WO2016016654A1

WO2016016654A1 - Matrix for immunoadsorption of anti-pepmhigus antigen antibodies

Info

Publication number: WO2016016654A1
Application number: PCT/GB2015/052209
Authority: WO
Inventors: Jonathan Douglas Lane; Michael KILLEEN
Original assignee: Pemphimmune Solutions Limited
Priority date: 2014-07-31
Filing date: 2015-07-31
Publication date: 2016-02-04
Also published as: GB201413612D0

Abstract

The present invention defines an immunoadsorption matrix comprising pemphigus antigen immobilised by covalent attachment to a substrate. The immunoadsorption matrix allows pemphigus antigens to retain their native conformation and to therefore bind to anti- pemphigus antibodies. This is of use in the treatment of pemphigus as it allows the autoimmune antibodies to be removed from patient serum.

Description

MATRIX FOR IMMUNOADSORPTION OF ANTI-PEPMHIGUS ANTIGEN

ANTIBODIES

Field of the Invention

This invention relates to methods for the treatment of subjects suffering from pemphigus.

Pemphigus is a group of autoimmune skin disorders in which there is blistering of the skin and/or mucosal surfaces. The blisters are superficial and confined to the epidermal layer in contrast to bullous pemphigoid where blisters are subepidermal. Pemphigus can occur at any age, but often strikes people in middle age or older. The condition is usually chronic and is currently best controlled by early diagnosis and treatment, which may include medications or treatments similar to those used for severe bums.

There are three major variants of pemphigus with each having characteristic features. Pemphigus vulgaris (PV), the most common variant, accounts for 70% of cases of pemphigus and is associated with antibodies that recognise desmoglein 3 (dsg3) polypeptides. Pemphigus foliaceus (PF) is characterised by lesions which occur only in the skin and associated with antibodies to desmoglein 1 (dsg1 ) polypeptides. The least common variant is paraneoplastic pemphigus (PNP) which presents in association with a tumour that may be occult.

Pemphigus vulgaris is a rare disease. There are no patient registers, but there are estimated to be about 10,000-12,000 serum-positive patients in the USA and 25,000-45,000 patients worldwide. Patients produce an autoimmune antibody that recognises and binds to a protein responsible for cell-to-cell adhesion in the dermis. The pathogenic antibody binds to the protein known as desmoglein 3 (dsg3), preventing the protein from binding to neighbouring cells. This interruption of binding capability in the dermis leads to a painful excoriation of the skin in various areas, exposing the tissue below to infection. This disruption is widely recognized as blistering. Before treatments became available, the disease was generally lethal due to increased infection rates. Nowadays there are fewer than 20 deaths per year attributed to the disease across the world, but the side-effects from current treatments can be terminal and the cost of treating the disease can reach 6 figures on a per patient basis.

Pemphigus treatment typically involves immunosuppressing the patient in order to reduce the levels of pathogenic autoimmune antibody. The side-effects of this approach are numerous, and they can include nephrotoxicity, diabetogenesis, cholesterol and lipid problems, bone marrow damage, and anemia. In some cases the side-effects may be worse than the pemphigus disease being treated.

The most widely practiced treatment is administration of high dose corticosteroids. Although steroid treatment is inexpensive the cost of treating the side-effects can be high, and the costs associated with treating the side-effects of high dose steroid treatment can reach about US$120,000 per patient per annum.

A second, more experimental and much more costly treatment, is administration of intravenous immunoglobulin (IGIV). This treatment involves the removal of patient IgG followed by intravenous replacement with normal IgG. Normal IgG is produced from pooled donor blood, and consequently there are risks of blood-borne diseases inherent in this therapeutic approach. This treatment does not cause significant side-effects, but the cost of the IgG is high, contributing to treatment costs of about US$75,000 per patient per annum in the United States.

Other emerging therapeutic approaches include intravenous immunoadsorption (IVIA), which involves extracorporeal treatment of plasma to remove total IgG, and administration of the anti-inflammorty arthritis drug Rituximab has been used.

The underlying mechanism of pemphigus is now understood as involving an autoimmune antibody to a pemphigus antigen. Pemphigus foliaceous involves an anti-desmogiein 1 (dsg1 ) antibody, while pemphigus vulgaris involves an anti-desmogiein 3 (dsg3) antibody. The dsg1 and dsg3 proteins are understood to play a central role in cell-to-cell adhesion in the dermis.

Desmoglein (dsg) is a desmosomal transmembrane glycoprotein that belongs to the cadherin superfamily of cell-to-cell adhesion proteins. Dsg has three isotypes: dsgl dsg2 and dsg3. Dsgl and dsg3 are predominantly expressed in the stratified squamous epiderma, whilst dsg3 is expressed in all desmosome-bearing cells.

There is a need for improved compositions, devices and methods for sequestering anti-pemphigus antigen antibodies and for the treatment of pemphigus. Summary of the invention

The present invention is based on the surprising finding that pemphigus antigens can be immobilised in vitro so that they retain their native conformation and are capable of binding autoimmune antibodies. The binding of autoimmune anti-pemphigus antigen antibodies is sufficiently robust to allow their removal from serum isolated from pemphigus patients. The present invention further provides an important step forward in pemphigus treatment and provides a valuable addition to the range of options available to the physician treating pemphigus patients.

The ability to immobilise pemphigus antigens in their native conformation on immunoadsorption matrices, to manufacture solid-state devices containing those immunoadsorption matrices and to use them to remove autoimmune antibodies from patient serum, allows pemphigus to be treated effectively with reduced side-effects and reduced costs.

Thus, in a first aspect, the present invention is an immunoadsorption matrix comprising a pemphigus antigen immobilised by covaient attachment to a substrate.

In a second aspect, the present invention is a solid-state device comprising an immunoadsorption matrix comprising a pemphigus antigen immobilised by covaient attachment to a substrate.

In a third aspect, the present invention is a method of making an immunoadsorption matrix comprising preparation of a reaction mixture containing amine-functionaiised polymer beads having di-giycine on their surface, pemphigus antigen and sortase A, incubation of the reaction mixture under conditions that permit sortase A activity, and washing the polymer beads.

In a fourth aspect, the present invention is a method of using an immunoadsorption matrix comprising a pemphigus antigen immobilised by covaient attachment to a substrate, or a solid-state device comprising such an immunoadsorpiion matix, to remove anti-pemphigus antigen antibodies from serum comprising contacting serum isolated from a subject with the immunoadsorption matrix to allow anti-pemphigus antigen antibodies bind to the pemphigus antigen, and separation of the serum from the immunoadsorption matrix. Description of the Figures

Figure 1 shows the transmembrane nature of desmogiein polypeptides organisation and illustrates the extracellular domains of desmogiein 1 (dsgi ) and desmogiein 3 (dsg3) polypeptides as comprising distinct domains (EC1 -5).

Figure 2 shows a graph of index values of the patient serum samples before and after imunoadsorption using dsg3 polypeptide determined using an ELISA sandwich assay. Values are the average (mean) of three repeats and the error bars are the standard deviation, P = protein (free dsg3 polypeptide), N = new conjugation. B1 = beads with 4.5μg dsg3 polypeptide conjugated (17.5 μg/mg beads); B2 = beads with 2,5μg dsg3 polypeptide conjugated (10 μg/mg beads); B3 = beads with 1 .3μg dsg3 polypeptide conjugated (5,2 μg/mg beads); B4 = beads with 0.625μg dsg3 polypeptide conjugated (2.5 μg/mg beads); B5 = beads with 0.225μg dsg3 polypeptide conjugated (0,9 μg/mg beads); B1 N = newly conjugated beads with 4.5μg dsg3 polypeptide (17.5μg/mg beads); B5N = newly conjugated beads with 0.225μg dsg3 polypeptide (17.5μg/mg beads); P1 = 4.5μg dsg3 polypeptide free polypeptide; P2 = 2.5μg dsg3 polypeptide free polypeptide; P5 = 0.25μg dsg3 polypeptide free polypeptide; Serum = unreacted serum used as a control; and Beads = unconjugated beads used as a control.

Figure 3 shows a graph of the % decrease in index values of the patient serum samples plotted against μg dsg3 polypeptide in each conjugated bead sample used. Values are the average (mean) of three repeats and the error bars are the standard deviation. A linear trend line was added to each data set to produce an equation of the line.

Figure 4 shows a graph of index values of the undiluted patient serum samples after incubation for increasing lengths of time compared to diluted serum incubated at 4°C for 24 hours. Values are the average (mean) of three repeats and the error bars are the standard deviation. Index value calculated using the formula: index value = (average optical density [OD] of test serum - average OD N3 C)/(average OD of M3 C - average OD N3 C) x 100. All bead samples contained 17,5μg dsg3 polypeptide/mg beads (except control), details on x-axis show length of time of incubation, temperature of incubation and if the samples were diluted. RT = room temperature, 4 = 4°C, 10, 20, 30, 60 = minutes of incubation, dil = serum diluted 1 in 2, old = previously prepared conjugation, beads = unconjugated beads control, serum = unreacted serum used as a control (equivalent to TO).

Figure 5 shows a graph of the % decrease in index value after adsorption at room temperature with increasing incubation times. Values are the average (mean) of three repeats and the error bars are the standard deviation, index value calculated using the formula: index value = (average optical density [OD] of test serum - average OD N3 C)/(average OD of M3 C - average OD N3 C) x 100. AN bead samples contained 17.5μg dsg3 polypeptide/mg beads (except control), details on x-axis show length of time of incubation and 0 = unreacted serum used as a control.

Figure 6 shows a graph of index values of the undiluted patient serum samples after adsorption, with index value calculated for each class of patient serum. Values are the average (mean) of three repeats and the error bars are the standard deviation, index value = (average optical density [OD] of test serum - average OD N3 C)/(average OD of positive control - average OD N3 C) x 100, where positive control = serum control (H2 C, M3 C, L2 C or N3 C). All bead samples contained 17.5μg dsg3 polypeptide/mg beads (except control), details on x-axis show length of time of incubation, temperature of incubation and if the samples were diluted. RT = room temperature, 4 = 4°C, 10, 20, 30, 80 = minutes of incubation, dil = serum diluted 1 in 2, old = previously prepared conjugation, beads = unconjugated beads control, serum = unreacted serum used as a control (equivalent to TO).

Detailed Description of the Invention

The present invention is based on an appreciation of the role of anti- pemphigus antigen antibodies in the pathology of pemphigus and on effective removal of those antibodies from serum isolated from subjects suffering from pemphigus. The present inventors have developed a method of covalently immobilising pemphigus antigens dsg1 and dsg3 to substrates. This key feature of the present invention provides for manufacture of immunoadsoprtion matrices and solid-state devices comprising immunoadsorption matrices that can be used to remove anti-pemphigus antigen antibodies from serum. Additionally, the present invention discloses the immobilisation of pemphigus antigen at high density on substrate by covaient attachment, and this allows for effective removal of anti-pemphigus antigen antibodies from serum even when they are present at very high concentrations. This is a significant breakthrough in pemphigus treatment as it allows patients to be treated effectively at much lower cost and with much fewer side-effects than current standard therapies.

The following definitions apply to terms used throughout this description and in relation to any of the aspects of the invention described herein.

The terms "patient" and "subject" are used interchangeably herein and refer to any animal (e.g. mammal), including, but not limited to, humans, non- human primates, canines, felines, rodents and the like, which is to be the recipient of the diagnosis. Preferably, the subject or patient is a human.

The methods of the invention described herein are carried out ex vivo. For the avoidance of doubt, the term "ex vivo" has its usual meaning in the art, referring to methods that are carried out in or on a sample obtained from a subject in an artificial environment outside the body of the subject from whom the sample has been obtained.

The term "antibody" refers to an immunoglobulin which binds to a target antigen by specifically recognising an epitope on the target antigen as determined by the binding characteristics of the immunoglobulin variable domains of the heavy and light chains (V_HS and V_LS), more specifically the complementarity-determining regions (CDRs). Many potential antibody forms are known in the art, which may include, but are not limited to, a plurality of intact monoclonal antibodies or polyclonal mixtures comprising intact monoclonal antibodies, antibody fragments (for example

fragments, linear antibodies single chain antibodies and multi-specific antibodies comprising antibody fragments), single-chain variable fragments multi-specific

antibodies, chimeric antibodies, humanised antibodies and fusion proteins comprising the domains necessary for the recognition of a given epitope on a target. Preferably, references to antibodies in the context of the present invention refer to monoclonal antibodies. Antibodies may also be conjugated to various detectable labels to enable detection, including but not limited to radionuclides, fluoropbores, dyes or enzymes including, for example, horseradish peroxidase and alkaline phosphatase.

The term "epitope" refers to the portion of a target which is specifically recognised by a given antibody. In instances where the antigen is a protein, the epitope may be formed from either a contiguous or non-contiguous number of amino acids (linear' or 'conformation' epitopes respectively), whereby in the case of the latter, residues comprising the epitope are brought together in the three-dimensional fold of the polypeptide. An epitope typically comprises, but is not limited to, 3-10 amino acids in specific positions and orientations with respect to one another. Techniques known in the art for determining the epitope recognised by an antibody (specifically whether or not an epitope comprises a given residue) include but are not limited to, site-directed mutagenesis or the use of suitable homologous proteins to the target protein, in combination with techniques for determining specific recognition or lack thereof, as exemplified below. By way of example and not limitation, an epitope may be determined as comprising a given residue by comparative analysis with a control comprising specific recognition of the native (non-substituted) target protein by said antibody; wherein diminished binding and/or lack of specific recognition by said antibody when compared with said control identifies a given residue as forming part of an epitope. Furthermore, structural analyses of antibody-target protein complexes via x-ray crystallography and/or nuclear magnetic resonance (NMR) spectroscopy, or suitable derivatives thereof, may also be used to determine the residues which constitute an epitope.

The term "binds specifically", in the context of antibody-antigen interactions, refers to an interaction wherein the antibody and antigen associate more frequently or rapidly, or with greater duration or affinity, or with any combination of the above, than when either antibody or antigen is substituted for an alternative substance, for example an unrelated protein. Generally, but not necessarily, reference to binding means specific recognition. Furthermore, it is appreciated that a pemphigus antigen may be recognised and bound by more than one antibody specifically, for example, an antibody that specifically binds to dsg1 may also bind specifically to dsg3. Techniques known in the art for determining the specific binding of a target by an antibody or lack thereof include but are not limited to, FACS analysis, immunocytochemical staining, immunohistochemistry, western blotting/dot blotting, ELISA, affinity chromatography. By way of example and not limitation, specific binding, or lack thereof, may be determined by comparative analysis with a control comprising the use of an antibody which is known in the art to specifically recognise said target and/or a control comprising the absence of, or minimal, specific recognition of said target (for example wherein the control comprises the use of a non-specific antibody). Said comparative analysis may be either qualitative or quantitative. It is understood, however, that an antibody or binding moiety which demonstrates exclusive specific recognition of a given target is said to have higher specificity for said target when compared with an antibody which, for example, specifically recognises both the target and a homologous protein.

in producing an immunoadsorption matrix by attaching an antigen to a substrate if is preferable to attach the antigen to the substrate covaienfly. Covalent attachment may be achieved using conventional chemical means, but preferably it is achieved using sortase A.

Covalent attachment of the antigen to the substrate can be monitored using a Lowry protein assay to ensure that the reaction proceeds effectively and efficiently (see Tables 1 and 2).

A first aspect of the present invention provides an immunoadsorption matrix comprising a pemphigus antigen immobilised by covalent attachment to a substrate.

The pemphigus antigen may be a desmogiein 3 (dsg3) polypeptide or a desmoglein 1 (dsg1 ) polypeptide. This allows the immunoadsorption matrix to be used to remove autoantibodies from serum isolated from subjects suffering from pemphigus foiiaceus (dsg1 ) or from subjects suffering from pemphigus vulgaris (dsg3). The immunoadsorption matrix may comprise dsg1 and dsg3 polypeptides.

The pemphigus antigen may comprise an extracellular domain of dsg1 or dsg3 to ensure that the immunoadsorption matrix effectively binds to anti- pemphigus antigen antibodies in serum isolated from subjects suffering from pemphigus. The pemphigus antigen may comprise extracellular domains 1 to 5 of dsg1 or dsg3 to allow the immune adsorption matrix to bind all anti-pemphigus antigen antibodies in the serum isolated from subjects suffering from pemphigus. The pemphigus antigen may be produced by mammalian cells so that it is produced in its native conformation with representative human post-translational modifications. This will serve to ensure that the immunoadsorption matrix comprises a pemphigus antigen capable of binding antibodies that recognise conformational epitopes.

The pemphigus antigen may comprise a sortase A recognition motif to allow its covaienf attachment to the substrate using the sortase A enzyme.

The substrate can be a polymer bead that is suitable for use in a chromatography column to allow removal of bound antibodies from serum that has been isolated from subjects suffering from pemphigus.

The pemphigus antigen may be bound to between 5 and 40% of the surface of the substrate to ensure effective binding of anti-pemphigus antigen antibodies when in contact with serum isolated from subjects suffering from pemphigus.

The pemphigus antigen may be bound to 10% of the surface of the substrate to conserve pemphigus antigen protein when making the immunoadsorption matrix while also ensuring that it can effectively bind anti- pemphigus antigen antibodies when in contact with serum isolated from subjects suffering from pemphigus.

A second aspect of the present invention provides a solid-state device comprising an immunoadsorption matrix comprising a pemphigus antigen immobilised by covalent attachment to a substrate.

A third aspect of the present invention provides an apheresis column comprising an immunoadsorption matrix comprising a pemphigus antigen immobilised by covalent attachment to a substrate.

A fourth aspect of the present invention provides a method of making an immunoadsorption matrix comprising a pemphigus antigen immobilised by covalent attachment to a substrate comprising the steps of reacting a functionalised substrate and the pemphigus antigen and washing the substrate to remove unreacted pemphigus antigen.

The functionalised substrate may be an amine-functionalised substrate having di-glycine on its surface, and the reaction may further comprises sortase A enzyme and may be carried out under conditions that permit sortase A activity. The pemphigus antigen used in the method may be a desmoglein 3 (dsg3) polypeptide or a desmoglein 1 (dsg1 ) polypeptide. This allows the immunoadsorption matrix to be used to remove autoantibodies from serum isolated from subjects suffering from pemphigus foliaceus (dsg1 ) or from subjects suffering from pemphigus vulgaris (dsg3). Additionally, the immunoadsorption matrix used in the method may comprise dsg1 and dsg3 polypeptides.

The pemphigus antigen used in the method may comprise an extracellular domain of dsg1 or dsg3 to ensure that the immunoadsorption matrix effectively binds to anti-pemphigus antigen antibodies in serum isolated from subjects suffering from pemphigus. The pemphigus antigen used in the method may comprise extracellular domains 1 to 5 of dsg1 or dsg3 to allow the immune adsorption matrix to bind all anti-pemphigus antigen antibodies in the serum isolated from subjects suffering from pemphigus.

The pemphigus antigen used in the method may be produced by mammalian cells so that it is produced in its native conformation with representative human post-translationai modifications. This will serve to ensure that the immunoadsorption matrix comprises a pemphigus antigen capable of binding antibodies that recognise conformational epitopes.

The pemphigus antigen used in the method may comprise a sortase A recognition motif to allow its covalent attachment to the substrate using the sortase A enzyme.

The substrate can be a polymer bead that maximises surface area of the immunoadsorption matrix used in the method allowing effective binding of anti- pemphigus antigen antibodies when in contact with serum isolated from subjects suffering from pemphigus and that is suitable for use in a chromatography column to allow removal of bound antibodies from serum that has been isolated from subjects suffering from pemphigus.

The pemphigus antigen used in the method may be bound to between 5 and 40% of the surface of the substrate to ensure effective binding of anti- pemphigus antigen antibodies when in contact with serum isolated from subjects suffering from pemphigus.

The pemphigus antigen used in the method may be bound to 10% of the surface of the substrate to conserve pemphigus antigen protein when making the immunoadsorption matrix while also ensuring that is can effectively bind anti- pemphigus antigen antibodies when in contact with serum isolated from subjects suffering from pemphigus.

The pemphigus antigen may be included in the reaction mixture at 17,5 mg per mg of beads, at 10mg per mg of beads, at 5mg per mg of beads, at 2.5mg per mg of beads or at 1 mg per mg of beads. Adjusting the concentration of antigen in the reaction mixture allows for optimisation of the density of antigen on the surface of the immunoadsorption matrix so as to allow effective binding of anti-pemphigus antigen antibodies when in contact with serum isolated from subjects suffering from pemphigus.

A fifth aspect of the present invention provides a method of removing anti-pemphigus antigen antibodies from serum isolated from a subject suffering from pemphigus comprising the steps of contacting the isolated serum with an immunoadsorption matrix comprising a pemphigus antigen immobilised by covaient attachment to a substrate under conditions that allow anti-pemphigus antigen antibodies to bind to the pemphigus antigen, and separating the serum from the immunoadsorption matrix. This provides immunoadsorbed serum that can be reintroduced into the patient so as to prevent further symptoms of pemphigus such as blistering fo the skin or mucosa.

The pemphigus antigen used in the method may be a desmoglein 3 (dsg3) polypeptide. This allows binding of anti-dsg3 antibodies found in serum of subjects suffering from pemphigus vulgaris.

The pemphigus antigen used in the method may be a desmoglein 1 (dsg1 ) polypeptide. This allows binding of anti-dsg1 antibodies found in serum of subjects suffering from pemphigus foliaceus.

The immunoadsorption matrix used in the method may comprise dsg1 and dsg3 polypeptides.

The substrate can be a polymer bead that maximises surface area of the immunoadsorption matrix used in the method allowing effective binding of anti- pemphigus antigen antibodies when in contact with serum isolated from subjects suffering from pemphigus and that is suitable for use in a chromatography column to allow removal of bound antibodies from serum that has been isolated from subjects suffering from pemphigus. The pemphigus antigen used in the method may be bound to between 5 and 40% of the surface of the substrate to ensure effective binding of anti- pemphigus antigen antibodies when in contact with serum isolated from subjects suffering from pemphigus.

The method may further comprise the step of measuring anti-pemphigus antigen antibody levels in the isolated serum. This allows for optimisation of the conditions for antibody binding to the immunoadsorption matrix in the method. The method may also comprise a step of optimising the time of contact between the isolated serum and the imunoadsorpfion matrix so that more than 60% of anti-pemphigus antigen antibodies are removed from the serum.

The method is suitable for use with serum isolated from human patients suffering from pemphigus vulgaris and/or pemphigus foiiaceus.

A sixth aspect of the present invention provides a method of treating a subject suffering from pemphigus by removing anti-pemphigus antigen antibodies from serum isolated from the subject comprising the steps of isolating serum from the subject, contacting the isolated serum with an immunoadsorption matrix comprising a pemphigus antigen immobiiised by covaient attachment to a substrate under conditions that allow anti-pemphigus antigen antibodies to bind to the pemphigus antigen, separating the serum from the immunoadsorption matrix, and returning the serum to the subject. This provides immunoadsorbed serum to the patient that lacks anti-pemphigus antigen antibodies so as to prevent further symptoms of pemphigus such as blistering fo the skin or mucosa.

The pemphigus antigen used in the method may be a desmoglein 1 (dsg1 ) polypeptide. This allows binding of anti-dsg1 antibodies found in serum of subjects suffering from pemphigus foiiaceus. The immunoadsorption matrix used in the method may comprise dsg1 and dsg3 polypeptides.

The substrate can be a polymer bead that maximises surface area of the immunoadsorpiion matrix used in the method allowing effective binding of anti- pemphigus antigen antibodies when in contact with serum isolated from subjects suffering from pemphigus and that is suitable for use in a chromatography column to allow removal of bound antibodies from serum that has been isolated from subjects suffering from pemphigus.

The method is suitable for use with serum isolated from human patients suffering from pemphigus vulgaris and/or pemphigus foliaceus.

The invention is further described with reference to the following non- limiting example: !XAMPLES

Example 1

Transient expression of his tagged native dsg3.

This report describes the transient expression of HIS-tagged human dsg3 polypeptide protein in suspension HEK 293-8E ceils using serum free medium, followed by one-step purification.

HEK 293-6E cells were maintained in serum-free Freestyle 293 expression medium (invitrogen, Carlsbad, CA, USA) in Erlenmeyer Flasks at 37°C with 5% CO2 (Corning Inc., Acton, MA) on an orbital shaker (VWR Scientific, Chester, PA). One day before fransfection, the cells were seeded at an appropriate density, and on the day of transfection, DNA and PEI (Polysciences, Eppeibeim, Germany) were mixed at an optimal ratio and added to the cells. Cell supernatant collected on day 8 was used for further purification.

Cell culture broth was centrifuged and filtered before loading onto His Trap™ FF crude 5 mL column (GE Healthcare, Uppsala, Sweden). After washing, elution and desalting, the purified protein was analyzed by SDS-PAGE and Western blot by using standard protocols for molecular weight and purity.

The plasmid DNA encoding HIS-tagged human dsg3 polypeptide was transiently transfected into 3L suspension HEK 293-8E cells. The target protein was captured from the cell culture supernatant and analyzed by SDS-PAGE and Western blot. The primary antibody for Western blot was Mouse-anti-his mAb (GenScript, Cat. No. A00188).

We have successfully expressed and purified human dsg3 protein in suspension HEK 293-8E cells. The target protein was detected with an estimated molecular weight of -70 kDa (Cal.M.W. -64.5 kDa) on SDS-PAGE and confirmed by Western blot.

Conjugation of dsg3 to beads.

Amine-functionalised polymer beads were purchased form Bangs Laboratories. The amino groups were functionalised with Fmoc-GG-OH by EDC chemistry, before site-specific conjugation of protein to the beads was carried out using Sortase A. Functionalization of amine-beads with Glyciiglycine

1 mL of beads (1.2g/mL) was pelleted (10,000xg for 15 min) and washed with water (x3). The beads were resuspended in DMF (400μL) and reacted with by Fmoc-GG-OH (1.58 μmol), TBTU (1.58 μmol), DIPEA (3.08 μmol) and HOBt (0.77 μmol). The beads were mixed for 1 hour at room temperature, and then pelleted (10,000xg for 15 min). The supernatant was removed and the beads reacted again with Fmoc-GG-OH. The beads were then pelleted (10,000xg for 15 min) and washed with DMF (x3) and then water (x3). δ0μΙ_ of the bead suspension was freeze-dried and used for the determination of the Fmoc-loading. The rest of the beads were resuspended in a 20% piperidine in DMF solution for the Fmoc group removal (20 min). The beads were pelleted (10,000xg for 15 min) and washed with DMF (x3), then with water five times (x3).

Conjugation of functionalised beads and increasing concentrations of DSG3

DSG3 incorporating the LPETGG C-terminus sequence was expressed and purified by GenScript. Purity was checked by Western blot and protein concentration was determined by Lowry assay.

Reactions were set up in a total volume of 2mL and included 7mg functionalised beads, one of 10μ1, 20μl, 40μl, 80μl, 1 10μl dsg3 (0.1654mM), 100μl Sortase A (4.463mM) and sortase buffer (50mM Tris, 100mM NaCi, 5mM CaCl₂ pH 7.5). A control reaction of 0.5mg functionalised beads, 10μl DSG3 (0.1654mM) and sortase buffer (50mM Tris, 100mM NaC!, 5mM CaCl₂ pH 7.5) in 1 mL was run. The reaction mixtures were incubated at room temperature for 24h with shaking at 250rpm.

Purification of Conjugated beads

After conjugation the beads were centrifuged for 10 minutes at 13,000xg. The supernatant was removed and kept then the beads were re-suspended in sortase buffer (up to 2m L). This process was repeated up to 4 times and the beads were finally resuspended in 1 mL sortase buffer.

By using the results for the determination of Fmoc-loading and calculation of bead surface saturation (Bangs Laboratories, TechNote 205) the amount of dsg3 polypeptide needed to produce different levels of surface coverage was determined. Due to the size of the protein and to allow for antibody binding, a 10% surface coverage was chosen for the initial investigation using the optimised eGFP conditions.

The amount of protein conjugated to the beads and in the wash steps was determined by Lowry assay (Table 1) and indicated the reaction produced a protein concentration per mg of beads slightly higher than expected. The unbound protein was successfully removed from the beads during the wash steps as no protein can be detected in the final wash step.

Table 1 : DSG3 concentration using Lowry assay - initial conjugation.

Beads = un-reacted beads; reaction = reaction including Sortase A; control = without Sortase A; FT = un-reacted DSG3 and Sortase A removed after reaction completed; W1 , 2, 3, 4 = reaction wash steps 1 -4 (2 mL wash volume). FT and Wash results are μg/gml of protein, which is equivalent to μg/mg beads.

As the initial conjugation was successful, reactions to achieve higher and lower bead surface coverage were set up. For each sample (Table 2) the protein concentration was determined by Lowry assay and % surface coverage was recorded. The reactions were set up order to achieve 2, 5, 10, 20 and 40% surface coverage, all of them were achieved apart from the 40%, where the reaction only reached 35%, but this is still of use for further investigation of antibody binding to dsg3 polypeptide.

Table 2: DSG3 concentration and corresponding per cent surface coverage.

These results indicate the conditions chosen were suitable for use with the dsg3 polypeptide, leading to production of a set of 5mg samples of beads with increasing concentrations of dsg3 polypeptide conjugated to the surface.

Conclusions

The beads were modified to add a di-giycine to the surface of the beads to allow site specific attachment of proteins using Sortase A. This allowed a set of beads conjugated with increasing concentrations of dsg3 polypeptide to be produced (5mg of beads for each concentration including un-reacted). These samples are ready for use in the investigation of the antibody binding capability of the dsg3 polypeptide conjugated to the functionalised beads.

Example 2

Immunoabsorption of anti-dsg3 antibodies using free and bound dsq3 protein

The aim of this project was to use increasing concentrations of free and bound dsg3 polypeptide to investigate if the anti-dsg3 antibodies in serum isolated from pemphigus patients could be reduced by adsorption to dsg3 protein. Four samples of patient serum were selected to represent the different levels of anti-dsg3 antibodies seen in patient samples (high, medium, low and normal). The amount of anti-dsg3 antibodies in the patient samples before and after adsorption was measured using a previously optimised sandwich ELISA assay.

Adsorption of anti-Dsg3 antibodies to free and bound Dsq3

Previously, 5mg of beads were prepared for each of the five protein concentrations chosen. This meant that not all the patient serum samples available could be used to investigate the effect of absorption as there would not be enough beads to carry out replicates. Therefore four samples were chosen to represent the different levels of antibodies seen within the patient groups: H3 for the high level of antibodies, M2 for the medium, L3 for the low and N3 for the normal patients (control group).

Pure dsg3 protein was diluted in dilution buffer (PBS, 1 mM CaCl₂) to create the same range of concentrations as the samples conjugated to the beads. The samples of beads (old and new) were centrifuged at 13,000xg for 10 minutes, the supernatant was removed and the pellet of beads were resuspended in dilution buffer by vortexing to give a final concentration of 5mg/ml of beads. Samples of serum were diluted to give a final dilution of 1 in 2 or 1 in 10 in dilution buffer after the addition of the relevant volume of free or bound protein. Once all the reaction components had been added, samples were vortexed for 1 minute to ensure the beads were dispersed throughout the solution, incubated at room temperature for 1 hour with shaking at 250 rpm, and then the samples were incubated for 24 hours at 4°C with shaking at 250 rpm. Unreacted serum samples were treated the same as the adsorption samples to ensure a fair comparison.

Adsorption reactions were set up in triplicate as three independent repeats for each different protein concentration and each dilution as follows:

After incubation the samples were vortexed for 1 minute then centrifuged at 13,000xg for 10 minutes to pellet the beads. A sample of the supernatant was removed and diluted to a final dilution of 1 in 200 (1 in 2 samples were diluted 1 in 100, 1 in 10 samples were diluted 1 in 20) before addition to the EL!SA plate, see the method detailed below. A further plate was run to determine the background of the assay with 3 repeats of one of: no primary antibody, no Dsg3 protein, no serum or no secondary antibody. The highest values of these samples were used as the background. ELISA method

Basic sandwich ELISA method adapted from Abeam sandwich ELISA protocol (Abeam) and previously published methods for defection of anti-dsg3 antibodies by ELISA (Amagai M, et a!., (1999) Usefulness of enzyme-linked immunosorbent assay using recombinant desogleins 1 and 3 for serodiagnosis of pemphigus. British J. Dematoi. 140: 351 , Ishii K et al., (1997) Characterisation of Autoantibodies in Pemphigus using antigen-specific enzyme-linked immunosorbent assays with Baculovirus-expressed recombinant desmogleins. J. Immunol. 159: 2010). Unless otherwise stated, all solutions or washes were removed by flicking the plate over the sink and removing the remaining drops by patting the plate on a paper towel. During incubations the plate was covered with adhesive plastic to stop loss of liquid. 1 mM CaCl₂ was added to the buffers to help maintain the correct conformation of Dsg3 (Ishii K et a!., (1997) Characterisation of Autoantibodies in Pemphigus using antigen-specific enzyme- linked immunosorbent assays with Baculovirus-expressed recombinant desmogleins. J. Immunol. 159: 2010).

Coating the plate with capture antibody

The wells of a PVC 96-well microtiter plate were coated with 100μ! of the capture antibody (0.2μg/mi) in dilution buffer (PBS, 1 mM CaCl₂). The plate was covered and incubated overnight at 4°C. The coating solution was removed and the wells were washed three times with 200 μl wash buffer (0.05% Tween, PBS, 1 mM CaCl₂).

Blocking and adding the protein

The remaining protein-binding sites were blocked by coating the wells with 200μl of blocking buffer (1 % BSA PBS) per well. The plate was incubated overnight at 4°C. The plate was washed three times with 200μl of wash buffer per well. 100μl of dsg3 protein (5μg/ml) in dilution buffer was added to each well (apart from the blanks) and the plate was incubated for 90 minutes at 37°C. The plate was washed three times using 200μl of wash buffer per well.

Adding the serum and secondary antibody

100μl of serum after adsorption treatment (1 in 200 dilution) in dilution buffer was added to each well and the plate was incubated for two hours at room temperature. After incubation the plate was washed four times with wash buffer.

100μl of secondary antibody (1 in 15,000 dilution) in blocking buffer was added to each well and the plate was incubated for 2 hours at room temperature. The plate was washed four times with 200μl of wash buffer per well. Detection

The 1MB solution was used undiluted. Avoiding direct light, 100μl of solution was added to each well, the plate was covered with adhesive plastic, wrapped in foil and incubated at room temperature for 20 minutes. 100μl of stop solution (0.1 M H₂S0₄) was added to each well and the absorbance at 450nm was measured using a plate reader. The index value was calculated using the following formula: index = (optical density [OD] of test serum - OD of negative controi)/OD of positive control - OD negative control) x 100.

Results

New conjugation of Dsg3 to amide functionalised beads

After the conjugated beads were washed, the protein content of the beads was measured using the Lowry assay. The results of the assay showed that the beads reacted with the higher concentration of dsg3 polypeptide had a 17.5μg of protein per mg of beads, which is the same as before. The beads reacted with the lower concentration of dsg3 polypeptide had 1.0μg of protein per mg of beads, which is slightly higher than the previous reaction. Both samples of beads were suitable for use in the investigation of adsorption. ELISA assay of patient serum samples after adsorption

Using the optimised assay conditions, the samples of patient serum after adsorption using increasing levels of free or bound dsg3 protein were analysed for anti-dsg3 antibodies. The average (mean) index values were calculated and plotted, the results (Figure 2) show clear differences between all the different samples after adsorption, the lower the index value the smaller the number of anti-dsg3 antibodies present. Note that results for N3 1 :2 and 1 : 100 not shown as all had values of 0 after correction for background. After the subtraction of the background absorbance readings, the normal (N3) samples show no anti- dsg3 antibodies as expected, so they were not plotted on the graph. The index values for the untreated serum are similar to those found previously; suggesting the ELISA assay has good reproducibility. For all the samples investigated the error bars are not very large, which shows good correlation between the independent repeats. There are no significant differences between the results for the untreated serum samples and the beads only samples, suggesting nonspecific binding of the antibodies to the beads is very small.

By analysing the two dilutions it can be seen that there are differences between the two sets of data; more antibodies are removed (lower index value) in the 1 in 10 dilution than the 1 in 2 dilution. The results for the new beads preparations do give a slightly lower index value than the previously prepared samples, suggesting more antibodies are removed, but the difference is not significant. This suggests that the conjugated beads are stable when stored at 4°C. For all the protein concentrations investigated there was a small increase in antibody adsorption for the samples of bound protein, suggesting that conjugation of the protein to the beads does not impede the antibody binding and is actually beneficial.

By calculating the percent decrease in index value for each sample (Table 3), this shows the actual decrease in antibody concentration for the adsorption samples, correcting for the different starting index values of the patient serum samples.

Table 3: Results for the average % decrease in index value after adsorption.

The results show that the highest concentration of protein conjugated to the beads removed 97.2% of the antibodies for the low antibody titre (L3) sample diluted 1 in 10, which is nearly all of the antibodies in the sample. For the medium antibody titre (M2) sample 88.65% of the antibodies were removed for the 1 in 10 dilution, and for the high antibody titre (H3) sample 79.54% of the antibodies were removed (1 in 10 dilution). These results show that the method is effective and that by increasing the concentration of protein used (by increasing the amount of beads), all the antibodies could potentially be removed from the samples of patient serum.

To calculate how much protein would be needed to remove all the anti- dsg3 antibodies for the patient serum samples, the percentage decrease in index value was plotted against μg of protein conjugated to beads used in each adsorption experiment (Figure 3). A linear trend line was added to each data set and the equation of the line was used to calculate the concentration of protein needed to remove all of the antibodies (100% decrease in index value). Overall the data for each dilution of the patient serum samples fit the line well, with all the R² values being above 0.991.

The results for the calculation of the concentration of protein needed to remove all of the antibodies (Table 4) shows the amount of protein required (and mg of beads) depends on the which serum sample and dilution is used.

Table 4: Titration of dsg3 polypeptide matrix against pemphigus vulgaris serum

Using these values the amount of protein (and conjugated beads) needed to remove the all the antibodies from 1 ml of the different classes of patient serum was calculated. For H3 (based on 1 in 2 dilution) to remove all the antibodies in 1 ml of patient serum would require 145.87μg of dsg3 polypeptide, which would be 8,34mg of conjugated beads (17.5μg dsg3 polypeptide/mg beads). For M2 (1 in 2 dilution) it would take 123.91 μg dsg3 polypeptide or 7.08mg beads (17.5μg dsg3 polypeptide/mg), and for L3 (1 in 2 dilution) it would need 1 15,13μg dsg3 polypeptide or 6.58 mg beads (17.5μg dsg3 polypeptide/mg). This suggests that different amounts of conjugated beads could be prepared for the different levels of antibodies found in different patients, or the highest amount of conjugated beads could be used as a treatment for all patients.

Conclusions

The results for the ELISA of the untreated serum samples produced similar index values to those detected previously, suggesting that the specific sandwich ELISA previously optimised is robust and reproducible. This is also confirmed by the ability of the ELISA assay to detect changes in antibody concentration after adsorption in all of the samples of patient serum investigated.

The ability of the dsg3 polypeptide conjugated to the functionalised beads to bind antibodies suggests that the process of conjugation does not cause the protein to be denatured, and the concentrations of protein used on the beads allow the antibodies to access binding sites on the surface of the protein. The fact that there are only small differences between the freshly conjugated and previously conjugated samples shows that the conjugated beads can be stored at 4°C for months without loss of protein structure. This could be useful for scale up and storage in the future.

From the adsorption results it is clear that the amount of antibodies present in the sample and the dilution used has an effect on how well the protein conjugated to the beads can remove the antibodies from solution, with the lower concentrations and higher dilutions giving the largest reduction in antibodies present. This needs to be taken into account when designing further experiments.

The calculation of how much protein (or mg of conjugated beads) is needed to remove all the antibodies from 1 ml of serum shows how use of the beads can be scaled up to remove more antibodies from larger volumes of solution. For example for the patient serum with the highest antibody concentration (H3) it would take 8.34mg of conjugated beads (17.5μg dsg3 polypeptide/mg beads) to remove all the antibodies from 1 ml. This amount could potentially be reduced by increasing the concentration of protein on the beads, but this could cause a reduction in the efficiency of binding as the antibodies may have restricted access to the binding sites on the protein. Further investigations could determine the maximum concentration of protein per mg of beads that would allow efficient binding of the antibodies.

Overall, this work has shown that adsorption using either free or bound dsg3 polypeptide does reduce the concentration of anti-dsg3 antibodies in patient serum. Using the protein conjugated to beads has advantages over using the free protein as the protein and bound antibodies are no longer present in the serum sample.

Example 3

Immunoabsorption of anti-dsg3 antibodies using free and bound dsg3 protein

The aim of this project was to use undiluted patient serum and evaluate the ability of conjugated dsg3 polypeptide to remove anti-dsg3 antibodies when incubated at room temperature for increasing lengths of time. To compare to the previously obtained results, samples of serum were diluted and allowed to bind overnight at 4°C to either the newly conjugated beads or a previously prepared sample. Four samples of patient serum were selected to represent the different levels of anti-dsg3 antibodies seen in patient samples (high, medium, low and normal). The amount of anti-dsg3 antibodies in the patient samples before and after adsorption was measured using the previously optimised sandwich ELISA assay.

New conjugation of dsq3 polypeptide to amide functionaiised beads

To assess the ability of dsg3 polypeptide conjugated to the beads to remove anti-dsg3 antibodies from undiluted serum, a new conjugation was carried out as there were not enough conjugated beads left from the previous conjugation reactions.

First, the beads needed to be functionalized with glycilgiycine. 200μl of beads (1 .2g/mL) was pelleted (10,000xg for 15 min) and washed with water (x3). The beads were resuspended in DMF (40Όμl_) and reacted with by Fmoc-GG- OH (1 .58μmol), TBTU (1 .58μΐ7ΐοΙ), DIPEA (3.08μmol) and HOBt (0.77μmol). The beads were mixed for 1 hour at room temperature, and then pelleted (10,000xg for 15 min). The supernatant was removed and the beads reacted again with Fmoc-GG-OH. The beads were then pelleted ( 10,000xg for 15 min) and washed with DMF (x3) and then water (x3). 60μL of the beads were freeze-dried and used for the determination of the Fmoc-loading. The rest of the beads were resuspended in a 20% piperidine in DMF solution for the Fmoc group removal (20 min). The beads were pelleted (10,000xg for 15 min) and washed with DMF (x3), then with water five times (x3).

Then the beads were conjugated with dsg3 polypeptide using Sortase A. Based on the previously optimized conditions, a reaction was set up in a total volume of 2mL and included 15 mg functionalised beads, 200μl dsg3 polypeptide (0.1854mM), 100μl Sortase A (4,463mM) and sortase buffer (5GmM Tris, 100mlvl NaCl, 5mM CaCi₂ pH 7,5), A control reaction of 0,5mg functionaiised beads, 10μl dsg3 polypeptide (0.1654mM) and sortase buffer (50mM Tris, 100mM NaCI, 5mM CaCl₂ pH 7.5) in 1 mL was run. The reaction mixtures were incubated at room temperature for 24 h with shaking at 250 rpm. After the reaction the beads were washed 3 times with sortase buffer: samples were vortexed for 1 minute, centrifuged at 13,000xg for 10 minutes and the supernatant was removed. The beads were resuspended in a final volume of 1 ml sortase buffer. The protein concentration of the beads was measured using the Lowry assay.

Adsorption of anfi-Dsq3 antibodies from undiluted patient serum

To investigate the ability of the conjugated beads to remove anti-dsg3 antibodies form undiluted patient serum at room temperature over increasing lengths of incubation time, reactions were set up in triplicate (three independent repeats) and included controls of samples reacted with just beads, samples incubated with either undiluted or diluted serum (1 in 2) at 4°C for 24 hours (to allow comparison to previous results) and also a set of reactions to calculate the background of the ELISA reaction (Table 1 ). For each time point 0.25mg of conjugated beads and 50μl of undiluted (or 1 in 2 diluted) serum were used, for the just beads control 0.25mg of unconjugated beads were used.

Before the adsorption reactions were set up, the beads were centrifuged 13,000xg for 10 minutes and all of the supernatant was removed. For the 1 in 2 serum dilution samples, the beads were resuspended in 50μl dilution buffer (PBS with 1 mM CaCl₂) before use. Ail reactions had the relevant serum sample added, were mixed by vortexing for 1 minute and then the samples were incubated either at room temperature or 4°C for the desired length of time with shaking at 250 rpm. Samples with longer incubation times were set up first so all the reactions could be terminated at the same time and applied to the ELISA plates. Unreacfed serum samples (HC, MC, LC and NC) were treated the same as the adsorption samples to ensure a fair comparison.

After incubation the samples were vortexed for 1 minute then centrifuged at 13,000xg for 10 minutes to pellet the beads. A sample of the supernatant was removed and diluted to a final dilution of 1 in 200 before addition to the ELISA plate, see the method detailed below. ELISA method

Basic sandwich ELISA method adapted from Abeam sandwich ELISA protocol (Abeam) and previously published methods for detection of anti-dsg3 antibodies by ELISA (Amagai M, et a/., (1999) Usefulness of enzyme-linked immunosorbent assay using recombinant desogleins 1 and 3 for serodiagnosis of pemphigus. British J. Dematol. 140: 351 , Ishii K et al., (1997) Characterisation of Autoantibodies in Pemphigus using antigen-specific enzyme-linked immunosorbent assays with Baculovirus-expressed recombinant desmogleins. J. Immunol. 159: 2010). Unless otherwise stated, all solutions or washes were removed by flicking the plate over the sink and removing the remaining drops by patting the plate on a paper towel. During incubations the plate was covered with adhesive plastic to stop loss of liquid. 1 mM CaCl₂ was added to the buffers to help maintain the correct conformation of Dsg3 (Ishii K et ai., (1997) Characterisation of Autoantibodies in Pemphigus using antigen-specific enzyme- linked immunosorbent assays with Baculovirus-expressed recombinant desmogleins. J. Immunol. 159: 2010).

Coating the plate with capture antibody

The wells of a PVC 96-well microtiter plate were coated with 100μl of the capture antibody (0.2μg/mi) in dilution buffer (PBS, 1 mM CaCl₂). The plate was covered and incubated overnight at 4°C. The coating solution was removed and the wells were washed three times with 200μl wash buffer (0.05% Tween, PBS, 1 mM CaCl₂). Blocking and adding the protein

The remaining protein-binding sites were blocked by coating the wells with 200μl of blocking buffer (1 % BSA PBS) per well. The plate was incubated overnight at 4°C. The plate was washed three times with 200μl of wash buffer per well.

100μl of dsg3 protein (5μg/ml) in dilution buffer was added to each well (apart from the blanks) and the plate was incubated for 90 minutes at 37°C. The plate was washed three times using 200μl of wash buffer per well.

Adding the serum and secondary antibody

100μl of secondary antibody (1 in 15,000 dilution) in blocking buffer was added to each well and the plate was incubated for 2 hours at room temperature. The plate was washed four times with 200μ! of wash buffer per well.

Detection

The TMB solution was used undiluted. Avoiding direct light, 100μl of solution was added to each well, the plate was covered with adhesive plastic, wrapped in foil and incubated at room temperature for 20 minutes. 100μl of stop solution (0.1 M H₂S0₄) was added to each well and the absorbance at 450nm was measured using a plate reader. The average reading from the background measurements were subtracted from all of the assay values before the index value was calculated. The index value was calculated using the following formula: index = (optical density [OD] of test serum - OD of negative control)/(OD of positive control - OD negative control) x 100, the negative control was the average of N3 C and the positive control was the average of M3 C, so the formula can be rewritten as: index value = (average optical density [OD] of test serum - average OD N3 C)/(average OD of M3 C - average OD N3 C) x 100. Results

New conjugation of dsg3 polypeptide to amide functionalised beads

After the conjugated beads were washed, the protein content of the beads was measured using the Lowry assay. The results of the assay showed that the beads had a 17,5μg of protein per mg of beads, which is the same as before, allowing the adsorption results for the two sets of beads to be compared. ELISA assay of undiluted patient serum samples after adsorption

Using the optimised assay conditions, the samples of patient serum after incubation were analysed for anti-dsg3 antibodies. The average (mean) index values were calculated (after the background absorbance was subtracted) and plotted, the results (Figure 4) show clear differences between the increasing times of incubation at room temperature, whereas the 24 hour samples show a similar pattern across all the different conditions. As before, after subtraction of the background absorbance readings the normal (N2) samples show no anti- dsg3 antibodies and the index values for the untreated serum are similar to those found previously. There is a small reduction in index value of the just beads samples compared to the serum only samples suggesting there is some non-specific binding, but this is not at a significant level when compared to the results for the dsg3 polypeptide conjugated beads.

As the incubation time at room temperature increases the concentration of anti-dsg3 antibodies decreases, after 60 minutes at room temperature the reduction of anti-dsg3 antibodies is very similar to the results obtained for incubation for 24 hours at 4°C. The results for the diluted samples are very similar, suggesting the newly conjugated beads have similar adsorption ability to those created previously.

By comparing the percent decrease in index value for all the different incubation times and conditions, the actual reduction in anti-dsg3 antibodies can be seen (Table 5). The results show that even after 10 minutes incubation at room temperature there is a significant reduction in index value for all of the different samples of serum, with a 78 % reduction for the sample with the lowest anti-dsg3 antibody count (L2). After 60 minutes at room temperature even the sample with the highest anti-dsg3 antibody count has over a 75% reduction in antibodies, suggesting room temperature incubation is a feasible method for the reduction of anfi-dsg3 antibodies in patient serum. The largest reduction in percent index value for all the sample was seen for the new sample of beads after 24 hours incubation at 4°C, suggest this offers a small advantage over the undiluted samples, but the difference is not significant.

Table 5: Results for the % decrease in index value after adsorption.

By plotting the % reduction in absorbance against time of incubation at room temperature (Figure 5), the reduction in anti-dsg3 antibodies over time for each class of patient serum can be seen. The results show that for the low patient serum (L2) and medium patient serum (M3) samples the majority of the anti-dsg3 antibodies are removed after 10 minutes incubation with only a small increase in removal as the incubation time increased; whereas for the high patient serum (H3) sample there was a significant increase in removal of anti- dsg3 antibodies at the longer incubation times compared to the 10 minute incubation.

If the index value is calculated for each class of serum (high, medium, low and normal) instead of as an assay as a whole (just using M3 C as the positive control); all the control serum only samples will all have an index value of 100 and the reduction in index value due to adsorption can be calculated (Table 6) and plotted as a graph (Figure 6). The results show the same pattern as the % decrease results (Table 5), with the shorter time points and undiluted serum allowing for significant adsorption of anti-dsg3 antibodies.

Table 6: Index values after adsorption calculated for each patient class

Overall these results suggest that using undiluted serum has no significant effect on the ability of the dsg3 conjugated beads to bind anti-dsg3 antibodies when compared to the 1 in 2 diluted serum samples. The samples incubated at room temperature for increasing lengths of time show that the dsg3 polypeptide conjugated beads can bind at room temperature and do so at shorter time points, suggesting these conjugated beads have the potential to be used as an adsorption therapy in real time.

Conclusions

This investigation has shown that the conjugated beads can bind anti- Dsg3 antibodies in undiluted patient serum at room temperature. As the incubation time increases so too does the concentration of antibodies bound, but even at the shortest time point (10 minutes) there was a significant reduction on the antibody concentration for all of the different sample groups of patient serum (high, medium and low).

The latest conjugation of dsg3 polypeptide to the functionalised beads was successful and produced similar (slightly improved) results for the removal of anti-dsg3 antibodies from diluted patient serum to those of the previously conjugated beads. This suggests that the conjugation process is reproducible and that storage of the conjugated beads at 4°C as no detrimental effect on the protein.

Overall, these results suggest that these conjugated beads could be used with undiluted patient serum and a relatively short incubation time at room temperature as part of a patient therapy.

Claims

1. An immunoadsorption matrix comprising a pemphigus antigen immobilised by covalent attachment to a substrate,

2. An immunoadsorption matrix according to claim 1 , wherein the pemphigus antigen is a desmogiein 3 (dsg3) polypeptide.

3. An immunoadsorption matrix according to claim 1 , wherein the pemphigus antigen is a desmogiein 1 (dsgi) polypeptide.

4. An immunoadsorption matrix according to any preceding claim, wherein a dsgi and a dsg3 polypeptide are immobilised by covalent attachment to the substrate.

5. An immunoadsorption matrix according to any preceding claim, wherein the substrate is a polymer bead.

6. An immunoadsorption matrix according to any preceding claim, wherein the pemphigus antigen is bound to between 5 and 40% of the surface of the substrate.

7. An immunoadsorption matrix according to claim 6, wherein the pemphigus antigen is bound to 10% of the surface of the substrate

8. An immunoadsorption matrix according to any preceding claim, wherein the pemphigus antigen comprises an extracellular domain of dsgi or dsg3,

9. An immunoadsorption matrix according to claim 8, wherein the pemphigus antigen comprises extracellular domains 1 to 5 of dsgi or dsg3.

10. An immunoadsorption matrix according to any preceding claim, wherein the pemphigus antigen is produced by mammalian cells.

11. An immunoadsorption matrix according to any preceding claim, wherein the pemphigus antigen further comprises a sortase A recognition motif.

12. A solid-state device comprising an immunoadsorption matrix according to any preceding claim.

13. An apheresis column comprising an immunoadsorption matrix according to any preceding claim.

14. A method of making an immunoadsorption matrix according to any preceding claim comprising the steps of reacting a functionalised substrate and the pemphigus antigen and washing the substrate to remove unreacted pemphigus antigen.

15. A method according to claim 14, wherein the functionalised substrate is an amine-functionaiised substrate having di-glycine on its surface, the reaction further comprises sortase A enzyme, and the reaction is carried out under conditions that permit sortase A activity.

16. A method according to claim 14 or claim 15, wherein the pemphigus antigen is a desmogiein 3 (dsg3) polypeptide.

17. A method according to claim 14 or claim 15, wherein the pemphigus antigen is a desmogiein 1 (dsg1 ) polypeptide.

18. A method according to any one of claims 14 to 17, wherein a dsg1 and a dsg3 polypeptide are immobilised by covalent attachment to the substrate.

19. A method according to any one of claims 14 to 18, wherein the substrate is a polymer bead.

20. A method according to any one of claims 14 to 19, wherein the pemphigus antigen is bound to between 5 and 40% of the surface of the substrate.

21 , A method according to any one of claims 14 to 20, wherein the pemphigus antigen is bound to 10% of the surface of the substrate

22, A method according to any one of claims 14 to 21 , wherein the pemphigus antigen comprises an extracellular domain of dsg1 or dsg3.

23, A method according to claim 22, wherein the pemphigus antigen comprises extracellular domains 1 to 5 of dsg1 or dsg3.

24, A method according to any one of claims 14 to 23, wherein the pemphigus antigen is produced by mammalian cells.

25, A method according to any one of claims 14 to 24, wherein the pemphigus antigen further comprises a sortase A recognition motif.

26, A method according to any one of claims 14 to 25, wherein the pemphigus antigen is present in the reaction mixture at a concentration of 17.5mg per mg of beads, 10mg per mg of beads, 5mg per mg of beads, 2.5mg per mg of beads or 1 mg per mg of beads.

27, A method of removing anti-pemphigus antigen antibodies from serum isolated from a subject suffering from pemphigus comprising the steps of contacting the isolated serum with an immunoadsorption matrix comprising a pemphigus antigen immobilised by covalent attachment to a substrate under conditions that allow anti-pemphigus antigen antibodies to bind to the pemphigus antigen, and separating the serum from the immunoadsorption matrix.

28, A method according to claim 27, wherein the pemphigus antigen is a desmoglein 3 (dsg3) polypeptide.

29, A method according to claim 27, wherein the pemphigus antigen is a desmoglein 1 (dsg1 ) polypeptide.

30. A method according to any one of claims 27 to 29, wherein a dsg1 and a dsg3 polypeptide are immobilised by covalent attachment to the substrate.

31. A method according to any one of claims 27 to 30, wherein the substrate is a polymer bead.

32. A method according to any one of claims 27 to 31 , wherein the pemphigus antigen is bound to between 5 and 40% of the surface of the substrate.

33. A method according to claim 32, wherein the pemphigus antigen is bound to 10% of the surface of the substrate

34. A method according to any one of claims 27 to 33, wherein the pemphigus antigen comprises an extracellular domain of dsg1 or dsg3.

35. A method according to claim 34, wherein the pemphigus antigen comprises extracellular domains 1 to 5 of dsg1 or dsg3.

36. A method according to any one of claims 27 to 35, wherein the pemphigus antigen is produced by mammalian cells.

37. A method according to any one of claims 27 to 36, wherein the pemphigus antigen further comprises a sortase A recognition motif.

38. A method according to any one of claims 27 to 37, further comprising measurement of anti-pemphigus antigen antibody levels in the isolated serum.

39. A method according to any one of claims 27 to 38, further comprising optimisation of the duration of contact between the serum and the immunoadsorption matrix such that more than 60% of anti-pemphigus antigen antibody is removed from the isolated serum.

40. A method according to any one of claims 27 to 39, wherein the subject is a human patient suffering from pemphigus vulgaris or pemphigus foiiaceus.