KR100759745B1 - -1 antibodies against interleukin-1 receptor and uses thereof - Google Patents

Abstract

Of IL-1 mediated secretion of IL-8 and IL-6 in human fibroblasts obtainable from the hybridoma cell line MAK <h-IL-1RI> 2D8 (DSM ACC 2601), or MRC5 (ATCC CCL 171) Human IL-1 that binds to human IL-1R and binds to IL-1R, characterized in that it is a chimeric, humanized or T cell epitope depleted antibody variant or fragment thereof of the antibody having an IC50 value of less than 35 pM for inhibition. Antibodies that inhibit the binding of have advantageous features for the treatment of inflammatory diseases.

Description

ANTIBODIES AGAINST INTERLEUKIN-1 RECEPTOR AND USES THEREOF

The present invention relates to antibodies against interleukin-1 receptor (IL-1R), methods for their preparation, pharmaceutical compositions containing said antibodies and their use.

Significant attention has been paid to the signal transduction pathways activated by the proinflammatory cytokine interleukin-1 (IL-1 alpha and IL-1 beta) because of the important role IL-1 plays in inflammatory diseases and inflammation and rheumatoid arthritis. Involved in joint destruction associated with

Numerous proteins have been described that participate in post-receptor activation of transcription factor nuclear factor κB (NF-κB), and stress-activated protein kinases such as p38 mitogen-activated protein kinase (MAPK). . Interleukin-1 receptor (IL-1R, Swiss Prot. P14778, CD 121a) is a member of this signaling which is an important determinant of the innate immune and inflammatory response. Therapeutic approaches to rheumatoid arthritis have evolved in recent years, in part due to the increased awareness of the severity of the condition and in part due to very significant advances in understanding the important role of cytokines in the immunological pathogenesis of the disease. The enemy has gone through a change. The main focus is based on rationale for targeting TNFα and IL-1. Recently published studies have confirmed that the use of various biological agents targeting TNFα leads to continuous improvement in the symptoms and signs of rheumatic disease, and further that TNFα blockage protects joints from structural injuries. Anakinra is an interleukin-1 receptor antagonist (IL-1Ra), which blocks IL-1 mediated action.

US Pat. No. 6,511,665 claims a monoclonal antibody that specifically binds to a human IL-1 receptor and blocks the binding of IL-1 to the IL-1 receptor.

It is an object of the present invention to provide novel antibodies against IL-1R, which are valuable means for the treatment of inflammatory diseases such as rheumatoid arthritis.

Summary of the Invention

Surprisingly, the presence of an antibody against IL-1R, indicating that the IC-50 value is 35 pM or less for IL-1 mediated inhibition of IL-8 (in human fetal lung fibroblasts such as MRC-5 cells). did.

The antibody according to the invention has epitope specificity for natural IL-1R and denatured IL-1R, binding of IL-1 to IL-1R and subsequent signal transduction (nuclear factor κ B (NF-κB) Activation). The antibodies bind to the soluble domain of IL-1R in its glycosylated form and exhibit an affinity of 300 pM, preferably up to 200 pM (K _D ). The antibodies show markedly reduced affinity for binding to deglycosylated IL-1R.

Antibodies according to the invention inhibit the binding of IL-1 to IL-1R in vitro and in vivo, and accordingly IL-1, IL-1 receptor and IL-1Racp (interleukin-1 receptor accessory protein; Q9NPH3) Inhibits the formation of tertiary complexes.

The present invention provides an IC-50 value for IL-1 mediated inhibition of Il-8 in human fibroblast MRC5 (ATCC CCL 171) of 35 pM or less, preferably hybridoma cell line MAK <h-IL-1RI> Human IL-binding to and binding to IL-1R, which is obtainable from 2D8 (DSM ACC 2601) or is a chimeric, humanized or T cell epitope depleted antibody variant or fragment thereof of said antibody. Antibodies that inhibit the binding of 1 are included.

The antibodies according to the invention preferably do not exhibit effector functions (ADCC and CDC) and are therefore IgG4 isotypes. Mutation of serine 228 to proline is particularly preferred (Angal, S., et al., Mol. Immunol. 30 (1993) 105-108). Alternatively, the antibodies are IgG1 isoform, preferably modified in the hinge region of about aa 220 to 240 between CH1 and CH2 and / or in the second interdomain region of about aa 330 between CH2 and CH3. (Numbering is based on Kabat's literature; references, for example, Johnson, G., and Wu, TT, Nucleic Acids Res. 28 (2000) 214-218) avoid agonist sensory. Switching of the IgG class can be easily carried out by the exchange of the constant heavy and light chains of the antibody by heavy and light chains from antibodies of the desired class, such as IgG ₁ or IgG ₄ . Such methods are well known in the art.

Antibodies according to the invention show advantages for patients in need of anti-inflammatory therapy. The antibodies according to the invention have novel and inventive features which provide advantages for patients suffering from such diseases, in particular rheumatoid arthritis. The antibody according to the invention is characterized by the abovementioned features.

More preferred are antibodies of rat origin and comprising the antibody sequence frame of the rat antibody according to Kabat. In the Kabat sequence amino acid 10 (serine), preferably the VL chain (DEL10) is deleted and / or the amino acid 26 (glycine) of the VH chain is changed to glutamic acid (G26E). Preferably, the antibody is a T cell epitope depleted using the method described in WO 98/08097.

IL-1R antagonists (Arend WP, J. Clin. Invest. 88 (1991) 1445-1451) do not inhibit the binding of IL-1R (10 nM) to immobilized antibodies according to the invention at concentrations up to 100 μM. More preferred (IL-1R antagonist), for example the binding of the antibody according to the invention to IL-1R is not inhibited by Il-1R antagonist.

The constant region is preferably a human IgG1 or human IgG4 constant region according to Kabat, E. (see literature below). Preferred constant regions are shown in FIGS. 14, 15 and 16.

The invention also encompasses antibody encoding nucleic acids. Coated polypeptides may form aggregates with each other antibody chain defined below:

Antibody heavy chain comprising CDR1 (aa 45-54), CDR2 (aa 69-84) and CDR3 (aa 117-123) of SEQ ID NO: 1 as CDR, CDR1 (aa 43-57) of SEQ ID NO: 2 as CDR , Antibody light chain comprising CDR2 (aa 73-79) and CDR3 (aa 112-120). CDR numbering and definitions, including signal sequences, were according to Kabat, E. (see, eg, Johnson, G., and Wu, T. T., Nucleic Acids Res. 28 (2000) 214-218 for reference).

Preferably, the nucleic acid encodes a polypeptide which is a heavy chain consisting of the variable region (VH) of SEQ ID NO: 1 and a light chain consisting of the variable region of SEQ ID NO: 2.

The antibody is preferably a monoclonal antibody, in addition, a chimeric antibody (human constant chain), a humanized antibody, and particularly preferably a T cell epitope depleting antibody.

The antibody binds to human IL-1R in competition with an antibody characterized by the variable region of SEQ ID NO: 2.

The antibody is further characterized by an affinity of 300 pM or less, preferably 200 pM (K _D ) or less, more preferably about 70 to 200 pM.

Accordingly, the present invention also encompasses polypeptides and coding nucleic acids selected from the group consisting of CDR1, CDR2, CDR3 of the heavy chain and CDR1, CDR2, CDR3 of the light chain as described above.

The present invention further provides a hybridoma cell line providing the antagonist monoclonal antibody according to the present invention.

The hybridoma cell line MAK <h-IL-1RI> 2D8, the preferred hybridoma cell line according to the present invention, was developed in 2003 under the accession number DSM ACC 2601, in accordance with the Budapest Treaty on the International Regulation of Microbial Deposits for the purpose of the patent procedure. It was deposited on July 10 in Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) in Germany.

Antibodies obtainable from such cell lines are preferred embodiments of the present invention. It can also be combined from the variable and constant regions shown in FIGS. 7-10 and 14-16, wherein IC-50 values for IL-1 mediated secretion inhibition of Il-8 in human fibroblast MRC5 (ATCC CCL 171) All antibodies that are 35 pM or less are also preferred. The sequences are examples of sequences obtained by modifying the sequence of antibody 2D8 to obtain improved antibodies that possess superior characteristics of antibody 2D8 that are IC50 and / or epitope characteristic. In such antibodies, the light and heavy chains from FIGS. 7 and 8 (T cell epitope depletion) or from FIGS. 9 and 10 (humanization) are combined with the constant regions from FIGS. 14 and 15 or 16. Antibodies DEI5 / 7, DEI4 / 7, DEI2 / 4, DEI5 / 4, DEI4 / 5, DEI5 / 5, HUM2 / 2, HUM2 / 3, DEI1 / 8, DEI2 / 8, DEI2 / 9, DEI4 / 9, DEI5 / 8 and DEI5 / 9 are particularly preferred.

The invention further provides a method for the identification and / or preparation of antibodies to IL-1R having improved characteristics according to the invention. In this method, the polypeptide sequence of antibody 2D8 is modified by mutation, deletion or addition of amino acids to make the antibody less immunogenic when administered to humans. In general, up to about 50 amino acids are modified in variable heavy and light chains to reduce immunogenicity. The modification is performed by comparing the polypeptide sequence of 2D8 with the sequence of the human antibody provided by Kabat (in the above quotation) and / or identifying and removing T cell epitopes in the variable chain. Examples of such modifications are shown in FIGS. 7-10. Useful antibodies can be generated with one or more changes in amino acids shown in the frame in the figure. Preferably, about 20-50 amino acids are modified in the region indicated by the frame of the variable chain.

Thus, the present invention mutates, deletes or adds one or more amino acids for each amino acid (s) shown in the frames shown in FIGS. 7 to 10 in the sequence of the variable region of antibody 2D8, wherein the expression vector is a continuous reading frame. An expression vector is prepared to contain within said modified antibody variable region and preferably a nucleic acid encoding an additional human constant region, as shown in FIGS. 14-16, wherein expression is performed in a host cell and recombinantly Provided is a method for producing an antibody according to the present invention, wherein the produced antibody light and heavy chains form an aggregate with the antibody according to the present invention.

The invention further provides a nucleic acid encoding said antibody, an expression vector comprising said nucleic acid and a host cell comprising said vector for recombinant production of said antibody.

The present invention further provides a method for recombinant production of such antibodies.

The present invention further comprises administering to a patient diagnosed with rheumatoid arthritis (hence the following therapy) an effective amount of an antagonist antibody against IL-1R according to the present invention, and / or rheumatoid arthritis Provided are methods of treating osteoarthritis. The antibody is preferably administered in a pharmaceutical composition.

The invention further comprises the use of an antibody according to the invention for the treatment of rheumatoid arthritis and for the preparation of a pharmaceutical composition according to the invention. In addition, the present invention includes a method for preparing a pharmaceutical composition according to the present invention.

In addition, the present invention encompasses pharmaceutical compositions containing a pharmaceutically effective amount of the antibody according to the invention, optionally with buffers and / or additional excipients useful in the formulation of the antibody for pharmaceutical purposes.

The invention further provides a pharmaceutical composition comprising said antibody in a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition may be included in an article of manufacture or kit.

The invention further comprises a vector comprising the nucleic acid according to the invention, which is capable of expressing said nucleic acid in a prokaryotic or eukaryotic host cell.

The invention further comprises prokaryotic or eukaryotic host cells comprising the vector according to the invention.

The invention further comprises a method for producing a recombinant human antibody according to the invention, characterized by expressing the nucleic acid according to the invention in a prokaryotic or eukaryotic host cell and recovering said antibody from said cell. The present invention further includes an antibody obtainable by the above recombinant method.

Detailed description of the invention

The term “antibody” includes, but is not limited to, intact antibodies, antibody fragments, humanized antibodies, chimeric antibodies, T cell epitope depleting antibodies, and additional genetically engineered antibodies so long as the characteristic features of the present invention are maintained. To encompass various types of antibodies.

"Antibody fragments" comprise a portion of a full length antibody, generally at least an antigen binding portion or variable region thereof. Examples of antibody fragments include diabodies, single chain antibody molecules, immunotoxins and multispecific antibodies formed from antibody fragments. In addition, the antibody fragment has a characteristic of a VH chain, ie can form an aggregate with the VL chain, or has a characteristic of a VL chain capable of binding to IL-1R, ie a functional antigen binding that binds to a pocket. And single chain polypeptides that can form aggregates with the VH chains against, thereby providing the property of inhibiting binding of IL-1 to IL-1R.

The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to the preparation of antibody molecules of single amino acid composition. The term “chimeric antibody” includes variable regions from rats, such as binding regions and one or more constant regions from different sources or species, and are generally prepared by recombinant DNA technology. Particular preference is given to chimeric antibodies comprising rat variable regions and human constant regions. The rat / human chimeric antibody is the product of an expressed immunoglobulin gene comprising a DNA segment encoding a rat immunoglobulin variable region and a DNA segment encoding a human immunoglobulin constant region. Other forms of "chimeric antibodies" encompassed by the present invention are those whose class or subclass has been modified or changed from that of the original antibody. Such "chimeric" antibodies are also referred to as "class-switched antibodies". Methods of making chimeric antibodies involve conventional recombinant DNA and gene transfection techniques currently well known in the art. References include, for example, Morrison, S. L., et al., Proc. Natl. Acad Sci. USA 81 (1984) 6851-6855; US Patents 5,202, 238 and 5,204,244.

The term “humanized antibody” refers to an antibody wherein the framework or “complementarity determining region” (CDR) is modified to include CDRs of immunoglobulins of different specificity compared to that of the parent immunoglobulin. In a preferred embodiment, the rat CDRs are grafted into the framework regions of human antibodies to produce "humanized antibodies." References include, for example, Riechmann, L., et al., Nature 332 (1988) 323-327; And Neuberger, M. S., et al., Nature 314 (1985) 268-270. Particularly preferred CDRs correspond to those representing sequences recognizing the antigens indicated above for chimeric and bifunctional antibodies. Examples of humanized antibodies according to the invention are shown in FIGS. 9 and 10.

The term “T cell epitope depleting antibody” refers to an antibody that has been modified or eliminated or reduced immunogenicity by removing human T cell epitopes (peptide sequences in proteins with a capacity to bind MHC class II molecules). In this way, the interaction between the amino acid side chains of the peptide and the specific binding pocket with MHC class II binding grooves is identified. The identified immunogenic regions are mutated for immunogenic elimination. The method is generally described, for example, in WO 98/52976. Examples of T cell epitope depleted antibody variable regions useful in the present invention and thus are shown in FIGS. 7 and 8.

As used herein, “binding” refers to antibody binding to IL-1R, having an affinity of about 300 pM or less, preferably about 200 pM or less (K _D ), more preferably about 70 to 200 pM. Refers to.

The term “nucleic acid molecule”, as used herein, is intended to include DNA molecules and RNA molecules. The nucleic acid molecule may be single stranded or double stranded, but is preferably double stranded DNA.

As used herein, “variable region” (variable region of the light chain (VL), variable region of the heavy chain (VH)) means each pair of light and heavy chains that are directly involved in binding of the antibody to the antigen. The domains of the variable light and heavy chains have the same general structure, each domain having four broadly conserved (FR) regions with broadly conserved sequences that are linked to three “high-variable regions” (or complementarity determining regions, CDRs). It includes. The framework region adopts a β-sheet structure, and the CDRs may form a loop connecting the β-sheet structure. The CDRs in each chain are captured within their three-dimensional structure by the framework regions, and together with the CDRs from other chains form antigen binding sites. The antibody heavy and light chain CDR3 regions play a particularly important role in the binding specificity / affinity of the antibodies according to the invention, thus providing a further object of the invention.

The term “solid region” or “antigen binding portion of an antibody” when used herein refers to an amino acid residue of an antibody that contributes to antigen binding. The high variant region includes amino acid residues derived from "complementarity determining regions" or "CDRs". "Framework" or "FR" regions are those of the variable domain region other than the high variant region residues defined herein. Thus, the light and heavy chains of the antibody comprise the N- to C-terminal domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. In particular, CDR3 of the heavy chain is a region that contributes mostly to antigen binding. CDR and FR regions are determined by the standard definition of Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) and / or "variable loops". Is a residue from.

As used herein, the term “binding to IL-1R” refers to IL-1R in an in vitro ELISA assay, preferably in a binding assay using microtiter plates coated with biotinylated human IL-1R and streptavidin. Means the binding affinity of the antibody. Binding affinity for IL-1R can also be studied by the Biacore assay (Biacore AB, Uppsala, Sweden). The affinity of binding is defined by the terms k _on (rate constant for association of antibody from antibody / antigen complex), k _off (dissociation constant) and K _D (k _on / k _off ). The antibody according to the present invention has a K _D of 300 pM or less, preferably 200 pM or less, more preferably 70 to 200 pM, and inhibits binding to IL-1R and preferably IL-1, preferably IL-1. It binds to IL-1R at the same position where it binds.

The term "IL-1R expressing cell" refers to a cell expressing an IL-1 receptor. The cells are, for example, human fibroblasts such as MRC5 cells.

Antibodies according to the invention exhibit binding to the same epitope of IL-1R as antibody 2D8, or are inhibited in binding to IL-1R due to steric hindrance interference of binding. Binding inhibition can be detected by competitive assays with immobilized IL-1R and antibody 2D8. A signal reduction of at least 50% indicates that the antibody competes with antibody 2D8.

The term "epitope" refers to protein determinants capable of specific binding to an antibody. Epitopes generally consist of chemically active surface groups of molecules such as amino acids or sugar side chains and generally have specific three dimensional structural characteristics as well as specific charge characteristics.

The conformational and nonstereotropic epitopes are distinguished by the loss of binding to the former but not the latter in the presence of a denatured solvent. Epitopes of IL-1R that specifically bind to antibodies according to the invention are recognized by antibodies according to the invention in both natural and denatured IL-1R. Antibodies according to the invention are at least about 50 times, preferably at least 100 times stronger than human deglycosylated IL-1R (measured by Western blot after treatment with N-glycosidase F). (Glycosylation, soluble extracellular domain). Thus, the antibodies according to the invention show much reduced affinity for binding to deglycosylated IL-1R.

Antibodies according to the invention further include antibodies, nucleotides and amino acid sequence modifications having "constant sequence modifications" which do not affect or alter the above mentioned features of the antibodies according to the invention. Modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include the replacement of amino acid residues with amino acid residues having similar side chains. Family of amino acids with similar side chains are defined in the art. The family includes amino acids with basic side chains (eg lysine, arginine, histidine), amino acids with acidic side chains (eg aspartic acid, glutamic acid), amino acids with uncharged polar side chains (eg glycine , Asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with beta-branched side chains For example, threonine, valine, isoleucine) and amino acids with aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine). Accordingly, predetermined non-essential amino acid residues in human anti-IL-1R antibodies may preferably be substituted with another amino acid residue from the same side chain family.

Amino acid substitutions are described in Riechmann, L., et al., Nature 332 (1988) 323-327 and Queen, C., et al., Proc. Natl. Acad. Sci. USA 86 (1989) 10029-10033, by mutagenesis based on molecular modeling as described.

Antibodies according to the invention preferably exhibit an in vivo serum half-life (Cynomolgus or human) of at least about 5 days, preferably 8 to 15 days.

Antibodies according to the invention are preferably prepared by recombinant means. Such methods are well known in the art and include protein expression in prokaryotic and eukaryotic cells, subsequent isolation of antibody polypeptides, and purification to generally pharmaceutically acceptable purity. For protein expression, nucleic acids or fragments thereof that encode light and heavy chains are inserted into expression vectors by standard methods. Expression is carried out in suitable prokaryotic or eukaryotic host cells, such as CHO cells, NSO cells, SP2 / 0 cells, HEK293 cells, COS cells, yeast, or E. coli cells, and the antibody is fused to the cells (lysed). Then supernatant or cells).

Recombinant production of antibodies is well known in the art and described, for example, in Makrides, S. C., Protein Expr. Purif. 17 (1999) 183-202; Geisse, S., et al., Protein Expr. Purif. 8 (1996) 271-282; Kaufman, R. J., Mol. Biotechnol. 16 (2000) 151-161; Werner, R. G., Drug Res. 48 (1998) 870-880.

Antibodies may be present in whole cells, cell lysates, or in particular in purified or substantially pure form. Purification is a standard technique, including alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other known in the art, and includes other cellular components or other contaminants such as other cellular nucleic acids or This is done to remove the protein. For references, see Ausubel, F., et al., Ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

Expression in NSO cells is described, for example, in Barnes, L. M., et al., Cytotechnology 32 (2000) 109-123; And Barnes, L. M., et al., Biotech. Bioeng. 73 (2001) 261-270. Transient expression is described, for example, in Durocher, Y., et al., Nucl. Acids. Res. 30 (2002) E9. Cloning of various domains is described by Orlandi, R., et al., Proc. Natl. Acad. Sci. USA 86 (1989) 3833-3837; Carter, P., et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; And Norderhaug, L., et al., J. Immunol. Methods 204 (1997) 77-87. Preferred transient expression systems (HEK 293) are described in Schlaeger, E.-J., and Christensen, K., Cytotechnology 30 (1999) 71-83 and by Schlaeger, E.-J., in J. Immunol . Methods 194 (1996) 191-199.

Suitable control sequences for prokaryotes include, for example, promoters, optionally effector sequences, and ribosomal binding sites. Eukaryotic cells are known to utilize promoters, enhancers and polyadenylation signals.

Nucleic acids are “operably linked” when located in a functioning relationship with another nucleic acid sequence. For example, the DNA for a presequence or secretory leader is operably linked to the DNA for a polypeptide when expressed as a preprotein that participates in the secretion of the polypeptide; A promoter or enhancer is operably linked to a coding sequence when affecting the transcription of the sequence; Or the ribosome binding site is operably linked to a coding sequence when positioned to facilitate translation. In general, “operably linked” means that the DNA sequences are linked in series, and in the case of a secretion leader, it is continuous and linked within the reading frame. However, enhancers are not necessarily continuous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice.

Monoclonal antibodies are suitably isolated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A-sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography. DNA and RNA encoding monoclonal antibodies are readily isolated and sequenced using conventional procedures. Hybridoma cells can be provided as a source of such DNA and RNA. Once isolated, the DNA can be inserted into an expression vector, followed by transfection with a host cell, such as HEK 293 cells, CHO cells, or myeloma cells that do not produce immunoglobulin proteins, thereby synthesizing recombinant monoclonal antibodies in the host cell. To obtain.

Amino acid sequence variants of human IL-1R antibodies are prepared by introducing appropriate nucleotide changes into the antibody DNA or by peptide synthesis. The modification can be carried out but can only be carried out in a very limited range, for example as described above. For example, modifications do not alter the above mentioned antibody characteristics, such as IgG isoforms and epitope binding, and may improve the yield of recombinant products, protein stability or facilitate purification.

Any cysteine residue that is not involved in maintaining the proper placement of the anti-IL-1R antibody may also be generally substituted with serine, improving the oxidative stability of the molecule and preventing variant crosslinking. In contrast, cysteine binding (s) can be added to the antibody to improve its stability (especially when the antibody is an antibody fragment such as an Fv fragment).

Another type of amino acid variant of an antibody alters the original glycosylation pattern of the antibody. By altering is meant deleting one or more carbohydrate moieties found in the antibody and / or adding one or more glycosylation sites present in the antibody. Glycosylation of antibodies is typically N-linked. N-linking refers to the binding of a carbohydrate residue to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, wherein X is any amino acid except proline, are recognition sequences for the enzymatic binding of carbohydrate moieties to the asparagine side chains. Thus, the presence of any such tripeptide sequence in a polypeptide creates a potential glycosylation site. The addition of glycosylation sites to the antibody is simply accomplished by altering the amino acid sequence to include one or more tripeptide sequences (N-linked glycosylation sites) described above.

Nucleic acid molecules encoding amino acid sequence variants of the anti-IL-1R are prepared using a variety of methods known in the art. Such methods include, but are not limited to, preparation by PCR from natural sources (for naturally occurring amino acid sequence variants) or oligonucleotide-mediated (or site directed) mutagenesis, PCR mutagenesis, and humanized or T cell epitopes. Earlier prepared variant or non-variant versions of depleted anti-IL-1R antibodies are included.

Another type of covalent modification involves the chemical or enzymatic coupling of glycosides to the antibody. This process is advantageous because it does not require antibody production in host cells with glycosylation capacity for N- or O-linked glycosylation. Depending on the coupling embodiment employed, the sugar (s) can be (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as cysteine, (d) free hydroxyl groups such as serine , Threonine or hydroxyproline, (e) aromatic residues such as phenylalanine, tyrosine or tryptophan, or (f) glutamine amide groups. The methods are described in WO 87/05330 and in Aplin, J. D., and Wriston, J. C. Jr., CRC Crit. Rev. Biochem. 4 (1981) 259-306.

Removal of any carbohydrate moiety present on the antibody can be performed chemically or enzymatically. Chemical deglycosylation requires exposing the antibody to compound trifluoromethanesulfonic acid or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugars (N-acetylglucosamine or N-acetylgalactosamine), while at the same time liberating the antibody intact. Chemical deglycosylation is described by Sojahr, H. T., and Bahl, O. P., Arch. Biochem. Biophys. 259 (1987) 52-57 and Edge, A., et al., Anal. Biochem. 118 (1981) 131-137. Enzymatic cleavage of the carbohydrate moiety on an antibody is described by Thotakura, N. R., and Bahl, O. P., Meth. Enzymol. 138 (1987) 350-359, can be accomplished with the use of various endo- and exo-glycosidases.

Another type of covalent modification of an antibody is described in US Pat. No. 4,640,835; No. 4,496,689; No. 4,301,144; No. 4,670,417; Linking the antibody to one of a variety of nonproteinaceous polymers, such as polyethylene glycol, polypropylene glycol or polyoxyalkylene, by the methods described in US Pat. No. 4,791,192 or 4,179,337.

The present invention preferably encodes a polypeptide that binds to IL-1R, selected from the group consisting of a heavy chain consisting of the variable region (VH) of SEQ ID NO: 1 and a light chain consisting of the variable region (VL) of SEQ ID NO: 2 Including nucleic acid fragments inhibits the binding of IL-1 to IL-1R.

The reconstructed heavy and light chain variable regions are combined with sequences of promoters, translation initiation, constant regions, 3′-untranslated, polyadenylation, and transcription termination to form expression vector constructs. The heavy and light chain expression constructs can be combined into a single vector, co-transfected, serially transfected, or separately transfected into host cells, followed by fusion It is possible to form a single host cell that expresses all chains.

Accordingly, the present invention encodes a heavy chain consisting of the variable region (VH) of SEQ ID NO: 1 and a light chain consisting of the variable region (VL) and human light chain constant region (CL) of SEQ ID NO: 2 in prokaryotic or eukaryotic host cells. Provided is a method for producing a recombinant human antibody according to the present invention, characterized by expression of a nucleic acid and recovery of the antibody from the cell.

The invention further comprises the use of the antibody of the invention for the in vitro diagnosis of IL-1R by measuring the binding between IL-1R of the sample and the antibody according to the invention, preferably by immunological assay. .

In another aspect, the present invention provides a composition, eg, a pharmaceutical composition, comprising one or more combinations of the monoclonal antibodies or antigen binding portions thereof of the invention formulated together with a pharmaceutically acceptable carrier.

As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg injection or infusion).

"Pharmaceutically acceptable salt" refers to salts which retain the desired biological activity of the antibody and which exhibit no undesirable toxicological effects (see, eg, Berge, SM, et al., J. Pharm. Sci. 66 (1977) 1-19). Such salts are included in the present invention. Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloride.

The composition of the present invention can be administered by various methods known in the art. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired result.

In order to administer a compound of the present invention by a particular route of administration, it may be necessary to coat the compound with, or to administer with, a substance that prevents its inactivation. For example, the compound may be administered to the subject in a suitable carrier, such as liposomes, or diluents. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the provisional formulation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art.

As used herein, the phrases “parenteral administration” and “administering parenterally” refer to modes of administration other than oral and topical administration, generally by injection, including but not limited to intravenous, muscle Intra, intraarterial, intradural, intracapsular, orbital, intracardia, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intrathecal, intradural and episternal (intrasternal) ) Injections and infusions.

The composition may also include excipients such as preservatives, wetting agents, emulsifiers and dispersants. Prevention of the presence of microorganisms can be ensured by sterile processes or aseptic conditions and by inclusion of various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, delayed absorption of the injectable pharmaceutical form may be enabled by the inclusion of reagents that delay absorption such as aluminum monostearate and gelatin.

Regardless of the route of administration chosen, the compounds of the present invention can be used in suitable hydrated forms and / or in pharmaceutical formulations of the invention, and formulated in pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art. Become

The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention may be variable to obtain an amount of the active ingredient which is not toxic to the patient and which is effective to achieve the desired therapeutic response for the particular patient, composition and mode of administration. have. The dosage level selected is the activity of the particular composition, ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of treatment, other drugs, compounds used in combination with the particular composition employed and And / or various pharmacokinetic factors, including factors known in the medical arts, such as materials, age, sex, weight, condition, general health, and past medical history of the patient to be treated.

The composition must be sterile and must be liquid to the extent that the composition can be delivered by a syringe. In addition to water, the carrier may be isotonic buffered saline, ethanol, polyols (eg, glycerol, propylene glycol and liquid polyethylene glycols, etc.) and suitable mixtures thereof.

Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, by the use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be made possible by the inclusion of a reagent in the composition which delays absorption, for example aluminum monostearate or gelatin.

If the active compound is suitably protected as described above, the compound may be administered orally, for example using an inert carrier or an assimilable edible carrier.

The antibodies according to the invention can be used for the treatment of patients suffering from rheumatoid arthritis and in need of such therapy. Accordingly, the present invention includes a method of treating a patient suffering from rheumatoid arthritis.

The invention further provides a process for the preparation of a pharmaceutical composition containing an effective amount of an antibody according to the invention together with a pharmaceutically acceptable carrier and the use of the antibody according to the invention for said method.

The following examples, references and sequence listings are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It will be appreciated that modifications may be made in the process without departing from the spirit of the invention.

Brief description of the drawings

Figure 1 ELISA binding assay for 2D8 antibodies

Figure 2 Determination of affinity of 2D8 antibodies to IL-1R

Figure 3 Competition of 2D8 Antibodies with Binding of Ligand IL-1 to IL-1R

Figure 4 Inhibition of ternary component complex formation with hIL-1 / hIL-1R / hIL-1R AcP by 2D8 antibody

Upper line: binding of hIL-1 / hIL-1R to hIL-1R Acp

Lower Stroke Line: Addition of Antibody 2D8

Inhibition of IL-1 Inducible Production of IL-8 in MRC-5 Cells

6 Inhibition of IL-1 Inducible Production of IL-8 in Human Blood

Figure 7 Amino acid sequence of the variable region of the T cell epitope depleted heavy chain

Amino acid sequence of the variable region of the T cell epitope depletion light chain

9 amino acid sequence of the variable region of the humanized heavy chain

Figure 10 Amino acid sequence of the variable region of the humanized light chain

11 Amino acid sequence of IL-1R

Coarse N: Potential Glycosylation Sites

Figure 12 SDS PAGE analysis after treatment with glycosylated IL-1R and glycosidase

Figure 13 Western blot, glycosylated and deglycosylated IL-1R with 2D8

14 constant regions of light chains

15 constant region of heavy chain (IgG4)

16 constant region of heavy chain (IgG1)

Figure 17 heavy and light chains of DEI 5/8

Description of the sequence

The variable region of the heavy chain of SEQ ID NO: 1 rat 2D8; Aa1-19 signal sequence, 20-134 variable region, 135-139 terminal fragment of rat origin

The variable region of the light chain of SEQ ID NO 2 rat 2D8; Aa 1-20 signal sequence, 21-129 variable region, 130-138 terminal fragment, of rat origin

Amino acid sequence of SEQ ID NO: 3 2D8 chimeric H-chain (rat / human) (IgG1)

Amino acid sequence of SEQ ID NO: 4 2D8 chimeric H-chain (rat / human) (IgG4)

SEQ ID NO: 5 amino acid sequence of the 2D8 chimeric L-chain (rat / human)

Amino acid sequence of SEQ ID NO 6 HURVH (FIGS. 7 and 9)

Amino Acid Sequence of SEQ ID NO: 7 HURDIVHvl (FIG. 7)

SEQ ID NO: 8 HURDIVHv2 (FIG. 7)

SEQ ID NO: 9 HURDIVHv3 (FIG. 7)

Amino acid sequence of SEQ ID NO: 10 HURDIVHv4 (FIG. 7)

Amino acid sequence of SEQ ID NO: 11 HURDIVHv5 (FIG. 7)

Amino acid sequence of SEQ ID NO: 12 HURDIVHv6 (FIG. 7)

Amino Acid Sequence of SEQ ID NO: 13 HURVK (Figures 8 and 10)

Amino acid sequence of SEQ ID NO: 14 HURDIVKv4 (FIG. 8)

Amino acid sequence of SEQ ID NO: 15 HURDIVKv5 (FIG. 8)

Amino acid sequence of SEQ ID NO: 16 HURDIVKv7 (FIG. 8)

Amino acid sequence of SEQ ID NO: 17 HURDIVKv8 (FIG. 8)

Amino acid sequence of SEQ ID NO: 18 HURDIVKv9 (FIG. 8)

Amino acid sequence of SEQ ID NO 19 19 HURDIVKv10 (FIG. 8)

Amino Acid Sequence of SEQ ID NO: 20 HUR HuVH v1 (FIG. 9)

Amino Acid Sequence of SEQ ID NO: 21 HUR HuVH v2 (FIG. 9)

Amino acid sequence of SEQ ID NO: 22 HUR HuVH v3 (FIG. 9)

Amino Acid Sequence of SEQ ID NO: 23 AAB67785-1 (FIG. 9)

Amino Acid Sequence of SEQ ID NO: 24 HUR HuVK vl (FIG. 10)

Amino acid sequence of SEQ ID NO: 25 HUR HuVK v2 (FIG. 10)

Amino acid sequence of SEQ ID NO 26 HUR HuVK v3 (FIG. 10)

Amino acid sequence of SEQ ID NO: 27 CAD43025 (FIG. 10)

Amino Acid Sequence of SEQ ID NO: 28 IL-1R (FIG. 11)

SEQ ID NO: 29 sequence of the constant region of the light chain (FIG. 14)

Sequence of SEQ ID NO: 30 heavy chain IgG4 (FIG. 15)

Sequence of constant region of SEQ ID NO 31 31 IgG1 (FIG. 16)

The sequence of the heavy chain of SEQ ID NO: 32 DEI 5/8 (FIG. 17)

Sequence of the light chain of SEQ ID NO: 33 DEI 5/8 (FIG. 17)

Example

Example  One

h-IL-1R Rat Monoclonal  Generation of Antibodies

Hybridoma  culture

The resulting rat monoclonal antibodies were incubated at 37 ° C. and 5% CO ₂ in RPMI 1640 and 10% Hyclone medium (BioWhittaker) without serum.

Immunization process and Hybridoma  Occur

Five Sprague Dawley rats were immunized with recombinant human IL-1R producing inSF9 insect cells. Initial immunization (100 μg protein) was performed intraperitoneally with Freunds' adjuvant. All other immunizations were performed with incomplete Freund's adjuvant. Monoclonal antibodies were prepared by fusing NSO cells with non-cells derived from rats.

Example  2

IL-1R specific ELISA

(Intergen Company) 용액을 이용하여 15 내지 20 분 동안 RT 에서 암실 내의 쉐이커 상에서 검정을 진행했다다. 흡광도는 492 nm 를 참고 파장으로 하여 370 nm 에서 측정했다. 임의로는, 발색 반응은 20 ㎕ 의 1M H₂S0₄ 를 각각의 웰에 첨가하여 정지시킬 수 있고; 상기의 경우, 측정은 690 nm 을 참고 파장으로 하여 450 nm 에서 수행했다. 결과를 도 1 에 나타냈다. Titers of antibodies in anti-IL-1R or culture supernatants in serum of immunized mice were measured by antigen specific ELISA. Soluble biotinylated h-IL-1R at a concentration of 0.125 μg / ml in PBSBSA (PBS / 1% BSA) was coated in a 96 well plate (precoated with streptavidin) on a shaker at RT for 1 hour. The wells were then blocked with PBSBSA for 30 minutes at RT. Serum was previously diluted to 1/100 in PBSBSA and serially diluted to 1/6400. Supernatants were diluted by counting in the range from 1/100 to 1/10000 in PBSBSA. Diluted serum or supernatant was added to the wells and incubated on a shaker at RT for 2 hours. Pre-tap serum or culture medium was used as a negative control. 125 ng / ml rat anti-human-IL-1R-antibody (clone 2D8) was used as a positive control. Subsequently, the plates were washed twice with PBS / 0.05% Tween20, incubated with horseradish peroxidase (HRP) -conjugated rabbit-anti-rat IgG (Bethyl Laboratories Inc.) and at 1: 18750 in PBSBSA. Dilute on shaker with RT for 1 hour. Wash wells 4 times with PBS / 0.05% Tween20 and freshly prepared TM Blue

The assay was run on a shaker in the dark at RT for 15-20 minutes using the Intergen Company solution. The absorbance was measured at 370 nm using 492 nm as a reference wavelength. Optionally, the color reaction can be stopped by adding 20 μl of 1M H ₂ SO ₄ to each well; In this case, the measurement was performed at 450 nm with 690 nm as the reference wavelength. The results are shown in FIG.

Example  3

term- hIL Of binding properties of -1R antibody

3000 상에서 CM5 칩을 이용하여 수행했다. 커플링은 아민 커플링으로서 수행했다. 사용한 완충액은 PBST (PBS + 0.05% Tween) pH 7.4, 25℃) 였다. Emotion is BIACORE

It was performed using a CM5 chip on 3000. Coupling was performed as amine coupling. The buffer used was PBST (PBS + 0.05% Tween) pH 7.4, 25 ° C.).

a) Affinity emotion of anti-hIL-1R antibody against IL-1R

For affinity measurements, anti-Fcγ antibodies (rabbit-anti-human) were coupled to the chip surface for antibody presentation to IL-1R. Anti-IL-1R antibodies were bound to anti-Fcγ antibodies and recombinant human IL-1R extracellular domain was added to the solution at various concentrations. Association was measured by 60 seconds of IL-1R injection and dissociation was measured by washing the chip surface with buffer for 3 minutes. The affinity constant K _D (k _on / k _off ) for the antibody is as follows:

Antibody 2D8 100 pM

Chimeric 2D8 (IgG1) 93 pM

Chimeric 2D8 (IgG4) 105 pM

The data range for all measurements of all antibodies is between 70 and 120 pM, thus the affinity of the three antibodies is in the same range.

b) Binding of ligand IL-1 to IL-1R and competition of anti-hIL-1R antibodies

For this measurement, the same method as in a) was used. When hIL-1R binds to anti-hIL-1R antibody 2D8, hIL-1 no longer binds to the receptor. The antibodies block the binding site of hIL-1 on the receptor (FIG. 3).

c) Inhibition of ternary complex formation hIL-1 / hIL-1R / hIL-1R AcP by antibody anti-hIL-1R 2D8

For ternary complex detection, polyclonal anti-IgG antibodies were coupled to the chip surface for presentation of the fusion protein Fc / hIL-1R AcP (Swiss Prot Q9NPH3; R & D Sytems). The binding of hIL-1 / hIL-1R complexes in solution to immobilized hIL-1R AcP was detected. The antibody anti-hIL-1R 2D8 inhibited the formation of the ternary complex hIL-1 / hIL-1R / hIL-1R AcP when incubated with hIL-1R (FIG. 4).

Example  4

a) inhibition of IL-1 inducible production of IL-8 in MRC-5 cells

Inhibition of IL-1 inducible production of IL-8 in human fibroblasts was assessed in cellular bioassay. Human embryonic lung fibroblasts MRC-5 were stimulated with h-IL1β and production of IL-8 was assessed in a single step ELISA. Inhibition of stimulation by the addition of anti-h-IL-1R antibodies has been shown to block the function of the antibodies.

Bioassay was conducted according to the following assay procedure:

On day 1 MRC-5 cells were seeded at a density of 2.5 × 10 ³ cells per well in 1001 culture medium (10% FBS) and incubated for 24 hours in an incubator.

On day 2 the culture medium was removed and samples were added to 50 μl assay medium (1% FBS) (if testing purified antibody) or hybridoma-medium SF (if testing hybridoma supernatant). . Optimal dilution of hybridoma supernatant is from 1: 1000 to 1: 1,000,000). Incubation was performed in the incubator for 30 minutes. Subsequently, h-IL-1β, 50 μl / well (2 × concentrated!) Was added to assay medium (1% FBS, or 2% FBS when testing hybridoma supernatants), resulting in final FBS concentration. Is 1%. Additional incubation was performed at 37 ° C. for 7 hours and the supernatant (80 μl) was transferred to another 96-well plate.

b) h-IL-8-ELISA

Assays (Miller, MD, and Krangel, MS, crit. Rev. Immunol. 12 (1992) 17-46) were performed using undiluted supernatant according to the manufacturer's protocol (R & D Systems, USA) (blood and cerebrospinal fluid samples). Protocol). Maximum stimulus (antibody control) was about 750 pg / ml IL-8. IC 50 for inhibition of IL-8 secretion by antibodies is shown in FIG. 5 and below:

Antibody 2D8 7.2 pM (1.08 ng / ml)

Chimeric 2D8 (IgG1) 4.7 pM (0.71 ng / ml)

Chimeric 2D8 (IgG4) 5.1 pM (0.77 ng / ml)

The range for all experiments using the antibodies according to the invention was 4.0 to 35.0 pM.

A comparison of 2D8 using commercially available antibodies to Il-1R in this MRC-5 bioassay is shown in Table 1:

Based on the amino acid sequences of FIGS. 7-10, panels of humanized T cell antigen depleting antibodies were constructed by mutation and examined for IC50 as h-IL-8-ELISA. The results are shown in Table 2 for antibody 2D8 (1.0).

Explanation of Abbreviations Used:

HUM: combination of the sequences of FIGS. 9 and 10

DEI: Combination of Sequences of FIGS. 7 and 8

HUM2 / 2: combination of sequences HURHuVHv 2 and HURHuVKv 2

DEI1 / 8: combination of sequences HURDIVHv 1 and HURDIVKv 8

c) Determination of immune activity by ELISA

Relative immune activity of antibodies compared to 2D8 antibody was measured by ELISA. Soluble biotinylated human IL-1R was coated in 96-well-plates (precoated with streptavidin) for 1 hour at room temperature. After blocking with BSA, the antibodies were diluted and added to the wells and incubated for 2 hours at room temperature. Plates were washed three times and incubated with horseradish peroxidase (POD) conjugated rabbit-anti-rat-IgG for 1 hour at room temperature. After further washing, the amount of bound antibody was determined by addition of TM-Blue ^R and absorbance measurement at 370 nm.

The results are shown in Table 3.

Example  5

IL- in human blood 8 of  Inhibition of IL-1 Inducible Production

To assess the blocking of the ability of antibody 2D8 to inhibit IL-1-induced production of IL-8 in human blood, a bioassay was performed. Human blood collected with heparin (19 U / ml) was stimulated with 10 ng / ml of h-IL1β and production of IL-8 was measured in a single stage ELISA. Inhibition of stimulation by the addition of anti-h-IL-1R antibody demonstrates the functional blocking of the antibody in human ex vivo assays. Bioassay performed incubation with IL-1 in the absence or presence of antibody for 1 hour at 37 ° C., and the 1: 5 diluted sample was subjected to the presence of IL-8 according to the manufacturer's protocol (R & D Systems, USA). It was carried out in accordance with the description of Example 4 except that the test was carried out. IL-1 induced IL-8 production in human blood is inhibited by ˜50% at a concentration of 100 ng / ml 2D8 antibody (see FIG. 6).

Example  6

Antibody Recombinant  Produce

Vectors were constructed for the expression of chimeric antibodies consisting of human constant regions linked to murine variable regions. Two chimeric heavy chain expression vectors of anti-IL1R rat VH linked to human IgG1 and human IgG4 constant regions were constructed in the expression vector pSVgpt. A chimeric light chain vector consisting of anti-IL1R rat VK linked to human C κ in the expression vector pSVhyg was constructed. 5 proximity sequences comprising the leader signal peptide, leader intron and murine immunoglobulin promoter and 3 proximity sequences comprising the splice site and the intron sequence were introduced into the chimeric expression vector. Heavy and light chain expression vectors were introduced into NSO cells (ECACC No 85110503, non-immunoglobulin producing mouse myeloma) by electrophoresis. Transfected cell clones were screened for production of human antibodies by ELISA against human IgG.

Example  7

Of IL-1R Deglycosylation

5 μg of recombinant soluble human type 1 IL-1 receptor (IL-1R, amino acid sequence (cf. FIG. 11), purified from supernatant of transformed SF9 insect cells, respectively, was 3 mU neuraminidase (N) (Roche Diagnostics GmbH , DE; No. 269611), 0.3 U N-glycosidase F (Roche Diagnostics GmbH, DE; No. 1365185) and 0.15 mU U O-glycosidase (Roche Diagnostics GmbH, DE; No. 1347101) 37 Incubation was for 17 hours at ° C. After centrifugation, samples were analyzed by SDS-PAGE.

SDS-PAGE analysis (FIG. 12) showed that the molecular weight of IL-1R (lane 1) decreased about 9 kDa with the introduction of N-glycosidase F (lane 3), while neuraminidase (lane 2) and O-glyco It was proved that no significant difference in molecular weight was observed upon incubation with the cedase (lane 4). The combination of N-glycosidase F and neuraminidase (lane 5), N-glycosidase F and O-glycosidase (lane 6), or all three (lane 8), may be combined with any of the carbohydrate side chains. It did not result in further cleavage. The combination of neuraminidase and O-glycosidase does not remove the carbohydrate side chain (lane 7) at all.

The data demonstrate that IL-1R is glycosylated at one or more potential N-glycosylation sites and that N-linked glycosylation can be completely eliminated by incubation with N-glycosidase F. O-linked glycosylation was not detected at all.

Western blot data for 2D8 antibodies are shown in FIG. 13 (lanes as in FIG. 12).

Example  8

Epitope  decision

The binding region (epitope) of the antibody on the antibody molecule was determined by blotting and pepscan analysis.

For blot analysis, native or denatured recombinant, soluble human IL-1R (rshIL-1R) samples were spotted directly on the membrane, incubated with anti-hIL1R antibodies, and chemiluminescent detection using anti-FC gamma-POD antibodies. To detect. In addition, rshIL-1R was deglycosylated using N-glycosidase F, and both glycosylated and deglycosylated rshIL-1R were separated under reduced denaturation conditions on SDS-PAGE. Proteins are blotted onto PVDF-membrane, incubated with anti-hIL-1R antibody and detected by chemiluminescence detection using anti-FCγ-POD-antibody.

Dot blot analysis shows that the antibodies according to the invention specifically bind rshIL-1R under natural and denaturing conditions that specifically bind. Finally, Western blot analysis showed that the antibodies according to the invention recognize only glycosylated rshIL1R (natural or denatured). Deglycosylated hIL1R could not be detected by Western blot analysis. Other glycosylated proteins (eg erythropoietic factor, carboxypeptidase Y) could not be recognized as antibodies according to the present invention, so the recognition of glycosylated rshIL-1R was very specific.

In addition, biotinylated peptides (20 amino acids) representing additional extracellular domains of rshIL1R were synthesized for use in anti-IL-1R / rshIL-1R pepscan analysis. The peptide overlaps by 10 amino acids and comprises an inert N-terminal biotinylated spacer. Biotinylated peptides were coupled to micro titer plates coated with streptavidin, incubated with anti-hILIR antibodies and detected with anti-FCγ-antibodies. Bound peptides represent the recognized sequence epitopes of IL-1R.

Example  9

IL-1R Antagonist  In existence Il 2 for -1R D8  And DEI5 Of 8 of  Combination

Protein-protein interactions of anti-IL-1R antibodies with IL-1R are analyzed by surface plasmon resonance using the Biacore 3000 System. Association rate is measured by infusion at 2 minute intervals; The dissociation rate is measured in three steps of washing steps. Negative control data are subtracted from the original curve to correct the overall intrinsic baseline flow and reduce the noise signal. Two assay formats using antibodies immobilized on the chip surface and IL-1R were used in this study.

a) Anti-IL-1R antibody 2D8 is covalently fixed to the chip surface (CM5) by amine coupling. IL-1R is injected at a concentration of 10 nM under mass transfer conditions to determine the maximum signal to be provided as standard in inhibition studies. Inhibition of IL-1R (10 nM) binding was preincubated with IL-1R with increased concentrations (0.78 to 100 nM) IL-1R antagonists, IL-1 beta or DEI5 / 8 (HBS- over 20 minutes at room temperature). In P buffer). Subsequently, the sample is injected into flow cells and the interaction with the immobilized 2D8 antibody is measured.

b) IL-1 beta is covalently anchored to the chip surface (CM5) by amine coupling. IL-1R is injected at a concentration of 10 nM under mass transfer conditions to determine the maximum signal provided as a standard value. Inhibition of rshIL-1R (1OnM) binding is measured by preincubation of IL-1R with increased concentrations (0.78 to 100 nM) of IL-1R antagonist, rhIL-1 beta or DEI5 / 8. Subsequently, the sample is injected into flow cells and the interaction with immobilized rhIL-1 beta is measured.

IL-1R antagonists showed no inhibition of IL-1R binding at concentrations of 100 μM in the first assay. IL-1 beta showed half inhibition of the maximum (IC50) at 8 nM and DEI5 / 8 at 2 nM. In the second assay, IL-1R antagonists had an inhibitory activity (IC50) of 4 nM, DEI5 / 8 of 2 nM, and 2D8 of 2 nM.

Since IL-1R antagonists do not inhibit the binding of IL-1R to 2D8 at concentrations up to 100 μM, it was concluded that 2D8 and IL-1R antagonists do not bind IL-1R in a competitive manner. 2D8 induces a drastic structural change in IL-1R upon binding. This structural change in IL-1R no longer allows IL-1R antagonists to bind to the receptor. 2D8 and DEI5 / 8 show allosteric inhibition compared to IL-1R antagonists in binding to the receptor. IL-1 beta inhibits IL-1R binding to 2D8, which means competitive binding of IL-1 beta and 2D8 to IL-1R.

List of References

Attach sequence list electronic file

Claims (18)

IL-1 mediated secretion of IL-8 and IL-6 in human fibroblasts, such as antibodies obtainable from the hybridoma cell line MAK <h-IL-1RI> 2D8 (DSM ACC 2601), or MRC5 (ATCC CCL 171) Human IL-binding to IL-1R and binding to human IL-1R, characterized in that it is a chimeric, humanized or T cell epitope depleted antibody variant or fragment thereof of the antibody having an IC50 value of less than 35 pM for inhibition of An antibody that inhibits the binding of 1. The antibody of claim 1, which is produced by the hybridoma cell line MAK <h-IL-1RI> 2D8 (DSM ACC 2601). 3. The antibody of claim 1, wherein the antibody is IgG4 isotype or IgG1 isoform and is modified in the hinge region at about aa 220-240 between CH1 and CH2. 4. The antibody of claim 1, wherein the antibody comprises variable and constant regions selected from FIGS. 7 to 10 and 14 to 16. The antibody of claim 1 or 2, wherein the affinity is about 300 pM or less (K _D ). The antibody of claim 1 or 2, comprising the following sequence as the complementarity determining region (CDR): a) CDR1 (aa 45-54), CDR2 (aa 69-84) and CDR3 (aa 117-123) of SEQ ID NO: 1 as CDRs in the heavy chain, and b) CDR1 (aa 43-57), CDR2 (aa 73-79) and CDR3 (aa 112-120) of SEQ ID NO: 2 as CDRs in the light chain. The antibody according to claim 1 or 2, wherein the antibody is of rat origin. The antibody according to claim 1 or 2, obtainable from the hybridoma cell line MAK <h-IL-1RI> 2D8 (DSM ACC 2601). delete A pharmaceutical composition for anti-inflammatory therapy, comprising the antibody of claim 1 or 2 in a pharmaceutically effective amount. Hybridoma cell line MAK <h-IL-1RI> 2D8 (DSM ACC 2601). A method for preparing a pharmaceutical composition containing the antibody of claim 1 or 2 in a pharmaceutically effective amount. Nucleic acids encoding a polypeptide of any one of the following: a) or b), wherein each of the polypeptides can be sampled from each other: a) an antibody heavy chain comprising CDR1 (aa 45-54), CDR2 (aa 69-84) and CDR3 (aa 117-123) of SEQ ID NO: 1 as CDRs, or b) an antibody light chain comprising CDR1 (aa 43-57), CDR2 (aa 73-79) and CDR3 (aa 112-120) of SEQ ID NO: 2 as CDRs. An expression vector comprising the nucleic acid according to claim 13 and capable of expressing said nucleic acid in a prokaryotic or eukaryotic host cell. A prokaryotic or eukaryotic host cell comprising the vector of claim 14. A nucleic acid encoding a light chain according to claim 13 and a nucleic acid encoding a heavy chain are expressed in a prokaryotic or eukaryotic host cell, and the polypeptide is recovered from the cell. A method for producing a polypeptide that inhibits binding of IL-1. delete One or more amino acids in the sequence of the variable region of the antibody produced by the hybridoma cell line MAK <h-IL-1RI> 2D8 (DSM ACC 2601) are mutated, deleted or deleted for each indicated amino acid shown in FIGS. In addition, an expression vector comprising the modified antibody variable region and a nucleic acid encoding an additional human constant region as shown in FIGS. 14-16 is prepared within a continuous reading frame, and expression is performed in a host cell. A method for producing an antibody according to claim 1 or 2, characterized in that the recombinantly prepared antibody light and heavy chains are aggregated together with the antibody according to claim 1 or 2.

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