Combinatorial antibody libraries and uses thereof

A library of nucleic acid molecules encoding a combinatorial human antibody library, wherein the encoded combinatorial human antibody library is an addressable library and comprises a plurality of antibody or antigen-binding antibody fragments, wherein each member in the library is a functional antibody or functional antigen-binding antibody fragment, and:

• a) each antibody or antigen-binding antibody fragment contains a variable light (VL) chain and a variable heavy (VH) chain or a sufficient portion thereof to form an antigen binding site;
• wherein:
• i) each VL chain is encoded by a nucleic acid molecule that comprises a Vκ and a Jκ human germline segment or degenerate codons thereof or a Vλ and a Jλ human germline segment or degenerate codons thereof, wherein the segments are linked in-frame without a stop codon;
• ii) each VH chain is encoded by a nucleic acid molecule that comprises a human VH, DH, and a JH germline segment or degenerate codons thereof, wherein the segments are linked in-frame without a stop codon;
• and
• b) the library comprises at least about or 50 or 100 or more different antibody or antigen-binding antibody fragments.

A method of generating a combinatorial human antibody library, comprising:

• a) combining a VH, a DH, and a JH human germline segment or portion thereof in frame to generate a sequence of a nucleic acid molecule encoding a VH chain or a portion thereof;
• b) combining a Vκ and a Jκ human germline segment or portion thereof, or a Vλ and a Jλ human germline segment or portion thereof in frame to generate a sequence of a nucleic acid molecule encoding a VL chain or a portion thereof, wherein:
• in step a) and b) each of the portions of the VH, DH, JH, Vκ, Jκ, Vλ or Jλ are
• sufficient to produce an antibody or portion thereof containing a VH or VL or portion thereof that forms a sufficient antigen binding site;
• c) repeating step a) and b) a plurality of times to generate sequences of a plurality of different nucleic acid molecules;
• d) synthesizing the nucleic acid molecules to produce two libraries, wherein:
• the first library comprises nucleic acid molecules encoding a VH chain or a portion thereof;
• and
• the second library comprises nucleic acid molecules encoding a VL chain or a portion thereof;
• e) expressing the nucleic acid molecules of the first library and the second library of part (d) and generating an antibody or antigen-binding antibody fragment formed by the VH chains and VL chains or portions thereof, thereby generating the combinatorial human antibody library.

A method of screening a combinatorial human antibody library for binding or activity against a target protein, comprising:

• 1) contacting one or more members of a combinatorial human antibody library with a target protein, wherein:
• the combinatorial human antibody library is an addressable library and comprises a plurality of antibody or antigen-binding antibody fragments, wherein each member in the library is a functional antibody or functional antigen-binding antibody fragment, and:
• a) each antibody or antigen-binding antibody fragment contains a variable light (VL) chain and a variable heavy (VH) chain or a sufficient portion thereof to form an antigen binding site;
• wherein:
• i) each VL chain is encoded by a nucleic acid molecule that comprises a Vκ and a Jκ human germline segment or degenerate codons thereof or a Vλ and a Jλ human germline segment or degenerate codons thereof, wherein the segments are linked in-frame without a stop codon;
• ii) each VH chain is encoded by a nucleic acid molecule that comprises a human VH, DH, and a JH germline segment or degenerate codons thereof, wherein the segments are linked in-frame without a stop codon;
• and
• b) the library comprises at least about or 50 or 100 or more different antibody or antigen-binding antibody fragments;
• and
• the target protein is a membrane-bound protein, cell surface receptor (CSR), or a CSR ligand, a cytokine receptor, a receptor kinase, a receptor phosphatase, a receptor involved in cell-cell interactions, and a cellular adhesion molecule, wherein the target protein is selected from the group consisting of VEGFR-1, VEGFR-2, VEGFR-3, a epidermal growth factor receptor (EGFR), ErbB-2, ErbB-3, IGF-R1, C-Met, TNF-R1, TNF-R2, BTLA, HVEM, LT-13R, CD20, CD3, CD25, NOTCH, DLL4, G-CSF-R, GM-CSF-R, EPO-R, a cadherin, an integrin, CD52 and CD44, a VEGF-A, VEGF-B, VEGF-C, VEGF-D, PIGF, EGF, HGF, TNF-a, LIGHT, lymphotoxin (LT), IgE, G-CSF, GM-CSF, and EPO;
• and
• 2) determining whether any member of the combinatorial human antibody library binds to or modulates a functional activity of the target protein, wherein the functional activity is selected from the group consisting of cellular proliferation, lymphoma apoptosis, chemotaxis, cancer cell invasion, matrigel, endothelial proliferation, tube formation, and signal transduction;
• wherein the method further comprises identifying the antibody or antigen-binding antibody fragment that binds the target protein.