WO2024006820A1

WO2024006820A1 - Anti-siglec-10 antibodies and methods of use thereof

Info

Publication number: WO2024006820A1
Application number: PCT/US2023/069249
Authority: WO
Inventors: Bradford A. YOUNGBLOOD; Julia SCHANIN; John Leung; Thuy LUU; Wouter Korver
Original assignee: Allakos Inc.
Priority date: 2022-06-29
Filing date: 2023-06-28
Publication date: 2024-01-04

Abstract

The present disclosure provides anti-Siglec-10 antibodies and their use in treating or delaying progression of cancer, as well as compositions and kits comprising the anti-Siglec-10 antibodies.

Description

ANTI- SIGLEC- 10 ANTIBODIES AND METHODS OF USE THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Nos. 63/356,846, filed June 29, 2022, and 63/422,725, filed November 4, 2022, each of which is hereby incorporated by reference in its entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (701712001740SEQLIST.xml; Size: 82,280 bytes; and Date of Creation: June 16, 2023) are herein incorporated by reference in their entirety.

FIELD

[0003] This invention relates to anti-human Siglec-10 antibodies and methods of treating or preventing cancers or diseases mediated by cells expressing Siglec-10.

BACKGROUND

[0004] Siglecs (sialic acid-binding immunoglobulin-like lectins) are single-pass transmembrane cell surface proteins found predominantly on leukocytes and that are characterized by their specificity for sialic acids attached to cell-surface glycoconjugates. The Siglec family contains at least 15 members that are found in mammals (Pillai et al., Annu Rev Immunol., 2012, 30:357-392). These members include sialoadhesion (Siglec- 1), CD22 (Siglec- 2), CD33 (Siglec-3), myelin associated glycoprotein (Siglec-4), Siglec-5, OBBP1 (Siglec-6), AIRMI (Siglec-7), SAF-2 (Siglec-8), and CD329 (Siglec-9).

[0005] Siglec-10 (also known as SLG2, PRO940, and SIGLEC 10) is an inhibitory receptor that is expressed by immune cells spanning both the myeloid and lymphoid lineages. Siglec-10 binds to its ligands, CD24 and CD52, to induce inhibitory intracellular signaling cascades within immune cells. Tumor cells overexpress CD24 in a HIF la-dependent mechanism. The binding of tumor-expressed CD24 or CD52 to immune-expressed Siglec-10 is thought to lead to immune evasion. See, e.g., Barkal, A. A. et al. (2019) Nature 572:392-396; Xiao, N. et al. (2021) Exp Hematol Oncology 10:36; Bandala- Sanchez, E. et al. (2013) Nat. Immunol. 14:741-748;

Whitney, G. et al. (2001) Eur. J. Biochem 268:6083-6096; Chen, G.Y. et al. (2009) Science 323: 1722-1725; Bandala-Sanchez, E. et al. (2018) Proc. Natl. Acad. Sci. 115:7783-7788; and International Publication No. WO2017/085166.

[0006] There remains a need for antibodies that bind to human Siglec-10 with high affinity and have other properties of interest (e.g., ligand blocking/non-blocking, induction of Siglec-10 internalization, anti-tumor activity, etc.).

[0007] All references cited herein, including patent applications, patent publications, and scientific literature, are herein incorporated by reference in their entirety, as if each individual reference were specifically and individually indicated to be incorporated by reference.

BRIEF SUMMARY

[0008] To meet this and other needs, the present disclosure provides, inter alia, antibodies that bind to human Siglec-10, as well as compositions, uses, and methods related thereto.

[0009] Accordingly, certain aspects of the present disclosure relate to antibodies that bind to human Siglec-10. In some embodiments, the antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region. In some embodiments, the antibody is a humanized antibody.

[0010] In some embodiments, the antibody comprises a VH region that comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:79, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:80, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:81; and/or a VL region that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:82, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:83, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 84. In some embodiments, the antibody comprises a VH region that comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:79, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:80, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:81; and a VL region that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 82, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:83, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:84. In some embodiments, the antibody comprises a VH region that comprises the amino acid sequence of SEQ ID NO:85 and/or a VL region that comprises the amino acid sequence of SEQ ID NO:86. In some embodiments, the antibody comprises a VH region that comprises the amino acid sequence of SEQ ID NO:85 and a VL region that comprises the amino acid sequence of SEQ ID NO:86. In some embodiments, the antibody comprises a heavy chain that comprises the amino acid sequence of SEQ ID NO:87 or 88 and/or a light chain that comprises the amino acid sequence of SEQ ID NO:89. In some embodiments, the antibody comprises a heavy chain that comprises the amino acid sequence of SEQ ID NO:87 or 88 and a light chain that comprises the amino acid sequence of SEQ ID NO:89.

[0011] In some embodiments, the antibody comprises a VH region that comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 19, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:20, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:21; and/or a VL region that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:22, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:23, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:24. In some embodiments, the antibody comprises a VH region that comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:25, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:26, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:27; and/or a VL region that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:28, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:30. In some embodiments, the antibody comprises a VH region that comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:31, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:32, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:33; and/or a VL region that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:34, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:35, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:36. In some embodiments, the antibody comprises a VH region that comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:37, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:38, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:39; and/or a VL region that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:40, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:41, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:42. In some embodiments, the antibody comprises a VH region that comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:43, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:44, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:45; and/or a VL region that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:46, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:47, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:48. In some embodiments, the antibody comprises a VH region that comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:49, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:50, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:51; and/or a VL region that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:52, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:53, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:54. In some embodiments, the antibody comprises a VH region that comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:55, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 56, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:57; and/or a VL region that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:58, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:59, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:60. In some embodiments, the antibody comprises a VH region that comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:61, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:62, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:63; and/or a VL region that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:64, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:65, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:66. In some embodiments, the antibody comprises a VH region that comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:67, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:68, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:69; and/or a VL region that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:70, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:71, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:72.

[0012] In some embodiments, the antibody competes for binding to human Siglec-10 with a reference antibody that comprises a VH region comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 19, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:20, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:21 and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:22, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:23, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:24. In some embodiments, the antibody competes for binding to human Siglec-10 with a reference antibody that comprises a VH region comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO:25, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:26, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:27 and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:28, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:30. In some embodiments, the antibody competes for binding to human Siglec-10 with a reference antibody that comprises a VH region comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO:31, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:32, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:33 and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:34, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:35, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:36. In some embodiments, the antibody competes for binding to human Siglec-10 with a reference antibody that comprises a VH region comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO:37, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:38, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:39 and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:40, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:41, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:42. In some embodiments, the antibody competes for binding to human Siglec-10 with a reference antibody that comprises a VH region comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO:43, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:44, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:45 and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:46, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:47, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:48. In some embodiments, the antibody competes for binding to human Siglec-10 with a reference antibody that comprises a VH region comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO:49, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 50, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:51 and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:52, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:53, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 54. In some embodiments, the antibody competes for binding to human Siglec-10 with a reference antibody that comprises a VH region comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO:55, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:56, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:57 and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:58, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:59, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:60. In some embodiments, the antibody competes for binding to human Siglec-10 with a reference antibody that comprises a VH region comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO:61, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:62, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:63 and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:64, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:65, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:66. In some embodiments, the antibody. In some embodiments, the antibody competes for binding to human Siglec-10 with a reference antibody that comprises a VH region comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO:67, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:68, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:69 and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:70, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:71, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:72. In some embodiments, the antibody competes for binding to human Siglec-10 with a reference antibody that comprises a VH region comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO:79, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:80, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:81 and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:82, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:83, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:84.

[0013] In some embodiments, the antibody comprises one, two, or three HVR sequences from the VH region sequence of SEQ ID NO: 1 and/or one, two, or three HVR sequences from the VL region sequence of SEQ ID NO:2. In some embodiments, the antibody comprises one, two, or three HVR sequences from the VH region sequence of SEQ ID NO:3 and/or one, two, or three HVR sequences from the VL region sequence of SEQ ID NON. In some embodiments, the antibody comprises one, two, or three HVR sequences from the VH region sequence of SEQ ID NO:5 and/or one, two, or three HVR sequences from the VL region sequence of SEQ ID NO:6. In some embodiments, the antibody comprises one, two, or three HVR sequences from the VH region sequence of SEQ ID NO:7 and/or one, two, or three HVR sequences from the VL region sequence of SEQ ID NO:8. In some embodiments, the antibody comprises one, two, or three HVR sequences from the VH region sequence of SEQ ID NOV and/or one, two, or three HVR sequences from the VL region sequence of SEQ ID NO: 10. In some embodiments, the antibody comprises one, two, or three HVR sequences from the VH region sequence of SEQ ID NO: 11 and/or one, two, or three HVR sequences from the VL region sequence of SEQ ID NO: 12. In some embodiments, the antibody comprises one, two, or three HVR sequences from the VH region sequence of SEQ ID NO: 13 and/or one, two, or three HVR sequences from the VL region sequence of SEQ ID NO: 14. In some embodiments, the antibody comprises one, two, or three HVR sequences from the VH region sequence of SEQ ID NO: 15 and/or one, two, or three HVR sequences from the VL region sequence of SEQ ID NO: 16. In some embodiments, the antibody comprises one, two, or three HVR sequences from the VH region sequence of SEQ ID NO: 17 and/or one, two, or three HVR sequences from the VL region sequence of SEQ ID NO: 18. In some embodiments, the antibody comprises one, two, or three HVR sequences from the VH region sequence of SEQ ID NO:85 and/or one, two, or three HVR sequences from the VL region sequence of SEQ ID NO:86.

[0014] In some embodiments, an anti-Siglec-10 antibody of the present disclosure comprises a VH region comprising 1, 2, or all 3 CDR or HVR sequences present in the VH region sequence of AK01 as described herein (see, e.g., Tables 2 and 3) and/or a VL region comprising 1, 2, or all 3 CDR or HVR sequences present in the VL region sequence of AK01 as described herein (see, e.g., Tables 2 and 3). In some embodiments, an anti-Siglec-10 antibody of the present disclosure comprises a VH region comprising 1, 2, or all 3 CDR or HVR sequences present in the VH region sequence of AK02 as described herein (see, e.g., Tables 2 and 3) and/or a VL region comprising 1, 2, or all 3 CDR or HVR sequences present in the VL region sequence of AK02 as described herein (see, e.g., Tables 2 and 3). In some embodiments, an anti-Siglec-10 antibody of the present disclosure comprises a VH region comprising 1, 2, or all 3 CDR or HVR sequences present in the VH region sequence of AK03 as described herein (see, e.g., Tables 2 and 3) and/or a VL region comprising 1, 2, or all 3 CDR or HVR sequences present in the VL region sequence of AK03 as described herein (see, e.g., Tables 2 and 3). In some embodiments, an anti-Siglec-10 antibody of the present disclosure comprises a VH region comprising 1, 2, or all 3 CDR or HVR sequences present in the VH region sequence of AK04 as described herein (see, e.g., Tables 2 and 3) and/or a VL region comprising 1, 2, or all 3 CDR or HVR sequences present in the VL region sequence of AK04 as described herein (see, e.g., Tables 2 and 3). In some embodiments, an anti-Siglec-10 antibody of the present disclosure comprises a VH region comprising 1, 2, or all 3 CDR or HVR sequences present in the VH region sequence of AK05 as described herein (see, e.g., Tables 2 and 3) and/or a VL region comprising 1, 2, or all 3 CDR or HVR sequences present in the VL region sequence of AK05 as described herein (see, e.g., Tables 2 and 3). In some embodiments, an anti-Siglec-10 antibody of the present disclosure comprises a VH region comprising 1, 2, or all 3 CDR or HVR sequences present in the VH region sequence of AK06 as described herein (see, e.g., Tables 2 and 3) and/or a VL region comprising 1, 2, or all 3 CDR or HVR sequences present in the VL region sequence of AK06 as described herein (see, e.g., Tables 2 and 3). In some embodiments, an anti-Siglec-10 antibody of the present disclosure comprises a VH region comprising 1, 2, or all 3 CDR or HVR sequences present in the VH region sequence of AK07 as described herein (see, e.g., Tables 2 and 3) and/or a VL region comprising 1, 2, or all 3 CDR or HVR sequences present in the VL region sequence of AK07 as described herein (see, e.g., Tables 2 and 3). In some embodiments, an anti-Siglec- 10 antibody of the present disclosure comprises a VH region comprising 1, 2, or all 3 CDR or HVR sequences present in the VH region sequence of AK08 as described herein (see, e.g., Tables 2 and 3) and/or a VL region comprising 1, 2, or all 3 CDR or HVR sequences present in the VL region sequence of AK08 as described herein (see, e.g., Tables 2 and 3). In some embodiments, an anti-Siglec-10 antibody of the present disclosure comprises a VH region comprising 1, 2, or all 3 CDR or HVR sequences present in the VH region sequence of AK09 as described herein (see, e.g., Tables 2 and 3) and/or a VL region comprising 1, 2, or all 3 CDR or HVR sequences present in the VL region sequence of AK09 as described herein (see, e.g., Tables 2 and 3). In some embodiments according to any of the embodiments described herein, the antibody is a humanized antibody. In some embodiments, an anti-Siglec-10 antibody of the present disclosure comprises a VH region comprising 1, 2, or all 3 CDR or HVR sequences present in the VH region sequence of AKIO as described herein (see, e.g., Tables 2 and 3) and/or a VL region comprising 1, 2, or all 3 CDR or HVR sequences present in the VL region sequence of AKIO as described herein (see, e.g., Tables 2 and 3).

[0015] In some embodiments, the antibody binds to Domain 1 of human Siglec-10. In some embodiments, binding of the antibody to human Siglec-10 blocks interaction between human Siglec-10 and human CD24. In some embodiments, binding of the antibody to human Siglec-10 does not block interaction between human Siglec-10 and human CD24. In some embodiments, binding of the antibody to human Siglec-10 blocks interaction between human Siglec-10 and human CD52. In some embodiments, binding of the antibody to human Siglec-10 does not block interaction between human Siglec-10 and human CD52. In some embodiments, the antibody binds to the extracellular domain of human Siglec-10 when expressed on a surface of a human myeloid cell. In some embodiments, the human myeloid cell is a human macrophage, dendritic cell, or monocyte. In some embodiments, binding of the antibody to the extracellular domain of human Siglec-10 when expressed on the surface of a human myeloid cell induces internalization of Siglec-10.

[0016] In some embodiments according to any of the embodiments described herein, the antibody comprises an Fc region. In some embodiments, the Fc region is a human Fc region. In some embodiments, the Fc region is a human IgGl, human IgG2, or human IgG4 Fc region. In some embodiments, the Fc region is a human IgG4 Fc region comprising the amino acid substitution S228P, numbering according to EU index. In some embodiments, the Fc region comprises one or more mutation(s) that reduce effector function. In some embodiments, the antibody comprises a human IgGl Fc region with a substitution or deletion at one or more of the following position(s), numbering based on EU index: (a) L234 and/or L235; (b) A327, A330, and/or P331; (c) E233, L234, L235, and/or G236; (d) E233, L234, and/or L235; (e) E233, L234, L235, G236, A327, A330, and/or P331; (f) E233, L234, L235, A327, A330, and/or P331; (g) N297; (h) L242, N297, and/or K334; (i) A287, N297, and/or L306; (j) R292, N297, and/or V302; (k) N297, V323, and/or 1332; (1) V259, N297, and/or L306; (m) L234, L235, K322, M252, S254, and/or T256; (n) L234, L235, and/or P329; or (o) L234, L235, and/or K322. In some embodiments, the antibody comprises a human IgGl Fc region with one or more of the following mutation(s), numbering based on EU index: (a) L234A and/or L235A; (b) A327G, A330S, and/or P33 IS; (c) E233P, L234V, L235A, and/or G236del; (d) E233P, L234V, and/or L235A; (e) E233P, L234V, L235A, G236del, A327G, A330S, and/or P33 IS; (f) E233P, L234V, L235A, A327G, A330S, and/or P33 IS; (g) N297A; (h) N297G; (i) N297Q; (j) L242C, N297C, and/or K334C; (k) A287C, N297G, and/or L306C; (1) R292C, N297G, and/or V302C; (m) N297G, V323C, and/or I332C; (n) V259C, N297G, and/or L306C; (o) L234F, L235Q, K322Q, M252Y, S254T, and/or T256E; (p) L234A, L235A, and/or P329G; or (q) L234A, L235Q, and/or K322Q. In some embodiments, the antibody comprises a human IgGl Fc region with L234A, L235Q, and K322Q substitutions, numbering based on EU index. In some embodiments, the antibody comprises a human IgG2 Fc region with a substitution or deletion at one or more of the following position(s), numbering based on EU index: (a) A330 and/or P331; (b) V234, G237, P238, H268, V309, A330, and/or P331; or (c) V234, G237, H268, V309, A330, P331, C232, C233, S267, L328, M252, S254, and/or T256. In some embodiments, the antibody comprises a human IgG2 Fc region with one or more of the following mutation(s), numbering based on EU index: (a) A330S and/or P33 IS; (b) V234A, G237A, P238S, H268A, V309L, A330S, and/or P331S; or (c) V234A, G237A, H268Q, V309L, A330S, P331S, C232S, C233S, S267E, L328F, M252Y, S254T, and/or T256E. In some embodiments, the antibody comprises a human IgG4 Fc region with a substitution or deletion at one or more of the following position(s), numbering based on EU index: (a) E233, F234, L235, and/or G236; (b) E233, F234, and/or L235; or (c) S228 and/or L235. In some embodiments, the antibody comprises a human IgG4 Fc region with one or more of the following mutation(s), numbering based on EU index: (a) E233P, F234V, L235A, and/or G236del; (b) E233P, F234V, and/or L235A; (c) S228P and/or L235E; or (d) S228P and/or L235A. In some embodiments, the Fc region comprises one or more mutation(s) that enhance effector function. In some embodiments, the antibody comprises a human IgGl Fc region with a substitution or deletion at one or more of the following position(s), numbering based on EU index: (a) F243, R292, Y300, V305, and/or P396; (b) S239 and/or 1332; (c) S239, 1332, and/or A330; (d) S298, E333, and/or K334; (e) G236, S239, and/or 1332; (f) K326 and/or E333; (g) S267, H268, and/or S324; or (h) E345, E430, and/or S440. In some embodiments, the antibody comprises a human IgGl Fc region with one or more of the following mutation(s), numbering based on EU index: (a) F243L, R292P, Y300L, V305I, and/or P396L; (b) S239D and/or I332E; (c) S239D, I332E, and/or A330L; (d) S298A, E333A, and/or K334A; (e) G236A, S239D, and/or I332E; (f) K326W and/or E333S; (g) S267E, H268F, and/or S324T; or (h) E345R, E430G, and/or S440Y.

[0017] In some embodiments according to any of the embodiments described herein, at least one or two of the heavy chains of the antibody is non-fucosylated. In some embodiments, the antibody is produced in a cell line having an alpha- 1,6-fucosyltransferase (Fut8) knockout. In some embodiments, the antibody is produced in a cell line overexpressing 1,4-N- acetylglucosaminyltransferase III (GnT-III). In some embodiments, the antibody is produced in a cell line overexpressing 01,4-N-acetylglucosaminyltransf erase III (GnT-III) that additionally overexpresses Golgi p- mannosidase II (Manll).

[0018] In some embodiments according to any of the embodiments described herein, the antibody is an antibody fragment selected from the group consisting of a Fab, F(ab’)2, Fab’-SH, Fv, and scFv fragment. In some embodiments, the antibody comprises a light chain constant (CL) domain. In some embodiments, the CL domain is a human kappa CL domain. In some embodiments, the light chain comprises the amino acid sequence of SEQ ID NO:78. In some embodiments, the antibody comprises a heavy chain that comprises the amino acid sequence of SEQ ID NO:87 or 88 and/or a light chain that comprises the amino acid sequence of SEQ ID NO:89. In some embodiments, the antibody comprises a heavy chain that comprises the amino acid sequence of SEQ ID NO:87 or 88 and a light chain that comprises the amino acid sequence of SEQ ID NO:89. In some embodiments, the antibody comprises a heavy chain that comprises the amino acid sequence of SEQ ID NO:87 and a light chain that comprises the amino acid sequence of SEQ ID NO:89. In some embodiments, the antibody comprises a heavy chain that comprises the amino acid sequence of SEQ ID NO:88 and a light chain that comprises the amino acid sequence of SEQ ID NO:89. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a multispecific antibody. In some embodiments, the antibody is a bispecific antibody. In some embodiments, the antibody is conjugated to an agent. In some embodiments, the agent is a cytotoxic agent or label.

[0019] Other aspects of the present disclosure relate to compositions comprising the antibody according to any one of the above embodiments. In some embodiments, the antibody comprises a Fc region and N-glycoside-linked carbohydrate chains linked to the Fc region, wherein less than 50% of the N-glycoside-linked carbohydrate chains contain a fucose residue. In some embodiments, substantially none of the N-glycoside-linked carbohydrate chains contain a fucose residue.

[0020] Other aspects of the present disclosure relate to polynucleotides encoding the antibody according to any one of the above embodiments. Other aspects of the present disclosure relate to vectors comprising one or more polynucleotides encoding the antibody according to any one of the above embodiments. Other aspects of the present disclosure relate to host cells comprising the polynucleotide(s) and/or vector(s) according to any one of the above embodiments. In some embodiments, the host cell is a mammalian or insect cell. In some embodiments, the host cell is Chinese hamster ovary (CHO) cell. In some embodiments, the host cell comprises a Fut8 knockout. In some embodiments, the host cell overexpresses GnT-III. In some embodiments, the host cell overexpresses GnT-III and additionally overexpresses Manll. Other aspects of the present disclosure relate to methods of producing an antibody, comprising culturing the host cell according to any one of the above embodiments under a condition that produces the antibody. In some embodiments, the methods further comprise recovering the antibody produced by the host cell. Other aspects of the present disclosure relate to antibodies produced by the method according to any one of the above embodiments. Other aspects of the present disclosure relate to pharmaceutical compositions comprising the antibody according to any one of the above embodiments and a pharmaceutically acceptable carrier. Other aspects of the present disclosure relate to kits or articles of manufacture comprising a medicament comprising a composition or antibody according to any one of the above embodiments. In some embodiments, the kits or articles of manufacture further comprise a package insert comprising instructions for administration of the medicament in an individual in need thereof, e.g., according to any one of the methods disclosed herein.

[0021] Other aspects of the present disclosure relate to methods of treating or delaying progression of cancer in an individual (e.g., in need thereof), comprising administering to the individual an effective amount of the antibody or composition according to any one of the above embodiments. Other aspects of the present disclosure relate to uses of the antibody or composition according to any one of the above embodiments in the manufacture of a medicament, e.g., for treating or delaying progression of cancer in an individual (e.g., in need thereof). Other aspects of the present disclosure relate to the antibody or composition according to any one of the above embodiments for use as a medicament. Other aspects of the present disclosure relate to the antibody or composition according to any one of the above embodiments for use in treating or delaying progression of cancer in an individual (e.g., in need thereof). In some embodiments, the individual has or has been diagnosed with cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a blood, liquid, or hematological cancer. In some embodiments, the cancer is selected from the group consisting of gastric cancer, breast cancer, lung cancer, ovarian cancer, cervical cancer, prostate cancer, bladder cancer, colorectal cancer, pancreatic cancer, liver cancer, renal cancer, thyroid cancer, brain cancer, head and neck cancer, leukemia, lymphoma, myeloma, carcinoma, melanoma, leiomyoma, leiomyosarcoma, glioma, glioblastoma, and esophageal cancer. In some embodiments, the antibody or composition is administered to the individual in combination with an additional anti-cancer agent.

[0022] Other aspects of the present disclosure relate to methods of treating or delaying progression of an autoimmune disease or disorder in an individual (e.g., in need thereof), comprising administering to the individual an effective amount of the antibody or composition according to any one of the above embodiments. Other aspects of the present disclosure relate to uses of the antibody or composition according to any one of the above embodiments in the manufacture of a medicament, e.g., for treating or delaying progression of an autoimmune disease or disorder in an individual (e.g., in need thereof). Other aspects of the present disclosure relate to the antibody or composition according to any one of the above embodiments for use as a medicament. Other aspects of the present disclosure relate to the antibody or composition according to any one of the above embodiments for use in treating or delaying progression of an autoimmune disease or disorder in an individual (e.g., in need thereof). In some embodiments, the individual has or has been diagnosed with an autoimmune disease or disorder. In some embodiments, the autoimmune disease or disorder is inflammatory bowel disease (IBD), rheumatoid arthritis (RA), multiple sclerosis (MS), psoriasis, or systemic lupus erythematosus.

[0023] Other aspects of the present disclosure relate to methods of depleting cells expressing Siglec-10 in an individual in need thereof, comprising administering to the individual an effective amount of the antibody or composition according to any one of the above embodiments. Other aspects of the present disclosure relate to uses of the antibody or composition according to any one of the above embodiments in the manufacture of a medicament, e.g., for depleting cells expressing Siglec-10 in an individual in need thereof. Other aspects of the present disclosure relate to the antibody or composition according to any one of the above embodiments for use as a medicament. Other aspects of the present disclosure relate to the antibody or composition according to any one of the above embodiments for use in depleting cells expressing Siglec-10 in an individual in need thereof. In some embodiments, the individual has or has been diagnosed with an autoimmune disease or disorder. In some embodiments, the autoimmune disease or disorder is inflammatory bowel disease (IBD), rheumatoid arthritis (RA), multiple sclerosis (MS), psoriasis, or systemic lupus erythematosus. [0024] It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present disclosure. These and other aspects of the present disclosure will become apparent to one of skill in the art. These and other embodiments of the present disclosure are further described by the detailed description that follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG. 1A shows an overview of the mechanism by which an anti-Siglec-10 antibody is thought to increase antitumor immunity by rescuing the phagocytic and cytotoxic T-cell priming activities of intratumoral myeloid cells (e.g., tumor-associated macrophages).

[0026] FIGS. IB & 1C show that Siglec-10 expression is upregulated in multiple human solid cancers. FIG. IB shows SIGLEC10 mRNA expression levels in human cancers from the TCGA (The Cancer Genome Atlas; right for each tissue) and normal matched-tissue from the GTEX (Genotype Tissue Expression Project; left for each tissue). Boxes show the median and whiskers indicate min to max expression, * <0.01, **** <0.0001, unpaired, Mann-Whitney U test. BLCA, bladder urothelial carcinoma; KICH, kidney chromophobe; KIRC, kidney renal cell carcinoma; KIRP, kidney renal papillary carcinoma; OV, ovarian adenocarcinoma; PAAD, pancreatic adenocarcinoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma. FIG. 1C shows SIGLEC10 expression data from colon adenocarcinoma cohort (TCGA) compared to tissue-matched healthy individuals (TCGA and GTEX cohorts). Upper box shows SIGLEC10 high; lower box shows SIGLEC10 low. , **** <0.0001, unpaired, Mann-Whitney U test.

[0027] FIG. ID shows that individuals with SIGLEC10 high tumor expression (from colon adenocarcinoma cohort in FIG. 1C) had lower survival probability than those with SIGLEC10 low tumor expression (44% 5-year survival vs. 68%, respectively). Tumor samples were separated into two cohorts based on expression level of SIGLEC10 with cut-off of 2.75 FPKM. Kaplan-Meier survival curves were plotted for both cohorts and 5-year survival were determined.

[0028] FIG. 2 shows that Siglec-10 is expressed on macrophages in normal tissues and upregulated on TAMs in tumor tissues, and expressed on dendritic cells (cDCl and cDC2) in normal and tumor tissues. MFI, median fluorescence intensity. * p<0.05, one-way anova test. For all tissues, expression in normal tissues is shown on left, and expression in tumor tissues is shown on right.

[0029] FIG. 3 shows that the indicated anti-Siglec-10 monoclonal antibodies (mAbs) induce Siglec-10 internalization on myeloid cells.

[0030] FIG. 4 shows that the majority of anti-Siglec-10 mAbs tested, but not all anti-Siglec-10 mAbs, block the binding of biotinylated Siglec-10 extracellular domain (ECD) to its ligand on MCF-7 cancer cells. [0031] FIGS. 5 shows an overview of the study design for testing the anti-tumor effect of anti- Siglec-10 mAbs in a pancreatic syngeneic mouse tumor model (Panc02).

[0032] FIG. 6A & 6B show that the treatment of Panc02-tumor bearing mice with anti-Siglec- 10 mAb leads to a reduction in tumor burden in vivo. Shown are effects on tumor size (mm³; FIG. 6A) and weight (g; FIG. 6B), as compared to isotype control mAb (ISO). Data are plotted as individual mice (n=l l/group). * p = <0.05; ** p = <0.01.

[0033] FIG. 7A summarizes the design of a pre-clinical study examining the effects of anti- Siglec-10 antibody treatment in a colorectal syngeneic mouse tumor model (MC38 cells) using mice expressing human Siglec-10.

[0034] FIG. 7B shows tumor growth in mice expressing human Siglec-10 implanted with MC38 tumor cells treated with anti-Siglec-10 antibody or isotype control. Treatment with anti- Siglec-10 mAb significantly reduced tumor size compared to isotype control treated mice. Data are plotted as individual mice (n=l 1/group). **** p = <0.0001.

[0035] FIGS. 7C & 7D show numbers of tumor-associated macrophages (TAMs; FIG. 7C) and levels of MHC class II expression on TAMs (FIG.7D) in mice expressing human Siglec-10 implanted with MC38 tumor cells treated with anti-Siglec-10 antibody or isotype control. Treatment with anti-Siglec-10 mAb significantly increased the percentage of TAMs compared to isotype control treated mice and led to upregulation of MHC class II on TAMs from anti-Siglec- 10-treated mice compared to isotype control. Data are plotted as individual mice (n=l 1/group). ** p = <0.01; *** p = <0.001. In each graph, results from isotype control are shown on left, and results from anti-Siglec-10 antibody treatment are shown on right.

[0036] FIGS. 7E & 7F show numbers of CD 103+ dendritic cells (DCs; FIG. 7E) and levels of MHC class II expression on CD 103+ DCs (FIG.7F) in mice expressing human Siglec-10 implanted with MC38 tumor cells treated with anti-Siglec-10 antibody or isotype control. Treatment with anti-Siglec-10 mAb significantly increased the percentage of CD 103+ DCs compared to isotype control treated mice and led to upregulation of MHC class II on CD 103+ DCs from anti-Siglec-10-treated mice compared to isotype control. Data are plotted as individual mice (n=l 1/group). ** p = <0.01. In each graph, results from isotype control are shown on left, and results from anti-Siglec-10 antibody treatment are shown on right.

[0037] FIGS. 7G-7I show numbers of CD8+ T cells (as a percentage of CD45+ cells; FIG. 7G), levels of PD-1 expression on CD8+ T cells (FIG. 7H), and numbers of CD4+ T cells (as a percentage of CD45+ cells; FIG. 71) in mice expressing human Siglec-10 implanted with MC38 tumor cells treated with anti-Siglec-10 antibody or isotype control. Treatment with anti-Siglec- 10 mAh significantly increased the percentage of both CD8+ and CD4+ T cells in the tumor compared to isotype control treated mice and led to upregulation of PD-1 expression on CD8+ T cells from anti-Siglec-10-treated mice compared to isotype control. Data are plotted as individual mice (n=l 1/group). ** p = <0.01; *** p = <0.001. In each graph, results from isotype control are shown on left, and results from anti-Siglec-10 antibody treatment are shown on right. [0038] FIG. 7J shows a volcano plot of differentially expressed genes from MC38 tumors from mice treated with anti-Siglec-10 mAb vs. MC38 tumors from mice treated with isotype control, as assessed by RNA-sequencing analysis. Siglec-10 mAb treatment induced upregulation of a significant number of genes associated with type-1 inflammation, T cell activation, antigen presentation and macrophages, as indicated.

[0039] FIG. 7K shows tumor growth in mice treated with isotype control or anti-Siglec-10 mAb with inactive Fc (mlgGl DA).

[0040] FIG. 8A summarizes the design of a pre-clinical study examining the effects of anti- Siglec-10 antibody treatment in an experimental TLR-mediated lung model using mice expressing human Siglec-10.

[0041] FIG. 8B shows Siglec-10 expression levels on CD 103+ DCs (left) and Ly6C^h'^8h MHC+ inflammatory monocytes (right) in lung tissue from mice intranasally challenged with poly (EC) and dosed with a Siglec-10 mAb or isotype control, compared to PBS challenged mice. In both plots, left bars indicate PBS challenged mice, middle bars indicate mice administered poly EC and isotype control antibody, and right bars indicate mice administered poly EC and anti-Siglec- 10 antibody. Data are plotted as means +/- SEM for individual mice (n=6-7/group). ** p = <0.01; *** p = <0.001; **** p = <0.0001 as determined by one-way ANOVA with Tukey’s multiple comparisons.

[0042] FIG. 8C shows levels of cytokines and chemokines in serum of mice challenged intranasally with poly (EC) or PBS control. In all plots, left bars indicate PBS challenged mice, middle bars indicate mice administered poly EC and isotype control antibody, and right bars indicate mice administered poly EC and anti-Siglec-10 antibody. Data are plotted as means +/- SEM for individual mice (n=6-7/group). ** p = <0.01; *** p = <0.001; **** p = <0.0001 as determined by one-way ANOVA with Tukey’s multiple comparisons.

[0043] FIG. 9 shows the expression of Siglec-10 ligands in multiple human cancer cell lines but not normal human tissue. Human cancer cell lines were cultured and incubated with fluorophore conjugated Siglec-10 extracellular domain (ECD). Siglec-10 ligand levels were assessed by flow cytometry.

[0044] FIG. 10 shows that anti-Siglec-10 mAh treatment blocks interaction between Siglec-10 ECD and Siglec-10 ligands expressed by multiple human cancer cell lines. Cancer cells OVCAR and SK-BR-3 were pretreated with indicated antibodies, followed by incubation with Siglec-10 ECD (hlgGl-FC biotin) and streptavidin-APC detection. Antibody-dependent blockade of the Siglec-10 ligand interaction corresponds with the reduction of geometric mean fluorescence intensity (gMFI). Shown are dose-response curves of Siglec-10 ECD (extracellular domain) binding to OVCAR ovarian cancer (left) or SK-BR-3 breast cancer (right) cell lines in presence of AK01 (circles) or isotype control antibody (squares) ranging from 0.15 -20 pg/ml. [0045] FIGS. 11A-11C show stimulation of antibody-dependent cellular phagocytosis (ADCP) by macrophages in the presence of an opsonizing antibody using anti-Siglec-10 mAb treatment. FIG. 11A shows a schematic diagram of macrophage-mediated phagocytosis of tumor cells by tumor-opsonizing antibodies (e.g., anti-HER2 antibody trastuzumab) stimulated by anti-Siglec-10 mAb. FIG. 11B shows phagocytosis of breast cancer cells (SK-BR-3) by human macrophages following treatment with trastuzumab, in presence of AKIO antibodies or isotype control. SK-BR-3 cells were stained with CFSE cell tracer and treated with anti-HER2 trastuzumab (0.1 pg/ml). Next, CFSE-labeled tumor cells were incubated with human polarized macrophages previously incubated with 10 pg/mL AKIO Fc inert (middle), AKIO active Fc (right), or isotype control (left). Phagocytosis levels were assessed by flow cytometry and determined as frequency of CD14⁺ CFSE⁺ cells. FIG. 11C shows CD163 expression as assessed by flow cytometry after treatment with 10 pg/mL AKIO Fc inert (middle), AKIO active Fc (right), or isotype control (left). Data are representative of 3 independent experiments and showed as mean ± s.e.m of n = 6 donors. ** P< 0.001, *** P<0.0001 as determined by unpaired t-test with Welch’s correction.

[0046] FIGS. 12A-12C show the effect of anti-Siglec-10 mAb treatment on pro-inflammatory cytokine levels in the colorectal syngeneic mouse tumor model (MC38 cells) using mice expressing human Siglec-10 described above. Shown are production of CD80 and IL-6 from tumor-associated macrophages from mice treated with anti-Siglec-10 (right in both panels) or isotype control (left in both panels) (FIG. 12A); production of CD80 and IL-12 from CD103+ dendritic cells from mice treated with anti-Siglec-10 (right in both panels) or isotype control (left in both panels) (FIG. 12B); and production of IL-2 and co-production of TNF-a and IFN-y from CD8+ T cells from mice treated with anti-Siglec-10 (right in both panels) or isotype control (left in both panels) (FIG. 12C). Data are plotted as mean ± SD (n=9-10 mice/group). TAMs were gated on CD45 ⁺/CD1 lb ⁺/F4/80⁺, CD 103 ⁺ DCs were gated on CD45 ⁺ /CD1 lb ⁺ /CD1 lc ⁺ /MHCII ⁺ /CD103 ⁺ and CD8 ⁺ T cells were gated on CD45 ⁺ /CD1 lb ’/CD3⁺/CD8⁺. *P<0.05; ** P<0.001,*** P<0.005, **** P<0.0001 as determined by a Mann-Whitney test.

[0047] FIG. 13 shows a heatmap of the cytokine profile for Ml -polarized macrophages in presence of Siglec-10 mAb with inert Fc or isotype control, and MC38 tumor cell line. MC38 cells were pre-treated with mitomycin (50 pM) for 48 hours and co-cultured with bone marrow- derived macrophages (BMDM) treated with antibodies (10 pg/mL) in presence of LPS+IFN-y for 16 hours. Supernatant was collected and analyzed by multiplex cytokine assay from Meso Scale discovery (MSD), for the release of IL-ip, IL-6, IL-12/23p40, TNF, KC, MCP-1, MIP- a, MIP-ip and IL- 10. Assay was performed in quadruplicate and raw data was normalized to isotype control and presented as mean of fold induction. Significance was determined by two- way ANOVA with Tukey’s multiple comparison test. * P <0.05.

DETAILED DESCRIPTION

I. Definitions

[0048] It is to be understood that the present disclosure is not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes a combination of two or more such molecules, and the like.

[0049] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.

[0050] It is understood that aspects and embodiments of the present disclosure include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments.

[0051] The term “antibody” includes polyclonal antibodies, monoclonal antibodies (including full length antibodies which have an immunoglobulin Fc region), antibody compositions with poly epitopic specificity, multispecific antibodies e.g., bispecific antibodies, diabodies, and single-chain molecules), as well as antibody fragments (e.g., Fab, F(ab')2, and Fv). The term “immunoglobulin” (Ig) is used interchangeably with “antibody” herein.

[0052] The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. An IgM antibody consists of 5 of the basic heterotetramer units along with an additional polypeptide called a J chain, and contains 10 antigen binding sites, while IgA antibodies comprise from 2-5 of the basic 4-chain units which can polymerize to form polyvalent assemblages in combination with the J chain. In the case of IgGs, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the a and y chains and four CH domains for p and 8 isotypes. Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CHI). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VH and VL together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see e.g., Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6.

[0053] The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chains designated a, 5, s, y and p, respectively. The y and a classes are further divided into subclasses on the basis of relatively minor differences in the CH sequence and function, e.g., humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. IgGl antibodies can exist in multiple polymorphic variants termed allotypes (reviewed in Jefferis and Lefranc 2009. mAbs Vol 1 Issue 4 1-7) any of which are suitable for use in the present disclosure. Common allotypic variants in human populations are those designated by the letters a, f, n, z. [0054] An “isolated” antibody is one that has been identified, separated and/or recovered from a component of its production environment (e.g., naturally or recombinantly). In some embodiments, the isolated polypeptide is free of association with all other components from its production environment. Contaminant components of its production environment, such as that resulting from recombinant transfected cells, are materials that would typically interfere with research, diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the polypeptide is purified: (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody’s natural environment will not be present. Ordinarily, however, an isolated polypeptide or antibody is prepared by at least one purification step.

[0055] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, z.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or posttranslation modifications (e.g., isomerizations, amidations) that may be present in minor amounts. In some embodiments, monoclonal antibodies have a C-terminal cleavage at the heavy chain and/or light chain. For example, 1, 2, 3, 4, or 5 amino acid residues are cleaved at the C- terminus of heavy chain and/or light chain. In some embodiments, the C-terminal cleavage removes a C-terminal lysine from the heavy chain. In some embodiments, monoclonal antibodies have an N-terminal cleavage at the heavy chain and/or light chain. For example, 1, 2, 3, 4, or 5 amino acid residues are cleaved at the N-terminus of heavy chain and/or light chain. In some embodiments, monoclonal antibodies are highly specific, being directed against a single antigenic site. In some embodiments, monoclonal antibodies are highly specific, being directed against multiple antigenic sites (such as a bispecific antibody or a multispecific antibody). The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present disclosure may be made by a variety of techniques, including, for example, the hybridoma method, recombinant DNA methods, phage-display technologies, and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences.

[0056] The term “naked antibody” refers to an antibody that is not conjugated to a cytotoxic moiety or radiolabel.

[0057] The terms “full-length antibody,” “intact antibody” or “whole antibody” are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Specifically whole antibodies include those with heavy and light chains including an Fc region. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. In some cases, the intact antibody may have one or more effector functions.

[0058] An “antibody fragment” comprises a portion of an intact antibody, the antigen binding and/or the variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 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.

[0059] Papain digestion of antibodies produced two identical antigen-binding fragments, called “Fab” fragments, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain along with the variable region domain of the H chain (VH), and the first constant domain of one heavy chain (CHI). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment which roughly corresponds to two disulfide linked Fab fragments having different antigen-binding activity and is still capable of cross-linking antigen. Fab' fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of the CHI domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0060] The Fc fragment comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, the region which is also recognized by Fc receptors (FcR) found on certain types of cells.

[0061] “Fv” is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0062] “Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. In some embodiments, the sFv polypeptide further comprises a polypeptide linker between the VH and VL regions which enables the sFv to form the desired structure for antigen binding. For a review of the sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0063] “Functional fragments” of the antibodies of the present disclosure comprise a portion of an intact antibody, generally including the antigen binding or variable region of the intact antibody or the Fv region of an antibody which retains or has modified FcR binding capability. Examples of antibody fragments include linear antibody, single-chain antibody molecules and multispecific antibodies formed from antibody fragments.

[0064] The monoclonal antibodies herein specifically include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is (are) identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)). Chimeric antibodies of interest herein include PRIMATIZED® antibodies wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with an antigen of interest. As used herein, “humanized antibody” is used as a subset of “chimeric antibodies.”

[0065] “Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from an HVR of the recipient are replaced by residues from an HVR of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and/or capacity. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody performance, such as binding affinity. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all of the FR regions are those of a human immunoglobulin sequence, although the FR regions may include one or more individual FR residue substitutions that improve antibody performance, such as binding affinity, isomerization, immunogenicity, etc. In some embodiments, the number of these amino acid substitutions in the FR are no more than 6 in the H chain, and in the L chain, no more than 3. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, for example, Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1 : 105-115 (1998); Harris, Biochem. Soc. Transactions 23: 1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409. In some embodiments, humanized antibodies are directed against a single antigenic site. In some embodiments, humanized antibodies are directed against multiple antigenic sites. An alternative humanization method is described in U.S. Pat. No. 7,981,843 and U.S. Patent Application Publication No. 2006/0134098.

[0066] The “variable region” or “variable domain” (terms used interchangeably herein) of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domains of the heavy chain and light chain may be referred to as “VH” and “VL”, respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen binding sites.

[0067] The term “hypervariable region,” “HVR,” or “HV,” when used herein refers to the regions of an antibody-variable domain that are hypervariable in sequence and/or form structurally defined loops. Generally, antibodies comprise six HVRs; three in the VH (Hl, H2, H3), and three in the VL (LI, L2, L3). In native antibodies, H3 and L3 display the most diversity of the six HVRs, and H3 in particular is believed to play a unique role in conferring fine specificity to antibodies. See, e.g., Xu et al. Immunity 13:37-45 (2000); Johnson and Wu in Methods in Molecular Biology 248: 1-25 (Lo, ed., Human Press, Totowa, NJ, 2003)). Indeed, naturally occurring camelid antibodies consisting of a heavy chain only are functional and stable in the absence of light chain. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993) and Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0068] A number of HVR delineations are in use and are encompassed herein. The HVRs that are Kabat complementarity-determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5^th Ed. Public Health Service, National Institute of Health, Bethesda, MD (1991)). Chothia HVRs refer instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The “contact” HVRs are based on an analysis of the available complex crystal structures. The residues from each of these HVRs are noted below.

Loop _ Kabat _ Chothia _ Contact

LI L24-L34 L26-L34 L30-L36

L2 L50-L56 L50-L56 L46-L55

L3 L89-L97 L91-L96 L89-L96

Hl H31-H35B H26-H32 H30-H35B (Kabat Numbering)

Hl H31-H35 H26-H32 H30-H35 (Chothia Numbering)

H2 H50-H65 H53-H56 H47-H58

H3 H95-H102 H95-H102 H93-H101

[0069] Unless otherwise indicated, the variable-domain residues (HVR residues and framework region residues) are numbered according to Kabat et al., supra.

[0070] “Framework” or “FR” residues are those variable-domain residues other than the HVR residues as herein defined.

[0071] The expression “variable-domain residue-numbering as in Kabat” or “amino-acid- position numbering as in Kabat,” and variations thereof, refers to the numbering system used for heavy-chain variable domains or light-chain variable domains of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain. For example, a heavy-chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy-chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence.

[0072] An “acceptor human framework” for the purposes herein is a framework comprising the amino acid sequence of a VL or VH framework derived from a human immunoglobulin framework or a human consensus framework. An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes are 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.

[0073] “Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y where X is the number of amino acid residues scored as identical matches by the sequence in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A.

[0074] An antibody that “binds to”, “specifically binds to” or is “specific for” a particular a polypeptide or an epitope on a particular polypeptide is one that binds to that particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope. In some embodiments, binding of an anti-Siglec-10 antibody described herein (e.g., an antibody that binds to human Siglec-10) to an unrelated non-Siglec-10 polypeptide is less than about 10% of the antibody binding to Siglec-10 as measured by methods known in the art (e.g., enzyme-linked immunosorbent assay (ELISA)). In some embodiments, an antibody that binds to a Siglec-10 (e.g., an antibody that binds to human Siglec-10) has a dissociation constant (Kd) of < IpM, < 100 nM, < 10 nM, < 2 nM, < 1 nM, < 0.7 nM, <0 .6 nM, < 0.5 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g. 10'⁸M or less, e.g. from 10'⁸M to 10'¹³ M, e.g., from 10'⁹M to 10'¹³ M), about 250pM or less, about lOOpM or less, about lOpM or less, or about IpM or less.

[0075] The term “anti-Siglec-10 antibody” or “an antibody that binds to human Siglec-10” refers to an antibody that binds to a polypeptide or an epitope of human Siglec-10 without substantially binding to any other polypeptide or epitope of an unrelated non-Siglec-10 polypeptide.

[0076] The term “Siglec-10” as used herein refers to a human Siglec-10 protein. The term also includes naturally occurring variants of Siglec-10, including splice variants or allelic variants. Siglec-10, the sialic acid binding Ig-like lectin 10, is also known as SLG2, PRO940, and SIGLEC-10. In some embodiments, a human Siglec-10 protein is any protein or polypeptide expressed by a human SIGLEC10 gene. An exemplary human SIGLEC10 gene is described by NCBI Ref. Seq. Gene ID No. 89790. Amino acid sequences of exemplary human Siglec-10 proteins and domains thereof are described herein. For example, in some embodiments, a human Siglec-10 protein comprises an extracellular domain (ECD) comprising the amino acid sequence MDGRFWIRVQESVMVPEGLCISVPCSFSYPRQDWTGSTPAYGYWFKAVTETTKGAPVA TNHQSREVEMSTRGRFQLTGDPAKGNCSLVIRDAQMQDESQYFFRVERGSYVRYNFM NDGFFLKVTALTQKPDVYIPETLEPGQPVTVICVFNWAFEECPPPSFSWTGAALSSQGTK PTTSHFSVLSFTPRPQDHNTDLTCHVDFSRKGVSAQRTVRLRVAYAPRDLVISISRDNTP ALEPQPQGNVPYLEAQKGQFLRLLCAADSQPPATLSWVLQNRVLSSSHPWGPRPLGLEL PGVKAGDSGRYTCRAENRLGSQQRALDLSVQYPPENLRVMVSQANRTVLENLGNGTS LPVLEGQSLCLVCVTHSSPPARLSWTQRGQVLSPSQPSDPGVLELPRVQVEHEGEFTCH ARHPLGSQHVSLSLSVHYSPKLLGPSCSWEAEGLHCSCSSQASPAPSLRWWLGEELLEG NS SQDSFEVTPS S AGPWANS SLSLHGGLS SGLRLRCEAWNVHGAQSGSILQLPDKKGLIS T (SEQ ID NO:73).

[0077] Antibody “effector functions” refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell- mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0078] “Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies “arm” the cytotoxic cells and are required for killing of the target cell by this mechanism. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII. Fc expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9: 457- 92 (1991). In some embodiments, an anti-Siglec-10 antibody (e.g., an antibody that binds to human Siglec-10) described herein enhances ADCC. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., PNAS USA 95:652-656 (1998). Other Fc variants that alter ADCC activity and other antibody properties include those disclosed by Ghetie et al., Nat Biotech. 15:637-40, 1997; Duncan et al, Nature 332:563-564, 1988; Lund et al., J. Immunol 147:2657-2662, 1991; Lund et al, Mol Immunol 29:53-59, 1992; Alegre et al, Transplantation 57: 1537-1543, 1994; Hutchins et al., Proc Natl. Acad Sci USA 92: 11980-11984, 1995; Jefferis et al, Immunol Lett. 44: 111-117, 1995; Lund et al., FASEB J9: 115-119, 1995; Jefferis et al, Immunol Lett 54: 101-104, 1996; Lund et al, J Immunol 157:4963-4969, 1996; Armour et al., Eur J Immunol 29:2613-2624, 1999; Idusogie et al, J Immunol 164:4178-4184, 200; Reddy et al, J Immunol 164: 1925-1933, 2000; Xu et al., Cell Immunol 200: 16-26, 2000; Idusogie et al, J Immunol 166:2571-2575, 2001; Shields et al., J Biol Chem 276:6591-6604, 2001; Jefferis et al, Immunol Lett 82:57-65. 2002; Presta et al., Biochem Soc Trans 30:487-490, 2002; Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005-4010, 2006; U.S. Pat. Nos. 5,624,821; 5,885,573; 5,677,425; 6,165,745; 6,277,375; 5,869,046; 6,121,022; 5,624,821; 5,648,260; 6,194,551; 6,737,056; 6,821,505; 6,277,375;

7,335,742; and 7,317,091.

[0079] The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy-chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. Suitable native-sequence Fc regions for use in the antibodies of the present disclosure include human IgGl, IgG2, IgG3 and IgG4. A single amino acid substitution (S228P according to Kabat numbering; designated IgG4Pro) may be introduced to abolish the heterogeneity observed in recombinant IgG4 antibody. See Angal, S. et al. (1993) Mol Immunol 30, 105-108.

[0080] “Non-fucosylated” or “fucose-deficient” antibody refers to a glycosylation antibody variant comprising an Fc region wherein a carbohydrate structure attached to the Fc region has reduced fucose or lacks fucose. In some embodiments, an antibody with reduced fucose or lacking fucose has improved ADCC function. Non-fucosylated or fucose-deficient antibodies have reduced fucose relative to the amount of fucose on the same antibody produced in a cell line. In some embodiments, a non-fucosylated or fucose-deficient antibody composition contemplated herein is a composition wherein less than about 50% of the N-linked glycans attached to the Fc region of the antibodies in the composition comprise fucose.

[0081] The terms "fucosylation" or “fucosylated” refers to the presence of fucose residues within the oligosaccharides attached to the peptide backbone of an antibody. Specifically, a fucosylated antibody comprises a (l,6)-linked fucose at the innermost N-acetylglucosamine (GlcNAc) residue in one or both of the N-linked oligosaccharides attached to the antibody Fc region, e.g. at position Asn 297 of the human IgGl Fc region (EU numbering of Fc region residues). Asn297 may also be located about + 3 amino acids upstream or downstream of position 297, i.e. between positions 294 and 300, due to minor sequence variations in immunoglobulins.

[0082] The "degree of fucosylation" is the percentage of fucosylated oligosaccharides relative to all oligosaccharides identified by methods known in the art e.g., in an N-glycosidase F treated antibody composition assessed by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS). In a composition of a "fully fucosylated antibody" essentially all oligosaccharides comprise fucose residues, i.e. are fucosylated. In some embodiments, a composition of a fully fucosylated antibody has a degree of fucosylation of at least about 90%. Accordingly, an individual antibody in such a composition typically comprises fucose residues in each of the two N-linked oligosaccharides in the Fc region. Conversely, in a composition of a "fully non-fucosylated" antibody essentially none of the oligosaccharides are fucosylated, and an individual antibody in such a composition does not contain fucose residues in either of the two N-linked oligosaccharides in the Fc region. In some embodiments, a composition of a fully non- fucosylated antibody has a degree of fucosylation of less than about 10%. In a composition of a "partially fucosylated antibody" only part of the oligosaccharides comprise fucose. An individual antibody in such a composition can comprise fucose residues in none, one or both of the N- linked oligosaccharides in the Fc region, provided that the composition does not comprise essentially all individual antibodies that lack fucose residues in the N-linked oligosaccharides in the Fc region, nor essentially all individual antibodies that contain fucose residues in both of the N- linked oligosaccharides in the Fc region. In one embodiment, a composition of a partially fucosylated antibody has a degree of fucosylation of about 10% to about 80% (e.g., about 50% to about 80%, about 60% to about 80%, or about 70% to about 80%).

[0083] “Binding affinity” as used herein refers to the strength of the non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). In some embodiments, the binding affinity of an antibody for a Siglec-10 polypeptide or sub-domain thereof (e.g., the ECD, Domain 1, Domain 2, Domain 3, or Domain 4, e.g., as described herein) can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein.

[0084] “Binding avidity” as used herein refers to the binding strength of multiple binding sites of a molecule (e.g, an antibody) and its binding partner (e.g, an antigen). [0085] An “isolated” nucleic acid molecule encoding the antibodies herein is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which it was produced. In some embodiments, the isolated nucleic acid is free of association with all components associated with the production environment. The isolated nucleic acid molecules encoding the polypeptides and antibodies herein is in a form other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from nucleic acid encoding the polypeptides and antibodies herein existing naturally in cells.

[0086] The term “pharmaceutical formulation” refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective, and that contains no additional components that are unacceptably toxic to an individual to which the formulation would be administered. Such formulations are sterile.

[0087] “ Carriers” as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, histidine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

[0088] As used herein, the term “treatment” or “treating” refers to clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. An individual is successfully “treated”, for example, if one or more symptoms associated with a disease (e.g., viral infection) are mitigated or eliminated. For example, an individual is successfully “treated” if treatment results in increasing the quality of life of those suffering from a disease, decreasing the dose of other medications required for treating the disease, reducing the frequency of recurrence of the disease, lessening severity of the disease, delaying the development or progression of the disease, and/or prolonging survival of individuals.

[0089] As used herein, “in conjunction with” or “in combination with” refers to administration of one treatment modality in addition to another treatment modality. As such, “in conjunction with” or “in combination with” refers to administration of one treatment modality before, during or after administration of the other treatment modality to the individual.

[0090] As used herein, the term “prevention” or “preventing” includes providing prophylaxis with respect to occurrence or recurrence of a disease in an individual. An individual may be predisposed to a disease, susceptible to a disease, or at risk of developing a disease, but has not yet been diagnosed with the disease.

[0091] An “effective amount” refers to at least an amount effective, at dosages and for periods of time necessary, to achieve the desired or indicated effect, including a therapeutic or prophylactic result. An effective amount can be provided in one or more administrations. An effective amount may also differ when a drug is used in combination with another therapeutic agent or therapy; for example, a smaller dose of a drug (e.g., antibody) may be required to achieve an effective amount when administered in combination with an additional agent or therapy. A “therapeutically effective amount” is at least the minimum concentration required to effect a measurable improvement of a particular disease. A therapeutically effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the antibody to elicit a desired response in the individual. A therapeutically effective amount may also be one in which any toxic or detrimental effects of the antibody are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at the dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in individuals prior to or at the earlier stage of disease, the prophylactically effective amount can be less than the therapeutically effective amount.

[0092] “ Chronic” administration refers to administration of the medicament(s) in a continuous as opposed to acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time. “Intermittent” administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.

[0093] The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.

[0094] As used herein, an “individual” or a “subject” is a mammal. A “mammal” for purposes of treatment includes humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats, etc. In some embodiments, the individual or subject is a human.

II. Anti-Siglec-10 Antibodies and Compositions

[0095] Certain aspects of the present disclosure relate to antibodies that bind to human Siglec- 10, z.e., anti-Siglec-10 antibodies. In some embodiments, an anti-Siglec-10 antibody of the present disclosure comprises a heavy chain variable (VH) region and a light chain variable (VL) region. In some embodiments, the anti-Siglec-10 antibody is a humanized antibody.

[0096] In some embodiments, an anti-Siglec-10 antibody of the present disclosure comprises a VH region that comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 19, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:20, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:21; and/or a VL region that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:22, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:23, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:24.

[0097] In some embodiments, an anti-Siglec-10 antibody of the present disclosure comprises a VH region that comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:25, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:26, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:27; and/or a VL region that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:28, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:30.

[0098] In some embodiments, an anti-Siglec-10 antibody of the present disclosure comprises a VH region that comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:31, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:32, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:33; and/or a VL region that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:34, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:35, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:36.

[0099] In some embodiments, an anti-Siglec-10 antibody of the present disclosure comprises a VH region that comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:37, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:38, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:39; and/or a VL region that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:40, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:41, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:42.

[0100] In some embodiments, an anti-Siglec-10 antibody of the present disclosure comprises a VH region that comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:43, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:44, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:45; and/or a VL region that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:46, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:47, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:48.

[0101] In some embodiments, an anti-Siglec-10 antibody of the present disclosure comprises a VH region that comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:49, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:50, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:51; and/or a VL region that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:52, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:53, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 54.

[0102] In some embodiments, an anti-Siglec-10 antibody of the present disclosure comprises a VH region that comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:55, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:56, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:57; and/or a VL region that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:58, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 59, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:60.

[0103] In some embodiments, an anti-Siglec-10 antibody of the present disclosure comprises a VH region that comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:61, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:62, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:63; and/or a VL region that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:64, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:65, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:66.

[0104] In some embodiments, an anti-Siglec-10 antibody of the present disclosure comprises a VH region that comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:67, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:68, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:69; and/or a VL region that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:70, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:71, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:72.

[0105] In some embodiments, an anti-Siglec-10 antibody of the present disclosure comprises a VH region that comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:79, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:80, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:81; and/or a VL region that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:82, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:83, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 84. In some embodiments, an anti-Siglec-10 antibody of the present disclosure comprises a VH region that comprises the amino acid sequence of SEQ ID NO:85 and/or a VL region that comprises the amino acid sequence of SEQ ID NO:86. In some embodiments, an anti-Siglec-10 antibody of the present disclosure comprises a VH region that comprises the amino acid sequence of SEQ ID NO: 85 and a VL region that comprises the amino acid sequence of SEQ ID NO:86.

[0106] In some embodiments, an anti-Siglec-10 antibody of the present disclosure comprises a heavy chain that comprises the amino acid sequence of SEQ ID NO:87 or 88 and/or a light chain that comprises the amino acid sequence of SEQ ID NO:89. In some embodiments, an anti- Siglec-10 antibody of the present disclosure comprises a heavy chain that comprises the amino acid sequence of SEQ ID NO:87 or 88 and a light chain that comprises the amino acid sequence of SEQ ID NO:89. In some embodiments, one or both of the antibody Fc regions or heavy chains do not have a C-terminal lysine. As is known in the art, the C-terminal lysine of some antibody heavy chain species may be cleaved off in some fraction of molecules. In some embodiments, an anti-Siglec-10 antibody of the present disclosure comprises a heavy chain that comprises the amino acid sequence of SEQ ID NO:87 or 88 and a light chain that comprises the amino acid sequence of SEQ ID NO: 89. In some embodiments, an anti-Siglec-10 antibody of the present disclosure comprises a heavy chain that comprises the amino acid sequence of SEQ ID NO:87 and a light chain that comprises the amino acid sequence of SEQ ID NO:89. In some embodiments, an anti-Siglec-10 antibody of the present disclosure comprises a heavy chain that comprises the amino acid sequence of SEQ ID NO:88 and a light chain that comprises the amino acid sequence of SEQ ID NO:89. In some embodiments, a composition of the present disclosure comprises a mixture of anti-Siglec-10 antibody species, wherein each species comprises a heavy chain that comprises the amino acid sequence of SEQ ID NO: 87 or 88 and a light chain that comprises the amino acid sequence of SEQ ID NO:89.

AKIO heavy chain without C-terminal lysine QVQLQESGPGLVKPSETLSLTCTVSGFSLTSYGIHWIRQPPGKGLEWIGVIWSDAGPAYNS ALRSRLTISKDTSKNQVSLKLSSVTAADTAVYYCARQGHYDYDFDYWGQGTTVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAQGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCQVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:87)

AKIO heavy chain with C-terminal lysine QVQLQESGPGLVKPSETLSLTCTVSGFSLTSYGIHWIRQPPGKGLEWIGVIWSDAGPAYNS ALRSRLTISKDTSKNQVSLKLSSVTAADTAVYYCARQGHYDYDFDYWGQGTTVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAQGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCQVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:88) AK10 light chain DIQMTQSPSSLSASVGDRVTITCRASQDISNHLNWYQQKPGKVPKLLIYYTSRLHPGVPS RFSGSGSGTDFTLTISSLQPEDVATYFCQKGNMFPPTFGGGTKLEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:89)

[0107] In some embodiments, an anti-Siglec-10 antibody provided herein competes for binding to human Siglec-10 (e.g., an ECD or sub-domain thereof of a human Siglec-10 protein) with a reference antibody, e.g., an anti-Siglec-10 antibody of the present disclosure. In some embodiments, an anti-Siglec-10 antibody provided herein competes for binding to human Siglec-10 (e.g., an ECD or sub-domain thereof of a human Siglec-10 protein) with one or more of the following anti-Siglec-10 antibodies described herein: AK01, AK02, AK03, AK04, AK05, AK06, AK07, AK08, AK09, and AKIO. In some embodiments, an anti-Siglec-10 antibody provided herein competes for binding to human Siglec-10 with a reference antibody that comprises a VH region comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 19, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:20, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:21 and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:22, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:23, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:24. In some embodiments, an anti-Siglec-10 antibody provided herein competes for binding to human Siglec-10 with a reference antibody that comprises a VH region comprising an HVR- H1 comprising the amino acid sequence of SEQ ID NO:25, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:26, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:27 and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:28, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:30. In some embodiments, an anti-Siglec- 10 antibody provided herein competes for binding to human Siglec-10 with a reference antibody that comprises a VH region comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO:31, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:32, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:33 and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:34, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:35, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:36. In some embodiments, an anti-Siglec-10 antibody provided herein competes for binding to human Siglec-10 with a reference antibody that comprises a VH region comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO:37, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:38, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:39 and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:40, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:41, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:42. In some embodiments, an anti-Siglec-10 antibody provided herein competes for binding to human Siglec-10 with a reference antibody that comprises a VH region comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO:43, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:44, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:45 and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:46, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:47, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:48. In some embodiments, an anti-Siglec-10 antibody provided herein competes for binding to human Siglec-10 with a reference antibody that comprises a VH region comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO:49, an HVR- H2 comprising the amino acid sequence of SEQ ID NO:50, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:51 and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 52, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:53, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:54. In some embodiments, an anti-Siglec-10 antibody provided herein competes for binding to human Siglec-10 with a reference antibody that comprises a VH region comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO:55, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:56, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:57 and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:58, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:59, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:60. In some embodiments, an anti-Siglec- 10 antibody provided herein competes for binding to human Siglec-10 with a reference antibody that comprises a VH region comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO:61, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:62, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:63 and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:64, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:65, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:66. In some embodiments, an anti-Siglec-10 antibody provided herein competes for binding to human Siglec-10 with a reference antibody that comprises a VH region comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO:67, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:68, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:69 and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:70, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:71, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:72. In some embodiments, an anti-Siglec-10 antibody provided herein competes for binding to human Siglec-10 with a reference antibody that comprises a VH region comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO:79, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:80, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:81 and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:82, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:83, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:84.

[0108] In some embodiments, the anti-Siglec-10 antibody comprises 1, 2, 3, 4, 5, or all 6 HVR sequences of a single anti-Siglec-10 antibody as set forth in Table 2. In some embodiments, the anti-Siglec-10 antibody comprises a VH region comprising 1, 2, or all 3 HVR sequences of a VH region of a single anti-Siglec-10 antibody as set forth in Table 2. In some embodiments, the anti-Siglec-10 antibody comprises a VL region comprising 1, 2, or all 3 HVR sequences of a VL region of a single anti-Siglec-10 antibody as set forth in Table 2.

Table 2. Anti-Siglec-10 antibody HVR sequences.

[0109] In some embodiments, an anti-Siglec-10 antibody of the present disclosure comprises a VH region comprising 1, 2, or all 3 CDR or HVR sequences present in the VH region sequence of AK01 as described herein (see, e.g., Tables 2 and 3) and/or a VL region comprising 1, 2, or all 3 CDR or HVR sequences present in the VL region sequence of AK01 as described herein (see, e.g., Tables 2 and 3). In some embodiments, an anti-Siglec-10 antibody of the present disclosure comprises a VH region comprising 1, 2, or all 3 CDR or HVR sequences present in the VH region sequence of AK02 as described herein (see, e.g., Tables 2 and 3) and/or a VL region comprising 1, 2, or all 3 CDR or HVR sequences present in the VL region sequence of AK02 as described herein (see, e.g., Tables 2 and 3). In some embodiments, an anti-Siglec-10 antibody of the present disclosure comprises a VH region comprising 1, 2, or all 3 CDR or HVR sequences present in the VH region sequence of AK03 as described herein (see, e.g., Tables 2 and 3) and/or a VL region comprising 1, 2, or all 3 CDR or HVR sequences present in the VL region sequence of AK03 as described herein see, e.g., Tables 2 and 3). In some embodiments, an anti-Siglec-10 antibody of the present disclosure comprises a VH region comprising 1, 2, or all 3 CDR or HVR sequences present in the VH region sequence of AK04 as described herein (see, e.g., Tables 2 and 3) and/or a VL region comprising 1, 2, or all 3 CDR or HVR sequences present in the VL region sequence of AK04 as described herein (see, e.g., Tables 2 and 3). In some embodiments, an anti-Siglec-10 antibody of the present disclosure comprises a VH region comprising 1, 2, or all 3 CDR or HVR sequences present in the VH region sequence of AK05 as described herein (see, e.g., Tables 2 and 3) and/or a VL region comprising 1, 2, or all 3 CDR or HVR sequences present in the VL region sequence of AK05 as described herein (see, e.g., Tables 2 and 3). In some embodiments, an anti-Siglec-10 antibody of the present disclosure comprises a VH region comprising 1, 2, or all 3 CDR or HVR sequences present in the VH region sequence of AK06 as described herein (see, e.g., Tables 2 and 3) and/or a VL region comprising 1, 2, or all 3 CDR or HVR sequences present in the VL region sequence of AK06 as described herein (see, e.g., Tables 2 and 3). In some embodiments, an anti-Siglec-10 antibody of the present disclosure comprises a VH region comprising 1, 2, or all 3 CDR or HVR sequences present in the VH region sequence of AK07 as described herein (see, e.g., Tables 2 and 3) and/or a VL region comprising 1, 2, or all 3 CDR or HVR sequences present in the VL region sequence of AK07 as described herein (see, e.g., Tables 2 and 3). In some embodiments, an anti-Siglec- 10 antibody of the present disclosure comprises a VH region comprising 1, 2, or all 3 CDR or HVR sequences present in the VH region sequence of AK08 as described herein (see, e.g., Tables 2 and 3) and/or a VL region comprising 1, 2, or all 3 CDR or HVR sequences present in the VL region sequence of AK08 as described herein (see, e.g., Tables 2 and 3). In some embodiments, an anti-Siglec-10 antibody of the present disclosure comprises a VH region comprising 1, 2, or all 3 CDR or HVR sequences present in the VH region sequence of AK09 as described herein (see, e.g., Tables 2 and 3) and/or a VL region comprising 1, 2, or all 3 CDR or HVR sequences present in the VL region sequence of AK09 as described herein (see, e.g., Tables 2 and 3). In some embodiments according to any of the embodiments described herein, the antibody is a humanized antibody. In some embodiments, an anti-Siglec-10 antibody of the present disclosure comprises a VH region comprising 1, 2, or all 3 CDR or HVR sequences present in the VH region sequence of AKIO as described herein (see, e.g., Tables 2 and 3) and/or a VL region comprising 1, 2, or all 3 CDR or HVR sequences present in the VL region sequence of AKIO as described herein (see, e.g., Tables 2 and 3).

Table 3. Anti-Siglec-10 antibody variable region sequences.

[0110] Many definitions for CDR or HVR sequences of an antibody variable region are known in the art and may be used to describe an antibody of the present disclosure, e.g., by CDR/HVR sequences. In some embodiments, antibody CDR/HVR sequences are defined as in Kabat (see, e.g., Kabat el al., Sequences of Proteins of Immunological Interest, 5^th Ed. Public Health Service, National Institute of Health, Bethesda, MD (1991)). In some embodiments, antibody CDR/HVR sequences are defined as in Chothia (see, e.g., Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In some embodiments, antibody CDR/HVR sequences are defined as in IMGT (see, e.g., Lefranc, M.P. (1999) The Immunologist 7 : 132-136). In some embodiments, CDR/HVR sequences of a single antibody are defined as by mixing two or more definitions, e.g., Kabat, Chothia, and/or IMGT.

[OHl] In some embodiments, an anti-Siglec-10 antibody of the present disclosure binds to an extracellular domain (ECD) of a human Siglec-10 protein. In some embodiments, the Siglec-10 ECD comprises the amino acid sequence MDGRFWIRVQESVMVPEGLCISVPCSFSYPRQDWTGSTPAYGYWFKAVTETTKGAPVA TNHQSREVEMSTRGRFQLTGDPAKGNCSLVIRDAQMQDESQYFFRVERGSYVRYNFM NDGFFLKVTALTQKPDVYIPETLEPGQPVTVICVFNWAFEECPPPSFSWTGAALSSQGTK PTTSHFSVLSFTPRPQDHNTDLTCHVDFSRKGVSAQRTVRLRVAYAPRDLVISISRDNTP ALEPQPQGNVPYLEAQKGQFLRLLCAADSQPPATLSWVLQNRVLSSSHPWGPRPLGLEL PGVKAGDSGRYTCRAENRLGSQQRALDLSVQYPPENLRVMVSQANRTVLENLGNGTS LPVLEGQSLCLVCVTHSSPPARLSWTQRGQVLSPSQPSDPGVLELPRVQVEHEGEFTCH ARHPLGSQHVSLSLSVHYSPKLLGPSCSWEAEGLHCSCSSQASPAPSLRWWLGEELLEG NSSQDSFEVTPSSAGPWANSSLSLHGGLSSGLRLRCEAWNVHGAQSGSILQLPDKKGLIS T (SEQ ID NO:73).

[0112] In some embodiments, an anti-Siglec-10 antibody described herein binds to Domain 1, Domain 2, Domain 3, or Domain 4 of a human Siglec-10 protein (e.g., an ECD of a human Siglec-10 protein). In some embodiments, an anti-Siglec-10 antibody described herein binds to Domain 1 of a human Siglec-10 protein (e.g., an ECD of a human Siglec-10 protein). In some embodiments, Domain 1 comprises the amino acid sequence DGRFWIRVQESVMVPEGLCISVPCSFSYPRQDWTGSTPAYGYWFKAVTETTKGAPVATN HQSREVEMSTRGRFQLTGDPAKGNCSLVIRDAQMQDESQYFFRVE (SEQ ID NO:74). In some embodiments, an anti-Siglec-10 antibody described herein binds to Domain 2 of a human Siglec-10 protein (e.g., an ECD of a human Siglec-10 protein). In some embodiments, Domain 2 comprises the amino acid sequence

PDVYIPETLEPGQPVTVICVFNWAFEECPPPSFSWTGAALSSQGTKPTTSHFSVLSFTPRP QDHNTDLTCHVDFSRKGVSAQRTVR (SEQ ID NO:75). In some embodiments, an anti- Siglec-10 antibody described herein binds to Domain 3 of a human Siglec-10 protein (e.g., an ECD of a human Siglec-10 protein). In some embodiments, Domain 3 comprises the amino acid sequence

PALEPQPQGNVPYLE AQKGQFLRLLC AAD SQPPATL S WVLQNRVLS S SHPWGPRPLGLE LPGVKAGDSGRYTCRAENRLGSQQRALDLS (SEQ ID NO:76). In some embodiments, an anti-Siglec-10 antibody described herein binds to Domain 4 of a human Siglec-10 protein (e.g., an ECD of a human Siglec-10 protein). In some embodiments, Domain 4 comprises the amino acid sequence PENLRVMVSQANRTVLENLGNGTSLPVLEGQSLCLVCVTHSSPPARLSWTQRGQVLSPS QPSDPGVLELPRVQVEHEGEFTCHARHPLGSQHVSLSLS (SEQ ID NO:77). In some embodiments, an anti-Siglec-10 antibody described herein binds to Domain 3 and Domain 4 of a human Siglec-10 protein (e.g., an ECD of a human Siglec-10 protein).

[0113] In some embodiments, an anti-Siglec-10 antibody provided herein binds the same epitope on human Siglec-10 (e.g., an ECD or sub-domain thereof of a human Siglec-10 protein) as an anti-Siglec-10 antibody of the present disclosure, e.g., AK01, AK02, AK03, AK04, AK05, AK06, AK07, AK08, AK09, and/or AKIO. Exemplary assays for epitope mapping are known in the art. For example, epitope mapping can be performed using cross-linking mass spectrometry (XL-MS), X-ray crystallography, or alanine scanning mutagenesis.

[0114] In some embodiments, an anti-Siglec-10 antibody provided herein binds to human Siglec-10 (e.g., the extracellular domain of human Siglec-10) when expressed on the surface of a cell. In some embodiments, an anti-Siglec-10 antibody provided herein binds to human Siglec- 10 (e.g., the extracellular domain of human Siglec-10) when expressed on the surface of a human myeloid cell, e.g., a human macrophage, dendritic cell, or monocyte.

[0115] In some embodiments, binding of an anti-Siglec-10 antibody provided herein to human Siglec-10 expressed on the surface of a cell (such as a human myeloid cell) induces internalization of Siglec-10. In some embodiments, binding of an anti-Siglec-10 antibody provided herein to human Siglec-10 expressed on the surface of a cell (such as a human myeloid cell) leads to reduced levels of Siglec-10 on the cell surface. Binding of the antibody can lead to reduced levels of Siglec-10 on the cell membrane, e.g., through Siglec-10 internalization, endocytosis, shedding, cleaving, etc. Assays for assessing levels of Siglec-10 surface expression are known in the art and exemplified herein. In some embodiments, Siglec-10 surface expression is measured by flow cytometry, e.g., using an anti-Siglec-10 antibody with a detectable (e.g., fluorescent) tag. For example, cells can be contacted with an anti-Siglec-10 antibody, and surface expression can be measured by flow cytometry with a fluorescent-tagged anti-Siglec-10 antibody that binds to a different epitope on Siglec-10 than the test antibody. Reduced fluorescence in the presence of the test antibody can indicate a reduced level of Siglec- 10 surface expression. In some embodiments, binding of the antibody to the extracellular domain of human Siglec-10 when expressed on a surface of a human mast cell leads to reduced levels of Siglec-10 surface expression regardless of the presence/absence of an antibody Fc region, or regardless of the ability of the antibody Fc region to bind an Fc receptor (e.g., expressed on an effector cell). In some embodiments, binding of the antibody to the extracellular domain of human Siglec-10 when expressed on a surface of a human cell leads to an expression level Siglec-10 on the cell membrane that is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%, e.g., as compared to surface expression of Siglec-10 in the absence of an antibody, or in the absence of an antibody that does not bind to Siglec-10.

[0116] In some embodiments, binding of an anti-Siglec-10 antibody provided herein to human Siglec-10 (e.g., human Siglec-10 expressed on the surface of a cell, such as a human myeloid cell) blocks interaction between human Siglec-10 and human CD24. In some embodiments, binding of an anti-Siglec-10 antibody provided herein to human Siglec-10 (e.g., human Siglec- 10 expressed on the surface of a cell, such as a human myeloid cell) blocks interaction between human Siglec-10 and human CD52.

[0117] In some embodiments, binding of an anti-Siglec-10 antibody provided herein to human Siglec-10 (e.g., human Siglec-10 expressed on the surface of a cell, such as a human myeloid cell) does not block interaction between human Siglec-10 and human CD24. In some embodiments, binding of an anti-Siglec-10 antibody provided herein to human Siglec-10 (e.g., human Siglec-10 expressed on the surface of a cell, such as a human myeloid cell) does not block interaction between human Siglec-10 and human CD52.

[0118] Assays for assessing interaction between Siglec-10 and another protein (e.g., CD24 or CD52) are known in the art and exemplified herein. For example, cells expressing a ligand of Siglec-10 (e.g., CD24 or CD52) can be incubated with a biotinylated Siglec-10 ECD that binds to the Siglec-10 ligand and a test antibody (e.g., that binds Siglec-10). After washing and staining (e.g., with a detection agent such as a fluorescently labeled streptavidin), interaction between the ECD and ligand-expressing cells can be assessed by flow cytometry. Ability of Siglec-10 ECD to bind to its ligand on cells is therefore assayed in the presence of a test anti- Siglec-10 antibody to determine if the antibody interferes with the interaction between the ECD and the ligand (which would lead to a decrease in measured fluorescence from the ECD associated with the ligand-expressing cell, as compared to co-incubation of the ECD with a control antibody).

[0119] In one aspect, an anti-Siglec-10 antibody described herein is a monoclonal antibody. In one aspect, an anti-Siglec-10 antibody described herein is an antibody fragment (including antigen-binding fragment), e.g., a Fab, Fab'-SH, Fv, scFv, or (Fab '^ fragment. In one aspect, an anti-Siglec-10 antibody described herein is a chimeric, humanized, or human antibody. In one aspect, any of the anti-Siglec-10 antibodies described herein are purified.

[0120] An anti-Siglec-10 antibody described herein may comprise any suitable framework variable domain sequence, provided that the antibody retains the ability to bind human Siglec- 10. As used herein, heavy chain framework regions are designated "HC-FR1-FR4," and light chain framework regions are designated "LC-FR1-FR4."

[0121] There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chains designated a, 5, 8, y and p, respectively. The y and a classes are further divided into subclasses e.g., humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. IgGl antibodies can exist in multiple polymorphic variants termed allotypes (reviewed in Jefferis and Lefranc 2009. mAbs Vol 1 Issue 4 1-7) any of which are suitable for use in some of the embodiments herein. Common allotypic variants in human populations are those designated by the letters a,f,n,z or combinations thereof.

[0122] In any of the embodiments herein, the antibody may comprise a heavy chain Fc region, e.g., a human Fc region or human IgG Fc region. In further embodiments, the human IgG Fc region comprises a human IgGl or IgG4 Fc region. In some embodiments, the human IgG4 Fc region comprises the amino acid substitution S228P, wherein the amino acid residues are numbered according to the EU index as in Kabat. In some embodiments, the human Fc region comprises one or more mutation(s) that reduce effector function.

[0123] In some embodiments, the human IgGl Fc region comprises one or more mutation(s) that reduce effector function. In some embodiments, the human IgGl Fc region comprises a substitution or deletion at one or more of the following position(s), numbering based on EU index: (a) L234 and/or L235; (b) A327, A330, and/or P331; (c) E233, L234, L235, and/or G236; (d) E233, L234, and/or L235; (e) E233, L234, L235, G236, A327, A330, and/or P331; (f) E233, L234, L235, A327, A330, and/or P331; (g) N297; (h) L242, N297, and/or K334; (i) A287, N297, and/or L306; (j) R292, N297, and/or V302; (k) N297, V323, and/or 1332; (1) V259, N297, and/or L306; (m) L234, L235, K322, M252, S254, and/or T256; or (n) L234, L235, and/or P329. In some embodiments, the antibody comprises a human IgGl Fc region with one or more of the following mutation(s), numbering based on EU index: (a) L234A and/or L235A; (b) A327G, A330S, and/or P33 IS; (c) E233P, L234V, L235A, and/or G236del; (d) E233P, L234V, and/or L235A; (e) E233P, L234V, L235A, G236del, A327G, A330S, and/or P331S; (f) E233P, L234V, L235A, A327G, A330S, and/or P33 IS; (g) N297A; (h) N297G; (i) N297Q; (j) L242C, N297C, and/or K334C; (k) A287C, N297G, and/or L306C; (1) R292C, N297G, and/or V302C; (m) N297G, V323C, and/or I332C; (n) V259C, N297G, and/or L306C; (o) L234F, L235Q, K322Q, M252Y, S254T, and/or T256E; (p) L234A, L235A, and/or P329G; or (q) L234A, L235Q, and/or K322Q. See, e.g., Schlothauer, T. et al. (2016) Protein Eng. Des. SeL 29:457-466; Armour, K.L. et al. (2003)Mol. Immunol. 40:585-593; Jacobsen, F.W. et al. (2017) J. Biol. Chem. 292: 1865- 1875; and Borrok, M.J. et al. (2017) J. Pharm. Sci. 106: 1008-1017.

[0124] In some embodiments, the human IgGl Fc region comprises one or more mutation(s) that increase or enhance effector function. In some embodiments, the human IgGl Fc region comprises a substitution or deletion at one or more of the following position(s), numbering based on EU index: (a) F243, R292, Y300, V305, and/or P396; (b) S239 and/or 1332; (c) S239, 1332, and/or A330; (d) S298, E333, and/or K334; (e) G236, S239, and/or 1332; (f) K326 and/or E333; (g) S267, H268, and/or S324; or (h) E345, E430, and/or S440. In some embodiments, the human IgGl Fc region comprises one or more of the following mutation(s), numbering based on EU index: (a) F243L, R292P, Y300L, V305I, and/or P396L; (b) S239D and/or I332E; (c) S239D, I332E, and/or A330L; (d) S298A, E333A, and/or K334A; (e) G236A, S239D, and/or I332E; (f) K326W and/or E333S; (g) S267E, H268F, and/or S324T; or (h) E345R, E430G, and/or S440Y. See, e.g., Stavenhagen, J.B. et al. (2007) Cancer Res. 67:8882-8890; Lazar, G.A. et al. (2006) roc. Natl. Acad. Sci. USA 103:4005-4010; Shields, R.L. et al. (2001) J. Biol.

Chem. 276:6591-6604; Richards, J.O. et al. (2008)Afo/. Cancer Ther. 7:2517-2527; Idusogie, E.E. et al. (2001) J. Immunol. 166:2571-2575; Moore, G.L. et al. (2010) MAbs 2: 181-189; and Diebolder, C.A. et al. (2014) Science 343: 1260-1263. In some embodiments, the human IgGl Fc region comprises L234A, L235Q, and K322Q substitutions, numbering based on EU index. [0125] In some embodiments, the human IgG2 Fc region comprises one or more mutation(s) that reduce effector function. In some embodiments, the human IgG2 Fc region comprises a substitution or deletion at one or more of the following position(s), numbering based on EU index: (a) A330 and/or P331; (b) V234, G237, P238, H268, V309, A330, and/or P331; or (c) V234, G237, H268, V309, A330, P331, C232, C233, S267, L328, M252, S254, and/or T256. In some embodiments, the human IgG2 Fc region comprises one or more of the following mutation(s), numbering based on EU index: (a) A330S and/or P331S; (b) V234A, G237A, P238S, H268A, V309L, A330S, and/or P331S; or (c) V234A, G237A, H268Q, V309L, A330S, P331S, C232S, C233S, S267E, L328F, M252Y, S254T, and/or T256E. See, e.g, Armour, K.L. et al. (2003) Afo/. Immunol. 40:585-593 and US PG Pub. Nos. 20170204193 and 20170240631. [0126] In some embodiments, the human IgG4 Fc region comprises one or more mutation(s) that reduce effector function. In some embodiments, the human IgG4 Fc region comprises a substitution or deletion at one or more of the following position(s), numbering based on EU index: (a) E233, F234, L235, and/or G236; (b) E233, F234, and/or L235; or (c) S228 and/or L235. In some embodiments, the human IgG4 Fc region comprises one or more of the following mutation(s), numbering based on EU index: (a) E233P, F234V, L235A, and/or G236del; (b) E233P, F234V, and/or L235A; (c) S228P and/or L235E; or (d) S228P and/or L235A. See, e.g., Schlothauer, T. et al. (2016) Protein Eng. Des. Sei. 29:457-466; and Armour, K.L. et al. (2003) Mol. Immunol. 40:585-593.

[0127] In some embodiments, an anti-Siglec-10 antibody of the present disclosure comprises an antibody light chain comprising a light chain constant (CL) domain, e.g., a human kappa or lambda CL domain. In some embodiments, the CL domain is a human kappa CL domain. In some embodiments, the CL domain comprises the amino acid sequence of RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:78). [0128] In one aspect, the present disclosure provides anti-Siglec-10 antibodies with reduced or eliminated fucosylation, e.g., as described infra. For example, in some embodiments, at least one or two of the heavy chains of the antibody is non-fucosylated. In some embodiments, the Fc region is non-fucosylated. In some embodiments, the antibody comprises a non-fucosylated human IgGl Fc region. Exemplary assays for measuring antibody fucosylation, as well as methods and cell lines for producing antibodies with altered, reduced, or eliminated fucosylation, are provided herein. [0129] In one aspect, polynucleotides encoding anti-Siglec-10 antibodies are provided. In certain embodiments, vectors comprising polynucleotides encoding anti-Siglec-10 antibodies are provided. In certain embodiments, host cells comprising such vectors are provided. In another aspect of the invention, compositions comprising anti-Siglec-10 antibodies or polynucleotides encoding anti-Siglec-10 antibodies are provided. In certain embodiments, a composition of the present disclosure is a pharmaceutical formulation for the treatment of cancer, such as those enumerated herein.

1. Antibody Affinity

[0130] In some embodiments, the anti-Siglec-10 antibody binds to the ECD of human Siglec- 10 with an equilibrium dissociation constant (KD) of about 150pM or less, about 125pM or less, about lOOpM or less, about 90pM or less, about 80pM or less, about 70pM or less, about 60pM or less, about 50pM or less, about 40pM or less, about 30pM or less, about 20pM or less, about lOpM or less, or about IpM or less. In some embodiments, the anti-Siglec-10 antibody binds to Domain 1 of the ECD of human Siglec-10 with an equilibrium dissociation constant (KD) of about 150pM or less, about 125pM or less, about lOOpM or less, about 90pM or less, about 80pM or less, about 70pM or less, about 60pM or less, about 50pM or less, about 40pM or less, about 30pM or less, about 20pM or less, about lOpM or less, or about IpM or less. In some embodiments, the anti-Siglec-10 antibody binds to Domain 3 and/or 4 of the ECD of human Siglec-10 with an equilibrium dissociation constant (KD) of about 150pM or less, about 125pM or less, about lOOpM or less, about 90pM or less, about 80pM or less, about 70pM or less, about 60pM or less, about 50pM or less, about 40pM or less, about 30pM or less, about 20pM or less, about lOpM or less, or about IpM or less.

[0131] Exemplary assays for determining binding affinity of an antibody for human Siglec-10, its ECD, or a sub-domain thereof are known in the art and exemplified herein. In one embodiment, the binding affinity of the anti-Siglec-10 antibody can be determined by a surface plasmon resonance assay. For example, the Kd or Kd value can be measured by using a BIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C with immobilized antigen CM5 chips at ~10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore® Inc.) are activated with N-ethyl-N'-(3-dimethylaminopropyl)- carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Capture antibodies (e.g., anti-human-Fc) are diluted with 10 mM sodium acetate, pH 4.8, before injection at a flow rate of 30 pl/minute and further immobilized with an anti- Siglec-10 antibody. For kinetics measurements, two-fold serial dilutions of dimeric Siglec-10 are injected in PBS with 0.05% Tween 20 (PBST) at 25° C at a flow rate of approximately 25 pl/min. Association rates (kon) and dissociation rates (koff) are calculated using a simple one-to- one Langmuir binding model (BIAcore® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is calculated as the ratio koff/kon. See, e.g., Chen, Y., et al., (1999) J. Mol. Biol. 293:865-881. [0132] In another embodiment, biolayer interferometry may be used to determine the affinity of anti-Siglec-10 antibodies against Siglec-10. In an exemplary assay, Siglec-10-Fc tagged protein is immobilized onto anti-human capture sensors, and incubated with increasing concentrations of mouse, chimeric, or humanized anti-Siglec-10 Fab fragments to obtain affinity measurements using an instrument such as, for example, the Octet Red 384 System (ForteBio). [0133] The binding affinity of the anti-Siglec-10 antibody can, for example, also be determined by the Scatchard analysis described in Munson et al., Anal. Biochem., 107:220 (1980) using standard techniques well known in the relevant art. See also Scatchard, G., Ann. N.Y. Acad. Sci. 51 :660 (1947).

2. Competition Assays [0134] Competition assays can be used to determine whether two antibodies bind the same epitope by recognizing identical or sterically overlapping epitopes or one antibody competitively inhibits binding of another antibody to the antigen. These assays are known in the art. Typically, antigen or antigen expressing cells is immobilized on a multi-well plate and the ability of unlabeled antibodies to block the binding of labeled antibodies is measured. Common labels for such competition assays are radioactive labels or enzyme labels. In some embodiments, an anti-Siglec-10 antibody described herein competes with a reference antibody described herein for binding to a Siglec-10 polypeptide or an ECD or domain thereof, e.g., expressed on the cell surface of a cell (e.g., a myeloid cell).

III. Antibody Preparation

[0135] The antibody described herein is prepared using techniques available in the art for generating antibodies, exemplary methods of which are described in more detail in the following sections. 1. Antibody Fragments

[0136] The present invention encompasses antibody fragments. Antibody fragments may be generated by traditional means, such as enzymatic digestion, or by recombinant techniques. In certain circumstances there are advantages of using antibody fragments, rather than whole antibodies. For a review of certain antibody fragments, see Hudson et al. (2003) Nat. Med. 9: 129-134.

[0137] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992); and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and ScFv antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coll and chemically coupled to form F(ab')2 fragments (Carter et al., Bio/Technology 10: 163-167 (1992)). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab')2 fragment with increased in vivo half-life comprising salvage receptor binding epitope residues are described in U.S. Pat. No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In certain embodiments, an antibody is a single chain Fv fragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and 5,587,458. Fv and scFv are the only species with intact combining sites that are devoid of constant regions; thus, they may be suitable for reduced nonspecific binding during in vivo use. scFv fusion proteins may be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an scFv. See Antibody Engineering, ed. Borrebaeck, supra. The antibody fragment may also be a “linear antibody”, e.g., as described in U.S. Pat. No. 5,641,870, for example. Such linear antibodies may be monospecific or bispecific.

2. Humanized Antibodies

[0138] The present disclosure encompasses humanized antibodies. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody can have one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed following the method of Winter (Jones et al. (1986) Nature 321 :522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239: 1534-1536), by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0139] The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies can be important to reduce antigenicity. According to the so-called “best- fit” method, the sequence of the variable domain of a rodent (e.g., mouse) antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework for the humanized antibody (Sims et al. (1993) J. Immunol. 151 :2296; Chothia et al. (1987) J. Mol. Biol. 196:901. Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) J. Immunol., 151 :2623.

[0140] It is further generally desirable that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, according to one method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those, skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding. 3. Human A ntibodies

[0141] Human anti-Siglec-10 antibodies of the invention can be constructed by combining Fv clone variable domain sequence(s) selected from human-derived phage display libraries with known human constant domain sequences(s). Alternatively, human monoclonal anti-Siglec-10 antibodies of the invention can be made by the hybridoma method. Human myeloma and mousehuman heteromyeloma cell lines for the production of human monoclonal antibodies have been described, for example, by Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).

[0142] It is possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255 (1993); Bruggermann et al., Year in Immunol., 7: 33 (1993).

[0143] Gene shuffling can also be used to derive human antibodies from non-human (e.g., rodent) antibodies, where the human antibody has similar affinities and specificities to the starting non-human antibody. According to this method, which is also called “epitope imprinting”, either the heavy or light chain variable region of a non-human antibody fragment obtained by phage display techniques as described herein is replaced with a repertoire of human V domain genes, creating a population of non-human chain/human chain scFv or Fab chimeras. Selection with antigen results in isolation of a non-human chain/human chain chimeric scFv or Fab wherein the human chain restores the antigen binding site destroyed upon removal of the corresponding non-human chain in the primary phage display clone, z.e., the epitope governs the choice of the human chain partner. When the process is repeated in order to replace the remaining non-human chain, a human antibody is obtained (see PCT WO 93/06213 published Apr. 1, 1993). Unlike traditional humanization of non-human antibodies by CDR grafting, this technique provides completely human antibodies, which have no FR or CDR residues of non- human origin. 4. Multispecific Antibodies

[0144] Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different antigens. Bispecific antibodies may refer to antibodies that have binding specificities for two different antigens, or two different epitopes on the same antigen. In certain embodiments, bispecific antibodies are human or humanized antibodies. In certain embodiments, one of the binding specificities is for Siglec-10 and the other is for any other antigen. In certain embodiments, bispecific antibodies may bind to two different epitopes of Siglec-10. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express Siglec-10. Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab')2bispecific antibodies).

[0145] Methods for making bispecific antibodies are known in the art. See Milstein and Cuello, Nature, 305: 537 (1983), WO 93/08829 published May 13, 1993, and Traunecker et al., EMBO J., 10: 3655 (1991). For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121 :210 (1986). Bispecific antibodies include cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Heteroconjugate antibodies may be made using any convenient cross-linking method. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.

5. Single-Domain Antibodies

[0146] In some embodiments, an antibody of the invention is a single-domain antibody. A single-domain antibody is a single polyeptide chain comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 Bl). In one embodiment, a singledomain antibody consists of all or a portion of the heavy chain variable domain of an antibody.

6. Antibody Variants

[0147] In some embodiments, amino acid sequence modification(s) of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of the antibody may be prepared by introducing appropriate changes into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics. The amino acid alterations may be introduced in the subject antibody amino acid sequence at the time that sequence is made.

[0148] A useful method for identification of certain residues or regions of the antibody that are preferred locations for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244: 1081-1085. Here, a residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to affect the interaction of the amino acids with antigen. Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenesis is conducted at the target codon or region and the expressed immunoglobulins are screened for the desired activity.

[0149] Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme or a polypeptide which increases the serum half-life of the antibody.

[0150] In certain embodiments, an antibody of the invention is altered to increase or decrease the extent to which the antibody is glycosylated. Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X- threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5- hydroxyproline or 5-hydroxylysine may also be used.

[0151] Addition or deletion of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that one or more of the above-described tripeptide sequences (for N-linked glycosylation sites) is created or removed. The alteration may also be made by the addition, deletion, or substitution of one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

[0152] Where the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. For example, antibodies with a mature carbohydrate structure that lacks fucose attached to an Fc region of the antibody are described in US Pat Appl No US 2003/0157108 (Presta, L.). See also US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Antibodies with a bisecting N- acetylglucosamine (GlcNAc) in the carbohydrate attached to an Fc region of the antibody are referenced in WO 2003/011878, Jean-Mairet et al. and U.S. Pat. No. 6,602,684, Umana et al. Antibodies with at least one galactose residue in the oligosaccharide attached to an Fc region of the antibody are reported in WO 1997/30087, Patel et al. See, also, WO 1998/58964 (Raju, S.) and WO 1999/22764 (Raju, S.) concerning antibodies with altered carbohydrate attached to the Fc region thereof. See also US 2005/0123546 (Umana et al.) on antigen-binding molecules with modified glycosylation.

[0153] In certain embodiments, a glycosylation variant comprises an Fc region, wherein a carbohydrate structure attached to the Fc region lacks fucose or has reduced fucose. Such variants have improved ADCC function. Optionally, the Fc region further comprises one or more amino acid substitutions therein which further improve ADCC, for example, substitutions at positions 298, 333, and/or 334 of the Fc region (Eu numbering of residues). Examples of publications related to “defucosylated” or “fucose-deficient” antibodies include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; W02005/053742; Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines producing defucosylated antibodies include Lecl3 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US 2003/0157108 Al, Presta, L; and WO 2004/056312 Al, Adams et al., especially at Example 11), and knockout cell lines, such as alpha- 1,6-fucosyltransf erase gene, FUT8, knockout CHO cells (Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004)), and cells overexpressing pi,4-N-acetylglucosaminyltransferase III (GnT-III) and Golgi p-mannosidase II (Manll).

[0154] Antibodies are contemplated herein that have reduced fucose relative to the amount of fucose on the same antibody produced in a wild-type CHO cell. For example, the antibody has a lower amount of fucose than it would otherwise have if produced by native CHO cells (e.g., a CHO cell that produce a native glycosylation pattern, such as, a CHO cell containing a native FUT8 gene). In certain embodiments, an anti-Siglec-10 antibody provided herein is one wherein less than about 50%, 40%, 30%, 20%, 10%, 5% or 1% of the N-linked glycans thereon comprise fucose. In certain embodiments, an anti-Siglec-10 antibody provided herein is one wherein none of the N-linked glycans thereon comprise fucose, i.e., wherein the antibody is completely without fucose, or has no fucose or is non-fucosylated or is afucosylated. The amount of fucose can be determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn297 (e.g., complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. In some embodiments, at least one or two of the heavy chains of the antibody is non-fucosylated.

[0155] In one embodiment, the antibody is altered to improve its serum half-life. To increase the serum half-life of the antibody, one may incorporate a salvage receptor binding epitope into the antibody (especially an antibody fragment) as described in U.S. Pat. No. 5,739,277, for example. As used herein, the term “salvage receptor binding epitope” refers to an epitope of the Fc region of an IgG molecule (e.g., IgGl, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule (US 2003/0190311, U.S. Pat. No. 6,821,505; U.S. Pat. No. 6,165,745; U.S. Pat. No. 5,624,821; U.S. Pat. No. 5,648,260; U.S. Pat. No. 6,165,745; U.S. Pat. No. 5,834,597).

[0156] Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule replaced by a different residue. Sites of interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are shown in Table 1 under the heading of “preferred substitutions.” If such substitutions result in a desirable change in biological activity, then more substantial changes, denominated “exemplary substitutions” in Table 1, or as further described below in reference to amino acid classes, may be introduced and the products screened.

Table 1.

[0157] Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or c) the bulk of the side chain. Amino acids may be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)):

(1) non-polar: Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M)

(2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q) (3) acidic: Asp (D), Glu (E)

(4) basic: Lys (K), Arg (R), H s (H)

[0158] Alternatively, naturally occurring residues may be divided into groups based on common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

[0159] Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Such substituted residues also may be introduced into the conservative substitution sites or, into the remaining (non-conserved) sites.

[0160] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further development will have modified (e.g., improved) biological properties relative to the parent antibody from which they are generated. A convenient way for generating such substitutional variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibodies thus generated are displayed from filamentous phage particles as fusions to at least part of a phage coat protein (e.g., the gene III product of M13) packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity). In order to identify candidate hypervariable region sites for modification, scanning mutagenesis (e.g., alanine scanning) can be performed to identify hypervariable region residues contributing significantly to antigen binding.

Alternatively, or additionally, it may be beneficial to analyze a crystal structure of the antigenantibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues are candidates for substitution according to techniques known in the art, including those elaborated herein. Once such variants are generated, the panel of variants is subjected to screening using techniques known in the art, including those described herein, and antibodies with superior properties in one or more relevant assays may be selected for further development. [0161] Nucleic acid molecules encoding amino acid sequence variants of the antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the antibody. [0162] It may be desirable to introduce one or more amino acid modifications in an Fc region of antibodies of the present disclosure, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions including that of a hinge cysteine. In some embodiments, the Fc region variant comprises a human IgGl, IgG2, or IgG4 Fc region. Exemplary Fc region variants are provided herein.

[0163] In accordance with this description and the teachings of the art, it is contemplated that in some embodiments, an antibody of the invention may comprise one or more alterations as compared to the wild type counterpart antibody, e.g. in the Fc region. These antibodies would nonetheless retain substantially the same characteristics required for therapeutic utility as compared to their wild type counterpart. For example, it is thought that certain alterations can be made in the Fc region that would result in altered (i.e., either improved or diminished) Clq binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in WO99/51642. See also Duncan & Winter Nature 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO94/29351 concerning other examples of Fc region variants. WO00/42072 (Presta) and WO 2004/056312 (Lowman) describe antibody variants with improved or diminished binding to FcRs. The content of these patent publications are specifically incorporated herein by reference. See, also, Shields et al. J. Biol. Chem. 9(2): 6591-6604 (2001). Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol.

117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005/0014934A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Polypeptide variants with altered Fc region amino acid sequences and increased or decreased Clq binding capability are described in U.S. Pat. No. 6,194,551B1, WO99/51642. The contents of those patent publications are specifically incorporated herein by reference. See, also, Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

7. Vectors, Host Cells, and Recombinant Methods

[0164] For recombinant production of an antibody of the invention, the nucleic acid encoding it is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. DNA encoding the antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Many vectors are available. The choice of vector depends in part on the host cell to be used. Generally, host cells are of either prokaryotic or eukaryotic (generally mammalian) origin. It will be appreciated that constant regions of any isotype can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE constant regions, and that such constant regions can be obtained from any human or animal species.

Generating Antibodies Using Prokarvotic Host Cells: a) Vector Construction

[0165] Polynucleotide sequences encoding polypeptide components of the antibody of the invention can be obtained using standard recombinant techniques. Desired polynucleotide sequences may be isolated and sequenced from antibody producing cells such as hybridoma cells. Alternatively, polynucleotides can be synthesized using nucleotide synthesizer or PCR techniques. Once obtained, sequences encoding the polypeptides are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in prokaryotic hosts. Many vectors that are available and known in the art can be used for the purpose of the present invention. Selection of an appropriate vector will depend mainly on the size of the nucleic acids to be inserted into the vector and the particular host cell to be transformed with the vector. Each vector contains various components, depending on its function (amplification or expression of heterologous polynucleotide, or both) and its compatibility with the particular host cell in which it resides. The vector components generally include, but are not limited to: an origin of replication, a selection marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, the heterologous nucleic acid insert and a transcription termination sequence.

[0166] In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species. pBR322 contains genesencoding ampicillin (Amp) and tetracycline (Tet) resistance and thus provides easy means for identifying transformed cells. pBR322, its derivatives, or other microbial plasmids or bacteriophage may also contain, or be modified to contain, promoters which can be used by the microbial organism for expression of endogenous proteins. Examples of pBR322 derivatives used for expression of particular antibodies are described in detail in Carter et al., U.S. Pat. No. 5,648,237.

[0167] In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts. For example, bacteriophage such as XGEM.TM.-l 1 may be utilized in making a recombinant vector which can be used to transform susceptible host cells such as E. coli LE392. [0168] The expression vector of the invention may comprise two or more promoter-ci stron pairs, encoding each of the polypeptide components. A promoter is an untranslated regulatory sequence located upstream (5') to a cistron that modulates its expression. Prokaryotic promoters typically fall into two classes, inducible and constitutive. Inducible promoter is a promoter that initiates increased levels of transcription of the cistron under its control in response to changes in the culture condition, e.g. the presence or absence of a nutrient or a change in temperature.

[0169] A large number of promoters recognized by a variety of potential host cells are well known. The selected promoter can be operably linked to cistron DNA encoding the light or heavy chain by removing the promoter from the source DNA via restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the invention. Both the native promoter sequence and many heterologous promoters may be used to direct amplification and/or expression of the target genes. In some embodiments, heterologous promoters are utilized, as they generally permit greater transcription and higher yields of expressed target gene as compared to the native target polypeptide promoter.

[0170] Promoters suitable for use with prokaryotic hosts include the PhoA promoter, the P- galactamase and lactose promoter systems, a tryptophan (trp) promoter system and hybrid promoters such as the tac or the trc promoter. However, other promoters that are functional in bacteria (such as other known bacterial or phage promoters) are suitable as well. Their nucleotide sequences have been published, thereby enabling a skilled worker operably to ligate them to cistrons encoding the target light and heavy chains (Siebenlist et al. (1980) Cell 20: 269) using linkers or adaptors to supply any required restriction sites.

[0171] In one aspect of the invention, each cistron within the recombinant vector comprises a secretion signal sequence component that directs translocation of the expressed polypeptides across a membrane. In general, the signal sequence may be a component of the vector, or it may be a part of the target polypeptide DNA that is inserted into the vector. The signal sequence selected for the purpose of this invention should be one that is recognized and processed (i.e. cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the signal sequences native to the heterologous polypeptides, the signal sequence is substituted by a prokaryotic signal sequence selected, for example, from the group consisting of the alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II (STII) leaders, LamB, PhoE, PelB, OmpA and MBP. In one embodiment of the invention, the signal sequences used in both cistrons of the expression system are STII signal sequences or variants thereof.

[0172] In another aspect, the production of the immunoglobulins according to the invention can occur in the cytoplasm of the host cell, and therefore does not require the presence of secretion signal sequences within each cistron. In that regard, immunoglobulin light and heavy chains are expressed, folded and assembled to form functional immunoglobulins within the cytoplasm. Certain host strains (e.g., the E. coll trxB-strains) provide cytoplasm conditions that are favorable for disulfide bond formation, thereby permitting proper folding and assembly of expressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

[0173] Antibodies of the invention can also be produced by using an expression system in which the quantitative ratio of expressed polypeptide components can be modulated in order to maximize the yield of secreted and properly assembled antibodies of the invention. Such modulation is accomplished at least in part by simultaneously modulating translational strengths for the polypeptide components.

[0174] One technique for modulating translational strength is disclosed in Simmons et al., U.S. Pat. No. 5,840,523. It utilizes variants of the translational initiation region (TIR) within a cistron. For a given TIR, a series of amino acid or nucleic acid sequence variants can be created with a range of translational strengths, thereby providing a convenient means by which to adjust this factor for the desired expression level of the specific chain. TIR variants can be generated by conventional mutagenesis techniques that result in codon changes which can alter the amino acid sequence. In certain embodiments, changes in the nucleotide sequence are silent. Alterations in the TIR can include, for example, alterations in the number or spacing of Shine-Dalgarno sequences, along with alterations in the signal sequence. One method for generating mutant signal sequences is the generation of a “codon bank” at the beginning of a coding sequence that does not change the amino acid sequence of the signal sequence (i.e., the changes are silent). This can be accomplished by changing the third nucleotide position of each codon; additionally, some amino acids, such as leucine, serine, and arginine, have multiple first and second positions that can add complexity in making the bank. This method of mutagenesis is described in detail in Yansura et al. (1992) METHODS: A Companion to Methods in Enzymol. 4: 151-158.

[0175] In one embodiment, a set of vectors is generated with a range of TIR strengths for each cistron therein. This limited set provides a comparison of expression levels of each chain as well as the yield of the desired antibody products under various TIR strength combinations. TIR strengths can be determined by quantifying the expression level of a reporter gene as described in detail in Simmons et al. U.S. Pat. No. 5,840,523. Based on the translational strength comparison, the desired individual TIRs are selected to be combined in the expression vector constructs of the invention.

[0176] Prokaryotic host cells suitable for expressing antibodies of the invention include Archaebacteria and Eubacteria, such as Gram-negative or Gram-positive organisms. Examples of useful bacteria include Escherichia (e.g., E. coll), Bacilli (e.g., B. subtilis), Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or Paracoccus. In one embodiment, gramnegative cells are used. In one embodiment, E. coli cells are used as hosts for the invention. Examples of E. coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.: American Society for Microbiology, 1987), pp. 1190-1219; ATCC Deposit No. 27,325) and derivatives thereof, including strain 33D3 having genotype W3110 AfhuA (AtonA) ptr3 lac Iq lacL8 AompTA(nmpc-fepE) degP41 kanR (U.S. Pat. No. 5,639,635). Other strains and derivatives thereof, such as E. coli 294 (ATCC 31,446), E. coli B, E. coli/. 1776 (ATCC 31,537) and A. coli RV308(ATCC 31,608) are also suitable. These examples are illustrative rather than limiting. Methods for constructing derivatives of any of the above- mentioned bacteria having defined genotypes are known in the art and described in, for example, Bass et al., Proteins, 8:309-314 (1990). It is generally necessary to select the appropriate bacteria taking into consideration replicability of the replicon in the cells of a bacterium. For example, E. coli, Serratia, or Salmonella species can be suitably used as the host when well known plasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supply the replicon. Typically the host cell should secrete minimal amounts of proteolytic enzymes, and additional protease inhibitors may desirably be incorporated in the cell culture. b) Antibody Production

[0177] Host cells are transformed with the above-described expression vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[0178] Transformation means introducing DNA into the prokaryotic host so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integrant. Depending on the host cell used, transformation is done using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride is generally used for bacterial cells that contain substantial cell-wall barriers. Another method for transformation employs polyethylene glycol/DMSO. Yet another technique used is electroporation.

[0179] Prokaryotic cells used to produce the polypeptides of the invention are grown in media known in the art and suitable for culture of the selected host cells. Examples of suitable media include luria broth (LB) plus necessary nutrient supplements. In some embodiments, the media also contains a selection agent, chosen based on the construction of the expression vector, to selectively permit growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to media for growth of cells expressing ampicillin resistant gene.

[0180] Any necessary supplements besides carbon, nitrogen, and inorganic phosphate sources may also be included at appropriate concentrations introduced alone or as a mixture with another supplement or medium such as a complex nitrogen source. Optionally the culture medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycollate, di thioerythritol and dithiothreitol.

[0181] The prokaryotic host cells are cultured at suitable temperatures. In certain embodiments, for A. coll growth, growth temperatures range from about 20° C. to about 39° C.; from about 25° C. to about 37° C.; or about 30° C. The pH of the medium may be any pH ranging from about 5 to about 9, depending mainly on the host organism. In certain embodiments, for A. coli. the pH is from about 6.8 to about 7.4, or about 7.0.

[0182] If an inducible promoter is used in the expression vector of the invention, protein expression is induced under conditions suitable for the activation of the promoter. In one aspect of the invention, PhoA promoters are used for controlling transcription of the polypeptides. Accordingly, the transformed host cells are cultured in a phosphate-limiting medium for induction. In certain embodiments, the phosphate-limiting medium is the C.R.A.P. medium (see, e.g., Simmons et al., J. Immunol. Methods (2002), 263: 133-147). A variety of other inducers may be used, according to the vector construct employed, as is known in the art.

[0183] In one embodiment, the expressed polypeptides of the present invention are secreted into and recovered from the periplasm of the host cells. Protein recovery typically involves disrupting the microorganism, generally by such means as osmotic shock, sonication or lysis. Once cells are disrupted, cell debris or whole cells may be removed by centrifugation or filtration. The proteins may be further purified, for example, by affinity resin chromatography. Alternatively, proteins can be transported into the culture media and isolated therein. Cells may be removed from the culture and the culture supernatant being filtered and concentrated for further purification of the proteins produced. The expressed polypeptides can be further isolated and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assay.

[0184] In one aspect of the invention, antibody production is conducted in large quantity by a fermentation process. Various large-scale fed-batch fermentation procedures are available for production of recombinant proteins. Large-scale fermentations have at least 1000 liters of capacity, and in certain embodiments, about 1,000 to 100,000 liters of capacity. These fermentors use agitator impellers to distribute oxygen and nutrients, especially glucose. Small scale fermentation refers generally to fermentation in a fermentor that is no more than approximately 100 liters in volumetric capacity, and can range from about 1 liter to about 100 liters.

[0185] In a fermentation process, induction of protein expression is typically initiated after the cells have been grown under suitable conditions to a desired density, e.g., an OD550 of about 180-220, at which stage the cells are in the early stationary phase. A variety of inducers may be used, according to the vector construct employed, as is known in the art and described above. Cells may be grown for shorter periods prior to induction. Cells are usually induced for about 12-50 hours, although longer or shorter induction time may be used.

[0186] To improve the production yield and quality of the polypeptides of the invention, various fermentation conditions can be modified. For example, to improve the proper assembly and folding of the secreted antibody polypeptides, additional vectors overexpressing chaperone proteins, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (a peptidylprolyl cis, trans-isomerase with chaperone activity) can be used to co-transform the host prokaryotic cells. The chaperone proteins have been demonstrated to facilitate the proper folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al. (1999) J. Biol. Chem. 274: 19601-19605; Georgiou et al., U.S. Pat. No. 6,083,715; Georgiou et al., U.S. Pat. No. 6,027,888; Bothmann and Pluckthun (2000) J. Biol. Chem. 275: 17100-17105; Ramm and Pluckthun (2000) J. Biol. Chem. 275: 17106-17113; Arie et al. (2001) Mol. Microbiol. 39: 199-210.

[0187] To minimize proteolysis of expressed heterologous proteins (especially those that are proteolytically sensitive), certain host strains deficient for proteolytic enzymes can be used for the present invention. For example, host cell strains may be modified to effect genetic mutation(s) in the genes encoding known bacterial proteases such as Protease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V, Protease VI and combinations thereof. Some / . coll protease-deficient strains are available and described in, for example, Joly et al. (1998), supra; Georgiou et al., U.S. Pat. No. 5,264,365; Georgiou et al., U.S. Pat. No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72 (1996).

[0188] In one embodiment, E. coli strains deficient for proteolytic enzymes and transformed with plasmids overexpressing one or more chaperone proteins are used as host cells in the expression system of the invention. c) Antibody Purification

[0189] In one embodiment, the antibody protein produced herein is further purified to obtain preparations that are substantially homogeneous for further assays and uses. Standard protein purification methods known in the art can be employed. The following procedures are exemplary of suitable purification procedures: fractionation on immunoaffinity or ion-exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica or on a cationexchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration using, for example, Sephadex G-75.

[0190] In one aspect, Protein A immobilized on a solid phase is used for immunoaffinity purification of the antibody products of the invention. Protein A is a 41 kD cell wall protein from Staphylococcus aureas which binds with a high affinity to the Fc region of antibodies. Lindmark et al (1983) J. Immunol. Meth. 62: 1-13. The solid phase to which Protein A is immobilized can be a column comprising a glass or silica surface, or a controlled pore glass column or a silicic acid column. In some applications, the column is coated with a reagent, such as glycerol, to possibly prevent nonspecific adherence of contaminants.

[0191] As the first step of purification, a preparation derived from the cell culture as described above can be applied onto a Protein A immobilized solid phase to allow specific binding of the antibody of interest to Protein A. The solid phase would then be washed to remove contaminants non-specifically bound to the solid phase. Finally the antibody of interest is recovered from the solid phase by elution.

Generating Antibodies Using Eukaryotic Host Cells:

[0192] A vector for use in a eukaryotic host cell generally includes one or more of the following non-limiting components: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. a) Signal Sequence Component

[0193] A vector for use in a eukaryotic host cell may also contain a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide of interest. The heterologous signal sequence selected may be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In mammalian cell expression, mammalian signal sequences as well as viral secretory leaders, for example, the herpes simplex gD signal, are available. The DNA for such a precursor region is ligated in reading frame to DNA encoding the antibody. b) Origin of Replication

[0194] Generally, an origin of replication component is not needed for mammalian expression vectors. For example, the SV40 origin may typically be used only because it contains the early promoter. c) Selection Gene Component

[0195] Expression and cloning vectors may contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, where relevant, or (c) supply critical nutrients not available from complex media. [0196] One example of a selection scheme utilizes a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene produce a protein conferring drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin. [0197] Another example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the antibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-I and -II, primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.

[0198] For example, in some embodiments, cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR. In some embodiments, an appropriate host cell when wild-type DHFR is employed is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCC CRL-9096).

[0199] Alternatively, host cells (particularly wild-type hosts that contain endogenous DHFR) transformed or co-transformed with DNA sequences encoding an antibody, wild-type DHFR protein, and another selectable marker such as aminoglycoside 3 '-phosphotransferase (APH) can be selected by cell growth in medium containing a selection agent for the selectable marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Pat. No.

4,965,199. Host cells may include NSO, CHOK1, CHOK1SV or derivatives, including cell lines deficient in glutamine synthetase (GS). Methods for the use of GS as a selectable marker for mammalian cells are described in U.S. Pat. No. 5,122,464 and U.S. Pat. No. 5,891,693. d) Promoter Component

[0200] Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to nucleic acid encoding a polypeptide of interest (e.g., an antibody). Promoter sequences are known for eukaryotes. For example, virtually all eukaryotic genes have an AT -rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region where N may be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition of the poly A tail to the 3' end of the coding sequence. In certain embodiments, any or all of these sequences may be suitably inserted into eukaryotic expression vectors.

[0201] Transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, from heatshock promoters, provided such promoters are compatible with the host cell systems.

[0202] The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hindlll E restriction fragment. A system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in U.S. Pat. No. 4,419,446. A modification of this system is described in U.S. Pat. No. 4,601,978. See also Reyes et al., Nature 297:598-601 (1982), describing expression of human P-interferon cDNA in mouse cells under the control of a thymidine kinase promoter from herpes simplex virus. Alternatively, the Rous Sarcoma Virus long terminal repeat can be used as the promoter. e) Enhancer Element Component

[0203] Transcription of DNA encoding an antibody of this invention by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, a-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the human cytomegalovirus early promoter enhancer, the mouse cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature 297: 17-18 (1982) describing enhancer elements for activation of eukaryotic promoters. The enhancer may be spliced into the vector at a position 5' or 3' to the antibody polypeptide-encoding sequence, but is generally located at a site 5' from the promoter. f) Transcription Termination Component

[0204] Expression vectors used in eukaryotic host cells may also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding an antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94/11026 and the expression vector disclosed therein. g) Selection and Transformation of Host Cells [0205] Suitable host cells for cloning or expressing the DNA in the vectors herein include higher eukaryote cells described herein, including insect or vertebrate host cells. Propagation of insect or vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful insect cell lines are Sf-9 and Sf-21 of Spodoptera frugiperda, DS2 cells of Drosophila melanogaster, or High Five cells (BTI-TN-5B1-4) of Trichopulsia ni. See, e.g., Frenzel, A. el al. (2013) Front. Immunol. 4:217. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/- DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3 A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; CHOK1 cells, CHOK1SV cells or derivatives and a human hepatoma line (Hep G2).

[0206] Host cells are transformed with the above-described-expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. h) Culturing the Host Cells

[0207] The host cells used to produce an antibody of this invention may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. No. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan. i) Purification of Antibody

[0208] When using recombinant techniques, the antibody can be produced intracellularly, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, may be removed, for example, by centrifugation or ultrafiltration. Where the antibody is secreted into the medium, supernatants from such expression systems may be first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

[0209] The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being a convenient technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc region that is present in the antibody. Protein A can be used to purify antibodies that are based on human yl, y2, or y4 heavy chains (Lindmark et al., J. Immunol. Methods 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and for human y3 (Guss et al., EMBO J. 5: 15671575 (1986)). The matrix to which the affinity ligand is attached may be agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABX™ resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™ chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered. [0210] Following any preliminary purification step(s), the mixture comprising the antibody of interest and contaminants may be subjected to further purification, for example, by low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, performed at low salt concentrations e.g., from about 0-0.25M salt).

[0211] In general, various methodologies for preparing antibodies for use in research, testing, and clinical use are well-established in the art, consistent with the above-described methodologies and/or as deemed appropriate by one skilled in the art for a particular antibody of interest.

Production of non-fucosylated antibodies

[0212] Provided herein are methods for preparing antibodies with a reduced degree of fucosylation. For example, methods contemplated herein include, but are not limited to, use of cell lines deficient in protein fucosylation (e.g., Lecl3 CHO cells, alpha- 1,6-fucosyltransferase gene knockout CHO cells, cells overexpressing pi,4-N-acetylglucosaminyltransferase III and further overexpressing Golgi p-mannosidase II, etc.), and addition of a fucose analog(s) in a cell culture medium used for the production of the antibodies. See Ripka et al. Arch. Biochem.

Biophys. 249:533-545 (1986); US Pat Appl No US 2003/0157108 Al, Presta, L; WO 2004/056312 Al, Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); and US Pat. No. 8,574,907. Additional techniques for reducing the fucose content of antibodies include Glymaxx technology described in U.S. Patent Application Publication No. 2012/0214975. Additional techniques for reducing the fucose content of antibodies also include the addition of one or more glycosidase inhibitors in a cell culture medium used for the production of the antibodies.

Glycosidase inhibitors include a-glucosidase I, a-glucosidase II, and a-mannosidase I. In some embodiments, the glycosidase inhibitor is an inhibitor of a-mannosidase I (e.g., kifunensine). [0213] As used herein, “core fucosylation” refers to addition of fucose (“fucosylation”) to N- acetylglucosamine (“GlcNAc”) at the reducing terminal of an N-linked glycan. Also provided are antibodies produced by such methods and compositions thereof.

[0214] In some embodiments, fucosylation of complex N-glycoside-linked sugar chains bound to the Fc region (or domain) is reduced. As used herein, a “complex N-glycoside-linked sugar chain” is typically bound to asparagine 297 (according to the number of Kabat), although a complex N-glycoside linked sugar chain can also be linked to other asparagine residues. A “complex N-glycoside-linked sugar chain” excludes a high mannose type of sugar chain, in which only mannose is incorporated at the non-reducing terminal of the core structure, but includes 1) a complex type, in which the non-reducing terminal side of the core structure has one or more branches of galactose-N-acetylglucosamine (also referred to as “gal-GlcNAc”) and the non-reducing terminal side of Gal-GlcNAc optionally has a sialic acid, bisecting N- acetylglucosamine or the like; or 2) a hybrid type, in which the non-reducing terminal side of the core structure has both branches of the high mannose N-glycoside-linked sugar chain and complex N-glycoside-linked sugar chain.

[0215] In some embodiments, the “complex N-glycoside-linked sugar chain” includes a complex type in which the non-reducing terminal side of the core structure has zero, one or more branches of galactose-N-acetylglucosamine (also referred to as “gal-GlcNAc”) and the nonreducing terminal side of Gal-GlcNAc optionally further has a structure such as a sialic acid, bisecting N-acetylglucosamine or the like.

[0216] According to the present methods, typically only a minor amount of fucose is incorporated into the complex N-glycoside-linked sugar chain(s). For example, in various embodiments, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 1% of the antibody has core fucosylation by fucose in a composition. In some embodiments, substantially none (z.e., less than about 0.5%) of the antibody has core fucosylation by fucose in a composition. In some embodiments, more than about 40%, more than about 50%, more than about 60%, more than about 70%, more than about 80%, more than about 90%, more than about 91%, more than about 92%, more than about 93%, more than about 94%, more than about 95%, more than about 96%, more than about 97%, more than about 98%, or more than about 99% of the antibody is nonfucosylated in a composition.

[0217] In some embodiments, provided herein is an antibody wherein substantially none (z.e., less than about 0.5%) of the N-glycoside-linked carbohydrate chains contain a fucose residue. In some embodiments, provided herein is an antibody wherein at least one or two of the heavy chains of the antibody is non-fucosylated.

[0218] As described above, a variety of mammalian host-expression vector systems can be utilized to express an antibody. In some embodiments, the culture media is not supplemented with fucose. In some embodiments, an effective amount of a fucose analog is added to the culture media. In this context, an “effective amount” refers to an amount of the analog that is sufficient to decrease fucose incorporation into a complex N-glycoside-linked sugar chain of an antibody by at least about 10%, at least about 20%, at least about 30%, at least about 40% or at least about 50%. In some embodiments, antibodies produced by the instant methods comprise at least about 10%, at least about 20%, at least about 30%, at least about 40% or at least about 50% non-core fucosylated protein (e.g., lacking core fucosylation), as compared with antibodies produced from the host cells cultured in the absence of a fucose analog.

[0219] The content (e.g., the ratio) of sugar chains in which fucose is not bound to N- acetylglucosamine in the reducing end of the sugar chain versus sugar chains in which fucose is bound to N-acetylglucosamine in the reducing end of the sugar chain can be determined, for example, as described in the Examples. Other methods include hydrazinolysis or enzyme digestion (see, e.g., Biochemical Experimentation Methods 23: Method for Studying Glycoprotein Sugar Chain (Japan Scientific Societies Press), edited by Reiko Takahashi (1989)), fluorescence labeling or radioisotope labeling of the released sugar chain and then separating the labeled sugar chain by chromatography. Also, the compositions of the released sugar chains can be determined by analyzing the chains by the HPAEC-PAD method (see, e.g., J. Liq Chromatogr. 6: 1557 (1983)). (See generally U.S. Patent Application Publication No. 2004/0110282.).

IV. Compositions

[0220] In some aspects, also provided herein are compositions (e.g., pharmaceutical composition) comprising any of the anti-Siglec-10 antibodies described herein. In some aspects, provided herein is a composition comprising an anti-Siglec-10 antibody described herein, wherein the antibody comprises a Fc region and N-glycoside-linked carbohydrate chains linked to the Fc region, wherein less than about 50% of the N-glycoside-linked carbohydrate chains contain a fucose residue. In some aspects, provided herein is a composition comprising an anti- Siglec-10 antibody described herein, wherein the antibody comprises a Fc region and N- glycoside-linked carbohydrate chains linked to the Fc region, wherein substantially none of the N-glycoside-linked carbohydrate chains contain a fucose residue.

[0221] Therapeutic formulations are prepared for storage by mixing the active ingredient having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington: The Science and Practice of Pharmacy, 20th Ed., Lippincott Williams & Wikiins, Pub., Gennaro Ed., Philadelphia, Pa. 2000). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers, antioxidants including ascorbic acid, methionine, Vitamin E, sodium metabi sulfite; preservatives, isotonicifiers, stabilizers, metal complexes (e.g. Zn-protein complexes); chelating agents such as EDTA and/or non-ionic surfactants.

[0222] Buffers can be used to control the pH in a range which optimizes the therapeutic effectiveness, especially if stability is pH dependent. Buffers can be present at concentrations ranging from about 50 mM to about 250 mM. Suitable buffering agents for use with the present invention include both organic and inorganic acids and salts thereof. For example, citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate. Additionally, buffers may be comprised of histidine and trimethylamine salts such as Tris.

[0223] Preservatives can be added to prevent microbial growth, and are typically present in a range from about 0.2%-1.0% (w/v). Suitable preservatives for use with the present invention include octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium halides (e.g., chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol, 3 -pentanol, and m-cresol.

[0224] Tonicity agents, sometimes known as “stabilizers” can be present to adjust or maintain the tonicity of liquid in a composition. When used with large, charged biomolecules such as proteins and antibodies, they are often termed “stabilizers” because they can interact with the charged groups of the amino acid side chains, thereby lessening the potential for inter and intramolecular interactions. Tonicity agents can be present in any amount between about 0.1% to about 25% by weight or between about 1 to about 5% by weight, taking into account the relative amounts of the other ingredients. In some embodiments, tonicity agents include polyhydric sugar alcohols, trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.

[0225] Additional excipients include agents which can serve as one or more of the following: (1) bulking agents, (2) solubility enhancers, (3) stabilizers and (4) and agents preventing denaturation or adherence to the container wall. Such excipients include: polyhydric sugar alcohols (enumerated above); amino acids such as alanine, glycine, histidine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycol; sulfur containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, a- monothioglycerol and sodium thio sulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin or other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose; disaccharides (e.g., lactose, maltose, sucrose); trisaccharides such as raffinose; and polysaccharides such as dextrin or dextran.

[0226] Non-ionic surfactants or detergents (also known as “wetting agents”) can be present to help solubilize the therapeutic agent as well as to protect the therapeutic protein against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody. Non-ionic surfactants are present in a range of about 0.05 mg/ml to about 1.0 mg/ml or about 0.07 mg/ml to about 0.2 mg/ml. In some embodiments, non-ionic surfactants are present in a range of about 0.001% to about 0.1% w/v or about 0.01% to about 0.1% w/v or about 0.01% to about 0.025% w/v.

[0227] Suitable non-ionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), poly oxamers (184, 188, etc.), PLURONIC® polyols, TRITON®, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, sucrose fatty acid ester, methyl celluose and carboxymethyl cellulose. Anionic detergents that can be used include sodium lauryl sulfate, dioctyle sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0228] In order for the formulations to be used for in vivo administration, they must be sterile. The formulation may be rendered sterile by filtration through sterile filtration membranes. The therapeutic compositions herein generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0229] The route of administration is in accordance with known and accepted methods, such as by single or multiple bolus or infusion over a long period of time in a suitable manner, e.g., injection or infusion by subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional or intraarticular routes, topical administration, inhalation or by sustained release or extended-release means.

[0230] The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may comprise a cytotoxic agent, cytokine or growth inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

V. Methods of Treatment

[0231] Provided herein are methods for treating or delaying progression of cancer in an individual. In some embodiments, the methods comprise administering to a subject an effective amount of an anti-Siglec-10 antibody or composition thereof described herein. In some embodiments, the individual is a human. In some embodiments, the anti-Siglec-10 antibody is used in treating, delaying the progression of, preventing relapse of or alleviating a symptom of a cancer or other neoplastic condition, as a monotherapy, or in combinations with an additional anti-cancer agent(s) or therapy, e.g., as a combination therapy.

[0232] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a liquid cancer, such as a blood or hematological cancer, e.g., leukemia, lymphoma, myeloma, etc. In some embodiments, the cancer is selected from the group consisting of gastric cancer, breast cancer, lung cancer, ovarian cancer, cervical cancer, prostate cancer, bladder cancer, colorectal cancer, pancreatic cancer, liver cancer, renal cancer, thyroid cancer, brain cancer, head and neck cancer, leukemia, lymphoma, myeloma, carcinoma, melanoma, leiomyoma, leiomyosarcoma, glioma, glioblastoma, and esophageal cancer. In some embodiments, administration of the antibody or composition comprising an anti-Siglec-10 antibody of the present disclosure reduces or alleviates one or more symptom(s) of cancer. Symptoms associated with cancers and other neoplastic disorders include, but are not limited to, inflammation, fever, general malaise, pain, loss of appetite, weight loss, edema, headache, fatigue, rash, anemia, muscle weakness and muscle fatigue.

[0233] In some embodiments, the antibody or composition comprising an anti-Siglec-10 antibody of the present disclosure is administered in combination with an additional anti-cancer agent. In some embodiments, the additional anti-cancer agent is a chemotherapeutic agent, anti- neoplastic agent, cytokine, growth factor inhibitor, immunosuppressant, anti-inflammatory agent, metabolic inhibitor, enzyme inhibitor, and/or cytotoxic or cytostatic agent. In some embodiments, the additional anti-cancer agent is a vaccine, e.g., a cancer vaccine. In some embodiments, the additional anti-cancer agent is an immunotherapeutic agent. In some embodiments, the chemotherapeutic agent comprises aldesleukin, altretamine, amifostine, asparaginase, bleomycin, capecitabine, carboplatin, carmustine, cladribine, cisapride, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, docetaxel, doxorubicin, dronabinol, duocarmycin, etoposide, filgrastim, fludarabine, fluorouracil, gemcitabine, granisetron, hydroxyurea, idarubicin, ifosfamide, interferon alpha, irinotecan, lansoprazole, levamisole, leucovorin, megestrol, mesna, methotrexate, metoclopramide, mitomycin, mitotane, mitoxantrone, omeprazole, ondansetron, paclitaxel (Taxol™), pilocarpine, prochloroperazine, saproin, tamoxifen, taxol, topotecan hydrochloride, vinblastine, vincristine, or vinorelbine tartrate. In some embodiments, the additional anti-cancer agent is an antibody, e.g., an antibody approved or under investigation for use in cancer. Such antibodies include, without limitation, rituximab (Rituxan®, CD20: chimeric IgGl), trastuzumab (Herceptin®, HER2: chimeric IgGl), alemtuzumab (Campath®, CD52: humanized IgGl), ibritumomab tiuxetan (Zevalin®, CD20: murine, IgGl, radiolabeled (Yttrium 90), tositumomab-I-131 (Bexxar®: CD20, murine, IgG2a, radiolabeled (Iodine 131)), cetuximab (Erbitux®, EGFR: chimeric, IgGl), bevacizumab (VEGF: humanized, IgG4), panitumumab (Vectibix®, EGFR: human IgG2), ofatumumab (Arzerra®, CD20: human IgGl), ipilimumab (Ypervoy®, CTLA-4: human IgGl), brentiuximab vedotin (Adectris®, CD30: chimeric, IgGl, drug-conjugate), pertuzumab (Perjecta®, HER2: humanized IgGl, drug conjugate), adotrastuzumab ematansine (Kadcyla®, HER2: humanized, IgGl, drugconjugate), obinutuzumab (Gazyva®, CD20: humanized and glycol-engineered), nivolumab, atezolizumab, and pembrolizumab. In some embodiments, the antibody binds a cancer- expressed or tumor-associated antigen. In some embodiments, the antibody modulates activity of the immune system, e.g., an immune checkpoint inhibitor (ICI) such as antibodies targeting CTLA4, PD1, or PDL1, or an immune modulator that acts on macrophages or dendritic cells. [0234] Provided herein are methods for treating or delaying progression of an autoimmune disease or disorder in an individual. In some embodiments, the methods comprise administering to a subject an effective amount of an anti-Siglec-10 antibody or composition thereof described herein. In some embodiments, the individual is a human. Examples of autoimmune diseases or disorders include, without limitation, inflammatory bowel disease (IBD), rheumatoid arthritis (RA), multiple sclerosis (MS), psoriasis, and systemic lupus erythematosus.

[0235] Provided herein are methods for depleting cells expressing Siglec-10 in an individual, e.g., in need thereof. In some embodiments, the methods comprise administering to a subject an effective amount of an anti-Siglec-10 antibody or composition thereof described herein. In some embodiments, the individual is a human. In some embodiments, the cells are myeloid cells. In some embodiments, the individual has or has been diagnosed with an autoimmune disease or disorder, including without limitation inflammatory bowel disease (IBD), rheumatoid arthritis (RA), multiple sclerosis (MS), psoriasis, or systemic lupus erythematosus.

[0236] For the prevention or treatment of disease, the appropriate dosage of an active agent, will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the agent is administered for preventive or therapeutic purposes, previous therapy, the subject's clinical history and response to the agent, and the discretion of the attending physician. The agent is suitably administered to the subject at one time or over a series of treatments. In some embodiments of the methods described herein, an interval between administrations of an anti-Siglec-10 antibody described is about one month or longer. In some embodiments, the interval between administrations is about two months, about three months, about four months, about five months, about six months or longer. As used herein, an interval between administrations refers to the time period between one administration of the antibody and the next administration of the antibody. As used herein, an interval of about one month includes four weeks. Accordingly, in some embodiments, the interval between administrations is about four weeks, about eight weeks, about twelve weeks, about sixteen weeks, about twenty weeks, about twenty four weeks, or longer. In some embodiments, the treatment includes multiple administrations of the antibody, wherein the interval between administrations may vary. For example, the interval between the first administration and the second administration is about one month, and the intervals between the subsequent administrations are about three months. In some embodiments, the interval between the first administration and the second administration is about one month, the interval between the second administration and the third administration is about two months, and the intervals between the subsequent administrations are about three months. In some embodiments, an anti-Siglec-10 antibody described herein is administered at a flat dose. In some embodiments, an anti-Siglec-10 antibody described herein is administered to a subject at a dosage from about 150 to about 450 mg per dose. In some embodiments, the anti- Siglec-10 antibody is administered to a subject at a dosage of about any of 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, and 450 mg per dose. In some embodiments, an anti-Siglec-10 antibody described herein is administered at a weight-based dose. In some embodiments, an anti-Siglec-10 antibody described herein is administered to a subject at a dosage from about 0.1 mg/kg to about 10 mg/kg or about 1.0 mg/kg to about 10 mg/kg. In some embodiments, an anti- Siglec-10 antibody described herein is administered to a subject at a dosage of about any of 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 3.0 mg/kg, 3.5 mg/kg, 4.0 mg/kg, 4.5 mg/kg, 5.0 mg/kg, 5.5 mg/kg, 6.0 mg/kg, 6.5 mg/kg, 7.0 mg/kg, 7.5 mg/kg, 8.0 mg/kg, 8.5 mg/kg, 9.0 mg/kg, 9.5 mg/kg, or 10.0 mg/kg. Any of the dosing frequency described above may be used.

VI. Articles of Manufacture or Kits

[0237] In another aspect, an article of manufacture or kit is provided which comprises an anti- Siglec-10 antibody or composition described herein. The article of manufacture or kit may further comprise instructions for use of the antibody or composition in the methods of the present disclosure. Thus, in certain embodiments, the article of manufacture or kit comprises instructions for the use of an anti-Siglec-10 antibody or composition in methods for treating or delaying progression of cancer and/or treating a disease or condition characterized by increased activity and/or number of cells expressing Siglec-10, e.g., in a subject in need thereof. In certain embodiments, the individual is a human.

[0238] The article of manufacture or kit may further comprise a container. Suitable containers include, for example, bottles, vials (e.g., dual chamber vials), syringes (such as single or dual chamber syringes) and test tubes. The container may be formed from a variety of materials such as glass or plastic. The container holds the formulation.

[0239] The article of manufacture or kit may further comprise a label or a package insert, which is on or associated with the container, may indicate directions for reconstitution and/or use of the formulation. The label or package insert may further indicate that the formulation is useful or intended for subcutaneous, intravenous, or other modes of administration for treating or preventing a mast cell-mediated disorder in an individual. The container holding the formulation may be a single-use vial or a multi-use vial, which allows for repeat administrations of the reconstituted formulation. The article of manufacture or kit may further comprise a second container comprising a suitable diluent. The article of manufacture or kit may further include other materials desirable from a commercial, therapeutic, and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0240] In a specific embodiment, the present invention provides kits for a single doseadministration unit. Such kits comprise a container of an aqueous formulation of therapeutic antibody, including both single or multi-chambered pre-filled syringes. Exemplary pre-filled syringes are available from Vetter GmbH, Ravensburg, Germany. 10241] The article of manufacture or kit herein optionally further comprises a container comprising a second medicament, wherein the anti-Siglec-10 antibody is a first medicament, and which article or kit further comprises instructions on the label or package insert for treating the subject with the second medicament (e.g., an additional anti-cancer agent of the present disclosure), in an effective amount.

[0242] In another embodiment, provided herein is an article of manufacture or kit comprising the formulations described herein for administration in an auto-injector device. An auto-injector can be described as an injection device that upon activation, will deliver its contents without additional necessary action from the patient or administrator. They are particularly suited for self-medication of therapeutic formulations when the delivery rate must be constant and the time of delivery is greater than a few moments.

[0243] The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

EXAMPLES

[0244] The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

Example 1: Generation and characterization of anti-Siglec-10 antibodies

[0245] Siglec-10 (also known as SLG2, PRO940, and SIGLEC10) is an inhibitory receptor that is expressed by immune cells spanning both the myeloid and lymphoid lineages. Siglec-10 binds to its ligand, CD-24, to induce inhibitory intracellular signaling cascades within immune cells. Tumor cells overexpress CD24 in a HIF la-dependent mechanism. The binding of tumor- expressed CD24 to immune-expressed Siglec-10 is thought to lead to immune evasion, as depicted in FIG. 1A. However, by treating intratumoral myeloid cells (e.g. tumor associated macrophages, TAMs) with anti-Siglec-10 antibody to block this interaction, the myeloid cell processes of phagocytosing tumor cells and priming cytotoxic lymphocytes are thought to be restored, potentially leading to improved antitumor immunity (FIG. 1A). See, e.g., Barkal, A. A. et al. (2019) Nature 572:392-396; Xiao, N. et al. (2021) Exp Hematol Oncology 10:36; Bandala- Sanchez, E. et al. (2013) Nat. Immunol. 14:741-748; Whitney, G. et al. (2001) Eur. J. Biochem 268:6083-6096; Chen, G.Y. et al. (2009) Science 323: 1722-1725; Bandala-Sanchez, E. et al. (2018) Proc. Natl. Acad. Sci. 115:7783-7788; and International Publication No.

WO20 17/085166.

[0246] This example describes the generation and characterization of antibodies that bind various epitopes of human Siglec-10 with high affinity and show potent inhibition of myeloid cell Siglec-10 binding to its ligand, CD24, in tumor cells both in vitro and in vivo.

Materials and Methods

Expression of Siglec-10

[0247] Fresh human tumor and normal (i.e. non-diseased) tissue samples were dissociated into single cells and immunophenotyped to identify the immune cell populations that express Siglec-10 and to evaluate expression levels and patterns in healthy and tumor tissues. Following tissue digestion, macrophage and dendritic cell populations were identified by flow cytometry and stained with an anti-Siglec-10 mAb (BioLegend, clone 5G6) to evaluate expression.

Generation of antibodies

[0248] Mice were immunized with Siglec-10 ECD-Fc and boosted with Siglec-10 ECD comprising the amino acid sequence of SEQ ID NO:73. Following immunization, mice with high titer tail bleeds were selected and spleens and lymph nodes were harvested and fused with myeloma cells to generate hybridomas. Supernatants from hydridoma clones were screened against Siglec-10 ECD using ELISA and high affinity clones were selected for variable region sequencing. Variable regions were then cloned into a mouse IgGl plasmid, recombinantly expressed, and purified for biochemical and functional characterization. Antibody characterization

[0249] Bivalent binding affinities of IgG for Siglec-10 mAbs were measured by biolayer interferometry using a ForteBio Octet Red 384 instrument using immobilized Siglec-10 ECD Fc protein.

[0250] Domain mapping was assessed by ELISA using recombinantly expressed Siglec-10 extracellular domains fused to a human Fc containing the full ECD, domains 1, 2, and 3, domain 1 and 2, or domain 1. Siglec-10 mAbs were tested for binding to each of these domain fusion proteins and assigned domains based on their specific binding properties. Siglec-10 mAbs that bound to all four fusion proteins were assigned domain 1 binders, mAbs that bound to full ECD and domains 1, 2, and 3 were assigned domain 3 binders, mAbs that bound to full ECD and domains 1 and 2 were assigned domain 2 binders, and mAbs that bound to only the full ECD protein were assigned domain 4 binders.

[0251] Epitope binning was assessed by biolayer interferometry using a ForteBio Octet Red 384 instrument using immobilized Siglec-10 ECD Fc protein. A panel of 9 Siglec-10 mAbs were tested in pairwise fashion by saturating Siglec-10 ECD with one Siglec-10 mAb followed by binding evaluation of a second Siglec-10 mAb. Siglec-10 mAbs were assigned the same bin if binding was reduced or blocked. mAbs were assigned different bins if the no blocking occurred.

Siglec-10 internalization

[0252] Human non-classical monocytes were isolated from healthy donors (n=5) and cultured overnight in the presence of anti-SigleclO mAbs (5 pg/mL) or an isotype control (1 pg/mL). Siglec-10 internalization was assessed the next day by flow cytometry using a non-blocking, fluorophore conjugated Siglec-10 mAb. Data from each mAb condition were normalized to isotype control and represented as the percent of Siglec-10 on the cell surface.

Siglec-10 ligand binding assays

[0253] MCF-7 cancer cells were cultured in the presence of a biotinylated Siglec-10 extracellular domain (ECD) that binds to Siglec-10 ligand expressed on the MCF-7 cells. Cells were co-incubated with Siglec-10 ECD (10 pg/mL) and with either anti-Siglec-10 mAbs (5 pg/mL) or an isotype control (1 pg/mL) for 30 mins to evaluate the ability of each anti-Siglec-10 mAb to block Siglec-10 ECD/ligand binding. Post-incubation, MCF-7 cells were washed and stained with fluorescent streptavidin and then analyzed by flow cytometry.

Siglec-10 mouse assays

[0254] Mice expressing humanized Siglec-10 were implanted with pancreatic syngeneic Panc02 tumor cells. At days 7 and 14 post-implantation, mice were given intraperitoneal (i.p.) injections of either a Siglec-10 mAh or isotype control at a dose of 10 pg/kg. Tumor growth was measured using standard caliper measurement practices. On day 21 post-implantation, tumors were harvested and weighed. An overview of the experimental timeline is provided in FIG. 5.

Results

[0255] Siglec-10 mRNA expression was examined in human cancers from the TCGA (The Cancer Genome Atlas) and normal matched-tissue from the GTEX (Genotype Tissue Expression Project) (FIG. IB). These results indicated that Siglec-10 expression is upregulated in multiple human solid cancers, as compared to Siglec-10 expression in normal matched-tissue. Siglec-10 expression was further examined in the colon adenocarcinoma cohort and stratified into Siglec- 10 high and Siglec-10 low cohorts (FIG. 1C). 2.75 FPKM was used as a cutoff to separate tumor samples into Siglec-10 high and Siglec-10 low cohorts, and survival probability of each cohort was examined (FIG. ID) These results indicated that Siglec-10 high expression in tumors was associated with significantly lower survival probability than Siglec-10 low expression (44% 5-year survival for Siglec-10 high cohort vs. 68% 5-year survival for Siglec-10 low cohort; n=338 for Siglec-10 low; n=100 for Siglec-10 high; p=0.026).

[0256] Flow cytometry was also used to assess Siglec-10 expression from human normal and tumor tissues. Siglec-10 expression was confirmed on myeloid cells including macrophages and cDCl and cDC2 dendritic cells (FIG. 2). Siglec-10 expression was identified to be upregulated in TAMs in tumor tissues compared to normal tissue.

[0257] Antibodies that bind to various epitopes across the extracellular domain of human Siglec-10 were generated. Newly generated antibodies were characterized, along with a previous antibody from BioLegend (clone # 5G6). The epitope of each new anti-Siglec-10 antibody was mapped to Domain 1, 3, or 4 of the human Siglec-10 ECD and classified into bins (Table A). Affinity of binding to human Siglec-10 and ligand blocking of human Siglec-10 were also determined for each antibody, as shown in Table A.

[0258] Next, the effect of antibody binding on monocyte internalization of Siglec-10 was examined. Internalization was assessed by flow cytometry using a fluorophore-conjugated anti- Siglec-10 mAb. Each anti-Siglec-10 mAb tested induced significant internalization of the Siglec-10 receptor compared to isotype control (FIG. 3). Without wishing to be bound to theory, it is thought that induction of Siglec-10 internalization may be advantageous, e.g., in preventing interaction between Siglec-10 and one or more of its ligand(s), since it would not be accessible on the cell surface.

[0259] The ability of Siglec-10 to bind to its ligand on MCF-7 tumor cells in the presence of various anti-Siglec-10 antibodies was assessed by flow cytometry in order to evaluate if anti- Siglec-10 mAbs blocked the interaction between Siglec-10 ECD and the MCF-7 tumor cells (FIG. 4). Most anti-Siglec-10 mAbs blocked ligand binding; however, several clones did not (Table A). Anti-Siglec-10 mAbs AK09, AK02, and BL5G6 showed no ability to block ligand binding.

[0260] Activity of anti-Siglec-10 mAb treatment was also tested in the humanized mouse Panc02 tumor model described above (see also FIG. 5). In FIG. 6A, mice implanted with Panc02 tumor cells that received two doses of anti-Siglec-10 mAb (AK01) showed significant reduction in tumor growth over time compared to mice that received isotype control. In corroboration, data presented in FIG. 6B show that the final weight of tumors from mice given anti-Siglec- 10 mAb was lower than the final weight of tumors from mice given isotype control. These results demonstrate an improved tumor outcome upon in vivo administration of anti- Siglec-10 mAb as a potential pancreatic cancer treatment.

[0261] Taken together, these results demonstrate the generation of new anti-Siglec-10 antibodies with high-affinity binding that cover a range of unique epitopes on Siglec-10. In addition, these results identify anti-Siglec-10 antibodies as a potential anti-cancer therapeutic.

Example 2: Activity of anti-Siglec-10 antibodies in a mouse colorectal tumor model [0262] Anti -tumor activity of anti-Siglec-10 mAb treatment was next investigated in a syngeneic model of colorectal cancer using MC38 tumor cells in mice expressing human Siglec- 10.

Materials and Methods

[0263] The study design is shown in FIG. 7A. Mice expressing human Siglec-10 were implanted with MC38 tumor cells and followed for 16 days. On days 1, 5, 10 and 15, mice were dosed (i.p.) with either anti-Siglec-10 mAb (AK01) or isotype control at lOmg/kg. Tumor growth was monitored over two weeks and measured using standard practices. On day 16, tumors were harvested. For cellular analysis, tumors were processed into single cells and immuno-phenotyped using flow cytometry. Tumor associated macrophages (TAMs) were identified as Ly6C MHC class II⁺ F480⁺; tumor associated dendritic cells were identified as CD 11 c^hlgh MHC class II⁺ CD 103⁺ .

Results

[0264] Tumor growth over time is shown in FIG. 7B. Treatment with a Siglec-10 mAb significantly reduced tumor size compared to isotype control treated mice.

[0265] Treatment with anti-Siglec-10 mAb significantly increased the percentage of TAMs compared to isotype control treated mice (FIG. 7C). In addition, MHC class II was upregulated on TAMs from anti-Siglec-10 mAb-treated mice, indicative of an activated phenotype (FIG. 7D).

[0266] Treatment with anti-Siglec-10 mAb significantly increased the percentage of tumor- associated dendritic cells (CD 103+) compared to isotype control treated mice (FIG. 7E). MHC class II was upregulated on CD103+ dendritic cells from anti-Siglec-10 mAb-treated mice, indicative of an activated phenotype (FIG. 7F).

[0267] Treatment anti-Siglec-10 mAb significantly increased the percentage of both CD8+ (FIG. 7G) and CD4+ (FIG. 71) T cells in the tumor compared to isotype control treated mice. PD-1 expression was upregulated on CD8+ T cells from anti-Siglec-10 mAb-treated mice, indicative of an activated phenotype (FIG. 7H).

[0268] MC38 tumors from mice treated with an anti-Siglec-10 mAb or isotype control were subjected to RNA-sequencing analysis. Differentially expressed genes are shown in FIG. 7 J. Siglec-10 mAb treatment induced upregulation of a significant number of genes associated with type-1 inflammation, T cell activation, antigen presentation and macrophages.

[0269] An anti-Siglec-10 mAb (AK01 variable regions) with an inactive Fc region (mlgGl D265A, or “DA;” see Baudino, L. et al. (2008) J. Immunol. 181 :6664-6669) was also tested. MC38 tumors were established in mice through subcutaneous injection. When tumors reached 100mm³, mice were administered an anti-Siglec-10 mlgGl DA or isotype control at 10 mg/kg every 2 days until study takedown. Tumor growth was assessed every 2 days. The results indicated that using a Siglec-10 mAb with an inactive Fc had enhanced anti -tumor activity (FIG.

7K).

[0270] These results demonstrate that treatment with anti-Siglec-10 mAb significantly reduced tumor growth and led to upregulation and activation of tumor-associated macrophages, dendritic cells, and T cells in a preclinical model of colorectal cancer.

Example 3: Antibody blockade of the immunoinhibitory receptor Siglec-10 induces receptor internalization and proinflammatory responses

[0271] Anti -tumor activity of anti-Siglec-10 mAb treatment was next investigated in a model of TLR-mediated lung inflammation in mice expressing human Siglec-10.

Materials and Methods

[0272] The study design is shown in FIG. 8A. Mice expressing human Siglec-10 were challenged intranasally with poly I:C or PBS on days 0, 1, 2, and 3. Prior to poly I:C administration, mice were dosed (i.p.) with either anti-Siglec-10 mAb (AK01) or isotype control at lOmg/kg. Lungs were obtained after study termination and processed into single cells.

Siglec-10 expression levels were measured on CD 103+ dendritic cells and Ly6high MHC+ inflammatory monocytes from processed lung tissue using flow cytometry. Levels of cytokines and chemokines were measured in serum using Meso Scale Discovery.

Results

[0273] Siglec-10 expression levels were measured on CD103+ DCs and Ly6C^h'^8h MHC+ inflammatory monocytes in lung tissue from mice intranasally challenged with poly (EC) and dosed with a Siglec-10 mAh or isotype control, compared to PBS challenged mice (FIG. 8B). Administration of the Siglec-10 mAh induced receptor internalization, leading to lower expression of Siglec-10 on CD103+ dendritic cells and Ly6C^h'^8h monocytes. Administration of the Siglec-10 mAh also led to enhanced TLR-mediated inflammation, as evidenced by increased levels of serum cytokines and chemokines such as IL-12p40, TNF, CCL2, and CCL4 (FIG. 8C). [0274] These results indicate that anti-Siglec-10 antibody treatment resulted in polarization of myeloid cells toward an inflammatory phenotype, suggesting that Siglec-10 represents a promising myeloid cell target for enhancing inflammation and anti-tumor immunity in solid tumors.

Example 4: Further characterization of anti-tumor response to anti-Siglec-10 antibody treatment

[0275] In order to determine a potential role for the ligands of Siglec-10 (e.g., CD24 and CD52) in cancer, human cancer cell lines were tested for levels of Siglec-10 ligand expression. Human cancer cell lines were cultured and incubated with fluorophore conjugated Siglec-10 extracellular domain (ECD). The following human cancer cell lines were used: OVCAR and SK-OV-3 for ovarian cancer; A549 for lung cancer; MCF-7, T47D, and SK-BR-3 for breast cancer; and SKMEL28 for melanoma. Siglec-10 ligand levels were assessed by flow cytometry. As shown in FIG. 9, siglec-10 ligand expression was observed in all human cancer cell lines assessed and was not observed in normal human tissue (skin).

[0276] Anti-Siglec-10 mAb was also tested for the ability to block binding between the Siglec- 10 ECD and human cancer cell lines. Cancer cell lines OVCAR and SK-BR-3 were pretreated with indicated antibodies, followed by incubation with Siglec-10 ECD (hlgGl-FC biotin) and streptavidin-APC detection. Antibody-dependent blockade of the Siglec-10 ligand interaction corresponded with the reduction of geometric mean fluorescence intensity (gMFI). As shown in FIG. 10, anti-Siglec-10 mAb AK01 blocked the interaction between Siglec-10 ECD and ligands expressed by these cancer cell lines.

[0277] Next, anti-Siglec-01 mAb AKIO (having VH and VL region sequences of SEQ ID Nos: 85 and 86, respectively) was tested for ability to increase antibody-dependent cellular phagocytosis (ADCP) by macrophages in the presence of an opsonizing antibody. A diagram of macrophage-mediated phagocytosis of tumor cells by tumor-opsonizing antibodies (e.g., anti- HER2 antibody trastuzumab) stimulated by anti-Siglec-10 mAb is shown in FIG. 11A. SK-BR- 3 cells were stained with CFSE cell tracer and treated with anti-HER2 trastuzumab (0.1 pg/ml). Next, CFSE-labeled tumor cells were incubated with human polarized macrophages previously incubated with 10 pg/mL AKIO Fc inert, AKIO active Fc, or isotype control. Phagocytosis levels were assessed by flow cytometry and determined as frequency of CD14⁺ CFSE⁺ cells. FIG. 11B shows phagocytosis of breast cancer cells (SK-BR-3) by human macrophages following treatment with trastuzumab, in presence of anti-Siglec-10 mAbs AKIO or isotype control. Treatment with AKIO having an inert Fc led to stimulation of phagocytosis of breast cancer cells. CD 163 expression was also assessed by flow cytometry. As shown in FIG. 11C, CD163 expression was downregulated after treatment with AKIO Fc inert, indicating repolarization of the macrophages.

[0278] Siglec-10 mAb treatment was also examined in the colorectal syngeneic mouse tumor model (MC38 cells) using mice expressing human Siglec-10 described above. Briefly, siglec-10 transgenic mice were challenged with MC38 cells, and tumor volume was monitored twice weekly. Once a tumor was established, mice were treated with Siglec-10 mAb (AK01 variable domain sequences with mlgGl having D265A Fc mutation to render it inert) or isotype control every 2 days for a total of 6 doses. On day 14, tissues (tumor and draining lymph nodes) were harvested and dissociated in single cells and immune profiled by flow cytometry. TAMs were gated on CD45 ⁺/CD1 lb ⁺/F4/80⁺, CD 103 ⁺ DCs were gated on CD45 ⁺ /CD1 lb ⁺ /CD1 lc ⁺ /MHCir /CD I 03 ⁺ and CD8 ⁺ T cells were gated on CD45 ⁺ /CD1 lb 7CD3⁺/CD8⁺. The results indicated that tumor-bearing mice following treatment with Siglec-10 mAb induced strong production of the pro-inflammatory cytokines IL-6 and IL-12 from TAMs (FIG. 12A) and migratory dendritic cells (FIG. 12B), respectively, and significantly upregulated co-stimulatory molecule CD80 on both TAMs and dendritic cells. Additionally, Siglec-10 mAb increased coproduction of TNF-a and IFN-y on CD8⁺ T cells (FIG. 12C). [0279] The reprogramming effect of anti-Siglec-10 mAb treatment on macrophages in the presence of tumor cells was further examined. FIG. 13 shows a heatmap of the cytokine profile for Ml -polarized macrophages in presence of Siglec-10 mAb (AK01 variable domain sequences with mlgGl having D265A Fc mutation to render it inert) or isotype control, and MC38 tumor cell line. MC38 cells were pre-treated with mitomycin (50 pM) for 48 hours and co-cultured with bone marrow-derived macrophages (BMDM) treated with antibodies (10 pg/ml) in presence of LPS+IFN-y for 16 hours. Supernatant was collected and analyzed by multiplex cytokine assay from Meso Scale discovery (MSD), for the release of IL-ip, IL-6, IL-12/23p40, TNF, KC, MCP-1, MIP- a, MIP-ip and IL-10. Multiple pro-inflammatory cytokines and chemokines such as IL-ip, TNF-a, MIP- a and MIP-ip were upregulated in presence of Siglec- 10 mAb (FIG. 13), suggesting its potential effect on reprograming macrophages on the tumor microenvironment (TME).

Claims

CLAIMS What is claimed is:

1. An antibody that binds to human Siglec-10, wherein the antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:79, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:80, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:81; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:82, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:83, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:84.

2. The antibody of claim 1, wherein the VH region comprises the amino acid sequence of SEQ ID NO:85 and/or the VL region comprises the amino acid sequence of SEQ ID NO:86.

3. An antibody that binds to human Siglec-10, wherein the antibody competes for binding to human Siglec-10 with a reference antibody that comprises a VH region comprising an HVR- H1 comprising the amino acid sequence of SEQ ID NO:79, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:80, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:81 and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:82, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:83, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:84.

4. An antibody that binds to human Siglec-10, wherein the antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 19, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:20, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:21; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:22, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:23, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:24.

5. An antibody that binds to human Siglec-10, wherein the antibody competes for binding to human Siglec-10 with a reference antibody that comprises a VH region comprising an HVR- H1 comprising the amino acid sequence of SEQ ID NO: 19, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:20, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:21 and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:22, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:23, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:24.

6. An antibody that binds to human Siglec-10, wherein the antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:25, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:26, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:27; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:28, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:30.

7. An antibody that binds to human Siglec-10, wherein the antibody competes for binding to human Siglec-10 with a reference antibody that comprises a VH region comprising an HVR- H1 comprising the amino acid sequence of SEQ ID NO:25, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:26, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:27 and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:28, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:29, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:30.

8. An antibody that binds to human Siglec-10, wherein the antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:31, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:32, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:33; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:34, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:35, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:36.

9. An antibody that binds to human Siglec-10, wherein the antibody competes for binding to human Siglec-10 with a reference antibody that comprises a VH region comprising an HVR- H1 comprising the amino acid sequence of SEQ ID NO:31, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:32, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:33 and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:34, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:35, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:36.

10. An antibody that binds to human Siglec-10, wherein the antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:37, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:38, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:39; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:40, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:41, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:42.

11. An antibody that binds to human Siglec-10, wherein the antibody competes for binding to human Siglec-10 with a reference antibody that comprises a VH region comprising an HVR- H1 comprising the amino acid sequence of SEQ ID NO:37, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:38, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:39 and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:40, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:41, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:42.

12. An antibody that binds to human Siglec-10, wherein the antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:43, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:44, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:45; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:46, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:47, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:48.

13. An antibody that binds to human Siglec-10, wherein the antibody competes for binding to human Siglec-10 with a reference antibody that comprises a VH region comprising an HVR- H1 comprising the amino acid sequence of SEQ ID NO:43, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:44, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:45 and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:46, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:47, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:48.

14. An antibody that binds to human Siglec-10, wherein the antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:49, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:50, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:51; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:52, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:53, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:54.

15. An antibody that binds to human Siglec-10, wherein the antibody competes for binding to human Siglec-10 with a reference antibody that comprises a VH region comprising an HVR- H1 comprising the amino acid sequence of SEQ ID NO:49, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:50, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:51 and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:52, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:53, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:54.

16. An antibody that binds to human Siglec-10, wherein the antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:55, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:56, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:57; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:58, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:59, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:60.

17. An antibody that binds to human Siglec-10, wherein the antibody competes for binding to human Siglec-10 with a reference antibody that comprises a VH region comprising an HVR- H1 comprising the amino acid sequence of SEQ ID NO:55, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:56, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:57 and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:58, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:59, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:60.

18. An antibody that binds to human Siglec-10, wherein the antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:61, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:62, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:63; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:64, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:65, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:66.

19. An antibody that binds to human Siglec-10, wherein the antibody competes for binding to human Siglec-10 with a reference antibody that comprises a VH region comprising an HVR- H1 comprising the amino acid sequence of SEQ ID NO:61, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:62, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:63 and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:64, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:65, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:66.

20. An antibody that binds to human Siglec-10, wherein the antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:67, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:68, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:69; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:70, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:71, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:72.

21. An antibody that binds to human Siglec-10, wherein the antibody competes for binding to human Siglec-10 with a reference antibody that comprises a VH region comprising an HVR- H1 comprising the amino acid sequence of SEQ ID NO:67, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:68, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:69 and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:70, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:71, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:72.

22. The antibody of any one of claims 1-19, wherein the antibody binds to Domain 1 of human Siglec-10.

23. The antibody of any one of claims 1-5 and 8-19, wherein binding of the antibody to human Siglec-10 blocks interaction between human Siglec-10 and human CD24.

24. The antibody of any one of claims 6, 7, 20, and 21, wherein binding of the antibody to human Siglec-10 does not block interaction between human Siglec-10 and human CD24.

25. The antibody of any one of claims 1-5 and 8-19, wherein binding of the antibody to human Siglec-10 blocks interaction between human Siglec-10 and human CD52.

26. The antibody of any one of claims 6, 7, 20, and 21, wherein binding of the antibody to human Siglec-10 does not block interaction between human Siglec-10 and human CD52.

27. The antibody of any one of claims 1-26, wherein the antibody binds to the extracellular domain of human Siglec-10 when expressed on a surface of a human myeloid cell.

28. The antibody of claim 27, wherein the human myeloid cell is a human macrophage, dendritic cell, or monocyte.

29. The antibody of claim 27 or claim 28, wherein binding of the antibody to the extracellular domain of human Siglec-10 when expressed on the surface of a human myeloid cell induces internalization of Siglec-10.

30. The antibody of any one of claims 1 and 3-26, wherein the antibody is a humanized antibody.

31. The antibody of any one of claims 1-30, wherein the antibody comprises an Fc region.

32. The antibody of claim 31, wherein the Fc region is a human Fc region.

33. The antibody of claim 32, wherein the Fc region is a human IgGl or human IgG4 Fc region.

34. The antibody of claim 33, wherein the Fc region is a human IgG4 Fc region comprising the amino acid substitution S228P, numbering according to EU index.

35. The antibody of any one of claims 31-34, wherein the Fc region comprises one or more mutation(s) that reduce effector function.

36. The antibody of claim 35, wherein the antibody comprises a human IgGl Fc region with one or more of the following mutation(s), numbering based on EU index:

(a) L234A and/or L235A;

(b) A327G, A330S, and/or P33 IS;

(c) E233P, L234V, L235A, and/or G236del;

(d) E233P, L234V, and/or L235A;

(e) E233P, L234V, L235A, G236del, A327G, A330S, and/or P33 IS;

(f) E233P, L234V, L235A, A327G, A330S, and/or P331S;

(g) N297A;

(h) N297G;

(i) N297Q;

(j) L242C, N297C, and/or K334C;

(k) A287C, N297G, and/or L306C;

(l) R292C, N297G, and/or V302C;

(m) N297G, V323C, and/or I332C;

(n) V259C, N297G, and/or L306C;

(o) L234F, L235Q, K322Q, M252Y, S254T, and/or T256E;

(p) L234A, L235A, and/or P329G; or

(q) L234A, L235Q, and K322Q.

37. The antibody of claim 35, wherein the antibody comprises a human IgG2 Fc region with one or more of the following mutation(s), numbering based on EU index: (a) A330S and/or P331S;

(b) V234A, G237A, P238S, H268A, V309L, A330S, and/or P33 IS; or

(c) V234A, G237A, H268Q, V309L, A330S, P331S, C232S, C233S, S267E, L328F, M252Y, S254T, and/or T256E.

38. The antibody of claim 35, wherein the antibody comprises a human IgG4 Fc region with one or more of the following mutation(s), numbering based on EU index:

(a) E233P, F234V, L235A, and/or G236del;

(b) E233P, F234V, and/or L235A;

(c) S228P and/or L235E; or

(d) S228P and/or L235A.

39. The antibody of any one of claims 31-34, wherein the Fc region comprises one or more mutation(s) that enhance effector function.

40. The antibody of claim 39, wherein the antibody comprises a human IgGl Fc region with one or more of the following mutation(s), numbering based on EU index:

(a) F243L, R292P, Y300L, V305I, and/or P396L;

(b) S239D and/or I332E;

(c) S239D, I332E, and/or A330L;

(d) S298A, E333A, and/or K334A;

(e) G236A, S239D, and/or I332E;

(f) K326W and/or E333S;

(g) S267E, H268F, and/or S324T; or

(h) E345R, E430G, and/or S440Y.

41. The antibody of any one of claims 31-40, wherein at least one or two of the heavy chains of the antibody is non-fucosylated.

42. The antibody of claim 41, wherein the antibody is produced in a cell line having an alpha- 1,6-fucosyltransferase (Fut8) knockout.

43. The antibody of claim 41, wherein the antibody is produced in a cell line overexpressing pi,4-N-acetylglucosaminyltransf erase III (GnT-III).

44. The antibody of claim 43, wherein the cell line additionally overexpresses Golgi p- mannosidase II (Manll).

45. The antibody of any one of claims 1-30, wherein the antibody is an antibody fragment selected from the group consisting of a Fab, F(ab’)2, Fab’-SH, Fv, and scFv fragment.

46. The antibody of any one of claims 1-45, wherein the antibody comprises a light chain constant (CL) domain.

47. The antibody of claim 46, wherein the CL domain is a human kappa CL domain.

48. The antibody of any one of claims 1-3 and 41-43, wherein the antibody comprises a heavy chain that comprises the amino acid sequence of SEQ ID NO:87 or 88 and/or a light chain that comprises the amino acid sequence of SEQ ID NO: 89.

49. The antibody of any one of claims 1-48, wherein the antibody is a monoclonal antibody.

50. The antibody of any one of claims 1-49, wherein the antibody is a multispecific antibody.

51. The antibody of any one of claims 1-50, wherein the antibody is conjugated to an agent.

52. The antibody of claim 51, wherein the agent is a cytotoxic agent or label.

53. A composition comprising the antibody of any one of claims 1-52.

54. The composition of claim 53, wherein the antibody comprises a Fc region and N- glycoside-linked carbohydrate chains linked to the Fc region, wherein less than 50% of the N- glycoside-linked carbohydrate chains contain a fucose residue.

55. The composition of claim 54, wherein substantially none of the N-glycoside-linked carbohydrate chains contain a fucose residue.

56. A polynucleotide encoding the antibody of any one of claims 1-55.

57. A vector comprising the polynucleotide of claim 56.

58. A host cell comprising the polynucleotide of claim 56 or the vector of claim 57.

59. The host cell of claim 58, wherein the host cell is a mammalian or insect cell.

60. The host cell of claim 59, wherein the host cell is Chinese hamster ovary (CHO) cell.

61. The host cell of claim 59 or claim 60, wherein the host cell comprises a Fut8 knockout.

62. The host cell of claim 59 or claim 60, wherein the host cell overexpresses GnT-III.

63. The host cell of claim 62, wherein the host cell additionally overexpresses Manll.

64. A method of producing an antibody, comprising culturing the host cell of any one of claims 58-63 under a condition that produces the antibody.

65. The method of claim 64, further comprising recovering the antibody produced by the host cell.

66. An anti-Siglec-10 antibody produced by the method of claim 64 or claim 65.

67. A pharmaceutical composition comprising the antibody of any one of claims 1-55 and 66 and a pharmaceutically acceptable carrier.

68. A method of treating or delaying progression of cancer in an individual, comprising administering to the individual an effective amount of the antibody of any one of claims 1-55 and 66 or the composition of claim 67.

69. The method of claim 68, wherein the cancer is a solid tumor.

70. The method of claim 68, wherein the cancer is a blood cancer.

71. The method of claim 68, wherein the cancer is selected from the group consisting of gastric cancer, breast cancer, lung cancer, ovarian cancer, cervical cancer, prostate cancer, bladder cancer, colorectal cancer, pancreatic cancer, liver cancer, renal cancer, thyroid cancer, brain cancer, head and neck cancer, leukemia, lymphoma, myeloma, carcinoma, melanoma, leiomyoma, leiomyosarcoma, glioma, glioblastoma, and esophageal cancer.

72. The method of any one of claims 68-71, wherein the antibody or composition is administered to the individual in combination with an additional anti-cancer agent.

73. A method of treating or delaying progression of an autoimmune disease or disorder in an individual, comprising administering to the individual an effective amount of the antibody of any one of claims 1-55 and 66 or the composition of claim 67.

74. The method of claim 73, wherein the autoimmune disease or disorder is inflammatory bowel disease (IBD), rheumatoid arthritis (RA), multiple sclerosis (MS), psoriasis, or systemic lupus erythematosus.

75. A method of depleting cells expressing Siglec-10 in an individual in need thereof, comprising administering to the individual an effective amount of the antibody of any one of claims 1-55 and 66 or the composition of claim 67.

76. The method of claim 75, wherein the individual has or has been diagnosed with an autoimmune disease or disorder.

77. The method of claim 76, wherein the autoimmune disease or disorder is inflammatory bowel disease (IBD), rheumatoid arthritis (RA), multiple sclerosis (MS), psoriasis, or systemic lupus erythematosus.