Anti-SIRP-β1 antibodies and methods of use thereof

Abstract

The present disclosure generally relates to compositions comprising antibodies (eg, monoclonal antibodies, antibody fragments, etc.) that specifically bind to SIRPβ1 polypeptides (eg, human SIRPβ1), and the use of such compositions in preventing, reducing risk, or treating individuals in need thereof.

Description

[0001] Cross-citation of related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 691,913, filed June 29, 2018, which is incorporated herein by reference in its entirety for any purpose. Invention Field

[0003] This disclosure relates to anti-SIRPβ1 antibodies and the therapeutic use of such antibodies. Background of the Invention

[0005] Signal regulatory protein β (SIRPβ) belongs to the SIRP family of transmembrane receptors, expressed in myeloid cell lineages (including monocytes, macrophages, granulocytes, and dendritic cells) and in neurons. SIRP family proteins are characterized by an extracellular domain containing two juxtamembranous IgC domains and one distal IgV domain. Unlike SIRPα receptors, SIRPβ proteins contain a short cytoplasmic domain lacking the cytoplasmic sequence motifs that recruit protein tyrosine phosphatases SHP-2 and SHP-1. SIRPβ1 isoform 1 associates with the adaptor protein DAP12, which contains a single cytoplasmic immune receptor-based tyrosine activation (ITAM) motif. See, for example, Dietrich et al., 2000, J Immunol 164:9-12; Liu et al., 2005, J Biol Chem 280:36132-36140. SIRPβ has been shown to be a microglial regulator of phagocytosis in Alzheimer's disease. See, for example, Gaikwad et al., 2009, Am J Pathol 175:2528-2539.

[0006] Therefore, therapeutic anti-SIRPβ1 antibodies are needed to treat diseases, symptoms, and conditions associated with SIRPβ1 activity. Invention Overview

[0008] In some embodiments, an isolated antibody binding to human SIRPβ1 is provided, wherein the antibody has at least one, at least two, at least three, at least four, or at least five properties selected from the following:

[0009] a) Binds to human SIRPβ1 isotype 1, but not human SIRPα;

[0010] b) Binds to human SIRPβ1 isotype 1, but not human SIRPγ;

[0011] c) Binds to human SIRPβ1 isotype 1, but not to human SIRPβ1 isotype 3;

[0012] d) Binds to human SIRPβ1 isotype 1, but not to mouse SIRPβ1;

[0013] e) Combined with human SIRPβ1 isotype 1, but not with cynomolgus monkey SIRPβ1 isotype 1;

[0014] f) Activate SIRPβ1 activity in CD14-positive monocytes in vitro and / or in vivo;

[0015] g) Inducing or enhancing respiratory bursts in immune cells, such as neutrophils and / or monocytes, in vitro and / or in vivo;

[0016] h) Inducing or enhancing IL-8 expression in monocytes in vitro and / or in vivo;

[0017] i) Inducing or enhancing TNFα expression in macrophages and / or dendritic cells in vitro and / or in vivo;

[0018] j) Inducing neutrophil-mediated phagocytosis, such as phagocytosis by tumor cells, in vitro and / or in vivo;

[0019] k) Enhance neutrophil-mediated tumor cell clearance in vivo;

[0020] l) Enhance TREM2 expression on macrophages in vitro and / or in vivo;

[0021] m) Enhances macrophage viability in vitro and / or in vivo, alone and / or in combination with agonistic anti-TREM2 antibodies; and

[0022] n) Enhance the viability of dendritic cells in vitro and / or in vivo.

[0023] In some embodiments, the antibody binds to human SIRPβ1 isotype 1 but not human SIRPα or human SIRPβ1 isotype 3. In some embodiments, the antibody binds to human SIRPβ1 isotype 1 but not human SIRPα, SIRPγ, or human SIRPβ1 isotype 3. In some embodiments, the antibody has at least one, at least two, or at least three of the following properties:

[0024] a) Activating SIRPβ1 activity in CD14-positive monocytes in vitro and / or in vivo;

[0025] b) Inducing or enhancing respiratory bursts in immune cells, such as neutrophils and / or monocytes, in vitro and / or in vivo;

[0026] c) Inducing or enhancing IL-8 expression in monocytes in vitro and / or in vivo;

[0027] d) Inducing or enhancing TNFα expression in macrophages and / or dendritic cells in vitro and / or in vivo;

[0028] e) Inducing neutrophil-mediated phagocytosis, such as phagocytosis by tumor cells, in vitro and / or in vivo;

[0029] f) Enhance neutrophil-mediated tumor cell clearance in vivo;

[0030] g) Enhance TREM2 expression on macrophages in vitro and / or in vivo;

[0031] h) Enhances macrophage viability in vitro and / or in vivo, alone and / or in combination with agonistic anti-TREM2 antibodies; and

[0032] i) Enhance dendritic cell viability in vitro and / or in vivo.

[0033] In some embodiments, an isolated antibody that binds to human SIRPβ1 is provided, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises (a) HVR-H1 comprising the HVR-H1 amino acid sequence shown in Table 4 and / or Table 9; (b) HVR-H2 comprising the HVR-H2 amino acid sequence shown in Table 4 and / or Table 9; (c) HVR-H3 comprising the HVR-H3 amino acid sequence shown in Table 4 and / or Table 9; the light chain variable region comprises (d) HVR-L1 comprising the HVR-L1 amino acid sequence shown in Table 3 and / or Table 8; (e) HVR-L2 comprising the HVR-L2 amino acid sequence shown in Table 3 and / or Table 8; and (f) HVR-L3 comprising the HVR-L3 amino acid sequence shown in Table 3 and / or Table 8. In some embodiments, the heavy chain variable region comprises one, two, three, or four frame regions selected from VH FR1, VH FR2, VH FR3, and VH FR4 shown in Table 6. In some embodiments, the light chain variable region comprises one, two, three, or four frame regions selected from VL FR1, VL FR2, VL FR3, and VL FR4 shown in Table 5.

[0034] In some embodiments, the antibody comprises SB-1, SB-2, SB-3, SB-4, SB-5, SB-6, SB-7, SB-8, SB-9, SB-10, SB-11, SB-12, SB-13, SB-14, SB-15, SB-16, SB-17, SB-18, SB-19, SB-20, SB-21, SB-22, SB-23, SB-24, SB-25, SB-26, SB-27, SB-28, SB-29, SB-30, SB-31, SB-32, SB-33, SB-34, SB-35, SB-36, SB-37, SB-38, S HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 of antibodies of B-39, SB-40, SB-41, SB-42, SB-43, SB-44, SB-45, SB-46, SB-47, SB-48, SB-49, SB-50, SB-1-2, SB-1-3, SB-1-4, SB-1-5, SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21. In some embodiments, the antibody comprises a heavy chain variable region, which contains HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 selected from SEQ ID NO. NO:268,270,272,274,276,279,281,283,285,287,289,291,293,295,297,299,301,303 ,305,307,309,311,313,315,317,319,321,323,325,327,329,331,333,335,337,339,34 The sequence 1,343,345,347,349,351,353,355,357,359,361,363,365,366,367,368,369,371,372,373,374,375,376,377,378,379,380,381, or 382 is at least 90%, at least 95%, at least 97%, or at least 99% identical to the same amino acid sequence.In some embodiments, the antibody comprises a subset selected from SEQ ID NO:268,270,272,274,276,279,281,283,285,287,289,291,293,295,297,299,301,303,305,307,309,311,313,315,317,319,321,323,325,327,329,331,333. Heavy chain variable regions of 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 366, 367, 368, 369, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, or 382. In some embodiments, the antibody comprises a light chain variable region containing a heavy chain variable region selected from SEQ ID NO. The sequence NO:267,269,271,273,275,277,278,280,282,284,286,288,290,292,294,296,298,300,302,304,306,308,310,312,314,316,318,320,322,324,326,328,330,332,334,336,338,340,342,344,346,348,350,352,354,356,358,360,362,364, or 370 is at least 90%, at least 95%, at least 97%, or at least 99% identical to the same amino acid sequence. In some embodiments, the antibody comprises a light chain variable region selected from SEQ ID NO:267,269,271,273,275,277,278,280,282,284,286,288,290,292,294,296,298,300,302,304,306,308,310,312,314,316,318,320,322,324,326,328,330,332,334,336,338,340,342,344,346,348,350,352,354,356,358,360,362,364, or 370.In some embodiments, the antibody comprises an antibody selected from SB-1, SB-2, SB-3, SB-4, SB-5, SB-6, SB-7, SB-8, SB-9, SB-10, SB-11, SB-12, SB-13, SB-14, SB-15, SB-16, SB-17, SB-18, SB-19, SB-20, SB-21, SB-22, SB-23, SB-24, SB-25, SB-26, SB-27, SB-28, SB-29, SB-30, SB-31, SB-32, SB-33, SB-34, SB-35, SB-36, SB-37, SB-38, SB-29. The heavy chain variable region of antibodies of SB-39, SB-40, SB-41, SB-42, SB-43, SB-44, SB-45, SB-46, SB-47, SB-48, SB-49, SB-50, SB-1-2, SB-1-3, SB-1-4, SB-1-5, SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21 is at least 90%, at least 95%, at least 97%, or at least 99% identical. The heavy chain variable region, and is selected from the group consisting of SB-1, SB-2, SB-3, SB-4, SB-5, SB-6, SB-7, SB-8, SB-9, SB-10, SB-11, SB-12, SB-13, SB-14, SB-15, SB-16, SB-17, SB-18, SB-19, SB- 20,SB-21,SB-22,SB-23,SB-24,SB-25,SB-26,SB-27,SB-28,SB-29,SB-30,SB-31,SB-32,SB-33,SB-34,SB-35,SB-36,SB-37,SB-38,SB-39,SB The light chain variable regions of antibodies SB-40, SB-41, SB-42, SB-43, SB-44, SB-45, SB-46, SB-47, SB-48, SB-49, SB-50, SB-1-2, SB-1-3, SB-1-4, SB-1-5, SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21 are at least 90%, at least 95%, at least 97%, or at least 99% of the same light chain variable region.In some embodiments, the antibody comprises a protein selected from SB-1, SB-2, SB-3, SB-4, SB-5, SB-6, SB-7, SB-8, SB-9, SB-10, SB-11, SB-12, SB-13, SB-14, SB-15, SB-16, SB-17, SB-18, SB-19, SB-20, SB-21, SB-22, SB-23, SB-24, SB-25, SB-26, SB-27, SB-28, SB-29, SB-30, SB-31, SB-32, SB-33, SB-34, SB-35, SB-36, and SB-37. The heavy chain variable region and light chain variable region of the antibodies of SB-38, SB-39, SB-40, SB-41, SB-42, SB-43, SB-44, SB-45, SB-46, SB-47, SB-48, SB-49, SB-50, SB-1-2, SB-1-3, SB-1-4, SB-1-5, SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21.

[0035] In some embodiments, the antibody comprises HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 of antibodies selected from (shown in Tables 3, 4, 8, and 9) SB-1, SB-2, SB-3, SB-4, SB-5, SB-6, SB-7, SB-8, SB-9, SB-14, SB-28, SB-39, SB-40, SB-49, SB-1-2, SB-1-3, SB-1-4, SB-1-5, SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21. In some embodiments, the antibody comprises a heavy chain variable region that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the heavy chain variable region of the antibody selected from SB-1, SB-2, SB-3, SB-4, SB-5, SB-6, SB-7, SB-8, SB-9, SB-14, SB-28, SB-39, SB-40, SB-49, SB-1-2, SB-1-3, SB-1-4, SB-1-5, SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21. In some embodiments, the antibody comprises a heavy chain variable region of an antibody selected from SB-1, SB-2, SB-3, SB-4, SB-5, SB-6, SB-7, SB-8, SB-9, SB-14, SB-28, SB-39, SB-40, SB-49, SB-1-2, SB-1-3, SB-1-4, SB-1-5, SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21.In some embodiments, the antibody comprises a light chain variable region that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the light chain variable region of the antibody selected from SB-1, SB-2, SB-3, SB-4, SB-5, SB-6, SB-7, SB-8, SB-9, SB-14, SB-28, SB-39, SB-40, SB-49, SB-1-2, SB-1-3, SB-1-4, SB-1-5, SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21. In some embodiments, the antibody comprises a light chain variable region of an antibody selected from SB-1, SB-2, SB-3, SB-4, SB-5, SB-6, SB-7, SB-8, SB-9, SB-14, SB-28, SB-39, SB-40, SB-49, SB-1-2, SB-1-3, SB-1-4, SB-1-5, SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21.In some embodiments, the antibody comprises at least 90%, at least 95%, at least 97%, or at least 99% of the heavy chain variable region of antibodies selected from SB-1, SB-2, SB-3, SB-4, SB-5, SB-6, SB-7, SB-8, SB-9, SB-14, SB-28, SB-39, SB-40, SB-49, SB-1-2, SB-1-3, SB-1-4, SB-1-5, SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21. The heavy chain variable region, and the light chain variable region of the antibody selected from SB-1, SB-2, SB-3, SB-4, SB-5, SB-6, SB-7, SB-8, SB-9, SB-14, SB-28, SB-39, SB-40, SB-49, SB-1-2, SB-1-3, SB-1-4, SB-1-5, SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21 are at least 90%, at least 95%, at least 97%, or at least 99% identical to the same light chain variable region. In some embodiments, the antibody comprises heavy chain variable regions and light chain variable regions of antibodies selected from SB-1, SB-2, SB-3, SB-4, SB-5, SB-6, SB-7, SB-8, SB-9, SB-14, SB-28, SB-39, SB-40, SB-49, SB-1-2, SB-1-3, SB-1-4, SB-1-5, SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21.

[0036] In some embodiments, the antibody comprises HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 of antibodies selected from (shown in Tables 8 and 9) SB-1-2, SB-1-3, SB-1-4, SB-1-5, SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21. In some embodiments, the antibody comprises a heavy chain variable region that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the heavy chain variable region of the antibody selected from SB-1-2, SB-1-3, SB-1-4, SB-1-5, SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21. In some embodiments, the antibody comprises a heavy chain variable region of an antibody selected from SB-1-2, SB-1-3, SB-1-4, SB-1-5, SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21. In some embodiments, the antibody comprises a light chain variable region that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the light chain variable region of the antibody selected from SB-1-2, SB-1-3, SB-1-4, SB-1-5, SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21. In some embodiments, the antibody comprises a light chain variable region of an antibody selected from SB-1-2, SB-1-3, SB-1-4, SB-1-5, SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21.In some embodiments, the antibody comprises at least 90%, at least 95%, at least 97%, or at least 99% of the heavy chain variable region of antibodies selected from SB-1-2, SB-1-3, SB-1-4, SB-1-5, SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21. The heavy chain variable region, and the light chain variable region of the antibody selected from SB-1-2, SB-1-3, SB-1-4, SB-1-5, SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21 is at least 90%, at least 95%, at least 97%, or at least 99% identical to the same light chain variable region. In some embodiments, the antibody comprises heavy chain variable regions and light chain variable regions of antibodies selected from SB-1-2, SB-1-3, SB-1-4, SB-1-5, SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21.

[0037] In some embodiments, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 80, 228, or 229; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 101, 239, 239, 240, or 241; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 125; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 383; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 31. In some embodiments, the antibody comprises a heavy chain variable region that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 366, 367, 368, or 369. In some embodiments, the antibody comprises a light chain variable region that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO:267. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:366, 367, 368, or 369. In some embodiments, the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:267.

[0038] In some embodiments, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 81, 99, 230, 231, or 232; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 102, 242, 243, 244, or 245; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 126 or 253; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 383; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 32. In some embodiments, the antibody comprises a heavy chain variable region that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 371, 372, 373, 374, or 375. In some embodiments, the antibody comprises a light chain variable region that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 269 or 370. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 371, 372, 373, 374, or 375. In some embodiments, the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 269 or 370. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 371, 372, or 373 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 370, or the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 374 or 375 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 269.

[0039] In some embodiments, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 85, 233, or 234; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 107, 246, 247, or 248; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 131 or 254; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 383; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 19; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 37. In some embodiments, the antibody comprises a heavy chain variable region that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 376, 377, or 378. In some embodiments, the antibody comprises a light chain variable region that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 280. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 376, 377, or 378. In some embodiments, the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 280.

[0040] In some embodiments, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 97, 235, 236, or 237; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 121, 249, 250, 251, or 252; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 163; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 2; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 22; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 69. In some embodiments, the antibody comprises a heavy chain variable region that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 379, 380, 381, or 382. In some embodiments, the antibody comprises a light chain variable region that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 344. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 379, 380, 381, or 382. In some embodiments, the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 344.

[0041] In some embodiments, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:229; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:239; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:125; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:383; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:16; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:31. In some embodiments, the antibody comprises a heavy chain variable region that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO:367. In some embodiments, the antibody comprises a light chain variable region that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO:267. In some embodiments, the antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO:367. In some embodiments, the antibody comprises a light chain variable region containing the amino acid sequence of SEQ ID NO:267.

[0042] In some embodiments, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:231; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:243; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:126; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:383; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:16; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:32. In some embodiments, the antibody comprises a heavy chain variable region that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO:372. In some embodiments, the antibody comprises a light chain variable region that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO:370. In some embodiments, the antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO:372. In some embodiments, the antibody comprises a light chain variable region containing the amino acid sequence of SEQ ID NO:270.

[0043] In some embodiments, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:233; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:246; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:131; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:383; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:19; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody comprises a heavy chain variable region that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO:376. In some embodiments, the antibody comprises a light chain variable region that is at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequence of SEQ ID NO:280. In some embodiments, the antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO:376. In some embodiments, the antibody comprises a light chain variable region containing the amino acid sequence of SEQ ID NO:280.

[0044] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is of the IgG, IgM, or IgA class. In some embodiments, the antibody is of the IgG class and has IgG1, IgG2, or IgG4 isotypes. In some embodiments, the antibody has an IgG1 isotype. In some embodiments, the antibody comprises E430G and P331S substitutions according to EU numbering schemes.

[0045] In some embodiments, the antibody is an antibody fragment. In some embodiments, the fragment is a Fab, Fab', Fab'-SH, F(ab')2, Fv, or scFv fragment.

[0046] In some embodiments, the antibody has an affinity (K0.1 nM to 50 nM, or 0.5 nM to 10 nM, or 0.5 nM to 5 nM for human SIRPβ1 isotype 1. D In some implementations, affinity is achieved using ForteBio. System measurements.

[0047] In some embodiments, the antibody recognizes a first antigen and a second antigen, wherein the first antigen is SIRPβ1 and the second antigen is:

[0048] (a) Antigens that facilitate transport across the blood-brain barrier;

[0049] (b) Selected antigens that drive transport across the blood-brain barrier from a group consisting of transferrin receptor (TR), insulin receptor (HIR), insulin-like growth factor receptor (IGFR), low-density lipoprotein receptor-associated proteins 1 and 2 (LPR-1 and 2), and diphtheria toxin receptor.

[0050] (c) Pathogenic agents selected from the group consisting of a pathogenic peptide, protein, or nucleic acid, wherein the pathogenic nucleic acid is an antisense GGCCCC(G2C4) repeat amplified RNA, and the pathogenic protein is selected from amyloid β, oligoamyloid β, amyloid β plaque, amyloid precursor protein or fragment thereof, Tau, IAPP, α-synuclein, TDP-43, FUS protein, C9orf72 (chromosome 9 open reading frame 72), c9RAN protein, prion protein, PrPSc, huntingtin protein, calcitonin, superoxide dismutase, ataxia protein, ataxia protein 1, ataxia protein 2, ataxia protein 3, ataxia protein 7, and ataxia protein 6. 8, ataxia protein 10, Lewy body, atrial natriuretic factor, pancreatic amyloid peptide, insulin, apolipoprotein AI, serum amyloid A, medin, prolactin, thyroxine transporter, lysozyme, β2 microglobulin, coagulant, corneal epithelial protein, cysteine ​​protease inhibitor, immunoglobulin light chain AL, S-IBM protein, repeat-related non-ATG (RAN) translation products, dipeptide repeat (DPR) peptide, glycine-alanine (GA) repeat peptide, glycine-proline (GP) repeat peptide, glycine-arginine (GR) repeat peptide, proline-alanine (PA) repeat peptide, ubiquitin, and a group consisting of proline-arginine (PR) repeat peptide;

[0051] (d) Ligands and / or proteins expressed on immune cells, wherein the ligands and / or proteins are selected from the group consisting of CD40, OX40, ICOS, CD28, CD137 / 4-1BB, CD27, GITR, PD-L1, CTLA-4, PD-L2, PD-1, B7-H3, B7-H4, HVEM, BTLA, KIR, GAL9, TIM3, A2AR, LAG-3, and phosphatidylserine; and

[0052] (e) Proteins, lipids, polysaccharides, or glycolipids expressed on one or more tumor cells.

[0053] In some embodiments, an isolated antibody that binds to human SIRPβ1 is provided, wherein the antibody competes with one or more antibodies provided herein for binding to human SIRPβ1 isotype 1. In some embodiments, an isolated antibody that binds to human SIRPβ1 isotype 1 epitopes that are substantially the same as or overlap with one or more antibodies provided herein.

[0054] In some embodiments, an isolated nucleic acid is provided that comprises a nucleic acid sequence encoding the anti-SIRPβ1 antibody provided herein. In some embodiments, a vector is provided that contains the nucleic acid. In some embodiments, an isolated host cell is provided that contains the vector. In some embodiments, an isolated host cell is provided that expresses the anti-SIRPβ1 antibody provided herein. In some embodiments, a method for generating an antibody binding to human SIRPβ1 is provided, comprising culturing a host cell expressing the anti-SIRPβ1 antibody provided herein, thereby generating the antibody. In some embodiments, the method further comprises recovering the antibody generated from the cell.

[0055] In some embodiments, a pharmaceutical composition is provided comprising the anti-SIRPβ1 antibody and a pharmaceutically acceptable carrier provided herein.

[0056] In some embodiments, a method of treating cancer is provided, comprising administering a therapeutically effective amount of the anti-SIRPβ1 antibody provided herein to an individual in need. In some embodiments, the cancer is selected from sarcoma, bladder cancer, brain cancer, breast cancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer, renal pelvis cancer, leukemia, lung cancer, small cell lung cancer, melanoma, lymphoma, pancreatic cancer, prostate cancer, ovarian cancer, and fibrosarcoma, glioblastoma multiforme, clear cell renal cell carcinoma, adrenocortical carcinoma, urothelial carcinoma of the bladder, diffuse large B-cell lymphoma, lung adenocarcinoma, pancreatic adenocarcinoma, renal cell carcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, painless B-cell lymphoma, aggressive B-cell lymphoma, T-cell lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia, and chronic lymphocytic leukemia. Cellular leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma, myelodysplastic syndrome, myeloproliferative neoplasms, invasive breast cancer, cervical squamous cell carcinoma, cervical adenocarcinoma, bile duct cancer, colon adenocarcinoma, diffuse large B-cell lymphoma, esophageal cancer, head and neck squamous cell carcinoma, renal chromophobe carcinoma, renal papillary cell carcinoma, low-grade glioma, hepatocellular carcinoma, lung squamous cell carcinoma, mesothelioma, ovarian serous cystadenocarcinoma, pancreatic adenocarcinoma, pheochromocytoma and paraganglioma, prostate adenocarcinoma, rectal adenocarcinoma, skin melanoma, gastric adenocarcinoma, testicular germ cell tumor, thyroid cancer, thymoma, endometrial cancer, uterine carcinosarcoma, and uveal melanoma.

[0057] In some embodiments, the treatment further comprises administering an agent that inhibits or activates PD1, PDL1, CD40, OX40, ICOS, CD28, CD137 / 4-1BB, CD27, GITR, CTLA4, PD-L2, B7-H3, B7-H4, HVEM, LIGHT, BTLA, CD30, TIGIT, VISTA, KIR, GAL9, TIM1, TIM3, TIM4, A2AR, LAG3, DR-5, CD2, CD5, CD39, or CD73. In some embodiments, the treatment further comprises administering to the individual at least one antibody that specifically binds to an inhibitory checkpoint molecule, and / or one or more standard or investigative anticancer therapies.

[0058] In some embodiments, the treatment method further comprises administering, in combination with the anti-SIRPA antibody, at least one antibody that specifically binds to an inhibitory checkpoint molecule. In some embodiments, the at least one antibody that specifically binds to an inhibitory checkpoint molecule is selected from anti-PD-L1 antibody, anti-CTLA4 antibody, anti-PD-L2 antibody, anti-PD-1 antibody, anti-B7-H3 antibody, anti-B7-H4 antibody, and anti-HVEM antibody, anti-B and T lymphocyte attenuator (BTLA) antibody, anti-killer inhibitory receptor (KIR) antibody, anti-GAL9 antibody, anti-TIM-1 antibody, anti-TIM3 antibody, anti-TIM-4 antibody, anti-A2AR antibody, and anti-CD4 antibody. Antibody 39, anti-CD73, anti-LAG-3, anti-phosphatidylserine, anti-CD27, anti-CD30, anti-TNFα, anti-CD33, anti-Siglec-5, anti-Siglec-7, anti-Siglec-9, anti-Siglec-11, antagonistic anti-TREM1, antagonistic anti-TREM2, anti-TIGIT, anti-VISTA, anti-CD2, anti-CD5, and any combination thereof.

[0059] In some implementations, the treatment method further includes administering one or more standard or investigational anticancer therapies selected from radiotherapy, cytotoxic chemotherapy, targeted therapy, imatinib therapy, trastuzumab therapy, etanercept therapy, adoptive cell transfer (ACT) therapy, chimeric antigen receptor T-cell transfer (CAR-T) therapy, vaccine therapy, and cytokine therapy.

[0060] In some embodiments, the treatment method further comprises administering to the individual an antibody that specifically binds to at least one inhibitory cytokine. In some embodiments, the antibody that specifically binds to the inhibitory cytokine is selected from anti-CCL2 antibody, anti-CSF-1 antibody, anti-IL-2 antibody, and any combination thereof.

[0061] In some embodiments, the treatment further comprises administering to the individual at least one agonist antibody that specifically binds to a stimulating checkpoint protein. In some embodiments, the at least one agonist antibody that specifically binds to a stimulating checkpoint protein is selected from agonist anti-CD40 antibodies, agonist anti-OX40 antibodies, agonist anti-ICOS antibodies, agonist anti-CD28 antibodies, agonist anti-TREM1 antibodies, agonist anti-TREM2 antibodies, agonist anti-CD137 / 4-1BB antibodies, agonist anti-CD27 antibodies, agonist anti-glucocorticoid-induced TNFR-related protein GITR antibodies, agonist anti-CD30 antibodies, agonist anti-BTLA antibodies, agonist anti-HVEM antibodies, agonist anti-CD2 antibodies, agonist anti-CD5 antibodies, and any combination thereof.

[0062] In some embodiments, the treatment further comprises administering at least one stimulating cytokine to the individual. In some embodiments, the at least one stimulating cytokine is selected from IFN-α4, IFN-β, IL-1β, TNF-α, IL-6, IL-8, CRP, IL-20 family members, LIF, IFN-γ, OSM, CNTF, GM-CSF, IL-11, IL-12, IL-15, IL-17, IL-18, IL-23, CXCL10, IL-33, MCP-1, MIP-1-β, and any combination thereof.

[0063] In some embodiments, the use of the anti-SIRPβ1 antibody provided herein is provided for the preparation of a medicament. In some embodiments, the medicament is used to treat cancer. In some embodiments, the anti-SIRPβ1 antibody provided herein is provided for the treatment of cancer.

[0064] In some embodiments, a method for treating a neurodegenerative disease or condition is provided, comprising administering a therapeutically effective amount of the anti-SIRPβ1 antibody provided herein to an individual in need. In some embodiments, the neurodegenerative disease or condition is selected from dementia, frontotemporal dementia, progressive supranuclear palsy, Alzheimer's disease, vascular dementia, Nasu-Hakola disease, cognitive impairment, memory loss, seizures, retinal dystrophy, amyotrophic lateral sclerosis, traumatic brain injury, spinal cord injury, stroke, Parkinson's disease, acute disseminated encephalomyelitis, retinal degeneration, age-related macular degeneration, glaucoma, and multiple sclerosis.

[0065] In some embodiments, the use of the anti-SIRPβ1 antibody provided herein is provided for the preparation of a medicament for the treatment of neurodegenerative diseases or conditions. In some embodiments, the neurodegenerative disease or condition is selected from dementia, frontotemporal dementia, progressive supranuclear palsy, Alzheimer's disease, vascular dementia, Nasu-Hakola disease, cognitive impairment, memory loss, seizures, retinal dystrophy, amyotrophic lateral sclerosis, traumatic brain injury, spinal cord injury, stroke, Parkinson's disease, acute disseminated encephalomyelitis, retinal degeneration, age-related macular degeneration, glaucoma, and multiple sclerosis. In some embodiments, the anti-SIRPβ1 antibody provided herein is provided for use in the treatment of neurodegenerative diseases or conditions. In some implementations, the neurodegenerative disease or condition is selected from dementia, frontotemporal dementia, progressive supranuclear palsy, Alzheimer's disease, vascular dementia, Nasu-Hakola disease, cognitive impairment, memory loss, seizures, retinal dystrophy, amyotrophic lateral sclerosis, traumatic brain injury, spinal cord injury, stroke, Parkinson's disease, acute disseminated encephalomyelitis, retinal degeneration, age-related macular degeneration, glaucoma, and multiple sclerosis.

[0066] It should be understood that one, some, or all of the features of the various embodiments described herein can be combined to form other embodiments of the invention. These and other aspects of the invention will become apparent to those skilled in the art. The following detailed description further illustrates these and other embodiments of the invention. Brief description of the attached diagram

[0068] Figure 1A The image shows an alignment of the amino acid sequences of SIRPβ1 isotype 1 (SEQ ID NO:1; Uniprotocol O00241) and SIRPβ1 isotype 3 (SEQ ID NO:384; Uniprotocol Q5TFQ8). Figure 1B The amino acid sequence alignment of SIRPβ1 isoform 1 (SEQ ID NO:1) with SIRPα (SEQ ID NO:385; Uniprot accession number P78324) shows high homology within the extracellular domain.

[0069] Figure 2 The image shows an alignment of the amino acid sequences of human SIRPβ1 isotype 1 (SEQ ID NO: 1) and mouse SIRPβ1 (SEQ ID NO: 386; Uniprot accession number Q8BFX8).

[0070] Figures 3A-3BThis demonstrates the induction of human SIRPβ1-dependent luciferase expression in a cell-based reporter assay. Cells were stimulated with plate-bound anti-SIRPβ1 antibody (3A), anti-SIRPα (SA-9C2) (3B), or a human IgG1 isotype control. Results are expressed as folds relative to background. Background levels were set to 1 on the y-axis.

[0071] Figure 4A This diagram illustrates the expression patterns of two DAP12-associated receptors (SIRPβ1 and TREM1) on human monocytes. Shaded histograms represent background fluorescence from isotype-stained cells. Black-outlined histograms represent receptor expression levels in target-specific antibody-stained cells. Figure 4B This shows the expression of SIRPβ1 on M1 and M2 polarized macrophages from two healthy donors. The shaded histograms represent background fluorescence from isotype-stained cells. The black-outlined histograms represent the SIRPβ1 expression level on M2 macrophages, while the dashed histograms represent the SIRPβ1 expression level on M1 macrophages. Figure 4C The expression of SIRPβ1 and TREM1 on human monocyte-derived dendritic cells (DCs) is shown with and without LPS stimulation. Shaded histograms represent background fluorescence from isotype-stained cells. Black-outlined histograms represent receptor expression levels in target-specific antibody-stained cells.

[0072] Figure 5A We depict the RNA expression patterns of SIRPβ1 isoform 1 in various tissues from human donors. Figure 5B We depicted the RNA expression patterns of SIRPβ1 isoform 3 in various tissues from human donors. Peak expression of SIRPβ1 isoform 3 occurred in the brain, with limited expression in peripheral tissues.

[0073] Figure 6A This demonstrates a SIRPβ1-mediated respiratory burst from primary human neutrophils obtained from two different donors. Figure 6B This shows SIRPβ1-mediated respiratory burst (top inset) or IL-8 release (bottom inset) from primary human monocytes. Figure 6C This shows SIRPβ1-mediated release of TNFα cytokines from primary human macrophages or dendritic cells (DCs).

[0074] Figure 7 The antitumor activity of primary human neutrophils stimulated with plate-bound anti-SIRPβ1 antibody is shown. Luminescence values ​​are presented on a relative scale, with the signal of Raji-Luc cells co-cultured with neutrophils in the absence of regulatory antibodies set to 1 on the y-axis.

[0075] Figure 8The induction of human SIRPβ1-dependent luciferase expression by affinity maturation antibody in a cell-based reporter assay is shown. Results are presented as raw luminescence values. Background levels are set to 10,000 on the y-axis.

[0076] Figure 9 This shows SIRPβ1-mediated TNFα cytokine release from primary human dendritic cells (DCs).

[0077] Figures 10A-10B The cross-reactivity of anti-SIRPβ1 antibodies to multiple SIRPβ1 antigens was demonstrated. Soluble human SIRPβ1-Fc isotype 1 (O00241) or isotype 3 (Q5TFQ8) or cynomolgus monkey SIRPβ1-Fc (XM005568541) were coated onto plates and incubated with increasing concentrations of the parent and affinity-mature forms of anti-SIRPβ1 antibodies SB-1, SB-1-2, SB-2, SB-2-7, SB-8, SB-8-13, SB-40, and SB-40-21. All anti-SIRPβ1 antibodies bound only to human SIRPβ1-Fc isotype 1 antigen.

[0078] Figure 11A The full-length anti-SIRPβ1 antibodies SB-1-3, SB-2-8, SB-8-13, SB-8-15, and SB-40-20, conjugated to the display plate, upregulated TREM2 expression on monocyte-derived human macrophages from three different healthy donors. Baseline TREM2 expression was determined by incubating cells on human IgG1 isotype control antibodies. TREM2 was detected using an anti-TREM2 antibody (clone ADI-22) conjugated to the DyLight650 fluorophore. Figure 11B The viability of monocyte-derived human macrophages cultured on plate-bound full-length anti-SIRPβ1 antibodies SB-1-3, SB-2-8, SB-8-13, SB-8-15, and SB-40-20 was demonstrated. Viable cells were quantified by measuring luminescence values ​​after the addition of Cell Titer Glo substrate. Baseline cell viability was established on cells cultured on human IgG1 isotype controls or in the absence of plate-bound antibodies (antibody-free).

[0079] Figure 12 The viability of monocyte-derived macrophages from two different healthy donors cultured on increasing concentrations of plate-bound full-length anti-SIRPβ1 antibodies SB-1-3 and SB-2-8 or a human IgG1 isotype control is shown. Viable cells were quantified by measuring luminescence values ​​after addition of Cell Titer Glo substrate.

[0080] Figure 13The study demonstrated that anti-SIRPβ1 antibody enhanced the agonistic activity of anti-TREM2 antibody in a macrophage viability assay. Monocyte-derived macrophages were cultured on plate-bound full-length SB-1-3, SB-2-8, or huIgG1 isotype controls in the presence or absence of soluble anti-TREM2 antibody. Viable cells were quantified by measuring luminescence values ​​after addition of Cell Titer Glo substrate.

[0081] Figure 14A The viability of bone marrow-derived macrophages obtained from human SIRPβ1 BAC transgenic mice cultured on plate-bound full-length anti-SIRPβ1 antibodies SB-1-3, SB-2-8, and SB-8-13 or human IgG1 isotype control was demonstrated. Figure 14B Viability of bone marrow-derived dendritic cells obtained from human SIRPβ1 BAC transgenic mice cultured on plate-bound full-length anti-SIRPβ1 antibodies SB-1-3, SB-2-8, and SB-8-13 or human IgG1 isotype controls was demonstrated. Viable cells were quantified by measuring luminescence values ​​after the addition of Cell TiterGlo substrate. Baseline cell viability was established for cells cultured on human IgG1 isotype controls or in the absence of plate-bound antibodies (antibody-free).

[0082] Figures 15A-15C This demonstrates the cross-reactivity of the anti-SIRPβ1 antibody with different receptors in the SIRP family. Figure 15A and Figure 15B Titration curves of various anti-SIRP receptor antibodies binding to BW5147.G.1.4 cells overexpressing recombinant human SIRPβ1 or recombinant human SIRPα are shown. Figure 15C The titration curves of the antibody binding to Jurkat cells and immortalized human T cell lines endogenously expressing SIRPγ are shown. Positive control antibodies were anti-SIRPα / β clone 18D5, anti-SIRPα / β / γ clone KWAR23, and anti-SIRPγ clone LSB2.20. Invention Details

[0084] This disclosure relates to anti-SIRPβ1 antibodies (e.g., monoclonal antibodies); methods for generating and using such antibodies; pharmaceutical compositions containing such antibodies; nucleic acids encoding such antibodies; and host cells containing nucleic acids encoding such antibodies.

[0085] The techniques and procedures described or mentioned in this article are generally well understood and used by those skilled in the art, and are commonly employed by conventional methods, such as those widely used in Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (FMAusubel et al., eds., 2003); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000).

[0086] Therefore, all references cited in this article, including patent applications and publications, are cited in their entirety.

[0087] I. Definition

[0088] The terms “signal regulatory protein β1”, “SIRPβ1”, and “SIRPβ1 polypeptide” are used interchangeably herein and refer to any naturally occurring SIRPβ1 from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats), unless otherwise specified. In some embodiments, the term covers both wild-type sequences and naturally occurring variant sequences, such as splice variants or allelic variants. In some embodiments, the term covers “full-length”, unprocessed SIRPβ1, and any form of processed SIRPβ1 derived from cells. In some embodiments, SIRPβ1 is human SIRPβ1. In some embodiments, the amino acid sequence of an exemplary SIRPβ1 is Uniprotocol 000241, dated February 28, 2018. In some embodiments, the amino acid sequence of an exemplary human SIRPβ1 is SEQ ID NO:1. In some embodiments, an exemplary SIRPβ1 isotype 3 amino acid sequence is Uniprotocol Q5TFQ8, dated January 31, 2018. In some embodiments, an exemplary human SIRPβ1 isotype 3 amino acid sequence is SEQ ID NO:384. Unless otherwise specifically indicated, “SIRPβ1” as used herein refers to SIRPβ1 isotype 1.

[0089] The terms "anti-SIRPβ1 antibody," "SIRPβ1-binding antibody," and "SIRPβ1-specifically binding antibody" refer to antibodies capable of binding SIRPβ1 with sufficient affinity, making the antibody useful as a diagnostic and / or therapeutic agent targeting SIRPβ1. In one embodiment, the anti-SIRPβ1 antibody binds to unrelated, non-SIRPβ1 peptides to less than about 10% of the antibody's binding to SIRPβ1, as measured, for example, by radioimmunoassay (RIA). In some embodiments, the SIRPβ1-binding antibody has a concentration of <1 μM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 10 μM). -8 M or smaller, such as 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 The dissociation constant (KD) of M). In some embodiments, the anti-SIRPβ1 antibody binds to a conserved SIRPβ1 epitope in a SIRPβ1 peptide from a different species.

[0090] Regarding antibody binding to target molecules, the terms "specific binding" to a specific polypeptide or epitope on a specific polypeptide target, or "specific to it," imply a binding that is measurable in contrast to nonspecific interactions. Specific binding can be measured, for example, by comparing the binding of the molecule to that of a control molecule. For instance, specific binding can be measured by competition with a control molecule similar to the target, such as an excess of unlabeled target. In this case, specific binding is indicated if the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target. The terms "specific binding" to a specific polypeptide or epitope on a specific polypeptide target, or "specific to it," as used herein, can be, for example, derived from antibodies having approximately 10... -4 M or lower, 10 -5 M or lower, 10 -6 M or lower, 10 -7 M or lower, 10 -8 M or lower, 10 -9 M or lower, 10 -10 M or lower, 10 -11 M or lower, 10 -12 M or lower of either KD or 10 -4 M to 10 -6 M or 10 -6 M to 10 -10 M or 10 -7 M to 10 -9Molecular representation of KD against a target within the M range. As those skilled in the art will understand, affinity and KD value are negatively correlated. High affinity against an antigen is measured by a low KD value. In one embodiment, the term "specific binding" refers to binding in which a molecule binds to a specific polypeptide or an epitope on a specific polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

[0091] The term “immunoglobulin” (Ig) is used interchangeably with “antibody” in this document. The term “antibody” is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) (including those formed from at least two complete antibodies), and antibody fragments, provided they exhibit the desired biological activity.

[0092] "Natural antibodies" are typically heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two identical light ("L") chains and two identical heavy ("H") chains. Each light chain is linked to the heavy chain by a covalent disulfide bond, the number of which varies among heavy chains of different immunoglobulin isoforms. Each heavy and light chain also has regularly spaced intrachain disulfide linkages. Each heavy chain has a variable domain (V) at one end. H ), followed by some constant fields. Each light chain has a variable field (V) at one end. L The light chain's variable domain is aligned with the first constant domain of the heavy chain, while the light chain's variable domain is aligned with the heavy chain's variable domain. It is assumed that specific amino acid residues form the interface between the light and heavy chain variable domains.

[0093] For information on the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th ed., Daniel P. Stites, Abba I. Terr and Tristram G. Parslow, Appleton & Lange, Norwalk, CT, 1994, p. 71 and Chapter 6.

[0094] Based on their constant domain amino acid sequences, light chains from any vertebrate species can be classified into two distinct types, called kappa (“κ”) and lambda (“λ”). Immunoglobulins can be classified into different classes or isotypes based on their heavy chain constant domain (CH) amino acid sequences. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, each with a heavy chain called alpha (“α”), delta (“δ”), epsilon (“ε”), gamma (“γ”), and muon (“μ”), respectively. Based on minor differences in CH sequences and function, the γ and α classes can be further subdivided into subclasses (isotypes), for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well-known and generally described in, for example, Abbas et al., Cellular and Molecular Immunology, 4 th ed. (WBSaunders Co., 2000).

[0095] The "variable region" or "variable domain" of an antibody (such as the anti-SIRPβ1 antibody disclosed herein) refers to the amino-terminal domain of the antibody's heavy or light chain. The heavy chain variable domain and the light chain variable domain may be referred to as "V" and "V" respectively. H " and "V L These domains are generally the most variable parts of an antibody (relative to other antibodies in the same class) and contain antigen-binding sites.

[0096] The term "variable" refers to the fact that certain segments within a variable domain exhibit significant sequence differences between antibodies (such as the anti-SIRPβ1 antibody of this disclosure). Variable domains mediate antigen binding and define the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed across the entire span of the variable domain. Instead, it is concentrated in three segments within both the light and heavy chain variable domains, called hypervariable regions (HVRs). More highly conserved portions of the variable domain are called frame regions (FRs). The variable domains of the natural heavy and light chains each contain four FRs, which mostly adopt a β-sheet conformation and are linked by three HVRs that form a loop and, in some cases, part of a β-sheet structure. The HVRs in each chain are held together very closely by the FRs and, together with the HVRs of the other chain, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Immunological Interest, 5th ed., National Institute of Health, Bethesda, MD. (1991)). The constant domain does not directly participate in the binding of antibodies to antigens, but exhibits a variety of effector functions, such as the participation of antibodies in the cytotoxicity of antibody-dependent cells.

[0097] The term "monoclonal antibody," as used herein, refers to an antibody derived from a substantially homogeneous group of antibodies (such as the monoclonal anti-SIRPβ1 antibody of this disclosure), meaning that the individual antibodies constituting the group are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation, etc.) that may be present in very small amounts. Monoclonal antibodies are highly specific, targeting a single antigenic site. Unlike typical polyclonal antibody preparations that contain different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they are synthesized from hybridoma cultures and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristic that the antibody is derived from a substantially homogeneous group of antibodies and should not be construed as requiring the antibody to be generated by any particular method. For example, monoclonal antibodies intended for use according to the invention can be generated by a variety of techniques, including, for example, hybridoma methods, recombinant DNA methods, and techniques for generating human or human-like antibodies in animals having partial or complete human immunoglobulin loci or genes encoding human immunoglobulin sequences.

[0098] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably to refer to an antibody in essentially its complete form (such as the anti-SIRPβ1 antibody of this disclosure), as opposed to antibody fragments. Specifically, intact antibodies include those that have both heavy and light chains (including the Fc region). The constant domain can be a native sequence constant domain (e.g., a human native sequence constant domain) or a variant of its amino acid sequence. In some cases, intact antibodies may have one or more effector functions.

[0099] "Antibody fragment" refers to a molecule that is distinct from the intact antibody but contains a portion of the intact antibody to which the intact antibody binds. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; biantibodies; linear antibodies (see U.S. Patent 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062(1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0100] Digestion of antibodies (such as the anti-SIRPβ1 antibody disclosed herein) with papain produces two identical antigen-binding fragments, termed the "Fab" fragment, and a residual "Fc" fragment, the names reflecting its ease of crystallization. The Fab fragment consists of a complete light chain and a variable region of the heavy chain (V... H ) and the first constant field of the heavy chain (C H1) Composition. Each Fab fragment is monovalent for antigen binding, meaning it has only one antigen-binding site. Pepsin treatment of the antibody produces a larger F(ab')2 fragment, which is roughly equivalent to two Fab fragments linked by disulfide bonds, possessing different antigen-binding activities while still being able to cross-link the antigen. The Fab' fragment, due to its C... H The 1 domain differs from the Fab fragment by the addition of a few residues at its carboxyl terminus, including one or more cysteine ​​residues from the antibody hinge region. Fab'-SH is the terminology used herein for Fab' where the constant-domain cysteine ​​residues carry free thiol groups. The F(ab')2 antibody fragment was initially generated as a pair of Fab' fragments with a hinge cysteine ​​residue between them. Other chemical conjugations of antibody fragments are also known.

[0101] The Fc fragment contains the carboxyl-terminal portions of two heavy chains held together by disulfide bonds. The effector function of an antibody is determined by the sequence in the Fc region, which is also recognized by Fc receptors (FcRs) found on certain cell types.

[0102] The “functional fragment” of the antibodies of this invention (such as the anti-SIRPβ1 antibody of this disclosure) comprises a portion of the complete antibody, generally including the antigen-binding or variable region of the complete antibody or the retained or modified Fc region of the antibody having FcR binding ability. Examples of antibody fragments include linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0103] The term "double antibody" refers to antibodies produced by transvaginal administration of V... H and V L Small antibody fragments are prepared by constructing sFv fragments (see previous paragraph) using short linkers (approximately 5-10 residues) between the domains. Due to the short linkers, the variable domains pair interchain rather than intrachain, resulting in bivalent fragments, i.e., fragments with two antigen-binding sites. Bispecific biantibodies are heterodimers of two "crossover" sFv fragments, where the V of each antibody... H and V L Domains exist on different polypeptide chains.

[0104] As used herein, "chimeric antibody" refers to antibodies (immunoglobulins), such as the chimeric anti-SIRPβ1 antibody of this disclosure, wherein a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and fragments of such antibodies, provided they exhibit the desired biological activity. Chimeric antibodies of interest herein include "primate-derived" antibodies, wherein the antigen-binding region of the antibody is derived from an antibody generated by immunizing a macaque monkey with, for example, an antigen of interest. As used herein, "humanized antibody" is a subset of "chimeric antibody".

[0105] "Humanized" forms of non-human (e.g., mouse) antibodies, such as the humanized form of the anti-SIRPβ1 antibody of this disclosure, are chimeric antibodies comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In some embodiments, the humanized antibody may comprise substantially all of at least one and typically two variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to those of non-human antibodies, and all or substantially all FRs correspond to those of human antibodies. Optionally, the humanized antibody may comprise at least a portion of an antibody constant region derived from a human antibody. The term "humanized form" of an antibody, such as a non-human antibody, refers to an antibody that has undergone humanization.

[0106] "Human antibody" refers to an antibody having an amino acid sequence corresponding to that of antibodies generated by humans and / or generated using any technology disclosed herein for generating human antibodies, such as the anti-SIRPβ1 antibody of this disclosure. This definition of human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be generated using a variety of technologies known in the art, including phage display libraries and yeast-based platform technologies. Human antibodies can be prepared by administering antigens to transgenic animals that have been modified to generate human antibodies in response to antigenic stimulation, such as immunized xenomic mice, or by human B-cell hybridoma technology.

[0107] The term "hypervariant region" or "HVR," as used herein, refers to a region in the antibody variable domain (such as the variable domain of the anti-SIRPβ1 antibody of this disclosure) that is highly variable in sequence and / or forms a structurally defined loop. Typically, antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). In native antibodies, H3 and L3 exhibit the greatest diversity of these six HVRs, and H3 is considered to play a unique role, particularly in conferring precise specificity to the antibody. Naturally occurring camel-like antibodies, consisting only of heavy chains, are functional and stable in the absence of light chains.

[0108] This document uses and covers many descriptions of HVRs. In some embodiments, the HVR may be the Kabat complementarity-determining region (CDR), which is based on sequence variability and is the most commonly used (Kabat et al., see above). In some embodiments, the HVR may be the Chothia CDR. Chothia instead refers to the location of the structural loop (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). In some embodiments, the HVR may be the AbM HVR. AbM HVR represents a compromise between the Kabat CDR and the Chothia structural loop and is derived from the use of the AbM antibody modeling software from Oxford Molecular. In some embodiments, the HVR may be a “contact” HVR. A “contact” HVR is based on the analysis of the available crystal structure of the complex. The residues of each of these HVRs are described below.

[0109]

[0110] HVRs may include the following “extended HVRs”: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in VL and 26-35 (H1), 50-65 or 49-65 (a preferred embodiment) (H2) and 93-102, 94-102, or 95-102 (H3) in VH. For each of these extended HVR definitions, the variable domain residues are as numbered according to Kabat et al., see above.

[0111] "Frame" or "FR" residues refer to those residues in the variable domain other than the HVR residues defined herein.

[0112] As used in this article, the "recipient human framework" refers to V derived from the human immunoglobulin framework or the human common framework. L or V H The frame of amino acid sequences. Receptor human frames or human common frames “derived” from human immunoglobulin frames may contain their same amino acid sequences, or they may contain previously existing amino acid sequence variations. In some embodiments, the number of previously existing amino acid variations is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In cases where pre-existing amino acid variations exist in VH, those variations are preferably present at only three, two, or one position at 71H, 73H, and 78H; for example, the amino acid residues at those positions may be 71A, 73T, and / or 78A. In one embodiment, the VL receptor human frame is sequence-related to V...L The human immunoglobulin framework sequence or the human common framework sequence is the same.

[0113] "Human Common Framework" refers to the framework representing human immunoglobulin V. L or V H The frame sequence selection is based on the most common amino acid residues. Typically, human immunoglobulin V... L or V H The sequence selection is derived from a variable-domain sequence subgroup. Typically, sequence subgroups are those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991). Examples include those for V. L Subgroups can be subgroups κI, κII, κIII, or κIV, as in Kabat et al., see above. Additionally, for V... H Subgroups can be subgroup I, subgroup II, or subgroup III, as in Kabat et al., see above.

[0114] (For example, in the anti-SIRPβ1 antibody of this disclosure) "Amino acid modification" at a specified position refers to the substitution or deletion of a specified residue, or the insertion of at least one amino acid residue near the specified residue. An insertion "near" the specified residue means an insertion within one or two residues. The insertion can be at the N-terminus or C-terminus of the specified residue. The preferred amino acid modification herein is substitution.

[0115] "Affinity-matured" antibodies (such as the affinity-matured anti-SIRPβ1 antibody of this disclosure) refer to antibodies with one or more alterations in one or more HVRs, resulting in an improved affinity of the antibody for the antigen compared to a parent antibody without these alterations. In one embodiment, the affinity-matured antibody has an affinity for the target antigen on the nanomolar or even picomolar scale. Affinity-matured antibodies can be generated using procedures known in the art. For example, Marks et al., Bio / Technology 10:779-783 (1992) describes a method using V... H and V LAffinity maturation occurs through domain shuffling. The following literature documents the random mutagenesis of HVR and / or framework residues: for example, Barbas et al., Proc. Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al., Gene 169:147-155 (1995); Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0116] "Fv" is the smallest antibody fragment containing both a complete antigen recognition and binding site. This fragment consists of a dimer of a tightly bound, non-covalently bound heavy chain variable region and a light chain variable region. Six hypervariable rings (three rings each from the heavy and light chains) emanate from the folds of these two domains, contributing the amino acid residues for antigen binding and conferring antigen-binding specificity to the antibody. However, even a single variable domain (or half an Fv containing only the three antigen-specific HVRs) can recognize and bind antigens, although with lower affinity than the complete binding site.

[0117] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is a type of Fv containing V molecules linked together to form a single polypeptide chain. H and V L Antibody fragments containing antibody domains. Preferably, the sFv peptide is in V H With V L The domains further contain peptide linkers, which enable sFv to form the desired structure for binding antigens.

[0118] Antibody "effector function" refers to those biological activities that can be attributed to the Fc region of the antibody (the native Fc region or the Fc region of an amino acid sequence variant) and vary with antibody isotype.

[0119] The term "Fc region" is used herein to define the C-terminal region of the immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. While the boundaries of the immunoglobulin heavy chain Fc region can vary, the human IgG heavy chain Fc region is generally defined as the segment from its Cys226 or Pro230 position to the carboxyl terminus. The C-terminal lysine (residue 447, according to the EU numbering system) of the Fc region can be eliminated, for example, during antibody production or purification, or through recombinant engineering of the nucleic acid encoding the antibody heavy chain. Thus, compositions of complete antibodies may include antibody groups with all K447 residues eliminated, antibody groups with none of the K447 residues eliminated, and antibody groups that are a mixture of antibodies with and without K447 residues. Suitable native sequence Fc regions for use in the antibodies of this disclosure include human IgG1, IgG2, IgG3, and IgG4.

[0120] The “natural sequence Fc region” contains the same amino acid sequence as the Fc region found in nature. The natural sequence human Fc regions include the natural sequence human IgG1 Fc region (non-A and A allotypes); the natural sequence human IgG2 Fc region; the natural sequence human IgG3 Fc region; and the natural sequence human IgG4 Fc region, as well as their naturally occurring variants.

[0121] The “variant Fc region” comprises an amino acid sequence that differs from the amino acid sequence of the native Fc region due to at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution compared to the native Fc region or the Fc region of the parent polypeptide, for example, about one to about ten amino acid substitutions in the native Fc region or the Fc region of the parent polypeptide, and more preferably about one to about five amino acid substitutions. The variant Fc region as described herein preferably has at least about 80% homology with the native Fc region and / or the Fc region of the parent polypeptide, and most preferably at least about 90% homology with it, more preferably at least about 95% homology with it.

[0122] The term "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. Preferred FcRs are native human FcRs. Furthermore, preferred FcRs are FcRs (γ receptors) that bind IgG antibodies, including receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splicing forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor-based tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor-based tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. The term "FcR" herein encompasses other FcRs, including those to be identified in the future. FcRs can also prolong the serum half-life of antibodies.

[0123] As used herein, “percentage (%) amino acid sequence identity” and “homology” with respect to peptide, polypeptide, or antibody sequences refer to the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a specific peptide or polypeptide sequence, after sequence alignment and, where necessary, the introduction of gaps to achieve maximum percentage sequence identity, and without regard to any conserved substitutions as part of sequence identity. Alignments used to determine percentage amino acid sequence identity can be performed in a variety of ways within the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or MEGALIGN. TM (DNASTAR) software. Those skilled in the art can determine suitable parameters for measuring alignment, including any algorithms known in the art that are needed to achieve maximum alignment across the full length of the sequences being compared.

[0124] When used in the context of antibodies competing for the same epitope (e.g., neutralizing antibodies), the term "competition" implies competition between antibodies, as determined by assays in which the antibody being tested blocks or inhibits (e.g., reduces) the specific binding of a reference molecule (e.g., a ligand, or a reference antibody) to a common antigen (e.g., SIRPβ1 or a fragment thereof). Numerous types of competitive binding assays can be used to determine whether antibodies compete with each other, such as: solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich-style competitive assays (see, for example, Stahli et al., 1983, Methods in Enzymology 9:242-253); solid-phase direct biotin-avidin EIA (see, for example, Kirkland et al., 1986, J. Immunol. 137:3614-3619); solid-phase direct labeling assays; and solid-phase direct labeling sandwich assays (see, for example, Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Springs). Harbor Press); solid-phase direct labeling of RIAs using I-125 (see, for example, Morel et al., 1988, Molec. Immunol. 25:7-15); solid-phase direct biotin-avidin EIAs (see, for example, Cheung et al., 1990, Virology 176:546-552); and direct labeling of RIAs (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). Typically, such assays involve the use of purified antigens or cells carrying either of these bound to a solid surface, an unlabeled test antibody, and a labeled reference antibody. Competitive inhibition is measured by determining the amount of the label bound to the solid surface or cells in the presence of the test antibody. Typically, an excess of the test antibody is present. Antibodies identified by competitive assays (competitive antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to adjacent epitopes that are sufficiently close to the epitope bound by the reference antibody to cause steric hindrance. Further details regarding methods for determining competitive binding are provided in the examples herein. Typically, when an excess of the competitive antibody is present, it will inhibit (e.g., reduce) the specific binding of the reference antibody to the common antigen by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97.5%, and / or close to 100%.

[0125] As used herein, the “interaction” between the SIRPβ1 peptide and the second peptide encompasses, but is not limited to, protein-protein interactions, physical interactions, chemical interactions, binding, covalent binding, and ionic binding. As used herein, an antibody “inhibits the interaction between the two peptides” when it disrupts, reduces, or completely eliminates the interaction between the two peptides. When an antibody binds to one of the two peptides, the antibody of this disclosure “inhibits the interaction between the two peptides.” In some embodiments, the interaction may be inhibited by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97.5%, and / or close to 100%.

[0126] The term "epitaxy" encompasses any epitope capable of being bound by an antibody. An epitope is a region of an antigen that is bound by an antibody targeting that antigen, and when the antigen is a peptide, it includes a specific amino acid that directly contacts the antibody. Most commonly, epitopes are located on peptides, but in some cases, they can be located on other types of molecules, such as nucleic acids. Epitope epitopes can comprise groups of chemically active surfaces of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and can have specific three-dimensional structural features and / or specific charge features. Generally, antibodies specific to a particular target antigen preferentially recognize epitopes on the target antigen in complex mixtures of peptides and / or macromolecules.

[0127] "Agonistic" or "activating" antibodies are antibodies that induce (e.g., enhance) one or more activities or functions of an antigen after binding to it.

[0128] "Antagonistic" antibodies, "blocking" antibodies, or "inhibitory" antibodies are antibodies that, upon binding to an antigen, reduce, inhibit, and / or eliminate (e.g., decrease) the binding of the antigen to one or more ligands, and / or reduce, inhibit, and / or eliminate (e.g., decrease) one or more activities or functions of the antigen. In some embodiments, antagonistic antibodies, or blocking antibodies, or inhibitory antibodies substantially or completely inhibit the binding of the antigen to one or more ligands and / or one or more activities or functions of the antigen.

[0129] "Isolated" antibodies, such as the isolated anti-SIRPβ1 antibody of this disclosure, are antibodies that have been identified, separated, and / or recovered from components of their environment of origin (e.g., native or recombinant). Preferably, the isolated antibody is not associated with any other contaminating components from its environment of origin. Contaminating components from its environment of origin, such as those derived from recombinant transfected cells, are typically substances that would interfere with the research, diagnostic, or therapeutic use of the antibody, and may include enzymes, hormones, and other protein- or non-protein-like solutes. In a preferred embodiment, the antibody is purified to: (1) greater than 95% by weight of the antibody, as determined by, for example, the Lowry method, and in some embodiments, greater than 99% by weight; (2) sufficient to obtain an N-terminal or internal amino acid sequence of at least 15 residues using a turn-cup sequencer; or (3) homogenized, according to SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or preferred silver staining. The isolated antibody includes in situ antibodies within recombinant T cells, since at least one component of the antibody's native environment is not present. However, isolated peptides or antibodies are usually prepared through at least one purification step.

[0130] The “isolated” nucleic acid molecule encoding the antibody, such as the anti-SIRPβ1 antibody of this disclosure, is a nucleic acid molecule identified and separated from at least one contaminant nucleic acid molecule normally associated with it in the environment in which it is generated. Preferably, the isolated nucleic acid is not associated with all components related to the environment in which it is generated. The isolated nucleic acid molecule encoding the polypeptide and antibody described herein is in a different form or setting than that found in nature. Therefore, the isolated nucleic acid molecule is distinct from the nucleic acid encoding the polypeptide and antibody described herein that naturally occurs in cells.

[0131] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which is a circular double-stranded DNA in which an additional DNA segment can be linked. Another type of vector is a bacteriophage vector. Yet another type of vector is a viral vector, in which an additional DNA segment can be linked into a viral genome. Some vectors are capable of autonomous replication in the host cell in which they are introduced (e.g., bacterial vectors with bacterial origins of replication and episome mammalian vectors). Other vectors (e.g., non-epithelial mammalian vectors) can integrate into the host cell's genome after introduction into the host cell and thereby replicate along with the host genome. Furthermore, some vectors can direct the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors." Generally, expression vectors used in recombinant DNA technology are often in the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably because plasmids are the most common form of vector.

[0132] As used interchangeably in this document, “polynucleotide” or “nucleic acid” refers to a polymer of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogues, or any substrate that can be incorporated into the polymer by DNA or RNA polymerases or through synthetic reactions.

[0133] "Host cell" includes an individual cell or cell culture that can or is already a recipient of a vector to incorporate a polynucleotide insert. Host cells include the offspring of a single host cell, and due to natural, accidental, or intentional mutations, the offspring may not need to be completely identical to the original parent cell (morphologically or in terms of genomic DNA complements). Host cells include cells transfected in vivo with the polynucleotides of this invention.

[0134] When used in this article, “carrier” includes pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to the cells or mammals exposed to them at the doses and concentrations employed.

[0135] As used herein, the term “prevention” includes providing prevention with respect to the occurrence or recurrence of a particular disease, condition, or ailment in an individual. An individual may be susceptible to or at risk of developing a particular disease, condition, or ailment, but has not yet been diagnosed with that disease, condition, or ailment.

[0136] As used herein, an individual “at risk” for a particular disease, condition, or condition may or may not have a detectable disease or disease symptom, and may or may not exhibit a detectable disease or disease symptom prior to the treatments described herein. “At risk” means that an individual possesses one or more risk factors, which are measurable parameters associated with the occurrence of a particular disease, condition, or condition, as known in the art. Individuals possessing one or more of these risk factors have a higher probability of developing a particular disease, condition, or condition than individuals without one or more of these risk factors.

[0137] As used in this article, “treatment / management” refers to a clinical intervention designed to alter the natural progression of an individual during clinicopathological processes. The expected outcomes of treatment include a reduced rate of progression, improvement or alleviation of the pathological condition, and resolution or improvement of the prognosis for a particular disease, symptom, or condition. For example, an individual is considered successfully “treated” if one or more symptoms associated with a particular disease, symptom, or condition are reduced or eliminated.

[0138] "Effective amount" refers to the amount that is effective at least for the necessary dose and time period to achieve the desired therapeutic or preventative outcome. An effective amount may be provided in one or more administrations. The effective amount as used herein may vary with factors such as an individual's disease state, age, sex, and weight, and the ability of the treatment to elicit the desired response in the individual. An effective amount is also the amount in which the beneficial effect of treatment outweighs any toxic or adverse effects of treatment. For prophylactic use, beneficial or desired outcomes include results such as elimination or reduction of risk, reduction of severity, or delay of disease onset, including biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes presented during disease development. For therapeutic use, beneficial or desired outcomes include clinical outcomes such as reduction of one or more symptoms arising from the disease, improvement of the quality of life of those suffering from the disease, reduction of the dose of other drugs required to treat the disease, enhancement of the effect of another drug (e.g., via targeted therapy), delay of disease progression, and / or prolongation of survival. An effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve a preventative or therapeutic treatment. As understood in a clinical context, an effective amount of a drug, compound, or drug composition may be achieved with or without another drug, compound, or drug composition. Thus, an "effective amount" can be considered in the context of administering one or more therapeutic agents, and a single agent can be considered to be administered in an effective amount if, together with one or more other agents, the desired result can be achieved.

[0139] "Individual" for the purpose of treatment, prevention, or risk reduction means any animal classified as a mammal, including humans, domesticated and farm animals, and zoo, sport, or pet animals such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats, and so on. In some implementations, the individual is a human.

[0140] As used herein, “combination” administration with another compound or composition includes simultaneous administration and / or administration at different times. Combination administration also encompasses administration as a co-prepared formulation or as separate compositions, including administration at different frequencies or intervals, and using the same or different routes of administration. In some embodiments, combination administration is administration as part of the same treatment regimen.

[0141] As used herein, the term “about” refers to the typical range of error for a corresponding value that is readily known to those skilled in the art. References to “about” values ​​or parameters herein include (and describe) embodiments relating to that value or parameter itself.

[0142] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise. For example, referring to an “antibody” means referring to one or more antibodies, such as in molar amounts, and includes their equivalents as known to those skilled in the art, and so on.

[0143] It is understood that the descriptions of various aspects and embodiments of this disclosure herein include aspects and embodiments that are “comprising,” “composed of,” and “substantially composed of.”

[0144] I. Anti-SIRPβ1 antibody

[0145] This article provides information on anti-SIRPβ1 antibodies. These antibodies are useful, for example, for the diagnosis or treatment of SIRPβ1-mediated conditions.

[0146] In some embodiments, the anti-SIRPβ1 antibody of this disclosure is an agonist of SIRPβ1 activity. In some embodiments, an anti-SIRPβ1 antibody that binds to human SIRPβ1 isotype 1 but not human SIRPα is provided. In some embodiments, an anti-SIRPβ1 antibody that binds to human SIRPβ1 isotype 1 but not human SIRPγ is provided. In some embodiments, an anti-SIRPβ1 antibody that binds to human SIRPβ1 isotype 1 but not human SIRPβ1 isotype 3 is provided. In some embodiments, an anti-SIRPβ1 antibody that binds to human SIRPβ1 isotype 1 but not mouse SIRPβ1 and / or cynomolgus monkey SIRPβ1 is provided. In some embodiments, the anti-SIRPβ1 antibody of this disclosure activates SIRPβ1 activity on CD14-positive monocytes. In some embodiments, the anti-SIRPβ1 antibody of this disclosure induces a respiratory burst in immune cells, such as neutrophils, monocytes, and / or macrophages. In some embodiments, the anti-SIRPβ1 antibody of this disclosure induces or enhances IL-8 expression in monocytes. In some embodiments, the anti-SIRPβ1 antibody of this disclosure induces or enhances TNFα expression in macrophages and / or dendritic cells. In some embodiments, the anti-SIRPβ1 antibody of this disclosure induces neutrophil-mediated phagocytosis, such as phagocytosis by tumor cells. In some embodiments, the anti-SIRPβ1 antibody of this disclosure enhances neutrophil-mediated tumor cell clearance. In some embodiments, the anti-SIRPβ1 antibody of this disclosure enhances or strengthens the antitumor properties or activity of neutrophils.

[0147] In some embodiments, the anti-SIRPβ1 antibody of this disclosure recruits immune cells. In some embodiments, the anti-SIRPβ1 antibody of this disclosure induces syk phosphorylation. In some embodiments, the anti-SIRPβ1 antibody of this disclosure induces syk phosphorylation upon passage through adjacent cell clusters expressing Fcγ receptors.

[0148] In some embodiments, the SIRPβ1 antibody of this disclosure downregulates SIRPβ1 expression on the cell surface. In some embodiments, the SIRPβ1 antibody of this disclosure does not downregulate SIRPβ1 expression on the cell surface. In some embodiments, the SIRPβ1 antibody of this disclosure blocks ligand binding to SIRPβ1. In some embodiments, the SIRPβ1 antibody of this disclosure does not block ligand binding to SIRPβ1.

[0149] In some embodiments, an anti-SIRPβ1 antibody is provided having one or more of the following properties: a) binding to human SIRPβ1 isotype 1, but not binding to human SIRPα;

[0150] b) Binds to human SIRPβ1 isotype 1, but not human SIRPγ;

[0151] c) Binds to human SIRPβ1 isotype 1, but not to human SIRPβ1 isotype 3;

[0152] d) Binds to human SIRPβ1 isotype 1, but not to mouse SIRPβ1;

[0153] e) Combined with human SIRPβ1 isotype 1, but not with cynomolgus monkey SIRPβ1;

[0154] f) Activate SIRPβ1 activity in CD14-positive monocytes in vitro and / or in vivo;

[0155] g) Inducing or enhancing respiratory bursts in immune cells, such as neutrophils and / or monocytes, in vitro and / or in vivo;

[0156] h) Inducing or enhancing IL-8 expression in monocytes in vitro and / or in vivo;

[0157] i) Inducing or enhancing TNFα expression in macrophages and / or dendritic cells in vitro and / or in vivo;

[0158] j) Inducing or enhancing neutrophil-mediated phagocytosis, such as phagocytosis by tumor cells, in vitro and / or in vivo;

[0159] k) Inducing or enhancing neutrophil-mediated tumor cell clearance in vivo;

[0160] l) Upregulate TREM2 expression on macrophages in vitro and / or in vivo;

[0161] m) Enhances macrophage viability in vitro and / or in vivo, alone and / or in combination with agonistic anti-TREM2 antibodies; and

[0162] n) Enhance the viability of dendritic cells in vitro and / or in vivo.

[0163] In some implementations, anti-SIRPβ1 antibodies with the following properties are provided:

[0164] a) Binds to human SIRPβ1 isotype 1, but not human SIRPα;

[0165] b) Binds to human SIRPβ1 isotype 1, but not human SIRPγ;

[0166] c) Binds to human SIRPβ1 isotype 1, but not to human SIRPβ1 isotype 3;

[0167] d) Binds to human SIRPβ1 isotype 1, but not to mouse SIRPβ1;

[0168] e) Combined with human SIRPβ1 isotype 1, but not with cynomolgus monkey SIRPβ1;

[0169] f) Activate SIRPβ1 activity in CD14-positive monocytes in vitro and / or in vivo;

[0170] g) Inducing or enhancing respiratory bursts in immune cells, such as neutrophils and / or monocytes, in vitro and / or in vivo;

[0171] h) Inducing or enhancing IL-8 expression in monocytes in vitro and / or in vivo;

[0172] i) Inducing or enhancing TNFα expression in macrophages and / or dendritic cells in vitro and / or in vivo;

[0173] j) Inducing or enhancing neutrophil-mediated phagocytosis, such as phagocytosis by tumor cells, in vitro and / or in vivo;

[0174] k) Inducing or enhancing neutrophil-mediated tumor cell clearance in vivo;

[0175] l) Upregulate TREM2 expression on macrophages in vitro and / or in vivo;

[0176] m) Enhances macrophage viability in vitro and / or in vivo, alone and / or in combination with agonistic anti-TREM2 antibodies; and

[0177] n) Enhance the viability of dendritic cells in vitro and / or in vivo.

[0178] A. Exemplary antibody and certain other antibody implementation schemes

[0179] In some embodiments, the present invention provides an anti-SIRPβ1 antibody comprising at least one, two, three, four, five, or six HVRs selected from the following: (a) HVR-H1 comprising the HVR-H1 amino acid sequence shown in Table 4 and / or Table 9; (b) HVR-H2 comprising the HVR-H2 amino acid sequence shown in Table 4 and / or Table 9; (c) HVR-H3 comprising the HVR-H3 amino acid sequence shown in Table 4 and / or Table 9; (d) HVR-L1 comprising the HVR-L1 amino acid sequence shown in Table 3 and / or Table 8; (e) HVR-L2 comprising the HVR-L2 amino acid sequence shown in Table 3 and / or Table 8; and (f) HVR-L3 comprising the HVR-L3 amino acid sequence shown in Table 3 and / or Table 8.

[0180] In some embodiments, the present invention provides an anti-SIRPβ1 antibody comprising HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, wherein: (a) HVR-H1 comprises the HVR-H1 amino acid sequence shown in Table 4 and / or Table 9; (b) HVR-H2 comprises the HVR-H2 amino acid sequence shown in Table 4 and / or Table 9; (c) HVR-H3 comprises the HVR-H3 amino acid sequence shown in Table 4 and / or Table 9; (d) HVR-L1 comprises the HVR-L1 amino acid sequence shown in Table 3 and / or Table 8; (e) HVR-L2 comprises the HVR-L2 amino acid sequence shown in Table 3 and / or Table 8; and (f) HVR-L3 comprises the HVR-L3 amino acid sequence shown in Table 3 and / or Table 8.

[0181] In some embodiments, the present invention provides an anti-SIRPβ1 antibody comprising (shown in Tables 3, 4, 8, and 9) SB-1, SB-2, SB-3, SB-4, SB-5, SB-6, SB-7, SB-8, SB-9, SB-10, SB-11, SB-12, SB-13, SB-14, SB-15, SB-16, SB-17, SB-18, SB-19, SB-20, SB-21, SB-22, SB-23, SB-24, SB-25, SB-26, SB-27, SB-28, SB-29, SB-30, SB-31, SB-32, SB-33, and SB-34. ,SB-35,SB-36,SB-37,SB-38,SB-39,SB-40,SB-41,SB-42,SB-43,SB-44,SB-45,SB-46,SB-47,SB-48,SB-49,SB-50,SB-1-2,SB-1-3,SB-1-4,SB Six HVRs for antibodies to -1-5, SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21.

[0182] In some embodiments, the present invention provides an anti-SIRPβ1 antibody comprising six HVRs of antibodies selected from (shown in Tables 3, 4, 8, and 9) SB-1, SB-2, SB-3, SB-4, SB-5, SB-6, SB-7, SB-8, SB-9, SB-14, SB-28, SB-39, SB-40, SB-49, SB-1-2, SB-1-3, SB-1-4, SB-1-5, SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21.

[0183] In some embodiments, the present invention provides an anti-SIRPβ1 antibody comprising six HVRs selected from the antibodies of SB-1-2, SB-1-3, SB-1-4, SB-1-5, SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21 (shown in Tables 8 and 9).

[0184] In some implementations, the present invention provides an anti-SIRPβ1 antibody comprising six HVRs selected from antibodies of SB-1-3, SB-2-8, and SB-8-13 (shown in Tables 8 and 9).

[0185] In some embodiments, the present invention provides an anti-SIRPβ1 antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:228; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:238; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:125; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:383; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:16; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:31.

[0186] In some embodiments, the present invention provides an anti-SIRPβ1 antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:229; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:239; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:125; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:383; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:16; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:31.

[0187] In some embodiments, the present invention provides an anti-SIRPβ1 antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:229; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:240; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:125; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:383; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:16; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:31.

[0188] In some embodiments, the present invention provides an anti-SIRPβ1 antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:229; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:241; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:125; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:383; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:16; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:31.

[0189] In some embodiments, the present invention provides an anti-SIRPβ1 antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:230; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:242; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:126; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:383; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:16; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:32.

[0190] In some embodiments, the present invention provides an anti-SIRPβ1 antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:231; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:243; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:126; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:383; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:16; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:32.

[0191] In some embodiments, the present invention provides an anti-SIRPβ1 antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:232; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:244; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:126; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:383; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:16; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:32.

[0192] In some embodiments, the present invention provides an anti-SIRPβ1 antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 99; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 245; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 126; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 383; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 32.

[0193] In some embodiments, the present invention provides an anti-SIRPβ1 antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:230; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:242; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:253; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:383; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:16; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:32.

[0194] In some embodiments, the present invention provides an anti-SIRPβ1 antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:233; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:246; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:131; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:383; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:19; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:37.

[0195] In some embodiments, the present invention provides an anti-SIRPβ1 antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:234; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:247; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:131; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:383; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:19; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:37.

[0196] In some embodiments, the present invention provides an anti-SIRPβ1 antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:233; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:248; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:131; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:383; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:19; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:37.

[0197] In some embodiments, the present invention provides an anti-SIRPβ1 antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:233; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:246; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:254; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:383; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:19; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:37.

[0198] In some embodiments, the present invention provides an anti-SIRPβ1 antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:235; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:249; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:163; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:2; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:69.

[0199] In some embodiments, the present invention provides an anti-SIRPβ1 antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:236; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:250; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:163; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:2; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:69.

[0200] In some embodiments, the present invention provides an anti-SIRPβ1 antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:237; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:251; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:163; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:2; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:69.

[0201] In some embodiments, the present invention provides an anti-SIRPβ1 antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:236; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:252; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:163; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:2; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:22; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:69.

[0202] In some embodiments, the present invention provides an anti-SIRPβ1 antibody comprising at least one, at least two, or all three selected from the following V HHVR sequences: (a) HVR-H1 containing the HVR-H1 amino acid sequence shown in Table 4 and / or Table 9; (b) HVR-H2 containing the HVR-H2 amino acid sequence shown in Table 4 and / or Table 9; (c) HVR-H3 containing the HVR-H3 amino acid sequence shown in Table 4 and / or Table 9. In some embodiments, the anti-SIRPβ1 antibody comprises SB-1, SB-2, SB-3, SB-4, SB-5, SB-6, SB-7, SB-8, SB-9, SB-10, SB-11, SB-12, SB-13, SB-14, SB-15, SB-16, SB-17, SB-18, SB-19, SB-20, SB-21, SB-22, SB-23, SB-24, SB-25, SB-26, SB-27, SB-28, SB-29, SB-30, SB-31, SB-32, SB-33, SB-34, SB-35, SB-36, SB-37, SB-28, SB-29, SB-30, SB-31, SB-32, SB-33, SB-34, SB-35, SB-36, SB-37, SB-38, SB-3 ... -37,SB-38,SB-39,SB-40,SB-41,SB-42,SB-43,SB-44,SB-45,SB-46,SB-47,SB-48,SB-49,SB-50,SB-1-2,SB-1-3,SB-1-4,SB-1-5,SB-2-7,SB- 2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21 antibodies to HVR-H1, HVR-H2, and HVR-H3. In some embodiments, the anti-SIRPβ1 antibody comprises HVR-H1, HVR-H2, and HVR-H3 of antibodies selected from (shown in Tables 4 and 9) SB-1, SB-2, SB-3, SB-4, SB-5, SB-6, SB-7, SB-8, SB-9, SB-14, SB-28, SB-39, SB-40, SB-49, SB-1-2, SB-1-3, SB-1-4, SB-1-5, SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21.In some embodiments, the anti-SIRPβ1 antibody comprises HVR-H1, HVR-H2, and HVR-H3 of antibodies selected from SB-1-2, SB-1-3, SB-1-4, SB-1-5, SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21. In some embodiments, the anti-SIRPβ1 antibody comprises HVR-H1, HVR-H2, and HVR-H3 of antibodies selected from SB-1-3, SB-2-8, and SB-8-13. In some embodiments, the anti-SIRPβ1 antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:229; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:239; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:125. In some embodiments, the anti-SIRPβ1 antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:231; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:243; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:126. In some embodiments, the anti-SIRPβ1 antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:233; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:246; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:131.

[0203] In some embodiments, the present invention provides an anti-SIRPβ1 antibody comprising at least one, at least two, or all three selected from the following V LHVR sequences: (a) HVR-L1 containing the HVR-L1 amino acid sequence shown in Table 3 and / or Table 8; (b) HVR-L2 containing the HVR-L2 amino acid sequence shown in Table 3 and / or Table 8; (c) HVR-L3 containing the HVR-L3 amino acid sequence shown in Table 3 and / or Table 8. In some embodiments, the anti-SIRPβ1 antibody comprises SB-1, SB-2, SB-3, SB-4, SB-5, SB-6, SB-7, SB-8, SB-9, SB-10, SB-11, SB-12, SB-13, SB-14, SB-15, SB-16, SB-17, SB-18, SB-19, SB-20, SB-21, SB-22, SB-23, SB-24, SB-25, SB-26, SB-27, SB-28, SB-29, SB-30, SB-31, SB-32, SB-33, SB-34, SB-35, SB-36, SB-37, SB-28, SB-29, SB-30, SB-31, SB-32, SB-33, SB-34, SB-35, SB-36, SB-37, SB-28, SB-3 ...38, SB-39, SB-30, SB-31, SB-32, SB-33, SB-34, SB- -37,SB-38,SB-39,SB-40,SB-41,SB-42,SB-43,SB-44,SB-45,SB-46,SB-47,SB-48,SB-49,SB-50,SB-1-2,SB-1-3,SB-1-4,SB-1-5,SB-2-7,SB- 2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21 antibodies to HVR-L1, HVR, L2, and HVR-L3. In some embodiments, the anti-SIRPβ1 antibody comprises HVR-L1, HVR, L2, and HVR-L3 of antibodies selected from SB-1, SB-2, SB-3, SB-4, SB-5, SB-6, SB-7, SB-8, SB-9, SB-14, SB-28, SB-39, SB-40, SB-49, SB-1-2, SB-1-3, SB-1-4, SB-1-5, SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21.In some embodiments, the anti-SIRPβ1 antibody comprises HVR-L1, HVR, L2, and HVR-L3 of antibodies selected from SB-1-2, SB-1-3, SB-1-4, SB-1-5, SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21. In some embodiments, the anti-SIRPβ1 antibody comprises HVR-L1, HVR, L2, and HVR-L3 of antibodies selected from SB-1-3, SB-2-8, and SB-8-13 (shown in Table 8). In some embodiments, the anti-SIRPβ1 antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:383; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:16; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:31. In some embodiments, the anti-SIRPβ1 antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:383; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:16; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:32. In some embodiments, the anti-SIRPβ1 antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:383; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:19; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:37.

[0204] In some implementations, an anti-SIRPβ1 antibody is provided herein, which comprises (a)V H The domain comprises at least one, at least two, or all three of the following VH HVR sequences: (i) HVR-H1 comprising the HVR-H1 amino acid sequence shown in Table 4 and / or Table 9, (ii) HVR-H2 comprising the HVR-H2 amino acid sequence shown in Table 4 and / or Table 9, and (iii) HVR-H3 comprising the HVR-H3 amino acid sequence shown in Table 4 and / or Table 9; and (b) V L The domain contains at least one, at least two, or all three selected from the following V. L HVR sequences: (i) HVR-L1 containing the HVR-L1 amino acid sequence shown in Table 3 and / or Table 8, (ii) HVR-L2 containing the HVR-L2 amino acid sequence shown in Table 3 and / or Table 8, and (iii) HVR-L3 containing the HVR-L3 amino acid sequence shown in Table 3 and / or Table 8.

[0205] In some implementations, an anti-SIRPβ1 antibody is provided herein, which comprises (a)V H The domain includes (i) HVR-H1 containing the HVR-H1 amino acid sequence shown in Table 4 and / or Table 9, (ii) HVR-H2 containing the HVR-H2 amino acid sequence shown in Table 4 and / or Table 9, and (iii) HVR-H3 containing the HVR-H3 amino acid sequence shown in Table 4 and / or Table 9; and (b) V L The domain includes (i) HVR-L1 containing the HVR-L1 amino acid sequence shown in Table 3 and / or Table 8, (ii) HVR-L2 containing the HVR-L2 amino acid sequence shown in Table 3 and / or Table 8, and (iii) HVR-L3 containing the HVR-L3 amino acid sequence shown in Table 3 and / or Table 8.

[0206] In some embodiments, the present invention provides an anti-SIRPβ1 antibody comprising V containing HVR-H1, HVR-H2, and HVR-H3. H Domains, and V containing HVR-L1, HVR-L2, and HVR-L3 L Domains, where HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 are selected from (shown in Tables 3, 4, 8, and 9) SB-1, SB-2, SB-3, SB-4, SB-5, SB-6, SB-7, SB-8, SB-9, SB-10, SB-11, SB-12, SB-13, SB-14, SB-15, SB-16, SB-17, SB-18, SB-19, SB-20, SB-21, SB-22, SB-23, SB-24, SB-25, SB-26, SB-27, SB-28, SB-29, SB-30, SB-31, SB-32, S B-33,SB-34,SB-35,SB-36,SB-37,SB-38,SB-39,SB-40,SB-41,SB-42,SB-43,SB-44,SB-45,SB-46,SB-47,SB-48,SB-49,SB-50,SB-1-2,SB-1-3,SB- HVR of antibodies to 1-4, SB-1-5, SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21.

[0207] In some embodiments, the present invention provides an anti-SIRPβ1 antibody comprising V containing HVR-H1, HVR-H2, and HVR-H3. H Domains, and V containing HVR-L1, HVR-L2, and HVR-L3 L The domains, wherein HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 are HVRs of antibodies selected from (shown in Tables 3, 4, 8, and 9) SB-1, SB-2, SB-3, SB-4, SB-5, SB-6, SB-7, SB-8, SB-9, SB-14, SB-28, SB-39, SB-40, SB-49, SB-1-2, SB-1-3, SB-1-4, SB-1-5, SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21.

[0208] In some embodiments, the present invention provides an anti-SIRPβ1 antibody comprising V containing HVR-H1, HVR-H2, and HVR-H3. H Domains, and V containing HVR-L1, HVR-L2, and HVR-L3 L The domains, wherein HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 are HVRs of antibodies selected from (shown in Tables 8 and 9) SB-1-2, SB-1-3, SB-1-4, SB-1-5, SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21.

[0209] In some embodiments, the present invention provides an anti-SIRPβ1 antibody comprising V containing HVR-H1, HVR-H2, and HVR-H3. H Domains, and V containing HVR-L1, HVR-L2, and HVR-L3 L The domains, wherein HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 are HVRs of antibodies selected from (shown in Tables 8 and 9) SB-1-3, SB-2-8, and SB-8-13.

[0210] In some implementations, an anti-SIRPβ1 antibody is provided herein, which comprises (a)V H The domain comprises (i) HVR-H1 containing the amino acid sequence of SEQ ID NO:229; (ii) HVR-H2 containing the amino acid sequence of SEQ ID NO:239; (iii) HVR-H3 containing the amino acid sequence of SEQ ID NO:125; and (b) V L The domain includes (iv) HVR-L1 containing the amino acid sequence of SEQ ID NO:383; (v) HVR-L2 containing the amino acid sequence of SEQ ID NO:16; and (vi) HVR-L3 containing the amino acid sequence of SEQ ID NO:31.

[0211] In some implementations, an anti-SIRPβ1 antibody is provided herein, which comprises (a)V H The domain comprises (i) HVR-H1 containing the amino acid sequence of SEQ ID NO:231; (ii) HVR-H2 containing the amino acid sequence of SEQ ID NO:243; (iii) HVR-H3 containing the amino acid sequence of SEQ ID NO:126; and (b) V L The domain includes (iv) HVR-L1 containing the amino acid sequence of SEQ ID NO:383; (v) HVR-L2 containing the amino acid sequence of SEQ ID NO:16; and (vi) HVR-L3 containing the amino acid sequence of SEQ ID NO:32.

[0212] In some implementations, an anti-SIRPβ1 antibody is provided herein, which comprises (a)V H The domain comprises (i) HVR-H1 containing the amino acid sequence of SEQ ID NO:233; (ii) HVR-H2 containing the amino acid sequence of SEQ ID NO:246; (iii) HVR-H3 containing the amino acid sequence of SEQ ID NO:131; and (b) V L The domain includes (iv) HVR-L1 containing the amino acid sequence of SEQ ID NO:383; (v) HVR-L2 containing the amino acid sequence of SEQ ID NO:19; and (vi) HVR-L3 containing the amino acid sequence of SEQ ID NO:37.

[0213] In another aspect, the anti-SIRPβ1 antibody comprises the antibodies corresponding to SEQ ID NO:268,270,272,274,276,279,281,283,285,287,289,291,293,295,297,299,301,303,305,307,309,311,313,315,317,319,321,323,325,327,329,331,333,335,337,339,341,343,345,347,34 The heavy chain variable domain (V) of amino acid sequences 9, 351, 353, 355, 357, 359, 361, 363, 365, 366, 367, 368, 369, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, or 382 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. H (SEQ ID) sequence. In some embodiments, with SEQ ID The amino acid sequences of NO:268,270,272,274,276,279,281,283,285,287,289,291,293,295,297,299,301,303,305,307,309,311,313,315,317,319,321,323,325,327,329,331,333,335,337,339,341,343,345,347,349,351,353,355,357,359,361,363, or 365 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. HThe sequence contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to the reference sequence, but the anti-SIRPβ1 antibody containing this sequence retains its ability to bind SIRPβ1. In some embodiments, the sequence is already present in SEQ ID NO: 268,270,272,274,276,279,281,283,285,287,289,291,293,295,297,299,301,303,305,307,309,311,313,315,317,319,321,323,325,327,329,331,333,335,337. Replace, insert, and / or delete a total of 1 to 10 amino acids in SEQ ID NO. 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 366, 367, 368, 369, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, or 382. In some embodiments, SEQ ID NO. 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 359, 361, 363, 365, 366, 367, 368, 369, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, or 382. NO:268,270,272,274,276,279,281,283,285,287,289,291,293,295,297,299,301, 303,305,307,309,311,313,315,317,319,321,323,325,327,329,331,333,335,337 The substitutions, insertions, and / or deletions of a total of 1 to 5 amino acids are made in the sequences 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 366, 367, 368, 369, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, or 382. In some embodiments, the substitutions, insertions, or deletions occur in regions outside the HVR (i.e., in the FR). Optionally, the anti-SIRPβ1 antibody contains SEQ ID [SEQ ID]. NO:268,270,272,274,276,279,281,283,285,287,289,291,293,295,297,299 ,301,303,305,307,309,311,313,315,317,319,321,323,325,327,329,331,3 33,335,337,339,341,343,345,347,349,351,353,355,357,359,361,363,365,366,367,368,369,371,372,373,374,375,376,377,378,379,380,381, or 382 of VH The sequence, including its post-translational modifications. In one particular implementation, V H It contains one, two or three HVRs selected from the following: (a) HVR-H1 containing the HVR-H1 amino acid sequence shown in Table 4 and / or Table 9; (b) HVR-H2 containing the HVR-H2 amino acid sequence shown in Table 4 and / or Table 9; (c) HVR-H3 containing the HVR-H3 amino acid sequence shown in Table 4 and / or Table 9.

[0214] In another aspect, an anti-SIRPβ1 antibody is provided, wherein the antibody comprises the same components as SEQ ID NO: 267,269,271,273,275,277,278,280,282,284,286,288,290,292,294,296,298,300,302,304,306,308,310,312,314,316,318,320,322,324,326,328,330,33 The light chain variable domain (V) of amino acid sequences 2,334,336,338,340,342,344,346,348,350,352,354,356,358,360,362,364, or 370 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. L In some embodiments, the SEQ ID NO: 267,269,271,273,275,277,278,280,282,284,286,288,290,292,294,296,298,300,302,304,306,308,310,312,314,316,318,320,322,324,326,328, The amino acid sequences 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, or 370 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. LThe sequence contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to the reference sequence, but the anti-SIRPβ1 antibody containing that sequence retains its ability to bind SIRPβ1. In some implementations, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted in SEQ ID NO:267,269,271,273,275,277,278,280,282,284,286,288,290,292,294,296,298,300,302,304,306,308,310,312,314,316,318,320,322,324,326,328,330,332,334,336,338,340,342,344,346,348,350,352,354,356,358,360,362,364, or 370. In some implementations, a total of 1 to 5 amino acids have been substituted, inserted, and / or deleted in SEQ ID NO:267,269,271,273,275,277,278,280,282,284,286,288,290,292,294,296,298,300,302,304,306,308,310,312,314,316,318,320,322,324,326,328,330,332,334,336,338,340,342,344,346,348,350,352,354,356,358,360,362,364, or 370. In some implementations, substitution, insertion, or deletion occurs in regions outside the HVR (i.e., in the FR). Optionally, the anti-SIRPβ1 antibody comprises V of SEQ ID NO: 267, 269, 271, 273, 275, 277, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, or 370. L The sequence, including its post-translational modifications. In one particular implementation, V LIt contains one, two, or three HVRs selected from the following: (a) HVR-L1 containing the HVR-L1 amino acid sequence shown in Table 3 and / or Table 8; (b) HVR-L2 containing the HVR-L2 amino acid sequence shown in Table 3 and / or Table 8; and (c) HVR-L3 containing the HVR-L3 amino acid sequence shown in Table 3 and / or Table 8.

[0215] In some embodiments, an anti-SIRPβ1 antibody is provided, wherein the antibody comprises V as described in any of the embodiments provided above. H And V in any of the implementation schemes provided above L In some embodiments, an anti-SIRPβ1 antibody is provided herein, wherein the antibody comprises V as described in any of the embodiments provided above. H And V in any of the implementation schemes provided above L In one embodiment, the antibody comprises a subset selected from SEQ ID NO:268,270,272,274,276,279,281,283,285,287,289,291,293,295,297,299,301,303,305,307,309,311,313,315,317,319,321,323,325,327,329,331,3 33,335,337,339,341,343,345,347,349,351,353,355,357,359,361,363,365,366,367,368,369,371,372,373,374,375,376,377,378,379,380,381, and 382 of V H Sequences; and V selected from SEQ ID NO: 267, 269, 271, 273, 275, 277, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, and 370 LThe sequence, including post-translational modifications of those sequences. In some embodiments, the antibody comprises a sequence selected from SB-1, SB-2, SB-3, SB-4, SB-5, SB-6, SB-7, SB-8, SB-9, SB-10, SB-11, SB-12, SB-13, SB-14, SB-15, SB-16, SB-17, SB-18, SB-19, SB-20, SB-21, SB-22, SB-23, SB-24, SB-25, SB-26, SB-27, SB-28, SB-29, SB-30, SB-31, SB-32, SB-33, SB-34, SB-35, SB-36. SB-37,SB-38,SB-39,SB-40,SB-41,SB-42,SB-43,SB-44,SB-45,SB-46,SB-47,SB-48,SB-49,SB-50,SB-1-2,SB-1-3,SB-1-4,SB-1-5 , SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21 antibodies. H Sequence and V L Sequence. In some embodiments, the antibody comprises the V of an antibody selected from SB-1, SB-2, SB-3, SB-4, SB-5, SB-6, SB-7, SB-8, SB-9, SB-14, SB-28, SB-39, SB-40, SB-49, SB-1-2, SB-1-3, SB-1-4, SB-1-5, SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21. H Sequence and V L Sequence. In some embodiments, the antibody comprises the V sequence of an antibody selected from SB-1-2, SB-1-3, SB-1-4, SB-1-5, SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21. H Sequence and V L Sequence. In some embodiments, the antibody comprises the V sequence of an antibody selected from SB-1-3, SB-2-8, and SB-8-13. H Sequence and VL Sequence. In some embodiments, the antibody comprises V of SEQ ID NO:367. H Sequence and V of SEQ ID NO:267 L Sequence. In some embodiments, the antibody comprises V of SEQ ID NO:372. H Sequence and V of SEQ ID NO:370 L Sequence. In some embodiments, the antibody comprises V of SEQ ID NO:376. H Sequence and V of SEQ ID NO:280 L sequence.

[0216] In some embodiments, the anti-SIRPβ1 antibody is combined with a compound selected from SEQ ID NO:268,270,272,274,276,279,281,283,285,287,289,291,293,295,297,299,301,303,305,307,309,311,313,315,317,319,321,323,325,327,329,331,3 33,335,337,339,341,343,345,347,349,351,353,355,357,359,361,363,365,366,367,368,369,371,372,373,374,375,376,377,378,379,380,381, and 382 of V H The sequence and V selected from SEQ ID NO:267,269,271,273,275,277,278,280,282,284,286,288,290,292,294,296,298,300,302,304,306,308,310,312,314,316,318,320,322,324,326,328,330,332,334,336,338,340,342,344,346,348,350,352,354,356,358,360,362,364, and 370 LAntibodies competitively bind to sequences. In some embodiments, the anti-SIRPβ1 antibody binds to a sequence selected from SB-1, SB-2, SB-3, SB-4, SB-5, SB-6, SB-7, SB-8, SB-9, SB-10, SB-11, SB-12, SB-13, SB-14, SB-15, SB-16, SB-17, SB-18, SB-19, SB-20, SB-21, SB-22, SB-23, SB-24, SB-25, SB-26, SB-27, SB-28, SB-29, SB-30, SB-31, SB-32, SB-33, SB-34, SB-35, SB-36. 6,SB-37,SB-38,SB-39,SB-40,SB-41,SB-42,SB-43,SB-44,SB-45,SB-46,SB-47,SB-48,SB-49,SB-50,SB-1-2,SB-1-3,SB-1-4,SB-1-5 , SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21 antibodies compete for binding. In some implementations, the anti-SIRPβ1 antibody competitively binds to antibodies selected from SB-1, SB-2, SB-3, SB-4, SB-5, SB-6, SB-7, SB-8, SB-9, SB-14, SB-28, SB-39, SB-40, SB-49, SB-1-2, SB-1-3, SB-1-4, SB-1-5, SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21. In some embodiments, the anti-SIRPβ1 antibody competitively binds to antibodies selected from SB-1-2, SB-1-3, SB-1-4, SB-1-5, SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21. In some embodiments, the anti-SIRPβ1 antibody competitively binds to antibodies selected from SB-1-3, SB-2-8, and SB-8-13. In some embodiments, the anti-SIRPβ1 antibody binds to V containing SEQ ID NO:367. H Sequence and V of SEQ ID NO:267 LAntibodies competitively bind to the sequence. In some embodiments, the anti-SIRPβ1 antibody binds to V containing SEQ ID NO:372. H Sequence and V of SEQ ID NO:370 L Antibodies competitively bind to the sequence. In some embodiments, the anti-SIRPβ1 antibody binds to V containing SEQ ID NO:376. H Sequence and V of SEQ ID NO:280 L Antibodies compete for binding to the sequence.

[0217] In some embodiments, the antibody binds to an antibody containing a number selected from SEQ ID NO: 268, 270, 272, 274, 276, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 3 33,335,337,339,341,343,345,347,349,351,353,355,357,359,361,363,365,366,367,368,369,371,372,373,374,375,376,377,378,379,380,381, and 382 of V H The sequence and V selected from SEQ ID NO:267,269,271,273,275,277,278,280,282,284,286,288,290,292,294,296,298,300,302,304,306,308,310,312,314,316,318,320,322,324,326,328,330,332,334,336,338,340,342,344,346,348,350,352,354,356,358,360,362,364, and 370 LThe anti-SIRPβ1 antibody binds to the same or overlapping human SIRPβ1 epitopes. In some embodiments, the antibody binds to epitopes selected from SB-1, SB-2, SB-3, SB-4, SB-5, SB-6, SB-7, SB-8, SB-9, SB-10, SB-11, SB-12, SB-13, SB-14, SB-15, SB-16, SB-17, SB-18, SB-19, SB-20, SB-21, SB-22, SB-23, SB-24, SB-25, SB-26, SB-27, SB-28, SB-29, SB-30, SB-31, SB-32, SB-33, SB-34, SB-35, SB-36, SB-37, and SB-38. The anti-SIRPβ1 antibodies of SB-39, SB-40, SB-41, SB-42, SB-43, SB-44, SB-45, SB-46, SB-47, SB-48, SB-49, SB-50, SB-1-2, SB-1-3, SB-1-4, SB-1-5, SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21 bind to the same or overlapping human SIRPβ1 epitopes.

[0218] In some embodiments, the antibody binds to human SIRPβ1 epitopes that are the same as or overlap with those of anti-SIRPβ1 antibodies selected from SB-1, SB-2, SB-3, SB-4, SB-5, SB-6, SB-7, SB-8, SB-9, SB-14, SB-28, SB-39, SB-40, SB-49, SB-1-2, SB-1-3, SB-1-4, SB-1-5, SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21. In some embodiments, the antibody binds to human SIRPβ1 epitopes that are identical to or overlap with the epitopes bound to anti-SIRPβ1 antibodies selected from SB-1-2, SB-1-3, SB-1-4, SB-1-5, SB-2-7, SB-2-8, SB-2-9, SB-2-10, SB-2-11, SB-8-13, SB-8-14, SB-8-15, SB-8-16, SB-40-18, SB-40-19, SB-40-20, and SB-40-21. In some embodiments, the antibody binds to human SIRPβ1 epitopes that are identical to or overlap with the epitopes bound to anti-SIRPβ1 antibodies selected from SB-1-3, SB-2-8, and SB-8-13. In some embodiments, the anti-SIRPβ1 antibody binds to V containing SEQ ID NO:367. H Sequence and V of SEQ ID NO:267 L The anti-SIRPβ1 antibody binds to the same or overlapping human SIRPβ1 epitopes. In some embodiments, the anti-SIRPβ1 antibody binds to V containing SEQ ID NO:372. H Sequence and V of SEQ ID NO:370 L The anti-SIRPβ1 antibody binds to the same or overlapping human SIRPβ1 epitopes. In some embodiments, the anti-SIRPβ1 antibody binds to V containing SEQ ID NO:376. H Sequence and V of SEQ ID NO:280 L The anti-SIRPβ1 antibody in the sequence binds to the same or overlapping human SIRPβ1 epitope. In some embodiments, the human SIRPβ1 epitope is the same as the epitope bound by the anti-SIRPβ1 antibody.

[0219] In some embodiments, the anti-SIRPβ1 antibody binds to epitopes from amino acids 30 to 148 of SEQ ID NO:1. In some embodiments, the anti-SIRPβ1 antibody binds to epitopes from amino acids 30 to 136 of SEQ ID NO:1. In some embodiments, the anti-SIRPβ1 antibody binds to epitopes from amino acids 30 to 80 of SEQ ID NO:1. In some embodiments, the anti-SIRPβ1 antibody binds to epitopes from amino acids 40 to 90 of SEQ ID NO:1. In some embodiments, the anti-SIRPβ1 antibody binds to epitopes from amino acids 50 to 100 of SEQ ID NO:1. In some embodiments, the anti-SIRPβ1 antibody binds to epitopes from amino acids 60 to 110 of SEQ ID NO:1. In some embodiments, the anti-SIRPβ1 antibody binds to epitopes from amino acids 70 to 120 of SEQ ID NO:1. In some embodiments, the anti-SIRPβ1 antibody binds to epitopes from amino acids 80 to 130 of SEQ ID NO:1. In some implementations, the anti-SIRPβ1 antibody binds to epitopes within amino acids 90 to 140 of SEQ ID NO:1.

[0220] In some embodiments, the anti-SIRPβ1 antibody according to any of the above embodiments is a monoclonal antibody, including humanized and / or human antibodies. In some embodiments, the anti-SIRPβ1 antibody is an antibody fragment, such as Fv, Fab, Fab', scFv, a biantibody, or an F(ab')2 fragment. In some embodiments, the anti-SIRPβ1 antibody is a substantial full-length antibody, such as an IgG1 antibody, an IgG2a antibody, or other antibody class or isotype as defined herein.

[0221] In some embodiments, the anti-SIRPβ1 antibody according to any of the above embodiments may be incorporated, alone or in combination, with any of the features described in Sections 1-7 below:

[0222] (1) Anti-SIRPβ1 antibody binding affinity

[0223] In some embodiments of any antibody provided herein, the antibody has a concentration of <1 μM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 10 μM). -8 M or smaller, such as 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 - 13 The dissociation constant (K) of M) DThe dissociation constant can be determined by any analytical technique, including any biochemical or biophysical technique such as ELISA, surface plasmon resonance (SPR), biolayer interferometry (see, for example, Octet System, ForteBio), isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), circular dichroism (CD), stop-flow analysis, and colorimetric or fluorescent protein melting analysis. In one embodiment, K D It is measured by radiolabeled antigen binding assay (RIA). In some embodiments, RIA is performed using a Fab-type antibody of interest and its antigen, as described, for example, in Chen et al., J. Mol. Biol. 293:865-881 (1999). In some embodiments, K D The measurement was performed using the BIACORE surface plasmon resonance assay, for example, at 25°C with ~10 response units (RU) using an immobilized antigen CM5 chip with a BIACORE-2000 or BIACORE-3000 (BIAcore, Inc., Piscataway, NJ). In some embodiments, K D It uses ForteBio Measurements were taken using a Red384 system (ForteBio, Menlo Park, CA), as discussed in the examples herein.

[0224] In some implementation schemes, K D It is determined using monovalent antibodies (e.g., Fab) or full-length antibodies. In some implementations, K D It is measured using full-length antibodies in monovalent form.

[0225] (2) Antibody fragments

[0226] In some embodiments of any antibody provided herein, the antibody is an antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, and other fragments described below. For a review of some antibody fragments, see Hudson et al., Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues and having an extended in vivo half-life, see U.S. Patent No. 5,869,046.

[0227] A biantibody is an antibody fragment having two antigen-binding sites, and it can be bivalent or bispecific. See, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003). Triantibodies and tetraantibodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003). A single-domain antibody is an antibody fragment containing the entire or part of the heavy chain variable domain, or the entire or part of the light chain variable domain. In some embodiments, the single-domain antibody is a human single-domain antibody (see, for example, U.S. Patent No. 6,248,516).

[0228] Antibody fragments can be prepared using various techniques, including but not limited to the proteolytic digestion of intact antibodies and the production of them through recombinant host cells (such as E. coli or bacteriophages), as described herein.

[0229] (3) Chimeric and humanized antibodies

[0230] In some embodiments of any antibody provided herein, the antibody is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567. In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In yet another example, a chimeric antibody is a "class-switched" antibody, wherein the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include their antigen-binding fragment.

[0231] In some embodiments of any antibody provided herein, the antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce its immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, the humanized antibody is substantially non-immunogenic in humans. In some embodiments, the humanized antibody has substantially the same affinity for the target as an antibody from another species from which the humanized antibody is derived. See, for example, U.S. Patent Nos. 5,530,101, 5,693,761, 5,693,762, and 5,585,089. In some embodiments, amino acids in the antibody variable domain are identified that can be modified without reducing the native affinity of the antigen-binding domain while simultaneously reducing its immunogenicity. See, for example, U.S. Patent Nos. 5,766,886 and 5,869,619. Generally, a humanized antibody comprises one or more variable domains, wherein the HVR (or a portion thereof) is derived from a non-human antibody, and the FR (or a portion thereof) is derived from a human antibody sequence. Optionally, the humanized antibody may also comprise at least a portion of a human constant region. In some implementations, some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., an antibody derived from HVR residues), for example, to restore or improve antibody specificity or affinity.

[0232] Humanized antibodies and their generation methods are reviewed, for example, by Almagro et al., Front. Biosci. 13:1619-1633 (2008), and further described, for example, by U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409. Human frame regions that can be used for humanization include, but are not limited to: frame regions selected using a "best-fit" method (see, for example, Sims et al., J. Immunol. 151:2296 (1993)); frame regions derived from the common sequences of specific subgroups of human antibodies from the light or heavy chain variable regions (see, for example, Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992); and Presta et al., J. Immunol. 151:2623). (1993)); human mature (somatic mutation) framework regions or human germline framework regions (see, for example, Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); and framework regions derived by screening FR libraries (see, for example, Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271: 22611-22618 (1996)).

[0233] (4) Human antibodies

[0234] In some embodiments of any antibody provided herein, the antibody is a human antibody. Human antibodies can be generated using a variety of techniques known in the art. Generally, human antibodies are described in van Dijk et al., Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0235] Human antibodies can be prepared by administering immunogens to transgenic animals modified to generate complete human antibodies or complete antibodies with human variable regions in response to antigenic challenge. Mouse strains deficient in antibody production can be engineered with large fragments of human Ig loci, with the expectation that these mice will generate human antibodies in the absence of mouse antibodies. Large fragments of human Ig preserve significant variable gene diversity and appropriate regulation of antibody production and expression. By utilizing mouse mechanisms for antibody diversification and selection and the lack of immunological tolerance to human proteins, the complete set of human antibodies reproduced in these mouse strains can generate high-affinity complete human antibodies against any antigen of interest, including human antigens. Using hybridoma technology, antigen-specific human monoclonal antibodies with desired specificity can be generated and selected. Some illustrative methods are described in U.S. Patent Nos. 5,545,807, 546,073, and 546,073. See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE. TM Technology; US Patent No. 5770429, its description Technology; US Patent No. 7041870, which describes KM Technology; and U.S. Patent Application Publication No. US2007 / 0061900, which describes The technology allows for the extraction of human variable regions from complete antibodies generated by such animals, for example, through further modification by combining them with different human constant regions.

[0236] Human antibodies can also be generated using hybridoma-based methods. Human myeloma and mouse-human heterologous myeloma cell lines for generating human monoclonal antibodies have been described (see, for example, Kozbor, J. Immunol. 133:3001 (1984) and Boerner et al., J. Immunol. 147:86 (1991)). Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA 103:3557-3562 (2006). Other methods include those described, for example, in U.S. Patent No. 7,189,826 (which describes the generation of monoclonal human IgM antibodies from hybridoma cell lines). Human hybridoma technology (Trioma technology) is also documented in Vollmers et al., Histology and Histopathology 20(3):927-937 (2005) and Vollmers et al., Methods and Findings in Experimental and Clinical Pharmacology 27(3):185-91 (2005). Human antibodies can also be generated by isolating variable domain sequences from Fv clones selected from a human-derived phage display library. These variable domain sequences can then be combined with the desired human constant domain. The technique for selecting human antibodies from an antibody library is described below.

[0237] In some embodiments of any antibody provided herein, the antibody is a human antibody isolated by in vitro methods and / or by screening a combinatorial library for antibodies with one or more desired activities. Suitable examples include, but are not limited to, phage display (CAT, Morphosys, Dyax, Biosite / Medarex, Xoma, Symptomogen, Alexion (formerly Proliferon), Affimed), ribosome display (CAT), yeast-based platform technology (Adimab), etc. In some phage display methods, the complete sets of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, which can then be screened for antigen-binding phages, as described in Winter et al., Ann. Rev. Immunol. 12:433-455 (1994). For example, various methods for generating phage display libraries and screening such libraries for antibodies with desired binding characteristics are known in the art. See also Sidhu et al., J. Mol. Biol. 338(2):299-310, 2004; Lee et al., J. Mol. Biol. 340(5):1073-1093, 2004; Fellouse Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(-2):119-132 (2004). Phages typically present antibody fragments as single-chain Fv (scFv) fragments or as Fab fragments. High-affinity antibodies against immunogens are provided from immunogenic libraries without the need for hybridoma construction. Alternatively, a non-immunized library can be cloned (e.g., from human) to provide a single source of antibodies against a large number of non-self and self-antigens in the absence of any immunization, as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, a non-immunized library can also be synthesized by cloning an unrearranged V gene segment from stem cells and using PCR primers containing random sequences to encode a highly variable HVR3 region and achieve rearrangement in vitro, as described by Hoogenboom et al., J. Mol. Biol. 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373 and U.S. Patent Publications Nos. 2007 / 0292936 and 2009 / 0002360. Antibodies isolated from human antibody libraries are considered to be human antibodies or fragments of human antibodies as described herein.

[0238] (5) Constant region including Fc region

[0239] In some embodiments of any antibody provided herein, the antibody comprises an Fc. In some embodiments, the Fc is a human IgG1, IgG2, IgG3, and / or IgG4 isotype. In some embodiments, the antibody is an IgG class, IgM class, or IgA class.

[0240] In some embodiments of any antibody provided herein, the antibody has an IgG2 isotype. In some embodiments, the antibody contains a human IgG2 constant region. In some embodiments, the human IgG2 constant region includes an Fc region. In some embodiments, the antibody induces one or more SIRPβ1 activities or independently of binding to an Fc receptor. In some embodiments, the antibody binds to an inhibitory Fc receptor. In some embodiments, the inhibitory Fc receptor is an inhibitory Fcγ receptor IIB (FcγIIB).

[0241] In some embodiments of any antibody provided herein, the antibody has an IgG1 isotype. In some embodiments, the antibody contains a mouse IgG1 constant region. In some embodiments, the antibody contains a human IgG1 constant region. In some embodiments, the human IgG1 constant region includes an Fc region. In some embodiments, the antibody binds to an inhibitory Fc receptor. In some embodiments, the inhibitory Fc receptor is an inhibitory Fcγ receptor IIB (FcγIIB).

[0242] In some embodiments of any antibody provided herein, the antibody has an IgG4 isotype. In some embodiments, the antibody contains a human IgG4 constant region. In some embodiments, the human IgG4 constant region includes an Fc region. In some embodiments, the antibody binds to an inhibitory Fc receptor. In some embodiments, the inhibitory Fc receptor is an inhibitory Fcγ receptor IIB (FcγIIB).

[0243] In some embodiments of any antibody provided herein, the antibody has a heterozygous IgG2 / 4 isotype. In some embodiments, the antibody comprises an amino acid sequence comprising amino acids 118 to 260 of human IgG2 according to EU numbering schemes and amino acids 261-447 of human IgG4 according to EU numbering schemes (WO 1997 / 11971; WO 2007 / 106585).

[0244] In some embodiments, the Fc region enhances clustering but does not activate complement compared to corresponding antibodies containing or not containing the amino acid substitution in the Fc region. In some embodiments, the antibody induces one or more activities of a target specifically bound by the antibody. In some embodiments, the antibody binds to SIRPβ1.

[0245] It may also be desirable to modify the anti-SIRPβ1 antibody of this disclosure to modify the antibody's effector function and / or increase its serum half-life. For example, the Fc receptor binding site on the constant region may be modified or mutated to remove or reduce binding affinity for certain Fc receptors, such as FcγRI, FcγRII, and / or FcγRIII, to reduce antibody-dependent cell-mediated cytotoxicity. In some embodiments, effector function is weakened by removing N-glycosylation of the antibody's Fc region (e.g., in the CH2 domain of IgG). In some embodiments, effector function is weakened by modifying regions such as 233-236, 297, and / or 327-331 of human IgG, as described in WO 99 / 58572 and Armour et al., Molecular Immunology 40:585-593 (2003); Reddy et al., J. Immunology 164:1925-1933 (2000). In other embodiments, it may be desirable to modify the anti-SIRPβ1 antibody of this disclosure to modify effector function to improve search selectivity for ITIM-containing FcγRIIb (CD32b) to improve SIRPβ1 antibody clustering on adjacent cells without activating humoral responses, including antibody-dependent cell-mediated cytotoxicity and antibody-dependent cell phagocytosis.

[0246] To increase the serum half-life of an antibody, a rescue receptor-binding epitope can be incorporated into the antibody (especially an antibody fragment), as described, for example, in U.S. Patent 5,739,277. As used herein, the term "rescue receptor-binding epitope" refers to an epitope in the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) responsible for increasing the in vivo serum half-life of the IgG molecule. Other amino acid sequence modifications.

[0247] (6) Multispecific antibodies

[0248] Multispecific antibodies are antibodies that have binding specificity against at least two different epitopes, including those on the same or another polypeptide (e.g., one or more SIRPβ1 polypeptides of this disclosure). In some embodiments, multispecific antibodies are bispecific antibodies. In some embodiments, multispecific antibodies are trispecific antibodies. In some embodiments, multispecific antibodies are tetraspecific antibodies. Such antibodies can be derived from full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). In some embodiments, a multispecific antibody includes a first antigen-binding region that binds to the first site on SIRPβ1 and a second antigen-binding region that binds to the second site on SIRPβ1. In some embodiments, a multispecific antibody includes a first antigen-binding region that binds to SIRPβ1 and a second antigen-binding region that binds to a second polypeptide.

[0249] This document provides a multispecific antibody comprising a first antigen-binding region that binds to SIRPβ1 (wherein the first antigen-binding region contains one of the six HVRs of the antibody described herein) and a second antigen-binding region that binds to a second polypeptide. In some embodiments, the first antigen-binding region contains one of the V of the antibody described herein. H or V L .

[0250] In some embodiments of any multispecific antibody, the second peptide is an antigen that facilitates transport across the blood-brain barrier. In some embodiments, the antibody described herein is conjugated to a peptide that facilitates transport across the blood-brain barrier. Numerous antigens and peptides that facilitate transport across the blood-brain barrier are known in the art (see, for example, Gabathuler R., Neurobiol. Dis. 37:48-57 (2010)). Such second antigens and peptides include, but are not limited to, transferrin receptor (TR), insulin receptor (HIR), insulin-like growth factor receptor (IGFR), low-density lipoprotein receptor-associated proteins 1 and 2 (LPR-1 and 2), diphtheria toxin receptors, including CRM197 (a non-toxic mutant of diphtheria toxin), TMEM 30(A) (a flipper enzyme), protein transduction domains such as TAT, Syn-B, or penetratin, polyarginine or generally positively charged peptides, angiopep peptides such as ANG1005 (see, for example, Gabathuler, 2010), and other cell surface proteins enriched on blood-brain barrier endothelial cells (see, for example, Daneman et al., PLoS One 5(10):e13741(2010)). In some embodiments, the second polypeptide is transferrin.

[0251] In some embodiments of any multispecific antibody, the second polypeptide is a pathogenic protein selected from amyloid β, oligoamyloid β, amyloid β plaque, amyloid precursor protein or fragment thereof, Tau, IAPP, α-synuclein, TDP-43, FUS protein, C9orf72 (chromosome 9 open reading frame 72), c9RAN protein, prion protein, PrPSc, huntingtin protein, calcitonin, superoxide dismutase, ataxia protein, ataxia protein 1, ataxia protein 2, ataxia protein 3, ataxia protein 7, ataxia protein 8, ataxia protein 10, and Lewy body. The second polypeptide includes: atrial natriuretic factor, pancreatic amyloid peptide, insulin, apolipoprotein AI, serum amyloid A, medin, prolactin, thyroxine transporter, lysozyme, β2-microglobulin, coagulant protein, corneal epithelial protein, cysteine ​​protease inhibitor, immunoglobulin light chain AL, S-IBM protein, repeat-associated non-ATG (RAN) translation products, dipeptide repeat (DPR) peptide, glycine-alanine (GA) repeat peptide, glycine-proline (GP) repeat peptide, glycine-arginine (GR) repeat peptide, proline-alanine (PA) repeat peptide, ubiquitin, and proline-arginine (PR) repeat peptide. In some embodiments, the second polypeptide is Tau. In some embodiments, the second polypeptide is Aβ. In some embodiments, the second polypeptide is TREM2. In some embodiments, the second polypeptide is α-synuclein.

[0252] In some embodiments of any multispecific antibody, the second polypeptide is a ligand and / or protein expressed on immune cells, wherein the ligand and / or protein is selected from CD40, OX40, ICOS, CD28, CD137 / 4-1BB, CD27, GITR, PD-L1, CTLA-4, PD-L2, PD-1, B7-H3, B7-H4, HVEM, BTLA, KIR, GAL9, TIM3, A2AR, LAG-3, and phosphatidylserine.

[0253] In some implementations of any multispecific antibody, the second polypeptide is a protein, lipid, polysaccharide, or glycolipid and any combination thereof expressed on one or more tumor cells.

[0254] In some embodiments of any multispecific antibody, the second polypeptide is an immunoglobulin-like receptor, such as TREM2. In some embodiments of any multispecific antibody, the second polypeptide is an immunoglobulin-like receptor expressed on myeloid lineage cells.

[0255] Multivalent antibodies contain at least one polypeptide chain (preferably two polypeptide chains), wherein the one or more polypeptide chains contain two or more variable domains. For example, one or more polypeptide chains may contain VD1-(X1). n -VD2-(X2) n -Fc, where VD1 is the first variable domain, VD2 is the second variable domain, Fc is a polypeptide chain in the Fc region, X1 and X2 represent amino acids or polypeptides, and n is 0 or 1. Similarly, one or more polypeptide chains may contain V H -C H 1-Flexible Joint-V H -C H 1-Fc chain; or V H -C H 1-V H -C H 1-Fc region chain. The multivalent antibodies described herein preferably further comprise at least two (and preferably four) light chain variable domain peptides. The multivalent antibodies described herein may, for example, comprise about two to about eight light chain variable domain peptides. The light chain variable domain peptides covered herein comprise light chain variable domains and optionally further comprise CL domains.

[0256] Techniques for generating multispecific antibodies include, but are not limited to, recombinant co-expression of two pairs of immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983), WO 93 / 08829, and Traunecker et al., EMBO J.10:3655 (1991)), and “segment-entry-cavity” engineering (see, for example, U.S. Patent No. 5,731,168). See also WO 2013 / 026833 (CrossMab). Multispecific antibodies can also be generated by engineered electrostatic manipulation effects used to generate antibody Fc-heterodimeric molecules (WO 2009 / 089004A1); crosslinking two or more antibodies (see, for example, U.S. Patent No. 4,676,980); using leucine; using a “biantibody” technique for generating bispecific antibody fragments (see, for example, Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (see, for example, Gruber et al., J. Immunol., 152:5368 (1994)); and by preparing trispecific antibodies as described, for example, Tutt et al., J. Immunol. 147:60 (1991).

[0257] This article also includes engineered antibodies having three or more functional antigen-binding sites, including “octopus antibodies” (see, for example, US 2006 / 0025576). Antibodies described herein also include “dual-action FAbs” or “DAFs” containing antigen-binding sites that bind multiple SIRPβ1 (see, for example, US 2008 / 0069820).

[0258] (7) Antibody variants

[0259] In some implementations of any antibody provided herein, amino acid sequence variants of the antibody are covered. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody.

[0260] (i) Substitution, insertion, and deletion variants

[0261] In some embodiments of any antibody provided herein, antibody variants with one or more amino acid substitutions are provided. Amino acid sequence variants of the antibody can be prepared by introducing suitable modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, residue deletions and / or insertions and / or substitutions within the antibody amino acid sequence.

[0262] Table 1: Amino Acid Substitutions

[0263]

[0264]

[0265] Substantial modification of the biological properties of antibodies is achieved by selecting substitutions that differ significantly in maintaining the following: (a) the structure of the polypeptide backbone in the substituted region, such as sheet or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site; or (c) the volume of the side chains. Naturally occurring residues are grouped based on common side chain characteristics:

[0266] (1) Hydrophobic: Leucine, Met, Ala, Val, Leu, Ile;

[0267] (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0268] (3) Acidic: Asp, Glu;

[0269] (4) Alkaline: His, Lys, Arg;

[0270] (5) Residues affecting chain orientation: Gly, Pro; and

[0271] (6) Aromatics: Trp, Tyr, Phe.

[0272] For example, non-conserved substitutions can involve the exchange of a member of one of these classes for a member of another class. Such substitution residues can be introduced, for example, into regions homologous to non-human antibodies in human antibodies or into non-homologous regions of molecules.

[0273] When modifying the peptides or antibodies described herein, the hydrophobic index of the amino acids may be considered according to certain embodiments. A hydrophobic index has been assigned to each amino acid based on its hydrophobic and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0274] The importance of the hydrophobic amino acid index in conferring biological function to protein interactions is understood in the art (Kyte et al., J. Mol. Biol., 157:105-131 (1982)). It is known that certain amino acids can substitute for other amino acids with similar hydrophobic indices or scores while still retaining similar biological activity. In some embodiments, substitutions based on the hydrophobic index include amino acids whose hydrophobic index is within ±2. In some embodiments, substitutions are included for those within ±1, and in some embodiments, those within ±0.5.

[0275] It is also understood in the art that similar amino acid substitutions can be effectively made based on hydrophobicity, particularly in cases where the resulting biologically functional proteins or peptides are intended for use in immunological embodiments, such as in this case. In some embodiments, the maximum local average hydrophilicity of a protein (as determined by the hydrophilicity of its adjacent amino acids) is related to its immunogenicity and antigenicity, i.e., the biological characteristics of the protein.

[0276] The following hydrophilicity values ​​have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0±1); aspartic acid (+3.0±1); glutamic acid (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). When modifications are made based on similar hydrophilicity values, in some embodiments, this includes substitutions of amino acids with hydrophilicity values ​​within ±2, in others within ±1, and in still others within ±0.5. Epitopes can also be identified based on the primary amino acid sequence based on hydrophilicity. These regions are also referred to as "epitope core regions."

[0277] In some embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, as long as such changes do not materially reduce the antibody's ability to bind to the antigen. For example, conserved changes (e.g., conserved substitutions, as provided herein) may be made in the HVR that do not materially reduce binding affinity. Such changes may, for example, be outside the antigen-contacting residues in the HVR. In some embodiments of the variant VH and VL sequences provided above, each HVR is either unchanged or contains no more than one, two, or three amino acid substitutions.

[0278] Amino acid sequence insertions include fusion of the amino and / or carboxyl ends of peptides ranging in length from one residue to 100 or more residues, as well as intra-sequence insertions of one or more amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of the N- or C-terminus of the antibody with an enzyme (e.g., for ADEPT) or a peptide that extends the serum half-life of the antibody.

[0279] Cysteine ​​residues that are not involved in maintaining the correct conformation of the antibody can also be substituted, typically with serine, to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine ​​bonds can be added to the antibody to improve its stability (especially when the antibody is an antibody fragment, such as an Fv fragment).

[0280] (ii) Glycosylation variants

[0281] In some implementations of any antibody provided herein, the antibody is modified to increase or decrease the degree of antibody glycosylation. Adding or deleting glycosylation sites on the antibody can be conveniently achieved by altering the amino acid sequence to create or eliminate one or more glycosylation sites.

[0282] Antibody glycosylation is typically N-linked or O-linked. N-linking refers to the attachment of a carbohydrate module to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid other than proline) are recognition sequences for the enzymatic attachment of the carbohydrate module to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in the polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyl amino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

[0283] Adding glycosylation sites to antibodies can be conveniently achieved by altering the amino acid sequence to include one or more of the tripeptide sequences described above (for N-linked glycosylation sites). Alternatively, alterations can be made by adding or substituting one or more serine or threonine residues (for O-linked glycosylation sites) to the original antibody sequence.

[0284] In cases where the antibody contains an Fc region, the carbohydrates attached to the Fc region can be modified. Naturally occurring antibodies generated from mammalian cells typically contain branched, biantennary oligosaccharides, generally linked by an N-terminal linker to the CH2 domain of the Fc region, designated Asn297 according to Kabat numbering conventions. Oligosaccharides can include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose of GlcNAc attached to the "backbone" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the present invention can be modified to create antibody variants with certain modified properties.

[0285] In one embodiment, an antibody variant is provided having a carbohydrate structure lacking (direct or indirect) fucose attachment to the Fc region. See, for example, U.S. Patent Publications Nos. 2003 / 0157108 and 2004 / 0093621. Examples of publications involving “defucosylated” or “fucosylated” antibody variants include: US 2003 / 0157108; US2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US ​​2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; Okazaki et al., J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004). Examples of cell lines capable of generating defucosylation antibodies include Led 3CHO cells with protein fucosylation defects (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US 2003 / 0157108), and knockout cell lines, such as α-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, for example, Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004); Kanda et al., Biotechnol. Bioeng. 94(4):680-688 (2006)).

[0286] (iii) Modified constant region

[0287] In some embodiments of any antibody provided herein, the antibody Fc is an antibody Fc isotype and / or modification. In some embodiments, the antibody Fc isotype and / or modification is capable of binding to the Fcγ receptor.

[0288] In some embodiments of any antibody provided herein, the modified antibody Fc is an IgG1-modified Fc. In some embodiments, the IgG1-modified Fc contains one or more modifications. For example, in some embodiments, the IgG1-modified Fc contains one or more amino acid substitutions (e.g., relative to the wild-type Fc region of the same isotype). In some embodiments, one or more amino acid substitutions are selected from N297A (Bolt S et al., (1993) Eur J Immunol 23:403-411), D265A (Shields et al., (2001) RJBiol.Chem.276,6591-6604), L234A, L235A (Hutchins et al., (1995) Proc Natl Acad Sci USA, 92:11980-11984; Alegre et al., (1994) Transplantation 57:1537-1543.31; Xu et al., (2000) Cell Immunol, 200:16-26), G237A (Alegre et al., (1994) Transplantation 57:1537-1543.31; Xu et al., (2000) Cell Immunol, 200:16-26), C226S, C229S, E233P, L234V, L234F, L235E (McEarchern et al., (2007) Blood, 109:1185-1192), P331S (Sazinsky et al., (2008) Proc Natl Acad Sci USA 2008, 105:20167-20172), S267E, L328F, A330L, M252Y, S254T, and / or T256E, wherein the amino acid positions are in accordance with EU numbering rules.

[0289] In some embodiments of any IgG1-modified Fc, the Fc contains the N297A mutation according to EU numbering. In some embodiments of any IgG1-modified Fc, the Fc contains the D265A and N297A mutations according to EU numbering. In some embodiments of any IgG1-modified Fc, the Fc contains the D270A mutation according to EU numbering. In some embodiments, the IgG1-modified Fc contains the L234A and L235A mutations according to EU numbering. In some embodiments of any IgG1-modified Fc, the Fc contains the L234A and G237A mutations according to EU numbering. In some embodiments of any IgG1-modified Fc, the Fc contains the L234A, L235A, and G237A mutations according to EU numbering. In some embodiments of any IgG1-modified Fc, the Fc contains one or more (inclusive) of the following mutations according to EU numbering: P238D, L328E, E233, G237D, H268D, P271G, and A330R. In some embodiments of any IgG1-modified Fc, the Fc contains one or more of the following mutations according to EU numbering: S267E / L328F. In some embodiments of any IgG1-modified Fc, the Fc contains the following mutations according to EU numbering: P238D, L328E, E233D, G237D, H268D, P271G, and A330R. In some embodiments of any IgG1-modified Fc, the Fc contains the following mutations according to EU numbering: P238D, L328E, G237D, H268D, P271G, and A330R. In some embodiments of any IgG1-modified Fc, the Fc contains mutations according to EU numbering: P238D, S267E, L328E, E233D, G237D, H268D, P271G, and A330R. In some embodiments of any IgG1-modified Fc, the Fc contains mutations according to EU numbering: P238D, S267E, L328E, G237D, H268D, P271G, and A330R. In some embodiments of any IgG1-modified Fc, the Fc contains mutations according to EU numbering: C226S, C229S, E233P, L234V, and L235A. In some embodiments of any IgG1-modified Fc, the Fc contains mutations according to EU numbering: L234F, L235E, and P331S. In some embodiments of any IgG1-modified Fc, the Fc contains the S267E and L328F mutations according to EU numbering schemes. In some embodiments of any IgG1-modified Fc, the Fc contains the S267E mutation according to EU numbering schemes.In some embodiments of any IgG1-modified Fc, the Fc comprises replacing the constant weight 1 (CH1) and hinge region of IgG1, and the carpa light chain, with the CH1 and hinge region of IgG2 (amino acids 118-230 of IgG2 according to EU numbering scheme).

[0290] In some embodiments of any IgG1-modified Fc, the Fc includes two or more amino acid substitutions that enhance antibody clustering but do not activate complement compared to a corresponding antibody having an Fc region that does not include the two or more amino acid substitutions. Thus, in some embodiments of any IgG1-modified Fc, the IgG1-modified Fc is an antibody containing an Fc region, wherein the antibody includes an amino acid substitution at position E430G and one or more amino acid substitutions at residue positions selected from L234F, L235A, L235E, S267E, K322A, L328F, A330S, P331S, and any combination thereof in the Fc region according to EU numbering schemes. In some embodiments, the IgG1-modified Fc includes amino acid substitutions at positions E430G, L243A, L235A, and P331S according to EU numbering schemes. In some embodiments, the IgG1-modified Fc includes amino acid substitutions at positions E430G and P331S according to EU numbering schemes. In some embodiments, the IgG1-modified Fc contains amino acid substitutions at positions E430G and K322A according to EU numbering. In some embodiments, the IgG1-modified Fc contains amino acid substitutions at positions E430G, A330S, and P331S according to EU numbering. In some embodiments, the IgG1-modified Fc contains amino acid substitutions at positions E430G, K322A, A330S, and P331S according to EU numbering. In some embodiments, the IgG1-modified Fc contains amino acid substitutions at positions E430G, K322A, and A330S according to EU numbering. In some embodiments, the IgG1-modified Fc contains amino acid substitutions at positions E430G, K322A, and P331S according to EU numbering.

[0291] In some embodiments of any IgG1-modified Fc, the IgG1-modified Fc described herein may further be combined with one or more of the following mutations according to EU numbering rules: A330L (Lazar et al., Proc Natl Acad Sci USA, 103:4005-4010 (2006)), or L234F, L235E, and / or P331S mutations (Sazinsky et al., Proc Natl Acad Sci USA, 105:20167-20172 (2008)) to eliminate complement activation. In some embodiments of any IgG1-modified Fc, the IgG1-modified Fc may further comprise one or more of the following mutations according to EU numbering rules: A330L, A330S, L234F, L235E, and / or P331S. In some embodiments of any IgG1-modified Fc, the IgG1-modified Fc may further contain one or more mutations to enhance the antibody's half-life in human serum (e.g., one or more of the M252Y, S254T, and T256E mutations according to EU numbering rules (including all)). In some embodiments of any IgG1-modified Fc, the IgG1-modified Fc may further contain one or more of the E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, and / or S440W according to EU numbering rules.

[0292] Other aspects of this disclosure relate to antibodies having a modified constant region (i.e., the Fc region). Antibodies that rely on binding to the FcγR receptor to activate the target receptor may lose their agonist activity if modified to eliminate FcγR binding (see, for example, Wilson et al., Cancer Cell 19:101-113 (2011); Armour et al., Immunology 40:585-593 (2003); and White et al., Cancer Cell 27:138-148 (2015)). Accordingly, it is believed that when an antibody has an Fc domain derived from the human IgG2 isotype (CH1 and hinge region) or an Fc domain of another type that preferentially binds to the inhibitory FcγRIIB receptor, or a variation thereof, the anti-SIRPβ1 antibody of this disclosure with proper epitope specificity can activate the target antigen with minimal adverse effects.

[0293] In some embodiments of any antibody provided herein, the modified antibody Fc is an IgG2-modified Fc. In some embodiments, the IgG2-modified Fc contains one or more modifications. For example, in some embodiments, the IgG2-modified Fc contains one or more amino acid substitutions (e.g., relative to the wild-type Fc region of the same isotype). In some embodiments of any IgG2-modified Fc, one or more amino acid substitutions are selected according to EU numbering rules: V234A (Alegre et al., Transplantation 57:1537-1543 (1994); Xu et al., Cell Immunol, 200:16-26 (2000)); G237A (Cole et al., Transplantation, 68:563-571 (1999)); H268Q, V309L, A330S, P331S (US 2007 / 0148167; Armour et al., Eur J Immunol 29:2613-2624 (1999); Armour et al., The Haematology Journal 1 (Suppl. 1):27 (2000); Armour et al., The Haematology Journal 1 (Suppl. 1): 27 (2000)), C219S, and / or C220S (White et al., Cancer Cell 27, 138-148 (2015)); S267E, L328F (Chu et al., Mol Immunol, 45: 3926-3933 (2008)); and M252Y, S254T, and / or T256E. In some embodiments of any IgG2-modified Fc, the Fc contains amino acid substitutions at positions V234A and G237A according to EU numbering. In some embodiments of any IgG2-modified Fc, the Fc contains amino acid substitutions at positions C219S or C220S according to EU numbering. In some embodiments of any IgG2-modified Fc, the Fc contains amino acid substitutions at positions A330S and P331S according to EU numbering. In some embodiments of any IgG2-modified Fc, the Fc contains amino acid substitutions at positions S267E and L328F according to EU numbering scheme.

[0294] In some embodiments of any IgG2-modified Fc, the Fc contains a C127S amino acid substitution according to EU numbering rules (White et al., (2015) Cancer Cell 27, 138-148; Lightle et al., Protein Sci. 19: 753-762 (2010); and WO 2008 / 079246). In some embodiments of any IgG2-modified Fc, the antibody has an IgG2 isotype and a carpa light chain constant domain containing a C214S amino acid substitution according to EU numbering rules (White et al., Cancer Cell 27: 138-148 (2015); Lightle et al., Protein Sci. 19: 753-762 (2010); and WO 2008 / 079246).

[0295] In some embodiments of any IgG2-modified Fc, the Fc contains a C220S amino acid substitution according to EU numbering rules. In some embodiments of any IgG2-modified Fc, the antibody has an IgG2 isotype and a carpa light chain constant domain containing a C214S amino acid substitution according to EU numbering rules.

[0296] In some embodiments of any IgG2-modified Fc, the Fc contains a C219S amino acid substitution according to EU numbering rules. In some embodiments of any IgG2-modified Fc, the antibody has an IgG2 isotype and a carpa light chain constant domain containing a C214S amino acid substitution according to EU numbering rules.

[0297] In some embodiments of any IgG2-modified Fc, the Fc comprises an IgG2 isotype heavy chain constant domain 1 (CH1) and a hinge region (White et al., Cancer Cell 27:138-148 (2015)). In some embodiments of any IgG2-modified Fc, the IgG2 isotype CH1 and hinge region contain an amino acid sequence of 118-230 according to EU numbering rules. In some embodiments of any IgG2-modified Fc, the antibody Fc region contains an S267E amino acid substitution, an L328F amino acid substitution, or both, and / or an N297A or N297Q amino acid substitution according to EU numbering rules.

[0298] In some embodiments of any IgG2-modified Fc, the Fc further comprises one or more amino acid substitutions at positions E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, and S440W according to EU numbering rules. In some embodiments of any IgG2-modified Fc, the Fc may further comprise one or more mutations to enhance the antibody's half-life in human serum (e.g., one or more of the mutations M252Y, S254T, and T256E according to EU numbering rules). In some embodiments of any IgG2-modified Fc, the Fc may further comprise A330S and P331S.

[0299] In some embodiments of any IgG2-modified Fc, the Fc is an IgG2 / 4 hybrid Fc. In some embodiments, the IgG2 / 4 hybrid Fc comprises IgG2 aa 118 to 260 and IgG4 aa 261 to 447. In some embodiments of any IgG2-modified Fc, the Fc comprises one or more amino acid substitutions at positions H268Q, V309L, A330S, and P331S according to EU numbering scheme.

[0300] In some embodiments of any IgG1 and / or IgG2 modified Fc, the Fc contains one or more additional amino acid substitutions selected from A330L, L234F; L235E, or P331S according to EU numbering scheme; and any combination thereof.

[0301] In some embodiments of any IgG1 and / or IgG2 modified Fc, the Fc comprises one or more amino acid substitutions at residue positions selected from C127S, L234A, L234F, L235A, L235E, S267E, K322A, L328F, A330S, P331S, E345R, E430G, S440Y, and any combination thereof, according to EU numbering schemes. In some embodiments of any IgG1 and / or IgG2 modified Fc, the Fc comprises amino acid substitutions at positions E430G, L243A, L235A, and P331S, according to EU numbering schemes. In some embodiments of any IgG1 and / or IgG2 modified Fc, the Fc comprises amino acid substitutions at positions E430G and P331S, according to EU numbering schemes. In some embodiments of any IgG1 and / or IgG2 modified Fc, the Fc contains amino acid substitutions at positions E430G and K322A according to EU numbering. In some embodiments of any IgG1 and / or IgG2 modified Fc, the Fc contains amino acid substitutions at positions E430G, A330S, and P331S according to EU numbering. In some embodiments of any IgG1 and / or IgG2 modified Fc, the Fc contains amino acid substitutions at positions E430G, K322A, and A330S according to EU numbering. In some embodiments of any IgG1 and / or IgG2 modified Fc, the Fc contains amino acid substitutions at positions E430G, K322A, and A330S according to EU numbering. In some embodiments of any IgG1 and / or IgG2 modified Fc, the Fc contains amino acid substitutions at positions E430G, K322A, and P331S according to EU numbering. In some embodiments of any IgG1 and / or IgG2 modified Fc, the Fc contains amino acid substitutions at positions S267E and L328F according to EU numbering. In some embodiments of any IgG1 and / or IgG2 modified Fc, the Fc contains amino acid substitutions at position C127S according to EU numbering. In some embodiments of any IgG1 and / or IgG2 modified Fc, the Fc contains amino acid substitutions at positions E345R, E430G, and S440Y according to EU numbering.

[0302] In some embodiments of any antibody provided herein, the modified antibody Fc is an IgG4-modified Fc. In some embodiments, the IgG4-modified Fc contains one or more modifications. For example, in some embodiments, the IgG4-modified Fc contains one or more amino acid substitutions (e.g., relative to the wild-type Fc region of the same isotype). In some embodiments of any IgG4-modified Fc, the one or more amino acid substitutions are selected from L235A, G237A, S229P, L236E (Reddy et al., J Immunol 164:1925-1933 (2000)), S267E, E318A, L328F, M252Y, S254T, and / or T256E according to EU numbering rules. In some embodiments of any IgG4-modified Fc, the Fc may further contain L235A, G237A, and E318A according to EU numbering rules. In some embodiments of any IgG4-modified Fc, the Fc may further comprise S228P and L235E in accordance with EU numbering rules. In some embodiments of any IgG4-modified Fc, the IgG4-modified Fc may further comprise S267E and L328F in accordance with EU numbering rules.

[0303] In some implementations of any IgG4-modified Fc, the IgG4-modified Fc may be combined with one or more mutations described in accordance with EU numbering rules as described in S228P mutation (Angal et al., Mol Immunol. 30: 105-108 (1993)) and / or (Peters et al., J Biol Chem. 287 (29): 24525-33 (2012)) to enhance antibody stabilization.

[0304] In some embodiments of any IgG4-modified Fc, the IgG4-modified Fc may further include one or more mutations to enhance the antibody’s half-life in human serum (e.g., one or more of the M252Y, S254T, and T256E mutations according to EU numbering rules, including all of them).

[0305] In some embodiments of any IgG4-modified Fc, the Fc comprises L235E according to EU numbering. In some embodiments of any IgG4-modified Fc, the Fc comprises one or more amino acid substitutions at residue positions selected from C127S, F234A, L235A, L235E, S267E, K322A, L328F, E345R, E430G, S440Y, and any combination thereof according to EU numbering. In some embodiments of any IgG4-modified Fc, the Fc comprises amino acid substitutions at positions E430G, L243A, L235A, and P331S according to EU numbering. In some embodiments of any IgG4-modified Fc, the Fc comprises amino acid substitutions at positions E430G and P331S according to EU numbering. In some embodiments of any IgG4-modified Fc, the Fc comprises amino acid substitutions at positions E430G and K322A according to EU numbering. In some embodiments of any IgG4-modified Fc, the Fc contains an amino acid substitution at position E430 according to EU numbering. In some embodiments of any IgG4-modified Fc, the Fc region contains amino acid substitutions at positions E430G and K322A according to EU numbering. In some embodiments of any IgG4-modified Fc, the Fc contains amino acid substitutions at positions S267E and L328F according to EU numbering. In some embodiments of any IgG4-modified Fc, the Fc contains an amino acid substitution at position C127S according to EU numbering. In some embodiments of any IgG4-modified Fc, the Fc contains amino acid substitutions at positions E345R, E430G, and S440Y according to EU numbering.

[0306] (8) Other antibody modifications

[0307] In some embodiments of any antibody, the antibody is a derivative. The term "derivative" refers to a molecule that includes chemical modifications other than the insertion, deletion, or substitution of amino acids (or nucleic acids). In some embodiments, the derivative contains covalent modifications, including but not limited to chemical bonds with polymers, lipids, or other organic or inorganic modules. In some embodiments, the chemically modified antigen-binding protein may have a longer circulating half-life than the unmodified antigen-binding protein. In some embodiments, the chemically modified antigen-binding protein may have improved targeting ability against desired cells, tissues, and / or organs. In some embodiments, the derivative antigen-binding protein is covalently modified to include attachment to one or more water-soluble polymers, including but not limited to polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol. See, for example, U.S. Patent Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, and 4,179,337. In some embodiments, the derivative antigen-binding protein comprises one or more polymers, including but not limited to monomethoxy-polyethylene glycol, dextran, cellulose, ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, polyethylene pyrrolidone, poly-1,3-dioxacyclopentane, poly-1,3,6-trioxacyclohexane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), poly-(N-vinylpyrrolidone)-polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethyleneized polyols (e.g., glycerol), and polyvinyl alcohol, as well as mixtures of such polymers.

[0308] In some embodiments, the derivative is covalently modified with polyethylene glycol (PEG) subunits. In some embodiments, one or more water-soluble polymers are bonded at one or more specific sites on the derivative, such as at the amino terminus. In some embodiments, one or more water-soluble polymers are randomly attached to one or more side chains of the derivative. In some embodiments, PEG is used to improve the therapeutic capacity of antigen-binding proteins. In some embodiments, PEG is used to improve the therapeutic capacity of humanized antibodies. Certain such methods are discussed, for example, in U.S. Patent No. 6,133,426, and are incorporated herein by reference for any purpose.

[0309] Peptide analogs are frequently used in the pharmaceutical industry as non-peptide drugs that possess properties similar to those of a template peptide. These types of non-peptide compounds are called peptide mimetics (or peptidomimetics) (Fauchere, J. Adv. Drug Res., 15:29 (1986); and Evans et al., J. Med. Chem., 30:1229 (1987), which are incorporated herein by reference for any purpose). Such compounds are often developed using computer molecular modeling. Peptide mimetics, structurally similar to therapeutically useful peptides, can be used to produce similar therapeutic or preventative effects. Generally, peptide mimics are structurally similar to exemplary polypeptides (i.e., polypeptides with biochemical properties or pharmacological activity), such as human antibodies, but have one or more peptide links optionally replaced by methods known in the art with links selected from: -CH2NH-, -CH2S-, -CH2-CH2-, -CH=CH- (cis and trans), -COCH2-, -CH(OH)CH2-, and -CH2SO-. In some embodiments, a more stable peptide can be generated by systematically replacing one or more amino acids of the common sequence with the same type of D-amino acid (e.g., D-lysine instead of L-lysine). Alternatively, a constrained peptide containing a common sequence or substantially identical variations of the common sequence can be generated by methods known in the art (Rizo and Gierasch, Ann. Rev. Biochem., 61:387 (1992), incorporated herein by reference for any purpose); for example, by adding an internal cysteine ​​residue capable of forming an intramolecular disulfide bridge of a cyclized peptide.

[0310] Drug conjugation involves conjugating a biologically active cytotoxic (anticancer) payload or drug to an antibody that specifically targets certain tumor markers (e.g., ideally, peptides found only in or on tumor cells). The antibody tracks these proteins in the body and attaches itself to the surface of cancer cells. A biochemical reaction between the antibody and the target protein (antigen) triggers a signal in the tumor cell, which then absorbs or internalizes the antibody along with the cytotoxic agent. Once internalized, the cytotoxic drug is released and kills the cancer. Due to this targeting, the drug ideally has fewer side effects and provides a wider therapeutic window than other chemotherapeutic agents. Techniques for conjugating antibodies are disclosed and known in the art (see, for example, Jane de Lartigue OncLive July 5, 2012; ADC Review on antibody-drug conjugates; and Ducry et al., Bioconjugate Chemistry 21(1):5-13(2010)).

[0311] II. Antibody activity

[0312] In some embodiments, an anti-SIRPβ1 antibody that binds to human SIRPβ1 isotype 1 but not human SIRPα is provided. In some embodiments, an anti-SIRPβ1 antibody that binds to human SIRPβ1 isotype 1 but not human SIRPγ is provided. In some embodiments, an anti-SIRPβ1 antibody that binds to human SIRPβ1 isotype 1 but not human SIRPβ1 isotype 3 is provided. In some embodiments, an anti-SIRPβ1 antibody that binds to human SIRPβ1 isotype 1 but not mouse SIRPβ1 is provided. In some embodiments, an anti-SIRPβ1 antibody that binds to human SIRPβ1 isotype 1 but not cynomolgus monkey SIRPβ1 is provided. In some embodiments, an anti-SIRPβ1 antibody that binds to human SIRPβ1 isotype 1 but not cynomolgus monkey SIRPβ1 isotype 1 is provided.

[0313] In some implementations, ForteBio can be used. The Red384 system (ForteBio, MenloPark, CA) measures the binding of anti-SIRPβ1 antibodies to antigens, as described in Example 1. Antibody binding can be measured using, for example, Fab fragments of the antibody (for monovalent binding) or full-length antibodies such as IgG (for bivalent binding, or affinity). ForteBio is used. An exemplary binding assay using the Red384 system for full-length antibodies is as follows: The antibody is loaded onto an AHQ sensor. The loaded antibody is then exposed to the antigen, and the dissociation rate is measured in assay buffer at multiple time intervals (e.g., 3 minutes). The kinetic data can then be fitted using a binding model in the data analysis software provided with the system. For antibody Fab fragment affinity measurements, in some embodiments, the antigen-Fc can be loaded onto the AHQ sensor and then exposed to the antibody Fab fragment. The dissociation rate is measured as described above, and the data is analyzed using the software provided with the system.

[0314] In some embodiments, an anti-SIRPβ1 antibody is provided that stimulates SIRPβ1 activity on CD14-positive monocytes in vitro and / or in vivo. In some embodiments, an anti-SIRPβ1 antibody is provided that stimulates SIRPβ1 activity on CD14-positive monocytes in vitro. In some embodiments, an anti-SIRPβ1 antibody is provided that stimulates SIRPβ1 activity on CD14-positive monocytes in vivo. In some embodiments, the anti-SIRPβ1 antibody stimulates SIRPβ1 activity on CD14-positive monocytes in vitro in an assay in which the anti-SIRPβ1 antibody is immobilized on a solid support, such as a culture plate, or bound to an anti-IgG secondary antibody, or bound to an Fcγ receptor on helper cells. In some embodiments, an anti-SIRPβ1 antibody stimulates SIRPβ1 activity on CD14-positive monocytes in vivo. Examples 3 and 4 describe a non-limiting illustrative assay for determining whether an anti-SIRPβ1 antibody stimulates SIRPβ1 activity on CD14-positive monocytes in vitro. For example, isolated monocytes can be starved for a period of time, such as 4 hours, and then incubated with an anti-SIRPβ1 antibody, for example, in the presence of an anti-IgG antibody. After incubation, the cells are lysed and phosphorylation of one or more of Syk, ERK, AKT, SIRPβ1, and / or DAP12 is measured, for example, using anti-phosphotyrosine and / or anti-phosphoserine antibodies. An increase in phosphorylation of one or more of Syk, ERK, AKT, SIRPβ1, and / or DAP12 indicates an increase in SIRPβ1 activity (i.e., an agonist of SIRPβ1 activity). As another example, monocytes can be incubated with an anti-SRPβ1 antibody in the presence of helper cells expressing the Fcγ receptor, such as B cells. After incubation, the cells are lysed and phosphorylation of one or more of Syk, ERK, AKT, SIRPβ1, and / or DAP12 is measured as described above. An increase in phosphorylation of one or more of Syk, ERK, AKT, SIRPβ1, and / or DAP12 indicates an increase in SIRPβ1 activity (i.e., an anti-SIRPβ1 antibody is an agonist of SIRPβ1 activity). A non-limiting illustrative assay for determining whether an anti-SIRPβ1 antibody stimulates SIRPβ1 activity in vivo on CD14-positive monocytes involves administering an anti-SIRPβ1 antibody to human SIRPβ1 BAC transgenic C57BL / 6 mice and isolating CD14-positive monocytes, for example, by FACS. The monocytes are then lysed and phosphorylation of one or more of Syk, ERK, AKT, SIRPβ1, and / or DAP12 is measured as described above. An increase in phosphorylation of one or more of Syk, ERK, AKT, SIRPβ1, and / or DAP12 indicates an increase in SIRPβ1 activity (i.e., an agonist of SIRPβ1 activity).

[0315] In some embodiments, anti-SIRPβ1 antibodies are provided that induce or enhance respiratory bursts in immune cells, such as neutrophils and / or monocytes, in vitro and / or in vivo. In some embodiments, anti-SIRPβ1 antibodies are provided that induce or enhance respiratory bursts in immune cells, such as neutrophils and / or monocytes, in vitro. In some embodiments, anti-SIRPβ1 antibodies are provided that induce or enhance respiratory bursts in immune cells, such as neutrophils and / or monocytes, in vivo. Examples 5 and 6 describe non-limiting illustrative in vitro assays for determining whether anti-SIRPβ1 antibodies induce or enhance respiratory bursts in immune cells, such as neutrophils and / or monocytes. For example, primary neutrophils are contacted with an anti-SIRPβ1 antibody, and reactive oxygen species (ROS) generation is detected using a universal oxidative stress indicator, such as the fluorescent dye CM-H2DCFDA. Alternatively, an assay can be performed using an anti-SIRPβ1 antibody immobilized on a solid support, such as a culture plate, or bound to an anti-IgG secondary antibody, or bound to an Fcγ receptor on helper cells.

[0316] In some embodiments, an anti-SIRPβ1 antibody is provided that induces or enhances IL-8 expression in monocytes in vitro and / or in vivo. In some embodiments, an anti-SIRPβ1 antibody is provided that induces or enhances IL-8 expression in monocytes in vitro. In some embodiments, an anti-SIRPβ1 antibody is provided that induces or enhances IL-8 expression in monocytes in vivo. Example 5 describes a non-limiting illustrative in vitro assay for determining whether an anti-SIRPβ1 antibody induces or enhances IL-8 expression in monocytes. For example, primary monocytes can be immobilized on a solid support, such as a culture plate, or stimulated with an anti-IgG secondary antibody, or with an anti-SIRPβ1 antibody that binds to Fcγ receptors on helper cells, for example, overnight. The supernatant can be collected to measure IL-8 release. A non-limiting illustrative assay for determining whether an anti-SIRPβ1 antibody induces or enhances IL-8 expression in monocytes in vivo comprises administering an anti-SIRPβ1 antibody to human SIRPβ1 BAC transgenic C57BL / 6 mice and obtaining one or more blood samples. Commercial assays, such as the Duoset ELISA kit (R&D Systems), can be used to measure serum IL-8 concentrations.

[0317] In some embodiments, an anti-SIRPβ1 antibody is provided that induces or enhances TNFα expression in macrophages and / or dendritic cells in vitro and / or in vivo. In some embodiments, an anti-SIRPβ1 antibody is provided that induces or enhances TNFα expression in macrophages and / or dendritic cells in vitro. In some embodiments, an anti-SIRPβ1 antibody is provided that induces or enhances TNFα expression in macrophages and / or dendritic cells in vivo. Example 6 describes a non-limiting illustrative in vitro assay for determining whether an anti-SIRPβ1 antibody induces or enhances TNFα expression. For example, monocyte-derived macrophages or dendritic cells can be stimulated with LPS, e.g., overnight, immobilized on a solid support, such as a culture plate, or in the presence of an anti-SIRPβ1 antibody bound to an anti-IgG secondary antibody or to an Fcγ receptor on helper cells. The supernatant can be collected to measure TNFα release. A non-restrictive illustrative assay for determining whether anti-SIRPβ1 antibodies induce or enhance TNFα expression in macrophages and / or dendritic cells in vivo involves administering the anti-SIRPβ1 antibody, along with LPS (such as 5 μg LPS / mouse), to human SIRPβ1 BAC transgenic C57BL / 6 mice. The mice are then sacrificed, for example by CO2 asphyxiation, and the peritoneal fluid is recovered and clarified by centrifugation. TNF-α concentrations can be measured using commercially available assays, such as the Duoset ELISA kit (R&D Systems).

[0318] In some embodiments, anti-SIRPβ1 antibodies are provided to induce or enhance neutrophil-mediated phagocytosis, such as phagocytosis of tumor cells, in vitro and / or in vivo. In some embodiments, anti-SIRPβ1 antibodies are provided to induce or enhance neutrophil-mediated phagocytosis, such as phagocytosis of tumor cells, in vitro. In some embodiments, anti-SIRPβ1 antibodies are provided to induce or enhance neutrophil-mediated tumor cell clearance in vivo. Example 7 describes a non-limiting illustrative in vitro assay for determining whether an anti-SIRPβ1 antibody induces or enhances neutrophil-mediated phagocytosis. For example, primary neutrophils are contacted with an anti-SIRPβ1 antibody immobilized on a solid support, such as a culture plate, or subjected to anti-IgG secondary antibodies, or subjected to Fcγ receptors on helper cells. Cancer cells, such as Raji B-cell lymphoma cells, were co-cultured with neutrophils and immobilized anti-SIRPβ1 antibodies in the presence of modulating antibodies, such as anti-CD20 antibodies. The viability of Raji cells was quantified by measuring luciferase activity. A reduction in viable Raji cells in the presence of anti-SIRPβ1 antibodies compared to an IgG control antibody indicated that anti-SIRPβ1 antibodies induced or enhanced neutrophil-mediated phagocytosis. A non-restrictive illustrative assay to determine whether anti-SIRPβ1 antibodies induce or enhance neutrophil-mediated tumor cell clearance in vivo involved intravenously injecting GFP-expressing B16F10 melanoma cells into human SIRPβ1-modified BAC transgenic mice. The mice were then treated with anti-SIRPβ1 antibodies along with modulating anti-gp75 antibodies. Peripheral blood was collected, erythrocytes were lysed, and leukocytes were resuspended in FACS buffer. Neutrophils were stained with anti-CD11b and anti-Ly6G antibodies, and the green fluorescence signal acquired from the ingested tumor cells was analyzed by flow cytometry. An increase in green fluorescence signal in neutrophils compared to the same experiment using a type-matched control antibody indicates that anti-SIRPβ1 antibody induces or enhances neutrophil-mediated phagocytosis.

[0319] Another non-restrictive illustrative assay for determining whether anti-SIRPβ1 antibodies induce or enhance neutrophil-mediated tumor cell clearance in vivo uses a lung metastasis model, for example, as follows: B16F10 melanoma cells were intravenously injected into Fc receptor γ chain-deficient C57BL / 6 mice lacking activating FcγR expression. Then, bone marrow neutrophils isolated from human SIRPβ1 BAC transgenic mice were intravenously injected together with a modulating anti-gp75 antibody and an anti-SIRPβ1 antibody. After a period of time, the mice were euthanized, and the lungs were harvested and fixed. The number of metastatic tumor nodules with dark pigmentation was visually counted. A decrease in the number of metastatic tumor nodules compared to the same experiment using an isotype-matched control antibody indicated that the anti-SIRPβ1 antibody induced or enhanced neutrophil-mediated tumor cell clearance in vivo.

[0320] In some embodiments, an anti-SIRPβ1 antibody is provided that enhances TREM2 expression on macrophages in vitro and / or in vivo. In some embodiments, an anti-SIRPβ1 antibody is provided that enhances TREM2 expression on macrophages in vitro. In some embodiments, an anti-SIRPβ1 antibody is provided that enhances TREM2 expression on macrophages in vivo. Example 16 describes a non-limiting illustrative in vitro assay for determining whether an anti-SIRPβ1 antibody enhances TREM2 expression on macrophages. For example, monocyte-derived macrophages differentiated in culture with M-CSF are incubated together with an anti-SIRPβ1 antibody immobilized on a solid support, such as a culture plate, or subjected to anti-IgG secondary antibody binding, or subjected to Fcγ receptor binding on helper cells. TREM2 expression on macrophages is analyzed, for example, by FACS analysis. A non-limiting illustrative assay for determining whether an anti-SIRPβ1 antibody enhances TREM2 expression on macrophages in vivo is as follows. Human SIRPβ1 BAC transgenic C57BL / 6 mice were challenged with aged, sterile thioglycolate medium injected into the peritoneum. Following challenge, the mice were treated with anti-SIRPβ1 antibody. The mice were then sacrificed, and peritoneal macrophages were harvested post-euthanasia, for example, by flushing the peritoneal cavity with PBS. Macrophages were defined as CD11b+Ly6C-F4 / 80+ cells, and TREM2 expression levels were determined by FACS analysis.

[0321] In some embodiments, an anti-SIRPβ1 antibody is provided, alone and / or in combination with an agonist anti-TREM2 antibody, to enhance macrophage viability in vitro and / or in vivo. In some embodiments, an anti-SIRPβ1 antibody is provided, alone and / or in combination with an agonist anti-TREM2 antibody, to enhance macrophage viability in vitro. In some embodiments, an anti-SIRPβ1 antibody is provided, alone and / or in combination with an agonist anti-TREM2 antibody, to enhance macrophage viability in vivo. In some embodiments, an anti-SIRPβ1 antibody is provided to enhance dendritic cell viability in vitro and / or in vivo. In some embodiments, an anti-SIRPβ1 antibody is provided to enhance dendritic cell viability in vitro. In some embodiments, an anti-SIRPβ1 antibody is provided to enhance dendritic cell viability in vivo. Examples 17, 18, and 19 describe non-limiting illustrative in vitro assays for determining whether an anti-SIRPβ1 antibody, alone or in combination with an agonist anti-TREM2 antibody, enhances macrophage viability in vitro. For example, monocyte-derived macrophages differentiated in M-CSF culture are incubated with anti-SIRPβ1 antibodies immobilized on a solid support, such as a culture plate, or subjected to anti-IgG secondary antibodies, or to Fcγ receptors on helper cells, with or without anti-TREM2 antibodies. Cell viability is measured, for example, using the Cell Titer Glo kit (Promega), a reagent that generates a luminescent signal relative to the ATP concentration in the sample. A similar assay can be used to determine whether anti-SIRPβ1 antibodies enhance the viability of bone marrow-derived macrophages or dendritic cells in vitro. For this assay, bone marrow cells are cultured with M-CSF (for macrophage differentiation) or GM-CSF (for dendritic cell differentiation). Macrophages or dendritic cells are incubated with anti-SIRPβ1 antibodies immobilized on a solid support, such as a culture plate, or subjected to anti-IgG secondary antibodies, or to Fcγ receptors on helper cells, with or without anti-TREM2 antibodies. As described above, cell viability is measured, for example using the Cell Titer Glo kit (Promega), a reagent that generates a luminescent signal relative to the ATP concentration in a sample. A non-limiting illustrative assay for determining whether anti-SIRPβ1 antibody enhances macrophage viability in vivo is as follows. Peritoneal and tissue-resident F4 / 80+ macrophages are reduced due to the elimination of M-CSF-mediated survival signals by prolonged treatment with a blocking antibody against CSF1R. For this assay, anti-CSF1R antibody was administered to human SIRPβ1BAC transgenic C57BL / 6 mice along with the anti-SIRPβ1 antibody. Following treatment, the mice were euthanized, for example by CO2 asphyxiation, and peripheral blood was collected via cardiac puncture. Peritoneal macrophages were collected by irrigating with PBS.Macrophage population counting was performed via FACS by gating CD11b+Ly6C-F4 / 80+ cells in blood and peritoneum.

[0322] III. Nucleic acids, vectors, and host cells

[0323] The disclosed anti-SIRPβ1 antibodies can be generated using recombinant methods and compositions, such as those described in U.S. Patent No. 4,816,567. In some embodiments, isolated nucleic acids having a nucleotide sequence encoding any of the anti-SIRPβ1 antibodies of this disclosure are provided. Such nucleic acids may encode V containing an anti-SIRPβ1 antibody. L The amino acid sequence and / or containing V H The amino acid sequence (e.g., the light and / or heavy chain of an antibody). In some embodiments, one or more vectors (e.g., expression vectors) containing such nucleic acids are provided. In some embodiments, a host cell containing such nucleic acids is also provided. In some embodiments, the host cell contains (e.g., via transduction): (1) a protein encoding a V protein containing an antibody. L The amino acid sequence and V containing the antibody H A vector of nucleic acid containing an amino acid sequence, or (2) containing a V encoding an antibody. L The first vector of nucleic acid containing the amino acid sequence and containing V encoding antibodies. H The second carrier of nucleic acid containing the amino acid sequence. In some embodiments, the host cell is eukaryotic, such as Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NSO, Sp20 cells). The host cells disclosed herein also include, but are not limited to, isolated cells, cells cultured in vitro, and cells cultured in vitro.

[0324] A method for producing the disclosed anti-SIRPβ1 antibody is provided. In some embodiments, the method includes culturing a host cell containing nucleic acid encoding an anti-SIRPβ1 antibody under conditions suitable for antibody expression. In some embodiments, the antibody is subsequently recovered from the host cell (or host cell culture medium).

[0325] To recombinantly generate the publicly disclosed anti-SIRPβ1 antibody, the nucleic acid encoding the anti-SIRPβ1 antibody is isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using standard procedures, such as by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody.

[0326] Suitable vectors containing nucleic acid sequences encoding any anti-SIRPβ1 antibody disclosed herein, or polypeptides (including antibodies) described herein, or cell surface expression fragments thereof, are included, but are not limited to, cloning vectors and expression vectors. Suitable cloning vectors can be constructed according to standard techniques or can be selected from a wide variety of cloning vectors available in the art. While the chosen cloning vector can vary depending on the intended host cell, useful cloning vectors generally possess self-replicating capabilities, may have a single target for a specific restriction endonuclease, and / or carry a gene that can be used to select a clone containing that vector. Suitable examples include plasmids and bacterial viruses such as pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors such as pSA3 and pAT28. These and many other cloning vectors are available from suppliers such as BioRad, Stragene, and Invitrogen.

[0327] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells. For example, the disclosed anti-SIRPβ1 antibody can be generated in bacteria, particularly when glycosylation and Fc effector function are not required. Antibody fragments and peptides can be expressed in bacteria (e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523). After expression, the antibody can be separated from the bacterial cell clump in a soluble fraction and can be further purified.

[0328] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been “humanized”, resulting in the production of antibodies with partially or fully human glycosylation patterns (e.g., Gerngross, Nat. Biotech. 22:1409-1414 (2004); and Li et al., Nat. Biotech. 24:210-215 (2006)).

[0329] Suitable host cells for expressing glycosylated antibodies can also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified that can be used with insect cells, particularly for transfecting Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts (e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429, which describe PLATNIBODIES for generating antibodies in transgenic plants). TM technology).

[0330] Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension can be useful. Other examples of useful mammalian host cell lines are: monkey kidney CV1 line (COS-7) transformed with SV40; human embryonic kidney line (293 or 293 cells, as described in, for example, Graham et al., J. Gen Virol. 36:59 (1977)); juvenile hamster kidney cells (BHK); mouse setoli cells (TM4 cells, as described in, for example, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumors (MMT). 060562); TRI cells, such as those described in, for example, Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR - CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NSO, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (edited by BKCLo, Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0331] IV. Pharmaceutical Compositions / Formulations

[0332] This document provides pharmaceutical compositions and / or pharmaceutical formulations comprising the anti-SIRPβ1 antibody and a pharmaceutically acceptable carrier of the present disclosure.

[0333] In some embodiments, pharmaceutically acceptable carriers are preferably non-toxic to the recipient at the dose and concentration used. The antibodies described herein can be formulated into solid, semi-solid, liquid, or gaseous forms. Examples of such formulations include, but are not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalers, gels, microspheres, and aerosols. Depending on the desired formulation, pharmaceutically acceptable carriers may include pharmaceutically acceptable non-toxic carrier diluents, which are commonly used as a medium for formulating pharmaceutical compositions for administration to animals or humans. In some embodiments, the pharmaceutical composition may contain formulation materials for altering, maintaining, or preserving, for example, the composition's pH, permeability, viscosity, clarity, color, isotensivity, odor, sterility, stability, rate of dissolution or release, absorption, or permeation.

[0334] In some embodiments, pharmaceutically acceptable carriers include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antibacterial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids); extenders (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyethylene glycol, β-cyclodextrin, or hydroxypropyl β-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colorants, flavorings, and diluents; emulsifiers; and hydrophilic polymers (such as poly(acetic acid)). Vinylpyrrolidone; low molecular weight peptides; salt-forming anti-charge ions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (such as glycerol, propylene glycol, or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as Pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, Triton, tromethamine, lecithin, cholesterol, terbufotasol); stability enhancers (such as sucrose or sorbitol); tension enhancers (such as alkali metal halides, preferably sodium or potassium chloride, mannitol, sorbitol); delivery media; diluents; excipients and / or pharmaceutical adjuvants. Other examples of formulations suitable for various types of administration can be found in Remington: The Science and Practice of Pharmacy, Pharmaceutical Press 22nd ed. (2013). A brief review of methods for drug delivery can be found in Langer, Science 249:1527-1533 (1990).

[0335] Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injectable solutions, which may contain antioxidants, buffers, antibacterial agents, and solutes, making the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions, which may include suspending agents, solubilizers, thickeners, stabilizers, and preservatives.

[0336] Formulations can be optimized for retention and stability in the brain or central nervous system. When a drug is administered into a cranial compartment, it is desirable that the drug remain within the compartment rather than diffuse or otherwise cross the blood-brain barrier. Stabilization techniques include cross-linking, polymerization, or attachment to groups such as polyethylene glycol, polyacrylamide, neutral protein carriers, etc., to increase the molecular weight.

[0337] Other strategies for increasing retention include encapsulating antibodies (such as the anti-SIRPβ1 antibody disclosed herein) in biodegradable or bioerectable implants. The release rate of the therapeutic agent is controlled by the transport rate across the polymer matrix and the biodegradation of the implant. Implants can be particles, sheets, patches, plaques, fibers, microcapsules, etc., and can be of any size or shape compatible with the chosen insertion site. The biodegradable polymer compositions that can be used can be organic esters or ethers that produce physiologically acceptable degradation products, including monomers, upon degradation. Anhydrides, amides, orthoesters, etc., can be used, either independently or in combination with other monomers. The polymer will be a condensation polymer. The polymer can be crosslinked or non-crosslinked. Polymers of hydroxyaliphatic carboxylic acids (homopolymers or copolymers) and polysaccharides are of particular interest. Polyesters of interest include D-lactic acid, L-lactic acid, racemic lactic acid, glycolic acid, polycaprolactone, and polymers of combinations thereof. Among the polysaccharides of interest are calcium alginate and functionalized cellulose, particularly carboxymethyl cellulose ester, characterized by being insoluble in water and having a molecular weight of about 5 kD to 500 kD, etc. Biodegradable hydrogels can also be used in the implants of the present invention. Hydrogels are typically copolymer materials characterized by their ability to absorb liquids.

[0338] V. Therapeutic Uses

[0339] As disclosed herein, the anti-SIRPβ1 antibody of this disclosure can be used to prevent, reduce risk, or treat diseases and conditions. In various embodiments, the anti-SIRPβ1 antibody provided herein has one or more activities selected from: activating SIRPβ1 activity on CD14-positive monocytes in vitro and / or in vivo; inducing respiratory burst in immune cells, such as neutrophils and / or monocytes, in vitro and / or in vivo; inducing IL-8 expression in monocytes in vitro and / or in vivo; inducing TNFα expression in macrophages and / or dendritic cells in vitro and / or in vivo; inducing neutrophil-mediated phagocytosis, such as phagocytosis by tumor cells, in vitro and / or in vivo; enhancing neutrophil-mediated tumor cell clearance in vivo; upregulating TREM2 expression on macrophages in vitro and / or in vivo; enhancing macrophage viability in vitro and / or in vivo, alone and / or in combination with an agonist anti-TREM2 antibody; and enhancing dendritic cell viability in vitro and / or in vivo.

[0340] In various implementations, the anti-SIRPβ1 antibody provided herein can be used to prevent, reduce risk, or treat dementia, frontotemporal dementia, Alzheimer's disease, vascular dementia, mixed dementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus, amyotrophic lateral sclerosis, Huntington's disease, Tau disease, Nasu-Hakola disease, stroke, acute trauma, chronic trauma, lupus, acute and chronic colitis, wound healing, Crohn's disease, inflammatory bowel disease, ulcerative colitis, obesity, malaria, essential tremor, central nervous system lupus, and beta-lactamase. Chetter's disease, Parkinson's disease, Lewy body dementia, multiple system atrophy, Chetter's syndrome, progressive supranuclear palsy, corticobasal ganglia degeneration, acute disseminated encephalomyelitis, granulomatous diseases, sarcoidosis, age-related diseases, seizures, spinal cord injury, traumatic brain injury, age-related macular degeneration, glaucoma, retinitis pigmentosa, retinal degeneration, respiratory infections, sepsis, eye infections, systemic infections, lupus, arthritis, multiple sclerosis, low bone mineral density, osteoporosis, osteoporosis, osteosclerotic diseases, Paget's disease of bone, and / or cancer. In some such implementations, the anti-SIRPβ1 antibody is an agonist antibody.

[0341] In some implementation methods, this document provides for the prevention, risk reduction, or treatment of dementia, frontotemporal dementia, Alzheimer's disease, vascular dementia, mixed dementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus, amyotrophic lateral sclerosis, Huntington's disease, Tau disease, Nasu-Hakola disease, stroke, acute trauma, chronic trauma, lupus, acute and chronic colitis, wound healing, Crohn's disease, inflammatory bowel disease, ulcerative colitis, obesity, malaria, essential tremor, central nervous system lupus, Bechtel's disease, Parkinson's disease, Lewy body dementia, and multiple system atrophy. Methods for treating individuals with Hedyotis diffraction syndrome, progressive supranuclear palsy, corticobasal ganglia degeneration, acute disseminated encephalomyelitis, granulomatous diseases, sarcoidosis, age-related diseases, seizures, spinal cord injury, traumatic brain injury, age-related macular degeneration, glaucoma, retinitis pigmentosa, retinal degeneration, respiratory infections, sepsis, ocular infections, systemic infections, lupus, arthritis, multiple sclerosis, low bone mineral density, osteoporosis, osteoproliferation, osteosclerotic diseases, Paget's disease of bone, and / or cancer, by administering a therapeutically effective amount of the disclosed anti-SIRPβ1 antibody to the individual. In some such embodiments, the anti-SIRPβ1 antibody is, for example, an agonist that induces or enhances SIRPβ1 activity.

[0342] As disclosed herein, the anti-SIRPβ1 antibody of this disclosure can also be used to induce and / or promote the survival of innate immune cells. In some embodiments, this disclosure provides a method for inducing or promoting the survival of innate immune cells in an individual in need by administering a therapeutically effective amount of the agonistic anti-SIRPβ1 antibody of this disclosure to the individual.

[0343] As disclosed herein, the anti-SIRPβ1 antibody of this disclosure can also be used to induce and / or promote wound healing, such as after injury. In some embodiments, wound healing may be the repair of a colonic wound following injury. In some embodiments, this disclosure provides a method for inducing or promoting wound healing in an individual in need by administering to that individual a therapeutically effective amount of the agonistic anti-SIRPβ1 antibody of this disclosure.

[0344] In some implementations, the subject or individual is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). In some implementations, the subject or individual is a human.

[0345] The antibodies (and any other therapeutic agents) provided herein may be administered by any suitable means, including parenteral, intrapulmonary, intranasal, intralesional, intracerebrospinal, intracranial, intraspinal, intrasynovial, intrathecal, oral, topical, or inhalation routes. Parenteral infusion includes intramuscular, intravenous (as a bolus or via continuous infusion over a period of time), intra-articular, intra-articular, intraperitoneal, or subcutaneous administration. In some embodiments, administration is intravenous. In some embodiments, administration is subcutaneous. Dosage administration may be via any suitable route, such as by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. Various dosing schedules are covered herein, including but not limited to multiple administrations at single or multiple time points, bolus administration, and pulsatile infusion.

[0346] The antibodies described herein are formulated, dosed, and administered in accordance with good medical practice. Factors considered in this context include the specific disease being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the disease, the site of administration, the method of administration, the pace of administration, and other factors known to the medical practitioner. Antibodies are not required but may be formulated with one or more currently used agents for the prevention or treatment of the disease in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disease or treatment, and other factors discussed above. These are generally used at the same doses and routes of administration as described herein, or approximately 1 to 99% of the doses described herein, or any dose and route determined empirically / clinically to be appropriate.

[0347] For the prevention or treatment of disease, the appropriate dosage of the antibody of the present invention (alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, prior therapy, the patient's clinical history and response to the antibody, and the attending physician's discretion. The antibody may be administered to the patient in a single dose or as part of a series of treatments.

[0348] Depending on the type and severity of the disease, an antibody dose of approximately 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg–10 mg / kg) may be the initial candidate dose for administration to a patient, whether by single or multiple separate administrations or by continuous infusion. A typical daily dose range may be approximately 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, treatment generally continues until the desired disease symptoms are contained, depending on the condition. An exemplary dose of antibody may be in the range of approximately 0.05 mg / kg to approximately 10 mg / kg. Thus, a patient may be given one or more doses of approximately 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof). Such doses may be administered intermittently, for example weekly or every three weeks (e.g., so that the patient receives approximately 2 to approximately 20, or for example, approximately 6 doses of antibody). In some implementations, the dosing frequency is three times daily, twice daily, once daily, every other day, once a week, once every two weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, or once a month, once every two months, once every three months, or longer. An initial higher loading dose may be administered, followed by one or more lower doses. However, other dosing regimens may be useful. Progression of this therapy can be easily monitored using routine techniques and assays.

[0349] dementia

[0350] Dementia is a nonspecific syndrome (i.e., a range of signs and symptoms) characterized by a severe loss of overall cognitive abilities in a previously unimpaired person, exceeding what might be expected based on normal aging. Dementia can be static, as a result of isolated overall brain damage. Alternatively, dementia can be progressive, leading to a long-term decline due to damage or disease in the body. While dementia is much more common in older populations, it can also occur before age 65. Cognitive areas affected by dementia include, but are not limited to, memory, attention span, language, and problem-solving. Generally, symptoms must be present for at least six months before an individual is diagnosed with dementia.

[0351] Exemplary forms of dementia include, but are not limited to, frontotemporal dementia, Alzheimer's disease, vascular dementia, semantic dementia, and Lewy body dementia.

[0352] In some embodiments, administration of the anti-SIRPβ1 antibody of this disclosure can prevent, reduce the risk, and / or treat dementia. In some embodiments, administration of the anti-SIRPβ1 antibody can induce one or more SIRPβ1 activities in an individual with dementia.

[0353] Frontotemporal dementia

[0354] Frontotemporal dementia (FTD) is a condition caused by the gradual deterioration of the frontal lobe of the brain. Over time, the degeneration may progress to the temporal lobe. It is second only to Alzheimer's disease (AD) in prevalence, accounting for 20% of early-onset dementia cases. Clinical features of FTD include memory deficits, behavioral abnormalities, personality changes, and language decline (Cruts, M. & Van Broeckhoven, C., Trends Genet. 24:186-194 (2008); Neary, D. et al., Neurology 51:1546-1554 (1998); Ratnavalli, E., Brayne, C., Dawson, K. & Hodges, JR, Neurology 58:1615-1621 (2002)).

[0355] A significant proportion of FTD cases are inherited in an autosomal dominant manner, but even within a single family, symptoms can range from behavioral disturbances to primary progressive aphasia to corticobasal ganglia degeneration. Like most neurodegenerative diseases, FTD can be characterized by the pathological presence of specific protein aggregates in the affected brain. Historically, the first description of FTD identified the presence of neuronal accumulation of hyperphosphorylated Tau protein in neurofibrillary tangles or the pickl body. The identification of mutations in the gene encoding Tau protein in several families supports the causative role of the microtubule-associated protein Tau (Hutton, M. et al., Nature 393:702-705 (1998)). However, most FTD brains show no accumulation of hyperphosphorylated Tau, but do exhibit immunoreactivity to ubiquitin (Ub) and TAR DNA-binding protein (TDP43) (Neumann, M. et al., Arch. Neurol. 64:1388-1394 (2007)). Most FTD cases with Ub inclusions (FTD-U) show mutations in the pregranulosome protein gene.

[0356] In some embodiments, administration of the anti-SIRPβ1 antibody of this disclosure can prevent, reduce risk, and / or treat FTD. In some embodiments, administration of the anti-SIRPβ1 antibody can induce one or more SIRPβ1 activities in individuals with FTD.

[0357] Alzheimer's disease

[0358] Alzheimer's disease (AD) is the most common form of dementia. The disease is incurable, progresses, and eventually leads to death. Most commonly, AD is diagnosed in people over 65 years of age. However, the less common early-onset Alzheimer's disease can occur much earlier.

[0359] Common symptoms of Alzheimer's disease include behavioral symptoms such as difficulty remembering recent events; cognitive symptoms such as confusion, irritability and aggression, mood instability, language difficulties, and long-term memory loss. As the disease progresses, bodily functions decline, eventually leading to death. Alzheimer's disease develops over an unknown and variable period before becoming fully apparent, and it can progress for many years without being diagnosed.

[0360] In some embodiments, administration of the anti-SIRPβ1 antibody of this disclosure can prevent, reduce the risk, and / or treat Alzheimer's disease. In some embodiments, administration of the anti-SIRPβ1 antibody can induce one or more SIRPβ1 activities in an individual with Alzheimer's disease.

[0361] Nasu-Hakola disease

[0362] Nasu-Hakola disease (NHD), alternatively known as polycystic fat membrane osteodysplasia and sclerotic leukoencephalopathy (PLOSL), is a rare inherited leukodystrophy characterized by progressive early-onset dementia associated with recurrent fractures resulting from multicystic bone damage in the lower or upper limbs. The NHD disease process generally progresses through four stages: latency, skeletal, early neurological, and late neurological. After normal development during childhood (latency stage), NHD begins to manifest during adolescence or early adulthood (typical onset age 20-30 years), with pain in the hands, wrists, ankles, and feet. Patients then begin to experience recurrent fractures due to multicystic bone damage and osteoporotic lesions in the limbs (skeletal stage). During the third or fourth decade of life (early neurological stage), patients exhibit marked frontal lobe syndrome-like personality changes (e.g., euphoria, inattention, impaired judgment, and social inhibition). Patients also typically experience progressive memory impairment. Frequent seizures are also observed. Finally (in the late neurological stage), patients progress to severe dementia, becoming unable to speak or move, and typically die by age 50.

[0363] In some embodiments, administration of the anti-SIRPβ1 antibody of this disclosure can prevent, reduce risk, and / or treat Nasu-Hakola disease (NHD). In some embodiments, administration of the anti-SIRPβ1 antibody can induce one or more SIRPβ1 activities in individuals with NHD.

[0364] Parkinson's disease

[0365] Parkinson's disease, also known as idiopathic or primary Parkinsonian syndrome, hypokinetic rigidity syndrome (HRS), or Parkinson's palsy, is a neurodegenerative brain disorder affecting motor control. The progressive death of dopamine-producing cells in the brain leads to the main symptoms of Parkinson's disease. Most commonly, Parkinson's disease is diagnosed in people over 50 years of age. In most cases, Parkinson's disease is idiopathic (with an unknown cause). However, genetic factors also play a role in the disease.

[0366] Symptoms of Parkinson's disease include, but are not limited to, tremors in the hands, arms, legs, jaw, and face; muscle rigidity in the limbs and trunk; slow movement (bradykinesia); postural instability; difficulty walking; neuropsychiatric problems; changes in speech or behavior; depression; anxiety; pain; psychosis; dementia; hallucinations; and sleep problems.

[0367] In some embodiments, administration of the anti-SIRPβ1 antibody of this disclosure can prevent, reduce the risk, and / or treat Parkinson's disease. In some embodiments, administration of the anti-SIRPβ1 antibody can induce one or more SIRPβ1 activities in an individual with Parkinson's disease.

[0368] Amyotrophic Lateral Sclerosis (ALS)

[0369] As used in this article, amyotrophic lateral sclerosis (ALS), motor neuron disease, or Lou Gehrig's disease are used interchangeably to refer to a weakening disease with different etiologies, characterized by rapidly progressive weakness, muscle atrophy and fasciculations, muscle spasms, difficulty speaking (dysarthria), difficulty swallowing (dysphagia), and difficulty breathing (dyspnea).

[0370] Pre-granulosome proteins have been shown to play a role in ALS (Schymick, JC et al., (2007) J Neurol Neurosurg Psychiatry, 78:754-6) and to provide protection against damage caused by proteins that induce ALS, such as TDP-43 (Laird, AS et al., (2010). PLoS ONE 5:e13368). Pro-NGF has also been shown to induce p75-mediated oligodendrocyte and cortical spinal cord neuronal death following spinal cord injury (Beatty et al., Neuron (2002), 36, pp.375-386; Giehl et al., Proc. Natl. Acad. Sci USA (2004), 101, pp.6226-30).

[0371] In some embodiments, administration of the anti-SIRPβ1 antibody of this disclosure can prevent, reduce risk, and / or treat ALS. In some embodiments, administration of the anti-SIRPβ1 antibody can induce one or more SIRPβ1 activities in an individual with ALS.

[0372] Huntington's disease

[0373] Huntington's disease (HD) is a hereditary neurodegenerative disorder caused by an autosomal dominant mutation in the huntingtin protein gene (HTT). An expansion of the cytosine-adenine-guanine (CAG) triplet repeat in the huntingtin protein gene leads to the generation of a mutant form of the gene-encoded huntingtin protein (Htt). This mutant huntingtin protein (mHtt) is toxic and promotes neuronal death. Symptoms of Huntington's disease most commonly appear between the ages of 35 and 44, although they can appear at any age.

[0374] Symptoms of Huntington's disease include, but are not limited to, motor control problems, cramps, random movements (chorea), abnormal eye movements, impaired balance, seizures, difficulty chewing, difficulty swallowing, cognitive problems, language changes, memory loss, difficulty thinking, insomnia, fatigue, dementia, personality changes, depression, anxiety, and compulsive behaviors.

[0375] In some embodiments, administration of the anti-SIRPβ1 antibody of this disclosure can prevent, reduce the risk, and / or treat Huntington's disease (HD). In some embodiments, administration of the anti-SIRPβ1 antibody can induce one or more SIRPβ1 activities in an individual with HD.

[0376] Tau disease

[0377] Tau disease, or Tau syndrome, is a group of neurodegenerative diseases caused by the accumulation of the microtubule-associated protein Tau in the brain. Alzheimer's disease (AD) is the most well-known Tau disease and involves the accumulation of Tau protein in the form of insoluble neurofibrillary tangles (NFTs) within neurons. Other Tau diseases and symptoms include progressive supranuclear palsy, boxer's dementia (chronic traumatic encephalopathy), frontotemporal dementia and chromosome 17-linked Parkinson's syndrome, Lytico-Bodig disease (Guam-type Parkinson's-dementia complex), tangle-predominant dementia, ganglioglioma and gangliocytoma, meningioma, subacute sclerosing panencephalitis, lead encephalitis, tuberous sclerosis, H. Schereschi disease, lipofuscinosis, Pick's disease, cortical-basal degeneration, argyrophilic granuloma (AGD), Huntington's disease, frontotemporal dementia, and frontotemporal degeneration.

[0378] In some embodiments, administration of the anti-SIRPβ1 antibody of this disclosure can prevent, reduce risk, and / or treat Tau disease. In some embodiments, administration of the anti-SIRPβ1 antibody can induce one or more SIRPβ1 activities in an individual with Tau disease.

[0379] Multiple sclerosis

[0380] Multiple sclerosis (MS), also known as disseminated cerebral sclerosis or disseminated encephalomyelitis, is an inflammatory disease in which the fatty myelin sheath surrounding axons in the brain and spinal cord is destroyed, leading to demyelination, scarring, and a range of signs and symptoms. MS affects the ability of nerve cells in the brain and spinal cord to communicate effectively with each other. Nerve cells communicate by sending electrical signals called action potentials along long fibers called axons, which are contained within an insulating material called myelin. In MS, the body's own immune system attacks and destroys myelin. When myelin is lost, axons can no longer transmit signals effectively. MS attacks typically occur in young adults and are more common in women.

[0381] Symptoms of MS include, but are not limited to, sensory changes such as loss of sensitivity or tingling; numbness or tingling, such as hypoesthesia and paresthesia; muscle weakness; clonic spasms; muscle cramps; difficulty moving; difficulty coordinating and balancing, such as ataxia; speech problems such as dysarthria, or swallowing problems such as dysphagia; visual problems such as nystagmus, optic neuritis including photopsia, and diplopia; fatigue; acute or chronic pain; and bladder and bowel problems; varying degrees of cognitive impairment; depressive or mood-stiff symptoms; Utov's phenomenon, which is the exacerbation of existing symptoms upon exposure to temperatures higher than normal; and Lermit's sign, which is an electric shock that travels down the back when the neck is bent.

[0382] In some embodiments, administration of the anti-SIRPβ1 antibody of this disclosure can prevent, reduce the risk, and / or treat multiple sclerosis. In some embodiments, administration of the anti-SIRPβ1 antibody can induce one or more SIRPβ1 activities in an individual with multiple sclerosis.

[0383] cancer

[0384] Other aspects of this disclosure provide methods for preventing, reducing risk, or treating an individual with cancer, comprising administering to the individual a therapeutically effective amount of the isolated anti-SIRPβ1 antibody of this disclosure. Any isolated antibody of this disclosure may be used in these methods. In some embodiments, the isolated antibody is an agonist antibody of this disclosure.

[0385] The tumor microenvironment is known to contain heterogeneous immune infiltrators, including T lymphocytes, macrophages, and cells of the myeloid / granulocytic lineage. In particular, the presence of M2 macrophages in tumors is associated with poor prognosis. Therapies that reduce the number of these cells in tumors (such as CSF1R blockers) have shown beneficial effects in preclinical models and early-stage clinical studies. A pioneering preclinical study has also shown synergy between drugs targeting tumor-associated macrophages (e.g., CSF1 / CSF1R blocking antibodies) and checkpoint blocking antibodies targeting T cells, indicating that manipulating these two cell types may be effective in tumor models where individual therapies are less effective (Zhu Y., Cancer Res. 2014 Sep 15; 74(18):5057-69). Without being bound by any particular theory, it is believed that inducing SIRPβ1 signaling in tumor-associated macrophages and / or neutrophils can suppress the suppression of immune responses in the tumor microenvironment, leading to therapeutic antitumor immune responses.

[0386] In some embodiments, the cancers to be prevented or treated by the methods of this disclosure include, but are not limited to, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer, small cell lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), adenocarcinoma of the lung, squamous carcinoma of the lung, non-squamous NSCLC, glioma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, gastric cancer or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal carcinoma), kidney cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone-resistant prostate adenocarcinoma), thyroid cancer, neuroblastoma, pancreatic cancer, Glioblastoma (glioblastoma multiforme), cervical cancer, gastric cancer, bladder cancer, liver tumor, breast cancer, colon cancer, and head and neck cancer (or carcinoma), gastric cancer, germ cell tumors, pediatric sarcomas, sinus natural killer, melanoma (e.g., metastatic malignant melanoma, such as cutaneous or intraocular malignant melanoma), bone cancer, skin cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small bowel cancer, endocrine system cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, solid tumors in children, ureteral cancer, renal pelvis cancer, central nervous system (CNS) vegetations, primary Primary CNS lymphomas, myelodysplastic syndromes, colorectal vegetations, solid tumors, tumor angiogenesis, spinal tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphomas, environmentally induced cancers (including those induced by asbestos), virus-associated cancers (e.g., human papillomavirus (HPV)-associated tumors), and hematologic malignancies derived from either of the two main blood cell lineages—myeloid (which produces granulocytes, erythrocytes, platelets, macrophages, and mast cells) or lymphoid (which produces B, T, NK, and plasma cells)—such as all types of leukemia, lymphoma, and myeloma, e.g., acute myeloma. Chronic, lymphocytic and / or myeloid leukemias, such as acute leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), undifferentiated AML (M0), myeloblastic leukemia (M1), myeloblastic leukemia (M2; with mature cells), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), solitary granulocytic sarcoma, and chloroma;Lymphomas, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), painless lymphoma, large B-cell diffuse lymphoma, B-cell hematologic malignancies such as B-cell lymphoma, T-cell lymphoma, lymphoplasmacytic lymphoma, monocytic B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki 1+) large cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, angioblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorders, true histiocytic lymphoma, primary central nervous system lymphoma. Systemic lymphoma, primary effusion lymphoma, lymphoblastic lymphoma (LBL), lymphoid spectrum hematopoietic tumors, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B-cell lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also known as mycosis fungoides or Cezari syndrome), and Waldenström's macroglobulinemia. Lymphoplasmacytic lymphoma (LPL); myeloma, such as IgG myeloma, light chain myeloma, non-secreting myeloma, suffocating myeloma (also known as painless myeloma), solitary plasmacytoma, and multiple myeloma; chronic lymphocytic leukemia (CLL), pilocellular lymphoma; hematopoietic tumors of the myeloid lineage; mesenchymal tumors, including fibrosarcoma and rhabdomyosarcoma; seminoma, teratoma; tumors of the central and peripheral nervous systems, including astrocytoma and Schwann cell tumor; Mesenchymal tumors, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid carcinoma, and teratoma; hematopoietic tumors of the lymphoid spectrum, such as T-cell and B-cell tumors, including but not limited to T-cell disorders such as T-prolymphocytic leukemia (T-PLL), including small cell and gyral cell types; large granular lymphocytic leukemia (LGL), preferably T-cell type; a / d T-NHL heptasyllabic lymphoma; peripheral / postthymic T-cell lymphoma (pleomorphic and immunoblastic subtypes); angiocentric (nasal) T-cell lymphoma; head and neck cancer, renal cancer, rectal cancer, thyroid cancer; acute myeloid lymphoma; and any combination of said cancers. The anti-SIRPβ1 antibody disclosed herein can also be used to treat metastatic cancers.

[0387] In some embodiments, the cancer is selected from sarcoma, bladder cancer, brain cancer, breast cancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer, renal pelvis cancer, leukemia, lung cancer, small cell lung cancer, melanoma, lymphoma, pancreatic cancer, prostate cancer, ovarian cancer, and fibrosarcoma. In some embodiments, the cancer is triple-negative breast cancer. In some embodiments, the cancer can be early-stage or late-stage cancer. In some embodiments, the cancer can be a primary tumor. In some embodiments, the cancer can be a metastatic tumor derived from any of the above-mentioned types of cancer at a second site.

[0388] In some implementation schemes, the cancer is selected from glioblastoma multiforme; clear cell renal cell carcinoma; adrenocortical carcinoma; urothelial carcinoma of the bladder; diffuse large B-cell lymphoma; lung adenocarcinoma; pancreatic adenocarcinoma; renal cell carcinoma; Hodgkin's lymphoma; non-Hodgkin's lymphoma; painless B-cell lymphoma; aggressive B-cell lymphoma; T-cell lymphoma; acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); chronic lymphocytic leukemia (CLL); chronic myeloid leukemia (CML); multiple myeloma; myelodysplastic syndrome. Symptoms include myeloproliferative growths, invasive breast cancer, cervical squamous cell carcinoma, cervical adenocarcinoma, bile duct cancer, colon adenocarcinoma, diffuse large B-cell lymphoma, esophageal cancer, head and neck squamous cell carcinoma, renal chromophobe carcinoma, renal papillary cell carcinoma, low-grade glioma, hepatocellular carcinoma, lung squamous cell carcinoma, mesothelioma, ovarian serous cystadenocarcinoma, pancreatic adenocarcinoma, pheochromocytoma and paraganglioma, prostate adenocarcinoma, rectal adenocarcinoma, skin melanoma, gastric adenocarcinoma, testicular germ cell tumor, thyroid cancer, thymoma, endometrial cancer, uterine carcinosarcoma, and uveal melanoma.

[0389] In some embodiments, this disclosure provides methods for preventing, reducing risk, or treating an individual with cancer by administering to the individual a therapeutically effective amount of the anti-SIRPβ1 antibody of this disclosure.

[0390] In some embodiments, this disclosure provides methods for preventing, reducing risk, or treating an individual with cancer, wherein the individual is not responding to checkpoint inhibitor therapy, which is administered to the individual in a therapeutically effective amount of the anti-SIRPβ1 antibody of this disclosure.

[0391] In some embodiments, the anti-SIRPβ1 antibody of this disclosure may be administered in combination with a therapeutic agent that acts as a checkpoint inhibitor. In some embodiments, the method further includes administering to the individual at least one antibody that specifically binds to an inhibitory immune checkpoint molecule, and / or another standard or investigative anticancer therapy. In some embodiments, the inhibitory checkpoint molecule is selected from PD1, PDL1, CD40, OX40, ICOS, CD28, CD137 / 4-1BB, CD27, GITR, CTLA4, PD-L2, B7-H3, B7-H4, HVEM, LIGHT, BTLA, CD30, TIGIT, VISTA, KIR, GAL9, TIM1, TIM3, TIM4, A2AR, LAG3, DR-5, CD2, CD5, CD39, and CD73. In typical embodiments, the therapeutic agent is an antibody against a checkpoint inhibitor selected from PD1, PDL1, CD40, OX40, ICOS, CD28, CD137 / 4-1BB, CD27, GITR, CTLA4, PD-L2, B7-H3, B7-H4, HVEM, LIGHT, BTLA, CD30, TIGIT, VISTA, KIR, GAL9, TIM1, TIM3, TIM4, A2AR, LAG3, DR-5, CD2, CD5, CD39, or CD73. In some embodiments, at least one antibody that specifically binds to an inhibitory checkpoint molecule is administered in combination with the anti-SIRPβ1 antibody of this disclosure. In some embodiments, a combination of antibodies against checkpoint inhibitors is administered in combination with the anti-SIRPβ1 antibody of the present invention.

[0392] In some embodiments, the anti-SIRPβ1 antibody of this disclosure can be administered in combination with at least one agonist antibody that specifically binds to a stimulating checkpoint protein, such as an agonist anti-CD40 antibody, an agonist anti-OX40 antibody, an agonist anti-ICOS antibody, an agonist anti-CD28 antibody, an agonist anti-TREM1 antibody, an agonist anti-TREM2 antibody, an agonist anti-CD137 / 4-1BB antibody, an agonist anti-CD27 antibody, an agonist anti-glucocorticoid-induced TNFR-related protein GITR antibody, an agonist anti-CD30 antibody, an agonist anti-BTLA antibody, an agonist anti-HVEM antibody, an agonist anti-CD2 antibody, an agonist anti-CD5 antibody, and any combination thereof.

[0393] In some embodiments, the method further includes administering to the individual at least one antibody that specifically binds to an inhibitory immune checkpoint molecule, and / or another standard or investigational anticancer therapy. In some embodiments, the antibody that specifically binds to an inhibitory checkpoint molecule is administered in combination with the anti-SIRPβ1 antibody of this disclosure. In some embodiments, the antibody that specifically binds to an inhibitory checkpoint molecule is selected from anti-PD-L1 antibody, anti-CTLA4 antibody, anti-PD-L2 antibody, anti-PD-1 antibody, anti-B7-H3 antibody, anti-B7-H4 antibody, and anti-HVEM antibody, anti-B and T lymphocyte attenuator (BTLA) antibody, anti-killer inhibitory receptor (KIR) antibody, anti-GAL9 antibody, anti-TIM3 antibody, anti-A2AR antibody, anti-LAG-3 antibody, anti-phosphatidylserine antibody, anti-CD27 antibody, and any combination thereof. In some embodiments, the standard or investigational anticancer therapy is selected from radiotherapy, cytotoxic chemotherapy, targeted therapy, imatinib, etc. Trastuzumab One or more of adoptive cell transfer (ACT), chimeric antigen receptor T-cell transfer (CAR-T), vaccine therapy, and cytokine therapy. In some embodiments, the method further includes administering to the individual an antibody that specifically binds to at least one inhibitory cytokine. In some embodiments, the antibody that specifically binds to at least one inhibitory cytokine is administered in combination with the anti-SIRPβ1 antibody of this disclosure. In some embodiments, the antibody that specifically binds to at least one inhibitory cytokine is selected from anti-CCL2 antibody, anti-CSF-1 antibody, anti-IL-2 antibody, and any combination thereof.

[0394] In some embodiments, the method further includes administering to the individual at least one agonist antibody that specifically binds to a stimulating immune checkpoint protein. In some embodiments, the at least one agonist antibody that specifically binds to a stimulating checkpoint protein is administered in combination with the anti-SIRPβ1 antibody of this disclosure. In some embodiments, the at least one agonist antibody that specifically binds to a stimulating checkpoint protein is selected from agonist anti-CD40 antibodies, agonist anti-OX40 antibodies, agonist anti-ICOS antibodies, agonist anti-CD28 antibodies, agonist anti-CD137 / 4-1BB antibodies, agonist anti-CD27 antibodies, agonist anti-glucocorticoid-induced TNFR-related protein GITR antibodies, and any combination thereof.

[0395] In some embodiments, the anti-SIRPβ1 antibody of the present invention is administered in combination with radiotherapy and / or chemotherapy agents. Chemotherapy agents include, for example, the group consisting of: antimetabolites / anticancer agents, such as pyrimidine analogs (5-fluorouracil, fluorouracil, capecitabine, gemcitabine, and cytarabine) and purine analogs, folic acid antagonists and related inhibitors (methotrexate, pemetrexed, mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine (cladribine)); antiproliferative / antimitotic agents, including natural products, such as vinca alkaloids (vinblastine, vincristine, and vinorelbine); microtubule disruptors, such as taxanes (palitaxetine, docetaxel), vincristine, vinblastine, etc. Nocodazole, epormycin, eribulin, and novibone; epipodophyllotoxin (etoposide, teniposide); DNA disruptors (actinomycin, acridine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, Cytoxan, daunorubicin, doxorubicin, epirubicin, hexamethylmelamine, oxaliplatin, ifosfamide, mefenamic acid, dichloroethylmethylamine, mitomycin, mitoxantrone, nitrosourea, procainamide, procarbazine, tisulosin, tesole, temozolomide, teniposide, trimethylphosphamide, and etoposide (VP) 16); DNA methyltransferase inhibitors (azytidine); antibiotics, such as daunorubicin (actinomycin D), daunorubicin, doxorubicin (doxorubicin), idarubicin, anthracycline antibiotics, mitoxantrone, bleomycin, procainox (scintillan), and mitomycin; enzymes (L-asparaginase, which systemically metabolizes L-asparagine and deprives cells unable to synthesize their own asparagine); antiplatelet agents; antiproliferative / antimitotic alkylating agents, such as nitrogen mustard (dichloroethyl methylamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethyleneimine and methylmelamine (hexamethylmelamine and thiotepa), alkyl sulfonates / esters (busulin), nitrosoureas (carmustine (BCNU) and analogs, streptozotocin), triazines (dacarbazine (DTI)). C)); Antiproliferative / antimitotic antimetabolites, such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogens, tamoxifen, goserelin, bicalutamide, niglutethimide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other thrombin inhibitors); fibrinolytic agents (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigration agents; antisecretory agents (brefidobacterium); immunosuppressants (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil);Anti-angiogenic compounds (TNP470, genistein, pomalidomide) and growth factor inhibitors (vascular endothelial growth factor (VEGF) inhibitors, such as Ziv-aflibercept; fibroblast growth factor (FGF) inhibitors); apoptosis protein inhibitor (IAP) antagonists (birinapant); histone deacetylase (HDAC) inhibitors (vorinostat, romidepsin, chidamide, panobinostat, mocetinostat, abexinostat, belinostat, entinostat, resminostat, etc.). givinostat, quisinostat, SB939; proteasome inhibitors (ixazomib); angiotensin receptor blockers; nitric oxide donors; antisense oligonucleotides; antibodies (trastuzumab, panitumumab, pertuzumab, cetuximab, adalimumab, golimumab, infliximab, rituximab, ozoglucomannab, olfamumab, ozoglucomannab, alenzumab, abciximab, atlizumab, dalizumab, denosumab, efazolinumab, erlotuzumab, rovitazolinumab, rulizumab, eutecticumab, vexizumab, ozoglucomannab, ozoglucomannab, brentuximab) Vedotin; chimeric antigen receptors; cell cycle inhibitors (flavopiridol, roscovitine, lichenstatin-1) and differentiation inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin, acridine, camptothecin, daunorubicin, daunorubicin, teniposide, epirubicin, etoposide, idarubicin, irinotecan (CPT-11), mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, and prednisolone); PARP inhibitors (niraparib, olaparib); focal adhesion. kinase (FAK) inhibitors (defactinib (VS-6063), VS-4718, VS-6062, GSK2256098); growth factor signal transduction kinase inhibitors (sildenafil, galunisertib, rociletinib, vandetanib, afatinib, EGF816, AZD4547); c-Met inhibitors (carmatinib, INC280); ALK inhibitors (celitinib, crizotinib); mitochondrial dysfunction inducers; toxins such as cholera toxin, ricin, Pseudomonas exotoxin, Bordetella pertussis adenylate cyclase toxin, or diphtheria toxin, and caspase activators.And chromatin-destroying agents. In some embodiments, the chemotherapeutic agents are β-Raf inhibitors, MEK inhibitors, VEGF inhibitors, VEGFR inhibitors, tyrosine kinase inhibitors, antimitotic agents, or any combination thereof.

[0396] In some embodiments, the anti-SIRPβ1 antibody of this disclosure is administered in combination with adoptive cell transfer (ACT) therapy, chimeric antigen receptor T-cell transfer (CAR-T) therapy, vaccine therapy, and / or cytokine therapy.

[0397] In some embodiments, the anti-SIRPβ1 antibody of this disclosure is administered in combination with at least one antibody that specifically binds to an inhibitory cytokine, such as an anti-CCL2 antibody, an anti-CSF-1 antibody, or an anti-IL-2 antibody.

[0398] In some embodiments, the anti-SIRPβ1 antibody of this disclosure is administered in combination with at least one stimulating cytokine. In some embodiments that can be combined with any of the foregoing embodiments, the at least one stimulating cytokine is selected from IFN-α4, IFN-β, IL-1β, TNF-α, IL-6, IL-8, CRP, members of the IL-20 family, LIF, IFN-γ, OSM, CNTF, GM-CSF, IL-11, IL-12, IL-15, IL-17, IL-18, IL-23, CXCL10, IL-33, MCP-1, MIP-1-β, and any combination thereof.

[0399] VI. Diagnostic Uses

[0400] In some embodiments of any antibody, any anti-SIRPβ1 antibody provided herein is useful for detecting the presence of SIRPβ1 in a sample or individual. As used herein, the term "detection" encompasses both quantitative and qualitative detection. Methods for using this disclosed antibody for diagnostic purposes, such as detecting SIRPβ1 in or from tissue samples derived from an individual, are provided herein. In some embodiments, the individual is a person.

[0401] The detection method may involve quantifying antibodies bound to antigens. Antibody detection in biological samples can be performed using any method known in the art, including immunofluorescence microscopy, immunocytochemistry, immunohistochemistry, ELISA, FACS analysis, immunoprecipitation, or micropositron emission tomography. In some embodiments, the antibodies are radiolabeled, for example using... 18F, subsequently detected using micro-positron emission tomography analysis. Antibody binding can also be quantified in patients using non-invasive techniques such as positron emission tomography (PET), X-ray computed tomography, single-photon emission computed tomography (SPECT), computed tomography (CT), and computed axial computed tomography (CAT).

[0402] VII. Products

[0403] This document describes products (e.g., kits) containing the anti-SIRPβ1 antibody described herein. Products may include one or more containers containing the antibody described herein. Containers may be any suitable packaging, including but not limited to tubular vials, bottles, cans, flexible packaging (e.g., sealed Mylar or plastic bags), etc. Containers may be unit dose, bulk (e.g., multi-dose packaging), or subunit dose.

[0404] In some embodiments, the kit may further include a second pharmaceutical agent. In some embodiments, the second pharmaceutical agent is a pharmaceutically acceptable buffer or diluent, including but not limited to bactericidal water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextran solution. In some embodiments, the second pharmaceutical agent is a pharmaceutically active agent. In some embodiments, the second pharmaceutical agent is the pharmaceutically active agent described herein.

[0405] In some embodiments of any product, the product further includes instructions for use according to the methods disclosed herein. The instructions generally include information regarding the dosage, dosing schedule, and route of administration for the intended treatment. In some embodiments, these instructions contain instructions for administering an isolated antibody (e.g., the anti-SIRPβ1 antibody described herein) according to any method disclosed herein to prevent, reduce risk, or treat individuals with a disease, condition, or lesion selected from: dementia, frontotemporal dementia, Alzheimer's disease, vascular dementia, mixed dementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus, amyotrophic lateral sclerosis, Huntington's disease, Tau disease, Nasu-Hakola disease, stroke, acute trauma, chronic trauma, lupus, acute and chronic colitis, rheumatoid arthritis, wound healing, Crohn's disease, inflammatory bowel disease. Ulcerative colitis, obesity, malaria, essential tremor, lupus, Bechtel's disease, Parkinson's disease, Lewy body dementia, multiple system atrophy, Hedlock syndrome, progressive supranuclear palsy, corticobasal ganglia degeneration, acute disseminated encephalomyelitis, granulomatous diseases, sarcoidosis, age-related diseases, seizures, spinal cord injury, traumatic brain injury, age-related macular degeneration, glaucoma, retinitis pigmentosa, retinal degeneration, respiratory infections, sepsis, eye infections, systemic infections, lupus, arthritis, multiple sclerosis, low bone mineral density, osteoporosis, osteoporosis, osteosclerotic diseases, Paget's disease of bone, and cancer. In some implementations, the instructions for use include instructions for use of the anti-SIRPβ1 antibody and the second agent (e.g., a second pharmaceutically active agent).

[0406] This disclosure will be more fully understood by referring to the following embodiments. However, they should not be construed as limiting the scope of this disclosure. Accordingly, all references made throughout this disclosure are expressly included. Example

[0407] The following embodiments are provided by way of example only and not by way of limitation. Those skilled in the art will readily recognize that various non-critical parameters can be changed or modified to produce substantially similar results.

[0408] Example 1: Generation, identification, and characterization of agonistic anti-SIRPβ1 antibodies

[0409] The amino acid sequence of human SIRPβ1 isoform 1 protein (SIRPβ1) is listed in SEQ ID NO:1 below. Human SIRPβ1 contains a signal peptide located at amino residues 1-29 of SEQ ID NO:1; an extracellular immunoglobulin-like variable (IgV) domain located at amino residues 30-136 of SEQ ID NO:1; two immunoglobulin-like constant (IgC) domains located at amino residues 147-246 and 253-347 of SEQ ID NO:1; a transmembrane domain located at amino residues 372-392 of SEQ ID NO:1; and an intracellular domain located at amino residues 393-398 of SEQ ID NO:1.

[0410] Human SIRPβ1 amino acid sequence (SEQ ID NO:1):

[0411]

[0412] The human SIRPβ1 amino acid sequence contains a lysine residue (aa380) within a transmembrane domain, which interacts with aspartic acid in DAP12, a key adaptor protein that transduces signaling from SIRPβ1, as well as TREM1, TREM2, and other related IgV family members. BLAST analysis of human SIRPβ1 identified several isoforms that may be derived from alternative splicing. Among these transcript variants, SIRPβ1 isoform 3 shares the greatest sequence identity with SIRPβ1 isoform 1. Figure 1A Human SIRPβ1 is also related to human SIRPα. The amino acid sequences of human SIRPβ1 and human SIRPα were aligned using bidirectional BLAST. Figure 1B The two receptors share 85% sequence identity in their extracellular regions. In contrast, human SIRPβ1 shows poor homology with mouse SIRPβ1. Figure 2 This suggests that the SIRP locus in the two species experiences different selective pressures.

[0413] Antibodies binding to the extracellular domain of human SIRPβ1, particularly the extracellular IgV domain (amino acid residues 30-136 of SEQ ID NO: 1), were generated using mouse hybridoma technology, phage display technology, and a yeast-based platform technology. The antibodies were screened for their ability to bind to cells expressing SIRPβ1 and their ability to activate SIRPβ1 signaling, activity, and function in vivo in cells and in intact animals, as described in Examples 2-20 below. For example, agonistic anti-SIRPβ1 antibodies targeting the IgV domain (amino acid residues 30-136) were generated. Based on similarity to SIRPα, it was predicted that the IgV domain of SIRPβ1 would bind to an unknown ligand and, via receptor polymerization, lead to activation.

[0414] Generation of His-tagged and Fc-conjugated human SIRPα and human SIRPβ1 IgV domains

[0415] To achieve mammalian expression of human SIRPα and human SIRPβ1 IgV domain (also referred to as "domain 1") antigens (SEQ ID NO: 387 and 388, respectively), a cDNA-based synthetic gene was cloned into a mammalian expression vector, followed by transient transfection and expression in HEK293 / T cells. The construct includes a heterologous signal peptide and a C-terminal human IgG1 Fc and / or His tag. In short, the expression was achieved by conjugating the transfection reagent with the vector, followed by exposure to HEK293 / T cells for 1 hour, and then dilution to a final density of 4 x 10⁻⁶ cells in culture medium. 6 Cells were transfected at 100 cells / mL with expression vectors containing antigens of interest. Cells were then cultured for 7 days with fresh feed every 48 hours. After 7 days, the supernatant was collected after centrifugation and purified using Ni-Sepharose and, in some cases, SEC column chromatography to achieve >95% non-aggregated monomer content. SIRPα and SIRPβ1 monomeric antigens were prepared by fragmentation of SIRPα / β1 Fc fusion antigens with modified hinge regions (Lynaugh et al., MAbs. 2013 Oct; 5(5): 641-45) with FabRICATOR (IdeS) protease (Genovis, catalog number A2-FR2-1000), followed by protein A affinity purification to remove undigested Fc fusion protein and SEC purification to remove aggregated monomers.

[0416] Library screening for anti-SIRPβ1 antibodies

[0417] As previously described (see, for example, Xu et al., 2013; WO2009036379; WO2010105256; WO2012009568; Xu et al., Protein Eng Des Sel 2013 Oct; 26(10):663-70), eight non-immunized (pre-immunized) human synthetic yeast libraries, each approximately 10 9 Diversity. Ten parallel selections were performed, with eight non-immunized libraries used for human SIRPβ1-Fc fusion antigen selection and two sets of eight libraries used for human SIRPβ1 monomer selection. For the first two rounds of selection, essentially as described (Siegel et al., J Immunol Methods 2004 Mar; 286(1-2):141-53), magnetic bead sorting using the Miltenyi MACs system was implemented. In short, yeast cells (approximately 10 9 Cells / library, total density approximately 10 10The yeast cells were incubated for 15 minutes at room temperature in FACS wash buffer with 3 ml of 10 nM biotinylated SIRPβ1-Fc fusion antigen or 100 nM biotinylated SIRPβ1 monomer antigen in PBS containing 0.1% BSA. After washing once with 50 ml of ice-cold wash buffer, the cell clumps were resuspended in 40 ml of wash buffer, and 500 μL of streptavidin beads (Miltenyi Biotec, Bergisch Gladbach, Germany, catalog number 130-048-101) were added to the yeast cells and incubated at 4°C for 15 minutes. The yeast cells were then clumped, resuspended in 5 ml of wash buffer, and loaded onto a MACS LS column (Miltenyi Biotec, Bergisch Gladbach, Germany, catalog number 130-042-401). After loading 5 mL, the column was washed three times with 3 ml of FACS wash buffer. The column was then removed from the magnetic field and the yeast was eluted with 5 mL of growth medium, followed by overnight growth. The following four rounds of sorting were performed using flow cytometry.

[0418] For the first round of FACS selection, approximately 1x10 8 Yeast cells were clumped and washed three times with washing buffer, then incubated at room temperature for 10 minutes with either 10 nM biotinylated SIRPβ1-Fc fusion antigen or 100 nM biotinylated SIRPβ1 monomer antigen. The yeast cells were then washed twice and stained at 4°C for 15 minutes with either a 1:100 dilution of goat anti-human F(ab')2κ-FITC (Southern Biotech, Birmingham, Alabama, catalog 2062-02) or a 1:500 dilution of streptavidin-Alexa Fluor633 (Life Technologies, Grand Island, NY, catalog S21375) or a 1:50 dilution of Extravidin-phycoerythrin (Sigma-Aldrich, St Louis, catalog E4011). After washing twice with ice-cold washing buffer, the cell clumps were resuspended in 0.4 mL of washing buffer and transferred to sorting tubes with filter caps. Sorting was performed using a FACS ARIA sorter (BD Biosciences) and sorting gates were determined to select only SIRPβ1-binding clones. In the following rounds of selection, approximately 2 x 10⁻⁶ clones were prepared as described above. 7Yeast cells were incubated with a multispecific reactive reagent for negative selection to reduce multispecific conjugates (Xu et al., PEDS. 2013 Oct; 26(10): 663-70), and conjugates of the His-tagged human SIRPα monomer against the control protein. The next round used 10 nM human SIRPβ1-Fc fusion antigen and 100 nM human SIRPβ1 monomer antigen for labeling. After the final round of sorting, yeast cells were plated and individual colonies were picked for characterization. The final round used 100 nM and 10 nM human SIRPβ1 monomer antigen for labeling.

[0419] Heavy chains from the output of the second and fourth rounds of FACS sorting were used to prepare light chain diversity libraries for further selection. For these selections, the first round utilized Miltenyi MACs beads labeled with 10 nM human SIRPβ1-Fc fusion antigen. This was followed by four rounds of FACS sorting. The first round used 100 nM human SIRPβ1 monomeric antigen. The second round of FACS was negative sorting to reduce the number of binding reagents, multispecific bindings, and bindings to His-tagged monomers targeting the control protein human SIRPα. The final two rounds used human SIRPβ1 monomer titrations (100 nM, 10 nM, and 1 nM) to select the highest affinity binding, 100 nM human SIRPα monomer, and competition with the control AM4-5 antibody binding the SIRPβ1 IgV domain to assess the representativeness of the competitor in the rich clusters (see U.S. Patent Application Publication No. US2014 / 0242095). After the final round of sorting, yeast cells were plated and individual colonies were picked for characterization.

[0420] Antibody IgG and Fab generation and purification

[0421] Yeast clones were grown to saturation and then induced at 30°C with shaking for 48 hours. After induction, yeast cells were clumped and the supernatant was harvested for purification. IgG was purified using a protein A column and eluted with acetic acid at pH 2.0. The Fab fragment was generated by papain digestion and purified on CaptureSelect IgG-CH1 affinity matrix (LifeTechnologies, catalog number 1943200250).

[0422] Combined with experiments

[0423] By using ForteBio The Red384 system (ForteBio, Menlo Park, CA) was used to measure the dissociation constant (Ki) of anti-SIRPβ1 antibody. DTo determine their affinity, the procedure is generally as described previously (Estep et al., MAbs. 2013 Mar-Apr; 5(2):270-8). In short, Octet affinity measurements are performed as follows: IgG is loaded onto the AHQ sensor. The sensor is equilibrated offline in assay buffer for 30 minutes, then monitored online for 60 seconds to establish a baseline. For affinity binding measurements, the IgG-loaded sensor is exposed to 100 nM antigen (human SIRPα domain 1(D1)Fc fusion or SIRPβ1 domain 1(D1)Fc fusion) for 3 minutes, then transferred to assay buffer for 3 minutes for dissociation rate measurement. Additional affinity binding is determined by loading biotinylated SIRPβ1 monomers onto the SA sensor and exposing them in solution to 100 nM IgG. Monovalent binding measurements are obtained by loading human SIRPα- or SIRPβ1-Fc fusion antigens onto the AHQ sensor and then exposing them to 100 nM anti-SIRPβ1 antibody Fab. Additional monovalent measurements were performed by loading biotinylated human SIRPα or SIRPβ1 monomers onto the SA sensor and then exposing it in solution to a 100 nM Fab. Kinetic data were fitted using a 1:1 binding model in ForteBio's data analysis software (ForteBio Data Analysis Software 7.0).

[0424] Sub-warehouse

[0425] Using the standard sandwich-type compartmentalization measurement method in ForteBio Epitope compartmentation of anti-SIRPβ1 antibodies was performed on a Red384 system (ForteBio, MenloPark, CA). Control anti-target IgG was loaded onto an AHQ sensor, and unoccupied Fc binding sites on the sensor were blocked with an unrelated human IgG1 antibody. The sensor was then exposed to 100 nM of the target antigen, followed by a second anti-target antibody. Data were processed using ForteBio data analysis software 7.0. Additional binding of the second antibody after antigen conjugation indicated unoccupied epitopes (non-competitors), while no binding indicated epitope blocking (competitors). This process was repeated for two reference antibodies that bound the SIRPβ1 IgV domain: (i) AM4-5, defining compartment 1; and (ii) SB-17, defining compartment 2.

[0426] The final set of anti-SIRPβ1 antibodies was selected based on antigen-binding affinity. Antibodies that were positive for binding to human SIRPβ1 were tested for cross-reactivity with human SIRPα. The binaries for each antibody are listed in Table 2 below. In Table 2, “ND” indicates an antibody whose binary is undetermined; “NB” indicates an antibody that did not bind to the antigen shown; and “PF” indicates an antibody whose antigen-binding kinetics showed a poor fit to the 1:1 binding model. No detectable binding to mouse SIRPβ1D1-Fc, human SIRPαD1-Fc, or mouse SIRPαD1-Fc was observed for any of the anti-SIRPβ1 antibodies.

[0427] Table 2: Biochemical characterization of anti-SIRPβ1 antibody

[0428]

[0429]

[0430] Antibody heavy and light chain variable domain sequences

[0431] The amino acid sequences encoding the light chain variable domain and heavy chain variable domain of the antibody were determined using standard techniques. The EU or Kabat sequences of the antibody are listed in Tables 3-6, as follows. The EU or Kabat light chain HVR sequences of the antibody are listed in Table 3. The EU or Kabat heavy chain HVR sequences of the antibody are listed in Table 4. The EU or Kabat light chain framework (FR) sequences of the antibody are listed in Table 5. The EU or Kabat heavy chain framework (FR) sequences of the antibody are listed in Table 6.

[0432] Table 3: EU or Kabat light chain HVR sequences of anti-SIRPβ1 antibodies

[0433]

[0434]

[0435]

[0436] Table 4: EU or Kabat heavy chain HVR sequences of anti-SIRPβ1 antibodies

[0437]

[0438]

[0439]

[0440] Table 5: EU or Kabat light chain framework sequences of anti-SIRPβ1 antibodies

[0441]

[0442]

[0443]

[0444]

[0445]

[0446]

[0447] Table 6: EU or Kabat heavy chain framework sequences of anti-SIRPβ1 antibodies

[0448]

[0449]

[0450]

[0451]

[0452]

[0453]

[0454]

[0455]

[0456] Characterization of SIRPβ1 antibody binding

[0457] Initial characterization of anti-SIRPβ1 antibodies involved determining their ability to bind to cell lines expressing human or mouse SIRPβ1. Cells were harvested and cultured in 96-well plates at 10... 6 Dispense cells per ml, wash, and incubate on ice for 0.5 h in 100 μl of FACS buffer containing 1 μg / ml anti-SIRPβ1 antibody. Then wash cells twice and incubate on ice for 30 min in 100 μl of FACS buffer containing 0.5 μg / ml PE-conjugated secondary antibody. Wash cells twice in cold FACS buffer and acquire them on BD FACSCanto. Perform mean fluorescence intensity (MFI) values ​​or % of positive cells data analysis and calculation using FlowJo (TreeStar) software version 10.0.7.

[0458] Table 7 shows the mean fluorescence intensity (MFI) values ​​of anti-SIRPβ1 antibodies binding to Chinese hamster ovary (CHO) cell lines expressing low levels of recombinant human SIRPβ1. A background fluorescence signal set to 1 was established for the human IgG1 isotype control. Of the 50 anti-SIRPβ1 antibody clones tested, 32 clones bound cells with an MFI ≥2-fold above the background. As a negative control, anti-SIRPβ1 antibody screening was also performed to detect surface binding of CHO cells overexpressing recombinant mouse SIRPβ1. As expected, none of the tested antibodies bound mouse SIRPβ1. Given the high sequence similarity among SIRP family receptors, anti-SIRPβ1 antibody screening was also performed to detect cross-reactivity with human and mouse SIRPα. In the cell binding assay, none of the anti-SIRPβ1 antibodies bound cells overexpressing either human or mouse SIRPα.

[0459] Table 7: Cell binding characterization of anti-SIRPβ1 antibody

[0460]

[0461]

[0462] In addition, antigen specificity was screened for anti-SIRPβ1 antibodies using reporter cell lines expressing the luciferase gene under the control of the NFAT (nuclear factor of activated T cells) promoter. The cell line BW5147.G.1.4, derived from mouse thymic lymphoma T lymphocytes, was infected with Cignal Lenti NFAT-luciferase virus (Qiagen). TIB48 TM BWZ / NFAT-luciferase reporter cells were generated. Subsequently, cells were transduced with either a lentivirus expressing the human SIRPα-DAP12 chimeric compound (where the intracellular ITIM motif of SIRPα was replaced by the intracellular ITAM motif of DAP12) or two lentiviruses expressing human SIRPβ1 and human DAP12. Test antibodies and human IgG1 isotype controls were adsorbed onto 96-well plates at 10 μg / mL. After washing, 10... 5 NFAT-luciferase reporter cells expressing either the huSIRPα / DAP12 chimera (BWZ-huSIRPα) or co-expressing huSIRPβ1 and DAP12 (BWZ-huSIRPβ1) were seeded onto plates and incubated overnight at 37°C. Luciferase activity was measured by adding OneGlo reagent (Promega) to each well and incubating the sample at room temperature for 3 minutes on a shaker. GEN5 was used. TM Software 2.04 uses BioTek Synergy TM Microplate reader quantizes light emission signals. For example... Figure 3AAs shown, 48 out of 50 anti-SIRPβ1 clones induced luciferase expression at a fold >5 against the background in BWZ-huSIRPβ1 reporter cells. In contrast, when BWZ-huSIRPα reporter cells were added to antibody-coated wells, the anti-SIRPβ1 antibody failed to induce luciferase expression. Figure 3B In summary, only reporter cells expressing human SIRPβ1 induced luciferase expression in the presence of immobilized anti-SIRPβ1 antibodies, as measured by luminescence signals. These results establish that most anti-SIRPβ1 antibodies capable of binding to membrane-bound antigens exhibit specificity for their target antigens and do not cross-react with other SIRP receptors.

[0463] Example 2: Overview of SIRPβ1 expression on myeloid cells

[0464] The SIRP family comprises several transmembrane glycoproteins primarily expressed within the compartments of myeloid cells. The expression pattern of SIRPβ1 was validated in primary human cells isolated from healthy human donors. Primary human monocytes were isolated from heparinized human peripheral blood obtained from two healthy donors (Blood Centers of the Pacific) using the RosetteSep Human Monocyte Enrichment Mixture (STEMCELL Technologies) according to the manufacturer's protocol. Both SIRPβ1 and TREM1 are preferentially expressed on CD14. 高 Expression on monocytes. Monocytes were seeded in RPMI (Invitrogen) containing 10% fetal bovine serum (Hyclone) and 50 ng / ml M-CSF or GM-CSF (Peprotech) to induce the differentiation of M2-like or M1-like macrophages, respectively. Macrophages were harvested by scraping the cells attached to the plastic after 5-6 days. Alternatively, monocytes were seeded in RPMI medium containing 10% fetal bovine serum (Hyclone) and 20 ng / ml IL-4 and GM-CSF (Peprotech) to induce the differentiation of immature dendritic cells. Dendritic cells were harvested by scraping the cells attached to the plastic after 6-7 days.

[0465] like Figure 4A As shown, CD14 expression defines a subset of primary human monocytes. Classical and intermediate monocytes express high levels of CD14, while non-classical monocytes lack CD14 expression. Like TREM1, SIRPβ1 is abundantly expressed in CD14+ monocytes. Monocytes isolated from peripheral blood as described above were cultured in M-CSF or GM-CSF for 5 days to generate M2 or M1 macrophages, respectively. SIRPβ1 expression decreased upon differentiation of primary monocytes into macrophages, and M1-like macrophages expressed higher levels of the receptor ( ) compared to M2-like macrophages. Figure 4BSimilarly, SIRPβ1 expression decreased during the differentiation of primary monocytes into immature dendritic cells. LPS-induced dendritic cell maturation further downregulated SIRPβ1 expression, contrasting with the increased expression observed with TREM1. Figure 4C Unlike TREM1, LPS treatment downregulates SIRPβ1 expression on dendritic cells (DCs). Peak SIRPβ1 expression occurred in blood, spleen, and lung. Observation of SIRPβ1 expression patterns from RNA-seq datasets (from various tissue samples collected from healthy donors) confirmed that SIRPβ1 transcript levels were predominant in blood, consistent with high receptor expression in circulating monocytes and neutrophils. Figure 5A However, RNA-seq data revealed that the expression pattern of SIRPβ1 isoform 3 transcripts differs from that of SIRPβ1 isoform 1; SIRPβ1 isoform 3 is mostly expressed in the brain rather than peripheral tissues. Figure 5B (Data were obtained from the Genotype-Tissue Expression (GTEx) Combined Database.)

[0466] Example 3: SIRPβ1 antibody induces Syk phosphorylation

[0467] Spleen tyrosine kinase (Syk) is an intracellular signaling molecule that functions downstream of SIRPβ1. It promotes the formation of signal transduction complexes by phosphorylating several substrates, leading to cell activation and inflammatory processes. The ability of agonistic SIRPβ1 antibodies to induce Syk activation was determined by culturing mouse monocytes and measuring the phosphorylation status of Syk protein in cell extracts. In these experiments, secondary antibodies were used to cross-link anti-SIRPβ1 antibodies on cells to induce intracellular signaling.

[0468] Bone marrow-derived mononuclear cells (BMDM) from wild-type (WT) mice, human SIRPβ1 BAC transgenic mice, and mice lacking functional Fc receptor co-γ chain gene expression (FcgR KO; REF: Takai T, 1994, Cell, 76(3):519-29) were starved in 1% serum RPMI for 4 hours, then removed from tissue culture dishes with PBS-EDTA, washed with PBS, and counted. Cells were coated on ice with full-length SIRPβ1 antibody or huIgG1 isotype control for 15 minutes. After washing with cold PBS, cells were incubated at 37°C for the indicated time period in the presence of goat anti-human IgG. Following stimulation, cells were lysed with lysis buffer (1% v / v NP-40%, 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1.5 mM MgCl2, 10% glycerol, with protease and phosphatase inhibitors), followed by centrifugation at 16,000 g for 10 minutes at 4°C to remove insoluble material. The lysates were then immunoprecipitated with anti-Syk antibody (N-19 for BMDM or 4D10 for human DCs, Santa Cruz Biotechnology). Protein fractionation by SDS-PAGE was performed, transferred to a PVDF membrane, and probed with anti-phosphotyrosine antibody (4G10, Millipore). To confirm adequate immunoprecipitation of all substrates, the immunoblot was re-probeged with anti-Syk antibody (Abcam, for BMDM) or anti-Syk antibody (Novus Biological, for human DCs). As described (e.g., Peng et al., (2010) SciSignal., 3(122): ra38), visualization was performed using an enhanced chemiluminescence (ECL) system (GE Healthcare). These studies support the anti-SIRPβ1 antibody-induced syk phosphorylation of the present invention.

[0469] Example 4: When expressed by Fc γ SIRPβ1 antibody induces Syk phosphorylation when the receptor is clustered in adjacent cells.

[0470] Activation of spleen tyrosine kinase (Syk) is driven by antibody-mediated cross-linking of two or more SIRPβ1 receptors, thereby promoting the formation of signal transduction complexes, leading to cellular activation and inflammatory processes. In vivo cross-linking is mediated by neighboring cells expressing Fc receptors (FcRs), such as B cells and other leukocytes (White AL, Cancer Immunol Immunother, 62:941-948, 2013; Wilson NS, Cancer Cell 19:101-113, 2011; Bartholomaeus P, J Immunol, 192:2091-2098, 2014). In these experiments, helper cells expressing Fcγ receptors (i.e., B cells) were used to cross-link anti-SIRPβ1 antibodies to induce intracellular signal transduction.

[0471] The ability of the Fc receptor to induce Syk activation via antibody clusters was determined by culturing mouse monocytes in the presence of Fc receptor-expressing cells and measuring the phosphorylation status of Syk protein in cell extracts. Bone marrow-derived monocytes (BMDM) from wild-type (WT) mice and human SIRPβ1 BAC transgenic mice were starved in 1% serum RPMI for 4 hours, then removed from tissue culture dishes with PBS-EDTA, washed with PBS, and counted. Cells were coated on ice with full-length SIRPβ1 antibody or huIgG1 isotype control for 15 minutes. After washing with cold PBS, the cells were incubated at 37°C for 5 minutes with glutaraldehyde-fixed Fc receptor-expressing cells prepared previously as described below. In short, Fc receptor-expressing cells were prepared using MACS microbeads (CD19). + B cell isolation kit (Miltenyi Biotec) isolates B cells from mouse spleens according to the manufacturer's protocol, or from the P815 cell line overexpressing FcR2b and FcR3. Use 2x10 6Cells were fixed at room temperature for 1 minute with 0.05% glutaraldehyde, and the reaction was stopped with 1 μM glycine. Cells were then thoroughly washed with PBS. After stimulation, cells were lysed with lysis buffer (1% v / v NP-40%, 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1.5 mM MgCl2, 10% glycerol, with protease and phosphatase inhibitors). The lysate was then centrifuged at 16,000 g for 10 minutes at 4°C to remove insoluble material. The lysate was then immunoprecipitated with anti-Syk antibody (N-19 for BMDM or 4D10 for human DC, Santa Cruz Biotechnology). The precipitated proteins were fractionated by SDS-PAGE, transferred to a PVDF membrane, and probed with anti-phosphotyrosine antibody (4G10, Millipore). To confirm that all substrates were adequately immunoprecipitated, the immunoblot was re-examined with an anti-Syk antibody (Abcam, for BMDM) or an anti-Syk antibody (Novus Biological, for human DCs). As described (e.g., Peng et al., (2010) SciSignal., 3(122):ra38), visualization was performed using an enhanced chemiluminescence (ECL) system (GE Healthcare). These studies support the induction of syk phosphorylation by the anti-SIRPβ1 antibody of this invention when it is generated by adjacent cell clusters expressing the Fcγ receptor.

[0472] Example 5: SIRPβ1 antibody induces respiratory burst in immune cells

[0473] The agonistic function of the SIRPβ1 antibody disclosed herein was evaluated in primary human innate immune cells (e.g., monocytes and neutrophils).

[0474] Anti-SIRPβ1 and allotype control antibodies were diluted in serum-free RPMI medium and mixed with 100,000 neutrophils at a concentration of 10 μg / ml in a 96-well plate. The mixture was then processed using EasySep. TMPrimary neutrophils were isolated from peripheral blood obtained on the same day using the Direct Human Neutrophil Isolation Kit (STEMCELL) according to the manufacturer's instructions. To detect reactive oxygen species (ROS) generation, cells were labeled with the 2 μM fluorescent dye CM-H2DCFDA. Cells were stimulated with soluble full-length human IgG1 isotype control or anti-SIRPβ1 antibodies SB-1, -2, -3, -4, -5, -6, -7, -8, -9, -11, -14, -15, -17, -27, -28, -31, -39, -40, -41, -45, -46, and -49. After antibody-mediated stimulation at 37°C in the presence of CM-H2DCFDA for 1 hour, relative fluorescence units in the cells were measured at an excitation wavelength of 495 nm and an emission wavelength of 530 nm. The specific fluorescence index of stimulated cells was obtained by subtracting the background fluorescence of labeled cells incubated alone in culture medium and / or with an allotype control antibody (huIgG1). This was achieved using BioTek Synergy. TM Microplate reader uses GEN5 TM 2.04 Software reads the board.

[0475] exist Figure 6A In this study, primary human neutrophils from two healthy donors were stimulated with 10 μg / mL soluble full-length anti-SIRPβ1 antibody or human IgG1 isotype control and labeled with 2 μM CM-H2DCFDA at 37°C for 1 hour to monitor SIRPβ1-mediated ROS generation. SIRPβ1 antibodies SB-1, -2, -3, and -5 demonstrated high agonism in solution. In contrast, SIRPβ1 antibodies SB-15, -17, -27, -28, -31, -39, -40, -41, -45, -46, and -49 weakly activated SIRPβ1-mediated respiratory burst in solution.

[0476] Cells were left untreated or stimulated with soluble full-length human IgG1 isotype control or anti-SIRPβ1 antibody SB-1,-2,-3,-4,-5,-6,-7,-8,-9,-11,-12,-13,-14,-15,-17,-23,-26,-27,-28,-31,-33,-34,-35,-36,-37,-39,-40,-41,-45,-46, and-49. Figure 6BPrimary human monocytes were stimulated with 20 μg / mL soluble full-length anti-SIRPβ1 antibody or human IgG1 isotype control and labeled with 2 μM CM-H2DCFDA at 37°C for 1 hour to monitor SIRPβ1-mediated ROS generation and IL-8 production. SIRPβ1 antibodies SB-2, -3, -4, -5, -6, -7, -28, and -49 showed high activating activity in solution. In contrast, SIRPβ1 antibodies SB-14, -15, -17, -27, -41, -45, and -46 weakly activated SIRPβ1-mediated respiratory burst in solution. Additionally, primary monocytes were stimulated overnight at 37°C with 10 μg / mL anti-SIRPβ1 antibody adsorbed onto 96-well plates. The supernatant was then collected, fractionated, and IL-8 release was measured. SIRPβ1 antibodies SB-1, -2, -3, -7, -8, -9, -14, 28, and -49 demonstrated high agonism as plate-binding antibodies. In contrast, SIRPβ1 antibodies SB-4, -12, -23, -26, -33, -34, -35, -36, and -37 weakly activated SIRPβ1-mediated cytokine release.

[0477] Example 6: SIRPβ1 enhances the secretion of inflammatory cytokines from macrophages

[0478] Published literature describes bone marrow-derived macrophages (BMDM) or primary peritoneal macrophages exhibiting altered TLR responses in cases of DAP12 deficiency. To determine whether the SIRPβ1 antibody of this disclosure induces changes in inflammatory cytokine production, primary human monocyte-derived macrophages and dendritic cells were cultured with a plate-bound test antibody in combination with unsaturated levels of TLR stimulants, and cytokine levels were measured after 24 hours. To generate monocyte-derived macrophages and dendritic cells, primary human monocytes were isolated from heparinized human blood (Blood Centers of the Pacific) using RosetteSep Human Monocyte Enrichment Mixture (STEMCELL Technologies) according to the manufacturer's protocol. Monocytes were seeded in RPMI (Invitrogen) containing 10% fetal bovine serum (Hyclone) and 50 ng / ml M-CSF to induce macrophage differentiation. Macrophages were harvested after 5–6 days by scraping cells attached to plastic. Alternatively, mononuclear cells were seeded in RPMI medium containing 10% fetal bovine serum (Hyclone) and 20 ng / ml IL-4 and GM-CSF (Peprotech) to induce differentiation of immature dendritic cells. After 6–7 days, dendritic cells were harvested by scraping the cells attached to the plastic. 5Macrophages or dendritic cells were distributed per cell / well in a 96-well plate coated with the antibody shown and incubated at 37°C for 24 hours. Cells were co-stimulated with the TLR4 agonist LPS (Salmonella abortus equi).

[0479] like Figure 6C As shown, monocyte-derived macrophages and dendritic cells were stimulated overnight at 37°C with 0.5 ng / mL LPS in the presence of anti-SIRPβ1 antibodies (SB-1, -2, -3, -4, -5, -6, -7, -8, -9, -11, -12, -14, -15, -16, -17, -18, -19, -20, -21, -28, -32, -40, and -49) adsorbed onto 96-well plates. The supernatant was then collected, fractionated, and TNFα release was measured. As a negative control, dendritic cells (DCs) were stimulated with LPS in the presence of plate-bound anti-SIRPα antibody SA-56-90 (which inhibits TNFα release). In all experiments measuring respiratory burst, reactive oxygen species (ROS) generation was monitored by labeling cells with the 2 μM fluorescent indicator CM-H2DCFDA. SIRPβ1 antibodies SB-1, -2, -6, -8, and -49, as plate-bound antibodies, demonstrated high agonism. In contrast, most of the remaining SIRPβ1 antibodies weakly activated SIRPβ1-mediated cytokine release. Similarly, plate-bound SB-1 and SB-40, but not SB-32, enhanced LPS-induced TNFα release from monocyte-derived dendritic cells. In contrast, LPS-induced TNFα release was reduced in dendritic cells stimulated with the plate-bound anti-SIRPα antibody SA-90, confirming that agonistic anti-SIRPβ1 antibodies activate cells, while agonistic SIRPα antibodies inhibit cellular activity.

[0480] Example 7: Antitumor activity of SIRPβ1-stimulated neutrophils

[0481] While neutrophils are efficient phagocytics for antibody or complement opsonization targets, in the context of the tumor microenvironment, they generally contribute to tumor progression, invasion, and angiogenesis. However, recent publications suggest that tumor-associated neutrophils, like other myeloid cells, retain the potential for polarization toward an anti-tumor phenotype. Because neutrophils express high levels of SIRPβ1, their ability to induce neutrophil-mediated tumor cell clearance in vitro was assessed using anti-SIRPβ1 antibodies. (Using EasySep...) TMPrimary neutrophils were isolated from peripheral blood of healthy donors obtained on the same day, following the manufacturer's instructions using the Direct Human Neutrophil Isolation Kit (STEMCELL). The isolated human neutrophils were then added to 96-well plates previously coated with 10 μg / mL anti-SIRPβ1 antibody or an allotype control. Subsequently, Raji B-cell lymphoma cells, modified to stably express luciferase, were mixed with neutrophils at a 1:1 ratio, with or without a regulatory antibody (anti-CD20 human IgG1). The co-cultured cells were incubated overnight at 37°C, and viable Raji cells were quantified by measuring luciferase activity after adding OneGlo reagent (Promega) and incubating the sample at room temperature for 3 minutes on a shaker. GEN5 was used. TM Software 2.04 uses BioTek Synergy TM Microplate reader detects light emission signals.

[0482] like Figure 7 As shown, human neutrophils failed to eliminate Raji cells when co-cultured in the presence of immobilized isotype control antibodies. The addition of anti-CD20 huIgG1 did not restore tumor cell clearance with unstimulated neutrophils. Similarly, neutrophils stimulated with immobilized anti-SIRPβ1 antibodies failed to significantly eliminate Raji cells. However, SIRPβ1-stimulated neutrophils cleared anti-CD20-opsonized Raji cells. Agonistant anti-SIRPβ1 antibodies SB-1, SB-2, SB-3, and SB-4 reduced the luminescent signal from viable Raji cells by approximately 50% compared to isotype control-treated neutrophils. Anti-SIRPβ1 antibodies SB-28 and SB-49 exhibited weak activity against optimized Raji cells. These results suggest that agonist anti-SIRPβ1 antibodies enhance the antitumor properties of neutrophils.

[0483] Example 8: Affinity maturation of anti-SIRPβ1 antibody

[0484] Affinity maturation was performed on five anti-SIRPβ1 antibodies, SB-1, SB-2, SB-8, and SB-40 (referred to as "parental" antibodies), each possessing diverse physical and functional properties. In short, a diverse antibody library was created in yeast for each starting parental antibody. Diversity was created (referred to as "H1 / H2" optimization) by combining the parental heavy chain CDR-H3 and light chain (LC) with existing genetically diverse heavy chains (HC) in the CDR-H1 and CDR-H2 regions using standard molecular cloning techniques. This yielded six libraries, roughly 10 in size. 5One clone; 1:755-768 was used to select antibodies with improved affinity. The selection pressures used for screening the library included balanced titration of human SIRPα and SIRPβ1 antigens, parental antibody Fab competitive kinetics, and the use of multispecific reagents for selection (as described in, for example, WO 2014 / 179363; Xu et al., Protein Eng Des Sel, 26(10):663-670). Antibodies were then visualized and selected using standard techniques with FACS flow cytometry (see, for example, Chao et al., Nature Protocols, 2006; 1:755-768). The desired population was then moved forward to another selection round. After 6 rounds of enrichment, yeast cells were plated to obtain monoclonal antibody isolates, which were then generated and characterized as described in Example 1. Thus, 17 affinity-improved antibodies were obtained from 4 of the 5 starting parental antibodies.

[0485] Antibody IgG and Fab generation and purification

[0486] Yeast clones were allowed to grow to saturation, and then induced at 30°C for 48 hours with shaking. After induction, the yeast cells were clumped together and the supernatant was harvested for purification.

[0487] Immunoglobulins were purified using a Protein A column and eluted with acetic acid at pH 2.0. Fab fragments were generated by papain digestion and purified on CaptureSelect IgG-CH1 affinity matrix (Life Technologies).

[0488] Affinity Measurement

[0489] By using ForteBio Meso Scale Discovery (MSD) instrument measures K D The affinity of the anti-SIRPβ1 antibody was determined using the value. Generally, as previously described (Estep et al., MAbs. 2013 Mar-Apr; 5(2):270-8), the assay was performed at room temperature. Affinity measurement. In short, it is implemented as follows: Affinity measurements involved loading IgG onto the AHQ sensor. The sensor was equilibrated offline in assay buffer for 30 minutes, followed by online monitoring for 60 seconds to establish a baseline. For affinity binding measurements, the IgG-loaded sensor was exposed to 100 nM antigen (human SIRPα or SIRPβ1 Fc fusion) for 3 minutes, followed by transfer to assay buffer for 3 minutes to measure the dissociation rate. Additional affinity binding was measured by loading biotinylated SIRPβ1 monomer onto the SA sensor and exposing it in solution to 100 nM IgG. Monovalent binding measurements were obtained by loading human SIRPβ1 Fc fusion antigen onto the AHQ sensor and then exposing it to 100 nM anti-SIRPβ1 antibody Fab. Additional monovalent measurements were performed by loading biotinylated human SIRPβ1 monomer onto the SA sensor and then exposing it in solution to 100 nM Fab. Kinetic data were fitted using a 1:1 binding model in data analysis software provided by ForteBio.

[0490] For MSD-SET K D Measurements were performed using solution equilibration titration (SET) in PBS containing recombinant human SIRPβ1 and 0.1% IgG-free BSA (PBSF), maintained at a constant 100 pM and incubated with 3 to 5-fold serial dilutions of the antibody, starting at approximately 50 nM. The antibody (20 nM, in PBS) was coated onto a standard binding MSD-ECL plate and incubated overnight at 4°C or for 30 minutes at room temperature. The plate was then blocked with 1% BSA for 30 minutes with shaking at 700 rpm, followed by three washes with washing buffer (PBSF + 0.05% Tween 20). SET samples were applied and incubated on the plate for 150 seconds with shaking at 700 rpm, followed by one wash. Antigens captured on the plate were detected by incubating on the plate for 3 minutes in PBSF with 250 ng / ml sulfur-tagged streptavidin. The plate was washed three times with washing buffer and then read using 1x reading buffer T with surfactant on an MSD Sector Imager 2400 instrument. In Prism, the percentage of free antigen as a function of titrated antibody was plotted and a quadratic equation was fitted to extract KD. To improve throughput, a liquid manipulation robot was used throughout the MSD-SET experiments, including SET sample preparation.

[0491] Cell binding affinity measurements were performed at 4°C using BWZ reporter cells expressing human SIRPα or SIRPβ1. Briefly, cells were harvested, washed in PBS, and incubated with progressively increasing concentrations of anti-SIRPβ1 antibody or an allotype control. The antibody was diluted in FACS buffer (PBS + 2% FBS). After incubation on ice for 30 min, cells were washed twice in FACS buffer and incubated on ice for 30 min with PE-conjugated anti-human secondary antibody (BD Biosciences). Cells were then washed twice in 200 μl of FACS buffer and analyzed on a FACS Canto screener (BD). Epigenetic K was determined by nonlinear curve fitting (modified OneSiteTotal, Graph Pad Prism). D value.

[0492] Anti-SIRPβ1 antibody selection

[0493] Further characterization revealed affinity-matured anti-SIRPβ1 antibody clones with improved affinity compared to their respective parent antibodies. Following initial screening of all affinity-matured antibody clones, clones of each parent antibody were selected for further analysis.

[0494] Antibody heavy and light chain variable domain sequences

[0495] The amino acid sequences encoding the variable domains of the light chain and heavy chain of the affinity maturation antibody were determined using standard techniques. The Kabat light chain HVR sequence of the affinity maturation antibody is listed in Table 8. The Kabat heavy chain HVR sequence of the antibody is listed in Table 9. The Kabat heavy chain framework (FR) sequence of the antibody is listed in Table 10. The Kabat light chain framework (FR) sequence of the antibody is listed in Table 11.

[0496] Table 8: Kabat light chain HVR sequence of affinity-matured anti-SIRPβ1 antibody

[0497]

[0498] (p) indicates the parental antibody sequence.

[0499] Table 9: Kabat heavy chain HVR sequence of affinity-matured anti-SIRPβ1 antibody

[0500]

[0501] (p) indicates the parental antibody sequence.

[0502] Table 10: Kabat light chain FR sequence of affinity-matured anti-SIRPβ1 antibody

[0503]

[0504]

[0505]

[0506]

[0507] (p) indicates the parental antibody sequence.

[0508] Table 11: Kabat heavy chain FR sequence of affinity-matured anti-SIRPβ1 antibody

[0509]

[0510]

[0511]

[0512] (p) indicates the parental antibody sequence.

[0513] Characterization of affinity maturation and anti-SIRPβ1 antibody binding

[0514] The final set of affinity-matured anti-SIRPβ1 antibodies was selected based on antigen-binding affinity. Cross-reactivity with human SIRPα was tested on antibodies that were positive for binding to human SIRPβ1. The biochemical characteristics of each antibody are listed in Table 12 below. In Table 12, “NB” indicates an antibody that does not bind to the antigen shown; “PF” indicates an antibody whose antigen-binding kinetics show a poor fit to the 1:1 binding model; and “NM” indicates unmeasurable.

[0515] Table 12: Biochemical charact...

Claims

1. An isolated antibody that binds to human SIRPβ1, wherein the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, the light chain variable region comprises HVR-L1, HVR-L2, and HVR-L3, (a) HVR-L1 is represented by the amino acid sequence of SEQ ID NO: 383, HVR-L2 is shown in the amino acid sequence of SEQ ID NO: 16, HVR-L3 is shown in the amino acid sequence of SEQ ID NO:31, HVR-H1 is shown in the amino acid sequence of SEQ ID NO:80, HVR-H2 is represented by the amino acid sequence of SEQ ID NO: 101, and HVR-H3 is shown in the amino acid sequence of SEQ ID NO:125; (b) HVR-L1 is represented by the amino acid sequence of SEQ ID NO: 383, HVR-L2 is shown in the amino acid sequence of SEQ ID NO: 16, HVR-L3 is shown in the amino acid sequence of SEQ ID NO:31, HVR-H1 is shown in the amino acid sequence of SEQ ID NO: 228, HVR-H2 is represented by the amino acid sequence of SEQ ID NO: 238, and HVR-H3 is shown in the amino acid sequence of SEQ ID NO:125; (c) HVR-L1 is represented by the amino acid sequence of SEQ ID NO: 383, HVR-L2 is shown in the amino acid sequence of SEQ ID NO: 16, HVR-L3 is shown in the amino acid sequence of SEQ ID NO:31, HVR-H1 is shown in the amino acid sequence of SEQ ID NO: 229, HVR-H2 is represented by the amino acid sequence of SEQ ID NO: 239, and HVR-H3 is shown in the amino acid sequence of SEQ ID NO:125; (d) HVR-L1 is represented by the amino acid sequence of SEQ ID NO: 383, HVR-L2 is shown in the amino acid sequence of SEQ ID NO: 16, HVR-L3 is shown in the amino acid sequence of SEQ ID NO:31, HVR-H1 is shown in the amino acid sequence of SEQ ID NO: 229, HVR-H2 is represented by the amino acid sequence of SEQ ID NO: 240, and HVR-H3 is as shown in the amino acid sequence of SEQ ID NO:125; or (e) HVR-L1 is represented by the amino acid sequence of SEQ ID NO: 383, HVR-L2 is shown in the amino acid sequence of SEQ ID NO: 16, HVR-L3 is shown in the amino acid sequence of SEQ ID NO:31, HVR-H1 is shown in the amino acid sequence of SEQ ID NO: 229, HVR-H2 is represented by the amino acid sequence of SEQ ID NO: 241, and HVR-H3 is shown in the amino acid sequence of SEQ ID NO:

125.

2. The antibody of claim 1, wherein the light chain variable region comprises one, two, three or four framework regions selected from VL FR1, VL FR2, VL FR3, and VL FR4, wherein: VL FR1 is as shown in the amino acid sequence of SEQ ID NO: 174, VL FR2 is as shown in the amino acid sequence of SEQ ID NO: 186, VL FR3 is as shown in the amino acid sequence of SEQ ID NO: 192, and / or VL FR4 is as shown in the amino acid sequence of SEQ ID NO:

200.

3. The antibody of claim 1, wherein the heavy chain variable region comprises one, two, three or four framework regions selected from VH FR1, VH FR2, VH FR3, and VH FR4, wherein: (a) when HVR-H1 is as shown in the amino acid sequence of SEQ ID NO:80, HVR-H2 is as shown in the amino acid sequence of SEQ ID NO:101, and HVR-H3 is as shown in the amino acid sequence of SEQ ID NO:125, VH FR1 is as shown in the amino acid sequence of SEQ ID NO:201, VH FR2 is as shown in the amino acid sequence of SEQ ID NO:210, VH FR3 is as shown in the amino acid sequence of SEQ ID NO:215, and / or VH FR4 is as shown in the amino acid sequence of SEQ ID NO:222; (b) when HVR-H1 is as shown in the amino acid sequence of SEQ ID NO:228, HVR-H2 is as shown in the amino acid sequence of SEQ ID NO:238, and HVR-H3 is as shown in the amino acid sequence of SEQ ID NO:125, VH FR1 is as shown in the amino acid sequence of SEQ ID NO:256, VH FR2 is as shown in the amino acid sequence of SEQ ID NO:210, VH FR3 is as shown in the amino acid sequence of SEQ ID NO:215, and / or VH FR4 is as shown in the amino acid sequence of SEQ ID NO:222; (c) when HVR-H1 is as shown in the amino acid sequence of SEQ ID NO:229, HVR-H2 is as shown in the amino acid sequence of SEQ ID NO:239, and HVR-H3 is as shown in the amino acid sequence of SEQ ID NO:125, VH FR1 is as shown in the amino acid sequence of SEQ ID NO:257, VH FR2 is as shown in the amino acid sequence of SEQ ID NO:210, VH FR3 is as shown in the amino acid sequence of SEQ ID NO:215, and / or VH FR4 is as shown in the amino acid sequence of SEQ ID NO:222; (d) when HVR-H1 is as shown in the amino acid sequence of SEQ ID NO:229, HVR-H2 is as shown in the amino acid sequence of SEQ ID NO:240, and HVR-H3 is as shown in the amino acid sequence of SEQ ID NO:125, VH FR1 is as shown in the amino acid sequence of SEQ ID NO:258, VH FR2 is as shown in the amino acid sequence of SEQ ID NO:210, VH FR3 is as shown in the amino acid sequence of SEQ ID NO:215, and / or VH FR4 is as shown in the amino acid sequence of SEQ ID NO:222; or (e) when HVR-H1 is as shown in the amino acid sequence of SEQ ID NO:229, HVR-H2 is as shown in the amino acid sequence of SEQ ID NO:241, and HVR-H3 is as shown in the amino acid sequence of SEQ ID NO:125, VH FR1 is shown in the amino acid sequence of SEQ ID NO:202, VH FR2 is shown in the amino acid sequence of SEQ ID NO:210, VH FR3 is shown in the amino acid sequence of SEQ ID NO:215, and / or VH FR4 is shown in the amino acid sequence of SEQ ID NO:

222.

4. The antibody of claim 1, Wherein the antibody comprises: (a) a heavy chain variable region that is at least 90% identical to the amino acid sequence of SEQ ID NO: 268; (b) a heavy chain variable region that is at least 90% identical to the amino acid sequence of SEQ ID NO: 366; (c) a heavy chain variable region that is at least 90% identical to the amino acid sequence of SEQ ID NO: 367; (d) a heavy chain variable region that is at least 90% identical to the amino acid sequence of SEQ ID NO: 368; or (e) a heavy chain variable region that is at least 90% identical to the amino acid sequence of SEQ ID NO: 369, and / or, Wherein the antibody comprises: A light chain variable region that is at least 90% identical to the amino acid sequence of SEQ ID NO:

267.

5. The antibody of claim 1, Wherein the antibody comprises: (a) a heavy chain variable region that is at least 95% identical to the amino acid sequence of SEQ ID NO: 268; (b) a heavy chain variable region that is at least 95% identical to the amino acid sequence of SEQ ID NO: 366; (c) a heavy chain variable region that is at least 95% identical to the amino acid sequence of SEQ ID NO: 367; (d) a heavy chain variable region that is at least 95% identical to the amino acid sequence of SEQ ID NO: 368; or (e) a heavy chain variable region that is at least 95% identical to the amino acid sequence of SEQ ID NO: 369, and / or, Wherein the antibody comprises: A light chain variable region that is at least 95% identical to the amino acid sequence of SEQ ID NO:

267.

6. The antibody of claim 1, Wherein the antibody comprises: (a) a heavy chain variable region that is at least 97% identical to the amino acid sequence of SEQ ID NO: 268; (b) a heavy chain variable region that is at least 97% identical to the amino acid sequence of SEQ ID NO: 366; (c) a heavy chain variable region that is at least 97% identical to the amino acid sequence of SEQ ID NO: 367; (d) a heavy chain variable region that is at least 97% identical to the amino acid sequence of SEQ ID NO: 368; or (e) a heavy chain variable region that is at least 97% identical to the amino acid sequence of SEQ ID NO: 369, and / or, Wherein the antibody comprises: A light chain variable region that is at least 97% identical to the amino acid sequence of SEQ ID NO:

267.

7. The antibody of claim 1, Wherein the antibody comprises: (a) a heavy chain variable region that is at least 99% identical to the amino acid sequence of SEQ ID NO: 268; (b) a heavy chain variable region that is at least 99% identical to the amino acid sequence of SEQ ID NO: 366; (c) a heavy chain variable region that is at least 99% identical to the amino acid sequence of SEQ ID NO: 367; (d) a heavy chain variable region that is at least 99% identical to the amino acid sequence of SEQ ID NO: 368; or (e) a heavy chain variable region that is at least 99% identical to the amino acid sequence of SEQ ID NO: 369, and / or, Wherein the antibody comprises: A light chain variable region that is at least 99% identical to the amino acid sequence of SEQ ID NO:

267.

8. The antibody of claim 1, Wherein the antibody comprises: (a) a heavy chain variable region as shown in the amino acid sequence of SEQ ID NO: 268; (b) a heavy chain variable region as shown in the amino acid sequence of SEQ ID NO: 366; (c) a heavy chain variable region as shown in the amino acid sequence of SEQ ID NO: 367; (d) the heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 368; or (e) the heavy chain variable region as shown in the amino acid sequence of SEQ ID NO:

369.

9. The antibody of claim 1, Wherein the antibody comprises: The light chain variable region is shown in the amino acid sequence of SEQ ID NO:

267.

10. The antibody of claim 1, Wherein the antibody comprises: (a) a heavy chain variable region as shown in the amino acid sequence of SEQ ID NO: 268 and a light chain variable region as shown in the amino acid sequence of SEQ ID NO: 267; (b) a heavy chain variable region as shown in the amino acid sequence of SEQ ID NO: 366 and a light chain variable region as shown in the amino acid sequence of SEQ ID NO: 267; (c) a heavy chain variable region as shown in the amino acid sequence of SEQ ID NO: 367 and a light chain variable region as shown in the amino acid sequence of SEQ ID NO: 267; (d) the heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 368 and the light chain variable region shown in the amino acid sequence of SEQ ID NO: 267; or (e) the heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 369 and the light chain variable region shown in the amino acid sequence of SEQ ID NO:

267.

11. The antibody of any one of claims 1-10, wherein the antibody is a monoclonal antibody.

12. The antibody of claim 11, wherein the antibody is of the IgG class, the IgM class, or the IgA class.

13. The antibody of claim 12, wherein the antibody is of the IgG class and has an IgG1, IgG2, or IgG4 isotype.

14. The antibody of claim 13, wherein the antibody has an IgG1 isotype.

15. The antibody of claim 14, wherein the antibody comprises an E430G substitution and a P331S substitution according to EU numbering.

16. The antibody of any one of claims 1-10, wherein the antibody is an antibody fragment.

17. The antibody of claim 16, wherein the fragment is a Fab, Fab', Fab'-SH, F(ab')2, Fv or scFv fragment.

18. The antibody of any one of claims 1-10, wherein the antibody binds to human SIRPβ1 isoform 1 but does not bind to human SIRPβ1 isoform 3.

19. The antibody of any one of claims 1-10, wherein the antibody binds to human SIRPβ1 isoform 1 but does not bind to human SIRPα.

20. The antibody of any one of claims 1-10, wherein the antibody binds to human SIRPβ1 isoform 1 but does not bind to human SIRPγ.

21. The antibody of any one of claims 1-10, wherein the antibody binds to human SIRPβ1 isoform 1 but does not bind to mouse SIRPβ1.

22. The antibody of any one of claims 1-10, wherein the antibody binds to human SIRPβ1 isoform 1 but does not bind to cynomolgus monkey SIRPβ1 isoform 1.

23. The antibody of any one of claims 1-10, wherein the antibody has at least one property selected from the group consisting of: a) stimulating SIRPβ1 activity on CD14-positive monocytes in vitro and / or in vivo; b) inducing or increasing the respiratory burst in immune cells in vitro and / or in vivo; c) inducing or increasing IL-8 expression in monocytes in vitro and / or in vivo; d) inducing or increasing TNFα expression in macrophages and / or dendritic cells in vitro and / or in vivo; e) inducing neutrophil-mediated phagocytosis in vitro and / or in vivo; f) enhancing neutrophil-mediated tumor cell clearance in vivo; g) increasing TREM2 expression on macrophages in vitro and / or in vivo; h) increasing the viability of macrophages in vitro and / or in vivo when alone and / or in combination with an agonistic anti-TREM2 antibody; and i) Improving the survival of dendritic cells in vitro and / or in vivo.

24. The antibody of any one of claims 1-10, wherein the antibody has at least two properties selected from the group consisting of: a) stimulating SIRPβ1 activity on CD14-positive monocytes in vitro and / or in vivo; b) inducing or increasing the respiratory burst in immune cells in vitro and / or in vivo; c) inducing or increasing IL-8 expression in monocytes in vitro and / or in vivo; d) inducing or increasing TNFα expression in macrophages and / or dendritic cells in vitro and / or in vivo; e) inducing neutrophil-mediated phagocytosis in vitro and / or in vivo; f) enhancing neutrophil-mediated tumor cell clearance in vivo; g) increasing TREM2 expression on macrophages in vitro and / or in vivo; h) increasing the viability of macrophages in vitro and / or in vivo when alone and / or in combination with an agonistic anti-TREM2 antibody; and i) Improving the survival of dendritic cells in vitro and / or in vivo.

25. The antibody of any one of claims 1-10, wherein the antibody has at least three properties selected from the following: a) stimulating SIRPβ1 activity on CD14-positive monocytes in vitro and / or in vivo; b) inducing or increasing the respiratory burst in immune cells in vitro and / or in vivo; c) inducing or increasing IL-8 expression in monocytes in vitro and / or in vivo; d) inducing or increasing TNFα expression in macrophages and / or dendritic cells in vitro and / or in vivo; e) inducing neutrophil-mediated phagocytosis in vitro and / or in vivo; f) enhancing neutrophil-mediated tumor cell clearance in vivo; g) increasing TREM2 expression on macrophages in vitro and / or in vivo; h) increasing the viability of macrophages in vitro and / or in vivo when alone and / or in combination with an agonistic anti-TREM2 antibody; and i) Improving the survival of dendritic cells in vitro and / or in vivo.

26. The antibody of any one of claims 1-10, wherein the antibody has at least four properties selected from the group consisting of: a) stimulating SIRPβ1 activity on CD14-positive monocytes in vitro and / or in vivo; b) inducing or increasing the respiratory burst in immune cells in vitro and / or in vivo; c) inducing or increasing IL-8 expression in monocytes in vitro and / or in vivo; d) inducing or increasing TNFα expression in macrophages and / or dendritic cells in vitro and / or in vivo; e) inducing neutrophil-mediated phagocytosis in vitro and / or in vivo; f) enhancing neutrophil-mediated tumor cell clearance in vivo; g) increasing TREM2 expression on macrophages in vitro and / or in vivo; h) increasing the viability of macrophages in vitro and / or in vivo when alone and / or in combination with an agonistic anti-TREM2 antibody; and i) Improving the survival of dendritic cells in vitro and / or in vivo.

27. The antibody of any one of claims 1-10, wherein the antibody does not bind to human SIRPα or human SIRPγ.

28. The antibody of any one of claims 1-10, wherein the antibody has an affinity (K affinity) for human SIRPβ1 isoform 1 of 0.1 nM to 50 nM. D ).

29. The antibody of claim 28, wherein the antibody has an affinity (K affinity) for human SIRPβ1 isoform 1 of 0.5 nM to 10 nM. D ).

30. The antibody of claim 29, wherein the antibody has an affinity (K affinity) for human SIRPβ1 isoform 1 of 0.5 nM to 5 nM. D ).

31. The antibody of claim 28, wherein affinity is determined using ForteBio System measured.

32. The antibody of any one of claims 1-10, wherein the antibody recognizes a first and a second antigen, wherein the first antigen is SIRPβ1 and the second antigen is: (a) Antigens that promote transport across the blood-brain barrier; (b) an antigen that promotes transport across the blood-brain barrier selected from the group consisting of transferrin receptor (TR), insulin receptor (HIR), insulin-like growth factor receptor (IGFR), low-density lipoprotein receptor-related proteins 1 and 2 (LPR-1 and 2), and diphtheria toxin receptor; (c) a pathogenic agent selected from the group consisting of a pathogenic peptide or protein or a pathogenic nucleic acid, wherein the pathogenic nucleic acid is an antisense GGCCCC (G2C4) repeat expansion RNA, and the pathogenic protein is selected from amyloid β, oligomeric amyloid β, amyloid β plaques, amyloid precursor protein or a fragment thereof, Tau, IAPP, α-synuclein, TDP-43, FUS protein, C9orf72 (chromosome 9 open reading frame 72), c9RAN protein, prion protein, PrPSc, huntingtin protein, calcitonin, superoxide dismutase, ataxin 1, ataxin 2, ataxin 3, ataxin 7, ataxin 8, ataxin the group consisting of dysregulated protein 10, Lewy bodies, atrial natriuretic factor, islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, β2-microglobulin, gelsolin, corneal epithelial protein, cystatin, immunoglobulin light chain AL, S-IBM protein, repeat-associated non-ATG (RAN) translation products, dipeptide repeat (DPR) peptides, glycine-alanine (GA) repeat peptides, glycine-proline (GP) repeat peptides, glycine-arginine (GR) repeat peptides, proline-alanine (PA) repeat peptides, ubiquitin, and proline-arginine (PR) repeat peptides; and (d) a ligand and / or protein expressed on an immune cell, wherein the ligand and / or protein is selected from the group consisting of CD40, OX40, ICOS, CD28, CD137 / 4-1BB, CD27, GITR, PD-L1, CTLA-4, PD-L2, PD-1, B7-H3, B7-H4, HVEM, BTLA, KIR, GAL9, TIM3, A2AR, LAG-3, and phosphatidylserine.

33. An isolated nucleic acid comprising a nucleic acid sequence encoding the antibody of any one of claims 1-32.

34. A vector comprising the nucleic acid of claim 33.

35. An isolated host cell comprising the vector of claim 34.

36. An isolated host cell expressing the antibody of any one of claims 1-32.

37. A method for producing an antibody that binds to human SIRPβ1, comprising culturing the cell of claim 36 to allow the antibody to be produced.

38. The method of claim 37, further comprising recovering antibodies produced by the cells.

39. A pharmaceutical composition comprising the antibody of any one of claims 1-32 and a pharmaceutically acceptable carrier.

40. Use of an antibody according to any one of claims 1 to 32 for the preparation of a medicament for use in a method for treating colon cancer, wherein the method comprises administering a therapeutically effective amount of an antibody according to any one of claims 1 to 32 to an individual in need thereof.

41. The use of claim 40, wherein the method further comprises administering a therapeutic agent that inhibits or agonizes PD1, PDL1, CD40, OX40, ICOS, CD28, CD137 / 4-1BB, CD27, GITR, CTLA4, PD-L2, B7-H3, B7-H4, HVEM, LIGHT, BTLA, CD30, TIGIT, VISTA, KIR, GAL9, TIM1, TIM3, TIM4, A2AR, LAG3, DR-5, CD2, CD5, CD39, or CD73.

42. The use of claim 40, wherein the method further comprises administering to the individual at least one antibody that specifically binds to an inhibitory checkpoint molecule, and / or one or more standard or investigational anti-cancer therapies.

43. The use of claim 42, wherein the method further comprises administering at least one antibody that specifically binds to an inhibitory checkpoint molecule in combination with the antibody.

44. The use of claim 42, wherein the at least one antibody that specifically binds to an inhibitory checkpoint molecule is selected from anti-PD-L1 antibodies, anti-CTLA4 antibodies, anti-PD-L2 antibodies, anti-PD-1 antibodies, anti-B7-H3 antibodies, anti-B7-H4 antibodies, and anti-HVEM antibodies, anti-B and T lymphocyte attenuator (BTLA) antibodies, anti-killer inhibitory receptor (KIR) antibodies, anti-GAL9 antibodies, anti-TIM-1 antibodies, anti-TIM3 antibodies, anti-TIM-4 antibodies, anti-A2AR antibodies, Anti-CD39 antibodies, anti-CD73 antibodies, anti-LAG-3 antibodies, anti-phosphatidylserine antibodies, anti-CD27 antibodies, anti-CD30 antibodies, anti-TNFα antibodies, anti-CD33 antibodies, anti-Siglec-5 antibodies, anti-Siglec-7 antibodies, anti-Siglec-9 antibodies, anti-Siglec-11 antibodies, antagonistic anti-TREM1 antibodies, antagonistic anti-TREM2 antibodies, anti-TIGIT antibodies, anti-VISTA antibodies, anti-CD2 antibodies, anti-CD5 antibodies, and any combination thereof.

45. The use of claim 42, wherein the one or more standard or investigational anti-cancer therapies are selected from radiation therapy, cytotoxic chemotherapy, targeted therapy, imatinib therapy, trastuzumab therapy, etanercept therapy, adoptive cell transfer (ACT) therapy, chimeric antigen receptor T cell transfer (CAR-T) therapy, vaccine therapy, and cytokine therapy.

46. ​​The use of claim 40, wherein the method further comprises administering to the individual at least one antibody that specifically binds to an inhibitory cytokine.

47. The use of claim 46, wherein the at least one antibody that specifically binds to an inhibitory cytokine is selected from an anti-CCL2 antibody, an anti-CSF-1 antibody, an anti-IL-2 antibody, and any combination thereof.

48. The use of claim 40, wherein the method further comprises administering to the individual at least one agonistic antibody that specifically binds to a stimulatory checkpoint protein.

49. The use of claim 48, wherein the at least one agonist antibody that specifically binds to a stimulatory checkpoint protein is selected from an agonist anti-CD40 antibody, an agonist anti-OX40 antibody, an agonist anti-ICOS antibody, an agonist anti-CD28 antibody, an agonist anti-TREM1 antibody, an agonist anti-TREM2 antibody, an agonist anti-CD137 / 4-1BB antibody, an agonist anti-CD27 antibody, an agonist anti-glucocorticoid-induced TNFR-related protein GITR antibody, an agonist anti-CD30 antibody, an agonist anti-BTLA antibody, an agonist anti-HVEM antibody, an agonist anti-CD2 antibody, an agonist anti-CD5 antibody, and any combination thereof.

50. The use of claim 40, wherein the method further comprises administering to the individual at least one stimulatory cytokine.

51. The use of claim 50, wherein the at least one stimulatory cytokine is selected from IFN-α4, IFN-β, IL-1β, TNF-α, IL-6, IL-8, CRP, IL-20 family members, LIF, IFN-γ, OSM, CNTF, GM-CSF, IL-11, IL-12, IL-15, IL-17, IL-18, IL-23, CXCL10, IL-33, MCP-1, MIP-1-β, and any combination thereof.

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