Human antibodies to il-β1

FIELD: immunology, biology.

SUBSTANCE: invention relates to variants of IL-1β-binding molecule having common functionally active sites (CDR sites) and may bind human IL-1β. Said molecules have neutralizing activity IC50 of approximately 50 pM and binding constant KD of approximately 30 pM. Amino acid sequence is described in description of present invention. Variants of DNA constructs encoding of heavy chain and light chain of IL-1β-binding molecule are disclosed. Expression vectors carrying at least one abovementioned nuclear acid and method for production of IL-1β-binding molecules by using the same also are described.

EFFECT: IL-1β-binding molecules against human IL-1β with high neutralizing activity and binding constant useful in suppression of HAMA response.

10 cl, 1 dwg, 3 tbl, 4 ex

 

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

Interleukin 1 (IL-1) is a factor produced by cells of the immune system, which acts as a mediator of the acute phase inflammatory response. The disruption or excessive production of IL-1, particularly IL-1βassociated with the pathology of various diseases and disorders, such as septicaemia, septic or endotoxic shock, allergies, asthma, bone loss, ischemia, "stroke", rheumatoid arthritis and other inflammatory diseases. Antibodies to IL-1β proposed for treatment mediated IL-1 diseases and disorders (see, for example, WO 95/01997 and discussion given in the introduction).

When creating inventions received superior antibodies to human IL-1βintended for the treatment mediated IL-1 diseases and disorders.

Thus, the invention relates to IL-1β-binding molecule, which contains antigennegative centre antibodies, comprising at least one variable region of the heavy chain (VH) immunoglobulin, which consists of arranged in sequence hypervariable sites CDR1, CDR2 and CDR3, where CDR1 has the amino acid sequentially shall be Val-Tyr-Gly-Met-Asn, CDR2 has the amino acid sequence Ile-Ile-Trp-Tyr-Asp-Gly-Asp-Asn-Gln-Tyr-Tyr-Ala-Asp-Ser-Val-Lys-Gly, and CDR3 has the amino acid sequence Asp-Leu-Arg-Thr-Gly-Pro; and direct equivalents.

Thus, the invention relates to IL-1β-binding molecule that contains at least one variable region light chain (VL,) immunoglobulin, which consists of arranged in sequence hypervariable sites CDR1', CDR2' and CDR3', where CDR1' has the amino acid sequence Arg-Ala-Ser-Gln-Ser-Ile-Gly-Ser-Ser-Leu-His, CDR2' has the amino acid sequence Ala-Ser-Gln-Ser-Phe-Ser and CDR3' has the amino acid sequence His-Gln-Ser-Ser-Ser-Leu-Pro, and its direct equivalents.

The first object of the invention is a single domain of IL-1β-binding molecules containing the selected heavy chain immunoglobulin which comprises the above-mentioned variable region heavy chain (VH).

The second object of the invention is also IL-1β-binding molecule that contains variable regions and heavy (VH) and light chain (VL), with the indicated IL-1β-binding molecule comprises at least one antigennegative centre antibodies, including:

a) the variable region of the heavy chain (VH) immunoglobulin, which is located in the specified n the coherence hypervariable sites CDR1, CDR2 and CDR3, where CDR1 has the amino acid sequence Val-Tyr-Gly-Met-Asn, CDR2 has the amino acid sequence Ile-Ile-Trp-Tyr-Asp-Gly-Asp-Asn-GIn-Tyr-Tyr-Ala-Asp-Ser-Val-Lys-Gly, and CDR3 has the amino acid sequence Asp-Leu-Arg-Thr-Gly-Pro, and

b) variable region light chain (VL) immunoglobulin, which consists of arranged in sequence hypervariable sites CDR1', CDR2' and CDR3', where CDR1' has the amino acid sequence Arg-Ala-Ser-Gln-Ser-Ile-Gly-Ser-Ser-Leu-His, CDR2' has the amino acid sequence Ala-Ser-Gln-Ser-Phe-Ser and CDR3' has the amino acid sequence His-Gln-Ser-Ser-Ser-Leu-Pro;

and its direct equivalents.

Unless otherwise stated, any presents in the description of the polypeptide chain has an amino acid sequence that starts at the N-end and ends at the C-end. If antigennegative center contains antibodies and VH-, and VL-the field, they can be located on the same polypeptide molecule or preferably each area can be on different circuits, VHthe area may be part of the heavy chain of the immunoglobulin or its fragment, and VLthe region is part of the light chain of the immunoglobulin or its fragment.

The term "IL-1β-binding molecule" refers to any molecule that has the ability to bind to the antigen IL-1; either individually or in combination with other molecules. The reaction of binding can be identified using standard methods (qualitative analysis), which include, for example, biological analysis of inhibition of binding of IL-1β with its receptor or analyses of the binding of any type in comparison with the negative control, in which the use of the antibody of unrelated specificity, but preferably belong to the same isotype, for example an antibody to CD25. To analyze the binding of IL-1β-binding molecules according to the invention with IL-1β it is advisable to apply competitive analysis of the binding.

Examples antigenspecific molecules include antibodies, produced by b-cells or hybridomas, and chimeric antibodies, antibodies with built by transplantation hypervariable sites (CDR) or human antibodies, or any fragments thereof such as F(ab')2- and Fab-fragments, and single chain or single domain antibodies.

Single-chain antibody consists of the variable regions of the heavy and light chains of the antibody, covalently linked with a peptide linker, which usually consists of 10 to 30 amino acids, preferably from 15 to 25 amino acids. Thus, this structure does not include a constant region heavy and light chains, and we can assume that a small peptide spacer should oblad shall be less pronounced antigenic properties, than all of a constant region. The term "chimeric antibody" refers to an antibody in which the constant region of the heavy and light chain or both chains derived from human antibodies and the variable region of both heavy and light chains derived from antibodies of the body except the person (e.g., mouse) or of the antibodies of the human body, but which is a different human antibody. The term "antibody with built-in by transplantation hypervariable area" means an antibody in which hypervariable sites (CDR) derived from antibody-donor, such as an antibody of the body except the person (e.g., mouse) or more human antibody, while all or almost all other parts of immunoglobulin, such as a constant region and a highly conserved parts of the variable regions, i.e. wireframe plots come from antibodies-acceptor, such as human antibodies. However, the antibody with built-in by transplantation hypervariable area may include a small number of amino acid sequences of the donor in the solid areas, such as parts of the frame adjacent to the hypervariable sites. The term "human antibody" means an antibody in which all constant and variable region of both heavy and light CE is she come from human antibodies or almost identical sequences of human antibodies but not necessarily derived from the same antibody, and incorporate in the body of the mouse antibody, in which murine genes variable and constant regions of immunoglobulin are replaced by their human copies, for example, as described generally in EP 0546073 B1, US patents 5545806, 5569825, 5625126, 5633425, 5661016, 5770429, EP 0438474 B1 and EP 0463151 B1.

Particularly preferred IL-1β-binding molecules according to the invention are human antibodies, primarily 5 antibody ACZ885 described below in the examples.

Thus, in a preferred chimeric antibody variable region of both heavy and light chains derived from human antibodies, such as antibody ACZ885, the sequence of which is presented in Seq. Id. No.1 and Seq. Id. No.2. Areas of constant regions also contain acceptable parts of the human constant regions, for example, as described in "Sequences of Proteins of Immunological Interest", Kabat E.A. and others, US Department of Health and Human Services, Public Health Service, National Institute of Health.

Hypervariable sites can be obtained from the frame sections of any type, although preferably they are derived from human antibodies. Acceptable frame sections described in Kabat E.A. and others, ibid. The preferred frame plot heavy chain is a framed area of the human heavy chain, for example antibody ACZ885, the sequence of which is presented in Seq Id. No.1. It consists of arranged in sequence FR1-, FR2-, FR3 and FR4 regions. Similarly, the sequence shown in Seq. Id. No.2, is preferred as the frame area light chain ACZ885 and consists of arranged in sequence FR1'-, FR2'-, FR3'FR4'parcels.

Thus, the invention relates to IL-1β-binding molecule that contains at least one antigennegative centre antibody, comprising either a first domain, the amino acid sequence of which is practically identical to the sequence presented in Seq. Id. No.1, which begins with amino acids in position 1 and ending with amino acid at position 118 or above the first domain and a second domain, the amino acid sequence of which is practically identical to the sequence presented in Seq. Id. No.2, which begins with amino acids in position 1 and ending with amino acid at position 107.

Monoclonal antibodies that are produced against the protein, found in all people, as a rule, get in the body than humans, for example in mice, and therefore they usually are proteins not found in the human body. The direct result is that obtained by hybridoma xenogen the second antibody when administered to humans cause unwanted immune response, which is mainly mediated constant region xenogeneic immunoglobulin. This significantly limits the use of such antibodies, because they cannot be entered in for long periods of time. As a consequence, the particularly preferable to apply the single-stranded, bearing a single domain, chimeric antibodies, antibodies with built-in by transplantation hypervariable area or, above all, human antibodies, which when administered to humans, apparently, should not cause pronounced allogeneic reaction.

In light of the above more preferred IL-1β-binding molecule according to the invention is chosen from the group comprising human antibody to IL-1βthat includes at least

a) a heavy chain immunoglobulin or its fragment, which includes (I) variable region comprising spaced in sequence hypervariable sites CDR1, CDR2 and CDR3, and (II) a constant region of a human heavy chain or fragment; where CDR1 has the amino acid sequence Val-Tyr-Gly-Met-Asn, CDR2 has the amino acid sequence Ile-Ile-Trp-Tyr-Asp-Gly-Asp-Asn-GIn-Tyr-Tyr-Ala-Asp-Ser-Val-Lys-Gly, and CDR3 has the amino acid the sequence Asp-Leu-Arg-Thr-Gly-Pro, and

b) the light chain immunoglobulin or its fragment, which includes (I) variable region containing the located is in sequence hypervariable sites and also optional hypervariable sites CDR1', CDR2' and CDR3', and (II) a constant region of human light chain or a fragment, which CDR1' has the amino acid sequence Arg-Ala-Ser-Gln-Ser-Ile-Gly-Ser-Ser-Leu-His, CDR2' has the amino acid sequence Ala-Ser-Gln-Ser-Phe-Ser and CDR3' has the amino acid sequence His-Gln-Ser-Ser-Ser-Leu-Pro;

and its direct equivalents.

In another embodiment, IL-1β-binding molecule according to the invention can be selected from the group comprising single-stranded binding molecule that contains antigennegative centre antibodies, including

a) the first domain, which consists of arranged in sequence hypervariable sites CDR1, CDR2 and CDR3, where

hypervariable sites have the amino acid sequence represented in Seq. Id. No.1,

b) a second domain containing hypervariable sites CDR1', CDR2' and CDR3'hypervariable sites have the amino acid sequence represented in Seq. Id. No.2, and

C) a peptide linker which is bound either to the N-end of the first domain and the C-end of the second domain or To the end of the first domain and the N-end of the second domain;

and its direct equivalents.

As is well known, minor substitutions in the amino acid sequence, such as a deletion, addition or substitution of one, a few or even several amino acids, can PR is led to receive allelic forms of the original protein, which has almost identical properties.

Thus, the notion of "direct equivalent" means either any one domain of IL-1β-binding molecules (molecule X),

(I) in which hypervariable sites CDR1, CDR2 and CDR3 overall homology of at least 80%, preferably homologous to at least 90%, more preferably homologous, at least 95% of hypervariable sites, the sequence of which is presented in Seq. Id. No.1, and

(II) which has the ability to inhibit the binding of IL-1β with its receptor in almost the same degree, that is a molecule with which a comparison is made, the carrier frame parts, identical frame parts of the molecule X, and having hypervariable sites CDR1, CDR2 and CDR3 identical to the above sections, the sequence of which is presented in Seq. Id. No.1,

or any IL-1β-binding molecule having at least two domains in each binding site (molecule X'),

(I) in which hypervariable sites CDR1, CDR2, CDR3, CDR1', CDR2' and CDR3' overall homology of at least 80%, preferably homologous to at least 90%, more preferably homologous, at least 95% of hypervariable sites, the sequence of which is presented in Seq. Id. No.1 and 2, and

(II) which has the ability of ingibiruet the binding of IL-1β with its receptor in almost the same degree, that is a molecule with which a comparison is made, the carrier frame sections and a constant region that is identical frame sections and constant regions of a molecule X', and having hypervariable sites CDR1, CDR2, CDR3, CDR1', CDR2' and CDR3', identical to the above sections, the sequence of which is presented in Seq. Id. No.1 and 2.

In the context of the present description assumes that the amino acid sequence at least 80% homologous to each other if they have at least 80% identical amino acid residues in a similar situation, according to the optimal comparative analysis, in which gaps or insertions in the amino acid sequences are considered as non-identical residues.

Inhibition of binding of IL-1β with its receptor is easy to test using various analyses, including analyses, which are described below. The concept of "equally" means that a molecule with which a comparison is made, and the equivalent molecule give almost identical from the point of view of statistics curves of inhibition of binding of IL-1β in the study by using one of the methods above. For example, IL-1β-binding molecules according to the invention, as a rule, gain values IC50for inhibition of binding of IL-1β, Rotz the torus, which are within +/-x5 are preferably almost the same) relative to the IC50corresponding molecule with which a comparison is made, in the analysis using the above methods.

For example, applied analysis may represent an analysis of the competitive inhibition of binding of IL-1β with soluble binding of IL-1β receptor and IL-1β-binding molecules according to the invention.

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

a) one heavy chain, which carries variable region amino acid sequence of which is virtually identical to the sequence presented in Seq. Id. No.1, which begins with amino acids in position 1 and ending with amino acid at position 118, and a constant region of a human heavy chain; and

b) one light chain, which carries variable region amino acid sequence of which is virtually identical to the sequence presented in Seq. Id. No.2, which begins with amino acids in position 1 and ending with amino acid at position 107, and a constant region of human light chain.

The constant region of a human heavy chain may be γ1-, γ2-, γ3-, γ4-, μ-, α1-, α2-, δand the ε -type, preferably γ-type, more preferably γ1-type, and the constant region of human light chain may be κor λ-type (including subtypes λ1that λ2and λ3); but preferably κ-type. Amino acid sequences of all these constant regions described in Kabat, and others, ibid.

IL-1β-binding molecule according to the invention can be obtained by using methods of recombinant DNA. Thus it is necessary to construct one or more DNA molecules encoding the binding molecule, placed under the control of the corresponding sequences and transform the body-master, which can happen expression.

Thus, in its broadest sense, the invention relates to

(I) DNA molecules, encoding a single domain of IL-1β-binding molecules according to the invention, single-chain IL-1β-binding molecule according to the invention, a heavy or light chain of IL-1β-binding molecules according to the invention or their fragments, and

(II) the application of DNA molecules according to the invention to obtain by recombinant IL-1β-binding molecules according to the invention.

The existing level of technology is such that allows the experts to synthesize DNA molecules according to the invention on the basis presented in the present description information is then, i.e. the amino acid sequences of hypervariable sites and their coding DNA sequences. Method of constructing gene variable regions is described, for example, in EPA 239400, and generally consists of the following: clone the gene encoding the variable region of the Mat, regardless of its specificity. Define segments of DNA encoding frame section and hypervariable sites, and the DNA segments encoding the hypervariable sites removed so that the segments of DNA that encode frame sections, merged into the joints with appropriate restriction enzymes cut sites. Restriction enzymes cut sites can be created in the required positions by mutagenesis of DNA using standard procedures. Cartridge bearing double-stranded synthetic CDR produced by DNA synthesis based on the sequences disclosed in Seq. Id. No.1 or 2. These tapes are manufactured so that they had a "sticky" ends for installation by ligating into the joints of the skeleton.

In addition, to obtain the structure of DNA that encodes IL-1β-binding molecules according to the invention does not require the use of mRNA generates hybridoma cell line. So, in PCT application WO 90/07861 presents complete instructions for obtaining antibodies using methods of recombinant DNA based only written information, for example about nucleotide the th gene sequence. The method consists in the synthesis of multiple oligonucleotides, amplification by PCR and their splicing with obtaining the desired DNA sequence.

The scientific community is known expression vectors containing acceptable promoters or genes encoding the constant region of the heavy and light chains. So, after receiving the DNA molecules according to the invention it can be taken way to transfer into the appropriate expression vector. DNA molecules encoding single-chain antibodies, can also be obtained by standard methods, for example, described in WO 88/1649.

In light of the above, to meet the criteria of completeness is not required to use the deposited hybridoma or cell line.

A specific embodiment of the invention are described below the first and second structures of the DNA intended for production of IL-1β-1-binding molecules:

The first design DNA encodes a heavy chain or a fragment and includes

a) a first fragment that encodes the variable region containing alternative frame and hypervariable sites, where

hypervariable sites consist of consecutive CDR1, DR2 and CDR3, the amino acid sequence presented in Seq. Id. No.1; this is the first fragment starts with a codon that encodes p is pout amino acid variable region, and ends with a codon that encodes the last amino acid variable region, and

b) a second fragment that encodes the constant region of the heavy chain or a fragment, which starts with a codon that encodes the first amino acid of the constant region of the heavy chain, and ends with a codon that encodes the last amino acid of the constant region or fragment, after which there is a stop codon.

Preferably the first fragment encodes the variable region amino acid sequence of which is virtually identical to the amino acid sequence represented in SEQ ID No.1, which begins with amino acids in position 1 and ending with amino acid at position 118. More preferably the first fragment has the nucleotide sequence presented in SEQ ID No.1, which begins with the nucleotide in position 1 and ends at the nucleotide in position 354. It is preferable that the second fragment encodes a constant region of a human heavy chain, more preferably a constant region of a human chain γ1type. This second fragment is a DNA fragment of the genomic origin (which contains introns) or cDNA fragment (without introns).

The second design DNA encodes a light chain or fragment of it and sod RIT

a) a first fragment that encodes the variable region containing alternative frame section and hypervariable sites; where hypervariable sites are CDR1', CDR2' and CDR3', the amino acid sequence presented in Seq. Id. No.2; this is the first fragment starts with a codon that encodes the first amino acid variable region and ends with a codon that encodes the last amino acid variable region, and

b) a second fragment that encodes the constant region light chain or a fragment, which starts with a codon encoding the first amino acid of the constant region of the light chain, and ending with a codon that encodes the last amino acid of the constant region or fragment, after which there is a stop codon.

Preferably the first fragment encodes the variable region amino acid sequence of which is virtually identical to the amino acid sequence represented in SEQ ID No.2, which begins with amino acids in position 1 and ending with amino acid at position 107. More preferably the first fragment has the nucleotide sequence presented in SEQ ID No.2, which begins with the nucleotide in position 1 and ends at the nucleotide in position 321. Preferably also, the second fragment encodes a constant region of human light chain, more preferably, the constant region of the human chain κtype.

The invention relates also to IL-1β-binding molecules in which one or more, typically, only a small number of residues of CDR1, CDR2, CDR3, CDR1', CDR2 or CDR3' or frame sections, as a rule, only a small number (e.g., 1-4), replaced compared with the remnants of the sequences disclosed in SEQ ID No.1 and SEQ ID No.2; for example, by mutation, for example, catapaulting mutagenesis of the corresponding DNA sequences. The invention relates to DNA sequences encoding such modified IL-1β-binding molecules. The invention relates also to IL-1β-binding molecules in which one or more residues of CDR1 or CDR2' replaced compared with the remnants of the sequences disclosed in SEQ ID No.2.

In both structures the DNA of the first and second fragments can be separated by an intron, as well as appropriate, when in the intron between the first and second fragment is the enhancer. The presence of the enhancer, which is transcribed but not translated, can increase the efficiency of transcription. According to a specific implementation options both design DNA contain enhancer gene of the heavy chain, preferably a human gene.

Each design DNA is placed under the control of mimih controlling the expression of sequences, first of all, under the control of appropriate promoters. You can use any type of promoter, provided that it is adapted to the body-master, in which the structure of DNA must be transferred to the expression. However, if the expression occurs in the cell of a mammal, it is particularly preferable to apply the immunoglobulin gene promoter.

The desired antibody can be obtained in cell culture or in transgenic organism of the animal. The corresponding transgenic animal can be obtained using standard methods, which include microinjection into oocytes of the first and second structures of the DNA is placed under the control of an acceptable regulatory sequences, transfer thus obtained eggs in respective of pseudoharmonic females and selection of progeny expressing the desired antibody.

When the chain antibody receive in cell culture, constructs, DNA must first be built either in a single expression vector, or two different, but compatible expression vector, the latter option is preferred.

Thus, the invention relates also to an expression vector that can replicate in line prokaryotic or eukaryotic cells and contains at least one of the above-described structures of DNA.

Each the expression vector, contains the design DNA is then transferred into an acceptable master-organism. When the structure of DNA is integrated individually in two expression vector, they can be moved separately, i.e. one type of the vector to a cell, or by cotransformation, the last option specified is preferred. Acceptable master-organism may be a line of cells, bacteria, yeast or mammals, the last option specified is preferred. More preferably the cell line of mammalian derived from lymphomas, such as myeloma, hybridoma or healthy immortalized b-cells, which preferably does not Express the heavy or light chain of any of endogenous antibodies.

For expression in mammalian cells the coding sequence of IL-1β-binding molecule preferably is integrated into the DNA of the host cell at a locus, which may be provided with a high level of expression of IL-1β-binding molecule, or which is preferred to ensure a high level of expression of IL-1β-binding molecules. Cells in which there are such loci is preferable to integrate the coding sequence of IL-1β-binding molecules can be identified and selected on the basis of data on the levels of expression of IL-1β-binding molecules is. To obtain host cells containing the coding sequence of IL-1β-binding molecules, it is possible to apply any of breeding marker; for example, you can use gene dhfr/methotrexate, or an equivalent system selection. System for the expression of IL-1β-binding molecules of the invention include systems of amplification/selection based on the application of GS, for example, described in EP 0256055 IN, ER 0323997 and IN EP-A 89303964.4.

Another object of the invention is a method for IL-1β-binding molecule, which provides for (I) culturing an organism transformed by the above-described expression vector, and (II) the secretion of IL-1β-binding molecule from the culture.

In the claimed invention, it was found that the antibody ACZ885 probably has the ability to specific binding to the epitope of human IL-1βthat includes a loop, carrying the Glu residue 64 of Mature human IL-1β. (Glu residue 64 of Mature human IL-1β corresponds to residue 180 predecessor of the human IL-1β). This epitope is probably off-site recognition receptor of IL-1 and therefore, quite unexpectedly, it was found that antibodies to this epitope, such as antibody ACZ885, have the ability to inhibit the binding of IL-1β with its receptor. Antibodies, in particular Chimera ant the body and antibodies with built by transplantation hypervariable sites (CDR) and above all, human antibodies, capable of specific binding to an antigenic epitope of Mature human IL-1βthat contains the loop that carries the residue Glu 64 and which is able to inhibit the binding of IL-1β with its receptor; and the use of such antibodies for the treatment of mediated IL-1 diseases and disorders that are new and included in the scope of the present invention.

Thus, another object of the invention is an antibody to IL-1βwhich is capable of specific binding to an antigenic epitope of human IL-1βthat contains a loop, carrying the Glu residue 64 of Mature human IL-1βand possessing the ability to inhibit the binding of IL-1β with its receptor.

The objects of the invention are:

I) the use of antibodies to IL-1βwhich is capable of specific binding to an antigenic epitope of Mature human IL-1βcontaining a loop, which carries the residue Glu 64 and which is able to inhibit the binding of IL-1β with its receptor, for the treatment of mediated IL-1 disease or disorder;

II) a method of treatment mediated IL-1 disease or disorder of a patient, introducing a patient an effective amount of antibodies to IL-1βwhich is capable of specific binding to an antigenic epitope maturely what about the human IL-1β that contains the loop that carries the residue Glu 64 and which is able to inhibit the binding of IL-1β with its receptor;

III) a pharmaceutical composition comprising an antibody to IL-1βwhich is capable of specific binding to an antigenic epitope of Mature human IL-1βcontaining a loop, which carries the residue Glu 64 and which is able to inhibit the binding of IL-1β with its receptor, in combination with a pharmaceutical acceptable excipient, diluent or carrier; and

IV) the use of antibodies to IL-1βwhich is capable of specific binding to an antigenic epitope of Mature human IL-1βcontaining a loop, which carries the residue Glu 64 and which is able to inhibit the binding of IL-1β with its receptor, for the preparation of medicines intended for the treatment of mediated IL-1 diseases or disorders.

For the purposes of the present description, the term "antibody having the ability to inhibit the binding of IL-1β" means that the antibody has the ability to inhibit the binding of IL-1β with its receptor in almost the same extent as the antibody ACZ885, where the notion of " equally" has the above value.

The affinity for binding to IL-1β, antibody ACZ885 is higher than the affinity of previously opican the x antibodies to IL-1β for example antibodies to human IL-1β. So, for ACZ885 is the equilibrium constant for dissociation ToDcharacterizing the binding of IL-1βis less than about 50 PM, for example approximately 35 PM. This high affinity binding makes the antibody ACZ most preferred for use in therapy.

Thus, another object of the invention is an antibody to IL-1βwhere the value of KDcharacterizing the binding of IL-1βis about 50 PM or less. This object of the invention also includes methods and compositions of antibodies with high affinity, which are described for antibodies to IL-1βwith high specificity against antigenic determinants of Mature human IL-1βthat contains a loop, the carrier Glu 64.

In the context of describing the phrase "mediated IL-1 disease includes all diseases and medical conditions that are associated with IL-1, with a direct or indirect role of IL-1 includes the origination, development, progress, persistence or pathology of the disease or medical condition.

In the present description the terms "treatment" or "treat" refer to both prophylactic or preventive treatment, and healing, or alter the course of disease treatment, including treatment of the patient, which has the risk of infection b is Lesney or for which there is a possibility of disease infection, as well as patients who are already ill or diagnosed with a disease or medical condition, and includes the suppression of clinical relapse.

IL-1β-binding molecule, as defined above, in particular IL-1β-binding molecules, described in the first and second objects of the invention, i.e. antibodies that are capable of specific binding to an antigenic epitope of Mature human IL-1βthat contains a loop, the carrier Glu 64, in particular antibodies, which have the ability to inhibit the binding of IL-1β with its receptor; and antibodies to IL-1βthat is ToDfor the binding of IL-1β is about 50 PM or less, indicated in the description as antibodies according to the invention.

Preferably the antibody according to the invention are IL-1β-binding molecules, described in the first and second objects of the invention. Preferably the antibody according to the invention are human antibodies, most preferably the antibody ACZ 885 or its direct equivalent.

Antibodies of the invention block the action of IL-1β on its target cells and therefore can be used to treat mediated IL-1 diseases and disorders. These and other pharmacological activity of the antibodies according to the invention can be demonstrated with the aid of the using standard methods, for example, as described below:

Neutralization-dependent IL-1β production of PGE2and interleukin-6 in primary human fibroblasts

The production of PGE2(prostaglandin E2) and IL-6 in primary human skin fibroblasts is dependent on IL-1β. One TNF-α could not effectively induce the production of these inflammatory mediators, but when applied together with IL-1 has increased due to synergism activity. Primary dermal fibroblasts used as a surrogate model for estimating induced IL-1 cell activation.

Primary human fibroblasts stimulated with recombinant IL-1β or with conditioned medium obtained from stimulated LPS (lipopolysaccharide) human RVMS (blood mononuclear peripheral blood) in the presence of various concentrations of antibodies according to the invention or IL-1RA in the range from 6 to 18000 PM. Chimeric antibody to CD25 Simulect® (basiliximab) is used as the corresponding isotype control. Select the supernatant after stimulation for 16 h and analyzed for the presence of IL-6 using ELISA. As a rule, in the evaluation method described above values IC50antibodies according to the invention in relation to inhibition of production of IL-6 is about 1 nm or lower (for example, from about 0.1 to about 1 nm.

According to the above method, the antibodies according to the invention is effective in blocking the actions of IL-1β. Thus, antibodies of the invention can be found the following applications in the pharmaceutical industry:

Antibodies according to the invention can be used for prevention and treatment mediated IL-1 diseases or medical conditions, such as inflammatory conditions, allergies and allergic conditions, hypersensitive reactions, autoimmune diseases, severe infections and rejection of the transplant organ or tissue.

For example, antibodies of the invention can be used for the treatment of recipients after transplantation of heart, lung, systems, heart-lung, liver, kidney, pancreatic, skin or corneal, including allograft rejection or xenograft rejection, and for the prevention of graft versus host, for example, occur after bone marrow transplantation, and is associated with arteriosclerosis rejection of an organ transplant.

Antibodies according to the invention can be used to treat, prevent or alleviate autoimmune diseases and inflammatory conditions, in particular inflammatory conditions, etiology including an autoimmune component such as arthritis (for example rheumatoid arthritis, a chronic progressive arthritis and deforming the th arthritis), and rheumatic diseases, including inflammatory conditions and rheumatic diseases associated with bone loss associated with inflammation, pain, hypersensitivity (including both the hypersensitivity of the respiratory tract and dermal hypersensitivity) and allergies. Specific autoimmune diseases for which treatment can be applied to the antibodies of the invention include autoimmune haematological diseases (such as hemolytic anemia, aplastic anemia, true anemia erythrocytes and idiopathic thrombocytopenia), systemic lupus erythematosus, polyhedric, sclerodema, granulomatous's granulomatosis, dermatomyositis, chronic active hepatitis, severe pseudoparalysis the gravis, psoriasis, and syndrome of Stevens-Johnson, idiopathic sprue, autoimmune inflammatory bowel disease (such as ulcerative colitis, Crohn's disease and irritable bowel syndrome), endocrine ophthalmopathy, graves disease, sarcoidosis, multiple sclerosis, primary biliary cirrhosis, juvenile diabetes (diabetes mellitus type I), uveitis (anterior and posterior), keratoconjunctivitis sicca and vernal keratoconjunctivitis, collagen disorders of the lung, psoriatic arthritis and glomerulonephritis (with and without nephrotic syndrome or nephrotic syndrome, for example, the key idiopathic nephrotic syndrome or nephropathy minimal change).

Antibodies according to the invention can be used to treat, prevent or alleviate asthma, bronchitis, pneumoconiosis, pulmonary eczema and other obstructive or inflammatory diseases of the respiratory tract.

Antibodies according to the invention can be used to treat undesirable acute and hyperstretch inflammatory reactions, which are mediated by IL-1 or trigger the production of IL-1, primarily IL-1βor increased secretion of TNF under the action of IL-1, for example, acute infections, for example, septic shock (such as endotoxic shock and respiratory distress syndrome in adults), meningitis, pneumonia; and severe burns; for treatment of cachexia or wasting syndrome associated with morbid secretion of TNF, which is a consequence of infection, cancer or organ dysfunction, primarily for the treatment of AIDS-related cachexia, for example, associated with HIV infection or which is its consequence.

Antibodies according to the invention particularly preferably be used for the treatment-related metabolic diseases of bone and cartilage tissue, including osteoarthritis, osteoporosis and other inflammatory arthritis, and loss of bone tissue as a whole, including associated with age, the loss of bone tissue, and is primarily a disease of the periodontium.

The appropriate dose, which can be used to treat the above display is s, naturally should vary depending on, for example, specific antibodies used according to the invention, host, route of administration and the nature and severity of the condition to be treated. At the same time to prevent satisfactory results are usually obtained using doses of from about 0.05 to about 10 mg per kg body weight, more preferably from about 0.1 to about 5 mg per kg of body weight. Frequency of doses when used in the preventive purposes, typically ranges from about once per week to about once every 3 months, more preferably from about once every 2 weeks to about 1 time every 10 weeks, for example, once every 4 or 8 weeks. The antibody according to the invention it is expedient to introduce parenteral, intravenous, for example, subcutaneously or intramuscularly in in front from the elbow or other peripheral vein. For example, prophylactic treatment includes, as a rule, the introduction of antibodies according to the invention from once a month to once every 2-3 months or less.

The pharmaceutical compositions according to the invention can be prepared in the usual way. The composition according to the invention is preferably in dried form. For direct injection lyophilisate is dissolved in a suitable aqueous carrier, for example in the article is Riley water for injection, or sterile buffered saline solution. If you want a larger volume of solution for administration by infusion, for example, rapid intravenous administration of large volumes of fluid (bolus injection), it is useful in physiological solution during preparation of the composition to add bovine serum albumin or heparinised the patient's own blood. The presence of an excess of such physiologically inert protein prevents the loss of antibodies in the adsorption container walls and tubes, which are used together with a solution for infusion. If used albumin, the acceptable concentration range from 0.5 to 4.5% in recalculation on weight of saline.

The invention is additionally described using the following examples are presented only to illustrate that made reference to the attached drawing, which shows graphs of dose-response for inhibition of binding of IL-1β soluble receptors of IL-1 type I and II.

Examples

Created a transgenic mouse line, which was the expression of the spectrum of human IgG/mouse instead of the spectrum of immunoglobulins (Fishwild, etc., Nat. Biotechnol, 14, 845-851, 1996), used to generate antibodies to human IL-1β. B cells of these mice immortalized using standard methods based on hybrid and receive cells of the mouse hibrido is s, which secrete human antibody ACZ885 related to IgGl/κ.

Example 1: Getting hybridoma and purification of antibodies

Obtained through genetic engineering of the mouse 18077 (company Medarex Inc., Annadale, new Jersey, US) subjected to immunization by subcutaneous injection (s.c.) in several different areas of recombinant human IL-1βmade with KLH (hemocyanin lymph snails, 50 mcg)in Freund. The mouse is subjected to an additional booster injection, the latter carried out for 3 days before fusion. On the day of fusion, the mouse 18077 death by inhalation of CO2and spleen cells (4,1×107) are merging with the conventional method using PEG 4000, with the same number of cells of the mouse myeloma line PAI-O. Fused cells were seeded in 624 wells (1 ml/well)containing the supply sublayer mouse peritoneal cells (mouse strain Balb C), GAT-environment (gipoksantin-aminopterin-thymidine), supplemented RPMI medium 1640, 10% heat inactivated processing fetal calf serum, 5×10-5M β-mercaptoethanol. Supernatant collected and subjected to screening for IL-1β-reactive monoclonal antibodies using ELISA. Identify 5 monoclonal antibodies of the IgG subclass/K. Perform cloning using four 96-well titration microplate, sowing each well of rascheta,5 cells/well. After 2 weeks, the clones analyzed using an inverted microscope. Supernatant collected from wells positive for growth, and production of monoclonal antibodies to IL-1β and analyzed by ELISA. Get 1-2 l of conditioned supernatant 4 subclones originally identified hybridoma No. 657 and antibodies purified by affinity chromatography on a column of protein A.

The purity of the antibodies and partial amino acid sequences of the heavy and light chain

Sequencing of the amino acid sequences

Light and heavy chain purified antibodies ACZ 885 share with LTO-page and N-terminal amino acids was determined by splitting Admino. Using sequencing established that the purity of the antibodies used in these experiments is ≥90%. Sequence of cDNA encoding the variable regions of the heavy and light chains, create using PCR amplification of cDNA derived from mRNA cloned cells hybridoma and is sequenced completely. Aminobenzene sequence of the variable regions of the heavy and light chains and the corresponding DNA sequence presented in Seq. Id No.1 and Seq Id No.2, below, where the CDR is shown in bold.

Variable region of the heavy chain ACZ885 is presented in Seq. Id. No.1

Variable on the region of light chain ACZ 885 presented in Seq. Id. No.2

Italic: Leader sequence (absent in the Mature antibody)

Bold: CDR (hypervariable sites)

Construction of expression vectors of the heavy and light chain

Apply based on the GS system amplification/selection, for example, described in EP 0256055 IN, ER 0323997 IN or in the application for the European patent 45 89303964.4, whose breeding marker use the coding sequence GS.

Example 2: the Results of biochemical and biological assays

It is established that the antibody ACZ885 neutralizes the activity of Il-1β in vitro. Monoclonal antibody is additionally characterized based on its ability to bind to recombinant human IL-1βobtained using Biacore analysis. The mechanism of neutralizing activity is assessed through experiments on competitive binding to soluble receptors of IL-1. Biological activity of antibodies ACZ 885 respect and recombinant produced in vivo IL-1β evaluate the culture of primary human cells (example 3), sensitive to stimulation by IL-1β.

Determination of equilibrium constants of dissociation

The rate constants of Association and dissociation for the binding of recombinant human IL-1β with ACZ885 assess the try using BIAcore analysis. ACZ885 immobilized and the binding of recombinant IL-1β in the concentration range of 1-4 nm assessed using surface plasmon resonance. The selected format corresponds to the monovalent interaction and therefore allows you to provide the number of cases the binding of IL-1β with ACZ885, corresponding to the stoichiometric ratio of 1:1. Data analysis performed using the software BIAevaluation.

Table 1
kon[105/MS]koff[10-5/s]KD[RM]
Human IL-1β11,0±0,233,3±0,2730,5±2,6n=22

Conclusion: the Binding of ACZ885 with recombinant human IL-1β characterized by a very high affinity.

Study competitive binding to soluble IL-1--*receptor type I and II

Competitive binding ACZ885 with soluble human IL-1 receptor type I and type II assessed using Biacore analysis. ACZ885 immobilized on the surface of the biosensor chip and injected by injection of human IL-β (1Nm) to estimate binding with ACZ885 in the presence of increasing concentrations of recombinant human soluble cocktail recipes. is a type I or type II (0-12 nm; each concentration estimate using 4 independent replicates) or without addition of receptors. The obtained results are shown in the drawing.

The binding of NVP-ACZ885 with human IL-1β assess in the presence of recombinant human soluble IL-1 receptor type I or type II. The values that make up half of the maximum binding (IC50), assessed graphically using the software Origin 6.0. The results are presented as averages ± the standard deviation (n=4).

Conclusion: ACZ885 competes with IL-1β for binding to IL-1-receptor-like type I and type II.

Profile of reactivity against human IL-1αhuman IL-1RA and IL-1β other species

Profile of reactivity ACZ885 in relation to human IL-1αhuman IL-1RA and IL-1β monkey (cynomolgous) rabbit, mouse and rat assessed using Biacore analysis. ACZ885 immobilizer and studied cytokines contribute to the concentration of 8 nm (6 independent replicates.)

Table 2

The cross-reactivity NVP-ACZ885 with IL-1β, IL-1α and IL-1RA
% binding (mean ± RMS)
Rivers. human IL-1β (n=6)100
Rivers. monkey IL-1β (n=11)7,8±and 1.0
Rivers. rabbit IL-1β (n=6)-0,5±0,2
Rivers. mouse IL-1β (n=6)-2,6±0,6
Rivers. rat IL-1β (n=6)is 6.2±1,0
Rivers. human IL-1α (n=6)8,4±2,4
Rivers. human IL-1Ra (n=6)was 3.7±1,7

Resonance signals recorded during 1000 s after the start of administration; data obtained during the injection of the buffer for analysis shall be deducted from all sensogram and the basic level after immobilization of the antibody to Fcγ accept as zero. Binding is expressed as the percent of accumulated resonance signals for human IL-1β.

Conclusion: ACZ885 does not have a pronounced cross-reactivity against human IL-1αhuman IL-1RA or IL-1β monkey (cynomolgous), rabbit, mouse or rat.

Example 3: Neutralization with ACZ885 release of IL-6 from human dermal fibroblasts

To study the biological activity of ACZ885 in relation to neutralise human IL-1β use the following method:

1. Preparation containing IL-1β air-conditioned environment

Preparation of conditioned medium for mononuclear cells of peripheral blood is as follows: mononuclear cells receive the Ute from the peripheral blood of monkeys by dividing the density gradient picola-vipaka according to Hansel [Hansel TT etc. An improved immunomagnetic procedure for the isolation of highly purified human blood eosinophils. J. Imm. Methods.145: 105-110 (1991)]; they are used at a concentration of 105cells/well in medium RPMI/10% FCS. Add IFNβ (100 units/ml) and LPS (5 μg/ml) and the cells are then incubated for 6 hours Incubation stop by centrifugation at 1200 rpm for 10 minutes, the Content of IL-1β in the supernatant assessed quantitatively using ELISA.

2. Analysis of neutralization

Dermal foreskin fibroblasts person receive from the company Clonetics (CC-2509) and grown in medium FBM (firm Clonetics, CC-3131 ), which contains bFGF (1 ng/ml, SS-4065), insulin (5 βg/ml, SS-4021) and 2% FCS (SS-4101).

For the induction of IL-6 cells were seeded with a density of 104cells per well in 48-well cluster tissue culture. The next day, cells incubated for 6-7 h in incomplete medium FBM containing 2% FCS, and then add citizin. To stimulate the culture medium is replaced by an environment FBM+2% FCS containing a number of air-conditioned environment necessary for injection of 50 PG/ml IL-1β. Alternatively, use of recombinant human IL-1β at a final concentration of 50 PG/ml

Neutralizing antibody to IL-1β titrated in diluted air-conditioned environment, then add the cells. As a positive control, use of recombinant IL-1Ra (firm R&D Systems # 280-RA-010).

Through 16-17 is after stimulation obtain the supernatant of cells and the amount of released IL-6 assessed using a sandwich ELISA.

3. Analysis of IL-6 using ELISA

Titration microplates for ELISA sensibiliser mouse Mat to human IL-6 [314-14 (Novartis Pharma; party EN23,961; 5,5 mg/ml); 100 μl, 3 mg/ml] in SFR, supplemented with 0.02% NaN3and incubated over night at +4°C. the next day tetralonia the microplate is washed 4 times with a mixture SFR/0.05% tween/0.02% of NaN3and blocked with 300 μl of the mixture SFR/3% bovine serum albumin (BSA)/0,02% NaN3within 3 hours the Tablets again washed (4 times) and duplication add 100 ál of supernatant (final dilution 1:20) or recombinant human IL-6 as standard [(Novartis Pharma, No. 91902), tetrazona curve obtained using concentrations from 1 to 0,0156 ng/ml in serial two breeding]. After incubation over night at room temperature (RT) tablets washed (4 times) and add different mouse Mat to human IL-6 [(110-14, Novartis Pharma; 6,3 mg/ml); 100 μl, 1 μg/ml; 3 h at room temperature]. After 4 washes add labeled with Biotin goat anticigarette to mouse IgG2b (Southern company Biotechnology; No. 1090-08) end 1/10000 dilution (100 µl/well; 3 h at room temperature). After incubation tablets washed 4 times and add stitched with streptavidin alkaline phosphatase (firm Jackson Immunoresearch, No. 016-050-084) end 1/3000 dilution(100 µl/well; 30 min at room temperature). After washing (4 times) for 30 min add substrate (para-nitrophenylphosphate in diethanolamine buffer; 100 μl). The reaction stopped by adding 50 µl/well of 1,5M NaOH. The tablets analyzed using a tablet reader for titration microplate (firm Bio-Rad), using filters at wavelengths of 405 and 490 nm.

The levels of IL-6 in supernatant culture count in comparison with standard curves, using the approximation of cubic curves.

Statistical analysis and determination of the values of IC50carried out on the basis of approximation of the sigmoid curve.

The results:

Table 3

The inhibition induced by IL-1β the secretion of IL - 6
NVP-ACZ885, part 1 IC50[RM]±SKONVP-ACZ885, part 2 IC50[RM]±SKOIL-1ra IC50[RM]±CKO
The secretion of IL-6, air-conditioned environment54±6,1 (9,1±1.0 ng/ml) (n=6)44,6±3,6 (7,4±0,6 ng/ml) (n=6)30±3,1 (0,51±0.05 ng/ml) (n=5)
The secretion of IL-6, and rivers. human IL-1β42±3,4 (7,1±of 0.56 ng/ml) (n=4)63±2,8 (10,5±0,5) (n=6)H/o

Presents the values of the IC50for inhibition inducir the bathtub IL-1β the secretion of IL-6 from human skin fibroblasts. Fibroblasts stimulated with recombinant human IL-1β or with conditioned medium containing 50-100 PG/ml IL-1β.

Example 4: identification of the epitope for ACZ885

ACZ885 binds to human IL-1β with high affinity, but cannot recognize the high level of homology of IL-1βobtained from rhesus monkeys. One of the most pronounced differences between the amino acid sequences of IL-1β rhesus monkeys and humans is the amino acid at position 64 of Mature IL-1β. In human IL-1β in this position is glutamic acid; and rhesus monkeys - alanine. Mutant human IL-1β with appropriate replacement Glu64Ala loses its ability to communicate with ACZ885 with a noticeable level of affinity. From this we can conclude that Glu64 in human IL-1β is important for recognition by the antibody ACZ885. Glu64 localized in the loop of IL-1βthat is not part of the surface to bind to the IL-1β-receptor type I, or close to it. Thus, the antibodies involved in binding to the epitope, which includes Glu64, can neutralize the biological activity of human IL-1β.

1. IL-1β-Binding molecule containing the variable region of the heavy (VH) and light (VL) circuits, when it is specified IL-1β -binding molecule includes at least one antigennegative centre, containing

(a) the variable region of the heavy chain (VH) immunoglobulin, which includes consistently located hypervariable sites CDR1, CDR2 and CDR3, where CDR1 has the amino acid sequence Val-Tyr-Gly-Met-Asn, CDR2 has the amino acid sequence Ile-Ile-Trp-Tyr-Asp-Gly-Asp-Asn-Gln-Tyr-Tyr-Ala-Asp-Ser-Val-Lys-Gly, and CDR3 has the amino acid sequence Asp-Leu-Arg-Thr-Gly-Pro; and

(b) variable region light chain (VL) immunoglobulin, which consists of arranged in sequence hypervariable sites CDR1', CDR2' and CDR3', where CDR1' has the amino acid sequence Arg-Ala-Ser-Gln-Ser-Ile-Gly-Ser-Ser-Leu-His, CDR2' has the amino acid sequence Ala-Ser-Gln-Ser-Phe-Ser and CDR3' has the amino acid sequence His-Gln-Ser-Ser-Ser-Leu-Pro.

2. IL-1β-Binding molecule according to claim 1, which represents a human antibody.

3. IL-1β-Binding molecule, which includes at least one antigennegative centre, including either the first domain, the amino acid sequence of which is identical to the sequence presented in Seq. Id. No.1, which begins with amino acids in position 1 and ending with amino acid at position 118 or above the first domain and a second domain, the amino acid is of the selected identical sequence, presented in Seq. Id. No.2, which begins with amino acids in position 1 and ending with amino acid at position 107.

4. Design DNA, which encodes the heavy chain of IL-1β-binding molecule or its fragment, including

a) a first fragment that encodes the variable region containing alternative frame and hypervariable areas where hypervariable sites consist of consecutive CDR1, CDR2 and DR3, amino acid sequence presented in Seq. Id. No.1; this is the first fragment starts with a codon that encodes the first amino acid variable region, and ends with a codon that encodes the last amino acid variable region, and

b) a second fragment that encodes the constant region of the heavy chain or a fragment, which starts with a codon that encodes the first amino acid of the constant region of the heavy chain, and ends with a codon that encodes the last amino acid of the constant region or fragment, after which there is a stop codon.

5. Design DNA, which encodes the light chain of IL-1β-binding molecule or its fragment, including

a) a first fragment that encodes the variable region containing alternative frame section and hypervariable areas where hype the variable sites are CDR1' and CDR3', amino acid sequence of which is presented in Seq. Id. No.2; this is the first fragment starts with a codon that encodes the first amino acid variable region and ends with a codon that encodes the last amino acid variable region, and

b) a second fragment that encodes the constant region light chain or a fragment, which starts with a codon encoding the first amino acid of the constant region of the light chain, and ending with a codon that encodes the last amino acid of the constant region or fragment, after which there is a stop codon.

6. The expression vector is capable of replication in line prokaryotic or eukaryotic cells, which contains at least one design DNA according to claim 4.

7. The expression vector is capable of replication in line prokaryotic or eukaryotic cells, which contains at least one design DNA according to claim 5.

8. The method of obtaining IL-1β-binding molecule, which provides for (I) culturing an organism transformed by the expression vector according to claim 6, and (II) the secretion of IL-1β-binding molecule from the culture.

9. The method of obtaining IL-1β-binding molecule, which provides for (I) culturing an organism, transformed expre the pension vector according to claim 7, and (II) the secretion of IL-1β-binding molecule from the culture.

10. IL-1β-Binding molecule having antigennegative specificity in relation to the epitope of Mature human IL-1βcontaining loop carrier residue Glu 64, which includes at least one antigennegative centre, comprising as a first domain having an amino acid sequence that is identical represented in Seq. Id. No. 1, and a second domain having the amino acid sequence that is identical represented in Seq.Id. No. 2.



 

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11 cl, 1 tbl, 6 ex

FIELD: genetic engineering, virology, medicine.

SUBSTANCE: invention relates to method for production of modified Vaccinia virus Ankara (MVA). Claimed method includes contamination of mammalian continuous cell line with Vaccinia virus Ankara (MVA) of wild type, followed by viruses cultivation and collection. Further fresh cells of the same cell line are infected with newly formed viruses. Abovementioned steps optionally are repeated. Also disclosed are strains of modified Vaccinia virus Ankara (MVA) and utilization thereof. Said strains are capable to growth in continuous cell lines.

EFFECT: strains having decreased virulence in relates to mammalians.

20 cl, 5 tbl

The invention relates to the field of molecular biology and genetic engineering and can be used in medicine
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