iatpsnfr2 a95a a119l halotag  (Addgene inc)

Journal: bioRxiv

Article Title: An approach for single-amino-acid resolution epitope mapping by kinetic affinity screening of antibody drugs against biosensor on-chip library of deep mutationally-scanned target variants

doi: 10.64898/2026.04.30.722015

Figure Lengend Snippet: Schematic representation of CD20 showing (A) Primary amino acid sequence of the partial CD20 expressed in this study. Residues involved in rituximab and ocrelizumab binding are indicated. Non-membrane and membrane-spanning residues are unshaded and shaded in gray, respectively. Cysteine residues responsible for disulfide bond formation are highlighted in red. Only the underlined residues were targeted for partial mutational scanning. (B) PyMOL-generated 3D structures of the partial CD20 sequence and fused HaloTag protein as covalently immobilized on SPOC biosensor. The first five residues (TQSFF) and the last nine residues (GIVENEWKR) of the partial CD20 sequence in (a) were not displayed in this PyMol structure. The membrane-spanning region of CD20 is shaded in gray, whereas non-membrane regions are highlighted in white. CD20 PyMol structure was adapted from an online PDB crystal structure (ID: 6VJA). The crystal structure of the SPOC CD20 has not been obtained. Also, the lipid bilayer is shown for visual representation only – CD20 was not displayed on SPOC chip with a lipid bilayer. (C) Orientation of CD20 on the lipid bilayer, highlighting the binding epitopes targeted by rituximab and ocrelizumab (adapted from Delgado et al. ). Residues in the functional epitope regions indicated by the broken box were substituted with alanine, aspartate, lysine, and serine to generate CD20 DMS library.

Article Snippet: The print mix consisted of 1.46 mg/ml BS3 in DMSO, 7.4 mg/mL BSA, and limited amounts, 0 to 1 mM, of amine-terminated chloroalkane HaloTag ligand (Iris Biotech; RL-3680) in nuclease-free water.

Techniques: Sequencing, Binding Assay, Membrane, Generated, Functional Assay

Journal: bioRxiv

Article Title: An approach for single-amino-acid resolution epitope mapping by kinetic affinity screening of antibody drugs against biosensor on-chip library of deep mutationally-scanned target variants

doi: 10.64898/2026.04.30.722015

Figure Lengend Snippet: (A) In silico recombinant plasmid map of CD20 extracellular domain. Each gene variant was subcloned onto a custom cell-free expression-compatible pT7CFE1 plasmid for expression with an N-terminal HaloTag. (B) Graphical depiction of the SPOC chip design used in his study. CD20 ECD domains (gray) were expressed as HaloTag (blue) fusion proteins synthesized cell-free and captured onto SPOC chips. Various amino acid substitutions (colored circles) were introduced within regions of CD20 spanning the epitopes of the therapeutic antibodies, rituximab and ocrelizumab. The SPOC chip was then screened against these antibodies to assess the impacts of epitope mutagenesis on the antibody binding. (C) SPR flowcell image of a SPOC biosensor, showing distinct protein spots as well as a hockey-shaped fiducial (highlighted in red square) for locating each member protein.

Article Snippet: The print mix consisted of 1.46 mg/ml BS3 in DMSO, 7.4 mg/mL BSA, and limited amounts, 0 to 1 mM, of amine-terminated chloroalkane HaloTag ligand (Iris Biotech; RL-3680) in nuclease-free water.

Techniques: In Silico, Recombinant, Plasmid Preparation, Variant Assay, Expressing, Synthesized, Mutagenesis, Binding Assay

Journal: bioRxiv

Article Title: An approach for single-amino-acid resolution epitope mapping by kinetic affinity screening of antibody drugs against biosensor on-chip library of deep mutationally-scanned target variants

doi: 10.64898/2026.04.30.722015

Figure Lengend Snippet: Heat map of protein expression and capture validation, showing the mean binding response levels of SPOC biosensor proteins to 133 nM mouse anti-HaloTag. Columns show the native amino acid residues of CD20 targeted for substitution. Rows indicate the type of amino acid side-chain substitution. This heat map compares the capture levels of the CD20 mutant library with the wildtype and few control proteins, and validates the presence of CD20 mutants on the chip. Protein capture level was also validated using a mouse anti-HaloTag and anti-p53 antibodies (see for all sensorgrams). Overall, the capture validation result indicates that all CD20 mutants were relatively well-expressed when compared to the wildtype CD20 and controls except variants T159K and I162S, which produced comparatively lower RU values. Therefore, these mutants were ignored in subsequent binding kinetics computation. Crossed cell indicate no substitution for that wildtype residue.

Article Snippet: The print mix consisted of 1.46 mg/ml BS3 in DMSO, 7.4 mg/mL BSA, and limited amounts, 0 to 1 mM, of amine-terminated chloroalkane HaloTag ligand (Iris Biotech; RL-3680) in nuclease-free water.

Techniques: Expressing, Biomarker Discovery, Binding Assay, Mutagenesis, Control, Produced, Residue

Journal: bioRxiv

Article Title: An approach for single-amino-acid resolution epitope mapping by kinetic affinity screening of antibody drugs against biosensor on-chip library of deep mutationally-scanned target variants

doi: 10.64898/2026.04.30.722015

Figure Lengend Snippet: Sensorgrams of capture validation of SPOC protein chip using anti-HaloTag VHH, mouse anti-HaloTag, and mouse anti-p53

Article Snippet: The print mix consisted of 1.46 mg/ml BS3 in DMSO, 7.4 mg/mL BSA, and limited amounts, 0 to 1 mM, of amine-terminated chloroalkane HaloTag ligand (Iris Biotech; RL-3680) in nuclease-free water.

Techniques: Biomarker Discovery

Journal: bioRxiv

Article Title: An approach for single-amino-acid resolution epitope mapping by kinetic affinity screening of antibody drugs against biosensor on-chip library of deep mutationally-scanned target variants

doi: 10.64898/2026.04.30.722015

Figure Lengend Snippet: Sensorgrams of capture validation of each CD20 mutant protein and some controls on the SPOC protein chip using mouse anti-HaloTag antibody only

Article Snippet: The print mix consisted of 1.46 mg/ml BS3 in DMSO, 7.4 mg/mL BSA, and limited amounts, 0 to 1 mM, of amine-terminated chloroalkane HaloTag ligand (Iris Biotech; RL-3680) in nuclease-free water.

Techniques: Biomarker Discovery, Mutagenesis

Journal: bioRxiv

Article Title: An approach for single-amino-acid resolution epitope mapping by kinetic affinity screening of antibody drugs against biosensor on-chip library of deep mutationally-scanned target variants

doi: 10.64898/2026.04.30.722015

Figure Lengend Snippet: Schematic of SPOC platform application in antibody discovery pipelines. Initial antibody library screening (Phases 1.1 & 1.2) is accomplished by cloning antibody sequences in scFv or VHH format as HaloTag fusion constructs for cell-free expression and downstream SPOC chip capture (Chip A). In Phase 1.1, this diverse library of clones is screened with the desired target as analyte (gray) for antibody ligands that bind productively (dotted red circles) and with desirable interaction kinetics. In Phase 1.2, Chip A can be re-screened with target once-again, yet this time pre-bound with either a partner protein or competitive antibody that blocks the desired epitope. In this manner, competitive inhibition of binding to antibody ligands on the biosensor chip can be assessed as a means of functional characterization and epitope clustering. In Phase 2, a second chip is designed taking top clones from Phase 1 and performing deep-mutation scanning (DMS) of the paratopes (Chip B). The target can be screened and paratope substitutions that further tune binding identified. Finally, in Phase 3, a third chip is manufactured where the target itself is expressed and captured as a HaloTag fusion containing various amino-acid substitutions by DMS (Chip C). Top clones identified from Phase 2 can then be screened, and clones with desired epitope engagement (e.g. specificity to certain mutations, or resistance to certain epitope substitutions) can be selected as top lead candidates for further engineering and functional screening in cell-based or in vivo experimentation (e.g. neutralization assays).

Article Snippet: The print mix consisted of 1.46 mg/ml BS3 in DMSO, 7.4 mg/mL BSA, and limited amounts, 0 to 1 mM, of amine-terminated chloroalkane HaloTag ligand (Iris Biotech; RL-3680) in nuclease-free water.

Techniques: Library Screening, Cloning, Construct, Expressing, Clone Assay, Inhibition, Binding Assay, Functional Assay, Mutagenesis, In Vivo, Neutralization