of being recognized and targeted by T cells. To examine the ability of combination therapy (gemcitabine + FGK45) to induce T cell dependent anti-tumor immunity against spontaneously arising PDA, KPC mice treated with combination therapy were challenged subcutaneously two weeks later with PDA cells. However, no immune protection to tumor challenge was observed suggesting a failure to induce tumor-specific T cell immunity. To determine whether systemic immune suppression within KPC mice may inhibit the ability to induce productive T cell immunity, we next transplanted PDA tumor cells subcutaneously into KPC mice with ultrasound-confirmed PDA tumors. We found that combination therapy (gemcitabine + FGK45) delivered to these KPC mice bearing two tumors (i.e. implanted tumor plus a spontaneously arising PDA tumor) could elicit an anti-tumor T cell immune response capable of inducing tumor regression of the subcutaneously implanted tumor (Control – 0%; gemcitabine + FGK45 – 100%; p <0.001). Moreover, by delivering combination therapy to KPC animals in this “two tumor” system, we observed by immunohistochemistry an unprecedented and robust T cell infiltrate, comprised of both CD4 and CD8 T cells, into the spontaneously arising PDA tumor. Our findings suggest that ineffective T cell priming rather than immunoediting may limit T cell immunosurveillance in PDA – a finding that has important implications into the design of cancer immunotherapy for this disease. This abstract is also presented as Poster B84. Citation Format: Gregory L. Beatty, Rafi Winograd, Rebecca Evans, Santiago Lombo Luque, Patrick Guirnalda, Robert H. Vonderheide. Restoring T cell immuno-surveillance in pancreatic carcinoma using CD40 agonists. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr PR4.