Multi-specific monoclonal antibodies

Abstract

The present invention is relevant to the generation of multi-specific antibodies, antibodies that are distinguished by their ability to bind to multiple antigens with specificity and with affinity. In particular, the present invention is related to bi-specific antibodies.

1 - 3 . (canceled) 
     
     
         4 . The method of  claim 15 , wherein step (d) further comprises screening for one or more optimized characteristics selected from the group consisting of equilibrium dissociation constant (K D ); stability; melting temperature (T m ); pI; solubility; expression level; reduced immunogenicity; and improved effector function. 
     
     
         5 - 9 . (canceled) 
     
     
         10 . The method of  claim 15 , wherein the multi-specific antibody of step (b) specifically binds 3 epitopes. 
     
     
         11 . The method of  claim 15 , wherein the multi-specific antibody of step (b) specifically binds 4 epitopes. 
     
     
         12 - 14 . (canceled) 
     
     
         15 . A method of identifying and modifying a multi-specific antibody, the method comprising:
 a. generating a library of antibodies;   b. screening the library to identify a multi-specific antibody that binds to two or more epitopes;   c. evolving the multi-specific antibody to produce a set of modified antibodies;   d. screening the modified antibodies for optimized binding to one or more of the two or more epitopes; and   e. modifying the multi-specific antibody such that the multi-specific antibody comprises an organic moiety,   wherein step (c) employs one or more of comprehensive positional evolution (CPE); comprehensive positional insertion evolution (CPI); comprehensive positional deletion evolution (CPD); comprehensive positional evolution (CPE) followed by combinatorial protein synthesis (CPS); and comprehensive positional deletion evolution (CPD) followed by combinatorial protein synthesis (CPS).   
     
     
         16 . The method of  claim 15 , further comprising manufacturing the modified, multi-specific antibody protein. 
     
     
         17 . The method of  claim 15 , wherein:
 In step (c) the multi-specific antibody is evolved in a manufacturing host; and   further comprising a step of
 f. producing the modified, multi-specific antibody protein in the manufacturing host. 
   
     
     
         18 . The method of  claim 17  wherein the manufacturing host is selected from a member of the group consisting of 3T3 mouse fibroblast cells; BHK21 Syrian hamster fibroblast cells; MDCK, dog epithelial cells; Hela human epithelial cells; PtK1 rat kangaroo epithelial cells; SP2/0 mouse plasma cells; and NS0 mouse plasma cells; HEK 293 human embryonic kidney cells; COS monkey kidney cells; CHO, Chinese hamster ovary cells; R1 mouse embryonic cells; E14.1 mouse embryonic cells; H1 human embryonic cells; H9 human embryonic cells; PER C.6, human embryonic cells;  S. cerevisiae  yeast cells; and pichia yeast cells. 
     
     
         19 . The method of  claim 15  wherein the screening steps comprise fluorescence-activated cell sorting (FACS). 
     
     
         20 . The method of  claim 15  wherein the library of antibodies is generated from an approved ethical protein therapeutic drug. 
     
     
         21 . (canceled) 
     
     
         22 . The method of  claim 15  wherein the screening steps employ a technique selected from the group consisting of quantitative ELISA; affinity ELISA; ELISPOT; flow cytometry, immunocytology, Biacore® surface plasmon resonance analysis, Sapidyne KinExA™ kinetic exclusion assay; SDS-PAGE; Western blot, and HPLC. 
     
     
         23 . The method of  claim 15  wherein the multi-specific antibody of step (b) specifically binds 5 or more epitopes. 
     
     
         24 . The method of  claim 17 , wherein the manufacturing host is selected from CHO, HEK293 and COS-7. 
     
     
         25 . The method of  claim 17 , wherein the manufacturing host uses cell surface display. 
     
     
         26 . The method of  claim 15 , wherein the organic moiety is selected from a linear hydrophilic polymeric group, a branched hydrophilic polymeric group, a fatty acid group, and a fatty acid ester group. 
     
     
         27 . The method of  claim 15 , wherein the organic moiety is a linear hydrophilic polymeric group or a branched hydrophilic polymeric group selected from a polyalkane glycol group, a carbohydrate polymeric group, an amino acid polymeric group and a polyvinyl pyrrolidone group. 
     
     
         28 . The method of  claim 27 , wherein the organic moiety is the carbohydrate polymeric group and is selected from a dextran group, a cellulose group, an oligosaccharide group, and a polysaccharide group. 
     
     
         29 . The method of  claim 27 , wherein the organic moiety is the amino acid polymeric group and is selected from a polylysine group, a polyarginine group, and a polyaspartate group. 
     
     
         30 . The method of  claim 26 , wherein the organic moiety is the fatty acid group or the fatty acid ester group and the organic moiety contains from about eight to about forty carbon atoms. 
     
     
         31 . The method of  claim 26 , wherein the organic moiety is the fatty acid group and is selected from an n-dodecanoate group, an n-tetradecanoate group, an n-octadecanoate group, an n-eicosanoate group, an n-docosanoate group, an n-triacontanoate group, an n-tetracontanoate group, a cis-δ 9-octadecanoate group, cis-δ 5,8,11,14-eicosatetraenoate groups, an octanedioic acid group, a tetradecanedioic acid group, an octadecanedioic acid group, and a docosanedioic acid group.