Antibodies to human sigle-9 and their use for immunotherapy

Abstract

The present invention provides compositions comprising anti-Siglecc antibodies and compositions comprising nucleic acid molecules encoding the antibodies, and methods of treating or preventing diseases or disorders using the same.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 325,865, filed on March 31, 2022, which is hereby incorporated by reference in its entirety. Background Art

[0003] Cancer manipulates multiple immune mechanisms to ensure a permissive local microenvironment that promotes tumor progression. Glycosylation is often considered a hallmark of cancer. Altered surface glycosylation of tumor / infected cells appears to be a key feature of cancer and different infectious diseases. Sialyl glycans found on both glycoproteins and glycolipids are recognized by Siglecs, a family of lectins expressed on the surface of many immune cell subtypes. The interaction of cancer cell / infected cell-bound sialic acid with Siglecs can therefore modulate immune cell phenotypes and enable them to escape the immune system.

[0004] Siglecs contain an N-terminal V-set domain that binds Sia, followed by a C2-set Ig domain that is thought to act as a spacer or regulator of oligomerization. Most of them function as transmembrane receptors in the immune system by recognizing Sia residues. Each type of Siglec generally recognizes a specific set of sialylated structures.

[0005] Siglec 9 is an important inhibitory sialic acid-binding immunoglobulin-like lectin (Siglec) receptor expressed by NK cells (CD16pos / CD56dim), B cells, and monocytes. The interaction between Siglec 9 and Sia leads to phosphorylation of the immunoreceptor tyrosine-based inhibition motif (ITIM), which inhibits NK cell cytotoxicity.

[0006] Studies have shown that abnormally glycosylated mucins (including MUC1 and MUC16) on cancer cells bind to Siglec-9 on immune cells, thereby inhibiting the anti-tumor function of natural killer (NK) and T cells and regulating the immune function of myeloid cells. Siglec-9 / sialic acid binding can generate inhibitory signals, leading to impaired immune responses in the tumor microenvironment.

[0007] There remains a need in the art for novel cancer treatment methods that effectively treat cancer while minimizing negative side effects. The present invention meets this unmet need. Summary of the invention

[0008] In one embodiment, the invention relates to an antibody or fragment thereof that specifically binds to a sialic acid binding receptor. In one embodiment, the sialic acid binding receptor is Siglec-9.

[0009] In one embodiment, the antibody comprises a variable heavy chain sequence comprising the following CDR sequences: SEQ ID NO: 1-3; SEQ ID NO: 17-19; SEQ ID NO: 33-35; SEQ ID NO: 49-51; SEQ ID NO: 65-67; ​​SEQ ID NO: 81-83; SEQ ID NO: 97-99; SEQ ID NO: 113-115; SEQ ID NO: 129-131; SEQ ID NO: 145-147; SEQ ID NO: 161-163; SEQ ID NO: 177-179; or SEQ ID NO: 193-195.

[0010] In one embodiment, the antibody comprises a variable heavy chain sequence comprising the following CDR sequences: SEQ ID NO:9-11; SEQ ID NO:25-27; SEQ ID NO:41-43; SEQ ID NO:57-59; SEQ ID NO:73-75; SEQ ID NO:89-91; SEQ ID NO:105-107; SEQ ID NO:121-123; SEQ ID NO:137-139; SEQ ID NO:153-155; SEQ ID NO:169-171; SEQ ID NO:185-187; or SEQ ID NO:201-203.

[0011] In one embodiment, the antibody comprises a variable heavy chain sequence of SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, SEQ ID NO:84, SEQ ID NO:100, SEQ ID NO:116, SEQ ID NO:132, SEQ ID NO:148, SEQ ID NO:164, SEQ ID NO:180, or SEQ ID NO:196.

[0012] In one embodiment, the antibody comprises a variable light chain sequence of SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 44, SEQ ID NO: 60, SEQ ID NO: 76, SEQ ID NO: 92, SEQ ID NO: 108, SEQ ID NO: 124, SEQ ID NO: 140, SEQ ID NO: 156, SEQ ID NO: 172, SEQ ID NO: 188, or SEQ ID NO: 204.

[0013] In one embodiment, the antibody includes a variable heavy chain sequence comprising a CDR sequence of SEQ ID NO: 1-3 and a variable light chain sequence comprising a CDR sequence of SEQ ID NO: 9-11. In one embodiment, the antibody includes a variable heavy chain sequence comprising a CDR sequence of SEQ ID NO: 17-19 and a variable light chain sequence comprising a CDR sequence of SEQ ID NO: 25-27. In one embodiment, the antibody includes a variable heavy chain sequence comprising a CDR sequence of SEQ ID NO: 33-35 and a variable light chain sequence comprising a CDR sequence of SEQ ID NO: 41-43. In one embodiment, the antibody includes a variable heavy chain sequence comprising a CDR sequence of SEQ ID NO: 49-51 and a variable light chain sequence comprising a CDR sequence of SEQ ID NO: 57-59. In one embodiment, the antibody includes a variable heavy chain sequence comprising a CDR sequence of SEQ ID NO: 65-67 and a variable light chain sequence comprising a CDR sequence of SEQ ID NO: 73-75. In one embodiment, the antibody includes a variable heavy chain sequence comprising a CDR sequence of SEQ ID NO:81-83 and a variable light chain sequence comprising a CDR sequence of SEQ ID NO:89-91. In one embodiment, the antibody includes a variable heavy chain sequence comprising a CDR sequence of SEQ ID NO:97-99 and a variable light chain sequence comprising a CDR sequence of SEQ ID NO:105-107. In one embodiment, the antibody includes a variable heavy chain sequence comprising a CDR sequence of SEQ ID NO:113-115 and a variable light chain sequence comprising a CDR sequence of SEQ ID NO:121-123. In one embodiment, the antibody includes a variable heavy chain sequence comprising a CDR sequence of SEQ ID NO:129-131 and a variable light chain sequence comprising a CDR sequence of SEQ ID NO:137-139. In one embodiment, the antibody includes a variable heavy chain sequence comprising a CDR sequence of SEQ ID NO:145-147 and a variable light chain sequence comprising a CDR sequence of SEQ ID NO:153-155. In one embodiment, the antibody comprises a variable heavy chain sequence comprising a CDR sequence of SEQ ID NO: 161-163 and a variable light chain sequence comprising a CDR sequence of SEQ ID NO: 169-171. In one embodiment, the antibody comprises a variable heavy chain sequence comprising a CDR sequence of SEQ ID NO: 177-179 and a variable light chain sequence comprising a CDR sequence of SEQ ID NO: 185-187.In one embodiment, the antibody comprises a variable heavy chain sequence comprising the CDR sequences of SEQ ID NOs: 193-195 and a variable light chain sequence comprising the CDR sequences of SEQ ID NOs: 201-203.

[0014] In one embodiment, the antibody comprises a variable heavy chain sequence of SEQ ID NO:4 and a variable light chain sequence of SEQ ID NO:12. In one embodiment, the antibody comprises a variable heavy chain sequence of SEQ ID NO:20 and a variable light chain sequence of SEQ ID NO:28. In one embodiment, the antibody comprises a variable heavy chain sequence of SEQ ID NO:36 and a variable light chain sequence of SEQ ID NO:44. In one embodiment, the antibody comprises a variable heavy chain sequence of SEQ ID NO:52 and a variable light chain sequence of SEQ ID NO:60. In one embodiment, the antibody comprises a variable heavy chain sequence of SEQ ID NO:68 and a variable light chain sequence of SEQ ID NO:76. In one embodiment, the antibody comprises a variable heavy chain sequence of SEQ ID NO:84 and a variable light chain sequence of SEQ ID NO:92. In one embodiment, the antibody comprises a variable heavy chain sequence of SEQ ID NO:100 and a variable light chain sequence of SEQ ID NO:108. In one embodiment, the antibody comprises a variable heavy chain sequence of SEQ ID NO:116 and a variable light chain sequence of SEQ ID NO:124. In one embodiment, the antibody comprises a variable heavy chain sequence of SEQ ID NO: 132 and a variable light chain sequence of SEQ ID NO: 140. In one embodiment, the antibody comprises a variable heavy chain sequence of SEQ ID NO: 148 and a variable light chain sequence of SEQ ID NO: 156. In one embodiment, the antibody comprises a variable heavy chain sequence of SEQ ID NO: 164 and a variable light chain sequence of SEQ ID NO: 172. In one embodiment, the antibody comprises a variable heavy chain sequence of SEQ ID NO: 180 and a variable light chain sequence of SEQ ID NO: 188. In one embodiment, the antibody comprises a variable heavy chain sequence of SEQ ID NO: 196 and a variable light chain sequence of SEQ ID NO: 204.

[0015] In one embodiment, the antibody comprises a sequence that is at least 95% identical to one or more of the variable heavy chain sequences of SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, SEQ ID NO:84, SEQ ID NO:100, SEQ ID NO:116, SEQ ID NO:132, SEQ ID NO:148, SEQ ID NO:164, SEQ ID NO:180, or SEQ ID NO:196; a sequence that is at least 95% identical to one or more of the variable light chain sequences of SEQ ID NO:12, SEQ ID NO:28, SEQ ID NO:44, SEQ ID NO:60, SEQ ID NO:76, SEQ ID NO:92, SEQ ID NO:108, SEQ ID NO:124, SEQ ID NO:140, SEQ ID NO:156, SEQ ID NO:172, SEQ ID NO:188, or SEQ ID NO:204; : or a fragment comprising at least 80% of the full-length sequence of a variable heavy chain sequence of SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, SEQ ID NO:84, SEQ ID NO:100, SEQ ID NO:116, SEQ ID NO:132, SEQ ID NO:148, SEQ ID NO:164, SEQ ID NO:180, or SEQ ID NO:196; or a fragment comprising at least 80% of the full-length sequence of a variable light chain sequence of SEQ ID NO:12, SEQ ID NO:28, SEQ ID NO:44, SEQ ID NO:60, SEQ ID NO:76, SEQ ID NO:92, SEQ ID NO:108, SEQ ID NO:124, SEQ ID NO:140, SEQ ID NO:156, SEQ ID NO:172, SEQ ID NO:188, or SEQ ID NO:204, or a combination thereof.

[0016] In one embodiment, the present invention relates to a nucleic acid molecule encoding an antibody or fragment thereof that specifically binds to a sialic acid binding receptor. In one embodiment, the sialic acid binding receptor is Siglec-9.

[0017] In one embodiment, the nucleic acid molecule encodes an antibody comprising: a variable heavy chain sequence selected from SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, SEQ ID NO:84, SEQ ID NO:100, SEQ ID NO:116, SEQ ID NO:132, SEQ ID NO:148, SEQ ID NO:164, SEQ ID NO:180, or SEQ ID NO:196; a variable light chain sequence selected from SEQ ID NO:12, SEQ ID NO:28, SEQ ID NO:44, SEQ ID NO:60, SEQ ID NO:76, SEQ ID NO:92, SEQ ID NO:108, SEQ ID NO:124, SEQ ID NO:140, SEQ ID NO:156, SEQ ID NO:172, SEQ ID NO:188, or SEQ ID NO:204; and a variable light chain sequence selected from SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, SEQ ID NO:84, SEQ ID NO:100, SEQ ID NO:116, SEQ ID NO:132, SEQ ID NO:148, SEQ ID NO:164, SEQ ID NO:180, or SEQ ID NO:196; NO:52, SEQ ID NO:68, SEQ ID NO:84, SEQ ID NO:100, SEQ ID NO:116, SEQ ID NO:132, SEQ ID NO:148, SEQ ID NO:164, SEQ ID NO:180 or SEQ ID NO:196. A sequence having at least 95% identity to one or more of the variable heavy chain sequences of SEQ ID NO:12, SEQ ID NO:28, SEQ ID NO:44, SEQ ID NO:60, SEQ ID NO:76, SEQ ID NO:92, SEQ ID NO:108, SEQ ID NO:124, SEQ ID NO:140, SEQ ID NO:156, SEQ ID NO:172, SEQ ID NO:188 or SEQ ID NO:204; A sequence having at least 95% identity to one or more of the variable light chain sequences of SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, SEQ ID NO:84, SEQ ID NO:100, SEQ ID NO:116, SEQ ID NO:132, SEQ ID NO:148, SEQ ID NO:164, SEQ ID NO:180 or SEQ ID NO:196. A fragment of at least 80% of the full-length sequence of a variable heavy chain sequence of SEQ ID NO: 100, SEQ ID NO: 116, SEQ ID NO: 132, SEQ ID NO: 148, SEQ ID NO: 164, SEQ ID NO: 180, or SEQ ID NO: 196;or a fragment comprising at least 80% of the full-length sequence of one or more variable light chain sequences selected from SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 44, SEQ ID NO: 60, SEQ ID NO: 76, SEQ ID NO: 92, SEQ ID NO: 108, SEQ ID NO: 124, SEQ ID NO: 140, SEQ ID NO: 156, SEQ ID NO: 172, SEQ ID NO: 188 or SEQ ID NO: 204, or a combination thereof. ;

[0018] In some embodiments, a fragment of SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, SEQ ID NO:84, SEQ ID NO:100, SEQ ID NO:116, SEQ ID NO:132, SEQ ID NO:148, SEQ ID NO:164, SEQ ID NO:180, or SEQ ID NO:196 comprises at least all three CDR regions of SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, SEQ ID NO:84, SEQ ID NO:100, SEQ ID NO:116, SEQ ID NO:132, SEQ ID NO:148, SEQ ID NO:164, SEQ ID NO:180, or SEQ ID NO:196. In some embodiments, a variant of SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, SEQ ID NO:84, SEQ ID NO:100, SEQ ID NO:116, SEQ ID NO:132, SEQ ID NO:148, SEQ ID NO:164, SEQ ID NO:180, or SEQ ID NO:196 comprises 100% identity to all three SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, SEQ ID NO:84, SEQ ID NO:100, SEQ ID NO:116, SEQ ID NO:132, SEQ ID NO:148, SEQ ID NO:164, SEQ ID NO:180, or SEQ ID NO:196.

[0019] In some embodiments, a fragment of SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 44, SEQ ID NO: 60, SEQ ID NO: 76, SEQ ID NO: 92, SEQ ID NO: 108, SEQ ID NO: 124, SEQ ID NO: 140, SEQ ID NO: 156, SEQ ID NO: 172, SEQ ID NO: 188, or SEQ ID NO: 204 comprises at least all three CDR regions of SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 44, SEQ ID NO: 60, SEQ ID NO: 76, SEQ ID NO: 92, SEQ ID NO: 108, SEQ ID NO: 124, SEQ ID NO: 140, SEQ ID NO: 156, SEQ ID NO: 172, SEQ ID NO: 188, or SEQ ID NO: 204. In some embodiments, a variant of SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 44, SEQ ID NO: 60, SEQ ID NO: 76, SEQ ID NO: 92, SEQ ID NO: 108, SEQ ID NO: 124, SEQ ID NO: 140, SEQ ID NO: 156, SEQ ID NO: 172, SEQ ID NO: 188, or SEQ ID NO: 204 comprises 100% identity to all three CDR regions in SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 44, SEQ ID NO: 60, SEQ ID NO: 76, SEQ ID NO: 92, SEQ ID NO: 108, SEQ ID NO: 124, SEQ ID NO: 140, SEQ ID NO: 156, SEQ ID NO: 172, SEQ ID NO: 188, or SEQ ID NO: 204.

[0020] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the variable heavy chain CDR sequence of SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, SEQ ID NO:84, SEQ ID NO:100, SEQ ID NO:116, SEQ ID NO:132, SEQ ID NO:148, SEQ ID NO:164, SEQ ID NO:180 or SEQ ID NO:196. In one embodiment, the nucleic acid molecule comprises SEQ ID NOs:5-7; SEQ ID NOs:21-23; SEQ ID NOs:37-39; SEQ ID NOs:53-55; SEQ ID NOs:69-71; SEQ ID NOs:85-87; SEQ ID NOs:101-103; SEQ ID NOs:117-119; SEQ ID NOs:133-135; SEQ ID NOs:149-151; SEQ ID NOs:165-167; SEQ ID NOs:181-183; or SEQ ID NOs:197-199. In one embodiment, the nucleic acid molecule comprises SEQ ID NO:8; SEQ ID NO:24; SEQ ID NO:40; SEQ ID NO:56; SEQ ID NO:72; SEQ ID NO:88; SEQ ID NO:104; SEQ ID NO:120; SEQ ID NO:136; SEQ ID NO:152; SEQ ID NO:168; SEQ ID NO:184; or SEQ ID NO:200. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO:8; SEQ ID NO:24; SEQ ID NO:40; SEQ ID NO:56; SEQ ID NO:72; SEQ ID NO:88; SEQ ID NO:104; SEQ ID NO:120; SEQ ID NO:136; SEQ ID NO:152; SEQ ID NO:168; SEQ ID NO:184; or SEQ ID NO:200.In one embodiment, the nucleic acid molecule comprises a fragment comprising at least 80% of the full-length sequence of SEQ ID NO:8; SEQ ID NO:24; SEQ ID NO:40; SEQ ID NO:56; SEQ ID NO:72; SEQ ID NO:88; SEQ ID NO:104; SEQ ID NO:120; SEQ ID NO:136; SEQ ID NO:152; SEQ ID NO:168; SEQ ID NO:184; or SEQ ID NO:200.

[0021] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the variable light chain CDR sequence of SEQ ID NO:12, SEQ ID NO:28, SEQ ID NO:44, SEQ ID NO:60, SEQ ID NO:76, SEQ ID NO:92, SEQ ID NO:108, SEQ ID NO:124, SEQ ID NO:140, SEQ ID NO:156, SEQ ID NO:172, SEQ ID NO:188 or SEQ ID NO:204. In one embodiment, the nucleic acid molecule comprises SEQ ID NOs: 13-15; SEQ ID NOs: 29-31; SEQ ID NOs: 45-47; SEQ ID NOs: 61-63; SEQ ID NOs: 77-79; SEQ ID NOs: 93-95; SEQ ID NOs: 109-111; SEQ ID NOs: 125-127; SEQ ID NOs: 141-143; SEQ ID NOs: 157-159; SEQ ID NOs: 173-175; SEQ ID NOs: 189-191; or SEQ ID NOs: 205-207. In one embodiment, the nucleic acid molecule comprises SEQ ID NO: 16; SEQ ID NO: 32; SEQ ID NO: 48; SEQ ID NO: 64; SEQ ID NO: 80; SEQ ID NO: 96; SEQ ID NO: 112; SEQ ID NO: 128; SEQ ID NO: 144; SEQ ID NO: 160; SEQ ID NO: 176; SEQ ID NO: 192; or SEQ ID NO: 208. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 16; SEQ ID NO: 32; SEQ ID NO: 48; SEQ ID NO: 64; SEQ ID NO: 80; SEQ ID NO: 96; SEQ ID NO: 112; SEQ ID NO: 128; SEQ ID NO: 144; SEQ ID NO: 160; SEQ ID NO: 176; SEQ ID NO: 192; or SEQ ID NO: 208.In one embodiment, the nucleic acid molecule comprises a fragment comprising at least 80% of the full-length sequence of SEQ ID NO:16; SEQ ID NO:32; SEQ ID NO:48; SEQ ID NO:64; SEQ ID NO:80; SEQ ID NO:96; SEQ ID NO:112; SEQ ID NO:128; SEQ ID NO:144; SEQ ID NO:160; SEQ ID NO:176; SEQ ID NO:192; and SEQ ID NO:208.

[0022] In one embodiment, the invention provides a combination of nucleic acid molecules, wherein the first nucleic acid molecule comprises a heavy chain CDR encoding sequence of SEQ ID NOs: 5-7; SEQ ID NOs: 21-23; SEQ ID NOs: 37-39; SEQ ID NOs: 53-55; SEQ ID NOs: 69-71; SEQ ID NOs: 85-87; SEQ ID NOs: 101-103; SEQ ID NOs: 117-119; SEQ ID NOs: 133-135; SEQ ID NOs: 149-151; SEQ ID NOs: 165-167; SEQ ID NOs: 181-183; or SEQ ID NOs: 197-199, and the second nucleic acid molecule comprises a heavy chain CDR encoding sequence of SEQ ID NOs: 13-15; SEQ ID NOs: 29-31; SEQ ID NOs: 45-47; SEQ ID NOs: 61-63; SEQ ID NOs: 77-79; SEQ ID NOs: 93-95; SEQ ID NOs: 109-111; SEQ ID NOs: NO: 125-127; SEQ ID NO: 141-143; SEQ ID NO: 157-159; SEQ ID NO: 173-175; SEQ ID NO: 189-191; or SEQ ID NO: 205-207. In one embodiment, the combination of nucleic acid molecules comprises a first nucleic acid molecule and a second nucleic acid molecule, wherein the first nucleic acid molecule comprises a heavy chain coding sequence or a fragment or variant thereof of SEQ ID NO:8; SEQ ID NO:24; SEQ ID NO:40; SEQ ID NO:56; SEQ ID NO:72; SEQ ID NO:88; SEQ ID NO:104; SEQ ID NO:120; SEQ ID NO:136; SEQ ID NO:152; SEQ ID NO:168; SEQ ID NO:184; or SEQ ID NO:200, and the second nucleic acid molecule comprises a light chain coding sequence or a fragment or variant thereof of SEQ ID NO:16; SEQ ID NO:32; SEQ ID NO:48; SEQ ID NO:64; SEQ ID NO:80; SEQ ID NO:96; SEQ ID NO:112; SEQ ID NO:128; SEQ ID NO:144; SEQ ID NO:160; SEQ ID NO:176; SEQ ID NO:192; or SEQ ID NO:208.

[0023] In one embodiment, the present invention relates to a composition comprising a sialic acid binding receptor antibody. In one embodiment, the composition further comprises a pharmaceutically acceptable excipient. In one embodiment, the composition further comprises an adjuvant. In some embodiments, the composition further comprises a tumor antigen or a nucleotide sequence encoding a tumor antigen.

[0024] In one embodiment, the present invention relates to a composition comprising a nucleic acid molecule encoding a sialic acid binding receptor antibody. In one embodiment, the nucleic acid molecule comprises an expression vector. In one embodiment, the nucleic acid molecule is incorporated into a viral particle. In one embodiment, the composition further comprises a pharmaceutically acceptable excipient. In one embodiment, the composition further comprises an adjuvant. In some embodiments, the composition further comprises a nucleotide sequence encoding a tumor antigen.

[0025] In one embodiment, the present invention relates to a method for treating or preventing a disease or condition in a subject in need thereof, the method comprising administering a sialic acid binding receptor antibody, a nucleic acid molecule encoding a sialic acid binding receptor antibody, or a composition comprising a sialic acid binding receptor antibody or a nucleic acid molecule encoding a sialic acid binding receptor antibody. In one embodiment, the disease or condition is cancer, or a disease or condition associated with cancer. In one embodiment, the disease or condition is an infectious disease or condition.

[0026] In one embodiment, the present invention relates to a method of increasing natural killer cell function in a subject in need thereof, the method comprising administering a sialic acid binding receptor antibody, a nucleic acid molecule encoding a sialic acid binding receptor antibody, or a composition comprising a sialic acid binding receptor antibody or a nucleic acid molecule encoding a sialic acid binding receptor antibody.

[0027] In one embodiment, the present invention relates to an immunogenic composition comprising an antibody or fragment thereof that specifically binds to a sialic acid binding receptor and a nucleic acid molecule encoding a tumor antigen.

[0028] In one embodiment, the disease or condition is cancer, or a disease or condition associated with cancer. In one embodiment, the cancer has increased sialic acid levels. In one embodiment, the cancer is ovarian cancer, melanoma, renal cell carcinoma, prostate cancer, colon cancer, breast cancer, head and neck squamous cell carcinoma, or oral cancer.

[0029] In one embodiment, the invention relates to a method of increasing natural killer cell function in a subject in need thereof, the method comprising administering an immunogenic composition comprising an antibody or fragment thereof that specifically binds to a sialic acid binding receptor and a nucleic acid molecule encoding a tumor antigen.

[0030] In one embodiment, the present invention relates to a method of increasing natural killer cell function in a subject in need thereof, the method comprising administering an immunogenic composition comprising a nucleic acid molecule encoding an antibody or fragment thereof that specifically binds to a sialic acid binding receptor, and a nucleic acid molecule encoding a tumor antigen. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Binding of anti-Siglec-9 clones to human Siglec 9 is shown. Specificity and binding activity of generated hybridoma supernatants are shown.

[0032] Figure 2 Depicted are exemplary experimental results demonstrating the cytotoxicity of Siglec-9 antibody clones to OVISE cells.

[0033] Figure 3 Depicted are exemplary experimental results demonstrating the cytotoxicity of Siglec-9 antibody clones against OVCAR10 cells. DETAILED DESCRIPTION

[0034] The present invention relates to sialic acid receptor antibodies, fragments thereof, variants thereof, or nucleic acid molecules encoding the same, and methods for increasing the activity of natural killer cells in subjects in need thereof.

[0035] In one aspect, the present invention relates to a composition that can be used to increase or enhance an immune response, i.e., produce a more effective immune response, by administering a sialic acid receptor antibody, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same. In one embodiment, the sialic acid receptor antibody is an antibody against Siglec-9.

[0036] In one aspect, the present invention relates to a sialic acid receptor antibody, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same in combination with a nucleic acid molecule encoding a tumor antigen.

[0037] In one aspect, the present invention relates to a method for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a sialic acid receptor antibody, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same. In one embodiment, the disease or condition is cancer. In one embodiment, the disease or condition is an infectious disease.

[0038] In one embodiment, the invention relates to a method of treating cancer or a disease or condition associated therewith in a subject in need thereof, the method comprising administering to the subject a sialic acid receptor antibody, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same in combination with a nucleic acid molecule encoding a tumor antigen. In one embodiment, the cancer has increased sialic acid levels.

[0039] definition

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice and testing of the present invention, exemplary methods and materials are described.

[0041] As used herein, the following terms each have the meaning associated with them in this section.

[0042] The articles "a" and "an" are used herein to refer to one or more than one (i.e., at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0043] As used herein, "about" when referring to a measurable value (e.g., an amount, a time interval, etc.) is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are suitable for performing the disclosed methods.

[0044] "Antibody" may refer to an antibody of the IgG, IgM, IgA, IgD or IgE class, or a fragment, fragment or derivative thereof, including Fab, F(ab')2, Fd and single-chain antibodies and derivatives thereof. The antibody may be an antibody isolated from a serum sample of a mammal, a polyclonal antibody, an affinity purified antibody or a mixture thereof, which exhibits sufficient binding specificity for the desired epitope or a sequence derived therefrom.

[0045] "Antigen" refers to a protein that has the ability to produce an immune response in a host. Antigens can be recognized and bound by antibodies. Antigens can originate from within the body or from the external environment.

[0046] "CDR" is defined as the complementary determining region amino acid sequence of an antibody, which is a hypervariable region of an immunoglobulin heavy chain and light chain. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, US Department of Health and Human Services, National Institutes of Health (1987). The variable portion of an immunoglobulin has three heavy chain CDRs (or CDR regions) and three light chain CDRs (or CDR regions). Therefore, "CDR" as used herein refers to all three heavy chain CDRs, or all three light chain CDRs (or all heavy chain CDRs and all light chain CDRs, if appropriate). The structure and protein folding of antibodies may mean that other residues are considered part of the antigen binding region, and the skilled person will understand this. See, e.g., Chothia et al., (1989) Conformations of immunoglobulin hypervariable regions; Nature 342, p 877-883.

[0047] "Antibody fragment" or "fragment of an antibody", used interchangeably herein, refers to a portion of an intact antibody including the antigen binding site or variable region. This portion does not include the constant heavy chain domains (i.e., CH2, CH3, or CH4, depending on the antibody isotype) of the Fc region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv) molecules, single-chain polypeptides containing only one light chain variable domain, single-chain polypeptides containing three CDRs of a light chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing three CDRs of a heavy chain variable region.

[0048] As used herein, "adjuvant" means any molecule added to the vaccines described herein to enhance the immunogenicity of the antigen.

[0049] As used herein, "coding sequence" or "encoding nucleic acid" may refer to a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence encoding an antibody as described herein. The coding sequence may also comprise a DNA sequence encoding an RNA sequence. The coding sequence may further include start and stop signals operably linked to regulatory elements, including a promoter and polyadenylation signal capable of directing expression in cells of an individual or mammal to which the nucleic acid is administered. The coding sequence may further include a sequence encoding a signal peptide.

[0050] As used herein, "complementary" or "complementary" may mean that a nucleic acid may have Watson-Crick (eg, AT / U and CG) or Hoogsteen base pairing between nucleotides or nucleotide analogs in the nucleic acid molecule.

[0051] "Disease" is a health condition in an animal in which the animal is unable to maintain homeostasis and in which the animal's health continues to deteriorate if the disease is not ameliorated.

[0052] In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Without treatment, the disorder does not necessarily cause a further decline in the animal's state of health.

[0053] A disease or condition is "relieved" if the signs or symptoms of the disease or condition are reduced in severity, if a patient experiences such signs or symptoms less frequently, or both.

[0054] "Encoding" refers to the inherent property of a specific nucleotide sequence in a polynucleotide (e.g., a gene, cDNA, or mRNA) as a template for synthesizing other polymers and macromolecules in biological processes having a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and the resulting biological properties. Thus, if the transcription and translation of the mRNA corresponding to a gene produces a protein in a cell or other biological system, the gene encodes the protein. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing) and the non-coding strand (used as a template for transcription of a gene or cDNA) may be referred to as encoding a protein or other product of the gene or cDNA.

[0055] An "effective amount" of a compound is an amount of the compound sufficient to provide an effect on the subject or system to which the compound is administered.

[0056] "Expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements for expression; other elements for expression may be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating recombinant polynucleotides.

[0057] As used herein, a "feedback mechanism" may refer to a process performed by software or hardware (or firmware) that receives the impedance of the desired tissue (before, during, and / or after energy pulse delivery) and compares it to a current value (preferably current) and adjusts the delivered energy pulse to achieve a preset value. The feedback mechanism may be performed by an analog closed-loop circuit.

[0058] "Fragment" may mean a polypeptide fragment of an antibody that is functional, i.e., can bind to a desired target and have the same intended effect as a full-length antibody. A fragment of an antibody may be 100% identical to the full length except for the deletion of at least one amino acid from the N and / or C terminus (with or without a signal peptide and / or methionine at position 1 in each case). A fragment may comprise more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% of the length of a particular full-length antibody, excluding any heterologous signal peptide added. The fragment may comprise a fragment of a polypeptide that is 95% or more, 96% or more, 97% or more, 98% or more identical to the antibody, and additionally comprises an N-terminal methionine or a heterologous signal peptide that is not included when calculating the percent identity. The fragment may further comprise an N-terminal methionine and / or a signal peptide, such as an immunoglobulin signal peptide, e.g., an IgE or IgG signal peptide. The N-terminal methionine and / or signal peptide may be attached to a fragment of the antibody.

[0059] A fragment of a nucleic acid sequence encoding an antibody may be 100% identical to the full length except for the deletion of at least one nucleotide from the 5' and / or 3' end (in each case with or without the sequence encoding a signal peptide and / or a methionine at position 1). A fragment may comprise more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% of the length of a particular full-length coding sequence, excluding any added heterologous signal peptide. The fragment may include a fragment encoding a polypeptide that is 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to an antibody, and optionally includes a sequence encoding an N-terminal methionine or a heterologous signal peptide that is not included when calculating percent identity. The fragment may further include a coding sequence for an N-terminal methionine and / or a signal peptide (such as an immunoglobulin signal peptide, e.g., an IgE or IgG signal peptide). The coding sequence encoding an N-terminal methionine and / or a signal peptide may be connected to a fragment of the coding sequence.

[0060] As used herein, "gene construct" refers to a DNA or RNA molecule containing a nucleotide sequence encoding a protein, such as an antibody. A gene construct may also refer to a DNA molecule that transcribes RNA. The coding sequence includes start and stop signals operably linked to regulatory elements, including promoters and polyadenylation signals that can direct expression in the cells of the individual to which the nucleic acid molecule is administered. As used herein, the term "expressible form" refers to a gene construct containing the necessary regulatory elements operably linked to the coding sequence encoding the protein, so that when present in the cells of the individual, the coding sequence will be expressed.

[0061] "Homologous" refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When one position in both of the two comparison sequences is occupied by the same base or amino acid monomer subunit, for example, if one position in each of the two DNA molecules is occupied by adenine, the two molecules are homologous at that position. The homology percentage between the two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in the two sequences are matched or homologous, the two sequences are 60% homologous. For example, the DNA sequences ATTGCC and TATGGC share 50% homology. Typically, comparison is performed when the two sequences are aligned to provide maximum homology.

[0062] As used herein in the context of two or more nucleic acid or polypeptide sequences, "same" or "identity" means that the sequence has a specified percentage of identical residues in a specified region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences in a specified region, determining the number of positions where the identical residues appear in the two sequences to derive the number of matching positions, dividing the number of matching positions by the total number of positions in the specified region, and multiplying the result by 100 to derive the percentage of sequence identity. In the case where the lengths of the two sequences are different or the alignment produces more than one staggered end and the specified comparison region includes only a single sequence, the residues of the single sequence are included in the denominator of the calculation but not in the numerator. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered to be equivalent. Identity can be performed manually or by using a computer sequence algorithm (e.g., BLAST or BLAST2.0).

[0063] "Isolated" means changed or removed from the natural state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated", but the same nucleic acid or peptide is "isolated" when it is partially or completely separated from the coexisting materials of its natural state. An isolated nucleic acid or protein can exist in a substantially purified form or can exist in a non-natural environment, such as a host cell.

[0064] In the context of the present invention, the following abbreviations for common nucleic acid bases are used: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0065] Unless otherwise indicated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The phrase "nucleotide sequence encoding a protein or RNA" may also include introns, to the extent that the nucleotide sequence encoding the protein contains introns (one or more) in some versions.

[0066] As used herein, “impedance” may be used when discussing a feedback mechanism and may be converted to a current value according to Ohm's law so that it can be compared with a preset current.

[0067] As used herein, "immune response" may refer to the activation of a host immune system (eg, a mammal's immune system) in response to the introduction of one or more nucleic acids and / or peptides. The immune response may be in the form of a cellular or humoral response, or both.

[0068] The terms "patient", "subject" and "individual" and the like are used interchangeably herein to refer to any animal or cell thereof (whether in vitro or in situ) suitable for the methods described herein. In some embodiments, the patient, subject or individual is a human.

[0069] "Parenteral" administration of the compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intradermal injection, or infusion techniques.

[0070] As used herein, "nucleic acid" or "oligonucleotide" or "polynucleotide" may mean at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Therefore, nucleic acid also encompasses the complementary strand of the depicted single strand. Many variants of nucleic acid can be used for the same purpose as a given nucleic acid. Therefore, nucleic acid also encompasses substantially identical nucleic acids and their complements. The single strand provides a probe that can hybridize with a target sequence under stringent hybridization conditions. Therefore, nucleic acid also encompasses probes that hybridize under stringent hybridization conditions.

[0071] Nucleic acids may be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. Nucleic acids may be DNA (both genomic and cDNA), RNA, or hybrids (wherein the nucleic acid may contain a combination of deoxyribonucleotides and ribonucleotides), and a combination of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acids may be obtained by chemical synthesis or recombinant methods.

[0072] As used herein, "operably linked" may mean that the expression of a gene is controlled by a promoter that is spatially linked thereto. A promoter may be located 5' (upstream) or 3' (downstream) of the gene it controls. The distance between the promoter and the gene may be about the same as the distance between the promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variations in this distance may be accommodated without loss of promoter function.

[0073] As used herein, "peptide," "protein," or "polypeptide" may mean a linked sequence of amino acids, and may be natural, synthetic, or a modification or combination of natural and synthetic.

[0074] As used herein, "promoter" means a synthetic or naturally derived molecule that can confer, activate or enhance the expression of nucleic acids in cells. A promoter may contain more than one specific transcriptional regulatory sequence to further enhance expression and / or change its spatial expression and / or temporal expression. A promoter may also contain distal enhancer or repressor elements, which may be located up to several thousand base pairs from the transcription start site. Promoters may be derived from sources including viruses, bacteria, fungi, plants, insects and animals. A promoter may constitutively or differentially regulate the expression of a genomic component with respect to the cell, tissue or organ in which expression occurs, or with respect to the developmental stage in which expression occurs, or in response to external stimuli (e.g., physiological stress, pathogens, metal ions or inducers). Representative examples of promoters include bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 late promoter and CMV IE promoter.

[0075] As used herein, the term "promoter / regulatory sequence" means a nucleic acid sequence required for the expression of a gene product that is operably linked to the promoter / regulatory sequence. In some cases, the sequence may be a core promoter sequence, while in other cases, the sequence may also include enhancer sequences and other regulatory elements required for the expression of the gene product. The promoter / regulatory sequence may be, for example, a sequence that expresses a gene product in a tissue-specific manner.

[0076] A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0077] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell substantially only when an inducer corresponding to the promoter is present in the cell.

[0078] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is of the tissue type corresponding to the promoter.

[0079] "Signal peptide" and "leader sequence" are used interchangeably herein and refer to an amino acid sequence that can be attached to the amino terminus of a protein described herein. The signal peptide / leader sequence generally directs the localization of the protein. The signal peptide / leader sequence used herein can promote the secretion of a protein from the cell in which it is produced. The signal peptide / leader sequence is generally cleaved from the remainder of the protein (generally referred to as the mature protein) when it is secreted from the cell. The signal peptide / leader sequence is attached to the N-terminus of the protein.

[0080] As used herein, "stringent hybridization conditions" may refer to conditions under which a first nucleic acid sequence (e.g., a probe) will hybridize to a second nucleic acid sequence (e.g., a target), for example in a complex nucleic acid mixture. Stringent conditions are sequence-dependent and will be different in different circumstances. Stringent conditions may be selected to be higher than the thermal melting point (T ) of a specific sequence at a defined ionic strength pH. m ) is about 5-10℃ lower. m It can be the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (due to the presence of excess target sequence, at T m Stringent conditions may be salt concentrations less than about 1.0 M sodium ions, such as about 0.01-1.0 M sodium ion concentrations (or other salts) at pH 7.0 to 8.3, and temperatures of at least about 30° C. for short probes (e.g., about 10-50 nucleotides) and at least about 60° C. for long probes (e.g., greater than about 50 nucleotides). Stringent conditions may also be achieved by the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal may be at least 2 to 10 times background hybridization. Exemplary stringent hybridization conditions include the following: 50% formamide, 5x SSC, and 1% SDS, incubated at 42° C., or 5xSSC, 1% SDS, incubated at 65° C., and washed in 0.2x SSC and 0.1% SDS at 65° C.

[0081] As used herein, "subject" and "patient" refer interchangeably to any vertebrate, including, but not limited to, mammals (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats and mice, non-human primates (e.g., monkeys, such as cynomolgus or rhesus monkeys, chimpanzees, etc.), and humans). In some embodiments, the subject may be human or non-human. The subject or patient may be receiving other forms of therapy.

[0082] As used herein, "substantially complementary" can mean that a first sequence is at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the complement of a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides or amino acids, or that the two sequences hybridize under stringent hybridization conditions.

[0083] As used herein, “substantially identical” may mean that the first and second sequences are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 80 97%, 98%, or 99% identical over a region of at least 10, 100, 200, 300, 400, 500, 600, 700, 8 ...

[0084] As used herein, a "synthetic antibody" refers to an antibody encoded by a recombinant nucleic acid sequence described herein and produced in a subject.

[0085] As used herein, "treat" or "treatment" may mean protecting a subject from the effects of a disease by means of preventing, inhibiting, suppressing, or completely eliminating the disease. Preventing a disease involves administering a vaccine of the present invention to a subject prior to the onset of the disease. Suppressing a disease involves administering a vaccine of the present invention to a subject after the induction of the disease but prior to the clinical appearance of the disease. Suppressing a disease involves administering a vaccine of the present invention to a subject after the clinical appearance of the disease.

[0086] "Therapeutic" treatment is treatment administered to a subject exhibiting signs or symptoms of a disease or condition, with the goal of reducing or eliminating the frequency or severity of those signs or symptoms.

[0087] As used herein, "treating a disease or condition" means reducing the frequency or severity, or both, of at least one sign or symptom of the disease or condition experienced by a patient.

[0088] As used herein, the phrase "therapeutically effective amount" refers to an amount sufficient or effective to prevent or treat (delay or prevent onset, prevent progression, inhibit, reduce or reverse) a disease or condition, including alleviating the signs and / or symptoms of such diseases and conditions.

[0089] As used herein, the term "treating" a disease or condition means reducing the frequency or severity of at least one sign or symptom of the disease or condition experienced by a subject.

[0090] As used herein, "variant" with respect to nucleic acids means (i) a portion or fragment of a reference nucleotide sequence; (ii) the complement of a reference nucleotide sequence or a portion thereof; (iii) a nucleic acid substantially identical to a reference nucleic acid or its complement; or (iv) a nucleic acid that hybridizes to a reference nucleic acid, its complement, or a sequence substantially identical thereto under stringent conditions.

[0091] Variants can be further defined as peptides or polypeptides whose amino acid sequences differ due to insertion, deletion or conservative substitution of amino acids but retain at least one biological activity. Representative examples of "biological activity" include the ability to be bound by a specific antibody or to promote an immune response. Variants can also refer to proteins having substantially the same amino acid sequence as a reference protein having an amino acid sequence retaining at least one biological activity. Conservative substitution of amino acids, i.e., replacing amino acids with different amino acids of similar properties (e.g., hydrophilicity, degree and distribution of charged regions), is considered in the art to generally involve minor changes. These minor changes can be identified in part by considering the hydropathic index of amino acids, as understood in the art. Kyte et al., J. Mol. Biol. 157: 105-132 (1982). The hydropathic index of amino acids is based on considerations of their hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids with a hydropathic index of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that will result in proteins retaining biological function. Considering the hydrophilicity of amino acids in the context of peptides allows calculation of the maximum local average hydrophilicity of the peptide, which is a useful measurement method that is reported to correlate well with antigenicity and immunogenicity. Substitution of amino acids with similar hydrophilicity values ​​can result in peptides retaining biological activity, such as immunogenicity, as understood in the art. Amino acids with hydrophilicity values ​​within ± 2 of each other can be substituted. Both the hydrophobicity index and the hydrophilicity value of amino acids are affected by the specific side chain of the amino acid. Consistent with this observation, amino acid substitutions compatible with biological function are understood to depend on the relative similarity of amino acids, particularly those side chains, as revealed by hydrophobicity, hydrophilicity, charge, size and other properties.

[0092] Variants can be substantially identical nucleic acid sequences over the full length of the entire gene sequence or its fragment. The nucleic acid sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical over the full length of the gene sequence or its fragment. Variants can be substantially identical amino acid sequences over the full length of the amino acid sequence or its fragment. The amino acid sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical over the full length of the amino acid sequence or its fragment.

[0093] "Vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Various vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ions or amphiphilic compounds, plasmids and viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that promote the transfer of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral vectors include but are not limited to adenoviral vectors, adeno-associated viral vectors, and retroviral vectors.

[0094] Range: Throughout this disclosure, various aspects of the invention may be presented in range format. It should be understood that the description in range format is only for convenience and brevity and should not be interpreted as an inflexible limitation on the scope of the invention. Therefore, the description of a range should be considered to have explicitly disclosed all possible sub-ranges and individual numerical values ​​within the range. For example, a description of a range such as from 1 to 6 should be considered to have explicitly disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within the range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0095] illustrate

[0096] The present invention provides antibodies or antibody-like molecules that specifically bind to Siglec-9. In one embodiment, the present invention provides an immunogenic composition comprising an antibody or antibody-like molecule that specifically binds to Siglec-9 of the present invention, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same. The immunogenic composition of the present invention can be used to protect against diseases or conditions associated with glycosylation changes, including but not limited to cancer and infectious diseases.

[0097] Therefore, in some embodiments, the present invention provides a composition comprising one or more antibodies, fragments thereof, or variants thereof, or nucleic acid molecules encoding the same, directed against Siglec-9.

[0098] In some embodiments, the present invention provides a method for treating or preventing a disease or disorder, comprising administering to a subject an antibody or antibody-like molecule, fragment thereof, variant thereof, or nucleic acid molecule encoding the same that specifically binds to Siglec-9 of the present invention.

[0099] In some embodiments, the present invention provides a method for treating or preventing cancer associated with increased Siglec-9, the method comprising administering to a subject an antibody or antibody-like molecule of the present invention that specifically binds to Siglec-9, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same.

[0100] Antibody composition

[0101] In one embodiment, the invention relates to a composition comprising at least one Siglec-9 antibody, or a fragment or variant thereof.

[0102] In one embodiment, the anti-siglec-9 antibody or fragment thereof comprises a heavy chain variable region having the sequence of SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, SEQ ID NO:84, SEQ ID NO:100, SEQ ID NO:116, SEQ ID NO:132, SEQ ID NO:148, SEQ ID NO:164, SEQ ID NO:180, or SEQ ID NO:196, or a fragment or variant thereof. In one embodiment, the anti-Siglec-9 antibody or fragment thereof comprises a light chain variable region having a sequence of SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 44, SEQ ID NO: 60, SEQ ID NO: 76, SEQ ID NO: 92, SEQ ID NO: 108, SEQ ID NO: 124, SEQ ID NO: 140, SEQ ID NO: 156, SEQ ID NO: 172, SEQ ID NO: 188 or SEQ ID NO: 204, or a fragment or variant thereof.

[0103] In one embodiment, the antibody includes a variable heavy chain sequence comprising a CDR sequence of SEQ ID NO: 1-3 and a variable light chain sequence comprising a CDR sequence of SEQ ID NO: 9-11. In one embodiment, the antibody includes a variable heavy chain sequence comprising a CDR sequence of SEQ ID NO: 17-19 and a variable light chain sequence comprising a CDR sequence of SEQ ID NO: 25-27. In one embodiment, the antibody includes a variable heavy chain sequence comprising a CDR sequence of SEQ ID NO: 33-35 and a variable light chain sequence comprising a CDR sequence of SEQ ID NO: 41-43. In one embodiment, the antibody includes a variable heavy chain sequence comprising a CDR sequence of SEQ ID NO: 49-51 and a variable light chain sequence comprising a CDR sequence of SEQ ID NO: 57-59. In one embodiment, the antibody includes a variable heavy chain sequence comprising a CDR sequence of SEQ ID NO: 65-67 and a variable light chain sequence comprising a CDR sequence of SEQ ID NO: 73-75. In one embodiment, the antibody includes a variable heavy chain sequence comprising a CDR sequence of SEQ ID NO:81-83 and a variable light chain sequence comprising a CDR sequence of SEQ ID NO:89-91. In one embodiment, the antibody includes a variable heavy chain sequence comprising a CDR sequence of SEQ ID NO:97-99 and a variable light chain sequence comprising a CDR sequence of SEQ ID NO:105-107. In one embodiment, the antibody includes a variable heavy chain sequence comprising a CDR sequence of SEQ ID NO:113-115 and a variable light chain sequence comprising a CDR sequence of SEQ ID NO:121-123. In one embodiment, the antibody includes a variable heavy chain sequence comprising a CDR sequence of SEQ ID NO:129-131 and a variable light chain sequence comprising a CDR sequence of SEQ ID NO:137-139. In one embodiment, the antibody includes a variable heavy chain sequence comprising a CDR sequence of SEQ ID NO:145-147 and a variable light chain sequence comprising a CDR sequence of SEQ ID NO:153-155. In one embodiment, the antibody comprises a variable heavy chain sequence comprising a CDR sequence of SEQ ID NO: 161-163 and a variable light chain sequence comprising a CDR sequence of SEQ ID NO: 169-171. In one embodiment, the antibody comprises a variable heavy chain sequence comprising a CDR sequence of SEQ ID NO: 177-179 and a variable light chain sequence comprising a CDR sequence of SEQ ID NO: 185-187.In one embodiment, the antibody comprises a variable heavy chain sequence comprising the CDR sequences of SEQ ID NOs: 193-195 and a variable light chain sequence comprising the CDR sequences of SEQ ID NOs: 201-203.

[0104] In one embodiment, the antibody comprises a variable heavy chain sequence of SEQ ID NO:4 and a variable light chain sequence of SEQ ID NO:12. In one embodiment, the antibody comprises a variable heavy chain sequence of SEQ ID NO:20 and a variable light chain sequence of SEQ ID NO:28. In one embodiment, the antibody comprises a variable heavy chain sequence of SEQ ID NO:36 and a variable light chain sequence of SEQ ID NO:44. In one embodiment, the antibody comprises a variable heavy chain sequence of SEQ ID NO:52 and a variable light chain sequence of SEQ ID NO:60. In one embodiment, the antibody comprises a variable heavy chain sequence of SEQ ID NO:68 and a variable light chain sequence of SEQ ID NO:76. In one embodiment, the antibody comprises a variable heavy chain sequence of SEQ ID NO:84 and a variable light chain sequence of SEQ ID NO:92. In one embodiment, the antibody comprises a variable heavy chain sequence of SEQ ID NO:100 and a variable light chain sequence of SEQ ID NO:108. In one embodiment, the antibody comprises a variable heavy chain sequence of SEQ ID NO:116 and a variable light chain sequence of SEQ ID NO:124. In one embodiment, the antibody comprises a variable heavy chain sequence of SEQ ID NO: 132 and a variable light chain sequence of SEQ ID NO: 140. In one embodiment, the antibody comprises a variable heavy chain sequence of SEQ ID NO: 148 and a variable light chain sequence of SEQ ID NO: 156. In one embodiment, the antibody comprises a variable heavy chain sequence of SEQ ID NO: 164 and a variable light chain sequence of SEQ ID NO: 172. In one embodiment, the antibody comprises a variable heavy chain sequence of SEQ ID NO: 180 and a variable light chain sequence of SEQ ID NO: 188. In one embodiment, the antibody comprises a variable heavy chain sequence of SEQ ID NO: 196 and a variable light chain sequence of SEQ ID NO: 204.

[0105] In some embodiments, variants of the amino acid sequences as described herein comprise at least about 60% identity, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over a specified region when compared to a defined amino acid sequence. In some embodiments, the variants of the amino acid sequences as described herein are over the full length of the variable heavy chain having one or more of the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 36, SEQ ID NO: 52, SEQ ID NO: 68, SEQ ID NO: 84, SEQ ID NO: 100, SEQ ID NO: 116, SEQ ID NO: 132, SEQ ID NO: 148, SEQ ID NO: 164, SEQ ID NO: 180, or SEQ ID NO: 196, or over the full length of the variable heavy chain having one or more of the amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 44, SEQ ID NO: 60, SEQ ID NO: 76, SEQ ID NO: 92, SEQ ID NO: 108, SEQ ID NO: 124, SEQ ID NO: 140, SEQ ID NO: 156, SEQ ID NO: 172, SEQ ID NO: 188, or SEQ ID NO: 197. %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%, 100%,

[0106] In some embodiments, a variant of SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, SEQ ID NO:84, SEQ ID NO:100, SEQ ID NO:116, SEQ ID NO:132, SEQ ID NO:148, SEQ ID NO:164, SEQ ID NO:180, or SEQ ID NO:196 comprises 100% identity to all three CDR regions of SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, SEQ ID NO:84, SEQ ID NO:100, SEQ ID NO:116, SEQ ID NO:132, SEQ ID NO:148, SEQ ID NO:164, SEQ ID NO:180, or SEQ ID NO:196. In some embodiments, a variant of SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 44, SEQ ID NO: 60, SEQ ID NO: 76, SEQ ID NO: 92, SEQ ID NO: 108, SEQ ID NO: 124, SEQ ID NO: 140, SEQ ID NO: 156, SEQ ID NO: 172, SEQ ID NO: 188, or SEQ ID NO: 204 comprises 100% identity to all three CDR regions of SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 44, SEQ ID NO: 60, SEQ ID NO: 76, SEQ ID NO: 92, SEQ ID NO: 108, SEQ ID NO: 124, SEQ ID NO: 140, SEQ ID NO: 156, SEQ ID NO: 172, SEQ ID NO: 188, or SEQ ID NO: 204.

[0107] As used herein, the term "antibody" or "immunoglobulin" refers to proteins (including glycoproteins) of the immunoglobulin (Ig) superfamily of proteins. An antibody or immunoglobulin (Ig) molecule may be a tetramer comprising two identical light chain polypeptides and two identical heavy chain polypeptides. The two heavy chains are linked together by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. Each full-length Ig molecule contains at least two binding sites for a specific target or antigen.

[0108] Sialic acid binding receptor antibodies or antigen-binding fragments thereof include, but are not limited to, polyclonal antibodies, monoclonal fusion proteins, antibodies or fragments thereof, chimeric or chimeric fusion proteins, antibodies or fragments thereof, humanized fusion proteins, antibodies or fragments thereof, deimmunized humfusion proteins, antibodies or fragments thereof, fully humfusion proteins, antibodies or fragments thereof, single chain antibodies, single chain Fv fragments (scFv), Fv, Fd fragments, Fab fragments, Fab' fragments, F(ab')2 fragments, diabodies or antigen-binding fragments thereof, minibodies or antigen-binding fragments thereof, triabodies or antigen-binding fragments thereof, domain fusion proteins, antibodies or fragments thereof, camelid fusion proteins, antibodies or fragments thereof, dromedary fusion proteins, antibodies or fragments thereof, phage displayed fusion proteins, antibodies or fragments thereof, or antibodies or antigen-binding fragments thereof recognized by a repeating backbone array (e.g., repeating antigen display).

[0109] The immune system produces several different classes of Ig molecules (isotypes), including IgA, IgD, IgE, IgG, and IgM, each distinguished by the presence of a specific class of heavy chain polypeptides: alpha (a) present in IgA, delta (δ) present in IgD, epsilon (ε) present in IgE, gamma (γ) present in IgG, and mu (μ) present in IgM. There are at least five different gamma heavy chain polypeptides (isotypes) present in IgG. In contrast, there are only two light chain polypeptide isotypes, called kappa (κ) chains and lambda (λ) chains. The unique characteristics of an antibody isotype are defined by the sequence of the constant domain of the heavy chain.

[0110] IgG molecules contain two light chains (κ or λ forms) and two heavy chains (γ forms) held together by disulfide bonds. The κ and λ forms of the IgG light chain each contain a domain of relatively variable amino acid sequence, called the variable region (variably referred to as the "V L -","V κ -" or "V λ -region) and a relatively conserved amino acid sequence domain, called the constant region (C L Similarly, each IgG heavy chain contains a variable region (V H -region) and one or more conserved regions: The complete IgG heavy chain contains three constant domains ("C H 1-", "C H 2-" and "C H 3-region”) and hinge region. At each V L- or V H -region, hypervariable regions (also called complementarity determining regions ("CDR")) are interspersed between relatively conserved framework regions ("FR"). Typically, the variable region of a light or heavy chain polypeptide contains four FRs and three CDRs, which are arranged along the polypeptide in the following order: NH2-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-COOH. CDRs and FRs together determine the three-dimensional structure of the IgG binding site, thereby determining the specific target protein or antigen to which the IgG molecule binds. Each IgG molecule is a dimer and can bind to two antigen molecules. Cleavage of dimeric IgG with the protease papain produces two identical antigen-binding fragments ("Fab") and an "Fc" fragment or Fc domain, so named because it is easy to crystallize.

[0111] As used throughout this disclosure, the term "antibody" also refers to a whole or complete antibody (e.g., IgM, IgG, IgA, IgD or IgE) molecule produced by any of the various methods known in the art and described herein. The term "antibody" includes polyclonal antibodies, monoclonal antibodies, chimeric or chimeric antibodies, humanized antibodies, deimmunized human antibodies and fully human antibodies. Antibodies can be made in any of a variety of species or derived from any of a variety of species, such as mammals such as humans, non-human primates (e.g., monkeys, baboons or chimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats and mice. The antibody can be a purified antibody or a recombinant antibody.

[0112] As used herein, the term "epitope" refers to a site on a protein to which an antibody binds."Overlapping epitopes" include at least one (eg, two, three, four, five or six) common amino acid residues.

[0113] In one embodiment, the antibody of the present invention specifically binds to a Siglec polypeptide. As used herein, the term "specific binding" or "specifically binds" refers to two molecules forming a relatively stable complex under physiological conditions. Generally, when the binding constant (K a ) is higher than 10 6 M -1 Thus, an antibody can bind specifically to a target with a Ka of at least (or greater than) 10 6 (For example, at least or greater than 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 or 1015 or higher)M -1 .

[0114] In one embodiment, the antibodies of the invention specifically bind to Siglec-9.

[0115] Methods for determining whether an antibody binds to a protein antigen and / or the affinity of an antibody for a protein antigen are known in the art. For example, the binding of an antibody to a protein antigen can be detected and / or quantified using various techniques, such as, but not limited to, Western blot, dot blot, surface plasmon resonance methods (e.g., BIAcore system; Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ) or enzyme-linked immunosorbent assay (ELISA). See, for example, Harlow and Lane (1988) "Antibodies: A Laboratory Manual," Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Benny KCLo (2004) "Antibody Engineering: Methods and Protocols," Humana Press (ISBN: 1588290921); Borrebaek (1992) "Antibody Engineering, A Practical Guide," WH Freeman and Co., NY; Borrebaek (1995) "Antibody Engineering," 2nd ed., Oxford University Press, NY, Oxford; Johne et al. (1993) J. Immunol. Meth. 160: 191-198; Jonsson et al. (1993) Ann. Biol. Clin. 51: 19-26; and Jonsson et al. (1991) Biotechniques 11:620-627. See also U.S. Patent No. 6,355,245.

[0116] Immunoassays that can be used to analyze the immunospecific binding and cross-reactivity of antibodies include, but are not limited to, competitive and non-competitive assay systems using techniques such as Western blots, RIA, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays. Such assays are routine and well known in the art.

[0117] Antibodies can also be measured using any surface plasmon resonance (SPR)-based assay known in the art to characterize the kinetic parameters of the interaction of an antibody with its target or epitope. Any commercially available SPR instrument includes, but is not limited to, a BIAcore instrument (Biacore AB; Uppsala, Sweden); an IAsys instrument (Affinity Sensors; Franklin, Massachusetts); an IBIS system (Windsor Scientific Limited; Berks, UK), an SPR-CELLIA system (Nippon Laser and Electronics Lab; Hokkaido, Japan), and an SPR Detector Spreeta (Texas Instruments; Dallas, Texas) can be used for the methods described herein. See, e.g., Mullett et al. (2000) Methods 22:77-91; Dong et al. (2002) Reviews in Mol Biotech 82:303-323; Fivash et al. (1998) Curr Opin Biotechnol 9:97-101; and Rich et al. (2000) Curr Opin Biotechnol 11:54-61.

[0118] In some embodiments, antibodies and fragments thereof may be "chimeric". Chimeric antibodies and antigen-binding fragments thereof contain portions from two or more different species (e.g., mouse and human). Chimeric antibodies can be produced by splicing mouse variable regions of desired specificity onto human constant domain gene segments (see, e.g., U.S. Patent No. 4,816,567). In this way, non-human antibodies can be modified to make them more suitable for human clinical applications (e.g., methods for treating or preventing complement-associated diseases in human subjects).

[0119] The monoclonal antibodies disclosed herein include "humanized" forms of non-human (e.g., mouse) antibodies. Humanized or CDR-transplanted mAbs are particularly useful as therapeutic agents for humans because they are not rapidly cleared from the circulation like mouse antibodies and generally do not cause adverse immune responses. Methods for preparing humanized antibodies are generally well known in the art. For example, humanization can be performed substantially following the method of Winter and colleagues (see, e.g., Jones et al. (1986) Nature 321: 522-525; Riechmann et al. (1988) Nature 332: 323-327; and Verhoeyen et al. (1988) Science 239: 1534-1536)), by replacing the corresponding sequences of human antibodies with rodent CDRs or CDR sequences. See also, e.g., Staelens et al. (2006) Mol Immunol 43: 1243-1257. In some embodiments, the humanized form of non-human (e.g., mouse) antibodies is a human antibody (receptor antibody), wherein the hypervariable (CDR) region residues of the receptor antibody are replaced by the hypervariable region residues of mice, rats, rabbits, or non-human primates from non-human species (donor antibodies), for example, with the desired specificity, affinity, and binding capacity. In some cases, the framework region residues of human immunoglobulins are also replaced by corresponding non-human residues (so-called "back mutations"). In addition, phage display libraries can be used to change the amino acid at the selected position in the antibody sequence. The characteristics of humanized antibodies are also affected by the selection of human frameworks. In addition, humanized and chimeric antibodies can be modified to be included in residues not present in the receptor antibody or the donor antibody, to further improve antibody properties, such as affinity or effector functions.

[0120] The disclosure also provides fully human antibodies.The term "human antibody" includes antibodies with variable regions and constant regions (if present) derived from human germline immunoglobulin sequences.Human antibodies can include amino acid residues not encoded by human germline immunoglobulin sequences (for example, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, the term "human antibody" does not include antibodies (i.e., humanized antibodies) in which the CDR sequences derived from germlines of other mammalian species such as mice have been transplanted to human framework sequences. Fully human antibodies or human antibodies can be derived from transgenic mice carrying human antibody genes (carrying variable (V), diversity (D), connection (J) and constant (C) exons) or from human cells. For example, transgenic animals (such as mice) can be produced now, which can produce complete human antibody repertoires in the absence of endogenous immunoglobulin production after immunity. (See, e.g., Jakobovits et al. (1993) Proc. Natl. Acad. Sci. USA 90:2551; Jakobovits et al. (1993) Nature 362:255-258; Bruggemann et al. (1993) Year in Immunol. 7:33; and Duchosal et al. (1992) Nature 355:258.) Transgenic mouse strains can be engineered to contain gene sequences from unrearranged human immunoglobulin genes. The human sequences can encode the heavy and light chains of human antibodies and will function correctly in mice, rearranging to provide a broad antibody repertoire similar to that of humans. Transgenic mice can be immunized with a target protein (to produce a diverse array of specific antibodies and their encoding RNAs. Nucleic acids encoding the antibody chain components of such antibodies can then be cloned from the animal into a display vector. Typically, separate populations of nucleic acids encoding heavy and light chain sequences are cloned and then inserted into a vector followed by recombination so that any given copy of the vector receives a random combination of heavy and light chains. The vector is designed to express the antibody chains so that they can be assembled and displayed on the outer surface of a display package containing the vector. For example, the antibody chains can be expressed as fusion proteins with a phage coat protein from the outer surface of a phage. Thereafter, the display package can be screened to display antibodies bound to the target.

[0121] Thus, in some embodiments, the present disclosure provides, for example, humanized, deimmunized, or primatized antibodies comprising one or more complementarity determining regions (CDRs) of the mouse monoclonal antibodies described herein that retain the ability of the mouse monoclonal antibody counterpart to bind to its antigen (e.g., at least 50, 60, 70, 80, 90, or 100%, or even greater than 100%).

[0122] In addition, human antibodies can be derived from phage display libraries (Hoogenboom et al. (1991) J. Mol. Biol. 227: 381; Marks et al. (1991) J. Mol. Biol, 222: 581-597; and Vaughan et al. (1996) Nature Biotech 14: 309 (1996)). Synthetic phage libraries can be created using random combinations of synthetic human antibody V regions. By selecting for antigen, fully human antibodies can be prepared in which the V regions are very similar to those of humans in nature. See, for example, U.S. Pat. Nos. 6,794,132, 6,680,209, 4,634,666 and Ostberg et al. (1983), Hybridoma 2: 361-367, the contents of each of which are hereby incorporated by reference in their entirety.

[0123] For the generation of human antibodies, see also Mendez et al. (1998) Nature Genetics 15: 146-156 and Green and Jakobovits (1998) J. Exp. Med. 188: 483-495, the disclosures of which are hereby incorporated by reference in their entireties. Human antibodies are further discussed and described in U.S. Patent Nos.: 5,939,598; 6,673,986; 6,1 14,598; 6,075,181; 6,162,963; 6,150,584; 6,713,610; and 6,657,103 and U.S. Patent Application Publication Nos. 2003-0229905A1, 2004-0010810A1, US 2004-0093622A1, 2006-0040363A1, 2005-0054055A1, 2005-0076395A1, and 2005-0287630A1. See also International Publication Nos. WO 94 / 02602, WO 96 / 34096 and WO 98 / 24893, and European Patent No. EP 0 463 151 Bl. The disclosures of each of the above patents, applications and references are hereby incorporated by reference in their entirety.

[0124] In another approach, other companies, including GenPharm International, Inc., have adopted the "minilocus" approach. In the minilocus approach, a foreign Ig locus is mimicked by including fragments (single genes) from the Ig locus. Thus, one or more VH genes, one or more DH genes, one or more JH genes, a mu constant region, and a second constant region (preferably a gamma constant region) form a construct for insertion into an animal. This method is described in, for example, U.S. Patent Nos.: 5,545,807; 5,545,806; 5,625,825; 5,625,126; 5,633,425; 5,661,016; 5,770,429; 5,789,650; and 5,814,318; 5,591,669; 5,612,205; 5,721,367; 5,789,215; 5,643,763; 5,569,825; 5,877,397; 6,300,129; 5,874,299; 6,255,458; and 7,041,871, the disclosures of which are hereby incorporated herein by reference. See also European Patent No. 0 546 073 Bl, International Patent Publication Nos. WO 92 / 03918, WO 92 / 22645, WO 92 / 22647, WO 92 / 22670, WO 93 / 12227, WO 94 / 00569, WO 94 / 25585, WO 96 / 14436, WO 97 / 13852 and WO 98 / 24884, the disclosures of each of which are hereby incorporated by reference in their entirety. See further Taylor et al. (1992) Nucleic Acids Res. 20:6287; Chen et al. (1993) Int. Immunol. 5:647; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90:3720-4; Choi et al. (1993) Nature Genetics 4:1 17; Lonberg et al. (1994) Nature 368:856-859; Taylor et al. (1994) International Immunology 6:579-591; Tuaillon et al. (1995) J. Immunol. 154:6453-65; Fishwild et al. (1996) Nature Biotechnology 14:845; and Tuaillon et al. (2000) Eur. J. Immunol. 10:2998-3005, the disclosures of each of which are hereby incorporated by reference in their entirety.

[0125] In some embodiments, a deimmunized antibody or antigen binding fragment thereof is provided. A deimmunized antibody or antigen binding fragment thereof is a modified antibody so that the antibody or its antigen binding fragment is non-immunogenic or less immunogenic to a given species (e.g., to humans). Deimmunization can be achieved by modifying fusion proteins, antibodies, or fragments thereof using any of a variety of techniques known to those skilled in the art (see, e.g., PCT Publication No. WO 04 / 108158 and WO 00 / 34317). For example, a fusion protein, an antibody, or its fragment can be deimmunized by identifying potential T cell epitopes and / or B cell epitopes in the amino acid sequence of a fusion protein, an antibody, or its fragment and removing more than one potential T cell epitope and / or B cell epitope from the fusion protein, an antibody, or its fragment (e.g., using recombinant technology). The modified antibody or its antigen binding fragment can then be optionally produced and tested to identify antibodies or its antigen binding fragments that retain more than one desired biological activity (e.g., such as binding affinity) but have reduced immunogenicity. Methods for identifying potential T cell epitopes and / or B cell epitopes can be performed using techniques known in the art, such as computational methods (see, e.g., PCT Publication No. WO 02 / 069232), in vitro or computer techniques, and biological assays or physical methods (e.g., such as determining the binding of a peptide to an MHC molecule, determining the binding of a peptide:MHC complex to a T cell receptor from a species that accepts a fusion protein, antibody, or fragment thereof, testing a protein or its peptide portion using a transgenic animal having an MHC molecule of a species that accepts an antibody or antigen-binding fragment thereof, or testing using a transgenic animal reconstituted with immune system cells from a species that accepts a fusion protein, antibody, or fragment thereof, etc.). In various embodiments, the deimmunized antibodies described herein include deimmunized antigen-binding fragments, Fab, Fv, scFv, Fab' and F(ab')2, monoclonal antibodies, murine antibodies, engineered antibodies (e.g., chimeric, single chain, CDR-grafted, humanized, fully human antibodies, and artificially selected antibodies), synthetic antibodies, and semisynthetic antibodies.

[0126] In some embodiments, the present disclosure also provides bispecific antibodies. Bispecific antibodies are monoclonal antibodies, preferably human antibodies or humanized antibodies with binding specificity to at least two different antigens. For example, in one embodiment, the bispecific antibody of the present invention comprises a domain with binding specificity to a Siglec protein or polypeptide, and a domain with binding specificity to an alternative protein or polypeptide. In one embodiment, the bispecific antibody of the present invention comprises a domain with binding specificity to a Siglec protein or polypeptide, and a domain with binding specificity to an alternative Siglec protein or polypeptide.

[0127] The method for preparing bispecific antibodies is within the knowledge of those skilled in the art. Traditionally, the recombinant production of bispecific antibodies is based on the coexpression of two heavy chain immunoglobulin / light chain pairs, wherein the two heavy chain / light chain pairs have different specificities (Milstein and Cuello (1983) Nature 305:537-539). The antibody variable domains (antibody-antigen binding sites) with the desired binding specificity can be fused to the immunoglobulin constant domain sequence. The fusion of the heavy chain variable region is preferably fused with the immunoglobulin heavy chain constant domain, including at least a portion of the hinge region, CH2 region and CH3 region. The DNA encoding the immunoglobulin heavy chain fusion and (if necessary) immunoglobulin light chain is inserted into a separate expression vector and cotransfected into a suitable host organism. For more details on exemplary currently known methods of producing bispecific antibodies, see Suresh et al. (1986) Methods in Enzymology 121:210; PCT Publication No. WO96 / 27011; Brennan et al. (1985) Science 229:81; Shalaby et al., J Exp Med (1992) 175:217-225; Kostelny et al. (1992) J Immunol 148(5):1547-1553; Hollinger et al. (1993) Proc Natl Acad Sci USA 90:6444-6448; Gruber et al. (1994) J Immunol 152:5368; and Tutt et al. (1991) J Immunol 147:60. Bispecific antibodies also include cross-linked antibodies or heteroconjugate antibodies. Heteroconjugate antibodies may be prepared using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in US Pat. No. 4,676,980, along with a number of cross-linking techniques.

[0128] Various techniques for preparing and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. See, for example, Kostelny et al. (1992) J Immunol 148(5):1547-1553. Leucine zipper peptides from Fos and Jun proteins can be connected to the Fab' portion of two different antibodies by gene fusion. Antibody homodimers can be reduced in the hinge region to form monomers and then oxidized to form antibody heterodimers. This method can also be used to produce antibody homodimers. The "double antibody" technology described by Hollinger et al. Proc Natl Acad Sci USA 90:6444-6448 provides an alternative mechanism for preparing bispecific antibody fragments. The fragment comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) by a linker, and the linker is too short to allow pairing between the two domains on the same chain. Thus, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen binding sites. Another strategy for preparing bispecific antibody fragments by using single-chain Fv (scFv) dimers has also been reported. See, for example, Gruber et al. (1994) J Immunol 152:5368. Alternatively, the antibody may be a "linear antibody", such as described in Zapata et al. (1995) Protein Eng. 8 (10): 1057-1062. In short, these antibodies comprise a pair of tandem Fd fragments (VH-CH1-VH-CH1) that form a pair of antigen binding regions. Linear antibodies may be bispecific or monospecific.

[0129] Antibodies with more than two valencies (eg, trispecific antibodies) are also contemplated and are described, for example, in Tutt et al. (1991) J Immunol 147:60.

[0130] The disclosure also encompasses variant forms of multispecific antibodies, such as Wu et al. (2007) Nat Biotechnol 25 (11): Dual variable domain immunoglobulin (DVD-1g) molecules described in 1290-1297. DVD-1g molecules are designed so that two different light chain variable domains (VL) from two different parent antibodies are directly or via a short linker connected in series by recombinant DNA technology, followed by a light chain constant domain. Similarly, the heavy chain comprises two different heavy chain variable domains (VH) connected in series, followed by a constant domain CH1 and an Fc region. For example, PCT Publication No. WO 08 / 024188 and WO 07 / 024715 further describe methods for making DVD-Ig molecules from two parent antibodies.

[0131] The present disclosure also provides camelid or dromedary antibodies (e.g., antibodies derived from Bactrianus bactrianus, Calelus dromaderius, or lama paccos). Unlike typical two-chain (fragments) or four-chain (whole antibodies) antibodies from most mammals, such antibodies generally lack light chains. See U.S. Pat. No. 5,759,808; Stijlemans et al. (2004) J Biol Chem 279: 1256-1261; Dumoulin et al. (2003) Nature 424: 783-788; and Pleschberger et al. (2003) Bioconjugate Chem 14: 440-448.

[0132] Engineered libraries of camelid antibodies and antibody fragments are commercially available, for example, from Ablynx (Ghent, Belgium). Like other non-human derived antibodies, the amino acid sequence of camelid antibodies can be recombinantly altered to obtain a sequence that is more closely similar to the human sequence, i.e., nanobodies can be "humanized", thereby further reducing the potential immunogenicity of the antibody.

[0133] In some embodiments, the disclosure also provides antibodies or their antigen-binding fragments, which are variants of peptides, proteins or antibodies described herein. In some embodiments, such variant peptides, proteins or antibodies maintain the binding or inhibitory ability of parent peptides, proteins or antibodies. Methods for preparing variants of known proteins, peptides or antibodies are known in the art. In some embodiments, such variants include at least a single amino acid substitution, deletion, insertion or other modification. In some embodiments, fusion proteins, antibodies or their fragments described herein include more than two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acid modifications (e.g., amino acid substitutions, deletions or additions). In some embodiments, fusion proteins, antibodies or their fragments described herein do not contain amino acid modifications in CDR. In some embodiments, the fusion proteins, antibodies, or fragments thereof described herein contain more than one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acid modifications in the CDRs.

[0134] As used herein, the term "antibody fragment", "antigen-binding fragment", "antigen binding fragment" or similar terms refers to an antibody fragment that retains the ability to bind to an antigen, wherein the antigen binding fragment may optionally include other components that are not part of the original antibody (such as different framework regions or mutations) as well as fragments (more than one) from the original antibody. Examples include, but are not limited to, single-chain antibodies, single-chain Fv fragments (scFv), Fd fragments, Fab fragments, Fab' fragments, or F(ab')2 fragments. The scFv fragment is a single polypeptide chain that includes both the heavy chain and light chain variable regions of the antibody from which the scFv is derived. In addition, diabodies (Poljak (1994) Structure 2(12):1121-1123; Hudson et al. (1999) J. Immunol. Methods 23(1-2):177-189, the disclosures of each of which are hereby incorporated by reference in their entireties), minibodies, triabodies (Schoonooghe et al. (2009) BMC Biotechnol 9:70), and domain antibodies (also known as "heavy chain immunoglobulins" or camelid antibodies; Holt et al. (2003) Trends Biotechnol 21(11):484-490) that bind to complement component proteins (the disclosures of each of which are hereby incorporated by reference in their entireties) can be incorporated into the compositions described herein and used in the methods described herein. In some embodiments, any antigen-binding fragment described herein may be included in "antigen-binding fragment thereof" or equivalent terms, when referring to fragments associated with antibodies, whether or not such fragments are actually derived from antibodies, or are antigen-binding fragments that bind to the same epitope or overlapping epitopes or epitopes contained in antibody epitopes. Antigen-binding fragments thereof may include antigen-binding fragments that bind to the same or overlapping antigens as the original antibody, wherein the antigen-binding fragment includes a portion that is a fragment of the original antibody (e.g., more than one CDR, more than one variable region, etc.).

[0135] In some embodiments, a fragment of SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, SEQ ID NO:84, SEQ ID NO:100, SEQ ID NO:116, SEQ ID NO:132, SEQ ID NO:148, SEQ ID NO:164, SEQ ID NO:180, or SEQ ID NO:196 comprises at least all three CDR regions of SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, SEQ ID NO:84, SEQ ID NO:100, SEQ ID NO:116, SEQ ID NO:132, SEQ ID NO:148, SEQ ID NO:164, SEQ ID NO:180, or SEQ ID NO:196. In some embodiments, a fragment of SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 44, SEQ ID NO: 60, SEQ ID NO: 76, SEQ ID NO: 92, SEQ ID NO: 108, SEQ ID NO: 124, SEQ ID NO: 140, SEQ ID NO: 156, SEQ ID NO: 172, SEQ ID NO: 188, or SEQ ID NO: 204 comprises at least all three CDR regions of SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 44, SEQ ID NO: 60, SEQ ID NO: 76, SEQ ID NO: 92, SEQ ID NO: 108, SEQ ID NO: 124, SEQ ID NO: 140, SEQ ID NO: 156, SEQ ID NO: 172, SEQ ID NO: 188, or SEQ ID NO: 204.In some embodiments, the fragment of the sialic acid binding receptor comprises a scFv antibody fragment comprising the heavy chain of SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, SEQ ID NO:84, SEQ ID NO:100, SEQ ID NO:116, SEQ ID NO:132, SEQ ID NO:148, SEQ ID NO:164, SEQ ID NO:180, or SEQ ID NO:196, or all three CDR regions of SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, SEQ ID NO:84, SEQ ID NO:100, SEQ ID NO:116, SEQ ID NO:132, SEQ ID NO:148, SEQ ID NO:164, SEQ ID NO:180, or SEQ ID NO:196, and all three CDR regions of SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, SEQ ID NO:84, SEQ ID NO:100, SEQ ID NO:116, SEQ ID NO:132, SEQ ID NO:148, SEQ ID NO:164, SEQ ID NO:180, or SEQ ID NO:196. NO: 44, SEQ ID NO: 60, SEQ ID NO: 76, SEQ ID NO: 92, SEQ ID NO: 108, SEQ ID NO: 124, SEQ ID NO: 140, SEQ ID NO: 156, SEQ ID NO: 172, SEQ ID NO: 188 or SEQ ID NO: 204, or all three CDR regions of SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 44, SEQ ID NO: 60, SEQ ID NO: 76, SEQ ID NO: 92, SEQ ID NO: 108, SEQ ID NO: 124, SEQ ID NO: 140, SEQ ID NO: 156, SEQ ID NO: 172, SEQ ID NO: 188 or SEQ ID NO: 204.

[0136] In some embodiments, the antibodies described herein comprise altered or mutated sequences that result in changes in stability or half-life compared to the parent antibody. This includes, for example, increased stability or half-life to obtain higher affinity or longer in vitro or in vivo clearance time, or reduced stability or half-life to obtain lower affinity or faster clearance. In addition, the antibodies described herein may comprise more than one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) amino acid substitutions, deletions or insertions that result in altered post-translational modifications, including, for example, altered glycosylation patterns (e.g., adding more than one sugar component, losing more than one sugar component, or changing the composition of more than one sugar component).

[0137] In some embodiments, the antibodies described herein include reduced (e.g., or no) effector functions. The altered effector functions include, for example, modulation of one or more of the following activities: antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), apoptosis, binding to one or more Fc receptors, and proinflammatory responses. Modulation refers to an increase, decrease, or elimination of the effector function activity exhibited by the subject antibody containing the altered constant region compared to the activity of the unaltered form of the constant region. In specific embodiments, modulation includes situations where the activity is abolished or completely absent.

[0138] Antibodies with altered or no effector function can be produced by engineering or producing antibodies with variant constant regions, Fc regions, or heavy chain regions; antibodies with altered function and / or activity can be produced using recombinant DNA techniques and / or cell culture and expression conditions. For example, one or more amino acid substitutions, deletions, or insertions in regions (e.g., such as Fc regions or constant regions) that affect antibody function (including effector function) can be engineered using recombinant DNA techniques. Alternatively, changes in post-translational modifications (e.g., glycosylation patterns) can be achieved by manipulating cell culture and expression conditions that produce the antibody. Suitable methods for introducing one or more substitutions, additions or deletions into the Fc region of an antibody are well known in the art and include, for example, standard DNA mutagenesis techniques as described in Sambrook et al. (1989) "Molecular Cloning: A Laboratory Manual, 2nd Edition", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane (1988), supra; Borrebaek (1992), supra; Johne et al. (1993), supra; PCT Publication No. WO 06 / 53301; and U.S. Pat. No. 7,704,497.

[0139] Nucleic acid molecules

[0140] Provided herein are polynucleotides encoding Siglec-9 antibodies or fragments thereof of the present invention. In some embodiments, the polynucleotide further comprises a sequence encoding a signal peptide operably linked to the 5' end of the coding sequence. In some embodiments, the polynucleotide further comprises a sequence encoding a linker sequence.

[0141] In one embodiment, the nucleic acid molecule encodes an antibody comprising a variable heavy chain sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 36, SEQ ID NO: 52, SEQ ID NO: 68, SEQ ID NO: 84, SEQ ID NO: 100, SEQ ID NO: 116, SEQ ID NO: 132, SEQ ID NO: 148, SEQ ID NO: 164, SEQ ID NO: 180, or SEQ ID NO: 196; a variable light chain sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 44, SEQ ID NO: 60, SEQ ID NO: 76, SEQ ID NO: 92, SEQ ID NO: 108, SEQ ID NO: 124, SEQ ID NO: 140, SEQ ID NO: 156, SEQ ID NO: 172, SEQ ID NO: 188, or SEQ ID NO: 204; and a variable light chain sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 36, SEQ ID NO: 52, SEQ ID NO: 68, SEQ ID NO: 84, SEQ ID NO: 100, SEQ ID NO: 116, SEQ ID NO: 132, SEQ ID NO: 148, SEQ ID NO: 164, SEQ ID NO: 180, or SEQ ID NO: 196; NO:52, SEQ ID NO:68, SEQ ID NO:84, SEQ ID NO:100, SEQ ID NO:116, SEQ ID NO:132, SEQ ID NO:148, SEQ ID NO:164, SEQ ID NO:180 or SEQ ID NO:196. A sequence having at least 95% identity to one or more of the variable heavy chain sequences of SEQ ID NO:12, SEQ ID NO:28, SEQ ID NO:44, SEQ ID NO:60, SEQ ID NO:76, SEQ ID NO:92, SEQ ID NO:108, SEQ ID NO:124, SEQ ID NO:140, SEQ ID NO:156, SEQ ID NO:172, SEQ ID NO:188 or SEQ ID NO:204; A sequence having at least 95% identity to one or more of the variable light chain sequences of SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, SEQ ID NO:84, SEQ ID NO:100, SEQ ID NO:116, SEQ ID NO:132, SEQ ID NO:148, SEQ ID NO:164, SEQ ID NO:180 or SEQ ID NO:196. A fragment of at least 80% of the full-length sequence of a variable heavy chain sequence of SEQ ID NO: 100, SEQ ID NO: 116, SEQ ID NO: 132, SEQ ID NO: 148, SEQ ID NO: 164, SEQ ID NO: 180, or SEQ ID NO: 196;or a fragment comprising at least 80% of the full length of one or more variable light chain sequences selected from SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 44, SEQ ID NO: 60, SEQ ID NO: 76, SEQ ID NO: 92, SEQ ID NO: 108, SEQ ID NO: 124, SEQ ID NO: 140, SEQ ID NO: 156, SEQ ID NO: 172, SEQ ID NO: 188 or SEQ ID NO: 204, or a combination thereof;

[0142] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the variable heavy chain CDR sequence of SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, SEQ ID NO:84, SEQ ID NO:100, SEQ ID NO:116, SEQ ID NO:132, SEQ ID NO:148, SEQ ID NO:164, SEQ ID NO:180 or SEQ ID NO:196. In one embodiment, the nucleic acid molecule comprises SEQ ID NOs:5-7; SEQ ID NOs:21-23; SEQ ID NOs:37-39; SEQ ID NOs:53-55; SEQ ID NOs:69-71; SEQ ID NOs:85-87; SEQ ID NOs:101-103; SEQ ID NOs:117-119; SEQ ID NOs:133-135; SEQ ID NOs:149-151; SEQ ID NOs:165-167; SEQ ID NOs:181-183; or SEQ ID NOs:197-199. In one embodiment, the nucleic acid molecule comprises SEQ ID NO:8; SEQ ID NO:24; SEQ ID NO:40; SEQ ID NO:56; SEQ ID NO:72; SEQ ID NO:88; SEQ ID NO:104; SEQ ID NO:120; SEQ ID NO:136; SEQ ID NO:152; SEQ ID NO:168; SEQ ID NO:184; or SEQ ID NO:200.

[0143] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the variable light chain CDR sequence of SEQ ID NO:12, SEQ ID NO:28, SEQ ID NO:44, SEQ ID NO:60, SEQ ID NO:76, SEQ ID NO:92, SEQ ID NO:108, SEQ ID NO:124, SEQ ID NO:140, SEQ ID NO:156, SEQ ID NO:172, SEQ ID NO:188 or SEQ ID NO:204. In one embodiment, the nucleic acid molecule comprises SEQ ID NOs: 13-15; SEQ ID NOs: 29-31; SEQ ID NOs: 45-47; SEQ ID NOs: 61-63; SEQ ID NOs: 77-79; SEQ ID NOs: 93-95; SEQ ID NOs: 109-111; SEQ ID NOs: 125-127; SEQ ID NOs: 141-143; SEQ ID NOs: 157-159; SEQ ID NOs: 173-175; SEQ ID NOs: 189-191; or SEQ ID NOs: 205-207. In one embodiment, the nucleic acid molecule comprises SEQ ID NO:16; SEQ ID NO:32; SEQ ID NO:48; SEQ ID NO:64; SEQ ID NO:80; SEQ ID NO:96; SEQ ID NO:112; SEQ ID NO:128; SEQ ID NO:144; SEQ ID NO:160; SEQ ID NO:176; SEQ ID NO:192; or SEQ ID NO:208.

[0144] In some embodiments, variants of the nucleotide sequences described herein comprise at least about 60% identity, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over a specified region when compared to a defined nucleotide sequence. In some embodiments, variants of the nucleotide sequences described herein comprise at least about 60% identity, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over the entire length of the nucleotide sequence. In some embodiments, a variant of SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, SEQ ID NO:84, SEQ ID NO:100, SEQ ID NO:116, SEQ ID NO:132, SEQ ID NO:148, SEQ ID NO:164, SEQ ID NO:180, or SEQ ID NO:196 comprises 100% identity to all three SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, SEQ ID NO:84, SEQ ID NO:100, SEQ ID NO:116, SEQ ID NO:132, SEQ ID NO:148, SEQ ID NO:164, SEQ ID NO:180, or SEQ ID NO:196.In some embodiments, a variant of SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 44, SEQ ID NO: 60, SEQ ID NO: 76, SEQ ID NO: 92, SEQ ID NO: 108, SEQ ID NO: 124, SEQ ID NO: 140, SEQ ID NO: 156, SEQ ID NO: 172, SEQ ID NO: 188, or SEQ ID NO: 204 comprises 100% identity to all three CDR regions of SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 44, SEQ ID NO: 60, SEQ ID NO: 76, SEQ ID NO: 92, SEQ ID NO: 108, SEQ ID NO: 124, SEQ ID NO: 140, SEQ ID NO: 156, SEQ ID NO: 172, SEQ ID NO: 188, or SEQ ID NO: 204. Thus, in some embodiments, the variability is in regions outside of the CDR coding regions.

[0145] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO:8; SEQ ID NO:24; SEQ ID NO:40; SEQ ID NO:56; SEQ ID NO:72; SEQ ID NO:88; SEQ ID NO:104; SEQ ID NO:120; SEQ ID NO:136; SEQ ID NO:152; SEQ ID NO:168; SEQ ID NO:184; or SEQ ID NO:200. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO:16; SEQ ID NO:32; SEQ ID NO:48; SEQ ID NO:64; SEQ ID NO:80; SEQ ID NO:96; SEQ ID NO:112; SEQ ID NO:128; SEQ ID NO:144; SEQ ID NO:160; SEQ ID NO:176; SEQ ID NO:192; or SEQ ID NO:208.

[0146] In some embodiments, a fragment of a nucleotide sequence described herein comprises at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the full-length sequence of the defined nucleotide sequence. In some embodiments, a fragment of a nucleotide sequence described herein comprises at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the full-length nucleotide sequence. In some embodiments, a fragment of SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, SEQ ID NO:84, SEQ ID NO:100, SEQ ID NO:116, SEQ ID NO:132, SEQ ID NO:148, SEQ ID NO:164, SEQ ID NO:180, or SEQ ID NO:196 comprises at least all three CDR regions of SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, SEQ ID NO:84, SEQ ID NO:100, SEQ ID NO:116, SEQ ID NO:132, SEQ ID NO:148, SEQ ID NO:164, SEQ ID NO:180, or SEQ ID NO:196.In some embodiments, a fragment of SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 44, SEQ ID NO: 60, SEQ ID NO: 76, SEQ ID NO: 92, SEQ ID NO: 108, SEQ ID NO: 124, SEQ ID NO: 140, SEQ ID NO: 156, SEQ ID NO: 172, SEQ ID NO: 188, or SEQ ID NO: 204 comprises at least all three CDR regions of SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 44, SEQ ID NO: 60, SEQ ID NO: 76, SEQ ID NO: 92, SEQ ID NO: 108, SEQ ID NO: 124, SEQ ID NO: 140, SEQ ID NO: 156, SEQ ID NO: 172, SEQ ID NO: 188, or SEQ ID NO: 204.

[0147] In one embodiment, the nucleic acid molecule comprises a fragment comprising at least 80% of the full-length sequence of SEQ ID NO:8; SEQ ID NO:24; SEQ ID NO:40; SEQ ID NO:56; SEQ ID NO:72; SEQ ID NO:88; SEQ ID NO:104; SEQ ID NO:120; SEQ ID NO:136; SEQ ID NO:152; SEQ ID NO:168; SEQ ID NO:184; or SEQ ID NO:200. In one embodiment, the nucleic acid molecule comprises a fragment comprising at least 80% of the full-length sequence of SEQ ID NO:16; SEQ ID NO:32; SEQ ID NO:48; SEQ ID NO:64; SEQ ID NO:80; SEQ ID NO:96; SEQ ID NO:112; SEQ ID NO:128; SEQ ID NO:144; SEQ ID NO:160; SEQ ID NO:176; SEQ ID NO:192; or SEQ ID NO:208.

[0148] In one embodiment, the invention provides a combination of nucleic acid molecules, wherein the first nucleic acid molecule comprises a heavy chain CDR encoding sequence of SEQ ID NOs: 5-7; SEQ ID NOs: 21-23; SEQ ID NOs: 37-39; SEQ ID NOs: 53-55; SEQ ID NOs: 69-71; SEQ ID NOs: 85-87; SEQ ID NOs: 101-103; SEQ ID NOs: 117-119; SEQ ID NOs: 133-135; SEQ ID NOs: 149-151; SEQ ID NOs: 165-167; SEQ ID NOs: 181-183; or SEQ ID NOs: 197-199, and the second nucleic acid molecule comprises a heavy chain CDR encoding sequence of SEQ ID NOs: 13-15; SEQ ID NOs: 29-31; SEQ ID NOs: 45-47; SEQ ID NOs: 61-63; SEQ ID NOs: 77-79; SEQ ID NOs: 93-95; SEQ ID NOs: 109-111; SEQ ID NOs: NO: 125-127; SEQ ID NO: 141-143; SEQ ID NO: 157-159; SEQ ID NO: 173-175; SEQ ID NO: 189-191; or SEQ ID NO: 205-207. In one embodiment, the combination of nucleic acid molecules comprises a first nucleic acid molecule and a second nucleic acid molecule, wherein the first nucleic acid molecule comprises a heavy chain coding sequence or a fragment or variant thereof of SEQ ID NO:8; SEQ ID NO:24; SEQ ID NO:40; SEQ ID NO:56; SEQ ID NO:72; SEQ ID NO:88; SEQ ID NO:104; SEQ ID NO:120; SEQ ID NO:136; SEQ ID NO:152; SEQ ID NO:168; SEQ ID NO:184; or SEQ ID NO:200, and the second nucleic acid molecule comprises a light chain coding sequence or a fragment or variant thereof of SEQ ID NO:16; SEQ ID NO:32; SEQ ID NO:48; SEQ ID NO:64; SEQ ID NO:80; SEQ ID NO:96; SEQ ID NO:112; SEQ ID NO:128; SEQ ID NO:144; SEQ ID NO:160; SEQ ID NO:176; SEQ ID NO:192; or SEQ ID NO:208.

[0149] The isolated nucleic acid can include any type of nucleic acid, including but not limited to DNA, cDNA and RNA. For example, in one embodiment, the composition includes an isolated DNA molecule encoding a protein inhibitor or a functional fragment thereof, including, for example, an isolated cDNA molecule. In one embodiment, the composition includes an isolated RNA molecule encoding a protein inhibitor or a functional fragment thereof.

[0150] In some embodiments, the invention provides a combination of nucleic acid molecules comprising a first nucleic acid molecule comprising a nucleotide sequence encoding an antibody heavy chain and a second nucleic acid molecule encoding an antibody light chain. Thus, in some embodiments, the invention provides a combination of a first nucleic acid molecule comprising a nucleotide sequence encoding a variable heavy chain of SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, SEQ ID NO:84, SEQ ID NO:100, SEQ ID NO:116, SEQ ID NO:132, SEQ ID NO:148, SEQ ID NO:164, SEQ ID NO:180, or SEQ ID NO:196, and a second nucleic acid molecule encoding a variable light chain of SEQ ID NO:12, SEQ ID NO:28, SEQ ID NO:44, SEQ ID NO:60, SEQ ID NO:76, SEQ ID NO:92, SEQ ID NO:108, SEQ ID NO:124, SEQ ID NO:140, SEQ ID NO:156, SEQ ID NO:172, SEQ ID NO:188, or SEQ ID NO:204.

[0151] The nucleic acid molecules of the present invention can be modified to improve their stability in serum or in a growth medium for cell culture. Modifications can be added to enhance the stability, functionality and / or specificity of the nucleic acid molecules of the present invention and to minimize their immunostimulatory properties. For example, in order to enhance stability, 3'-residues can be stabilized to prevent degradation, for example, they can be selected so that they consist of purine nucleotides (particularly adenosine or guanosine nucleotides). Alternatively, substitution of pyrimidine nucleotides by modified analogs, for example substitution of uridine by 2'-deoxythymidine is acceptable and does not affect the function of the molecule.

[0152] In one embodiment of the invention, the nucleic acid molecule may contain at least one modified nucleotide analog. For example, the termini may be stabilized by incorporating modified nucleotide analogs.

[0153] Non-limiting examples of nucleotide analogs include sugar- and / or backbone-modified ribonucleotides (i.e., including modifications to the phosphate-sugar backbone). For example, the phosphodiester bond of a natural RNA can be modified to include at least one nitrogen or sulfur heteroatom. In an exemplary backbone-modified ribonucleotide, the phosphate group connected to an adjacent ribonucleotide is replaced by a modified group (e.g., a phosphorothioate group).

[0154] Other examples of modifications are nucleobase-modified ribonucleotides, i.e., ribonucleotides containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. The base can be modified to block the activity of adenosine deaminase. Exemplary modified nucleobases include, but are not limited to, uridine and / or cytidine modified at the 5-position, such as 5-(2-amino)propyluridine, 5-bromouridine; adenosine and / or guanosine modified at the 8 position, such as 8-bromoguanosine; deazanucleotides, such as 7-deaza-adenosine; O- and N-alkylated nucleotides, such as N6-methyladenosine are suitable. The above modifications can be combined.

[0155] In some cases, the nucleic acid molecule comprises at least one of the following chemical modifications: 2'-H, 2'-O-methyl or 2'-OH modification of more than one nucleotide. In some embodiments, the nucleic acid molecule of the present invention may have enhanced resistance to nucleases. In order to increase nuclease resistance, the nucleic acid molecule may include, for example, 2'-modified ribose units and / or thiophosphate bonds. For example, 2'hydroxyl (OH) can be modified or replaced with many different "oxygen" or "deoxy" substituents. In order to increase nuclease resistance, the nucleic acid molecule of the present invention may include 2'-O-methyl, 2'-fluoro, 2'-O-methoxyethyl, 2'-O-aminopropyl, 2'-amino and / or thiophosphate bonds. The inclusion of locked nucleic acids (LNA), ethylene nucleic acids (ENA), such as 2'-4'-ethylene-bridged nucleic acids, and certain nucleobase modifications such as 2-amino-A, 2-thio (e.g., 2-thio-U), G-clamp modifications, may also increase binding affinity to the target.

[0156] In one embodiment, the nucleic acid molecule includes 2'-modified nucleotides, such as 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE) or 2'-ON-methylacetamido (2'-O-NMA). In one embodiment, the nucleic acid molecule includes at least one 2'-O-methyl modified nucleotide, and in some embodiments, all nucleotides of the nucleic acid molecule include a 2'-O-methyl modification.

[0157] The nucleic acid agents discussed herein include other unmodified RNA and DNA and RNA and DNA that have been modified (e.g., to improve efficacy), as well as polymers of nucleoside substitutes. Unmodified RNA refers to molecules in which the components of nucleic acid (i.e., sugar, base and phosphate moieties) are identical or substantially identical to those existing in nature (e.g., naturally occurring in the human body). Rare or rare but naturally occurring RNA has been referred to as modified RNA in the art, see, for example, Limbach et al. (Nucleic Acids Res., 1994, 22: 2183-2196). Such rare or rare RNA (often referred to as modified RNA) is usually the result of post-transcriptional modification and belongs to the term unmodified RNA used herein. As used herein, modified RNA refers to molecules in which one or more components of nucleic acid (i.e., sugar, base and phosphate moieties) are different from the components existing in nature (e.g., different from the components existing in the human body). Although they are referred to as "modified RNA", due to modification, they certainly include molecules that are not RNA in the strict sense. Nucleoside surrogates are molecules in which the ribose phosphate backbone is replaced with a non-ribose phosphate construct, which allows the bases to be presented in the correct spatial relationship such that hybridization is substantially similar to that seen when the ribose phosphate backbone is used, e.g., a non-charged mimetic of the ribose phosphate backbone.

[0158] Modifications of the nucleic acids of the invention may occur at one or more of the phosphate group, sugar group, backbone, N-terminus, C-terminus, or nucleobase.

[0159] The present invention also includes vectors into which the isolated nucleic acid of the present invention is inserted. The art is rich in suitable vectors that can be used in the present invention.

[0160] Therefore, in another aspect, the present invention relates to a vector comprising a nucleotide sequence of the present invention or a construct of the present invention. The selection of the vector will depend on the host cell to be subsequently introduced. In some embodiments, the vector of the present invention is an expression vector. Suitable host cells include a variety of prokaryotic and eukaryotic host cells. In a specific embodiment, the expression vector is selected from the group consisting of viral vectors, bacterial vectors and mammalian cell vectors. Systems based on prokaryotes and / or eukaryotic vectors can be used in the present invention to produce polynucleotides or their homologous polypeptides. Many such systems are commercially and widely available.

[0161] In some embodiments, expression of a synthetic nucleic acid encoding a protein is typically achieved by operably linking a nucleic acid encoding a protein or portion thereof to a promoter and incorporating the construct into an expression vector. The vector used is suitable for replication and optionally integration in eukaryotic cells. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters for regulating expression of the desired nucleic acid sequence.

[0162] The recombinant nucleic acid sequence construct may include more than one transcription termination region. The transcription termination region may be located downstream of the coding sequence to provide effective termination. The transcription termination region may be obtained from the same gene as the above-mentioned promoter, or may be obtained from more than one different gene.

[0163] The recombinant nucleic acid sequence construct may include more than one start codon. The start codon may be located upstream of the coding sequence. The start codon may be in frame with the coding sequence. The start codon may be associated with more than one signal required for effective translation initiation, such as, but not limited to, a ribosome binding site.

[0164] The recombinant nucleic acid sequence construct may include more than one termination or stop codon. The termination codon may be located downstream of the coding sequence. The termination codon may be in frame with the coding sequence. The termination codon may be associated with more than one signal required for effective translation termination.

[0165] The recombinant nucleic acid sequence construct may include more than one polyadenylation signal. The polyadenylation signal may include more than one signal required for efficient polyadenylation of the transcript. The polyadenylation signal may be located downstream of the coding sequence. The polyadenylation signal may be an SV40 polyadenylation signal, an LTR polyadenylation signal, a bovine growth hormone (bGH) polyadenylation signal, a human growth hormone (hGH) polyadenylation signal, or a human β-globin polyadenylation signal. The SV40 polyadenylation signal may be a polyadenylation signal from a pCEP4 plasmid (Invitrogen, San Diego, CA).

[0166] The recombinant nucleic acid sequence construct may include more than one leader sequence. The leader sequence may encode a signal peptide. The signal peptide may be an immunoglobulin (Ig) signal peptide, such as, but not limited to, an IgG signal peptide and an IgE signal peptide.

[0167] The vectors of the present invention can also be used for nucleic acid immunization using standard gene delivery protocols. Gene delivery methods are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are hereby incorporated by reference in their entirety.

[0168] The isolated nucleic acid of the present invention can be cloned into various types of vectors. For example, the nucleic acid can be cloned into vectors including but not limited to plasmids, phagemids, phage derivatives, animal viruses and cosmids. Particularly interesting vectors include expression vectors, replication vectors, probe generation vectors and sequencing vectors.

[0169] In addition, the vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described in, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. The virus that can be used as a vector includes but is not limited to retrovirus, adenovirus, adeno-associated virus, herpes virus and slow virus. In general, suitable vectors contain a replication origin, a promoter sequence, a convenient restriction endonuclease site and more than one selective marker (for example, WO 01 / 96584 with function in at least one organism; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0170] In addition, the expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described in, for example, Sambrook et al. (2012) and Ausubel et al. (1997) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and slow viruses. In general, suitable vectors contain a replication origin, a promoter sequence, a convenient restriction endonuclease site, and more than one selective marker that has a function in at least one organism. (See, for example, WO 01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193.

[0171] As an illustration, the vector into which the nucleic acid sequence is introduced may be a plasmid, which may or may not be integrated into the genome of the host cell when introduced into the cell. Exemplary, non-limiting examples of vectors into which the nucleotide sequence of the invention or the gene construct of the invention may be inserted include tet-on inducible vectors for expression in eukaryotic cells.

[0172] The vector can be obtained by conventional methods known to those skilled in the art (Sambrook et al., 2012). In a specific embodiment, the vector is a vector for transforming animal cells.

[0173] In one embodiment, the recombinant expression vector may also contain a nucleic acid molecule encoding a peptide or protein of the present invention as described elsewhere herein.

[0174] Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject's cells, either in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0175] For example, vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow long-term stable integration of transgenics and their propagation in daughter cells. Lentiviral vectors have additional advantages over vectors derived from oncogenic retroviruses such as murine leukemia viruses because they can transduce non-proliferating cells, such as hepatocytes. They also have the additional advantage of low immunogenicity. In one embodiment, the composition includes a vector derived from adeno-associated virus (AAV). Adeno-associated virus (AAV) vectors have become powerful gene delivery tools for treating various diseases. AAV vectors have many features that make them very suitable for gene therapy, including lack of pathogenicity, minimal immunogenicity, and the ability to transduce post-mitotic cells in a stable and effective manner. By selecting an appropriate combination of AAV serotypes, promoters, and delivery methods, the expression of specific genes contained in AAV vectors can be specific to more than one type of cell.

[0176] In some embodiments, the vector also includes conventional control elements that are operably linked to the transgene in a manner that allows the transgene to be transcribed, translated, and / or expressed in cells transfected with a plasmid vector or infected with a virus produced by the present invention. As used herein, "operably linked" sequences include expression control sequences adjacent to the gene of interest and expression control sequences that act in trans or remotely to control the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; effective RNA processing signals, such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and sequences that enhance secretion of the encoded product when desired. A large number of expression control sequences, including natural, constitutive, inducible, and / or tissue-specific promoters, are known in the art and can be used.

[0177] The promoter may be a promoter naturally associated with a gene or polynucleotide sequence, such as one that can be obtained by isolating a 5′ non-coding sequence upstream of a coding segment and / or exon. Such a promoter may be referred to as “endogenous”. Similarly, an enhancer may be an enhancer naturally associated with a polynucleotide sequence, located downstream or upstream of the sequence. Alternatively, certain advantages may be obtained by placing the coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not usually associated with a polynucleotide sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer that is not usually associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, as well as promoters or enhancers isolated from any other prokaryotic, viral or eukaryotic cell, and non-“naturally occurring” promoters or enhancers, i.e., different elements containing different transcriptional regulatory regions and / or mutations that alter expression. In addition to synthetically generating nucleic acid sequences of promoters and enhancers, sequences can also be generated using recombinant cloning and / or nucleic acid amplification techniques (including PCR) in conjunction with the compositions disclosed herein (U.S. Pat. No. 4,683,202, U.S. Pat. No. 5,928,906). In addition, it is believed that control sequences can also be utilized that direct the transcription and / or expression of sequences within non-nuclear organelles such as mitochondria and chloroplasts.

[0178] Of course, it is very important to utilize promoters and / or enhancers that effectively direct the expression of the DNA fragment in the cell type, organelle, and organism selected for expression. Those skilled in the art of molecular biology generally know how to use promoters, enhancers, and cell type combinations for protein expression, for example, see Sambrook et al. (2012). The promoter utilized can be constitutive, tissue-specific, inducible, and / or can be used to direct high-level expression of the introduced DNA fragment under appropriate conditions, so as to facilitate large-scale production of recombinant proteins and / or peptides. The promoter can be heterologous or endogenous.

[0179] The recombinant expression vector can also contain a selective marker gene, which helps select a transformed or transfected host cell. Suitable selective marker genes are genes encoding proteins such as G418 and hygromycin, which confer resistance to certain drugs, beta-galactosidase, chloramphenicol acetyltransferase, firefly luciferase or immunoglobulin or its part such as the Fc part of an immunoglobulin such as IgG. Selective markers can be directed to a carrier different from the nucleic acid of interest.

[0180] Additional promoter elements such as enhancers regulate the frequency of transcription initiation. Typically, they are located in the region of 30-110bp upstream of the start site, although recently many promoters have been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is usually flexible, so when elements are inverted or relatively moved to each other, promoter function is retained. In the thymidine kinase (tk) promoter, before activity begins to decline, the spacing between promoter elements can be increased to 50bp away. Depending on the promoter, each element seems to work synergistically or independently to activate transcription.

[0181] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive high-level expression of any polynucleotide sequence operably connected thereto. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, hemoglobin promoter and creatine kinase promoter. In addition, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also considered as part of the present invention. Use of an inducible promoter provides a molecular switch that can turn on the expression of a polynucleotide sequence to which it is operably linked when such expression is desired, or can turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0182] The enhancer sequence found on the vector also regulates the expression of the gene contained in the vector. Generally, the enhancer is combined with a protein factor to enhance the transcription of the gene. The enhancer can be located upstream or downstream of the gene it regulates. The enhancer can also be tissue-specific to enhance the transcription in a specific cell or tissue type. In one embodiment, the vector of the present invention comprises more than one enhancer to strengthen the transcription of the gene present in the vector.

[0183] In order to evaluate the expression of protein inhibitors, the expression vector to be introduced into the cell may also contain a selective marker gene or a reporter gene or both, so as to identify and select expressing cells from a cell population that is attempted to be transfected or infected by a viral vector. In other aspects, the selective marker may be carried on a separate DNA fragment and used for a co-transfection procedure. Both the selective marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in host cells. Useful selective markers include, for example, antibiotic resistance genes, such as neo, etc.

[0184] Reporter gene is used to identify potential transfected cells and to evaluate the function of regulatory sequences. Generally speaking, reporter gene is a gene that does not exist or is not expressed in a receptor organism or tissue, and the expression of its encoded polypeptide is shown as some properties that are easy to detect, such as enzyme activity. After DNA is introduced into the receptor cell, the expression of the reporter gene is detected at the appropriate time. Suitable reporter gene can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein gene (for example, Ui-Tei et al., 2000FEBS Letters 479:79-82). Suitable expression system is well-known, and known technology can be used to prepare or commercially obtain. Generally speaking, the construct with the minimum 5' flanking region showing the highest level reporter gene expression is identified as a promoter. This type of promoter region can be connected to a reporter gene, and is used to evaluate the ability of a reagent to regulate the promoter to drive transcription.

[0185] Methods for introducing genes into cells and expressing them are known in the art. In the context of expression vectors, vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical or biological means.

[0186] Physical methods for introducing peptides or proteins into host cells include calcium phosphate precipitation, lipofection, particle bombardment, and microinjection. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0187] Biological methods for introducing peptides or proteins of interest into host cells include the use of DNA and RNA vectors. Viral vectors, especially retroviral vectors, have become the most widely used methods for inserting genes into mammalian cells such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0188] Chemical means for introducing peptides or proteins into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0189] In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. It is contemplated that a lipid formulation is used to introduce nucleic acid into a host cell (in vitro, in vitro or in vivo). On the other hand, nucleic acid can be associated with a lipid. Nucleic acid associated with a lipid can be encapsulated in the aqueous interior of a liposome, dispersed in the lipid bilayer of the liposome, attached to the liposome via a linker associated with both a liposome and an oligonucleotide, embedded in the liposome, compounded with the liposome, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, included in lipids as a suspension, included or compounded with micelles, or otherwise associated with lipids. Lipid, lipid / DNA or lipid / expression vector related compositions are not limited to any specific structure in the solution. For example, they can exist with a double-layer structure, as micelles or using a "collapse" structure. They can also just be dispersed in the solution, and may form aggregates of uneven size or shape. Lipid is a fatty substance, which can be a naturally occurring or synthetic lipid. For example, lipids include naturally occurring fat droplets in the cytoplasm as well as the class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0190] Lipids suitable for use are available from commercial sources. For example, dimyristoylphosphatidylcholine ("DMPC") is available from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") is available from K&K Laboratories (Plainview, NY); cholesterol ("Choi") is available from Calbiochem-Behring; dimyristoylphosphatidylglycerol ("DMPG") and other lipids are available from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the only solvent because it evaporates more easily than methanol. "Liposome" is a general term covering various monolayer and multilayer lipid carriers formed by producing closed lipid bilayers or aggregates. Liposomes are characterized by having a vesicle structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. When phospholipids are suspended in an excess of aqueous solution, they form spontaneously. The lipid components rearrange themselves before forming a closed structure, and trap water and dissolved solutes between lipid bilayers (Ghosh et al., 1991 Glycobiology 5:505-10). However, compositions having structures different from normal vesicle structures in solution are also contemplated. For example, lipids may present micellar structures or exist only as non-uniform aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0191] ScFv Antibody

[0192] In one embodiment, the antibody fragment comprises an scFv fragment. In one embodiment, the ScFv antibody fragment relates to a Fab fragment without CH1 and CL regions. Therefore, in one embodiment, the scFv antibody fragment relates to a Fab fragment comprising VH and VL. In one embodiment, the scFv antibody fragment comprises a linker between VH and VL. In one embodiment, the scFv antibody fragment comprises VH, VL and CH2 and CH3 regions. In one embodiment, the scFv antibody fragment of the present invention has modified expression, stability, half-life, antigen binding, heavy chain-light chain pairing, tissue penetration or a combination thereof compared to the parent MAb.

[0193] In one embodiment, the scFv antibody fragment of the invention has at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 2.1-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6-fold, at least 2.7-fold, at least 2.8-fold, at least 2.9-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 8.5-fold, at least 9-fold, at least 9.5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, or more than 50-fold higher expression than the parent MAb.

[0194] In one embodiment, the scFv antibody fragment of the invention has at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 2.1-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6-fold, at least 2.7-fold, at least 2.8-fold, at least 2.9-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 8.5-fold, at least 9-fold, at least 9.5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, or more than 50-fold higher antigen binding than the parent MAb.

[0195] In one embodiment, the scFv antibody fragment of the invention has a half-life that is at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 2.1-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6-fold, at least 2.7-fold, at least 2.8-fold, at least 2.9-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 8.5-fold, at least 9-fold, at least 9.5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, or more than 50-fold longer than that of the parent MAb.

[0196] In one embodiment, the scFv antibody fragment of the invention has at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 2.1-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6-fold, at least 2.7-fold, at least 2.8-fold, at least 2.9-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 8.5-fold, at least 9-fold, at least 9.5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, or more than 50-fold greater stability than the parent MAb.

[0197] In one embodiment, the scFv antibody fragment of the invention has at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 2.1-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6-fold, at least 2.7-fold, at least 2.8-fold, at least 2.9-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 8.5-fold, at least 9-fold, at least 9.5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, or more than 50-fold greater tissue penetration than the parent MAb.

[0198] In one embodiment, the scFv antibody fragment of the invention has at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 2.1-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6-fold, at least 2.7-fold, at least 2.8-fold, at least 2.9-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 8.5-fold, at least 9-fold, at least 9.5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, or more than 50-fold greater heavy chain-light chain pairing than that of the parent MAb.

[0199] Delivery vehicle

[0200] In one embodiment, the invention provides a composition comprising a delivery vehicle comprising a sialic acid binding receptor antibody, a fragment thereof, or a nucleic acid molecule encoding the same, as described herein.

[0201] Exemplary delivery vehicles include, but are not limited to, microspheres, microparticles, nanoparticles, polymerosomes, liposomes, and micelles. For example, in some embodiments, the delivery vehicle is loaded with an anti-Siglec-9 polypeptide, a fragment thereof, or a nucleic acid molecule encoding it. In some embodiments, the delivery vehicle provides controlled release, delayed release, or sustained release of the cargo it carries. In some embodiments, the delivery vehicle includes a targeting portion that targets the delivery vehicle to a treatment site.

[0202] Pharmaceutical composition

[0203] The present invention also provides pharmaceutical compositions comprising more than one composition described herein. The preparations can be mixed with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier materials suitable for application to the treatment site. The pharmaceutical compositions can be sterilized and, if necessary, mixed with adjuvants such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure buffers, colorants and / or aromatic substances. They can also be combined with other active agents (e.g., other analgesics) as needed.

[0204] Administration of the compositions of the invention may be carried out, for example, by parenteral, intravenous, subcutaneous, intramuscular or intraperitoneal injection, or by infusion or by any other acceptable systemic method.

[0205] As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients; surfactants; dispersants; inert diluents; granulating agents and disintegrants; binders; lubricants; colorants; preservatives; physiologically degradable compositions, such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersants or wetting agents; emulsifiers, demulcents; buffers; salts; thickeners; fillers; emulsifiers; antioxidants; antibiotics; antifungal agents; stabilizers; and pharmaceutically acceptable polymers or hydrophobic materials. Other "additional ingredients" that may be included in the pharmaceutical compositions of the present invention are known in the art and are described, for example, in Genaro, ed. (1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA), which is incorporated herein by reference.

[0206] The composition of the present invention may contain about 0.005% to 2.0% preservatives, based on the total weight of the composition. Preservatives are used to prevent spoilage in the event of exposure to pollutants in the environment. Examples of preservatives useful according to the present invention include, but are not limited to, preservatives selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidazole lignocerae, and combinations thereof.

[0207] In one embodiment, the composition includes an antioxidant and a chelating agent that inhibits the degradation of one or more components of the composition. Exemplary antioxidants for some compounds are BHT, BHA, alpha-tocopherol, and ascorbic acid. Exemplary chelating agents include ethylenediaminetetraacetic acid salts (e.g., disodium edetate) and citric acid. Chelating agents can be used to chelate metal ions in the composition that may be harmful to the shelf life of the formulation. Although BHT and disodium edetate can be antioxidants and chelating agents for some compounds, respectively, other suitable and equivalent antioxidants and chelating agents can therefore be substituted as known to those skilled in the art.

[0208] Liquid suspensions can be prepared using conventional methods so that the compounds of the present invention or other compositions are suspended in aqueous or oily solvents. Aqueous solvents include, for example, water and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethanol, vegetable oils such as peanut oil, olive oil, sesame oil or coconut oil, fractionated vegetable oils and mineral oils such as liquid paraffin. Liquid suspensions may also include more than one additional component, including but not limited to suspending agents, dispersants or wetting agents, emulsifiers, mitigating agents, preservatives, buffers, salts, flavoring agents, coloring agents and sweeteners. Oily suspensions may also include thickeners. Known suspending agents include but are not limited to sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinyl pyrrolidone, tragacanth gum, gum arabic and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose. Known dispersants or wetting agents include, but are not limited to, naturally occurring phospholipids, such as lecithin, condensation products of alkylene oxides and fatty acids, condensation products with long-chain fatty alcohols, condensation products with partial esters derived from fatty acids and hexitol, or condensation products with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitol monooleate, respectively). Known emulsifiers include, but are not limited to lecithin and gum arabic. Known preservatives include, but are not limited to methyl, ethyl or n-propyl p-hydroxybenzoate, ascorbic acid and sorbic acid.

[0209] For oral use, tablets, dragees, liquids, drops, suppositories or capsules, caplets and gelcaps are particularly suitable. Other formulations suitable for oral administration include, but are not limited to, powder or granular formulations, aqueous or oily suspensions, aqueous or oily solutions, pastes, gels, toothpastes, mouthwashes, coatings, oral rinses, chewing gums, varnishes, sealants, oral and dental "dissolving strips" or emulsions. Compositions for oral use may be prepared according to any method known in the art, and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutical excipients suitable for making tablets. Such excipients include, for example, inert diluents such as lactose; granulating agents and disintegrants such as corn starch; binders such as starch; and lubricants such as magnesium stearate.

[0210] Tablets may be non-coated, or they may be coated using known methods to achieve delayed disintegration in the gastrointestinal tract of a subject, thereby providing sustained release and absorption of the active ingredient. For example, materials such as glyceryl monostearate or glyceryl distearate may be used to coat tablets. Further, for example, tablets may be coated using the methods described in U.S. Patent Nos. 4,256,108, 4,160,452, and 4,265,874 to form osmotic controlled release tablets. Tablets may also include sweeteners, flavoring agents, coloring agents, preservatives, or some combination of these to provide pharmaceutically elegant and palatable preparations.

[0211] Hard capsules containing the active ingredient can be made using physiologically degradable compositions such as gelatin. Such hard capsules contain the active ingredient and may also contain other ingredients including, for example, inert solid diluents such as calcium carbonate, calcium phosphate or kaolin.

[0212] Soft gelatin capsules containing the active ingredient can be made using physiologically degradable compositions such as gelatin. Such soft capsules contain the active ingredient, which can be mixed with water or an oily medium such as peanut oil, liquid paraffin or olive oil.

[0213] For oral administration, the compositions of the present invention may be in the form of tablets or capsules prepared by conventional means using pharmaceutically acceptable excipients such as binders; fillers; lubricants; disintegrants; or wetting agents. If desired, tablets may be coated using suitable methods and coating materials such as OPADRY available from Colorcon, West Point, Pa. TM Film coating systems (eg OPADRY TM OY type, OYC type, organic enteric-coated OY-P type, aqueous enteric-coated OY-A type, OY-PM type and OPADRY TM White, 32K18400) for coating.

[0214] Liquid preparations for oral administration may be in the form of solutions, syrups or suspensions. Liquid preparations can be prepared by conventional means using pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methylcellulose or hydrogenated edible fats); emulsifiers (e.g., lecithin or gum arabic); non-aqueous solvents (e.g., almond oil, oily esters or ethanol); and preservatives (e.g., methyl or propyl parabens or sorbic acid). Liquid preparations of the pharmaceutical compositions of the present invention suitable for oral administration can be prepared, packaged and sold in liquid form or in the form of dry products intended to be reconstituted with water or another suitable solvent before use.

[0215] Tablets containing active ingredients can be prepared, for example, by compressing or forming the active ingredient (optionally with one or more additional ingredients). Compressed tablets can be prepared by compressing a free-flowing form of the active ingredient, such as a powder or granular formulation (optionally mixed with one or more binders, lubricants, excipients, surfactants and dispersants) in a suitable device. Formed tablets can be prepared by forming a mixture of an active ingredient, a pharmaceutically acceptable carrier and a liquid at least sufficient to wet the mixture in a suitable device. Pharmaceutically acceptable excipients for making tablets include, but are not limited to, inert diluents, granulators and disintegrants, binders and lubricants. Known dispersants include, but are not limited to, potato starch and sodium starch glycolate. Known surfactants include, but are not limited to, sodium lauryl sulfate. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate and sodium phosphate. Known granulators and disintegrants include, but are not limited to, corn starch and alginic acid. Known binders include, but are not limited to, gelatin, gum arabic, pregelatinized corn starch, polyvinyl pyrrolidone and hydroxypropyl methylcellulose. Known lubricants include, but are not limited to, magnesium stearate, stearic acid, silicon dioxide, and talc.

[0216] The preparation of the pharmaceutical composition suitable for parenteral administration comprises an active ingredient combined with a pharmaceutically acceptable carrier (e.g., sterile water or sterile isotonic saline). Such preparations can be prepared, packaged or sold in a form suitable for bolus administration or continuous administration. Injectable preparations can be prepared, packaged or sold in a unit dose form such as an ampoule or a multi-dose container containing a preservative. Preparations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable preparations. Such preparations may also include more than one additional ingredient, including but not limited to suspending agents, stabilizers or dispersants. In one embodiment of a preparation for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granule) form for reconstitution with a suitable solvent (e.g., sterile pyrogen-free water) before parenteral administration of the reconstructed composition.

[0217] Pharmaceutical compositions can be prepared, packaged or sold in the form of sterile injectable aqueous or oily suspensions or solutions. The suspension or solution can be prepared according to known techniques and, in addition to the active ingredient, can also include additional ingredients such as dispersants, wetting agents or suspending agents as described herein. Such sterile injectable preparations can be prepared using nontoxic parenteral acceptable diluents or solvents (e.g., water or 1,3-butylene glycol). Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic monoglycerides or diglycerides. Other useful parenteral formulations include preparations including active ingredients including microcrystalline forms, liposome preparations, or as components of biodegradable polymer systems. Compositions for sustained release or implantation can include pharmaceutically acceptable polymers or hydrophobic materials, such as emulsions, ion exchange resins, poorly soluble polymers, or poorly soluble salts.

[0218] Excipients and other ingredients of the composition

[0219] Compositions can also include a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient can be a functional molecule, such as a solvent, adjuvant, carrier or diluent. A pharmaceutically acceptable excipient can be a transfection facilitator, which can include a surfactant, such as an immunostimulating complex (ISCOMS), Freund's incomplete adjuvant, LPS analogs (including monophosphoryl lipid A), muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations or nanoparticles, or other known transfection facilitators.

[0220] The transfection facilitator is a polyanion, a polycation (including poly-L-glutamic acid (LGS)), or a lipid. The transfection facilitator is poly-L-glutamic acid, and the concentration of the poly-L-glutamic acid in the composition may be less than 6 mg / ml. The transfection facilitator may also include a surfactant, such as an immunostimulating complex (ISCOMS), Freund's incomplete adjuvant, an LPS analog (including monophosphoryl lipid A), a muramyl peptide, a quinone analog, and a vesicle such as squalene and squalene, and hyaluronic acid, which may also be administered in combination with the composition. The composition may further include a transfection facilitating agent, such as a lipid, a liposome (including a phosphatidylcholine liposome or other liposomes known in the art, such as a DNA-liposome mixture (see, e.g., WO9324640), a calcium ion, a viral protein, a polyanion, a polycation or a nanoparticle, or other known transfection facilitating agents. The transfection facilitating agent is a polyanion, a polycation (including poly-L-glutamic acid (LGS)), or a lipid. The concentration of the transfection agent in the composition is less than 4 mg / ml, less than 2 mg / ml, less than 1 mg / ml, less than 0.750 mg / ml, less than 0.500 mg / ml, less than 0.250 mg / ml, less than 0.100 mg / ml, less than 0.050 mg / ml or less than 0.010 mg / ml.

[0221] Pharmaceutically acceptable excipients may be adjuvants other than the checkpoint inhibitor antibodies of the present invention. Additional adjuvants may be other genes expressed in alternative plasmids or delivered as proteins in combination with the above-mentioned plasmids in the composition. Adjuvants may be selected from the group consisting of: alpha-interferon (IFN-α), beta-interferon (IFN-β), gamma-interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), skin T cell attraction chemokine (CTACK), epithelial thymus expression chemokine (TECK), mucosa-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86, including IL-15 with deleted signal sequences and optionally including a signal peptide from IgE. The adjuvant can be IL-12, IL-15, IL-28, CTACK, TECK, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, PD-1, IL-10, IL-12, IL-18, or a combination thereof.

[0222] In addition to the antibodies of the present invention, other genes that can be used as adjuvants include genes encoding MCP-1, MIP-1a, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-C SF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, IL-22, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-1, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANKLIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 and their functional fragments.

[0223] The composition may also include a genetic enhancer as described in US Serial No. 021,579, filed April 1, 1994, which is incorporated by reference in its entirety.

[0224] The composition may contain DNA in an amount of about 1 nanogram to 100 milligrams; about 1 microgram to about 10 milligrams; or preferably about 0.1 microgram to about 10 milligrams; or more preferably about 1 milligram to about 2 milligrams. In some preferred embodiments, the composition according to the present invention contains DNA in an amount of about 5 nanograms to about 1000 micrograms. In some preferred embodiments, the composition may contain DNA in an amount of about 10 nanograms to about 800 micrograms. In some preferred embodiments, the composition may contain DNA in an amount of about 0.1 to about 500 micrograms. In some preferred embodiments, the composition may contain DNA in an amount of about 1 to about 350 micrograms. In some preferred embodiments, the composition may contain about 25 to about 250 micrograms, about 100 to about 200 micrograms, about 1 nanogram to 100 milligrams; about 1 microgram to about 10 milligrams; about 0.1 microgram to about 10 milligrams; about 1 milligram to about 2 milligrams, about 5 nanograms to about 1000 micrograms, about 10 nanograms to about 800 micrograms, about 0.1 to about 500 micrograms, about 1 to about 350 micrograms, about 25 to about 250 micrograms, about 100 to about 200 micrograms of DNA.

[0225] The composition can be formulated according to the mode of administration to be used. The injectable pharmaceutical composition can be sterile, pyrogen-free and particle-free. Isotonic preparations or solutions can be used. Isotonic additives can include sodium chloride, dextrose, mannitol, sorbitol and lactose. The composition can include a vasoconstrictor. The isotonic solution can include phosphate buffered saline. The composition can also include stabilizers, including gelatin and albumin. Stabilizers can stabilize the formulation for a long time at room temperature or ambient temperature, including LGS or polycations or polyanions.

[0226] Application method

[0227] The present invention provides a method for increasing the function or activity of natural killer (NK) cells. This can be measured, for example, in a standard NK cell or T cell-based cytotoxicity assay, in which the ability of a therapeutic compound to stimulate Siglec-positive lymphocytes to kill sialic acid ligand-positive cells is measured. In one embodiment, the antibody preparation causes at least a 10% increase in the cytotoxicity of Siglec-restricted lymphocytes, optionally at least a 40% or 50% increase in lymphocyte cytotoxicity, or optionally at least a 70% increase in NK cell cytotoxicity, and reference is made to the cytotoxicity assay. In one embodiment, the antibody preparation causes at least a 10% increase in cytokine release from Siglec-restricted lymphocytes, optionally at least a 40% or 50% increase in cytokine release, or optionally at least a 70% increase in cytokine release, and reference is made to the cytotoxicity assay. In one embodiment, the antibody preparation results in at least a 10% increase in cell surface expression of cytotoxic markers (e.g., CD107 and / or CD137) of Siglec-restricted lymphocytes, optionally results in at least a 40% or 50% increase in cell surface expression of cytotoxic markers (e.g., CD107 and / or CD137), or optionally results in at least a 70% increase in cell surface expression of cytotoxic markers (e.g., CD107 and / or CD137).

[0228] The present invention also relates to a method of increasing an immune response in a subject. Increasing the immune response can be used to treat and / or prevent a disease in a subject. The method may include administering a vaccine disclosed herein to a subject. The subject administered the vaccine may have an increased or boosted immune response compared to a subject administered only the antigen. In some embodiments, the immune response may increase by about 0.5 times to about 15 times, about 0.5 times to about 10 times, or about 0.5 times to about 8 times. Alternatively, the immune response in a subject administered the vaccine may be increased by at least about 0.5-fold, at least about 1.0-fold, at least about 1.5-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 5.5-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 10.5-fold, at least about 11.0-fold, at least about 11.5-fold, at least about 12.0-fold, at least about 12.5-fold, at least about 13.0-fold, at least about 13.5-fold, at least about 14.0-fold, at least about 14.5-fold, or at least about 15.0-fold.

[0229] In still other alternative embodiments, the immune response in a subject administered the vaccine may be increased by about 50% to about 1500%, about 50% to about 1000%, or about 50% to about 800%. In other embodiments, the immune response in a subject administered the vaccine may be increased by at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, at least about 500%, at least about 550%, at least about 600%, at least about 650%, at least about 700%, at least about 750%, at least about 800%, at least about 850%, at least about 900%, at least about 950%, at least about 1000%, at least about 1050%, at least about 1100%, at least about 1150%, at least about 1200%, at least about 1250%, at least about 1300%, at least about 1350%, at least about 1450%, or at least about 1500%.

[0230] The vaccine dose may be between 1 μg and 10 mg of active ingredient / kg body weight / time, or may be between 20 μg and 10 mg of active ingredient / kg body weight / time. The vaccine may be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 days. The number of vaccine doses for effective treatment may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0231] Combination vaccines

[0232] In one embodiment, the present invention relates to the combined administration of the Siglec-9 antibody of the present invention or the nucleic acid molecule encoding it and one or more other therapeutic agents. In some embodiments, one or more other therapeutic agents may be used to induce or increase an immune response. Therefore, in some embodiments, the present invention relates to an immunogenic composition, such as a vaccine, which comprises a Siglec-9 antibody of the present invention or a nucleic acid molecule encoding it and an antigenic protein, a fragment thereof or a variant thereof, a nucleic acid molecule encoding it, or a combination thereof. Therefore, in some embodiments, the present invention relates to an immunogenic composition, such as a vaccine, which comprises a Siglec-9 antibody of the present invention or a nucleic acid molecule encoding it and a PD-(L)1 axis inhibitor, a fragment thereof or a variant thereof, a nucleic acid molecule encoding it, or a combination thereof.

[0233] The combination vaccine can significantly induce an immune response in a subject to which the vaccine is administered, thereby protecting against or treating a disease or disorder.

[0234] The immunogenic composition may be a DNA vaccine, a peptide vaccine, or a combined DNA and peptide vaccine. A DNA vaccine may include a nucleic acid sequence encoding an antigen. The nucleic acid sequence may be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The nucleic acid sequence may also include an additional sequence encoding a linker, a leader or a tag sequence that is linked to the antigen by a peptide bond. A peptide vaccine may include an antigenic peptide, an antigenic protein, a variant thereof, a fragment thereof, or a combination thereof. A combined DNA and peptide vaccine may include the above-mentioned nucleic acid sequence encoding an antigen and an antigenic peptide or protein.

[0235] The vaccine may induce a humoral immune response in a subject to which the vaccine is administered. The induced humoral immune response may be specific to the antigen. The induced humoral immune response may be reactive with the antigen. The humoral immune response may be induced in a subject to which the vaccine is administered by about 1.5 to about 16 times, about 2 to about 12 times, or about 3 to about 10 times. The humoral immune response can be induced in a subject administered the vaccine by at least about 1.5 times, at least about 2.0 times, at least about 2.5 times, at least about 3.0 times, at least about 3.5 times, at least about 4.0 times, at least about 4.5 times, at least about 5.0 times, at least about 5.5 times, at least about 6.0 times, at least about 6.5 times, at least about 7.0 times, at least about 7.5 times, at least about 8.0 times, at least about 8.5 times, at least about 9.0 times, at least about 9.5 times, at least about 10.0 times, at least about 10.5 times, at least about 11.0 times, at least about 11.5 times, at least about 12.0 times, at least about 12.5 times, at least about 13.0 times, at least about 13.5 times, at least about 14.0 times, at least about 14.5 times, at least about 15.0 times, at least about 15.5 times, or at least about 16.0 times.

[0236] The humoral immune response induced by the vaccine may include an increase in the level of neutralizing antibodies associated with the subject to which the vaccine is administered compared to a subject not administered the vaccine. The neutralizing antibodies may be specific for the antigen. The neutralizing antibodies may be reactive with the antigen. The neutralizing antibodies may provide protection against a disease or condition and / or treatment of a disease or condition in a subject to which the vaccine is administered.

[0237] The humoral immune response induced by the vaccine may include an increase in the level of IgG antibodies associated with the subject to which the vaccine is administered compared to a subject to which the vaccine is not administered. These IgG antibodies may be specific to tumor antigens. These IgG antibodies may be reactive with antigens. The level of IgG antibodies associated with the subject to which the vaccine is administered may increase by about 1.5 times to about 16 times, about 2 times to about 12 times, or about 3 times to about 10 times compared to a subject to which the vaccine is not administered. The level of IgG antibodies associated with subjects administered the vaccine may be increased by at least about 1.5 times, at least about 2.0 times, at least about 2.5 times, at least about 3.0 times, at least about 3.5 times, at least about 4.0 times, at least about 4.5 times, at least about 5.0 times, at least about 5.5 times, at least about 6.0 times, at least about 6.5 times, at least about 7.0 times, at least about 7.5 times, at least about 8.0 times, at least about 8.5 times, at least about 9.0 times, at least about 9.5 times, at least about 10.0 times, at least about 10.5 times, at least about 11.0 times, at least about 11.5 times, at least about 12.0 times, at least about 12.5 times, at least about 13.0 times, at least about 13.5 times, at least about 14.0 times, at least about 14.5 times, at least about 15.0 times, at least about 15.5 times, or at least about 16.0 times compared to subjects not administered the vaccine.

[0238] The vaccine may induce a cellular immune response in a subject to which the vaccine is administered. The induced cellular immune response may be specific to the antigen. The induced cellular immune response may be reactive to the antigen. The induced cellular immune response may include eliciting CD8 + T cell response. Triggered CD8 + T cell responses can be reactive to antigens. + The T cell response may be multifunctional. The induced cellular immune response may include eliciting CD8 + T cell responses, including CD8 + T cells produce interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-2 (IL-2), or a combination of IFN-γ and TNF-α.

[0239] The induced cellular immune response may include an increase in CD8 associated with the vaccine-administered subjects compared to subjects not administered the vaccine. + T cell responses increased. CD8 + The T cell response can be increased by about 2-fold to about 30-fold, about 3-fold to about 25-fold, or about 4-fold to about 20-fold. The CD8 +The T cell response can be increased by at least about 1.5 fold, at least about 2.0 fold, at least about 3.0 fold, at least about 4.0 fold, at least about 5.0 fold, at least about 6.0 fold, at least about 6.5 fold, at least about 7.0 fold, at least about 7.5 fold, at least about 8.0 fold, at least about 8.5 fold, at least about 9.0 fold, at least about 9.5 fold, at least about 10.0 fold, at least about 10.5 fold, at least about 11.0 fold, at least about 11.5 fold, at least about 12.0 fold, at least about 12.5 fold, at least about 13.0 times, at least about 13.5 times, at least about 14.0 times, at least about 14.5 times, at least about 15.0 times, at least about 16.0 times, at least about 17.0 times, at least about 18.0 times, at least about 19.0 times, at least about 20.0 times, at least about 21.0 times, at least about 22.0 times, at least about 23.0 times, at least about 24.0 times, at least about 25.0 times, at least about 26.0 times, at least about 27.0 times, at least about 28.0 times, at least about 29.0 times, or at least about 30.0 times.

[0240] The induced cellular immune response may include CD3 producing IFN-γ + CD8 + The frequency of T cells increased in the subjects who received the vaccine compared to those who did not receive the vaccine. + CD8 + IFN-γ + The frequency of T cells may be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold.

[0241] The induced cellular immune response may include CD3+ production of TNF-α + CD8 + The frequency of T cells increased in the subjects who received the vaccine compared to those who did not receive the vaccine. + CD8 + TNF-α + The frequency of T cells may be increased at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, or 14-fold.

[0242] The induced cellular immune response may include CD3 + CD8 + The frequency of T cells increased in the subjects who received the vaccine compared to those who did not receive the vaccine. + CD8 + IL-2 +The frequency of T cells may be increased by at least about 0.5-fold, 1.0-fold, 1.5-fold, 2.0-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, or 5.0-fold.

[0243] The induced cellular immune response may include CD3+ producing both IFN-γ and TNF-α + CD8 + The frequency of T cells increased in the subjects who received the vaccine compared to those who did not receive the vaccine. + CD8 + IFN-γ + TNF-α + The frequency of T cells can be increased by at least about 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 170-fold, or 180-fold.

[0244] Vaccine-induced cellular immune responses may include eliciting CD4 + T cell response. Initiation of CD4 + T cell responses can be reactive to antigens. + The T cell response may be multifunctional. The induced cellular immune response may include eliciting CD4 + T cell responses, including CD4 + T cells produce IFN-γ, TNF-α, IL-2, or a combination of IFN-γ and TNF-α.

[0245] The induced cellular immune response may include CD3 producing IFN-γ + CD4 + The frequency of T cells increased in the subjects who received the vaccine compared to those who did not receive the vaccine. + CD4 + IFN-γ + The frequency of T cells may be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold.

[0246] The induced cellular immune response may include CD3+ production of TNF-α + CD4 + The frequency of T cells increased in the subjects who received the vaccine compared to those who did not receive the vaccine. + CD4 + TNF-α +The frequency of T cells may be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, or 22-fold.

[0247] The induced cellular immune response may include CD3 + CD4 + The frequency of T cells increased in the subjects who received the vaccine compared to those who did not receive the vaccine. + CD4 + IL-2 + The frequency of T cells can be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold, 39-fold, 40-fold, 45-fold, 50-fold, 55-fold, or 60-fold.

[0248] The induced cellular immune response may include CD3+ producing both IFN-γ and TNF-α + CD4 + The frequency of T cells increased in the subjects who received the vaccine compared to those who did not receive the vaccine. + CD4 + IFN-γ + TNF-α + The frequency of can be increased by at least about 2 times, 2.5 times, 3.0 times, 3.5 times, 4.0 times, 4.5 times, 5.0 times, 5.5 times, 6.0 times, 6.5 times, 7.0 times, 7.5 times, 8.0 times, 8.5 times, 9.0 times, 9.5 times, 10.0 times, 10.5 times, 11.0 times, 11.5 times, 12.0 times, 12.5 times, 13.0 times, 13.5 times, 14. 4.0 times, 14.5 times, 15.0 times, 15.5 times, 16.0 times, 16.5 times, 17.0 times, 17.5 times, 18.0 times, 18.5 times, 19.0 times, 19.5 times, 20.0 times, 21 times, 22 times, 23 times, 24 times, 25 times, 26 times, 27 times, 28 times, 29 times, 30 times, 31 times, 32 times, 33 times, 34 times or 35 times.

[0249] The vaccines of the present invention may have the characteristics desired for an effective vaccine, such as safety so that the vaccine itself does not cause disease or death; protection against disease caused by exposure to live pathogens such as viruses or bacteria; induction of neutralizing antibodies to protect cells; induction of protective T cells against intracellular pathogens; and providing ease of administration, few side effects, biological stability, and low cost per dose.

[0250] The immune response can be further induced when the vaccine is injected into different tissues such as muscle or skin.

[0251] Tumor Antigens

[0252] As described above, in some embodiments, the combination vaccine of the present invention comprises a combination of an anti-Siglec-9 antibody or a nucleic acid molecule encoding the same and a tumor antigen, a fragment thereof, a variant thereof, a nucleic acid molecule encoding the same, or a combination thereof.

[0253] Therefore, in some embodiments, the present invention includes a composition for enhancing an immune response to an antigen in a subject in need thereof, comprising a sialic acid binding receptor antibody of the present invention in combination with a synthetic antigen capable of generating an immune response in the subject, or a biologically functional fragment or variant thereof, or a nucleic acid molecule encoding the same.

[0254] In one embodiment, the antigen is a tumor-associated surface antigen. Illustrative examples of tumor-associated surface antigens are CD10, CD19, CD20, CD22, CD33, Fms-like tyrosine kinase 3 (FLT-3, CD135), chondroitin sulfate proteoglycan 4 (CSPG4, melanoma-associated chondroitin sulfate proteoglycan), epidermal growth factor receptor (EGFR), Her2neu, Her3, IGFR, CD133, IL3R, fibroblast activation protein (FAP), CDCP1, Derlin1, Tenascin, Frizzled protein 1-10, vascular antigen VEGFR2 (KDR / FLK1), VEGFR3 (FLT4, CD309), PDGFR-α (CD140a), PDGFR-β (CD140b), Endoglin, CLEC14, Tem1-8 and Tie2. Further examples may include A33, CAMPATH-1 (CDw52), carcinoembryonic antigen (CEA), carbonic anhydrase IX (MN / CA IX), CD21, CD25, CD30, CD34, CD37, CD44v6, CD45, CD133, de2-7EGFR, EGFRvIII, EpCAM, Ep-CAM, folate-binding protein, G250, Fms-like tyrosine kinase 3 (FLT-3, CD135), c-Kit (CD117), CSF1R (CD115), HLA-DR, IGFR, IL-2 receptor, IL3R, MCSP (melanoma-associated cell surface chondroitin sulfate proteoglycan), Muc-1, prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), prostate-specific antigen (PSA) and TAG-72. Examples of antigens expressed on the extracellular matrix of tumors are tenascin and fibroblast activation protein (FAP).

[0255] In the context of the present invention, a "tumor antigen" or "hyperproliferative disorder antigen" or "antigen associated with a hyperproliferative disorder" refers to an antigen that is common to a particular hyperproliferative disorder, such as cancer. The antigens discussed herein are included by way of example only. This list is not intended to be exclusive, and other examples will be apparent to those skilled in the art.

[0256] Tumor antigens are proteins produced by tumor cells that elicit an immune response, particularly a T cell mediated immune response.The choice of antigen binding portion of the invention will depend on the particular type of cancer to be treated. Tumor antigens are well known in the art and include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostate cancer-associated protein (prostein), PSMA, Her2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin.

[0257] In one embodiment, tumor antigens include more than one antigenic cancer epitope associated with a malignant tumor. Malignant tumors express many proteins that can be used as target antigens for immune attack. These molecules include but are not limited to tissue-specific antigens, such as MART-1, tyrosinase and GP 100 in melanoma and prostate acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the transformation-related molecule group, such as oncogene HER-2 / Neu / ErbB-2. Another group of target antigens is onco-fetal antigen (onco-fetal antigen), such as carcinoembryonic antigen (CEA). In B cell lymphoma, tumor-specific idiotype immunoglobulin constitutes a true tumor-specific immunoglobulin antigen, which is unique to a single tumor. B cell differentiation antigens such as CD19, CD20 and CD37 are other candidates for target antigens in B cell lymphoma. Some of these antigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targets for passive immunotherapy using monoclonal antibodies, but the success rate is limited.

[0258] The types of tumor antigens mentioned in the present invention may also be tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs). TSAs are specific to tumor cells and do not appear on other cells in the body. TAA-related antigens are not specific to tumor cells, but are also expressed on normal cells under conditions where a state of immune tolerance to the antigen cannot be induced. The expression of antigens on tumors can occur under conditions that enable the immune system to respond to the antigens. TAAs may be antigens expressed on normal cells during fetal development, when the immune system is immature and unable to respond, or they may be antigens that are usually present at very low levels on normal cells but expressed at much higher levels on tumor cells.

[0259] Non-limiting examples of TSA or TAA antigens include the following: differentiation antigens, such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multi-lineage antigens, such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens, such as CEA; overexpressed oncogenes and mutated tumor suppressor genes, such as p53, Ras, HER-2 / neu; unique tumor antigens caused by chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA19-9, CA72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP and TPS.

[0260] PD-(L)1 axis inhibitors

[0261] In some embodiments, the present invention provides a combination of an anti-Siglec-9 antibody of the present invention and a PD-(L)1 axis inhibitor. In various embodiments, the composition comprises an inhibitor of more than one gene or protein in the PD-(L)1 axis. In various embodiments, the present invention includes compositions and methods for reducing the level or activity of more than one gene or protein in the PD-(L)1 axis.

[0262] Based on the disclosure provided herein, those skilled in the art will understand that the reduction in the level or activity of one or more genes or proteins in the PD-(L)1 axis encompasses a reduction in the expression of a biomarker, including transcription, translation, or both. Once a skilled person in the art has mastered the teachings of the present invention, it will also be understood that the reduction in the level or activity of one or more genes or proteins in the PD-(L)1 axis includes a reduction in the amount of a polypeptide, a reduction in the amount of mRNA, a reduction in transcription, a reduction in translation, or a combination thereof; and it also includes reducing any activity of one or more genes or proteins in the PD-(L)1 axis.

[0263] Exemplary inhibitors of the PD-(L)1 axis include, but are not limited to, small interfering RNA (siRNA), microRNA, antisense nucleic acid, ribozyme, expression vector encoding a transdominant negative mutant, antibody, antibody fragment, fusion protein, aptamer, peptide and small molecule.

[0264] Based on the disclosure provided herein, one skilled in the art will appreciate that one method of reducing the mRNA and / or protein levels of more than one PD-(L)1 axis protein in a cell is to reduce or inhibit the expression of a nucleic acid encoding the PD-(L)1 axis protein. Therefore, molecules or compounds that inhibit or reduce gene expression (e.g., such as siRNA, antisense molecules, or ribozymes) can be used to reduce the protein level of the PD-(L)1 axis protein in a cell. However, the present invention should not be limited to these examples.

[0265] In one embodiment, RNAi is used to reduce the level or activity of PD-(L)1 axis protein. RNA interference (RNAi) is a phenomenon in which the introduction of double-stranded RNA (dsRNA) into a diverse range of organisms and cell types results in the degradation of complementary mRNA. In cells, long dsRNAs are cut into short 21-25 nucleotide small interfering RNAs or siRNAs by a ribonuclease called Dicer. The siRNA is then assembled with protein components into an RNA-induced silencing complex (RISC), unwinding in the process. The activated RISC then binds to the complementary transcript through base pairing interactions between the siRNA antisense strand and the mRNA. The bound mRNA is cut, and sequence-specific degradation of the mRNA leads to gene silencing. Chemical modification of siRNA can facilitate intravenous systemic delivery. Optimizing siRNA involves consideration of the overall G / C content, the C / T content at the end, the Tm, and the nucleotide content of the 3' overhang. Therefore, the present invention also includes a method for reducing the level of more than one PD-(L)1 axis protein using RNAi technology.

[0266] In some embodiments, the present invention includes an isolated nucleic acid encoding an inhibitor, such as a protein, antibody, siRNA, or antisense molecule, which is operably linked to a nucleic acid comprising a promoter / regulatory sequence such that the nucleic acid is preferably capable of directing the expression of the inhibitor encoded by the nucleic acid. Thus, the present invention encompasses expression vectors and methods for introducing exogenous DNA into cells and concomitantly expressing the exogenous DNA in cells.

[0267] In order to evaluate the expression of inhibitor, the expression vector to be introduced into the cell can also contain a selective marker gene or a reporter gene or both, so that from attempting to identify and select expressing cells by viral vector transfection or infected cell mass. In other embodiments, the selective marker can be carried on an independent DNA fragment and used for cotransfection procedures. Both sides of the selective marker and the reporter gene can have suitable regulatory sequences so that it can be expressed in host cells. Useful selective markers are known in the art and include, for example, antibiotic resistance genes, such as neomycin.

[0268] When the inhibitor of the present invention is a small molecule, standard methods known to those skilled in the art can be used to obtain small molecule antagonists. Such methods include chemical organic synthesis or biological means. Biological means include purification from biological sources, recombinant synthesis and in vitro translation systems using methods well known in the art.

[0269] Combinatorial libraries of molecularly diverse chemical compounds potentially useful for treating various diseases and conditions are well known in the art, as are methods for preparing the libraries. The methods can use various techniques well known to those skilled in the art, including solid phase synthesis, solution methods, parallel synthesis of single compounds, synthesis of chemical mixtures, rigid core structures, flexible linear sequences, deconvolution strategies, labeling techniques, and generation of unbiased molecular landscapes for lead discovery relative to biased structures for lead development.

[0270] In the general method of small library synthesis, the activated core molecule is condensed with many building blocks to obtain a combinatorial library of covalently linked core-building blocks. The shape and rigidity of the core determine the orientation of the building blocks in shape space. The library can be biased towards the biological structure characterized by targeting ("focused library") by changing the core, connection or building blocks, or a flexible core can be used to synthesize the library with less structural bias.

[0271] In another aspect of the invention, one or more proteins in the PD-(L)1 axis can be inhibited by inactivating and / or sequestering the protein(s). Thus, the effect of inhibiting one or more proteins in the PD-(L)1 axis can be achieved by using a trans-dominant negative mutant.

[0272] In one embodiment, antibodies specific for more than one protein in the PD-(L)1 axis can be used. As will be appreciated by those skilled in the art, any antibody that can recognize and bind to an antigen of interest can be used in the present invention. Methods for preparing and using antibodies are well known in the art. For example, polyclonal antibodies useful in the present invention are produced by immunizing rabbits according to standard immunological techniques well known in the art. Such techniques include immunizing animals with chimeric proteins comprising a portion of another protein, such as a maltose binding protein or glutathione (GSH) tag polypeptide portion, and / or a portion that makes the antigen protein of interest immunogenic (e.g., an antigen of interest conjugated to keyhole limpet hemocyanin (KLH)) and a portion comprising amino acid residues of the corresponding antigen protein. The chimeric protein is produced by cloning a suitable nucleic acid encoding the marker protein into a plasmid vector suitable for this purpose (e.g., but not limited to, pMAL-2 or pCMX).

[0273] Exemplary anti-PD-(L)1 axis antibodies include, but are not limited to, nivolumab Pembrolizumab sintilimab, cemiplimab tripolibamab, tislelizumab, spartalizumab, camrelizumab, dostralimab, genolimzumab, or cetrelimab, or an antibody that binds to PD-L1, such as envafolimab, atezolizumab, or Durvalumab Avelumab REGN2810, pidilizumab, MEDI0680, PDR001, PF-06801591, BGB-A317, TSR-042, and SHR-1210.

[0274] Methods of delivering the composition

[0275] The present invention also relates to a method of delivering a composition to a subject in need thereof. The delivery method may include administering the composition to the subject. Administration may include, but is not limited to, DNA injection with or without in vivo electroporation, liposome-mediated delivery, and nanoparticle-facilitated delivery.

[0276] The mammal to which the composition is delivered can be a human, a primate, a non-human primate, a cow, a cattle, a sheep, a goat, an antelope, a bison, a buffalo, a bison, bovids, a deer, a hedgehog, an elephant, a camel, an alpaca, a mouse, a rat, and a chicken.

[0277] The composition can be administered by different routes, including oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, by inhalation, buccal administration, intrapleural, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal, intranasal, intrathecal and intraarticular or a combination thereof. For veterinary use, the composition can be administered in a suitable acceptable formulation in accordance with normal veterinary practice. Veterinarians can easily determine the dosage regimen and route of administration that is most suitable for a particular animal. The composition can be administered by conventional syringes, needle-free injection devices, "microparticle bombardment gene guns" or other physical methods such as electroporation ("EP"), "hydrodynamic methods" or ultrasound.

[0278] Treatment

[0279] In one embodiment, the invention provides a method of treating or preventing a disease or condition that would benefit from an increase in NK cell function or activity. Exemplary diseases and conditions that can be treated using the compositions and methods of the invention include, but are not limited to, cancer and infectious diseases.

[0280] The following are non-limiting examples of cancers that can be diagnosed or treated by the disclosed methods and compositions: acute lymphocytic leukemia, acute myeloid leukemia, adrenocortical carcinoma, appendix cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain and spinal cord tumors, brain stem glioma, brain tumors, breast cancer, bronchial tumors, Burkitt's lymphoma, carcinoid tumors, atypical teratoid / rhabdoid tumors of the central nervous system, embryonal tumors of the central nervous system, lymphomas of the central nervous system, cerebellar astrocytoma, cerebral astrocytoma / glioblast ... cell tumors, extrahepatic bile duct cancer, extrahepatic cancer, eye cancer, fungoides, gallbladder cancer, gastric cancer, gastrointestinal cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (gist), germ cell tumors, pregnancy cancer, gestational trophoblastic tumors, glioblastoma, glioma, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway gliomas, hypothalamic tumors, Intraocular (eye) cancer, intraocular melanoma, islet cell tumor, Kaposi's sarcoma, kidney (renal cell) cancer, Langerhans cell carcinoma, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer, lymphoma, macroglobulinemia, malignant fibrous histiocytoma and osteosarcoma of bone, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, mesothelioma, primary occult metastatic squamous neck cancer, mouth cancer (mouth cancer), multiple endocrine neoplasia syndrome, multiple myeloma, mycosis, myelodysplastic syndrome, myelodysplastic / myeloproliferative disease, myeloid leukemia, myeloid leukemia, myeloma, myeloproliferative disease, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oral cavity cancer, oropharyngeal cancercancer), osteosarcoma and malignant fibrous histiocytoma, osteosarcoma and malignant fibrous histiocytoma of bone, ovary, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low-grade malignant potential tumor, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, moderately differentiated pineal parenchymal tumor, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasma cell tumor, plasma cell tumor / multiple myeloma, pleuropulmonary blastoma, primary central nervous system cancer, primary central nervous system lymphoma, prostate cancer, colorectal cancer, renal cell (kidney) cancer, renal pelvis and ureter cancer, respiratory tract cancer involving the nut gene on chromosome 15, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sezary syndrome, skin cancer (melanoma), skin cancer (non-melanoma), skin cancer (skin carcinoma), small cell lung cancer, small intestine cancer, soft tissue cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer, gastric cancer, supratentorial primitive neuroectodermal tumor, supratentorial primitive neuroectodermal tumor and pineoblastoma, T-cell lymphoma, testicular cancer, laryngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumor, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, optic pathway and hypothalamic gliomas, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

[0281] In one embodiment, the composition is used to treat cancers with high sialic acid levels. Cancers associated with high sialic acid levels include, but are not limited to, ovarian cancer, melanoma, renal cell carcinoma, prostate cancer, colon cancer, breast cancer, head and neck squamous cell carcinoma, skin cancer, and oral cancer.

[0282] Bacterial infection

[0283] In one embodiment, the infectious disease or condition is associated with bacteria. In some embodiments, the bacteria can be from any of the following phyla: Acidobacteria, Actinobacteria, Aquificae, Bacteroidetes, Caldiserica, Chlamydiae, Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus-Thermus, Dictyoglomi, Elusimicrobium, The phylum includes the following: bia, Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes, Lentisphaerae, Nitrospira, Planctomycetes, Proteobacteria, Spirochaetes, Synergistetes, Tenericutes, Thermodesulfobacteria, Thermotogae, and Verrucomicrobia.

[0284] The bacteria may be gram-positive or gram-negative. The bacteria may be aerobic or anaerobic. The bacteria may be autotrophic or heterotrophic. The bacteria may be mesophilic, neutrophile, extremophilic, acidophilic, alkaliphilic, thermophilic, psychrophilic, halophilic or osmophilic.

[0285] The bacteria may be anthrax bacteria, antibiotic-resistant bacteria, pathogenic bacteria, food poisoning bacteria, infectious bacteria, Salmonella bacterium, Staphylococcus bacterium, Streptococcus bacterium or tetanus bacterium. The bacteria may be mycobacteria, Clostridium tetani, Yersinia pestis, Bacillus anthracis, methicillin-resistant Staphylococcus aureus (MRSA), Clostridium difficile.

[0286] Viral infection

[0287] In one embodiment, the infectious disease or condition is associated with bacteria. In some embodiments, the virus is from one of the following families: Adenoviridae, Arenaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Papovaviridae, Paramyxoviridae, Parvoviridae, Picornaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, or Togaviridae. Viral antigens may be derived from human immunodeficiency virus (HIV), chikungunya virus (CHIKV), dengue virus, papillomaviruses such as human papillomavirus (HPV), poliovirus, hepatitis viruses such as hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV) and hepatitis E virus (HEV), smallpox virus (variola major and variola minor), vaccinia virus, influenza virus, rhinovirus, equine encephalitis virus, rubella virus, yellow fever virus, Norwalk virus (Norwalk virus), swine flu virus, typhoid virus, leukemia virus, leukopenia virus, leukemia ... virus), hepatitis A virus, human T-cell leukemia virus (HTLV-I), hairy cell leukemia virus (HTLV-II), California encephalitis virus, hantavirus (hemorrhagic fever), rabies virus, Ebola virus, Marburg virus, measles virus, mumps virus, respiratory syncytial virus (RSV), herpes simplex 1 (oral herpes), herpes simplex 2 (genital herpes), herpes zoster (varicella-zoster, also known as chickenpox), cytomegalovirus (CMV) such as human CMV, Epstein-Barr virus (EBV), flavivirus, foot-and-mouth disease virus, Lassa virus, arenavirus, severe acute respiratory syndrome-associated coronavirus (SARS), Middle East respiratory syndrome-associated coronavirus (MERS), severe acute respiratory syndrome-associated coronavirus 2 (SARS CoV 2), or oncoviruses.

[0288] parasitic infection

[0289] In one embodiment, the infectious disease or condition is associated with a parasite. In some embodiments, the parasite may be a protozoan, a helminth, or an ectoparasite. A helminth may be a flatworm (e.g., a fluke and a tapeworm), an acanthocephalic worm, or a round worm (e.g., a pinworm). An ectoparasite may be a louse, a flea, a tick, and a mite.

[0290] The parasite may be any parasite that causes any of the following diseases: Acanthamoeba keratitis, Amoebiasis, Ascariasis, Babesiosis, Balantidiasis, Baylisascariasis, Chagas disease disease), Clonorchiasis, Cochliomyia, Cryptosporidiosis, Diphyllobothriasis, Dracunculiasis, Echinococcosis, Elephantiasis, Enterobiasis, Fascioliasis, Fasciolopsiasis, Filariasis, Giardiasis, Gnathostomiasis, Hymenolepiasis, Isosporiasis, Katayama fever, Leishmaniasis, Lyme disease disease, Malaria, Metagonimiasis, Myiasis, Onchocerciasis, Pediculosis, Scabies, Schistosomiasis, Sleepingsickness, Strongyloidiasis, Taeniasis, Toxocariasis, Toxoplasmosis, Trichinosis, and Trichuriasis.

[0291] The parasites may be Acanthamoeba, Anisakis, Ascarislumbricoides, Botfly, Balantidium coli, Bedbug, Cestoda (tapeworms), Chiggers, Cochliomyia hominivorax, Entamoeba histolytica, Fasciola hepatica, Giardialamblia, Hookworm, Leishmania, Linguatula serrata, Liverfluke, Loa loa, Paragonimus-lung fluke, Pinworm, Plasmodium falciparum falciparum, Schistosoma, Strongyloides stercoralis, mites, tapeworms, Toxoplasma gondii, Trypanosoma, whipworms, or Wuchereria bancrofti.

[0292] Fungal infections

[0293] In one embodiment, the infectious disease or condition is associated with a fungus. In some embodiments, the fungus may be an Aspergillus species, Blastomyces dermatitidis, a Candida yeast (e.g., Candida albicans), Coccidioides, Cryptococcus neoformans, Cryptococcus gattii, a dermatophyte, a Fusarium species, Histoplasma capsulatum, Mucoromycotina, Pneumocystis jirovecii, Sporothrix schenckii, Exserohilum, or Cladosporium.

[0294] Cancer Therapy

[0295] In one embodiment, the invention provides a method for treating or preventing cancer, or a method for treating and preventing the growth or metastasis of a tumor. Related aspects of the invention provide methods for preventing, assisting in the prevention and / or reducing the metastasis of hyperplastic or tumor cells in an individual.

[0296] In one embodiment, the composition is used to treat cancers with high sialic acid levels, including but not limited to ovarian cancer, melanoma, renal cell carcinoma, prostate cancer, colon cancer, breast cancer, head and neck squamous cell carcinoma, and oral cancer.

[0297] One aspect of the invention provides a method of inhibiting metastasis in an individual in need thereof, the method comprising administering to the individual an effective amount of a nucleic acid molecule encoding a multivalent antibody of the invention, wherein the multivalent antibody is specific for the cancer to be treated. The invention also provides a method of inhibiting metastasis in an individual in need thereof, the method comprising administering to the individual an effective metastasis-inhibiting amount of a nucleic acid molecule encoding a multivalent antibody of the invention, wherein the multivalent antibody is specific for the cancer to be treated.

[0298] In some embodiments of treating or preventing cancer in an individual in need thereof or treating and preventing tumor metastasis in an individual in need thereof, a second agent, such as an anti-tumor agent, is administered to the individual. In some embodiments, the second agent comprises a second metastasis inhibitor, such as a plasminogen antagonist or an adenosine deaminase antagonist. In other embodiments, the second agent is an angiogenesis inhibitor.

[0299] The compositions of the present invention can be used to prevent, alleviate, minimize, control and / or reduce cancer in humans and animals. The compositions of the present invention can also be used to slow the growth rate of primary tumors. The compositions of the present invention can be used to prevent the spread of cancer cells when administered to a subject in need of treatment. Therefore, an effective amount of nucleic acid molecules encoding the multivalent antibodies of the present invention (wherein the multivalent antibodies are specific to the cancer to be treated) can be administered as part of a combination therapy with more than one drug or other agent. When used as part of a combination therapy, the reduction in metastasis and reduction in primary tumor growth provided by the compositions of the present invention allow for more effective and efficient use of any drug or drug therapy used to treat a patient. In addition, the control of metastasis by the compositions of the present invention provides a greater possibility for the subject to concentrate the disease in one location.

[0300] In one embodiment, the invention provides a method of treating cancer metastasis, the method comprising treating the subject with a complementary cancer therapy such as surgery, chemotherapy, chemotherapeutic agents, radiation therapy or hormonal therapy, or a combination thereof, prior to, concurrently with, or after treatment with a composition of the invention.

[0301] Chemotherapeutic agents include cytotoxic agents (e.g., 5-fluorouracil, cisplatin, carboplatin, methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, oxorubicin, carmustine (BCNU), lomustine (CCNU), cytarabine USP, cyclophosphamide, estramustine sodium phosphate, altretamine, hydroxyurea, ifosfamide, procarbazine, mitomycin, busulfan, cyclophosphamide, mitoxantrone, carboplatin, cisplatin, interferon alpha-2a recombinant, paclitaxel, teniposide, and streptozocin), cytotoxic alkylating agents (e.g., busulfan, chlorambucil, cyclophosphamide, melphalan, or ethionine), alkylating agents (e.g., asaley, AZQ, BCNU, busulfan, bisulphan, carboxyphthalate carboxyphthalatoplatinum, CBDCA, CCNU, CHIP, chlorambucil, chlorozotocin, cis-platinum, clomesone, cyanomorpholinodoxorubicin, cyclodisone, cyclophosphamide, dianhydrogalactitol, fluorodopan, hepsulfam, hydroxylamine lucanthone, ifosfamide, melphalan, methyl CCNU, mitomycin C, mitozolamide, nitrogen mustard, PCNU, piperazine, piperazine dione, piperobroman, porphyromycin, spirohydantoin mustard), streptozotocin, thiroxilon, tetraplatin, thiotepa, triethylene melamine, uracil nitrogen mustard, and Yoshi-864), antimitotic agents (e.g., allocolchicine, halichondrin M, colchicine, colchicine derivatives, Aplysia 10, maytansine, rhizoxin, paclitaxel derivatives, paclitaxel, thiocolchicine, tritylcysteine, vinblastine sulfate, and vincristine sulfate), plant biopharmaceuticals Bases (e.g., actinomycin D, bleomycin, L-asparaginase, idarubicin, vinblastine sulfate, vincristine sulfate, mitramycin, mitomycin, daunomycin, VP-16-213, VM-26, vinorelbine, and taxotere), biologics (e.g., interferon alpha, BCG, G-CSF, GM-CSF, and interleukin-2), topoisomerase I inhibitors (e.g., camptothecin, camptothecin derivatives, and morpholinodoxorubicin), topoisomerase II inhibitors (e.g.,mitoxantrone, aminafide, m-AMSA, anthrapyrazole derivatives, pyrazoloacridine, bisantrene hydrochloride, daunomycin, deoxydoxorubicin, menolide, N,N-dibenzyldaunomycin, oxanthrazole, rubidazone, VM-26 and VP-16), and synthetic preparations (e.g., hydroxyurea, procarbazine, o,p'-DDD, dacarbazine, CCNU, BCNU, cis-diaminedichloroplatinum, mitoxantrone, CBDCA, levamisole, hexamethylmelamine, all-trans retinoic acid, gliadel and porfimer sodium).

[0302] Antiproliferative agents are compounds that reduce the proliferation of cells. Antiproliferative agents include alkylating agents, antimetabolites, enzymes, biological response modifiers, miscellaneous agents, hormones and antagonists, androgen inhibitors (e.g., flutamide and leuprolide acetate), antiestrogens (e.g., tamoxifen citrate and its analogs, toremifene, droloxifene and raloxifene), and other examples of specific antiproliferative agents include, but are not limited to, levamisole, gallium nitrate, granisetron, sargramostim strontium-89 chloride, filgrastim, pilocarpine, dexrazoxane and ondansetron.

[0303] The compounds of the present invention can be administered alone or in combination with other anti-tumor agents, which include cytotoxic / antitumor agents and anti-angiogenic agents. Cytotoxic / antitumor agents are defined as agents that attack and kill cancer cells. Some cytotoxic / antitumor agents are alkylating agents that alkylate the genetic material in tumor cells, such as cisplatin, cyclophosphamide, nitrogen mustard, trimethylene thiophosphoramide, carmustine, busulfan, chlorambucil, belustine, uracil mustard, chlomaphazin and dacarbazine. Other cytotoxic / antitumor agents are antimetabolites for tumor cells, such as cytosine arabinoside, fluorouracil, methotrexate, mercaptopurine, thiopurine and methyl procarbazine. Other cytotoxic / antitumor drugs include antibiotics, such as doxorubicin, bleomycin, actinomycin, daunorubicin, mithramycin, mitomycin, mitomycin C and daunorubicin. There are many commercially available liposomal formulations of these compounds. Other cytotoxic / antitumor agents are mitotic inhibitors (vinca alkaloids). These include vincristine, vinblastine, and etoposide. Other cytotoxic / antitumor agents include paclitaxel and its derivatives, L-asparaginase, antitumor antibodies, dacarbazine, azacytidine, amsacrine, melphalan, VM-26, ifosfamide, mitoxantrone, and vindesine.

[0304] Anti-angiogenic agents are well known to those skilled in the art. Anti-angiogenic agents suitable for use in the methods and compositions of the present invention include anti-VEGF antibodies (including humanized and chimeric antibodies), anti-VEGF aptamers, and antisense oligonucleotides. Other known angiogenesis inhibitors include angiostatin, endostatin, interferon, interleukin 1 (including α and β), interleukin 12, retinoic acid, and tissue inhibitors of metalloproteinases-1 and -2 (TIMP-1 and -2). Small molecules, including topoisomerases, such as razoxane, a topoisomerase II inhibitor with anti-angiogenic activity, may also be used.

[0305] Other anticancer agents that can be used in combination with the compositions of the present invention include, but are not limited to: acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; binasafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride hydrochloride); decitabine; dexormaplatin;dezaguanine; dezaguanine mesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflornithine hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine sodium phosphate; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride hydrochloride); ifosfamide; ilmofosine; interleukin II (including recombinant interleukin II, or rIL2), interferon α-2a; interferon α-2b; interferon α-n1; interferon α-n3; interferon β-I a; interferon γ-I b; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate;liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride; megestrolacetate; melengestrol acetate acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid acid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium sodium); porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safingol;Safingol hydrochloride; semustine; simtrazene; sparrosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalansodium; tegafur; teloxantrone hydrochloride hydrochloride; temoporfin; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; hydrochloride; uracilmustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole;Zeniplatin; zinostatin; zorubicin hydrochloride. Other anticancer drugs include, but are not limited to: 20-epi-1,25-dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; acid; amrubicin; amsacrine; anagrelide; anastrozole; and andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen, prostate cancer; antiestrogens; antineoplastons; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene regulator; apoptosis regulator; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1 1); acistatin 2; acistatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR / ABL antagonists; benzochlorins; benzoylstaurosporine; β-lactam derivatives; β-alethine; betaclamycin B; betulinic acid; bFGF inhibitors; bicalutamide; bisantrene;bisaziridinylspermine; bischafide; bistratene A; bizelesin; breflate; bropirimine; budotitane; buthioninesulfoximine; calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage-derived inhibitor; carzelesin; casein kinase inhibitor (ICOS); castanospermine; cecropin B B); cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogs; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogs; conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A A); cyclopentanthraquinones; cycloplatam; cypemycin; cytarabineocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docetaxel; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA SA; ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride; estramustine analogs; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezlastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadoliniumtexaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylenebisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene;idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulatory peptides; insulin-like growth factor-1 receptor inhibitors; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N-triacetate triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibitory factor; interferon-alpha; leuprolide + estrogen + progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogs; lipophilic disaccharide peptides; lipophilic platinum compounds; lissoclinamide 7 7); lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; cleaved peptide; maitansine; mannostatin A A); marimastat; masoprocol; maspin; matrixlysin inhibitor; matrix metalloproteinase inhibitor; menogaril; merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double-stranded RNA; mitoguaone; mitolactol; mitomycin analogs; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotropin; monophosphoryl lipid A+ mycobacterial cell wall sk; mopidamol; multidrug resistance gene inhibitor; multiple tumor suppressor 1-based therapy; nitrogen mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyl narelin; N-substituted benzamides; nafarelin; nagrestip; naloxone + pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidants; nitrullyn; O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducers; ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogs; paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; sodium pentosan polysulfate; pentostatin;pantoprazole; perflubron; perfosfamide; perillylalcohol; phenazinomycin; phenyl acetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitors; platinum complexes; platinum compounds; platinum-triamine complexes; porfimer sodium sodium; porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immunomodulators; protein kinase C inhibitors; protein kinase C inhibitors, microalgae; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitors; demethylated retelliptine demethylated; rhenium etidronate Re186; rhizoxin; ribozyme; RII retinamide; rogletimide; rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetic; semustine; senescence-derived inhibitor 1; sense oligonucleotide; signal transduction inhibitor; signal transduction modulator; single-chain antigen binding protein; sizofiran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin;sparrosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem cell division inhibitor; stipiamide; stromelysin inhibitor; sulfinosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetics; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene dichloride bichloride; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector systems, erythrocyte gene therapy; velaresol; veramine; verdins;Verteporfin; vinorelbine; vinxaltine; vitamin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer. In one embodiment, the anticancer drug is 5-fluorouracil, paclitaxel, or leucovorin. ;

[0306] Combination

[0307] In some embodiments, the present invention provides a method of treating, protecting against and / or preventing cancer or a cancer-related disease or condition in a subject in need thereof by administering a combination of a composition comprising a synthetic Siglec-9 antibody, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same and a composition comprising a tumor antigen, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same.

[0308] In one embodiment, a composition comprising a synthetic anti-Siglec-9 antibody, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same and a composition comprising a tumor antigen, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same can be administered using any suitable method so that the combination of anti-Siglec-9 and tumor antigen are both present in the subject. In one embodiment, the method can include administering a first composition comprising a synthetic Siglec-9 antibody, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same of the present invention by any method detailed elsewhere herein, and administering a second composition comprising a tumor antigen, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same less than 1 day, less than 2 days, less than 3 days, less than 4 days, less than 5 days, less than 6 days, less than 7 days, less than 8 days, less than 9 days, or less than 10 days after administration of the synthetic Siglec-9 antibody, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same. In one embodiment, the method may include administering a first composition comprising a synthetic Siglec-9 antibody, fragment thereof, variant thereof, or nucleic acid molecule encoding the same of the present invention by any of the methods detailed above, and administering a second composition comprising a tumor antigen, fragment thereof, variant thereof, or nucleic acid molecule encoding the same more than 1 day, more than 2 days, more than 3 days, more than 4 days, more than 5 days, more than 6 days, more than 7 days, more than 8 days, more than 9 days, or more than 10 days after administering the synthetic anti-Siglec-9 antibody, fragment thereof, variant thereof, or nucleic acid molecule encoding the same. In one embodiment, the method may include administering a first composition comprising a tumor antigen, fragment thereof, variant thereof, or nucleic acid molecule encoding the same, and administering a second composition comprising a synthetic anti-Siglec-9 antibody, fragment thereof, variant thereof, or nucleic acid molecule encoding the same by any of the methods detailed above less than 1 day, less than 2 days, less than 3 days, less than 4 days, less than 5 days, less than 6 days, less than 7 days, less than 8 days, less than 9 days, or less than 10 days after administering the composition comprising the tumor antigen, fragment thereof, variant thereof, or nucleic acid molecule encoding the same. In one embodiment, the method may include administering a first composition comprising a tumor antigen, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same, and administering a second composition comprising a synthetic anti-Siglec-9 antibody, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same by any of the methods detailed above more than 1 day, more than 2 days, more than 3 days, more than 4 days, more than 5 days, more than 6 days, more than 7 days, more than 8 days, more than 9 days, or more than 10 days after administering the composition comprising the tumor antigen, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same. In one embodiment, the method may include simultaneously administering a first composition comprising a synthetic anti-Siglec-9 antibody, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same by any of the methods detailed above and a second composition comprising a tumor antigen, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same.In one embodiment, the method may include administering a single composition comprising a synthetic anti-Siglec-9 antibody, fragment thereof, variant thereof, or nucleic acid molecule encoding the same of the present invention and a tumor antigen, fragment thereof, variant thereof, or nucleic acid molecule encoding the same.

[0309] The present invention is further illustrated in the following examples. It should be understood that these examples, although showing exemplary embodiments of the present invention, are given only by way of illustration. From the above discussion and these examples, those skilled in the art can determine the essential features of the present invention, and without departing from its spirit and scope, various changes and modifications can be made to the present invention to adapt it to various uses and conditions. Therefore, according to the above description, in addition to those shown and described herein, various modifications of the present invention will be apparent to those skilled in the art. Such modifications are also intended to fall within the scope of the appended claims.

[0310] Example

[0311] The present invention will be described in further detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise stated. Therefore, the present invention should never be construed as being limited to the following examples, but should be construed as covering any and all variations that become apparent due to the teachings provided herein.

[0312] Without further description, it is believed that one skilled in the art can use the foregoing description and the following illustrative examples to make and use the present invention and practice the claimed methods. Therefore, the following working examples should not be construed as limiting the remainder of the disclosure in any way.

[0313] Example 1: Generation of Siglec-9 antibodies using H2L2 mice

[0314] Sialic acid-binding receptors such as Siglecs and selectins are readily exploited by cancer hypersialylation. Sialic acid (Sias) is a monosaccharide that is typically located at the outermost end of complex glycan chains that decorate the cell surface of all vertebrates. Sias are essential for embryonic development and mediate important intrinsic organismal functions. Seventy human genes are known to be involved in Sias biology, of which more than 10 have been shown to be human-specific. Most of these human-specific genetic changes occur in the Sia-recognizing Ig-like lectin (SIGLEC) gene, which is present in most types of white blood cells and is associated with the immune synapse. Upregulation of sialosides has long been associated with poor tumor prognosis and reduced immunogenicity.

[0315] Siglec-9 is expressed on monocytes, macrophages, natural killer (NK) cells, dendritic cells, neutrophils, and subsets of T cells. Siglec-9 is an inhibitory receptor whose signaling 1) inhibits the anti-tumor function of NK and T cells, and 2) regulates the immune function of myeloid cells.

[0316] The experiments described here illustrate the development and characterization of Siglec-9 antibodies ( Figure 1 ). In these experiments, hybridoma supernatants demonstrated specificity for and binding to human Siglec-9.

[0317] The antibody was observed to be cytotoxic to OVISE cells ( Figure 2 ). OVISE cells are ovarian clear cell adenocarcinomas derived from metastatic sites: pelvis; innominate bone of a 40-year-old Japanese woman. The experiment was designed to test the activity of the antibody. Target cells: OVISE (20,000 cells / well); Effector cells: human PBMC; E(effector cells):T(target cells)=5:1; Effector cells and antibodies (1:10) were added at time points 20 and 56 hours. Images were taken at 100 hours, whereby Siglec9 antibody-driven killing of OVISE cells was observed.

[0318] It was also observed that the antibody exhibited cytotoxicity against OVCAR10 cells ( Figure 3 ). OVCAR10 is an ovarian human cancer cell line. The experiment was designed to test the activity of the antibody. Target cells: OVCAR10 (10,000 cells / well); Effector cells: human PBMC E:T=5:1; Effector cells and antibodies were added at 28.67 hours. Images were taken at 53 hours, whereby Siglec9 antibody-driven killing of OVCAR10 cells was observed. See Figure 3 .

[0319] The antibody showed no cytotoxicity in TC-1 cells, which were used as a control target. TC-1 is a mouse cell line in which Siglec9 is absent on the cells.

[0320] Example 2: Sequences - CDR sequences are underlined

[0321] HB9-4G4(S9-DB-2)

[0322] Heavy chain

[0323] (SEQ ID NO:4)

[0324] EVQLVESGGGLVKPGGSLRLSCAAS GFTFSNAW MSWVRQAPGKGLEWVGR IKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYC TTGWELQDYYYYGMDV WGQGTTVTVSS

[0325] HB9-4G4(S9-DB-2)

[0326] Heavy chain

[0327] (SEQ ID NO:8)

[0328] GAGGTGCAGCTGGTGGAGTCGGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTTAGACTCTCCTGTGCAGCCTCT GGATTCACTTTCAGTAACGCCTGG ATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCCGT ATTAAAAGCAAAACTGATGGTGGGACAACA GACTACGCTGCACCCGTGAAAGGCAGATTCACCATCTCAAGAGATGATTCAAAAAACACGCTGTATCTGCAAATGAACAGCCTGAAAACCGAGGACACAGCCGTGTATTACTGT ACCACA GGGTGGGAGCTACAGGACTACTACTACGGTATGGACGTC TGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

[0329] HB9-4G4(S9-DB-2)

[0330] Light chain

[0331] (SEQ ID NO:12)

[0332] IVMTQSPATLSVSPGERATLSCRAS QSVSSN LAWYQQKPGQAPRLLIY GAS TRATGIPARFSGSGSGTEFTLTI SSLQSEDFAVYYC QQYNNWPLT FGGGTKVEIK

[0333] HB9-4G4(S9-DB-2)

[0334] Light chain

[0335] (SEQ ID NO:16)

[0336] ATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGT CAGAGTGTTAGCAGCAAC TTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT GG TGCATCC ACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGT CAGCAGTATAATAACTGGCCGCTCACT TTCGGCGGAGGGACCAAGGTGGAGATCAAA

[0337] HB9-6A8(S9-DB-3)

[0338] Heavy chain

[0339] (SEQ ID NO:20)

[0340] QVQLQQWGAGLLKPSETLSLTCAVY GGSFSGYY WSWIRQPPGKGLEWIGE INHSGST NYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYC ARGDCSGGSCPYWYFDL WGRGTLVTVSS

[0341] HB9-6A8(S9-DB-3)

[0342] Heavy chain

[0343] (SEQ ID NO:24)

[0344] CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTAT GGTGGGTCCTTCAGTGGTTACTACT GGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAA ATCAATCATAGTGGAAGCACC AACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGT GCGAGAGGTGATTGT AGTGGTGGTAGCTGTCCTTACTGGTACTTCGATCTC TGGGGCCGTGGCACCCTGGTCACTGTCTCCTCA

[0345] HB9-6A8(S9-DB-3)

[0346] Light chain

[0347] (SEQ ID NO:28)

[0348] DIVMTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC MQALQTPRT FGQGTKVEIK

[0349] HB9-6A8(S9-DB-3)

[0350] Light chain

[0351] (SEQ ID NO:32)

[0352] GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGCTCTACAAACTCCT CGGACG TTCGGCCAAGGGACCAAGGTGGAAATCAAA

[0353] HB9-6F3(S9-DB-4)

[0354] Heavy chain

[0355] (SEQ ID NO:36)

[0356] EVQLLESGGGLVQPGGSLRLSCEAS GFTFRNYA MSWVRQAPGKGLEWVSA IRGSGSRT YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC AKDEGFGDLLAHYVMDA WGQGASVTVSS

[0357] HB9-6F3(S9-DB-4)

[0358] Heavy chain

[0359] (SEQ ID NO:40)

[0360] GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGAAGCCTCA GGATTCACCTTTAGAAACTATGCC ATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCT ATTCGTGGTAGTGGTAGTAGAACA TACTACGCAGACTCTGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGC GCGAAAGATGAG GGGTTCGGGGACTTATTAGCGCACTATGTTATGGATGCC TGGGGTCAAGGAGCTTCAGTCACTGTCTCCTCA

[0361] HB9-6F3(S9-DB-4)

[0362] Light chain

[0363] (SEQ ID NO:44)

[0364] EIVMTQSPATLSVSPGERATLSCRAS QSVSSN LAWYQQKPGQAPRLLIY GAS TRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYC QQYNNWPIT FGQGTRLEIK

[0365] HB9-6F3(S9-DB-4)

[0366] Light chain

[0367] (SEQ ID NO:48)

[0368] GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGT CAGAGTGTTAGCAGCAAC TTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT GGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGT CAGCAGTATAATAACTGGCCGATCACC TTCGGCCAAGGGACACGACTGGAGATTAAA

[0369] HB9-9B11(S9-DB-5)

[0370] Heavy chain

[0371] (SEQ ID NO:52)

[0372] EVQLLESGGGLVQPGGSLRLSCAAS GFTFSNYA MNWVRQAPGKGLEWVSA ISMSGGST YYADSVKGRFTISRDNSKNTLYLQMNTLRAEDTAVYYC AKDEYSSGWYQFDY WGQGTLVTVSS

[0373] HB9-9B11(S9-DB-5)

[0374] Heavy chain

[0375] (SEQ ID NO:56)

[0376] GAGGTGCAGTTGTTGGAGTCTGGGGGGGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCT GGATTCACCTTTAGCAACTATGCC ATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCT ATTAGTATGAGTGGTGGTAGCACA TACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACGGCCGTATATTACTGT GCGAAAGACGAA TATAGCAGTGGCTGGTACCAATTTGACTAT TGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0377] HB9-9B11(S9-DB-5)

[0378] Light chain

[0379] (SEQ ID NO:60)

[0380] DIQMTQSPSSLSASVGDRVTITCRAS QSISSY LNWYQQKPGKAPKLLIY AAS SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSYSTPLT FGGGTKVEIK

[0381] HB9-9B11(S9-DB-5)

[0382] Light chain

[0383] (SEQ ID NO:64)

[0384] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGT CAGAGCATTAGCAGCTAT TTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTAT GCTGCATCC AGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGT CAACAGAGTTACAGTACCCCGCTCACT TTCGGCGGAGGGACCAAGGTGGAGATCAAA

[0385] HB9-10B6(S9-DB-6)

[0386] Heavy chain

[0387] (SEQ ID NO:68)

[0388] QVQLVESGGGVVQPGRSLRLSCAAS GFTFSTYG MHWVRQAPGKGLEWVAV TWYDEYNK YYADSVKGRFTISRDNSKNMLYLQMNSLRAEDTAMYYC <h2 style=";text-align:left;direction:ltr">ARNVLRYFDWTLDY WGQGTLVTVSS

[0389] HB9-10B6(S9-DB-6)

[0390] Heavy chain

[0391] (SEQ ID NO:72)

[0392] CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCT <h2 style=";text-align:left;direction:ltr"> GGATTCACCTTCAGTACCTATGGC ATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTT <h2 style=";text-align:left;direction:ltr"> ACATGGTATGATGAATATAATAAA TACTATGCAGACTCCGTGAAGGGCCGCTTCACCATCTCCAGAGACAATTCCAAGAACATGTTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTATGTATTACTGT <h2 style=";text-align:left;direction:ltr"> GCGAGGAACGTA <h2 style=";text-align:left;direction:ltr"> TTACGATATTTTGACTGGACCCTTGACTAC TGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG

[0393] HB9-10B6(S9-DB-6)

[0394] Light chain

[0395] (SEQ ID NO:76)

[0396] IVLTQSPATLSLSPGERATLSCRAS <h2 style=";text-align:left;direction:ltr"> QSVSSY LAWYQQKPGQAPRLLIY <h2 style=";text-align:left;direction:ltr"> DAS NRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC <h2 style=";text-align:left;direction:ltr"> QQRSNWPPT FGQGTKVEIK

[0397] HB9-10B6(S9-DB-6)

[0398] Light chain

[0399] (SEQ ID NO:80)

[0400] ATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGT <h2 style=";text-align:left;direction:ltr"> CAGAGTGTTAGCAGCTAC TTAGCCTGGTATCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT <h2 style=";text-align:left;direction:ltr"> GA <h2 style=";text-align:left;direction:ltr"> TGCATCC AACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGT<h2 style=";text-align:left;direction:ltr"> CAGCAGCGTAGCAACTGGCCTCCCACT TTTGGCCAGGGGACCAAGGTGGAGATCAAA

[0401] HB9-10E3(S9-DB-7)

[0402] Heavy chain

[0403] (SEQ ID NO:84)

[0404] EVQLLESGGGLVQPGGSLRLSCAAS <h2 style=";text-align:left;direction:ltr"> GFTFSSYA MSWVRQAPGKGLEWVSA <h2 style=";text-align:left;direction:ltr"> ISGSGGST YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC <h2 style=";text-align:left;direction:ltr"> AKAGIAVAGGWYFDL WGRGTLVTVSS

[0405] HB9-10E3(S9-DB-7)

[0406] Heavy chain

[0407] (SEQ ID NO:88)

[0408] GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCT <h2 style=";text-align:left;direction:ltr"> GGATTCACCTTTAGCAGCTATGCC ATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCT <h2 style=";text-align:left;direction:ltr"> ATTAGTGGTAGTGGTGGTAGCACA TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGT <h2 style=";text-align:left;direction:ltr"> GCGAAAGCCGGT <h2 style=";text-align:left;direction:ltr"> ATAGCAGTGGCTGGGGGATGGTACTTCGATCTC TGGGGCCGTGGCACCCTGGTCACTGTCTCCTCA

[0409] HB9-10E3(S9-DB-7)

[0410] Light chain

[0411] (SEQ ID NO:92)

[0412] IVLTQSPATLSLSPGERATLSCRAS <h2 style=";text-align:left;direction:ltr"> QSVSSY LAWYQQKPGQAPRLLIY <h2 style=";text-align:left;direction:ltr"> DASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC <h2 style=";text-align:left;direction:ltr"> QQRSNWPPT FGQGTKLEIK

[0413] HB9-10E3(S9-DB-7)

[0414] Light chain

[0415] (SEQ ID NO:96)

[0416] ATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGT <h2 style=";text-align:left;direction:ltr"> CAGAGTGTTAGCAGCTAC TTAGCCTGGTATCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT <h2 style=";text-align:left;direction:ltr"> GA <h2 style=";text-align:left;direction:ltr"> TGCATCC AACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGT <h2 style=";text-align:left;direction:ltr"> CAGCAGCGTAGCAACTGGCCTCCCACT TTTGGCCAGGGGACCAAGCTGGAGATCAAA

[0417] HB9-11B10(S9-DB-8)

[0418] Heavy chain

[0419] (SEQ ID NO:100)

[0420] EVQLLESGGGLVQPGGSLRLSCAAS <h2 style=";text-align:left;direction:ltr"> GFTFSNYA MNWVRQAPGKGLEWVSA <h2 style=";text-align:left;direction:ltr"> ISMSGGST YYADSVKGRFTISRDNSKNTLYLQMNTLRAEDTAVYYC <h2 style=";text-align:left;direction:ltr"> AKDEYSSGWY QFDYWGQGTLVTVSS

[0421] HB9-11B10(S9-DB-8)

[0422] Heavy chain

[0423] (SEQ ID NO:104)

[0424] GAGGTGCAGTTGTTGGAGTCTGGGGGGGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCT<h2 style=";text-align:left;direction:ltr"> GGATTCACCTTTAGCAACTATGCC ATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCT <h2 style=";text-align:left;direction:ltr"> ATTAGTATGAGTGGTGGTAGCACA TACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACGGCCGTATATTACTGT <h2 style=";text-align:left;direction:ltr"> GCGAAAGACGAA <h2 style=";text-align:left;direction:ltr"> TATAGCAGTGGCTGGTAC CAATTTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0425] HB9-11B10(S9-DB-8)

[0426] Light chain

[0427] (SEQ ID NO:108)

[0428] EIVLTQSPATLSLSPGERATLSCRAS <h2 style=";text-align:left;direction:ltr"> QSVNSY LAWYQQKPGQAPRLLIY <h2 style=";text-align:left;direction:ltr"> DAS IRATGIPARFSGSGSGTDFTLTISSLEPEDFAFYYC <h2 style=";text-align:left;direction:ltr"> QQRSNWPPT FGQGTKVEIK

[0429] HB9-11B10(S9-DB-8)

[0430] Light chain

[0431] (SEQ ID NO:112)

[0432] GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGT <h2 style=";text-align:left;direction:ltr"> CAGAGTGTTAACAGCTAC TTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT <h2 style=";text-align:left;direction:ltr"> GATGCATCC ATCAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCATTTTATTACTGT <h2 style=";text-align:left;direction:ltr"> CAACAGCGTAGTAACTGGCCTCCGACG TTCGGCCAAGGGACCAAGGTGGAAATCAAA

[0433] HB9-1F11

[0434] Heavy chain:

[0435] (SEQ ID NO:116)

[0436] QVQLQQPGAELVKPGASVKLSCKAS <h2 style=";text-align:left;direction:ltr"> GYTFTSYW MHWVKQRPGRGLEWIGR <h2 style=";text-align:left;direction:ltr"> IDPNSGGT KYNEKFKSKATLTVDKPSSTAYMQLSSLTSEDSAVYYC <h2 style=";text-align:left;direction:ltr"> ARYDYYGSSYFDY WGQGTTVTVSS

[0437] HB9-1F11

[0438] (SEQ ID NO:120)

[0439] CAAGTGCAGCTGCAGCAGCCTGGCGCCGAGCTGGTGAAACCTGGAGCCTCCGTGAAGCTGAGCTGTAAGGCCAGC <h2 style=";text-align:left;direction:ltr"> GGCTACACCTTCACCAGCTACTGG ATGCACTGGGTCAAGCAGCGGCCAGGAAGAGGCCTGGAATGGATCGGCAGA <h2 style=";text-align:left;direction:ltr"> ATCGACCCCAACAGCGGCGGCACA AAGTACAACGAGAAGTTCAAGAGCAAGGCTACACTGACAGTGGACAAGCCTAGCTCCACCGCCTACATGCAGCTGTCTAGCCTGACCAGCGAAGATAGCGCCGTGTACTACTGC <h2 style=";text-align:left;direction:ltr"> GCCAGATACGAC <h2 style=";text-align:left;direction:ltr"> TACTACGGCAGCTCTTATTTTGATTAC TGGGGCCAGGGCACCACCGTGACCGTGTCCAGC

[0440] HB9-1F11

[0441] Light chain:

[0442] (SEQ ID NO:124)

[0443] QAVVTQESALTTSPGETVTLTCRSS <h2 style=";text-align:left;direction:ltr"> TGAVTTSNY ANWVQEKPDHLFTGLIG <h2 style=";text-align:left;direction:ltr"> GTN NRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFC <h2 style=";text-align:left;direction:ltr"> ALWYSNHWV FGGGTKLTVL

[0444] HB9-1F11

[0445] (SEQ ID NO:128)

[0446] CAAGCCGTGGTGACACAGGAGAGCGCCCTGACCACATCCCCCGGCGAGACAGTGACCCTGACCTGTAGAAGCAGC <h2 style=";text-align:left;direction:ltr"> ACAGGCGCTGTGACCACCTCTAACTAC GCCAACTGGGTCCAGGAAAAACCTGACCACCTGTTTACCGGCCTGATCGGCG <h2 style=";text-align:left;direction:ltr"> GAACCAACA ACCGGGCCCCTGGAGTGCCAGCCAGATTCAGCGGCTCTCTGATCGGCGACAAGGCCGCTCTCACCATCACCGGCGCCCAGACAGAAGATGAGGCCATCTACTTCTGC <h2 style=";text-align:left;direction:ltr"> GCCCTGTGGTACAGCAATCACTGGGTG TTCGGCGGAGGCACCAAGCTGACAGTGCTG

[0447] HB9-4C10

[0448] Heavy chain:

[0449] (SEQ ID NO:132)

[0450] EVQLQQSGPELVKPGASVKISCKAS <h2 style=";text-align:left;direction:ltr"> GYTFTDYY MNWVKQSHGKSLEWIGG <h2 style=";text-align:left;direction:ltr"> INPNNGGT SYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYC <h2 style=";text-align:left;direction:ltr"> ARNDGYRGYAMDY WGQGTSVTVSS

[0451] HB9-4C10

[0452] (SEQ ID NO:136)

[0453] GAGGTGCAGCTGCAGCAATCTGGACCTGAGCTGGTGAAGCCTGGCGCTTCTGTGAAGATCTCTTGTAAAGCCAGC <h2 style=";text-align:left;direction:ltr"> GGCTACACCTTCACCGATTACTAC ATGAACTGGGTCAAGCAGAGCCACGGCAAAAGCCTGGAATGGATCGGCGGC <h2 style=";text-align:left;direction:ltr"> ATCAACCCCAACAACGGCGGAACA AGCTACAACCAGAAGTTCAAGGGCAAGGCTACCCTGACCGTGGACAAGAGCAGCTCCACCGCCTACATGGAACTGCGGAGCCTGACAAGCGAGGACAGCGCCGTGTACTACTGC <h2 style=";text-align:left;direction:ltr"> GCCAGAAATGAT <h2 style=";text-align:left;direction:ltr"> GGCTATAGAGGCTACGCCATGGACTACTGGGGACAGGGCACCAGCGTGACAGTGTCCTCC

[0454] HB9-4C10

[0455] Light chain:

[0456] (SEQ ID NO:140)

[0457] DIVMSQSPSSLAVSVGEKVTMSCKSS <h2 style=";text-align:left;direction:ltr"> QSLLYSSNQKNY LAWYQQKPGQSPKLLIY <h2 style=";text-align:left;direction:ltr"> WAS TRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYCC <h2 style=";text-align:left;direction:ltr"> QQYYSNPPLT FGAGTKLELK

[0458] HB9-4C10

[0459] (SEQ ID NO:144)

[0460] GACATCGTGATGAGCCAGTCTCCATCTAGCCTGGCCGTGTCCGTGGGCGAGAAGGTGACCATGTCTTGTAAAAGCTCC <h2 style=";text-align:left;direction:ltr"> CAGAGCCTGCTGTACAGCAGCAACCAGAAAAACTAC CTGGCCTGGTATCAGCAAAAGCCCGGCCAGAGCCCCAAGCTCCTGATCTAC <h2 style=";text-align:left;direction:ltr"> TGGGCCTCC ACACGGGAAAGCGGAGTGCCTGATAGATTCACCGGCTCTGGCAGCGGAACCGACTTTACCCTGACCATCAGCAGCGTCAAGGCCGAGGACCTGGCTGTGTACTGCTGC <h2 style=";text-align:left;direction:ltr"> CAGCAGTACTACAGCAAT <h2 style=";text-align:left;direction:ltr"> CCTCCTCTGACA TTCGGCGCCGGCACAAAGCTGGAACTGAAG

[0461] HB9-5B7

[0462] Heavy chain:

[0463] (SEQ ID NO:148)

[0464] EVQLVESGGGLVQPGRSLRLSCAAS <h2 style=";text-align:left;direction:ltr"> GFTFDDYA MHWVRQAPGKGLEWVSA <h2 style=";text-align:left;direction:ltr"> ITWNSGHI DYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC <h2 style=";text-align:left;direction:ltr"> AKVSYLSTASSLDY WGQGTLVTVSS

[0465] HB9-5B7

[0466] (SEQ ID NO:152)

[0467] GAGGTGCAGCTGGTGGAAAGCGGAGGAGGCCTGGTTCAACCCGGCCGGAGCCTGAGACTGAGCTGTGCCGCTTCT GGCTTTACATTCGACGACTACGCC ATGCACTGGGTCAGACAGGCCCCTGGCAAGGGCCTCGAGTGGGTGTCCGCC ATCACCTGGAATAGCGGCCACATC GACTATGCTGATAGCGTGGAAGGCAGATTCACCATCAGCAGAGATAACGCCAAGAACTCCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGC GCCAAAGTGTCC TACCTGTCTACAGCCAGCAGCCTGGACTAC TGGGGCCAGGGCACCCTGGTGACCGTGTCTAGC

[0468] HB9-5B7

[0469] Light chain:

[0470] (SEQ ID NO:156)

[0471] DIQMTQSPSSLSASVGDRVTITCRAS QGISNY LAWYQQKPGQVPKLLIY AAS ALQSGVPSRFSGSGSGTDFTLTITSLQPEDVATYYC HKYNSAPWT FGQGTEVEIK

[0472] HB9-5B7

[0473] (SEQ ID NO:160)

[0474] GACATCCAGATGACCCAGTCCCCTAGCTCTCTGAGCGCCAGCGTGGGCGACCGGGTGACCATCACCTGTAGAGCCTCT CAGGGCATCAGCAACTAC CTGGCCTGGTATCAGCAGAAACCCGGACAGGTGCCAAAGCTGCTGATCTAC GCCGCTTCT GCTCTGCAGAGCGGAGTCCCCAGCAGATTCAGCGGCTCCGGCAGCGGCACAGATTTTACCCTGACCATTACAAGCCTGCAACCTGAGGACGTGGCCACATACTACTGC CACAAGTACAACAGCGCCCCTTGGACC TTCGGCCAGGGCACCGAGGTGGAAATCAAG

[0475] HB9-7B8

[0476] Heavy chain:

[0477] (SEQ ID NO:164)

[0478] ELQLLESGGGLVQPGGSLRLSCAAS GFTFTNYA MNWVRQAPGKGLEWVSA ISGSGGRT YYADSVKGRFTISRDNSRNTLFLQMNSLRPEDTAVYYC AKDQTSGTTGYPYFAY WGQGTLVTVSS

[0479] HB9-7B8

[0480] (SEQ ID NO:168)

[0481] GAGCTGCAGCTGCTGGAGTCTGGCGGCGGCCTGGTGCAGCCCGGCGGCTCCCTGAGGCTGAGCTGCGCCGCCTCC GGCTTTACATTCACAAATTACGCC ATGAATTGGGTGCGGCAGGCCCCAGGCAAGGGCCTGGAGTGGGTGTCCGCC ATCTCTGGCTCCGGCGGCAGAACC TATTACGCCGATTCCGTGAAGGGCAGATTCACCATCAGCAGAGACAACTCTCGCAATACCCTGTTTCTGCAGATGAATAGCCTGAGGCCCGAGGATACAGCCGTGTACTACTGC GCCAAGGATCAG ACATCTGGCACAACAGGCTACCCTTATTTCGCCTAC TGGGGCCAGGGCACCCTGGTGACCGTGTCTTCC

[0482] HB9-7B8

[0483] Light chain:

[0484] (SEQ ID NO:172)

[0485] ETVMTQSPATLSVSPGERAILSCRAS QSVSSN LVWYQQKPGQAPRLFIY GAS TRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYC QQYNNWPRT FGQGTKVEIK

[0486] HB9-7B8

[0487] (SEQ ID NO:176)

[0488] GAGACAGTGATGACCCAGAGCCCAGCCACCCTGTCCGTGTCTCCAGGCGAGAGGGCCATCCTGTCCTGTAGGGCCTCC CAGAGCGTGTCTTCCAAT CTGGTGTGGTACCAGCAGAAGCCAGGCCAGGCCCCTAGGCTGTTTATCTAC GGCGCCTCC ACAAGGGCCACCGGCATCCCTGCCAGATTCTCTGGCTCTGGCTCCGGCACAGAGTTCACCCTGACAATCTCTTCCCTGCAGTCTGAGGACTTTGCCGTGTACTATTGC CAGCAGTATAATAACTGGCCTAGGACC TTTGGCCAGGGCACAAAGGTGGAGATCAAGCA

[0489] HB9-14D4

[0490] Heavy chain:

[0491] (SEQ ID NO:180)

[0492] QVQLVESGGGVVQPGRSLRLSCAAS GFAFSSYG MHWVRQAPGKGLEWVAV IWFDGTKK YYTDSVKGRFTISRDNSKNTLYLQMNTLRAEDTAVYYC ARDRGIGARRGPYYMDV WGKGTTVTVSS

[0493] HB9-14D4

[0494] (SEQ ID NO:184)

[0495] CAAGTGCAGCTGGTTGAGAGCGGCGGAGGCGTGGTGCAGCCCGGCAGAAGCCTGAGACTGTCTTGTGCTGCTTCT GGATTTGCCTTCAGCAGCTACGGC ATGCACTGGGTGCGGCAGGCCCCTGGCAAGGGCCTGGAATGGGTCGCCGTG ATCTGGTTCGACGGCACAAAGAAG TACTATACAGACAGCGTGAAGGGCAGATTCACCATCAGCAGAGATAATAGCAAGAACACCCTGTACCTGCAGATGAACACCCTGCGGGCCGAGGACACCGCCGTGTACTACTGC GCCAGAGATAGA GGCATCGGCGCCAGGCGGGGACCTTACTACATGGACGTG TGGGGCAAAGGCACCACAGTGACCGTGTCCTCC

[0496] HB9-14D4

[0497] Light chain:

[0498] (SEQ ID NO:188)

[0499] DIQMTQSPSSLTASVGDRVTITCRAS QSISSY VNWYQQKPGKAPKVLIF AAS SLQSGVPSRFSGSGSGTDFTLTISSLQAEDVAVYYC QQYYSTPLT FGGGTKVEIK

[0500] HB9-14D4

[0501] (SEQ ID NO:192)

[0502] GATATCCAGATGACACAGAGCCCCAGCAGCCTGACCGCCTCTGTGGGCGACCGGGTGACCATCACCTGTAGAGCCAGC CAGTCCATCAGCTCCTAC GTGAACTGGTATCAGCAGAAACCTGGCAAGGCCCCTAAGGTGCTGATCTTT GCTGCCAGC AGCCTGCAGAGCGGAGTCCCATCTAGATTCAGCGGCTCTGGCAGCGGCACCGACTTCACCCTGACAATTAGCTCTCTGCAGGCCGAGGACGTGGCCGTGTACTACTGC CAGCAATACTACAGCACCCCTCTGACC TTCGGCGGCGGAACAAAGGTGGAAATCAAG

[0503] HB9-14H12

[0504] Heavy chain:

[0505] (SEQ ID NO:196)

[0506] QVQLKESGPGLVAPSQSLSITCTVS GFLLISNG VHWVRQPPGKGLEWLGV IWAGGNTN YNSALMSRVSISKDNSKSQVFLKMKSLQTDDTAMYYC ARDFYDYDVFYYAMDY WGQGTSVTVSS

[0507] HB9-14H12

[0508] (SEQ ID NO:200)

[0509] CAAGTCCAGCTGAAGGAAAGCGGCCCAGGCCTGGTGGCCCCTAGCCAGAGCCTGAGCATCACATGTACCGTGTCC GGCTTCCTGCTGATCAGCAACGGC GTGCACTGGGTGCGGCAGCCTCCCGGCAAGGGCCTCGAGTGGCTGGGAGTG ATCTGGGCTGGCGGAAATACCAAC TACAACAGCGCCCTGATGTCTAGAGTGTCTATCTCTAAGGACAACAGCAAGTCCCAGGTGTTCCTGAAGATGAAAAGCCTGCAGACCGACGACACCGCCATGTACTACTGC GCCAGAGATTTCTAC GATTATGACGTTTTTTACTACGCCATGGACTAC TGGGGCCAGGGCACCAGCGTGACAGTGTCCAGC

[0510] Light chain:

[0511] HB9-14H12

[0512] (SEQ ID NO:204)

[0513] QAVVTQESALTTSPGETVPLTCRSS TGTVTTSNF ANWVQEKPDHLFTGLIG GTN NRAPGLPARFSGSLIGDKAALTITGAQTEDEAIFFC ALWYSNHFV FGGGSQLTVL

[0514] HB9-14H12

[0515] (SEQ ID NO:208)

[0516] CAAGCCGTGGTGACACAGGAATCCGCCCTCACAACCTCTCCAGGCGAGACAGTGCCTCTGACCTGTAGAAGCAGC ACCGGCACCGTGACCACCAGCAACTTC GCCAACTGGGTCCAGGAAAAGCCCGACCACCTGTTCACCGGCCTGATCGGC GGAACAAAC AACCGGGCCCCTGGCCTGCCTGCTAGATTCAGCGGCAGCCTGATCGGAGATAAGGCCGCTCTGACAATCACCGGCGCCCAGACCGAGGACGAGGCCATCTTTTTCTGC GCCCTGTGGTACAGCAATCACTTTGTG TTCGGCGGCGGATCTCAGCTGACCGTGCTG

[0517] Table 1: Sequences

[0518]

[0519]

[0520]

[0521] The disclosure of each patent, patent application and publication cited herein is incorporated herein by reference in its entirety. Although the present invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of the present invention can be designed by those skilled in the art without departing from the true spirit and scope of the present invention. The appended claims are intended to be interpreted as including all such embodiments and equivalent variations.

Claims

1. A Siglec-9 binding molecule comprising a Siglec-9 antibody, a Siglec-9 binding antibody fragment, or a variant of a Siglec-9 antibody.

2. The Siglec-9 binding molecule according to claim 1, wherein the antibody comprises an amino acid sequence selected from the group consisting of: a) a variable heavy chain sequence comprising a CDR sequence selected from the group consisting of: SEQ ID NOs: 1-3; SEQ ID NOs: 17-19; SEQ ID NOs: 33-35; SEQ ID NOs: 49-51; SEQ ID NOs: 65-67; ​​SEQ ID NOs: 81-83; SEQ ID NOs: 97-99; SEQ ID NOs: 113-115; SEQ ID NOs: 129-131; SEQ ID NOs: 145-147; SEQ ID NOs: 161-163; SEQ ID NOs: 177-179; and SEQ ID NOs: 193-195; b) a variable light chain sequence comprising a CDR sequence selected from the group consisting of: SEQ ID NOs: 9-11; SEQ ID NOs: 25-27; SEQ ID NOs: 41-43; SEQ ID NOs: 57-59; SEQ ID NOs: 73-75; SEQ ID NOs: 89-91; SEQ ID NOs: 105-107; SEQ ID NOs: 121-123; SEQ ID NOs: 137-139; SEQ ID NOs: 153-155; SEQ ID NOs: 169-171; SEQ ID NOs: 185-187; and SEQ ID NOs: 201-203; c) a sequence that is at least 95% identical to one or more of the variable heavy chain sequences of SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, SEQ ID NO:84, SEQ ID NO:100, SEQ ID NO:116, SEQ ID NO:132, SEQ ID NO:148, SEQ ID NO:164, SEQ ID NO:180, and SEQ ID NO:196; d) a sequence that is at least 95% identical to one or more of the variable light chain sequences of SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 44, SEQ ID NO: 60, SEQ ID NO: 76, SEQ ID NO: 92, SEQ ID NO: 108, SEQ ID NO: 124, SEQ ID NO: 140, SEQ ID NO: 156, SEQ ID NO: 172, SEQ ID NO: 188, and SEQ ID NO: 204; e) a fragment comprising at least 80% of the full-length sequence of a variable heavy chain sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, SEQ ID NO:84, SEQ ID NO:100, SEQ ID NO:116, SEQ ID NO:132, SEQ ID NO:148, SEQ ID NO:164, SEQ ID NO:180, and SEQ ID NO:196; and f) a fragment comprising at least 80% of the full-length sequence of a variable light chain sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 44, SEQ ID NO: 60, SEQ ID NO: 76, SEQ ID NO: 92, SEQ ID NO: 108, SEQ ID NO: 124, SEQ ID NO: 140, SEQ ID NO: 156, SEQ ID NO: 172, SEQ ID NO: 188 and SEQ ID NO:

204.

3. The Siglec-9 binding molecule or fragment thereof according to claim 1, wherein the Siglec-9 binding molecule comprises an amino acid sequence selected from the group consisting of: a) a variable heavy chain sequence comprising the CDR sequences of SEQ ID NOs: 1-3 and a variable light chain sequence comprising the CDR sequences of SEQ ID NOs: 9-11; b) a variable heavy chain sequence comprising the CDR sequences of SEQ ID NOs: 17-19 and a variable light chain sequence comprising the CDR sequences of SEQ ID NOs: 25-27; c) a variable heavy chain sequence comprising the CDR sequences of SEQ ID NOs: 33-35 and a variable light chain sequence comprising the CDR sequences of SEQ ID NOs: 41-43; d) a variable heavy chain sequence comprising the CDR sequences of SEQ ID NOs: 49-51 and a variable light chain sequence comprising the CDR sequences of SEQ ID NOs: 57-59; e) a variable heavy chain sequence comprising the CDR sequences of SEQ ID NOs: 65-67 and a variable light chain sequence comprising the CDR sequences of SEQ ID NOs: 73-75; f) a variable heavy chain sequence comprising the CDR sequences of SEQ ID NOs: 81-83 and a variable light chain sequence comprising the CDR sequences of SEQ ID NOs: 89-91; g) a variable heavy chain sequence comprising the CDR sequences of SEQ ID NOs: 97-99 and a variable light chain sequence comprising the CDR sequences of SEQ ID NOs: 105-107; h) a variable heavy chain sequence comprising the CDR sequences of SEQ ID NOs: 113-115 and a variable light chain sequence comprising the CDR sequences of SEQ ID NOs: 121-123; i) a variable heavy chain sequence comprising the CDR sequences of SEQ ID NOs: 129-131 and a variable light chain sequence comprising the CDR sequences of SEQ ID NOs: 137-139; j) a variable heavy chain sequence comprising the CDR sequences of SEQ ID NOs: 145-147 and a variable light chain sequence comprising the CDR sequences of SEQ ID NOs: 153-155; k) a variable heavy chain sequence comprising the CDR sequences of SEQ ID NOs: 161-163 and a variable light chain sequence comprising the CDR sequences of SEQ ID NOs: 169-171; 1) a variable heavy chain sequence comprising the CDR sequences of SEQ ID NOs: 177-179 and a variable light chain sequence comprising the CDR sequences of SEQ ID NOs: 185-187; and m) a variable heavy chain sequence comprising the CDR sequences of SEQ ID NOs: 193-195 and a variable light chain sequence comprising the CDR sequences of SEQ ID NOs: 201-203.

4. The Siglec-9 binding molecule or fragment thereof according to claim 1, wherein the Siglec-9 binding molecule comprises an amino acid sequence selected from the group consisting of: a) the variable heavy chain sequence of SEQ ID NO: 4 and the variable light chain sequence of SEQ ID NO: 12; b) the variable heavy chain sequence of SEQ ID NO: 20 and the variable light chain sequence of SEQ ID NO: 28; c) the variable heavy chain sequence of SEQ ID NO: 36 and the variable light chain sequence of SEQ ID NO: 44; d) the variable heavy chain sequence of SEQ ID NO: 52 and the variable light chain sequence of SEQ ID NO: 60; e) the variable heavy chain sequence of SEQ ID NO: 68 and the variable light chain sequence of SEQ ID NO: 76; f) the variable heavy chain sequence of SEQ ID NO: 84 and the variable light chain sequence of SEQ ID NO: 92; g) the variable heavy chain sequence of SEQ ID NO: 100 and the variable light chain sequence of SEQ ID NO: 108; h) the variable heavy chain sequence of SEQ ID NO: 116 and the variable light chain sequence of SEQ ID NO: 124; i) the variable heavy chain sequence of SEQ ID NO: 132 and the variable light chain sequence of SEQ ID NO: 140; j) the variable heavy chain sequence of SEQ ID NO: 148 and the variable light chain sequence of SEQ ID NO: 156; k) the variable heavy chain sequence of SEQ ID NO: 164 and the variable light chain sequence of SEQ ID NO: 172; 1) a variable heavy chain sequence of SEQ ID NO: 180 and a variable light chain sequence of SEQ ID NO: 188; and m) the variable heavy chain sequence of SEQ ID NO: 196 and the variable light chain sequence of SEQ ID NO:

204.

5. The Siglec-9 binding molecule or fragment thereof according to claim 1, wherein the Siglec-9 binding molecule comprises a fusion protein comprising a Siglec-9 antibody, a Siglec-9 binding antibody fragment or a variant of a Siglec-9 antibody.

6. A nucleic acid molecule, or a combination of multiple nucleic acid molecules, comprising one or more nucleotide sequences encoding the Siglec-9 binding molecule according to any one of claims 1 to 5.

7. The nucleic acid molecule according to claim 6, wherein the nucleic acid molecule comprises one or more nucleotide sequences selected from the group consisting of: a) a nucleotide sequence encoding a variable heavy chain sequence, wherein the nucleotide sequence comprises a CDR encoding sequence selected from the group consisting of: SEQ ID NOs: 5-7; SEQ ID NOs: 21-23; SEQ ID NOs: 37-39; SEQ ID NOs: 53-55; SEQ ID NOs: 69-71; SEQ ID NOs: 85-87; SEQ ID NOs: 101-103; SEQ ID NOs: 117-119; SEQ ID NOs: 133-135; SEQ ID NOs: 149-151; SEQ ID NOs: 165-167; SEQ ID NOs: 181-183; and SEQ ID NOs: 197-199; b) a nucleotide sequence encoding a variable light chain sequence, wherein the nucleotide sequence comprises a CDR encoding sequence selected from the group consisting of: SEQ ID NOs: 13-15; SEQ ID NOs: 29-31; SEQ ID NOs: 45-47; SEQ ID NOs: 61-63; SEQ ID NOs: 77-79; SEQ ID NOs: 93-95; SEQ ID NOs: 109-111; SEQ ID NOs: 125-127; SEQ ID NOs: 141-143; SEQ ID NOs: 157-159; SEQ ID NOs: 173-175; SEQ ID NOs: 189-191; and SEQ ID NOs: 205-207; c) a nucleotide sequence having at least 95% identity to a variable heavy chain encoding sequence selected from the group consisting of SEQ ID NO:8; SEQ ID NO:24; SEQ ID NO:40; SEQ ID NO:56; SEQ ID NO:72; SEQ ID NO:88; SEQ ID NO:104; SEQ ID NO:120; SEQ ID NO:136; SEQ ID NO:152; SEQ ID NO:168; SEQ ID NO:184; and SEQ ID NO:200; d) a nucleotide sequence having at least 95% identity to a variable light chain encoding sequence selected from the group consisting of SEQ ID NO: 16; SEQ ID NO: 32; SEQ ID NO: 48; SEQ ID NO: 64; SEQ ID NO: 80; SEQ ID NO: 96; SEQ ID NO: 112; SEQ ID NO: 128; SEQ ID NO: 144; SEQ ID NO: 160; SEQ ID NO: 176; SEQ ID NO: 192; and SEQ ID NO: 208; e) a fragment comprising at least 80% of the full-length sequence of a variable heavy chain encoding sequence selected from the group consisting of: SEQ ID NO:8; SEQ ID NO:24; SEQ ID NO:40; SEQ ID NO:56; SEQ ID NO:72; SEQ ID NO:88; SEQ ID NO:104; SEQ ID NO:120; SEQ ID NO:136; SEQ ID NO:152; SEQ ID NO:168; SEQ ID NO:184; and SEQ ID NO:200; and f) a fragment comprising at least 80% of the full-length sequence of a variable light chain encoding sequence selected from the group consisting of SEQ ID NO: 16; SEQ ID NO: 32; SEQ ID NO: 48; SEQ ID NO: 64; SEQ ID NO: 80; SEQ ID NO: 96; SEQ ID NO: 112; SEQ ID NO: 128; SEQ ID NO: 144; SEQ ID NO: 160; SEQ ID NO: 176; SEQ ID NO: 192; and SEQ ID NO:

208.

8. The nucleic acid molecule or molecules according to claim 7, wherein the nucleic acid molecule or molecules comprises a nucleotide sequence selected from the group consisting of: a) a first nucleotide sequence encoding a variable heavy chain sequence comprising SEQ ID NOs: 5-7, and a second nucleotide sequence encoding a variable light chain sequence comprising SEQ ID NOs: 13-15; b) a first nucleotide sequence encoding a variable heavy chain sequence comprising SEQ ID NOs: 21-23, and a second nucleotide sequence encoding a variable light chain sequence comprising SEQ ID NOs: 29-31; c) a first nucleotide sequence encoding a variable heavy chain sequence comprising SEQ ID NOs: 37-39, and a second nucleotide sequence encoding a variable light chain sequence comprising SEQ ID NOs: 45-47; d) a first nucleotide sequence encoding a variable heavy chain sequence comprising SEQ ID NOs: 53-55, and a second nucleotide sequence encoding a variable light chain sequence comprising SEQ ID NOs: 61-63; e) a first nucleotide sequence encoding a variable heavy chain sequence comprising SEQ ID NOs: 69-71, and a second nucleotide sequence encoding a variable light chain sequence comprising SEQ ID NOs: 77-79; f) a first nucleotide sequence encoding a variable heavy chain sequence comprising SEQ ID NOs: 85-87, and a second nucleotide sequence encoding a variable light chain sequence comprising SEQ ID NOs: 93-95; g) a first nucleotide sequence encoding a variable heavy chain sequence comprising SEQ ID NOs: 101-103, and a second nucleotide sequence encoding a variable light chain sequence comprising SEQ ID NOs: 109-111; h) a first nucleotide sequence encoding a variable heavy chain sequence comprising SEQ ID NOs: 117-119, and a second nucleotide sequence encoding a variable light chain sequence comprising SEQ ID NOs: 125-127; i) a first nucleotide sequence encoding a variable heavy chain sequence comprising SEQ ID NOs: 133-135, and a second nucleotide sequence encoding a variable light chain sequence comprising SEQ ID NOs: 141-143; j) a first nucleotide sequence encoding a variable heavy chain sequence comprising SEQ ID NOs: 149-151, and a second nucleotide sequence encoding a variable light chain sequence comprising SEQ ID NOs: 157-159; k) a first nucleotide sequence encoding a variable heavy chain sequence comprising SEQ ID NOs: 165-167, and a second nucleotide sequence encoding a variable light chain sequence comprising SEQ ID NOs: 173-175; 1) a first nucleotide sequence encoding a variable heavy chain sequence comprising SEQ ID NOs: 181-183, and a second nucleotide sequence encoding a variable light chain sequence comprising SEQ ID NOs: 189-191; and m) a first nucleotide sequence encoding a variable heavy chain sequence comprising SEQ ID NOs: 197-199, and a second nucleotide sequence encoding a variable light chain sequence comprising SEQ ID NOs: 205-207.

9. The nucleic acid molecule or molecules according to claim 7, wherein the nucleic acid molecule or molecules comprises a nucleotide sequence selected from the group consisting of: a) a first nucleotide sequence encoding a variable heavy chain sequence comprising SEQ ID NO: 8, and a second nucleotide sequence encoding a variable light chain sequence comprising SEQ ID NO: 16; b) a first nucleotide sequence encoding a variable heavy chain sequence comprising SEQ ID NO: 24, and a second nucleotide sequence encoding a variable light chain sequence comprising SEQ ID NO: 32; c) a first nucleotide sequence encoding a variable heavy chain sequence comprising SEQ ID NO: 40, and a second nucleotide sequence encoding a variable light chain sequence comprising SEQ ID NO: 48; d) a first nucleotide sequence encoding a variable heavy chain sequence comprising SEQ ID NO: 56, and a second nucleotide sequence encoding a variable light chain sequence comprising SEQ ID NO: 64; e) a first nucleotide sequence encoding a variable heavy chain sequence comprising SEQ ID NO: 72, and a second nucleotide sequence encoding a variable light chain sequence comprising SEQ ID NO: 80; f) a first nucleotide sequence encoding a variable heavy chain sequence comprising SEQ ID NO: 88, and a second nucleotide sequence encoding a variable light chain sequence comprising SEQ ID NO: 96; g) a first nucleotide sequence encoding a variable heavy chain sequence comprising SEQ ID NO: 104, and a second nucleotide sequence encoding a variable light chain sequence comprising SEQ ID NO: 112; h) a first nucleotide sequence encoding a variable heavy chain sequence comprising SEQ ID NO: 120, and a second nucleotide sequence encoding a variable light chain sequence comprising SEQ ID NO: 128; i) a first nucleotide sequence encoding a variable heavy chain sequence comprising SEQ ID NO: 136, and a second nucleotide sequence encoding a variable light chain sequence comprising SEQ ID NO: 144; j) a first nucleotide sequence encoding a variable heavy chain sequence comprising SEQ ID NO: 152, and a second nucleotide sequence encoding a variable light chain sequence comprising SEQ ID NO: 160; k) a first nucleotide sequence encoding a variable heavy chain sequence comprising SEQ ID NO: 168, and a second nucleotide sequence encoding a variable light chain sequence comprising SEQ ID NO: 176; 1) a first nucleotide sequence encoding a variable heavy chain sequence comprising SEQ ID NO: 184, and a second nucleotide sequence encoding a variable light chain sequence comprising SEQ ID NO: 192; and m) a first nucleotide sequence encoding a variable heavy chain sequence comprising SEQ ID NO: 200, and a second nucleotide sequence encoding a variable light chain sequence comprising SEQ ID NO:

208.

10. A composition comprising the Siglec-9 binding molecule according to any one of claims 1-5. The composition according to claim 10 , further comprising at least one selected from the group consisting of pharmaceutically acceptable excipients and adjuvants.

12. The composition according to claim 10, further comprising a PD-(L)1 axis inhibitor.

13. The composition according to claim 10, further comprising a tumor antigen or a nucleic acid molecule encoding a tumor antigen.

14. The composition of claim 10, comprising a delivery vehicle comprising a Siglec-7 binding molecule.

15. The composition of claim 14, wherein the delivery vehicle is a lipid nanoparticle.

16. The composition according to any one of claims 10 to 15, further comprising at least one selected from the group consisting of pharmaceutically acceptable excipients and adjuvants.

17. A composition comprising one or more nucleic acid molecules encoding Siglec-9 binding molecules according to any one of claims 6 to 9.

18. The composition of claim 17, wherein the composition comprises a combination of a first expression vector and a second expression vector, the first expression vector comprising a nucleotide sequence encoding a heavy chain sequence, and the second expression vector comprising a nucleotide sequence encoding a light chain sequence.

19. The composition of claim 17, wherein the composition comprises a single expression vector comprising a first nucleotide sequence encoding a heavy chain sequence and a second nucleotide sequence encoding a light chain sequence.

20. The composition of claim 17, further comprising a PD-(L)1 axis inhibitor.

21. The composition according to claim 17, further comprising a tumor antigen or a nucleic acid molecule encoding a tumor antigen.

22. The composition of claim 17, comprising a delivery vehicle comprising the nucleic acid molecule or molecules.

23. The composition of claim 22, wherein the delivery vehicle is a lipid nanoparticle.

24. The composition according to any one of claims 17 to 23, further comprising at least one selected from the group consisting of pharmaceutically acceptable excipients and adjuvants.

25. A method for treating or preventing a disease or disorder in a subject in need thereof, the method comprising administering to the subject a Siglec-9 binding molecule according to any one of claims 1-5, one or more nucleic acid molecules encoding a Siglec-9 binding molecule according to any one of claims 6-9, or a composition according to any one of claims 10-24.

26. The method of claim 25, wherein the disease or condition is cancer or a disease or condition associated with cancer.

27. The method of claim 26, wherein the cancer has increased sialic acid levels.

28. The method of claim 27, wherein the cancer is selected from the group consisting of ovarian cancer, melanoma, renal cell carcinoma, prostate cancer, colon cancer, breast cancer, head and neck squamous cell carcinoma, skin cancer, and oral cancer.

29. The method of claim 25, wherein the disease or condition is an infectious disease or condition.

30. A method of increasing natural killer cell function in a subject in need thereof, the method comprising administering to the subject a Siglec-9 binding molecule according to any one of claims 1-5, one or more nucleic acid molecules encoding a Siglec-9 binding molecule according to any one of claims 6-9, or a composition according to any one of claims 10-24.

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