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Sino Biological recombinant monkey pd l1 ecd
Recombinant Monkey Pd L1 Ecd, supplied by Sino Biological, used in various techniques. Bioz Stars score: 95/100, based on 69 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/monkey+pd1/Human+PD1+%2F+PDCD1+Protein/us12590155-433-37-43
Average 95 stars, based on 69 article reviews

recombinant monkey pd l1 ecd - by Bioz Stars, 2026-10

95/100 stars

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Derivative Assay:

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1
Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Co-culture Assay:

Expressing:

Variant Assay:

Inhibition:

Transduction:

Staining:

Generated:

Control:

Purification:

Labeling:

Isolation:

Blocking Assay:

Imaging:

Sequencing:

Reconstitution Assay:

Fluorescence:

Selection:

Adoptive Transfer Assay:

Flow Cytometry:

Image Search Results

(A) Left, cartoon depicting a human-derived coculture assay containing Jurkat expressing TCR, CD28 and mGFP-tagged hu PD1 or mo PD1, and SEE-pulsed Raji APCs expressing MHCII, CD86, and mCherry-tagged hu PDL1 or mo PDL1. Right, FACS histograms showing the expression of indicated PD1-mGFP or PDL1-mCherry variant on the Jurkat or Raji cells. (B) Atezolizumab dose-responses of secreted IL-2 in the indicated Jurkat:Raji coculture in the presence of 0.1 ng/mL SEE. Data were normalized to the IL-2 amounts at the highest [atezolizumab]. (C) Superantigen dose-responses of % IL-2 inhibition mediated by either hu PD1: hu PDL1 or mo PD1: mo PDL1 pair in the Jurkat:Raji coculture assay depicted in A . Raji cells were pre-treated with 0 or 120 µg/mL Atezolizumab. (D) Left, cartoon depicting a mouse-derived coculture assay containing DO11.10 cells expressing TCR, CD28 and mGFP-tagged hu PD1 or mo PD1, and OVA 323-339 -pulsed A20 APCs expressing MHCII, CD86, and mCherry-tagged hu PDL1 or mo PDL1. Right, FACS histograms showing the expression of indicated PD1-mGFP or PDL1-mCherry variant on the DO11.10 or A20 cell. (E) OVA 323-339 dose-responses of % IL-2 inhibition mediated by either hu PD1: hu PDL1 or mo PD1: mo PDL1 pair in the DO11.10:A20 coculture assay depicted in D . Data are mean ± SD from three independent experiments. ***P < 0.001; two-way ANOVA.

Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet: (A) Left, cartoon depicting a human-derived coculture assay containing Jurkat expressing TCR, CD28 and mGFP-tagged hu PD1 or mo PD1, and SEE-pulsed Raji APCs expressing MHCII, CD86, and mCherry-tagged hu PDL1 or mo PDL1. Right, FACS histograms showing the expression of indicated PD1-mGFP or PDL1-mCherry variant on the Jurkat or Raji cells. (B) Atezolizumab dose-responses of secreted IL-2 in the indicated Jurkat:Raji coculture in the presence of 0.1 ng/mL SEE. Data were normalized to the IL-2 amounts at the highest [atezolizumab]. (C) Superantigen dose-responses of % IL-2 inhibition mediated by either hu PD1: hu PDL1 or mo PD1: mo PDL1 pair in the Jurkat:Raji coculture assay depicted in A . Raji cells were pre-treated with 0 or 120 µg/mL Atezolizumab. (D) Left, cartoon depicting a mouse-derived coculture assay containing DO11.10 cells expressing TCR, CD28 and mGFP-tagged hu PD1 or mo PD1, and OVA 323-339 -pulsed A20 APCs expressing MHCII, CD86, and mCherry-tagged hu PDL1 or mo PDL1. Right, FACS histograms showing the expression of indicated PD1-mGFP or PDL1-mCherry variant on the DO11.10 or A20 cell. (E) OVA 323-339 dose-responses of % IL-2 inhibition mediated by either hu PD1: hu PDL1 or mo PD1: mo PDL1 pair in the DO11.10:A20 coculture assay depicted in D . Data are mean ± SD from three independent experiments. ***P < 0.001; two-way ANOVA.

Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Techniques: Derivative Assay, Co-culture Assay, Expressing, Variant Assay, Inhibition

Left, FACS histograms showing hu PD1 expressions on anti-CD3ε/anti-CD28 stimulated human primary CD4 + T cells and on stable Jurkat cells transduced with hu PD1 driven by either an dSV40 (weak) or an SFFV (strong) promoter. Right, PD1 densities on human primary CD4 + T cells plotted against duration of stimulation by anti-CD3/anti-CD28 beads. Dashed lines indicate the densities of SFFV- and dSV40-driven PD1 expressions on Jurkat cells. PD1 densities were calculated using molecular numbers quantified with a Quantum PE MESF kit and diameters of cells (human primary CD4 + T cell: 4.8 µm, Jurkat cells: 15 µm).

Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet: Left, FACS histograms showing hu PD1 expressions on anti-CD3ε/anti-CD28 stimulated human primary CD4 + T cells and on stable Jurkat cells transduced with hu PD1 driven by either an dSV40 (weak) or an SFFV (strong) promoter. Right, PD1 densities on human primary CD4 + T cells plotted against duration of stimulation by anti-CD3/anti-CD28 beads. Dashed lines indicate the densities of SFFV- and dSV40-driven PD1 expressions on Jurkat cells. PD1 densities were calculated using molecular numbers quantified with a Quantum PE MESF kit and diameters of cells (human primary CD4 + T cell: 4.8 µm, Jurkat cells: 15 µm).

Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Techniques: Transduction

(A) FACS histograms showing expressions of endogenous mo PD1, mo PDL1, mo CD28, mo CD80, and mo CD86 on DO11.10 T cell hybridoma and A20 cells before and after the KO of indicated genes. Isotype antibodies were used as controls. (B) FACS histograms showing mo CD80 and mo CD86 levels on A20 cells transduced with indicated mCherry-tagged PDL1 variants. Controls for mo CD80 and mo CD86 staining were generated with isotype antibodies. Control for PDL1-mCherry corresponded to untransduced PDL1 −/− A20 cells.

Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet: (A) FACS histograms showing expressions of endogenous mo PD1, mo PDL1, mo CD28, mo CD80, and mo CD86 on DO11.10 T cell hybridoma and A20 cells before and after the KO of indicated genes. Isotype antibodies were used as controls. (B) FACS histograms showing mo CD80 and mo CD86 levels on A20 cells transduced with indicated mCherry-tagged PDL1 variants. Controls for mo CD80 and mo CD86 staining were generated with isotype antibodies. Control for PDL1-mCherry corresponded to untransduced PDL1 −/− A20 cells.

Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Techniques: Transduction, Staining, Generated, Control

(A) FACS histograms showing the expressions of indicated mGFP-tagged PD1 or mCherry-tagged PDL1 variants on Jurkat or Raji cells used for cocultures. (B) Left, a cartoon depicting the PD1:PDL1 pairs tested in five parallel Jurkat:Raji coculture assays. Right, bar graphs showing the % inhibition of IL-2 secretion and of CD69 expression by the PD1:PDL1 pairs indicated on the left (n = 6 independent experiments). (C) FACS histograms showing the expressions of indicated mGFP-tagged PD1 or mCherry-tagged PDL1 variants on DO11.10 or A20 cells used for cocultures. (D) Left, a cartoon depicting the PD1:PDL1 pairs tested in five parallel A20:DO11.10 coculture assays. Right, a bar graph showing the % inhibition of IL-2 secretion by the PD1:PDL1 pairs indicated on the left (n = 3 independent experiments). (E) Left, diagram showing the purification method for human primary CD4 + T cells. Right, FACS histograms of CD3 and CD4 expressions on hu PBMC and CD4 + T cells. (F) Top, cartoon depicting a coculture assay containing CD4 + T cells expressing TCR, CD28, and mGFP ECD -TMD-PD1 ICD , and SEB-pulsed Raji APCs expressing MHCII, CD86, and GFPNb-TM-TagBFP. Bottom, FACS histograms showing the expression of indicated PD1 chimera or GFPNb-TM-TagBFP on CD4 + T or Raji cells, respectively. (G) A dot plot showing % inhibition of IL-2 secretion by the PD1 chimera:GFPNb association in CD4 + T cell:Raji coculture depicted in F (n = 3 independent experiments). Data are mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant; student’s t-test.

Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet: (A) FACS histograms showing the expressions of indicated mGFP-tagged PD1 or mCherry-tagged PDL1 variants on Jurkat or Raji cells used for cocultures. (B) Left, a cartoon depicting the PD1:PDL1 pairs tested in five parallel Jurkat:Raji coculture assays. Right, bar graphs showing the % inhibition of IL-2 secretion and of CD69 expression by the PD1:PDL1 pairs indicated on the left (n = 6 independent experiments). (C) FACS histograms showing the expressions of indicated mGFP-tagged PD1 or mCherry-tagged PDL1 variants on DO11.10 or A20 cells used for cocultures. (D) Left, a cartoon depicting the PD1:PDL1 pairs tested in five parallel A20:DO11.10 coculture assays. Right, a bar graph showing the % inhibition of IL-2 secretion by the PD1:PDL1 pairs indicated on the left (n = 3 independent experiments). (E) Left, diagram showing the purification method for human primary CD4 + T cells. Right, FACS histograms of CD3 and CD4 expressions on hu PBMC and CD4 + T cells. (F) Top, cartoon depicting a coculture assay containing CD4 + T cells expressing TCR, CD28, and mGFP ECD -TMD-PD1 ICD , and SEB-pulsed Raji APCs expressing MHCII, CD86, and GFPNb-TM-TagBFP. Bottom, FACS histograms showing the expression of indicated PD1 chimera or GFPNb-TM-TagBFP on CD4 + T or Raji cells, respectively. (G) A dot plot showing % inhibition of IL-2 secretion by the PD1 chimera:GFPNb association in CD4 + T cell:Raji coculture depicted in F (n = 3 independent experiments). Data are mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant; student’s t-test.

Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Techniques: Inhibition, Expressing, Purification, Co-culture Assay

(A) Representative BLI data obtained for the indicated PD1:ligand pairs with the PDL1 or PDL2 immobilized on the sensor chip and increasing concentrations of PD1-ECD presented in the solution. (B) K d values of the indicated PD1:ligand pairs based on the three independent BLI experiments. (C) Left, representative confocal images of a conjugate between Jurkat expressing the indicated PD1 variants (green) and Raji expressing the indicated PDL1 variants (red) with or without anti-PDL1 (atezolizumab). Right, a dot plot showing the synaptic enrichment score of each PD1 variant in the presence or absence of atezolizumab (n = 40 cells). (D) A cartoon depicting a cell-SLB assay, in which Jurkat cells expressing mGFP-tagged PD1 variant interacted with a SLB functionalized with anti- hu CD3ε and hu / mo PDL1-His. PD1 microclusters were visualized via TIRF-M. (E) Top, representative TIRF images showing microclusters of indicated mGFP-tagged PD1 variant in a Jurkat cell that contacted the SLB functionalized with 3 nM hu PDL1 or mo PDL1 that matched the species of the ECD of the corresponding PD1 variant. Bottom, dot plots showing the clustering indices of all five PD1 variants (See methods) (n = 40 cells). (F) Dose-response curves showing PD1 clustering indices of the indicated PD1 variant plotted against [PDL1] (n = 3 independent experiments). Scale bars: 5 µm. Data are mean ± SD. **P < 0.01; ***P < 0.001; ns, not significant; student’s t-test.

Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet: (A) Representative BLI data obtained for the indicated PD1:ligand pairs with the PDL1 or PDL2 immobilized on the sensor chip and increasing concentrations of PD1-ECD presented in the solution. (B) K d values of the indicated PD1:ligand pairs based on the three independent BLI experiments. (C) Left, representative confocal images of a conjugate between Jurkat expressing the indicated PD1 variants (green) and Raji expressing the indicated PDL1 variants (red) with or without anti-PDL1 (atezolizumab). Right, a dot plot showing the synaptic enrichment score of each PD1 variant in the presence or absence of atezolizumab (n = 40 cells). (D) A cartoon depicting a cell-SLB assay, in which Jurkat cells expressing mGFP-tagged PD1 variant interacted with a SLB functionalized with anti- hu CD3ε and hu / mo PDL1-His. PD1 microclusters were visualized via TIRF-M. (E) Top, representative TIRF images showing microclusters of indicated mGFP-tagged PD1 variant in a Jurkat cell that contacted the SLB functionalized with 3 nM hu PDL1 or mo PDL1 that matched the species of the ECD of the corresponding PD1 variant. Bottom, dot plots showing the clustering indices of all five PD1 variants (See methods) (n = 40 cells). (F) Dose-response curves showing PD1 clustering indices of the indicated PD1 variant plotted against [PDL1] (n = 3 independent experiments). Scale bars: 5 µm. Data are mean ± SD. **P < 0.01; ***P < 0.001; ns, not significant; student’s t-test.

Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Techniques: Expressing, Variant Assay

Left, cartoon depicting a TIRF-M assay visualizing PD1 ECD -attached, bodipy-labeled LUVs captured by PDL1 ECD -attached SLB. Middle, representative TIRF images of LUVs (bodipy) and SLBs (DiD) in the presence or absence of anti-PDL1 blockade antibody (atezolizumab). Right, normalized F.I. of LUVs in the TIRF field under indicated conditions. Scale bars: 5 µm. Data are mean ± SD from three independent experiments. **P < 0.01; student’s t-test.

Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet: Left, cartoon depicting a TIRF-M assay visualizing PD1 ECD -attached, bodipy-labeled LUVs captured by PDL1 ECD -attached SLB. Middle, representative TIRF images of LUVs (bodipy) and SLBs (DiD) in the presence or absence of anti-PDL1 blockade antibody (atezolizumab). Right, normalized F.I. of LUVs in the TIRF field under indicated conditions. Scale bars: 5 µm. Data are mean ± SD from three independent experiments. **P < 0.01; student’s t-test.

Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Techniques: Labeling

(A) A cartoon depicting a cell-SLB assay using Jurkat expressing PD1-mGFP. (B) Time lapse TIRF images visualizing microclusters of indicated PD1 variants. Scale bars: 5 µm.

Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet: (A) A cartoon depicting a cell-SLB assay using Jurkat expressing PD1-mGFP. (B) Time lapse TIRF images visualizing microclusters of indicated PD1 variants. Scale bars: 5 µm.

Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Techniques: Expressing

(A) Left, IL-2 secretion from human CD8 + T cells stimulated by SEB-loaded hu PDL1-expressing Raji cells, with or without the presence of anti-PD1 blockade antibody (pembrolizumab), with or without 5 µM SHP099. Right, degree of PD1-mediated inhibition of IL-2 secretion with or without 5 µM SHP099, calculated based on data on the left. (B) Same as A except using mouse CD8 + T cells isolated from the splenocytes of P14 mice, and using anti- mo PD1 RMP1-14 to block PD1 signaling. (C) Degrees of SHP099 mediated inhibition of endogenous PD1 function in human and mouse CD8 + T cells calculated based on data from A and B . (D) Same as B except using Pdcd1 −/− P14 mouse CD8 + T cells engineered to express ICD-humanized mo PD1. (E) Same as B except using Pdcd1 −/− P14 mouse CD8 + T cells reconstituted with WT mo PD1. (F) Degrees of SHP099 mediated inhibition of exogenous PD1 function in Pdcd1 −/− P14 mouse CD8 + T cells calculated based on data from D and E . Data are mean ± SD from three independent experiments. **P<0.01, ***P < 0.001, ns, not significant; student’s t-test.

Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet: (A) Left, IL-2 secretion from human CD8 + T cells stimulated by SEB-loaded hu PDL1-expressing Raji cells, with or without the presence of anti-PD1 blockade antibody (pembrolizumab), with or without 5 µM SHP099. Right, degree of PD1-mediated inhibition of IL-2 secretion with or without 5 µM SHP099, calculated based on data on the left. (B) Same as A except using mouse CD8 + T cells isolated from the splenocytes of P14 mice, and using anti- mo PD1 RMP1-14 to block PD1 signaling. (C) Degrees of SHP099 mediated inhibition of endogenous PD1 function in human and mouse CD8 + T cells calculated based on data from A and B . (D) Same as B except using Pdcd1 −/− P14 mouse CD8 + T cells engineered to express ICD-humanized mo PD1. (E) Same as B except using Pdcd1 −/− P14 mouse CD8 + T cells reconstituted with WT mo PD1. (F) Degrees of SHP099 mediated inhibition of exogenous PD1 function in Pdcd1 −/− P14 mouse CD8 + T cells calculated based on data from D and E . Data are mean ± SD from three independent experiments. **P<0.01, ***P < 0.001, ns, not significant; student’s t-test.

Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Techniques: Expressing, Inhibition, Isolation, Blocking Assay

( A ) A cartoon showing a cell-SLB assay for TIRF imaging of PD1:Shp2 association in Jurkat or human CD4 + T cells. ( B ) Left, representative TIRF images of the indicated mGFP-tagged PD1 variants (green) and anti-Shp2 (magenta) at the interface of a Jurkat cell and the SLB as depicted in A . Right, dot plots showing the anti-Shp2 F.I. normalized to PD1 F.I. (n = 40 cells). ( C ) Same as B , except that human CD4 + T cells expressing the indicated PD1 variants were imaged (n = 40 cells). ( D, E ) Same as A, B , except that DO11.10 cells were observed. ( F ) AA sequence alignment of hu PD1 ICD and mo PD1 ICD , with ITIM, PRS, PEQ/H, and ITSM highlighted. ( G ) Cartoon of a liposome reconstitution assay for measuring PD1 ICD :Shp2 tSH2 interaction. ( H ) Left, representative time courses of SC505 (Shp2 tSH2 ) F.I. at increasing concentrations of hu PD1 ICD . Right, % SC505 quenching 30 min after ATP addition plotted against the [ hu PD1 ICD ] and [ mo PD1 ICD ] (n = 3 independent experiments). ( I ) Bar graphs summarizing apparent K d of Shp2 tSH2 interaction with indicated PD1 ICD variants determined via assays shown in G and H (n = 3 independent experiments). ( J ) Left, representative TIRF images showing Shp2 recruitment to microclusters of indicated PD1 variants in a Jurkat-SLB assays. Right, dot plots showing anti-Shp2 F.I. normalized to PD1 F.I. (n = 40 cells). Scale bars: 5 µm. Data are mean ± SD. **P < 0.01; ***P < 0.001; ns, not significant; student’s t-test.

Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet: ( A ) A cartoon showing a cell-SLB assay for TIRF imaging of PD1:Shp2 association in Jurkat or human CD4 + T cells. ( B ) Left, representative TIRF images of the indicated mGFP-tagged PD1 variants (green) and anti-Shp2 (magenta) at the interface of a Jurkat cell and the SLB as depicted in A . Right, dot plots showing the anti-Shp2 F.I. normalized to PD1 F.I. (n = 40 cells). ( C ) Same as B , except that human CD4 + T cells expressing the indicated PD1 variants were imaged (n = 40 cells). ( D, E ) Same as A, B , except that DO11.10 cells were observed. ( F ) AA sequence alignment of hu PD1 ICD and mo PD1 ICD , with ITIM, PRS, PEQ/H, and ITSM highlighted. ( G ) Cartoon of a liposome reconstitution assay for measuring PD1 ICD :Shp2 tSH2 interaction. ( H ) Left, representative time courses of SC505 (Shp2 tSH2 ) F.I. at increasing concentrations of hu PD1 ICD . Right, % SC505 quenching 30 min after ATP addition plotted against the [ hu PD1 ICD ] and [ mo PD1 ICD ] (n = 3 independent experiments). ( I ) Bar graphs summarizing apparent K d of Shp2 tSH2 interaction with indicated PD1 ICD variants determined via assays shown in G and H (n = 3 independent experiments). ( J ) Left, representative TIRF images showing Shp2 recruitment to microclusters of indicated PD1 variants in a Jurkat-SLB assays. Right, dot plots showing anti-Shp2 F.I. normalized to PD1 F.I. (n = 40 cells). Scale bars: 5 µm. Data are mean ± SD. **P < 0.01; ***P < 0.001; ns, not significant; student’s t-test.

Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Techniques: Imaging, Expressing, Sequencing, Reconstitution Assay

(A) FACS histograms showing the expressions of indicated mGFP-tagged PD1 variant and mCherry-tagged huShp2 in human primary CD4 + T cells. T cells expressing both mCherry-huShp2 and the indicated PD1 variant were sorted and used in cell-SLB assays. (B) Left, representative TIRF images showing microclusters of indicated PD1-mGFP variants and mCherry-Shp2. Right, dot plots showing mCherry (Shp2) F.I. normalized to (mGFP) PD1 F.I. in TIRF images. Scale bars: 5 µm. Data are mean ± SD from n = 30 cells. ***P < 0.001; student’s t-test.

Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet: (A) FACS histograms showing the expressions of indicated mGFP-tagged PD1 variant and mCherry-tagged huShp2 in human primary CD4 + T cells. T cells expressing both mCherry-huShp2 and the indicated PD1 variant were sorted and used in cell-SLB assays. (B) Left, representative TIRF images showing microclusters of indicated PD1-mGFP variants and mCherry-Shp2. Right, dot plots showing mCherry (Shp2) F.I. normalized to (mGFP) PD1 F.I. in TIRF images. Scale bars: 5 µm. Data are mean ± SD from n = 30 cells. ***P < 0.001; student’s t-test.

Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Techniques: Variant Assay, Expressing

(A) Cartoon of a liposome reconstitution assay for measuring PD1 ICD :Shp1 tSH2 interaction. Same as except using hu Shp1 tSH2 rather than hu Shp2 tSH2 . (B) % ATP-triggered quenching of Shp1 tSH2 fluorescence plotted against either [ hu PD1-ICD] or [ mo PD1-ICD]. (C) K d values of PD1:Shp1 tSH2 interaction determined via fitting the data in B . Data are mean ± SD from three independent experiments. *P < 0.05; student’s t-test.

Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet: (A) Cartoon of a liposome reconstitution assay for measuring PD1 ICD :Shp1 tSH2 interaction. Same as except using hu Shp1 tSH2 rather than hu Shp2 tSH2 . (B) % ATP-triggered quenching of Shp1 tSH2 fluorescence plotted against either [ hu PD1-ICD] or [ mo PD1-ICD]. (C) K d values of PD1:Shp1 tSH2 interaction determined via fitting the data in B . Data are mean ± SD from three independent experiments. *P < 0.05; student’s t-test.

Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Techniques: Reconstitution Assay, Fluorescence

( A ) Phylogeny of 236 vertebrate species, color-coded based on the pre-ITSM sequence of PD1. ( B ) Left, a phylogenetic tree of pre-ITSM sequence found in PD1 orthologs. Middle, representative TIRF images of microclusters of GFP-tagged hu PD1 variant bearing the indicated pre-ITSM sequence (green) and endogenous Shp2 (magenta) in an anti- hu CD3ε/ hu PDL1 stimulated Jurkat cell. Right, dot plots showing anti-Shp2 F.I. normalized to PD1 F.I. in Jurkat cells expressing the indicated hu PD1 variant (n = 40 cells). ( C ) The subtree of the 107-species mammal phylogeny spanning the rodent clade and the primate clade. The branch lengths were inferred based on PD1 codon sequence evolution. The marked nodes are mouse, human or the ones with reconstructed ancestral sequences. The color codes show the inhibition ability of the sequences at respective nodes, based on data in D. ( D ) Left, FACS histograms showing the expressions of hu PD1 chimera harboring an ICD corresponding to the indicated node, on Jurkat cells. Right, % inhibition of IL-2 secretion by the indicated hu PD1 chimera upon stimulating Jurkat ( hu PD1 chimera) with Raji ( hu PDL1). The ITIM and pre-ITSM type is labeled: hu , human-like, mo , mouse like (n = 5 independent experiments). Data are mean ± SD. Scale bars: 5 µm. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant; student’s t-test.

Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet: ( A ) Phylogeny of 236 vertebrate species, color-coded based on the pre-ITSM sequence of PD1. ( B ) Left, a phylogenetic tree of pre-ITSM sequence found in PD1 orthologs. Middle, representative TIRF images of microclusters of GFP-tagged hu PD1 variant bearing the indicated pre-ITSM sequence (green) and endogenous Shp2 (magenta) in an anti- hu CD3ε/ hu PDL1 stimulated Jurkat cell. Right, dot plots showing anti-Shp2 F.I. normalized to PD1 F.I. in Jurkat cells expressing the indicated hu PD1 variant (n = 40 cells). ( C ) The subtree of the 107-species mammal phylogeny spanning the rodent clade and the primate clade. The branch lengths were inferred based on PD1 codon sequence evolution. The marked nodes are mouse, human or the ones with reconstructed ancestral sequences. The color codes show the inhibition ability of the sequences at respective nodes, based on data in D. ( D ) Left, FACS histograms showing the expressions of hu PD1 chimera harboring an ICD corresponding to the indicated node, on Jurkat cells. Right, % inhibition of IL-2 secretion by the indicated hu PD1 chimera upon stimulating Jurkat ( hu PD1 chimera) with Raji ( hu PDL1). The ITIM and pre-ITSM type is labeled: hu , human-like, mo , mouse like (n = 5 independent experiments). Data are mean ± SD. Scale bars: 5 µm. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant; student’s t-test.

Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Techniques: Sequencing, Variant Assay, Expressing, Inhibition, Labeling

(A) AA sequence alignment of ITIM-ITSM region in PD1 of five selected species, with the pre-ITSM region annotated with a box. (B) Left, FACS histograms showing the expressions of mGFP-tagged hu PD1 variants that harbored the indicated PD1 ICD in five engineered Jurkat lines. Right, bar graphs showing % inhibition of IL-2 secretion mediated by the indicated hu PD1 variants. The five Jurkat lines were stimulated in parallel using SEE-pulsed, hu PDL1-expressing Raji cells, with or without Atezo (n = 3 independent experiments). Data are mean ± SD. *P < 0.05; ***P < 0.001; ns, not significant; student’s t-test.

Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet: (A) AA sequence alignment of ITIM-ITSM region in PD1 of five selected species, with the pre-ITSM region annotated with a box. (B) Left, FACS histograms showing the expressions of mGFP-tagged hu PD1 variants that harbored the indicated PD1 ICD in five engineered Jurkat lines. Right, bar graphs showing % inhibition of IL-2 secretion mediated by the indicated hu PD1 variants. The five Jurkat lines were stimulated in parallel using SEE-pulsed, hu PDL1-expressing Raji cells, with or without Atezo (n = 3 independent experiments). Data are mean ± SD. *P < 0.05; ***P < 0.001; ns, not significant; student’s t-test.

Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Techniques: Sequencing, Inhibition, Expressing

Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet:

Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Techniques: Selection

( A ) Schematic of an adoptive transfer experiment using Pdcd1 −/− P14 CD8 + T cells and B16.gp33 cells. Pdcd1 −/− P14 cells were retrovirally transduced with exoPD1 and Thy1.1, and adoptively transferred to mice bearing B16.gp33 melanoma. ( B ) Surface staining of indicated mo PD1 variants and Thy1.1 on P14 cells transferred to mice in A . ( C ) Tumor growth curves in mice received 1 million Pdcd1 −/− P14 cells expressing either mo PD1 or mo PD1 hu ICD (n = 2-4 mice). ( D ) Number of Thy1.1+ intratumoral CD8 + T cells (n = 5 tumors). ( E ) % TCF-1+, % TIM-3+, % IFNγ+, and % GzmB+ population within TOX+ intratumoral P14 cells containing either mo PD1 or mo PD1 hu ICD , based on flow cytometry; n = 4-5 tumors. ( F ) Expressions of the indicated genes in Tox + intratumoral P14 cells containing either mo PD1 or mo PD1 hu ICD , based on scRNAseq. ( G ) UMAP showing the Tox + intratumoral P14 cells containing either mo PD1 or mo PD1 hu ICD and their cell clusters. ( H ) Dot plot of indicated gene expressions in the cell subsets identified in G . ( I ) % population of the indicated subsets of intratumoral P14 cells containing either mo PD1 or mo PD1 hu ICD ; n = 4-6 tumors. ( J ) B16.gp33 tumor growth curves in mice that received 1.2 million P14 cells containing mo PD1 WT (left) or mo PD1 hu ICD (right), in response to treatment of either anti-PD1 (blue) or isotype control (magenta); n = 7 mice per group. ( K ) Model showing how PD1 humanization affects the precursor-to-terminal differentiation of Tox + intratumoral CD8+ T cells. Data are mean ± SEM ( C , D , E , I , and J ). For violin-box plots ( F ), black bars are median, the boxes are 25% to 75% interquartile range, and the whiskers stand for minimum to maximum values excluding outliers. *P < 0.5; **P < 0.01; ***P < 0.001; ns, not significant; two-way ANOVA ( C and J ), student’s t-test ( D , E , and I ), or Wilcoxon signed-rank test ( F ).

Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet: ( A ) Schematic of an adoptive transfer experiment using Pdcd1 −/− P14 CD8 + T cells and B16.gp33 cells. Pdcd1 −/− P14 cells were retrovirally transduced with exoPD1 and Thy1.1, and adoptively transferred to mice bearing B16.gp33 melanoma. ( B ) Surface staining of indicated mo PD1 variants and Thy1.1 on P14 cells transferred to mice in A . ( C ) Tumor growth curves in mice received 1 million Pdcd1 −/− P14 cells expressing either mo PD1 or mo PD1 hu ICD (n = 2-4 mice). ( D ) Number of Thy1.1+ intratumoral CD8 + T cells (n = 5 tumors). ( E ) % TCF-1+, % TIM-3+, % IFNγ+, and % GzmB+ population within TOX+ intratumoral P14 cells containing either mo PD1 or mo PD1 hu ICD , based on flow cytometry; n = 4-5 tumors. ( F ) Expressions of the indicated genes in Tox + intratumoral P14 cells containing either mo PD1 or mo PD1 hu ICD , based on scRNAseq. ( G ) UMAP showing the Tox + intratumoral P14 cells containing either mo PD1 or mo PD1 hu ICD and their cell clusters. ( H ) Dot plot of indicated gene expressions in the cell subsets identified in G . ( I ) % population of the indicated subsets of intratumoral P14 cells containing either mo PD1 or mo PD1 hu ICD ; n = 4-6 tumors. ( J ) B16.gp33 tumor growth curves in mice that received 1.2 million P14 cells containing mo PD1 WT (left) or mo PD1 hu ICD (right), in response to treatment of either anti-PD1 (blue) or isotype control (magenta); n = 7 mice per group. ( K ) Model showing how PD1 humanization affects the precursor-to-terminal differentiation of Tox + intratumoral CD8+ T cells. Data are mean ± SEM ( C , D , E , I , and J ). For violin-box plots ( F ), black bars are median, the boxes are 25% to 75% interquartile range, and the whiskers stand for minimum to maximum values excluding outliers. *P < 0.5; **P < 0.01; ***P < 0.001; ns, not significant; two-way ANOVA ( C and J ), student’s t-test ( D , E , and I ), or Wilcoxon signed-rank test ( F ).

Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Techniques: Adoptive Transfer Assay, Transduction, Staining, Expressing, Flow Cytometry, Control

(A) A diagram depicting an adoptive transfer experiment using Cas9 +/- P14 CD8 + T cells and B16.gp33 cells. Cas9 +/- P14 CD8 + T cells were retrovirally transduced with sgRNA targeting Pdcd1 , exoPD1, and Thy1.1 to KO the Pdcd1 and reconstitute with exoPD1. Thy1.1 + CD8 + T cells were sorted and adoptively transferred to mice bearing B16.gp33 melanoma. (B) Pdcd1 KO scores of Cas9 +/- P14 CD8 + T cells calculated as described in Methods (n = 3 independent experiments). (C) Surface staining of indicated mo PD1 variants and Thy1.1 on T cells transferred to mice in A . (D) Tumor growth curves in individual mice received 0.5 million Cas9 +/- Pdcd1 −/− P14 CD8 + T cells expressing the indicated mo PD1 variant. Averaged tumor growth curves are shown in the bottom-right graph (n = 2-4 tumors). Data are mean ± SD ( B ) or SEM ( D ). *P < 0.05; **P < 0.01; ns, not significant; two-way ANOVA

Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet: (A) A diagram depicting an adoptive transfer experiment using Cas9 +/- P14 CD8 + T cells and B16.gp33 cells. Cas9 +/- P14 CD8 + T cells were retrovirally transduced with sgRNA targeting Pdcd1 , exoPD1, and Thy1.1 to KO the Pdcd1 and reconstitute with exoPD1. Thy1.1 + CD8 + T cells were sorted and adoptively transferred to mice bearing B16.gp33 melanoma. (B) Pdcd1 KO scores of Cas9 +/- P14 CD8 + T cells calculated as described in Methods (n = 3 independent experiments). (C) Surface staining of indicated mo PD1 variants and Thy1.1 on T cells transferred to mice in A . (D) Tumor growth curves in individual mice received 0.5 million Cas9 +/- Pdcd1 −/− P14 CD8 + T cells expressing the indicated mo PD1 variant. Averaged tumor growth curves are shown in the bottom-right graph (n = 2-4 tumors). Data are mean ± SD ( B ) or SEM ( D ). *P < 0.05; **P < 0.01; ns, not significant; two-way ANOVA

Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Techniques: Adoptive Transfer Assay, Transduction, Staining, Expressing, Variant Assay

(A) % IFNγ+, % GzmB+, and %TOX+ of the intratumoral P14 T cells expressing either mo PD1 WT or mo PD1 hu ICD measured by flow cytometry (n = 4-6 tumors). (B) Schematic showing scRNA-seq comparing intratumoral P14 Thy1.1+ CD8+ T cells expressing either mo PD1 WT or mo PD1 hu ICD . Each mouse was adoptively transferred with 2 million P14 cells at 7 days after B16.gp33 inoculation. Another 7 days later, intratumoral P14 cells were purified, hash-tagged, and subjected to scRNA-seq. (C) UMAP showing the intratumoral P14 Thy1.1+ cells expressing mo PD1 WT (orange) or hu PD1 hu ICD (blue). (D) Expressions of the indicated transcripts in the intratumoral P14 Thy1.1+ cells expressing either mo PD1 WT or mo PD1 hu ICD . (E) UMAP showing cell clusters (left) and the indicated gene expressions (right) of intratumoral P14 cells. Stem-like exhausted cells (cluster 2; Tox high , Tcf7 high ), Tex1 (cluster 0; Tcf7 low , Mki67 high ), Tex2 (cluster 1; Tcf7 low , Mki67 low ), Prf1 -high effector cells (cluster 3; Tox low , Pfr1 high , Gzmb high ), and Gzma/k -high effector cells (cluster 4; Tox low , Gzmb high , Gzma high , Gzmk high ) are annotated. (F) Left, % population of the indicated cell clusters in E for intratumoral P14 cells expressing either mo PD1 or mo PD1 hu ICD . Right, summarized % population of Tox high cell clusters shown in the left (n = 4-6 tumors). Data are mean ± SEM ( A and F ), or violin-box plot ( D ), in which the black bars are median, boxes are 25% to 75% interquartile range, whiskers are minimum to maximum values excluding outliers. *P < 0.05; ns, not significant; FDR, false discovery rate; student’s t-test ( A and F ), or Wilcoxon signed-rank test ( D ).

Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet: (A) % IFNγ+, % GzmB+, and %TOX+ of the intratumoral P14 T cells expressing either mo PD1 WT or mo PD1 hu ICD measured by flow cytometry (n = 4-6 tumors). (B) Schematic showing scRNA-seq comparing intratumoral P14 Thy1.1+ CD8+ T cells expressing either mo PD1 WT or mo PD1 hu ICD . Each mouse was adoptively transferred with 2 million P14 cells at 7 days after B16.gp33 inoculation. Another 7 days later, intratumoral P14 cells were purified, hash-tagged, and subjected to scRNA-seq. (C) UMAP showing the intratumoral P14 Thy1.1+ cells expressing mo PD1 WT (orange) or hu PD1 hu ICD (blue). (D) Expressions of the indicated transcripts in the intratumoral P14 Thy1.1+ cells expressing either mo PD1 WT or mo PD1 hu ICD . (E) UMAP showing cell clusters (left) and the indicated gene expressions (right) of intratumoral P14 cells. Stem-like exhausted cells (cluster 2; Tox high , Tcf7 high ), Tex1 (cluster 0; Tcf7 low , Mki67 high ), Tex2 (cluster 1; Tcf7 low , Mki67 low ), Prf1 -high effector cells (cluster 3; Tox low , Pfr1 high , Gzmb high ), and Gzma/k -high effector cells (cluster 4; Tox low , Gzmb high , Gzma high , Gzmk high ) are annotated. (F) Left, % population of the indicated cell clusters in E for intratumoral P14 cells expressing either mo PD1 or mo PD1 hu ICD . Right, summarized % population of Tox high cell clusters shown in the left (n = 4-6 tumors). Data are mean ± SEM ( A and F ), or violin-box plot ( D ), in which the black bars are median, boxes are 25% to 75% interquartile range, whiskers are minimum to maximum values excluding outliers. *P < 0.05; ns, not significant; FDR, false discovery rate; student’s t-test ( A and F ), or Wilcoxon signed-rank test ( D ).

Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Techniques: Expressing, Flow Cytometry, Purification

(A) UMAP showing the cell cycle phases of the five subclusters of Tox + P14 cells expressing either mo PD1 WT or mo PD1 huICD . (B) Violin plots showing the cell cycle phase of the five subclusters of Tox + P14 cells.

Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet: (A) UMAP showing the cell cycle phases of the five subclusters of Tox + P14 cells expressing either mo PD1 WT or mo PD1 huICD . (B) Violin plots showing the cell cycle phase of the five subclusters of Tox + P14 cells.

Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Techniques: Expressing

(A) Schematic of the adoptive transfer experiment. Pdcd1 −/− P14 cells were retrovirally transduced with exoPD1 and Thy1.1, and adoptively transferred to mice bearing B16.gp33 melanoma. The host mice then received anti-PD1 or isotype control on day 10, 14, and 17. (B) Tumor growth curves of individual mice that received 1.2 million Pdcd1 −/− P14 T cells expressing either mo PD1 or mo PD1 hu ICD , and treated with either anti-PD1 or isotype control antibody.

Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet: (A) Schematic of the adoptive transfer experiment. Pdcd1 −/− P14 cells were retrovirally transduced with exoPD1 and Thy1.1, and adoptively transferred to mice bearing B16.gp33 melanoma. The host mice then received anti-PD1 or isotype control on day 10, 14, and 17. (B) Tumor growth curves of individual mice that received 1.2 million Pdcd1 −/− P14 T cells expressing either mo PD1 or mo PD1 hu ICD , and treated with either anti-PD1 or isotype control antibody.

Article Snippet: cDNA of rat PD1 (#RG80448-G), dog PD1 (#DG70109-G), and monkey PD1 (#KG90305-G) were purchased from Sino Biological.

Techniques: Adoptive Transfer Assay, Transduction, Control, Expressing

ELISA assay comparing the binding of clG4 and Nivolumab reference to human PD-1 (a). ELISA assay comparing the ability of clG4 and Nivolumab reference to block binding of human PD-Ll to human PD-1 (b). Data points are means ± SD.

Journal: PLoS ONE

Article Title: Structural basis of HLX10 PD-1 receptor recognition, a promising anti-PD-1 antibody clinical candidate for cancer immunotherapy

doi: 10.1371/journal.pone.0257972

Figure Lengend Snippet: ELISA assay comparing the binding of clG4 and Nivolumab reference to human PD-1 (a). ELISA assay comparing the ability of clG4 and Nivolumab reference to block binding of human PD-Ll to human PD-1 (b). Data points are means ± SD.

Article Snippet: Recombinant cynomolgus monkey PD-1-ECD-Fc (catalog# 90311-C02H), mouse PD-1-ECD-Fc (catalog# 50124-M02H) and rat PD-1 ECD-Fc (catalog# 80448-R02H) were purchased from Sino Biological Inc. Recombinant human PD-1-ECD-Fc was expressed in CHO-S cells by pairing human PD-1 (Leu25-Thr168) to human IgG1 Fc and purified with protein A agarose (MabSelect SuRe; GE Healthcare Life Sciences).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Blocking Assay

The cross-reactivity of HLX10 binding to PD-1 extracellular domains (ECDs) of (a) human, (b) cynomolgus monkey, (c) mouse and (d) rat were determined using ELISA. HLX04, an unrelated human antibody mAb, was used as a negative control. All data points represent the mean man antibody human.

Journal: PLoS ONE

Article Title: Structural basis of HLX10 PD-1 receptor recognition, a promising anti-PD-1 antibody clinical candidate for cancer immunotherapy

doi: 10.1371/journal.pone.0257972

Figure Lengend Snippet: The cross-reactivity of HLX10 binding to PD-1 extracellular domains (ECDs) of (a) human, (b) cynomolgus monkey, (c) mouse and (d) rat were determined using ELISA. HLX04, an unrelated human antibody mAb, was used as a negative control. All data points represent the mean man antibody human.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Negative Control

(a) The binding of HLX10 antibody to either CHO-S or PD-1 transfected CHO-S cells was assessed by flow cytometry. The reference anti PD-1 antibody (Nivolumab) and anti-PD-L1 were used as the positive control and negative control (mAb control), respectively. Binding is determined as the mean fluorescent intensity (MFI) of staining. (b) The binding of HLX10 to PHA activated human T-cells was tested by flow cytometry. The ability of HLX10 to inhibit either PD-L1 (c) or PD-L2 (d) binding to cell surface PD-1 was assessed using flow cytometry. Anti-VEGF humanized antibody was used as the negative control (mAb control). All data points represent the means ± SD of triplicate. (e) PD-L1 blocking reporter assay. PDL-1 blocking activity is presented as increase in luciferase signal upon blocking by either HLX10 or Nivolumab reference antibody. Each datapoint represents mean ± SD of duplicate (n = 2).

Journal: PLoS ONE

Article Title: Structural basis of HLX10 PD-1 receptor recognition, a promising anti-PD-1 antibody clinical candidate for cancer immunotherapy

doi: 10.1371/journal.pone.0257972

Figure Lengend Snippet: (a) The binding of HLX10 antibody to either CHO-S or PD-1 transfected CHO-S cells was assessed by flow cytometry. The reference anti PD-1 antibody (Nivolumab) and anti-PD-L1 were used as the positive control and negative control (mAb control), respectively. Binding is determined as the mean fluorescent intensity (MFI) of staining. (b) The binding of HLX10 to PHA activated human T-cells was tested by flow cytometry. The ability of HLX10 to inhibit either PD-L1 (c) or PD-L2 (d) binding to cell surface PD-1 was assessed using flow cytometry. Anti-VEGF humanized antibody was used as the negative control (mAb control). All data points represent the means ± SD of triplicate. (e) PD-L1 blocking reporter assay. PDL-1 blocking activity is presented as increase in luciferase signal upon blocking by either HLX10 or Nivolumab reference antibody. Each datapoint represents mean ± SD of duplicate (n = 2).

Techniques: Binding Assay, Transfection, Flow Cytometry, Positive Control, Negative Control, Staining, Blocking Assay, Reporter Assay, Activity Assay, Luciferase

(a) Binding of Nivolumab is mainly located on the top (N-terminal extension, BC loop, and FG loop) of h-PD-1, whereas Pembrolizumab and HLX10 is located on CC’FG sheets of h-PD-1. hPD-1 is represented as surface in grey. PD-L1, Pembrolizumab, Nivolumab, and HLX10 are colored in slate, deep teal, lime and magenta ribbon, respectively (b) Residues of hPD-1 contribute to the interactions with different binders. hPD-1 is represented as white surface with binding epitope colored in slate. The residues which are involved in hydrogen bond, salt bridge and hydrophobic interaction are colored in blue, red and black, respectively.

Journal: PLoS ONE

Article Title: Structural basis of HLX10 PD-1 receptor recognition, a promising anti-PD-1 antibody clinical candidate for cancer immunotherapy

doi: 10.1371/journal.pone.0257972

Figure Lengend Snippet: (a) Binding of Nivolumab is mainly located on the top (N-terminal extension, BC loop, and FG loop) of h-PD-1, whereas Pembrolizumab and HLX10 is located on CC’FG sheets of h-PD-1. hPD-1 is represented as surface in grey. PD-L1, Pembrolizumab, Nivolumab, and HLX10 are colored in slate, deep teal, lime and magenta ribbon, respectively (b) Residues of hPD-1 contribute to the interactions with different binders. hPD-1 is represented as white surface with binding epitope colored in slate. The residues which are involved in hydrogen bond, salt bridge and hydrophobic interaction are colored in blue, red and black, respectively.

Techniques: Binding Assay

(a) Mean Serum drug concentration-time curves of HLX10 following a single IV-infusion at 3, 10 and 30 mg/kg (N = 3 ). (b) Receptor occupancy rate of HLX10 on PD-1 on the surface of T cells following a single IV-infusion at 3, 10 and 30 mg/kg, which was assessed by flow cytometry.

Journal: PLoS ONE

Article Title: Structural basis of HLX10 PD-1 receptor recognition, a promising anti-PD-1 antibody clinical candidate for cancer immunotherapy

doi: 10.1371/journal.pone.0257972

Figure Lengend Snippet: (a) Mean Serum drug concentration-time curves of HLX10 following a single IV-infusion at 3, 10 and 30 mg/kg (N = 3 ). (b) Receptor occupancy rate of HLX10 on PD-1 on the surface of T cells following a single IV-infusion at 3, 10 and 30 mg/kg, which was assessed by flow cytometry.

Techniques: Concentration Assay, Flow Cytometry

In vitro receptor occupancy rate of HLX10 on PD-1 on the surface of human activated T cells, which was assessed by flow cytometry.

Journal: PLoS ONE

Article Title: Structural basis of HLX10 PD-1 receptor recognition, a promising anti-PD-1 antibody clinical candidate for cancer immunotherapy

doi: 10.1371/journal.pone.0257972

Figure Lengend Snippet: In vitro receptor occupancy rate of HLX10 on PD-1 on the surface of human activated T cells, which was assessed by flow cytometry.

Techniques: In Vitro, Flow Cytometry

a Overview of the tumour cell/T cell assay system. A549-SC3 cells engage the TCR on T cells via a single chain αCD3 antibody. MHC-II and PD-L1 expressed on A549-SC3 cells suppress T cell proliferation by engaging LAG3 and PD1, respectively. b Baseline T cell proliferation with negative control treatment. Percent proliferation as shown by Ki67 positivity relative to total CD4+ and CD8+ T cell populations with HEL-MSA on board in ex vivo blood samples from 22 patients. Boxes represent quartiles, the dotted black line is the mean value, the solid vertical bar is the 95% confidence interval of the mean. ‘Count’ is the number of patients. c Proliferation of NSCLC patient T cells after ex vivo stimulation with A549-SC3 cells in the presence of various potential checkpoint modulators: Lg-Lg: two Lag3-binding V H connected by a glycine/serine linker; Pd: one PD1-binding V H ; Lg-Lg + Pd: Lg-Lg and Pd co-administered; nivo: nivolumab analogue mAb; Lg-Lg-Pd: LAG3-LAG3-PD1 triple V H construct. All values represent the patient-specific paired difference in %Ki67+ T cells with respect to the negative control V H construct HEL-MSA for each given patient. Boxes represent quartiles, the dotted black line is the mean value, the solid vertical bar is the 95% confidence interval of the mean. ‘Count’ is the number of patients. Asterisk indicates P > 0.05 by two-tailed Wilcoxon signed-rank test vs. negative control; asterisk above square bracket shows P > 0.05 by two-tailed unpaired Mann–Whitney test for nivolumab vs. LAG3-LAG3-PD1 in CD8+ T cells.

Journal: British Journal of Cancer

Article Title: The multi-specific V H -based Humabody CB213 co-targets PD1 and LAG3 on T cells to promote anti-tumour activity

doi: 10.1038/s41416-021-01684-4

Figure Lengend Snippet: a Overview of the tumour cell/T cell assay system. A549-SC3 cells engage the TCR on T cells via a single chain αCD3 antibody. MHC-II and PD-L1 expressed on A549-SC3 cells suppress T cell proliferation by engaging LAG3 and PD1, respectively. b Baseline T cell proliferation with negative control treatment. Percent proliferation as shown by Ki67 positivity relative to total CD4+ and CD8+ T cell populations with HEL-MSA on board in ex vivo blood samples from 22 patients. Boxes represent quartiles, the dotted black line is the mean value, the solid vertical bar is the 95% confidence interval of the mean. ‘Count’ is the number of patients. c Proliferation of NSCLC patient T cells after ex vivo stimulation with A549-SC3 cells in the presence of various potential checkpoint modulators: Lg-Lg: two Lag3-binding V H connected by a glycine/serine linker; Pd: one PD1-binding V H ; Lg-Lg + Pd: Lg-Lg and Pd co-administered; nivo: nivolumab analogue mAb; Lg-Lg-Pd: LAG3-LAG3-PD1 triple V H construct. All values represent the patient-specific paired difference in %Ki67+ T cells with respect to the negative control V H construct HEL-MSA for each given patient. Boxes represent quartiles, the dotted black line is the mean value, the solid vertical bar is the 95% confidence interval of the mean. ‘Count’ is the number of patients. Asterisk indicates P > 0.05 by two-tailed Wilcoxon signed-rank test vs. negative control; asterisk above square bracket shows P > 0.05 by two-tailed unpaired Mann–Whitney test for nivolumab vs. LAG3-LAG3-PD1 in CD8+ T cells.

Article Snippet: A FortéBio OCTET instrument was used to study the interaction between CB213 with human (R&D Systems 2319-L3) and recombinant cynomolgus monkey LAG3-Fc tagged proteins (R&D System, 8578-PD-050) and similarly for recombinant human and cynomolgus monkey Fc-tagged PD1 (R&D Systems: 1086-PD, 8578-PD-050), and for albumin from human (Sigma Aldrich A3782), cynomolgus monkey (Abcam ab184894) and mouse (Sigma Aldrich A3559).

Techniques: Negative Control, Ex Vivo, Binding Assay, Construct, Two Tailed Test, MANN-WHITNEY

a CB213 PK and ADA in two female cynomolgus macaques: animals 102 and 103. b Cartoon of CB213 structure. A Schematic of CB213 is shown. Individual V H are shown as globular domains, with stylised glycine/serine linkers intervening in dark grey. The diagram is not to scale. c Schematic of the MSD-based CB213 PK bridging assay. Molecules that can simultaneously bind to LAG3 and to PD1 are detected. d Schematic of the MSD-based ADA assay. Molecules that can bind to CB213 when bound to LAG3 that contain an IgG light chain are detected.

Journal: British Journal of Cancer

Article Title: The multi-specific V H -based Humabody CB213 co-targets PD1 and LAG3 on T cells to promote anti-tumour activity

doi: 10.1038/s41416-021-01684-4

Figure Lengend Snippet: a CB213 PK and ADA in two female cynomolgus macaques: animals 102 and 103. b Cartoon of CB213 structure. A Schematic of CB213 is shown. Individual V H are shown as globular domains, with stylised glycine/serine linkers intervening in dark grey. The diagram is not to scale. c Schematic of the MSD-based CB213 PK bridging assay. Molecules that can simultaneously bind to LAG3 and to PD1 are detected. d Schematic of the MSD-based ADA assay. Molecules that can bind to CB213 when bound to LAG3 that contain an IgG light chain are detected.

Techniques:

Affinity of CB213 to cross-species targets ( K D ).

Journal: British Journal of Cancer

Article Title: The multi-specific V H -based Humabody CB213 co-targets PD1 and LAG3 on T cells to promote anti-tumour activity

doi: 10.1038/s41416-021-01684-4

Figure Lengend Snippet: Affinity of CB213 to cross-species targets ( K D ).

Techniques:

Cell-based assessment of T cell activation measured by induction of an NFAT response element-driven luminescent reporter gene. Relative light units (arbitrary scale) are reported. a Relief of LAG3/MHC-II-mediated suppression. Standard errors of the mean are indicated. Approximate half-maximal stimulation concentrations: CB213: 20 nM, Lg-Lg-Pd (LAG3-LAG3-PD1): 22 nM, relatlimab: 9.7 nM, aLAG3 17B4 (anti LAG3 mouse monoclonal): 1.6 nM. MSA-MSA-MSA is three interlinked V H recognising mouse serum albumin as a negative control. b Relief of PD1/PD-L1-mediated suppression. Standard errors of the means are indicated. Approximate half-maximal stimulation concentrations: CB213: 105 nM, Lg-Lg-Pd (LAG3-LAG3-PD1): 115 nM, nivolumab: 6 nM, DART (MGD-013): 1.4 nM. MSA-MSA-MSA is three interlinked V H recognising mouse serum albumin as a negative control.

Journal: British Journal of Cancer

Article Title: The multi-specific V H -based Humabody CB213 co-targets PD1 and LAG3 on T cells to promote anti-tumour activity

doi: 10.1038/s41416-021-01684-4

Figure Lengend Snippet: Cell-based assessment of T cell activation measured by induction of an NFAT response element-driven luminescent reporter gene. Relative light units (arbitrary scale) are reported. a Relief of LAG3/MHC-II-mediated suppression. Standard errors of the mean are indicated. Approximate half-maximal stimulation concentrations: CB213: 20 nM, Lg-Lg-Pd (LAG3-LAG3-PD1): 22 nM, relatlimab: 9.7 nM, aLAG3 17B4 (anti LAG3 mouse monoclonal): 1.6 nM. MSA-MSA-MSA is three interlinked V H recognising mouse serum albumin as a negative control. b Relief of PD1/PD-L1-mediated suppression. Standard errors of the means are indicated. Approximate half-maximal stimulation concentrations: CB213: 105 nM, Lg-Lg-Pd (LAG3-LAG3-PD1): 115 nM, nivolumab: 6 nM, DART (MGD-013): 1.4 nM. MSA-MSA-MSA is three interlinked V H recognising mouse serum albumin as a negative control.

Techniques: Activation Assay, Negative Control

End-of study in vivo pharmacology syngeneic tumours disaggregated and analysed by flow cytometry. Treatments were HEL-MSA (negative control) or CB213 dosed 10 mg/kg. Datapoints represent single tumours from individual animals. a Tumour-infiltrating lymphocytes as a percentage of leucocytes (CD45+) in disaggregated tumours. b Expression of PD1 and/or LAG3 on CD4+ or CD8+ tumour-infiltrating lymphocytes, respectively.

Journal: British Journal of Cancer

Article Title: The multi-specific V H -based Humabody CB213 co-targets PD1 and LAG3 on T cells to promote anti-tumour activity

doi: 10.1038/s41416-021-01684-4

Figure Lengend Snippet: End-of study in vivo pharmacology syngeneic tumours disaggregated and analysed by flow cytometry. Treatments were HEL-MSA (negative control) or CB213 dosed 10 mg/kg. Datapoints represent single tumours from individual animals. a Tumour-infiltrating lymphocytes as a percentage of leucocytes (CD45+) in disaggregated tumours. b Expression of PD1 and/or LAG3 on CD4+ or CD8+ tumour-infiltrating lymphocytes, respectively.

Techniques: In Vivo, Flow Cytometry, Negative Control, Expressing

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recombinant monkey pd l1 ecd (Sino Biological)

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Derivative Assay:

Co-culture Assay:

Expressing:

Variant Assay:

Inhibition:

Transduction:

Staining:

Generated:

Control:

Purification:

Labeling:

Isolation:

Blocking Assay:

Imaging:

Sequencing:

Reconstitution Assay:

Fluorescence:

Selection:

Adoptive Transfer Assay:

Flow Cytometry:

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