Structure-based selection and affinity maturation of antibody library

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 (VH) or light chain (VL) 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 VH or VL 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.

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 (VH) or light chain (VL) 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 VH or VL 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 VH or VL 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.

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 (VH) or light chain (VL) 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 VH or VL 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 1×106, repeating steps j) through l) until the diversity of the diversity of the nucleic acid library is equal to or lower than 1×106;
• 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 106 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 106 M−1.

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 (VH) or light chain (VL) 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 VH or VL 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 1×106, repeating steps n) through p) until the diversity of the diversity of the nucleic acid library is equal to or lower than 1×106;
• 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 cells of the host organism;
• s) selecting the recombinant antibody that binds to a target antigen with affinity higher than 106 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 106 M−1.