Express humanization of antibodies
AssigneeBioAtla, Inc.
InventorJay M. Short
Life of this patent
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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.
Claims20
A method of producing a humanized antibody from a template antibody, the method comprising:
- (a) synthesizing immunoglobulin heavy chain (HC) double stranded DNA fragment libraries comprising complementarity determining region (CDR) fragment encoding libraries and framework (FW) fragment encoding libraries, wherein at least one CDR fragment encoding library comprises a plurality of CDR-encoding DNA fragments derived from the template antibody using an evolution technique independently selected from Comprehensive Positional Evolution, Comprehensive Protein Synthesis, Flex Evolution, Synergy Evolution, Comprehensive Positional Insertion evolution, Comprehensive Positional Deletion evolution, and combinations thereof, and each FW fragment encoding library is selected from a human framework pool obtained from functionally expressed human antibodies;
- (b) synthesizing immunoglobulin light chain (LC) double stranded DNA fragment libraries comprising CDR fragment encoding libraries and FW fragment encoding libraries, wherein at least one CDR fragment encoding library comprises a plurality of CDR-encoding DNA fragments derived from the template antibody using an evolution technique independently selected from Comprehensive Positional Evolution, Comprehensively Protein Synthesis, Flex Evolution, Synergy Evolution, Comprehensive Positional Insertion evolution, Comprehensive Positional Deletion evolution, and combinations thereof, and each FW fragment encoding library is selected from a human framework pool obtained from functionally expressed human antibodies;
- (c) assembling from the HC fragment libraries by stepwise liquid phase ligation of heavy chain FW encoding fragments and CDR encoding fragments in the order of:
- FW1-CDR1-FW2-CDR2-FW3-CDR3 to produce a humanized HC variable domain encoding library comprising one or more polynucleotides;
- (d) assembling from the LC fragment libraries by stepwise liquid phase ligation of light chain FW encoding fragments and CDR encoding fragments in the order of:
- FW1-CDR1-FW2-CDR2-FW3-CDR3 to produce a humanized LC variable domain encoding library comprising one or more polynucleotides;
- (e) cloning the one or more polynucleotides in the assembled humanized heavy chain variable domain library and the one or more polynucleotides in the assembled light chain variable domain library into an expression vector to create a humanization library;
- (f) transfecting the humanization library into cells;
- and
- (g) expressing humanized antibodies in the cells to create a humanized antibody library.
Dependencies are inferred from the claim text.