Recruitment of Intratumoral CD103+ Dendritic Cells by a CXCR4 Antagonist-Armed Virotherapy Enhances Antitumor Immunity.

Intratumoral dendritic cells play an important role in stimulating cytotoxic T cells and driving antitumor immunity. Using a metastatic ovarian tumor model in syngeneic mice, we explored whether therapy with a CXCR4 antagonist-armed oncolytic vaccinia virus activates endogenous CD103+ dendritic cell responses associated with the induction of adaptive immunity against viral and tumor antigens. The overall goal of this study was to determine whether expansion of CD103+ dendritic cells by the virally delivered CXCR4 antagonist augments overall survival and in situ boosting with a tumor antigen peptide-based vaccine. We found that locoregional delivery of the CXCR4-A-armed virus reduced the tumor load and the immunosuppressive network in the tumor microenvironment, leading to infiltration of CD103+ dendritic cells that were capable of phagocytic clearance of cellular material from virally infected cancer cells. Further expansion of tumor-resident CD103+ DCs by injecting the FMS-related tyrosine kinase 3 ligand, the formative cytokine for CD103+ DCs, provided a platform for a booster immunization with the Wilms tumor antigen 1 peptide-based vaccine delivered intraperitoneally with polyriboinosinic:polyribocytidylic acid as an adjuvant. The vaccine-induced antitumor responses inhibited tumor growth and increased overall survival, indicating that expansion of intratumoral CD103+ dendritic cells by CXCR4-A-armed oncovirotherapy treatment can potentiate in situ cancer vaccine boosting.

Reprogramming antitumor immunity against chemoresistant ovarian cancer by a CXCR4 antagonist-armed viral oncotherapy.

Ovarian cancer remains the most lethal gynecologic malignancy owing to late detection, intrinsic and acquired chemoresistance, and remarkable heterogeneity. Here, we explored approaches to inhibit metastatic growth of murine and human ovarian tumor variants resistant to paclitaxel and carboplatin by oncolytic vaccinia virus expressing a CXCR4 antagonist to target the CXCL12 chemokine/CXCR4 receptor signaling axis alone or in combination with doxorubicin. The resistant variants exhibited augmented expression of the hyaluronan receptor CD44 and CXCR4 along with elevated Akt and ERK1/2 activation and displayed an increased susceptibility to viral infection compared with the parental counterparts. The infected cultures were more sensitive to doxorubicin-mediated killing both in vitro and in tumor-challenged mice. Mechanistically, the combination treatment increased apoptosis and phagocytosis of tumor material by dendritic cells associated with induction of antitumor immunity. Targeting syngeneic tumors with this regimen increased intratumoral infiltration of antitumor CD8+ T cells. This was further enhanced by reducing the immunosuppressive network by the virally-delivered CXCR4 antagonist, which augmented antitumor immune responses and led to tumor-free survival. Our results define novel strategies for treatment of drug-resistant ovarian cancer that increase immunogenic cell death and reverse the immunosuppressive tumor microenvironment, culminating in antitumor immune responses that control metastatic tumor growth.

CXCL12/CXCR4 blockade by oncolytic virotherapy inhibits ovarian cancer growth by decreasing immunosuppression and targeting cancer-initiating cells.

Signals mediated by the chemokine CXCL12 and its receptor CXCR4 are involved in the progression of ovarian cancer through enhancement of tumor angiogenesis and immunosuppressive networks that regulate dissemination of peritoneal metastasis and development of cancer-initiating cells (CICs). In this study, we investigated the antitumor efficacy of a CXCR4 antagonist expressed by oncolytic vaccinia virus (OVV) against an invasive variant of the murine epithelial ovarian cancer cell line ID8-T. This variant harbors a high frequency of CICs that form multilayered spheroid cells and express the hyaluronan receptor CD44, as well as stem cell factor receptor CD117 (c-kit). Using an orthotopic ID8-T tumor model, we observed that i.p. delivery of a CXCR4 antagonist-expressing OVV led to reduced metastatic spread of tumors and improved overall survival compared with oncolysis alone. Inhibition of tumor growth with the armed virus was associated with efficient killing of CICs, reduced expression of ascitic CXCL12 and vascular endothelial growth factor, and decreases in i.p. numbers of endothelial and myeloid cells, as well as plasmacytoid dendritic cells. These changes, together with reduced recruitment of T regulatory cells, were associated with higher ratios of IFN-γ(+)/IL-10(+) tumor-infiltrating T lymphocytes, as well as induction of spontaneous humoral and cellular antitumor responses. Similarly, the CXCR4 antagonist released from virally infected human CAOV2 ovarian carcinoma cells inhibited peritoneal dissemination of tumors in SCID mice, leading to improved tumor-free survival in a xenograft model. Our findings demonstrate that OVV armed with a CXCR4 antagonist represents a potent therapy for ovarian CICs with a broad antitumor repertoire.

Targeting CXCL12/CXCR4 signaling with oncolytic virotherapy disrupts tumor vasculature and inhibits breast cancer metastases.

Oncolytic viruses hold promise for the treatment of cancer, but their interaction with the tumor microenvironment needs to be elucidated for optimal tumor cell killing. Because the CXCR4 receptor for the stromal cell-derived factor-1 (SDF-1/CXCL12) chemokine is one of the key stimuli involved in signaling interactions between tumor cells and their stromal microenvironment, we used oncolytic virotherapy with a CXCR4 antagonist to target the CXCL12/CXCR4 signaling axis in a triple-negative 4T1 breast carcinoma in syngeneic mice. We show here that CXCR4 antagonist expression from an oncolytic vaccinia virus delivered intravenously to mice with orthotopic tumors attains higher intratumoral concentration than its soluble counterpart and exhibits increased efficacy over that mediated by oncolysis alone. A systemic delivery of the armed virus after resection of the primary tumor was efficacious in inhibiting the development of spontaneous metastasis and increased overall tumor-free survival. Inhibition of tumor growth with the armed virus was associated with destruction of tumor vasculature, reductions in expression of CXCL12 and VEGF, and decrease in intratumoral numbers of bone marrow-derived endothelial and myeloid cells. These changes led to induction of antitumor antibody responses and resistance to tumor rechallenge. Engineering an oncolytic virus armed with a CXCR4 antagonist represents an innovative strategy that targets multiple elements within the tumor microenvironment. As such, this approach could have a significant therapeutic impact against primary and metastatic breast cancer.

Targeting a mimotope vaccine to activating Fcgamma receptors empowers dendritic cells to prime specific CD8+ T cell responses in tumor-bearing mice.

A major challenge for inducing antitumor immune responses with native or modified tumor/self-Ags in tumor-bearing hosts relates to achieving efficient uptake and processing by dendritic cells (DCs) to activate immune effector cells and limit the generation of regulatory T cell activity. We analyzed the ability of therapeutic DC vaccines expressing a CD166 cross-reactive mimotope of the GD2 ganglioside, 47-LDA, to selectively expand adoptively transferred, tumor-specific T cells in NXS2 neuroblastoma tumor-bearing syngeneic mice. Before the adoptive cell transfer and DC vaccination, the tumor-bearing mice were lymphodepleted by nonmyeloablative total body irradiation or a myeloablative regimen that required bone marrow transplantation. The 47-LDA mimotope was presented to DCs either as a linear polypeptide in conjunction with universal Th epitopes or as a fusion protein with the murine IgG2a Fc fragment (47-LDA-Fcgamma2a) to deliver the antigenic cassette to the activating Fcgamma receptors. We demonstrate that immunization of adoptively transferred T cells in tumor-bearing mice with the 47-LDA mimotope expressed in the context of the activating Fc fusion protein induced higher levels of antitumor immune responses and protection than the 47-LDA polypeptide-DC vaccine. The antitumor efficacy of the therapeutic 47-LDA-Fcgamma2a-DC vaccine was comparable to that achieved by a virotherapy-associated cancer vaccine using a recombinant oncolytic vaccinia virus expressing the 47-LDA-Fcgamma2a fusion protein. The latter treatment, however, did not require total body irradiation or adoptive cell transfer and resulted in induction of antitumor immune responses in the setting of established tolerance, paving the way for testing novel anticancer treatment strategies.

Immunization with a mimotope of GD2 ganglioside induces CD8+ T cells that recognize cell adhesion molecules on tumor cells.

The GD2 ganglioside expressed on neuroectodermal tumor cells has been used as a target for passive and active immunotherapy in patients with malignant melanoma and neuroblastoma. We have reported that immunization of mice with a 47-LDA mimotope of GD2, isolated from a phage display peptide library with anti-GD2 mAb 14G2a, induces MHC class I-restricted CD8(+) T cell responses to syngeneic neuroblastoma tumor cells. The cytotoxic activity of the vaccine-induced CTLs was independent of GD2 expression, suggesting recognition of a novel tumor-associated Ag cross-reacting with 47-LDA. Glycan microarray and immunoblotting studies using 14G2a mAb demonstrated that this Ab is highly specific for the entire carbohydrate motif of GD2 but also cross-reacts with a 105 kDa glycoprotein expressed by GD2(+) and GD2(-) neuroblastoma and melanoma cells. Functional studies of tumor cells grown in three-dimensional collagen cultures with 14G2a mAb showed decreases in matrix metalloproteinase-2 activation, a process regulated by the 105 kDa-activated leukocyte cell adhesion molecule (ALCAM/CD166). A recombinant CD166 glycoprotein was shown to be recognized by 14G2a Ab and inhibition of CD166 expression by RNA interference ablated the cell sensitivity to lysis by 47-LDA-induced CD8(+) T cells in vitro and in vivo. The binding of 14G2a to CD166 was not disruptable by a variety of exo- and endo-glycosidases, implying recognition of a non-glycan epitope on CD166. These results suggest that the vaccine-induced CTLs recognize a 47-LDA cross-reactive epitope expressed by CD166, and reveal a novel mechanism of induction of potent tumor-specific cellular responses by mimotopes of tumor-associated carbohydrate Ags.

Induction of protective immune responses against NXS2 neuroblastoma challenge in mice by immunotherapy with GD2 mimotope vaccine and IL-15 and IL-21 gene delivery.

The GD2 ganglioside expressed on neuroectodermal tumor cells is weakly immunogenic in tumor-bearing patients and induces predominantly IgM antibody responses in the immunized host. Using a syngeneic mouse challenge model with GD2-expressing NXS2 neuroblastoma, we investigated novel strategies for augmenting the effector function of GD2-specific antibody responses induced by a mimotope vaccine. We demonstrated that immunization of A/J mice with DNA vaccine expressing the 47-LDA mimotope of GD2 in combination with IL-15 and IL-21 genes enhanced the induction of GD2 cross-reactive IgG2 antibody responses that exhibited cytolytic activity against NXS2 cells. The combined immunization regimen delivered 1 day after tumor challenge inhibited subcutaneous (s.c.) growth of NXS2 neuroblastoma in A/J mice. The vaccine efficacy was reduced after depletion of NK cells as well as CD4(+) and CD8(+) T lymphocytes suggesting involvement of innate and adaptive immune responses in mediating the antitumor activity in vivo. CD8(+) T cells isolated from the immunized and cured mice were cytotoxic against syngeneic neuroblastoma cells but not against allogeneic EL4 lymphoma, and exhibited antitumor activity after adoptive transfer in NXS2-challenged mice. We also demonstrated that coimmunization of NXS2-challenged mice with the IL-15 and IL-21 gene combination resulted in enhanced CD8(+) T cell function that was partially independent of CD4(+) T cell help in inhibiting tumor growth. This study is the first demonstration that the mimotope vaccine of a weakly immunogenic carbohydrate antigen in combination with plasmid-derived IL-15 and IL-21 cytokines induces both innate and adaptive arms of the immune system leading to the generation of effective protection against neuroblastoma challenge.