Readily isolated bispecific antibodies with native immunoglobulin format

Abstract

The invention relates to antigen-binding proteins or antibodies having heterodimers of heavy chains, i.e., two immunoglobulin heavy chains that differ by at least one or two amino acid(s) that allows for isolation of the antigen-binding protein based on a differential affinity of an immunoglobulin heavy chain and a modified/mutated immunoglobulin heavy chain toward an affinity reagent. The invention also relates antigen-binding proteins, including bispecific antibodies, having IgG CH1 regions with different affinities with respect to affinity reagent(s) that allows rapid isolation by differential binding of the IgG regions to the affinity reagent(s).

1 .- 8 . (canceled) 
     
     
         9 . A method for making a multispecific antibody comprising:
 a) obtaining a nucleic acid sequence encoding a first immunoglobulin heavy chain comprising a first variable domain that recognizes a first epitope, wherein the first immunoglobulin heavy chain comprises an IgG1, IgG2, IgG3 or IgG4 isotype constant domain;   b) obtaining a second nucleic acid sequence encoding a second immunoglobulin heavy chain comprising a second variable domain that recognizes a second epitope, wherein the second immunoglobulin heavy chain comprises an IgG1, IgG2, IgG3 or IgG4 isotype constant domain, or a chimeric isotype constant domain thereof, that comprises a modification in its CH1 domain that eradicates or reduces binding to the CaptureSelect® IgG-CH1 affinity reagent;   c) obtaining a third nucleic acid sequence encoding an immunoglobulin a light chain that pairs with the first and the second immunoglobulin heavy chain;   d) introducing the first, second, and third nucleic acid sequences into a mammalian cell;   e) allowing the cell to express an immunoglobulin; and   f) isolating the immunoglobulin using the CaptureSelect® IgG-CH1 affinity reagent, or any affinity reagent targeting the human IgG1, IgG2, IgG3 and IgG4 CHI domain.   
     
     
         10 . The method of  claim 9 , wherein the cell is selected from a CHO, COS, 293, HeLa, and a retinal cell expressing a viral nucleic acid sequence (e.g., a PERC.6™ cell). 
     
     
         11 . The method of  claim 9 , wherein the method comprises a step of isolating from a disrupted cell or a mixture of antibodies a multispecific antibody having differentially modified IgG1, IgG2, IgG3 or IgG4 CH1 domains, wherein the differentially modified CH1 domains are non-immunogenic or substantially non-immunogenic in a human, and wherein the modification results in a multispecific antibody with heterodimeric heavy chains whose monomers have a differential affinity for an affinity reagent, and the multispecific antibody is isolated from the disrupted cell or the mixture using an affinity reagent. 
     
     
         12 . The method of  claim 9 , wherein the multispecific antibody can be preferentially purified at specific pH range and salt concentration. 
     
     
         13 . The method of  claim 9 , wherein the multispecific antibody is composed of two different heavy chains, one modified at positions 40 and 47 (IMGT® numbering), or at positions 40, 45 and 47 (IMGT® numbering), on its CH1 domain; and the other one lacks modification at positions 40 and 47 (IMGT® numbering), or at positions 40, 45 and 47 (IMGT® numbering), on its CH1 domain. 
     
     
         14 . The method of  claim 9 , wherein the modification in the second CH1 domain comprises an S40T mutation in the IMGT exon numbering system, a T47S mutation in the IMGT exon numbering system or a combination thereof. 
     
     
         15 . The method of  claim 14 , wherein the first CH1 domain of the multispecific antibody, the second CH1 domain or both the first and second CH1 domains are non-immunogenic or substantially non-immunogenic in a human. 
     
     
         16 . The method of  claim 9 , wherein the multispecific antibody is isolated on a solid support comprising a CaptureSelect® IgG-CH1 affinity reagent, or any affinity reagent targeting the human IgG1, IgG2, IgG3 and IgG4 CH1 domain. 
     
     
         17 . The method of  claim 16 , wherein the solid support comprises a CaptureSelect® IgG-CH1 affinity column, or any affinity reagent targeting the human IgG1, IgG2, IgG3 and IgG4 CH1 domain, and the multispecific antibody is isolated employing a pH gradient. 
     
     
         18 . The method of  claim 17 , wherein the pH gradient is a step gradient comprising one or more pH steps between pH 3 and pH 5. 
     
     
         19 . The method of  claim 9 , wherein the first immunoglobulin heavy chain comprises a mutation or modification altering its binding properties to an affinity chromatography resin. 
     
     
         20 . The method of  claim 19 , wherein the first immunoglobulin heavy chain comprises a mutation altering its binding properties to Protein A. 
     
     
         21 . The method of  claim 20 , wherein the first immunoglobulin heavy chain comprises a H435R mutation altering its binding properties to Protein A.