GB2160544A - Monoclonal Antibody to HLI and its use for purifying HLI - Google Patents

Abstract

The present invention provides a monoclonal antibody which is directed against natural or recombinant human leukocyte interferon (HLI), characterised in that the antibody is of IgG isotype, it has a gamma 1, heavy chain, and it binds to recombinant HLI A and D but not to recombinant HLI B and F, which is suitable for use in purifying HLI, particularly by affinity chromatography.

Description

1 GB 2 160 544 A 1

SPECIFICATION

Monoclonal antibody to HLI and its use for purifying HLI The present invention is concerned with antibodies proteins, namely against human leucocyte interferon 5 (HLI) which is natural or obtained by recombinant DNAtechnology.

HLI is possibly a valuable therapeutic forthe treatment of neoplastic and viral illnesses. The evaluation of this substance and its possible developmentto a widely-used medicament is restricted by the difficulty of purifying and of characterising the active substance in the requisite manner. A test which could be carried out in less than 24 hours would be a valuable aid for the desired purification and the requisite characterization of HLI.

In the scope of the present invention it has now been found that forthe purification of HLI in an excellent mannerthere is suitable a particular monoclonal antibodywhich is directed against leucocyte interferon which is natural or obtained by recombinant DNA technology.

According to the present invention, there is provided a monoclonal antibody which is directed against 15 natural or recombinant human leukocyte interferon (HLI) characterised in that the antibody is of 19G isotype, it has a -yj heavy chain, and it binds to recombinant HLI A and D but not to recombinant HLI B and F.

This antibody is suitable for use in purification of HLI, preferably by affinity chromatography. In a second aspect, the present invention provides a process for the purification of HLI characterised in that HLI-containing raw material is purified by affinity chromatography using the monoclonal antibody defined 20 above. Forthe purification in a commercial scale the aff inity should be high enough so that the interferon is practically quantitatively retained also with great volumes which pass the column with a not too low speed.

With a single antibody this is not possible for all sub-classes of interferon. For this purpose a collection of monoclonal antibodies which individual members show a high affinity for particular sub-classes of interferon is especially suitable.

The preparation of the monoclonal antibodies is effected according to cell fusion technology known per se, in which the myeloma cells are fused with spleen cells of mice, which have been immunized with HLI. The hybridomas obtained secrete monoclonal antibodies against leucocyte interferon which is natural or obtained by recombinant DNA technology.

The purification of HLI by means of a monoclonal antibody can be effected according to methods known 30 per se, whereby the aff inity chromatography has been found to be particularly suitable. In this affinity chromatography Aff i-Gel 10 (BiroRad Laboratories, Richmond, California) Affi-Gel is a Registered Trade Mark. is preferably used as the carrier.

On the other hand, it has been shown that human leucocyte interferon obtained by recombinant DNA technology can be purified with a monoclonal antibody in accordance with the present invention in a simple 35 manner such that it is usable for clinical trials.

In the following Examples, the present invention is illustrated with reference to the accompanying drawings, in which Figure 1 is a graph showing the binding of various monoclonal antibodies to various types of HLI; and Figure 2 is a graph showing the binding of various monoclonal antibodies to various sub-types of HLI. 40 In the Examples indicates a registered Trade Mark.

EXAMPLE 1 Manufacture of the monoclonal antibodies 45 A) Interferon preparation Partially purified human leucocyte interferon (HLI) was available in sufficient amountforthe immunization of the mice. 5 HLI preparations are used.

a) HLI y(a) main peak of the fraction in accordance with step 8 of Table 3 of DOS No. 29 47 134 with an approximate purity of 10-15% (estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis) and specific activity in the antiviral test; b) HLI a; c) HLI p; cl) HLI y; and e) HLI 8.

The HLI et, P and y fractions consist in each case of mixtures of the individual ((Xl, "2), P1, P2, 133) and (^11, Y2, ^13, ^14, Y5) species. These are shoulder fractions of the corresponding purified species, which are pooled from different preparations (see step 9 of Table 3 of the previously mentioned DOS). The HLI 6 fraction is the shoulder of the 8-species. Besides the species et, p and y on the Lichrosorb-diol column (see step 8 of Table 55 3 of the previously mentioned DOS) there is occasionally seen a 8-fraction. This is purified as in step 9 in accordance with Table 3 of the previously mentioned DOS, whereby it is evident that the species 8 shows no sub-species and is uniform. The purity of b), c) and d) is between 20 and 40% in accordance with specific biological activity and in accordance with sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The purity of e) is at most 5%.

Determination of interferon activity The interferon activity is determined by means of the "Cytopathic-effect- inhibition" (CPE) test in accordance with U.S. Patent Specification No. 4, 241 174 (Serial No. 963,256).

2 GB 2 160 544 A B) Immunization of the mice 3 eightweeks old Balb c/J female mice arefirstly immunized with HLI -y(a) in Freund's complete adjuvant. Each mouse receives approximately 150 Lg total protein containing 20-25 [Lg of interferon in 0.25 ml at 5 different positions (0.05 ml in each position): subcutaneously in the left and right iliac region and in the left and right axilliary region as well as an intraperitoneal injection.

53 days later a second immunization is carried out as follows: interferon HLI -y(a) is separated by preparative sodium dodecyl sulphatepolyacrylamide (15%) gel electrophoresis in three 0.6 x 11 cm cylindrical gels. Approximately 300 Lg total protein in 0.3 ml (dialyzed against assay buffer) containing approximately 40-50 jig of interferon are loaded per gel. [This purification is effected essentially according to the method of Laemmli, U.K. (1970) Nature 227, 680-6851. After the electrophoresis, the gels are sliced into 2 10 mm thick discs. These are immersed for 10 minutes in 0.5 ml of phosphate- buffered sodium chloride solution (0.01 M potassium phosphate buffer, pH 7.3; 0.14 M sodium chloride) containing 0.1 % Triton X-l 00 in propylene test tubes. The buff er is investigated for interferon activity in serial dilution according to the CPE inhibition test. The discs 6 and 7 (numbered from the lower end of each gel) show the highest interferon activity (in each case approximately 25% of the activity of the total gel). In each case one disc with highest 15 activity is finely sliced with a razor blade on a glass plate, transferred into a 1 ml syringe containing 0.2 ml of 0.15 M sodium chloride and injected into each mouse. The gel suspension is injected i ntra peritonea I ly, whereupon 0.2 ml of BCG (Bacillus Calmette-Gu6rin; Serum Institute Berne) are injected.

After 12 days, the serum of each mouse is investigated for interferon neutralization activity. The serum of mouse No. 3 shows a 50z neutralization 10 units/ml of HLI -y(a) at a dilution of 1:72 000, but does not 20 neutralize the same concentration of crude HLI (< 0.1% purity) even at a dilution of 1:100. The mice No. land No. 2 show neutralization titres of less than 1:1000 against HLI -y(a).

days after the second immunization mouse No. 3 receives on three successive days an intraperitoneal booster injection with a mixture of HLI a-, P and -y containing approximately 50 I.Lg of interferon and 15 1A of normal mouse serum as the carrier protein (in 0.1 ml of 0.15 M sodium chloride). 48 hours after the last injection the mouse is killed and the spleen is removed for the preparation of the monoclonal antibodies.

2 C) Cell cultures and cell fusions The following materials and media are used. Iscove's modification of Dulbecco's modified Eagle medium (IMDMEM) is obtainable from Gibco. It is made up freshly with sodium pyruvate (1 mM), glutamine (1.5 MM), 30 2-mercaptoethanol (5 x 10-5M), penicillin (100 units/mO and streptomycin (100 jig mi). Complete HAT medium consists of thus-completed IMDMEM as well as hypoxanthine (1 0AM), aminopterin (4x 10-7M), thymidine (1.5 x 10-5M) and 15% foetal calf serum. A 50% (wlv) solution of polyethyleneglycol 4000 (PEG 4000, Merck) in IM13MEM is prepared.

The fusion with a non-producing azaguanine-resistant myeloma cell line is carried out, with small 35 modifications, according to the method of St5Inli et al. in J. Immunol. Methods 32, 297-304. In this fusion 48 x 106 nucleated cells are fused with 25 x 106 myeloma cells of the line FO (Fazekas de St. Groth and Scheidegger in J. Immunol. Methods 35,1-25 (1980). The spleen cells and myeloma cells are washed in serum-free IMDMEM. They are then re-suspended in the same medium, mixed in the above-mentioned ratio for the fusion and sedimented in 40 m] of serum-free IM13MEM in a 50 mi conical polypropylene test tube at 40 x 9 for 15 minutes, followed by complete sucking off the supernatant. To the cell sediment there are added dropwise with constant stirring 0.5 m] of 50% PEG 4000 in order to re-suspend and to disperse the cells. After approximately 90 seconds, 10 mi of serum-free IMDMEM are added dropwise at room temperature with constant stirring during 4-5 minutes. After a further 15 minutes without stirring, large cell clumps are separated by cautious pipetting with a 10 m] pipette. The fusion mixture is diluted to 250 mi with 45 complete HAT medium and then placed in 240 "Costar cluster wells (1 m[lwell)", which already contain 1 m] of complete HAT medium and 101 peritonea[ mouse cells as the nutritive layer. The cultures are incubated in a 5% C02195% air atmosphere at 85% humidity. The cultures are fed twice a week by replacing half of the medium (1 ml) with fresh HAT medium.

so D) Detection of interferon-specific hybridomas by a solidphase-antibody binding test (SABA) This method is adapted from the principle described by Catt and Tregear [Science 158,1570-1572 (1967)] and is suitable for the detection of hybridoma antibodies (see also St5hli et al in J. Immunol. Methods 32, 297-304 (1980) as well as Kennet, R.H. in Current Topics in Microbiology and Immunology, Vol. 81, p. 77-91 (1978). Interferon preparations of HLI a-, P, -y and 6 are individually diluted in polypropylene test tubes with 55 phosphate-buffered sodium chloride solution (PBS) to an end concentration of approximately 0.2 to 0.5 Lg interferon/ml (approximately 1-3 Rg total protein per ml; except for HLI 8 where approximately 10-15 Rg of total protein are present per ml), in order to coat therewith polyvinyl chloride microtitre plates (Cooke Laboratory Products Division, Dynatech Laboratories, Inc.) as in the case of St6hli et aL loc. cit.. 50 0 of the interferon solution is placed in each well and left for at least 4 hours at room temperature in a humid chamber or held at (sic) 4C for several weeks. Before use the wells are filled with 3% bovine serum albumin (BSA) in PBS and leftfor 30 to 60 minutes in orderto block all protein binding positions. The plates are then washed fourtimes with PBS. 50 0 of supernatant of the hybridoma cultures are incubated in in each case two wells for at least 4 hours at room temperature. Control experiments (unspecific binding) are carried out on plates which are coated only with 3% BSA. After four-fold washing with PBS, there is added to each well 65 3 GB 2 160 544 A 3 jjI of sheep-anti-mouse Ig-antibody (purified by affinity chromatography and labelled with 1251; 50,000 to 100 000 cpm per 20 to 30 ng of antibody in PBS containing 1 % BSA) and it is incubated at room temperature for at least 4 hours. The plates are washed four times to five times with PBS and then divided into the individual wells, which are then tested for radioactivity in a gamma-scintillation spectrometer E) Results a) Initial screening by antibody binding (SABA) againstpartia/typurified-HU fractions 152 of the 240 cultures show growth of hybridomas. The supernatants are tested for interferon-specific antibodies in the SABA test, when the cultures are approximately 20-30% confluent (12-29 days after the fusion). Antibody binding is tested parallel with HLI a, HLI 0, HLI -y and HLU 6. Of the 152 cultures, 13 supernatants show high antibody binding against all four partially purified interferon preparations. The medium of four further hybriclomas shows lower binding activity (3-4 times greater than unspecific binding) against all four interferon preparations. These cultures are discarded. The media of all other hybriclomas (135 growing cultures) yield unspecific bindings against all four HLI preparations. Of the 13 specific hybridomas, 9 are characterized representative forthe present invention. Their designation is LI-1, LI-2, LI-3, 15 LI-5, LI-6, LI-7, LI-8, LI-9 and LI-12.

As is evidentfrom Figure 1, the antibody binding of the 9 specific hybridomas shows a different behaviour to the 4 partially purified interferon preparations. LI-1, LI-2, LI-3, LI-5, LI-6, LI-7 and LI-8 show an identical characterization with the following binding sequence: HLI a < HLI 8 < HLI, < HLI P. For LI-9 the binding order isHLIB< HLI-y< HLIP. LI-12 isclifferentfrom all others.

b) Antibody binding (SABA) against highlypurifiedleukocyte interferon Six purified (: 80% pure) interferon species (see step 9 of Table 3 of DOS 29 47 134) are immobilized by adsorption on polyvinyl chloride microtitre plates. In this case there are concerned HLI (1, 02y 13202 andY3 as well as HLI-K of the cell line KG-1 [Koeffler, H.P. et al. Science 200, 1153-1154 (19780)], which is purifed in 25 analogy to the steps 1 to 9 of Table 3 of the mentioned DOS. Since no clear separation into individual species was to be observed in the steps 8 and 9, the main fraction with the interferon activity was pooled. It accordingly represents a mixture of different species. The adsorption of interferon (50 Ll/per depression) is carried out overnight with approximately 0.5 jig of interferon and 2 jig of BSA per ml of PBS followed by blocking off of the protein binding positions with 3% PBS as described previously.

The antibody binding with the culture medium of the 9 specific hybridomas against the 6 different, purified interferons is shown in Figure 2. There can again be differentiated various groups of antibodies with different characteristic binding behaviour. The group 1 embraces the antibodies LI1 and LI-2, which have identical binding characteristics, but which do not recognize HLI'Y3 The group 2 embraces the antibodies LI-3, LI-5, LI-6, LI-7 and LI-8 with a possible differentiation of two sub-groups. Sub-group 2a (LI-3, LI-5 and LI-6) shows 35 overall higher binding (Figure 1 and Figure 2) than sub-group 2b (LI-7 and LI-8), whereas the latter sub-group binds relatively stronger to purified HLI al and HLI K than the first. According to the interferon binding behaviour of the remaining antibodies in accordance with Figure 2 LI-9 has the most characteristics of sub-group 2b. LI-1 2 appears, in turn, to be unique.

Having regard to the fact of the high antibody binding to different purified interferons it is very unlikely that 40 any of these antibodies was not directed against interferon, but against contaminants. This is indicated for most of these antibodies also by the capability of neutralizing the biological activity of interferon. LI-1, LI-2, LI-3, LI-5, LI-6, LI-7, LI-8 and LI-9 annul the inhibition of the viral CPE on MDBK cells induced by interferon.

The interferon neutralization is tested with partially purified HLI -y(a) and/or purified HLI K, HLI-yj, HLI ot2 and crude leucocyte interferon. None of the antibodies was in a position to neutralize crude leucocyte interferon. 45 LI-1 2 neutralized none of the tested interferons (see Table 1 for this).

2) Isotypes of the heavy and light chains of the monoclonal interferon antibodies Monoclonal antibodies of hybridoma culture medium are firstly bound to interferon-coated microtitre plates as has been described previously for the SABA test. After washing, they are incubated with isotype-specific rabbit anti-mouse Ig antiserurn (Nordic). For the detection there is used goat anti-rabbit IgG, which is labelled with horseradish-peroxiclase. As the enzyme substrate there are used 2,2'-azino-di-(3-ethyI benzothiazoline sulphate) and hydrogen peroxide. The results are compiled in Table 1, second column.

Seven of the tested antibodies have the immunoglobulin chainsyl/k, one hasY2b/k and one has I.L/k. All seven -yllk antibodies as well as also theY2b/k neutralize interferon, whereas the R/k does not neutralize interferon, 55 as is likewise evident from Table 1.

The ability of the antibodies according to the present invention to bind with high affinityto interferons obtained by recombinant DNA technology is evident from Table 3.

3) Cloning and stabilization of the interferon-specific hybridomas For the preparation of stable hybridoma lines, all 9 specific hybridomas are cloned by limiting dilution in microtitre plates with mouse-peritoneal cells as the nutritive layer [Hengartner H. et al. in Current Topics in Microbiology and Immunology, Vol. 81,92-99 (1978)1. The cloning was begun at the point in time of the transfer of the original cultures into flasks or shortly thereafter. The clones were tested against HLI P with he SABA test. The results of this first cloning of the 9 hybridomas is shown in Table 2.

4 GB 2 160 544 A From each hybridoma there are intensively cultivated two to four strongly positive clones and not only injected i.p. into mice for ascites production but also frozen. In most cases the selected clones of a hybridoma are pooled for simplification and reduction of work. From all 9 hybridomas there is obtained ascites liquor for the production of antibodies on a large scale. Ascites cells are likewise transferred into 5 cultures and frozen.

4) Grouping of the antibodies according to their interactions with HLI The main group of the seven hybridomas, which produceyl/k immunoglobulin, can be divided into two groups on the basis of their antibody interaction with purified interferons. The first group (LI-1 and LI-2) does not recognize purified HLI _Y3, whereas the second group (LI-3 and LI-5 to LI-8) recognizes HLI"Y3. The relative, quantitative binding of the monoclonal antibodies to six different purified interferons (see Figure 2) and the difference between neutralizing and non-neutralizing antibodies yields information for the differentiation of different epitopes (antigenic determinants) of HLI and forthe demonstration of structural differences between different purified interferons. From the binding behaviour of the antibodies (see Figure 2) it is clear that LI-1 and LI-2 recognize a different structure than all other antibodies and that HLI 'Y3 is structurally different from all other tested interferons, since it does not exhibitthe epitope recognized by LI- 1 and LI-2. LI-12, a non-neutralizing antibody, recognizes with approximately the same, although perhaps lower affinity, a less variable determinant which is present in all tested interferons. The results in respect of the antibody binding and of the interferon neutralization indicate that at least three different epitopes of HLI can be recognized and defined by the collection of the monoclonal antibodies.

EXAMPLE 2 Purification of leucocyte interferon obtained by recombinant DNA technology (interferon A, c.f. Goeddel et al. Nature, vol. 20, March 1981).

a) pre-purification of the human leucocyte interferon obtained by recombinant DNA technology All steps are carried out 4C. Frozen bacteria cells (1 kg) are broken up by pressure disintigration. The extract is worked up according to the standard method, i.e. by polyethyl enei mine (Polymin P) and ammonium sulphate precipitation, followed by a dialysis of the 65% ammonium sulphate precipitate against 25 mM Tris HCI, pH 7.8; 0.01% thiodiglycol; 0.1% Triton X-1 00; 10 gM phenyl fluoride. A centrifugation follows for the removal of insoluble constituents.

4 b) Preparations of the immunoadsorbent Purified antibody solution containing LI-8 is dialyzed at room temperature against 0.2 M sodium carbonate/0.3 M sodium chloride buffer. The dialyzed solution is centrifuged at 20 000 x g for 15 minutes in order to remove insoluble material. The protein concentration is adjusted to about 25 mg/ml with the above-mentioned buffer. Affi-gel 10 (BioRad Laboratories, Richmond, California) is washed three times with ice-cold isopropanol on a sintered glass filter and then washed three times with ice-cold distilled water. The gel is transferred into plastic test tubes and sedimented by brief centrifugation. The supernatant is sucked off. The gel is mixed with an equal volume of purified antibody solution and rotated head overfoot at 40C for 5 hours. After the reaction, the gel is centrifuged and then washed twice with buffer (0. 1 M NaHC03/0.15 M 40 NaCI) in orderto remove unbound antibody. Protein determination of the combined wash-water shows that about 24 mg of antibody were bound per ml of gel.

c) Affinity-chromatographicalpurification of the pre-purifiedleucocyte interferon obtainedby recombinant DNA technology The solution obtained in step a) (700 ml containing 37 g of protein) is placed at 50 ml/h on the immuno-absorbent column (2.5 x 3.5 cm; 17 ml bed volume containing about 400 mg of purified monoclonal antibody LI-8) equilibrated with PBS. The column is washed with 20 column volumes of buffer (0.5 M NaCl; 0.02 M Tris. HCI, pH 7.5; 0.2% Triton X-100), then the column is washed with 5 column volumes of 0.15 M NaCl, 0.1% Triton X-100 and then eluted with 0.2 M acetic acid, 0.15 M NaCl, 0.1% Triton X-1 00 (pH 2.5). 50 The interferon activity is eluted in about 30 ml in an average concentration of approximately 1 mg protein/ml.

This solution is adjusted to i)H 4.5 with 1 M Tris base and diluted fourfold with water. The sample is loaded on to a carboxymethy1cel lu lose (CM 52 Whatman) column (1.3 x 3 cm), which is equilibrated with 0.1 M ammonium acetate (pH 5.0). The column is washed with 20 ml of 0.1 M ammonium acetate (pH 5.0) and the interferon is eluted with 20 ml of 0.5 M ammonium acetate (pH 5.0). The result of the purification is reproduced in Table 4.

GB 2 160 544 A TABLE 1

Epitope Monoclonal Isotypes Chains Neutralization antibody 5 LI-1 -yllk Yes U-2 yl/k Yes LI-3 yl/k Yes LI-5 -yl/k Yes 10 LI-6 -yllk Yes Ii f LI-7 19G ^illk Yes LI-8 -yl/k Yes ,Li-g -L Y2b/k Yes Ill ( LI-12 Igm li/k No 15 TABLE 2

Culture Cells per 96 -wells - Testedclones Number Number or -wellspositive negative LI-1 100 6 6 0 25 LI-2 100 11 6 5 LI-3 100 7 7 0 LI-5 100 13 13 0 LI-6 100 12 12 0 LI-7 100 10 8 2 30 LI-8 100 9 8 1 LI-9 100 10 8 2 LI-12 100 4 0 4 LI-1 2 300 48 6 42 35 TABLE 3

Antibody Interferon Ll- 1 L1-3 L1-5 L1-6 LI-7 L1-8 L1-9 A + + + + + + + B + 45 D + + + + + + F 5 Legend: 50 The interferons designated as A-F are those obtained by the DNA recombinant technology and described in the publication of David V. Goeddel et al. in Nature Vol. 290, March 1981. A + designates that a given interferon was completely absorbed, a -;: partially absorbed and a - not absorbed by the columns loaded with the respective antibody.

6 GB 2 160 544 A 6 TABLE 4

Step Volume TotalProtein Total Specific Purification Yield (M0 (M9) activity activity factor (OW 5 (units) (unitslmg) Ammonium sulphate 700 37,100 7.4 X 109 2 X 105 1.0 100 precipitation Antibody column 30 30 7 x 109 2.3 x 1013 1150 95 10 pool CM-52 20 20 6 x 109 3 x 108 1500 81 The specific activity was determined according to the WE inhibition procedure mentioned earlier. In accordance with sodium dodecyl sulphate-polyacrylamide gel electrophoresis the interferon after the immunoadsorption step is over 90% pure. According to the CM-52 column it appears to be practically homogeneous.

Claims (4)

1. A monoclonal antibody which is directed against natural or recombinant human leukocyte interferon (HLI), characterised in that the antibody is of IgG isotype, it has a -yj heavy chain, and it binds to recombinant HLI Aand D butnotto recombinant HLI Band F.

2. Use of the antibody of claim 1 for the purification of HLI.

3. A process for the purification of HLI characterised in that the HLIcontaining raw material is purified by 25 affinity chromatography using the monoclonal antibody of claim 1.

4. A process for the purification of HLI substantially as hereinbefore described with reference to Example 2.

Printed in the UK for HMSO, D8818935, 11185, 7102. Published by The Patent Office, 25 Southampton Buildings, London, WC2A lAY, from which copies may be obtained.