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Cited by 342Cancer Immunotherapy and BiomarkersTGF-β signaling in diseasesT-cell and B-cell Immunology

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Bifunctional immune checkpoint-targeted antibody-ligand traps that simultaneously disable TGFβ enhance the efficacy of cancer immunotherapy

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Ravi, Rajani, Noonan, Kimberly A., Pham, Vui, Bedi, Rishi, Zhavoronkov, Alex, Ozerov, Ivan V., Makarev, Eugene, V. Artemov, Artem, Wysocki, Piotr T., Mehra, Ranee, Nimmagadda, Sridhar, Marchionni, Luigi, Sidransky, David, Borrello, Ivan M., Izumchenko, Evgeny, Bedi, Atul·2018·Nature Communications
DOI 10.1038/s41467-017-02696-6PMC PMC5821872
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Abstract

Abstract A majority of cancers fail to respond to immunotherapy with antibodies targeting immune checkpoints, such as cytotoxic T-lymphocyte antigen-4 (CTLA-4) or programmed death-1 (PD-1)/PD-1 ligand (PD-L1). Cancers frequently express transforming growth factor-β (TGFβ), which drives immune dysfunction in the tumor microenvironment by inducing regulatory T cells (Tregs) and inhibiting CD8+ and TH1 cells. To address this therapeutic challenge, we invent bifunctional antibody–ligand traps (Y-traps) comprising an antibody targeting CTLA-4 or PD-L1 fused to a TGFβ receptor II ectodomain sequence that simultaneously disables autocrine/paracrine TGFβ in the target cell microenvironment (a-CTLA4-TGFβRIIecd and a-PDL1-TGFβRIIecd). a-CTLA4-TGFβRIIecd is more effective in reducing tumor-infiltrating Tregs and inhibiting tumor progression compared with CTLA-4 antibody (Ipilimumab). Likewise, a-PDL1-TGFβRIIecd exhibits superior antitumor efficacy compared with PD-L1 antibodies (Atezolizumab or Avelumab). Our data demonstrate that Y-traps counteract TGFβ-mediated differentiation of Tregs and immune tolerance, thereby providing a potentially more effective immunotherapeutic strategy against cancers that are resistant to current immune checkpoint inhibitors.

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Figures

Fig. 1 TGFβ pathway activation correlates with elevated TGFB1 and FOXP3 mRNA expression in TCGA-SKCM and TCGA-BRCA data sets. iPANDA software suite for analysis of intracellular signaling pathway activation based on transcriptomic data was used to estimate the level of TGFβ signaling in melanoma or breast samples from TCGA-SKCM (n = 472) and TCGA-BRCA data set, respectively (n = 776). TCGA transcriptomic data from normal breast tissue (n = 114) or skin biopsy of healthy women (n = 122) was used as a reference after proper normalization. The TGFβ pathway activation scores for a 472 melanomas (metastatic and primary) and c 776 breast tumors (ER/PR positive, HER2 positive, and TNBC) are sorted from low to high. The corresponding expression of TGFB1 gene, FOXP3, and CD274 is shown for each patient. The units on the color bar represent the pathway activation score or the fold change in the gene expression on the logarithmic scale in comparison with the average normal control level. Correlation matrix for melanoma (b) and breast cancer (d) are shown (numbers in plot indicate correlation coefficient value). Pairwise correlations between TGFβ pathway activation and message for TGFB1, FOXP3, and CD274 were computed using the Pearson's correlation coefficient. The p-values were calculated with the null hypothesis that the tested samples are unrelated. The p-values for all correlation < 0.05
Fig. 1 TGFβ pathway activation correlates with elevated TGFB1 and FOXP3 mRNA expression in TCGA-SKCM and TCGA-BRCA data sets. iPANDA software suite for analysis of intracellular signaling pathway activation based on transcriptomic data was used to estimate the level of TGFβ signaling in melanoma or breast samples from TCGA-SKCM (n = 472) and TCGA-BRCA data set, respectively (n = 776). TCGA transcriptomic data from normal breast tissue (n = 114) or skin biopsy of healthy women (n = 122) was used as a reference after proper normalization. The TGFβ pathway activation scores for a 472 melanomas (metastatic and primary) and c 776 breast tumors (ER/PR positive, HER2 positive, and TNBC) are sorted from low to high. The corresponding expression of TGFB1 gene, FOXP3, and CD274 is shown for each patient. The units on the color bar represent the pathway activation score or the fold change in the gene expression on the logarithmic scale in comparison with the average normal control level. Correlation matrix for melanoma (b) and breast cancer (d) are shown (numbers in plot indicate correlation coefficient value). Pairwise correlations between TGFβ pathway activation and message for TGFB1, FOXP3, and CD274 were computed using the Pearson's correlation coefficient. The p-values were calculated with the null hypothesis that the tested samples are unrelated. The p-values for all correlation < 0.05CC BY · PubMed Central
Fig. 2 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. 2 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 experimentsCC BY · PubMed Central
Fig. 3 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
Fig. 3 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 shownCC BY · PubMed Central
Fig. 4 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 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 miceCC BY · PubMed Central
Fig. 5 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. 5 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)CC BY · PubMed Central
Fig. 6 Design and bifunctional target binding ability of anti-PDL1-TGFβRII. a Schematic representation of PD-L1 and TGFβ entrained independent and cooperative mechanisms of immune tolerance. Schematic structure and targets of a-PDL1-TGFβRII are shown. a-PDL1-TGFβRII was designed by fusing the C terminus of the heavy chain of a human a-PD-L1 antibody with a ligand-binding sequence of the extracellular domain of TGFβ Receptor II (TGFβRII ECD) via a flexible linker peptide, (GGGGS)3. b SDS-PAGE under non-reducing (NR) and reducing (R) conditions was used to compare the full-length, heavy chain and light chain of a-PDL1-TGFβRII and a-PD-L1 antibody. Figure shows the results of SDS-PAGE analyses of each of two separate a-PDL1-TGFβRII Y-traps (Ab1 and Ab2) and their respective human a-PD-L1 antibody (atezolizumab and avelumab). c SEC-HPLC analysis of purified a-PDL1-TGFβRII; d ELISA showing the comparative ability of a-PDL1-TGFβRII and a-PD-L1 antibody to bind PD-L1. Biotinylated recombinant human PD-L1 (rh B7-H1-biotin; 0–100 ng ml−1) was added to plates coated with a-PDL1-TGFβRII or a-PD-L1 antibody (1 μg ml−1), followed by detection with HRP-Avidin. Plates coated with non-specific IgG-TGFβRII showed no binding to PD-L1 and served as a negative control to analyze the binding ability of the test samples. e ELISA showing the comparative ability of a-PDL1-TGFβRII and a-PD-L1 antibody to bind TGFβ. Recombinant human TGFβ (rhTGFβ1; 0–2,000 pg ml−1) was added to plates coated with a-PDL1-TGFβRII or a-PD-L1 antibody (1 μg ml−1), which was detected by biotinylated a-TGFβ1 and HRP-Avidin. Plates coated with nonspecific IgG-TGFβRII and rhTGFβRII-Fc served as positive controls to analyze the binding ability of the test samples to TGFβ. f ELISA showing the ability of a-PDL1-TGFβRII to simultaneously bind both PD-L1 and TGFβ. Anti-PDL1-TGFβRII or a-PD-L1 antibody (0–100 ng ml−1) was added to PD-L1-Fc coated plates (1 μg ml−1), followed by rhTGFβ1 (100 ng ml−1) that was detected by a biotinylated anti-human TGFβ1 antibody. 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. 6 Design and bifunctional target binding ability of anti-PDL1-TGFβRII. a Schematic representation of PD-L1 and TGFβ entrained independent and cooperative mechanisms of immune tolerance. Schematic structure and targets of a-PDL1-TGFβRII are shown. a-PDL1-TGFβRII was designed by fusing the C terminus of the heavy chain of a human a-PD-L1 antibody with a ligand-binding sequence of the extracellular domain of TGFβ Receptor II (TGFβRII ECD) via a flexible linker peptide, (GGGGS)3. b SDS-PAGE under non-reducing (NR) and reducing (R) conditions was used to compare the full-length, heavy chain and light chain of a-PDL1-TGFβRII and a-PD-L1 antibody. Figure shows the results of SDS-PAGE analyses of each of two separate a-PDL1-TGFβRII Y-traps (Ab1 and Ab2) and their respective human a-PD-L1 antibody (atezolizumab and avelumab). c SEC-HPLC analysis of purified a-PDL1-TGFβRII; d ELISA showing the comparative ability of a-PDL1-TGFβRII and a-PD-L1 antibody to bind PD-L1. Biotinylated recombinant human PD-L1 (rh B7-H1-biotin; 0–100 ng ml−1) was added to plates coated with a-PDL1-TGFβRII or a-PD-L1 antibody (1 μg ml−1), followed by detection with HRP-Avidin. Plates coated with non-specific IgG-TGFβRII showed no binding to PD-L1 and served as a negative control to analyze the binding ability of the test samples. e ELISA showing the comparative ability of a-PDL1-TGFβRII and a-PD-L1 antibody to bind TGFβ. Recombinant human TGFβ (rhTGFβ1; 0–2,000 pg ml−1) was added to plates coated with a-PDL1-TGFβRII or a-PD-L1 antibody (1 μg ml−1), which was detected by biotinylated a-TGFβ1 and HRP-Avidin. Plates coated with nonspecific IgG-TGFβRII and rhTGFβRII-Fc served as positive controls to analyze the binding ability of the test samples to TGFβ. f ELISA showing the ability of a-PDL1-TGFβRII to simultaneously bind both PD-L1 and TGFβ. Anti-PDL1-TGFβRII or a-PD-L1 antibody (0–100 ng ml−1) was added to PD-L1-Fc coated plates (1 μg ml−1), followed by rhTGFβ1 (100 ng ml−1) that was detected by a biotinylated anti-human TGFβ1 antibody. 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 experimentsCC BY · PubMed Central
Fig. 7 a-PDL1-TGFβRII exhibits superior antitumor efficacy compared to a-PD-L1 antibodies. Effect of the indicated treatments on melanoma and TNBC tumor xenografts in NSG mice reconstituted with human CD34+ HSC. Mice engrafted with human CD3+ cells were inoculated subcutaneously with either human melanoma (A375) or TNBC (MDA-MB-231-Luc) tumor cells. At 10d following tumor inoculation, mice were randomized into groups and treated i.p. with the indicated antibodies (5 mg kg−1 weekly) or vehicle alone (untreated control). Tumor growth curves in A375 tumor-bearing mice (a) or MDA-MB-231-Luc tumor-bearing mice (c) are shown (mean ± SEM of five to six mice in each indicated group). In TNBC model, independent experiments were conducted to compare two different a-PDL1-TGFβRII antibody–ligand traps with their respective a-PD-L1 antibodies, Atezolizumab (c, left) (and Avelumab (c, right). b, d Flow cytometric analysis of the comparative in vivo effect of the indicated treatments on infiltrating FOXP3-expressing Tregs and tumor-reactive IFN-γ-expressing CD8+ T cells in melanoma (b) and TNBC (d) models
Fig. 7 a-PDL1-TGFβRII exhibits superior antitumor efficacy compared to a-PD-L1 antibodies. Effect of the indicated treatments on melanoma and TNBC tumor xenografts in NSG mice reconstituted with human CD34+ HSC. Mice engrafted with human CD3+ cells were inoculated subcutaneously with either human melanoma (A375) or TNBC (MDA-MB-231-Luc) tumor cells. At 10d following tumor inoculation, mice were randomized into groups and treated i.p. with the indicated antibodies (5 mg kg−1 weekly) or vehicle alone (untreated control). Tumor growth curves in A375 tumor-bearing mice (a) or MDA-MB-231-Luc tumor-bearing mice (c) are shown (mean ± SEM of five to six mice in each indicated group). In TNBC model, independent experiments were conducted to compare two different a-PDL1-TGFβRII antibody–ligand traps with their respective a-PD-L1 antibodies, Atezolizumab (c, left) (and Avelumab (c, right). b, d Flow cytometric analysis of the comparative in vivo effect of the indicated treatments on infiltrating FOXP3-expressing Tregs and tumor-reactive IFN-γ-expressing CD8+ T cells in melanoma (b) and TNBC (d) modelsCC BY · PubMed Central

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Cites

Research works printed but not linked: 2

  1. Bedi, A. & Ravi, R. Compositions and methods for targeted immunomodulatory antibodies and fusion proteins. US patent 8,993,524 (2011).
  2. Bedi, A. & Ravi, R. Compositions and methods for targeted immunomodulatory antibodies and fusion proteins. US patent 9,850,306 (2011).

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Metadata

Authors
Ravi, R., Noonan, K. A., Pham, V., Bedi, R., Zhavoronkov, A., Ozerov, I. V., Makarev, E., V. Artemov, A., Wysocki, P. T., Mehra, R., Nimmagadda, S., Marchionni, L., Sidransky, D., Borrello, I. M., Izumchenko, E., & Bedi, A.
Affiliations
  • Johns Hopkins University (US)
  • Stanford University (US)
Corresponding author
Bedi, Atul
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2018-02-21
Volume
9
Issue
1
Pages
741-741
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-017-02696-6
PMC
PMC5821872
OpenAlex
W2789421415
Cited by
325
References
74
Citations by year
2026: 13 · 2025: 26 · 2024: 37 · 2023: 57 · 2022: 45 · 2021: 52 · 2020: 56 · 2019: 17 · 2018: 21
FWCI
13.78
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • National Institutes of Health (DE019032)
  • National Cancer Institute (R01CA184199)
  • National Institute of Dental and Craniofacial Research (P50DE019032)
Topics
  • Health Sciences › Medicine › Oncology › Cancer Immunotherapy and Biomarkers
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › TGF-β signaling in diseases
  • Life Sciences › Immunology and Microbiology › Immunology › T-cell and B-cell Immunology
Keywords
TGFBR2, CTLA-4, PD-L1, immune checkpoint inhibitors, regulatory T cells, tumor microenvironment, cancer immunotherapy, Treg differentiation, immune tolerance, antitumor efficacy
MeSH terms
Animals, Antibodies, Breast Neoplasms, Female, Humans, Immunotherapy, Melanoma, Cohort Studies, Transforming Growth Factor beta, Lymphocytes, Tumor-Infiltrating, Mice, Inbred NOD, Receptors, Transforming Growth Factor beta, Th1 Cells, T-Lymphocytes, Regulatory, Mice, Forkhead Transcription Factors, Th17 Cells, B7-H1 Antigen, CTLA-4 Antigen

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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