Antibodies that bind to leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2) and uses thereof

Abstract

Antibodies (anti-LILRB2 antibodies) that bind to human leukocyte immunoglobulin-like receptor 2 (LILRB2) and uses thereof for modulating anti-tumor immune responses. Also provided herein are nucleic acids encoding an anti-LILRB2 antibody, host cells comprising the encoding nucleic acids, and methods for producing the anti-LILRB2 antibody.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of the filing date of U.S. Provisional Application No. 63 / 377,416, filed September 28, 2022, the entire contents of which are incorporated herein by reference.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy was created on September 27, 2023, is named 112124-0025-70003US00_SEQ.XML, and is 397,137 bytes in size. Background Art

[0005] The presence of tumor-associated myeloid cells is often associated with a poor prognosis in solid tumors. Tumor-associated myeloid cells maintain an immunosuppressive microenvironment within the tumor and promote immune evasion. There are two major groups of suppressive myeloid cells within the tumor microenvironment: tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs).

[0006] Macrophages are highly plastic immune cells that dynamically integrate microenvironmental signals to shape their functional phenotype. There are two major macrophage subsets: M1 and M2, representing classically activated macrophages and alternatively activated macrophages, respectively. M2 macrophages play an immunosuppressive role in the tumor microenvironment, while M1 macrophages act as immunostimulatory and cytotoxic effector cells targeting tumor cells.

[0007] Most TAMs and MDSCs are very similar to M2 macrophages, which play a suppressive role in the tumor microenvironment. Converting M2 macrophages to the M1 subtype is a promising approach for the treatment of solid tumors. Summary of the Invention

[0008] The present disclosure is based, at least in part, on the development of antibodies that bind to leukocyte immunoglobulin-like receptor 2 (LILRB2) (anti-LILRB2 antibodies). Such antibodies have high binding specificity for human LILRB2 and are capable of blocking HLA-G and / or HLA-A2 binding to LILRB2 receptors on the cell surface, thereby reducing tumor immune escape. Therefore, the anti-LILRB2 antibodies disclosed herein are expected to be beneficial for tumor treatment.

[0009] In some cases, provided herein are antibodies that bind to human leukocyte immunoglobulin-like receptor 2 (LILRB2) (anti-LILRB2 antibodies). Such anti-LILRB2 antibodies include:

[0010] (a) Heavy chain variable region (VH ), which contains

[0011] (ai) a heavy chain CDR1 comprising GX1SITSGYX2WX3 (SEQ ID NO: 192), wherein X1 is Y or G, X2 is Y, S, A or H, and X3 is N, S or W;

[0012] (aii) a heavy chain CDR2 comprising X4ISYDGNX5X6 (SEQ ID NO: 194), wherein X4 is S, T, or Y, X5 is A, I, K, L, M, N, P, S, T, V, or W, and X6 is H, L, N, S, or W; optionally, wherein the heavy chain CDR2 comprises X4ISYDGNX5X6YNPSLKN (SEQ ID NO: 416);

[0013] (aiii) heavy chain CDR3 comprising EEX7TMX8TTX9WFX 10 Y (SEQ ID NO: 196), wherein X7 is A or S, X8 is F or I, X9 is A or G, and X 10 is A, G, Q, or S; and

[0014] (b) a light chain variable region (VL), comprising

[0015] (bi) light chain CDR1 comprising X 11 X 12 SEX 13 IX 14 SNX 15 A (SEQ ID NO: 199), wherein X 11 is E, G, L, N, Q, R, S, T or V, X 12 Is A or G, X 13 is K, N, T or V, X 14 is F or Y, and X 15 is L, N or Q;

[0016] (bii) Light chain CDR2, which contains GATX 16 X 17 X 18 X 19 (SEQ ID NO:201), wherein X16 is E, N, W or Y, X17 is F, H, L, P or R, X18 is A, N, R or S, and X19 is A, G, K, L, R, S or V; and

[0017] (biii) light chain CDR3 comprising QX 20 FWf 21 PYX 22 (SEQ ID NO: 203), wherein X20 It is H or Q, X 21 is L, S, T, or Y, and X 22 It is I, M, R, T, Y or V.

[0018] In some embodiments, the X variables in the heavy chain CDRs can be: X1 is Y, X2 is Y, X3 is N, X4 is Y, X5 is I, X6 is N, X7 is S, X8 is I, X9 is A, and / or X 10 It’s A.

[0019] Alternatively or additionally, the X variable in the light chain CDR may be: X 11 It's R, X 12 It's A, X 13 It is N, X 14 It's Y, X 15 It's L, X 16 It is N, X 17 It's L, X 18 It's A, X 19 It's S, X 20 It is H, X 21 It's Y, X 22 is T, or a combination thereof.

[0020] In some instances, the heavy chain CDR3 is EESTMITTAWFAY (SEQ ID NO: 11); and / or the light chain CDR3 is QHFWDYPYT (SEQ ID NO: 247).

[0021] In some examples, the anti-LILRB2 antibody can comprise the same heavy chain CDR1, CDR2, and CDR3 as the antibodies listed in Table 2, and the same light chain CDR1, CDR2, and CDR3.

[0022] In some embodiments, the anti-LILRB2 antibody comprises the same heavy chain CDRs as clone 2E1_FC11 (i.e., heavy chain CDR1, CDR2, and CDR3 as set forth in SEQ ID NOs: 9, 243, and 11, respectively), and the same light chain CDRs as clone 2E1_FC11 (i.e., light chain CDR1, CDR2, and CDR3 as set forth in SEQ ID NOs: 13, 14, and 247, respectively). In other embodiments, the heavy chain CDR1, CDR2, and CDR3 of the anti-LILRB2 antibody comprise up to five amino acid residue variations relative to the heavy chain CDRs of clone 2E1_FC11 (SEQ IDs: 9, 243, and 11), and / or the light chain CDR1, CDR2, and CDR3 of the anti-LILRB2 antibody comprise up to five amino acid variations relative to the light chain CDRs of clone 2E1_FC11 (SEQ IDs: 13, 14, and 247).

[0023] In some embodiments, any anti-LILRB2 antibody provided herein can be a humanized antibody. H Contains (i)V H Framework 1 (FR1), which is represented by EVQLVESGGGLVQPGGSLRLSCAZ1S (SEQ ID NO: 191), wherein Z1 is A or V, (ii) V H Framework 2 (FR2), which is represented by WZ2RQAPGKGLEWVA (SEQ ID NO: 193), wherein Z2 is I or V, and / or (iii) V H Framework 3 (FR3) which is represented by RFTISRDZ3SKNTLZ4LQMNSLRAEDTAVYYCZ5R (SEQ ID NO: 195), wherein Z3 is A, D or T, Z4 is F, L or V, and Z5 is A or V, and / or (iv) V H Framework 4 (FR4), which is represented by WGQGTLVTVSS (SEQ ID NO: 197). Alternatively or in addition, V L Contains (i)V L FR1, which is shown to comprise DIQZ6TQSPSSLSASVGDRVTITC (SEQ ID NO: 198), wherein Z6 is L or M, (ii) V L FR2, which is represented by WYQQKPGKAPKLLIY (SEQ ID NO: 200), (iii) V L FR3, which is represented by GVPSRFSGSGSGTDZ7TLTISSLQPEDFATYYC (SEQ ID NO: 202), wherein Z7 is F or Y, and / or (iv) V L FR4, which is represented by FGQGTKVEIK (SEQ ID NO: 204).

[0024] Exemplary anti-LILRB2 antibodies provided herein are listed in Table 2, each of which is within the scope of the present disclosure.

[0025] In some examples, the V of the anti-LILRB2 antibody is H Contains the same V as clone 2E1_FC11 H (SEQ ID NO: 244) is at least 85% identical to an amino acid sequence; and / or wherein V L Contains the same V as clone 2E1_FC11 L (SEQ ID NO: 248) is at least 85% identical to an amino acid sequence. In a specific example, V of the anti-LILRB2 antibody is H Contains the same V as 2E1_FC11H (comprising the amino acid sequence of SEQ ID NO: 244); and / or wherein the V L Contains the same V as 2E1_FC11 L (comprising the amino acid sequence SEQ ID NO: 248).

[0026] Any of the anti-LILRB2 antibodies disclosed herein may be a full-length antibody or an antigen-binding fragment thereof.

[0027] In other aspects, provided herein are nucleic acids or collections of nucleic acids comprising a nucleotide sequence encoding any of the anti-LILRB2 antibodies provided herein (e.g., clone 2E1_FC11 or a functional variant thereof). In some embodiments, the nucleic acid or collection of nucleic acids can be a vector or a collection of vectors comprising a nucleotide sequence encoding an anti-LILRB2 antibody. In some examples, the vector can be an expression vector.

[0028] In addition, provided herein are host cells or host cell collections comprising nucleic acids encoding any of the anti-LILRB2 antibodies provided herein (e.g., clone 2E1_FC11 or a functional variant thereof). Such host cells can be mammalian cells, yeast cells, or bacterial cells.

[0029] Additionally, the disclosure features pharmaceutical compositions comprising (a) an anti-LILRB2 antibody as disclosed herein (e.g., clone 2E1_FC11 or a functional variant thereof), or a nucleic acid or collection of nucleic acids encoding the antibody, and a pharmaceutically acceptable carrier.

[0030] In other aspects, the disclosure features methods for modulating an immune response, comprising administering to a subject in need thereof an effective amount of an anti-LILRB2 antibody disclosed herein (e.g., clone 2E1_FC11 or a functional variant thereof), a nucleic acid or collection of nucleic acids encoding the antibody, or a pharmaceutical composition comprising the antibody or nucleic acid encoding the antibody. In some cases, the subject is a human patient having or suspected of having cancer.

[0031] In addition, the present disclosure provides a method for preparing an anti-LILRB2 antibody, comprising: culturing a host cell or a collection of host cells carrying a coding sequence for any anti-LILRB2 antibody disclosed herein (e.g., clone 2E1_FC11 or a functional variant thereof) under conditions permitting antibody expression, and harvesting the antibodies produced thereby.

[0032] Additional embodiments are provided below, all of which are within the scope of the present disclosure.

[0033] Embodiment 1: An anti-LILRB2 antibody comprising: (a) a heavy chain variable region (V H), which (i) comprises the same heavy chain complementary determining region 1 (CDR1), complementary determining region 2 (CDR2) and complementary determining region 3 (CDR3) as the reference antibody; or (ii) comprises up to five amino acid variations in total in CDR1, CDR2 and CDR3 compared to the reference antibody; and (b) a light chain variable region (V L ), which (i) comprises the same light chain CDR1, CDR2 and CDR3 as the reference antibody; or (ii) comprises up to five amino acid variations in total in CDR1, CDR2 and CDR3 compared to the reference antibody. Reference antibodies include those listed in Table 1 below, such as 11B12, 9B6, 14F1, 10B11, 11D9, 4A9, 10D7, 13H5, 15A6, 6E8, 6H8, 13C4, 13D12, 2C1, 3C12, 7C12, 7G4, 8A4, 9H6, 12D8, 13C5, 15E2, 2F1, 3B5, 14C11, SLL-1, SLL-2, SLL-3, SLL-3, SLL-4 or SLL-5. In some cases, the anti-LILRB2 antibody comprises V H and V L , the V H comprising the same heavy chain CDRs as the reference antibody, the V L Contains the same light chain CDRs as the reference antibody.

[0034] Embodiment 2: The anti-LILRB2 antibody of embodiment 1 is one of those listed in Table 1.

[0035] Embodiment 3: The anti-LILRB2 antibody of embodiment 1 comprises a V sequence comprising a total of up to five amino acid variations in heavy chain CDR1, CDR2, and CDR3 relative to the reference antibody 11B12. H , and / or V comprising a total of up to five amino acid variations in the light chain CDR1, CDR2, and CDR3 relative to the reference antibody 11B12 L .

[0036] Embodiment 4: The anti-LILRB2 antibody of embodiment 3, comprising:

[0037] (ai) a heavy chain CDR1 comprising GX1SITSGYX2WX3 (SEQ ID NO: 192), wherein X1 is Y or G, X2 is Y, S, A or H, and X3 is N, S or W;

[0038] (aii) a heavy chain CDR2 comprising X4ISYDGNX5X6 (SEQ ID NO: 194), wherein X4 is S, T, or Y, X5 is A, I, K, L, M, N, P, S, T, V, or W, and X6 is H, L, N, S, or W; optionally, wherein the heavy chain CDR2 comprises X4ISYDGNX5X6YNPSLKN (SEQ ID NO: 416);

[0039] (aiii) heavy chain CDR3 comprising EEX7TMX8TTX9WFX 10 Y (SEQ ID NO: 196), wherein X7 is A or S, X8 is F or I, X9 is A or G, and X10 is A, G, Q or S;

[0040] (bi) light chain CDR1 comprising X 11 X 12 SEX 13 IX 14 SNX 15 A (SEQ ID NO: 199), wherein X 11 is E, G, L, N, Q, R, S, T or V, X 12 Is A or G, X 13 is K, N, T or V, X 14 is F or Y, and X 15 is L, N or Q;

[0041] (bii) Light chain CDR2, which contains GATX 16 X 17 X 18 X 19 (SEQ ID NO:201), wherein X16 is E, N, W or Y, X17 is F, H, L, P or R, X18 is A, N, R or S, and X19 is A, G, K, L, R, S or V; and

[0042] (biii) light chain CDR3 comprising QX 20 FWf 21 PYX 22 (SEQ ID NO: 203), wherein X20 is H or Q, X21 is L, S, T or Y, and X22 is I, M, R, T, Y or V.

[0043] Embodiment 5: The anti-LILRB2 antibody of any one of Embodiments 1 to 4 is a humanized antibody.

[0044] Embodiment 6: The humanized antibody of embodiment 5 may comprise V H and V L , the V H Contains VH Frame 1 (FR1), V H Frame 2 (FR2), V H Framework 3 (FR3) and V H Frame 4 (FR4), and the V L Contains V L FR1, V L FR2, V L FR3 and V L FR4, each of which can be shown as follows:

[0045] V H FR1 comprises EVQLVESGGGLVQPGGSLRLSCAZ1S (SEQ ID NO: 191), wherein Z1 is A or V,

[0046] V H FR2 comprises WZ2RQAPGKGLEWVA (SEQ ID NO: 193), wherein Z2 is I or V,

[0047] V H FR3 comprises RFTISRDZ3SKNTLZ4LQMNSLRAEDTAVYYCZ5R (SEQ ID NO: 195), wherein Z3 is A, D, or T, Z4 is F, L, or V, and Z5 is A or V, and

[0048] V H FR4 comprises WGQGTLVTVSS (SEQ ID NO: 197); and wherein:

[0049] V L FR1 comprises DIQZ6TQSPSSLSASVGDRVTITC (SEQ ID NO: 198), wherein Z6 is L or M,

[0050] V L FR2 comprises WYQQKPGKAPKLLIY (SEQ ID NO: 200),

[0051] V L FR3 comprises GVPSRFSGSGSGTDZ7TLTISSLQPEDFATYYC (SEQ ID NO: 202), wherein Z7 is F or Y, and

[0052] V L FR4 comprises FGQGTKVEIK (SEQ ID NO: 204).

[0053] Embodiment 7: The anti-LILRB2 antibody of embodiment 3 comprises the same heavy chain CDR1, CDR2 and CDR3 as the antibodies listed in Table 2, and the same light chain CDR1, CDR2 and CDR3.

[0054] Embodiment 8: The anti-LILRB2 antibody of Embodiment 7 may be any one of those listed in Table 2.

[0055] Embodiment 9: The anti-LILRB2 antibody of embodiment 1 or 2 disclosed herein may comprise the same heavy chain CDRs and / or the same light chain CDRs as the reference antibody 9B6.

[0056] Embodiment 10: The anti-LILRB2 antibody of embodiment 8 may comprise the same heavy chain CDR1, CDR2 and CDR3 as the antibodies listed in Table 3, and the same light chain CDR1, CDR2 and CDR3.

[0057] Embodiment 11: The anti-LILRB2 antibody of any one of Embodiments 9 to 10 is a humanized antibody.

[0058] Embodiment 12: The humanized antibody of embodiment 11 may comprise V H and V L , the V H Contains V H Frame 1 (FR1), V H Frame 2 (FR2), V H Framework 3 (FR3) and V H Frame 4 (FR4), and the V L Contains V L FR1, V L FR2, V L FR3 and V L FR4, each of which can be shown as follows:

[0059] V H FR1 comprises EVQLVESGGZ1LVQPGGSLRLSCAZ2S (SEQ ID NO: 354), wherein Z1 is D or G, and Z2 is A, T or V,

[0060] V H FR2 comprises WZ3RQAPGKGLEWVA (SEQ ID NO: 193), wherein Z3 is I or V,

[0061] V HFR3 comprises RZ4TISRDZ5SKNTLYLQMNSLRAEDTAVYYCZ6R (SEQ ID NO: 355), wherein Z4 is A, F, I, L, S, or V, Z5 is D or N, and Z6 is A or S, and

[0062] V H FR4 comprises WGQGTLVTVSS (SEQ ID NO: 197); and wherein:

[0063] V L FR1 comprises DIQZ7TQSPSSLSASVGDRVTITC (SEQ ID NO: 198), wherein Z7 is L or M,

[0064] V L FR2 comprises WZ8QQKPGKAPKLLIY (SEQ ID NO: 356), wherein Z8 is F or Y,

[0065] V L FR3 contains GVPSRFSGSGSGTDZ9TLTISSLQPEDZ 10 ATYYC (SEQ ID NO: 357), wherein Z9 is F or Y, and Z 10 is F or L; and

[0066] V L FR4 comprises FGQGTKVEIK (SEQ ID NO: 204).

[0067] Embodiment 13: The anti-LILRB2 antibody of any one of Embodiments 9 to 12 is one of those listed in Table 3.

[0068] Embodiment 14: The anti-LILRB2 antibody of embodiment 1 or embodiment 2 disclosed herein may comprise the same heavy chain CDRs and / or the same light chain CDRs as the reference antibody 14F1.

[0069] Embodiment 15: The anti-LILRB2 antibody of Embodiment 14 may comprise the same heavy chain CDR1, CDR2 and CDR3 as the antibodies listed in Table 4, and the same light chain CDR1, CDR2 and CDR3.

[0070] Embodiment 16: The anti-LILRB2 antibody of embodiment 14 or embodiment 15 is a humanized antibody.

[0071] Embodiment 17: The humanized antibody of embodiment 16 may comprise V H and V L , the V H Contains VH Frame 1 (FR1), V H Frame 2 (FR2), V H Framework 3 (FR3) and V H Frame 4 (FR4), and the V L Contains V L FR1, V L FR2, V L FR3 and V L The VL of FR4 can be shown as follows:

[0072] V H FR1 comprises EVQLVESGGGLVQPGGSLRLSCAZ1S (SEQ ID NO: 379), wherein Z1 is A, I or V,

[0073] V H FR2 comprises WZ2RQAPGKGLEWVA (SEQ ID NO: 193), wherein Z2 is I or V,

[0074] V H FR3 comprises RZ3TISZ4DZ5SKZ6TZ7YLQMNSLRAEDTAVYYCAZ8 (SEQ ID NO: 381), wherein Z3 is A, F, I, T, or V, Z4 is K or R, Z5 is D or N, Z6 is A, L, or V, Z7 is A, L, or V, and Z8 is K or R, and

[0075] V H FR4 comprises WGQGTLVTVSS (SEQ ID NO: 197); and wherein:

[0076] V L FR1 comprises DIQZ9TQSPSSLSASVGDRVTITC (SEQ ID NO: 198), wherein Z9 is L or M,

[0077] V L FR2 comprises WYQQKPGKAPKLLIY (SEQ ID NO: 200),

[0078] V L FR3 contains GVPSRFSGSGSGTDFTLTISSLQPEDZ 10 ATYYC (SEQ ID NO: 383), wherein Z 10 is F or L, and

[0079] V L FR4 comprises FGQGTKVEIK (SEQ ID NO: 204).

[0080] Embodiment 18: The anti-LILRB2 antibody of embodiment 14 is one of those listed in Table 4.

[0081] Embodiment 19: Any of the anti-LILRB2 antibodies of embodiments 1 to 18 disclosed herein may be a full-length antibody.

[0082] Embodiment 20: Any of the anti-LILRB2 antibodies of embodiments 1 to 18 disclosed herein may be an antigen-binding fragment thereof.

[0083] Embodiment 21: A nucleic acid or a collection of nucleic acids comprising a nucleotide sequence encoding any anti-LILRB2 antibody of any one of embodiments 1 to 20 as disclosed herein.

[0084] Embodiment 21: The nucleic acid or collection of nucleic acids of embodiment 21 is a vector or a collection of vectors comprising a nucleotide sequence encoding an anti-LILRB2 antibody.

[0085] Embodiment 22: The vector of Embodiment 21 is an expression vector.

[0086] Embodiment 23: A host cell or a collection of host cells comprising a nucleic acid encoding any anti-LILRB2 antibody of embodiments 1 to 20 as disclosed herein.

[0087] Embodiment 24: The host cell or host cell collection of embodiment 23 is a mammalian cell.

[0088] Embodiment 25: The host cell or host cell collection of embodiment 23 is a yeast cell.

[0089] Embodiment 26: The host cell or host cell collection of embodiment 23 is a bacterial cell.

[0090] Embodiment 27: A method for modulating an immune response, comprising administering to a subject in need thereof an effective amount of an anti-LILRB2 antibody, a nucleic acid encoding the antibody, or a collection of nucleic acids, or a pharmaceutical composition comprising the antibody or encoding nucleic acid, as disclosed herein, according to any one of embodiments 1 to 20.

[0091] Embodiment 28: The subject treated by the method of embodiment 27 is a human patient having or suspected of having cancer.

[0092] Embodiment 29: A method for preparing an anti-LILRB2 antibody, comprising: (a) culturing a host cell or a collection of host cells containing a nucleic acid encoding any of the anti-LILRB2 antibodies of Embodiments 1 to 20 disclosed herein under conditions that permit antibody expression, and (b) harvesting the antibodies thereby produced.

[0093] Also within the scope of the present disclosure are pharmaceutical compositions comprising (a) an anti-LILRB2 antibody as disclosed herein, or a nucleic acid or collection of nucleic acids encoding the antibody, and (b) a pharmaceutically acceptable carrier. Furthermore, provided herein are pharmaceutical compositions for use in treating cancer (e.g., solid tumors) or any of the anti-LILRB2 antibodies disclosed herein for use in the manufacture of a medicament for the treatment of cancer.

[0094] The details of one or more embodiments of the present invention are set forth in the description below. Other features and advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and from the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] The following drawings form part of this specification and are included to further illustrate certain aspects of the present disclosure, which may be better understood by reference to the drawings in combination with the detailed description of specific embodiments presented herein.

[0096] Figure 1A and Figure 1B Included are graphs showing the binding specificity of exemplary anti-LILRB2 antibodies to LILRB isoforms as detected by ELISA. Figure 1A : Binds to LILRB1, LILRB2, and LILRB3 expressed on 293T cells. Figure 1B : Binds to His-tagged LILRB1, LILRB2, and LILRB3.

[0097] Figure 2A and Figure 2B Included are graphs showing the binding specificity of exemplary anti-LILRB2 antibodies to human and cynomolgus monkey LILRB2 as detected by flow cytometry. Figure 2A : Binds to human LILRB1, human LILRB2, human LILRB3, and cynomolgus monkey LILRB2 expressed on 293T cells. Figure 2B : Binds to human LILRB2 expressed in CHO-K1 cells.

[0098] Figures 3A to 3F Included are graphs showing the blocking activity of exemplary anti-LILRB2 antibodies. Figures 3A to 3B : Blocking activity on HLA-A2 binding to CHO-K1-huLILRB2 cells, as shown by MFI values ​​(3A) and percentage of HLA-A2 pentamer binding (3B). Figures 3C to 3D : Blocking activity on HLA-G binding to CHO-K1-huLILRB2 cells, as shown by MFI values ​​(3C) and percentage of HLA-G tetramer binding (3D). Figures 3E to 3F: Blocking activity on ANGPTL2 binding to human LILRB2, as shown by MFI values ​​(3E) and percentage of LILRB2-Fc binding (3F).

[0099] Figure 4A and Figure 4B Included are graphs showing the effects of exemplary anti-LILRB2 antibodies on cytokine secretion by LPS-stimulated PBMCs. Figure 4A : Enhanced TNFα expression. Figure 4B : Inhibited IL-10 expression.

[0100] Figures 5A to 5C Included are graphs showing the binding activity of exemplary humanized anti-LILRB2 antibodies derived from parental clone 11B12 to 293T-LILRB2 cells. Figure 5A : Humanized clones h#2E1, h#2E2, h#2B1 and chimeric 11B12. Figure 5B : Humanized clones h#2E1, h#2D2 and H#2C1. Figure 5C : Humanized clones h#2E1, h#2D4, h#2D1 and h#2G6.

[0101] Figure 6 is a graph showing the binding activity of exemplary humanized anti-LILRB2 antibodies derived from the parental clone 9B6 to 293T-LILRB2 cells.

[0102] 7A to 7C Included are graphs showing the blocking activity of exemplary humanized anti-LILRB2 antibodies on HLA-G binding to 293T-LILRB2 cells. Figure 7A : Clones h#2E1, h#2E2, h#2B1, and h#2D2. Figure 7B : Clones h#2C1, h#2D4, h#2D1, and h#2G6. Figure 7C : Clone h#12a and chimeric 9B6.

[0103] Figure 8 is a graph showing the blocking activity of an exemplary humanized anti-LILRB2 antibody h#2E1 on HLA-A2 binding to 293T-LILRB2 cells.

[0104] Figures 9A to 9C Included are graphs showing the blocking activity of exemplary mature anti-LILRB2 antibodies on HLA-G binding to 293T-LILRB2 cells. Figure 9A : Clones 2E1, 2E1_FC1, 2E1_FG9, 2E1_FD3, 2E1_FC3, 2E1_FF4, 2E1_FF5, and 2E1_FC11. Figure 9B: Clones 2E1_FC11, 2E1_FG8, 2E1_GB5, 2E1_GC6, 2E1_GB11, 2E1_GF1, 2E1_GD6, and 2E1_GH6. Figure 9C : clones h#2E1, 2E1_GC6, 2E1_FG9, and 2E1_FC11.

[0105] Figure 10 is a graph showing the blocking activity of exemplary mature anti-LILRB2 antibodies on HLA-A2 binding to 293T-LILRB2 cells as indicated.

[0106] 11A to 11D Included are graphs showing induction of macrophage M1 polarization in the PBMC / LPS model by humanized anti-LILRB2 antibodies as indicated. Figure 11A : TNFα secretion. Figure 11B :IL-10 secretion. Figure 11C : TNFα secretion in PBMC cultures treated with clone 2E1_FC11. Figure 11B : IL-10 secretion in PBMC cultures treated with clone 2E1_FC11.

[0107] Figure 12 is a graph showing the induction of macrophage M1 polarization by clone 2E1_FC11 in human monocyte-derived macrophages as observed in the HMDM / LPS model.

[0108] 13A to 13D Included are graphs showing activation of CD8+ T cells by clone 2E1_FC11 compared to PD1 inhibitors. Figure 13A : IFNγ secretion of immune cells treated with clone 2E1_FC11 compared to the PD1 inhibitor A105 (single domain anti-PDL1 antibody). Figure 13B : GM-CSF secretion by immune cells treated with clone 2E1_FC11 compared with the PD1 inhibitor A105. Figure 13C : IFNγ secretion by immune cells treated with clone 2E1_FC11 compared with the PD1 inhibitor nivolumab. Figure 13D : GM-CSF secretion by immune cells treated with clone 2E1_FC11 compared with the PD1 inhibitor nivolumab. DETAILED DESCRIPTION

[0109] During tumor progression, tumor-associated myeloid cells can directly promote tumor cell proliferation and support epithelial-mesenchymal transition in tumors through the production of growth factors. In the more advanced stage, M2-like macrophages can be found in metastatic cell niches, in which they promote the cellular immune clearance escape caused by tumors. The immunosuppressive mechanism adopted by TAM and MDSC is mainly through suppressing the activity of the adaptive immune system. Suppressive myeloid cells achieve this by direct cell-cell interaction with target cells or by secretory factors. They can induce signal transduction to induce T cell apoptosis and anergy via immune checkpoint inhibitors PD1 and CTLA4. In addition, they can deprive T cells of the local environment of nutrients necessary for activation and function. In addition, they produce nitrogen oxides, reactive nitrogen species and reactive oxygen species to induce T cell exhaustion. These mechanisms ultimately lead to a reduction in the effect of anti-tumor T cells and a reduction in number, while enhancing the colony of tumor-supported regulatory T cells. Targeting TAM can significantly improve the efficacy of both conventional treatment and immunotherapy.

[0110] The remarkable functional plasticity of macrophages is the basic principle for developing methods to convert cells from M2-like immunosuppressive TAMs to M1-like immunostimulatory and anti-tumor cytotoxic effectors. Tumor-associated myeloid cells can be reprogrammed to a proinflammatory state through direct intervention via small molecules and antibodies targeting key immunomodulatory receptors. Among them, leukocyte immunoglobulin-like receptors (LILRs) play an important role in coordinating immune responses in various immune cells. Two reprogramming strategies can be used - blocking receptors that normally transduce inhibitory intracellular signals or using exogenous ligands to activate receptors that stimulate proinflammatory intracellular cascades. LILR includes inhibitory receptors LILRB and activating receptors LILRA, which regulate immune responses and inflammatory processes associated with disease progression. LILRA and LILRB members are highly homologous in their extracellular regions and are different in their intracellular regions. The six members of LILRA (LIRA 1-6) associate with membrane adaptors to transmit signals via immunoreceptor tyrosine-based activation motifs (ITAMs), and the LILRB (LILRB 1-5) members transmit signals via multiple cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). LILR is expressed on various immune cells, including NK, T, B and myeloid mononuclear cells (monocytes, macrophages, dendritic cells and granulocytes). The function of LILRB in cancer has been extensively studied. Activation of LILRB signaling in cancer contributes to immune escape and supports cancer development. The dual role of LILRB1 and LIRB2 in cancer biology as immune checkpoint molecules and as tumor-supporting factors suggests that LILRB may represent an attractive target for cancer therapy.

[0111] Among the LILRB family, LILRB2 is primarily expressed on myeloid cells and hematopoietic stem cells. It is an important homeostatic surface regulator for myeloid cell maturation and has great therapeutic value as a promising myeloid immune checkpoint target that specifically targets myeloid cell functional determinants. The LILRB2 molecule consists of four immunoglobulin (Ig)-like domains and a long cytoplasmic tail with an ITIM motif. ITIM-dependent recruitment of LILRB2 by SHP1 / SHP2 negatively regulates immune activation. LILRB2 ligands include class 1 HLA (HLA-A, HLA-B, and HLA-C) without the β2m form, non-classical class 1 MHC molecules (HLA-E, HLA-F, HLA-G, and HLA-H), and the angiopoietin-like protein family (ANGPTL). LILRB2 on immune cells regulates cancer development through interactions with its ligands. Class 1 HLA molecules, aberrantly expressed on a variety of human malignant cells, interact with LILRB2 expressed on immune cells. This interaction is involved in tumor immune evasion. HLA-G is initially expressed selectively on cytotrophoblasts at the maternal-fetal interface and contributes to maternal-fetal tolerance. Numerous studies have shown that HLA-G gene transcription and protein translation are turned on in various tumor tissues and remain off in surrounding normal areas. The HLA-G / LILRB pathway inhibits dendritic cell maturation and differentiation, promotes macrophage differentiation into M2-like macrophages, and allows MDSC expansion. During macrophage maturation, LILRB2 antagonism inhibits AKT and STAT6 activation in response to M-CSF and IL4 treatment; and enhances NFκB and STAT1 activation in response to LPS / IFN-γ stimulation.

[0112] Provided herein are antibodies that bind to LILRB2 (anti-LILRB2 antibodies), nucleic acids encoding such antibodies, methods for producing the anti-LILRB2 antibodies disclosed herein, and their uses for therapeutic, diagnostic, and / or research purposes.

[0113] I. Anti-LILRB2 Antibodies

[0114] Leukocyte immunoglobulin-like receptor subfamily B, member 2 (LILRB2) is a member of the leukocyte immunoglobulin-like receptor (LIR) family. LILRB2 belongs to the class B subfamily of the LIR family and contains two or four extracellular immunoglobulin domains, a transmembrane domain, and two to four cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). The amino acid sequence of the LILRB2 protein is known in the art. As an example, the amino acid sequence of human LILRB2 can be found under GenBank accession number NP_001074447.

[0115] Transcriptome analysis revealed that LILRB2 antagonism alters genes involved in cytoskeletal remodeling, lipid / cholesterol metabolism, and endosomal sorting pathways, and shifts differentiation gene networks to polarize TAMs toward an inflammatory phenotype. High LILRB2 expression in DCs promotes DC tolerance, inhibits Th1 and CTL differentiation, and enhances the generation of type 2 cytokine-secreting Th2 and Tc2 cells. LILRB2 on DCs reduces the cytotoxicity of CTLs by competing with CD8 for class 1 MHC or upregulating HLA-G in CTLs. These findings suggest that LILRB2 is a promising myeloid immune checkpoint target. LILRB2 antagonism through antibody-mediated ligand-blocking could reprogram and deregulate tumor-associated myeloid cells in the tumor microenvironment, thereby eliciting antitumor immunity in cancer therapy.

[0116] The present disclosure provides antibodies that bind to LILRB2, for example, antibodies that bind to human LILRB2. In some embodiments, the anti-LILRB2 antibodies disclosed herein are capable of binding to LILRB2 expressed on the surface of a cell. Therefore, the antibodies disclosed herein can be used for therapeutic or diagnostic purposes to target LILRB2-positive cells (e.g., immune cells such as macrophages to enhance anti-tumor immune responses). As used herein, the term "anti-LILRB2 antibody" refers to any antibody that is capable of binding to a LILRB2 receptor (e.g., a LILRB2 receptor expressed on the surface of a cell), or a fragment thereof, which may be of a suitable origin, for example, a human or non-human mammal (e.g., a mouse, rat, rabbit, primate such as a monkey, etc.). In some cases, the anti-LILRB2 antibodies disclosed herein are capable of binding to the extracellular domain of LILRB2.

[0117] Antibodies (used interchangeably in the plural) are immunoglobulin molecules that are capable of specifically binding to a target (e.g., carbohydrates, polynucleotides, lipids, polypeptides, etc.) through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" (e.g., anti-LILRB2 antibody) encompasses not only complete (e.g., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single-chain antibodies (scFv), fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, single-domain antibodies (e.g., nanobodies), single-domain antibodies (e.g., V-only antibodies), and fragments thereof. HAntibodies), multispecific antibodies (e.g., bispecific antibodies) and any other modified immunoglobulin molecule configuration that contains a desired specific antigen recognition site, including glycosylation variants of antibodies, amino acid sequence variants of antibodies and covalently modified antibodies. Antibodies (e.g., anti-LILRB2 antibodies) include antibodies of any class, such as IgD, IgE, IgG, IgA or IgM (or their subclasses), and the antibodies do not necessarily have to be of any particular class. Immunoglobulins can be divided into different classes based on the antibody amino acid sequence of the constant region of their heavy chains. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and some of these can be further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0118] A typical antibody molecule includes a heavy chain variable region (V H ) and light chain variable region (V L ), which are usually involved in antigen binding. H and V L The V domains can be further subdivided into regions of hypervariability, also called "complementarity determining regions" ("CDRs"), interspersed with regions that are more conserved, called "framework regions" ("FRs"). H and V LTypically, it consists of three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The boundaries of the framework regions and CDRs can be precisely identified using methods known in the art, for example, by the Kabat definition, the Chothia definition, the AbM definition, and / or the contact definition, all of which are well known in the art. See, e.g., Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242, Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al (1997) J. Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk / abs).

[0119] The anti-LILRB2 antibodies described herein can be full-length antibodies containing two heavy chains and two light chains, each comprising a variable domain and a constant domain. Alternatively, the anti-LILRB2 antibodies can be antigen-binding fragments of full-length antibodies. Examples of binding fragments encompassed within the term "antigen-binding fragment" of a full-length antibody include (i) a Fab fragment, which is comprised of a V L 、V H 、C L and C H 1 domain; (ii) F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragment, which consists of V H and C H 1 domain; (iv) Fv fragment, which consists of the V L and V H domains, (v)dAb fragments (Ward et al., (1989) Nature 341: 544-546), which are composed of V H domains; and (vi) separate complementary determining regions (CDRs) that retain functionality. In addition, although the two domains VL and V H are encoded by separate genes, but they can be joined using recombinant methods by synthetic linkers that enable them to be made into a single protein chain, where V L and V H The two regions pair to form a monovalent molecule known as a single-chain Fv (scFv). See, e.g., Bird et al. (1988) Science 242:423-426; and Hustone et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883.

[0120] The antibodies described herein can be of a suitable origin, for example, mouse, rat, or human. Such antibodies are non-naturally occurring, i.e., they are not produced in animals without human intervention (e.g., immunization of such animals with a desired antigen or fragment thereof or isolation from an antibody library). Any antibody described herein (e.g., an anti-LILRB2 antibody) can be monoclonal or polyclonal. "Monoclonal antibody" refers to a homogenous antibody population, and "polyclonal antibody" refers to a heterogenous antibody population. These two terms do not limit the source of the antibody or how it is prepared.

[0121] In some embodiments, the anti-LILRB2 antibody is a human antibody that can be isolated from a human antibody library or produced in a transgenic mouse. For example, fully human antibodies can be obtained by using commercially available mice that have been engineered to express specific human immunoglobulins. Transgenic animals designed to produce a more desirable (e.g., fully human antibodies) or more robust immune response can also be used to produce humanized or human antibodies. An example of such technology is the Xenomouse® from Amgen, Inc (Fremont, CA). TM and HuMAb-Mouse from Medarex, Inc (Princeton, NJ) TM and TC Mouse TM In another alternative, antibodies can be produced recombinantly by phage display or yeast technology. See, for example, U.S. Patent Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winter et al., (1994) Annu. Rev. Immunol. 12: 433-455. Alternatively, antibody library display technologies known in the art, such as phage, yeast display, mammalian cell display, or mRNA display technology, can be used to produce human antibodies and antibody fragments in vitro from immunoglobulin variable (V) domain gene repertoires of unimmunized donors.

[0122] In other embodiments, the anti-LILRB2 antibody can be a humanized antibody or a chimeric antibody. Humanized antibodies refer to forms of non-human (e.g., murine) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or antigen-binding fragments thereof that contain minimal sequence derived from a non-human immunoglobulin. Typically, humanized antibodies are human immunoglobulins (recipient antibodies) in which the residues of the receptor CDRs are replaced with residues of a CDR from a non-human species (donor antibody) (e.g., mouse, rat, or rabbit) with the desired specificity, affinity, and capacity. In some cases, one or more Fv framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues.

[0123] In addition, humanized antibodies can comprise residues that are neither present in receptor antibodies nor in the CDR or framework sequences introduced, but the residues are included in order to further improve and optimize antibody performance. In some cases, humanized antibodies can comprise substantially all of the following: at least one, and usually two variable domains, wherein all or substantially all of the CDR regions correspond to those of non-human immunoglobulins, and all or substantially all of the FR regions are those of human immunoglobulin consensus sequences. Humanized antibodies optimally also comprise at least a portion of an immunoglobulin constant region or domain (Fc), which is typically a constant region or domain of a human immunoglobulin. Antibodies can have the Fc region modified as described in WO 99 / 58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five or six) that change relative to the original antibody, also referred to as "derived from" one or more CDRs from the original antibody. Humanized antibodies can also participate in affinity maturation. It is also well known in the art to construct methods for humanized antibodies. See, e.g., Queen et al., Proc. Natl. Acad. Sci. USA, 86: 10029-10033 (1989).

[0124] In some embodiments, the anti-LILRB2 antibodies disclosed herein may be chimeric antibodies. A chimeric antibody refers to an antibody having a variable region or a portion of a variable region from a first species and a constant region from a second species. Typically, in these chimeric antibodies, the variable regions of both the light and heavy chains mimic the variable regions of an antibody derived from one mammalian species (e.g., a non-human mammal such as mouse, rabbit, and rat), while the constant portion is homologous to sequences in an antibody derived from another mammal (e.g., human). In some embodiments, amino acid modifications may be made in the variable and / or constant regions. Techniques developed for producing "chimeric antibodies" are well known in the art. See, for example, Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81, 6851; Neuberger et al. (1984) Nature 312, 604; and Takeda et al. (1984) Nature 314: 452.

[0125] In some embodiments, the anti-LILRB2 antibodies described herein specifically bind to the corresponding target antigen (e.g., human LILRB2) or an epitope thereof. "Specific binding" of an antibody to an antigen or epitope is a term well known in the art. A molecule is said to exhibit "specific binding" if it reacts more frequently, more rapidly, for a longer duration, and / or with a stronger affinity to a particular target antigen than it reacts to an alternative target antigen. An antibody "specifically binds" to a target antigen or epitope if it binds to it with a stronger affinity, a stronger avidity, more readily, and / or for a longer duration than it binds to other substances. For example, an antibody that specifically (or preferentially) binds to an antigen (LILRB2, such as human LILRB2) or an antigenic epitope therein is an antibody that binds to the target antigen with a stronger affinity, a stronger avidity, more readily, and / or for a longer duration than it binds to other antigens or other epitopes on the same antigen. This definition can also be understood to mean that, for example, an antibody that specifically binds to a first target antigen may or may not specifically or preferentially bind to a second target antigen. Thus, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. In some instances, an antibody that "specifically binds" to a target antigen or an epitope thereof may not bind to other antigens or other epitopes within the same antigen (i.e., only baseline binding activity may be detected in conventional methods).

[0126] In some instances, the anti-LILBR2 antibodies disclosed herein specifically bind to LILRB2. In some instances, the anti-LILBR2 antibodies disclosed herein do not bind to another member of the LILRB family (e.g., LILRB1 or LILBR3) (i.e., no significant binding is detected in conventional methods). In other instances, the anti-LILBR2 antibodies disclosed herein are capable of binding to another member of the LILRB family (e.g., LILRB1 or LILBR3), but with a much lower affinity than their binding affinity to LILRB2.

[0127] In some embodiments, an anti-LILRB2 antibody as described herein has a suitable binding affinity for a target antigen (e.g., human LILRB2) or an antigenic epitope thereof. As used herein, "binding affinity" refers to the apparent association constant or K A .K A is the dissociation constant (K D The anti-LILRB2 antibodies described herein can have a binding affinity (K) of at least 100 nM, 10 nM, 1 nM, 0.1 nM or less for LILRB2 (e.g., human LILRB2). D ). Increased binding affinity corresponds to a decreased K D Higher affinity binding of an antibody to a first antigen relative to a second antigen can be explained by a higher K for binding to the first antigen. A (or smaller value K D ) rather than K binding to the second antigen A (or value K D ). In such cases, the antibody is specific for the first antigen (e.g., the first protein or a mimetic thereof in a first conformation) relative to the second antigen (e.g., the same first protein or a mimetic thereof in a second conformation; or the second protein). The difference in binding affinity (e.g., for specificity or other comparisons) can be at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 37.5-fold, 50-fold, 70-fold, 80-fold, 90-fold, 100-fold, 500-fold, 1000-fold, 10,000-fold, or 10 5 In some embodiments, any anti-LILRB2 antibody may also be affinity matured to increase the binding affinity of the antibody to the target antigen or its antigenic epitope.

[0128] Binding affinity (or binding specificity) can be measured by a variety of methods, including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance or spectroscopy (e.g., using fluorescence determination). Exemplary conditions for evaluating binding affinity are in HBS-P buffer (10 mM HEPES, 150 mM NaCl, 0.005% (v / v) surfactant P20 at pH 7.4). These techniques can be used to measure the concentration of bound binding protein as a function of target protein concentration. The concentration of bound binding protein ([bound]) is typically related to the concentration of free target protein ([free]) by the following equation:

[0129] [Bound] = [Free] / (Kd + [Free])

[0130] Although it is not always necessary to determine K A , because sometimes it is sufficient to obtain a quantitative measure of affinity (e.g., determined using methods such as ELISA or FACS analysis) in conjunction with K A is proportional and can therefore be used for comparison, e.g., to determine whether a higher affinity is, e.g., 2-fold higher, to obtain a qualitative measure of affinity, or to obtain an inference of affinity, e.g., by activity in a functional assay (e.g., in vitro or in vivo).

[0131] Any anti-LILRB2 antibody as described herein (e.g., an exemplary anti-LILRB2 antibody provided herein) can bind to and inhibit (e.g., reduce or eliminate) the activity of LILRB2-positive cells (e.g., immune cells, such as macrophages expressing LILRB2). In some embodiments, an anti-LILRB2 antibody as described herein can bind to and inhibit the activity of LILRB2-positive cells (e.g., cancer cells) by at least 30% (e.g., 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increments therein). The inhibitory activity of the anti-LILRB2 antibodies described herein can be determined by conventional methods known in the art, for example, by measuring K i, app Determination of the value.

[0132] In some examples, the K of an antibody is i, app The value can be determined by measuring the extent of inhibition of the reaction of interest by varying concentrations of the antibody; fitting the change in the pseudo-first-order rate constant (v) as a function of inhibitor concentration to a modified Morrison equation (Equation 1) to obtain an estimate of the apparent Ki value. For competitive inhibitors, Ki app You can get it from K i, appThe y-intercept was obtained by extracting the linear regression analysis of the plots against substrate concentration.

[0133]

[0134] Where A is equal to v o / E, the initial velocity (v) of the enzymatic reaction in the absence of an inhibitor (I) o ) divided by the total enzyme concentration (E). In some embodiments, the anti-LILRB2 antibodies described herein may have a Ki of 1000 pM, 500 pM, 100 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 5 pM or less for the target antigen or antigenic epitope. app value.

[0135] Exemplary Anti-LILRB2 Antibodies

[0136] A number of exemplary anti-LILRB2 antibodies are provided in Table 1 below (Table 1 also provides the CDRs of each exemplary anti-LILRB2 antibody, as determined by Kabat numbering. See Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242. See also www2.mrc-lmb.cam.ac.uk / vbase / alignments2.php). Each of these exemplary anti-LILRB2 antibodies disclosed herein, as well as their functional equivalents, are within the scope of the present disclosure.

[0137] Table 1. Exemplary anti-LILRB2 antibodies

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146] In some embodiments, the anti-LILRB2 antibodies described herein bind to the same epitope of the LILRB2 polypeptide as any of the exemplary (reference) antibodies described herein, or compete with the exemplary antibodies for binding to the LILRB2 antigen. An "epitope" refers to a site on a target antigen that is recognized and bound by an antibody. A site can be composed entirely of amino acid components, entirely of chemical modifications of the amino acids of a protein (e.g., a glycosyl moiety), or a combination thereof. Overlapping epitopes contain at least one common amino acid residue. An epitope can be linear, typically 6 to 15 amino acids in length. Alternatively, an epitope can be conformational. The epitope to which an antibody binds can be determined by conventional techniques, such as epitope mapping methods (see, for example, the description below). An antibody that binds to the same epitope as an exemplary antibody described herein can bind to exactly the same epitope or a substantially overlapping epitope (e.g., containing less than 3 non-overlapping amino acid residues, less than 2 non-overlapping amino acid residues, or only 1 non-overlapping amino acid residue) as an exemplary antibody. Whether two antibodies compete with each other for binding to a cognate antigen can be determined by competition assays, which are well known in the art.

[0147] In some examples, the anti-LILRB2 antibodies comprise the same V domains as the exemplary antibodies described herein. H and / or V L CDR. With the same V H and / or V L The same CDRs of two antibodies mean that their CDRs are identical when determined by the same method (e.g., the Kabat method, the Chothia method, the AbM method, the Contact method, or the IMGT method, as known in the art. See, e.g., bioinf.org.uk / abs / ). Such anti-LILRB2 antibodies may have the same V CDRs as the exemplary antibodies described herein. H , the same V L or both.

[0148] Exemplary humanized antibodies

[0149] In some embodiments, the anti-LILRB2 antibodies disclosed herein may be humanized antibodies as disclosed herein, which may be derived from any of the exemplary antibodies provided in Table 1 above.

[0150] In some cases, humanized antibodies can be derived from the parent antibody 11B12. Such humanized antibodies can comprise the same heavy and light chains as antibody 11B12 (see Table 1 above) and the heavy and light chain framework regions (HC FR1-F4 and LC FR1-FR4) provided in Table 2 below.

[0151] Table 2. Humanized and affinity matured variants of clone 11B12

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163] In some examples, the anti-LILRB2 antibody is h#2E1.

[0164] In some cases, the humanized antibody can be derived from the parent antibody 9B6. Such a humanized antibody can comprise the same heavy and light chain CDRs as antibody 9B6 (see Table 1 above and Table 3 below) and the heavy and light chain framework regions (HC FR1-F4 and LC FR1-FR4) provided in Table 3 below.

[0165] Table 3. Humanized variants of clone 9B6

[0166]

[0167]

[0168]

[0169]

[0170]

[0171] In some cases, the humanized antibody can be derived from the parent antibody 14F1. Such a humanized antibody can comprise the same heavy and light chain CDRs as antibody 14F1 (see Table 1 above and Table 4 below) and the heavy and light chain framework regions (HC FR1-F4 and LC FR1-FR4) provided in Table 4 below.

[0172] Table 4. Humanized variants of clone 14F1

[0173]

[0174]

[0175]

[0176]

[0177]

[0178] Functional variants

[0179] Functional variants of any of the exemplary anti-LILRB2 antibodies disclosed herein are also within the scope of the present disclosure. Such functional variants are substantially similar in structure and function to the exemplary antibodies. Functional variants comprise V sequences substantially identical to those of the exemplary antibodies. H and V L CDR. For example, it may comprise up to 8 (e.g., 8, 7, 6, 5, 4, 3, 2, or 1) amino acid variations in only the total CDR regions of the antibody and bind with substantially similar affinity (e.g., with a K of the same order of magnitude). D In some cases, the functional variant may have the same heavy chain CDR3 as the exemplary antibody, and optionally the same light chain CDR3 as the exemplary antibody. Alternatively or in addition, the functional variant may have the same heavy chain CDR2 as the exemplary antibody. H In contrast, such anti-LILRB2 antibodies may comprise V variants having CDR amino acid residue variations only in the heavy chain CDR1, the heavy chain CDR2, or both. H In some examples, the anti-LILRB2 antibody may further comprise a V L Fragment, the V L The fragment may have the same V as the exemplary antibody. L In some examples, the antibody may have the same V CDR3 as the exemplary antibody. L CDR1 or V L CDR2. Alternatively, the V LIn contrast, the antibody may comprise a V variant having CDR amino acid residue variations only in the light chain CDR1, the light chain CDR2, or both. L fragment.

[0180] Alternatively or in addition, the amino acid residue variation can be a conservative amino acid residue substitution. As used herein, a "conservative amino acid substitution" refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods known to those of ordinary skill in the art for altering polypeptide sequences, such as those found in references to such methods, for example, Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, FM Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative amino acid substitutions include substitutions between amino acids in the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0181] In some embodiments, an anti-LILRB2 antibody may comprise a V domain that is identical to the exemplary antibodies described herein. H The heavy chain CDRs may have at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity compared to the CDRs of the respective heavy chain CDRs (individually or collectively). Alternatively or in addition, the anti-LILRB2 antibody may comprise V sequences identical to those of the exemplary antibodies described herein. L The light chain CDRs of the exemplary antibodies are compared (individually or collectively) to have at least 80% (e.g., 85%, 90%, 95% or 98%) sequence identity. As used herein, "individually" means that one CDR of an antibody shares the indicated sequence identity with respect to the corresponding CDR of the exemplary antibody. "Collectively" means that the three V CDRs of an antibody share the indicated sequence identity with respect to the corresponding CDR of the exemplary antibody. H or V L The CDRs are combined relative to the corresponding three V H or V L The CDRs are combined to share the sequence identity shown.

[0182] The "percent identity" of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, as modified from Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program using a score of 50 and a word length of 3 to obtain amino acid sequences homologous to a protein molecule of interest. In cases where gaps exist between the two sequences, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0183] In some embodiments, the heavy chain of any anti-LILRB2 antibody as described herein may further comprise a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region may be of any suitable origin, for example, human, mouse, rat, or rabbit. Alternatively or in addition, the light chain of the anti-LILRB2 antibody may further comprise a light chain constant region (CL), which may be any CL known in the art. In some examples, CL is a kappa light chain. In other examples, CL is a lambda light chain. Antibody heavy and light chain constant regions are well known in the art, for example, those provided in the IMGT database (www.imgt.org) or at www.vbase 2.org / vbstat.php, both of which are incorporated herein by reference.

[0184] In some cases, the anti-LILRB2 antibodies disclosed herein are affinity matured variants of the exemplary antibodies provided in Table 1 above. In some examples, the anti-LILRB2 antibodies disclosed herein are affinity matured variants of antibody 11B12. Such 11B12 variants may comprise the HC CDR1-CDR3 and LC CDR1-CDR3 motifs listed in Table 2 above. In some cases, such 11B12 variants may also comprise one or more of the HC FR1-FR4 and LC FR1-FR4 motifs also provided in Table 2 above. Alternatively, the 11B12 variant may comprise the same heavy and light chain frameworks as 11B12 (see Table 1 above).

[0185] Also provided herein are functional variants derived from affinity-matured h#2E1 (a humanized form of clone 11B12). Exemplary h#2E1 affinity-matured variants are provided in Table 2 above, each of which is within the scope of the present disclosure. In a specific example, the anti-LILRB2 antibody provided herein is 2E1_FC11.

[0186] Such functional variants share a high degree of sequence homology with the parental clone h#2E1 in the heavy and / or light chain CDR regions. For example, relative to the heavy chain CDRs of the parental clone, the functional variant may contain up to five amino acid variations (commonly) in the heavy chain CDRs. Alternatively or in addition, relative to the heavy chain CDRs of the parental clone, the functional variant may contain up to five amino acid variations (commonly) in the light chain CDRs.

[0187] In some examples, the anti-LILRB2 antibodies are affinity-matured variants of h#2E1, such as clone 2E1_FC11 or those having substantially similar biological activity to 2E1_FC11. Such anti-LILRB2 antibodies exhibit excellent binding affinity for human LILRB2 in a range of binding assays, including ELISA, BLI, SPR, and intracellular binding assays. Furthermore, they exhibit comparable ability to block the interaction of LILRB2 with its primary ligands, such as HLA-G and HLA-A2. When compared to known antibodies such as MK4830, JTX8064, and NGM707, 2E1_FC was found to be superior to these known antibodies in its functional activity, for example, in polarizing M2 macrophages toward the pro-inflammatory M1 phase, as observed in multiple healthy human donors in the PBMC / LPS model and the HMDM / LPS model.

[0188] The anti-LILRB2 antibody 2E1_FC11 or a variant thereof may comprise a heavy chain variable region (V H ), which comprises (ai) a heavy chain CDR1 comprising GX1SITSGYX2WX3 (SEQ ID NO: 192), wherein X1 is Y or G, X2 is Y, S, A or H, and X3 is N, S or W; (aii) a heavy chain CDR2 comprising X4ISYDGNX5X6 (SEQ ID NO: 194), wherein X4 is S, T or Y, X5 is A, I, K, L, M, N, P, S, T, V or W, and X6 is H, L, N, S or W; (aiii) a heavy chain CDR3 comprising EEX7TMX8TTX9WFX 10Y (SEQ ID NO: 196), wherein X7 is A or S, X8 is F or I, X9 is A or G, and X10 is A, G, Q or S. In some examples, the heavy chain CDR2 may comprise X4ISYDGNX5X6YNPSLKN (SEQ ID NO: 416), each X variable being as defined herein. In addition, the anti-LILRB2 antibody 2E1_FC11 or a variant thereof may comprise (b) a light chain variable region (V L ), which comprises (bi) light chain CDR1, which comprises X 11 X 12 SEX 13 IX 14 SNX 15 A (SEQ ID NO: 199), wherein X 11 is E, G, L, N, Q, R, S, T or V, X 12 Is A or G, X 13 is K, N, T or V, X 14 is F or Y, and X 15 is L, N or Q; (bii) light chain CDR2, which contains GATX 16 X 17 X 18 X 19 (SEQ ID NO: 201), wherein X 16 is E, N, W or Y, X 17 is F, H, L, P or R, X 18 is A, N, R, or S, and X 19 is A, G, K, L, R, S or V; and (biii) a light chain CDR3 comprising QX 20 FWf 21 PYX 22 (SEQ ID NO: 203), wherein X 20 It is H or Q, X 21 is L, S, T, or Y, and X 22 It is I, M, R, T, Y or V.

[0189] In some embodiments, the X variables in the heavy chain CDRs can be the same as one or more of the following in clone 2E1_FC11: X1 is Y, X2 is Y, X3 is N, X4 is Y, X5 is I, X6 is N, X7 is S, X8 is I, X9 is A, and / or X 10 is A. Alternatively or in addition, the X variable in the light chain CDR may be the same as one or more of 2E1_FC11: X 11 It's R, X 12 It's A, X 13 It is N, X 14 It's Y, X 15It's L, X 16 It is N, X 17 It's L, X 18 It's A, X 19 It's S, X 20 It is H, X 21 It's Y, X 22 is T, or a combination thereof.

[0190] The anti-LILRB2 antibody 2E1_FC11 or a variant thereof may comprise the heavy and light chain CDRs as described above and the human framework regions also disclosed herein, e.g., the heavy and light chain framework regions provided in Table 2 above. In some cases, the anti-LILRB2 antibody 2E1_FC11 or a variant thereof may comprise a V domain that is identical to that of 2E1_FC11. H (SEQ ID NO: 244) is at least 80% (e.g., at least 80%, at least 90%, at least 95% or more) identical to V H ; and / or with 2E1_FC11's V L (SEQ ID NO: 248) is at least 80% (e.g., at least 80%, at least 90%, at least 95% or more) identical to V L .

[0191] II. Methods for Generating Anti-LILRB2 Antibodies

[0192] Any anti-LILRB2 antibody described herein can be prepared by any method known in the art. See, for example, Harlow and Lane, (1998) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. In some embodiments, anti-LILRB2 antibodies can be produced by conventional hybridoma technology. Alternatively, anti-LILRB2 antibodies can be identified from a suitable library (e.g., a human antibody library). In some cases, high-affinity fully human LILRB2 binders can be obtained from a human antibody library (e.g., an affinity maturation library (e.g., with variations in one or more CDR regions)). See also the Examples below. There are many conventional methods known in the art to identify and isolate antibodies capable of binding to the target antigens described herein, including phage display, yeast display, ribosome display, or mammalian display technology.

[0193] As needed, the antibody of interest (monoclonal or polyclonal) (e.g., produced by a hybridoma cell line or isolated from an antibody library) can be sequenced and the polynucleotide sequence can then be cloned into a vector for expression or propagation. The sequence encoding the antibody of interest can be maintained in a vector in a host cell, which can then be amplified and frozen for future use. Alternatively, the polynucleotide sequence can be used for genetic manipulation, for example, to humanize the antibody or improve the affinity (affinity maturation) or other properties of the antibody. For example, if the antibody is from a non-human source and is used in human clinical trials and treatments, the constant region can be engineered to be more similar to a human constant region to avoid an immune response. Alternatively or in addition, it may be desirable to genetically manipulate the antibody sequence to obtain greater affinity and / or specificity for the target antigen and greater efficacy in enhancing LILRB2 activity. It will be apparent to those skilled in the art that one or more polynucleotide changes can be made to the antibody and still maintain its binding specificity to the target antigen.

[0194] Antigen-binding fragments of intact antibodies (full-length antibodies) can be prepared by conventional methods. For example, F(ab')2 fragments can be produced by pepsin digestion of antibody molecules, and Fab fragments can be produced by reducing the disulfide bridges of F(ab')2 fragments.

[0195] Genetically engineered antibodies (such as humanized antibodies, chimeric antibodies, single-chain antibodies and bispecific antibodies) can be produced via, for example, conventional recombinant techniques. In one example, conventional procedures can be used (for example, by using oligonucleotide probes that can specifically bind to the heavy and light chains of the monoclonal antibody encoding the DNA encoding the monoclonal antibody specific for the target antigen). After separation, the DNA can be placed in one or more expression vectors, which are then transfected into host cells (such as Escherichia coli cells, ape COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not originally produce immunoglobulins) to obtain the synthesis of monoclonal antibodies in recombinant host cells. See, for example, PCT Publication No. WO 87 / 04462. The DNA can then be modified, for example, by replacing the homologous mouse sequence (Morrison et al., (1984) Proc. Nat. Acad. Sci. 81: 6851) with the coding sequence of the human heavy and light chain constant regions, or by covalently linking all or part of the coding sequence of the immunoglobulin coding sequence and the non-immunoglobulin polypeptide. In this manner, genetically engineered antibodies, eg, "chimeric" or "hybrid" antibodies, can be prepared that have binding specificity for the target antigen.

[0196] Techniques developed for producing "chimeric antibodies" are well known in the art. See, for example, Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81, 6851; Neuberger et al. (1984) Nature 312, 604; and Takeda et al. (1984) Nature 314: 452.

[0197] Methods for constructing humanized antibodies are also well known in the art. See, for example, Queen et al., Proc. Natl. Acad. Sci. USA, 86: 10029-10033 (1989). In one example, the V of the parent non-human antibody is modified according to methods known in the art. H and V L The variable regions of the parent V were subjected to three-dimensional molecular modeling analysis. Next, the same molecular modeling analysis was used to identify framework amino acid residues that were predicted to be important for the formation of correct CDR structures. H and V L Sequence as a search query to identify human V antibodies from any antibody gene database that have amino acid sequences homologous to those of the parent non-human antibody. H and V L Then select Person V H and V L Receptor gene. The CDR region in the selected human receptor gene can be replaced by the CDR region from the parent non-human antibody or its functional variant. When necessary, the residues in the framework region of the parent chain predicted to be important in interacting with the CDR region (see description above) can be used to replace the corresponding residues in the human receptor gene.

[0198] Single-chain antibodies can be prepared by recombinant techniques by linking the nucleotide sequence encoding the heavy chain variable region and the nucleotide sequence encoding the light chain variable region. Preferably, a flexible linker is incorporated between the two variable regions. Alternatively, the techniques described for producing single-chain antibodies (U.S. Patent Nos. 4,946,778 and 4,704,692) can be adapted to produce phage-displayed, yeast-displayed, mammalian cell-displayed, or mRNA-displayed scFv libraries, and scFv clones specific for LILRB2 can be identified from the libraries according to conventional procedures.

[0199] Antibodies obtained according to methods known in the art and described herein can be characterized using methods well known in the art. For example, one method is to identify the epitope bound by the antigen, or "epitope mapping". Many methods for mapping and characterizing the position of epitopes on proteins are known in the art, including crystal structure analysis of antibody-antigen complexes, competitive assays, gene fragment expression assays, and assays based on synthetic peptides, for example, as described in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999. In an additional example, epitope mapping can be used to determine the sequence to which the antibody binds. The epitope can be a linear epitope (i.e., contained in a single fragment of amino acids), or a conformational epitope formed by the three-dimensional interaction of amino acids that may not necessarily be contained in a single fragment (primary structure linear sequence). Peptides of different lengths (e.g., at least 4 to 6 amino acids long) can be separated or synthesized (e.g., by recombinant means) and used for binding analysis with antibodies. In another example, the epitope to which the antibody binds can be determined in a systematic screening by using overlapping peptides derived from the target antigen sequence and measuring the binding of the antibody. In some embodiments, the present invention provides the method for the preparation of the antibody of the present invention.Then, the antibody of the present invention is expressed in the form of a fragment of a gene.The open reading frame of the target antigen is randomly or fragmented by specific genetic construction, and the expression fragment of the antigen and the reactivity of the antibody to be tested are determined.Gene fragment can, for example, be produced by PCR, then be transcribed in vitro and translated into protein in the presence of radioactive amino acids.Then, the combination of antibody and radiolabeled antigen fragment is determined by immunoprecipitation and gel electrophoresis.Some epi-position can also be identified by using the large library (phage library) of the random peptide sequence displayed on the phage particle surface.

[0200] Alternatively, a defined library of overlapping peptide fragments can be tested for binding to a test antibody in a simple binding assay. In additional examples, mutagenesis of the antigen binding domain, domain swapping experiments, and alanine scanning mutagenesis can be performed to identify residues that are required, sufficient, and / or essential for epitope binding. For example, domain swapping experiments can be performed using mutants of the target antigen in which various fragments of LILRB2 have been replaced (exchanged) with sequences from a closely related but antigenically distinct protein (e.g., another member of the tumor necrosis factor receptor family). By evaluating antibody binding to mutant LILRB2, the importance of specific antigen fragments for antibody binding can be assessed.

[0201] Alternatively, competition assays can be performed using other antibodies known to bind to the same antigen to determine whether the antibody binds to the same epitope as the other antibody. Competition assays are well known to those skilled in the art. In some examples, the anti-LILRB2 antibodies or bispecific antibodies disclosed herein can be prepared by recombinant techniques as exemplified below.

[0202] The nucleic acids encoding the heavy and light chains of the anti-LILRB2 antibodies or bispecific antibodies described herein can be cloned into an expression vector, with each nucleotide sequence operably linked to a suitable promoter. In one example, each of the nucleotide sequences encoding the heavy and light chains is operably linked to a different promoter. Alternatively, the nucleotide sequences encoding the heavy and light chains can be operably linked to a single promoter such that both heavy and light chains are expressed from the same promoter. When necessary, an internal ribosome entry site (IRES) can be inserted between the heavy and light chain coding sequences.

[0203] In some examples, the nucleotide sequences encoding the two chains of an antibody are cloned into two vectors, which can be introduced into the same or different cells. When the two chains are expressed in different cells, each of them can be isolated from the host cell expressing such a chain, and the isolated heavy and light chains can be mixed and incubated under appropriate conditions that allow antibody formation.

[0204] Generally, the nucleic acid sequence of one or all chains of the antibody encoding the antibody can be cloned into a suitable expression vector using methods known in the art and operably connected with a suitable promoter. For example, the nucleotide sequence and the carrier can be contacted with a restriction endonuclease under suitable conditions to produce complementary ends on each molecule, and the complementary ends can be paired with each other and connected together using a ligase. Alternatively, a synthetic nucleic acid connexon can be connected to the end of the gene. These synthetic connexons contain the nucleic acid sequence corresponding to a specific restriction site in the carrier. The selection of the expression vector / promoter depends on the type of the host cell that is used to produce the antibody.

[0205] Various promoters can be used to express the antibodies described herein, including, but not limited to, the cytomegalovirus (CMV) intermediate early promoter, viral LTRs (e.g., Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR), the simian virus 40 (SV40) early promoter, the Escherichia coli (E. coli) lac UV5 promoter, and the herpes simplex tk virus promoter.

[0206] Regulatable promoters can also be used. Such regulatable promoters include those that use the lac repressor from E. coli as a transcriptional regulator to regulate transcription from mammalian cell promoters carrying the lac operon [Brown, M. et al., Cell, 49:603-612 (1987)], and those that use the tetracycline repressor (tetR) [Gossen, M., and Bujard, H., Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992); Yao, F. et al., Human Gene Therapy, 9:1939-1950 (1998); Shockelt, P., et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)]. Other systems include FK506 dimers, VP16, or p65 using estradiol, RU486, diphenolmulesterone, or rapamycin. Inducible systems are available from Invitrogen, Clontech, and Ariad.

[0207] A regulatable promoter comprising a repressor and an operator can be used. In one embodiment, the lac repressor from Escherichia coli can be used as a transcriptional regulator to regulate transcription from a mammalian cell promoter carrying the lac operon [M. Brown et al., Cell, 49: 603-612 (1987); Gossen and Bujard (1992); M. Gossen et al., Natl. Acad. Sci. USA, 89: 5547-5551 (1992)], the tetracycline repressor (tetR) is combined with a transcription activator (VP16) to produce the tetR-mammalian cell transcription activator fusion protein tTa (tetR-VP16), the tetracycline repressor (tetR) is combined with a minimal promoter carrying tetO derived from the major early promoter of human cytomegalovirus (hCMV) to produce a tetR-tet operator system to control gene expression in mammalian cells. In one embodiment, a tetracycline-inducible switch is used. When the tetracycline operator is properly positioned downstream of the TATA element of the CMVIE promoter, the tetracycline repressor (tetR) alone, rather than tetR-mammalian cell transcription factor fusion derivatives, can act as an effective trans-regulator to regulate gene expression in mammalian cells (Yao et al., Human Gene Therapy, 10(16):1392-1399(2003)). A particular advantage of this tetracycline-inducible switch is that it does not require the use of tetracycline repressor-mammalian cell transactivator or repressor fusion proteins, which may be toxic to cells in some cases (Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551(1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526(1995)) to achieve its regulatable effect.

[0208] In addition, the vector may contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfection in mammalian cells; an enhancer / promoter sequence from the immediate early gene of human CMV for high-level transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; the SV40 polyoma origin of replication and ColE1 for correct episomal replication; an internal ribosome binding site (IRES) for universal multiple cloning sites; and T7 and SP6 RNA promoters for sense and antisense RNA in vitro transcription. Suitable vectors and methods for generating vectors containing transgenes are well known and available in the art.

[0209] Examples of polyadenylation signals for practicing the methods described herein include, but are not limited to, the human collagen I polyadenylation signal, the human collagen II polyadenylation signal, and the SV40 polyadenylation signal.

[0210] One or more vectors (e.g., expression vectors) comprising nucleic acids encoding any antibody can be introduced into suitable host cells to produce the antibody. Host cells can be cultured under suitable conditions for expressing the antibody or any of its polypeptide chains. Such antibodies or their polypeptide chains can be recovered (e.g., from cells or culture supernatant) by cultured cells via conventional methods (e.g., affinity purification). If necessary, the polypeptide chains of the antibody can be incubated under suitable conditions for a suitable period of time to allow for the production of the antibody.

[0211] In some embodiments, the method for preparing the antibodies described herein comprises a recombinant expression vector encoding both the heavy and light chains of an anti-LILRB2 antibody, or encoding two chains of a two-chain bispecific antibody as also described herein. The recombinant expression vector can be introduced into a suitable host cell (e.g., dhfr-CHO cells) by conventional methods (e.g., calcium phosphate-mediated transfection). Positive transformant host cells can be selected and cultured under suitable conditions that allow expression of the two polypeptide chains that form the antibody, and the antibody can be recovered from the cells or from the culture medium. When necessary, the two chains recovered from the host cells can be incubated under suitable conditions that allow antibody formation.

[0212] In one example, two recombinant expression vectors are provided, one encoding the heavy chain of an anti-LILRB2 antibody disclosed herein or one of the two chains of two bispecific antibodies, and the other encoding the light chain of an anti-LILRB2 antibody or the other chain of a bispecific antibody. Both recombinant expression vectors can be introduced into a suitable host cell (e.g., dhfr-CHO cells) by conventional methods (e.g., calcium phosphate-mediated transfection). Alternatively, each of the expression vectors can be introduced into a suitable host cell. Positive transformants can be selected and cultured under suitable conditions that allow expression of the polypeptide chain of the antibody. When the two expression vectors are introduced into the same host cell, the antibodies produced therein can be recovered from the host cell or from the culture medium. If necessary, the polypeptide chain can be recovered from the host cell or from the culture medium and then incubated under suitable conditions that allow the formation of the antibody. When the two expression vectors are introduced into different host cells, each of them can be recovered from the corresponding host cell or from the corresponding culture medium. The two polypeptide chains can then be incubated under suitable conditions for forming the antibody.

[0213] Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select the transformants, culture the host cells and recover the antibodies from the culture medium. For example, some antibodies can be isolated by affinity chromatography using a matrix coupled to protein A or protein G.

[0214] Any nucleic acid encoding the heavy chain, light chain, or both of an anti-LILRB2 antibody or any bispecific antibody as described herein, a vector (e.g., an expression vector) containing such a nucleic acid; and a host cell comprising the vector are within the scope of the present disclosure.

[0215] III. Uses of anti-LILRB2 antibodies

[0216] Any of the anti-LILRB2 antibodies disclosed herein can be used for therapeutic, diagnostic, and / or research purposes, all of which are within the scope of the present disclosure.

[0217] Pharmaceutical composition

[0218] Antibodies as described herein, as well as encoding nucleic acids or nucleic acid collections, vectors comprising such nucleic acids or nucleic acid collections, or host cells comprising the vectors can be mixed with pharmaceutically acceptable carriers (excipients) to form pharmaceutical compositions for treating target diseases. "Acceptable" means that the carrier must be compatible with the active ingredient of the composition (and preferably, capable of stabilizing the active ingredient) and harmless to the subject to be treated. Pharmaceutically acceptable excipients (carriers) include buffers well known in the art. See, for example, Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KE Hoover.

[0219] The pharmaceutical composition used in the method of the present application may comprise a pharmaceutically acceptable carrier, excipient or stabilizer in the form of a lyophilized formulation or an aqueous solution. (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KE Hoover). Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations used and may include buffers such as phosphoric acid, citric acid and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethylammonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) (e.g., sucrose, mannitol, trehalose, or sorbitol); salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN®. TM 、PLURONICS TM or polyethylene glycol (PEG).

[0220] In some examples, the pharmaceutical compositions described herein comprise liposomes containing antibodies (or encoding nucleic acids), which can be prepared by methods known in the art, such as Epstein, et al., Proc. Natl. Acad. Sci. USA 82: 3688 (1985); Hwang, et al., Proc. Natl. Acad. Sci. USA 77: 4030 (1980); and as described in U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556. Particularly useful liposomes can be produced by a reverse phase evaporation method using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derived phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter of a defined pore size to produce liposomes with a desired diameter.

[0221] Antibodies or encoding nucleic acids can also be embedded in microcapsules, such as those prepared by coacervation techniques or by interfacial polymerization, for example, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules in macroemulsions. Such technology is known in the art, see, for example, Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

[0222] In other examples, pharmaceutical compositions as described herein can be formulated in a sustained release form. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing antibodies, which are in the form of molded articles, such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol), polylactic acid (U.S. Patent number 3,773,919), copolymers of L-glutamic acid and 7 ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT®, and the like. TM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0223] Pharmaceutical compositions for in vivo administration must be sterile. This is easily accomplished, for example, by filtration through a sterile filtration membrane. Therapeutic antibody compositions are typically placed in a container with a sterile access port, for example, an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle.

[0224] Pharmaceutical compositions as described herein can be unit dosage forms, such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories for oral, parenteral or rectal administration, or for administration by inhalation or insufflation. In order to prepare solid compositions such as tablets, the main active ingredient can be mixed with a pharmaceutical carrier to form a solid preformulated composition containing a uniform mixture of the compounds of the present invention or its nontoxic pharmaceutically acceptable salt, wherein the pharmaceutical carrier is, for example, conventional tableting ingredients (such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or colloid) and other pharmaceutical diluents (such as water). When referring to these preformulated compositions as uniform, this means that the active ingredient is evenly dispersed throughout the composition so that the composition can be easily subdivided into equivalent unit dosage forms, such as tablets, pills and capsules. The solid preformulation composition is then subdivided into unit dosage forms of the above type containing 0.1 mg to about 500 mg of the active ingredient of the present invention. The tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form that provides an advantage of prolonged action. In some embodiments, the present invention provides the composition of the present invention.For example, tablet or pill can comprise inner dosage and outer dosage component, and the latter is the coating form on the former.Two kinds of components can be separated by enteric layer, and described enteric layer is used for resisting the disintegration in stomach and allows inner component to enter duodenum or delay release intactly.Various materials can be used for such enteric layer or coating, and such material comprises the mixture of multiple polymeric acid and polymeric acid and the material of for example shellac, spermol and cellulose acetate.

[0225] Suitable surfactants include, in particular, nonionic agents such as polyoxyethylene sorbitol (e.g., Tween TM 20, 40, 60, 80 or 85) and other sorbitols (e.g., Span TM 20, 40, 60, 80 or 85). Compositions with surfactants typically contain 0.05% to 5% surfactant, and can be 0.1% to 2.5%. It should be understood that other ingredients, such as mannitol or other pharmaceutically acceptable carriers, can be added if necessary.

[0226] Suitable emulsions can be prepared using commercially available fat emulsions such as Intralipid TM , Liposyn TM Infonutrol TM 、Lipofundin TM and Lipiphysan TM. The active ingredient can be dissolved in a premixed emulsion composition, or alternatively can be dissolved in an oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and form an emulsion when mixed with a phospholipid (e.g., lecithin, soybean lecithin or soybean lecithin) and water. It will be appreciated that other ingredients (e.g., glycerol or glucose) can be added to adjust the tonicity of the emulsion. Suitable emulsions typically contain up to 20% (e.g., 5% to 20%) oil. The fat emulsion may comprise fat droplets of 0.1 μm to 1.0 μm, particularly 0.1 μm to 0.5 μm, and have a pH of 5.5 to 8.0.

[0227] The emulsion composition can be prepared by combining the antibody with the intralipid TM or those prepared by mixing soybean oil, lecithin, glycerol, and water or its components. Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents or mixtures thereof, as well as powders. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic action.

[0228] Compositions in preferably sterile pharmaceutically acceptable solvents can be aerosolized using gases. The aerosolized solution can be breathed directly from the aerosol device, or the aerosol device can be attached to a mask, tent, or intermittent positive pressure breathing machine. Solutions, suspensions, or powder compositions can be administered preferably orally or nasally from a device that delivers the formulation in an appropriate manner.

[0229] Therapeutic applications

[0230] To practice the methods disclosed herein, an effective amount of a pharmaceutical composition comprising any anti-LILRB2 antibody described herein can be administered to a subject (e.g., a human) in need of treatment via a suitable route, such as intravenous administration, for example, as a bolus injection or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, inhalation, or topical route.

[0231] The subject to be treated by the methods described herein can be a mammal, more preferably a human or non-human primate. Mammals include, but are not limited to, farm animals, sports animals, pets, primates, horses, dogs, cats, mice, and rats. The human subject in need of treatment can be a human patient suffering from a target disease / disorder (cancer, such as a solid tumor), a human patient at risk for a target disease / disorder (cancer, such as a solid tumor), or a human patient suspected of having a target disease / disorder (cancer, such as a solid tumor) who benefits from regulating the expression of LILRB2. +The invention relates to a method for treating a variety of cancers, including but not limited to: treating a variety of cancers, including but not limited to treating a variety of ovarian cancers, treating a variety of colorectal cancers, ...

[0232] Subjects with the target cancer can be identified by routine medical examinations (e.g., laboratory tests, organ function tests, CT scans, or ultrasound). In some embodiments, the subject to be treated by the methods described herein can be a human cancer patient who has undergone or is undergoing anti-cancer therapy (e.g., chemotherapy, radiotherapy, immunotherapy, or surgery).

[0233] A subject suspected of having any such disease / condition of interest may display one or more symptoms of the disease / condition.A subject at risk of a disease / condition may be a subject having one or more risk factors for the disease / condition.

[0234] As used herein, "effective amount" refers to the amount of each active agent required for the therapeutic effect imparted to a subject, either alone or in combination with one or more other active agents. It will be apparent to those skilled in the art that the amount of the antibody is effective. As will be appreciated by those skilled in the art, the effective amount varies according to the specific condition being treated, the severity of the condition, individual patient parameters (including age, physical condition, size, sex, and weight), the duration of treatment, the nature of concurrent treatment (if any), the specific route of administration, and similar factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be resolved only by routine experimentation. It is generally preferred to use a maximum dose of a single component or a combination thereof, that is, the highest safe dose according to reasonable medical judgment.

[0235] The dosage of the present invention can be determined by the method for the treatment of a disease or illness. ...

[0236] In one example, the dosage of an antibody described herein can be determined empirically in an individual who has been given one or more administrations of the antibody. The individual is given increasing doses of the agonist. To assess the efficacy of the agonist, indicators of the disease / condition can be tracked.

[0237] Typically, for the administration of any antibody described herein, the initial candidate dose can be about 2 mg / kg. For the purposes of this disclosure, the range of a typical daily dose can be about 0.1 μg / kg to 3 μg / kg to 30 μg / kg to 300 μg / kg to 3 mg / kg, to 30 mg / kg to 100 mg / kg or higher, depending on the above factors. For repeated administration over several days or longer, depending on the condition, continued treatment occurs until desired symptom suppression occurs or until sufficient therapeutic levels are reached to alleviate the target disease or condition or their symptoms. An exemplary dosing regimen includes administering an initial dose of about 2 mg / kg, followed by a weekly maintenance dose of about 1 mg / kg of the antibody, or subsequently administering a maintenance dose of about 1 mg / kg every other week. However, other dosage regimens may be useful depending on the pattern of pharmacokinetic attenuation that the practitioner wishes to achieve. For example, administration once to four times a week is contemplated. In some embodiments, a dosage ranging from about 3 μg / mg to about 2 mg / kg (e.g., about 3 μg / mg, about 10 μg / mg, about 30 μg / mg, about 100 μg / mg, about 300 μg / mg, about 1 mg / kg, and about 2 mg / kg) can be used. In some embodiments, the frequency of administration is once a week, once every 2 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, or once every 10 weeks; or once a month, once every 2 months, or once every 3 months, or longer. The progress of this treatment can be easily monitored by conventional techniques and assays. The dosage regimen (including the antibody used) can vary over time.

[0238] The specific dosage regimen, ie, dose, timing, and repetition, depends on the specific individual and the individual's medical history, as well as the properties of the individual agent (eg, half-life of the agent, and other considerations well known in the art).

[0239] For the purposes of this disclosure, the appropriate dosage of an antibody as described herein depends on the specific antibody, multiple antibodies and / or non-antibody peptides (or their compositions) used, the type and severity of the disease / disorder, whether the antibody is administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to agonists, and the judgment of the attending physician. Typically, the clinician administers the antibody until a dosage that achieves the desired result is reached. In some embodiments, the desired result is an increase in the anti-tumor immune response in the tumor microenvironment. Methods for determining whether a dosage produces the desired result are obvious to those skilled in the art. Administration of one or more antibodies can be continuous or intermittent, depending on, for example, the physiological condition of the recipient, whether the purpose of administration is treatment or prevention, and other factors known to technical practitioners. Administration of the antibody can be substantially continuous over a preselected time period, or can be a series of intervals of administration, for example, before, during, or after the development of the target disease or disorder.

[0240] As used herein, the term "treat," "treat," or "treat" refers to the application or administration of a composition comprising one or more active agents to a subject having a target disease or condition, symptoms of a disease / condition, or susceptibility to a disease / condition, with the intention of curing, rehabilitating, alleviating, eliminating, altering, remedying, ameliorating, improving, or otherwise affecting the condition, symptoms of the disease, or susceptibility to the disease or condition.

[0241] Alleviate target disease / illness and comprise delaying the development or progress of disease or reducing disease severity or prolonging survival.Alleviate disease or prolong survival and do not necessarily need to cure result.As used herein, "delaying" the development of target disease or illness means to postpone, hinder, slow down, slow down, stabilize and / or postpone the progress of disease.This delay can be different lengths of time, depending on the history of disease and / or the individual being treated.The method of "delaying" or alleviating the development of disease, or delaying the onset of disease is when compared with not using this method, reducing the possibility of one or more symptom development of disease and / or reducing the degree of symptom in a given time frame within a given time frame.Such comparison is usually based on clinical research, using many objects that are enough to obtain statistically significant results.

[0242] "Development" or "progression" of a disease refers to the initial manifestation and / or subsequent progression of the disease. The progression of a disease can be detectable and can be assessed using standard clinical techniques as are well known in the art. However, progression also refers to progression that may not be detectable. For the purposes of this disclosure, development or progression refers to the biological course of a symptom. "Development" includes onset, recurrence, and onset. As used herein, "onset" or "occurrence" of a disease or condition of interest includes initial onset and / or recurrence.

[0243] According to the type of disease to be treated or the site of disease, conventional methods known to those of ordinary skill in the medical field can be used to administer pharmaceutical compositions to an object. This composition can also be used via other conventional routes, for example, orally, parenterally, by inhalation spray, topically, rectum, nose, cheek, vaginal or via implantable reservoir. As used herein, term "parenteral" includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion technology. In addition, compositions can be administered to an object via an injectable depot route of administration, for example, using 1 month, 3 months or 6 months depot injectable or biodegradable materials and methods. In some examples, pharmaceutical compositions are administered intraocularly or intravitreally.

[0244] Injectable compositions can contain various carriers, such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol and polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). For intravenous injection, water-soluble antibodies can be administered by a drip method, whereby a pharmaceutical preparation containing the antibody and a physiologically acceptable excipient is infused. A physiologically acceptable excipient can include, for example, 5% dextran, 0.9% saline, Ringer's solution or other suitable excipients. Intramuscular preparations (e.g., sterile preparations of suitable soluble salt forms of the antibody) can be dissolved and administered in pharmaceutical excipients such as water for injection, 0.9% saline or 5% glucose solution.

[0245] In one embodiment, the antibody is administered via site-specific or targeted local delivery technology. Examples of site-specific or targeted local delivery technology include various implantable reservoir sources or local delivery catheters (e.g., infusion catheters, indwelling catheters or needle catheters, synthetic grafts, adventitial wraps, shunts and stents or other implantable devices) of the antibody, site-specific carriers, direct injection or direct application. See, e.g., PCT Publication No. WO 00 / 53211 and U.S. Patent No. 5,981,568.

[0246] Targeted delivery of therapeutic compositions containing one or more nucleic acids (e.g., expression vectors for producing any anti-LILRB2 antibody or bispecific antibody) can also be used. Receptor-mediated DNA delivery techniques are described, for example, in Findeis et al., Trends Biotechnol. (1993) 11:202; Chiou et al., Gene Therapeutics: Methods And Applications Of Direct Gene Transfer (JA Wolff, ed.) (1994); Wu et al., J. Biol. Chem. (1988) 263:621; Wu et al., J. Biol. Chem. (1994) 269:542; Zenke et al., Proc. Natl. Acad. Sci. USA (1990) 87:3655; Wu et al., J. Biol. Chem. (1991) 266:338.

[0247] For local administration in a gene therapy regimen, therapeutic compositions containing polynucleotides (e.g., those encoding the antibodies described herein) are administered in a range of about 100 ng to about 200 mg of DNA. In some embodiments, concentrations ranging from about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg or more of DNA may also be used during a gene therapy regimen.

[0248] The therapeutic polynucleotides and polypeptides described herein can be delivered using gene delivery vehicles. The gene delivery vehicle can be viral or non-viral in origin (see generally, Jolly, Cancer Gene Therapy (1994) 1: 51; Kimura, Human Gene Therapy (1994) 5: 845; Connelly, Human Gene Therapy (1995) 1: 185; and Kaplitt, Nature Genetics (1994) 6: 148). The expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters and / or enhancers. The expression of coding sequences can be constitutive or regulated.

[0249] Viral-based vectors for delivery of a desired polynucleotide and expression in a desired cell are well known in the art. Exemplary viral-based vectors include, but are not limited to, recombinant retroviruses (see, e.g., PCT Publication Nos. WO 90 / 07936; WO 94 / 03622; WO 93 / 25698; WO 93 / 25234; WO 93 / 11230; WO 93 / 10218; WO 91 / 02805; U.S. Patent Nos. 5,219,740 and 4,777,127; British Patent No. 2,200,651; and European Patent No. 0 345 242), alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246), and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-1250). VR-532)), and adeno-associated virus (AAV) vectors (see, e.g., PCT Publication Nos. WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655). Administration of DNA linked to a killed adenovirus (as described in Curiel, Hum. Gene Ther. (1992) 3:147) can also be used.

[0250] Non-viral delivery vehicles and methods can also be used, including but not limited to polycation-condensed DNA linked to or not linked to adenovirus alone (see, e.g., Curiel, Hum. Gene Ther. (1992) 3: 147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989) 264: 16985); eukaryotic cell delivery vehicle cells (see, e.g., U.S. Patent No. 5,814,482; PCT Publication No. WO 95 / 07994; WO 96 / 17072; WO 95 / 30763; and WO 97 / 42338) and nucleic acid charge neutralization or fusion with cell membranes. Naked DNA can also be used. Exemplary naked DNA introduction methods are described in PCT Publication No. WO 90 / 11092 and U.S. Patent No. 5,580,859. Liposomes that can serve as gene delivery vehicles are described in U.S. Patent No. 5,422,120; PCT Publication Nos. WO 95 / 13796; WO 94 / 23697; WO 91 / 14445; and European Patent No. 0 524 968. Additional methods are described in Philip, Mol. Cell. Biol. (1994) 14:2411 and Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

[0251] The particular dosage regimen, ie, dosage, timing, and repetition, used in the methods described herein will depend on the particular subject and the subject's medical history.

[0252] In some embodiments, more than one antibody or a combination of an antibody and another suitable therapeutic agent may be administered to a subject in need of treatment. Antibodies may also be used in conjunction with other agents for enhancing and / or supplementing the effectiveness of the agent. The therapeutic efficacy of a target disease / disorder (e.g., those disclosed herein) may be assessed by methods well known in the art.

[0253] In some embodiments, any anti-LILRB2 antibody disclosed herein can be used in combination with other anti-cancer agents, such as chemotherapeutic agents, immunotherapeutic agents, or combinations thereof. As used herein, "combination," "combination," and related terms refer to the simultaneous or sequential administration of multiple therapeutic agents according to the present disclosure. For example, any anti-LILRB2 antibody or any bispecific antibody described herein can be administered simultaneously with another therapeutic agent, or administered sequentially in different dosage forms, or administered together in a single dosage form.

[0254] Diagnostic applications

[0255] Any of the anti-LILRB2 antibodies disclosed herein can be used to detect and quantify LILRB2 protein levels in biological samples using conventional methods (e.g., any immunohistological method known to those skilled in the art) (see, e.g., Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen et al., J. Cell Biol. 105:3087-3096 (1987)). Other antibody-based methods that can be used to detect LILRB2 protein expression include immunoassays, such as enzyme-linked immunosorbent assays (ELISAs), immunoprecipitations, or Western blotting. Suitable assays are described in more detail elsewhere herein.

[0256] The term "biological sample" means any biological sample obtained from an individual, cell line, tissue culture, or other cell source potentially expressing LILRB2. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art.

[0257] To perform the methods disclosed herein, any of the anti-LILRB2 antibodies disclosed herein can be combined with a mouse suspected of containing a target antigen disclosed herein (e.g., human LILRB2 protein or LILRB2 protein). + In general, the term "contacting" or "in contact" refers to contacting an anti-LILRB2 antibody disclosed herein with a sample suspected of containing a target antigen under suitable conditions and for a suitable period of time sufficient to form a complex between the anti-LILRB2 antibody and the target antigen in the sample, if any. The antibody-antigen complex (if any) so formed can be determined by conventional methods. Detection of such an antibody-antigen complex after incubation indicates the presence of the target antigen in the sample. When desired, the amount of the antibody-antigen complex can be quantified, which is indicative of the level of the target antigen in the sample.

[0258] In some examples, the anti-LILRB2 antibodies described herein can be conjugated to a detectable label, which can be any agent capable of directly or indirectly releasing a detectable signal. The presence or signal intensity of such a detectable signal indicates the presence or amount of the target antigen in the sample. Alternatively, a secondary antibody specific for the anti-LILRB2 antibody or for the target antigen can be used in the methods disclosed herein. For example, when the anti-LILRB2 antibody used in the method is a full-length antibody, the secondary antibody can bind to the constant region of the anti-LILRB2 antibody. In other cases, the secondary antibody can bind to an epitope of the target antigen that is different from the binding epitope of the anti-LILRB2 antibody. Any secondary antibody disclosed herein can be conjugated to a detectable label.

[0259] Any suitable detectable label known in the art can be used in the assay methods described herein. In some embodiments, the detectable label can be a label that directly releases a detectable signal. Examples include fluorescent labels or dyes. Fluorescent labels include fluorophores, which are fluorescent chemical compounds that can re-emit light after light excitation. Examples of fluorescent labels include, but are not limited to, xanthene derivatives (e.g., fluorescein, rhodamine, Oregon Green, Eosin, and Texas Red), cyanine derivatives (e.g., cyanine, indocarbocyanine, oxocarbocyanine, thiocarbocyanine, and merocyanine), squaric acid derivatives and ring-substituted squaric acids (e.g., Seta and Square dyes), squaric acid rotaxane derivatives such as SeTau dyes, naphthalene derivatives (e.g., dansyl and sodium fluorosilicate derivatives), coumarin derivatives, oxadiazole derivatives (e.g., The detectable label may be a molecule comprising a chromophore, which is responsible for the color of the dye. In some examples, the detectable label may be fluorescein isothiocyanate (FITC), phycoerythrin (PE), biotin, allophycocyanin (APC), or Alexa Fluor. 488.

[0260] In some embodiments, the detectable label can be a molecule that releases a detectable signal indirectly, for example, by converting the reagent into a product that directly releases a detectable signal. In some examples, such a detectable label can be an enzyme (e.g., β-galactosidase, HRP, or AP) that can produce a colored product from a colorless substrate.

[0261] IV. Kits containing anti-LILRB2 antibodies and their uses

[0262] The present disclosure also provides a product package comprising any anti-LILRB2 antibody disclosed herein. Such a product package can be used for any application of such an antibody disclosed herein, for example, for treating or ameliorating a target disease (such as a cancer disclosed herein), or for detecting LILRB2 protein or LILRB2 in a biological sample. + Such a kit can comprise one or more containers comprising an anti-LILRB2 antibody or bispecific antibody as described herein.

[0263] In some embodiments, the kit may include instructions for use according to any of the methods described herein. The included instructions may include a description of administering an anti-LILRB2 antibody or bispecific antibody as described herein to treat, delay the onset of, or alleviate the target disease. The kit may also include instructions for selecting an individual suitable for treatment based on identifying whether the individual has the target disease. In other embodiments, the instructions include a description of administering the antibody to an individual at risk for the target disease.

[0264] Instructions for use of an anti-LILRB2 antibody typically include information about the dosage, dosing schedule, and route of administration for the intended treatment. The container can be a unit dose, bulk package (e.g., a multi-dose package), or a subunit dose. The instructions provided in the packaged products of the present disclosure are typically written on a label or package insert (e.g., a paper included in the packaged product), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0265] The label or package insert indicates that the composition is used to treat, delay the onset of, and / or alleviate a disease, such as cancer or an immune disorder (eg, an autoimmune disease).Instructions for practicing any of the methods described herein can be provided.

[0266] The set of products of the present invention is in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, cans, flexible packaging (e.g., sealed Mylar or plastic bags), etc. Also contemplated are packaging used in combination with specific devices (e.g., inhalers, nasal administration devices (e.g., nebulizers), or infusion devices (e.g., mini pumps). The set of products can have a sterile access port (e.g., the container can be an intravenous solution bag or vial with a stopper pierceable by a hypodermic injection needle). The container can also have a sterile access port (e.g., the container can be an intravenous solution bag or vial with a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an anti-LILRB2 antibody or bispecific antibody as described herein.

[0267] The package product can optionally provide additional components such as buffers and explanatory information. Typically, the package product comprises a container and a label or package insert that is positioned on or associated with the container. In some embodiments, the invention provides an article of manufacture comprising the contents of the package product described above.

[0268] General techniques

[0269] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are fully explained in the following documents: for example, Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ Gait, ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JECellis, ed., 1989) Academic Press; Animal Cell Culture(RIFreshney,ed.1987);Introduction to Cell and Tissue Culture(JPMather and PERoberts,1998)Plenum Press;Cell and Tissue Culture:Laboratory Procedures(A.Doyle,JBGriffiths,and DGNewell,eds.1993-8)J.Wiley and Sons;Methods inEnzymology(Academic Press,Inc.);Handbook of Experimental Immunology(DMWeirand CCBlackwell, eds.): Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos, eds., 1987); Current Protocols in Molecular Biology (FMAusubel, et al. eds. 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994); Current Protocols in Immunology (JEColigan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P.Shepherd and C.Dean, eds., Oxford University Press, 2000); Using antibodies: alaboratory manual (E.Harlow and D.Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M.Zanetti and JDCapra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (DNGlover ed.1985); Nucleic Acid Hybridization (BD Hames & S.J. Higgins, eds. (1985); Transcription and Translation (BD Hames & S.J. Higgins, eds. (1984); Animal Cell Culture (RI Freshney, ed. (1986)); Immobilized Cells and Enzymes (lRL Press, (1986)); and B. Perbal, A practical Guide To Molecular Cloning (1984); FMAusubel et al. (eds.).

[0270] Without further elaboration, it is believed that one skilled in the art can utilize the present invention to the greatest extent possible based on the above description. Therefore, the following specific examples should be interpreted as merely illustrative and not limiting in any way the remainder of the present disclosure. All disclosures cited herein are incorporated by reference for the purposes or subject matter cited herein.

[0271] Example 1 : Generation and characterization of mouse anti-LILRB2 antibodies

[0272] This example demonstrates the generation of mouse anti-LILRB2 antibodies via conventional hybridoma technology and the characterization of exemplary clones for binding specificity and affinity to the target antigen.

[0273] A. Generation of Anti-LILRB2 Antibodies

[0274] The plasmid encoding full-length human LILRB2 was mixed with jetPEI in vivo TM The cells were mixed with 50 μg of plasmid (plasmid 50 μg) of 5 μg of plasmid (plasmid 50 μg) of 5 μg of plasmid (plasmid 50 μg) of 5 μg of plasmid (plasmid 50 μg) of 5 μg of plasmid) and injected intravenously into female BALB / c mice via the tail vein according to the manufacturer's instructions. Three injections were performed at 3-week intervals. Ten days after the final DNA injection, an additional 40 μg of plasmid was injected intravenously using the same method as above, and 5 million 293T cells overexpressing human LILRB2 (HEK293T-LILRB2) were also injected intraperitoneally for a boost. Three days later, the mice were euthanized, and splenocytes were collected for fusion.

[0275] To generate monoclonal hybridomas, mouse myeloma cells Sp2 / 0 were grown to the logarithmic growth phase and fused with immune mouse spleen cells at a ratio of 1:2 or 1:3 in the presence of polyethylene glycol / dimethyl sulfoxide (PEG / DMSO; 45% / 5%) solution (Hybri-max, Sigma, P7181, D2650). The cells were seeded in 96-well plates and selectively cultured with hypoxanthine-aminopterin-thymidine (HAT) medium (Sigma, H0262) for 5 to 7 days. The HAT medium was then replaced with HT medium and cultured for another 5 to 7 days. When hybridoma cell clones were present in the 96-well plates, the culture supernatant was used for cell ELISA of HEK293T-LILRB2 and HEK293T simultaneously. Clones that reacted only with HEK293T-LILRB2 were routinely subcloned. After 3 to 5 rounds of subcloning, until all single clones reacted positively with HEK293T-LILRB2 alone, clones were selected to establish hybridoma cell lines.

[0276] To generate monoclonal antibodies for characterization, selected monoclonal hybridoma cells were injected into the peritoneal cavity of Balb / C mice to produce monoclonal antibodies in the ascites. Monoclonal antibodies were purified from the ascites using protein G for further characterization.

[0277] B. Characterization of Exemplary Anti-LILRB2 Antibodies

[0278] (i) ELISA to detect the binding specificity of LILRB1, LILRB2 and LILRB3

[0279] 2x10 4 293T cells, 293T-LILRB1 cells, 293T-LILRB2 cells or 293T-LILRB3 cells were placed in each well of a 96-well plate and cultured for 18 hours. The cells were then fixed with glutaraldehyde for 2 hours at room temperature in the presence of 5% skim milk. Anti-LILRB2 antibody (2 μg / ml) was added to each well and incubated at room temperature for 2 hours. HRP-labeled goat anti-mouse secondary antibody was then added to each well and incubated at room temperature for 1 hour. HRP substrate o-phenylenediamine dihydrochloride (OPD) was then added to each well. The plate was incubated at room temperature for 15 minutes to allow color development and the OD was measured. 490 value.

[0280] like Figure 1A As shown, most clones showed high binding specificity for LILRB2 relative to LILRB1 and / or LILRB3 (e.g., for clone 9B6, the OD490 for LILRB2, LILRB1 and LILRB3 binding were 1.426, 0.1215 and 0.1255, respectively; for clone 14F1, the OD490 for LILRB2, LILRB1 and LILRB3 binding were 1.2115, 0.1715 and 0.1725, respectively; for clone 9H6, the OD490 values ​​for LILRB2, LILRB1 and LILRB3 binding were 1.8625, 0.177 and 0.139, respectively), and clones such as 13C4 showed some cross-reactivity on the LILRB antigen.

[0281] Binding specificity was also determined in ELISA assays using recombinant LILRB1, LILRB2, and LILRB3 proteins. Similar results were observed, as shown in Figure 5. Figure 1B As shown in .

[0282] (ii) Flow cytometry analysis of the binding specificity of human and nonhuman primate LILRB2

[0283] 2x10 5 293T cells, 293T-LILRB1 cells, 293T-LILRB2 cells, 293T-LILRB3 cells, or 293T-cynomolgus monkey-LILRB2 cells were placed in each well of a 96-well plate and cultured for 18 hours. The cells were then fixed with glutaraldehyde in the presence of 5% skim milk at room temperature for 2 hours. Anti-LILRB2 antibody (5 μg / ml) was added to each well and incubated at room temperature for 1 hour. Alex Labeled goat anti-mouse secondary antibody (1:2,000 dilution) was added to each well and incubated at room temperature for 30'. After staining, the cells were washed twice with PBS. The fluorescence intensity (MFI) was measured. The results are shown in Figure 2ACertain clones (e.g., 9B6 and 9H6) showed high binding specificity for LILRB2 and cross-reacted with both human and cynomolgus monkey LILRB2 antigens.

[0284] (iii) Binding activity was detected by flow cytometry

[0285] 2x10 5 CHO-K1 cells and CHO-K1 cells expressing human LILRB2 (CHO-K1-LILRB2 cells) were placed in each well of a 96-well plate and cultured for 18 hours. The cells were then fixed with glutaraldehyde in the presence of 5% skim milk at room temperature for 2 hours. Anti-LILRB2 antibody (5 μg / ml) was added to each well and incubated at room temperature for 1 hour. Alex Labeled goat anti-mouse secondary antibody was added to each well and incubated at room temperature for 30'. The cells were then analyzed by flow cytometry. The results are shown in Figure 2B All clones tested showed no binding to parental CHO-K1 cells and all displayed high binding activity to CHO-K1-LILRB2 cells.

[0286] (iv) Binding affinity of exemplary anti-LILRB2 antibodies was tested by ELISA

[0287] Different concentrations of anti-LILRB2 antibodies were used in the ELISA assay as described above. Binding affinity was determined according to conventional methods and is provided in Table 5 below.

[0288] Table 5. Binding affinities of exemplary anti-OLILRB2 antibodies

[0289]

[0290]

[0291] (v) Detection of Binding Affinity of Exemplary Anti-LILRB2 Antibodies via SPR

[0292] Surface plasmon resonance (SPR) was used to detect the binding activity of the exemplary anti-LILBR2 antibodies disclosed herein. Briefly, the anti-LILBR2 antibodies were diluted to a concentration of 30 μg / ml in coating buffer and loaded onto a CM5 chip at 10 μl / min to allow the antibody to be coated on the surface of the CM5 chip. His-tagged human LILBR2 polypeptides were diluted in loading buffer to produce samples with antigen concentrations ranging from 125 nM to 0.244 nM. Binding affinities were measured based on a 1:1 binding model. The results are shown in Table 6 below.

[0293] Table 6. Anti-LILBR2 antibody binding activity determined by SPR

[0294]

[0295] (vi) Exemplary anti-LILBR2 antibodies block HLA-A2 binding in CHO-K1-huLILRB2 cells

[0296] A preliminary experiment was performed to determine the parameters for this assay. PE-labeled HLA-A2 tetramers at a concentration of 0.25 μg / ml and anti-LILRB2 antibodies at a concentration of 2.5 μg / ml were incubated with CHO-K1-huLILRB2 cells for 10 minutes. The cells were washed twice and then analyzed by flow cytometry. Many anti-LILBR2 antibodies showed high levels of blocking HLA-A2 binding to CHO-K1-huLILRB2 cells. Figure 3A .

[0297] Clones 11B12, 9B6, and 9H6 were selected for dose-dependent blocking assays of HLA-A2 binding to CHO-K1-huLILRB2 cells. Mouse IgG (mIgG) antibody was used as a negative control. The results are shown in Figure 3B Determination of the IC values ​​of clones 11B12, 9B6, and 9H6 50 The values ​​were 0.475 nM, 0.34 nM and 4.972 nM, respectively.

[0298] (vi) Exemplary anti-LILBR2 antibodies block HLA-G binding in 293T-huLILRB2 and THP1-huLILRB2 cells combine

[0299] A preliminary experiment was performed to determine the parameters for this assay. PE-labeled HLA-G tetramers at a concentration of 0.3 μg / ml and anti-LILRB2 antibodies at concentrations of 0.02 μg / ml or 0.0002 μg / ml were incubated with 293T-huLILBR2 cells or THP1-huLILRB2 cells for 10 minutes. The cells were washed twice and then analyzed by flow cytometry. Many anti-LILBR2 antibodies showed high levels of blocking HLA-A2 binding to CHO-K1-huLILRB2 cells. Figure 3C .

[0300] Clones 11B12, 9B6, 9H6, and 14F1 were selected for dose-dependent blocking assays of HLA-G binding to CHO-K1-huLILRB2 cells. Mouse IgG (mIgG) antibody was used as a negative control. The results are shown in Figure 3D All anti-LILBR2 clones tested showed dose-dependent blocking of HLA-G tetramer binding to 293T-huLILBR2 cells. The IC50 values ​​for clones 11B12, 9B6, 9H6, and 14F1 were 0.8566 nM, 0.6181 nM, 3.121 nM, and 0.82 nM, respectively. Clone 9B6 showed the best blocking effect.

[0301] (viii) Exemplary anti-LILBR2 antibodies block ANGPTL2 binding to human LILRB2

[0302] His-tagged ANGPTL2 protein (1 μg / ml) was placed in a 96-well plate (100 μg / well) and incubated overnight at 4°C. Then, 0.25 mg / ml of LILRB2-Fc fusion protein was incubated with various concentrations of anti-LILRB2 antibody at room temperature for 1 hour and then added to the His-ANGPTL2-coated plate. The plate was placed in a 500 rpm shaker at room temperature for 2 hours. HRP-labeled goat anti-human Fc antibody (1:10,000 dilution) was added to the plate (100 μl / well). After incubation at room temperature for 1 hour, TMB was added for color development. Figure 3E As shown, many of the exemplary anti-LILRB2 antibodies tested showed blocking effects on the binding of ANGPTL2 to LILRB2.

[0303] Clones 11B12, 9B6, 9H6, and 14F1 were selected for dose-dependent blocking assays. Mouse IgG (mIgG) antibody was used as a negative control. The results are shown in Figure 3F All anti-LILBR2 clones tested showed a dose-dependent blockade of HLA-G tetramer binding to 293T-huLILBR2 cells. 50 The IC50 values ​​of clones 11B12, 9B6, 9H6, and 14F1 were 1.001 nM, 1.007 nM, 0.945 nM, and 0.942 nM, respectively.

[0304] (ix) Exemplary anti-LILBR2 antibodies enhance TNFα expression and inhibit IL-10 expression in LPS-stimulated PBMCs

[0305] Freshly isolated human peripheral blood mononuclear cells (PBMCs) were cultured in RPMI1640 supplemented with 10% fetal bovine serum (FBS). Cells were first treated with exemplary anti-OLILRB2 antibodies 11B12, 9B6, 9H6, or 14F1 and then stimulated with 100 ng / ml lipopolysaccharide (LPS). Supernatants were collected from the cell cultures and subjected to ELISA assays to measure levels of human tumor necrosis factor alpha (TNFα) and IL-10. Mouse IgG isotype antibodies were used as controls.

[0306] like Figure 4A and Figure 4B As shown, exemplary anti-LILRB2 antibodies enhanced the level of TNFα secreted by LPS-stimulated PBMCs, but inhibited IL-10 expression by LPS-stimulated PBMCs.

[0307] Example 2 : Generation and characterization of humanized anti-LILRB2 antibodies

[0308] This example demonstrates the generation of humanized anti-LILRB2 antibodies and the characterization of their binding and biological activity.

[0309] A. Generation of Humanized Antibodies

[0310] Clones 11B12, 9B6, and 14F1 were selected as exemplary parent mouse anti-LILRB2 antibodies for humanization according to conventional antibody humanization methods. See, e.g., the disclosure herein. Briefly, the heavy and light chain complementarity determining regions of the mouse parent clones were grafted onto appropriate human V H and V L In some cases, back mutations were performed at selected framework positions to return the amino acid residues in the human acceptor chain to those of the mouse parent clone. Exemplary humanized antibodies of clones 11B12, 9B6, and 14F1 are provided in Tables 2 to 4 above.

[0311] B. Characterization of Humanized Antibodies

[0312] (i) Binding activity to cell surface LILRB2

[0313] The binding affinity of humanized LILRB2 antibodies to 293T-LILRB2 cells was measured by flow cytometry. Briefly, serially diluted humanized anti-LILRB2 antibodies were incubated with 293T-LILRB2 cells at room temperature for 30 minutes, followed by staining with a secondary APC-labeled anti-human IgG antibody. The cells were washed and then subjected to flow cytometry to measure the level of fluorescent signal.

[0314] The binding activity of an exemplary humanized antibody derived from parental clone 11B12 is shown in Figures 5A to 5C middle. Figure 6 The binding activity of an exemplary humanized antibody derived from the parental clone 9B6 is shown.

[0315] (ii) Blockade of HLA-G binding to 293T-LILRB2 cells

[0316] 0.2 μg of His-tagged HLA-G tetramers were incubated with 293T-LILRB2 cells at room temperature for 45 minutes to allow HLA-G / LILRB2 binding. Serially diluted humanized LILRB2 antibodies were then added to the mixture and incubated for an additional 45 minutes at room temperature. HLA-G / LILRB2 binding was then detected by flow cytometry using a secondary PE-labeled anti-His tag antibody.

[0317] Figure 7A and Figure 7B It was shown that the exemplary humanized antibodies derived from 11B12 all blocked HLA-G binding to 293T-LILRB2 cells. Figure 7CAn exemplary humanized antibody derived from 9B6 is shown to block HLA-G binding to 293T-LILRB2 cells.

[0318] (iii) Blockade of HLA-A2 binding to 293T-LILRB2 cells

[0319] 0.2 μg of His-tagged HLA-A2 tetramers were incubated with 293T-LILRB2 cells at room temperature for 45 minutes to allow HLA-G / LILRB2 binding. Serially diluted h#2E1 antibody was then added to the mixture and incubated for an additional 45 minutes at room temperature. HLA-G / LILRB2 binding was then detected by flow cytometry using a secondary PE-labeled anti-His tag antibody.

[0320] Figure 8 An exemplary humanized antibody derived from 11B12 is shown to block HLA-A2 binding to 293T-LILRB2 cells.

[0321] Example 3 : Affinity Maturation and Characterization

[0322] Humanized antibody h#2E1 (derived from mouse parent 11B12) was selected for affinity maturation. Briefly, an affinity maturation library containing mutations at certain positions in the heavy and light chain CDRs was constructed using conventional methods. Exemplary mature anti-LILRB2 antibodies are provided in Table 2 above.

[0323] (i) Binding activity

[0324] This affinity maturation library was screened against human LILRB2 to identify antibodies with high binding affinity for LILRB2. Briefly, ELISA plates were coated with 0.1 μg / well recombinant human LILRB2 protein. Serial dilutions of mature h#2E1 antibody were incubated in the LILRB2-coated plates for 2 hours. The plates were washed, and a secondary HRP-labeled anti-human IgG was added to each well. After incubation at room temperature for 1 hour, a colorimetric ELISA signal was generated by adding TMB substrate, and the reaction was terminated with 1 M H3PO4. Table 7 below provides the binding activity of exemplary mature antibodies to recombinant LILRB2 protein.

[0325] In addition, flow cytometry was used to detect the binding of mature antibodies to 293T-LILRB2 cells as follows. Serial dilutions of exemplary mature anti-LILRB2 antibodies were incubated with 293T-LILRB2 cells and then incubated with a secondary APC-labeled anti-human IgG antibody. Fluorescence signals were detected by flow cytometry. Table 8 below provides the binding activity of exemplary mature antibodies to 293T-LILRB2 cells.

[0326] (ii) Blockade of HLA-G binding to 293T-LILRB2 cells

[0327] 0.2 μg of His-tagged HLA-G tetramers were incubated with 293T-LILRB2 cells at room temperature for 45 minutes to allow HLA-G / LILRB2 binding. Serially diluted humanized LILRB2 antibodies were then added to the mixture and incubated for an additional 45 minutes at room temperature. HLA-G / LILRB2 binding was then detected by flow cytometry using a secondary PE-labeled anti-His-tag antibody.

[0328] All tested mature anti-LILRB2 antibodies successfully blocked HLA-G binding to 293T-LILRB2 cells, as Figures 9A to 9C shown.

[0329] Table 7. Binding activity of exemplary mature anti-LILRB2 antibodies to recombinant LILRB2 protein

[0330]

[0331] Table 8. Binding activity of exemplary mature anti-LILRB2 antibodies to 293T-LILRB2 cells

[0332]

[0333]

[0334] (iii) Blockade of HLA-A2 binding to 293T-LILRB2 cells

[0335] 0.2 μg of His-tagged HLA-A2 tetramers were incubated with 293T-LILRB2 cells at room temperature for 45 minutes to allow HLA-G / LILRB2 binding. Serially diluted h#2E1 antibodies were then added to the mixture and incubated for an additional 45 minutes at room temperature. HLA-G / LILRB2 binding was then detected by flow cytometry using a secondary PE-labeled anti-His tag antibody.

[0336] All tested mature anti-LILRB2 antibodies successfully blocked HLA-A2 binding to 293T-LILRB2 cells, as shown in Tables 9 and Figure 10 shown.

[0337] Table 9. Blocking activity of exemplary mature anti-LILRB2 antibodies in binding of HLA-A2 to 293T-LILRB2 cells

[0338]

[0339] Example 4. Humanized anti-LILRB2 antibody induces macrophage M1 polarization in the PBMC / LPS model

[0340] This example explores the ability of exemplary humanized anti-LILRB2 antibodies to induce macrophage M1 polarization in a PBMC / LPS model. Briefly, freshly isolated human peripheral blood mononuclear cells (PBMCs) were cultured in RPMI1640 supplemented with 10% FBS. Cells were treated with exemplary humanized anti-LILRB2 antibodies (including clones 2E1, 2E1_FC11 (A16), 2E1_GC6 (A26), and 2E1_FG9 (A3)) for 48 hours and then stimulated with 100 ng / ml lipopolysaccharide (LPS) for 24 hours. Culture supernatants were collected and the levels of hTNFα and IL10 were measured using ELISA. 11A to 11D As shown, the exemplary humanized anti-LILRB2 antibodies tested, particularly clone 2E1_FC11 (A16), induced macrophage polarization to the pro-inflammatory M1 phase, as evidenced by increased TNFα levels and decreased IL10 levels.

[0341] Example 5. Humanized anti-LILRB2 antibody induces macrophage M1 polarization in the human monocyte-derived macrophage HMDM / LPS model

[0342] Human peripheral blood mononuclear cells (PBMC) were isolated from human donors according to conventional practice. Anti-CD33 beads were used to isolate monocytes from PBMC. The isolated monocytes were incubated for 6 days in a culture medium containing M-CSF to allow differentiation into macrophages. The cells were then treated for 24 hours using clone 2E1_FC11 as an example or with human IgG4 as a control. The cells were stimulated with 100 ng / ml LPS for 24 hours, and the level of TNFα in the culture supernatant was measured by ELISA as a reading of M1 polarization.

[0343] like Figure 12 As shown, clone 2E1_FC11 successfully induced macrophage M1 polarization, as indicated by the secretion of TNFα in the culture supernatant.

[0344] Example 6. CD8+ T cell activation by humanized anti-LILRB2 antibodies in an autologous mixed lymphocyte reaction assay

[0345] Human peripheral blood mononuclear cells (PBMCs) were isolated from human donors according to routine practice. CD33+ monocytes were isolated and differentiated into macrophages as 6-day cultures cultured with CSF and test antibodies. CD8+ T cells were isolated and added to the differentiated macrophages and cultured for 4 days with test antibodies and anti-CD3 antibodies. Single-domain anti-PDL1 antibody A105 or anti-PD1 antibody nivolumab were used as controls to activate T cells in an automated MLR system. The levels of IFNγ and GM-CSF in the culture supernatant were determined by ELISA as a readout of T cell activation.

[0346] like 13A to 13D As shown, clone 2E1_FC11 displayed better activity in inducing CD8+ T cell activation compared with the PD1 inhibitor A105 and nivolumab.

[0347] Other implementation plans

[0348] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Therefore, unless expressly stated otherwise, each feature disclosed is merely an example of a general series of equivalent or similar features.

[0349] From the above description, those skilled in the art can easily determine the essential characteristics of the present invention, and without departing from the spirit and scope of the present invention, various changes and modifications can be made to the present invention to adapt it to various uses and conditions. Therefore, other embodiments are also within the claims.

[0350] Equivalent solutions

[0351] Although several embodiments of the present invention have been described and illustrated herein, a person of ordinary skill in the art will readily recognize various other means and / or structures for performing the functions described herein and / or obtaining the results and / or one or more advantages, and each of such variations and / or modifications is considered to be within the scope of the embodiments of the present invention described herein. More generally, a person of ordinary skill in the art will readily understand that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations depend on the specific application or applications in which the teachings of the present invention are used. Using only routine experimentation, a person skilled in the art will recognize or be able to ascertain many equivalents to the specific embodiments of the present invention described herein. Therefore, it should be understood that the foregoing embodiments are presented by way of example only, and that within the scope of the appended claims and their equivalents, embodiments of the present invention may be practiced in a manner other than that specifically described and claimed. The embodiments of the present invention disclosed herein are directed to each individual feature, system, article, material, package, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, packages, and / or methods is included within the scope of the present invention disclosed herein, provided that such features, systems, articles, materials, packages, and / or methods are not mutually inconsistent.

[0352] All definitions, as defined and used herein, should be understood to limit to dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0353] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which they are cited, which in some cases may be incorporated in their entirety.

[0354] As used herein in the specification and claims, the indefinite articles "a" and "an" should be understood to mean "at least one," unless explicitly stated to the contrary.

[0355] As used herein in the specification and claims, the phrase "and / or" should be understood to mean "either or both" of the elements so combined, i.e., elements that are present in combination in some cases and separately in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more elements" of the elements so combined. In addition to the elements specifically identified by the "and / or" clause, other elements may optionally be present, whether related or unrelated to those specifically identified. Thus, as a non-limiting example, when used in conjunction with open language such as "comprising," a reference to "A and / or B" may refer to only A (optionally including elements other than B) in one embodiment; to only B (optionally including elements other than A) in another embodiment; to both A and B (optionally including other elements) in yet another embodiment; etc.

[0356] As used herein in the specification and claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when the items in the list are separated, "or" or "and / or" should be interpreted as inclusive, that is, including at least one element in many elements or element lists, but also including more than one element and optionally other unlisted items. Only explicitly point out that the opposite term such as "only one" or "just one" or when used in the claims, "consisting of ... " refers to including just one element in many elements or element lists. Usually, when there is an exclusive term before, such as "any one", "one in ... ", "only one in ... " or "just one in ... ", the term "or" as used herein should only be interpreted as indicating exclusive alternatives (that is, "one or another rather than two"). When used in the claims, "substantially consisting of ... " should have the common meaning as used in the field of patent law.

[0357] As used herein in the specification and claims, the phrase "at least one" with respect to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one element from each element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows for the optional presence of elements in addition to the elements specifically identified in the list of elements to which the term "at least one" refers, whether related or unrelated to the elements specifically identified. Thus, as a non-limiting example, in one embodiment, "at least one of A and B" (or equivalently, "at least one of A or B," or equivalently, "at least one of A and / or B") can refer to at least one that optionally includes more than one A, is absent B (and optionally includes elements other than B); in another embodiment, can refer to at least one that optionally includes more than one B, is absent A (and optionally includes elements other than A); in yet another embodiment, can refer to at least one that optionally includes more than one A and at least one that optionally includes more than one B (and optionally includes other elements); etc.

[0358] It should also be understood that in any method claimed herein that includes more than one step or action, the order of the method steps or actions is not necessarily limited to the order of the method steps or actions recited unless explicitly stated to the contrary.

Claims

1. An antibody that binds to human leukocyte immunoglobulin-like receptor 2 (LILRB2) (anti-LILRB2 antibody), wherein the anti-LILRB2 antibody comprises: (a) Heavy chain variable region (V H ), which contains (ai) a heavy chain CDR1 comprising GX1SITSGYX2WX3 (SEQ ID NO 192), wherein X1 is Y or G, X2 is Y, S, A or H, and X3 is N, S or W; (aii) a heavy chain CDR2 comprising X4ISYDGNX5X6 (SEQ ID NO: 194), wherein X4 is S, T, or Y, X5 is A, I, K, L, M, N, P, S, T, V, or W, and X6 is H, L, N, S, or W; optionally, wherein the heavy chain CDR2 comprises X4ISYDGNX5X6YNPSLKN (SEQ ID NO: 416); (aiii) heavy chain CDR3 comprising EEX7TMX8TTX9WFX 10 Y (SEQ ID NO: 196), wherein X7 is A or S, X8 is F or I, X9 is A or G, and X10 is A, G, Q or S; and (b) Light chain variable region (V L ), which contains (bi) light chain CDR1 comprising X 11 X 12 SEX 13 IX 14 SNX 15 A (SEQ ID NO: 199), wherein X 11 is E, G, L, N, Q, R, S, T or V, X 12 Is A or G, X 13 is K, N, T or V, X 14 is F or Y, and X 15 is L, N or Q; (bii) Light chain CDR2, which contains GATX 16 X 17 X 18 X 19 (SEQ ID NO:201), wherein X16 is E, N, W or Y, X17 is F, H, L, P or R, X18 is A, N, R or S, and X19 is A, G, K, L, R, S or V; and (biii) light chain CDR3 comprising QX 20 FWf 21 PYX 22 (SEQ ID NO: 203), wherein X 20 It is H or Q, X 21 is L, S, T, or Y, and X 22 It is I, M, R, T, Y or V.

2. The antibody of claim 1, wherein X1 is Y, X2 is Y, X3 is N, X4 is Y, X5 is I, X6 is N, X7 is S, X8 is I, X9 is A, X 10 is A, or a combination thereof; and / or wherein X 11 It's R, X 12 It's A, X 13 It is N, X 14 It's Y, X 15 It's L, X 16 It is N, X 17 It's L, X 18 It's A, X 19 It's S, X 20 It's H, X 21 It's Y, X 22 is T, or a combination thereof.

3. The antibody of claim 1 or claim 2, wherein the heavy chain CDR3 is EESTMITTAWFAY (SEQ ID NO: 11); and / or the light chain CDR3 is QHFWDYPYT (SEQ ID NO: 247).

4. The antibody of claim 1, wherein the anti-LILRB2 antibody comprises the same heavy chain CDR1, CDR2, and CDR3 as the antibodies listed in Table 2, and the same light chain CDR1, CDR2, and CDR3.

5. The antibody of claim 1, wherein the anti-LILRB2 antibody comprises heavy chain CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 9, 243, and 11, respectively, and light chain CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 13, 14, and 247, respectively.

6. The antibody of claim 1 , wherein the heavy chain CDR1, CDR2 and CDR3 of the anti-LILRB2 antibody comprise up to five amino acid residue variations relative to SEQ IDs: 9, 243 and 11, and / or the light chain CDR1, CDR2 and CDR3 of the anti-LILRB2 antibody comprise up to five amino acid variations relative to SEQ IDs: 13, 14 and 247.

7. The antibody of any one of claims 1 to 6, wherein the anti-LILRB2 antibody is a humanized antibody.

8. The antibody according to claim 7, wherein the V H Contains V H Frame 1 (FR1), V H Frame 2 (FR2), V H Framework 3 (FR3) and V H Frame 4 (FR4), and the V L Contains V L FR1, V L FR2, V L FR3 and V L FR4; in: The V H FR1 contains EVQLVESGGGLVQPGGSLRLSCAZ1S, where Z1 is A or V, The V H FR2 contains WZ2RQAPGKGLEWVA, where Z2 is I or V, The V H FR3 comprises RFTISRDZ3SKNTLZ4LQMNSLRAEDTAVYYCZ5R, wherein Z3 is A, D, or T, Z4 is F, L, or V, and Z5 is A or V, and The V H FR4 includes WGQGTLVTVSS; as well as in: The V L FR1 contains DIQZ6TQSPSSLSASVGDRVTITC, where Z6 is L or M, The V L FR2 contains WYQQKPGKAPKLLIY, The V L FR3 contains GVPSRFSGSGSGTDZ7TLTISSLQPEDFATYYC, where Z7 is F or Y, and The V L FR4 contains FGQGTKVEIK.

9. The antibody of claim 5, wherein the V of the anti-LILRB2 antibody is H comprising an amino acid sequence at least 85% identical to SEQ ID NO: 244; and / or wherein the V L Comprising an amino acid sequence at least 85% identical to SEQ ID NO:

248.

10. The antibody of claim 9, wherein the V of the anti-LILRB2 antibody is H comprising the amino acid sequence of SEQ ID NO: 244; and / or wherein the V L Comprising the amino acid sequence of SEQ ID NO:

248.

11. The antibody of claim 1, wherein the anti-LILRB2 antibody is one of those listed in Table 2.

12. The antibody of any one of claims 1 to 11, wherein the anti-LILRB2 antibody is a full-length antibody or an antigen-binding fragment thereof.

13. A nucleic acid or a collection of nucleic acids comprising a nucleotide sequence encoding the anti-LILRB2 antibody of any one of claims 1 to 12. 14 . The nucleic acid or a collection of nucleic acids according to claim 13 , which is a vector or a collection of vectors comprising a nucleotide sequence encoding the anti-LILRB2 antibody.

15. The nucleic acid or collection of nucleic acids according to claim 14, wherein the vector is an expression vector.

16. A host cell or a collection of host cells comprising the nucleic acid of any one of claims 13 to 15.

17. The host cell or host cell collection according to claim 16, which is a mammalian cell, a yeast cell or a bacterial cell.

18. A pharmaceutical composition comprising (a) the anti-LILRB2 antibody of any one of claims 1 to 12, or a nucleic acid or a collection of nucleic acids encoding the antibody, and a pharmaceutically acceptable carrier.

19. A method for modulating an immune response, comprising administering to a subject in need thereof an effective amount of the anti-LILRB2 antibody of any one of claims 1 to 12, a nucleic acid or a collection of nucleic acids encoding the antibody, or a pharmaceutical composition comprising the antibody or the encoding nucleic acid.

20. The method of claim 19, wherein the subject is a human patient having or suspected of having cancer.

21. A method for preparing an anti-LILRB2 antibody, comprising: Culturing the host cell or collection of host cells according to claim 16 or claim 17 under conditions that allow expression of the antibody, and The antibodies thus produced are harvested.

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