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: Antibodies

Article Title: Development of a Bispecific IgG1 Antibody Targeting BCMA and PDL1

doi: 10.3390/antib13010015

Figure Lengend Snippet: Structure and specificity of binding of chimeric BCMA×PDL1 bsAb. ( A ) Starting from the N-terminus, the fused heavy chain is composed of the anti-BCMA J22.9 antibody VH sequence-CH1 hinge1-A1linker-anti-PDL1 atezolizumab VH sequence-CH1 hinge2-CH2-CH3. The CH1 hinge-CH2 and CH3 sequences are from human IgG1. The two light chains (both k) are anti-BCMA J22.9 VL-CL and anti-PDL1 atezolizumab VL-CL. The red dot indicates the paired complementary mutations on CH1 and CL of the anti-PDL1 moiety to drive correct light chain pairing . ( B ) Specificity of binding of the purified bsAb and respective mAbs was tested by flow cytometry on mBCMA + and PDL1 + single-positive cell lines, KMS11 and HDLM2, respectively. MFI: mean fluorescence intensity. %: percentage of mBCMA-positive cells.

Article Snippet: After chip rotation, the following analytes, diluted in SPR running buffer (Dulbecco’s Phosphate-Buffered Saline with 0.005% Tween-20), were injected simultaneously on all the immobilized antibodies: recombinant human BCMA extracellular domain Fc chimera (recBCMA, R&D system, Minneapolis, MN, USA) and recombinant human PDL1 extracellular domain-Fc chimera (recPDL1, R&D system).

Techniques: Binding Assay, Sequencing, Purification, Flow Cytometry, Fluorescence

Journal: Antibodies

Article Title: Development of a Bispecific IgG1 Antibody Targeting BCMA and PDL1

doi: 10.3390/antib13010015

Figure Lengend Snippet: Relative binding affinity of BCMA×PDL1 bsAb and respective mAbs for target antigens. The relative affinity of the BCMA×PDL1 bsAb, anti-BCMA, and anti-PDL1 mAbs was tested by flow cytometry, using increasing concentrations of primary antibodies and detection with anti-human Fc-FITC secondary antibody. ( A ) Binding of bsAb and mAbs to mBCMA + KMS11 cell line. ( B ) Binding of bsAb and mAbs to PDL1 + HDM2 cell line. ( C ) Relative binding affinities (IC 50 ) for each antigen; NA: not applicable.

Article Snippet: After chip rotation, the following analytes, diluted in SPR running buffer (Dulbecco’s Phosphate-Buffered Saline with 0.005% Tween-20), were injected simultaneously on all the immobilized antibodies: recombinant human BCMA extracellular domain Fc chimera (recBCMA, R&D system, Minneapolis, MN, USA) and recombinant human PDL1 extracellular domain-Fc chimera (recPDL1, R&D system).

Techniques: Binding Assay, Flow Cytometry

Journal: Antibodies

Article Title: Development of a Bispecific IgG1 Antibody Targeting BCMA and PDL1

doi: 10.3390/antib13010015

Figure Lengend Snippet: Binding constants determined by SPR studies.

Article Snippet: After chip rotation, the following analytes, diluted in SPR running buffer (Dulbecco’s Phosphate-Buffered Saline with 0.005% Tween-20), were injected simultaneously on all the immobilized antibodies: recombinant human BCMA extracellular domain Fc chimera (recBCMA, R&D system, Minneapolis, MN, USA) and recombinant human PDL1 extracellular domain-Fc chimera (recPDL1, R&D system).

Techniques: Binding Assay

Journal: Antibodies

Article Title: Development of a Bispecific IgG1 Antibody Targeting BCMA and PDL1

doi: 10.3390/antib13010015

Figure Lengend Snippet: Surface plasmon resonance analysis. The sensorgrams shown were obtained by injecting recPDL1 or recBCMA, alone ( A – C ) or in succession ( D ) over immobilized anti-PDL1 ( A ), anti-BCMA ( B ), or BCMA×PDL1 bsAb ( C , D ). The antigens were flowed for 3 min, as indicated by the dashed lines.

Article Snippet: After chip rotation, the following analytes, diluted in SPR running buffer (Dulbecco’s Phosphate-Buffered Saline with 0.005% Tween-20), were injected simultaneously on all the immobilized antibodies: recombinant human BCMA extracellular domain Fc chimera (recBCMA, R&D system, Minneapolis, MN, USA) and recombinant human PDL1 extracellular domain-Fc chimera (recPDL1, R&D system).

Techniques: SPR Assay

Journal: Antibodies

Article Title: Development of a Bispecific IgG1 Antibody Targeting BCMA and PDL1

doi: 10.3390/antib13010015

Figure Lengend Snippet: The BCMA×PDL1 bsAb blocks APRIL binding to mBCMA and PD1-PDL1 interaction. ( A ) To test the ability of bsAb to block APRIL binding to mBCMA, we used CEM-mBCMA + cell line, increasing concentrations of bsAb and anti-BCMA mAb, a Flag-tagged APRIL protein, and an anti-Flag antibody. *: p < 0.05. ( B ) For assessing the inhibition of the PD1-PDL1 axis, we tested increasing concentrations of BCMA×PDL1 bsAb or anti-PDL1 mAb in the cell-based PD1/PDL1 Blockade Bioassay. ( C ) Cell-based PD1/PDL1 Blockade Bioassay in presence of recBCMA. Atezolizumab and cetuximab were used as positive and negative controls, respectively. *: p < 0.05 vs. PDL1.

Article Snippet: After chip rotation, the following analytes, diluted in SPR running buffer (Dulbecco’s Phosphate-Buffered Saline with 0.005% Tween-20), were injected simultaneously on all the immobilized antibodies: recombinant human BCMA extracellular domain Fc chimera (recBCMA, R&D system, Minneapolis, MN, USA) and recombinant human PDL1 extracellular domain-Fc chimera (recPDL1, R&D system).

Techniques: Binding Assay, Blocking Assay, Inhibition, Bioassay

Journal: Antibodies

Article Title: Development of a Bispecific IgG1 Antibody Targeting BCMA and PDL1

doi: 10.3390/antib13010015

Figure Lengend Snippet: The BCMA×PDL1 bsAb mediates CDC of mBCMA + cells. ( A ) The BJAB-mBCMA + cell line was incubated with increasing concentrations of bsAb, mAbs, or RTX as positive control and in the presence of 50% HS as a source of complement. CDC was measured after 4 h by 7-AAD staining and flow cytometry. *: p < 0.05 and **: p < 0.01. ( B ) Flow cytometry histograms showing the expression of mBCMA of BJAB cells stably expressing BCMA in presence or absence of DAPT. MFI: mean fluorescence intensity. %: percentage of mBCMA-positive cells.

Article Snippet: After chip rotation, the following analytes, diluted in SPR running buffer (Dulbecco’s Phosphate-Buffered Saline with 0.005% Tween-20), were injected simultaneously on all the immobilized antibodies: recombinant human BCMA extracellular domain Fc chimera (recBCMA, R&D system, Minneapolis, MN, USA) and recombinant human PDL1 extracellular domain-Fc chimera (recPDL1, R&D system).

Techniques: Incubation, Positive Control, Staining, Flow Cytometry, Expressing, Stable Transfection, Fluorescence

Journal: Biomedicines

Article Title: Evaluation of Anti-CAR Linker mAbs for CAR T Monitoring after BiTEs/bsAbs and CAR T-Cell Pretreatment

doi: 10.3390/biomedicines12081641

Figure Lengend Snippet: Schematical representation of CAR T-cell detection with the classical ligand-based assay and anti-CAR linker mAb. ( A ) A schematical representation of CD19 and BCMA tumor antigens and their targeting CARs are shown. The recombinantly produced and biotinylated ligands and APC-conjugated anti-biotin Abs are used to visualize CAR expression on the surface of T-cells. ( B ) The general structure of a chimeric antigen receptor (CAR) is depicted as an illustration. The single-chain variable fragment (scFv) of CARs contains either a Whitlow linker (GSTSGSGKPGSGEGSTKG) or a (G 4 S) 3 linker (GGGGSGGGGSGGGGS), targeted by anti-Whitlow mAb or by anti-G 4 S monoclonal antibody (mAb), respectively. Biotinylated CAR-linker mABs and APC-conjugated anti-biotin Abs are used for the universal visualization of CAR expression on the surface of T-cells.

Article Snippet: Biotin-labeled BCMA CAR detection reagent, biotin-labeled CD19 CAR detection reagent, and anti-biotin APC were obtained from Miltenyi Biotec (Bergisch-Gladbach, Germany).

Techniques: Produced, Expressing

Journal: Biomedicines

Article Title: Evaluation of Anti-CAR Linker mAbs for CAR T Monitoring after BiTEs/bsAbs and CAR T-Cell Pretreatment

doi: 10.3390/biomedicines12081641

Figure Lengend Snippet: Anti-CAR linker mAbs allows for specific and sensitive CAR T monitoring in patients treated with any clinically approved CAR T therapies except Cilta-cel. ( A ) The structures of BCMA- and CD19-specific CARs are depicted in the illustration. CD19 antigen-specific CARs: Brexu-cel, Liso-cel, Axi-cel, and Tisa-cel; BCMA-specific CARs: Ide-cel and Cilta-cel. ( B ) Flow cytometry-based CAR detection analyses are shown, performed with biotinylated anti-CAR linker mAbs and APC-conjugated anti-biotin Abs to all approved BCMA and CD19 CAR T-cell products using isolated lymphocyte specimens from patients’ blood (n = 3). ( C ) Flow cytometry-based CAR detection analyses are shown, performed with Alexa 647-conjugated anti-CAR linker mAbs to all approved BCMA and CD19 CAR T-cell products using isolated lymphocyte specimens from patients’ blood (n = 3). The CAR-positive cells were determined from the CD3-positive cells and significances calculated based on BCMA- and CD19-antigen-based CAR detection reagent (Miltenyi = 100%). Statistical analysis was performed using Student’s t -test. * p ≤ 0.05%; ** p ≤ 0.01%; *** p ≤ 0.001%.

Article Snippet: Biotin-labeled BCMA CAR detection reagent, biotin-labeled CD19 CAR detection reagent, and anti-biotin APC were obtained from Miltenyi Biotec (Bergisch-Gladbach, Germany).

Techniques: Flow Cytometry, Isolation

Journal: Biomedicines

Article Title: Evaluation of Anti-CAR Linker mAbs for CAR T Monitoring after BiTEs/bsAbs and CAR T-Cell Pretreatment

doi: 10.3390/biomedicines12081641

Figure Lengend Snippet: Anti-CAR linker mAbs allows for specific CAR T monitoring in patients pretreated with BiTE/bsAb, targeting the same antigen as the CAR. ( A ) The structure of the blinatumomab (CD19×CD3 BiTE) and teclistamab (BCMA×CD3 bsAb) are illustrated. ( B ) Shown is a schematic overview of CAR T-cell detection strategies based on the CD19 or BCMA CAR detection reagent (Miltenyi) that leads to a false positive staining in patients pretreated with BiTE/bsAb targeting the same antigen as the CAR. The structure of CD19- or BCMA-specific CAR and CD3 on the cell surface of a T-cell is depicted as an illustration. ( C ) Shown is a schematic overview of CAR T-cell detection strategies based on anti-CAR linker mAbs. The specificity of the CAR detection is not affected by BiTE/bsAb targeting the same antigen as the CAR. ( D ) Detection of CAR-positive cells in CD3-positive blood T-lymphocytes from patients, treated either with Brexu-cel, Liso-cel, and Axi-cel (anti-CD19 CAR T-cells) simultaneously in the presence or absence of blinatumomab BiTE (CD19×CD3) or treated either with Ide-cel (anti-BCMA CAR T-cells) simultaneously in the presence or absence of teclistamab bsAb (BCMA×CD3). Representative clinical blood specimens of a Lymphoma or MM patient are shown as dot plots. CAR-negative and CAR-positive cells are visualized using blue and green dots, respectively. Green and red checkmarks denote for specific or artificial CAR T-cell detection, respectively. Shown are the CD3-positive cells. Note that CD19- and BCMA-based CAR detection reagents (Miltenyi) interfere with blinatumomab and teclistamab, respectively, and bind to CD3 on the surface of all T-cells, irrespective of whether they carry a CAR or not. This leads to false positive results (denoted by dots shown in orange or red). The problem is solved using anti-CAR linker mAbs targeting the artificial linker sequence between the variable heavy- and light-chain domains of the scFv.

Article Snippet: Biotin-labeled BCMA CAR detection reagent, biotin-labeled CD19 CAR detection reagent, and anti-biotin APC were obtained from Miltenyi Biotec (Bergisch-Gladbach, Germany).

Techniques: Staining, Sequencing