The SpyBLI cell-free pipeline for the rapid quantification of binding kinetics from crude samples

Abstract

Accurate measurements of binding kinetics, encompassing equilibrium dissociation constant (K _D), association rate (k _on), and dissociation rate (k _off), are critical for the development and optimisation of high-affinity binding proteins. However, such measurements require highly purified material and precise ligand immobilisation, limiting the number of binders that can be characterised within a reasonable timescale and budget. Here, we present the SpyBLI method, a rapid and cost-effective biolayer interferometry (BLI) pipeline that leverages the SpyCatcher003-SpyTag003 covalent association, eliminating the need for both binder purification and concentration determination. This approach allows for accurate binding-kinetic measurements to be performed directly from crude mammalian-cell supernatants or cell-free expression blends. We also introduce a linear gene fragment design that enables reliable expression in cell-free systems without any PCR or cloning steps, allowing binding kinetics data to be collected in under 24 hours from receiving inexpensive DNA fragments, with minimal hands-on time. We demonstrate the method's broad applicability using a range of nanobodies and single-chain antibody variable fragments (scFvs), with affinity values spanning six orders of magnitude. By minimising sample preparation and employing highly controlled, ordered sensor immobilisation, our workflow delivers reliable kinetic measurements from crude mixtures without sacrificing precision. We expect that the opportunity to carry out rapid and accurate binding measurements in good throughput should prove especially valuable for binder engineering, the screening of next-generation sequencing-derived libraries, and computational protein design, where large numbers of potential binders for the same target must be rapidly and accurately characterised to enable iterative refinement and candidate selection.

Fig. 1. Overview of the SpyBLI pipeline. (A) Binders of interest are encoded in linear gene fragments, which are either used directly in cell-free expression, or Golden-Gate-cloned into vectors for expression in mammalian cell media. (B) Overview of the full BLI assay set up for a single assay sensor, with all steps highlighted (see legend). (C) Schematic of the fully loaded BLI assay sensor, forming a uniform surface of similarly oriented binders. (D) Example of a single-cycle kinetics binding curve (blue) obtained from an assay sensor probing increasing concentrations of antigen during the various association steps. This curve is fitted with a binding model (black line) to extract kinetic rate constants (k_on, k_off) and equilibrium dissociation constant (K_D = k_off/k_on).

Fig. 2. Consistency of binding kinetic measurements between purified and non-purified antibody fragments. (A) BLI sensorgrams of a scFv (PDB ID 7seg) binding to CD16a. The monovalent antigen was purified and used as analyte at increasing concentrations (6.25, 12.5, 25, 50, 100 nM) in each association phase. The SpyTagged scFv is used as ligand, and it was loaded either as purified scFv in buffer or from unpurified mixtures (see legend and Fig. S4, ESI†). Different experiments (coloured lines) were carried out on different days, and the minor differences in R_max (maximum signal) observed can be rationalised by minor differences in loading. The solid lines correspond to a fit with a 1 : 1 standard binding model (fit RMSEs between 0.015 and 0.018 nm, ≤2.3% of the response window). (B) Same as (A) but for the nanobody Nb24 (PDB ID 4kdt) binding to purified β₂-microglobulin used as analyte, which was present at 25, 50, 100, 200, 400 nM in each association phase, respectively. The solid lines correspond to a fit with a 1 : 1 partial dissociation binding model (fit RMSEs between 0.0035 and 0.0042 nm, ≤4% of the response window). Results of all fits are in Table 1.

Fig. 3. Characterisation of nanobodies spanning a broad range of affinities. (A) Binding sensorgrams of SpyTagged nanobody cAb-Lys3 (PDB ID 1mel) loaded from cell-free-expression blend and binding to purified hen egg-white lysozyme, which was used as analyte at increasing concentrations of 0.625,1.25, 2.5, 5, 10 nM. The solid lines correspond to a fit with a 1 : 1 standard binding model (fit RMSE ≅ 0.007 nm, ≅ 2.8% of the response window). (B) Binding sensorgram of SpyTagged nanobody Nb.B201 (PDB ID 5vnw) loaded from a mammalian-cell supernatant and binding to HSA, which was used as analyte at increasing concentrations of 62.5, 125, 250, 500, 1000 nM. The solid lines correspond to a fit with a 1 : 1 standard binding model (fit RMSE ≅ 0.006 nm, ≅ 6.4% of the response window).

Fig. 4. Characterisation of scFv binding in the pico-molar range. (A) Binding sensorgrams of SpyTagged scFv HyHEL10 (PDB ID 2znw) loaded from cell-free-expression mixture and binding to purified hen egg-white lysozyme, which was used as analyte at increasing concentrations of 0.31, 0.625, 1.25, 2.5, and 5 nM. The solid lines correspond to a fit with a 1 : 1 standard binding model (fit RMSE ≅ 0.007 nm, ≅ 2.7% of the response window). (B) Binding sensorgrams of SpyTagged scFv Ixekizumab and Secukinumab (see legend) loaded from a mammalian-cell supernatant and binding to IL-17A, which was used as analyte at increasing concentrations of 0.25, 0.5, 1, 2, and 4 nM. The association phase is plotted on a linear scale (x-axis), while the much longer dissociation phase on a log 10 scale. The solid lines correspond to a fit with a 1 : 1 standard binding model (fit RMSE ≅ 0.005 nm and 0.004 nm, ≅ 1.7% and 3.5% of the response window, respectively for Ixekizumab and Secukinumab).