EP3953381A1 - Fusion protein comprising nanoluciferase inserted between immunoglobulin variable domains - Google Patents

Abstract

The present invention provides polypeptides comprising the antibody domains held together by an in-frame single or tandem nanoluciferase(s), retaining antigen binding and the nanoluciferase activity. These recombinant antibodies may be suitable for the detection of anti-drug antibody antibodies or other analytes.

Description

FUSION PROTEIN COMPRISING NANOLUCIFERASE INSERTED BETWEEN IMMUNOGLOBULIN VARIABLE DOMAINS

Although immunoassay, the mainstay of clinical diagnostic laboratories has changed formats considerably over the past 60 years, the basic principles have remained fundamentally the same: i.e., antibody-antigen interaction determination, either qualitative (yes/no), or quantitative (how much), and various combinations of these metrics. The scope for the application of immunoassays is wide, from over-the-counter pregnancy tests, HIV testing, substance of abuse monitoring, detecting mycotoxins in food and feed, measuring markers of cardiac health to the detection of anti-drug antibody antibodies.

In this connection, during this time, biologic drug therapies have been developing and, in particular those based on monoclonal antibodies, which may result in the development of anti-drug antibodies (ADA). ADA can reduce the drug efficacy and possibly lead to an adverse event (i.e., immune allergic reaction) and eventually to loss of response to the biologic therapy. Monitoring for the emergence of the ADA qualitatively and quantitatively can inform when a treatment is failing, or likely to fail allowing cessation of the drug and switching to an alternative.

The monitoring of the ADA is currently ad hoc requiring the therapeutic drug to capture and detect the anti-drug antibodies. There are various methods for achieving this monitoring, all which have disadvantages. All methods require a sandwich between the capture antibody (immobilised) and detection with a labelled antibody (by attaching a covalent label, biotin/digoxin/fluorescent/enzyme molecules) with the ADA in-between. Currently, the clinical development of novel therapeutic antibodies requires the evaluation of their potential immunogenicity by appropriate assays (Kaliyaperumal, A. and Jing, S., Curr. Pharm. Biotechnol. 10 (2009) 352-358). The ADA testing usually involves an assay for ADA detection and a separate assay for ADA characterization.

The ADA detection assays include screening and specificity confirmation (confirmatory) assays. Microtiter plate-based enzyme-linked immunosorbent assays (ELISAs) are the most widely used format to screen for ADAs due to their high-throughput efficiency, simplicity and high sensitivity (Geng, D., et al, J. Pharm. Biomed. Anal. 39 (2005) 364-375). ADA ELISAs are most often designed in a bridge format which provides high selectivity, detection of all isotypes and pan-species ADA detection capability (Mire-Sluis, A.R., et al, J. Immunol. Methods 289 (2004) 1-16). However, the bridging format assay (coating with drug and detecting with labelled drug) has limitations since immobilizing drug antibody on a solid surface may mask or alter epitopes, the conjugating of a reporter may mask epitopes, the chemistry involved in conjugation may alter the molecular structure, and the bridging format is affected more by drug interference than other ELISA formats.

The Direct ELISA format (coating with drug and detecting with labelled anti-lgG) also has limitations, since immobilizing drug antibody on a solid surface may mask or alter epitopes, isotype detection is also determined by conjugate, likewise the species specificity determined by conjugate and the detection reagents may be different between control and sample.

The Indirect ELISA (coating with a specific mAb or biotin to capture the drug and orient with the Fab portion available for binding) requires extensive studies to demonstrate the mAb does not alter epitope accessibility, isotype detection is determined by conjugate, likewise the species specificity determined by conjugate.

The disadvantage of the Radio Immuno-precipitation assay (RIP) is the use of radioactivity short lived tracers such as I¹²⁵ (half life of ~60 days) whereby conjugating chemistry may degrade or alter the molecule. Further, the radio label decay may affect molecule stability.

With surface plasmon resonance, the linking chemistry to immobilise the drug may affect the molecule, coupling to dextran may mask epitopes, the regeneration step may degrade molecules, the reagents/technology are not generic but vendor specific and it is a low throughput and is often less sensitive than ELISA or RIP.

Finally, Electrochemiluminescence bridging format has the need to prepare two conjugates (biotin and TAG). It, therefore, requires more material, conjugations may mask or alter epitopes, the conjugation chemistry may degrade or alter the molecule, if the target molecule has two of the same antigen epitopes, it may give rise to false positives and the reagent/technology is vendor specific. As set out above, the current approaches all have inherent limitations as a generic platform since chemical labeling of therapeutic antibodies be it for conjugation or immobilization will be heterogeneous for a single antibody and vary with different antibodies. Thus, a generic platform for ADA is at best qualitative, but not quantitative due to the inherent variation of detection for each therapeutic antibody. Each ADA assay would require optimisation to develop a quantitative assay. In addition, assays results may vary due to the presence of the antibody drug in the sample competing for the binding site thus giving an underestimate of the true ADA level.

An object of the present invention is to attempt to provide a test for ADA which is qualitative and quantitative.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a single polypeptide comprising a nanoluciferase domain that possesses a N-terminus and a C-terminus, a first antibody domain linked to the N-terminus by a covalent linker, and a second antibody domain linked to the C-terminus by a second covalent linker.

By incorporating the enzyme reporter, nanoluciferase, with stoichiometry of either 1 antibody binding site with 1 nanoluciferase enzyme (as shown in Figure 1) or 1 antibody binding site with 2 nanoluciferase enzymes (as shown in Figure 2) we have generated reagents that can be used in, for example, an assay which allows, for example, the ADA in the sample to be bound to a reporter molecule containing nanoluciferase, and then captures all the antibodies present in a sample along with bound reporter. The amount of bound reporter is then proportional to the ADA in the sample. Accordingly, any number of nanoluciferase polypeptides or active parts thereof could be included in the nanoluciferase domain and the relevant stoichiometric calculation carried out.

The use of a single polypeptide allows the polypeptide to have full nluc activity and the binding activity of VH/VL. In this single form it can, for example, be secreted into the periplasmic space of E.coli which is an oxidising environment that aids folding. It will be appreciated that any suitable host or processing environment could be used to produce the single polypeptide of the present invention. It will also be appreciated that when processing a polypeptide with antibody domains, it is important to ensure that the correct configuration of the antigen binding site is achieved and, therefore, production of a single polypeptide is advantageous.

As the nanoluciferase domain is placed between the two antibody domains rather than merely on the end of one domain, the antibody domains in the polypeptide of the present invention can pair together to form an antibody binding site.

The modular nature of the nanoluciferase fusion polypeptides described herein permit great design flexibility and the possibility of exploiting the known binding affinity and specificity of any known therapeutic antibody. The antibody binding site is configured to be identical or functionally similar to an antibody binding site for an analyte, for example, drug antibody VH and VL.

As used herein, an antibody domain is that portion of the fusion polypeptide that exhibits affinity interaction with a ligand/analyte that is to some degree specific As used herein, "specific" and variations thereof refer to having a differential or a non-general affinity, to any degree, for a particular target. In some embodiments, an antibody domain can include any suitable portion of an immunoglobulin. As immunoglobulin structure and function are well characterized, those of skill in the art are well equipped to determine the amount and the particular portions of an immunoglobulin necessary to provide desired target recognition.

The polypeptide of the present invention contains all the potential paratopes, but lacks the constant light and constant heavy domains that could facilitate binding to Protein G, Protein A, Protein L, Protein A/G, anti-kappa constant, anti-lambda constant or anti-Fc antibodies. The lack of binding to Protein G, Protein A, Protein L, Protein A/G, anti-kappa constant, antilambda constant or anti-Fc antibodies allows the reagent to be used to detect IgG from sera captured on Protein G as exemplified or by the other capture reagents namely Protein A, Protein L, Protein A/G, or anti-Fc antibodies. The antibody domains may be derived from any suitable monoclonal or polyclonal antibody. Accordingly, the polypeptide of the present invention can be configured to recognise any conventional therapeutic antibody that has a V_H, or VL or combinations of VH and VL domains (i.e., single domain VHH, SCFV, Fab, IgG, CAR-T cell and any format of bispecific antibodies). Further, the polypeptide of the present invention can be configured to recognise nonbiologic drugs such as Bicycle· drugs.

When the term "GloBody" is used herein, it is intended to refer to the polypeptide of the present invention and is not intended to refer to any product which may become known under the trade mark.

The polypeptide can include a linker that provides the covalent linkage between the nanoluciferase domain and one of the antibody domains. In such embodiments, the polypeptide can include more than one linker so that a linker provides the link between the nanoluciferase domain and each of the antibody domains. The linker may be of any suitable length. In some embodiments, the linker assists in providing the stability of the molecule by contributing to spacing between the C-terminus of one antibody domain and the N-terminus of the second antibody domain that reflects the spacing natively found in a Fab fragment or full IgG. In such embodiments, the length of the linker may be no more than 10 amino acids such as, for example, no more than nine amino acids, no more than eight amino acids, no more than seven amino acids, no more than six amino acids, no more than five amino acids, no more than four amino acids, no more than three amino acids, or no more than two amino acids. When two linkers are present, the length (and sequence) of one linker may be identical to or different from the length (and sequence) of the other linker. In one embodiment, the linker can include five amino acids such as, for example, Ala-Ser-Thr-Gly- SerJSEQ ID NO:7), Gly-Gly-Gly-Gly-Ser (SEQ ID No:8), Ser-Gly-Ser-Gly-Ser (SEQ ID NO:9), Ala- Thr-Ser-Gly-Ser (SEQ ID No:10).

Conveniently at least one of the covalent linkers comprises no more than 5 amino acids. In this way the structure of the polypeptide of the present invention is relatively rigid. If the linkers are too long the structure of the polypeptide would be flexible and a solution containing the polypeptide may not be stable such that the polypeptide domains could disassociate and aggregate. We have devised an approach that can capture all the IgG in a sample and then selectively detect the ADA. Although exemplified for IgG capture via Protein G, it should be feasible to capture individual isotypes (i.e., IgE, IgA, IgM Igx, IgA) using Fc specific or light chain specific antibodies. In the embodiment described in Example 1 (Figure 1), the VH C-terminus is fused to the N- terminus of nanoLuc, replacing the CHI domain. The C-terminus of the nanoLuc is fused to the N-terminus of the VL, providing an alternative anchor for the V_L and thus eliminating a need for the CL. This example used the VH and VL domain pairing for the recombinant anti- CD52 antibody based on Campath-1H (also known as Alemtuzumab). The exemplary Alem snluc described in Example 1 has the following characteristics: readily made and isolated from E. coli; the enzyme activity remains unaltered; it is monomeric; it is stable; it retains the specificity of the parental antibody; and it is simple to use as a single reagent in immunoassays. In the embodiment described in Example 2 (FIG 2), the VH C-terminus is fused to the N- terminus of a tandem dual nanoLuc, replacing the CHI domain. The C-terminus of the tandem dual nanoLuc is fused to the N-terminus of the V_u providing an alternative anchor for the VL and thus eliminating a need for the C_L. The exemplary Alem dnluc described in Example 2 has the following characteristics: readily made and isolated from E. coli; the enzyme activity remains unaltered; it is monomeric; it is stable; it retains the specificity of the parental antibody; and it is simple to use as a single reagent in immunoassays.

The present invention involves novel fusion polypeptides/proteins constructs, polynucleotides that encode the constructs, and methods, including diagnostic, and detection methods, employing such constructs. The present invention includes the design, assembly, bacterial production, and affinity enrichment of a modular assembly that may be used generally to produce novel antibody-nanoluciferase protein fusion molecules. In one particular embodiment, the insertion of nanoluciferase proteins in-between the VH/VL regions of anti-CD52 antibody Alemtuzumab resulted in VH/VL interface interactions to create a polypeptide that is designated herein as "AlemGloBody". The bacterially expressed monomeric molecule used in luminescence capture assay for the detection of ADA against Alemtuzumab. The molecular model may be generalized beyond the use of a single nanoLuc and the Alemtuzumab and may, instead, use the VH/VL of any therapeutic or targeting antibody, such as CART and use the tandem nanoluciferase domains nanoluciferase, as described in more detail below. Thus, references to the polypeptide of the present invention which we have designated as GloBody are merely an exemplary and should not be construed as limiting.

We have engineered a fusion polypeptide that uses a single nanoluciferase or dual/tandem nanoluciferase domain to bridge the Vn and the Viand resulting in functional binding sites.

Conveniently, at least one of the nanoluciferase domain, the first antibody domain, and/or the second antibody domain comprises an affinity tag.

Affinity tags are routinely used to assist with the isolation and/or collection of recombinant polypeptides. Affinity tags, their use, and the methods of isolating polypeptides equipped with an affinity tag are well known to those of skill in the art. Exemplary affinity tags include, for example, a six-histidine tag (His-tag). A recombinant protein containing a His-tag can be purified and detected easily because the string of histidine residues binds to several types of immobilized metal ions such as, for example, nickel, cobalt or copper, under specific buffer conditions. In addition, anti-His-tag antibodies are commercially available for use in assay methods involving His-tagged proteins. In either case, the tag provides a means of specifically purifying or detecting the recombinant protein without a protein-specific antibody or probe. It is also possible to use alternative conventional tags including, for example, tags that include three or more amino acids, which bind to known corresponding affinity acceptors. Conveniently, the first antibody domain and the second antibody domain specifically bind to a single target molecule.

If both antibody domains specifically bind to the same target molecule, the polypeptide of the present invention is characterised as mono-specific. If, for example, the polypeptide is based on a therapeutic antibody, the specific nature of the polypeptide of the present invention ensures that the test is accurate and minimises false positive results. Alternatively, the first antibody domain and the second antibody domain bind to more than one molecule, (i.e., a polyclonal response). In this regard, in some instances the body can produce a polyclonal response to, for example, the antibody therapy.

Conveniently the first antibody domain comprises a variable heavy chain (VH) comprising an N-terminus. Alternatively, the first antibody domain comprises a variable light chain (VL) comprising an N-terminus.

Conveniently, the second antibody domain comprises a VL comprising a C-terminus. Alternatively, the second antibody domain comprises a VH comprising a C-terminus.

Conveniently, the C-terminus (or N-terminus) of first antibody domain and N-terminus (or C- terminus) of the second antibody domain are separated by a distance of no less than 30 Å and no more than 40 Å. Conveniently, the C-terminus (or N-terminus) of first antibody domain and N-terminus (or C-terminus) of the second antibody domain are separated by a distance of no less than 33 Å and no more than 36 Å.

Conveniently, nanoludferase domain comprises at least a portion of a nanoluciferase polypeptide/protein sufficient to have nanoluciferase activity.

Accordingly, not all of the nanoluciferase protein needs to be present in the polypeptide of the present invention provided that the portion present can produce a sufficient signal, such as luminescence, in the presence of a substrate, such as furimazine. The structure and function of nanoluciferase polypeptides are well characterized. Thus, a person of ordinary skill in the art can readily determine the portion of a nanoluciferase polypeptide that is required to maintain nanoluciferase functionality.

The nanoluciferase domain can optionally provide structural integrity to the polypeptide in addition to providing a source for the nanoluciferase signal. Thus, in some embodiments, the nanoluciferase domain can include a portion of the nanoluciferase polypeptide sufficient to retain enzyme activity to generate a nanoluciferase signal and to provide desired steric stability.

Conveniently, the polypeptide of the present invention can include at least a portion of a monomeric nanoluciferase protein sufficient to have nanoluciferase activity. Conveniently, the nanoluciferase domain comprises a portion of more than one nanoluciferase protein wherein at least one of the portions is sufficient to have nanoluciferase activity.

Conveniently the nanoluciferase domain comprises two (or dual) nanoluciferase portions. Conveniently, the nanoluciferase portions are in tandem.

Accordingly, more than one nanoluciferase active portion can be present. If more than one nanoluciferase active portion is present and active then a more sensitive test can be produced. A person skilled in the art would be able to use such a polypeptide of the present invention using stoichiometric calculations. In some embodiments, the nanoluciferase domain can include an amino acid sequence that bears a specified level of amino acid sequence similarity to a reference polypeptide SEQ ID NO:6 or SEQ ID NO:24 or an active part thereof.

Conveniently, the nanoluciferase domain is reference sequence SEQ ID NO:5, SEQ ID NO:6 or reference sequence SEQ ID NO:23 or SEQ ID NO:24. Conveniently, the nanoluciferase domain can include a polypeptide with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence similarity to the reference amino acid sequence SEQ ID No:6 or SEQ ID No:24 provided that it is still at least partly active.

Conveniently, the active portions in the tandem or dual nanoluciferase proteins/polypeptides have a different nucleic acid sequence. In this regard, the different nucleic acid sequences can lead to improved processing during assembly of the polypeptide of the present invention.

Conveniently, the tandem or dual nanoluciferase proteins/polypeptides are linked by a suitable covalent linker. The linker may be of any suitable length. In some embodiments, the linker assists in providing the stability of the molecule by contributing to spacing between the C-terminus of one antibody domain and the N-terminus of the second antibody domain that reflects the spacing natively found in a Fab fragment or full IgG.

Conveniently, the length of the linker may be no more than 10 amino acids such as, for example, no more than nine amino acids, no more than eight amino acids, no more than seven amino acids, no more than six amino acids, no more than five amino acids, no more than four amino acids, no more than three amino acids, or no more than two amino acids. When two linkers are present, the length of one linker may be identical or different than the length of the other linker.

Conveniently, the linker can include five amino acids such as, for example, Ala-Ser-Thr-Gly- SerjSEQ ID NO:7), Gly-Gly-Gly-Gly-Ser (SEQ ID No:8, Ser-Gly-Ser-Gly-Ser (SEQ ID NO:9), Ala- Thr-Ser-Gly-Ser (SEQ ID No:10). Conveniently, the nano covalent linker is Ser-Gly-Ser-Gly-Ser (SEQ ID NO:9).

Conveniently, the nanoluciferase domain comprises the sequence - Gly-Ser-nlucl-Ser-Gly- Ser-Gly-Ser-nluc2-Gly-Gly-Glu-Gly-Ser (SEQ ID No: 20). Using this or a similar asymmetric sequence allows the nanoluciferase domain to be inserted directionally between the VH and

V_L.

Portions of certain dual, tandem or dimeric nanoluciferase polypeptides may also be suitable for use as the nanoluciferase domain. Such nanoluciferase polypeptides may naturally orient so that they can provide steric stability as described above while avoiding steric interference with the antigen-binding site formed by the antibody domains.

We have inserted a single and tandem dual nanoluciferase between the VH and VL domains of a range of recombinant therapeutic antibodies such as, for example, single chain FVs (scFv's) based on Alemtuzumab, Infliximab, Adalimumab, Rituximab, Trastuzumab and both arms of Emicizumab by replacing the flexible peptide linker. This general approach can be used to produce nanoluciferase fusion polypeptides that include a portion of VH and VL domains of any antibody of interest. In another aspect, the invention provides a method of using the nanoluciferase fusion polypeptide as a reporter.

In another aspect, the invention provides a composition comprising at least one polypeptide of the present invention.

Conveniently, the composition of the present invention comprises a first and a second polypeptide of the present invention. Conveniently, the nanoluciferase domain of the first polypeptide can have nanoluciferase activity and the nanoluciferase domain of the second polypeptide can also have nanoluciferase activity.

Conveniently, each polypeptide in the composition can either be designed to target a single analyte or multiple analytes. Accordingly, the composition can be used to identify a monoclonal or polyclonal response, or a monoclonal response to one or more analytes.

In another aspect, the invention provides a polynucleotide that encodes any one of the polypeptides as herein described.

In another aspect of the present invention there is provided a method for detecting an analyte, the method comprising: contacting a sample comprising the analyte with a polypeptide according to the present invention wherein at least one of the first antibody domain and the second antibody domain specifically binds to the analyte; removing unbound polypeptides; and detecting a luminescent signal produced by the polypeptide specifically bound to the analyte, thereby detecting presence of the analyte in the sample.

Conveniently, the invention provides a method that generally includes providing a sample that comprises the analyte, contacting the sample with any one or more of the polypeptides summarized above, wherein at least one antibody domain specifically binds to the analyte, capturing the complex by Protein G, removing unbound polypeptides, and detecting a nanoluciferase signal produced by the polypeptide specifically bound to the analyte, thereby detecting presence of the analyte in the sample. The total IgG in the sera sample is captured on Protein G; if ADA are present, they will also be captured along with the bound polypeptide of the present invention. The unbound polypeptide of the present invention can be washed away and the amount of polypeptide retained being directly proportional to the ADA in the sample.

When the polypeptide of the present invention is added to a serum sample, if an analyte, such as an ADA, is present in the sample that recognises the drug VH/V_L combination, it will bind to the polypeptide of the present invention since it has the same VH/VL combination. To capture ADA in the presence of the drug antibody, a brief acidification step to dissociate any preformed complexes between the drug and the ADA prior to adding the polypeptide in excess in neutralising solution allows the total ADA to be determined. Accordingly, the method of the present invention can further comprise an acidification step or any other method to dissociate any preformed complexes between the drug and the ADA. Conveniently, the methods of the present invention can further include immobilizing at least a portion of the sample on an affinity resin.

Conveniently, the methods of the present invention can further include quantifying the nanoluciferase signal.

The methods of the present invention further comprise the step of quantifying the analyte using stochiometric calculations known to those in the art.

Conveniently, the methods of the present invention further comprise immobilising at least a portion of the sample on a substrate. The step of immobilising a portion of the sample on a substrate will be well known to those in the art. The immobilising step aids the separation of the bound polypeptide and the unbound polypeptide.

Conveniently, the analyte is an anti-drug antibody.

In another aspect, the invention provides a method of making a fusion polypeptide. Generally, the method includes creating an expression vector that comprises a polynucleotide operably linked to a promoter, wherein the polynucleotide encodes an fusion polypeptide comprising: a nanoluciferase domain comprising a N-terminus and a C-terminus, a first antibody domain covalently linked to the N-terminus, and a second antibody domain covalently linked to the C-terminus; introducing the expression vector into a host cell; and growing the host cell comprising the expression vector in conditions effective for the host cell to express the fusion polypeptide.

In another aspect, the invention provides a method of making a fusion polypeptide. Generally, the method includes creating an expression vector that comprises a polynucleotide operably linked to a promoter, wherein the polynucleotide encodes an fusion polypeptide comprising: a tandem nanoluciferase domain comprising a N-terminus and a C- terminus, a first antibody domain covalently linked to the N-terminus, and a second antibody domain covalently linked to the C-terminus; introducing the expression vector into a host cell; and growing the host cell comprising the expression vector in conditions effective for the host cell to express the fusion polypeptide. For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list

The present invention is illustrated by the following figures and examples. It is to be understood that the particular figures, examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

Figure. 1: A 3D model of the anti-CAM PATH-lH/NanoLuc luciferase fusion antibody. Molecular model (ribbon representation) of the CAMPATH-1 antigen-binding fragment (Fv) (PDB 1BEY) fused with the NanoLuc luciferase (PDB 5IBO). The Fv variable region heavy chain (VH) and the Fv variable region light chain (VL) are labelled. NanoLuc is fused in between these domains and is separated by two amino acid linker sequences.

Figure. 2: A 3D model of the anti-CAM PATH-lH/NanoLuc luciferase fusion antibody. Molecular model (ribbon representation) of the CAMPATH-1 antigen-binding fragment (Fv) (PDB 1BEY) fused with the two NanoLuc luciferase (PDB 5IBO) separated by a short linker sequence. The Fv variable region heavy chain (VH) and the Fv variable region light chain (VL) are labelled. First nanoluc and the second nanoluc are inserted in-frame in between the VH and VL domains and are separated by amino acid linker sequences.

Figure. 3: ADA Assay format Serum sample acidified to dissociate preformed drug-ADA complexes. Excess AlemGloBody with neutralising solution added to the acidified serum sample. Excess Protein G agarose added to capture the IgG in the neutralized sample. Protein G agarose washed to remove any unbound AlemGloBody. Bound IgG and bound AlemGlobody eluted from protein G agarose and assayed by adding Furimazine in Glo buffer and light emission read on a luminometer plate reader. Assayed sera samples include a blank sera and 50ug/ml standard ADA monoclonal spiked into blank sera.

Figure. 4: Anti-Alemtuzumab Antibody Assay using AlemGloBody

Baseline sample before drug treatment treatment; Pla sample after 1st dosing of Alemtuzumab; Plb sample after 1st dosing of Alemtuzumab but before 2nd dose; P2 sample after 2nd dosing of Alemtuzumab but before 3rd dose; P3a sample after 3rd dosing of Alemtuzumab; P3b sample 6 years after 3rd dosing of Alemtuzumab. Blank sera (penultimate column); Standard 50ug/ml spiked ADA into Blank sera (solid column).

Figure. 5: Anti-Alemtuzumab Antibody Assay using AlemGloBody

PI sample after 1st dosing of Alemtuzumab; P2 sample after 2nd dosing of Alemtuzumab but before 3rd dose; P3a sample after 3rd dosing of Alemtuzumab; P3b sample 3 years after 3rd dosing of Alemtuzumab; Blank sera (penultimate column); 50ug/ml spiked ADA into Blank sera (solid column).

Figure. 6: Anti-Alemtuzumab Antibody Assay using AlemGloBody Baseline sample before drug treatment treatment; PI sample after 1st dosing of Alemtuzumab; P2 sample after 2nd dosing of Alemtuzumab but before 3rd dose; P3 sample after 3rd dosing of Alemtuzumab; Blank sera(penultimate column); Standard 50ug/ml spiked ADA into Blank sera )solid column). Figure. 7: Anti-Alemtuzumab Antibody Assay using AlemGloBody

PI sample after 1st dosing of Alemtuzumab; P2 sample after 2nd dosing of Alemtuzumab but before 3rd dose; P3 sample after 3rd dosing of Alemtuzumab, but before 4th dose; P4 sample after 4th dosing of Alemtuzumab, but before 5th dose; P5 sample after 5th dosing of Alemtuzumab, but before 6th dose; P6 sample after 6th dosing of Alemtuzumab; Blank sera (penultimate column); and Standard 50ug/ml spiked ADA into Blank sera (solid column).

Figure. 8: Illustration of the recombinant vectors. (A) The Alem scFv variable regions are connected with a short glycine-serine rich linker region. All three constructs have a 6xHis- tag. The pK constructs include (B) single nanoluc / (C) dual nanoluc as reporter genes both of which are between the variable chains. The Ncol and Notl restriction sites flank the Alem VH and VL sequence respectively. The BamHI restriction sites flank the single nLuc and dual nluc sequences within Alem scFv variable regions.

Figure. 9: Generation of E. coll expression cassette. Generation of E. coll expression cassette. (A) The single chain Fv encoding fragment flanked by Ncol and Notl sites with an inframe BamHI site between the VH and VL was inserted into pKlO vector to generate pKlOAIem. The single nanoluc (snluc) (B) or dual tandem nanoluc (dnluc) (C) encoding fragments flanked by BamHI sites were inserted into the pKlOAIem to generate ALEM VH link snluc link VL His Tag (D) and ALEM VH link dnluc link VL His Tag (E) respectively

Figure. 10: Shows the nucleic acid (SEQ ID NO:l) and amino acid sequence (SEQ ID NO:2) for the VH, VL, and single nanoluc of Alemsnluc. The tertiary structure is illustrated in Figure. 1. Italic correspond to the VH domain; bold text corresponds to the nanoluciferase domain and underlined text correspond to the VL domain.

Figure. 11: Shows the nucleic acid (SEQ ID NO:3) and amino acid sequence (SEQ ID NO:4) for the VH, VL, and the dual nanoluc of Alemdnluc. The tertiary structure is illustrated in Figure. 2, italic letters correspond to the Alemtuzumab VH domain; bold text corresponds to the nanoluciferase domains and underlined text correspond to the Alemtuzumab VL domain. The removal of the BamHI site between the two nanoluc is in italics and underlined. Figure. 12: Shows the nucleic acid (SEQ ID NO:5) and amino acid sequence (SEQ ID NO:6) of the dual tandem nanoluciferase domain in which the internal Bam HI site had been removed by replacing the TCC codon for serine with AGT which also codes for serine (TCC AGT) is underlined shown in bold. Figure. 13: Is a bar chart illustrating the luciferase activity during AlemGloBody expression and purification. Single colony NEB* Express Iq transformed with plasmid pK AlemdnLuc added to 5ml auto-induction media with lOOug/ml kanamycin, grown with shaking at 275rpm for 25hr. Cells pelleted at 3000 rpm for 15 min 0.5ml of the clear supernatant (SUP) retained. The pellet lysed with the addition of 567ul of NPI-10 and 63ul of BugBuster (lOx) and 15 units of Benzonase on ice for 30 min and then centrifuged at 12000g for 30 min at 4"C. 20ul of Soluble Extract (SE) retained for SDS PAGE. 600ul loaded onto NI2+NTA Spin Colum (Qiagen) and centrifuged 1600rpm for 5min and the Flow Through (FT) retained. Washed with N PI-20600ul at 3000rpm for 2 min and 1st wash discarded, repeated wash and the Final Wash (FW) retained. The bound proteins eluted with 2 x 300ul of N PI-500 added for 3min then centrifuged at 3000rpm for 2 min Elution 1 (El) and Elution 2 (E2). lul of each sample added to lOOul of furimazine in Glo Buffer (49ul PBS, 49ul GloBuffer and 2ul furimazine and the luminescence measured after 10 minutes (with a gain setting of 1900).

Figure. 14: Single colony NEB* Express Iq transformed with pK AlemGloBody added to 5ml autoinduction media with lOOug/ml kanamycin, grown with shaking at 275rpm for 25hr. Cells pelleted at 3000 rpm for 15 min 0.5ml of the clear supernatant (SUP) retained. The pellet lysed with the addition of 567ul of NPI-10 and 63ul of BugBuster (lOx) and 15 units of Benzonase on ice for 30 min and then centrifuged at 12000g for 30 min at 4"C. 20ul of Soluble Extract (SE) retained for SDS PAGE. 600ul loaded onto NΪ2+NTA Spin Colum (Qiagen) and centrifuged 1600rpm for 5min and the flow through (FT) retained. Washed with N PI-20 600ul at 3000rpm for 2 min and 1st wash discarded, repeated wash and the final wash (FW) retained. The bound proteins eluted with 2 x 300ul of N PI-500 added for 3min then centrifuged at 3000rpm for 2 min (El).

LDS 4x loading gel with 10% b-mecaptoethanol 15ul, was added to 30ul of each samples (SE, FT, FW and El), heated to 98"C for 10 min and 25ul run on a 4-12% gradient gel in MES buffer at 200v for 60 min. PageRuler prestained marker was include. The resolved proteins were transferred to a PVDF membrane and processed as outlined in Figure 15. The gel was then stained with Coomassie Blue for lhr and destained in water for lhr and then left overnight with a change of water. The 70 kDa and 50 kDa are shown in bold. The expected band is in the vicinity of 65 kDa.

Figure. 15: Single colony NEB· Express Iq transformed with pK AlemGlobody added to 5ml autoinduction media with lOOug/ml kanamycin, grown with shaking at 275rpm for 25hr. Cells pelleted at 3000 rpm for 15 min 0.5ml of the clear supernatant (SUP)retained. The pellet lysed with the addition of 567ul of NPI-10 and 63ul of BugBuster (lOx) and 15 units of Benzonase on ice for 30 min and then centrifuged at 12000g for 30 min at 4"C. 20ul of Soluble Extract (SE) retained for SDS PAGE. 600ul loaded onto NI2+NTA Spin Colum (Qiagen) and centrifuged 1600rpm for Smin and the flow through (FT) retained. Washed with NPI-20 600ul at 3000rpm for 2 min and 1st wash discarded, repeated wash and the final wash (FW) retained. The bound proteins eluted with 2 x 300ul of NPI-500 added for 3min then centrifuged at 3000rpm for 2 min (El).

LDS 4x loading gel with 10% b-mecaptoethanol 15ul, was added to 30ul of each samples (SE, FT, FW and El), heated to 98"C for 10 min and 25ul run on a 4-12% gradient gel in MES buffer at 200v for 60 min. PageRuler prestained marker was include. The resolved proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for lhr at ambient temperature and then probed with a goat anti-6xHis tag antibody conjugated to HRP (diluted 1:25,000) for lhr at ambient temperature, washed 5x with PBS 0.05% Tween-20 for Smin per wash and developed with Pierce ECL kit. The developed film was aligned with the membrane to locate the 70 kDa and 50 kDa.

Figure. 16: Representations of possible assemblies of the present invention (GloBody) in VH- VL orientation: (A) Monomeric with a single NanoLuc; (B) Monomeric is a dual NanoLuc; (C) Dimeric with a single NanoLuc and (D) Dimeric with a dual NanoLuc. Figure. 17: Representations of possible assemblies of the present invention (GloBody) in VL- VH orientation: (E) Monomeric with a single NanoLuc; (F) Monomeric is a dual NanoLuc; (G) Dimeric with a single NanoLuc and (H) Dimeric with a dual NanoLuc.

Figure 18: Anti-Emicizumab Antibody Assay using EmidaXIaGloBody and EmidaXGlobody

Panel A: ADA against the anti-(FIXa) arm of Emicizumab: Negative control A serum sample haemophilia patient treated with Emicizumab not making ADA; Negative control B serum sample haemophilia patient treated with Emicizumab not making ADA; Serum sample taken from the same haemophilia patient treated with Emicizumab at sample time point 1 and 2. Column 3 control sera from a haemophilia patient not treated with Emicizumab.

Panel B: ADA against the anti-(FX) arm of Emicizumab: negative control A serum sample haemophilia patient treated with Emicizumab not making ADA; Negative control B serum sample haemophilia patient treated with Emicizumab not making ADA; Serum sample taken from the same haemophilia patient treated with Emicizumab at sample time point 1 and 2. Column 3 control sera from a haemophilia patient not treated with Emicizumab.

Figure. 19: Anti-Adalimumab and Anti-Infliximab Antibody Assay using AdaliGloBody and InflixiGloBody. Panel A: ADA against Adalimumab Serum sample 1-10; Standard human monoclonal anti - adalimumab IgG (Bio-Rad HCA-204 Affinity KD, 0.06 nM) added to blank sera ~50ug/mL; Blank sera. Sample 9 is higher than the blank, but lower that the spiked standard.

Panel B: ADA against Infliximab Serum sample 1-10; Standard human monoclonal antiinfliximab IgG (Bio-Rad HCA233 Affinity KD 0.12 nM) added to blank sera ~50ug/mL; Blank sera. Sample 6 is higher than the blank, but lower that the spiked standard.

Figure 20 sets out various sequence listings.

SEQ ID NO: 11- DNA sequence of pK Adali scFv

SEQ ID NO: 12- DNA sequence of pK Inflixi scFv

SEQ ID NO: 13- DNA sequence of pK Goli scFv SEQ ID NO: 14- DNA sequence of pK Rituxi scFv

SEQ ID NO: 15- DNA sequence of pKTrast scFv

SEQ ID NO: 16- DNA sequence of pK Emici IXa scFv

SEQ ID NO: 17- DNA sequence of pK Emici X scFv

SEQ ID NO: 18- DNA sequence of vector pK SEQ ID NO: 19- DNA sequence of vector pKamp

SEQ ID NO: 23- DNA sequence of single nanoluciferase domain SEQ ID NO: 24- Amino acid sequence of single nanoluciferase polypeptide

Example 1

Molecular Biology

The VH-dnluc-VL-His-Tag (GloBody) constructs were assembled in a modified pET-26b vector in which the T7 promoter region was replaced with a Lac promoter and designated pK. A pelB leader sequence inserted to direct secretion of the expressed protein to the periplasm of E. coli (Figure 8). The synthetic genes of the VH-VL SCFV antibodies were designed to encode an in-frame Bam HI site IGGA TCC encoding Gly Ser) between the VH and VL regions and the whole flanked by Ncol and Notl sites for in-frame directional doning to generate pK Alem scFv. The dnluc or nluc gene was inserted into each of these plasmids to make expression plasmids pK Alemsnluc or pK Alemdnluc (GloBody) as shown in Figure 9.

The pK Adali scFv (SEQ ID NO: 11), pK Inflixi scFv, (SEQ ID NO: 12), pK Goli scFv, (SEQ ID NO: 13) pK Rituxi scFv, (SEQ ID NO:14 ) pKTrast scFv, (SEQ ID NO: 15), pK Emici IXa scFv (SEQ ID NO:16 ) and pK Emici X scFv (SEQ ID NO: 17) were assembled in a similar manner. The dnluc or nluc gene were inserted into each of these plasmids to make the corresponding expression plasmids with dnluc or nluc for each antibody. The expression cassettes start from pelB leader sequence followed by VH chain, snluc or dnluc, VL chain and an hexa-His-tag at the C terminus of the resulting protein sequence as shown for Alem constructs in Figure 8. pK (SEQ ID: 18) carries the resistance marker for kanamycin. We also prepared a vector similar to pK in which the resistance marker for kanamycin was replaced with the marker for resistance to ampicillin and this was designated pKamp (SEQ ID: 19). Protein Expression & Purification

The recombinant GloBody proteins were expressed in NEB· Express l^q E. coli strain and recovered from the soluble extract via Ni NTA affinity chromatography. Protein expression and purification processes were monitored by luciferase activity (Figure 13) and by SDS- PAGE/western blot analysis. The predicted molecular weights of the GloBody recombinant protein was approximately 65 kDa as shown in Figure 14.

Luminescent Measurement The emission peaks of nanoLuc is 460nm. The opaque 96 well plates were read on a Clariostar plate reader under the pre-installed nanoLuc settings, with the gain setting at 3600 unless otherwise stated. Moreover, this platform may be applied to other existing and future therapeutic mAbs to create anti-drug-antibody diagnostic molecules. The platform may also be used to create libraries of VH and VL domains that may be readily accessed using high throughput nonisotopic screening and not rely on phage or other types of display selection technologies. Construction of the Expression Cassette of pKAIemGloBody Vector.

Construction of an exemplary vector expressing an exemplary scFv-nanoLuc fusion is shown in Figure 8 and Figure 9. A 724 bp Ncol/Notl fragment encoding the Alemtuzumab scFv VH-VL orientation with a five amino acid (Gly Gly Gly Gly Ser) linker (SEQ ID NO: 21) incorporating an in-frame BamHI (GGA TCC) restriction site (encoding amino acids Gly Ser) was inserted into pK vector. Modified dnluc with similar (Gly Ser) linkers on both ends was inserted using Gibson Assembly.

One-way to retain as much ligand-binding activity and as much enzyme activity as possible can involve a modular approach where the two functionalities reside in distinct, nonoverlapping regions of a single molecule. However, recombinant scFv with linkers can be prone to disassociation and aggregation (Worn and Pluckthun, 2001 J Mol Biol 305(5):989- 1010). Yet, in the context of a Fab molecule, the VH/VL remain associated. Nature has provided two solutions for stabilizing VH/VL pairs. In conventional antibodies, the CHi and the constant light chain orient and hold the Vn and the Viin place for optimal interface pairing. In the other case, the VL is not required and only the VH is required for ligand binding, as is the case in camelid antibodies (Hamers-Casterman et al., 1993 Nature 363(6428):446-448). Although making direct enzyme fusions with engineered VHH domains is possible, the vast majority of characterized therapeutic monoclonal antibodies consist of both VH and VL domains in the context of a whole IgG molecule. Thus, an approach that could conserve the Fab-like VH/VL pairing and permit fusion with a nanoluciferase protein can produce a nanoluciferase antibody that possesses reliable enzyme properties and high affinity and specificity characteristics. A novel modular approach has been devised that generates a stable nanoluciferase antibody that lack the constant domains. We have inserted the nanoluciferase between the variable domains. These modular constructs not only introduce the enzyme into the fusion molecule, but also stabilizes the positional orientation of the variable domain interfaces, resulting in Fab-like VH/VL pairing and, therefore, Fab-like ligand binding. These molecules do not bind to Protein G.

Examole 2

Plasmids and Synthetic DNA

Plasmid pK (SEQ ID NO: 18) and pKamp (SEQ ID NO: 19) were previously constructed in our laboratory are based on pET-26b vector, with the T7 promoter replaced by the lac promoter. The plasmids pK and pKamp have kanamycin and carbenicillin resistance markers respectively. All primers were purchased from Merck. Synthetic DNA sequences of Alemtuzumab, Adalimumab and Infliximab Golimumab, Rituximab, Emicizumab antibody variable domains in VH-VL orientation were codon optimized for E. coli expression and purchased from Genewiz or Genscript as plasmid pUC57 inserts, with Ncol, Bam HI and Notl restriction sites to facilitate construction of the expression vectors. Trastuzumab was also in the VH-VL orientation as described in Markiv et ah, (2011b). The snluc was codon optimised for E.coli expression. The dnluc was based on the nluc encoded in pNLl (Promega) and the E.coli optimised nluc linked via Ser Gly Ser Gly Ser linker (SEQ ID No:22).

Bacterial Strains, Growth Media and Recombinant DNA Technique NEB 5a Escherichia coil strain (NEB) was used for plasmid construction steps. To express recombinant antibodies NEB· Express l^q E. coli (NEB) was used. E. coli cells were grown in Lysogeny Broth (LB) (or LB agar plates, with either kanamycin at 50 mg/mL or carbenicillin, at 100 mg/mL Plasmid DNA was isolated using Sigma Spin Miniprep Kit and DNA from gel bands were purified using NEB Kit Escherichia coli cells were transformed using standard heat shock methods. Restriction and modification enzymes were purchased from New England Biolabs, Inc. Final plasmid constructs were confirmed by DNA sequence analysis.

Construction of the Expression Plasmids Antibody scFv encoding fragments were designed and optimised for E.coli codon usage and the synthetic DNA ordered from either Genewiz or Genscript with in-frame Ncol and Notl flanking sites in pUC57. The inserts were digested directly from pU57 or preexisting assembled VH and VL domains. The competent E. coli NEB5a cells were transformed using ligation mixtures and the clones were selected on the LB plates containing kanamycin 50ug/ml. Positive clones were confirmed by DNA sequencing. To make GloBody chimeras in V_H-nanoLuc-Vu orientation, plasmids pK containing the scFv were digested with BamHI restriction enzyme and using Gibson assembly the snluc or dnluc were inserted. Colonies were screened for luciferase activity and selected clones confirmed by plasmid DNA sequencing.

Protein Expression and Purification.

Small-scale expression (5m L Culture)

NEB· Express l^q cells transformed with plasmid (based on pK i.e., kan resistance marker) plated onto LB agar supplemented with kanamycin (50 mg/ mL final concentration), and incubated at 30"C for 16 hours. A single colony was inoculated into 5 mL of Auto-induction media (base broth: 6g Na2HP04, 3g KH2P04, 20g Tryptone, 5g Yeast Extract, 5g NaCI made up to 1L with dH20 and autoclaved; sugar mix: 150mL glycerol, 12.5g glucose, 50g lactose and made up to 1L with dH20 filter sterilised through 0.45um filter. Added 0.2mL sugar mix to 5mL of base broth in 50mL tube (lOOug/ml kanamycin) and incubated at room temperature for 25 hours with shaking at 275 rpm. The bacterial cultures were centrifuged for 15 minutes, 3000 rpm at 4^*C, an aliquot of the supernatant was retained and the pellets stored at -80"C. Small scale affinity chromatography with His tag

As in Example 1, after thawing on ice, each of the bacterial cell pellets from 5mL cultures were resuspended in 0.57ml (50mM Sodium Phosphate pHB.0, 0.3M NaCI (NP) with lOmM imidazole) (NPI-10) 0.063mL BugBuster and 0.07mL Lysozyme (from lOmg/mL stock) and 15 units of Benzonase. The cells were incubated on ice for 30 minutes and centrifuged at 12000g for 30 minutes at 4ºC. The soluble extract (0.6mL) was applied on a Ni-NTA spin column (Qiagen) following the instructions provided, washed with NPI-20 (20m M Imidazole) and eluted with NPI-500 (500m M Imidazole). Aliquots of the soluble extract, flow through from the column the final wash and the eluted fractions were analysed by luminescence activity (Figure 13) and by SDS-PAGE (Figure 14 and western blot (Figure 15).

Large scale Expression (200mL Culture)

NEB* Express l^q cells transformed with plasmid (based on pK i.e., kan resistance marker) plated onto LB agar supplemented with kanamycin (50 mg/ mL final concentration), and incubated at 30ºC for 16 hours. A single colony was inoculated into 10 mL of LB media (with antibiotics) and grown at 37*C (with shaking at 250 rpm) for 1 hour. Then the whole 10ml culture added to 200 mL of Auto-induction media (base broth: 6g Na2HP04, 3g KH2P04, 20g Tryptone, 5g Yeast Extract, 5g NaCI made up to 1L with dH20 and autoclaved; sugar mix: 150m L glycerol, 12.5g glucose, 50g lactose and made up to 1L with dH20 filter sterilised through 0.45um filter. Added BmL sugar mix to 200mL of base broth in 2 L conical flasks (lOOug/ml kanamycin) and incubated at room temperature for 24 hours with shaking at 250 rpm. Aliquots (50mL) of bacterial cultures were centrifuged for 20 minutes, 4000 rpm at 4"C. Pellets were retained, weighed and stored at -80ºC. After thawing on ice, each of the bacterial cell pellets from 50 mL cultures were resuspended in 2.5 mL of periplasmic extraction buffer (30 mM Tris-base, pH 8.0, 20% sucrose and 1 mM EDTA) supplemented to a final concentration of 100 nM phenylmethylsulfonyl fluoride (PMSF) just before use. The cells were incubated on ice for 10 minutes and centrifuged at 3000 rpm for 15 minutes at 4ºC. The supernatants were collected and stored on ice, whilst cell pellets were resuspended in 1.75ml of 5 mM MgCI2 (4^*C). After incubation for five minutes on ice, bacterial cells were pelleted by centrifugation as described before, and the supernatants combined to give the periplasmic fraction.

Large Scale Affinity Chromatography with His tag

Equilibrated HisPur Co-NTA 3ml column to room temperature. Drained storage buffer by gravity. Applied 6ml 50m M Tris, 0.5M NaCI (pH 8.0) 10 mM imidazole, allowed to enter the resin bed then drained by gravity applied another 6ml 50m M Tris 0.5M NaCI 10 mM imidazole allow to enter the resin bed then drained by gravity. Added clear filtered periplasmic extract to 3ml HisPur Co-NTA and allow to pass by gravity. Retained the Flow Through FT), washed with 5 x 5m L 50mM Tris pH8.0, 0.5M NaCI 10 mM imidazole. Repeated wash and collected the 1ml of the Final wash (FW). ElutedlO x 1.4ml 50 mM Tris pH8.0, 0.5M NaCI, 500mM imidazole collected El-10. The soluble periplasmic protein enriched by immobilized metal ion affinity chromatography (IMAC) on cobalt resin and the eluted fractions analyzed for nanoLuc activity, pooled and 5xl0⁷ lux activity aliquots stored in 30% glycerol at -80°C until required.

Example 3

Alemtuzumab treatment.

People taking part in a long-running population study of MS had received five daily 12mg infusions at baseline and three daily 12mg infusions where administered twelve months later. Following disease activity (typically 1 relapse and/or 2 unique lesions defined as either new/enlarging T2 hyper-intense and/or gadolinium-enhancing brain and/or spinal cord lesions via magnetic resonance imaging), additional cycles of three daily 12 mg infusions could be administered at least 12 months apart.

Samples were obtained from the Welsh Neuroscience Research Tissue Bank (WNRTB) in relation to the aforementioned people. Assays were applied to bioarchived serum samples from 32 PwMS (patients with MS) who had all received three or more cycles of alemtuzumab. Analysis of ADA was performed blinded to clinical and laboratory data from the WNRTB. Individuals had received either three (n=24), four (n=3), five (n=4) or six (n=l) cycles of alemtuzumab. Absolute lymphocyte counts, taken from routine laboratory reports where available, from time points immediately before, and 1-2 month after each infusion were used to calculate relative depletion rates. Apparent lymphocyte depletion was defined as ³ 35% reduction in absolute lymphocyte count pre- to post infusion and/or depletion below the lower limit of normal.

Ant!-Alemtuzumab Drug Antibody Assay

Baseline serum samples were available for 17/32 PwMS and, therefore, an anti- alemtuzumab drug antibody assay was performed using the following methodology: the IgG fraction of the sera (20 ul ~has 150-300ug IgG average range) if associated with ADA, is disassociated by the addition of 50 ul 0.1M Glycine at pH 2.7 for 5 minutes at ambient temperature. An aliquot of O.lmL of the GloBody (based on alemtuzumab VH and VL sequences with a dual nanoluciferase linker and prepared using the method of Examples 1 and 2) AlemGloBody (~5 x10⁷ lux units) in PBS with 0.05% Tween 20 (PBST) is mixed with 6 ul Tris 0.1M pH 9.0 and added to the acidic serum sample. This neutralises the acid and in the presence of an excess of AlemGloBody allows anti-alemtuzumab antibody to bind the GloBody. The addition of 60 ul Protein G Sepharose 50% slurry in PBS followed by the addition of 1.064 ml PBST allows the capture of IgG in the sample along with bound GloBody (30ul settled Protein G sepharose (AbCam, abl93259) ~ has the capacity to capture >600 ug IgG). The Protein G sepharose is washed to remove unbound AlemGloBody and transferred to microspin columns for ease of removing residual wash buffer and the bound complexes eluted as follow. To each 1.5 ml collection, tube 6.0 ul of 1M Tris pH9.0 is added. To the Protein G sepharose resin in the spin column 50ul 0.1M Glycine pH2.7 added ensuring the resin is in contact with the acidic solution and then placed into a collection tube containing 6 ul 0.1M Tris pH 9.0 for 1 minute before centrifugation 13000 rpm fori minute. This ensures that the IgG bound to the Protein G is released along with the retained GloBody. The neutralized eluate is assayed by adding 15 ul of neutralised eluate IgG-AlemGloBody complex in triplicate into 0.1 ml nanoLuc buffer consisting of 49 ul Glo Buffer and 49 ul PBS 2ul furimazine substrate as provided by Promega and nanoluciferase activity determined after 10 minutes at ambient temperature. In this example a putative blank sera sample (Sigma H4522 from human male AB plasma, USA origin, sterile-filtered) was assayed with and without a spike of a known human anti-alemtuzumab monoclonal antibody at 50ug/ml (Bio Rad Cat No HCA175) as shown in Figures 4 to 7.

Example 4

The polypeptide sequences of emicizumab variable regions (VH/VL) were accessed from DrugBank via accession number DB13923. The VL sequence is common to both arms and the VH differ. The VH/VL polypeptides assembled as a single chain Fv (scFv) and reverse translated and codon optimised for E.coli expression using The Sequence Manipulation Suite and the scFv synthetic constructs synthesized. The assembly of Globody based on alemtuzumab has been described earlier. Using the same approach Emici F(IXa) Globody and Emici F(X) Globody constructs were assembled.

Plasmid pKamp, previously constructed in our laboratory based on pK (modified by replacing the kanamycin resistance with ampicillin resistance). To produce Emici GloBody, E. coli NEB Express Iq (NEB) cells were transformed with the plasmid and plated onto LB agar supplemented with carbenicillin (100 mg/mL). A single colony NEB Express Iq transformed with the plasmid added to 200 mL Overnight Express Auto-Induction media (Studier, 2005) with 200 mg/mL carbenicillin, grown with shaking at 275 rpm for 24 hr. 50ml aliquots of culture pelleted at 877 x g for 20 min and the pellet retained. The pellets were resuspended in 30mM Tris pHB.O 20% sucrose w/v, 10 mL/g pellet, on ice for 20 minutes. The resuspension transferred 1.8 mL/2 mL Eppendorf tubes and centrifuged at 10,000 x g at 4ºC for another 20 min and the supernatant retained. Each pellet was then resuspended in 1.8 mL 5 mM MgCI2 on ice for 10 minutes and centrifuged at 10,000 x g at 4*C for another 20 min and the supernatants pooled and filtered through a

0.45 mm filter. The nanoluciferase activity in the pooled supernatants determined and ~7 million lux generating units aliquots (in 15mM Tris pH8.O, 10% sucrose w/v and 2.5 mM MgCI2) prepared and stored at -80'C. The assay with serum using either Emici Globody were carried out as described for the Alem GloBody as in Examples 1 and 2.

Example 5

The amino acid sequence of the variable domains of infliximab and adalimumab were accessed from Protein Data Base pdb 4g3y and DrugBank via accession number and DB00051 respectively and scFv assembled with the antibody variable domains in VH-VL orientation as described for alemtuzumab. Synthetic DNA sequences of Infliximab and Adalimumab antibody scFv's and assembled as pK InflixiGlobody and pK AdaliGlobody following the process described for assembling pKAIemGlobody in Examples 1 and 2. Cloning and protein expression and assay were carried out as described for AlemGloBody in Examples 1 and 2.

The technology should be applicable to any conventional therapeutic antibody that has a V_H, or VL or combinations of VH and VL domains (i.e., single domain VHH, SCFV, Fab, IgG, CAR-T cell and any format of bispecific antibodies). Moreover, it should be possible to modify this platform to accommodate non-biologic drugs such as Bicycle™ drugs for ADA testing.

The assay format described above and in the figures is but an example, and may be modified for use on different formats and devices. It is noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the," include plural referents unless expressly and unequivocally limited to one referent. As used herein, the term "include" and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or other items that can be added to the listed items.

Upon studying the disclosure, it will be apparent to those skilled in the art that various modifications and variations can be made in the devices and methods of various embodiments of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification and practice of the embodiments disclosed herein. It is intended that the specification and examples be considered as examples only. The various embodiments are not necessarily mutually exclusive, as some embodiments can be combined with one or more other embodiments to form new embodiments. CITATIONS

1. Engineering recombinant antibodies

Markiv A, Beatson R, Burchell J, Durvasula RV, Kang AS. Expression of recombinant multicoloured fluorescent antibodies in gor -/ trxB- E. coli cytoplasm. BMC Biotechnol. 2011 Nov 30;11(1):117.

Markiv A, Anani B, Durvasula RV, Kang AS. Module based antibody engineering: a novel synthetic REDantibody. J Immunol Methods. 2011 Feb l;364(l-2):40-9.

US 9,566,353 Fluorescent fusion polypeptides and methods of use Markiv A, Durvasula RV, Kang AS

US 8,877,898 Fluorescent fusion polypeptides and methods of use Markiv A, Durvasula RV, Kang AS

2. Anti-Drug-Antibody Assays (overview and reccommendations)

Mire-Sluis AR, Barrett YC, Devanarayan V, Koren E, Uu H, Mala M, Parish T, Scott G, Shankar G, Shores E, Swanson SJ, Taniguchi G, Wierda D, Zuckerman LA. Recommendations for the design and optimization of immunoassays used in the detection of host antibodies against biotechnology products. J Immunol Methods. 2004 Jun;289(l-2):1-16.

Shalini Gupta, Viswanath Devanarayan, Deborah Finco, George R. Gunn III, Susan Kirshner, Susan Richards, Bonita Rup, An Song, Meena Subramanyam. Recommendations for the validation of cell-based assays used for the detection of neutralizing antibody immune responses elicited against biological therapeutics. Journal of Pharmaceutical and Biomedical Analysis, Volume 55, Issue 5, 2011, pp. 878-888

Meenu Wadhwa, Ivana Knezevic, Hye-Na Kang, Robin Thorpe. Immunogenidty assessment of biotherapeutic products: An overview of assays and their utility. Biologicals, Volume 43, Issue 5, 2015, pp. 298-306

United States Patent Application Publication (10) Pub. No.: US 2015/0253338A1

ANT-DRUG ANTIBODY ASSAY Hoesel et al. (43) Pub. Date:

US 20150253338A1 Sep. 10, 2015

Nath N, Flemming R, Godat B, Urh M. Development of NanoLuc bridging immunoassay for detection of anti-drug antibodies.

J Immunol Methods. 2017 Nov;450:17-26. doi: 10.1016/j.jim.2017.07.006. Epub 2017 Jul 19.

Development of NanoLuc bridging immunoassay for detection of anti-drug antibodies.

Claims

1. A single polypeptide comprising a nanoluciferase domain that possesses a N-terminus and a C-terminus, a first antibody domain linked to the N-terminus by a covalent linker, and a second antibody domain linked to the C-terminus by a second covalent linker.

2. A polypeptide according to claim 1 wherein at least one of the covalent linkers comprises no more than 5 amino acids.

3. A polypeptide according to either claim 1 or 2 wherein at least one of the nanoluciferase domain, the first antibody domain, and/or the second antibody domain comprises an affinity tag.

4. A polypeptide according to any preceding claim wherein the first antibody domain and the second antibody domain specifically bind to a single target molecule.

5. A polypeptide according to any preceding claim wherein the first antibody domain and the second antibody domain bind to more than one molecule.

6. A polypeptide according to any preceding claim wherein the first antibody domain comprises a variable heavy chain (VH) comprising an N-terminus.

7. A polypeptide according to any preceding claim wherein the second antibody domain comprises a variable light chain (VL) comprising a C-terminus.

8. A polypeptide according to any of claims 1 to 5 wherein the first antibody domain comprises a VL comprising an N-terminus.

9. A polypeptide according to claim 8 wherein the second antibody domain comprises a VH comprising a C-terminus

10. A polypeptide according to any preceding claim wherein the C-terminus of first antibody domain and the N-terminus of the second antibody domain are separated by a distance of no less than 30 Å and no more than 40 Å.

11. A polypeptide according to any preceding claim wherein the nanoluciferase domain comprises at least a portion of a nanoluciferase protein sufficient to have nanoluciferase activity.

12. A polypeptide according to any preceding claim wherein the nanoluciferase domain comprises at least a portion of more than one nanoluciferase protein wherein at least one of the portions is sufficient to have nanoluciferase activity.

13. A polypeptide according to claim 12 wherein the nanoluciferase domain comprises two nanoluciferase portions.

14. A polypeptide according to either claim 12 or 13 wherein the nanoluciferase portions are in tandem.

15. A composition comprising at least one polypeptide according to any of claims 1 to 14.

16. A composition according to claim 15 comprising a first polypeptide according to any of claims 1 to 14 and a second polypeptide according to any of claims 1 to 14.

17. A polynucleotide that encodes any one of the polypeptides of claims 1 to 14.

18. A method for detecting an analyte, the method comprising: contacting a sample comprising the analyte with a polypeptide according to any of claims 1 to 14 wherein at least one of the first antibody domain and the second antibody domain specifically binds to the analyte; removing unbound polypeptides; and detecting a luminescent signal produced by the polypeptide specifically bound to the analyte, thereby detecting presence of the analyte in the sample.

19. A method according to claim 18 further comprising immobilizing at least a portion of the sample on a substrate.

20. A method according to either claim 18 or 19 wherein the analyte is an anti-drug antibody.