HK1061403B - ANTIBODIES TO HUMAN IL-1β - Google Patents

Description

Antibodies to human IL-1 beta

The present invention relates to antibodies to human interleukin-1 beta (IL-1 beta) and the use of such antibodies in the treatment of IL-1 mediated diseases and disorders.

Interleukin 1(IL-1) is an activity produced by cells of the immune system, which is a mediator of the acute phase inflammatory response. Inappropriate or excessive production of IL-1, and in particular IL-1 β, has been implicated in the pathology of a variety of diseases and disorders, examples of which are septicemia, septic or endotoxic shock, allergy, asthma, bone loss, ischemia, stroke, rheumatoid arthritis and other inflammatory diseases. Antibodies to IL-1 β have been proposed for the treatment of IL-1 mediated diseases and disorders; see, for example, WO 95/01997 and its introduction.

We have now prepared improved antibodies to human IL-1 β which may be useful in the treatment of IL-1 mediated diseases and disorders.

Accordingly, the present invention provides IL-1 β binding molecules comprising an antigen binding site comprising at least one immunoglobulin heavy chain variable region (V) comprising, in order, the hypervariable regions CDR1, CDR2 and CDR3_H) The CDR1 having the amino acid sequence Val-Tyr-Gly-Met-Asn, the CDR2 having the amino acid sequence Ile-Ile-Trp-Tyr-Asp-Gly-Asp-Asn-Gln-Tyr-Tyr-Ala-Asp-Ser-Val-Lys-Gly and the CDR3 having the amino acid sequence Asp-Leu-Arg-Thr-Gly-Pro; and direct equivalents thereof.

Accordingly, the present invention also provides an immunoglobulin light chain variable region (V) comprising at least one hypervariable region CDR1 ', CDR2 ' and CDR3 ' in sequence_L) The IL-1 β binding molecule of (a), the CDR1 ' having the amino acid sequence Arg-Ala-Ser-Gln-Ser-Ile-Gly-Ser-Leu-His, the CDR2 ' having the amino acid sequence Ala-Ser-Gln-Ser-Phe-Ser, and the CDR3 ' having the amino acid sequence His-Gln-Ser-Leu-Pro; and direct equivalents thereof.

In a first aspect, the invention provides a single domain IL-1 β binding molecule comprising a heavy chain variable region (V) as defined above_H) The isolated immunoglobulin heavy chain of (a).

In a second aspect, the invention also provides a polypeptide comprising a heavy chain (V)_H) And light chain (V)_L) An IL-1 β binding molecule of the variable region and direct equivalents thereof, wherein said IL-1 β binding molecule comprises at least one antigen binding site comprising:

a) immunoglobulin heavy chain variable region (V) comprising in sequence the hypervariable regions CDR1, CDR2 and CDR3_H)，The CDR1 has the amino acid sequence Val-Tyr-Gly-Met-Asn, the CDR2 has the amino acid sequence Ile-Ile-Trp-Tyr-Asp-Gly-Asp-Asn-Gln-Tyr-Tyr-Ala-Asp-Ser-Val-Lys-Gly, and the CDR3 has the amino acid sequence Asp-Leu-Arg-Thr-Gly-Pro, and

b) immunoglobulin light chain variable region (V) comprising in sequence the hypervariable regions CDR1 ', CDR 2' and CDR3_L) The CDR1 ' has the amino acid sequence Arg-Ala-Ser-Gln-Ser-Ile-Gly-Ser-Ser-Leu-His, the CDR2 ' has the amino acid sequence Ala-Ser-Gln-Ser-Phe-Ser, and the CDR3 ' has the amino acid sequence His-Gln-Ser-Ser-Ser-Leu-Pro.

Unless otherwise indicated, all polypeptide chains herein are described as having an amino acid sequence that starts at the N-terminus and ends at the C-terminus. When the antigen binding site comprises V_HAnd V_LWhere domains are provided, these domains may be located on the same polypeptide molecule or, preferably, each domain may be located on a different chain, i.e.V_HThe domain is part of an immunoglobulin heavy chain or a fragment thereof, and V_LIs part of an immunoglobulin light chain or fragment thereof.

"IL-1 beta binding molecule" refers to any molecule capable of binding to the IL-1 beta antigen alone or in conjunction with other molecules. The binding reaction can be reflected by standard methods (qualitative assays) including, for example, bioassay methods for determining the inhibition of the binding of IL-1 β to its receptor with reference to a negative control assay in which antibodies with unrelated specificity but of the same isotype, e.g., anti-CD 25 antibodies, are used, or any type of binding assay. Advantageously, the binding of the IL-1. beta. binding molecules of the invention to IL-1. beta. can be demonstrated in a competitive binding assay.

Examples of antigen binding molecules include antibodies produced by B cells or hybridomas as well as chimeric, CDR grafted, or human antibodies, or any fragment thereof, such as F (ab')₂And Fab fragments, as well as single chain or single domain antibodies.

Single chain antibodies consist of the variable regions of the heavy and light chains of an antibody, which are covalently bound together by a peptide linker, which usually consists of 10 to 30 amino acids, preferably 15 to 25 amino acids. Thus, the structure does not include the constant portions of the heavy and light chains, and the small spacer peptide is believed to be less antigenic than the entire constant portion. "chimeric antibody" refers to an antibody in which either the heavy chain constant region or the light chain constant region, or both, are of human origin, while the variable regions of both the heavy and light chains are of non-human (e.g., murine) origin or of human origin but are derived from different humans. By "CDR-grafted antibody" is meant an antibody in which the hypervariable regions (CDRs) are derived from a donor antibody, such as a non-human (e.g., murine) antibody or a different human antibody, while all or substantially all other portions of the immunoglobulin, e.g., the constant regions and highly conserved portions of the variable regions (i.e., framework regions), are derived from an acceptor antibody, such as a human antibody. However, a CDR-grafted antibody may contain a few amino acids of the donor sequence in the framework regions, for example in the portion of the framework regions adjacent to the hypervariable regions. "human antibody" refers to antibodies in which the constant and variable regions of both the heavy and light chains are of human origin, or are substantially identical to sequences of human origin, but not necessarily from the same antibody, including antibodies produced by mice in which the genes for the variable and constant parts of an immunoglobulin of murine origin have been replaced by their human-derived counterparts, as described in general terms in, for example, EP 0546073B 1, USP 5545806, USP 5569825, USP 5625126, USP 5633425, USP5661016, USP 5770429, EP 0438474B 1 and EP 0463151B 1.

Particularly preferred IL-1. beta. binding molecules of the invention are human antibodies, particularly ACZ885 antibodies, as described in the examples below.

Thus, in preferred chimeric antibodies, the variable regions of both the heavy and light chains are derived from human, such as the variable regions of ACZ885 antibodies shown in seq.id No.1 and seq.id No. 2. The constant region domain preferably also comprises suitable human constant region domains, see for example "sequences of proteins of immunological interest", Kabat, E.A. et al, US Department of Health and Humanservices, Public Health Service, National Institute of Health.

Hypervariable regions can be linked to any type of framework regions, but preferably the framework regions are of human origin. Suitable framework regions are described in Kabat e.a. et al, supra. Preferred heavy chain frameworks are human heavy chain frameworks, such as those of the ACZ885 antibody shown in seq. It consists of FR1, FR2, FR3 and FR4 regions in sequence. In a similar manner, seq. id.no.2 shows a preferred ACZ885 light chain framework consisting of FR1 ', FR 2', FR3 'and FR 4' regions in sequence.

Accordingly, the present invention also provides an IL-1 β binding molecule comprising at least one antigen binding site comprising a first domain having an amino acid sequence substantially identical to the sequence set forth in seq.id No.1 starting at amino acid 1 and ending at amino acid 118 or a first domain as described above and a second domain having an amino acid sequence substantially identical to the sequence set forth in seq.id No.2 starting at amino acid 1 and ending at amino acid 107.

Monoclonal antibodies directed against proteins naturally occurring in all humans are typically produced in non-human systems such as mice and are thus typically non-human proteins. This directly results in the induction of an adverse immune response when xenogenous antibodies produced by hybridomas are administered to humans, which is mediated primarily by the constant regions of xenogenous immunoglobulins. This clearly limits the utility of such antibodies, since they cannot be administered for long periods of time. Thus, it is particularly preferred to use single chain, single domain, chimeric, CDR-grafted antibodies, or in particular human antibodies, which are unlikely to elicit a substantial allogeneic response upon administration to humans.

In view of the foregoing, more preferred IL-1 β binding molecules of the invention are selected from the group consisting of human anti-IL-1 β antibodies and direct equivalents thereof, wherein said human anti-IL-1 β antibody comprises at least

a) An immunoglobulin heavy chain or fragment thereof comprising (i) a variable region comprising the hypervariable regions CDR1, CDR2 and CDR3 in sequence and (ii) a constant portion of a human heavy chain or fragment thereof; the CDR1 has the amino acid sequence Val-Tyr-Gly-Met-Asn, the CDR2 has the amino acid sequence Ile-Ile-Trp-Tyr-Asp-Gly-Asp-Asn-Gln-Tyr-Tyr-Ala-Asp-Ser-Val-Lys-Gly, and the CDR3 has the amino acid sequence Asp-Leu-Arg-Thr-Gly-Pro, and

b) an immunoglobulin light chain or fragment thereof comprising (i) a variable region comprising a hypervariable region and optionally further comprising hypervariable regions of CDR1 ', CDR 2' and CDR3 ', in sequence, and (ii) a constant portion of a human light chain or fragment thereof, the CDR 1' having the amino acid sequence Arg-Ala-Ser-Gln-Ser-Ile-Gly-Ser-Leu-His, the CDR2 'having the amino acid sequence Ala-Ser-Gln-Ser-Phe-Ser, and the CDR 3' having the amino acid sequence His-Gln-Ser-Leu-Pro.

Alternatively, the IL-1. beta. binding molecules of the invention may be selected from single chain binding molecules comprising an antigen binding site comprising

a) A first domain comprising in sequence the hypervariable region CDR1, CDR2 and CDR3, said hypervariable region having the amino acid sequence shown in seq. Id.No.1,

b) a second domain comprising the hypervariable region CDR1 ', CDR2 ' and CDR3 ', said hypervariable region having the amino acid sequence set forth in seq. ID.No.2, and

c) a peptide linker bound to the N-terminus of the first domain and the C-terminus of the second domain or to the C-terminus of the first domain and the N-terminus of the second domain.

As is well known, minor changes in the amino acid sequence, such as deletion, insertion or substitution of one, several or even more amino acids, may result in an allelic form of the original protein having substantially identical properties.

Thus, the term "direct equivalent thereof refers to any single domain IL-1. beta. binding molecule (molecule X),

(i) wherein the hypervariable regions CDR1, CDR2 and CDR3 as a whole are at least 80% homologous, preferably at least 90% homologous, more preferably at least 95% homologous to the hypervariable regions of seq. ID.No.1, and

(ii) it is capable of inhibiting the binding of IL-1 β to its receptor to substantially the same extent as a reference molecule having the same framework regions as molecule X but having the same hypervariable regions CDR1, CDR2 and CDR3 as set out in seq.id No. 1;

or any IL-1. beta. binding molecule (molecule X') having at least two domains per binding site,

(i) wherein the hypervariable regions CDR1, CDR2, CDR3, CDR1 ', CDR2 ' and CDR3 ' as a whole are at least 80% homologous, preferably at least 90% homologous, more preferably at least 95% homologous to the hypervariable regions depicted in seq. ID.No.1 and 2, and

(ii) it is capable of inhibiting the binding of IL-1 β to its receptor to substantially the same extent as a reference molecule having the same framework regions and constant parts as molecule X 'but having the same hypervariable regions CDR1, CDR2, CDR3, CDR 1', CDR2 'and CDR 3' as shown in seq.id.no.1 and 2.

In the present specification, two amino acid sequences are at least 80% homologous to each other if they have at least 80% identical amino acid residues at the same position when optimally aligned (gaps or insertions in the amino acid sequences are counted as non-identical residues).

Inhibition of IL-1 β binding to its receptor can be conveniently tested in a variety of assays, including those described hereinafter. The term "to the same extent" means that the reference molecule and the equivalent molecule exhibit a substantially consistent IL-1 β binding inhibition curve on a statistical basis in one of the assays described above. For example, inhibition of binding of IL-1 β to its receptor, typically the IL-1 β binding molecules of the invention have an IC, when measured as described above_50sIC at the corresponding reference molecule₅₀Preferably substantially the same as +/-. times.5.

For example, the assay used may be an IL-1 β binding competitive inhibition assay performed by soluble IL-1 receptor and the IL-1 β binding molecules of the invention.

Most preferably, the human IL-1 β antibody comprises at least

a) A heavy chain comprising a variable region having an amino acid sequence substantially identical to the sequence set forth in seq.id No.1 starting at amino acid 1 and ending at amino acid 118 and a human heavy chain constant portion; and

b) a light chain comprising a variable region having an amino acid sequence substantially identical to the sequence set forth in seq.id No.2 starting at amino acid 1 and ending at amino acid 107 and a constant portion of a human light chain.

The human heavy chain constant moiety may be gamma₁、γ₂、γ₃、γ₄、μ、α₁、α₂Delta or epsilon, preferably gamma, more preferably gamma₁Type, whereas the human light chain constant portion may be of the kappa or lambda type (which includes lambda)₁、λ₂And λ₃Subtype) but is preferably the kappa type. The amino acid sequences of all of these constant portions can be found in Kabat et al, supra.

The IL-1. beta. binding molecules of the invention may be prepared by recombinant DNA techniques. In view of this, one or more DNA molecules encoding the binding molecule must be constructed, placed under the appropriate control sequences and transferred to a suitable host organism for expression.

Accordingly, the present invention provides in a very general sense

(i) DNA molecules encoding the single domain IL-1. beta. binding molecules of the invention, single chain IL-1. beta. binding molecules of the invention, heavy or light chains of the IL-1. beta. binding molecules of the invention or fragments thereof, and

(ii) use of a DNA molecule of the invention for the preparation of an IL-1. beta. binding molecule of the invention by recombinant means.

The state of the art is that the person skilled in the art is able to synthesize the DNA molecules of the invention on the basis of the information provided herein, i.e.the amino acid sequences of the hypervariable regions and the DNA sequences coding for them. Methods for constructing variable region genes are described, for example, in EPA 239400 and can be briefly summarized as follows: the genes encoding the variable regions of the MAb with any specificity were cloned. The DNA segments encoding the framework and hypervariable regions are identified and the DNA segments encoding the hypervariable regions are removed therefrom so that the DNA segments encoding the framework regions are fused together with appropriate restriction sites at the junctions. These restriction sites can be generated at the appropriate positions by means of mutagenesis of the DNA molecule by standard methods. Synthetic double-stranded CDR cassettes were prepared by DNA synthesis according to the sequence shown in seq. Id.No.1 or 2. The cassettes are provided with adhesive ends so that they can be attached at the joints of the framework.

Furthermore, it is not necessary to obtain mRNA from a producing hybridoma cell line in order to obtain a DNA construct encoding an IL-1. beta. binding molecule of the invention. Thus, PCT application WO90/07861 gives full teaching as to how antibodies can be prepared by recombinant DNA techniques with only written information about the nucleotide sequence of the antibody gene. The method involves synthesizing a large number of oligonucleotides, amplifying them by PCR methods, and splicing them into the desired DNA sequence.

Expression vectors containing suitable promoters or genes encoding the constant portions of the heavy and light chains are available from public sources. Thus, once the DNA molecule of the invention is prepared, it can be conveniently transferred to an appropriate expression vector. DNA molecules encoding single chain antibodies may also be prepared by standard methods, as described in WO 88/1649.

In view of the above, it is not necessary for the present application that a deposit of hybridomas or cell lines be made to meet the requirements of the full disclosure of the specification.

In a specific embodiment, the invention includes first and second DNA constructs for making IL-1 β binding molecules as described below:

the first DNA construct encodes a heavy chain or fragment thereof and comprises

a) A first portion encoding a variable region comprising alternately a hypervariable region and a framework region, which hypervariable region is in sequence a CDR1, a CDR2 and a CDR3 whose amino acid sequences are shown in seq.id No. 1; the first portion begins at a codon encoding the first amino acid of the variable region and ends at a codon encoding the last amino acid of the variable region, and

b) a second part encoding a heavy chain constant portion or fragment thereof, which starts with a codon encoding the first amino acid of the heavy chain constant portion and ends with a codon encoding the last amino acid of the constant portion or fragment thereof,

followed by a stop codon.

Preferably, the first portion encodes a variable region having an amino acid sequence substantially identical to the sequence set forth in seq.id No.1 starting at amino acid 1 and ending at amino acid 118. More preferably, the first portion has the nucleotide sequence of seq.id No.1 starting at nucleotide 1 and ending at nucleotide 354. Also preferably, the second portion encodes the constant part of a human heavy chain, more preferably the constant part of a human gamma 1 chain. The second part may be a DNA fragment (containing introns) or cDNA fragment (without introns) of genomic origin.

The second DNA construct encodes a light chain or fragment thereof and comprises

a) Encoding a first portion of variable regions comprising hypervariable regions and framework regions alternately; the hypervariable regions are CDR3 ' and optionally CDR1 ' and CDR2 ', their amino acid sequences are shown in seq.id No. 2; the first portion begins at a codon encoding the first amino acid of the variable region and ends at a codon encoding the last amino acid of the variable region, and

b) a second portion encoding a light chain constant portion or fragment thereof, which starts with a codon encoding the first amino acid of the light chain constant portion and ends with a codon encoding the last amino acid of the constant portion or fragment thereof,

followed by a stop codon.

Preferably, the first portion encodes a variable region having an amino acid sequence substantially identical to the amino acid sequence set forth in seq.id No.2 starting at amino acid 1 and ending at amino acid 107. More preferably, the first portion has the nucleotide sequence of seq.id No.2 starting at nucleotide 1 and ending at nucleotide 321. Also preferably, the second portion encodes a constant portion of a human light chain, more preferably a constant portion of a human kappa chain.

The invention also includes IL-1 β binding molecules in which one or more, typically only a few (e.g. 1 to 4) residues, of CDR1, CDR2, CDR3, CDR1 ', CDR2 ' or CDR3 ' or the framework are altered compared to the residues shown in seq.id No.1 and seq.id No. 2; this alteration can be achieved, for example, by mutation, such as site-directed mutagenesis, of the corresponding DNA sequence. The invention includes DNA sequences encoding such altered IL-1 β binding molecules. In particular, the invention includes IL-1. beta. binding molecules in which one or more residues of CDR1 'or CDR 2' are altered from the residues set forth in seq. Id.No. 2.

In the first and second DNA constructs, the first and second portions may be separated by an intron, and it may be convenient to place an enhancer in the intron between the first and second portions. This enhancer is transcribed but not translated, and its presence can help transcription proceed efficiently. In a particular embodiment, the first and second DNA constructs comprise an enhancer of the heavy chain gene, advantageously of human origin.

Each DNA construct is placed under the control of suitable control sequences, particularly a suitable promoter. Any type of promoter may be used, as long as it is suitable for the host organism into which the DNA construct is to be transferred for expression. However, if expression occurs in mammalian cells, it is particularly preferred to use the promoter of an immunoglobulin gene.

The desired antibody can be produced in cell culture or in transgenic animals. Suitable transgenic animals may be obtained according to standard procedures which include microinjection of the first and second DNA constructs placed under suitable control sequences into eggs, transfer of the eggs so prepared to suitable pseudopregnant female individuals and selection of offspring expressing the desired antibody.

When preparing antibody chains in cell culture, these DNA constructs must first be inserted into a single expression vector, or into two separate but compatible expression vectors, the latter possibility being preferred.

Thus, the present invention also provides an expression vector capable of replication in a prokaryotic or eukaryotic cell line and comprising at least one of the above-described DNA constructs.

The respective expression vector containing the DNA construct is then transferred into a suitable host organism. When the DNA constructs are inserted separately on two expression vectors, they can be transferred separately, i.e.one type of vector per cell, or co-transferred, this latter possibility being preferred. Suitable host organisms may be bacterial, yeast or mammalian cell lines, the latter being preferred. More preferably, the mammalian cell line is of lymphoid origin, such as myeloma, hybridoma or normal immortalised B cell, which advantageously does not express any endogenous antibody heavy or light chain.

For expression in mammalian cells, the coding sequence for the IL-1 β binding molecule is preferably integrated into the host cell DNA at a locus that allows or facilitates high level expression of the IL-1 β binding molecule. Cells in which the coding sequence for the IL-1 β binding molecule is integrated at such a favorable locus can be identified and selected based on the level of IL-1 β binding molecule expressed. Any suitable selectable marker may be used to prepare host cells containing the coding sequence for the IL-1 β binding molecule; for example, the dhfr gene/methotrexate or equivalent selection systems can be used. Other alternative systems for expression of the IL-1 β binding molecules of the invention include GS-based amplification/selection systems, such as those described in EP 0256055B, EP 0323997B and european patent application 89303964.4.

In a further aspect of the invention, there is provided a method of producing an IL-1 β binding molecule, comprising (i) culturing an organism transformed with an expression vector as described above and (ii) recovering the IL-1 β binding molecule from the culture.

According to the present invention, it has been found that the ACZ885 antibody exhibits binding specificity for an antigenic epitope of human IL-1 β that includes a loop containing Glu64 residues of mature human IL-1 β. (the Glu64 residue of mature human IL-1 β corresponds to residue 180 of the precursor human IL-1 β.) this epitope appears outside the recognition site of the IL-1 receptor, and it is therefore most surprising that antibodies directed against this epitope, such as ACZ885, are capable of inhibiting the binding of IL-1 β to its receptor. Antibodies, particularly chimeric and CDR grafted antibodies and particularly human antibodies, having binding specificity for an epitope of mature human IL-1 β comprising a loop containing residue Glu64 and capable of inhibiting the binding of IL-1 β to its receptor; and the use of these antibodies in the treatment of IL-1 mediated diseases and disorders are novel and are included within the scope of the present invention.

Thus, in a further aspect, the invention includes an anti-IL-1 β antibody having antigen binding specificity for an epitope of human IL-1 β that includes a loop containing residue Glu64 of mature human IL-1 β and capable of inhibiting the binding of IL-1 β to its receptor.

In other aspects, the invention comprises:

i) use of an anti-IL-1 β antibody in the treatment of an IL-1 mediated disease or disorder, wherein said antibody has antigen binding specificity to an epitope of mature human IL-1 β comprising a loop comprising residue Glu64 and is capable of inhibiting the binding of IL-1 β to its receptor;

ii) a method of treating an IL-1 mediated disease or disorder in a patient, the method comprising administering to the patient an effective amount of an anti-IL-1 β antibody, wherein the antibody has antigen-binding specificity for an epitope of mature human IL-1 β comprising a loop containing residue Glu64 and is capable of inhibiting the binding of IL-1 β to its receptor;

iii) a pharmaceutical composition comprising an anti-IL-1 β antibody, wherein said antibody has antigen-binding specificity for an epitope of mature human IL-1 β comprising a loop containing residue Glu64 and is capable of inhibiting the binding of IL-1 β to its receptor, and a pharmaceutically acceptable excipient, diluent or carrier; and

iv) use of an anti-IL-1 β antibody in the manufacture of a medicament for the treatment of an IL-1 mediated disease or disorder, wherein the antibody has antigen binding specificity to an epitope of mature human IL-1 β comprising a loop comprising residue Glu64 and is capable of inhibiting the binding of IL-1 β to its receptor.

For the purposes of this specification, an antibody is "capable of inhibiting the binding of IL-1 β" if it is capable of inhibiting the binding of IL-1 β to its receptor to substantially the same extent as compared to the ACZ885 antibody, wherein "to the same extent" has the meaning defined above.

The binding affinity of the ACZ885 antibody to IL-1 is higher than that of previously reported anti-IL-1 β antibodies, such as anti-human IL-1 β antibodies, to IL-1 β. Thus, the dissociation equilibrium constant K for the binding of ACZ885 to IL-1 β_DLess than about 50pM, for example about 35 pM. This high binding affinity makes ACZ antibodies particularly useful for therapeutic applications.

Thus, in a further aspect, the invention provides a K that binds to IL-1 β_DAn anti-IL-1 β antibody of about 50pM or less. As with the uses, methods and compositions described above for anti-IL-1 β antibodies having binding specificity for an antigenic determinant of mature human IL-1 β that includes a loop containing Glu64, this aspect of the invention also includes uses, methods and compositions involving these high affinity antibodies.

In this specification, the term "IL-1 mediated disease" includes all diseases and conditions in which IL-1 plays a direct or indirect role in a disease or condition, including the cause, development, progression, persistence, or pathology of the disease or condition.

In this specification, the term "treatment" refers to both prophylactic or preventative treatment as well as curative or disease modifying treatment, including treatment of patients susceptible to or suspected of having an infected disease, as well as patients who are ill or have been diagnosed with a disease or condition, and includes inhibition of clinical relapse.

As described aboveAn IL-1 β binding molecule, in particular an IL-1 β binding molecule according to the first and second aspects of the invention, having binding specificity for an epitope of mature human IL-1 β comprising a loop containing Glu64, in particular an antibody capable of inhibiting the binding of IL-1 β to its receptor; and a K of about 50pM or less for binding to IL-1 beta_DThe anti-IL-1. beta. antibodies of (a) are referred to herein as antibodies of the invention.

Preferably, the antibody of the invention is an IL-1 β binding molecule according to the first and second aspects of the invention. Advantageously, the antibodies of the invention are human antibodies, most preferably ACZ885 antibody or direct equivalent thereof.

The antibodies of the invention block the effects of IL-1 β on their target cells and are therefore useful in the treatment of IL-1 mediated diseases and disorders. These and other pharmacological activities of the antibodies of the invention may be demonstrated in standard test methods, for example as follows:

IL-1 beta-dependent PGE on primary human fibroblasts₂And neutralization of interleukin-6 production

PGE in Primary human skin fibroblasts₂And IL-6 production is dependent on IL-1. beta. TNF- α alone is not effective in inducing these inflammatory mediators but can act synergistically with IL-1. Primary skin fibroblasts can be used as a surrogate model for IL-1 induced cell activation.

Primary human fibroblasts were stimulated with recombinant IL-1 β or conditioned media obtained from LPS-stimulated human PBMC in the presence of various concentrations of the antibody of the invention or IL-1RA (6-18,000 pM). Use of chimeric anti-CD 25 antibodies(basiliximab) as isotype-matched control. Supernatants were removed 16 hours after stimulation and analyzed for IL-6 by ELISA. For inhibition of IL-6 production, the antibodies of the invention typically have an IC of about 1nM or less (e.g., about 0.1 to about 1nM) when tested as described above_50S。

As indicated in the above experiments, the antibodies of the present invention can potently block the effect of IL-1 β. Therefore, the antibody of the present invention has the following pharmaceutical uses:

the antibodies of the invention can be used for the prevention and treatment of IL-1 mediated diseases or conditions, such as inflammation, allergic and anaphylactic diseases, hypersensitivity reactions, autoimmune diseases, severe infections and organ or tissue transplant rejection.

For example, the antibodies of the invention may be used to treat recipients of cardiac, pulmonary, cardiopulmonary, hepatic, renal, pancreatic, skin or corneal transplants, including allograft rejection or xenograft rejection, and to prevent graft-versus-host disease (e.g., bone marrow post-transplant of the disease) and organ transplantation associated with atherosclerosis.

The antibodies of the invention are particularly useful in the treatment, prevention or amelioration of autoimmune diseases and inflammation, particularly inflammation whose etiology includes an autoimmune component, such as arthritis (e.g., rheumatoid arthritis, chronic progressive arthritis, and osteoarthritis) and rheumatic diseases, including inflammation and rheumatic diseases involving bone loss, inflammatory pain, hypersensitivity (including respiratory hypersensitivity and skin hypersensitivity) and allergy. Specific autoimmune diseases to which the antibodies of the invention may be applied include autoimmune hematologic disorders (including, e.g., hemolytic anemia, aplastic anemia, pure red cell anemia, and idiopathic thrombocytopenia), systemic lupus erythematosus, multiple osteomalacia, scleroderma, wegener's granulomatosis, dermatophytosis, chronic active hepatitis, myasthenia gravis, psoriasis, sjogren's syndrome, idiopathic sprue, autoimmune inflammatory bowel disease (including, e.g., ulcerative colitis, crohn's disease, and irritable bowel syndrome), endocrine ocular disease, graves' disease, sarcoidosis, multiple sclerosis, primary biliary cirrhosis, juvenile diabetes mellitus (type I diabetes), uveitis (anterior and posterior uveitis), keratoconjunctivitis sicca and vernal keratoconjunctivitis, interstitial pulmonary fibrosis, psoriatic arthritis, and glomerulonephritis (with or without nephrotic syndrome, including, for example, idiopathic nephrotic syndrome or minimal change nephropathy).

The antibodies of the invention may also be used to treat, prevent or ameliorate asthma, bronchitis, pneumoconiosis, emphysema, and other obstructive or inflammatory respiratory diseases.

The antibodies of the invention may be used to treat adverse acute and hyperacute inflammatory responses mediated by IL-1 or involving IL-1 (especially IL-1 β) production or IL-1-promoted TNF release, for example acute infections such as septic shock (e.g. endotoxic shock and adult respiratory distress syndrome), pia-arachnoiditis, pneumonia; and severe burns; it can also be used for the treatment of cachexia or wasting syndrome associated with pathological TNF release caused by infection, cancer or organ dysfunction, especially AIDS-related cachexia, such as cachexia associated with or caused by HIV infection.

The antibodies of the invention are particularly useful for the treatment of diseases of bone metabolism including osteoarthritis, osteoporosis and other inflammatory arthritides, as well as bone loss in general, including bone loss associated with aging, and periodontal disease in particular.

For these indications, the appropriate dosage will, of course, vary with, for example, the particular antibody of the invention to be administered, the host, the mode of administration and the nature and severity of the disease being treated. However, in prophylactic applications, satisfactory results have been shown to be obtained at dosages of from about 0.05mg to about 10mg per kilogram of body weight, more typically from about 0.1mg to about 5mg per kilogram of body weight. For prophylactic use, the frequency of administration will normally be from about once a week to no more than about once every 3 months, more usually from about once every 2 weeks to no more than about 1 time every 10 weeks, for example once every 4 to 8 weeks. The antibodies of the invention may conveniently be administered by parenteral, intravenous (e.g. into the antecubital or other peripheral intravenous), intramuscular or subcutaneous routes. Prophylactic treatment typically includes administration of an antibody of the invention once a month to once every 2 to 3 months, or less frequently.

The pharmaceutical compositions of the present invention may be prepared in a conventional manner. Preferably, the compositions of the present invention are provided in lyophilized form. For direct administration, it may be dissolved in a suitable aqueous carrier, such as sterile water for injection or sterile buffered saline. If it is desired to formulate a large volume solution for perfusion, more precisely for bolus administration, it is advantageous to add human serum albumin or the patient's own heparinized blood to this saline at the time of formulation. The presence of an excess of such physiologically inert proteins can prevent loss of antibodies due to adsorption by the walls of the containers and tubes used for the perfusion solution. If albumin is used, a suitable concentration is 0.5 to 4.5% by weight salt solution.

The invention is further described in the following examples by way of illustration with reference to the accompanying drawing which shows dose response curves for the inhibition of IL-1 β binding by soluble IL-1 receptor types I and II.

Examples

We used constructed transgenic mice (expressing all components of human IgG/kappa but not all components of the murine immunoglobulin) (Fishwild et al, 1996, Nat Biotechnol., Vol.14, p.845-851) to make antibodies against human IL-1. beta. B cells from these mice were immortalized by standard hybridoma techniques and murine hybridoma cells secreting human IgG 1/kappa antibody ACZ885 were obtained.

Example 1: hybridoma production and antibody purification

Genetically engineered mouse 18077(Medarex inc. anadale, NJ) was immunized subcutaneously at several locations with recombinant human IL-1 β (50 μ g) coupled to KLH in adjuvant. Mice were given 5 more boosts, with the last injection being performed 3 days before fusion. On the day of fusion, with CO₂Mice were sacrificed by inhalation 18077 and spleen cells (4.1X 10) by conventional methods using PEG 4000⁷One) were fused with an equal number of mouse myeloma cell line PAI-O cells. In 624 wells containing a mouse peritoneal cell (Balb C mice) feeder layerFused cells (1 ml/well) with HAT supplemented RPMI 1640, 10% heat-inactivated fetal bovine serum, 5X10^-5M β -mercaptoethanol. The supernatant was collected, and IL-1. beta. reactive monoclonal antibodies were detected and screened by enzyme-linked immunosorbent assay (ELISA). Monoclonal antibodies of 5 IgG/kappa subtypes were identified. Cloning was performed by seeding 0.5 cells per well using 4 × 96 well microtiter plates. After two weeks the wells were observed using an inverted microscope. Supernatants were collected from growth positive wells and evaluated for anti-IL-1. beta. monoclonal antibody production by ELISA. 1-2L conditioned supernatants were prepared from 4 subclones of the initially identified hybridoma #657 and the antibody was purified by affinity chromatography on a protein A column.

Purification and partial amino acid sequence of heavy and light chains

Amino acid sequencing

The light and heavy chains of the purified antibody ACZ885 were separated by SDS-PAGE and the amino acid at the amino terminus was determined by Edman degradation. The purity of the antibodies used in these studies was ≥ 90% by sequencing. Obtaining mRNA from the cloned hybridoma cell and further obtaining cDNA, amplifying the cDNA by PCR to obtain cDNA sequences encoding the heavy chain variable region and the light chain variable region, and performing full sequencing. The amino-terminal sequences of the heavy and light chain variable regions and the corresponding DNA sequences are given in seq.Id No.1 and Seq Id No.2 below, with the CDRs shown in bold.

Seq. Id.No.1 of ACZ885 heavy chain variable region

ATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGCTCTTTTAAGAGGTGTCCAGTGTCAG

-19 M E F G L S W V F L V A L L R G V Q C Q -1

GTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCC

V Q L V E S G G G V V Q P G R S L R L S -21

TGTGCAGCGTCTGGATTCACCTTCAGTGTTTATGGCATGAACTGGGTCCGCCAGGCTCCA

C A A S G F T F SW V R Q A P -41

GGCAAGGGGCTGGAGTGGGTGGCAATTATTTGGTATGATGGAGATAATCAATACTATGCA

G K G L E W V A-61

GACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTG

R F T I S R D N S K N T L Y L -81

CAAATGAACGGCCTGAGAGCCGAGGACACGGCTGTGTATTATTGTGCGAGAGATCTTAGG

Q M N G L R A E D T A V Y Y C A R-101

ACTGGGCCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTC

F D Y W G Q G T L V T V S S -118

Seq. Id.No.2 of ACZ885 light chain variable region

ATGTTGCCATCACAACTCATTGGGTTTCTGCTGCTCTGGGTTCCAGCCTCCAGGGGTGAA

-19 M L P S Q L I G F L L L W V P A S R G E -1

ATTGTGCTGACTCAGTCTCCAGACTTTCAGTCTGTGACTCCAAAGGAGAAAGTCACCATC

I V L T Q S P D F Q S V T P K E K V T I -21

ACCTGCCGGGCCAGTCAGAGCATTGGTAGTAGCTTACACTGGTACCAGCAGAAACCAGAT

T CW Y Q Q K P D -41

CAGTCTCCAAAGCTCCTCATCAAGTATGCTTCCCAGTCCTTCTCAGGGGTCCCCTCGAGG

Q S P K L L I K YG V P S R -61

TTCAGTGGCAGTGGATCTGGGACAGATTTCACCCTCACCATCAATAGCCTGGAAGCTGAA

F S G S G S G T D F T L T I N S L E A E -81

GATGCTGCAGCGTATTACTGTCATCAGAGTAGTAGTTTACCATTCACTTTCGGCCCTGGG

D A A A Y Y CF T F G P G -101

ACCAAAGTGGATATCAAA -107

T K V D I K

Italic: leader sequence (not in mature antibody)

Bold: CDR

Construction of heavy and light chain expression vectors

GS-based amplification/screening systems are used, such as those described in EP 0256055B, EP 0323997B or european patent application 89303964.4, wherein the selection marker used is the GS coding sequence.

Example 2: biochemical and biological data

The human monoclonal antibody ACZ885 was found to neutralize interleukin-1 β activity in vitro. The monoclonal antibody and recombinant human IL-1 beta binding by Biacore analysis to further characterization. The mode of neutralization was evaluated by competitive binding studies using the soluble IL-1 receptor. The biological activity of the antibody ACZ885 on recombinant and naturally occurring IL-1 β was determined in primary human cells in response to IL-1 β stimulation (example 3).

Determination of dissociation equilibrium constant

BIAcore analysis was used to determine the association and dissociation rate constants of recombinant human IL-1. beta. with ACZ 885. ACZ885 was immobilized and its binding to recombinant IL-1. beta. was measured by Surface plasmon resonance (Surface plasmon resonance) at a concentration ranging from 1 to 4 nM. The selected format is a monovalent interaction, thereby allowing processing of binding events of IL-1 β to ACZ885 according to a 1: 1 stoichiometry. Data analysis was performed using BIA evaluation software.

	K[10/Ms]	K[10/s]	K[pM]
					Human IL-1 beta	11.0+/-0.23	3.3+/-0.27	30.5+/-2.6	n＝22

And (4) conclusion: ACZ885 binds to recombinant human IL-1 β with high affinity.

Binding competition studies using soluble type I and II IL-1 receptors

Competition between ACZ885 and soluble human type I and II IL-1 receptors was measured by Biacore. ACZ885 was immobilized on the chip surface and recombinant human IL- β (1nM) was injected to bind to ACZ885 with or without increasing concentrations of recombinant human soluble type I or type II receptors (0-12 nM; 4 experiments were performed independently). The obtained results are shown in the attached figure.

NVP-ACZ885 binding to human IL-1 β was determined in the presence of recombinant human soluble type I or type II IL-1 receptor. Half of the maximum value was determined graphically using Origin 6.0 software (IC 50). Mean ± SEM (n ═ 4) is given.

And (4) conclusion: ACZ885 competes with type I and type II IL-1 receptors for binding to IL-1 β.

Reactivity profile with human IL-1 alpha, human IL-1RA and IL-1 beta from other species

Reactivity of ACZ885 with human IL-1 α, human IL-1RA and IL-1 β of cynomolgus monkeys, rabbits, mice and rats was determined using Biacore analysis. ACZ885 was fixed and the tested cytokines were applied at 8nM concentration (6 runs independently).

Table 3: cross-reactivity of NVP-ACZ885 with IL-1 β, IL-1 α, and IL-1Ra

	Combined% (mean. + -. SEM)
		Recombinant human IL-1 β (n ═ 6)	100
Recombinant cynomolgus monkey IL-beta (n ═ 11)	7.8+/-1.0
		Recombinant rabbit IL-1 beta (n ═ 6)	-0.5+/-0.2
Recombinant mouse IL-1 β (n ═ 6)	-2.6+/-0.6
		Recombinant rat IL-1 beta (n ═ 6)	-6.2+/-1.0
Recombinant human IL-1 α (n ═ 6)	8.4+/-2.4
		Recombinant human IL-1Ra (n ═ 6)	-3.7+/-1.7

Reading the resonance units at 1000s after the start of the injection; injections of experimental run buffer were subtracted from all sensorgrams (sensorgram) and baseline was set to zero after immobilization of anti-Fc γ. Binding is expressed as a percentage of cumulative resonance units of human IL-1 β.

And (4) conclusion: ACZ885 does not significantly cross-react with human IL-1 α, human IL-1RA, and IL-1 β of cynomolgus monkeys, rabbits, mice, and rats.

Example 3

Neutralization of IL-6 release from ACZ885 to human dermal fibroblasts

The following methodology was used to evaluate the biological activity of ACZ885 in neutralizing the effects of human IL-1 β:

1. preparation of conditioned Medium containing IL-1 beta

Conditioned medium was prepared from human peripheral blood mononuclear cells as follows: according to Hansel's method [ Hansel, t.t. et al (1991), a modified immunomagnetic method for the isolation of highly pure human blood eosinophils, j.imm.methods 145: 105-110](ii) preparing mononuclear cells from peripheral blood of monkeys using Ficoll-Hypaque density separation; at 10⁵Concentration of individual cells/well these cells were used in RPMI/10% FCS. Adding IFN beta (100U/ml) and LPS (5. mu.g/ml), followed by incubation of the cells for 6 hours. Incubations were terminated by centrifugation at 1200RPM for 10 minutes. IL-1. beta. in the supernatant was quantified using ELISA.

2. Neutralization test

Human foreskin skin fibroblasts (CC-2509) were obtained from Clonetics and cultured in FBM (Clonetics, CC-3131) containing bFGF (1ng/ml, CC-4065), insulin (5 β g/ml, CC-4021) and 2% FCS (CC-4101).

To induce IL-6, cells were seeded in a Tissue Cluster at a density of 10 per well in 48 wells⁴And (4) cells. The following day, cells were starved for 6-7 hours in FBM containing 2% FCS, after which cytokines were added. For stimulation, the medium was replaced with FBM + 2% FCS containing the appropriate amount of conditioned medium to achieve approximately 50pg/ml IL1 β. Alternatively, recombinant human IL-1. beta. was used at a final concentration of 50 pg/ml.

anti-IL-1. beta. neutralizing antibodies were titrated in this diluted conditioned medium prior to addition to the cells. Recombinant IL-1Ra (R & D Systems #280-RA-010) was used as a positive control.

Cell supernatants were taken 16 to 17 hours after stimulation and the amount of IL-6 released was determined in a sandwich ELISA.

3.IL-6 ELISA

With PBS 0.02% NaN₃The mouse anti-human IL-6 MAb (314-14(Novartis Pharma; batch No. EN23,961, 5.5 mg/ml); 100. mu.l, 3. mu.g/ml) was coated onto ELISA microtiter plates and incubated overnight at +4 ℃. The following day, with PBS/0.05% Tween/0.02% NaN₃The microtiter plate was washed 4 times and then washed with 300. mu.l of PBS/3% Bovine Serum Albumin (BSA)/0.02% NaN₃And sealing for 3 hours. The microtiter plates were washed again (4 times) and 100. mu.l of supernatant (1: 20 final dilution) or 100. mu.l of recombinant human IL-6 standard ((Novartis Pharma #91902) were added in duplicate, the titration curve following a 2-fold dilution step from 1 to 0.0156 ng/ml). After overnight incubation at RT, the plates were washed (4 times) and different mouse anti-human IL-6 MAbs (110-14, Novartis Pharma; 6.3mg/ml) were added; 100 μ l, 1 μ gPer ml; room temperature for 3 hours). After 4 additional washes, biotin-labeled goat anti-mouse IgG2b antiserum (Southern Biotechnology; #1090-08) was added at a final concentration of 1/10000 (100. mu.l/well; room temperature for 3 hours). After incubation, the plates were washed 4 times and streptavidin-conjugated alkaline phosphatase (Jackson Immuoresearch, # 016-. After washing (4 times), substrate (p-nitrophenyl phosphate in diethanolamine buffer; 100. mu.l) was added for 30 minutes. The reaction was blocked by adding 50. mu.l of 1.5M NaOH per well. Plates were read on a microtiter reader (Bio-Rad) using filters at 405 and 490 nm.

The IL-6 levels in the culture supernatants were calculated using a cubic curve fit with reference to a standard curve. Statistical evaluation and IC50 determination were performed based on sigmoidal curve fitting.

As a result:

table: inhibition of IL-1 beta-induced IL-6 secretion

	NVP-ACZ885 batch 1IC[pM]±SEM	NVP-ACZ885 batch 2IC[pM]±SEM	IL-1raIC[pM]±SEM
				IL-6 secretion conditioned medium	54±6.1(9.1±1.0ng/ml)(n＝6)	44.6±3.6(7.4±0.6ng/ml)(n＝6)	30±3.1(0.51±0.05ng/ml)(n＝5)
IL-6 secreting recombinant human IL-1 beta	42±3.4(7.1±0.56ng/ml)(n＝4)	63±2.8(10.5±0.5)(n＝6)	nd

IC for inhibition of IL-1 beta-induced IL-6 secretion from human dermal fibroblasts₅₀The value is obtained.

Fibroblasts are stimulated with recombinant human IL-1 β or with conditioned medium containing 50-100pg/ml IL-1 β.

Example 4

Determination of epitopes for ACZ885

ACZ885 binds to human IL-1 β with high affinity, but does not recognize highly homologous IL-1 β from the rhesus. One of the most prominent differences in amino acid sequence between the rhesus IL-1 β and human IL-1 β is at position 64 of the mature IL-1 β. At this position human IL-1. beta. has glutamic acid, while rhesus IL-1. beta. has alanine. Mutant human IL-1 β with the corresponding substitution Glu64Ala lost its ability to bind ACZ885 with detectable affinity. We conclude that: glu64 in human IL-1 β is required for recognition by antibody ACZ 885. Glu64 is located on a loop of IL-1 β that is not part of, nor in close proximity to, a surface that binds to IL-1 β type I receptors. Thus, antibodies directed against the binding epitope containing Glu64 have the potential to neutralize the biological activity of human IL-1 β.

Claims (9)

1. Comprising a heavy chain variable region V_HAnd light chain variable region V_LThe IL-1. beta. antibody of (1), wherein the variable region of the heavy chain is V_HThe amino acid sequence of (1) is 1-118 of seq. Id.No.1, the variable region of light chain V_LIs 1-107 of seq. Id.No.2 and is in V_HThe amino acid sequence of the CDR1 is Val-Tyr-Gly-Met-Asn, the amino acid sequence of the CDR2 is Ile-Ile-Trp-Tyr-Asp-Gly-Asp-Asn-Gln-Tyr-Tyr-Ala-Asp-Ser-Val-Lys-Gly, the amino acid sequence of the CDR3 is Asp-Leu-Arg-Thr-Gly-Pro, and the amino acid sequence of the CDR3 is V_LThe amino acid sequence of the CDR 1' is Arg-Ala-Ser-Gln-Ser-Ile-Gly-Ser-Ser-Leu-His, the amino acid sequence of CDR2 'is Ala-Ser-Gln-Ser-Phe-Ser, and the amino acid sequence of CDR 3' is His-Gln-Ser-Ser-Ser-Leu-Pro.

2. The IL-1 β antibody according to claim 1, which is a human antibody.

3. An IL-1 β antibody comprising an antigen binding site, wherein the antigen binding site has a first domain with an amino acid sequence identical to the sequence set forth in seq.id No.1 starting at amino acid 1 and ending at amino acid 118 and a second domain with an amino acid sequence identical to the sequence set forth in seq.id No.2 starting at amino acid 1 and ending at amino acid 107.

4. A DNA combination construct for the preparation of an IL-1 β antibody, consisting of a first DNA construct encoding a heavy chain or fragment thereof and a second DNA construct encoding a light chain or fragment thereof, wherein the first DNA construct comprises a) a first portion encoding a variable region comprising alternatively hypervariable regions and framework regions, which hypervariable regions are in sequence the CDR1, CDR2 and CDR3 of the amino acid sequences shown in seq.id No. 1; the first portion starting at a codon encoding the first amino acid of the variable region and ending at a codon encoding the last amino acid of the variable region, and b) a second portion encoding the constant part of the heavy chain or a fragment thereof, starting at a codon encoding the first amino acid of the constant part of the heavy chain and ending at a codon encoding the last amino acid of the constant part or a fragment thereof, followed by a stop codon; the second DNA construct comprises a) a first portion encoding a variable region comprising hypervariable regions and framework regions in alternation; the hypervariable regions are in sequence CDR1 ', CDR2 ' and CDR3 ' with the amino acid sequences shown in seq.id No. 2; the first portion begins with a codon encoding the first amino acid of the variable region and ends with a codon encoding the last amino acid of the variable region, and b) a second portion encoding the constant portion of the light chain or a fragment thereof, which begins with a codon encoding the first amino acid of the constant portion of the light chain and ends with a codon encoding the last amino acid of the constant portion or a fragment thereof, followed by a stop codon.

5. A single expression vector capable of replication in a prokaryotic or eukaryotic cell line, wherein the DNA combination construct according to claim 4 is comprised in said single expression vector.

6. A method for producing an IL-1 β antibody, comprising (i) culturing an organism transformed with an expression vector according to claim 5 and (ii) recovering the IL-1 β antibody from the culture.

7. The IL-1 β antibody according to claim 1, which IL-1 β antibody has antigen binding specificity to an epitope of mature human IL-1 β comprising a loop containing residue Glu64 and is capable of inhibiting the binding of IL-1 β to its receptor.

8. A pharmaceutical composition comprising an IL-1 β antibody according to any one of claims 1-3 and 7, together with a pharmaceutically acceptable excipient, diluent or carrier.

9. Use of an IL-1 β antibody according to any one of claims 1-3 and 7 in the manufacture of a medicament for the treatment of an IL-1 β mediated disease or disorder.