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We employed a genetically defined human cancer model to investigate the contributions of two genes upregulated in several cancers to phenotypic changes associated with late stages of tumorigenesis. Specifically, tumor cells expressing two structurally unrelated bonerelated genes, osteonectin and osteoactivin, acquired a highly invasive phenotype when implanted intracranially in immunocompromised mice. Mimicking a subset of gliomas, tumor cells invaded brain along blood vessels and developed altered vasculature at the brain-tumor interface, suggesting that production of those two proteins by tumor cells may create a complex relationship between invading tumor and vasculature co-opted during tumor invasion. Interestingly, the same tumor cells formed massive spontaneous metastases when implanted subcutaneously. This dramatic alteration in tumor phenotype indicates that cellular microenvironment plays an important role in defining the specific effects of those gene products in tumor behavior. In vitro examination of tumor cells expressing either osteonectin or osteoactivin revealed that there was no impact on cellular growth or death but increased invasiveness and expression of MMP-9 and MMP-3. Specific pharmacologic inhibitors of MMP-2/9 and MMP-3 blocked the increased in vitro invasion associated with osteoactivin expression, but only MMP-3 inhibition altered the invasive in vitro phenotype mediated by osteonectin. Results from this genetically defined model system are supported by similar findings obtained from several established tumor cell lines derived originally from human patients. In sum, these results reveal that the expression of a single bonerelated gene can dramatically alter or modify tumor cell behavior and may confer differential growth characteristics in different microenvironments. Genetically defined human cancer models offer useful tools in functional genomics to define the roles of specific genes in late stages of carcinogenesis.

Bispecific antibodies (bscAbs), particularly those of the bispecific T-cell engager (BiTE) subclass, have been shown to effectively redirect T cells against cancer. Previous efforts to target antigens expressed in both tumors and normal tissues have produced significant toxicity, however. Moreover, like other large molecules, bscAbs may be restricted from entry into the “immunologically privileged” CNS. A tumor-specific mutation of the epidermal growth factor receptor, EGFRvIII, is a constitutively activated tyrosine kinase not found in normal tissues but frequently expressed in glioblastomas and many other neoplasms. Because it is localized solely to tumor tissue, EGFRvIII presents an ideal target for immunotherapy. Here we report the preclinical evaluation of an EGFRvIII-targeted BiTE, bscEGFRvIIIxCD3. Our results show that bscEGFRvIIIxCD3 activates T cells to mediate potent and antigen-specific lysis of EGFRvIII-expressing gliomas in vitro ( P < 0.001) at exceedingly low concentrations (10 ng/mL) and effector-to-target ratios (2.5:1). Treatment with i.v. bscEGFRvIIIxCD3 yielded extended survival in mice with well-established intracerebral tumors ( P < 0.05) and achieved durable complete cure at rates up to 75%. Antitumor efficacy was significantly abrogated on blockade of EGFRvIII binding, demonstrating the need for target antigen specificity both in vitro and in vivo. These results demonstrate that BiTEs can be used to elicit functional antitumor immunity in the CNS, and that peptide blockade of BiTE-mediated activity may greatly enhance the safety profile for antibody-redirected T-cell therapies. Finally, bscEGFRvIIIxCD3 represents a unique advancement in BiTE technology given its exquisite tumor specificity, which enables precise elimination of cancer without the risk of autoimmune toxicity.

Epidermal Growth Factor Receptor (EGFR) is frequently over-expressed in head and neck squamous cell carcinoma (HNSCC) where aberrant signaling downstream of this receptor contributes to tumor growth. EGFR variant III (EGFRvIII) is the most commonly altered form of EGFR and contains a truncated ligand-binding domain. We previously reported that EGFRvIII is expressed in up to 40% of HNSCC tumors where it is associated with increased proliferation, tumor growth and chemoresistance to anti-tumor drugs including the EGFR targeting monoclonal antibody cetuximab. Cetuximab was FDA-approved in 2006 for HNSCC but has not been shown to prevent invasion or metastasis. The present study was undertaken to evaluate the mechanisms of EGFRvIII-mediated cell motility and invasion in HNSCC. We found that EGFRvIII induced HNSCC cell migration and invasion in conjunction with increased STAT3 activation, which was not abrogated by cetuximab treatment. Further investigation demonstrated that EGF-induced expression of the STAT3 target gene HIF1-α, was abolished by cetuximab in HNSCC cells expressing wild-type EGFR under hypoxic conditions, but not in EGFRvIII-expressing HNSCC cells. These results suggest that EGFRvIII mediates HNSCC cell migration and invasion via increased STAT3 activation and induction of HIF1-α, which contribute to cetuximab resistance in EGFRvIII-expressing HNSCC tumors.

Introduction-Podoplanin/Aggrus is a mucin-like sialoglycoprotein that is highly expressed in malignant gliomas. Podoplanin has been reported to be a novel marker to enrich tumor-initiating cells, which are thought to resist conventional therapies and to be responsible for cancer relapse. The purpose of this study is to determine whether an anti-podoplanin antibody is suitable to target radionuclides to malignant gliomas. Results-The dissociation constant (K D ) of NZ-1 was determined to be 1.2 × 10 -10 M by surface plasmon resonance, and 9.8 × 10 -10 M for D397MG glioblastoma cells by Scatchard analysis. Pairedlabel internalization assays in LN319 glioblastoma cells indicated that [ 131 I]SGMIB-NZ-1 resulted in higher intracellular retention of radioactivity (26.3 ± 0.8% of initially bound radioactivity at 8 hr) compared to that from the 125 I-NZ-1(Iodogen) (10.0 ± 0.1% of initially bound radioactivity at 8 hr). Likewise, tumor uptake of [ 131 I]SGMIB-NZ-1 (39.9 ± 8.8%ID/g at 24 hr) in athymic mice bearing D2159MG xenografts in vivo was significantly higher than that of 125 I-NZ-1(Iodogen) (29.7 ± 6.1% ID/g at 24 hr). Methods-TheConclusions-The overall results suggest that an anti-podoplanin antibody NZ-1 warrants further evaluation for antibody-based therapy against glioblastoma.

Purpose The epidermal growth factor receptor gene (EGFR) is most frequently amplified and overexpressed, along with its deletion mutant, EGFRvIII, in glioblastoma. We tested the preclinical efficacy of the recombinant immunotoxin, D2C7-(scdsFv)-PE38KDEL, which is reactive with a 55-amino acid (AA) region present in the extracellular domain of both EGFRwt (583–637 AAs) and EGFRvIII (292–346 AAs) proteins. Experimental Design The binding affinity and specificity of D2C7-(scdsFv)-PE38KDEL for EGFRwt and EGFRvIII were measured by surface-plasmon resonance and flow cytometry. In vitro cytotoxicity of D2C7-(scdsFv)-PE38KDEL was measured by protein synthesis inhibition in human EGFRwt-transfected NR6 (NR6W), human EGFRvIII-transfected NR6 (NR6M), EGFRwt-overexpressing A431-epidermoid-carcinoma, and glioblastoma xenograft cells (43, D08-0493MG, D2159MG and D270MG). In vivo anti-tumor efficacy of D2C7-(scdsFv)-PE38KDEL was evaluated with 43, NR6M, and D270MG orthotopic tumor models. Results The KD of D2C7-(scdsFv)-PE38KDEL for EGFRwt and EGFRvIII was 1.6×10−9 M and 1.3×10−9 M, respectively. Flow cytometry with NR6W and NR6M cells confirmed the specificity of D2C7-(scdsFv)-PE38KDEL for EGFRwt and EGFRvIII. The D2C7-(scdsFv)-PE38KDEL IC50 was 0.18–2.5 ng/ml on cells expressing EGFRwt (NR6W, A431, 43, and D08-0493MG). The D2C7-(scdsFv)-PE38KDEL IC50 was ≈0.25 ng/ml on EGFRvIII-expressing cells (NR6M) and on EGFRwt- and EGFRvIII-expressing glioblastoma xenograft cells (D2159MG and D270MG). Significantly, in intracranial tumor models of 43, NR6M, and D270MG, D2C7-(scdsFv)-PE38KDEL treatment by convection-enhanced delivery (CED) prolonged survival by 310% (P=0.006), 28% (P=0.002), and 166% (P=0.001), respectively. Conclusions In preclinical studies, the D2C7-(scdsFv)-PE38KDEL immunotoxin exhibited significant potential for treating brain tumors expressing EGFRwt, EGFRvIII, and both EGFRwt and EGFRvIII.

Epidermal growth factor receptor variant III (EGFRvIII) is a glycoprotein uniquely expressed in glioblastoma, but not in normal brain tissues. To develop targeted therapies for brain tumors, we selected RNA aptamers against the histidine-tagged EGFRvIII ectodomain, using an Escherichia coli system for protein expression and purification. Representative aptamer E21 has a dissociation constant (Kd) of 33×10−9 m, and exhibits high affinity and specificity for EGFRvIII in ELISA and surface plasmon resonance assays. However, selected aptamers cannot bind the same protein expressed from eukaryotic cells because glycosylation, a post-translational modification present only in eukaryotic systems, significantly alters the structure of the target protein. By transfecting EGFRvIII aptamers into cells, we find that membrane-bound, glycosylated EGFRvIII is reduced and the percentage of cells undergoing apoptosis is increased. We postulate that transfected aptamers can interact with newly synthesized EGFRvIII, disrupt proper glycosylation, and reduce the amount of mature EGFRvIII reaching the cell surface. Our work establishes the feasibility of disrupting protein post-translational modifications in situ with aptamers. This finding is useful for elucidating the function of proteins of interest with various modifications, as well as dissecting signal transduction pathways.

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