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

Journal: Antibodies

Article Title: Novel Humanized Anti-HER3 Antibodies: Structural Characterization and Therapeutic Activity

doi: 10.3390/antib14040084

Figure Lengend Snippet: Identification of variants binding HER3. ( A ) Binding activity of TK-hu A3 and TK-hu A4 antibody variants to human HER3, as measured by ELISA. Six combinations of TK-hu A3 heavy (H) and light (L) chains and nine combinations of TK-hu A4 variants were transiently expressed in HEK293 cells. Supernatants were harvested 72 h post-transfection and assessed for HER3 binding. Bars represent mean absorbance ± SD of triplicate measurements. H/L ratio: DNA heavy chain to DNA light chain ratio. ( B ) Expression and integrity of recombinant mAbs H1L1 (lane 1), H2L1 (lane 2) from TK-hu A3, and H3L1 (lane 3) from TK-hu A4 were analyzed by SDS-PAGE under reducing conditions (1 mM DTT) followed by Coomassie blue staining. Molecular weight markers corresponding to the heavy chain (~50 kDa) and light chain (~25 kDa) are indicated.

Article Snippet: The interactions of HER3 Fc chimera protein (R&D Systems, Minneapolis, MN, USA) with TK-murine A3, TK-murine A4, TK-hu A3, and TK-hu A4 antibodies were studied by surface plasmon resonance detection using a Biacore X100 instrument (Cytiva, Marlborough, MA, US).

Techniques: Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Transfection, Expressing, Recombinant, SDS Page, Staining, Molecular Weight

Journal: Antibodies

Article Title: Novel Humanized Anti-HER3 Antibodies: Structural Characterization and Therapeutic Activity

doi: 10.3390/antib14040084

Figure Lengend Snippet: Antibodies species cross-reactivity. Binding of TK-murine A3, TK-hu A3, and TK-hu A4 purified antibody variants to recombinant mouse, rat, human, and rhesus HER3 was determined in a dose–response experiment by ELISA. Data represent mean absorbance ± SD from triplicate wells.

Article Snippet: The interactions of HER3 Fc chimera protein (R&D Systems, Minneapolis, MN, USA) with TK-murine A3, TK-murine A4, TK-hu A3, and TK-hu A4 antibodies were studied by surface plasmon resonance detection using a Biacore X100 instrument (Cytiva, Marlborough, MA, US).

Techniques: Binding Assay, Purification, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Antibodies

Article Title: Novel Humanized Anti-HER3 Antibodies: Structural Characterization and Therapeutic Activity

doi: 10.3390/antib14040084

Figure Lengend Snippet: Binding activity of TK-hu A3 and TK-hu A4 antibody variants. ( A ) TK-murine A3 and TK-murine A4 antibodies, as well as TK-hu A3 and TK-hu A4 purified antibody variants, were analyzed for their binding to members of the ErbB tyrosine kinase receptor family: EGFR, ErbB2, HER3, and ErbB4. Antibodies were used at a concentration of 10 µg/mL, and binding was analyzed by ELISA. Absorbance values are plotted on the y-axis with respect to the antibody tested on the different recombinant proteins. ( B ) Serial dilutions of TK-murine A3, TK-hu A3, and TK-hu A4 purified antibody variants were analyzed by ELISA to assess their binding to human HER3 in a dose-dependent manner. Absorbance values are plotted on the y-axis with respect to the antibody tested at different dilutions.

Article Snippet: The interactions of HER3 Fc chimera protein (R&D Systems, Minneapolis, MN, USA) with TK-murine A3, TK-murine A4, TK-hu A3, and TK-hu A4 antibodies were studied by surface plasmon resonance detection using a Biacore X100 instrument (Cytiva, Marlborough, MA, US).

Techniques: Binding Assay, Activity Assay, Purification, Concentration Assay, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: Antibodies

Article Title: Novel Humanized Anti-HER3 Antibodies: Structural Characterization and Therapeutic Activity

doi: 10.3390/antib14040084

Figure Lengend Snippet: Epitope mapping of murine and humanized anti-HER3 antibodies. ( A ) Peptide array binding assays for TK-hu A3-H1L1, TK-hu A3-H2L1, and the murine antibody TK-A3. Reactivity was measured by ELISA using overlapping 15-mer peptides covering the HER3 extracellular domain (ECD). Each colored bar represents the optical density (OD) at 405 nm for an individual peptide. Strong binding was observed for peptides #54 and #55 in TK-hu A3-H1L1 and TK-A3, indicating conserved epitope recognition. In contrast, TK-hu A3-H2L1 showed loss of binding to these peptides and gained reactivity for peptide #66, suggesting a shift in epitope specificity. C+ = positive control; C− = negative control. ( B ) Structural mapping of the identified epitopes onto the HER3 ECD (PDB entry: 1M6B). Left: location of peptide #54 and #55 (orange) shared by TK-A3 and TK-hu A3-H1L1. Right: location of peptide #66 (purple), recognized exclusively by TK-hu A3-H2L1. Protein domains are indicated with Roman numerals. Only HER3 domain II is rendered as surface. ( C ) Amino acid sequences of the relevant peptides with conserved regions highlighted in bold. Peptides #54 and #55 share the core motif HCFGPNPNQCC, while peptide #66 contains a distinct sequence (QPLVYNKLTFQLEPN), reflecting altered antigen recognition in TK-hu A3-H2L1.

Article Snippet: The interactions of HER3 Fc chimera protein (R&D Systems, Minneapolis, MN, USA) with TK-murine A3, TK-murine A4, TK-hu A3, and TK-hu A4 antibodies were studied by surface plasmon resonance detection using a Biacore X100 instrument (Cytiva, Marlborough, MA, US).

Techniques: Peptide Microarray, Binding Assay, Enzyme-linked Immunosorbent Assay, Positive Control, Negative Control, Sequencing

Journal: Antibodies

Article Title: Novel Humanized Anti-HER3 Antibodies: Structural Characterization and Therapeutic Activity

doi: 10.3390/antib14040084

Figure Lengend Snippet: Structure of HER3::TK-hu A3 Fab complex. ( A ) Front view (left) and side view (right) of HER3 (cyan) in complex with TK-hu A3 Fab (light chain, V L , in light green and heavy chain, V H , in coral). Only HER3 domain II is rendered as surface; domains I–IV are indicated with Roman numerals. The domain II dimerization arm (residues 242–257, chain B) is shown in magenta. HER3 domain II surface colored by Fab contacts (atom–atom cutoff 4.0 Å). ( B ) Residues contacting V L only are light green, residues contacting V H only are coral, and residues contacting both chains are yellow. The HER3 cartoon is shown underneath (cyan); the Fab is omitted for clarity. (Contact patches computed with PDBePISA). Atomic details of HER3::TK-hu A3 Fab interface region. ( C , D ) TK-hu A3 Fab binds to the N-terminus region of domain II of HER3 mostly via hydrogen bonds, except for R84, which is involved in a π-cation interaction with residue Y33/F. ( C ): interactions between V L (variable light chain, light green) and HER3 domain II (cyan). ( D ): interactions between V H (variable heavy chain, coral) and HER3 domain II (cyan) (PDB entry: 9I1Q).

Article Snippet: The interactions of HER3 Fc chimera protein (R&D Systems, Minneapolis, MN, USA) with TK-murine A3, TK-murine A4, TK-hu A3, and TK-hu A4 antibodies were studied by surface plasmon resonance detection using a Biacore X100 instrument (Cytiva, Marlborough, MA, US).

Techniques: Residue

Journal: Antibodies

Article Title: Novel Humanized Anti-HER3 Antibodies: Structural Characterization and Therapeutic Activity

doi: 10.3390/antib14040084

Figure Lengend Snippet: TK-hu A3 and TK-hu A4 antibodies variants bind HER3 receptor in its native conformation. MCF7 cells were incubated on ice for 1 h in the presence or absence of TK-murine A3 ( A ), TK-hu A3-H1L1 ( B ), TK-hu A3-H2L1 ( C ), and TK-hu A4-H3L1 ( D ) antibody variants at concentrations of 10 µg/mL and 100 µg/mL. After incubation, cells were washed, and antibody binding was detected using anti-mouse or anti-human IgG antibody conjugated to Alexa Fluor 488 (gray bars). Cells were analyzed using a CytoFLEX flow cytometer platform. Histograms display the percentage of HER3-positive cells. CTRL antibody: The APC anti-human HER3/HER-3 Antibody was used as a positive control (white bars).

Article Snippet: The interactions of HER3 Fc chimera protein (R&D Systems, Minneapolis, MN, USA) with TK-murine A3, TK-murine A4, TK-hu A3, and TK-hu A4 antibodies were studied by surface plasmon resonance detection using a Biacore X100 instrument (Cytiva, Marlborough, MA, US).

Techniques: Incubation, Binding Assay, Flow Cytometry, Positive Control

Journal: Antibodies

Article Title: Novel Humanized Anti-HER3 Antibodies: Structural Characterization and Therapeutic Activity

doi: 10.3390/antib14040084

Figure Lengend Snippet: TK-hu A3 and TK-hu A4 antibodies inhibit NRG-driven HER3 signaling and reduce cancer cell viability. ( A ) Neuregulin competition assay. SK-BR-3 cells were incubated for 1 h at 4 °C with increasing concentrations of TK-hu A3 or TK-hu A4, either in the presence or absence of recombinant neuregulin (NRG). Binding to cell-surface HER3 was assessed by flow cytometry. The plot shows representative binding curves: TK-hu A3 ± NRG (purple and yellow), and TK-hu A4 ± NRG (light green and dark green). The percentage of HER3-positive cells is plotted on the y-axis versus antibody concentration (log10 [µg/mL], x-axis). ( B ) Western blot analysis of HER3 signaling pathway activation in MCF7, FaDu, and BxPC-3 cells. Cells were pretreated for 6 h with increasing concentrations of TK-hu A3 or TK-hu A4 antibodies, then stimulated with NRG. The Control (Ctrl) lane represents untreated, unstimulated cells, while the NRG lane represents NRG-stimulated cells in the absence of antibody. Blots were probed for phosphorylated HER3 (pHER3, Tyr1289), total HER3, phosphorylated Akt (pAkt, Ser473), and phosphorylated p42/44 MAPK (Thr202/Tyr204). β-tubulin served as the loading control. ( C ) Colony-formation assay. BxPC-3 cells were seeded in 6-well plates, treated or not with increasing concentrations of TK-hu A3-H1L1 or TK-hu A4-H3L1 and cultured to allow colony formation; colonies were then fixed, stained, and counted. Only colonies comprising >50 cells were scored as survival colonies. The percentage of inhibition of colony formation (% inh) relative to untreated controls is plotted against the logarithmic antibody concentration (log 10 [µg/mL]), and data are fitted with a four-parametric non-linear regression curve (GraphPad v8.0).

Article Snippet: The interactions of HER3 Fc chimera protein (R&D Systems, Minneapolis, MN, USA) with TK-murine A3, TK-murine A4, TK-hu A3, and TK-hu A4 antibodies were studied by surface plasmon resonance detection using a Biacore X100 instrument (Cytiva, Marlborough, MA, US).

Techniques: Competitive Binding Assay, Incubation, Recombinant, Binding Assay, Flow Cytometry, Concentration Assay, Western Blot, Activation Assay, Control, Colony Assay, Cell Culture, Staining, Inhibition

Journal: mAbs

Article Title: Discovery of potent allosteric antibodies inhibiting EGFR

doi: 10.1080/19420862.2024.2406548

Figure Lengend Snippet: Biophysical data of anti-egfr antibodies. Affinities and binding assays were performed by BLI. Recombinant human (rh), recombinant cynomolgus (rc) and recombinant mouse (rm).

Article Snippet: Lastly, cross reactivity binding assays were performed using rhHER2 (Sino Biological 10,004-H08H), rhHER3 (10368-RB-050), rhHER4 (1131-ER-050), recombinant cynomolgus monkey EGFR (10405-ER-050) and recombinant mouse EGFR (1280-ER-050) all from R&D Systems.

Techniques: Binding Assay, Recombinant

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