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

Journal: Cancer immunology research

Article Title: Identification of Immunogenic MHC Class II Human HER3 Peptides that Mediate Anti-HER3 CD4 + Th1 Responses and Potential Use as a Cancer Vaccine.

doi: 10.1158/2326-6066.CIR-21-0454

Figure Lengend Snippet: Figure 1. HER3 expression in cancer, and HER3 peptide screening of ECD and ICD class II peptide libraries. A, Expression of HER3 mRNA in normal (N) and tumor (T) tissues obtained from RNA-seq data from the GDC across cancer types (see Materials and Methods). B, Correlation between percentage of HER3 expression and overall patient survival (in months) in breast cancer. Samples were sorted in the descending order of HER3 expression and put into two groups: high HER3 (red) and low HER3 (blue). C, Correlation of overall patient survival with high HER3 (red) versus low HER3 (blue) expression in melanoma. P value indicated in individual graphs. D and E, IFN-g production at each screening step for sample 1 [normal donor (ND) 8; D] and sample 2 (ND 9; E) when stimulated with ECD peptides. F and G, IFN-g production at each screening step for sample 3 (ND 3; F) and sample 4 (ND 5; G) upon stimulation with ICD peptides. D–G, IFN-g response to negative peptide control (black) compared with HER3 peptides (red) with an immunogenic response threshold of ≥1.5-fold increase. Data represented as mean SEM with statistical significance determined using a multiple t test without correction for multiple comparisons. Each row was analyzed individually, without assuming consistent SD. , P < 0.05; , P < 0.01; , P < 0.001.

Article Snippet: Whole HER3 ECD/ICD protein restimulation Immunogenicity of the identified HER3 class II epitopes was confirmed through sensitization of CD4þ T cells with HER3-DC1 as described above, followed by restimulation with iDCs pulsed with the corresponding HER3 class II peptide, a negative peptide control (BRAF class II p8), native HER3 ECD (cat. #NBP2-52128-0.05 mg, Novus Biologicals) or ICD (cat. #10201-H20B1, SinoBiological) whole protein sequence, or a negative whole protein control (HemocyaninKeyhole Limpet Native protein; cat. #SRP6195, Sigma).

Techniques: Expressing, RNA Sequencing, Control

Journal: Cancer immunology research

Article Title: Identification of Immunogenic MHC Class II Human HER3 Peptides that Mediate Anti-HER3 CD4 + Th1 Responses and Potential Use as a Cancer Vaccine.

doi: 10.1158/2326-6066.CIR-21-0454

Figure Lengend Snippet: Figure 4. Intratumoral HER3-DC1 administration elicits peptide-specific immune responses and delays tumor growth. A, Tumor growth in the 4T1 murine mammary carcinoma model. BALB/c mice bearing subcutaneous 4T1 tumors received either intratumoral PBS (black), unpulsed mature DC1 (blue), or HER3 peptide–pulsed DC1 (red; n ¼ 10 mice/group), starting on day 7 when tumors were palpable. Tumor growth was monitored until endpoint and was compared between control and HER3-DC1, as well as between unpulsed DC1 and HER3-DC1. , control versus HER3-DC1; #, unpulsed DC1 versus HER3-DC1. B, Individual tumor growth for each mouse from control (black)-, unpulsed DC1 (blue)–, and HER3-DC1 (red)–treated groups. C, Percent survival in the 4T1 mouse model. Control, black; unpulsed DC1, blue; HER3-DC1, red. D, Intratumoral CD3þCD4þ and CD3þCD8þ T-cell infiltration per milligram of tumor in control (black)-, unpulsed DC1 (blue)–, and HER3-DC1 (red)–treated mice. Absolute number of immune cells was compared between control and HER3-DC1 groups. E, Frequency of CD62LþCD44þ central memory (CM), CD62LCD44þ

Article Snippet: Whole HER3 ECD/ICD protein restimulation Immunogenicity of the identified HER3 class II epitopes was confirmed through sensitization of CD4þ T cells with HER3-DC1 as described above, followed by restimulation with iDCs pulsed with the corresponding HER3 class II peptide, a negative peptide control (BRAF class II p8), native HER3 ECD (cat. #NBP2-52128-0.05 mg, Novus Biologicals) or ICD (cat. #10201-H20B1, SinoBiological) whole protein sequence, or a negative whole protein control (HemocyaninKeyhole Limpet Native protein; cat. #SRP6195, Sigma).

Techniques: Control

Journal: Cancer immunology research

Article Title: Identification of Immunogenic MHC Class II Human HER3 Peptides that Mediate Anti-HER3 CD4 + Th1 Responses and Potential Use as a Cancer Vaccine.

doi: 10.1158/2326-6066.CIR-21-0454

Figure Lengend Snippet: Figure 5. Intratumoral HER3-DC1 delays tumor growth and enhances immune infiltration in a HER2pos TUBO therapeutic model in a CD4-dependent manner. A, Tumor growth in the TUBO murine mammary carcinoma model. BALB/c mice were injected with TUBO tumor cells, and on day 7, mice received either PBS control (black), unpulsed mature DC1 (blue), or HER3-DC1 (red) intratumorally once weekly for six doses (n ¼ 10 mice/group). Tumor growth was monitored until endpoint and compared in control versus HER3-DC1 (*) and unpulsed DC1 versus HER3-DC1 (#) mice. B, Percent survival in TUBO mouse model. Control: black; unpulsed DC1: blue; HER3-DC1: red. C, CD3þCD4þ and CD3þCD8þ T cells per milligram of tumors from mice (A) after intratumoral DC injectionwas compared between control (black) and HER3-DC1 (red) groups. No statistical analyses were performed for the unpulsed DC1 (blue) mice (n ¼ 3/group). D, Abundance of CD4þ central memory (CD62LþCD44þ CM), effector memory (CD62LCD44þ EM), and effector (CD62LCD44 Eff) T cells in control (black) versus HER3-DC1 mice (red) per milligram of tumor tissue. Data shown are the representative from three independent experiments. E, Tumor growth of TUBO tumors after CD4 depletion. BALB/c mice were injected with anti-CD4 antibodies 3 days before subcutaneous TUBO tumor injection. When tumors were palpable, mice received either PBS control (black), intratumoral HER3-DC1 once weekly (red) for six doses, CD4 depletion antibody alone (blue; continued twice weekly until endpoint), or HER3-DC1 (green) with CD4 depletion. Tumor growth was monitored until endpoint. F and G, Percentage of CD4þIFN-gþ (F) and CD8þIFN-gþ (G) TILs in the tumors from control (black) versus HER3-DC1 (red) mice from E. H, Coculture of the lymph node immune cells with HER3 peptide–pulsed DC1 for 72 hours to detect IFN-g via ELISA. Control: black bar; unpulsed DC1: blue bar; HER3-DC1: red bar. I, Western blot for HER3, phosphorylated AKT (phAKT), and cleaved caspase-3 (clCasp-3) with total protein isolated from control- and HER3-DC1–treated TUBO tumors. b-Actin: loading control. J, Western blot for HER3 and phosphorylated p44/42 MAPK (ph-p44/42 MAPK) from control-, unpulsed DC1–, and HER3- DC1–treated TUBO tumors. b-Actin: loading control. Data represented as mean SEM with statistical significance determined using multiple t test without correction for multiple comparisons. Each row was analyzed individually, without assuming a consistent SD. A log-rank (Mantel–Cox) test was used to determine differences between the survival curves. , P ≤0.05; , P ≤0.01; , P ≤0.001; #, P ≤0.01.

Article Snippet: Whole HER3 ECD/ICD protein restimulation Immunogenicity of the identified HER3 class II epitopes was confirmed through sensitization of CD4þ T cells with HER3-DC1 as described above, followed by restimulation with iDCs pulsed with the corresponding HER3 class II peptide, a negative peptide control (BRAF class II p8), native HER3 ECD (cat. #NBP2-52128-0.05 mg, Novus Biologicals) or ICD (cat. #10201-H20B1, SinoBiological) whole protein sequence, or a negative whole protein control (HemocyaninKeyhole Limpet Native protein; cat. #SRP6195, Sigma).

Techniques: Injection, Control, Enzyme-linked Immunosorbent Assay, Western Blot, Isolation