Preclinical Efficacy of Endoglin-Targeting Antibody–Drug Conjugates for the Treatment of Ewing Sarcoma

Ewing sarcoma is the second most common primary bone and soft tissue sarcoma of children, adolescents, and young adults. It results from pathognomonic balanced t(11;22)(q24;q12) chromosomal translocations between EWSR1 and one of several genes in the ETS family of transcription factors. Micrometastatic spread is nearly universal at the time of diagnosis, and about one-quarter of patients with Ewing sarcoma present with radiologically-evident metastatic disease at that time.

Although the long-term survival rate has improved over the last few decades for those who present with localized disease, mainly due to the introduction of multimodal therapeutic strategies, the 5-year survival rate in patients with metastatic disease remains below 20%–30% (1). Poor clinical outcomes can, in part, be attributed to tumor cell heterogeneity and varied expression of the fusion-protein expression (2), which influences the epigenomic (3, 4), transcriptomic, and proteomic profiles (5) of the cells. Ultimately, those changes regulate cell phenotype and drug sensitivity (3, 6).

This implies a critical need to shift from an outdated 'one fits all' therapeutic approach to a more customized treatment strategy directed towards the unique molecular traits that exist within each tumor. Toward that end, ADCs have been used with increasing frequency in the last few decades as a precision-guided treatment to combat cancer and are now an essential weapon in the oncologists' arsenal of antineoplastic therapies (7). ADCs consist of an mAb joined to a cytotoxic agent by a chemical linker. Among a wide range of cytotoxic molecules, we evaluated the type-2 ribosome-inactivating protein nigrin-b A chain and the microtubule-disrupting synthetic small molecule cytolysin (8, 9). Irrespective of the type of cytotoxic payload fused to the mAb, the targeting antibody recognizes tumor tissues by specifically binding to the cognate cell surface protein. Once the ADC is internalized via receptor-mediated endocytosis, the drug is released into the late endosome compartment and becomes active (7, 10). The mechanism of action of ADCs can be incredibly specific, and off-target effects can often be limited to tissues that selectively express the target of interest (10).

ENG is a homodimeric glycoprotein that acts as a coreceptor of TGFβ family of proteins. It occurs in two isoforms and exists at the tumor cell surface and tumor microenvironment within the extracellular matrix (11). ENG is also expressed in endothelial cells (EC), activated macrophages, normal marrow cells, some leukemic cells, and other types of tumor cells. Transmembrane ENG usually exists as part of a multiprotein complex that can include TGFβ receptor 1 (ALK1, ALK5), TGFβ receptor 2, and BMP receptors (12, 13). Soluble ENG (sENG), however, is predominantly distributed in the extracellular matrix (ECM) following cleavage by MMP14 (14). Its expression plays a key role during embryogenesis, angiogenesis, vascular development and homeostasis (15, 16). Of particular relevance to our work, ENG is highly expressed by a range of human sarcomas and by human mesenchymal stem cells (MSC), the putative Ewing sarcoma cell of origin (17). Notably, its expression has been linked to cell plasticity, an especially-important finding given that Ewing sarcoma is a poorly differentiated neoplasm with no normal tissue counterpart. Further, the sentinel trait of Ewing sarcoma plasticity and epigenetic dysregulation appears to facilitate vasculogenic mimicry and promote tumor cell invasiveness (18–20).

Herein, we sought to better understand how ENG contributes to Ewing sarcoma biology while teasing apart the direct and indirect contribution of ENG-targeting. Critically, with an eye towards a future clinical development, our research leverages the strength of two independent laboratories to validate the preclinical results. Independently, we establish that human-specific anti-ENG ADCs are superior to unconjugated ENG-targeting therapies in their capacity to suppress cell proliferation and hinder tumor growth. Furthermore, we demonstrate a number of heretofore unreported tumor-centric effects that have a major impact on critical cell signaling pathways. Although naked anti-ENG MAbs (e.g., TRC105) are already being tested across a diverse window of tumor types, our data suggest that ADCs could significantly improve the likelihood of clinical benefit.

A4573, CADO, ES8, RDES, RM82, TC32, VH64, and TC71 ES cell lines were cultured in complete RPMI medium. Other Ewing sarcoma cell lines, A673 and SKNMC, were cultured in DMEM 4.5 g/L glucose and EMEM medium, respectively. All the cell culture media were supplemented with 1% penicillin/streptomycin, 1% glutamine, and 10% FBS (all from Gibco, Thermo Fisher). All parental cell lines were tested twice per year for mycoplasma contamination using the MycoAlert Detection Kit (Lonza Group Ltd.) according to the manufacturer's protocol and authenticated using either short tandem repeats (STR) fingerprinting with an AmpFLSTR Identifier Kit as previously described or STR analysis (CLS, Cell Lines Service GmbH). Cell line individual characteristics and origin are depicted in Supplementary Table S1. All commercially available cell lines were purchased from ATCC, and each were used for no longer than 3 months.

Plasmas from patients with Ewing sarcoma (n = 27) were kindly provided by Dr. Arjan Lankester from the Leiden University Medical Center. Patient samples, (plasma, tumor) were obtained under patient consent and in accordance to Declaration of Helsinki. Plasmas from healthy donors (n = 7) were obtained under patient consent by the Centro de Investigación del Cáncer in Salamanca, Spain. A cohort of 22 Ewing sarcoma samples embedded in Tissue-Tek O.C.T. compound (Sakura Finetek USA Inc.) and an additional cohort of 43 FFPE samples [used to generate tissue microarray (TMA)] were obtained from the Hospital Universitario Virgen del Rocío-Instituto de Biomedicina de Sevilla Biobank (Andalusian Public Health System Biobank and ISCIII-Red de Biobancos PT13/0010/0056). All identified patients and collected data were in accordance with guidelines of MDACC and IBIS's institutional review board. Authors followed the recommendations suggested by REMARK guidelines.

All experiments were conducted in accordance with protocols and conditions approved by the European guidelines (EU Directive 2010/63/EU) and by the University of Texas MDACC (Houston, Texas) Institutional Animal Care and Committee (eACUF Protocol #00000928-RN01). In vivo binding assay, dose-ranging–study and RM82 xenograft models were approved by the local institution and the Dirección General de la producción Agrícola y ganadera de la Junta de Andalucía in Spain. PDX studies were approved by the local animal care and use committee (Comité Ético de Experimentación Animal at Universidad de Barcelona, protocol 419/15). ES8 xenograft model was approved by the University of Texas MD Anderson Cancer Center Institutional Animal Care and Use Committee (ACUF Protocol#000928-RN01). Additional material and methods are provided in the Supplementary Information section.

ENG expression was evaluated in a set of Ewing sarcoma cell lines, using MSCs and an endothelial cell line (HUVEC) as positive controls. The Ewing sarcoma panel comprises cell lines bearing various EWS–ETS fusion variants (Supplementary Table S1). ENG mRNA and protein levels varied considerably across the Ewing sarcoma cell lines (Fig. 1A and B). The CADO cell line lacked expression of ENG, both at the mRNA (Fig. 1A), and at the protein levels (Fig. 1B). This heterogeneous expression of ENG prompted us to analyze the methylation status of the CpG islands present in ENG promoter in Ewing sarcoma cell lines. In fact, the ENG promoter was only hypermethylated in the CADO cell line, suggesting that ENG expression is epigenetically regulated in Ewing sarcoma (Fig. 1C; Supplementary Fig. S1A). In addition, compared with CADO cell line, the ENG was expressed in RM82 (high level) and TC71 (intermediate-low level) cell lines, as confirmed by immunofluorescence (Supplementary Fig. S1B) and FACS analyses (Supplementary Fig. S1C). When cleaved by MMP14, ENG is shed into the extracellular matrix in a soluble form (sENG), which can also be detected in the supernatants of Ewing sarcoma cell lines (n = 7) by ELISA (Fig. 1D). Here, sENG concentration positively correlated with the mRNA levels of ENG (Pearson's correlation: r = 0.7747, P = 0.0408; Supplementary Fig. S1D). sENG was also evaluated in human plasma from healthy donors and patients with Ewing sarcoma; however, no significant differences were observed between healthy donors/Ewing sarcoma-patient, localized/metastatic disease, or low/high tumor volume groups (Supplementary Fig. S2A–S2C). Finally, no link between sENG and clinical parameters was observed (Supplementary Fig. S2F). One possible explanation for this finding could be that the constitutive concentration of sENG in the organism may be overlapping the differential tumor-derived sENG concentration, as ENG is expressed in physiologic conditions.

In parallel, ENG expression was characterized by flow cytometry analysis in a panel of five Ewing sarcoma cell lines (ES8, TC32, TC71, A4573, and A673) using the OMTX003 vehicle antibody at their cellular membrane and intracellular levels. OMTX003 exhibited linear dose-dependent binding and effectively discriminated between low-expressing (TC71 and A673) and high-expressing (ES8, TC32, and A4573) cell lines (Fig. 1E). ENG expression was also evaluated by IHC analysis in a set of Ewing sarcoma xenograft (n = 3, Fig. 1F) and PDX models (n = 9, Fig. 1G), altogether confirmed heterogeneous expression of ENG. In the group of PDX tumors, this heterogeneous ENG expression was also confirmed ranging from 11.1% low, 22.2% intermediate to 66.7% high levels (Fig. 1G).

We investigated the expression of MMP14 within a panel of Ewing sarcoma cell lines (Fig. 1B; Fig. 2A and B) and determined whether this could be useful as a prognostic factor. The CADO cell line, which exhibited no ENG expression (Fig. 1B; Fig. 2B), showed a lack of MMP14 expression and hypermethylation of its promoter (Fig. 1C; Supplementary Fig. S1A). To clarify if MMP14 is the metalloprotease implicated in the extracellular domain cleavage of ENG in Ewing sarcoma, we could not find any correlation between MMP14 and ENG at their mRNA expression levels (r = 0.6053; P = 0.1498 by Pearson's-Supplementary Fig. S2D); however, we observed a positive correlation between the mRNA levels of MMP14 and the concentration of sENG in Ewing sarcoma cell line supernatants (n = 5; r = 0.9531; P = 0.0121 by Pearson's-Supplementary Fig. S2E). Furthermore, frozen tissue samples from one cohort of patients with Ewing sarcoma (n = 22) were used to evaluate the mRNA expression of ENG and MMP14 (Fig. 2C) and FFPE tissues from another cohort (n = 43) of patients were used to evaluate protein expression of ENG and MMP14 (Fig. 2D; Supplementary Table S3) by IHC. High ENG expression was found in 51.16% of the analyzed samples (Fig. 2F). In both cohorts, we found a significant correlation between ENG and MMP14 at their mRNA and protein levels (r = 0.8331, P < 0.0001, Fig. 2E; and r = 0.4614, P = 0.0021, Fig. 2D, respectively). These results were in agreement with the in vitro data in Ewing sarcoma cell lines and PDX models, demonstrating heterogeneous expression of ENG and MMP14 in Ewing sarcoma samples. The pattern of ENG and MMP14 protein expression did not correlate with the clinical parameters either in the presence of primary, metastatic, or relapse tumors.

Having shown that ENG is highly expressed not only in the endothelium, but also variably-expressed in tumor cells, we sought to isolate the relative contribution of targeting ENG within the vascular stroma versus tumor. As a nontumoral in vitro cell-based assay for OMTX003 drug efficacy, HUVEC cells were exposed to this antibody. It was shown previously that OMTX003 binds to human ENG extracellular domain (amino acids 26-584) in a concentration-dependent manner, with high specificity and affinity (Supplementary Fig. S3C). Incubation of OMTX003 with human rhENG or human HT1080 cells showed OD₅₀ at 0.3 and MFI₅₀ at 0.4 nmol/L by ELISA and flow cytometry analyses, respectively (Supplementary Fig. S3C and S3D).

Incubation of OMTX003 (100 μg/mL, 699.3 nmol/L) with HUVEC cells for 72 hours was able to inhibit tube formation (Supplementary Fig. S4B). ES cell lines presenting the ability to form vasculogenic mimicry (Supplementary Fig. S4A) were also exposed to OMTX003 within the same conditions (Supplementary Fig. S4C), however no effects were observed. Because ENG has been reported to enhance Ewing sarcoma cell proliferation, we assumed that the maximal antiproliferative effect of OMTX003 would occur in Ewing sarcoma cells that abundantly express ENG. To test that hypothesis, three Ewing sarcoma cell lines with varied ENG expression were chosen for experimental validation: RM82 (high expression), TC71 (medium-low expression), and CADO (no expression) cell lines. Surprisingly, we observed little to no effect of OMTX003 in these cell lines (Fig. 3A). Although OMTX003 was able to achieve more than 50% suppression in the ENG-high expressing RM82 cells, the requirement for micromolar range drug concentrations suggested that ENG targeting using naked MAbs such as OMTX003 may not adequately impair Ewing sarcoma cell proliferation.

Considering the promise of ADC technology, we assessed whether drug conjugation to OMTX003 could enhance its antiproliferative effect in Ewing sarcoma cells. Two ADCs were generated by cystein-based conjugation of OMTX003 to either the ribosome-inactivating protein nigrin-b A chain (OMTX503), using the 4-succynimidyloxycarbonyl-α-methyl-α-(2-pyridyl-dithio)toluene (SMPT) chemical linker, or the microtubule disrupting cytotoxic drug cytolysin (OMTX703), using the protease-cleavable vcPABA optimized linker (Fig. 3B). Similarly to OMTX003, both ADCs were highly stable and able to bind with high specificity and affinity to human ENG on the surface of ENG-expressing cells and got internalized efficiently to allow specific intracellular release and activation of their payload moieties (data not shown). As anticipated, OMTX503 demonstrated a compelling anti-proliferative effect in the assessed Ewing sarcoma cells: RM82 (high ENG expression; IC₅₀ = 1.18 × 10⁻¹⁰ M), TC71 (medium-low ENG expression; IC₅₀ = 9.155 × 10⁻⁹ M), and CADO cell lines (no ENG expression; IC₅₀ ≈ 1.738 × 10⁻⁸ M; Fig. 3C). As shown in Fig. 3D, the sensitivity of Ewing sarcoma cell lines to this agent positively correlated with ENG expression (r = −0.9999; P = 0.0082). A parallel evaluation was performed to assess the in vitro efficacy of OMTX703 by choosing ES8 cell line, which presents the highest expression of ENG among other Ewing sarcoma cell lines to validate the assay (Fig. 1E). This high expression level of ENG in ES8 correlated with efficient internalization (data not shown), efficacy, and cytotoxic effects in vitro through cell proliferation assays (Fig. 3E, IC₅₀ = 260.6 nmol/L). Collectively, these data indicate that ADCs impair in vitro Ewing sarcoma cell proliferation in an ENG expression-dependent manner.

To initiate animal studies, we determined first whether high ENG expression by Ewing sarcoma xenografts (RM82 and CADO) is required to achieve an in vivo effect, as our in vitro data suggested would be the case. Importantly, as assessed by IHC, the expression of ENG in the xenograft tumors was maintained at levels comparable to the in vitro parental cell lines (Fig. 1F). As a second pivotal control, the in vivo binding assay confirmed that OMTX003 binds specifically to human ENG, which was only present in tumor cells, but not in the murine-derived microvessels (Fig. 4A). A murine OMTX003 MAb version (OMTX003-mu) targeting ENG was also used to detect its binding to murine-derived vessels expressing ENG, as it was observed in the lower panel of Fig. 4A. To evaluate the adequate therapeutic window of the drugs, a dose-ranging study (DRS) was performed. OMTX003 (unconjugated MAb) and OMTX503 (nigrin-b A chain-ADC) were tested in vivo in dose-escalated manner and the animal weight loss (defined as a loss >20% of the initial animal weight) was assessed as an indirect metric of general drug toxicity (Fig. 4B). The vehicle OMTX003 MAb elicited no toxicity when tested at 10 mg/kg in both RM82, CADO (Fig. 4B), and ES8 (Fig. 5A and B) xenograft models. OMTX503 dosing at 0.5 mg/kg also appeared safe. Following euthanasia, careful histopathologic analyses of the lung, liver, and kidney were performed, showing no significant deviation from normality.

The efficacy of OMTX003 and OMT503 was evaluated in RM82 xenografts in parallel with irinotecan (Supplementary Table S4), which was used as a positive control of drug efficacy (Supplementary Fig. S5A). The evolution of tumor growth in each individual group was assessed throughout the 21-day treatment period. The irinotecan-treated group was maintained in a long-term survival study until day 41, when all animals relapsed and were sacrificed (Supplementary Fig. S5A). Animal weight was also evaluated during this time as a measure of treatment-induced toxicity (Supplementary Fig. S5B). Notably, significant delays in tumor growth were observed in OMTX503-treated mice by day 13 (P = 0.0218, Fig. 4C), and tumor growth inhibition (TGI) was higher than 50% after 15 days posttreatment (Fig. 4D; Supplementary Table S5). ENG and MMP14 expressions (Supplementary Fig. S5C and S5D) were similar in tumor samples from all groups, as evaluated by IHC (Supplementary Tables S6–S9). H&E staining on the tumor samples revealed a loss of tumor viability in those groups treated with OMTX003 and OMTX503 after day 15 of treatment (Fig. 4E). Systemic signs of toxicity were found in some mice from the OMTX503-treated groups. The macroscopic toxicity observed was not translated into weight loss, as the tumor growth masked the global weight change (Supplementary Fig. S5B). Collectively, these results suggest that the internalization of an immunotoxin by targeting ENG in Ewing sarcoma xenografts impaired tumor growth.

To test the efficacy of OMTX703 (cytolysin-ADC) in ES8 xenograft, an additional preclinical study was conducted. Tumor growth and Kaplan–Meier curves revealed only modest antitumor activity of OMTX703 at 30 mg/kg, as shown in Fig. 5A–C. Tumor growth was markedly impaired using the highest dose of OMTX703 (60 mg/kg, P = 0.019). Having determined an optimal dose for OMTX703, an additional experiment was conducted to assess the mechanism(s) of OMTX703 action and its potential mechanism(s) of resistance following a 2-week exposure to OMTX703 at 0, 10, 30, and 60 mg/kg; 246 proteins were assessed by reverse-phase protein lysate array (RPPA, GSE#114866). ANOVA, Pearson's correlation as distance metric and Ward's linkage as the clustering method using an FDR of 0.01, identified 60 proteins that discriminated between treatment groups. By principal component analysis (PCA), tumor specimens from mice treated with OMTX703 at 10 mg/kg clustered tightly with the placebo-treated group, suggesting that this dose had limited effect (Supplementary Fig. S6C). Surprisingly, two specimens from the OMTX703 group treated at 60 mg/kg nearly overlapped the placebo-treated cluster (Fig. 5D); further scrutiny of these specimens suggested that they had acquired resistance and begun to progress (Supplementary Fig. S6A). RPPA analysis of the xenograft-treated specimens demonstrated two major and five minor clusters. Cluster 1 was associated with proteins significantly downregulated by OMTX703 at 30 and 60 mg/kg compared with a heterogeneously-treated group of tumors from mice that received placebo control, a naked anti-endoglin Ab (OMTX003), or a subtherapeutic (i.e., 10 mg/kg) dose of the OMTX703 ADC. Gene set enrichment analysis (GSEA) indicated marked suppression of proteins linked to insulin receptor signaling or downstream effectors such as PI3K and mTOR, pathways of known importance in Ewing sarcoma (Supplementary Fig. S7). In contrast, the most differentially upregulated proteins resulting from OMTX703 treatment (cluster 2, Supplementary Fig. S8) were linked to genes associated with nerve growth factor (NGF) and its cognate receptor (TRKA). Obviously GSEA relies upon gene set enrichment and fails to reflect the site-specific protein phosphorylation states that can markedly affect protein function. Thus, the collective effect that individual phospho-proteins have upon pathway activation must be further scrutinized.

To investigate the proteomic changes associated with the heightened clinical activity of the 60 mg/kg dose, a secondary analysis was performed, which grouped the 10 mg/kg OMTX703 samples and the 10 mg/kg OMTX003 ones with the placebo-treated samples (Supplementary Fig. S6B). Using a FDR of 0.0001, an absolute log2-fold change of 1.5, Pearson's correlation as distance metric and Ward's linkage as the clustering method, 22 proteins were discriminately identified between the three treatment groups (Supplementary Fig. S6B). Notably, a protein regulator of altered metabolism (RPS6) was exclusively upregulated following OMTX703 (60 mg/kg), and a second metabolism biomarker (LDHA) was downexpressed in the 30 and 60 mg/kg-treated groups.

Finally, we evaluated the efficacy of OMTX703 by using a PDX model that many believe predicts drug activity better than cell lines. Considering the in vitro data that indicate ENG expression is required to elicit an antiproliferative effect, an initial Ewing sarcoma PDX panel was screened to identify a suitable model to validate the therapeutic effect of OMTX703. HSJD-ES-009 was considered the best candidate because of its relatively high ENG expression as well as its high rate of successful engraftment in mice as compared with the rest of the PDX models (Fig. 1G; Supplementary Fig. S9A and S9B). Irinotecan was used as reference drug because of its potent activity as single agent in preclinical models of pediatric solid tumors (Supplementary Table S10; ref. 21).

Criteria to quantify drug response are shown in Supplementary Table S11. Although animals did not experience weight loss during treatment (Fig. 6A), OMTX703 at 60 mg/kg produced an acute toxic death rate of 50%, with three animals dying at days 21, 22, and 28 from treatment initiation. Regarding this fact, we proceeded to thoroughly examine the drug-induced toxicity in FFPE samples of lung, liver, and kidney from the PDXs. However, by histopathological analyses, no significant differences were observed between the drug-treated and the control groups (Supplementary Tables S12–S14). Animals treated with OMTX003 experienced progressive tumor growth, as the control animals did. We found a dose-dependent antitumor response with OMTX703 at 30 and 60 mg/kg. Interestingly, OMTX703 at 60 mg/kg induced a complete response (CR) rate of 60% at day 21 of treatment, in comparison to the reference treatment (irinotecan) which induced CR in 11% of tumors (Fig. 6B and D).

To quantify the long-term effects of drug treatment upon survival, a second study was conducted. After cessation of treatments all groups experienced progressive tumor growth, with relapse occurring even in mice that had shown complete tumor response (Fig. 6C; Supplementary Fig. S9B). As with prior studies, no weight loss was observed during the survival study and weight gain was mainly due to the increase in tumor volume (Supplementary Fig. S9C). Median time to achieve endpoint is shown in Fig. 6E and F, with significant longer median time in OMTX703 (60 mg/kg) and irinotecan-treated animals as compared with control-treated mice [60 days, P = 0.0065 for OMTX703 (60 mg/kg); and 46 days, P = 0.0018 for irinotecan]. Finally, IHC was performed to evaluate the possible changes on ENG and MMP14 expression amongst groups (Supplementary Fig. S10A and S10B; Supplementary Tables S15 and S16) as well as proliferation rate by Ki67 staining (Supplementary Fig. S10C) and viability by H&E staining (Supplementary Fig. S9D). Compared with placebo-treated mice, we found a significant increase of ENG expression in mice treated with OMTX003 at 10 mg/kg (P = 0.0005; Supplementary Fig. S10A) and a significant increase of MMP14 expression in the groups treated with OMTX703 at 30 mg/kg (P = 0.0316), OMTX703 at 60 mg/kg (P = 0.0163), and irinotecan at 10 mg/kg (P = 0.0240) as reflected in Supplementary Fig. S10B.

Cell viability, measured by H&E, was not affected by the treatment in any case (Supplementary Fig. S9D). However, regarding the cell proliferation evaluated by Ki67 staining, we found a significant decrease in the group treated with OMTX003 10 mg/kg (P = 0.0479) and a nonsignificant decrease in the groups treated with OMTX703 (30 mg/kg; 60 mg/kg) and irinotecan 10 mg/kg (Supplementary Fig. S10C). From this result, we could hypothesize that cells with stable proliferation the ENG upregulation as a way to compensate its activity was impaired by the antibody blockade. MMP14 overexpression could be due to the strong blockade of cell proliferation, because this protein also plays an important role in cell cycle and proliferation.

The present investigation is the first work to show the antitumor activity of ENG-targeting ADC therapies in Ewing sarcoma. We propose that this novel approach might provide improved tumor selectivity as compared with cytotoxic agents in a subset of patients with Ewing sarcoma expressing high levels of ENG. Most cytotoxic chemotherapies used today exhibit a narrow therapeutic index dependent upon a higher proliferative rate of tumor cells compared with normal cells. The implications of Ewing sarcoma heterogeneity in response to treatment also represent a challenge when it comes to defining upfront clinical strategies. These facts stress the need to selectively target the unique molecular features that exist in tumor cells while preventing off-target effects in normal cells. In this context, precision medicine represents an appealing yet rather challenging paradigm. Toward that end, ADCs have shown clinical promise given their ability to harness the specificity of monoclonal antibodies to more selectively deliver apoptosis-inducing molecules to epitope-bearing tumor cells (10, 22–24).

Although the pathognomonic hallmarks of Ewing sarcoma (i.e., the EWS–FLI1 fusion protein and CD99 overexpression) have been known for decades, only in the last years have Ewing sarcoma-specific trials taken direct aim at the oncogenic fusion protein or the ETS subcomponent (25). Although we eagerly await the clinical results from trials testing the first-generation EWS-FLI1 modulators, such as TK-216, the issue of innate and acquired intratumoral heterogeneity is likely to remain a significant hurdle that prevents—or at least substantially reduces—the likelihood that any single therapy will eradicate every tumor cell. Substantiating this concern, recent evidence from two laboratories suggests that Ewing sarcoma cells can oscillate between high- and low-expressing states with respect to the EWS–FLI1 fusion protein. Intriguingly, low levels of EWS–FLI1 might actually result in increased migration and invasion by tumor cells, questioning the effectiveness of exclusively targeting the ES fusion protein without cotargeting of other oncogenic drivers (5, 26). Although CD99 is highly expressed by Ewing sarcoma cells, MSCs and other cell types also express it in physiologic conditions (27). So far, no specific membrane receptors have been described for Ewing sarcoma, with the exception of LINGO1. Nonetheless, it was also expressed in the brain and its expression was highly variable in Ewing sarcoma when compared with that of CD99 (28).

The ENG receptor, as mentioned previously, is not tumor-specific and it is also expressed by normal vascular tissues. Mutations in ENG are associated with hereditary hemorrhagic telangiectasia type 1 (HHT1). HHT1 patients present massive vascular malformation (17). In cancer, the transmembrane form of ENG has been associated with poor clinical outcomes, high intratumoral microvessel densities, and an aggressive/invasive phenotype in certain tumor types (18, 29, 30). Interestingly, the soluble form of ENG (sENG) has been described as an antagonist of transmembrane ENG and a biomarker of hypertension, diabetes, and preeclampsia (31, 32). sENG has also been pointed as a circulating biomarker of poor prognosis in patients with solid tumors (33, 34). However, our analysis of sENG in plasma showed no significant differences between healthy donors and patients with Ewing sarcoma, and no significant correlations with clinical parameters. To sum up, ENG presents two major roles in cancer biology: (i) modulating tumor angiogenesis and (ii) promoting tumor progression in certain tumor types; hence, it represents an attractive therapeutic target.

Previous works from other groups, hereby reproduced (Supplementary Fig. S4), have, in fact, shown that ENG-targeting with a humanized MAb impairs the ability of endothelial cells to form functional tubular structures, which are essential for tumor neo-angiogenesis (11). ENG-targeting MAbs are currently being used in clinical trials for solid tumors, mainly in combination with other anti-angiogenic agents (35–37). Expression of ENG in Ewing sarcoma was previously described by our group in a small set of tumor samples (27). The role of ENG on Ewing sarcoma phenotype and malignancy has already been studied in in vitro and in vivo models, where ENG was associated with increased invasiveness and metastasis formation (18). In fact, ENG is described as a “gold” receptor protein in a recent work focused on the study of the surfaceome of Ewing sarcoma for the search of targetable membrane receptors (28). Our finding of a possible epigenetic downregulation of ENG by methylation of its promoter region in the CADO cell line is consistent with other studies in solid tumors, where the hypermethylation of ENG promoter region results in gene silencing, and was reactivated after in vitro methylation reversal assays (38, 39). Moreover, the positive correlation between ENG and MMP14 observed in Ewing sarcoma cell lines and patients' samples, suggests that there might be an important highly regulated balance between both proteins.

A major strength of our work stems from the concurrent evaluation of ENG as a therapeutic target against Ewing sarcoma by two academic labs, based respectively in Europe and the United States, which focus on Ewing sarcoma drug development. As initial proof-of-concept, we evaluated OMTX503, a humanized MAb engaging ENG linked to a nontoxic type 2 ribosome-inactivating protein nigrin-b A chain, in vitro and within murine models. The development of an anti-ENG-nigrin-b-based ADC has already shown activity towards melanoma mouse tumors, both in vitro and in vivo (40). This work showed that targeting human ENG in a xenograft model with the ADC resulted in tumor shrinkage and inhibition of neo-angiogenesis (40). It is important to mention that this MAb engaging ENG is linked to the nontoxic A chain of nigrin-b.

Although OMTX503 demonstrated moderate preclinical activity, we subsequently transitioned our research focus to ENG-targeting ADC with cytolysin, OMTX703, given its lower toxicity and broader therapeutic window. OMTX503 and OMTX703 share the same mechanism of action and both lead to cell-death after drug internalization. Our in vitro results confirmed an ENG-dependent effect for both molecules. Ewing sarcoma cell line-derived xenografts with a high expression of ENG were used as a first in vivo approach. At this point, we found differences of response to treatment, depending on the drug administered, OMTX503 or OMTX703. We suggest that the response to treatment could be mainly influenced by the therapeutic window of each ADC.

Recognizing the potential importance of human tumor stroma in evaluating the anti-ENG ADC response, we sought to validate the xenograft results using Ewing sarcoma PDX models that likely exhibit a wider breadth of intratumoral heterogeneity that better reflects the clinical setting. Only OMTX703 was comprehensively assessed in PDXs. Notably, the frequency of CR (60%) after 21 days of treatment in PDXs treated with OMTX703 reinforces the promising efficacy of this drug. Given that all mice relapsed in the long-term survival study, we hereby suggest the importance of drug combinations to avoid acquired drug resistance.

Taken together, the antineoplastic effects observed in our Ewing sarcoma PDXs are highly encouraging. Although additional research is certainly required before one would propose using ENG expression in order to select patients receiving ENG-targeting therapies, the prospect of using ENG expression as a predictive biomarker of response would clearly represent a crucial step to identify patients with Ewing sarcoma most likely to benefit from anti-ENG ADC therapies and, additionally, enable tissue-based companion diagnostics. Similarly, the toxicity profiles associated to these molecules, need be to further analyzed in specific regulatory toxicologic studies in additional preclinical animal models. Although this study focused primarily on the direct effect of targeting ENG in tumor cells, future research must decipher how anti-ENG ADCs affect vascular stroma and the processes responsible for endothelial cell function and angiogenesis. In that regard, the capacity for OMTX703 to jointly target the tumor and stroma raises the tantalizing possibility that OMTX703 might delay tumor growth even when ENG expression is low or absent (36, 37). With the addition of more comprehensive toxicity data, ENG-targeting ADCs represent a promising therapeutic alternative that may someday help patients with ES suffering from ENG-positive tumors.

R.E. Kontermann reports receiving commercial research grants from and is a consultant/advisory board member for Oncomatryx. K. Pfizenmaier reports receiving commercial research grants from and is a consultant/advisory board member for Oncomatryx. L. Simon is an employee of and holds ownership interest (including patents) in Permedika Entrepeneurs, Quimatryx, Sunrock Biopharma, Patia Biopharma, Nelum, Patia Europe, Stemtek Therapeutics, and Helenes Ventures. J.A. Ludwig is a consultant/advisory board member for Oncomatryx. E. de Álava reports receiving speakers bureau honoraria from Roche, Lilly, and Pfizer, and is a consultant/advisory board member for Roche. No potential conflicts of interest were disclosed by the other authors.

Conception and design: P. Puerto-Camacho, A.T. Amaral, S.-E. Lamhamedi-Cherradi, S. Domínguez, K. Schadler, L. Simón, M. Fabre, Á.M. Carcaboso, J.A. Ludwig, E. de Álava

Development of methodology: P. Puerto-Camacho, A.T. Amaral, S.-E. Lamhamedi-Cherradi, B.A. Menegaz, J.L. Ordóñez, C. Jordan-Perez, J. Diaz-Martin, M. Lopez-Alvarez, K. Schadler, R. Kontermann, K. Pfizenmaier, J.A. Ludwig, E. de Álava

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): P. Puerto-Camacho, A.T. Amaral, S.-E. Lamhamedi-Cherradi, B.A. Menegaz, H. Castillo-Ecija, J.L. Ordóñez, C. Jordan-Perez, G. Civantos-Jubera, M.J. Robles-Frías, C. Ferrer, J. Mora, B. Cuglievan, Á.M. Carcaboso, J.A. Ludwig, E. de Álava

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): P. Puerto-Camacho, A.T. Amaral, S.-E. Lamhamedi-Cherradi, B.A. Menegaz, H. Castillo-Ecija, J.L. Ordóñez, S. Domínguez, J. Diaz-Martin, L. Romero-Pérez, C. Ferrer, O. Seifert, M. Fabre, Á.M. Carcaboso, J.A. Ludwig, E. de Álava

Writing, review, and/or revision of the manuscript: P. Puerto-Camacho, A.T. Amaral, S.-E. Lamhamedi-Cherradi, B.A. Menegaz, S. Domínguez, J. Mora, O. Seifert, R. Kontermann, K. Pfizenmaier, M. Fabre, Á.M. Carcaboso, J.A. Ludwig, E. de Álava

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): P. Puerto-Camacho, A.T. Amaral, S.-E. Lamhamedi-Cherradi, B.A. Menegaz, S. Domínguez, M. Biscuola, J. Mora, O. Seifert, R. Kontermann, K. Pfizenmaier, J.A. Ludwig

Study supervision: S.-E. Lamhamedi-Cherradi, S. Domínguez, L. Simón, Á.M. Carcaboso, J.A. Ludwig, E. de Álava

This research has been conducted using samples from the Hospital Universitario Virgen del Rocío - Instituto de Biomedicina de Sevilla Biobank (Andalusian Public Health System Biobank and ISCIII-Red de Biobancos PT13/0010/0056). The authors thank the donors and the Hospital Universitario Virgen del Rocío - Instituto de Biomedicina de Sevilla Biobank (Andalusian Public Health System Biobank and ISCIII-Red de Biobancos PT13/0010/0056) and Xarxa de Bancs de Tumors de Catalunya (XBTC) sponsored by Pla Director d'Oncologia de Catalunya for the human specimens used in this study. The authors thank Dr. Arjan Lankester, from LUMC, for providing the plasmas and clinical data from Ewing sarcoma patients.

The authors thank Drs. Ying Wang and Jing Wang from Bioinformatics and Computational Biology Department at MDACC for their contribution in performing the bioinformatic analyses regarding the RPPA profiling.

EDA's lab is supported by the RETOS COLABORACIÓN project (RTC-2014-2102-1); the AECC project (GCB13131578DEÁ); ISCIII-FEDER (PI14/01466 and PI17/00464); CIBERONC (CB16/12/00361 and CB16/12/00316 Fundación CRIS contra el cáncer); and Asociación Pablo Ugarte. PPC is sponsored by the Fundación María García Estrada. AMC lab is supported by ISCIII-FEDER (CP13/00189). MJRF is supported by Consejería de Salud de la Junta de Andalucía (ECAIF2-0176-2013). J.A. Ludwig lab is supported by the NIH (R01 CA180279) and the University of Texas MD Anderson Cancer Center's Support Grant CA016672.

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