ISOLATED ANTIBODY OR FRAGMENT OF THE SAME THAT SPECIFICALLY LINKS TO HUMAN IL-17A, ITS USE, AND PHAR

专利摘要:
il-17a antagonists. the present invention relates to interleukin-17a (il-17a) antibody antagonists, polynucleotides encoding il-17a antibody antagonists or fragments thereof, and methods of preparing and using them.
公开号:BR112012010280B1
申请号:R112012010280-0
申请日:2010-10-29
公开日:2020-09-24
发明作者:Merle Elloso；Jinquan Luo；Michael Naso；Raymond Sweet；Sheng-Jiun Wu；Daniela Della Ducata；Robert Rauchenberger；Mark Rutz；Juan Carlos Almagro；Susann Taudte；Biugyan Wu；Galina Obmolova；Thomas Malia
申请人:Janssen Biotech, Inc.；
IPC主号:

专利说明:

[0001] [0001] The present invention relates to interleukin-17A (IL-17A) antibody antagonists, polynucleotides encoding IL-17A antibody antagonists, or fragments thereof, and methods of preparing and using them. BACKGROUND OF THE INVENTION
[0002] [0002] Interleukin-17A (IL-17A, CTLA-8, IL-17) is a cytokine secreted by activated Th17 cells, CD8 + T cells, T □□ cells and NK cells in response to cytokines such as IL-23 and TGF- β, which regulates the production of mediators such as microbicidal peptides (defensins), cytokines and pro-inflammatory chemokines from multiple cell types such as fibroblasts and synoviocytes that are involved in neutrophil biology, inflammation, organ destruction and host defense ( reviewed in Weaver et al., Annu. Rev. Immunol. 25: 82152, 2007; Aggarwal et al., J. Biol. Chem. 278: 1910-4, 2003; Mangan et al., Nature 441: 231-4, 2006). IL-17A acts in synergy with other cytokines, such as TNF-α and IL-Ιβ to enhance the pro-inflammatory environment.
[0003] [0003] The IL-17A cytokine family consists of six homologues designated IL-17A, B, C, D, E and F, each with divergent and distinct biological roles (Kawaguchi et al., J. Allergy Clin. Immunol. 114: 1265-73, 2004; Kolls and Linden, Immunity 21: 467-76, 2004; Moseley et al., Cytokine Growth Factor Rev. 14: 155-74, 2003). Of the family members, IL-17F is the most homologous to IL-17A and shares many similar functional properties such as induction of neutrophilia in the lungs and induction of pro-inflammatory cytokines; however, in humans, IL-17F is about 10 times less potent than IL-17A (Moseley et al., Cytokine Growth Factor Rev. 14: 155-74, 2003; Kolls et al., Immunity, 21: 467-76, 2004; McAllister et al., J. Immunol. 175: 404-12, 2005). IL-17A and IL-17F can also form hetero-dimers, which have intermediate bioactivity in vitro (Wright et al., J. Biol. Chem. 282: 13447-55, 2007).
[0004] [0004] IL-17A mediates its effects through interaction with interleukin-17 A receptor (IL-17RA) and C receptor (IL-17RC) (Moseley et al., Cytokine Growth Factor Rev. 14: 155- 74, 2003; Toy et al., J. Immunol. 177: 36-9, 2006). IL-17F signals through the same receptors, although IL-17F's affinity for receptors is significantly less (Kuestner et al., J. Immunol. 179: 5462-73, 2007). Crystal structures of human IL-17F and the human IL-17F / IL-17RA complex identified a putative receptor binding cavity in the IL-17F homodimer (Hymowitz et al., EMBO J. 20: 5332-41, 2001; Ely et al., Nat. Immunology 10: 1245-51, 2009). A similar cavity was identified in the crystalline structure of human IL-17A in the complex with a neutralizing Fab, although the cavity was partially occupied (Gerhardt et al., J. Mol. Biol. 394: 905-21,2009).
[0005] [0005] Inadequate or excessive production of IL-17A is associated with the pathology of several diseases and disorders, including rheumatoid arthritis (Lubberts, Cytokine 41: 84-91, 2008), airway hypersensitivity including allergic airway disease such as asthma (reviewed in Linden, Curr. Opin. Investig. Drugs. 4: 1304-12, 2003; Ivanov, Trends Pharmacol. Sci. 30: 95-103, 2009), psoriasis (Johansen et al., Br. J. Dermatol. 160: 319-24, 2009), dermal hypersensitivity including atopic dermatitis (Toda et al., J. Allergy Clin. Immunol. 111: 875-81, 2003), systemic sclerosis (Fujimoto et al., J. Dermatolog. Sci. 50: 240-42, 2008), inflammatory bowel diseases including ulcerative colitis and Crohn's disease (Holtta et al., Inflamm. Bowel Dis. 14: 1175-84, 2008; Zhang et al., Inflamm. Bowel Dis. 12 : 382-88, 2006), and lung diseases including chronic obstructive pulmonary disease (Curtis et al., Proc. Am. Thorac. Soc. 4: 512-21,2007).
[0006] [0006] Antibodies to IL-17A have been proposed for use in the treatment of IL-17A-mediated diseases and disorders (PCT application numbers: WO08 / 021156, WO07 / 070750, WO07 / 149032, WO06 / 054059, WO06 / 013107, WO08 / 001063, WO10 / 034443; US patent applications No. US2008 / 095775, US2009 / 0175881;). As the pharmacokinetic profiles, efficacy and safety of antibody therapy will depend on specific compositions, there is a need for enhanced antibodies to human IL-17A that are suitable for use in the treatment of IL-17A-mediated diseases and disorders. BRIEF DESCRIPTION OF THE DRAWINGS
[0007] [0007] Figure 1. A-H. CDR sequences from family 2, 6a, 6b, 19a and 19b of IL-17A antibody antagonists.
[0008] [0008] Figure 2. Exemplifier A) Genes of germline lineage IGLV3 and IGLJ; and B) IGHV3 and IGHJ as frameworks for grafting paratope residues. The sequence mAb6785 is shown above. The CDR regions are underlined and the core contact sites in the light chain (Y31, D49, Y90, F92, F93) and in the heavy chain (S52, T54, F57, Y59, Q99, L100 and T101) of mAb6785 are denoted by an asterisk "*". The regions of structure 4 in B) are underlined twice. The sequence shown are * 01 alleles except where specifically indicated otherwise.
[0009] [0009] Figure 3. Kabat and Chothia numbering for the selection of antibody A) and heavy B) chains. The locations of Kabat CDRs and Chothia HVs are highlighted in gray.
[0010] [00010] Figure 4. Competitive binding tests for A) and B) mAb1926; C) mAb317; D) mAb3171; E) and F) IL-17A-labeled mAb7357 in an ELISA format.
[0011] [00011] Figure 5. A) H / D exchange maps of IL-17A complexed with different anti-IL-17A mAbs. The numbering above the protection blocks corresponds to the sequence numbering of the mature IL-17A (SEQ ID No. 105).
[0012] [00012] Figure 6. A) The overall molecular structure of the IL-17A / Fab6468 complex. The dimer of IL-17A is shown in dark gray and light gray. The two Fab molecules are shown in dark gray and light gray, respectively; B) comparison between IL-17A monomer (light gray) and IL-17F (dark gray); C) IL-17A dimer (light gray and dark gray); D) dimer of IL-17F (light gray and dark gray).
[0013] [00013] Figure 7. The two binding sites and the core epitope on IL-17A for Fab6468. Protomers 1 and 2 are dark gray and light gray, respectively. The nucleus epitope is indicated by the black oval. The segmented line represents disordered waste.
[0014] [00014] Figure 8. Comparison between IL-17A and IL-17F putative receptor binding pockets. A) Anterior and posterior views of the IL-17A P1 and P2 pockets. The FF theme of the mAb6468 light chain CDR3 is shown in pocket P2. B) IL-17F with FF N-terminal theme in pocket P2. C) IL-17A and IL-17F sequence alignment and conservation of P1 and P2 pockets.
[0015] [00015] Figure 9. Specificity of binding of mAb1926 with different species of IL-17A proteins in an ELISA format. SUMMARY OF THE INVENTION
[0016] [00016] One aspect of the invention is an isolated antibody or fragment thereof, the antibody specifically binding to human IL-17A having the sequence shown in SEQ ID No. 105 in amino acid residues 56-68 (SEQ ID No. 157) and 100-116 (SEQ ID No. 158); or in residues L26, R55, E57, P59, E60, R61, Y62, S64, V65, W67, R101, E102, P103 and F110.
[0017] [00017] Another aspect of the invention is an isolated antibody or fragment thereof, the antibody specifically binding to a pocket cavity P2 in human IL-17A, the pocket cavity P2 comprising amino acid residues V22, V24, L26, I28, Y62, L99, R101, F110 and L112 of SEQ ID No. 105.
[0018] [00018] Another aspect of the invention is an isolated antibody or fragment that specifically binds to human IL-17A that competes for the binding of human IL-17A with a monoclonal antibody that comprises the amino acid sequences of certain complementarity determining regions (CDRs) ) 1, 2 and 3 heavy chain (HCDR1, HCDR2, HCDR3), the amino acid sequences of certain complementarity determining regions (CDRs) 1, 2 and 3 of light chain (LCDR1, LCDR2, LCDR3), the amino acid sequences of certain heavy chain variable regions (VH), or the amino acid sequences of certain light chain variable regions (VL).
[0019] [00019] Another aspect of the invention is an isolated antibody or fragment that specifically binds to human IL-17A, which comprises certain amino acid residues from the heavy chain variable region and certain amino acid residues from the heavy chain variable region. that interact with certain residues of human IL-17A having the amino acid sequence shown in SEQ ID No. 105.
[0020] [00020] Another aspect of the invention is an isolated antibody or fragment that specifically binds to human IL-17A, which comprises a heavy chain variable region and a light chain variable region, the antibody comprising a variable region parope. heavy chain selected from Chothia F56 and Y58 residues; and a light chain variable region parotope selected from Chothia Y91, F93 and F94 residues.
[0021] [00021] Another aspect of the invention is an isolated antibody or fragment that specifically binds to human IL-17A, which comprises a heavy chain variable region (VH) and a light chain variable region (VL), the antibody comprises the amino acid sequences of certain complementarity determining regions of (CDRs) 1, 2 and 3 heavy chain (HCDR1, HCDR2, HCDR3), the amino acid sequences of certain complementarity determining regions (CDRs) 1, 2 and 3 of light chain (LCDR1, LCDR2, LCDR3), the amino acid sequences of certain variable regions of heavy chain (VH), or the amino acid sequences of certain variable regions of light chain (VL).
[0022] [00022] Another aspect of the invention is an isolated antibody or fragment that specifically binds to human IL-17A, the antibody comprising the amino acid sequences of certain heavy chains and the amino acid sequences of certain light chains.
[0023] [00023] Another aspect of the invention is a pharmaceutical composition that comprises the isolated antibody or fragment of the invention and a pharmaceutically acceptable carrier.
[0024] [00024] Another aspect of the invention is an isolated antibody heavy chain comprising the amino acid sequence shown in SEQ ID No. 67, 68, 69, 81, 82, 83, 84, 85, 86, 92, 93, 94, 95, 96, 97, 98, 99 or 100.
[0025] [00025] Another aspect of the invention is an isolated antibody light chain comprising the amino acid sequence shown in SEQ ID NOs 76, 77, 78, 79, 80, 87, 88, 89, 90 or 91.
[0026] [00026] Another aspect of the invention is an isolated polynucleotide that encodes an antibody heavy chain comprising the amino acid sequence shown in SEQ ID No. 67, 68, 69, 81, 82, 83, 84, 85, 86, 92, 93, 94, 95, 96, 97, 98, 99 or 100.
[0027] [00027] Another aspect of the invention is an isolated polynucleotide that encodes an antibody light chain comprising the amino acid sequence shown in SEQ ID No. 76, 77, 78, 79, 80, 87, 88, 89, 90 or 91.
[0028] [00028] Another aspect of the invention is a vector that comprises at least one polynucleotide of the invention.
[0029] [00029] Another aspect of the invention is a host cell that comprises the vector of the invention.
[0030] [00030] Another aspect of the invention is a method of inhibiting the interaction of human IL-17A with IL-17RA comprising: providing human IL-17A and IL-17RA; and contacting human IL-17A with an antagonist that binds human IL-17A to at least one amino acid residue selected from the group consisting of V22, V24, L26, I28, Y62, L99, R101, F110 and L112.
[0031] [00031] Another aspect of the invention is a method of inhibiting the biological activity of IL-17A, which comprises: providing human IL17-A and IL-17RA; and placing human IL-17A in contact with an antagonist that binds human IL-17A to at least one amino acid residue selected from the group consisting of V22, V24, L26, I28, Y62, L99, R101, F110 and L112 .
[0032] [00032] Another aspect of the invention is a method for treating an inflammatory condition which comprises administering a therapeutically effective amount of the isolated antibody according to claim 3 or 7 to a patient who has been in need of it long enough to treat the condition inflammatory. DETAILED DESCRIPTION
[0033] [00033] All publications, including but not limited to patents and patent applications, cited in this specification are hereby incorporated by reference as if they were described in full.
[0034] [00034] It should be understood that the terminology used here is only intended to describe particular modalities and is not intended to be limiting. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning, as commonly understood by one skilled in the art to which the invention belongs.
[0035] [00035] Although any methods and materials similar or equivalent to those described herein can be used in the practice of the tests of the present invention, exemplary materials and methods are described here. For the description and claims of the present invention, the following terminology will be used.
[0036] [00036] The term "antagonist", for use in the present invention, means a molecule that partially or completely inhibits, by any mechanism, the activity of IL-17A. Examples of antagonists are antibodies, fusion proteins, peptides, peptidomimetics, nucleic acids, oligonucleotides and small molecules. The agent can be identified using well-known tests for IL-17A activity as described below.
[0037] [00037] The term "IL-17A antibody antagonist" or an "IL-17A antibody reactive" for use in the present invention, refers to an antibody that is capable of, directly or indirectly, reducing or inhibiting the biological activity of IL-17A, blocking the binding of IL-17A to its receptor, or inhibiting the activation of the IL-17A receptor. For example, an antibody reactive with IL-17A can bind directly to IL-17A and neutralize IL-17A activity, that is, block IL-17A signaling to reduce cytokine and chemokine release.
[0038] [00038] The term "IL-17A" (CTLA-8, IL-17, interleukin-17A) refers to a human IL-17A polypeptide that has an amino acid sequence shown in GenBank Acc. No. NP_002181. SEQ ID No. 105 shows the amino acid sequence of mature human IL-17A. IL-17A in vivo forms homodimers of two monomers, which are called monomer A and monomer B, or protomer A and protomer B, or protomer 1 and protomer 2, or chain A and chain B. IL-17A can also form a heterodimer with IL-17F. The term "IL-17A" comprises the monomer, homodimer and heterodimer forms. The term "IL-17Amut6" refers to a variant of IL-17A that has A70Q and A132Q substitutions. The amino acid sequence of mature IL-17Amut6 is shown in SEQ ID No. 106, and the cDNA sequence in SEQ ID No. 112. IL-17A and IL-17Amut6 have comparable activities (PCT. International No. WO09 / 003096).
[0039] [00039] The term "IL-17A receptor", for use in the present invention, comprises both receptor polypeptides, IL-17RA (Gen-Bank Acc no: NP_055154, SEQ ID No. 107) and IL-17RC (GenBank Acc No. NP_703191, SEQ ID No. 113), and the homodimers or heterodimers of the two polypeptides.
[0040] [00040] The term "antibodies" for use in the present invention is used in a broad sense and includes immunoglobulin molecules that include polyclonal antibodies, monoclonal antibodies including murine, human monoclonal antibodies, adapted to humans, humanized and chimeric, fragments of antibodies, multi-specific antibodies formed from at least two intact antibodies, dimeric, tetrameric or multimeric antibodies.
[0041] [00041] The term "monoclonal antibody" (mAb), for use in the present invention, means an antibody (or antibody fragment) obtained from a population of substantially homogeneous antibodies. Monoclonal antibodies are highly specific, typically directed against a single epitope. The "monoclonal" modifier indicates the substantially homogeneous character of the antibody and does not require production of the antibody by any particular method.
[0042] [00042] Immunoglobulins can be assigned to five main classes, namely, IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub-classified as the IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4 isotypes. Antibody light chains of any vertebrate species can be attributed to one of two clearly distinct types, namely, kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.
[0043] [00043] The term "antibody fragments" comprises at least a portion of an immunoglobulin molecule, as a heavy chain complementarity determining region (HCDR), a light chain complementarity determining region (LCDR), a variable region of heavy chain (VH), a light chain variable region (VL), a heavy chain constant region (CH), a light chain constant region (CL), or a heavy or light chain structure (FR) region of antibody. An antibody can be a Fab, F (ab '), F (ab') 2, scFv, dsFv, or diabody. The structures of the aforementioned antibody fragments, and the techniques for preparing and using the antibodies and fragments thereof, are well known in the art.
[0044] [00044] An antibody variable region consists of a "backbone" region interrupted by three "antigen binding sites". Antigen binding sites are defined using several terms: (i) The complementarity determining regions (CDRs), three in the VH (HCDR1, HCDR2, HCDR3), and three in the VL (LCDR1, LCDR2, LCDR3), are based on sequence variability (Wu and Kabat, J. Exp. Med. 132: 211-250, 1970; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). (ii) The term "hypervariable regions", "HVR", or "HV", three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3), refer to the regions of the domains antibody variables that have a hypervariable structure, as defined by Chothia and Lesk (Chothia and Lesk, Mol. Biol. 196: 901-917, 1987). Other terms include "IMGT CDRs" (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003) and "use of specificity-determining residues" (SDRU) (Almagro, Mol. Recognit. 17: 132- 143, 2004). The International ImMunoGeneTics (IMGT) database (http: // www_imgt_org) provides standardized numbering and definition of antigen-binding sites. The correspondence between CDRs, HVs and IMGT designs is described in Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003.
[0045] [00045] The term "Chothia residues" for use in the present invention are the VL and VH residues of the antibody numbered according to Al-Lazikani (Al-Lazikani et al., J. Mol. Biol. 273: 927- 48, 1997). The correspondence between the two most used numbering systems, Kabat (Kabat et al., Sequences of Immunological Interest, 5th Ed., Public Health Service, NIH, Bethesda, MD, 1991) and Chothia (Chothia and Lesk, Mol. Biol. 196: 901-17, 1987) in relation to the sequential polypeptide numbering is shown in Figure 3 for exemplifying antibodies of the invention.
[0046] [00046] "Structure" or "structural sequences" are the remaining sequences from a variable region other than those defined to be the antigen binding site. Because the antigen-binding site can be defined by several terms, as described above, the exact amino acid sequence of a structure depends on how the antigen-binding site was defined.
[0047] [00047] The term "substantially identical", for use in the present invention, means that the two amino acid sequences of the antibody or antibody fragment being compared are identical or have "insubstantial differences". Insubstantial differences are substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in an antibody amino acid sequence or antibody fragment that do not adversely affect the properties of the antibody. Amino acid sequences substantially identical to the sequences presented here are also part of that patent application. In some embodiments, the sequence identity can be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. The identity percentage can be determined, for example, by aligning sequence pairs using the default settings of the Vector NTI v.9.0.0 AlignX module (Invitrogen, Cars-lbad, CA, USA). The protein sequences of the present invention can be used as a query string to perform a search on public or patent databases to, for example, identify related sequences. Exemplary programs used to perform such searches are the XBLAST or BLASTP programs (http _ // www_ncbi_nlm / nih_gov), or the set of integrated GenomeQuest ™ programs (GenomeQuest, Westborough, MA, USA) using the default settings.
[0048] [00048] The term "in combination with" as used herein means that the agents described can be administered to an animal together in a mixture, simultaneously as single agents or sequentially as single agents in any order.
[0049] [00049] The term "inflammatory condition", for use in the present invention, refers to acute or chronic responses, localized or systemic, to harmful stimuli, such as pathogens, damaged cells, physical injuries or irritating agents, which are partially mediated by the activity of cytokines, chemokines, or inflammatory cells (for example, neutrophils, monocytes, lymphocytes, macrophages) and is characterized, in most cases, by pain, redness, swelling and impaired tissue function.
[0050] [00050] The term "IL-17A-mediated inflammatory condition" for use in the present invention refers to an inflammatory condition resulting, at least in part, from the biological activity of IL-17A, or caused by the activity of IL-17A . Examples of inflammatory conditions mediated by IL-17A are psoriasis and rheumatoid arthritis.
[0051] [00051] The term "IL-17A-mediated condition", for use in the present invention, encompasses all diseases and medical conditions in which IL-17A plays a role, directly or indirectly, in the disease and medical condition, including cause, development, progress, persistence or pathology of the disease or condition.
[0052] [00052] The term "epitope" as used here means a portion of an antigen to which an antibody specifically binds. Epitopes typically consist of chemically active surface clusters (such as polar, non-polar or hydrophobic) of portions such as amino acids or polysaccharide side chains and may have specific three-dimensional structural characteristics as well as specific charge characteristics. An epitope can be composed of one or both contiguous or noncontiguous amino acids that form a conformational spatial unit. For a noncontiguous epitope, amino acids from different portions of the linear antigen sequence are closely approximated in three-dimensional space by flexing the protein molecule.
[0053] [00053] The term "paratope", for use in the present invention, means a portion of an antibody to which an antigen specifically binds. A paratope can be linear in nature or it can be discontinuous, formed by a spatial relationship between non-contiguous amino acids of an antibody and not for a linear series of amino acids. A "light chain paragraph" and a "heavy chain paragraph" or "light chain paragraph amino acid residues" and "heavy chain paragraph amino acid residues" refer to light chain and heavy chain residues of a antibody in contact with an antigen, respectively.
[0054] [00054] The term "specific binding" as used here, refers to the antibody that binds to a predetermined antigen with greater affinity than other antigens or proteins. Typically, the antibody binds with a dissociation constant (Kd) of 10-7 M or less, and binds to the predetermined antigen with a Kd that is at least ten times less than its Kd to bind to a non-specific antigen. -specific (for example, BSA, casein, or any other specified polypeptide), in addition to the predetermined antigen. For the present invention, the phrases "an antibody that recognizes an antigen" and "an antibody specific to an antigen" are used interchangeably with the term "an antibody that specifically binds an antigen" or "a specific antibody antigen ", for example, an IL-17A specific antibody. The dissociation constant can be measured using standard procedures.
[0055] [00055] The term "biological activity of IL-17A" or "activation of IL-17A", for use in the present invention, refers to any activity that occurs as a result of binding of IL-17A with the IL-17A receptor 17A. Examples of biological activities of IL-17A result in increased secretion of IL-6 or IL-8, activation of NF-kB, or regulation of downstream kinases such as ERK1, ERK2 and p38 by binding to the IL-17A receptor . The release of cytokines and chemokines from cells, tissues or in circulation, the activation of NF-kB, or kinase phosphorylation events can be measured using well-known methods, for example, immunoassays, immunoblots, or gene-reporter (Yao et al., Immunity 3: 811-21, 1995; Awane et al., J. Immunol. 162: 5337-44, 1999).
[0056] [00056] The term "vector" means a polynucleotide capable of being duplicated within a biological system or that can be moved between those systems. Vector polynucleotides typically contain elements, such as the origins of replication, polyadenylation signals or selection markers, that work to facilitate the duplication or maintenance of these polynucleotides in a biological system. Examples of such biological systems may include a cell, virus, animal, plant, and reconstituted biological systems that use biological components capable of duplicating a vector. The polynucleotide that comprises a vector can be DNA or RNA molecules or a hybrid thereof.
[0057] [00057] The term "expression vector" means a vector that can be used in a biological system or in a reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.
[0058] [00058] The term "polynucleotide" means a molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent chemical covalent bond. Double-stranded and single-stranded DNAs and RNAs are typical examples of polynucleotides.
[0059] [00059] The term "polypeptide" or "protein" means a molecule that comprises at least two amino acid residues linked by a peptide bond to form a polypeptide. Small polypeptides with less than 50 amino acids can be called "peptides".
[0060] [00060] Conventional one-letter and three-letter amino acid codes are used here as follows:
[0061] [00061] The present invention features IL-17A antibody antagonists capable of inhibiting the biological activity of IL-17A and the uses of such antibodies. Exemplary mechanisms by which the activation of IL-17A can be inhibited by such antibodies include inhibition in vitro, in vivo or in situ of homo- or heterodimerization of IL-17A, and blocking the binding of IL-17A with the receptor of IL-17A, inhibition of receptor dimerization, inhibition of kinase activity in downstream signaling pathways, or inhibition of IL-17A mRNA transcription. Other antibody antagonists capable of inhibiting the activation of IL-17A by other mechanisms are also within the scope of the various aspects and modalities of the invention. These antagonists are useful as research reagents, diagnostic reagents and therapeutic agents.
[0062] [00062] The invention provides innovative antigen-binding sites derived from human immunoglobulin gene libraries. The structure for transporting an antigen binding site is, in general, a heavy or light chain of antibody or a portion thereof.
[0063] [00063] The invention provides an isolated antibody or fragment thereof that specifically binds to human IL-17A, which comprises a heavy chain variable region (VH) and a light chain variable region (VL), the antibody comprises the complementarity determining region (CDRs) 1, 2 and 3 heavy chain amino acids (HCDR1, HCDR2 and HCDR3) and the light chain complementarity determining region (CDR) 1, 2 and 3 amino acid sequences (LCDR1, LCDR2 and LCDR3), as shown in Table 1a.
[0064] [00064] In certain embodiments, the invention provides an isolated antibody or fragment that specifically binds to human IL-17A, comprising a VH and a VL, the antibody comprising the amino acid sequences HCDR1, HCDR2 and HCDR3 shown in SEQ ID No. 23, 35 and 52, the HCDR2 of SEQ ID No. 35 is further defined as shown in formula (I): XaaH-IPWFG-Xaa2-T-Xaa3-YAQKFQG, (I)
[0065] [00065] where
[0066] [00066] Xaa1 can be His, Met, Arg, Ser or Tyr;
[0067] [00067] Xaa2 can be Trp, Thr or Tyr; and
[0068] [00068] Xaa3 can be Tyr, Phe, Ser or Asp.
[0069] [00069] In other embodiments, the invention provides an isolated antibody or fragment that specifically binds to human IL-17A, comprising a VH and a VL, the antibody comprising the amino acid sequences LCDR1, LCDR2 and LCDR3 shown in SEQ ID No. 2, 5 and 11, the LCDR3 of SEQ ID No. 11 is further defined as shown in formula (II): Xaa4-Q-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10, (II)
[0070] [00070] where
[0071] [00071] Xaa4 can be His or Gln;
[0072] [00072] Xaa5 can be Phe or Gly;
[0073] [00073] Xaa6 can be Thr, Val or Asn;
[0074] [00074] Xaa7 can be Ile, Thr or Tyr;
[0075] [00075] Xaa8 can be Pro or Arg;
[0076] [00076] Xaa9 can be Ser or Pro; and
[0077] [00077] Xaa10 can be His, Phe or Leu.
[0078] [00078] In other embodiments, the invention provides an isolated antibody or fragment that specifically binds to human IL-17A binding, comprising a VH and a VL, the antibody comprising the amino acid sequences LCDR1, LCDR2 and LCDR3 shown in SEQ ID No. 2, 5 and 17, the LCDR3 of SEQ ID No. 17 is further defined as shown in formula (III): Xaan -Q-Xaa12-Xaa13-Xaa14-Xaa 15-Xaa 16-Xaa17-Xaa18-T, (III)
[0079] [00079] where
[0080] [00080] Xaan can be Gln or Thr;
[0081] [00081] Xaa12 can be Ser or Tyr;
[0082] [00082] Xaa13 can be Asn, Arg, Val or Tyr;
[0083] [00083] Xaa14 can be His or Ser;
[0084] [00084] Xaa15 can be Ile, Thr, Leu, Ala or Ser;
[0085] [00085] Xaa16 can be Pro, Leu or Ser;
[0086] [00086] Xaa17 can be Pro, Ser, Phe or Leu; and
[0087] [00087] Xaai8 can be Ala, Leu or Asp.
[0088] [00088] In other embodiments, the invention provides an isolated antibody or fragment that specifically binds to human IL-17A, comprising a VH and a VL, the antibody comprising the amino acid sequences HCDR1, HCDR2 and HCDR3 shown in SEQ ID No. 24, 36 and 57, the HCDR3 of SEQ ID No. 57 is further defined as shown in formula (IV): EVDS-Xaa19-YYSYFDI, (IV)
[0089] [00089] where
[0090] [00090] Xaa19 is Met, Ile, Leu or Thr.
[0091] [00091] In other embodiments, the invention provides an isolated antibody or fragment that specifically binds to human IL-17A, comprising a VH and a VL, the antibody comprising the amino acid sequences LCDR1, LCDR2 and LCDR3 shown in SEQ ID No. 3, 6 and 22, the LCDR3 of SEQ ID No. 22 is further defined as shown in formula (V): GSYDFFLG-Xaa20-IV, (V)
[0092] [00092] where
[0093] [00093] Xaa20 is Met, Leu, Thr or Tyr.
[0094] [00094] In other embodiments, the invention provides an isolated antibody or fragment that specifically binds to human IL-17A, comprising a VH and a VL, the antibody comprising the amino acid sequences HCDR1, HCDR2 and HCDR3 shown in SEQ ID No. 25, 46 and 61, the HCDR2 of SEQ ID No. 46 is further defined as shown in formula (VI): Xaa21-I-Xaa22-Xaa23-Xaa24-Xaa25-Xaa26-Xaa27-Xaa28-Xaa29-YADSVKG, (SAW)
[0095] [00095] where
[0096] [00096] Xaa21 can be Ala, Gly, Thr or Val;
[0097] [00097] Xaa22 can be Asn or Ser;
[0098] [00098] Xaa23 can be Gly, Met, Lys, Ile, Leu or His;
[0099] [00099] Xaa24 can be Leu, Asp, Ala, His, Thr, Gly or Ser;
[0100] [000100] Xaa25 can be Gly or Ser;
[0101] [000101] Xaa26 can be Thr, Gly, Tyr or Asp;
[0102] [000102] Xaa27 can be His, Trp, Tyr or Phe;
[0103] [000103] Xaa28 can be Lys, Thr or Ile; and
[0104] [000104] Xaa29 can be Tyr, Phe or Asn, and
[0105] [000105] the HCDR3 of SEQ ID No. 61 is defined as shown in formula (VII): QL-Xaa30-LDV, (VII)
[0106] [000106] where
[0107] [000107] Xaa30 can be Met, Leu or Thr.
[0108] [000108] In other embodiments, the invention provides an isolated antibody or fragment that specifically binds to human IL-17A, comprising a VH and a VL, the antibody comprising the amino acid sequences HCDR1, HCDR2 and HCDR3 shown in SEQ ID No. 25, 51 and 58, the HCDR2 of SEQ ID No. 51 is further defined as shown in formula (VIII): VTS-Xaa31-Xaa32-Xaa33-Xaa34-TYYA-Xaa35-SVKG, (VIII)
[0109] [000109] where
[0110] [000110] Xaa31 can be Ala, Lys, Met or His;
[0111] [000111] Xaa32 can be Asn, Met, Thr or Arg; Xaa33 can be either Gly or Asp; Xaa34 can be Arg, His or Asn; and Xaa35 can be Asp or Gly.
[0112] [000112] Antibodies whose amino acid sequences of antigen binding sites are substantially identical to those shown in Table 1a (SEQ ID NO: 1-61) are within the scope of the invention. Typically, this involves one or more substitutions of amino acids with an amino acid having similar hydrophobic or stereochemical charges or characteristics, and are done to optimize the properties of the antibody such as, for example, stability or affinity. For example, a conservative substitution may involve replacing a native amino acid residue with a non-native residue so that there is little or no effect on the polarity or charge of the amino acid residue at that position. In addition, any native residue in the polypeptide can also be replaced with alanine, as previously described for alanine scan mutagenesis (MacLennan et al., Acta Physiol. Scand. Suppl. 643: 55 to 67, 1998; Sasaki et al. , Adv. Biophys. 35: 1-24, 1998). Conservative substitutions will produce molecules with functional and chemical characteristics similar to those of the molecule from which such modifications are made. Non-conservative substitutions in the functional and / or chemical characteristics of the molecules can be made by selecting the substitutions in the amino acid sequence that differ significantly in their effect on the maintenance (1) of the molecular main chain structure in the substitution area, for example example, as a blade or helical conformation, (2) the charge or hydrophobic capacity of the molecule at the target site, or (3) the size of the molecule. The desired amino acid substitutions (conservative or non-conservative) can be determined by those skilled in the art at the time such substitutions are desired. For example, amino acid substitutions can be used to identify residues important for antibody function, such as residues that affect affinity, or residues that confer undesired properties such as aggregation. Examples of amino acid substitutions are shown in Table 1b, and in Figure 1.
[0113] [000113] Substitutions in the framework regions, in contrast to antigen binding sites, can also be made as long as they do not adversely affect the properties of the antibody. Structure replacements can be made, for example, in the residues of the Vernier Zone (US patent No. 6,649,055) to optimize the affinity or stability of the antibody. Substitutions can also be made at antibody structure positions that differ in sequence when compared to sequences of homologous human germline genes to reduce possible immunogenicity. Such modifications can be made, for example, to antibodies derived from new antibody libraries, such as pIX libraries.
[0114] [000114] Conservative amino acid substitutions also include non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis, rather than by synthesis in biological systems. Amino acid substitutions can be made, for example, by PCR mutagenesis (US patent No. 4,683,195). Variant libraries can be generated using well-known methods, for example, using random (NNK) or non-random codons, for example, DVK codons, which encode 11 amino acids (ACDEGKNRSYW), and screening of the libraries or variants with desired properties, as shown in Example 1. Figure 1 shows substitutions made on five parental IL-17A antibody antagonists in the LCDR3, HCDR2 and HCDR3 regions to optimize the antibody properties. Optimized properties, such as affinity or stability, can be measured using well-known methods.
[0115] [000115] In other embodiments, the invention provides an isolated antibody or fragment that specifically binds to human IL-17A, which comprises a VH and a VL, the antibody comprising certain VH and VL sequences, and also provides each VH and VL isolated, as shown in Table 2.
[0116] [000116] Although the modalities illustrated in the examples comprise pairs of variable regions, pairs of full-length antibody chains, or pairs of CDR1, CDR2 and CDR3 regions, one from a heavy chain and one from a light chain, one skilled in the art will recognize that alternative modalities further comprise isolated heavy chain variable regions or isolated light chain variable regions, isolated full length antibody chains, or CDR1, CDR2 and CDR3 regions of an antibody chain, heavy or light. The isolated variable region, the full length antibody chain or the CDR1, CDR2 and CDR3 regions of a chain can be used to separate corresponding domains in another chain, the two chains being able to form an antibody that specifically binds to IL -17A. Screening can be done using "phage display" screening methods using, for example, a hierarchical double combination approach presented in PCT application no. WO92 / 01047. In this approach, an individual colony containing an H chain or L chain clone is used to infect a complete clone library encoding the other chain (L or H), and the resulting two chain specific antigen binding domain is selected according to the phage presentation techniques, as described.
[0117] [000117] In other embodiments, the invention provides an isolated antibody or fragment that specifically binds to human IL-17A, comprising a VH and a VL with amino acid sequences at least 90% identical to the VH and VL amino acid sequences shown in Table 2.
[0118] [000118] In other embodiments, the invention provides an isolated antibody or fragment that specifically binds to human IL-17A, comprising a VH and a VL with amino acid sequences at least 95% identical to the VH and VL amino acid sequences shown in Table 2.
[0119] [000119] In another aspect, the invention provides an isolated antibody or fragment with certain heavy chain and light chain amino acid sequences as shown in Table 2. In addition to sequentially numbering the antibody residues, polypeptides that encode chains of antibodies based on Kabat or Chothia numbering (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Md., 1991; Chothia and Lesk, Mol. Biol 196: 901917, 1987). Examples of the correspondence between Kabat and Chothia sequential numbering for a selected antibody chain are shown in Figure 3. The gray highlighted positions indicate CDR antibody regions.
[0120] [000120] In other embodiments, the invention features an isolated antibody or fragment that specifically binds to human IL-17A, comprising a VH and a VL, the antibody comprising a heavy chain variable region paratope selected from the residues of Chothia S51, T53, F56, Y58, Q95, L96 and T97 and a parapet of variable region of light chain selected from residues of Chothia Y32, D50, Y91, F93 and F94. The Chothia residues of the heavy chain and the light chain parameters correspond to the heavy chain residues S52, T54, F57, Y59, Q99, L100 and T101 of SEQ ID No. 86, and to the light chain residues Y31, D49 , Y90, F92 and F93 of SEQ ID No. 79.
[0121] [000121] In other embodiments, the invention provides an isolated antibody or fragment that specifically binds to human IL-17A, comprising amino acid residues from the variable region of the heavy chain that interact with human IL-17A residues having the amino acid sequence shown in SEQ ID No. 105, which comprises: a first threonine residue that interacts with human IL-17A R55 or E57; a glutamine residue that interacts with human IL-17A R55 or E57; a lysine residue that interacts with human IL-17A E57; a tyrosine residue that interacts with human IL-17A P59, E60 or R101; a phenylalanine residue that interacts with human IL-17A E60, R101, E102 or P103; a serine residue that interacts with human IL-17A E60; and a second threonine residue that interacts with human IL-17A E60.
[0122] [000122] In other embodiments, the invention provides an isolated antibody or fragment that specifically binds to human IL-17A, comprising amino acid residues from the paratope of the light chain variable region that interact with residues of human IL-17A having the sequence of amino acids shown in SEQ ID No. 105, which comprises: a first phenylalanine residue that interacts with human IL-17A L26; an aspartic acid residue that interacts with human IL-17A R55 or W67; a first tyrosine residue that interacts with human IL-17A P59, S64 or R101; a second phenylalanine residue that interacts with human IL-17A P59, E60, R61, Y62, R101 or F110; and a second tyrosine residue that interacts with V65 of human IL-17A.
[0123] [000123] In another embodiment, the invention provides an isolated antibody or fragment that specifically binds to human IL-17A, comprising amino acid residues from the heavy chain variable region paratope and amino acid residues from the light chain variable region paratope that interact with human IL-17A residues having the amino acid sequence shown in SEQ ID No. 105, which comprises: a tyrosine residue in the heavy chain variable region that interacts with human IL-17A R101; a phenylalanine residue in the heavy chain variable region that interacts with human IL-17A R101; a first phenylalanine residue in the light chain variable region that interacts with human IL-17A Y62 and R101; a second phenylalanine residue in the light chain variable region that interacts with human IL-17A L26 and F110; and a tyrosine residue in the light chain variable region that interacts with human IL-17A R101.
[0124] [000124] In another embodiment, the invention provides an isolated antibody or fragment that specifically binds to human, human IL-17A, comprising a heavy chain variable region and a light chain variable region, the antibody comprising: a heavy chain variable region parotope selected from Chothia F56 and Y58 residues; and a light chain variable region parotope selected from Chothia Y91, F93 and F94 residues.
[0125] [000125] The Chothia F56 and Y58 residues of the heavy chain parotope and the Chothia Y91, F92 and F94 residues of the light chain parotope are residues in direct contact with the IL-17A residues L26, Y62, R101 and F110. These IL-17A residues are part of the Fab6468 epitope and pocket cavity P2 (see below). Without sticking to any specific theory, it is believed that the interaction between Fab6468 and IL-17A in these selected residues may be sufficient for the antibody to block IL-17A activity.
[0126] [000126] fully human mAbs devoid of any non-human sequences can be prepared and optimized from "phage display" libraries using techniques mentioned in, for example, Knappik et al., J. Mol. Biol. 296: 57 to 86, 2000; and Krebs et al., J. Immunol. Meth. 254: 67-84 2001. In an exemplifying method, the antibodies of the invention are isolated from libraries that express variable regions of heavy and light chain antibodies such as fusion proteins with bacteriophage pIX coat protein. The antibody libraries are screened to bind to human IL-17mut6 (SEQ ID No. 105), and the positive clones obtained are further characterized, the Fabs isolated from the clone lysates, and expressed as full-length IgGs. Examples of antibody libraries and screening methods are described in Shi et al., J. Mol. Biol. 397: 385-96, 2010; PCT patent application No. WO09 / 085462, and U.S. serial number 12/546850; U.S. Patent Nos. 5,223,409, 5,969,108 and 5,885,793).
[0127] [000127] The resulting mAbs can be further modified in their structure regions to alter certain structure residues in those present in a corresponding human germline, as exemplified here.
[0128] [000128] Antibodies of the invention that bind to specific IL-17A epitopes can be produced by immunizing humanized mice expressing human immunoglobulin sites (Lonberg et al., Nature 368: 856-9, 1994; Fishwild et al. , Nature Biotechnology 14: 845-51, 1996; Mendez et al., Nature Genetics 15: 146-56, 1997, US Patent No. 5,770,429, 7,041,870 and 5,939,598) or Balb / c mice with the peptides which encode the epitopes, for example peptide 56NEDPERYPSVIWE68 (SEQ ID No. 157) or 100RREPPHCPNSFRLEKIL116 (SEQ ID No. 158) and using the hybrodoma method of Kohler et al., Nature 256: 495-97. The resulting antibodies are evaluated for their attachment to the epitope using standard methods. The identified mAbs can be further modified by incorporating altered structure support residues to preserve binding affinity using techniques such as those presented in Queen et al., Proc. Natl. Acad. Sci. (USA), 86: 10029-32, 1989 and Hodgson et al., Bio / Technology, 9: 421, 1991.
[0129] [000129] Isolated antibodies containing certain paratope residues (for example, the nucleotide residues defined in Table 10) that specifically bind to human IL-17A can be produced, for example, by grafting the paratope residues in a suitable framework, grouping the projected frameworks into complete antibodies, expressing the resulting antibodies, and testing the antibodies for binding to IL-17A or for an effect on the biological activity of IL-17A. Exemplary frameworks are amino acid sequences from variable regions of human antibody encoded by human germline genes. The frameworks can be selected based, for example, on the global sequence homology, on the percentage of identity between the residues of the paratope, or on the class identity of the canonical structure between the framework and an exemplary antibody, such as mAb6785. Germ line genes of human antibodies are presented, for example, in Tomlinson et al., J. Mol. Biol 227: 776 to 798, and in the international database ImMunoGeneTics (IMGT) (http_: // _ www_imgt_org). Consensus human structural regions can also be used, for example, as described in U.S. Patent No. 6,054,297. The selection of a suitable framework can be made, for example, according to methods described in PCT publication No. WO10 / 045340.
[0130] [000130] Examples of human germline genes that can be used as scaffolds on which the paratope residues are grafted are the genes encoded by the structures VA3, Vh3, JA and Jh. Examples of Vk3 genes are IGLV3-1, IGLV3-9, IGLV3-10, IGLV3-12, IGLV3-16, IGLV3-19, IGLV3-21, IGLV3-22, IGLV3-25, IGLV3-27 and IGLV3-32 (nomenclature IMGT, alleles * 01), (SEQ ID No. 117-127, respectively). Examples of JA genes are IGLJ1, IGLJ2, IGLJ3, IGLJ4, IGLJ5, IGLJ6 and IGLJ7 (SEQ ID No. 128134, respectively). Examples of Vh3 genes are IGHV3-7, IGHV3-9, IGHV3-11, IGHV3-16, IGHV3-19, IGHV3-20, IGHV3-21, IGHV3-23, IGHV3-30, IGHV3-30 * 03, IGHV3-33 , IGHV3-45, IGHV3-48, IGHV3-64 and IGHV3-74 (IMGT nomenclature, alleles * 01, except when a different allele is specified) (SEQ ID No. 135-150, respectively). Examples of Jh genes are IGHJ1, IGHJ2, IGHJ3, IGHJ4, IGHJ5 and IGHJ6 (SEQ ID No. 151-156, respectively). Germline J regions are used in whole or in part to select FR4 sequences. For example, light chain mAb6785 residues can be grafted into an IGLV3-1-encoded VA3 protein structure (SEQ ID No. 117) that is joined to the IGLJ2-encoded J region sequence (SEQ ID No. 129) with the insertion of a single amino acid residue between the sequences IGLV3-1 and IGLJ2, for example, methionine. The IGLV3-1-encoded VA3 protein structure may contain additional substitutions, for example a replacement of cysteine residue at position 33 of SEQ ID No. 117 ("ACW") with, for example, asparagine; and replacing residues 1-3 of SEQ ID No. 117 ("SYE") with an amino-terminal sequence common to other lambda chain families, such as "QSV" of the IGLV1 family. The sequences of other examples of functional genes VA3 and JA can be used for grafting the light chain mAb6785 residues with the insertion of zero, one, or two amino acid residues between the carboxy-terminal encoded by the VA3 genes and the amino- terminal encoded by the JA genes, so that the length of the CDR3 region is 11 amino acids. For example, methionine and isoleucine can be inserted between IGLV3-22 (SEQ ID No. 124) and IGLJ2 (SEQ ID No. 129). Figure 2A shows the alignment of exemplary light chain frameworks that can be used for grafting. The residues of the mAb6785 heavy chain paragraph can be grafted, for example, onto a Vh3 structure encoded by IGHV3-23 (SEQ ID No. 142), which is joined to the FR4 sequence of region J (11 C-terminal amino acids, for example , "WGQGTLVTVSS") of IGHJ1 (SEQ ID No. 151), with the insertion of about 5-7 residues, for example 6 residues, constituting HCDR3, between regions V and J. The approximately 5-7 residues of HCDR3 inserted include the insertion of glu-tamine, leucine and threonine, for example 3 of the paratope residues from mAb6785 Vh (Table 10). The sequences of other exemplary functional Vh3 and Jh genes can be used for grafting residues of the heavy chain mAb6785 parope. In some cases, a C-terminal amino acid from the Vh3 gene can be excluded before the insertion of the approximately 5-7 residues that make up HCDR3 so that only FR3 sequences are included in the framework. Sequences from other Vh3 genes encoding a 17-residue CDR2 (residues 50-66 of IGHV3-23 (SEQ ID No. 142) can also be used, and FR4 sequences from other Jh genes can be substituted in place of IGJH1.
[0131] [000131] The specific binding to human IL-17A and the biological activity of the resulting antibody can be assessed using standard methods. The alignments of the light chain variable regions and the heavy chain variable regions of mAb6785 with the exemplary Vh3, VA3, JA or Jh genes are shown in Figures 2A and 2B. Alternatively, residues from the extended paraben of mAb6785, as defined in Table 10, can be used in place of residues from the nucleus paratope. The projected antibodies grafted with paratope can be further modified by replacing residues in the Vernier Zone (US patent No. 6,639,055) or affinity-determining residues (US patent application No. 2010/0261620; Cobaugh et al., J Mol Biol. 378: 622-33, 2008) to optimize the properties of the antibody, for example affinity. As long as the graft-grafted antibody retains binding to IL-17A, the amino acid sequence of the structure in the graft-grafted antibody can be 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97 %, 98% or 99% identical to the mAb6785 structural sequences. Allele variants of the exemplifying germline gene structures can be used in place of protein sequences from regions V and J. The sequences of allele variants are well known and can be obtained from the International ImMu-noGeneTics database ( IMGT) (http_: // _ www_imgt_org).
[0132] [000132] Sequences of the antigen-binding sites can be grafted in addition to the paratope residues using standard methods. For example, a complete HCDR3 or LCDR3 can be grafted.
[0133] [000133] Another embodiment of the invention is an isolated antibody or fragment that specifically binds to human IL-17A that competes for binding human IL-17A with a monoclonal antibody that comprises certain amino acid sequences HCDR1, HCDR2 and HCDR3, and LCDR1 , LCDR2 and LCDR3. Examples of the monoclonal antibodies of the invention are an isolated antibody comprising amino acid sequences HCDR1, HCDR2 and HCDR3 as shown in SEQ ID No. 25, 43 and 60 and the amino acid sequences LCDR1, LCDR2 and LCDR3 as shown in SEQ ID No. 3, 6 and 18.
[0134] [000134] The competition between specific binding to IL-17A can be assessed in vitro using well-known methods. For example, the binding of NHS MSD Sulfo-Tag ™ ester labeled antibody to IL-17A in the presence of an unlabeled antibody can be assessed by the ELISA method.
[0135] [000135] One embodiment of the invention is an isolated antibody or antibody or fragment thereof, the antibody specifically binding to human IL-17A having the sequence shown in SEQ ID No. 105 in amino acid residues 56-68 (SEQ ID No. 157) and 100116 (SEQ ID No. 158); or in residues L26, R55, E57, P59, E60, R61, Y62, S64, V65, W67, R101, E102, P103 and F110.
[0136] [000136] Several well-known methodologies can be employed in order to determine the binding epitope of the antibodies of the invention. For example, when the structures of both individual components are known, protein-protein coupling in silicon can be performed in order to identify compatible sites of interaction. The hydrogen-deuterium (H / D) exchange can be performed with the antibody and antigen complex in order to map the regions in the antigen that can be linked through the antibody. Mutagenesis of the antigen spot or segment can be used to locate the amino acids important for antibody binding. The co-crystalline structure of the antibody-antigen complex is used to identify residues that contribute to the epitope and paratope.
[0137] [000137] The anti-IL-17A antibodies described above bind to IL-17A epitopes other than the Fab6468 epitope described in the present invention. Antibodies that bind to human IL-17A residues (SEQ ID No. 105) 74-85, 46-53, 71-87, 80-86, 11-18, 29-41 or 54-62 have been described (orders PCT No. WO08 / 021156, WO07 / 106769, WO07 / 149032, WO07 / 070750; US patent application No. US2008 / 095775, respectively). Conformational epitopes are described in PCT application No. WO09 / 130459 and in Gerhardt et al., J. Mol. Biol: 394: 901-21,2009.
[0138] [000138] Another embodiment of the invention is an isolated antibody or fragment thereof, the antibody specifically binding to a pocket cavity P2 on IL-17A, with pocket pocket P2 comprising amino acid residues V22, V24 , L26, I28, Y62, L99, R101, F110 and L112 of SEQ ID No. 105.
[0139] [000139] The co-crystalline structure of the IL-17A homodimer with the anti-IL-17A Fab6468 identified a hydrophobic pocket in the P2 pocket on the surface of the IL-17A homodimer, which is probably involved in binding to IL-17RA ( see Examples). The term "pocket cavity P2" for use in the present invention refers to a tertiary hydrophobic structural cavity in the IL-17A homodimer, where the exposed surface residues in pocket P2 are V24, L26, I28, Y62, L99, R101, F110 and L112 in monomer A and V22, V24 and L112 in monomer B, and vice versa. The selected antibodies of the invention reactive with IL-17A, for example Fab6468, have direct contacts with residues L26, Y62, R101 and F110 from pocket cavity P2, whose residues are also part of the Fab6468 epitope. Without adhering to any specific theory, it is assumed that the antibodies of the invention that bind to the residues of the selected IL-17A pocket cavity block the interaction between IL-17A and IL-17RA. Based on the co-crystalline structure, the phenylalanine (FF) theme in residues 93 and 94 in a light chain (SEQ ID No. 79) of Fab6468 blocks the interaction between IL-17A and IL-17RA, and thus , is a pocket cavity blocker P2. Also within the scope of this invention are other blocking antagonists of the P2 pocket cavity, such as innovative peptides or small molecules. These can be modeled based on the IL-17A / Fab6468 co-structure, and screened for their ability to replace the Fab6468 binding to IL-17A. For example, peptide inhibitors can be screened from random peptide libraries that have incorporated the FF theme (for example, XXXXFFXX libraries; X indicated by any amino acid; F = phenylalanine) and presented in bacteriophage as a fusion with , for example, pIII, pVII or pIX coating protein (US patent No. 5,223,409; Gao et al., Proc. Natl. Acad. Sci. USA, 96: 602530, 1999, Tornetta et al., J. Immunol Methods 360: 39-46, 2010; Shi et al., J. Mol. Biol. 397: 385-96, 2010) and subsequently evaluated for their inhibition of Fab6468 binding to IL-17A, and inhibition of activity of IL-17A.
[0140] [000140] Small molecules can be screened using libraries of synthetic or natural compounds, or any combination thereof, and the resulting primary positive hits can be readily modified to produce structural analogues of the agents. Methods of preparing libraries of peptides and pIX fusions, and screening the resulting libraries are well known.
[0141] [000141] Another embodiment of the invention of a method of inhibiting the interaction of human IL-17A with IL-17RA comprises: providing human IL-17A and IL-17RA; and placing human IL-17A in contact with an antagonist that specifically binds human IL-17A to at least one amino acid residue selected from the group consisting of: V22, V24, L26, I28, Y62, L99, R101, F110 and L112.
[0142] [000142] Another embodiment of the invention is a method of inhibiting the biological activity of human IL-17A, comprising: providing human IL17-A and IL-17RA; and placing human IL-17A in contact with an antagonist that specifically binds human IL-17A to at least one amino acid residue selected from the group consisting of: V22, V24, L26, I28, Y62, L99, R101, F110 and L112 .
[0143] [000143] Human IL-17A and IL-17RA can be supplied as isolated proteins or fusion proteins. The homodimer of human IL-17A can be purified from activated Th17 cell media prepared by in vitro stimulation of native CD4 T cells by stimulation of two anti-CD3 / anti-CD28 in the presence of IL-2, IL-23 and IL-1 β. IL-17RA can be associated with cells or cell membranes, it can be native or clearly expressed, or it can be a fragment of IL-17RA, for example the extracellular domain of the receptor. IL-17RA can be a human IL-17RA, or IL-17RA from other species such as mice, rats or monkeys. Antagonists that bind to residues of human IL-17A V22, V24, L26, I28, Y62, L99, R101, F110 and L112 can be identified by the antagonist's ability to replace the binding of Fab6468 to IL-17A, via mutagenesis or by co-crystalline structures. Fusion proteins of human IL-17A and IL-17RA can be prepared using well-known methods. An exemplary fusion protein is a soluble IL-17RA fused to an immunoglobulin Fc domain.
[0144] [000144] Another aspect of the invention is an isolated polynucleotide that encodes any of the antibody heavy chains or antibody light chains or fragments thereof or the complement thereof. Certain exemplary polynucleotides are presented here, however, other polynucleotides that, given the degeneracy of the genetic code or codon preferences in a given expression system, encode the antibody antagonists of the invention are also within the scope of the invention. Examples of polynucleotides are shown in SEQ ID No. 101, 102, 103 and 104.
[0145] [000145] Examples of antibody antagonists may be antibodies of the IgG, IgD, IgE, IgA or IgM isotypes. In addition, such antibody antagonists can be modified post-translationally by processes such as glycosylation, isomerization, deglycosylation or non-naturally occurring covalent modification, such as the addition of portions of polyethylene glycol ("PEG") (pegylation) and lipidation. Such modifications can occur in vivo or in vitro. For example, the antibodies of the invention can be conjugated to polyethylene glycol (pegylated) to optimize their pharmacokinetic profiles. Conjugation can be performed by techniques known to those skilled in the art. The combination of therapeutic antibodies with PEG has been shown to improve pharmacodynamics while not interfering with function. See Deckert et al., Nt. J. Cancer 87: 382-90, 2000; Knight et al., Platelets 15: 409-18, 2004; Leong et al., Cytokine 16: 106-19, 2001; and Yang et al., Protein Eng. 16: 761-70, 2003.
[0146] [000146] The pharmacokinetic properties of the antibodies of the invention can be enhanced through Fc modifications using techniques known to those skilled in the art. An antibody's "Fc" is not directly involved in binding an antibody to an antigen, but it does have several effector functions. An antibody "Fc" is a well-known term and is defined based on the papain cleavage of antibodies. An antibody's Fc is directly involved in ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity) based on complement activation, C1q binding and Fc receptor binding. Such complement and Fc receptor binding sites are well known and include, for example, L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbering according to the EU Kabat index) (Brek- ke et al., Eur. J. Immunol. 24: 2542-7, 1995; US Patent Nos. 5,624,821, 7,597,889, Canfield and Morrison, J. Exp. Med. 173: 1483-91, 1991) . For example, the Leu234 / Leu235 mutation in the IgG1 to L234A / L235A or Phe235 / Leu236 region in the IgG4 to P235A / L236A articulation region minimizes FcR binding and reduces immunoglobulin's ability to mediate complement-dependent cytotoxicity and ADCC. A Ser to Pro substitution in the Cys-Pro-Ser-Cys (CPSC) theme in the IgG4 heavy chain articulation region capable of forming inter- or intra heavy chain disulfide bonds in vivo through the action of isomerases (Aalberse and Schuur -man, Immunology 105: 9-19, 2002), results in "IgG1-like behavior", that is, Pro-substituted molecules are not capable of forming heavy intra-chain disulfide bonds. The location of the CPSC motif is typically found at residue 228 of a mature heavy chain, but it can change, depending on the lengths of CDR. An example of the IgG1 Fc region containing the Leu234 / Leu235 residues has an amino acid sequence shown in SEQ ID No. 114, with residues L117 and L118 corresponding to Leu234 / Leu235 residues in the mature heavy chain. An example of an IgG4 Fc region containing the Cys-Pro-Ser-Cys (CPSC) theme and the Leu234 / Leu235 residues has an amino acid sequence shown in SEQ ID No. 115, with the CPSC theme being located at residues 106- 109 and Leu234 / Leu235 residues in positions 122 and 123.
[0147] [000147] Antibodies or fragments thereof modified to optimize stability, selectivity, cross-reactivity, affinity, immunogenicity or other desired biological or biophysical properties are contained within the scope of the invention. The stability of an antibody is influenced by a number of factors, including (1) core packaging of individual domains that affects its intrinsic stability, (2) protein / protein interface interactions that impact HC and LC pairing, ( 3) disposal of polar and charged waste, (4) connection network H for polar and charged waste; and (5) distribution of surface charge and polar residues among other intra- and inter-molecular forces (Worn et al., J. Mol. Biol. 305: 989-1010, 2001). Residues with the potential to destabilize structures can be identified based on the crystal structure of the antibody or through molecular modeling in certain cases, and the effect of residues on antibody stability can be assessed through the generation and evaluation of variants that harbor mutations in identified waste. One way to increase antibody stability is to raise the thermal transition midpoint (Tm) as measured by differential scanning calorimetry (CVD)). In general, the Tm protein is correlated with its stability and inversely correlated with its susceptibility to unfolding and denaturation in solution and to the degradation processes that depend on the tendency of the protein to unfold (Remmele et al., Biopharm. 13: 36-46, 2000). Numerous studies have revealed the correlation between the classification of the physical stability of the formulations measured as thermal stability by differential scanning calorimeter and physical stability measured by other methods (Gupta et al., AAPS PharmSci. 5E8, 2003; Zhang et al., J Pharm. Sci. 93: 3076-89, 2004; Maa et al., Int. J. Pharm., 140: 155-68, 1996; Bedu-Addo et al., Pharm. Res., 21: 1353-61 , 2004; Remmele et al., Pharm Res., 15: 200-8, 1997). Formulation studies suggest that a Fab Tm implies the long-term physical stability of a corresponding mAb. Differences in amino acids both in structure and within CDRs could have significant effects on the thermal stability of the Fab domain (Yasui, et al., FEBS Lett. 353: 143-6, 1994).
[0148] [000148] The antibody antagonists of the invention can bind to IL-17A with a Kd less than or equal to about 10-7, 10-8, 10-9, 1010, 10-11 or 10-12 M. The affinity of a given molecule for IL-17A, as an antibody, can be determined experimentally using any suitable method. Such methods may use Biacore or KinExA instrumentation, ELISA or competitive binding tests known to those skilled in the art.
[0149] [000149] Antagonist antibodies that bind to human IL-17A with a desired affinity can be selected from libraries of variants or fragments using techniques that include antibody affinity maturation. Antibody antagonists can be identified based on their inhibition of the biological activity of IL-17A using any suitable method. Such methods can use reporter gene tests or tests that measure cytokine production using well known methods and as described in the application.
[0150] [000150] Another embodiment of the invention is a vector that comprises at least one polynucleotide of the invention. Such vectors may be plasmid vectors, viral vectors, vectors for bacu-lovirus expression, transposon-based vectors or any other vectors suitable for introducing the polynucleotides of the invention into a given organism or genetic background by any means.
[0151] [000151] Another embodiment of the invention is a host cell that comprises any of the polynucleotides of the invention as a polynucleotide that encodes a polypeptide that comprises an immunoglobulin heavy chain variable region having the amino acid sequence shown in SEQ ID No. 67 -75 and 81-86 or an immunoglobulin light chain variable region having the amino acid sequence shown in SEQ ID No. 62-66 and 76-80 or an immunoglobulin heavy chain having the amino acid sequence shown in SEQ ID No. 92-100 ° or an immunoglobulin light chain having the amino acid sequence shown in SEQ ID No. 8791. Such host cells can be eukaryotic cells, bacterial cells, plant cells or archaea cells. Exemplary eukaryotic cells can be of mammalian, insect, bird or other animal origin. Mammalian eukaryotic cells include cell lines immortalized as hybridoma or myeloma cell lines as murine cell lines SP2 / 0 (American Type Culture Collection (ATCC), Manassas, VA, USA CRL-1581), NS0 (European Collection of Cell Cultures (ECACC), Salisbury, Wiltshire, United Kingdom, ECACC No. 85110503), FO (ATCC CRL-1646) and Ag653 (ATCC CRL-1580). An exemplary human myeloma cell line is U266 (ATTC CRL-TIB-196). Other useful cell lines include those derived from Chinese hamster ovary (CHO) cells such as CHO-K1SV (Lonza Biologics, Walkersville, MD,), CHO-K1 (ATCC CRL-61) or DG44.
[0152] [000152] Another embodiment of the invention is a method of producing an antibody reactive with IL-17A which comprises culturing a host cell of the invention and recovering the antibody produced by the host cell. Methods of making antibodies and purifying them are well known in the art. For expression, the heavy chain sequences of the projected families 2, 6a, 6b, 19a and 19b can include an N-terminal leader sequence such as MAWVWTLLFLMAAAQSIQA (SEQ ID NO: 109). Examples of nucleotide sequences encoding the mAb6785 candidate heavy chain (family 19) with a leader sequence and the mature form (without a leader sequence) are shown in SEQ ID No. 101 and 102, respectively. Similarly, for expression, antibody light chain sequences of families 2, 6a, 6b of the invention can include an N-terminal leader sequence such as MGVPTQVL-GLLLLWLTDARC (SEQ ID NO: 110) and antibody light chain sequences 19a and 19b of the family of the invention may include an N-terminal leader sequence such as MAWSPLLLTLLAHCTGSWA (SEQ ID No. 116). Examples of nucleotide sequences encoding the codon mAb6785 light chain optimized with a leader sequence and the mature form (without a leader sequence) are shown in SEQ ID NOs 103 and 104, respectively.
[0153] [000153] Another embodiment of the invention is a hybridoma cell line that produces an antibody of the invention. Treatment methods
[0154] [000154] The IL-17A antagonists of the invention, for example IL-17A antibody antagonists, can be used in any therapy where it is desired to reduce the effects of IL-17A on the animal patient. IL-17A may circulate through the body or may be present at an undesirably high level in a particular location on the body, for example, a site of inflammation. Without adhering to any specific theory, the antagonists of the invention provide beneficial therapy by preventing or reducing the binding of IL-17A to its receptor, or the homo- or hetero-rhodimerization of IL-17A. The methods of the invention can be used to treat an animal patient belonging to any classification. Examples of such animals include mammals such as humans, rodents, dogs, cats and farm animals.
[0155] [000155] The antibodies of the invention may be useful for the prophylaxis and treatment of conditions mediated by IL-17A, such as inflammatory conditions, allergies and allergic conditions, reactions to hypersensitivity, autoimmune diseases, serious infections, and transplant rejection. organs or tissues. The antibodies of the invention are also useful in the preparation of a medicament for such treatment, the medicament being prepared for administration in the dosages defined herein. Examples of conditions mediated by IL-17A are inflammatory conditions, immunological and proliferative disorders, including rheumatoid arthritis (RA), ankylosing spondylitis, psoriatic arthritis, os-teoarthritis, osteoporosis, uveitis, inflammatory fibrosis (eg, scleroderma, pulmonary fibrosis , and cirrhosis), inflammatory bowel disorders (eg, Crohn's disease, ulcerative colitis and inflammatory bowel disease), asthma (including allergic asthma), allergies, COPD, multiple sclerosis, psoriasis, systemic Lupus erythematosus, diabetes and cancer. Positive results in patients treated with human an-ti-IL-17A therapies are described for rheumatoid arthritis, psoriasis and non-infectious uveitis (Genovese et al., Arthritis Rheum. 62: 929-39, 2010; Hueber et al., Sci Transl. Med. 2: 52ra72., 2010).
[0156] [000156] The pulmonary inflammatory condition is an example of an inflammatory condition. Examples of pulmonary inflammatory conditions include infection-induced lung conditions including those associated with viral, bacterial, fungal, parasitic or prion infections; allergen-induced lung conditions; pulmonary conditions induced by pollutants such as asbestosis, silicosis, or berylliosis; lung conditions induced by gastric aspiration, immune dysregulation, inflammatory conditions with genetic predisposition such as cystic fibrosis, and lung conditions induced by physical trauma, such as injuries caused by the ventilator. These inflammatory conditions also include asthma, emphysema, bronchitis, chronic obstructive pulmonary disease (COPD), sarcoidosis, histiocytosis, lymphangiomyomatosis, acute lung injuries, acute respiratory distress syndrome, chronic lung disease, bronchopulmonary dysplasia, community-acquired pneumonia, pneumonia nosocomial, ventilator-associated pneumonia, sepsis, viral pneumonia, influenza infection, parainfluenza infections, rotavirus infections, human metapneumovirus infection, respiratory syncytial virus infection and Aspergillus or other fungal infections. Exemplary inflammatory diseases associated with infection may include viral or bacterial pneumonia, including severe pneumonia, cystic fibrosis, bronchitis, airway exacerbations, and acute respiratory distress syndrome (ARDS). Such conditions associated with infection can involve multiple infections such as a primary viral infection and a secondary bacterial infection. The production of deregulated IL-17A may play a role in the pathology of lung diseases such as asthma and chronic obstructive pulmonary disease (COPD) (reviewed in Alcorn et al., Annu. Rev. Physiol. 72: 495-516, 2010). IL-17A has been shown to regulate neutrophilic inflammation in the lungs - a milestone in the treatment of severe asthma, as well as COPD - due to IL-17A's ability to induce important factors in neutrophil recruitment, survival and activation from cells epithelial cells resident in the lungs (for example, IL-6, IL-8, GM-CSF, G-CSF). The antibodies of the present invention suppress the secretion of IL-6, IL-8 and GM-CSF by the epithelial cells of the lungs, and, therefore, can be beneficial in the therapeutic or prophylactic treatment of patients with inflammatory lung conditions, such as asthma and COPD . Commonly used animal models of asthma and airway inflammation include the ovalbumin challenge model and methacholine sensitization models (Hessel et al., Eur. J. Pharmacol. 293: 401-12, 1995). Inhibition of cytokine and chemokine production from cultured human epithelial and bronchial cells, bronchial fibroblasts or airway smooth muscle cells can also be used as in vitro models. The administration of antagonists of the present invention in any of these models can be used to evaluate the use of these antagonists to improve symptoms and alter the course of asthma, airway inflammation, COPD and the like.
[0157] [000157] Psoriasis is another example of an inflammatory condition. Psoriasis is characterized by hyperproliferation mediated by keratinocyte T cells coupled with an inflammatory infiltrate. Inflammation and hyperproliferation of psoriatric tissue are associated with a histological, antigenic and cytokine profile that differs from normal skin. Among the cytokines associated with psoriasis are: TNFa, IL-19, IL-18, IL-15, IL-12, IL-7, IFNy, IL-17A and IL-23 (Gudjonsson et al., Clin. Exp. Immunol 135: 1-8, 2004). IL-17A has been found to be clearly expressed in psoriatric lesions (US Patent No. 7,776,540) and positive results are reported in patients treated with anti-human IL-17A therapies (Hueber et al., Sci. Transl. Med 2: 52ra72., 2010).
[0158] [000158] Arthritis, including osteoarthritis, rheumatoid arthritis, arthritic joints resulting from injuries, and the like, are common inflammatory conditions that would benefit from the therapeutic use of anti-inflammatory proteins, such as the antagonists of the present invention. Activation of IL-17A signaling can perpetuate inflammation and further damage tissue in the inflamed joint. Several animal models of rheumatoid arthritis are known. For example, in the collagen-induced arthritis (ASD) model, mice develop chronic inflammatory arthritis that is very similar to human rheumatoid arthritis. Administration of the IL-17A antibodies of the present invention to the CIA mouse model can be used to evaluate the use of these antagonists to improve symptoms and alter the course of diseases.
[0159] [000159] Examples of inflammatory gastrointestinal conditions are inflammatory bowel disease (IBD), ulcerative colitis (UC) and Crohn's disease (CD), colitis induced by environmental aggressions (eg, gastrointestinal inflammation (eg, colitis) caused by or associated with (for example, as a side effect) a therapeutic regimen, such as administration of chemotherapy, radiation therapy, and the like), infection colitis, ischemic colitis, collagen or lymphocytic colitis, necrotizing enterocolitis, colitis in conditions such as granuloma-tosa disease or celiac disease, food allergies, gastritis, gastritis or infectious endo-rocolitis (for example, chronic active gastritis infected with Helicobacter pylori) and other forms of gastrointestinal inflammation caused by an infectious agent. There are several animal models for gastrointestinal inflammatory conditions. Some of the most used models are the ethanol / 2,4,6-trinitrobenesulfonic acid (TNBS) colitis model or the oxazalone model, which induces chronic ulceration and inflammation in the colon (Neurath et al., Intern. Rev. Immunol 19 : 51 to 62, 2000). Another model uses sodium dextran sulfate (DSS), which induces an acute colitis manifested by blood diarrhea, weight loss, colon shrinkage and mucosal ulceration with neutrophil infiltration. Another model involves the adoptive transfer of naive (virgin) T cells from high CD45RB CD4 to RAG or SCID mice. In this model, virgin donor T cells attack the recipient intestine causing chronic inflammation of the digestive system and symptoms similar to human inflammatory bowel diseases (Read and Powrie, Curr. Protoc. Immunol. Chapter 15 unit 15.13, 2001). The administration of antagonists of the present invention in any of these models can be used to assess the potential effectiveness of those antagonists in improving symptoms and altering the course of diseases associated with inflammation in the intestine, such as inflammatory bowel disease.
[0160] [000160] Renal fibrosis can develop from an acute aggression (for example, ischemia / reperfusion graft) (Freese et al., Nephrol. Dial. Transplant. 16: 2401-6, 2001) or chronic condition (for example , diabetes) (Ritz et al., Nephrol. Dial. Transplant. 11 Suppl 9: 38-44, 1996). Pathogenesis is typically characterized by an initial inflammatory response followed by sustained fibrogenesis of the glomerular filtration apparatus and tubular interstitium (Liu, Kidney Int. 69: 213-7, 2006). Tubulointerstitial fibrosis has been shown to play a critical role in the pathogenesis of renal lesions for end-stage renal failure and the proximal tubule cell has proved to be a central mediator (Phillips and Steadman, Histol. Histopa-thol. 17: 247- 52, 2002; Phillips, Chang Gung Med. J. 30: 2-6, 2007). Fibrogenesis in the tubulointerstitial compartment is mediated in part by the activation of resident fibroblasts that secrete pro-inflammatory cytokines that stimulate the proximal tubule epithelium to secrete local inflammatory and fibrogenic mediators. Additionally, chemotactic cytokines are secreted by fibroblasts and epithelial cells and provide a directional gradient that guides the infiltration of monocytes / macrophages and T cells into the tubulointerstitium. The inflammatory infiltrate produces additional fibrogenic and inflammatory cytokines that further activate the release of fibroblasts and epithelial cytokine while stimulating the submission of the epithelium to a phenotypic transition in which cells deposit excess extracellular matrix components (Simonson, Kidney Int. 71: 846-54, 2007). IL-17A has been shown to be activated during human kidney transplant rejection (Van Kooten et al., J. Am. Soc. Nephrol. 9: 1526-34, 1998; Loong et al., J. Path. 197 : 322-32, 2002). IL-17A stimulates the production of the pro-inflammatory mediators IL-6, IL-8, component of complement C3, and RANTES by proximal tubular epithelium (Van Kooten et al., J. Am. Soc. Nephrol. 9: 1526- 34, 1998; Woltman et al., J. Am. Nephrol. 11: 2044-55, 2000). These factors, in turn, mediate the recruitment of other types of inflammatory cells into the interstitium, which contribute to the maintenance of the inflammatory / immune response and, if not suppressed, to the onset of fibrosis and chronic transplant necropathy ( Racusen et al., Kidney Int. 55: 71323, 1999; Mannon, Am. J. Transpl. 6: 867-75, 2006).
[0161] [000161] Other exemplary fibrotic conditions may include fibrosis of the liver (including, but not limited to, alcohol-induced cirrhosis, virus-induced cirrhosis, autoimmune-induced hepatitis); fibrosis of the lungs (including, but not limited to, scleroderma, idiopathic pulmonary fibrosis); kidney fibrosis (including, but not limited to, scleroderma, diabetic nephritis, glomerular nephritis, lupus nephritis); dermal fibrosis (including, but not limited to, scleroderma, hypertrophic and keloid scarring, burns); myelofibro-se; neurofibromatosis; fibroma; intestinal fibrosis; and fibrotic adhesions that result from surgical procedures. Fibrosis can be specific organ fibrosis or systemic fibrosis. Organ-specific fibrosis can be associated with fibrosis of the lungs, fibrosis of the liver, fibrosis of the kidney, fibrosis of the heart, vascular fibrosis, fibrosis of the skin, fibrosis of the eye or bone marrow fibrosis. Lung fibrosis may be associated with idiopathic pulmonary fibrosis, drug-induced pulmonary fibrosis, asthma, sarcoidosis or chronic obstructive pulmonary disease. Liver fibrosis can be associated with cirrhosis, schistosomiasis or cholangitis. Cirrhosis can be selected from alcoholic cirrhosis, post-hepatitis C cirrhosis, primary biliary cirrhosis. Cholangitis can be sclerosing cholangitis. Kidney fibrosis may be associated with diabetic nephropathy or lupus glomeruloscelerosis. Heart fibrosis may be associated with myocardial infarction. Vascular fibrosis may be associated with arterial restenosis after angioplasty or atherosclerosis. Skin fibrosis can be associated with burn healing, hypertrophic, keloid scarring or nephrogenic fibrosing dermatology. Fibrosis of the eye may be associated with retro-orbital fibrosis after cataract surgery or proliferative vitreous-retinopathy. Bone marrow fibrosis may be associated with idiopathic myelofibrosis or drug-induced myelofibrosis. Systemic fibrosis can be systemic sclerosis or graft-versus-host disease.
[0162] [000162] Other inflammatory conditions and neuropathies that can be prevented or treated by the methods of the invention are those caused by autoimmune diseases. These conditions and neuropathies include multiple sclerosis, erythematous systemic lupus, and neuro-degenerative and central nervous system (CNS) disorders including Alzheimer's disease, Parkinson's disease, Huntington's disease, bipolar disorder and amyotrophic lateral sclerosis (ALS), liver disease including primary biliary cirrhosis, primary sclerosing cholangitis, steatohepatitis / non-alcoholic fatty liver disease, fibrosis, hepatitis C virus (HCV) and hepatitis B virus (HBV), diabetes and insulin-resistant cardiovascular disorders including atherosclerosis, cerebral hemorrhage, stroke and myocardial infarction, arthritis, rheumatoid arthritis, psoriatic arthritis and juvenile rheumatoid arthritis (JRA), osteoporosis, osteoarthritis, pancreatitis, fibrosis, encephalitis, psoriasis, giant cell arthritis, ankylosing spondolitis, autoimmune hepatitis, virus (HIV), inflammatory skin conditions, transplantation, cancer, allergies, endocrine diseases, skin repair injuries, other autoimmune disorders, airway hyperresponsiveness, and cell, virus and prion-mediated infections or disorders. Pharmaceutical Administration / Compositions
[0163] [000163] The "therapeutically effective amount" of the agent effective in treating conditions where suppression of IL-17A activity is desirable can be determined using standard screening techniques. For example, the dosage of the agent that will be effective in treating an inflammatory condition such as asthma, Crohn's disease, ulcerative colitis or rheumatoid arthritis can be determined by administering the agent in relevant animal models, such as the models described herein.
[0164] [000164] In addition, in vitro tests can optionally be employed to assist in the identification of ideal dosage ranges. The selection of a particular effective dosage can be determined (for example, through clinical tests) by those skilled in the art based on consideration of several factors. Such factors include the disease to be treated or prevented, the symptoms involved, the patient's body mass, the patient's immune status and other factors known to those skilled in the art. The dosage that needs to be used in the formulation will also depend on the route of administration and the severity of the disease, and should be decided according to the judgment of the professional and the circumstances of each patient. Effective dosages can be extrapolated from these dosage response curves derived from animal model or in vitro test systems.
[0165] [000165] The mode of administration for therapeutic use of the agent of the invention can be any suitable route that delivers the agent to the host. The pharmaceutical compositions of these agents are particularly useful for parenteral administration, for example, in-translation, intramuscular, intraperitoneal, intravenous, subcutaneous or intranasal.
[0166] [000166] The agent of the invention can be prepared as a pharmaceutical composition that contains an effective amount of the agent as an active ingredient in a pharmaceutically acceptable carrier. The term "vehicle" refers to a diluent, adjuvant, excipient or vehicle with which the active compound is administered. Such pharmaceutical vehicles can be liquids, such as water and oils, including those derived from petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. For example, 0.4% saline and 0.3% glycine can be used. These solutions are sterile and, in general, free of particulate matter. They can be sterilized using conventional well-known sterilization techniques (for example, filtration). The compositions can contain pharmaceutically acceptable auxiliary substances as required to bring them closer to physiological conditions such as buffering and pH adjusting agents, stabilizing agents, thickeners, lubricants and dyes, etc. The concentration of the agent of the invention in such a pharmaceutical formulation can vary widely, that is, from less than about 0.5%, usually at or at least about 1% to as much as 15 or 20% by weight, and will be selected primarily based on the required dosage, fluid volume, viscosities, etc., according to the particular mode of administration selected.
[0167] [000167] Thus, a pharmaceutical composition of the invention for intramuscular injection could be prepared to contain 1 ml of sterile buffered water, and between about 1 ng to about 100 mg, for example, about 50 ng to about 30 mg or, more preferably, about 5 mg to about 25 mg, of an IL-17A antagonist antibody of the invention. Similarly, a pharmaceutical composition of the invention for intravenous infusion could be prepared to contain about 250 ml of sterile Ringer's solution, and about 1 mg to about 30 mg and, preferably, 5 mg to about 25 mg of an antagonist of the invention. Current methods for preparing parenterally administrable compositions are well known and are described in more detail in, for example, "Remington's Pharmaceutical Science", 15th edition, Mack Publishing Company, Easton, PA, USA.
[0168] [000168] The antibody antagonists of the invention can be lyophilized for storage and reconstituted in a suitable vehicle before use. This technique has been shown to be effective with conventional immunoglobulins and in protein preparations, and the reconstitution and lyophilization techniques known in the art can be employed.
[0169] [000169] The present invention will now be described with reference to the following specific non-limiting examples. Example 1 Identification of anti-human IL-17A antagonist mAbs
[0170] [000170] The MorphoSys Human Combinatorial Antibody Library (HuCAL®) Gold "phage display" library (Morphosys AG, Mar-tinsried, Germany) was used as a source of human antibody fragments and was screened using the "panning" technique in solution subgroups. In the first round of "panning", the sub-libraries were selected according to the mature biotinylated IL-17A A132Q, identified as His6 and variant A70Q (IL-17Amut6) (SEQ ID No. 106). In the second round, the amplified result from round 1 was selected on the basis of biotinylated IL-17Amut6, identified as His6, in the presence or absence of other members of the IL-17A family as a competitor to polarize against antibodies that were specific to IL -17A. The amplified result of round 2 was divided into two groups. The first group was screened using the "panning" technique as in round 1. The clones in the second group were additionally diversified into HCDR2 or LCDR3, depending on the sub-library used in the initial selections and then subjected to 2 additional rounds of "panning. "based on IL-17Amut6 to provide a second source of clones for screening. Fabs from clone lysates were captured in ELISA plate wells coated with sheep anti-human Fd antibody and screened for binding to biotinylated IL-17Amut6. Crude lysates from positive clones were screened for inhibition of binding of IL-17Amut6 to the recombinant human IL-17RA receptor (SEQ ID No. 107).
[0171] [000171] The selected clones were chosen for further characterization as purified Fabs based on the score ("scoring"), affinity and sequence representation of all sequence families, and were designated with MOR numbers. Additional variants for MOR7708, MOR7785, MOR7706, MOR7775 and MOR7700 have been generated to replace Trp or Met resident in HCDR2, HCDR3 or LCDR3. Table 3 shows the generated variants.
[0172] [000172] The Fabs were assessed for their inhibition of IL-17Amut6 and cynoIL-17A (crab monkey) binding to the recombinant human IL-17RA receptor, and for their binding to IL-17Amut6. All evaluated Fabs inhibited the binding of IL-17Amut6 and cynoIL-17A to IL-17RA. The Fabs' affinity for IL-17Amut6 was measured using the SET test (Table 4). From the identified Fabs, candidates from families 2, 6a, 6b, 19a and 19b were selected for further characterization.
[0173] [000173] The selected MOR number Fabs were converted and expressed as mAbs in a human IgG1 format, and were given the corresponding MORmAb designation. The generated MORmAbs were evaluated for expression and aggregation, their ability to inhibit the binding of human IL-17A and cyno (crab monkey) to human IL-17RA, and for IL-8 secretion from NHDF cells. Table 5 shows the IC50 values for the selected MORmAbs tests. None of the evaluated MORmAbs (MORmAbs numbers 7702, 7708, 7785, 7786, 7706, 7775, 7700, 8095, 8096, 8097, 8098, 7768) cross-reacted with other members of the IL-17 family.
[0174] [000174] Based on biophysical and biochemical activity and properties, the selected MORmAbs were additionally designed in their variable regions to alter certain structure residues in those present in a corresponding human germline and to alter codons in those with a higher frequency of occurrence in proteins of highly expressed mammals. In families 2 VL, L11V and V85T (linear sequence) substitutions were made, converting the structure into an exact pair with the Vb-L5 (IGKV1-12 * 01) of VK-1 germline. A variable region with the exemplary V11V and V85T substitutions is the variable region having the amino acid sequence shown in SEQ ID No. 76. In families 6a and 6b VL, D1E, V59I and T86V substitutions were made (linear sequence) converting the structure in an exact pair with Vb-L6 (IGKV3-11 * 01) of germline Vk-3. A variable region with the exemplary D1E, V59I and T86V substitutions is the variable region having the amino acid sequence shown in SEQ ID No. 77. In families 6a and 6b VH, a G44S (linear sequence) substitution was made to match the Vb 6-01 (IGHV6-1 * 01) of Vh-6 germline. A variable region with the exemplary G44S substitution is the variable region having the amino acid sequence shown in SEQ ID No. 81. In families 19a and 19b VL, amino acids 1-3 (DIE) have been replaced by QSV to replace the kappa N - artificial terminal for that of a lambda chain. A variable region with exemplary QSV substitution is a variable region having the amino acid sequence shown in SEQ ID No. 79. In families 19a and 19b VH, a V5L substitution was made to provide a close match for Vb 3-23 (IGHV3 -23 * 01 of germline Vh-3. In addition, in this process, the residues of the amino acid sequence of the heavy chain constant region 353-357 (REEMT) were replaced by RDELT, a variable region with the substitution of exemplary V5L is a variable region having the amino acid sequence shown in SEQ ID No. 86. An example of a heavy chain with substitutions 353-357 REEMT -> RDELT of the constant region is a heavy chain having the amino acid sequence shown in SEQ ID No. 100 Projected antibodies were assigned mAb numbers.
[0175] [000175] The corresponding designations and sequence listings of the projected and original variable regions and that of the full length antibodies are shown in Table 2. The CDR sequences for each family are shown in Figure 1.
[0176] [000176] The projected mAbs were characterized as described above for MORmAbs. IC50 values (pM) measured using the indicated tests are shown in Table 6.
[0177] [000177] The affinity of mAbs was assessed using Biacore. The measurement results are shown in Table 7.
[0178] [000178] IL-17A has been shown to regulate neutrophilic inflammation in the lungs, a milestone in the treatment of severe asthma, as well as COPD, due to the ability of IL-17A to induce important factors in neutrophil recruitment, survival and activation (for example, IL-6, IL-8, GM-CSF). To determine whether the anti-IL-17A antibodies of the invention can inhibit IL-17A-induced changes in cells resident in the lungs, normal human bronchial epithelial cells (NHBE) were stimulated with human IL-17A for 48 hours in the presence of mAb6785. MAb6785 inhibited IL-17A-induced IL-6 and GM-CSF production by NHBE cells with IC50 = 619.0 ± 64.0 pM and 564 ± 86 pM, respectively. The anti-IL-17 antibody inhibits the biological activity of the IL-17A / F heterodimer
[0179] [000179] Normal human dermal fibroblast cells (NHDF; Lonza) were implanted in a 48-well flat-bottom tissue culture plate at a rate of 10,000 cells per well in FGM-2 medium (Lonza) and incubated for one day for the other (37 °, 5% CO2). After incubation, 50 ng / mL of final concentration (1.47 nM) of rhIL-17A / F heterodimer (R&D Systems) was preincubated with a series of dilutions (30 μg / mL - 0.5 ng / mL ) of mAb6785 or control antibodies for 10 minutes at room temperature, and added to the cells. The cells were incubated for 48 hours (37 °, 5% CO2) and culture supernatants were collected and evaluated via ELISA for IL-6 content using double sets of human IL-6 (R&D Systems, Inc.) according to the manufacturer's instructions. IC50 values were determined by non-linear regression using the GraphPad Prism software (GraphPad Software, Inc). MAb6785 inhibited the production of IL-6 induced by the IL-17A / F heterodimer by NHDF cells with EC50 2 ± 2.5 nM. Methods Determination of picomolar affinities using solution equilibrium titration (SET)
[0180] [000180] For the determination of Kd by solution balance titration (SET), fractions of monomers (at least 90% monomer content, analyzed by analytical SEC; Super-dex75 column, GE) of the Fab protein were used.
[0181] [000181] The affinity determination based on the electrochemiluminescence (ECL) in solution and the data evaluation were performed basically as previously described (Haenel et al., Anal Biochem 339: 182-4, 2005). A defined fixed concentration of purified Fab (~ 10-100 pM) was incubated with increasing concentrations of IL-17Amut6 (highest concentration of 5 nM) in solution until chemical equilibrium was reached. To quantify unbound Fab in solution, the samples were transferred to a 384-well streptavidin MSD microtiter plate (Meso Scale Discovery, Gaithersburg, MD, USA) with coated biotinylated IL-17Amut6. For detection, an anti-human Fab / IgG antibody labeled with ruthenium complex was applied and the plates were read with Sector ™ Imager 6000 (MSD). The titration curves (free Fab concentration as a function of antigen concentration) were plotted and adjusted with Excel / XLfit software using the model described below.
[0182] [000182] For the evaluation of data for Kd the determination of Fab molecules, the following adjustment model was used (modified according to Abraham et al. J Mol Recognit. 9: 456-461, 1996): y = Bmax- (Bmax / (2 * cFab) * (x + cFab + KD-sqrt ((x + cFab + KD) * (x + cFab + KD) -4 * x * cFab)))
[0183] [000183] where:
[0184] [000184] Bmax: maximum binding signal (with antigen concentration = 0)
[0185] [000185] cFab: applied Fab concentration
[0186] [000186] x: total soluble concentration of applied antigen (binding sites)
[0187] [000187] sqrt: square root
[0188] [000188] Kd: equilibrium dissociation constant Inhibition of IL-17A binding to IL-17RA (for example, "IL-17RA inhibition" test
[0189] [000189] Clear maxisorp plates were coated with 100 pl / well of 2.5 pg / ml human IL-17RA-Fc (R&D Systems, Minneapolis, MN) in 0.1 M sodium carbonate-bicarbonate buffer, pH 9.4 and incubated overnight at 4 ° C. After blocking and washing, 25 ng / ml of human IL-17mut6 (SEQ ID No. 106) or biotinylated IL-17A cynomolgus (SEQ ID No. 108) was pre-incubated with tested mAbs or control mAbs (30 to 0 pg / ml final concentration) in a combined volume of 100 pl for 5-10 minutes and then added to the plates. The signal was detected with 100 µl dilution 1: 10,000 of 1 mg / ml SA-HRP (Jackson Immunorese-arch, West Grove, PA) for 20 minutes at room temperature (RT) followed by 100 µl / well of OPD substrate (Sigma-Aldrich Corp., St. Louis, MO). The plates were read at 492 nm (Envision, Per-kinElmer, Waltham, MA). Fab binding to IL-17RA was tested as described for mAbs. Inhibition of the production of IL-8 and IL-6 from NHDF cells (for example, "IL-8 production" and "IL-6 production" tests)
[0190] [000190] The effect of inhibiting anti-IL-17A mAbs on IL-8 and IL-6 production was evaluated in normal human dermal fibroblasts (NHDF). The cells were placed in a 48-well flat-bottom tissue culture plate at a rate of 0.1 x 105 cells per well, 250 µl per well in FGM-2 medium and incubated overnight (37 °, 5 ° C). % CO2). After incubation, 0.1 ng / ml of human TNF-α was added to all wells. 10 ng / ml IL-17mut6 or 25 ng / ml IL-17A cynomolgus were preincubated with tested mAbs or control mAbs (30-0 μg / ml final concentration) in a combined volume of 250 μl for 10 minutes RT and then added to 250 μl of cells. In the tests, samples of IL-17mut6 without the addition of antibodies were included as control samples, while samples consisting of TNF-α or culture medium were only included as negative controls. The cells were incubated for 24 hours (37 °, 5% CO2) and conditioned media were collected and evaluated by ELISA for IL-6 and IL-8 content using double IL-6 & IL- ELISA sets 8 human in accordance with the manufacturer's instructions (R&D Systems, Minneapolis, MN). Fabs were assessed as described for mAbs. Inhibition of IL-6 and G-CSF production by NHBE cells
[0191] [000191] Normal human bronchial epithelial cells (NHBE; Lonza) were implanted at a rate of 20,000 cells per well in BEGM media (Lonza) and incubated overnight (37 °, 5% CO2). After incubation, cells were stimulated with IL-17Amut6 for 48 hours in the presence of the tested antibodies in a range of concentrations (30 μg / mL - 0.5 ng.mL). Supernatants were collected after incubation and assessed for IL-6 or G-CSF content using an ELISA specific for human IL-6- or G-CSF (R&D Systems, Inc.). IC50 values were determined by non-linear regression using the GraphPad Prism software (GraphPad Software, Inc). Cross-reactivity with members of the IL-17A family
[0192] [000192] Clear maxisorp plates were coated with 100 μl / well of 5 μg / ml mAbs or mAbs isotype control in PBS, and incubated overnight at 4 ° C. The plates were blocked with 200 μl / well for 1 hour with ELISA block buffer (1% BSA, 5% sucrose in PBS with 0.05% NaN3) and washed three times with wash buffer (PBS, 0, 01% Tween-20). The competing cytokines were titrated in test diluent buffer (1% BSA in PBS) at 2x the final concentration, and the biotinylated cytokine was prepared at 2x the final concentration. 100 μl of cytokines at 2x the final concentration were mixed (30-0 μg / ml final concentration) with 100 μl of biotinylated IL-17mut6 at 2x the final concentration (25 ng / ml final concentration) in test buffer. The recombinant human IL-23 (R&D Systems, Minneapolis, MN) was used as a negative control, buffer-only sample as the base control, and IL-17mut6 as a positive control. 100 μl per well in duplicate biotinylated cytokine / IL-17mut6 mixture was added to the plate and incubated for 1-2 hours. The plates were washed three times with wash buffer, and incubated with 100 μl of a 1: 10,000 dilution of 1 mg / ml SA-HRP (Jackson Immunoresearch, West Grove, PA) for 20 minutes at RT. The plates were washed three times with ELISA wash buffer. After washing, 100 μl / well of OPD substrate (Sigma-Aldrich Corp., St. Louis, MO) was added to each well and incubated until an appropriate color change was detected. The reaction was stopped with the addition of 50 μl of 2N sulfuric acid, and the plate reading was 492 nm using the Envision instrument. Affinity measurements - Biacore test
[0193] [000193] Affinity measurements using surface plasmon resonance (SPR) were performed using a Biacore 3000 optical biosensor (Biacore). The selected Fabs (~ 30 RU) or mAbs (~ 50 RU) were captured on the surface of the sensor's integrated circuit using an anti-sheep Fd antibody or an anti-human Fc antibody to capture the Fabs or mAbs, respectively. The capture of Fab or mAb was followed by the injection of huIL-17mut6 or cyno IL-17A in solution (0.2 to 49 nM). Example 4 Epitope Mapping
[0194] [000194] The antibody epitopes were elicited by a combination of competitive binding, H / D exchange analysis, and IL-17A antibody co-structure (see Example 5). The following antibodies were used: mAb1926, MORmAb7700, MORmAb7706, MORmAb7708, mAb7357 (a mouse anti-human IL-17A neutralizing antibody derived from the C1863 hybridoma hybridoma), mAb2832 (a mouse chimeric anti-human IL-17A neutralizing antibody / human derived from hybridoma C1861), mAb317 (mouse anti-human anti-IL-17A antibody, R&D Systems, Minneapolis, MN) and mAb3171 (mouse anti-human IL-17A antibody, R&D Systems, Minneapolis, MN ), and mAbeBIO16-7178 (a mouse anti-human IL-17A antibody, e-Bioscience, San Diego, CA). The three commercial antibodies showed varying degrees of neutralizing activity. Competitive Epitope Liaison
[0195] [000195] For competitive ELISA, 5 μΙ (20 μg / ml) of IL-17Amut6 protein was used as coated on an MSD HighBind plate (Meso Scale Discovery, Gaithersburg, MD, USA) per well for 2 hours at room temperature . 150 μ! 5% of MSD blocking buffer A (Meso Scale Discovery, Gaithersburg, MD, USA) was added to each well and incubated for 2 hours at room temperature. The plates were washed with 0.1 M HEPES buffer (pH 7.4). 10 nM of labeled antibodies (MDS fluorescence dye) were incubated with increasing concentrations of competing antibodies (1 nM - 2 μM), and 25 μl of the mixture was added to the designated wells. After 2 hours of incubation with gentle shaking at RT, the plates were washed as described above, 150 μΙ of diluted T reading buffer from the MSD was added, and the plates were read with an MDS Sector Imager 6000.
[0196] [000196] The tests were performed with mAb1926, mAb317, mAb3171, or labeled mAb7357 (Figure 4). Based on the competition tests, anti-IL-17A antibodies were assigned to four different trays. tray A: mAb1926, MORmAb7706 and MOR-mAb7708; tray B: eBio16-7178 and mAb7357; tray C: mAb317; tray D: mAb3171. H / D exchange analysis:
[0197] [000197] For H / D exchange, the procedure used to analyze the antibody disturbance was similar to that previously described (Hamuro et al., J. Biomol. Techniques, 14: 171-82, 2003; Horn et al., Biochemistry, 45: 8488-98, 2006) with some modifications. The recombinant IL-17Amut6 (expressed in HEK293E cells with His-tag C-terminal) was incubated in a solution of deuterated water for predetermined periods resulting in the incorporation of deuterium in exchangeable hydrogen atoms. The deuterated IL-17Amut6 was captured in a column containing individual immobilized anti-IL-17A mAbs and then washed with aqueous buffer. The swapped IL-17Amut6 protein was eluted from the column and the location of deuterium-containing fragments was determined by protease digestion and mass spectrometry analysis. The regions bound to the antibody were inferred to be the sites relatively protected against exchange and, therefore, containing a greater fraction of deuterium, compared to IL-17Amut6 not complexed with antibody. The IL-17Amut6 H / D exchange disturbance maps are shown in Figure 5. The numbers above the bars refer to IL-17Amut6 residues.
[0198] [000198] The antibodies MORmAb7700, MORmAb7706 and MOR-mAb7708 showed varying degrees of differential exchange for three segments of IL-17A (SEQ ID No. 105) 45NRSTSPWNLH54 (SEQ ID No. 159), 56NEDPERYPSVIWE68 (SEQ ID No. 157) and 100RREPPHCPNSFRLEKIL116 (SEQ ID n ° 158), indicating protection by antibodies. The 56NEDPERYPSVIWE68 fragment (SEQ ID No. 157) was strongly protected by MORmAb7708, weakly protected by MORmAb7700, and not protected by MORmAb7706. The overlap in protection patterns of fragments of these antibodies is consistent with their cross-inhibition in the competition tests described above.
[0199] [000199] For both mAb7357 and mAbeBio16-7178, strong protection was observed for 71CRHLGCINADGNVDYHM87 (SEQ ID No. 160) consistent with its cross-inhibition in the competition tests described above. Weak, and therefore inconclusive, differential exchange was observed for other fragments with mAb7357, mAb2832, mAb317 and mAb3171.
[0200] [000200] H / D exchange studies have located the binding sites for two of the four competition groups defined above. Antibodies from tray A (MORmAb7700, MORmAb7706 and MOR-mAb7708) bound to the region of the 45NRSTSPWNLH54 peptide segments, (SEQ ID No. 159), 56NEDPERYPSVIWE68 (SEQ ID No. 157) and 100RREPPHCPNSFRLEKIL116 (SEQ ID No. ° 158) , from SEQ ID No. 105, and antibodies from tray B (mAb7357 and mAbeBio16-7178) bound in the 71CRHLGCINADGNVDYHM87 peptide segment region (SEQ ID No. 160). The antibodies mAb317 and mAb3171 bound to sites distinct from each other and from the antibodies in tray A and tray B. However, the weak signals in the H / D exchange studies with both antibodies did not provide enough evidence to locate their epitopes in the IL-17A. Example 5 Co-crystalline structure of IL-17A and anti-IL-17A antibody
[0201] [000201] The co-structure of IL-17Amut6 with Fab6468, a His6 re-combining Fab of mAb6785, was determined by X-ray crystallography. The light chain amino acid sequence of Fab6468 is shown in SEQ ID No. 90 , and the heavy chain amino acid sequence is shown in SEQ ID No. 111. In Example 5, the mentioned IL-17A amino acid residues indicate residues according to SEQ ID No. 105, and the mentioned Fab6468 residues indicate residues of the light chain variable region according to SEQ ID No. 79 and residues of the heavy chain variable region according to SEQ ID No. 86. The expression, refolding and purification of recombinant human IL-17Amut6 has been described ( Wu et al., Cytokine, electronic publication prior to printing in July / 29). Fab6468 was expressed in HEK-293Fe cells purified using a similar method as described (Zhao et al., Protein Expr Purif, 67: 182-9, 2009). Crystallization of the IL-17A / Fab6468 complex
[0202] [000202] The IL-17A / Fab6468 complex was prepared by mixing IL-17Amut6 and Fab6468 at a molar ratio of 1: 1.1 in 20 mM methyl ester sulfonate (MES), pH 6.5, 0.2 M NaCl, and 10% glycerol and incubated overnight at 4 ° C. The complex was purified from excess non-complexed Fab using size exclusion chromatography (SEC) on a Superdex 200 10/300 GL column (GE Healthcare, Piscataway, NJ) in 20 mM MES, pH 6 , 5, 0.2 M NaCl, and 10% glycerol. The fractions corresponding to the complex were pooled and concentrated with an Amicon Ultra 10000 PMC device at 4.6 mg / ml.
[0203] [000203] An automated crystallization screening was done using the robot for automatic crystallization of Oryx4 protein (Douglas Instruments, East Garston, UK) dispensing equal volumes of protein and reservoir solution in a droplet format in re-landing with the use of a Corning 3550 plate (Corning Inc., Corning, NY, USA). The initial screening was performed with a Hampton HT crystal screen (HR2-130, Hampton Research) and produced needle-like crystals from various conditions containing ammonium sulfate, PEGs at a pH of 4.5-4.6. These small crystals were used to produce a seed stock for screening in a micro-seed matrix (MMS) (D’Arcy et al., Acta Crystallographica, section D, 63: 550-4, 2007). Diffraction quality crystals were obtained from the MMS sorting in 0.1 M sodium acetate with a pH of 5.5, 12% PEG MME 5000 and 0.2 M lithium sulfate. X-ray data collection of the IL-17A / Fab6468 complex
[0204] [000204] For X-ray data collection, the crystals were immersed for a few seconds in the mother liquid supplemented with 24% glycerol, and quickly frozen in the nitrogen flow at 95 ° K. X-ray diffraction data was collected and processed using the Rigaku MicroMax ™ 007HF microfocus X-ray generator equipped with Osmic ™ Vari-Max ™ confocal optical elements, Saturn 944 CCD detector, and an X-stream cooling system ™ 2000 (Rigaku, Woodlands, TX, USA). Diffraction intensities were detected in a crystal rotation of 254 ° with an exposure time of 3 min per half-degree image up to the maximum resolution of 2.2 Å. The X-ray data were processed with the D * TREK program (Pflugrath, J., Acta Crystallographica, section D, 55: 1718-25, 1999). The crystal belonged to the monoclinic space group P2i with a = 73.40 Â, b = 64.04 Â, c = 145.61 Â and β = 95.39 °. Statistics on X-ray data are given in Table 8.
[0205] [000205] The crystal structure of IL-17A / Fab6468 was determined by molecular substitution with the use of Phaser (Read, Acta Crystallorg D Biol Crystallorg, 57: 1373-82, 2001). The research models were IL-17F (PDB ID 1JPY) (Hymowitz et al., EMBO J., 20: 5332-41, 2001) and a homology model for Fv (VH / VL), which was built based on in the anti-IL-13 CNTO607 antibody (PDB ID 3G6A) (Teplyakov et al., J. Mol. Biol. 389: 115-23. 2009) for both VH and VL, using a styler (Accelrys , CA). The two CL / CH1 constant domains were obtained from PDB ID 8FAB (Strong et al., Biochemistry, 30: 3739-48, 1991). The structure was refined using a PHENIX radioactive synchroton (Adams et al., J. 11: 53-5, 2004). Twice the non-crystallographic symmetry was initially imposed in the early stages of refining, but was relaxed in the final stages based on Rlivre. Manual adjustment and reconstruction of the model was performed using COOT (Emsley et al., Acta Crystallogr. D. Biol. Crystallogr. 60: 2126-32, 2004). The final Rcrist and Rlivre were 23.4% and 29.7%, respectively, for all 61,706 independent reflections at 2.2Â. Refining statistics are given in Table 9.
[0206] [000206] The structure of the complex was determined with high resolution (~ 2.2 A). IL-17A was shown to be an almost symmetrical homodimer in the crystal and bound to two Fab molecules. Antibody-antigen interactions were shown to be largely hydrophobic and in contrast to most antibodies, light chain CDRs made numerous important contacts . The overall molecular structure of the IL-17A / Fab6468 complex is shown in Figure 6A. The monomer of the IL-17A dimer adopted the general topology of a cystine node (Figure 6B). The two monomers were very similar with a RMSD Ca of 0.54 Á for the Cα atoms of the main chain. The general architecture of the IL-17A monomer cystine node was very similar to the IL-17F architecture with an RMSD of 0.71 Á for Ca 76 atoms (Figure 6B). Each IL-17A monomer was stabilized by three disulfide bonds. For chain B, three intra-chain disulfide bonds (C10-C106, C71-C121, C76-C123) were observed, while for chain A, the C10-C106 disulfide bond was not observed due to the disorder in these segments of the monomer. These last two disulfide bonds (C71-C121, C76-C123) stabilized the cystine knot architecture, analogous to IL-17F and NGF. The IL-17A chain B structural model included all residues 10-128 (residues 1-9 were disordered), while for residues in chain A only residues 21-29, 41104 and 109-127 were observed and the other residues 1-20, 30-40, 105-108 and 128 were absent due to disorder in the structure. For both Fabs, residues 1-2 of both light chains were disordered or had low electron density. The 3 C-terminal residues of the heavy and light chains, including the inter-chain disulfide bonds and the heavy-chain His-tag were disordered.
[0207] [000207] The ordered N-terminal segment of IL-17A (chain B) contained a short helical element (residues 8-12). The segment was folded back towards loop 3-4 of the same monomer and formed an intra-chain disulfide bond (C10-C106). In contrast, the equivalent segment of IL-17F reached the other monomer of the dimer and formed an inter-chain disulfide bond and linked the two mo-numbers covalently. The ordered parts of segments 17-39 of the two IL-17A monomers were exchanged, as in IL-17F. This exchange resulted in a recombination of these parts of the IL-17A dimer. Combined with the intra-molecular disulfide bond (C10-C106), the two N-terminal segments of IL-17A formed two inter-blocked monomers, which also gave rise to an apparent 26 kD dimer in the non-reducing SDS-Page.
[0208] [000208] The dimer of IL-17A was almost symmetrical for the four main β helices (helices 1-4) (Figure 6C). The Ca rmsd for residues 76 is 0.71 Á. The slight asymmetry originated from the two sources. First, chain A contained numerous disordered segments, mainly at the N-terminal. Only a small β helix (helix 0, residues 2226) was apparently ordered, while residues 10-40 of chain B were ordered with a helical segment (residues 12-16) and a helix β (helix 0, residues 21- 25). Second, although the four main β helices, connected by cystine, the two mo-numbers (40-128) of IL-17A were related by twice as much symmetry of rotation, the ordered parts of helix 0 did not overlap well when the main body was overlapped (not shown). Without further investigation, it is not clear whether this was an artifact of refolding the protein or if such a disposition exists in nature. The bioactivity of this species was similar to that of a reference IL-17A from a commercial source (also produced in E. coli) (R&D Systems, Minneapolis, MN) suggesting that the inter- or intra-chain disulfide bond of C10 -C106 is not important for your receiver connection. However, the current structure suggests that the N-terminal (1-20) and (30-39) segments are very flexible and their structures do not impact the activity of the folded dimer of IL-17A. The epitope and the paratope
[0209] [000209] The residues involved in the binding between IL-17A and Fab6468 are shown in Table 10. Due to the residues absent in protomer A in IL-17A and the slightly asymmetric nature of the IL-17A dimer, all epitope residues the two contact sites were not identical (Table 10 and Figure 7). However, there was a core set of residues that were identical, as well as their interactions. These residues were L26, R55, E57, P59, E60, R61, Y62, S64, V65, W67, R101, E102, P103 and F110 of IL-17A SEQ ID No. 105 (highlighted in black in Table 10), and constitute the core epitope for Fab6468. Table 10. Residues from the nucleus epitope are highlighted in black. The residues of the nucleus parotope are indicated in bold. Residues from the extended parotope are shown in parentheses.
[0210] [000210] Similarly, the contact residues of the antibodies at the two sites were not all identical. The residues involved in identical contacts for the residues of the nucleus epitope are referred to as the "nucleus paratope", and were composed of the following residues: light chain (LC): Y31, D49, Y90, F92, F93 (SEQ ID no 79); and heavy chain (HC): S52, T54, F57, Y59, Q99, L100 and T101 (SEQ ID No. 86) (Table 10). The residues of the nucleus paratope are shown in bold in Table 10. The additional "extended paratope" residues identified in a monomer binding to a specific IL-17A residue are shown in parentheses.
[0211] [000211] The H / D protection data for MORmAb7700 was in accordance with co-crystal studies, since all residues of the core epitope identified in the co-crystalline structure, except L26, were found within or along the edges of two of the protected segments identified by H / D exchange, 56NEDPERYPSVIWE68 (SEQ ID No. 157) and 100RREPPHCPNSFRLEKIL116 (SEQ ID No. 158) for MORmAb7700. All derivatives of the MORmAb7700 antibody, including MORmAb8302 and mAb1926, are assumed to have the same binding specificity as Fab6468, since they differ at most in one residue in the VH N-terminal region (see Example 1), 3 residues at the N-terminal of the VL (see Example 1), and 3 CDR residues (one in each H2, H3 and L3, Table 1a), none of which are part of the antibody parameter.
[0212] [000212] The structure of IL-17A characterized in this invention is very similar to the structure previously published, except that, due to the missing segments, the pocket cavity P2 (see below) was not identified in the previous work (structure 2VXS, available in Protein DataBank, http _ // www_rcsb_org / pdb / home / home_do; Gerhardt et al., J. Mol. Biol. 394: 905-21,2009).
[0213] [000213] The crystal structure of human IL-17F in the complex with IL-17RA has been mentioned (Ely et al., Nat. Immunology, 10: 1245-51, 2009). Due to the sequence and structural similarities of IL-17A and IL-17F, it is likely that IL-17A will interact with IL-17RA in a similar way to IL-17F. The molecular modeling by overlapping the structure of IL-17A in the Fab6468 complex obtained in this study on IL-17F in the mentioned IL-17F / IL-17RA complex showed that the Fab6468 segments would have steric conflicts with IL-17RA. One of these segments is located around the FF theme (residues 92 and 93 of SEQ ID No. 79) in the Fab6468 light chain CDR3. Thus, without sticking to any specific theory, it is suggested that Fab6468 would inhibit the function of IL-17A by blocking its interactions with IL-17RA and, by analogy, IL-17RC, although the mode of interaction between IL-17RC and IL-17A is not known at the molecular level.
[0214] [000214] Significant differences in the affinities of IL-17A and IL-17F for IL-17RA suggest that there may be significant differences in the details of the interactions of IL-17A and IL-17RA, the extent of which will be available only when the structure co-crystalline of IL-17A / IL-17RA is determined. This is implied by the identification of the pocket cavity P2 in this study, which is only partially identified in the IL-17F analog region in the mentioned IL-17F / IL-17RA crystalline structure (Ely et al., Nat. Immunology, 10: 1245- 51,2009).
[0215] [000215] Two deep, largely hydrophobic pockets have been identified on the surface of IL-17A along the dimer interface (Figure 8A, 5B). Pocket P1, which is analogous to a pocket initially discovered on IL-17F (Hymowitz et al., EMBO J, 20: 5332-41, 2001), consists of residues Q94, E95, L97 and K114 from monomer A and L53 , Y62, P63, V65, I66, W67, I96, V117 and V119 of monomer B, and vice versa. On one side of the dimer, pocket P1 is partially covered by segment 30-40, while on the other side it was completely open due to the fact that the segment is disordered. Since this segment appears to be flexible, pocket P1 would be accessible by other molecules. Pocket P2 is also composed of residues from both chains: V24, L26, I28, Y62, L99, R101, F110 and L112 of monomer A and V22, V24 and L112 of monomer B, and vice versa.
[0216] [000216] Although the details of the P2 pockets are slightly different due to the asymmetry of the IL-17A dimer as described above, the general geometry of the two P2 pockets is very similar. The two sets of waste that line the P1 and P2 pockets are well maintained between IL-17A and IL-17F (Figure 8C). However, in the IL-17F structure, pocket P2 is occupied by residues F10 and F11 (theme F10F11) (Figure 8B) (Hymowitz et al., EMBO J, 20: 5332-41, 2001). The FF theme is absent in human IL-17A; instead, the corresponding amino acid residues are I4 and P5 (residues 4 and 5 in SEQ ID No. 105) (Figure 8C). These residues have little chance of binding to the P2 pocket, as well as the FF theme, because they are much smaller than the phenylalanine residues, and most likely will not have enough affinity for the P2 pocket. Thus, the FF theme of IL-17F is probably a structural discriminant for the interactions of human IL-17A and IL-17F with the IL-17RA and IL-17RC receptors. These two largely hydrophobic pockets (P1 and P2) are likely to be necessary for inhibiting the binding of IL-17A to IL-17RA. The recent crystalline structure of the IL-17F / IL-17RA complex shows that the FF theme is displaced by the IL-17RA (Ely et al., Nat. Immunology 10: 1245-51, 2009). The energy penalty for expelling the FF theme from the P2 pocket probably results in a lower binding affinity. This is consistent with observations that IL-17RA binds to IL-17A with a very high degree of affinity, but to IL-17F with a very low degree of affinity in humans (Kuestner et al., J Immunol, 179: 5462-73, 2007), and could potentially explain the differences in IL-17A and IL-17F powers. In mice, the FF theme is absent in IL-17A in IL-17F, and is replaced by IP and AL residues, respectively. The AL and IP residue pairs are small and probably have a very low degree of affinity for the P2 pocket. Thus, P2 would be available on IL-17A and IL-17F from mice for binding to IL-17RA. Mouse IL-17A and IL-17F bind mouse IL-17RA with similar affinity (Kuestner et al., J Immunol, 179: 5462-73, 2007), consistent with the present suggestion that availability of the P2 connection pocket increases the affinity of the ligands.
[0217] [000217] In general, the structural differences observed between IL-17A and IL-17F provide a basis for dissecting their interactions with the respective receptors. In addition, it is conceivable that peptides, peptidomimetics and small molecules could be designed to bind in one or both pockets to block IL-17A and / or IL-17F preventing their interaction with their receptors. As the FF theme present in Fab6468 (residues F92 and F93 in SEQ ID No. 79) links to residues L26, R61, L99, R101 and R102 in pocket P2, the Fab 6468 structure could be used to select and optimize antagonists additional IL-17A as peptides from randomized or projected peptide libraries using a "phage display".
[0218] [000218] The residues lining the P1 and P2 pockets are well kept between IL-17A and IL-17F and molecular modeling suggests that an IL-17A / F heterodimer would adopt an almost identical overall structure when compared to the IL homodimer -17A alone. Therefore, it is likely that pockets P1 and P2 are present in the IL-17A / F heterodimer with a similar general topology and constitute the binding sites of its receptors. Thus, IL-17A antagonists that bind to the P2 pocket residues could bind and antagonize the IL-17A / F heterodimer. Example 6 Species specificity between species
[0219] [000219] To assess the binding specificity between mAbtr1926 species, a binding ELISA was performed with different IL-17A proteins applied as a coating on micro-grinding plates. The human, mouse and rat IL-17A proteins were applied as a coating on the microtiter plates. Serial dilutions of labeled mAb1926 were incubated at 37 ° C for 2 hours. After incubation, the microtiter plates were washed thoroughly, and bound labeled mAb1926 was detected. The 1926 mAb bound to human IL-17A much more strongly than rat or mouse IL-17A proteins (Figure 9). This reduced binding to rat and mouse IL-17A is consistent with the fact that both of these proteins differ from human IL-17A at positions 7 of the Fab6468 extended epitope (Table 10). In addition, there is an amino acid insertion in rat and mouse IL-17A between residues 40 and 41 of human IL-17A, a position close to the Fab 6468 epitope part.

权利要求:
Claims (10)
[0001]
Isolated antibody or fragment thereof, characterized by the fact that the antibody specifically binds to human IL-17A and comprises a heavy chain complementarity determining region 1 (HCDR1), an HCDR2 amino acid sequence and HCDR3 amino acid sequence as shown in SEQ ID NOs: 25, 43 and 60, respectively, and a light chain complementarity determining region 1 (LCDR1), an LCDR2 amino acid sequence and an LCDR3 amino acid sequence as shown in SEQ ID NOs: 3, 6 and 18, respectively.
[0002]
Isolated antibody or fragment thereof according to claim 1, characterized by the fact that it comprises a heavy chain variable region (VH) and a light chain variable region (VL), in which VH comprises an amino acid sequence shown in SEQ ID NO: 86 and the VL comprises an amino acid sequence shown in SEQ ID NO: 79.
[0003]
Isolated antibody or fragment thereof according to claim 2, characterized in that it comprises an antibody heavy chain of SEQ ID NO: 100 and an antibody light chain of SEQ ID NO: 90.
[0004]
Isolated antibody or fragment thereof according to any one of claims 1 to 3, characterized by the fact that the antibody is entirely human.
[0005]
Isolated antibody or fragment thereof according to any one of claims 1 to 4, characterized in that the antibody is conjugated to polyethylene glycol.
[0006]
Isolated antibody or fragment thereof according to any one of claims 1 to 5, characterized by the fact that it has an IgG1 or IgG4 isotype.
[0007]
Pharmaceutical composition, characterized in that it comprises the isolated antibody or fragment thereof as defined in any one of claims 1 to 6 and a pharmaceutically acceptable carrier.
[0008]
Use of an isolated antibody, as defined in any of claims 1 to 6, characterized by the fact that it is for the preparation of a pharmaceutical composition to treat an inflammatory condition.
[0009]
Use according to claim 8, characterized by the fact that the inflammatory condition affects the respiratory tract, lung, gastrointestinal tract, small intestine, large intestine, colon, rectum, joint, bone and synovial tissue, cartilage, epithelium, endothelium, tissue liver or skin
[0010]
Use according to claim 8, characterized by the fact that the inflammatory condition is an inflammatory lung condition, asthma, chronic obstructive pulmonary disease (COPD), inflammatory bowel disease, autoimmune disease, rheumatoid arthritis, psoriasis or systemic sclerosis.

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NZ599737A|2015-02-27|

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MX2012005086A|2012-09-28|

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AU2010313304A1|2012-05-24|

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EA029283B1|2018-03-30|

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CR20120298A|2014-01-09|

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PE20121363A1|2012-10-15|

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ECSP12011871A|2012-08-31|

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法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-05-28| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-06-25| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2020-05-05| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-09-24| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 29/10/2010, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US25686209P| true| 2009-10-30|2009-10-30|

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US61/256,862|2009-10-30|

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US31091910P| true| 2010-03-05|2010-03-05|

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US61/310,919|2010-03-05|

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PCT/US2010/054662|WO2011053763A2|2009-10-30|2010-10-29|Il-17a antagonists|

---