Journal: New biotechnology

Article Title: Affibody-based hBCMA x CD16 dual engagers for NK cell-mediated killing of multiple myeloma cells.

doi: 10.1016/j.nbt.2023.09.002

Figure Lengend Snippet: Fig. 1. Anti-CD16a affibody affinity proteins. (a) Structure of the 58 aa (6.5 kDa) three-helix bundle affibody affinity protein scaffold used to construct combinatorial libraries from affibody molecules to CD16a were selected via phage display. The positions highlighted in red correspond to the 14 surface-located positions in helices 1 and 2 subjected to randomization for the construction of the library (see Supplementary information). The image was produced based on PDB entry 1q2n.pdb. (b) Structure of the extracellular domains (ECD) of human CD16a (hFcγRIIIa) protein. Position 158 (red) is a polymorphic position that can be occupied by either phenylalanine or valine, depending on the hCD16a allotype. Position 158 is located within the region of CD16a that interacts with the ligand IgG Fc (hinge) (dashed line). The image was produced based on the PBD entry 3ay4.pdb. (c) Left: Schematic figure showing the interpretation from direct binding and binning experiments of the relative epitope preferences for the A10, H09, and A11 affibody clones, where clone A10 binds to a distinct epitope different from an epitope shared by clones H09 and A11. Right: Sensorgrams obtained after injection of three selected anti-CD16a affibody affinity proteins (A10, H09, and A11, produced as gene fusions to an albumin-binding domain, ABD) at different concentrations (5 nM to 2.56 μM) over sensor chip surfaces containing either CD16a 158F or CD16a 158V ligands. Amino acid sequences of the A10, H09 and A11 affibodies are available in the PCT filing PCT/EP2023/064624 with sequence ID numbers 1, 75 and 74, respectively.

Article Snippet: Four cycles of biopanning were performed using recombinant, biotinylated hCD16a 158V (biotinylated human CD16a (F176) Avi tag, His tag, cat. no. CDA-H82E8, Acro Biosystems, Cambridge, USA), corresponding to residues 17–208 of Uniprot entry P08637, as the target protein, starting at 80 nM and decreasing in concentration with each cycle.

Techniques: Construct, Produced, Binding Assay, Clone Assay, Injection, Sequencing

Journal: New biotechnology

Article Title: Affibody-based hBCMA x CD16 dual engagers for NK cell-mediated killing of multiple myeloma cells.

doi: 10.1016/j.nbt.2023.09.002

Figure Lengend Snippet: Fig. 2. Bi-paratopic binding of heterodimeric anti-CD16a affibody constructs. Top panel: Schematic illustration of a biosensor-based experiment to investigate the binding characteristics of monovalent, homodimeric and heterodimeric anti-hCD16a affibody affinity protein constructs based on the A10, H09, and A11 clones, previously characterized in an epitope binning experiment, as indicated. All constructs were produced as equipped with a unique end C-terminal cysteine residue, allowing for a directed immobilization onto the sensor chip using thiol chemistry. Injection of a 200 nM concentration of hCD16a 158F or 158V protein over all ligands revealed differences in apparent affinities and binding kinetics for the different ligands. Bottom panel: Resulting sensorgrams from the experiment, showing higher response levels and slower off-rate kinetics for heterodimeric variants composed of affibody affinity protein units recognizing different epitopes, suggesting co- operative bi-paratopic binding to the injected hCD16a analytes (see text for details).

Article Snippet: Four cycles of biopanning were performed using recombinant, biotinylated hCD16a 158V (biotinylated human CD16a (F176) Avi tag, His tag, cat. no. CDA-H82E8, Acro Biosystems, Cambridge, USA), corresponding to residues 17–208 of Uniprot entry P08637, as the target protein, starting at 80 nM and decreasing in concentration with each cycle.

Techniques: Binding Assay, Construct, Clone Assay, Produced, Residue, Injection, Concentration Assay

Journal: New biotechnology

Article Title: Affibody-based hBCMA x CD16 dual engagers for NK cell-mediated killing of multiple myeloma cells.

doi: 10.1016/j.nbt.2023.09.002

Figure Lengend Snippet: Fig. 3. Assembly and simultaneous target binding activity of hBCMA x hCD16a affibody dual engager constructs. (a) Schematic illustration of a tri-partite anti- hBCMA x hCD16a affibody affinity protein-based dual engager containing one hBCMA-binding affibody (blue) and two different anti-CD16a affibody molecules (orange and brown), capable of bridging hBCMA expressing multiple myeloma (MM) tumor cells and hCD16a expressing natural killer (NK) cells, leading to the killing of MM cells by activated NK cells. (b) Block diagrams of constructed affibody affinity protein-based hBCMA x hCD16a dual engager candidates anti-hBCMA- A10-His6, anti-hBCMA-A10-A10-His6, and anti-hBCMA-H09-A10-His6. Affibody units were connected via flexible 15 aa long (GGGSG)3 linkers. (c) Schematic description of a biosensor-based experiment to demonstrate simultaneous hBCMA and CD16a binding by the hBCMA x CD16a dual engager constructs. In a first injection, one of the dual engagers at a time was injected at a concentration of 200 nM over a sensor chip surface containing immobilized hBCMa-Rabbit Fc fusion protein (Injection I). After a short period of buffer flow, a second injection was performed (Injection II) using 200 nM solutions of either hCD16a 158F/V proteins. (d) Overlay plot of the resulting sensorgrams from the experiment, showing that all three anti-hBCMA-hCD16a dual engagers were capable of simultaneous binding to both the sensor chip-immobilized hBCMA protein and the finally injected hCD16a 158F and 158V proteins. The dual engager construct anti-hBCMA-H09-A10-His6 containing a combination of the H09 and A10 affibody units, earlier shown to be capable of bi-paratopic binding to hCD16a 158F/V (brown traces), showed the highest responses and the slowest off-rate kinetics after the second injection (combined off-rate from both involved interactions).

Article Snippet: Four cycles of biopanning were performed using recombinant, biotinylated hCD16a 158V (biotinylated human CD16a (F176) Avi tag, His tag, cat. no. CDA-H82E8, Acro Biosystems, Cambridge, USA), corresponding to residues 17–208 of Uniprot entry P08637, as the target protein, starting at 80 nM and decreasing in concentration with each cycle.

Techniques: Binding Assay, Activity Assay, Construct, Expressing, Blocking Assay, Injection, Concentration Assay