CCR8 is a chemokine receptor expressed principally on regulatory T cells (Treg) 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 patients with breast, colon, and lung cancer when compared with normal tissue-resident Tregs. Therefore, CCR8 tumor-resident Tregs are rational targets for cancer immunotherapy. Here, we demonstrate that 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 CD4CCR8 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 induction and suppressive function of Tregs without affecting CD8 T cells. Initial studies explored a combinatorial regimen using anti-CCR8 mAb therapy and a -based immunotherapy. Anti-CCR8 mAb therapy synergized with-based immunotherapy to significantly delay growth of established tumors and to 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. 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. .
Introduction: Virtually all tumors contain somatic mutations that can result in novel antigenic sequences that may be targeted by the host cellular immune response. Some of these mutations occur in preferential regions of specific genes commonly referred to as hotspot mutations. Hotspot mutations are commonly shared by cancer patients both within and across multiple tumor types. These hotspot mutations often confer loss or gain of function contributing to oncogenesis, which makes them promising therapeutic targets. One such mutation commonly found in several human tumor types is an aspartic acid substitution for glycine at position 12 (G12D) in KRAS. This same mutation occurs in the CT26 murine colorectal tumor model. To determine if expression of the KRAS G12D sequence in a bacterial immunotherapy vector can control tumor growth in the CT26 murine model, the Advaxis Listeria monocytogenes (Lm)-based platform was engineered to express a 21-amino acid KRAS sequence peptide containing the G12D mutation (Lm-Hot KRAS_G12D). In addition, we evaluated control of tumor growth using an ADXS-HOT construct (ADXS-503) that expresses multiple shared human hotspot and tumor-associated antigens, including the G12D KRAS. The ADXS-HOT clinical program is comprised of several Lm-based immunotherapies designed to target multiple shared hotspot and tumor-associated antigens commonly found in specific cancer types. In this study, we demonstrate control of tumor growth in a mouse model by targeting a commonly shared hotspot mutation using an Lm-based immunotherapy. Results: We show that the Lm-HOT KRAS_G12D therapy significantly delayed tumor growth and improved long-term survival in the murine CT26 colon carcinoma model. This response was associated with an increase in the frequency of tumor infiltrating antigen-specific CD8 T cells and γδ T cells within the tumor microenvironment and a decrease in the frequency of intratumoral regulatory T cells (Tregs). Furthermore, tumor-specific CD8 T cells displayed lower expression of exhaustion markers as well as increased functionality upon restimulation. Interestingly, our proprietary ADXS-503 (a clinical ADXS-HOT construct) which includes KRAS G12D as one of its multiple targets, was also capable of significantly suppressing tumor growth in the CT26 tumor model. Conclusion: These results suggest that our ADXS-HOT platform is a promising approach to target shared hotspot mutations. That ADXS Lm constructs targeting a single hotspot mutation can significantly control tumor growth whether it is in a single or multi-target construct. These data describe an exciting translatable discovery with the potential for broad utility across multiple tumor types and patients who share common hotspot mutations. Citation Format: Daniel Villarreal, Brandon Coder, Susan Armington, Andrew L'Huillier, Cristina Mottershead, Elena Filippova, Nithya Thambi, Kim Ramos, David Balli, Robert Petit, Michael Princiotta. Targeting shared hotspot cancer mutations with a Listeria monocytogenes immunotherapy induce potent anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-149.
Introduction: Neoantigens derived from tumor-specific mutations have been shown to drive tumor specific CD8+ T cell responses leading to tumor regression and extending overall survival. Frameshift mutations are estimated to generate up to nine times more neoantigens per mutation compared to in-frame mutations. However, it is not clear if vaccination against frameshift mutations induces neoantigen-specific CD8+ T cell responses that result in control of tumor growth. ADXS-NEO is a personalized Listeria monocytogenes (Lm)-based immunotherapy designed to target mutation-derived tumor-specific neoantigens. Advaxis' Lm-based immunotherapies consist of live attenuated bacterial vectors that are bioengineered to secrete an antigen-adjuvant fusion protein consisting of a truncated non-hemolytic fragment of listeriolysin O, which has adjuvant properties, and tumor-specific antigens. Here, we demonstrate the feasibility of using the ADXS-NEO platform to target tumor-specific frameshift mutations in order to generate neoantigen-specific T cells that control tumor growth. Results: Whole-exome sequencing of the CT26 and MC38 mouse tumor cell lines identified 30 and 31 unique frameshift mutations respectively. Individual frameshift mutations ranged in size from 12 to as many as 150 amino acids (aa). Lm vectors targeting the two longest frameshift mutations were constructed for each tumor model. The therapeutic efficacy of Lm vectors expressing either a single 57 aa (Lm-57) or a single 150 aa (Lm-150) MC38 frameshift mutation were evaluated in C57BL/6J mice. Both Lm vectors generated multiple unique frameshift-specific TILs and slowed tumor growth. Furthermore, we evaluated the tumor microenvironment following Lm-57 or Lm-150 treatment and observed a decrease in the frequency and absolute number of Tregs, TAMs, and MDSCs and an increase in the frequency and absolute number of total cytotoxic granzyme A+ effector CD8+ T cells. Similarly, Lm vectors expressing either a 64 aa (Lm-64) or a 93 aa (Lm-93) CT26 frameshift mutation were evaluated in the CT26 tumor model. Both Lm-64 and Lm-93 significantly controlled tumor growth. Additionally, an influx of neoantigen-specific TILs and a significant decrease in the frequency of intratumoral Tregs was observed. Conclusion: ADXS-NEO induced potent immune responses against tumor-specific frameshift mutations and controlled tumor growth. Advaxis' Lm platform is able to target frameshift mutations ≥150 aa and generate multiple neoantigen-specific T cells per frameshift. ADXS-NEO controls tumor growth via multiple mechanisms, including the generation of tumor-specific cytotoxic TILs, by secreting tumor-derived neoantigens directly into dendritic cells and by attenuating the suppressive tumor microenvironment. Citation Format: Brandon Coder, Daniel O. Villarreal, Susan Armington, Elena Filippova, Andrew L'Huillier, Dipti Kelkar, Xiaoming Ju, Cristina Mottershead, David Balli, Kim Ramos, Hyewon Phee, Jim Johnston, Robert Petit, Michael Princiotta. Targeting frameshift mutations with a Listeria monocytogenes immunotherapy drives neoantigen-specific antitumor immunity in the MC38 and CT26 mouse tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-148.
<p>The frequency of peripheral Tregs in tumor-bearing αCCR8-treated mice and the effects of αCCR8 on ex vivo tumor CD8+ and CD4+ T cells.</p>
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