Libraries of genetic packages comprising novel HC CDR1, CDR2, and CDR3 and novel LC CDR1, CDR2, and CDR3 designs

• US 9,388,510 B2
• Filed: 04/24/2009
• Issued: 07/12/2016
• 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.
• Primary Examiner(s): BOESEN, CHRISTIAN C
• Application Number:
  US12/989,307
• Publication Number:
  US 20110172125A1
• Time in Patent Office: 2,636 Days
• Field of Search: None
• US Class Current: 1/1
• 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, comprising variegated DNA sequences each encoding an antibody heavy chain (HC) variable region that comprises a HC CDR1, a HC CDR2, and a HC CDR3, wherein the HC CDR3 regions encoded by the variegated DNA sequences are 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, respectively;

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

1;

1;

1;

1;

1 ratio, respectively; 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, respectively.

2. The library of claim 1, 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, respectively.

3. A library of vectors or genetic packages, comprising variegated DNA sequences each encoding an antibody heavy chain (HC) variable region that comprises a HC CDR1, a HC CDR2, and a HC CDR3, wherein the HC CDR3 regions encoded by the variegated DNA sequences are 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, respectively;

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

1;

1;

1;

1 ratio, respectively; 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, respectively.

4. A library of vectors or genetic packages, comprising variegated DNA sequences each encoding an antibody heavy chain (HC) variable region that comprises a HC CDR1, a HC CDR2, and a HC CDR3, wherein the HC CDR3 regions encoded by the variegated DNA sequences are four amino acids in length set forth as X₁X₂X₃X₄ (SEQ ID NO:

996), wherein;

residue X₁ of HC CDR3 varies among Y, S, D R, and L in a 4;

1;

1;

1;

1 ratio, respectively;

residue X₂ of HC CDR3 varies among F, S, Y, D, R, and L in a 4;

1;

1;

1;

1;

1 ratio, respectively;

residue X₃ of HC CDR3 varies among D, R, S, Y, and L in a 4;

1;

1;

1;

1 ratio, respectively; and

residue X₄ of HC CDR3 varies among L, S, Y, D, and R in a 4;

1;

1;

1;

1 ratio, respectively.

5. A library of vectors or genetic packages, comprising variegated DNA sequences each encoding an antibody heavy chain (HC) variable region that comprises a HC CDR1, a HC CDR2, and a HC CDR3, wherein the HC CDR3 regions encoded by the variegated DNA sequences are four amino acids in length set forth as X₁X₂X₃X₄ (SEQ ID NO:

997), wherein;

residue X₁ of HC CDR3 varies among L, S, Y, D, and R in a 4;

1;

1;

1;

1 ratio, respectively;

residue X₂ of HC CDR3 varies among L, S, Y, D, and R in a 4;

1;

1;

1;

1 ratio, respectively;

residue X₃ of HC CDR3 varies among W, S, Y, D, and R in a 4;

1;

1;

1;

1 ratio, respectively; and

residue X₄ of HC CDR3 varies among F, S, Y, D, and R in a 4;

1;

1;

1;

1 ratio, respectively.

6. The library of claim 5, wherein a FR3 is located at the amino terminus of the HC CDR3, and the last residue of the FR3 varies between K and R in a 4:

1 ratio, respectively.

7. A library of vectors or genetic packages, comprising variegated DNA sequences each encoding an antibody heavy chain (HC) variable region that comprises a HC CDR1, a HC CDR2, and a HC CDR3, wherein the HC CDR3 regions encoded by the variegated DNA sequences are sixteen amino acids in length set forth as X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆ (SEQ ID NO:

998), wherein;

residue X₁ of HC CDR3 varies among Y, S, R, D, and L in a 3;

1;

1;

1;

1 ratio, respectively;

residue X₂ of HC CDR3 varies among Y, S, R, D, and L in a 3;

1;

1;

1;

1 ratio, respectively;

residue X₃ of HC CDR3 varies among Y, S, R, D, and L in a 3;

1;

1;

1;

1 ratio, respectively;

residue X₄ of HC CDR3 varies among D, Y, S, R, and L in a 3;

1;

1;

1;

1 ratio, respectively;

residue X₅ of HC CDR3 varies among S, Y, R, D, and L in a 3;

1;

1;

1;

1 ratio, respectively;

residue X₆ of HC CDR3 varies among S, Y, R, D, and L in a 3;

1;

1;

1;

1 ratio, respectively;

residue X₇ of HC CDR3 varies among G, A, S, Y, R, D, and L in a 3;

1;

1;

1;

1;

1;

1 ratio, respectively;

residue X₈ of HC CDR3 varies among Y, S, R, D, and L in a 3;

1;

1;

1;

1 ratio, respectively;

residue X₉ of HC CDR3 varies among Y, S, R, D, and L in a 3;

1;

1;

1;

1 ratio, respectively;

residue X₁₀ of HC CDR3 varies among Y, S, R, D, and L in a 3;

1;

1;

1;

1 ratio, respectively;

residue X₁₁ of HC CDR3 varies among A, S, Y, R, and Din a 3;

1;

1;

1;

1 ratio, respectively;

residue X₁₂ of HC CDR3 varies among E, R, S, Y, and L in a 3;

1;

1;

1;

1 ratio, respectively;

residue X₁₃ of HC CDR3 varies among Y, S, R, D, and L in a 3;

1;

1;

1;

1 ratio, respectively;

residue X₁₄ of HC CDR3 varies among F, Y, S, R, and Din a 3;

1;

1;

1;

1 ratio, respectively;

residue X₁₅ of HC CDR3 varies among Q, E, R, S, and Yin a 3;

1;

1;

1;

1 ratio, respectively; and

residue X₁₆ of HC CDR3 varies among H, E, R, S, Y, and L in a 3;

1;

1;

1;

1;

1 ratio, respectively.

8. The library of claim 7, wherein a FR3 is located at the amino terminus of the HC CDR3, and the last residue of the FR3 varies between K and R in a 3:

1 ratio, respectively.

9. A library of vectors or genetic packages, comprising variegated DNA sequences each encoding an antibody heavy chain (HC) variable region that comprises a HC CDR1, a HC CDR2, and a HC CDR3, wherein the HC CDR3 regions encoded by the variegated DNA sequences are sixteen amino acids in length set forth as X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆ (SEQ ID NO:

999), wherein;

residue X₁ of HC CDR3 varies among G, S, Y, D, R, and L in a 3;

1;

1;

1;

1;

1 ratio, respectively;

residue X₂ of HC CDR3 varies among Y, S, D, R, and L in a 3;

1;

1;

1;

1 ratio, respectively;

residue X₃ of HC CDR3 is C;

residue X₄ of HC CDR3 varies among S, Y, R, D, and L in a 3;

1;

1;

1;

1 ratio, respectively;

residue X₅ of HC CDR3 varies among S, Y, R, D, and L in a 3;

1;

1;

1;

1 ratio, respectively;

residue X₆ of HC CDR3 varies among T, Y, R, D, and L in a 3;

1;

1;

1;

1 ratio, respectively;

residue X₇ of HC CDR3 varies among S, Y, R, D, and L in a 3;

1;

1;

1;

1 ratio, respectively;

residue X₈ of HC CDR3 is C;

residue X₉ of HC CDR3 varies among Y, S, D, R, and L in a 3;

1;

1;

1;

1 ratio, respectively;

residue X₁₀ of HC CDR3 varies among T, Y, R, D, and L in a 3;

1;

1;

1;

1 ratio, respectively;

residue X₁₁ of HC CDR3 varies among A, S, Y, D, R, and L in a 3;

1;

1;

1;

1;

1 ratio, respectively;

residue X₁₂ of HC CDR3 varies among E, R, S, Y, and L in a 3;

1;

1;

1;

1 ratio, respectively;

residue X₁₃ of HC CDR3 varies among Y, S, D, R, and L in a 3;

1;

1;

1;

1 ratio, respectively;

residue X₁₄ of HC CDR3 varies among F, Y, S, R, D, and L in a 3;

1;

1;

1;

1;

1 ratio, respectively;

residue X₁₅ of HC CDR3 varies among Q, E, R, S, Y, and L in a 3;

1;

1;

1;

1;

1 ratio, respectively; and

residue X₁₆ of HC CDR3 varies among H, D, R, S, Y, and L in a 3;

1;

1;

1;

1;

1 ratio, respectively.

10. The library of claim 9, wherein a FR3 is located at the amino terminus of the HC CDR3, and the last residue of the FR3 varies between K and R in a 3:

1 ratio, respectively.

11. A library of vectors or genetic packages, comprising variegated DNA sequences each encoding an antibody heavy chain (HC) variable region that comprises a HC CDR1, a HC CDR2, and a HC CDR3, wherein the HC CDR3 is between 1 and 35 amino acids in length, wherein at least one position of the CDR3 is selected from the group consisting of Tyr, Gly, Asp, Ser, and Arg residues, and wherein more than about 20% of the HC CDR3 regions encoded by the variegated DNA sequences contain Tyr;

the library further comprising variegated DNA sequences that encode an A27 light chain (LC), wherein CDR3 of the LC comprises X₉₁X₉₂X₉₃X₉₄X₉₅X₉₆ (SEQ ID NO;

1001) and is diversified, and wherein

position X₉₁ is about 64% Y, and about 9% each of F, E, R, and S;

position X₉₂ is about 52% G and about 8% each of A, D, R, S, T, and Y;

position X₉₃ is about 52% S and about 8% each of D, F, N, R, T, and Y;

position X₉₄ is about 55% S and about 9% each of W, E, R, Y, and S;

position X₉₅ is about 64% P and about 9% each of E, R, Y, and S, and about 8% with no amino acid; and

position X₉₆ is about 55% L and about 9% each of E, R, P, Y, and S.

13. The library of claim 11, wherein about 50% of all the positions of the HC CDR3 regions encoded by the variegated DNA sequences are Tyr, Gly, Asp, Ser, or Arg.

14. The library of claim 11, wherein an Arg residue is present in a filler region between HC V and HC D, between HC D and HC J, or between HC V and HC J.

15. The library of claim 11, wherein a framework region (FR) 3 is located at the amino terminus of the HC CDR3, and a FR4 portion of a JH region is located at the carboxy terminus of the HC CDR3.

16. The library of claim 15, wherein the FR3 is a 3-23 VH FR3.

17. The library of claim 11, wherein HC D regions and HC J stumps of the HC CDR3 consist of more than about 20% of Tyr residues.

18. The library of claim 11, wherein CDR1 of the LC comprises X₂₇X₂₈X₂₉X₃₀X_30aX₃₁X₃₂X₃₃X₃₄ (SEQ ID NO:

1000) and is diversified, and wherein

position X₂₇ is about 55% Q, and about 9% each of E, R, Y, S, and L;

position X₂₈ is about 46% S and about 9% each of N, T, Y, E, R, and L;

position X₃₀ is about 55% S and about 9% each of D, N, R, T, and Y;

position X_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 X₃₁ is about 44% S and about 8% each of D, F, G, N, R, T, and Y;

position X₃₂ is about 44% Y and about 7% each of F, D, L, N, Q, R, S, and Y; and

position X₃₄ is about 70% A and about 15% each of S and Y.

19. The library of claim 11, 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.

20. The library of claim 18, wherein the library further comprises LC CDR2 diversity of:

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

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

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

12. A library of vectors or genetic packages, comprising variegated DNA sequences each encoding an antibody heavy chain (HC) variable region that comprises a HC CDR1, a HC CDR2, and a HC CDR3, wherein the HC CDR3 is between 1 and 35 amino acids in length, wherein at least one position of the CDR3 is selected from the group consisting of Tyr, Gly, Asp, Ser, and Arg residues, and wherein more than about 20% of the HC CDR3 regions encoded by the variegated DNA sequences contain Tyr;

the library further comprising variegated DNA sequences encoding light chains having diversity at LC CDR1, LC CDR2, and LC CDR3; and

where in

(i) the CDR1 comprises X₂₇X₂₈X₂₉X₃₀X_30aX₃₁X₃₂X₃₃X₃₄ (SEQ ID NO;

1000) and is diversified, and wherein

position X₂₇ is about 55% Q, and about 9% each of E, R, Y, S, and L;

position X₂₈ is about 46% S and about 9% each of N, T, Y, E, R, and L;

position X₃₀ is about 55% S and about 9% each of D, N, R, T, and Y;

position X_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 X₃₁ is about 44% S and about 8% each of D, F, G, N, R, T, and Y;

position X₃₂ is about 44% Y and about 7% each of F, D, L, N, Q, R, S, and Y; and

position X₃₄ is about 70% A and about 15% each of S and Y, (

ii) the CDR2 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;

or

(iii) the CDR3 comprises X₉₁X₉₂X₉₃X₉₄X₉₅X₉₆ (SEQ ID NO;

1001) and is diversified, and wherein

position X₉₁ is about 64% Y, and about 9% each of F, E, R, and S;

position X₉₂ is about 52% G and about 8% each of A, D, R, S, T, and Y;

position X₉₃ is about 52% S and about 8% each of D, F, N, R, T, and Y;

position X₉₄ is about 55% S and about 9% each of W, E, R, Y, and S;

position X₉₅ is about 64% P and about 9% each of E, R, Y, and S, and about 8% with no amino acid; and

position X₉₆ is about 55% L and about 9% each of E, R, P, Y, and S.

21. The library of claim 12, wherein about 50% of all the positions of the HC CDR3 regions encoded by the variegated DNA sequences are Tyr, Gly, Asp, Ser, or Arg.

22. The library of claim 12, wherein an Arg residue is present in a filler region between HC V and HC D, between HC D and HC J, or between HC V and HC J.

23. The library of claim 12, wherein a framework region (FR) 3 is located at the amino terminus of the HC CDR3, and a FR4 portion of a JH region is located at the carboxy terminus of the HC CDR3.

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

25. The library of claim 12, wherein HC D regions and HC J stumps of the HC CDR3 consist of more than about 20% of Tyr residues.