Anti‐PD‐1/Her2 Bispecific Antibody IBI315 Enhances the Treatment Effect of Her2‐Positive Gastric Cancer through Gasdermin B‐Cleavage Induced Pyroptosis

Abstract The majority of patients with human epidermal growth factor receptor 2 (Her2)‐positive gastric cancer develop refractory to Her2‐targeted therapy, where upregulation of immune checkpoints plays an essential role. Herein, a recombinant fully human IgG1 bispecific antibody IBI315 targeting both PD‐1 and Her2 is developed and its antitumor efficacy as well as the underlying mechanism is investigated. IBI315 crosslinks the physical interaction between Her2‐positive tumor cells and PD‐1‐positive T cells, resulting in significantly enhanced antitumor effects compared to each parent antibody or their combination, both in vitro and in vivo mouse tumor models reconstituted with human immune cells using patient‐derived xenografts and organoids. Moreover, IBI315 treatment also induces the recruitment and activation of immune cells in tumors. Mechanistically, IBI315 triggers gasdermin B (GSDMB)‐mediated pyroptosis in tumor cells, leading to the activation and recruiments of T cells. The activated T cells secret IFNγ, enhancing GSDMB expression and establishing a positive feedback loop of T cell activation and tumor cell killing. Notably, GSDMB is found to be elevated in Her2‐positive gastric cancer cells, providing a rationale for IBI315's efficacy. IBI315 is supported here as a promising bispecific antibody‐based immunotherapy approach for Her2‐positive gastric cancer in preclinical studies, broadening the therapeutic landscape of this patient population.


Figure S1
Elevated PD-1 expression on T cells following co-culture with gastric cancer cells.After co-culture with N87 cells or alone for 3 days, CD8 + T cells were analyzed for the proportion of PD-1 + cells.A) Representative flow cytometry plots, B) statistical analysis of the data.Statistical significance is indicated as ****P<0.0001.

Figure S2
Establishment tumor cells or organoids/T cells co-culture cytotoxicity system.A) Schematic diagram of the system.PBMCs were activated with anti-CD3 antibody, anti-CD28 antibody, and IL-2 for 7-10 days, and T cells were subsequently enriched using magnetic beads.T cells were then co-cultured with gastric cancer cells or organoids at an effector-to-target ratio of 10:1, with the addition of indicated antibodies.After 24 hours, the supernatant was collected to measure the level of LDH, and the cytotoxicity of each group was compared (detailed calculation methods are described in the experimental section).B) Morphological changes of PBMCs after activation were observed under a microscope.Figure S14 GSDMB expression as a predictive biomarker for immunotherapy response.The TIDE database was used to evaluate the predictive value of GSDMB expression for immunotherapy response in various cancer types.The area under the curve (AUC) was calculated for each cohort.TIDE is an algorithm that analyzes the association between gene expression and T cell function with treatment outcomes.Cohorts without GSDMB transcription data were excluded from the analysis.

Figure S3
Figure S3 IBI315 mediates T cell-mediated cytotoxicity against Her2-positive gastric cancer cells.A-D) Her2-positive gastric cancer cells (N87 and SNU-216) were pre-labeled with CFSE and treated with IBI315, its parent antibody, or their combination for 24 hours in the presence of T cells.Representative images and quantification of tumor cell death were analyzed by flow cytometry, and the percentage of PI + tumor cells (PI + /CFSE + ) were measured in N87/T cells (A, B) and SNU-216/T cells (C, D) co-culture systems.Statistical significance is indicated as **P<0.01.

Figure S4
Figure S4 IBI315 retains the ADCC activity of its parental trastuzumab.A-B) N87 cells were treated with IBI315, its parent antibody, or their combination in the presence of PBMCs or NK cells.Quantification of tumor cell death by measuring LDH release as indicated treatment of N87/PBMCs co-culture system is presented (A).Comparison of NK cell-mediated cytotoxicity of N87 cells by indicated treatment (B).Statistical significance is indicated as *P<0.05 and **P<0.01.

Figure S5
Figure S5 IBI315 mediates T cell-mediated cytotoxicity against Her2-positive breast cancer cells.A-B) Her2-positive breast cancer cells (BT-474 and SK-BR-3) were treated with IBI315, its parent antibody, or their combination for 24 hours in the presence of T cells.Quantification of tumor cell death by measuring LDH release as indicated treatment of BT-474/T cells (A) and SK-BR-3/T cells (B) co-culture system is presented.Statistical significance is indicated as **P<0.01.

Figure S6
Figure S6 IBI315 elicits a minimal cytokine release syndrome.A) Comparative assessment of cytokine levels in the supernatant of human PBMCs treated with IgG (765nM), anti-CD3 antibody (positive control, 30nM), anti-PD-1 antibody (765nM), and IBI315 (765nM) for 24 and 48 hours.B-D) Serial dilutions of anti-PD-1 antibody and IBI315 were applied to human PBMCs for 24 and 48 hours, and the supernatant was analyzed for IL-6 (B), IFNγ (C), and TNFα (D) levels.The red dashed line represents the average cytokine concentration in the supernatant after treatment with 30nM anti-CD3 antibody (positive control) under corresponding conditions.The average cytokine concentration in the supernatant after treatment with 765nM IgG under corresponding conditions are overlapping with the x-axis.Statistical significance is indicated as ****P<0.0001.

Figure S8
Figure S8IBI315 induces pyroptosis-like morphological changes in N87 cells in the N87/T cells coculture system.N87 cells were treated with IBI315, its parent antibody, or their combination for 6 hours in the presence of T cells, and images were captured.Pyroptotic cells are marked with black arrows.Scale bar: 100 μm.Abbreviations: TZB: trastuzumab, αPD1: anti-PD1 antibody (sintilimab), T+P: trastuzumab+sintilimab.

Figure S9
Figure S9 Expression of GSDM family in gastric cancer.A) Relative expression levels of GSDM family members (DFNA5 refers to GSDME) in gastric cancer and paracancer tissues from the TCGA database.B) Immunoblotting analysis of the expression of GSDMB, GSDMD and GSDME in N87 cells, with positive control represented by gastric cancer tissues (P).

Figure S11
Figure S11Proportion of GSDMB amplification in ERBB2-amplified gastric cancer.Analysis of the TCGA database (n=441) revealed a higher frequency of GSDMB amplification in ERBB2-amplified gastric cancers.

Figure S12
Figure S12 Association of GSDMB expression with improved prognosis in immunotherapy.A-C) TIDE database analysis of the correlation between GSDMB expression and treatment outcomes in melanoma (A), bladder cancer (B), and kidney cancer (C) immunotherapy cohorts.

Figure S13
Figure S13Analysis of GSDMB expression revealed an association with altered T cell function and increased responsiveness to immunotherapy.Using the TIDE database, we evaluated the expression of GSDMs family members and their association with treatment outcomes in various immunotherapy cohorts.