Structure-Based Selection and Affinity Maturation of Antibody Library

Abstract

The present invention provides a structure-based methodology for efficiently generating and screening a library of recombinant antibodies for optimized antibodies with desirable functions, such as higher binding affinity or low immunogenicity. In one embodiment, a method is provided for constructing a library of antibody sequences based on a three dimensional structure of a lead antibody. The method comprises: providing a lead structural template comprising the amino acid sequence of the variable region of the heavy chain (V H ) or light chain (V L ) of a lead antibody, comparing the lead template sequence with a plurality of tester protein sequences; selecting a hit library from the tester protein sequences; determining if a member of the hit library is structurally compatible with the lead structural template using a scoring function; selecting members for the hit library that score equal to or better than the lead sequence and screening members for improved function(s).

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         47 . A method for constructing a library of antibody sequences, the method comprising the steps of:
 providing an amino acid sequence of the variable region of the heavy chain (V H ) or light chain (V L ) of a lead antibody, the lead antibody having a known three dimensional structure which is defined as a lead structural template;   identifying the amino acid sequences in the CDRs of the lead antibody;   selecting one of the CDRs in the V H  or V L  region of the lead antibody;   providing an amino acid sequence that comprises at least 3 consecutive amino acid residues in the selected CDR, the selected amino acid sequence being a lead sequence;   comparing the lead sequence with a plurality of tester protein sequences;   selecting from the plurality of tester protein sequences at least two peptide segments that have at least 10% sequence identity with lead sequence, the selected peptide segments forming a hit library;   building an amino acid positional variant profile of the hit library based on frequency of amino acid variant appearing at each position of the lead sequence;   combining the amino acid variants in the hit library to produce a combination of hit variants which form a hit variant library;   determining if a member of the hit variant library is structurally compatible with the lead structural template using a scoring function; and   selecting the members of the hit variant library that score equal to or better than the lead sequence.   
     
     
         48 . The method of  claim 47 , wherein the step of combining the amino acid variants in the hit library comprises the step of:
 selecting the amino acid variants with frequency of appearance higher than 4 times.   
     
     
         49 . The method of  claim 47 , wherein the step of combining the amino acid variants in the hit library comprises the step of:
 selecting the amino acid variants with frequency of appearance higher than 6 times.   
     
     
         50 . The method of  claim 47 , wherein the step of combining the amino acid variants in the hit library comprises the step of:
 selecting the amino acid variants with frequency of appearance higher than 5% out of the total variants at each position.   
     
     
         51 . The method of  claim 47 , wherein the step of combining the amino acid variants in the hit library comprises the steps of:
 selecting the amino acid variants with frequency of appearance higher than 10% out of the total variants at each position; and   combining the selected amino acid variants in the hit library to produce a combination of hit variants which form a hit variant library.   
     
     
         52 . The method of  claim 47 , wherein the step of combining the amino acid variants in the hit library comprises the step of:
 selecting the amino acid variants with frequency of appearance higher than 5% out of the total variants at each position;   selecting the amino acid of the lead sequence if its frequency of appearance is equal to or lower than 5% out of the total variants at each position; and   combining the selected amino acid variants in the hit library to produce a combination of hit variants which form the hit variant library.   
     
     
         53 . The method of  claim 47 , wherein the scoring function is an energy scoring function selected from the group consisting of electrostatic interactions, van der Waals interactions, electrostatic solvation energy, solvent-accessible surface solvation energy, and conformational entropy. 
     
     
         54 . The method of  claim 47 , wherein the scoring function is a scoring function that incorporates a forcefield selected from the group consisting of the Amber forcefield, Charmm forcefield, the Discover cvff forcefields, the ECEPP forcefields, the GROMOS forcefields, the OPLS forcefields, the MMFF94 forcefield, the Tripos forcefield, the MM3 forcefield, the Dreiding forcefield, and UNRES forcefield, and other knowledge-based statistical forcefield (mean field) and structure-based thermodynamic potential functions. 
     
     
         55 . The method of  claim 47 , further comprising the step of:
 constructing a nucleic acid library comprising DNA segments encoding the amino acid sequences of the selected members of the hit variant library.   
     
     
         56 . The method of  claim 47 , further comprising the step of:
 partitioning the parsing the selected members of hit variant library into at least two sub-hit variant libraries;   selecting a sub-hit variant library;   building an amino acid positional variant profile of the selected sub-hit variant library;   converting the amino acid positional variant profile of the selected sub-hit variant library into a nucleic acid positional variant profile by back-translating the amino acid positional variants into their corresponding genetic codons; and   constructing a degenerate nucleic acid library of DNA segments by combinatorially combining the nucleic acid positional variants.   
     
     
         57 . The method of  claim 56 , wherein the step of parsing the hit variant library comprises the step of:
 randomly selecting 10-30 members of the hit variant library that score equal to or better than the lead sequence, the selected members forming a sub-variant library.   
     
     
         58 . The method of  claim 56 , wherein the step of parsing the hit variant library comprises the step of:
 building an amino acid positional variant profile of the hit variant library, resulting a hit variant profile; and   Route V. Library construction by parsing the hit variant profile into segments of sub-variant profile based on the contact maps of the Cα, Cβ or heavy atoms of the structure or structure ensembles of a lead sequence within certain distance cutoff (8 A to 4.5 A). A structural model or lead structural template within a distance of 4.5 Å.   
     
     
         59 . The method of  claim 56 , wherein the step of parsing the hit variant library comprises the step of:
 building an amino acid positional variant profile of the hit variant library, resulting a hit variant profile; and   Route V. Library construction by parsing the hit variant profile into segments of sub-variant profile based on the contact maps of the Cα, Cβ or heavy atoms of the structure or structure ensembles of a lead sequence within certain distance cutoff (8 A to 4.5 A). A structural model or lead structural template within a distance of 6-8 Å.   
     
     
         60 . A method for constructing a library of antibody based on a structural ensemble of multiple antibodies, the method comprising the steps of:
 providing an amino acid sequence of the variable region of the heavy chain (V H ) or light chain (V L ) of a lead antibody, the lead antibody having a known three dimensional structure;   providing 3D structures of one or more antibodies with different sequences in V H  or V L  region than that of the lead antibody;   forming a structure ensemble by combining the structures of the lead antibody and the one or more antibodies; the structure ensemble being defined as a lead structural template;   identifying the amino acid sequences in the CDRs of the lead antibody;   selecting one of the CDRs in the V H  or V L  region of the lead antibody;   providing an amino acid sequence that comprises at least 3 consecutive amino acid residues in the selected CDR, the selected amino acid sequence being a lead sequence;   comparing the lead sequence with a plurality of tester protein sequences;   selecting from the plurality of tester protein sequences at least two peptide segments that have at least 10% sequence identity with lead sequence, the selected peptide segments forming a hit library;   building an amino acid positional variant profile of the hit library based on frequency of amino acid variant appearing at each position of the lead sequence;   combining the amino acid variants in the hit library to produce a combination of hit variants which form a hit variant library;   determining if a member of the hit variant library is structurally compatible with the lead structural template using a scoring function; and   selecting the members of the hit variant library that score equal to or better than the lead sequence.   
     
     
         61 . A method for constructing a library of antibody based on a structure of a lead antibody, the method comprising the steps of:
 a) providing an amino acid sequence of the variable region of the heavy chain (V H ) or light chain (V L ) of a lead antibody, the lead antibody having a known three dimensional structure;   b) identifying the amino acid sequences in the CDRs of the lead antibody;   c) selecting one of the CDRs in the V H  or V L  region of the lead antibody;   d) providing an amino acid sequence that comprises at least 3 consecutive amino acid residues in the selected CDR, the selected amino acid sequence being defined as a lead sequence;   e) comparing the lead sequence with a plurality of tester protein sequences;   f) selecting from the plurality of tester protein sequences at least two peptide segments that have at least 10% sequence identity with lead sequence, the selected peptide segments forming a hit library;   g) building an amino acid positional variant profile of the hit library based on frequency of amino acid variant appearing at each position of the lead sequence;   h) combining the amino acid variants in the hit library to produce a combination of hit variants which form a hit variant library;   i) determining if a member of the hit variant library is structurally compatible with the lead structural template using a scoring function;   j) selecting the members of the hit variant library that score equal to or better than the lead sequence;   k) constructing a degenerate nucleic acid library comprising DNA segments encoding the amino acid sequences of the selected members of the hit variant library;   l) determining the diversity of the nucleic acid library, if the diversity is higher than 1×10 6 , repeating steps j) through l) until the diversity of the diversity of the nucleic acid library is equal to or lower than 1×10 6 ;   m) introducing the DNA segments in the degenerate nucleic acid library into cells of a host organism;   n) expressing the DNA segments in the host cells such that recombinant antibodies containing the amino acid sequences of the hit library are produced in the cells of the host organism;   o) selecting the recombinant antibody that binds to a target antigen with affinity higher than 10 6  M −1 ; and   p) repeating steps e) through o) if no recombinant antibody is found to bind to the target antigen with affinity higher than 10 6  M −1 .   
     
     
         62 . A method for constructing a library of antibody based on a structure of a lead antibody, the method comprising the steps of:
 a) providing an amino acid sequence of the variable region of the heavy chain (V H ) or light chain (V L ) of a lead antibody, the lead antibody having a known three dimensional structure which is defined as a lead structural template;   b) identifying the amino acid sequences in the CDRs of the lead antibody;   c) selecting one of the CDRs in the V H  or V L  region of the lead antibody;   d) providing an amino acid sequence that comprises at least 3 consecutive amino acid residues in the selected CDR, the selected amino acid sequence being defined as a lead sequence;   e) mutating the lead sequence by substituting one or more of the amino acid residues of the lead sequence with one or more different amino acid residues, resulting in a lead sequence mutant library;   f) determining if a member of the lead sequence mutant library is structurally compatible with the lead structural template using a first scoring function;   g) selecting the lead sequence mutants that score equal to or better than the lead sequence;   h) comparing the lead sequence with a plurality of tester protein sequences;   i) selecting from the plurality of tester protein sequences at least two peptide segments that have at least 10% sequence identity with lead sequence, the selected peptide segments forming a hit library;   j) building an amino acid positional variant profile of the hit library based on frequency of amino acid variant appearing at each position of the lead sequence;   k) combining the amino acid variants in the hit library to produce a combination of hit variants;   l) combining the selected lead sequence mutants with the combination of hit variants to produce a hit variant library;   m) determining if a member of the hit variant library is structurally compatible with the lead structural template using a second scoring function;   n) selecting the members of the hit variant library that score equal to or better than the lead sequence;   o) constructing a degenerate nucleic acid library comprising DNA segments encoding the amino acid sequences of the selected members of the hit variant library;   p) determining the diversity of the nucleic acid library, and if the diversity is higher than 1×10 6 , repeating steps n) through p) until the diversity of the diversity of the nucleic acid library is equal to or lower than 1×10 6 ;   q) introducing the DNA segments in the degenerate nucleic acid library into cells of a host organism;   r) expressing the DNA segments in the host cells such that recombinant antibodies containing the amino acid sequences of the hit library are produced in the cell of the host organism;   s) selecting the recombinant antibody that binds to a target antigen with affinity higher than 10 6  M −1 ; and   t) repeating steps e) through s) if no recombinant antibody is found to bind to the target antigen with affinity higher than 10 6  M −1 .