FI79121B - ANTIKROPPAR MOT LE-FORMIGA MAENNISKOINTERFERONPROTEINERS IMMUNOLOGISKA DETERMINANTER OCH FOERFARANDE FOER PRODUCERING AV DESSA ANTIKROPPAN. - Google Patents

Description

1 79121

Antibodies to immunological determinants of human Le interferon proteins and method for their production

This invention relates to a purified form of human interferon antibodies and to methods of making the same.

In the description and claims of the invention, the term "protein" also includes "glycoprotein".

Many attempts have been made to purify human interferon. The aim of these purification efforts has also been to define the characteristics of interferon species for standardization purposes. To date, however, no attempts to purify Le interferon human interferon have been entirely successful.

The Le form of interferon is defined in: E.A. Havell, B. Berman, C.A. Ogburn, K. Berg, K. Paucker, and J. Vilcek, Proc. Nat. Acad. Sci. USA, 72, 2185-2187 (1975).

According to the invention according to strain application FI-801 215, pure human leukocyte interferon proteins have been prepared from crude human leukocyte interferon through several special purification steps, and pure human leukocyte interferons have been characterized by stained protein bands in the SDS PAGE electrosulphide decileyl

The specific experimental conditions used for the preparation and characterization of pure human leukocyte interferon proteins are described later in "Materials and Methods" and in "Experimental Part 2 79121". The pure human interferon proteins, and in particular the pure Le interferon proteins, made available and characterized by patent application FI-801 215, are the key to the new developments which are aspects of the present invention and which are described in this specification.

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

Several repeated experiments have shown that under the SDS PAGE and staining conditions described in "Materials and Methods" and with a total interferon of 0.9 x 10 ^ IFU, pure human leukocyte interferon shows essentially only two sharp colored protein bands at 18400 ± 200 and 20100 ± 200 Daltons and a smaller colored protein band between 20300 ± 200 and 20400 ± 200 Daltons. As stated in the protein assay described later, the specific activity of pure human leukocyte interferon is about 10 μg IFU / mg protein, but the specific activity obtained may vary somewhat depending on the protein assay method used, and thus the specific activity of the protein is concluded to be between 2x 10 ^ - 2 x 10 ^ IFU / mg protein. The presence of two strong, clearly distinguishable bands in the spectrum of pure interferon is consistent with previous observations using unpurified or partially purified interferon preparations, indicating that human leukocyte interferon comprises at least two major species. With a higher total amount of interferon, e.g., 3.8 x 10 10 IFU, the aforementioned SDS PAGE system has been found to be able to present a more differentiated protein pattern comprising six interferon protein bands, namely two strongly stained bands at 18410 ± 200 Dalton and 20180 ± 200 Daltons, at 20420 ± 200 Daltons on the medium-colored strip (corresponding to the minimum

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3 79121 dyed ribbons) and barely visible protein bands at 19500 ± 200, 21130 ± 200 and 23440 ± 200 Dalton. The individual components of each SDS PAGE acrylic gradient gel in the above bands have been shown to show biological interferon activities: antiviral activity, the ability to neutralize only non-human leukocyte interferon (but not the corresponding fibro-blast interferon), and cellular interferon activity. . virus-free activities, such as strengthening "Natural Killer" cells, strengthening MLC-CML, adding HLA antigens, etc.

Complete purification of interferon proteins makes it possible, for the first time, to produce an anti-interferon that is precisely specific for active interferon species simply by immunizing animals with the interferon preparation alone or one or more of its components. Such a highly monospecific anti-interferon is very useful in antibody affinity chromatography for the purification of unpurified or partially purified interferon, so as to provide large amounts of pure or highly purified interferon in a simple and economical manner for clinical purposes, standardization, chemical studies and serial studies, for the repetition of monospecific anti-interferon.

Monospecific anti-interferon is also useful in forming a genetic building system for interferon protein production in a manner known per se: according to known methods in genetic engineering, the first step is to isolate RNA-Messenger from interferon-producing cells in which interferon synthesis is initiated by interferon-inducing protein. the degree to which the immunological determinants (or portions thereof) of the interferon are expressed, while the interferon remains bound to the ribosomes and the RNA-Messenger. The most suitable interferon-producing cells are Namalva cell suspension, which produces a large number of clones, grown conventionally or in a so-called "Buffy coats" (i.e., lymphocytes isolated by the Ficoll method). The RNA messenger is isolated from these cells by dissolving the cells in a manner known per se and passing the solvent through an antibody affinity column in which the covalently bound antibody is a monospecific anti-interferon. The antibody column selectively retains not only the interferon but also the RNA-Messenger bound to it. By known methods, such as salt elution, the RNA messenger is isolated from the column eluate and treated according to known methods by a "reverse transcriptase" procedure to generate the corresponding DNA. Alternatively, immunoprecipitation methods known per se can be used, possibly in combination with double immunoprecipitation methods. According to methods known in the art of genetic engineering, such DNA encoded for interferon or important parts thereof is ligated into a suitable cloning vector, preferably a mini-plasmid, and transformed into a microorganism whose culture produces interferon and / or interferon derivatives which are released into the culture medium from which interferon is obtained. Purification of such an interferon obtained by culturing the microorganism can be suitably carried out in the same manner as described above, namely by passing the crude preparation through an antibody affinity column prepared with monospecific anti-interferon. A radiolabeled monospecific anti-interferon can be an important tool in determining which clones of a microorganism have received DNA and are capable of producing interferon or portions or derivatives thereof.

At an interferon supply of 0.9 x 10 ^UU, pure human white cell interferon proteins are present in the above

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5,79,121 stacked as three separate protein bands on an SDS PAGE acrylamide gradient gel along with five or six biological peaks. Whether five or six biological bands are present depends on the intersection of the gel slice. Figure 1 shows a stained SDS PAGE gradient gel plate prepared with this interferon feed as described in "Materials and Methods". Each of the protein bands has been shown to have clear interferon activity. Figure 2 is a drawing of a second experiment with an SDS plate with the same amount of interferon, Figure 2 also showing the interferon activity profile associated with the bands, determined as described later in "Materials and Methods". Five biological interferon peaks are seen along with three distinct colored proteins. It is clear from Figure 2 that the protein bands coincide exactly at the peaks of interferon activity. This indicates that the proteins are interferon proteins. It is important to note that the interferon activity profile will, of course, depend on the exact position of the individual gel slices. In Figure 2, the interferon activity of the smaller band at 20410 ± 200 Dalton is not very clear, but experiments with the same interferon feed have shown that this smaller band has interferon activity, and experiments with the larger feed (see description below) have shown that the lower band has clear interferon activity. The amount of interferon activity obtained from the SDS PAGE system observed in the corresponding interferon protein slices corresponds linearly to the amount of protein determined from the intensity of staining of the protein bands. Thus, the clear existence of two larger interferon bands and a smaller band has been proven in the experiments described in Figures 1 and 2. In experiments where the interferon feed was higher, the more detailed band pattern mentioned above has been shown, as shown in Figure 3 (six interferon proteins together with six biological peaks 6 79121 determined after staining and decolorization). Because it is known that SDS-treated interferon retains its immunological determinants, it even more clearly expresses (or maintains) its antigenicity than SDS-untreated interferon (as demonstrated by immunizing mice with human leukocyte interferon-la Paucker et al. [Dalton, BF, Ogburn, CA, Paucker, K., Production of antibodies to human interferons in mice Infect Immun. 19 (2), 570-574 (1978), pp. 4; 25-30]), preparative SDS PAGE allows not only the intake of each component in isolated form, but also to perform immunization with isolated components, as described below.

Pure interferon proteins can preferably be expressed as Le-form human interferon proteins, which under the SDS PAGE and staining conditions defined in this specification, with a total interferon feed of 0.9 x 10 10 IFU, reveal two major sharp colored, antiviral interferons. protein band at 18400 and 20400 Dalton and a smaller colored band with antiviral interferon activity at 20300-20400 Dalton along with smaller peaks with antiviral interferon activity at 19500, 21130 and 23440 Digron ( without showing substantially any other colored protein regions; or human Le interferon proteins which, under the SDS PAGE and staining conditions defined in this specification, with a total interferon feed of 3.8 x 10 6 IFU, produce six colored protein bands with antiviral interferon activity, namely, strong bands at 10 sites. 20180 Dalton, medium band at 20420 Dalton and barely visible bands at 19500, 21130 and 23440 Dalton (these Dalton molecular weights have a test accuracy of ± 200 Daltons), these viral

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7 79121 peaks of interferon activity accurately at the colored protein bands, with an SDS PAGE acrylamide gradient showing essentially no other colored protein regions.

It is important to note that the private components in the aforementioned SDS PAGE gel strips show biological interferon activity, the ability to neutralize non-human leukocyte interferon, and anti-cellular activity, etc.

A particularly useful preparation of pure interferon proteins is an aqueous solution. When the stabilized preparation is an aqueous solution of pure Le interferon protein of Form Le or such proteins for use in immunizing animals for the production of monospecific anti-interferon, stabilization of SDS (sodium dodecyl sulfate) to form pure human interferon protein of Form Le or such proteins preferred, given the above-mentioned fact that SDS increases the antigenicity and / or stability of interferon.

The pH of a solution of pure interferon protein for the immunization of animals for the production of monospecific anti-interferon is preferably about 7.2, with PBS (phosphate buffered saline) as a suitable buffer.

The stabilized preparation of pure interferon protein for immunization of animals may further comprise an adjuvant to further increase its antigenicity, the suitable adjuvant being a Freund's adjuvant.

Also within the scope of the invention is the increase and / or stabilization of the antigenicity of pure interferon proteins of the Le form or of each individual protein by incorporation into an immunogenic carrier (such that the pure interferon protein or such proteins will exist as a kind of "hapten") according to known principles. Examples of immunogenic carriers include PPD (Purified Protein Derivative) and BCG (Bacille Calmette Guärin). However, the use of such immunogenic carriers is not currently considered desirable.

For immunization purposes, mice, rabbits, goats and sheep are the most suitable animals, but other animals are within the scope of the invention, and as described below, porcine IgG globulins have been shown to have special advantages for certain purposes.

Immunization of animals against pure interferon is in principle carried out in accordance with known methods for the preparation of anti-interferon, such as those described, for example, in Acta Path. Microbiol. Scand. Section B, 83, 443-460 (1975), but the fact that the interferon proteins of the invention are pure causes minor changes in immunogen concentration, immunization time, and immunization intervals. Examples of immunization programs are provided in the experimental section of this specification.

Blood collection from animals and isolation of antiserum are performed according to known methods.

The antibodies prepared as described above, except for the insignificant fact that they show a characteristic background of the immunized animal, are essentially specific for the interferon proteins described by the aforementioned SDS PAGE bands. A very small amount of impurities that are not seen as colored bands on the SDS-PAGE system together with the interferon protein cannot be eliminated. Such proteins, which may be present in small amounts corresponding to about 1-5% of the total protein content in pure interferon protein,

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9 79-21 in the iin preparation, may potentially release antibodies against the corresponding impurities. One way to control this possibility is to construct an anti-interferon column from the antiserum in question prepared by immunizing a rabbit with pure interferon (i.e., an interferon having the characteristics described above that provides visible interferon protein bands in the SDS PAGE system). IFU). The column is constructed without any absorption. Crude human leukocyte interferon is loaded onto a column, and normal antibody affinity chromatography is performed as described later. The eluate is analyzed on an SDS PAGE system (see below), in which case only the interferon bands should be visible, possibly together with 1-4 other proteins (impurities). This (3 proteins) was in fact observed in rabbit antiserum at a titer of 500,000 IFU-NU / ml on a 2 ml column and at a feed rate of 2-3 x 10 6 IFU of crude human white cell interferons.

"Foreign" proteins can also be caused by a simple spontaneous cross-reaction that occurs between them.

The above method for controlling whether certain antibodies are monospecific is believed to be novel in itself and constitutes an aspect of the present invention. This aspect is a method for controlling whether a particular antibody preparation (e.g., antiserum) is monospecific for a particular antigen, the aspect comprising constructing an antibody affinity chromatography column with a controlled antibody preparation, feeding the solution containing the antigen and impurities to the column, and feeding the column to the column. to indicate the presence of a protein other than an antigen. This assay is preferably performed on a 10 791 21 SDS PAGE gradient in the same manner as described for the method used to determine anti-interferon monospecificity, and the presence of bands corresponding to up to four impurity proteins in the eluate is generally considered a satisfactory indication of monospecific antibody. for most uses.

Since the colored interferon protein bands in the SDS PAGE system have fully or substantially retained their antigenicity, it is also possible to use the colored interferon proteins immediately removed from the SDS PAGE system as antigen preparations for immunizing animals that can be immunized, such as rabbits. When a colored strip cut from an SDS PAGE system is used for immunization (after the preparation described below), it is less likely that a possible cross-reaction (or contamination by very small amounts of impurities) would occur, as mentioned above, (compared to the whole eluate corresponding to 5 interferon species). Antibodies with optimal specificity against private interferon species (primarily two sites against the corresponding major species of about 18400 and 20100 Dalton) can be prepared according to the following protocol: 1. 1.4-5 x 10 ^ IFU human white cell interferon (such as CIF) is completely purified (by tandem affinity chromatography described below) and subjected to SDS PAGE.

2. The gel is stained for only 10-15 minutes at room temperature, and the decolorization is performed partially for 10 minutes, followed by rinsing with distilled water 3 times for 1-2 minutes at 0 ° C. The exact location of the protein bands is recorded (e.g., by polaroid photography, and the two major species of interferon proteins are specifically removed by cutting with a sharp knife. Each slice is cut with a Teflon stick into 1 ml of 0.01% SDS solution (PBSr). pH 7.2) and injected subcutaneously into the rabbit.When this procedure is performed every other week, low titer antibodies against human white blood cell interferon proteins develop within 2-4 months.As soon as a low titer against interferon is detected, Freund’s is added to the immunogenic mixture. adjuvant every four weeks (every four to six weeks) depending on the development of titers, this procedure is continued for 3 to 12 months and anti-interferon anti-interferon species develop (10,000 to 1,000,000 IFU-NU / ml). "Monospecific anti-interferon" is used for both anti-interferon produced by pure interferon proteins, as described above. it has been described, without the SDS PAGE excision step, that of antibodies raised against the colored interferon band or such bands that have been excised from the SDS PAGE system.

Another method for producing monospecific antibodies against interferon proteins is the so-called hybridoma domitekniikka. The hybridoma technique is a known method for producing antibodies and involves the production of monoclonal antibody-producing lymphocyte / myeloma hybrids (cf. e.g., Current Topics in Microbiology and Immunology, Vol. 81, Lymphocyte Hybridomas, Eds. F Melchors, M. Potter and NL Warner, Springer Verlag, 1978). Prior to this invention, however, it was not known or apparent that it would be possible to provide an anti-interferon-producing hybridoma cell clone. In a hybridoma method, e.g., using a mouse as an immunizing animal, mice are immunized with human interferon Form Le, and spleen cells from immunized mice are fused to myeloma cells, after which the fused hybridomas are cloned, antibody-producing clones are selected, and cultured. antibodies are obtained from the culture medium.

12 79121

Antibodies prepared by the hybridoma method in a mouse system are completely monospecific and therefore highly preferred in radioactive immunoassays or other similar experiments.

In the hybridoma method, one way to produce an antibody is to culture selected clones in vivo in the animal species from which the spleen cells are derived and to collect the antibody from the aqueous humor of the animal, this embodiment being within the scope of this invention.

Selection of positive hybridoma clones can be performed by a conventional interferon neutralization assay. However, because a conventional interferon neutralization assay requires that the interferon antigenic determinant be located very close to the center or centers of biological activity (within a distance of about 1 IgG molecular length), it is likely that antigenic determinants located farther from the center or centers of biological activity or activities. are not detected in this experiment, and thus it is likely that "positive" hybridomic clones (which produce antibodies against antigenic determinants of the interferon protein located farther from the biological center than the length of one IgG molecule) will not be detected in the experiment. A more preferred method for testing positive hybridoma clones is therefore to use the radiolabeled pure human interferon proteins of the invention of the invention in a radioimmunoassay. Pure radiolabeled human Le interferon proteins can be prepared by radiolabeling Le human interferon, e.g., a gel filtrate prepared by the gel filtration method described below using a standard method, such as using lactoperoxidase and iodine 135 and then purifying the interferon proteins herein. as described in U.S. Pat. No. 7,712,121, subjecting the purified interferon proteins to an SDS PAGE system and eluting the radiolabeled pure interferon proteins from the SDS PAGE gel. Another method for selecting positive hybridoma clones in a manner that detects clones that are not detected in a conventional interferon neutralization assay involves subjecting a certain amount, e.g., 500 μΐ, of fluid from each clone culture to immobilization from the matrix, e.g., immobilization in CNBr-activated Sepharose. Materials and Methods "according to the method described, by introducing a Le form interferon, e.g. crude human leukocyte interferon, into the resulting treated matrix, e.g. by mixing a matrix gel suspension corresponding to each clone with interferon and allowing the mixture to stand for a period of time, e.g. 37 ° C, by separating unbound interferon from the matrix material, e.g. by centrifugation and washing with PBS, and then eluting each batch of matrix gel to release all bound interferon, e.g. by mixing with elution buffer (pH 2.4) and centrifuging, and selecting clones corresponding to those matrix gels from which the elution buffer batches, especially the last elution buffer batches, contain interferon, because the release of interferon during elution is indicative of a positive clone. The above-mentioned preferred methods for detecting positive hybridomic clones can be applied not only to anti-interferon-producing hybridomiclones but, with certain modifications, also to the detection of positive hybridomiclones that produce an antibody directed against other proteins.

According to known principles, the monospecific anti-interferon of the invention can be used for the determination of the corresponding interferon or interferon component in biological fluids, such as radioimmunoassay or the like. However, as mentioned above, monospecific antibodies have an interesting and important utility in the purification of interferon-containing solutions in antibody affinity chromatography. For this purpose, the antibodies are immobilized in a matrix in a known manner, preferably covalently bound to a suitable antibody affinity chromatography matrix, such as cross-linked agarose, e.g. Sepharose 4B. Purification of interferon-containing solutions by antibody affinity chromatography can be performed according to any known method, either highly or, preferably, using matrix-immobilized antibody applied to the column.

Because interferon preparations that are applied to monospecific anti-interferon columns usually contain interferon at very low concentrations by weight and because it is necessary to isolate as much valuable interferon as possible, it is important to minimize the potential degradation of interferon proteins that may result from with which the interferon comes into contact, and one aspect of the present invention involves the removal of all proteolytic activity from each biological agent with which the interferon to be purified is in contact.

An important benefit of this aspect is the removal of proteolytic activity according to the invention from anti-interferon antibodies (immunoglobulins). According to the invention, this removal is preferably performed by treating the antibodies with a matrix-immobilized enzyme inhibitor or enzyme degrader that does not damage the immunoglobulins (or important fragments thereof) prior to binding to the matrix. Thus, the antibodies can be passed through a column containing matrix-immobilized poly-L-lysine and / or matrix-immobilized

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is 79121

Soyabean Trypsin inhibitor and / or matrix-immobilized kallikrein inactivator. An example of a suitable antibody treatment is transport through a column containing covalently bound poly-L-lysine to a cross-linked agarose such as Sepharose 4B, followed by transport to the same matrix through a column containing covalently bound Soyabean-Trypsin inhibitor. It has been found that such removal of proteolytic activity increases the yield of interferon activity upon purification of interferon-containing solutions by antibody affinity chromatography.

When monospecific anti-interferon is covalently bound to a matrix such as cross-linked agarose, it is preferably bound in an amount such that the total amount of antibody covalently bound to the matrix will not exceed 85% of the amount of immunoglobulins used in the covalent binding step as described in this patent application. J. Immunolog., 6, 77-86 (1977). This provides the highest possible interferon yield from the column.

When an eluate from an affinity chromatography column containing a monospecific anti-interferon is to be used for administration to humans, it is important that the eluate does not contain any component that could be immunogenic in humans. The risk associated with antibody affinity chromatography could be that immunoglobulins or fragments thereof are released from the column and co-eluted with the desired protein or proteins.

Such immunoglobulins or fragments thereof which are immunogenic in humans are removed by passing the eluate through an antibody affinity column in which the antibodies are directed against anti-interferon immunoglobulins and are of a species which is not immunogenic parenterally in humans. When diluting. (Before passing the eluate through the column, it must be adjusted to a neutral pH, eg by dialysis against PBS, pH 7.2).

Immunoglobulins that are non-immunogenic when administered parenterally to humans are primate immunoglobulins, but the intake of primate immunoglobulins directed against the immunoglobulins of an animal used to produce a monospecific anti-interferon may be legal or ethical. limited or completely impossible for some reasons. It is therefore important to note that porcine IgG immunoglobulins have been found to be non-immunogenic in humans, as described in U.S. Patent 4,132,769. Antibodies produced in the human hybridoma system, when available, provide an interesting alternative.

According to the invention, the removal of all or part of the anti-interferon immunoglobulin from the anti-interferon affinity chromatography eluate is preferably carried out by passing the eluate (after adjusting the pH to neutral) through a matrix-immobilized porcine IgG column, porcine IgG against anti-interferon immunoglobulins.

Porcine IgG immunoglobulins directed against anti-interferon immunoglobulins can be prepared in a generally known manner by immunizing porcine anti-interferon immunoglobulin-producing animal species with immunoglobulins and isolating the porcine IgG fraction from the porcine antiserum selected from the above-described 132 methods. in accordance with

Other general aspects related to this aspect include the use of porcine IgG immunoglobulins as immunoglobulins in antibody affinity chromatography using matrix-immobilized antibodies, matrix-immobilized

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Porcine IgG immunoglobulins for antibody affinity chromatography and a method for purifying proteinaceous solutions for parenteral administration to humans by antibody affinity chromatography using matrix immunoglobulin immobilized on antibody affinity chromatography. These general aspects, their applicability and their practical embodiments will become apparent from the above description.

As described in the experimental section of this specification, the eluate from the affinity chromatography column was subjected to final purification in the initial experiments leading to pure interferon by passing it through an absorbed antibody affinity column in which the antibodies are immunoglobulin-purified from which antibodies against contaminating proteins have since been removed by passing it several times through columns containing matrix-immobilized crude human leukocyte interferon. As will be explained in more detail below, covalent binding of crude interferon to a matrix (such as Sepharose 4B) eliminates the immunological determinants of the interferon itself (> 98%), but apparently not the determinants of most impurities, and this means that when partially purified interferon interferon the immunoglobulins produced are transported (normally several times) through the column, the anti-impurities in them becoming trapped on the column as the anti-interferon passes through the column. This multi-absorbed anti-interferon was used in the antibody affinity chromatography step following affinity chromatography.

As can be seen from the experimental part, the preferred method using affinity columns, i.e. a Blue Dextran-Sepharose column and an antibody affinity column, ie 79121, is to combine the two columns so that the eluate leaving the Blue Dextran column is fed simultaneously to the antibody affinity column. This avoids any losses that might otherwise occur if the eluate fractions from the Blue Dextran column were treated separately.

Materials and methods

Interferon assays were performed according to a known standard method [Berg K., Sequential Antibody Affinity Chromatography of Human Leukocyte Interferon, Scand. J. Immunol. 6, 77-86 (1977)] using VERO cells (monkey kidney cells) and Vesicular Stomatitis virus (VSV) as a challenge virus. All interferon units (IFU) are reported in International Standard Units (69/19 B units) (69/19 B standard was obtained from MRC, Mill Hill, England).

Interferon. Crude human leukocyte interferon was prepared according to the method of Cantell (Cantell, K., Hirvonen, S., Mogensen, KE and Py-hälä, L., Human leukocyte Interferon: production, purification, stability and animal experiments. In: The Production and Use of Interferon for the Treatment and Prevention of Human Virus Infections, pp. 35-38, Way-mouth, C. (ed.), Proceedings of a Tissue Culture Association Workshop held at Lake Placid, 1973 (In Vitro Monograph , volume 3) .Tissue Culture Association, Rockville Md.) using Sendai virus as an interferon producer. Partially purified interferon (PIF) with a specific activity of 5 x 10 10 IFU / mg protein was prepared from crude concentrated human leukocyte interferon (CIF) by doping with ethanol as described in: Cantell; K., Hirvonen, S., Mogensen, K.E. and Pyhälä, L., loc. cit.

Unpurified Namalva interferon was prepared essentially as described in: 19,791 21

Strader et al., Production of human lymphoblastoid Interferon, J. Clin. Microbiol. 1, 116-124 (1975), using Sendai virus as interferon producers.

Neutralization of interferon to determine anti-interferon was performed in a microassay system as follows: 20,000 VERO cells per well were seeded in 100 μl of medium and maintained in a humidification chamber containing 5% CO 2 in the atmosphere. On the second day, the medium was removed from the cells, and 100 μΐ (with medium) diluted antiserum containing 6-8 IFU / ml (with serum and interferon pre-incubated at 37 ° C for 1 hour) was added to each well. On the third day, the medium was removed and 100 μΐ of virus VSV (diluted to 10-3.5 in medium) was added to all wells. On day 4, CPE (cytopathogenic effect) was determined, and 50% degradation was considered the endpoint of the anti-interferon titer assay. Titers are expressed in Interferon Neutralization Units (IFU-NU) per milliliter.

A non-monospecific anti-interferon against PIF was prepared according to Mogensen, K.E., Pyhälä, Liisa and Cantell, K., Acta path, Microbiol. Scand. Sect B, 83, 443-450 (1975), partly in sheep and partly in rabbits. The ovine anti-interferon titer was 100-250000 IFU-NU / ml. To prepare rabbit anti-interferon, PIF (2 x 10 ^ IFU) was injected subcutaneously into the rabbit weekly for more than 2 years. The rabbit anti-interferon titer ranged from 15,000 to 30,000 IFU-NU / ml. All immunoglobulins were isolated by mixing with 50% ammonium sulfate solution and then dialysis against phosphate buffered saline (PBS) (pH 7.2).

Chemicals. CNBr was from Fluka (stored at -20 ° C). Sodium dodecyl sulfate (SDS), for high purity electrophoresis, was purchased through British Drug House (BDH). Soyabean Trypsin Inhibitor (STI) and L-lysine were purchased from Sigma. Sepharose 4B, CNBr-activated Sepharose 4B, CH-activated Sepharose 4B and epoxy-activated Sepharose 6B were all from Pharmacia (Denmark).

Binding method. Covalent binding of immunoglobulins to Sepharose 4B was performed as previously described in K. Berg, Scand. J. Immunolog., 6, 77-86 (1977). Only 80-85% of the immunoglobulins were intentionally bound.

Protein assays were performed using a modification of the Lowry method [Berg. K., Sequential Antibody Affinity Chromatography of Human Leukocyte Interferon, Scand. J. Immunolog., 6, 77-86 (1977)], which makes it possible to detect 1-2 pg / ml as the lowest level of detectable proteins (using the "LKB Calculation Absorptioner Ultralab System"). Crystalline bovine serum albumin was used as the standard protein. The following procedure was used to determine the protein content of purified interferon (1-5 pg in total): SDS was added to 0.1%. The lyophilized protein assay was further examined by SDS-polyacrylamide electrophoresis (SDS PAGE, see below) after dialysis against distilled water. The intensities of the stained protein bands were compared to known standards in various amounts (compare SDS PAGE below), and total amounts of proteins were estimated. The deviations ranged from 5 to 10% and the lowest detectable protein level was 0.1 pg (overall). The results of this method are approximate estimates and not from an accurate measurement.

Affinity chromatographies were performed at 4 ° C. The gel suspensions were degassed before being packed into columns. The packaging was performed by rinsing with 3-5 times the bed volume of the filling buffer using a peristaltic pump. Experiments (100 pi)

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2i 79121 for interferon titrations were taken from either whole batches or private fractions and titrated on the same day or frozen in plastic tubes (-20 ° C) and titrated later. Dilutions were performed in medium containing 10% calf serum.

Antibody affinity chromatography was performed essentially as described in Berg, K., Sequential Antibody Affinity Chromatography of Human Leukocyte Interferon, Scand. J. Immunol., 6, 77-86 (1977). 0.1 M NaOA / 0.3 M NaCl, pH 7.2 (flow rate 40 ml / h) was used as feed buffer. Stepwise elution was performed with a solution of 0.1 M HOAc / 0.3 M NaCl with the addition of a small amount of citric acid (sufficient to maintain a pH of 2.4). When not in use, the column was stored at 4 ° C in 1 M NaCl in PBS containing penicillin, streptomycin, gentamicin and chloramphenicol (1% each). Before using the column for purification purposes, it was first rinsed with 100 ml of feed buffer and then with 10 ml of elution buffer, and finally equilibrated with 2-30 ml of feed buffer. This rinsing step was necessary to avoid "spontaneous" proteins, especially for interferons with specific activities greater than 10 IFU / mg protein. The plastic tubes used to collect the interferon eluate were pre-wetted with a solution of 100 μΐ of 1% SDS.

SDS PAGE. Purified concentrated interferon preparations were analyzed for polypeptide components on SDS PAGE plate gels using 20 cm long and 0.75 mm thick separation gels (Bio Rad type 221: double vertical plate gel electrophoresis cell) and a 7-10 cm long layer gel. Exponential gradient gels containing about 9-22% polyacrylamide were prepared by mixing 11 ml of a 22% acrylamide solution and about 32 ml of a 9% solution in a simple ice-cooled gradient apparatus, as described in Knight, E., Interfer. Purification and initial characterization from human diploid cells, Proc. Natn. Acad. Sci. USA 73, 520-523 (1976). The discontinuous buffer system described in Laenomli, U.K., Cleavage of Structural Proteins During Assembly of the Head of Bacteriophage T4, Nature 227, 680-685 (1970) was used. The gel was precooled for 2 hours (10eC) before starting the actual electrophoresis. Electrophoresis was continued overnight (10eC) at constant power (LKB power supply) with an initial current of 10 mA (and a voltage of about 20 V). The assays to be analyzed were dissolved (or diluted) in 0.1 M Tris, HCl (pH 6.8) containing 2.5% SDS and 5% glucose and trace dye (assay buffer). The gel was stained with Coomassie Blue (1.25 mg / ml mixture containing 50% methanol, 40% water and 10% acetic acid) without prior fixation for 15 minutes at room temperature with constant shaking, and the color was removed in 7% acetic acid. (5% methanol). The gels were dried on good quality paper [e.g. Whatman Chromatographic paper (17 mm)] on under vacuum heating using a gel dryer (Bio Rad, gel slab dryer, model 224). Solutions of five different molecular markers containing 0.1 Ι-ΙΟ pg of each marker per 20 μl - labeled lactalbumin (14400 Dalton), Soyabean Trypsin Inhibit (20100 Dalton), Carbonic Anhydrase (30,000 Dalton), Dalbumin (43,000 , bovine serum albumin (67,000 Daltons) and phosphorylase [obtained as an electrophoretic calibration device (Pharmacia, Denmark)] - were subjected to SDS PAGE and stained. It should be noted that the experimental accuracy of the molecular weights determined in this way is about ± 200 Daltons. The stained protein bands were compared to the corresponding bands obtained from a parallel SDS PAGE experiment with a purified interferon preparation, and the total interferon protein content was evaluated. To obtain a biological profile from the SDS PAGE assay, the portion of the gel used for the interferon assay was

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23,791 21 were covered separately from the rest of the gel and kept on a glass plate at 4 ° C (humidity chamber). Most of the gel was stained for 15 minutes, and after decolorization for 3-5 minutes, clearly weak bands were seen on a blue background, allowing the exact location of the protein bands corresponding to 14,000 and 30,000 to be determined. The unstained portion of the gel was cut off to contain proteins only between 14,000 and 30,000 Daltons and further divided into 1 mm thick slices with sharp knives. Interferon was eluted from these slices in 0.5 ml of 0.1 M SDS solution after complete reduction with a Teflon rod. After the experiment was kept for 5 hours at room temperature (shaking), the interferon activity of the liquid was determined. The private fractions were frozen at -20 ° C without additives.

Experimental part

Preparation of pure human leukocyte and lymphoblastoid interferon proteins

Concentration of crude human white blood cell interferon.

To 3 liters of crude human white cell interferon was added to a KSCN concentration of 0.5 M at pH 7.2. The pH was lowered to 4.5 by the addition of 1 N HCl solution (magnetic stirring) to give a protein precipitate containing interferon (and some impurities). The precipitate was dissolved in 150 ml of PBS (phosphate buffered saline, pH 7.2) containing 1 M NaCl and 25% v / v ethylene glycol and subjected to thorough dialysis 3 times against a 2 liter batch of the same buffer at 4 ° C. The specific activity of crude concentrated human leukocyte interferon (HuLeCIF) was 5-10 x 10 ^ IFU / mg protein. The yield was about 98%.

Concentration of crude Namalva interferon.

To 1 liter of crude Namalva interferon with a titer of about 8000 IFU / ml was added KSCN

24,791 21 up to a concentration of 0.5 M at pH 7.2. The pH was lowered to 4.5 by the addition of 1 N HCl solution (magnetic stirring) to give a protein precipitate containing interferon (and some of the impurities). The precipitate was dissolved in 50 ml of PBS solution (pH 7.2) containing 1 M NaCl solution and 25% by volume of ethylene glycol, and thorough dialysis was performed 3 times against a 2-liter batch of the same buffer at 4 ° C. The specific activity of crude concentrated Namalva interferon (Na-CIF) was 10-12 x 10 ^ IFU / mg protein. The yield was about 98%.

Gel filtration. A 100 cm long column (diameter 2.6 cm, Pharmacia K 2.6 / 100) was packaged with Ultrogel AcA 5/4 (LKB Denmark) in PBS containing 1 M NaCl and 25% v / v ethylene glycol at 4 ° C ( pH 7.2). After rinsing the column with 3 μl of bed volume buffer, the column was stabilized. 10-15 ml of HuLeCIF (prepared as above in 25% v / v ethylene glycol, 1 M NaCl in PBS, pH 7.4) was charged to the column, and the column was "eluted" with feed buffer, followed by determination of the interferon activity of the fractions. The interferon-containing fractions were pooled, and approximately 95% of the original interferon activity was recovered. The specific activity of the gel-filtered human leukocyte interferon-containing eluates was nearly 1,000,000 IFU / mg protein, corresponding to a purification factor of 200. As determined by molecular markers, the molecular weight of interferon corresponds to the range of 10,000-20,000 Daltons. Titration of the private fractions revealed only one broad peak, with a maximum at 18,000 Daltons.

In the same manner as described above, 10 ml of NaCl (prepared as above in 25% v / v ethylene glycol, 1 M NaCl in PBS, pH 7.4) were charged to the column, and "elution" was performed as above. The yield was about 90%. The specific activity of the gel-filtered eluate containing Namalva interferon was almost 1,000,000 IFU / mg protein, which corresponds to a purification factor of 100. As determined by molecular markers, the molecular weight of the interferon corresponds to the range of 10,000-20,000 Daltons. Titration of the private fractions revealed only one broad peak, with a maximum at 18,000 Daltons.

The gel filtration curves obtained from the gel filtration of HuLeCIF and NaCIF described above are shown in Figures 5 and 6, respectively, where "HULEIF" means human white cell interferon and "NALYIF" means Namalva (lymphoblastoid) interferon. It is clearly seen that interferon activity is effectively differentiated from the majority of proteins.

Blue Dextran chromatography. The gel-filtered human white cell interferon solution, prepared as described above, was thoroughly dialyzed against 200 volumes of a solution of 20 mM PB (pH 7.2). Dialysis was performed twice, with a total dialysis time of about 24 hours. The dialyzed solution (25 ml, containing 1.8 x 10 ^ IFU) was applied to a Blue Dextran-Sepharose 4B column. The column was 1 cm in diameter and 10 cm long. The column was prewashed with 200-300 ml of a solution of 20 mM PB (phosphate buffer, pH 7.4). The dialyzed interferon preparation was fed to an equilibrated column, and the column was rinsed with 75 ml of PB solution. 4500 IFU was found in the rinsing fluid. The column was eluted with 0.6 M NaCl, 20 mM PB (pH 7.2) to recover more than 95% of the interferon activity in 6 ml of eluate, as determined by interferon titration.

In exactly the same manner, the above-mentioned gel-filtered Namalva interferon solution was thoroughly dialyzed and then subjected to Blue Dextran chromatography. The input for Blue Dextran chromatography was 1.6 million IFU. 50 ml of rinsing fluid comprised 70,000 IFU. The eluate was formed by adding 0.6 M NaCl to PB-26,791 21 solution (pH 7.2). Blue Dextran chromatography of Namalva interferon is shown in Figure 7. The fibroblast portion of Namalva interferon did not elute from the column under these conditions, but is expected to elute using e.g. 25% ethylene glycol in 1 M NaCl (pH 7.2).

The above-mentioned Blue Dextran column was a column containing Blue Dextran [Cibacron Blue F3GA immobilized on Dextran 2000 (molecular weight 2 million)] coupled to cyanogen bromide-activated agarose (Sepharose 4B). Thus, the more complete designation of the column is Blue Dextran-Sepharose 4B. A column of this type is described in Bollin et al., Loc. cit. After elution, the column was purified by eluting with a solution containing 20-30 ml of 25% ethylene glycol and 1.5 M NaCl in 20 mM PB. The column was stored in this buffer at 4 ° C when not in use. As mentioned above, the feed conditions may also involve the use of hydrophobic reagents such as alcohols in varying amounts (0-50%).

0.6 M NaCl eluates from Blue Dextran chromatography show specific activity of 70 x 10 10 IFU / mg protein for both human white blood cell interferon and Namalva interferon. Thus, they are both suitable for parenteral administration to humans for therapeutic purposes and are in this respect much purer preparations than the commonly used PIF preparations. For such use, the eluates are stabilized as described above, e.g. with 1% human albumin.

The eluates from the Blue Dextran column are immediately transferred to an antibody affinity chromatography column for further purification and preparation of pure interferon. In the most preferred embodiment, the antibody affinity chromatography column is combined 27 7 91 21

With the Blue Dextran column as a "tandem system", as described below:

Tandem-affiniteettlkromatografia. Instead of eluting the Blue Dextran column described above, the Blue Dextran column is combined with the equilibrated antibody column prior to elution by connecting the outlet tube of the Blue Dextran column to the inlet tube of the antibody column. In this way, the eluate leaving the Blue Dextran column is immediately passed to the antibody column. This combination takes advantage of the fact that elution conditions (0.6 M NaCl, 20 mM PB pH 7.2) can also be used as antibody column feed conditions. After elution / feeding using 20 ml of eluate / "feed buffer" (this "feed buffer" of course containing interferon eluted from the Blue Dextran column, of course), the columns are separated and the antibody column is further rinsed before elution as described above. The human white cell interferon eluate obtained from the antibody column contains pure interferon proteins with a specific activity greater than 10 IFU / mg protein (determined by the method described above). To stabilize the pure interferon proteins, the tubes in which the eluate is collected from the antibody column (fraction size 2 ml) are each pre-moistened with 100 of a 1% SDS solution. After combining the interferon-containing eluates, the compound SDS is added to a total concentration of up to 0.1% by weight.

The combined interferon-containing eluates, stabilized with 0.1% SDS, are transferred to a 20 ml stainless steel tube precooled to 0 ° C in an ice bath. After 15 minutes a precipitate forms. The precipitate is isolated by centrifugation at 20,000 rpm at 4 ° C for 20 minutes. The separated liquid is discarded (no interferon activity), and the precipitate is dissolved in 4 M urea solution and transferred to a Millipore concentration cell, volume 8 791 21 ml, with a filter cut at molecular weight of 10,000, and concentrated to about 100 μl: at room temperature. Then, 4 ml of the analyzed 4 M urea solution was added to the concentrate, and the solution was concentrated to about 100 silicon at room temperature.

1-3 ml of distilled water was added, and the solution was again concentrated to a volume of 20 μΐ and mixed with 20 μl of SDS electrophoresis buffer. 20 of the obtained solution was used for characterization as described in "SDS PAGE" below.

The above-mentioned antibody affinity chromatography column was prepared according to "binding procedures" using a non-monospecific anti-PIF preparation absorbed as follows: total amount of anti-interferon-immunoglobulins 106 IFU-NU (corresponding to 4 ml of sheep anti-interferon serum) was absorbed 3 times on a 150 ml human serum column bound to Sepharos® 4B, followed by 4 absorbations on a CIF-epoxy-Sepharose column and 2 absorbances on a CIF-CH-activated Sepharos® 4B column as described in Scand. J. Immunol, 8, 429-436 (1978). Finally, immunoglobulins were absorbed on a poly-L-lysine-Sepharose column (once) and a Soyabean Trypsin inhibitor-Sepharose column (2 times).

The eluate from Namalva interferon Blue Dextran chromatography was divided into two portions. One aliquot was used for SDS PAGE electrophoresis as described below. 250,000 IFUs were applied to the absorbed antibody column as shown in Figure 8. No interferon was detected in the rinsing fluid. Interferon was eluted conventionally by lowering the pH to 2.4, and 235,000 IFU (collected in the presence of 0.1% SDS) was recovered. This eluate was concentrated as described above and further examined on an SDS PAGE system.

29 791 21 SDS PAGE. SDS PAGE electrophoresis was performed as described in "Materials and Methods" above. The stained plate obtained from pure human white cell interferon proteins depicted in Figures 1 and 2 shows schematically a stained plate from another experiment together with the corresponding interferon activity eluted from an unstained parallel gel strip. Significant reproducibility is evident from these figures, with the difference between 20100 and 20180 being within the limits of experimental accuracy. As mentioned above, the biological peaks coincide exactly with the protein peaks.

Figure 1 shows that the interferon preparation is completely pure as determined by SDS PAGE. No other protein bands are visible.

Figure 9 shows stained SDS PAGE plates (feed 0.9 x 10 6) of pure Namalva interferon proteins (A) and eluate from a Blue Dextran column (B). Comparing Figure 1, it is found that the bands of pure Namalva interferon are substantially identical to the bands of pure human white cell interferon exported in the same amount.

Generation of hybridoma cells with interferon activity 3 female Balb / c mice, 2 months old, were immunized with human leukocyte interferon as follows:

The first injection (40,000 IFU) was performed subcutaneously in the back of each mouse. Immunization was continued weekly by subcutaneous injection (70,000 IFU). The last injection was given intravenously at week 9 (mouse 1) and week 10 (mice 2 and 3).

Anti-interferon development was determined from serum assays taken from mice using an interferon neutralization assay. In addition, a conventional 30 7 9 121 anti-interferon IgG preparation (produced by injecting rabbits with partially purified human white cell interferon preparations) was used for laboratory control of the interferon neutralization test system. Serum experiments from mice showed no anti-interferon activity at all during the first six weeks. Subsequently, clear anti-interferon activity was observed:

Table I. IFU-NU / ml Week 7 Week 8 Week 9 Week 10 Week Mouse 1 160 160 120

Mouse 2 200 1280 2500 1200-2500

Mouse 3 80 40 40 5-10

Mice were sacrificed by folding their necks 2-4 days after the last injection, and their spleens were removed under sterile conditions. After each spleen was homogenized in PBS, the homogenized cell suspension was transferred to centrifuge tubes, which were centrifuged at 170 g at 4 ° C. The cells were suspended in PBS, and after the second centrifugation, they were suspended in serum-free DMEM (approximately 0.5 ml per spleen). The total number of cells was 10® (mouse 1), 0.8 x 10® (mouse 2), and 0.8 x 10® (mouse 3). The viability was about 85-90%.

The spleen cell suspension obtained from each mouse by treatment with polyethylene glycol as described above was fused with 107 X63Ag8 (HPRT) myeloma cells as follows: 10® mouse spleen cells and 107 8-azaguanine-resistant myeloma cells (X63Ag8; NS-I / 1Ag 4-1; 0-Ag 14) were mixed together in a 50 ml conical plastic centrifuge tube (Falcon 2070). The tubes were filled with serum-free DMEM and centrifuged at 170-200 g for 10 minutes at 4 ° C. The separated liquid was carefully removed, and at 37 ° C, 0.7 ml of a 50% polyethylene glycol solution at 37 ° C was added dropwise over 1 minute with gentle stirring. After incubation for 90 31 79121 seconds at 37 ° C, 15 ml of warm serum-free DMEM solution was added very slowly (over 1-2 minutes). The mixture was then centrifuged at 200 g for 10 minutes, and the cell pellet was suspended in 50 ml of DMEM-FCS solution for seeding on Costar plates.

Of each fusion, 48 cultures, 1 ml each, were seeded in Costar plates (2 plates, each with 24 holes per spleen, i.e., 48 cultures per mouse). After 10–15 days, growth was observed in 21 cultures (mouse 1), 0 cultures (mouse 2), and (after further inoculation) in 150 cultures (mouse 3).

Cells were transferred to 5 ml cultures in 25 ml NUNC flasks, which, like Costar plates, contained a "nutrient layer" of macrophages. By changing the medium, supernatants were obtained, and the cells obtained from these concentrated cultures were frozen in liquefied nitrogen.

Fluids from private cultures obtained from mouse 1 were examined for positive clones using an interferon neutralization assay. In this case, one positive clone was found, which, however, has a very low titer value (about 2-3 iFU-NU / ml).

Preparation of anti-interferon by pure interferon proteins (pure by SDS PAGE) The eluate from the tandem affinity chromatography described above, characterized by SDS PAGE, was used to immunize rabbits as follows:

About 1,000,000 IFU units were concentrated to about 1 ml, and the concentrate was dialyzed against PBS at 10 ° C overnight. Two rabbits were injected subcutaneously with 10,000,000 IFU of concentrate prepared in this way. The injection was repeated 32 79121 every other week. The development of antibodies is shown in Table 11:

Table II

In Neutralization Units (IFU-NU)

Freund's adjuvant

Rabbit I

xx) 1 3 5 7 ψ, 9 11 13 15 xxx) 002 100 2000 NDX> 20000 20000

Freund's adjuvant

Rabbit I

xx) 17 V 19 21 23 25 27 xxx) 20000 800000 600000 500000 600000 600000

Freund's adjuvant

Rabbit II

xx) 1 3 5 7 ^ 7 9 11 13 15 xxx) 0 0 0 20 500 NDX> 20000 10000

Freund's adjuvant

Rabbit II

xx) 17 77 19 21 23 25 27 xxx) 8000 100000 150000 200000 180000 185000 x) not determined xx) weeks xxx) anti-interferon titers (IFU-NU / ml)

Preparation of anti-interferons from pure stained interferon proteins excised from the SDS PAGE system

Immunization was performed according to the procedure previously described in this specification using a comminuted interferon-containing (partially rinsed and decolorized) gel suspension directly as an immunogenic preparation. One rabbit (III) was immunized with 18400 ± 200 Dalton species and the other rabbit (IV) with 20100 ± 200 Dalton species (died after 15 weeks). Good results were obtained as shown in Table III:

Table III

neutralization

Freund's adjuvant

Rabbit III weeks 1 3 5 7 9 11V 15 (18400 Dalton species) IFU-NU / ml 0 0 0 1-2 2 2 200

Rabbit IV weeks 13579 11_ 15

(20100 Dal- V

ton species) IFU-NU / ml 000012 200 18400 Antigenicity of Dalton species against 20100 Dalton species and vice versa

To demonstrate that the above two species are identical, the following cross-neutralization experiments were performed:

Interferon protein was eluted from 18400 ± 200 Dalton species SDS PAGE and 20100 ± 200 Dalton species SDS PAGE as described above, and solutions containing 5-10 IFUs of these two species were prepared. Anti-interferon solutions from these two species, prepared in rabbits as described above, were diluted to contain a total of 20 IFU-NU / ml. Aliquots (1 ml) of pure interferon species containing 10 IFU 18400 Dalton interferon species and 10 IFU 20100 Dalton interferon species, respectively, were mixed with 1 ml of 18400 Dalton anti-interferon solution and 1 ml of 20100 Dalton species, respectively. -in- 34 79 1 21 terferone solution using all possible variations, i.e. that the anti-interferon of each species was mixed separately with the interferon of each species. After maintaining the mixtures at 37 ° C for 1 hour, any residual interferon activity was determined by conventional interferon titration (compare "Materials and Methods" above). No interferon activity was observed. Thus, when 18400 ± 200 Dalton species and 20100 ± 200 Dalton species were mixed separately with each anti-18400 ± 200 Dalton species and anti-20100 ± 200 Dalton species, respectively, and vice versa, no interferon was detected, i.e. complete neutralization had occurred. . Thus, it can be concluded that these two species of interferon express the same antigenic determinants. This means that the anti-18400 ± 200 Dalton species can be used as a monospecific antibody to purify both interferon species, and that the same is true for the anti-20100 ± 200 Dalton species and the mixture of these species. Additional experiments performed in the same manner showed that each antiserum produced against these two main species completely neutralized the said six biological peaks.

It is very likely that these two main species isolated from the Namalva-SDS PAGE system give the same result, i.e. that they also cross-react and show identity with HuLelF in terms of antigenicity, (so that HuLelF 18400 ± 200 is identical to Namalva 18400 ± 200 antigenicity and molecular weight, and that HuLelF 20100 ± 200 is similarly identical to Namalva 20100 ± 200).

Biological effects of pure interferon proteins Antiviral activity

The antiviral activity of each of the six stained protein bands shown in Figure 3 was determined. The gel was fed into two slits, each of which was stained. The colored strips in one of the slits are shown

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35 79121 at point A in Figure 3. The second slit was rapidly decolorized (in a mixture of 50% methanol, 45% water and 5% acetic acid), the exact location of the interferon proteins on the wet gel was recorded and the gel was rinsed with water and cut into slices as shown in Figure 3. B. The number of gel slices is shown in point C of Figure 3. In this way, each interferon protein band was accurately excised from the gel without mixing with the adjacent band. Each slice was eluted as described in "Materials and Methods", and the biological profile shown in Figure 3 was constructed using the conventional interferon titration described in "Materials and Methods". The neutralizing activity of each of the six species excised and eluted from the SDS PAGE system was controlled against anti-leukocyte interferon, and it was found that all species were completely neutralized by the same antiserum. The yield of interferon shown in Figure 3 was quite low (20%) compared to that obtained with normal "SDS PAGE elution" without pre-staining (except for 18400 ± 200 Dalton species), indicating that the biological activity of most interferon species was selectively destroyed compared to antigenicity. . In neutralization experiments against anti-leukocyte interferon, the "eluted from Figure 3" interferon proteins were able to neutralize anti-leukocyte interferon 3-5 times more efficiently than native (crude) human leukocyte interferon, based on interferon activity, indicating selectivity of determinants corresponding to biological activity.

Non-viral effects

The antiviral effects of pure human leukocyte interferon species were controlled in 3 systems: 1) Anti-cellular activity 36,791 21

The anti-cellular activity of pure interferon proteins was examined by incubating Daudi cells at 1: 1000 dilutions (in medium) of pure interferon proteins obtained from eluted SDS PAGE fractions as shown in Figure 2 and confirming the relative reduction of tritiated thymidine [ Heron and K. Berg, The actions of Interferon are potentiated at elevated temperature, Nature, 274, 508-510 (1978)] compared to controls without interferon (top of Figure 2, where "% G-I" means percent growth inhibition). It is clearly seen that the "anti-cellular curve" follows very closely the anti-viral curve. This proves that all 5 species of pure native human white cell interferon contain both antiviral activity and anti-cellular activity. The size of the various "anti-cellular peaks" does not vary linearly with the size of the "interferon peaks", which probably reflects the sensitivity of the Daudi cell system [J. Hilfenhaus, H. Damm, H.E. Karges and K.E. Manthey, Growth inhibition of human lymphoblastoid Daudi cells in vitro by Interferon preparations, Arc. Virol. 5, 87-97 (1976)]. The small interferon peak at 19500 Dalton did not cause a similar peak in the anti-cell curve. However, at a 10-fold lower dilution, a small but clear peak of anti-cellular activity (not shown in the figure) was observed.

2) Expression of major histocompatibility antigen (MHC) by lymphocytes and monocytes

A selective increase in P2-associated MHC expression was observed using partially purified human leukocyte interferon, as described, for example, in I. Heron, M. Hokland, and K. Berg, "Enhanced expression of β2 microglobulin and HLA on human lymphoid cells by Interferon." , Proc. Natl. Acad. Sci., 75, 6215-6222 (1978) (hereinafter referred to as PNAS 75). For each of the two major species of human white blood cell interferon (18400 and 20100 Dalton, comparator

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37 79121 Fig. 1) was performed at doses of about 100 IFU / ml of culture medium. The above effect was observed using these pure molecular species, whereas the eluates obtained from gel slices outside the regions where antiviral activity was observed had no effect. Thus, it has been demonstrated that the effect of selective addition of MHC antigen expression on lymphoid cells is an inherent property of interferon molecules.

3) Potentiation of the Natural Killer cell system (NK system)

Figure 10 shows the antiviral profile (determined by the SDS PAGE system in the same manner as described in connection with Figure 2). Each of the gel species was tested for NK-enhancing activity using the method described in PNAS 75. Fractions with antiviral activity, as shown in the lower curve, gave the increased NK value described in the upper curve, whereas the "baseline" fractions instead. did not increase the NK value. One arrow indicates the case where saline alone has been added as a negative control, and two arrows indicate the case where partially purified human white cell interferon (PIF) has been used as a positive control. Approximately 100 IFU antiviral units of each interferon preparation were added per ml.

Claims (20)

1. Antibodies, characterized in that they are produced or directed substantially against immunological determinants of Le-shaped human cointerferon proteins, which are proteins in SDS PAGE using an exponential gradient gel, with a total of interferon gel of 0.9 x 10 IPU, showing two single speckled colored protein bands, each of which has antiviral interferon activity at sites 18400 and 20100 Dalton, respectively, and a small colored protein band with antiviral interferon activity between 20300-20400 Dalton, to eeammane with less antiviral interferon activity 19500, 21130 and 23440 Dalton (with teet precision of desea Dalton molecular weights of +. 200 Dalton), wherein stained protein regions of SDS PAGE acrylamide gradient are essentially the only stained interferon proteins. 2. Antibodies according to claim 1, characterized in that they are produced or directed substantially against immunological determinants of Le-shaped mannick cointerferon proteins which proteins in SDS PAGE using an exponential gradient gel, with total interferon loading of 3.8 x 10 10 IFU, viear eex colored protein bands, which antiviral interferon activity, namely the etheric bands, are also found on sites 18410 and 20180 Dalton, respectively, the medium-heavy band on site 20400 Dalton and the highly visible bands on sites 19500 Dalton and 21130 Dalton, 21130 , respectively, (with teet precision of Deeea Dalton molecular weights of + 200 Dalton), where antiviral interferon activity peaks meet exactly beside the stained protein bands, stained protein areas of the SDS PAGE acrylamide gradient are essentially the only interferon stained dyes. 3. Antibodies, characterized in that they are produced or directed essentially against immunological determinants of which, as a whole, single protein or a combination thereof, which proteins have antiviral interferon activity, which are components of the Le-shaped human co-interferons. the proteins set forth in claim 1 or claim 2. 4. Antibodies according to claim 3, characterized in that they are obtained by immunizing an immunized animal with a Le-shaped human interferon protein component of the proteins according to claims 1 or 2, which component corresponds to a value of 1,400 +. 200 Dalton, value 20100 +. 200 Daltons or a combination of deeea values. Method for producing antibodies as claimed in claims 1-4, characterized by immunizing an immunizable animal with Le-shaped human interferon proteins as claimed in claims 1-4, and collecting anti-serum from the animal. Method according to claim 5, characterized in that the Le-shaped human interferon proteins used for immunization are obtained from the corresponding band or bands cut from the SDS PAGE gel. Process according to claim 6, characterized in that the strips are cut from the SDS PAGE gel after staining and brief washing in distilled water of said gel. Θ. Process according to any of claims 5-7, characterized in that the immunization is carried out with a stabilized aqueous solution of the interferon protein (s), the stabilizing substance present in the stabilized aqueous solution being sodium dodecyl sulfate. Method according to claim 8, characterized in that the stabilized aqueous solution of the interferon protein / proteins is buffered with a phosphate buffered physiological aqueous solution to a pH of about 7.2. 79121 Method according to claim 8 or 9, characterized in that the stabilized aqueous solution contains adjuvant. Method according to claim 10, characterized in that the adjuvant is Freund's adjuvant. A method for producing antibodies according to any of claims 1-4, characterized in that it comprises culturing such a hybridoma cell clone derived from a myeloma cell line X 63 Ag 8; NSI / 1 Ag 4-1; SP2 / 0-Ag 14, which produces antibodies directed against immunological determinants of Le-shaped human interferon proteins according to the whole of claims 1-4, and recovery of the antibodies from the culture medium. Antibodies according to which as a whole of claims 1-4, characterized in that they are immobilized in a matrix. Antibodies according to claim 13, characterized in that they are covalently linked to a matrix. Antibodies according to claim 14, characterized in that the matrix is a cross-linked agarose, such as Sepharose 4B. 16. In a matrix immobilized antibodies according to which as a whole of claims 13-15, characterized in that they do not substantially have proteolytic enzymatic activity. 17. In a matrix immobilized antibody according to claim 16, characterized in that the proteolytic enzymatic activity has been substantially totally removed from it by treatment with enzyme inhibitors or enzyme destroying agents. 18. In a matrix immobilized antibodies according to claim 17, characterized in that their treatment with