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JP-7856719-B2 - Dynamic Human Heavy Chain Antibody Library

JP7856719B2JP 7856719 B2JP7856719 B2JP 7856719B2JP-7856719-B2

Inventors

  • ルオ， ピーター ペイチー
  • リー， ヤン
  • トゥー， ファンヨン

Assignees

  • アダジーン インコーポレイテッド

Dates

Publication Date
20260511
Application Date
20240920

Claims (20)

  1. A library containing polynucleotide members, all of which encode an antibody heavy chain variable region consisting of three hypervariable regions (HVR) and four framework (FW) regions arranged in the order (FW-H1)-(HVR-H1)-(FW-H2)-(HVR-H2)-(FW-H3)-(HVR-H3)-(FW-H4), All polynucleotide members encode the antibody heavy chain variable region, including HVR-H1 and HVR-H2 as described below. The aforementioned HVR-H1 is, (Formula I) X 1 TFX 2 X 3 YX 4 IHWV (Sequence ID 198) (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) YSIX 1 SGX 2 X 3 WX 4 WI (Sequence ID 199) (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) FSLSTX 1 GVX 2 VX 3 WI (Sequence ID 200) (wherein X 1 is G or S, X 2 is A or G, and X 3 is A, G, S, or T) It includes an amino acid sequence that follows a formula selected from the group consisting of the following: The aforementioned HVR-H2 is, (Formula IV) LAX 1 IX 2 WX 3 X 4 DKX 5 YSX 6 SLKSRL (Sequence ID 201) (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) IGX 1 IX 2 X 3 SGSTYYSPSLKSRV (Sequence ID 202) (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) IGX 1 IYX 2 SGX 3 TX 4 YNPSLKSRV (Sequence ID 203) (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) VSX 1 ISGX 2 GX 3 X 4 TYYADSVKGRF (Sequence ID 204) (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) IGX 1 INPNX 2 GX 3 TX 4 YAQKFQGRV (Sequence ID 205) (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)IGX 1 IX 2 PSX 3 GX 4 TX 5 YAQKFQGRV (Sequence ID 206) (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)VGRIX 1 SKX 2 X 3 GX 4 TTX 5 YAAX 6 VKGRF (Sequence ID 207) (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) It includes an amino acid sequence that follows a formula selected from the group consisting of the following: The heavy chain variable region includes FW-H1 containing the amino acid sequence of SEQ ID NO: 165, FW-H2 containing the amino acid sequence of SEQ ID NO: 166, FW-H3 containing the amino acid sequence of SEQ ID NO: 167, and FW-H4 containing the amino acid sequence of SEQ ID NO: 168. The library further comprises at least one polynucleotide encoding the antibody light chain variable region. Library.
  2. The library according to claim 1, wherein the polynucleotide comprises a unique combination of HVR-H1 and HVR-H2 sequences less than 6.5 * 10⁴ .
  3. a. The library according to claim 2, wherein the polynucleotide comprises a unique combination of HVR-H1 sequences and HVR-H2 sequences of less than 6700, or b. The polynucleotide comprises a unique combination of HVR-H1 sequences and HVR-H2 sequences of 6660 or less.
  4. a. A library according to any one of claims 1 to 3, wherein the heavy chain variable region comprises HVR-H1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 52 and 137 to 158, and/or b. A library according to any one of claims 1 to 3, wherein the heavy chain variable region comprises HVR-H2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 136 and 159 to 164.
  5. HVR-H1 and HVR-H2 are, HVR-H1 containing the amino acid sequence of formula (II) and HVR-H2 containing the amino acid sequence of formula (IX), HVR-H1 containing the amino acid sequence of formula (II) and HVR-H2 containing the amino acid sequence of formula (VII), HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (VII), HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (IX), HVR-H1 containing the amino acid sequence of formula (II) and HVR-H2 containing the amino acid sequence of formula (IV), HVR-H1 containing the amino acid sequence of formula (II) and HVR-H2 containing the amino acid sequence of formula (V), HVR-H1 containing the amino acid sequence of formula (II) and HVR-H2 containing the amino acid sequence of formula (VI), formula (I HVR-H1 containing the amino acid sequence of formula (VI) and HVR-H2 containing the amino acid sequence of formula (VI), HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (VI), HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (VII), HVR-H1 containing the amino acid sequence of formula (II) and HVR-H2 containing the amino acid sequence of formula (VIII), HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (V), and HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (VIII), A library according to claim 1, selected from the group consisting of the following.
  6. HVR-H1 and HVR-H2 are, HVR-H1 containing the amino acid sequence of SEQ ID NO: 157 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 63, HVR-H1 containing the amino acid sequence of SEQ ID NO: 1 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 122, HVR-H1 containing the amino acid sequence of SEQ ID NO: 138 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 63, HVR-H1 containing the amino acid sequence of SEQ ID NO: 154 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 63, HVR-H1 containing the amino acid sequence of SEQ ID NO: 158 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 161, HVR-H1 containing the amino acid sequence of SEQ ID NO: 158 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 63, HVR-H1 containing the amino acid sequence of sequence number 145 and HVR-H2 containing the amino acid sequence of sequence number 128, HVR-H1 containing the amino acid sequence of sequence number 22 and HVR-H2 containing the amino acid sequence of sequence number 61, HVR-H1 containing the amino acid sequence of sequence number 31 and HVR-H2 containing the amino acid sequence of sequence number 63, HVR-H1 containing the amino acid sequence of sequence number 153 and HVR-H2 containing the amino acid sequence of sequence number 63, HVR-H1 containing the amino acid sequence of sequence number 155 and HVR-H2 containing the amino acid sequence of sequence number 67, HVR-H1 containing the amino acid sequence of sequence number 156 and HVR-H2 containing the amino acid sequence of sequence number 100, sequence HVR-H1 containing the amino acid sequence of number 51 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 162, HVR-H1 containing the amino acid sequence of SEQ ID NO: 138 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 123, HVR-H1 containing the amino acid sequence of SEQ ID NO: 139 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 110, HVR-H1 containing the amino acid sequence of SEQ ID NO: 8 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 126, HVR-H1 containing the amino acid sequence of SEQ ID NO: 13 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 129, HVR-H1 containing the amino acid sequence of SEQ ID NO: 31 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 124, sequence HVR-H1 containing the amino acid sequence of number 25 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 130, HVR-H1 containing the amino acid sequence of SEQ ID NO: 150 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 132, HVR-H1 containing the amino acid sequence of SEQ ID NO: 158 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 162, HVR-H1 containing the amino acid sequence of SEQ ID NO: 12 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 82, HVR-H1 containing the amino acid sequence of SEQ ID NO: 149 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 117, and HVR-H1 containing the amino acid sequence of SEQ ID NO: 7 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 134 A library according to claim 1, selected from the group consisting of the following.
  7. The library according to any one of claims 1 to 6, wherein the heavy chain variable region comprises HVR-H3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 223 to 256.
  8. The library according to claim 1, wherein the heavy chain variable region includes sequences selected from the group consisting of SEQ ID NOs: 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, and 195.
  9. The library according to any one of claims 1 to 8, wherein the polynucleotides encode full-length antibody heavy chains.
  10. a. The antibody light chain variable region comprises HVR-L1, HVR-L2, and HVR-L3, wherein HVR-L1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 257 to 264, and/or HVR-L3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 265 to 274; and/or b. The polynucleotide encoding the antibody light chain variable region comprises at least one unique sequence, at least 280 unique sequences, or at least 10⁵ unique sequences. The library according to any one of claims 1 to 9.
  11. When at least one of the HVR-H1 and HVR-H2 in the antibody heavy chain variable region is evaluated by structural determination and/or computer modeling, it takes on multiple three-dimensional structures. The library according to any one of claims 1 to 10.
  12. The library according to any one of claims 1 to 11, wherein at least one polynucleotide encoding an antibody heavy chain variable region is present in the vector.
  13. The library according to claim 12, wherein the vector is an expression vector or a display vector.
  14. The library according to any one of claims 1 to 13, wherein at least one polynucleotide encoding the antibody heavy chain variable region is present in the cell.
  15. The library according to claim 14, wherein the cells are bacterial, yeast, or mammalian cells.
  16. A library according to any one of claims 1 to 15, comprising polynucleotides encoding multiple unique antibodies.
  17. The library according to claim 16, wherein the heavy chain variable regions of each of the multiple antibodies contain the same sequence.
  18. A kit comprising the library described in any one of claims 1 to 17.
  19. A method for preparing a library, comprising preparing and assembling the polynucleotide sequences of the library according to any one of claims 1 to 17.
  20. A non-human animal comprising the library described in any one of claims 1 to 17.

Description

This disclosure relates to a library containing synthetic polynucleotides encoding antibody heavy chains (e.g., heavy chains of dynamic human antibodies), as well as antibody heavy chains, antibodies, cells, animals, methods, and kits relating thereto. Monoclonal antibodies have become extremely useful in a wide range of fields, including biological research, medical diagnostics, and pharmaceuticals. Their potential diversity in binding specificity allows for the creation of antibodies with beneficial specificity and capabilities. However, this diversity makes screening a vast number of antibodies to identify one or more with the desired properties a difficult and time-consuming process. One method for identifying a target antibody is to screen antibody libraries, such as libraries of cloned B cell sequences, phage display libraries, and yeast display libraries. These libraries allow for screening a large number of antibodies representing numerous unique antibody sequences, enabling the identification of antibodies with desired specific properties, such as binding to a particular target, binding affinity, and selectivity. However, current libraries have certain limitations. Libraries derived from biological sources, such as the human B cell repertoire, are limited to antibody sequences that can be cloned from that source. Synthetic libraries, while potentially containing non-natural sequences compared to biologically obtained libraries, are also limited by the amount of antibodies that can be synthesized within a given timeframe. Furthermore, very large libraries require more time and a more comprehensive screening approach; otherwise, only a small fraction of the library can be effectively screened for the target antibody. Therefore, to design and construct potentially highly functionally relevant dynamic antibodies, there is a need to develop dynamic antibody libraries containing a sufficient set of dynamic units with clearly defined, developable sequence profiles. Such libraries would significantly improve the efficiency of antibody screening, not only in terms of the diversity of antibody-binding sites on the antibodies within the library, but also in terms of novel epitopes and/or structural epitopes on a given antigen. Furthermore, such libraries would increase the likelihood of identifying specific antibodies of interest with high affinity and developability profiles. All references cited herein, including patent applications, patent publications, non-patent literature, and UniProtKB/Swiss-Prot accession numbers, are incorporated herein by reference in their entirety, as is indicated in each individual reference where such reference is specifically and individually referred to. To satisfy the above and other needs, disclosed herein are antibody sequences such as heavy chain hypervariable regions (HVRs) and heavy chain variable regions (e.g., VH regions) that enable dynamic human antibodies. These sequences are designed to produce antibodies with highly flexible HVR sequence loops that can bind to targets with high ability and/or recognize multiple useful epitopes and/or cross-react with epitopes shared across different species with low sequence identity (approximately 60% or less). Advantageously, these antibody sequences enable the creation of libraries that contain a large number of useful antibodies in smaller libraries and/or have much greater diversity for a given library size. Such libraries can be used to identify novel antibodies of interest that are specific to a wide range of targets, or in some cases, novel antibodies that cross-react with multiple targets of interest. Furthermore, novel concepts and methodologies for designing and constructing dynamic antibody libraries that capture the flexibility of a wide range of three-dimensional structures of antibody binding sites in physically compact libraries using newly identified dynamic units are introduced and implemented herein. Furthermore, results using such antibodies (described below) highlight the possibility of identifying antibodies from these libraries that target three-dimensional structural epitopes and/or evolutionarily conserved sites on a given heterologous antigen with low sequence identity (e.g., less than 60% to 70%). Accordingly, in one embodiment, provided herein are one or more HVR-H1 amino acid sequences and/or one or more polynucleotides (e.g., synthetic polynucleotides) encoding said amino acid sequences, wherein the HVR-H1 is given by formula (I): X1TFX2X3YX4IHWV (SEQ ID NO: 198) (wherein X1 is F or Y, X2 is S or T, X3 is D, G, N, or S, and X4 is A, G, or W); The amino acid sequence includes an amino acid sequence selected from the group consisting of formula (II): YSIX1SGX2X3WX4WI (SEQ ID NO: 199) (wherein X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N, S, or T); and formula (III): FSLSTX1GVX2VX3WI (SEQ ID NO: 200) (wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T). In some embodiments, HVR