NIH Public Access Author Manuscript Cancer Res. Author manuscript; available in PMC 2011 January 1.
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Published in final edited form as: Cancer Res. 2010 January 1; 70(1): 109. doi:10.1158/0008-5472.CAN-09-2326.
CCL2 Blockade Augments Cancer Immunotherapy Zvi G. Fridlender1, George Buchlis1, Veena Kapoor1, Guanjun Cheng1, Jing Sun1, Sunil Singhal1, Cecilia Crisanti1, Liang-Chuan S Wang1, Daniel Heitjan2, Linda A. Snyder3, and Steven M. Albelda1 1 Thoracic Oncology Research Laboratory, 1016B ARC, University of Pennsylvania, 3615 Civic Center Blvd., Philadelphia, PA 19104-6160 2
Department of Epidemiology and Biostatistics, University of Pennsylvania, PA
3
Ortho Biotech Oncology Research and Development, Centocor, Inc., 145 King of Prussia Road, Radnor, PA 19087
Abstract NIH-PA Author Manuscript NIH-PA Author Manuscript
Since an immuno-inhibitory environment exists within tumors, successful vaccines will likely require additional approaches to alter the tumor microenvironment. Monocyte chemoattractant proteins (such as CCL2) are produced by many tumors and have both direct and indirect immuno-inhibitory effects. We hypothesized that CCL2 blockade would reduce immunosuppression and augment vaccine immunotherapy. Anti-murine-CCL2/CCL12 monoclonal antibodies were administered in three immunotherapy models: one aimed at the HPV-E7 antigen expressed by a non-small cell lung cancer line, one targeted to mesothelin expressed by a mesothelioma cell line, and one using an adenovirus expressing Interferon-α to treat a non-immunogenic, non-small cell lung cancer line. We evaluated the effect of the combination treatment on tumor growth and assessed the mechanism of these changes by evaluating cytotoxic T cells, immunosuppressive cells, and the tumor microenvironment. Administration of anti-CCL2/CCL12 antibodies along with the vaccines markedly augmented efficacy with enhanced reduction in tumor volume and cures of approximately half of the tumors. The combined treatment generated more total intra-tumoral CD8+ T-cells that were more activated and more anti-tumor antigen specific, as measured by tetramer evaluation. Another important potential mechanism was reduction in intratumoral T-regulatory (T-reg) cells. CCL2 appears to be a key proximal cytokine mediating immunosuppression in tumors. Its blockade augments CD8+ T cell immune response to tumors elicited by vaccines via multifactorial mechanisms. These observations suggest that combining CCL2 neutralization with vaccines should be considered in future immunotherapy trials.
Keywords CCL2; Cancer immunotherapy; Lung Cancer; Mesothelioma; T-lymphocytes
Introduction Current immunotherapies are primarily aimed at initiating or boosting T cell responses to tumors and their antigens. However, the effectiveness of these therapies may be limited by
Corresponding author: Zvi G. Fridlender, Thoracic Oncology Research Laboratory, University of Pennsylvania, Philadelphia, PA.
[email protected],
[email protected], Tel: 1-215-573-9849, Fax: 1-215-573-4469. Potential conflicts of interest: Dr. Snyder is an employee of Centocor, Inc. This study was partially funded by a research grant from Centocor, Inc.
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systemic and local tumor-induced immunosuppression (1). It is therefore becoming more widely accepted that successful immunotherapy will require a second approach to alter tumor microenvironment and/or decrease immune-suppression (2). Several approaches have been used such as blockade of TGF-β or TGF-β signaling (3), use of Cox-2 inhibitors (4), depletion of T-regulatory cells (5), or blocking CTLA-4 (6). Another immunomodulatory factor secreted from tumor cells and the associated tumor stromal cells is monocyte chemoattractant protein 1 (MCP-1, CCL2), a CC (β) member of the cytokine/ chemokine superfamily. Although first identified as a chemokine that could induce the migration of monocytes (7), CCL2 has a number of other chemotactic properties that include attraction of subsets of lymphocytes (including T-regulatory cells) and endothelial cells into sites of inflammation (7–9). Importantly, it has also been observed to directly affect T-cell function, specifically inhibiting CD8+ T cell effector functions (10–12).
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Because of these immunosuppressive properties, we hypothesized that CCL2 was acting as an inhibitor of the effect of cancer immunotherapy, and its blockade might thus be benficial. In the mouse, there are two human CCL2 orthologues – CCL2 (MCP-1) and CCL12 (MCP-5). We initially evaluated the effect of blocking either one of these orthologues with antibodies that specifically neutralize these chemokines (8,13), and found that mAb against each one of them had a modest effect alone on tumor growth. We therefore used a mixture of the two mAb in further clinical and mechanistic experiments (which we will heretofore refer to as “αCCL2”). Our data suggest that CCL2/12 is an endogenous barrier to cancer immunotherapy, and that blockade could be a promising approach to augment CD8+ T-cell-mediated immunotherapy.
Materials and Methods Animals Female C57BL/6 mice were purchased from Charles River Laboratories (Wilmington, MA). Female C57BL/6J X 129P3/J hybrids (B6-129/J1) were purchased from Jackson Labs (Bar Harbor, ME). The Animal Use Committee of the University of Pennsylvania approved all protocols in compliance with the Guide for the Care and Use of Laboratory Animals. Cell lines
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TC1 cells were derived from mouse lung epithelial cells of a C57B6 mouse, immortalized with HPV-16 E6 and E7 and transformed with the c-Ha-ras oncogene (14). The murine lung cancer line LKR was derived from an explant of a pulmonary tumor from an activated Kras G12D mutant mouse that had been induced in an F1 hybrid of 129Sv.J and C57BL/6 (15). The murine malignant mesothelioma cell line AE17 was derived from the peritoneal cavity of C57BL/6J mice injected with asbestos (crocidolite) fibers, and given to us by Dr. Delia Nelson (16). Human mesothelin was transfected into the AE17 cell line using a lentiviral construct (AE17hmeso). Anti-CCL2/CCL12 monoclonal antibodies C1142 is a rat/mouse chimeric monoclonal antibody (mAb) that neutralizes mouse CCL2/JE (MCP-1) and C1450 is a human/mouse chimeric mAb that neutralizes the second mouse homolog CCL12 (MCP-5) (8,13,17). Both mAb were produced at Centocor Inc. (Malvern, PA). In most experiments mice were treated with a mixture of 250 μg per mouse of each mAb (α-CCL2),, in a total volume of 200 μl normal saline intra-peritoneally (IP), twice per week. Control mice were treated with an equal volume of normal saline.
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Immunotherapy models
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We used three different immunotherapy models. For the TC1 tumor model, we used an E1/ E3-deleted type 5 adenoviral vector expressing the HPV-E7 protein under control of a cytomegalovirus promoter as previously described (Ad.E7) (4). Animals bearing TC1 tumors were vaccinated subcutaneously (S.Q.) with 1×109 pfu of Ad.E7 vector, followed by a booster after seven days. For the LKR cell line, we used an Adenovirus (Ad) expressing a hybrid Interferon-α2α1 (Ad.IFNα) with activity in mice, received from Schering-Plough Inc. (17). One dose of 1×109 pfu of virus was injected intratumorally. For the AE17.hmeso tumor model, we used a modified, live attenuated Listeria monocytogenes vector expressing human mesothelin (Lm.Meso) provided by Drs. Dirk Brockstedt and Thomas Dubinsky of Anza Corporation. Mesothelin is a tumor-associated antigen highly expressed in human malignant mesotheliomas (18). Lm.Meso was constructed by inserting a mesothelin expression cassette integrated at the inlB locus. Animal flank tumor models
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Mice were injected on the right flank with 1×106 TC1, LKR or AE17-h.meso tumor cells in the appropriate mouse strain. The flank tumors were allowed to reach an average size of 200– 250 mm3 (approximately 12–15 days). Mice were treated in one of 4 groups: 1) controluntreated, 2) α-CCL2/CCL12 mAbs 3) Immunotherapy alone, or 4) Combination of immunotherapy and α-CCL2/CCL12 mAbs. All experiments had at least 5 mice per group and were repeated at least once. When needed for analysis (i.e. for FACS, RNA, cell subsets isolation, etc.), flank tumorswere harvested from the mice, and digested with 2 mg/mL DNase I (Sigma, St. Louis, MO) and 4 mg/mL collagenase type IV (Sigma) at 37°C for 1 hour. Flow cytometric analysis of tumors and spleens Splenocytes, lymph nodes and tumor cells were studied by FACS analysis as previously described (4). All fluorescently labeled antibodies used were purchased from BD Biosciences (San Jose, CA) except for: CD206-PE, obtained from Serotec (Oxford, UK); 4-1BB (CD137)PE, obtained from Abcam (Cambridge, UK); and GR-1-FITC, obtained from eBioscience (San Diego, CA). Flow cytometry was done using a Becton Dickinson FACS Calibur flow cytometer (San Jose, CA). Data analysis was done using FlowJo software (Ashland, OR). The allophycocyanin-labeled H-2Db tetramer loaded with E7 peptide (RAHYNIVTF) was obtained fromthe National Institute of Allergy and Infectious Diseases tetramercore. Intracellular staining for FoxP3 was done using the PE anti mouse/rat FoxP3 staining set (eBioscience). RNA isolation and real-time, reverse transcription-PCR
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Mice with tumors were treated with either one of the four treatments detailed above, removed 2 days after the Ad.E7 boost vaccine, flash frozen, and the RNA from each tumor isolated. For each treatment condition, a pool of RNA was created by adding the same amount of RNA from each of the tumors within the group. cDNA was made from each pool, RNA levels were normalized to β-actin levels, and quantification of tumor mRNA levels was performed as previously described (19). Relative levels of expression of each of the selected genes (fold change versus control) were determined. Each sample was run in quadruplicate and the experiment was repeated at least once. Primer sequences are given in supplemental Table 1. Immunohistochemical staining of tumors Animals bearing flanktumors, treated with each of the treatments as above, were euthanized 2–3 days after the booster Ad.E7 vaccine. The tumors were immediately placedin Tissue-Tek OCT compound (Sakura Finetek USA, Inc., Torrance,CA) to be stored at 80°C. Staining was done as previously described (4).
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Evaluation of secretion of cell products (TNF-α) from explants
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Mice with tumors were treated with one of the four treatment options as above. Tumors were removed 2 days after the boost vaccine, cut into pieces of about 5 × 5 mm, weighed and placed in a 24 well plate with 800 μl of culture media. After 24 hours, the media was collected, and spun to remove cellular debris (5 min, 1500 rpm). The amountof TNF-α secreted by tumors (corrected for weight) was quantifiedusing an ELISA kit according to the instructionsof the manufacturer (BD OptEIA ELISA set, BD Biosciences). Statistical analyses For the RT-PCR, FACS studies, and flank tumor studies comparing differences between two groups, we used unpaired Student t-tests. For FACS and flank tumor studies comparing more than two groups, we used one sided ANOVA with appropriate post hoc testing. Differences were considered significant when P
