Express humanization of antibodies

Abstract

The disclosure provides a method for generation of humanized full length antibodies in mammalian cells. A library of humanized variants is provided with high, validated human framework diversity without requiring back-mutations to retain original affinity. Synthetic CDR encoding fragment libraries derived from a template antibody are ligated to human framework region encoding fragments from a human framework pool limited only to germline sequences from a functionally expressed antibodies. The vector comprises a nucleic acid sequence encoding HC framework region 4. No CDR grafting or phage display is required.

We claim: 
     
         1 . A method of producing a humanized antibody or a humanized antibody fragment, the method comprising:
 (a) synthesizing immunoglobulin heavy chain double stranded DNA fragment libraries comprising complementarity determining region fragment encoding libraries and framework fragment encoding libraries, wherein at least one complementarity determining region fragment library includes at least one double stranded DNA fragment encoding at least a portion of a heavy chain complementarity determining region having at least 90% sequence identity to a complementarity determining region of a non-human template antibody and each framework fragment library includes at least one double stranded DNA fragment encoding at least a portion of a heavy chain framework derived from a human framework pool obtained from functionally expressed human antibodies having framework regions that have a sequence identity of at least 85% with a framework region of the template antibody;   (b) synthesizing immunoglobulin light chain double stranded DNA fragment libraries comprising complementarity determining region fragment encoding libraries and framework fragment encoding libraries, wherein at least one complementarity determining region fragment library includes at least one double strand DNAed fragment encoding at least a portion of a light chain complementarity determining region having at least 90% sequence identity to a complementarity determining region of the template antibody and each framework fragment library comprising at least one double stranded DNA fragment encoding at least a portion of a light chain framework derived from a human framework pool obtained from functionally expressed human antibodies having framework regions that have a sequence identity at least 85% with a framework region of the template antibody;   (c) assembling from the heavy chain fragment libraries by stepwise liquid phase ligation of heavy chain framework encoding fragments from the heavy chain framework fragment encoding libraries and heavy chain complementarity determining region encoding fragments from the heavy chain complementarity determining region fragment encoding libraries in the order of: framework1-complementarity determining region1-framework2-complementarity determining region2-framework3-complementarity determining region3 to produce a humanized heavy chain variable domain encoding library;   (d) assembling from the light chain fragment libraries by stepwise liquid phase ligation of light chain framework encoding fragments from the light chain framework fragment encoding libraries and light chain complementarity determining region encoding fragments from the light chain complementarity determining region fragment encoding libraries in the order of: framework1-complementarity determining region1-framework2-complementarity determining region2-framework3-complementarity determining region3 to produce a humanized light chain variable domain encoding library;   (e) cloning the assembled humanized heavy chain variable domain encoding library and the assembled light chain variable domain encoding library into an expression vector to create a humanization library;   (f) transfecting the humanization library into cells;   (g) expressing humanized antibodies or humanized antibody fragments in the cells to create a humanized antibody library; and   (h) screening the humanized antibody library to determine an affinity of the humanized antibodies or humanized antibody fragments for an antigen compared to an affinity of the template antibody to the same antigen.   
     
     
         2 . The method of  claim 1  wherein the expression vector comprises a nucleic acid sequence encoding heavy chain framework region 4. 
     
     
         3 . The method of  claim 2  wherein the nucleotide sequence encoding heavy chain framework 4 is derived from a human heavy chain variable domain of a functionally expressed human antibody. 
     
     
         4 . The method of  claim 1  wherein the expression vector comprises a nucleic acid sequence encoding light chain framework region 4. 
     
     
         5 . The method of  claim 4  wherein the nucleotide sequence encoding light chain framework 4 is derived from a human light chain variable domain of a functionally expressed human antibody. 
     
     
         6 . The method of  claim 1  wherein the humanized antibody library has 10,000,000 members or fewer. 
     
     
         7 . The method of  claim 6  wherein the humanized antibody library has 1,000,000 members or fewer. 
     
     
         8 . The method of  claim 7  wherein the humanized antibody library has 100,000 members or fewer. 
     
     
         9 . The method of  claim 1  wherein the cloning step comprises cloning the assembled humanized heavy chain variable domain encoding library into the expression vector to create a vector-heavy chain variable domain DNA library, and ligating the assembled light chain variable domain encoding library into the vector-heavy chain carriable domain DNA library to create the humanization library. 
     
     
         10 . The method of  claim 1  wherein the expression step comprises expressing both the humanized heavy chain variable domain and the humanized light chain variable domain from a single promoter. 
     
     
         11 . The method of  claim 1  further comprising a step of screening for a humanized antibody or humanized antibody fragment having one or more additional improved characteristics when compared to the template antibody; the one or more 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 relative to the template antibody. 
     
     
         12 . The method of  claim 1  wherein the cells are selected from a eukaryotic cell production host cell line selected from 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; COS monkey kidney cells; CHO, CHO-S Chinese hamster ovary cells; R1 mouse embryonic cells; E14.1 mouse embryonic cells; PER C.6, human embryonic cells;  S. cerevisiae  yeast cells; and picchia yeast cells. 
     
     
         13 . The method of  claim 12  wherein the eukaryotic cell production host cell line is CHO-S. 
     
     
         14 . The method of  claim 12  wherein the eukaryotic cell production host cell line is CHOK1SV or NS0. 
     
     
         15 . The method of  claim 1  wherein the cell is a eukaryotic cell production host with antibody cell surface display. 
     
     
         16 . The method of  claim 15  wherein one or both of the screening steps is performed in the eukaryotic cell production host. 
     
     
         17 . The method of  claim 1  wherein one or both of the screening steps are selected from quantitative ELISA; affinity ELISA; ELISPOT; flow cytometry, immunocytology, Biacore® surface plasmon resonance analysis, Sapidyne KinExA™ kinetic exclusion assay; SDS-PAGE; Western blot, and HPLC. 
     
     
         18 . The method of  claim 1 , wherein the at least one double strand DNA fragment encoding at least a portion of a heavy chain complementarity determining region is derived from the template antibody through evolving a complementarity determining region of the template antibody. 
     
     
         19 . The method of  claim 1 , wherein the at least one double strand DNA fragment encoding at least a portion of a light chain complementarity determining region is derived from the template antibody through evolving a complementarity determining region of the template antibody. 
     
     
         20 . The method of  claim 18 , wherein the evolving a complementarity determining region is accomplished by substitutions, insertions and deletions. 
     
     
         21 . The method of  claim 18 , wherein the evolving a complementarity determining region is accomplished by Comprehensive Positional Evolution (CPE™), Comprehensive Protein Synthesis (CPS™), Flex Evolution, Synergy Evolution, Comprehensive Positional Insertion evolution (CPI™), or Comprehensive Positional Deletion evolution (CPD™). 
     
     
         22 . The method of  claim 1 , wherein the at least a portion of a heavy chain complementarity determining region has a sequence at least 90% identical to a complementarity determining region of the template antibody.