Rationally Designed, Synthetic Antibody Libraries and Uses Therefor

• US 20090181855A1
• Filed: 09/12/2008
• Published: 07/16/2009
• Est. Priority Date: 09/14/2007
• Status: Active Grant

• Current Assignee: Adimab, LLC
• Original Assignee: Adimab, LLC
• Inventors: Feldhaus, Michael, Vasquez, Maximiliano, Wittrup, K. Dane, Gerngross, Tillman U.
• Granted Patent: US 8,691,730 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C07K 16/005 constructed by phage libraries
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C07K 16/40 against enzymes
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/565 Complementarity determining region [CDR]
• CPC Class Codes: C07K 2317/567 Framework region [FR]
• CPC Class Codes: C40B 40/10 Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms

Abstract

The present invention overcomes the inadequacies inherent in the known methods for generating libraries of antibody-encoding polynucleotides by specifically designing the libraries with directed sequence and length diversity. The libraries are designed to reflect the preimmune repertoire naturally created by the human immune system and are based on rational design informed by examination of publicly available databases of human antibody sequences.

Claims

1. A library of synthetic polynucleotides, wherein said polynucleotides encode at least 10⁶ unique antibody CDRH3 amino acid sequences comprising:

(i) an N1 amino acid sequence of 0 to about 3 amino acids, wherein each amino acid of the N1 amino acid sequence is among the 12 most frequently occurring amino acids at the corresponding position in N1 amino acid sequences of CDRH3 amino acid sequences that are functionally expressed by human B cells;

(ii) a human CDRH3 DH amino acid sequence, N- and C-terminal truncations thereof, or a sequence of at least about 80% identity to any of them;

(iii) an N2 amino acid sequence of 0 to about 3 amino acids, wherein each amino acid of the N2 amino acid sequence is among the 12 most frequently occurring amino acids at the corresponding position in N2 amino acid sequences of CDRH3 amino acid sequences that are functionally expressed by human B cells; and

(iv) a human CDRH3 H3-JH amino acid sequence, N-terminal truncations thereof, or a sequence of at least about 80% identity to any of them.

2. An antibody isolated from the polypeptide expression products of the library of claim 1.

3. The library of claim 1, wherein one or more CDRH3 amino acid sequences further comprise an N-terminal tail residue.

4. The library of claim 3, wherein the N-terminal tail residue is selected from the group consisting of G, D, and E.

5. The library of claim 1, wherein the N1 amino acid sequence is selected from the group consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL, GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV, RP, AP, SP, LP, TP, VP, GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG, PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT, GGV, D, E, F, H, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA, EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL, QT, RA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK, SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS, AAE, AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL, SGL, SSE, TGL, WGT, and combinations thereof.

6. The library of claim 1, wherein the N2 amino acid sequence is selected from the group consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL, GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV, RP, AP, SP, LP, TP, VP, GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG, PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT, GGV, D, E, F, H, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA, EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL, QT, RA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK, SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS, AAE, AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL, SGL, SSE, TGL, WGT, and combinations thereof.

7. The library of claim 1, wherein the H3-JH amino acid sequence is selected from the group consisting of AEYFQH, EYFQH, YFQH, FQH, QH, H, YWYFDL, WYFDL, YFDL, FDL, DL, L, AFDV, FDV, DV, V, YFDY, FDY, DY, Y, NWFDS, WFDS, FDS, DS, S, YYYYYGMDV, YYYYGMDV, YYYGMDV, YYGMDV, YGMDV, GMDV, and MDV.

8. The library of claim 1, wherein the polynucleotides further comprise a 5′

polynucleotide sequence and a 3′

polynucleotide sequence that facilitate homologous recombination.

9. The library of claim 1, wherein the polynucleotides further encode one or more heavy chain chassis amino acid sequences that are N-terminal to the CDRH3 amino acid sequences, and the one or more heavy chain chassis amino acid sequences are selected from the group consisting of about Kabat amino acid 1 to about Kabat amino acid 94 encoded by IGHV1-2, IGHV1-3, IGHV1-8, IGHV1-18, IGHV1-24, IGHV1-45, IGHV1-46, IGHV1-58, IGHV1-69, IGHV2-5, IGHV2-26, IGHV2-70, IGHV3-7, IGHV3-9, IGHV3-11, IGHV3-13, IGHV3-15, IGHV3-20, IGHV3-21, IGHV3-23, IGHV3-30, IGHV3-33, IGHV3-43, IGHV3-48, IGHV3-49, IGHV3-53, IGHV3-64, IGHV3-66, IGHV3-72, IGHV3-73, IGHV3-74, IGHV4-4, IGHV4-28, IGHV4-31, IGHV4-34, IGHV4-39, IGHV4-59, IGHV4-61, IGHV4-B, IGHV5-51, IGHV6-1, and IGHV7-4-1, or a sequence of at least about 80% identity to any of them.

10. The library of claim 1, wherein the polynucleotides further encode one or more FRM4 amino acid sequences that are C-terminal to the CDRH3 amino acid sequences, wherein the one or more FRM4 amino acid sequences are selected from the group consisting of a FRM4 amino acid sequence encoded by IGHJ1, IGHJ2, IGHJ3, IGHJ4, IGHJ5, and IGHJ6, or a sequence of at least about 80% identity to any of them.

11. The library of claim 10, wherein the polynucleotides further encode one or more immunoglobulin heavy chain constant region amino acid sequences that are C-terminal to the FRM4 amino acid sequence.

12. The library of claim 11, wherein the CDRH3 amino acid sequences are expressed as part of full-length heavy chains.

13. The library of claim 12, wherein the full-length heavy chains are selected from the group consisting of an IgG1, IgG2, IgG3, and IgG4, or combinations thereof.

14. The library of claim 1, wherein the polynucleotides further encode an alternative scaffold.

15. The library of claim 1, wherein the CDRH3 amino acid sequences are from about 2 to about 30, from about 8 to about 19, or from about 10 to about 18 amino acid residues in length.

16. The library of claim 1, wherein the synthetic polynucleotides of the library encode from about 10⁶ to about 10¹⁴, from about 10⁷ to about 10¹³, from about 10⁸ to about 10¹², from about 10⁹ to about 10¹², or from about 10¹⁰ to about 10¹² unique CDRH3 amino acid sequences.

17. A library of polypeptides encoded by the synthetic polynucleotide library of claim 1.

18. A library of vectors comprising the polynucleotide library of claim 1.

19. A population of cells comprising the vectors of claim 18.

20. The population of cells of claim 19, wherein the doubling time of the population of cells is from about 1 to about 3 hours, from about 3 to about 8 hours, from about 8 to about 16 hours, from about 16 to about 20 hours, or from 20 to about 30 hours.

21. The population of cells of claim 19, wherein the cells are yeast cells.

22. The yeast cells of claim 21, wherein the yeast is Saccharomyces cerevisiae.

40. A library of synthetic polynucleotides, wherein said polynucleotides encode a plurality of antibody proteins comprising:

(i) a CDRH3 amino acid sequence of claim 1; and

(ii) a VKCDR3 amino acid sequence comprising about 1 to about 10 of the amino acids found at Kabat positions 89, 90, 91, 92, 93, 94, 95, 95A, 96, and 97, in selected VKCDR3 amino acid sequences derived from a particular IGKV or IGKJ germline sequence.

41. The library of claim 40, wherein the VKCDR3 amino acid sequence comprises one or more of the amino acid sequences listed in Table 33 or a sequence at least about 80% identical to any of them.

42. An antibody isolated from the library of claim 40.

43. The library of claim 40, wherein the antibody proteins are expressed in a heterodimeric form.

44. The library of claim 40, wherein the antibody proteins are expressed as antibody fragments.

45. The library of claim 44, wherein the antibody fragments are selected from the group consisting of Fab, Fab′

, F(ab′

)₂, Fv fragments, diabodies, linear antibodies, and single-chain antibodies.

46. A library of synthetic polynucleotides, wherein said polynucleotides encode a plurality of antibody proteins comprising:

(i) a CDRH3 amino acid sequence of claim 1; and

(ii) a VKCDR3 amino acid sequence of at least about 80% identity to a amino acid sequence represented by the following formula;

[VK_Chassis]-[L3-VK]-[X]-[JK*], wherein;

(a) VK_Chassis is an amino acid sequence selected from the group consisting of about Kabat amino acid 1 to about Kabat amino acid 88 encoded by IGKV1-05, IGKV1-06, IGKV1-08, IGKV1-09, IGKV1-12, IGKV1-13, IGKV1-16, IGKV1-17, IGKV1-27, IGKV1-33, IGKV1-37, IGKV1-39, IGKV1D-16, IGKV1D-17, IGKV1D-43, IGKV1D-8, IGKV2-24, IGKV2-28, IGKV2-29, IGKV2-30, IGKV2-40, IGKV2D-26, IGKV2D-29, IGKV2D-30, IGKV3-11, IGKV3-15, IGKV3-20, IGKV3D-07, IGKV3D-11, IGKV3D-20, IGKV4-1, IGKV5-2, IGKV6-21, and IGKV6D-41, or a sequence of at least about 80% identity to any of them;

(b) L3-VK is the portion of the VKCDR3 encoded by the IGKV gene segment; and

(c) X is any amino acid residue; and

(d) JK* is an amino acid sequence selected from the group consisting of amino acid sequences encoded by IGJK1, IGJK2, IGJK3, IGJK4, and IGJK5, wherein the first residue of each IGJK amino acid sequence is not present.

47. An antibody isolated from the library of claim 46.

60. A method of preparing a synthetic polynucleotide library comprising providing and assembling the polynucleotide sequences of claim 1.

67. A method of isolating one or more host cells expressing one or more antibodies, the method comprising:

(i) expressing the antibodies of claim 40 in one or more host cells,

(ii) contacting the host cells with one or more antigens; and

(iii) isolating one or more host cells having antibodies that bind to the one or more antigens.

68. The method of claim 67, further comprising isolating one or more antibodies from the one or more host cells.

70. The method of claim 67, further comprising the step of isolating one or more polynucleotide sequences encoding one or more antibodies from the one or more host cells.

71. A kit comprising the library of synthetic polynucleotides of claim 1.

72. The CDRH3 amino acid sequences encoded by the library of synthetic polynucleotides of claim 1 in computer readable form.

23. A library of synthetic polynucleotides, wherein said library has a theoretical total diversity of N unique CDRH3 sequences, wherein N is about 10⁶ to about 10¹⁵;

and

wherein the physical realization of the theoretical total CDRH3 diversity has a size of at least about 3N, thereby providing a probability of at least about 95% that any individual CDRH3 sequence contained within the theoretical total diversity of the library is present in the actual library.

24. A library of synthetic polynucleotides, wherein said polynucleotides encode at least about 10⁶ unique antibody CDRH3 amino acid sequences comprising:

(i) an N1 amino acid sequence of 0 to about 3 amino acids, wherein;

(a) the most N-terminal N1 amino acid, if present, is selected from a group consisting of R, G, P, L, S, A, V, K, I, Q, T and D;

(b) the second most N-terminal N1 amino acid, if present, is selected from a group consisting of G, P, R, S, L, V, E, A, D, I, T and K; and

(c) the third most N-terminal N1 amino acid, if present, is selected from the group consisting of G, R, P, S, L, A, V, T, E, D, K and F;

(ii) a human CDRH3 DH amino acid sequence, N- and C-terminal truncations thereof, or a sequence of at least about 80% identity to any of them;

(iii) an N2 amino acid sequence of 0 to about 3 amino acids, wherein;

(a) the most N-terminal N2 amino acid, if present, is selected from a group consisting of G, P, R, L, S, A, T, V, E, D, F and H;

(b) the second most N-terminal N2 amino acid, if present, is selected from a group consisting of G, P, R, S, T, L, A, V, E, Y, D and K; and

(c) the third most N-terminal N2 amino acid, if present, is selected from the group consisting of G, P, S, R, L, A, T, V, D, E, Wand Q; and

(iv) a human CDRH3 H3-JH amino acid sequence, N-terminal truncations thereof, or a sequence of at least about 80% identity to any of them.

25. An antibody isolated from the polypeptide expression products of the library of claim 24.

26. A library of synthetic polynucleotides, wherein said polynucleotides encode at least about 10⁶ unique antibody CDRH3 amino acid sequences that are at least about 80% identical to an amino acid sequence represented by the following formula:

[X]-[N1]-[DH]-[N2]-[H3-JH], wherein;

(i) X is any amino acid residue or no amino acid residue;

(ii) N1 is an amino acid sequence selected from the group consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL, GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV, RP, AP, SP, LP, TP, VP, GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG, PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT, GGV, D, E, F, H, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA, EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL, QT, RA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK, SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS, AAE, AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL, SGL, SSE, TGL, WGT, and combinations thereof;

(iii) DH is an amino acid sequence selected from the group consisting of all possible reading frames that do not include a stop codon encoded by IGHD1-1, IGHD1-20, IGHD1-26, IGHD1-7, IGHD2-15, IGHD2-2, IGHD2-21, IGHD2-8, IGHD3-10, IGHD3-16, IGHD3-22, IGHD3-3, IGHD3-9, IGHD4-17, IGHD4-23, IGHD4-4, IGHD-4-11, IGHD5-12, IGHD5-24, IGHD5-5, IGHD-5-18, IGHD6-13, IGHD6-19, IGHD6-25, IGHD6-6, and IGHD7-27, and N- and C-terminal truncations thereof,

(iv) N2 is an amino acid sequence selected from the group consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL, GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV, RP, AP, SP, LP, TP, VP, GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG, PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT, GGV, D, E, F, H, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA, EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL, QT, RA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK, SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS, AAE, AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL, SGL, SSE, TGL, WGT, and combinations thereof, and

(v) H3-JH is an amino acid sequence selected from the group consisting of AEYFQH, EYFQH, YFQH, FQH, QH, H, YWYFDL, WYFDL, YFDL, FDL, DL, L, AFDV, FDV, DV, V, YFDY, FDY, DY, Y, NWFDS, WFDS, FDS, DS, S, YYYYYGMDV, YYYYGMDV, YYYGMDV, YYGMDV, YGMDV, GMDV, and MDV, or a sequence of at least 80% identity to any of them.

27. An antibody isolated from the polypeptide expression products of the library of claim 26.

28. A library of synthetic polynucleotides, wherein said library consists essentially of a plurality of polynucleotides encoding CDRH3 amino acid sequences that are at least about 80% identical to an amino acid sequence represented by the following formula:

[X]-[N1]-[DH]-[N2]-[H3-JH], wherein;

(i) X is any amino acid residue or no amino acid residue;

(ii) N1 is an amino acid sequence selected from the group consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL, GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV, RP, AP, SP, LP, TP, VP, GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG, PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT, GGV, D, E, F, H, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA, EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL, QT, RA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK, SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS, AAE, AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL, SGL, SSE, TGL, WGT, and combinations thereof,

(iii) DH is an amino acid sequence selected from the group consisting of all possible reading frames that do not include a stop codon encoded by IGHD1-1, IGHD1-20, IGHD1-26, IGHD1-7, IGHD2-15, IGHD2-2, IGHD2-21, IGHD2-8, IGHD3-10, IGHD3-16, IGHD3-22, IGHD3-3, IGHD3-9, IGHD4-17, IGHD4-23, IGHD4-4, IGHD-4-11, IGHD5-12, IGHD5-24, IGHD5-5, IGHD-5-18, IGHD6-13, IGHD6-19, IGHD6-25, IGHD6-6, and IGHD7-27, and N- and C-terminal truncations thereof,

(iv) N2 is an amino acid sequence selected from the group consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL, GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV, RP, AP, SP, LP, TP, VP, GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG, PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT, GGV, D, E, F, H, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA, EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL, QT, RA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK, SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS, AAE, AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL, SGL, SSE, TGL, WGT, and combinations thereof, and

(v) H3-JH is an amino acid sequence selected from the group consisting of AEYFQH, EYFQH, YFQH, FQH, QH, H, YWYFDL, WYFDL, YFDL, FDL, DL, L, AFDV, FDV, DV, V, YFDY, FDY, DY, Y, NWFDS, WFDS, FDS, DS, S, YYYYYGMDV, YYYYGMDV, YYYGMDV, YYGMDV, YGMDV, GMDV, and MDV, or a sequence of at least 80% identity to any of them.

29. An antibody isolated from the polypeptide expression products of the library of claim 28.

30. A library of synthetic polynucleotides, wherein said polynucleotides encode one or more antibody heavy chain amino acid sequences, and wherein the unique CDRH3 amino acid sequences of the heavy chain comprise:

(i) an N1 amino acid sequence of 0 to about 3 amino acids, wherein each amino acid of the N1 amino acid sequence is among the 12 most frequently occurring amino acids at the corresponding position in N1 amino acid sequences of CDRH3 amino acid sequences that are functionally expressed by human B cells;

(ii) a human CDRH3 DH amino acid sequence, N- and C-terminal truncations thereof, or a sequence of at least about 80% identity to any of them;

(iii) an N2 amino acid sequence of 0 to about 3 amino acids, wherein each amino acid of the N2 amino acid sequence is among the 12 most frequently occurring amino acids at the corresponding position in N2 amino acid sequences of CDRH3 amino acid sequences that are functionally expressed by human B cells; and

(iv) a human CDRH3 H3-JH amino acid sequence, N-terminal truncations thereof, or a sequence of at least about 80% identity to any of them.

31. An antibody isolated from the polypeptide expression products of the library of claim 30.

32. A library of synthetic polynucleotides, wherein said polynucleotides encode a plurality of antibody VKCDR3 amino acid sequences comprising about 1 to about 10 of the amino acids found at Kabat positions 89, 90, 91, 92, 93, 94, 95, 95A, 96, and 97, in selected VKCDR3 amino acid sequences derived from a particular IGKV or IGKJ germline sequence.

33. The library of claim 32, wherein the synthetic polynucleotides encode one or more of the amino acid sequences listed in Table 33 or a sequence at least about 80% identical to any of them.

34. An antibody isolated from the polypeptide expression products of the library of claim 32.

35. A library of synthetic polynucleotides, wherein said polynucleotides encode a plurality of unique antibody VKCDR3 amino acid sequences that are of at least about 80% identity to an amino acid sequence represented by the following formula:

[VK_Chassis]-[L3-VK]-[X]-[JK*], wherein;

(i) VK_Chassis is an amino acid sequence selected from the group consisting of about Kabat amino acid 1 to about Kabat amino acid 88 encoded by IGKV1-05, IGKV1-06, IGKV1-08, IGKV1-09, IGKV1-12, IGKV1-13, IGKV1-16, IGKV1-17, IGKV1-27, IGKV1-33, IGKV1-37, IGKV1-39, IGKV1D-16, IGKV1D-17, IGKV1D-43, IGKV1D-8, IGKV2-24, IGKV2-28, IGKV2-29, IGKV2-30, IGKV2-40, IGKV2D-26, IGKV2D-29, IGKV2D-30, IGKV3-11, IGKV3-15, IGKV3-20, IGKV3D-07, IGKV3D-11, IGKV3D-20, IGKV4-1, IGKV5-2, IGKV6-21, and IGKV6D-41, or a sequence of at least about 80% identity to any of them;

(ii) L3-VK is the portion of the VKCDR3 encoded by the IGKV gene segment; and

(iii) X is any amino acid residue; and

(iv) JK* is an amino acid sequence selected from the group consisting of amino acid sequences encoded by IGJK1, IGJK2, IGJK3, IGJK4, and IGJK5, wherein the first amino acid residue of each amino acid sequence is not present.

36. An antibody isolated from the polypeptide expression products of the library of claim 35.

37. The library of claim 35, wherein X is selected from the group consisting of F, L, I, R, W, Y, and P.

38. A library of synthetic polynucleotides, wherein said polynucleotides encode a plurality of Vλ

CDR3 amino acid sequences that are of at least about 80% identity to an amino acid sequence represented by the following formula;

[Vλ

_Chassis]-[L3-Vλ

]-[Jλ

], wherein;

(i) Vλ

_Chassis is an amino acid sequence selected from the group consisting of about Kabat amino acid 1 to about Kabat amino acid 88 encoded by IG/V1-36, IG/V1-40, IG/V1-44, IG/V1-47, IG/V1-51, IGλ

V10-54, IGλ

V2-11, IGλ

V2-14, IGλ

V2-18, IGλ

V2-23, IGλ

V2-8, IGλ

V3-1, IGλ

V3-10, IGλ

V3-12, IGλ

V3-16, IGλ

V3-19, IGλ

V3-21, IGλ

V3-25, IGλ

V3-27, IGλ

V3-9, IGλ

V4-3, IGλ

V4-60, IGλ

V4-69, IGλ

V5-39, IGλ

V5-45, IGλ

V6-57, IGλ

V7-43, IGλ

V7-46, IGλ

V8-61, IGλ

V9-49, and IGλ

V10-54, or a sequence of at least about 80% identity to any of them;

(ii) L3-Vλ

is the portion of the Vλ

CDR3 encoded by the IGλ

V segment; and

(iii) JR is an amino acid sequence selected from the group consisting of amino acid sequences encoded by IGλ

J1-01, IGλ

J2-01, IGλ

J3-01, IGλ

J3-02, IGλ

J6-01, IGλ

J7-01, and IGλ

J7-02, and wherein the first amino acid residue of each sequence may or may not be deleted.

39. An antibody isolated from the polypeptide expression products of the library of claim 38.

48. A library of synthetic polynucleotides encoding a plurality of antibody CDRH3 amino acid sequences, wherein the percent occurrence within the central loop of the CDRH3 amino acid sequences of at least one of the following i-i+1 pairs in the library is within the ranges specified below:

Tyr-Tyr in an amount from about 2.5% to about 6.5%;

Ser-Gly in an amount from about 2.5% to about 4.5%;

Ser-Ser in an amount from about 2% to about 4%;

Gly-Ser in an amount from about 1.5% to about 4%;

Tyr-Ser in an amount from about 0.75% to about 2%;

Tyr-Gly in an amount from about 0.75% to about 2%; and

Ser-Tyr in an amount from about 0.75% to about 2%.

49. The library of claim 48, wherein at least 2, 3, 4, 5, 6, or 7 of the specified i-i+1 pairs in the library are within the specified ranges.

50. An antibody isolated from the polypeptide expression products of the library of claim 48.

51. The library of claim 48, wherein the polynucleotides encode at least about 10⁶ unique CDRH3 amino acid sequences.

52. A library of synthetic polynucleotides encoding a plurality of antibody CDRH3 amino acid sequences, wherein the percent occurrence within the central loop of the CDRH3 amino acid sequences of at least one of the following i-i+2 pairs in the library is within the ranges specified below:

Tyr-Tyr in an amount from about 2.5% to about 4.5%;

Gly-Tyr in an amount from about 2.5% to about 5.5%;

Ser-Tyr in an amount from about 2% to about 4%;

Tyr-Ser in an amount from about 1.75% to about 3.75%;

Ser-Gly in an amount from about 2% to about 3.5%;

Ser-Ser in an amount from about 1.5% to about 3%;

Gly-Ser in an amount from about 1.5% to about 3%; and

Tyr-Gly in an amount from about 1% to about 2%.

53. The library of claim 52, wherein at least 2, 3, 4, 5, 6, 7, or 8 of the specified i-i+2 pairs in the library are within the specified ranges.

54. A antibody isolated from the polypeptide expression products of the library of claim 52.

55. The library of claim 52, wherein the polynucleotides encode at least about 10⁶ unique CDRH3 amino acid sequences.

56. A library of synthetic polynucleotides encoding a plurality of antibody CDRH3 amino acid sequences, wherein the percent occurrence within the central loop of the CDRH3 amino acid sequences of at least one of the following i-i+3 pairs in the library is within the ranges specified below:

Gly-Tyr in an amount from about 2.5% to about 6.5%;

Ser-Tyr in an amount from about 1% to about 5%;

Tyr-Ser in an amount from about 2% to about 4%;

Ser-Ser in an amount from about 1% to about 3%;

Gly-Ser in an amount from about 2% to about 5%; and

Tyr-Tyr in an amount from about 0.75% to about 2%.

57. The library of claim 56, wherein at least 2, 3, 4, 5, or 6 of the specified i-i+3 pairs in the library are within the specified ranges.

58. An antibody isolated from the polypeptide expression products of the library of claim 56.

59. The library of claim 56, wherein the polynucleotides encode at least about 10⁶ unique CDRH3 amino acid sequences.

61. A method of preparing a library of synthetic polynucleotides encoding a plurality of antibody CDRH3 amino acid sequences, the method comprising:

(i) providing polynucleotide sequences encoding;

(a) one or more N1 amino acid sequences of 0 to about 3 amino acids, wherein each amino acid of the N1 amino acid sequence is among the 12 most frequently occurring amino acids at the corresponding position in N1 amino acid sequences of CDRH3 amino acid sequences that are functionally expressed by human B cells;

(b) one or more human CDRH3 DH amino acid sequences, N- and C-terminal truncations thereof, or a sequence of at least about 80% identity to any of them;

(c) one or more N2 amino acid sequences of 0 to about 3 amino acids, wherein each amino acid of the N1 amino acid sequence is among the 12 most frequently occurring amino acids at the corresponding position in N2 amino acid sequences of CDRH3 amino acid sequences that are functionally expressed by human B cells; and

(d) one or more human CDRH3 H3-JH amino acid sequences, N-terminal truncations thereof, or a sequence of at least about 80% identity to any of them; and

(ii) assembling the polynucleotide sequences to produce a library of synthetic polynucleotides encoding a plurality of human antibody CDRH3 amino acid sequences represented by the following formula;

[N1]-[DH]-[N2]-[H3-JH].

62. The method of claim 61, wherein one or more of the polynucleotide sequences are synthesized via split-pool synthesis.

63. The method of claim 61, further comprising the step of providing a 5′

polynucleotide sequence and a 3′

polynucleotide sequence that facilitate homologous recombination.

64. The method of claim 61, further comprising the step of recombining the assembled synthetic polynucleotides with a vector comprising a heavy chain chassis and a heavy chain constant region, to form a full-length heavy chain.

65. The method of claim 64, wherein the step of recombining is performed in yeast.

66. The method of claim 65, wherein the yeast is S. cerevisiae.