COMBINATORIAL ANTIBODY LIBRARIES AND USES THEREOF

1 . A combinatorial human antibody library, wherein the library is an addressable library and is selected from the group consisting of:

A. a combinatorial human antibody addressable library comprising 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 V κ and a J κ human germline segment or degenerate codons thereof, or a V λ and a J λ human germline segment or degenerate codons thereof, wherein the segments are linked in-frame without a stop codon; and

ii) each VH chain is encoded by a nucleic acid molecule that comprises a human V H and a human J H germline segment and any sequence of nucleotides between the V H and J H germline segments, 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;

B. a combinatorial human antibody addressable library comprising 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 modified variable light (VL) chain and/or a modified variable heavy chain (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 J κ human germline segment or degenerate codons thereof or a V λ and a J λ 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 V H , D H and a J H human germline segment or degenerate codons thereof, wherein the segments are linked in-frame without a stop codon; and

b) the VL chain and/or VH chain is/are modified by replacement or insertion of at least one amino acid into at least one complementarity determining region (CDR); and

c) the library comprises at least about or 50 or 100 or more different antibody or antigen-binding antibody fragments.

2 . The combinatorial human antibody library according to claim 1 , wherein:

the sequence of nucleotides between the V H and J H germline segments encodes a peptide mimetic; or

the amino acids that are inserted or replaced correspond to a peptide mimetic.

3 . The combinatorial human antibody library according to claim 2 , wherein:

the peptide mimetic is selected from the group consisting of a peptide mimetic of TPO, EPO, G-CSF, IL-5, human brain natriuretic peptide (hBNP-32), exendin 4, GLP-1, GLP-2, glucagon, PACAP-38, CD209L, TNF, VEGF, MMP inhibitor, and CTLA-4; or

the peptide mimetic has the sequence of amino acids selected from among any of SEQ ID NOS: 891 and 987-1014.

4 . The combinatorial human antibody library according to claim 1 , wherein the antibody or antigen-binding antibody fragment at each address is the same antibody or antigen-binding antibody fragment and is different from the antibody or antigen-binding antibody fragment at all other addresses.

5 . The combinatorial human antibody library according to claim 1 , wherein:

all or a subset of germline V H segments are linked with all or a subset of D H segments which are linked with all or a subset of germline J H segments to generate the plurality of nucleic acid molecules encoding a VH chain; and

all or a subset of germline V κ segments are linked with all or a subset of germline J κ segments, or all or a subset of germline V λ segments are linked to all or a subset of germline J λ segments to generate a plurality of nucleic acid molecules encoding a VL chain.

6 . The combinatorial human antibody library according to claim 1 , wherein

each V H germline segment is selected from the group consisting of IGHV1, IGHV2, IGHV3, IGHV4, IGHV5, IGHV6, IGHV7, and an allelic variant of any of the foregoing; and/or

each D H germline segment is selected from the group consisting of IGHD1, IGHD2, IGHD3, IGHD4, IGHD5, IGHD6, IGHD7, and an allelic variant of any of the foregoing; and/or

each J H germline segment is selected from the group consisting of IGHJ1, IGHJ2, IGHJ3, IGHJ4, IGHJ5, IGHJ6, and an allelic variant of any of the foregoing; and/or

each V κ germline segment is selected from the group consisting of IGKV1, IGKV2, IGKV3, IGKV4, IGKV5, IGKV6, and an allelic variant of any of the foregoing; and/or

each J κ germline segment is selected from the group consisting of IGKJ1, IGKJ2, IGKJ3, IGKJ4, IGKJ5, and an allelic variant of any of the foregoing and/or

each V λ germline segment is selected from the group consisting of IGLV1, IGLV2, IGLV3, IGLV4, IGLV5, IGLV6, IGLV7, IGLV8, IGLV9, IGLV10, IGLV11, and an allelic variant of any of the foregoing; and/or

each J λ germline segment selected from the group consisting of IGLJ1, IGLJ2, IGLJ3, IGLJ4, IGLJ5, IGLJ6, IGLJ7, and an allelic variant of any of the foregoing.

7 . The combinatorial human antibody library according to claim 1 , wherein the plurality of nucleic acid molecules encoding a VH chain and/or a VL chain is/are generated from a subset of germline segments selected based on sequence similarities or differences, gene family, length, composition, CDR length or composition, species, functionality, specificity, group, or subgroup.

8 . The combinatorial human antibody library according to claim 1 that comprises at or about or more than at or about 50, 10 2 , 10 3 , 10 4 , 2×10 4 , 3×10 4 , 4×10 4 , 5×10 4 , 6×10 4 , 7×10 4 , 8×10 4 , 9×10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 different antibody or antigen-binding antibody fragments.

9 . The combinatorial human antibody library of claim 1 , wherein the CDR is CDRH3.

10 . The combinatorial human antibody library of claim 1 , wherein the sequence of nucleotides between the V H and J H germline segments is a DH germline segment or degenerate codons thereof or is an inverted D—H germline segment.

11 . The combinatorial human antibody library of claim 1 , wherein the antibodies or antigen-binding fragments are arranged in a spatial array.

12 . The combinatorial human antibody library of claim 1 , wherein the antibodies or antigen-binding fragments are immobilized on a solid support or are in solution.

13 . The combinatorial human antibody library of claim 1 , wherein the antibodies or antigen-binding fragments are identifiably labeled.

14 . The combinatorial human antibody library of claim 6 , wherein:

each V H germline segment is selected from among any of SEQ ID NOS: 10-238; and/or

each D H germline segment is selected from among any of SEQ ID NOS: 239-272; and/or

each J H germline segment is selected from among any of SEQ ID NOS: 273-285; and/or

each V κ germline segment is selected from among any of SEQ ID NOS: 286-355 and 868; and/or

each J κ germline segment is selected from among any of SEQ ID NOS: 356-364; and/or

each V λ germline segment is selected from among any of SEQ ID NOS: 365-441; and/or

each J λ germline segment is selected from among any of SEQ ID NOS: 442-451.

15 . The combinatorial human antibody library of claim 1 , wherein the antibody or antigen-binding antibody fragments are full length antibodies.

16 . The combinatorial human antibody library of claim 1 , wherein the antibody or antigen-binding antibody fragments are antigen-binding antibody fragments.

17 . The combinatorial human antibody library of claim 16 , wherein the antigen-binding antibody fragment is a Fab.

18 . A library of nucleic acid molecules that encodes the antibody and/or antigen-binding antibody fragments of a combinatorial human antibody library according to claim 1 .

19 . The library of nucleic acid molecules of claim 18 , wherein the library of nucleic acid molecules are carried in vectors.

20 . The library of nucleic acid molecules of claim 19 , wherein the vector is an expression vector.

21 . 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 V κ and a J κ human germline segment or degenerate codons thereof or a V λ and a J λ 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 V H , D H , and a J H 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.

22 . The nucleic acid library of claim 21 , wherein in the encoded combinatorial human antibody library the antibody or antigen-binding antibody fragment at each address is the same antibody or antigen-binding antibody fragment and is different from the antibody or antigen-binding antibody fragment at all other addresses.

23 . The nucleic acid library according to claim 21 , wherein in the encoded combinatorial human antibody library:

all or a subset of germline V H segments are linked with all or a subset of D H segments which are linked with all or a subset of germline J H segments to generate the plurality of nucleic acid molecules encoding a VH chain; and

all or a subset of germline V κ segments are linked with all or a subset of germline J κ segments, or all or a subset of germline V λ segments are linked to all or a subset of germline J λ segments to generate a plurality of nucleic acid molecules encoding a VL chain.

24 . The nucleic acid library according to claim 21 , wherein in the encoded combinatorial human antibody library:

each V H germline segment is selected from the group consisting of IGHV1, IGHV2, IGHV3, IGHV4, IGHV5, IGHV6, IGHV7, and an allelic variant of any of the foregoing; and/or

each D H germline segment is selected from the group consisting of IGHD1, IGHD2, IGHD3, IGHD4, IGHD5, IGHD6, IGHD7, and an allelic variant of any of the foregoing; and/or

each J H germline segment is selected from the group consisting of IGHJ1, IGHJ2, IGHJ3, IGHJ4, IGHJ5, IGHJ6, and an allelic variant of any of the foregoing; and/or

each V κ germline segment is selected from the group consisting of IGKV1, IGKV2, IGKV3, IGKV4, IGKV5, IGKV6, and an allelic variant of any of the foregoing; and/or

each J κ germline segment is selected from the group consisting of IGKJ1, IGKJ2, IGKJ3, IGKJ4, IGKJ5, and an allelic variant of any of the foregoing and/or

each V λ germline segment is selected from the group consisting of IGLV1, IGLV2, IGLV3, IGLV4, IGLV5, IGLV6, IGLV7, IGLV8, IGLV9, IGLV10, IGLV11, and an allelic variant of any of the foregoing; and/or

each J λ germline segment selected from the group consisting of IGLJ1, IGLJ2, IGLJ3, IGLJ4, IGLJ5, IGLJ6, IGLJ7, and an allelic variant of any of the foregoing.

25 . The nucleic acid library of claim 21 , wherein the encoded combinatorial human antibody library comprises at or about or more than at or about 50, 10 2 , 10 3 , 10 4 , 2×10 4 , 3×10 4 , 4×10 4 , 5×10 4 , 6×10 4 , 7×10 4 , 8×10 4 , 9×10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 different antibodies or antigen-binding antibody fragments.

26 . The nucleic acid library of claim 21 , wherein in the encoded combinatorial human antibody library:

each V H germline segment is selected from among any of SEQ ID NOS: 10-238; and/or

each D H germline segment is selected from among any of SEQ ID NOS: 239-272; and/or

each J H germline segment is selected from among any of SEQ ID NOS: 273-285; and/or

each V κ germline segment is selected from among any of SEQ ID NOS: 286-355 and 868; and/or

each J κ germline segment is selected from among any of SEQ ID NOS: 356-364; and/or

each V λ germline segment is selected from among any of SEQ ID NOS: 365-441; and/or

each J λ germline segment is selected from among any of SEQ ID NOS: 442-451.

27 . The nucleic acid library of claim 21 , wherein in the encoded combinatorial human antibody library each of the antibodies or antigen-binding antibody fragments are full length antibodies.

28 . The nucleic acid library of claim 21 , wherein in the encoded combinatorial human antibody library each of the antibody or antigen-binding antibody fragments are antigen-binding antibody fragments.

29 . The nucleic acid library of claim 28 , wherein the antigen-binding antibody fragment is a Fab.

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

a) combining a V H , a D H , and a J H 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 V κ and a J κ human germline segment or portion thereof, or a V λ and a J λ 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 V H , D H , J H , V κ , J κ , V λ or J λ 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.

31 . The method of generating a combinatorial human antibody library of claim 30 , wherein expressing the nucleic acid molecules comprises introducing a nucleic acid molecule from the first library and from the second library into a cell and growing the cells under conditions to express the antibodies or portions thereof in each cell, and repeating this a plurality of times to produce a library of cells, wherein each cell contains nucleic acid molecules encoding a different combination of VH and VL from every other cell in the library of cells.

32 . The method of generating a combinatorial human antibody library of claim 30 , wherein the library is an addressable library, wherein in step d) the synthesized nucleic acid sequences are individually addressed, thereby generating a first addressed nucleic acid library and a second addressed nucleic acid library.

33 . The method of generating a combinatorial human antibody library of claim 30 , wherein the expressed antibody is a full length antibody, or a fragment or portion thereof sufficient to form an antigen binding site.

34 . The method of generating a combinatorial human antibody library of claim 30 , wherein the expressed antibody is a Fab.

35 . The method of generating a combinatorial human antibody library of claim 30 , further purifying the antibodies or portions thereof.

36 . A human combinatorial antibody library produced by the method of claim 30 .

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

a) contacting one or more members of a combinatorial human antibody library according to claim 1 with a target protein, wherein 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

b) 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.

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

a) contacting one or more members of a combinatorial human antibody library of claim 36 with a target protein, wherein 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

b) 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.

39 . 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 V κ and a J κ human germline segment or degenerate codons thereof or a V λ and a J λ 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 V H , D H , and a J H 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.