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. viral oncotherapy | vascular targeting

Signals mediated by the chemokine CXCL12 and its receptor CXCR4 are involved in progression of ovarian cancer by enhancing tumor angiogenesis and immunosuppressive networks that regulate dissemination of peritoneal metastasis and development of cancer initiating cells (CICs). Here, 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 intraperitoneal delivery of a CXCR4 antagonist-expressing OVV led to reduced metastatic spread of tumors and improved overall survival over that mediated by oncolysis alone. Inhibition of tumor growth with the armed virus was associated with efficient killing of CICs, reductions in expression of ascitic CXCL12 and VEGF, and decreases in intraperitoneal numbers of endothelial and myeloid cells as well as plasmacytoid dendritic cells (pDCs). 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.

We have analyzed the ability of human gamma+/delta+ T cells to recognize a nominal antigen in association with MHC molecules. A TT-specific T cell line with approximately 40% gamma+/delta+ T cells was established from a hyperimmunized donor, D.F., by stimulation with antigen and autologous APC. Three DF-derived gamma+/delta+ clones were CD8+ as determined by immunofluorescence staining, and by Southern and Northern blotting with probes detecting delta chain rearrangement and delta and gamma chain transcripts, respectively. The gamma+/delta+ clones responded to stimulation with TT, but not TNP-BSA, and autologous APC by proliferation and IFN-gamma production. No proliferation or IFN-gamma production was detected when TT-specific T cell clones were stimulated with either TT or autologous APC only. The response to TT was enhanced by addition of exogenous IL-2. The use of allogeneic APC from 19 donors sharing one HLA-determinant with the autologous donor D.F., showed that the gamma+/delta+ T cells responded to TT with HLA-DR4-related restriction as measured by proliferation and IFN-gamma production. These results demonstrate that gamma/delta receptors can recognize non-MHC-encoded foreign antigen in a self-MHC-restricted fashion.

A 6-thioguanine-resistant, human lymphoblastoid B-cell line (GM1500 6TG A-ll; IgG secreting) was mutagentreated with low-level V-irradiation and selected for ouabain resistance. One line showing 10,000-fold higher drug resistance, designated KR4, was fused with an Epstein-Barr virus-transformed, cloned, B-lymphocyte cell line (B6) producing antitetanus toxoid (TT) antibody (IgM), and the hybrids were selected in hypoxanthine/aminopterin/thymidine medium containing 10 JIM ouabain. Surviving cells, which arose at an optimal frequency of i0-5, were subcloned by limiting dilution and screened for anti-TT production. Out of395 final subclones, 372 were found positive for anti-TT, and seven that were selected for further study secreted specific antibody (IgM, K chain) at a maximum concentration of 3-6 #g/ml. The differential rate of anti-TT production during the logarithmic phase of cell growth was 15-fold higher in the hybridomas than in the original B6 line. The hybrid nature of the clones was confirmed by karyotype analysis, histocompatibility antigen typing, and expression of secreted and membrane-bound Ig classes. Biosynthetic labeling of the cells revealed that all hybrids secreted both IgM and IgG but that only the IgM class had specificity for TT. Because Epstein-Barr virus is a polyclonal Blymphocyte activator, the technique we applied here may be useful for increasing the recovery of rare antigen-specific B cells in the peripheral blood and for improving the frequency and stability of hybridomas secreting a given antibody.The production of human monoclonal antibodies expressing a desired specificity has proved successful with methods that use either the lymphotropic Epstein-Barr virus (EBV) to immortalize antigen-specific B cells (1)(2)(3)(4)(5) or the classical hybridoma technique, whereby human blood lymphocytes are fused with murine (6) or human myeloma cell lines (7,8). To overcome difficulties inherent in each technique (9), we examined the possibility of combining them. We have described a technique whereby lymphocytes from normal donors immunized with tetanus toxoid (TT) were preselected for antigen-binding cells, subsequently transformed with EBV, and cloned (10). In the present study, an EBV-transformed clone B6 was fused with a human lymphoblastoid cell line ofthe B-cell type (B-LCL), KR-4, to rescue high amounts of anti-TT antibody production. The resulting hybridomas were found to be more stable, to have a higher cloning efficiency, and to secrete =8-fold more specific antibody compared with the parental B6 clone. In addition KR-4 had a 25-fold higher frequency of hybridization with EBVtransformed B cells compared with nontransformed B cells. MATERIALS AND METHODSCell Lines. B6 is an anti-TT antibody-producing, EBV-transformed, cloned cell line that was previously established in our laboratory (10). GM 1500 6TG A-li is a 6-thioguanine-resistant (SGuaR) [hypoxanthine/aminopterin/thymidine (HAT)-sensitive] human B-cell line, which was generously provided by H. Koprowski at The Wistar Insti...

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