Materials and methods relating to modifying the binding of antibodies

Abstract

The invention relates to materials and methods for modifying the binding of antibodies, and more particularly to antibodies that are obtainable by inserting an amino acid sequence capable of binding to a target into a complementarity determining region of a parent antibody so that the antibody thus obtained is capable of binding to the target. The invention further relates to the uses of the antibodies for therapy, diagnosis or imaging, and to methods of producing the antibodies.

1 .- 48 . (canceled) 
     
     
         49 . An anti-αvβ6 integrin antibody as obtainable by inserting an amino acid sequence of between 12 and 20 amino acids in length, which comprises RGDLX 5 X 6 X 7 , wherein X 5  and X 6  represent any amino acid and X 7  is Leu or Ile between adjacent amino acid residues Thr98 an Gly99 (Kabat numbering) of heavy chain complementary determining region (CDR) 3 of a parent antibody, wherein the parent antibody comprises heavy chain CDRs 1 to 3 and light chain CDRs 1 to 3, and wherein the parent heavy chain CDR 3 has the sequence (i) Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr (SEQ ID NO: 55) or (ii) Gly Thr Pro Thr Gly Pro Tyr Pro Phe Asp Tyr (SEQ ID NO: 56). 
     
     
         50 . The anti-αvβ6 integrin antibody of  claim 49 , wherein the parent antibody comprises the following CDR sequences:
 (a) Heavy Chain CDR 1: Asp Ser Tyr Met His (SEQ ID NO: 53); and 
 (b) Heavy Chain CDR 2: Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe Gln Gly (SEQ ID NO: 54); and 
 (c) Heavy Chain CDR 3: Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr (SEQ ID NO: 55), or (ii) Gly Thr Pro Thr Gly Pro Tyr Pro Phe Asp Tyr (SEQ ID NO: 56); and 
 (d) Light Chain CDR 1: (i) Ser Ala Ser Ser Ser Val Pro Tyr Met His (SEQ ID NO: 57), or (ii) Ser Ala Ser Ser Ser Val Ser Tyr Met His (SEQ ID NO: 58); and 
 (e) Light Chain CDR 2: (i) Ser Thr Ser Asn Leu Ala Ser (SEQ ID NO: 59), or (ii) Leu Thr Ser Asn Leu Ala Ser (SEQ ID NO: 60); and 
 (f) Light Chain CDR 3: Gln Gln Arg Ser Ser Tyr Pro Leu Thr (SEQ ID NO: 61). 
 
     
     
         51 . The anti-αvβ6 integrin antibody of  claim 49 , wherein the antibody is humanised. 
     
     
         52 . The anti-αvβ6 integrin antibody of  claim 49 , wherein antibody is a scFv or a diabody. 
     
     
         53 . The anti-αvβ6 integrin antibody of  claim 52 , wherein the scFv or diabody comprises a linker having the sequence (Gly 4 Ser) n , wherein n is between 1 and 4 (SEQ ID NOs: 26-29), respectively. 
     
     
         54 . The anti-αvβ6 integrin antibody of  claim 49 , wherein the antibody is a chimeric molecule comprising an immunoglobulin binding domain comprising said CDRs, wherein the immunoglobulin binding domain is fused to another polypeptide. 
     
     
         55 . The anti-αvβ6 integrin antibody of  claim 49 , wherein the parent antibody comprises the mutation G44C in the heavy chain, A100C in the light chain and/or Y100bP in the heavy chain (using Kabat nomenclature). 
     
     
         56 . The anti-αvβ6 integrin antibody of  claim 49 , wherein X 5  and X 6  are independently selected from Glu, Ala, Leu, Met, Gln, Lys, Arg, Val, Ile, Trp, Phe, Asp, His and Thr. 
     
     
         57 . The anti-αvβ6 integrin antibody of  claim 49 , wherein the inserted amino acid sequence consists of the amino acid sequence AVPNLRGDLQVLAQKVA (SEQ ID NO: 19). 
     
     
         58 . The anti-αvβ6 integrin antibody of  claim 49 , wherein the antibody is conjugated to a detectable moiety. 
     
     
         59 . The anti-αvβ6 integrin antibody of  claim 58 , wherein the detectable moiety is detectable by Magnetic Resonance Imaging (MRI), Magnetic Resonance Spectroscopy (MRS), Single Photon Emission Computed Tomography (SPECT), Positron Emission Tomography (PET) or optical imaging. 
     
     
         60 . The anti-αvβ6 integrin antibody of  claim 58 , wherein the detectable moiety is a radioactive moiety. 
     
     
         61 . The anti-αvβ6 integrin antibody of  claim 49 , wherein the antibody is conjugated to a therapeutically active moiety. 
     
     
         62 . The anti-αvβ6 integrin antibody of  claim 61 , wherein the therapeutically active moiety is an anti-cancer agent. 
     
     
         63 . The anti-αvβ6 integrin antibody of  claim 49 , wherein the antibody is conjugated to a tagging group for use in purification or detection. 
     
     
         64 . The anti-αvβ6 integrin antibody of  claim 63 , wherein the tagging group is c-Myc or a poly-histidine group. 
     
     
         65 . The anti-αvβ6 integrin antibody of  claim 49 , wherein the antibody is directly or indirectly conjugated or linked to one or more of a cytotoxic moiety, an agent capable of converting a prodrug to a cytotoxic moiety, a radiosensitiser and/or a radioactive atom. 
     
     
         66 . The anti-αvβ6 integrin antibody of  claim 65 , wherein the cytotoxic moiety is a chemotherapeutic agent or a cytotoxic polypeptide. 
     
     
         67 . The anti-αvβ6 integrin antibody of  claim 65 , wherein the agent capable of converting a prodrug to a cytotoxic moiety is an enzyme for use in antibody directed enzyme prodrug therapy (ADEPT). 
     
     
         68 . The anti-αvβ6 integrin antibody of  claim 49 , wherein the parent antibody comprises the amino acid sequence of residues 1 to 244 of SEQ ID NO: 2. 
     
     
         69 . A nucleic acid molecule that encodes an anti-αvβ6 integrin antibody of  claim 49 . 
     
     
         70 . An expression vector comprising a nucleic acid molecule of  claim 69 , operably linked to control sequences to direct its expression. 
     
     
         71 . An isolated host cell transformed with the expression vector of  claim 70 . 
     
     
         72 . A method of producing the anti-αvβ6 integrin antibody of  claim 49 , comprising culturing the host cell of claim  23  and isolating the antibody thus produced. 
     
     
         73 . A method of modifying a parent antibody to an antibody capable of binding αvβ6 integrin, wherein the parent antibody comprises heavy chain CDRs 1 to 3 and light chain CDRs 1 to 3, and wherein the parent heavy chain CDR 3 has the sequence (i) Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr (SEQ ID NO: 55) or (ii) Gly Thr Pro Thr Gly Pro Tyr Pro Phe Asp Tyr (SEQ ID NO: 56), and wherein the method comprises inserting an amino acid sequence of between 12 and 20 amino acids in length, which comprises RGDLX 5 X 6 X 7 , wherein X 5  and X 6  represent any amino acid and X 7  is Leu or Ile, between adjacent amino acid residues Thr98 and Gly99 (Kabat numbering) of heavy chain CDR 3 of the parent antibody. 
     
     
         74 . The method of  claim 73 , wherein the parent antibody comprises the following CDR sequences:
 (a) Heavy Chain CDR 1: Asp Ser Tyr Met His (SEQ ID NO: 53); and   (b) Heavy Chain CDR 2: Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe Gln Gly (SEQ ID NO: 54); and   (c) Heavy Chain CDR 3: Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr (SEQ ID NO: 55), or (ii) Gly Thr Pro Thr Gly Pro Tyr Pro Phe Asp Tyr (SEQ ID NO: 56); and   (d) Light Chain CDR 1: (i) Ser Ala Ser Ser Ser Val Pro Tyr Met His (SEQ ID NO: 57), or (ii) Ser Ala Ser Ser Ser Val Ser Tyr Met His (SEQ ID NO: 58); and   (e) Light Chain CDR 2: (i) Ser Thr Ser Asn Leu Ala Ser (SEQ ID NO: 59), or (ii) Leu Thr Ser Asn Leu Ala Ser (SEQ ID NO: 60); and   (f) Light Chain CDR 3: Gln Gln Arg Ser Ser Tyr Pro Leu Thr (SEQ ID NO: 61).   
     
     
         75 . The method of  claim 73 , wherein the method further comprises humanizing the antibody capable of binding αvβ6 integrin or the parent antibody by mutating one or more amino acid residues. 
     
     
         76 . A method for diagnosing or imaging a condition characterised by diseased cells which express αvβ6 integrin and/or which is a disease mediated by αvβ6 integrin, the method comprising administering to a subject suspected of having the condition the antibody conjugated to a detectable moiety according to  claim 58  and detecting the detectable moiety to diagnose or image the condition. 
     
     
         77 . The method of  claim 76 , wherein the imaging or diagnosis is for a cancer. 
     
     
         78 . A method of treating an αvβ6 integrin-mediated disease or a disease in which cells overexpress αvβ6 integrin, said disease being selected from the group consisting of: cancer, chronic fibrosis, chronic obstructive pulmonary disease (COPD), lung emphysemia and epidermolysis bullosa, the method comprising administering to a patient in need thereof a therapeutically effective amount of an antibody according to  claim 49 . 
     
     
         79 . The method of  claim 78 , wherein said disease is an epithelial cancer.