InVivoMAb anti-human GPC3

Catalog #BE0404
Clone:
YP7

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Product Details

The YP7 monoclonal antibody reacts with the C-lobe (within amino acids 521ā€“530) of cell surface-associated glypican-3 (GPC3), also called GTR2-2, intestinal protein OCI-5, and MXR7. GPC3 is an oncofetal heparan sulfate proteoglycan, and its C-terminal domain attaches to the cell membrane through a glycophosphatidylinositol anchor. The 70-kDa precursor GPC3 protein is cleaved by furin to produce an N-terminal 40-kDa protein and a membrane-bound C-terminal 30-kDa protein. The GPC3 protein is prominently upregulated in hepatocellular carcinoma (HCC) tissues. The expression of GPC3, however, is highly heterogeneous, and it is expressed at relatively low levels in other cancers such as ovarian clear cell carcinoma and melanoma. The elevated expression of GPC3 in HCC triggers Wnt/Ī²-catenin activation (a hallmark of cancer), thereby promoting cancer cell proliferation, invasion, and metastasis. Because of the cancer-specific expression of GPC3, this glycoprotein is an appealing HCC immunotherapy target, and the in vivo experimental outcomes have been promising with GPC3-targeting bispecific antibodies, CAR T cells, and ADC immunotherapies. Among the GPC3 antibodies developed so far, the YP7 monoclonal antibody has been found to be more specific for HCC-overexpressed GPC3, and this antibody does not bind normal tissues or other forms of primary liver cancers (e.g., cholangiocarcinoma). The YP7 monoclonal antibody and its humanized version, hYP7, have been documented for antitumor activity in in vivo HCC xenograft models. When conjugated to the photosensitizing phthalocyanine dye IR700 (i.e., IR700-YP7) and exposed to near-infrared light, the YP7 antibody showed an antitumor effect in vivo in photoimmunotherapy experiments involving GPC3-positive tumor-bearing mice.

Specifications

Isotype Mouse IgG1, Īŗ
Recommended Isotype Control(s) InVivoMAb mouse IgG1 isotype control, unknown specificity
Recommended Dilution Buffer InVivoPure pH 7.0 Dilution Buffer
Conjugation This product is unconjugated. Conjugation is available via our Antibody Conjugation Services.
Reported Applications in vitro photoimmunotherapy
in vivo anti-tumor activity
in vivo imaging
in vivo photoimmunotherapy
Flow cytometry
Immunohistochemistry (paraffin)
Immunofluorescence
Western blot
Immunoprecipitation
ELISA
Formulation PBS, pH 7.0
Contains no stabilizers or preservatives
Endotoxin <2EU/mg (<0.002EU/Ī¼g)
Determined by LAL gel clotting assay
Purity >95%
Determined by SDS-PAGE
Sterility 0.2 Āµm filtration
Production Purified from cell culture supernatant in an animal-free facility
Purification Protein G
Molecular Weight 150 kDa
Storage The antibody solution should be stored at the stock concentration at 4Ā°C. Do not freeze.
Flow Cytometry
Ma L, Heinrich S, Wang L, Keggenhoff FL, Khatib S, Forgues M, Kelly M, Hewitt SM, Saif A, Hernandez JM, Mabry D, Kloeckner R, Greten TF, Chaisaingmongkol J, Ruchirawat M, Marquardt JU, Wang XW. (2022). "Multiregional single-cell dissection of tumor and immune cells reveals stable lock-and-key features in liver cancer" Nat Commun 13(1):7533. PubMed

Intratumor heterogeneity may result from the evolution of tumor cells and their continuous interactions with the tumor microenvironment which collectively drives tumorigenesis. However, an appearance of cellular and molecular heterogeneity creates a challenge to define molecular features linked to tumor malignancy. Here we perform multiregional single-cell RNA sequencing (scRNA-seq) analysis of seven liver cancer patients (four hepatocellular carcinoma, HCC and three intrahepatic cholangiocarcinoma, iCCA). We identify cellular dynamics of malignant cells and their communication networks with tumor-associated immune cells, which are validated using additional scRNA-seq data of 25 HCC and 12 iCCA patients as a stable fingerprint embedded in a malignant ecosystem representing features of tumor aggressiveness. We further validate the top ligand-receptor interaction pairs (i.e., LGALS9-SLC1A5 and SPP1-PTGER4 between tumor cells and macrophages) associated with unique transcriptome in additional 542 HCC patients. Our study unveils stable molecular networks of malignant ecosystems, which may open a path for therapeutic exploration.

Immunohistochemistry (paraffin), Flow Cytometry
Li N, Spetz MR, Ho M. (2020). "The Role of Glypicans in Cancer Progression and Therapy" J Histochem Cytochem 68(12):841-862. PubMed

Glypicans are a family of heparan sulfate proteoglycans that are attached to the cell membrane via a glycosylphosphatidylinositol anchor. Glypicans interact with multiple ligands, including morphogens, growth factors, chemokines, ligands, receptors, and components of the extracellular matrix through their heparan sulfate chains and core protein. Therefore, glypicans can function as coreceptors to regulate cell proliferation, cell motility, and morphogenesis. In addition, some glypicans are abnormally expressed in cancers, possibly involved in tumorigenesis, and have the potential to be cancer-specific biomarkers. Here, we provide a brief review focusing on the expression of glypicans in various cancers and their potential to be targets for cancer therapy.

Immunohistochemistry (paraffin), Flow Cytometry
Li D, Li N, Zhang YF, Fu H, Feng M, Schneider D, Su L, Wu X, Zhou J, Mackay S, Kramer J, Duan Z, Yang H, Kolluri A, Hummer AM, Torres MB, Zhu H, Hall MD, Luo X, Chen J, Wang Q, Abate-Daga D, Dropulic B, Hewitt SM, Orentas RJ, Greten TF, Ho M. (2020). "Persistent Polyfunctional Chimeric Antigen Receptor T Cells That Target Glypican 3 Eliminate Orthotopic Hepatocellular Carcinomas in Mice" Gastroenterology 158(8):2250-2265.e20. PubMed

Background and aims: Glypican 3 (GPC3) is an oncofetal antigen involved in Wnt-dependent cell proliferation that is highly expressed in hepatocellular carcinoma (HCC). We investigated whether the functions of chimeric antigen receptors (CARs) that target GPC3 are affected by their antibody-binding properties. Methods: We collected peripheral blood mononuclear cells from healthy donors and patients with HCC and used them to create CAR T cells, based on the humanized YP7 (hYP7) and HN3 antibodies, which have high affinities for the C-lobe and N-lobe of GPC3, respectively. NOD/SCID/IL-2Rgcnull (NSG) mice were given intraperitoneal injections of luciferase-expressing (Luc) Hep3B or HepG2 cells and after xenograft tumors formed, mice were given injections of saline or untransduced T cells (mock control), or CAR (HN3) T cells or CAR (hYP7) T cells. In other NOD/SCID/IL-2Rgcnull (NSG) mice, HepG2-Luc or Hep3B-Luc cells were injected into liver, and after orthotopic tumors formed, mice were given 1 injection of CAR (hYP7) T cells or CD19 CAR T cells (control). We developed droplet digital polymerase chain reaction and genome sequencing methods to analyze persistent CAR T cells in mice. Results: Injections of CAR (hYP7) T cells eliminated tumors in 66% of mice by week 3, whereas CAR (HN3) T cells did not reduce tumor burden. Mice given CAR (hYP7) T cells remained tumor free after re-challenge with additional Hep3B cells. The CAR T cells induced perforin- and granzyme-mediated apoptosis and reduced levels of active Ī²-catenin in HCC cells. Mice injected with CAR (hYP7) T cells had persistent expansion of T cells and subsets of polyfunctional CAR T cells via antigen-induced selection. These T cells were observed in the tumor microenvironment and spleen for up to 7 weeks after CAR T-cell administration. Integration sites in pre-infusion CAR (HN3) and CAR (hYP7) T cells were randomly distributed, whereas integration into NUPL1 was detected in 3.9% of CAR (hYP7) T cells 5 weeks after injection into tumor-bearing mice and 18.1% of CAR (hYP7) T cells at week 7. There was no common site of integration in CAR (HN3) or CD19 CAR T cells from tumor-bearing mice. Conclusions: In mice with xenograft or orthoptic liver tumors, CAR (hYP7) T cells eliminate GPC3-positive HCC cells, possibly by inducing perforin- and granzyme-mediated apoptosis or reducing Wnt signaling in tumor cells. GPC3-targeted CAR T cells might be developed for treatment of patients with HCC.

Immunohistochemistry (paraffin), Flow Cytometry, ELISA
Liu X, Gao F, Jiang L, Jia M, Ao L, Lu M, Gou L, Ho M, Jia S, Chen F, Gao W. (2020). "32A9, a novel human antibody for designing an immunotoxin and CAR-T cells against glypican-3 in hepatocellular carcinoma" J Transl Med 18(1):295. PubMed

Background: Treatment of hepatocellular carcinoma (HCC) using antibody-based targeted therapies, such as antibody conjugates and chimeric antigen receptor T (CAR-T) cell therapy, shows potent antitumor efficacy. Glypican-3 (GPC3) is an emerging HCC therapeutic target; therefore, antibodies against GPC3 would be useful tools for developing immunotherapies for HCC. Methods: We isolated a novel human monoclonal antibody, 32A9, by phage display technology. We determined specificity, affinity, epitope and anti-tumor activity of 32A9, and developed 32A9-based immunotherapy technologies for evaluating the potency of HCC treatment in vitro or in vivo. Results: 32A9 recognized human GPC3 with potent affinity and specificity. The epitope of 32A9 was located in the region of the GPC3 protein core close to the modification sites of the HS chain and outside of the Wnt-binding site of GPC3. The 32A9 antibody significantly inhibited HCC xenograft tumor growth in vivo. We then pursued two 32A9-based immunotherapeutic strategies by constructing an immunotoxin and CAR-T cells. The 32A9 immunotoxin exhibited specific cytotoxicity to GPC3-positive cancer cells, while 32A9 CAR-T cells efficiently eliminated GPC3-positive HCC cells in vitro and caused HCC xenograft tumor regressions in vivo. Conclusions: Our study provides a rationale for 32A9 as a promising GPC3-specific antibody candidate for HCC immunotherapy.

ELISA
Li N, Wei L, Liu X, Bai H, Ye Y, Li D, Li N, Baxa U, Wang Q, Lv L, Chen Y, Feng M, Lee B, Gao W, Ho M. (2019). "A Frizzled-Like Cysteine-Rich Domain in Glypican-3 Mediates Wnt Binding and Regulates Hepatocellular Carcinoma Tumor Growth in Mice" Hepatology 70(4):1231-1245. PubMed

Wnt signaling is one of the key regulators of hepatocellular carcinoma (HCC) tumor progression. In addition to the classical receptor frizzled (FZD), various coreceptors including heparan sulfate proteoglycans (HSPGs) are involved in Wnt activation. Glypican-3 (GPC3) is an HSPG that is overexpressed in HCC and functions as a Wnt coreceptor that modulates HCC cell proliferation. These features make GPC3 an attractive target for liver cancer therapy. However, the precise interaction of GPC3 and Wnt and how GPC3, Wnt, and FZD cooperate with each other are poorly understood. In this study, we established a structural model of GPC3 containing a putative FZD-like cysteine-rich domain at its N-terminal lobe. We found that F41 and its surrounding residues in GPC3 formed a Wnt-binding groove that interacted with the middle region located between the lipid thumb domain and the index finger domain of Wnt3a. Mutating residues in this groove significantly inhibited Wnt3a binding, Ī²-catenin activation, and the transcriptional activation of Wnt-dependent genes. In contrast with the heparan sulfate chains, the Wnt-binding groove that we identified in the protein core of GPC3 seemed to promote Wnt signaling in conditions when FZD was not abundant. Specifically, blocking this domain using an antibody inhibited Wnt activation. In HCC cells, mutating residue F41 on GPC3 inhibited activation of Ī²-catenin in vitro and reduced xenograft tumor growth in nude mice compared with cells expressing wild-type GPC3. Conclusion: Our investigation demonstrates a detailed interaction of GPC3 and Wnt3a, reveals the precise mechanism of GPC3 acting as a Wnt coreceptor, and provides a potential target site on GPC3 for Wnt blocking and HCC therapy.

Immunohistochemistry (paraffin)
Chahoud J, Zhang M, Pisters LL, Lin SC, Lin SH, Tu SM. (2018). "Identification of Glypican-3 (GPC3) Expression in a Lethal Subgroup of Refractory Cisplatin-Resistant Testicular Germ-Cell Tumors" Clin Genitourin Cancer 16(5):325-327. PubMed

Flow Cytometry
Li W, Guo L, Rathi P, Marinova E, Gao X, Wu MF, Liu H, Dotti G, Gottschalk S, Metelitsa LS, Heczey A. (2017). "Redirecting T Cells to Glypican-3 with 4-1BB Zeta Chimeric Antigen Receptors Results in Th1 Polarization and Potent Antitumor Activity" Hum Gene Ther 28(5):437-448. PubMed

T cells engineered to express CD19-specific chimeric antigen receptors (CARs) have shown breakthrough clinical successes in patients with B-cell lymphoid malignancies. However, similar therapeutic efficacy of CAR T cells in solid tumors is yet to be achieved. In this study we systematically evaluated a series of CAR constructs targeting glypican-3 (GPC3), which is selectively expressed on several solid tumors. We compared GPC3-specific CARs that encoded CD3Ī¶ (Gz) alone or with costimulatory domains derived from CD28 (G28z), 4-1BB (GBBz), or CD28 and 4-1BB (G28BBz). All GPC3-CARs rendered T cells highly cytotoxic to GPC3-positive hepatocellular carcinoma, hepatoblastoma, and malignant rhabdoid tumor cell lines in vitro. GBBz induced the preferential production of Th1 cytokines (interferon Ī³/granulocyte macrophage colony-stimulating factor) while G28z preferentially induced Th2 cytokines (interleukin-4/interleukin-10). Inclusion of 4-1BB in G28BBz could only partially ameliorate the Th2-polarizing effect of CD28. 4-1BB induced superior expansion of CAR T cells in vitro and in vivo. T cells expressing GPC3-CARs incorporating CD28, 4-1BB, or both induced sustained tumor regressions in two xenogeneic tumor models. Thus, GBBz CAR endows T cells with superior proliferative potential, potent antitumor activity, and a Th1-biased cytokine profile, justifying further clinical development of GBBz CAR for immunotherapy of GPC3-positive solid tumors.

Flow Cytometry, Western Blot, ELISA
Gao W, Tang Z, Zhang YF, Feng M, Qian M, Dimitrov DS, Ho M. (2015). "Immunotoxin targeting glypican-3 regresses liver cancer via dual inhibition of Wnt signalling and protein synthesis" Nat Commun . PubMed

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 signalling, whereas the second antibody recognizes a C-terminal epitope but does not inhibit Wnt signalling. Both are fused to a fragment of Pseudomonas exotoxin A (PE38) to create immunotoxins. Interestingly, the immunotoxin based on HN3 (HN3-PE38) has superior antitumor activity as compared with 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 tumour xenografts in mice. This study establishes glypican-3 as a promising candidate for immunotoxin-based liver cancer therapy. Our results demonstrate immunotoxin-induced tumour regression via dual mechanisms: inactivation of cancer signalling via the antibody and inhibition of protein synthesis via the toxin.

in vitro photoimmunotherapy, in vivo photoimmunotherapy, in vivo imaging, Immunofluorescence, Flow Cytometry
Hanaoka H, Nakajima T, Sato K, Watanabe R, Phung Y, Gao W, Harada T, Kim I, Paik CH, Choyke PL, Ho M, Kobayashi H. (2015). "Photoimmunotherapy of hepatocellular carcinoma-targeting Glypican-3 combined with nanosized albumin-bound paclitaxel" Nanomedicine (Lond) 10(7):1139-47. PubMed

Aim: Effectiveness of Glypican-3 (GPC3)-targeted photoimmunotherapy (PIT) combined with the nanoparticle albumin-bound paclitaxel (nab-paclitaxel) for hepatocellular carcinoma was evaluated. Materials & methods: GPC3 expressing A431/G1 cells were incubated with a phthalocyanine-derivative, IRDye700DX (IR700), conjugated to an anti-GPC3 antibody, IR700-YP7 and exposed to near-infrared light. Therapeutic experiments combining GPC3-targeted PIT with nab-paclitaxel were performed in A431/G1 tumor-bearing mice. Results: IR700-YP7 bound to A431/G1 cells and induced rapid target-specific necrotic cell death by near-infrared light exposure in vitro. IR700-YP7 accumulated in A431/G1 tumors. Tumor growth was inhibited by PIT compared with nontreated control. Additionally, PIT dramatically increased nab-paclitaxel delivery and enhanced the therapeutic effect. Conclusion: PIT targeting GPC3 combined with nab-paclitaxel is a promising method for treating hepatocellular carcinoma.

in vitro photoimmunotherapy, in vivo photoimmunotherapy, Flow Cytometry
Hanaoka H, Nagaya T, Sato K, Nakamura Y, Watanabe R, Harada T, Gao W, Feng M, Phung Y, Kim I, Paik CH, Choyke PL, Ho M, Kobayashi H. (2015). "Glypican-3 targeted human heavy chain antibody as a drug carrier for hepatocellular carcinoma therapy" Mol Pharm 12(6):2151-7. PubMed

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.

Western Blot
Gao W, Kim H, Ho M. (2015). "Human Monoclonal Antibody Targeting the Heparan Sulfate Chains of Glypican-3 Inhibits HGF-Mediated Migration and Motility of Hepatocellular Carcinoma Cells" PLoS One 10(9):e0137664. PubMed

Heparan sulfate proteoglycans (HSPGs) participate in many processes related to tumor development, including tumorigenesis and metastasis. HSPGs contain one or more heparan sulfate (HS) chains that are covalently linked to a core protein. Glypican-3 (GPC3) is a cell surface-associated HSPG that is highly expressed in hepatocellular carcinoma (HCC). GPC3 is involved in Wnt3a-dependent HCC cell proliferation. Our previous study reported that HS20, a human monoclonal antibody targeting the HS chains on GPC3, inhibited Wnt3a/Ī²-catenin activation. In the current study, we showed that the HS chains of GPC3 could mediate HCC cells' migration and motility. Knocking down GPC3 or targeting the HS chains by HS20 inhibited HCC cell migration and motility. However, HS20 had no effect on GPC3 knockdown cells or GPC3 negative cells. In addition, an antibody that recognizes the core protein of GPC3 did not change the rate of cell motility. HCC cell migration and motility did not respond to either canonical or non-canonical Wnt induction, but did increase under hepatocyte growth factor (HGF) treatment. HS20-treated HCC cells exhibited less ability for HGF-mediated migration and motility. Furthermore, HS20 inhibited in vitro HCC spheroid formation and liver tumor growth in mice. GPC3 interacted with HGF; however, a mutant GPC3 lacking the HS chain showed less interaction with HGF. Blocking the HS chains on GPC3 with HS20 reduced c-Met activation in HGF-treated HCC cells and 3D-cultured spheroids. Taken together, our study suggests that GPC3 is involved in HCC cell migration and motility through HS chain-mediated cooperation with the HGF/Met pathway, showing how HS targeting has potential therapeutic implications for liver cancer.

Immunohistochemistry (paraffin), Flow Cytometry, Western Blot, Immunoprecipitation
Gao W, Kim H, Feng M, Phung Y, Xavier CP, Rubin JS, Ho M. (2014). "Inactivation of Wnt signaling by a human antibody that recognizes the heparan sulfate chains of glypican-3 for liver cancer therapy" Hepatology 60(2):576-87. PubMed

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/Ī²-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/Ī²-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/Ī²-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.

in vivo anti-tumor activity, Immunohistochemistry (paraffin), Flow Cytometry, Western Blot, Immunoprecipitation, ELISA
Phung Y, Gao W, Man YG, Nagata S, Ho M. (2012). "High-affinity monoclonal antibodies to cell surface tumor antigen glypican-3 generated through a combination of peptide immunization and flow cytometry screening" MAbs 4(5):592-9. PubMed

Isolating high-affinity antibodies against native tumor antigens on the cell surface is not straightforward using standard hybridoma procedures. Here, we describe a combination method of synthetic peptide immunization and high-throughput flow cytometry screening to efficiently isolate hybridomas for cell binding. Using this method, we identified high-affinity monoclonal antibodies specific for the native form of glypcian-3 (GPC3), a target heterogeneously expressed in hepatocellular carcinoma (HCC) and other cancers. We isolated a panel of monoclonal antibodies (YP6, YP7, YP8, YP9 and YP9.1) for cell surface binding. The antibodies were used to characterize GPC3 protein expression in human liver cancer cell lines and tissues by flow cytometry, immunoblotting and immunohistochemistry. The best antibody (YP7) bound cell surface-associated GPC3 with equilibrium dissociation constant, KD = 0.3 nmol/L and was highly specific for HCC, not normal tissues or other forms of primary liver cancers (such as cholangiocarcinoma). Interestingly, the new antibody was highly sensitive in that it detected GPC3 in low expression ovarian clear cell carcinoma and melanoma cells. The YP7 antibody exhibited significant HCC xenograft tumor growth inhibition in nude mice. These results describe an improved method for producing high-affinity monoclonal antibodies to cell surface tumor antigens and represent a general approach to isolate therapeutic antibodies against cancer. The new high-affinity antibodies described here have significant potential for GPC3-expressing cancer diagnostics and therapy.