Fig 2: a-CTLA4-TGFβRII inhibits tumor growth more effectively than the combination of a-CTLA-4 and a-PD1. NSG mice immune reconstituted with tumor-matched HLA A2+ human CD34+ BM cells and bearing MDA-MB231-Luc human TNBC tumor xenografts were treated (5 mg kg−1 i.p. weekly) with vehicle alone (untreated control) or the following antibodies (either alone or in combination), as indicated: a-CTLA4-TGFβRII; a-CTLA-4; a-TGFβ (1D11); a-PD1 (Pembrolizumab); (five to six mice per group). a In vivo tumor growth curves (mean ± SEM). p-values were derived using unpaired, two-sided t-test. b Bioluminescence assay of primary tumors in untreated controls or the indicated treatment groups at 10 and 25 days after tumor cell inoculation. c Bioluminescence assay of lung metastases in untreated controls or the indicated treatment groups at 40 days post tumor inoculation. d Immunophenotype analysis of the effect of the indicated treatments on the percentage of Tregs (CD4+/CD25high/CD127low/FOXP3+), tumor-reactive IFN-γ-expressing CD8+ T cells, and central memory CD4+ and CD8+ T cells (CD45ROhighCD62Lhigh). Bars represent mean ± SEM of three animals per treatment group. Asterisks above each bar denote the statistical significance of the difference between the indicated group and a-CTLA4-TGFβRII (blue bar)

Fig 3: a-CTLA4-TGFβRII exhibits superior antitumor efficacy with elevation tumor-reactive IFNγ-expressing and central memory T cells. Effect of the indicated treatments on melanoma tumor xenografts in NSG mice that were immune reconstituted with tumor-matched HLA A2+ human CD34+ BM cells. Mice engrafted with human CD3+ cells were inoculated subcutaneously with either human melanoma tumor cells (A375) or patient-derived melanoma tumor xenografts. At 10d following tumor inoculation, mice were randomized into groups and treated with the indicated antibodies (5 mg kg−1 i.p. weekly) or vehicle alone (untreated control). a Representative images and in vivo tumor growth curves in A375 tumor-bearing mice: (mean + SEM of 12 mice in each indicated group). b Representative images and mass of tumors at the end of treatment in A375 tumor-bearing mice: (seven mice in each indicated group). c Flow cytometric analysis of the comparative in vivo effect of the indicated treatments on tumor-reactive IFN-γ expression in CD8+ T cells in A375 tumor-bearing mice. Representative flow data is shown (mean ± SEM). d, e Comparative antitumor efficacy of a-CTLA4-TGFβRII, a-CTLA-4, nonspecific IgG-TGFβRII, and the combination of a-CTLA-4 and IgG-TGFβRII in A375 tumor-bearing mice. d Tumor growth curves (mean ± SEM of five mice). The p-value (p < 0.02, Student’s unpaired t-test) denotes significant difference between a-CTLA4-TGFβRII and each other treatment group. e Flow cytometric analysis of infiltrating FOXP3-expressing Tregs (left) and tumor-reactive IFN-γ expressing CD8+ T cells (right). f Tumor growth curves in melanoma PDX-bearing mice: (mean ± SEM of five mice). g Representative flow data of in vivo effect of a-CTLA4-TGFβRII and a-CTLA-4 on tumor-specific IFN-γ expression in CD8+ T cells in PDX-bearing mice. h Flow cytometric analysis of the comparative in vivo effect of a-CTLA4-TGFβRII and a-CTLA-4 on tumor-infiltrating central memory T cells (CD45ROhighCD62Lhigh) in PDX-bearing mice

Fig 4: Design and bifunctional target binding ability of a-CTLA4-TGFβRII. a Autocrine/paracrine TGFβ-induced expression of FOXP3, the signature transcription factor of the Treg lineage. FOXP3 induces the expression of CTLA-4 and Galectin-9, a ligand that engages TIM-3 and triggers exhaustion or apoptosis of effector T cells. Galectin-9 further interacts with TGFβ receptors to drive FOXP3 expression in a positive-feed forward autocrine loop involving SMAD3 activation to induce and maintain Tregs. Schematic representation of the structure and targets of a-CTLA4-TGFβRII are shown. a-CTLA4-TGFβRII was designed by fusing the C terminus of the heavy chain of a human a-CTLA-4 antibody with a ligand-binding sequence of the extracellular domain of TGFβ Receptor II (TGFβRII ECD) via a flexible linker peptide, (GGGGS)3. b Amino acid sequences of the heavy chain and light chain of a-CTLA4-TGFβRII. c SDS-PAGE (unreduced-NR and reduced-R) comparing the molecular weight of a-CTLA4-TGFβRII and a-CTLA-4 antibody (FL, full-length; HC, heavy chain; LC, light chain). d,e Bifunctional ability of a-CTLA4-TGFβRII to simultaneously bind CTLA-4 and TGFβ1 using a ‘double-sandwich’ ELISA, wherein a-CTLA4-TGFβRII or a-CTLA-4 antibody was added to CTLA-4-Fc-coated plates, followed by rhTGFβ1 that was detected by a biotinylated anti-human TGFβ1 antibody. TGFβRII-Fc-coated plates were used as a TGFβ-binding positive control. f Standard ELISA showing the ability of a-CTLA4-TGFβRII to compete with a TGFβ capture antibody for binding to TGFβ1. For d–f, the data show the optical density (OD) values (mean of three replicate wells for each assay condition) from a representative of two independent experiments

Fig 5: a-CTLA4-TGFβRII counteracts tumor-infiltrating Tregs and inhibits TH17 differentiation. a Immunoblot analyses of the effect of a-CTLA4-TGFβRII or a-CTLA-4 on TGFβ-induced SMAD-2/3 phosphorylation and expression of FOXP3 in human PBMC. PBMCs were cultured for 24–48 h with rhIL-2 (100 IU ml−1) and anti-CD3/anti-CD28-coated Dynabeads in the presence or absence of rhTGFβ1 (2.5 ng ml−1) with or without either a-CTLA4-TGFβRII or a-CTLA-4 (5 μg ml−1). b, c Flow cytometric analysis of the in vivo effect of a-CTLA4-TGFβRII or a-CTLA-4 on bone marrow (BM) and tumor-infiltrating Tregs in human melanoma tumor-bearing NSG mice that were immune reconstituted with tumor-matched HLA A2+ human CD34+ bone marrow cells. Mice engrafted with human CD3+ cells were inoculated subcutaneously with either human melanoma tumor cells (A375 or SK-MEL-5) or patient-derived melanoma tumor xenografts (PDX-1 and PDX-2). Tumor-bearing mice (A375; SK-MEL-5; PDX-1; PDX-2) were randomized into groups and treated with either a-CTLA4-TGFβRII or a-CTLA-4 (5 mg kg−1 i.p. weekly), or vehicle alone (untreated control). b Representative flow data of FOXP3 expression in BM CD4+CD25+ T cells in A375 or SK-MEL-5 tumor-bearing mice from each treatment or control group. c The percentage of Tregs (CD4+/CD25high/CD127low/FOXP3+) in tumor-infiltrating lymphocytes in A375- or PDX-bearing mice (five animals per treatment group for A375 and four mice for PDXs). d Effect of a-CTLA4-TGFβRII or a-CTLA-4 on the suppression of tumor-reactive T cells by autologous Tregs in tumor-infiltrated BM from a patient. Marrow-infiltrating lymphocytes (MILs) stimulated with anti-CD3/anti-CD28-beads and rhIL-2 were carboxyfluorescein N-hydroxysuccinimidyl ester (CFSE) labeled and added to autologous BM that had been pulsed with either myeloma cell lysate (tumor-specific antigen) or nonspecific antigen in the presence or absence of autologous CD4+/CD25+ MILs, with or without either a-CTLA4-TGFβRII or a-CTLA-4 (5 μg ml−1) for 3 days. Tumor antigen-reactive T cells (CD3+/CFSElow/IFNγ+) were quantified by flow cytometry (three in vitro replicates for each experimental group). e PBMCs were stimulated with anti-CD3/CD28 beads in the presence of IL17-skewing cytokines (10 ng ml−1 IL-6, 5 ng ml−1 TGFβ, 10 ng ml−1 anti–IFN-γ, and 10 ng ml−1 anti–IL-4) with or without either a-CTLA4-TGFβRII or a-CTLA-4 (5 μg ml−1) for 3 days. Following addition of Leukocyte Activation Cocktail (2 μl ml−1 for 4 h), cells were analyzed by flow cytometry for intracellular expression of IL-17 and IFN-γ in CD4 + T cells (three in vitro replicates for each experimental group). Asterisks represent statistical significance between the a-CTLA4-TGFβRII and a-CTLA-4 treatments. Representative flow cytometric dot plots are shown