LIBRARIES OF GENETIC PACKAGES COMPRISING NOVEL HC CDR1, CDR2, AND CDR3 AND NOVEL LC CDR1, CDR2, AND CDR3 DESIGNS

• US 20110172125A1
• Filed: 04/24/2009
• Published: 07/14/2011
• Est. Priority Date: 04/24/2008
• Status: Active Grant

• Current Assignee: Takeda Pharmaceutical Company Limited
• Original Assignee: Dyax Corporation (Shire plc)
• Inventors: Ladner, Robert C.
• Granted Patent: US 9,388,510 B2
• Time in Patent Office: Days
• US Class Current: 506/17
• CPC Class Codes: C07K 16/005 constructed by phage libraries
• CPC Class Codes: C07K 2317/565 Complementarity determining region [CDR]
• CPC Class Codes: C40B 40/02 Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
• CPC Class Codes: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• CPC Class Codes: C40B 40/10 Libraries containing peptides or polypeptides, or derivatives thereof

Abstract

Provided are compositions and methods for preparing and identifying antibodies having CDR3s that vary in sequence and in length from very short to very long which in certain embodiments may bind to a carbohydrate moiety or the active site of an enzyme. Libraries coding for antibodies with the CDR3s are also provided. The libraries can be provided by modifying a pre-existing nucleic acid library.

Claims

1. A library of vectors or genetic packages that display, display and express, or comprise a member of a diverse family of human antibody related peptides, polypeptides and proteins and collectively display, display and express, or comprise at least a portion of the diversity of the antibody family, wherein the vectors or genetic packages comprise variegated DNA sequences that encode a heavy chain (HC) CDR3, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 of the positions in the HC CDR3 of the family are varied among Tyr, Gly, Asp, Ser, and Arg residues.

2. The library of claim 1, wherein about 50% of all the positions of the HC CDR3 are Tyr, Gly, Asp, Ser, or Arg.

3. The library of claim 1, wherein an Arg residue is present in a filler region between V and D, between D and J, or between V and J.

26. The library of any of claims 1-19 further comprising a light chain having diversity at one or more of LC CDR1, LC CDR2, and LC CDR3.

28. A method of preparing the library of any one of claims 1 to 27, the method comprising dobbling.

4. A library of vectors or genetic packages that display, display and express, or comprise a member of a diverse family of human antibody related peptides, polypeptides and proteins and collectively display, display and express, or comprise at least a portion of the diversity of the antibody family, wherein the vectors or genetic packages comprise variegated DNA sequences that encode a heavy chain (HC) CDR3 consisting of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 residues, each of which independently vary among Tyr, Asn, Asp, Ser, and Arg residues, wherein a framework region (FR) 3 is located at the amino terminus of the CDR3, and a FR4 portion of a JH region is located at the carboxy terminus of the CDR3.

5. The library of claim 4, wherein the FR3 is a 3-23 VH FR3.

6. A library of vectors or genetic packages that display, display and express, or comprise a member of a diverse family of human antibody related peptides, polypeptides and proteins and collectively display, display and express, or comprise at least a portion of the diversity of the antibody family, wherein the vectors or genetic packages comprise variegated DNA sequences that encode a heavy chain (HC) CDR3, wherein more than about 20% of the HC CDR3 sequence consists of Tyr residues.

7. The library of claim 6, wherein D regions and J stumps of the HC CDR3 consist of more than about 20% of Tyr residues.

8. A library of vectors or genetic packages that display, display and express, or comprise a member of a diverse family of human antibody related peptides, polypeptides and proteins and collectively display, display and express, or comprise at least a portion of the diversity of the antibody family, wherein the vectors or genetic packages comprise variegated DNA sequences that encode a heavy chain (HC) CDR3, wherein less than about 20% of the HC CDR3 sequence consists of Tyr residues.

9. The library of claim 8, wherein leadin or DJ filler positions of the HC CDR3 consist of less than about 20% of Tyr residues.

10. A library of vectors or genetic packages that display, display and express, or comprise a member of a diverse family of human antibody related peptides, polypeptides and proteins and collectively display, display and express, or comprise at least a portion of the diversity of the antibody family, wherein the vectors or genetic packages comprise variegated DNA sequences that encode a heavy chain (HC) CDR3, wherein the HC CDR3 is three amino acids in length and:

the first residue of HC CDR3 varies among F, S, Y, D, and R in a 3;

1;

1;

1;

1 ratio;

the second residue of HC CDR3 varies among Q, E, R, S, Y, and L in a 3;

1;

1;

1;

1;

1 ratio; and

the third residue of HC CDR3 varies among H, D, R, S, Y, and L in a 3;

1;

1;

1;

1;

1 ratio.

11. The library of claim 10, wherein a FR3 is located at the amino terminus of the CDR3, and the last residue of the FR3 varies between K and R in a 3:

1 ratio.

12. A library of vectors or genetic packages that display, display and express, or comprise a member of a diverse family of human antibody related peptides, polypeptides and proteins and collectively display, display and express, or comprise at least a portion of the diversity of the antibody family, wherein the vectors or genetic packages comprise variegated DNA sequences that encode a heavy chain (HC) CDR3, wherein the HC CDR3 is three amino acids in length and:

the first residue of HC CDR3 varies among T, Y, R, D, and L in a 5;

1;

1;

1;

1 ratio;

the second residue of HC CDR3 varies among T, Y, R, D, and L in a 5;

1;

1;

1;

1 ratio; and

the third residue of HC CDR3 varies among G, S, Y, R, D, and L in a 5;

1;

1;

1;

1;

1 ratio.

13. A library of vectors or genetic packages that display, display and express, or comprise a member of a diverse family of human antibody related peptides, polypeptides and proteins and collectively display, display and express, or comprise at least a portion of the diversity of the antibody family, wherein the vectors or genetic packages comprise variegated DNA sequences that encode a heavy chain (HC) CDR3, wherein the HC CDR3 is four amino acids in length and:

the first residue of HC CDR3 varies among Y, S, D R, and L in a 4;

1;

1;

1;

1 ratio;

the second residue of HC CDR3 varies among F, S, Y, D, R, and L in a 4;

1;

1;

1;

1;

1 ratio;

the third residue of HC CDR3 varies among D, R, S, Y, and L in a 4;

1;

1;

1;

1 ratio; and

the fourth residue of HC CDR3 varies among L, S, Y, D, and R in a 4;

1;

1;

1;

1 ratio.

14. A library of vectors or genetic packages that display, display and express, or comprise a member of a diverse family of human antibody related peptides, polypeptides and proteins and collectively display, display and express, or comprise at least a portion of the diversity of the antibody family, wherein the vectors or genetic packages comprise variegated DNA sequences that encode a heavy chain (HC) CDR3, wherein the HC CDR3 is four amino acids in length and:

the first residue of HC CDR3 varies among L, S, Y, D, and R in a 4;

1;

1;

1;

1 ratio;

the second residue of HC CDR3 varies among L, S, Y, D, and R in a 4;

1;

1;

1;

1 ratio;

the third residue of HC CDR3 varies among W, S, Y, D, and R in a 4;

1;

1;

1;

1 ratio; and

the fourth residue of HC CDR3 varies among F, S, Y, D, and R in a 4;

1;

1;

1;

1 ratio.

15. The library of claim 14, wherein a FR3 is located at the amino terminus of the CDR3, and the last residue of the FR3 varies between K and R in a 4:

1 ratio.

16. A library of vectors or genetic packages that display, display and express, or comprise a member of a diverse family of human antibody related peptides, polypeptides and proteins and collectively display, display and express, or comprise at least a portion of the diversity of the antibody family, wherein the vectors or genetic packages comprise variegated DNA sequences that encode a heavy chain (HC) CDR3, wherein the HC CDR3 is sixteen amino acids in length and:

the first residue of HC CDR3 varies among Y, S, R, D, and L in a 3;

1;

1;

1;

1 ratio;

the second residue of HC CDR3 varies among Y, S, R, D, and L in a 3;

1;

1;

1;

1 ratio;

the third residue of HC CDR3 varies among Y, S, R, D, and L in a 3;

1;

1;

1;

1 ratio;

the fourth residue of HC CDR3 varies among D, Y, S, R, and L in a 3;

1;

1;

1;

1 ratio;

the fifth residue of HC CDR3 varies among S, Y, R, D, and L in a 3;

1;

1;

1;

1 ratio;

the sixth residue of HC CDR3 varies among S, Y, R, D, and L in a 3;

1;

1;

1;

1 ratio;

the seventh residue of HC CDR3 varies among G, A, S, Y, R, D, and L in a 3;

1;

1;

1;

1;

1;

1 ratio;

the eighth residue of HC CDR3 varies among Y, S, R, D, and L in a 3;

1;

1;

1;

1 ratio;

the ninth residue of HC CDR3 varies among Y, S, R, D, and L in a 3;

1;

1;

1;

1 ratio;

the tenth residue of HC CDR3 varies among Y, S, R, D, and L in a 3;

1;

1;

1;

1 ratio;

the eleventh residue of HC CDR3 varies among A, S, Y, R, and D in a 3;

1;

1;

1;

1 ratio;

the twelfth residue of HC CDR3 varies among E, R, S, Y, and L in a 3;

1;

1;

1;

1 ratio;

the thirteenth residue of HC CDR3 varies among Y, S, R, D, and L in a 3;

1;

1;

1;

1 ratio;

the fourteenth residue of HC CDR3 varies among F, Y, S, R, and D in a 3;

1;

1;

1;

1 ratio;

the fifteenth residue of HC CDR3 varies among Q, E, R, S, and Yin a 3;

1;

1;

1;

1 ratio; and

the sixteenth residue of HC CDR3 varies among H, E, R, S, Y, and L in a 3;

1;

1;

1;

1;

1 ratio.

17. The library of claim 16, wherein a FR3 is located at the amino terminus of the CDR3, and the last residue of the FR3 varies between K and R in a 3:

1 ratio.

18. A library of vectors or genetic packages that display, display and express, or comprise a member of a diverse family of human antibody related peptides, polypeptides and proteins and collectively display, display and express, or comprise at least a portion of the diversity of the antibody family, wherein the vectors or genetic packages comprise variegated DNA sequences that encode a heavy chain (HC) CDR3, wherein the HC CDR3 is sixteen amino acids in length and:

the first residue of HC CDR3 varies among G, S, Y, D, R, and L in a 3;

1;

1;

1;

1;

1 ratio;

the second residue of HC CDR3 varies among Y, S, D, R, and L in a 3;

1;

1;

1;

1 ratio;

the third residue of HC CDR3 is C;

the fourth residue of HC CDR3 varies among S, Y, R, D, and L in a 3;

1;

1;

1;

1 ratio;

the fifth residue of HC CDR3 varies among S, Y, R, D, and L in a 3;

1;

1;

1;

1 ratio;

the sixth residue of HC CDR3 varies among T, Y, R, D, and L in a 3;

1;

1;

1;

1 ratio;

the seventh residue of HC CDR3 varies among S, Y, R, D, and L in a 3;

1;

1;

1;

1 ratio;

the eighth residue of HC CDR3 is C;

the ninth residue of HC CDR3 varies among Y, S, D, R, and L in a 3;

1;

1;

1;

1 ratio;

the tenth residue of HC CDR3 varies among T, Y, R, D, and L in a 3;

1;

1;

1;

1 ratio;

the eleventh residue of HC CDR3 varies among A, S, Y, D, R, and L in a 3;

1;

1;

1;

1;

1 ratio;

the twelfth residue of HC CDR3 varies among E, R, S, Y, and L in a 3;

1;

1;

1;

1 ratio;

the thirteenth residue of HC CDR3 varies among Y, S, D, R, and L in a 3;

1;

1;

1;

1 ratio;

the fourteenth residue of HC CDR3 varies among F, Y, S, R, D, and L in a 3;

1;

1;

1;

1;

1 ratio;

the fifteenth residue of HC CDR3 varies among Q, E, R, S, Y, and L in a 3;

1;

1;

1;

1;

1 ratio; and

the sixteenth residue of HC CDR3 varies among H, D, R, S, Y, and L in a 3;

1;

1;

1;

1;

1 ratio.

19. The library of claim 19, wherein a FR3 is located at the amino terminus of the CDR3, and the last residue of the FR3 varies between K and R in a 3:

1 ratio.

20. A library of vectors or genetic packages that display, display and express, or comprise a member of a diverse family of human antibody related peptides, polypeptides and proteins and collectively display, display and express, or comprise at least a portion of the diversity of the antibody family, wherein the vectors or genetic packages comprise variegated DNA sequences that encode an A27 light chain (LC), wherein CDR1 of the LC is diversified, and wherein

position 27 is about 55% Q, and about 9% each of E, R, Y, S, and L;

position 28 is about 46% S and about 9% each of N, T, Y, E, R, and L;

position 30 is about 55% S and about 9% each of D, N, R, T, and Y;

position 30a is about 46% S and about 9% each of G, N, R, T, Y, and D, and about 8% with no amino acid;

position 31 is about 44% S and about 8% each of D, F, G, N, R, T, and Y;

position 32 is about 44% Y and about 7% each of F, D, L, N, Q, R, S, and Y; and

position 34 is about 70% A and about 15% each of S and Y.

23. The library of claim 20, wherein the library further comprises LC CDR2 diversity of claim 21.

24. The library of claim 20, wherein the library further comprises LC CDR3 diversity of claim 22.

27. The library of claim 26, wherein the library comprises LC CDR1, LC CDR2, or LC CDR3 of claim 20, 21, or 22, respectively, or combinations thereof.

21. A library of vectors or genetic packages that display, display and express, or comprise a member of a diverse family of human antibody related peptides, polypeptides and proteins and collectively display, display and express, or comprise at least a portion of the diversity of the antibody family, wherein the vectors or genetic packages comprise variegated DNA sequences that encode an A27 light chain (LC), wherein CDR2 of the LC is diversified, and wherein

position 50 is about 55% G, and about 9% each of D, R, S, Y, and L;

position 53 is about 52% S and about 8% each of N, T, S, Y, E, and R; and

position 56 is about 64% T and about 9% each of E, R, S, and Y.

25. The library of claim 21, wherein the library further comprises LC CDR3 diversity of claim 22.

22. A library of vectors or genetic packages that display, display and express, or comprise a member of a diverse family of human antibody related peptides, polypeptides and proteins and collectively display, display and express, or comprise at least a portion of the diversity of the antibody family, wherein the vectors or genetic packages comprise variegated DNA sequences that encode an A27 light chain (LC), wherein CDR3 of the LC is diversified, and wherein

position 91 is about 64% Y, and about 9% each of F, E, R, and S;

position 92 is about 52% G and about 8% each of A, D, R, S, T, and Y;

position 93 us about 52% S and about 8% each of D, F, N, R, T, and Y;

position 94 is about 55% S and about 9% each of W, E, R, Y, and S;

position 95 is about 64% P and about 9% each of E, R, Y, and S, and about 8% with no amino acid; and

position 96 is about 55% L and about 9% each of E, R, P, Y, and S.