Focused libraries of genetic packages

• US 8,399,384 B2
• Filed: 06/15/2011
• Issued: 03/19/2013
• 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): Marschel, Ardin
• Assistant Examiner(s): BOESEN, CHRISTIAN C
• Application Number:
  US13/161,441
• Publication Number:
  US 20120028840A1
• Time in Patent Office: 643 Days
• Field of Search: None
• US Class Current: 506/14
• 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 vectors or genetic packages, comprising a first set of variegated DNA molecules, each of which comprises a DNA sequence that encodes an antibody heavy chain (FTC) variable region comprising a CDR1 region, a CDR2 region, and a CDR3 region, wherein the first set of variegated DNA molecules collectively encode a plurality of:

(a) 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) V_IS₂G₃G₄S₅I₆S₇<

<

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;

(b) 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, 5, 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;

or

(c) HC CDR3 regions selected from the group consisting of;

(1) YYCA21111YFDYWG (SEQ ID NO;

6), 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; and

2 is an equimolar mixture of K and R;

(2) YYCA2111111YFDYWG (SEQ ID NO;

7), 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; and

2 is an equimolar mixture of K and R;

(3) YYCA211111111YFDAYTG (SEQ ID NO;

8), 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; and

2 is an equimolar mixture of K and R;

(4) YYCAR111S2S3111YFDYWG (SEQ ID NO;

9), 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; and

2 is an equimolar mixture of S and G; and

3 is an equimolar mixture of Y and W;

(5) YYCA2111CSG11CY1YFDYWG (SEQ ID NO;

10), 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; and

2 is an equimolar mixture of K and R;

(6) YYCA211SITIFG11111YFDYWG (SEQ ID NO;

11), 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; and

2 is an equimolar mixture of K and R;

(7) YYCAR111YY2S3344111YFDYWG (SEQ ID NO;

12), 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;

2 is an equimolar mixture of D and S; and

3 is an equimolar mixture of S and G;

(8) YYCAR1111YC2231CY111YFDYWG (SEQ 1D NO;

13), 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;

2 is an equimolar mixture of S and G; and

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

(9) a mixture of any of (1) to (8) set forth above.

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

0.17;

0.02.

3. The focused library according to claim 1, 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.

4. The focused library according to claim 1, wherein 1 in one or all of HC CDR3s (1) through (8) is 0.095 of each of G and Y and 0.048 of each of A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, and W.

5. The focused library according to claim 1, wherein HC CDR3s (1) through (8) are present in the library in the following proportions:

(1) 0.10 (2) 0.14 (3) 0.25 (4) 0.13 (5) 0.13 (6) 0.11 (7) 0.04 and (8) 0.10.

6. The focused library according to claim 1, wherein the HC CDR3s (1) through (8) are present in the library in the following proportions:

(1) 0.02 (2) 0.14 (3) 0.25 (4) 0.14 (5) 0.14 (6) 0.12 (7) 0.08 and (8) 0.11.

7. The focused library of vectors or genetic packages according to claim 1, further comprising a second set of variegated DNA molecules each of which comprises a DNA sequence that encodes an antibody kappa light chain (LC_κ

) comprising a LC_κ

CDR1 region, a LC_κ

CDR2 region, and a LC_κ

CDR3 region, wherein the second set of variegated DNA molecules collectively encode a plurality of;

(a) LC_κ

CDR1 regions selected from the group consisting of;

(1) RASQ<

1>

V<

2>

<

2>

<

3>

LA (SEQ ID NO;

14),

(2) RASQ<

1>

V<

2>

<

2>

<

2>

<

3>

LA (SEQ ID NO;

15);

wherein <

1>

is an equimolar mixture of amino acid residues ADEFGHIKLMNPQRSTVWY;

<

2>

is 0.2 S and 0.044 of each of ADEFGHIKLMNPQRTVWY; and

<

3>

is 0.2Y and 0.044 each of ADEFGHIKLMNPQRTVW and S; and

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

(b) LC_κ

CDR2 regions <

1>

AS<

2>

R<

4>

<

1>

(SEQ ID NO;

102), wherein <

1>

is an equimolar mixture of amino acid residues ADEFGHIKLMNPQRSTVWY;

<

2>

is 0.2 S and 0.044 of each of ADEFGHIKLMNPQRTVWY; and

<

4>

is 0.2 A and) 0.044 each of DEFGHIKLMNPQRSTVWY;

or

(c) 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;

<

3>

is 0.2 Y and 0.044 each of ADEFGHIKLMNPQRTVW;

(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 ADEFGHIKLMNPQRTVWY; and

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

8. The focused library of claim 7, wherein LC_κ

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

0.32.

9. The focused library according to claim 7, wherein the LC_κ

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

0.1;

0.25.

10. The focused library of vectors or genetic packages according to claim 1, further comprising a third set of variegated DNA molecules each of which comprises a DNA sequence that encodes an antibody lambda light chain (LC_λ

) chain comprising a LC_λ

CDR1 region, a LC_λ

CDR2 region, and a LC_λ

CDR3 region, wherein the third set of variegated DNA molecules collectively encodes a plurality of;

(a) 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 ADEFHIKLMNPQRSVWY, <

2>

is 0.27 D, 0.27 N and 0.027 each of AEFGHIKLMPQRSTVWY, and <

3>

is 0.36 Y and 0.036 each of ADEFGHIKLMNPQRSTVW;

(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 ADEFGHIKLMNPQRSTVWY; and

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

(b) 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 AEFGHIKLMPQRSTVWY and <

4>

is an equimolar mixture of amino acid residues ADEFGHIKLMNPQRSTVW, or

(c) 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 AEFGHIKLMPQRSTVWY;

<

4>

is an equimolar mixture of amino acid residues ADEFGHIKLMNPQRSTVW; and

<

5>

is 0.36 S and 0.0355 each of ADEFGHIKLMNPQRTVWY;

(2) <

5>

SY<

1>

<

5>

S<

5>

<

1>

<

4>

V (SEQ ID NO;

19), wherein <

1>

is an equimolar mixture of ADEFGHIKLMNPQRSTVWY; and

<

4> and

<

5>

are as defined in (1) above; and

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

11. The focused library according to claim 10, where LC_λ

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

0.33.

12. The focused library according to claim 10, wherein LC_λ

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

13. The focused library according to claim 1, wherein the first set of variegated DNA molecules collectively encode a plurality of HC CDR1s set forth in (a), HC CDR2s set forth in (b), and HC CDR3s set forth in (c).

14. The focused library according to claim 7, wherein the second set of variegated DNA molecules collectively encode a plurality of LC_κ

CDR1s set forth in (a), LC_κ

CDR2s set forth in (b), and LC, CDR3s set forth in (c).

15. The focused library according to claim 10, wherein the third set of variegated DNA molecules collectively encode a plurality of LC_λ

CDR1s set forth in (a), LC_λ

CDR2s set forth in (b), and LC_λ

CDR3s set forth in (c).

16. The library of vectors or genetic packages according to claim 1, wherein the library is a library of vectors.

17. The library of vectors or genetic packages according to claim 16, wherein the vectors are yeast vectors.

18. The library of vectors or genetic packages according to claim 1, wherein the library is a library of genetic packages.

19. The library of vectors or genetic packages according to claim 18, wherein the genetic packages are cells, spores, or viral particles.

20. The library of vectors or genetic packages according to claim 19, wherein the genetic packages are phage particles.

21. The library of vectors or genetic packages according to claim 19, wherein the genetic packages are yeast cells.

22. The library of vectors or genetic packages according to claim 21, wherein the yeast cells display the antibody heavy chains encoded by the first set of variegated DNA molecules.