Journal: Journal for immunotherapy of cancer

Article Title: Anti-CTGF/PD-1 bispecific antibody Y126S restrains desmoplastic and immunosuppressive microenvironment in pancreatic cancer.

doi: 10.1136/jitc-2025-012144

Figure Lengend Snippet: Figure 1 The basic characteristics of CAFs-targeting anti-CTGF/PD-1 bispecific antibody (Y126S). (A) Y126S was designed with a structure comprizing two anti-CTGF regions and two anti-PD-1 regions. Schematic representation of Y126S bispecific binding via CTGF and PD-1 in the tumor site. (B) The results of non-reduced and reduced SDS-PAGE assay. A single band was found in the lane of non-reduced Y126S monomers, and two bands were observed in the lane of Y126S. (C–H) Binding affinity to hCTGF, mCTGF, and mPD-1 for neutralizing antibodies at the indicated concentrations was assessed by surface plasmon resonance. (C, D) Binding affinity to hCTGF antigen for Y126S and α-CTGF at the indicated concentrations by surface plasmon resonance. (E, F) Binding affinity to mCTGF antigen for Y126S and α-CTGF at the indicated concentrations by surface plasmon resonance. (G, H) Binding affinity to mPD-1 antigen for Y126S and α-PD-1 at the indicated concentrations by surface plasmon resonance. (I, J) Indirect anti-hIgG ELISA showed the binding affinity of serially diluted Y126S, binding controls, or negative controls to mCTGF and mPD-1. (K) Double-antigen sandwich ELISA showed that Y126S simultaneously bound mCTGF and mPD-1 at indicated concentrations. n = 3 replicates in I–K. CAF, cancer-associated fibroblast; CTGF, connective tissue growth factor; hCTGF, human CTGF antigen; mCTGF, mouse CTGF antigen; mPD-1, mouse programmed cell death 1 antigen; OD, optimal density; PD-L1, programmed cell death ligand 1; SDS-PAGE, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Article Snippet: Three replicates of hCAFs isolated from patients with PDAC were treated with recombinant human CTGF (rhCTGF) (HY- P70106, MedChemExpress) for 2 days.

Techniques: Binding Assay, SDS Page, SPR Assay, Enzyme-linked Immunosorbent Assay, Sandwich ELISA, Polyacrylamide Gel Electrophoresis

Journal: Journal for immunotherapy of cancer

Article Title: Anti-CTGF/PD-1 bispecific antibody Y126S restrains desmoplastic and immunosuppressive microenvironment in pancreatic cancer.

doi: 10.1136/jitc-2025-012144

Figure Lengend Snippet: Figure 2 Y126S reversed CTGF-mediated immunosuppressive TME. (A) Correlation analysis of CTGF with PD-L1 expression and exhausted CD8+ T cells in TCGA database (n=179). (B) Gene set enrichment analysis (GSEA) correlates CAFs-CTGF function with T cell differentiation, immune response regulation, and immune response regulating cell surface receptor signaling pathway in TCGA database (n=179). (C, D) Primary hCAFs isolated from patients with PDAC were treated with 20 ng/mL rhCTGF for 48 hours and subjected to RNA-seq analysis. Heatmap of differentially expressed genes (C) and top pathways in GO enrichment (D). (E, F) PD-L1 expression of hCAFs (E) and KPC-CAFs (F) after treatment with CM from cancer cells or CTGF (20 ng/mL) with α-CTGF or Y126S for 48 hours. (G–P) The effects of α-CTGF injection in orthotopic PDAC-bearing mice were evaluated. Schematic illustration for the design (G), bioluminescence images and average radiance (H), tumor weight (I), and representative images and correlation analysis between CTGF and PD-L1 expression in IHC staining (J). Quantification for the percentage of CD3+ T cells (K), CD8+ T cells (L), PD-1+ TIM-3+ T cells in CD8+ T cells (M), and expression of CD206+ by TAMs (N) of tumors treated with α-CTGF by flow cytometry. Representative images and quantification of anti-CD8 (O) and anti-Ki67 (P) IHC staining in tumors treated with α-CTGF or IgG group. 7 mice per group (n = 7) in G–P. Data were represented as mean ± SEM. Statistical analysis was performed with two-tailed Student’s t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, and ns = not significant. Scale bar: 20 µm. AOD, average optimal density; CM, conditioned media; CTGF, connective tissue growth factor; FACS, fluorescence-activated cell sorting; FDR, false discovery rate; GO, gene oncology; hCAFs, human cancer-associated fibroblasts; IHC, immunohistochemistry; IVIS, In Vivo Imaging System; KPC-CAFs, KPC’s cancer-associated fibroblasts; mCTGF, mouse CTGF protein; NES, nonparametric estimation statistics; PDAC, pancreatic ductal adenocarcinoma; PD-L1, programmed cell death ligand 1; rhCTGF, recombinant human CTGF protein; TAMs, tumor-associated macrophages; TCGA, The Cancer Genome Atlas; TME, tumor microenvironment.

Article Snippet: Three replicates of hCAFs isolated from patients with PDAC were treated with recombinant human CTGF (rhCTGF) (HY- P70106, MedChemExpress) for 2 days.

Techniques: Expressing, Cell Differentiation, Cell Surface Receptor Assay, Isolation, RNA Sequencing, Injection, Immunohistochemistry, Flow Cytometry, Two Tailed Test, Fluorescence, FACS, In Vivo Imaging, Recombinant

Journal: Journal for immunotherapy of cancer

Article Title: Anti-CTGF/PD-1 bispecific antibody Y126S restrains desmoplastic and immunosuppressive microenvironment in pancreatic cancer.

doi: 10.1136/jitc-2025-012144

Figure Lengend Snippet: Figure 3 Y126S counteracts CTGF-caused immunosuppression by downregulating PD-L1 expression and reactivating T cell immune response. (A) KPC-CAFs were pretreated with the stimulation of 20 ng/mL mCTGF plus multiple antibodies for 48 hours and then co-cultured with CD8+ T cells isolated from C57BL/6J spleen for 18 hours (effector/target ratio = 2:1). Representative flow cytometric images and corresponding quantifications of PD-L1 expression by KPC-CAFs (top), ratio of PD-1+ TIM-3+ T cells in CD8+ T cells (center), and apoptosis of CD8+ T cells by flow cytometry (bottom). (B) Level of IL-2, IFN-γ, and TNF-α secreted by T cells in a co-culture system. 3 replicates per group (n = 3) in all experiments. Data were represented as mean ± SEM. Statistical analysis was performed with ANOVA followed by Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001, and ns = not significant. ANOVA, analysis of variance; comb, combination of α-CTGF and α-PD-1; IFN-γ, interferon- gamma; KPC-CAFs, KPC’s cancer-associated fibroblasts; mCTGF, mouse connective tissue growth factor protein; PD-1, programmed cell death 1; PD-L1, programmed cell death ligand 1; TNF-α, tumor necrosis factor-alpha.

Article Snippet: Three replicates of hCAFs isolated from patients with PDAC were treated with recombinant human CTGF (rhCTGF) (HY- P70106, MedChemExpress) for 2 days.

Techniques: Expressing, Cell Culture, Isolation, Flow Cytometry, Co-Culture Assay, Comparison

Journal: Journal for immunotherapy of cancer

Article Title: Anti-CTGF/PD-1 bispecific antibody Y126S restrains desmoplastic and immunosuppressive microenvironment in pancreatic cancer.

doi: 10.1136/jitc-2025-012144

Figure Lengend Snippet: Figure 4 Y126S inhibited desmoplasia induced by CAF activation through targeting CTGF. (A–E) The desmoplasia of tumors derived from PDAC mice treated with α-CTGF or IgG in figure 2G. Representative images (A), quantification of anti-α-SMA (B), anti-collagen I (C), Masson (D) staining, and tissue stiffness (E). (F, G) Relative expression of specific genes of hCAFs after treatment with CM from PANC-1 cells or rhCTGF (20 ng/mL) plus α-CTGF or Y126S for 48 hours by qRT-PCR (F) and western blotting (G). (H) Representative images of hCAFs stimulated by rhCTGF (20 ng/mL) plus α-CTGF or Y126S for 48 hours for α-SMA (green), collagen I (red), and DAPI (blue). (I, J) Relative expression of specific genes of KPC-CAFs after treatment with CM from Panc-02 cells or mCTGF (20 ng/mL) plus α-CTGF or Y126S for 48 hours by qRT-PCR (I) and western blotting (J). (K– M) Representative fast force MAP AFM image (K), Young’s modulus of hCAFs (L), and KPC-CAFs (M) by AFM with a CSC38 cantilever (spring constant: 0.02–0.04 N/m). (N) Gel contraction of hCAFs treated with 20 ng/mL rhCTGF plus α-CTGF or Y126S. 7 replicates per group (n = 7) in B–E, 3 replicates per group (n = 3) in F, J, N, and 5 replicates per group (n = 5) in L, M. Data were represented as mean ± SEM. Statistical analysis in B–E was performed with two-tailed Student’s t-tests, and in F, J, L–N was performed with ANOVA followed by Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Scale bar: 20 µm in A, 50 µm in H, and 10 µm in K. AFM, atomic force microscopy; ANOVA, analysis of variance; α-SMA, alpha- smooth muscle actin; CM, conditioned media; CTGF, connective tissue growth factor; DAPI, 4',6-diamidino-2-phenylindole; FAP, fibroblast activation protein; hCAFs, human cancer-associated fibroblasts; KPC-CAFs, KPC’s cancer-associated fibroblasts; mCTGF, mouse CTGF protein; mRNA, messenger RNA; PDAC, pancreatic ductal adenocarcinoma; qRT-PCR, quantitative reverse transcription PCR; rhCTGF, recombinant human CTGF protein.

Article Snippet: Three replicates of hCAFs isolated from patients with PDAC were treated with recombinant human CTGF (rhCTGF) (HY- P70106, MedChemExpress) for 2 days.

Techniques: Activation Assay, Derivative Assay, Staining, Expressing, Quantitative RT-PCR, Western Blot, Two Tailed Test, Comparison, Microscopy, Reverse Transcription, Recombinant

Journal: Journal for immunotherapy of cancer

Article Title: Anti-CTGF/PD-1 bispecific antibody Y126S restrains desmoplastic and immunosuppressive microenvironment in pancreatic cancer.

doi: 10.1136/jitc-2025-012144

Figure Lengend Snippet: Figure 5 Y126S exhibits potent antitumor efficiency in PDAC mouse model by remodeling TME. (A–C) The effects of Y126S with different dosages in orthotopic PDAC mice. Schematic illustration for the design (A), tumor weight (B), and survival curve (C) of PDAC-bearing mice after receiving IgG, low (4 mg/kg), middle (8 mg/kg), and high dose (16 mg/kg) of Y126S treatment. (D–Q) The effects of IgG, α-CTGF, α-PD-1, a combination of monospecific antibodies, and Y126S in orthotopic PDAC mice were evaluated. Schematic illustration (D), bioluminescence images (E), average radiance (F), and tumor weight (G) of mice after the antibody treatment as indicated. Representative images (H), quantification of anti-α-SMA (I), anti-collagen I (J), Masson (K) staining, and tissue stiffness (L) in PDAC tumors. Proportion of CD3+ T cells (M) and CD8+ T cells (N) in live cells, PD-1+ TIM- 3+ T cells in CD8+ T cells (O), and expression of CD206+ by CD11b+ F4/80+ TAMs (P) in tumors of different treatment groups by flow cytometry. (Q) The level of IFN-γ in tumor tissues by ELISA assay. 8 mice per group (n = 8) in all experiments. Data were represented as mean ± SEM. Statistical analysis was performed with ANOVA followed by Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001, and ns = not significant. Scale bar: 20 µm. ANOVA, analysis of variance; α-SMA, alpha smooth muscle actin; comb, combination of α-CTGF and α-PD-1; CTGF, connective tissue growth factor; IFN-γ, interferon-gamma; i.p., intraperitoneal; IVIS, In Vivo Imaging System; PD-1, programmed cell death 1; PDAC, pancreatic ductal adenocarcinoma; TAMs, tumor-associated macrophages; TME, tumor microenvironment.

Article Snippet: Three replicates of hCAFs isolated from patients with PDAC were treated with recombinant human CTGF (rhCTGF) (HY- P70106, MedChemExpress) for 2 days.

Techniques: Staining, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Comparison, In Vivo Imaging

Journal: Journal for immunotherapy of cancer

Article Title: Anti-CTGF/PD-1 bispecific antibody Y126S restrains desmoplastic and immunosuppressive microenvironment in pancreatic cancer.

doi: 10.1136/jitc-2025-012144

Figure Lengend Snippet: Figure 6 Y126S yields a robust antitumor response and prolongs survival in the KPC model. The effects of IgG, α-CTGF, α-PD-1, a combination of monospecific antibodies, and Y126S were validated in the KPC mouse model. (A) Schematic illustration of study design. (B) 68Ga-FAPI PET MIP images of different groups before and after treatment. The white dotted circles and red arrows indicate the tumor sites. ΔSUVmax of each tumor was analyzed by subtracting the SUVmax measured pretreatment from the SUVmax after treatment. (C, D) The tumor weight (C) and overall survival (D) of KPC mice in different groups. (E–N) Tumors were then further evaluated to understand the change in TME of KPC mice. Representative images (E) and quantification of anti-α-SMA (F), anti-collagen I (G), Masson (H) staining, and tissue stiffness (I). The proportion of CD3+ T cells (J) and CD8+ T cells (K) in live cells, PD-1+ TIM-3+ T cells in CD8+ T cells (L), and expression of CD206+ by CD11b+ F4/80+ TAMs (M) by flow cytometry. (N) The level of IFN-γ in tumor tissues by ELISA assay. 3 mice in IgG, α-CTGF, α-PD-1 group, 4 in the combination group, and 6 in Y126S group in B due to survival statement in D, 6 mice per group (n = 6) in C, F–N. Data were represented as mean ± SEM. Statistical analysis was performed with ANOVA followed by Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar: 20 µm. ANOVA, analysis of variance; α-SMA, alpha-smooth muscle actin; comb, a combination of α-CTGF and α-PD-1; CTGF, connective tissue growth factor; IFN-γ, interferon-gamma; i.p., intraperitoneal; PD-1, programmed cell death 1; PET, positron emission tomography; SUVmax, maximum of standard uptake values; TAMs, tumor- associated macrophages; TME, tumor microenvironment.

Article Snippet: Three replicates of hCAFs isolated from patients with PDAC were treated with recombinant human CTGF (rhCTGF) (HY- P70106, MedChemExpress) for 2 days.

Techniques: Staining, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Comparison, Positron Emission Tomography

Journal: Journal for immunotherapy of cancer

Article Title: Anti-CTGF/PD-1 bispecific antibody Y126S restrains desmoplastic and immunosuppressive microenvironment in pancreatic cancer.

doi: 10.1136/jitc-2025-012144

Figure Lengend Snippet: Figure 7 Y126S exhibits tumor-specific accumulation through targeting CTGF. (A–C) The expression of CTGF in tumor tissues and adjacent normal tissue from patients with PDAC by RT-qPCR (A), western blotting (B), and IHC staining (C). For α-CTGF staining in C, representative histological plots were captured both in 100× and 200× (left panel), and quantification was conducted by measuring the positive area percentage of CTGF. n=5 (individual donors, illustrated as three technical replicates) (right panel). (D) Schematic illustration of the procedure of mice sacrifices and sample collection over time (0.5 hour, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, and 48 hours) after receiving a single administration of Y126S or α-PD-1. (E) Concentration curve of Y126S in serum. (F, G) Molecular weight-normalized-tumor concentration of Y126S and α-PD-1 in orthotopic tumor- bearing mice over time (F) and at 2 hours (G), expressed as molar concentration. (H–K) Further mass-normalized antibody levels of Y126S in tumor compared with liver (H) or lung (I), and of α-PD-1 in tumor compared with liver (J) or lung (K) were expressed as the percentage of molar concentration per gram of tissue. (L) Schematic illustration depicting the mechanism of Y126S treatment repressing desmoplastic and immunosuppressive TME in PDAC (created in BioRender.com). 3 replicates per group (n = 3) in A, 3 mice per group at each time point (n = 3) in D–K. Data were represented as mean ± SEM. Statistical analysis was performed with two-tailed Student’s t-tests. *p < 0.05, **p < 0.01, ***p < 0.001. CAF, cancer-associated fibroblast; CTGF, connective tissue growth factor; ECM, extracellular matrix; IHC, immunohistochemistry; mRNA, messenger RNA; PD- 1, programmed cell death 1; PDAC, pancreatic ductal adenocarcinoma; PD-L1, programmed cell death ligand 1; RT-qPCR, reverse transcription quantitative PCR; TAM, tumor-associated macrophage; TME, tumor microenvironment.

Article Snippet: Three replicates of hCAFs isolated from patients with PDAC were treated with recombinant human CTGF (rhCTGF) (HY- P70106, MedChemExpress) for 2 days.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Staining, Concentration Assay, Molecular Weight, Two Tailed Test, Reverse Transcription, Real-time Polymerase Chain Reaction