WO2017137613A1

WO2017137613A1 - Immunoassay for detection and monitoring of inflammatory responses

Info

Publication number: WO2017137613A1
Application number: PCT/EP2017/053086
Authority: WO
Inventors: Kim Suzanne Midwood; David Pritchard
Original assignee: Nascient Limited
Priority date: 2016-02-10
Filing date: 2017-02-10
Publication date: 2017-08-17
Also published as: GB201602413D0

Abstract

The present disclosure relates to an immunoassay for the detection and monitoring of inflammatory and/or autoimmune diseases. The immunoassay detects the presence of a tenascin in a sample and comprises a capture and conjugate antibody, of which at least one antibody has a human VH specific to the Tenascin. Also provided is a kit comprising said immunoassay.

Description

IMMUNOASSAY FOR DETECTION AND MONITORING OF INFLAMMATORY RESPONSES

The present disclosure relates an immunoassay suitable for the detection and monitoring of inflammatory and/or autoimmune diseases, such as rheumatoid arthritis and kits for use in the said assay.

BACKGROUND

Rheumatoid Arthritis (RA) is a progressive autoimmune disorder which can be debilitating. It is estimated that RA affects 0.5-1.0% of the global adult population. The symptoms of RA include swollen and painful joints. Each patient presenting symptoms which vary in severity. In addition the response to treatment varies considerably across the patient population.

RA is diagnosed using a defined set of clinical features, including a selection of biomarkers: such as auto-antibodies against citrullinated peptides (anti-CCP) and rheumatoid factor (RF). After diagnosis C-reactive protein (CRP) levels and erythrocyte sedimentation rate (ESR) are employed to measure disease progression.

The pathology of RA is complex. It is characterised by a chronic synovial inflammation and destruction of joint cartilage and bone, which is mediated by the recruitment and invasion of immune cells into the acellular synovium (the joint lining). This cellular invasion is thought to be a result of the production of a variety of cytokines, matrix-degrading enzymes and small molecule mediators such as prostaglandins and nitric oxide. A selection of immune cells, including CD4⁺ T cells, B cells and macrophages, are activated by certain synoviocyte cells which exhibit macrophage and fibroblast-like properties. The RA synoviocytes also recruit surrounding cells.

Whilst the healthy synovium protects the joints and facilitates joint movement; in RA, the synoviocytes show increased proliferation, the ability to proliferate in anchorage-independent conditions and defective contact inhibition. This results in a hyperplastic intimal lining and the formation of aggressive fronts of tissue called pannus, which lead the destruction of the articular structures within the joint.

Thus the synoviocytes in RA have are thought to be oligoclonality, particularly the synoviocytes involved in the pannus regions. In contrast to RA synoviocytes, synoviocytes taken from osteoarthritis patients (OA) show normal behaviour. This has resulted in the hypothesis that the expanding synovium is a locally invasive tumour.

The destruction of cartilage and joint tissue in RA involves a complicated interaction of proinflammatory and anti-inflammatory signalling, which recruits many different immune cells. The resulting pathology is particularly challenging to understand and treat. RA treatments are multifaceted, with a variety of different targets. The responses to treatment ranges from near elimination of disease signs and symptoms in some patients to almost complete unresponsiveness in others. Currently, there is a significant demand for an assay which can diagnose RA and perhaps subsets of patients, within the general category of patients with RA. It would also be useful to have an assay that provides information about a patient's prognosis and progression to allow informed decisions to made in relation to suitable therapies.

The present inventors have data to suggest that the employing at least a VH from a fully antibody specific to a tenascin polypeptide may provide equivalent or better results when employed in an immunoassay to results generated with current commercially available kits. SUMMARY OF THE PRESENT DISCLOSURE

Thus the present disclosure provides an immunoassay, for the detection of a Tenascin in a biological sample, comprising at least a capture antibody and a conjugate antibody, wherein at least one of said antibodies has a human VH specific to the Tenascin. In one embodiment the sample is an ex vivo patient sample.

In one embodiment the immunoassay is a method, for the detection of a Tenascin in a biological sample, comprising at least a capture antibody and a conjugate antibody, said method comprising the step of contacting an ex vivo patient derived sample with said antibodies, wherein at least one of said antibodies has a human VH specific to the Tenascin.

The immunoassay of the present disclosure is suitable for identifying levels of Tenascin (or specific forms thereof) indicative of an autoimmune disease and/or an inflammatory disease, such as rheumatoid arthritis.

In one embodiment the immunoassay is for identifying the presence or levels of Tenascin (or specific forms thereof) indicative chronic inflammatory responses.

In one embodiment the capture antibody is specific to the Tenascin, for example is specific to a region of Tenascin selected from the group comprising domain B, EGF-like repeats, FBG domain (in particular a conserved portion, section, fragment thereof), FNIII 1-3 region (in particular a conserved section thereof), such as the FBG domain and FNIII 1-3 region, in particular the FBG domain.

In one embodiment the conjugate antibody is specific to the capture antibody.

In one embodiment the conjugate antibody is specific to Tenascin, for example is specific to the FBG domain of Tenascin.

In one embodiment the capture antibody is specific to an FBG region in the Tenascin.

In one embodiment the Tenascin is Tenascin C.

In one embodiment the at least one human VH is specific to the FBG region.

In one embodiment the at least one human VH is specific to the FBG region of Tenascin-C.

In one embodiment the human VH is selected from a sequence shown in SEQ ID NO: 4, 12, 19,

In one embodiment the capture antibody is from a non-human mammal, for example a mouse, a rat, a rabbit or a camelid (others non-human mammals include sheep, cows, llamas, camels, goats, donkey and pigs), for example one or more, such as a six CDRs from the murine monoclonal antibody 9F8, in particular the VH domain and VL domain from said antibody.

In one embodiment the conjugate antibody comprises a human variable domain, for example the antibody or binding domain (VH and VL) thereof is fully human.

In one embodiment the conjugate antibody is from a non-human mammal, for example mouse, rat, rabbit or camelid (others non-human mammals include sheep, cows, llamas camels, goats, donkey and pigs), for example one or more, such as a six CDRs from the murine monoclonal antibody 9F8, in particular the VH domain and VL domain from said antibody.

In one embodiment the capture and/or conjugate antibody is humanized.

In one embodiment both the capture antibody and the conjugate antibody comprise a human VH, for example a VH of SEQ ID NO: 12. In one embodiment both the capture antibody and the conjugate antibody comprise a VH with the same amino acid sequence, for example a VH disclosed herein such as SEQ ID NO: 12.

In one embodiment the immunoassay of the present disclosure is capable of detecting and/or differentiating oligomeric Tenascin molecules.

In one embodiment the immunoassay of the present disclosure is capable of detecting

Tenascin and citrullinated forms thereof, for example as disclosed in WO2015/104564 incorporated herein by reference.

In one embodiment the assay of the present disclosure is capable of detecting and/or differentiating a Tenascin form associated with pathogenesis of rheumatoid arthritis.

In one embodiment the immunoassay is an ELISA assay.

In one embodiment the immunoassay is a self-ELISA.

In one embodiment there is provided a kit for use in an immunoassay defined herein, comprising a capture antibody and a conjugate antibody wherein at least one of said antibodies has a human VH specific to the Tenascin, in particular as described herein.

p

In one embodiment the immunoassay according to the present disclosure is not specific for citrullinated Tenascin, for example an antibody employed in the present immunoassay is not specific to citrullinated Tenascin-C.

In one embodiment the immunoassay according to the present disclosure does not identify the presence or levels of patient antibodies specific to citrullinated Tenascin (such as citrullinated Tenascin C).

In one independent aspect of there is provided an antibody or binding fragment as specifically disclosed herein or a composition comprising the same, for example for use in diagnosis (including in vivo analysis) and/or treatment. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a graph of serum concentration in a sample against the percentage of maximum absorbance generated by each assay, demonstrating the linearity of each reaction. IBL is a commercially available assay available from IBL (Tenascin-C large (FNIII-B) Assay Kit Code No: 27767). 9F8 is a murine antibody specific to the FNIII

1-3 region of Tenascin (see for example Hu et el Neuroscience 2015, 284(0) : 412- 421). B12-VH refers to a human antibody C3 disclosed herein.

Notably the combination of 9F8 as the capture antibody and B12-VH as the conjugate antibody provides a linear response. In contrast the commercially available assay does not provide a linear response, even when 9F8 is employed as the capture antibody.

Figure 2 shows a graph of absorbance response generated in two analysis's by incubation of sera samples (n=40) with 9F8 as the capture antibody and B12-VH as the conjugate antibody. Also shown on the plot is the mean of these two plots.

Figure 3 shows a graph of absorbance response generated by incubation of sera samples

(n=20) against B12 (capture antibody):9F8 (conjugate antibody), with IBL (FNIII-C) and 9F8:B12-VH data for comparison.

Figure 4 shows normalised results for Figure 3

Figure 5 shows a graph of absorbance response generated by incubation of sera samples

(n=20) against the B12-VH (capture antibody) :9F8 (conjugate antibody, with IBL

(FNIII-C) and 9F8:B12-VH data for comparison

Figure 6 shows normalised results for Figure 5

Figure 7 shows a graph of absorbance response generated by incubation of sera samples

(n=40) against the IBL (FNIII-B) assay kit, with IBL (FNIII-C) and 9F8 (capture antibody):B12-VH (conjugate antibody) assay data for comparison

Figure 8 shows normalised results for Figure 7

Figure 9 shows complete sera testing with self-sandwich assay employing a B12 antibody or

VH therefrom.

Figure 10 shows a ROC curve comparison of 9F8 (capture antibody): B12-VH (conjugate antibody) and IBL (FNIII-C) assay. After about 0.5 on the X-axis the IBL assay is incapable of discriminating normal samples from RA samples. In contrast the 9F8:B12-VH assay is capable of discriminating sample at all points bar one (at about 0.9).

Figure 11 shows a ROC curve of serum testing including single antibody sandwich assays

Also in included in this specification is a sequence listing.

DETAILED DISCLOSURE

Generally antibodies employed in detection, assays and diagnostics are generated from human hosts because there are no issues in relation to immunogenicity for in vitro analysis. Surprisingly the present inventors have established that a fully human VH region employed in an immunoassay of the present disclosure provides better results than employing commercially available murine antibodies. Further the analysis employing a human VH provides at least comparable results to currently available commercial kits for the analysis of Tenascin (such as the kit available from IBL). What is more employing a human VH in the analysis of Tenascin, may allow identification of nuances in the data, which are not detected by currently available commercial kits. The immunoassays of the present disclosure may provide more linear correlation than one or more currently available kits.

Furthermore, the detectable ranges vary widely between currently available assay kits, as does the particular splice being detected. As a result, there is no currently available "one size fits all" assay kit which can be used for all forms of tenascin. In contrast, the immunoassay of the present disclosure may be able to assess multiple forms of tenascin.

The information generated from the immunoassays of the present disclosure may have significant implications for the diagnosis and analysis of the prognosis of inflammatory diseases, such as RA.

A further surprising aspect of the disclosure herein is that an ELISA assay comprising the same human VH in the capture antibody and conjugate antibody (referred to herein as a self- sandwich/self-ELISA/Single antibody sandwich) gave good discrimination between healthy samples and RA samples. This may allow, for example for oligomeric forms of Tenascin to be analysed. Immunoassay as employed herein refers to is a biochemical test that measures the presence or concentration of a macromolecule, in this instance Tenascin, in a sample using an antibody.

In one embodiment the immunoassay of the present disclosure is a sandwich assay. In one embodiment an immunoassay of the present disclosure is an ELISA assay. ELISA assay as employed herein refers to an enzyme-linked immunosorbent assay. ELISA assays are well known in the art. Capture antibody as employed herein refers to an antibody which binds the Tenascin. In one or more embodiments the capture antibody is labelled, for example wherein binding to the antigen (in this case Tenascin) can be detected as the colour or change in colour of the label.

Conjugate antibody as employed herein refers to an antibody which is conjugated to a label, wherein the antibody in the conjugate binds the antigen or binds to and epitope on the capture antibody. Binding of the conjugate antibody is generally detected as the colour or change in colour of the label.

In one embodiment one or more photochromic molecules or compounds are employed as labels, for example a fluorescent label is employed. Alternatively, labels such radiolabels can be employed. However, radiolabels are generally less convenient to use than fluorescent labels.

Fluorescent labels can be detected by measuring absorbance or a change in absorbance.

Tenascin as employed herein refers to the family of extracellular matrix glycoproteins, which includes Tenascin C, Tenascin R, Tenascin X and Tenascin W, in particular a human form of said protein or proteins.

Tenascin C is a large hexameric protein of 1.5 million Da. Each chain comprises different domains, including an assembly domain (TA), EGF-like repeats (EGF-L), fibronectin type Ill-like repeats (TNIII) and the fibrinogen-like globe (FBG).

Human tenascin C has the Uniprot number P24821. Human tenascin R has the Uniprot number Q992752. Human Tenascin X has the Uniprot number P22105. Antibody as employed herein refers to an immunoglobulin, a molecule comprising an immunoglobulin, such as a multispecific antibody molecule, or a binding fragment thereof.

A "binding fragment" as employed herein refers to a fragment capable of binding a target peptide or antigen with sufficient affinity to characterise the fragment as specific for the peptide or antigen. "Binding fragments" include but are not limited to Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, single domain antibodies (VH or VLs), scFv, Fv, bi, tri or tetra-valent antibodies, Bis-scFv, diabodies, triabodies, tetrabodies and epitope-binding fragments of any of the above (see for example Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews - Online 2(3), 209-217).

Linkers suitable for use in recombinant antibodies or binding fragments include but are not limited to hinge linkers as shown in SEQ ID NOs: 160 to 168, flexible linkers as shown in SEQ ID NOs: 169 to 208 and rigid linkers PPP and as shown in SEQ ID NOs: 209 210.

"Multispecific antibody molecule" as employed herein refers to a molecule with the ability to specifically bind at least two distinct antigens, for example different antigens. In one embodiment the multispecific molecule is a bispecific, trispecific or tetraspecific molecule, in particular a bispecific or trispecific molecule.

VH as employed herein refers to a variable heavy domain sequence, i.e. a heavy framework in combination with CDRHl, CDRH2 and CDRH3, for example B12-VH refers to the variable heavy domain of the antibody B12, as shown in SEQ ID NO: 12.

VL as employed herein refers to a variable light domain sequence, i.e. a light framework in combination with CDRL1, CDRL2 and CDRL3.

The disclosure also extends to use of a variant of a variable domain which is disclosed herein. A variant in this context is intended to refer to where one, two, three, four or five amino acids in a naturally occurring sequence have been replaced or deleted, for example to optimize the properties of the domain such as by eliminating undesirable properties but wherein the characterizing feature(s) of the domain is/are retained. Examples of modifications are those to remove glycosylation sites, GPI anchors, or solvent exposed lysines. These modifications can be achieved by replacing the relevant amino acid residues with a conservative amino acid substitution.

reference.

Highly similar as employed herein is intended to refer to an amino acid sequence which over its full length is 95% similar or more, such as 96, 97, 98 or 99% similar. The present disclosure extends to antibodies having at least 95% sequence identity, such as 96, 96, 98 or 99% identity to the sequences disclosed herein.

Antibodies generated against the antigen polypeptide (in this case tenascin) may be obtained, where immunisation of an animal is necessary, by administering the polypeptides to an animal, preferably a non-human animal, using well-known and routine protocols, see for example Handbook of Experimental Immunology, D. M. Weir (ed.), Vol 4, Blackwell Scientific Publishers, Oxford, England, 1986). Many warm-blooded animals, such as rabbits, mice, rats, sheep, cows, camels or pigs may be immunized. However, mice, rabbits, pigs and rats are generally most suitable.

Monoclonal antibodies may be prepared by any method known in the art such as the hybridoma technique (Kohler & Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today, 4:72) and the EBV- hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp77-96, Alan R Liss, Inc., 1985).

Antibodies may also be generated using single lymphocyte antibody methods by cloning and expressing immunoglobulin variable region cDNAs generated from single lymphocytes selected for the production of specific antibodies by, for example, the methods described by Babcook, J. et al., 1996, Proc. Natl. Acad. Sci. USA 93(15):7843-78481; WO92/02551; WO2004/051268 and WO2004/106377.

The antibodies for use in the present disclosure can also be generated using various phage display methods known in the art and include those disclosed by Brinkman et al. (in J. Immunol. Methods, 1995, 182: 41-50), Ames et al. (J. Immunol. Methods, 1995, 184:177-186), Kettleborough

Humanised (which include CDR-grafted antibodies) as employed herein refers to molecules having one or more complementarity determining regions (CDRs) from a non-human species and a framework region from a human immunoglobulin molecule (see, e.g. US 5,585,089; WO91/09967). It will be appreciated that it may only be necessary to transfer the specificity determining residues of the CDRs rather than the entire CDR (see for example, Kashmiri et al., 2005, Methods, 36, 25-34). Humanised antibodies may optionally further comprise one or more framework residues derived from the non-human species from which the CDRs were derived.

As used herein, the term 'humanised antibody molecule' refers to an antibody molecule wherein the heavy and/or light chain contains one or more CDRs (including, if desired, one or more modified CDRs) from a donor antibody (e.g. a murine monoclonal antibody) grafted into a heavy and/or light chain variable region framework of an acceptor antibody (e.g. a human antibody). For a review, see Vaughan et al, Nature Biotechnology, 16, 535-539, 1998. In one embodiment rather than the entire CDR being transferred, only one or more of the specificity determining residues from any one of the CDRs described herein above are transferred to the human antibody framework (see for example, Kashmiri et al., 2005, Methods, 36, 25-34). In one embodiment only the specificity determining residues from one or more of the CDRs described herein above are transferred to the human antibody framework. In another embodiment only the specificity determining residues from each of the CDRs described herein above are transferred to the human antibody framework.

When the CDRs or specificity determining residues are grafted, any appropriate acceptor variable region framework sequence may be used having regard to the class/type of the donor antibody from which the CDRs are derived, including mouse, primate and human framework regions. Suitably, the humanised antibody according to the present disclosure has a variable domain comprising human acceptor framework regions as well as one or more of the CDRs provided herein.

Examples of human frameworks which can be used in the present disclosure are KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (Rabat et al., supra). For example, KOL and NEWM can be used for the heavy chain, REI can be used for the light chain and EU, LAY and POM can be used for both the heavy chain and the light chain. Alternatively, human germline sequences may be used; these are available at: http://vbase.rnrc-cpe.carn.ac.uk/

In a humanised antibody of the present disclosure, the acceptor heavy and light chains do not necessarily need to be derived from the same antibody and may, if desired, comprise composite chains having framework regions derived from different chains.

The framework regions need not have exactly the same sequence as those of the acceptor antibody. For instance, unusual residues may be changed to more frequently-occurring residues for that acceptor chain class or type. Alternatively, selected residues in the acceptor framework regions may be changed so that they correspond to the residue found at the same position in the donor antibody (see Reichmann et al., 1998, Nature, 332, 323-324). Such changes should be kept to the minimum necessary to recover the affinity of the donor antibody. A protocol for selecting residues in the acceptor framework regions which may need to be changed is set forth in WO91/09967. Fully human antibody as used herein is intended to describe antibodies in which the variable regions and the constant regions (where present) of both the heavy and the light chains are all of human origin, or substantially identical to sequences of human origin, not necessarily from the same antibody.

Examples of fully human antibodies may include antibodies produced, for example by the phage display methods described above and antibodies produced by mice in which the murine immunoglobulin variable and optionally the constant region genes have been replaced by their human counterparts eg. as described in general terms in EP0546073 Bl, US 5,545,806, US 5,569,825, US 5,625,126, US 5,633,425, US 5,661,016, US5,770,429, EP 0438474 and EP0463151.

Antibody 163 02 Dll (derived from 2 A5)

EXAMPLES ABBREVIATIONS

B12-B12 refers to full-length IgG B12 as the capture antibody and as the conjugate antibody.

Series 2 -is complete sera testing with B12-VH -B12-VH self sandwich (B12-VH is C3 variable heavy domain).

Plate Coating:

The plate coating procedure was based on using pH 9.6 bicarbonate coating buffer and a coating concentration of 10μg/ml. 100μΙ of coating solution was dispensed into the wells of a high-binding microtitre plate (Biomat, Sir), and incubated for 24hrs at room temperature. The buffer was decanted, the plate was blotted dry and 100μΙ blocking buffer (lOmM PBS, 1% BSA) was dispensed into the well before another 24hr incubation time at room temperature. The blocking buffer was decanted, the wells were thoroughly washed x3 with overcoat buffer (lOmM PBS, 0.1% BSA, 5% sucrose) and the plate was placed in a 37°C incubator for at least 2 hrs until completely dry. The plate was then stored in a sealed bag with desiccant at 2-8°C until use.

Conjugation Procedure:

Lightning- Link® Horseradish Peroxidase kits (LL-HRP) (Innova Biosciences Ltd.) were chosen for use due to the simplicity, time efficiency and reported reproducibility of the procedure. 20 μg of antibody in solution, mixed with LL-modifier reagent, as per instruction, was placed in the glass vial of Lightning- Link® mix. The solution was disturbed to allow full resuspension of the freeze-dried conjugation mix, and allowed to incubate at room temperature overnight, before the appropriate amount of LL-quencher reagent was added to the glass vial. The resulting conjugate stock solution was then stored at 2-8°C to be diluted to working strength as desired for later use.

Antigen Solutions:

Standard solutions were made up to concentration using Human Tenascin-C Purified Protein (Merck Millipore Corporation, Cat No: CC065) in a lOmM PBS 1%, BSA buffer. This was the only full protein extraction available on market at initiation of project work. The protein had been extracted from Human glioma cell line (U251) after induced expression, and is quoted to migrate at 280-300kDa by SDS-PAGE analysis.

Standard Assay Format:

100 μΐ of antigen solution was placed in the wells of a coated plate and incubated at room temperature for 1 hr. The plate was decanted, and washed x3 with wash buffer (FMHBP160), ensuring that each well was completely filled each time. The plate was blotted dry. ΙΟΟμΙ of conjugate solution (0.1ug/ml, in lOmM PBS, 1% BSA, 0.5% Tween20) was placed into each well and the plate was incubated, once again, at room temperature for 1 hr. The plate was decanted, and washed x3 with the wash buffer, ensuring that each well was completely filled each time. The plate was blotted dry. ΙΟΟμΙ of TMB substrate solution (FMHBP130) was added to each well, and the plate was incubated at room temperature for 10 mins, or until sufficient colour had developed. ΙΟΟμΙ of stop solution (FMHBP140) was added to each well and the plate was read at A450nm on the FC plate reading using Skanit software.

Standard deviation (S.D.) and coefficient of variance (C.V.) was calculated each time. The C.V. values were monitored, and if they crept above 15% then the test was repeated.

Antigen Binding Test:

The wells of a plate were coated with either Human TNC Purified Protein (Merck Millipore) or BSA. ΙΟΟμΙ of each conjugate antibody solution (at [^g/ml], in lOmM PBS, 1% BSA) was placed in each well and incubated at room temperature for 1 hr. The plate was washed. ΙΟΟμΙ of substrate solution (FMHBP130) was placed in each well, and the plate was incubated at room temperature until sufficient colour had developed, with the incubation time noted. ΙΟΟμΙ of stop solution (FMHBP140) was added to each well, and the plate was read with a plate reader.

Conjugate Activity Tests:

Initial Free Conjugate Activity Test ΙΟΟμΙ of substrate solution (FMHBP130) was spiked with conjugate antibody by submerging the end of a pipette tip into the conjugate stock solution and then swabbing into the substrate solution, and the reaction was allowed to occur for 5 seconds before being quenched by the addition of ΙΟΟμΙ stop solution (FMHBP140). The reaction mix was then diluted up to 1ml by the addition of 800μ1 RO water before being read at A45 Onm on a spectrophotometer.

Bound Conjugate Activity Test

Conjugate antibody was coated on a plate (at [Ιμΐ/ml]), before being incubated with ΙΟΟμΙ of substrate solution (FMHBP130) at room temperature for 1 hr. ΙΟΟμΙ of stop solution (FMHBP140) was added to each well and the plate was read with a plate reader.

Further Free Conjugate Activity Test

ΙΟΟμΙ of TMB substrate solution (FMHBP130) was dispensed into an Eppendorf tube. 5μ1 of conjugate solution ([^g/ml] in lOmM PBS, 1% BSA) was added to the tube and the contents were mixed by inversion. The reaction mix was allowed to incubate for 2 minutes at room temperature before ΙΟΟμΙ of stop solution (FMHBP140) was added to the mix. The solution was then made up to 1ml by the addition of 800μ1 of RO water, mixed by inversion, and read at A450nm on a spectrophotometer.

Antibody Pair Testing Against Buffer:

Each antibody was coated on a plate, and tested against increasing concentrations of TNC solutions (0, 10, 50 and 100ng/ml), with each different antibody as conjugate (at [^g/ml] in lOmM PBS, 1% BSA). Substrate incubation times varied, to allow sufficient colour to develop. Results were graphed, and ratings were generated based on the absorbance response of each antibody pair before analysis with respect to dose response and strength of reaction.

Serum Dilution Assay:

Serum sample N2 (ProMedDx, Lie), selected after preliminary testing using the IBL (FNIII-C) assay was diluted using a double-down serial dilution with buffer (lOmM PBS, 1% BSA, 0.5% Tween 20) to 3.125% and tested against the 9F8:B12-VH and IBL (FNIII-C) assays, with the addition of a 1/50 dilution point on the IBL (FNIII-C) assay. Simultaneously, the IBL (FNIII-C) kit conjugate and plate was tested against the 9F8 coated plate and B12-VH conjugate, respectively.

Tenascin-C Spiking of Buffer and Serum:

400 μΐ of lOmM PBS, 1% BSA buffer and sera samples were made up to 500 μΐ by the addition of 100 μΐ of increasing concentrations of TNC spike solution. Spike solutions were made up using TNC purified protein and sample diluent (lOmM PBS, 1% BSA, 0.5% Tween20) at concentrations of 0, 25, 50, 75, 100, 200ng/ml. The spiked samples were then simultaneously tested against the 9F8:B12-VH and IBL (FNIII-C) assays.

Tenascin-C Spiking of Buffer and Serum:

400μ1 of lOmM PBS, 1% BSA buffer and sera samples were made up to 500μl by the addition of ΙΟΟμΙ of increasing concentrations of TNC spike solution. Spike solutions were made up using TNC purified protein and sample diluent (lOmM PBS, 1% BSA, 0.5% Tween20) at concentrations of 0, 25, 50, 75, 100, 200ng/ml. The spiked samples were then simultaneously tested against the 9F8:B12-VH and IBL (FNIII-C) assays.

Serum Samples: A selection of serum samples (n=40) (ProMedDx, Lie.) were used to carry out the initial sera testing. Disease state samples (n=20) were diagnosed based on physicians diagnosis and provided in 1ml aliquots; where normal samples (n=20), provided at a volume of 3 - 5ml, were screened for viral reactivity using FDA approved methods and a clean physical examination results.

Serum was tested on IBL (FNIII-C) and (FNIII-B) kits (protocols available online,), purchased from Takara Bio, Inc. (Cat. No: 27751A and 27767A, respectively), as well as against a selection of antibody pairs. The results of the 9F8:B12-VH and IBL (FNIII-C) assays were used as comparison when performing any analysis to somewhat compensate for the lack of suitable standard solutions.

ROC Curve analysis was generated using Analyse-it software (Analyse-it Software, Ltd).

Test for Native Tenascin-C Antibody in Sera:

Plates were coated with TNC purified protein and incubated with sera samples for 1 hr, before washing and blotting dry. The plates were then incubated with ΙΟΟμΙ of w/s anti-Human IgG-HRP conjugate (FCCP620) from the ASD anti-CCP EIA (FCCP600) for 1 hr, before washing and blotting. ΙΟΟμΙ ofTMB substrate (FMHBP130) was placed in each well and incubated at room temperature for 105 mins to allow colour to develop. ΙΟΟμΙ of stop solution (FMHBP140) was added and the plate was read at A450nm on the plate reader.

Self- Sandwich of the B12 Antibodies:

Plates were coated with each of the antibodies B12 and B12-VH. ΙΟΟμΙ of sera samples, at a 1/8 dilution, was tested against the same conjugate as capture antibody, resulting in B12:B12 and B12- VH:B12-VH assays. The assays were run with a substrate incubation time of 45 mins.

Conjugate Performance

The LL-HRP kits were chosen for a variety of reasons, including the economy and ease of use, and due to the elegance of HRP as a small conjugate enzyme with a variety of available substrates, which are both fast-acting and affordable.

Example 1

Initial testing of the ProMedDx samples was completed for at least 20 samples (10 normal, 10 RA confirmed) with each of a number of antibody pairs.

Error bars are included, with the "error" of each being the S.D. of each sample performance in assay. Where the mean results of two different assay runs are represented, the error bars are the mean S.D. produced by the sample, in place of representing the full spread of each sample alongside the mean each time.

Standard solutions were found to be non-commutable with TNC in serum, with significantly weaker reactions than those seen in serum. For example, in the case of the 9F8:B12-VH assay, where sample N2 gives an absorbance value of 0.74 units, and a standard solution made up to be[24 ng/ml] TNC gives an absorbance value of 0.34 units. This would make the calculated concentration of TNC in sample N2 to be 402.60 ng/ml (concentrations calculated using the IBL assay protocol). The mean TNC concentration in normal serum is quoted as being 20.34 ng/ml², suggesting that this result is extremely exaggerated.

The results of the IBL (FNIII-C) and 9F8:B12-VH assays were used as a basis for comparison between preliminary assay results.

"Normalised" graphs present each absorbance value as a percentage of the maximum absorbance achieved by each assay, to adjust for variance in sensitivity and N.S.B between assays. RESULTS

The data generated in shown in Figures 1 to 11.

Figure 1 suggests that the an assay based on murine capture antibody 9F8 and the conjugate antibody B12 (or the VH form thereof) provides a more linear correlation to the levels of Tenascin in samples than commercially available assays.

Figure 2 shows that the data generated with the capture antibody 9F8 and the conjugate antibody B12-VH is reproducible.

Figures 3 and 4 show that normalised data for B12 (capture antibody) and 9F8 (conjugate antibody) shows similar trends to the commercially available assay IBL (FNIII-C) for many samples.

Figures 5 and 6 show that normalised data for B12-VH (capture antibody) and 9F8 (conjugate antibody) shows similar trends to the commercially available assay IBL (FNIII-C) for many samples, especially normal samples.

In Figure 7 and 8 the IBL (FNIII-B) assay shows very high background absorbance most likely due to the 1/8 serum concentration being too high for the sensitivity of the assay. Substrate incubation time was 4 minutes.

Figures 9 shows that the B12-B12 self-sandwich assay shows a significant difference in between the normal and RA samples. The data generated with the self -sandwich assays may suggest that polymeric forms of Tenascin-C exist in the RA serum samples.

Testing using the same antibody B12 or B12-VH on both sides of the ELISA sandwich appeared to give much better discrimination between normal and RA samples, with a significantly improved AUC on the ROC curve compared to other assay formats comprising murine antibodies.

Figure 10 associated data.

g

References

Firestein, G.S. "Evolving concepts of rheumatoid arthritis." Nature, 2003 May, 15;423(6937):356-61. Midwood, K.S., ISIS Innovation, Ltd. "Tenascin-C as a Biomarker and Use Thereof". Int. Patent; WO 2013/088140 A2, June 20, 2013.

Page, T.H., Charles, P.J. et al. "Raised circulating tenascin-C in rheumatoid arthritis." Arthritis Research & Therapy, 2012 Nov 29;14(6):R260.

Koenders, M.I., Rolls, J.K., et al. "Interleukin-17 receptor deficiency results in impaired synovial expression of interleukin-1 and matrix metalloproteinases 3, 9, and 13 and prevents cartilage destruction during chronic reactivated streptococcal cell wall-induced arthritis." Arthritis & Rheumatology, 2005 Oct;52(10):3239-47.

Imamura, F., Aono, H., et al. "Monoclonal expansion of synoviocytes in rheumatoid arthritis." Arthritis & Rheumatology, 1998 Nov;41(ll):1979-86.

Yamanishi, Y., Firestein G.S. "Pathogenesis of rheumatoid arthritis: the role of synoviocytes." Rheumatic Disease Clinics North America, 2001 May;27(2):355-71.

Xue, C, Takahashi, M., et al. "Characterisation of fibroblast-like cells in pannus lesions of patients with rheumatoid arthritis sharing properties of fibroblasts and chondrocytes." Annals of the Rheumatic Diseases, 1997 Apr; 56(4): 262-267.

Midwood, K.S., Orend, G. "The role of tenascin-C in tissue injury and tumorigenesis." Journal of Cell Communication and Signalling, 2009 Dec;3(3-4):287-310.

Midwood, K.S., Hussenet, T., et al. "Advances in tenascin-C biology." Cellular and Molecular Life Sciences, 2011 Oct; 68(19): 3175-3199.

Giblin, S.P., Midwood, K.S. "Tenascin-C: Form versus function." Cell Adhesion & Migration, 2015;9(1- 2):48-82.

Kayser, T. "On-Market Product Cost List" on ASD Internal Network. N:\New Marker Group\Tenascin C\On Market Product Cost Listxlsx

Bartok, B., Firestein, G.S. "Fibroblast-like synoviocytes: key effector cells in rheumatoid arthritis." Immunology Review, 2010 Jan; 233(1): 233-255.

Sokolove, J., Zhao, X., et al. "Immune complexes containing citrullinated fibrinogen costimulate macrophages via Toll-like receptor 4 and Fey receptor". Arthritis & Rheumatism, 2011 Jan, 63(1) :53- 62.

Claims

1. An immunoassay for the detection of a Tenascin in a biological sample comprising at least a capture antibody and a conjugate antibody, contacting an ex vivo patient derived sample with said antibodies, wherein at least one of said antibodies has a human VH specific to the Tenascin.

2. An immunoassay according to claim 1, wherein the immunoassay is for identifying levels of tenascin indicative of an inflammatory disease, such as rheumatoid arthritis.

3. An immunoassay according to claim 1 or 2, wherein the capture antibody is specific to the Tenascin.

4. An immunoassay according to claim 3, wherein the capture antibody is specific to a region of Tenascin selected from the group comprising domain B, EGF-like repeats, FBG domain (in particular a conserved portion, section, fragment thereof), FNIII 1-3 regionfjn particular a conserved section thereof), such as the FBG domain and FNIII 1-3 region, in particular the FBG domain.

5. An immunoassay according to any one of claims 1 to 4, wherein the conjugate antibody is specific to the capture antibody.

6. An immunoassay according to any one claims 1 to 4, wherein the conjugate antibody is specific to Tenascin.

7. An immunoassay according to claim 6, wherein the conjugate antibody is specific to the FBG domain of Tenascin.

8. An immunoassay according to any one of claims 1 to 7, wherein the Tenascin is Tenascin C.

9. An immunoassay according to any one of claims 1 to 8, wherein the at least one human VH is specific to the FBG region.

10. An immunoassay according to any one of claims 1 to 6, wherein the human VH is from antibody disclosed herein, for example B12 (i.e. the VH has an amino acid sequence shown in SEQ ID NO: 12).

11. An immunoassay according to any one of claims 1 to 10, where in the capture antibody has the human VH.

12. An immunoassay according to claim 11, wherein the capture antibody is fully human.

13. An immunoassay according to any claim 11 or 12, wherein the conjugate antibody is from non-human mammal, for example mouse, rat, rabbit or camelid.

14. An immunoassay according to any one of claims 1 to 10, wherein the capture antibody is from a non-human mammal, for example mouse, rat, rabbit or camelid.

15. An immunoassay according to any one f claims 1 to 14, wherein the conjugate antibody comprises a human variable domain.

16. An immunoassay according to claim 15, wherein the conjugate antibody is fully human.

17. An immunoassay according to any one of claims 1 to 13, 15 and 16, wherein both the capture antibody and the conjugate antibody comprise a VH with the same amino acid sequence.

18. An immunoassay according to claim 17, wherein the VH has the amino acid sequence shown in SEQ ID NO: 12.

19. An immunoassay according to claim 17 or 18 wherein the capture antibody and the conjugate antibody comprise a VL with the same amino acid sequence.

20. An immunoassay according to any one of claims 1 to 19, wherein the assay is capable of detecting and/or differentiating oligomeric Tenascin molecules.

21. An immunoassay according to any one of claims 1 to 20, wherein the assay is capable of detecting and/or differentiating a Tenascin form associated with pathogenesis of rheumatoid arthritis.

22. An immunoassay according to any one of claims 1 to 21, wherein the immunoassay is an ELISA assay.

23. A kit for use in an immunoassay defined herein, comprising a capture antibody and a conjugate antibody wherein at least one of said antibodies has a human VH specific to the tenascin.