Journal: JCI Insight

Article Title: Trastuzumab/pertuzumab combination therapy stimulates antitumor responses through complement-dependent cytotoxicity and phagocytosis

doi: 10.1172/jci.insight.155636

Figure Lengend Snippet: ( A ) HER2 + tumor model cell-lines (KPL-4, Au565, SKOV3, BT-474, 3T3-HER2) were treated with 20 μg/mL of HER2 mAbs, as indicated, and cultured in vitro for 6 days. Cell viability were analyzed by CellTiter-Glo assay. n = 6. ( B ) KPL-4 cells were implanted into mammary fat pads of SCID-beige Balb/c mice (5 × 10 5 cells). HER2 mAbs (100 μg each) or control human IgG1 were administered weekly starting on day 21, and tumor volume was measured. n = 10. ( C ) SKOV3 (HER2 + /HER3 – ovarian cancer line) were implanted into the flank of SCID-beige Balb/c mice (1 × 10 6 cells each). HER2 mAbs (100 μg each) were administered weekly. n = 5. ( D ) NIH/3T3 cells stably expressing HER2 were implanted into the flank of SCID-beige Balb/c mice (2 × 10 5 cells). HER2 mAbs (200 μg each) or control human IgG1 were administered weekly. n = 5. ( A – D ) Data are shown as mean ± SEM. Two-way ANOVA with Tukey’s multiple-comparison post hoc test; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ( E ) Experiment using an immunocompetent, human HER2Δ16 transgenic mouse model treatment with murinized HER2 mAbs. Spontaneous breast tumors in the transgenic mice were induced with doxycycline diet. Four treatment arms were set up: Control mouse IgG (200 μg weekly, n = 15), 4D5-IgG2A (murinized Trastuzumab, 200 μg weekly, n = 16), 2C4-IgG2A (murinized Pertuzumab, 200 μg weekly, n = 10), and 4D5-IgG2A combined with 2C4-IgG2A ( n = 14). Individual animals were consecutively enrolled into a specific treatment arm as soon as palpable breast tumors were detected (~100 mm 3 ). Day 0 is the day of palpable tumor detection and treatment enrollment. Log-rank (Mantel-Cox) test for survival analysis, **** P < 0.0001 of treatment group vs control group, ## P < 0.01 significant difference observed between 4D5-IgG2A and 4D5-IgG2A + 2C4-IgG2A groups.

Article Snippet: Human HER2 + BC cell lines (BT-474, SKBR3, and Au565) and HER2 + ovarian cancer cell line SKOV3 were obtained from ATCC and cultured as described by their ATCC protocol.

Techniques: Cell Culture, In Vitro, Glo Assay, Control, Stable Transfection, Expressing, Comparison, Transgenic Assay

Journal: JCI Insight

Article Title: Trastuzumab/pertuzumab combination therapy stimulates antitumor responses through complement-dependent cytotoxicity and phagocytosis

doi: 10.1172/jci.insight.155636

Figure Lengend Snippet: ( A and B ) Mouse FCGR signaling activation assay. KPL-4 cells were plated and treated with indicated HER2 mAbs concentrations for 1 hour. Jurkat cells containing NFAT-luciferase reporter and expressing mouse FCGR1 ( A ) and FCGR4 ( B ) were added to the KPL-4 cells containing antibodies and cocultured for 4 hours. FCGR signaling activation were assessed by luciferase activity quantification. ( C and D ) Human FCGR signaling activation was similarly quantified with Jurkat-NFAT-Luciferase reporter cells expressing human FCGR1 ( C ) and FCGR3A ( D ). ( E – G ) Mouse FCGR signaling activation assays were repeated using murinized HER2 mAbs with the IgG2A isotype. In addition to mFCGR1 ( E ) and mFCGR4 ( F ), mouse FCGR3 ( G ) was tested here since mFCGR3 can be activated by murine antibodies but not human antibodies. All data represents mean ± SEM; n = 4. ( H – J ) Antigen-binding-fragment F(ab’) 2 of the HER2 mAbs were generated and their therapeutic efficacy against HER2 + BC in vivo were compared with the parental antibody, respectively. As described before, KPL-4 cells (5 × 10 5 cells) were implanted in mammary fat pads of SCID-beige mice and treated with the indicated HER2 mAbs or F(ab’) 2 (100 μg per week). ( H ) Comparison between Trastuzumab versus Trastuzumab-F(ab’) 2 . ( I ) Comparison between Pertuzumab versus Pertuzumab-F(ab’) 2 . ( J ) Comparison between T+P versus Trastuzumab + Pertuzumab-F(ab’) 2 versus Trastuzumab-F(ab’) 2 + Pertuzumab. Numbers of mice showing total tumor regression are displayed on the right of the graph. ( H – J ) n = 8–10 for all groups. Two-way ANOVA with Tukey’s multiple-comparison post hoc test. All data represent mean ± SEM; * P < 0.05, *** P < 0.001, **** P < 0.0001.

Article Snippet: Human HER2 + BC cell lines (BT-474, SKBR3, and Au565) and HER2 + ovarian cancer cell line SKOV3 were obtained from ATCC and cultured as described by their ATCC protocol.

Techniques: Activation Assay, Luciferase, Expressing, Activity Assay, Binding Assay, Generated, Drug discovery, In Vivo, Comparison

Journal: JCI Insight

Article Title: Trastuzumab/pertuzumab combination therapy stimulates antitumor responses through complement-dependent cytotoxicity and phagocytosis

doi: 10.1172/jci.insight.155636

Figure Lengend Snippet: ( A ) Representative FACS quantification plots of human complement 3b (C3b) surface deposition on KPL-4 cells after 30 minutes of incubation with normal human serum with indicated HER2 mAbs. ( B ) Graphical summary of C3b depositions staining described in A , confirmed on several HER2 + BC lines: KPL-4, BT-474, and SKBR3. n = 3; 1-way ANOVA with Tukey’s multiple-comparison post hoc test. Assay were repeated using normal human serum from 3 different donor sources. ( C ) Complement-dependent cytotoxicity (CDC) killing assays were performed on HER2 + cell lines in vitro (BT-474, KPL-4, NIH-3T3-HER2, MM3MG-HER2Δ16). Tumor cells were incubated with the indicated HER2 mAbs and with 25% of non-heat-inactivated normal rabbit serum. Cell viability were analyzed 2–4 hours after serum incubation, using CellTiter-Glo luminescent assay. Heat-inactivated (HI) serum was used as negative control. n = 3–4; 1-way ANOVA with Tukey’s multiple-comparison post hoc test. All data represent mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Human HER2 + BC cell lines (BT-474, SKBR3, and Au565) and HER2 + ovarian cancer cell line SKOV3 were obtained from ATCC and cultured as described by their ATCC protocol.

Techniques: Incubation, Staining, Comparison, In Vitro, Luminescence Assay, Negative Control

Journal: JCI Insight

Article Title: Trastuzumab/pertuzumab combination therapy stimulates antitumor responses through complement-dependent cytotoxicity and phagocytosis

doi: 10.1172/jci.insight.155636

Figure Lengend Snippet: Quantification of C3 and C5 depositions on HER2 + BC cells in vivo. KPL-4 cells were labeled with Vybrant DiD dye and implanted into SCID-beige mice. Once tumor volume reaches ~300 mm 3 , mice were treated with HER2 mAbs (200 μg each) or control IgG. The next day, tumors were harvested and surface stained for mouse C3 and C5, and quantified by FACS. C3 + staining was gated on live CD45 – DiD + cells. C5 + staining was gated on dying CD45 – cells. ( A ) Representative FACS quantification plot of C3 deposition on live KPL-4 tumor cells in vivo. ( B ) Representative FACS quantification plots of C5 staining on dying KPL-4 tumor cells in vivo. ( C ) Representative FACS quantification plots of dying KPL-4 tumor cells in vivo. ( D ) Summary of in vivo mouse C3 deposition on KPL-4 tumors. ( E ) Summary of in vivo mouse C5 deposition on KPL-4 tumors. ( F ) Summary of dying KPL-4 tumors cells frequency from above experiment. ( D – F ) n = 7–8; 1-way ANOVA with Tukey’s multiple-comparison post hoc test. All data represent mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Human HER2 + BC cell lines (BT-474, SKBR3, and Au565) and HER2 + ovarian cancer cell line SKOV3 were obtained from ATCC and cultured as described by their ATCC protocol.

Techniques: In Vivo, Labeling, Control, Staining, Comparison

Journal: JCI Insight

Article Title: Trastuzumab/pertuzumab combination therapy stimulates antitumor responses through complement-dependent cytotoxicity and phagocytosis

doi: 10.1172/jci.insight.155636

Figure Lengend Snippet: ( A ) HER2 mAb–induced antibody-dependent cellular phagocytosis (ADCP) of HER2 + BC cells by mouse BM-derived macrophages (BMDM) in vitro. KPL-4 or BT-474 cells were labeled with Brilliant Violet 450 Dye (BV450), and cocultured with BMDM with control or HER2 mAbs (10 μg/mL). ADCP rates were measured by percentage of BMDM uptake of labeled tumor cells (CD45 + and BV450 + ). n = 3. ( B ) In vivo ADCP experiment. KPL-4 cells were labeled with Vybrant DiD dye and implanted (1 × 10 6 cells) into mammary fat pads of SCID-beige mice. Once tumor volume reached ~300 mm 3 , mice were treated with HER2 mAbs (200 μg each) or control IgG. The next day, tumors were harvested and analyzed by FACS. TAMs that had phagocytosed DiD-labeled tumor cells were quantified for ADCP. ( C ) Tumor-associated macrophages (TAMs), identified as CD11b + F480 + LY6G – LY6C – , were quantified. n = 7–8; 1-way ANOVA with Tukey’s multiple-comparison post hoc test. ( D ) HER2 mAb–induced C3b deposition is dependent on C1q activation. KPL-4 cells were incubated with HER2 mAbs and NHS to promote C3b deposition as described in Figure 3. Where indicated, cells were treated with or without C1 inhibitor (C1-INH) to inhibit C1 activation and C3b deposition. n = 3. ( E ) C3b-opsonized antibody-bound KPL-4 in D were cocultured with human macrophages to assess complement-enhanced ADCP. n = 3. ( F ) KPL-4 cells were cultured with BMDMs derived from WT or C1q-deficient mice, and ADCP activity with HER2 mAbs were quantified as before. n = 3. ( A and D – F ) Data are shown as mean ± SEM, 2-way ANOVA with Tukey’s multiple-comparison post hoc test.

Article Snippet: Human HER2 + BC cell lines (BT-474, SKBR3, and Au565) and HER2 + ovarian cancer cell line SKOV3 were obtained from ATCC and cultured as described by their ATCC protocol.

Techniques: Derivative Assay, In Vitro, Labeling, Control, In Vivo, Comparison, Activation Assay, Incubation, Cell Culture, Activity Assay

Journal: JCI Insight

Article Title: Trastuzumab/pertuzumab combination therapy stimulates antitumor responses through complement-dependent cytotoxicity and phagocytosis

doi: 10.1172/jci.insight.155636

Figure Lengend Snippet: ( A ) Immunodeficient C1q-KO mice (SCID/C1q –/– ) were implanted with KPL-4 cells in the mammary fat pads (5 × 10 5 cells). HER2 mAbs (100 μg each) or control human IgG1 was administered weekly, and tumor volume was measured. Data are shown as mean ± SEM. Control IgG group, n = 5; Trastuzumab group, n = 14; T+P group, n = 14. ( B ) Same experiment as in A was repeated in SCID/C1q +/+ mice. Data are shown as mean ± SEM. Control IgG group, n = 5; Trastuzumab group, n = 14; T+P group, n = 14. ( C ) KPL-4 cells were implanted into mammary fat pads of SCID-beige mice as before (1 × 10 6 cells). When average tumor volume reached ~100 mm 3 , mice were divided into 4 treatment groups: Control IgG, T+P (100 μg each, weekly), C1-INH (200 μg, twice per week), or T+P and C1-INH. Data are shown as mean ± SEM; n = 10 for all groups. ( A – C ) Two-way ANOVA with Tukey’s multiple-comparison post hoc test. All data represent mean ± SEM; * P < 0.05, *** P < 0.001. ( D ) The prognostic value of C1QA expression in BC patients. BC patients in KM plotter database were classified into HER2 + , Luminal A, Luminal B, and Basal subtypes using St. Gallen classification. For each subtype, patients were split into 2 groups by their expression levels of C1QA , and the overall survival (OS) was compared between patients with high and low C1QA gene expression.

Article Snippet: Human HER2 + BC cell lines (BT-474, SKBR3, and Au565) and HER2 + ovarian cancer cell line SKOV3 were obtained from ATCC and cultured as described by their ATCC protocol.

Techniques: Control, Comparison, Expressing, Gene Expression

Journal: JCI Insight

Article Title: Trastuzumab/pertuzumab combination therapy stimulates antitumor responses through complement-dependent cytotoxicity and phagocytosis

doi: 10.1172/jci.insight.155636

Figure Lengend Snippet: ( A and B ) The prognostic value of CD55 and CD59 expression in breast cancer patients. BC patients in KM plotter database were classified into HER2 + , Luminal A, Luminal B, and Basal subtypes using St. Gallen classification. For each subtype, patients were split into 2 groups by their expression levels of CD55 or CD59 , and the relapse-free survival (RFS) was compared between patients with high and low gene expression. ( C ) Control KPL-4 or CD55/CD59 double-knockdown KPL-4 cells were implanted into mammary fat pads of SCID-beige mice (5 × 10 5 cells). When average tumor volume reached ~100 mm 3 , mice were divided into 2 treatment groups: Control IgG and T+P (25 μg each, weekly). Data are shown as mean ± SEM; n = 9 for all groups. Two-way ANOVA with Tukey’s multiple-comparison post hoc test. ( D – F ) In vivo complement deposition and killing of control and knockdown KPL-4 tumors from C . Data are shown as mean ± SEM; n = 6–9 for all groups. One-way ANOVA with Tukey’s multiple-comparison post hoc test. ( D ) Summary of in vivo C3 deposition. ( E ) Summary of in vivo C5 deposition. ( F ) Summary of dying KPL-4 tumor cell frequency. All data are shown as mean ± SEM; ** P < 0.01, **** P < 0.0001.

Article Snippet: Human HER2 + BC cell lines (BT-474, SKBR3, and Au565) and HER2 + ovarian cancer cell line SKOV3 were obtained from ATCC and cultured as described by their ATCC protocol.

Techniques: Expressing, Gene Expression, Control, Knockdown, Comparison, In Vivo