InVivoMAb anti-human GPC-2
Product Details
The CT3 monoclonal antibody reacts with tumor-associated exons 3 and 10 of glypicans-2 (GPC2), a cell surface glycoprotein differentially expressed in neuroblastomas compared with normal tissues. GPC2 is also known as cerebroglycan, and it is one of the six members of glypicans, which are glycosylphosphatidylinositol (GPI)-anchored cell surface coreceptors. GPCs interact with multiple growth factors and chemokines to regulate the processes of cell proliferation, death, and differentiation. N-myc proto-oncogene protein (MYCN) controls the expression of GPC2 at its transcriptional level. Enhanced GPC2 expression in neuroblastoma promotes the Wnt/Ī²-catenin pathway and upregulates N-Myc expression to drive tumorigenesis. Among various GPC2 antibodies, the CT3 antibody is known to recognize over-expressed GPC2 in neuroblastoma, and it does not show reactivity in normal peripheral nerves or other normal tissues. The CT3 antibody does not bind to any other GPCs. CT3-based CAR T cells directed towards GPC2 show potent anti-tumor activity in vivo in preclinical mouse models of localized as well as metastatic neuroblastoma. GPC2 antibody-drug conjugates (e.g., D3-GPC2-PBD) have been documented to deliver cytotoxicity in neuroblastoma cell lines and robust antitumor effects in human neuroblastoma patient-derived xenograft (PDX) in vivo models.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 |
Immunohistochemistry (paraffin) Flow cytometry ELISA Western blot |
| 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. |
Recommended Products
-
Recommended Isotype Control(s)
InVivoMAb mouse IgG1 isotype control, unknown specificity
-
Recommended Dilution Buffer
InVivoPure pH 7.0 Dilution Buffer
Flow Cytometry
Sun M, Cao Y, Okada R, Reyes-GonzƔlez JM, Stack HG, Qin H, Li N, Seibert C, Kelly MC, Ruppin E, Ho M, Thiele CJ, Nguyen R. (2023). "Preclinical optimization of a GPC2-targeting CAR T-cell therapy for neuroblastoma" J Immunother Cancer 11(1):e005881. PubMed
Background: Although most patients with newly diagnosed high-risk neuroblastoma (NB) achieve remission after initial therapy, more than 50% experience late relapses caused by minimal residual disease (MRD) and succumb to their cancer. Therapeutic strategies to target MRD may benefit these children. We developed a new chimeric antigen receptor (CAR) targeting glypican-2 (GPC2) and conducted iterative preclinical engineering of the CAR structure to maximize its anti-tumor efficacy before clinical translation. Methods: We evaluated different GPC2-CAR constructs by measuring the CAR activity in vitro. NOD-SCID mice engrafted orthotopically with human NB cell lines or patient-derived xenografts and treated with human CAR T cells served as in vivo models. Mechanistic studies were performed using single-cell RNA-sequencing. Results: Applying stringent in vitro assays and orthotopic in vivo NB models, we demonstrated that our single-chain variable fragment, CT3, integrated into a CAR vector with a CD28 hinge, CD28 transmembrane, and 4-1BB co-stimulatory domain (CT3.28H.BBĪ¶) elicits the best preclinical anti-NB activity compared with other tested CAR constructs. This enhanced activity was associated with an enrichment of CD8+ effector T cells in the tumor-microenvironment and upregulation of several effector molecules such as GNLY, GZMB, ZNF683, and HMGN2. Finally, we also showed that the CT3.28H.BBĪ¶ CAR we developed was more potent than a recently clinically tested GD2-targeted CAR to control NB growth in vivo. Conclusion: Given the robust preclinical activity of CT3.28H.BBĪ¶, these results form a promising basis for further clinical testing in children with NB.
Flow Cytometry
Tian M, Cheuk AT, Wei JS, Abdelmaksoud A, Chou HC, Milewski D, Kelly MC, Song YK, Dower CM, Li N, Qin H, Kim YY, Wu JT, Wen X, Benzaoui M, Masih KE, Wu X, Zhang Z, Badr S, Taylor N, Croix BS, Ho M, Khan J. (2022). "An optimized bicistronic chimeric antigen receptor against GPC2 or CD276 overcomes heterogeneous expression in neuroblastoma" J Clin Invest 132(16):e155621. PubMed
Chimeric antigen receptor (CAR) T cell therapies targeting single antigens have performed poorly in clinical trials for solid tumors due to heterogenous expression of tumor-associated antigens (TAAs), limited T cell persistence, and T cell exhaustion. Here, we aimed to identify optimal CARs against glypican 2 (GPC2) or CD276 (B7-H3), which were highly but heterogeneously expressed in neuroblastoma (NB), a lethal extracranial solid tumor of childhood. First, we examined CAR T cell expansion in the presence of targets by digital droplet PCR. Next, using pooled competitive optimization of CAR by cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq), termed P-COCC, we simultaneously analyzed protein and transcriptome expression of CAR T cells to identify high-activity CARs. Finally, we performed cytotoxicity assays to identify the most effective CAR against each target and combined the CARs into a bicistronic "OR" CAR (BiCisCAR). BiCisCAR T cells effectively eliminated tumor cells expressing GPC2 or CD276. Furthermore, the BiCisCAR T cells demonstrated prolonged persistence and resistance to exhaustion when compared with CARs targeting a single antigen. This study illustrated that targeting multiple TAAs with BiCisCAR may overcome heterogenous expression of target antigens in solid tumors and identified a potent, clinically relevant CAR against NB. Moreover, our multimodal approach integrating competitive expansion, P-COCC, and cytotoxicity assays is an effective strategy to identify potent CARs among a pool of candidates.
Immunohistochemistry (paraffin), Flow Cytometry, Western Blot, ELISA
Li N, Torres MB, Spetz MR, Wang R, Peng L, Tian M, Dower CM, Nguyen R, Sun M, Tai CH, de Val N, Cachau R, Wu X, Hewitt SM, Kaplan RN, Khan J, St Croix B, Thiele CJ, Ho M. (2021). "CAR T cells targeting tumor-associated exons of glypican 2 regress neuroblastoma in mice" Cell Rep Med 2(6):100297. PubMed
Targeting solid tumors must overcome several major obstacles, in particular, the identification of elusive tumor-specific antigens. Here, we devise a strategy to help identify tumor-specific epitopes. Glypican 2 (GPC2) is overexpressed in neuroblastoma. Using RNA sequencing (RNA-seq) analysis, we show that exon 3 and exons 7-10 of GPC2 are expressed in cancer but are minimally expressed in normal tissues. Accordingly, we discover a monoclonal antibody (CT3) that binds exons 3 and 10 and visualize the complex structure of CT3 and GPC2 by electron microscopy. The potential of this approach is exemplified by designing CT3-derived chimeric antigen receptor (CAR) T cells that regress neuroblastoma in mice. Genomic sequencing of T cells recovered from mice reveals the CAR integration sites that may contribute to CAR T cell proliferation and persistence. These studies demonstrate how RNA-seq data can be exploited to help identify tumor-associated exons that can be targeted by CAR T cell therapies.
Flow Cytometry
Li N, Nguyen R, Thiele CJ, Ho M. (2021). "Preclinical testing of chimeric antigen receptor T cells in neuroblastoma mouse models" STAR Protoc 2(4):100942. PubMed
The translation of chimeric antigen receptor (CAR) T cell therapy for pediatric solid tumors is limited by the lack of preclinical models that fully recapitulate solid tumor biology. We describe steps to implement neuroblastoma metastatic and orthotopic mouse models. We delineate an analysis pipeline to quantify the efficacy and determine the immunological characteristics of both CAR T and tumor cells in these models. Both mouse models can be applied to evaluate other experimental therapies for neuroblastoma. For complete details on the use and execution of this protocol, please refer to Li et al. (2021).
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.