Hailan Piao

Construction of an Immunotoxin, D2C7-(scdsFv)-PE38KDEL, Targeting EGFRwt and EGFRvIII for Brain Tumor Therapy

Purpose: The EGF 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 inhibition of protein synthesis 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 antitumor efficacy of D2C7-(scdsFv)-PE38KDEL was evaluated using 43, NR6M, and D270MG orthotopic tumor models. Results: The KD of D2C7-(scdsFv)-PE38KDEL for EGFRwt and EGFRvIII was 1.6 × 10−9 mol/L and 1.3 × 10−9 mol/L, 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 to 2.5 ng/mL on cells expressing EGFRwt (NR6W, A431, 43, and D08-0493MG). The D2C7-(scdsFv)-PE38KDEL IC50 was approximately 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, treatment with D2C7-(scdsFv)-PE38KDEL by convection-enhanced delivery 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, or both. Clin Cancer Res; 19(17); 4717–27. ©2013 AACR.

Affinity-matured recombinant immunotoxin targeting gangliosides 3′-isoLM1 and 3′,6'-isoLD1 on malignant gliomas

About 60 percent of glioblastomas highly express the gangliosides 3′-isoLM1 and 3′,6′-isoLD1 on the cell surface, providing ideal targets for brain tumor immunotherapy. A novel recombinant immunotoxin, DmAb14m-(scFv)-PE38KDEL (DmAb14m-IT), specific for the gangliosides 3′-isoLM1 and 3′,6′-isoLD1, was constructed with improved affinity and increased cytotoxicity for immunotherapeutic targeting of glioblastoma. We isolated an scFv parental clone from a previously established murine hybridoma, DmAb14, that is specific to both 3′-isoLM1 and 3′,6′-isoLD1. We then performed in vitro affinity maturation by CDR hotspot random mutagenesis. The binding affinity and specificity of affinity-matured DmAb14m-IT were measured by surface-plasmon resonance, flow cytometry, and immunohistochemical analysis. In vitro cytotoxicity of DmAb14m-IT was measured by protein synthesis inhibition and cell death assays in human cell lines expressing gangliosides 3′-isoLM1 and 3′,6′-isoLD1 (D54MG and D336MG) and xenograft-derived cells (D2224MG). As a result, the KD of DmAb14m-IT for gangliosides 3′-isoLM1 and 3′,6′-isoLD1 was 2.6 × 10−9M. Also, DmAb14m-IT showed a significantly higher internalization rate in cells expressing 3′-isoLM1 and 3′,6′-isoLD1. The DmAb14m-IT IC50 was 80 ng/mL (1194 pM) on the D54MG cell line, 5 ng/ml (75 pM) on the D336MG cell line, and 0.5 ng/ml (7.5 pM) on the D2224MG xenograft-derived cells. There was no cytotoxicity on ganglioside-negative HEK293 cells. Immunohistochemical analysis confirmed the specific apparent affinity of DmAb14m-IT with 3′-isoLM1 and 3′,6′-isoLD1. In conclusion, DmAb14m-IT showed specific binding affinity, a significantly high internalization rate, and selective cytotoxicity on glioma cell lines and xenograft-derived cells expressing 3′-isoLM1 and 3′,6′-isoLD1, thereby displaying robust therapeutic potential for testing the antitumor efficacy of DmAb14m-IT at the preclinical level and eventually in the clinical setting.

Benzimidazole inhibitors from the Niclosamide chemotype inhibit Wnt/β-catenin signaling with selectivity over effects on ATP homeostasis

The Wnt signaling pathway plays a key role in organ and tissue homeostasis, and when dysregulated, can become a major underlying mechanism of disease, particularly cancer. We reported previously that the anthelmintic drug Niclosamide inhibits Wnt/β-catenin signaling and suppresses colon cancer cell growth in vitro and in vivo. To define Niclosamides mechanism of Wnt/β-catenin inhibition, and to improve its selectivity and pharmacokinetic properties as an anticancer treatment, we designed a novel class of benzimidazole inhibitors of Wnt/β-catenin signaling based on SAR studies of the Niclosamide salicylanilide chemotype. Niclosamide has multiple biological activities. To address selectivity in our design, we interrogated a protonophore SAR model and used the principle of conformational restriction to identify novel Wnt/β-catenin inhibitors with less effect on ATP cellular homeostasis. These studies led to the identification of 4-chloro-2-(5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl) phenol (4) and related derivatives with greater selectivity for Wnt/β-catenin signaling inhibition vs. differential effects on cellular ATP homeostasis. This is the first report that the Wnt signaling inhibitory activity of Niclosamide can be translated into a new chemical class and to show that its effects on ATP homeostasis can be separated from its inhibitory effects on Wnt signaling. These compounds could be useful tools to elucidate the mechanism of Niclosamides inhibition of Wnt signaling, and aid the discovery of inhibitors with improved pharmacologic properties to treat cancer and diseases in which Niclosamide has important biological activity.

Validation of an Immunohistochemistry Assay for Detection of CD155, the Poliovirus Receptor, in Malignant Gliomas.

CONTEXT - The oncolytic polio-rhinovirus recombinant (PVSRIPO) has demonstrated promise in currently ongoing phase I/II clinical trials against recurrent glioblastoma and was granted breakthrough therapy designation by the Food and Drug Administration/Center for Biologics Evaluation and Research. A reliable clinical assay to document expression of the poliovirus receptor, CD155, in routinely available patient tumor samples is needed for continued clinical development of PVSRIPO oncolytic immunotherapy in primary brain tumors and beyond. OBJECTIVES - To validate a novel anti-CD155 antibody for immunohistochemistry and develop a robust, reliable, and specific protocol for detecting CD155 expression in glioblastoma formalin-fixed, paraffin-embedded (FFPE) tissue samples. To characterize the expression of CD155 in human glioblastoma cells as well as to evaluate the influence of CD155 expression levels on tumor cell susceptibility to PVSRIPO infection and killing. DESIGN - Immunohistochemical staining on glioblastoma FFPE tissue sections and immunoblot of corresponding frozen tissues were performed. Positive controls were confirmed sites of poliovirus propagation, spinal cord anterior horn, and tonsils; negative controls were vascular smooth muscle in patient samples and FFPE sections from a confirmed CD155-negative Burkitt lymphoma line (Raji). RESULTS - We succeeded in developing a reliable assay to specifically detect CD155 by immunohistochemistry in glioblastoma FFPE sections. Our data suggest widespread, virtually universal expression of CD155 in glioblastoma cells at levels commensurate with susceptibility to PVSRIPO infection and killing. CONCLUSIONS - Anti-CD155 antibody D3G7H achieves monospecific detection of CD155 in immunoblots of tumor homogenates and immunohistochemistry of tumor FFPE sections. Our assay has utility in defining appropriate use of PVSRIPO in oncolytic immunotherapy against malignant glioma and other cancer histotypes.

Elevated expression of podoplanin and its clinicopathological, prognostic, and therapeutic values in squamous non-small cell lung cancer

Background Squamous non-small cell lung cancer (SqNSCLC), as a leading cause of cancer-related deaths worldwide, has limited treatment options and poor prognosis. Thus, novel targeted therapies are desperately needed. Materials and methods SqNSCLC cases from derivation and validation cohorts were ana-lyzed for podoplanin (PDPN) expression, and its clinicopathological correlation and prognostic prediction. The Human Proteome Map database was used to compare the expression of different lung cancer targets in normal human tissues. Two human lung cancer cell lines, H226 (a SqNSCLC line) and A549 (a non-SqNSCLC line), were examined for PDPN expression. The in vitro cytotoxicity of an anti-PDPN therapy (NZ-1-immunotoxin [NZ-1-IT]) was tested against both lines. The in vivo therapeutic effect of NZ-1-IT was examined in subcutaneous non-small cell lung cancer (NSCLC) xenograft mouse models. Results In the derivation cohort, 40% (28/70) were PDPN positive. There was significantly increasing pleural invasion (46.4% vs 9.5%, p=0.001), lymphovascular invasion (25.0% vs 9.5%, p=0.08), and lymph node involvement (53.6% vs 33.3%, p=0.09) in PDPN-positive vs PDPN-negative patients, along with poorer progression-free survival in PDPN-positive patients (p=0.07). The validation cohort with 224 randomly matched cases from The Cancer Genome Atlas data set also displayed significantly shorter overall survival in the group with elevated PDPN mRNA (p=0.05). However, PDPN showed limited expression in normal tissues. PDPN was highly and specifically expressed on the surface of H226 cells instead of A549 cells. Subsequently, PDPN-positive H226 cells were around 800 times more sensitive to anti-PDPN NZ-1-IT therapy than PDPN-negative A549 cells in vitro. Furthermore, NZ-1-IT significantly delayed tumorigenesis only in the H226 subcutaneous mouse model (p<0.05). Conclusion Our results demonstrate a distinctively elevated expression of PDPN in SqNSCLC, which is significantly associated with worse clinicopathological features and poorer prognosis. With promising preclinical therapeutic results, anti-PDPN targeted therapy can thus be a robust potential strategy for future SqNSCLC treatment.

Immunotoxin Therapy for Lung Cancer

introduction Lung cancer is the leading cause for cancer‐related deaths in both genders throughout the world. In the United States alone, there were 224,390 estimated new lung cancer cases and 158,080 estimated deaths in 2016.[1] As conventional chemotherapy has reached a plateau of effectiveness in lung cancers and fails in those tumors whose growth and metabolism can hardly be distinguished from normal tissues, innovative therapeutic strategies have been explored. The use of novel agents, for example, immunotherapies, has started to show promising potential in the field.