Targeting CCR8 Induces Protective Antitumor Immunity and Enhances Vaccine-Induced Responses in Colon Cancer

Author Manuscript Published OnlineFirst on July 19, 2018; DOI: 10.1158/0008-5472.CAN-18-1119 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Targeting CCR8 induces protective antitumor immunity and enhances vaccine-induced responses in colon cancer Daniel O Villarreal1, Andrew L’Huillier1, Susan Armington1, Cristina Mottershead1, Elena V Filippova1, Brandon D Coder1, Robert G. Petit1, Michael F Princiotta1 1Advaxis Immunotherapies, Princeton, NJ, 08540, USA To whom correspondence should be addressed: Daniel O Villarreal, email: [email protected] Running Title: CCR8 is a novel target for cancer immunotherapy Key words: CCR8, Vaccines, Cancer Immunotherapy, CD8 T cells, Tregs Total figures: 5 Word Count: 5,000 Disclosure: No potential conflicts of interest were disclosed by the other authors. 1 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 19, 2018; DOI: 10.1158/0008-5472.CAN-18-1119 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract CCR8 is a chemokine receptor expressed principally on regulatory T cells (Tregs) and is known to be critical for CCR8+ Treg-mediated immunosuppression. Recent studies have demonstrated that CCR8 is uniquely upregulated in human tumor-resident Tregs of breast, colon, and lung cancer patients when compared to normal tissue-resident Tregs. Therefore, CCR8+ tumor-resident Tregs are rational targets for cancer immunotherapy. Here we demonstrate that monoclonal antibody (mAb) therapy targeting CCR8 significantly suppresses tumor growth and improves long-term survival in colorectal tumor mouse models. This antitumor activity correlated with increased tumor-specific T cells, enhanced infiltration of CD4+ and CD8+ T cells, and a significant decrease in the frequency of tumor-resident CD4+CCR8+ Tregs. Tumor-specific CD8+ T cells displayed lower expression of exhaustion markers as well as increased functionality upon restimulation. Treatment with anti-CCR8 mAb prevented de novo induction and suppressive function of Tregs without affecting CD8+ T cells. Initial studies explored a combinatorial regimen using anti-CCR8 mAb therapy and a Listeria monocytogenes (Lm)-based immunotherapy. Anti- CCR8 mAb therapy synergized with Lm-based immunotherapy to significantly delay growth of established tumors and prolong survival. Collectively, these findings identify CCR8 as a promising new target for tumor immunotherapy and provide a strong rationale for further development of this approach, either as a monotherapy or in combination with other immunotherapies. Statement of Significance Inhibition of CCR8 represents a promising new cancer immunotherapy strategy that modulates tumor- resident regulatory T cells to enhance antitumor immunity and prolong patient survival. 2 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 19, 2018; DOI: 10.1158/0008-5472.CAN-18-1119 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction Tumor-infiltrating CD4+Foxp3+ regulatory T cells (Tregs) are a major immune cell population that contribute to the establishment of an immunosuppressive tumor microenvironment (TME) (1-4). Infiltration of large numbers of Tregs into the tumors hamper the development of effective antitumor immunity (1-4) and is often associated with poor prognosis (2-5). Treg modulation strategies have been shown to increase anti-tumor immunity and reduce tumor burden in both preclinical and clinical settings (4-7). However, although these strategies have demonstrated enhanced antitumor immune responses, there are drawbacks, such as autoimmunity and specificity of targeting (2-4, 8-13). Because Tregs and activated effector lymphocytes both express surface molecules that can be used as therapeutic targets (e.g. anti-CD25), there is the potential for ablation of essential tumor-specific effector cells required to control tumor progression in these types of antibody mediated immunotherapies (13-14). Therefore, the development of a more effective approach to specifically and selectively target tumor-infiltrating Tregs is required. In both human and mouse, the chemokine receptor CCR8 is predominantly expressed on Tregs and on a small portion of Th2 cells, but not on Th1 cells (15-16). This subset of CD4+Foxp3+ Tregs expressing CCR8, (CCR8+ Tregs), has been demonstrated to be a major driver of immunosuppression and is critical for Treg function and suppression (15-17). More recently, two independent studies characterizing the distinct molecular signature of tumor-resident Tregs demonstrated that CCR8 was a specific marker selectively upregulated by tumor-resident Tregs in several tumor types (18-19). Interestingly, Plitas, et al. highlighted that high expression of CCR8+ Tregs was associated with poor prognosis in breast cancer patients (19). These studies suggest CCR8 may be an effective therapeutic target by which to selectively and specifically modulate a subpopulation of tumor-resident Tregs in the TME to augment antitumor immunity. The extent of antitumor effects resultant from targeting CCR8 and its potential as a promising cancer immunotherapy remains to be determined. Here, we demonstrate that an anti-CCR8 (αCCR8) blocking monoclonal antibody (mAb) treatment impaired the suppressive character of the TME, markedly reducing tumor-resident CCR8+ Tregs. Ultimately, this contribution enhanced effector T cell and antitumor immunity in the CT26 colorectal tumor model. Furthermore, our study demonstrates that αCCR8 therapy synergizes with an Lm-based immunotherapy to enhance optimal antitumor efficacy. Collectively, these novel findings highlight CCR8 as a promising new target for cancer immunotherapy and its potential for broad clinical application. 3 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 19, 2018; DOI: 10.1158/0008-5472.CAN-18-1119 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Materials and Methods Mice and tumor cell lines Female, 6-8 week old BALB/c and C57BL/6 (B6) mice were purchased from Jackson Laboratories. All mouse procedures were performed in accordance with protocols approved by Advaxis Immunotherapies IACUC. The CT26.WT (CRL-2638) were purchased from the American Type Culture Collection (ATCC) and authenticated by ATCC using COI analysis. The MC38 colon carcinoma cells were purchased from Kerafast and were authenticated by Simple Sequence Length Polymorphism (SSLP). CT26 cells were maintained in RPMI medium supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere with 5% CO2 at 37°C. The MC38 cell lines were maintained in DMEM medium supplemented with 10% FBS. All cell lines were passaged twice prior to storage and thawed and passaged twice prior to implantation for all described tumor experiments. All cell lines were determined to be free of Mycoplasma (Sigma-Aldrich). Tumor models, tumor vaccine, and αCCR8 treatment CT26 (300,000) and MC38 (300,000) cells were implanted subcutaneously (s.c.) in the right flank of mice. Tumor vaccine consisted of LmddA-274 (1x108) or AH1-21mer (1x108) diluted in PBS. Details of plasmid construction are described in the supplemental material and methods. For therapeutic treatments, mice were treated with intraperitoneal (i.p.) injections of purified αCCR8 (4 µg; clone: SA214G2; Biolegend) and control antibody (rat IgG2b,k, Clone RTK4530; Biolegend). Tumor growth was monitored using electronic calipers and calculated according to the formula: V = (length x width2)/2. Mice were immunized with 200 µl of vaccine intravenous (i.v.) on day +12 and +19 post-tumor implantation. To analyze tumor-infiltrating T cells upon vaccine/anti-CCR8 combination treatment, tumors from all groups were harvested for analysis +22 days post-tumor implantation. For survival experiments, mice were euthanized when tumor size reached 2000 mm3 or when tumors become necrotic. In Vitro Assays Treg Induction: Naïve 2 x 106 CD4+ T cells were isolated from spleens of BALB/c mice through negative selection using the StemCell EasySep Mouse Naïve CD4+ T Cell Isolation Kit (Cat. 19765) according to manufacturer’s instructions. The cells were seeded on plates pretreated with 2 µg/ml αCD3. Cells were incubated for 3 to 5 days with αCD28 (1µg/mL), IL-2 (100U/mL), and TGF-β1 at 5ng/ml. The percent of converted Tregs was evaluated by flow cytometry on the third day. For inhibition of conversion, αCCR8 was added at 10 µg/ml. 4 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 19, 2018; DOI: 10.1158/0008-5472.CAN-18-1119 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Proliferation of CD8+ and CD4+ T cells from αCCR8 treated mice: The 2 x 106 CD8+ and CD4+ T cells were isolated from spleens of BALB/c mice through negative selection using the StemCell EasySep Mouse CD8+ or Naïve CD4+ T Cell Isolation Kits (Cat. 19853; Cat. 19765), respectively. To measure proliferation, both CD8+ and CD4+ T cells were stained with CFSE at a concentration of 0.5µM for 10 minutes. Cells

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