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: Theranostics

Article Title: Precision radiolabeled B-cell maturation nanobody for targeted PET imaging and radioligand therapy of disseminated multiple myeloma

doi: 10.7150/thno.126920

Figure Lengend Snippet: Schematic of BCMA-Nb radiolabeling with (A) [ 18 F]FPy and (B) [ 131 I]I.

Article Snippet: Biolayer interferometry studies were conducted similarly to the published procedure using 1ug/mL of biotinylated human BCMA protein (ACROBiosystems, BCA-H82E4) and 100nM concentration of BCMA-Nb in PBS as an eluent .

Techniques: Radioactivity

Journal: Theranostics

Article Title: Precision radiolabeled B-cell maturation nanobody for targeted PET imaging and radioligand therapy of disseminated multiple myeloma

doi: 10.7150/thno.126920

Figure Lengend Snippet: (A) Kinetic binding assay of BCMA nanobody to human BCMA protein measured by biolayer interferometry using 100nM anti-BCMA-nanobody (B) Representative flow cytometry histograms of BCMA expression in MC38-BCMA and MC38-WT cells (C) Immunohistochemistry and Immunofluorescent staining of BCMA in excised tumors (D) Saturation binding assays of [ 18 F]FPy-BCMA Nb in MC38-BCMA cells (E) Representative PET/CT coronal images of MC38-BCMA and MC38-WT tumor model (150-250 mm 3 ), injected with 3.7 MBq [ 18 F]FPy-BCMA-Nb at 2 h post-injection. PET scans are normalized to the same scale. Data are presented as mean ± SD (n = 6 mice per group).

Article Snippet: Biolayer interferometry studies were conducted similarly to the published procedure using 1ug/mL of biotinylated human BCMA protein (ACROBiosystems, BCA-H82E4) and 100nM concentration of BCMA-Nb in PBS as an eluent .

Techniques: Binding Assay, Flow Cytometry, Expressing, Immunohistochemistry, Staining, Positron Emission Tomography-Computed Tomography, Injection

Journal: Theranostics

Article Title: Precision radiolabeled B-cell maturation nanobody for targeted PET imaging and radioligand therapy of disseminated multiple myeloma

doi: 10.7150/thno.126920

Figure Lengend Snippet: (A) Flow cytometry analysis of BCMA expression in H929 and RPMI8226 cell lines. (B) Immunofluorescent staining of H929 cells using anti-BCMA antibody (green) and DAPI (blue). Saturation binding assay to assess the specific binding and dissociation constant of [ 18 F]FPy-BCMA-Nb in (C) H929 and (D) RPMI8226 cells. (E) Internalization of [ 18 F]FPy-BCMA-Nb in H929 cells over 180 minutes. (F) 3 h post-injection uptake of [ 18 F]FPy-BCMA-Nb in the femur/knee region and (G) spine of the human H929 and RPMI8226 MM model compared to healthy mice and blocking. White arrows indicate tumor cells in the bone marrow. Relative quantitative uptake of the tracer in the H929 human MM model with higher BCMA expression in spine, femur, arm, skull bone marrow, and kidney under (H) pre-blocking and (I) post-blocking conditions . Data are presented as mean ± SD (n = 6 mice per group).

Article Snippet: Biolayer interferometry studies were conducted similarly to the published procedure using 1ug/mL of biotinylated human BCMA protein (ACROBiosystems, BCA-H82E4) and 100nM concentration of BCMA-Nb in PBS as an eluent .

Techniques: Flow Cytometry, Expressing, Staining, Saturation Assay, Binding Assay, Injection, Blocking Assay

Journal: Theranostics

Article Title: Precision radiolabeled B-cell maturation nanobody for targeted PET imaging and radioligand therapy of disseminated multiple myeloma

doi: 10.7150/thno.126920

Figure Lengend Snippet: (A) Schematic illustration of the timeline, treatment regimen and efficacy study for the radiotherapy study. (B) Representative BLI images of mice pre- and post-treatment regimen and (C) overall survival percentage across all groups, including Control (PBS), 7.4 MBq and 18.5 MBq of [ 131 I]I-BCMA-Nb, compared to 18.5 MBq of [¹³¹I]I-non-specific-Nb treated cohorts, as determined by Kaplan-Meier analysis. Data are presented as mean ± SD (n = 8 mice per group).

Article Snippet: Biolayer interferometry studies were conducted similarly to the published procedure using 1ug/mL of biotinylated human BCMA protein (ACROBiosystems, BCA-H82E4) and 100nM concentration of BCMA-Nb in PBS as an eluent .

Techniques: Control

Journal: Theranostics

Article Title: Precision radiolabeled B-cell maturation nanobody for targeted PET imaging and radioligand therapy of disseminated multiple myeloma

doi: 10.7150/thno.126920

Figure Lengend Snippet: (A) Representative Sagittal [ 18 F]FDG PET images of healthy mice compared to treatment groups (PBS, 7.4 MBq and 18.5 MBq of [ 131 I]I-BCMA-Nb,) acquired pre- and post-treatment regimen. Quantitative analysis of [ 18 F]FDG uptake in bone marrow of the (B) spine, (C)femur, (D) arm, and (E) skull across all groups pre-, during and post-treatment. Gray areas are defined as baseline based on [ 18 F]FDG of healthy mice. Data are presented as mean ± SD (n = 8 mice per group).

Article Snippet: Biolayer interferometry studies were conducted similarly to the published procedure using 1ug/mL of biotinylated human BCMA protein (ACROBiosystems, BCA-H82E4) and 100nM concentration of BCMA-Nb in PBS as an eluent .

Techniques:

Journal: Theranostics

Article Title: Precision radiolabeled B-cell maturation nanobody for targeted PET imaging and radioligand therapy of disseminated multiple myeloma

doi: 10.7150/thno.126920

Figure Lengend Snippet: Changes in (A) body weight during the treatment regimen. (B) Complete cell blood counts for mice pre- and post-treatment in all groups including PBS, injected with 7.4 MBq and 18.5 MBq of [ 131 I]I-BCMA-Nb and 18.5 MBq of [ 131 I]I-non-specific-Nb. Orange-shaded cells indicate values significantly different from standard NSG mouse ranges (p<0.05). Data are presented as mean ± SD (n = 8 mice per group).

Article Snippet: Biolayer interferometry studies were conducted similarly to the published procedure using 1ug/mL of biotinylated human BCMA protein (ACROBiosystems, BCA-H82E4) and 100nM concentration of BCMA-Nb in PBS as an eluent .

Techniques: Injection

Journal: Theranostics

Article Title: Precision radiolabeled B-cell maturation nanobody for targeted PET imaging and radioligand therapy of disseminated multiple myeloma

doi: 10.7150/thno.126920

Figure Lengend Snippet: (A) Immunohistochemistry staining and (B) quantitative analysis of BCMA, (C) immunofluorescence staining for and (D) quantitative analysis of Ki67 proliferation marker in femur and spine bone marrow sections of H929 human MM-bearing mice, before and after treatment, compared with healthy mice.

Article Snippet: Biolayer interferometry studies were conducted similarly to the published procedure using 1ug/mL of biotinylated human BCMA protein (ACROBiosystems, BCA-H82E4) and 100nM concentration of BCMA-Nb in PBS as an eluent .

Techniques: Immunohistochemistry, Staining, Immunofluorescence, Marker