Rationally designed, synthetic antibody libraries and uses therefor

1. A method of preparing a library comprising synthetic polynucleotides that encode an antibody heavy chain variable domain containing at least 10 6 unique CDRH3 amino acid sequences represented by the formula [N1]-[DH]-[N2]-[H3-JH], the method comprising:

(i) providing a set of N1, DH, N2, and H3-JH segments representative of an immune repertoire, wherein

(a) the N1 and N2 segments are provided by:

obtaining a set of antibody amino acid sequences;

identifying the amino acid residues at the V-D junction and the D-J junction of the antibody sequences;

analyzing the amino acid residues at the identified V-D and D-J junctions, respectively, to determine patterns of length and composition of the amino acid residues present at the identified V-D and D-J junctions; and

generating amino acid sequences of rationally designed composition and length based on the determined patterns of length and composition of the amino acid residues present at the identified V-D and D-J junctions

wherein N1 and N2 are a fixed length of 0, 1, 2, or 3 amino acid residues;

(b) the DH segments are provided by:

obtaining a set of polynucleotide sequences of IGHD genes and alleles from a non-human vertebrate species;

translating the polynucleotide sequences of the IGHD genes and alleles; and

progressively deleting one or more amino acids from either or both of the N-terminus and C-terminus of the resultant IGHD amino acid sequences; and

(c) the H3-JH segments are provided by:

obtaining a set of polynucleotide sequences and amino acid sequences of human IGHJ genes and alleles;

analyzing the amino acid sequences expressed from such IGHJ genes and alleles to determine patterns and length of progressive deletion of nucleotides at the N-terminus; and

generating amino acid sequences of rationally designed composition and length based on the determined patterns and length of progressive deletion of nucleotides at the N-terminus,

wherein the H3-JH segments comprise 1 to 9 amino acids;

(ii) synthesizing polynucleotide sequences that encode each of the N1, DH, N2, and H3-JH segments provided in step (i); and

(iii) assembling the polynucleotide sequences of step (ii) to produce a library of synthetic polynucleotides encoding a plurality of antibody CDRH3 amino acid sequences represented by the following formula: [N1]-[DH]-[N2]-[H3-JH],

wherein the diversity of the polynucleotides encoding the unique CDRH3 amino acid sequences is created by polynucleotides having CDRH3 sequences that are different from the CDRH3 sequences of other polynucleotides; and

(iv) assembling the CDRH3 from step (iii) into an antibody heavy chain variable domain, wherein the antibody heavy chain variable domain comprises framework (FRM) and complementary determining regions (CDR) of the formula FRMH1-CDRH1-FRMH2-CDRH2-FRMH3-CDRH3-FRMH4.

2. The method of claim 1 , wherein the set of N1, N2, and H3-JH segments provided in step (i) comprise the most frequently occurring amino acids at the corresponding regions of antibody sequences expressed by human B cells.

3. The method of claim 1 , further comprising the step of recombining the assembled polynucleotide sequences with a vector comprising a heavy chain chassis and a heavy chain constant region to form a full-length heavy chain.

4. The method of claim 3 , wherein the step of recombining is performed in yeast.

5. The method of claim 4 , wherein the yeast is S. cerevisiae.

6. The method of claim 1 , wherein the DH segment is selected according to one or more of diversity in length and/or sequence, maximal or minimal human string content, and absence or minimization of T cell epitopes.

7. The method of claim 1 , wherein the DH segment is about 1 to about 10 amino acids in length.

8. The method of claim 1 , wherein the vertebrate species is selected from the group consisting of Mus musculus, Camelus sp., Llama sp., Camelidae sp., Raja sp., Ginglymostoma sp., Carcharhinus sp., Heterodontus sp., Hydrolagus sp., Ictalurus sp., Gallus sp., Bos sp., Marmaronetta sp., Aythya sp., Netta sp., Equus sp., Pentalagus sp., Bunolagus sp., Nesolagus sp., Romerolagus sp., Brachylagus sp., Sylvilagus sp., Oryctolagus , sp., Poelagus sp., Ovis sp., Sus sp., Gadus sp., Salmo sp., Oncorhynchus sp, Macaca sp., Rattus sp., Pan sp., Hexanchus sp., Heptranchias sp., Notorynchus sp., Chlamydoselachus sp., Heterodontus Sp. Pristiophorus Sp., Pliotrema Sp., Squatina Sp., Carcharia Sp., Mitsukurina Sp., Lamma sp., Isurus sp., Carcharodon sp., Cetorhinus sp., Alopias sp., Nebrius sp., Stegostoma sp., Orectolobus sp., Eucrossorhinus sp., Sutorectus sp., Chiloscyllium sp., Hemiscyllium sp., Brachaelurus sp., Heteroscyllium sp., Cirrhoscyllium sp., Parascyllium sp., Rhincodon sp., Apristurus sp., Atelomycterus sp., Cephaloscyllium sp., Cephalurus sp., Dichichthys sp., Galeus sp., Halaelurus sp., Haploblepharus sp., Parmaturus sp., Pentanchus sp., Poroderna sp., Schroederichthys sp., Scyliorhinus sp., Pseudotriakis sp., Scylliogaleus sp., Furgaleus sp., Hemitriakis sp., Mustelus sp., Triakis sp., Iago sp., Galeorhinus sp., Hypogaleus sp., Chaenogaleus sp., Hemigaleus sp., Paragaleus sp., Galeocerdo sp., Prionace sp., Sciolodon sp., Loxodon sp., Rhizoprionodon sp., Aprionodon sp., Negaprion sp., Hypoprion sp., Carcharhinus sp., Isogomphodon sp., Triaenodon sp., Sphyrna sp., Echinorhinus sp., Oxynotus sp., Squalus sp., Centroscyllium sp., Etmopterus sp., Centrophorus sp., Cirrhigaleus sp., Deania sp., Centroscymnus sp., Scymnodon sp., Dalatias sp., Euprotomicrus sp., Isistius sp., Squaliolus sp., Heteroscymnoides sp., Somniosus sp. and Megachasma sp.

9. The method of claim 1 , wherein one or more N1 segments is selected from a non-human sequence.

10. The method of claim 1 , wherein one or more N2 segments is selected from a non-human sequence.

11. The method of claim 1 , wherein one or more polynucleotide sequences are synthesized via split-pool synthesis.

12. The method of claim 1 , wherein the degree of deletion of the one or more amino acids on the N and/or C termini of the DH segment is determined by analogy with human sequences.

13. The method of claim 1 , wherein the plurality of CDRH3 amino acid sequences are further represented by the formula [X]-[N1]-[DH]-[N2]-[H3-JH], wherein [X] is any amino acid residue.

14. The method of claim 13 , wherein [X] is selected from the group consisting of G, D, and E.

15. The method of claim 1 , further comprising mutagenizing one or more of the provided N1, DH, N2, and H3-JH segments.

16. The method of claim 15 , wherein mutagenizing includes one or more of site-directed mutagenesis, error-prone PCR mutagenesis, cassette mutagenesis, and random PCR mutagenesis.

17. The method of claim 1 , further comprising a step of progressively deleting one or more amino acids from the C-terminus of the resultant IGHD amino acid sequences.