Conjugated compounds comprising cysteine-engineered antibodies

Abstract

This disclosure provides conjugate compounds comprising antibodies and fragments thereof engineered with one or more reactive cysteine residues and more specifically to conjugate compounds with therapeutic or diagnostic applications. The conjugate compounds comprise cysteine-engineered antibodies or fragments thereof conjugated, for example, with chemotherapeutic drugs, toxins, and detection labels such as radionuclides or fluorophores. The disclosure also provides methods of using the disclosed conjugate compounds for in vitro, in situ, ex vivo, and in vivo diagnosis or treatment of mammalian cells, or associated pathological conditions.

What is claimed is: 
     
         1 . A method for making a conjugate compound comprising:
 (i) mutagenizing at least a nucleic acid sequence encoding an antibody or Fc fusion protein by inserting a codon encoding for a cysteine (C) between the codons encoding the amino acids at positions 239 and 240, wherein the amino acid position numbering is according to the EU index as set forth in Kabat;   (ii) expressing the cysteine-engineered antibody or Fc fusion protein;   (iii) isolating the cysteine-engineered antibody or Fc fusion protein; and   (iv) reacting at least one engineered cysteine group of the cysteine-engineered antibody or Fc fusion protein with a heterologous moiety.   
     
     
         2 . A method for making a conjugate compound comprising:
 (i) operably linking a nucleic acid sequence encoding a variable heavy chain region or a heterologous protein to a nucleic acid sequence encoding an Fc region protein, wherein the nucleic acid sequence encoding the Fc region protein comprises:   at least a codon encoding a cysteine inserted between the codons encoding the amino acid at positions 239 and 240, wherein the amino acid position numbering is according to the EU index as set forth in Kabat;   (ii) expressing the cysteine-engineered antibody or Fc fusion protein;   (iii) isolating the cysteine-engineered antibody or Fc fusion protein; and   (iv) reacting at least one engineered cysteine group of the cysteine-engineered antibody or Fc fusion protein with a heterologous moiety.   
     
     
         3 . The method according to  claim 1 , wherein the cysteine-engineered antibody or Fc fusion protein further comprises at least one engineered cysteine amino acid selected from cysteine amino acid substitutions at amino acid position 241, 243, 251, 253, 258, 264, 269, 271, 272, 274, 280, 281, 285, 288, 291, 293, 294, 296, 301, 307, 309, 311, 318, 329, 340, 341, 345, 357, 385, 386, 387, 401, 402, 411, 417, 433, 435, or 439 wherein the amino acid position numbering is according to the EU index as set forth in Kabat. 
     
     
         4 . The method according to  claim 3 , wherein the engineered cysteine amino acid is selected from cysteine amino acid substitutions at amino acid positions 241, 243, 251, 253, 258, 264, 271, 285, 288, 291, 296, 301, 307, 309, 311, 329, 385, 387, 433, or 435 and combinations thereof. 
     
     
         5 . The method according to  claim 4 , wherein the engineered cysteine amino acid is selected from cysteine amino acid substitutions at amino acid positions 258, or 435 and combinations thereof. 
     
     
         6 . The method according to  claim 1 , wherein the cysteine-engineered antibody or Fc fusion protein comprises:
 a Cysteine (C) inserted between the Serine (S) located at position 239 and the Valine (V) located at position 240; and   (a) a Cysteine (C) substituting the Glutamic acid (E) located at position 258;   (b) a Cysteine (C) substituting the Histidine (H) located at position 435;   (c) a Cysteine (C) substituting the Arginine (R) located at position 435; or   (d) a combination thereof,   wherein the amino acid position numbering is according to the EU index as set forth in Kabat.   
     
     
         7 . The method according to  claim 1 , wherein the Fc domain further comprises at least one engineered cysteine residue selected from cysteine amino acid substitutions at amino acid positions 239, 248, 254, 273, 279, 282, 284, 286, 287, 289, 297, 298, 312, 324, 326, 330, 335, 337, 339, 350, 355, 356, 359, 360, 361, 375, 383, 384, 389, 398, 400, 413, 415, 418, 422, 440, 441, 442, 443 and 446. 
     
     
         8 . The method according to  claim 1 , wherein each one of the heterologous moieties is conjugated to an engineered cysteine. 
     
     
         9 . The method according to  claim 1 , wherein the Fc domain of the antibody or Fc fusion protein is a human IgG Fc domain, optionally selected from the group consisting of the IgG Fc domain of an IgG1, IgG2, IgG3, or IgG4. 
     
     
         10 . The method according to  claim 1 , wherein the Fc fusion protein comprises an antigen binding domain selected from the group consisting of: (a) an scFv; (b) a diabody; (c) an Fd fragment; (d) an Fv fragment; (e) a TANDAB®; (f) a F(ab′)2 fragment; (g) a FCAB™, and (h) a F(ab) fragment. 
     
     
         11 . The method according to  claim 1 , wherein the antibody is a monoclonal antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, a human antibody, or a humanized antibody. 
     
     
         12 . The method according to  claim 1 , wherein at least one heterologous moiety is a toxin, drug, radionuclide, immunomodulator, cytokine, lymphokine, chemokine, growth factor, tumor necrosis factor, hormone, hormone antagonist, enzyme, oligonucleotide, DNA, RNA, siRNA, RNAi, microRNA, peptide nucleic acid, photoactive therapeutic agent, anti-angiogenic agent, pro-apoptotic agent, non-natural amino acid, peptide, lipid, carbohydrate, scaffolding molecule, fluorescent tag, visualization peptide, biotin, serum half-life extender, capture tag, chelating agent, solid support, or a combination thereof, and wherein conjugation is at one of the engineered cysteines. 
     
     
         13 . The method according to  claim 12 , wherein the drug is an auristatin, a tubulysin, a pyrrolobenzodiazepine (PBD), or a maytansinoid. 
     
     
         14 . The method according to  claim 13 , wherein the drug is a pyrrolobenzodiazepine (PBD). 
     
     
         15 . The method according to  claim 1 , wherein the cysteine-engineered antibody or Fc fusion protein specifically binds to at least one target. 
     
     
         16 . The conjugate compound according to  claim 15 , wherein the target is a tumor antigen and the heterologous moiety is an antitumor agent. 
     
     
         17 . A nucleic acid encoding a cysteine-engineered antibody or Fc fusion protein, wherein the Fc domain of the antibody or Fc fusion protein comprises a cysteine amino acid insertion between positions 239 and 240, wherein the amino acid position numbering is according to the EU index as set forth in Kabat. 
     
     
         18 . A vector comprising the nucleic acid of  claim 17 . 
     
     
         19 . A host cell comprising the nucleic acid of  claim 17 . 
     
     
         20 . A host cell comprising the vector of  claim 18 .