Anti-siglec-9 antibody molecules

Abstract

Anti-Siglec-9 antibody molecules or binding fragments thereof are disclosed. These anti-Siglec-9 antibody molecules or binding fragments can be used to treat cancer, acute or chronic hepatitis B.

Description

Technical Field

[0001] This disclosure relates to anti-Siglec-9 antibody molecules or their binding fragments. This disclosure further relates to nucleic acids encoding antibody molecules or their binding fragments, expression vectors, host cells, and methods for preparing antibody molecules or their binding fragments. Pharmaceutical compositions comprising antibody molecules or their binding fragments are also provided. The anti-Siglec-9 antibody molecules or their binding fragments of this disclosure can be used (alone or in combination with other agents or treatments) to treat cancer, acute or chronic hepatitis B. Therefore, this disclosure further relates to anti-Siglec-9 antibody molecules or their binding fragments, or pharmaceutical compositions comprising anti-Siglec-9 antibody molecules or their binding fragments, for the treatment of cancer, acute or chronic hepatitis B. Background of the Invention

[0003] During malignant transformation, glycosylation changes significantly compared to healthy tissues due to differential expression of glycosyltransferases, glycosidases, and monosaccharide transporters in the cancer microenvironment. Upregulation and alteration of terminal sialic acid structure are hallmarks of cancer.

[0004] Sialic acid structural patterns are recognized by a receptor called Siglec. Siglec is an immunomodulatory receptor that binds to sialic acid structures and is typically an inhibitory receptor preferentially expressed on immune cells. In recent years, several experimental models have provided evidence that Siglec is associated with cancer progression and immune evasion. These models have also shown that targeting Siglec, such as Siglec-9, can enhance anti-tumor immunity.

[0005] Given the ongoing need for improved strategies for targeting diseases such as cancer, there is a great need for novel compositions for modulating Siglec-9 activity. Invention Overview

[0007] Aspect A

[0008] In one aspect A1, this disclosure relates to an anti-Siglec-9 antibody molecule or an anti-Siglec-9 binding fragment thereof.

[0009] Structural characteristics

[0010] In some embodiments of aspect A1, this disclosure relates to an anti-Siglec-9 antibody molecule or an anti-Siglec-9 binding fragment thereof, wherein the antibody molecule or binding fragment comprises at least one, two, three, four, five or six complementarity-determining regions (CDRs) (or collectively all CDRs) from a heavy chain variable region (VH) and / or a light chain variable region (VL), the CDRs comprising the amino acid sequences shown in Table 6, wherein one or more CDRs (or collectively all CDRs) may have one, two, three, four, five, six or more variations relative to the amino acid sequences shown in Table 6, such as amino acid substitutions (e.g., conserved amino acid substitutions), insertions or deletions.

[0011] In some embodiments of aspect A1, this disclosure relates to an anti-Siglec-9 antibody molecule or an anti-Siglec-9 binding fragment thereof, wherein the antibody molecule or binding fragment comprises at least one, two, three, four, five or six complementarity-determining regions (CDRs) (or collectively all CDRs) from a heavy chain variable region (VH) and / or a light chain variable region (VL) comprising the amino acid sequence shown in Table 5, wherein one or more CDRs (or collectively all CDRs) may have one, two, three, four, five, six or more variations relative to the amino acid sequence shown in Table 5, such as amino acid substitutions (e.g., conserved amino acid substitutions), insertions or deletions.

[0012] In some embodiments of aspect A1, this disclosure relates to an anti-Siglec-9 antibody molecule or an anti-Siglec-9 binding fragment thereof, wherein the antibody molecule or binding fragment comprises at least one, two, three, four, five or six complementarity-determining regions (CDRs) (or collectively all CDRs) from a heavy chain variable region (VH) and / or a light chain variable region (VL) comprising the amino acid sequences shown in Table 1, wherein one or more CDRs (or collectively all CDRs) may have one, two, three, four, five, six or more variations relative to the amino acid sequences shown in Table 1, such as amino acid substitutions (e.g., conserved amino acid substitutions), insertions or deletions.

[0013] In some embodiments of aspect A1, this disclosure relates to an anti-Siglec-9 antibody molecule or an anti-Siglec-9 binding fragment thereof, comprising:

[0014] The heavy chain variable region (VH) comprises one, two, or three of the following: the amino acid sequence of heavy chain complementarity-determining region 1 (VHCDR1) of SEQ ID NO:1 or a sequence having one, two, three, or four amino acid substitutions (e.g., conserved amino acid substitutions), insertions, or deletions; the amino acid sequence of heavy chain complementarity-determining region 2 (VHCDR2) of SEQ ID NO:51 or a sequence having one, two, three, or four amino acid substitutions (e.g., conserved amino acid substitutions), insertions, or deletions; and the amino acid sequence of heavy chain complementarity-determining region 3 (VHCDR3) of SEQ ID NO:3 or a sequence having one, two, three, or four amino acid substitutions (e.g., conserved amino acid substitutions), insertions, or deletions; and / or the light chain variable region (VL) comprises one, two, or three of the following: the amino acid sequence of light chain complementarity-determining region 1 (VLCDR1) of SEQ ID NO:52 or a sequence having one, two, three, or four amino acid substitutions (e.g., conserved amino acid substitutions), insertions, or deletions; SEQ ID NO:1; VHCDR2; VHCDR3; VHCDR2 ... The amino acid sequence of light chain complementarity-determining region 2 (VLCDR2) of SEQ ID NO:5 or a sequence having one, two, three or four amino acid substitutions (e.g., conserved amino acid substitutions), insertions or deletions, and the amino acid sequence of light chain complementarity-determining region 3 (VLCDR3) of SEQ ID NO:6 or a sequence having one, two, three or four amino acid substitutions (e.g., conserved amino acid substitutions), insertions or deletions.

[0015] In some embodiments of aspect A1, this disclosure relates to an anti-Siglec-9 antibody molecule or an anti-Siglec-9 binding fragment thereof, comprising:

[0016] The heavy chain variable region (VH) comprises one, two, or three of the following: the amino acid sequence of heavy chain complementarity-determining region 1 (VHCDR1) of SEQ ID NO:1 or a sequence having one, two, three, or four amino acid substitutions (e.g., conserved amino acid substitutions), insertions, or deletions; the amino acid sequence of heavy chain complementarity-determining region 2 (VHCDR2) of SEQ ID NO:45 or a sequence having one, two, three, or four amino acid substitutions (e.g., conserved amino acid substitutions), insertions, or deletions; and the amino acid sequence of heavy chain complementarity-determining region 3 (VHCDR3) of SEQ ID NO:3 or a sequence having one, two, three, or four amino acid substitutions (e.g., conserved amino acid substitutions), insertions, or deletions; and / or the light chain variable region (VL) comprises one, two, or three of the following: the amino acid sequence of light chain complementarity-determining region 1 (VLCDR1) of SEQ ID NO:46 or a sequence having one, two, three, or four amino acid substitutions (e.g., conserved amino acid substitutions), insertions, or deletions; SEQ ID NO:1; VHCDR2; VHCDR3; VHCDR2 ... The amino acid sequence of light chain complementarity-determining region 2 (VLCDR2) of SEQ ID NO:5 or a sequence having one, two, three or four amino acid substitutions (e.g., conserved amino acid substitutions), insertions or deletions, and the amino acid sequence of light chain complementarity-determining region 3 (VLCDR3) of SEQ ID NO:6 or a sequence having one, two, three or four amino acid substitutions (e.g., conserved amino acid substitutions), insertions or deletions.

[0017] In some embodiments of aspect A1, this disclosure relates to an anti-Siglec-9 antibody molecule or an anti-Siglec-9 binding fragment thereof, comprising:

[0018] The heavy chain variable region (VH) comprises one, two, or three of the following: the amino acid sequence of heavy chain complementarity-determining region 1 (VHCDR1) of SEQ ID NO:1 or a sequence having one, two, three, or four amino acid substitutions (e.g., conserved amino acid substitutions), insertions, or deletions; the amino acid sequence of heavy chain complementarity-determining region 2 (VHCDR2) of SEQ ID NO:2 or a sequence having one, two, three, or four amino acid substitutions (e.g., conserved amino acid substitutions), insertions, or deletions; and the amino acid sequence of heavy chain complementarity-determining region 3 (VHCDR3) of SEQ ID NO:3 or a sequence having one, two, three, or four amino acid substitutions (e.g., conserved amino acid substitutions), insertions, or deletions; and / or the light chain variable region (VL) comprises one, two, or three of the following: the amino acid sequence of light chain complementarity-determining region 1 (VLCDR1) of SEQ ID NO:4 or a sequence having one, two, three, or four amino acid substitutions (e.g., conserved amino acid substitutions), insertions, or deletions; SEQ ID NO:1; VHCDR2; VHCDR3; VHCDR2 ...3; VHCDR2; VHCDR3; VHCDR2; VHCDR3; VHCDR3; VHCDR4; VHCDR2; VHCDR3; VHCDR3; VHCDR4; VHCDR2; VHCDR3; VHCDR3; VHCDR4; VHCDR2; VHCDR The amino acid sequence of light chain complementarity-determining region 2 (VLCDR2) of SEQ ID NO:5 or a sequence having one, two, three or four amino acid substitutions (e.g., conserved amino acid substitutions), insertions or deletions, and the amino acid sequence of light chain complementarity-determining region 3 (VLCDR3) of SEQ ID NO:6 or a sequence having one, two, three or four amino acid substitutions (e.g., conserved amino acid substitutions), insertions or deletions.

[0019] In some embodiments of aspect A1, this disclosure relates to an anti-Siglec-9 antibody molecule or an anti-Siglec-9 binding fragment thereof, comprising:

[0020] Heavy chain variable region (VH) comprising the heavy chain complementarity-determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO:3, or a sequence having one, two, three, or four amino acid substitutions (e.g., conserved amino acid substitutions); and / or

[0021] The light chain variable region (VL) contains the amino acid sequence of the light chain complementarity-determining region 3 (VLCDR3) of SEQ ID NO:6, or a sequence having one, two, three or four amino acid substitutions (e.g., conserved amino acid substitutions).

[0022] In some embodiments of aspect A1, this disclosure relates to an anti-Siglec-9 antibody molecule or an anti-Siglec-9 binding fragment thereof, comprising:

[0023] Heavy chain variable regions (VH) comprising the amino acid sequence of heavy chain complementarity-determining region 1 (VHCDR1) of SEQ ID NO:1 or a sequence having one, two, three, or four amino acid substitutions (e.g., conserved amino acid substitutions), the amino acid sequence of heavy chain complementarity-determining region 2 (VHCDR2) of SEQ ID NO:51 or a sequence having one, two, three, or four amino acid substitutions (e.g., conserved amino acid substitutions), and the amino acid sequence of heavy chain complementarity-determining region 3 (VHCDR3) of SEQ ID NO:3 or a sequence having one, two, three, or four amino acid substitutions (e.g., conserved amino acid substitutions); and / or

[0024] The light chain variable region (VL) comprises the amino acid sequence of light chain complementarity-determining region 1 (VLCDR1) of SEQ ID NO:52 or a sequence having one, two, three or four amino acid substitutions (e.g., conserved amino acid substitutions), the amino acid sequence of light chain complementarity-determining region 2 (VLCDR2) of SEQ ID NO:5 or a sequence having one, two, three or four amino acid substitutions (e.g., conserved amino acid substitutions), and the amino acid sequence of light chain complementarity-determining region 3 (VLCDR3) of SEQ ID NO:6 or a sequence having one, two, three or four amino acid substitutions (e.g., conserved amino acid substitutions).

[0025] In some embodiments of aspect A1, this disclosure relates to an anti-Siglec-9 antibody molecule or an anti-Siglec-9 binding fragment thereof, comprising:

[0026] Heavy chain variable regions (VH) comprising the amino acid sequence of heavy chain complementarity-determining region 1 (VHCDR1) of SEQ ID NO:1 or a sequence having one, two, three, or four amino acid substitutions (e.g., conserved amino acid substitutions), the amino acid sequence of heavy chain complementarity-determining region 2 (VHCDR2) of SEQ ID NO:45 or a sequence having one, two, three, or four amino acid substitutions (e.g., conserved amino acid substitutions), and the amino acid sequence of heavy chain complementarity-determining region 3 (VHCDR3) of SEQ ID NO:3 or a sequence having one, two, three, or four amino acid substitutions (e.g., conserved amino acid substitutions); and / or

[0027] The light chain variable region (VL) comprises the amino acid sequence of light chain complementarity-determining region 1 (VLCDR1) of SEQ ID NO:46 or a sequence having one, two, three or four amino acid substitutions (e.g., conserved amino acid substitutions), the amino acid sequence of light chain complementarity-determining region 2 (VLCDR2) of SEQ ID NO:5 or a sequence having one, two, three or four amino acid substitutions (e.g., conserved amino acid substitutions), and the amino acid sequence of light chain complementarity-determining region 3 (VLCDR3) of SEQ ID NO:6 or a sequence having one, two, three or four amino acid substitutions (e.g., conserved amino acid substitutions).

[0028] In some embodiments of aspect A1, this disclosure relates to an anti-Siglec-9 antibody molecule or an anti-Siglec-9 binding fragment thereof, comprising:

[0029] The heavy chain variable region (VH) comprises the amino acid sequence of heavy chain complementarity-determining region 1 (VHCDR1) of SEQ ID NO:1 or a sequence having one, two, three, or four amino acid substitutions (e.g., conserved amino acid substitutions), the amino acid sequence of heavy chain complementarity-determining region 2 (VHCDR2) of SEQ ID NO:2 or a sequence having one, two, three, or four amino acid substitutions (e.g., conserved amino acid substitutions), and the amino acid sequence of heavy chain complementarity-determining region 3 (VHCDR3) of SEQ ID NO:3 or a sequence having one, two, three, or four amino acid substitutions (e.g., conserved amino acid substitutions); and / or

[0030] The light chain variable region (VL) comprises the amino acid sequence of light chain complementarity-determining region 1 (VLCDR1) of SEQ ID NO:4 or a sequence having one, two, three or four amino acid substitutions (e.g., conserved amino acid substitutions), the amino acid sequence of light chain complementarity-determining region 2 (VLCDR2) of SEQ ID NO:5 or a sequence having one, two, three or four amino acid substitutions (e.g., conserved amino acid substitutions), and the amino acid sequence of light chain complementarity-determining region 3 (VLCDR3) of SEQ ID NO:6 or a sequence having one, two, three or four amino acid substitutions (e.g., conserved amino acid substitutions).

[0031] In some embodiments of aspect A1, this disclosure relates to an anti-Siglec-9 antibody molecule or an anti-Siglec-9 binding fragment thereof, comprising:

[0032] Heavy chain variable region (VH) comprising the amino acid sequence of heavy chain complementarity-determining region 1 (VHCDR1) of SEQ ID NO:1, the amino acid sequence of heavy chain complementarity-determining region 2 (VHCDR2) of SEQ ID NO:2 or a sequence having one, two or three amino acid substitutions, and the amino acid sequence of heavy chain complementarity-determining region 3 (VHCDR3) of SEQ ID NO:3; and / or

[0033] The light chain variable region (VL) comprises the amino acid sequence of light chain complementarity-determining region 1 (VLCDR1) of SEQ ID NO:4 or a sequence having one or two amino acid substitutions, the amino acid sequence of light chain complementarity-determining region 2 (VLCDR2) of SEQ ID NO:5, and the amino acid sequence of light chain complementarity-determining region 3 (VLCDR3) of SEQ ID NO:6 or a sequence having one, two, three or four amino acid substitutions.

[0034] In some embodiments of aspect A1, the anti-Siglec-9 antibody molecule or its anti-Siglec-9 binding fragment comprises:

[0035] The heavy chain variable region (VH) comprises the amino acid sequence of heavy chain complementarity-determining region 1 (VHCDR1) of SEQ ID NO:1, the amino acid sequence of heavy chain complementarity-determining region 2 (VHCDR2) of SEQ ID NO:51, and the amino acid sequence of heavy chain complementarity-determining region 3 (VHCDR3) of SEQ ID NO:3; and

[0036] The light chain variable region (VL) comprises the amino acid sequence of light chain complementarity-determining region 1 (VLCDR1) of SEQ ID NO:52, the amino acid sequence of light chain complementarity-determining region 2 (VLCDR2) of SEQ ID NO:5, and the amino acid sequence of light chain complementarity-determining region 3 (VLCDR3) of SEQ ID NO:6.

[0037] In some embodiments of aspect A1, the anti-Siglec-9 antibody molecule or its anti-Siglec-9 binding fragment comprises:

[0038] The heavy chain variable region (VH) comprises the amino acid sequence of heavy chain complementarity-determining region 1 (VHCDR1) of SEQ ID NO:1, the amino acid sequence of heavy chain complementarity-determining region 2 (VHCDR2) of SEQ ID NO:45, and the amino acid sequence of heavy chain complementarity-determining region 3 (VHCDR3) of SEQ ID NO:3; and

[0039] The light chain variable region (VL) comprises the amino acid sequence of light chain complementarity-determining region 1 (VLCDR1) of SEQ ID NO:46, the amino acid sequence of light chain complementarity-determining region 2 (VLCDR2) of SEQ ID NO:5, and the amino acid sequence of light chain complementarity-determining region 3 (VLCDR3) of SEQ ID NO:6.

[0040] In some embodiments of aspect A1, the anti-Siglec-9 antibody molecule or its anti-Siglec-9 binding fragment comprises:

[0041] The heavy chain variable region (VH) comprises the amino acid sequence of heavy chain complementarity-determining region 1 (VHCDR1) of SEQ ID NO:1, the amino acid sequence of heavy chain complementarity-determining region 2 (VHCDR2) of SEQ ID NO:2, and the amino acid sequence of heavy chain complementarity-determining region 3 (VHCDR3) of SEQ ID NO:3; and

[0042] The light chain variable region (VL) comprises the amino acid sequence of light chain complementarity-determining region 1 (VLCDR1) of SEQ ID NO:4, the amino acid sequence of light chain complementarity-determining region 2 (VLCDR2) of SEQ ID NO:5, and the amino acid sequence of light chain complementarity-determining region 3 (VLCDR3) of SEQ ID NO:6.

[0043] In some embodiments of aspect A1, the anti-Siglec-9 antibody molecule or its anti-Siglec-9 binding fragment comprises:

[0044] The heavy chain variable region (VH) comprises the amino acid sequence of heavy chain complementarity-determining region 1 (VHCDR1) of SEQ ID NO:1, the amino acid sequence of heavy chain complementarity-determining region 2 (VHCDR2) of SEQ ID NO:51, and the amino acid sequence of heavy chain complementarity-determining region 3 (VHCDR3) of SEQ ID NO:3; and

[0045] The light chain variable region (VL) comprises the amino acid sequence of light chain complementarity-determining region 1 (VLCDR1) of SEQ ID NO:52, the amino acid sequence of light chain complementarity-determining region 2 (VLCDR2) of SEQ ID NO:5, and the amino acid sequence of light chain complementarity-determining region 3 (VLCDR3) of SEQ ID NO:6.

[0046] One, two, three, four, five, six, seven, or eight amino acids in the CDR have been inserted, deleted, or substituted.

[0047] In some embodiments of aspect A1, the anti-Siglec-9 antibody molecule or its anti-Siglec-9 binding fragment comprises:

[0048] The heavy chain variable region (VH) comprises the amino acid sequence of heavy chain complementarity-determining region 1 (VHCDR1) of SEQ ID NO:1, the amino acid sequence of heavy chain complementarity-determining region 2 (VHCDR2) of SEQ ID NO:45, and the amino acid sequence of heavy chain complementarity-determining region 3 (VHCDR3) of SEQ ID NO:3; and

[0049] The light chain variable region (VL) comprises the amino acid sequence of light chain complementarity-determining region 1 (VLCDR1) of SEQ ID NO:46, the amino acid sequence of light chain complementarity-determining region 2 (VLCDR2) of SEQ ID NO:5, and the amino acid sequence of light chain complementarity-determining region 3 (VLCDR3) of SEQ ID NO:6.

[0050] One, two, three, four, five, six, seven, or eight amino acids in the CDR have been inserted, deleted, or substituted.

[0051] In some embodiments of aspect A1, the anti-Siglec-9 antibody molecule or its anti-Siglec-9 binding fragment comprises:

[0052] The heavy chain variable region (VH) comprises the amino acid sequence of heavy chain complementarity-determining region 1 (VHCDR1) of SEQ ID NO:1, the amino acid sequence of heavy chain complementarity-determining region 2 (VHCDR2) of SEQ ID NO:2, and the amino acid sequence of heavy chain complementarity-determining region 3 (VHCDR3) of SEQ ID NO:3; and

[0053] The light chain variable region (VL) comprises the amino acid sequence of light chain complementarity-determining region 1 (VLCDR1) of SEQ ID NO:4, the amino acid sequence of light chain complementarity-determining region 2 (VLCDR2) of SEQ ID NO:5, and the amino acid sequence of light chain complementarity-determining region 3 (VLCDR3) of SEQ ID NO:6.

[0054] One, two, three, four, five, six, seven, or eight amino acids in the CDR have been inserted, deleted, or substituted.

[0055] In some embodiments of aspect A1, the anti-Siglec-9 antibody molecule or its anti-Siglec-9 binding fragment includes a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO:47, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:47.

[0056] In some embodiments of aspect A1, the anti-Siglec-9 antibody molecule or its anti-Siglec-9 binding fragment includes a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:7.

[0057] In some embodiments of aspect A1, the anti-Siglec-9 antibody molecule or its anti-Siglec-9 binding fragment includes a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:48, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:48.

[0058] In some embodiments of aspect A1, the anti-Siglec-9 antibody molecule or its anti-Siglec-9 binding fragment includes a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:8, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:8.

[0059] In some embodiments of aspect A1, the anti-Siglec-9 antibody molecule or its anti-Siglec-9 binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising the amino acid sequence of SEQ ID NO:47, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:47, and the light chain variable region comprising the amino acid sequence of SEQ ID NO:48, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:48.

[0060] In some embodiments of aspect A1, the anti-Siglec-9 antibody molecule or its anti-Siglec-9 binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:7, and the light chain variable region comprising the amino acid sequence of SEQ ID NO:8, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:8.

[0061] In some embodiments of aspect A1, the anti-Siglec-9 antibody molecule or its anti-Siglec-9 binding fragment comprises a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO:47 and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO:48.

[0062] In some embodiments of aspect A1, the anti-Siglec-9 antibody molecule or its anti-Siglec-9 binding fragment comprises a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO:7 and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO:8.

[0063] In some embodiments of aspect A1, the anti-Siglec-9 antibody molecule or its anti-Siglec-9 binding fragment is a humanized anti-Siglec-9 antibody molecule or its anti-Siglec-9 binding fragment.

[0064] Features

[0065] In some embodiments of aspect A1, the anti-Siglec-9 antibody molecule or its anti-Siglec-9 binding fragment comprises one or more of the following properties (e.g., 2, 3, 4, 5, 6, 7, 8, or 9):

[0066] (i) Binding to human Siglec-9 with EC50 values ​​of less than about 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM or 0.05 nM, for example, as described in Example 3, when the antibody molecule or its binding fragment is used as a bivalent molecule in an ELISA test;

[0067] (ii) Binding to human Siglec-9 with a dissociation constant (KD) of less than about 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM or 0.1 nM, for example, as described in Example 4, when the antibody molecule or its binding fragment is tested as a divalent molecule using surface plasmon resonance, such as Carterra LSA;

[0068] (iii) Binding of the antibody molecule or its binding fragment to human CD14+ monocytes at EC50 levels of less than about 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM or 0.1 nM, for example, as described in Example 5, when the antibody molecule or its binding fragment is tested as a bivalent molecule using flow cytometry;

[0069] (vi) Inhibit the interaction between Siglec-9 and one or more Siglec-9 ligands, for example, inhibit the binding of A549 tumor cells expressing sialic acid to Siglec-9, for example, as described in Example 6, when the antibody molecule or its binding fragment is used as a bivalent molecule for testing by ELISA;

[0070] (v) Increase the proliferation of CD8+ T cells stimulated by anti-CD3 / anti-CD28, for example, as described in Example 7, when the antibody molecule or its binding fragment is tested as a bivalent molecule in a T cell activation assay using Cell Trace Violet and flow cytometry;

[0071] (vi) Upregulate the activation markers CD69 and CD25 of anti-CD3 / anti-CD28 stimulated CD8+ T cells, for example, as described in Example 7, when the antibody molecule or its binding fragment is tested as a bivalent molecule in a T cell activation assay using staining and flow cytometry.

[0072] (vii) Increase the proliferation of CD4+ T cells stimulated by anti-CD3 / anti-CD28, for example, as described in Example 7, when the antibody molecule or its binding fragment is tested as a bivalent molecule in a T cell activation assay using Cell Trace Violet and flow cytometry.

[0073] (viii) Upregulate the activation markers CD69 and CD25 of anti-CD3 / anti-CD28 stimulated CD4+ T cells, for example, as described in Example 7, when the antibody molecule or its binding fragment is tested as a bivalent molecule in a T cell activation assay using staining and flow cytometry; or

[0074] (ix) Increase NK cell-mediated killing activity, for example, increase the killing of K562 cells by NK92 cells overexpressing Siglec-9, for example, as described in Example 8, when antibody molecules or their binding fragments are tested as bivalent molecules using Calcein-AM labeled K562 cells.

[0075] In one aspect A2, this disclosure relates to an antibody molecule or its binding fragment thereof that competes with the antibody molecule or its binding fragment thereof described herein for binding to human Siglec-9. In some embodiments of aspect A2, this disclosure relates to an antibody molecule or its binding fragment thereof that competes with an antibody molecule or its binding fragment thereof that comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the amino acid sequence of heavy chain complementarity-determining region 1 (VHCDR1) of SEQ ID NO:1, the amino acid sequence of heavy chain complementarity-determining region 2 (VHCDR2) of SEQ ID NO:2, and the amino acid sequence of heavy chain complementarity-determining region 3 (VHCDR3) of SEQ ID NO:3, and the light chain variable region (VL) comprises the amino acid sequence of light chain complementarity-determining region 1 (VLCDR1) of SEQ ID NO:4, the amino acid sequence of light chain complementarity-determining region 2 (VLCDR2) of SEQ ID NO:5, and the amino acid sequence of light chain complementarity-determining region 3 (VLCDR3) of SEQ ID NO:6. In some embodiments of aspect A2, this disclosure relates to an antibody molecule or a binding fragment thereof that competes with an antibody molecule comprising a heavy chain variable region (VH) and a light chain variable region (VL) for binding to human Siglec-9, the heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:7 and the light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:8.

[0076] In one aspect A3, this disclosure relates to pharmaceutical compositions comprising an antibody molecule or a binding fragment thereof described herein and a pharmaceutically acceptable carrier, excipient, or stabilizer.

[0077] In one aspect A4, this disclosure relates to the anti-Siglec-9 antibody molecule described herein or a binding fragment thereof, or a pharmaceutical composition comprising the anti-Siglec-9 antibody molecule described herein or a binding fragment thereof, for the treatment of a disease selected from cancer, acute hepatitis B, and chronic hepatitis B. In some embodiments of aspect A4, the cancer is selected from non-small cell lung cancer, colorectal cancer, breast cancer, epithelial ovarian cancer, hepatocellular carcinoma, and prostate cancer.

[0078] In one aspect A5, this disclosure relates to nucleic acids encoding antibody heavy chain variable regions and / or light chain variable regions of the antibody molecule described herein or its binding fragments.

[0079] In one aspect A6, this disclosure relates to expression vectors comprising the nucleic acids described herein.

[0080] In one aspect A7, this disclosure relates to a host cell comprising the nucleic acid or expression vector described herein.

[0081] In one aspect A8, this disclosure relates to a method for producing antibody molecules, the method comprising culturing the host cells described herein under conditions suitable for gene expression.

[0082] Aspect B

[0083] In one aspect B1, this disclosure relates to an anti-Siglec-9 antibody molecule or an anti-Siglec-9 binding fragment thereof.

[0084] Structural characteristics

[0085] In some embodiments of aspect B1, this disclosure relates to an anti-Siglec-9 antibody molecule or an anti-Siglec-9 binding fragment thereof, wherein the antibody molecule or binding fragment comprises at least one, two, three, four, five or six complementarity-determining regions (CDRs) (or collectively all CDRs) from a heavy chain variable region (VH) and / or a light chain variable region (VL) comprising the amino acid sequences shown in Table 2, wherein one or more CDRs (or collectively all CDRs) may have one, two, three, four, five, six or more variations relative to the amino acid sequences shown in Table 2, such as amino acid substitutions (e.g., conserved amino acid substitutions), insertions or deletions.

[0086] In some embodiments of aspect B1, this disclosure relates to an anti-Siglec-9 antibody molecule or an anti-Siglec-9 binding fragment thereof, comprising:

[0087] The heavy chain variable region (VH) comprises one, two, or three of the following: the amino acid sequence of heavy chain complementarity-determining region 1 (VHCDR1) of SEQ ID NO:30 or a sequence having one, two, three, or four amino acid substitutions (e.g., conserved amino acid substitutions), insertions, or deletions; the amino acid sequence of heavy chain complementarity-determining region 2 (VHCDR2) of SEQ ID NO:31 or a sequence having one, two, three, or four amino acid substitutions (e.g., conserved amino acid substitutions), insertions, or deletions; and the amino acid sequence of heavy chain complementarity-determining region 3 (VHCDR3) of SEQ ID NO:32 or a sequence having one, two, three, or four amino acid substitutions (e.g., conserved amino acid substitutions), insertions, or deletions; and / or the light chain variable region (VL) comprises one, two, or three of the following: the amino acid sequence of light chain complementarity-determining region 1 (VLCDR1) of SEQ ID NO:33 or a sequence having one, two, three, or four amino acid substitutions (e.g., conserved amino acid substitutions), insertions, or deletions; SEQ ID NO:30 ... The amino acid sequence of light chain complementarity-determining region 2 (VLCDR2) of SEQ ID NO:34 or a sequence having one, two, three or four amino acid substitutions (e.g., conserved amino acid substitutions), insertions or deletions, and the amino acid sequence of light chain complementarity-determining region 3 (VLCDR3) of SEQ ID NO:35 or a sequence having one, two, three or four amino acid substitutions (e.g., conserved amino acid substitutions), insertions or deletions.

[0088] In some embodiments of aspect B1, this disclosure relates to an anti-Siglec-9 antibody molecule or an anti-Siglec-9 binding fragment thereof, comprising:

[0089] Heavy chain variable region (VH) comprising the heavy chain complementarity-determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO:32, or a sequence having one, two, three, or four amino acid substitutions (e.g., conserved amino acid substitutions); and / or

[0090] The light chain variable region (VL) contains the amino acid sequence of the light chain complementarity-determining region 3 (VLCDR3) of SEQ ID NO:35, or a sequence having one, two, three or four amino acid substitutions (e.g., conserved amino acid substitutions).

[0091] In some embodiments of aspect B1, this disclosure relates to an anti-Siglec-9 antibody molecule or an anti-Siglec-9 binding fragment thereof, comprising:

[0092] The heavy chain variable region (VH) comprises the amino acid sequence of heavy chain complementarity-determining region 1 (VHCDR1) of SEQ ID NO:30 or a sequence having one, two, three, or four amino acid substitutions (e.g., conserved amino acid substitutions), the amino acid sequence of heavy chain complementarity-determining region 2 (VHCDR2) of SEQ ID NO:31 or a sequence having one, two, three, or four amino acid substitutions (e.g., conserved amino acid substitutions), and the amino acid sequence of heavy chain complementarity-determining region 3 (VHCDR3) of SEQ ID NO:32 or a sequence having one, two, three, or four amino acid substitutions (e.g., conserved amino acid substitutions); and / or

[0093] The light chain variable region (VL) comprises the amino acid sequence of light chain complementarity-determining region 1 (VLCDR1) of SEQ ID NO:33 or a sequence having one, two, three or four amino acid substitutions (e.g., conserved amino acid substitutions), the amino acid sequence of light chain complementarity-determining region 2 (VLCDR2) of SEQ ID NO:34 or a sequence having one, two, three or four amino acid substitutions (e.g., conserved amino acid substitutions), and the amino acid sequence of light chain complementarity-determining region 3 (VLCDR3) of SEQ ID NO:35 or a sequence having one, two, three or four amino acid substitutions (e.g., conserved amino acid substitutions).

[0094] In some embodiments of aspect B1, this disclosure relates to an anti-Siglec-9 antibody molecule or an anti-Siglec-9 binding fragment thereof, comprising:

[0095] Heavy chain variable region (VH) comprising the amino acid sequence of heavy chain complementarity-determining region 1 (VHCDR1) of SEQ ID NO:30, the amino acid sequence of heavy chain complementarity-determining region 2 (VHCDR2) of SEQ ID NO:31 or a sequence having one, two or three amino acid substitutions, and the amino acid sequence of heavy chain complementarity-determining region 3 (VHCDR3) of SEQ ID NO:32; and / or

[0096] The light chain variable region (VL) comprises the amino acid sequence of light chain complementarity-determining region 1 (VLCDR1) of SEQ ID NO:33 or a sequence having one or two amino acid substitutions, the amino acid sequence of light chain complementarity-determining region 2 (VLCDR2) of SEQ ID NO:34, and the amino acid sequence of light chain complementarity-determining region 3 (VLCDR3) of SEQ ID NO:35 or a sequence having one, two, three or four amino acid substitutions.

[0097] In some embodiments of aspect B1, the anti-Siglec-9 antibody molecule or its anti-Siglec-9 binding fragment comprises:

[0098] The heavy chain variable region (VH) comprises the amino acid sequence of heavy chain complementarity-determining region 1 (VHCDR1) of SEQ ID NO:30, the amino acid sequence of heavy chain complementarity-determining region 2 (VHCDR2) of SEQ ID NO:31, and the amino acid sequence of heavy chain complementarity-determining region 3 (VHCDR3) of SEQ ID NO:32; and

[0099] The light chain variable region (VL) comprises the amino acid sequence of light chain complementarity-determining region 1 (VLCDR1) of SEQ ID NO:33, the amino acid sequence of light chain complementarity-determining region 2 (VLCDR2) of SEQ ID NO:34, and the amino acid sequence of light chain complementarity-determining region 3 (VLCDR3) of SEQ ID NO:35.

[0100] In some embodiments of aspect B1, the anti-Siglec-9 antibody molecule or its anti-Siglec-9 binding fragment comprises:

[0101] The heavy chain variable region (VH) comprises the amino acid sequence of heavy chain complementarity-determining region 1 (VHCDR1) of SEQ ID NO:30, the amino acid sequence of heavy chain complementarity-determining region 2 (VHCDR2) of SEQ ID NO:31, and the amino acid sequence of heavy chain complementarity-determining region 3 (VHCDR3) of SEQ ID NO:32; and

[0102] The light chain variable region (VL) comprises the amino acid sequence of light chain complementarity-determining region 1 (VLCDR1) of SEQ ID NO:33, the amino acid sequence of light chain complementarity-determining region 2 (VLCDR2) of SEQ ID NO:34, and the amino acid sequence of light chain complementarity-determining region 3 (VLCDR3) of SEQ ID NO:35.

[0103] One, two, three, four, five, six, seven, or eight amino acids in the CDR have been inserted, deleted, or substituted.

[0104] In some embodiments of aspect B1, the anti-Siglec-9 antibody molecule or its anti-Siglec-9 binding fragment includes a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO:36, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:36.

[0105] In some embodiments of aspect B1, the anti-Siglec-9 antibody molecule or its anti-Siglec-9 binding fragment includes a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:37, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:37.

[0106] In some embodiments of aspect B1, the anti-Siglec-9 antibody molecule or its anti-Siglec-9 binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising the amino acid sequence of SEQ ID NO:36, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:36, and the light chain variable region comprising the amino acid sequence of SEQ ID NO:37, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:37.

[0107] In some embodiments of aspect B1, the anti-Siglec-9 antibody molecule or its anti-Siglec-9 binding fragment comprises a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO:36 and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO:37.

[0108] In some embodiments of aspect B1, the anti-Siglec-9 antibody molecule or its anti-Siglec-9 binding fragment is a humanized anti-Siglec-9 antibody molecule or its anti-Siglec-9 binding fragment.

[0109] Features

[0110] In some embodiments of aspect B1, the anti-Siglec-9 antibody molecule or its anti-Siglec-9 binding fragment comprises one or more of the following properties (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10):

[0111] (i) Binding to human Siglec-9 with EC50 values ​​of less than about 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM or 0.05 nM, for example, as described in Example 3, when the antibody molecule or its binding fragment is used as a bivalent molecule in an ELISA test;

[0112] (ii) Binding to human Siglec-9 with a dissociation constant (KD) of less than about 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM or 0.1 nM, for example, as described in Example 4, when the antibody molecule or its binding fragment is tested as a divalent molecule using surface plasmon resonance, such as Carterra LSA;

[0113] (iii) Binding of the antibody molecule or its binding fragment to human CD14+ monocytes at EC50 levels of less than about 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM or 0.1 nM, for example, as described in Example 5, when the antibody molecule or its binding fragment is tested as a bivalent molecule using flow cytometry;

[0114] (vi) Inhibit the interaction between Siglec-9 and one or more Siglec-9 ligands, for example, inhibit the binding of A549 tumor cells expressing sialic acid to Siglec-9, for example, as described in Example 6, when the antibody molecule or its binding fragment is used as a bivalent molecule for testing by ELISA;

[0115] (v) Increase the proliferation of CD8+ T cells stimulated by anti-CD3 / anti-CD28, for example, as described in Example 7, when the antibody molecule or its binding fragment is tested as a bivalent molecule in a T cell activation assay using Cell Trace Violet and flow cytometry;

[0116] (vi) Upregulate the activation markers CD69 and CD25 of anti-CD3 / anti-CD28 stimulated CD8+ T cells, for example, as described in Example 7, when the antibody molecule or its binding fragment is tested as a bivalent molecule in a T cell activation assay using staining and flow cytometry.

[0117] (vii) Increase the proliferation of CD4+ T cells stimulated by anti-CD3 / anti-CD28, for example, as described in Example 7, when the antibody molecule or its binding fragment is tested as a bivalent molecule in a T cell activation assay using Cell Trace Violet and flow cytometry.

[0118] (viii) Upregulate the activation markers CD69 and CD25 of anti-CD3 / anti-CD28 stimulated CD4+ T cells, for example, as described in Example 7, when the antibody molecule or its binding fragment is tested as a bivalent molecule in a T cell activation assay using staining and flow cytometry; or

[0119] (ix) Increase NK cell-mediated killing activity, for example, increase the killing effect of Siglec-9-overexpressing NK92 cells on K562 cells, for example, as described in Example 8, when the antibody molecule or its binding fragment is used as a bivalent molecule in Calcein-AM-labeled K562 cells for testing; or

[0120] (x) binds to human Siglec-7.

[0121] In one aspect B2, this disclosure relates to an antibody molecule or its binding fragment thereof that competes with the antibody molecule or its binding fragment thereof described herein for binding to human Siglec-9. In some embodiments of aspect B2, this disclosure relates to an antibody molecule or its binding fragment thereof that competes with an antibody molecule or its binding fragment thereof that comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the amino acid sequence of heavy chain complementarity-determining region 1 (VHCDR1) of SEQ ID NO:30, the amino acid sequence of heavy chain complementarity-determining region 2 (VHCDR2) of SEQ ID NO:31, and the amino acid sequence of heavy chain complementarity-determining region 3 (VHCDR3) of SEQ ID NO:32, and the light chain variable region (VL) comprises the amino acid sequence of light chain complementarity-determining region 1 (VLCDR1) of SEQ ID NO:33, the amino acid sequence of light chain complementarity-determining region 2 (VLCDR2) of SEQ ID NO:34, and the amino acid sequence of light chain complementarity-determining region 3 (VLCDR3) of SEQ ID NO:35. In some embodiments of aspect B2, this disclosure relates to an antibody molecule or a binding fragment thereof that competes with an antibody molecule comprising a heavy chain variable region (VH) and a light chain variable region (VL) for binding to human Siglec-9, the heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:36 and the light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:37.

[0122] In one aspect B3, this disclosure relates to pharmaceutical compositions comprising an antibody molecule or a binding fragment thereof described herein and a pharmaceutically acceptable carrier, excipient, or stabilizer.

[0123] In one aspect B4, this disclosure relates to the anti-Siglec-9 antibody molecule described herein or a binding fragment thereof, or a pharmaceutical composition comprising the anti-Siglec-9 antibody molecule described herein or a binding fragment thereof, for the treatment of a disease selected from cancer, acute hepatitis B, and chronic hepatitis B. In some embodiments, the cancer is selected from non-small cell lung cancer, colorectal cancer, breast cancer, epithelial ovarian cancer, hepatocellular carcinoma, and prostate cancer.

[0124] In one aspect B5, this disclosure relates to nucleic acids encoding antibody heavy chain variable regions and / or light chain variable regions that encode antibody molecules or their binding fragments described herein.

[0125] In one aspect B6, this disclosure relates to expression vectors comprising the nucleic acids described herein.

[0126] In one aspect B7, this disclosure relates to a host cell comprising the nucleic acid or expression vector described herein.

[0127] In one aspect B8, this disclosure relates to a method for producing antibody molecules, the method comprising culturing the host cells described herein under conditions suitable for gene expression. Attached Figure Description

[0128] Figure 1 The reactivity of monoclonal antibodies (68D4 and 224B1; comparative monoclonal antibodies mAbA and 5C6; and negative control 53C3) to Siglec-5, Siglec-7, Siglec-8, Siglec-15, and Siglec-9 was analyzed by ELISA. The reactivity was determined by measuring the accumulated colored product at 450 nm after the reaction was stopped (n=3).

[0129] Figure 2 Binding curves of monoclonal antibodies (68D4 and 224B1; comparative monoclonal antibodies mAbA and 5C6; and negative control 53C3) to Siglec-9 were analyzed by ELISA. Binding of the monoclonal antibodies to Siglec-9 was determined by measuring the accumulated colored product at 450 nm after the reaction was stopped and plotted against the concentration of the monoclonal antibody being evaluated. EC50 values ​​determined by three-parameter analysis in GraphPad Prism (GraphPad Software) are given in the lower right corner of the graph (n=3).

[0130] Figure 3 Binding curves of monoclonal antibodies (68D4 and 224B1; comparative monoclonal antibodies mAbA and 5C6 and negative control 53C3) to Siglec-9 cells displayed on human CD14+ monocytes, analyzed by flow cytometry. The mean fluorescence signal of the secondary antibody binding to the CD14+ population is plotted against the concentration of the monoclonal antibody being evaluated. The EC50 values ​​determined by three-parameter analysis in GraphPad Prism (GraphPad Software) are shown in the lower right corner of the graph.

[0131] Figure 4Monoclonal antibodies (68D4 and 224B1; comparative monoclonal antibodies mAbA and 5C6, and negative control 53C3) were titrated to determine the blocking effect of sialic acid-displaying A549 tumor cells on the binding of Siglec-9. Prior to the binding assay, tumor cells were loaded with Calcein-AM fluorescence, and binding was determined by the fluorescence released upon cell lysis. Fluorescence released in the absence of antibodies was set as 100% binding. Fitted data were analyzed using a three-parameter analysis in GraphPad Prism (GraphPad Software); n=3.

[0132] Figure 5 CD8+ T cell activation assay. The titration of monoclonal antibodies (68D4 and 224B1; comparative monoclonal antibodies mAbA and 5C6 and negative control 53C3) and their effects on the proliferation (A), activation markers CD69 (B), and CD25 (C) of CD8+ T cells (co-stimulated with anti-CD3 and anti-CD28 antibodies) from a Siglec-9 transgenic mouse model. The levels of CD8+ T cell proliferation and activation markers were analyzed by flow cytometry.

[0133] Figure 6 CD4+ T cell activation assay. The titration of monoclonal antibodies (68D4 and 224B1; comparative monoclonal antibodies mAbA and 5C6 and negative control 53C3) and their effects on the proliferation (A), activation markers CD69 (B), and CD25 (C) of CD4+ T cells (co-stimulated with anti-CD3 and anti-CD28 antibodies) from a Siglec-9 transgenic mouse model were investigated. The levels of CD4+ T cell proliferation and activation markers were analyzed by flow cytometry.

[0134] Figure 7 Monoclonal antibodies (68D4 and 224B1; comparative monoclonal antibodies mAbA and 5C6; and negative control 53C3) were titrated to determine the cytotoxic effect of NK92 cells on Calcein-AM-labeled K562 cells. Cytotoxicity was determined by fluorescence released upon K562 cell lysis. Specific lysis was calculated as: (lysis test wells - spontaneous lysis) / (maximum lysis - spontaneous lysis) x 100%. Fluorescence released after adding detergent Triton X-100 and after incubation of K562 cells alone were used to determine maximum and spontaneous lysis, respectively. Data were analyzed using a single-site (total) analysis in GraphPad Prism (GraphPad Software); n = 3.

[0135] Figure 8Humanization of rabbit antibody 68D4. The following comparisons are shown: light chain variable region of human IgG 263E11 (SEQ ID NO:54), light chain variable region of human germline sequence KV1-5 / J4 (SEQ ID NO:55 and SEQ ID NO:56), light chain variable region of rb 68D4 (SEQ ID NO:8), and light chain variable region of the resulting graft 68D4-hum (SEQ ID NO:48); and heavy chain variable region of human IgG 263E11 (SEQ ID NO:53), heavy chain variable region of human germline sequence VH3-23 / J4 (SEQ ID NO:57 and SEQ ID NO:58), heavy chain variable region of rb 68D4 (SEQ ID NO:7), and heavy chain variable region of the resulting graft 68D4-hum (SEQ ID NO:47).

[0136] Figure 9 Binding curves of monoclonal antibodies (rabbit 68D4; humanized 68D4 and comparative monoclonal antibody mAbA) to Siglec-9 were analyzed by ELISA. The binding of monoclonal antibodies to Siglec-9 was determined by measuring the accumulated colored product at 450 nm after the reaction was stopped and plotted against the concentration of the monoclonal antibody being evaluated. Invention Details

[0138] The invention described below by way of example may be suitably practiced without any single or multiple elements or single or multiple limitations not specifically disclosed herein.

[0139] The present invention will be described with reference to specific embodiments and certain accompanying drawings, but the invention is not limited thereto by the claims alone.

[0140] When the term "comprising" is used in this specification and claims, it does not exclude other elements. For the purposes of this invention, the term "consisting of" is considered a preferred embodiment of the term "comprising...". If a group is defined below as comprising at least a certain number of embodiments, this should also be understood as disclosing a group preferably consisting only of those embodiments.

[0141] Unless otherwise expressly stated, the indefinite or definite articles used with respect to singular nouns, such as “a,” “one,” or “the,” include the plural form of the noun. In the context of this invention, the terms “about” or “approximately” indicate a range of accuracy that, as will be understood by those skilled in the art, still ensures the technical effect of the features in question. This term indicates a deviation from the indicated values ​​of ±20%, preferably ±10%, and more preferably ±5%.

[0142] Technical terms are used according to their common sense. If a specific meaning is to be conveyed to certain terms, the definition of the term will be given in the context in which it is used below.

[0143] Human Siglec-7 (NCBI reference sequence: Q9Y286.1) has the following amino acid sequence (SEQ ID NO:60):

[0144]

[0145] Human Siglec-9 (NCBI reference sequence: NP_055256.1) is a member of the CD33-associated Siglec family, also known as sialic acid-binding Ig-like lectin 9, CD329, CDw329, FOAP-9, and OBBP-LIKE. Human Siglec-9 contains 463 amino acids and has the following amino acid sequence (SEQ ID NO:9):

[0146]

[0147] Siglec-9 is an inhibitory immune receptor expressed in the tumor microenvironment by cells of the adaptive and innate immune systems, such as NK cells, CD8+ and CD4+ T cells, and macrophages. Inhibitory signaling via this receptor is shown to be triggered by upregulated ligand expression on tumor cells and the tumor stroma. Stanczak et al., Proc Natl Acad Sci U SA. 2014 Sep 30; 111(39):14211-6. Importantly, Siglec-9 upregulation was observed in CD8+ and CD4+ tumor-infiltrating lymphocytes (TILs) of patients with CRC (colorectal cancer) and NSCLC (non-small cell lung cancer), particularly in those TILs expressing high levels of PD-1 (Stanczak et al., J Clin Invest. 2018 Nov 1; 128(11):4912-4923). Furthermore, Siglec-9 blockade has been suggested as a potential approach to control hepatitis B virus replication (Zhao et al., Front Immunol. 2018 May 30; 9:1124).

[0148] Certain aspects of this disclosure are based, at least in part, on the identification of an antibody molecule or a binding fragment thereof against Siglec-9:

[0149] - Inhibit the interaction between Siglec-9 and one or more Siglec-9 ligands; and / or

[0150] - Increase the proliferation of anti-CD3 / anti-CD28 stimulated CD8+ T cells; and / or

[0151] - Upregulate CD69 and CD25, activation markers of anti-CD3 / anti-CD28 stimulated CD8+ T cells; and / or

[0152] - Increase the proliferation of anti-CD3 / anti-CD28 stimulated CD4+ T cells; and / or

[0153] - Upregulate activation markers of anti-CD3 / anti-CD28 stimulated CD4+ T cells, CD69 and CD25; and / or

[0154] -Increases NK cell-mediated killing activity.

[0155] As described above, this disclosure considers anti-Siglec-9 antibody molecules or their binding fragments. Full-length antibodies include constant regions and variable regions. The constant region need not be present in the antigen-binding fragment of the antibody.

[0156] Therefore, binding fragments can include portions of a full-length antibody, such as the antigen-binding region or variable region of a full-length antibody. Examples of antibody fragments include Fab, F(ab')2, Id, and Fv fragments; diabody; linear antibody; single-chain antibody molecules (e.g., scFv); multispecific antibody fragments, such as bispecific, trispecific, and multispecific antibodies (e.g., bispecific, trispecific, and tetraspecific antibodies); minibody; chelated recombinant antibody; trispecific or bispecific antibody; intracellular antibody; nanobody; small modular immunopharmaceutical (SMIP); binding domain immunoglobulin fusion protein; camelified antibody; antibody containing VHH; and any other polypeptide formed from antibody fragments. Those skilled in the art know that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0157] Humanized antibodies are also within the scope of this disclosure.

[0158] This document discloses polypeptides having a specified sequence or a sequence substantially the same as or similar to that specified sequence, such as polypeptides having a sequence identity of at least about 85%, 90%, 95% or 99% with the specified sequence.

[0159] The preferred mathematical algorithm for determining the percentage of identity between two sequences is that of Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. This algorithm is incorporated into the BLASTp (Protein BLAST) program available on NCBI by Altschul et al. (1990) J. Mol. Biol. 215:403-410. https: / / blast.ncbi.nlm.nih.gov / The identity percentage can be determined using the standard parameters of the BLASTp program. For general parameters, the "Maximum Target Sequence" box can be set to 100, the "Short Query" box can be checked, the "Expected Threshold" box can be set to 10, the "Word Length" box can be set to "3", and the "Maximum Matches in Query Range" box can be set to "0". For scoring parameters, the "Matrix" box can be set to "BLOSUM62", the "Empty Cost" box can be set to "Existence: 11 Expansion: 1", and the "Composition Adjustment" box can be set to "Conditional Composition Score Matrix Adjustment". For filter and masking parameters, the "Low Complexity Region" box, the "Mask Only for Lookup Tables" box, and the "Mask Lowercase" box can be left unchecked.

[0160] According to this disclosure, "conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0161] As mentioned, this disclosure also relates in some embodiments to nucleic acids encoding antibody molecules or their binding fragments, vectors containing such nucleic acids, and host cells containing such nucleic acids or vectors.

[0162] Antibody molecules or their binding fragments can be encoded by a single nucleic acid (e.g., a single nucleic acid containing a nucleotide sequence encoding both the light and heavy chain polypeptides of an antibody), or by two or more separate nucleic acids, each encoding a different part of the antibody molecule or antibody fragment. Nucleic acids can be DNA, cDNA, RNA, etc.

[0163] The nucleic acids described herein can be inserted into vectors. A "vector" is any molecule or composition that has the ability to carry a nucleic acid sequence into a suitable cell in which the encoded polypeptide can be synthesized.

[0164] This disclosure further provides, in some respects, host cells (e.g., isolated or purified cells) comprising the nucleic acids or vectors of the present invention. The host cell can be any type of cell capable of being transformed with the nucleic acids or vectors of the present invention to produce polypeptides encoded therefrom.

[0165] Anti-Siglec-9 antibody molecules or their anti-Siglec-9 binding fragments can be formulated into compositions, particularly pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of the antibody or its binding fragment, mixed with a pharmaceutically acceptable carrier, excipient, or stabilizer.

[0166] Furthermore, the anti-Siglec-9 antibody molecule or its anti-Siglec-9 binding fragment and pharmaceutical composition described herein can be administered in methods of treating patients with a disease selected from cancer, acute hepatitis B, and chronic hepatitis B. This disease can be characterized by upregulation of one or more Siglec-9 ligands.

[0167] Treatment of non-small cell lung cancer, colorectal cancer, breast cancer, epithelial ovarian cancer, hepatocellular carcinoma, and prostate cancer may be particularly effective when using anti-Siglec-9 antibody molecules or anti-Siglec-9 binding fragments or pharmaceutical compositions as described herein.

[0168] Preferred embodiments of aspects B1 to B8 of the present invention relate to:

[0169] 1. An anti-Siglec-9 antibody molecule or its anti-Siglec-9 binding fragment, comprising:

[0170] The heavy chain variable region (VH) comprises the amino acid sequence of heavy chain complementarity-determining region 1 (VHCDR1) of SEQ ID NO:30 or a sequence having one, two, three, or four amino acid substitutions; the amino acid sequence of heavy chain complementarity-determining region 2 (VHCDR2) of SEQ ID NO:31 or a sequence having one, two, three, or four amino acid substitutions; and the amino acid sequence of heavy chain complementarity-determining region 3 (VHCDR3) of SEQ ID NO:32 or a sequence having one, two, three, or four amino acid substitutions; and

[0171] The light chain variable region (VL) comprises the amino acid sequence of light chain complementarity-determining region 1 (VLCDR1) of SEQ ID NO:33 or a sequence having one, two, three or four amino acid substitutions, the amino acid sequence of light chain complementarity-determining region 2 (VLCDR2) of SEQ ID NO:34 or a sequence having one, two, three or four amino acid substitutions, and the amino acid sequence of light chain complementarity-determining region 3 (VLCDR3) of SEQ ID NO:35 or a sequence having one, two, three or four amino acid substitutions.

[0172] 2. The antibody molecule or binding fragment in item 1 includes:

[0173] The heavy chain variable region (VH) comprises the amino acid sequence of heavy chain complementarity-determining region 1 (VHCDR1) of SEQ ID NO:30, the amino acid sequence of heavy chain complementarity-determining region 2 (VHCDR2) of SEQ ID NO:31, and the amino acid sequence of heavy chain complementarity-determining region 3 (VHCDR3) of SEQ ID NO:32; and

[0174] The light chain variable region (VL) comprises the amino acid sequence of light chain complementarity-determining region 1 (VLCDR1) of SEQ ID NO:33, the amino acid sequence of light chain complementarity-determining region 2 (VLCDR2) of SEQ ID NO:34, and the amino acid sequence of light chain complementarity-determining region 3 (VLCDR3) of SEQ ID NO:35.

[0175] 3. The antibody molecule or its binding fragment of the first or second item, comprising a heavy chain variable region (VH), said heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:36 or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:36.

[0176] 4. An antibody molecule or its binding fragment of any one of items 1-3, comprising a light chain variable region (VL), said light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:37 or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:37.

[0177] 5. The antibody molecule or its binding fragment of any one of items 1-4, comprising a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO:36 and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO:37.

[0178] 6. An antibody molecule or its binding fragment thereof from any of items 1-5, comprising one or more of the following characteristics (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10):

[0179] (i) Binding to human Siglec-9 with EC50 values ​​of less than about 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM or 0.05 nM, for example, as described in Example 3, when the antibody molecule or its binding fragment is used as a bivalent molecule in an ELISA test;

[0180] (ii) Binding to human Siglec-9 with a dissociation constant (KD) of less than about 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM or 0.1 nM, for example, as described in Example 4, when the antibody molecule or its binding fragment is tested as a divalent molecule using surface plasmon resonance, such as Carterra LSA;

[0181] (iii) The antibody molecule or its binding fragment is bound to human CD14+ monocytes at EC50 concentrations of less than about 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM or 0.1 nM, for example, as described in Example 5, when the antibody molecule or its binding fragment is tested as a bivalent molecule using flow cytometry;

[0182] (vi) Inhibit the interaction between Siglec-9 and one or more Siglec-9 ligands, for example, inhibit the binding of A549 tumor cells expressing sialic acid to Siglec-9, for example, as described in Example 6, when the antibody molecule or its binding fragment is tested as a bivalent molecule using an ELISA.

[0183] (v) Increase the proliferation of CD8+ T cells stimulated by anti-CD3 / anti-CD28, for example, as described in Example 7, when the antibody molecule or its binding fragment is tested as a bivalent molecule in a T cell activation assay using Cell Trace Violet and flow cytometry;

[0184] (vi) Upregulate the activation markers CD69 and CD25 of anti-CD3 / anti-CD28 stimulated CD8+ T cells, for example, as described in Example 7, when the antibody molecule or its binding fragment is tested as a bivalent molecule in a T cell activation assay using staining and flow cytometry;

[0185] (vii) Increase the proliferation of CD4+ T cells stimulated by anti-CD3 / anti-CD28, for example, as described in Example 7, when the antibody molecule or its binding fragment is tested as a bivalent molecule in a T cell activation assay using Cell Trace Violet and flow cytometry;

[0186] (viii) Upregulate the activation markers CD69 and CD25 of anti-CD3 / anti-CD28 stimulated CD4+ T cells, for example, as described in Example 7, when the antibody molecule or its binding fragment is tested as a bivalent molecule in a T cell activation assay using staining and flow cytometry; or

[0187] (ix) Increase NK cell-mediated killing activity, for example, increase the killing effect of Siglec-9-overexpressing NK92 cells on K562 cells, for example, as described in Example 8, when the antibody molecule or its binding fragment is tested as a bivalent molecule using Calcein-AM-labeled K562 cells; or

[0188] (x) binds to human Siglec-7.

[0189] 7. The antibody molecule or its binding fragment that competes with any of the antibody molecules or their binding fragments in items 1-6 for binding to human Siglec-9.

[0190] 8. The antibody molecule or its binding fragment in item 7, which competes with the antibody molecule or its binding fragment in any of items 1-6 for binding to the same epitope of human Siglec-9.

[0191] 9. A pharmaceutical composition comprising an antibody molecule or a binding fragment thereof from any one of items 1-8 and a pharmaceutically acceptable carrier, excipient or stabilizer.

[0192] 10. An antibody molecule or its binding fragment from any of items 1-8 or a pharmaceutical composition from item 9, for the treatment of a disease selected from cancer, acute hepatitis B, and chronic hepatitis B.

[0193] 11. An antibody molecule or its binding fragment from any of the items 1-8 used in claim 10, or a pharmaceutical composition of claim 9, wherein the cancer is selected from non-small cell lung cancer, colorectal cancer, breast cancer, epithelial ovarian cancer, hepatocellular carcinoma, and prostate cancer.

[0194] 12. Nucleic acid that encodes the antibody heavy chain variable region and / or light chain variable region or a binding fragment thereof of an antibody molecule of any one of items 1-8.

[0195] 13. An expression vector containing the nucleic acid described in item 12.

[0196] 14. A host cell containing the nucleic acid described in item 12 or the expression vector described in item 13.

[0197] 15. A method for producing antibody molecules, the method comprising culturing the host cells of item 14 under conditions suitable for gene expression. Example

[0198] Antibody discovery

[0199] Example 1a

[0200] NZW rabbits were immunized with Siglec-9-Fc protein via DNA immunization. Antibody libraries were derived from lymph node and spleen B cells by cloning the variable regions of immunoglobulin light and heavy chains into expression cassettes providing the constant heavy region of rabbit immunoglobulins and a transmembrane domain derived from human CD8. After transduction of the libraries into HEK 293T cells, antibody libraries were screened and then antigen-specifically sorted using fluorescently labeled Siglec-9. This sorting yielded over 1000 Siglec-9-specific antibody-expressing cell clones, which were further propagated for downstream analysis of antibody characterization. Siglec-9-specific antibodies with high affinity and no cross-reactivity to Siglec-7 were then subcloned into expression vectors for soluble antibody expression and expressed after transient transfection into HEK 293F cells. The antibodies were then characterized in various assays based on protein G purification.

[0201] The Siglec-9 DNA sequence used for gene immunization has the following sequence (codon-optimized compared to the human sequence):

[0202]

[0203] The Siglec-9-Fc protein used for protein immunization has the following amino acid sequence:

[0204]

[0205] Table 1 - Amino acid sequence of anti-Siglec-9 antibody 68D4.

[0206]

[0207] Example 1b

[0208] NZW rabbits were immunized with Siglec-9-Fc protein via DNA immunization. Antibody libraries were derived from lymph node and spleen B cells by cloning the variable regions of immunoglobulin light and heavy chains into expression cassettes providing the constant heavy region of rabbit immunoglobulins and a transmembrane domain derived from human CD8. After transduction of the libraries into HEK 293T cells, antibody libraries were screened and then antigen-specifically sorted using fluorescently labeled Siglec-9. This sorting yielded over 1000 Siglec-9-specific antibody-expressing cell clones, which were further propagated for downstream analysis of antibody characterization. Siglec-9-specific antibodies with high affinity and cross-reactivity to Siglec-7 were then subcloned into expression vectors for soluble antibody expression and expressed after transient transfection into HEK 293F cells. The antibodies were then characterized in various assays based on protein G purification.

[0209] The Siglec-9 DNA sequence used for gene immunization and the Siglec-9-Fc protein used for protein immunization are identical to those described in Example 1a (SEQ ID NO:10 and SEQ ID NO:11).

[0210] Table 2 - Amino acid sequence of anti-Siglec-9 antibody 224B1.

[0211]

[0212]

[0213] Measurement

[0214] Comparative antibodies

[0215] mAbA (WO 2017 / 153433) and 5C6 (WO 2017 / 075432); mAbA is cloned as a mouse-human chimera as described in WO 2017 / 153433. The mouse VH and VL domains are fused with the human constant region for IgG expression, and the resulting mAbA antibody is detected by secondary antibody against human IgG. 5C6 (WO 2017 / 075432) is cloned as a mouse-rabbit chimera. The mouse VH and VL domains are fused with the rabbit constant region for IgG expression. The resulting 5C6 antibody is detected by secondary antibody against rabbit, the same as the rabbit antibody described herein. Negative control: 53C3. 53C3 is generated by immunizing rabbits with an unrelated peptide.

[0216] Table 3 - Comparative amino acid sequences of anti-Siglec-9 antibody mAbA (WO 2017 / 153433) and 5C6 (WO 2017 / 075432)

[0217]

[0218]

[0219]

[0220] Example 2 - Specificity / cross-reactivity of Siglec-9 specific antibodies against Siglec-5, -7, -8, -9, and -15

[0221] Materials and methods

[0222] Specificity for Siglec-5, Siglec-7, Siglec-8, Siglec-15, and Siglec-9 was determined using a 96-well half-area ELISA plate (Corning) in enzyme-linked immunosorbent assay (ELISA). Siglec-5-Fc (R&Dsystems) and Siglec-7-Fc (in-house manufactured; the Siglec-7-Fc protein used has the following amino acid sequence):

[0223] Siglec-8-Fc (R&D systems), Siglec-15-Fc (R&D systems), and Siglec-9-Fc (in-house manufactured, SEQ ID NO: 11) were coated at 1 μg / ml in 50 mM carbonate-bicarbonate buffer (pH 9.6) for 15–20 hours at 4°C. The antigen was eluted (Tecan Hydrospeed) and the plates were blocked with 5% skim milk in phosphate-buffered saline (PBS). After washing the plates, antibody was added in triplicate at a concentration of 5 μg / ml (33 nM) in 0.5% milk PBS. The antibody-containing plates were incubated at room temperature for 1 hour. The plates were washed, and the bound antibody was detected using horseradish peroxidase (HRP)-labeled polyclonal anti-rabbit IgG antibody (Biolegend) or anti-human Fab fragment against mAbA (Sigma Aldrich). Development of 3,3',5,5'-tetramethylbenzidine (TMB; Sigma Aldrich) was terminated by adding sulfuric acid, and absorbance was measured at 450 nm (Tecan, CM INFINITE MONO 200).

[0224] result

[0225] Monoclonal antibody 68D4 showed specific binding to Siglec-9, but no cross-reactivity with closely related Siglec-5, Siglec-7, Siglec-8, and Siglec-15. Two comparative antibodies, mAbA and 5C6, exhibited similar behavior. The negative control 53C3 was unreactive to any of the test antigens. Monoclonal antibody 224B1 showed specific binding to Siglec-9 and Siglec-7, but no cross-reactivity with closely related Siglec-5, Siglec-8, and Siglec-15. Figure 1 ).

[0226] Example 3 - Measurement of EC50 of Siglec-9 protein by Siglec-9 specific antibody in enzyme-linked immunosorbent assay (ELISA)

[0227] Materials and methods

[0228] The binding affinity of the antibody to Siglec-9 was measured by serially diluting the fixed concentration of Siglec-9 protein with the analytical antibody in an ELISA. For this purpose, Siglec-9 was coated at a concentration of 1 μg / mL in 50 mM carbonate-bicarbonate buffer (pH 9.6) for 15–20 hours at 4 °C. The antigen solution (Tecan Hydrospeed) was eluted, and the plates were blocked for 1 hour at room temperature with 5% skim milk in phosphate-buffered saline (PBS). The plates were washed, and antibodies were added at concentrations of 0–420 nM. After incubation at room temperature for 1 hour, the plates were washed again. The binding antibody was detected by adding a horseradish peroxidase (HRP)-conjugated secondary antibody. Rabbit antibody was detected using polyclonal anti-rabbit IgG antibody (Biolegend), and mAbA was detected using anti-human Fab fragment (SigmaAldrich). After the final washing step, the plates were developed by adding 3,3',5,5'-tetramethylbenzidine (TMB; SigmaAldrich). The reaction was terminated by adding sulfuric acid. Absorbance was measured at 450 nm (Tecan, CM INFINITEMONO 200). The data were fitted and the EC50 value was determined by three-parameter analysis in GraphPad Prism (GraphPad Software).

[0229] result

[0230] Compared to mAbA and 5C6, monoclonal antibodies 68D4 and 224B1 showed stronger affinity for Siglec-9. Figure 2 ).

[0231] Example 4 - Affinity Measurement via Surface Plasmon Resonance (SPR)

[0232] Materials and methods

[0233] Antibodies were individually coated onto the HC2000M chip via SPR and sulfonyl-NHS / EDC coupling chemistry for K-cell assay. D Assay. The chip was then blocked with ethanolamine. The antigen was applied at one of three different concentrations (20 nM, 100 nM, and 500 nM) with an contact time of 240 seconds followed by a 120-second dissociation period. After each antigen incubation, the bound antigen was removed from the antibody with 10 mM glycine at pH 2.0. All kinetic parameters (k) were determined using a kinetic fitting program (LSA Kinetics, Carterra). on k off and K D ).

[0234] result

[0235] Compared to the comparative antibody mAbA, monoclonal antibody 68D4 exhibited strong affinity with a faster binding rate and a slower dissociation rate, resulting in an approximately 16.7-fold improvement in the dissociation constant. Monoclonal antibody 224B1 also showed strong affinity.

[0236] Table 4 - Kinetic parameters determined by SPR

[0237] Antibody <![CDATA[K D (M)]]> <![CDATA[k on (M -1 s -1 )]]> <![CDATA[k off (s -1 )]]> 68D4 1.50E-10 6.40E+05 9.60E-05 224B1 5.10E-09 2.30E+05 1.20E-03 mAbA 2.50E-09 2.10E+05 5.30E-04

[0238] Example 5 - Binding of Siglec-9 specific antibody to human monocytes

[0239] Materials and methods

[0240] Since monocytes express the target Siglec-9 strongly in a consistent manner, the binding of antibodies to human monocytes was evaluated. Therefore, human monocytes are an excellent and suitable in vitro model for characterizing the binding of Siglec-9-specific antibodies.

[0241] Human PBMCs were stained in vitro with a fluorescently labeled antibody against the monocyte marker CD14 (Biolegend) (allophycocyanin, APC) in assay buffer (0.5% (v / v) heat-inactivated fetal bovine serum (Gibco), 2 mM EDTA phosphate solution). Cells were then co-incubated with different concentrations (67 nM – 0.38 pM) of the antibodies to be evaluated. After washing, cells were stained with a fluorescently labeled antibody against human IgG (mAbA) or rabbit IgG (all other monoclonal antibodies to be evaluated; both secondary antibodies were from Biolegend) (Brilliant Violet 421). TM The two antibodies were co-incubated.

[0242] The binding of the Siglec-9 specific antibody to monocytes (CD14-positive live cells) was analyzed using a CytoFLEX flow cytometer (Beckham Coulter's). The mean fluorescence intensity of secondary antibody staining in the CD14-positive population at each dilution was measured using FlowJo software (BD). Data points generated from this analysis were fitted, and EC50 values ​​were determined using three-parameter analysis in GraphPad Prism (GraphPad Software).

[0243] result

[0244] In the relevant cellular environment of human CD14+ monocytes, monoclonal antibody 68D4 showed approximately 3-fold stronger affinity for Siglec-9 expressed on monocytes compared to mAbA. Compared to monoclonal antibody 5C6, 68D4 showed approximately 22-fold stronger binding. In the relevant cellular environment of human CD14+ monocytes, monoclonal antibody 224B1 showed similar affinity for Siglec-9 expressed on monocytes compared to mAbA. Compared to monoclonal antibody 5C6, 224B1 showed approximately 10-fold stronger binding. Figure 3 ).

[0245] Example 6 - Blocking of ligand binding to Siglec-9

[0246] Materials and methods

[0247] To analyze whether anti-Siglec-9 antibody recognizes the ligand-binding domain and blocks the binding of Siglec-9 to its ligand sialic acid, a cell-based assay was performed, in which the binding of sialic acid-expressing A549 tumor cells to Siglec-9-Fc was assessed by ELISA. ELISA plates were coated overnight at 4°C with 3 μg / ml protein A (Thermo Fisher Scientific). The plates were blocked with PBS and 1% BSA for 1 hour and incubated with 5 μg / ml Siglec-9-Fc for 1 hour at room temperature. Different concentrations of anti-Siglec-9 antibody (starting from 8 μg / ml or 53 nM) were mixed 1:1 with Calcein-AM (Thermo Fisher Scientific)-labeled A549 tumor cells. The mixture was added to ELISA plates and incubated at 4°C for 1 hour. After washing the plate, cells bound to the plate were lysed with PBS and 1% Triton-X 100, and the emitted fluorescence was measured using Synergy H1 (BioTek) at 485 nm excitation and 535 nm emission. Fitted data were analyzed using three-parameter analysis in GraphPad Prism (GraphPad Software).

[0248] result

[0249] Monoclonal antibodies 68D4 and 224B1 blocked the binding of sialic acid-expressing A549 tumor cells to Siglec-9 in a concentration-dependent manner, to a degree comparable to that of the comparative monoclonal antibodies mAbA and 5C6. Figure 4 ).

[0250] Example 7 - T cell activation assay

[0251] Materials and methods

[0252] For the T cell activation assay, cells from Siglec-9 transgenic mice were used (Stanczak et al., J Clin Invest. 2018 Nov 1; 128(11):4912-4923). In these mice, Siglec-9 expression was induced with Cre recombinase by excision of the GFP terminator before the Siglec-9 gene. Cre expression was driven by the CD4 promoter. Siglec-9 expression in these T cells resulted in a phenotype in which activation of cells was not achieved by stimulation of the T cell receptor alone and by co-stimulatory signaling via the CD28 receptor, but by disruption of a third inhibitory signal mediated by Siglec-9.

[0253] For T cell activation assays, 96-well plates were coated overnight at 4°C or for 2 hours at 37°C with 0.5 μg / ml anti-CD3 and 1 μg / ml anti-CD28. Spleen cells and lymph node cells were isolated from Siglec-9 transgenic mice. Red blood cells were lysed and labeled with Cell Trace Violet (Thermo Fisher Scientific). 250,000 cells / well were added with or without the antibody to be evaluated at various concentrations (0.25 μg / ml–0.0125 μg / ml; 1.7 nM–83 pM). Cells were cultured for 48 hours and then stained for T cell markers CD3, CD4, and CD8, as well as activation markers CD69 and CD25. Cell proliferation was assessed by analyzing Cell Trace Violet dilutions. Cells were analyzed using a CytoFLEX flow cytometer (Beckman Coulter's). The amplification index was calculated using FlowJo software (BD). Cell activation was measured by the mean fluorescence intensity of CD69 and CD25 on T cells. Data were described using GraphPad Prism (GraphPad Software).

[0254] result

[0255] CD8+ T cells co-stimulated with monoclonal antibodies 68D4 or 224B1 responded in a concentration-dependent manner via upregulation of proliferation and activation markers CD69 and CD25. Monoclonal antibody 68D4 exhibited stronger proliferation and activation at lower concentrations compared to the comparative antibody mAbA. Monoclonal antibody 224B1 exhibited stronger proliferation at higher concentrations compared to the comparative antibody mAbA. The activating factors of antibodies 224B1 and mAbA appeared to be comparable. Monoclonal antibody 5C6 showed neither stimulation nor activation. Figure 5 ).

[0256] Similarly, in CD4+ T cells, monoclonal antibody 68D4 exhibited stronger proliferation at lower concentrations compared to mAbA, while monoclonal antibody 224B1 showed stronger proliferation at higher concentrations compared to mAbA. In CD8+ T cells, monoclonal antibody 5C6 failed to elicit a stimulus or activation response. In all cases, stronger proliferation was also accompanied by stronger upregulation of activation markers CD69 and CD25. Figure 6 ).

[0257] Example 8 - Activation of Natural Killer Cell Activity

[0258] Materials and methods

[0259] NK cell killing assays were performed using a human NK cell line that overexpresses Siglec-9.

[0260] NK92 cells were stably transfected with a plasmid containing Siglec-9 and GFP (NK92-Siglec-9). Control cells were stably transfected with GFP only (NK92-GFP). NK92 cells were activated overnight with 20 ng / ml IL-15 and co-cultured for 4 h in 96-well V-plates with Calcein-AM-labeled K562 cells at a 3:1 ratio, with or without serially diluted antibody. Cells were precipitated by centrifugation and the supernatant was transferred to black 96-well flat-bottomed plates. K562 cell lysis was measured by Calcein-AM release in the supernatant using a Synergy H1 (BioTek) at 485 nm excitation and 535 nm emission. Specific lysis was calculated as follows: (lysis test wells - spontaneous lysis) / (maximum lysis - spontaneous lysis) x 100%. For spontaneous cell lysis, only K562 cells were used. To maximize lysis, Triton X-100 was added to K562 cells. Analyze data in GraphPad Prism (GraphPad Software).

[0261] result

[0262] Monoclonal antibody 68D4 showed concentration-dependent activation of NK cell-mediated cytotoxic activity. Specific cell lysis was achieved at lower concentrations compared to comparative monoclonal antibodies mAbA and 5C6. The latter antibody, 5C6, induced cell lysis only at the highest concentration tested. Monoclonal antibody 224B1 showed concentration-dependent activation of NK cell-mediated cytotoxic activity. Specific cell lysis reached values ​​comparable to comparative monoclonal antibody mAbA. Figure 7 ).

[0263] Example 9 - Humanization of Rabbit Antibody 68D4

[0264] Human IgG 263E11 was selected from the Memo Therapeutics AG human IgG database due to its close similarity to 68D4. First, the CDR of rabbit immunoglobulin 68D4 was transplanted into the framework of the variable domain of 263E11. Further adjustments were made to the framework positions: in the heavy chain, Kabat positions 27, 28, and 29, and position 94, were replaced with rabbit amino acids (comparison shown in...). Figure 8 In addition, some rabbit CDR positions were changed to human amino acids. In the light chain variable domain, positions 24 and 30 of CDR1 were replaced by human amino acid side chains. Figure 8 In the heavy chain CDR2, positions 61, 62, and 63 of the Kabat sequence were substituted with human amino acids. The resulting heavy and light chain sequences were synthesized and cloned into the IgG1 / kappa expression vector. Humanized 68D4 (68D4-hum) was expressed and purified from HEK293F cells. 68D4-hum was compared with its parent rb 68D4 and mAbA in ELISA, essentially following the protocol described in Example 3.

[0265] As an antigen, the extracellular domain of Siglec-9 fused with the C-terminal his-tag is used:

[0266] Binding IgG was detected using a polyclonal anti-rabbit IgG antibody (68D4; Biolegend, #406401) or an anti-human Fcγ fragment (humanized 68D4 and mAbA; Jackson Immuno Research Laboratory, #109-035-098). The results showed that humanization of 68D4 resulted in a slightly reduced binding affinity to the high-affinity Siglec-9 binder compared to rabbit 68D4. Figure 9 Siglec-9 binding capacity is comparable to mAbA (a mouse variable domain expressed with human IgG Fc).

[0267] Table 5 - Amino acid sequence of humanized anti-Siglec-9 antibody 68D4.

[0268]

[0269]

[0270] Table 6 - CDR amino acid sequence of rabbit / humanized anti-Siglec-9 antibody 68D4.

[0271] sequence list <110> Memorandum Therapy Inc. University of Basel <120> Anti-Siglec-9 antibody molecules <130> M11467WO <160> 60 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Heavy chain complementarity determination region 1 <400> 1 Ser Tyr Asp Met Thr 1 5 <210> 2 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Heavy chain complementarity determination region 2 <400> 2 Ile Ile Tyr Thr Gly Gly Leu Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 1 5 10 15 <210> 3 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Heavy chain complementarity determination region 3 <400> 3 Glu Val Gly Asp Asn Asn Asn Phe 1 5 <210> 4 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Light chain complementarity determination region 1 <400> 4 Gln Ala Ser Glu Ser Val Asp Ser Gly Leu Ala 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Light chain complementarity determination region 2 <400> 5 Asp Asp Thr Asp Leu Ala Ser 1 5 <210> 6 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Light chain complementarity determination region 3 <400> 6 Gln Tyr Thr Asp Tyr Gly Arg Thr Tyr Val Gly Thr 1 5 10 <210> 7 <211> 113 <212> PRT <213> Artificial sequence <220> <223> Heavy chain variable region <400> 7 Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Lys Pro Asp Glu Thr 1 5 10 15 Leu Thr Ile Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Ser Tyr Asp 20 25 30 Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Ile Ile Tyr Thr Gly Gly Leu Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Ile Thr 65 70 75 80 Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Glu Val 85 90 95 Gly Asp Asn Asn Asn Phe Trp Gly Pro Gly Thr Leu Val Thr Val Ser 100 105 110 Ser <210> 8 <211> 110 <212> PRT <213> Artificial Sequence <22​​​​​​​​​​​​​Leu Ala Trp Tyr His Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Asp Thr Asp Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly 50 55 60 Ser Arg Ser Gly Thr Ala His Thr Leu Thr Ile Ser Asp Leu Glu Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Tyr Thr Asp Tyr Gly Arg Thr 85 90 95 Tyr Val Gly Thr Phe Gly Gly Gly Thr Glu Val Val Ile Lys 100 105 110 <210> 9 <211> 463 <212> PRT <213> Homo sapiens <400> 9 Met Leu Leu Leu Leu Leu Pro Leu Leu Trp Gly Arg Glu Arg Ala Glu 1 5 10 15 Gly Gln Thr Ser Lys Leu Leu Thr Met Gln Ser Ser Val Thr Val Gln 20 25 30 Glu Gly Leu Cys Val His Val Pro Cys Ser Phe Ser Tyr Pro Ser His 35 40 45 Gly Trp Ile Tyr Pro Gly Pro Val Val His Gly Tyr Trp Phe Arg Glu 50 55 60 Gly Ala Asn Thr Asp Gln Asp Ala Pro Val Ala Thr Asn Asn Pro Ala 65 70 75 80 Arg Ala Val Trp Glu Glu Thr Arg Asp Arg Phe His Leu Leu Gly Asp 85 90 95 Pro His Thr Lys Asn Cys Thr Leu Ser Ile Arg Asp Ala Arg Arg Ser 100 105 110 Asp Ala Gly Arg Tyr Phe Phe Arg Met Glu Lys Gly Ser Ile Lys Trp 115 120 125 Asn Tyr Lys His His Arg Leu Ser Val Asn Val Thr Ala Leu Thr His 130 135 140 Arg Pro Asn Ile Leu Ile Pro Gly Thr Leu Glu Ser Gly Cys Pro Gln 145 150 155 160 Asn Leu Thr Cys Ser Val Pro Trp Ala Cys Glu Gln Gly Thr Pro Pro 165 170 175 Met Ile Ser Trp Ile Gly Thr Ser Val Ser Pro Leu Asp Pro Ser Thr 180 185 190 Thr Arg Ser Ser Val Leu Thr Leu Ile Pro Gln Pro Gln Asp His Gly 195 200 205 Thr Ser Leu Thr Cys Gln Val Thr Phe Pro Gly Ala Ser Val Thr Thr 210 215 220 Asn Lys Thr Val His Leu Asn Val Ser Tyr Pro Pro Gln Asn Leu Thr 225 230 235 240 Met Thr Val Phe Gln Gly Asp Gly Thr Val Ser Thr Val Leu Gly Asn 245 250 255 Gly Ser Ser Leu Ser Leu Pro Glu Gly Gln Ser Leu Arg Leu Val Cys 260 265 270 Ala Val Asp Ala Val Asp Ser Asn Pro Pro Ala Arg Leu Ser Leu Ser 275 280 285 Trp Arg Gly Leu Thr Leu Cys Pro Ser Gln Pro Ser Asn Pro Gly Val 290 295 300 Leu Glu Leu Pro Trp Val His Leu Arg Asp Ala Ala Glu Phe Thr Cys 305 310 315 320 Arg Ala Gln Asn Pro Leu Gly Ser Gln Gln Val Tyr Leu Asn Val Ser 325 330 335 Leu Gln Ser Lys Ala Thr Ser Gly Val Thr Gln Gly Val Val Gly Gly 340 345 350 Ala Gly Ala Thr Ala Leu Val Phe Leu Ser Phe Cys Val Ile Phe Val 355 360 365 Val Val Arg Ser Cys Arg Lys Lys Ser Ala Arg Pro Ala Ala Gly Val 370 375 380 Gly Asp Thr Gly Ile Glu Asp Ala Asn Ala Val Arg Gly Ser Ala Ser 385 390 395 400 Gln Gly Pro Leu Thr Glu Pro Trp Ala Glu Asp Ser Pro Pro Asp Gln 405 410 415 Pro Pro Pro White White White Arg White Val Gly Glu Gly Glu Leu Gln 420 425 430 Tyr Ala Ser Leu Ser Phe Gln Met Vallys Pro Trp Asp Ser Arg Gly 435 440 445 Gln Glu Ala Thr Asp Thr Glu Tyr Ser Glu Ile Lys Ile His Arg 450 455 460 <210> 10 <211> 1431 <212> DNA <213> The snowstorm <220> <223> Recombinant Siglec‑9 DNA fragment <400> 10 cctgcaggcc gctagcacca tgctgctgct gctgctgccc ctgctgtggg gccgcgagcg cgccgagggc cagacctcca agctgctgac catgcagtcc tccgtgaccg tgcaggaggg 120 cctgtgcgtg cacgtgccct gctccttctc ctacccctcc cacggctgga tctaccccgg 180 ccccgtggtg cacggctact ggttccgcga gggcgccaac accgaccagg acgccccgt 240 ggccaccaac aaccccgccc gcgccgtgtg ggaggagacc cgcgaccgct tccacctgct 300 gggcgacccc cacaccaaga actgcaccct gtccatccgc gacgcccgcc gctccgacgc 360 cggccgctac ttcttccgca tggagaaggg ctccatcaag tggaactaca agcaccaccg 420 cctgtccgtg aacgtgaccg ccctgaccca ccgccccaac atcctgatcc ccggcaccct 480 ggagtccggc tgcccccaga acctgacctg ctccgtgccc tgggcctgcg agcagggcac 540 cccccccatg atctcctgga tcggcacctc cgtgtccccc ctggacccct ccaccacccg 600 ctcctccgtg ctgaccctga tcccccagcc ccaggaccac ggcacctccc tgacctgcca 660 ggtgaccttc cccggcgcct ccgtgaccac caacaagacc gtgcacctga acgtgtccta 720 ccccccccag aacctgacca tgaccgtgtt ccagggcgac ggcaccgtgt ccaccgtgct 780 gggcaacggc tcctccctgt ccctgcccga gggccagtcc ctgcgcctgg tgtgcgccgt 840 ggacgccgtg gactccaacc cccccgcccg cctgtccctg tcctggcgcg gcctgaccct 900 gtgcccctcc cagccctcca accccggcgt gctggagctg ccctgggtgc acctgcgcga 960 cgccgccgag ttcacctgcc gcgcccagaa ccccctgggc tcccagcagg tgtacctgaa 1020 cgccgccgag ttcacctgcc gcgcccagaa ccccctgggc tcccagcagg tgtacctgaa 1020 cgtgtccctg cagtccaagg ccacctccgg cgtgacccag ggcgtggtgg gcggcgccgg 1080 cgtgtccctg cagtccaagg ccacctccgg cgtgacccag ggcgtggtgg gcggcgccgg 1080 cgccaccgcc ctggtgttcc tgtccttctg cgtgatcttc gtggtggtgc gctcctgccg 1140 cgccaccgcc ctggtgttcc tgtccttctg cgtgatcttc gtggtggtgc gctcctgccg 1140 caagaagtcc gcccgccccg ccgccggcgt gggcgacacc ggcatcgagg acgccaacgc 1200 caagaagtcc gcccgccccg ccgccggcgt gggcgacacc ggcatcgagg acgccaacgc 1200 cgtgcgcggc tccgcctccc agggccccct gaccgagccc tgggccgagg actccccccc 1260 cgtgcgcggc tccgcctccc agggccccct gaccgagccc tgggccgagg actccccccc 1260 cgaccagccc ccccccgcct ccgcccgctc ctccgtgggc gagggcgagc tgcagtacgc 1320 cgaccagccc ccccccgcct ccgcccgctc ctccgtgggc gagggcgagc tgcagtacgc 1320 ctccctgtcc ttccagatgg tgaagccctg ggactcccgc ggccaggagg ccaccgacac 1380 ctccctgtcc ttccagatgg tgaagccctg ggactcccgc ggccaggagg ccaccgacac 1380 cgagtactcc gagatcaaga tccaccgcta atctagaacg cgtttaatta a 1431 cgagtactcc gagatcaaga tccaccgcta atctagaacg cgtttaatta a 1431 <210> 11<210> 11 <211> 556<211> 556 <212> PRT<212> PRT <213> 人工序列 <213> Artificial sequence <220> <220> <223> 用于蛋白免疫的Siglec‑9‑Fc蛋白 <223> Siglec-9-Fc protein for protein immunization <400> 11 <400> 11 Gln Thr Ser Lys Leu Leu Thr Met Gln Ser Ser Val Thr Val Gln Glu Gln Thr Ser Lys Leu Leu Thr Met Gln Ser Ser Val Thr Val Gln Glu 1 5 10 15 1 5 10 15 [[ID=20 25 30 Trp Ile Tyr Pro Gly Pro Val Val His Gly Tyr Trp Phe Arg Glu Gly 35 40 45 Ala Asn Thr Asp Gln Asp Ala Pro Val Ala Thr Asn Asn Pro Ala Arg 50 55 60 Ala Val Trp Glu Glu Thr Arg Asp Arg Phe His Leu Leu Gly Asp Pro 65 70 75 80 His Thr Lys Asn Cys Thr Leu Ser Ile Arg Asp Ala Arg Arg Ser Asp 85 90 95 Ala Gly Arg Tyr Phe Phe Arg Met Glu Lys Gly Ser Ile Lys Trp Asn 100 105 110 Tyr Lys His His Arg Leu Ser Val Asn Val Thr Ala Leu Thr His Arg 115 120 125 Pro Asn Ile Leu Ile Pro Gly Thr Leu Glu Ser Gly Cys Pro Gln Asn 130 135 140 Leu Thr Cys Ser Val Pro Trp Ala Cys Glu Gln Gly Thr Pro Pro Met 145 150 155 160 Ile Ser Trp Ile Gly Thr Ser Val Ser Pro Leu Asp Pro Ser Thr Thr 165 170 175 Arg Ser Ser Val Leu Thr Leu Ile Pro Gln Pro Gln Asp His Gly Thr 180 185 190 Ser Leu Thr Cys Gln Val Thr Phe Pro Gly Ala Ser Val Thr Thr Asn 195 200 205 Lys Thr Val His Leu Asn Val Ser Tyr Pro Pro Gln Asn Leu Thr Met 210 215 220 Thr Val Phe Gln Gly Asp Gly Thr Val Ser Thr Val Leu Gly Asn Gly 225 230 235 240 Ser Ser Leu Ser Leu Pro Glu Gly Gln Ser Leu Arg Leu Val Cys Ala 245 250 255 Val Asp Ala Val Asp Ser Asn Pro Pro Ala Arg Leu Ser Leu Ser Trp 260 265 270 Arg Gly Leu Thr Leu Cys Pro Ser Gln Pro Ser Asn Pro Gly Val Leu 275 280 285 Glu Leu Pro Trp Val His Leu Arg Asp Ala Ala Glu Phe Thr Cys Arg 290 295 300 Ala Gln Asn Pro Leu Gly Ser Gln Gln Val Tyr Leu Asn Val Ser Leu 305 310 315 320 Gln Ser Lys Ala Thr Ser Gly Val Thr Gln Gly Val Asp Lys Thr His 325 330 335 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 340 345 350 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 355 360 365 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 370 375 380 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 385 390 395 400 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 405 410 415 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 420 425 430 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 435 440 445 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 450 455 460 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Asn Leu Thr Cys Leu 465 470 475 480 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 485 490 495 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 500 505 510 Asp Gly Ser Phe Phe Leu Asn Ser Thr Leu Thr Val Asp Lys Ser Arg 515 520 525 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 530 535 540 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 545 550 555 <210> 12 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Heavy chain complementarity determination region 1 <400> 12 Ser Tyr Trp Met His 1 5 <210> 13 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Heavy chain complementarity determination region 2 <400> 13 Glu Ile Asn Pro Ser Asn Gly His Thr Asn Tyr Asn Glu Lys Phe Glu 1 5 10 15 Ser <210> 14 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Heavy chain complementarity determination region 3 <400> 14 Gly Val Glu Ser Tyr Asp Phe Asp Asp Ala Leu Asp Tyr 1 5 10 <210> 15 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Light chain complementarity determination region 1 <400> 15 Arg Ala Ser Gln Asp Ile Asn Asn Tyr Leu Asn 1 5 10 <210> 16 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Light chain complementarity determination region 2 <400> 16 Tyr Thr Ser Arg Leu His Ser 1 5 <210> 17 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Light chain complementarity determination region 3 <400> 17 Gln Gln Gly Asn Thr Leu Pro Phe Thr 1 5 <210> 18 <211> 122 <212> PRT <213> Artificial sequence <220> <223> Heavy chain variable region <400> 18 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ser 1 5 10 15 Pro Val Lys Leu Ser Cys Lys Ala Ser Tyr Phe Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn Pro Ser Asn Gly His Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Glu Ser Lys Ala Thr Leu Thr Val Asp Arg Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Phe Tyr Cys 85 90 95 Ala Asn Gly Val Glu Ser Tyr Asp Phe Asp Asp Ala Leu Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 19 <211> 107 <212> PRT <213> Synthetic Sequence <220> <223> Light Chain Variable Region <400> 19 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Asn Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Ile Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Asn Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Phe 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 20 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Heavy chain complementarity determination region 1 <400> 20 Thr Asp Tyr Asn Ile His 1 5 <210> twenty one <211> 17 <212> PRT <213> Artificial sequence <220> <223> Heavy chain complementarity determination region 2 <400> twenty one Tyr Ile Tyr Pro Tyr Asn Gly Asp Thr Gly Tyr Asn Gln Lys Phe Gln 1 5 10 15 Asn <210> twenty two <211> 11 <212> PRT <213> Artificial sequence <220> <223> Heavy chain complementarity determination region 3 <400> twenty two Gly Asn Tyr Leu Tyr Tyr Tyr Ala Met Asp Tyr 1 5 10 <210> twenty three <211> 16 <212> PRT <213> Artificial sequence <220> <223> Light chain complementarity determination region 1 <400> twenty three Ser Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His 1 5 10 15 <210> twenty four <211> 7 <212> PRT <213> Artificial sequence <220> <223> Light chain complementarity determination region 2 <400> twenty four Lys Val Ser Asn Arg Phe Ser 1 5 <210> 25 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Light chain complementarity determination region 3 <400> 25 Ser Gln Asn Thr His Val Pro Leu Thr 1 5 <210> 26 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Heavy chain variable region <400> 26 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Arg Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Asn Ile His Trp Val Lys Gln Ser Gln Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Pro Tyr Asn Gly Asp Thr Gly Tyr Asn Gln Lys Phe 50 55 60 Gln Asn Lys Ala Thr Leu Thr Val Asp Asn Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Gly Asn Tyr Leu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 27 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region <400> 27 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Val Ser Ile Ser Cys Ser Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Ser Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Arg Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Asn 85 90 95 Thr His Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 <210> 28 <211> 436 <212> PRT <213> Synthetic Sequence <220> <223> Full - length heavy chain <400> 28 Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Lys Pro Asp Glu Thr 1 5 10 15 Leu Thr Ile Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Ser Tyr Asp 20 25 30 Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Ile Ile Tyr Thr Gly Gly Leu Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Ile Thr 65 70 75 80 Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Glu Val 85 90 95 Gly Asp Asn Asn Asn Phe Trp Gly Pro Gly Thr Leu Val Thr Val Ser 100 105 110 Ser Gly Gln Pro Lys Ala Pro Ser Val Phe Pro Leu Ala Pro Cys Cys 115 120 125 Gly Asp Thr Pro Ser Ser Thr Val Thr Leu Gly Cys Leu Val Lys Gly 130 135 140 Tyr Leu Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Thr Leu Thr 145 150 155 160 Asn Gly Val Arg Thr Phe Pro Ser Val Arg Gln Ser Ser Gly Leu Tyr 165 170 175 Ser Leu Ser Ser Val Val Ser Val Thr Ser Ser Ser Gln Pro Val Thr 180 185 190 Cys Asn Val Ala His Pro Ala Thr Asn Thr Lys Val Asp Lys Thr Val 195 200 205 Ala Pro Ser Thr Cys Ser Lys Pro Thr Cys Pro Pro Pro Glu Leu Leu 210 215 220 Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu 225 230 235 240 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 245 250 255 Gln Asp Asp Pro Glu Val Gln Phe Thr Trp Tyr Ile Asn Asn Glu Gln 260 265 270 Val Arg Thr Ala Arg Pro Pro Leu Arg Glu Gln Gln Phe Asn Ser Thr 275 280 285 Ile Arg Val Val Ser Thr Leu Pro Ile Ala His Gln Asp Trp Leu Arg 290 295 300 Gly Lys Glu Phe Lys Cys Lys Val His Asn Lys Ala Leu Pro Ala Pro 305 310 315 320 Ile Glu Lys Thr Ile Ser Lys Ala Arg Gly Gln Pro Leu Glu Pro Lys 325 330 335 Val Tyr Thr Met Gly Pro Pro Arg Glu Glu Leu Ser Ser Arg Ser Val 340 345 350 Ser Leu Thr Cys Met Ile Asn Gly Phe Tyr Pro Ser Asp Ile Ser Val 355 360 365 Glu Trp Glu Lys Asn Gly Lys Ala Glu Asp Asn Tyr Lys Thr Thr Pro 370 375 380 Ala Val Leu Asp Ser Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Ser 385 390 395 400 Val Pro Thr Ser Glu Trp Gln Arg Gly Asp Val Phe Thr Cys Ser Val 405 410 415 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Ile Ser Arg 420 425 430 Ser Pro Gly Lys 435 <210> 29 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Full - length light chain <400> 29 Asp Val Val Met Thr Gln Thr Pro Ala Ser Val Ser Glu Pro Val Gly 1 5 10 15 Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Glu Ser Val Asp Ser Gly 20 25 30 Leu Ala Trp Tyr His Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Asp Thr Asp Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly 50 55 60 Ser Arg Ser Gly Thr Ala His Thr Leu Thr Ile Ser Asp Leu Glu Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Tyr Thr Asp Tyr Gly Arg Thr 85 90 95 Tyr Val Gly Thr Phe Gly Gly Gly Thr Glu Val Val Ile Lys Gly Asp 100 105 110 Pro Val Ala Pro Thr Val Leu Ile Phe Pro Pro Ala Ala Asp Gln Val 115 120 125 Ala Thr Gly Thr Val Thr Ile Val Cys Val Ala Asn Lys Tyr Phe Pro 130 135 140 Asp Val Thr Val Thr Trp Glu Val Asp Gly Thr Thr Gln Thr Thr Gly 145 150 155 160 Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser Ala Asp Cys Thr Tyr Asn 165 170 175 Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr Gln Tyr Asn Ser His Lys 180 185 190 Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr Thr Ser Val Val Gln Ser 195 200 205 Phe Asn Arg Gly Asp Cys 210 <210> 30 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Heavy chain complementarity determination region 1 <400> 30 Ser Tyr Tyr Met Ser 1 5 <210> 31 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Heavy chain complementarity determination region 2 <400> 31 Tyr Ile Trp Thr Asp Gly Ser Ala His Tyr Ala Thr Trp Thr Lys Gly 1 5 10 15 <210> 32 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Heavy chain complementarity determination region 3 <400> 32 Ala Asn Ser Gly Ser His Thr Tyr Ile Glu Lys Phe Asn Leu 1 5 10 <210> 33 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Light chain complementarity determination region 1 <400> 33 Gln Ala Ser Pro Ser Ile Asn Asp Glu Leu Ser 1 5 10 <210> 34 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Light chain complementarity determination region 2 <400> 34 Glu Ala Ser Ala Leu Glu Ser 1 5 <210> 35 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Light chain complementarity determination region 3 <400> 35 Gln Cys Thr Tyr Gly Ser Arg Ser Asp Ile Ser Tyr Gly Asn Thr 1 5 10 15 <210> 36 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Heavy chain variable region <400> 36 Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Glu Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Ser Tyr Tyr 20 25 30 Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Tyr Ile Trp Thr Asp Gly Ser Ala His Tyr Ala Thr Trp Thr Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Asp Leu Lys Ile 65 70 75 80 Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Val Arg Ala 85 90 95 Asn Ser Gly Ser His Thr Tyr Ile Glu Lys Phe Asn Leu Trp Gly Pro 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 37 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> Light Chain Variable Region <400> 37 Asp Val Val Met Thr Gln Thr Pro Ala Ser Val Glu Ala Thr Val Gly 1 5 10 15 Gly Ser Val Thr Ile Lys Cys Gln Ala Ser Pro Ser Ile Asn Asp Glu 20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Thr Leu Leu Ile 35 40 45 [[ID=​​​​​​​Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Cys Thr Tyr Gly Ser Arg Ser 85 90 95 Asp Ile Ser Tyr Gly Asn Thr Phe Gly Gly Gly Thr Glu Val Val Val 100 105 110 Lys Gly <210> 38 <211> 443 <212> PRT <213> Artificial Sequence <220> <223> Full - length heavy chain <400> 38 Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Glu Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Ser Tyr Tyr 20 25 30 Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Tyr Ile Trp Thr Asp Gly Ser Ala His Tyr Ala Thr Trp Thr Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Asp Leu Lys Ile 65 70 75 80 Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Val Arg Ala 85 90 95 Asn Ser Gly Ser His Thr Tyr Ile Glu Lys Phe Asn Leu Trp Gly Pro 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Gly Gln Pro Lys Ala Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Cys Cys Gly Asp Thr Pro Ser Ser Thr Val Thr 130 135 140 Leu Gly Cys Leu Val Lys Gly Tyr Leu Pro Glu Pro Val Thr Val Thr 145 150 155 160 Trp Asn Ser Gly Thr Leu Thr Asn Gly Val Arg Thr Phe Pro Ser Val 165 170 175 Arg Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Ser Val Thr 180 185 190 Ser Ser Ser Gln Pro Val Thr Cys Asn Val Ala His Pro Ala Thr Asn 195 200 205 Thr Lys Val Asp Lys Thr Val Ala Pro Ser Thr Cys Ser Lys Pro Thr 210 215 220 Cys Pro Pro Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 245 250 255 Cys Val Val Val Asp Val Ser Gln Asp Asp Pro Glu Val Gln Phe Thr 260 265 270 Trp Tyr Ile Asn Asn Glu Gln Val Arg Thr Ala Arg Pro Pro Leu Arg 275 280 285 Glu Gln Gln Phe Asn Ser Thr Ile Arg Val Val Ser Thr Leu Pro Ile 290 295 300 Ala His Gln Asp Trp Leu Arg Gly Lys Glu Phe Lys Cys Lys Val His 305 310 315 320 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Arg 325 330 335 Gly Gln Pro Leu Glu Pro Lys Val Tyr Thr Met Gly Pro Pro Arg Glu 340 345 350 Glu Leu Ser Ser Arg Ser Val Ser Leu Thr Cys Met Ile Asn Gly Phe 355 360 365 Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Lys Asn Gly Lys Ala Glu 370 375 380 Asp Asn Tyr Lys Thr Thr Pro Ala Val Leu Asp Ser Asp Gly Ser Tyr 385 390 395 400 Phe Leu Tyr Ser Lys Leu Ser Val Pro Thr Ser Glu Trp Gln Arg Gly 405 410 415 Asp Val Phe Thr Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 420 425 430 Thr Gln Lys Ser Ile Ser Arg Ser Pro Gly Lys 435 440 <210> 39 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> Full - length light chain <400> 39 Asp Val Val Met Thr Gln Thr Pro Ala Ser Val Glu Ala Thr Val Gly 1 5 10 15 Gly Ser Val Thr Ile Lys Cys Gln Ala Ser Pro Ser Ile Asn Asp Glu 20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Thr Leu Leu Ile 35 40 45 Tyr Glu Ala Ser Ala Leu Glu Ser Gly Val Pro Ser Arg Phe Lys Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Gly Asp Leu Glu Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Cys Thr Tyr Gly Ser Arg Ser<0​​​ 100 105 110 Lys Gly Asp Pro Val Ala Pro Thr Val Leu Ile Phe Pro Pro Ala Ala 115 120 125 Asp Gln Val Ala Thr Gly Thr Val Thr Ile Val Cys Val Ala Asn Lys 130 135 140 Tyr Phe Pro Asp Val Thr Val Thr Trp Glu Val Asp Gly Thr Thr Gln 145 150 155 160 Thr Thr Gly Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser Ala Asp Cys 165 170 175 Thr Tyr Asn Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr Gln Tyr Asn 180 185 190 Ser His Lys Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr Thr Ser Val 195 200 205 Val Gln Ser Phe Asn Arg Gly Asp Cys 210 215 <210> 40 <211> 452 <212> PRT <213> Artificial Sequence <220> <223> Full - length heavy chain <400> 40 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ser 1 5 10 15 Pro Val Lys Leu Ser Cys Lys Ala Ser Tyr Phe Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn Pro Ser Asn Gly His Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Glu Ser Lys Ala Thr Leu Thr Val Asp Arg Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Phe Tyr Cys 85 90 95 Ala Asn Gly Val Glu Ser Tyr Asp Phe Asp Asp Ala Leu Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly Lys 450 <210> 41 <21l> 214 <212> PRT <213> Artificial Sequence <220> <223> Full - length light chain <400> 41 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Asn Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Ile Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Asn Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Phe 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 42 <211> 443 <212> PRT <213> Synthetic Sequence <220> <223> Full - length heavy chain<00%1493><400> 42 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Arg Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Asn Ile His Trp Val Lys Gln Ser Gln Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Pro Tyr Asn Gly Asp Thr Gly Tyr Asn Gln Lys Phe 50 55 60[[ID=4l]] Gln Asn Lys Ala Thr Leu Thr Val Asp Asn Ser Ser Ser Thr Ala Tyr<0x)01503>65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Gly Asn Tyr Leu Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser Gly Gln Pro Lys Ala Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Cys Cys Gly Asp Thr Pro Ser Ser Thr Val Thr 130 135 140 Leu Gly Cys Leu Val Lys Gly Tyr Leu Pro Glu Pro Val Thr Val Thr 145 150 155 160 Trp Asn Ser Gly Thr Leu Thr Asn Gly Val Arg Thr Phe Pro Ser Val 165 170 175 Arg Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Ser Val Thr 180 185 190 Ser Ser Ser Gln Pro Val Thr Cys Asn Val Ala His Pro Ala Thr Asn 195 200 205 Thr Lys Val Asp Lys Thr Val Ala Pro Ser Thr Cys Ser Lys Pro Thr 210 215 220 Cys Pro Pro Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 245 250 255 Cys Val Val Val Asp Val Ser Gln Asp Asp Pro Glu Val Gln Phe Thr 260 265 270 Trp Tyr Ile Asn Asn Glu Gln Val Arg Thr Ala Arg Pro Pro Leu Arg 275 280 285 Glu Gln Gln Phe Asn Ser Thr Ile Arg Val Val Ser Thr Leu Pro Ile 290 295 300 Ala His Gln Asp Trp Leu Arg Gly Lys Glu Phe Lys Cys Lys Val His 305 310 315 320 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Arg 325 330 335 Gly Gln Pro Leu Glu Pro Lys Val Tyr Thr Met Gly Pro Pro Arg Glu 340 345 350 Glu Leu Ser Ser Arg Ser Val Ser Leu Thr Cys Met Ile Asn Gly Phe 355 360 365 Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Lys Asn Gly Lys Ala Glu 370 375 380 Asp Asn Tyr Lys Thr Thr Pro Ala Val Leu Asp Ser Asp Gly Ser Tyr 385 390 395 400 Phe Leu Tyr Ser Lys Leu Ser Val Pro Thr Ser Glu Trp Gln Arg Gly 405 410 415 Asp Val Phe Thr Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 420 425 430 Thr Gln Lys Ser Ile Ser Arg Ser Pro Gly Lys 435 440 <210> 43 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> Full - length light chain <400> 43 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Val Ser Ile Ser Cys Ser Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Ser Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Arg Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Asn 85 90 95 Thr His Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 Gly Asp Pro Val Ala Pro Thr Val Leu Ile Phe Pro Pro Ala Ala Asp 115 120 125 Gln Val Ala Thr Gly Thr Val Thr Ile Val Cys Val Ala Asn Lys Tyr 130 135 140 Phe Pro Asp Val Thr Val Thr Trp Glu Val Asp Gly Thr Thr Gln Thr 145 150 155 160 Thr Gly Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser Ala Asp Cys Thr 165 170 175 Tyr Asn Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr Gln Tyr Asn Ser 180 185 190 His Lys Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr Thr Ser Val Val 195 200 205 Gln Ser Phe Asn Arg Gly Asp Cys 210 215 <210> 44 <211> 339 <212> PRT <213> Artificial sequence <220> <223> Siglec-9 fused to the C-terminal his tag <400> 44 Gln Thr Ser Lys Leu Leu Thr Met Gln Ser Ser Val Thr Val Gln Glu 1 5 10 15 Gly Leu Cys Val His Val Pro Cys Ser Phe Ser Tyr Pro Ser His Gly 20 25 30 Trp Ile Tyr Pro Gly Pro Val Val His Gly Tyr Trp Phe Arg Glu Gly 35 40 45 Ala Asn Thr Asp Gln Asp Ala Pro Val Ala Thr Asn Asn Pro Ala Arg 50 55 60 Ala Val Trp Glu Glu Thr Arg Asp Arg Phe His Leu Leu Gly Asp Pro 65 70 75 80 His Thr Lys Asn Cys Thr Leu Ser Ile Arg Asp Ala Arg Arg Ser Asp 85 90 95 Ala Gly Arg Tyr Phe Phe Arg Met Glu Lys Gly Ser Ile Lys Trp Asn 100 105 110 Tyr Lys His His Arg Leu Ser Val Asn Val Thr Ala Leu Thr His Arg 115 120 125 Pro Asn Ile Leu Ile Pro Gly Thr Leu Glu Ser Gly Cys Pro Gln Asn 130 135 140 Leu Thr Cys Ser Val Pro Trp Ala Cys Glu Gln Gly Thr Pro Pro Met 145 150 155 160 Ile Ser Trp Ile Gly Thr Ser Val Ser Pro Leu Asp Pro Ser Thr Thr 165 170 175 Arg Ser Ser Val Leu Thr Leu Ile Pro Gln Pro Gln Asp His Gly Thr 180 185 190 Ser Leu Thr Cys Gln Val Thr Phe Pro Gly Ala Ser Val Thr Thr Asn 195 200 205 Lys Thr Val His Leu Asn Val Ser Tyr Pro Pro Gln Asn Leu Thr Met 210 215 220 Thr Val Phe Gln Gly Asp Gly Thr Val Ser Thr Val Leu Gly Asn Gly 225 230 235 240 Ser Ser Leu Ser Leu Pro Glu Gly Gln Ser Leu Arg Leu Val Cys Ala 245 250 255 Val Asp Ala Val Asp Ser Asn Pro Pro Ala Arg Leu Ser Leu Ser Trp 260 265 270 Arg Gly Leu Thr Leu Cys Pro Ser Gln Pro Ser Asn Pro Gly Val Leu 275 280 285 Glu Leu Pro Trp Val His Leu Arg Asp Ala Ala Glu Phe Thr Cys Arg 290 295 300 Ala Gln Asn Pro Leu Gly Ser Gln Gln Val Tyr Leu Asn Val Ser Leu 305 310 315 320 Gln Ser Lys Ala Thr Ser Gly Val Thr Gln Gly Val Asp His His His 325 330 335 His His His <210> 45 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Heavy chain complementarity determination region 2 <400> 45 Ile Ile Tyr Thr Gly Gly Leu Thr Tyr Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 46 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Light chain complementarity determination region 1 <400> 46 Arg Ala Ser Glu Ser Val Ser Ser Gly Leu Ala 1 5 10 <210> 47 <211> 116 <212> PRT <213> Artificial sequence <220> <223> Heavy chain variable region <400> 47 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Ile Ser Cys Ala Ala Ser Gly Ile Asp Leu Ser Ser Tyr 20 25 30 Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ile Ile Tyr Thr Gly Gly Leu Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Glu Val Gly Asp Asn Asn Asn Phe Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 48 <211> 110 <212> PRT <213> artificial sequence <220> <223> Chain link <400> 48 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ser Gly 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Val 35 40 45 Tyr Asp Asp Thr Asp Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Phe Tyr Cys Gln Tyr Thr Asp Tyr Gly Arg Thr 85 90 95 Tyr Val Gly Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 49 <211> 446 <212> PRT <213> Artificial Sequence <220> <223> Full - length heavy chain <400> 49 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Ile Ser Cys Ala Ala Ser Gly Ile Asp Leu Ser Ser Tyr 20 25 30 Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ile Ile Tyr Thr Gly Gly Leu Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Glu Val Gly Asp Asn Asn Asn Phe Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 50 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> Full - length light chain <400> 50 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ser Gly 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Val 35 40 45 Tyr Asp Asp Thr Asp Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Phe Tyr Cys Gln Tyr Thr Asp Tyr Gly Arg Thr 85 90 95 Tyr Val Gly Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 51 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain Complementary Determining Region 2 <220> <221> variants <222> (12)..(12) <223> Xaa is either Asp or Ser <220> <221> variants <222> (13)..(13) <223> Xaa is either Ser or Trp <220> <221> variants <222> (14)..(14) <223> Xaa is Val or Ala <400> 51 Ile Ile Tyr Thr Gly Gly Leu Thr Tyr Tyr Ala Xaa Xaa Xaa Lys Gly 1 5 10 15 <210> 52 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Light chain complementarity determinant 1 <220> <221> variants <222> (1)..(1) <223> Xaa is Arg or Gln <220> <221> variants <222> (7)..(7) <223> Xaa is either Ser or Asp <400> 52 Xaa Ala Ser Glu Ser Val Xaa Ser Gly Leu Ala 1 5 10 <210> 53 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Heavy chain variable region <400> 53 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Ile Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Asp Ser Gly Val Arg Thr Tyr Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Phe Val Gly Ser Tyr Tyr Asp Tyr Trp Ser Gly Ser Phe Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 54 <211> 107 <212> PRT <213> Synthetic Sequence <220> <223> Light Chain Variable Region <400> 54 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 天冬氨酸-精氨酸-缬氨酸-苏氨酸-异亮氨酸-苏氨酸-半胱氨酸-精氨酸-丙氨酸-丝氨酸-谷氨酰胺-苏氨酸-异亮氨酸-丝氨酸-丝氨酸-色氨酸 20 25 30 亮氨酸-丙氨酸-色氨酸-酪氨酸-谷氨酰胺-谷氨酰胺-赖氨酸-脯氨酸-甘氨酸-赖氨酸-丙氨酸-脯氨酸-赖氨酸-亮氨酸-亮氨酸-缬氨酸 35 40 45 酪氨酸-赖氨酸-丙氨酸-丝氨酸-苏氨酸-亮氨酸-谷氨酸-丝氨酸-甘氨酸-缬氨酸-脯氨酸-丝氨酸-精氨酸-苯丙氨酸-丝氨酸-甘氨酸 50 55 60 丝氨酸-甘氨酸-丝氨酸-甘氨酸-苏氨酸-谷氨酸-苯丙氨酸-苏氨酸-亮氨酸-苏氨酸-异亮氨酸-丝氨酸-丝氨酸-亮氨酸-谷氨酰胺-脯氨酸 65 70 75 80 天冬氨酸-天冬氨酸-苯丙氨酸-丙氨酸-苏氨酸-苯丙氨酸-酪氨酸-半胱氨酸-谷氨酰胺-谷氨酰胺-酪氨酸-天冬酰胺-苏氨酸-酪氨酸-脯氨酸-亮氨酸 85 90 95 苏氨酸-苯丙氨酸-甘氨酸-甘氨酸-甘氨酸-苏氨酸-赖氨酸-缬氨酸-谷氨酸-异亮氨酸-赖氨酸 100 105 <210> 55 <211> 94 <212> PRT <213> Homo sapiens <400> 55 天冬氨酸-异亮氨酸-谷氨酰胺-甲硫氨酸-苏氨酸-谷氨酰胺-丝氨酸-脯氨酸-丝氨酸-苏氨酸-亮氨酸-丝氨酸-丙氨酸-丝氨酸-缬氨酸-甘氨酸 1 5 10 15 天冬氨酸-精氨酸-缬氨酸-苏氨酸-异亮氨酸-苏氨酸-半胱氨酸-精氨酸-丙氨酸-丝氨酸-谷氨酰胺-丝氨酸-异亮氨酸-丝氨酸-丝氨酸-色氨酸 20 25 30 亮氨酸-丙氨酸-色氨酸-酪氨酸-谷氨酰胺-谷氨酰胺-赖氨酸-脯氨酸-甘氨酸-赖氨酸-丙氨酸-脯氨酸-赖氨酸-亮氨酸-亮氨酸-异亮氨酸 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr 85 90 <210> 56 <211> 12 <212> PRT <213> Homo sapiens <400> 56 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 1 5 10 <210> 57 <211> 98 <212> PRT <213> Homo sapiens <400> 57 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys <210> 58 <211> 13 <212> PRT <213> Homo sapiens <400> 58 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 59 <211> 560 <212> PRT <213> Artificial Sequence <220> <223> Siglec-7-Fc <400> 59 Gln Lys Ser Asn Arg Lys Asp Tyr Ser Leu Thr Met Gln Ser Ser Val 1 5 10 15 Thr Val Gln Glu Gly Met Cys Val His Val Arg Cys Ser Phe Ser Tyr 20 25 30 Pro Val Asp Ser Gln Thr Asp Ser Asp Pro Val His Gly Tyr Trp Phe<000193rd]]35 40 45 Arg Ala Gly Asn Asp Ile Ser Trp Lys Ala Pro Val Ala Thr Asn Asn 50 55 60 Pro Ala Trp Ala Val Gln Glu Glu Thr Arg Asp Arg Phe His Leu Leu 65 70 75 80 Gly Asp Pro Gln Thr Lys Asn Cys Thr Leu Ser Ile Arg Asp Ala Arg 85 90 95 Met Ser Asp Ala Gly Arg Tyr Phe Phe Arg Met Glu Lys Gly Asn Ile 100 105 110 Lys Trp Asn Tyr Lys Tyr Asp Gln Leu Ser Val Asn Val Thr Ala Leu 115 120 125 Thr His Arg Pro Asn Ile Leu Ile Pro Gly Thr Leu Glu Ser Gly Cys 130 135 140 Phe Gln Asn Leu Thr Cys Ser Val Pro Trp Ala Cys Glu Gln Gly Thr 145 150 155 160 Pro Pro Met Ile Ser Trp Met Gly Thr Ser Val Ser Pro Leu His Pro 165 170 175 Ser Thr Thr Arg Ser Ser Val Leu Thr Leu Ile Pro Gln Pro Gln His 180 185 190 His Gly Thr Ser Leu Thr Cys Gln Val Thr Leu Pro Gly Ala Gly Val 195 200 205 Thr Thr Asn Arg Thr Ile Gln Leu Asn Val Ser Tyr Pro Pro Gln Asn 210 215 220 Leu Thr Val Thr Val Phe Gln Gly Glu Gly Thr Ala Ser Thr Ala Leu 225 230 235 240 Gly Asn Ser Ser Ser Leu Ser Val Leu Glu Gly Gln Ser Leu Arg Leu 245 250 255 Val Cys Ala Val Asp Ser Asn Pro Pro Ala Arg Leu Ser Trp Thr Trp 260 265 270 Arg Ser Leu Thr Leu Tyr Pro Ser Gln Pro Ser Asn Pro Leu Val Leu 275 280 285 Glu Leu Gln Val His Leu Gly Asp Glu Gly Glu Phe Thr Cys Arg Ala 290 295 300 Gln Asn Ser Leu Gly Ser Gln His Val Ser Leu Asn Leu Ser Leu Gln 305 310 315 320 Gln Glu Tyr Thr Gly Lys Met Arg Pro Val Ser Gly Val Leu Leu Val 325 330 335 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 340 345 350 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 355 360 365 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 370 375 380 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 385 390 395 400 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 405 410 415 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 420 425 430 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 435 440 445 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 450 455 460 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Asn 465 470 475 480 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 485 490 495 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 500 505 510 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Asn Ser Thr Leu Thr Val 515 520 525 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 530 535 540 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 545 550 555 560 <210> 60 <211> 467 <212> PRT <213> Homo sapiens <400> 60 Met Leu Leu Leu Leu Leu Leu Pro Leu Leu Trp Gly Arg Glu Arg Val 1 5 10 15 Glu Gly Gln Lys Ser Asn Arg Lys Asp Tyr Ser Leu Thr Met Gln Ser 20 25 30 Ser Val Thr Val Gln Glu Gly Met Cys Val His Val Arg Cys Ser Phe 35 40 45 Ser Tyr Pro Val Asp Ser Gln Thr Asp Ser Asp Pro Val His Gly Tyr 50 55 60 Trp Phe Arg Ala Gly Asn Asp Ile Ser Trp Lys Ala Pro Val Ala Thr 65 70 75 80 Asn Asn Pro Ala Trp Ala Val Gln Glu Glu Thr Arg Asp Arg Phe His 85 90 95 Leu Leu Gly Asp Pro Gln Thr Lys Asn Cys Thr Leu Ser Ile Arg Asp 100 105 110 Ala Arg Met Ser Asp Ala Gly Arg Tyr Phe Phe Arg Met Glu Lys Gly 115 120 125 Asn Ile Lys Trp Asn Tyr Lys Tyr Asp Gln Leu Ser Val Asn Val Thr 130 135 140 Ala Leu Thr His Arg Pro Asn Ile Leu Ile Pro Gly Thr Leu Glu Ser 145 150 155 160 Gly Cys Phe Gln Asn Leu Thr Cys Ser Val Pro Trp Ala Cys Glu Gln 165 170 175 Gly Thr Pro Pro Met Ile Ser Trp Met Gly Thr Ser Val Ser Pro Leu 180 185 190 His Pro Ser Thr Thr Arg Ser Ser Val Leu Thr Leu Ile Pro Gln Pro 195 200 205 Gln His His Gly Thr Ser Leu Thr Cys Gln Val Thr Leu Pro Gly Ala 210 215 220 Gly Val Thr Thr Asn Arg Thr Ile Gln Leu Asn Val Ser Tyr Pro Pro 225 230 235 240 Gln Asn Leu Thr Val Thr Val Phe Gln Gly Glu Gly Thr Ala Ser Thr 245 250 255 Ala Leu Gly Asn Ser Ser Ser Leu Ser Val Leu Glu Gly Gln Ser Leu 260 265 270 Arg Leu Val Cys Ala Val Asp Ser Asn Pro Pro Ala Arg Leu Ser Trp 275 280 285 Thr Trp Arg Ser Leu Thr Leu Tyr Pro Ser Gln Pro Ser Asn Pro Leu 290 295 300 Val Leu Glu Leu Gln Val His Leu Gly Asp Glu Gly Glu Phe Thr Cys 305 310 315 320 Arg Ala Gln Asn Ser Leu Gly Ser Gln His Val Ser Leu Asn Leu Ser 325 330 335 Leu Gln Gln Glu Tyr Thr Gly Lys Met Arg Pro Val Ser Gly Val Leu 340 345 350 Leu Gly Ala Val Gly Gly Ala Gly Ala Thr Ala Leu Val Phe Leu Ser 355 360 365 Phe Cys Val Ile Phe Ile Val Val Arg Ser Cys Arg Lys Lys Ser Ala 370 375 380 Arg Pro Ala Ala Asp Val Gly Asp Ile Gly Met Lys Asp Ala Asn Thr 385 390 395 400 Ile Arg Gly Ser Ala Ser Gln Gly Asn Leu Thr Glu Ser Trp Ala Asp 405 410 415 Asp Asn Pro Arg His His Gly Leu Ala Ala His Ser Ser Gly Glu Glu 420 425 430 Arg Glu Ile Gln Tyr Ala Pro Leu Ser Phe His Lys Gly Glu Pro Gln 435 440 445 Asp Leu Ser Gly Gln Glu Ala Thr Asn Asn Glu Tyr Ser Glu Ile Lys 450 455 460 Ile Pro Lys 465

Claims

1. An anti-Siglec-9 antibody molecule or its anti-Siglec-9 binding fragment, wherein the anti-Siglec-9 antibody molecule or its anti-Siglec-9 binding fragment comprises: (i) The heavy chain variable region (VH) comprising the amino acid sequence of heavy chain complementarity-determining region 1 (VHCDR1) of SEQ ID NO: 1, the amino acid sequence of heavy chain complementarity-determining region 2 (VHCDR2) of SEQ ID NO: 2, and the amino acid sequence of heavy chain complementarity-determining region 3 (VHCDR3) of SEQ ID NO: 3; and The light chain variable region (VL) comprises the amino acid sequence of light chain complementarity-determining region 1 (VLCDR1) of SEQ ID NO: 4, the amino acid sequence of light chain complementarity-determining region 2 (VLCDR2) of SEQ ID NO: 5, and the amino acid sequence of light chain complementarity-determining region 3 (VLCDR3) of SEQ ID NO:

6. or (ii) a heavy chain variable region (VH) comprising the amino acid sequence of heavy chain complementarity-determining region 1 (VHCDR1) of SEQ ID NO: 1, the amino acid sequence of heavy chain complementarity-determining region 2 (VHCDR2) of SEQ ID NO: 45, and the amino acid sequence of heavy chain complementarity-determining region 3 (VHCDR3) of SEQ ID NO: 3; and The light chain variable region (VL) comprises the amino acid sequence of light chain complementarity-determining region 1 (VLCDR1) of SEQ ID NO: 46, the amino acid sequence of light chain complementarity-determining region 2 (VLCDR2) of SEQ ID NO: 5, and the amino acid sequence of light chain complementarity-determining region 3 (VLCDR3) of SEQ ID NO:

6.

2. The molecule of claim 1, wherein the molecule comprises: a heavy chain variable region (VH) comprising the amino acid sequence of heavy chain complementarity-determining region 1 (VHCDR1) of SEQ ID NO: 1, the amino acid sequence of heavy chain complementarity-determining region 2 (VHCDR2) of SEQ ID NO: 2, and the amino acid sequence of heavy chain complementarity-determining region 3 (VHCDR3) of SEQ ID NO: 3; and a light chain variable region (VL) comprising the amino acid sequence of light chain complementarity-determining region 1 (VLCDR1) of SEQ ID NO: 4, the amino acid sequence of light chain complementarity-determining region 2 (VLCDR2) of SEQ ID NO: 5, and the amino acid sequence of light chain complementarity-determining region 3 (VLCDR3) of SEQ ID NO: 6; The heavy chain variable region (VH) comprises the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 7; and the light chain variable region (VL) comprises the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:

8.

3. The molecule of claim 1, wherein the molecule comprises: a heavy chain variable region (VH) comprising the amino acid sequence of heavy chain complementarity-determining region 1 (VHCDR1) of SEQ ID NO: 1, the amino acid sequence of heavy chain complementarity-determining region 2 (VHCDR2) of SEQ ID NO: 45, and the amino acid sequence of heavy chain complementarity-determining region 3 (VHCDR3) of SEQ ID NO: 3; and a light chain variable region (VL) comprising the amino acid sequence of light chain complementarity-determining region 1 (VLCDR1) of SEQ ID NO: 46, the amino acid sequence of light chain complementarity-determining region 2 (VLCDR2) of SEQ ID NO: 5, and the amino acid sequence of light chain complementarity-determining region 3 (VLCDR3) of SEQ ID NO: 6; The heavy chain variable region (VH) comprises the amino acid sequence of SEQ ID NO: 47, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 47; and the light chain variable region (VL) comprises the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:

48.

4. The molecule of claim 2, wherein the molecule comprises: a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:

8.

5. The molecule of claim 3, wherein the molecule comprises: a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 47 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:

48.

6. A pharmaceutical composition comprising the molecule of any one of claims 1-5 and a pharmaceutically acceptable carrier, excipient, or stabilizer.

7. A nucleic acid encoding the antibody heavy chain variable region and light chain variable region of the molecule according to any one of claims 1-5.

8. An expression vector comprising the nucleic acid of claim 7.

9. A host cell comprising the nucleic acid of claim 7 or the expression vector of claim 8.

10. A method for producing antibody molecules, the method comprising culturing the host cell of claim 9 under conditions suitable for gene expression.

11. Use of the molecule according to any one of claims 1-5 or the pharmaceutical composition according to claim 6 in the preparation of a medicament for treating non-small cell lung cancer.

Images