Production of heteromultimeric proteins

Abstract

Described herein are methods for the efficient production of antibodies and other multimeric protein complexes (collectively referred to herein as heteromultimeric proteins) capable of specifically binding to more than one target. The targets may be, for example, different epitopes on a single molecule or located on different molecules. The methods combine efficient, high gene expression level, appropriate assembly, and ease of purification for the heteromultimeric proteins. The invention also provides methods of using these heteromultimeric proteins, and compositions, kits and articles of manufacture comprising these antibodies.

1 - 89 . (canceled) 
     
     
         90 . A method of generating an antibody library,
 wherein each antibody in the antibody library comprises a first antibody heavy chain paired with a first antibody light chain and a second antibody heavy chain paired with a second antibody light chain, wherein the first and second antibody heavy chains are linked by at least one interchain disulfide bond, and wherein at least two antibodies in the antibody library comprise two different second antibody heavy chains and/or two different second antibody light chains, the method comprising the steps of:   (a) culturing a first host cell comprising one or more nucleic acid(s) encoding the first antibody heavy chain and the first antibody light chain under conditions where a first half antibody comprising the first antibody heavy chain and the first antibody light chain is expressed;   (b) culturing at least two additional host cells, each comprising one or more nucleic acid(s) encoding a second antibody heavy chain and a second antibody light chain under conditions where at least two second half antibodies each comprising the second antibody heavy chain and the second antibody light chain are expressed, wherein the at least two second half antibodies comprise two different second antibody heavy chains and/or two different second antibody light chains;   (c) combining the culture of the first host cell and the cultures of the at least two additional host cells to produce a combined culture comprising the first host cell and the at least two additional host cells, wherein the antibodies are formed in the combined culture; and   (d) recovering the antibodies without prior recovery or purification of the first and second half antibodies from separate cultures.   
     
     
         91 . The method of  claim 90 , wherein at least two of the antibodies in the antibody library comprise two different second antibody heavy chains and two different second antibody light chains. 
     
     
         92 . The method of  claim 90 , wherein said at least one interchain disulfide bond is in a hinge region of the antibody. 
     
     
         93 . The method of  claim 90 , wherein said at least one interchain disulfide bond is between hinge regions of the first and second antibody heavy chains. 
     
     
         94 . The method of  claim 90 , wherein the first antibody heavy chain and first antibody light chain are encoded on separate expression plasmids. 
     
     
         95 . The method of  claim 90 , wherein the first antibody heavy chain and first antibody light chain are encoded on the same expression plasmid. 
     
     
         96 . The method of  claim 90 , wherein the second antibody heavy chains and second antibody light chains are encoded on separate expression plasmids. 
     
     
         97 . The method of  claim 90 , wherein the second antibody heavy chain and second antibody light chain of the corresponding second half antibody are encoded on the same expression plasmid. 
     
     
         98 . The method of  claim 90 , wherein the first host cell and the at least two additional host cells are selected from the group consisting of prokaryotic cells, eukaryotic cells, mammalian cells, and plant cells. 
     
     
         99 . The method of  claim 98 , wherein the first host cell and the at least two additional host cells are prokaryotic cells, and wherein the prokaryotic cells are  E. coli  cells. 
     
     
         100 . The method of  claim 99 , wherein said  E. coli  cells are Ipp deficient (Ipp − ). 
     
     
         101 . The method of  claim 98 , wherein the first host cell and the at least two additional host cells are mammalian cells, and wherein the mammalian cells are Chinese hamster ovary (CHO) cells. 
     
     
         102 . The method of  claim 90 , wherein the recovering step (d) further comprises at least one purification step. 
     
     
         103 . The method of  claim 102 , wherein the at least one purification step comprises:
 (i) capturing the antibodies on a column comprising Protein A, and   (ii) eluting the antibodies from said column.   
     
     
         104 . The method of  claim 90 , wherein said antibodies are human antibodies or humanized antibodies. 
     
     
         105 . The method of  claim 90 , wherein the antibodies in the antibody library are bispecific antibodies or multispecific antibodies. 
     
     
         106 . The method of  claim 105 , wherein the antibodies in the antibody library are bispecific antibodies. 
     
     
         107 . The method of  claim 106 , wherein the first antibody heavy chain comprises a first Fc domain or a variant thereof, and the at least two second antibody heavy chains each comprises a second Fc domain or a variant thereof. 
     
     
         108 . The method of  claim 107 , wherein the first and second Fc domains are both human IgA, IgG, or IgD Fc domains or variants thereof. 
     
     
         109 . The method of  claim 108 , wherein the first and second Fc domains are both human IgG Fc domains or variants thereof, and wherein the human IgG Fc domains or variants thereof are both IgG1 or IgG2 Fc domains or variants thereof. 
     
     
         110 . The method of  claim 107 , wherein the first Fc domain is a first Fc domain variant and the second Fc domain is a second Fc domain variant, wherein the first Fc domain variant and the second Fc domain variant meet at an interface, wherein the interface of the first Fc domain variant comprises a cavity, and wherein the interface of the second Fc domain variant comprises a protuberance which is positionable in the cavity in the interface of the first Fc domain variant. 
     
     
         111 . The method of  claim 106 , wherein said bispecific antibodies are capable of specifically binding two different antigens or two epitopes on the same antigen. 
     
     
         112 . The method of  claim 90 , further comprising a step of adding a reductant. 
     
     
         113 . The method of  claim 90 , wherein the first half antibody and the second half antibodies are secreted into the culture medium of the combined culture comprising the first host cells and the at least two additional host cells. 
     
     
         114 . The method of  claim 113 , wherein the antibodies are formed in the culture medium of the combined culture comprising the first host cells and the at least two additional host cells. 
     
     
         115 . The method of  claim 90 , further comprising disrupting the cell membrane in the combined culture comprising the first host cells and the at least two additional host cells to form the antibodies. 
     
     
         116 . The method of  claim 115 , wherein disrupting the cell membrane comprises permeabilizing the cell membrane or cell membrane disintegration. 
     
     
         117 . The method of  claim 116 , wherein disrupting the cell membrane is selected from the group consisting of: (i) enzymatic lysis, (ii) cell lysis, (iii) sonication, (iv) osmotic shock, (v) passage through a microfluidizer, (vi) addition of EDTA, detergents, solvents, surfactants, and/or hypotonic buffers, (vii) use of freeze/thaw techniques, (viii) electroporation, and (ix) passage through a stainless steel ball homogenizer.