Synthetic immunoglobulin domains with binding properties engineered in regions of the molecule different from the complementarity determining regions

Abstract

Method for engineering an immunoglobulin comprising at least one modification in a structural loop region of said immunoglobulin and determining the binding of said immunoglobulin to an epitope of an antigen, wherein the unmodified immunoglobulin does not significantly bind to said epitope, comprising the steps of:—providing a nucleic acid encoding an immunoglobulin comprising at least one structural loop region,—modifying at least one nucleotide residue of at least one of said structural loop regions,—transferring said modified nucleic acid in an expression system,—expressing said modified immunoglobulin,—contacting the expressed modified immunoglobulin with an epitope, and—determining whether said modified immunoglobulin binds to said epitope, as well as modified immunoglobulins.

1 . Method for engineering an immunoglobulin comprising at least one modification in a structural loop region of said immunoglobulin and determining the binding of said immunoglobulin to an epitope of an antigen, wherein the unmodified immunoglobulin does not significantly bind to said epitope, comprising the steps of:
 providing a nucleic acid encoding an immunoglobulin comprising at least one structural loop region,   modifying at least one nucleotide residue of at least one of said structural loop regions,   transferring said modified nucleic acid in an expression system,   expressing said modified immunoglobulin,   contacting the expressed modified immunoglobulin with an epitope, and   determining whether said modified immunoglobulin binds to said epitope.   
     
     
         2 . Method according to  claim 1 , wherein said immunoglobulin is binding specifically to at least two different epitopes. 
     
     
         3 . Method according to any of  claim 1  or  claim 2 , comprising at least one modification in at least one structural loop region of said immunoglobulin and determining the specific binding of said at least one loop region to at least one molecule selected from the group consisting of allergens, tumor associated antigens, self antigens, enzymes, bacterial antigens, fungal antigens, viral antigens and protozoal antigens, wherein the immunoglobulin containing an unmodified structural loop region does not specifically bind to said at least one molecule. 
     
     
         4 . Method for manufacturing an immunoglobulin or a pharmaceutical preparation thereof comprising at least one modification in a structural loop region of said immunoglobulin and determining the binding of said immunoglobulin to an epitope of an antigen, wherein the unmodified immunoglobulin does not significantly bind to said epitope, comprising the steps of:
 providing a nucleic acid encoding an immunoglobulin comprising at least one loop region,   modifying at least one nucleotide residue of at least one of said loop regions,   transferring said modified nucleic acid in an expression system,   expressing said modified immunoglobulin,   contacting the expressed modified immunoglobulin with an   epitope,   determining whether said modified immunoglobulin binds to said epitope, and   providing the modified immunoglobulin binding to said epitope and optionally finishing it to a pharmaceutical preparation.   
     
     
         5 . Method for manufacturing a multi-specific immunoglobulin binding specifically to at least one first molecule or a pharmaceutical preparation thereof comprising at least one modification in at least one structural loop region of said immunoglobulin and determining the specific binding of said at least one loop region to at least one second molecule selected from the group consisting of allergens, tumor associated antigens, self antigens, enzymes, bacterial antigens, fungal antigens, viral antigens and protozoal antigens, wherein the immunoglobulin containing an unmodified structural loop region does not specifically bind to said at least one second molecule, comprising the steps of:
 providing a nucleic acid encoding an immunoglobulin binding specifically to at least one first molecule comprising at least one structural loop region,   modifying at least one nucleotide residue of at least one of said loop regions encoded by said nucleic acid,   transferring said modified nucleic acid in an expression system,   expressing said modified immunoglobulin,   contacting the expressed modified immunoglobulin with said at least one second molecule, and   determining whether said modified immunoglobulin binds specifically to the second molecule and   providing the modified immunoglobulin specifically to said at least one second molecule and optionally finishing it to a pharmaceutical preparation.   
     
     
         6 . Method according to  claim 1 , characterised in that the immunoglobulin is of human or murine origin. 
     
     
         7 . Method according to  claim 6 , characterised in that the human immunoglobulin is selected from the group consisting of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 and IgM. 
     
     
         8 . Method according to  claim 6 , characterised in that the murine immunoglobulin is selected from the group consisting of IgA, IgD, IgE, IgG1, IgG2A, IgG2B, IgG2C, IgG3 and IgM. 
     
     
         9 . Method according to  claim 7  or  8 , characterised in that the immunoglobulin comprises a heavy and/or light chain of the immunoglobulin or a part thereof. 
     
     
         10 . Method according to  claim 1 , characterised in that the immunoglobulin comprises at least one constant domain and/or at least one variable domain of the immunoglobulin, or a part thereof including a minidomain. 
     
     
         11 . Method according to  claim 10 , characterised in that the constant domain is selected from the group of CH1, CH2, CH3, CH4, Igk-C, Igl-C and combinations thereof. 
     
     
         12 . Method according to  claim 11 , characterised in that the modified loop regions of CH1, CH2, CH3 and CH4 comprise amino acids 7 to 21, amino acids 25 to 39, amino acids 41 to 81, amino acids 83 to 85, amino acids 89 to 103 and amino acids 106 to 117. 
     
     
         13 . Method according to  claim 11 , characterised in that the loop regions of Igk-C and Igl-C of human origin comprise amino acids 8 to 18, amino acids 27 to 35, amino acids 42 to 78, amino acids 83 to 85, amino acids 92 to 100, amino acids 108 to 117 and amino acids 123 to 126. 
     
     
         14 . Method according to  claim 11 , characterised in that the loop regions of Igk-C and Igl-C of murine origin comprise amino acids 8 to 20, amino acids 26 to 36, amino acids 43 to 79, amino acids 83 to 85, amino acids 90 to 101, amino acids 108 to 116 and amino acids 122 to 125. 
     
     
         15 . Method according to  claim 10 , characterised in that the structural loop regions of the variable domain of the immunoglobulin of human origin comprise amino acids 8 to 20, amino acids 44 to 50, amino acids 67 to 76 and amino acids 89 to 101. 
     
     
         16 . Method according to  claim 10 , characterised in that the structural loop regions of the variable domain of the immunoglobulin of murine origin comprise amino acids 6 to 20, amino acids 44 to 52, amino acids 67 to 76 and amino acids 92 to 101. 
     
     
         17 . Method according to  claim 1 , characterised in that the immunoglobulin is of camel origin. 
     
     
         18 . Method according to  claim 17 , characterised in that the immunoglobulin comprises at least one constant domain selected from the group consisting of CH1, CH2 and CH3. 
     
     
         19 . Method according to  claim 18 , characterised in that the loop regions of CH1, CH2 and CH3 comprise amino acids 8 to 20, amino acids 24 to 39, amino acids 42 to 78, amino acids 82 to 85, amino acids 91 to 103 and amino acids 108 to 117. 
     
     
         20 . Method according to  claim 1 , characterised in that the modification of at least one nucleotide results in a substitution, deletion and/or insertion of the immunoglobulin encoded by said nucleic acid. 
     
     
         21 . Method according to  claim 1 , characterised in that the amino acids of at least one loop region are modified by site-directed random mutation. 
     
     
         22 . Method according to  claim 21 , characterised in that the randomly modified nucleic acid molecule comprises at least one nucleotide repeating unit having the sequence 5′-NNS-3′, 5′-NNN3′ or 5′-NNK-5′. 
     
     
         23 . Method according to  claim 1 , characterized in that the expression system comprises a vector. 
     
     
         24 . Method according to  claim 1  characterised in that the modified immunoglobulin is expressed in a host, preferably in a bacterial, a yeast, a plant cell, in an animal cell or in a plant or animal. 
     
     
         25 . Method according to  claim 1 , characterised in that the specific binding of the modified immunoglobulin to the molecule is determined by a binding assay selected from the group consisting of immunological assays, preferably enzyme linked immunosorbent assays (ELISA), surface plasmon resonance assays, saturation transfer difference nuclear magnetic resonance spectroscopy, transfer NOE (trNOE) nuclear magnetic resonance spectroscopy, competitive assays, tissue binding assays, live cell binding assays and cellular extract assays. 
     
     
         26 . Method according to  claim 1 , characterised in that the modified immunoglobulin is conjugated to a label selected from the group consisting of organic molecules, enzyme labels, radioactive labels, colored labels, fluorescent labels, chromogenic labels, luminescent labels, haptens, digoxigenin, biotin, metal complexes, metals, colloidal gold and mixtures thereof. 
     
     
         27 . Immunoglobulin consisting of a constant domain selected from the group consisting of CH1, CH2, CH3, CH4, Igk-C, Igl-C, or a part thereof including a minidomain, with at least one loop region, characterised in that said at least one loop region comprises at least one amino acid modification forming at least one modified loop region, wherein said at least one modified loop region binds specifically to at least one epitope of an antigen. 
     
     
         28 . Immunoglobulin consisting of a variable domain of a heavy or light chain, or a part thereof including a minidomain, with at least one loop region, characterised in that said at least one loop region comprises at least one amino acid modification forming at least one modified loop region, wherein said at least one modified loop region binds specifically to at least one epitope of an antigen. 
     
     
         29 . Immunoglobulin according to  claim 27  or  28 , characterised in that the antigen is selected from the group consisting of proteinaceous molecules, nucleic acids, and carbohydrates. 
     
     
         30 . Immunoglobulin according to  claim 27 , characterised in that the antigen is selected from the group consisting of tumor associated antigens, in particular EpCAM, tumor-associated glycoprotein-72 (TAG-72), tumor-associated antigen CA 125, Prostate specific membrane antigen (PSMA), High molecular weight melanoma-associated antigen (HMW-MAA), tumor-associated antigen expressing Lewis Y related carbohydrate, Carcinoembryonic antigen (CEA), CEACAM5, HMFG PEM, mucin MUC1, MUC18 and cytokeratin tumor-associated antigen, bacterial antigens, viral antigens, allergens, fluorescein, lysozyme, toll-like receptor 9, erythropoietin, CD2, CD3, CD3E, CD4, CD11, CD11a, CD14, CD18, CD19, CD20, CD22, CD23, CD25, CD28, CD29, CD30, CD33 (p67 protein), CD38, CD40, CD40L, CD52, CD54, CD56, CD80, CD147, GD3, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-6R, IL-8, IL-12, IL-15, IL-18, IL-23, interferon alpha, interferon beta, interferon gamma; TNF-alpha, TNFbeta2, TNF.alpha., TNFalphabeta, TNF-R1, TNF-R11, FasL, CD27L, CD30L, 4-1BBL, TRAIL, RANKL, TWEAK, APRIL, BAFF, LIGHT, VEG1, OX40L, TRAIL Receptor-1, A1 Adenosine Receptor, Lymphotoxin Beta Receptor, TACI, BAFF-R, EPO; LFA-3, ICAM-1, ICAM-3, integrin beta1, integrin beta2, integrin alpha4/beta7, integrin alpha2, integrin alpha3, integrin alpha4, integrin alpha5, integrin alpha6, integrin alphav, alphaVbeta3 integrin, FGFR-3, Keratinocyte Growth Factor, VLA-1, VLA-4, L-selectin, anti-Id, E-selectin, HLA, HLADR, CTLA-4, T cell receptor, B7-1, B7-2, VNRintegrin, TGFbeta1, TGFbeta2, eotaxin1, BLyS (B-lymphocyte Stimulator), complement C5, IgE, factor VII, CD64, CBL, NCA 90, EGFR (ErbB-1), Her2/neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB4), Tissue Factor, VEGF, VEGFR, endothelin receptor, VLA-4, carbohydrates such as blood group antigens and related carbohydrates, Galili-Glycosylation, Gastrin, Gastrin receptors, tumor associated carbohydrates, Hapten NP-cap or NIP-cap, T cell receptor alpha/beta, E-selectin, digoxin, placental alkaline phosphatase (PLAP) and testicular PLAP-like alkaline phosphatase, transferrin receptor, Heparanase I, human cardiac myosin, Glycoprotein IIb/IIIa (GPIIb/IIIa), human cytomegalovirus (HCMV) gH envelope glycoprotein, HIV gpl20, HCMV, respiratory syncital virus RSV F, RSVF Fgp, VNRintegrin, Hep B gp120, CMV, gpIIbIIIa, HIV IIIB gp120 V3 loop, respiratory syncytial virus (RSV) Fgp, Herpes simplex virus (HSV) gD glycoprotein, HSV gB glycoprotein, HCMV gB envelope glycoprotein,  Clostridium perfringens  toxin and fragments thereof. 
     
     
         31 . Immunoglobulin according to  claim 27 , characterised in that amino acid residues of positions 15 to 17, 29 to 34, 85.4 to 85.3, 92 to 94, 97 to 98 and/or 108 to 110 of CH3 are modified. 
     
     
         32 . Immunoglobulin according to  claim 27 , characterised in that the modification is a deletion, a substitution, a insertion or a combination thereof. 
     
     
         33 . Immunoglobulin according to  claim 27 , characterised in that the CH3 region comprises SEQ ID No. 16 or SEQ ID No. 18, when EpCam binds to said immunoglobulin, SEQ ID No. 20, when fluorescein binds to said immunoglobulin, SEQ ID No. 22, 24, 26, 28, 30 or 32, when lysozyme binds to said immunoglobulin, SEQ ID No. 34, 36, 38 or 40, when TLR9 binds to said immunoglobulin, and SEQ ID No. 42, when lysozyme and/or erythropoietin bind to said immunoglobulin. 
     
     
         34 . Immunoglobulin according to  claim 27 , characterised in that it comprises SEQ ID No. 44 or SEQ ID No. 46, when lysozyme and gp41 bind to said immunoglobulin. 
     
     
         35 . Immunoglobulin according to  claim 27 , characterised in that the modified immunoglobulin is conjugated to a label or reporter molecule selected from the group consisting of organic molecules, enzyme labels, radioactive labels, colored labels, fluorescent labels, chromogenic labels, luminescent labels, haptens, digoxigenin, biotin, metal complexes, metals, colloidal gold and mixtures thereof. 
     
     
         36 . Use of an immunoglobulin according to  claim 27  or obtainable by the method according to  claim 1  for the preparation of a vaccine for active immunization. 
     
     
         37 . Use of an immunoglobulin according to  claim 27  or obtainable by the method according to  claim 1  for the preparation of a protein library of immunoglobulins. 
     
     
         38 . Protein library comprising an immunoglobulin obtainable by the method according to  claim 27  or obtainable by the method according to  claim 1 . 
     
     
         39 . Method for specifically binding and/or detecting a molecule comprising the steps of:
 (a) contacting a modified immunoglobulin according to  claim 27  or a modified immunoglobulin obtainable by a method according to  claim 1  with a test sample suspected to contain said molecule, and   (b) detecting the potential formation of a specific immunoglobulin/molecule complex.   
     
     
         40 . Method for isolating specifically a molecule comprising the steps of:
 (a) contacting a modified immunoglobulin according to  claim 27  or a modified immunoglobulin obtainable by a method according to  claim 1  with a sample containing said molecule,   (b) separating the specific immunoglobulin/molecule complex formed, and   (c) optionally isolating the molecule from said complex.   
     
     
         41 . Method for targeting a compound to a target comprising the steps of:
 (a) contacting a modified immunoglobulin according to  claim 27  or a modified immunoglobulin obtainable by a method according to  claim 1  binding specifically to said compound,   (b) delivering the specific immunoglobulin/compound complex to the target.   
     
     
         42 . A modified immunoglobulin having an antigen binding site foreign to the unmodified immunoglobulin and incorporated in one or more structural loops. 
     
     
         43 . Immunoglobulin according to  claim 42 , wherein said antigen is selected from the group consisting of allergens, tumor associated antigens, self antigens, enzymes, bacterial antigens, fungal antigens, viral antigens and protozoal antigens. 
     
     
         44 . Immunoglobulin according to  claim 42 , wherein said immunoglobulin comprises at least two antigen binding sites, the first site binding to a first epitope, and the second site binding to a second epitope. 
     
     
         45 . Immunoglobulin according to  claim 42 , wherein said immunoglobulin comprises at least two loop regions, the first loop region binding to a first epitope, and the second loop region binding to a second epitope. 
     
     
         46 . Immunoglobulin according to  claim 45 , wherein said loop regions contain structural loops. 
     
     
         47 . Immunoglobulin according to  claim 42  being composed of at least two immunoglobulin domains, or a part thereof including a minidomain, and each domain contains at least one antigen binding site. 
     
     
         48 . Immunoglobulin according to  claim 42 , wherein the immunoglobulin comprises at least one domain of the constant region and/or at least one domain of the variable region of the immunoglobulin, or a part thereof including a minidomain. 
     
     
         49 . Immunoglobulin according to  claim 42 , wherein said immunoglobulin comprises a domain that has at least 50% homology with the unmodified domain. 
     
     
         50 . Immunoglobulin according to  claim 42 , wherein said immunoglobulin is a bispecific antibody or a bispecific single chain antibody. 
     
     
         51 . Immunoglobulin according to  claim 42 , wherein said immunoglobulin comprises a bispecific domain or a part thereof including a minidomain. 
     
     
         52 . Immunoglobulin according to  claim 42 , for therapeutic and prophylactic use. 
     
     
         53 . Immunoglobulin according to  claim 42 , for preparative and analytic use. 
     
     
         54 . Immunoglobulin according to  claim 42 , for diagnostic use. 
     
     
         55 . Library consisting of at least 100 immunoglobulins according to  claim 27  with different modifications in the structural loops. 
     
     
         56 . Library according to  claim 55 , wherein said immunoglobulins are immunoglobulin selected from the group consisting of domains of an immunoglobulin, minidomains or derivatives thereof. 
     
     
         57 . Kit of binding partners containing
 (a) a modified immunoglobulin having an antigen binding site foreign to the immunoglobulin incorporated in one or more structural loops, and   (b) a binding molecule containing an epitope of said antigen.   
     
     
         58 . Use of the binding molecule of a kit according to  claim 57  for identifying the binding specificity of said modified immunoglobulin thereof. 
     
     
         59 . Use of the binding molecule of a kit according to  claim 57  for determining the potency of said modified immunoglobulin. 
     
     
         60 . Use of the binding molecule of a kit according to  claim 57  for selecting said modified immunoglobulin from a library consisting of at least 100 immunoglobulins with different modifications in the structural loops.