Journal: Frontiers in Immunology

Article Title: Comparative characterization of two monoclonal antibodies targeting canine PD-1

doi: 10.3389/fimmu.2024.1382576

Figure Lengend Snippet: PD1-1.1 and PD1-2.1 both inhibit the PD-1/PD-L1 binding in ELISA, yet differ in their blocking capacity at the higher concentration, suggesting distinct binding sites or mechanisms; IC50 [PD1-1.1] = 0.0002, IC50 [PD1-2.1] = 0.0006; error bars represent standard error of the mean (SEM).

Article Snippet: The recombinant canine PD-1 protein (rcPD-1) with a C-terminal polyhistidine tag (Sino Biological, #70109-D08H) and recombinant canine PD-L1 (rcPD-L1) extracellular domain (ECD) with a C-terminal Fc-tag (Sino biological, #70110-D02H) lyophilized from PBS with protectants were reconstituted in sterile water according to the manufacturer’s instruction, reaching 0.25mg/mL protein concentration.

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

Journal: Frontiers in Immunology

Article Title: Comparative characterization of two monoclonal antibodies targeting canine PD-1

doi: 10.3389/fimmu.2024.1382576

Figure Lengend Snippet: Putative epitopes of PD1-1.1 and PD1-2.1 on canine PD-1 (cPD-1) were identified by molecular modeling and docking. Receptor residues participating in the protein-protein interaction were visualized on the cPD-1 structure and sequence for PD1-1.1 (A) and PD1-2.1 (B) . Green – residues participating in the interface; magenta – interface residues contributing hydrogen/disulphide bonds, salt bridges or covalent links. For comparison, the cPD-1 regions corresponding to the human PD-1 interaction sites for PD-L1 , Nivolumab (Nivo ) and Pembrolizumab (Pembro were marked with red (C) , cyan and yellow (D) , respectively.

Article Snippet: The recombinant canine PD-1 protein (rcPD-1) with a C-terminal polyhistidine tag (Sino Biological, #70109-D08H) and recombinant canine PD-L1 (rcPD-L1) extracellular domain (ECD) with a C-terminal Fc-tag (Sino biological, #70110-D02H) lyophilized from PBS with protectants were reconstituted in sterile water according to the manufacturer’s instruction, reaching 0.25mg/mL protein concentration.

Techniques: Sequencing, Comparison

Journal: International Journal of Molecular Sciences

Article Title: An Anti-BCMA Affibody Affinity Protein for Therapeutic and Diagnostic Use in Multiple Myeloma

doi: 10.3390/ijms26115186

Figure Lengend Snippet: First-generation anti-B Cell Maturation Antigen (BCMA) affibodies. ( a ) Overlay of single 200 nM concentration surface plasmon resonance (SPR) sensorgrams for five recombinantly expressed BCMA-targeting affibody candidates (His 6 -affibody-ABD format), binding to immobilised human BCMA-rFc. ( b ) Amino acid sequence alignment of the five clones, showing the amino acid occupancies in the variable positions, compared to the library gene (variable positions denoted X in the library gene sequence). Note the cysteines (red) in position 32 in clones Fa-B3 and Ft-H11. The numbers above the alignment indicate the amino acid residue numbering of the affibody 58-amino-acid scaffold sequence. The number to the right of each sequence indicates its frequency of appearance during the sequencing of 54 ELISA-positive candidates.

Article Snippet: Recombinant, biotinylated human BCMA (human TNFRS/BCMA/CD269 Protein (His & Fc tag), Biotinylated, cat. no. 10620-H03H-B, Sino Biological, Eschborn, Germany), corresponding to residues 1–54 of Uniprot entry Q02223 , was used as the target antigen.

Techniques: Concentration Assay, SPR Assay, Binding Assay, Sequencing, Clone Assay, Residue, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: An Anti-BCMA Affibody Affinity Protein for Therapeutic and Diagnostic Use in Multiple Myeloma

doi: 10.3390/ijms26115186

Figure Lengend Snippet: Alanine scanning of the candidate BCMA-binding clone Fa-G6. ( a ) The amino acid residues in the 14 variable positions (blue) located on the first two helices of the Fa-G6 clone were individually substituted to alanine. The numbers correspond to the amino acid residue numbering of the affibody 58-amino-acid scaffold sequence, from the N-terminus (N) to the C-terminus (C). The image is based on 5U4Y.pdb. ( b ) Single concentration (200 nM) SPR sensorgrams of the 14 alanine variants binding to immobilised human BCMA-rFc, compared to the Fa-G6 wild-type clone.

Article Snippet: Recombinant, biotinylated human BCMA (human TNFRS/BCMA/CD269 Protein (His & Fc tag), Biotinylated, cat. no. 10620-H03H-B, Sino Biological, Eschborn, Germany), corresponding to residues 1–54 of Uniprot entry Q02223 , was used as the target antigen.

Techniques: Binding Assay, Residue, Sequencing, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: An Anti-BCMA Affibody Affinity Protein for Therapeutic and Diagnostic Use in Multiple Myeloma

doi: 10.3390/ijms26115186

Figure Lengend Snippet: Library design and selection output. Amino acid distributions and frequencies of the second-generation library gene designs compared to the selection output, in the 15 randomised positions located in helices 1 ( a ) and 2 ( b ). Based on alanine scanning of the BCMA binding clone Fa-G6 two second-generation libraries, Library A and Library B, were designed. Library A was designed to be more conserved than Library B. The dataset for the distribution and frequencies of amino acids in the 15 variable positions in the output of the second-generation selections is based on sequencing data of 107 ELISA-positive unique clones (56 clones originating from Library A and 51 clones originating from Library B). Codons are represented by the three-letter abbreviation of respective amino acid, which here include all naturally occurring amino acids, except Gly, Pro, and Cys (not included in the library designs).

Article Snippet: Recombinant, biotinylated human BCMA (human TNFRS/BCMA/CD269 Protein (His & Fc tag), Biotinylated, cat. no. 10620-H03H-B, Sino Biological, Eschborn, Germany), corresponding to residues 1–54 of Uniprot entry Q02223 , was used as the target antigen.

Techniques: Selection, Binding Assay, Sequencing, Enzyme-linked Immunosorbent Assay, Clone Assay

Journal: International Journal of Molecular Sciences

Article Title: An Anti-BCMA Affibody Affinity Protein for Therapeutic and Diagnostic Use in Multiple Myeloma

doi: 10.3390/ijms26115186

Figure Lengend Snippet: Candidate clones from the second-generation selection campaign output, compared to the parental Fa-G6 clone. ( a ) SDS-PAGE gels of Escherichia coli ( E. coli ) produced monomeric affibodies. ( b ) Overlay of single concentration (100 nM) SPR sensorgrams of clones binding to immobilised human BCMA-rFc. ( c ) Overlay of thermal denaturation profiles recorded at 221 nm. ( d ) Approximate melting temperatures (Tm) of respective clone (0.2 mg/mL), based on thermal denaturing from 20 °C to 90 °C (5 °C/min) measured at 221 nm. ( e ) Sequence alignment of the four candidate second-generation clones, compared to the parental Fa-G6 clone, showing the amino acid distribution in the 15 variable positions (underlined in the Fa-G6 sequence). The numbers above the alignment indicate the amino acid residue numbering of the affibody 58-amino-acid scaffold sequence.

Article Snippet: Recombinant, biotinylated human BCMA (human TNFRS/BCMA/CD269 Protein (His & Fc tag), Biotinylated, cat. no. 10620-H03H-B, Sino Biological, Eschborn, Germany), corresponding to residues 1–54 of Uniprot entry Q02223 , was used as the target antigen.

Techniques: Clone Assay, Selection, SDS Page, Produced, Concentration Assay, Binding Assay, Sequencing, Residue

Journal: International Journal of Molecular Sciences

Article Title: An Anti-BCMA Affibody Affinity Protein for Therapeutic and Diagnostic Use in Multiple Myeloma

doi: 10.3390/ijms26115186

Figure Lengend Snippet: Kinetic analysis of the BCMA-binding affibody clone 1-E6. One representative serial dilution (1.1–90 nM) of the second-generation clone 1-E6, injected in duplicate over immobilised human BCMA-rFc. Kinetic constants K D (dissociation equilibrium constant), k a (association rate constant) and k d (dissociation rate constant) were estimated from the resulting sensorgrams using BIAevaluation software (Cytiva, Uppsala, Sweden) and assuming 1:1 binding.

Article Snippet: Recombinant, biotinylated human BCMA (human TNFRS/BCMA/CD269 Protein (His & Fc tag), Biotinylated, cat. no. 10620-H03H-B, Sino Biological, Eschborn, Germany), corresponding to residues 1–54 of Uniprot entry Q02223 , was used as the target antigen.

Techniques: Binding Assay, Serial Dilution, Injection, Software

Journal: International Journal of Molecular Sciences

Article Title: An Anti-BCMA Affibody Affinity Protein for Therapeutic and Diagnostic Use in Multiple Myeloma

doi: 10.3390/ijms26115186

Figure Lengend Snippet: Epitope binning studies through SPR-based blocking assay. The response signal of BCMA-binding analyte APRIL ( a ) or belantamab ( b ) with no prior blocking (binding interaction is denoted by double-headed arrows) was compared to blocking (blocking is denoted by a red cross) with 1-E6 affibody. In the non-blocking response, running buffer was injected over the surface containing immobilised human BCMA-rFc, followed BCMA-binding analyte, 100 nM APRIL or 25 nM belantamab. Assessment of potential blocking by 1-E6 was done by first injecting 1 µM 1-E6, followed by either BCMA-binding analyte (sample run) or running buffer (reference run). The plotted blocking response corresponds to the response obtained after subtracting the reference run from the sample run.

Article Snippet: Recombinant, biotinylated human BCMA (human TNFRS/BCMA/CD269 Protein (His & Fc tag), Biotinylated, cat. no. 10620-H03H-B, Sino Biological, Eschborn, Germany), corresponding to residues 1–54 of Uniprot entry Q02223 , was used as the target antigen.

Techniques: Blocking Assay, Binding Assay, Injection

Journal: International Journal of Molecular Sciences

Article Title: An Anti-BCMA Affibody Affinity Protein for Therapeutic and Diagnostic Use in Multiple Myeloma

doi: 10.3390/ijms26115186

Figure Lengend Snippet: Binding activity of anti-BCMA 1-E6 affibody to BCMA + and BCMA − cell lines. Flow cytometry staining of MM.1s (BCMA + /HER2 − ) cells and SKBR3 (BCMA − /HER2 + ) cells with AlexaFluor647 (AF647)-labelled anti-BCMA 1-E6 affibody (1-E6-1-E6-His 6 ) or anti-HER2 affibody control ( a ), or PE-labelled anti-BCMA antibodies (monoclonal antibody (mAb) or polyclonal antibody (pAb) or isotype control antibodies ( b ). Unstained cells were used to set the negative population.

Article Snippet: Recombinant, biotinylated human BCMA (human TNFRS/BCMA/CD269 Protein (His & Fc tag), Biotinylated, cat. no. 10620-H03H-B, Sino Biological, Eschborn, Germany), corresponding to residues 1–54 of Uniprot entry Q02223 , was used as the target antigen.

Techniques: Binding Assay, Activity Assay, Flow Cytometry, Staining, Control

Journal: International Journal of Molecular Sciences

Article Title: An Anti-BCMA Affibody Affinity Protein for Therapeutic and Diagnostic Use in Multiple Myeloma

doi: 10.3390/ijms26115186

Figure Lengend Snippet: Fluorescence and brightfield microscopy of MM.1s cells stained with anti-BCMA 1-E6 affibody. AF647-labelled 1-E6 affibody (1-E6-1-E6-His 6 ) (red) ( a ) demonstrated clear binding to BCMA-positive cell line MM.1s (BCMA + /HER2 − ). PE-labelled anti-BCMA mAb (red) ( b ) and pAb (red) ( c ) also demonstrated binding to the MM.1s cells. In ( a.ii – c.ii ) , the fluorescence signal of each reagent is overlapped with an image acquired with brightfield microscopy, to visualise the shape of the cells. A merge of ( i , ii ) is shown in ( a.iii – c.iii ). Nuclei are stained with DAPI (blue). Scale bars: 20 µm.

Article Snippet: Recombinant, biotinylated human BCMA (human TNFRS/BCMA/CD269 Protein (His & Fc tag), Biotinylated, cat. no. 10620-H03H-B, Sino Biological, Eschborn, Germany), corresponding to residues 1–54 of Uniprot entry Q02223 , was used as the target antigen.

Techniques: Fluorescence, Microscopy, Staining, Binding Assay

Journal: FEBS Open Bio

Article Title: Development and characterization of a novel human CD137 agonistic antibody with anti‐tumor activity and a good safety profile in non‐human primates

doi: 10.1002/2211-5463.13494

Figure Lengend Snippet: Binding, specificity, and blocking profiles of PE0116. (A) The binding activity of PE0116, Urelumab, and Utomilumab toward CHOK1‐hCD137 and CHOK1‐cynoCD137 cells was analyzed as concentration‐mean fluorescence intensity (MFI) curve. (B) Specificity of PE0116 to CD137 was conducted by ELISA after capturing 1 μg·mL −1 humanOX40, GITR, and CD137 as the antigen. All assays were performed in duplicate, and all error bars indicate the SD. (C) Cell‐based blocking assay was performed to evaluate PE0116, Urelumab, and Utomilumab using CHOK1‐hCD137 stable cell line and CD137L‐hFc‐Biotin with streptavidin‐Alexa Fluor 488 conjugate.

Article Snippet: Flow cytometry assay was performed to evaluate the ability of anti‐hCD137 to block the binding of CD137 and CD137L based on CHOK1 cell overexpressing hCD137 and CD137L‐hFc‐Biotin (15693‐H01H‐B; Sino Biological, Beijing, China) protein.

Techniques: Binding Assay, Blocking Assay, Activity Assay, Concentration Assay, Fluorescence, Enzyme-linked Immunosorbent Assay, Stable Transfection

Journal: FEBS Open Bio

Article Title: Development and characterization of a novel human CD137 agonistic antibody with anti‐tumor activity and a good safety profile in non‐human primates

doi: 10.1002/2211-5463.13494

Figure Lengend Snippet: Characterization of PE0116 in vitro functionality. (A) Luminescence value changed by HEK293‐CD137‐NF‐κB reporter cells treated with serially diluted PE0116, Urelumab, or Utomilumab (cross‐linking or not cross‐linking, antibodies were cross‐linked by anti‐Fc F(ab') 2 fragment, the molar ratio was 1 : 1.5). (B) Levels of IFN‐γ released by activated CD3 + T cells from three donors after 72 h of incubation with serially diluted PE0116, Urelumab, or Utomilumab in the presence of OKT3‐scFv displayed on the CHOK1 cell surface (cross‐linking or not cross‐linking). The EC50 value of Utomilumab non‐crosslink was blank in (B) (upper right panel), representing that the dates could not be analyzed by curve fitting. **** P < 0.0001 (hIgG4 vs. PE0116 at 6.67 n m in donor#2 or at 0.67 n m in donor#3 with cross‐linking) and *** P < 0.001 (hIgG4 vs. PE0116 at 66.7 n m in donor#2 without cross‐linking), as determined by two‐way ANOVA. All assays were performed in duplicate, and all error bars indicate the SEM.

Article Snippet: Flow cytometry assay was performed to evaluate the ability of anti‐hCD137 to block the binding of CD137 and CD137L based on CHOK1 cell overexpressing hCD137 and CD137L‐hFc‐Biotin (15693‐H01H‐B; Sino Biological, Beijing, China) protein.

Techniques: In Vitro, Incubation

Journal: eLife

Article Title: Misfolded proteins bind and activate death receptor 5 to trigger apoptosis during unresolved endoplasmic reticulum stress

doi: 10.7554/eLife.52291

Figure Lengend Snippet: ( A ) A peptide array tiled with sequences from the ectodomain of myelin protein zero (MPZ) and extracellular loops from rhodopsin (RHO) was incubated with Fc-tagged DR5 ectodomain domain (long isoform, 500 nM). Signal was obtained by probing with anti-Fc. ( B ) Coomassie stained SDS-PAGE gel of pulldown on Fc-tagged DR5L ECD (55 kDa) or TNFR1 ECD (65 kDa) incubated with increasing concentrations of the MPZ-ecto VD peptide (apparent MW of 10 kDa, see 'Amino acid sequences of MPZ-derived peptides' for sequence). ( C ) Fluorescence quenching of AlexaFluor647-DR5L (green) or TNFR1 ECD (blue) was measured with increasing MPZ-ecto peptide to quantify the binding affinity, whereas quenching was not observed with the mutated MPZ-ecto Tyr→Glu peptide (magenta) (N = 3, error bars are SD). DR5L ECD binds to the MPZ-ecto peptide with a K 1/2 of 109 ± 11 μM with a hill coefficient of 2.6 ± 0.5. ( D ) SDS-PAGE of recombinant FLAG-tagged DR5L ECD (25 kDa, 10 μM) incubated with MPZ-ecto peptide at the noted concentrations and treated with the amine crosslinker BS3 (100 μM), probed with anti-FLAG. ( E ) Size exclusion chromatographs of absorbance at 280 nm for 25 μM recombinant DR5L ECD alone (black), pre-incubated with 100 μM fluorescein-conjugated MPZ-ecto peptide (green) or 100 μM fluorescein-conjugated MPZ-ecto Tyr→Glu peptide (magenta). ( F ) SDS-PAGE gels scanned for fluorescence and then stained with Coomassie for eluted size exclusion fractions in ( E ). Green outlines (top pair) correspond to fractions from DR5L pre-incubated with MPZ-ecto peptide, and magenta outlines (bottom pair) correspond to DR5L with MPZ-ecto Tyr→Glu peptide. Lane marked by “-“ denotes a blank lane between the input and 7 ml fraction to minimize spillover of signal from input sample. Arrowheads mark detectable peptide fluorescence in the indicated fractions. Figure 3—source data 1. Sequences and quantification of peptides probed with Fc-DR5 ECD on the peptide array. This excel file contains the peptide sequences of the peptide array shown in , the quantification of DR5 ECD detected for each spot, and the analysis for enriched amino acids in .

Article Snippet: Peptide, recombinant protein , Fc-tagged DR5 ECD , Genentech , recombinant protein , .

Techniques: Peptide Microarray, Incubation, Staining, SDS Page, Derivative Assay, Sequencing, Fluorescence, Binding Assay, Recombinant

Journal: eLife

Article Title: Misfolded proteins bind and activate death receptor 5 to trigger apoptosis during unresolved endoplasmic reticulum stress

doi: 10.7554/eLife.52291

Figure Lengend Snippet:

Article Snippet: Peptide, recombinant protein , Fc-tagged DR5 ECD , Genentech , recombinant protein , .

Techniques: Transfection, Construct, Expressing, Mutagenesis, Sequencing, Plasmid Preparation, Recombinant, Purification, Electron Microscopy, Labeling, Incubation, SYBR Green Assay, Software, Peptide Microarray, Fluorescence, Fractionation

Journal: eLife

Article Title: Misfolded proteins bind and activate death receptor 5 to trigger apoptosis during unresolved endoplasmic reticulum stress

doi: 10.7554/eLife.52291

Figure Lengend Snippet:

Article Snippet: Peptide, recombinant protein , Fc-tagged DR5 ECD , Genentech , recombinant protein , .

Techniques: Plasmid Preparation, Construct