DK161098B - Antibodies directed against modified beta2-microglobulin, processes for detecting modified beta2-microgloblin, and pharmaceutical preparations comprising such antibodies - Google Patents

Description

in

DK 161098 B

The invention generally relates to antibodies directed against modified B2 microglobulins (nd32m) of the formula set forth below, which antibodies may be poly- or monoclonal, methods for detecting m / 2m, and pharmaceutical compositions containing anti-microf2m antibodies.

The modified S2 microglobulins against which antibodies are raised in accordance with the invention have the general formula I:

RrCys-R 2 -X

10 I I

Y-R3-Cys-R4 wherein R1 is a 24 amino acid group having the sequence: Ile-Gln-Arg-Thr-Pro-Lys-Ile-Gln-Val-Tyr-Ser-Arg-His-Pro-Ala-Glu-Asn Gly-Lys-Ser-Asn-Phe-Leu-Asn-, R2 is a 30 amino acid group having the sequence: -Tyr-Val-Ser-Gly-Phe-His-Pro-Ser-Asp-Ile-Glu-Val-Asp -Leu-Leu-

Lys-Asn-Gly-Glu-Arg-Ile-Gly-Lys-Val-Glu-His-Ser-Asp-Leu-Ser-, R3 is a 20-amino acid group having the sequence: -Trp-Ser-Phe-Tyr-Leu -Leu-Tyr-Tyr-Thr-Glu-Phe-Thr-Pro-Thr-Glu-20 Lys-Asp-Glu-Tyr-Ala, R4 is a 19 amino acid group having the sequence: -Arg-Val-Asn-His- Val-Thr-Leu-Ser-Gln-Pro-Lys-Ile-Val-Lys-Trp-Asp-Arg-Asp-Met, -X is -Phe, -Phe-Ser, or -Phe-Ser-Lys, preferably -25 Phe and Y- are Asp-, Lys-Asp-, Ser-Lys-Asp-, preferably Asp-, with the sum of the number of amino acid residues in X and Y being at most 4.

Modified fi2 microglobulin (mB2m) is a variant of ft2 microglobulin (B2m) which has been discovered in connection with a number of cancers and immune system disorders.

Its precursor B2m is a serum protein with a molecular weight of 11,800 Daltons consisting of a single chain polypeptide of 99 amino acid groups with a disulfide bridge between cysteine groups.

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2 at positions 25 and 80, and with a known amino acid sequence (1). Structurally, B2m exhibits a marked homology to the constant region of IgG, especially the CH3 region and the a3 region of the heavy chain HLA-B7. B2m is part of the large histocompatibility complex on cell membranes and is present in the free form in body fluids such as serum, spinal fluid, saliva, semen and carbon (2, 3). In healthy individuals, the serum concentration of B2 m is 50 - 200 nmol / l (4).

The serum concentration of B2m is elevated by a number of diseases e.g. rheumatoid arthritis (RA), systemic lupus erythomatosus (SLE), malignant lymphoma (ML), and certain types of lung cancer such as small cell lung cancer (SLC) (5-7). High serum B2m levels in ML decrease under the influence of chemotherapeutic β1, whereas relapse is generally not accompanied by increasing serum B2m.

Small cell lung cancer (SLC) is almost always disseminated at the time of diagnosis, and the main treatment is therefore combination chemotherapy. With chemotherapy, it is possible to induce remission in more than 80% of all patients, but in most cases the tumor will later escape control. Accordingly, the earliest possible diagnosis, preferably at a stage where the disease is located, may be crucial in improving the survival rate of patients suffering from SLC.

Acquired Immunodeficiency Syndrome (AIDS) is the ultimate culmination of a disease evident in other forms, such as AIDS-related complex or lymphadenopathy syndrome, and in less expressed conditions, including the carrier state. Blood donor screening with an antibody to the putative generating virus (HTLV-III / LAV) has begun. Such a study for the virus only discovers an exposure to it, not the presence or prognosis of the progression of the disease. Furthermore, the antibody test for HTLV-III sometimes fails to detect the presence of the virus (1). Thus, there is a need for another study that will allow for a more quantitative assessment.

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3 shows the immune system's response to exposure to HTLV-III.

B2m has received much attention as a possible marker that could be used to diagnose and monitor diseases such as those mentioned above.

Thus, it has been reported that most AIDS patients studied have B2m levels above normal. It has also been reported that increased B2m levels have been shown in a number of cases even two years before the clinical diagnosis of AIDS.

In vitro incubation of serum from patients with RA, SLE, germ cell tumors and ML has been shown to result in the occurrence of a 2 µm fraction of "α-electrophoretic mobility" by crossed radioimmune electrophoresis (CRIE). In patients with ML, an inverse correlation between the amount of this α fraction and response to chemotherapy has been found.

Rhavi B. Bhalla et al .: Clinical Chemistry 31 (1985) 20 p.1411 reported that the α-electrophoretic form of B2m or modified B2m was present in all their AIDS patients including those with normal B2m levels, and in addition, a patient who was HTLV-III negative.

Swanson et al .: Clinical Chemistry 28 (1982) pp. 2033-25 2039, on page 2038, describes that their B2 microglobulin assays show 5 different forms by 2-dimensional electrophoresis, all with different pi · but uniform molecular weights.

30 Thus, it is not yet determined which fractions are of interest for use in the above methods.

The α-electrophoretic form or modified 92m 35 (mj82m) therefore appears to be highly attractive as a marker for a variety of immunological disorders. mj82m is apparently not

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4 specific to any of the above diseases, but measuring them will nevertheless be a valuable tool for examining a large number of samples or for monitoring the development of these diseases.

Accordingly, the invention aims to develop means of using m / 2m as a general marker in the diagnosis and assessment of the prognosis of cancer and immunological disorders manifesting at abnormal m / 32m levels-10. This is achieved according to the invention by specifying an effective method for determining and measuring m / 32 m in body fluids. The method uses specific polyclonal antibodies obtained in antisera against mjS2m or specific epitopes thereon, or mjS2m specific monoclonal antibodies obtained by fusing such antibody producing cells with myeloma cells to obtain hybridomas and selection of hybridoma cells producing monoclonal antibodies which produce with m /? 2m.

To date, no biological significance of any of the modified forms of µm has been described in the literature, but during work on the invention, it was surprisingly found that mB2m is biologically active as a biological response modifier that enhances immunological responses that are already present. mB2m increased the cytotoxic activity of mixed lymphocyte cultures and stimulated the production of the lymphokine interleukin-2, and B2m in this respect acts as a precursor of the active substance mB2m, with B2m apparently degrading into the organism. Furthermore, it was found that mjS2m specific antibodies were able to block the stimulatory effect of mjS2m.

In view of these discoveries, it is also an object of the invention to disclose pharmaceutical compositions.

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5 containing anti-mj82m antibodies for use as biological response modifiers.

In its first aspect, the invention relates to an antibody which is characterized in that it specifically responds to modified B2 microglobulin of the general formula I:

R-R-Cys-X

I I

Y-R3-Cys-R4 wherein R1 is a 24 amino acid group having the sequence:

Ile-Gln-Arg-Thr-Pro-Lys-Ile-Gln-Val-Tyr-Ser-Arg-Hi s-Pro-Ala-Glu-Asn-Gly-Lys-Ser-Asn-Phe-Leu-Asn-, R 2 is a 30 amino acid group having the sequence: -Tyr-Val-Ser-Gly-Phe-His-Pro-Ser-Asp-Ile-Glu-Val-15 Asp-Leu-Leu-Lys-Asn-Gly-Glu-Arg Ile-Gly-Lys-Val-Glu-His-Ser-Asp-Leu-Ser-, R3 is a 20-amino acid group having the sequence: -Trp-Ser-Phe-Tyr-Leu-Leu-Tyr-Tyr-Thr-Glu -Phe-Thr-Pro-Thr-Glu-Lys-Asp-Glu-Tyr-Ala-20 R4 is a 19 amino acid group having the sequence: -Arg-Val-Asn-His-Val-Thr-Leu-Ser-Gln-Pro -Lys-Ile-Val-Lys-Trp-Asp-Arg-Asp-Met, -X is -Phe, -Phe-Ser, or -Phe-Ser-Lys, preferably -Phe, and Y is Asp, Lys-Asp-, Ser-Lys-Asp-, preferably Asp-, with the sum of the number of amino acid residues in X and Y being at most 4.

Furthermore, the invention relates to an antibody of the aforementioned kind, characterized in that it is a polyclonal antibody and can be prepared by immunizing an animal with an antigen selected from modified B2 microglobulin of the general formula I, wherein R1f R2, R3, R4, X and Y have the above meanings and compounds of the general formula II RZ II

Wherein R is a carrier protein, preferably keyhole limped hemocyanin, and Z is a peptide of formula R2'-X or R3'-Y wherein

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6 X and Y are as defined above, R2 'is a peptide having from 1 to 30 amino acid groups corresponding to a fragment from the c-terminal end of R2 as defined above, and R3' is a peptide having from 1 to 20 amino acid groups corresponding to a fragment from the 5 N-terminal end of R3 as defined above, serum is isolated and purified to full specificity against modified B2 microglobulin.

In a further aspect, the invention relates to a monoclonal anti-m / 2m antibody which can be produced by culturing a hybridoma cell line obtained by fusion of myeloma cells with spleen cells from animals immunized with an antigen selected from modified B2 microglobulin of general formula I wherein R 1, R 2, R 3, R 4, X and Y have the above meanings and compounds of the general formula II, wherein R and Z have the above meanings.

In a further aspect, the invention relates to a method for detecting mjS2m in human body fluids, in which method the body fluid containing or suspected to contain modified B2 microglobulin is contacted with an antibody as set forth above, the antibodies thus formed. antigen complexes are separated from uncomplexed antibody / antigen and it is detected whether antibody-antigen complexes are formed.

In yet another aspect, the invention relates to pharmaceutical compositions which are characterized in that it contains one or more antibodies as set forth above, in particular a biological residue. nmodifying agent.

The invention is described in detail below with reference to the drawing, in which

FIG. 1 shows the results of titration by ELISA of an antiserum of the invention, 35

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7

FIG. 2 shows the results of the primary study of anti-mB-jin antibody producing cultures,

FIG. 3 shows the results of the secondary ELISA study of cultures selected from the primary study in FIG. 2, and

FIG. 4 shows the result of an assay for mB2m using one of the FIG. 3 monoclonal antibodies.

10

Modified B2 microglobulin is defined as the fraction of B2m that has a relative mobility of 2.0 as compared to the native protein by CRIE analysis.

15

The modified B2 microglobulin of the invention was obtained by isolating it together with B2m from serum samples from patients with histologically verified small cell anaplastic lung cancer and subsequent separation from B2m.

20

Isolation of mB2m and B2m from the serum was performed by gel filtration after 5 days of incubation at 20 ° C.

Fractions containing mB2m and B2m were chromatofocused at pH 7-4 to separate mB2m from B2m, and the mB2m containing fraction was subsequently purified by gel filtration.

mB2m was characterized physicochemically and biochemically by CIE, CRIE, sodium dodecyl sulfate polyacrylamide gel electrophoresis 30 (SDS-PAGE) and analytical isoelectric focusing (IEF) and was found to correspond to the α-electrophoretic form of B2m or mB2m as indicated in Figs. 4 B, p. 630, in Piesner: Allergy 35 (1980) pp. 627-637.

35 MB2m had an apparent molecular weight of 15,000 in non-reduced form, and split into two smaller peptides with

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8 mol weight less than 12,000 in reduced form by SDS-PAGE analysis. In non-reduced form, mB2m had a pI of 5.3 at analytical IEF.

The isolated mfi2m was subjected to amino acid sequencing and was found to consist of a mixture of peptides of general formula I

R ^ R ^ Cys-X

I I

Y-R3-Cys-R4 wherein R1 is a 24 amino acid group having the sequence:

Ile-Gln-Arg-Thr-Pro-Lys-Ile-Gln-Val-Tyr-Ser-Arg-His-Pro-Ala-Glu-Asn-Gly-Lys-Ser-Asn-Phe-Leu-Asn,

Rg is a 30 amino acid group having the sequence: Tyr-Val-Ser-Gly-Phe-His-Pro-Ser-Asp-Ile-Glu-Val-Asp-Leu-Leu-15 Lys-Asn-Gly-Glu-Arg-Ile -Gly-Lys-Val-Glu-His-Ser-Asp-Leu-Ser, R3 is a 20-amino acid group having the sequence: Trp-Ser-Phe-Tyr-Leu-Leu-Tyr-Tyr-Thr-Glu-Phe- Thr-Pro-Thr-Glu-Lys-Asp-Glu-Tyr-Ala R4 is a 19 amino acid group having the sequence: Arg-Val-Asn-His-Val-Thr-Leu-Ser-Gln-Pro-Lys-Ile -Val-Lys-Trp

Asp-Arg-Asp-Met, -X is -Phe, -Phe-Ser, or -Phe-Ser-Lys, preferably -Phe, and Y- is Asp-, Lys-Asp-, Ser-Lys-Asp-, preferably Asp-, with the sum of the number of amino acid residues in X and Y being at most 4.

The amino acid sequence of the R1-Cys-R2-X chain (A chain) is identical to the sequence published in (1) of groups 1-30 56 (58) of the native B2m with the only exception of amino acid residue 42, wherein the remainder of the invention is Asn, whereas the published sequence indicates Asp. However, the sequence of the invention is consistent with the sequence derived from the cloned cDNA of Suggs et al: Proc. Natl.

35 Acad. Sci. United States 78 (1981) pp. 6613-6617, which also indicates Asn at position 42.

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9

The sequence of the C-terminal end of the R ^-Cys-R2-X chain is determined both by Edman degradation from the N-terminal end and by carboxypeptidase degradation from the C-terminal end, and it was found that the product consisted predominantly of the peptide where X is Phe. The C-terminal determination was complicated by the presence of srin residues at positions 52, 55 and 57.

The amino acid sequence of the Y-R 2 -Cys-R 2 j chain (B chain) is identical to residues (57) 59 - 99 in the sequence published in (1) from native B2m, with Y being predominantly Asp.

The antigen used for immunization may be m62m, but other antigens selected for their ability to raise 15 anti-m62m antibodies that will specifically bind to m32m epitopes may also be used. In general, antigens of general formula II can be used

R-Z II

Wherein R is a carrier protein and Z is a peptide of formula R2'-X or R4 '- Y wherein X and Y are defined as in formula I, R21 is a peptide having from 1 to 30 amino acid residues corresponding to a fragment from the C-terminal end of R 2, as defined in formula I, and R 1 'is a peptide having from 1 to 20 amino acid residues corresponding to a fragment from the N-terminal end of R 1, as defined in formula I.

in one case, an antigen comprising the amino acid groups -Lys-Val-Glu-His-Ser-Asp-Leu-Ser-Phe with a free C-30 terminal end and the N-terminal end coupled to keyhole limpet hemocyanin (KLH) with success in producing anti-sera as well as monoclonal antibodies that bind specifically to epitopes in the nine C-terminal amino acid groups of the A-chain of m32m.

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10

Immunization was performed in mice by subcutaneous injection of the antigen several times and subsequently boosting intravenously with the antigen. After appropriate time, the mice were sacrificed and their spleens removed for fusion with myeloma cells. The fused cells were derived after R.T. Taggart and I.M. Samloff:

Science 219 (1983) pp. 1228 - 1230 (APRT + selection), as described below.

The study of the selected hybridoma cultures for specific monoclonal antibodies was performed with an ELISA and a number of clones reacted specifically with mf32m without cross-reaction with f32m.

The antibodies of the invention can be used in methods for detecting the presence of mf µm and also for estimating the quantities present quantitatively.

The detection of the presence of the complete 20 µm antigen or its characteristic epitope can be performed in sources or samples of body fluids such as serum, blood, saliva, urine, semen, spinal fluid, colostrum, tissue, tissue samples, bronchial lavage fluid and the like.

The detection of m82m on the basis of specific antibodies can be performed by immunoassays such as the competitive or immunometric ("sandwich") types. In a competitive assay, the purified m | 32m antigen or molecule, comprising its characteristic epitope, is labeled with a detectable label. The sample suspected of containing the mf32m antibody or epitope is incubated with the m | 32m antibody and labeled m82m antigen and after the formation of immune complexes separation and detection, the level of m32m antigen is readily determined.

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11

Another type of competitive assay involves immobilized m32m antigen or epitope and labeled 11162111 antibody. The presence of 11162 µl of antigen in the source or sample prevents binding of antibody to immobilized antigen or epitope, thereby providing an inverse target for the presence of antigen.

In a competitive type assay, the antibody can also be immobilized on a solid phase either before or after the formation of the immune complex. Eg. For example, another, anti-mouse IgG, antibody 10 immobilized on a solid phase can be added to the mixture by what is known as a "double technique".

In an immunometric (sandwich) assay, one antibody is detectable. Another antibody is insolubilized on a solid phase. The incubation of the sample with labeled and insolubilized antibody leads to a sandwich in which the amount of label after separation from unbound antibody (s) is proportional to the amount of antigen. Immunometric assays may be performed in a straightforward, reverse or simultaneous manner depending on the order of addition of the insolubilised and / or labeled antibodies.

The assay can also be a so-called "dot-blot" analysis.

For increased sensitivity in the sandwich system, the 25 described procedures can be modified with biotinylated mB2m antibody reacting with avidin-peroxidase conjugates.

Of course, other steps such as washing, stirring, shaking, filtering or pre-assay extraction of antigen and the like can be added to the assays as desired or whatever is needed for a particular situation.

The specific concentrations, temperature and incubation time, and other assay conditions may be varied depending on such factors as the concentration of antigen in the sample, the nature of the sample and the like. Those skilled in the art will be able to determine the

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12 ve and optimal assay conditions for each assay using routine experiments. Eg. For example, the immunoassay may be performed at 4 - 37 ° C, preferably 26 ° C, and each incubation step may last as long as 72 hours.

Instead of the antigen or characteristic epitope, it is possible to use, by assays, anti-idiotypic antibodies or immunologically active fragments thereof produced by raising antibodies against the binding site of monoclonal 10 mB2m antibody. Instead of the specific µm antibody described herein, it is also possible to use equivalent antibodies made by examining hybridomas with antigen according to routine methods. The hybridomas are prepared by fusion of lymphocytes sensitized to suitably purified antigen and 15 appropriate myelomas.

There are many carriers to which the antibody (s) can be bound and which can be used in the invention. Well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amyloses, natural and modified cellulose, polyacrylamide, agaroses and magnetite. The nature of the carrier may be either soluble to some extent or insoluble for the purposes of the invention. Those of skill in the art will know many other suitable carriers for binding the antibody fatty substance (s) or will be able to determine such by routine experiments.

Depending on the particular embodiment of the present assay, the antigen or one or more of the antibodies may be coupled with a detectable label such as an enzyme, radioactive isotope, fluorescent compound or metal, chemiluminescent compound or bioluminescent compound. Furthermore, the binding of these labels to the desired molecule can be carried out according to standard techniques well known to those skilled in the art.

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13

One of the ways in which the desired molecule in any immunoassay can be detectable is by attaching it to an enzyme. This enzyme, for its part, when subsequently exposed to its substrate, reacts with the substrate in such a way as to produce a chemical unit detectable by e.g. bac-trophotometry (ELISA system) or fluorometry. Examples of enzymes that can be used as detectable labels are horseradish peroxidase, maleate dehydrogenase, staphylococ nuclease, delta-5 steroid isomerase, yeast alcohol hydrogenase, α-glycerophosphate dehydrogenase, triosephosphate isomerase, alkaline phosphatase, asparagine galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.

The presence of antigen or antibody (and, as a result, its corresponding m ^ 111 antigen) can also be detected by labeling the desired molecule with a radioactive isotope. The presence of the radioactive isotope is then determined by means such as gamma counter or scintillation counter. Isotopes that are particularly on - -, · 3 125T 131_ 32_. 35 „140 51- 36-.

20 useful are Η, I, I, P, S, C, Cr, Cl, 57. 58- 59_ 75c 111_ 99m ,,, 67_ 90v

Co, Co, Fe, Se, In, Tc, Ga, and Y.

It is also possible to detect the presence of the antigen or antibody by labeling the desired molecule with a fluorescent compound. When the fluorescently labeled molecule is exposed to light of the appropriate wavelength, its presence can be detected by the fluorescence of the dye. Among the most important fluorescence-labeling compounds are fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

Fluorescence emitting metal atoms such as Eu (europium) and other lanthanides can also be used. These can be linked to the desired molecule with metal chelating groups such as DTPA or EDTA.

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14

Another way in which the antigen or antibody can be detectably labeled is by coupling it to a chemiluminescent compound. The presence of the chemiluminescent labeled immunological molecule is then determined by detecting the presence of luminescence that occurs during a chemical reaction course. Examples of particularly useful chemilusminizing labeling compounds are luminol, isoluminol, aromatic acridine ester, imidazole, acridine salt, and oxalate ester.

Similarly, a bioluminescent compound can also be used as a label. Bioluminescence is a particular type of chemiluminescence found in biological systems, whereby a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent molecule can be determined by detecting the presence of luminescence. Important bioluminescent compounds for labeling purposes are luciferin, luciferase and aequorin.

Another possible assay technique for m32m antigen or its corresponding characteristic epitope is by agglutination, where mP2m antibody binds to latex beads, and the presence of m32m antigen is measured by ball aggregation.

Alternatively, the presence of m32m antibody can be detected by agglutination of particles coated with m 2m antigen or the relevant epitope.

An alternative use of the mf32m antibody is by immunohistochemical analysis using labeled antibody.

30

In the aforementioned methods for detecting the m32m antigen, it is possible to use a specific anti-m32m antibody. However, it is also possible to increase the selectivity of the method to use combinations of two or more specific anti-m32m antibodies, e.g. an antibody specific to one

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15 epitope at the C-terminal end of the A chain in combination with an antibody specific for an epitope at the N-terminal end of the B chain.

The materials used in the present assay are well suited for the preparation of sets. Such a set may comprise a box of compartments for one or more containers such as glass, tubes and the like, each of these containers comprising one of the individual elements to be used by any desired method.

For example, For example, one of these containers useful in immunometric assays may comprise monoclonal µm antibody bound to a carrier. Another container may contain soluble detectably labeled monoclonal anti-µm antibody in lyophilized form or in solution.

A kit useful for a competitive assay would contain a first container containing a detectable labeled antigen, and a second container containing anti-mf ^ 111 antibody free or carrier bound.

In addition, the box may also contain a number of containers, each containing different amounts of antigen given. These latter containers can then be used to prepare a standard curve 25 for interpolating the results obtained from the sample containing the unknown amount of antigen.

During the work of the invention, purified µm was found to increase the cytotoxic activity of mixed 30 lymphocyte cultures (MLC) as well as stimulate the production of the lymphoquine interleukin-2 (IL-2). The interaction of n m with the large histocompatibility complex (MHC), in which 02 m is a normal component, could lead to activation of cells resulting in e.g. an increased cellular receptor expression for general stimulatory activity such as interleukin-1 (IL-1) and an increased expression of receptors for IL-2 and increased endogenous production of this lymphoma.

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16 kin. This biological activity at mi111 indicates a pharmaceutical utility of both polynomial and monoclonal antibodies to m32m as a biological response modifier, the pharmacological effect being related to the state of the immune system.

Pharmacological effects of the aforementioned antibodies may, as mentioned earlier, be as biological response blocking agents of mg2 / l / which may be related both to the effect at the membrane level due to item interaction with MHC or could be related to specific stimulatory effect 10 on IL-2 production.

The invention is further illustrated in the following examples.

Example 1

Preparation of polyclonal antisera RBF / Dn strain mice (from Jackson Laboratory, Bar Harbor, 20 Maine, USA) was immunized three times at two week intervals with the synthetic peptide Lys-Val-Glu-His-Ser-Asp-Leu-Ser. Phe coupled via the N-terminal end to keyhole limpet hemocyanin (KLH).

The peptide conjugate (25 μg per mouse in PBS, 100 μΐ) was emulsified 1: 1 in Freund's complete adjuvant at the first immunization, and in 25 Freund's incomplete adjuvant in the following immunizations and a total of 200 μΐ subcutaneously (sc) was administered. . Blood samples from immunized mice were taken from the retroorbital plexus or eye, and serum samples were obtained after centrifugation of the samples

after coagulation. The specificity of the serum samples was assessed by ELISA

30 with immunoplates (NUNC, Roskilde, Denmark) coated with 1 μg / ml of either native 3 ~ m or m39m. Serum samples were diluted in tidal dilutions (10, 10, 10, and 10) and incubated with the coated plates and antibody activity to the respective antigen was measured colorimetrically with a rabbit anti-mouse immunoglobulin conjugated with horseradish peroxidase. (Dakopatts, Denmark) and the o-phenylenediamine (OPD) substrate reaction.

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17

FIG. Figure 1 illustrates the titration of a representative mouse serum analyzed for binding of (shaded bars) and native 8 µm (filled bars). The antiserum reacts selectively with * o-4 and appears to be specific for mf ^ m at dilutions of 10 5 under these conditions. A completely specific polyclonal antiserum against m32m could be obtained by further absorption with native e2m.

Example 2 10

Immunization and Fusion Experiments 1 mg of the peptide Lys-Val-Glu-His-Ser-Asp-Leu-Ser-Phe was mixed with 3 mg of KLH (Keyhole Limpet Hemocyanin, Calbiochem) and 15 glutaraldehyde (Merck) added to the final concentration of 0, 25% v / v a total of 1 ml. The pH was adjusted to 8.0. The reaction was carried out for one hour at room temperature followed by 18 hours at 4 ° C. Then, the mixture was dialyzed against PBS to obtain the conjugate used as antigen for the following immunization.

20

Preparation of RBF / Dn Monoclonal Antibody Mice containing RB (8.12) 5Bnr Robertsonian translocation chromosome (from Jackson Laboratory, Bar Harbor, Maine, USA) was immunized three times at two week intervals with the peptide Lys-Val-Glu-His-Ser -Asp-Leu-Ser-Phe connected via the N-terminal to KLH. The conjugate (25 μg per mouse in PBS, 100 μΐ) was emulsified 1: 1 in Freund's complete adjuvant for the first injection and incomplete Freund's adjuvant for the following 30 injections and 200 μΐ was administered subcutaneously (s.c). The mice were boosted intravenously (i.v.) with 10 μg of conjugate in 100 μΐ PBS 30 days after the last s.c. immunization. After another three days, the mice were sacrificed and their spleens removed for fusion with myeloma cells.

35 7

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18

Spleen cells from one RBF / Dn mouse (13.3 x 10 6 cells) were fused to 3.5 x 10 Laboratories, Logan, Utah, USA) This eliminates exposure of the cell fusion mixtures to a medium requiring APRT activity (APRT + selection) for both non-fused APRT myelomas and APRT hybridomas (RT Taggart and IM Samloff: Science 219 (1983)). 1228 - 1230). The molten cells were seeded on Balb / c-10 strain mouse macrophage feeds in a total of 10 96-well microtiter plates (NUNC, Roskilde, Denmark) in a medium consisting of RPMI-1640 with 15% w / v fetal calf serum (Gibco) supplemented with adenine (7.5 x 10 7 M), aminoterin (8 x 10 7 M) and thymidine (1.6 x 10 M), (AAT).

15 Cultures were incubated for 7 days at 37 ° C in air containing the 5% CC> 2 before medium was replaced with fresh medium, and after a 4-day incubation period, the cultures were subjected to a primary study by an ELISA.

20 Investigation Procedure (ELISA)

Microtiter plates (NUNC Immunoplates, NUNC, Roskilde, Denmark) were coated with antigen. The ELISA used was a modification of the procedure described by A. Voller and E.

25 de Savigny (in R.A. Thompson: Techniques in Clinical Immunology, 2nd ed. (1981) pp. 157 - 169. Blackwell Scientific Publications, Boston, Mass.). The coating was performed overnight with the antigen in phosphate buffered saline (PBS), 50 μΐ / ml. well. The plates were emptied and blocked with PBS containing 2% w / v bovine serum albumin 30 (BSA) 200 μΐ / ml. well at 20 ° C for 1 hour, followed by three washes with PBS-Tween-20 (0.05% v / v Tween-20 in PBS). The undiluted supernatant from a hybridoma culture (50 μΐ per well) was applied at 20 ° C for 1 hour followed by washing the plates as described above. The antibody activity against the coating antigen used was measured colorimetrically by incubation at 20 ° C for 1 hour at 100 µg per day. well rabbit anti-mouse immunoglobulin conjugated with

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19 horseradish peroxidase (Dakopatts, Denmark) diluted to 1: 1000 in PBS containing 0.5% w / v BSA and after a further 3 washing in 0.1 M citrate-phosphate buffer of pH 5.0 (citric acid monohydrate 7.3 g, Na2HPO1.12H2O: 23.87 g diluted to 1 liter) and then incubated as described above with o-phenylenediamine (OPD) substrate (o-phenylenediamine, 2HC1: 8 mg; citrate buffer: 15 ml / v): 5 μΐ). The reaction was stopped after 3 minutes by the addition of 150 μΐ 1 M H2 ^ 4 ° 9 axor edge ve ^ 492 nm read with a double beam KONTRON SLT-210 photometer (SLT, Zurich, Switzerland) with the 620 nm 10 reading as a reference.

Primary examination

Using 10 microtiter plates coated with the nonapeptide used for immunization, but not coupled to KLH (1 μg / ml) by the above method, 960 hybridoma culture supernatants were examined. Twenty-seven cultures reacted significantly positively and underwent further testing.

FIG. 2 gives an example of the results of ELISA in the primary study. The results from one of the ten microtiter plates are listed. The horizontal line represents an arbitrary background level selected for the primary study. Only the cultures labeled with x were selected for further testing.

Secondary examination

Microtiter plates were coated with either the nonapeptide, 30 82m (native) or n ^ m (variant) at concentrations of 1 μg / ml, 10 μg / ml and 10 pg / ml, respectively. The 27 culture supernatants were added and the study was performed according to the aforementioned study procedure.

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In FIG. 3, the results of the secondary study are shown for the cultures selected from the primary study except cultures FI, F3 and F7 which were negative in the sense that they did not react with any of the antigens listed above. From the 5 figures it appears that 3 clones (F2, F10, F25) reacted exclusively with the peptide 15 reacted with the peptide and m32m and 3 (F5, F8, F26) reacted with the peptide, m $ 2m and 02m (native). The remaining 3 clones (F12, F18, F21) had lost reactivity.

10 Selection of hybridomas

All the hybridoma antibodies reacting with m32m and / or 82m were further found to be of the mouse immunoglobulin class IgM. This was found in studies using the aforementioned ELISA procedure with a modification consisting of replacing the peroxidase-labeled rabbit anti-mouse immunoglobulin with labeled antibodies specific for the murine classes and subclasses.

Example 3

Enzyme immunoassay for m82m

To test the utility of the obtained monoclonal antibodies against m82m in assay to determine the m82m content in biological samples, an assay was constructed using antibody F19 in FIG. Third

The assay was performed according to the following procedure:

Rabbit anti-human 82m antibody (Dakopatts, Copenhagen, Denmark) was applied to the bottom of microtiter plates. After blocking the plates by adding phosphate buffer (pH 7.2) containing 1% BSA and incubating for 1 hour at room temperature with subsequent 3 times washing with phosphate buffer (pH 7.2) containing 0.1% Tween®, samples containing 82m were added. or m32m to the microtiter plates at various concentrations of 20, 61, 185, 555, 1600 and 5500 ng / ml. After 1 hour of incubation at room temperature, antibody F19 was added and the samples incubated for 1 hour at room temperature.

In the next step, the plates are incubated with a peroxidase label

DK 161098 B

21 mouse immunoglobulin antibody (Tago, Inc., Burlingame, California, USA) for one hour at room temperature, and finally the plates were induced by incubation for 1 hour with a peroxidase substrate consisting of OPD in citrate buffer (pH 4.5), after which the 5 color reaction was produced. was measured by the absorbance at 492 nm.

The results of this assay are given in FIG. 4, showing the measured absorbance at 492 nm as a function of the concentration of É> 2m (dashed line) and n ^ m (full line, respectively).

10 From FIG. 4 it is clear that this assay gives a clear indication of the presence of m ^ 111 / without a content of unmodified (32m affecting the measurement.

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22

Literature References: 5 1. Cunningham, B.A., Wang, J.L., Berggård, I. & Peterson, P.A. (1973) Biochemistry 12, 4811 - 4822.

2. Suggs, S.V., Wallace, R.B., Hirose, T., Kawashima, E.H. & Itakura, K. (1981) Proc.Natl.Acad.Sci. U.S.A. 78, 6613 - 6617.

3. Berggård I. Bearn A.G. (1968) J. Biol.Chem. 243, 4095 - 4103.

4. Piesner T. & Bjerrum O.J. (1980) Scand.J. Immunol. 11, 341 - 351.

5. Solheim, B.G. & Thorsby, E. (1974) Nature (Lond) 249, 36 - 38.

6. Peterson P.A., Rask, L. & Lindblom, J.B. (1974) Proc.Natl.- Acad. Sci. U.S.A. 71, 35 - 39.

7. Cotner, J., Mashimo, H., Kung, P.G. Goldstein, G. &

Strominger, J.L. (1981) Proc.Natl.Acad.Sci. U.S.A. 78, 3858 -3862.

8. Terhorst, C., Agthoven, v. A., LeClair, K., Snow, P., Reinherz, E. & Schlossman, S. (1981) Cell 23, 771 - 780.

9. Peterson, P.A., Cunningham, B.A., Berggård, I. & Edelman, G.M.

30 (1972) Proc.Natl.Acad.Sci. U.S.A. 69, 1697 - 1701.

18. Piesner, T., Wilken, M., Bjerrum, O.J. & Hansen, M.M. (1982) J.Clin.Lab.Immunol. 8, 137 - 414.

II

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23 19. Weeke, B. (1973) A Manual of Quantitative Immunoelectrophoresis. Methods and applications. 1st edn. (Axelsen, N.H., Krøll, J. & Weeke, B., eds.) Pp. 47 - 56, Universitetsforlaget, Oslo.

5 20. Piesner, T., Nørgaard-Pedersen, B. & Boenisch T. (1975)

Scand.J. Clin.Lab.Invest 35, 729 - 735.

22. Wycoff, M., Rodbard, A. & Chrambach, A. (1977) Anal. Biochem.

78, 459 - 482.

10 23. Bury, A.F. (1981) J. Chromatography 213, 491-500.

25. Moody, A. J., Thim, L. & Valverde, I. (1984) FEBS Lett. 172, 142 - 148.

Claims (10)

24 DK 16109 8 B An antibody, characterized in that it reacts specifically with modified B2 microglobulin of the general formula I: R., - Cys-R2-X I I Y-R3-Cys-R4 wherein R1 is a 24 amino acid group having the sequence: Ile-Gln-Arg-Thr-Pro-Lys-Ile-Gln-Val-Tyr-Ser-Arg-His-Pro-Ala-Glu-Asn-Gly-Lys-Ser-Asn-Phe-Leu-Asn- R2 is an amino acid group having the sequence: -Tyr-Val-Ser-Gly-Phe-His-Pro-Ser-Asp-Ile-Glu-Val-Asp-Leu-Leu-Lys-Asn-Gly-Glu-Arg-Ile -Gly-Lys-Val-15 Glu-His-Ser-Asp-Leu-Ser-, R3 is a 20-amino acid group having the sequence: -Trp-Ser-Phe-Tyr-Leu-Leu-Tyr-Tyr-Thr-Glu -Phe-Thr-Pro-Thr-Glu-Lys-Asp-Glu-Tyr-Ala-R4 is a 19 amino acid group having the sequence: -Arg-Val-Asn-His-Val-Thr-Leu-Ser-Gln-Pro-Lys-Ile-Val-Lys-Trp-Asp-Arg-Asp-Met, -X is -Phe, -Phe-Ser , or -Phe-Ser-Lys, preferably -Phe, and Y- are Asp-, Lys-Asp-, Ser-Lys-Asp-, preferably Asp-, with the sum of the number of amino acid residues in X and Y being at most 4 . An antibody according to claim 1, characterized in that it is a polyclonal antibody and can be prepared by immunizing an animal with an antigen selected from modified B2 microglobulin of the general formula i, wherein R 1, R 2, R 3, R 4, X and Y have the meanings referred to in claim 1 and compounds of the general formula II RZ II 35 wherein R is a carrier protein, preferably keyhole limpet hemocyanin, and Z is a peptide of formula R2'-X or R3 ' -Y, wherein BX and Y are as defined in claim 1, R2 'is a peptide having from 1 to 30 amino acid groups corresponding to a fragment from the C-terminal end of R2 as defined in claim 1, and R3 is a peptide having from 1 to 20 amino acid groups corresponding to a 5 fragment from the N-terminal end of R3 as defined in claim 1, serum is isolated and purified to full specificity against modified B2 microglobulin. The antibody of claim 1, characterized in that it is a monoclonal antibody and can be produced by culturing a hybridoma cell line obtained by fusion of myeloma cells with spleen cells from animals immunized with an antigen selected from modified B2 microglobulin of the general formula I, wherein R 1f R 2, R 3, R 4, X and Y have the meanings referred to in claim 1 and compounds of the general formula II wherein R and Z have the meanings mentioned in claim 2. An antibody according to claim 1, 2, or 3, characterized in that it is coupled to a detectable label, such as an enzyme, radioactive isotope, fluorescent compound, or metal, chemiluminescent or bioluminescent compound, preferably an enzyme. An antibody according to claim 1, 2, or 3, characterized in that it is bound to an insoluble solid phase. A method of detecting modified B2 microglobulins in human body fluids, characterized in that the body fluid containing or suspected to contain modified B2 microglobulin is contacted with an antibody according to claims 1, 4 or 5, any antibody-antigen complexes formed are separated from non-complexed antibody / antigen and that it is detected whether antibody-antigen complexes are formed. in DK 161098 B 26 Method according to claim 6, characterized in that the method is a competitive assay. Method according to claim 6, characterized in that the method is an immunometric assay. Pharmaceutical composition, characterized in that it contains one or more antibodies according to any one of claims 1 to 5. Pharmaceutical composition according to claim 9, characterized in that it is a biological response modifier.