Peptide or peptide complex binding to alpha2 integrin and methods and uses involving the same

Abstract

The present invention relates to a peptide or peptide complex binding to α2 integrin, to one or more nucleic acid(s) coding for the peptide or peptide complex, a recombinant cell producing the peptide or peptide complex, a method for producing the peptide or peptide complex, a pharmaceutical composition comprising the peptide or peptide complex or the nucleic acid(s) for use as a medicament, a method for detecting α2 integrin and a screening method.

1 . A peptide or peptide complex, preferably isolated monoclonal antibody or antigen binding fragment thereof, wherein said peptide or peptide complex, antibody or fragment specifically binds to the I-domain of a human α2-integrin, said antibody or fragment comprising a heavy chain variable region (VH) domain and a light chain variable region (VL) domain, wherein said antibody or fragment cross-reacts with a non-human primate α2-integrin but does not cross-react with a non-primate α2-integrin. 
     
     
         2 . The peptide or peptide complex according to  claim 1 , wherein said antibody or fragment competes with a reference antibody for binding to the epitope of the reference antibody, said reference antibody comprising a light chain encoded by the plasmid as deposited with the DSMZ under accession No. DSM 23944 and a heavy chain encoded by either (i) the plasmid as deposited with the DSMZ under accession DSM 23946 or (ii) the plasmid as deposited with the DSMZ under accession No. DSM 23945. 
     
     
         3 . A peptide or peptide complex preferably an isolated monoclonal antibody or antigen binding fragment thereof, comprising one or more of the following components a to f:
 a) LCDR1, wherein LCDR1 is RASESVESYGNSFIY (SEQ ID NO:6) or a functionally active variant thereof,   b) LCDR2, wherein LCDR2 is LASNLAS (SEQ ID NO:7) or a functionally active variant thereof,   c) LCDR3, wherein LCDR3 is QQNNEDPYT (SEQ ID NO:8) or a functional active variant thereof,   d) HCDR1, wherein HCDR1 is (GYTFTSYWMN, (SEQ ID NO:3) or a functionally active variant thereof,   e) HCDR2, wherein HCDR2 is RIDPSDSETHYNQKFK (SEQ ID NO:4) or a functionally active variant thereof, and   f) HCDR3, wherein HCDR3 is VGRGYFDY (SEQ ID NO:5) or a functional active variant thereof,   and wherein the one or more of the components a) to f) are arranged to allow for binding of the peptide or peptide complex to α2 integrin.   
     
     
         4 . The peptide or peptide complex according to  claim 1 ,
 (i) wherein components a) to c) are comprised in a variable domain of a light chain (VL); and/or   (ii) wherein components d) to f) are comprised in a variable domain of a heavy chain (VH); and/or   (iii) wherein the peptide or peptide complex is an antibody; and/or   (iv) wherein the peptide or peptide complex is a monoclonal antibody, a chimeric antibody, a humanized antibody, a Fab, a Fab′, a F(ab′)2, a Fv, a disulfide linked Fv, a scFv, a (scFv) 2 , a single domain antibody, a diabody, a multispecific antibody, a dual specific antibody, a isotype antibody, a dual variable domain antibody and a bispecific antibody; and/or   (v) wherein the peptide or peptide complex comprises a heavy chain immunoglobulin constant domain selected from the group consisting of: a human IgM constant domain, a human IgG1 constant domain, a human IgG2 constant domain, a human IgG3 constant domain, domain, a human IgG4 constant domain, a human IgE constant domain, and a human IgA constant domain; and/or   (vi) wherein the functionally active variant is a functionally active fragment consisting of at least 90% sequence identity to any of the amino acid sequences of SEQ ID NOS: 3 to 8; and/or   (vii) wherein the functionally active variant is a functionally active variant having at least 70%, preferably at least 80%, more preferably at least 90% sequence identity to an amino acid sequence of any of SEQ ID NOS: 3 to 8, particularly wherein the functionally active variant is derived from the amino acid sequences of any of SEQ ID NOS: 3 to 8 one or more conservative amino acid substitution; and/or   (viii) comprising the amino acid sequence of
 SEQ ID NO: 1, or a functionally active variant thereof, and/or 
 SEQ ID NO: 2, or a functionally active variant thereof, and/or 
 SEQ ID NO: 9, or a functionally active variant thereof, and/or 
 SEQ ID NO: 10, or a functionally active variant thereof, and/or 
 SEQ ID NO: 11, or a functionally active variant thereof, or 
   (ix) consisting of the amino acid sequence of
 SEQ ID NO: 1, or a functionally active variant thereof, and 
 SEQ ID NO: 2, or a functionally active variant thereof, and 
 optionally 50 additional amino acid residue(s), preferably 1 to 40, more preferably 1 to 30, even more preferably at most 1 to 25, still more preferably at most 1 to 10, most preferably 1, 2, 3, 4 or 5 additional amino acids residue(s) or 
   (x) consisting of the amino acid sequence of
 SEQ ID NO: 9, or a functionally active variant thereof, and 
 SEQ ID NO: 10, or a functionally active variant thereof, and 
 optionally 50 additional amino acid residue(s), preferably 1 to 40, more preferably 1 to 30, even more preferably at most 1 to 25, still more preferably at most 1 to 10, most preferably 1, 2, 3, 4 or 5 additional amino acids residue(s) or 
   (xi) consisting of the amino acid sequence of
 SEQ ID NO: 9, or a functionally active variant thereof, and 
 SEQ ID NO: 11, or a functionally active variant thereof, and 
 optionally 50 additional amino acid residue(s), preferably 1 to 40, more preferably 1 to 30, even more preferably at most 1 to 25, still more preferably at most 1 to 10, most preferably 1, 2, 3, 4 or 5 additional amino acids residue(s) and further, optionally 
 wherein the functionally active variant of SEQ ID NO: 1 comprises one or more mutations at amino acid positions 9, 12, 15, 22, 34, 46, 47, 80, 83, 85, 87 and/or 89, particularly selected from the group consisting of 9Ala→Ser, 12Ala→Ser, 15Leu→Val, 15Leu→Pro, 22Ser→Thr, 34Asn→Gln, 46Gln→Lys, 47Ala→Pro, 80Asp→Asn, 83Glu→Gln, 85Asp→Glu, 87Ala→Thr and 89Thr→Asn; and/or 
   (xiii) wherein the functionally active variant of SEQ ID NO: 2 comprises one or more mutations at amino acids positions 5, 7, 11, 12, 17, 20, 38, 40, 43, 55, 61, 65, 66, 67, 76, 81, 82, 87, 91, 93, 112, 113 and/or 116, particularly selected from the group consisting of 5His→Val, 7Pro→Ser, 11Leu→Val, 12Val→Lys, 17Pro→Ser, 20Leu→Val, 38Lys→Arg, 40Arg→Ala, 43Arg→Gln, 55Asp→Glu, 61Asn→Ala, 65Lys→Gln, 66Asp→Gly, 67Lys→Arg, 76Ser→Thr, 81Ile→Met, 82Gln→Glu, 87Thr→Arg, 91Ser→Thr, 93Val→Lys, 112Thr→Leu, 113Leu→Val and 116Ser→Val.   
     
     
         5 . The peptide or peptide complex according to  claim 3 :
 wherein the functionally active variant of LCDR1 comprises the mutation at amino acid position 11, particularly 11Asn→Gln; and/or   wherein the functionally active variant of HCDR2 comprises the mutation at amino acid position 6, particularly 6Asp→Glu.   
     
     
         6 . The Peptide or peptide complex according to  claim 1 , wherein said antibody or fragment specifically binds to the I-domain of the human α2-integrin with nM binding affinity; or
 wherein said peptide or peptide complex inhibits the interaction of the human α2-integrin with collagen in vitro, thereby inhibiting the activation of platelets due to adhesion of said platelets to said collagen. 
 
     
     
         7 . (canceled) 
     
     
         8 . The Peptide or peptide complex according to  claim 1 ,
 wherein said heavy chain variable region domain comprising the heavy chain HCDR3 of SEQ ID NO:5   wherein said heavy chain variable region domain comprising the heavy chain CDRs of SEQ ID NO:3 (HCDR1), SEQ ID NO:4 (HCDR2), and SEQ ID NO:5 (HCDR3), or functionally active variants thereof, optionally wherein the functionally active variant of HCDR2 comprises the mutation Asp→Glu at amino acid position 6;   wherein said light chain variable region domain comprising the light chain LCDR3 of SEQ ID NO:8; and/or   wherein said light chain variable region domain comprising the light chain CDRs of SEQ ID NO:6 (LCDR1), SEQ ID NO:7 (LCDR2), and SEQ ID NO:8 (LCDR3), or functionally active variants thereof, optionally wherein the functionally active variant of LCDR1 comprises the mutation Asn→Gln at amino acid position 11.   
     
     
         9 .- 13 . (canceled) 
     
     
         14 . The Peptide or peptide complex according to  claim 1 , said heavy chain variable region (VH) domain having at least 90%, 95%, 97% or 99% sequence identity to the VH sequence of SEQ ID NO: 2, optionally wherein said heavy chain variable region (VH) domain comprises the sequence of SEQ ID NO:2 or a functionally active variant thereof. 
     
     
         15 . (canceled) 
     
     
         16 . The Peptide or peptide complex according to  claim 1 , said light chain variable region (VL) domain having at least 90%, 95%, 97% or 99% sequence identity to the VL sequence of SEQ ID NO: 1, optionally wherein said light chain variable region (VL) domain comprises the sequence of SEQ ID NO:1 or a functionally active thereof. 
     
     
         17 . (canceled) 
     
     
         18 . The Peptide or peptide complex according to  claim 1 , wherein said heavy chain variable region (VH) domain comprises one or more amino acid substitutions at positions selected from the group consisting of H5, H7, H11, H12, H17, H20, H38, H40, H43, H55, H61, H65, H66, H67, H76, H81, H82, H87, H91, H93, H112, H113 and H116, optionally wherein the one or more amino acid substitutions are selected from the group consisting 5His→Val, 7Pro→Ser, 11Leu→Val, 12Val→Lys, 17Pro→Ser, 20Leu→Val, 38Lys→Arg, 40Arg→Ala, 43Arg→Gln, 55Asp→Glu, 61Asn→Ala, 65Lys→Gln, 66Asp→Gly, 67Lys→Arg, 76Ser→Thr, 81Ile→Met, 82Gln→Glu, 87Thr→Arg, 91Ser→Thr, 93Val→Lys, 112Thr→Leu, 113Leu→Val and 116Ser→Val; and/or
 wherein said light chain variable region (VL) domain comprises one or more amino acid substitutions at positions selected from the group consisting of L9, L12, L15, L22, L34, L46, L47, L80, L83, L85, L87, and L89, optionally wherein the one or more amino acid substitutions are selected from the group consisting of 9Ala→Ser, 12Ala→Ser, 15Leu→Val, 15Leu→Pro, 22Ser→Thr, 34Asn→Gln, 46Gln→Lys, 47Ala→Pro, 80Asp→Asn, 83Glu→Gln, 85Asp→Glu, 87Ala→Thr and 89Thr→Asn. 
 
     
     
         19 .- 21 . (canceled) 
     
     
         22 . The Peptide or peptide complex according to  claim 1 , said heavy chain variable region (VH) domain having at least 90%, 95%, 97% or 99% sequence identity or having 100% identity to a VH sequence selected from the group consisting of SEQ ID NO: 38 (HC1), SEQ ID NO:39 (HC2), SEQ ID NO:40 (HC3), SEQ ID NO:41 (HC4), SEQ ID NO:42 (HC5), SEQ ID NO:43 (HC6), and SEQ ID NO:44 (HC7); and/or said light chain variable region (VL) domain having at least 90%, 95%, 97% or 99% sequence identity or having 100% identity to a VL sequence selected from the group consisting of SEQ ID NO: 33 (LC1), SEQ ID NO:34 (LC2), SEQ ID NO:35 (LC3), SEQ ID NO:36 (LC4), and SEQ ID NO:37 (LC5). 
     
     
         23 .- 25 . (canceled) 
     
     
         26 . The Peptide or peptide complex according to  claim 1 ,
 wherein said antibody or binding portion is a chimeric antibody or humanized antibody;   wherein the antigen binding portion is selected from the group consisting of a Fab, a Fab′, a F(ab′)2, a Fv, a disulfide linked Fv, a scFv, and a (scFv) 2 ;   which is selected from the group consisting of a multispecific antibody, a dual specific antibody, a isotype antibody, a dual variable domain antibody and a bispecific antibody;   comprising a heavy chain immunoglobulin constant domain selected from the group consisting of: a human IgM constant domain, a human IgG1 constant domain, a human IgG2 constant domain, a human IgG3 constant domain, domain, a human IgG4 constant domain, a human IgE constant domain, and a human IgA constant domain; and/or   wherein the peptide or peptide complex comprises or consists of an isolated monoclonal antibody or antigen binding fragment thereof.   
     
     
         27 .- 31 . (canceled) 
     
     
         32 . One or more nucleic acid(s) coding for the peptide or peptide complex according to  claim 1 , optionally wherein the nucleic acid(s) is/are located in a vector. 
     
     
         33 .- 34 . (canceled) 
     
     
         35 . A cell heterologously expressing one of the nucleic acids according to  claim 32 . 
     
     
         36 . A method for producing a peptide or peptide complex comprising culturing the cell according to  claim 35  under conditions permitting expression of the peptide or peptide complex and optionally recovering the peptide or peptide complex from the host cell. 
     
     
         37 . A pharmaceutical composition comprising at least one peptide or peptide complex according to  claim 1 . 
     
     
         38 . A method of treating or preventing an α2 integrin-related disease or disorder, the method comprising
 administering the pharmaceutical composition of  claim 37  to a subject in need thereof, wherein the α2 integrin-related disease or disorder is optionally selected from the group consisting of thrombosis, a vascular disease, cancer, including neo-angiogenesis and metastasis, inflammation, inflammatory disease, autoimmune disease and a disease characterized by abnormal or increase angiogenesis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, reactions to transplant, optical neuritis, spinal cord trauma, rheumatoid arthritis, systemic lupus erythematosus (SLE), multiple sclerosis, Reynaud's syndrome, experimental autoimmune encephalomyelitis, Sjorgen's syndrome, scleroderma, cardiovascular disease, psoriasis, and infections that induce an inflammatory response. 
 
     
     
         39 . (canceled) 
     
     
         40 . A method of diagnosing a disease associated with altered α2 integrin, the method comprising
 a) contacting a sample comprising α2 integrin with the peptide or peptide complex of  claim 1 ; 
 b) detecting binding of α2 integrin to the peptide or peptide complex; and 
 c) comparing the binding of step b) with a reference, 
 wherein altered α2 integrin binding in the sample relative to the reference is indicative of the disease. 
 
     
     
         41 . An article of manufacture comprising
 a) a packaging material,   b) a peptide or peptide complex according to  claim 1 , and   c) a label or a package insert, the insert contained within said packaging material, indicating that said peptide or peptide complex is effective for treatment of a disease or disorder.   
     
     
         42 . A diagnostic kit for the diagnosis of an α 2-integrin related disorder or disease comprising a peptide or peptide complex according to  claim 1 , a suitable packaging, and possibly suitable instructions for using said peptide or peptide complex in the detection of α 2 integrin. 
     
     
         43 .- 44 . (canceled)