IP Library › Granted Patent US 12,385,162

Granted Patent B2

US 12,385,162 · App. 18/153,070 · Granted Aug 12, 2025

Dynamic human heavy chain antibody libraries

Inventors: Peter Peizhi Luo (Lansdale, PA); Yan Li (Jiangsu, CN); Fangyong Du (Jiangsu, CN)

Assignee: Adagene Inc.

C40B40/08 C07K16/00 C07K2317/51 C07K2317/56 C07K2317/565

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Quick Facts

Patent No.: US 12,385,162
App. No.: 18/153,070
Granted: Aug 12, 2025
Kind: B2

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Abstract

Provided herein are libraries containing polynucleotides, where one of the polynucleotides encodes an antibody heavy chain with specific hypervariable regions HVR-H1 and HVR-H2. Further provided herein are libraries containing polynucleotides encoding a plurality of unique antibodies, wherein each antibody comprises a heavy chain variable region and a light chain variable region. Also provided are antibodies, polypeptide libraries, vector libraries, cells, non-human animals, antibody heavy chains, methods of making an antibody library, kits, and methods of generating a bispecific antibody related thereto.

Claims (166)

1. A non-human animal comprising polynucleotides that encode antibody heavy chain variable domains (V H s), wherein each of the V H s comprises a HVR-H1, a HVR-H2 and a HVR-H3, and wherein at least one V H comprises

an HVR-H1 that comprises an amino acid sequence according to a formula selected from the group consisting of:

(Formula I)

(SEQ ID NO: 198)

X 1 TFX 2 X 3 YX 4 IHWV,

wherein X 1 is F or Y, X 2 is S or T, X 3 is D,

G, N, or S, and X 4 is A, G, or W;

(Formula II)

(SEQ ID NO: 199)

YSIX 1 SGX 2 X 3 WX 4 WI, wherein X 1 is S or T, X 2 is H or

Y, X 3 is H or Y, and X 4 is A, D, G, N, S, or T;

and

(Formula III)

(SEQ ID NO: 200)

FSLSTX 1 GVX 2 VX 3 WI, wherein X 1 is G or

S, X 2 is A or G, and X 3 is A, G, S, or T;

and

an HVR-H2 that comprises an amino acid sequence according to a formula selected from the group consisting of:

(Formula IV)

(SEQ ID NO: 201)

LAX 1 IX 2 WX 3 X 4 DKX 5 YSX 6 SLKSRL, wherein X 1 is

L or R, X 2 is D or Y, X 3 is A, D, S, or Y, X 4

is D or G, X 5 is R, S, or Y, and X 6 is P or T;

(Formula V)

(SEQ ID NO: 202)

IGX 1 IX 2 X 3 SGSTYYSPSLKSRV, wherein X 1

is A, D, E, S, or Y, X 2 is S or Y, and X 3 is

H or Y;

(Formula VI)

(SEQ ID NO: 203)

IGX 1 IYX 2 SGX 3 TX 4 YNPSLKSRV, wherein X 1 is

D, E, R, S, or Y, X 2 is Hor Y, X 3 is N or S,

and X 4 is N or Y;

(Formula VII)

(SEQ ID NO: 204)

VSXIISGX 2 GX 3 X 4 TYYADSVKGRF, wherein X 1 is

A, G, S, V, or Y, X 2 is A, D, S, or Y, X 3 is

D, G, or S, and X 4 is S or T;

(Formula VIII)

(SEQ ID NO: 205)

IGX 1 INPNX 2 GX 3 TX 4 YAQKFQGRV, wherein X 1 is

I, R, or W, X 2 is For R, X 3 is D, G, or S,

and X 4 is K or N;

(Formula IX)

(SEQ ID NO: 206)

IGX 1 IX 2 PSX 3 GX 4 TXYAQKFQGRV, wherein X 1 is

I, R, or W, X 2 is S or Y, X 3 is G or S, X 4 is

D, G, or S, and X 5 is K or N;

and

(Formula X)

(SEQ ID NO: 207)

VGRIX 1 SKX 2 X 3 GX 4 TTX 5 YAAX 6 VKGRF, wherein X 1 is

K or R, X 2 is A or T, X 3 is D or Y, X 4 is G or

Y, X 5 is D or E, and X 6 is P or S.

2. The non-human animal of claim 1 , wherein at least two, at least three, at least four, at least five or at least ten of the V H s comprise,

an HVR-H1 that comprises an amino acid sequence according to a formula selected from the group consisting of:

(Formula I)

(SEQ ID NO: 198)

X 1 TFX 2 X 3 YX 4 IHWV, wherein X 1 is F or Y,

X 2 is S or T, X 3 is D, G, N, or S, and X 4 is A,

G, or W;

(Formula II)

(SEQ ID NO: 199)

YSIX 1 SGX 2 X 3 WX 4 WI, wherein X 1 is S or T,

X 2 is H or Y, X 3 is Hor Y, and X 4 is A, D, G, N,

S, or T;

and

(Formula III)

(SEQ ID NO: 200)

FSLSTX 1 GVX 2 VX 3 WI, wherein X 1 is G or S,

X 2 is A or G, and X 3 is A, G, S, or T;

and

an HVR-H2 that comprises an amino acid sequence according to a formula selected from the group consisting of:

(Formula IV)

(SEQ ID NO: 201)

LAX 1 IX 2 WX 3 X 4 DKX 5 YSX 6 SLKSRL, wherein X 1 is L or R,

X 2 is D or Y, X 3 is A, D, S, or Y, X 4 is D or G,

X 5 is R, S, or Y, and X 6 is P or T;

(Formula V)

(SEQ ID NO: 202)

IGX 1 IX 2 X 3 SGSTYYSPSLKSRV, wherein X 1 is A, D, E,

S, or Y, X 2 is S or Y, and X 3 is H or Y;

(Formula VI)

(SEQ ID NO: 203)

IGX 1 IYX 2 SGX 3 TX 4 YNPSLKSRV, wherein X 1 is D, E, R,

S, or Y, X 2 is H or Y, X 3 is N or S, and X 4 is

N or Y;

(Formula VII)

(SEQ ID NO: 204)

VSX 1 ISGX 2 GX 3 X 4 TYYADSVKGRF, wherein X 1 is A, G, S,

V, or Y, X 2 is A, D, S, or Y, X 3 is D, G, or S,

and X 4 is S or T;

(Formula VIII)

(SEQ ID NO: 205)

IGX 1 INPNX 2 GX 3 TX 4 YAQKFQGRV, wherein X 1 is I, R, or

W, X 2 is F or R, X 3 is D, G, or S, and X 4 is K

or N;

(Formula IX)

(SEQ ID NO: 206)

IGX 1 IX 2 PSX 3 GX 4 TXsYAQKFQGRV, wherein X 1 is I, R,

or W, X 2 is S or Y, X 3 is G or S, X 4 is D, G, or

S, and X 5 is K or N;

and

(Formula X)

(SEQ ID NO: 207)

VGRIX 1 SKX 2 X 3 GX 4 TTX 5 YAAX 6 VKGRF, wherein X 1 is K or R,

X 2 is A or T, X 3 is D or Y, X 4 is G or Y, X 5 is D

or E, and X 6 is P or S.

3. The non-human animal of claim 1 , wherein the HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of:

(Formula XI)

(SEQ ID NO: 208)

IGX 1 IX 2 X 3 SGSTYYSPSLKSRV, wherein X 1 is A, D, or

E, X 2 is S or Y, and X 3 is H or Y;

(Formula XII)

(SEQ ID NO: 209)

IGXIIYX 2 SGX 3 TX 4 YNPSLKSRV, wherein X 1 is D, E, or

S, X 2 is H or Y, X 3 is N or S, and X 4 is N or Y;

and

(Formula XIII)

(SEQ ID NO: 210)

VGRIX1SKX 2 X 3 GX 4 TTEYAAX 5 VKGRF, wherein X 1 is K or

R, X 2 is A or T, X 3 is D or Y, X 4 is G or Y, X 5

is P or S.

4. The non-human animal of claim 1 , wherein each of the V H s comprises an HVR-H1 that comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:1-52 and 137-158.

5. The non-human animal of claim 1 , wherein at least one of the V H s comprises an HVR-H1 that comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:1-52.

6. The non-human animal of claim 1 , wherein each of the V H s comprises an HVR-H2 that comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:53-136 and 159-164.

7. The non-human animal of claim 1 , wherein at least one of the V H s comprises an HVR-H2 that comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:53-136.

8. The non-human animal of claim 1 , wherein the V H s contain fewer than about 6.5*10 4 unique combinations of HVR-H1 and HVR-H2 sequences.

9. The non-human animal of claim 8 , wherein the V H s contain fewer than about 6700 unique combinations of HVR-H1 and HVR-H2 sequences.

10. The non-human animal of claim 9 , wherein the V H s contain about 6660 or contain fewer unique combinations of HVR-H1 and HVR-H2 sequences.

11. The non-human animal of claim 1 , wherein each of the V H s comprises a HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:1-52 and 137-158, and a HVR-H2 of the antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:53-136 and 159-164.

12. The non-human animal of claim 1 , wherein at least one of the V H s comprises a HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:1-52, and a HVR-H2 of the antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:53-136.

13. The non-human animal of claim 1 , wherein the HVR-H1 and HVR-H2 of the at least one V H are selected from the group consisting of:

a HVR-H1 comprising the amino acid sequence of Formula (II) and a HVR-H2 comprising the amino acid sequence of Formula (IX);

a HVR-H1 comprising the amino acid sequence of Formula (II) and a HVR-H2 comprising the amino acid sequence of Formula (VII);

a HVR-H1 comprising the amino acid sequence of Formula (I) and a HVR-H2 comprising the amino acid sequence of Formula (VII);

a HVR-H1 comprising the amino acid sequence of Formula (I) and a HVR-H2 comprising the amino acid sequence of Formula (IX);

a HVR-H1 comprising the amino acid sequence of Formula (II) and a HVR-H2 comprising the amino acid sequence of Formula (IV);

a HVR-H1 comprising the amino acid sequence of Formula (II) and a HVR-H2 comprising the amino acid sequence of Formula (V);

a HVR-H1 comprising the amino acid sequence of Formula (II) and a HVR-H2 comprising the amino acid sequence of Formula (VI);

a HVR-H1 comprising the amino acid sequence of Formula (I) and a HVR-H2 comprising the amino acid sequence of Formula (VI);

a HVR-H1 comprising the amino acid sequence of Formula (III) and a HVR-H2 comprising the amino acid sequence of Formula (VI);

a HVR-H1 comprising the amino acid sequence of Formula (III) and a HVR-H2 comprising the amino acid sequence of Formula (VII);

a HVR-H1 comprising the amino acid sequence of Formula (II) and a HVR-H2 comprising the amino acid sequence of Formula (VIII);

a HVR-H1 comprising the amino acid sequence of Formula (I) and a HVR-H2 comprising the amino acid sequence of Formula (V);

a HVR-H1 comprising the amino acid sequence of Formula (III) and a HVR-H2 comprising the amino acid sequence of Formula (V); and

a HVR-H1 comprising the amino acid sequence of Formula (I) and a HVR-H2 comprising the amino acid sequence of Formula (VIII).

14. The non-human animal of claim 1 , wherein the HVR-H1 and HVR-H2 of the at least one V H are selected from the group consisting of: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:157, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:122; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:138, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:154, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:158, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:161; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:158, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:145, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:128; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:22, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:61; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:31, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:153, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:155, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:67; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:156, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:100; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:51, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:162; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:138, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:123; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:139, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:110; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:8, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:126; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:129; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:31, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:124; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:25, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:130; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:150, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:132; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:158, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:162; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:12, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:82; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:149, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:117; and a HVR-H1 comprising the amino acid sequence of SEQ ID NO:7, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:134.

15. The non-human animal of claim 1 , wherein the at least one V H comprises a HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 223-256.

16. The non-human animal of claim 1 , wherein the at least one V H comprises a FW-H1 comprising the amino acid sequence of SEQ ID NO:165, a FW-H2 comprising the amino acid sequence of SEQ ID NO:166, a FW-H3 comprising the amino acid sequence of SEQ ID NO:167, and/or a FW-H4 comprising the amino acid sequence of SEQ ID NO:168.

17. The non-human animal of claim 1 , wherein the at least one V H comprises a sequence selected from the group consisting of SEQ ID NOs: 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, and 195.

18. The non-human animal of claim 1 , wherein the polynucleotides that encode V H s encode full-length antibody heavy chains.

19. The non-human animal of claim 1 , further comprising polynucleotides that encode antibody light chain variable regions.

20. The non-human animal of claim 19 , wherein at least one light chain variable region comprises a HVR-L1, a HVR-L2 and a HVR-L3, wherein the HVR-L1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 257-264 and/or the HVR-L3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 265-274.

21. The non-human animal of claim 19 , wherein the polynucleotides that encode the light chain variable regions include at least one unique light chain variable region sequence.

22. The non-human animal of claim 19 , wherein the the polynucleotides that encode the light chain variable regions include at least about 280 unique light chain variable region sequences.

23. The non-human animal of claim 19 , wherein the polynucleotides that encode the light chain variable regions include at least about 10 5 unique light chain variable region sequences.

24. The non-human animal of claim 19 , wherein the polynucleotides that encode V H s and the polynucleotides that encode the light chain variable regions together encode a plurality of unique antibodies, wherein the V H s of each antibody of the plurality comprise an identical sequence.

25. The non-human animal of claim 1 , wherein at least one of the HVR-H1 and HVR-H2 of the at least one V H adopts multiple conformations, as assayed by structural determination and/or computational modeling.

26. The non-human animal of claim 1 , wherein at least one of the polynucleotides encoding the V H s is in a vector.

27. The non-human animal of claim 26 , wherein the vector is an expression vector.

28. The non-human animal of claim 26 , wherein the vector is a display vector.

29. The non-human animal of claim 1 , wherein at least one of the polynucleotides encoding the V H s is in a cell.

30. The non-human animal of claim 16 , wherein all of the VHs comprise a FW-H1 comprising the amino acid sequence of SEQ ID NO:165, a FW-H2 comprising the amino acid sequence of SEQ ID NO:166, a FW-H3 comprising the amino acid sequence of SEQ ID NO:167, and/or a FW-H4 comprising the amino acid sequence of SEQ ID NO:168.

31. The non-human animal of claim 26 , wherein each of the polynucleotides encoding the V H s is in a vector.

32. The non-human animal of claim 1 , wherein the non-human animal is a mammal.

Assignments (1)

ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Mar 7, 2023

From: LUO, PETER PEIZHI; LI, YAN; DU, FANGYONG

To: ADAGENE INC.

Reel/Frame 062912/0509 →

Continuity (2)

Division 16640679

Related Publication 20240240358A1 · Jul 18, 2024

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