Xuhui Bao

Recombinant anti-podoplanin (NZ-1) immunotoxin for the treatment of malignant brain tumors

Our study demonstrates the glioma tumor antigen podoplanin to be present at very high levels (>90%) in both glioblastoma (D2159MG, D08‐0308MG and D08‐0493MG) and medulloblastoma (D283MED, D425MED and DAOY) xenografts and cell line. We constructed a novel recombinant single‐chain antibody variable region fragment (scFv), NZ‐1, specific for podoplanin from the NZ‐1 hybridoma. NZ‐1‐scFv was then fused to Pseudomonas exotoxin A, carrying a C‐terminal KDEL peptide (NZ‐1‐PE38KDEL). The immunotoxin (IT) was further stabilized by a disulfide (ds) bond between the heavy‐chain and light‐chain variable regions as the construct NZ‐1‐(scdsFv)‐PE38KDEL. NZ‐1‐(scdsFv)‐PE38KDEL exhibited significant reactivity to glioblastoma and medulloblastoma cells. The affinity of NZ‐1‐(scdsFv), NZ‐1‐(scdsFv)‐PE38KDEL and NZ‐1 antibody for podoplanin peptide was 2.1 × 10−8 M, 8.0 × 10−8 M and 3.9 × 10−10 M, respectively. In a protein stability assay, NZ‐1‐(scdsFv)‐PE38KDEL retained 33–98% of its activity, whereas that of NZ‐1‐PE38KDEL declined to 13% of its initial levels after incubation at 37°C for 3 days. In vitro cytotoxicity of the NZ‐1‐(scdsFv)‐PE38KDEL was measured in cells isolated from glioblastoma xenografts, D2159MG, D08‐0308MG and D08‐0493MG, and in the medulloblastoma D283MED, D425MED and DOAY xenografts and cell line. The NZ‐1‐(scdsFv)‐PE38KDEL IT was highly cytotoxic, with an 50% inhibitory concentration in the range of 1.6–29 ng/ml. Significantly, NZ‐1‐(scdsFv)‐PE38KDEL demonstrated tumor growth delay, averaging 24 days (p < 0.001) and 21 days (p < 0.001) in D2159MG and D283MED in vivo tumor models, respectively. Crucially, in the D425MED intracranial tumor model, NZ‐1‐(scdsFv)‐PE38KDEL caused a 41% increase in survival (p ≤ 0.001). In preclinical studies, NZ‐1‐(scdsFv)‐PE38KDEL exhibited significant potential as a targeting agent for malignant brain tumors.

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.

EGFR/EGFRvIII-targeted immunotoxin therapy for the treatment of glioblastomas via convection-enhanced delivery

Glioblastoma is the most aggressive malignant brain tumor among all primary brain and central nervous system tumors. The median survival time for glioblastoma patients given the current standard of care treatment (surgery, radiation, and chemotherapy) is less than 15 months. Thus, there is an urgent need to develop more efficient therapeutics to improve the poor survival rates of patients with glioblastoma. To address this need, we have developed a novel tumor-targeted immunotoxin (IT), D2C7-(scdsFv)-PE38KDEL (D2C7-IT), by fusing the single chain variable fragment (scFv) from the D2C7 monoclonal antibody (mAb) with the Pseudomonas Exotoxin (PE38KDEL). D2C7-IT reacts with both the wild-type epidermal growth factor receptor (EGFRwt) and EGFR variant III (EGFRvIII), two onco-proteins frequently amplified or overexpressed in glioblastomas. Surface plasmon resonance and flow cytometry analyses demonstrated a significant binding capacity of D2C7-IT to both EGFRwt and EGFRvIII proteins. In vitro cytotoxicity data showed that D2C7-IT can effectively inhibit protein synthesis and kill a variety of EGFRwt-, EGFRvIII-, and both EGFRwt- and EGFRvIII-expressing glioblastoma xenograft cells and human tumor cell lines. Furthermore, D2C7-IT exhibited a robust anti-tumor efficacy in orthotopic mouse glioma models when administered via intracerebral convection-enhanced delivery (CED). A preclinical toxicity study was therefore conducted to determine the maximum tolerated dose (MTD) and no-observed-adverse-effect-level (NOAEL) of D2C7-IT via intracerebral CED for 72 hours in rats. Based on this successful rat toxicity study, an Investigational New Drug (IND) application (#116855) was approved by the Food and Drug Administration (FDA), and is now in effect for a Phase I/II D2C7-IT clinical trial (D2C7 for Adult Patients with Recurrent Malignant Glioma, https://clinicaltrials.gov/ct2/show/NCT02303678). While it is still too early to draw conclusions from the trial, results thus far are promising.

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.

XIAP Regulation by MNK Links MAPK and NFκB Signaling to Determine an Aggressive Breast Cancer Phenotype

Hyperactivation of the NFκB pathway is a distinct feature of inflammatory breast cancer (IBC), a highly proliferative and lethal disease. Gene expression studies in IBC patient tissue have linked EGFR (EGFR/HER2)-mediated MAPK signaling to NFκB hyperactivity, but the mechanism(s) by which this occurs remain unclear. Here, we report that the X-linked inhibitor of apoptosis protein (XIAP) plays a central role in linking these two pathways. XIAP overexpression correlated with poor prognoses in breast cancer patients and was frequently observed in untreated IBC patient primary tumors. XIAP drove constitutive NFκB transcriptional activity, which mediated ALDH positivity (a marker of stem-like cells), in vivo tumor growth, and an IBC expression signature in patient-derived IBC cells. Using pathway inhibitors and mathematical models, we defined a new role for the MAPK interacting (Ser/Thr)-kinase (MNK) in enhancing XIAP expression and downstream NFκB signaling. Furthermore, targeted XIAP knockdown and treatment with a MNK inhibitor decreased tumor cell migration in a dorsal skin fold window chamber murine model that allowed for intravital imaging of local tumor growth and migration. Together, our results indicate a novel role for XIAP in the molecular cross-talk between MAPK and NFκB pathways in aggressive tumor growth, which has the potential to be therapeutically exploited.Significance: Signaling by the MNK kinase is essential in inflammatory breast cancer, and it can be targeted to inhibit XIAP-NFκB signaling and the aggressive phenotype of this malignancy. Cancer Res; 78(7); 1726-38. ©2018 AACR.

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.

Abstract LB-82: MLL2 maintains neoplastic cell growth and global histone H3 lysine 4 monomethylation

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA One striking theme emerging from recent findings in cancer genetics is that chromatin remodeling and histone methylation modifiers are frequently altered in human cancers. Among these newly identified cancer genes, one of the best examples is the gene encoding MLL2 (KMT2D, a.k.a. ALR/MLL4), a histone lysine methyltransferase that plays an important role in regulating gene transcription. Genetic alterations suggestive of a functional deficiency in MLL2 and other genes in the same pathways are common. The identification of alterations in MLL2 suggests potential new opportunities for therapeutics, and highlights an urgent need to understand the underlying tumorigenic mechanism. Filling such a knowledge gap has been challenging, due to the lack of appropriate assays for the gigantic (∼600 kDa), understudied MLL2 protein. To overcome the difficulty, we have used innovative somatic gene editing-based assays to determine the effect of an MLL2 deficiency on neoplastic cells. In particular, we have used homologous recombination- and nuclease-mediated gene editing approaches to generate a panel of isogenic human cancer cell lines that differ with respect to their endogenous MLL2 status. Our studies found that an MLL2 deficiency results in attenuated cancer cell proliferation and defective cell migration. We identified direct transcriptional target genes and revealed the connection of MLL2 to multiple cellular signaling pathways. Analysis of histone H3 modifications revealed that MLL2 is essential for maintaining the level of global histone H3 lysine 4 (H3K4) monomethylation and that its enzymatic SET domain is directly responsible for this function. Furthermore, we found that a majority of MLL2 binding sites are located in regions of potential enhancer elements. The finding concerning enhancer elements is significant, as enhancer elements have increasingly been recognized as critically involved in tumorigenesis. Together, these findings revealed the role of MLL2 in mediating diverse signaling pathways and regulating enhancer elements in human cells, and shed light on the tumorigenic role of its deficiency. Our study supports that MLL2 has distinct roles in neoplastic cells, as opposed to pre-neoplastic cells, and that inhibiting MLL2 may be a viable strategy for cancer therapeutics. Citation Format: Changcun Guo, Lee H. Chen, Yafen Huang, Chun-chi Chang, Ping Wang, Christopher J. Pirozzi, Xiaoxia Qin, Xuhui Bao, Paula K. Greer, Roger E. McLendon, Hai Yan, Stephen T. Keir, Darell D. Bigner, Yiping He. MLL2 maintains neoplastic cell growth and global histone H3 lysine 4 monomethylation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-82. doi:10.1158/1538-7445.AM2014-LB-82