Journal: Molecular Therapy Oncolytics

Article Title: CXCR5 guides migration and tumor eradication of anti-EGFR chimeric antigen receptor T cells

doi: 10.1016/j.omto.2021.07.003

Figure Lengend Snippet: The specificity of EGFR recognition by EGFR scFv and the construction of EGFR-CXCR5 chimeric antigen receptor (CAR)-Ts (A) Graphical representation of the CAR designed using the anti-EGFR scFv, CD8a hinge, and transmembrane domain, 4-1BB and CD3zeta endodomain. EGFR-CXCR5 was constructed with an additional CXCR5 sequence after the CD3zeta endodomain. (B) ELISA of anti-EGFR scFv with recombinant human immunoglobulin G1 (IgG1) Fc-conjugated EGFR (ErbB1), HER2 (ErbB2), HER3 (ErbB3), MUC1, Flk1 (VEGFR2), and FLT4 (VEGFR3). Recombinant proteins were coated in the plate wells at 5 μg/mL. Anti-EGFR scFv concentration started from 5,000 pg/mL and was diluted 5-fold repeatedly until 8 pg/mL. (C) FACs analysis of A549 and PC9 (LUAD cell lines), H929 (myeloma cell line), Raji (human Burkitt’s lymphoma cell line), and K562 (human myelogenous leukemia cell line) stained with anti-EGFR scFv. Concentration started from 20,000 ng/mL and was diluted 10-fold repeatedly until 0.2 ng/mL. (D) The expression of transgenes in lentivirus-transduced T cells was analyzed by flow cytometry using protein L and anti-CXCR5 antibody. Single dot represents individual sample. Error bars represent mean ± SD for each T cell population (n = 4).

Article Snippet: Recombinant human HER3, VEGFR2 (Flk1), and VEGFR3 (Flt4) were purchased from Sino Biological.

Techniques: Construct, Sequencing, Enzyme-linked Immunosorbent Assay, Recombinant, Concentration Assay, Staining, Expressing, Flow Cytometry

Journal: Molecular Cancer Therapeutics

Article Title: A Novel Therapeutic Anti-ErbB3, ISU104 Exhibits Potent Antitumorigenic Activity by Inhibiting Ligand Binding and ErbB3 Heterodimerization

doi: 10.1158/1535-7163.mct-20-0907

Figure Lengend Snippet: Figure 1. ISU104 binds to domain 3 of ErbB3 with high affinity/avidity, specificity, and selectivity. A, Binding of fluorescently conjugated ISU104 to ErbB3-expressing cancer cells. B, Kinetic value of ISU104 and ISU104-Fab binding to human ErbB3-ECD measured by surface plasmon resonance analysis. C, Cross-reactivity of ISU104. D, No binding of ISU104 to EGFR or ErbB2. E, Domain 3 of ErbB3 is required for ISU104 binding. F, The two peptides (374-ITGYLNIQ-381 and 405-YNRGFSLL-412) in domain 3 were hindered by ISU104 from hydrogen-deuterium exchange.

Article Snippet: Recombinant human ErbB3 ECD-Fc proteins (R&D Systems) were immobilized on the surface of a CM5 chip and ISU104 or ISU104-Fab was injected over the surface at a constant flow rate (Osong Medical Innovation Foundation).

Techniques: Binding Assay, Expressing, SPR Assay

Journal: Molecular Cancer Therapeutics

Article Title: A Novel Therapeutic Anti-ErbB3, ISU104 Exhibits Potent Antitumorigenic Activity by Inhibiting Ligand Binding and ErbB3 Heterodimerization

doi: 10.1158/1535-7163.mct-20-0907

Figure Lengend Snippet: Figure 2. Structural analysis of the interaction between ErbB3 and ISU104. A, Crystal structure of the ErbB3-domain 3::ISU104-Fab complex (PDB ID: 7D85). B, Superimposition results of previously reported ErbB3 ECD and ISU104-Fab complex on tethered ErbB3 surface (PDB ID: 1M6B). C, Open-book representation of ErbB3-domain 3:: ISU104-Fab complex. The epitope of ISU104-Fab is highlighted either as a blue surface [recognized by Fab heavy chain (HC)] or a pink surface [recognized by Fab light chain (LC)] within the ErbB3-domain 3 surface. Similarly, the paratope of ISU104-Fab is denoted as a blue surface (Fab HC) and a pink surface (Fab LC) on the surface of ISU104-Fab. D, Detailed epitope residues in ErbB3 that interact with ISU104-Fab. The epitope recognized by Fab HC is colored blue, and that recognized by Fab LC is shown in red. E, Multiple interactions of ErbB3 Arg407 residue with ISU104-Fab. The guanidinium group of ErbB3 Arg407 interacts with the CDR (HC, Asp33H, Asp99H, Thr50H, and Ser35H and LC, Trp92L and His99L) of ISU104-Fab through a hydrogen bond or salt bridge (red dashed line). F, Hydrophobic interactions on the buried surface area between ErbB3-domain 3 and ISU104 Fab. Met102H of the Fab HC is buried on the hydrophobic core (arc line). ErbB3 Tyr436 forms a hydrogen bond with His101H of the Fab HC (red dashed line). All H superscripts represent HC residuesof ISU104-Fab; all L superscripts represent LC residues of ISU104-Fab.

Article Snippet: Recombinant human ErbB3 ECD-Fc proteins (R&D Systems) were immobilized on the surface of a CM5 chip and ISU104 or ISU104-Fab was injected over the surface at a constant flow rate (Osong Medical Innovation Foundation).

Techniques: Residue

Journal: Molecular Cancer Therapeutics

Article Title: A Novel Therapeutic Anti-ErbB3, ISU104 Exhibits Potent Antitumorigenic Activity by Inhibiting Ligand Binding and ErbB3 Heterodimerization

doi: 10.1158/1535-7163.mct-20-0907

Figure Lengend Snippet: Figure 3. ISU104 blocks NRG1 binding to ErbB3 directly and inhibits conformational changes for dimerization. A, Predicted NRG binding site (yellow surface) on the ErbB3-domain 3 surface (white surface) based on modeling of interaction between NRG (orange cartoon) and ErbB3. Modeling was performed on the basis of the crystal structure of ErbB4 in complex with NRG1 (PDB ID: 3U7U). B, Overlap between ISU104 epitope and NRG1bindingsite.Theburiedsurfacearea of ISU104-Fab is colored blue, and the NRG1 binding site is colored yellow. The overlapping site is colored red. C, Sche- maticrepresentationofthemechanism of action of ISU104 inhibiting both ligand binding to ErbB3 and heterodimerization with other ErbB family proteins. Tethered ErbB3 ECD is stretched by binding to NRG1 (yellow; the NRG1 binding site is indicated as a red star). Fully activated ErbB3 interacts with EGFR or ErbB2 to form a heterodimer complex (black arrow). As a result, cellular signal trans- duction is induced by these complexes. However, ISU104-Fab (brown) is a com- petitive inhibitor that binds to the NRG binding site within ErbB3-domain 3, and, thus, prevents the binding of NRG (red arrow). Conclusively, the tethered con- formation of ErbB3 is forced by ISU104- Fab, which blocks the formation of ErbB3 heterodimeric complex with other mem- bers of the EGFR family.

Article Snippet: Recombinant human ErbB3 ECD-Fc proteins (R&D Systems) were immobilized on the surface of a CM5 chip and ISU104 or ISU104-Fab was injected over the surface at a constant flow rate (Osong Medical Innovation Foundation).

Techniques: Binding Assay, Ligand Binding Assay

Journal: Molecular Cancer Therapeutics

Article Title: A Novel Therapeutic Anti-ErbB3, ISU104 Exhibits Potent Antitumorigenic Activity by Inhibiting Ligand Binding and ErbB3 Heterodimerization

doi: 10.1158/1535-7163.mct-20-0907

Figure Lengend Snippet: Figure 4. ISU104 prevents ErbB3 signaling and cancer cell proliferation by inhibiting ligand binding, heterodimerization between ErbB2 and ErbB3, and ErbB3 internalization. ISU104 prevents binding of NRG1 to ErbB3 (A) and NRG1-induced heterodimerization between ErbB3 and ErbB2 in cells (B). ISU104 binding induced internalization of ErbB3 as demonstrated by the reduction in ErbB3 levels in plasma membrane: FACS (C) and Western blotting (biotin-labeled plasma membrane cell extract; D). E and F, Inhibition of NRG1-induced activation of PI3K–AKT signaling. NRG1-dependent (G) and -independent cell proliferation (H). IB, immunoblot; IP, immunoprecipitation.

Article Snippet: Recombinant human ErbB3 ECD-Fc proteins (R&D Systems) were immobilized on the surface of a CM5 chip and ISU104 or ISU104-Fab was injected over the surface at a constant flow rate (Osong Medical Innovation Foundation).

Techniques: Ligand Binding Assay, Binding Assay, Clinical Proteomics, Membrane, Western Blot, Labeling, Inhibition, Activation Assay, Immunoprecipitation