Combinatorial antibody libraries and uses thereof
Methods for making a combinatorial antibody library from human germline segments are provided. Also provided are libraries of nucleic acid molecules compiled from germline segments encoding VL chains and libraries of nucleic acid molecules encoding VH chains, and resulting antibody libraries. The libraries are provided as addressable libraries. Methods for screening antibody libraries against a target protein antigen, and the identified or selected antibodies are provided.
1. A method of generating a combinatorial human antibody library comprising a plurality of antibody or antigen-binding antibody fragment species, comprising:
a) for each antibody or antigen-binding antibody fragment species in the library, combining a human V H and a J H or a human V H , a D H , and a J H germline segment or portion thereof in frame to generate a nucleic acid molecule encoding a VH chain or a portion thereof, wherein, optionally, the VH chain is modified by replacement or insertion of one amino acid, optionally the replacement or insertion of at least one amino acid into at least one complementarity determining region (CDR) selected from among a CDRH1, CDRH2, CDRH3, wherein the CDR optionally is CDRH3;
b) for each antibody or antigen-binding antibody fragment species in the library, combining a human V κ and a J κ germline segment or portion thereof, or a human V λ and a J λ germline segment or portion thereof in frame to generate a nucleic acid molecule encoding a VL chain or a portion thereof, wherein, optionally, the VL chain is modified by replacement or insertion of one amino acid, optionally the replacement or insertion of at least one amino acid into at least one complementarity determining region (CDR) selected from among a CDRL1, CDRL2, and CDRL3,
wherein each nucleic acid molecule of part (a) encoding the VH chain or portion thereof comprises a different combination of V H and J H or V H , D H and J H germline segments yielding a different nucleic acid sequence encoding the VH chain or portion thereof and/or each nucleic acid molecule of part (b) encoding the VL chain or portion thereof comprises a different combination of V κ and J κ germline segments or V λ and J λ germline segments yielding a different nucleic acid sequence encoding the VL chain; and
c) expressing nucleic acid molecules of parts (a) and (b) and generating the antibody or antigen-binding antibody fragment species that contain a VL chain and a VH chain or a sufficient portion thereof to form an antigen binding site, thereby generating the combinatorial human antibody library,
wherein the library comprises at least about 50 or 100 or more different antibody or antigen-binding antibody fragment species in an addressable format, wherein the antibody or antigen-binding antibody fragment species at each address is the same antibody or antigen-binding antibody fragment species and is different from the antibody or antigen-binding antibody fragment species at all other addresses, and wherein the amino acid sequence identity of each antibody or antigen-antibody fragment species is known based on the address of the antibody or antigen-binding antibody fragment species; and optionally further comprising,
d) purifying the antibody or antigen-binding antibody fragment species, optionally to about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% purity.
2. A method according to claim 1 , wherein the antibody species are full-length antibody species and the antigen-binding antibody fragment species are Fab fragments.