mg50714 ut  (Sino Biological)

Journal: Molecular Cancer Therapeutics

Article Title: TPP-45142—an Anti-HER2 T-cell Engager—Designed for Selective HER2-Low Cancer Immunotherapy

doi: 10.1158/1535-7163.MCT-25-0654

Figure Lengend Snippet: TPP-45142 is a bispecific molecule that binds with a novel epitope of HER2. A, Schematic representation of TPP-45142. Green, two HER2-binding NANOBODY domains; orange, anti-TCRαβ NANOBODY domain; and gray, Fc domain with effectorless function. B, Cryo-EM structure of the complex HER2–29E09–Fab was obtained at 2.78 Å resolution. Left, colored electron density map. Right, full model. C, Cryo-EM structure of the 27A05–HER2–47D05–Fab complex was obtained at 2.66 Å resolution. Left, colored electron density map. Center, full model. Right, 27A05–HER2 interface. D, Structural superposition showing the relative location of pertuzumab and trastuzumab (based on PDB 6OGE) versus 27A05 and 29E09 as observed using cryo-EM. E, Structural superposition of 29E09 and 27A05. [ A, Created in BioRender. Vintem, A.P. (2026) https://BioRender.com/lk4spzo .]

Article Snippet: Human, cyno, and mouse HER2 Fc proteins were immobilized on a ProteOn GLC sensor chip (BioRad Laboratories, Inc. cat. #176-5011; 20 μg/mL, 10 mmol/L acetate pH 4.0, 120 seconds, 30 μL/minute).

Techniques: Binding Assay, Cryo-EM Sample Prep

Journal: Molecular Cancer Therapeutics

Article Title: TPP-45142—an Anti-HER2 T-cell Engager—Designed for Selective HER2-Low Cancer Immunotherapy

doi: 10.1158/1535-7163.MCT-25-0654

Figure Lengend Snippet: Cytotoxicity of TPP-45142 and its mechanism of action toward HER2-low breast cancer cell lines. A–D, TDCC of TPP-45142 for three T-cell donors compared with that of non-HER2 negative control (TPP-45161); co-cultures of human T cells with HCC1954, ZR-75-1, BT-20, or BT-549 cells were used at an E:T ratio of 5:1. E, TDCC of TPP-45142 for three T-cell donors compared with that of TPP-45161 in a co-culture of human T cells with BT20 3D spheroids at an E:T ratio of 1:5. F, T-cell activation induced by TPP-45142 as measured by expression of CD25 and CD69 expression on both CD4 + and CD8 + T cells as per FC analysis of ZR-75-1 and BT20 cells. G, Production of IFN-γ, IL2, IL6, IL8, IL10, and TNF-α cytokines in the culture supernatants obtained in the T-cell activation assay was measured using electrochemiluminescence assays.

Article Snippet: Human, cyno, and mouse HER2 Fc proteins were immobilized on a ProteOn GLC sensor chip (BioRad Laboratories, Inc. cat. #176-5011; 20 μg/mL, 10 mmol/L acetate pH 4.0, 120 seconds, 30 μL/minute).

Techniques: Negative Control, Co-Culture Assay, Activation Assay, Expressing, Electrochemiluminescence

Journal: Molecular Cancer Therapeutics

Article Title: TPP-45142—an Anti-HER2 T-cell Engager—Designed for Selective HER2-Low Cancer Immunotherapy

doi: 10.1158/1535-7163.MCT-25-0654

Figure Lengend Snippet: PK profiles and antitumor efficacy of TPP-45142 in the ZR-75-1 HER2-low breast cancer mouse model. A, TPP-45142 PK behavior in the ZR-75-1 xenograft model. Human T cells were administered to female NGS mice bearing intramammary ZR-75-1 tumors, and they were treated once with 89 Zr-TPP-45142 or the non-HER2 negative control 89 Zr-TPP-45161 ( n = 3). Microsamples (5 µL/time point) of blood were collected, and radioactivity was measured extemporaneously using a gamma counter (time: after radiolabeled-compound injection). B, Tumor accumulation of 89 Zr-TPP-45142 or non-HER2 negative control 89 Zr-TPP-45161 as measured by PET/CT imaging ( n = 3; time: after radiolabeled-compound injection). C, Antitumor activity of TPP-45142 in the ZR-75-1 xenograft model. Human T cells (10 × 10 6 ) were administered to female NSG mice bearing ZR-75-1 tumors, and they were treated on days 22 and 29 with TPP-45142 (500, 100, 50, and 10 μg/kg) and non-HER2 negative control TPP-45161 (500 μg/kg; n = 10 per group). ID, injected dose; MAD, median absolute deviation.

Article Snippet: Human, cyno, and mouse HER2 Fc proteins were immobilized on a ProteOn GLC sensor chip (BioRad Laboratories, Inc. cat. #176-5011; 20 μg/mL, 10 mmol/L acetate pH 4.0, 120 seconds, 30 μL/minute).

Techniques: Negative Control, Radioactivity, Injection, Positron Emission Tomography-Computed Tomography, Imaging, Activity Assay

Journal: Molecular Cancer Therapeutics

Article Title: TPP-45142—an Anti-HER2 T-cell Engager—Designed for Selective HER2-Low Cancer Immunotherapy

doi: 10.1158/1535-7163.MCT-25-0654

Figure Lengend Snippet: Safety profile of TPP-45142. A, T cell–mediated lysis of human cardiomyocytes was measured by impedance using xCELLigence. For donor 3, nine doses were tested in the range of 3 × 10 −11 to 5 × 10 −7 mol/L, whereas for donors 1 and 2, only the highest three doses were tested. B and C, HER2 distribution on the cell surface of BT-549 and HCM, respectively, via immunofluorescence. Arrowheads point to large HER2-enriched areas. D, Comparison of HER2 distribution between BT-549 ( B ) and HCM ( C ) cells via frequency distribution analysis of objects sorted by area. E, Heatmap of cytokine concentration as measured using the Luminex multiplex array after MIMIC CRA.

Article Snippet: Human, cyno, and mouse HER2 Fc proteins were immobilized on a ProteOn GLC sensor chip (BioRad Laboratories, Inc. cat. #176-5011; 20 μg/mL, 10 mmol/L acetate pH 4.0, 120 seconds, 30 μL/minute).

Techniques: Lysis, Immunofluorescence, Comparison, Concentration Assay, Luminex, Multiplex Assay

Journal: iScience

Article Title: From byproduct to biotherapeutic: Comparative study of buffy coats and leukoreduction system chambers for NK cell-based immunotherapies

doi: 10.1016/j.isci.2026.114907

Figure Lengend Snippet: Functional characterization of NK cells (A) Degranulation capacity of NK cells following a 2 h co-culture with K562 leukemia cells, measured by surface CD107a expression ( n = 5). (B and C) Europium-based cytotoxicity assay assessing the specific lysis of K562 target cells at E:T ratios of 10:1, 5:1, 3:1, 1:1, and 0.5:1 on day 7 (B) and day 14 (C) of culture to monitor functional decline over time ( n = 6 for both time points). Here, all comparisons were not significant and are summarized as such (n.s.: p > 0.05). (D) IFN-γ release following 16 h of co-culture of NK cells with K562 cells, quantified via Luminex assay ( n = 5). (E) Kinetic live-cell killing assay using the Incucyte monitoring of Nuclight Red signal loss in K562 cells over 72 h at an E:T ratio of 1:1 ( n = 6). (F) Proportion of donors reaching 25%, 50%, and 75% cumulative lysis thresholds during Incucyte co-culture with K562 cells. (G) CD107a degranulation assay of NK cells with or without pre-incubation with trastuzumab (anti-HER2 antibody) during a 2 h co-culture with MDA-MB-453 cells ( n = 5). (H) Incucyte-based cytotoxicity assay shows the real-time lysis of MDA-MB-453 cells by trastuzumab-loaded NK cells at an E:T ratio of 1:1 over 72 h ( n = 6). (I) Proportion of donors reaching 25%, 50%, and 75% cumulative lysis during the ADCC assay. (n varies as not all samples reached the benchmark values within the observation period). All ADCC experiments have been performed after 14 days of in vitro NK cell expansion. N/A indicates that statistical analysis was not possible for this group. 9J) CD107a degranulation of unmodified NK cells and ErbB2-CAR-NK cells after 2 h co-culture with MDA-MB-453 cells ( n = 5). (K) Incucyte-based cytotoxicity assay showing killing dynamics of ErbB2-CAR-NK cells against MDA-MB-453 cells at an E:T ratio of 1:1 over 72 h ( n = 6). (L) Proportion of donors reaching 25%, 50%, and 75% cumulative lysis during the CAR-mediated cytotoxicity assay. (n varies as not all samples reached the benchmark values within the observation period). All CAR-killing experiments have been performed after 14 days of in vitro NK cell expansion. Unless otherwise stated, n refers to biologically independent donor samples and is indicated per group. Data are shown as mean ± SEM. p-values were determined by Student’s t test with Welsh correction (A, D, F, G, I, J, and L) or by one-way ANOVA (B and C) with Tukey post-test (K and L).

Article Snippet: Mouse Monoclonal anti-CD340 (Her2) , Miltenyi Biotec , Cat# 130-124-474.

Techniques: Functional Assay, Co-Culture Assay, Expressing, Cytotoxicity Assay, Lysis, Luminex, Degranulation Assay, Incubation, ADCC Assay, In Vitro

Journal: Advanced Science

Article Title: PD‐L1‐Binding Antigen Presenters: Redirecting Vaccine‐Induced Antibodies for Cancer Immunotherapy

doi: 10.1002/advs.202519574

Figure Lengend Snippet: PBAP Conjugated with Tumor‐Specific Antigens Enhances Synergistic Anti‐Tumor Activity When Combined with Clinical Antibodies and Antibody‐Drug Conjugates (ADCs) In Vitro. A) Schematic representation of the design of sPD‐1‐HER2 and PBAP‐HER2 (sPD‐1‐HER2‐Fc). PBAP‐HER2 was engineered via the fusion of extracellular domain of human PD‐1 (sPD‐1) with Domain IV of HER2 protein, followed by the incorporation of an Fc region to enhance protein stability and prolong in vivo half‐life. vB) Structural modeling of PBAP‐HER2 with AlphaFold 3. (C) Pharmacokinetic profiles of sPD‐1‐HER2 and PBAP‐HER2 following intravenous injection into C57BL/6J mice (n=3 mice/group, 100 µg/mice). Data are presented as the mean ± SD (n = 3). (D) The binding inhibition of PBAP‐HER2 on PD‐L1/PD‐1 interaction was assessed by ELISA. The absorbance was measured at 450 nm to determine the blocking effect. Data are presented as the mean ± SD (n = 3). (E) The fluorescence intensity of the antibody‐cell binding was analyzed using a flow cytometry to assess the blocking effect on the PD‐1/PD‐L1 pathway. (F) Diagram illustrating the mechanism by which PBAP‐HER2 synergizes with Herceptin and Kadcyla to kill PD‐L1‐positive target cells. Created with BioRender.com. (G) ADCC and ADCP activities were assessed using Jurkat‐FcγR reporter systems: ADCC (FcγRIIIa‐V158 variant) and ADCP (FcγRIIa‐R131 variant) in response to PBAP‐HER2/PBAP‐gE combined with Herceptin. PBAP‐HER2 in combination with Herceptin significantly enhanced ADCC and ADCP activities against HER2‐negative MDA‐MB‐231 cells. Representative of 3 independent experiments. Data are presented as mean ± SD (n = 3). (H) NK cells were co‐incubated with PBAP‐Her2 and Herceptin, against MDA‐MB‐231‐IFN‐γ (IFN‐γ induced, PD‐L1 + ) tumor cells and MDA‐MB‐231‐WT cells. NK cells, NK cells co‐incubated with PBAP‐Her2, NK cells co‐incubated with Herceptin, and all groups treated with anti‐FcγRIII blocking antibody were used as controls. Data are presented as mean ± SD of 3 independent experiments, each performed in triplicate. (I) Flow cytometry analysis of perforin, granzyme B, IFN‐γ and CD107a in NK cells. Representative of 3 independent experiments. (J) The CCK8 assay was used to evaluate the cytotoxicity of commercial ADCs (Kadcyla and Adcetris) combined with PBAP‐HER2. MDA‐MB‐231‐PD‐L1‐OE cells were treated with PBAP‐HER2 (10 µg/well) for 4 h, followed by ADC drugs (Kadcyla or Adcetris) at various concentrations (0.1, 1, 10, 100, 1000 ng/mL). After 24 h of incubation, cell viability was measured using the CCK8 assay. PBAP‐HER2 with Adcetris and HER2 protein with Kadcyla were used as controls. Representative of 3 independent experiments. Data are presented as mean ± SD (n = 3).

Article Snippet: For the ELISA blocking activity detection of PBAP‐gE, the same procedure was followed with the following modifications: human PD‐L1 (ECD, His Tag, SinoBiological, 10084‐H08H) protein was replaced with mouse PD‐L1 (ECD, His Tag, SinoBiological, 50010‐M08H), PBAP‐HER2 (1 μg/well and 10 μg/well) was replaced with PBAP‐gE (1 μg/well and 10 μg/well), PD‐L1 monoclonal antibody (SinoBiological, 10084‐MM33) was replaced with PD‐L1 monoclonal antibody (SinoBiological, B50010 ‐R678), and Goat anti‐Mouse IgG, HRP secondary antibody (SinoBiological, SSA007) was replaced with Goat anti‐Rabbit IgG, HRP secondary antibody (SinoBiological, SSA004).

Techniques: Activity Assay, In Vitro, In Vivo, Injection, Binding Assay, Inhibition, Enzyme-linked Immunosorbent Assay, Blocking Assay, Fluorescence, Flow Cytometry, Variant Assay, Incubation, CCK-8 Assay

Journal: Advanced Science

Article Title: PD‐L1‐Binding Antigen Presenters: Redirecting Vaccine‐Induced Antibodies for Cancer Immunotherapy

doi: 10.1002/advs.202519574

Figure Lengend Snippet: PBAP‐HER2 Synergizes with Antibody‐Drug Conjugates to Enhance Anti‐tumor Efficacy in NSG Mice Bearing Subcutaneous Tumors. (A) Overview of Experimental Design. NSG mice were subcutaneously inoculated with MDA‐MB‐231 cells. Once the tumors reached approximately 100 mm 3 , mice were assigned to one of four treatment groups (n=5 mice/group): PBAP‐HER2 alone, Kadcyla alone, PBAP‐HER2 + Adcetris, and PBAP‐HER2 + Kadcyla. PBAP‐HER2 (150 µg/mouse) was administered intraperitoneally, followed by tail vein injections of Kadcyla (3 mg/kg) or Adcetris (3 mg/kg) 24 h later. Treatments were administered once a week for two consecutive cycles, with tumor growth monitored throughout the study. At the experimental endpoint, mice were euthanized, and tumor and tissue samples were collected for further analysis. Created with BioRender.com. (B) Representative tumor images for each experimental group are displayed in the left panel, with tumor volumes at the experimental endpoint shown in the right panel. Notably, the PBAP‐HER2 + Kadcyla group exhibited the most significant tumor regression, accompanied by robust tumor control, relative to all other groups. Data are presented as the mean ± SD (n = 5). Statistical significance was determined using one‐way ANOVA. Statistically significant differences were observed (p < 0.05). (C) Immunohistochemistry (IHC) analysis was performed to evaluate the intratumoral infiltration of PBAP‐HER2 and ADC drugs. The results demonstrated that PBAP‐HER2 effectively infiltrated the tumor tissue. Furthermore, Kadcyla was found to exhibit intratumoral infiltration exclusively when co‐administered with PBAP‐HER2. In contrast, Adcetris failed to infiltrate tumor tissues in the PBAP‐HER2 plus Adcetris combination group. Scale bars, 50 µm. (D) Immunofluorescence analysis further confirmed the specific efficacy of the PBAP‐HER2 + Kadcyla combination. Tumor sections revealed clear co‐localization of PBAP‐HER2 with PD‐L1 on tumor cells. Kadcyla was observed to enter tumor cells exclusively in the PBAP‐HER2 + Kadcyla group. In contrast, no intracellular ADC uptake was detected in the control groups (PBAP‐HER2 + Adcetris or Kadcyla only). Scale bars, 20 µm. (E) H&E staining showed no significant histopathological damage to major organs (heart, liver, spleen, lungs) in the experimental group, indicating a favorable safety profile. An increased presence of multinucleated giant cells was observed in the spleens, particularly in the PBAP‐HER2 + Kadcyla group, as indicated by white arrows. Scale bars, 60 µm.

Article Snippet: For the ELISA blocking activity detection of PBAP‐gE, the same procedure was followed with the following modifications: human PD‐L1 (ECD, His Tag, SinoBiological, 10084‐H08H) protein was replaced with mouse PD‐L1 (ECD, His Tag, SinoBiological, 50010‐M08H), PBAP‐HER2 (1 μg/well and 10 μg/well) was replaced with PBAP‐gE (1 μg/well and 10 μg/well), PD‐L1 monoclonal antibody (SinoBiological, 10084‐MM33) was replaced with PD‐L1 monoclonal antibody (SinoBiological, B50010 ‐R678), and Goat anti‐Mouse IgG, HRP secondary antibody (SinoBiological, SSA007) was replaced with Goat anti‐Rabbit IgG, HRP secondary antibody (SinoBiological, SSA004).

Techniques: Control, Immunohistochemistry, Immunofluorescence, Staining