Antibody humanization by framework assembly
View Patent ↗An improved method for producing humanized antibody or an antigen binding fragment thereof is described. The method, designated framework-assembly, bypasses the reliance on structural biology and the construction of large libraries. It is easier to implement and more efficient than the rational design and empirical methods. Also described are humanized antibodies produced by the method and related framework-assembly library.
1. A method of producing a humanized antibody or an antigen binding fragment thereof for a non-human antibody to an antigen, comprising:
(1) constructing a heavy chain variable domain (VH) framework (FR) library comprising nucleic acids encoding a diversity of VHs, each of the VHs comprising FR1, FR2, FR3 and FR4 independently selected from corresponding FRs of a first number of homologous human germline VHs, and corresponding complementarity determining regions (CDRs) of the VH of the non-human antibody;
(2) constructing a light chain variable domain (VL) FR library comprising nucleic acids encoding a diversity of VLs, each of the VLs comprising FR1, FR2, FR3 and FR4 independently selected from corresponding FRs of a second number of homologous human germline VLs, and corresponding CDRs of the VL of the non-human antibody;
(3) constructing a framework-assembly library comprising nucleic acids encoding a diversity of Fabs or fragments thereof, each of the Fabs or the fragments thereof comprising a VH encoded by a nucleic acid in the VH FR library and a VL encoded by a nucleic acid in the VL FR library;
(4) expressing the framework-assembly library in host cells;
(5) identifying from the expressed framework-assembly library an Fab or a fragment thereof that binds to the antigen;
(6) identifying the VH and VL in the identified Fab or the fragment thereof as a humanized VH and a humanized VL, respectively, for the humanized antibody or the antigen binding fragment thereof; and
(7) producing the humanized antibody or the antigen binding fragment thereof comprising the identified humanized VH and the humanized VL,
wherein the sequences of FR1, FR2, FR3, and FR4 in the VH FR library and VL FR library are natural human germline sequences.
2. The method of claim 1 , wherein the framework-assembly library is expressed and the Fab or the fragment thereof is identified using phage-display.
3. The method of claim 1 , wherein the host cells are bacterial cells.
4. The method of claim 1 , wherein more than one Fab or fragments thereof that bind to the antigen are identified, and the method further comprises measuring expression level of each of the more than one Fabs or fragments thereof in an expression cell, and identifying the VH and VL within the Fab or fragment thereof that has the highest expression level as the humanized VH and the humanized VL, respectively.
5. The method of claim 4 , wherein the expression level is measured using a Fast Screen for Expression, Biophysical-properties and Affinity system.
6. The method of claim 1 , wherein more than one Fab or fragments thereof that bind to the antigen are identified, and the method further comprises measuring expression level of each of the more than one Fabs or fragments thereof in an expression cell, measuring the binding affinity of each of the more than one Fabs or fragments therein to the antigen, and identifying the VH and VL within the Fab or fragment thereof that has a high expression level and strong binding affinity as the humanized VH and the humanized VL, respectively.
7. The method of claim 1 , wherein the non-human antibody is a rodent antibody.
8. The method of claim 1 , wherein each of the CDRs of the homologous human germline VHs has the same length as each of the corresponding CDRs in the VH of the non-human antibody, each of the CDRs of the homologous human germline VLs has the same length as each of the corresponding CDRs in the VL of the non-human antibody, and each of the homologous human germline VHs and VLs does not contain a proline, a cysteine or a N-glycosylation site.
9. The method of claim 1 , wherein each of the homologous human germline VHs has at least about 60% sequence identity to the VH amino acid sequence of the non-human antibody, and each of the homologous human germline VLs has at least about 70% sequence identity to the VL amino acid sequence of the non-human antibody.
10. The method of claim 1 , wherein the first number and the second number are same or different.
11. The method of claim 1 , wherein the diversity of the VH FR library and the diversity of the VL FR library are same or different.
12. The method of claim 1 , wherein the diversity of the framework assembly library is less than 10,000.