PT99114B - PROCESS FOR THE PREPARATION OF MONOCLONAL ANTIBODIES DIRECTED TO TROMBIN-FORMED COMPLEXES AND TROMBIN INHIBITORS - Google Patents

Abstract

The present invention relates to a process for the preparation of monoclonal antibodies which are directed against the thrombin/hirudin complex and derivatives thereof and of cell lines of hybridomas which secrete said monoclonal antibodies. The invention is also related to the use of monoclonal antibodies and/or derivatives thereof for the determination of the thrombin/hirudin complexes as well as test kits containing said monoclonal antibodies and/or derivatives thereof. More concretely, the present invention essentially relates to a process for the preparation of a monoclonal antibody which is directed against the thrombin/hirudin complex, and of a derivative thereof which maintains the specificity of the antibodies from which they are derived, which consists in the cells of a hybridoma cell line which secretes said antibody being propagated in vitro or in vivo and, when necessary, the monoclonal antibody obtained being isolated and/or converted into a derivative thereof.

Description

The invention concerns monoclonal antibodies that are directed against the thrombin / hirudin complex and its derivatives, processes for their preparation, hybridoma cell lines secreting said monoclonal antibodies and processes for the preparation of hybridoma cell lines. In addition, the invention relates to the use of monoclonal antibodies and / or their derivatives for the determination of the thrombin / hirudin complex as well as test kits comprising said monoclonal antibodies and / or their derivatives.

Background of the invention

A hemostatic system that works effectively is a vital necessity for the mammalian organism. In healthy organisms, defects in the blood vascular system, eg vascular lesions, are repaired in a two-step process: aggregation of thrombocytes is followed by the formation of a fibrin clot in an enzyme cascade with the participation of various blood clotting factors . Most of these factors are serine proteases, for example thrombin that catalyzes the reaction of fibrinogen to give fibrin. The coagulation system is counteracted by the fibrinolytic system involving, among others, the plasmin protease that cleaves fibrin. Coagulation and fibrinolytic systems are generally in dynamic balance. However, in cases where the organism's fibrinolytic potential is disturbed or insufficient, for example in patients suffering from thromboembolisms or post-operative complications, it is essential to give the body anticoagulants to prevent the formation of fibrin and thrombolytic agents to dissolve thrombi formed.

Hirudin, an anticoagulant that occurs naturally in leeches (Hirudi medicinalis), is a potent inhibitor and i

specific for thrombin, preventing fibrinogen cleavage and subsequent formation of fibrin clots. Hirudin reacts very quickly with α-thrombin to form a very strong non-covalent complex (K ^ «1-0.01 pM) that is extremely stable and enzymatically totally inactive. Several closely related hirudin variants have been described, each containing 65 or 66 amino acids, for example the variants designated hirudin variant 1 (HV1), hirudin variant 2 (HV2), hirudin variant PA (HV3) and des- (Val) ₂ -hirudin. The variants differ from one another in a series of amino acids, but all have in common an accumulation of hydrophobic amino acids at the N-terminus, an accumulation of polar amino acids at the C-terminus, a tyrosine residue (Tir 63) present as a sulphate mono-ester, three disulfide bridges and anticoagulant activity. Recently, cDNAs and synthetic genes encoding hirudin have been cloned and expressed in microbial hosts. Recombinant hirudin variants do not have the sulfate monoester group in Tir63 and are therefore also referred to as desulfate-hirudins. However, they have biological properties at least equivalent to those of sulfated natural hirudins.

Hirudin has great potential for future therapeutic use due to its selective inhibition of thrombin in conjunction with its low toxicity and the absence of immunological side effects. However, successful therapeutic application of hirudin also requires a system for controlling activity as well as the progress of therapy. Furthermore, it is desirable to be able to determine the real need for treatment with hirudin. A solution to these problems would be the development of antibodies against the complex formed by thrombin and hirudin that can be used to detect the formation of thrombin even in small amounts.

Purpose of the invention

It is an object of the present invention to produce monoclonal antibodies that are directed against complexothrombin / hirudin. This objective is achieved by using hirudin coupled to a suitable vehicle or the thrombin / hirudin complex to immunize a suitable mammal and fuse the antibody-secreting cells of said mammal with cells from a continuous cell line, thereby producing hybridoma cells that secrete monoclonal antibodies. intended. The monoclonal antibodies of the invention are useful for a number of diagnostic and therapeutic purposes, for example for the early detection of the formation of thrombin and thrombosis.

Description of the invention Invention concerns monoclonal antibodies directed against the thrombin / hirudin complex and its derivatives which retain the specificity of the antibody from which they are derived.

In the present patent application, the term hirudin, when not otherwise specified, is intended to encompass (1) all natural or synthetic variants and derivatives of hirudin, such as fragments of hirudin and (2) all variants of recombinant hirudin (desulfate-hirudin) and recombinant hirudin derivatives (desulfate-hirudin), such as C-shortened desulfate-hirudins, which are described in the literature iu are obtained by recombinant DNA technology methods.

Examples of such hirudins are:

- χ ’- (a) a variant of hirudin type HV1 with the formula

H-Val-Val-Tyr-Thr-Asp-Cys-Thr-Glu-Ser-Gly20

-Gln-Asn-Leu-Cys-Leu-Cys-Glu-Gly-Ser-Asn30

-Val-Cys-Gly-Gln-Gly-Asn-Lys-Cys-Ile-Leu40

-Gly-Ser-Asp-Gly-Glu-Lys-Asn-Gln-Cys-Val50

-Thr-Gly-Glu-Gly-Thr-Pro-Lys-Pro-Gln-Ser60

-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr (R) -Leu-Gln-OH, (I) where (desulfate-hirudin)

- (R) is the phenolic hydroxyl group of Tir

or a -O-SO ₃ H group, and / or - LEIS27 is replaced by Ile or Glu or - Lis36 is replaced by Ile or Glu or - LEAD47 is replaced by Ile or Glu or - His51 is replaced by Leu or Asp or - Vall-Val2 are replaced by Tre or

- Vali is replaced by Leu and Val2 by Tre

- the complete molecule is shortened by Gln65 or or by Leu64 and Gln65;

(b) a variant of HV2 type hirudin with the formula

H-Ile-Thr-Tyr-Thr-Asp-Cys-Thr-Glu-Ser-Gly20

-Gln-Asn-Leu-Cys-Leu-Cys-Glu-Gly-Ser-Asn30

-Val-Cys-Gly-Lys-Gly-Asn-Lys-Cys-Ile-Leu40 (II)

-Gly-Ser-Asn-Gly-Lys-Gly-Asn-Gln-Cys-Val50

-Thr-Gly-Glu-Gly-Thr-Pro-Asn-Pro-Glu-Ser60

-His-Asn-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr (R) -Leu-Gln-OH, where

- (R) is the phenolic hydroxyl group of Tir (desulfate-hirudin) or an -O-SO ₃ H group, and / or

- Ilel is replaced by Vai and Tre2 by Vai or

- Asn47 is replaced by Lis or Arg or His or

- Tir63 is replaced by Glu or Asp;

(c) a variant of hirudin type PA (HV3) with the formula ** ι ...

H-Ile-Thr-Tyr-Thr-Asp-Cys-Thr-Glu-Ser-Gly20

-Gln-Asn-Leu-Cys-Leu-Cys-Glu-Gly-Ser-Asn30

-Val-Cys-Gly-Lys-Gly-Asn-Lys-Cys-Ile-Leu40 (wm)

-Gly-Ser-Gln-Gly-Lys-Asp-Asn-Gln-Cys-Val50

-Thr-Gly-Glu-Gly-Thr-Pro-Lys-Pro-Gln-Ser60

-His-Asn-Gln-Gly-Asp-Phe-Glu-Pro-Ile-Pro-Glu-Asp-Ala-Tyr (R) -Asp-Glu-OH, where (R) is the phenolic hydroxyl group of Tir (desulfate-hirudin or a group -O-SC> ₃ H, and / or

- the polypeptide chain is shortened at the C end by 18, 10, 9,

6, 4 or 2 amino acids or

- the polypeptide chain is shortened at the N end by 1 or 2 amino acids.

The monoclonal antibodies of the invention have been tested for desired properties, preferably by radioimmunoassay or enzyme immunoassay. For example, an enzyme immunoassay can be performed in which a suitable vehicle such as a microtiter plate is coated with an anti-a-thrombin monoclonal antibody, eg an anti-a-thrombin monoclonal antibody, eg an anti-a monoclonal antibody -thrombin that does not interfere with the binding of hirudin to thrombin and the thrombin / hirudin complex. Then an enzyme-labeled monoclonal antibody of the invention and a substrate solution and the binding of the monoclonal antibody according to the invention were detected by determining the enzyme label of the immune complex bound to the vehicle.

Monoclonal antibodies that are directed against the thrombin / hirudin complex in which the hirudin is hirudin variant 1 (HV1) and its derivatives are preferred. Also preferred are monoclonal antibodies that are directed against the thrombin / hirudin complex in which the hirudin is recombinant hirudin (rHV) and its derivatives. Monoclonal antibodies directed against the thrombin / hirudin complex in which the hirudin is recombinant variant 1 hirudin (rHV1) and its derivatives are especially preferred.

Since the thrombin / hirudin complex contains the two components thrombin and hirudin, monoclonal antibodies reactive with the complex can recognize the hirudin or thrombin fraction of the complex or a determinant comprising parts of both fractions. In addition, monoclonal antibodies to the thrombin / hirudin complex can recognize the so-called specific neo-determinants of the complex resulting from conformational changes that occur during the formation of the thrombin / hirudin complex.

A preferred embodiment of the present invention concerns monoclonal antibodies that are directed against the thrombin / hirudin complex and that recognize determinants of the hirudin fraction and its derivatives. Monoclonal antibodies that are directed against the thrombin / hirudin complex in which the hirudin is hirudin variant 1 (HV1) and which recognize determinants of the HV1 fraction in the complex are preferred. Monoclonal antibodies are also preferred which are directed against the thrombin / hírudin complex in which hirudin is recombinant hirudin (rHV) and which recognize determinants of the rHV fraction in the complex and its derivatives. Monoclonal antibodies which are directed against the thrombin / hirudin complex in which the hirudin is recombinant hirudin variant 1 (rHV1) and which recognizes the rHV1 fraction in the complex and its derivatives are particularly preferred. In the latter group of monoclonal antibodies, monoclonal antibodies that recognize determinants within the central N-terminal domain of the rHVl fraction in the thrombin / rHVl complex are preferred, preferably determinants comprising amino acid residues 1 to 43 of the rHVl fraction and its derivatives. An example of a particularly preferred monoclonal antibody and its derivatives is the monoclonal antibody designated MAb 4158-81-7 and its derivatives.

Another preferred embodiment of the present invention concerns monoclonal antibodies that are directed against the thrombin / hirudin complex and that recognize determinants of the thrombin fraction in the thrombin / hirudin complex and its derivatives. Monoclonal antibodies which are directed against the thrombin / hirudin complex in which the hirudin is hirudin variant 1 (HV1) and which recognizes determinants in the thrombin fraction in the complex are preferred. Also preferred are monoclonal antibodies that are directed against the thrombin / hirudin complex in which the hirudin is recombinant hirudin (rHV) and which recognize determinants of the thrombin fraction in the complex and its derivatives. Monoclonal antibodies which are directed against the thrombin / hirudin complex in which the hirudin is recombinant hirudin variant 1 (rHV1) and which recognize the thrombin fraction of the complex and its derivatives are particularly preferred. In the latter group of monoclonal antibodies, monoclonal antibodies which recognize determinants of the thrombin fraction are especially preferred and are exposed for binding to the antibody only when thrombin is bound to hirudin or a solid surface but do not recognize free thrombin in solution and its derivatives. An example of a particularly preferred monoclonal antibody and its derivatives is the monoclonal antibody designated MAb 4107-76-1 and its derivatives.

Derivatives of a monoclonal antibody of the invention retain the specificity of the antibody from which they are derived, i.e. they retain the binding pattern characteristic of the parental antibody. Examples of such derivatives are conjugates of monoclonal antibodies to an enzyme, a fluorescent tag, a chemiluminescent tag, a metal chelate, paramagnetic particles, avidin, biotin or the like or radiolabeled monoclonal antibodies or fragments of monoclonal antibodies.

Enzymes used for antibody conjugates of the invention are, for example, horseradish peroxidase, alkaline phosphatase, β-D-galactosidase, glucose-oxidase, glucoamylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate-dehydro-genase or glucose-6- phosphate dehydrogenase.

Fluorescent tags conjugated to antibodies of the invention can be fluorescein, fluorochrome, rhodamine and the like

Chemiluminescent tags are organic molecules that emit light upon modification of the chemical structure, e.g. luminol, isoluminol, pyrogallol, luciferin and the like.

Examples of metal chelates are ethylenediamine natetracetic acid (EDTA), diethylene triaminopentacetic acid (DPTA), 1,4,8,11-tetrazatetradecane,

1,4,8,11-tetrazatetradecane-1, 4,8,11-tetracetic, 1-oxa-4,7,12,15-tetraza-heptadecan-4,7,12,15-tetracetic acid or similar

In such conjugates the antibody is linked to the conjugation partner directly or by means of a spacer or linker group.

The radioactively labeled antibodies of the invention contain eg radioactive iodine ( ¹²³ I, ¹²⁵ I, ¹³¹ I), tritium ( ³ H),

35. 90. 99m carbon (C), sulfur (S), yttrium (Y), technecium (Tc) or similar.

The antibody fragments of the invention are for example Fab, Fab 'or F (ab') ₂ fragments

The monoclonal antibodies of the invention and their derivatives are obtained by procedures known per se in which cells of a hybridoma cell line secreting the desired monoclonal antibodies are multiplied in vitro or in vivo and, when necessary, the obtained monoclonal antibodies are isolated and / or converted into its derivatives.

In vitro multiplication is performed in suitable culture media, which are the conventional culture media, for example Dulbecco's Modified Eagle's Medium (DMEM) or RPMI 1640 medium, optionally supplemented with a mammalian serum, eg fetal calf serum or micronutrients and supplements that maintain growth, eg support cells such as mouse peritoneal exudate cells, spleen cells, bone marrow macrophages, 2-aminoethanol, insulin, transferrin, low density lipoptrotein, oleic acid or the like.

'χ

In vitro production provides relatively pure antibody preparations and allows scaling to give larger amounts of the desired antibodies. Techniques for culturing mammalian cells under tissue culture conditions are known and include homogeneous suspension culture, eg in a reactor with air inlet or a reactor with continuous agitation or immobilized or trapped cell culture, eg in hollow fibers , microcapsules, in agarose microspheres or ceramic cassettes.

For isolation of monoclonal antibodies, immunoglobulins in culture supernatants are first concentrated e.g. by precipitation with ammonium sulfate, dialysis against hygroscopic material such as polyethylene glycol (PEG), filtration through selective membranes or the like. If necessary and / or desired, the concentrated antibodies are purified by conventional chromatography methods ·, for example gel filtration, ion exchange chromatography, DEAE-cellulose or Protein A chromatography or immunoaffinity chromatography.

Large amounts of the desired monoclonal antibodies can also be obtained by multiplying the cells in vivo. To this end, hybridoma cells producing the desired antibodies are injected into histocompatible mammals in order to cause the growth of antibody-producing tumors. Optionally, animals are sensitized with a hydrocarbon, especially mineral oils such as pristane (tetramethylpentadecane), before injection. After one to three weeks, antibodies are isolated from the fluids of those mammals' bodies. For example, hybridoma cells derived from Balb / c mice that produce the desired monoclonal antibodies are injected intraperitoneally into mice

Balb / c optionally previously treated with pristane and after one to two weeks the ascitic fluid is removed from the animals.

The monoclonal antibody conjugates of the invention are prepared by known methods, eg by reaction of an antibody prepared as described above in the presence of a coupling agent, eg glutaraldehyde, periodate, N, N'-o-phenylenedimleimide, N- (m -maleimidobenzoyloxy) -succinimide, N- (3- [2'-pyridyldithio] -propionoxy) -succinimide, N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide or the like. Biotin conjugates are prepared e.g. by reacting antibodies with an activated biotin ester such as the N-hydroxy succinimide ester. Conjugates with fluorescent or guimioluminescent labels are prepared in the presence of a coupling agent, preferably fluorescein isothiocyanate.

Iodine (I, I, I) radiolabeled monoclonal antibodies were obtained from the antibodies of the invention by iodination known per se, for example with radioactive sodium or potassium iodide and an oxidizing chemical such as sodium hypochlorite, chloramine T or the like or an oxidizing enzyme agent, such as lactoperoxidase or glucose oxidase and glucose. The antibodies according to the invention are labeled with yttrium (Y) for example by chelation with diethylenetriaminepentacetic acid (DPTA). Antibodies labeled with technetium-99m are prepared by ligand exchange processes, for example by reducing tertechnate (TcO ₄ ) with stannous ion solution, chelating the reduced technetium in a Sephadex column and applying the antibodies to this column or by direct labeling, eg by pertechnate incubation, a reducing agent such as SnCl ₂ , a buffer solution such as sodium and potassium phthalate solution and antibodies.

Fragments of the monoclonal antibodies according to the invention, for example Fab, Fab 'or F (ab') ₂ can be obtained from monoclonal antibodies prepared as described above by methods known per se, eg by digestion with enzymes such as papain or pepsin and / or cleavage of disulfide bonds by chemical reduction.

The invention also relates to hybridoma cell lines that secrete the monoclonal antibodies of the invention, in particular hybridoma cell lines that secrete monoclonal antibodies directed against the thrombin / HVl complex or against the thrombin / rHVl complex, preferably against the thrombin / rHVl complex. .

In particular, the invention relates to hybridoma cell lines that secrete the desired antibodies which are hybrids of myeloma cells and B lymphocytes from a mammal immunized with hirudin coupled to a suitable vehicle or with the thrombin / hirudin complex. Suitable mammals and vehicles are those described in detail below. Hybridoma cell lines according to the invention which are hybrids of mouse myeloma cells and B lymphocytes of a mouse immunized with recombinant hirudin variant 1 (rHV1) coupled to a suitable vehicle or to the thrombin / rHV1 complex are preferred.

Hybridoma cell lines designated 4158-81-7 and 4107-76-1 which are deposited with the European Collection of Animal Cell Cultures (ECACC), PHLS Center for Applied Microbiology & Research, Porton Down, Salisbury, Wilts are particularly preferred. SP4 OJG, U.K., with deposit number 90061405 and 90061404, respectively on June 14, 1990.

The hybridoma cell lines of the invention are genetically stable, secrete the monoclonal antibodies of the invention with constant specificity and can be maintained in frozen cultures and can be reactivated by thawing and optionally recloning.

The invention also relates to a process for the preparation of hybridoma cell lines that secrete the monoclonal antibodies of the invention in which a suitable animal is immunized with hirudin coupled to a suitable vehicle or with the thrombin / hirudin complex, the cell-producing cells. mouse antibodies are fused with cells from a continuous cell line, the hybrid cells obtained in the fusion are cloned and the cell clones that secrete the desired monoclonal antibodies are selected.

The immunogen used to induce the monoclonal antibodies of the invention is an immunogenic conjugate of hirudin, in particular hirudin variant HV1, recombinant hirudin (rHV) or preferably recombinant hirudin HV1 (rHVl) or the thrombin / hirudin complex, in particular the thrombin complex / HV1 or the thrombin / rHV1 complex, or preferably the thrombin / rHV1 complex.

Coupling of hirudin to a vehicle to form an immunogenic hirudin conjugate is necessary to increase the immunogenicity of hirudin which is only weakly immunogenic in itself.

Suitable carrier molecules are, for example, lysine-rich proteins with free amine groups available for coupling, especially high molecular weight proteins such as bovine serum albumin (BSA; PM 66200) alpha-amylase derived from

Bacillus subtilis (PM 58000) or keyhole limpet hemocyanin (KLH; PM> 1000000) that can be purchased commercially in large quantities. Porcine thyroglobulin, toxins such as tetanus, cholera or diphtheria toxins, human serum albumin (HSA), beta-2 microglobulin and the like can also be used as vehicles. The fraction of rabbit IgG purified against IgG (H + L) (Kawamura & Berzofsky,

J. Immunol. 136, 58, 1986) can also be employed as a vehicle. Other possible carrier molecules include polysaccharides, natural or synthetic lipopolysaccharides, synthetic polypeptides such as polylysine, activated membranes, latex particles, bacteria such as Salmonella and the like.

An immunogenic hirudin conjugate, particularly hirudin variant HV1, is coupled with keyhole limpet hemocyanin (KLH). Particularly preferred is an immunogenic hirudin conjugate in which the recombinant hirudin, particularly HV1 variant recombinant hirudin (rHV1) is coupled to KHL.

Hirudin conjugates are prepared by methods known per se, either by adsorption of hirudin to the vehicle or by coupling using periodate, glutaraldehyde, carbodimides, e.g.

φ N, N'-o-phenylenediimaleimide, N- (m-maleimidobenzoyloxy) -succinimide, N- (3- [2'-pyridyldithio] -propionoxy) -succinimide, N-ethyl-N '- (3-dimethylaminopropyl) - carbodiimide or the like. If coupling via carboxyl groups is desired, the amine groups of the hirudin can be protected first, e.g. by acylation, for example with tertiary acetyl or butoxycarbonyl groups.

thrombin / hirudin complex used for immunization was prepared by mixing hirudin with thrombin, in particular by mixing an excess of hirudin with thrombin. The thrombin / hirudin complex can be separated from free hirudin for example by gel filtration by high pressure liquid chromatography (HPLC).

immunogenic hirudin conjugate or the thrombin / hirudin complex can be mixed with adjuvants, i.e. agents that will further enhance the immune response, for the immunization process. Adjuvants possible are Freund's complete adjuvant (water and oil emulsion only), mineral gels, e.g. aluminum hydroxide gels, surfactants such as lysolecithin, polyanions, peptides, BCG (Bacillus Calmette-Guerin), etc.,

Immunogens are used to immunize suitable mammals that recognize them as foreign molecules, for example mice, rats, rabbits, donkeys, goats, sheep, horses, pigs or chimpanzees, especially mice or rats, preferably mice. Balb / c mice are particularly preferred.

The immunization pathways include, among other intradermal, subcutaneous, intramuscular, intraperitoneal, intravascular and intracranial injections. Since high antibody titers are desired, a series of injections are usually given. Immunization is carried out, for example, by injection of the immunogenic hirudin conjugate, optionally mixed with Freund's incomplete or complete adjuvant, three to eight times parenterally, eg intraperitoneally and / or subcutaneously, in amounts of 10-50 / ig in Balb / mice. c at intervals of 1-3 weeks, followed by a booster injection of approximately 50-500 / ig 1-3 months after the last immunization.

The antibody-producing cells of the immunized mice, preferably lymphoid cells such as spleen lymphocytes, removed for example one to five days after the fianl injection, are fused with the cells of a continuous cell line, ie a cell clone that replicates continuously. which gives this ability to replicate the hybrid cells resulting from the fusion. An example for such a cell line is the tumor cell line (myeloma) which by itself does not produce immunoglobulins or their fragments but which has the potential to produce and secrete large amounts of antibody and which carries a genetic marker so that hybrid cells can be selected against unfused parental cells. Several suitable myeloma cell lines are known. Myeloma cell lines without the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT) or the enzyme thymidine kinase (TK) are preferred, which therefore do not survive in a selective culture medium containing hypoxanthine, aminopterin and thymidine (HAT medium). Particularly preferred are myeloma cells and derived cell lines that do not survive in HAT medium and do not secrete immunoglobulins or their fragments, particularly Sp2 / O-Agl4 mouse myeloma cell lines (Shulman et al., Nature 276, 269, 1978) or X63-Ag8.653 (Kearney et al., J. Immunol. 123. 1548, 1979) that can be purchased commercially (Flow) or the PAI mouse myeloma cell line (Stocker et al., Research Disclosure 21713 , 1982).

The fusion is carried out in the presence of a fusion promoter, for example Sendai virus or other paramyxoviruses, optionally in the form inactivated by UV or chemical fusion agents such as calcium ions, surfactant lipids, eg lysolecithin or polyethylene glycol (PEG) or by electrofusion, Preferably, myeloma cells are fused with a three to twenty-fold excess of spleen cells derived from immunized mammals in a solution containing about 30% to about 60% polyethylene glycol of a molecular weight between 1000 and 4000.

After fusion, the cells were resuspended and cultured in a selective medium chosen depending on the genetic selection marker, for example HAT medium. In this medium, only hybridoma cells will survive, as they combine the ability to grow and replicate in vitro inherited from parental myeloma cells and the absence of the HGPRT or TK genes essential to survival in HAT medium inherited from derived antibody-producing spleen cells. immunized mammals.

Suitable culture media for the expansion of hybridoma cells are conventional culture media, such as Dulbecco's Modified Eagle Medium (DMEM), minimal essential medium, RPMI 1640 and the like, optionally supplemented with a mammalian serum, eg 10 to 15% of fetal calf serum. Preferably, support cells, eg normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages or the like, are added at the beginning of cell growth immediately after the fusion step to feed the hybridoma cells and support their growth , especially whenever cell densities are low, providing growth factors and the like. If phagocytic cells such as macrophages or monocytes are used, they can play a useful role in cleaning up the debris from dead myeloma cells that are always found after treatment with aminopterin. The culture media are supplemented with selective media to prevent myeloma cells from growing more than hybridoma cells.

Supernatants from hybridoma cell cultures are tested for the desired monoclonal antibodies with an immunoassay, preferably a radioimmunoassay or enzyme immunoassay. For example, a suitable vehicle such as a microtiter plate is coated with the thrombin / hirudin complex and incubated with the hybridoma supernatant to be tested. Then, enzyme-labeled antibodies that recognize the antibodies bound to the thrombin / hirudin complex in the vehicle are added and a substrate solution and the labeled antibodies bound to the antibody / thrombin / hirudin complex are detected by determining the enzyme tag attached to the vehicle.

The positive hybridoma cells were cloned, e.g. by limit dilution or on soft agar, preferably two or more times. Optionally, the hybridoma cells are passed on animals, e.g. mice, by intraperitoneal injection and collection of ascites, which stabilizes hybridomas and improves growth characteristics. The cloned cell lines can be frozen in a conventional manner.

The monoclonal antibodies of the invention and their derivatives are useful for a number of medical purposes in vitro and in vivo, all of which are based on the qualitative and / or quantitative determination of the thrombin / hirudin complex. In particular, the monoclonal antibodies of the invention and their derivatives can be used in the early diagnosis of thrombosis, especially in high-risk groups and patients with thrombotic disorders. This is achieved by detecting small amounts of thrombin spontaneously generated by injecting hirudin as a mark followed by measuring the complex formed, thus determining when treatment with hirudin is necessary or preventive therapy with hirudin is advisable. In addition, the monoclonal antibodies of the invention and their derivatives are useful for controlling hirudin therapy by determining hirudin activity, for establishing the correct dosage of hirudin and for recording possible disease progression.

For the quantitative determination of the thrombin / hirudin complex, the minoclonal antibodies of the invention and / or its derivatives can be used, for example, in any of the known immunoassays that are based on the interaction of the link between the antigenic determinants of the thrombin / hirudin complex molecule and paratopes of monoclonal antibodies. Examples of such assays are immunoassays that use radioactive labeling, enzymes, fluorescence, chemiluminescence, immunoprecipitation, agglutination with latex and hemegglutination, laser beam deviation or tests with evanescent light.

The monoclonal antibodies of the invention can be used as such or in the form of radiolabeled derivatives (RIA). Such immunoassays also include testing procedures in which the radiolabeled antibodies known per se that recognize and bind an epitope of the antibodies of the invention are used. Any of the recognized modifications of RIA can be used, for example RIA in soluble phase (homogeneous), RIA in solid phase (heterogeneous), single RIA or double RIA (sandwich) with direct or indirect determination of the thrombin / hirudin complex.

A sandwich RIA is preferred in which a suitable vehicle, for example the plastic surface of a microtiter plate or test tube, eg polystyrene, polypropylene or polyvinyl chloride, glass or plastic beads, filter paper, dextran, etc., cellulose acetate or nitrocellulose filters, magnetic particles or the like, is coated with a monoclonal antibody of the invention, preferably MAb 4107-76-1. Then the solutions are added to

testing, for example, a patient's plasma, containing the thrombin / hirudin complex and finally the second polyclonal or monoclonal antibody that recognizes an antigen epitope different from that recognized by the first monoclonal antibody bound to the vehicle. . 125 and which are radiolabeled, e.g. with I, preferably radiolabeled monoclonal antibodies such as MAb 4158-81-7.The amount of the thrombin / hirudin complex in | The test solution is directly proportional to the amount of second antibody bound and is determined by measuring the radioactivity bound to the vehicle.

The monoclonal antibodies according to the invention can be used as such or in the form of enzyme-conjugated derivatives in an enzyme immunoassay. Such an immunoassay also includes test procedures in which enzyme-labeled antibodies known per se that recognize and bind to an epitope of the antibodies of the invention are used. As described above for radioimmunoassays, any of the known modifications of enzyme immunoassays can be used.

The tests are carried out in a manner analogous to the radioimmunoassays described above using a nezymphatic mark instead of a radioactive mark. The amount of immune complex formed that φ corresponds to the amount of the thrombin / hirudin complex present in the solution to be tested is determined by adding an enzyme substrate solution. The reaction of the substrate with the enzyme results, for example, in a color change that can be observed with the naked eye or with optical measurement systems.

An enzyme-linked immunosorbent assay (ELISA) is preferred in which a vehicle such as that described above for RIA is coated with a monoclonal antibody of the invention, preferably MAb 4107-76-1, incubated with test solutions containing the thrombin / hirudin complex. , with a second polyclonal or monoclonal antibody that recognizes an epitope of the antigen different from that recognized by the first monoclonal antibody bound to the vehicle and which is conjugated to the enzyme, preferably monoclonal antibodies conjugated to enzymes of the invention such as MAb 4158-81-7 and with a solution of substrate. The reaction of the enzyme and substrate results, for example, in a color change and can be observed with the naked eye or with optical measurement systems, so that the amount of bound enzyme can be determined, which is proportional to the amount of the thrombin / hirudin complex. in the solution to be tested. An alternative is an immunoassay that is carried out essentially as described above but in which the first monoclonal antibody bound to the vehicle of the invention is coupled to a small chemical group such as biotin and is detected by a labeled reagent, eg a biotin-binding protein as be it avidin or streptavidin.

The monoclonal antibodies according to the invention can be used as such or in the form of derivatives conjugated to chemiluminescent labels in a chemiluminescent immunoassay. Such immunoassays also include test methods in which antibodies known per se are used that recognize and bind to an epitope of the antibodies of the invention and which are conjugated to chemiluminescent labels. As described above for radioimmunoassays, any of the known modifications of a chemiluminescent immunoassay can be used.

The tests are carried out in a manner similar to the radioimmunoassays described above using a chemiluminescent marker instead of a radioactive marker. The amount of immune complex formed that corresponds to the amount of antibodies directed against the thrombin / hirudin complex present in the solutions to be tested is determined by adding a compound that captures luminescence, eg H ₂ O ₂ and NaOH and measuring the light emission with an optical measuring system.

The use according to the invention of monoclonal antibodies and their derivatives as described herein for the qualitative and quantitative determination of the thrombin / hirudin complex also includes other immunoassays known per se. for example immunofluorescence assays using antibodies or antibody derivatives conjugated with fluorescent tags such as fluorescein, latex agglutination with antibody-coated or antigen-coated latex particles, hemagglutination with antibody-coated or antigen-coated red blood cells, tests with evanescent light using an antibody coated optical fiber and other direct acting immuno-sensors that convert the binding event to an electrical or optical signal or the like.

The application of the monoclonal antibodies of the invention and / or its derivatives as already described allows the determination of the presence and / or the concentration of the thrombin / hirudin complex in the buffer, urine and plasma in concentrations ranging between 1 and 200 ng / ml.

The invention also relates to test kits for the qualitative and quantitative determination of the thrombin / hirudin complex comprising monoclonal antibodies of the invention and / or its derivatives and, optionally, other monoclonal or polyclonal antibodies and / or additives.

Test kits according to the invention for the radioimmunoassay contain, for example, a suitable vehicle, eg microtiter plates or nitrocellulose sheets, uncoated or coated with a monoclonal antibody of the invention, optionally lyophilized or concentrated solutions of a second antibody radioactively labeled against a thrombin / hirudin complex epitope other than that recognized by the vehicle-bound monoclonal antibody and / or a radioactively labeled derivative, standard thrombin / hirudin complex solutions, buffer and optionally, polypeptides and detergents to prevent unspecific adsorption and formation of aggregates, pipettes, reaction vessels, calibration curves, instruction manuals and the like.

The test kits according to the invention for an enzyme immunoassay contain, for example, a suitable vehicle, coated or not with a monoclonal antibody of the invention, optionally lyophilized or concentrated solutions of a second enzyme-labeled antibody directed against an epitope of the complex thrombin / hirudin other than that recognized by the first monoclonal antibody and / or an enzyme-labeled derivative, enzyme substrates in solid or dissolved form, standard solutions of the thrombin / hirudin complex, buffer solutions and, optionally, polypeptides and detergents to prevent adsorption nonspecific and formation of aggregates, pipettes, reaction vessels, calibration curves, instruction manuals and the like.

The following examples illustrate the invention, but do not limit it in any way.

Abbreviations

HAT - hypoxanthine / aminopterin / thymidine HPLC - high pressure liquid chromatography MAb - monoclonal antibody

MES - 2- [N-morpholino] ethanesulfonic acid PBS - PEG saline phosphate buffer - polyethylene glycol

I rHVl - HV1 variant recombinant hirudin

RT - room temperature

THC - thrombin-hirudin complex

Examples

Example 1 Preparation of monoclonal antibodies against the thrombin / hirudin complex

1.1 Preparation of immunogens

As immunogens can be used: (1) a conjugate of recombinant hirudin HV1 (rHVl) with (rHVl) with keyhole limpet hemocyanin (KLH) and (2) the thrombin / rHVl complex.

rHVl (Plantorgan / Ciba-Geigy) is coupled to KLH (Calbiochem) by the carbodiimide method as described below: 2 mg of rHVl in 560 μΐ of buffer O, 1M MES pH 4.75 is mixed with 100 μΐ of KLH (10 mg / ml) and 200 μΐ of N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (20 mg / ml). After 2 hours at 22 ° C, the mixture was dialyzed in PBS.

The thrombin / rHV1 complex was prepared by mixing an excess of rHV1 with thrombin in PBS (1.7: 1 molar ratio).

1.2 Immunization protocol

Two groups of five female Balb / c mice (4-6 weeks of age) were given three series of injections with rHVl conjugated to KLH (30 Mg / injection; group I) or with the thrombin / rHVl complex (15 Mg / injection group II). The first injection consists of 0.1 ml of the respective immunogen in PBS in a 1: 1 molar ratio with 0.1 ml of Freund's complete adjuvant (Difco);

μΐ were injected intraperitoneally and 150 μΐ subcutaneously. On the second (14th) and third (30th) of the injections, Freund's complete adjuvant was replaced by Freund's incomplete adjuvant. One week after the last injection, serum was collected and antibody titers determined by enzyme-linked immunosorbent assay (ELISA) as described in example 1.3.

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1.3 Enzyme-linked immunosorbent assay (ELISA) for determination of antibody titer in serum

Serum antibody titers were determined by ELISA as described below.

A two-fold molar excess of thrombin was mixed with rHV1 (0.2 yg and 0.02 µg / well, respectively). The microtiter plates (Dynatech) were coated with 100 μΐ per well of a solution of the thrombin / rHVl complex in coating buffer (50 mM sodium carbonate buffer pH 9.6: 477 mg Na ₂ C0 ₃ , 1.8 ml NaN 0.5M ₃ ad 300 ml H ₂ 0). The plates were incubated overnight at 4 ° C in a humid chamber and washed five times with PBS-Tween 0.1% (1 ml Tween-20, Serva, 1000 ml PBS). The wells were allowed to dry, filled with 200 ml per well of 1% PBS-BSA (1g BSA, 3 ml 0.5M NaN ₃ , ad 100 ml of PBS), incubated for two hours at rt and washed five times with PBS- 0.1% Tween. Then 100 μΐ of the respective serum diluted in PBS-Tween 0.1% was added. After 2 hours of incubation at room temperature, the plates were washed five times with PBS-TweenO, 1%. In the next step, 100 µl goat antibody affinity purified against mouse IgG and conjugated to alkaline phosphatase (Kikegaard & Perry Laboratories) diluted 1: 1500 in PBS-Tween 0.1% was added to each well. Finally, 150 μΐ was added per well of substrate solution (1 mg of p-nitrophenylphosphate [Sigma] per ml of diethanolamine buffer pH 9.8: 97 ml of diethanolamine [Merck], 6 ml NaN ^ 0.5M, 100 mg MgC ^ .ôB ^ O, adjusted to pH 9.8 with concentrated HCl, ad 1000 ml H ^ O), the reaction was stopped by adding 50 μΐ of 3M NaOH and reading the optical density at 405 nm after 2 hours of incubation in the dark.

* -1 ui ·

1.4 Fusion protocol

After a rest period of more than two months, the mice received an intraperitoneal boost injection with 300 mg of rHV1-KLH conjugate (group I) or 70 mg of the thrombin / rHV1 complex (group II). Three to four days later the mice were sacrificed, the spleen cells were fused with PAI murine myeloma cell lines (Stocker et al., Research Disclosure Ne 21713, p.155, 1982), using PEG 4000 (Merck), by a modification of the original method by Kohler and Milstein (Galfre et al., Nature 266, 550, 1977) and the cells were distributed into microtiter plate wells containing HAT medium (Boehringer). After 2 to 4 weeks, the wells containing hybridomas to be grown were tested for specific monoclonal antibodies by the ELISA of Example 1.5.

1.5 Enzyme-linked immunosorbent assay (ELISA) for hybridoma screening 'Hybridomas that have grown have been tested for the presence of specific antibodies by an ELISA as described in Example 1.3. Instead of serum, 200 µl hybridoma supernatants diluted 1: 2 in PBS Tween 0.1% were added to φ microtiter plates coated with the thrombin / rHV1 complex.

From these three fusions performed as a group of mice I (fusion efficiency 100%), six MAbs secretors reactive with the thrombin / rHV1 complex were obtained. The three fusions with the group II mice result in four such hybridomas, one of which secretes MAbs that cross-react with rHV1, while the MAbs secreted by the remaining three hybridomas cross-react with thrombin. Two lines of hybridoma cells, one from each group, were selected for later jjw

I characterization of secreted MAbs. Both secreted lines were deposited on June 14, 1990 at the European Collection of Animal Cell Cultures (ECACC), under deposit numbers 90061405 for hybridoma 4158-81-7 (group I) and 90061404 for hybridoma 4107-76- 1 (group II). The monoclonal antibodies secreted by these hybridomas were designated by the prefix "MAb" and the internal number of the respective hybridoma, e.g. MAb 4107-76-1.

1.6 Hybridoma storage and processing

The selected hybridoma cells can be grown in culture, frozen at -80 ° C and kept in liquid nitrogen and then reactivated. The cells were cloned by the limit dilution method (J.W. Goding, J. Immunol. Methods 39., 285, 1980) and expanded to form ascites in pristane-sensitized Balb / c mice (see Example 2.1).

Example 2 Production, isolation and purification of anti-THC monoclonal antibodies

2.1 Expansion of hybridomas in vivo and purification of monoclonal antibodies

For the production of ascites, female Balb / c mice (20-25 g) (Tierfarm Sisseln, Switzerland) were pretreated with 0.3 ml of pristane oil (Aldrich) intraperitoneally. 1 At 3 weeks later, the mice received a second injection of pristane (0.2 ml i.p.) and were simultaneously inoculated i.p. with 2 x 10 hybridoma cells in 0.2 ml PBS. After 8-10 days, the resulting ascites fluid was collected by centrifugation at 800 g and stored at -20 ° C or -80 ° C.

I

The thawed ascites fluid was clarified by centrifugation at 30000g for 1h. After removing the top layer containing lipids, the protein concentration was determined and adjusted to 10-12 mg / ml with PBS. The immunoglobulin G (IgG) faction was precipitated by adding dropwise 0.9 volumes of saturated ammonium sulfate at 0 ° C. After 1 hour, the IgG fraction was pelleted by centrifugation for 1 hour at 22000g. The pellet was dissolved in 20 mM Tris-HCl pH 7.9 buffer containing 50 mM NaCl and was dialyzed against the same buffer overnight at 4 ° C. The IgG fraction was further purified by anion exchange chromatography on a DE52 diethylaminoethylcellulose column (Whatman). The sample was diluted 1: 2 (v / v) in 20 mM Tris-HCl pH 7.9 to a final concentration of 25 mM NaCl and 10 mg of protein per ml of gel were applied to the column. The elution was obtained by increasing the sodium chloride concentration from 25 mM to 200 mM (linear gradient). In general, MAbs were eluted around 80 mM NaCl. The fractions were dialyzed against PBS overnight at 4 ° C and stored at -70 ° C. Purity was tested by SDS-PAGE and isoelectric focusing. Purity was more than 90%.

2.2 Expansion of hybridomas in vitro

A pre-culture of any of the cell lines was obtained by culturing the hybridoma cells at physiological temperature (about 37 ° C) in RPMI1640 medium (Seromed) containing 10% fetal calf serum (FCS) to a final density of 5 x 10 at 10 ° cells per ml. The entire pre-culture was used to fill Bellco culture flasks and adjusted to a total volume of 1500 ml with fresh RPMI / 10% FCS medium. The culture was stirred at 37 ° C with 5% CO ₂ at 30 rpm for two to three days, then diluted to a total volume of 3000 ml with RPMI 1640/10% FCS and stirred for another seven to ten days. After this time, 95% of the cells were dead. The culture broth was centrifuged at 100xg for 20 minutes at 4 ° C. The supernatant was filtered through a 0.2 µm pore filter under sterile conditions. The crude immunohglobulin was precipitated by the slow dropwise addition of 0.9 volumes of saturated ammonium sulfate at 0 ° C. This precipitate was purified as described in Example 2.1.

Example 3 Determination of the class and subclass of anti-THC monoclonal antibodies

The class and subclass of monoclonal antibodies was determined in a Bio-Rad enzyme-linked immunosorbent assay kit (ELISA). MAb 4158-81-7 and MAb 4107-76-1 are of the IgGl class.

Example 4 Preparation of rHV1 analogs

4.1 Preparation of synthetic rHV1 peptides

Synthetic rHV1 peptides representing sequences 52-65, 40-65, 29-38 and 1-15 were synthesized by the known solid phase method (Rink, Tetrahedron Letters 28., 3787, 1987). The peptide representing the sequence 1-43 was synthesized as described in Chang et al., FEBS Letters 260, 209, 1990.

4.2 Reducing S-carboxymethylation

RHV1-reducing S-carboxymethylation was performed according to Hirs (Methods in Enzymol-11, 199, 1967). It results in the complete destruction of the three-dimensional structure of rHV1.

4.3 Digestion of rHVl by V8 staphylococcal protease of native rHVl dissolved in 90 (NH ₄ ) 50 mM HCO ₃ , pH 8.0, supplemented with EDTA mixed with 20 μ% (in 20 μΐ) of aureus protease strain V8 (Sigma) and incubated for 30 minutes, 2 h or 4 h at 37 ° C. The reaction was stopped by adding 5 μΐ of 0.01M diisopropyl fluorophosphate (Sigma). The degree of digestion was tested by reverse phase HPLC as described by Schlaeppi et al. (Eur. J. Biochem. 188, 463, 1990) or by amino-terminal analysis (Chang,

Analytical Biochem. 170, 542, 1988) and by SDS-PAGE.

μΐ of 2 mM buffer, were Staphylococus

4.4 Digestion of rHVl by lysyl endopeptidase

To 70 µg of rHVl dissolved in 97.5 μΐ of 25 mM Tris / HCl pH 8.3, 2.5 Mg in 2.5 μΐ of lysyl endopeptidase (Wako Chemicals) was added and incubated for different periods of time at 37 ° C. The proteolytic fragments were separated by HPLC as described by Schlaeppi et al., (Loc. Cit.) And the degree of digestion determined by quantitative N-terminal analysis.

Example 5 Test processes used for the characterization of anti-THC monoclonal antibodies »

5.1 Biotinylation of rHVl, thrombin and monoclonal antibodies

Thrombin (1.1 nmol in 2 μΐ DMSO) for 3 hours at 4 ° C. Then 360 μΐ of PBS was added before dialysis overnight against PBS.

Biotinylation of monoclonal antibodies was performed essentially as described by Bayer et al. (Methods Enzymol. 62, 308, 1979). Biotin-X-N-hydroxysuccinimide ester (200 μg in 200 μΐ DMSO) was added to 5 mg of purified MAb in 5 ml of PBS (pH 7.0) (13: 1 molar ratio). After 4 h at 4 ° C the mixture was extensively dialyzed in PBS.

0.5 mg of rHVl in 200 μΐ of acetate buffer (20 mM, pH 6.0) were mixed with 100 μg of the biotin-XN-hydroxis succinimide ester (Calbiochem) dissolved in 40 g of ethanol / water (1: 1 , v / v) so that the molar ratio of biotin to rHV1 is 3.2: 1 and the mixture was incubated for 20 minutes at Then, 260 μΐ of PBS was added and the solution dialyzed overnight against PBS at 4 ° C.

5.2 Competitive ELISA epitope mapping was performed by competitive ELISA experiments using rHV1, rHV1 analogs as described in example 4 and recombinant hirudin variant PA (rHV3; Dodt et al., Biol. Chem. Hoppe-Seyler 367. 803, 1983) . The competitive two-step ELISA was performed as described below.

rHVl (0.2 μ9 / 100 μΐ of 50 mM coating buffer) was attached to mierotiter plates. After overnight incubation at 4 ° C, the plates were washed with 0.1% PBS-Tween and the remaining free sites on the solid support were blocked by incubation with PBS / BSA (1% w / v). In separate test tubes, 50 μΐ of each of the purified MAbs (40 to 400 ng / ml) were incubated with 650 μΐ of a standard solution containing increasing amounts of rHVl or rHVl analogs. After overnight incubation at 4 ° C, 200 μΐ of the antibody-antigen mixture was added to each well and incubated for another hour. The wells were then washed five times and 100 μΐ per well of goat anti-mouse antibody conjugated with alkaline phosphatase (dilution 1: 1500) was added over 1.5 h. After washing,

150 μΐ was added to the wells per well of the substrate p-nitrophenylphosphate (1 mg / ml in diethanolamine buffer pH 9.8, supplemented with 0.5 mM MgCl ₂ .H ₂ O). The color change, which is proportional to the amount of antibody that reacts with the antigen bound to the solid phase was controlled at 405 nm. All samples were run in triplicate. Typical inhibition curves were plotted representing B / Bo x 100 (bound percentage) vs. the concentration of inhibitor present (Bo represents the absorbance measured without rHV1 added to the antibody and B the absorbance measured with various concentrations of rHV1). The value of ΙΟ ^ θ represents the concentration of antigen that inhibits 50% of antibody binding to the solid phase. IC _5Q was calculated using an adaptation of the ENZFITTER curve fitting program (RJ Leatherbarrow, Elsevier) based on a curve with four logistical parameters: U = (DC) / 1 + (z / A) b) + C, where U is the expected response for a z dose of the standard. The four parameters describe the shape of the curve, D and C give the upper and lower asymptote and A is the average asymptote dose (Raab, Clin. Chem. 29., 1757, 1983).

5.3 Double sandwich ELISA with MAbs selective for free hirudin to determine overlapping epitopes

An ELISA with two antibodies was performed on sandwiches using MAbs selective for free hirudin as described below to determine whether monoclonal antibodies recognize overlapping epitopes.

The microtiter plates were coated with 0.5 µg / well of purified group I anti-THC MAb prepared in sodium carbonate buffer (50 mM, pH 9.6) and incubated overnight at 4 ° C. After the blocking and washing steps (by 1% BSA and 0.1% PBS-Tween, respectively), increasing concentrations of rHV1 prepared in 0.1% PBS-Tween (0 to 100 ng / well) were added to the bound monoclonal antibody and incubated for 1 hour at After washing, a second biotinylated MAb selective for free rHV1 (0.5 µg / well) was added and incubated for 2 h. As the second monoclonal antibody, MAb 4114-96-1, MAb 4120-37-7 and MAb 4049-83-12 respectively, were used, as described by Schlaeppi et al., (Loc. Cit.). Then, after washing, 100 μΐ of streptavidin conjugated to alkaline phosphatase (diluted 1: 2500) was added and incubated for 1.5 h at r.t. After washing, the substrate (see Example 5.2) was added and absorbance at 405 nm was measured after different time intervals, starting 15 minutes after adding the substrate.

5.4 Sandwich ELISA to determine the rHVl / thrombin complex thrombin / rHVl complex was prepared by mixing 3 pmoles of a-thrombin (3000 NIH units / mg; CBR Laboratories) with 15 pmoles of rHVl in 100 μΐ of 0.1% PBS Tween containing 0.1% BSA or in 100 μΐ of human plasma diluted 1: 3 in PBS-Tween 0.1% containing 0.1% BSA or in 100 μΐ of human plasma diluted 1: 3 in PBS-Tween.

The microtiter plates were coated with the MAb EST6 anti-thrombin MAb (0.2 µg / 100 μΐ; Bioscot, Edinburgh, UK). After overnight incubation at 4 ° C, blocking with PBS-BSA (0.1%) and washing with PBS-Tween, 100 μΐ of a series of two-fold dilutions of the thrombin / rHVl complex prepared as described above (molar ratio 1: 5) were added to the well and incubated for 2 hours at room temperature. Then the following reagents were added consecutively: (1) 0.5 μg / 100 μΐ of biotinylated MAb 4107-76-1 and MAb 4158-81-7, respectively, for 2 h, (2) streptavidin conjugated to alkaline phosphatase (diluted 1: 2500) for 1.5 hours, (3) substrate (see example 5.2). Incubations were carried out at 22 ° C. Between each step the plates were washed with PBS-0.1% Tween. Absorbance was controlled at 405 nm after 15 to 30 minutes.

A simplified assay was developed using microtiter plates coated with the chosen MAb. Biotinylated thrombin (0.2 µg / well) complexed with rHV1 (1: 1 molar ratio) was added to the wells and the biotinylated thrombin was revealed directly by streptavidin conjugated to alkaline phosphatase.

5.5 Gel filtration by HPLC

HPLC gel filtration (high performance liquid chromatography was used to analyze the thrombin / rHVl complex or antigen-antibody complexes. Samples containing thrombin (0.3 nmole) and rHVl (1.5 nmoles) were incubated for 15 minutes The tested anti-THC MAb or anti-hirudin control MAb 4049-83-12 (0.2 nmole; Schlaeppi et al., Loc. Cit) was added (total volume 45 μΐ) and further incubated for 5 h at room temperature The samples were injected into a TSK-G3000SW gel filtration column (LKB; 8 x 300 mm) and eluted with PBS at a flow rate of 0.3 ml / min The protein absorbance was controlled at 280 nm.

Example 6 Characterization of anti-THC monoclonal antibodies

6.1 Group I monoclonal antibodies the results of the competitive ELISA of example 5.2 MAb 4158-81-7 (group I) are shown in Table 1 below.

- »ί Λ

Table 1: Characterization of MAb 4158-81-7 by competitive ELISA

I 1- variant / analogous hirudin | I I IC _5O (ng / ml) ^A | 1 1 | rHVl | 3.5 | | rHV3 ^B | 1000 | | rHVl 1-43 | 1.0 | | HV1 peptide 52-65 | > 1000 | | HV1 peptide 40-65 | > 1000 | | HV1 peptide 29-38 [ > 1000 | | HV1 peptide 1-15 | > 1000 | | S-CM-rHVl ^C | 200 | | rHVl + V8 protease (2h) ° | 0.5 | | rHVl + lysyl endopeptidase (1.5 h) ^E | 1 1 11.8 I

Subtitle:

A- inhibitor concentration for 50% solid phase MAb inhibition

B- rHV3 residues that are different from rHVl: Ile, Tre 2, Lis 24, Gin 33, Lis 35, Asp 36, Gin 53, Pro 58, Asp 62, Ala 63,

Asp 65, Glu 66

Reduced C- rHVl and S-carboxymethylated

D- 97% cleavage on Glu 43; 50% cleavage in Glu 61

E- 100% cleavage in Lys 47; 100% cleavage in Lis 36

MAb 4158-81-7 binds rHVl solution with an IC _5Q value of 3.5 ng / ml but did not cross-react with the four rHVl peptides 1-15, 29-38, 40-65 and 52- 65 that make up most of the rHV1 sequence. In addition, the binding of reduced rHV1 and S-carboxymethylated decreased dramatically compared to unmodified rHV1, suggesting an epitope-dependent conformation. MAb 4158-81-7 fully cross-reacts with the central N-terminal domain of rHV1, as well as with rHV1 digested with protease V8 in which the central domain of rHV1 remained intact. The affinity of MAb for rHVl 1-43 and for rHVl digested with V8 is higher than for the complete rHVl molecule, suggesting that some spatial impediment of the C-terminal domain occurs, which alters its native conformation.

In the double sandwich ELISA of example 5.3, MAb 4158-81-7 combines with any of the anti-hirudin monoclonal antibodies that bind to the C-terminal domain (see Schlaeppi et al., Loc. Cit.), Indicator of non-overlapping epitopes. However, ten to twenty times higher rHV1 concentrations are required when MAb 4158-81-7 was combined with MAb 4049-83-12 which binds to residues within the 41-47 domain. This indicates a partial overlap between the epitopes of both MAbs which was further confirmed by limited proteolysis of the rHH1 / antibody complex by protease V8. Cleavage between Glu 43 and Gli 44 was prevented by binding MAb 4158-81-7 to rHV1.

All other group I MAbs also cross-react with the rHV1 1-43 peptide, suggesting that group I MAbs bind to only the central N-terminal domain of rHV1. These results also indicate that the main antigenic determinants of rHVl accessible on the surface of the thrombin / rHVl complex are located exclusively in the N-terminal domain, as suggested by the crystalline structural of the thrombin / hirudin complex (Grutter et al., EMBO Journal 9, 2361, nineteen ninety).

MAb 4158-81-7 does not bind to the thrombin / rHV1 complex in solution, but binds to the complex adsorbed on a solid surface such as a microtiter plate or presented by an antibody such as MAb EST6 or MAb 4107-76-1. In addition, MAb 4158-81-7 does not bind to the biotinylated thrombin / rHVl complex (when

contrary to MAb 4107-76-1, see below). Therefore, it was concluded that the conformation of the thrombin / rHVl complex in solution is different from the behavior on a solid support and that the central domain of rHVl, to which MAb 4158-81-7 is attached, is exposed only in the last conformation. It follows that MAb 4158-81-7 cross-reacts with the thrombin / rHV1 complex only under restricted conditions but not in general.

6.2 Group II monoclonal antibodies

Among the four MAbs in group II, only one MAb cross-reacts with rHV1, however with low affinity. As shown by the competitive ELISA in example 5.2, this MAb also cross-reacts with the central rHV1 domain (residues 1-43), indicating that it is closely related to group I MAbs. Among the remaining three MAbs, MAb 4107-76-1 has been studied in more detail as it binds very strongly to the thrombin / rHV1 complex. Neither rHVl nor thrombin in solution binds. However, this MAb binds to the thrombin adsorbed to the microtiter plates. Several lines of evidence suggest that binding occurs only when thrombin is adsorbed to a solid surface but not when it is in solution. First of all in HPLC gel filtration experiments as described in example 5.6, MAb 4107-76-1 binds to the complex, but not to free thrombin. MAb 4049-83-12 anti-rHV1, which only recognizes free rHV1, is used as a control. Second, using biotinylated thrombin, MAb 4107-76-1 binds only to the complex and not to free biotinylated thrombin. Finally by the double sandwich ELISA with two MAb using MAb EST6 to avoid direct adsorption of thrombin to the plate, the binding of MAb 4107-76-1 occurs only with the complex but not with thrombin. When the 40-65 rHVl C-terminal peptide replaces rHVl in the complex, MAb 4107-76-1 does not bind, suggesting that the entire rHVl molecule is

158, 1988), according to (Grutter et al., Loc.

necessary for the connection. However, the replacement of rHV1 by the rHV3 variant reduces the binding of MA 4107-76-1 to the complex only partially, suggesting that MAbs recognize a common determinant. Neither low molecular weight inhibitors nor heparin could induce the binding of MAb to thrombin. These results suggest that MAb 4107-76-1 recognizes a neoepitope in thrombin expressed upon binding of rHV1 or adsorbing thrombin to a solid phase. Thrombin undergoes conformational changes when rHV1 is bound (S. Konno et al., Arch. Biochem.Biophys. 267.

confirmed by cit. crystallography data). This conformational change must create immunodominant neo-epitopes. Conformational change or even denaturation of an antigen upon adsorption to a solid surface is well documented and may explain the binding of MAb 4107-76-1 to thrombin adsorbed to the surface.

Some test results for the group II monoclonal antibody MAb 4107-76-1 are shown in Table 2.

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Table 2: ELISA determination of the binding of the thrombin / rHVl complex to the MAbs absorbance (405 nm) added reagents

MAb4107-76-l | MAb EST6

biotinylated thrombin 1 0.040 | 0.719 biotinylated thrombin and rHVl 1 1,027 | 1,018 biotinylated rHVl 1 0.013 [0.020 PBS-Tween 1 0.009 | 0.017

A: The reagents were added to the MAbs-coated microtiter plates and developed with streptavidin conjugated to alkaline phosphatase

Example 7 Sandwich ELISA for the determination of the thrombin / hirudin complex in plasma

Specific determination of the thrombin / hirudin complex in plasma was achieved by sandwich ELISA using MAb4107-76-1 as the capture antibody and MAb 4158-81-7 as the second antibody. ELISA is a modification of the ELISA described in example 5.4 by replacing MAb EST6 with MAb 4107-76-1 and was performed as described below.

Microtiter plates were coated with MAb 4107-76-1 (0.2 gg / 100 gl). After blocking with PBS-BSA 1% and washing with PBS-Tween 0.1%, 200 g of plasma to be tested were added to the well and incubated for 2 hours. Then the following reagents were added consecutively: (1) 0.5 gg / 100 g of biotinylated MAb 4158-81-7 for 2 hours, (2) streptavidin conjugated to alkaline phosphatase (diluted 1: 2500) for 1.5 hours h, (3) substrate (see example 5.2). Incubations were performed

22 ° c. Between each step the plates were washed with

0.1% PBS-Tween. Absorbance was controlled at 405 nm after 15 minutes.

The test allows the determination of the presence and / or concentration of the thrombin / hirudin complex in concentrations ranging from 1 to 200 ng / ml.

Example 8 Test kit for an ELISA for determining the thrombin / hirudin complex

An ELISA test kit described in example 7 contains:

. microtiter plates in polyvinyl chloride;

. 20 ml of the MAb 4107-76-1 monoclonal antibody (10 µg / ml) in coating buffer;

. 20 ml of biotinylated MAb 4158-81-7 in PBS-Tween 0.1% (5 μ9 / ιη1); . 2 ml of standard solution containing 5 µg rHV1;

. 300 ml of 0.1% PBS-Tween;

. 300 ml of 1% PBS-BSA;

. 0.5 ml streptavidin conjugated to alkaline phosphatase;

. 50 ml of p-nitrophenylphosphate (1 mg / ml) in diethanolamine buffer (10%, 0.5 mM MgCl ₂ , 0.02% NaN ^, adjusted to pH 8.9 with HCl);

. calibration curve;

. color intensity scale;

. instruction manual.

Claims (19)

l ^â . - Process for the preparation of a monoclonal antibody that is directed against the thrombin / hirudin complex, and a derivative thereof that maintains the specificity of the antibodies from which they are derived, characterized by the cells of a hybridoma cell line that secrete said antibody to be multiplied in vitro or in vivo and, when necessary, the monoclonal antibody obtained will be isolated and / or converted into a derivative thereof. 2 ^â . Process according to claim 1 for the preparation of a monoclonal antibody and a derivative thereof, characterized in that the monoclonal antibody is directed against the thrombin / hirudin complex in which the hirudin is hirudin variant 1 (HV1). 3a. Process according to claim 1 for the preparation of a monoclonal antibody and a derivative thereof, characterized in that the monoclonal antibody is directed against the thrombin / hirudin complex in which the hirudin is recombinant hirudin (rHV). 4S. Process according to any one of claims 1 to 3 for preparing a monoclonal antibody and a derivative thereof, characterized in that the monoclonal antibody is directed against the thrombin-hirudin complex in which the hirudin is recombinant hirudin variant 1 (rHVl). 5th. cations 1 to 4 its derivative, Process according to any one of the claims for the preparation of a monoclonal antibody and one characterized in that the monoclonal antibody recognizes * * determinants of the hirudin fraction in the thrombin-hirudin complex. 6th. Process according to claim 4 for the preparation of a monoclonal antibody and a derivative thereof, characterized in that the monoclonal antibody recognizes determinants of the recombinant hirudin fraction variant 1 (rHV1) in the thrombin / rHV1 complex. 7th. process according to claim 6 for the preparation of a monoclonal antibody and a derivative thereof, characterized in that the monoclonal antibody recognizes determinants within the N-terminal domain in the nucleoid of the variant 1 recombinant hirudin fraction (rHVl) in the thrombin / rHVl complex . 8§. Process according to claim 6 or 7 for the preparation of a monoclonal antibody and a derivative thereof, characterized in that the monoclonal antibody is designated MAb 4158-81-7. 9§. Process according to any one of claims 1 to 4 for the preparation of a monoclonal antibody and a derivative thereof, characterized in that the monoclonal antibody recognizes determinants of the thrombin fraction in the thrombin / hirudin complex. IOã. Process according to claim 4 for the preparation of a monoclonal antibody and a derivative thereof, characterized in that the monoclonal antibody recognizes determinants of the thrombin fraction in the thrombin / rHV1 complex. llâ. Process according to claim 10 for the preparation of a monoclonal antibody and a derivative thereof, characterized in that the monoclonal antibody recognizes determinants of the thrombin fraction in the thrombin / rHV1 complex that are exposed for binding to the antibody only when thrombin is bound to hirudin or a solid surface but does not recognize the free thrombin in solution. 12th. Process according to claim 10 or 11 for the preparation of a monoclonal antibody and a derivative thereof, characterized in that the monoclonal antibody is called MAb 4107-76-1. 3. A process according to any one of claims 1 to 12, characterized in that a derivative of a monoclonal antibody is prepared, which is conjugated with an enzyme, with a fluorescent mark, with a chemiluminescent mark, with a metal chelate. , with avidin, with biotin or similar. 14a. Process according to any one of claims 1 to 12, characterized in that a derivative of a monoclonal antibody is prepared which is radiolabelled. 15th. Process according to claims 1 to 12, characterized in that a derivative of a monoclonal antibody is prepared which is a fragment. » 163. - Process for the preparation of a hybridoma cell line that secretes monoclonal antibodies directed against the thrombin / hirudin complex, characterized in that a suitable animal is immunized with hirudin coupled to a suitable vehicle or with the thrombin / hirudin complex, producing cells of mouse antibodies are fused with cells of a continuous cell line, the hybrid cells obtained in the fusion are cloned and the clones of cells that secrete the desired monoclonal antibodies are selected. 17th. Process according to claim 16 for the preparation of a hybridoma cell line, characterized in that the hybridoma cell line is a hybrid of myeloma cells and B lymphocytes from a mammal immunized with hirudin coupled to a suitable vehicle or with the thrombin / hirudin complex. 18 ^â . Process according to claim 17 for the preparation of a hybridoma cell line, characterized in that the hybridoma cell line is a hybrid of mouse myeloma cells and B lymphocytes from a mouse immunized with recombinant l variant hirudin (rHVl) to a suitable vehicle or thrombin / rHV1 complex. 19th. Process according to any one of claims 16 to 18 for the preparation of a hybridoma cell line, characterized in that the hybridoma cell line is designated 4158-81-7 and has been deposited with the European Collection of Animal Cell Cultures (ECACC) number 90061405 on June 14, 1990. Process according to any one of claims 16 to 18 for the preparation of a hybridoma cell line, characterized in that the hybridoma cell line is designated 4107-76-1 and has been deposited with the European Collection of Animal Cell Cultures (ECACC ) under number 90061404 on June 14, 1990. 21§. Method for the use of a monoclonal antibody and / or a derivative thereof prepared by a process according to any one of claims 1 to 15, characterized in that said antibody is used for the qualitative and quantitative determination of the thrombin / hirudin complex in vitro or in vivo. 22s. - Test equipment (kit) for the qualitative and quantitative determination of the thrombin / hirudin complex, characterized in that it comprises a monoclonal antibody and / or a derivative thereof prepared by a process according to any one of claims 1 to 15 and, optionally, other antibodies monoclonal or polyclonal and / or additives.