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PDF file - 981KB, Liver metastasis, C5aR expression on myeloid, 4T1 and T cells, infiltration of CD8+ T cells and MDSCs in livers of breast tumor-bearing mice (S1). MDSCs infiltrate lungs of breast tumor bearing mice prior to metastases (S2). Complement production and activation in tumor bearing mice and C5aR expression in lymph nodes of breast cancer patients (S3). Gating for regulatory T (Treg) cells (S4). Gating for cytokine producing myeloid cells (S5).

Syngeneic mouse tumor models are critical to investigating new immunotherapeutic modalities and providing a rationale for clinical translation. Oncolytic herpes simplex virus (oHSV) is an emerging treatment strategy for breast cancer. Genetically modified oHSV selectively replicates in cancer cells (sparring healthy cells), induces immunogenic cancer cell death, and stimulates innate/adaptive anti-tumor immune responses. The therapeutic success of oHSV depends on a dynamic interaction between oHSV, the tumor, and the host. Here, we evaluated the anti-tumor effects of G47Δ-IL12, a genetically engineered oHSV expressing interleukin-12 (IL-12), in two orthotopic immunocompetent mouse models of breast cancer, E0771 (some classified this model as luminal B subtype and some as triple-negative) and EMT6 (triple-negative subtype), which are syngeneic to C57BL/6 and BALB/c mice, respectively. Intratumoral G47Δ-IL12 treatment led to complete eradication of orthotopic E0771 and EMT6 mammary tumors and prevention of lung metastases, resulting in 100% long-term survivors, which remained tumor-free during the observation period (>100 days following the last G47Δ-IL12 treatment). In a contralateral orthotopic mammary tumor model, G47Δ-IL12 treatment of primary tumors led to the eradication of both treated and untreated distal tumors, confirming the abscopal and anti-metastatic effect of G47Δ-IL12 virotherapy. All cured mice from G47Δ-IL12-treated groups rejected lethal tumor re-challenge in the contralateral naïve mammary fat pad, indicating successful generation of a protective memory response. In both E0771 and EMT6 models, cell-specific IFN-γ responses and the presence of effector memory T cells (CD4+CD44highCD62Llow phenotype) in spleens of long-term survivors, possibly contributing to tumor relapse prevention. G47Δ-IL12-mediated anti-tumor efficacy was associated with significant increases in CD4+ and CD8+ tumor-infiltrating lymphocytes (TILs) (vs. mock group) in both E0771 and EMT6 tumor lesions. G47Δ-IL12 monotherapy also led to a significantly increased CD8+/FoxP3+ ratio (an important parameter for immunotherapy success) in both tumor models. Interestingly, immune cell depletion studies demonstrated that efficacy requires both CD4+ and CD8+ T cells in the E0771 model while only CD8+ T cells were required in the EMT6 model. Conclusions: G47Δ-IL12 monotherapy was effective in eradicating both orthotopic and metastatic tumors and generating long-term protective memory responses in two syngeneic breast cancer models. Treatment efficacy of G47Δ-IL12 was associated with significant immunologic alterations of the TME. G47Δ-IL12 utilized distinct immunologic mechanisms of action to eradicate tumors: CD8+ T cell-dependent action in the EMT6 model, whereas CD4+ and CD8+ T cell-dependent efficacy in the E0771 model. Altogether, our data suggest that G47Δ-IL12 is an efficient inflammatory modulator and beneficially exploits the immune system to eradicate breast tumors. These studies provide the necessary supporting evidence for the clinical translation of this agent into breast cancer patients. Citation Format: Hong-My Nguyen, Othon Almanza, Dipongkor Saha. Oncolytic herpes simplex virus immunovirotherapy eradicates breast cancer and prevents tumor relapse [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-11-01.

This report describes a novel HLA/peptide complex with potential prognostic and therapeutic roles for invasive breast cancer. Macrophage migration inhibitory factor (MIF) mediates inflammation and immunity, and MIF overexpression is observed in breast cancer. We hypothesized that the HLA class I of cancerous breast epithelial cells would present MIF-derived peptides. Consistent with this hypothesis, the peptide FLSELTQQL (MIF19–27) was eluted from the HLA-A*0201 (HLA-A2) of breast cancer cell lines. We posited that if this MIF19–27/HLA-A2 complex was exclusively found in invasive breast cancer, it could be a useful prognostic indicator. To assess the presentation of MIF peptides by the HLA of various cells and tissues, mice were immunized with the MIF19–27/HLA-A2 complex. The resulting mAb (RL21A) stained invasive ductal carcinoma (IDC) but not ductal carcinoma in situ, fibroadenoma, or normal breast tissues. RL21A did not stain WBCs (total WBCs) or normal tissues from deceased HLA-A2 donors, substantiating the tumor-specific nature of this MIF/HLA complex. As this MIF/HLA complex appeared specific to the surface of IDC, RL21A was tested as an immunotherapeutic for breast cancer in vitro and in vivo. In vitro, RL21A killed the MDA-MB-231 cell line via complement and induction of apoptosis. In an in vivo orthotopic mouse model, administration of RL21A reduced MDA-MB-231 and BT-20 tumor burden by 5-fold and by >2-fold, respectively. In summary, HLA-presented MIF peptides show promise as prognostic cell surface indicators for IDC and as targets for immunotherapeutic intervention.

The impact of complement on cancer metastasis has not been well studied. In this report, we demonstrate in a preclinical mouse model of breast cancer that the complement anaphylatoxin C5a receptor (C5aR) facilitates metastasis by suppressing effector CD8+ and CD4+ T-cell responses in the lungs. Mechanisms of this suppression involve recruitment of immature myeloid cells to the lungs and regulation of TGFβ and IL10 production in these cells. TGFβ and IL10 favored generation of T regulatory cells (Treg) and Th2-oriented responses that rendered CD8+ T cells dysfunctional. Importantly, pharmacologic blockade of C5aR or its genetic ablation in C5aR-deficient mice were sufficient to reduce lung metastases. Depletion of CD8+ T cells abolished this beneficial effect, suggesting that CD8+ T cells were responsible for the effects of C5aR inhibition. In contrast to previous findings, we observed that C5aR signaling promoted Treg generation and suppressed T-cell responses in organs where metastases arose. Overall, our findings indicated that the immunomodulatory functions of C5aR are highly context dependent. Furthermore, they offered proof-of-concept for complement-based immunotherapies to prevent or reduce cancer metastasis. Cancer Res; 74(13); 3454–65. ©2014 AACR.

The role of complement in cancer metastasis has not yet been recognized. In addition, involvement of adaptive immunity in preventing metastasis to the vital organs is unclear. Here we show that the complement anaphylatoxin C5a receptor (C5aR) facilitates metastasis by suppressing effector CD8+ and CD4+ T cell responses in the lungs. Mechanisms of this suppression involve recruitment of immature myeloid cells co-expressing TGF-β and IL-10 to this organ. TGF- β and IL-10 favored generation of inducible T regulatory (iTreg) cells and Th2 responses that rendered CD8+ T cells dysfunctional. Importantly, pharmacological blockade of C5aR or its genetic ablation in C5aR-deficient mice prevented metastases in a syngeneic model of breast cancer. CD8+ T cell depletion abolished beneficial effect of C5aR blockade on metastasis suggesting that presence of CD8+ T cells is required for C5aR blockade-mediated protection from metastasis. These data reveal a surprising and contrasting role of C5aR-signaling in cancer metastasis that enables iTreg cell generation and suppress T cell responses. Furthermore, present study opens a new avenue for developing complement-based immunotherapies to prevent or reduce cancer metastasis.