Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6854—Immunoglobulins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins

Definitions

• the invention relates to assay kits and methods for detecting or monitoring IgG4-related disease, using antibodies or binding agents such as antibodies specific for immunoglobulins, the immunoglobulins comprising an IgG class and a heavy chain subclass, further characterised by being bound to a kappa or lambda light chain class or multiple light chain classes.
• Compositions and methods of using the assay for example in the detection of an IgG4-related disease, are also provided.
• Antibody molecules also known as immunoglobulins
• the variable domains of the heavy and light chains combine to form an antigen- binding site, so that both chains contribute to the antigen-binding specificity of the antibody molecule.
• the basic tetrameric structure of antibodies comprises two heavy chains covalently linked by a disulphide bond. Each heavy chain is in turn attached to a light chain, again via disulphide bond. This produces a substantially "Y"-shaped molecule.
• Heavy chains are the larger of the two types of chain found in antibodies, with typical molecular weight of 50,000-77,000 D, compared with the smaller light chain (25,000 D).
• IgG immunoglobulin of normal human serum, accounting for 70-75% of the total immunoglobulin pool. This is the major antibody of secondary immune responses. It forms a single tetramer of two heavy chains plus two light chains.
• IgM accounts for approximately 10% of the immunoglobulin pool.
• the individual heavy chains have a molecular weight of approximately 65,000 and the whole molecule has a molecular weight of about 970,000.
• IgM is largely confined to the intravascular pool and is the dominant antibody produced in the primary immune response.
• IgA represents 15-20% of human serum immunoglobulin pool. More than 80% of IgA occurs as a monomer. However, some of the IgA (secretory IgA) exists as a dimeric form. IgD is present on the plasma membranes of mature B-lymphocytes and accounts for less than 1% of the total immunoglobulin in the plasma.
• IgE is the least common serum immunoglobulin isotype, and IgE is most commonly found bound to Fc receptors on surface of mast cells and basophils.
• IgG IgG
• IgG2 IgG2
• IgG3 and IgG4 subclasses for IgA
• IgAl and IgA2 In healthy adult serum, IgGl, IgG2, IgG3 and IgG4 account for 60-70%, 14-20%, 4-8% and 2-6% of the total IgG pool respectively. These percentages may be altered in certain disease types.
• IgG4 is therefore the least abundant IgG subclass in human plasma but has a 100-fold normal concentration range of 0.01-2. lg/1, and a mean range of 0.35-0.51 mg/ml. IgG4 constant regions show 95% amino acid sequence similarity with other IgG subclasses. IgG4 has a half- life of approximately 21 days and is recycled by a neonatal fragment crystallisable receptor (FcRn) mediated process.
• FcRn neonatal fragment crystallisable receptor
• the classic antibody paradigm is that a single mature plasma cell produces one type of immunoglobulin heavy chain ⁇ , ⁇ , ⁇ , ⁇ and ⁇ in humans) and one type of immunoglobulin light chain ( ⁇ or ⁇ ). These are combined within the cell to produce a tetrameric molecule composed of two identical heavy chains and two identical light chains.
• IgG4 molecules are dynamic and can exchange half molecules to become hybrid bi-specific (monovalent) antibodies. This process may also be known as "Fab arm exchange" and was first characterised in a study which focused on the anti-inflammatory properties of IgG4 in potential immunotherapy (van der Neut Kolfschoten et al 2007).
• IgG4 does not activate the classical and alternative complement pathways and has only a weak affinity for macrophage and phagocyte Fc receptors.
• Fab arm exchange effectively renders the antibody monovalent and thus, for the proportion of IgG4 that has undergone Fab arm exchange, limits the ability of IgG4 to cross link antigens.
• IgG4 molecules that have undergone Fab arm exchange may have two different kappa light chains one bound to each heavy chain, two different lambda light chains one bound to each heavy chain or one kappa light chain and one lambda light chain bound to their respective heavy chains. The latter is referred to as the IgG4K/ mixed hybrid molecule. Therefore, upregulation of IgG4 could potentially suppress an immune response.
• IgG4 production is normally associated with prolonged exposure to antigens and the increase of T helper 2 (Th2) cytokines which mediate allergic responses and IgE production. Unlike IgE, IgG4 does not exhibit high affinity binding to mast cells and basophils. IgG4 has been reported to interact with antibodies of the IgG and IgE classes through their Fc domains; analogous to rheumatoid factor interactions.
• Th2 T helper 2
• Fab arm exchange involves the swapping of a heavy-light chain pair (half antibody) between different IgG4 molecules, possibly reforming new disulphides in the hinge region but without disruption of the heavy-light chain disulphide bond.
• an asymmetrical or hybrid immunoglobulin can be produced as described above (also see Figure 1).
• a serine residue at position 228 in the core hinge of IgG4 that allows the formation of intra-chain rather than inter-chain disulphides, and an arginine at position 409 within the CH 3 domain have been implicated in the control of this mechanism.
• Alternative amino acids at both of these positions as found in other IgG subclasses, and in low abundance in IgG4 isoallotype molecules, abolishes or reduces the exchange process.
• IgG4 has isoallotypes at position 309, whereby leucine is changed to a valine (L309/V309), and at position 409, whereby arginine is changed to a lysine (R409/K409).
• the arginine residue R409, in the R409 IgG4 isoallotype, enables the IgG4 Fab arm exchange, whereas the lysine residue K409, in the K409IgG4 isoallotype results in it resembling IgGl , IgG2 and IgG3 which do not undergo Fab arm exchange.
• Half molecule exchange may play a physiological role as these naturally produced bi- specific molecules cannot cross link antigens or elicit lymphoid responses, which may dampen the inflammatory response.
• the Fab arm exchange effectively renders the IgG4 antibody monovalent and thus limiting the ability to cross link antigens in vitro.
• the ⁇ light chains usage bias in IgG4 is much higher than for the IgG class per se, and is reported to be approximately 5: 1 respectively (Young et al. 2014).
• the IgG heavy chain specific class, IgG4 can therefore exist as three different forms determined by the light chains bound to the heavy chains.
• the first form exists as an IgG4 molecule bound to two ⁇ light chains, one attached to each of the two heavy chains (IgG4K).
• the second form exists as an IgG4 molecule bound to two ⁇ light chains, one attached to each of the two heavy chains (IgG ).
• the other isoform exists as an IgG4 molecule bound to two light chains, one being a ⁇ light chain and the other being a ⁇ light chain (IgGK/ ⁇ ), as a so-called IgGK/ ⁇ mixed hybrid molecule.
• IgGK/ ⁇ mixed hybrid molecules The exact proportion of IgGK/ ⁇ mixed hybrid molecules to IgGK and IgG molecules that have not undergone fab arm exchange varies widely between healthy subjects, but is expected to represent a significant proportion of total IgG4.
• IgG4K/ mixed hybrid molecules are reported to be represent between 20-30% of the total IgG4 immunoglobulins; similar values are reported in both Young et al. 2014 (21-33%) and Silva et al. 2015 (28%).
• IgG4 antibody production is associated with a number of proliferative, autoimmune and chronic inflammatory diseases which feature infiltration of target organs by IgG4- expressing cells. These diseases are known collectively as IgG4-related diseases. Chronic exposure to an antigen also results in the increase of serum IgG4. It has been proposed, in Karagiannis et al. 2013, that the increase in IgG4 levels diverts the humoral immune response away from an IgE-dominated response. However, it is unknown if IgG4 is pathogenic or mediates the host's response in IgG4-related diseases. The epidemiology of IgG4-related diseases is relatively unknown, although the incidence in the Japanese population is reported to be 0.28- 1.08 per 100,000 individuals.
• IgG4-related diseases comprise a collection of disorders which share specific pathological, serological and clinical features.
• the common features may comprise tumour- like swelling of organs, a lymphoplasmacytic infiltrate enriched in IgG4-positive plasma cells and CD4+T lymphocytes, obliterative phlebitis and a degree of fibrosis which has a characteristic "storiform" pattern typified by a cartwheel appearance of arranged fibroblasts and inflammatory cells. Modest tissue eosinophilia is also common. Subjects often present with the development of a mass in an organ or diffuse enlargement of an organ. Multiple organs are affected in 60 to 90% of subjects.
• Elevated serum concentrations of IgG and IgG4 are found in 60 to 70% of subjects with IgG4-related disease.
• the upper limit of normal for IgG4 for an adult human is approximately 2. lg/1.
• the increased severity of IgG4-related disease correlates with an increased amount of serum IgG4.
• the sensitivity of IgG4 measurement in IgG4-related diseases ranges from approximately 50-100%, although the sensitivity of IgG4 measurement in many of these diseases has not been determined.
• subjects are responsive to glucocorticoids, therefore glucocorticoid responsiveness has been considered one diagnostic criteria for these disorders.
• IgG4-levels decrease in response to glucocorticoid treatment.
• the common organs affected in IgG4-related disease in order of frequency are: pancreas, salivary gland, kidney, lacrimal gland, aorta, bile duct, lung, paravertebra, orbita, retroperitoneum and artery (Inoue et al. 2015).
• IgG4-related disorders occur more commonly in middle-aged and older men. However, IgG4-related disorders of organs of the head and neck are seen equally in both males and females.
• the major disorders contained within IgG4-related disease are Type 1 IgG4-related autoimmune pancreatitis, salivary gland disease (Mikilukz's disease), orbital disease and retroperitoneal fibrosis. IgG4-related disease also appears to increase the risk of malignancy, particularly lymphomas and gastric cancers.
• IgG4-related disorders The pathogenesis of IgG4-related disorders remains largely unknown. Elevations in serum and tissue IgG4 are not specific to IgG4-related diseases; this may also occur in other disorders such as multicentric Castleman's disease, allergic disorders and Churg-Strauss syndrome. An allergic response will often include increased serum levels of IgE, and increased levels of Th2 cytokines, IL-10 and TGF-beta in affected tissues. Additionally, subjects with IgG4-related disease have an increased prevalence of allergic rhinitis and bronchial asthma. Up to 40% of subjects with an IgG4-related disease have a peripheral eosinophilia.
• IgG4-related disease is currently diagnosed upon a radiological finding or histopathological examination of a tissue sample.
• the tissue sample is extracted using an invasive core needle biopsy.
• the tissue sample is observed for signs of lymphoplasmacytic tissue infiltration by IgG4-positive plasma cells and lymphocytes, accompanied by fibrosis which has storiform features and obliterative phlebitis.
• Tissue IgG4-positive cell counts and the ratio of IgG4- negative to IgG4-positive cells are also considered. However, cell counts vary widely between tissues and subjects. PET, CT and MRI investigation is also recommended to identify areas of fibrotic tissue. Serum total IgG4 levels may be measured but are not regarded for diagnosis alone, as they are considered neither sufficiently sensitive or specific for IgG4-related disease.
• IgG4-related disease were made in patients with autoimmune pancreatitis, which can be mistaken for pancreatic cancer. Elevated IgG4 may be seen in pancreatic cancer, although to a lesser degree than in autoimmune pancreatitis. Steroid treatment for type 1 autoimmune pancreatitis results in a decrease in IgG4, therefore differentiating the disease from pancreatic cancer. Therefore, measuring IgG4 may be beneficial in ensuring pancreatic cancer is not autoimmune pancreatitis and vice versa.
• IgG4-related sclerosing cholangitis but not primary sclerosing cholangitis and/or cholangiocarcinoma, which may be beneficial in diagnosis as biopsies are seldom deep enough to define the histological features of IgG4-related sclerosing cholangitis.
• IgG4 has also been shown to be present in IL-10-driven Th2 immune response in some inflammatory diseases. Th2-mediated inflammation is also usually indicative of tumour growth. IgG4 has been shown to infiltrate tumour cells and accumulate around tumours in melanoma (Karagiannis et al 2014). IgG4 has also been shown to inhibit the antitumor function of IgGl, possibly due to IgG4 binding to tumour antigens in the absence of Fc receptor binding and effectively dampening the anti-tumour immune function of IgGl by blocking further non IgG4 antibody binding to the tumour antigens.
• IgG4 may also inhibit the anti-tumour function of IgGl via antagonism of Fc receptors (Karagiannis JCI 2013).
• This study is focused on the ratio of IgG4:total IgG as indicative of a switch to production of the IgG4 class in melanoma.
• the study also highlights the need to avoid stimulating IgG4 immunoregulation in designing future therapeutics. IgG4 is therefore regarded as having immunosuppressive effects and low cytotoxic potential.
• the Applicants have previously developed a sensitive assay that can detect the free ⁇ light chains and separately, the free ⁇ light chains.
• This method uses a polyclonal antibody directed towards either the free ⁇ or the free ⁇ light chains.
• the possibility of raising such antibodies was also discussed as one of a number of different possible antigens, in W097/17372.
• This document discloses methods of tolerising an animal to allow it to produce desired antibodies that are more specific than prior art techniques could produce.
• the free light chain assay (FreeliteTM) uses the antibodies to bind to free ⁇ or free ⁇ light chains.
• the concentration of the free light chains is determined by nephelometry or turbidimetry. This form of assay is highly sensitive. Indication of clonal production of light chains, such as in light chain multiple myeloma, can be provided by identifying an abnormal ratio of free kappa to free lambda light chains in the serum.
• Measuring antibody ratios assists in the diagnosis and monitoring of diseases. Furthermore, if the disease is treated, the technique allows the progression of the disease to be monitored. If the disease is successfully being treated, then the concentrations of the free light chains, which have a relatively short life span within the blood will change and move more towards the normal concentrations observed for normal sera.
• the Applicants have also produced antibodies and assays (known as HevyliteTM) that would be able to distinguish between, for example, IgG ⁇ and IgG ⁇ , as disclosed in EP 1842071 and EP2306202. They produced antibodies which are specific for intact immunoglobulins and which had specificity for both a heavy chain class and a light chain type. They have also produced assays that allow the rapid quantitative measurement of, e.g. IgG and IgGK ratios to allow the rapid identification and/or follow the progression of diseases associated with production of a specific heavy chain class, or even heavy chain subclass, in conjunction with a bound ⁇ or ⁇ chain, as disclosed in WO2011021041.
• HevyliteTM antibodies and assays that would be able to distinguish between, for example, IgG ⁇ and IgG ⁇ , as disclosed in EP 1842071 and EP2306202. They produced antibodies which are specific for intact immunoglobulins and which had specificity for both a heavy chain class and a light chain type. They have also
• the inventors By determining the composition of immunoglobulins using an antibody specific to a heavy chain class at the same time as a light chain type or by using a first antibody against a heavy chain class and a second antibody to determine the light chain type bound to the heavy chain, the inventors produced a sensitive assay for specific immunoglobulin related diseases.
• the assays developed allow more sensitive progression of the diseases than, for example, by SPE.
• Heavy chain-light chain specific assays have been produced by the Applicant as HevyliteTM.
• HevyliteTM uses a capture antibody which is heavy chain-light chain specific and is able to differentiate between immunoglobulins with a particular heavy chain class bound to either ⁇ or ⁇ light chains.
• HevyliteTM is unable to differentiate on its own between IgG4 mixed hybrid molecules from IgG4 molecules with only ⁇ or ⁇ light chains as it requires a highly specific anti-IgG4 capture antibody. If HevyliteTM was used to measure the amount of IgG4 molecules bound to ⁇ light chains in a sample, it would give a positive result for every IgG4 molecule that is bound to a K chain. This would include the single type ⁇ light chain IgG4 and the mixed hybrid IgG4, which also contains a ⁇ light chain and is therefore an entirely different class of molecule.
• HevyliteTM is not able to discriminate on its own between the hybrid IgG4 and the single type light chain IgG4, leading to a misrepresentative measurement of the increase of the single type light chain IgG4.
• HevyliteTM is also concerned with the detection of expansion of monoclonal heavy-chain-light-chain specific antibodies in malignant plasma cell diseases. IgG4 only represents a very small proportion of total IgG (-4%) and it is therefore highly rare that a malignant plasma cell disease will result in an expansion of IgG4 molecules. Therefore, HevyliteTM when used on its own would not be suitable for use in diagnosing or monitoring an IgG4-related disease.
• both HevyliteTM assays yielded a positive result upon detecting the hybrid, causing those molecules to be counted twice.
• Both the HevyliteTM assay with an anti- ⁇ capture antibody and the HevyliteTM assay with an anti- ⁇ capture antibody showed detection of the IgG4K/ mixed hybrid and neither equated to the total amount, thus demonstrating an impact on the assay calibration.
• the Applicant has realised that there are advantages in having an assay which is able to detect immunoglobulins in a sample from a subject which does not require initial purification steps. This reduces the amount of time taken to achieve a result and is less labour intensive. It also removes the need to possess specialist equipment and techniques such as affinity columns and HPLC, and is therefore ideal for use in the clinic.
• IgG4K/ mixed hybrids Although the presence and measurement of IgG4K/ mixed hybrids is known in the field, there has been no evidence that their measurement is useful. It has been reported that individuals with IgG4-related disease do not have a substantially increased frequency of the K409 variant of IgG4 which compromises Fab arm exchange (Ahmad et al. 2014). This suggests that mixed hybrid molecule dynamics are unaffected in IgG4-related disease, and would not, therefore, be selected in the diagnosis or monitoring of an IgG4-related disease. Another study has reported that therapeutic antibodies engage in Fab arm exchange with endogenous human IgG4 in vivo, but does not comment on the clinical relevance of this (Labrijn et al.
• IgG4 antibodies are known to antagonise the complement fixing action of IgGl antibodies, thereby dampening down inflammation, the Applicants speculate that the proportion of 3 ⁇ 4 ⁇ 4 ⁇ / ⁇ hybrids may alter this effect. IgG4 antibodies also appear to interfere with antibody mediated cell killing via Fc receptor binding (Karagiannis et al. 2014).
• IgG4 mixed hybrid antibodies One approach to purify and analyse IgG4 mixed hybrid antibodies has been recently reported (Yang et al. 2015). Purified human IgG was mixed with IgG4 monoclonal antibodies and the amount of half molecule exchange that occurred was detected by the separation of IgG4 mixed hybrid antibodies by mixed mode chromotography. IgG4 mixed hybrid antibodies which underwent half molecule exchange with the IgG4 monoclonal antibodies were then quantitatively measured using UV absorption or protein fluorescence. This method involves several purification steps of both the IgG sample from a human subject and the production of monoclonal IgG4 antibodies.
• the Applicants realised it was possible to develop a highly specific quantitative serological assay to detect the presence of IgG4i ⁇ A. hybrids from an unpurified sample directly from a subject. All the previous attempts in the prior art to analyse the percentage of IgG4i ⁇ A. have used lengthy protocols involving purified fractions of IgG4 and subsequent SDS-PAGE or immunoprecipitation analysis. Using their knowledge of heavy light chain specific antibodies, the Applicants have developed an immunoassay which identifies the ratio of IgG4K/ hybrids to total IgG4 in order to diagnose or monitor an IgG4-related disease such as autoimmune pancreatitis or pemphigus.
• a first aspect of the invention provides a method of detecting or monitoring an IgG4-related disease comprising detecting in a sample the ratio between the relative amounts of antibodies having: (i) an IgG heavy chain class; and
• immunoglobulins having the same heavy chain class but bound to either ⁇ light chains only or ⁇ light chains only;
• the method comprising the quantitative detection of said immunoglobulins comprising:
• the method preferably quantitatively measures the amounts of the immunoglobulins in the sample.
• Immunoglobulins bound to binding agent may be washed to remove unbound immunoglobulins.
• Step (iii), as described above, may be used to determine the total amount of an IgG4 heavy chain class which is bound to either kappa light chains or lambda light chains.
• the total amount of IgG4 is determined using step (i).
• the total amount of an IgG4 heavy chain class which is bound to both a kappa and a lambda light chain is also determined using step (ii) and is subtracted from the total amount of IgG4 from step (i).
• the remaining fraction indicates the total amount of an IgG4 heavy chain class which is bound to either kappa light chains or lambda light chains.
• the method of the invention may be determined using:
• binding agent specific for the IgG heavy chain class (i) at least one binding agent specific for the IgG heavy chain class; and a) a binding agent specific for the IgG heavy chain class bound to ⁇ light chains in combination with a binding agent specific for ⁇ light chains; and/or b) a binding agent specific for the IgG heavy chain class bound to ⁇ light chains in combination with a binding agent specific for ⁇ light chains; and/or c) a binding agent specific for the IgG heavy chain class bound to ⁇ light chains in combination with a binding agent specific for the IgG heavy chain class bound to ⁇ light chains.
• An IgG4 - specific binding agent may be bound to the IgG4, which is also bound to an IgG4K- specific binding agent.
• the amount of the binding may be configured to a calibration curve obtained for predetermined concentrations of IgG4i ⁇ A. mixed hybrid.
• Figure 2 shows two worked examples of the combination of an anti-IgG4K capture antibody and an anti- ⁇ detecting antibody, or an anti-IgG4 capture antibody and an anti- ⁇ detecting antibody to detect the IgG4i ⁇ A. mixed hybrid.
• the binding agent specific for the immunoglobulin to be detected is preferably an antibody or fragment thereof, or an aptamer.
• the fragments of antibody used in all aspects of the invention may be Fab or F(ab')2 fragments.
• Aptamers are short single stranded DNA or RNA molecules with high affinity and specificity, and may be referred to as "nucleic acid antibodies”.
• the heavy chain class to be detected may be selected from IgGl, IgG2, IgG3 and IgG4, most preferably IgG4. A combination of all classes may also be detected as total IgG.
• the sample may optionally be enriched for the IgG heavy chain subclass before use in the detection method by affinity purification or adsorption.
• the method of the invention may also be used using one or more of the following methods wherein the binding of the binding agents to the antibodies in the sample is determined by using an ELISA (Enzyme Linked Immunosorbent Assay), flow cytometry or fluorescently labelled beads such as LuminexTM beads.
• ELISA Enzyme Linked Immunosorbent Assay
• flow cytometry fluorescently labelled beads
• fluorescently labelled beads such as LuminexTM beads.
• assay methods may be singleplex (measures one analyte of interest) or multiplex (measures multiple analytes of interest).
• a microarray assay may be produced using the specific binding agents.
• the total amount of a particular IgG heavy chain class, or the total amount of IgG detected as described above is preferably measured as part of a multiplex ELISA assay, or as a singleplex assay to be carried out alongside the detection steps of either a), b) and/or c) as described above.
• the ratio of IgG -K/ mixed hybrid to total IgG4, or IgG4-K/ mixed hybrid to total IgG is determined immunologically, most preferably via ELISA.
• ELISA-type assays per se are well known in the art. They use specific binding agents such as antibodies to detect blood groups, cell surface markers, drugs and toxins. In the case of the current invention, this type of assay has been used for the method of the invention.
• ELISA uses antibodies or other binding agents such as aptamers, or fragments of antibodies to detect specific antigens.
• One or more of the antibodies, aptamers or fragments of antibodies used in the assay may be labelled with an enzyme capable of converting a substrate into a detectable analyte.
• enzymes include horseradish peroxidase, alkaline phosphatase and other enzymes known in the art. Alternatively, other detectable tags or labels may be used.
• radioisotopes include radioisotopes, a wide range of coloured and fluorescent labels known in the art, including fluorescein, Alexa fluor, Oregon Green, BODIPY, rhodamine red, Cascade Blue, Marina Blue, Pacific Blue, Cascade Yellow, gold; and conjugates such as biotin (available from, for example, Invitrogen Ltd, United Kingdom).
• Dye sols, metallic sols or coloured latex may also be used.
• One or more of these labels may be used in the ELISA assays according to the various inventions described herein, or alternatively in the other assays, labelled antibodies or kits described herein.
• a "binding antibody” specific for the antigen is immobilised on a substrate.
• the antigen is an antibody comprising an IgG heavy chain, or an IgG heavy chain of a particular subclass, attached to either a ⁇ light chain or a ⁇ light chain, or both a ⁇ light chain or a ⁇ light chain.
• the "binding antibody” may be immobilised onto the substrate by methods which are well known in the art. Antigens in the sample are bound by the "binding antibody” which binds the antigen to the substrate via the "binding antibody”. Unbound antibodies may be washed away.
• the presence of bound antibodies may be determined by using a labelled "detecting antibody” specific to a different part of the antigen of interest than the binding antibody.
• Flow cytometry may be used to detect the binding of the antibodies of interest and measure the ratios. This technique is well known in the art for, e.g. cell sorting. However, it can also be used to detect labelled particles, such as beads, and to measure their size. Numerous text books describe flow cytometry, such as Practical Flow Cytometry, 3rd Ed. (1994), H. Shapiro, Alan R. Liss, New York, and Flow Cytometry, First Principles (2nd Ed.) 2001, A.L. Given, Wiley Liss.
• One of the binding antibodies such as the antibody specific for the heavy chain class, is bound to a bead, such as a polystyrene or latex bead.
• the beads are mixed with the sample and the second detecting antibody, such as antibody specific for ⁇ light chains.
• the detecting antibody is preferably labelled with a detectable label, which binds the antibody to be detected in the sample. This results in a labelled bead when antibody to be assayed is present.
• Labelled beads may then be detected via flow cytometry.
• Different labels such as different fluorescent labels may be used for, for example, the anti- ⁇ and anti- ⁇ antibodies. This allows the amount of each type of antibody bound to be determined simultaneously and allows the rapid identification of the ⁇ / ⁇ hybrid:single type ⁇ or ⁇ ratio for a given heavy chain class when carried out in combination with the identification of the total amount of a given heavy chain class.
• different sized beads may be used for different antibodies, for example for different class specific antibodies. Flow cytometry can distinguish between different sized beads and hence can rapidly determine the amount of each heavy chain class in a sample.
• An alternative method uses the antibodies bound to, for example, fluorescently labelled beads such as commercially available LuminexTM beads. Different beads are used with different antibodies. Different beads are labelled with different fluorophore mixtures, thus allowing the single type or hybrid type ratio for a particular heavy chain class or subclass to be determined by the fluorescent wavelength.
• LuminexTM beads are available from LuminexTM Corporation, Austin, Texas, United States of America.
• the immunoglobulin of interest may also be detected using a homogenous time resolved fluorescence (HTRF) assay platform.
• Homogenous assays require a simple mix and read procedure without the necessity for multiple processing steps such as separation or washing.
• HTRF technology combines fluorescence resonance energy transfer (FRET) with time resolved measurement (TR) of fluorescence.
• FRET relies on the energy transfer between two fluorophores which come into close contact upon the interaction of one protein with another, both of which have either a donor fluorophore attached, or an acceptor fluorophore attached.
• the donor fluorophore which has a long duration of fluorescence
• the protein of interest from the sample, the 3 ⁇ 4 ⁇ 4 ⁇ / ⁇ mixed hybrid antibody, would then bind to the donor labelled anti-IgG4K antibody.
• An anti- ⁇ antibody could then be added, which may have an acceptor fluorophore attached (which has a short duration of fluorescence), which would also bind to the 3 ⁇ 4 ⁇ 4 ⁇ / ⁇ mixed hybrid antibody.
• the anti-IgG4K antibody and the anti- ⁇ antibody come into close proximity, such as that upon binding to the IgG4i ⁇ A. mixed hybrid, the level of energy transfer between the donor and acceptor fluorophores can be detected as the emission of fluorescence. This information can be used to indicate the amount of a particular protein of interest in a sample.
• the immunoglobulin of interest may also be detected using a lateral flow assay platform, preferably a lateral flow sandwich assay platform.
• a lateral flow sandwich assay uses coloured or fluorescent particles which are used to label binding agents, typically an antibody.
• the labelled antibody is used to detect the antigen of interest from a sample which passes through a series of capillary beds.
• the labelled antibody is usually immobilised to the surface of one of the capillary beds and forms an antigen-antibody complex upon encounter of the antigen of interest in the sample.
• the matrix immobilising the antibody to the capillary bed dissolves and the antigen-antibody complex is free to migrate further where it becomes bound to a second specific binding agent, typically an antibody.
• a coloured/fluorescent band may be observed as the accumulation of the antigen- antibody particles as an indicator of the presence or amount of the antigen in the sample.
• the sample is obtained from tissue or fluid, such as whole blood, plasma or serum from the blood of an animal, such as a mammal, preferably a human and additionally rhesus monkeys (Macaca mulatta), cynomolgus macaques (Macaca fascicularis), rabbits
• tissue or fluid such as whole blood, plasma or serum from the blood of an animal, such as a mammal, preferably a human and additionally rhesus monkeys (Macaca mulatta), cynomolgus macaques (Macaca fascicularis), rabbits
• mice may exhibit light chain mixed hybrid antibodies of another class, such as IgG3.
• tissue or fluid may be taken from a specific area within the body, such as the local tissue or fluid surrounding a tumour, or fluid within a skin blister. Preferably the sample is assayed in vitro.
• human antibodies may be determined using anti-human antibodies, e.g. from sheep, horse, goat, donkey, rabbit, chicken, mouse or rat.
• Measurement of the heavy chain-light chain specific pair or hybrid pair is capable of being automated. Furthermore, the technique is more sensitive and allows the quantitative determination of the amount of different immunoglobulins. It can be used both to aid diagnosis of a disease and also to monitor the response of the disease to treatment, such as glucocorticoid treatment. It may be used to detect the presence of a tumour in an IgG4-related disease. It may also be used in prognosis of future disease progression and the risk of survival of a subject. These values may be calculated as a percentage of risk. The method may also be used to monitor the response of an allergy to treatment. It may also be used to indicate whether a specific antigen has been removed from the environment of a subject.
• IgG4-related disease may be known as: IgG4-related systemic disease, IgG4- syndrome, IgG4-associated disease, IgG4-related sclerosing disease, IgG4-related systemic sclerosing disease, IgG4-related autoimmune disease, IgG4-positive multiorgan lymphoproliferative syndrome, hyper- IgG4 disease, systemic IgG4-related plasmacytic syndrome, , IgG4-related multifocal systemic fibrosis, multifocal fibrosclerosis, or multifocal idiopathic fibrosclerosis.
• the IgG4-related disease is selected from: Type 1 (IgG4-related) autoimmune pancreatitis, IgG4-related sclerosing cholangitis, Mikulicz's disease (or IgG4-related dacryoadenitis and sialadenitis), sclerosing sialadenitis (or Kiittner's tumor, IgG4-related submandibular gland disease), IgG4-related orbital inflammation or IgG4-related orbital inflammatory pseudotumor, chronic sclerosing dacryoadenitis (or lacrimal gland enlargement, IgG4-related dacryoadenitis), retroperitoneal fibrosis (or Ormond's disease) and related disorders (IgG4-related retroperitoneal fibrosis, IgG4-related mesenteritis), chronic sclerosing aortitis and periaortitis (or IgG4-related a) autoimmune
• IgG4-related diseases The common organs affected in IgG4-related disease in order of frequency are: pancreas, salivary gland, kidney, lacrimal gland, aorta, bile duct, lung, paravertebra, orbita, retroperitoneum and artery. Additionally, cancers such as melanomas, or allergies, may exhibit elevated IgG4 and may be considered IgG4-related diseases.
• the method or kit described herein may include co-assaying for IgG4-related disease inflammatory markers such as VEGF, IL-10, TGF- ⁇ or Th2 cytokines.
• the presence of the IgG4 isoallotype K409 is usually tested for using a method such as PCR and post-PCR analysis. If the subject has a diploid K409 mutation, there will be no IgG4 mixed hybrid molecules to detect. If the subject has a haploid K409 mutation, the proportion of 3 ⁇ 4 ⁇ 4 ⁇ / ⁇ mixed hybrid molecules and IgG4 single type light chain molecules will be disrupted and reduced.
• Figure 6A shows clear outliers when detecting the ratio of IgG4 to IgG4K/ mixed hybrid molecules. Therefore, the identification of these outliers may be used to identify subjects with either a haploid or diploid K409 mutation.
• the method may also be used to determine the amount or proportion of an IgG4 heavy chain class bound to both a ⁇ light chain and a ⁇ light chain antibody in synthetic antibody preparations, such as monoclonal antibodies produced as biologicals for immunotherapy, comprising detecting in a sample of the synthetic antibody preparation the ratio of two or more of the relative amounts of immunoglobulins having:
• immunoglobulins having the same heavy chain class but bound to either ⁇ light chains only or ⁇ light chains only; the method comprising the quantitative detection of said immunoglobulins by a binding assay comprising:
• Polyclonal antibodies are preferably used, additionally monoclonal antibodies may also be used. Polyclonal antibodies allow an improved assay to be produced to monitor different immunoglobulins of, for example, the same class. Polyclonal antibodies allow a plurality of different antibodies to be raised against different epitopes for the specific IgG heavy chain- light chain combination. This allows for the slight variations between different immunoglobulins, but which nevertheless comprise the same IgG heavy chain-light chain combination.
• the polyclonal antibodies used in the various aspects of the invention may be capable of being produced by the method shown in WO 97/17372. This allows the production of highly specific polyclonal antibodies.
• the sample may be further characterised by measuring the amount of free ⁇ or free ⁇ light chains in the sample.
• the total amount of ⁇ and ⁇ free light chains may also be measured.
• a ratio may also be produced of ⁇ to ⁇ free light chains. This is preferably carried out using antibodies specific for free ⁇ or free ⁇ light chains, such as those sold under the trade mark FreeliteTM and CombyliteTM by The Binding Site Ltd, Birmingham, UK.
• the resultant ratio value from the detection of total IgG or IgG heavy chain subclass specific versus IgG heavy chain subclass specific bound to both ⁇ and ⁇ light chains may be compared to a normal range or a reference value for each IgG4-related disease type.
• the normal range will typically have a median ratio of 0.328, a mean ratio of 0.358 with a standard deviation of 0.1272.
• the ratio value may also be used to further characterise an IgG4-related disease type according to a range specific for that disease.
• a further aspect of the invention provides an assay kit, preferably an ELISA, comprising:
• a further aspect of the kit may comprise a predetermined amount of an IgG4i ⁇ A. mixed hybrid calibrator.
• the opposite light chain class is considered to be kappa when the binding agent specific for the IgG4 heavy chain class is bound to a lambda light chain, and the opposite light chain is considered to be lambda when the binding agent specific for the IgG4 heavy chain class is bound to a kappa light chain.
• a further aspect of the kit is for use in a method wherein the ratio of IgG4K/ mixed hybrid to total IgG4, or IgG4i ⁇ A. mixed hybrid to total IgG, is detected between two or more of the relative amounts of immunoglobulins having: (i) at least one binding agent specific for the IgG heavy chain class; and
• kits for use in a method wherein the amount of IgG4-K/ mixed hybrid molecule is detected and compared to a calibrator, wherein the kit comprises :- a) a binding agent specific for ⁇ light chains; and
• the calibrator is used to calibrate the assay.
• substantially pure hybrid IgGK/ ⁇ is diluted with a suitable buffer to form the calibrator agent.
• the binding agent or detecting agent provided in the kit may be an antigen specific aptamer, an antibody or antigen-specific fragment thereof.
• the antibodies, labels, etc. are preferably as described above.
• the antibody specific for the IgG heavy chain class or the antibody specific for the IgG4 heavy chain class bound to a specific light chain class is immobilised to a substrate.
• the substrate may be a bead, but preferably is a microtitre plate well.
• One or more of the antibodies preferably comprises a detectable label.
• Indirect labels such as enzymes (e.g. alkaline phosphatase and horseradish peroxidase) can be used, as can radioactive labels such as 35 S.
• Each type of detecting antibody may be labelled with a different detectable label if used in combination, for example, in a multiplex assay.
• One or more controls such as a known amount of a predetermined monoclonal protein, such as purified IgG, IgG4 or IgG4K/ , or a fragment thereof, may be provided in this and indeed other ELISA, flow cytometry, LuminexTM or other assays described herein.
• the calibrator is typically linked to an international standard concentration.
• the assay may report in standard units compared to these values.
• the fragments, when used will retain, e.g. antigenic determinants for detecting class and/or light chain type.
• kits with capture and Luminex beads are also provided.
• these kits comprise an antibody or fragments thereof, or additionally aptamers, specific for the IgG heavy chain class, and/or an IgG heavy chain subclass, and/or specific for the IgG4 heavy chain class bound to a specific light chain class, and each of the types of different antibodies, if used in combination, are attached to a different size of bead.
• the kit additionally comprises a labelled antibody for detecting the presence of antibodies from a sample bound, via the antibodies of the method, to the bead.
• kits of the invention may additionally comprise antibodies specific for free ⁇ or free ⁇ light chains.
• anti-total free light chain (FLC) antibodies for measuring total FLC concentration may be provided or used to additionally measure total FLC, for example against a predetermined standard.
• kits may additionally comprise one or more of instructions for using the kit, substrate, a buffer, label, a preservative or a control.
• Figure 1 shows a schematic diagram of IgG4 half-molecule exchange.
• Table 1 shows immunoassay quantification of the purified polyclonal IgG4 Kappa, IgG4 Lambda and mixed light-chain IgG4 hybrids from normal human sera (taken from Young et al 2014). IgG4 ⁇ / ⁇ hybrids are positively detected by both Hevylite G assays.
• Figure 2 shows two worked examples of the combination of an anti-IgG4K capture antibody and an anti- ⁇ detecting antibody, or an anti-IgG4 capture antibody and an anti- ⁇ detecting antibody to detect the IgG4i ⁇ A. mixed hybrid.
• Figure 3 shows a 5 point calibration curve using the current optimised assay parameters.
• the working range is 18.5-1500mg/L using a 1/5000 sample dilution.
• the calibrator is pure IgG4MM purified (quantified by Optilite G4 assay), the calibrator diluent is IgG4 depleted serum diluted 1/5000, the sample diluent is standard ELISA buffer T271, the conjugate is a- K-Perox diluted 1/3000. All steps are carried out at 30mins at RTP.
• Figure 4 shows three samples corresponding to the lower middle and higher portions of the calibration curve which were tested. The percentage CV within sets of samples is shown. This demonstrates good inter-assay precision. Three regions of the calibration curve were targeted (25, 85, 580 mg/L IgG4MM) and there were 16 replicates per sample.
• Figure 5A shows total IgG4 levels in 90 healthy controls (outliers removed) using EIA.
• Figure 5B shows total IgG4 ⁇ / ⁇ mixed hybrid levels in 90 healthy controls (outliers removed) using EIA.
• Figure 5A shows the ratio of total IgG4 ⁇ / ⁇ mixed hybrid levels to total IgG4 levels in 90 healthy controls (outliers removed) using EIA.
• Figure 6A shows a linear plot of total IgG4 and IgG4 ⁇ / ⁇ mixed hybrid with the inclusion of identified outliers.
• Figure 6B shows a linear plot of total IgG4 and IgG4 ⁇ / ⁇ mixed hybrid without the inclusion of identified outliers.
• Figure 7 shows the ratios of total IgG4 to total IgG4 ⁇ / ⁇ mixed hybrid using EIA from clinical samples from patients with known IgG4-related diseases; Pemphigus/Pemphigoid and Autoimmune pancreatitis type l(AIP).
• EIA Enzyme immunoassay
• IgG4 ⁇ / ⁇ hybrid levels were measured using antigen capture EIA.
• EIA plates MaxisorpTM flat-bottom, clear, 96-well plates; Nunc, Roskilde, Denmark
• PBS phosphate-buffered saline
• the plates were blocked with StabilcoatTM, (SurModics, Eden Prairie, MN, USA) for 30 min at 22°C.
• StabilcoatTM (SurModics, Eden Prairie, MN, USA) for 30 min at 22°C. After removal of the block, the plates were dried under vacuum, before sealing in a foil pouch containing desiccant sacs. The diluted patient sample was added across all the strips in duplicate.
• IgG4 ⁇ / ⁇ hybrid in IgG4 depleted serum (1/5000) was used as a calibrator and triply diluted from 300ng/ml to 3.7ng/ml.
• Serum samples were diluted at 1/5000 sample dilution in PBS containing 0.1% TweenTM (PBS-T) sample diluent and incubated for 30 min at 22°C. After washing (Bio-Teck instruments Inc, VT, USA) with PBS containing 0.1% TweenTM-T bound IgG4 ⁇ / ⁇ hybrids were detected by 1/3000 dilution of anti-kappa-HRP (BindingSite, UK) in 10% StabliZyme-HRP (SurModics) in saline.
• TMB tetramethylbenzidine
• IgG4 levels were measured using antigen capture EIA.
• EIA plates were coated overnight with monoclonal anti-IgG4 clone HP6025 (Sigma Aldrich) at 5.0 ⁇ g/ml in phosphate-buffered saline (PBS) in a moist box at 22 °C. Following removal of the coat solution the plates were blocked with StabilcoatTM, for 30 min at 22°C. After removal of the block, the plates were dried under vacuum, before sealing in a foil pouch containing desiccant sacs. The diluted patient sample was added across all the strips in duplicate.
• PBS phosphate-buffered saline
• a turbimetric IgG4 calibrator fluid was diluted in sample diluent to produce a range from 339.33ng/ml to 1.40ng/ml.
• Serum samples were diluted at 1/20000 sample dilution in PBS containing 0.1% TweenTM (PBS-T) sample diluent and incubated for 30 min at 22°C. After washing with PBS containing 0.1% TweenTM-T bound IgG4 was detected by 1/8000 dilution of anti-IgG-HRP (Binding Site, UK) in 10% StabliZyme-HRP in saline. After a further 30 min incubation and washing, the plates were developed using TMB solution, and the absorbance was measured at 450 nm (Bio-Teck EL800 micro plate reader). The calibration range obtained is equivalent to 27.93 to 6787 mg/1 Results
• the 95% reference range as determined by a non- parametric percentile method (CLSI C28-A3) was 28.9 to 1603 mg/L, which is within the expected published range for serum IgG4.
• the IgG4 ⁇ / ⁇ hybrid levels were between 16.309mg/L and 494.3mg/L with a median of 84.085mg/L , a mean of 108.284mg/L and a standard deviation of 82.7324 (Figure 5B).
• the 95% reference range as determined by a non- parametric percentile method (CLSI C28-A3) was 20.3 to 460 mg/L.
• the IgG4 ⁇ / ⁇ hybrid to total IgG4 ratio had a range between 0.163 and 0.9450 with a median of 0.328, a mean of 0.353 and a standard deviation of 0.127525 (Figure 5C).
• the Total IgG4 and IgG4 ⁇ / ⁇ hybrid EIAs were also used with clinical samples from known IgG4-related diseases; Pemphigus/Pemphigoid and Autoimmune pancreatitis type l(AIP) ( Figure 7).
• Pemphigus/Pemphigoid was found to be between 141.3mg/L and 429.3mg/L with a median of 234.2mg/L, a mean of 257.9mg/L and a standard deviation of 118.7.
• the IgG4 ⁇ / ⁇ hybrid levels were found to be between 28.35mg/L and 111.2mg/L with a median of 57.33mg/L , a mean of 66.18mg/L and a standard deviation of 30.89.
• the IgG4 ⁇ / ⁇ hybrid to total IgG4 ratio had a range between 0.2000 and 0.3400 with a median of 0.2500, a mean of 0.2580 and a standard deviation of 0.05119.
• IgG4 and IgG4 ⁇ / ⁇ hybrid EIA Nine AIP samples were analysed using the IgG4 and IgG4 ⁇ / ⁇ hybrid EIA.
• the total IgG4 was found to be between 16138mg/L and 59478mg/L with a median of 26405mg/L, a mean of 35421mg/L and a standard deviation of 117514.
• the IgG4 ⁇ / ⁇ hybrid levels were found to be between 4100mg/L and 18711mg/L with a median of 7929mg/L, a mean of 9996mg/L and a standard deviation of 5359.
• the IgG4 ⁇ / ⁇ hybrid to total IgG4 ratio had a range between 0.2400 and 0.3100 with a median of 0.2800, a mean of 0.2778 and a standard deviation of 0.02635.

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