Journal: JCI Insight

Article Title: Target arm affinities determine preclinical efficacy and safety of anti-HER2/CD3 bispecific antibody

doi: 10.1172/jci.insight.133757

Figure Lengend Snippet: (A) Representation of anti-HER2/CD3 TDB. (B) Binding to human CD8+ cells was analyzed by flow cytometry (n = 1). (C) TDB-induced (1 μg/mL TDBs) TCR signaling pathway activation in splenic CD8+ cells extracted from huCD3 transgenic mice was analyzed by phos–SLP-76 Western blot (see complete unedited blots in the supplemental material; lanes were run on the same gel but were noncontiguous; n = 1). (D) TDB-induced activation of human CD8+cells was analyzed by flow cytometry (n = 1). (E) In vitro killing activity of HER2-amplified SKBR3 (solid lines) and low-HER2–expressing MCF7 (dotted lines) cells was analyzed by Cell Titer Glo viability assay (n = 3). Data presented as mean ± SD.

Article Snippet: Recombinant human (Genentech) and cynomolgus monkey (Sino Biologicals) HER2 extracellular domain (ECD) were used to determine the binding affinity of anti-HER2/CD3 TDB for HER2 ECD by surface plasmon resonance (SPR) technology using a Biacore T200 instrument (GE Healthcare).

Techniques: Binding Assay, Flow Cytometry, Activation Assay, Transgenic Assay, Western Blot, In Vitro, Activity Assay, Amplification, Expressing, Viability Assay

Journal: JCI Insight

Article Title: Target arm affinities determine preclinical efficacy and safety of anti-HER2/CD3 bispecific antibody

doi: 10.1172/jci.insight.133757

Figure Lengend Snippet: (A) Individual tumor volume response of HER2-amplified KPL4 breast cancer xenografts to HER2–TDB 2 (higher CD3 affinity; groups 4–7) and HER2–TDB 1 (lower CD3 affinity; groups 8–11) in NSG mice supplemented with human PBMCs. Mice with established tumors received a single i.v. dose at day 0 at indicated dose levels. Trellis plots of individual and fitted tumor volumes are presented, with study day on the x axis and tumor volume on the y axis. Each panel in the trellis depicts 1 dose group (panel headers indicate group numbers). Bold, solid black lines indicate the fitted tumor volume for each dose group. Dashed blue lines indicate the fitted tumor volume for the control group (vehicle, histidine buffer). Gray lines indicate the tumor response over time in individual animals present through the course of the study. Red lines indicate the tumor response over time in mice that were removed from the study due to tumor progression beyond prespecified limits. n = 8–9 for each treatment group. (B and C) Tumor-bearing MMTVhuHER2.huCD3.TG mice were treated with a single dose of HER2–TDB 2 (red), HER2–TDB 1 (blue), or vehicle (black) at day 0. (B) Effect of 0.5 mg/kg anti-HER2/CD3 TDB on tumor growth. n = 6–11 for each treatment group. (C) Effect of 0.25 mg/kg (filled symbols) or 0.5 mg/kg (open symbols) dose on T cell activation and proliferation markers on tumor-infiltrating CD8+ cells analyzed by flow cytometry 6 days after dose. Error bars represent mean ± SEM. n = 5–8 for each treatment group. Statistical analysis using unpaired t test with Welch’s correction.

Article Snippet: Recombinant human (Genentech) and cynomolgus monkey (Sino Biologicals) HER2 extracellular domain (ECD) were used to determine the binding affinity of anti-HER2/CD3 TDB for HER2 ECD by surface plasmon resonance (SPR) technology using a Biacore T200 instrument (GE Healthcare).

Techniques: Amplification, Activation Assay, Flow Cytometry

Journal: JCI Insight

Article Title: Target arm affinities determine preclinical efficacy and safety of anti-HER2/CD3 bispecific antibody

doi: 10.1172/jci.insight.133757

Figure Lengend Snippet: (A) To analyze cytokine release in vitro, human healthy donor whole blood was cocultured with MCF7 cells and HER2–TDB 1 (lower CD3 affinity; blue) or HER2–TDB 2 (higher CD3 affinity; red) for 20 hours, and cytokine levels in media were analyzed by Bio-Plex Pro Human Cytokine Assay. Data presented as mean (n = 3) ± SD. (B and C) Tumor-bearing MMTVhuHER2.huCD3.TG mice were treated with 0.5 mg/kg dose of indicated anti-HER2/CD3 variant (n = 6–7). (B) Serum cytokines were analyzed at indicated time points by Luminex. (C) Body weight change after TDB treatment (n = 6–9). Statistical analysis (unpaired t test with Welch’s correction) of body weights 4 days after treatment are presented in the right panel. Error bars represent mean ± SEM. (D) In vivo cytokine levels from cynomolgus monkeys treated with a single dose of 0.5 mg/kg. Data points represent individual animals (n = 2 for TDB 1; n = 1 for TDB 2).

Article Snippet: Recombinant human (Genentech) and cynomolgus monkey (Sino Biologicals) HER2 extracellular domain (ECD) were used to determine the binding affinity of anti-HER2/CD3 TDB for HER2 ECD by surface plasmon resonance (SPR) technology using a Biacore T200 instrument (GE Healthcare).

Techniques: In Vitro, Cytokine Assay, Variant Assay, Luminex, In Vivo

Journal: JCI Insight

Article Title: Target arm affinities determine preclinical efficacy and safety of anti-HER2/CD3 bispecific antibody

doi: 10.1172/jci.insight.133757

Figure Lengend Snippet: (A) SKBR3 cells were treated with HER2 affinity variants of anti-HER2/CD3 TDB. (B) CHO cells were transfected to express cyno-HER2 and treated with HER2–TDB 1 (red) or HER2–TDB 3 (blue). Viability was measured using Cell Titer Glo. Data presented as mean ± SD (n = 3).

Article Snippet: Recombinant human (Genentech) and cynomolgus monkey (Sino Biologicals) HER2 extracellular domain (ECD) were used to determine the binding affinity of anti-HER2/CD3 TDB for HER2 ECD by surface plasmon resonance (SPR) technology using a Biacore T200 instrument (GE Healthcare).

Techniques: Transfection

Journal: JCI Insight

Article Title: Target arm affinities determine preclinical efficacy and safety of anti-HER2/CD3 bispecific antibody

doi: 10.1172/jci.insight.133757

Figure Lengend Snippet: Cynomolgus monkeys were treated with 0.5 and 1.5 mg/kg of HER2–TDB 1 (lower HER2 affinity; blue and magenta, respectively) or HER2–TDB 3 (higher HER2 affinity; red and green, respectively) on days 0 and 7. (A–C) Peripheral blood was sampled at indicated time points and analyzed for T cell activation (CD69) (A), systemic cytokine levels (B), and number of CD8+ and CD4+ lymphocytes (C). Data are presented as mean ± SEM (A and C) or individual animals (B). Arrowheads indicate time of dosing (A and C) or point out individual animals where dosing was not tolerated (3503 and 4502) or tolerated (4503 and 4504) (B). n = 3–4 for each treatment group.

Article Snippet: Recombinant human (Genentech) and cynomolgus monkey (Sino Biologicals) HER2 extracellular domain (ECD) were used to determine the binding affinity of anti-HER2/CD3 TDB for HER2 ECD by surface plasmon resonance (SPR) technology using a Biacore T200 instrument (GE Healthcare).

Techniques: Activation Assay

Journal: JCI Insight

Article Title: Target arm affinities determine preclinical efficacy and safety of anti-HER2/CD3 bispecific antibody

doi: 10.1172/jci.insight.133757

Figure Lengend Snippet: (A) Effect of dose fractionation of HER2–TDB 3 on systemic exposure in cynomolgus monkey. Animals in group 1A (0.2 mg/kg on days 0 and 1, 0.4 mg/kg on day 7; n = 1) and group 1B (0.1 mg/kg on days 0 and 1, 0.4 mg/kg on day 7; n = 2) were dosed using dose fractionation. Animals in group 2 (n = 4) received 0.5 mg/kg on days 0 and 7. Blood samples were collected at indicated time points, and human IgG was detected by ELISA. Data are presented as mean ± SD for group 2 and as exposure for individual animal for group 1. PK parameters are presented in Supplemental Table 1. (B) Serum cytokine analysis using Luminex from cynomolgus monkeys dosed using the dose fractionation strategy (n = 3, individual animal data depicted). (C) Individual tumor volume response of HER2-amplified KPL4 breast cancer xenografts to anti-HER2/CD3 TDB in NSG mice supplemented with human PBMCs. Mice were treated with 0.05 mg/kg (once a week x2) dose (blue). Alternatively, the initial dose was fractionated to 2 doses of 0.025 mg/kg administered on days 0 and 1 (red). n = 8–9 for each dose group. Arrows indicate time of dosing.

Article Snippet: Recombinant human (Genentech) and cynomolgus monkey (Sino Biologicals) HER2 extracellular domain (ECD) were used to determine the binding affinity of anti-HER2/CD3 TDB for HER2 ECD by surface plasmon resonance (SPR) technology using a Biacore T200 instrument (GE Healthcare).

Techniques: Fractionation, Enzyme-linked Immunosorbent Assay, Luminex, Amplification

Journal: JCI Insight

Article Title: Target arm affinities determine preclinical efficacy and safety of anti-HER2/CD3 bispecific antibody

doi: 10.1172/jci.insight.133757

Figure Lengend Snippet: (A) In the 2-tumor model, each NSG mouse was implanted with HER2-amplified KPL4 tumors and HT55 tumors, which express low levels of HER2. Mice were further supplemented with human PBMCs by i.p. injection. HER2 expression was analyzed by Western blot. Lanes were run on the same gel but were noncontiguous (n = 1). (B) Mice with 2 established tumors were treated with single 0.05 mg/kg dose of HER2–TDB 1. n = 5 for each group. Data represented as mean ± SEM.

Article Snippet: Recombinant human (Genentech) and cynomolgus monkey (Sino Biologicals) HER2 extracellular domain (ECD) were used to determine the binding affinity of anti-HER2/CD3 TDB for HER2 ECD by surface plasmon resonance (SPR) technology using a Biacore T200 instrument (GE Healthcare).

Techniques: Amplification, Injection, Expressing, Western Blot