IE904273A1 - Monoclonal antibodies and method of diagnosis - Google Patents

Abstract

A decapeptide of the amino acid sequence: H-X<1>-Leu-Thr-Ile-Asp-Lys-Lys-Gly-Thr-Gly-Ala-X<2>-Y where X<1> and X<2> each represent an optional coupling-facilitating amino acid residue and Y represents -NH2 or -OH, is disclosed. Also disclosed are monoclonal antibodies specifically binding to only one epitode of Lys<342>- alpha 1-antitrypsin, i.e. Z-protein, comprising Lys<342>; a hybridoma cell line producing said monoclonal antibodies; and a method for preparing the hybridoma cell line. Further disclosed are a method for in vitro diagnosis of alpha 1-antitrypsin deficiency, i.e. the presence in the blood of Lys<342>- alpha 1-antitrypsin, in man, and an immunoassay kit for said diagnosis.

Description

MONOCLONAL ANTIBODIES AND METHOD OF DIAGNOSIS The present invention relates to a decapeptide cor342 responding to the amino acids 338-347 of Lys -a^-anti5 trypsin (Z-protein), and the use of this decapeptide coupled to an inert carrier, in a method for obtaining a hybridoma cell line producing monoclonal antibodies speci342 fically binding to only one epitope of Lys -a -anti342 1 trypsin comprising Lys . The invention further encom10 passes monoclonal antibodies specifically binding to said epitope and a hybridoma cell line producing said monoclonal antibodies. In addition, the invention is directed to a method for in vitro diagnosis of α^-antitrypsin deficiency in man, using said monoclonal antibodies, and an immunoassay kit for said diagnosis.

Background In 1955, an -glycoprotein was isolated and designated ct^-antitrypsin (a^AT) after its ability to bind trypsin had been observed (Schultze, H.E., Heide, K., and Haupt, H. , a1-Antitrypsin aus Humanserum. Klin. Wochenschr. 40, 427-429 (1962)). As the name α^-antitrypsin indicates, it is the main component in the α^-electrophoretic band of human plasmaproteins and acts as an inhibitor of proteolytic enzymes. It is now known that α^-antitrypsin is one of the major extracellular serine protease inhibitors in man. In molar respect, a^AT is the most important extracellular protease inhibitor whose main function is to inactivate elastase liberated from leucocytes and macrophages. Therefore, α^-antitrypsin has also been referred to as α^-protease inhibitor (a^PI) which better indicates its function (Johnson, D.R., Pannell, R.N., and Travies, J., The molecular stoichiometry of trypsin inhibition by human α^-proteinase inhibitor. Biochem. Biophys. Res. Commun. 57, 584-589 (1974)). -antitrypsin has a polypeptide chain of 394 amino acid residues, and three carbohydrate side chains, giving a molecular weight of 51 kDa (Carrel, R.W., Jeppsson, J-0 and Laurell C-B. Structure and variation of human -antitrypsin. Nature (1982) 298, 329). ct^AT exhibits a considerable genetic variation with more than 60 variants (Jeppsson, J-0 and Franzen, B. Typing of Genetic variants of a^-antitryp5 sin by Electrofocusing. Clin Chemistry (1982) 28, 219). These variants or, in other words, alleles are designated by letters according to their electrophoretic mobility, primarily in starch gel electrophoresis and later according to their appearance in isoelectric focusing. As to the al10 phabetical classification of the variants in Pi (protease inhibitor) nomenclature, reference is here made to Fagerhol, M.K. Ser. Haemat. 1^, Suppl. 1, 153-161 ( 1968).

The PI* M-allele is the most common and corresponds to a normal amount of ct^AT in plasma. Of clinical interest are three alleles giving lower concentrations of a^AT in plasma.

Variants of particular interest are the common Zand S-mutants giving low concentrations of oc^-antitrypsin in plasma; the product of the Z-allele gives concentra20 tions corresponding to 15% of the concentration of the normal M-allele, while the product of the S-allele gives concentrations of 60% of the normal allele. Mixed heterozygotes, such as MZ, MS and SZ, give proportionally changed plasma levels. The genotypes giving such a deficiency of a1-antitrypsin in plasma, which is an evident health hazard, are PiZZ (15% of normal concentration), PiSZ (35% of normal concentration) and combinations with an uncommon 0 (-)gene, such as PiZ- (8% of normal concentration).

The difference in charge between M-, S- and Z-pro30 teins depends on amino acid substitutions. Glutamic acid in position 342 of the M-protein is replaced by lysine in the Z-protein, and glutamic acid in position 264 of the M-protein is replaced by valine in the S-protein.

Deficiency in, or rather low concentration of, the plasmaprotein -antitrypsin is correlated with the presence of lung emphysema (Eriksson, S. Acta Med. Scand. •Ε 904273 175, 197-205 (1964)) and certain liver diseases (Sharp. H.L. Hosp. Pract. 6. 83-96 (1971)).

Persons having a hereditary low content of antitrypsin in plasma (PiZZ-individuals) exhibit earlier aging of the lung tissue than normal. This is very strongly pronounced when they are exposed to air pollution, especially tobacco smoke, inducing an increased invasion of phagocytes in the lungs (Pierce J. Antitrypsin and emphysema. JAMA (1988), 259, 2890). The presence of Z-protein in ho10 mozygous form is equivalent to the classic form of a^AT deficiency. This means in actual clinical practice that about 80% of these individuals will contract a chronic obstructive lung disease in the form of lung emphysema and/or chronic bronchitis, while 20% of them will develop hepatic cirrhosis in adult age, and 10% of neonates will develop neonatal icterus and later juvenile cirrhosis (James 0. Morse, New England, J. Med. part I 299, 10451048, 1978 and part II, 299, 1099-1105, 1978; H.L. Sharp, Gastroenterology, 70, 611-621, 1976; Chr. Larsson, Acade20 mic thesis. Liver and Lung Manifestations in a.^-Antitrypsin Deficiency, Malmo 1979).

In non-smokers, dyspnea appears at the age of 40-50 while in smokers it appears 15-20 years earlier. Consequently, screening for PiZZ individuals should be per25 formed at the age of puberty at the latest, when smoking habits and choice of profession are generally being established. If such young people are informed of their abnormal sensitivity to air pollution, they will have the possibility of protecting themselves (Sveger T. et al.

Screening for α^-antitrypsinbrist. Lakartidningen (1987), 84, 343). Also augmentation therapy with isolated human a^-antitrypsin might be offered to prevent chronic destruction of the lung (Hubbard R.C. et al. Biochemical efficacy and safety of monthly augmentation therapy for -antitrypsin deficiency. JAMA (1988) 260, 1259).

Diagnosing ot^-antitrypsin deficiency has hitherto been done by quantitative determination of ct^-antitrypsin in plasma, supplemented with electrofocusing at low levels. This is a relatively laborious and complicated tech5 nique. Therefore, there is a need for a simpler and faster technique allowing routine analysis of blood samples with an aim to locate at an early stage individuals having -antitrypsin deficiency, in order to prevent in various ways the development of lung diseases. To that end, Wallmark A. and co-workers produced an antibody against α^-antitrypsin isolated from the liver of a PiZZ individual and used said antibody in ELISA for the detection of PiZ gene carriers (Wallmark A., et al. Monoclonal antibody specific for the mutant PiZ -antitrypsin and its appli15 cation in an ELISA procedure for identification of PiZ gene carriers. Proc.Natl.Acad.Sci. USA, Vol. 81, pp 5690-5693 (1984)). However, said antibody produced against the whole a.-antitrypsin protein does not seem to be spe1 342 cific for only one epitope of Lys -antitrypsin corn342 1 prising Lys , since it does not always distinguish between heterozygote and homozygote PiZ gene individuals, respectively. (Cf. Fig. 3 of the above reference). So, there is still a need for a reliable method for the diagnosis of oc^-antitrypsin deficiency and distinguishing between heterozygote and homozygote PiZ gene individuals. Description of the invention The present invention is based on a decapeptide corresponding to amino acids 338-347 in the amino acid sequence of the Z-protein. With the aid of this decapeptide, it has been possible to produce monoclonal antibodies spe342 cifically binding to only one epitope of Lys -ct.. -anti342 . 1 . trypsin comprising Lys . Said monoclonal antibodies have then been used in a method of in vitro diagnosis of ct^antitrypsin deficiency, that is the presence in the blood of Lys^^-a^-antitrypsin, in man, and distinguishing between a heterozygote and a homozygote PiZ gene carrier.

One aspect of the invention is directed to a decapeptide having the amino acid sequence H-X^-Leu-Thr-Ile-Asp-Lys-Lys-Gly-Thr-Gly-Ala-X^-Y 2 where X and X each represent an optional coupling-facilitating amino acid residue and Y represents -NH9 or -OH. 2 1 The optional coupling-facilitating amino acid residues X 2 and X may be e.g. -Lys- or -Cys-, such an amino acid re10 sidue facilitating the coupling of the decapeptide to an inert carrier. The peptide according to the invention can be produced by suitable known methods for synthesising peptides; for instance, an amino acid can be coupled to the next one to form a peptide bond, either in solution or by so-called solid phase technique. The peptide according to the invention can be synthesised either in amide form or as a free acid, which is indicated by Y representing -NH2 or -OH.

Another aspect of the invention is directed to mono20 clonal antibodies specifically binding to only one epitope 342 342 of Lys -ct. -antitrypsin comprising Lys . The expression 342 1 Lys -antitrypsin is equivalent to the expression Z-protein as used in the literature. Thus, the monoclonal antibodies should specifically bind to only one epitope of the Z-protein, which epitope comprises the amino acid residue -Lys- which is in position 342 in the Z-protein and which is considered to be the determining amino acid residue distinguishing the Z-protein from the most commonly existing M-protein.

Yet another aspect of the invention is directed to a hybridoma cell line producing monoclonal antibodies speci342 fically binding to only one epitope of Lys -a1-antitryp342 Χ sin comprising Lys . The hybridoma cell line can then be used for producing unlimited amounts of said monoclonal antibodies.

A further aspect of the invention is directed to a method for producing a hybridoma cell line producing monoclonal antibodies specifically binding to only one 342 342 epitope of Lys -α^-antitrypsin comprising Lys . This method consists in that spleen cells derived from a mouse previously immunised with a decapeptide having the amino acid sequence H-X1-Leu-Thr-Ile-Asp-Lys-Lys-Gly-Thr-Gly-Ala-X2-Y 2 where X and X each represent an optional coupling-facilitating amino acid residue and Y represents or -OH, coupled to an inert carrier, are fused with myeloma cells, followed by selection of the hybridoma cells producing said monoclonal antibodies. Spleen cells from other mammals or poultry might be used, but in practice use is made of spleen cells from mouse for producing hybridoma cell lines. The decapeptide coupled to an inert carrier and used for immunising a mouse can be coupled to any inert carrier usable for immunisation purposes. For example, the decapeptide, suitably extended by -Cys-, may be coupled to an inert protein, such as bovine serum albumin or hemocyanin, or plastic particles. The fusion of the spleen cells with myeloma cells can be effected in conventional manner, and a known myeloma cell line can be used. Also the selection of the hybridoma cell lines producing the desired monoclonal antibodies can be effected in known manner.

A further aspect of the invention is directed to a method for in vitro diagnosis of a.-antitrypsin deficien1 342 cy, that is the presence in the blood of Lys -a^-antitrypsin, in man, and distinguishing between a heterozygote and a homozygote PiZ gene carrier. This method involves subjecting a blood sample from a human to an immunoassay using monoclonal antibodies specifically binding to only 342 342 one epitope of Lys -α^-antitrypsin comprising Lys as diagnostic antibody. In principle, any suitable immunoIE 904273 assay using a diagnostic antibody can be used for the purposes of the invention, especially suitable methods being radioimmunoassay (RIA), enzyme linked immunosorbent assay (ELISA) and Time-Resolved-Fluorescence. The analysed presence of antigen-antibody complexes confirms the pre342 sence of Lys -d^^-antitrypsin in the blood sample, that is α^-antitrypsin deficiency in the human from whom the blood sample was taken and analysed and the amount of said complexes revealing if said human is a homozygote or heterozygote PiZ gene carrier. If no antigen-antibody complex is detected by the method according to the invention, this means that the blood sample does not contain any Z-protein. The analysed presence of antigen-antibody complexes also indicates whether the blood sample contains homozygous protease inhibitor ZZ or heterozygous protease inhibitor comprising only one Z when the concentration is compared with references. The currently most preferred immunoassay techniques are enzyme linked immunosorbent assay and Time-Resolved-Fluorescence.

A final aspect of the invention is directed to an immunoassay kit for diagnosing α^-antitrypsin deficiency in man, which kit comprises a container holding monoclonal antibodies specifically binding to only one epitope of 342 342 Lys -α^-antitrypsin comprising Lys , in an inert medium. The inert medium may be e.g. a phosphate buffer or a physiological saline solution. The immunoassay kit may optionally contain further reagents, diluents etc. in accordance with the immunoassay method to be used. Preparation of peptide coupled to inert carrier A synthetic peptide comprising 11 amino acids was obtained from Peninsula Laboratories, San Fransisco, California : H-Leu-Thr-Ile-Asp-Lys-Lys-Gly-Thr-Gly-Ala-Cys-OH This peptide was coupled C-terminally to an inert carrier, viz. hemocyanin. The hemocyanin (Sigma) was activated with maleimidobenzoyl-n-hydroxy succinic acid ester (MBS) (Pierce). Activated hemocyanin was gel-fil5 tered on a G-25 column in 10 mM sodium phosphate, pH 7.0. The protein fraction from the gel filtration was mixed with the peptide under agitation for 3 h at room temperature and was thereafter dialysed against 50 mM Tris-HCl, 0.15 M NaCl, pH 7.5. The resulting coupling product was used for immunisation.

Immunisation of mouse pg of the above-mentioned coupling product in sterile physiological saline solution was mixed with Freund's complete adjuvant. The mixture was injected subcutaneously (10-20 urticas) during week 1, week 2 and week 4. To the mice which gave a good antibody response was administered a booster dose of 200 pg intraperitoneally 3 days before the planned fusion.

Preparation of a hybridoma cell line The immunised mice were killed and the spleens taken 6 out, whereupon 10 spleen cells were fused with 12x10 myeloma cells, cell line (Sp 2/0 Ag 14) from Balb/c mice. The fusion was carried out in polyethylene glycol/dimethyl sulphoxide/water in proportions 45:7:48. The fused cells 7 were mixed with 10 macrophages derived from peritoneal exudate after mineral oil challenge in the same type of mice. The cells were seeded in a microtiter plate in dilutions corresponding to 1.0, 0.5 and 0.3 cells/well. Master clones were detected with ELISA technique (see below).

Production of monoclonal antibodies 5χ10θ cells of a master clone were injected intraperitoneally in Balb/c mice after an injection of 0.5 ml Pristine (2,6,10,14-tetramethylpentadecane. After 8 days, all ascites fluid was aspirated, and the resulting immu35 noglobulins were precipitated with 50% ammonium sulphate. The precipitate was dissolved in water and dialysed against 50 mM Tris-HCl, pH 7.4, and purified on a Mono-Q (Pharmacia) column equilibrated with 20 mM Tris-HCl, pH 8.0. Immunoglobulin fractions, which had been detected with agarose electrophoresis, were pooled and frozen at -70°C.

Detection with ELISA technique Wells in a microtiter plate (Nunc) were prepared with 342 0.5 pg Z-protein produced in pure form, i.e. Lys -antitrypsin, as antigen. Antibodies from mouse serum diluted 1:1000 or ascites fluid diluted 1:10,000 were reacted, and antigen-antibody complexes were detected with rabbit antimouse globulin coupled to peroxidase (Dakopatts A/S, Denmark ).

Detection with Time-Resolved-Fluorescence Technique Monoclonal antibodies according to the invention were labelled in known manner with the earth metal Europium giving a strong fluorescence of short duration. (See e.g. Hemmila, I. et al. Europium as a Label in Time-Resolved Immunofluorometric Assays, Analytical Biochemistry 137, 335-343 (1984)).

Wells in a microtiter plate were incubated at pH 9.6 with a polyclonal antibody against cr^-antitrypsin derived from rabbit (Dako No. A 012). To avoid unspecific adsorption, the remaining surfaces were blocked with 0.1% Tween 20 in phosphate buffer, pH 8.0. After washing, incubation with patient serum diluted 1:100 was performed. Incubation was conducted for 2 h, and washing with phosphate buffer was done 5 times. The wells were finally incubated with the 342 Europium-conjugated monoclonal antibody against Lys -i -antitrypsin. After incubation for 1 h, fluoroscence was read in an Arcus fluorometer (Wallac, Finland).

In vitro diagnosis of a^-antitrypsin deficiency in man For testing the method, serum was collected from previously known antitrypsin-deficient patients ZZ (20 patients), antitrypsin predisposed patients MZ (20 patients), and from normal blood donors MM (20 patients). All 60 individuals were examined with a specific electrofocusing technique, pH range 4.2-4.9. ΪΕ 904273 Results obtained with Time-Resolved-Fluorescence Technique TABLE Cps (counts per second) for 60 individuals in respect of antitrypsin-deficient patients (homozygotes) (ZZ), anti- trypsin predisposed patients mal patients (MM). (heterozygotes) (MZ) and nor- Patient serum Pi-type ZZ cps Pi-type MZ cps Pi-type MM cps 1 63594 11682 678 2 57546 17573 568 3 52404 17478 456 4 50258 20789 789 5 58322 15406 698 6 61751 16646 478 7 64353 19148 589 8 65982 15038 854 9 63225 14971 596 10 65051 15947 832 11 64216 15926 652 12 72724 18430 746 13 62605 11280 567 14 54454 16353 931 15 50877 16076 832 16 61356 16435 753 17 59658 14195 951 18 58619 12484 456 19 61586 13285 851 20 61176 14470 751 These results unambiguously show that the method can differentiate between homozygous ZZ antitrypsin patients, predisposed MZ and normal MM.

Results obtained with ELISA technique Results similar to those stated above have been achieved with conventional ELISA technique. The principle is basically the same as for Time-Resolved-Fluorescence, with the exception of the detection. This requires a further step, namely a peroxidase-conjugated rabbit antimouse antibody.

Claims (12)

1. A decapeptide, characterised by the 5 amino acid sequence H-X 1 -Leu-Thr-Ile-Asp-Lys-Lys-Gly-Thr-Gly-Ala-X 2 -Y 1 2 where X and X each represent an optional coupling-faci10 litating amino acid residue and Y represents -NH 2 or -OH.

2. Monoclonal antibodies, characterised in that they specifically bind to only one epitope of 342 342 Lys -ot^-antitrypsin comprising Lys

3. A hybridoma cell line, characteris15 e d in that it produces monoclonal antibodies according to claim 2.

4. A method for preparing a hybridoma cell line producing monoclonal antibodies specifically binding to only 342 342 one epitope of Lys -α^-antitrypsin comprising Lys , 20 characterised in that spleen cells derived from a mouse previously immunised with a decapeptide having the amino acid sequence H-X^-Leu-Thr-Ile-Asp-Lys-Lys-Gly-Thr-Gly-Ala-X^-Y 1 2 where X and X each represent an optional coupling-facilitating amino acid residue and Y represents -NH 2 or -OH, coupled to an inert carrier, are fused with myeloma cells, followed by selection of the hybridoma cells producing 30 said monoclonal antibodies.

5. A method for in vitro diagnosis of co-antitrypsin 1 342 deficiency, i.e the presence in the blood of Lys -a i~ -antitrypsin, in man, and distinguishing between a heterozygote and a homozygote PiZ gene carrier, c h a r a c 35 terised in that a blood sample from a human is subjected to an immunoassay using monoclonal antibodies 342 specifically binding to only one epitope of Lys -oc i~ 342 -antitrypsin comprising Lys as diagnostic antibody, the analysed presence of an antigen-antibody complex confirm342 ing the presence of Lys -antitrypsin in the blood sample, i.e. -antitrypsin deficiency in said human, and 5 the amount of said complex revealing if said human is a homozygote or heterozygote PiZ gene carrier.

6. Method as claimed in claim 5, characterised in that enzyme linked immunosorbent assay (ELISA) is used as immunoassay. 10

7. Method as claimed in claim 5, characterised in that Time-Resolved-Fluorescence is used as immunoassay.

8. An immunoassay kit for diagnosing a^-antitrypsin deficiency in man, characterised in that it 15 comprises a container holding monoclonal antibodies specifical342 ly binding to only one epitope of Lys -a.-anti342 1 trypsin comprising Lys , in an inert medium. 20

9. A decapeptide substantially as described herein with reference to the Examples.

10. Monoclonal antibodies substantially as described herein with reference to the Examples.

11. A method for in vitro diagnosis of a-antitrypsin 25 1 deficiency substantially as described herein with reference to the Examples.

12. An immunoassay kit for diagnosing a^-antitrypsin deficiency in man comprising a monoclonal antibody as claimed 30 in claim 2 or claim 10. DATED THIS 27th day of November, 1990 BY: