SI9500249A - Monoclonal antibodies versus soluble tnf-alpha receptors p55 and p75 and tnf-alpha and its analogues - Google Patents

Abstract

The submitted invention belongs to the field of pharmaceutical industry and refers to new monoclonal antibodies and their fragments which bind to an accessible surface of human recombinant TNF-Alpha, TNF-Alpha analogues and TNF-Alpha soluble receptors. The invention also refers to stable cell lines of hybridomas which are capable of producing such monoclonal antibodies, to the procedure for their preparation and to pharmaceutical preparations which contain them. Further, the invention refers to the method for a quantitative determination of a human TNF-Alpha, free and complex with soluble TNF-Alpha receptors p55 and p75 as well as putty which is used in this procedure. It was not possible to simultaneously determine free TNF-Alpha macromolecules and their soluble receptors as well as their complexes by means of monoclonal antibodies known so far and the reagents developed from them. This invention also refers to TNF-Alpha analogue TNF-Alpha His 107/108, Cys95/148 which is used in the complex with TNF-Alpha receptors as an antigen for the immunization of mice.

Description

The present invention relates to the pharmaceutical industry and relates to novel monoclonal antibodies and fragments thereof that bind to the accessible surface of human recombinant TNF-α, TNF-α analogs and TNF-α soluble receptors p55 and p75. The invention further relates to stable cell lines of hybridomas capable of producing such monoclonal antibodies, the process for their preparation and the pharmaceutical compositions containing them.

The invention also relates to a method for the quantitative determination of human TNF-α, free or complexed with soluble receptor, its analogues in biological samples, and the kit used therein. Furthermore, the invention also provides a TNF-α analogue of TNF-α His107 / 108, Cys95 / 148, which in combination with TNF-α receptors p55 and / or p75 is used as an antigen for immunization of mice.

A technical problem

With the monoclonal antibodies described so far and the reagents developed therefrom, it is not possible to simultaneously determine both the free macromolecules of TNF-α and their soluble receptors as well as their complexes. This is very important from the point of view of the evaluation of the disease state or the success of the treatment, as the development of the disease in many cases also increases the level of soluble TNF-α receptors that bind TNF-α, thereby reducing the true content of both receptors and TNF-α.

The state of the art

Tumor necrosis factor alpha (TNF-α) is a cytokine primarily produced by monocytes and macrophages in response to stimulation by endotoxin or other stimuli. Soluble TNF-α can also be produced by other cells: granulocytes, T and B lymphocytes, and NK cells. The soluble TNF-α is in the trimeric form; the molecular weight of the subunits is 17 kDa and that of the membranes is 26 kDa. An overview of TNF-α is described in many journals, e.g. B.Beutler et al., Nature 320, 584, 1986 and G. Serša, Healthy, Conscientious. 63, Suppl. II, 35, 1994.

Due to the antitumor activity, TNF-a was originally planned to be used in the treatment of cancer. Subsequent studies have shown that TNF-α is a pluripotent and pleitropic cytokine: its function reflects e.g. in reduced lipase activity, inhibition of virus propagation, activation and chemotaxis of neutrophils, cell growth - proliferation and differentiation - different cells, increased production of prostaglandin E2 and collagenase, decreased collagen synthesis, procoagulant activity, inhibition of contractility and increased expression of major histocompatibil Classes I and II. Many negative effects of TNF-α are associated with the development of various serious pathological conditions such as meningitis, septic shock and other bacterial, viral or fungal infections, various acute and chronic inflammations, tissue necrosis, neoplasm, cachexia, autoimmune diseases, etc. e.g. (LE Junming et al., WO 92/16553; G. Serša, Healthy News 63, Suppl. II, 35, 1994).

From a therapeutic point of view, apart from the preparation of TNF-α and its less harmful analogues, the development of products that neutralize TNF-α activity is also interesting. Such an opportunity is provided by, for example, specific monoclonal antibodies. Specific monoclonal antibodies also make it possible to detect TNF-α in certain body fluids in various conditions, thus contributing significantly to the diagnosis of a variety of serious diseases.

TNF-α achieves its activity through receptors located on different cells. There are two types of receptors: one having a molecular weight of 55 kDa and the other having 75 kDa (M. Brockhaus et al., Off Natl Acad Sci USA 87, 3127, 1990). The extra-membrane regions of the receptors are similar (J. Vilček and TH Lee, J Biol. Chem. 166, 7313, 1991) and the intracellular ones are different. This is probably related to different stimulation in the cell (Z. Dembic et al., Cytokine 2, 231, 1990).

The cleaved extra-membrane portions of the serum and plasma receptors are called soluble TNF-α receptors (P. Seckinger et al., Natl. Acad. Sci. USA 87, 5188, 1990). These proteins were sometimes called TNF inhibitory peptides (US Pat. 5359037). The plurality of soluble receptors is e.g. in some diseases (I. Olson et al., Eur. Cytokine Netw. 4, 169, 1993) depends on the intensity of the disease and their treatment, and their amount in the serum is likely to depend on the kinetics of TNF-α and thus its efficacy (Kus et al., Int. J. Cancer 55, 110, 1993). Determination of soluble TNF-α receptors is therefore important both in terms of diagnosing the severity of the disease and determining the efficacy and effectiveness of the treatment. Soluble TNF-α receptors can be determined by specific monoclonal antibodies.

Antibodies to the selected antigen can be prepared by immunizing the animal with the antigen and isolating the resulting antibodies from the serum of the animals. The antibodies thus obtained are heterogeneous - polyclonal; the antibodies have different affinities for the antigen, and they also bind to different antigenic determinants of the epitope.

Koehler and Milstein (Nature 256, 495, 1975) have developed a cell hybridization method that allows the production of monoclonal antibodies, that is, antibodies of the same structure, that bind all to the same site and react with a single antigenic determinant. Such cells - hybridomas - are formed by the fusion of lymphoblasts and myeloma cells. This method allows unlimited synthesis of identical monoclonal antibodies; these properties make monoclonal antibodies indispensable in many fields of therapy and diagnostics.

Cerami et al. EP 212489 described a product isolated from macrophages for the treatment of septic shock and a dagnostic based on it. Rubin et al. have developed hybridomas for the production of monoclonal antibodies and antibodies for the determination of TNF-α and purification of TNF-α (EP 2218868).

A. Moeller and F. Emlig have developed monoclonal antibodies with different heavy chains, giving the antibodies a different neutralizing ability and thus more specific utility (EP 260610).

Yone et al. have described in EP 288088 a method for determining TNF-α in the body fluids of patients with Kawasaki's disease, with epitope recognition at TNF-α sites from 68 (Gly) to 97 (lle). These monoclonal antibodies can neutralize the cytotoxic effects of TNF-α on L9929 cells, effects associated with fat metabolism and binding to the TNF-α receptor. They also describe monoclonal antibodies that recognize sites on TNF-α from 7 (Thr) to 37 (Leu) and sites from 113 (Pro) to 127 (Glu).

E. Barbanti described, in EP 492448, monoclonal antibodies or fragments thereof for the neutralization of TNF-α in a molar ratio of 2: 1 and also for the detection of human TNF-α in body fluids.

In WO 92/16553, Junming et al. In addition to conventional monoclonal antibodies to TNF-α, chimeric, i.e. humanized antibodies of various structures with high affinity and neutralizing ability to TNF-α have been described; in these cases, the monoclonal antibodies can bind to the amino acids of recombinant TNF-α 59-80 and 87-108, whereas to the TNF-α portions that are important for receptor binding: 11-13, 37-42, 49-57 or 155157, do not bind. The substances are useful in the diagnosis of TNF-α and for the treatment of diseases with increased synthesis of TNF-α.

Partially humanized antibodies to TNF-α have also been described for therapy and diagnosis by Adair et al. in WO 92/11383. These antibodies are characterized by the fact that three specific binding sites on the variable part have heavy and light chains, allowing multiple and safer parenteral use.

US 5223395 describes a method for immunometric determination of TNF-α in biological samples using two types of monoclonal antibodies simultaneously, which in turn react with the same and non-repeating epitope on TNF-a monomers.

Y. Ohomoto et al. In US 5360716, high-specificity monoclonal antibodies have been prepared that can be used to purify TNF-α or to quantify it in biological samples. Among them is a series of antibodies that react with SDS-denatured TNF-α.

In addition to TNF-α antibodies themselves, Fab fragments have been developed that are capable of preventing septic shock due to binding to TNF-α (KJ Tracy et al., Nature 330, 662, 1987).

Bispecific monoclonal antibodies to TNF-α and horseradish peroxidase are also used for the qualitative and quantitative determination of human TNF-α in biological fluids and bacterial lysates (N. Berkova et al., DE 4104787).

In patent application WO 91/02756, M. Kriegler describes antibodies of various origin that prevent the cleavage of pro-TNF-α into larger or smaller fragments. The combination of both antibodies can determine the TNF-α content in different body fluids and can be used for various prophylactic and therapeutic purposes in patients with pathological TNFα content.

P. Boyle et al. have developed monoclonal antibodies that are useful in diagnostics and capable of binding to TNF-α on the cell surface and preventing its secretion induced by LPS on monocytes (EP 614984).

Polyclonal and monoclonal antibodies to TNF-α are also the basic constituents of the reagent that allow selective determination of oligomeric TNF-α but not of presumably biologically inactive monomers or TNF-α-bound TNF receptors (A. Corti, WO 93/06489).

Antibodies against TNF-α and its neutralizing fragments of different origin and nature are also found in combination with various xanthine derivatives intended for the treatment of various diseases with increased amounts of TNF-α (H. Anagnostopulos, WO 92/07585).

For the treatment of diseases associated with the immune system, such as. in autoimmune diseases and in graft rejection, a combination of antibodies to TNF-α and interferon γ is proposed (Jonker. and PH Van der Meide, WO 90/10707).

Combinations of antibodies to TNF-α with various antibiotics have been proposed for the treatment of bacterial meningitis (RF Hector and MS Collins, EP 585705).

Various literature describes a species for recombinant TNF-α specific monoclonal antibodies, eg: CH Liang, Biochem. Biophys. Really. Comm. 137, 847, 1986; A. Meger et al., Hybridoma 6, 305, 1987; Fendly et al. Hybridoma 6, 359, 1987, TS Bringman et al .; Hybridoma 6, 489, 1987; M. Hirai et al., J. Immunol. Meth. 96, 57, 1987; A Mooler et al., Cytokine 2, 162, 1990. They have been used either to determine TNF-α epitopes or to develop immune methods for TNF-α determination or TNF-α isolation. However, none of these works directly addresses the field of therapy or in vivo diagnostics (LE Junming et al., WO 92/16553).

In view of the development of antibodies to TNF-α having different binding or neutralizing ability, it is worth mentioning the work of PR Temest et al. Hybridoma 13, 183, 1994, which also describes such monoclonal antibodies that are partially competitive antagonists of unmodified TNF-α antibody produced prior to structural alterations of TNF-α.

Monoclonal antibodies and biologically active fragments against human TNF-α receptors isolated from HL-60 cells were developed by Brockhaus and Loetscher (EP 334165). These agents serve either to purify the membrane receptors for TNF-α or for therapeutic or diagnostic purposes.

A more detailed definition of the use of monoclonal antibodies against soluble TNF receptors can be seen in patent application WO 94/09137 (Fendley et al.). They are used to treat patients with malignant tumors, HIV and T-cell-mediated autoimmune diseases.

In US 5359037 VVallash et al. describe polyclonal and monoclonal antibodies to the TNF-α receptor and their biologically active fragments; these cellular receptor proteins may stimulate or inhibit some of the effects of TNFα. They are also useful in diagnostics for detecting endogenously generated antibodies to TNF-α receptors and for determining TNF-α receptor levels in body fluids.

In the patent application WO 92/22666, Mr. Adolf presents monoclonal antibodies against the extra-membrane part of the TNF receptor. These antibodies are useful for the determination of soluble TNF-α p55 receptors in body fluids by the ELISA method.

Description of solution to a technical problem with implementation examples

Unlike the known monoclonal antibodies and the reagents developed for the determination of TNF-α or soluble receptors, the compositions of the present invention allow both the determination of free p55 and p75 receptors and TNF-α as well as the complexes of both soluble TNF-α receptors or TNFα analogues. This is very important from the point of view of assessing the disease status or the success of the treatment, as the development of the disease in many cases also increases the level of soluble TNF-α receptors that bind TNF-α, thereby reducing the true content of both receptors and TNF-α.

Such monoclonal antibodies were obtained by immunizing mice with TNF-α analog complexes His107 / 108, Cys95 / 148 with soluble TNF-α receptors p55 and / or p75.

The monoclonal antibodies of the present invention can be used in both diagnostics and prophylaxis and / or therapy in disease states where the amount of TNF-α is increased. Such typical disease states are e.g. septic shock, AIDS, cerebral malaria, graft-versus-host disease, rheumatoid arthritis, etc. The monoclonal antibodies of the present invention can be used for this purpose alone or as pharmaceutical preparations. The monoclonal antibodies in a suitable pharmaceutical preparation may be administered parenterally: subcutaneously, intravenously, intraarterially or intramuscularly.

The monoclonal antibodies of the present invention are also suitable for immunoassays in both liquid and solid phases.

Monoclonal antibodies and fragments thereof are prepared by immortalizing mouse B lymphocytes immunized with the selected antigen. This can be done either by transformation with an oncogenic virus, by electrofusion, or by fusion with an already immortalized myeloma or lymphoblastoid cell line. Plasmacytoma (myeloma) cells of mammalian origin lacking the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT) are most commonly used.

The cell lines were then cloned using one of the possible methods and then tested for the ability to produce the desired antibodies. There are various assays for determining specific antibodies, and their selection usually depends on the nature of the antigen itself and the desired properties of the antibodies. This stage is the most important in the whole process, because at this stage we select antibodies with high affinity constants and the desired specificity.

When we have a stable cell line of hybridomas that produce monoclonal antibodies with the desired specificity and affinity, that line represents the source of the genetic material encoding the monoclonal antibody.

Genes encoding selected monoclonal antibodies to TNF-α or its TNF-α analogue His107 / 108, Cys95 / 148 were isolated by preparing a cDNA library from mRNA. We then isolated the cDNA encoding the immunoglobulins and further manipulated it. Certain portions of the nucleotide sequence can be replaced and prepared e.g. humanized antibodies (thereby reducing their immunogenicity) that are suitable for therapeutic use. The cDNA clone can be inserted into a suitable prokaryotic or eukaryotic expression vector and used for potential production in large quantities.

The term stable cell line means that the line is viable for a long time, theoretically indefinitely, and at that time also produces the desired monoclonal antibody.

The term monoclonal antibody refers to an antibody selected from a mixture of different antibodies. All monoclonal antibodies of the same specificity and affinity are identical except for their natural mutants.

The term antibody refers to both intact immunoglobulin molecules and their fragments (Fab, F (ab ') 2, Fv, scFv) against the TNF analog.

The term neutralizing means the ability of antibodies to block the cytotoxic activity of TNF-α or its TNF-α analogue His107 / 108, Cys95 / 148 in vitro and / or in vivo.

For the preparation of the monoclonal antibodies of the present invention, we use the fusion method first described by Koehler and Milstein (Koehler, G. and Milstein, C., Nature 256: 496 (1975)). Use NS1 plazmacitomsko line which is obtained in BALB / c mice, the HGPRT ^- and does not produce and secrete immunoglobulins. Such immortalized cell line is fused with cells, some of which produce antibodies that bind to the TNF-α analogue His107 / 108, Cys95 / 148, and also to TNF-α. Cells that produce antibodies to the TNF-α analog were isolated from the spleen of BALB / c mice immunized with the TNF-α analog complex His107 / 108, Cys95 / 148. TNF analogue

His107 / 108, Cys95 / 148 was prepared using recombinant DNA technology. After fusion of both cell types, the presence of specific antibodies in the supernatants of the resulting hybridomas is tested. We choose those that react with either the TNF-α analogue or TNF-α itself. For testing, we use the ELISA method (enzyme-linked immusorbant assay), in which both recombinant proteins (TNF-α His107 / 108, Cys95 / 148 and TNF-a) are used to dress the plaque. Selected stable cell lines are grown in plastic bottles or in larger special bottles (spinner flasks). Monoclonal antibody concentrations of up to 0.1 mg / ml can be achieved in the supernatants in the appropriate medium and with appropriate cell line cultivation.

Such monoclonal antibodies can be isolated from the supernatants by precipitation with ammonium sulfate or by ion exchange chromatography or affinity chromatography on protein A or protein G Sepharosi.

Complexes with the composition AB _n - |, AB _n 2, or a mixture of AB _n iB _n 2 were selected and prepared for immunization.

A: means TNF-α analogue TNF-α His107 / 108, Cys95 / 148 everywhere

AB _n i: means a mixture of natural soluble TNF-α receptor complexes p55 with TNF-α analogue His107 / 108, Cys95 / 148

AB _n 2: means a mixture of complexes of naturally soluble TNF-α receptors p75, with TNF-α analogue His107 / 108, Cys95 / 148

ABniBn2, therefore, is a mixture of TNF-α analog complexes His107 / 108, Cys95 / 148 and various truncated forms of both naturally-soluble TNF-α receptors p55 and p75.

We are talking about the mixture because it is known that proteolytic cleavage of multiple amino acids occurs at the N-terminal portion of the soluble receptor and that one or the other soluble TNF receptor p55 or p75 can bind to the same TNF-α trimmer.

Recombinant soluble TNF-α receptors p55 or p75 may be used instead of naturally soluble TNF-α receptors.

The recombinant TNF-α analogue His107 / 108, Cys95 / 148 was selected for immunization for the following reasons:

1. The His107 / 108 double mutation allows sufficiently strong binding of the analogue to the Cu2 + ~ IDA chromatographic column and thus serves as an affinity ligand for single-step isolation of soluble TNF receptors from both natural sources (urine, plasma, pleural fluid, etc.) and from cultures of recombinant organisms, e.g. . E.coli, yeasts and insect or mammalian cell cultures. The complex is eluted from the column with an increased concentration of imidazole i.e. under mild conditions that do not damage the biological properties of the complex or its components or cause dissociation of the ozone denaturation proteins.

2. An additional double mutation of Cys95 / 148 allows the spontaneous association of subunits in TNF trimers with disulfide bonds. Individual mutations of Cys95 and Cys148 are located on contact surfaces that already naturally bind monomers into a relatively compact trimmer, but according to known data at low (eg physiological) concentrations, it nevertheless dissociates into monomers and thus denatures and loses its biological effect. (U.S. Pat. 5,223,395). The double mutation of Cys95 / 148 therefore prevented unwanted dissociation, while greatly improving the thermostability of the trimmer.

3. The introduction of these mutations did not affect in any way the sites that are important for receptor binding, which we conclude that cytotoxicity with respect to TNF-α did not change. Cytotoxicity was determined on an L929 cell line.

4. In addition to the TNF-α analogue His107 / 108, Cys95 / 148 serves for easy receptor isolation, it also represents a compact thermodynamically stable carrier core for soluble receptors. Such a complex is more stable, more long-lived and also more immunogenic than if the components were used separately.

5. Because the binding sites between TNF-α and the receptor are obscured, only monoclonal antibodies can be more likely to target exposed surfaces, which is important for the use of these antibodies for analytical purposes (ELISA), and is very important for the correct determination of TNF concentration. -α in the presence of a large excess of soluble receptors.

6. The isolation of single pure receptors is not required for the preparation of monoclonal antibodies. The use of the mixture produces equivalent or better results.

Some of the monoclonal antibodies of the present invention also have high neutralizing activity. They are used for therapeutic purposes. Other monoclonal antibodies allow the determination of both free macromolecules of TNF-α receptors p55, p75 and TNF-α, as well as their complexes.

EXECUTIVE EXAMPLES

The invention is explained, but in no way limited by the following embodiments:

EXAMPLE 1

Preparation of TNF-α Analogue His107 / 108, Cys95 / 148

Gene and expression system

We used a commercial synthetic TNF-alpha gene embedded in the carrier plasmid BBG4 (British Biotechnology, England). The gene has codons adapted for expression in E. coli.

The basic expression plasmid pCYTEXP1 (Belev T.N. et al. A fully modular vector system for optimization of gene expression in Escherichia coli. Plasmid 1991: 26: 147-150.) Was supplied by Medac (Hamburg). The enzymes used in our work were from Sigma, Amershen and Boehringer. When the origin of the reagents is not specifically mentioned, they meet the requirements for work in molecular biology in quality.

The gene was excised from the BBG4 plasmid with the Ndel / BamHI restriction pair and directed subcloned into the pCYTEXP1 expression plasmid pretreated with the same restriction pair.

The TNF gene is under the control of a thermo-inducible promoter in this expression plasmid, so fermentation must be conducted at 40-42 ° C. Under optimized fermentation conditions, TNF-α expression in this system was about 1-2% of total cellular proteins. Due to relatively low expression, we modified the expression plasmid so that:

a) The plasmid pCYTEXP1 was sectioned with the Hindlll enzyme, filled the gap gaps at both ends with the Klenov enzyme, and reattached to a circular plasmid with T4 DNA ligase; or

b) A portion of the repressor gene of the HindIII / Notl enzyme pair was cut from plasmid pCYTEXP1, supplemented with the Klenov enzyme, and reattached to a circular plasmid with T4 DNA ligase.

In both cases, plasmid DNA from ten clones was analyzed. The selected clone had to have the correct plasmid size and high expression at 30 ° C. Thus, the expression plasmid was obtained with the promoter no longer under the control of the repressor cl, and TNF-α expression increased to 15 in the optimal fermentation control in the laboratory fermenter. -25% of total cellular proteins, depending on the type of analogue. All TNF-α analogues listed here were obtained with the modified expression plasmid. Thus, it has been shown that constitutive expression of TNF or its analogues does not significantly affect the growth of E. coli, but at the same time, a sufficient amount of the desired protein is accumulated in the cytoplasm to allow economical isolation. Since expression is no longer related to temperature induction, cells can also be grown at a lower temperature (eg at 30 ° C), which has proven to be very favorable in cysteine mutants, where there is almost no protein accumulation at the normal temperature of induction. The addition of various expensive substances (inducers eg IPTG) is also eliminated. Most commercial expression systems use controlled promoters.

EXAMPLE 2

Preparation of mutant TNF-α genes

Appropriate mutations were introduced by oligonucleotide-directed mutagenesis on single-stranded plasmid DNA according to the known method (Kunkel.T.A. Rapid and efficient site specific mutagenesis whithout phenotypic selection. Off. Nat. Acad. Sci. USA 82, 488-492) in two steps. The oligonucleotides were synthesized on a DNA synthesizer (model 381 A) from Applied Biosystems with chemicals from the same company.

First, we prepared the TNF-α His 107/108 analog of origin and used the M28z31 oligo with the sequence to introduce the Glu107Gly108 -4- His107His108 mutations:

5'-CGC TTC TGC ATG GTG GGG AGT TTC ACG C-3 '

In the next stage, we introduced both cysteine mutations, using the M20z38 oligo with the sequence for the Ser95 -> Cys95 mutation

5'-CTT GAT AGC GCA CAG CAG GT-3 'and for the Gly148 -> Cys148 oligo M23z41 mutation with the sequence:

5'-GTA CAC CTG GCA AGA TTC AGC GA-3 '

EXAMPLE 3

Isolation and purification of TNF-α analogue His107 / 108, Cys95 / 148

From fresh E. coli NM522 colonies, 10 - 30 ml of overnight culture in a simple LB medium with 100 Bg ampicillin t ml medium at 30 ° C and 120 130 rpm were prepared in Erlenmayer bottles on a laboratory shaker. This culture served as a feedstock for the production of larger volume biomass (1 - 3 liters of LB with ampicillin). The bacterial culture grew to saturation under the indicated conditions (optical density at 550 nm ranged from 1 - 1.2). The cell pellet was separated from the medium by centrifugation (5000 rpm; 5 minutes) and washed. The washed bacterial pellet cells were mechanically shredded on a Brown MKS homogenizer. In the centrifuge, most of the nucleic acids were precipitated with 0.1% polyethyleneimine. Proteins were precipitated with ammonium sulfate (65% saturation), the precipitate was dissolved in 0.02 M K-phosphate buffer, 0.2 M NaCl, pH 7.1 and applied to a Zn ⁺⁺ -IDA Chelating Superose column (Pharmacia). It is possible to use other ions, e.g. Cu ++, Ni ++, Co ++. Elution was gradient with buffer of the same composition and addition of 50mM imidazole. Because our analogue has histidine mutations, it lingers on the column for the longest time, other proteins and most other impurities are eluted before. At the same time as the biologically active analogue, a smaller portion of the partially proteolytically degraded protein is eluted, which can be removed during the polishing phase on the Phenyl Superose HIC column (Pharmacia). The overall cleaning efficiency was 40 - 50%.

The purity and quality of the purified analogue was assessed by standard SDS-PAGE analysis (only 17 kDa major spots and barely visible spots covalently linked to mercaptoethanol non-reducible TNF-α dimers are visible in silver staining). With the implementation of SDS-PAGE without the addition of a reducing agent - that is, without mercaptoethanol - a single lysis of 40 - 42 kDa corresponding to a TNF-α trimmer is obtained, where all three subunits are compactly linked by disulfide bonds. The isoelectric point (pl) is increased to a value of about 7.85. One of the additional features of such a trimmer is its significantly increased thermostability.

A cytotoxicity test on a standard L929 mouse fibroplast cell line shows that biological activity is conserved and comparable to native TNF-α.

EXAMPLE 4

Isolation of soluble p55 and p75 receptors from urine and preparation of TNF-α analog complexes His107 / 108, Cys95 / 148 and soluble TNF-α receptors p55, p75

a) preparation of protein fraction from urine

From 50 liters of urine from patients with various cancers, 600 ml of concentrate were obtained by ultrafiltration (10,000 NMWL membrane polysulfone cat. No. PTGC0MP04, Minitan apparatus apparatus, Millipore). The proteins were precipitated by the addition of ammonium sulfate (up to 60% saturation) and stored until further processing at -70 ° C.

b) isolation of receptors on the affinity column with immobilized TNF-α analogue His107 / 108, Cys95 / 148

An affinity column of Chelating Superose (Pharmacia) with Cu2 ++ immobilized prepared according to the manufacturer's instructions was bound with 1mg of TNF-α analogue His107 / 108, Cys95 / 148 dissolved in 1ml 0.02 M Kphosphate buffer with 0.2 M NaCL, pH 7 , 1 (buffer A). The column prepared in this way was slowly applied (30 minutes) in buffer A dissolved ammonosulfate precipitate of proteins from urine. The unbound proteins were washed with buffer A. With a linear gradient of imidazole (from 0 dO 50 mM) in buffer A, the complexes between TNF-α His107 / 108, Cys95 / 148 and soluble receptors were eluted and TNF-α His107 / 108 , Cys95 / 148, all of which appear simultaneously in the same protein peak. In the same way we achieved e, learning the aforementioned protein complexes and the excess analog of TNF-α His107 / 108, Cys95 / 148 with a gradient of histidine and glycine, or only by lowering the pH of the initial buffer. In the case of pH-lowering elution, the eluates should be neutralized immediately after elution. Fractions containing protein complexes between TNF-α His107 / 108, Cys95 / 148 analogue and soluble receptors, and excess TNF-α His107 / 108, Cys95 / 148, were concentrated on the Amicon membrane ΥΜ10.

c) gel filtration

Separation of the excess analogue from the complexes between TNF-α His107 / 108, Cys95 / 148 analogue and soluble receptors was achieved by gel filtration on Superose 12 (Pharmacia) and Sephacryl 200 (Pharmacia) in the aforementioned buffer A, which was also used for equilibration columns.

EXAMPLE 5

Immunization of mice

After three BALB / c mice were immunized subcutaneously with a mixture of TNF-α analog complex His107 / 108, Cys95 / 148 and soluble human TNF-α receptor p55 or p75. Both proteins were incubated for 45 minutes at room temperature in complete Freund's adjuvant (Sigma, St.Louis, MO, USA). After one month, mice were immunized intraperitoneally with equal doses of both proteins in Freund's incomplete adjuvant (Sigma, St.Louis, MO, USA). Ten days after the second inoculation, blood was drawn from mice from the tail vein and the presence of polyclonal antibodies against both antigens was determined in all sera by ELISA. Mice with the best titer of specific antibodies were inoculated intravenously one month after the second immunization with the same doses of both complexes as in the first and second inoculations, this time in saline. After three days, the mice were sacrificed and her lymphoblasts were used for fusion.

EXAMPLE 6

Preparation of stable hybridoma lines

Lymphocytes were isolated from the spleen of sacrificed mice, while NS1 myeloma cells (> 99% of living cells) were prepared in the log growth phase. Both cell types were mixed at 10: 4 ratio and centrifuged. A 50% (v / v) solution of polyethylene glycol 1500 (PEG 1500; Sigma, St. Louis, MO, USA) in Eagle's Dulbecco modified medium (DMEM) was slowly added to the precipitate, and then the cell suspension was slowly diluted with DMEM medium (1 ml for one minute and 20 ml for 5 minutes). The DMEM medium consisted of DMEM (Flow, Scotland, UK), 10 mM HEPES (Flow, Scotland, UK) and 45 mM NaHCO3 (Sigma, St. Louis, MO, USA). After centrifugation, the cell pellet was resuspended in complete DMEM medium, which, in addition to DMEM, the composition of which was described above, contained 13% fetal calf serum (Hy Clone, Utah, USA), 100 pg / ml penicillin, 200 pg / ml streptomycin, and supplement 2mM L-glutamine (Gibco, Scotland, UK). Thymocyte suspension prepared from the thymus of two-month-old BALB / c mice was added as a nutrient medium. Cell suspension was deposited in eight 96-well microtiter plates (Costar, Cambridge, MA, USA), at 70 pl per well. The plates were stored overnight at 37 ° C in an incubator saturated with water vapor and 5% CO ₂ . The next day, cells were fed with HAT DMEM (Sigma, St. Louis, MO, USA) at 140 pl per well. In addition to the complete DMEM medium, HAT DMEM contains hypoxanthine (H), aminopterin (A) and thymidine (T). Only hybridomas grow in this medium, whereas NS1 cells in the presence of aminopterin collapse and unfused spleen cells have a limited lifespan under in vitro conditions. The plates were then stored at 37 ° C continuously in an incubator saturated with water vapor and 5% CO2- The plates were fed with HAT DMEM every third day. On the fourth day, we observed the first clones of the hybridomas. The supernatants were tested after seven days. First, the presence of murine immunoglobulins was determined in them, and then those supernatants in which highly potent reactions were obtained by ELISA were tested for the presence of antibodies against TNF-α analogue His107 / 108, Cys95 / 148 and recombinant human TNF-α. Hybridomas in those wells for which specific antibodies with the highest relative binding affinities were determined in the supernatants were multiplied to larger volumes (first in 24-well plates, and then in ever-larger bottles). The complete HAT DMEM medium was first replaced with the complete HT DMEM medium and then with the complete DMEM medium.

In order to ensure the stability and homogeneity of the selected cell lines, and to ensure that the antibodies produced by these lines are in fact monoclonal, part of the hybridomas was cloned using the restricted dilution method and part of them were frozen. The supernatants of the cloned hybridomas were again tested by ELISA. We have multiplied the selected hybridomas to larger volumes. Part of them were frozen and part was grown to maximum density. Such supernatants were used to determine the class and subclass of immunoglobulins, to determine the relative binding affinity of monoclonal antibodies, and to determine the concentration of immunoglobulins in depleted hybridoma supernatants.

EXAMPLE 7

ELISA for the determination of murine monoclonal antibodies against TNF-α analogue His107 / 108, Cys95 / 148, TNF-α, and p55 and p75 receptors

Flat-bottomed microtiter plates (Maxisorp, Nune, Denmark) were coated with 100 μΙ anti-mouse immunoglobulins (Dakopatts, Denmark) or. TNFα analogues His107 / 108, Cys95 / 148, TNF-α, p55 or p75 receptors, in 50 mM Nakarbonate / bicarbonate buffer pH 9.6 at a concentration of 1 μg / ml. The plates were incubated overnight at 4 ° C. They were then washed three times with isotonic phosphate buffer (PBS) with Tween 20 (PBS / Tween ₂ o) (Sigma, St. Louis, MO, USA). Vacancies were blocked with 100 μΙ of 1% bovine serum albumin (BSA) in PBS / Tween ₂ for 1 hour at 37 ° C. After washing three times with PBS / Tween ₂ g, 100 μΙ of the hybridoma supernatants were applied to the wells of the microtiter plate and incubated for 1 hour for 30 minutes at 37 ° C. The plates were then washed again with PBS / Tween ₂ o, and 100 μΙ of conjugate (goat anti-mouse peroxidase-labeled anti-mouse immunoglobulins; Jackson Immuno Research Laboratories, Dianova) diluted 1: 5000 in 1% BSA-PBS / Tween was added again. ₂ Q. After an hour and a half of incubation at 37 ° C, the plate was washed three times with an automatic plate washer and 100% of the substrate 0.1% 2,2'-azinobis- (3ethylbenzthiazoline sulfonic acid) (ABTS) and 0.012 were applied to each well. % H ₂ O2 in substrate buffer consisting of 0.1 M citric acid and 0.1 M phosphate buffer and its pH is 4.5). After 30 minutes, the absorbance at 410 nm was measured with an automatic tile reader.

Positive and negative controls were also included in the test.

EXAMPLE 8

ELISA for the quantification of mouse IgG antibodies

The wells of the microtiter plate (Maxisorp, Nunc, Denmark) were covered with 100 μ anti goat anti-mouse immunoglobulins (Dakopatts, Denmark) in 50 mM Nakarbonate / bicarbonate buffer pH = 9.6, final concentrations 1 pg / ml, and incubated overnight at 4 ° C. After rinsing, blocking the plate with 1% BSA in PBS / Tween ₂ o, and rinsing three times with PBS / Tween ₂ o, 100 μΙ mouse immunoglobulins G and M known concentrations were added to the wells (Sigma, St.Louis, MO, USA). and after 100 μΙ of appropriately diluted hybridoma supernatants in which monoclonal antibody concentrations were determined. Plates were applied after incubation for 1 hour for 30 minutes at 37 ° C and washing with 100 μΙ goat anti-mouse horseradish peroxidase (HRP) anti-mouse immunoglobulins (Jackson Immuno Research Laboratories, Dianova) and diluted 1: 5000 in 1% BSA-PBS / Tween ₂ o · After an hour and a half of incubation at 37 ° C and washing, 100 μΙ of substrate were added. Its composition is already described under point 7. After an hourly incubation at 37 ° C, the absorbance at 410nm was read with an automatic plate reader.

The concentration of monoclonal antibodies in the hybridoma supernatants was then calculated from the calibration curve obtained with known concentrations of mouse immunoglobulins.

EXAMPLE 9

Determination of class and subclass of monoclonal antibodies

The classes and subclasses of murine immunoglobulins were determined in two ways. One of them is an ELISA similar to the ELISA described in item 7, except that instead of goat peroxidase-labeled goat anti-mouse antibodies, optimal dilutions of specific immunoglobulins against mouse immunoglobulins M and against all four classes of mouse immunoglobulins G (IgGi, lgG _2a , lgG ₂ b, · 9θ3) (Nordic, The Netherlands) in 1% BSA-PBS / Tween ₂ Q. Positive and negative controls were also included in the test. All samples were measured in duplicate.

Mouse immunoglobulin classes and subclasses were also determined with a Sigma Immuno Type ^T ISO-1 kit (Sigma, St. Louis, MO, USA) according to the manufacturer's instructions.

EXAMPLE 10

Determination of the relative binding affinity of monoclonal antibodies

When the concentration of antibodies in the supernatants of the hybridomas is known or we already have pure monoclonal antibodies, we can apply the dilutions in 1% BSA-PBS / Tween to the microtiter plate instead of the supernatants of the hybridoma from the titration curve obtained in the indirect ELISA test described in point 7. ₂ o, or dilutions of pure monoclonal antibodies, determine the relative binding affinity. The antibody concentration at which the measured absorbance in the ELISA test equals half the absorbance value at saturation gives us, calculated in moles, the relative binding affinity of the antibodies, which is a measure of their affinity constant.

Positive and negative controls were also included in the test. All samples were measured in duplicate.

EXAMPLE 11

Purification of monoclonal antibodies

Monoclonal antibodies were purified on G-Sepharosi protein (Sigma, St. Louis, MO, USA). 25 times concentrated supernatants of hybridomas dialyzed in Centriprep 30 (Amicon, USA) against 0.02 M phosphate buffer pH 8.0 containing 0.5 M NaCl were loaded onto a column of G-Sepharose protein. The sample was bound for 30 minutes at room temperature. The unbound proteins were then washed with 0.02 M phosphate buffer pH 8.0 containing 0.5 M NaCl, bound immunoglobulin fractions, i.e. monoclonal antibodies, eluted with 0.1 M glycine / HCl pH 2.7. These fractions were immediately neutralized with 1 M Tris buffer pH 9.0. The presence of antibodies in individual fractions was determined by measuring the absorbance at 280 nm and their activity by an indirect ELISA assay. The positive fractions were concentrated and dialyzed on Centriprep 30, then divided into aliquots and frozen at -20 ° C.

EXAMPLE 12

Conjugation of horseradish peroxidase monoclonal antibodies

Monoclonal antibodies were conjugated to HRP (type VI, Sigma, St. Louis, MO, USA) using the glutaraldehyde method (Antibodies, A laboratory manual, Harlow, E., Lane, D., editors, 1988).

2.9 mg of HRP was dissolved in 0.1 ml of 1.25% gluteraldehyde / PBS pH 7.1 and incubated overnight at room temperature. Subsequently, excess glutaraldehyde was removed by filtration on a Sephadex G-25 Superfine column equilibrated with 0.15 M NaCl. The brown colored fractions were combined and dialyzed against 0.1 M Na carbonate / bicarbonate buffer pH 9.2 and concentrated to 0.65 ml in Centriprep 30.

1.5 mg of selected pure monoclonal antibodies were dialyzed in Centricon (Amicon, USA) against 0.1 M Na carbonate / bicarbonate buffer pH 9.2 and concentrated to 0.25 ml, then HRP was prepared and incubated overnight at room temperature.

The next day, 0.1 ml of 0.2 M lysine was added and incubated for 30 minutes at room temperature. The sample was dialyzed in Centricon against PBS pH 7.1 and concentrated to 1 ml. Conjugated antibodies were stored at -20 ° C in aliquots.

EXAMPLE 13

Optimization of sandwich ELISA assay for quantitative determination of TNF-α or its analogues (chessboard or chessboard)

To optimize the ELISA assay that can quantify TNF-α or its analogs, wells on a flat bottom microtiter plate (Maxisorp, Nune, Denmark) were coated with 100 μ 100 of selected monoclonal antibodies of different dilutions (10 μg / ml to 80 ng / ml; dilutions 1: 2 ⁿ ) in 50 mM Na carbonate / bicarbonate buffer pH 9.6. The same dilutions of monoclonal antibodies were applied to each of the eight rows. The plates were incubated overnight at + 4 ° C. The next day, they were washed three times with PBS / Tween20 · Free spots are 30 min. blocked with 100 μΙ of 1% BSA in PBS / Tween2o at 37 ° C. 100 wells of TNF-α or TNF-analog His107 / 108, Cys95 / 148 in 1% BSA-PBS / Tween2o at a concentration of 0.5 μg / ml were then applied to each well. After an hour and a half of incubation at 37 ° C and washing again with PBS / Tween2o, 100 μΙ of different conjugate dilutions were applied to each of the eight columns of the microtiter plate in 1% BSA-PBS / Tween2o (1:50 to 1: 6400; dilutions 1: 2 ⁿ ). After an hour and a half of incubation at 37 ° C and washing with PBS / Tween2o, 100 μl of ABTS substrate was added to each well (composition described under 7). After an hourly incubation at 37 ° C, the absorbance at 410 nm was read with an automatic plate reader. Positive and negative controls were also included in the test.

EXAMPLE 14

Calibration curve for the determination of TNF-α or its analogues

Flat-bottom microtiter wells (Maxisorp, Nune, Denmark) were coated with an optimal concentration (1 μg / ^^ nl) of selected monoclonal antibodies in 50 mM Na carbonate / bicarbonate buffer pH 9.6. After overnight incubation at + 4 ° C, the plates were washed three times with PBS / Tween ₂ o and blocked for 30 minutes at 37 ° C with 1% BSA-PBS / Tween ₂ 0 · After being rinsed again with PBS / Tween ₂ o Apply 100 μΙ of different concentrations of TNF-α or its analogs (from 10 μg / ml to 0.2 pg / ml) in 1% BSAPBS / Tween ₂ o. The plate was incubated for 1 hour for 30 minutes at 37 ° C, then washed three times with PBS / Tween ₂ o and applied to each well 100 μΙ of the optimal conjugate dilution (1: 1000), the preparation of which is described under 12, v. 1% BSA-PBS / Tween ₂ o. After an hour and a half of incubation at 37 ° C and washing the plate with PBS-Tween ₂ o, 100 μΙ of ABTS substrate was added (composition described under 7). After an hourly incubation at 37 ° C, the absorbance at 410 nm was read with an automatic plate reader. Positive and negative controls were also included in the test.

EXAMPLE 15

Optimization of sandwich ELISA assay for quantitative determination of both soluble TNF-α, p55 and p75 receptors (chessboard or checkerboard)

The checkerboard was performed in the same manner as described for TNF-α or. its TNF-α analogue His107 / 108, Cys95 / 148. Two horseradish peroxidase conjugates were prepared for each of the two antigens according to the method described in item 12. Each conjugate was then tested in combination with four monoclonal antibodies, which were used to cover the microtiter plate to determine which combination of monoclonal antibodies provide us with the most sensitive ELISA assay.

EXAMPLE 16

Calibration curves for the determination of soluble p55 and p75 receptors

The procedure and reagents are the same as for the calibration curve for TNF-α or. its analogue, except that the optimum concentration of monoclonal antibodies to cover the microtiter plate is 2 μg / ml and the optimal conjugate dilution is 1: 1000.

EXAMPLE 17

Pharmaceutical preparations with monoclonal antibodies

The pharmaceutical compositions contain a neutralizing monoclonal anti-TNF-α antibody. The formulations may be in a form suitable for parenteral use, i.e. in the form of injections or lyophilized powder, which, with the addition of a suitable solution, e.g. of water for injection to give a solution for injection. The amount of antibodies in the preparations depends on the purpose of administration, the mode of administration, the age and the patient's condition. Generally, preparations contain so much anti-TNF-α antibody that 0.1 to 5 mg of anti-TNF-α per kg body weight can be administered by a single administration. The compositions contain either the anti-TNF-α antibodies themselves, or various antihistamines and / or glucocorticoids are added. Various substances are added to the preparations for isotonizing, solubilizing, stabilizing and regulating pH.

As an example, mention the composition of the following preparation:

of anti-TNF-α 50.00 mg sodium chloride 112.00 mg sodium primary phosphate 11,25 mg sodium secondary phosphate 45,00 mg polysorbate 80 5.00 mg water for injections up to 5 ml

Hybridomic cell line deposit information:

Hybridoma cell lines bearing the codes C9 / 8, C7 / 12 and B12 / 1 have been deposited with European Collection of Whole Cultures under Nos. 95071216, 95071217 and

95071218.

acevtic and

Institute of the Republic of Slovenia for Blood Transfusion

Claims (25)

PATENT APPLICATIONS Claims 1. Monoclonal antibodies and fragments thereof, which bind to the accessible surface of human recombinant TNF-α, TNF-α analogs and both soluble TNF-α receptors p55 and p75, and their complexes. Monoclonal antibodies and fragments thereof according to claim 1, characterized in that the TNF-α analogue is His107 / 108, Cys 95/148. Monoclonal antibodies and fragments thereof according to claim 1, characterized in that they are subclasses of IgG1. Monoclonal antibodies according to claim 1, characterized in that their fragments are Fab, F (ab ') 2, Fv, scFv. Monoclonal antibodies and fragments thereof according to claim 1, characterized in that χ binds to sites 107 and 108 or only 107 of the analogue His 107/108, Cys 95/148 of human recombinant TNF. Monoclonal antibodies and fragments thereof according to claim 1, characterized in that they are obtained by growing a stable cell line of ECACC hybrids 95071216 - 95071218. Stable hybridoma cell lines capable of producing monoclonal antibodies or fragments thereof according to claims 1-6. Stable hybridoma cell lines according to claim 7, characterized in that they are a fusion product of murine spleen cells and myeloma cells. Stable hybridoma cell lines according to claim 7, characterized in that they are obtained by a process comprising: - immunization of mice with a mixture of TNF-α analog complex His107 / 108, Cys 95/148 and TNF-α soluble receptors p55 and p75 - fusion of isolated mouse spleen cells with a myeloma cell line - cloning of hybridomas producing monoclonal antibodies 10. ECACC 95071216 Stable Cell Hybridoma Cell Line. 11. ECACC 95071217 Stable Cell Hybridoma Cell Line. 12. Stable ECACC 9507121 hybridoma cell line. A method for preparing a stable hybridoma cell line according to claims 7-12, characterized in that it comprises: - immunization of mice with a mixture of TNF-α analog complex His107 / 108, Cys 95/148 and TNF-α soluble receptors p55 and p75 - fusion of isolated mouse spleen cells with a myeloma cell line - cloning of hybridomas producing monoclonal antibodies A method according to claim 13, characterized in that the TNF-α complex of His 107/108, Cys 95/148 and soluble TNF-α receptor p55 is used for immunization. A method according to claim 13, characterized in that the TNF-α complex of His 107/108, Cys 95/148 and soluble TNF-α receptor p75 is used for immunization. A method according to claim 13, characterized in that the TNF-α complex analog of His 107/108, Cys 95/148 and both soluble TNFα receptors p55 and p75 are used for immunization. A method for the preparation of monoclonal antibodies according to claims 1-6, characterized in that it comprises: - culturing hybridoma cell lines according to claims 7-12 - isolation of monoclonal antibodies 18. Pharmaceutical formulations containing monoclonal antibodies or fragments thereof according to claims 1-6 and pharmaceutically acceptable excipients as active ingredient. 19. Pharmaceutical formulations containing as mono-active agents the monoclonal antibodies or fragments thereof according to claims 1-6 in combination with antihistamines or glucocorticoids. The use of monoclonal antibodies and fragments thereof according to claims 1-6 for the preparation of medicaments for the treatment of TNF-α increased disease. 21. TNF-α analogue His107 / 108, Cys 95/148. A method for the preparation of a TNF-α analogue according to claim 21, characterized in that it comprises: - expression in the recombinant E.Coli strain and - one-step purification by chelating chromatography Use of the TNF-α analogue of claim 21 for the isolation of TNF-α soluble p55 and p75 receptors from human urine. 24. Method for the quantitative determination of human TNF-α. free and complexed with soluble TNF-α receptors p55 and p75, and analogues of TNF-α and both soluble TNF-α receptors p55 and p75 in biological samples using monoclonal antibodies or fragments thereof according to claims 1-6. A kit for the determination of human TNF-α, free and complexed with soluble TNF-α receptors p55 and 575, and TNF-α analogs in biological samples, characterized in that it contains monoclonal antibodies and fragments thereof according to claim 1-6.