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: Biotinylated human BCMA-rFc (human TNFRSF17/BCMA/CD269 Protein (His & human IgG1 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: Biotinylated human BCMA-rFc (human TNFRSF17/BCMA/CD269 Protein (His & human IgG1 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: Biotinylated human BCMA-rFc (human TNFRSF17/BCMA/CD269 Protein (His & human IgG1 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: Biotinylated human BCMA-rFc (human TNFRSF17/BCMA/CD269 Protein (His & human IgG1 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: Biotinylated human BCMA-rFc (human TNFRSF17/BCMA/CD269 Protein (His & human IgG1 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: Biotinylated human BCMA-rFc (human TNFRSF17/BCMA/CD269 Protein (His & human IgG1 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: Biotinylated human BCMA-rFc (human TNFRSF17/BCMA/CD269 Protein (His & human IgG1 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: Biotinylated human BCMA-rFc (human TNFRSF17/BCMA/CD269 Protein (His & human IgG1 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