GB2541027A - Treatment - Google Patents

Abstract

A method of identifying whether a subject having a B-cell disease is suitable to be treated with intravenous immunoglobulin (IVIg) or in need of treatment with IVIg. The method comprising detecting the presence or absence of a monoclonal immunoglobulin associated with the B-cell disease wherein if the monoclonal immunoglobulin is IgA, IgD, IgM, light chain only, the B-cell disease is non-secretory multiple myeloma (MM) or if the concentration of total IgG is below a pre-determined amount in a sample from the subject, the patient is suitable to be treated with IVIg or if the amount of polyclonal IgG is below a predetermined amount, the patient is in need of treatment with IVIg. Also claimed is a method of monitoring IVIg levels in a subject comprising monitoring the IgGλ lambda to IgGκ kappa ratio or the concentration of IgGλ lambda or IgGκ kappa in a subject.

Description

Treatment

The invention relates to methods of identifying whether a subject having a malignant plasma cell disease, such as multiple myeloma, is suitable to be treated with intravenous immunoglobulin (IVIg) and to methods of monitoring such treatments.

Antibody molecules (also known as immunoglobulins) have a twofold symmetry and are composed of two identical heavy chains and two identical light chains, each containing variable and constant domains. 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 a disulphide bond. This produces a substantially Ύ"-shaped molecule.

Heavy chains are the larger of the two types of chain found in antibodies, with typical molecular mass of 50,000-77,000 Da, compared with the smaller light chain (25,000 Da).

There are five main classes or isotypes of heavy chain which are y, a, μ, δ and ε which are the constituents heavy chains for: IgG, IgA, IgM, IgD and IgE respectively. IgG is the major 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.

There are two types of light chain: Lambda (λ) and Kappa (k). There are approximately twice as many κ as λ molecules produced in humans, but this is quite different in some mammals. Each chain contains approximately 220 amino acids in a single polypeptide chain that is folded into one constant and one variable domain. Plasma cells produce one of the five heavy chain types together with either κ or λ molecules. There is normally approximately 40% excess light chain production over heavy chain synthesis. Where the light chain molecules are not bound to heavy chain molecules, they are known as "free light chain molecules". The κ light chains are usually found as monomers. The λ light chains tend to form dimers.

There are a number of proliferative diseases associated with antibody producing cells. Figure 2 shows the development of the B-cell lineage and associated diseases. These diseases are known as B-cell diseases. They are summarised in detail in the book "Serum-free Light Chain Analysis plus Hevylite", 7th edition, (2015) available from The Binding Site Group Limited, Birmingham, UK (ISBN: 9780993219603).

In many such proliferative diseases a B-cell proliferates to form a monoclonal tumour of identical B-cells. This can result in production of large amounts of identical immunoglobulins and is known as a monoclonal gammopathy.

Diseases such as multiple myeloma and primary systemic amyloidosis (AL amyloidosis) account for approximately 1.5% and 0.3% respectively of cancer deaths in the United Kingdom. Multiple myeloma is the second-most common form of haematological malignancy after non-Hodgkin lymphoma. In Caucasian populations, the incidence is approximately 40 per million per year. Conventionally, the diagnosis of multiple myeloma is based on the presence of excess monoclonal plasma cells in the bone marrow, monoclonal immunoglobulins in the serum or urine and related organ or tissue impairment such as hypercalcaemia, renal insufficiency, anaemia or bone lesions. Normal plasma cell content of the bone marrow is about 1%, while in multiple myeloma the content is typically greater than 30%, but may be over 90%. AL amyloidosis is a protein conformation disorder characterised by the accumulation of monoclonal free light chains and fragments as amyloid deposits. Typically, these patients present with heart or renal failure but peripheral nerves and other organs may also be involved.

There are a number of other diseases which can be identified by the presence of monoclonal immunoglobulins within the blood stream, or indeed urine, of a patient. These include plasmacytoma and extramedullary plasmacytoma, a plasma cell tumour that arises outside the bone marrow and can occur in any organ. When present, the monoclonal protein is typically IgA. Multiple solitary plasmacytomas may occur with or without evidence of multiple myeloma. Waldenstrom's macroglobulinaemia is a low-grade lymphoproliferative disorder that is associated with the production of monoclonal IgM. There are approximately 1,500 new cases per year in the USA and 300 in the UK. Serum IgM quantification is important for both diagnosis and monitoring. B-cell non-Hodgkin lymphomas cause approximately 2.6% of all cancer deaths in the UK and monoclonal immunoglobulins have been identified in the serum of about 10-15% of patients using standard electrophoresis methods. Initial reports indicate that monoclonal free light chains can be detected in the urine of 60-70% of patients. In B-cell chronic lymphocytic leukaemia monoclonal proteins have been identified by free light chain immunoassay.

Additionally, there are so-called MGUS conditions. These are monoclonal gammopathy of undetermined significance. This term denotes the unexpected presence of a monoclonal intact immunoglobulin in individuals who have no evidence of multiple myeloma, AL amyloidosis, Waldenstrom's macroglobulinaemia, etc. MGUS may be found in 1% of the population over 50 years, 3% over 70 years and up to 10% over 80 years of age. Most of these are IgG- or IgM-related, although more rarely IgA-related or bi-clonal. Although most people with MGUS die from unrelated diseases, MGUS may transform into malignant monoclonal gammopathies.

In at least some cases for the diseases highlighted above, the diseases present abnormal concentrations of monoclonal immunoglobulins or free light chains. Where a disease produces the abnormal replication of a plasma cell, this often results in the production of more immunoglobulins by that type of cell as that "monoclone" multiplies and appears in the blood.

Under normal circumstances serum proteins that are too large for renal filtration (>65kDa) have a half life of 2 to 3 days and are removed by pinocytosis. IgG (and albumin) though has a prolonged half life of 20-25 days because of FcRn receptors. Such receptors are also known as neonatal or Brambell receptors (see Kim et al, Clin Immunol, (2007), 122, 146-55; Akilesh et al, J. Immunol, (2007) 179, 4580-8).

Heterodimeric FcRn receptors protect both IgG and albumin from acid digestion and recycle them both to cell surfaces to be released into the slightly alkaline environment of the blood. This process occurs many times under normal circumstances resulting in the half lives of IgG and albumin extending from about 3 to 21 days.

In contrast, other classes of immunoglobulins such as IgA and IgM, do not have such a recycling system so consequentially have shorter half lives. Serum free light chains (sFLC) have half lives 200-300 fold shorter than IgG and are predominantly cleared by renal clearance.

Patients producing high levels of IgG (though not other types of immunoglobulins, such as IgA), saturate the FcRn receptors causing excess IgG to be broken down in 3 days instead of the 20-25 days normally associated with IgG. Since increasing polyclonal IgG and monoclonal IgG both result in a progressively shortening half life, rising levels in for example MM, result in progressively shortening half life of IgG. A major problem associated with disease such as multiple myeloma (MM) is that up to 10% of newly diagnosed MM patients die within 60 days of their diagnosis, often due to infections. Myeloma patients are predisposed to infection because of immunoparesis with poor response to vaccination, neutropenia, lymphocytopenia, placement of catheters and impaired mucosal integrity owing to the effect of chemotherapy/radiotherapy (see for example Augustson et al J. Clin. Oncol. (2005), 23(36) pages 9219-9226, Perri et al Am. J. Med (1981) 71, 935-940). A number of techniques have been tried to reduce the incidence of infection, including using prophylactic antibiotics. The role of intravenous gamma globulin replacement (or intravenous immunoglobulin-IVIg) has been assessed for newly diagnosed patients with varying degrees of success.

Salmon et al (New England J. Med (1967) 1336-1340) reported trying immunoprophylaxis on newly diagnosed patients with MM using gamma globulin. Patients were compared to a control group who had been given a placebo. No significant difference in the rates of infection between the group treated with gamma globulin and that of the control group was reported.

Chapel et al (The Lancet (1994), 343, 1059-1063 and Clin. Exp. Immunol (1994) 97 (suppl. 1) Pages 21 - 24) disclosed treating a small number of patients in “plateau phase” MM. This is also known as stable phase MM and is when the myeloma becomes stable after chemotherapy and shows no sign of progressing. IVIg was observed to be protective against life threatening infections. Early, progressive or terminal MM were not included in that study. Moreover, the results of this trial were contradicted in a letter by Snowden et al (The Lancet (1994). 344, page 212). This stated that in true plateau phase MM, infections were low, so did not justify the use of prophylactic IVIg.

Since then IVIg has not been routinely utilised in treating myeloma patients.

At times IVIg can be a limiting resource, in the UK the Department of Health has provided clinical guidelines on immunoglobulin use (July 2011). The use of IVIg in patients with MM is rare and falls under the second tier utility in the demand management system, section ‘Secondary antibody deficiency (any cause)’. To qualify for IVIg treatment the patient must have hypogammoglobulinaemia associated with the MM, confirmed by a haematologist along with satisfying three further criteria: Recurrent or severe bacterial infection despite continuous oral antibiotic therapy for 3 months, IgG <5 g/L (excluding paraproteins) and documented failure of serum antibody response to unconjugated pneumococcal or other polysaccharide vaccine challenge. Newly diagnosed patients are treated as soon as possible following diagnosis, other than IgG concentration this would not allow time to determine the above criteria.

The Applicant has now identified why the administration of IVIg has not been successful. They believe it is due to high levels of monoclonal IgG, affecting the recycling system that, under normal circumstances, ensures a long lifespan for IVIg administered to patients.

If IVIg (which is essentially IgG) is put into a patient with an overloaded FcRn receptor recycling system when the patient has high levels of IgG in their serum, it results in the half life of the IVIg being 3 or so days, rather than the 21 days normally expected. This is because the IVIg is not recycled by the overloaded system.

The Applicant has realised that selecting patients according to the amount of monoclonal and polyclonal IgG in their blood, would allow those most suited to be treated with IVIg to be identified. Typically, patients having less than 5g/L polyclonal (functional) IgG would be anticipated to benefit from having this concentration increased and more so for those patients with < lg/L or < 0.5g/L. However, patients with >30g/L total IgG (for example) would only have a short-lived benefit from an IVIg infusion because the therapeutic IgG would be rapidly catabolised.

The invention provides a method of identifying whether a subject having a B cell disease is suitable to be treated with intravenous immunoglobulin (IVIg) or in need of treatment with IVIg, comprising detecting the presence or absence of a monoclonal immunoglobulin associated with the B cell disease, wherein if the monoclonal immunoglobulin is IgA, IgD, IgM, light chain only, the B cell disease is non-secretory multiple myeloma (MM) or if the concentration of total IgG is below a predetermined amount in a sample from the subject, the patient could benefit from being treated with ivlg because it will persist in the blood for a reasonable time. Additionally, if the polyclonal IgG is below a certain concentration it would indicate that the patient is in need of treatment.

If the polyclonal IgG of the HLC class not produced by the tumour (functional IgG) is below, for example, 5 g/L, or below 2 g/L or below 1 g/L or below 0.5 g/L the subject is in need of treatment. If the total IgG is below, for example, 20-30 g/L the subject is more likely to benefit from the treatment. The total IgG concentration also indicates how frequently they are likely to require treatment. The amount of polyclonal IgG in a patient with monoclonal IgG production can be inferred. For example, in a patient with monoclonal IgGK you would typically measure IgGX.

Typical normal reference ranges for healthy individuals are:

Normal adult serum Mean Median 95 Percentile Range

IgG kappa (g/L) 7.10 6.75 3.84-12.07

IgG lambda (g/L) 3.95 3.90 1.91-6.74

IgG kappa /IgG 1.84 1.74 1.12-3.21 lambda ratio

Normal adult serum Mean Median 95 Percentile Range

IgA kappa (g/L) 1.35 1.37 0.57-2.08

IgA lambda (g/L) 1.18 1.25 0.44-2.04

IgA kappa/ IgA 1.20 1.18 0.78-1.94 lambda ratio

Normal adult serum Mean Median 95 Percentile Range

IgM kappa (g/L) 0.71 0.63 0.19- 1.63

IgM lambda (g/L) 0.39 0.35 0.12- 1.01

IgM kappa/IgM 1.85 1.81 1.18-2.74 lambda ratio

The B cell disease may be selected from MGUS (monoclonal gammopathy of Undetermined Significance) AL amyloidosis, Waldenstrom’s macroglobulinaemia, plasmacytoma or multiple myeloma (MM), most preferably MM.

Having low polyclonal IgG levels means that the subject is prone to infection. Hence the invention also provides a method of identifying whether a subject with a B-ccll disease is prone to infection, comprising detecting the concentration of polyclonal IgG in a sample from the patient, wherein if the concentration of IgG is below a pre-determined amount, the patient is prone to infection.

The methods of the invention may additionally comprise monitoring the light chain types bound to the IgG, for example to monitor IgGicIgGX ratio in an IgG myeloma patient. This allows the persistence/longevity of the IVIg to be monitored, by virtue of the change in the ratio as IVIg is lost from the patient. An individual light chain type may be measured. For example in a subject with a monoclonal IgGK, the levels of IgGX may be measured.

The invention allows early identification of patients in need of treatment before they have infections or other complications. The patient may have had multiple myeloma diagnosed less than 2 or 3, or less than 12 months previously or has not had reoccurring bacterial infections for less than 1, 2 or less than 3 months.

Methods of monitoring the longevity of IVIg in IgG myeloma patients by monitoring the IgGicIgGX ratio or concentrations of IgGK or IgGX are also provided. This monitoring can be used to indicate intervention, ie further IVIg infusions, to ensure minimum IVIg trough levels are maintained.

The monoclonal immunoglobulin may be detected using specific binding entities such as antibodies. These include, for example, an anti-heavy chain class antibody, or an antibody having heavy chain-class (HLC), light chain type specificity, (Hevylite™ antibodies from The Binding Site, Birmingham, United Kingdom). Fragments of antibodies, such as F(ab)2 fragments may be used. WO 2006/079816, describes such antibodies.

The monoclonal immunoglobulin may be quantitatively detected, for example via a sandwich assay such as ELISA, internal flow devices, bead based systems such as Luminex by nephelometry, or turbidimetry or flow cytometers.

Preferably, the sample is obtained from tissue or fluid, such as blood, plasma or serum from blood of an animal, such as a mammal, preferably a human. Urine may also be used. Additionally, it may be possible to identify such proteins in urine. Preferably the sample is assayed in vitro.

The heavy chain class detected may be selected from IgA, IgG, IgM, IgD and IgE. The antibodies may also be subclass specific.

In an alternative aspect of the invention, two different parts of the immunoglobulin to be detected are bound by the antibodies used in the assay. One antibody binds a part of the heavy chain responsible for heavy chain class determination. The second binds a part of the light chain responsible for identifying it as a K or λ chain.

Hence, preferably the measurement is determined using: (i) at least one antibody, or a fragment thereof, specific for the heavy chain class; (ii) an antibody, or a fragment thereof, specific for λ light chains; and (iii) an antibody, or a fragment thereof, specific for K light chains.

The polyclonal levels of the relevant isotype, the same isotype of the monoclonal protein, may also be estimated by subtracting the measured monoclonal concentration (for example determined by SPE/CZE), from the total Ig concentration of the respective isotype (determined by immunoassay such as nephelometry). However, being a large amount (monoclonal concentration) with a little polyclonal Ig) such calculations are susceptible to error and challenge the accuracy of the methods employed. Therefore, typically either: (a) in the case of IgG this may be the estimated amount from the gammaglobulin region of the SPE (serum plasma electrophoresis) and subtracting the monoclonal concentration as calculated by densitometry. This can also be carried out by immunosubtraction using CZE (capillary zone electrophoresis).

Or, more preferably: (b) direct measurement of a component of the polyclonal isotype (uninvolved HLC pair) using, for example, Hevylite type antibodies as this may be a better indicated of polyclonal Ig production of the respective isotype.

The presence of an abnormal heavy chain class - light chain type ratio (HLC), such as IgAXdgAK ratio infers the production of monoclonal immunoglobulin.

The presence of the specific antibodies bound to these immunoglobulins may be determined using a labelled second antibody. For example, the binding antibody may be a sheep antibody. The immunoglobulins detected may be human immunoglobulins. Hence the presence of sheep antibodies bound to the human immunoglobulin may be determined using anti-sheep antibodies, e.g. from rabbit or horse.

The antibodies used in the assay may be heavy chain subclass specific. For example, anti-IgA (IgAl and IgA2) and anti-IgG (such as IgGl, IgG2, IgG3 or IgG4) antibodies are made by The Binding Site, Birmingham, United Kingdom, under the trademark Hevylite™. This gives more detailed knowledge of the disease being detected.

Monoclonal or polyclonal antibodies may be used. Polyclonal antibodies are preferably used. This allows an improved assay to be produced to monitor different monoclonal immunoglobulins of, for example, the same class. Polyclonal antibodies allow some variability between different heavy chains of the same class to be detected because they are raised against a number of parts of the heavy chain.

The method of the invention may also be used using one or more of the following methods wherein the binding of the antibodies to the immunoglobulins in the sample is determined by using a nephelometer, a turbidimeter, flow cytometry, ELISA or fluorescently labelled beads such as Luminex™ beads. Alternatively, a microarray assay may be produced using the antibodies.

Gel electrophoresis may be used to measure the monoclonal Ig immunoglobulin.

Preferably the measurement is determined by immunoassay, most preferably via an immunosorbent assay such as ELISA (Enzyme Linked Immunosorbent Assay). ELISA-type assays per se are well known in the art. They use antibodies, or fragments of 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 to detect specific antigens. One or more of the antibodies used in the assay may be labelled with an enzyme capable of converting a substrate into a detectable product. Such enzymes include horseradish peroxidase, alkaline phosphatase and other enzymes known in the art. Alternatively, other detectable tags or labels may be used instead of, or together with, the enzymes. These 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.

The construction of ELISA-type assays is itself well known in the art. For example, a "binding antibody" specific for the immunoglobulin is immobilised on a substrate. In this case, the immunoglobulin comprises a heavy chain of a particular class, or subclass, attached to either a λ light chain or a K light chain. The "binding antibody" may be immobilised onto the substrate by methods which are well known in the art. Immunoglobulins in the sample are bound by the "binding antibody" which binds the immunoglobulin to the substrate via the "binding antibody".

Unbound immunoglobulins may be washed away.

In ELISA assays the presence of bound immunoglobulins may be determined by using a labelled "detecting antibody" specific to a different part of the immunoglobulin of interest than the binding antibody.

Preferably, flow cytometry is used to detect the binding of the immunoglobulins of interest. 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 λ light chains. The detecting antibody is preferably labelled with a detectable label, which binds the immunoglobulin to be detected in the sample. This results in a labelled bead when the immunoglobulin 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-λ and anti-κ antibodies, or anti-IgG and anti-IgA antibodies. This allows the amount of each type of immunoglobulin bound to be determined simultaneously and allows the rapid identification of the κ:λ ratio or a given heavy chain class.

Alternatively, or additionally, 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.

Flow cytometry allows rapid identification of the κ/λ ratios or identifies a given heavy chain class or subclass.

An alternative method uses the antibodies bound to, for example, fluorescently labelled beads such as commercially available Luminex™ beads. Different beads are used with different antibodies. Different beads are labelled with different fluorophore mixtures, thus allowing the λ/κ ratio for a particular heavy chain class or subclass to be determined by the fluorescent wavelength. Luminex beads are available from Luminex Corporation, Austin, Texas, United States of America.

The amount of immunoglobulin may be compared to a predetermined normal amount of the protein. Typically, the predetermined amount of polyclonal IgG is 5g/L serum.

The sample may be serum, blood or plasma from the subject.

The invention also provides a method of treating a patient with B-cell disease comprising administering to a patient with a malignant plasma cell disease IVIg, wherein the patient has monoclonal immunoglobulin associated with the disease selected from:

IgA, IgD, IgM, light chain only, IgG below a predetermined amount in a sample of a subject, or the B cell disease is a non-secretory multiple myeloma .

The IVIg typically contains pooled IgG extracted from plasma of donors. It is generally known to be used to treat auto immune diseases such as dermatomyositis, Kawasaki’s disease, pediatric HIV infection and Guillain-Barre syndrome.

Typical dosages are 100 to 400 mg/kg body weight daily. A higher dose of up to 2g per kilogram of body weight may be used in acute cases, similar to prior uses of the treatment in other diseases.

Commercial sources include Baxter UK, who produce intravenous immunoglobulin under the trade name “Gammagard” or “KIOVIG”.

The monoclonal immunoglobulin may be IgA or IgG as described above.

The invention will now be described by way of example only.

Figure 1. Concentration/frequency plots for the uninvolved Igs for each patient group (black bars). Comparison is made with concentrations of the uninvolved HLC concentrations and total IgG, IgA or IgM concentrations in reference populations (grey bars). This illustrates the degree of suppression for all immunoglobulins and also indicates that the suppression of the alternate IgG heavy/light pair (eg. the suppression of IgG kappa production in IgG lambda patients) is proportionally greater.

Figure 2: Plot of IgGx v IgGk for 146 blood donor sera (Black diamonds) and 245 (166 IgGx (blue squares to the right of the parallel lines), 79 IgGX (red squares to the left of the parallel lines) presentation sera from the IFM 2005-01 multiple myeloma trial. The parallel lines indicate the 95%ile range for IgG*: / IgGk ratios. There was a strong negative correlation between the tumour produced immunoglobulin levels and the alternative HLC pair (IgG κ MM: Pearson’s -0.456, p<0.0001; IgG λ MM: Pearson’s -0.310, p=0.005). This illustrates in a different way, the extent of the heavy/light pair suppression in IgG patients that was also shown in figure 1.

Methods

Patients and serum samples. Patients from the Inter Groupe Francais du Myelome (IFM) 2005-01 MM trial were studied with the exclusion of those with FLC-only disease. All samples were taken at the time of initial clinical presentation and patients were monitored for progression-free survival (PFS) and overall survival (OS). A total of 339 patients were evaluated, comprising 245 expressing IgG (166 IgGK, 79 IgGk) and 94 expressing IgA (60 IgAK, 34 IgAk). Patients received bortezomib (Velcade®) and dexamethasone or VAD (vincristine, adriamycin, and dexamethasone) as induction therapy plus or minus dexamethasone, cyclophosphamide, etoposide and cisplatin (DCEP), followed by a stem cell autograft as first line therapy. Serum samples were collected at clinical presentation and stored at -80°C until analysis.

Laboratory methods

Total IgG, IgA and IgM plus IgGK, IgGk, IgAK and IgAk were measured by nephelometry using specific sheep antisera, and IgGK/IgGk, IgAK/IgAX ratios were calculated (Hevylite™, Binding Site Ltd., UK) (Bradwell et al, 2009). HLC normal ranges were obtained from analysis of blood donor samples. Serum FLCs were similarly measured by nephelometry using polyclonal sheep antisera bound on latex particles to enhance sensitivity (Freelite™, Binding Site Ltd., UK) (Bradwell et al, 2001). All analyses were performed on a Dade Behring BN™II nephelometer (Siemens GmbH, Germany) at Binding Site Ltd., in Birmingham, UK. Medians and 95% ranges were assessed. The degree of systemic humoral immune-suppression was determined from concentrations of non-tumour Igs (i.e. IgG and IgM in IgA patients and IgA and IgM in IgG patients). Reference ranges and medians for IgG, IgA and IgM were obtained from a general adult population (Gonzalez-Quintela et al, 2007). The following measurements were made in centres in France at the time of sample collection: β2-Μ and albumin by nephelometry, M-spike by SPEP densitometry (Sebia, France) and the cytogenetic markers Del: 13, t4:14, and Del:17p were analysed by fluorescence in-situ hybridisation (FISH) (Facon et al, 2001).

Ethics permission

All sera were studied under permission for analysing samples from the IFM trials.

Results

Reference ranges of blood donor samples used for the HLC assays are shown in Table I and were similar to those previously reported (Bradwell et al, 2009; Katzmann et al, 2009a).

Table I. Immunoglobulin concentrations in reference range samples. *IgG, IgA and IgM concentrations were from a published normal population study (Gonzalez-Quintela et al, 2007).

The concentrations of HLC Igs (with medians and 95% ranges) in the patient population, together with other protein measurements are shown in Table II. For individual patients a high degree of correlation existed between monoclonal Ig concentrations by SPEP densitometry, HLC assays for the tumour derived Igs (involved) and total Ig assays. Pearson’s correlations comprised: SPEP vs total Ig assays, 0.87, p=9xl0~5, SPEP vs HLC involved, 0.80, p<10'10, HLC involved vs total Ig assays, 0.87, p<10"10. However, median concentrations varied between methods, with monoclonal IgG results measured by nephelometry giving the highest estimates of monoclonal IgCiK and IgGX (Table II, line 5). For monoclonal IgAK and IgAX, the three assays produced more consistent results. For the four HLC assays IgGK, IgGX, IgAK and IgAX (Table II, line 2), median concentrations of the involved Igs were broadly similar (30.1g/L-36.7g/L) and the 95% ranges were consistent between patient groups (16- to 25-fold between lower and upper values). In contrast, concentrations of uninvolved HLC Igs (Table II line 3) were more variable (0.072g/L -0.41g/L) and the 95% ranges were wider at 28-fold for IgGK to 120-fold for IgAX.

Table II. Concentrations of serum immunoglobulins and other proteins in 339 patients with multiple myeloma showing median values together with 95% ranges. β:-Μ - serum p2-microglobulin. FLC - serum free light chains. HLC ratio - serum concentrations of IgK/IgX. Ig by SPEP-densitometry - monoclonal Ig concentrations from scanning densitometry of serum protein electrophoresis. NR - normal range. * see Table 1 for normal ranges

Discussion

This study shows the levels of IgGkappa, IgGlambda, IgAkappa and IgAlambda in patients with myeloma.

Serum IgG molecules are removed from the circulation by a concentration dependent process, so that measurements do not reliably relate to tumour production. IgG Fc receptors (FcRn), located on most nucleated cells, recycle IgG many times so that the half-life is extended from 3 to 21 days at normal serum concentrations. At high IgG concentrations, FcRn receptors are saturated, causing the excess IgG to be catabolised in 3 days. Consequently, the overall half-life of IgG is a balance between 21 days for the component that is recycled and 3 days for the component that is rapidly catabolised. Since polyclonal IgG and monoclonal IgG are affected equally, rising IgG concentrations in MM result in a progressively shortening half-life (Kim et al, 2007; Roopenian et al, 2007). This mechanism provides an explanation for the inverse relationship between IgGK and IgGk concentrations for both types of IgG patients seen in Fig 1. This figure also shows that fewer samples have uninvolved IgG concentrations above the median normal range value compared with IgA samples, which is further evidence supporting the concentration-dependent catabolism. A clinical consequence of these observations relates to the therapeutic use of intra venous Ig (IVIg). Studies have shown no reduction of infections in MM (Chapel et al, 1994). The half-life of the IVIg would be so short in the IgG patients that it would be effective for only a few days. This was proportional to the concentration of monoclonal IgG protein. In contrast, in the IgA patients, it would have a prolonged half-life and is likely to be useful.

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Claims (12)

1. A method of identifying whether a subject having a B-cell disease is suitable to be treated with intravenous immunoglobulin (IVIg) or in need of treatment with IVIg comprising detecting the presence or absence of a monoclonal immunoglobulin associated with the B-cell disease wherein if the monoclonal immunoglobulin is IgA, IgD, IgM, light chain only, the B-cell disease is non-secretory multiple myeloma (MM) or if the concentration of total IgG is below a pre-determined amount in a sample from the subject, the patient is suitable to be treated with IVIg or if the amount of polyclonal IgG is below a predetermined amount, the patient is in need of treatment with IVIg

2. A method of identifying whether a subject with a B-cell disease is prone to infection comprising detecting the concentration of polyclonal IgG in a sample from the patient, wherein if the concentration of IgG is below a pre-determined amount, the patient is prone to infection.

3. A method according to claim 1, wherein the B-cell disease is multiple myeloma.

4. A method according to any preceding claim wherein the monoclonal immunoglobulin or polyclonal IgG is identified by use of anti-heavy chain class antibodies and anti light chain antibodies, or antibodies having heavy chain-class, light chain type specificity, (HLC) and the presence of an abnormal HLC ratio.

5. A method according to any preceding claim, wherein the method comprises detecting the ratio or concentration, using anti-IgGX and anti-IgGK-specific antibodies.

6. A method according to any preceding claim where the method comprises using nephelometry or turbidimetry.

7. A method of treating patients with malignant plasma cell disease comprising administering to a patient IVIg, wherein the patient has monoclonal immunoglobins associated with the disease selected from: IgA, IgD, IgM, light chain only, total IgG below a predetermined amount, or the malignant plasma cell disease is a non-secretory multiple myeloma.

8. A method of monitoring a method of treatment according to any preceding claim, comprising monitoring the IgGX: IgGK ratio in the subject, or, the concentration of IgGX or IgGK in the subject.

9. A method according to any preceding claim wherein the monoclonal immunoglobin is IgA or IgG.

10. A method according to any preceding claim wherein the predetermined amount of polyclonal IgG is 5g/L serum.

11. A method according to any preceding claim, wherein the amounts of different immunoglobulins are detected by sandwich immunoassays.

12. A method of monitoring IVIg levels in a subject, comprising monitoring the IgG λ or IgG κ ratio in the subject, or the concentration of IgG λ or IgG κ in the subject.