HK1019339B - Antibodies against interferon alpha/beta receptor - Google Patents

Description

Anti-alpha/beta interferon receptor antibodies

Technical Field

The present invention relates to antibodies directed against ligand binding portions of the alpha/beta interferon receptor that selectively attenuate the activity of various type I interferons.

European Patent (EP) publication No.588,177 describes a soluble IFN-alpha receptor having a molecular weight of about 40,000 by reaction with¹²⁵I-IFN-alpha 2 cross-linking and immunoprecipitation with anti-IFN-alpha monoclonal antibodies. When such receptors are obtained from serum, their molecular weight is 50K. The patent also addresses the above-mentioned 40,000 IFN-. alpha.having a different sequence from any known protein obtained from urine in a homogeneous stateBinding proteins (hereinafter referred to as "IFNAB-BP" or "IFNAB-BPII") are described, IFNAB-BP binds to and blocks the activity of a variety of IFN-alpha subtypes as well as one IFN-beta.

EP publication No.676,413 describes the cloning and sequencing of two cDNA molecules encoding precursors of IFNAB-BP, possibly from the same gene, by different splicing. The patent also describes the production of two recombinant proteins called IFNAB-BPI and IFNAB-BPII in mammalian or other host cells. The patent also discloses that can be used to block IFN receptor, and used in IFNAB-BPI and IFNAB-BPII immunoassay and immunoprecipitation of anti-IFNAB-BP polyclonal and monoclonal antibodies.

The invention is described in two groups of IFNAB-BPII neutralizing antibody preparation. These neutralizing antibodies bind to the receptor for type I interferon on human cells. One group of antibodies can block the activity of each IFN-alpha subtype and IFN-beta. And another group of antibodies can selectively block the activity of various subtypes of interferon-alpha in human cells without affecting the activity of interferon-beta. Thus, one group of the above antibodies can inhibit the adverse effects of IFN- α with little effect on IFN- β activity.

Background

Type I Interferons (IFNs) (IFN- α, IFN- β, and ω) constitute a family of structurally related cytokines, generally defined as interferons by their ability to provide protection against viral infection. Many other biological activities have been reported for type I IFN including inhibition of cell proliferation, induction of MHC class I antigens and several other immunomodulatory activities (1). IFN-alpha and IFN-beta may be used to treat several viral diseases, including hepatitis C (2, 3) and viral warts, as well as certain malignancies such as hairy cell leukemia (6), chronic myelogenous leukemia (7), and Kaposis' sarcoma (8).

IFN- α is found in the serum of various patients with autoimmune diseases such as systemic lupus erythematosus (9) and AIDS (10) patients. IFN-alpha is associated with the development of juvenile diabetes (11). Moreover, IFN- α therapy in some cases has resulted in adverse side effects, including fever and neurological disorders, etc. (12). Therefore, in certain pathological conditions, it may be advantageous for the patient to neutralize the activity of IFN- α.

Like other cytokines, IFN- α exerts its biological activity by binding to cell surface receptors that are specific for all subtypes of IFN- α and IFN- β (13). Human IFN-alpha receptors (IFNAR) (14) were identified and cloned from Daudi cells. The cloned receptor has a single transmembrane region, an extracellular region and an intracellular region. When expressed in murine cells, the receptor responds to human IFN-. alpha.B, but not to other IFN-. alpha.and IFN-. beta.species, indicating that other components are involved in the response to each of the IFN-. beta.and IFN-. alpha.subtypes.

Other studies have shown that additional components or receptor subunits are involved in the binding of IFN- α and IFN- β (15-17). However, it has been reported that the above receptor (14) is involved in binding to all IFN-alpha and IFN-beta species (18). Indeed, a second receptor component, designated the IFN-. alpha./β receptor, has recently been identified and cloned (EP publication No.676,413 and reference 19). We show that the extracellular region sequence and IFNAB-BPI the same IFN-alpha/beta receptor is type I interferon receptor main ligand binding component; furthermore, the interferon- α/β receptor and IFNAR cooperate in ligand binding and form a ternary complex with type I interferon on the cell surface (20).

A number of monoclonal antibodies directed against IFNAR and capable of non-selectively blocking type I interferons have been reported (21). Other monoclonal antibodies directed against yet unidentified receptor components have also been described, which non-selectively block the activity of type I interferons.

Summary of The Invention

The present invention provides monoclonal antibodies against the IFN- α/β receptor, which is a universal receptor for IFN- α and IFN- β. These antibodies bind to various forms of receptors which may be expressed on human cells or are soluble IFNAF-BPI and IFNAF-BPI. Two classes of neutralizing monoclonal antibodies have been developed. One class of compounds exhibits high potency for blocking the antiviral activity of IFN-alpha and IFN-beta. Another group of compounds has unexpectedly found that the antiviral activity against IFN- α exhibits a high blocking titer, while the activity against blocking IFN- β exhibits a low blocking titer. Thus, surprisingly, a second class of monoclonal antibodies also permits use in selectively blocking IFN- α activity without affecting IFN- β activity over a range of concentrations.

The invention also provides anti-human cell expression of IFN-alpha/beta receptor (1FNAB-BPI and IFNAB-BPII) humanized monoclonal antibodies, humanized monoclonal antibodies are mouse-human hybrid antibody molecules, wherein the variable region from mouse antibody and constant region from human antibody.

The invention also provides DNA molecules encoding monoclonal antibodies and humanized monoclonal antibodies against IFN- α/β receptors (IFNAB-BPI and IFNAB-BPII).

The invention also provides replicable expression vectors containing the above DNA molecules, host cells transformed with the vectors, and proteins produced by the transformed host cells. The term "DNA molecule" includes genomic DNA, cDNA, synthetic DNA, and combinations thereof.

The invention also relates to DNA molecules which can hybridize under stringent conditions to the above-described DNA molecules and which encode proteins having the same biological activity as the monoclonal antibodies against IFN-. alpha./beta.receptors (i.e., IFNAB-BPI and IFNAB-BPII) and humanized monoclonal antibodies.

The invention also provides a method for producing host cells capable of producing functional monoclonal antibodies and humanized monoclonal antibodies against IFN-alpha/beta receptors (i.e., IFNAB-BPI and IFNAB-BPII).

The monoclonal antibody of the present invention inhibits the biological activity of human interferon-alpha in cells of human cells without affecting the biological activity of human interferon-beta. This biological activity was determined by quantitative evaluation of the inhibitory effect of the antibodies using a cytopathic inhibition assay with human WISH cells and vesicular stomatitis virus.

Detailed description of the invention

IFN-. alpha./β binding protein (IFNAB-BP) with a molecular weight of 40,000 was isolated from normal urine by two-step chromatography according to Israeli patent application No. 106591. The crude urine protein was loaded onto an IFN-. alpha.2 linked agarose chromatography column. Irrelevant proteins on the column were washed away, and then bound proteins were eluted at low pH. Analyzing the eluted protein by volume exclusion HPLC to obtain several protein peaks, one of which is characterized by specificity to¹²⁵I-IFN-alpha 2 reacts and blocks the antiviral activity of IFN-alpha and IFN-beta. The protein was further identified by N-terminal micro-sequencing analysis. The resulting sequence was compared with the known IFNAR receptor sequence (14) and found to be different. Furthermore, it was found by comparison with Swissport and Genebank databases using the FastA program (23) that it is different from any known protein and is not encoded by any known DNA sequence.

Homogeneous preparations of urinary IFNAB-BP were used as immunogens for antibody production.

The term "antibody" is meant to include polyclonal antibodies, monoclonal antibodies (mabs), chimeric antibodies, anti-idiotypic antibodies (anti-Id antibodies) and antibodies which can be labeled in soluble or bound form, as well as active fragments thereof produced by various known techniques, such as enzymatic cleavage, peptide synthesis or recombinant techniques.

Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of antigen-immunized animals. A monoclonal antibody is a population of substantially homogeneous antigen-specific antibody molecules that contain substantially the same epitope binding sites. Mabs can be obtained by methods known to those skilled in the art. See, for example, Kohler and Milstein,Nature256: 495-497 (1975); U.S. Pat. Nos. 4,376,110; edited by Ausubel et alCurrent Protocols in Molecular BiologyGreene Publishing Ashoc.and Wiley Interscience, N.Y. (1987-1996); the results of Harlow and Lane are,ANTIBODIES：A LABORATORY MANUALcold Spring Harbor Laboratory (1988); edited by Colligan et alCurrent Protocols in ImmunologyGreene Publishing Assoc. and Wiley Interscience. N.Y. (1992-. Such antibodies may be of the classes of immunoglobulins including IgG, IgM, IgE, IgA, GILD and their various subclasses. The hybridoma producing the Mab of the present invention can be cultured in vitro, in situ, or in vivo. The in vivo or in situ generation of high titers of mabs is the currently preferred method of production.

Chimeric antibodies are molecules whose different parts are derived from different species of animals, for example antibody molecules derived from the variable regions of a mouse Mab and the constant regions of a human immunoglobulin. Chimeric antibodies are used primarily to reduce immunogenicity and to increase production yield. For example, murine mabs produced by hybridomas are produced in high yields but are immunogenic in humans, so human/murine chimeric antibodies are used. Chimeric antibodies and methods for their preparation are well known in the art (Cabilly et al,Proc.Natl.Acad.Sci.USA81: 3273-3277 (1984); morrison et al, in a laboratory,Proc.Natl.Acad. Sci.USA81: 6851-6855 (1984); boulianne et al,Nature321: 643-646 (1984); cabilly et al, European patent application 125023 (published on 14/11/1984); neuberger et al, Nature 314: 268-270 (1985); taniguchi et al, European patent application 171496 (published on 19.2.1985); morrison et al, European patent application 173494 (published on 3/5/1986); neuberger et al, PCT application WO8601533 (published on 3/13/1986); kudo et al, European patent application 184187 (published on 11.6.1986); morrison et al, European patent application 173494 (published on 3/5/1986); sahagan et al, j.immunol.137: 1066-1074 (1986); robinson et al, International patent application WO9702671 (published on 7.5.1987); liu et al Proc.Natl.Acad Sci.USA 84: 3439-Proc.Natl.Acad.Sci.USA84: 214-218 (1987); better et al， Science240: 1041-1043 (1988); and Harlow and Lane, ANTIBODIES: ALABORATORY MANUALsupra. The contents of the above documents are fully incorporated herein by reference.

An anti-idiotypic (anti-Id) antibody is an antibody that recognizes an idiotypic determinant normally associated with the antigen-binding site of an antibody. Id antibodies can be prepared by immunizing an animal (e.g., mouse line) that is syngeneic and syngeneic with the Mab corresponding to the anti-Id to be prepared. The immunized animal will produce an antibody directed against the idiotypic determinant (anti-Id antibody) to recognize and react to the idiotypic determinant of the immunizing antibody. See, for example, U.S. Pat. No.4,699,880, the contents of which are incorporated herein by reference in their entirety.

anti-Id antibodies can also be used as "immunogens", in other animals induced immune responses, producing so-called anti-Id antibodies. The anti-Id may have the same epitope as the original Mab that induced the anti-Id. Thus, using antibodies directed against the idiotypic determinants of mabs, other clonally expressed antibodies of the same specificity can be identified.

Thus, the invention of anti IFNAB-BPI, IFNAB-BPII and related protein mabs, can be used in appropriate animals (such as BLAB/c mice) induced anti-Id antibody. Spleen cells of mice so immunized were used to produce anti-Id hybridomas secreting anti-Id MAbs. Furthermore, anti-Id mAbs can be coupled to a carrier such as keyhole limpet hemocyanin for immunization of other BLAB/c mice. The sera of these mice will contain anti-Id antibodies that have the binding properties of the original Mab (specific for IFNAB-BPI or IFNAB-BPII epitopes).

Thus, an anti-Id Mab has its own idiotypic epitope, or has an "epitope" that is structurally similar to the epitope being evaluated (e.g., IFNAB-BPI or IFNAB-BPII).

The term "antibody" is also meant to include whole antibody molecules, and active fragments thereof capable of binding antigen, such as Fab and F (ab')₂. Fab and F (ab')₂Fc fragment without intact antibody, cleared faster from circulation and better than intact antibodyAntibodies have less non-specific tissue binding (Wahl et al,J.Nucl.Med.24：316-325(1983))。

it will be appreciated that, in accordance with the methods described herein for intact antibody molecules, Fab and F (ab')₂And other fragments, can be used to selectively block the biological activity of IFN-alpha. These fragments are generally produced by proteolytic cleavage, e.g., papain to produce Fab fragments or pepsin to produce F (ab')₂And (3) fragment.

An antibody is said to be "capable of binding" a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody. The term "epitope" means any moiety on a molecule that is capable of being bound by and recognized by an antibody. Epitopes or "epitopes" are generally composed of chemically active surface groups of molecules (e.g., amino acids or sugar side chains) and have specific three-dimensional structural characteristics and specific charge characteristics.

An "antigen" is a molecule or portion of a molecule that is capable of being bound by an antibody and of inducing an animal to produce an antibody that binds to an epitope of the antigen. An antigen may have one or more epitopes. The above-mentioned specific reaction means that an antigen reacts with its corresponding antibody in a highly selective manner without reacting with many other antibodies elicited by other antigens.

The antibodies, including fragments of antibodies, used in the present invention can be used to block the biological activity of each subtype of IFN- α with little effect on IFN- β activity.

The invention also provides DNA molecules encoding any of the antibodies of the invention described above, replicable expression vectors containing such DNA molecules, and host cells (including prokaryotic and eukaryotic host cells) transformed with such expression vectors.

The present invention also includes a method for producing each antibody of the present invention by culturing the hybridoma of the present invention and recovering the monoclonal antibody secreted therefrom.

The invention also includes methods for producing the various antibodies of the invention by culturing the transformed cells of the invention and recovering the secreted antibodies encoded by the DNA molecules and expression vectors of the transformed host cells described above.

The invention also relates to active muteins and active fragments of the above antibodies, and to fusion proteins comprising a wild-type antibody, or an active mutein or an active fragment thereof, fused to another polypeptide or protein, and exhibiting a similar ability to block the biological activity of type I IFN.

The DNA encoding the above antibodies, active fragments thereof, muteins or fusion proteins, and operably linked transcriptional and translational regulatory signals are inserted into a eukaryotic vector capable of integrating the desired gene sequence into the chromosome of the host cell. To enable selection of cells stably incorporating the introduced DNA in the chromosome, one or more markers are used to allow selection of host cells containing the expression vector. The marker may provide prototrophy to an auxotrophic host, provide resistance to an antimicrobial agent (e.g., an antibiotic), or resistance to a heavy metal (e.g., copper), etc. The selectable marker gene can be linked directly to the DNA gene sequence to be expressed, or introduced into the same cell by co-transfection. Additional elements may also be required for optimal synthesis of single-stranded binding protein mRNA, which may include splicing signals, transcription promoters, enhancers, and termination signals (24).

For expression of the above-mentioned antibodies, active fragments or derivatives thereof, it is preferred to incorporate the DNA molecule to be introduced into the selected cell into a plasmid or viral vector capable of autonomous replication in the recipient host.

Important factors to consider in selecting a particular plasmid or viral vector include: whether it is easy to identify and select carrier-containing recipient cells from carrier-free recipient cells; whether the copy number of the desired vector in a particular host is desirable; whether it is desirable to have vectors that can "shuttle" between different germline host cells. Preferred prokaryotic vectors include those plasmids capable of replication in E.coli, such as pBR322, ColEl, pSC101, pACYC184, etc. (25); bacillus plasmids such as pC194, pC221, pT127 and the like (26); streptomyces plasmids, including pIJ101(27), Streptomyces bacteriophages such as IC31(28) and Pseudomonas plasmids (29, 30).

Preferred eukaryotic plasmids include BPV, vaccinia, SV40, 2-micron circle (2-micron circle), and the like, or derivatives thereof. These plasmids are well known in the art (31-35).

Once a vector or DNA sequence containing the above structure is prepared for expression, the expression vector can be introduced into a suitable host cell by a variety of suitable methods, including transformation, transfection, lipofection, conjugation, protoplast fusion, electroporation, calcium phosphate precipitation, direct microinjection, and the like.

The host cells used in the present invention may be prokaryotic or eukaryotic cells. Preferred prokaryotic host cells include bacteria belonging to the genera Escherichia coli, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, and the like. The most preferred prokaryotic host bacterium is E.coli. The bacterial host specifically includes Escherichia coli K12 strain 294(ATCC 31446), Escherichia coli X1776(ATCC 31537), Escherichia coli W3110 (F)^-，λ^-Phototrophic (ATCC 27325)), and other enterobacteria such as Salmonella typhimurium or Serratia marcescens and Pseudomonas. In this case, the protein will not be glycosylated. Prokaryotic host cells must be compatible with the replicon and regulatory sequences in the expression plasmid.

Preferred eukaryotic hosts are mammalian cells, such as human, monkey, mouse cells and Chinese Hamster Ovary (CHO) cells, because they are capable of post-translational modification of protein molecules, including proper folding, proper disulfide bond formation, and glycosylation at the correct position. In addition, yeast cells and insect cells are also capable of post-translational peptide modifications, including high mannose glycosylation. There are many recombinant DNA methods that employ strong promoter sequences and plasmids with high copy numbers, which can be used to produce desired proteins in yeast and insect cells. Yeast cells are capable of recognizing a leader sequence on a cloned mammalian gene product and secreting a peptide with the leader sequence. After introduction of the vector, the host cells are cultured on a selection medium to allow the growth of the vector-containing cells. Expression of the cloned gene sequence results in the production of the above-described antibody, fusion protein or mutein or an active fragment thereof. Then separating and purifying the expressed antibody by using conventional techniques such as extraction, precipitation, chromatography, electrophoresis and the like or affinity chromatography,

the term "mutein" as used herein refers to an analog of the above-mentioned antibody, i.e., one or several amino acid residues of the above-mentioned antibody or an active fragment thereof are substituted with different amino acid residues, or deleted, or one or several amino acid residues are added to the original sequence of the above-mentioned antibody, but the activity of the resulting product is not significantly altered as compared to the wild-type antibody or an active fragment thereof. These muteins are prepared by known synthetic methods and/or by site-directed mutagenesis techniques, or any other known suitable technique.

Any such mutein preferably has an amino acid sequence sufficiently similar to the above-described antibody so as to have substantially the same activity as the above-described antibody or an active fragment thereof. One group of the antibodies can block the antiviral activity of human INF-alpha 2 and human INF-beta. Another group of the above antibodies is capable of blocking the antiviral activity of human INF- α 2, while the antiviral activity of human INF- β remains substantially intact. Thus, it is possible to determine whether any given mutein has substantially the same activity as the above-described antibody using routine experiments, including simple antiviral assays on the above-described muteins, since a mutein capable of blocking any one type I interferon retains sufficient activity of the above-described antibody, has at least one use of the above-described antibody, and thus has substantially the same activity.

In a preferred embodiment, any of the above muteins has at least 40% identity or homology with the sequence of one of the above antibodies. More preferably, at least 50%, 60%, 70%, 80%, or, most preferably, at least 90% identity or homology.

Muteins of the above-mentioned antibodies or active fragments thereof, or nucleic acids encoding the same, which are used in the present invention, comprise a limited set of substantially corresponding sequences as substitution peptides or polynucleotides, and are routinely obtained without undue experimentation using ordinary skill in the art, in light of the teachings and guidance presented herein. For a detailed description of the Protein chemistry and Structure, see Schulz, G.E., et al, Principles of Protein Structure, Springer-Verlag, New York, 1978; and Greighton, t.e., Proteins: structure and Molecular Properties, w.h.freeman & co, San Francisco, 1983, the contents of which are incorporated herein by reference. For instructions on nucleotide sequence conversion, e.g., codon usage, see Ausubel et al, supra § A.1.1-A.1.24 and Sambrook et al, supra, appendices C and D.

According to the invention, the mutein changes are preferably so-called "conservative" transitions. Conservative amino acid exchanges for the above-described antibodies, polypeptides or proteins, or active fragments thereof, include the use of synonymous amino acids within the same group that are sufficiently similar in physicochemical properties (such that substitutions between members of the group will retain the biological function of the molecule), Grantham,Sciencevol.185, pp.862-864 (1). It is clear That insertions and deletions of amino acids can also be made in The above-defined sequences without altering their function, in particular insertions or deletions involving only a few (e.g.less than 30, preferably less than 10) amino acids, without removing or substituting amino acids which are critical for The functional conformation (e.g.cysteine residues), Anfinsen, "Princle That Goven The Folding of protein Chains",Sciencevol.181, pp.223-230 (1973). Proteins and muteins produced by the above deletions and/or insertions are also within the scope of the present invention.

Preferred groups of synonymous amino acids are those in Table I. More preferred are those in table II; most preferred are those in Table III.

TABLE I

Preferred synonymous amino acid grouping

Amino acid synonymous amino acid

Ser Ser，Thr，Gly，Asn

Arg Arg，Gln，Lys，Glu，His

Leu Ile，Phe，Tyr，Met，Val，Leu

Pro Gly，Ala，Thr，Pro

Thr Pro，Ser，Ala，Gly，His，Gln，Thr

Ala Gly，Thr，Pro，Ala

Val Met，Tyr，Phe，Il e，Leu，Val

Gly Ala，Thr，Pro，Ser，Gly

Ile Met，Tyr，Phe，Val，Leu，Ile

Phe Trp，Met，Tyr，Ile，Val，Leu，Phe

Tyr Trp，Met，Phe，Ile，Val，Leu，Tyr

Cys Ser，Thr，Cys

His Glu，Lys，Gln，Thr，Arg，His

Gln Glu，Lys，Asn，His，Thr，Arg，Gln

Asn Gln，Asp，Ser，Asn

Lys Glu，Gln，His，Arg，Lys

Asp Glu，Asn，Asp

Glu Asp，Lys，Asn，Gln，His，Arg，Glu

Met Phe，Ile，Val，Leu，Met

Trp Trp

TABLE II

More optimal grouping of synonymous amino acids

Amino acid synonymous amino acid

Ser Ser

Arg His，Lys，Arg

Leu Leu，Ile，Phe，Met

Pro Ala，Pro

Thr Thr

Ala Pro，Ala

Val Val，Met，Ile

Gly Gly

Ile Ile，Met，Phe，Val，Leu

Phe Met，Tyr，Ile，Leu，Phe

Tyr Phe，Tyr

Cys Cys，Ser

His His，Gln，Arg

Gln Glu，Gln，His

Asn Asp，Asn

Lys Lys，Arg

Asp Asp，Asn

Glu Glu，Gln

Met Met，Phe，Ile，Val，Leu

Trp Trp

TABLE III

Optimal synonymous amino acid grouping

Amino acid synonymous amino acid

Ser Ser

Arg Arg

Leu Leu，Ile，Met

Pro Pro

Thr Thr

Ala Ala

Val Val

Gly Gly

Ile Ile，Met，Leu

Phe Phe

Tyr Tyr

Cys Cys，Ser

His His

Gln Gln

Asn Asn

Lys Lys

Asp Asp

Glu Glu

Met Met，Ile，Leu

Trp Met

Examples of amino acid transformations in proteins useful for obtaining the above-described antibodies or active fragments thereof for use in the present invention include known process steps as provided in Mark et al, U.S. Pat. Nos. RE33,653, 4,959,314, 4,588,585 and 4,737,462; 5,116,943 by Koths et al, 4,965,195 by Namen et al; 4,879,111 to Chong et al; and 5,017,691 to Lee et al; and U.S. Pat. No.4,904,584 to Shaw et al.

In another preferred embodiment of the present invention, each mutein of the above-mentioned antibody or an active fragment thereof has an amino acid sequence substantially corresponding to the above-mentioned antibody. The term "substantially corresponding" is intended to mean a protein which has undergone minor changes in the sequence of the native protein without affecting its basic properties, in particular its ability to completely or selectively block IFN- α and IFN- β activities. It is generally accepted that the types of changes falling within the term "substantially corresponding" refer to those changes obtained by conventional mutagenesis techniques performed on the DNA encoding the antibody described above, which results in minor modifications of the antibody and screening for the desired activity in the manner described above.

The mutant proteins of the invention include those which react under stringent conditions with antibodies encoding the inventionDNA or RNA hybridizing to the protein encoded by the nucleic acid. The invention also includes nucleic acids that can also be used as probes to identify and purify desired nucleic acids. Moreover, such a nucleic acid would serve as a prime candidate for determining whether it encodes a polypeptide that retains the functional activity of the antibody of the invention. The term "stringent conditions" refers to hybridization and subsequent washing conditions. These common techniques are conventionally referred to in the art as "stringent". See, for example, Ausubel et al,Current Protocols in Molecular Biology，supra， InterscienceNY, § 6.3 and 6.4(1987, 1992), and Sambrook et al,supra. In addition, examples of the stringent conditions include the following washing conditions: washing in 2 SSC and 0.5% SDS for 5 minutes, and in 2 SSC and 0.1% SDS for 15 minutes at 12 to 20 ℃ below the experimentally calculated hybridization temperature Tm; washing in 0.1 × SSC and 0.5% SDS at 37 ℃ for 30 to 60 minutes; then washed in 0.1 × SSC and 0.5% SDS at 68 ℃ for 30 to 60 minutes. Those of ordinary skill in the art will recognize that stringency conditions will also depend on the length of the DNA sequence, oligonucleotide probe (e.g., 10 to 40 bases), or mixed oligonucleotide probe. If a mixed probe is used, it is preferable to replace SSC with tetramethylammonium chloride (TMAC). See also the examples in Ausubel for,supra。

the term "fusion protein" refers to a polypeptide comprising the above-described antibody or an active fragment thereof or a mutein thereof fused to another protein, which "other protein" has a longer residence time in body fluids. Thus, the above-mentioned antibody or an active fragment thereof may be fused with another protein, polypeptide or the like.

The term "salt" as used herein refers to the carboxyl salt and amino acid addition salt of the above-described antibody, active fragment thereof, mutein, or fusion protein thereof. The carboxylic acid salts may be formed by methods known in the art and include inorganic salts such as sodium, calcium, ammonium, ferric, or zinc salts, and the like, as well as salts with organic bases. For example, a salt with triethanolamine such as amine, arginine or lysine, piperidine, procaine, etc. Acid addition salts include, for example, salts with inorganic acids such as hydrochloric acid or sulfuric acid, and salts with organic acids such as acetic acid or oxalic acid. Of course, any such salt must have substantially the same activity as the above-described antibody or active fragment thereof.

"functional derivatives" as used herein include derivatives of the above antibodies or active fragments thereof and their muteins and fusion proteins, which can be prepared from functional groups as side chains of residues or from N-or C-terminal groups by methods known in the art and are also within the scope of the present invention as long as they remain pharmaceutically acceptable, i.e., do not destroy substantially the same activity of the protein as the above antibodies and do not render toxic the compositions containing them. For example, such derivatives include polyethylene glycol side chains that mask antigenic sites and prolong the humoral retention time of the above antibodies or active fragments thereof. Other derivatives include aliphatic esters of the carboxyl group, amides of the carboxyl group formed by reaction with ammonia gas or primary or secondary amines, N-acyl derivatives of the free amino group of the amino acid residue formed with an acyl group (e.g. an alkanoyl or carbocyclic aroyl group), or O-acyl derivatives of the free hydroxyl group formed with an acyl group (e.g. the hydroxyl group of a seryl or threonyl residue).

In the present invention, the "active fragments" of the above-mentioned antibody, mutein and fusion protein include fragments or precursors of the above-mentioned antibody polypeptide chains (either alone or in combination with molecules such as sugar or phosphate groups at amino acid residues), or fusion proteins containing such fragments of the above-mentioned antibody or aggregates of any of the above-mentioned antibodies, provided that these fragments have substantially the same activity as the above-mentioned antibody.

The invention also relates to pharmaceutical compositions comprising a pharmaceutically acceptable carrier and an antibody of the invention or a mutein, fusion protein or salt thereof, a functional derivative or active fragment thereof.

The above-mentioned antibody or a derivative thereof is mixed with a physiologically acceptable carrier and/or stabilizer and/or excipient, and a pharmaceutical composition for use in the present invention is prepared by dosage form (for example, prepared by low-temperature drying in a vial for filling). The method of use may be in any form of administration where similar drugs are approved and will depend on the therapeutic situation, e.g., intravenous, intramuscular, subcutaneous, topical injection or topical use, or continuous infusion, etc. The amount of active ingredient administered will depend on the route of administration, the disease being treated and the condition of the patient. For example, the amount of protein required for topical injection is lower on a weight basis than that required for intravenous infusion.

For example, the above antibodies can be used in type I diabetes, various autoimmune diseases, transplant rejection, AIDS and the like to attenuate or block the biological activity of various IFN- α subtypes, all of which have abnormal expression of IFN- α. That is, the above antibody can be used in various cases where IFN-. alpha.is excessive due to endogenous production or exogenous administration.

Accordingly, the above antibodies, humanized antibodies, active fragments thereof, muteins, fusion proteins and salts thereof, functional derivatives thereof, and active fragments of the above molecules are indicated for the treatment of autoimmune diseases, other inflammatory diseases in mammals, intoxication from the use of alpha or beta interferon, juvenile diabetes, lupus erythematosus and AIDS.

The invention will now be illustrated by way of non-limiting examples:

example 1: immunization of mice with IFNA-BP and fusion with myeloma cells

Female Balb/C mice (3 months old) were first injected with 2 micrograms of purified IFNA-BP Freund's complete adjuvant emulsion, and three weeks later with subcutaneous injection of Freund's incomplete adjuvant antigen emulsion. Antigen solutions in PBS were injected subcutaneously five times every 10 days. The binding titers of these mouse sera were determined to be 1: 100,000 by IsRIA (see below). The antiviral activity of antisera blocking IFN-alpha and IFN-beta was determined as follows: human WISH cell monolayers were pre-formed on 96-well test plates and incubated with two-fold dilutions of antisera (or monoclonal antibodies) for 2 hours at 37 ℃. IFN-. alpha.or IFN-. beta.was then added to all wells (10 units/ml (U/ml) final concentration was calibrated according to NIH standards), and the test plates were incubated for an additional 4 hours. Cells were challenged with Vesicular Stomatitis Virus (VSV) and incubated overnight until complete cytopathy occurred in control wells without IFN. The antibody neutralization titer of wells exhibiting 50% CPE was set to 9 units/ml. Thus, 1 blocking unit/ml is the concentration of antibody required to block 1 unit per ml of IFN under the test conditions described above. The serum of the immunized mice had a neutralizing titer of 120,000 units/ml for both IFN-. alpha.2 and IFN-. beta.s.

4 and 3 days before fusion, intraperitoneal injection of purified IFNAB-BP was performed for final booster immunization. The fusion was performed using NSO/l myeloma cell line and lymphocytes obtained from spleen and lymph nodes of immunized mice as fusion parents. The fused cells were seeded in a 96-well culture plate, and hybridomas were selectively cultured in DMEM medium supplemented with HAT and 15% horse serum.

Example 2: screening of hybridomas and identification of monoclonal antibodies

Screening of hybridomas producing anti-IFNAB-BP monoclonal antibodies was performed as follows, and the presence of anti-IFNAB-BP antibodies in the supernatants of the test hybridomas was determined by reverse solid phase radioimmunoassay (sRI): PVC microtiter plates (Dynatech Laboratories, Alexandria, Va.) goat anti-mouse serum F (ab) purified with affinity₂Antibodies (Jackson Labs, USA) (10 μ g/ml, 100 μ l/well) were coated. Incubated overnight at 4 ℃, the microtiter plates were washed twice with PBS containing BSA (0.5%) and Tween20 (0.05%) and blocked with wash solution for at least 2 hours at 37 ℃. Hybridoma culture supernatant (100. mu.l/well) was added. The microtiter plates were incubated at 37 ℃ for 4 hours. Then washing with washing solution for 3 times, adding¹²⁵I-IFNAB-BP (100. mu.l, 105cpm) was cultured at 4 ℃ for another 16 hours. The microplates were washed 3 times and the cut wells were counted in a gamma counter. Samples with a count value at least 5 times higher than the negative control value were considered positive (table IV). All positive clones were selected for subcloning. Each subcloned cell was injected into Balb/C mice pretreated with pristane to produce ascites fluid. Immunoglobulins were isolated from ascites fluid by precipitation with ammonium sulfate (50% saturation). The type of antibody was identified using a commercially available ELISA kit (Amersham, UK).

The ability to block the receptor and inhibit the antiviral activity of IFN-. alpha.2 and IFN-. beta.was further tested for various monoclonal antibodies, i.e., hybridoma supernatants, concentrated hybridoma supernatants, ascites fluid, or immunoglobulins isolated from ascites fluid, as described above for the determination of serum activity in immunized mice. The results are shown in Table IV. It was thus found that monoclonal antibodies 16.3, 35.2 and 392.1 blocked the antiviral activity of IFN-. alpha.2 and IFN-. beta.with a fairly high titer, but that the titers of monoclonal antibodies 35.9, 51.44 and 234.14 against IFN-. alpha.2 were surprisingly significantly higher than their titers against IFN-. beta.s. Thus, monoclonal antibodies 35.9, 51.44 and 234.14 can be used to selectively block IFN- α activity without affecting IFN- α activity over a range of concentrations.

TABLE IV

Characterization of anti-IFN-. alpha./beta receptor (IFNAB-BP) monoclonal antibodies

Antibody IsIRA (cpm) neutralization titer (U/ml) Ig class

IFN-α2 IFN-β

16.3 hybridoma supernatant 39,103 > 5,120 ND IgG1

16.3 ascites¹ ND² 60,000 60,000 IgG1

35.9 hybridoma supernatant 33,1001,280150 IgG1

35.9 ascites ND 60,00015,000 IgG1

51.44 hybridoma supernatant 6,3451,000 < 75 IgG2a

51.44Ig (5 mg/ml) ND 15,000 < 2500 IgG2a

53.2 hybridoma supernatant 26,7372,000 ND IgG1

53.2 ascites ND 120,00070,000 IgG1

117.7 hybridoma supernatant 38,9452,000 ND IgG1

117.7Ig (10 mg/ml) ND 28,800,0002,000,000 IgG1

234.14 hybridoma supernatant 21,812 > 5,120 < 200 IgG2a

234.14Ig (10 mg/ml) ND 1,440,00023,000 IgG2a

392.1 hybridoma supernatant 34,3902,400 ND IgG1

392.1 ascites ND 160,00070,000 IgG1

¹About 5 mg/ml Ig

²Non-test

Example 3: ELISA assay Using monoclonal antibodies for IFNAB-BPII

Microtiter plates (Dynatech or Maxisorb, bNunc) were coated with anti-IFNAB-BP monoclonal antibody overnight at 4 ℃. This first coating can be applied with monoclonal antibody No.46.10(Ig fragment, 120 μ l/well, 10 μ g/ml in PBS solution). Monoclonal antibody No.46.10 can be found in EP publication No.676,413.

Alternatively, monoclonal antibody No.117.7 may be used for the first coating. Microtiter plates were prepared with plates containing BSA (0.5%), Tween20 and NaN₃(0.02%) was washed with PBS (blocking solution) and blocked with this solution overnight at 37 ℃. Test samples were diluted two-fold serially (starting from 1: 4) in blocking solution containing 0.1% NP40 and 0.65M NaCl and added to the wells (100. mu.l) and incubated at 37 ℃ for 4 hours. The microtiter plates were then washed 3 times with PBS containing 0.05% Tween20 (PBS/Tween), followed by addition of biotinylated monoclonal antibody No.234.14 (1: 1000 in blocking solution, but without NaN)₃100. mu.l/well), and cultured at 4 ℃ overnight. The microtiter plates were washed 3 times with PBS/Tween (100. mu.l/well) and incubated for 2 hours at room temperature after addition of streptavidin-horseradish peroxidase conjugate (Jackson labs.1: 10,000 in PBS/Tween solution, 100. mu.l/well). The microtiter plates were washed 3 times with PBS/Tween and 100. mu.l of freshly prepared ABTS substrate solution (2, 2' -azo-bis (3-ethylbenzothiazoline sulfonic acid, Sigma, 10 mg; 6.4 ml H) was added to each well₂O; 2.2 ml of 0.2M Na₂HPO₄(ii) a 1.4 ml of 0.2M citric acid; 1 microliter H₂O₂) To develop color. After 30 minutes the reaction was stopped by addition of 100. mu.l/well 0.2M citric acid. The microtiter plates were read at 405 nm using an automated ELISA reader and corrected for non-specific readings at 630 nm. The lower limit of detection for this test is 30 pg/ml.

The foregoing description of the specific embodiments reveals the general nature of the invention so that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept. Thus, such modifications should and are intended to be understood as falling within the meaning and range of equivalents of the embodiments disclosed herein. The phraseology and terminology used herein should be regarded as illustrative and not as limiting the scope of the invention.

Hybridomas 46.10, 117.7, and 234.14 were deposited at Pasteur institute [ Pasteur institute (CNCM) ] at 23.4.1996, under accession numbers I-1697, I-1698, and I-1699, respectively.

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Claims (18)

1. An isolated polypeptide or salt thereof comprising the amino acid sequence of a monoclonal antibody produced by the hybridoma designated NUR 2117.7 with accession number I-1698 from Pasteur research institute or by the hybridoma designated NUR 2234.14 with accession number I-1699 from Pasteur research institute.

2. A hybridoma designated NUR 2117.7 deposited under the Pasteur institute as accession number I-1698.

3. A hybridoma designated NUR 2234.14 deposited under the Pasteur institute as accession number I-1699.

4. The polypeptide of claim 1, which is a chimeric antibody.

5. The polypeptide of claim 1, which is a humanized antibody.

6. The polypeptide of claim 1, wherein said amino acid sequence is Fab or F (ab')₂And (3) fragment.

7. An isolated DNA molecule comprising a DNA segment encoding the polypeptide of claim 1.

8. An isolated DNA molecule comprising a DNA segment encoding the polypeptide of claim 6.

9. The DNA molecule of claim 7, wherein said DNA segment is operably linked to transcriptional and translational regulatory signals.

10. The DNA molecule of claim 8, wherein said DNA segment is operably linked to transcriptional and translational regulatory signals.

11. The DNA molecule of claim 7 which is an expression vector comprising said DNA fragment.

12. The DNA molecule of claim 8 which is an expression vector comprising said DNA fragment.

13. A host cell capable of expressing a polypeptide that binds to an IFN- α/β receptor, said host cell carrying the vector of claim 11.

14. A host cell capable of expressing a polypeptide that binds to an IFN- α/β receptor, said host cell carrying the vector of claim 12.

15. A method of producing a polypeptide that binds to an IFN- α/β receptor, comprising: culturing the host cell of claim 13 and expressing the polypeptide encoded by the expression vector contained therein.

16. A method of producing a polypeptide that binds to an IFN- α/β receptor, comprising: culturing the host cell of claim 14 and expressing the polypeptide encoded by the expression vector contained therein.

17. A pharmaceutical composition for attenuating or blocking IFN- α biological activity comprising the polypeptide of claim 1 and a pharmaceutically acceptable carrier.

18. Use of a pharmaceutical composition according to claim 17 for the manufacture of a medicament for attenuating or blocking the biological activity of IFN- α.