Focused libraries of genetic packages

• US 9,803,190 B2
• Filed: 01/30/2013
• Issued: 10/31/2017
• Est. Priority Date: 12/18/2000
• Status: Expired due to Term

• Current Assignee: Takeda Pharmaceutical Company Limited
• Original Assignee: Dyax Corporation (Shire plc)
• Inventors: Ladner, Robert Charles
• Primary Examiner(s): BOESEN, CHRISTIAN C
• Application Number:
  US13/754,261
• Publication Number:
  US 20130178396A1
• Time in Patent Office: 1,735 Days
• Field of Search: None
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/005 constructed by phage libraries
• CPC Class Codes: C07K 2317/565 Complementarity determining region [CDR]
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• 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

Focused libraries of vectors or genetic packages that display, display and express, or comprise a member of a diverse family of antibody peptides, polypeptides or proteins and collectively display, display and express, or comprise at least a portion of the focused diversity of the family. The libraries have length and sequence diversities that mimic that found in native human antibodies.

Claims

1. A focused library of DNA plasmids or genetic packages comprising the DNA plasmids, the focused library comprising one or more sets of variegated DNA molecules, each of which comprises a DNA sequence that encodes an antibody heavy chain variable domain having both frame work (FW) regions and complementary determining regions (CDR), wherein each antibody heavy chain variable domain comprises antibody heavy chain FW1, antibody heavy chain CDR1, antibody heavy chain FW2, antibody heavy chain CDR2, antibody heavy chain FW3, antibody heavy chain CDR3 and antibody heavy chain FW4 in a DNA molecule arranged in the orientation of FW1-CDR1-FW2-CDR2-FW3-CDR3-FW4, wherein each of the one or more sets of the variegated DNA molecules collectively encodes a plurality of different antibody heavy chain variable domains, which share a common heavy chain CDR3 motif encompassed by one of the following components:

(i) YYCA21111YFDYWG (SEQ ID NO;

6), wherein 1 is any amino acid residue except C, and 2 is a mixture of K and R;

(ii) YYCA2111111YFDYWG (SEQ ID NO;

7), wherein 1 is any amino acid residue except C, and 2 is a mixture of K and R;

(iii) YYCA211111111YFDAYTG (SEQ ID NO;

8), wherein 1 is any amino acid residue except C, and 2 is a mixture of K and R;

(iv) YYCAR111S2S3111YFDYWG (SEQ ID NO;

9), wherein 1 is any amino acid residue except C, 2 is a mixture of S and G, and 3 is a mixture of Y and W;

(v) YYCA2111CSG11CY1YFDYWG (SEQ ID NO;

10), wherein 1 is any amino acid residue except C, and 2 is a mixture of K and R;

(vi) YYCA211S1TIFG11111YFDYWG (SEQ ID NO;

11), wherein 1 is any amino acid residue except C, and 2 is a mixture of K and R;

(vii) YYCAR111YY2S33YY111YFDYWG (SEQ ID NO;

12), wherein 1 is any amino acid residue except C, 2 is D or S, and 3 is a mixture of S and G; and

(viii) YYCAR1111YC2231CY111YFDYWG (SEQ ID NO;

13), wherein 1 is any amino acid residue except C, 2 is a mixture of S and G, and 3 is a mixture of T, D and G;

wherein the variation within the at least one set of the variegated DNA molecules, which collectively encodes the plurality of different antibody heavy chain variable domains, is due to variation in 1, 2, or 3 in the residue(s) of SEQ ID NOs 6-13;

wherein the one or more sets of variegated DNA molecules collectively encode one or more amino acid sequences selected from SEQ ID NOs 6 -13, and

wherein the one or more sets of the variegated DNA molecules comprise a human germline V_H gene as the FW1, FW2 and FW3 regions, and

wherein the diversity of the focused library is biased towards the natural diversity of heavy chain CDR3s found in the human antibody family antibodies, both in sequence and in length.

2. The library of claim 1, wherein:

the complexity of component (i) is 2×

10⁵,

the complexity of component (ii) is 9.4×

10⁷,

the complexity of component (iii) is 3.4×

10¹⁰,

the complexity of component (iv) is 1.9×

10⁸,

the complexity of component (v) is 9.4×

10⁷,

the complexity of component (vi) is 1.7×

10¹⁰,

the complexity of component (vii) is 3.8×

10⁸, and

the complexity of component (viii) is 2.0×

10¹¹.

3. The library of claim 1, wherein:

in one or more of components (i), (ii), (iii), (v), and (vi), 1 is (a) an equimolar mixture of amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y;

or (b) a mixture of 0.095 Y, 0.095 G, and 0.048 each of A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, and W; and

2 is an equimolar mixture of K and R;

in (iv), 1 is (a) an equimolar mixture of amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y;

or (b) a mixture of 0.095 Y, 0.095 G, and 0.048 each of A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, and W;

2 is an equimolar mixture of S and G; and

3 is an equimolar mixture of Y and W;

in (vii), 1 is (a) an equimolar mixture of amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y;

or (b) a mixture of 0.095 Y, 0.095 G, and 0.048 each of A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, and W;

2 is an equimolar mixture of D and G; and

3 is an equimolar mixture of S and G;

or

in (viii), 1 is (a) an equimolar mixture of amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y;

or (b) a mixture of 0.095 Y, 0.095 G, and 0.048 each of A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, and W;

2 is an equimolar mixture of S and G; and

3 is an equimolar mixture of T, D, and G.

4. The library of claim 1, wherein components (i) through (viii) are present in the library in the following proportions:

(i) 0.10, (ii) 0.14, (iii) 0.25, (iv) 0.13, (v) 0.13, (vi) 0.11, (vii) 0.04, and (viii) 0.10.

5. The library of claim 1, wherein components (i) through (viii) are present in the library in the following proportions:

(i) 0.02, (ii) 0.14, (iii) 0.25, (iv) 0.14, (v) 0.14, (vi) 0.12, (vii) 0.08, and (viii) 0.11.

6. The library of claim 1, wherein the one or more sets of the variegated DNA molecules further comprise a plurality of variegated HC CDR1 regions selected from the group consisting of:

(1) <

1>

₁Y₂<

1>

₃M₄<

1>

₅ (SEQ ID NO;

100), wherein <

1>

is an equimolar mixture of each of amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y;

(2) (S/T)₁(S/G/X)₂(S/G/X)₃Y₄Y₅W₆(S/G/X)₇ (SEQ ID NO;

101), wherein (S/T) is a 1;

1 mixture of S and T residues, (S/G/X) is a mixture of 0.2025 S, 0.2025, G and 0.035 of each of amino acid residues A, D, E, F, H, I, K, L, M, N, P, Q, R, T, V, W, and Y;

(3) V₁S₂G₃G₄S₅I₆S₇<

<

1>

₈<

1>

₉<

1>

₁₀Y₁₁Y₁₂W₁₃<

1>

₁₄ (SEQ ID NO;

1), wherein <

1>

is an equimolar mixture of each of amino acid residues A, D, E, F, G, H,. I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and

(4) a mixture of any of (1) to (3) set forth above.

7. The library of claim 1, wherein the one or more sets of the variegated DNA molecules further comprise a plurality of variegated HC CDR2 regions selected from the group consisting of:

(1) <

2>

I<

2>

<

3>

SGG<

1>

T<

1>

YADSVKG (SEQ ID NO;

2), wherein <

1>

is an equimolar mixture of each of amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y;

<

2>

is an equimolar mixture of each of amino acid residues Y, R, W, V, G, and S; and

<

3>

is an equimolar mixture of each of amino acid residues P, S, and G or an equimolar mixture of P and S;

(2) <

1>

I<

4>

<

1>

<

1>

<

G>

<

5>

<

1>

<

1>

−

<

1>

YADSVKG (SEQ ID NO;

3), wherein <

1>

is an equimolar mixture of each of amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y;

<

4>

is an equimolar mixture of residues D, I, N, S, W, Y; and

<

5>

is an equimolar mixture of residues S, G, D and N;

(3) <

1>

I<

4>

<

1>

<

1>

G<

5>

<

1>

<

1>

YNPSLKG (SEQ ID NO;

4), wherein <

1>

is an equimolar mixture of each of amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; and

<

4> and

<

5>

are as defined above;

(4) <

1>

I<

8>

S<

1>

<

1>

<

1>

GGYY<

1>

YAASVKG (SEQ ID NO;

5), wherein <

1>

is an equimolar mixture of each amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y;

<

8>

is 0.27 R and 0.027 of each of ADEFGHIKLMNPQSTVWY; and

(5) a mixture of any of (1) to (4) set forth above.

8. The library of claim 1, wherein the one or more sets of the variegated DNA molecules further comprise a plurality of variegated HC CDR2 regions selected from the group consisting of:

(1) <

2>

I<

2>

<

3>

SGG<

1>

T<

1>

YADSVKG (SEQ ID NO;

2), wherein <

1>

is an equimolar mixture of each of amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y;

<

2>

is an equimolar mixture of each of amino acid residues Y, R, W, V, G, and S; and

<

3>

is an equimolar mixture of each of amino acid residues P, S, and G or an equimolar mixture of P and S;

(2) <

1>

I<

4>

<

1>

<

1>

<

G>

<

5>

<

1>

<

1>

−

<

1>

YADSVKG (SEQ ID NO;

3), wherein <

1>

is an equimolar mixture of each of amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y;

<

4>

is an equimolar mixture of residues D, I, N, S, W, Y; and

<

5>

is an equimolar mixture of residues S, G, D and N;

(3) <

1>

I<

4>

<

1>

<

1>

G<

5>

<

1>

<

1>

YNPSLKG (SEQ ID NO;

4), wherein <

1>

is an equimolar mixture of each of amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; and

<

4> and

<

5>

are as defined above;

(4) <

1>

I<

8>

S<

1>

<

1>

<

1>

GGYY<

1>

YAASVKG (SEQ ID NO;

5), wherein <

1>

is an equimolar mixture of each amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y;

<

8>

is 0.27 R and 0.027 of each of A, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, and Y; and

(5) a mixture of any of (1) to (4) set forth above.

9. The focused library according to claim 6, wherein the HC CDR1s (1), (2) and (3) regions are present in the library in the ratio 0.80:

0.17;

0.02.

10. The focused library according to claim 7, wherein a mixture of HC CDR2s (1)/(2) (equimolar), (3) and (4) are present in the library in a ratio of 0.54:

0.43;

0.03.

11. The library of claim 1, further comprising an additional set of variegated DNA molecules encoding a collection of antibody light chains (LC), wherein each comprises a CDR1 region, a CDR2 region, and a CDR3 region.

12. The library of claim 11, wherein at least a light chain portion of the collection are kappa light chains, each of which comprises a LC_κ

CDR1 region, a LC_κ

CDR2 region, and a LC_κ

CDR3 region.

13. The library of claim 12, wherein the light chain portion comprises a plurality of variegated LC_κ

CDR3 regions selected from the group consisting of;

(1) QQ<

3>

<

1>

<

1>

<

1>

P<

1>

T (SEQ ID NO;

16), wherein <

1>

is an equimolar mixture of amino acid residues ADEFGHIKLMNPQRSTVWY; and

<

3>

is 0.2 Y and 0.044 each of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, and W;

(2) QQ33111P (SEQ ID NO;

103), wherein 1 and 3 are as defined in (1) above;

(3) QQ3211PP1T (SEQ ID NO;

17), wherein 1 and 3 are as defined in (1) above and 2 is 0.2 S and 0.044 each of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, and Y; and

(4) a mixture of any of (1) to (3) set forth above.

14. The library of claim 13, wherein the light chain portion further comprises a plurality of variegated LC_κ

CDR1 regions selected from the group consisting of;

(1) RASQ<

1>

V<

2>

<

2>

<

3>

LA (SEQ ID NO;

14), wherein <

1>

is an equimolar mixture of amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y;

<

2>

is 0.2 S and 0.044 of each of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, Y; and

<

3>

is 0.2Y and 0.044 each of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W and S; and

(2) RASQ<

1>

V<

2>

<

2>

<

2>

<

3>

LA (SEQ ID NO;

15);

wherein <

1>

, <

2>

, and <

3>

are as defined in (1) above; and

(3) a mixture of (1) and (2) set forth above.

15. The library of claim 13, wherein the light chain portion further comprises a plurality of variegated LC_κ

CDR2 regions <

1>

AS<

2>

R<

4>

<

1>

(SEQ ID NO;

102), wherein <

1>

is an equimolar mixture of amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y;

<

2>

is 0.2 S and 0.044 of each of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, and Y; and

<

4>

is 0.2 A and 0.044 each of D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

16. The library of claim 14, wherein the light chain portion further comprises a plurality of variegated LC_κ

CDR2 regions <

1>

AS<

2>

R<

4>

<

1>

(SEQ ID NO;

102), wherein <

1>

is an equimolar mixture of amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y;

<

2>

is 0.2 S and 0.044 of each of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, and Y; and

<

4>

is 0.2 A and 0.044 each of D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

17. The library of claim 14, wherein the LC_κ

CDR1s (1) and (2) are present in the library in a ratio of 0.68;

0.32.

18. The library of claim 13, wherein the LC_κ

CDR3s (1), (2) and (3) are present in the library in a ratio of 0.65;

0.1;

0.25.

19. The library of claim 11, wherein at least a light chain portion of the collection are lambda light chains, each of which comprises a LC_λ

CDR1 region, a LC_λ

CDR2 region, and a LC_λ

CDR3 region.

20. The library of claim 19, wherein the light chain portion further comprises a plurality of variegated LC_λ

CDR3 regions selected from the group consisting of;

(1) <

4>

<

5>

<

4>

<

2>

<

4>

S<

4>

<

4>

<

4>

<

4>

V (SEQ ID NO;

106), wherein <

2>

is 0.27 D, 0.27 N, and 0.027 each of A, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y;

<

4>

is an equimolar mixture of amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, and W; and

<

5>

is 0.36 S and 0.0355 each of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, and Y;

(2) <

5>

SY<

1>

<

5>

S<

5>

<

1>

<

4>

V (SEQ ID NO;

19), wherein <

1>

is an equimolar mixture of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and

<

4> and

<

5>

are as defined in (1) above; and

(3) a mixture of (1) and (2) set forth above.

21. The library of claim 20, wherein the light chain portion further comprises a plurality of variegated LC_λ

CDR1 regions selected from the group consisting of;

(1) TG<

1>

SS<

2>

VG<

1>

<

3>

<

2>

<

3>

VS (SEQ ID NO;

18), wherein <

1>

is 0.27 T, 0.27 G and 0.027 each of A, D, E, F, H, I, K, L, M, N, P, Q, R, S, V, W, and Y, <

2>

is 0.27 D, 0.27 N and 0.027 each of A, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y, and <

3>

is 0.36 Y and 0.036 each of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, and W;

(2) G<

2>

<

4>

L<

4>

<

4>

<

4>

<

3>

<

4>

<

4>

(SEQ ID NO;

104), wherein <

2>

is as defined in (1) above and <

4>

is an equimolar mixture of amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and

(3) a mixture of (1) and (2) set forth above.

22. The library of claim 20, wherein the light chain portion further comprises a plurality of variegated LC_λ

CDR2 regions <

4>

<

4>

<

4>

<

2>

RPS (SEQ ID NO;

105), wherein <

2>

is 0.27 D, 0.27 N, and 0.027 each of A, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y and <

4>

is an equimolar mixture of amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, and W.

23. The library of claim 21, wherein the light chain portion further comprises a plurality of variegated LC_λ

CDR2 regions <

4>

<

4>

<

4>

<

2>

RPS (SEQ ID NO;

105), wherein <

2>

is 0.27 D, 0.27 N, and 0.027 each of A, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, and Y and <

4>

is an equimolar mixture of amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, and W.

24. The library of claim 21, where the LC_λ

CDR1s (1) and (2) are present in the library in a ratio of 0.67;

0.33.

25. The library of claim 20, wherein the LC_λ

CDR3s (1) and (2) are present in the library in an equimolar mixture.

26. The library of claim 1, wherein the library is a library of vectors.

27. The library of claim 26, wherein the vectors are yeast vectors.

28. The library of claim 1, wherein the library is a library of genetic packages.

29. The library of claim 28, wherein the genetic packages are cells, spores, or viral particles.

30. The library of claim 29, wherein the genetic packages are phage particles or yeast cells.

31. The library of claim 30, wherein the genetic packages are yeast cells, which display the collection of antibody heavy chains encoded by the first set of variegated DNA molecules.

32. The library of claim 11, wherein the library is a library of vectors.

33. The library of claim 32, wherein the vectors are yeast vectors.

34. The library of claim 11, wherein the library is a library of genetic packages.

35. The library of claim 34, wherein the genetic packages are cells, spores, or viral particles.

36. The library of claim 35, wherein the genetic packages are phage particles or yeast cells.

37. The library of claim 36, wherein the genetic packages are yeast cells, which display the collection of antibody heavy chains encoded by the one or more sets of the variegated DNA molecules, the collection of antibody light chains encoded by the additional set of the variegated DNA molecules, or both.