Journal: Nature Communications

Article Title: An engineered oncolytic virus expressing PD-L1 inhibitors activates tumor neoantigen-specific T cell responses

doi: 10.1038/s41467-020-15229-5

Figure Lengend Snippet: a A schematic diagram of recombinant vaccinia virus (VV) shuttle vectors that express GM-CSF or/and iPDL1 (soluble PD-1-Fc). vTK, VV thymidine kinase gene; R and L, right and left flank sequences; RFP, red fluorescent protein. b Expression and secretion of iPDL1 from infected MC38 tumor cells infected with the indicated VVs. Anti-IgG Fc (Licor 926-32210; upper) or anti-PD-1 (Biolegend 114101; lower) was used for western blot with reducing or non-reducing loading buffer. The experiment was repeated twice. c , d Serum iPDL1 and GM-CSF levels in different VV-treated MC38-bearing mice at 2 days post-virus injection c . Kinetics of iPDL1 levels in injected tumors or sera of the VV-iPDL1/GM-treated mice d . n = 3 independent samples. Data are presented as the means ± SD. The experiment was repeated twice. e Purified iPDL1 binds to PD-L1 + tumor cell. Upper panel: flow cytometric analysis of PD-L1 expression on shPD-L1/MC38 tumor cells that were transduced with PD-L1-shRNA and wild-type MC38 cells. Lower panel: shPD-L1/MC38 cells and wild-type MC38 cells were incubated with 50 μg/mL of purified iPDL1, an irrelevant MAGE3-IgG Fc fusion protein, or IgG control, followed by staining with an anti-IgG Fc for flow cytometry. f Inhibition of PD-1/PD-L1 binding by purified iPDL1 protein using ELISA. An anti-PD-L1 antibody was used as a positive control; n = 3 independent samples. g iPDL1-mediated ADCC. ADCC Reporter Bioassays were performed in triplicate wells, and the concentrations of iPDL1 protein and control IgG Fc used for this assay are indicated; n = 3 independent samples. Data presented as the means ± SD. The experiment was repeated twice. Significant differences are indicated as *** P < 0.001, or **** P < 0.0001 using two-tailed student’s t -test. h CD11c + DC frequency in monocyte cultures in the presence of culture media of MC38 cells infected with VV-RFP, VV-GM, VV-iPDL1/GM, or GM-CSF as a positive control, and IL-4. i Viral replication in vitro; n = 3 independent samples. j Replication and biodistribution of VV after intratumor injections. Data presented as the means ± SD. The experiment was repeated twice.

Article Snippet: A total of 50 μL mixture of 20 ng mouse PD-1-biotin (Sino Biological, 50124-M08H-B) and purified iPDL1, IgG control (Sigma, I5381), or anti-PD-L1 antibody control (Biolegend, 124301) at indicated concentration, or 50 μL assay buffer (blank) was added into wells, and incubated at RT for 2 h. Diluted streptavidin–HRP was added to each well after wash and incubated at RT for 1 h with slow shaking.

Techniques: Recombinant, Expressing, Infection, Western Blot, Injection, Purification, Transduction, shRNA, Incubation, Staining, Flow Cytometry, Inhibition, Binding Assay, Enzyme-linked Immunosorbent Assay, Positive Control, Two Tailed Test, In Vitro

Journal: Nature Communications

Article Title: An engineered oncolytic virus expressing PD-L1 inhibitors activates tumor neoantigen-specific T cell responses

doi: 10.1038/s41467-020-15229-5

Figure Lengend Snippet: a MC38 tumor cells were infected with VV-RFP, VV-iPDL1/GM at an MOI = 0.5, or PBS for 24 h. The percentage of IgG Fc + population representing iPDL1 (soluble PD-1-IgG Fc)-bound VV-infected (RFP + ) or uninfected (RFP − ) PD-L1-expressing tumor cells was measured by flow cytometry. b MC38 tumor cells that were stimulated with IFN-γ (20 ng/mL) for 48 h were infected with the indicated VVs. PD-L1 expression of infected (RFP + ) or uninfected (RFP − ) cells was determined by flow cytometry. c – g MC38 cells were subcutaneously inoculated into the left (1 × 10 6 ) and right (5 × 10 5 ) flanks of C57BL/6 mice. When left flank tumor sizes reached ~100 mm 3 (counted as day 0), the tumors of the left flank were intratumorally injected with 50 μL of PBS, VV-RFP, VV-GM, or VV-iPDL1/GM (5 × 10 7 pfu per tumor), or 200 μg of anti-PD-L1 antibody (clone 10F.9G2) intravenously on days 0 and 3. Two days post-second VV treatment, VV-treated (primary tumor) and untreated, distant tumors were collected, weighed and digested with collagenase type I and DNase. Tumor cell suspensions were blocked with anti-CD16/32 antibody and then stained with antibodies against CD45, CD3, CD8, CD4, CD11c, CD11b, Gr-1, FoxP3, PD-L1, and IgG Fc to assess PD-L1 expression or IgG Fc + frequency on infected (RFP + ) or uninfected (RFP − ) tumor cells from the treated primary tumors c or untreated distant tumors (CD45 − cells) d , and PD-L1 expression on infiltrating immune cells from treated e or untreated distant tumors f , IgG Fc + frequency on infiltrating immune cells g . Infiltrating immune cells include cytotoxic T cells (CD45 + CD3 + CD8 + ), DCs (CD45 + CD11c + ), myeloid-derived suppressor cell MDSCs (CD45 + CD11c − CD11b + Gr-1 + ), and Treg (CD45 + CD3 + CD4 + FoxP3 + ); n = 5 mice. Significant differences are indicated as ** P < 0.01, *** P < 0.001, or **** P < 0.0001 determined by two-tailed Student’s t -test.

Article Snippet: A total of 50 μL mixture of 20 ng mouse PD-1-biotin (Sino Biological, 50124-M08H-B) and purified iPDL1, IgG control (Sigma, I5381), or anti-PD-L1 antibody control (Biolegend, 124301) at indicated concentration, or 50 μL assay buffer (blank) was added into wells, and incubated at RT for 2 h. Diluted streptavidin–HRP was added to each well after wash and incubated at RT for 1 h with slow shaking.

Techniques: Infection, Expressing, Flow Cytometry, Injection, Staining, Derivative Assay, Two Tailed Test

Journal: Nature Communications

Article Title: An engineered oncolytic virus expressing PD-L1 inhibitors activates tumor neoantigen-specific T cell responses

doi: 10.1038/s41467-020-15229-5

Figure Lengend Snippet: a Enhanced stimulatory potency of tumor-infiltrating DCs. Tumor-infiltrating DCs from VV-treated mice were loaded with neopeptide 4, 9, or 11, and cocultured with the neoantigens-primed T cells from mice immunized with the 11 neopeptide mixture to assess IFN-γ production; n = 3 mice. Data presented as the means ± SD. * P < 0.05, *** P < 0.001 by two-tailed Student’s t -test. b Enhanced maturation of tumor-infiltrating DCs. Using a similar treatment schedule as described in Fig. , cell suspensions prepared from VV-treated tumors were analyzed by flow cytometry. c Enhanced tumor infiltration of CD103 + DCs. Using the same treatment schedule as in Fig. , tumor cell suspensions from VV-treated mice were analyzed by FACS; n = 5 mice. Data presented as the means ± SD. ** P < 0.01 by two-tailed Student’s t -test. d Intracellular staining of IL-12 and CXCL9 of CD103 + DCs from VV-treated tumors. e qRT-PCR analysis of CXCL10 mRNA levels in CD103 + DCs isolated from VV-treated tumors; n = 5 mice. Data presented as the means ± SD. ** P < 0.01 by two-tailed Student’s t -test. f Neoantigens-primed T cells proliferated more efficiently in VV-iPDL1/GM-treated mice. The neoantigens-primed T cells were labeled with 5 μM CFSE and i.v. injected into VV-treated mice. Three days later, T cell proliferation was assessed by FACS. g Enhanced stimulatory effect of VV-iPDL1/GM-infected tumor cells. MC38 tumor cells infected with VVs at MOI = 1 were cocultured with the neoantigens-primed T cells. IFN-γ production (left) and T cell proliferation (right) were measured. Data presented as the means ± SD. * P < 0.05 by two-tailed Student’s t -test. h Serum of VV-iPDL1/GM-treated mice enhanced the cytolytic activity of neoantigens-primed T cells. MC38-Luc cells were cocultured with the neoantigen-specific T cells in the presence of the sera from treated MC38-bearing mice. Cytolytic activity was calculated using luciferase emission value. Data are presented as means ± SD. ** P < 0.01 by two-tailed Student’s t -test. i PD-1 + CD8 + T cells isolated from VV-treated MC38 tumors were cocultured with MC38 cells in the presence of purified iPDL1 or IgG. IFN-γ + frequencies of PD-1 + T cells were shown from one of two independent experiments.

Article Snippet: A total of 50 μL mixture of 20 ng mouse PD-1-biotin (Sino Biological, 50124-M08H-B) and purified iPDL1, IgG control (Sigma, I5381), or anti-PD-L1 antibody control (Biolegend, 124301) at indicated concentration, or 50 μL assay buffer (blank) was added into wells, and incubated at RT for 2 h. Diluted streptavidin–HRP was added to each well after wash and incubated at RT for 1 h with slow shaking.

Techniques: Two Tailed Test, Flow Cytometry, Staining, Quantitative RT-PCR, Isolation, Labeling, Injection, Infection, Activity Assay, Luciferase, Purification

Journal: Cell Reports Medicine

Article Title: Glycoengineering-based anti-PD-1-iRGD peptide conjugate boosts antitumor efficacy through T cell engagement

doi: 10.1016/j.xcrm.2024.101590

Figure Lengend Snippet: Synthesis and characterization of αPD-1-(iRGD) 2 (A) The pattern diagram of the structure and chemical synthesis procedures of αPD-1-(iRGD) 2 . (B) Production of core substrate GDP-fucose-iRGD. (C) ESI-MS characterization of αPD-1-(iRGD) 2 . (D and E) The binding affinity of αPD-1-(iRGD) 2 and unmodified antibody toward human (D) and murine (E) PD-1 protein by ELISA. (F) Mean fluorescence intensity (MFI) fold change of PD-1-Jurkat cells after being incubated with αPD-1 or αPD-1-(iRGD) 2 for 1 h. Data represent mean ± SEM; for (D)–(F), n = 3. (G–K) Flow cytometric analysis of changes in relative averaged fluorescence intensities of cancer cell lines (N87, HGC27, MFC, and B16) and normal cell line (293T) cocultured with 10 μg/mL αPD-1-(iRGD) 2 or 10 μg/mL αPD-1 before coculturing with anti-human IgG-PE (Abcam, #ab7005). The concentration of free iRGD (Genescript Biotech Corporation) was 100 μg/mL, and αNRP1 (Biolegend, #354502 and #145201) was 15 μg/mL. Data represent mean ± SEM; for (G)–(K), n = 3. For (G)–(J), one-way ANOVA test and Tukey’s multiple comparisons test. n.s., not significant; ∗ p < 0.5; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001.

Article Snippet: Recombinant Mouse PD-1 (HEK293, His) , MCE , HY-P73724.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Fluorescence, Incubation, Concentration Assay

Journal: Cell Reports Medicine

Article Title: Glycoengineering-based anti-PD-1-iRGD peptide conjugate boosts antitumor efficacy through T cell engagement

doi: 10.1016/j.xcrm.2024.101590

Figure Lengend Snippet: Antitumor efficacy of αPD-1-(iRGD) 2 (A) Schematic of the treatment regimen in MFC mouse gastric tumor model. Briefly, mice were treated with 1 × 10 6 MFC cells and injected intraperitoneally with PBS (100 μL), αPD-1 (5 mg/kg) alone, or with free iRGD (2.5 μg) and αPD-1-(iRGD) 2 (5 mg/kg) every 3 days. (B and C) Tumor growth profile (B) and weight (C) of tumors collected at the endpoint of (A). (D) Schematic of the treatment regimen in B16F10 mouse melanoma tumor model. Briefly, mice were treated with 1 × 10 5 B16F10 cells and injected intraperitoneally with PBS (100 μL control), αPD-1(5 mg/kg) alone, or with free iRGD (2.5 μg) and αPD-1-(iRGD) 2 (5 mg/kg) every 3 days. (E and F) Tumor growth profile (E) and weight (F) of tumors collected at the endpoint of (D). (G) Survival plot of MFC mouse gastric cancer model. Briefly, mice were treated with 1 × 10 6 MFC cells and injected intraperitoneally with PBS (100 μL control), αPD-1 (5 mg/kg) alone, or with free iRGD (4 μmol/kg) and αPD-1-(iRGD) 2 (5 mg/kg) every 3 days. A total of 4 injections were assigned to each mouse. (H–P) Quantitative flow cytometry results indicating the abundance and characteristics of TILs in an MFC model. 4- to 6-week-old 615 mice were treated with 1 × 10 6 MFC cells and injected intraperitoneally with PBS (100 μL), αPD-1 (5 mg/kg) alone, or with free iRGD(4 μmol/kg) and αPD-1-(iRGD) 2 (5 mg/kg) every 3 days. (H) CD3 + T cells, (I) CD4 + T cells, (J) CD8 + T cells, (K) PD-1 + CD8 + T cells, (L) IFNγ + CD8 + T cells, (M) CD137 + CD8 + T cells, (N) Ki67 + CD8 + T cells, (O) CD137 + CD4 + T cells, and (P) Ki67 + CD4 + T cells. Flow cytometry plots here utilized the following agents: anti-mouse CD3-FITC (Biolegend, #100204), anti-mouse CD3e-PC7 (Biolegend, #100320), anti-mouse CD4-PE (Biolegend, #100408), anti-mouse CD8-APC (Biolegend, #100712), anti-mouse CD8-FITC (Biolegend, #100706), anti-mouse Ki67-PC7 (Biolegend, #151217), anti-mouse PD-1-PerCP5.5 (Biolegend, #124334), anti-mouse IFNγ-PerCP5.5 (Biolegend, #505822), and anti-mouse CD137-PE (Biolegend, #106106). Data represent mean ± SEM; for (A)–(F), n = 6; for (G), n = 8; for (H)–(P), n = 5. For (B) and (E), two-way ANOVA test and Tukey’s multiple comparisons test. For (C), (F), and (H)–(P), one-way ANOVA test and Tukey’s multiple comparisons test; n.s., not significant; ∗ p < 0.5; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001.

Article Snippet: Recombinant Mouse PD-1 (HEK293, His) , MCE , HY-P73724.

Techniques: Injection, Control, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: Glycoengineering-based anti-PD-1-iRGD peptide conjugate boosts antitumor efficacy through T cell engagement

doi: 10.1016/j.xcrm.2024.101590

Figure Lengend Snippet: Antitumor efficacy of αPD-1-(iRGD) 2 depends on pre-existing intra-tumoral CD8 + T cells (A) Schematic of the T cell migration inhibition regimen in MFC mouse gastric tumor model. Briefly, mice were treated with 1 × 10 6 MFC cells and injected intraperitoneally with PBS (100 μL control), αPD-1-(iRGD) 2 (5 mg/kg), and FTY720 (MCE, #HY-12005, 20 μg) every 3 days. (B) Flow cytometry analysis of T cell abundance in peripheral blood to validate the T cell migration inhibition of FTY720. (C) Tumor growth profile of (A). (D) Schematic of the CD8 + T cell depletion regimen in MFC mouse gastric tumor model. Briefly, mice were treated with 1 × 10 6 MFC cells and injected intraperitoneally with PBS (100 μL control), αPD-1-(iRGD) 2 (5 mg/kg), and αCD8 (BioXCell, #BE0117, 200 μg) every 3 days. (E) Flow cytometry analysis of CD8 + T cell abundance in tumor to validate CD8 + T cell depletion. (F) Tumor growth profile of CD8 + T cell depletion assay. (G) Schematic of the treatment regimen in MFC mouse gastric tumor model. Briefly, mice were treated with 1 × 10 6 MFC cells and injected intraperitoneally with PBS (100 μL control), αPD-1 (0.1 mg/kg) alone, or with free iRGD (4 μmol/kg) and αPD-1-(iRGD) 2 (0.1 mg/kg). (H) Weight of murine subcutaneous tumors resected at the end of (G). (I) Tumor growth profile of (G). (J) Flow cytometry results indicating the abundance of CD8 + T cell (Biolegend, #100702) from resected tumor bulk at the endpoint of (G). (K) Flow cytometry quantification of PD-1 (Biolegend, #135206) expression on CD8 + T cell from resected tumor bulk at the endpoint of (G). (L) Flow cytometry quantification of CD39 (Biolegend, #143806) expression on PD-1 + CD8 + T cells from resected tumor bulk at the endpoint of (F). (M and N) Flow cytometry results indicating the abundance of CD4 + T cell (Biolegend, #100408) (M) and Treg (Biolegend, #320014 and #100412; eBioscience, #12-0251-83) (N) from resected tumor bulk at the endpoint of (G). (O) Flow cytometry analysis of propidium-iodide-positive tumor cells preloaded with GP33 (Genescript Biotech Corporation, 500 nM) or SIINFEKL peptide (Genescript Biotech Corporation, 500 nM) after simultaneously coculturing with OT-I cells. The ratio of unprimed, GP33-primed, SIINFEKL-primed B16F10 cells, and OT-I cells was 1:1:1:10. Concentration of αPD-1 and αPD-1-(iRGD) 2 was 10 μg/mL, and that of iRGD was 100 μg/mL (P) Flow cytometry analysis of activation markers (CD25 and CD69) on OT-I cells and spleen T cells simultaneously cocultured with SIINFEKL peptide (500 nM) preloaded tumor cells. The ratio of OT-I cells, non-specific spleen T cells, and SIINFEKL-primed B16 cells was 5:5:1. (Q) Histogram of the percentage of conjugated cells when 2 × 10 5 Dye 670-stained NY-ESO-1 157-165 -primed HLA-A∗0201-Raji cells were cocultured 1 h with mixed 2 × 10 4 CFSE-stained 1G4-PD-1-Jurkat cells and 2 × 10 4 Dye 450-stained PD-1-Jurkat cells. The concentration of αPD-1-(iRGD) 2 was 10 μg/mL, while blinatumomab (MCE, #HY-P9963) was 1 μg/mL (R) Histogram of CD69 (Biolegend, #319102) expression on 1G4-PD-1-Jurkat and PD-1-Jurkat under the same condition in (P). (S) Flow cytometry chart of (Q). (T) Flow cytometry chart of (R). Data represent mean ± SEM. For (B) and (O)–(T), n = 3. For (C), (E), (F), and (H)–(N), n = 5. For (B), (E), (H), and (J)–(M), one-way ANOVA test and Tukey’s multiple comparisons test. For (C), (F), (I), and (O)–(R), two-way ANOVA test and Tukey’s multiple comparisons test. n.s., not significant; ∗ p < 0.5; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001.

Article Snippet: Recombinant Mouse PD-1 (HEK293, His) , MCE , HY-P73724.

Techniques: Migration, Inhibition, Injection, Control, Flow Cytometry, Depletion Assay, Expressing, Concentration Assay, Activation Assay, Staining

Journal: Cell Reports Medicine

Article Title: Glycoengineering-based anti-PD-1-iRGD peptide conjugate boosts antitumor efficacy through T cell engagement

doi: 10.1016/j.xcrm.2024.101590

Figure Lengend Snippet:

Article Snippet: Recombinant Mouse PD-1 (HEK293, His) , MCE , HY-P73724.

Techniques: Recombinant, Activation Assay, Flow Cytometry, Membrane, Software