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: 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-γ 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-γ 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-γ 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: Immunogenicity of the identified HER3 class II epitopes was confirmed through sensitization of CD4 + 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 (Hemocyanin-Keyhole 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: HER3 class II peptides demonstrate anti-HER3 immune responses in vitro . A and B, ECD and ICD peptide libraries were screened sequentially in 10-peptide pools, 5-peptide pools, and individual peptides with an immunogenic response threshold of ≥1.5-fold increase in IFN-γ production between peptide and control restimulated CD4 + T cells (see ), ultimately identifying four ECD and five ICD HER3 class II peptides. Each schematic is representative of the combined responses across samples used in the peptide screening ( n = 3), indicating reproducible significant immunogenic response compared with the class II control in ≥2 samples and number of donor responses in parentheses (red). C and D, Peptide-primed CD4 + T cells were restimulated with matching class II peptide (HER3 ECD or HER3 ICD), class II–negative control (peptide control), whole HER3 domain protein (WP HER3 ECD or WP HER3 ICD), or whole protein control (WP 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. *, P < 0.05; **, P < 0.01; ***, P < 0.001. E, PBMCs from healthy donors (HD, black bar, n = 6) or breast cancer patients (Patient, red bar, n = 10) were individually stimulated with the nine HER3 class II peptides and analyzed via IFN-y ELISPOT. Left, percentage of subjects responding to ≥1 HER3 peptide (anti-HER3 responsivity). Middle, mean number of peptides inducing anti-HER3–specific immunity (response repertoire). Right, total IFN-γ spots (mean total SFC/1e6 cells) from stimulation with HER3 peptides (cumulative response). Data represented as mean ± SEM with statistical significance determined using Mann–Whitney test. ns, not significant.

Article Snippet: Immunogenicity of the identified HER3 class II epitopes was confirmed through sensitization of CD4 + 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 (Hemocyanin-Keyhole Limpet Native protein; cat. #SRP6195, Sigma).

Techniques: In Vitro, Control, Negative Control, Enzyme-linked Immunospot, MANN-WHITNEY

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: HER3-DC1 vaccination elicits peptide-specific immune response and delays tumor growth. A, Immunoblotting of murine tumor cell lines 4T1, TUBO, and M05 to detect HER3. β-Actin: loading control. B, Immunofluorescence for HER3 (red) and nucleus (DAPI, blue) in 4T1 and TUBO murine mammary tumor cells (image magnification: 1,200×). C and D, Individual HER3 peptide–specific immune responses in spleens ( C ) and lymph node–derived immune cells ( D ) from control (black), unpulsed mature DC1 (blue), and HER3-DC1 (red) vaccinated BALB/c mice ( n = 3). Spleens were processed, and splenocytes were restimulated with the HER3 peptides for 72 hours to detect IFN-γ by ELISA. Lymph node–derived lymphocytes were cocultured with DC1 pulsed with individual HER3 peptides for 72 hours to detect IFN-γ by ELISA. E, Tumor growth after 4T1 tumor challenge in control (black), unpulsed mature DC1 (blue), and HER3-DC1 (red) vaccinated mice ( n = 7–10 mice/group). Mice were challenged 2 weeks after the last vaccination and were monitored until endpoint. *, control versus HER3-DC1; #, unpulsed DC1 versus HER3-DC1. F, TUBO tumor growth in control (black) and HER3-DC1 (red) vaccinated mice ( n = 7–10 mice/group). Mice were challenged 2 weeks after the last vaccination. Data represented as mean ± SEM with statistical significance determined using multiple t test without correction for multiple comparisons. Each row analyzed individually, without assuming a consistent SD. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ## , P ≤ 0.01.

Article Snippet: Immunogenicity of the identified HER3 class II epitopes was confirmed through sensitization of CD4 + 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 (Hemocyanin-Keyhole Limpet Native protein; cat. #SRP6195, Sigma).

Techniques: Western Blot, Control, Immunofluorescence, Derivative Assay, Enzyme-linked Immunosorbent Assay

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: 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 = 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 CD3 + CD4 + and CD3 + CD8 + 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 CD62L + CD44 + central memory (CM), CD62L − CD44 + effector memory (EM), and CD62L − CD44 − effector (Eff) T-cell populations within intratumoral CD4 + cells between control- (black) and HER3-DC1–treated (red) tumors. The unpulsed DC1 (blue) group was not included in any statistical analyses. F, Absolute number of CD3 + CD4 + and CD3 + CD8 + T cells in lymph nodes of control (black)-, intratumoral unpulsed DC1 (blue)–, and HER3-DC1 (red)–treated mice. Cell numbers were compared in control versus HER3-DC1 groups. Data shown are the representative from three independent experiments. G, Absolute numbers of CD4 + CM, EM, and Eff T-cell populations in lymph nodes of control (black), unpulsed DC1 (blue), and HER3-DC1 (red) mice. Data shown are the representative from three independent experiments. H, Lymphocytes from the lymph nodes of control and treated mice were cocultured with DC1 pulsed with individual HER3 or OT-II (negative control) peptides. Culture supernatants were collected after 72 hours, and IFN-γ was measured by ELISA (control: black bar; HER3-DC1: red bar). I, Total protein isolated from in vivo tumor samples was analyzed by Western blotting to compare HER3 protein expression after intratumoral HER3-DC1 (green) administration with respect to the control (black). β-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. Unpaired two-tailed t test was performed to analyze Western blot data. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; # , P ≤ 0.01.

Article Snippet: Immunogenicity of the identified HER3 class II epitopes was confirmed through sensitization of CD4 + 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 (Hemocyanin-Keyhole Limpet Native protein; cat. #SRP6195, Sigma).

Techniques: Control, Negative Control, Enzyme-linked Immunosorbent Assay, Isolation, In Vivo, Western Blot, Expressing, Two Tailed Test

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: Intratumoral HER3-DC1 delays tumor growth and enhances immune infiltration in a HER2 pos 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 = 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, CD3 + CD4 + and CD3 + CD8 + T cells per milligram of tumors from mice ( A ) after intratumoral DC injection was compared between control (black) and HER3-DC1 (red) groups. No statistical analyses were performed for the unpulsed DC1 (blue) mice ( n = 3/group). D, Abundance of CD4 + central memory (CD62L + CD44 + CM), effector memory (CD62L − CD44 + EM), and effector (CD62L − CD44 − 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 CD4 + IFN-γ + ( F ) and CD8 + IFN-γ + ( 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-γ 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. β-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. β-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: Immunogenicity of the identified HER3 class II epitopes was confirmed through sensitization of CD4 + 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 (Hemocyanin-Keyhole Limpet Native protein; cat. #SRP6195, Sigma).

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

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: Vaccination prevents tumor development in a preventive HER3 + melanoma model and diminishes tumor growth and improves survival in a therapeutic HER3 + melanoma model. A, Immunofluorescence for HER3 (red) surface expression in M05 cells. DAPI (blue): nucleus (image magnification: 1,200×). B, HER3 peptide–specific immune responses ( n = 3–5/group). Splenocytes from control (black) and vaccinated (red) mice were restimulated with individual HER3 peptides for 72 hours to detect IFN-γ by ELISA. C, Lymph node lymphocytes from control (black) and HER3-DC1 (red) vaccinated mice were cocultured with individual HER3 peptide–pulsed DCs to detect antigen-specific immune response in HER3-DC1 vaccinated mice by ELISA. D, Preventive model: Two weeks after the last vaccine, C57BL/6 mice ( n = 10 mice/group) were challenged with M05 tumor cells. Tumor growth was monitored until endpoint in control (black) versus HER3-DC1 vaccinated (red) mice. E, Therapeutic setting: C57BL/6 mice were injected subcutaneously with the M05 murine melanoma cells in the left flank, and upon palpable tumor formation on day 10, mice were randomized into four groups ( n = 10 mice/group). Tumor growth was monitored in mice receiving PBS (black), unpulsed DC1 (blue), HER3-DC1 (red), and OT-II peptide–pulsed DC1 (green) intratumorally once weekly for 6 weeks. Tumor growth was compared between control and HER3-DC1 (*) and unpulsed DC1 vs. HER3-DC1 (#) groups. Individual tumor growth curve for each mouse shown on the right. F, Percent survival in M05 mouse model for control (black)-, unpulsed DC1 (blue)–, HER3-DC1 (red)–, and OT-II-DC1 (green)–treated mice. G, Intratumoral infiltration of CD3 + CD4 + and CD3 + CD8 + T cells was compared between control (black) and HER3-DC1 (red) mice from E . H, CD4 + central memory (CD62L + CD44 + CM) and effector memory (CD62L − CD44 + EM) T-cell infiltration per milligram of tumors in control (black) versus HER3-DC1 (red) mice from E . I, Absolute number of CD3 + CD4 + and CD3 + CD8 + T cells in the lymph nodes of control- versus HER3-DC1–treated mice from E . J, Abundance of CD4 + CM, EM, and effector (Eff) T cells in the lymph nodes of control versus treated mice. For G – J , Unpulsed DC1 (blue) and OT-II-DC1 (green) groups were not included in the statistical analyses. K, Lymph node lymphocytes from control and treated mice ( E ) were cocultured with individual HER3 peptide–pulsed DC1 for 72 hours to detect IFN-γ by ELISA. Responses were compared between control and HER3-DC1 groups for HER3 peptides, and control versus OT-II-DC1 groups to OT-II peptide–pulsed DCs. Data shown are the representative from three independent experiments and are 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 ≤ 0.0001; #### , P ≤ 0.0001.

Article Snippet: Immunogenicity of the identified HER3 class II epitopes was confirmed through sensitization of CD4 + 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 (Hemocyanin-Keyhole Limpet Native protein; cat. #SRP6195, Sigma).

Techniques: Immunofluorescence, Expressing, Control, Enzyme-linked Immunosorbent Assay, Injection