WO2023227893A1

WO2023227893A1 - Split reporter complex for protein-complementation assays

Info

Publication number: WO2023227893A1
Application number: PCT/GB2023/051374
Authority: WO
Inventors: Hope ADAMSON; Lars JEUKEN; Darren TOMLINSON; Christoph Walti; Michael Mcpherson
Original assignee: University Of Leeds
Priority date: 2022-05-26
Filing date: 2023-05-25
Publication date: 2023-11-30
Also published as: GB2619059A; GB202207769D0

Abstract

The present invention relates to a two part split reporter complex, method and kits.

Description

SPLIT REPORTER COMPLEX FOR PROTEIN-COMPLEMENTATION ASSAYS

Field of Invention

A split reporter complex, method and kits.

Background to the Invention

Split proteins have been used for the detection and/or quantification of protein interactions. Various names have been given to the processes used for such detection and/or quantification, such as protein-fragment complementation assays, split protein complementation, or bimolecular fluorescence complementation. In these split protein systems, each fragment of the split protein is individually inactive. However, when the fragments of a split protein are combined at high concentrations, the fragments can form an active protein complex. For example European Patent EP3705574 relates to a fusion protein system using split luciferase. The system can be used as a biosensor for screening substances that bind strongly to vitamin D receptors. US Patent US20210285031 relates to a biosensor for cyclic dinucleotides whereby the fusion proteins can comprise a functional cyclic dinucleotide binding domain linked to a first fluorescent domain and a second fluorescent domain. The first fluorescent domain and a second fluorescent domain can produce, as a FRET pair, a detectable signal when brought into sufficiently close proximity. The fusion protein can be used for in vitro or in cell assays. US Patent US20190256887 relates to a biosensor for monitoring and/or quantifying in vitro or in vivo activity of the Hippo signalling pathway. The biosensor can contain one or more fragments of firefly or NanoBiT luciferase or a functional equivalent thereof.

Clostridioides (formerly Clostridium) difficile is an anaerobic, Gram-positive bacillus that is a leading cause of healthcare-associated infections, with high morbidity and mortality. Transmission is via spores by the faecal -oral route and disruption of protective intestinal microbiota by prior antibiotic administration is a major risk factor for C. difficile infection (CDI). The main virulence factors are toxin A (TcdA) and toxin B (TcdB), which trigger a cascade of host cellular responses that can lead to significant intestinal damage. In the US alone the annual burden is estimated to be over 600000 episodes, 44500 deaths and $5.4 billion in costs, largely due to hospitalisation. The severity and frequency of CDI has increased over the last two decades and methods to reduce this burden are urgently required. Timely and accurate diagnosis of CDI is imperative to ensure effective treatment and implementation of infection control measures. Disease-free carriage of C. difficile is widespread and it is important to distinguish this from true CDI. Current clinical guidance is to first use a high sensitivity stool test (e.g. an enzyme immunoassay (EIA) for common C. difficile antigen glutamate dehydrogenase (GDH)), for which a negative result reliably rules out CDI. Positives are followed up with a high specificity stool test (e.g. EIA for disease causing toxins TcdA/TcdB), for which a positive result reliably confirms CDI. GDH and toxin immunoassays have been combined in lateral-flow tests (LFTs, e.g. C. diff Quik Chek Complete), offering a simple, rapid, point-of-care approach to CDI diagnosis. However, the toxin test has low sensitivity and GDH + / toxin - results can be due to CDI with low toxin levels or C. difficile carriage, so further clinical evaluation and testing are required. Thus, a more sensitive rapid toxin test would vastly improve CDI diagnosis and improve patient outcomes. Quantitative tests, rather than qualitative LFTs, may also prove useful for research into disease outcomes and optimal treatment.

Summary of Invention

The problem to be solved by the present invention is to provide a more sensitive, easy to use and produce split reporter complex, in which light emission increases when agonist binds, and the present invention aims to provide such a complex. The present invention provides a convenient method to screen a substance which binds strongly to the split reporter protein. The present invention provides a new 2-part split reporter complex, for example a split luciferase assay system, whereby a signal, for example (bio)luminescence, is produced when the two components of the system are brought into close proximity. Instead of being coupled with two proteins that interact, the split reporter components are instead coupled to either affimers or nanobodies, in the form of fusion proteins. These affimers or nanobodies can bind to specific antigens, thereby bringing the split reporter components into proximity, which results in a detectable signal such as fluorescence. The invention is based on the surprising finding that affimers or nanobodies can fuse with a two component split reporter protein and effectively detect an analyte.

This rapid, homogenous, wash-free assay has a simple mix-and-read format and the signal output, for example bioluminescent output, can even be read with a camera, therefore it is well suited to adoption in Point of Care Tests (POCTs).

Antibodies can be used in a similar way to detect the presence of antigens however, this requires the antibodies to be chemically conjugated to the split reporter components. By expressing the affimer or nanobody as fusion proteins, the split reporter components would already be bound to the affimer or nanobody when the proteins are purified, removing the need for chemical conjugation. Therefore the present invention provides a split reporter platform that is easier to produce and use and thereby lower costs is an additional advantage. In addition, chemical conjugation to antibodies can lead to more heterogeneity in the conjugated product, which could reduce sensitivity or reproducibility between batches.

In a first aspect, the present invention provides a two-part split reporter complex, the complex comprising a first agent, wherein the first agent comprises a first nanobody or affimer fused to the first peptide fragment of a split reporter protein and a second agent, wherein the second agent comprises a second nanobody or affimer fused to a second peptide fragment of a two- part split reporter protein.

In a second aspect, the present invention provides a two-part split reporter complex according to the first aspect of the invention, wherein the split reporter complex has an amino acid sequence comprising SEQ ID NO: 2, 4, 6 or 8.

In a third aspect, the present invention provides a DNA encoding an amino acid sequence according to the second aspect of the invention.

In a forth aspect, the present invention provides a plasmid comprising the DNA according to the third aspect of the invention in a vector.

In another aspect, the present invention provides a transformed cell wherein the cell comprises the plasmid according to the forth aspect of the invention.

In another aspect, the present invention provides a method of detecting an analyte in a mixture, the method comprising: delivering the two-part split reporter complex according to the first aspect of the invention, wherein the first agent and second agent are delivered as separate agents and wherein when the first and second nanobody or affimer bind an analyte, the first peptide fragment and second peptide fragment of the split reporter protein are brought into proximity and a detectable signal is produced.

In another aspect, a method for detecting an analyte in a cell, comprising expressing the two- part split reporter complex according to the first aspect of the invention in a cell and detecting a signal generated by the split reporter complex.

Brief Description of the Drawings Exemplary arrangements of the disclosure shall now be described with reference to the drawings in which:

Figure 1: Schematic of the NanoBiT split-luciferase assay. Fragments of the split NanoLuc enzyme, LgBiT and SmBiT, are attached to binding proteins that target different regions of the analyte. Analyte binding colocalises LgBiT and SmBiT, promoting reconstitution of the enzyme and bioluminescence upon addition ofNanoGlo substrate.

Figure 2: Direct ELISAs to select Affimers. (A) ELISA to assess binding of adsorbed Affimer (white) or TcdB (grey) to biotinylated TcdB or Affimer, respectively. (B) ELISA to assess binding of adsorbed Affimers to biotinylated GDH. Detection was with streptavidin- HRP and visualisation with TMB, read at 620 nm. Where error bars are present, data are the mean of duplicate measurements on the same plate and error bars indicate the standard deviation.

Figure 3: Sandwich ELISAs to select Affimers. (A) Sandwich ELISA to assess pairwise binding of adsorbed “capture Affimer” 18 (white) or 28 (grey) and biotinylated “detection Affimer” (including non-binding control Affimer C) with TcdB. (B) Sandwich ELISA to assess pairwise binding of adsorbed Affimer G4 (referred to as Affimer 4 in the main text) and biotinylated “detection Affimer” with GDH. Detection was with streptavidin- HRP and visualisation with TMB, read at 620 nm. Where error bars are present, data are the mean of duplicate measurements on the same plate and error bars indicate the standard deviation.

Figure 4: SPR binding curves for (A) Affimer 4 (B) Affimer 18 (C) Affimer 45 (D) Nanobody E3 and (E) Nanobody 7F. Affimers / nanobodies were biotinylated and immobilised on a streptavidin chip. Serial dilutions of analyte injected at 30 μl min ¹ for 3 min, followed by 10 min dissociation. Data normalised by subtraction of responses from unmodified reference cell and buffer only injection. A global fit to the SPR curves (thin lines) was made with a 1: 1 Langmuir model using the BIAevaluation software and used to determine the association (k_a) and dissociation (k_d) rate constants and equilibrium dissociation constant (K_D).

Figure 5: SDS-PAGE of purified split NanoLuc sensor proteins, as indicated in the Figure. Proteins each of the binding proteins (TcdB Affimers 18 and 45, TcdB nanobodies E3 and 7F and GDH Affimer 4) were genetically fused to the N- or C- terminus of LgBiT (L) or SmBiT101 (S) via a (GSG)7 linker peptide. L-45 and S-45 for an N-terminal fusion of LgBiT and SmBiT to Affimer 45, respectively and, similarly, E3-L and E3-S for a C-terminal fusion to Nanobody E3, etc. The linker, (GSG)₇ (SEQ ID NO: 33), is present in all constructs, but not included in this nomenclature. Of these 20 constructs 16 were successfully produced in E. coli and purified via a C-terminal 6x-Histag (Figure 5) with yields of up to 90 mg L-1. E3-L, E3-S, 7F-L and 7F-S are considered to have insufficient purity following metal affinity purification and were not taken forward.

Figure 6: Establishing optimal sensor protein combinations. (A) Fold gain in bioluminescence of TxB sensor proteins with 1 nM TxB vs. 0 nM TxB. Luminescence was read immediately after substrate addition and data are the mean of duplicates on the same plate. Different shades of grey indicate different binding protein pairs. (B) Dose response of TxB sensor proteins. Luminescence was read 4 mins after substrate addition and data are the mean of three independent measurements. (C) Dose response of GDH sensor proteins. Luminescence was read 4 mins after substrate addition and data are the mean of two sets of duplicates from two independent experiments. For all assays, analyte (final concentration indicated) and sensor proteins (final concentration = 2 nM each) were incubated for 30 mins, at 25 °C, with agitation prior to addition of NanoGlo substrate to a final dilution of 1: 1000. Error bars indicate standard deviation from the mean and solid lines are 5PL regression fits (B: 0.970<R2< 0.987 and C: 0.990<R2<0.999). For the nomenclature on sensor constructs, please see the legend of Figure 5.

Figure 7: Establishing optimal sensor protein concentrations. (A) Bio-luminescence and (B) Fold gain in bioluminescence of 0.125 - 2 nM each of S-E3 + L-45 with TxB. (C) Heat map of fold gain in bioluminescence of 0. 125 - 4 nM S-E3 + 0. 125 - 4 nM L-45 with 1 nM TxB. (D) Bioluminescence and (E) Fold gain in bioluminescence of 0.5 - 16 nM each of 4-S + 4-L with GDH (F) Heat map of fold gain in bioluminescence of 0.5 - 16 nM 4-S + 0.5 - 16 nM 4-L with 10 nM GDH. For all assays, analyte (final concentration indicated) and sensor proteins (final concentration indicated) were incubated for 30 mins, at 25°C, with agitation prior to addition of Nano-Glo substrate to a final dilution of 1: 1000 and bioluminescence was immediately read. For (A) and (B) data are the mean of triplicates on the same plate and error bars (which for most data lies with-in the point) indicate standard deviation from the mean. For (D) and (E) data are single measurements. For (C) and (F) data are the mean of duplicates on the same plate. For the nomenclature on sensor constructs, please see the legend of Figure 5.

Figure 8: Dose response of 0.5 - 1 nM S-E3 + 0.5 - 1 nM L-45 with TxB. Data are the mean of three independent measurements and error bars indicate standard deviation from the mean. For all assays, analyte (final concentration indicated) and sensor proteins (final concentration indicated) were incubated for 30 mins, at 25 °C, with agitation prior to addition of Nano-Glo substrate to a final dilution of 1: 1000 and bioluminescence was immediately read. For the nomenclature on sensor constructs, please see the legend of Figure 5.

Figure 9: Kinetics of the TxB split NanoLuc assay (A) Bioluminescence and (D) Fold gain in bioluminescence dose response curve, read immediately after substrate addition following a 0-60 mins pre-incubation. (B) Bioluminescence and (E) Fold gain in bioluminescence vs. 0 pM TxB over time after no pre-incubation. (C) Bioluminescence and (F) Fold gain in bioluminescence vs. 0 pM TxB over time after 15-60 mins pre-incubation. For all assays, TxB (final concentration indicated), S-E3 (0.5 nM final concentration) and L- 45 (1 nM final concentration) were incubated for the indicated length of time, at 25°C, with agitation prior to addition of Nano-Glo substrate to a final dilution of 1: 1000. Data are the mean of duplicates on the same plate and error bars indicate standard deviation from the mean. For the nomenclature on sensor constructs, please see the legend of Figure 5.

Figure 10: Kinetics of the GDH split NanoLuc assay (A) Bioluminescence and (C) Fold gain in bioluminescence dose response curve, read immediately after substrate addition following a 15-60 mins pre-incubation. (B) Bioluminescence and (D) Fold gain in bioluminescence vs. 0 nM GDH over time after 15-60 mins pre-incubation. For all assays, GDH (final concentration indicated), 4-S (8 nM final concentration) and 4-L (8 nM final concentration) were incubated for the indicated length of time, at 25°C, with agitation prior to addition of Nano-Glo substrate to a final dilution of 1: 1000. Data are the mean of duplicates on the same plate and error bars indicate standard deviation from the mean. For the nomenclature on sensor constructs, please see the legend of Figure 5.

Figure 11: Establishing optimal Nano-Glo substrate concentration. (A) Bioluminescence and (C) Fold gain in bioluminescence dose response curve read immediately after substrate addition (B) Bioluminescence and (D) Fold gain in bioluminescence vs. 0 pM TxB over time. For all assays, TxB (final concentration indicated), S-E3 (0.5 nM final concentration) and L-45 (1 nM final concentration) were incubated for 30 mins, at 25 °C, with agitation prior to addition of Nano-Glo substrate to a final dilution of 1: 100 - 1:4000. Data are the mean of duplicates on the same plate and error bars indicate standard deviation from the mean. For the nomenclature on sensor constructs, please see the legend of Figure 5.

Figure 12: Optimised dose response curves used to calculate intra-assay LOD, accuracy (% recovery) and precision (% CV). (A) Bio-luminescent response of TxB split- luciferase assay with 0.5 nM S-E3 + 1 nM L-45. Data are the mean of 6 (TxB, red) or 2 (TxA control, black) replicates on the same plate. (B) Bioluminescent response of GDH split- luciferase assay with 8 nM 4-S + 8 nM 4-L. Data are the mean of 6 (GDH, blue) replicates on the same plate or single measurements (TxB control, black). For all assays, analyte (final concentration indicated) and sensor proteins (final concentration indicated) were incubated for 30 mins, at 25°C, with agitation prior to addition of Nano-Glo substrate to a final dilution of 1: 1000 and bioluminescence was read after 2 mins. Error bars (which for most data he within the point) indicate standard deviation from the mean, solid lines are 5PL regression fits (R2=1.000 for both A and B) and LoD indicated by dash line. For the nomenclature on sensor constructs, please see the legend of Figure 5.

Figure 13: Effect of faeces on the split-luciferase assay dose response curves. (A) Bioluminescence and (B) Fold gain in bioluminescence of 0.5 nM S-E3 + 1 nM L-45 or 8 nM 4-S + 8 nM 4-L, with TxB or GDH, respectively, in the presence of 0 or 3.33 % w/v faeces. Sensor proteins (final concentration in the assay indicated) and analyte (final concentration in the assay indicated) incubated with C. difficile negative faecal sample or buffer for 30 mins, at 25°C, with agitation. Nano-Glo added to a final concentration of 1: 1000 and bioluminescence read after 2 mins. Data are the mean of duplicates on the same plate, error bars indicate standard deviation from the mean and solid lines are 5PL regression fits (A: 0.939<R2< 0.993 and B: 0.939<R2<1.000). For the nomenclature on sensor constructs, please see the legend of Figure 5.

Figure 14: SDS-PAGEs of purified split NanoBit sensor proteins (affimer fusion proteins with SmBiT and LgBiT), as indicated in the Figure. Affimers are either fused at the N-terminus (for example, Affimer-GSGv-SmBiT) or C-terminus (for example, SmBiT-GSGv Affimer) of the SmBiT and LgBit. Affimers are selected to bind to four different (therapeutic) antibodies (Ada = Adalimumab; Tra = Trastuzumab; Ipi = Ipillimumab and Rit = Rituximab).

Figure 15: Optimised dose response curves used to calculate intra-assay LOD, accuracy (% recovery) and precision (% CV). Bio-luminescent response of split-luciferase assay for four different therapeutic antibodies (Ipillimumab, Rituximab, Trastuzumab and Adalimumab as indicated) with 2 nM LgBiT and SmBiT fusion constructs with Affimers. Data are the mean of 3 replicates on the same plate. Error bars (which for most data lie within the point) indicate standard deviation from the mean, solid lines are 5PL regression fits and LoD indicated below the Figures. The quantitative ranges are as follows: Ipilimumab 6.7 nM - 1724 nM (9.7 ng/mL - 2500 ng/mL); Rituximab 0.84 nM - 1724 nM (1.22 ng/mL - 2500 ng/mL); Trastuzumab 6.7 nM - 215 nM (9.7 ng/mL - 312.5 ng/mL); Adalimumab 1.68 nM - 1724 nM (2.44 ng/mL - 2500 ng/mL). CV < 20% and recovery >80%.

Figure 16: (A) Establishing optimal Nano-Glo substrate concentration. (Left) Bioluminescence and (Right) Fold gain in bioluminescence dose response curve read 2 min after substrate addition. For all assays, Rituximab (final concentration indicated), LgBiT-GSG₇-affimer (2 nM final concentration) and SmBiT-GSG₇-affimer (2 nM final concentration) were incubated for 30 mins, at 25 °C, with agitation prior to the addition of Nano-Glo substrate to a final dilution of 1:50 - 1:2000. Data are the mean of duplicates on the same plate and error bars indicate standard deviation from the mean. (B) The same experiment as in A) with 1250 ng/mL Rituximub, but bioluminescence is measured continuously for 1800 s (30 min).

Figure 17: Effect of plasma on the split-luciferase assay dose response curves. (Left) Bioluminescence and (Right) Fold gain in bioluminescence of 2 nM LgBiT-GSG₇-Affimcr and SmBiT-GSG₇-Affimer, with Rituximab, in the presence of 0 - 1 % v/v human plasma. Sensor proteins incubated with Rituximab sample (at indicated concentration) for 30 mins, at 25 °C. Nano-Glo added to a final concentration of 1:500 and bioluminescence read after 2 mins.

Figure 18: Bio-luminescent response of split-luciferase assay for human C-reactive protein (hCRP) with 2 nM LgBiT and SmBit101 or SmBit114 fusion constructs with Affimers (Aff90). 8 Different combinations are compared as indicated in the legend with (A) LgBit- GSG₇-Aff90; (B) Aff90-GSG₇-LgBit; (C) SmBitl01-GSG₇-Aff90; (D) Aff90-GSG₇- SmBit101; (E) SmBit114-GSG₇-Aff90; (F) Aff90-GSG₇-SmBit114.

Figure 19: Four sensor combinations were trialled for each target TmAb and the optimal pair chosen for each. Raw luminescence data (top) and fold gain data (bottom) from NanoBiT assays performed on the selected optimal LgBiT / SmBit101 pairs for each target TmAb. Sigmoidal, 4 parameter logistic (4PL) fits were used on data points up to concentrations of 10 nM. Due to the evident hook effect at concentrations of 100 nM, these data points were not included in the fit and the curve was extrapolated for these points, n = 1.

Figure 20: The four optimal sensor combinations for each target TmAb are highly specific for their respective TmAb. Raw luminescence from NanoBiT assays performed on the selected optimal LgBiT / SmBit101 pairs for all four target TmAb. A final concentration of 2 nM LgBiT and SmBiT were used with 5 μg/mL (35 nM) of each TmAb. Data are presented as a mean of 3 repeats with error bars representing standard deviation.

Figure 21: All four anti -ID NanoBiT sensors can effectively detect within the therapeutic and trough concentration range of each respective TmAb. Data on all sensors at 2 nM in response to increasing concentrations of their respective TmAb. Assays were performed in 0. 1% pooled human serum, incubated for 5 minutes shaking at 25°C, and RLU measurements taken after 2 minutes. Standard curves were interpolated for all four sensors using sigmoidal, 4 parameter logistic (4PL) fits. Top Figure, n = 3, performed on the same day with the same reagents. Bottom Figure. N = 3, performed on separate days, using fresh reagents. Data is presented as fold gain activity and error bars represent ±SEM.

Specific Description

Current clinical diagnostics for CDI have major limitations especially in differentiating disease-free carriage of C. difficile from true CDI with toxin production. This leads to poor treatment and inappropriate antibiotic prescribing that in fact increases CDI and drug-resistant infection risk by dysbiosis. The reference test for true CDI (cell cytotoxity neutralisation assay, CCNA) sensitively detects toxin but is too slow and complex for routine use. Faster nucleic acid amplifications tests (NAATs) detect toxin genes, but not expression so lack clinical specificity, whilst EIAs and LFTs detect free toxin but lack the sensitivity of CCNA. No one test is sufficient and clinical guidance relies on multi-step algorithms, leading to delays and confusion with discordant results. Recent ultrasensitive toxin immunoassays offer promise as standalone tests for true CDI. However, commercial “single molecule counting” technology relies on specialised instrumentation for magnetic separation and fluorescent imaging detection, whilst electrochemical sandwich assays require multiple incubation and wash steps. Our homogenous wash-free assay is much simpler and amenable to adaptation into a POCT. The 12 μg ml-1 = 44 fM TxB (TcdB) LoD and 4-5 -log range in buffer compares favourably with clinically relevant concentrations and the ~ 20 pg ml-1 = 74 fM cut-off for optimal sensitivity and specificity vs. CCNA. Recently described POCTs based on a paper device and label-free electrochemical sensing have lower sensitivity and range, respectively. Therefore, our assay can be adapted into a POCT and after faecal sample matrix effects are reduced, it offers an urgently required improvement for ultrasensitive toxin detection and CDI diagnosis. Clinical TxB concentrations of CDI samples have an upper limit of 100 ng ml-1 with some samples going up to 1,000 ng ml-1 (370 pM and 3.7 nM, respectively). At the highest TxB concentrations (> InM TxB), the reading of the assay levels off and quantitative determination of the TxB concentration is less accurate. If a quantitative concentration determination at >lnM TxB is required, the sample can be diluted prior to the assay.

We have selected and characterised Affimers (13 kDa non-immunoglobulin binding proteins) targeting C. difficile biomarkers GDH and TcdB, which can be used in diagnostic assays for CDI. When incorporated alongside nanobodies (single domain antibodies) in the assays, they show advantages over antibodies in terms of ease of production and assay performance. The assays are applicable to other biomarkers and offer a promising underlying platform technology for POCTs.

The present disclosure relates generally to a two-part split reporter complex, including two- part split reporter complex for use in detecting and/or measuring an analyte.

A first aspect of the invention is a two-part split reporter complex, the complex comprising a first agent, wherein the first agent comprises a first nanobody or affimer fused to the first peptide fragment of a split reporter protein and a second agent, wherein the second agent comprises a second nanobody or affimer fused to a second peptide fragment of a two-part split reporter protein.

Both of the first agent and the second agent are recombinant fusion proteins. In some embodiments, the fusion proteins include a solubilizing protein or domain (e.g., HaloTag®, (Promega)).

The term “fusion” protein refers to a protein that contains at least two peptides or polypeptide domains or sub-sequences that do not naturally occur together and, thus, the fusion protein is artificial in the sense that human intervention is required to produce it. The fusion protein can be synthesized or produced recombinantly according to techniques known in the art. For example, recombinant expression comprises the arrangement of nucleic acid sequences encoding the two or more peptide or polypeptide sequences in a single expression cassette such that encoded domains are expressed together and in-frame in the final fusion protein.

The expression cassette can be introduced into a cell or other expression system, e.g., using an expression vector, to facilitate transcription and translation of the fusion protein.

As used herein, an “AFFIMER®” polypeptide (also referred to simply as an “AFFIMER”®) is a small, highly stable polypeptide (e.g., protein) that is a recombinantly engineered variant of stefin polypeptides. The term "Affimer" may be used interchangeably with AFFIMER®, etc., and any term may be used without limitation. A stefin polypeptide is a subgroup of proteins in the cystatin superfamily - a family that encompasses proteins containing multiple cystatin-like sequences. The stefin subgroup of the cystatin family are relatively small (~ 100 amino acids) single domain proteins. They receive no known post-translational modification, and lack disulfide bonds, suggesting that they will be able to fold identically in a wide range of extracellular and intracellular environments. Stefin A is a monomeric, single chain, single domain protein of 98 amino acids.

AFFIMER® polypeptides display two peptide loops and an N-terminal sequence that can all be randomized to bind to desired target proteins with high affinity and specificity, in a similar manner to monoclonal antibodies. Stabilization of the two peptide loops by the stefin A protein scaffold constrains the possible conformations that the peptide loops can take, increasing the binding affinity and specificity compared to libraries of free peptides. These engineered non-antibody binding proteins are designed to mimic the molecular recognition characteristics of monoclonal antibodies in different applications. Variations to other parts of the stefin A polypeptide sequence can be carried out, with such variations improving the properties of these affinity reagents, such as increase stability, make them robust across a range of temperatures and pH, for example. In some embodiments, an AFFIMER® polypeptide includes a sequence derived from stefin A, sharing substantial identity with a stefin A wild type sequence, such as human stefin A. In some embodiments, an AFFIMER® polypeptide has an amino acid sequence that shares at least 25%, 35%, 45%, 55% or 60% identity to the sequences corresponding to human stefin A. For example, an AFFIMER® polypeptide may have an amino acid sequence that shares at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95% identity, e.g., where the sequence variations do not adversely affect the ability of the scaffold to bind to the desired target, and e.g., which do not restore or generate biological functions such as those that are possessed by wild type stefin A, but which are abolished in mutational changes described herein. As used herein, the term AFFIMER® may be used interchangeably with "recombinantly engineered variant of stefin polypeptide".

As used herein, a “nanobody” is currently the smallest antibody molecule, whose binding domain is with a molecular weight of about 1/10 of that of a conventional monoclonal antibody. It was firstly discovered by Belgian scientist Hamers, R in camel blood. Nanobodies are recombinant, antigen-specific, single-domain, variable fragments of camelid heavy chain-only antibodies.

In addition to antigenic reactivity, nanobody also has some unique functional properties.

Nanobody has a small molecular weight, strong stability, good solubility, high expression, weak immunogenicity (with more than 70% similarity to human protein sequence), strong penetration, strong targeting, easy humanization, low preparation cost and so on.

It should be noted that, in general, the term Nanobody as used herein is not limited in the broadest sense to a specific biological material or a specific method of preparation. For example, the Nanobodies of the present invention comprise (1) isolation of a VHH domain of a natural heavy chain antibody, (2) expression of a nucleotide sequence encoding the natural VHH domain, (3) natural VHH Humanization of domains or expression of nucleic acids encoding the humanized VHH domains, (4) camelization of natural VH domains from any animal species, particularly mammals (e.g., humans), (5) Ward et al. (supra) "camel" of the "domain antibody" or "Dab" described, or expression of a nucleic acid encoding the camelized VH domain, ( 6) synthesis for the preparation of proteins, polypeptides, other amino acid sequences, use of semi-synthetic techniques, (7) Preparation of nucleic acids encoding Nanobodies using nucleic acid synthesis techniques, and thus obtained nucleic acid expression and / or (8) any combination of the above. Suitable methods and techniques for carrying out the above will be apparent to those skilled in the art based on the disclosure herein, including, for example, the methods and techniques detailed below.

It is noted that the terms Nanobody or Nanobodies are registered trademarks of Ablynx NV and can therefore be referred to as Nanobodies or Nanobody.

The polypeptide terms "polypeptide" and "peptide" and "protein" are used interchangeably herein to mean a polymer of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids and may be interrupted by non-amino acids. The term also includes amino acid polymers modified naturally or by intervention; other manipulations or modifications such as disulfide bond formation, glycosylation, lipoxylation, acetylation, phosphorylation, or conjugation with labelled components. Also includes the amino acid polymers that have been produced. Also included are, for example, polypeptides containing one or more analogs of amino acids (including, for example, unnatural amino acids), as well as other modifications known in the art.

In one embodiment the first peptide fragment of the split reporter protein and the second peptide fragment of the split reporter protein are complementary components, which when in proximity is capable of generating a detectable signal. In another embodiment, the first peptide fragment of the split reporter protein and the second peptide fragment of the split reporter protein are complementary components of a luminescent protein. In a further embodiment, the first peptide fragment of the split reporter protein and the second peptide fragment of the split reporter protein form a FRET pair. The FRET pair may be a blue/orange FRET pair, a cyan/yellow FRET pair, or a far-red FRET pair. In a further embodiment, the first peptide fragment of the split reporter protein and the second peptide fragment of the split reporter protein form a BRET pair.

In one embodiment the split reporter protein comprises β-lactamase, β-galactosidase, dihydrofolate reductase, green fluorescent protein, ubiquitin, and TEV protease. One split reporter protein that has been used to detect and quantify protein interactions is NanoBiT® (Promega®). NanoBiT® is a split and modified form of NanoLuc® (Promega®), an engineered luciferase derived from a deep sea luminous shrimp. The split NanoBiT® enzyme includes a relatively short peptide fragment (11 amino acids) and a relatively long peptide fragment (an 18 kDa polypeptide).

The embodiments disclosed herein involve the use of a two-part split reporter protein, which may also be referred to as a split reporter or a split reporter protein complex. The split reporter is a binary (i.e., two-part) reporter. The individual fragments of the split reporter may be individually inactive. However, when combined with complementary peptide fragment(s) of the split reporter, the peptide fragments may bind to one another to form an active protein complex. Examples of split reporter proteins include split green fluorescent protein, NanoBiT®, and split β-lactamase.

In another embodiment the present invention is a biosensor, biosensors are devices capable of providing specific quantitative or semi-quantitative analytical information using a biological recognition element that is combined with a transducing (detecting) element. The biological recognition element of a biosensor determines the selectivity, so that only the compound to be measured leads to a signal. The transducer translates the recognition of the biological recognition element into a semi-quantitative or quantitative signal. Possible transducer technologies are optical, electrochemical, acoustical/mechanical or colorimetric.

In some embodiments, the two-part split reporter protein is a split fluorescent protein, such as a split green fluorescent protein. In other embodiments, the split reporter protein is a split enzyme. The split enzyme may catalyze the conversion of a substrate to a product. The activity of the split enzyme on the substrate may result in the emission of a detectable (e.g., luminescent) signal. For example, in some embodiments, the split enzyme is a split luciferase. Luciferase is an enzyme oxidizing substance (generically referred to as luciferin) as substrate, and releases a part of the energy generated in the oxidation reaction as light emission, and various enzymes are known depending on its origin. Luciferase may include, for example, a luciferase selected from the group consisting of firefly luciferase, a Pyrophorus plagiophthalamus luciferase, an Emerald luciferase, a Renilla reniformis luciferase, a Cypridina noctiluca luciferase, and Gaussia princess luciferase, prawns Oplophorus gracilirostris (deep sea prawns) luciferase and a variant of Oplophorus gracilirostris luciferase. A substrate of firefly luciferase and Pyrophorus plagiophthalamus luciferase is firefly luciferin, and a substrate of Renilla reniformis luciferase, Cypridina noctiluca luciferase, Gaussia luciferase and Oplophorus gracilirostris luciferase is coelenterazine. Substrate of variant of Oplophorus gracilirostris luciferase is furimazine. The luciferase may be divided into N terminal domain LucN and C terminal domain LucC. In some embodiments, the substrate for the split enzyme is luciferin, furimazine, or some other luminogenic substrate or molecule. In some embodiments, the split enzyme catalyzes the conversion of furimazine to furimamide.

In another embodiment, the split fluorescent protein forms a Forster resonance energy transfer (FRET) pair. FRET is a mechanism whereby energy is transferred between two light-sensitive molecules (chromophores or fluors). In a FRET pair, typically one member of the pair serves as a donor that, in an excited state, transfers energy to the second member of the pair, which serves as the acceptor, through dipole-dipole coupling. The transfer of energy is detected by virtue of a unique wavelength of light that is released. The efficiency of the energy transfer is extremely sensitive to spatial distance and so the emission of the unique signal is only detected when the two pair members are in very close and precise proximity. In another embodiment, the split fluorescent protein forms a bioluminescence resonance energy transfer (BRET) pair. BRET is similar to FRET, but incorporates a biolumine scent donor instead of a fluorescent donor. A distinction with FRET is that the FRET donor must be excited, e.g., with an external light source, to initiate the energy transfer to the acceptor, whereas a bioluminescent donor does not require an external light source. BRET donor moieties or proteins are known and encompassed by the present disclosure and can be readily incorporated into a pairing appropriate for a BRET signal. BRET donor moieties or proteins can require a substrate to induce the initial bioluminescence. An exemplary BRET donor moiety or protein is a luciferase enzyme.

In another embodiment the nanobody or affimer binds an analyte. The analyte may be glutamate dehydrogenase (GDH), toxin B (TcdB), hCRP or an antibody. The analyte may be glutamate dehydrogenase (GDH) or toxin B (TcdB). More specifically the analyte may be glutamate dehydrogenase. In another embodiment the analyte may be toxin B (TcdB). In another aspect the present invention provides a two-part split reporter complex for use in detecting Clostridioides (formerly Clostridium) difficile. The present invention is particularly useful for detecting bacteria within body samples, for example faeces. In another aspect the present invention provides a two-part split reporter complex for use in detecting the toxins and markers of Clostridioides (formerly Clostridium) difficile. The present invention is particularly useful for detecting the toxins and markers of bacteria within body samples, for example faeces.

The term "binding proteins" refers to proteins that interact with specific analytes in a manner capable of providing or transducing a detectable and or reversible signal differentiable either from when analyte is not present, analyte is present in varying concentrations over time, or in a concentration-dependent manner, by means of the methods described.

The first nanobody or affimer may be configured to selectively bind to a first target region of an analyte, and the second nanobody or affimer may be configured to selectively bind to a second target region of the analyte. For example, a first nanobody or affimer may be configured to selectively bind to a first epitope of an analyte, while the second nanobody or affimer is configured to selectively bind to a second epitope of the analyte. The term "associates" or "binds", as used herein, refers to binding partners having a relative binding constant (Kd) sufficiently strong to allow detection of binding to the protein by a detection means. The Kd may be calculated as the concentration of free analyte at which half the protein is bound, or vice versa.

In some embodiments, the first target region and the second target region are different in structure (e.g., different epitopes). In other embodiments, the first target region and the second target region are identical or substantially identical in structure (e.g., identical or substantially identical epitopes), but are located at separate sites on the analyte (e.g., the protein). In some embodiments, the first target region and the second target region are separated by less than 300 (e.g., less than 150) angstroms. For example, in some embodiments, the first target region is separated from the second target region by 2-300 angstroms, 2-200 angstroms, 2-175 angstroms, 2-150 angstroms, 2-125 angstroms, 2-100 angstroms, 2-75 angstroms, 2-50 angstroms, 2-25 angstroms; 25-300 angstroms, 50-300 angstroms, 75-300 angstroms, 100-300 angstroms, 125-300 angstroms, 150-300 angstroms, 10-150 angstroms; 25-145 angstroms; 35-145 angstroms, 40-145 angstroms, 50-125 angstroms, or 60-100 angstroms. The analyte to be detected and/or measured may be any suitable analyte. In some embodiments, the analyte is a biomolecule, such as a protein, nucleic acid, carbohydrate, or lipid. In some embodiments, the analyte is a modified protein, such as a phosphorylated protein, a glycosylated protein, or an antibody-drug conjugate. In some embodiments, the analyte is an antibody, such as a natural, synthetic, or recombinant antibody (or a portion thereof). In some embodiments, the analyte is an antibody formed in response to an allergen, a bacterial infection, or a viral infection.

In some embodiments, the analyte is a viral or bacterial protein, DNA, or RNA. Stated differently, in some embodiments, the analyte is DNA, RNA, or a protein from a virus or a bacterium, such as a pathogenic virus or bacterium. Exemplary bacteria for the analyte can be Clostridioides (formerly Clostridium) difficile.

In one embodiment of the present invention, the split reporter complex is used for analyte sensing in vivo. In another embodiment of the present invention, the split reporter complex is used for analyte sensing in vitro.

In one embodiment the two-part split reporter complex, wherein the split reporter complex has an amino acid sequence comprising SEQ ID NO: 2, 4, 6 or 8. In another embodiment the polypeptide may have an amino acid sequence that shares at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95% identity with SEQ ID NO: 2, 4, 6 or 8, e.g., where the sequence variations do not adversely affect the ability of the complex to bind to the desired target.

The present invention encompasses DNAs encoding amino acid sequence of any one of the fused proteins of the present invention.

The present invention encompasses a plasmid which contains any vector DNAs encoding amino acid sequence of any one of fused proteins of the present invention. Vectors constituting the present plasmid are optional, and can be appropriately selected according to a cell to be transformed by introducing the present plasmid. It is preferable that the plasmid contains a promotor, for example, SV40, CMV, CAG promotor, or the like, because it is preferable that the fused protein of the present invention be expressed constitutively and highly in a cell.

Fused proteins of the present invention can be prepared by expressing in a cell (transformant), introduced with a plasmid which contains the DNA encoding amino acid sequence of the fused proteins of the present invention. The introduction of DNAs into a cell (transformation) and the expression of proteins in the transformants can be carried out as appropriate by conventional methods. Fused proteins of the present invention can also be synthesized by in vitro synthetic methods using DNAs encoding amino acid sequences of any of the fused proteins of the present invention. Synthesis of proteins in vitro can be carried out using a commercially available kit. Therefore there is provided another embodiment of a transformed cell wherein the cell comprises the plasmid of the present invention. The transformed cell may be a mammalian cell or bacterial cell. The transformed cell may be a human cell, a primate cell, or a murine cell. The transformed cell may be a cell derived from a mammal or an insect.

A cell into which the plasmid of the present invention has been introduced can be used regardless of whether it is adhesive or adherent, as long as it can contain plasmid DNAs in the cell. Specifically, any cells (NIH3T3 cell, a PC 12 cell, a HEK293 cell, a COS-7 cell, a CHO cell, a HeLa cell, a Sf-9 cell, a S2 insect) derived from mammals and insects can be used.

In some embodiments, the present invention provides a vector comprising the nucleic acid described above. The vector can be any construct that facilitates the delivery of the nucleic acid to the target cell and/or expression of the nucleic acid within the cell. The vectors can be viral vectors, circular nucleic acid constructs (e.g., plasmids), or nanoparticles (solid or lipid- based).

Plasmids are circular nucleic acid constructs that typically comprise the expression cassette, in addition to other sequence components to facilitate functionality, such as a gene encoding antibiotic resistance, origin of replication, restriction sites, and the like. A great variety of plasmids are known and are encompassed by this disclosure.

Related kits may be used to detect an analyte. In some embodiments, the kit includes a first vector. The first vector may include a first sequence that encodes a first peptide fragment of a split reporter complex. The vector may be configured to facilitate insertion of a nucleotide sequence for a first nanobody or affimer such that expression of the resulting vector yields a fusion protein that includes both the first affimer or nanobody and the first peptide fragment.

In some embodiments, the kit may additionally or alternatively include a second vector. The second vector may include a second sequence that encodes a second peptide fragment of a split reporter complex. The vector may be configured to facilitate insertion of a nucleotide sequence for a second affimer or nanobody such that expression of the resulting vector yields a fusion protein that includes both the second affimer or nanobody and the second peptide fragment.

In other embodiments, a kit may include a first agent and a second agent, as defined above. The first agent may include a first nanobody or affimer and a first peptide fragment of a split reporter protein. The second agent may include a second affimer or nanobody and a second peptide fragment of the split reporter protein. The first and second affimer or nanobody determine the antigen to be detected using the kit. Such a kit may optionally include the reagents necessary to generate a detectable signal when the first and second fragments of the split reporter protein are assembled into a functional reporter protein, such as a functional enzyme reporter. In such embodiments, the kit may optionally include the substrate for the assembled functional enzyme reporter, and instructions for use of the kit for detecting a specific antigen. In some embodiments of such kits, the specific analyte to be detected may be selected from any of the analytes disclosed herein. In some embodiments of such kits, the specific analyte to be detected may be selected from an antibody formed in response to an allergen, a bacterial infection, or a viral infection, a therapeutic antibody, or an autoantibody that binds a self-antigen.

In another aspect the present invention provides a method of detecting an analyte in a mixture, the method comprising: delivering the two-part split reporter complex according to the present invention, wherein the first agent and second agent are delivered as separate agents and wherein when the first and second nanobody or affimer bind an analyte, the first peptide fragment and second peptide fragment of the split reporter protein are brought into proximity and a detectable signal is produced. In one embodiment the detectable signal is a luminescence. In another embodiment the method is practiced in vitro.

In another aspect the present invention provides a method for detecting an analyte in a cell, comprising expressing the two-part split reporter complex of the present invention in a cell and detecting a signal generated by the split reporter complex.

In some embodiments, the analyte is detected in or from a cell lysate. In some embodiments the analyte is detected in a sample of faeces. In some embodiments, the method is carried out in a biological fluid. For example, some methods may be carried out in human serum or in a mixture that includes human serum. Other methods may be carried out in saliva or in a mixture that includes saliva. Other methods may be carried out in urine or in a mixture that includes urine. Detection of one or more analytes from other biological fluids is also possible. Detection of one or more analytes from breath condensate is also possible. In some embodiments, the method for detecting the analyte does not involve immobilization of the analyte. In other words, in some embodiments, the analyte may be in solution (e.g., a homogeneous solution) when detected. In other embodiments, the analyte is immobilized onto a surface. In other embodiments, the analyte is present on the surface of a tissue or tissue sample (e.g., a prepared tissue sample mounted on a microscope slide). In another embodiment the first and/or second agent is immobilized on a surface.

In another aspect, the present invention provides a cell comprising a fusion protein described herein.

In some embodiments, the cell is a mammalian cell or bacterial cell. In some embodiments, the cell is a human cell, a primate cell, or a murine cell.

In some embodiments, detecting the signal comprises performing flow cytometry, microscopy, in vivo imaging, or luminescence detection. In some embodiments, the fusion protein is localized in the cytoplasm, nucleus, or plasma membrane of the cell.

Preferred features and embodiments of the invention are as for the other aspects mutatis mutandis.

Aspects of the invention are demonstrated by the following non-limiting examples.

Examples

Example 1 Development of fusion proteins

For both GDH and TcdB, a pair of binding proteins targeting distinct regions of the biomarker are required for use in the split-luciferase assay (Figure 1). A literature search identified two nanobodies, E3 and 7F, which bind different regions of TcdB (TxB). We isolated Affimer binding proteins targeting GDH and TcdB, to complement the nanobody binders. An Affimer phage display library was screened against biotinylated GDH or TcdB with three rounds of panning. To improve the specificity of Affimers, phage for the GDH screen were prepanned against cell lysate and the third panning round for TcdB included a competitive incubation with TcdA to remove cross-reactive binders. After the third pan, individual clones were screened by phage ELISA and hits were classified as wells with more than 2-fold increase in signal relative to controls (TcdA for TcdB and cell lysate for GDH). All hits were sequenced and unique Affimer reagents were produced and purified. Binding to TcdB or GDH was assessed by ELISA (Figure 2). Affimers that showed the highest signal by ELISA were taken forward and pairwise binding of other Affimers was assessed by sandwich ELISA (Figure 3). Affimers 18 and 45 were identified as the best pair to bind distinct sites of TcdB. No binding was observed in negative controls or against TcdA. GDH is hexameric and Affimer 4 was demonstrated as the best capture and detection reagent.

Kinetic and equilibrium affinity constants of the binding proteins were determined by surface plasmon resonance (SPR) (Figure 4 and Table 1). Biotinylated Affimer / nanobody was immobilised on a streptavidin chip, titrated with serial dilutions of analyte and the association / dissociation response fitted to a 1: 1 Langmuir model. All binding proteins were specific for their target analyte (Figure 4) and displayed nM binding affinity (Table 1). Affimer selection and validation was with native toxin B (TcdB), whilst SPR and further sensor characterisation was with commercially available inactivated toxoid B (TxB) that maintains antigenicity.

Table 1 Kinetic and equilibrium affinity binding constants of Affimers and nanobodies derived from SPR data.

TxB sensor L-45 is nucleic acid SEQ ID NO: 1:

TxB sensor L-45 is amino acid SEQ ID NO: 2:

TxB sensor S-E3 is nucleic acid SEQ ID NO: 3:

TxB sensor S-E3 is amino acid SEQ ID NO: 4:

GDH sensor 4-L is nucleic acid SEQ ID NO: 5:

GDH sensor 4-L is amino acid SEQ ID NO: 6:

GDH sensor 4-S is nucleic acid SEQ ID NO: 7

GDH sensor 4-S is amino acid SEQ ID NO: 8

Development of split-luciferase assays

Sensor Expression and Purification

The construction of the phage library is described in literature Tiede, C.; Tang, A. A.; Deacon, S. E.; Mandal, U.; Nettleship, J. E.; Owen, R. L.; George, S. E.; Harrison, D. J.; Owens, R. J.; Tomlinson, D. C., Protein Eng. Des. Sei. 2014, 27, 145-155.

Phage display screening of the Affimer library was performed with an existing phage library as follows. Streptavidin coated wells (Pierce) were incubated with biotinylated target for 2 hours, washed with PBST then incubated with pre-panned phage for 2.5 hours. Panning wells were washed with PBST, eluted with 200 mM glycine-HCl (pH 2.2) for 10 min, neutralised with 1 M Tris-HCl (pH 9.1), further eluted with 100 mM triethylamine for 6 min and neutralised with 1 M Tris-HCl (pH 7). ER2738 cells were infected with eluted phage for 1 hour at 37°C, 90 rpm and were then plated onto LB agar (with 100 μg/ml carbenicillin) for overnight growth at 37°C. Colonies were scraped into 8 ml 2TY (with 100 μg/ml carbenicillin) to a dilution of A600 = 0.2, incubated for 1 hour at 37°C and 230 rpm, infected with M13K07 helper phage and grown for 30 mins at 37°C, 90 rpm. Then 25 μg/ml kanamycin was added prior to overnight incubation at 25 °C, 170 rpm. Phage were precipitated with 4 % (w/v) PEG 8000, 0.5 M NaCl and resuspended in 320 μl 10 mM Tris, pH 8.0, 1 mM EDTA (TE buffer). For panning round two, streptavidin magnetic beads (Dynabeads MyOne Streptavidin Tl, Invitrogen) were incubated with biotinylated target for 1 hour, washed and then incubated with pre-panned phage for 1 hour. They were then washed four times using a KingFisher instrument (Thermo Fisher), before elution and amplification of the phage as described above. The final pan was as described for panning round one but using Neutravidin coated wells (Pierce). For the toxin B screen there was an additional 24 hour incubation with toxin A and wash step prior to phage elution, in order to remove cross- reactive phage.

Affimer or Nanobody was amplified by PCR, incorporating the relevant restriction sites at the 5’ and 3’ ends. If not already available, the synthetic DNA encoding LgBiT, Affimers and/or nanobodies (all with the relevant restriction site) can be commercially synthesised in pUC57 vectors. Insert DNA encoding SmBiT 101 and (GSG)₇ linker sequences (see below) were generated by PCR of overlapping primers encoding appropriate restriction sites. Sequential restriction enzyme cloning was used to insert DNA encoding LgBiT, SmBiT 101, relevant Affimer or nanobody sequences between Nhel/Notl and Spel/Sall and a (GSG)₇ linker sequence between Notl and Spel. The vector was digested with appropriate restriction enzymes (NEB), dephosphorylated with antarctic phosphatase (NEB), separated on an agarose gel and then purified. Amplified insert DNA was purified, digested with appropriate restriction enzymes and then re-purified. The digested vector and insert were ligated with T4 DNA ligase (NEB) and transformed into E. coli XL-1 competent cells (Agilent Technologies). Plasmid DNA was purified using the Charge Switch Pro Plasmid Miniprep Kit (Invitrogen) and successful generation of constructs was confirmed by sequencing (Genewiz) with T7 / T7term primers.

The pET28a vectors with sensor constructs containing Affimers were transformed into E. coli BL21* (DE3) cells and those containing nanobodies were transformed into E. coli SHuffle T7 cells (NEB). A 1 ml starter culture was added to 50 ml LB media (with 50 μg ml-1 kanamycin) and grown at 37°C, 220 RPM before induction at OD600 ca. 0.6 with 0.3 mM isopropyl-β-D-thiogalactoside (IPTG) and overnight growth at 16°C, 180 RPM. Cells were harvested at ca. 4000 g for ca. 20 min, resuspended in 4 ml lysis buffer (pH 7.4, 50 mM Tris, 300 mM NaCl, 10 mM imid-azole, 0.1 mg ml-1 lysozyme, IX cOmplete EDTA-free protease inhibitor (Merck), 0.001 % v/v benzonase nuclease (Merck)) and incubated on a roller mixer for 1 hour at 4°C. Cells were lysed by sonication (UP50H, Hielscher) for 2 min (5s on / 5s off) at 100 % amplitude then pelleted at ca. 17000 g for 20 min. The supernatant was added to 250 μl Super Co-NTA resin (Generon) that had been pre-equilibrated with wash buffer (pH 7.4, 50 mM Tris, 300 mM NaCl, 10 mM imidazole) and was then incubated on a roller mixer for 1 hour at 4°C. The resin was washed thrice with 5 ml wash buffer and protein eluted with 3 x 0.5 ml elution buffer (pH 7.4, 50 mM Tris, 300 mM NaCl, 300 mM imidazole). Pure fractions (as assessed by SDS-PAGE) were buffer exchanged into storage buffer (50 mM Tris, 150 mM NaCl, pH 7.4) using Zeba spin desalting columns (ThermoFisher). Protein concentration was determined by BCA assay and aliquots stored at -80°C. The NanoBiT system consists of an 18 kDa LgBiT and a range of 11-13 amino acid SmBiT peptides that span a 5 order of magnitude binding affinity for LgBiT. Here we use SmBit101 (VTGYRLFEKES) with Kd = 2.5 μM, to provide a balance between minimising background complementation and maximising analyte induced reconstitution. To generate NanoBiT sensor proteins each of the binding proteins (TcdB Affimers, TcdB nanobodies E3 and 7F and GDH Affimer 4) were genetically fused to the N- or C- terminus of LgBiT (L) or SmBit101 (S) via a (GSG)₇ linker peptide. (Hereafter, we will use L-45 and S-45 for an N-terminal fusion of LgBiT and SmBiT to Affimer 45, respectively and, similarly, E3-L and E3-S for a C-terminal fusion to Nanobody E3, etc. The linker, (GSG)₇ (SEQ ID NO: 33), is present in all constructs, but not included in this nomenclature.) Of these 20 constructs 16 were successfully produced in E. coli and purified via a C-terminal 6x-Histag (Figure 5) with yields of up to 90 mg L-1, demonstrating the ease of production relative to antibody-based systems. All 4 constructs with N-terminal nanobodies (E3-L, E3-S, 7F-L and 7F-S) were produced with insufficient purity following metal affinity purification (Figure 5), so were not taken forward for use in assays.

Example 2 Sensor Characterisation

NanoBiT assay (in buffer)

All assays were performed in PBSB (pH 7.4, PBS + 1 mg ml-1 BSA) dilution buffer. 10 μl LgBiT sensor (5x final cone.), 10 μl SmBiT sensor (5x final cone.) and 5 μl TxB or GDH (10x final cone.) were added to a well of a white no-bind 384-well plate (Coming) and incubated, shaking at 25 °C, for the indicated length of time. Then 25 μl of diluted NanoGlo (2x final cone.) was added and the luminescence read (500 ms integration) on a Tecan Spark platereader. Data were fit to 5 parameter logistic (5PL) regression curves and interpolations made using GraphPad Prism 9 software.

NanoBiT assay (in faecal sample matrix)

C. difficile negative faecal samples were excess routinely collected diagnostic specimens from the Department of Microbiology, Leeds Teaching Hospitals NHS Trust. Samples were anonymised by the clinical team prior to storage of two 1 ml aliquots at -80°C, until transfer to the research team for testing.

All sample preparation and assays were performed in PBSBT (pH 7.4, PBS + 1 mg ml-1 BSA + 0.05 % Tween), unless otherwise stated. Faecal samples were homogenised in buffer, with 125 mg in 750 μl (16.67 % w/v). Particulates were pelleted by centrifugation at ca. 17000 g for 5 min (or allowed to settle for 10 minutes, if stated) and the supernatant was used as the faecal sample, which was added to the NanoBiT assay to give the indicated final concentrations (w/v). Typically, a 1:5 dilution was used to give 3.33% (w/v) faeces, as follows: 10 μl LgBiT + SmBiT sensor mix (5x final cone.), 5 μl TxB or GDH (10x final cone.) and 10 μl faecal sample were added to a well of a white no-bind 384-well plate (Coming) and incubated, shaking at 25 °C, for 30 mins (or the indicated length of time). Then 25 μl of diluted NanoGlo (2x final cone.) was added and the luminescence read (500 ms integration) on a Tecan Spark platereader. We note that a final stool concentration of 3.33% (w/v) is equivalent to that used in the in the commercial C. diff Quik Chek complete test (Alere). All data refer to the final concentration of analyte present in the final assay mixture.

To assess which combination of LgBiT and SmBiT sensor proteins have optimal TxB driven complementation, each pair was incubated with 0 or 1 nM TxB prior to addition of NanoGlo substrate and measurement of bioluminescence. The increase in bioluminescence with 1 nM TxB ranged from only 4-fold for sensor pairs containing Affimer 18 and nanobody 7F up to over 1000-fold for some containing Affimer 45 and nanobody E3 (Figure 6A). Fusion of binding proteins at the N- or C-terminus of either LgBiT (L) or SmBiT (S) affected the TxB driven signal increase. For example, L-45 + S-E3 and L-E3 + 45-S displayed 1000 and 230- fold increases, respectively, despite containing the same binding proteins. Sensors were also assayed for bioluminescence across a wide range of TxB concentrations to give dose response curves (Figure 6B). Thermodynamic modelling indicates that for binding protein Kd values between 2.5 and 33 nM (Table 1), the expected sensitivities are almost identical and differences in Kd do not explain the differences in sensitivity observed in Figure 6B. Instead, the molecular mechanism behind differences in signal may be due to some sensor pairs orienting more favourably for complementation of LgBiT and SmBiT. Alternatively, the engineering of LgBiT and/or SmBiT on the N- or C-terminus could alter the binding affinity of the Affimers or Nanobodies.

Sensors L-45 + S-E3 displayed the highest TxB driven signal increase across the entire concentration range, so were taken forward for further optimisation. All GDH sensor combinations displayed GDH-dependent bioluminescence (Figure 6C). The response was again dependent on Affimer placement at the N- or C-terminus of sensors, highlighting the importance of testing all combinations. Sensors 4-L + 4-S were optimal, so taken forward for further improvement.

Example 3 Optimal sensor protein concentration

The optimal concentrations of sensor proteins for TxB (L-45 + S-E3) and GDH (4-L + 4-S) were then established (Figure 7 and 8). Lower concentrations of LgBiT and SmBiT minimise background complementation (Figure 7A and 7D) but reduce the maximum amount of analyte driven reconstitution (Figure 7A and 7D). There is also a more pronounced “Hook” effect (loss in signal at high analyte concentrations), due to analyte binding each sensor protein individually rather than in a sandwich com-plex. Therefore, there is an optimal sensor concentration that maximises the ratio of analyte-induced to background bioluminescence (Figure 7B and 7E). Unequal concentrations of LgBiT and SmBiT were also tested (Figure 7C, 7F and 8A). For TxB, 0.5 nM S-E3 + 1 nM L-45 was established as the best combination as it is the most sensitive at low TxB concentrations (Figure 8). 8 nM 4-S + 8 nM 4-L performed the best for GDH (Figure 7E and 7F).

The kinetics of the NanoBiT assays were then studied (Figure 9 and 10) to optimise incubation and signal read times. When TxB sensors (S-E3 + L-45) were simultaneously mixed with TxB and substrate, the biolumine scent signal observed with 1 - 1000 pM TxB increased over a period of 30 mins (Figure 9B and 9E), indicating the timescale to approach equilibrium. A 30 min pre-incubation with TxB prior to substrate addition gave an optimal biolumine scent signal (Figure 9A and 9D) that was stable over 30 min (Figure 9C and 9F). A stable signal is possible due to the glow-type luminescence of NanoLuc and makes assay results less sensitive to the exact measurement time adopted by the user. Only a 15 min pre- incubation of GDH sensors (4-S and 4-L) with GDH was required to maximise the biolumine scent signal (Figure 10A and 10C), indicating slightly faster kinetics. The signal was however less stable over time (Figure 10B and 10D) and a read time of 0-2 minutes was optimal. We adopted a pre-incubation time of 30 min and read time of 2 min for a consistent protocol between TxB and GDH assays. It should be noted that the pre-incubation can be removed to give a simple mix-and-read protocol if needed for a POCT, but the signal will just require time to develop.

Finally, the concentration of substrate was optimised by performing the TxB assay with 1: 100 - 1:4000 Nano-Glo (Figure 11). Lower substrate concentrations mini-mised background luminescence (Figure 11A), so maximised the fold gain in bioluminescence with TxB (Figure 11C). However, the signal also decreased more quickly over time with low NanoGlo concentrations (Figure 11B and 11D), presumably due to depletion of substrate. A substrate concentration of 1: 1000 balanced maximising the signal increase with TxB and minimising signal loss over time.

Example 4: Quantification of C. difficile toxin B and GDH Each assay requires a target specific LgBiT and SmBiT sensor protein, confirming the expected need to form a sandwich complex with the analyte for luciferase reconstitution. The optimised split-luciferase assays for C. difficile TxB and GDH are specific for their target analytes, with no non-specific response with up to ten times the concentration of TxA and TxB, respectively (Figure 12). Many commercial assays detect toxin but do not differentiate TcdA and TcdB, so this assay offers an advantage in specifically quantifying the more clinically relevant TcdB.

The TxB and GDH assays were performed with nominal concentrations of TxB (0.01 - 1000 pM) and GDH (0.001 - 10 nM) to establish dose response curves and calculate sensitivity, accuracy and precision metrics (Figure 12). Responses were recorded as raw bioluminescence (RLU) (Figure 12) or fold gain in bioluminescence (RLU with analyte / RLU without analyte). There were approximately 250 and 1300-fold maximum signal gains for the GDH and TxB assays, respectively. To calculate intraassay metrics 6 replicates were performed on the same plate (Figure 12) and for inter-assay metrics 6 independent measurements were made for TxB and 3 for GDH. The logarithm of each dose response was fit to a 5 parameter logistic (5PL) regression standard curve. The limit of detection (LoD) was calculated by LoD = meanblank + 1.645(SDblank) + 1.645(SDlow cone.) as outlined by Armbruster and Pry (Clin Biochem Rev 2008, 29, S49-52) where blank = zero analyte and low cone. = 0.1 pM TxB or 0.01 nM GDH, to account for variability in both test and blank measurements. For each individual measurement at nominal concentrations, the concentration was interpolated back from the standard curve to assess accuracy (% recovery = (mean interpolated concentration / nominal concentration) x 100 %) and precision (% coefficient of variation (% CV) = (SD interpolated concentration / mean interpolated concentration) x 100%).

Intra-assay sensitivity, accuracy and precision metrics were optimal with raw bioluminescence rather than fold gain data, perhaps due to variability in low background luminescence. The intra-assay LoD was 44 fM for TxB and 4.5 pM for GDH (Figure 12, Table 2). Intra-assay recovery and CV values indicate good intra-assay accuracy and precision for analyte quantification over concentrations spanning 5 orders of magnitude for TxB (0.1-1000 pM) and 4 for GDH (0.01-10 nM). The inter-assay LoD was 190 fM for TxB and 14 pM for GDH (Table 2). Good inter-assay accuracy and precision metrics are maintained over 3 orders of magnitude (1-100 pM for TxB and 0.1-10 nM for GDH, Table 2). Inter-assay sensitivity, accuracy and precision metrics were further improved when using fold gain rather than raw bioluminescence data, as the zero analyte measurement acts as a calibrator for condition changes between assays. Table 2. Sensitivity (LoD), Accuracy (% recovery) and Precision (% CV) of TxB and GDH assays as determined from raw bioluminescence (RLU)

a %CV precision metrics >20 % only at limit of quantification

Example 5 : Assay performance in stool sample matrix

TxB and GDH are present in stool samples for patients with CDI, so the effect of this sample matrix on the split-luciferase assay was assessed. We homogenised solid C. difficile negative stool in buffer to 16.66 % w/v and added this matrix to the assay at the final % w/v indicated, along with analyte to the final concentration indicated. A currently used C. difficile POCT, C. diff Quik Chek complete (Quik Chek, Alere) uses a similar procedure to homogenise stool samples at 3.33 % w/v.

The TxB assay was initially performed with 0.66 % w/v stool in a number of different buffers and PBSBT (PBS + 1 mg ml-1 + 0.05 % Tween 20) was found to be optimal. Pelleting stool particulates with centrifugation, rather than allowing to settle by gravity prior to addition to the assay, minimised signal loss by scattering and absorption of light. NanoGlo substrate at 1 : 1000 and approx. 1 nM TxB sensor proteins were still optimal with a 0.66 % w/v stool matrix.

Whilst the assays are functional with stool, there are significant faecal sample matrix effects. For the TxB assay, increasing the concentration of stool from 0.0067 - 3.33 % w/v decreases both raw and fold gain in bioluminescence and increases signal loss over time. Nevertheless, the results indicate that the greatest sensitivity would be obtained with 3.33 % w/v stool, as for real samples further dilution would reduce the amount of TxB more than is compensated for by reduced matrix effects. At even higher stool concentrations the matrix effect then becomes too detrimental. The 2 pM LoD in 3.33 % w/v stool is significantly higher than the 44 fM LoD in buffer (Figure 13), but comparable to the 0.16 ng ml-1 = 0.6 pM cut off of Quik Chek (also in 3.33 % w/v stool matrix).

For the GDH assay, 3.33 % w/v faeces reduces raw bioluminescence (Figure 13A) and increases signal loss over time. Fold gain in bioluminescence with GDH is much less affected by 3.33 % w/v faeces (Figure 13B) and the 4.5 pM LoD is comparable to the 0.8 ng ml-1 = 3 pM cut off of Quik Chek. The sensitivity and time-to result (approx. 30 mins) of our GDH / TxB stool tests are comparable to this current POCT but less user steps (e.g. washing) are required and results are quantitative rather than qualitative. It will provide an important research tool to further investigate correlates of faecal toxin levels and disease severity, for prognosis and therapy guidance.

Example 6: Nanobit Assay for Therapeutic Antibodies

A fusion protein of affimer and Nanobit were developed which bind/target four different therapeutic antibodies (Ipillimumab, Rituximab, Trastuzumab and Adalimumab).

Affimer selection Sensor cloning

Phage display screening of the Affimer library was performed with an existing phage library as follows. Streptavidin coated wells (Pierce) were incubated with biotinylated target for 2 hours, washed with PBST then incubated with pre-panned phage for 2.5 hours. Panning wells were washed with PBST, eluted with 200 mM glycine-HCl (pH 2.2) for 10 min, neutralised with 1 M Tris-HCl (pH 9.1), further eluted with 100 mM triethylamine for 6 min and neutralised with 1 M Tris-HCl (pH 7). ER2738 cells were infected with eluted phage for 1 hour at 37°C, 90 rpm and were then plated onto LB agar (with 100 μg/ml carbenicillin) for overnight growth at 37°C. Colonies were scraped into 8 ml 2TY (with 100 μg/ml carbenicillin) to a dilution of A600 = 0.2, incubated for 1 hour at 37°C and 230 rpm, infected with M13K07 helper phage and grown for 30 mins at 37°C, 90 rpm. Then 25 μg/ml kanamycin was added prior to overnight incubation at 25°C, 170 rpm. Phage were precipitated with 4 % (w/v) PEG 8000, 0.5 M NaCl and resuspended in 320 μl 10 mM Tris, pH 8.0, 1 mM EDTA (TE buffer). For panning round two, streptavidin magnetic beads (Dynabeads My One Streptavidin Tl, Invitrogen) were incubated with biotinylated target for 1 hour, washed and then incubated with pre-panned phage for 1 hour. They were then washed four times using a KingFisher instrument (Thermo Fisher), before elution and amplification of the phage as described above. The final pan was as described for panning round one but using Neutravidin coated wells (Pierce).

All sensor constructs were generated in a pET28a vector containing Nhel, Notl, Spel and Sall restriction sites between the Ncol and Xhol sites of the vector, with an in frame 6xHistag sequence and stop-codon following Xhol. DNA from the LgBiT, SmBit101 , Affimer was amplified by PCR, incorporating the relevant restriction sites at the 5’ and 3’ ends. Insert DNA encoding SmBiT 101 and (GSG)₇ linker sequences were generated by PCR of overlapping primers encoding appropriate restriction sites. Sequential restriction enzyme cloning was used to insert DNA encoding LgBiT, SmBiT 101, relevant Affimer sequences between Nhel/Notl and Spel/Sall and a (GSG)₇ linker sequence between Notl and Spel. The vector was digested with appropriate restriction enzymes (NEB), dephosphorylated with antarctic phosphatase (NEB), separated on an agarose gel and then purified. Amplified insert DNA was purified, digested with appropriate restriction enzymes and then re-purified. The digested vector and insert were ligated with T4 DNA ligase (NEB) and transformed into E. coli XL-1 competent cells (Agilent Technologies). Plasmid DNA was purified using the Charge Switch Pro Plasmid Miniprep Kit (Invitrogen) and successful generation of constructs was confirmed by sequencing (Genewiz) with T7 / T7term primers.

Sensor Expression and Purification

The pET28a vectors with sensor constructs containing Affimers were transformed into E. coli BL21* (DE3) cells. A 1 ml starter culture was added to 50 ml LB media (with 50 μg ml-1 kanamycin) and grown at 37°C, 220 RPM before induction at OD600 ca. 0.6 with 0.3 mM isopropyl-β-D-thiogalactoside (IPTG) and overnight growth at 16°C, 180 RPM. Cells were harvested at ca. 4000 g for ca. 20 min, resuspended in 4 ml lysis buffer (pH 7.4, 50 mM Tris, 300 mM NaCl, 10 mM imidazole, 0.1 mg ml-1 lysozyme, IX complete EDTA-free protease inhibitor (Merck), 0.001 % v/v benzonase nuclease (Merck)) and incubated on a roller mixer for 1 hour at 4°C. Cells were lysed by sonication (UP50H, Hielscher) for 2 min (5s on / 5s off) at 100 % amplitude then pelleted at ca. 17000 g for 20 min. The supernatant was added to 250 μl Super Co-NTA resin (Generon) that is pre-equilibrated with wash buffer (pH 7.4, 50 mM Tris, 300 mM NaCl, 10 mM imidazole) and was then incubated on a roller mixer for 1 hour at 4°C. The resin was washed thrice with 5 ml wash buffer and protein eluted with 3 x 0.5 ml elution buffer (pH 7.4, 50 mM Tris, 300 mM NaCl, 300 mM imidazole). Pure fractions (as assessed by SDS-PAGE) were buffer exchanged into storage buffer (50 mM Tris, 150 mM NaCl, pH 7.4) using Zeba spin desalting columns (ThermoFisher). Protein concentration was determined by BCA assay and aliquots stored at -80°C. Figure 14 shows the cloned purified fusion proteins targeting specific antibodies (Ada = Adalimumab; Tra = Trastuzumab; Ipi = Ipillimumab and Rit = Rituximab). Affimers were cloned on both the N-terminus or C-terminus of the LgBiT and SmBiT with GSG₇ linkers.

Sensor Characterisation

NanoBiT assay (in buffer)

Assays were performed in PBSB (pH 7.4, PBS + 1 mg ml-1 BSA) dilution buffer. 10 μl LgBiT sensor (5x final cone.), 10 μl SmBiT sensor (5x final cone.) and 5 pl analyte (10x final cone.) are added to a well of a white no-bind 384-well plate (Coming) and incubated, shaking at 25°C. Then 25 μl of diluted NanoGlo (2x final cone.) was added and the luminescence read (500 ms integration) on a Tecan Spark platereader. Data can be fit to 5 parameter logistic (5PL) regression curves and interpolations made using GraphPad Prism 9 software.

A binding assay was performed for specific antibodies Rituximab, Trastuzumab, Adalimumab and Ipilimumab. All four possible combinations of sensor constructs were compared (with affimers of the N-terminus or C-terminus of either SmBiT and LgBiT). Optimal combinations (in terms of sensitivity) were:

Rituximab: LgBiT-GSG₇-Affimcr and SmBiT-GSG₇-Affimcr

Trastuzumab: LgBiT-GSG₇-Affimer and Affimer-GSGv-SmBiT

Adalimumab: LgBiT-GSG₇-Affimcr and SmBiT-GSG₇-Affimcr

Ipilimumab: Affimcr-GSG₇-LgBiT and Affimcr-GSG₇-SmBiT

Limit of detection and accuracy (CV) were determined for the best sensor fusion protein combinations and described in the legend in Figure 15.

Example 7 : Optimization Assay for Rituximab

An assay was performed to determine the optimum concentration of substrate and time for incubation. The results are shown in Figure 16A. The concentration of substrate was optimised by performing the Rituximab assay with 1:50 - 1:2000 Nano-Glo. Nano-Glo concentrations 1:200 - 1:2000 gave identical results when measured after 2 minutes (Fold gain). However, 1:500 gave the most consistent signal after various measuring times (Figure 16B) and hence 1:500 is the most optimal Nano-Glo concentration.

Example 8: Plasma effect on antibody binding assay An assay was performed to determine if plasma would interfere with antibody binding. The results are shown in Figure 17 and indicate that up 1% plasma (v/v), no significant effect is observed.

Example 9: CRP NanoBit assay versus BLA-BLIP assay

Finally, to further confirm the modularity of the assay, LgBiT and SmBiT sensor constructs were constructed with Affimer (Aff90) raised again human C-reactive protein (CRP). The construction was identical to those for Toxin B, GDH and the four therapeutic antibodies, with Aff90 fused to SmBiT and LgBiT at both the N-terminus and C-terminus using a GSG7 linker. To further optimise the sensor, two SmBiT sequences were trials with different affinities for LgBit (SmBit101 and SmBiT 114 ((VTGYRLFEEIL - SEQ ID NO: 31 }, where SmBiT has a lower affinity of LgBiT than SmBit101 ). Thus, 6 sensor constructs were designed, produced and purified:

(A) LgBit-GSG₇-Aff90

(B) Aff90-GSG₇-LgBit

(C) SmBit101-GSG₇-Aff90

(D) Aff90-GSG₇-SmBit101

(E) SmBit114-GSG₇-Aff90

(F) Aff90-GSG₇-SmBit114

Assay were performed with all possible combinations (AC, AD, AE, AF, BC, BD, BE and BF, where the letters refer to the list above) of the fusion constructs. Figure 18 shows the results (1:500 Nano-Glo and measured after 10 min) which show that in all cases the sensitivity with SmBit101 is better compared to SmBiT 114. All combinations with SmBit101 are similar with the BD combination (Aff90-GSG₇-LgBit and Aff90-GSG₇- SmBitlOl) showing the best results. As for the therapeutic antibody sensors, experiments in plasma confirmed the sensor (in terms of Fold gain) is not affected with up to 1% plasma.

Example 10:

Experimental Methods

Sensor Cloning

All sensor constructs were generated in a pET28a vector containing Nhel, Notl, Spel and Sall restriction sites between the Ncol and Xhol sites of the vector, with an in-frame 6xHistag sequence and stop-codon following Xhol. Sequential restriction enzyme cloning was used to insert DNA encoding LgBiT, SmBiT (101) or Affimer sequences between Nhel/Notl and Spel/Sall and a (GSG)7 linker sequence between Notl and Spel. The vector was digested with appropriate restriction enzymes (NEB), dephosphorylated with antarctic phosphatase (NEB), separated on an agarose gel, and then purified. All DNA was purified using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare). The synthetic DNA encoding LgBiT was purchased from Genscript in pUC57 vector. Affimers were encoded in a pEtLECTRA vector. This insert DNA was PCR amplified with primers encoding appropriate restriction sites, then treated with Dpnl (NEB) to remove parental vector DNA. Insert DNA encoding SmBit101 and (GSG)7 linker sequences were generated by PCR of overlapping primers encoding appropriate restriction sites. Amplified insert DNA was purified, digested with appropriate restriction enzymes and then re-purified. The digested vector and insert were ligated with T4 DNA ligase (NEB) and transformed into E. coli XL-1 competent cells (Agilent Technologies). Plasmid DNA was purified using the ChargeSwitch Pro Plasmid Miniprep Kit (Invitrogen) and successful generation of constructs was confirmed by sequencing (Genewiz) with T7 / T7term primers.

Sensor Expression and Purification

The pET28a vectors with sensor constructs were transformed into E. coli BL21* (DE3) cells. A 1 mL starter culture was added to 50 mL LB media (with 50 μg mL-1 kanamycin) and grown at 37°C, 220 RPM before induction at ODeoo ca. 0.6 with 0.3 mM isopropyl-β-D- thiogalactoside (IPTG) and grown overnight at 16°C, 180 RPM. Cells were harvested at 4000xg for ca. 20 min, resuspended in 4 mL lysis buffer (pH 7.4, 50 mM Tris, 300 mM NaCl, 10 mM imidazole, 0.1 mg mL-1 lysozyme, 1X complete EDTA-free protease inhibitor (Merck), 0.001% v/v benzonase nuclease (Merck)) and incubated on a roller mixer for 1 hour at 4°C. Cells were lysed by sonication (UP50H, Hielscher) for 2 min (5s on / 5s off) at 100% amplitude then pelleted at 17000xg for 20 min. The supernatant was added to 250 μL Super Co-NTA resin (Generon) that had been pre-equilibrated with wash buffer (pH 7.4, 50 mM Tris, 300 mM NaCl, 10 mM imidazole) and was then incubated on a roller mixer for 1 hour at 4°C. The resin was washed thrice with 5 mL wash buffer and protein eluted with 3 x 0.5 mL elution buffer (pH 7.4, 50 mM Tris, 300 mM NaCl, 300 mM imidazole). Pure fractions (as assessed by SDS-PAGE) were buffer exchanged into storage buffer (50 mM Tris, 150 mM NaCl, pH 7.4) using Zeba spin desalting columns (ThermoFisher). Protein concentration was determined by BCA assay and aliquots stored at -80°C.

Sensor Characterisation

Target mAbs

Biosimilars of each of the target mAbs were purchased from InvivoGen; Rituximab (Anti- hCD20-hIgG4 S228P), Adalimumab (Anti-hTNF-a-hlgGl), Trastuzumab (Anti-HER2-hIgG4 S228P) and Ipilimumab (Anti-hCTLA4-hIgGl). NanoBiT assay (in buffer)

Assays were performed in PBSB (pH 7.4, PBS + 1 mg mL-1 BSA) dilution buffer. 10 μL LgBiT sensor (5x final cone.), 10 μL SmBiT sensor (5x final cone.) and 5 μL mAb (InvivoGen) (lOx final cone.) were added to a well of a white no-bind 384-well plate (Coming) and incubated for 30 mins, 25°C, shaking. Then 25 μL of 1:500 Nano-Glo was added to give a final dilution of 1: 1000. Luminescence was read (500 ms integration) on a Tecan Spark plate reader.

NanoBiT assay (with plasma)

For experiments in 0 - 1% plasma, assays were performed in PBSB (pH 7.4, PBS + 1 mg/mL BSA) dilution buffer. 10 μL 10 nM LgBiT + 10 nM SmBiT in PBSB (2 nM each final cone.) and 5 μL mAb (InvivoGen; 10x final cone.) in PBSB were added to 10 μL human plasma (Clinical Trials Laboratory Services) at 5x the final concentration (in PBSB) in a white no- bind 384-well plate (Coming) and incubated for 30 mins, 25 °C, shaking.

For experiments in 5 and 50% plasma, assays were performed by first preparing stock solutions (LgBiT, SmBiT, mAb) in either 10% human plasma with PBSB or 100% human plasma. 10 μL of 10 nM LgBiT sensor (5x final cone.), 10 μL of 10 nM SmBiT sensor (5x final cone.) and 5 μL mAb (InvivoGen; various concentrations at 10x the final cone.) were added together in a well of a white no-bind 384-well plate (Coming) and incubated for 30 mins, 25 °C, shaking.

For all assays, bioluminescence was initiated by addition of 25 μL of 1 :500 Nano-Glo to give a final dilution of 1: 1000. Luminescence was read (500 ms integration) on a Tecan Spark plate reader. Parameter changes for optimisation are depicted in the results section.

Data analysis

All data analysis was performed using GraphPad, Prism software version 9.0. Graphs presented as fold gain refer to [RLU (X nM TmAb) / RLU (0 nM TmAb)]. Optimisation experiments presented as the mean of at least 2 repeats ± SEM unless specified otherwise. Intra- and inter-assays presented as n=3 or N=3 ± SEM. Statistical significance was determined as P<0.05, using one-tailed homoscedastic t-tests.

Limit of detection (LoD) was defined as the lowest concentration of analyte that produced a reading above a minimal value (RLUmin):

[RLU] _min= [RLU] _blank+1.645 ( [SD] _blank )+1.645( [SD] _(low conc.)) in which RLUblank is the average reading without analyte, SDblank = the standard deviation of samples without analyte and SDlow cone. = the standard deviation of samples with analyte at the lowest concentration for which a signal above baseline is produced.

Accuracy of assay performance was measured as percentage recovery:

% recovery = (mean interpolated concentration/nominal concentration) x 100%

And precision measured as percentage coefficient variation:

% CV = (SD interpolated concentration/mean interpolated concentration) x 100%

Selection and characterisation of binding proteins

An Affimer reagent phage display library was screened against target antibodies trastuzumab, rituximab, adalimumab, and ipilimumab. Briefly, the binding reagents selected for in three rounds of phage panning were subject to ELISA validation, and a lead candidate was chosen for each target TmAb and further characterised. Surface plasmon resonance (SPR) was used to determine the affinity of each anti-idiotypic Affimer protein. TmAb biosimilars were covalently immobilised onto an SPR chip and titrated with serial dilutions of respective Affimer reagents. Nanomolar affinities were confirmed for all antibody - Affimer reagent complexes (Table 3).

Table 3. KD values calculated from evaluation of Langmuir model fits of SPR curves. All anti-idiotypic Affimer proteins have nM affinity for their respective TmAb analytes and are within ~ 12-fold of one another. KD values are presented as a mean of three replicates ±SEM. (Aff-Ipi - ipilimumab n=2).

Split Luciferase sensor development

Luciferase enzymes are commonly used in split enzyme proximity switches with successful recombination seen from multiple luciferases. An engineered catalytic subunit of a luciferase from the deep-sea shrimp (Oplophorus gracilirostris) was isolated and termed NanoLuc. The small size and high stability of this luciferase subunit allows for splitting of NanoLuc into two inactive fragments that recover their enzymatic activity with reassembly, known as the NanoBiT system. Here we used the 18 kDa LgBiT and 11 amino acid SmBit101 peptide (VTGYRLFEKES - SEQ ID NO: 32) as the reporter fragments. The LgBiT and SmBit101 fragments were genetically fused to either the N-terminus or C-terminus of the anti-idiotypic binding reagents to produce four pair combinations for each TmAb. A (GSG)₇ (SEQ ID NO: 33) peptide linker was inserted between the NanoBiT fragment and the binding reagent. We will use one letter codes for the LgBiT (L) and SmBit101 (S) fragments, as well as for the four Affimers raised against the four TmAbs (adalimumab, A; ipilimumab, I; rituximab, R; and trastuzumab, T). The order of the one-letter codes represents N-terminal vs C-terminal constructs with, for instance, L-A denoting an Affimer against adalimumab with an N- terminal LgBiT fragment, connected via a (GSG)₇ linker. Full protein sequences are given below. All 16 possible combinations were constructed and expressed in E. coli and purified via a C-terminal 6xHis-tag.

Within the amino acid sequence, the Affimer variable region sequences are denoted as XXX. Within the nucleic acid sequence, the Affimer variable region sequences are denoted as NNN.

LgBit-GSG₇-AffX:

The amino acid SEQ ID NO: 9

Nucleic acid SEQ ID NO: 10

AffX-GSG₇-LgBit

The amino acid SEQ ID NO: 11

Nucleic acid SEQ ID NO: 12

SmBit101-GSG₇-AffX

The amino acid SEQ ID NO: 13

Nucleic acid SEQ ID NO: 14

AffX-GSG₇-SmBit101

The amino acid SEQ ID NO: 15

Nucleic acid SEQ ID NO: 16

Tables of Primers

Table SI. Primers used for Affimer amplification

Table S2. Primers used for LgBiT amplification

Table S3. Primers used to generate SmBiT (101)

Table S4. Primers used to generate GSG₇ linker

To determine the optimal sensor pair combinations for TmAb quantification, 2 nM of each sensor pair was incubated with a range of 0 - 100 nM respective TmAb, and after addition of substrate, bioluminescence was measured. Sensor pairs displayed responses ranging from 5- fold to 170-fold gain in bioluminescence. An obvious hook effect was observed at > 10 nM TmAb titres, which is likely due to the excess of TmAb relative to the split enzyme-Affimer constructs (2 nM). When TmAb is in excess, it is stochastically likely only one Affimer binds to each TmAb (Figure 19). Even without excess it is possible that two SmBiT or two LgBiT fragments bind to the same TmAb, which should reduce the ensemble signal. Still, the binding affinity between the SmBit101 and LgBiT fragments will provide a slight thermodynamic advantage to the formation of a SmBiT101-LgBiT-TmAb complex. The best sensitivity and activity fold gain was seen by the L-R / S-R sensor pair against rituximab and thus this rituximab sensor was chosen to further optimise the assay conditions. First, the ideal sensor concentration was determined. Higher concentrations of LgBiT and SmBiT components resulted in a larger RLU response but also contributed to much higher background complementation. In contrast, the lowest concentration had much lower background, but reduced the maximum analyte-driven reconstitution. To account for both extremes a mid-range concentration of 2 nM for each sensor component was selected to maximise the ratio of analyte-induced to background bioluminescence signal.

Optimal substrate concentration and incubation time before taking readings were then established. Increasing substrate concentration increases the RLU, but also the time required before maximum activity is observed. After an initial rise between 2 to 5 min, the signals slowly decayed over time. When analysed in terms of fold-gain, differences were significant but very small. To optimise the assay for speed and sensitivity, bioluminescence was recorded 2 minutes after addition of the substrate (NanoGlo) at a dilution factor of 1 : 1000 in the final assay.

Another aspect of the NanoBiT assay that could be optimised was the incubation time between the Affimer constructs and TmAb (at 25°C) prior to substrate addition. Maximum activity was already observed within 2.5 min., with the signal remaining stable thereafter. Therefore, and to allow time to prepare multiple tests at once, a 25°C incubation step of > 2.5 minutes should be used before adding the substrate. Importantly, when all reagents are prepared in advance, the time-to-results of the optimised assay is under 10 minutes.

The functionality of the NanoBiT sensor assay in pooled human serum was tested to establish its feasibility as a PoC TDM test. No significant difference in maximum fold gain of bioluminescence signal was detected in up to 1% pooled human serum (P>0.05). However, sensor activity was significantly diminished in 50% serum. With a range of functional serum percentages, we optimised the serum sample dilution so that the therapeutic range of rituximab was within the linear range of the interpolated curve. The therapeutic range of rituximab is approximately 150-500 nM. In 0. 1% serum the LOD of the rituximab NanoBiT assay is ~ 8 pM (see below), therefore, a lOOOx dilution of serum samples would result in TmAb concentrations quantifiable in this assay.

Before applying the optimised protocol to the other three sensor pairs, the specificity of each sensor was tested against the four TmAbs and blank buffer as a measurement of bioluminescence (Figure 20). All sensors are highly specific to their target TmAb, with no response to non-specific TmAb targets significantly higher than buffer (P>0.05).

Finally, performance and intra-assay and inter-assay variability were determined for the four best-performing sensor combinations for each of the TmAb. In the final optimised assay, 10 μL of 10 nM (2 nM final concentration) SmBit101 and 10 nM LgBiT was incubated with 10 μL 0.5% pooled human plasma (0.1% final concentration) and 5 μL of varying TmAb concentration from 0.005 - 2500 ng/mL for 5 minutes shaking at 25°C. 25 μL 1:500 dilution of NanoGlo substrate (final dilution 1: 1000) was then added and bioluminescence readings were taken after 2 minutes. For intra-assay assessment, measurements were taken as n = 3 performed on the same plate (Figure 21) and to determine inter-assay variation, 3 independent measurements were taken on separate dates for each target analyte, N = 3 (Figure 21). Concentrations from 0.005 - 2500 ng/mL were used to create a standard curve and concentrations interpolated back to determine percentage recovery and percentage coefficient of variance (CV) to assess sensitivity and precision respectively. Quantifiable ranges were subsequently calculated by percentage recovery between 80% and 111% and percentage CV < 25% (Table 4).

Table 4. Sensitivity (LoD), accuracy (% recovery), and precision (% CV) of TmAb NanoBiT assays, as determined from raw bioluminescence or fold gain data (Figure 21), to define a quantifiable range.

Discussion

The TmAb NanoBiT assays developed here measured TmAb drug levels accurately and precisely, down to reported trough concentrations for all four therapeutics with a time-to- result of under 10 minutes. Intra-assay variability for the sensors developed was low, with high sensitivity, accuracy and precision when detecting TmAb in 0. 1% pooled human serum. When assessing inter-assay variability, the sensors against rituximab and adalimumab exhibited high sensitivity, accuracy, and precision (Table 4). Although accuracy and sensitivity were adequate, the precision was insufficient for the sensors against trastuzumab and ipilimumab when assessing raw RLU data. Normalisation of the data against blank measurements to give fold-gain values recovered the %CV values of these sensors to provide an acceptable range of quantification. Substantial intra- and inter-assay variability is commonly seen in binding assays when using raw data due to a range of condition variations. Utilising blank or background signal to produce normalised ratio values works as a local control which limits confounding factors and specimen-to-specimen variability, giving more consistent results. The use of fold-gain data improved LoD and the upper limit of quantification (ULOQ) (Table 4).

The sensitivity of these biosensors would allow for substantial dilution of patient samples to keep analyte concentrations within the range of quantification. Trough concentrations of rituximab are reported between 8 - 400 nM45, with circulating levels < 84 nM after the first cycle of treatment associated with poor treatment outcome for follicular lymphoma patients. With a 1000x dilution of serum samples, the minimal effective concentration (C_min) falls within our NanoBit assay range of quantification. The current standard for serum rituximab measurements is an ELISA with a lower limit of detection (LoD) of 3 ng/mL (≈31 pM) in commercial kits using a 1000x diluted serum sample. Our LoD (4 pM) and LLOQ (8 pM) are thus in the same range with the significant advantage of a shorter timeframe, with sample collection to result within 10 minutes if reagents are preprepared. Similarly, the quantifiable range for our ipilimumab and trastuzumab sensors cover the reported trough concentration range and Cmin values for both drug therapies, while the LoD and LLOQ are in the same range as commercially available ELISA kits. Besides the improvement in time-to-result, homogenous assays as employed here do not require any wash steps and are thus ideally suited to be developed into bedside or PoC sensors.

Conclusion

We developed TmAb NanoBiT assays by combining anti-idiotypic Affimer proteins and NanoBiT split luciferase technology to provide a platform for rapid quantification of immunotherapies. Affimer proteins against four clinically relevant TmAb drugs were incorporated into the two component NanoBiT proximity switch and the best combinations selected. Assay conditions such as incubation time, sensor component and substrate concentration were optimised based on the rituximab NanoBiT sensor to improve assay performance. Sensitivity, precision, and accuracy metrics were then established for all four TmAb NanoBiT assays with intra- and inter-assay analysis. The rituximab sensor performed best with 4 pM LoD and a quantifiable range between 8 pM - 2 nM in 0. 1% serum. When adjusted for serum dilution, this falls within the clinically relevant range and compares favourably with the sensitivity of current ELISA standards. The sensors designed for ipilimumab and trastuzumab performed similarly when compared to the current ELISA kits available. The possibility of a time-to-result within 10 minutes without any wash steps make our sensors an appealing alternative to ELISA detection, with the prospect of improvement in sensitivity through further optimisation.

Overall, we have developed rapid assays for the quantification of rituximab, ipilimumab and trastuzumab that compete with current methods and have the potential to be applied in a PoC setting for therapeutic drug monitoring. The concentration-therapeutic efficacy relationship of therapeutic monoclonal antibodies means that serum drug concentrations outside of the therapeutic window can have negative impacts on patient health. TDM for immunotherapies is currently limited by centralised testing methods with long sample-collection to result timeframes. Our TmAb NanoBiT assays gives time-to-result within 10 minutes and could improve patient welfare by providing the opportunity for rapid, precise dose adjustments to improve treatment outcomes and prevent adverse reactions.

Claims

Claims:

1. A two-part split reporter complex, the complex comprising a first agent, wherein the first agent comprises a first nanobody or affimer fused to the first peptide fragment of a split reporter protein and a second agent, wherein the second agent comprises a second nanobody or affimer fused to a second peptide fragment of a two-part split reporter protein.

2. The two-part split reporter complex according to claim 1, wherein the first peptide fragment of the split reporter protein and the second peptide fragment of the split reporter protein are complementary components, which when in proximity is capable of generating a detectable signal.

3. The two-part split reporter complex according to any previous claim, wherein the first peptide fragment of the split reporter protein and the second peptide fragment of the split reporter protein are complementary components of a luminescent protein.

4. The two-part split reporter complex according to any previous claim, wherein the nanobody or affimer binds an analyte.

5. The two-part split reporter complex according to claim 4, wherein the analyte is glutamate dehydrogenase (GDH), toxin B (TcdB), hCRP or an antibody.

6. The two-part split reporter complex according to claim 5, wherein the analyte is glutamate dehydrogenase (GDH) or toxin B (TcdB).

7. The two-part split reporter complex according to any previous claim, wherein the split reporter complex has an amino acid sequence comprising SEQ ID NO: 2, 4, 6 or

8.

8. A DNA encoding an amino acid sequence of the split reporter complex according to claim 7.

9. A plasmid comprising the DNA according to claim 8 in a vector.

10. A transformed cell wherein the cell comprises the plasmid according to claim 9.

11. The transformed cell according to claim 10, wherein the cell is a mammalian cell or bacterial cell.

12. The transformed cell according to claim 10, wherein the cell is a human cell, a primate cell, or a murine cell.

13. The transformed cell according to claim 10, wherein said cell is a cell derived from a mammal or an insect.

14. A method of detecting an analyte in a mixture, the method comprising: delivering the two-part split reporter complex according to claims 1 to 7 into a mixture, wherein the first agent and second agent are delivered as separate agents and wherein when the first and second nanobody or affimer bind an analyte, the first peptide fragment and second peptide fragment of the split reporter protein are brought into proximity and a detectable signal is produced.

15. A method according to claim 14, wherein the method is practiced invitro.