Anti-Siglec-9 antibody molecules

Abstract

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

Description

[Technical Field]

[0001] The present disclosure relates to an anti-Siglec-9 antibody molecule or a binding fragment thereof. The present disclosure further relates to a nucleic acid encoding the antibody molecule or a binding fragment thereof, an expression vector, a host cell, and a method for producing the antibody molecule or a binding fragment thereof. Pharmaceutical compositions comprising the antibody molecule or a binding fragment thereof are also provided. The anti-Siglec-9 antibody molecule or a binding fragment thereof of the present disclosure can be used (alone or in combination with other drugs or treatment modalities) to treat cancer, acute, or chronic hepatitis B. Accordingly, the present disclosure further relates to an anti-Siglec-9 antibody molecule or a binding fragment thereof, or a pharmaceutical composition comprising the anti-Siglec-9 antibody molecule or a binding fragment thereof, for use in treating cancer, acute, or chronic hepatitis B. [Background technology]

[0002] During malignant transformation, glycosylation is known to be significantly altered compared to healthy tissue due to differential expression of glycosyltransferases, glycosidases, and monosaccharide transporters in the cancer microenvironment. Upregulation and alteration of terminal sialic acid structures are hallmarks of cancer.

[0003] Sialic acid structural patterns are recognized by recognition receptors called Siglecs. Siglecs are immunomodulatory receptors, often inhibitory receptors, that bind to sialic acid structures and are preferentially expressed on immune cells. In recent years, several experimental models have provided evidence that Siglecs are involved in cancer progression and immune evasion. These experimental models also suggest that targeting Siglecs, such as Siglec-9, may be useful in improving antitumor immunity.

[0004] In view of the need for improved strategies for targeting diseases such as cancer, new compositions for modulating Siglec-9 activity are highly desirable. Summary of the Invention

[0005] Aspect A In one aspect A1, the present disclosure relates to an anti-Siglec-9 antibody molecule or an anti-Siglec-9 binding fragment thereof.

[0006] Structural properties In some embodiments of aspect A1, the disclosure relates to an anti-Siglec-9 antibody molecule or 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 of the CDRs) from a heavy chain variable region (VH) and / or a light chain variable region (VL) comprising the amino acid sequences set forth in Table 6, wherein one or more of the CDRs (or collectively all of the CDRs) may have one, two, three, four, five, six, or more changes, e.g., amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions, relative to the amino acid sequences set forth in Table 6.

[0007] In some embodiments of aspect A1, the disclosure relates to an anti-Siglec-9 antibody molecule or 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 of the CDRs) from a heavy chain variable region (VH) and / or a light chain variable region (VL) comprising an amino acid sequence set forth in Table 5, wherein one or more of the CDRs (or collectively all of the CDRs) may have one, two, three, four, five, six, or more changes, e.g., amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions, relative to the amino acid sequences set forth in Table 5.

[0008] In some embodiments of aspect A1, the disclosure relates to an anti-Siglec-9 antibody molecule or 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 of the CDRs) from a heavy chain variable region (VH) and / or a light chain variable region (VL) comprising the amino acid sequences set forth in Table 1, wherein one or more of the CDRs (or collectively all of the CDRs) may have one, two, three, four, five, six, or more changes, e.g., amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions, relative to the amino acid sequences set forth in Table 1.

[0009] In some embodiments of aspect A1, the disclosure relates to an anti-Siglec-9 antibody molecule or anti-Siglec-9 binding fragment thereof, comprising: a heavy chain variable region (VH) comprising one, two, or three of the following: a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 1, or a sequence having one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions; a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 51, or a sequence having one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions; and a 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., conservative amino acid substitutions), insertions, or deletions; and / or or a light chain variable region (VL) comprising one, two or three of the following: a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 52, or a sequence having one, two, three or four amino acid substitutions (e.g., conservative amino acid substitutions), insertions or deletions; a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 5, or a sequence having one, two, three or four amino acid substitutions (e.g., conservative amino acid substitutions), insertions or deletions; and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 6, or a sequence having one, two, three or four amino acid substitutions (e.g., conservative amino acid substitutions), insertions or deletions.

[0010] In some embodiments of aspect A1, the disclosure relates to an anti-Siglec-9 antibody molecule or anti-Siglec-9 binding fragment thereof, comprising: a heavy chain variable region (VH) comprising one, two, or three of the following: a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 1, or a sequence having one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions; a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 45, or a sequence having one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions; and a 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., conservative amino acid substitutions), insertions, or deletions. and / or the light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 46, or a sequence having one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions, the light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 5, or a sequence having one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions, and the light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 6, or a sequence having one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions.

[0011] In some embodiments of aspect A1, the disclosure relates to an anti-Siglec-9 antibody molecule or anti-Siglec-9 binding fragment thereof, comprising: a heavy chain variable region (VH) comprising one, two, or three of the following: a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 1, or a sequence having one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions; a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 2, or a sequence having one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions; and a 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., conservative amino acid substitutions), insertions, or deletions. and / or the light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 4, or a sequence having one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions, the light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 5, or a sequence having one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions, and the light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 6, or a sequence having one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions.

[0012] In some embodiments of aspect A1, the disclosure relates to an anti-Siglec-9 antibody molecule or anti-Siglec-9 binding fragment thereof, comprising: a 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 with 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions); and / or A light chain variable region (VL) comprising the light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 6, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions).

[0013] In some embodiments of aspect A1, the disclosure relates to an anti-Siglec-9 antibody molecule or anti-Siglec-9 binding fragment thereof, comprising: a heavy chain variable region (VH) comprising the heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 1, or a sequence with one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), the heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 51, or a sequence with one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), and the heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 3, or a sequence with one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions); and / or A light chain variable region (VL) comprising the light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 52, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions), the light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 5, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions), and the light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 6, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions).

[0014] In some embodiments of aspect A1, the disclosure relates to an anti-Siglec-9 antibody molecule or anti-Siglec-9 binding fragment thereof, comprising: a heavy chain variable region (VH) comprising the heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 1, or a sequence with one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), the heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 45, or a sequence with one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), and the heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 3, or a sequence with one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions); and / or A light chain variable region (VL) comprising the light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 46, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions), the light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 5, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions), and the light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 6, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions).

[0015] In some embodiments of aspect A1, the disclosure relates to an anti-Siglec-9 antibody molecule or anti-Siglec-9 binding fragment thereof, comprising: a heavy chain variable region (VH) comprising the heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 1, or a sequence with one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), the heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 2, or a sequence with one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), and the heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 3, or a sequence with one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions); and / or A light chain variable region (VL) comprising the light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 4, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions), the light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 5, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions), and the light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 6, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions).

[0016] In some embodiments of aspect A1, the disclosure relates to an anti-Siglec-9 antibody molecule or anti-Siglec-9 binding fragment thereof, comprising: 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, or a sequence with 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 A light chain variable region (VL) comprising the light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 4, or a sequence having one or two amino acid substitutions, the light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 5, and the light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 6, or a sequence having one, two, three, or four amino acid substitutions.

[0017] In some embodiments of aspect A1, the anti-Siglec-9 antibody molecule or anti-Siglec-9 binding fragment thereof 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: 51, and the amino acid sequence of heavy chain complementarity determining region 3 (VHCDR3) of SEQ ID NO: 3; and A light chain variable region (VH) comprising 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 (VHCDR3) of SEQ ID NO: 6.

[0018] In some embodiments of aspect A1, the anti-Siglec-9 antibody molecule or anti-Siglec-9 binding fragment thereof 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 (VH) 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 (VHCDR3) of SEQ ID NO: 6.

[0019] In some embodiments of aspect A1, the anti-Siglec-9 antibody molecule or anti-Siglec-9 binding fragment thereof 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 (VHCDR3) of SEQ ID NO: 6.

[0020] In some embodiments of aspect A1, the anti-Siglec-9 antibody molecule or anti-Siglec-9 binding fragment thereof 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: 51, 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: 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; wherein 1, 2, 3, 4, 5, 6, 7, or 8 amino acids within the CDRs are inserted, deleted, or substituted.

[0021] In some embodiments of aspect A1, the anti-Siglec-9 antibody molecule or anti-Siglec-9 binding fragment thereof comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 1, the heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 45, and the heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 3; The light chain variable region (VL) comprises the light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 46, the light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 5, and the light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 6. wherein 1, 2, 3, 4, 5, 6, 7, or 8 amino acids within the CDRs are inserted, deleted, or substituted.

[0022] In some embodiments of aspect A1, the anti-Siglec-9 antibody molecule or anti-Siglec-9 binding fragment thereof comprises: a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 1, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 2, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 3; and a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 4, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 5, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 6; wherein 1, 2, 3, 4, 5, 6, 7, or 8 amino acids within the CDRs are inserted, deleted, or substituted.

[0023] In some embodiments of aspect A1, the anti-Siglec9 antibody molecule or anti-Siglec9-binding fragment thereof comprises a heavy chain variable region (VH) 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 to SEQ ID NO:47.

[0024] In some embodiments of aspect A1, the anti-Siglec9 antibody molecule or anti-Siglec9-binding fragment thereof comprises a heavy chain variable region (VH) 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 to SEQ ID NO:7.

[0025] In some embodiments of aspect A1, the anti-Siglec9 antibody molecule or anti-Siglec9-binding fragment thereof comprises 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 to SEQ ID NO:48.

[0026] In some embodiments of aspect A1, the anti-Siglec9 antibody molecule or anti-Siglec9-binding fragment thereof comprises 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 to SEQ ID NO:8.

[0027] In some embodiments of aspect A1, the anti-Siglec9 antibody molecule or anti-Siglec9-binding fragment thereof comprises a heavy chain variable region (VH) 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 to SEQ ID NO: 47, and 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 to SEQ ID NO: 48.

[0028] In some embodiments of aspect A1, the anti-Siglec9 antibody molecule or anti-Siglec9-binding fragment thereof comprises a heavy chain variable region (VH) 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 to SEQ ID NO:7, and 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 to SEQ ID NO:8.

[0029] In some embodiments of aspect A1, the anti-Siglec9 antibody molecule or anti-Siglec9-binding fragment thereof 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.

[0030] In some embodiments of aspect A1, the anti-Siglec9 antibody molecule or anti-Siglec9-binding fragment thereof 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.

[0031] In some embodiments of aspect A1, the anti-Siglec-9 antibody molecule or anti-Siglec-9 binding fragment thereof is a humanized anti-Siglec-9 antibody molecule or anti-Siglec-9 binding fragment thereof.

[0032] Functional properties In some embodiments of aspect A1, the anti-Siglec-9 antibody molecule or anti-Siglec-9 binding fragment thereof comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) of the following characteristics: (i) the antibody molecule or binding fragment thereof binds to human Siglec-9 with an EC50 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 when tested as a bivalent molecule using an ELISA, e.g., as described in Example 3; (ii) the antibody molecule, or binding fragment thereof, when tested as a bivalent molecule using surface plasmon resonance, e.g., Cartara LSA, e.g., as described in Example 4, binds 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; (iii) the antibody molecule or binding fragment thereof, when tested as a bivalent molecule using a flow cytometer, e.g., as described in Example 5, binds to human CD14+ monocytes with an EC50 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; (vi) inhibiting the interaction of Siglec-9 with one or more Siglec-9 ligands (e.g., inhibiting the binding of Siglec-9 to sialic acid-expressing A549 tumor cells) when the antibody molecule or binding fragment thereof is tested as a bivalent molecule using an ELISA, e.g., as described in Example 6; (v) the antibody molecule or binding fragment thereof increases the proliferation of anti-CD3 / anti-CD28 stimulated CD8+ T cells when tested as a bivalent molecule in a T cell activation assay using Cell Trace Violet and a flow cytometer, e.g., as described in Example 7; (vi) the antibody molecule or binding fragment thereof upregulates the activation markers CD69 and CD25 on anti-CD3 / anti-CD28-stimulated CD8+ T cells when tested as a bivalent molecule in a T cell activation assay using staining and a flow cytometer, e.g., as described in Example 7; (vii) the antibody molecule or binding fragment thereof increases the proliferation of anti-CD3 / anti-CD28 stimulated CD4+ T cells when tested as a bivalent molecule in a T cell activation assay using Cell Trace Violet and a flow cytometer, e.g., as described in Example 7; (viii) the antibody molecule or binding fragment thereof upregulates the activation markers CD69 and CD25 on anti-CD3 / anti-CD28 stimulated CD4+ T cells when tested as a bivalent molecule in a T cell activation assay using staining and a flow cytometer, e.g., as described in Example 7; or (ix) increasing NK cell-mediated killing activity (e.g., increasing killing of K562 cells by NK92 cells overexpressing Siglec-9) when the antibody molecule or binding fragment thereof is tested as a bivalent molecule using calcein-AM labeled K562 cells, e.g., as described in Example 8.

[0033] In one aspect A2, the disclosure relates to an antibody molecule or binding fragment thereof that competes for binding to human Siglec-9 with an antibody molecule or binding fragment thereof described herein. In some embodiments of aspect A2, the disclosure relates to an antibody molecule or binding fragment thereof that competes for binding to human Siglec-9 with an antibody molecule or binding fragment thereof comprising: a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 1, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 2, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 3; and a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 4, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 5, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 6. In some embodiments of aspect A2, the disclosure relates to an antibody molecule or binding fragment thereof that competes for binding to human Siglec-9 with an antibody molecule or binding fragment thereof comprising 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.

[0034] In one aspect A3, the present disclosure relates to a pharmaceutical composition comprising an antibody molecule or binding fragment thereof as described herein and a pharmaceutically acceptable carrier, excipient or stabilizer.

[0035] In one aspect A4, the disclosure relates to an anti-Siglec-9 antibody molecule or binding fragment thereof described herein, or a pharmaceutical composition comprising the anti-Siglec-9 antibody molecule or binding fragment thereof described herein, for use in treating a disease selected from the group consisting of cancer, acute and chronic hepatitis B. In some embodiments of aspect A4, the cancer is selected from the group consisting of non-small cell lung cancer, colorectal cancer, breast cancer, epithelial ovarian cancer, hepatocellular carcinoma, and prostate cancer.

[0036] In one aspect A5, the present disclosure relates to a nucleic acid encoding the antibody heavy and / or light chain variable region of an antibody molecule or binding fragment thereof described herein.

[0037] In one aspect A6, the present disclosure relates to an expression vector comprising a nucleic acid described herein.

[0038] In one aspect A7, the present disclosure relates to a host cell comprising a nucleic acid described herein or an expression vector described herein.

[0039] In one aspect A8, the present disclosure relates to a method for producing an antibody molecule, the method comprising culturing a host cell as described herein under conditions suitable for gene expression.

[0040] Aspect B In one aspect B1, the present disclosure relates to an anti-Siglec-9 antibody molecule or an anti-Siglec-9 binding fragment thereof.

[0041] Structural properties In some embodiments of aspect B1, the disclosure relates to an anti-Siglec-9 antibody molecule or 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 of the CDRs) from a heavy chain variable region (VH) and / or a light chain variable region (VL) comprising the amino acid sequences set forth in Table 2, wherein one or more of the CDRs (or collectively all of the CDRs) may have one, two, three, four, five, six, or more changes, e.g., amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions, relative to the amino acid sequences set forth in Table 2.

[0042] In some embodiments of aspect B1, the disclosure relates to an anti-Siglec-9 antibody molecule or anti-Siglec-9 binding fragment thereof, comprising: a heavy chain variable region (VH) comprising one, two, or three of the heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 30 or a sequence having one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions, the heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 31 or a sequence having one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions, and 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., conservative amino acid substitutions), insertions, or deletions; and / or or a light chain variable region (VL) comprising one, two, or three of the following: a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 33, or a sequence having one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions; a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 34, or a sequence having one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions; and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 35, or a sequence having one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), insertions, or deletions.

[0043] In some embodiments of aspect B1, the disclosure relates to an anti-Siglec-9 antibody molecule or anti-Siglec-9 binding fragment thereof, comprising: a 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 with 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions); and / or A light chain variable region (VL) comprising the light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 35, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions).

[0044] In some embodiments of aspect B1, the disclosure relates to an anti-Siglec-9 antibody molecule or anti-Siglec-9 binding fragment thereof, comprising: a heavy chain variable region (VH) comprising the heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 30, or a sequence with one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), the heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 31, or a sequence with one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions), and the heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 32, or a sequence with one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions); and / or A light chain variable region (VL) comprising the light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 33, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions), the light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 34, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions), and the light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 35, or a sequence having 1, 2, 3, or 4 amino acid substitutions (e.g., conservative amino acid substitutions).

[0045] In some embodiments of aspect B1, the disclosure relates to an anti-Siglec-9 antibody molecule or anti-Siglec-9 binding fragment thereof, comprising: a 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 with 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 A light chain variable region (VL) comprising the light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 33, or a sequence having one or two amino acid substitutions, the light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 34, and the light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 35, or a sequence having one, two, three, or four amino acid substitutions (e.g., conservative amino acid substitutions).

[0046] In some embodiments of aspect B1, the anti-Siglec-9 antibody molecule or anti-Siglec-9 binding fragment thereof comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 30, the heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 31, and the heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 32; and A light chain variable region (VL) comprising the light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 33, the light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 34, and the light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 35.

[0047] In some embodiments of aspect B1, the anti-Siglec-9 antibody molecule or anti-Siglec-9 binding fragment thereof comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 30, the heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 31, and the heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 32; and A light chain variable region (VL) comprising the light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 33, the light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 34, and the light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 35. wherein 1, 2, 3, 4, 5, 6, 7, or 8 amino acids within the CDRs are inserted, deleted, or substituted.

[0048] In some embodiments of aspect B1, the anti-Siglec9 antibody molecule or anti-Siglec9-binding fragment thereof comprises a 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 to SEQ ID NO:36.

[0049] In some embodiments of aspect B1, the anti-Siglec9 antibody molecule or anti-Siglec9-binding fragment thereof comprises 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 to SEQ ID NO: 37.

[0050] In some embodiments of aspect B1, the anti-Siglec9 antibody molecule or anti-Siglec9-binding fragment thereof comprises a 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 to SEQ ID NO: 36, and 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 to SEQ ID NO: 37.

[0051] In some embodiments of aspect B1, the anti-Siglec9 antibody molecule or anti-Siglec9-binding fragment thereof comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 37.

[0052] In some embodiments of aspect B1, the anti-Siglec-9 antibody molecule or anti-Siglec-9 binding fragment thereof is a humanized anti-Siglec-9 antibody molecule or anti-Siglec-9 binding fragment thereof.

[0053] Functional properties In some embodiments of aspect B1, the anti-Siglec-9 antibody molecule or anti-Siglec-9 binding fragment thereof comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) of the following characteristics: (i) the antibody molecule or binding fragment thereof binds to human Siglec-9 with an EC50 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 when tested as a bivalent molecule using an ELISA, e.g., as described in Example 3; (ii) the antibody molecule, or binding fragment thereof, when tested as a bivalent molecule using surface plasmon resonance, e.g., Cartara LSA, e.g., as described in Example 4, binds 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; (iii) the antibody molecule or binding fragment thereof, when tested as a bivalent molecule using a flow cytometer, e.g., as described in Example 5, binds to human CD14+ monocytes with an EC50 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; (vi) inhibiting the interaction of Siglec-9 with one or more Siglec-9 ligands (e.g., inhibiting the binding of Siglec-9 to sialic acid-expressing A549 tumor cells) when the antibody molecule or binding fragment thereof is tested as a bivalent molecule using an ELISA, e.g., as described in Example 6; (v) the antibody molecule or binding fragment thereof increases the proliferation of anti-CD3 / anti-CD28 stimulated CD8+ T cells when tested as a bivalent molecule in a T cell activation assay using Cell Trace Violet and a flow cytometer, e.g., as described in Example 7; (vi) the antibody molecule or binding fragment thereof upregulates the activation markers CD69 and CD25 on anti-CD3 / anti-CD28-stimulated CD8+ T cells when tested as a bivalent molecule in a T cell activation assay using staining and a flow cytometer, e.g., as described in Example 7; (vii) the antibody molecule or binding fragment thereof increases the proliferation of anti-CD3 / anti-CD28 stimulated CD4+ T cells when tested as a bivalent molecule in a T cell activation assay using Cell Trace Violet and a flow cytometer, e.g., as described in Example 7; (viii) the antibody molecule or binding fragment thereof upregulates the activation markers CD69 and CD25 on anti-CD3 / anti-CD28-stimulated CD4+ T cells when tested as a bivalent molecule in a T cell activation assay using staining and a flow cytometer, e.g., as described in Example 7; (ix) when the antibody molecule or binding fragment thereof is tested as a bivalent molecule using calcein-AM labeled K562 cells, e.g., as described in Example 8, it increases NK cell-mediated killing activity (e.g., it increases the killing of K562 cells by NK92 cells overexpressing Siglec-9); or (x) Binding to human Siglec-7.

[0054] In one aspect B2, the disclosure relates to an antibody molecule or binding fragment thereof that competes for binding to human Siglec-9 with an antibody molecule or binding fragment thereof described herein. In some embodiments of aspect B2, the disclosure relates to an antibody molecule or binding fragment thereof that competes for binding to human Siglec-9 with an antibody molecule or binding fragment thereof comprising: a heavy chain variable region (VH) comprising the heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 30, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 31, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 32; and a light chain variable region (VL) comprising the light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 33, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 34, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 35. In some embodiments of aspect B2, the disclosure relates to an antibody molecule or binding fragment thereof that competes for binding to human Siglec-9 with an antibody molecule or binding fragment thereof comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 37.

[0055] In one aspect B3, the present disclosure relates to a pharmaceutical composition comprising an antibody molecule or binding fragment thereof described herein and a pharmaceutically acceptable carrier, excipient or stabilizer.

[0056] In one aspect B4, the present disclosure relates to an anti-Siglec-9 antibody molecule or binding fragment thereof described herein, or a pharmaceutical composition comprising the anti-Siglec-9 antibody molecule or binding fragment thereof described herein, for use in treating a disease selected from the group consisting of cancer, acute and chronic hepatitis B. In some embodiments, the cancer is selected from the group consisting of non-small cell lung cancer, colorectal cancer, breast cancer, epithelial ovarian cancer, hepatocellular carcinoma, and prostate cancer.

[0057] In one aspect B5, the present disclosure relates to a nucleic acid encoding the antibody heavy and / or light chain variable region of an antibody molecule or binding fragment thereof described herein.

[0058] In one aspect B6, the present disclosure relates to an expression vector comprising a nucleic acid described herein.

[0059] In one aspect B7, the present disclosure relates to a host cell comprising a nucleic acid described herein or an expression vector described herein.

[0060] In one aspect B8, the present disclosure relates to a method for producing an antibody molecule, the method comprising culturing a host cell as described herein under conditions suitable for gene expression. [Brief explanation of the drawings]

[0061] [Figure 1] Reactivity of monoclonal antibodies (68D4 and 224B1; reference monoclonal antibodies mAbA and 5C6, negative control 53C3) to Siglec-5, Siglec-7, Siglec-8, Siglec-15, and Siglec-9 by ELISA. Binding was determined by measuring the accumulated colorimetric product at 450 nm after stopping the reaction (n=3).

[0062] [Figure 2] Binding curves of monoclonal antibodies (68D4 and 224B1, reference monoclonal antibodies mAbA and 5C6, and negative control 53C3) to Siglec-9 (analyzed by ELISA). After stopping the reaction, binding of the monoclonal antibodies to Siglec-9, as determined by measuring the accumulated colorimetric product at 450 nm, is plotted against the concentration of the monoclonal antibody being evaluated. EC50 values, calculated by three-parameter analysis using GraphPad Prism (GraphPad Software), are shown at the bottom right of the graph (n = 3).

[0063] [Figure 3]Binding curves of monoclonal antibodies (68D4 and 224B1; reference monoclonal antibodies mAbA and 5C6, and negative control 53C3) to Siglec-9 expressed on human CD14+ monocytes (analyzed by flow cytometry). The mean fluorescent signal of secondary antibody bound to the CD14+ population was plotted against the concentration of the monoclonal antibody being evaluated. EC50 values, determined by three-parameter analysis in GraphPad Prism (GraphPad Software), are indicated at the bottom right of the graph.

[0064] [Figure 4] Blocking of Siglec-9 binding to sialic acid-presenting A549 tumor cells was determined by titration of monoclonal antibodies (68D4 and 224B1; reference monoclonal antibodies mAbA and 5C6, and negative control 53C3). Tumor cells were pre-labeled with calcein-AM, and binding was determined by measuring the fluorescence emitted upon cell lysis. Fluorescence emitted in the absence of antibody was considered 100% binding. Data were fitted using a three-parameter analysis in GraphPad Prism (GraphPad Software); n = 3.

[0065] [Figure 5] CD8+ T cell activation assay. Titration of monoclonal antibodies (68D4 and 224B1; comparison monoclonal antibodies mAbA and 5C6, and negative control 53C3) and their effect on proliferation (A) and upregulation of activation markers CD69 (B) and CD25 (C) of CD8+ T cells from Siglec-9 transgenic mice (costimulated with anti-CD3 and anti-CD28 antibodies). CD8+ T cell proliferation and activation marker levels were analyzed by flow cytometry.

[0066] [Figure 6]CD4+ T cell activation assay. Titration of monoclonal antibodies (68D4 and 224B1; comparison monoclonal antibodies mAbA and 5C6 & negative control 53C3) and their effect on proliferation (A) and upregulation of activation markers CD69 (B) and CD25 (C) of CD4+ T cells (co-stimulated with anti-CD3 and anti-CD28 antibodies) derived from a Siglec-9 transgenic mouse model. CD4+ T cell proliferation and activation marker levels were analyzed by flow cytometry.

[0067] [Figure 7] Titration of monoclonal antibodies (68D4 and 224B1; reference monoclonal antibodies mAbA and 5C6 & negative control 53C3) to measure the killing of calcein-AM-labeled K562 cells by NK92 cells. Killing was determined by the fluorescence emitted upon cytolysis of K562 cells. Specific lysis was calculated as follows: (lysis test well - spontaneous lysis) / (maximum lysis - spontaneous lysis) × 100%. Fluorescence emitted upon addition of the detergent Triton X-100 and upon incubation of K562 cells alone was used to determine maximum lysis and spontaneous lysis, respectively. Data were analyzed using a single-site (sum) analysis in GraphPad Prism (GraphPad Software); n = 3.

[0068] [Figure 8] Humanization of rabbit antibody 68D4. Alignment of the light chain variable domain of human IgG 263E11 (SEQ ID NO:54), human germline KV1-5 / J4 (SEQ ID NO:55 and SEQ ID NO:56), rb 68D4 (SEQ ID NO:8) and the resulting graft 68D4-hum (SEQ ID NO:48); and the heavy chain variable domain of human IgG 263E11 (SEQ ID NO:53), human germline sequence VH3-23 / J4 (SEQ ID NO:57 and SEQ ID NO:58), rb 68D4 (SEQ ID NO:7) and the resulting graft 68D4-hum (SEQ ID NO:47).

[0069] [Figure 9]Binding curves of monoclonal antibodies (rabbit 68D4, humanized 68D4, and reference monoclonal antibody mAbA) to Siglec-9 (ELISA analysis). After stopping the reaction, binding of the monoclonal antibodies to Siglec-9, as determined by measuring the accumulated colored product at 450 nm, is plotted against the concentration of the monoclonal antibody being evaluated.

[0070] (Detailed explanation) The present invention, as illustratively described below, may suitably be practiced in the absence of any element or elements, limitation or limitations not specifically disclosed herein.

[0071] The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims.

[0072] In the present specification and claims, where the term "comprising" is used, it does not exclude other elements. For the purposes of the present invention, the term "consisting of" is considered to be a preferred embodiment of the term "comprising". Hereinafter, when a group is defined as comprising at least a certain number of embodiments, this is also understood to disclose a group that preferably consists only of these embodiments.

[0073] When an indefinite or definite article is used when referring to a singular noun, such as "a", "an", or "the", it also includes the plural of that noun, unless something else is clearly stated. The term "about" or "approximately" in the context of the present invention indicates an interval of precision that a person skilled in the art would understand to still ensure the technical effect of the feature in question. It indicates a deviation of ±20%, preferably ±10%, and more preferably ±5% from the indicated numerical value.

[0074] Terms are used according to their common general knowledge. Where a particular term has a specific meaning, the definition of that term is set forth below in the context in which it is used.

[0075] Human Siglec-7 (NCBI Reference Sequence: Q9Y286.1) has the following amino acid sequence (SEQ ID NO: 60): JPEG0007736682000001.jpg50170

[0076] Human Siglec-9 (NCBI Reference Sequence: NP#055256.1) is a member of the Siglec family related to CD33 and is also known as sialic acid-binding Ig-like lectin 9, CD329, CDw329, FOAP-9, or OBBP-LIKE. Human Siglec-9 consists of 463 amino acids and has the following amino acid sequence (SEQ ID NO: 9). JPEG0007736682000002.jpg57170

[0077] Siglec-9 is an inhibitory immune receptor expressed in the tumor microenvironment by cells of the adaptive and innate immune systems (e.g., NK cells, CD8+- and CD4+ T cells, macrophages, etc.). Inhibitory signaling through this receptor has been shown to be triggered by ligands upregulated in tumor cells and tumor stroma (Laubli et al., Proc Natl Acad Sci US A. 2014 Sep 30;111(39):14211-6). Importantly, CD8+ and CD4+ tumor-infiltrating lymphocytes (TILs) from CRC (colorectal cancer) and NSCLC (non-small cell lung cancer) cancer patients show upregulated Siglec-9, particularly in TILs expressing high levels of PD-1 (Stanczak et al., J Clin Invest. 2018 Nov 1;128(11):4912-4923). Furthermore, inhibition of Siglec-9 has been suggested as a way to control hepatitis B virus replication (Zhao et al., Front Immunol. 2018 May 30;9:1124).

[0078] Certain aspects of the present disclosure are based, at least in part, on the identification of the following anti-Siglec-9 antibody molecules or binding fragments thereof: Inhibiting the interaction between Siglec-9 and one or more Siglec-9 ligands; and / or Increases proliferation of anti-CD3 / anti-CD28 stimulated CD8+ T cells; and / or Upregulating the activation markers CD69 and CD25 on anti-CD3 / anti-CD28 stimulated CD8+ T cells; and / or Increases proliferation of anti-CD3 / anti-CD28 stimulated CD4+ T cells; and / or Upregulating the activation markers CD69 and CD25 on anti-CD3 / anti-CD28 stimulated CD4+ T cells; and / or Enhances the killing activity of NK cells.

[0079] As noted above, the present disclosure contemplates anti-Siglec-9 antibody molecules or binding fragments thereof. Full-length antibodies contain constant and variable domains. The constant domains need not be present in antigen-binding fragments of antibodies.

[0080] Thus, a binding fragment can include a portion of an intact, full-length antibody, such as the antigen-binding or variable region of the complete antibody. Examples of antibody fragments include Fab, F(ab'), Id, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); multispecific antibody fragments such as bispecific, trispecific, and multispecific antibodies (e.g., diabodies, triabodies, and tetrabodies); minibodies; chelating recombinant antibodies; tribodies or bibodies; intrabodies; nanobodies; small modular immunopharmaceuticals (SMIPs), binding domain immunoglobulin fusion proteins; camelized antibodies; VHH-containing antibodies; and other polypeptides formed from antibody fragments. Those skilled in the art will recognize that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0081] Humanized antibodies are also within the scope of this disclosure.

[0082] Disclosed herein are polypeptides having the designated sequences or sequences substantially identical or similar thereto, e.g., at least about 85%, 90%, 95%, or 99% sequence identity to the designated sequences.

[0083] The determination of percent identity between two sequences is preferably accomplished using the mathematical algorithm of Karlin and Altschul (1993) Proc. Natl. Acad. Sci USA 90:5873-5877. Such an algorithm is incorporated into the program Altschul et al. (1990) J. Mol. Biol. 215:403-410, available at NCBI (https: / / blast.ncbi.nlm.nih.gov / ). The determination of percent identity can be performed using the standard parameters of the BLASTp program. Typical parameters include setting the "Max Target Sequences" box to 100, checking the "Short queries" box, setting the "Expect threshold" box to 10, setting the "Word Size" box to "3," and setting the "Max matches in a query range" box to "0." For scoring parameters, the "Matrix" box may be set to "BLOSUM62", the "Gap Costs" box may be set to "Existence:11 Extension:1", and the "Compositional adjustments" box may be set to "Conditional compositional score matrix adjustment". For filter and masking parameters, the "Low complexity regions" box may be unchecked, the "Mask for lookup table only" box may be unchecked, and the "Mask lower case letters" box may be unchecked.

[0084] According to the present disclosure, a "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0085] As mentioned above, the present disclosure also relates in some embodiments to nucleic acids encoding antibody molecules or binding fragments thereof, vectors containing such nucleic acids, and host cells containing such nucleic acids or vectors.

[0086] An antibody molecule or binding fragment thereof may be encoded by a single nucleic acid (e.g., a single nucleic acid comprising nucleotide sequences encoding the antibody light and heavy chain polypeptides), or by two or more separate nucleic acids, each encoding a different portion of the antibody molecule or antibody fragment. The nucleic acid may be DNA, cDNA, RNA, etc.

[0087] The nucleic acids described herein can be inserted into a vector, a "vector" being any molecule or composition capable of transporting a nucleic acid sequence into an appropriate cell where synthesis of the encoded polypeptide can occur.

[0088] The present disclosure in some aspects further provides host cells (e.g., isolated or purified cells) comprising the nucleic acids or vectors of the invention. The host cells can be any type of cell that can be transformed with the nucleic acids or vectors of the invention so as to produce the polypeptides encoded thereby.

[0089] The anti-Siglec-9 antibody molecule or anti-Siglec-9 binding fragment thereof of the present invention can be formulated into a composition, particularly a pharmaceutical composition, comprising a therapeutically effective amount of the antibody or binding fragment thereof in admixture with a pharmaceutically acceptable carrier, excipient, or stabilizer.

[0090] Furthermore, the anti-Siglec-9 antibody molecules or anti-Siglec-9 binding fragments thereof and pharmaceutical compositions described herein can be administered in methods for treating patients with a disease selected from the group consisting of cancer, acute and chronic hepatitis B, which disease can be characterized by elevated expression of one or more Siglec-9 ligands.

[0091] Treatment of non-small cell lung cancer, colorectal cancer, breast cancer, epithelial ovarian cancer, hepatocellular carcinoma and prostate cancer is believed to be particularly effective using the anti-Siglec-9 antibody molecules or anti-Siglec-9 binding fragments or pharmaceutical compositions described herein.

[0092] Preferred embodiments of aspects B1 to B8 of the present invention relate to: 1. An anti-Siglec-9 antibody molecule or an anti-Siglec-9 binding fragment thereof, comprising: a heavy chain variable region (VH) comprising the heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 30, or a sequence with 1, 2, 3, or 4 amino acid substitutions, the heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 31, or a sequence with 1, 2, 3, or 4 amino acid substitutions, and the heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 32, or a sequence with 1, 2, 3, or 4 amino acid substitutions; and A light chain variable region (VL) comprising the light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 33, or a sequence having 1, 2, 3, or 4 amino acid substitutions, the light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 34, or a sequence having 1, 2, 3, or 4 amino acid substitutions, and the light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 35, or a sequence having 1, 2, 3, or 4 amino acid substitutions. 2. The antibody molecule or binding fragment thereof according to item 1, comprising: a heavy chain variable region (VH) comprising the heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 30, the heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 31, and the heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 32; and A light chain variable region (VL) comprising the light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 33, the light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 34, and the light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 35.

[0093] 3. The antibody molecule or binding fragment thereof described in item 1 or 2, comprising a 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 to SEQ ID NO: 36.

[0094] 4. The antibody molecule or binding fragment thereof according to any one of items 1 to 3, comprising 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 to SEQ ID NO: 37.

[0095] 5. The antibody molecule or binding fragment thereof according to any one of items 1 to 4, comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 36, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 37.

[0096] 6. The antibody molecule or binding fragment thereof according to any one of items 1 to 5, comprising one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) of the following properties: (i) the antibody molecule or binding fragment thereof binds to human Siglec-9 with an EC50 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 when tested as a bivalent molecule using an ELISA, e.g., as described in Example 3; (ii) the antibody molecule, or binding fragment thereof, when tested as a bivalent molecule using surface plasmon resonance, e.g., Cartara LSA, e.g., as described in Example 4, binds 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; (iii) the antibody molecule or binding fragment thereof, when tested as a bivalent molecule using a flow cytometer, e.g., as described in Example 5, binds to human CD14+ monocytes with an EC50 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; (vi) inhibiting the interaction of Siglec-9 with one or more Siglec-9 ligands (e.g., inhibiting the binding of Siglec-9 to sialic acid-expressing A549 tumor cells) when the antibody molecule or binding fragment thereof is tested as a bivalent molecule using an ELISA, e.g., as described in Example 6; (v) the antibody molecule or binding fragment thereof increases the proliferation of anti-CD3 / anti-CD28 stimulated CD8+ T cells when tested as a bivalent molecule in a T cell activation assay using Cell Trace Violet and a flow cytometer, e.g., as described in Example 7; (vi) the antibody molecule or binding fragment thereof upregulates the activation markers CD69 and CD25 on anti-CD3 / anti-CD28-stimulated CD8+ T cells when tested as a bivalent molecule in a T cell activation assay using staining and a flow cytometer, e.g., as described in Example 7; (vii) the antibody molecule or binding fragment thereof increases the proliferation of anti-CD3 / anti-CD28 stimulated CD4+ T cells when tested as a bivalent molecule in a T cell activation assay using Cell Trace Violet and a flow cytometer, e.g., as described in Example 7; (viii) the antibody molecule or binding fragment thereof upregulates the activation markers CD69 and CD25 on anti-CD3 / anti-CD28-stimulated CD4+ T cells when tested as a bivalent molecule in a T cell activation assay using staining and a flow cytometer, e.g., as described in Example 7; (ix) when the antibody molecule or binding fragment thereof is tested as a bivalent molecule using calcein-AM labeled K562 cells, e.g., as described in Example 8, it increases NK cell-mediated killing activity (e.g., it increases the killing of K562 cells by NK92 cells overexpressing Siglec-9); or (x) Binding to human Siglec-7.

[0097] 7. An antibody molecule or a binding fragment thereof that competes with the antibody molecule or binding fragment thereof according to any one of items 1 to 6 for binding to human Siglec-9.

[0098] 8. The antibody molecule or binding fragment thereof according to item 7, which competes with the antibody molecule or binding fragment thereof according to any one of items 1 to 6 for binding to the same epitope on human Siglec-9.

[0099] 9. A pharmaceutical composition comprising the antibody molecule or binding fragment thereof according to any one of items 1 to 8 and a pharmaceutically acceptable carrier, excipient or stabilizer.

[0100] 10. The antibody molecule or binding fragment thereof according to any one of items 1 to 8 or the pharmaceutical composition according to item 9 for use in treating a disease selected from the group consisting of cancer, acute and chronic hepatitis B.

[0101] 11. The antibody molecule or binding fragment thereof according to any one of items 1 to 8, or the pharmaceutical composition according to item 9 for use according to item 10, wherein the cancer is selected from the group consisting of non-small cell lung cancer, colorectal cancer, breast cancer, epithelial ovarian cancer, hepatocellular carcinoma, and prostate cancer.

[0102] 12. A nucleic acid encoding the antibody heavy chain and / or light chain variable region of the antibody molecule or binding fragment thereof according to any one of items 1 to 8.

[0103] 13. An expression vector comprising the nucleic acid according to item 12.

[0104] 14. A host cell comprising the nucleic acid of item 12 or the expression vector of item 13.

[0105] 15. A method for producing an antibody molecule, comprising culturing the host cell of item 14 under conditions suitable for gene expression. [Example]

[0106] Antibody search Example 1a NZW rabbits were immunized by DNA immunization or Siglec-9-Fc protein injection, and an antibody repertoire expression library was generated from lymph node and splenic B cells by cloning immunoglobulin light and heavy chain variable regions into an expression cassette containing a rabbit immunoglobulin constant heavy region and a transmembrane domain derived from human CD8. The antibody library was then transfected into HEK293T cells and then screened for antigen-specific sorting using fluorescently labeled Siglec-9. This selection yielded over 1,000 cell clones expressing Siglec-9-specific antibodies, which were then expanded and further characterized. Next, high-affinity Siglec-9-specific antibodies with no cross-reactivity to Siglec-7 were subcloned into a soluble antibody expression vector and transiently expressed in HEK293F cells. After protein G-based purification, the antibodies were characterized using various assays.

[0107] The DNA sequence of Siglec-9 used for genetic immunization was the following sequence (codon-optimized compared with the human sequence): JPEG0007736682000003.jpg155170

[0108] The Siglec-9-Fc protein used for protein immunization had the following amino acid sequence: JPEG0007736682000004.jpg61170

[0109] Amino acid sequence of anti-Siglec-9 antibody 68D4. [Table 1]

[0110] Example 1b NZW rabbits were immunized by DNA immunization or Siglec-9-Fc protein injection, and antibody repertoire expression libraries were generated from lymph node and splenic B cells by cloning immunoglobulin light and heavy chain variable regions into expression cassettes containing rabbit immunoglobulin constant heavy regions and a transmembrane domain derived from human CD8. The antibody library was then transfected into HEK 293T cells and then screened using fluorescently labeled Siglec-9 for antigen-specific sorting. This selection yielded over 1,000 cell clones expressing Siglec-9-specific antibodies, which were then expanded and further characterized. Next, Siglec-9-specific antibodies with high affinity and cross-reactivity to Siglec-7 were subcloned into a soluble antibody expression vector and expressed in HEK 293F cells by transient transfection. After protein G-based purification, the antibodies were characterized using various assays.

[0111] The Siglec-9 DNA sequence used for genetic immunization and the Siglec-9-Fc protein used for protein immunization were the same as those described for Example 1a (SEQ ID NO: 10 and SEQ ID NO: 11).

[0112] Amino acid sequence of anti-Siglec9 antibody 224B1. [Table 2]

[0113] Assay comparative antibody mAbA (WO 2017 / 153433), 5C6 (WO 2017 / 075432); mAbA was cloned as a mouse-human chimera as described in WO 2017 / 153433. The mouse VH and VL domains were fused to a human constant domain for IgG expression, and the resulting mAbA antibody was detected with a secondary anti-human IgG. 5C6 (WO 2017 / 075432) was cloned as a mouse-rabbit chimera. The mouse VH and VL domains were fused to a rabbit constant domain for IgG expression. The resulting 5C6 antibody was detected with the same secondary anti-rabbit antibody as the rabbit antibody described here. Negative control: 53C3. 53C3 was expressed in rabbits by immunization with an irrelevant peptide.

[0114] Amino acid sequences of comparative anti-Siglec-9 antibodies mAbA (WO 2017 / 153433) and 5C6 (WO 2017 / 075432) [Table 3] JPEG0007736682000008.jpg58170

[0115] Example 2 - Specificity / cross-reactivity of Siglec9-specific antibodies to Siglec5, -7, -8, -9 and -15 Materials and Methods The specificity for Siglec-5, Siglec-7, Siglec-8, Siglec-15, and Siglec-9 was measured by enzyme-linked immunosorbent assay (ELISA) using a 96-well half-area ELISA plate (Corning). Siglec-5-Fc (R&D Systems) and Siglec-7-Fc (manufactured in-house) were used. The Siglec-7-Fc protein used had the following amino acid sequence: JPEG0007736682000009.jpg69170Siglec-8-Fc (R&D systems), Siglec-15-Fc (R&D systems), and Siglec-9-Fc (in-house production, 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 washed off (Tecan Hydrospeed), and the plate was blocked with 5% nonfat milk in phosphate-buffered saline (PBS). After washing, antibodies were added in triplicates at 5 μg / ml (33 nM) in 0.5% milk in PBS. The antibody-containing plates were incubated at room temperature for 1 hour. Plates were washed, and bound antibodies were detected with horseradish peroxidase (HRP)-conjugated polyclonal anti-rabbit IgG antibody (Biolegend) or anti-human Fab fragment for mAbA (Sigma-Aldrich). 3,3',5,5'-Tetramethylbenzidine (TMB; Sigma-Aldrich) color development was stopped by adding sulfuric acid, and absorbance was measured at 450 nm (Tecan, CM INFINITE MONO 200).

[0116] result Monoclonal antibody 68D4 specifically binds to Siglec-9 but does not cross-react with the closely related antibodies Siglec-5, Siglec-7, Siglec-8, or Siglec-15. Two comparative antibodies, mAbA and 5C6, also exhibit similar behavior. The negative control, 53C3, shows no reactivity with any of the antigens tested. Monoclonal antibody 224B1 specifically binds to Siglec-9 and Siglec-7 but does not cross-react with the closely related antibodies Siglec-5, Siglec-8, or Siglec-15 (Figure 1).

[0117] Example 3 - Measurement of EC50 of Siglec-9 specific antibody against Siglec-9 protein in enzyme-linked immunosorbent assay (ELISA) Materials and Methods The binding affinity of antibodies to Siglec-9 was measured by ELISA using a dilution series of the assay antibody against a fixed concentration of Siglec-9 protein. To this end, Siglec-9 was coated at 1 μg / ml in 50 mM carbonate-bicarbonate buffer, pH 9.6, for 15–20 h at 4°C. The antigen solution was washed (Tecan Hydrospeed), and the plate was blocked with 5% nonfat milk in phosphate-buffered saline (PBS) for 1 h at room temperature. The plate was washed, and antibodies were added at concentrations ranging from 0–420 nM. After incubation for 1 h at room temperature, the plate was washed once more. Bound antibodies were detected by the addition of a horseradish peroxidase (HRP)-conjugated secondary antibody. Rabbit antibodies were detected with a polyclonal anti-rabbit IgG antibody (Biolegend), and mAbA was detected with an anti-human Fab fragment (Sigma-Aldrich). After a final wash step, the plate was developed by adding 3,3',5,5'-Tetramethylbenzidine (TMB; Sigma-Aldrich). The reaction was stopped by adding sulfuric acid. Absorbance was measured at 450 nm (Tecan, CM INFINITE MONO 200). Data were fitted and EC50 values ​​determined by three-parameter analysis in GraphPad Prism (GraphPad Software).

[0118] result Monoclonal antibodies 68D4 and 224B1 exhibited significantly stronger avidity for Siglec-9 than mAbA and 5C6 (Fig. 2).

[0119] Example 4 - Affinity measurement by surface plasmon resonance (SPR) Materials and Methods K DMeasurements were performed by SPR by individually coating antibodies onto an HC2000M chip using sulfo-NHS / EDC coupling chemistry. The chip was then blocked with ethanolamine. Antigen was applied at one of three concentrations (20 nM, 100 nM, or 500 nM) with a contact time of 240 s followed by a dissociation phase of 120 s. After each antigen incubation, bound antigen was removed from the antibody with 10 mM glycine at pH 2.0. All kinetic parameters (k on , k off , K D ) was determined using a kinetic fitting program (LSA Kinetics, Carterra).

[0120] result Monoclonal antibody 68D4 exhibited strong avidity with a faster on-rate and slower off-rate than the comparative antibody mAbA, resulting in an approximately 16.7-fold improvement in dissociation constant. Monoclonal antibody 224B1 also exhibited strong avidity. Kinetic parameters determined by SPR

[0121] [Table 4]

[0122] Example 5 – Binding of Siglec9-specific antibodies to human monocytes Materials and Methods We assessed antibody binding to human monocytes because monocytes strongly and uniformly express the target Siglec-9, and therefore represent an excellent and relevant ex vivo model for characterizing the binding of Siglec-9-specific antibodies.

[0123] Human ex vivo PBMCs were stained with a fluorescently labeled (allophycocyanin, APC) antibody (Biolegend) against the monocyte marker CD14 in assay buffer (0.5% (v / v) heat-inactivated fetal bovine serum (Gibco), 2 mM EDTA, phosphate-buffered saline). The cells were then incubated with various concentrations (67 nM - 0.38 pM) of the antibody to be evaluated. After a washing step, the cells were stained with a fluorescently labeled (Brilliant Violet 421) antibody against human IgG (mAbA) or rabbit IgG. TM ) and incubated with secondary antibody (all other monoclonal antibodies evaluated; both secondary antibodies were from Biolegend).

[0124] Binding of Siglec-9-specific antibodies to monocytes (live CD14-positive cells) was analyzed using a CytoFLEX flow cytometer (Beckham Coulter's). The mean fluorescence intensity of secondary antibody staining of the CD14-positive population at each dilution was calculated using Flow Jo software (BD). Data points obtained from this analysis were fitted to a three-parameter analysis in GraphPad Prism (GraphPad Software) to determine the EC50 value.

[0125] result In the relevant cellular context of human CD14+ monocytes, monoclonal antibody 68D4 exhibits approximately three-fold stronger avidity for monocyte-expressed Siglec-9 compared to mAb A. Compared to monoclonal antibody 5C6, 68D4 appears to bind approximately 22-fold stronger. In the relevant cellular context of human CD14+ monocytes, monoclonal antibody 224B1 exhibits similar avidity for monocyte-expressed Siglec-9 compared to mAb A. Compared to monoclonal antibody 5C6, 224B1 appears to bind approximately 10-fold stronger (Figure 3).

[0126] Example 6 – Inhibition of Ligand Binding to Siglec-9 Materials and Methods To analyze whether anti-Siglec-9 antibodies recognize the ligand-binding domain and inhibit the binding of Siglec-9 to its ligand, sialic acid, we performed a cell-based assay to evaluate the binding of Siglec-9-Fc to sialic acid-expressing A549 tumor cells by ELISA. ELISA plates were coated with 3 μg / ml Protein-A (Thermo Fisher Scientific) overnight at 4°C. The plates were blocked with PBS, 1% BSA for 1 hour and then incubated with 5 μg / ml Siglec-9-Fc for 1 hour at RT. Different concentrations of anti-Siglec-9 antibodies (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 the ELISA plate and incubated for 1 hour at 4°C. After washing the plate, bound cells were lysed with PBS, 1% Triton-X 100, and the emitted fluorescence was measured using a Synergy H1 (BioTek) at excitation 485 nm and emission 535 nm. Data were fitted using a three-parameter analysis in GraphPad Prism (GraphPad Software).

[0127] result Monoclonal antibodies 68D4 and 224B1 blocked the binding of Siglec-9 to sialic acid-expressing A549 tumor cells in a concentration-dependent manner, to a similar extent as comparative monoclonal antibodies mAbA and 5C6 (Fig. 4).

[0128] Example 7 – T cell activation assay Materials and Methods For T cell activation assays, we used cells from Siglec-9 transgenic mice (Stanczak et al, J Clin Invest. 2018 Nov 1;128(11):4912-4923). In these mice, Siglec-9 expression is induced by excising the GFP-stop cassette in front of the Siglec-9 gene using Cre recombinase. Cre expression is driven by the CD4 promoter. Expression of Siglec-9 in these T cells results in a phenotype in which T cell receptor triggering and costimulatory signals via the CD28 receptor are insufficient for cell activation; a third signal is required to release Siglec-9-mediated inhibition.

[0129] For T cell activation assays, 96-well flat-bottom plates were coated with 0.5 μg / ml anti-CD3 and 1 μg / ml anti-CD28 overnight at 4°C or for 2 hours at 37°C. Spleen and lymph node cells were isolated from Siglec-9 transgenic mice. Red blood cells were lysed, and cells were labeled with Cell Trace Violet (Thermo Fisher Scientific). 250,000 cells / well were added at various concentrations (0.25 μg / ml - 0.0125 μg / ml; 1.7 nM - 83 pM) with or without the antibody under evaluation. After 48 hours of culture, cells were stained for T cell markers CD3, CD4, and CD8, and activation markers CD69 and CD25. Cell proliferation was assessed by analyzing dilutions of Cell Trace Violet. Cells were analyzed using a CytoFLEX flow cytometer (Beckham Coulter's). Expansion index was calculated using Flow Jo software (BD). Cell activation was measured by the mean fluorescence intensity of CD69 and CD25 on T cells. Data were visualized using GraphPad Prism (GraphPad Software).

[0130] result CD8+ T cells costimulated with monoclonal antibodies 68D4 or 224B1 responded by proliferating in a concentration-dependent manner and upregulating the activation markers CD69 and CD25. Monoclonal antibody 68D4 showed stronger proliferation and activation at lower concentrations than the control antibody mAbA. Monoclonal antibody 224B1 showed stronger proliferation at higher concentrations than the control antibody mAbA. The activation factors appear to be equivalent between 224B1 and mAbA. Monoclonal antibody 5C6 did not appear to stimulate or activate (Figure 5).

[0131] Similarly, for CD4+ T cells, monoclonal antibody 68D4 induced much stronger proliferation at lower concentrations than mAb A, and monoclonal antibody 224B1 induced much stronger proliferation at higher concentrations than mAb A. As with CD8+ T cells, monoclonal antibody 5C6 failed to induce a stimulatory or activation response. In both cases, the stronger proliferation was accompanied by stronger expression of the activation markers CD69 and CD25 (Figure 6).

[0132] Example 8 - Stimulation of natural killer cell activity Materials and Methods NK cell killing assays were performed using a human NK cell line overexpressing Siglec-9.

[0133] NK92 cells were stably transfected with a plasmid containing Siglec-9 and GFP (NK92-Siglec-9). Control cells were stably transfected with GFP alone (NK92-GFP). NK92 cells were activated overnight with 20 ng / ml IL-15 and then cocultured with calcein-AM-labeled K562 cells at a 3:1 ratio with or without a dilution series of antibodies in a 96-well V-bottom plate for 4 hours. Cells were pelleted by centrifugation, and the supernatant was transferred to a black 96-well flat-bottom plate. K562 cell lysis was measured as Calcein-AM released into the supernatant using a Synergy H1 (BioTek) with excitation at 485 nm and emission at 535 nm. Specific lysis was calculated as follows: (lysis test wells - spontaneous lysis) / (maximum lysis - spontaneous lysis) × 100%. Only K562 cells were used for spontaneous cell lysis. For maximum lysis, Triton X-100 was added to K562 cells. Data were analyzed with GraphPad Prism (GraphPad Software).

[0134] result Monoclonal antibody 68D4 was shown to activate NK cell-mediated killing in a concentration-dependent manner. Compared to the comparative monoclonal antibodies mAbA and 5C6, it already achieved high cytolytic activity at lower concentrations. The latter antibody induced cytolysis only at the highest concentration tested. Monoclonal antibody 224B1 was shown to activate NK cell-mediated killing in a concentration-dependent manner. Specific cytolysis was comparable to that of the comparative monoclonal antibody mAbA (Figure 7).

[0135] Example 9 – Humanization of rabbit antibody 68D4 Human IgG 263E11 was selected from the Memo Therapeutics AG human IgG database due to its similarity to 68D4. As a first step, the CDRs of rabbit immunoglobulin 68D4 were grafted onto the 263E11 variable domain framework. Further modifications were made at framework positions, replacing Kabat positions 27, 28, 29, and 94 of the heavy chain with rabbit amino acids (see Figure 8). Additionally, several rabbit CDR positions were changed to human amino acids. In the light chain variable domain, Kabat positions 24 and 30 of CDR1 were replaced with human amino acid side chains (Figure 8). Kabat positions 61, 62, and 63 of heavy chain CDR2 were replaced with human amino acids. Genes for the resulting heavy and light chain sequences were synthesized and cloned into an IgG1 / kappa expression vector. Humanized 68D4 (68D4-hum) was expressed and purified from HEK293F cells. 68D4-hum was compared to its parent rb 68D4 and mAbA in an ELISA following the protocol essentially as described in Example 3.

[0136] The antigen used was a fusion of the extracellular domain of Siglec-9 with a C-terminal his-tag. JPEG0007736682000011.jpg41170

[0137] Bound 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 Laboratories, #109-035-098). Humanization of 68D4 resulted in a high-affinity Siglec-9 binder with slightly reduced binding affinity compared to rabbit 68D4 (Figure 9). The Siglec-9 binding affinity was comparable to that of mAbA, in which the mouse variable domain was expressed in the human IgG Fc region.

[0138] Amino acid sequence of humanized anti-Siglec-9 antibody 68D4. [Table 5]

[0139] CDR amino acid sequence of rabbit / humanized anti-Siglec-9 antibody 68D4. [Table 6]

Claims

1. An anti-Siglec-9 antibody molecule or anti-Siglec-9 binding fragment thereof, comprising: (i) the heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 1; the heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 2, and a heavy chain variable region (VH) comprising the heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 3; and light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 4; the light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 5, and a light chain variable region (VL) comprising the light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 6; or (ii) the heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 1; the heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO: 45, and a heavy chain variable region (VH) comprising the heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 3; and light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 46; the light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 5, and A light chain variable region (VL) comprising the light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO:

6.

2. The antibody molecule or binding fragment thereof of claim 1, comprising: (i) a heavy chain variable region (VH) 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 to SEQ ID NO: 7, and 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 to SEQ ID NO: 8; or (ii) a heavy chain variable region (VH) 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 to SEQ ID NO: 47, and 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 to SEQ ID NO:

48.

3. 3. The antibody molecule or binding fragment thereof of claim 1 or 2, comprising: (i) 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; or (ii) 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.

4. A pharmaceutical composition comprising the antibody molecule or binding fragment thereof according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier, excipient or stabilizer.

5. The antibody molecule or binding fragment thereof according to any one of claims 1 to 3, or the pharmaceutical composition according to claim 4, for use in the treatment of a disease selected from the group consisting of cancer, acute and chronic hepatitis B.

6. A nucleic acid encoding the antibody heavy chain variable region and light chain variable region of the antibody molecule or binding fragment thereof according to any one of claims 1 to 3.

7. An expression vector comprising the nucleic acid of claim 6.

8. A host cell comprising the nucleic acid of claim 6 or the expression vector of claim 7.

9. A method for producing an antibody molecule, comprising culturing the host cell of claim 8 under conditions suitable for gene expression.

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