Compositions and methods of treating diseases and disorders associating cytokine signal transmission
SUBSTANCE: there are offered versions of human IL22 specific antibodies. The antibodies are differed by the fact that they are produced of different hybridomas PTA-7312, PTA-7315, and PTA-7319. The PTA-7312 antibody is characterised by the fact that it inhibits STAT3 activation by reaching IC50 in the concentration 0.14 mcg/ml whereas the PTA-7315, PTA-7319 antibodies are characterised by Kd less than 1 nM.
EFFECT: use of the invention can find further application in therapy of the IL22 mediated immune disorders.
24 cl, 41 dwg, 1 tbl, 26 ex
This application is based on provisional application U.S. No. 60/741640, registered on December 2, 2005, and provisional application U.S. No. 60/822597, registered on August 16, 2006, the disclosure of which is reproduced in full in this document by reference.
The SCOPE of the INVENTION
The present invention relates to compositions and methods useful for the diagnosis and treatment of diseases and disorders associated with signaling by cytokines.
The LEVEL of TECHNOLOGY
Various diseases and disorders associated with inflammation. Inflammation is a process that is associated with recruitment of inflammatory cells (e.g. leukocytes) into the site of injury or infection. However, excessive or inappropriate inflammation can have a negative effect. Autoimmune disorders, for example, often cause inflammation, leading to the destruction of normal tissues of the body. Inflammation, in addition, is associated with cancer. See, for example, Coussens et al. (2002) Nature 420:860-867. For example, associated with inflammatory bowel disease (IBD) is a chronic inflammation strictly correlates with intestinal carcinogenesis. During the inflammatory response to certain inflammatory cells synthesize substances that stimulate angiogenesis, reduce the antitumor activity cytoxic T-cells and reduce the mutation of the DNA, thus creating the whirling accelerated tumour progression Id.
IL-23 is a heterodimeric cytokine that plays a dominant role in autoimmune/inflammatory disorders, and in particular in chronic inflammation. For example, in studies on mice it was shown that IL-23 is extremely important for the development of experimental allergic encephalomyelitis (autoimmune inflammation of the brain), which is a model of multiple sclerosis, collagen-induced arthritis, which is a model of rheumatoid arthritis; and hypersensitivity of the delayed type. IL-23 also functions to maintain the stable colitis (a form of IBD). Transgenic expression of IL-23 leads to a systemic inflammatory response and dysregulation of IL-23 leads to eczematous skin diseases (inflammatory skin condition). IL-23 activates specific populations of T cells (ThIL-17 cells), which in turn cause the production of ThIL-17 and anti-inflammatory cytokines. To explore the role of IL-23 in inflammation and autoimmune reactions, see, for example, Hunter (2005) Nat. Rev. Immunol. 5:521-531; and Holscher (2005) Curr. Opin. Invest. Drugs 6:489-495. It was shown that IL-23 also accelerates tumor growth by increasing angiogenesis and reduce tumor infiltration of cytotoxic T cells. Langowski et al. (2006)Nature 442:461-465.
SUMMARY of the INVENTION
Presents the compositions and methods useful for diagnostical the detention and treatment of inflammatory disorders and autoimmune disorders (for example, psoriasis). Below are compositions and methods useful for changing the signal transmission of IL-23 or IL-22. These and other embodiments of the invention presented in this document. The present invention is based in part on identification (definition) transmission signal, in which IL-23 acts through IL-22, stimulating expression of IL-22 in a recently discovered subset of T cells-helper cells (Th cells), that is, ThIL-17 line. In one aspect, presents an antibody that specifically binds to IL-22, in which the antibody is a (a) the antibody produced by hybridoma selected from 3F11.3 (ATCC catalog no PTA-7312), hybridomas 11H4.4 (ATCC catalog no PTA-7315), and hybridomas 8E11.9 (ATCC catalog no PTA-7319); (b) affine Mature form of the antibody (a); (c) antigennegative fragment antibodies (a) or (b); or (d) a humanized form of the antibody (a), (b) or (c).
In another aspect, presents an antibody that specifically binds to IL-22R, in which the antibody is a (a) the antibody produced by hybridoma selected from 7E9 (ATCC catalog no PTA-7313), hybridomas 8A12 (ATCC catalog no PTA-7318), and hybridomas 8H11 (ATCC catalog no PTA-7317); (b) affine Mature form of the antibody (a); (c) antigennegative fragment antibodies (a) or (b); or (d) a humanized form of the antibody (a), (b) or (c).
In another aspect, presents a method of treating autoimmune disorders, in which oterom autoimmune disorder is arthritis, the method involving the introduction of a mammal an effective amount of a pharmaceutical composition containing an antagonist of IL-22. In one embodiment, the antagonist of IL-22 is an antibody that specifically binds to IL-22. In one such embodiment, the antibody that specifically binds to IL-22, represents (a) the antibody produced by hybridoma selected from 3F11.3 (ATCC catalog no PTA-7312), hybridomas 11H4.4 (ATCC catalog no PTA-7315) and hybridomas 8E11.9 (ATCC catalog no PTA-7319); (b) affine Mature form of the antibody (a); (c) antigennegative fragment antibodies (a) or (b); or (d) a humanized form of the antibody (a), (b) or (c). In another embodiment, the antagonist of IL-22 is an antibody that specifically binds to IL-22R. In one such embodiment, the antibody that specifically binds to IL-22R, represents (a) the antibody produced by hybridoma selected from (ATCC catalog no PTA-7313), hybridomas 8A12 (ATCC catalog no PTA-7318) and hybridomas 8H11 (ATCC catalog no PTA-7317); (b) affine Mature form of the antibody (a); (c) antigennegative fragment antibodies (a) or (b); or (d) a humanized form of the antibody (a), (b) or (c). In one embodiment, the antagonist of IL-22 is an IL-22BP. In one embodiment, the autoimmune disease is an inflammatory Zab is levania intestine. In one embodiment, the autoimmune disease is a psoriasis. In one embodiment, the method further includes introducing at least one antibody selected from antibody, which specifically binds to IL20Ra, antibodies that specifically binds to IL20Rb, and antibody that specifically binds IL-22R. In one embodiment, the method further includes introducing at least one antibody selected from antibody that specifically binds IL-22 antibodies, which specifically binds IL20Ra, and antibody that specifically binds IL20Rb.
In another aspect, presents a method of treating inflammation in which inflammation is inflammation of arthritis, the method comprising the administration to a mammal an effective amount of a pharmaceutical composition comprising an antagonist of IL-22. In one embodiment, the antagonist of IL-22 is an antibody that specifically binds IL-22. In one such embodiment, the antibody that specifically binds IL-22, represents (a) the antibody produced by hybridoma selected from 3F11.3 (ATCC catalog no PTA-7312), hybridomas 11H4.4 (ATCC catalog no PTA-7315) and hybridomas 8E11.9 (ATCC catalog no PTA-7319); (b) affine Mature form of the antibody (a); (c) antigennegative fragment of the anti-Christ. ate (a) or (b); or (d) a humanized form of the antibody (a), (b) or (c). In one embodiment, the antagonist of IL-22 is an antibody that specifically binds IL-22R. In one such embodiment, the antibody that specifically binds IL-22R, represents (a) the antibody produced by hybridoma selected from 7E9 (ATCC catalog no PTA-7313), hybridomas 8A12 (ATCC catalog no PTA-7318) and hybridomas 8H11 (ATCC catalog no PTA-7317); (b) affine Mature form of the antibody (a); (c) antigennegative fragment antibodies (a) or (b); or (d) a humanized form of the antibody (a), (b) or (c). In one embodiment, the antagonist of IL-22 is an IL-22BP. In one embodiment, the inflammation is an autoimmune inflammation. In one embodiment, the inflammation is an inflammation of the skin. In one embodiment, the inflammation is a chronic inflammation.
In another aspect, presents a method of suppressing tumor progression, the method comprising the administration to a mammal an effective amount of a pharmaceutical composition containing an antagonist of IL-22. In one embodiment, the antagonist of IL-22 is an antibody that specifically binds IL-22. In another such embodiment, the antibody that specifically binds IL-22, is a (a) anti the ate, produced by hybridomas selected from 3F11.3 (ATCC catalog no PTA-7312), hybridomas 11H4.4 (ATCC catalog no PTA-7315) and hybridomas 8E11.9 (ATCC catalog no PTA-7319); (b) affine Mature form of the antibody (a); (c) antigennegative fragment antibodies (a) or (b); or (d) a humanized form of the antibody (a), (b) or (c). In one embodiment, the antagonist of IL-22 is an antibody that specifically binds IL-22R. In one such embodiment, the antibody that specifically binds IL-22R, represents (a) the antibody produced by hybridoma selected from 7E9 (ATCC catalog no PTA-7313), hybridomas 8A12 (ATCC catalog no PTA-7318) and hybridomas 8H11 (ATCC catalog no PTA-7317); (b) affine Mature form of the antibody (a); (c) antigennegative fragment antibodies (a) or (b); or (d) a humanized form of the antibody (a), (b) or (c). In one embodiment, the antagonist of IL-22 is an IL-22BP.
In another aspect, presents a method of stimulation of IL-23-mediated pathways of signal transmission in a biological system, the method comprising providing a biological system is an agonist of IL-22. In one embodiment, the agonist of IL-22 is an IL-22. In another aspect, presents a method of inhibiting IL-23-mediated pathways of signal transmission in a biological system, the method including the suppression of biological systems agonist of IL-22. In one embodiment, is sushestvennee, the agonist IL-22 is an antibody that specifically binds IL-22. In one embodiment, the agonist of IL-22 is an antibody that specifically binds IL-22R.
In another aspect, presents a method of stimulation of the function of ThIL-17 cells, the method including the impact of IL-22 agonist on ThIL-17 cells. In one embodiment, the agonist of IL-22 is an IL-22. In another aspect, presents a method of suppressing the functions of ThIL-17 cells, the method including the impact of IL-22 antagonist on ThIL-17 cells. In one embodiment, the antagonist of IL-22 is an antibody that specifically binds IL-22. In one embodiment, the antagonist of IL-22 is an antibody that specifically binds IL-22R.
BRIEF DESCRIPTION of DRAWINGS
Figure 1 shows the nucleotide sequence (SEQ ID NO: 1) cDNA encoding native IL-22.
Figure 2 shows the amino acid sequence (SEQ ID NO:2)derived from the coding sequence of SEQ ID NO: 1, shown in Fig. 1.
Figure 3 shows the amino acid sequence (SEQ ID NO:3) native IL-22R person.
Figure 4 shows the amino acid sequence (SEQ ID NO:4) native IL-22BP person.
Figure 5 presents a list of all of the obtained antibodies to IL-22 and their respective properties, as described in example 1. In Treklyano staining abbreviated as IC.
Figure 6 shows that anti-IL-22 antibodies capable of blocking the activation of STAT3, as described in example 2.
7 shows that three specific types of anti-IL-22 antibodies to block IL-22-dependent dose-dependent manner as described in example 3.
On Fig shown that three specific types of anti-IL-22 antibodies IL-22 is able to block murine IL-22-dependent dose-dependent manner as described in example 4.
Figure 9 presents the calculation of the affinity of anti-IL-22 antibodies to IL-22, as described in example 5.
Figure 10 shows that anti-IL-22 antibodies detect intracellular expression of IL-22, as described in example 6.
Figure 11 shows the intracellular FACS staining for IL-22 using a labeled anti-IL-22 antibodies, as described in example 6.
On Fig shows the expression of IL-22 in murine Th1 cells, as determined by 5' nuclease analysis, as described in example 7.
On Fig shows the expression of IL-22 in murine γδ T-cells, as determined by 5' nuclease analysis, as described in example 8.
On Fig shows the expression of IL-22 in activated T-cells, as defined microcephalum analysis, as described in example 9.
On Fig shows the level of expression of IL-22 in T-cells by FACS as described in example 10.
In Fig. 16 shows a study of anti-IL-22R antibodies on 293 cells expressing IL-22R, as described in example 11.
In Fig. 17 shows that anti-IL-22R antibodies can block induced IL-22 expression of STAT3 reporter construct as described in example 12.
In Fig. 18 shows the expression of IL-22R and IL-10R2 on the surface of primary keratinocytes, as described in example 13.
In Fig. 19 shows that IL-22 induces thinning of the epidermis of a person as described in example 14.
In Fig. 20 shows that IL-22 induces the expression of cytokeratin 16, marker keratinocyte metabolism, as described in example 14.
In Fig. 21 shows that the treatment of the epidermis of human IL-22 induces the induction of expression of psoriasin, vysokointensivnogo psoriasis gene, as described in example 14.
In Fig. 22 it was shown that treatment of keratinocytes with IL-22 increases the expression of several genes, including psoriasis as described in example 15.
In Fig. 23 shows that the expression of psoriasin decreases in the treatment of anti-IL-22 and anti-IL-22R antibodies as described in example 14.
In Fig. 24 shows that epidermal thinning decreases in the treatment of anti-IL-22 and anti-IL-22R antibodies as described in example 14.
In Fig. 25 it is shown that epidermal thinning decreases in the treatment of anti-IL-22 and anti-IL-22R antibodies as described in example 14.
In Fig. 26 it is shown that IL-23 and IL-12 induce epidermal thinning with distinct histological features, as described in the example is 16.
In Fig. 27 shows that IL-23 induces the expression of IL-22 and IL-22 induces skin inflammation and epidermal thinning in vivo as described in example 17 and 18.
In Fig. 28 shows that IL-12 and IL-23 induce the expression of specific groups of cytokines, as described in example 17.
In Fig. 29 shows that the treatment with anti-IL-22 monoclonal antibodies significantly reduces IL-23-induced epidermal acanthosis in vivo as described in example 20.
In Fig. 30 presents the strategy used for the destruction of the gene IL-22 in mice, and the evidence that the expression of IL-22 is absent in IL-22-/-mice as described in example 20.
In Fig. 31 it was shown that IL-23-induced acanthosis significantly reduced in IL-22 deficient mice as described in example 20.
In Fig. 32 it is shown that deficiency of IL-22 has no effect on IL-12-induced acanthosis, as described in example 20.
In Fig. 33 it is shown that IL-23 induces the production of IL-22 from different IL-23-activated lymphocytes, as described in example 21.
In Fig. 34 shows that IL-22 is a new effector cytokine of ThIL-17line, as described in example 22.
In Fig. 35 shows that IL-22 and IL-17 are produced by the same Th (ThIL-17as described in example 22.
In Fig. 36 shows that IL-23 stimulates the production of IL-22 on the basis of activation of T cells as described in example 22.
In Fig. 3 shows IL-19, IL-20, IL-22 and IL24 causes epidermal thinning, as described in example 23.
In Fig. 38 shows quantification of epidermal acanthosis, caused by IL-19, IL-20, IL-22 and IL24, as described in example 23.
On Fig shown that the components of the receptors for IL-19, IL-20 and IL-22 is expressed in human keratinocytes, as described in example 24.
In Fig. 40 shows that blocking antibodies to the components of the receptor for IL-19, IL-20 and IL-22 reduce the expression of psoriasin as described in example 24.
In Fig. 41 it was shown that antibodies to IL-20Ra and IL-22R, when used in combination, effectively blocking IL-20-induced expression of psoriasin.
DETAILED DESCRIPTION of embodiments
The term "IL-22 polypeptide" or "IL-22" refer to different interleukin-22 polypeptides (also designated as "interleukin-22 ligand or IL-22L" in this field). The term includes a native sequence IL-22 polypeptides, and their variants (defined later in this document). IL-22 polypeptides described herein, can be isolated from various sources, such as human tissue, or other source, or obtained recombinant or synthetic methods. Native IL-22 can be of any species, such as mouse ("mIL-22) or human ("hIL-22").
The terms "IL-22R polypeptide or IL-22R" refer to polypeptides component of heterodimer receptor interleukin-22 or heterodimer receptor interleukin-20. The term includes a native sequence IL-22R polypeptide and variants (which are defined later in this document). IL-22R polypeptide, described herein, can be isolated from different sources, such as human tissue, or other source, or obtained recombinant or synthetic methods. Native IL-22R can be any species, such as mouse ("mIL-22R") or human ("hIL-22R"). Native sequence IL-22R polypeptide, also known in this area as "IL-22R1 and IL22RA." "Native sequence IL-22 polypeptide" or "native sequence IL-22R polypeptide" refers to a polypeptide comprising the same amino acid sequence as the corresponding IL-22 or IL-22R polypeptide obtained in nature. Such native sequence IL-22 or IL-22R polypeptide can be purified natural or may be obtained by recombinant or synthetic methods. The terms, in particular, include naturally occurring truncated or secreted forms of the specific IL-22 or IL-22R polypeptide (for example, IL-22, not with its associated signal peptide), found in nature in various forms (for example, alternative forms of splashing), and naturally occurring allelic variants of the polypeptide. In a different implementation from the retene, native sequence IL-22 or IL-22R polypeptide, disclosed herein is a Mature or full-length native sequence polypeptides. Figure 2 and 3 shows a typical human full length IL-22 and IL-22R, respectively. Nucleic acid encoding the polypeptide shown in figure 2, is shown in figure 1. Start and stop codons shown in this drawing in bold and underlined. Although it was shown that IL-22 and IL-22R polypeptide sequence disclosed in the accompanying drawings, begin with methionine residues designated herein as amino acid position 1, it is possible and likely that other methionine residues located either in the reverse direction or in the forward direction from amino acid position 1 of the drawings, may be used as the starting amino acid residue for IL-22 or IL-22R polypeptide.
"IL-22 variant", "IL-22R option", "polypeptide IL-22 variant or polypeptide of IL-22R variant" means an active IL-22 or IL-22R polypeptide as defined above, having at least about 80% amino acid sequence identity with native sequence IL-22 full length or IL-22R polypeptide sequence. Usually the variant IL-22 or IL-22R polypeptide will have at least about 80% amino acid identity consequently the STI, alternatively, at least about 81% amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity, alternatively at least about 89% amino acid sequence identity, alternatively at least about 90% amino acid sequence identity, alternatively at least about 91% amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about 93% amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% identity AMI is kislotno sequence, alternatively, at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 98% amino acid sequence identity and alternatively, at least about 99% amino acid sequence identity to the full length or Mature native sequence IL-22 or IL-22R polypeptide sequence.
"Percent (%) amino acid sequence identity" with respect to IL-22 or IL-22R polypeptide sequences identified herein is defined as the percentage of amino acid residues in a suitable sequence that are identical with amino acid residues in the specific IL-22 or IL-22R polypeptide sequence, after the orientation of the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of identity sequences. Orientation for purposes of determining percent identity of amino acid sequence can be obtained in various ways known to the person skilled in the art, for example using a publicly available computer software on which especiany, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Specialists in this field can determine appropriate parameters for measuring orientation, including any algorithms needed to obtain the maximum orientation over the full length of the sequences used in the comparison. For comparisons of amino acid sequence % identity of amino acid sequences of the given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be referred to as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:
100 times the fraction X/Y,
where X represents the number of amino acid residues that are marked as identical pairs program orientation sequences in this software the orientation of A and B, and where Y represents the total number of amino acid residues in B. Taking into account that the length of amino acid sequence A is not equivalent to the length of amino acid sequence B, the % amino acid sequence identity of A to B is not equivalent to the % identity of amino acid posledovatel the surface B to A. As examples of computing % identity of amino acid sequences using this method, tables 2 and 3 shows how to calculate percent identity of the amino acid sequence designated "protein comparison of amino acid sequence to the amino acid sequence designated "IL-22 or IL-22R", where "IL-22 or IL-22R" represents the amino acid sequence of the polypeptide of interest IL-22 or IL-22R, "protein comparison" represents the amino acid sequence of a polypeptide against which compares the desired polypeptide IL-22 or IL-22R", and each "X, "Y" and "Z" represents a different amino acid residues.
|IL-22 or IL-22R||XXXXXXXXXXXXXXX||(length = 15 amino acids)|
|protein comparison||XXXXXYYYYYYY||(length = 12 amino acids)|
% amino acid sequence identity = (the number of identical pairs of amino acid residues between the two polypeptide sequences) divided by (the total number of amino acid residues of IL-22 or IL-22R polypeptide) = 5 divided by 15=33.3%of the
|IL-22 or IL-22R||XXXXXXXXXX||(length = 10 amino acids)|
|protein comparison||XXXXXYYYYYYZZYZ||(length = 15 amino acids)|
% amino acid sequence identity = (the number of identical pairs of amino acid residues between the two polypeptide sequences) divided by (the total number of amino acid residues of IL-22 or IL-22R polypeptide) = 5 divided by 10=50%
The term "IL-19" refers to any native IL-19 from any vertebrate animal, including mammals such as primates (e.g. humans and monkeys) and rodents (e.g. mice and rats), unless otherwise noted. The term encompasses "full-length", reprocessitems IL-19, and also any form of IL-19, which is obtained by processing in the cell. The term also encompasses naturally occurring variants of IL-19, for example, splice variants, allelic variants, and other isoforms. The term also encompasses fragments or variants of a native IL-19, which supports at least one biological activity of IL-19.
The term "IL-20" refers to any native IL-20 from any vertebrate animal, including mammals such as primates (e.g., l the di and monkeys) and rodents (e.g., mice and rats), unless otherwise noted. The term encompasses "full-length", reprocessitems IL-20, and any form of IL-20, which is obtained by processing in the cell. The term also encompasses naturally occurring variants of IL-20, for example, splice variants, allelic variants, and other isoforms. The term also encompasses fragments or variants of a native IL-20, which supports at least one biological activity of IL-20.
The term "IL-24" refers to any native IL-24 from any vertebrate animal, including mammals such as primates (e.g. humans and monkeys) and rodents (e.g. mice and rats), if no special instructions. The term encompasses "full-length", reprocessitems IL-24, and also any form IL-24, which is obtained by processing in the cell. The term also encompasses naturally occurring variants of IL-24, for example, splice variants, allelic variants, and other isoforms. The term also encompasses fragments or variants of a native IL-24, which supports at least one biological activity of IL-24.
The term "IL-22BP" or "IL-22 binding protein", as used herein, refers to any native IL-22BP from any vertebrate animal, including mammals such as primates (e.g. humans and monkeys) and rodents (e.g. mice and rats), unless otherwise noted. The term coverage is AET "full length", reprocessitems IL-22BP, and any form of IL-22BP, resulting from processing in the cell. The term also encompasses naturally occurring variants of IL-22BP, for example, splice variants, allelic variants, and other isoforms. The term also encompasses fragments or variants of a native IL-22BP, which supports at least one biological activity of IL-22BP. Native IL-22BP is also referred to as "IL-22RA2" in this field.
The term IL-20Ra refers to a polypeptide component of heterodimer receptor IL-19 or heterodimer receptor IL-20. The term includes any native IL-20Ra from any vertebrate animal, including mammals such as primates (e.g. humans and monkeys) and rodents (e.g. mice and rats), unless otherwise noted. The term encompasses "full-length", reprocessitems IL-20Ra and any form of IL-20Ra, resulting from processing in the cell. The term also encompasses naturally occurring variants of IL-20Ra, for example, splice variants, allelic variants, and other isoforms. The term also encompasses fragments or variants of a native IL-20Ra, which supports at least one biological activity of IL-20Ra. Native IL-20Ra is also referred to as "IL-20R1" in this field.
The term IL-20Rb refers to a polypeptide component of heterodimer receptor IL-19 or heterodimer receptor IL-20. The term includes uboy native IL-20Rb from any vertebrate animal including mammals such as primates (e.g. humans and monkeys) and rodents (e.g. mice and rats), unless otherwise noted. The term encompasses "full-length", reprocessitems IL-20Rb and any form IL-20Rb, resulting from processing in the cell. The term also encompasses naturally occurring variants of IL-20Rb, for example, splice variants, allelic variants, and other isoforms. The term also encompasses fragments or variants of a native IL-20Rb, which supports at least one biological activity of IL-20Rb. Native IL-20Rb also referred to as "IL-20R2 in this field.
The term "IL-10R2" refers to a polypeptide component of heterodimer receptor IL-22 or heterodimer receptor IL-20. The term includes any native IL-10R2 from any vertebrate animal, including mammals such as primates (e.g. humans and monkeys) and rodents (e.g. mice and rats), unless otherwise noted. The term encompasses "full-length", reprocessitems IL-10R2, and any form of IL-10R2, which is obtained by processing in the cell. The term also encompasses naturally occurring variants of IL-10R2, for example, splice variants, allelic variants, and other isoforms. The term also encompasses fragments or variants of a native IL-10R2, which supports at least one biological activity of IL-10R2. Native IL-10R2 is also called to the to IL-10Rb" in this field.
"Isolated" biological molecule, such as different polypeptides, polynucleotide and described in this document antibodies, refers to a biological molecule that has been identified and separated and/or removed from at least one component of the natural environment.
"Active" or "activity", in relation to IL-22 or IL-22R, refer to biological and/or immunological activity of native IL-22 or IL-22R, where "biological" activity refers to a biological function of the native IL-22 or IL-22R, eliminating the ability to cause the production of antibodies against antigenic epitope with native IL-22 or IL-22R. "Immunological" activity refers to the ability to induce the production of antibodies against antigenic epitope with native IL-22 or IL-22R.
The term "antagonist" is used in the broad sense of understanding and includes any molecule that partially or fully blocks, inhibits or neutralizes a biological activity of the polypeptide, such as native IL-22 or IL-22R polypeptide. Also included is the "antagonist" is a molecule that partially or fully inhibit the transcription or translation of mRNA encoding the polypeptide. Suitable molecule antagonists include, for example, antagonists, antibodies or antibody fragments, fragments or variants of aminokislotnykh sequences of the native polypeptide; peptides; antisense oligonucleotides, small organic molecules; and nucleic acids that encode the polypeptide antagonists or antagonists, antibodies. The reference to "an" antagonist includes a simple antagonist, or a combination of two or more different antagonists. The term "agonist" is used in the broad sense of understanding and includes any molecule that partially or fully reproduces the biological activity of the polypeptide, such as native IL-22 or IL-22R polypeptide. Also included "agonist" is a molecule that stimulates the transcription or translation of mRNA encoding the polypeptide. Suitable molecule agonists include, for example, agonists, antibodies or antibody fragments; native polypeptide, fragments or variants of the amino acid sequence of the native polypeptide; peptides; antisense oligonucleotides, small organic molecules; and nucleic acids that encode the polypeptide agonists or antibodies. The reference to "an" agonist includes a simple agonist or a combination of two or more different agonists.
"Relief" refers to therapeutic treatment and prophylactic or preventative measures, under which the individual is prevented or reduced rate (decreases) targeted pathologic condition or violation of Any in need of treatment include individuals with the violation, as well as individuals who are prone to disturbance, or individuals who have warned of the violation.
"Chronic" introduction refers to the introduction of substances(a) in continuous mode, in contrast to an acute mode, so as to maintain the initial therapeutic effect over an extended period of time. "Intermittent" introduction is a treatment that is not without a serial interrupt, but preferably is circular in nature.
The term "mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, rodents (e.g. mice and rats) and monkeys; Pets and farm animals; and the animals from the zoo, sports, laboratory or Pets, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. In some embodiments, implementation, mammals are selected from human, rodent or monkey.
Introduction "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and the subsequent introduction in any order. "Media"as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal, if exposed to it, in the action in the doses and concentrations. Often physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid; polypeptides of low molecular weight (less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; soleobrazutaya counterions such as sodium; and/or nonionic surfactants such as TWEENTM, polyethylene glycol (PEG) and PLURONICSTM. "Antibodies" (Abs) and "immunoglobulins" (Igs) are glycoproteins with similar structural characteristics.
Although antibodies inhibit the binding specificity of a specific antigen, the antibodies include antibodies and other antibody molecules, which, as a rule, devoid of antigenic specificity. The polypeptides of the latter type are, for example, produced by the lymphatic system in the low levels and myeloma at high level is X.
The terms "antibody" and "immunoglobulin" are used interchangeably in the broadest sense of understanding and include monoclonal antibodies (e.g., full length or intact monoclonal antibodies), polyclonal antibodies, monovalent antibodies, multivalent antibodies, multispecific antibodies (for example, bespecifically antibodies, because they exhibit the desired biological activity) and may also include certain antibody fragments (as described in detail in this document). The antibody may be chimeric, human, humanized and/or affinity Mature.
The antibody that specifically binds to a specific antigen, refers to the antibody that can bind antigen with sufficient affinato, so that the antibody is useful as a diagnostic and/or therapeutic substances in the target-antigen. Preferably, the degree of binding of such antibodies with anything the polypeptide represents less than about 10% of the binding of the antibody to the target antigen, as measured, for example, radioimmunological assay (RIA). In some embodiments, the implementation, the antibody that binds to the target antigen, has a dissociation constant (Kd)≤1 ám ≤100 nm ≤10 nm, ≤1 nm or ≤0.1 nm.
"Variable region" or "variable domain" antibody apply the to the amino-terminal domains of the heavy or light chain antibodies. Variable domain of the heavy chain can be named as "VH." Variable domain light chain can be named as "VL." These domains usually are the most variable part of the antibody containing the binding sites of the antigen.
The term "variable" refers to the fact that some parts of the variable domains differ significantly in sequence antibodies and are used in the binding and specificity of each particular antibody for a particular antigen. However, the variability is not evenly distributed on all variable domains of antibodies. It is concentrated in three segments called hypervariable sites (CDRs) or hypervariable sites (HVRs) in the variable domain and the light chain and heavy chain. More highly conserved areas of variable domains are called skeletal regions (FR). Each of the variable domains of native heavy and light chains contain four FR region, mainly taking beta-visible configuration, connecting three CDRs, which form a loop connection, and in some cases forming part of the beta-visible patterns. The CDRs in each chain are fastened in the immediate vicinity of the FR regions and, with the CDRs of the other circuit involved in the formation antigennegative site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Insttute of Health, Bethesda, MD (1991)). The constant domains are not directly involved in the binding of an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be attributed to one or two apparently distinct types, called Kappa (K) and lambda (λ), based on the amino acid sequences of their constant domains.
Depending on the amino acid sequence of the constant domain of their heavy chains of antibodies (immunoglobulins) can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of them can be further divided into subclasses (isotypes), e.g. IgG1, IgG2, IgG3, IgG4, IgA1and IgA2. The constant domains of the heavy chain, which correspond to the different classes of immunoglobulins are called α, δ, ε, γ and µ, respectively. Structures of subunits and spatial configuration of different classes of immunoglobulins are well known and widely described, for example, Abbas et al. Cellular and Mol. Immunology, 4th ed. (2000). The antibody may be part of a large complex molecule, formed by covalent or non-covalent coupling of antibodies with one or more other proteins or peptides.
The terms "shall titulo full length", "intact antibody" and "fully human antibody" is used in this document is equivalent to refer to antibodies in its core intact form, without fragments of antibodies, as described below. The terms, in particular, relate to the antibody heavy chains that contain an Fc region. "Fragments of antibodies include only the area of intact antibodies, in which the site supports at least one, and as many or all of the function, usually associated with land in the presentation of the intact antibody. In one embodiment, the antibody fragment includes antigennegative the site of the intact antibody and thus supports the ability to bind antigen. In another embodiment, the antibody fragment, for example, which includes the Fc region supports at least one of the biological functions normally associated with the Fc region in the presentation of the intact antibody, such as FcRn binding, regulation of time half-life of antibodies, ADCC function and binding of complement. In one embodiment, the fragment of the antibody is a monovalent antibody that is substantially similar half-life in vivo with intact antibody. For example, such an antibody fragment may lie on antigennegative shoulder associated with the Fc sequence, is able to impart stability fragment is in vivo.
Cleavage of antibodies with papain leads to the formation of two antigenspecific fragments, called "Fab" fragments, each with one antigennegative site, and a residual "Fc" fragment, whose name reflects its ability to crystallize. Product processing pepsin F(ab')2the fragment has two antigenspecific plot and is still able to cross-link the antigen.
"Fv" is the minimum antibody fragment that contains a binding site full of antigen. In one embodiment, double-stranded Fv types consist of a dimer of the variable domain of one heavy and one light chain in a stable non-covalent linkages. In single-chain Fv (scFv) species variable domain of one heavy and one light chain can be covalently linked to the free peptide bond, such that the light and heavy chains can be contacted in a "dimeric" structure analogous to the structure of double-stranded Fv species. It exists in this configuration that the three CDRs of each variable domain interact to recognition antigennegative area on the surface of the VH-VL dimer. In aggregate, the six CDRs give antigennegative the specificity of the antibody. However, even a single variable domain (or half of an Fv, containing only three CDRs specific for an antigen) has the ability of recognizing the ü and bind antigen, although with lower affinato than the entire binding site.
Fab fragment contains the variable domains of the heavy and light chains and also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl end of the domain of the heavy chain CH1, including one or more cysteines from the hinge region of the antibody. Fab'-SH in this document represents the designation of Fab'in which the cysteine residues constant domains associated with free Tilney group. F(ab')2fragments of antibodies originally producirovanie as pairs of Fab' fragments which have articulated cysteine between them. Also known other chemical interaction fragments of antibodies.
"Single-chain Fv" or "scFv fragments of antibodies contain the VH and VL domains of antibodies, where these domains are presented in a single polypeptide chain. As a rule, the scFv polypeptide further contains a polypeptide bond between VH and VL domains, which allow scFv to form the desired structure for antigen binding. For a review of scFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
The term "dimer" refers to small fragments of antibodies with two antihistamine parts, fragments which contain variable momentarily chain (VH), coupled with the variable domain of the light chain (VL) in the same polypeptide chain (VH-VL). Using a linker that is too short to mate with the two domains on the same chain, the domains must mate with complementary domains of another chain and form two antigenspecific plot. The dimers can be bivalent or bespecifically. More dimers described, for example, in EP 404097; WO93/1161; Hudson et al. (2003) Nat. Med. 9:129-134; and Hollinger et al, Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Trimers and tetramer also described in Hudson et al. (2003) Nat. Med. 9: 129-134.
As herein used, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical except for possible mutations that can be introduced in small quantities. Thus, the modifier "monoclonal" indicates the character of the antibody, which is not a mixture of separate antibodies. In some cases, the implementation of such monoclonal antibody generally includes the antibody containing a polypeptide sequence that binds a target, where the target-binding polypeptide sequence was obtained in a process that includes the selection of one target-binding polypeptide after which outermost of multiple polypeptide sequences. For example, the process of selection may be a selection of unique clone of many clones, such as a pool of hybridoma clones, phage clones or clones with recombinant DNA. It should be understood that the selected target-binding sequence may be further modified, for example, in order to improve the affinity to the target, to humanize the target-binding sequence, to improve its products in cell cultures, to reduce its immunogenicity in vivo, to get multispecific antibody and another, and that the antibody containing the modified target-binding sequence, is also a monoclonal antibody of this invention. Unlike drugs polyclonal antibodies, which usually include antibodies directed against different determinants (epitopes), each monoclonal antibody of the monoclonal antibody is directed against a single antigen determinants. In addition to their specificity, the preparations of monoclonal antibodies effective in that they typically do not contain other immunoglobulins.
The modifier "monoclonal" indicates the character of the antibody, derived largely from a homogeneous population of antibodies, and it cannot be considered as a desirable production of antibodies in any private way. In the example, to be used in accordance with the present invention, monoclonal antibodies can be obtained in various ways, including, for example, the hybridoma method (e.g., Kohler et al, Nature, 256: 495 (1975); Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), the methods of recombinant DNA (see, for example, U.S. patent No. 4816567), methods of phage display (see, for example, Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods. 284(1-2):119-132(2004) and technologies for human or human-like antibodies in animals that contain part or all of the loci or genes of the human immunoglobulin coding sequence of human immunoglobulin (see, for example, WO98/24893; WO96/34096; WO96/33735; WO91/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. patent№ 5545807; 5545806; 5569825; 5625126; 5633425; 5661016; Marks et al., Bio. Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-813 (1994); Fishwild et al., Nature Biotechnol. 14:845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995).
The monoclonal antibodies herein specifically include "chimeric" antibodies in which the plot heavy and/or light chain of similar or homologous ACC is stoysin sequences of antibodies received from a single species or belonging to a particular class or subclass of antibody, while the remainder of the chain (chain) similar or corresponding sequences of antibodies derived from other species or belonging to another class or subclass of antibody, as well as fragments of such antibodies, as they exhibit the desired biological activity (U.S. patent No. 416567; and Morrison et al, Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
"Humanized" forms of inhuman (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from a nonhuman immunoglobulin. In one embodiment, humanitariannet antibody is a human immunoglobulin (recipient antibody)in which residues from a hypervariable region of the recipient were replaced by residues from a hypervariable region of nonhuman species (donor antibody)such as mouse, rat, rabbit or nonhuman Primate having the desired specificity, affinitiy and/or ability. In some cases, the remains of the skeletal region (FR) of a human immunoglobulin are replaced by corresponding inhuman remnants. Furthermore, humanized antibodies may include residues that are not detected by the antibodies of the recipient or donor. This mod is the creativity you can get to further improve the productivity of antibodies. As a rule, humanitariannet antibody will contain almost all or at least one, and typically two, variable domain, in which all or nearly all of the hypervariable loops correspond to loops nonhuman immunoglobulin and all or nearly all FR represent the FR sequence of a human immunoglobulin. Additionally, humanized antibodies will also contain at least the plot of constant region of immunoglobulin (Fc), typically the area of human immunoglobulin. Further details, see Jones et al, Nature 321:522-525 (1986); Riechmann et al, Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). Cm. also described in this document, the following review articles and links: Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994).
"Human antibody" is an antibody that contains an amino acid sequence that corresponds to the sequence of the antibodies derived from human, and/or was obtained with the use of technology to generate antibodies of the person as shown in this document. This definition of human antibodies specifically excludes humanitariannet antibody containing inhuman antigennegative remains.
Depending on the amino acid sequence of the constant domain of their tagliata, immunoglobulins can be assigned to different classes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of them can be further divided into subclasses (isotypes), e.g. IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. "Affinity matured" antibody is an antibody with one or more transitions in one or more of the HVR, which increase the affinity of the antibody for antigen, compared to a parent antibody which does not contain such transitions. In one embodiment, affine Mature antibody possesses nanomolar or even picomolar affiniscape to the target antigen. Affine Mature antibodies can be obtained by methods known in this field. Marks et al. Bio/Technology 10:779-783 (1992) describes affinity maturation by shuffling VH and VL domains. Nonspecific mutagenesis HVR and/or skeletal remains described in: Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al, Gene 169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al, J. Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).
"Blocking" antibody, "neutralizing" antibody or antibody "antagonist" is an antibody that inhibits or reduces biological activity of binding antigen. Such antibodies can substantially or completely inhibit the biological activity of the antigen. As used herein, "antibody agonist" p is ecstasy an antibody, which partially or completely reproduces the biological activity of the polypeptide of interest.
"The effector functions of antibodies related to biological activities associated with the Fc region (Fc region of the native sequence or variant Fc region amino acid sequences) antibodies, and change depending on the isotype of the antibody. Examples of effector functions of antibodies include: C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., receptor B-cells); and activation of B-cells.
"The binding affinity of"generally refers to the strength of the aggregate non-covalent interactions between a single binding site of a molecule (e.g. antibody) and its binding partner (e.g., antigen). Unless otherwise indicated, as used herein, "binding affinity of" refers to the true binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y, as a rule, can be represented by a dissociation constant (Kd). The affinity can be measured by conventional means, well known in this area, including those described in this d is the document. Antibodies with low affinity, usually associated antigen slowly and tend to easily dissociate, whereas antibodies with high affinity, usually more quickly intended to bind the antigen and are likely to stay longer linked. Various ways to measure the affinity of binding known in this field, each of which can be used for the purposes of the present invention. Specific illustrative embodiments of described below. In one embodiment, the "Kd" or "Kd value" according to this invention is measured by analyzing the binding of radioactively labeled antigen (RIA)performed with the Fab version of specific antibody and its antigen as described by the following analysis. Dissolved the binding affinity of Fabs for antigen measured trim Fab with a minimal concentration of (125I)-labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound peroxidase antigen anti-Fab antibody coated tablet (Chen, et al., (1999) J. Mol. Biol. 293:865-881). To create conditions for analysis, microtitre plates (Dynex) are covered on the night of 5 µg/ml of captured anti-Fab antibody (Cappel Labs) in 50 mm sodium carbonate (pH 9,6), and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23°C). In readsorbing tablet (Nunc #269620), 00 PM or 26 PM [ 125I]-antigen are mixed with serial dilutions of interest to Fab (e.g., consistent with assessment of an anti-VEGF antibody, Fab-12, in Presta et al., (1997) Cancer Res. 57:4593-4599). Interested Fab then incubated over night; however, the incubation can be continued for a longer period (for example, approximately 65 hours), to ensure that the achieved balance. After this mixture is transferred to an exciting tablet for incubation at room temperature (for example, within one hour). Then remove the solution, and the plate is washed eight times with 0.1% Tween-20 in PBS. If the tablets were dried, then add 150 ál/well of scintillator (MicroScint-20; Packard), and tablets counted on Topcount gamma counter (Packard) for ten minutes. The concentration of each Fab, which gives less than or equal to 20% of maximum binding, choose to use in a competitive binding assay. According to another variant implementation, the Kd or Kd value was measured using surface plasmon resonance analysis using BIAcoreTM-2000 or a BIAcoreTM-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with immobilizerturning granules antigen CM5 ~10 response units (RU). Briefly, karboksimetilirovaniya dextranase biosensor granules (CM5, BIAcore Inc.) activated N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N - hydroxysuccinimide (NHS) according to the instructions about the voditelj. Antigen is diluted with 10 mm sodium acetate, pH of 4.8, to 5 μg/ml (~0.2 μm) before the introduction of the flow rate of 5 μl/minute to achieve approximately 10 response units (RU) of the associated protein. After injection of antigen injected with 1 M ethanolamine to block unreacted groups. For kinetics measurements, two serial cultivation of Fab (0.78 nm to 500 nm) are injected in PBS with 0.05% of Tween 20 (PBST) at 25°C at a flow rate of approximately 25 μl/min Rate of Association (kon) and dissociation rate (koff) calculated using a simple one-to-one Langmuir binding model (BIAcore Evaluation Software version 3,2), simultaneously aligning sensogram Association and dissociation. The equilibrium dissociation constant (Kd) is calculated as the ratio of the koff/kon. See, for example, Chen Y. et al., (1999) J. Mol. Biol. 293:865-881. If the on-rate exceeds 106M-1with-1using the aforementioned surface plasmon resonance analysis, then the on-rate can be determined using the method of fluorescence quenching, which measures the increase or decrease in fluorescence intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandwidth) at 25°C 20 nm antibody against the antigen (Fab form) in PBS, pH of 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a spectrophotometer, equipped ostanovlen the m stream (Aviv Instruments), or 8000 serial SLM-Aminco spectrophotometer (ThermoSpectronic) with a mixing cuvette.
"On-speed", "speed Association", "Association rate" or "kon" according to this invention can also be defined as described above using a BIAcoreTM-2000 or a BIAcoreTM-3000 system (BIAcore, Inc., Piscataway, NJ).
"Isolated" antibody is an antibody that was identified and isolated and/or extracted from a component of its natural environment. Unwanted components of its natural environment are substances that may prevent diagnostic and therapeutic application of antibodies and may include enzymes, hormones and other protein or non-protein solute. In preferred embodiments, the implementation, the antibody will be purified (1) to more than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) substantially to obtain at least 15 residues of N-terminal or internal amino acid sequence, using the sequencing machine with rotating cups, or (3) to homogeneity by SDS-PAGE in reducing or non conditions using Kumasi blue or, preferably, silver staining. Isolated antibody includes the antibody in situ within recomb nantah cells, since at least one component of the natural environment antibodies will be presented. Typically, however, an isolated antibody is obtained, at least one stage of the treatment.
The word "label" is used in this document to a detectable compound or composition that is conjugated directly or indirectly to a molecule (such as a nucleic acid, polypeptide, or antibody), as well as getting "tagged" molecules. A label can be defined by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a compound or composition of the substrate, leading to a detectable product.
By "solid phase" refers to non-aqueous matrix to which may stick molecule (such as a nucleic acid, polypeptide, or antibody). Examples of solid phases included in this document include phase forming partially or entirely of glass (e.g., glass, controlled pore size), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicone. In certain embodiments of the implementation, depending on the context, the solid phase may include the hole analyzed the tablet; in others it is a column for purification (for example the EP, column for affinity chromatography). This term also includes a heterogeneous solid phase of the scattered particles, such as described in U.S. patent No. 4275149.
"Liposome" is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug (such as a nucleic acid, polypeptide, antibody, agonist or antagonist) mammals. Components of liposomes are typically located in the two-layer formation, similar to the arrangement of lipids in biological membranes.
"Small molecule" or "small organic molecule" is defined herein as an organic molecule having a molecular weight below about 500 daltons.
"Oligopeptide", which binds the polypeptide-target is an Oligopeptide which is capable of binding polypeptide-target with sufficient affinity, so Oligopeptide useful as a diagnostic and/or therapeutic substance in the direction of the polypeptide. In certain embodiments of the exercise, the amount of binding of oligopeptides with unbound non-target the polypeptide is less than about 10% of the binding of oligopeptides with polypeptide-target, as measured, for example, surface plasmon resonance analysis. In certain variations the tah implementation Oligopeptide binds the polypeptide-target with a dissociation constant (Kd)≤1 ám ≤100 nm ≤10 nm, ≤1 nm or ≤0.1 nm.
"Organic molecule"which binds the polypeptide target, is an organic molecule, with the exception of oligopeptides or antibodies as defined herein that is capable of binding polypeptide-target with sufficient affinity, so that the organic molecule is useful as a diagnostic and/or therapeutic substance in the direction of the polypeptide. In certain embodiments of the implementation, the value of binding organic molecules from unbound non-target the polypeptide is less than about 10% binding organic molecules with the polypeptide-target, as measured, for example, surface plasmon resonance analysis. In certain embodiments of the implementation of the organic molecule binds to the polypeptide-target with a dissociation constant (Kd)≤1 ám ≤100 nm ≤10 nm, ≤1 nm or ≤0.1 nm.
"Biological systems" represent in vitro, ex vivo or in vivo system, including mammalian cells, which are involved in normal transmission of the signal.
The term "immune disease" means a disease in which component of the immune system in mammals causes, mediates or otherwise contributes to the incidence of mammals. Takakuni are diseases, in which stimulation or influence the immune response has a favorable effect on disease progression. Covered by this term are immune inflammatory diseases, immune inflammatory diseases, infectious diseases, immunodeficieny diseases and neoplasia.
The term "a disease mediated by T-cells" refers to a disease in which T cells directly or indirectly mediate or otherwise contribute to the incidence of mammals. The disease is mediated by T-cells, may be associated with mediated cell effects, mediated by lymphokines effects, and others, and even effects associated with B-cells, if, for example, b-cells are stimulated sekretiruemyi T-cells lymphokines.
As used herein, the term "psoriasis" is defined as a condition characterized by the eruption of limited, discrete and confluent, reddish, clean-calulcate macropapular certainly on the elbows, knees, scalp, or trunk. As used herein, the term "tumor" refers to all types of neoplastic growth and proliferation of cells, either benign or malignant, and all pre-cancerous and cancerous cells and tissues. The terms "cancer", "cancer", "violation of cell proliferation", "bre is the proliferation of" and "tumor" are not mutually exclusive, as mentioned in this document.
The term "tumor progression" refers to the growth and/or proliferation of a tumor.
The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, liver cell cancer, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colorectal cancer, rectal cancer, stomach cancer, endometrial or uterine carcinoma, carcinoma of the salivary glands, kidney cancer, liver cancer, prostate cancer, cancer-related external female genitals, thyroid cancer, liver cancer, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.
"Autoimmune disorders" or "autoimmunity" refers to the condition in which humoral or mediated by cells of the immune response increases, counteracting its own the public body tissue. "Autoimmune disorder mediated by IL-23" represents any autoimmune disorder that is caused by continuous or acute activity of IL-23.
"Inflammation" refers to the accumulation of leukocytes and dilation of blood vessels in the area of injury or infection, usually causing pain, swelling and redness.
"Chronic inflammation" refers to inflammation, which cause inflammation persists and it is difficult or impossible to remove.
"Autoimmune inflammation" refers to inflammation associated with autoimmune disorder.
"Arthritic inflammation" refers to inflammation associated with arthritis.
"Inflammatory bowel disease" or "IBD" refers to chronic disturbance, characterized by inflammation of the gastrointestinal track. IBD includes ulcerative colitis, which affects the colon and/or rectum, Crohn's disease, which can affect the entire gastrointestinal system, but more commonly affects the small intestine (the ileum) and possibly the colon.
"Arthritis" refers to the inflammation of the joints and include, but are not limited to, osteoarthritis, gout, infection-related arthritis, arthritis Reiter syndrome and arthritis associated with autoimmune disorders such as rheumatoid arthritis, psoriatic arthritis, associated with volcanoeafter, the vertebrae arthritis and scleroderma-associated arthritis.
The term "effective amount" is a concentration or number of molecules (e.g. nucleic acid, polypeptide, agonist or antagonist), which leads to the attainment of a specific objective. "Effective amount" can be determined empirically. "Therapeutically effective amount" is a concentration or number of molecules, which is effective for achieving a therapeutic effect. This number can also be determined empirically.
As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g., I131I125, Y90and Re186), therapeutic chemical substances and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof.
"Chemical therapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemical therapeutic substances include adriamycin, doxorubicin, epirubicin, 5-fluorouracil, cytosine arabinoside ("Ara-C"), cyclophosphamide, thiotepa, the bus is then, cytoxin, taxoid, for example, paclitaxel (Taxol, Bristol-Myers Squibb Oncology, Princeton, NJ), and docetaxel (Taxotere, Rhone-Poulenc Rorer, Antony, France), Taxotere, methotrexate, cisplatin, melphalan, vinblastine, bleomycin, etoposide, ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, carboplatin, teniposide, daunomycin, karminomitsin, aminopterin, dactinomycin, mitomycin, espiramicina (see U.S. patent No. 4675187), melphalan and other related nitrogen mustards. Also included in this definition are hormonal substances that affect the regulation and suppression of hormone action on tumors such as tamoxifen and onapristone.
"The vast growth agent" when used herein refers to a compound or composition which inhibits the growth of cells, particularly cancer cells, do overexpress any of the genes identified in this document, either in vitro or in vivo. Thus, the vast growth substance is a substance that significantly reduces the percentage of cells that do overexpress these genes in S phase. Examples inhibit the growth substances include substances that block the progress of the cell cycle (other than S phase), such as substances that cause a delay in G1 and the delay of the M-phase. Classic blockers M-phase include Vincas (vincristine and vinblastine), t is ksol and topo-II inhibitors, such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those substances that delay in G1 also spill over into the delay S-phase, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle regulation, oncogens, and antineoplastic drugs" Murakami et al. (WB Saunders: Philadelphia, 1995), in particular p. 13.
The term "cytokine" is a term specific to a certain kind of proteins secreted by a single population of cells, which acts on a different population of cells as intracellular mediators. Examples of such cytokines are lymphokines, Monokini and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, N-methionyl the human growth hormone and growth hormone in cattle; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prolactin; glycoprotein hormones such as folliculo-stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogenic; tumor necrosis factor-α and-β; müller inhibiting substance; mouse gonadotropin-associated is eptid; inhibin; activin; factor vascular endothelial growth; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet growth factor; transforming growth factors (TGFs)such as TGF-α and TGF-β; insulin-like growth factor-I and-II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-α, -β, and-γ; colony stimulating factors (CSFs)such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukine (ILs)such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12; a tumor necrosis factor such as TNF-α or TNF-β; and other polypeptide factors including LIF and ligand set (KL). As used herein, the term cytokine includes proteins from natural sources or culture of recombinant cells and biologically active equivalents of the native sequence cytokines.
As used herein, the term "cell inflammation" called cells, which amplify the inflammatory response, such as mononuclear cells, eosinophils, macrophages and polymorphonuclear neutrophils (PMN).
II. The COMPOSITIONS AND METHODS of the INVENTION
A. IL-22 or IL-22R polynucleotide and polypeptides
The present invention presents selected IL-22 or IL-22R polypeptide and a selected nucleotide sequence encoding such polypeptides. IL-22 or IL-22R polypeptide include is in itself a native full length or Mature IL-22 or IL-22R polypeptide, as well as variants of IL-22 or IL-22R. Variants of IL-22 or IL-22R can be obtained by introducing appropriate nucleotide changes into the DNA of IL-22 or IL-22R, and/or by synthesis of the desired IL-22 or IL-22R polypeptide. Specialists in the art will note that the amino acid changes may alter post-translational processing of IL-22 or IL-22R, such as changing the number or position of glycosylation sites or change the characteristics of the attached membrane.
Changes to native IL-22 or IL-22R or different domains of IL-22 or IL-22R, as described in this document can be obtained, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for example, in U.S. patent No. 5364934. Change can be a substitution, deletion or insertion of one or more codons encoding IL-22 or IL-22R, which lead to changes in the amino acid sequence of IL-22 or IL - 22R compared with the native sequence IL-22 or IL-22R. Additionally, the change is replacing at least one amino acid with any other amino acid in one or more domains of IL-22 or IL-22R. Guidance on determining which amino acid residue can be embedded, substitute or remove without adversely affecting the desired activity can be detected by comparing posledovatel the activity of IL-22 or IL-22R with a sequence homologous known protein molecules and minimizing the number of changes in amino acid sequence, made in regions of high homology. Amino acid substitutions can be the result of the substitution of one amino acid to another amino acid having similar structural and/or chemical properties, such as the substitution of leucine for serine, that is conservative amino acid substitutions. Insertions and deletions can be varied from approximately 1 to 5 amino acids. Acceptable changes can be defined systematically making insertions, deletions or substitutions of amino acids in the sequence and examining the received options to show activity on the primary or Mature native sequence.
In specific embodiments, the implementation of interest to conservative substitutions are shown in table 6 under the heading of preferred substitutions. If such substitutions result in a change in biological activity, then entered a more substantial changes, indicated by exemplary substitutions in table 6, or, as further described below in reference to classes of amino acids, and conduct screening products.
|The original balance||Approximate replacement||Preferred replacement|
|Ala (A)||val; leu; ile||Arg(R)||lys; gln; asn||lys|
|Asn (N)||gln; his; lys; arg||gln|
|His (H)||asn; gln; lys; arg||arg|
|Ile (I)||leu; val; met; ala; phe; norleucine||leu|
|Leu (L)||norleucine; ile; val; met; ala; phe||ile|
|Lys (K)||arg; gln; asn||arg|
|Met (M)||leu; phe; ile||leu|
|Phe (F)||leu; val; ile; ala; tyr||leu|
|Tyr (Y)||trp; phe; thr; ser||phe|
|Val (V)||ile; leu; met; phe; ala; norleucine||leu|
Substantial modifications in function or immunological identity of IL-22 or IL-22R polypeptide can be achieved by the selected substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as folded or helical conformation, (b) the charge or hydrophobicity of the molecule in the field of the target, or (c) the main part of the side chain. Naturally occurring residues are divided into groups based on common properties of the side chain:
(1) hydrophobic: norleucine, met, ala, val, leu, ile;
(2) neutral hydrophilic: cys, ser, thr;
(3) acidic: asp, glu;
(4) basic: asn, gin, his, lys, arg;
(5) residues that influence chain orientation: gly, pro; and
(6) aromaticheski is: trp, tyr, phe.
Non-conservative substitutions will cause the currency of a member of one of these classes for another class.
Such substituted residues can also be entered in a conservative replacement parts or, more preferably, in the rest (non-conservative) areas.
Changes can be obtained using methods known in this field, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. site-directed mutagenesis [Carter et al., Nucl. Acids Res., 12:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)], restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)] or other known methods can be performed on the cloned DNA to obtain a modified DNA IL-22 or IL-22R.
Polypeptide fragments of IL-22 or IL-22R is also presented in this paper. Such fragments can be subjected to processing at the N-end or C-end or may lack internal residues, for example, when comparing the primary native protein. Some fragments do not contain amino acid residues that are not essential for a desired biological activity of IL-22 or IL-22R polypeptide. Therefore, in some embodiments, the implementation of, a fragment of IL-22 or IL-22R is biologically active. In some embodiments, the implementation, the fragment is servicego IL-22 does not contain the N-end signal peptide sequence. In some embodiments, the implementation, the primary fragment of IL-22R is a soluble form of IL-22R, which is not membrane-associated, for example, the form IL-22R, which does not contain a transmembrane domain. For example, a soluble form of IL-22R person does not contain all or a considerable portion of the transmembrane domain from about 229-251 amino acids of SEQ ID NO:3.
Covalent modifications of IL-22 or IL-22R is included in the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of IL-22 or IL-22R polypeptide with an organic derivative of a substance which is capable of reacting with selected side chains or the N - or C-terminal residues of IL-22 or IL-22R. Obtaining derivatives with bifunctional agents is useful, for example, for cross-linking of IL-22 or IL-22R with a water-insoluble base carrier or surface for use in the method of purification of anti-IL-22 or IL-22R antibodies, and Vice versa. Common form cross-links substances include, for example, 1,1-bis(diazoacetate)-2-Penilaian, glutaraldehyde, ester imides N-hydroxyethanoic acid, esters of hydroxysuccinimide, for example, esters with 4-azidoaniline acid, homobifunctional imidiately, including succinimidylester, such as 3,3'-dithiobis(succinimide propionate), bifunctional maleimide, such as bis-N-maleimido-1,8-octane and agents such as methyl-3-[(p-azidophenyl)dithio]propionamide.
Other changes include desametasone glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of Proline and lysine, phosphorylation of hydroxyl groups was seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine and the side chains of histidine [T.E. Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.
Another type of covalent modification of IL-22 or IL-22R polypeptide, are included in the scope of this invention, includes the changing nature of the native glycosylation of the polypeptide. "The changing nature of the native glycosylation of the polypeptide" is intended for the purposes of this document, to indicate the deletion of one or more carbohydrate components found in native sequence IL-22 or IL-22R (or move the original glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence IL-22 or IL-22R. In addition, the phrase includes as the major changes in the glycosylation of the native proteins, involving a change in the nature and properties of various shows carbohydrate components.
IL-22 or IL-22R polypeptide of the present invention can also be modified in some way for the formation of chimeric molecules, including IL-22 or IL-22R, merged with other heterogeneous polypeptide or amino acid sequence. In one embodiment, the chimeric molecule comprises a fusion of IL-22 or IL-22R with a marker polypeptide, which represents the epitope, which can be selectively contact antimarket antibody. Marker epitope, usually located at the amino - or carboxy-ends of IL-22 or IL-22R. The presence of such epitope-marker forms of IL-22 or IL-22R can be identified using antibodies against the marker polypeptide. Providing a marker epitope facilitates IL-22 or IL-22R rapid purification by affinity purification using antimuscarinic antibodies or other affinity framework that binds the marker epitope. Different marker polypeptides and their respective antibodies are well known in this field. Examples include polyhistidine (poly-his) or polyhistidine-glycine (poly-his-gly) markers; the flu HA marker polypeptide and its antibody 12CA5 [Field et al, Mol. Cell. Biol, 8:2159-2165 (1988)]; the c-myc marker and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and Cellular Biology, 5:3610-3616 (1985)]; and marker glycoprotein D (gD) of the virus just what about herpes and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553 (1990)]. Other marker polypeptides include the Flag-peptide [Hopp et al., BioTechnology, 6:1204-1210 (1988)]; the peptide epitope KT3 [Martin et al., Science, 255: 192-194 (1992)]; alpha-tubulin peptide epitope [Skinner et al., J Biol. Chem., 266:15163-15166 (1991)]; and peptide marker protein T7 gene 10 [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)].
In another embodiment, the chimeric molecule may include the merger of IL-22 or IL-22R polypeptide with an immunoglobulin or a single region of the immunoglobulin. For a bivalent form of the chimeric molecule (also referred to as "immunoadhesin") such merger may be to the Fc region of IgG molecules. Merge Ig preferably include the substitution of a soluble form of IL-22 or IL-22R polypeptide in place of at least one variable region of the Ig molecule. In a particular preferred embodiment, the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 region of an IgG1 molecule. Receipt of immunoglobulin m, see also U.S. patent No. 5428130, published on June 27, 1995
1. Receiving IL-22 or IL-22R
IL-22 or IL-22R, you can get conventional recombinant methods, for example by transformation of cultured cells or vector transfection containing nucleic acid encoding IL-22 or IL-22R, for example, the nucleic acid shown in Fig. 1, which encodes IL-22. Also performance is aulani cells-owners, containing any such vector. As an example, the cells of the host can be a CHO cell, an E. coli or yeast. The process of obtaining any described in this document polypeptides presented herein and comprises culturing host cells under conditions suitable for expression of the desired polypeptide, and extracting the desired polypeptide from the cell culture.
In other variants of implementation, the invention presents a chimeric molecule, including any described in this document polypeptides, fused with heterogeneous polypeptide or amino acid sequence. Example of such chimeric molecules include any described in this document polypeptides, fused to the sequence of the epitope of the marker or the Fc region of immunoglobulin.
Of course, you can assume that you can use alternative methods that are well known in the field, to obtain IL-22 or IL-22R. For example, IL-22 or IL-22R sequence or their parts can be obtained by direct peptide synthesis using solid-phase methods [see, for example, Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, CA (1969); Merrifield, J. Am. Chem. Soc, 85:2149-2154 (1963)]. Protein synthesis in vitro can be accomplished by manual or automatic means. Automated synthesis can be performed, for example, using an Applied Biosystems Peptide Synthesizer (Foster City, CA), COI is lsua the manufacturer's instructions. Different parts of the IL-22 or IL-22R can be separated chemically synthesised and combined using chemical or enzymatic methods of obtaining primary IL-22 or IL-22R.
Recombinante expressed IL-22 or IL-22R can be extracted from culture medium or from cell extracts of host cells. The following processes are typical for the respective purification processes: fractionation on ionoobmennoi column; reversed-phase HPLC; chromatography on silica or cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; fractionation by ammonium sulfate; gel filtration using, for example, Sephadex G-75; protein sepharose columns to remove contaminants such as IgG; and metal hepatoblastoma columns to bind epitope-marker forms of IL-22 or IL-22R. You can use various methods of protein purification, and such methods are known in this field and are described for example in Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982). Selected stages of purification, for example, will depend on the nature of the used process of receiving and separately derived IL-22 or IL-22R.
2. Detection of gene expression
The expression of the gene encoding IL-22 or IL-22R, can be defined in various ways in this area, for example, determining the expression of mRNA encoding IL-22 or IL-22R. Ka is used in this document, the term "determination" includes quantitative or qualitative determination. By determining the gene expression of IL-22 or IL-22R can be identified, for example, those tissues that Express the gene of IL-22 or IL-22R. Gene expression can be measured using specific methods known to the person skilled in the art, for example Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 ); quantitative PCR; or in situ hybridization using appropriately labeled samples based on presented in this document sequences. Alternatively, gene expression can be measured by immunological methods, such as immunohistochemical staining of tissue sections and analysis of cell culture or body fluids, in order to quantify directly the expression of gene product. Suitable for immunohistochemical staining and/or analysis of the sample fluid antibodies include any presents in this document antibodies. Conveniently, the antibodies can be obtained against a native sequence IL-22 or IL-22R polypeptide; against a synthetic peptide containing a fragment of the IL-22 or IL-22R polypeptide sequence; or against exogenous sequence fused to IL-22 or IL-22R a polypeptide his fragment (including synthetic peptide).
Presents antibodies associated with any of the above or below described polypeptides. In one embodiment, the selected antibody that binds to IL-19, IL-20, IL-22, IL-24, IL-20Ra, IL-20Rb, IL-10R2 or IL-22R of the polypeptide. Typical antibodies include polyclonal, monoclonal, humanized, human, bespecifically and heteroconjugate antibodies. The antibody may be an antibody fragment, such as Fab, Fab'-SH, Fv, scFv, or (Fab')2the fragment. In one embodiment, presents the selected antibody that binds to IL-22 or IL-22R. In one such embodiment, the antibody is partially or completely blocks the activity of IL-22 or IL-22R polypeptide (i.e., "blocking" antibody).
This paper provides a typical monoclonal antibodies that bind IL-22 and IL-22R, and further described in the examples. Such antibodies include anti-IL-22 antibodies, labeled 3F11.3 ("3F11"), 11H4.4 ("11H4) and 8E11.9 ("8E11"), and anti-IL-22R antibodies labeled 7E9.10.8 ("7E9"), 8A12.32 ("8A12"), 8H11.33.28 ("8H11) and 12H5. In one embodiment, hybridoma that produces any such antibodies. In one embodiment, presents monoclonal antibodies that compete with 3F11.3, 11H4.4 or 8E11.9 for binding to IL-22. In another embodiment, presents monoclonal antibodies that are associated with that is they epitope, as 3F11.3, 11H4.4 or 8E11.9. In another embodiment, presents monoclonal antibodies that compete with 7E9, 8A12, 8H11 or 12H5 for binding to IL-22R. In one embodiment, presents monoclonal antibodies that bind to this epitope, as 7E9, 8A12, 8H11 or 12H5. Various embodiments of the antibodies provided below.
1. Polyclonal antibodies
Antibodies may comprise polyclonal antibodies. Methods of obtaining polyclonal antibodies are known to the person skilled in the art. Polyclonal antibodies can be obtained from mammals, for example, through one or more injection immunizing substances and tools, if necessary. Typically, the immunizing agent and/or adjuvant is administered to the mammal by multiple subcutaneous or intraperitoneal injections. Immunizing substance can include a polypeptide of interest or a hybrid protein. Can be useful for immunizing substance connected with protein, known as immunogenic in mammals after immunization. Examples of such immunogenic proteins include but are not limited to, hemocyanin lymph snails, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of assistive devices that can be used include complete adjuvant's adjuvant and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic dikarenakan trehalose). The immunization Protocol may be selected by a person skilled in the art without undue experimentation.
2. Monoclonal antibodies
Alternatively, the antibody can be a monoclonal antibody. Monoclonal antibodies can be produced using hybridoma methods, such as described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, a mouse, hamster, or other appropriate animal host is usually subjected to immunization immunization substance to obtain lymphocytes that produce or are capable of producing antibodies that specifically associated with immunizing agent. Alternatively, the lymphocytes can immunize in vitro.
Immunizing substance will typically include a polypeptide of interest or a hybrid protein. As a rule, are either peripheral blood lymphocytes ("PBLs"), if desired cells from a human source, or used splenocytes or cells of lymph nodes, if desired sources nonhuman mammals. Then lymphocytes are fused with immortalized cell line using a suitable agent for the merger, such as polyethylene glycol, to obtain a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103]. Immortalisation cell line usually is predstavlyaet a transformed mammalian cells, in particular, the myeloma cells from a source of rodent, bovine and human. Usually use a rat or mouse myeloma cell line. Hybridoma cells can be grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of non-hybrid immortalizing cells. For example, if the parental cells do not contain the enzyme gipoksantin guanine phosphoribosyl a transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include gipoksantin, aminopterin and thymidine ("HAT medium"), substances which prevent the growth of HGPRT-deficient cells.
Preferred immortalisation cell lines are lines that effectively merge, support stable high level expression of antibody selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalisation cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California, and the American Type Culture Collection, Manassas, Virginia. Cell lines of human myeloma and mouse-human heteromyinae have also been described for obtaining human monoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monocloal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63].
The cultural environment in which cultured hybridoma cells can then be examined for the presence of monoclonal antibodies that bind with the target polypeptide. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or analysis of binding in vitro, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such methods and known research in this area. Affinity binding of the monoclonal antibody can, for example, be determined using the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).
After identify the desired hybridoma cells, clones can be subclinical, limiting the methods of cultivation and growth of the standard methods [Goding, above]. Suitable cellular environment for this purpose includes, for example, Dulbecco modified Wednesday Needle and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo, intraperitoneally at a mammal.
Monoclonal antibodies isolated from subclones, you can split or peel from the culture medium or ascitic fluid by standard methods of purification of immunoglobulins, such as, for example, protein A-sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or AF is affine chromatography.
Monoclonal antibodies can be obtained by using combinatorial libraries to screen for antibodies with the desired activity or activities. For example, various methods known in the field to obtain libraries of phage display and screening such libraries for antibodies with the desired binding characteristics. Such methods are generally described in Hoogenboom et al. (2001) in Methods in Molecular Biology 178:1-37 (O'brien et al., ed., Human Press, Totowa, NJ), and in some embodiments, the implementation in Lee et al. (2004) J. Mol. Biol. 340:1073-1093.
Essentially clones of synthetic antibodies choose the screening of phage libraries containing the phage, which found various fragments of variable regions of antibodies (Fv), fused with the protein shell of the phage. Such phage libraries are separated by affinity chromatography, depending on the desired antigen. Clones expressing Fv fragments, capable of binding to the desired antigen, adsorbed to the antigen and thus separated from non-bound peroxidase clones in the library. Bound peroxidase clones then elute from the antigen and can be further enriched with additional cycles of adsorption/elution of the antigen. Any of the antibodies of the invention can be obtained by scheduling the appropriate method of screening antigens to select phage clone with the subsequent construction of a clone of the primary antibodies, the COI is lsua Fv sequences of the selected phage clone and sequence a suitable constant region (Fc), described in Kabat et al, Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3.
Monoclonal antibodies can also be obtained by means of recombinant DNA, such as the methods described in U.S. patent No. 4816567. DNA encoding the monoclonal antibodies of the invention can be easily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes that are capable of specific link to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells of the invention serve as a preferred source of such DNA. Once selected DNA can be placed in expressing vectors, which are then transferout cell host, such as COS cells, monkey cells Chinese hamster ovary (CHO) or myeloma cells that don't produce the protein of the immunoglobulin, to obtain the synthesis of monoclonal antibodies in the recombinant cell host. DNA can be modified, for example, by substituting the coding sequence for the constant domains of light and heavy chains of the person instead of the homologous murine sequences [U.S. patent No. 4816567; Morrison et al., above] or by covalent connection with the coding sequence of the immunoglobulin, the whole or part of the coding sequence for nimmanahaeminda p is dipeptide. This nimmanahaeminda polypeptide can be substituted for the constant domains of the antibodies of the invention or may be substituted on different domains antigennegative plot antibodies of the invention, to obtain a chimeric bivalent antibody.
3. Monovalent antibodies
Also presents monovalent antibodies. Methods of obtaining monovalent antibodies are well known in this field. For example, one method involves recombinant expression of the light chain of immunoglobulins and modified heavy chain. Typically, a heavy chain truncate at any point in the Fc region, in order to prevent cross-linking the heavy chains. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or deleted, to prevent cross-linking.
In vitro methods are also suitable for receiving monovalent antibodies. Cleavage of the antibodies for their fragments, in particular Fab fragments, can be produced using conventional methods known in this field.
4. Antibody fragments
Also presents fragments of antibodies. Antibody fragments can be obtained by conventional methods, such as enzymatic cleavage or by recombinant methods. In some cases there are advantages of using fragments of the antibodies is, than whole antibodies. The small size of the fragments promotes rapid removal and may lead to improved access to solid tumors. For an overview of specific fragments of antibodies, see Hudson et al. (2003) Nat. Med. 9:129-134.
To obtain fragments of antibodies have been developed in different ways. Traditionally, these fragments were obtained by proteolytic cleavage of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992); and Brennan et al., Science, 229:81 (1985)). Currently, however, these fragments can be obtained directly from recombinant host cells. Fab, Fv and ScFv antibody fragments can all be expressed and secretariats in E. coli, thus allowing to easily obtain large amounts of these fragments. Antibody fragments can be isolated from libraries of phage antibodies discussed above. Alternatively, Fab'-SH fragments can be extracted directly from E. coli and chemically combine for the formation of F(ab')2fragments (Carter et al., Bio/Technology 10: 163-167 (1992)). According to another approach, F(ab')2the fragments can be distinguished directly from recombinant cell culture-owners. Fab and F(ab')2fragment with increased half-life in vivo, contains the saved remnant receptornegative antigenic determinants described in U.S. patent No. 5869046. Other methods of obtaining fragments antic the l will be apparent to the skilled practitioner. In some embodiments, the implementation, the antibody is a single-chain Fv fragment (scFv). Cm. WO 93/16185; U.S. patent No. 5571894 and 5587458. Fv and scFv are the only species with intact binding sites that are devoid of constant regions; thus, they may be appropriate to reduce nonspecific binding when used in vivo. scFv hybrid proteins can be designed to receive the merger effector protein at either the amino-or carboxy-end of the scFv. Cm. Antibody Engineering, ed. Borrebaeck, above. The antibody fragment may also be a "linear antibody", e.g., as described in U.S. patent No. 5641870. Such linear antibodies can be a monospecific or bespecifically.
5. Humanized antibodies
Also presents humanized antibodies. In this area there are various ways of turning nonhuman antibodies in humanized. For example, humanitariannet antibody may contain one or more amino acid residue is introduced into it from a superhuman source. These inhuman amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can mainly be made by the method of Winter and colleagues (Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (198) Science 239:1534-1536), replacing the sequence of the hypervariable region on the corresponding sequences of human antibodies. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. patent No. 4816567), which is significantly less than an intact human variable domain, was replaced by the corresponding sequence from nonhuman species. In practice, humanized antibodies are typically human antibodies in which some hypervariable residues region and possibly some FR residues are substituted for residues from analogous sites of antibodies rodents.
The choice of human variable domains, both light and heavy for use in obtaining humanized antibodies may be important to reduce antigenicity. According to the so-called " best-matched" method, the sequence of the variable domain of the antibody of the rodent are relatively screening a library of known sequences of human variable domains. The human sequence which is closest to the sequence of the rodent is then accepted as the human framework for gumanitarnogo antibody (Sims et al. (1993) J. Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol. 196:901). In another method uses special cos the Wu, derived from the consensus sequence of all human antibodies of a separate subgroup of light or heavy chains. A similar framework can be used for several different humanized antibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) J. Immunol., 151:2623).
Further, generally, it is desirable that the antibodies were humanitarian with retention of high affinity for the antigen and other favorable biological properties. For this purpose, according to one method, has been humanized antibodies using the method of analysis of the parental sequences and various imaginary humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional models of immunoglobulins are generally available and well known to specialists in this field. There are computer programs that depict and show probable three-dimensional conformational structures of selected sequences candidates of immunoglobulins. The study of these images gives the opportunity to analyze the possible role of the residues in the functioning sequence of the candidate immunoglobulin, that is, the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. Thus, it is possible to choose R residues and combined with sequences of the recipient and imported, so there is achieved the desired characteristics of the antibodies, such as increased affinity for the target antigen. In General, the remnants of the hypervariable region are directly and most substantially affect binding to the antigen.
6. Human antibodies
Also presents human antibodies. You can create human antibodies, combining Fv clone sequences of the variable domain selected from derived from human libraries of phage display with known human sequences of the constant domains, as described above. Alternatively, human monoclonal antibodies of the invention can be obtained hybridoma method. Were described cell lines of human myeloma and mouse-human heteromyinae to obtain human monoclonal antibodies, e.g., Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).
Currently you can obtain transgenic animals (e.g. mice)that are capable of after immunization to produce a complete set of human antibodies in the absence of endogenous production of immunoglobulins. For example, it has been described that the homozygous deletion of the gene region binding heavy chain antibodies (JH) in chimeric and germ-line mutant mice leads to full the mu inhibition of the production of endogenous antibodies. Move group of the human germ-line immunoglobulin gene in such germ-line mutant mice will result in the production of human after antigenic stimulation. See, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255 (1993); Bruggermann et al., Year in Immunol., 7: 33 (1993).
Can also be used shuffling of genes for obtaining human antibodies from non-human, such as antibodies rodents, where the human antibody has similar affinity to the original non-human antibody. According to this method, also called "epitope imprinting", the variable region or a heavy or light chain fragment nonhuman antibodies, obtained by means of phage display as described in this document, move with genes of human V domain, creating a population of chimeras with inhuman chain/human chain scFv or Fab. Division with antigen leads to the secretion of chimeric scFv or Fab with inhuman chain/human chain, in which the human chain restores antigennegative land, destroyed after moving the corresponding non-human chain in the original clone phage display, i.e. the epitope determines (imprint) the choice of partner human chain. If the process is repeated to move the rest of lovecheck chain, you get a human antibody (see PCT WO 93/06213, published April 1, 1993). Unlike traditional humanitarian nonhuman antibodies by CDR merge in this way presents a fully human antibodies, which do not have any FR or CDR residues of human origin.
7. Bespecifically antibodies
Also presents bespecifically antibodies. Bespecifically antibodies are monoclonal antibodies that have specificnosti binding at least two different antigens. In some embodiments, implementation, bespecifically antibodies are human or humanized antibodies. In some embodiments, the implementation of one of specificdate binding is desired polypeptide, and the other is for any other antigen. In some embodiments, implementation, bespecifically antibodies can bind to two different epitopes of the polypeptide of interest. Bespecifically antibodies can also be used to localize cytotoxic substances in cells that Express a desired polypeptide, the polypeptide on the cell surface. These antibodies contain TAT226-binding arm that binds the cytotoxic agent, such as, for example, saporin, antiinterference-α, alkaloid barwing is(a), retinova A chain, methotrexate or isotope hapten. Bespecifically antibodies can be obtained in the form of primary antibodies or fragments of antibodies (e.g., F(ab')2bespecifically antibodies).
Methods of obtaining bespecifically antibodies known in the field. Traditionally, the recombinant production bespecifically antibodies is based on the co-expression of two pairs of heavy chain - light chain immunoglobulins, in which the two heavy chains have different specificnosti (Milstein and Cuello, Nature, 305: 537 (1983)). Due to independent differences in meiosis genes of heavy and light chains of immunoglobulins, these hybridoma (quadrigemina) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bespecifically structure. Purification of the correct molecule, which is usually performed on the stages of affinity chromatography, is time consuming, and the product yield is low. Similar methods are disclosed in WO 93/08829, published 13 may 1993, and in Traunecker et al., EMBO J., 10: 3655 (1991).
According to another approach, the variable domains of the antibodies with the desired specificnosti binding (antibody-antigen binding sites) merge with the sequences of the constant domains of immunoglobulins. The merge occurs, for example, with a constant domain of the heavy chain of immunoglobulin containing, according to what ina least part of the hinge, CH2 and CH3 regions. In some embodiments, implementation of the first constant region of the heavy chain (CH1)containing the necessary for binding to the light chain of the plot, is present at least in one of their mergers. DNA encoding the fusion heavy chain of immunoglobulins and, if necessary, light chain immunoglobulins, enter into a dedicated expressing vectors, and are cotransfection a suitable host organism. This provides significant flexibility in the regulation of the General proportions of the three polypeptide fragments in the variants of implementation, if used in the design of unequal ratios of the three polypeptide chains provide optimum output. However, it is possible to insert the coding sequences for two or all three polypeptide chains in one expressing vector when the expression of at least two polypeptide chains in equal ratios results in high output, or if the ratio has no special value.
In one embodiment of this approach, bespecifically antibodies are composed of a hybrid heavy chain immunoglobulin with a first binding specificity in one arm, and a hybrid pair of heavy-light chain immunoglobulin (providing a second binding specificity) in the other shoulder. It was found that this asymme the hex structure facilitates the separation of the desired especifismo connections from unwanted combinations of chains of immunoglobulins, since the presence of the light chain of immunoglobulin in only one half of bespecifically molecules provides an easy way of separation. This approach is disclosed in WO 94/04690. Further details of education bespecifically antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).
According to another approach, you can create a boundary surface between a pair of antibody molecules to maximize the percentage of heterodimers derived from recombinant cell culture. The surface of the partition contains at least a part of the CH3 domain of the constant domain of the antibody. In this way, one or more small amino acid side chains from the interface of the first antibody molecules as opposed to larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities" of the same or similar size with large side chains are on the surface section of the second molecule antibodies by replacing large amino acid side chains into smaller (e.g., alanine or threonine). This provides a mechanism to increase the output of heterodimer relative to other unwanted end-products such as homodimers.
Bespecifically antibodies include cross stitched or heteroconjugate" antibodies. For example, one of the antibodies in heteroconjugate can svjazyvaites is with Avidya, the other with Biotin. Assume that such antibodies, for example, direct the immune system cells to unwanted cells (U.S. patent No. 4676980) and for the treatment of HIV infection (WO 91/00360, WO 92/00373 and EP 03089). You can get heteroconjugate antibodies using any suitable cross-linking method. Suitable cross-linking compounds are known in this field and are disclosed in U.S. patent No. 4676980 together with a number of cross-linking methods.
Methods of education bespecifically antibodies, fragments of antibodies has also been described in the literature. For example, bespecifically antibodies can be obtained using chemical bonds. Brennan et al., Science, 229: 81 (1985) describe the way in which the intact antibody proteoliticeski split before the formation of F(ab')2the fragments. The presence of the dithiol complexing sodium arsenite reducyruet these fragments to stabilize adjacent dithioles and to prevent the formation of intermolecular disulfide bonds. Educated Fab' fragments are then converted into derivatives of dinitrobenzoate (TNB). One of the derivatives of Fab'-TNB then returns to the previous state of the Fab'-thiol, reducing mercaptoethylamine, and mixed with equimolar amounts of the other derived Fab'-TNB to get especifismo antibodies. Received bespecifically antibodies can use the VAT as substances for selective immobilization of enzymes.
Recent advances can help direct the receipt of Fab'-SH fragments from E. coli, which can chemically unite for education bespecifically antibodies. Shalaby et al., J. Exp. Med., 175:217-225 (1992) describe the production of a molecule completely gumanitarnogo especifismo antibody F(ab')2. Each Fab' fragment was separately secreted from E. coli and subjected to direct chemical bonding to education especifismo antibodies. Educated thus bespecifically antibodies were able to bind with cells that do overexpress the HER2 receptor, and normal T-cells, as well as system startup lytic activity of human cytotoxic lymphocytes relative to target tumors in the breast of man.
Also described different ways of obtaining and slice bespecifically antibodies directly from a culture of recombinant cells. For example, were obtained bespecifically antibodies using leucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). Peptides leucine zipper of Fos and Jun proteins were linked to the Fab' regions of two different antibodies by gene fusion. Homodimeric antibodies were cut in the hinge region to form monomers and then oxidized back to education heterodimeric antibodies. This method can also be used to obtain good the Mer antibodies. In the "diabody" method described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993), was presented an alternative mechanism for obtaining fragments bespecifically antibodies. The fragments contain the variable domain of the heavy chain (VH)associated with the variable domain of the light chain (VL) by a linker that is too short for the formation of pairs between the two domains on the homology of the chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming antigennegative plot. It was also reported another method of obtaining fragments bespecifically antibodies using single-chain Fv (sFv) dimers. Cm. Gruber et al., J. Immunol., 152:5368 (1994).
Discusses antibodies with more than two valencies. For example, you can get thespecification antibodies. Tutt et al., J. Immunol. 147: 60 (1991).
8. Multivalent antibodies
Also presents multivalent antibodies. Multivalent antibody can be internalized (and/or metabolizirovannom) faster than bivalent antibody, cellular expression of the antigen bound to the antibody. Antibodies of the present invention can be a multivalent antibodies (which differ from IgM) with three or more antihistamine areas (for example, tetravalent antibodies), which easily you can obtain the recombinant expression of nucleic acids, encoding the polypeptide chains of the antibody. Multivalent antibody may contain a dimerization domain, and three or more antigenspecific plot. In some embodiments, implementation, domain dimerization contains (or consists of) an Fc region or a hinge region. In this scenario, the antibody will contain the Fc region, and three or more antigenspecific site at the amino-end of the Fc region. In some embodiments, implementation, multivalent antibody contains (or consists of) three to about eight antigenspecific areas. In one such embodiment, the multivalent antibody contains (or consists of) four antigenspecific areas. Multivalent antibody comprises at least one polypeptide chain (for example, two polypeptide chains), in which the polypeptide chain(s) contains two or more variable domains. For example, the polypeptide chain(s) may contain VD1-(X1)n-VD2-(X2)n-Fc, where VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, X1 and X2 represent an amino acid or polypeptide, and n represents 0 or 1. For example, the polypeptide chain(s) may comprise: VH-CH1-flexible connectivity-VH-CH1-Fc chain; or VH-CH1-VH-CH1-Fc chain region. Multivalent antibody herein dleimages to contain, at least two (e.g., four) of the polypeptide of the variable domains of the light chains. Multivalent antibody herein may, for example, contain from about two to about eight polypeptides, variable domains of the light chains. The polypeptides of the variable domains of the light chains discussed in this document contain variable domain light chain and, optionally, further contain a CL domain.
9. Single-domain antibodies
Also presents a single domain antibodies. Single-domain antibody is a simple polypeptide chain containing all or a portion of a variable domain of the heavy chain or the whole or a portion of a variable domain of the light chain of the antibody. In some embodiments, the implementation, a single domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, for example, U.S. patent No. 6248516 B1). In one embodiment, a single domain antibody consists of the whole or part of the variable domain heavy chain antibodies.
10. Variants of antibodies
In some embodiments, the implementation considers the modification of the amino acid sequences of the antibodies described in this document. For example, it may be desirable to improve the binding affinity of and/or other biological properties of the antibody. Variants of the amino acid posledovatelnostyakh be obtained by introducing appropriate changes in the nucleotide sequence, encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues within the amino acid sequences of the antibodies. You can make any combination of deletions, insertions and substitutions to obtain the final construct, provided that the final construct possesses the desired characteristics. Amino acid changes can be introduced in the amino acid sequence of the antibodies of the individual at the time of receiving the order.
A convenient method for identification of certain residues or regions of the antibody that are preferred locations for mutagenesis is called "alanine scanning mutagenesis"as described by Cunningham and Wells (1989) Science, 244: 1081-1085. This document identified a residue or group of residues of target proteins (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine), in order to act on the interaction of amino acids with antigen. Such amino acid localization, demonstrating functional sensitivity to the substitutions then improve the introduction of the next, or in, or any of the other options in the parts replacement. Thus, a pre-defined area for the introduction of the variant amino acid follower of the spine, the nature of the mutation per se need not pre-defined. For example, to analyze the execution of a mutation at a given site, alanine scanning or non-specific mutagenesis is carried out in the codon of the target or area, and conduct screening of expressed immunoglobulin on the desired activity.
Insert amino acid sequences include amino - and/or carboxyl-end merger, ranging in length from one residue to the polypeptide consisting of a hundred or more residues, as well as insert into sequences of single or multiple amino acid residues. Examples of finite insertions include an antibody with a methionine residue at the N-end. Other insertional variants of the antibody molecules include the fusion to the N - or C-terminal end of the antibody to an enzyme (for example, with ADEPT) or a polypeptide which increases the half-life of antibodies in the serum.
In some embodiments, implement, modify the antibody of the invention to increase or decrease the amount of glycosylation of antibodies. Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of the hydrocarbon fragment to the side chain of aspartic residue. Tripeptide sequence asparagine-X-serine and asparagine-X-threonine, where X represents the t is any amino acid except Proline, represent different sequences for attachment of the enzyme hydrocarbon fragment to the side chain of asparagine. Thus, the presence of these Tripeptide sequences in the polypeptide leads to the formation of a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-atsetilgalaktozamin, galactose, or xylose to hydroxynicotinate, most often to serine or threonine, although you can also use 5-hydroxyproline or 5-hydroxylysine.
The insertion or deletion of glycosylation site in the antibody convenient to carry, changing amino acid sequence, so that it turns or moves one or more of the above Tripeptide sequences (for N-linked glycosylation sites). You can also make changes by insertion, deletion or substitution of one or more residues of serine or threonine in the sequence of the original antibody (for O-linked glycosylation sites).
If the antibody contains a Fc region, attached to the hydrocarbon can be changed. For example, antibodies with a Mature hydrocarbon structure, which do not contain fucose attached to the Fc region of the antibodies described in U.S. patent 2003/0157108 (Presta L.). Cm. also U.S. patent 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Antibodies to share what they N-acetylglucosamine (GlcNAc) in the hydrocarbon, attached to the Fc region of the antibody mentioned in WO 2003/011878, Jean-Mairet et al. and in patent No. 6602684, Umana et al. About the antibodies, at least one galactose residue in the oligosaccharide attached to the Fc region of antibodies, according to WO 1997/30087, Patel et al. Cm. also WO 1998/58964 (Raju, S.) and WO 1999/22764 (Raju, S.) on antibodies with altered attached hydrocarbon to their Fc region. Cm. also in U.S. patent 2005/0123546 (Umana et al.) antigennegative molecules with altered glycosylation.
In some embodiments, the implementation variant glycosylation contains a Fc region, in which a carbohydrate structure attached to the Fc region does not contain fucose. In such embodiments, improved ADCC function. Optionally, the Fc region further contains one or more amino acid substitutions which further improve ADCC, for example replacing the provisions of the Fc region 298, 333, and/or 334 (Eu numbering of residues). Examples of publications related to "deforsirovannym" or "photostability" antibodies include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; Okazaki et al., J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004). Examples of cell lines producing deoksigenirovanii antibodies include Lec13 CHO cells deficient in protein fokusirovanie (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); the patent is th application U.S. No. 2003/0157108 A1, Presta L.; and WO 2004/056312 A1, Adams et al., in particular, the example 11), and knockout cell lines, such as gene alpha-1,6-fucosyltransferase, FUT8, knockout CHO cells (Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004)).
Another option is a variant of amino acid replacement. These variants have at least one amino acid residue in the antibody molecule replaced by a different residue. Interest for substitutional mutagenesis areas include hypervariable region, but also assumed FR changes. Conservative substitutions are shown in table 6 above under the heading of "preferred substitutions". If such substitutions result in the desired change in biological activity, then you can enter more substantial changes, called "typical replacement" in table 6, or, as further described above with respect to classes of amino acids, and the resulting antibodies were skanirovali on the desired properties of the binding.
One type of substitution variant includes a substitution of one or more residues of the hypervariable region of the parent antibody (e.g., humanitariannet or human antibody). As a rule, received options selected for further development will be modified (for example, improved biological properties compared to the parent antibody from which they are derived. A convenient way to gaining the such substitution variants include affinity maturation using phage display. Briefly, several sections of hypervariable region (e.g., 6-7 sites) mutate for the education of all possible amino acid substitutions at each site. Thus obtained antibodies are portrayed from particles of filamentous phage, such as a merger, at least part of the envelope protein of a phage (e.g., the product of the gene III M13)that is packaged within each particle. This was followed by the screening of the variants of phage display for their biological activity (e.g. binding affinity of). In order to identify candidate areas hypervariable region for modification, it is possible to carry out scanning mutagenesis (for example, alanine scanning to identify residues of the hypervariable region, making a significant contribution to the binding to the antigen. Alternatively, or additionally, it may be beneficial to analyze a crystal structure of the complex antigen-antibody to identify contact points between the antibody and the antigen. Such contact residues and neighboring residues are candidates for replacement in accordance with methods known in this field, including the methods presented in this paper. If you create such options, then the group of variants is subjected to screening using methods known in this field, including the methods described in this document, and ant the body with the best properties in one or more of the relevant studies can be selected for further refinement.
Molecules of nucleic acids encoding amino acid sequence variants of antibodies, receive a variety of ways known in this field. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or the receipt by oligonucleotide-mediated (or site-specific) mutagenesis, PCR mutagenesis, and cassette mutagenesis obtained earlier variant or invariant version of the antibody.
It may be desirable that the introduction of one or more amino acid modifications in the Fc region of the antibodies of the invention has promoted the creation of the variant Fc region. The variant Fc region may contain human sequence Fc region (e.g., a human Fc region of IgG1, IgG2, IgG3 or IgG4), containing the amino acid modification (e.g. a substitution) at one or more amino acid positions include replacement hinge cysteine.
According to this description and teaching of the prior art, assume that in some embodiments, the implementation, the antibody of the invention may contain one or more changes in comparison with the variant antibody wild-type, for example in the Fc region. These antibodies probably still largely retain similar characteristics needed is for therapeutic use, in comparison with a variant of the wild type. For example, suppose that some changes can be obtained in the Fc region, which likely leads to a modified (i.e., either improved or diminished) C1q binding and/or complement-dependent cytotoxicity (CDC), for example, as described in WO99/51642. Cm. also Duncan & Winter Nature 322:738-40 (1988); U.S. patent No. 5648260; U.S. patent No. 5624821; and WO 94/29351 concerning other examples of variants of the Fc region. In WO 00/42072 (Presta) and WO 2004/056312 (Lowman) describe variants of antibodies with improved or diminished binding to FcRs. The contents of these patent publications are included in particular, in this document by reference. Cm. also Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001). Antibodies with increased half-life and enhanced binding to the neonatal Fc receptor (FcRn), which is responsible for transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), described in US2005/0014934A1 (Hinton et al.). These antibodies contain the Fc region with one or more substitutions, in which the improved binding of the Fc region with FcRn. Variants of polypeptides with altered amino acid sequences of the Fc region and improved or increased ability to bind C1q described in U.S. patent No. 6194551B1, WO99/51642. The contents of these patent publications are included, in particular, in this document by reference. Cm. also Idusogie et al., J. Immunol. 164: 4178-4184 (2000).p> In one aspect of the invention presents antibodies containing modifications in the contact area Fc polypeptide containing an Fc region, where modifications to facilitate and/or cause heterodimerization. These modifications include the introduction of a protrusion on the inside of the first Fc polypeptide and the cavity inside of the second Fc polypeptide, where the protrusion is installed in the cavity in order to cause the formation of a complex of first and second Fc polypeptides. Methods of antibody formation with these modifications known in this field, for example as described in U.S. patent No. 5731168.
11. Derived antibodies
Antibodies can be further modified to include additional non-protein components, which are known in this area and easily accessible. Preferably, suitable components for receiving derived antibodies are water-soluble polymers. Unlimited examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, a copolymer of ethylene/maleic anhydride, polyaminoamide (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, homopolymers p is piligrims, copolymers of polypropyleneoxide/ethylene oxide, polyoxyethylene polyhydric alcohols (e.g. glycerol), polyvinyl alcohol and mixtures thereof. Propionic aldehyde of the polyethylene glycol may have an advantage in production due to its stability in water. The polymer may have any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if attached to more than one polymer, they may be the same or different molecules. Typically, the number and/or type of polymers used to produce derivatives can be determined depending on factors including, but not limited to, special properties or functions of antibodies to improve, whether derived antibody used in the treatment of certain conditions, and so forth.
In another embodiment, the conjugates of the antibody and the protein fragment that selectively can be heated by the radiation. In one embodiment, the protein fragment is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. 102: 11600-11605 (2005)). The irradiation may be of any wavelength, and includes, but is not limited to, wavelengths that do not harm normal cells, but which damage the protein fragment at a temperature of at to the Torah killed cells, similar to the antibody-protein fragment.
In some embodiments, the implementation, the antibody can be labeled and/or may be mobilisierung on a solid basis. In a further aspect, the antibody is antiidiotypic antibody.
12. Heteroconjugate antibodies
Also presents heteroconjugate antibodies. Heteroconjugate antibodies contain two covalently linked antibodies. Such antibodies, for example, aimed at targeting of immune system cells to unwanted cells [U.S. patent No. 4676980] and for the treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089]. Assume that antibodies can be obtained in vitro using the methods of synthetic chemistry of proteins, including methods using cross-linking agents. For example, immunotoxins can be constructed using a disulfide reaction substitution or forming a thioester bond. Examples suitable for this purpose reagents include minotola and methyl-4-mercaptopyrimidine shown, for example, in U.S. patent No. 4676980.
13. Cytotoxic antibodies
Also presents cytotoxic antibodies. In some embodiments, implementation, cytotoxic antibody is an anti-IL22 antibody, such as the one below antibody that acts on effector function and/or causes destruction of cells. Some in the ways of implementation, cytotoxic anti-IL-22R antibody binds to the extracellular domain of IL-22R.
14. Design effector functions
It may be desirable to modify the antibody with the appropriate effector function, so as to increase, for example, the effectiveness of the antibodies in the treatment of diseases such as cancer. For example, cysteine residues can be introduced in the Fc region, thereby allowing to form a disulfide bond within the chain in this area. Thus obtained homodimeric antibodies may have an improved ability to internalize and/or increased complement-mediated cell death and antibody-dependent cellular cytotoxicity (ADCC). Cm. Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be obtained using heterobifunctional cross-linking substance, as described in Wolff et al., Cancer Research 53: 2560-2565 (1993). Alternatively, antibodies can be constructed to have dual Fc region and, therefore, could have increased the ability of complement lysis and ADCC. Cm. Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).
15. The vectors, cells of the host and recombinant methods
For recombinant obtain antibodies, in one embodiment, allocate encoding their nucleic acids and insert the replicable vector for further cloning (amplification of the DNA) or for expression. Encoding the antibody DNA is easily isolated and is sequenced using conventional methods (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the antibody). There are many vectors. The choice of vector depends partly on the used host cell. Typically, the cells of the host are cells, either prokaryotic or eukaryotic (usually mammals) source. It will be appreciated that the constant region of any isotype can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE constant region and that such constant region may be obtained from any person or animal species.
a) Obtaining antibodies using prokaryotic host cells
(1) Vector design
Polynucleotide sequences encoding polypeptide components of the antibodies can be obtained using standard recombinant methods. The desired polynucleotide sequence can be extracted and sequenced from producing antibodies cells, such as cells hybridoma. Alternatively, it is possible to synthesize polynucleotide using nucleotide synth or PCR methods. Once received, encoding the polypeptide sequence is inserted into a recombinant vector capable replic is to integrate and Express heterogeneous polynucleotide in prokaryotic hosts. Many vectors that exist and are known in this field can be used for the purpose of the present invention. The selection of the appropriate vector will depend mainly on the size of the nucleic acid inserted into the vector, and from a specific host cell transformed by the vector. Each vector contains various components, depending on its function (amplification or expression of heterogeneous polynucleotide, or both) and their compatibility with the specific cell of the host in which it is located. Typically, the components of the vector include, but are not limited to, the replication origin of the marker gene selection, promoter, ribosomal binding site (RBS), signal sequence, heterogeneous insert nucleic acid sequence and the termination of transcription.
Generally, plasmid vectors containing replicon and control sequences which are derived from species appropriate host cell, can be used in relation to these hosts. Typically, the vector is a replication site, as well as marking sequences which are capable of breeding phenotype in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from the species E. coli. pBR322 contains genes encoding resistance to ampic line (Amp) and tetracycline (Tet) and thus provides easy means of identifying transformed cells. pBR322, its derivatives, or other microbial plasmids or bacteriophage may also contain, or be modified to contain, promoters which can be used microbial organism for expression of endogenous proteins. Examples of derivatives of pBR322, was used for the expression of specific antibodies, are described in detail in Carter et al., U.S. patent No. 5648237.
In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transformation vectors in relation to these hosts. For example, bacteriophage such as λGEM.TM.-11 can be used to obtain a recombinant vector that can be used for transformation of competent host cells such as E. coli LE392.
The expression vector of the invention may contain two or more promoter-cistronic pairs, encoding each polypeptide components. The promoter is a noncoding regulatory sequence above (5') to cistron who changes her expression. Typically prokaryotic promoters are divided into two classes, inducible and constitutive. The inducible promoter is a promoter that causes an increase in the level of transcription of cistron under its control in response to changing conditions Kul is cultivated, for example, the presence or absence of nutrients or temperature changes.
It is well known a large number of promoters identified various potential cells of the owners. The selected promoter may be functionally linked to DNA cistron that encodes a light or heavy chain by moving the promoter from the source DNA by splitting restrictase and paste the selected sequence of the promoter in the vector of the invention. Both the native promoter sequence and many heterogeneous promoters can be used to direct amplification and/or expression of target genes. In some embodiments, implementation, heterogeneous promoters are used, as they generally permit greater transcription and higher yield of expressed target gene compared to the native polypeptide promoter of the target.
Suitable for use with prokaryotic hosts promoters include the PhoA promoter, the promoter system β-galactosi and lactose promoter system tryptophan (trp) and hybrid promoters such as the tac or the trc promoter. However, other promoters that function in bacteria (such as other known bacterial or phage promoters), are also suitable. Their nucleotide sequences have been published, thus the m allowing a qualified professional to quickly connect them with cisternae, encoding the light and heavy chains of the target (Siebenlist et al. (1980) Cell 20:269)using linkers or adapters to obtain any required restriction sites.
In one aspect of the invention, each cistron in the recombinant vector contains the secretory component of the signal sequence, which specifies the displacement of the expressed polypeptide through the membrane. Typically, the signal sequence may be a component of the vector, or may be part of a polypeptide DNA target, which is inserted into the vector. The signal sequence selected, for purposes of this invention should be selected and protestirovanny (i.e. split the signal peptidase) cells masters. For prokaryotic host cells that are not selected and not processed signal sequence, native to heterogeneous polypeptides, the signal sequence is substituted prokaryotic signal sequence selected, for example, from the group consisting of Alp, penitsillinazy, Ipp or leader sequences of thermostable enterotoxin II (STII), LamB, PhoE, PelB, OmpA and MBP. In one embodiment of the invention, the signal sequence is used in both cistronic expressing system represent the STII signal sequence or in the options.
In another aspect, the production of immunoglobulins according to the invention can be observed in the cytoplasm of the cells of the host, and therefore undesirable presence of secreted signal sequences in each cistron. At this light and heavy chains of immunoglobulins are expressed, are minimized and going for the formation of a functional immunoglobulin in the cytoplasm. Some strains of the host (e.g. E. coli trxB-strains) provide the status of the cytoplasm, which promotes the formation of disulfide bonds, thus, ensures the correct folding and Assembly of expressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).
Antibodies of the invention can also be obtained by using the expression system in which the quantitative ratio of the expressed polypeptide components can be modified to maximize the yield of secreted and correctly assembled antibodies of the invention. Such changes are at least partially simultaneous change in the efficiency of translation of the polypeptide components.
One way to change the translational efficiency described in Simmons et al., U.S. patent No. 5840523. It uses the options field of translation initiation (TIR) cistron. For this TIR, a number of variants with amino acid or nucleic acid sequence is th can be obtained with different translational efficiencies, therefore, providing the appropriate values, which govern this factor for the desired level of expression of a specific circuit. Options TIR can be obtained in the usual way mutagenesis, which leads to changes in the codons, which can alter the amino acid sequence. In some embodiments, implementation, changes in the nucleotide sequence of silent. Changes in TIR may include, for example, changes in the amount or distance between sequences Shine-Dalgarno along with changes in the signal sequence. One method of obtaining a mutant signal sequences represented by education Bank codons" in the beginning of the coding sequence that does not alter the amino acid sequence of the signal sequence (i.e. changes silent). This can be done by modifying the third nucleotide position of each codon; in addition, several amino acids, such as leucine, serine, and arginine, have many first and second positions, which can add complexity in the formation of the Bank. This method of mutagenesis is described in detail in Yansura et al. (1992) METHODS: A Companion to Methods in Enzymol. 4: 151-158.
In one embodiment, receive a set of vectors with different efficiencies TIR for each cistron. This limited number to ensure Ecevit comparing the level of expression of each chain, and the product yield of the desired antibodies at various combinations of the effectiveness of TIR. The effectiveness of TIR is possible to determine quantitatively measuring the level of expression of a reporter gene, as described in Simmons et al. U.S. patent No. 5840523. Based on a comparison of translational efficiency, choose a separate TIR to combine expressing vector constructs of the invention.
Prokaryotic cells are the owners that are suitable for expression of the antibodies of the invention include Archaebacteria and Eubacteria, such as gram-negative or gram-positive organisms. Examples of desired bacteria include Escherichia (e.g. E. coli), Bacilli (e.g., B. subtilis), Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or Paracoccus. In one embodiment, use gram-negative cells. In one embodiment, use of E. coli cells as hosts for the invention. Examples of E. coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.: American Society for Microbiology, 1987), pp. 1190-1219; ATCC Deposit No. 27325) and its derivatives, including strain 33D3 having genotype W3110 ΔfhuA (ΔtonA) ptr3 lac Iq lacL8 ΔompTΔ(nmpc-fepE) degP41 kanR (U.S. patent No. 5639635). Other strains and derivatives thereof, such as E. coli 294 (ATCC 31446), E. coli B, E. coliλ 1776 (ATCC 31537), and E. coli RV308(ATCC 31608) are also suitable. These examples illustrate but do not limit. FPIC who would design derivatives of any of the aforementioned bacteria, with certain genotypes are known in this field and are described, for example, Bass et al., Proteins, 8:309-314 (1990). As a rule, you must choose a suitable bacterium, have a rating of replenishement of replicons in the bacterial cells. For example, E. coli, Serratia, or Salmonella can appropriately be used as hosts as well known plasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to obtain replicon. Typically, cells of the hosts will secrete minimal amounts of proteolytic enzymes, and additional protease inhibitors may need to enter in cell culture.
(2) Obtaining antibodies
Cell owners transform the above expressing vectors and cultured in conventional nutrient medium, modified appropriately for inducing promoters, selecting transformants or amplificatoare genes encoding the desired sequences.
Transformation means introducing DNA into a prokaryotic host, so that the DNA is replicated either as necromany element or by chromosomal integration. Depending on the used host cell, transformation is done using standard methods, as appropriate to such cells. The processing of calcium using calcium chloride, usually used for bacterial cells that contain the is-standing barriers of the cell wall. In another method transformation using polyethylene glycol/DMSO. Another way to use electroporation.
Prokaryotic cells used to produce the polypeptides of the invention are grown in an environment known in the field and suitable for culture of the selected host cells. Examples of suitable environments include luria broth (LB) plus the necessary nutritional supplements. In some embodiments, the implementation also contains selectivity substance selected on the basis of constructions expressing vector to provide selective growth of prokaryotic cells containing expressing vector. For example, add ampicillin in the environment for the growth of cells expressing the gene of resistance to ampicillin.
Any necessary additives, in addition to sources of carbon, nitrogen and inorganic phosphate, may also be included at appropriate concentrations, administered separately or in the form of a mixture with other additives or environment, such as a complex nitrogen source. Optionally, the culture medium may contain one or more reducing substances selected from the group consisting of glutathione, cysteine, applied, thioglycolate, dithioerythritol and dithiothreitol.
Prokaryotic cells are the owners of cultivated under suitable temperatures. In some embodiments, is sushestvennee, for growth of E. coli, temperature growth range from about 20°C to about 39°C; from about 25°C to about 37°C; or about 30°C. the pH of the medium may be any pH ranging from about 5 to about 9, depending mainly on the host organism. In some embodiments, implement, for E. coli, pH is from about 6.8 to about 7.4 for, or approximately 7,0.
If expressed in the vector of the invention use an inducible promoter, expression of proteins occurs under conditions suitable for activation of the promoter. In one aspect of the invention, PhoA promoters used to control transcription of the polypeptide. Therefore, the transformed cell hosts grown in phosphate-limiting medium for induction. In some embodiments, implementation, phosphate-limiting medium is a C.R.A.P. environment (see, for example, Simmons et al., J. Immunol. Methods (2002), 263: 133-147). Many other inductors can be used in accordance with the vector construct, as is well known in this field.
In one embodiment, the expressed polypeptides of the present invention are secreted and out of periplasmic host cells. The selection of protein typically involves the destruction of the microorganism, as a rule, such as osmotic shock, destruction by ultrasound or lysis. Once the cells once asaut, cellular detritus or whole cells were removed by centrifugation or filtration. Further proteins can be cleaned, for example by chromatography on affinity resin. Alternatively, proteins can be transported into the cell medium and stand out there. Cells can be removed from the culture and the culture supernatant, filtered and concentrated for further purification the proteins. Expressed polypeptides then can be isolated and identified, using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot analysis.
In one aspect of the invention is to obtain antibodies in large quantities by fermentation. Various bulk fermentation methods with periodic recharge available for production of recombinant proteins. Bulk fermentation contain at least 1000 liters of volume, and in some embodiments, implementation, from about 1000 to 100000 liters of volume. In these fermenters are used blade mixer to distribute oxygen and nutrients, especially glucose (preferably carbon and energy source). Fermentation of a small number usually refers to fermentation in a fermenter, which contains no more than about 100 liters volume capacity and can vary from about 1 liter is up to about 100 liters.
In the method of fermentation, the induction of protein expression is usually called after the cells were grown in suitable conditions, until the desired density, for example OD550 of approximately 180-220, in which cells are in early stationary phase. You can use a number of inductors in accordance with the used vector construct, as is well known in this field and described above. Cells can be grown for more than short periods before induction. Usually cells are induced in approximately 12-50 hours, although you can use a longer or shorter induction time.
To improve the yield and quality of the polypeptides of the invention can be modified in different enzymatic conditions. For example, to get the proper Assembly and folding of secreted antibodies, polypeptides, added vectors that do overexpress protein chaperones, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD and/or DsbG) or FkpA (peptideprophet CIS,TRANS-isomerase with chaperone activity), can be used for co-transformation of prokaryotic host cells. It was shown that protein chaperones promote the proper folding and solubility of heterogeneous proteins derived from bacterial host cells. Chen et al. (1999) J. Biol. Chem. 274: 19601-19605; Georgiou et al., U.S. patent No. 6083715; Georgiou et al., U.S. patent No. 6027888; Bothmann and Pluckthun (2000) J. Biol. Chem. 275: 17100-17105 Ramm and Pluckthun (2000) J. Biol. Chem. 275: 17106-17113; Arie et al. (2001) Mol. Environ. 39:199-210.
To minimize proteolysis downregulation of heterogeneous proteins (especially those that are proteoliticeski sensitive), in the present invention can be used by some strains of the owners, is scarce proteolytic enzymes. For example, strains of host cells can be modified to cause genetic mutations in genes encoding known bacterial proteases such as protease III, OmpT, DegP, Tsp, protease I, protease Mi, protease V, protease VI and their combination. Some proteindeficient strains of E. coli are available and are described in, for example, Joly et al. (1998), above; Georgiou et al., U.S. patent No. 5264365; Georgiou et al., U.S. patent No. 5508192; Hara et al., Microbial Drug Resistance, 2:pp.63-72 (1996).
In one embodiment, the E. coli strains, deficient proteolytic enzymes and transformed with plasmids that do overexpress one or more proteins, chaperones, are used as host cells in expressing systems of the invention.
(3) Purification of antibodies
In one embodiment, received in this document antibodies are further purified to obtain preparations that are substantially homogeneous for further methods and applications. You can use the standard methods of protein purification known in this field. The following methods are sobytiyny appropriate cleaning methods: fractionation on immuno-affinity or ion-exchange columns, the ethanol precipitation, reverse phase HPLC, chromatography on silica or on a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE, fractionation with ammonium sulfate and gel filtration using, for example, Sephadex G-75.
In one aspect, protein A, immobilizovannyi on the solid phase, is used for immune-affinity purification of the products of the antibodies of the invention. Protein A is A protein of the cell wall 41 KD from Staphylococcus aureas, which binds with high affinity to the Fc region of antibodies. Lindmark et al (1983) J. Immunol. Meth. 62: 1-13. The solid phase, to which immobilized protein A, can be a column, including the surface of glass or silica, or glass column with a controlled pore size, or a column of silicic acid. In some applications, the column is coated with a reagent such as glycerin, for possible prevent nonspecific adhesion of impurities.
As the first stage of purification is obtained from a cell culture preparation, as described above, can be used in relation to protein A immobilized on a solid phase, to ensure specific binding of specific antibodies to the protein A. Then washed solid phase to remove impurities, nonspecific associated with the solid phase. Finally, the desired antibody are removed from the solid phase to ale the Oia.
b) Obtaining antibodies using eukaryotic host cells
Typically, the vector for use in eukaryotic cells-the owners comprises one or more of the following non-limiting components: a signal sequence, the starting point of replication, one or more marker genes, an enhancer element, a promoter and a sequence of termination of transcription.
(1) The signal sequence
Vector for use in eukaryotic cells-the owner may also contain a signal sequence or other polypeptide having a specific cleavage site at the N-end of the Mature protein or polypeptide of interest. Selected heterogeneous signal sequence can be recognized and protestirovanny (i.e. split the signal peptidase) the host-cell. Expression in mammalian cells, the signal sequence mammals, as well as viral secretory leader sequence, for example the signal herpes simplex gD, are available. The DNA for such areas predecessor closes in reading frame to DNA encoding the antibody.
(2) the replication origin
Generally, the starting point of replication is not necessary for expressing vectors mammals. For example, you can usually use the AMB only the beginning of SV40, because it contains the initial promoter.
(3) Selection gene component
Expressing and cloning vectors may contain a selection gene, also designated breeding marker. The usual selection genes encode proteins that (a) give resistance to antibiotics or other toxins, e.g. ampicillin, neomycin, methotrexate, or tetracycline, (b) auxotrophic deficiency of complement, where appropriate, or (c) provision of necessary nutrients, the lack of a complex environment.
In one example, the schema selection of used drug that stops the growth of the host cell. These cells that are successfully transformed heterogeneous gene produce a protein that gives drug resistance and thus preserve scheme selection. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin.
Another example of suitable breeding markers for mammalian cells is an example that allows you to identify competent cells to select nucleic acid antibodies, such as DHFR, timedancing, metallothionein-I and-II, preferably genes metallothionein primates, adenoidectomies, ornithindecarboxilase and others.
For example, in some embodiments, implementation, CL who weave transformed DHFR selectivity gene were first identified by culturing all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist DHFR. In some embodiments, the implementation of appropriate cell hosts when using wild-type DHFR are a cell line of Chinese hamster ovary (CHO), deficient in DHFR activity (e.g., ATCC CRL-9096).
Alternatively, cell owners (particularly wild-type hosts that contain endogeny DHFR)transformed or co-transformed with DNA sequences encoding the antibody, protein, wild-type DHFR and other breeding marker such as aminoglycoside 3'-phosphotransferase (APH)can be selected by cell growth in medium containing selectivity material for breeding marker such as aminoglycoside antibiotic, such as kanamycin, neomycin, or G418. Cm. U.S. patent No. 4965199.
(4) the Promoter component
Expressing and cloning vectors usually contain a promoter that is recognized by the host organism and is functionally linked to a nucleic acid that encodes a polypeptide of interest (e.g., antibody). Known promoter sequences for eukaryotes. For example, virtually all eukaryotic genes have an AT-rich region localized p is blithedale 25-30 bases above the site of transcription initiation. Another sequence found 70 to 80 bases above from the start of transcription of many genes is a CNCAAT region, where N can be any nucleotide. On the 3' end of most eukaryotic genes is AATAAA sequence that may be the signal for addition of poly A tail to the 3' end of the coding sequence. In some embodiments, implementation, any or all of these sequences can appropriately be inserted into eukaryotic expressing vectors.
Transcription from vectors in the cells of the host mammal is controlled, for example, by promoters obtained from the genomes of viruses such as virus polyoma, fowlpox virus, adenovirus (such as adenovirus 2), human papilloma virus of cattle, sarcoma virus of birds, cytomegalovirus, a retrovirus, hepatitis-B and the monkey virus 40 (SV40), from heterogenic mammalian promoters, e.g. the actin promoter or an immunoglobulin promoter, from the promoters of heat shock proteins provided such promoters are in accordance with the systems of host cells. Start and end the promoters of SV40 virus are conveniently obtained as SV40 restriction fragment which also contains the SV40 viral replication origin. The first promoter of the human cytomegalovirus is conveniently obtained in the form of a HindIII E restriction the second fragment. System for expression of DNA in mammalian hosts using the human papilloma virus of cattle represented as a vector, is described in U.S. Patent No. 4419446. Modification of this system is described in U.S. Patent No. 4601978. Cm. also Reyes et al., Nature 297:598-601 (1982), describe the expression of the cDNA of β-interferon in human cells of mice under control timeintensive promoter from herpes simplex virus. Alternatively, the long terminal repeat of rous sarcoma virus can be used as a promoter.
(5) The enhancer element
Transcription of DNA encoding the antibody of this invention, higher eukaryotes is often reinforce by inserting enhancer sequence into the vector. Currently known many enhancer sequences from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). Usually, however, will be used enhancer of virus eukaryotic cells. Examples include the SV40 enhancer on the last plot point early replication (bp 100-270), the initial promoter of the cytomegalovirus enhancer, enhancer of polyoma the last part of the starting point of replication and enhancers of adenoviruses. Cm. also Yaniv, Nature 297:17-18 (1982), describing enhancer elements activation of eukaryotic promoters. The enhancer may be inserted into the vector in which ogenyi 5' or 3' to the sequence, encodes a polypeptide antibodies, but, as a rule, are localized in the 5' promoter.
(6) The termination of transcription
Used in eukaryotic cells-the owners expressing vectors can also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Typically, such sequences are available from 5' and occasionally 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated section of mRNA that encodes the antibody. One convenient component termination of transcription is polyadenylation region of growth hormone in cattle. Cm. WO94/11026 and described therein expressing vector.
(7) Selection and transformation of host cells
Appropriate cell hosts for cloning or expression of the DNA in the vectors herein include cells of higher eukaryotes, as described in this document, including cells of vertebrate hosts. Propagation of vertebrate cells in culture (tissue culture) has become a common procedure. Examples of useful cell lines of mammalian hosts represent a line of CV1 monkey kidney transformed by SV40 (COS-7, ATCC CRL 1651); line kidney of a human embryo (293 or 293 cells, sub is donirovannye for growth in suspension culture, Graham et al, J. Gen Virol. 36:59 (1977)); the cells of the kidneys of dwarf hamster (BHK, ATCC CCL 10); the cells of the Chinese hamster ovary-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); the Sertoli cells of mice (TM4, Mather, Biol. Reprod 23:243-251 (1980)); kidney cells of monkeys (CV1 ATCC CCL 70); the kidney cells of the African green monkey (VERO-76, ATCC CRL-1587); cell carcinoma of the neck of human (HELA, ATCC CCL 2); cells of the kidneys of dogs (MDCK, ATCC CCL 34); liver cells Buffalo rats (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); the cells of the human liver (Hep G2, HB 8065); tumors of the mammary gland of the mouse (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N. Y. Acad. Sci. 383:44-68 (1982)); the cells, MRC 5; FS4 cells; and a line human hepatoma(Hep G2).
Cell owners transform the above expressing or cloning vectors to generate antibodies and cultivation in a suitable nutrient medium, modified as necessary for activating promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
(8) Culturing host cells
Used cell owners to obtain antibodies of this invention can be grown in different environments. Commercially available medium such as Ham''s F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco modified atmosphere Needle ((DMEM), Sigma) are suitable for culturing the host cells. In addition, you can use as a culture medium for cells households who s any of the environments, described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. patent No. 4767704; 4657866; 4927762; 4560655; or 5122469; WO 90/03430; WO87/00195; or U.S. Patent Re. 30985. Any of these environments, you can add necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as chloride and sodium phosphate, calcium, magnesium), buffers (such as HEPES), nucleotides (such as adenosine and timein), antibiotics (such as GENTAMYCINTMdrug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar level), and glucose or an equivalent energy source. Any other additions may also be included at appropriate concentrations that must be known to specialists in this field. Culturing conditions, such as temperature, pH, and others, are the conditions that previously used is selected for the expression of host cells, and will be obvious to the ordinary skilled specialist.
(9) Purification of antibodies
When using recombinant methods, antibodies can be obtained intracellular or secreted directly into the environment. If the antibody receive intracellularly, as a first stage, dispersed detritus, or cells of the host, or lysed fragments, can UDA is arranged, for example, by centrifugation or ultracentrifugation. If antibodies are secreted into the medium supernatant from expressing such systems source can be concentrated using a commercially available filter for concentrating the protein, for example, and Amicon Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF, may include any of the above mentioned steps to inhibit proteolysis and antibiotics may include the steps of preventing the growth of random impurities.
Obtained from the cell compositions of the antibodies can be cleaned using, for example, hydroxiapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography, which is a convenient way. Suitable protein A as an affinity ligand depends on the species and isotype of any Fc domain of immunoglobulin, which is present in the antibody. Protein A can be used for purification of antibodies, which are based on the human heavy chain γ1, γ2, or γ4 (Lindmark et al., J. Immunol. Methods 62:1-13 (1983)). Protein G is recommended for all isotypes mice and for human γ3 (Guss et al., EMBO J. 5:1567-1575 (1986)). The matrix to which is attached an affine ligand may be agarose, but other valid matrices. Mechanically solid matrices such as glass with controlled pore size or poly(stringifier)benzene, provide the t faster flow rates and shorter process time, than this will ensure that the agarose. If the antibody contains a CH3 domain, the Bakerbond ABXTMresin (J. T. Baker, Phillipsburg, NJ) easy to clean. Other methods of protein purification such as fractionation on an ion-exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSETM, chromatography on an anion or cation exchange resin (such as a column with polyaspartic acid), chromatofocusing, SDS-PAGE, and precipitation by ammonium sulfate is also available depending on the secreted antibodies.
After any pre-treatment mixture containing the desired antibody and impurities, can be subjected to further purification, for example by hydrophobic interaction chromatography at low pH using an eluting buffer at a pH between about 2.5 to 4.5, preferably produced at low salt concentrations (e.g., from about 0-0 .25 M salt).
Generally, various methods for producing antibodies for use in research, testing and clinical use are well established in this field, in accordance with the above-described method and/or, as it appears advisable by specialists in this field, for the individual interested antibodies.
Immunoconjugate, or "loaded Lakers the public by means of antibody", useful for delivery to the place of cytotoxic substances in the treatment of cancer. See, for example, Syrigos et al. (1999) Anticancer Research 19:605-614; Niculescu-Duvaz et al. (1997) Adv. Drug Deliv. Rev. 26:151-172; U.S. Patent No. 4975278. Immunoconjugate provide targeted delivery of a fragment of drugs to the tumor, because the system introduction unconjugated cytotoxic substances can lead to unacceptable levels of cytotoxicity in normal cells and tumor cells tend to suppress. Cm. Baldwin et al. (Mar. 15, 1986) Lancet pp. 603-05; Thorpe (1985) "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological and Clinical Applications (A. Pinchera et al., eds.) pp. 475-506.
In one aspect, immunoconjugate contains an antibody that binds IL-19, IL-20, IL-22, IL-24, IL22R, IL-20Ra, IL-20Rb or IL-10R2, such as antibodies, are presented in this document, and the cytotoxic substance such as a chemotherapeutic agent, a substance suppressing the growth, toxin (e.g., enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), or a radioactive isotope (i.e radioconjugates).
Chemotherapeutic substances, useful for obtaining such immunoconjugates were described above. Enzymatically active toxins and fragments thereof that can be used include chain And diphtheria, unbound active fragments of diphtheria toxin, CE the ü And exotoxin a (from Pseudomonas aeruginosa), chain A of ricin chain abrina, A chain of medecine, alpha sarcin, Aleurites fordii proteins, proteins diantin, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, krotin, sapaonaria officinalis inhibitor, gelonin, mitogillin, restrictocin, vanomycin, inomycin and tricothecene. A number of radionuclides available for radioconjugates antibodies. Examples include 212Bi, 131I, 131In, 90Y, and 186Re.
Conjugates of the antibody and cytotoxic substances can be obtained using a variety of bifunctional protein-binding substances, such as N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP), aminothiols (IT), bifunctional derivatives of imidapril (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), BIZ-azide compounds (such as bis-(p-azidobenzoyl)hexanediamine), bis-diezani derivatives (such as bis-(p-disoriented)Ethylenediamine), the diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-debtor-2,4-dinitrobenzene). For example, the immunotoxin of ricin can be obtained as described in Vitetta et al, Science, 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzene-3-metallitron triaminotoluene acid (MX-DTPA) is a typical chelate forming agent for conjugation of radionucleotide to the antibody. Cm. WO94/11026.
Conjugates of the antibody and one and the and more small molecules toxin, such as calicheamicin, maytansinoid, tricetin and CC1065, and the derivatives of these toxins that have activity of toxins, also proposed in this document.
1. Maytansine and maytansinoids
In one embodiment, immunoconjugate contain antibody conjugated with one or more molecules of maytansinoids. Maytansinoid are mycotoxicosis inhibitors, which act by inhibiting the polymerization of tubulin. Maytansine was first isolated from the East African shrub Maytenus serrata (U.S. patent No. 3896111). Subsequently it was discovered that certain microbes also produce maytansinoid, such as maytansine and C-3 maytansine esters (U.S. patent No. 4151042). Synthetic maytansines and derivatives and analogues are disclosed, for example, in U.S. patents№ 4137230; 4248870; 4256746; 4260608; 4265814; 4294757; 4307016; 4308268; 4308269; 4309428; 4313946; 4315929; 4317821; 4322348; 4331598; 4361650; 4364866; 4424219; 4450254; 4362663 and 4371533, the disclosure of which is expressly incorporated by reference.
When attempting to improve their therapeutic index, maytansine and maytansinoids were conjugated with antibodies that bind antigens on the surface of tumor cells. Immunoconjugate containing maytansinoid and their therapeutic use are disclosed, for example, in U.S. patent No. 5208020, 5416064 and European patent 0 425 235 B1, the disclosure of which is straight fluctoorisation links. In Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) described immunoconjugate containing maytansinoid designated DM1 associated with the monoclonal antibody C242 directed against human colorectal cancer. It was found that the conjugate is highly toxic towards cultured cells of colorectal cancer, and shows antitumor activity in the analysis of tumor growth in vivo. In Chari et al., Cancer Research 52:127-131 (1992) described immunoconjugate in which maytansinoid was anywhereman through disulfide bonds to the murine antibody A7 binding to the antigen on the cell lines of human colorectal cancer, or to another murine monoclonal antibody TA.1 that binds the HER-2/neu oncogene. Cytotoxicity TA.1-maytansinoid conjugate was tested in vitro on cell lines of human breast cancer SK-BR-3, which expresses 3×105HER-2 surface antigens per cell. Drug conjugate has reached a level of toxicity similar to free drug maytansinoid, which could be increased by increasing the number of molecules of maytansinoid molecule antibodies. A7-maytansinoid conjugate showed low systemic toxicity in mice.
Antibody-maytansinoid conjugates get through chemical binding of an antibody to a molecule maytansinoid without significant reduction Biol the environmental activity or antibodies, or molecules maytansinoid. Average 3-4 conjugated molecules of maytansinoids on the antibody shows the effectiveness of increasing the cytotoxicity of target cells without adversely affecting the function or the solubility of the antibody, although it is expected that even one molecule of the toxin to the antibody will increase the cytotoxicity more than when using pure antibodies. Maytansinoid well known in this field and can be synthesized using known methods or can be isolated from natural sources. Suitable maytansinoid disclosed, for example, in U.S. patent No. 5208020 and in other patents and non-patent publications referred to in this document above. Preferred maytansinoid are maytansines and modified analogues maytansine in the aromatic ring or in other positions of the molecule maytansine, such as various esters maytansine.
There are many linking groups known in the field, to obtain antibody-maytansinoid conjugates, including, for example, described in U.S. patent No. 5208020 or European patent 0 425 235 B1, and Chari et al, Cancer Research 52:127-131 (1992). Linking groups include disulfide groups, thioester groups, kislotolabilen group, photolabile group, peptidylarginine group or estradiollevelny group as disclosed is the above-mentioned patents, preferable are disulfide and thioester groups.
Conjugates of the antibody and maytansinoid can be obtained using a variety of bifunctional protein-binding substances, such as N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP), Succinimidyl-4-N-maleimidomethyl)cyclohexane-1-carboxylate, aminothiols (IT), bifunctional derivatives of imidapril (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis-(p-azidobenzoyl)hexanediamine), bis-derivatives diezani (such as bis-(p-disoriented)Ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-debtor-2,4-dinitrobenzene). Some binding agents, including N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson et al., Biochem. J. 173:723-737 (1978)) and N-Succinimidyl-4-(2-pyridylthio)pentanoate (SPP), provide disulfide bonds.
The binder can be attached to the molecule maytansinoid in different positions, depending on the type of communication. For example, the ester linkage can be formed by reaction with a hydroxyl group using conventional methods of binding. The reaction can occur in the C-3 position having a hydroxyl group, the C-14 position modified hydroxymethylene groups is th, C-15 position modified with hydroxyl group, and C-20 position having a hydroxyl group. In a preferred embodiment, the bond is formed in the C-3 position maytansine or similar maytansine.
2. Auristatin and dolastatin
In some embodiments, implementation, immunoconjugate contains antibody conjugated to dolastatin or peptide analogue or derivative dolastatin, such as auristatin (U.S. patent No. 5635483; 5780588). It was shown that dolastatin and auristatin prevent the change of microtubules, GTP hydrolysis, and nuclear and cellular division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584) and have anti-cancer (U.S. patent No. 5663149) and antifungal activity (Pettit et al. (1998) Antimicrob. Agents Chemother. 42:2961-2965). Fragment drugs with dolastatin or auristatin can be attached to the antibody through the N (amino) end or C (carboxyl) the end of the peptide drug fragment (WO 02/088172).
Typical embodiments of with auristatin include the N-terminal associated monomethylaniline medicinal components DE and DF, disclosed in "Monomethylvaline Compounds Capable of Conjugation to Ligands," patent application U.S. publication No. US 2005-0238649 A1, the disclosure of which is expressly incorporated by reference in full.
Typically, peptide drug components can be obtained through the formation of peptide is communication between two or more amino acids and/or peptide fragments. Such peptide bonds can be obtained, for example, according to the liquid-phase method for the synthesis (see E. Schroder and K. Lubke, "The Peptides", volume 1, pp 76-136, 1965, Academic Press)that is well known in the field of peptide chemistry. Auristatin/dolastatin drug components can be obtained according to the methods of: US 5635483; US 5780588; Pettit et al (1989) J. Am. Chem. Soc. 111:5463-5465; Pettit et al (1998) Anti-Cancer Drug Design 13:243-277; Pettit, G.R. et al. Synthesis, 1996, 719-725; Pettit et al (1996) J. Chem. Soc. Perkin Trans. 15:859-863. Cm. also Doronina (2003) Nat. Biotechnol. 21(7):778-784; patent application U.S. publication No. 2005-0238649 A1, the disclosure of which is incorporated by reference in full (revealing, for example, binding agents and methods of making compounds monomethylamine, such as MMAE and MMAF conjugated with a binding substance).
Another interesting immunoconjugate contains antibody conjugated to one or more molecules calicheamicin. Family of antibiotics calicheamicin capable of forming denitive DNA breaks in subpicomolar concentrations. To obtain conjugates family calicheamicin, see U.S. patent 5712374, 5714586, 5739116, 5767285, 5770701, 5770710, 5773001, 5877296 (everything from American Cyanamid). Structural analogues calicheamicin, which can be used include, but are not limited to, γ1Iα2Iα3IN-acetyl-γ1I, PSAG and θ1I(Hinman t al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998) and the aforementioned U.S. patents from American Cyanamid). Other antitumor drugs with which the antibody can form conjugates represent QFA, which antifolates. And calicheamicin, and QFA have intracellular sites of action and hardly penetrate through the plasma membrane. Therefore, the cellular accumulation of these substances through antibody-mediated internalization significantly enhances their cytotoxic effects.
4. Other cytotoxic substances
Other antitumor agents, which can konjugierte with the antibody include BCNU, streptozocin, vincristine and 5-fluorouracil, the family of substances known collectively as the LL-E33288 complex described in U.S. patents 5053394, 5770710 and espiramicina (U.S. patent 5877296).
Enzymatically active toxins and fragments thereof that can be used include chain And diphtheria, unbound active fragments of diphtheria toxin a chain of exotoxin a (from Pseudomonas aeruginosa), chain A of ricin chain abrina, A chain of medecine, alpha sarcin, Aleurites fordii proteins, proteins diantin, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, krotin, sapaonaria officinalis inhibitor, gelonin, mitogillin, restrictocin, vanomycin, inomycin and tricothecene. See, for example, WO 93/21232, opublikowany the th October 28, 1993
In another aspect, immunoconjugate may contain the antibody and the connection with nucleotidase activity (e.g., a ribonuclease or a DNA endonuclease such as a desoksiribonukleaza; Tenkasu).
For selective tumor destruction, immunoconjugate may contain anti-FGFR2 antibody and highly radioactive atom. A number of radioactive isotopes are available for obtaining radioconjugates anti-FGFR2 antibodies. Examples include At211I131I125, Y90That Re186That Re188Sm153Bi212, P32, Pb212and radioactive isotopes of Lu. If the conjugate is used for diagnosis, it may contain a radioactive atom for scintigraphic studies, for example Tc99mor I123or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as again, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.
Radio - or other labels can be embedded in the conjugate by known methods. For example, you can biosynthesizing peptide or you can synthesize chemical amino acid synthesis using suitable amino acid precursors, including, for example, fluorine-19 in place of hydrogen. Labels such as Tc99mor I123That Re186That Re188and In
D. Antagonists and agonists
Presents antagonists of IL-22. Such antagonists include antagonists that directly act on IL-22 (for example, anti-IL-22 antibody) and antagonists, which act indirectly on IL-22 activity (for example, anti-IL-22R antibody). Such antagonists are needed to, for example, 1) the treatment of inflammatory disorders and autoimmune disorders, and 2) changes in signal transmission of IL-23 or IL-22. In one specific embodiment, the composition containing an antagonist of IL-22 or IL-22R, the need to reduce the amount of psoriatic tissue in a mammal. In another specific embodiment, the composition containing an antagonist of IL-22 or IL-22R, the need for partial or complete suppression of proliferation of tumor cells.
In one aspect, the antagonist of IL-22 is an anti-IL-22 antibody or anti-IL-22R antibody. In some embodiments, implementation of anti-IL-22 antibody is a blocking antibody that partially or completely blocks the interaction between IL-22 with its receptor. In some embodiments, implementation of anti-L-22R antibody is a blocking antibody which partially or completely blocks the interaction between IL-22R and IL-22. In some embodiments, implementation of anti-IL-22R antibody binds to the extracellular landscapebased domain of IL-22R. For example, anti-IL-22R antibody can bind with extracellular landscapebased domain of human IL-22R, which is found in SEQ ID NO: 3 from about 18-228 of amino acids.
In another aspect, the antagonist of IL-22 is an Oligopeptide that binds to IL-22 or IL-22R. In one embodiment, the Oligopeptide binds to the extracellular landscapebased domain of IL-22R. The oligopeptides may be chemically synthesized using well-known method for the synthesis of oligopeptides or can be obtained and purified using recombinant methods. Such oligopeptides are usually at least about 5 amino acids in length, alternatively at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 amino acids in length. Such oligopeptides may be identified without undue experimentation using well known methods. It was also stated that the methods of screening Oligopeptide libraries for aigopad the Dov, which can specifically bind to a polypeptide target are well known in the art (see, for example, U.S. patents№ 5556762, 5750373, 4708871, 4833092, 5223409, 5403484, 5571689, 5663143; PCT publication no WO 84/03506 and WO84/03564; Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 81:3998-4002 (1984); Geysen et al. Proc. Natl. Acad. Sci. USA, 82: 178-182 (1985); Geysen et al., in Synthetic Peptides as Antigens, 130-149 (1986); Geysen et al., J. Immunol. Meth., 102:259-274 (1987); Schoofs et al., J. Immunol, 140:611-616 (1988), Cwirla, S. E. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6378; Lowman, H.B. et al. (1991) Biochemistry, 30: 10832; Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D. et al. (1991), J. Mol. Biol, 222:581; A.S. Kang et al. (1991) Proc. Natl. Acad. Sci. USA, 88:8363, and Smith, G. P. (1991) Current Opin. Biotechnol, 2:668). In some embodiments, implementation, Oligopeptide may conjugates with a cytotoxic substance.
In another aspect, the antagonist of IL-22 is an organic molecule that binds to IL-22 or IL-22R, except oligopeptides or antibodies as described in this document. The organic molecule can be, for example, a small molecule. In one embodiment, the organic molecule binds to the extracellular domain of IL-22R. Organic molecule that binds to IL-22 or IL-22R, may be identified and chemically synthesized using known methods (see, e.g., PCT publication No. WO00/00823 and WO00/39585). Such organic molecules are usually smaller than about 2000 daltons, alternatively less than about 1500, 750, 500, 250 or 200 daltons, wherein such organic molecules that are able to bind to IL-22 or IL-22R, can be identified without undue experimentation using well known methods. It was also stated that the methods of screening libraries of organic molecules for molecules that are capable of contacting a polypeptide target are well known in the art (see, e.g., PCT publication No. WO00/00823 and WO00/39585). In some embodiments, implementation of organic molecules can konjugierte with a cytotoxic substance.
In another aspect, an IL-22 antagonist is a soluble IL-22 receptor, for example, form IL-22R, which is not membrane-bound. Such soluble forms of IL-22R can compete with membrane-bound IL-22R for binding to IL-22. In some embodiments, the implementation of the soluble form of IL-22R may contain all or a ligand-binding site of the extracellular domain of IL-22R, for example all or a ligand-binding site of the polypeptide containing 18-228 amino acids of SEQ ID NO: 3. In some embodiments, the implementation of the soluble form of IL-22R has no transmembrane domain. For example, a soluble form of human IL-22R may not have all or a substantial plot transmembrane domain from about 229-251 amino acids of SEQ ID NO:3.
Reported found in the erode soluble receptor for IL-22. Cm. Dumoutier L. et al., "Cloning and characterization of IL-22 binding protein, a natural antagonist of IL-10-related T cell-derived inducible factor/IL-22," J. Immunol. 166:7090-7095 (2001); and W. Xu et al., "A soluble class II cytokine receptor, IL-22RA2, is a naturally occurring IL-22 antagonist," Proc. Natl. Acad. Sci. U.S.A. 98:9511-9516 (2001). This receptor is variously referred to as the "IL-22BP" or "IL-22RA2" in this field. The sequence of human IL-22BP is shown in Fig. 4. The term "IL-22BP" or "IL-22 binding protein", as used herein, refers to any native IL-22BP from any vertebrate source, including mammals such as primates (e.g. humans and monkeys) and rodents (e.g. mice and rats), unless otherwise noted.
In another aspect, the antagonist of IL-22 is an antisense nucleic acid that reduces expression of IL-22 or IL-22R (i.e., which reduces the transcription of the gene IL-22 or IL-22R and/or translation of mRNA of IL-22 or IL-22R). In some embodiments, implementation, antisense nucleic acid binds with a nucleic acid (DNA or RNA)that encodes IL-22 or IL-22R. In some embodiments, implementation, antisense nucleic acid is an oligonucleotide of about 10-30 nucleotides in length (including all points between those endpoints). In some embodiments, implementation, antisense oligonucleotide contains modified sugar-phosphodieterase frames (or other communication sugars, including phosphothioate communication as described in WO 91/06629), where such modified sugar-phosphodieterase skeletons are resistant to endogenous nucleases. In one embodiment, the antisense nucleic acid is oligodeoxyribonucleotide, which causes destruction and/or decrease transcription or translation of mRNA encoding IL-22 or IL-22R. In some embodiments, implementation, antisense nucleic acid is an RNA that reduces the expression of nucleic acid targets via RNA interference" ("RNAi"). For an overview of RNAi, see, for example, Novina et al. (2004) Nature 430: 161-164. Such RNA is obtained from, for example, short interfering RNA (siRNAs) and microRNAs. siRNAs, for example, can be synthesized in the form of double strand oligoribonucleotides of about 18-26 nucleotides in length.
In another aspect, the agonist IL-22. Typical agonists include, but are not limited to, native IL-22 or IL-22R, fragments, variants or modified forms of IL-22 or IL-22R, which retain at least one activity of the native polypeptide; substances which are able to bind and activate IL-22R; and substances that cause the overexpression of IL-22 or IL-22R, or nucleic acids encoding IL-22 or IL-22R.
E. Pharmaceutical compositions
The invention presents pharmaceutical compositions. In one embodiment, the headlamp is aseptically composition contains 1) an active substance, for example, any of the above polypeptides, antibodies, agonists or antagonists; and 2) a pharmaceutically acceptable carrier. In a further embodiment, the pharmaceutical composition further contains at least one additional therapeutic agent.
Pharmaceutical formulations are prepared for storage by mixing the substance with a certain degree of purification, with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington''s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations, and include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecylsilane ammonium chloride; hexamethonium chloride; benzalconi chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl para-aminobenzoic acid, such as methyl or propyl para-aminobenzoic acid; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); polypeptides of low molecular weight (less than about 10 residues); proteins such as serum albumin, m is tin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; hepatoblastoma agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; soleobrazutaya counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants such as TWEENTM, PLURONICSTMor polyethylene glycol (PEG).
You can also use lipofectin or liposomes for delivery of substances into the cell. If the substance is an antibody fragment, the smallest inhibitory fragment that specifically binds with the protein target is preferred. For example, on the basis of sequences of the variable regions of the antibodies can be constructed peptide molecules that retain the ability to bind the protein sequence. Such peptides can be synthesized chemically and/or to obtain a recombinant DNA methods (see, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA 90, 7889-7893 (1993)). The antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing the antibody are obtained by methods known in this field, as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al, Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. patent No. 4485045 and 4544545. Liposomes with enhanced circulation time are disclosed in U.S. patent No. 5013556. Particularly useful liposomes can be obtained by way of reverse-phase evaporation lipid composition containing phosphatidylcholine, cholesterol and PEG-derived phosphatidylethanolamine (PEG-PE). Liposomes are passed through filters with defined pore size to obtain liposomes of the desired diameter. Fab' fragments of antibodies of the present invention can be konjugierte with liposomes as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982) by the reaction of disulfide exchange. Chemotherapeutic agent (such as doxorubicin) optionally contained within the liposome. Cm. Gabizon et al., J. National Cancer Inst., 81(19): 1484 (1989).
The substance may also be enclosed in microcapsules obtained, for example, ways koatservatsii or interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and microcapsules of polymetylmetacrylate, respectively, in colloidal systems of drug delivery to the site of action (for example, liposomes, albumen microspheres, microemulsion, nanoparticles and nanocapsules) or in microemulsion. Such methods are disclosed in Remington''s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
You can get drugs with a slow release of the substance. Suitable examples of drugs with a slow release include semi-permeable matrices of solid hydrophobic polymers containing the substance matrix which is presented in the form of shaped articles, e.g. films, or microcapsules. Examples of matrices with a slow release include polyesters, hydrogels, for example, poly(2-hydroxyethylmethacrylate), or polyvinyl alcohol, polylactide (U.S. patent No. 3773919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene vinyl acetate, degradable copolymers of lactic acid and glycol acid, such as LUPRON DEPOTTM (injectable microspheres composed of a copolymer of lactic acid and glycol acid and acetate leuprolide), and poly-D-(-)-3-hydroxybutanoic acid. In addition, polymers such as ethylene vinyl acetate and lactic acid - glycol acid, contributing to the release of molecules for over 100 days, certain hydrogels release proteins for shorter time period. When encapsulated antibodies remain in the body for a long time, they can denaturing or aggregate as a result of exposure to water at 37°C, resulting in reduction of biological activity and possible changes in immunogenicity. Can be a rational strategy for Stabi is Itachi depending on the mechanism. For example, if you discovered that the mechanism of aggregation is the intramolecular formation of S-S linkages by ridiculing interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilised acidic solutions, adjusting the moisture content, using appropriate additives, and developing specific polymer compositions of matrices.
The pharmaceutical composition herein may also contain more than one active connection, if necessary, specify the treatment. For example, in one aspect, the pharmaceutical composition containing more than one active compound containing 1) at least one antagonist of IL-22, for example an antibody that binds to IL-22, and/or an antibody that binds to IL-22R; and 2) at least one antibody that binds to IL-19, IL-20, IL-24, IL20Ra, IL-20Rb or IL-10R2 (where any number of the antibodies listed in 2), you can choose any the combination). In another aspect, the pharmaceutical composition contains two or more active compounds with complementary activities. For example, in one embodiment, the pharmaceutical composition may contain 1) at least one antagonist of IL-22, for example an antibody that binds to IL-22, and/or an antibody that binds to IL-22R; and 2) an antagonist of TNF-α or IL-12. In one the aspect, pharmaceutical composition containing more than one active compound may contain a cytotoxic agent or a growth inhibitor.
F. Methods of treatment
There are therapeutic methods using the aforementioned compounds or pharmaceutical compositions. Such methods include in vitro, ex vivo and in vivo therapeutic methods, unless otherwise noted. In various aspects, the methods of stimulation or inhibition of IL-23-mediated pathways of signal transduction. Presents methods of stimulating or inhibiting function ThIL-17cells. Provides methods for treating inflammatory and/or autoimmune disorders. The following are methods of treating disorders associated with IL-23 or IL-22 signaling. Also presents methods of treatment ThIL-17-oposredovannyh violations. Below these and other aspects of the invention.
In one aspect, presents a method of stimulation of IL-23 mediated pathways of signal transmission in a biological system, the method including providing biological systems IL-22 agonist. Biological systems include, for example, mammalian cells in in vitro cell culture or in an organism in vivo. Typical biological systems that simulate the psoriasis presented in the examples and include the restored human is epidermis (RHE) (example 14) or animal models (example 16). In one embodiment, IL-22 agonist is an IL-22. In another aspect, presents a method of inhibiting IL-23-mediated pathways of signal transmission in a biological system, the method including providing biological systems IL-22 antagonist. In one embodiment, the antagonist of IL-22 is an antibody, such as neutralizing anti-IL-22 antibody and/or neutralizing anti-IL-22R antibody.
In another aspect, presents a method of stimulation of the function of ThIL-17cells, the method including the impact of IL-22 agonist on ThIL-17cells. In one embodiment, IL-22 agonist is an IL-22. In another aspect, presents a method of inhibiting the function of ThIL-17cells, the method including the impact of IL-22 antagonist on ThIL-17cells. In one embodiment, the IL-22 antagonist is an antibody, such as neutralizing anti-IL-22 antibody and/or neutralizing anti-IL-22R antibody. Typical functions of ThIL-17cells include, but are not limited to, stimulation of cell-mediated immunity (delayed-type hypersensitivity); recruiting characteristic of immune cells, such as myeloid cells (such as monocytes and neutrophils), to sites of inflammation; and the stimulation of infiltration of inflammatory cells in the tissue. In one Varian is e implementation function ThIL-17cells is mediated by IL-23.
In another aspect, presents a method of treating inflammation, the method comprising the administration to a mammal in need of such treatment an effective amount of a pharmaceutical composition comprising an antagonist of IL-22. In one embodiment, the antagonist of IL-22 is an antibody, such as neutralizing anti-IL-22 antibody and/or neutralizing anti-IL-22R antibody. Inflammation includes, but is not limited to, autoimmune inflammation (inflammation associated with autoimmune disorder), chronic inflammation, skin inflammation, arthritic inflammation (including inflammation associated with rheumatoid arthritis and systemic inflammatory response. In one embodiment, the inflammation is mediated by IL-23.
In another aspect, presents a method of treating autoimmune disorders, the method comprising the administration to a mammal in need of such treatment an effective amount of a pharmaceutical composition comprising an antagonist of IL-22. In one embodiment, the antagonist of IL-22 is an antibody, such as neutralizing anti-IL-22 antibody and/or neutralizing anti-IL-22R antibody. Autoimmune disorder includes, but is not limited to, connective tissue disease, multiple sclerosis, systemic Kras is th erythematosus, inflammatory arthritis (e.g. rheumatoid arthritis), autoimmune inflammation of the lungs, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitus, uveitis, myasthenia gravis, reaction of graft versus host disease", and autoimmune inflammatory eye disease, psoriasis, arthritis associated with an autoimmune reaction (e.g., rheumatoid arthritis), autoimmune inflammation of the brain and inflammatory bowel disease. In one embodiment, the autoimmune disorder is an IL-23-mediated autoimmune disorder.
In a separate aspect, the methods of treatment of psoriasis and/or disorders characterized by psoriatic symptoms. Psoriasis is considered as an autoimmune disease in which T cells of the immune system recognize a protein in the skin and attack the area in which discovered this protein, causing a very rapid growth of new skin cells and tenderness, swelling, superficial damage. These injuries are characterized by hyperproliferative keratinocytes and accumulation of activated T-cells in the epidermis of psoriatic lesions. Although the initial molecular cause of the disease is not known, were established genetic link to at least 7 locus susceptibility to psoriasis (Psor1 in 6p21,3, Psor2 in 17q, Psor3 in 4q, Psor4 1 cent-q21, Psor5 in 3q21, Psor6 in 19pl3, and Psor7 in p). Some of these loci are associated with other autoimmune/inflammatory diseases, including rheumatoid arthritis, atopic dermatitis and inflammatory bowel disease (IBD). Conventional approaches to the treatment of psoriasis include the administration of IL-12 or TNF-α antagonists. See, for example, Nickoloff et al. (2004) J.Clin. Invest. 113:1664-1675; Bowcock et al. (2005) Nat. Rev. Immunol. 5:699-711; Kauffman et al. (2004) J. Invest. Dermatol. 123:1037-1044. The data presented in this document, however, include a separate IL-23/IL-22 transmission signal in the pathogenesis of psoriasis. Accordingly, a treatment that modulates the transmission signal may be an alternative or can complement other approaches to the treatment of psoriasis.
In one embodiment, a method of treatment of psoriasis includes an introduction to the patient an effective amount of a pharmaceutical composition containing an IL-22 antagonist. In one embodiment, the antagonist of IL-22 is an antibody, such as neutralizing anti-IL-22 antibody and/or neutralizing anti-IL-22R antibody. In a different implementation, the method further includes the introduction (or in the same pharmaceutical composition or in separate pharmaceutical composition), at least one additional therapeutic agent. In one such embodiment, the additional therapeutic substances is about represents at least one antagonist of a cytokine, selected from IL-19, IL-20 and IL-24. Such antagonists include, but are they unlimited, the antibody that binds IL-19, IL-20, IL-24, IL-20Ra, IL-20Rb or IL-10R2. Any number of such antibodies can be selected in any combination. In another embodiment, the additional therapeutic substance is a substance that is known by effectiveness in the treatment of psoriasis. Some of these therapeutic substances are described, for example, Nickoloff et al. (2004) J. Clin. Invest. 113:1664-1675; Bowcock et al. (2005) Nat. Rev. Immunol. 5:699-711; Kauffman et al. (2004) J. Invest. Dermatol. 123:1037-1044. Such substances include, but are not limited to, a therapeutic agent, which targets T-cells, such as efalizumab and/or alefacept; antagonist of IL-12, for example, a blocking antibody that binds to IL-12 or its receptor; and the antagonist of TNF-α, such as a blocking antibody that binds TNF-α or its receptor.
In another aspect, presents a method for inhibiting tumor progression, the method comprising the administration to a mammal an effective amount of a pharmaceutical composition containing an antagonist of IL-22. In one embodiment, the antagonist of IL-22 is an antibody, such as neutralizing anti-IL-22 antibody and/or neutralizing anti-IL-22R antibody. In one embodiment, tumor progression is IL-23 mediated.
The compositions of N. the present invention (for example, the polypeptides, antibodies, antagonists, agonists and pharmaceutical compositions containing any of the above) is administered to a mammal, preferably a human, in accordance with known methods, such as intravenous bolus or by continuous infusion over time, intramuscular, intraperitoneal, nutrizionale, subcutaneous, intra-articular, vnutriobolochechnoe, oral, local, or inhalation (intranasal, intra-lungs) courses. Intravenous or inhalation introduction polypeptides and antibodies are preferred.
In some embodiments, implementation, introduction of anti-cancer substances can be combined with the introduction of the composition of this invention. For example, in order to be treated by the composition of the invention, the patient may also receive anti-cancer agent (chemotherapeutic agent) or radiation therapy. Graphics reception and dispensing of such chemotherapeutic agents can be used according to the manufacturer's instructions or as imperiously determined by a qualified technician. Graphics receiving and dispensing such chemotherapy are also described in Chemotherapy Service Ed., M. C. Perry, Williams & Wilkins, Baltimore, MD (1992). Chemotherapeutic substance may precede or follow the introduction of the composition or may be given simultaneously. Additionally, with the Association antiestrogen, such as tamoxifen, or antiprogesterone, such as onapristone (see, EP 616812) can be given in dosages known for such molecules.
Desirable may be the introduction of antibodies against antigens associated with other immune disease or tumor, such as antibodies that bind to CD20, CD11a, CD18, ErbB2, EGFR, ErbB3, ErbB4, or a growth factor, vascular endothelial (VEGF). Alternatively, or in addition, two or more antibodies that bind the same or two or more different antigen described herein, can be administered simultaneously to the patient. In some embodiments, implementation, favorable may also be the introduction of one or more cytokines to the patient. In some embodiments, the implementation, the compositions of the invention are introduced simultaneously with the growth inhibitor. For example, the growth inhibitor can be entered before, after or simultaneously with the introduction of the composition. Suitable doses of the inhibitor of growth are currently used doses and can be lowered due to the combined action (synergy) of the growth inhibitor and composition.
To treat or reduce the severity of immune diseases suitable dose of the composition of the invention will depend on the type of disease being treated, as defined above, the severity and the cause of the disease is introduced does the substance for the prevention or therapeutic purposes is, held earlier therapy, clinical history of the patient and response to the connection, and the discretion of the attending physician. Connections convenient way to give the patient a lump sum or during the entire period of treatment.
For example, depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg (for example, 0.1 to 20 mg/kg) of polypeptide or antibody is the starting dose for administration to a patient, or, for example, one or more separate injections, or by continuous infusion. The daily dose may vary from about 1 μg/kg to 100 mg/kg or more, depending on the above factors. For re-introduction in a few days or more depending on the condition of treatment to support the desired suppression of the observed symptoms. However, there may be used other modes of doses. The progress of this therapy is easily monitored by conventional means and research.
G. Diagnostic methods and methods of determination
In one aspect, presents a method of diagnosing psoriasis in a mammal, the method comprising determining the level of expression of the gene encoding IL-22 or IL-22R polypeptide in the test sample of tissue cells obtained from the mammal, where a higher level of expression in the test sample compared to a control sample (e.g., the image is C known normal tissue cells of the same cell type) shows the presence of psoriasis in a mammal, from which was obtained the sample. The determination may be quantitative or qualitative. In one embodiment, the sample contains blood or serum. In one embodiment, the level of expression of the gene encoding IL-22 or IL-22R polypeptide contains (a) the interaction of anti-IL-22 or anti-IL-22R antibody with a test sample obtained from the mammal, and (b) determination of the complex formation between the antibody and the polypeptide of IL-22 or IL-22R in the sample. The antibody may be associated with a detectable label. The complex formation can be observed, for example, using light microscopy, flow cytometry, fluorometry or other methods known in this field. The sample can be obtained from individuals suspected of having psoriasis.
In one embodiment, the level of expression of the gene encoding IL-22 or IL-22R polypeptide comprises determining the level of transcription of mRNA from the gene. The levels of transcription of mRNA can be determined either quantitatively or qualitatively, in a variety of ways known to the person skilled in the art. The levels of mRNA transcription can also be defined directly or indirectly by determining the levels of cDNA derived from mRNA. Conventional methods for determining the levels of transcription of mRNA include in the CE is I, but they are not limited to, quantitative real-time RT-PCR and the method based on hybridization, including microcephaly analysis and analysis based on filters such as Northern-blotty.
To another variant implementation of the present invention is a diagnostic kit containing an anti-IL-22 or anti-IL-22R antibody in a convenient package. Preferably, the kit contains instructions for using the antibodies for the determination of IL-22 or IL-22R polypeptide. In one aspect, a diagnostic kit is a diagnostic kit for psoriasis.
1. Research, cell-based and animal model
Research, cell-based and animal models for immune diseases, used in the practice of some embodiments of the invention. Some studies, cell-based, are presented in the examples below, using, for example, to study the effectiveness of IL-22 antagonists or agonists.
Also use animal models in vivo in the practice of some embodiments of the invention. Typical animal models are also described in the examples below. The nature of in vivo such models allows us to predict responses in human patients. Animal models immunostained diseases includes both non-recombinant and recombinant (transge the different animals. Non-recombinant animal models include, for example, rodents, for example, murine models. Such models can be obtained by introducing cells into syngeneic mice using standard methods, such as subcutaneous injection, injection into the tail vein, implantation of spleen, intraperitoneal implantation, the implantation of the capsule of the kidney and others.
Model of graft versus host are ways to evaluate T-cell reactivity against MHC antigens and minor transplant antigens. The reaction of "graft versus host disease" occurs when immune cells transplanted patients with a suppressed immune system or sustainable. Donor cells recognize and respond to antigens. The answer can range from life threatening severe inflammation to a mild degree of diarrhea and weight loss. Suitable evaluation method for graft-versus-host are described in detail in Current Protocols in Immunology, above, section 4.3.
Animal model rejection, skin allograft is a way to examine the ability of T cells to induce in vivo tissue destruction and evaluation of their role in rejection of the graft. The most common and recognized models use a skin graft of a mouse's tail. Repeated experiments showed that actor shall prolong skin allograft is mediated by T-cells, helper T-cells and killer T-cells, and not antibodies. Auchincloss, H. Jr. and Sachs, D. H., Fundamental Immunology, 2nd ed., W. E. Paul ed., Raven Press, NY, 1989, 889-992. A suitable method is described in detail in Current Protocols in Immunology, above, section 4.4. Other models rejection of transplantant that can be used to study the compounds of the invention represent a model of allogeneic heart transplant, described Tanabe, M. et al, Transplantation (1994) 58:23 and Tinubu, S. A. et al, J. Immunol. (1994) 4330-4338.
Contact hypersensitivity is a simple in vivo for cell-mediated immune functions (delayed-type hypersensitivity). In this way, the interaction of the skin with exogenous haptens leads to hypersensitivity reactions of the delayed type, which can be assessed and measured quantitatively. Contact hypersensitivity involves the initial phase of sensitization, followed by the phase of the receive output. Phase get o see if T-lymphocytes encounter antigen, with whom they had contact. Swelling and inflammation of the watch, making it the perfect model of allergic contact dermatitis of the person. A suitable method is described in detail in Current Protocols in Immunology, Eds. J. E. Cologan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober, John Wiley & Sons, Inc., 1994, unit 4.2. Cm. also Grabbe S. and Schwarz, T., Immun. Today 19 (1): 37-44 (1998).
Additionally, the compositions of which Britania can be investigated in animal models for parasponia diseases. For example, the compositions of the invention can be explored on the scid/scid mouse model described by Schon, M. P. et al, Nat. Med. (1997) 3: 183, in which mice show histopathological lesions similar to psoriasis. Another good model is a Chimera of human skin/scid mouse, obtained as described in Nickoloff, B. J. et al, Am. J. Path. (1995) 146:580. Another good model is described in Boyman et al., J. Exp. Med. (2004) 199(5):731-6, in which human skin prior to the development of psoriasis is grafted AGR129 mice, leading to the development of psoriatic skin lesions.
It is possible to construct a knock-out animals, which contain defective or altered gene encoding a polypeptide identified herein, as a result of homologous recombination between the endogenous gene encoding the polypeptide and the DNA molecule, in which this gene has been modified. For example, cDNA encoding a single polypeptide, can be used to clone genomic DNA encoding that polypeptide in accordance with established methods. The plot of the genomic DNA that encodes a single polypeptide can be deleted or moved to another gene such as the gene encoding breeding marker that can be used for controlled integration. Usually a few thousand base pairs of unaltered flanking DNA (both at the 5'and 3' ends) are included in the vector (see, the example Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologous recombinant vectors). The vector is introduced into a line of embryonic stem cells (e.g., by electroporation) and selected cells in which the introduced DNA homologous recombinable with endogenous DNA (see, e.g., Li et al., Cell 69:915 (1992)). The selected cells are then injected into a blastocyst of an animal (e.g. mouse or rat) to obtain aggregation chimeras (see, e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152). Then chimeric embryo can be implanted in a suitable pseudovirion female foster animal and the embryo assign the term "knock-out" animal. Descendant containing homologous nekombinirovannyh DNA in their germ cells can be identified by standard methods and used for breeding animals in which all cells of the animal contain homologous nekombinirovannyh DNA. Knockout animals can be characterized, for example, by their ability to resist certain pathological conditions and by their development of pathological conditions due to absence of the polypeptide.
2. Screening of drug candidates
Screening of drug candidates aimed to identify compounds that bind or form a complex with the polypeptides is, identified in this document, or their biologically active components, or otherwise prevent the interaction of the polypeptide with other cellular proteins. Such screening will include research, responsible for high-throughput screening of chemical libraries, making them more suitable for identifying small molecule drug candidates. Prospective small molecules include synthetic organic or inorganic compounds, including peptides, preferably soluble peptides, polypeptide - immunoglobulin fusion and, in particular, antibodies including, without limitation, poly - and monoclonal antibodies and antibody fragments, single-chain antibodies, anti-idiotypical antibodies, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and antibody fragments. Research can be done in a variety of formats, including studies of protein-protein binding studies of biochemical screening, immunological analysis and research-based cells, which are well characterized in this field. All studies are common in that they require contacting the compounds with the polypeptide identified in this document is those under the conditions and within the time, sufficient to allow the polypeptide to interact with the test substance.
In the analysis of the binding interaction is a binding, and the complex formed can be selected or determined in the reaction mixture. In a separate embodiment, the polypeptide or the test substance immobilized on a solid phase, for example on titration the microtiter plate, by covalent or non-covalent binding. Non-covalent binding, as a rule, is carried out by coating the solid surface with a solution of the polypeptide or the compounds and drying. Alternatively, immobilizovannoi antibody, such as monoclonal antibody specific for the polypeptide, in order to mobilitat, you can use a fixed polypeptide to a solid surface. The study carried out by adding neimmunizirovannah component that can be marked-defined label, to the immobilized component, for example a surface with a coating containing a fixed component. When the reaction, unreacted components are removed, for example, money laundering and define fixed on a solid surface. When initially neimmunizirovannah component shall be defined label, identification label, immobilizovannoi on the surface, which provides, what happened to the formation of the complex. Where initially neimmunizirovannah component does not bear the label, the formation of the complex can be detected, for example, using labeled antibodies that specifically bind the immobilized complex.
If the investigational compound interacts, but not associated with a single polypeptide identified herein, this interaction with this protein can explore the well-known methods for determining protein-protein interactions. Such studies include traditional methods such as crosslinking, co-immunoprecipitate and co-purification through gradients or chromatographic columns. In addition, protein-protein interactions can be observed, using the genetic system of yeast, described by Fields and co-workers (Fields and Song, Nature (London) 340, 245-246 (1989); Chien et al, Proc. Natl. Acad. Sci. USA 88, 9578-9582 (1991)), as disclosed Chevray and Nathans, Proc. Natl. Acad. Sci. USA 89, 5789-5793 (1991). Many activators of transcription, such as yeast GAL4, consist of two physically separate modular domains, one acting as the DNA-binding domain, while the other is functioning as a domain activation of transcription. Expressing the system of yeast, as described in the above publications (generally referred to as "two-hybrid system"), uses mostly esto this property and uses the two-hybrid proteins, one in which the protein is the target merges with the DNA-binding domain of GAL4, and the other in which the activating of the candidate proteins fused with the activation domain. The expression of GAL1-lacZ reporter gene under the control of a GAL4-activated promoter depends on recovery of GAL4 activity via protein-protein interactions. Colonies containing interacting polypeptides are determined by a chromogenic substrate for β-galactosidase. Full set (MATCHMAKERTMto identify protein-protein interactions between specific proteins using twohybrid method is commercially available from Clontech. This system can also spread to the mapped protein domains that are included in specific protein-protein interaction, and also for points identify amino acid residues that are important for these interactions.
For identification of compounds that inhibit the interaction of the polypeptide identified herein and other intra - or extracellular components can be prepared in the reaction mixture containing the polypeptide and the component under conditions suitable for the interaction of the polypeptide with the component. To study the ability of the compounds to inhibit the interaction of preparing the reaction mixture in the absence and presence in the AI of the investigated compounds. If there is a decrease in the interaction of the polypeptide with the component in the presence of the compounds, then they say that the test compound inhibits the interaction of the polypeptide with the component.
In some embodiments, implementation, methods of identifying agonists or antagonists of IL-22 or IL-22R polypeptide include contacting IL-22 or IL-22R polypeptide molecule with a candidate agonist or antagonist and evaluation of defined changes in one or more biological activities normally associated with IL-22 or IL-22R of the polypeptide. Such activity may include, but are not limited to that described in the examples below.
3. Analysis of antibody binding
Study of the binding of antibodies can be produced by any known method of study, such as competitive binding analysis, direct and indirect sandwich research and study immunoprecipitate. Zola, Monoclonal Antibodies: A Manual of Techniques, pp.147-158 (CRC Press, Inc., 1987).
Competitive binding analysis is based on the ability of a labeled standard to compete with the analyte sample for binding to a limited number of antibodies. The amount of protein target in the sample is inversely proportional to the number of standard, which binds to the antibody. To facilitate determining the amount of standard that communicates, EN is the body preferably transferred to an insoluble form before or after the competition, so that the standard and analyte that are associated with antibodies may conveniently be separated from the standard and analyte that are left unbound.
"Sandwich" the study include the use of two antibodies, each of which is able to communicate with various immunogenic site or epitope of the protein to be detected. In the "sandwich" the study of the analyte sample is bound to the first antibody that mobilisierung on a solid medium, and thereafter a second antibody binds to the analyte, thus forming an insoluble three-membered complex. See, for example, U.S. patent No. 4376110. The second antibody itself can be observed-defined fragment (direct sandwich studies) or may be estimated using antiimmunoglobulin antibodies, which observed-defined fragment (indirect sandwich research). For example, one type of "sandwich" research is an ELISA study, in which defined fragment is an enzyme.
Immunohistochemistry can also be used to determine the cellular localization of the antigen with which the antibody binds. For immunohistochemistry, the tissue sample may be fresh or frozen or may be placed in paraffin and fixed with a preservative substance, such as formalin, for example.
In another aspect, is redstavleny products containing the compositions used for the diagnosis and treatment of the disorders described above. Products contain a container and instructions. Suitable containers include, for example, bottles, vials, pipettes and test tubes. Containers can be made of various materials, such as glass or plastic. The container holds a composition which is effective for diagnosing or treating the condition and may have a sterile outlet (for example, the container may be an intravenous bag with a solution or a vial having a cap that can be puncturing needle for subcutaneous injection). The active substance in the composition is typically a polypeptide, antibody, agonist or antagonist of the invention. Manual or the label on, or associated with the container to indicate that the composition is used for diagnosis or treatment status of the options. The product can further contain a second container containing a pharmaceutically acceptable buffer, such as phosphate-buffered saline, ringer's solution and dextrose. Further, it may include other materials needed for commercial and consumer standpoint, including other buffers, solvents, filters, needles, syringes and medication with instructions for use.
In one embodiment, the invention presents the product, including:
a) chemical composition containing an agonist or antagonist of IL-22 or IL-22R;
b) a container containing the composition; and
c) a label affixed to the specified container, or information about the drug, vkluchaysya in themselves the specified container, referred to the use of this antagonist in the treatment of immune diseases or cancer. The composition can contain an effective amount of the antagonist.
The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way.
The disclosures of all patent and literature references referred to in the present description, incorporated by reference in full.
Commercially available reagents referred to in the examples were used according to the manufacturer's instructions, unless otherwise noted. The source of those cells identified in the following examples and throughout the description with ATCC accession numbers represents the American Type Culture Collection, Manassas, VA.
EXAMPLE 1: Receiving anti-IL-22 and anti-IL-22R antibodies
This example shows how to obtain monoclonal antibodies that specifically bind IL-22 or IL-22R. Used to obtain the methods were based on methods known in this field and are described, for instance, in Goding, supra. Used immunogen floor were razmernye purified human IL-22 (hIL-22) or full-purified human IL-22R (hIL-22R). Briefly, mice were immunized approximately 1-100 μg hIL-22 or hIL-22R immunogen, emulsified in the auxiliary substance. Immunized mice are then subjected to stimulation after 10-12 days additional immunogen, emulsified in the auxiliary substance. Periodically from mice were obtained serum samples for research methods ELISA to detect anti-IL-22 or IL-22R antibodies.
After determination of the desired antibody titer "positive" for antibodies dogs were killed and received spleen cells. The spleen cells are then fused (using 35% polyethylene glycol with mouse myeloma line cells. Merge were obtained hybridoma cells were cloned and cultured in medium containing HAT (gipoksantin, aminopterin and thymidine). Hybridoma cells were skanirovali ELISA for reactivity against IL-22 or IL-22R. (see figure 5). The list of antibodies derived from such a hybrid, and their relevant properties are shown in Fig. 5.
EXAMPLE 2: signaling IL-22 is blocked by anti-IL-22 antibodies
Activation of STAT3 is a hallmark of activation of the receptor for IL-22 and intracellular signal transduction. Raised against human IL-22 antibodies were tested for their ability to block IL-22-induced activation of STAT3. 293 T cells expressing heterodimer receptor is IL-22 (hIL-22R/hIL-10R2), were planted in 0.2×106/well of 24-well plate. The cells were transfusional STAT3 luciferase reporter (TK-SIE-SRE-S) using Lipofectamine 2000TM(Invitrogen). Consequently, upon activation of STAT3 cells will produce the luciferase, the enzyme activity which can be determined by adding luciferin. The decrease in luciferase activity means that blocked STAT3. The next day was added 0.5 nm (hIL-22 (R&D Systems) in each well with 20 μg/ml antibody. Sixteen hours later, cells were literally and determined readings of the samples on the luminometer. It is shown in Fig. 6 data represent luciferase activity relative to Renilla internal control, which is a measure of the relative activation of STAT3. As shown in Fig. 6, antibodies 3F11.3, 11H4.4 and 8E11.9 had a great blocking ability.
EXAMPLE 3: dose-response of anti-IL-22 antibodies
The number of doses of antibodies raised against human IL-22, were investigated for their ability to block human IL-22 in the study of activation of STAT3. 293 cells expressing hIL-22R/hIL-10R2 were planted in 0.2×106/well of 24-well plate. The cells were transfusional STAT3 luciferase reporter (TK-SIE-SRE-S) using Lipofectamine 2000TM (Invitrogen). The next day was added 0.5 nm (hIL-22 (R&D Systems) in each well together with different concentrations of anti-IL22 antibody 3F11, 8E11 or 11H4. The range of concentrations of antibodies began with 40 µg/ml with 2-multiple dilutions to a final concentration 0,012 mg/ml sixteen hours Later, cells were literally, and found evidence of design in the luminometer. Three antibodies showed a similar curve to the dose/response blocking STAT3 activation, as shown in Fig. 7.
EXAMPLE 4: dose-response of anti-IL-22 antibodies
The number of doses of antibodies raised against human IL-22, were investigated for their ability to block murine IL-22 (mIL-22) in the study of activation of STAT3. 293 cells expressing mIL-22R/mIL-10Rb, were planted in 0.2×106/well of 24-well plate. The cells were transfusional STAT3 luciferase reporter (TK-SIE-SRE-S) using Lipofectamine 2000TM(Invitrogen). The next day was added 0.5 nm (mIL-22 (polyhistidine labeled) in each well together with different concentrations of antibody 3F11, 8E11 or 11H4. The range of concentrations of antibodies began with 40 µg/ml with 2-multiple dilutions to a final concentration 0,012 mg/ml sixteen hours Later, cells were literally and determined readings of the samples on the luminometer. In Fig. 8 shows that anti-IL-22 antibodies cross-react with mouse IL-22, and shows a similar, but not rigidly, curve dose/response. This shows that anti-IL-22 antibodies can be used in mouse experiments.
EXAMPLE 5: the Affinity of anti-IL-22 human L-22
In Fig. 9 shows the affinity of anti-IL-22 to human IL-22. The affinity was assessed by BIACore analysis. Different amounts of anti-IL-22 IgG were immobilized on a CM-5 chip (845 EN (reference unit) for 11H4 IgG, 1933 EN for 8E11 IgG, & 7914 EN for 3F11 IgG) hydrochloride by N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) chemical compounds. A twofold serial dilution of IL-22 were prepared in the range of 0.5 to 250 nm. Samples of antigen were deposited on top of IgG-immobilized surface at a flow rate of 20 μl/min for 6 minutes, and bound complexes were subjected to dissociation for 10 minutes. Surface IgG was restored 10 mm Gly, pH of 1.5 after each period of introduction of the antigen. As negative control cells, an abnormal IgG (3A5 RF transplant) was immobilization to subtract the background level. Separating buffer, PBS containing 0.05% Tween 20 in 0.01% NaN3used for all dilutions of the samples and the binding experiment was carried out at 25°C. Data were analyzed by universal in accordance with the 1:1 binding model. These results indicate that anti-IL-22 antibodies have a very good affinity to human IL-22.
EXAMPLE 6: Anti-IL-22 antibodies detect IL-22 in the cell
Were investigated antibodies against IL-22 on the ability to detect intracellular IL-22. For intracellular FACS acrasia the Oia IL-22, we used the following line 293 cells: cells expressing hIL-22-GFP, mIL-22-GFP, mIL20-GFP and GFP only. Investigational antibody was an anti-human IL-22 antibody 3F11, 8E11 and 17F6. Murine anti-gp120 was used as isotype control. The secondary antibody used was antimachine IgG-PE from Jackson labs. Cells were incubated with Brefeldin a for 2 hours, washed in PBS and then fixed with 2% paraformaldehyde overnight at 4°C. Then cells were washed in PBS and incubated in 5 ml of 0.2% Tween-20 for 30 minutes at 37°C. Staining with antibodies was carried out for 30 minutes at 4°C, then washed with a solution of Tween-20. Cells resuspendable in FACS buffer and analyzed in a FACScan. In Fig. 10 shows the FACS results. FACS results show that antibody 3F11 and 8E11 cause a change in the pattern of staining cells, showing that these antibodies bind and mouse, and human intracellular IL-22.
Anti-IL-22 antibody 3F11 used in additional experiments staining cells. 3F11 antibody was congerville with Alexa 647, fluorophore phycoerythrin. Mouse IgG2a conjugated to Alexa 647 was used as isotype control (Caltag). 293 cell lines expressing hIL-22-GFP and GFP were examined for binding of an antibody 3F11. 293 cells were fixed with 2% paraformaldehyde for 30 minutes, then washed dwai the s PBS/2% FCS. Cells resuspendable in 0.5% saponin for 15 minutes. Normal mouse serum was added to the other 15 minutes, then added antibodies 0.5 μg/million cells for 30 minutes. Cells were washed and resuspendable in FACS buffer and analyzed in a FACScan. In Fig. 11 shown in the lower left pane of the change in the cells inside the upper right square. This result shows that conjugated antibody 3F11 associated with intracellular IL-22.
EXAMPLE 7: Expression of IL-22 in Th1 T-cells
When CD4+ T-cells Mature in the thymus and enter the peripheral lymphatic system, they typically retain their initial phenotype to contact with antigens specific to their T-cell receptor (TCR) (Sprent et al., Annu. Rev. Immunol. (2002); 20:551-79). The TCR binding with specific antigens presented by antigen-presenting cells (APC), causes activation of T-cells. Depending on the environment and cytokine stimulation, CD4+ T cells can differentiate into Th1 or Th2 phenotypes and become effector or memory cells (Sprent et al., Annu Rev Immunol. (2002); 20:551-79 and Murphy et al., Nat Rev Immunol. (2002) Dec;2(12):933-44). This process is known as primary activation. After experiencing the initial activation of CD4+ T cells become effector or cells, and they retain their phenotype as Th1 or Th2. After these cells had met again antigen, they were subjected to secondary activation is about this time of the response to the antigen will be faster, than the primary activation, and leads to the production of effector cytokines, as defined primary activation (Sprent et al., Annu Rev Immunol. (2002); 20:551-79 and Murphy et al., Annu Rev Immunol. 2000; 18:451-94). It was found in studies during primary and secondary activation of CD4+ T-cells that the expression of some genes different (Rogge et al., Nature Genetics. 25, 96-101 (2000) and Ouyang et al., Proc Natl Acad Sci USA. (1999) Mar 30;96(7):3888-93).
For state primary activation of native T cells can be activated Ova and APC. Isolated from the cells in this state RNA can provide information about what genes are differentially regulated during primary activation and that cytokines affect gene expression during Th1 and Th2 development. After initial activation, CD4+ T cells can be maintained in culture. Since the previous activation and processing of cytokines were detected in these cells, they become either effector or memory cells. During this period, because there is no APC or antigens, CD4+ T cells are entering a stage of rest. This phase of relaxation provides information about the differences between native and memory cells, and vacationing Th1 memory cells and resting cells of memory Th2. Resting memory cells Th1 and Th2 then subjected to secondary activation by anti-CD3/CD28 antibodies or stimulation of IL 12/IL 18 cytokines. These States provide information about p is sliced between native and activated activated T-cell memory and the differences between activated Th1 memory and activated Th2 memory cells.
For the experiment shown in Fig. F12, splenocytes were isolated DO11.10 mice and activated with OVA in certain Th1 conditions: [IL-12 (1 ng/ml), IFN-γ, and IL-4 (1 μg/ml)]; Th0 conditions: [(anti-IL12, anti-IFN-γ, and anti-IL4)]; or Th2 conditions: [(anti-IL-12 (0.5 μg/ml), anti-IFN-γ and IL-4 (5 ng/ml]). RNA was isolated after 48 hours (primary stimulation). The rest of the cells were kept in culture for up to 7 days, and then they re-stimulated (secondary stimulation) OVA and irradiated Balb/c splenocytes. A subpopulation of cells from a Th1 condition has also been stimulated by only IL-12 and IL-18. After 48 hours, RNA was isolated. The expression of IL-22, IFN-γ and IL-4 in these RNA samples were analyzed by 5'-nuclease analysis (TaqManTM). The expression was initially normalized to samples of HPRT autogrow, then written as fold increase compared with the level of expression of splenocytes. The result is shown in Fig. 12, and the data show that IL-22 vysokoagressivnyh in Th1 cells after re-stimulation. Therefore the treatment with anti-IL-22 may useful for targeting these cells or for the treatment of Th1-mediated disorders, when it is desirable to clear Th1 cells from the blood, or as diagnostic Th1 mediated disorders, when I suspect the important role of IL-22.
EXAMPLE 8: IL-22 is produced by γδ T-cells
In order to analyze the expression of IL-22 in γδ T-cells, cells were isolated from the CE is esence mouse and γδ T cells were separated MACS-sorting device. GL4 is an anti-γδ TCR antibody that specifically activate γδ T cells (Becton-Dickenson). Was used kit for RNA extraction Qiagen MINI to isolate RNA from cells to 5' nuclease analysis (TaqManTM). Master Mix for one-step RT-PCR Master Mix Reagent (Applied Biosystems; 4309169) was used, and autogeny RPL10 and SPF31 were used for normalization. Were used whole splenocytes to determine the relative level of expression of IL-22. In Fig. 13 shows that IL-22 vysokoaktivnye in γδ T-cells stimulated GL4 antibody.
EXAMPLE 9: IL-22 is produced by activated human T cells
The microchip for the nucleic acids are used to identify differentially expressed genes in diseased tissues compared with their normal counterparts. Using the microchip for nucleic acids, study and control samples of mRNA from the study and control tissue samples are subjected to reverse transcription and mark for obtaining cDNA samples. Sample cDNA then hybridized to chips of nucleic acids immobilized on solid media. Chips change conformation, so that the known sequence and position of each member of the chip. For example, the selected genes for which it is known that they are expressed in a disease state can be netwarcom media. Hybridization of the labeled sample with a specific member of the group shows that the sample from which was obtained the sample, expresses this gene. If the hybridization signal samples from the sample (in this example, the activated CD4+ T cells) more hybridization signal samples from the control sample (in this example, unstimulated CD4+ T cells)are identified gene or genes hyperexpression in the examined tissue. This result means that hyperexpression protein in the test tissue is not only used as a diagnostic marker to determine the status of the disease, but also as a therapeutic target for the treatment of a disease state.
The method of hybridization of nucleic acids and method of microarrays are well known in this field. For example, the specific preparation of nucleic acids for hybridization and sample preparations and conditions for hybridization are described in detail in PCT patent application number PCT/USO 1/10482, registered on March 30, 2001, and which is incorporated in this description by reference.
In this experiment, CD4+ T cells were purified from a single donor using RossetteSepTMProtocol from Stem Cell Technologies (Vancouver BC), in which use anti-CD8, anti-CD16, anti-CD19, anti-CD36 and anti-CD56 antibodies used to identify CD4+ T cells. Selected CD4+ T cells activated by anti-CD3 antibody is used at a concentration of, which did not stimulate proliferation) together with either ICAM-I or anti-CD28 antibody. After 24 or 72 hours cells were harvested, RNA was isolated and run the analysis on the microarray chips Affimax (Affymetrix Inc., Santa Clara, CA). Estimulando (resting) cells were collected immediately after treatment and subjected to the same analysis. Compared genes whose expression was increased in either one or the other of the two time points in activated compared with resting cells.
The results of this experiment are shown in Fig. 14. The microarray results confirm and approve the data in example 7. Th1 T cells produce large amounts of IL-22 stimulation in contrast, Th2 cells that produce IL-4 or IL-5. This result probably will allow you to split Th1 and Th2-related immune disorders, based on the cytokine profile. Th1 cells expressing IL-22 and IFN-γ, can be treated with therapy directed at these cytokines without affecting the population of Th2 cells.
EXAMPLE 10: Th1 cells Express intracellular IL-22
To determine the level of expression of IL-22 in T-cells by FACS, performed intracellular staining of mouse Th1/Th2 cells. Primary splenocytes were divided into Th1 or Th2. For FACS staining, 1 million cells were seeded per well of 96-hole tablet, and were treated with PMA/Ionomycin for 2 hours, then DL the other Brefeldin A for 2 hours. Used antibody was an anti-human IL-22 antibody 3F11.1) and anti-gp120 as a control. Antimachine IFN-γ-FITC and antimachine IL-4-PE were obtained from BD Bioscience (San Diego CA). PE-conjugated goat antimurine IgG (from BD Bioscience) were used as secondary antibodies. The cells were fixed with 2% paraformaldehyde for 30 minutes, then washed twice in PBS/2% FCS. Cells resuspendable in 0.5% saponin for 15 minutes, then added antibodies 0.5 μg/million cells for 30 minutes. The cells were then washed twice and added to the secondary antibody in 0.5% saponin for 15 minutes. Finally, cells were washed and resuspendable in FACS buffer and analyzed on FACScan. On the upper panels of Fig. 15 it is shown that Th1 cells can be distinguished from Th2 cells. Th1 cells are positive for IFN-γ, is negative for IL4 and positive for IL-22. Th2 cells are largely negative for IFN-γ, is positive for IL4 and negative for IL-22.
EXAMPLE 11: Receiving anti-IL-22 receptor (IL-22R)
To investigate the binding of anti-IL-22R antibodies, using 293 cells expressing ML-22R, and cells expressing GFP. One million cells were stained with various anti-hIL-22R antibodies at a concentration of 0.3 µg/million cells. The investigated antibodies represented 7E9, 8A12, 8H11 and 12H5. Secondary antibody was goat artemisinin PE conjugated (Jackson Las) used the dilution ratio is 1:200. Cells were washed and stained in FACS buffer (0,5% BSA/PBS). Staining of the investigated antibodies was carried out for 15 minutes at 4°C, then cells were washed and added to the secondary antibody for 15 minutes at 4°C. Cells were washed twice before FACScan analysis. The results are presented in Fig. 16. For each graph, where the peaks do not overlap, the peak on the left correspond to the control, and the peaks on the right correspond to the investigational antibody. In Fig. 16 shows that all four of the studied anti-IL-22R antibodies were positive for binding to IL-22R in tropicabana 293 cells. Antibodies 7E9, 8A12, 8H11 and 12H5 good contact with very little background.
EXAMPLE 12: IL-22R blocking antibodies
To study the blocking activity of anti-IL-22R antibodies, was used luciferase reporter construct (as described in example 2). If the antibody blocks the activity of STAT3 will not be activated, and the luciferase signal will be low. Cells expressing hIL-22R/hIL10Rb were planted in 0.2×106/well of a 24-hole tablet, and luciferase reporters TK-SIE-SRE-S (0.8 µg/well) and RL-TK-Luc (0.16 µg/well) were transliterowany in cells. The next day, was added hIL-22 0.5 nm in the wells, and each antibody was added 20 μg/ml Investigated anti-IL-22R antibodies were 7E9, 8A12, 8H11 and 12H5. Used control antibodies were G120 and 11H4, anti-hIL-22 antibody showed blocking activity in example 2. Sixteen hours later, cells were literally and the samples were evaluated on the luminometer to determine the luciferase activity. In Fig. 17 shows that all four investigated anti-IL-22R antibodies to block IL-22R-IL-22 interaction.
EXAMPLE 13: IL-22R is expressed in primary keratinocytes
Keratinocytes represent a cell population, which will hyperproliferate during psoriasis. Therapy targets keratinocytes is used to reduce psoriasis. The expression of IL-22R on primary human keratinocytes was determined by FACS analysis. Normal human epidermal keratinocytes (NHEK) donor 0526 were obtained from Cascade Biologies, passage #2, grown to 80% density, and were painted 300-600K cells in the sample. Anti-IL-22R serum was used at a dilution of 1:50 and pravidelnou serum was used at a dilution of 1:50 as the control. For IL10R2 staining, antibody from R&D (clone #90220, mouse IgG1) used 0.3 ág per sample with mouse IgG1-PE isotype control (BD Pharmingen #33815X). Secondary antibody for anti-IL-22R serum was mouse anti-mouse IgG1-PE (BD Pharmingen #550083)used 0.1 ág per sample. In Fig. 18 shows that IL-22R and IL10R2 is expressed in NHEK. Therefore, blocking of IL-22R or IL-22 may be useful to attenuate disturbances associated with hype the proliferation of keratinocytes, such as psoriasis.
EXAMPLE 14: Effect of IL-22 on epidermal culture
Restored human epidermis (RHE) can be used as a model for the action of cytokines on the skin. RHE and culture medium were obtained from MatTek Corporation (Ashland, MA). RHE balanced all night (20-22 hours) with 0.9 ml of medium at 37°C, 5% CO2to remove the load before the start of the experiment and then were cultured in the contact area of the air/liquid in 5 ml of medium at 37°C, 5% CO2. The effect of IL-22 on RHE investigated using three different States. IL-22 (1.2 nm) or epidermal growth factor (EGF-R&D systems) (1 nm) was added in Wednesday. The control consists of a raw environment. RHE were cultured for 4 days with changes of medium every two days, adding fresh EGF or IL-22. RHE was removed, fixed in 10% neutral buffered formalin (NBF) all night, were separated and stained with hematoxylin and eosin (H&E). In Fig. 19 shows that the processing of IL-22 induces thinning of the epidermis. This shows that IL-22 induces hyperplasia or proliferation of cells that form the epidermis.
If these parts were painted on the keratin 16 (K16), a marker of proliferation of keratinocytes, RHE treated IL-22 showed more vivid colouring to K16. K16 are only expressed in proliferating cells of the skin, such as psoriasis and wound healing (reviewed in Freedberg et al, Soc. Invest. Derm. 116:633-640 (2001)). The piano is, 20 shows K16 staining in the treated IL-22 RHE relative to RHE raw and processed EGF. IL-22 waste RHE showed K16 throughout the tissue, because the staining is localized in untreated and EGF-treated parts.
Processing RHE IL-22 promotes psoriasin, hyperexpression gene for psoriasis. Psoriasin (S100A7) was originally discovered as a protein expressed in psoriasis, but not in normal skin (Madsen P., et al., J. Invest. Derm. 97: 701-712 (1991)). Psoriasin is expressed in activated cultured and malignant keratinocytes and malignant epithelial breast cancer cells (Watson et al., Int. J. of Biochem. and Cell Bio. 30:567-571 (1998)). Current evidence supports the role of psoriasin in inflammatory diseases of the skin, chemotaxis and progression of breast tumors. Correlation of psoriasin with psoriazopodobne hyperplasia of the skin assumes the role of differentiation in keratinocytes. Psoriasis can also be chemotactic, stimulating the infiltration of neutrophils and CD4+ T-lymphocytes of the epidermis, which is the hallmark of psoriasin. In Fig. 21 shows that the processing of RHE IL-22 induces high levels of expression of psoriasin. This result confirmed that IL-22 and IL-22R play a role in psoriasis.
The inducing effect of IL-22 ways to psoriasin can be blocked by antibodies directed to IL-22 or IL-22R. Anti-IL-22 antibody 8E1, introduced at a concentration of 20 μg/ml, reduced the expression of psoriasin to undetectable levels (see, Fig. 23). When used at a concentration of 20 μg/ml, anti-IL-22R antibody (7E9) also significantly reduced the expression of psoriasin, as shown in Fig. 23.
Anti-IL-22 and anti-IL-22R antibodies were investigated to determine, could they reduce epidermal thinning observed in the processing of RHE IL-22. Anti-IL-22 antibody (8E11), introduced at a concentration of 20 μg/ml, showed a significant decrease in epidermal thinning (see Fig). RHE treated with IL-22, reaches a thickness of 80 to 90 μm, and processing of anti-IL-22(8E11) antibodies reduces RHE thickness of 50-60 μm (Fig. 25). Anti-IL-22R antibody (7E9) also reduces the thickness of the skin. When using a concentration of 20 µg/ml anti-IL-22R antibodies reduce RHE thickness of from 80-90 μm to 55-60 μm (Fig. 25). These data indicate that anti-IL-22 or anti-IL-22R antibodies can alleviate the symptoms associated with psoriasis, such as epidermal proliferation and thickness.
EXAMPLE 15: Use of microarray genes induced by IL-22
To determine what genes were stimulated by IL-22, normal human epidermal keratinocytes (NHEK) were obtained from a single donor, dissipated and was treated at 70% density within 24 hours and 20 ng/ml IL-22. Medium and supplements (EpiLifeR+HKGS) were obtained from Cascade BiologicsTM(Portland, OR). Cells were washed and were literally. Total RNA was purified from NHEK cells using Qiagen RNeasy Mini Kit. RNA was subjected microcinema analysis and quantified gene expression (see the above example 9 to describe microcapsule analysis).
Psoriasin stimulated 81 fold induction of IL-22. SPR-2G increased 11 fold (see Fig. 22.). These results show that IL-22 is involved in psoriasis. Therefore, antagonists and antagonists, antibodies directed against IL-22 or IL-22R, used for relief of psoriasis.
EXAMPLE 16: IL-23 causes the hallmarks of psoriasis in vivo
A murine model was used to compare the ability of IL-12 and IL-23 induce psoriatic skin symptoms. C57B1/6 mice were injected subcutaneously in the ear 500 ng of either recombinant IL-12 or recombinant IL-23 in total volume of 20 μl PBS. Control mice were injected 20 μl of PBS only. The mice were injected once every 2 days for 16 days. Each experimental group consisted of five mice. The ear thickness was measured before and at multiple time points after injection with a caliper (Mitutoyo America Corporation) and reported as mean +/- standard deviation. For this experiment and subsequent experiments, statistical significance was considered one way or two way ANOVA using Prism software (GraphPad). All p values <0,05 were considered significant. The ears of the mice were collected standard GI is tological analysis using hematoxylin-and-eosin (H& E) staining.
As shown in Fig. 26A, and IL-12 and IL-23 introduction causes a significant increase in the thickness of the ears within one week after the first injection. For mice, preserved IL-12, p was <0,001 (days 12, 14 and 16 against PBS control, respectively). For mice, preserved IL-23, p was <0,001 (days 8, 12, 14 and 16 against PBS control, respectively). Histological analysis showed that IL-12 and IL-23 entered on the ears develop prominent inflammatory cell infiltration and epidermal thinning (acanthosis) compared to PBS treated control group; however, there were several precise histological differences between the two groups. First, IL-12 caused a moderate change acanthosis with noticeable predominance dermal mononuclear inflammatory cell infiltration (Fig. 26 D, E) compared with the PBS control group (Fig. 26B, C), since IL-23 caused a marked acanthosis with a mixed dermal inflammatory cell infiltration many polymorphonuclear leukocytes (Fig. 26F, G), including neutrophils (arrows) and eosinophils. Epidermal hyperplasia and the presence of polymorphonuclear leukocytes are the histological hallmarks of psoriasis in humans, as well as the most simple histological detection in the mouse model of psoriasis. Cm. P. C. van de Kerkhof et al., Dermatologica 174: 224 (1987) and P. R. Mangan et l., Nature (2006) 441:235.
EXAMPLE 17: IL-22 acts after IL-23 in vivo
To identify cytokines that are potentially inhibit IL-12 or IL-23, used real time PCR to determine the expression of a panel of cytokines from skin samples of each ear, which was administered to IL-12 or IL-23. Injection into the skin of the ears and histological analysis was performed as described in the previous example. On the 8th day of the experiment, RNA was isolated from each mouse ear and conducted real-time PCR to quantify the levels of mRNA encoding IFN-γ, IL-17 and IL-22. Specifically, RNA was isolated RNeasy Mini Kit (Qiagen, Valencia, CA) according to manufacturer's instructions. RT-real time PCR was performed using the ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA) with primers and probes using TaqManTMOne-Step RT-PCR Master Mix reagents (Applied Biosystems). The reaction was carried out twice and samples were normalized to the control gene household RPL-19 and reported according to the ∆∆Ct method.
As shown in Fig. 27A, IL-12 caused a significant increase in IFN-γ expression in the ear, eight days after the first injection. IL-23 was caused by the production of IL-17 and inhibited IFN-γ production relative to PBS-treated control group (Fig. 27A). Interestingly, IL-22 was also significantly increased after injection of IL-23, but not after injection of IL-12 (Fig. 27A). These data suggested a link between IL-23 and IL-22.
H is usually used to confirm what cytokines were producirovanie lymphocytes, which were unfilterable ears, lymphocytes were suirvey of treated ears, and cytokine production was measured after activation by ELISA. In accordance with the data of RT-PCR in real time, cells from IL-23 injected ears preferably has produced IL-22 and IL-17, although cells from IL-12 injected ears secretively a large number of IFN-γ (Fig. 28).
EXAMPLE 18: IL-22 induces dermal inflammation and epidermal hyperplasia in vivo
To determine whether IL-22, like IL-23 that can cause psoriatic skin symptoms in vivo, mice were injected with subcutaneously in the ears IL-22 or PBS only, as described above in example 16. As shown in Fig. 27B, IL-22 has caused a significant increase in the thickness of the ears relative to the PBS-treated group. IL-20, another cytokine of the IL-10 family, causes only very mild and localized increase in thickness of the ears. This discovery contradicted the previous message, where epidermal transgenic overexpression of IL-20 caused a marked epidermal hyperplasia, which suggested that IL-20 can potentially play a role in epidermal function and psoriasis. Cm. Blumberg et al, Cell 104:9 (2001). Histological analysis showed that the treated IL-22 ears of mice have similar histological appearance and ears treated IL-23 group, shows the Oh in Fig. 26F and G, showing a marked acanthosis, and a mixed dermal inflammatory cell infiltration (Fig. 27G, H), including many neutrophils (arrows) and some eosinophils. In contrast, treated with IL-20 ears had only moderate focal acanthosis with moderate and highly focal mixed inflammation (Fig. 27D, E) relative to the PBS-treated group (Fig. 27C, F). These data suggested that IL-22 is essential for IL-23-induced dermal inflammation and acanthosis.
EXAMPLE 19: Anti-IL-22 blocking antibody significantly reduced IL-23-induced acanthosis
To confirm that IL-23 acts through IL-22 to cause psoriatic skin symptoms was investigated the effect of anti-IL-22 monoclonal antibody 8E11 on IL-23-induced dermal inflammation and acanthosis. The mice were injected subcutaneously in the ears IL-23, or PBS, as described above (example 16), except that the injection was made after 14 days. Mice were injected intraperitoneally 8E11 or control monoclonal antibody IgG1 isotype at a concentration of 200 μg per mouse, and the frequency of once every two days for 14 days. On day 14, collected the ears of mice for histological analysis using H&E staining.
As shown in Fig. 29A, 8E11 ("anti-IL-22 mAb") significantly reduced IL-23-induced epidermal acanthosis (*p<0,001) regarding the processing control is pushed IgG1 antibody. (Compare also figure 29D, and E (anti-IL-2 mAb) with B and C (control IgG1)). Next, mice treated with anti-IL-22 mAb also showed a moderate decrease in dermal inflammation. However, mice treated with anti-IL-22 mAb, still showed a moderate inflammatory cell infiltration compared to the skin of the ears treated with PBS. (Cf. Fig. 29D and E (anti-IL-22 mAb) with F and G (PBS)).
EXAMPLE 20: IL-23-induced acanthosis significantly decreased in IL-22 deficient mice
To further confirm that IL-23 acts through IL-22, causing psoriatic skin symptoms, the effects of IL-23 and on wild-type and IL-22 deficient mice was investigated. IL-22 deficient mice (e.g., homozygous IL-22 knockout mice, called "IL-22" mouse") were obtained by the destruction of the target genes according to the strategy depicted in Fig. 30A. The exons 1-4 (closed boxes) IL-22 coding sequences were replaced by cassette resistance to neomycin, flanked by loxP sites. Heterozygous mice carrying a specific allele were crossed with a transgenic line in which the Protamine 1 (Prm) the promoter ran the Cre recombinases. Dependent allele was cut during spermatogenesis in a mixture of heterozygous males (i.e. heterozygotes for the dependent allele and PrmCre of trangene). A mixture of heterozygous males were crossed with females of the wild type, and the resulting offspring was screenrow is but cross allele and reduced PrmCre transgene. Descendants were back crossed with C57B1/6 background for at least six generations. Mouse genotypes were confirmed by PCR using the primers shown in Fig. 30B.
IL-22 expression was investigated at the levels of mRNA and protein in Th cells of wild-type and IL-22-/-of mice. IL-22 mRNA expression was investigated in Th1, Th2 and ThIL-17cells of wild type ( + / + ) and IL-22-/-("-/-") mice (Fig. 30C), using RT-PCR, confirming that IL-22 mRNA is not detected in IL-22-/-of mice. The expression of IL-22, IL-17, IFN-γ and IL-4 was investigated in Th1, Th2 and ThIL-17cells from wild type ("WT") and IL-22-/-("KO") mice using ELISA. The results are shown in Fig. 30D for each IL-22, IL-17, IFN-γ and IL-4, as shown at the top of each graph, the shaded bars and open bars showing the levels of expression in WT and KO mice, respectively. Additionally, CD4 T cells from IL-22-/-mice were able to be activated and differentiate in all subpopulations of T-helper cells and is able to produce normal levels of IL-17, IFN-γ and IL-4 relative to wild-type CD4 T cells. As expected, however, IL-22 was absent in IL-22-/-CD4 T-cells. Received IL-22-/-mice that developed normally and had a similar composition of lymphocytes in all major lymphoid organs, considered in relation to wild-type mice (data not presented the ENES).
IL-22-/-mice and pups from the same litter wild type was administered by subcutaneous injection in the ears IL-23, or PBS, as described above (example 16). On day 16, the ears of the mice were analyzed by standard histological analysis. As shown in Fig. 31A and B, IL-23 caused a significantly lower ear epidermal thinning and thinning from IL-22-/-mice compared with control groups. (IL-22-/-a mouse named on this Fig. and Fig. 32 "KO" or "IL-22 KO"; mouse wild-type named in this Fig. and Fig. 32 "WT" or "IL-22 WT.") Histological staining, and epidermal acanthosis and dermal inflammation were significantly reduced in IL-22-/-mice (Fig. 31E and F, respectively) compared with IL-23-treated wild-type pups from the same litter (Fig. 31C and D, respectively). In contrast to these results, IL-22 deficiency does not affect IL-12-induced ear skin inflammation in all (Fig. 32). Therefore, the data show that IL-22 plays a significant role in dermal inflammation and epidermal acanthosis called IL-23 and not IL-12.
EXAMPLE 21: IL-23 induces IL-22 production in various IL-23-activated lymphocytes
Then investigated the ability of IL-23 to induce IL-22, distinct populations of lymphocytes were isolated and stimulated in vitro under the conditions shown in Fig. 33. Performed ELISA to determine IL-22 in the culture supernatant and reported Raza is tatah in Fig. 33 A as mean +/- standard deviation. The ability of IL-23 to induce IL-10 family of cytokines, but not IL-22, was also investigated. Splenocytes from DO 11.10 TCR transgenic mice were stimulated with 0.3 μm OVA peptide at the indicated conditions of polarization of T-helper cells for 4 days, then made a break for two days and re-stimulated associated with plate anti-CD3 (10 μg/ml) and soluble anti-CD28 (5 μg/ml) for the other 2 days. Conducted RT-real time PCR on RNA extracted from the cells in the specified conditions for the quantitative determination of expression of murine IL-19, IL-20 and IL-24. RNA from normal mouse splenocytes were also included as control. As shown in Fig. 33B, IL-23 did not cause the expression of any other investigational IL-10 family of cytokines.
EXAMPLE 22: IL-22 is a new effector cytokine of ThIL-17
Recently, IL-23 has been associated with the development of a new IL-17 producing effector CD4+ T-cell lines (ThIL-17). L. E. Harrington., Nat. Immunol 6:1123 (2005); H. Park., Nat. Immunol. 6:1133 (2005). IL-23 is able to induce ThIL-17cells from native CD4+ T cells in the presence of APC and antigen, but not able to induce IL-17 production when used natively purified T cells activated by anti-CD3/anti-CD28. L. E. Harrington et al., Nat. Immunol 6: 1123 (2005); M. Veldhoen et al, Immunity 24: 179 (2006). Moreover, it was suggested that TGF-β and IL-6 are de novo factors for which ifferentiate Th IL-17subpopulations. M. Veldhoen et al., Immunity 24:179 (2006).
Experiments can be performed to study whether IL-22 to be additional effector T cell cytokine induced IL-23, with additional stimulation of the TCR. CD4+ T cells from DO 11.10 TCR transgenic mice were activated by 0.3 μm OVA peptide for four days under the conditions of the Th1-polarizing (IL-12 and anti-IL-4), Th2-polarization (IL-4, anti-IL-12 and anti-IFN-γ), ThIL-17-polarization (IL-23, anti-IFN-γ and anti-IL-4) or Th0 (anti-IL 12/23 p40, anti-IFN-γ and anti-IL-4), as previously described. L. E. Harrington et al, Nat Immunol 6:1123 (2005). RNA was isolated from cells and performed real-time PCR to determine the expression of mRNA that encodes a different mouse cytokines (shown above the graphs in Fig. 34A). In addition, ELISA was performed on the culture supernatant to determine the expression of various cytokines at the protein level. As shown in Fig. 34A, IL-17 was induced IL-23, whereas IFN-γ and IL-4 were producirovanie Th1 and Th2 cells, respectively. IL-22 was obtained at the level of mRNA and the protein level of IL-17 producing ThIL-17cells.
To determine whether IL-22 new efactory cytokine from fully commitirovannah ThIL-17 line, polarized T cells, as described above, rested two days and then re-stimulated for two days associated with plate anti-CD3 (10 μg/ml) and soluble anti-CD28 (5 μg/ml) in the absence and presence of IL-23. Performed ELISA to determine the expression of murine cytokines shown above the graphs in Fig. 34B. The results show that IL-17 is produced specifically ThIL-17the subpopulation, even in the absence of IL-23, and IL-23 increased IL-17 production. IL-23 activates IL-17 production commitirovannah Th1 and Th2 cells. IL-22 showed identical to model the expression of both IL-17, indicating that IL-22 is really represented effector cytokine downregulation of this new ThIL-17the subpopulation.
Previously reported that IL-23 receptor can be expressed on activated T-cells memory. C. Parham et al, J Immunol 168:5699 (2002). The above experiments do not exclude the possibility that IL-23 is acted on T-memory cells for the production of IL-22. To treat this more critically, the above experiments were repeated using native CD4+ T cells isolated from DO 11.10 TCR transgenic mice. Specifically, CD4+ T cells from Rag2-/-DO 11.10. TCR-transgenic mice stimulated with OVA peptide-pulsed BALB/c cells of the channels of the spleen (irradiated depleted T-cells) for 72 hours in ThI-polarizing conditions (IL-12 and anti-IL-4), Th2-polarizing conditions (IL-4, anti-IL-12 and anti-IFN-γ), ThIL-17-polarizing (IL-23, anti-IFN-γ and anti-IL-4) or other conditions, as shown in Fig. 35A. As shown in this drawing, ThIL-17the cells were produced the highest levels of IL-22, although Th1 also secretarial certain levels of IL-22. Further, the addition of either IFN-γ or IL-4 completely stopped the production of IL-17; however, these two cytokines is only moderately inhibited the production of IL-22 (Fig. 35A). These data suggest potentially different ways of inducing the expression of IL-17 against IL-22. However, more fully shown ThIL-17the cells were produced and IL-17 and IL-22 after re-stimulation for 48 hours in these secondary conditions (Fig. 35B). IL-23 was further increased levels of these cytokines in the way that cannot be blocked, neither IFN-γ nor IL-4 (Fig. 35B). These data confirm the stability of this ThIL-17 line.
To further investigate whether produced IL-17 and IL-22 in the same cells during activation, ThIL-17cells were stimulated by PMA and ionomycin, and antibodies to IL-22 or IL-17 were used for intracellular staining. As shown in Fig. 35C, IL-17-producing cells mainly worked on ThIL-17axis (left panel). IL-22-producing cells also preferably detected from the line ThIL-17(right panel). The content and IL-22 and IL-17 showed that a significant portion of the cells of ThIL-17line and produces IL-22 and IL-17 at the same time, showing that IL-22 and IL-17 are produced by the same cells.
As discussed above, recent studies have also shown that other factors of the APC may be the ü primary driving force behind the differentiation of IL-17-producing T cells from native CD4+ T cells, since IL-23 is not able to induce de novo IL-17 production of purified native CD4 T cells. M. Veldhoen et al., Immunity 24: 179 (2006). Two of these factors are critical for the production of IL-17, from native CD4 T cells were identified as TGF-β and IL-6. Id. To determine was whether these factors are also critical for the production of IL-22 in mice, purified native CD4 T cells (>98%) stimulated associated with plates anti-CD3 (10 μg/ml) and soluble anti-CD28 (5 μg/ml). In accordance with published data, TGF-β and IL-6 but not IL-23 was caused by the production of IL-17 (Fig. 36A, right panel). Surprisingly, in contrast to the induction of IL-17, IL-22 could be the induction only in the presence of IL-23 and may not be the induction of TGF-β and IL-6 (Fig. 36A, left panel). These data suggest that transcription of IL-17 and IL-22 may be regulated differently. However, as previously reported, TGF-β and IL-6 can not maintain long-term IL-17-producing T line cells without IL-23 (Fig. 36B). Thus the data show that IL-23 may be one of the main factors stimulating the production of IL-22 subline T cells.
Next, we researched what similar IL-22 producing T line cells can be preserved from human CD4 T cells. We found that IL-23 can induce the secretion of IL-22 from purified native human CD4+ T cells stimulated with anti-CD3/anti-CD28 at ThIL-17-polarizing conditions (Fig. 36C, l is the first panel). These cells can produce IL-22 again when re-stimulation without the addition of exogenous IL-23 (Fig. 36C, right panel), showing the formation of a stable T-cell lines. Although these cells were cultured in similar conditions, such as the above studies in mice, we were unable to determine the production of IL-17 in connection with limitation (data not shown).
In conclusion, the data obtained for the first time that IL-23 can induce IL-22-producing T-cell subline from mouse and human native CD4 T cells. The production of IL-17 this line depends on other factors of the environment. Meanwhile, authentic antigen and APC stimulating conditions, IL-23 drives T-cell subline, producing and IL-22 and IL-17. IL-23 also stimulated IL-22 production, if native T cells were stimulated by anti-CD3 and anti-CD28. TGF-β and IL-6 can induce a temporary production of IL-17 from native T cells, but was short-lived commitlimit, not influencing the production of IL-22.
EXAMPLE 23: IL-19, IL-20 and IL-24 also cause epidermal thinning
IL-22 belongs to the family of cytokines, which include IL-19, IL-20 and IL-24, all of which show increased expression in psoriatic skin. These cytokines were also investigated to determine whether they are similar to IL-22, is able to induce epidermal hyperplasia and acanthosis. RHE cultiv is listed for four days and was treated with 20 ng/ml IL-19, IL20, IL-22 or IL-24 or 6 ng/ml EGF. Processed RHE were stained with H&E. the Results are shown in Fig. 37A. All cytokines caused contos viable containing nuclei of the epidermis, marked by the increased length of the double arrows. In accordance with previous observations (above), IL-22 induces hypogynous or decrease in the granular cell layer (arrow pointer), and hyalinization below the base (asterisks). IL-22 also caused parakeets in RHE, kultivirovanii within 7 days (data not shown). Hypogynous and parakeets are frequent histological signs of psoriasis. IL-19, IL-22 and IL-24 was the only epidermal acanthosis with a small or negligible effect on either granular cell layer, or on the ground. EGF caused epidermal acanthosis with hypergranulation and seal of keratinocytes in the stratum granulosum (arrow). Epidermal thinning, caused by IL-19, IL-20, IL-22 or IL-24, was quantified in independent experiments and are presented graphically in Fig. 38. IL-22 had the greatest effect. Inflammatory cytokines TNF-α, IFN-γ and IL-1β, which are believed to play a role in psoriasis, do not stimulate the proliferation of keratinocytes in this RHE system (data not shown). Thus, these cytokines may play a secondary role in psoriasis, or can play a role through hung the range of the path of the IL-19, IL-20, IL-22 and/or IL-24.
Used immunohistochemistry to determine the cytokeratin 16 (CK16), a marker of epidermal hyperplasia. IL-24, IL-22 and EGF induced CK16 the expression of all non-squamous epidermis, although only IL-19 and IL-20 induced CK16 expression in basal area (Fig. 37B).
The immunohistochemistry was also used to determine psoriasin (S100A7), one of several proteins of the S100 family, are increased when some of hyperproliferative and inflammatory skin conditions, including psoriasis. IL-19, IL-20, IL-22 and IL-24 are all induced S100A7 expression in suprabasal the epidermis, IL-22 and IL-24 has the greatest effect (Fig. 37C). S100A7 staining got in the nucleus and cytoplasm of keratinocytes, apparently, also with some protein, which is extracellular. The results shown in Fig. 37B and C were quantified and shown graphically in Fig. 37E and F.
The immunohistochemistry was also used to determine pY(705)-STAT3, transactionmode form of STAT3. It has been shown that activated STAT3 increased in damaged skin psoriasis. IL-19, IL-20, IL-22 and IL-24 are all induced persistent activation of STAT3 in the RHE keratinocytes found in all layers of living cells, showing nuclear localization (Fig. 37D).
EXAMPLE 24: Blocking antibodies, receptors for IL-20 and IL-22 reduce the expression of psoriasin
And IL-19 and IL-20 transmit signal is al by heterodimer receptor IL20Ra and IL20Rb. IL-20 also transmits a signal through heterodimer receptor IL-22R and IL-20Rb. IL-22 signals through heterodimer IL-22R and IL10R2. The expression of these components of the receptor on the surface of keratinocytes isolated from RHE or primary cultures of normal human epidermal keratinocytes (NHEK, from the donor neonatal foreskin), was investigated by flow cytometry. For flow cytometry were used following monoclonalny antibodies: anti-IL20Ra (obtained from mice for the purposes of this study); anti-IL20Rb (obtained from mice for the purposes of this study); anti-IL-22R antibody 7E9 (described above); and anti-IL-10R2 FAB874P (PE-conjugated) (R&D Systems, Minneapolis, MN). The results are shown in Fig. 39. Component of the receptor, which binds the antibody, shown in the upper right corner of each graph (IL-22R is referred to as "IL-22R1"). IL-20Rb and IL10R2 constantly expressionlist on the surface of NHEK, despite the degeneracy, the number of passages or levels of calcium in the medium (Fig. 39A.). In contrast, the expression of IL-20Ra and IL-22R1 on the surface of NHEK varied from donor to donor and was constantly low, but detectable levels (Fig. 39A and data not shown). Compared to levels of expression in monolayer NHEK, IL-20Ra and IL-22R expressibility in much higher levels in keratinocytes isolated from RHE (Fig. 39B). The reasons for this RA is unknown differences. However, it is obvious that all the analyzed receptor components are expressed in human keratinocytes. The expression of these receptor components on immune cells (T cells, B cells, natural killer cells and monocytes) was not determined (data not shown). Thus, apparently, the ligands for this receptor components provide a link between the immune system and disorders in keratinocytes.
To explore whether the above-described antibodies to block the effects of IL-19, IL-20 and IL-22, as described in the previous example, to RHE culture medium was added 20 organisms/ml anti-IL20Ra, anti-IL20Rb or anti-IL-22R for one hour before adding 20 ng/ml IL-19, IL-20 or IL-22. Then within four days the cells RHE cultured in a medium that was changed on the second day (of 4.5 ml of fresh medium, including cytokine and antibody). Then RHE immunohistochemically were stained for psoriasin (S100A7). The results are shown in Fig. 40. IL-19, IL-20 and IL-22-treated RHE shown in the first, second and third lines, respectively. The preprocessed anti-IL20Ra (αIL-20Ra), anti-IL20Rb (αIL-20Rb) or anti-IL-22R (αIL-22R1) cells RHE shown in the third, fourth and fifth columns, respectively. Controls without antibody and control antibody isotype shown in the first and second columns.
The result shows that either anti-IL20Ra, or anti-IL20Rb effectively lock is whether IL-19 induced expression of psoriasin. Similarly, anti-IL-22R effectively blocked IL-22-induced expression of psoriasin. Anti-IL20Rb effectively blocked IL-20-induced expression of psoriasin and anti-IL20Ra not. Similarly, anti-IL-22R could not block IL-20-induced expression of psoriasin.
To further investigate the effects of anti-IL-22R and anti-IL20Ra on IL-20-induced expression of psoriasin, spent preprocessing RHE these antibodies, either individually or in combination to the processing of IL-20. The results are shown in Fig. 41. As described above, neither anti-IL-22R or anti-IL20Ra themselves could not block IL-20-induced expression of psoriasin (second column, both panels). However, the combination and anti-IL20Ra, and anti-IL-22R effectively blocked IL-20-induced expression of psoriasin, indicating that IL-20Ra and IL-22R play an additional role in the signaling of IL-20 in human keratinocytes (left panel below).
EXAMPLE 25: IL-19, IL-20, IL-22 and IL-24 cause similar characters of gene expression
To identify genes induced by IL-19, IL-20, IL-22 and IL-24, was treated RHE 20 ng/ml IL-19, IL-20, IL-22 or IL-24 for four days. Was isolated RNA, and cDNA hybridized to Affymetrix U133 Plus Gene Chips (Affymetrix, Santa Clara, CA), which contain sets on 54675 samples. Data were analyzed genes, the expression of which is increased at least 2-fold. IL-20, IL-22 and IL-24 showed similar is ROFILE gene expression. Earlier it was reported that of the 20 leader of genes normally induced by IL-20, IL-22 and IL-24, seven genes associated with psoriasis. This genes psoriasin (S100A7), S100A12, SCCA2, SERPINB4, CCL20, CD36 and Stat3.
To explore increase if psoriasin genes induced by IL-20, IL-22 and IL-24, above microcephaly analyses compared with the previous microcephalum study of psoriatic cells (Zhou et al. (2003) Physiol. Genomics 13:69-78). Because this study was performed using different microcephaly chips, compared to only in General refseqs between this study and the present study. Of the 468 refseqs, which was increased in psoriatic skin, 356 were induced IL-20, IL-22 and IL-24, and 188 of them were significant (p<0,05). Taken together, the above studies show a significant overlap between genes that are induced by IL-20, IL-22 and IL-24, and genes that are increased in psoriatic skin.
EXAMPLE 26: Deposition of materials
The following hybridoma cell line deposited at the American Type Culture Collection, 10801 University Blvd., Manassas, VA 20110-2209, USA (ATCC).
|The name of hybridoma/antibodies||ATCC No.||Deposit date|
|Anti-IL-22 (3F11.3)||PTA-7312||January 13, 2006|
|Anti-IL-22 (11H4.4)||PTA-7315||January 13, 2006|
|Anti-IL-22(8E11.9)||PTA-7319||January 13, 2006|
|Anti-IL-22R (7E9.10.8)||PTA-7313||January 13, 2006|
|Anti-IL-22R (8A12.32)||PTA-7318||January 13, 2006|
|Anti-IL-22R (8H11.33.28)||PTA-7317||January 13, 2006|
This Deposit was made in accordance with the provisions of the Budapest Treaty on the International recognition of the Deposit of microorganisms for purposes of patent procedure (Budapest Treaty). This ensures the preservation of a viable culture for 30 years after the date of Deposit. Cell line will be provided by ATCC under the Budapest Treaty, and the agreement between Genentech, Inc. and ATCC, which ensures (a) that access to the culture will be provided during the examination of a patent application to a specific Commissioner, which the Commissioner under 37 CFR §1.14 and 35 U.S.C. §122, and (b) that all restrictions on the provision to the public thus stored culture will be irrevocably removed upon granting of the patent.
the Assignee of the present application has agreed, in case of death of stored crops on Deposit or lost, or destroyed when cultivated under appropriate conditions, it will be immediately replaced on notification viable specimen of the same culture. Providing the stored cell lines cannot be explained by the license to practice the invention in contravention of the rights conferred under the authorization of any government in accordance with its patent laws.
It is believed that the foregoing description is sufficient to enable a skilled person skilled in the art to apply the invention. The present invention is not limited to stored materials so stored as variants of implementation was intended as a simple illustration of some aspects of the invention and any constructs that are functionally equivalent are within the scope of the invention. The Deposit of the material here does not constitute an admission that the written description, enclosed in this document is insufficient to permit any aspect of the invention, including the best option, and cannot be explained by the limitation of the scope of claims for special instances in which it is presented. Indeed, various modifications of the invention in addition to presents and opican the m this description will become apparent to qualified specialists in this field from the above description and are within the scope of the attached claims.
1. The antibody that specifically binds to IL-22, comprising sequences of the variable regions specific to IL-22, where the antibody is produced by hybridoma 3F11.3 ADS Accession No. of the MOUTH-7312), where the antibody inhibits the activation of STAT3, reaching IC50at a concentration of 0.14 μg/ml.
2. The antibody according to claim 1, which is an affine Mature form of the antibody.
3. The antibody according to claim 2, which is an antigen-binding fragment of an antibody.
4. The antibody according to claim 3, which is humanitarian form antibodies.
5. The antibody according to claim 2, which is humanitarian form antibodies.
6. The antibody according to claim 1, which is an antigen-binding fragment of an antibody.
7. The antibody according to claim 6, which is humanitarian form antibodies.
8. The antibody according to claim 1, which is humanitarian form antibodies.
9. The antibody that specifically binds to IL-22 with affinity, which constitutes less than 1 nm, containing sequences of the variable regions specific to IL-22, where the antibody is produced by hybridoma N ATS Accession No. of the MOUTH-7315).
10. The antibody according to claim 9, which is an affine Mature form of the antibody.
11. The antibody of claim 10, which is an antigen-binding fragment of an antibody.
12. The antibody according to item 11, is AutoRAE is humanitarian form antibodies.
13. The antibody of claim 10, which represents humanitarian form antibodies.
14. The antibody according to claim 9, which is an antigen-binding fragment of an antibody.
15. The antibody according to 14, which represents humanitarian form antibodies.
16. The antibody according to claim 9, which is humanitarian form antibodies.
17. The antibody that specifically binds to IL-22 with affinity, which constitutes less than 1 nm, containing sequences of the variable regions specific to IL-22, where the antibody is produced by hybridoma E ATS Accession No. of the MOUTH-7319).
18. The antibody according to 17, which represents an affine Mature form of the antibody.
19. The antibody according p, which is an antigen-binding fragment of an antibody.
20. The antibody according to claim 19, which represents humanitarian form antibodies.
21. The antibody according p, which is humanitarian form antibodies.
22. The antibody according to 17, which represents an antigen-binding fragment of an antibody.
23. The antibody according to item 22, which represents humanitarian form antibodies.
24. The antibody according to 17, which represents humanitarian form antibodies.
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to biotechnology and represents OX40L antibodies which contain a Fc-fragment produced from a human body, and do not bind with a complement Clq factor. Besides vectors, cells, methods of producing the antibody, a composition and applications of the antibody for preparing a drug are presented.
EFFECT: antibodies exhibit new and patentable signs, particularly advantage to a patient which suffers inflammatory diseases.
19 cl, 7 tbl, 23 ex
SUBSTANCE: offered is a fused protein containing from an amino-terminus to carboxyl terminus: binding domain polypeptide which is bound with a biological target molecule; human one-cysteine "link" region IgGl peptide; immunoglobulin heavy chain CH2 constant region polypeptide and immunoglobulin heavy chain CH3 constant region polypeptide. The fused protein represents mixed monomers and dimers, has ADCC, CDC or both cytotoxicities. Also, described are a pharmaceutical composition and a method of treating a B-cell disorder with using the fused protein.
EFFECT: use of the invention can find the further application in medicine in treating B-cell disorders.
26 cl, 26 dwg, 9 ex
SUBSTANCE: claimed are versions of separated monoclonal antibody, specific to INNAR-1. Described are: bispecific molecule, immunoconjugate and compositions for treatment of IFNAR-1-mediated diseases and disorders based on monoclonal antibody. Also described are methods of inhibiting biological activity of type I interferons, method of treating diseases and disorders, mediated by type I interferon with application of antibody. Claimed are nucleic acid, which codes antibody, vector for antibody expression, cell, transformed by vector, as well as method of obtaining antibodies and antibody-producing hybridoma.
EFFECT: application of invention provides novel IFNAR-1 inhibiting antibodies, which block IFNAR-1 and bind its other epitope, in comparison with known antibody 64G12.
29 cl, 15 dwg, 6 tbl, 9 ex
SUBSTANCE: claimed are versions of polypeptides with Fc-segment from IgG, which possess increased binding FcRn due to introduction of mutation 308C, 308F, 308W, 308Y or modified binding with introduction of mutation 252Y/308F, 257L/308F, 257L/308Y, 257N/308Y, 279Q/308F, 279Y/308F, ^281S/308F, ^A281S/308Y, 284E/308F, 298A/308F/333A/334A, 308F/332E, 308F/311V, 308F/G385H, 308F/428L, and 308F/434Y. Described is antibody with said mutations in Fc region, as well as application of said Fc regions for obtaining fused protein. Application of the invention provides novel Fc-versions with modified FcRn binding.
EFFECT: possibility of application in medicine for obtaining various constructions, with prolonged time of preservation in blood serum in vivo or, vice versa, in case of therapy with application of radioactive medications, with reduced time of preservation in blood serum in vivo.
14 cl, 44 dwg, 4 ex
SUBSTANCE: binding molecule represents a CD45RO and CD45RB chimeric antibody. The molecule contains two domains with consistent hypervariable sites CDR1, CDR2 and CDR3, and CDR1', CDR2' and CDR3', CDR1 has amino acid sequence NYIIH, CDR2 has amino acid sequence YFNPYNHGTKYNEKFKG, and CDR3 has amino acid sequence SGPYAWFDT. CDR1' has amino acid sequence RASQNIGTSIQ, CDR2' has amino acid sequence SSSESIS, and CDR3' has amino acid sequence QQSNTWPFT. Related coding polynucleotide is described.
EFFECT: use of the invention allows to induce immunosuppression, to inhibit T-cell response and primary lymphocyte reaction in the mixed culture, to prolong survival time in mice with severe combined immunodeficiency SCID.
6 cl, 5 dwg, 2 tbl, 8 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: present invention relates to immunology and biotechnology. The invention discloses versions of a cytotoxically active CD3-specific binding structure. The structure comprises a first domain specifically binding to human CD3 and an Ig-derived second binding domain which is specific to molecules on the cell surface. The invention describes a coding nucleic acid, a vector for expressing the structure and an eukaryotic cell transformed by the vector. The invention discloses versions of compositions based on the structure for treating, preventing or alleviating various diseases and corresponding methods of treating the diseases. A method of obtaining the structure is disclosed.
EFFECT: use of the invention provides a structure with low immunological potency, which has cytotoxicity comparable to the initial structure, which may find further use in medicine.
60 cl, 18 dwg, 15 tbl, 8 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention relates to biotechnology and is an antibody which bonds with OX40L and variants of this antibody which contain certain Fc-fragments obtained from the human body and do not bond with the complement factor Clq. The monoclonal antibody is produced by a cell line selected from a group which includes cell lines deposited in the German collection of microorganisms and cell cultures (DSMZ) under inventory numbers No. DSM ACC 2685, DSM ACC 2686, DSM ACC 2688, DSM ACC 2689. The invention also relates to a method of obtaining such antibody, to nucleic acid molecules which code the disclosed antibody. The disclosed antibody has an advantage for patients suffering from inflammatory diseases.
EFFECT: antibody is used in diagnostic composition for detecting OX40L in vitro, in a pharmaceutical composition for preventing and treating inflammatory diseases, as well as in preparing a medicinal agent for preventing and treating inflammatory diseases.
9 cl, 20 dwg, 7 tbl, 23 ex
SUBSTANCE: proposed is a chimeric or humanised monoclonal antibody against hepatocyte growth factor, produced from L2G7 antibody. Invented is a mouse antibody L2G7, produced by hybridoma ATCC PTA-5162, and the said hydbridoma. Described is a cell line, producing a chimeric or humanised monoclonal antibody against hepatocyte growth factor. Proposed is a pharmaceutical composition and a method of treating tumours based on the said antibody.
EFFECT: use of the invention provides for a neutralising antibody against hepatocyte growth factor, which can be used in treating human cancer.
7 cl, 12 dwg, 1 tbl, 3 ex
SUBSTANCE: proposed is a recombinant single-strand trispecific antibody for treating tumours which express CEA. The said antibody consists of a series of three antibody fragments: anti-CEA-scFv, anti-CD3-scFv and VH CD28-antibody, linked by two intermediate linkers (intermediate linker Fc and intermediate linker HSA). If necessary, a c-myc-mark or (His)6-mark can be added at the C-end. Described is DNA, which codes the antibody, expression vector based on it and E.coli cell, containing the vector.
EFFECT: use of the invention is more beneficial in clinical use compared to bispecific antibodies and known trispecific antibodies, makes easier clearing and expression of an antibody, which can further be used in treating CEA-mediated tumours.
10 cl, 21 dwg, 11 ex
SUBSTANCE: present invention relates to biotechnology and immunology. Proposed here is a polynucleotide, encoding a cyclic single-stranded tri-specific antibody. The antibody is directed against human ovarian carcinoma in vitro, has mass of approximately 84 kD and consists of three components: an antibody against human ovarian carcinoma cells, anti-CD3 antibody and anti-CD28 antibody, which are joined together by peptide interlinks such that, they form a cyclic antibody. Invented is an expression vector, containing a coding polynucleotide and versions of E.coli host cell based on the polynucleotide and expression vector.
EFFECT: use of the invention provides for a stable antibody molecule, optimum for activation of T-cells, which can be used in curing human ovarian carcinoma.
8 cl, 12 dwg
SUBSTANCE: invention represents an IL-17-binding antibody or its active fragment. What is also presented is a DNA design coding the antibody, a method of producing the antibody, using the antibody, a pharmaceutical composition, an expression vector.
EFFECT: invention can be effectively used for preparing a drug for treating an IL-17 mediated disease or disorder.
14 cl, 2 dwg, 12 tbl, 4 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: humanised monoclonal antibody and its active fragment under the invention neutralises human IL2 activity by binding with said human IL2 prior to, during and/or after binding of said human IL2 with human IL2 receptor. A variable light chain region of said antibody contains an adherent amino acid KAPKA sequence in its second frame region, and in addition, in the CDR1-CDR3 regions, contains the amino acid sequences presented in SEQ ID NO 1-3 disclosed in the description, while a variable heavy chain region contains in the CDR1 - CDR3 regions, amino acid sequences presented in SEQ ID NO 4-6 disclosed in the description. The invention describes a polynecleotide molecule coding the antibody under the invention or its active fragment, a pharmaceutical composition based on said antibody or its active fragment exhibiting human IL2 neutralising action, and also an application of said antibody or its active fragment or the polynecleotide molecule coding it for preparing a drug which optionally contains an auxiliary anti-inflammatory or anticancer agent for treating inflammatory diseases or tumours, respectively.
EFFECT: production of the alternative specific IL2 activity inhibitors which directly bind with human IL2.
24 cl, 18 dwg, 4 tbl, 9 ex
SUBSTANCE: offered are versions of IL-13 antibodies each of which contains respectively three CDR heavy and three CDR light chains. A pharmaceutical composition of antibodies for asthma treatment is described.
EFFECT: use of the invention can find further application in therapy of the IL-13 mediated B-cell disorders.
11 cl, 6 tbl, 2 ex
SUBSTANCE: invention relates to biotechnology and represents stable and soluble scFv-antibody and Fab-fragment, specific with respect to TNFα, which contain specific sequences of light chain and heavy chain, optimised as to stability, solubility, in vitro and in vivo TNFα binding and low immunogenity. Also claimed are antibody-coding DNA-sequence, vectors, host-cell, as well as method of obtaining antibody with application of said cells. In addition claimed are therapeutic and diagnostic compositions and stable water pharmaceutically ready form based on antibodies, as well as method of treating TNFα-associated disease.
EFFECT: invention can be efficiently used for diagnostics and treatment of TNFα-associated disorders.
61 cl, 18 dwg, 5 tbl, 5 ex
SUBSTANCE: invention represents immunoglobulins, in particular antibodies, which specifically bind to human interleukin 13 (hIL-13). Claimed are recombinant or transfected mammalian host cell, for cloning, as well as for expression of vectors, coding antibodies. Claimed are antibody-containing pharmaceutical composition, set, containing said pharmaceutical composition and pharmaceutical composition, containing monoclonal antibody against IL-4, such as pascolizumab. Claimed is method ob obtaining antibody, and applications of said antibodies.
EFFECT: antibodies can be used for treatment of various diseases to disorders, responsible for modulation of hIL-13 interaction with human IL-13 receptor.
51 cl, 29 dwg, 22 tbl, 7 ex
SUBSTANCE: invention relates to molecular pharmacology and specifically to a peptide which is part of an interleukine-15 (IL-15) sequence which can inhibit biological activity of the said molecule.
EFFECT: obtaining a peptide which inhibits T cell proliferation induced by IL-15, and apoptosis caused by tumour necrosis factor when bonding with the alpha subunit of the (IL-15R) receptor.
8 cl, 4 dwg, 5 ex
SUBSTANCE: there are offered versions of human IL-13 antibodies, including based on CDR antibody BAK278D6. There is described a based composition, and also isolated nucleic acid, a host cell for preparing antibodies and versions of the method for preparing antibodies. There is disclosed application of antibodies for preparing a drug and a composition for treating various diseases mediated by IL-13 activity. Application of the invention provides antibodies neutralising IL-13.
EFFECT: applicable in medicine for preparing a vaccine.
52 cl, 32 dwg, 7 tbl, 29 ex
SUBSTANCE: there is offered a monoclonal antibody specific to human interleukine-4 (hIL-4) containing two domains with the related CDR1-3 region. There are described versions thereof that contain specified CDR, polynucleotide coding said antibody. There are described an expression vector and a host-cell for preparing the antibody to human interleukine-4 (hIL-4). There are opened: application of the antibody for preparing a pharmaceutical agent for treating the diseases mediated by interleukine-4 and/or IgE. There is discovered the pharmaceutical composition for treating the diseases mediated by interleukine-4 and/or IgE is opened.
EFFECT: application of the invention ensured the high-affinity neutralised monoclonal antibodies to human interleukine-4.
14 cl, 1 tbl, 6 ex
SUBSTANCE: invention concerns immunology and biotechnology. There is offered human monoclonal antibody specific to TNF-alpha containing light and heavy chain with appropriate CDR3 sites. There are described versions thereof including those based on heavy and light chains and coded by human genes VH3-33 and A30VK1 or VH3-53 and L2VK3 respectively. There are disclosed: the method for estimating the TNF-alpha content in the patient's sample with using specified antibodies, and application of antibodies for preparing a medical product. There are described: compositions for diagnostics and treatment of the conditions associated with TNF-alpha activity on the basis of antibodies. There is disclosed coding nucleic acid, a cell for making said antibodies and the method for making said antibodies.
EFFECT: application of the invention ensured high-affinity neutralizing monoclonal antibodies with improved Kd and IC50 in comparison with Infliximab, Adalimumab or Etanercept that can find application in medicine for treatment and diagnostics of the diseases associated with TNF-alpha hyperactivity.
35 cl, 13 dwg, 36 tbl, 14 ex
SUBSTANCE: there are disclosed polypeptide variants containing Fc-areas IgG, having amino acid modifications providing changed effector functions Fc in specified polypeptides. There is disclosed composition for antibody targeting on antigen, containing the specified polypeptide. There is described method for preparing the specified polypeptide. Also, there are disclosed the methods for treating V-cell tumour or a malignant disease characterised by V-cell expression of CD20, treating chronic lymphocytic leukosis, relieving the symptoms of the V-cell controlled autoimmune disease, treating a angiogenesis-associated disorder, treating HER2-expressing cancer, treating LFA-1-mediated involvement, treating IgE-mediated involvement wherein specified methods imply introduction to the patient of the therapeutically effective amount of said polypeptide.
EFFECT: higher clinical effectiveness.
63 cl, 6 ex, 13 dwg, 10 tbl
FIELD: biotechnology, medicine.
SUBSTANCE: Zalpha 11-ligand is isolated from cDNA library generated from activated cells of human peripheral blood that have been selected from CD3. Animal is inoculated with Zalpha 11-ligand and antibodies are prepared that are able to bind specifically with epitopes, peptides or polypeptides of Zalpha 11-ligand. Invention provides effective regulation and/or development of hemopoietic cells in vitro and in vivo. Invention can be used for preparing Zalpha 11-ligand and antibodies for it.
EFFECT: valuable properties of new cytokine.
18 cl, 5 tbl, 1 dwg, 55 ex