Recombinant monovalent antibodies and methods for production thereof

Abstract

The present invention provides monovalent antibodies with a long half-life when administered in vivo, methods of making such monovalent antibodies, pharmaceutical compositions comprising such antibodies, and uses of the monovalent antibodies.

1 - 228 . (canceled) 
     
     
         229 . A method for producing a monovalent antibody, said method comprising
 a) providing a nucleic acid construct encoding the light chain of said monovalent antibody, said construct comprising a nucleotide sequence encoding the V L  region of a selected antigen specific antibody and a nucleotide sequence encoding the constant C L  region of an Ig, wherein said nucleotide sequence encoding the V L  region of a selected antigen specific antibody and said nucleotide sequence encoding the C L  region of an Ig are operably linked together, and wherein, in case of an IgG1 subtype, the nucleotide sequence encoding the C L  region has been modified such that the C L  region does not contain any amino acids capable of forming disulfide bonds or covalent bonds with other peptides comprising an identical amino acid sequence of the C L  region in the presence of polyclonal human IgG or when administered to a human being;   b) providing a nucleic acid construct encoding the heavy chain of said monovalent antibody, said construct comprising a nucleotide sequence encoding the V H  region of a selected antigen specific antibody and a nucleotide sequence encoding a constant C H  region of a human Ig, wherein the nucleotide sequence encoding the C H  region has been modified such that the region corresponding to the hinge region and, as required by the Ig subtype, other regions of the C H  region, such as the C H 3 region, does not comprise any amino acid residues which participate in the formation of disulphide bonds or covalent or stable non-covalent inter-heavy chain bonds with other peptides comprising an identical amino acid sequence of the C H  region of the human Ig in the presence of polyclonal human IgG or when administered to a human being, wherein said nucleotide sequence encoding the V H  region of a selected antigen specific antibody and said nucleotide sequence encoding the C H  region of said Ig are operably linked together;   c) providing a cell expression system for producing said monovalent antibody; and   d) producing said monovalent antibody by co-expressing the nucleic acid constructs of (i) and (ii) in cells of the cell expression system of (iii).   
     
     
         230 . A nucleic acid construct for expression of a monovalent antibody for pharmaceutical use, comprising i) a nucleic acid sequence encoding the C H  region of an IgG4, wherein the nucleic acid sequence encoding the C H  region has been modified such that the region corresponding to the hinge region in said C H  region does not comprise any amino acid residues capable of participating in the formation of stable disulphide bonds with peptides comprising an amino acid sequence identical to the amino acid sequence of said C H  region in the presence of polyclonal human IgG or when administered to a human being or (ii) a nucleotide sequence encoding a constant C H  region of a human Ig, wherein the nucleotide sequence encoding the C H  region has been modified such that the region corresponding to the hinge region and, as required by the Ig subtype, other regions of the C H  region, such as the C H 3 region, does not comprise any amino acid residues which participate in the formation of disulphide bonds or covalent or stable non-covalent inter-heavy chain bonds with other peptides comprising an identical amino acid sequence of the C H  region of the human Ig in the presence of polyclonal human IgG or when administered to a human being or (iii) a sequence complementary thereto. 
     
     
         231 . A nucleic acid construct according to  claim 230 , wherein the nucleic acid sequence encoding the C H  region has been modified such that the region corresponding to the hinge region does not comprise any cysteine residues. 
     
     
         232 . A nucleic acid construct according to  claim 230 , wherein said construct comprises a nucleic acid sequence encoding the V H  region of an antigen specific antibody, or a sequence complementary thereto, wherein the nucleic acid sequence encoding the V H  region is operably linked to the nucleic acid sequence encoding the C H  region, or a sequence complementary thereto. 
     
     
         233 . A host cell comprising a nucleic acid construct according to  claim 230 . 
     
     
         234 . A method of treating a disease or disorder, wherein said method comprises administering to a subject in need of treatment a therapeutically effective amount of a monovalent antibody comprising a light chain and a heavy chain, wherein
 a) said light chain comprises the amino acid sequence of the variable (V L ) region of a selected antigen specific antibody and the amino acid sequence of the constant (C L ) region of an Ig, and wherein, in case of an IgG1 subtype, the amino sequence of the constant (C L ) region has been modified so that it does not contain any amino acids capable of participating in the formation of disulfide bonds or covalent bonds with other peptides comprising an identical amino acid sequence of the constant (C L ) region of the Ig in the presence of polyclonal human IgG or when administered to a human being, and   b) said heavy chain comprises the amino acid sequence of the variable (V H ) region of said selected antigen specific antibody and the amino acid sequence of the constant (C H ) region of human Ig, wherein the amino acid sequence of the constant (C H ) region has been modified so that the hinge region and, as required by the Ig subtype, other regions of the C H  region, such as the C H 3 region, does not contain any amino acid residues which participate in the formation of disulphide bonds or covalent or stable non-covalent inter-heavy chain bonds with other peptides comprising an identical amino acid sequence of the constant (C H ) region of the human Ig in the presence of polyclonal human IgG or when administered to a human being,   a pharmaceutical composition comprising said antibody, an immunoconjugate comprising said antibody, or a nucleic acid construct according to  claim 231 .   
     
     
         235 . The method of  claim 234 , wherein the antibody comprises a human IgG4 C H  region. 
     
     
         236 . The method of  claim 234 , wherein the heavy chain comprises the amino acid sequence of the heavy chain variable (VH) region of said selected antigen specific antibody and the amino acid sequence of SEQ ID NO: 16. 
     
     
         237 . The method of  claim 236 , wherein the CL region is the constant region of the kappa light chain of a human IgG. 
     
     
         238 . The method of  claim 232 , wherein the CL region comprises the amino acid sequence of SEQ ID NO: 2. 
     
     
         239 . The method of  claim 236 , wherein the CL region is the constant region of the lambda light chain of a human IgG. 
     
     
         240 . The method of  claim 239 , wherein the CL region comprises the amino acid sequence of SEQ ID NO: 4. 
     
     
         241 . The method of  claim 236 , wherein the light chain and the heavy chain are connected to each other via one or more disulphide bonds or via an amide bond. 
     
     
         242 . The method of  claim 234 , wherein the heavy chain comprises the amino acid sequence of the heavy chain variable (VH) region of the selected antigen specific antibody and the amino acid sequence of SEQ ID NO: 22. 
     
     
         243 . The method of  claim 234 , wherein the heavy chain comprises the amino acid sequence of the heavy chain variable (VH) region of said selected antigen specific antibody and the amino acid sequence of the heavy chain constant (CH) region of human IgG1, wherein the CH region comprises the amino acid sequence set forth in SEQ ID NO: 19, wherein Lys (K) in position 292 of SEQ ID NO: 19 has been replaced by Arg (R); and wherein the hinge region of the heavy chain lacks cysteine residues. 
     
     
         244 . The method of  claim 243 , wherein the CL region is a kappa light chain CL region comprising the amino acid sequence set forth in SEQ ID NO: 18, wherein the terminal cysteine residue in position 106 of SEQ ID NO: 18 has been replaced with a different amino acid residue or has been deleted. 
     
     
         245 . The method of  claim 243 , wherein the CL region is a lambda light chain CL region having the amino acid sequence set forth in SEQ ID NO: 17, wherein the cysteine residue in position 104 of SEQ ID NO: 17 has been replaced with a different amino acid residue or has been deleted. 
     
     
         246 . The method of  claim 234 , wherein the heavy chain comprises the amino acid sequence of the heavy chain variable (VH) region of said selected antigen specific antibody and the amino acid sequence of the heavy chain constant (CH) region of human IgG1, wherein the CH region comprises the amino acid sequence set forth in SEQ ID NO: 19, wherein Lys (K) in position 292 of SEQ ID NO: 19 has been replaced by Arg (R), and Ser (S) in position 14 of SEQ ID NO: 19 has been replaced by Cys (C); and wherein the hinge region of the heavy chain lacks cysteine residues. 
     
     
         247 . The method of  claim 234 , wherein the heavy chain comprises the amino acid sequence of the heavy chain variable (VH) region of said selected antigen specific antibody and the amino acid sequence of the heavy chain constant (CH) region of IgG2, wherein the CH region comprises the amino acid sequence set forth in SEQ ID NO: 20, wherein the following amino acid substitutions have been made: Arg (R) in position 234 of SEQ ID NO: 20 has been replaced by Gln (Q), Met (M) in position 276 of SEQ ID NO: 20 has been replaced by Val (V), Lys (K) in position 288 of SEQ ID NO: 20 has been replaced by Arg (R), Gln (Q) in position 298 of SEQ ID NO: 20 has been replaced by Glu (E), and Pro (P) in position 324 of SEQ ID NO: 20 has been replaced by Leu (L); and wherein the hinge region lacks cysteine residues. 
     
     
         248 . The method of  claim 234 , wherein the heavy chain comprises the amino acid sequence of the heavy chain variable (VH) region of said selected antigen specific antibody and the amino acid sequence of the heavy chain constant (CH) region of IgG3, wherein the CH region comprises the amino acid sequence set forth in SEQ ID NO: 21, wherein one or more of the following amino acid substitutions have been made: Arg (R) in position 285 of SEQ ID NO: 21 has been replaced by Gln (Q), Ser (S) in position 314 of SEQ ID NO: 21 has been replaced by Asn (N), Asn (N) in position 322 of SEQ ID NO: 21 has been replaced by Lys (K), Met (M) in position 327 of SEQ ID NO: 21 has been replaced by Val (V), Lys (K) in position 339 of SEQ ID NO: 21 has been replaced by Arg (R), Gln (Q) in position 349 of SEQ ID NO: 21 has been replaced by Glu (E), Ile (I) in position 352 of SEQ ID NO: 21 has been replaced by Val (V), Arg (R) in position 365 of SEQ ID NO: 21 has been replaced by His (H), Phe (F) in position 366 of SEQ ID NO: 21 has been replaced by Tyr (Y), and Pro (P) in position 375 of SEQ ID NO: 21 has been replaced by Leu (L); and wherein the hinge region of the heavy chain lacks cysteine residues.