Diffuse gliomas are the most common primary malignant brain tumor. Although extracranial metastases are rarely observed, recent studies have shown the presence of circulating tumor cells (CTCs) in the blood of glioma patients, confirming that a subset of tumor cells are capable of entering the circulation. The isolation and characterization of CTCs could provide a non-invasive method for repeated analysis of the mutational and phenotypic state of the tumor during the course of disease. However, the efficient detection of glioma CTCs has proven to be challenging due to the lack of consistently expressed tumor markers and high inter- and intra-tumor heterogeneity. Thus, for this field to progress, an omnipresent but specific marker of glioma CTCs is required. In this article, we demonstrate how the recombinant malaria VAR2CSA protein (rVAR2) can be used for the capture and detection of glioma cell lines that are spiked into blood through binding to a cancer-specific oncofetal chondroitin sulfate (ofCS). When using rVAR2 pull-down from glioma cells, we identified a panel of proteoglycans, known to be essential for glioma progression. Finally, the clinical feasibility of this work is supported by the rVAR2-based isolation and detection of CTCs from glioma patient blood samples, which highlights ofCS as a potential clinical target for CTC isolation.

Molecular similarities between embryonic and malignant cells can be exploited to target tumors through specific signatures absent in healthy adult tissues. One such embryonic signature tumors express is oncofetal chondroitin sulfate (ofCS), which supports disease progression and dissemination in cancer. Here, we report the identification and characterization of phage display-derived antibody fragments recognizing two distinct ofCS epitopes. These antibody fragments show binding affinity to ofCS in the low nanomolar range across a broad selection of solid tumor types in vitro and in vivo with minimal binding to normal, inflamed, or benign tumor tissues. Anti-ofCS antibody drug conjugates and bispecific immune cell engagers based on these targeting moieties disrupt tumor progression in animal models of human and murine cancers. Thus, anti-ofCS antibody fragments hold promise for the development of broadly effective therapeutic and diagnostic applications targeting human malignancies.

Early detection and monitoring of cancer progression is key to successful treatment. Therefore, much research is invested in developing technologies, enabling effective and valuable use of non-invasive liquid biopsies. This includes the detection and analysis of circulating tumor cells (CTCs) from blood samples. Recombinant malaria protein VAR2CSA (rVAR2) binds a unique chondroitin sulfate modification present on the vast majority of cancers and thereby holds promise as a near-universal tumor cell-targeting reagent to isolate CTCs from complex blood samples. This study describes a technical approach for optimizing the coupling of rVAR2 to magnetic beads and the development of a CTC isolation platform targeting a range of different cancer cell lines. We investigate both direct and indirect approaches for rVAR2-mediated bead retrieval of cancer cells and conclude that an indirect capture approach is most effective for rVAR2-based cancer cell retrieval.

Tumour‐derived extracellular vesicles (tdEVs) are widely studied for their contribution to tumour progression and metastasis. These studies are hampered by the lack of specific markers to identify the tdEVs. Here, we show that oncofoetal chondroitin sulphate, a malignancy‐associated glycosaminoglycan modification, is present on tdEVs and can be targeted by the malaria VAR2CSA protein or C9 antibody. Using a fluorescently labelled recombinant VAR2CSA protein, we identified EVs from cancer cells in vitro by super‐resolution fluorescence microscopy and flow cytometry, as well as in a proof‐of‐concept study using plasma samples from pancreatic adenocarcinoma patients. Thus, the binding of VAR2CSA offers a tool to identify tdEVs, and can be used to explore their function and biomarker potential in cancer.

Antibody-drug conjugates (ADCs) have emerged as promising targeted therapies for acute myeloid leukemia (AML). However, most ADCs exhibit off-target binding to normal hematopoietic stem and myeloid progenitor cells, resulting in adverse hematotoxicity and narrow therapeutic windows, thereby limiting their clinical application to young and fit patients with AML who are eligible for intensive curative therapies. Proteoglycans with high levels of the glycosaminoglycan oncofetal chondroitin sulfate (ofCS) are abundantly expressed in solid cancers but are absent or expressed at low levels in normal adult tissues. Here, we report high ofCS levels on bone marrow (BM) cells from patients with AML and patient-derived xenografts (PDXs) from these patients, whereas BM cells from healthy individuals showed low or undetectable ofCS levels. Consistently, an anti-ofCS antibody demonstrated binding to and internalization into AML cells, and anti-ofCS ADCs effectively killed AML cells in vitro. Moreover, anti-ofCS ADC treatment significantly prolonged survival of AML PDXs compared with controls and was associated with low toxicity. Thus, anti-ofCS ADCs could represent an effective therapy with acceptable toxicity for patients with AML, including those who are ineligible for or unresponsive to current intensive curative therapies. In conclusion, this study demonstrates, to our knowledge for the first time, that a glycosaminoglycan-like ofCS is a druggable target for the development of effective antibody-based AML therapies.