FI102115B - Monoclonal antibody composition to be used as diagnostic reagent - Google Patents

Description

102115

Diagnostic Monoclonal Antibody Composition and Test Kit Containing It 5 This application is separated from the parent application 901344 by division.

This invention relates to diagnostic monoclonal antibody compositions comprising monoclonal antibodies capable of reacting with human TNF.

10

Delivery of spreading stimuli, such as parasitic, bacterial, or viral infections, or neoplastic cells, to a susceptible host induces significant metabolic changes. If severe, the changes are likely to disrupt normal equilibrium mechanisms, both locally and systemically, leading to depletion of the host's energy stores leading to atrophy (Cachexia, morbid thinness), »# ·: · 'tissue destruction, multiple organ failure , shock and death.

• I> • »1» • · •, · · Until recently, clinicians believed that systematic models were mainly due to the functions of it-::: 20 it intrusive substances. However, it is now known that invasive stimuli caused the host to generate a variety of cytokines, the combined actions of which cause most unwanted biological responses. These host-derived inflammatory mediators represent new opportunities in the development of diagnostics for various inflammatory conditions.

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One of the most potent cytokines is cachectin, which is mainly released by macrophages. after the day stimulus. Cachectin, also known as tumor necrosis factor, TNF, is a protein of 157 amino acids normally found in vivo as a dimer or multimer. The calculated molecular weight of human TNF monomer is about 17 to 30,000 daltons.

TNF acts by inhibiting the biosynthesis of several adipocyte-specific proteins, such as lipoprotein lipase. It also acts by inducing the biosynthesis or release of a number of other proteins, including class I major histocompatibility factor 35, granule cell macrophage colony stimulating factor, and interleukin 1. (See generally Beutler and Cerami, New England J. of Med., 316: 379-385 (1987), which is incorporated herein by reference.) 102115 2

New cell lines are produced that produce monoclonal antibodies capable of binding to human TNF epitopes. In addition, the use of monoclonal antibodies in a diagnostic test kit is disclosed.

The essential features of the invention are set out in the appended claims.

According to the present invention, there are provided novel diagnostic monoclonal antibody compositions comprising neutralizing monoclonal antibodies having the desired affinities, with the compositions being capable of selectively recognizing epitopes in human TNF. Target cells have identifiable chromosomes in which their or the precursor cell's embryonic DNA has been rearranged to encode an antibody with a binding site for a desired epitope on human TNF. These monoclonal antibodies can be used in diagnostics.

• · '· 15 The production of monoclonal antibodies can be accomplished by immortalizing a cell line capable of expressing a nucleic acid sequence that *. encodes an antibody specific for a suitable epitope in human TNF. The depleted cell line may be a mammalian cell line transformed with oncogenes, transfection, mutation, or the like. Such cells include::: myeloma lines, lymphoma lines, or any other cell line capable of maintaining the expression and secretion of an immunoglobulin or binding fragment thereof in vitro. Immunology, y. the noglobulin or fragment may be a naturally occurring mammalian non-preferred murine or human source, an immunoglobulin produced by transforming a lymphocyte, especially a spleen cell, with a virus, or by fusion of a lymphocyte and a neo-plastic cell, e.g., to produce a myeloma hybrid cell line. Typically, a spleen cell is obtained from an animal immunized against TNF-like antigens: or fragments thereof having epitopic sites. Immunization protocols are well known and can vary considerably while still being effective. (See Go- • · Ding, Monoclonal Antibodies: Principles and Practice, 2nd Edition, Academic Press, 30 N.Y. [1986], which is incorporated herein by reference.)

Hybrid cell lines can be cloned and screened according to conventional techniques to detect antibodies in cell supernatants that are capable of binding to the desired determinants of human TNF with appropriate affinities. Suitable hybrid-35 cell lines are then grown in large scale culture or injected into the peritoneal cavity of a suitable host to produce antibodies to aqueous humor.

102115 3

In some cases, supernatants from subsequent experiments may be screened for the presence of antibodies of this invention specific for human TNF by a competitive assay with these monoclonal antibodies as a means of identifying further examples of the desired anti-human TNF monoclonal antibodies ( so-called "blocking antibodies"). Thus, hybrid cell lines can be readily produced from a variety of sources based on the availability of these antibodies, which are specific for affinity levels appropriate for specific TNF determinants.

Alternatively, when hybrid cell lines producing antibodies specific for the epitope sites of a target are available, these hybrid cell nodes can be fused with other neoplastic B cells, allowing such other B cells to serve as recipients for genomic DNA encoding the antibodies. These antibodies may be functional on the cell surface but not, e.g., as receptors. Although neoplastic B cells from a rodent, especially a mouse, are more commonly used, other mammalian species may be used, such as rabbits, cattle, sheep, horses, piglets, birds, or the like.

Monoclonal antibodies can be of any class or subclass of immunoglobulins, such as IgM, IgD, IgA, IgE, or subclasses of IgG known to any animal species. In general, monoclonal antibodies can be used as intact or binding fragments, such as Fv, Fab, F (ab ') 2, but generally intact Mana.

The cell lines of this invention can be used for the direct production of non-monoclonal antibodies. Genes encoding monoclonal antibodies can be isolated and used according to recombinant DNA techniques to produce a specific immunoglobulin in a variety of hosts. In particular, by preparing cDNA libraries from messenger RNA, a single cDNA clone encoding immunoglobulins and free introns can be isolated and inserted into a suitable prokaryotic or eukaryotic expression vector and then transformed into a host for final large-scale production. (See, generally, U.S. Patents 4,172,124; 4,350,683; 4,363,799; 4,381,292 and 4,423,147. See also Kennet, et al., Monoclonal Antibodies, Plenum New York [1980] and references cited therein). all of which are incorporated herein by reference.) More specifically, in accordance with hybrid DNA technology, the immunoglobulins or fragments of this invention may be produced in bacteria or yeast. (See Boss, et al., Nucl. Acid. Res., 12: 3791 and Wood et al., Nature 314: 446, both of which are incorporated herein by reference.) For example, a messenger RNA transcribed from genes that 102-105 The light and heavy chains of the monoclonal antibodies produced by the cell line of this invention can also be isolated by differential cDNA hybridization using cDNA from BALB / c lymphocytes other than the target clone. mRNAs that do not hybridize are rich in a messenger that encodes the desired immunoglobulin chains. If necessary, this process can be repeated to add more desired mRNA levels. The reduced mRNA composition can then be reverse transcribed to obtain a cDNA mixture enriched for the desired sequences. RNA can be hydrolyzed with an appropriate RNAse and ssDNA can be double-stranded with DNA polymerase I and random primers, e.g., randomly fragmented calf thymus DNA. The resulting dsDNA can then be cloned by insertion into a suitable vector, e.g., viral vectors such as lambda vectors or plasmid vectors (such as pBR322, pACY184, etc.). By developing probes based on known light and heavy chain constant region sequences, those cDNA clones having a gene encoding the desired light and heavy chain can be identified by hybridization. The genes can then be excised from the plasmid, manipulated by removing unnecessary DNA upstream of the start codon or constant region of the DNA, and then introduced into a suitable vector for transformation into a host and. , ‘i’ for extreme expression of the gene.

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Suitably, mammalian hosts (e.g., mouse cells) can be used to process the chain (e.g., to attach heavy and light light) to produce intact immunoglobulin and, in addition, to secrete an immunoglobulin with no leader sequence, if desired. Alternatively, unicellular microorganisms can be used to produce two • · · strands, which may require additional manipulations to remove DNA sequences encoding secretion and processing signals, while obtaining an «· · _ start codon at the 5 'end of the heavy chain coding sequence. . In this way you can:. ·. immunoglobulins can be prepared and processed to be assembled and glycosylated in non-mammalian cells. If desired, each of the chains can be truncated to leave at least a variable region, a region that can then be manipulated for other immunoglobulins or fragments specific for TNF epitopes (see, e.g., EP Patent Applications 0 239 400 and 0 125 023, which are incorporated herein by reference). attached for reference).

The monoclonal antibodies of this invention are particularly useful because of the affinity of their major human TNF epitopes.

The monoclonal antibodies of this invention can be widely used in vitro. By way of example, monoclonal antibodies can be used to purify native or recombinant human TNF, to selectively remove human TNF from a heterogeneous mixture of proteins, or the like.

For diagnostic purposes, monoclonal antibodies can be either labeled or unlabeled. Typically, diagnostic assays require the detection of complex formation by binding of a monoclonal antibody to a protein. Unlabeled antibodies can be used in agglutination assays. In addition, unlabeled antibodies can be used in conjunction with other labeled antibodies (other antibodies) that react with monoclonal antibodies, such as antibodies specific for an immunoglobulin. Alternatively, monoclonal antibodies can be labeled directly. Various labels can be used, such as radionuclides, fluorescent agents, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands (especially hapten), etc. Numerous immunoassays are available and, by way of example, some are described in U.S. Patents 3,817,827 3 / 15,850,752; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074 and 4,098,876, of which · '·' are all incorporated herein by reference.

The monoclonal antibodies of this invention are commonly used in immunoassays in which target antibodies or other antibodies from various species are conjugated to an enzyme. When a sample containing human TNF, such as human blood or a lysate thereof, is combined with target antibodies, binding occurs. ·. ·. between those and the molecules in which the desired epitope is present. Such complexes can then be separated from unbound reagents and another antibody (labeled with the enzyme) added. The presence of an antibody-enzyme conjugate that is specifically bound to the cells to be determined is then determined. Other suitable known methods may also be used by those skilled in the art.

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Assay kits can also be used with target antibodies to detect human TNF epitopes or human TNF epitopes in recombinant fractions. Thus, the monoclonal antibody composition of the present invention is usually obtained in lyophilized form, either alone or in combination with antibodies specific for endotoxins, exotoxins or gram-negative bacteria. Antibodies, which may be conjugated or unconjugated to the label, are included in test kits with buffers such as Tris, phosphate, carbonate, etc., stabilizers, biocides, inert proteins, e.g., bovine serum albumin, or the like. Usually, these materials are present in an amount of less than about 5% by weight based on the amount of active antibody, and usually in an total amount of at least about 0.001% by weight based on the antibody concentration. It is often desirable to include an inert extender or excipient to dilute the active ingredients, where the excipient may be from about 1% to about 99% by weight of the total composition. When another antibody capable of binding to a monoclonal antibody is used, this is usually in a separate ampoule. The second antibody is typically conjugated to a label and formulated in a similar manner with the antibody formulations described above.

The monoclonal antibodies of this invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional immunoglobulins, and lyophilization techniques known in the art and restoration techniques can be used. It will be appreciated by those skilled in the art that lyophilization and reconstitution may result in varying degrees of ... loss of antibody activity (e.g., conventional im-; 15 other globulins, IgM antibodies tend to have a greater loss of activity than IgG). antibodies) and use levels need to be adjusted to compensate.

. TNF production and purification • · • · · «·«: 20 A synthetic gene encoding methionine initiation and mature TNF sequence expresses recombinant human TNF (Pennica et al., Nature, 312: 724-729 (1984) and Shirai et al., Nature, 313: 803-806 (1985), both of which are incorporated herein by reference) in yeast as an intracellular protein as codons selected to reflect highly expressed yeast genes according to standard techniques. The synthetic sequences were placed downstream of the hybrid alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAPDH) promoter. TNF synthesis was induced: in transformed Saccharomyces cerevisiae cultures with glucose deficiency. About • · · * 5-10% of the total yeast protein was TNF.

TNF was purified from coarse yeast extracts by (a) ammonium sulfate fractional distillation, (b) Q-Sepharose flash flow column chromatography, (c) S-Sepharose flash flow column chromatography, and (d) Sepharciyl S-200 (HR) column chromatography.

This method yielded approximately 80% recovery in TNF activity as determined by actinomycin D-treated L929 cells (Ruff and Gifford, (1981) Tumor Necrosis Factor, Lymphokines, 2: 235-272). The purified TNF had a specific activity of 2-4 x ΙΟ ** units / mg and a purity greater than 98% as determined by SDS-polyacrylamide gel electrophoresis.

7 102115 Production of monoclonal antibodies against TNF BALB / c mice were immunized intraperitoneally with 5-25 μg of Freund's complete adjuvant and given an additional dose of twice the same dose of Freund's complete adjuvant every 3 weeks. A final additional intraperitoneal dose of the same dose in aqueous solution was given and the fusion was performed 3-4 days later.

Spleen cells from immunized mice (about 10 ** cells) were fused with about 2 x 10 6 myeloma cells (P3-X63-Ag.653) (Keamey et al., J. Immunol. (1979) 123: 1548-1550) using polyethylene glycol according to Kohler and Milstein (Nature 256: 495 (1979)). Cells were plated in microtiter plates and hybrids were selected on hypoxanthine aminopterin thymidine (HAT) medium.

; 15: · 5 fusions were performed with spleen cells from 5 TNF-immunized mice. Using solid phase enzyme-linked immunosorbent assays, 60 positive clones were obtained that produced antibodies to TNF.

• ««:. *: 20 Positive well hybrids were cloned 3-fold by limiting dilutions to ensure that each was a true clone. 14 TNF-positive hybrids were cloned. ·. ·. dooma and characterized them.

TNF / TNF Bioassay: L-292 Cellular Toxicity 25 • · · TNF activity was measured using the actinomycin D-hand cytolytic assay. L292 cells, as described by RufT and Gifford in Lymphokines 2: 235,]. ”(1981), which is incorporated herein by reference.

• 30 L292 cells (CCL1, American Type Culture Collection, Rockville, (MD)) are maintained in RPMI 1640 supplemented with 10 mM Hepe and 10% fetal bovine serum (or DME + 10% FBS). Confluent cultures (3-4 x 10 6 cells / 75 cm flask) are released by brief trypsin treatment (rinsed with 0.05% trypsin) in physiological saline with 5 mM EDTA and 10 mM Hepes, pH 7.4 and suspended. again to fresh medium with actimonine D (1 pg / ml). The cells are then plated in 96-well microtiter dishes (5-7x10 6 cells / well).

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After the cells have been cultured for 2 h, a dilution series of samples (less than 10-20% serum) are added to the wells and the plates are incubated overnight (5% CO2, 37 ° C). Samples were determined in quadruplicate. The next day after microscopic evaluation, the medium was decanted and the wells were filled with 0.2% crystal-5 violet solution, 10% formalin and 0.01 phosphate pH 7-7.5, 5 min, washed thoroughly with water and dried. The degree of lysis was determined spectrophotometrically (550-570 nM) with a microtiter reader. The results of the assay are expressed in U / ml, where 1 unit (U) is described as the amount of TNF that results in 50% lysis of the cells.

10 In Vitro Neutralization Assay

Recombinant human TNF (20 ng / ml in 0.02 M Tris NCl, pH 8.0, 0.15 M NaCl, 1 mg / ml BSA) is mixed with the corresponding diluted amounts of antibody and incubate at 37 ° C for 60 min. The mixture is diluted 1:10 with fresh medium, /; * 15 with actinomycin D (pg / ml); and add 0.1 ml of diluent (double) • in triplicate to the microtiter wells. Residual cytolytic activity is determined using an L292 cell cytotoxicity assay.

«::: Characterization of monoclonal antibodies: 20

Fourteen hybridomas that were TNF-positive were identified and subcloned.

The antibodies produced by these hybridomas were characterized by their ability to compete with monoclonal antibody 18-1-1. (Tracey, et al., Nature 330: 662-664 (1987), which is incorporated herein by reference) to bind TNF in an ELISA. As can be seen from Table 1:, i .: 25, the monoclonal antibodies fall into 3 categories; those who do not • · · J ... i compete with 18-1-1, those who compete and those who show average: * ·. competition. To ensure that monoclonal antibodies that did not compete with 18-1-1 were TNF-specific, a TNF capture assay was performed. Monoclonal antibodies to bind TNF were bound to the solid phase and monoclonal antibody 18-1-1 was used as a labeled antibody. All monoclonal antibodies were specific for TNF in this assay.

Experiments were also performed on an air-conditioned medium of hybridoma cells to determine the extent of neutralization of the TNF cell-killing activity of MAbs. All 14 monoclonal antibodies were produced in fluid accumulated in the mouse peritoneal cavity for further characterization. The efficacy of purified monoclonal antibodies to protect L292 cells against TNF killing was determined. The neutralizing activity of MAb 1- 2-4B1 9 102115 was confirmed with purified material. When incubated with an amount equivalent to MAb 2-2-3E3 (50 ng / ml) of recombinant human TNF (20 ng / ml) at 37 ° C for 60 min, it was shown that 50% of the cytolytic activity of TNF was neutralized. as determined by the L292 cell killing assay. As shown in tau-5 lock 1, MAb 2-2-3E3, which recognizes the same epitope as MAb 18-1-1, is required in 1/8 of 18-1-1 for cell protection. Table 1 also shows that most of the antibodies with the same specificity as 18-1-I neutralized TNF1 (100%) in the L292 cell killing assay. Other antibodies, such as 1-2-4B1, which have different epitope specificities than 18-1-1, also had significant neutralizing activity.

table 1

Review of Monoclonal Antibodies Reactive with Human TNF! '; * 15: Hybridoma MAb, Type Competition Assay Neutralization Assay with 18-1-ljj.,' · Substrate Purified Ig! : * i n8 rii: 18-1-1 IgG + + 400 1-2-4D4 IgG + +

1-2-4B2 IgM

1-2-4B1c IgG - + 400 1- 1-1F3 IgG - ± 2- 2-2FS IgG + +> 800 O 1-1-4C8 IgG - +:! ·. 2-2-3E3C IgG + + 50 1-2-2F8 igG + + 1-1-2E3 IgG + + 200 1- 2-2 AI IgG + + 2- 3-1A8 IgG + + 1-2-4C6 IgG + a) Determined by SDS-PAGE of MAb partially purified from intraperitoneal fluid by precipitation with ammonium sulfate.

102115 10 b) Determined solid phase competition in ELISA as follows. Aeration medium from 24 h hybridoma cultures (50 μΐ) and HPR-labeled 18-1-1 (25 μΐ, 0.2 pg / ml in PBS + 10% goat serum) were added to each well.

C) Cell lines 1-2-4B1 and 2-2-3E3 were deposited in A.T.C.C. and designated accession numbers HB9737 and HB9736, respectively.

From the foregoing, it will be appreciated that the cell lines of this invention provide the means to produce monoclonal antibodies and fragments reactive therewith with human TNF in low neutralizing amounts. Antibodies can be used in a variety of diagnostic methods.

Although the present invention has been described in detail in an illustrative manner and by way of example to clarify the understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

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Claims (4)

102115 π A monoclonal antibody composition for use as a diagnostic reagent, characterized in that the composition comprises (a) a monoclonal antibody produced by a cell line having a accession number ATCC HB9736, (b) a monoclonal antibody produced by a cell line having accession number ATCC HB9737, or an F (ab ') 2 fragment of a monoclonal antibody according to (a) or (b) capable of immunologically binding cachectin . 10 A monoclonal antibody composition according to claim 1, characterized in that the monoclonal antibody is conjugated to a label which gives a detectable signal. *> »• ·: **: 15 A monoclonal antibody composition according to claim 2, characterized in that the label is fluorescent or enzyme. A test kit for use in detecting an infection caused by an endotoxin-containing bacterium, characterized in that the test kit comprises a monoclonal antibody composition comprising monoclonal antibodies produced by (a) a cell line having accession number ATCC HB9736 and / or (b) a cell line having the accession number • · · ATCC HB9737, and a label giving a detectable signal, wherein the label is covalently bound to said antibodies (a) or (b) or to other antibodies; j1; which react with said antibodies (a) or (b). • · · «· • · ··· • · • · · • · ··· · · 102115