VT68.2: An Antibody to Chondroitin Sulfate Proteoglycan 4 (CSPG4) Displays Reactivity against a Tumor-Associated Carbohydrate Antigen.

The anti-CSPG4 monoclonal antibodies (mAbs) have shown anti-tumor activity and therapeutic potential for treating breast cancer. In addition, CSPG4 is a dominant tumor-associated antigen that is also involved in normal-tissue development in humans. Therefore, the potential for off-tumor activity remains a serious concern when targeting CSPG4 therapeutically. Previous work suggested that glycans contribute to the binding of specific anti-CSPG4 antibodies to tumor cells, but the specificity and importance of this contribution are unknown. In this study, the reactivity of anti-CSPG4 mAbs was characterized with a peptide mimetic of carbohydrate antigens expressed in breast cancer. ELISA, flow cytometry, and microarray assays were used to screen mAbs for their ability to bind to carbohydrate-mimicking peptides (CMPs), cancer cells, and glycans. The mAb VT68.2 displayed a distinctly strong binding to a CMP (P10s) and bound to triple-negative breast cancer cells. In addition, VT68.2 showed a higher affinity for N-linked glycans that contain terminal fucose and fucosylated lactosamines. The functional assays demonstrated that VT68.2 inhibited cancer cell migration. These results define the glycoform reactivity of an anti-CSPG4 antibody and may lead to the development of less toxic therapeutic approaches that target tumor-specific glyco-peptides.

Feeding Soy Protein Concentrates with Low and High Isoflavones Alters 9 and 18 Weeks Serum Isoflavones and Inflammatory Protein Levels in Lean and Obese Zucker Rats.

Soy's anti-inflammatory properties contribute to the health benefits of soy foods. This study was designed to investigate the bioavailability of soy isoflavones and whether the isoflavone content of soy protein concentrate diet would affect serum inflammatory proteins in an obese (fa/fa) Zucker rat model. Six-week-old male lean (L) and obese (O) Zucker rats were fed a casein control diet (C), soy protein concentrate with low isoflavones (SPC-LIF), or soy protein concentrate with high isoflavones (SPC-HIF) (7 rats/dietary group) before being killed at 9 and 18 weeks. Serum samples were analyzed for isoflavones and inflammatory proteins. At both time points, serum total (aglycone + conjugates) genistein, daidzein, and equol concentrations were significantly higher in L-SPC-HIF and O-SPC-HIF groups compared with L-SPC-LIF and O-SPC-LIF groups, respectively, and were not detectable in either L-C or O-C groups. At week 9, serum C-reactive protein (CRP) concentration was significantly lower in O-SPC-HIF group compared with O-C and O-SPC-LIF group, whereas proteins tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels did not differ between any groups. At week 18, serum CRP levels in both O-SPC-HIF and O-SPC-LIF groups were significantly lower compared with the O-C group. TNF-α level was higher in the O-SPC-LIF group compared with both O-C and O-SPC-HIF groups, whereas IL-6 levels were not different between any groups. Taken together, feeding Zucker rats SPC-LIF and SPC-HIF diets led to different serum isoflavone concentrations in both L and O Zucker rats and altered CRP and TNF-α levels in obese Zucker rats compared with controls.

Myxoma virus lacking the host range determinant M062 stimulates cGAS-dependent type 1 interferon response and unique transcriptomic changes in human monocytes/macrophages.

The evolutionarily successful poxviruses possess effective and diverse strategies to circumvent or overcome host defense mechanisms. Poxviruses encode many immunoregulatory proteins to evade host immunity to establish a productive infection and have unique means of inhibiting DNA sensing-dependent type 1 interferon (IFN-I) responses, a necessity given their dsDNA genome and exclusively cytoplasmic life cycle. We found that the key DNA sensing inhibition by poxvirus infection was dominant during the early stage of poxvirus infection before DNA replication. In an effort to identify the poxvirus gene products which subdue the antiviral proinflammatory responses (e.g., IFN-I response), we investigated the function of one early gene that is the known host range determinant from the highly conserved poxvirus host range C7L superfamily, myxoma virus (MYXV) M062. Host range factors are unique features of poxviruses that determine the species and cell type tropism. Almost all sequenced mammalian poxviruses retain at least one homologue of the poxvirus host range C7L superfamily. In MYXV, a rabbit-specific poxvirus, the dominant and broad-spectrum host range determinant of the C7L superfamily is the M062R gene. The M062R gene product is essential for MYXV infection in almost all cells tested from different mammalian species and specifically inhibits the function of host Sterile α Motif Domain-containing 9 (SAMD9), as M062R-null (ΔM062R) MYXV causes abortive infection in a SAMD9-dependent manner. In this study we investigated the immunostimulatory property of the ΔM062R. We found that the replication-defective ΔM062R activated host DNA sensing pathway during infection in a cGAS-dependent fashion and that knocking down SAMD9 expression attenuated proinflammatory responses. Moreover, transcriptomic analyses showed a unique feature of the host gene expression landscape that is different from the dsDNA alone-stimulated inflammatory state. This study establishes a link between the anti-neoplastic function of SAMD9 and the regulation of innate immune responses.

Myxoma Virus Optimizes Cisplatin for the Treatment of Ovarian Cancer In Vitro and in a Syngeneic Murine Dissemination Model.

A therapeutic approach to improve treatment outcome of ovarian cancer (OC) in patients is urgently needed. Myxoma virus (MYXV) is a candidate oncolytic virus that infects to eliminate OC cells. We found that in vitro MYXV treatment enhances cisplatin or gemcitabine treatment by allowing lower doses than the corresponding IC50 calculated for primary OC cells. MYXV also affected OC patient ascites-associated CD14+ myeloid cells, one of the most abundant immunological components of the OC tumor environment; without causing cell death, MYXV infection reduces the ability of these cells to secrete cytokines such as IL-10 that are signatures of the immunosuppressive tumor environment. We found that pretreatment with replication-competent but not replication-defective MYXV-sensitized tumor cells to later cisplatin treatments to drastically improve survival in a murine syngeneic OC dissemination model. We thus conclude that infection with replication-competent MYXV before cisplatin treatment markedly enhances the therapeutic benefit of chemotherapy. Treatment with replication-competent MYXV followed by cisplatin potentiated splenocyte activation and IFNγ expression, possibly by T cells, when splenocytes from treated mice were stimulated with tumor cell antigen ex vivo. The impact on immune responses in the tumor environment may thus contribute to the enhanced antitumor activity of combinatorial MYXV-cisplatin treatment.

An interaction domain in human SAMD9 is essential for myxoma virus host-range determinant M062 antagonism of host anti-viral function.

In humans, deleterious mutations in the sterile α motif domain protein 9 (SAMD9) gene are associated with cancer, inflammation, weakening of the immune response, and developmental arrest. However, the biological function of SAMD9 and its sequence-structure relationships remain to be characterized. Previously, we found that an essential host range factor, M062 protein from myxoma virus (MYXV), antagonized the function of human SAMD9. In this study, we examine the interaction between M062 and human SAMD9 to identify regions that are critical to SAMD9 function. We also characterize the in vitro kinetics of the interaction. In an infection assay, exogenous expression of SAMD9 N-terminus leads to a potent inhibition of wild-type MYXV infection. We reason that this effect is due to the sequestration of viral M062 by the exogenously expressed N-terminal SAMD9 region. Our studies reveal the first molecular insight into viral M062-dependent mechanisms that suppress human SAMD9-associated antiviral function.