Mingqian Feng

Therapeutically targeting glypican-3 via a conformation-specific single-domain antibody in hepatocellular carcinoma.

Glypican-3 (GPC3) has emerged as a candidate therapeutic target in hepatocellular carcinoma (HCC), but the oncogenic role of GPC3 in HCC is poorly understood. Here, we report a human heavy-chain variable domain antibody, HN3, with high affinity (Kd = 0.6 nM) for cell-surface–associated GPC3 molecules. The human antibody recognized a conformational epitope that requires both the amino and carboxy terminal domains of GPC3. HN3 inhibited proliferation of GPC3-positive cells and exhibited significant inhibition of HCC xenograft tumor growth in nude mice. The underlying mechanism of HN3 action may involve cell-cycle arrest at G1 phase through Yes-associated protein signaling. This study suggests a previously unrecognized mechanism for GPC3-targeted cancer therapy.

Glypican‐3 antibodies: A new therapeutic target for liver cancer

Glypican‐3 (GPC3) is an emerging therapeutic target in hepatocellular carcinoma (HCC), even though the biological function of GPC3 remains elusive. Currently human (MDX‐1414 and HN3) and humanized mouse (GC33 and YP7) antibodies that target GPC3 for HCC treatment are under different stages of preclinical or clinical development. Humanized mouse antibody GC33 is being evaluated in a phase II clinical trial. Human antibodies MDX‐1414 and HN3 are under different stages of preclinical evaluation. Here, we summarize current evidence for GPC3 as a new target in liver cancer, discuss both its oncogenic function and its mode of actions for current antibodies, and evaluate potential challenges for GPC3‐targeted anti‐cancer therapies.

Inactivation of Wnt signaling by a human antibody that recognizes the heparan sulfate chains of glypican‐3 for liver cancer therapy

Wnt signaling is important for cancer pathogenesis and is often up‐regulated in hepatocellular carcinoma (HCC). Heparan sulfate proteoglycans (HSPGs) function as coreceptors or modulators of Wnt activation. Glypican‐3 (GPC3) is an HSPG that is highly expressed in HCC, where it can attract Wnt proteins to the cell surface and promote cell proliferation. Thus, GPC3 has emerged as a candidate therapeutic target in liver cancer. While monoclonal antibodies to GPC3 are currently being evaluated in preclinical and clinical studies, none have shown an effect on Wnt signaling. Here, we first document the expression of Wnt3a, multiple Wnt receptors, and GPC3 in several HCC cell lines, and demonstrate that GPC3 enhanced the activity of Wnt3a/β‐catenin signaling in these cells. Then we report the identification of HS20, a human monoclonal antibody against GPC3, which preferentially recognized the heparan sulfate chains of GPC3, both the sulfated and nonsulfated portions. HS20 disrupted the interaction of Wnt3a and GPC3 and blocked Wnt3a/β‐catenin signaling. Moreover, HS20 inhibited Wnt3a‐dependent cell proliferation in vitro and HCC xenograft growth in nude mice. In addition, HS20 had no detectable undesired toxicity in mice. Taken together, our results show that a monoclonal antibody primarily targeting the heparin sulfate chains of GPC3 inhibited Wnt/β‐catenin signaling in HCC cells and had potent antitumor activity in vivo. Conclusion: An antibody directed against the heparan sulfate of a proteoglycan shows efficacy in blocking Wnt signaling and HCC growth, suggesting a novel strategy for liver cancer therapy. (Hepatology 2014;60:576–587)

A novel high-affinity human monoclonal antibody to mesothelin.

Mesothelin is a glycosylphosphatidylinositol‐anchored glycoprotein that is highly expressed on the cell surface of mesothelioma, ovarian cancer and other malignant tumors. The interaction between mesothelin and CA125 (also called MUC16) may facilitate the implantation and metastasis of tumors in the peritoneal cavity. A desirable therapeutic agent involves finding a fully human monoclonal antibody (mAb) that binds to mesothelin or CA125 and inhibits their interaction. Here, we report the identification of a novel human mAb to mesothelin. HN1, a human single‐chain Fv specific for mesothelin, was isolated from a naïve human single‐chain variable fragment (scFv) phage display library. To investigate HN1 as a potential therapeutic, we generated a fully human IgG with the γ 1 heavy chain and the κ light chain and an immuntoxin by fusing the HN1 scFv to a truncated Pseudomonas exotoxin A. The HN1 IgG kills cancer cells with very strong antibody‐dependent cell‐mediated cytotoxicity. HN1 binds a conformation‐sensitive epitope in human mesothelin with high affinity (KD = 3 nM). The HN1 epitope is different from that of SS1, a mouse Fv used to develop therapeutic antibodies that are currently in clinical trials. HN1 binds to cell surface‐associated mesothelin on human mesothelioma, ovarian cancer, lung adenocarcinoma and pancreatic cancer cells. In addition, HN1 can functionally block the interaction of mesothelin and CA125 on cancer cells. Most importantly, because the HN1 immuntoxin kills mesothelin‐expressing cancer cells with high cytotoxic activity, we believe that it has significant potential for mesothelin‐expressing cancer treatment and diagnosis. Published 2010 UICC. This article is a US Government work and, as such, is in the public domain of the United States of America.

The Development and Characterization of a Human Mesothelioma In Vitro 3D Model to Investigate Immunotoxin Therapy

BACKGROUND Tumor microenvironments present significant barriers to penetration by antibodies and immunoconjugates. Tumor microenvironments, however, are difficult to study in vitro. Cells cultured as monolayers exhibit less resistance to therapy than those grown in vivo and an alternative research model more representative of the in vivo tumor is more desirable. SS1P is an immunotoxin composed of the Fv portion of a mesothelin-specific antibody fused to a bacterial toxin that is presently undergoing clinical trials in mesothelioma. METHODOLOGY/PRINCIPAL FINDINGS Here, we examined how the tumor microenvironment affects the penetration and killing activity of SS1P in a new three-dimensional (3D) spheroid model cultured in vitro using the human mesothelioma cell line (NCI-H226) and two primary cell lines isolated from the ascites of malignant mesothelioma patients. Mesothelioma cells grown as monolayers or as spheroids expressed comparable levels of mesothelin; however, spheroids were at least 100 times less affected by SS1P. To understand this disparity in cytotoxicity, we made fluorescence-labeled SS1P molecules and used confocal microscopy to examine the time course of SS1P penetration within spheroids. The penetration was limited after 4 hours. Interestingly, we found a significant increase in the number of tight junctions in the core area of spheroids by electron microscopy. Expression of E-Cadherin, a protein involved in the assembly and sealing of tight junctions and highly expressed in malignant mesothelioma, was found significantly increased in spheroids as compared to monolayers. Moreover, we found that siRNA silencing and antibody inhibition targeting E-Cadherin could enhance SS1P immunotoxin therapy in vitro. CONCLUSION/SIGNIFICANCE This work is one of the first to investigate immunotoxins in 3D tumor spheroids in vitro. This initial description of an in vitro tumor model may offer a simple and more representative model of in vivo tumors and will allow for further investigations of the microenvironmental effects on drug penetration and tumor cell killing. We believe that the methods developed here may apply to the studies of other tumor-targeting antibodies and immunoconjugates in vitro.

A human single-domain antibody elicits potent antitumor activity by targeting an epitope in mesothelin close to the cancer cell surface.

Monoclonal antibodies against mesothelin are being evaluated for the treatment of mesothelioma and multiple forms of cancers, and show great promise for clinical development for solid cancers. Antibodies against mesothelin have been shown to act via immunotoxin-based inhibition of tumor growth and induction of antibody-dependent cell-mediated cytotoxicity (ADCC). However, complement-dependent cytotoxicity (CDC), considered an important additional mechanism of therapeutic antibodies against tumors, is inactive for such antibodies. Here, we used phage display antibody engineering technology and synthetic peptide screening to identify SD1, a human single-domain antibody to mesothelin. SD1 recognizes a conformational epitope at the C-terminal end (residues 539–588) of mesothelin close to the cell surface. To investigate SD1 as a potential therapeutic agent, we generated a recombinant human Fc (SD1-hFc) fusion protein. Interestingly, the SD1-hFc protein exhibits strong CDC activity, in addition to ADCC, against mesothelin-expressing tumor cells. Furthermore, it causes growth inhibition of human tumor xenografts in nude mice as a single agent. SD1 is the first human single-domain antibody targeting mesothelin-expressing tumors, shows potential as a cancer therapeutic candidate, and may improve current antibody therapy targeting mesothelin-expressing tumors. Mol Cancer Ther; 12(4); 416–26. ©2013 AACR.

Glypican-3 Targeted Human Heavy Chain Antibody as a Drug Carrier for Hepatocellular Carcinoma Therapy

Glypican-3 (GPC3) represents an attractive target for hepatocellular carcinoma (HCC) therapy because it is highly expressed in HCC but not in adult normal tissue. Recently, high affinity anti-GPC3 antibodies have been developed; however, full antibodies may not penetrate evenly into tumor parenchyma, reducing their effectiveness. In this study, we compared a whole IgG antibody, anti-GPC3 YP7, with an anti-GPC3 human heavy chain antibody, HN3, with regard to their relative therapeutic effects. Both YP7 and HN3 bound to GPC3-positive A431/G1 cells and were internalized by the cells by in vitro evaluation with (125)I- and (111)In-radiolabeling antibodies. In vivo biodistribution and tumor accumulation was performed with (111)In-labeled antibodies, and intratumoral microdistribution was evaluated using fluorescently labeled antibodies (IR700). HN3 showed similar high tumor accumulation but superior homogeneity within the tumor compared with YP7. Using the same IR700 conjugated antibodies photoimmunotherapy (PIT) was performed in vitro and in a tumor-bearing mouse model in vivo. PIT with IR700-HN3 and IR700-YP7 demonstrated that comparable results could be achieved despite of low reaccumulation 24 h after the first NIR light exposure. These results indicated that a heavy-chain antibody, HN3, showed more favorable characteristics than YP7, a conventional IgG, as a therapeutic antibody platform for designing molecularly targeted agents against HCC.

Immunotoxin targeting glypican-3 regresses liver cancer via dual inhibition of Wnt signalling and protein synthesis.

Glypican-3 is a cell surface glycoprotein that associates with Wnt in liver cancer. We develop two antibodies targeting glypican-3, HN3 and YP7. The first antibody recognizes a functional epitope and inhibits Wnt signaling, whereas the second antibody recognizes a C-terminal epitope but does not inhibit Wnt signaling. Both are fused to a fragment of Pseudomonas exotoxin A (PE38) to create immunotoxins. Interestingly, the immunotoxin based on HN3 (HN3-PE38) has superior anti-tumor activity as compared to YP7 (YP7-PE38) both in vitro and in vivo. Intravenous administration of HN3-PE38 alone, or in combination with chemotherapy, induces regression of Hep3B and HepG2 liver tumor xenografts in mice. This study establishes glypican-3 as a promising candidate for immunotoxin-based liver cancer therapy. Our results demonstrate immunotoxin-induced tumor regression via dual mechanisms: inactivation of cancer signaling via the antibody and inhibition of protein synthesis via the toxin.

Construction of an immunotoxin, HN3-mPE24, targeting glypican-3 for liver cancer therapy

Glypican-3 (GPC3) is overexpressed in hepatocellular carcinoma (HCC). We constructed a recombinant immunotoxin, HN3-mPE24, which contains a truncated form of Pseudomonas exotoxin A. The toxin portion lacks most of domain II and has seven point mutations in domain III to remove the B-cell epitopes thought to be responsible for causing off-target side effects and immunogenicity. We also fused a bivalent HN3 to mPE24. We tested these two molecules for GPC3 binding and cytotoxicity in HCC cell models. The KD values of HN3-mPE24 and HN3-HN3-mPE24 for GPC3-expressing tumor cells were 12 nM and 1.4 nM, respectively. The IC50 values of HN3-mPE24 and HN3-HN3-mPE24 for HCC cells were 0.2 nM and 0.4 nM, respectively. We also evaluated their toxicity and anti-tumor efficacy in mice. The maximum tolerated doses of HN3-mPE24 and HN3-HN3-mPE24 were 7 mg kg−1 and 3.6 mg kg−1, respectively. We treated mice with 5 mg kg−1 of HN3-mPE24 intravenously every other day for ten injections. The alpha-fetoprotein level of HN3-mPE24 treated group was approximately 700 fold less than that of the untreated group (1.1 μg ml−1 vs. 692.1 μg ml−1). In addition, 25% of the mice treated with HN3-mPE24 survived to the end of this study, which was 105 days after HCC tumor implantation. In conclusion, the HN3-mPE24 immunotoxin caused liver tumor regressions and extended survival with no significant side effects in mice. It is a promising candidate for the treatment of liver cancer that may be readily translated to humans.

Abstract 755: Synthesis and evaluation of sugar alcohol based molecules for drug conjugation and delivery

Hydrophilic linkers, which are available in different motifs, have been used as agents of drug delivery, as aqueous solubility enhancers for hydrophobic molecules, and as tethers or spacers in conjugation. Polyethylene glycol (PEG) is one of the most commonly used hydrophilic linkers and commercially available. Lower molecular weight (MW) heterobifunctional PEG linkers are the most frequently used linkers for linking hydrophobic compounds, whereas high MW polymers have been used to conjugate proteins, peptides, oligonucleotides, siRNA, and other therapeutic biopolymers. Here, we describe a new type of biomaterial based on sugar alcohols: AqueaTether™ (AqT™) molecules. Various AqT™ linkers with different lengths and end groups were synthesized and their hydrophilic nature and ability to enhance the water solubility of hydrophobic compounds compared to different commercial and PEG linkers. Various antibody drug conjugates (ADCs) with water soluble AqT™-drugs were prepared. An ADC made with AqT™-monomethyl auristatin E (MMAE) with only two sugar alcohol units attached and an average drug-to-antibody ratio (DAR) of 4 demonstrated significant improvements in minimizing the aggregation of antibodies and long-term stability during storage. Due to increased water solubility, AqT™ linkers also allow higher loading of drugs per antibody, enabling the use of less toxic drugs, potentially increasing the overall efficacy and safety profile of an ADC. For example, ADCs with average DARs of 8 and 15 have been made using AqT™-doxorubicin with only two sugar alcohol units attached. In peptide-based drug discovery, discrete high MW AqT™ linker is used to label peptide, resulting in an increased half-life in human serum. In summary, AqT™ molecules are by far the most hydrophilic and versatile linkers currently in development. AqT™ molecules represent a great tool for drug conjugation and delivery. Citation Format: Yumei Huang, Shuyan Chen, Binyuan Sun, Amanda Griffin, Nathanial Sweet, Mingqian Feng, Mitchell Ho. Synthesis and evaluation of sugar alcohol based molecules for drug conjugation and delivery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 755.