Antibodies Against Human Siglec-9 and Their Use for Immunotherapy - Patent application

Abstract

The present invention provides compositions comprising anti-Siglec antibodies and nucleic acid molecules encoding same, and methods for using them to treat or prevent a disease or disorder.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 325,865, filed March 31, 2022, which is incorporated by reference in its entirety herein. [Background technology]

[0002] Cancers manipulate several immunological mechanisms of action to ensure a permissive local microenvironment conducive to tumor progression. Glycosylation is frequently mentioned as a hallmark of cancer. Alterations in the surface glycosylation of tumor / infected cells appear as an important feature of cancer and different infectious diseases. Sialylated glycans found on both glycoproteins and glycolipids are recognized by Siglecs, a family of lectins expressed on the surface of many immune cell subtypes. Thus, the interaction of sialic acid-bound cancer / infected cells with Siglecs modulates the phenotype of immune cells, enabling them to evade the immune system.

[0003] Siglecs contain a Sia-binding N-terminal V-set domain followed by a C2-set Ig domain, which are thought to function as spacers or regulators of oligomerization. Most of them function as transmembrane receptors in the immune system through the recognition of Sia residues. Each type of Siglec broadly recognizes a specific set of sialylated structures.

[0004] Siglec-9 is an important inhibitory sialic acid-binding immunoglobulin-like lectin (Siglec) receptor expressed by NK cells (CD16pos / CD56dim), B cells, and monocytes. Interaction of Siglec-9 with Sia leads to phosphorylation of immunoreceptor tyrosine-based inhibitory motifs (ITIMs), which can suppress NK cell cytotoxicity.

[0005] Studies have shown that aberrantly glycosylated mucins (including MUC1 and MUC16) on cancer cells bind to Siglec-9 on immune cells, inhibiting the antitumor function of natural killer (NK) and T cells and modulating the immune function of myeloid cells. Siglec-9 / sialic acid binding can generate inhibitory signals that result in a reduced immune response in the tumor microenvironment.

[0006] There remains a need in the art for new cancer therapies that effectively treat cancer while minimizing adverse effects. The present invention fulfills this unmet need. Summary of the Invention

[0007] In one embodiment, the present invention relates to an antibody or fragment thereof that specifically binds to a sialic acid binding receptor, hi one embodiment, the sialic acid binding receptor is Siglec-9.

[0008] In one embodiment, the antibody comprises a variable heavy chain sequence comprising the CDR sequences of SEQ ID NOs: 1 to 3, SEQ ID NOs: 17 to 19, SEQ ID NOs: 33 to 35, SEQ ID NOs: 49 to 51, SEQ ID NOs: 65 to 67, SEQ ID NOs: 81 to 83, SEQ ID NOs: 97 to 99, SEQ ID NOs: 113 to 115, SEQ ID NOs: 129 to 131, SEQ ID NOs: 145 to 147, SEQ ID NOs: 161 to 163, SEQ ID NOs: 177 to 179, or SEQ ID NOs: 193 to 195.

[0009] In one embodiment, the antibody comprises a variable heavy chain sequence comprising the CDR sequences of SEQ ID NOs: 9 to 11, SEQ ID NOs: 25 to 27, SEQ ID NOs: 41 to 43, SEQ ID NOs: 57 to 59, SEQ ID NOs: 73 to 75, SEQ ID NOs: 89 to 91, SEQ ID NOs: 105 to 107, SEQ ID NOs: 121 to 123, SEQ ID NOs: 137 to 139, SEQ ID NOs: 153 to 155, SEQ ID NOs: 169 to 171, SEQ ID NOs: 185 to 187, or SEQ ID NOs: 201 to 203.

[0010] In one embodiment, the antibody comprises the 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.

[0011] In one embodiment, the antibody comprises the 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.

[0012] In one embodiment, the antibody comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 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. 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.

[0013] 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 the variable heavy chain sequence of SEQ ID NO: 148 and the variable light chain sequence of SEQ ID NO: 156. In one embodiment, the antibody comprises the variable heavy chain sequence of SEQ ID NO: 164 and the variable light chain sequence of SEQ ID NO: 172. In one embodiment, the antibody comprises the variable heavy chain sequence of SEQ ID NO: 180 and the variable light chain sequence of SEQ ID NO: 188. In one embodiment, the antibody comprises the variable heavy chain sequence of SEQ ID NO: 196 and the variable light chain sequence of SEQ ID NO:204.

[0014] In one embodiment, the antibody comprises a sequence having at least 95% identity 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 having at least 95% identity 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. a fragment comprising at least 80% of the full length of the 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; or a fragment comprising at least 80% of the full length of the 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.

[0015] In one embodiment, the present invention relates to a nucleic acid molecule encoding an antibody or a fragment thereof that specifically binds to a sialic acid binding receptor, hi one embodiment, the sialic acid binding receptor is Siglec-9.

[0016] In one embodiment, the nucleic acid molecule comprises 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 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 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 having at least 95% identity to one or more of the variable light chain sequences of 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 fragment comprising at least 80% of the full length sequence of the 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; or a fragment comprising at least 80% of the full length sequence of the variable light chain sequence selected from one or more 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.

[0017] 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 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.

[0018] 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 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.

[0019] 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 having at least 95% identity 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.

[0020] 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 NO: 13-15, SEQ ID NO: 29-31, SEQ ID NO: 45-47, SEQ ID NO: 61-63, SEQ ID NO: 77-79, SEQ ID NO: 93-95, SEQ ID NO: 109-111, SEQ ID 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 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 having at least 95% identity 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, or SEQ ID NO: 208.

[0021] In one embodiment, the present invention relates to a combination of nucleic acid molecules, wherein the first nucleic acid molecule comprises a heavy chain CDR coding sequence of SEQ ID NO:5 to 7, SEQ ID NO:21 to 23, SEQ ID NO:37 to 39, SEQ ID NO:53 to 55, SEQ ID NO:69 to 71, SEQ ID NO:85 to 87, SEQ ID NO:101 to 103, SEQ ID NO:117 to 119, SEQ ID NO:133 to 135, SEQ ID NO:149 to 151, SEQ ID NO:165 to 167, SEQ ID NO:181 to 183, or SEQ ID NO:197 to 199. and the second nucleic acid molecule comprises a light chain CDR coding sequence of SEQ ID NO: 13-15, SEQ ID NO: 29-31, SEQ ID NO: 45-47, SEQ ID NO: 61-63, SEQ ID NO: 77-79, SEQ ID NO: 93-95, SEQ ID NO: 109-111, SEQ ID 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 comprising a heavy chain coding 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, or a fragment or variant thereof, and a second nucleic acid molecule comprising a light chain coding 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, or a fragment or variant thereof.

[0022] 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 pharma- ceutically 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.

[0023] 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 pharma- ceutically 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.

[0024] In one embodiment, the present invention relates to a method for treating or preventing a disease or disorder in a subject in need thereof, 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 disorder is cancer or a cancer-related disease or disorder. In one embodiment, the disease or disorder is an infectious disease or disorder.

[0025] In one embodiment, the invention relates to a method for increasing natural killer cell function in a subject in need thereof, 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.

[0026] In one embodiment, the 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.

[0027] In one embodiment, the disease or disorder is cancer or a cancer-related disease or disorder. In one embodiment, the cancer has increased levels of sialic acid. In one embodiment, the cancer is ovarian cancer, melanoma, renal cell carcinoma, prostate cancer, colon cancer, breast cancer, squamous cell carcinoma of the head and neck, or oral cancer.

[0028] In one embodiment, the present invention relates to a method for increasing natural killer cell function in a subject in need thereof, 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.

[0029] In one embodiment, the present invention relates to a method for increasing natural killer cell function in a subject in need thereof, 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]

[0030]

Figure 1

Figure 2

Figure 3

[0031] The present invention relates to sialic acid receptor antibodies, fragments thereof, variants thereof, or nucleic acid molecules encoding same, and methods of use for increasing natural killer cell activity in a subject in need thereof.

[0032] In one aspect, the present invention relates to compositions that can be used to augment or enhance an immune response, i.e., generate a more effective immune response, by administering a sialic acid receptor antibody, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding same. In one embodiment, the sialic acid receptor antibody is an antibody against Siglec-9.

[0033] In one aspect, the invention relates to a combination of a sialic acid receptor antibody, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding same, with a nucleic acid molecule encoding a tumor antigen.

[0034] In one aspect, the present invention relates to a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a sialic acid receptor antibody, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding same. In one embodiment, the disease or disorder is cancer. In one embodiment, the disease or disorder is an infectious disease.

[0035] In one embodiment, the present invention relates to a method for treating cancer or a disease or disorder related thereto in a subject in need thereof, 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, hi one embodiment, the cancer has increased levels of sialic acid.

[0036] definition 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 this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described.

[0037] As used herein, each of the following terms has the meaning associated with it in this section.

[0038] The articles "a" and "an" are used herein to refer to one or to more than one (e.g., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0039] As used herein, "about" when referring to a measurable value, such as an amount, duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate for performing the methods of the present disclosure.

[0040] "Antibody" may mean IgG, IgM, IgA, IgD, or IgE class antibodies or fragments, fragments or derivatives thereof, including Fab, F(ab')2, Fd, and single chain antibodies, and derivatives thereof. The antibody may be an antibody isolated from a mammalian serum sample, a polyclonal antibody, an affinity purified antibody, or a mixture thereof, that exhibits sufficient binding specificity for a desired epitope.

[0041] "Antigen" refers to a protein capable of producing an immune response in a host. An antigen is capable of being recognized and bound by an antibody. An antigen may originate from within the body or from the external environment.

[0042] "CDR" is defined as the complementarity determining region amino acid sequences of an antibody that are the hypervariable regions of the immunoglobulin heavy and light chains. See, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., USDepartment of Health and Human Services, National Institutes of Health (1987). There are three heavy chain CDRs and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. Thus, as used herein, "CDR" refers to all three heavy chain CDRs or all three light chain CDRs (or both all heavy chain CDRs and all light chain CDRs, as appropriate). The structure and protein folding of an antibody may mean that other residues are considered to be part of the antigen binding region, and one of skill in the art would so understand. See, for example, Chothia et al., (1989) Conformations of immunoglobulin hypervariable regions; Nature 342, p877-883.

[0043] "Antibody fragment" or "fragment of an antibody" are used interchangeably herein and refer to a portion of an intact antibody that contains the antigen binding site or variable region. This portion does not include the constant heavy chain domains of the Fc region of the intact antibody (i.e., CH2, CH3, or CH4, depending on the antibody isotype). 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 comprising only one light chain variable domain, single-chain polypeptides comprising the three CDRs of a light chain variable domain, single-chain polypeptides comprising only one heavy chain variable region, and single-chain polypeptides comprising the three CDRs of a heavy chain variable region.

[0044] As used herein, "adjuvant" means any molecule added to the vaccines described herein to enhance the immunogenicity of an antigen.

[0045] As used herein, a "coding sequence" or "encoding nucleic acid" may refer to a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence encoding an antibody described herein. A coding sequence may also comprise a DNA sequence encoding an RNA sequence. A coding sequence may also comprise initiation and termination signals operably linked to regulatory elements comprising a promoter and a polyadenylation signal capable of directing expression in cells of an individual or mammal to which the nucleic acid is administered. A coding sequence may further comprise a sequence encoding a signal peptide.

[0046] As used herein, "complementary" or "complementary" may mean that a nucleic acid may have Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairing between nucleotides or nucleotide analogs of the nucleic acid molecule.

[0047] A "disease" is a condition in the health of an animal where the animal is unable to maintain homeostasis and where the animal's health continues to deteriorate if the disease is not ameliorated.

[0048] In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis but which is less favorable than if the disorder were not present. If untreated, the disorder does not necessarily cause the animal's health to deteriorate further.

[0049] A disease or disorder is "alleviated" if the severity of the signs and symptoms of the disease or disorder, the frequency with which a patient experiences such signs and symptoms, or both, are reduced.

[0050] "Encoding" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined nucleotide (i.e., rRNA, tRNA, mRNA) sequence or a defined amino acid sequence and the biological properties resulting therefrom. Thus, a gene encodes a protein when the protein is produced in a cell or other biological system by transcription and translation of the mRNA corresponding to that gene. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence, usually provided in a sequence listing) and the non-coding strand used as a template for transcription of a gene or cDNA can be referred to as encoding the protein or other product of that gene or cDNA.

[0051] An "effective amount" of a compound is an amount of the compound sufficient to confer a benefit on a subject or system to which the compound is administered.

[0052] "Expression vector" refers to a vector that contains a recombinant polynucleotide that includes an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can 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) that incorporate a recombinant polynucleotide.

[0053] As used herein, a "feedback mechanism" refers to a process implemented by software or hardware (or firmware) that receives a desired tissue impedance (before, during, and / or after delivery of an energy pulse), compares the impedance to a current value (preferably current), and adjusts the delivered energy pulse to achieve a preset value. The feedback mechanism may be implemented by an analog closed loop circuit.

[0054] "Fragment" may refer to a polypeptide fragment of an antibody that is functional, i.e., capable of binding to a desired target and having the same intended effect as the full-length antibody. An antibody fragment may be 100% identical to the full-length antibody except for the deletion of at least one amino acid from the N-terminus and / or C-terminus, in each case with or without a signal peptide and / or a methionine at position 1. A fragment may consist of 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more of the length of a particular full-length antibody excluding an added heterologous signal peptide. Fragments may include fragments of 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 may further include an N-terminal methionine or a heterologous signal peptide that is not included when calculating percent identity. Fragments may further include 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 the signal peptide may be linked to a fragment of an antibody.

[0055] 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 a signal peptide and / or a methionine at position 1. A fragment may comprise 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more of the length of the particular full length coding sequence excluding any added heterologous signal peptide. Fragments may include fragments that encode a polypeptide that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the antibody, and may optionally include sequences encoding an N-terminal methionine or a heterologous signal peptide that are not included when calculating percent identity. Fragments 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 the N-terminal methionine and / or a signal peptide may be linked to the fragment of the coding sequence.

[0056] As used herein, a "genetic construct" refers to a DNA or RNA molecule that includes a nucleotide sequence that encodes a protein, such as an antibody. A genetic construct may also refer to a DNA molecule that transcribes RNA. The coding sequence includes initiation and termination signals operably linked to regulatory elements, such as a promoter and a polyadenylation signal, that can direct expression in the cells of an individual to which the nucleic acid molecule is administered. As used herein, the term "expressible form" refers to a genetic construct that includes the necessary regulatory elements operably linked to a coding sequence that encodes a protein, such that the coding sequence is expressed when present in the cells of an individual.

[0057] "Homologous" refers to sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. If a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if a position in each of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage of homology between 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, multiplied by 100. For example, if 6 out of 10 positions in the two sequences are matching or homologous, then the two sequences are 60% homologous. As an example, the DNA sequences ATTGCC and TATGGC have 50% homology. Generally, the comparison is performed by aligning the two sequences to obtain maximum homology.

[0058] "Identical" or "identity" as used herein in the context of two or more nucleic acid or polypeptide sequences means that the sequences have a certain percentage of residues that are the same over a specified region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over a specified region, determining the number of positions where identical residues are present in both sequences to calculate 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 obtain the percentage of sequence identity. If the two sequences are of different lengths, or the alignment results in one or more staggered ends such that only one sequence is included in the specified comparison region, the residues of that one sequence are included in the denominator but not in the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identification can be done manually or using computer sequence algorithms such as BLAST or BLAST 2.0.

[0059] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from the coexisting materials of the natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a host cell.

[0060] In the context of the present invention, the following abbreviations for commonly occurring nucleobases are used: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine and "U" refers to uridine.

[0061] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate forms of each other and that 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 may contain introns in some form.

[0062] As used herein, "impedance" is used when discussing feedback mechanisms and can be converted to a current value according to Ohm's law, thus allowing comparison to a preset current.

[0063] As used herein, "immune response" may refer to activation of a host's immune system, e.g., a mammalian 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.

[0064] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal or cells thereof, whether in vitro or in situ, that can be amenable to the methods described herein. In some embodiments, the patient, subject, or individual is a human.

[0065] "Parenteral" administration of the compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intradermal injection or infusion techniques.

[0066] As used herein, "nucleic acid" or "oligonucleotide" or "polynucleotide" may mean at least two nucleotides covalently linked. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of the depicted single strand. Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and their complements. A single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.

[0067] 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, and may contain combinations of deoxyribonucleotides and ribonucleotides, and combinations 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.

[0068] As used herein, "operably linked" may mean that the expression of a gene is under the control of a promoter with which the gene is spatially connected. The promoter may be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene may be approximately 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, changes in distance can be accommodated without loss of promoter function.

[0069] As used herein, "peptide," "protein," or "polypeptide" can refer to a linked sequence of amino acids, which can be natural, synthetic, or a modified or combination of natural and synthetic.

[0070] A "promoter" as used herein may refer to a synthetic or naturally derived molecule that can confer, activate, or enhance expression of a nucleic acid in a cell. A promoter may contain one or more specific transcription control sequences to further enhance expression and / or alter its spatial and / or temporal expression. A promoter may also contain distal enhancer or repressor elements that can be located several thousand base pairs away 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 genetic components 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 such as physiological stress, pathogens, metal ions, or inducers. Representative examples of promoters include a bacteriophage T7 promoter, a bacteriophage T3 promoter, an SP6 promoter, a lac operator promoter, a tac promoter, an SV40 late promoter, an SV40 early promoter, an RSV-LTR promoter, a CMV IE promoter, an SV40 early promoter or an SV40 late promoter, and a CMV IE promoter.

[0071] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to the promoter / regulatory sequence. In some cases, this sequence may be a core promoter sequence, and in other cases, this sequence may also include an enhancer sequence and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may, for example, be one that provides tissue-specific expression of the gene product.

[0072] A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes production of a gene product in a cell under most or all physiological conditions of the cell.

[0073] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes production of the gene product in a cell substantially only when an inducer corresponding to the promoter is present in the cell.

[0074] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes production of the gene product in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0075] "Signal peptide" and "leader sequence" are used interchangeably herein and refer to an amino acid sequence that may be linked at the amino terminus of a protein described herein. A signal peptide / leader sequence typically directs the localization of a protein. As used herein, a signal peptide / leader sequence may facilitate secretion of a protein from the cell in which it is produced. A signal peptide / leader sequence is often cleaved from the remainder of the protein, often referred to as the mature protein, upon secretion from the cell. A signal peptide / leader sequence is linked at the N-terminus of a protein.

[0076] As used herein, "stringent hybridization conditions" may refer to conditions under which a first nucleic acid sequence (e.g., a probe) hybridizes to a second nucleic acid sequence (e.g., a target), such as in a complex mixture of nucleic acids. Stringent conditions are sequence-dependent and will be different in different circumstances. Stringent conditions may be selected to be about 5-10°C lower than the thermal melting point (Tm) of a particular sequence at a defined ionic strength pH. Tm is the temperature at which 50% of the probes complementary to the target hybridize (under defined ionic strength, pH, and nucleic acid concentration) to the target sequence at equilibrium (because the target sequence is present in excess, at Tm 50% of the probes are occupied at equilibrium). Stringent conditions are those in which the salt concentration is less than about 1.0 M sodium ion, for example, about 0.01 to 1.0 M sodium ion concentration (or other salt) at pH 7.0 to 8.3, and the temperature is at least about 30° C. for short probes (e.g., about 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., more than about 50 nucleotides). Stringent conditions can also be achieved by adding a destabilizing agent such as formamide. For selective or specific hybridization, a positive signal may be at least 2 to 10 times the background hybridization. Exemplary stringent hybridization conditions include: 50% formamide, 5×SSC and 1% SDS, incubation at 42° C., or 5×SSC, 1% SDS, incubation at 65° C., followed by washing with 0.2×SSC and 0.1% SDS at 65° C.

[0077] "Subject" and "patient", used interchangeably herein, refer 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, e.g., cynomolgus monkeys, 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.

[0078] As used herein, "substantially complementary" means that a first sequence is substantially complementary to 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. It may mean 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, or that the two sequences hybridize under stringent hybridization conditions.

[0079] As used herein, "substantially identical" means that a first sequence and a second sequence 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, 800, 900, 1000, 11 "complementary" means 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 over a region of 10, 00, or more nucleotides or amino acids, or that with respect to nucleic acids, a first sequence is substantially complementary to the complement of a second sequence.

[0080] As used herein, "synthetic antibody" refers to an antibody encoded by a recombinant nucleic acid sequence as described herein and generated in a subject.

[0081] As used herein, "treatment" or "treating" can mean protecting a subject from a disease by means of preventing, suppressing, arresting, or completely eliminating the disease. Preventing a disease involves administering a vaccine of the invention to a subject before the onset of the disease. Suppressing a disease involves administering a vaccine of the invention to a subject after induction of the disease but before it becomes clinically manifest. Suppressing a disease involves administering a vaccine of the invention to a subject after the disease becomes clinically manifest.

[0082] A "therapeutic" treatment is a treatment administered to a subject who exhibits signs and symptoms of a disease or disorder with the intent of reducing the frequency and severity of or eliminating those signs and symptoms.

[0083] As used herein, "treating a disease or disorder" means reducing the frequency or severity, or both, of at least one sign or symptom of the disease or disorder experienced by the patient.

[0084] As used herein, the phrase "therapeutically effective amount" refers to an amount sufficient or effective to prevent or treat (delay or prevent the onset of, prevent, inhibit, reduce, or reverse the progression of) a disease or disorder, including alleviating the signs and / or symptoms of the disease or disorder.

[0085] "Treating" a disease or disorder, as that term is used herein, means reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject.

[0086] A "variant," as used herein with respect to a nucleic acid, 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 that is substantially identical to a reference nucleic acid or its complement; or (iv) a nucleic acid that hybridizes under stringent conditions to a reference nucleic acid, its complement, or a sequence substantially identical thereto.

[0087] A variant is further defined as a peptide or polypeptide that differs in amino acid sequence by insertion, deletion, or conservative substitution of amino acids, but retains at least one biological activity. Representative examples of "biological activity" include the ability to bind to a specific antibody or to stimulate an immune response, etc. A variant can also refer to a protein having an amino acid sequence substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity. Conservative substitutions of amino acids, i.e., replacing an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree and distribution of charged regions), are recognized in the art as typically involving minor changes. These minor changes can be identified, in part, by considering the hydropathic index of an amino acid, as understood in the art (Kyte et al., J. Mol. Biol. 157:105-132 (1982)). The hydropathic index of an amino acid is based on a consideration of its 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 embodiment, amino acids with a hydropathic index of ±2 are substituted. The hydrophilicity of amino acids can also be used to identify substitutions that result in proteins that retain biological function. By considering the hydrophilicity of amino acids in the context of a peptide, the maximum local average hydrophilicity of the peptide can be calculated, which is a useful measure that has been reported to correlate well with antigenicity and immunogenicity. As is understood in the art, substitutions of amino acids with similar hydrophilicity values ​​can result in peptides that retain biological activity, e.g., immunogenicity. Substitutions can be made with amino acids that have hydrophilicity values ​​within ±2 of each other. Both the hydrophobic index and hydrophilicity value of an amino acid are influenced by the particular side chain of that amino acid. Consistent with this observation, it is understood that substitutions of amino acids that are compatible with biological function depend on the relative similarity of the amino acids, particularly the relative similarity of their side chains, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0088] A variant may be a nucleic acid sequence that is substantially identical over the entire length of the entire gene sequence or a fragment thereof. The nucleic acid sequence may 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 entire length of the gene sequence or a fragment thereof. A variant may be an amino acid sequence that is substantially identical over the entire length of the amino acid sequence or a fragment thereof. The amino acid sequence may 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 entire length of the amino acid sequence or a fragment thereof.

[0089] A "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid inside a cell. Numerous vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.

[0090] Ranges: Throughout this disclosure, various aspects of the invention can be presented in the form of a range. It should be understood that the description in the form of a range is merely for convenience and brevity and should not be construed as inflexibly limiting the scope of the invention. Thus, the description of a range should be considered to have all the possible subranges specifically disclosed and individual numbers within that range. For example, the description of a range such as 1-6 is considered to have the specifically disclosed subranges such as 1-3, 1 to 4, 1-5, 2-4, 2-6, 3-6, and individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0091] explanation Provided herein is an antibody or antibody-like molecule that specifically binds 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, or 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 disorders associated with altered glycosylation, including, but not limited to, cancer and infectious diseases.

[0092] Thus, in some embodiments, the invention provides compositions comprising one or more antibodies to Siglec-9, a fragment thereof, or a variant thereof, or a nucleic acid molecule encoding same.

[0093] 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 that specifically binds to Siglec-9, a fragment thereof, or a variant thereof of the present invention, or a nucleic acid molecule encoding same.

[0094] In some embodiments, the present invention provides a method for treating or preventing cancer associated with increased Siglec-9, comprising administering to a subject an antibody or antibody-like molecule that specifically binds to Siglec-9, a fragment thereof, or a variant thereof of the present invention, or a nucleic acid molecule encoding same.

[0095] antibody composition In one embodiment, the invention relates to a composition comprising at least one Siglec-9 antibody, or a fragment or variant thereof.

[0096] 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 the 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.

[0097] In one embodiment, the antibody comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 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. 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.

[0098] 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 the variable heavy chain sequence of SEQ ID NO: 148 and the variable light chain sequence of SEQ ID NO: 156. In one embodiment, the antibody comprises the variable heavy chain sequence of SEQ ID NO: 164 and the variable light chain sequence of SEQ ID NO: 172. In one embodiment, the antibody comprises the variable heavy chain sequence of SEQ ID NO: 180 and the variable light chain sequence of SEQ ID NO: 188. In one embodiment, the antibody comprises the variable heavy chain sequence of SEQ ID NO: 196 and the variable light chain sequence of SEQ ID NO:204.

[0099] In some embodiments, variants of the amino acid 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 designated region when compared to a defined amino acid sequence. In some embodiments, variants of the amino acid sequences described herein include a full-length 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 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: In some embodiments, the amino acid sequence of the variable light chain may be 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 variable light chain having one or more of the amino acid sequences of SEQ ID NO:172, SEQ ID NO:188, or SEQ ID NO:204.

[0100] 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.

[0101] As used herein, the term "antibody" or "immunoglobulin" refers to a protein (including glycoproteins) of the immunoglobulin (Ig) superfamily of proteins. An antibody or immunoglobulin (Ig) molecule may be a tetramer containing 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.

[0102] Sialic acid binding receptor antibodies or antigen-binding fragments thereof include polyclonal antibodies, monoclonal fusion proteins, antibodies or fragments thereof, chimerized 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 display fusion proteins, antibodies or fragments thereof, or repetitive backbone arrays (e.g., repetitive antigen display fusion proteins). The present invention is not limited to antibodies or antigen-binding fragments thereof identified in the above-mentioned examples.

[0103] The immune system produces several different classes of Ig molecules (isotypes), including IgA, IgD, IgE, IgG, and IgM, each distinguished by the particular class of heavy chain polypeptide present: alpha (a) found in IgA, delta (δ) found in IgD, epsilon (ε) found in IgE, gamma (γ) found in IgG, and mu (μ) found in IgM. At least five different gamma heavy chain polypeptides (isotypes) are found in IgG. In contrast, there are only two light chain polypeptide isotypes, termed kappa (K) and lambda (X) chains. The distinctive characteristics of an antibody isotype are defined by the sequence of the constant domain of the heavy chain.

[0104] An IgG molecule contains two light chains (either kappa or lambda types) and two heavy chains (gamma type) that are linked by disulfide bonds. Each of the kappa and lambda types of IgG light chains is referred to as a variable region ("V L Area”, “V κ Area" or "V λ The domains of relatively variable amino acid sequence and the constant regions (C L Similarly, each IgG heavy chain contains domains of relatively conserved amino acid sequences called variable regions (V H A complete IgG heavy chain contains three constant domains ("C H1 ", "C H2 " and "C H3 "region") and the hinge region. L Area or V H Within the region, hypervariable regions, also called complementarity determining regions ("CDRs"), are interspersed between relatively conserved framework regions ("FRs"). In general, the variable region of a light or heavy chain polypeptide comprises four FRs and three CDRs arranged in the following order along the polypeptide: NH2-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-COOH. Together, the CDRs and FRs determine the three-dimensional structure of the IgG binding site and thus the specific target protein or antigen to which the IgG molecule binds. Each IgG molecule is a dimer and can bind two antigen molecules. Cleavage of the 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 easily crystallized).

[0105] As used throughout this disclosure, the term "antibody" further refers to a whole or intact antibody (e.g., IgM, IgG, IgA, IgD, or IgE) molecule produced by any one of a variety of methods known in the art and described herein. The term "antibody" includes polyclonal antibodies, monoclonal antibodies, chimerized or chimeric antibodies, humanized antibodies, deimmunized human antibodies, and fully human antibodies. Antibodies may be made in or derived from any one of a variety of species, e.g., mammals such as humans, non-human primates (e.g., monkeys, baboons, or chimpanzees), horses, cows, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. Antibodies may be purified antibodies or recombinant antibodies.

[0106] As used herein, the term "epitope" refers to a site on a protein to which an antibody binds. "Overlapping epitopes" include at least one (e.g., 2, 3, 4, 5, or 6) common amino acid residues.

[0107] 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 that form a relatively stable complex under physiological conditions. Typically, the binding constant (Ka) is greater than or equal to 10. 6 M -1 Binding is considered specific if the antibody has a binding affinity of at least 10 6 (or more) (e.g., at least 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , or more)M -1 It can specifically bind to the target with a Ka of

[0108] In one embodiment, the antibody of the invention specifically binds to Siglec-9.

[0109] Methods for determining whether an antibody binds to a protein antigen and / or for determining the affinity of an antibody to a protein antigen are known in the art. For example, binding of an antibody to a protein antigen can be detected and / or quantified using a variety of techniques, including, but not limited to, Western blot, dot blot, surface plasmon resonance (e.g., BIAcore system, Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ), or enzyme-linked immunosorbent assay (ELISA). 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,"WHFreeman and Co., NY; Borrebaek (1995) "Antibody Engineering," 2nd Edition, Oxford University Press, NY, Oxford; Johne et al. (1993) J. Immunol. Meth. 160:191-198; Jonsson et al. (1993) Ann. Biol. Clin. al. (1991) Biotechniques 11:620-627. See also U.S. Patent No. 6,355,245.

[0110] 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, RIAs, ELISAs (enzyme-linked immunosorbent assays), "sandwich" immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays. Such assays are routinely performed and well known in the art.

[0111] The antibodies can also be assayed using any surface plasmon resonance (SPR)-based assay known in the art to characterize the kinetic parameters of the interaction of the antibody with its target or epitope. Any commercially available SPR instrument can be used in the methods described herein, including, but not limited to, the BIAcore instrument (Biacore AB, Uppsala, Sweden), the IAsys instrument (Affinity Sensors, Franklin, Massachusetts), the IBIS system (Windsor Scientific Limited; Berks, UK), the SPR-CELLIA system (Nippon Laser and Electronics Lab; Hokkaido, Japan), and the SPR Detector Spreeta (Texas Instruments, Dallas, Texas). 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.

[0112] Antibodies and fragments thereof may be "chimeric" in some embodiments. Chimeric antibodies and antigen-binding fragments thereof contain portions derived from two or more different species (e.g., mouse and human). Chimeric antibodies can be generated by joining mouse variable regions of desired specificity to human constant domain gene segments (see, e.g., U.S. Pat. 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-related diseases in human subjects).

[0113] The monoclonal antibodies of the present disclosure include "humanized" forms of non-human (e.g., murine) antibodies. Humanized or CDR-grafted mAbs are particularly useful as human therapeutics because they are not cleared from the circulation as rapidly as murine antibodies and do not typically provoke adverse immune responses. Methods for preparing humanized antibodies are generally well known in the art. For example, humanization can be essentially performed by following the method of Winter and colleagues, substituting rodent CDRs or CDR sequences with the corresponding sequences of a human antibody (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). See also Staelens et al. (2006) Mol Immunol 43:1243-1257. In some embodiments, humanized forms of non-human (e.g., murine) antibodies are human antibodies (recipient antibodies) in which hypervariable (CDR) region residues of the recipient antibody are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and binding capacity. In some cases, framework region residues of the human immunoglobulin are also replaced by corresponding non-human residues (so-called "back mutations"). Furthermore, phage display libraries can be used to alter amino acids at selected positions within the antibody sequence. The properties of the humanized antibody are also influenced by the choice of human framework. Furthermore, humanized and chimeric antibodies can be modified to include residues that are not found in the recipient or donor antibody to further improve antibody properties, such as affinity or effector function.

[0114] Fully human antibodies are also provided in the present disclosure. The term "human antibody" includes antibodies having variable 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 (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (i.e., humanized antibodies). Fully human or human antibodies may be derived from transgenic mice or human cells that have human antibody genes (with variable (V), diversity (D), joining (J), and constant (C) exons). For example, it is now possible to produce transgenic animals (e.g., mice) that can produce a full repertoire of human antibodies upon immunization without producing endogenous immunoglobulins. (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 may code for both heavy and light chains of human antibodies, which function correctly in the mouse and rearrange to provide a broad antibody repertoire similar to that in humans. The transgenic mice can be immunized with the target protein (to generate a variety of specific antibodies and the RNA encoding them). Nucleic acids encoding the antibody chain components of such antibodies are then cloned from the animal into a display vector.Typically, separate populations of nucleic acids encoding heavy and light chain sequences are cloned, and then the separate populations are recombined upon insertion into a vector, such that any given copy of the vector receives a random combination of heavy and light chains. The vector is designed to express antibody chains such that the antibody chains are 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 phage coat proteins on the extracellular surface. The display packages can then be screened for display of antibodies that bind to a target.

[0115] Thus, in some embodiments, the disclosure provides humanized, deimmunized, or primatized antibodies that comprise one or more of the complementarity determining regions (CDRs) of a murine monoclonal antibody described herein, e.g., that retain the ability (e.g., at least 50, 60, 70, 80, 90, or 100%, or more than 100%) of the murine monoclonal antibody counterpart to bind to its antigen.

[0116] Additionally, human antibodies can be obtained 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 generated that use random combinations of synthetic human antibody V-regions. By selecting antigens, fully human antibodies can be generated in which the V-regions are highly human in nature. See, e.g., 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 incorporated herein by reference.

[0117] 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 incorporated by reference in their entireties. Human antibodies are further discussed and described in U.S. Pat. Nos. 5,939,598, 6,673,986, 6,114,598, 6,075,181, 6,162,963, 6,150,584, 6,713,610, and 6,657,103, and U.S. Patent Application Publication Nos. 2003-0229905 A1, 2004-0010810 A1, US2004-0093622 A1, 2006-0040363 A1, 2005-0054055 A1, 2005-0076395 A1, and 2005-0287630 A1. See also International Publication Nos. WO 94 / 02602, WO 96 / 34096, WO 98 / 24893, and European Patent No. EP 0463151 Bl. The disclosures of each of the above patents, applications, and references are incorporated herein by reference in their entirety.

[0118] In an alternative approach, others, including GenPharm International, Inc., have utilized a "minilocus" approach. In the minilocus approach, an exogenous Ig locus is mimicked by including portions (individual 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) are formed in a construct for insertion into an animal. This approach is described, for example, in 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, 5,814,318, 5,591,669, 5,612,2 Nos. 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 incorporated herein by reference. See also European Patent No. 0546073Bl, International Patent Publication Nos. WO92 / 03918, WO92 / 22645, WO92 / 22647, WO92 / 22670, WO93 / 12227, WO94 / 00569, WO94 / 25585, WO96 / 14436, WO97 / 13852, and WO98 / 24884, the disclosures of each of which are incorporated herein by reference in their entirety.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. 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. See further, et al. (2000) Eur. J. Immunol. 10:2998-3005, the disclosures of each of which are incorporated herein by reference in their entireties.

[0119] In some embodiments, a deimmunized antibody or antigen-binding fragment thereof is provided. A deimmunized antibody or antigen-binding fragment thereof is an antibody that has been modified to render the antibody or antigen-binding fragment thereof non-immunogenic or less immunogenic for a given species (e.g., for humans). Deimmunization can be achieved by modifying the fusion protein, antibody, or fragment thereof using any of a variety of techniques known to those of skill in the art (see, e.g., PCT Publication Nos. WO04 / 108158 and WO00 / 34317). For example, a fusion protein, antibody, or fragment thereof may be deimmunized by identifying potential T-cell and / or B-cell epitopes within the amino acid sequence of the fusion protein, antibody, or fragment thereof, and removing one or more of the potential T-cell and / or B-cell epitopes from the fusion protein, antibody, or fragment thereof, e.g., using recombinant techniques. The modified antibodies or antigen-binding fragments thereof can then optionally be generated and tested to identify antibodies or antigen-binding fragments thereof that retain one or more desired biological activities, such as, for example, binding affinity, but have reduced immunogenicity. Methods for identifying potential T cell and / or B cell epitopes may be performed using techniques known in the art, such as computational methods (see, for example, PCT Publication No. WO02 / 069232), in vitro or in silico techniques, and biological assays or physical methods (e.g., determining binding of a peptide to an MHC molecule, determining binding of a peptide:MHC complex to a T cell receptor from the species receiving the fusion protein, antibody, or fragment thereof, testing of the protein or peptide portion thereof using transgenic animals having MHC molecules of the species receiving the antibody or antigen-binding fragment thereof, or testing using transgenic animals reconstituted with immune system cells from the species receiving the 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 (such as chimeric antibodies, single chain antibodies, CDR-grafted antibodies, humanized antibodies, fully human antibodies, and artificially selected antibodies), synthetic antibodies, and semi-synthetic antibodies.

[0120] In some embodiments, the present disclosure also provides bispecific antibodies. Bispecific antibodies are monoclonal antibodies, preferably human or humanized antibodies, that have binding specificities for at least two different antigens. For example, in one embodiment, a bispecific antibody of the present invention comprises one domain with binding specificity for a Siglec protein or polypeptide and one domain with binding specificity for an alternative protein or polypeptide. In one embodiment, a bispecific antibody of the present invention comprises one domain with binding specificity for a Siglec protein or polypeptide and one domain with binding specificity for an alternative Siglec protein or polypeptide.

[0121] Methods for producing bispecific antibodies are within the knowledge of those skilled in the art. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy / light chain pairs, with the two heavy / light chain pairs having different specificities (Milstein and Cuello (1983) Nature 305:537-539). Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion of the heavy chain variable region is preferably with an immunoglobulin heavy chain constant domain, including at least a part of the hinge, CH2, and CH3 regions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors and co-transfected into a suitable host organism. For details of exemplary methods currently known for generating bispecific antibodies, see, e.g., Suresh et al. (1986) Methods in Enzymology 121:210; PCT Publication No. WO 96 / 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 or heteroconjugate antibodies. Heteroconjugate antibodies may be made using any convenient cross-linking method. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Patent No. 4,676,980, along with a number of cross-linking techniques.

[0122] Various techniques have also been described for producing and isolating bispecific antibody fragments directly from recombinant cell culture. For example, leucine zippers are used to produce bispecific antibodies. See, for example, Kostelny et al. (1992) J Immunol 148(5):1547-1553. The leucine zipper peptides from the Fos and Jun proteins may be linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers may be reduced at the hinge region to form monomers and then reoxidized to form antibody heterodimers. This method can also be used to produce antibody homodimers. The "diabody" technology described by Hollinger et al. (1993) Proc Natl Acad Sci USA 90:6444-6448 provides an alternative mechanism for producing bispecific antibody fragments. The fragments contain a heavy chain variable domain (VH) connected to a light chain variable domain (VL) by a linker that 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 making bispecific antibody fragments by using single-chain Fv (scFv) dimers has also been reported. See, e.g., Gruber et al. (1994) J Immunol 152:5368. Alternatively, the antibodies may be "linear antibodies" as described, e.g., in Zapata et al. (1995) Protein Eng. 8(10):1057-1062. Briefly, these antibodies comprise tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions. Linear antibodies may be bispecific or monospecific.

[0123] 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.

[0124] The present disclosure also encompasses variant forms of multispecific antibodies, such as the dual variable domain immunoglobulin (DVD-lg) molecules described in Wu et al. (2007) Nat Biotechnol 25(11):1290-1297. DVD-lg molecules are designed such that two different light chain variable domains (VL) from two different parent antibodies are linked by recombinant DNA technology directly or via a short linker, followed by a light chain constant domain. Similarly, the heavy chain comprises two different heavy chain variable domains (VH) linked in tandem, followed by the constant domain CH1 and Fc region. Methods for generating DVD-lg molecules from two parent antibodies are further described, for example, in PCT Publication Nos. WO08 / 024188 and WO07 / 024715.

[0125] The disclosure also provides camelid or dromedary antibodies (e.g., antibodies from Camelus bactrianus, Calelus dromaderius, or lama paccos). Such antibodies differ from the typical two chain (fragments) or four chain (whole antibodies) antibodies from most mammals and 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.

[0126] Engineered libraries of camelid antibodies and antibody fragments are commercially available, for example from Ablynx (Ghent, Belgium). As with other antibodies of non-human origin, the amino acid sequences of camelid antibodies can be recombinantly altered to obtain sequences more similar to human sequences, i.e., the nanobodies can be "humanized", thereby further reducing the potential immunogenicity of the antibodies.

[0127] In some embodiments, the present disclosure also provides antibodies or antigen-binding fragments thereof that are variants of the peptides, proteins, or antibodies described herein. In some embodiments, such variant peptides, proteins, or antibodies retain the binding or inhibitory ability of the parent peptide, protein, or antibody. Methods for preparing variants of known proteins, peptides, or antibodies are known in the art. In some embodiments, such variants include at least one amino acid substitution, deletion, insertion, or other modification. In some embodiments, the fusion proteins, antibodies, or fragments thereof described herein include two or more (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, the fusion proteins, antibodies, or fragments thereof described herein do not include amino acid modifications in the CDRs. In some embodiments, the fusion proteins, antibodies, or fragments thereof described herein comprise one or more (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 a CDR.

[0128] As used herein, the terms "antibody fragment", "antigen-binding fragment", "antigen binding fragment" or similar terms refer to a fragment of an antibody that retains the ability to bind to an antigen, and may optionally include additional composition not part of the original antibody (e.g., different framework regions or mutations) and fragments derived 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. An scFv fragment is a single polypeptide chain that contains both the heavy and light chain variable regions of the antibody from which the scFv is derived. Additionally, 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 incorporated herein by reference in their entireties), minibodies, triabodies (Schoonooghe et al. (2009) BMC Biotechnol 9:70), and camelid-derived domain antibodies (also known as "heavy chain immunoglobulins"; Holt et al. (2003) Trends Biotechnol 21(1 1):484-490), the disclosures of each of which are incorporated herein by reference in their entireties, can be incorporated into the compositions and used in the methods described herein. In some embodiments, any of the antigen-binding fragments described herein may be included under the term "antigen-binding fragment thereof" or equivalent terms when referring to fragments related to an antibody, regardless of whether such fragments are actually derived from the antibody or bind to the same epitope or an overlapping epitope or epitopes contained within the epitope of the antibody. The antigen-binding fragments include antigen-binding fragments that bind to the same or overlapping antigens as the original antibody, and include portions that are fragments of the original antibody (e.g., one or more CDRs, one or more variable regions, etc.).

[0129] 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 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 the 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 the 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, or scFv antibody fragments comprising 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.

[0130] In some embodiments, the antibodies described herein contain altered or mutated sequences that result in altered stability or half-life compared to the parent antibody. This includes, for example, increased stability or half-life for higher affinity or longer clearance time in vitro or in vivo, or decreased stability or half-life for lower affinity or faster clearance. Additionally, the antibodies described herein may contain one or more (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., addition of one or more sugar moieties, loss of one or more sugar moieties, or a change in the composition of one or more sugar moieties).

[0131] In some embodiments, the antibodies described herein comprise reduced effector function (e.g., no effector function). Altered effector function includes, 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 response. Modulation refers to an increase, decrease, or elimination of an effector function activity exhibited by a subject antibody comprising an altered constant region compared to the activity of an unaltered form of the constant region. In certain embodiments, modulation includes a situation in which the activity is abolished or completely absent.

[0132] Antibodies with altered or no effector functions may be generated by engineering or producing antibodies with mutated constant regions, Fc regions, or heavy chain regions. Recombinant DNA technology and / or cell culture and expression conditions can be used to produce antibodies with altered function and / or activity. For example, recombinant DNA technology may be used to engineer the substitution, deletion, or insertion of one or more amino acids in a region (such as, for example, the Fc region or constant region) that affects antibody function, including effector function. Alternatively, changes in post-translational modifications, such as, for example, glycosylation patterns, may be achieved by manipulating cell culture and expression conditions in which the antibody is produced. Suitable methods for introducing one or more substitutions, additions, or deletions into an Fc region of an antibody are well known in the art and include, for example, 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.

[0133] nucleic acid molecule Provided herein is a polynucleotide encoding a Siglec-9 antibody or fragment thereof of the present invention. In some embodiments, the polynucleotide also comprises a sequence encoding a signal peptide operably linked to the 5' end of the coding sequence. In some embodiments, the polynucleotide also comprises a sequence encoding a linker sequence.

[0134] In one embodiment, the nucleic acid molecule comprises 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 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 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 having at least 95% identity to one or more of the variable light chain sequences of 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 fragment comprising at least 80% of the full length sequence of the 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; or a fragment comprising at least 80% of the full length sequence of the variable light chain sequence selected from one or more 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.

[0135] 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.

[0136] 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 NO: 13-15, SEQ ID NO: 29-31, SEQ ID NO: 45-47, SEQ ID NO: 61-63, SEQ ID NO: 77-79, SEQ ID NO: 93-95, SEQ ID NO: 109-111, SEQ ID 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 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.

[0137] 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 designated 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 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.Thus, in some embodiments, the variability is in regions outside of the CDR-encoding regions.

[0138] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence having at least 95% identity 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 having at least 95% identity 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.

[0139] In some embodiments, fragments of the nucleotide sequences described herein comprise 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, fragments of the nucleotide sequences described herein comprise 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.

[0140] 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.

[0141] In one embodiment, the present invention relates to a combination of nucleic acid molecules, wherein the first nucleic acid molecule comprises a heavy chain CDR coding sequence of SEQ ID NO:5 to 7, SEQ ID NO:21 to 23, SEQ ID NO:37 to 39, SEQ ID NO:53 to 55, SEQ ID NO:69 to 71, SEQ ID NO:85 to 87, SEQ ID NO:101 to 103, SEQ ID NO:117 to 119, SEQ ID NO:133 to 135, SEQ ID NO:149 to 151, SEQ ID NO:165 to 167, SEQ ID NO:181 to 183, or SEQ ID NO:197 to 199. and the second nucleic acid molecule comprises a light chain CDR coding sequence of SEQ ID NO: 13-15, SEQ ID NO: 29-31, SEQ ID NO: 45-47, SEQ ID NO: 61-63, SEQ ID NO: 77-79, SEQ ID NO: 93-95, SEQ ID NO: 109-111, SEQ ID 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 comprising a heavy chain coding 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, or a fragment or variant thereof, and a second nucleic acid molecule comprising a light chain coding 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, or a fragment or variant thereof.

[0142] The isolated nucleic acid may comprise any type of nucleic acid, including, but not limited to, DNA, cDNA, and RNA. For example, in one embodiment, the composition comprises an isolated DNA molecule, such as an isolated cDNA molecule, that encodes the protein inhibitor or a functional fragment thereof. In one embodiment, the composition comprises an isolated RNA molecule that encodes the protein inhibitor or a functional fragment thereof.

[0143] 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.

[0144] The nucleic acid molecules of the present invention can be modified to improve stability in serum or in growth medium for cell culture. Modifications can be made to enhance the stability, functionality, and / or specificity of the nucleic acid molecules of the present invention and minimize their immunostimulatory properties. For example, to enhance stability, the 3' residues can be stabilized against degradation, e.g., selected to be composed of purine nucleotides, particularly adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides with modified analogs, e.g., substitution of uridine with 2'-deoxythymidine, is tolerated and does not affect the function of the molecule.

[0145] In one embodiment of the invention, the nucleic acid molecule may comprise at least one modified nucleotide analogue, for example the termini may be stabilized by incorporation of modified nucleotide analogues.

[0146] Non-limiting examples of nucleotide analogs include sugar-modified and / or backbone-modified ribonucleotides (i.e., containing modifications to the phosphate-sugar backbone). For example, the phosphodiester linkages of natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom. In exemplary backbone-modified ribonucleotides, the phosphate ester group attached to adjacent ribonucleotides is replaced by a modified group such as a phosphothioate group.

[0147] Another example of modification is nucleobase-modified ribonucleotide, i.e., ribonucleotide containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. The base may 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-deazaadenosine; O- and N-alkylated nucleotides, such as N6-methyladenosine, and are preferred. The above modifications may also be combined.

[0148] In some cases, the nucleic acid molecule comprises at least one of the following chemical modifications of one or more nucleotides: 2'-H, 2'-O-methyl, or 2'-OH modification. In some embodiments, the nucleic acid molecule of the present invention can have enhanced resistance to nucleases. To increase nuclease resistance, the nucleic acid molecule can comprise, for example, 2' modified ribose units and / or phosphorothioate linkages. For example, the 2' hydroxyl group (OH) can be modified or replaced with many different "oxy" or "deoxy" substituents. To increase nuclease resistance, the nucleic acid molecule of the present invention can comprise 2'-O-methyl, 2'-fluorine, 2'-O-methoxyethyl, 2'-O-aminopropyl, 2'-amino, and / or phosphorothioate linkages. Inclusion of locked nucleic acids (LNA), ethylene nucleic acids (ENA), e.g., 2'-4'-ethylene bridged nucleic acids, and certain nucleobase modifications, such as 2-amino-A, 2-thio (e.g., 2-thio-U), and G-clamp modifications can increase binding affinity to the target.

[0149] In one embodiment, the nucleic acid molecule comprises a 2'-modified nucleotide, 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-methylacetamide (2'-O-NMA). In one embodiment, the nucleic acid molecule comprises at least one 2'-O-methyl modified nucleotide, and in some embodiments, all of the nucleotides of the nucleic acid molecule comprise a 2'-O-methyl modification.

[0150] Nucleic acid agents discussed herein include otherwise unmodified RNA and DNA, as well as RNA and DNA modified, such as to improve efficacy, and polymers of nucleoside surrogates. Unmodified RNA refers to molecules in which the components of the nucleic acid, i.e., sugar, base, and phosphate moieties, are the same or essentially the same as those naturally occurring, e.g., those naturally occurring in the human body. In the art, rare or unusual but naturally occurring RNAs are referred to as modified RNAs (see, e.g., Limbach et al. (Nucleic Acids Res., 1994, 22:2183-2196)). Such rare or unusual RNAs are often referred to as modified RNAs, typically the result of post-transcriptional modifications, and are within the scope of the term unmodified RNA as used herein. Modified RNAs as used herein refer to molecules in which one or more of the components of the nucleic acid, i.e., sugar, base, and phosphate moieties, are different from those naturally occurring, e.g., those that are different from those that occur in the human body. Although they are referred to as "modified RNAs", they of course include molecules that are not strictly speaking RNAs due to the modifications. A nucleoside surrogate is a molecule in which the ribophosphate backbone is replaced with a non-ribophosphate construct that is capable of presenting the bases in the correct spatial relationship such that hybridization is substantially similar to that found with the ribophosphate backbone (e.g., an uncharged mimic of the ribophosphate backbone).

[0151] Modifications of the nucleic acids of the invention may occur at one or more of the phosphate groups, sugar groups, backbone, N-terminus, C-terminus, or nucleobase.

[0152] The invention also includes vectors into which an isolated nucleic acid of the invention has been inserted. The art is replete with suitable vectors useful in the present invention.

[0153] Thus, in another aspect, the present invention relates to a vector comprising the nucleotide sequence of the present invention or the construct of the present invention. The choice of vector depends on the host cell into which the vector is subsequently introduced. In some embodiments, the vector of the present invention is an expression vector. Suitable host cells include a wide range of prokaryotic and eukaryotic host cells. In certain embodiments, the expression vector is selected from the group consisting of viral vectors, bacterial vectors, and mammalian cell vectors. Prokaryotic and / or eukaryotic vector-based systems can be utilized for use in the present invention to produce polynucleotides or their cognate polypeptides. Many such systems are commercially available and widely available.

[0154] In some embodiments, expression of synthetic nucleic acids encoding proteins is typically achieved by operably linking the nucleic acid encoding the protein or a portion thereof to a promoter and incorporating the construct into an expression vector. The vector used is suitable for replication and, optionally, integration into eukaryotic cells. Typical vectors include transcription and translation terminators, initiation sequences, and promoters useful for controlling expression of the desired nucleic acid sequence.

[0155] The recombinant nucleic acid sequence construct may include one or more transcription termination regions. The transcription termination region may be present downstream of the coding sequence to provide efficient termination. The transcription termination region may be obtained from the same gene as the promoter described above, or may be obtained from one or more different genes.

[0156] The recombinant nucleic acid sequence construct may include one or more start codons. 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 one or more signals required for efficient translation initiation (e.g., but not limited to, a ribosome binding site).

[0157] A recombinant nucleic acid sequence construct can include one or more termination or stop codons. The termination codon can be downstream of the coding sequence. The termination codon can be in-frame with the coding sequence. The termination codon can be associated with one or more signals required for efficient translation termination.

[0158] The recombinant nucleic acid sequence construct may include one or more polyadenylation signals. The polyadenylation signal may include one or more signals 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 pCEP4 plasmid (Invitrogen, San Diego, CA).

[0159] The recombinant nucleic acid construct can include one or more leader sequences. The leader sequence can encode a signal peptide. The signal peptide can be an immunoglobulin (Ig) signal peptide, such as, but not limited to, an IgG signal peptide and an IgE signal peptide.

[0160] The vector of the present invention can also be used for nucleic acid immunization using standard gene delivery protocols.Methods for gene delivery are known in the art.See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety.

[0161] The isolated nucleic acids of the present invention can be cloned into many types of vectors. For example, the nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0162] Furthermore, vectors may be provided to cells in the form of viral vectors. Viral vector technology is well known in the art and described, for example, in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, suitable vectors include an origin of replication that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers (e.g., WO01 / 96584, WO01 / 29058, and U.S. Patent No. 6,326,193).

[0163] Furthermore, the expression vector may be provided to the cell in the form of a viral vector.Viral vector technology is well known in the art and described, for example, in Sambrook et al. (2012) and Ausubel et al. (1997), and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, suitable vectors include an origin of replication that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers (see, for example, WO01 / 96584, WO01 / 29058, and U.S. Patent No. 6,326,193).

[0164] By way of example, the vector into which the nucleic acid sequence is introduced may be a plasmid that may or may not integrate into the genome of the host cell when introduced into the cell. Illustrative, non-limiting examples of vectors into which the nucleotide sequence of the invention or the genetic construct of the invention may be inserted include tet-on inducible vectors for expression in eukaryotic cells.

[0165] The vector may be obtained by conventional methods known to those skilled in the art (Sambrook et al., 2012). In certain embodiments, the vector is a vector useful for transforming animal cells.

[0166] In one embodiment, the recombinant expression vector may comprise a nucleic acid molecule encoding a peptide or protein of the invention as described elsewhere herein.

[0167] Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector using techniques known in the art and packaged into retroviral particles. The recombinant virus can then be isolated and delivered to cells of interest 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, lentiviral vectors are used.

[0168] For example, vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer, since they allow long-term stable integration of transgenes and their propagation in daughter cells. Lentivirus vectors have the added advantage of being able to transduce non-proliferating cells, such as hepatocytes, compared to vectors derived from oncogenic retroviruses, such as murine leukemia viruses. They also have the added advantage of being less immunogenic. In one embodiment, the composition comprises a vector derived from adeno-associated virus (AAV). Adeno-associated virus (AAV) vectors have become a powerful gene delivery tool for the treatment of various diseases. AAV vectors have many characteristics that make them ideally suited for gene therapy, including lack of pathogenicity, minimal immunogenicity, and the ability to stably and efficiently transduce post-mitotic cells. Expression of a particular gene contained within an AAV vector can be specifically targeted to one or more cell types by selecting a suitable combination of AAV serotype, promoter, and delivery method.

[0169] In some embodiments, the vector also includes conventional control elements operably linked to the transgene in a manner that allows transcription, translation, and / or expression in cells transfected with the plasmid vector or infected with the virus produced by the present invention. As used herein, "operably linked" sequences include both expression control sequences contiguous with the gene of interest and expression control sequences acting in trans or remotely to control the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient 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, if desired. Many expression control sequences, including native, constitutive, inducible, and / or tissue-specific promoters, are known in the art and may be utilized.

[0170] A promoter may be one that is naturally associated with a gene or polynucleotide sequence, which may be obtained by isolating 5' non-coding sequences located upstream of a coding segment and / or exon. Such a promoter may be referred to as "endogenous". Similarly, an enhancer may be one that is naturally associated with a polynucleotide sequence and located downstream or upstream of that sequence. Alternatively, certain advantages are obtained by placing a coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a polynucleotide sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer that is not normally associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, as well as promoters or enhancers that are not "naturally occurring", i.e., that contain different elements of different transcriptional regulatory regions and / or mutations that alter expression. In addition to producing promoter and enhancer nucleic acid sequences synthetically, the sequences may be produced using recombinant cloning and / or nucleic acid amplification techniques, including PCR, for the compositions disclosed herein (U.S. Patent No. 4,683,202; U.S. Patent No. 5,928,906).Furthermore, it is contemplated that control sequences that direct transcription and / or expression of sequences in non-nuclear organelles, such as mitochondria, chloroplasts, etc., may also be used.

[0171] Of course, it is important to use a promoter and / or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism selected for expression. Those skilled in the art of molecular biology generally know how to use a combination of promoters, enhancers, and cell types for protein expression (see, for example, Sambrook et al. (2012)). The promoter used may be constitutive, tissue-specific, inducible, and / or useful under appropriate conditions to direct high-level expression of the introduced DNA segment, which is advantageous for large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

[0172] The recombinant expression vector may also contain a selection marker gene to facilitate the selection of transformed or transfected host cells. Suitable selection marker genes are proteins such as G418 and hygromycin that confer resistance to certain drugs, β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or genes encoding immunoglobulins or portions thereof (such as the Fc portion of an immunoglobulin such as IgG). The selection marker may be introduced into a vector separate from the nucleic acid of interest.

[0173] Additional promoter elements, e.g. enhancers, regulate the frequency of transcription initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although it has recently been shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible, so that promoter function is preserved when elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased up to 50 bp apart before activity begins to decline. Depending on the promoter, individual elements appear to be able to function cooperatively or independently to activate transcription.

[0174] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked to the promoter sequence. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia 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. Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on expression of an operably linked polynucleotide sequence when such expression is desired, or turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0175] Enhancer sequences found on vectors also regulate the expression of genes contained in the vector. Typically, enhancers bind protein factors to enhance the transcription of genes. Enhancers may be located upstream or downstream of the gene they regulate. Enhancers may also be tissue-specific to enhance transcription in specific cell or tissue types. In one embodiment, the vectors of the present invention contain one or more enhancers to promote the transcription of genes present in the vector.

[0176] To evaluate the expression of protein inhibitors, the expression vector introduced into the cells can also contain either a selection marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from the cell population to be transfected or infected via the viral vector. In other embodiments, the selection marker can be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selection marker and the reporter gene can be flanked by suitable regulatory sequences to allow expression in the host cell. Useful selection markers include, for example, antibiotic resistance genes such as neo.

[0177] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Generally, reporter genes are genes that are not present in or expressed by the recipient organism or tissue and that encode a polypeptide whose expression is manifested by some easily detectable property, such as enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA is introduced into the recipient cells. Suitable reporter genes include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. Generally, the construct with the minimal 5' flanking region that exhibits the highest level of expression of the reporter gene is identified as the promoter. Such promoter regions may be linked to the reporter gene and used to evaluate drugs for their ability to modulate transcription driven by the promoter.

[0178] Methods for introducing and expressing genes into cells are known in the art. In the context of an expression vector, the vector can be easily introduced into a host cell, such as a mammalian, bacterial, yeast, or insect cell, by any method in the art. For example, the expression vector can be introduced into a host cell by physical, chemical, or biological means.

[0179] Physical methods for introducing peptides or proteins into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, etc. Methods for generating 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).

[0180] Biological methods for introducing peptides or proteins of interest into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian (e.g., 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.

[0181] 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 for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0182] When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for introducing nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another embodiment, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, bound to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution containing lipids, complexed with lipids, combined with lipids, contained in a suspension in lipids, contained in or complexed with micelles, or otherwise associated with lipids. The lipid, lipid / DNA, or lipid / expression vector-related compositions are not limited to any particular structure in solution. For example, they may exist in a bilayer structure such as a micelle, or in a "collapsed" structure. They may also simply be dispersed in the solution, which may form aggregates that are not uniform in size or shape.Lipids are fatty substances that may be naturally occurring lipids or synthetic lipids.For example, lipids include lipid droplets that naturally occur in cytoplasm, and a class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0183] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma (St. Louis, MO), dicetyl phosphate ("DCP") can be obtained from K&K Laboratories (Plainview, NY), cholesterol ("Choi") can be obtained from Calbiochem-Behring, and dimyristyl phosphatidylglycerol ("DMPG") and other lipids can be obtained 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 generic term that encompasses a variety of unilamellar and multilamellar lipid vesicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before forming a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5:505-10). However, compositions having structures in solution that differ from the normal vesicular structure are also contemplated. For example, lipids may adopt a micellar structure or simply exist as a heterogeneous aggregate of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0184] ScFv antibody In one embodiment, the antibody fragment comprises an scFv fragment. In one embodiment, the ScFv antibody fragment relates to a Fab fragment that does not have the CHI and CL regions. Thus, in one embodiment, the scFv antibody fragment relates to a Fab fragment that comprises a VH and a VL. In one embodiment, the scFv antibody fragment comprises a linker between the VH and the VL. In one embodiment, the scFv antibody fragment comprises a VH, a VL, and a CH2 and a CH3 region. In one embodiment, the scFv antibody fragment of the invention has modified expression, stability, half-life, antigen binding, heavy-light chain pairing, tissue penetration, or a combination thereof, compared to the parent MAb.

[0185] 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.

[0186] In one embodiment, an 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 antigen binding than the parent MAb.

[0187] In one embodiment, an 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 the parent MAb.

[0188] 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.

[0189] In one embodiment, an 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.

[0190] In one embodiment, an scFv antibody fragment of the invention has heavy chain-light chain pairing 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 greater than the parent MAb.

[0191] Delivery Vehicle 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 same, as described herein.

[0192] Exemplary delivery vehicles include, but are not limited to, microspheres, microparticles, nanoparticles, polymersomes, 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 the same. In some embodiments, the delivery vehicle provides controlled, delayed, or continuous release of the loaded cargo. In some embodiments, the delivery vehicle includes a targeting moiety that targets the delivery vehicle to a treatment site.

[0193] Pharmaceutical Compositions The present invention also provides pharmaceutical compositions comprising one or more of the compositions described herein.The formulations may be used in admixture with conventional excipients, i.e., pharma-ceutically acceptable organic or inorganic carrier substances suitable for administration to the treatment site.The pharmaceutical compositions are sterilized and, if desired, may be admixed with auxiliary substances, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring agents, and / or aromatic substances.They may also be combined with other active agents, such as other analgesics, if desired.

[0194] Administration of the compositions of the invention may be by, for example, parenteral, intravenous, subcutaneous, intramuscular, or intraperitoneal injection or infusion, or any other acceptable systemic method.

[0195] As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients, surfactants, dispersing agents, inert diluents, granulating and disintegrating agents, binders, lubricants, coloring agents, preservatives, physiologically degradable compositions such as gelatin, aqueous vehicles and solvents, oily vehicles and solvents, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, buffers, salts, thickening agents, fillers, emulsifiers, antioxidants, antibiotics, antifungal agents, stabilizers, and pharma- ceutically acceptable polymeric 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.

[0196] The composition of the present invention may contain a preservative in an amount of about 0.005% to 2.0% of the total weight of the composition. Preservatives are used to prevent spoilage when exposed to pollutants in the environment. Examples of preservatives useful according to the present invention include, but are not limited to, those selected from the group of benzyl alcohol, sorbic acid, parabens, imidurea, and combinations thereof.

[0197] In one embodiment, the composition includes an antioxidant and a chelating agent that inhibits the degradation of one or more components of the composition. Representative antioxidants for some compounds are BHT, BHA, alpha tocopherol, and ascorbic acid. Exemplary chelating agents include edetate (e.g., disodium edetate) and citric acid. Chelating agents are useful for chelating metal ions in the composition that may adversely affect the shelf life of the formulation. BHT and disodium edetate may be antioxidants and chelating agents for some compounds, respectively, but may be substituted with other suitable and equivalent antioxidants and chelating agents known to those skilled in the art.

[0198] Liquid suspensions may be prepared using conventional methods for suspending the compounds of the present invention or other compositions in aqueous or oily vehicles. Aqueous vehicles include, for example, water and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, 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 further comprise one or more additional ingredients, including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, colorings, and sweetening agents. Oily suspensions may further comprise a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats and oils, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, and hydroxypropylmethylcellulose. Known dispersing or wetting agents include, but are not limited to, naturally occurring phospholipids such as lecithin, condensation products of alkylene oxides with fatty acids, long chain aliphatic alcohols, partial esters derived from fatty acids and hexitols, or partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively), and the like. Known emulsifying agents include, but are not limited to, lecithin and acacia. Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl parahydroxybenzoates, ascorbic acid, and sorbic acid, and the like.

[0199] For oral administration, tablets, dragees, liquids, drops, suppositories, or capsules, caplets, and gel capsules are particularly suitable. Other formulations suitable for oral administration include, but are not limited to, powder or granule formulations, aqueous or oily suspensions, aqueous or oily solutions, pastes, gels, toothpastes, mouthwashes, coatings, mouthwashes, chewing gums, varnishes, sealants, oral and dental "dissolving strips", or emulsions. Compositions intended 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 and non-toxic pharma- ceutically acceptable excipients that are suitable for the manufacture of tablets. Such excipients include, for example, inert diluents such as lactose, granulating and disintegrating agents such as corn starch, binding agents such as starch, and lubricants such as magnesium stearate.

[0200] The tablets may be uncoated or may be coated using known methods to delay disintegration in the subject's gastrointestinal tract, 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 the tablets. For further example, the 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 osmotically controlled release tablets. The tablets may further comprise a sweetener, a flavoring agent, a coloring agent, a preservative, or a combination thereof to provide a pharma-ceutically elegant and palatable formulation.

[0201] Hard capsules containing the active ingredient may be prepared using a physiologically degradable composition such as gelatin. Such hard capsules contain the active ingredient and may further contain additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin.

[0202] Soft gelatin capsules containing the active ingredient may be prepared using a physiologically degradable composition such as gelatin. Such soft capsules contain the active ingredient, which may be mixed with water or an oil medium such as peanut oil, liquid paraffin, or olive oil.

[0203] For oral administration, the compositions of the present invention may be in the form of tablets or capsules prepared by conventional means, containing pharma- ceutically 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™ film coating systems available from Colorcon, WestPoint, Pastern (e.g., OPADRY™ OY type, OYC type, organic enteric OY-P type, aqueous enteric OY-A type, OY-PM type, and OPADRY™ White, 32K18400).

[0204] Liquid preparations for oral administration may be in the form of solution, syrup, or suspension. Liquid preparations can be prepared by conventional methods using pharma- ceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methylcellulose, or hydrogenated edible fats and oils), emulsifying agents (lecithin or acacia), non-aqueous vehicles (e.g., almond oil, oily esters, or ethyl alcohol), and preservatives (e.g., methyl or propyl p-hydroxybenzoate, or sorbic acid). Liquid formulations 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 a dry product that is intended to be reconstituted with water or other suitable vehicle before use.

[0205] A tablet containing an active ingredient may be produced, for example, by compressing or molding the active ingredient, optionally with one or more additional ingredients. Compressed tablets may be prepared by compressing in a suitable device the active ingredient in a free-flowing form, such as a powder or granular preparation, optionally mixed with one or more of a binder, lubricant, excipient, surfactant, and dispersant. Molded tablets may be produced by molding in a suitable device a mixture of the active ingredient, a pharma- ceutically acceptable carrier, and at least sufficient liquid to moisten the mixture. Pharmaceutically acceptable excipients used in the manufacture of tablets include, but are not limited to, inert diluents, granulating and disintegrating agents, binders, and lubricants. Known dispersing agents include, but are not limited to, potato starch and sodium starch glycolate. Known surfactants include, but are not limited to, sodium lauryl sulfate, and the like. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate. Known granulating and disintegrating agents include, but are not limited to, corn starch and alginic acid. Known binding agents include, but are not limited to, gelatin, acacia, pregelatinized corn starch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Known lubricants include, but are not limited to, magnesium stearate, stearic acid, silica, and talc.

[0206] A formulation of a pharmaceutical composition suitable for parenteral administration comprises the active ingredient combined with a pharma- ceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus or continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, for example in ampoules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional components, including, but not limited to, suspending agents, stabilizers, and dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granules) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0207] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. The suspension or solution may be formulated according to known techniques and may contain, in addition to the active ingredient, additional ingredients such as dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable preparations may be prepared using a non-toxic parenterally acceptable diluent or solvent, for example, water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or diglycerides. Other parenterally administered formulations that are useful include those that contain the active ingredient in microcrystalline form in a liposomal preparation or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may include pharma-ceutically acceptable polymeric or hydrophobic materials, such as emulsions, ion exchange resins, sparingly soluble polymers, or sparingly soluble salts.

[0208] Excipients and other components of the composition The composition may further comprise a pharma- ceutically acceptable excipient. The pharma- ceutically acceptable excipient may be a functional molecule such as a vehicle, an adjuvant, a carrier, or a diluent. The pharma- ceutically acceptable excipient may be a transfection-enhancing agent, which may include surfactants such as immune stimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogs including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalane, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection-enhancing agents.

[0209] The transfection facilitating agent is a polyanion, a polycation (including poly-L-glutamic acid (LGS)), or a lipid. The transfection facilitating agent is poly-L-glutamic acid, which may be present in the composition at a concentration of less than 6 mg / ml. The transfection facilitating agent may also include surfactants such as immune stimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogs including monophosphoryl lipid A, muramyl peptides, quinone analogs, and vesicles such as squalene and squalene, and hyaluronic acid may also be administered in combination with the composition. The composition may also include transfection facilitating agents such as lipids, liposomes (including lecithin liposomes or other liposomes known in the art as DNA-liposome mixtures (see, for example, WO9324640)), calcium ions, viral proteins, polyanions, polycations, or nanoparticles, 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.

[0210] The pharma- ceutically acceptable excipient may be an adjuvant in addition to the checkpoint inhibitor antibody of the present invention. The additional adjuvant may be another gene expressed in an alternative plasmid or a gene delivered as a protein in combination with the above plasmid in the composition. The adjuvant may be selected from the group consisting of α-interferon (IFN-α), β-interferon (IFN-β), γ-interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), cutaneous T-cell-attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosa-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86 (including IL-15 with deleted signal sequence and optionally with a signal peptide from IgE). The adjuvant can be IL-12, IL-15, IL-28, CTACK, TECK, platelet-derived growth factor (PDGF), TNFa, TNFP, 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.

[0211] In addition to the antibodies of the invention, other genes that may be useful as adjuvants include MCP-1, MIP-la, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, IL-18 variants, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, TL-22, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, L These include those encoding ARD, 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, SAPK, SAP-1, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK ligand, Ox40, Ox40 ligand, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2, and functional fragments thereof.

[0212] The composition further comprises a gene enhancer as described in US patent application Ser. No. 021,579, filed Apr. 1, 1994, which is incorporated by reference in its entirety.

[0213] The composition may comprise an amount of DNA from about 1 nanogram to about 100 milligrams, from about 1 microgram to about 10 milligrams, or preferably from about 0.1 microgram to about 10 milligrams, or more preferably from about 1 milligram to about 2 milligrams. In some preferred embodiments, the composition according to the present invention comprises about 5 nanograms to about 1000 micrograms of DNA. In some preferred embodiments, the composition can comprise about 10 nanograms to about 800 micrograms of DNA. In some preferred embodiments, the composition can comprise about 0.1 to about 500 micrograms of DNA. In some preferred embodiments, the composition can comprise about 1 to about 350 micrograms of DNA. In some preferred embodiments, the composition may comprise 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 micrograms 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, or about 100 to about 200 micrograms of DNA.

[0214] The composition can be formulated according to the method of administration used. The injectable pharmaceutical composition can be sterile, pyrogen-free, and particulate-free. An isotonic formulation or solution can be used. Additives for isotonicity 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 further include a stabilizer, including gelatin and albumin. The stabilizer can make the formulation stable for long periods at room or ambient temperature, and includes LGS or a polycation or polyanion.

[0215] Method of administration The present invention provides a method for increasing the function or activity of natural killer (NK) cells. This can be measured, for example, in standard NK cell or T cell-based cytotoxicity assays, which measure the ability of a therapeutic compound to stimulate the killing of sialic acid ligand-positive cells by Siglec-positive lymphocytes. In one embodiment, the antibody preparation causes at least a 10% increase in 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 cytotoxicity, with reference to the cytotoxicity assays described. In one embodiment, the antibody preparation causes at least a 10% increase in cytokine release by Siglec-restricted lymphocytes, optionally at least a 40% or 50% increase in cytokine release, or optionally at least a 70% increase in cytokine release, with reference to the cytotoxicity assays described. In one embodiment, the antibody preparation causes at least a 10% increase in cell surface expression of a marker of cytotoxicity (e.g., CD107 and / or CD137) by Siglec-restricted lymphocytes, optionally at least a 40% or 50% increase, or optionally at least a 70% increase in cell surface expression of a marker of cytotoxicity (e.g., CD107 and / or CD137).

[0216] The present invention also relates to a method of increasing an immune response in a subject. The increased immune response can be used to treat and / or prevent disease in a subject. The method can include administering to the subject a vaccine as disclosed herein. A subject administered the vaccine can have an increased or enhanced immune response compared to a subject administered the antigen alone. In some embodiments, the immune response can be increased by about 0.5-fold to about 15-fold, about 0.5-fold to about 10-fold, or about 0.5-fold to about 8-fold. Alternatively, the immune response in a subject to which the vaccine is administered can 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.

[0217] In still other alternative embodiments, the immune response in a subject to which the vaccine is administered 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 to which the vaccine has been administered 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%.

[0218] The vaccine dosage may be 1 μg to 10 mg of active ingredient / kg body weight / dose, and may be 20 μg to 10 mg of ingredient / kg body weight / dose. 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 times.

[0219] Combination vaccine In one embodiment, the present invention relates to administering a Siglec-9 antibody of the present invention or a nucleic acid molecule encoding the same in combination with one or more additional therapeutic agents. In some embodiments, one or more additional therapeutic agents may be used to induce or enhance an immune response. Thus, in some embodiments, the present invention relates to an immunogenic composition, such as a vaccine, comprising a Siglec-9 antibody of the present invention or a nucleic acid molecule encoding the same in combination with an antigenic protein, a fragment thereof, or a variant thereof, a nucleic acid molecule encoding the same, or a combination thereof. Thus, in some embodiments, the present invention relates to an immunogenic composition, such as a vaccine, comprising a Siglec-9 antibody of the present invention or a nucleic acid molecule encoding the same in combination with a PD-(L)1 axis inhibitor, a fragment thereof, or a variant thereof, a nucleic acid molecule encoding the same, or a combination thereof.

[0220] Combination vaccines can significantly induce an immune response in a subject to which the vaccine is administered, thereby preventing or treating a disease or disorder.

[0221] The immunogenic composition may be a DNA vaccine, a peptide vaccine, or a DNA and peptide combination vaccine. A DNA vaccine may comprise a nucleic acid sequence encoding an antigen. The nucleic acid sequence may be DNA, RNA, cDNA, variants thereof, fragments thereof, or a combination thereof. The nucleic acid sequence may also comprise additional sequences encoding linker, leader, or tag sequences linked to the antigen by a peptide bond. A peptide vaccine may comprise an antigenic peptide, an antigenic protein, variants thereof, fragments thereof, or a combination thereof. A DNA and peptide combination vaccine may comprise the above nucleic acid sequences encoding an antigen and an antigenic peptide or protein.

[0222] The vaccine can induce a humoral immune response in a subject to which the vaccine is administered. The induced humoral immune response can be specific to an antigen. The induced humoral immune response can be reactive to an antigen. The humoral immune response can be induced about 1.5-fold to about 16-fold, about 2-fold to about 12-fold, or about 3-fold to about 10-fold in a subject to which the vaccine is administered. A humoral immune response may be induced in a subject to which the vaccine is administered by 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, at least about 15.0-fold, at least about 15.5-fold, or at least about 16.0-fold.

[0223] The humoral immune response induced by the vaccine may include an increase in the level of neutralizing antibodies associated with a vaccinated subject compared to a non-vaccinated subject. The neutralizing antibodies may be specific to the antigen. The neutralizing antibodies may be reactive with the antigen. The neutralizing antibodies may provide protection against and / or treatment of a disease or disorder in a subject to which the vaccine is administered.

[0224] The humoral immune response induced by the vaccine includes an increase in the level of IgG antibodies associated with the vaccinated subject compared to a non-vaccinated subject. These IgG antibodies can be specific to a tumor antigen. These IgG antibodies can be reactive with the antigen. The level of IgG antibodies associated with the vaccinated subject can be increased by about 1.5-fold to about 16-fold, about 2-fold to about 12-fold, or about 3-fold to about 10-fold compared to a non-vaccinated subject. The level of IgG antibodies associated with the vaccinated subject can be increased by 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 9.0-fold, at least about 10 ... or may be increased by 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 10.0-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, at least about 15.0-fold, at least about 15.5-fold, or at least about 16.0-fold.

[0225] The vaccine can induce a cellular immune response in a subject to which the vaccine is administered. The cellular immune response induced can be specific to an antigen. The cellular immune response induced can be reactive to an antigen. The cellular immune response induced can include CD8 + The induction of a CD8+ T cell response may be antigen-reactive. + T cell responses can be polyfunctional. The cellular immune response induced includes CD8 + This includes the induction of a CD8 T cell response. +T cells produce interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin 2 (IL-2), or a combination of IFN-γ and TNF-α.

[0226] The induced cellular immune response included CD8 associated with vaccinated subjects compared to non-vaccinated subjects. + This may include an increase in CD8 T cell responses associated with the subject to which the vaccine is administered. + The T cell response may 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, as compared to a subject not administered the vaccine. The CD8+ T cell response associated with a vaccinated subject may 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 13.0-fold, at least about 14.0-fold, at least about 15.0-fold, at least about 16.0-fold, at least about 17.0-fold, at least about 18.0-fold, at least about 19.0-fold, at least about 20.0-fold, at least about 21.0-fold, at least about 22.0-fold, at least about 23.0-fold, at least about 24.0-fold, at least about 25.0-fold, at least about 26.0-fold, at least about 27.0-fold, at least about 28.0-fold, at least about 29.0-fold, at least about 30.0-fold, at least about 31.0-fold, at least about 32.0-fold, at least about 33.0-fold, at least about 34.0-fold, at least about 35. The increase may be 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, at least about 15.0-fold, at least about 16.0-fold, at least about 17.0-fold, at least about 18.0-fold, at least about 19.0-fold, at least about 20.0-fold, at least about 21.0-fold, at least about 22.0-fold, at least about 23.0-fold, at least about 24.0-fold, at least about 25.0-fold, at least about 26.0-fold, at least about 27.0-fold, at least about 28.0-fold, at least about 29.0-fold, or at least about 30.0-fold.

[0227] The induced cellular immune response includes CD3 + CD8 + This may include an increase in the frequency of CD3 T cells associated with the vaccinated subject. + CD8 + IFN-γ +The proportion 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, or 20-fold compared to a subject not administered the vaccine.

[0228] The induced cellular immune response includes CD3 + This may include an increase in the proportion of CD8+ T cells present in the vaccinated subject. + CD8 + TNF-α + The proportion 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, or 14-fold compared to a subject not administered the vaccine.

[0229] The induced cellular immune response includes CD3 + CD8 + The presence of CD3 T cells associated with the subject receiving the vaccine may be increased. + CD8 + IL-2 + The proportion 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 compared to a subject not administered the vaccine.

[0230] The induced cellular immune response includes CD3 T cells that produce both IFN-γ and TNF-α. + CD8 + The presence of CD3 T cells associated with the subject receiving the vaccine may be increased. + CD8 + IFN-γ + TNF-α + The proportion 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 compared to a subject not administered the vaccine.

[0231] Vaccine-induced cellular immune responses include CD4 + This may include the induction of a T cell response. + T cell responses are reactive to antigens. + T cell responses can be polyfunctional. The cellular immune response induced includes CD4 + This includes the induction of a CD4 T cell response. + The T cells produce IFN-γ, TNF-α, IL-2, or a combination of IFN-γ and TNF-α.

[0232] The induced cellular immune response includes CD3 + CD4 + The presence of CD3 T cells associated with the subject receiving the vaccine may be increased. + CD4 + IFN-γ + The proportion 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, or 20-fold compared to a subject not administered the vaccine.

[0233] The induced cellular immune response includes CD3 + CD4 + The presence of CD3 T cells associated with the subject receiving the vaccine may be increased. + CD4 + TNF-α + The proportion 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, or 22-fold compared to a subject not administered the vaccine.

[0234] The induced cellular immune response includes CD3 + CD4 +The presence of CD3 T cells associated with the subject receiving the vaccine may be increased. + CD4 + IL-2 + The proportion 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 compared to a subject not administered the vaccine.

[0235] The induced cellular immune response includes CD3 T cells that produce both IFN-γ and TNF-α. + CD4 + The presence of CD3 T cells associated with the subject receiving the vaccine may be increased. + CD4 + IFN-γ + TNF-α + The proportion of associated immunizations is at least about 2-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, 5.0-fold, 5.5-fold, 6.0-fold, 6.5-fold, 7.0-fold, 7.5-fold, 8.0-fold, 8.5-fold, 9.0-fold, 9.5-fold, 10.0-fold, 10.5-fold, 11.0-fold, 11.5-fold, 12.0-fold, 12.5-fold, or 13.0-fold greater than those in unvaccinated subjects , 13.5x, 14.0x, 14.5x, 15.0x, 15.5x, 16.0x, 16.5x, 17.0x, 17.5x, 18.0x, 18.5x, 19.0x, 19.5x, 20.0x, 21x, 22x, 23x, 24x, 25x, 26x, 27x, 28x, 29x, 30x, 31x, 32x, 33x, 34x, or 35x.

[0236] The vaccines of the present invention can have the characteristics necessary for an effective vaccine, such as being safe so that the vaccine itself does not cause illness or death, protecting against illness resulting from exposure to live pathogens such as viruses or bacteria, inducing neutralizing antibodies to prevent cellular proliferation, inducing protective T cells against intracellular pathogens, and providing ease of administration, few side effects, biological stability, and low cost per dose.

[0237] The vaccine can further induce an immune response when administered into a different tissue, such as muscle or skin.

[0238] Tumor antigens As described above, in some embodiments, the combination vaccine of the present invention comprises an anti-Siglec-9 antibody or a nucleic acid molecule encoding same in combination with a tumor antigen, a fragment thereof, a variant thereof, a nucleic acid molecule encoding same, or a combination thereof.

[0239] Thus, in some embodiments, the present invention includes compositions 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, or a biologically functional fragment or variant thereof, or a nucleic acid molecule encoding same, capable of generating an immune response in a subject.

[0240] In one embodiment, the antigen is a tumor-associated surface antigen. 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 1-10, vascular antigen VEGFR2 (KDR / FLK1), VEGFR3 (FLT4, CD309), PDGFR-α (CD140a), PDGFR-β (CD140b), endoglin, CLEC14, Tem1-8, and Tie2. Further examples include A33, CAMPATH-1 (CDw52), carcinoembryonic antigen (CEA), carboanhydrase IX (MN / CAIX), CD21, CD25, CD30, CD34, CD37, CD44v6, CD45, CD133, de2-7 EGFR, 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 include tenascin and fibroblast activation protein (FAP).

[0241] In the context of the present invention, "tumor antigen" or "hyperproliferative disorder antigen" or "antigen associated with a hyperproliferative disorder" refers to an antigen common to a particular hyperproliferative disorder, such as cancer. The antigens discussed herein are included as examples only. The list is not intended to be exhaustive, and further examples will be readily apparent to one of skill in the art.

[0242] Tumor antigens are proteins produced by tumor cells that elicit an immune response, in particular a T cell-mediated immune response. The choice of antigen-binding moiety of the invention depends on the particular type of cancer being 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, muthsp70-2, M-CSF, prostase, prostate specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.

[0243] In one embodiment, the tumor antigen comprises one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express many proteins that can serve as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and GP100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-associated molecules such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens are onco-fetal antigens such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotypic immunoglobulins constitute the truly tumor-specific immunoglobulin antigens that are unique to each individual tumor. B-cell differentiation antigens such as CD19, CD20, and CD37 are other potential target antigens in B-cell lymphomas. Several of these antigens (CEA, HER-2, CD19, CD20, idiotypes) have been used as targets for passive immunotherapy with monoclonal antibodies, but with limited success.

[0244] The type of tumor antigen referred to in the present invention may also be a tumor specific antigen (TSA) or a tumor associated antigen (TAA). A TSA is unique to tumor cells and is not present on other cells of the body. A TAA associated antigen is not unique to tumor cells, but instead is also expressed on normal cells under conditions that do not induce a state of immune tolerance to the antigen. Expression of an antigen on a tumor may occur under conditions that allow the immune system to respond to the antigen. A TAA may be an antigen that is expressed on normal cells during fetal development, when the immune system is immature and unable to respond, or an antigen that is usually present at very low levels on normal cells but is expressed at very high levels on tumor cells.

[0245] Non-limiting examples of TSA or TAA antigens include: differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel17), 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 resulting from 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, CAM17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BT These include AA, CA125, CA15-3\CA27.29\BCAA, CA195, 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-related protein, TAAL6, TAG72, TLP, and TPS.

[0246] PD-(L)1 axis inhibitors In some embodiments, the invention provides a combination of an anti-Siglec-9 antibody of the invention with a PD-(L)1 axis inhibitor. In various embodiments, the composition comprises an inhibitor of one or more genes or proteins in the PD-(L)1 axis. In various embodiments, the invention includes compositions and methods for reducing the level or activity of one or more genes or proteins in the PD-(L)1 axis.

[0247] Based on the disclosure provided herein, it will be understood by one of skill in the art that a decrease in the level or activity of one or more genes or proteins in the PD-(L)1 axis includes a decrease in expression of a biomarker, including transcription, translation, or both. A person of skill in the art, given the teachings of the present invention, will also understand that a decrease in the level or activity of one or more genes or proteins in the PD-(L)1 axis includes a decrease in the amount of polypeptide, a decrease in the amount of mRNA, a decrease in transcription, a decrease in translation, or a combination thereof, including a decrease in any activity of one or more genes or proteins in the PD-(L)1 axis.

[0248] Exemplary inhibitors of the PD-(L)1 axis include, but are not limited to, small interfering RNA (siRNA), microRNA, antisense nucleic acids, ribozymes, expression vectors encoding transdominant negative mutants, antibodies, antibody fragments, fusion proteins, aptamers, peptides, and small molecules.

[0249] Those skilled in the art will understand, based on the disclosure provided herein, that one way to reduce the mRNA and / or protein levels of one or more PD-(L)1 axis proteins in a cell is by reducing or inhibiting expression of a nucleic acid encoding the PD-(L)1 axis protein. Thus, the protein levels of a PD-(L)1 axis protein in a cell can be reduced using molecules or compounds that inhibit or reduce gene expression, such as, for example, siRNAs, antisense molecules, or ribozymes. However, the present invention should not be limited to these examples.

[0250] In one embodiment, RNAi is used to reduce the level or activity of PD-(L)1 axis proteins. RNA interference (RNAi) is a phenomenon in which double-stranded RNA (dsRNA) is introduced into a wide range of organisms and cell types, resulting in the degradation of complementary mRNA. In cells, long dsRNA is cleaved into 21-25 nucleotide small interfering RNAs or siRNAs by a ribonuclease known as Dicer. The siRNA then assembles with protein components to form an RNA-induced silencing complex (RISC), which unwinds 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 cleaved, resulting in gene silencing through sequence-specific degradation of the mRNA. Chemical modification of siRNA can aid in intravenous systemic delivery. Optimization of siRNA involves consideration of the overall G / C content, terminal C / T content, Tm, and nucleotide content of the 3' overhangs. Thus, the present invention also includes methods of reducing the levels of one or more PD-(L)1 axis proteins using RNAi technology.

[0251] In some embodiments, the invention includes isolated nucleic acids encoding inhibitors such as proteins, antibodies, siRNAs, or antisense molecules operably linked to a nucleic acid that preferably comprises a promoter / regulatory sequence such that the nucleic acid can direct expression of the inhibitor encoded by the nucleic acid. Thus, the invention encompasses expression vectors and methods for introducing exogenous DNA into cells and co-expressing the exogenous DNA in the cells.

[0252] To evaluate the expression of the inhibitor, the expression vector introduced into the cell may also contain either a selection marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from the cell population to be transfected or infected via the viral vector. In other embodiments, the selection marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selection marker and the reporter gene may be flanked by suitable regulatory sequences to allow expression in the host cell. Useful selection markers are known in the art and include, for example, antibiotic resistance genes such as neo.

[0253] When the inhibitor of the present invention is a small molecule, the small molecule antagonist may be obtained using standard methods known to those skilled in the art, including chemical organic synthesis or biological means, including purification from biological sources, recombinant synthesis, and in vitro translation systems, using methods well known in the art.

[0254] Combinatorial libraries of molecularly diverse compounds potentially useful in the treatment of various diseases and conditions are well known in the art, as are methods for generating the libraries. Methods may use a variety of techniques well known to those of skill 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, tagging techniques, and the generation of unbiased molecular landscapes for lead discovery versus biased structures for lead development.

[0255] In a common method of small library synthesis, an activated core molecule is enriched with many building blocks, generating a combinatorial library of covalently linked core building block assemblies. The shape and rigidity of the core determines the orientation of the building blocks in shape space. Libraries can be biased by altering the core, linkages, or building blocks to target distinct biological structures ("focused libraries"), or can be synthesized with less structural bias using flexible cores.

[0256] 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 proteins. Thus, inhibiting the effect of one or more proteins in the PD-(L)1 axis can be achieved by using transdominant negative mutants.

[0257] In one embodiment, antibodies specific for one or more proteins in the PD-(L)1 axis may be used. As will be appreciated by those skilled in the art, any antibody capable of recognizing and binding an antigen of interest is useful in the present invention. Methods for making and using antibodies are well known in the art. For example, polyclonal antibodies useful in the present invention are generated by immunizing rabbits according to standard immunological techniques well known in the art. Such techniques include immunizing animals with chimeric proteins that include a portion of another protein, such as a maltose binding protein or glutathione (GSH) tag polypeptide portion and / or a portion such that the antigen protein of interest is immunogenic (e.g., an antigen of interest conjugated to keyhole limpet hemocyanin (KLH)) and a portion that includes the respective antigen protein amino acid residues. Chimeric proteins are generated by cloning the appropriate nucleic acid encoding the marker protein into a plasmid vector suitable for this purpose, including, but not limited to, pMAL-2 or pCMX.

[0258] Exemplary anti-PD-(L)1 axis antibodies include nivolumab (OPDIVO®), pembrolimumab (KEYTRUDA®), sintilimab, cemiplimab (LIBTAYO®), tripolibamab, tislelizumab, spartalizumab, camrelizumab, dostralimab, genolimuzumab, or cetrelimab, or an antibody that binds to PD-L1 (e.g., Enbuf). These include, but are not limited to, PD-L1 antibodies, which are: olimab, atezolizumab (TECENTRIQ®), durvalumab (IMFINZI®), avelumab (BAVENCIO®), REGN2810, pidilizumab, MEDI0680, PDR001, PF-06801591, BGB-A317, TSR-042, and SHR-1210.

[0259] Methods for delivering the composition The present invention also relates to a method of delivering the composition to a subject in need thereof, comprising administering the composition to the subject, including, but not limited to, DNA injection with or without in vivo electroporation, liposome-mediated delivery, and nanoparticle facilitated delivery.

[0260] The mammal to which the composition is delivered may 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, a bovid, a deer, a hedgehog, an elephant, a llama, an alpaca, a mouse, a rat, and a chicken.

[0261] The composition may be administered by different routes, including orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, by inhalation, buccal administration, intrathoracically, intravenously, intraarterially, intraperitoneally, subcutaneously, intramuscularly, intranasally, intranasally, intrathecally, and intraarticularly, or combinations thereof. For veterinary use, the composition may be administered in a suitably acceptable formulation according to normal veterinary practice. A veterinarian can easily determine the most appropriate dosing regimen and route of administration for a particular animal. The composition may be administered by conventional syringes, needleless injection devices, "microprojectile bombardment guns," or other physical methods such as electroporation ("EP"), "hydrodynamic methods," or ultrasound.

[0262] Treatment method In one embodiment, the present invention provides a method of treating or preventing a disease or disorder that would benefit from increased NK cell function or activity. Exemplary diseases and disorders that can be treated using the compositions and methods of the present invention include, but are not limited to, cancer and infectious diseases.

[0263] The following are non-limiting examples of cancers that can be diagnosed or treated by the methods and compositions of the present disclosure: 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 tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, central nervous system atypical teratoid / rhabdoid tumor, central nervous system embryonal tumor, central nervous system lymphoma, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, cerebral astrocytoma / malignant lymphoma Glioma, cervical cancer, childhood visual pathway tumors, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorder, colon cancer, colorectal cancer, craniopharyngioma, skin cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, Ewing family tumors, extracranial cancer, extragonadal germ cell tumors, extrahepatic bile duct cancer, extrahepatic cancer, eye cancer, fungoides, gallbladder cancer, stomach cancer, gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (gist), germ cell tumor, gestational cancer cancer), gestational trophoblastic neoplasm, glioblastoma, glioma, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic tract glioma, hypothalamic tumor, intraocular (eye) cancer, intraocular melanoma, pancreatic 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, bone malignant fibrous histiocytoma and osteosarcoma, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell neck cancer of unknown primary, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis, myelodysplastic syndrome, myelodysplastic / myeloproliferative disorder, myeloid leukemia leukemia, myeloid leukemia, myeloma, myeloproliferative disorders, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cancer, cavity cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovary, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancercancer), pheochromocytoma, intermediate pineal parenchymal tumor, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasma cell neoplasm, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, primary central nervous system cancer, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter cancer, respiratory cancer related to the nut gene on chromosome 15, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sezary syndrome, skin cancer (melanoma), skin cancer (non-melanoma), skin cancer, small cell lung cancer, small intestine cancer, soft tissue cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell cervical cancer, gastric cancer, supratentorial primitive neuroectodermal tumor, supratentorial primitive neuroectodermal tumor and pineoblastoma, T-cell lymphoma, testicular cancer, pharyngeal cancer cancer), thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma, renal pelvis and ureteral transitional cell carcinoma, trophoblastic tumor, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, optic nerve hypothalamic glioma, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

[0264] In one embodiment, the composition is used to treat cancers with high levels of sialic acid. Cancers associated with high levels of sialic acid include, but are not limited to, ovarian cancer, melanoma, renal cell carcinoma, prostate cancer, colon cancer, breast cancer, squamous cell carcinoma of the head and neck, skin cancer, and oral cancer.

[0265] bacterial infection In one embodiment, the infectious disease or disorder is associated with bacteria. In some embodiments, the bacteria may be from any one of the following phyla: Acidobacteria, Actinobacteria, Aquificae, Bacteroidetes, Caldiserica, Chlamydiae, Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria. Cyanobacteria, Deferribacteres, Deinococcus-Thermus, Dictyoglomi, Elusimicrobia, Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonads, imonadetes, Lentisphaerae, Nitrospira, Planctomycetes, Proteobacteria, Spirochaetes, Synergistetes, Tenericutes, Thermodesulfobacteria, Thermotogae, and Verrucomicrobia.

[0266] 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, neutrophilic, extremophilic, acidophilic, alkaliphilic, thermophilic, psychrophilic, halophilic, or osmophilic.

[0267] The bacteria can be Bacillus anthracis, antibiotic-resistant bacteria, pathogenic bacteria, food poisoning bacteria, infectious bacteria, Salmonella, Staphylococcus, Streptococcus, or Clostridium tetani. The bacteria can be Mycobacterium tuberculosis, Clostridium tetani, Yersinia pestis, Bacillus anthracis, methicillin-resistant Staphylococcus aureus (MRSA), or Clostridium difficile.

[0268] Viral infections In one embodiment, the infectious disease or disorder is associated with bacteria. In some embodiments, the virus is from any of the following families: Adenoviridae, Arenaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, or Viruses. Orthomyxoviridae, Papovaviridae, Paramyxoviridae, Parvoviridae, Picornaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, or Togaviridae.Viral antigens include those from human immunodeficiency virus (HIV), chikungunya virus (CHIKV), dengue virus, papillomaviruses (e.g., human papillomavirus (HPV)), poliovirus, hepatitis viruses (e.g., 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, hepatitis A virus, human T-cell leukemia virus (HTLV-I), hairy cell leukemia virus (HEV), and poliovirus. TLV-II), California encephalitis virus, Hantavirus (hemorrhagic fever), Rabies virus, Ebola virus, Marburg virus, Measles virus, Mumps virus, Respiratory syncytial virus (RSV), Herpes simplex type 1 (oral herpes), Herpes simplex type 2 (genital herpes), Shingles (varicella, also known as chickenpox), Cytomegalovirus (CMV) (e.g., human CMV), Epstein-Barr virus (EBV), Flavivirus, Foot and mouth disease virus, Lassa virus, Arenavirus, Severe acute respiratory syndrome-related coronavirus (SARS), Middle East respiratory syndrome-related coronavirus (MERS), Severe acute respiratory syndrome-related coronavirus 2 (SARS CoV2), or a virus that causes cancer.

[0269] Parasitic infections In one embodiment, the infectious disease or disorder is associated with a parasite. In some embodiments, the parasite can be a protozoan, a helminth, or an ectoparasite. The helminth (i.e., worm) can be a flatworm (e.g., flukes and tapeworms), an anchovy, or a roundworm (e.g., pinworm). The ectoparasite can be a lice, a flea, a tick, and a mite.

[0270] The parasite may be any parasite that causes any one of the following diseases: Acanthamoeba keratitis, amebic dysentery, ascariasis, babesiosis, balantidiosis, Baylisascariasis, Chagas disease, clonorchiasis, Cochliomyiasis, Cryptosporidiosis, Diphyllobothriasis, and Guinea pig disease. Insect worms, echinococcosis, elephantiasis, enterobiasis, fascioliasis, filariasis, giardiasis, gnathostomiasis, hymenosteariasis, isosporosis, Katayama fever, leishmaniasis, Lyme disease, malaria, otrichosis, myiasis, onchocerciasis, pediculosis, scabies, schistosomiasis, sleeping sickness, strongyloidiasis, taeniasis, toxocariasis, toxoplasmosis, trichinosis, and trichuriasis.

[0271] Parasites include Acanthamoeba, Anisakis, Ascaris lumbricoides, Botfly, Balantidium coli, Bedbugs, Cestoda (tapeworms), Chiggers, Cochliomyia hominivorax, Entamoeba histolytica, Fasciola hepatica, Giardia lamblia, Hookworm, Leishmania, Linguatula serrata, Liver fluke, Loa loa, Paragonimus-lung fluke, Pinworm, Plasmodium falciparum, Schistosoma, Strongyloides stercoralis, Mite, Tapeworm, Toxoplasma gondii, Trypanosoma, Whipworm, and Wuchereria bancrofti.

[0272] fungal infection In one embodiment, the infectious disease or disorder is fungal associated. In some embodiments, the fungus is selected from the group consisting of Aspergillus species, Blastomyces dermatitidis, Candida yeast (e.g., Candida albicans), Coccidioides, Cryptococcus neoformans, Cryptococcus gattii, dermatophytes, Fusarium species, Histoplasma capsulatum, Mucoromycotina, Pneumocystis jirovecii, Sporothrix schenckii, and the like. schenckii, Exserohilum, or Cladosporium.

[0273] Cancer Treatment In one embodiment, the invention provides a method for treating or preventing cancer, or treating and preventing tumor growth or metastasis. An exemplary related aspect of the invention provides a method for preventing, assisting in the prevention of, and / or reducing metastasis of hyperplastic or tumor cells in an individual.

[0274] In one embodiment, the compositions are used to treat cancers that have high levels of sialic acid, including, but not limited to, ovarian cancer, melanoma, renal cell carcinoma, prostate cancer, colon cancer, breast cancer, squamous cell carcinoma of the head and neck, and oral cancer.

[0275] One aspect of the invention provides a method of inhibiting metastasis in an individual in need thereof, comprising administering to the individual an effective amount of a nucleic acid molecule encoding a multivalent antibody of the invention, where the multivalent antibody is specific for the cancer being treated. The invention further provides a method of inhibiting metastasis in an individual in need thereof, comprising administering to the individual an effective metastasis-inhibiting amount of a nucleic acid molecule encoding a multivalent antibody of the invention, where the multivalent antibody is specific for the cancer being treated.

[0276] In some embodiments of treating or preventing cancer or treating and preventing tumor metastasis in an individual in need thereof, a second agent, such as an anti-neoplastic 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.

[0277] The compositions of the present invention may be used to prevent, alleviate, minimize, control, and / or ameliorate cancer in humans and animals. The compositions of the present invention may also be used to slow the rate of growth of primary tumors. The compositions of the present invention, when administered to a subject in need of treatment, may be used to stop the spread of cancer cells. Thus, an effective amount of a nucleic acid molecule encoding a multivalent antibody of the present invention, where the multivalent antibody is specific to the cancer of the subject being treated, may be administered as part of a combination therapy with one or more drugs or other pharmaceutical agents. When used as part of a combination therapy, the reduction in metastasis and the reduction in growth of the primary tumor provided by the compositions of the present invention allows for more effective and efficient use of any pharmaceutical agent or drug therapy used to treat the patient. Furthermore, the control of metastasis by the compositions of the present invention provides the subject with a greater ability to consolidate the disease.

[0278] In one embodiment, the invention provides a method of treating cancer metastasis comprising treating a 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 following treatment with a composition of the invention.

[0279] Chemotherapeutic agents include cytotoxic agents (e.g., 5-fluorouracil, cisplatin, carboplatin, methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, oxorubicin, carmustine (BCNU), lomustine (CCNU), cytarabine USP, cyclophosphamide, estramucine phosphate sodium, altretamine, hydroxyurea, ifosfamide, procarbazine, mitomycin, busulfan, cyclophosphamide, mitoxantrone, carboplatin, cisplatin, interferon alpha-2a recombinant, paclitaxel, teniposide, and streptozocides, cytotoxic alkylating agents (e.g., busulfan, chlorambucil, cyclophosphamide, melphalan, or ethylsulfonic acid). , alkylating agents (e.g., Asar, AZQ, BCNU, busulfan, bisulfan, carboxyphthalate platinum, CBDCA, CCNU, CHIP, chlorambucil, chlorozotocin, cis-platinum, clomesone, cyanomorpholinodoxorubicin, cyclodizone, cyclophosphamide, dianhydrogalactitol, fluorodopan, hepsulfame, hycanthone, ifosfamide, melphalan, methyl CCNU, mitomycin C, mitozolamide , nitrogen mustard, PCNU, piperazine, piperazinedione, pipobroman, porfiromycin, spirohydantoin mustard, streptozotocin, teroxylon, tetraplatin, thiotepa, triethylenemelamine, uracil nitrogen mustard, and Yoshi-864), antimitotic agents (e.g., allocolchicine, halichondrin M, colchicine, colchicine derivatives, dolastatin 10, maytansine, rhizoxin, paclitaxel, cyclosporine ... taxel derivatives, paclitaxel, thiocolchicine, tritylcysteine, vinblastine sulfate, and vincristine sulfate), plant alkaloids (e.g., actinomycin D, bleomycin, L-asparaginase, idarubicin, vinblastine sulfate, vincristine sulfate, mithramycin, mitomycin, daunorubicin, VP-16-213, VM-26, navelbine, and taxotere), biological agents (e.g., alpha interferon,BCG, G-CSF, GM-CSF, and interleukin-2), topoisomerase I inhibitors (e.g., camptothecin, camptothecin derivatives, and morpholinodoxorubicin), topoisomerase II inhibitors (e.g., mitoxantrone, amonafide, m-AMSA, anthrapyrazole derivatives, pyrazoloacridine, bisantrene HCL, daunorubicin, deoxydoxorubicin, menogaril, N,N-dibenzyldaunomycin, oxantrazole, rubidazone, VM-26, and VP-16), and synthetic agents (e.g., hydroxyurea, procarbazine, o,p'-DDD, dacarbazine, CCNU, BCNU, cis-diamminedichloroplatinum, mitoxantrone, CBDCA, levamisole, hexamethylmelamine, all-trans retinoic acid, gliadel, and porfimer sodium).

[0280] Antiproliferative agents are compounds that reduce cell proliferation. Antiproliferative agents include alkylating agents, metabolic antagonists, 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 roloxifene). Additional examples of specific antiproliferative agents include, but are not limited to, levamisole, gallium nitrate, granisetron, sargramostim strontium-89 chloride, filgrastim, pilocarpine, dexrazoxane, and ondansetron.

[0281] The compounds of the present invention can be administered alone or in combination with other antitumor agents, including cytotoxic / antitumor agents and antiangiogenic agents. Cytotoxic / antitumor agents are defined as agents that attack and kill cancer cells. Some cytotoxic / antitumor agents are alkylating agents that alkylate genetic material in tumor cells, such as cisplatin, cyclophosphamide, nitrogen mustard, trimethylene thiophosphoramide, carmustine, busulfan, chlorambucil, verstine, uracil mustard, chromafazine, and dacabazine. Other cytotoxic / antitumor agents are metabolic antagonists against tumor cells, such as cytosine arabinoside, fluorouracil, methotrexate, mercaptopurine, azathioprime, and procarbazine. Other cytotoxic / antitumor agents are antibiotics, such as doxorubicin, bleomycin, dactinomycin, daunorubicin, mithramycin, mitomycin, mitomycin C, and daunomycin. There are numerous liposomal formulations of these compounds available commercially. Still other cytotoxic / antitumor agents are mitotic inhibitors (vinca alkaloids). These include vincristine, vinblastine, and etoposide. Other cytotoxic / antitumor agents include taxol and its derivatives, L-asparaginase, antitumor antibodies, dacarbazine, azacitidine, amsacrine, melphalan, VM-26, ifosfamide, mitoxantrone, and vindesine.

[0282] Antiangiogenic agents are well known to those skilled in the art. Suitable antiangiogenic agents 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 alpha and beta), interleukin 12, retinoic acid, and tissue inhibitor of metalloproteinases 1 and 2 (TIMP-1 and -2). Small molecules that contain topoisomerase, such as razoxane, a topoisomerase II inhibitor with antiangiogenic activity, can also be used.

[0283] Other anticancer drugs that can be used in combination with the compositions of the present invention include acivicin, aclarubicin, acodazole hydrochloride, acronine, adzelesin, aldesleukin, altretamine, ambomycin, amethanthrone acetate, aminoglutethimide, amsacrine, anastrozole, anthramycin, asparaginase, asperlin, azacytidine, azetepa, azotomycin, batimastat, benzodepa, bicalutamide, bissantrene hydrochloride, bisnafide dimesylate, bizelesin, bleomycin sulfate, brequinar sodium, bropirin, and bropyrimidine. amine, busulfan, cactinomycin, calsterone, caracemide, carbetimer, carboplatin, carmustine, carubicin hydrochloride, carzelesin, cedefingol, chlorambucil, ciloremycin, cisplatin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin hydrochloride, decitabine, dexorumaplatin, dezaguanine, dezaguanine mesylate, diaziquone, docetaxel, doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene citrate Salt, dromostanolone propionate, duazomycin, edatrexate, eflornithine hydrochloride, elsamitrucin, enloplatin, enpromate, epipropizine, epirubicin hydrochloride, elbrozole, esorubicin hydrochloride, estramustine, estramustine sodium phosphate, etanidazole, etoposide, etoposide phosphate, etopurine, fadrozole hydrochloride, fazarabine, fenretinide, floxuridine, fludarabine phosphate, fluorouracil, fluorocitabine, foskidone, fostriecin sodium, gemcitabine, hydrochloride Gemcitabine, hydroxyurea, idarubicin hydrochloride, ifosfamide, irmofosine, interleukin II (including recombinant interleukin II or rIL2), interferon alpha-2a, interferon alpha-2b, interferon alpha-nl, interferon alpha-n3, interferon beta-Ia, interferon gamma-Ib, iproplatin, irinotecan hydrochloride, lanreotide acetate, letrozole, leuprolide acetate, liarozole hydrochloride, lometrexol sodium, lomustine, losoxantrone hydrochloride,Masoprocol, maytansine, mechlorethamine hydrochloride, megestrol acetate, melengestrol acetate, melphalan, menogaril, mercaptopurine, methotrexate, methotrexate sodium, metoprine, meturedepa, mitindomide, mitocalcin, mitochromine, mitogillin, mitomarcine, mitomycin, mitospar, mitotane, mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxithra ;Paclitaxel;Pegaspargase;Periomycin;Pentamustine;Peplomycin sulfate;Perfosfamide;Pipobroman;Piposulfan;Piroxantrone hydrochloride;Plicamycin;Promestane;Porfimer sodium;Porfiromycin;Prednimustine;Procarbazine hydrochloride;Puromycin;Puromycin hydrochloride;Pyrazofurin;Riboprin;Rogletimide;Safingol;Safingol hydrochloride;Semustine;Simtrazene; Sparfosate sodium;Sparsomycin;Spirogermanium hydrochloride;Spiromustine;Spiroplatin;Streptonigrin;Streptozocin;Sulofenur;Tallysomycin,Tecogalan sodium,Tegafur,Teroxantrone hydrochloride,Temoporhum,Teniposide,Teroxylon,Testolactone,Thiamiprine,Thioguanine,Thiotepa,Tiazofurin,Tirapazamine,Toremifene citrate,Trestron acetate,Triciribine phosphate,Trimefen These include, but are not limited to, trexate, trimetrexate glucuronate, triptorelin, tuburozole hydrochloride, uracil mustard, uredepa, vapreotide, verteporfin, vinblastine sulfate, vincristine sulfate, vindesine, vindesine sulfate, vinepidine sulfate, vinglisinate sulfate, vinleurosine sulfate, vinorelbine tartrate, vinrocidine sulfate, vinzolidine sulfate, vorozole, zeniplatin, zinostatin, and zorubicin hydrochloride. Other anticancer drugs include 20-epi-1,25-dihydroxyvitamin D3, 5-ethynyluracil, abiraterone, aclarubicin, acylfulvene, adecypenol, adzelesin, aldesleukin, ALL-TK antagonists, altretamine, ambamustine, amidox, amifostine, aminolevulinic acid, amrubicin, amsacrine, anagrelide,anastrozole, andrographolide, angiogenesis inhibitors, antagonist D, antagonist G, antarelix, anti-dorsalizing morphogenetic protein-1, antiandrogens for prostate cancer, antiestrogens, antineoplastons, antisense oligonucleotides, aphidicolin glycinate, apoptosis gene regulators, apoptosis regulators, apurinic acid, ara-CDP-DL-PTBA, arginine deaminase, asulaculin, atamestane, atrimustine, axinastatin 1, axinastatin 2, axinastatin 3, azasetron, azatoxin, azatyrosine, baccatin III derivatives, balanol, batimastat, BCR / ABL a antagonists, benzochlorine, benzoylstaurosporine, beta-lactam derivatives, beta-arretin, beta-clamycin B, betulinic acid, bFGF inhibitors, bicalutamide, bisanthrene, bisaziridinylspermine, bisnafide, bisstraten A, bizelesin, brefulate, bropirimine, budotitane, buthionine sulfoximine, calcipotriol, calphostin C, camptothecin derivatives, canarypox IL-2, capecitabine, carboxamidotriazole, carboxyamidotriazole, CaRest M3, CARN700, cartilage derived inhibitor, carzelesin, casein kinase inhibitor (ICOS), castanospermine, cecropin B, cetrorelix, chlorin, chloroquinoxaline sulfonamide, cicaprost, cis-porphyrin, cladribine, clomiphene analogue, clotrimazole, collismycin A, collismycin B, combretastatin A4, combretastatin analogue, conagenin, crambecidin 816, crisnatol , cryptophycin 8, cryptophycin A derivative, curacin A, cyclopentaquinone, cycloplatam, sipemycin, cytarabine ocphosphate, cytolytic factor, cytostatin, dacliximab, decitabine, dehydrodidemnin B, deslorelin, dexamethasone, dexphosphamide, dexrazoxane, dexverapamil, diaziquone, didemnin B, didox, diethylnorspermine, dihydro-5-azacytidine,9-dihydrotaxol, dioxamycin, diphenylspiromustine, docetaxel, docosanol, dolasetron, doxifluridine, droloxifene, dronabinol, duocarmycin SA, ebselen, ecomustine, edelfosine, edrecolomab, eflomitine, elemene, emiteflu, epirubicin, epristeride, estramustine analogues, estrogen agonists, estrogen antagonists, etanidazole, etoposide phosphate, exemestane, fadrozole, fazarabine, fenretinide, filgrastim, finasteride hydrochloride, flavopiridol, flezelastine, fluasterone, fludarabine, fluorodaunornithine , forfenimex, formestane, fostriecin, fotemustine, gadolinium texaphyrin, gallium nitrate, gallocitabine, ganirelix, gelatinase inhibitors, gemcitabine, glutathione inhibitors, hepsulfame, heregulin, hexamethylene bisacetamide, hypericin, ibandronic acid, idarubicin, idoxifene, idramantone, ilmofosine, ilomastat, imidazoacridone, imiquimod, immunostimulating peptides, insulin-like growth factor-1 receptor inhibitors, interferon agonists, interferons, interleukins, iobenguane, iododoxorubicin, 4-ipomeanol, 4-), Ilopract, Irsogladine, Isobengazole, Isohomohalichondrin B, Itasetron, Jasplakinolide, Kahalalide F, Lamellarin-N Triacetate, Lanreotide, Leinamycin, Lenograstim, Lentinan Sulfate, Leptolstatin, Letrozole, Leukemia Inhibitory Factor, Leukocyte Alpha Interferon, Leuprorelin + Estrogen + Progesterone, Leuprorelin, Levamisole, Liarozole , linear polyamine analogs, lipophilic disaccharide peptides, lipophilic platinum compounds, lysoclinamide 7, lobaplatin, lombricin, lometrexol, lonidamine, losoxantrone, lovastatin, loxoribine, lurtotecan, lutetium texaphyrin, lysofylline, lytic peptides, maytansine, mannostatin A, marimastat, masoprocol, maspin, matrilysin inhibitors, matrix metalloproteinase inhibitors,Menogaril, mervalone, meterelin, methioninase, metoclopramide, MIF inhibitor, mifepristone, miltefosine, millimostim, mismatched double stranded RNA, mitoguazone, mitolactol, mitomycin analogue, mitonafide, mitotoxin fibroblast growth factor-saporin, mitoxantrone, mofalotene, molgramostim, monoclonal antibody, human chorionic gonadotropin, monophosphoryl lipid A + myobacterium cell wall sk sk), mopidamol, multidrug resistance gene inhibitors, multiple tumor suppressor 1-based therapy, mustard anticancer drugs, mycaperoxide B, mycobacterial cell wall extract, myriapolone, N-acetyldinaline, N-substituted benzamides, nafarelin, nagressip, naloxone + pentazocine, napavine, naphterpine, nartograstim, nedaplatin, nemorubicin, neridronic acid, neutral endopeptidase, nilutamide, nisamycin, nitric oxide regulators, nitroxide antioxidants, nitrulline, O6-benzylguanine, octreotide, oxenone, oligonucleotides, onapristone, ondansetron, ondansetron, oracin, oral cytokine inducers, ormaplatin, osaterone, oxaliplatin, oxaunomycin, paclitaxel, paclitaxel Cell analogues, paclitaxel derivatives, paraumin, palmitoyl rhizoxin, pamidronic acid, panaxytriol, panomyphen, parabactin, pazerliptin, pegaspargase, perdecin, pentosan polysulfate sodium, pentostatin, pentrozole, perflubron, perphosphamide, perillyl alcohol, phenazinomycin, phenyl acetate, phosphatase inhibitors, picibanil, pilocarpine hydrochloride, pirarubicin, piritrexim, prasetin A, prasetin B, plasminogen activator inhibitors, platinum complexes, platinum compounds, platinum triamine complexes, porfimer sodium, porfiromycin, prednisone, propyl bisacridone, prostaglandin J2, proteasome inhibitors, protein A-based immunomodulators, protein kinase C inhibitors,Protein kinase C inhibitors derived from microalgae (microalgal), protein tyrosine phosphatase inhibitors, purine nucleoside phosphorylase inhibitors, purpurin, pyrazoloacridine, pyridoxylated hemoglobin polyoxyethylene conjugates, raf antagonists, raltitrexed, ramosetron, ras farnesyltran, Spherase inhibitors, ras inhibitors, ras-GAP inhibitors, demethylated reterliptin, rhenium Re186 etidronate, rhizoxin, ribozyme, RII retinamide, logretimide, rohitukin, romurtide, roquinimex, rubiginone Bl, ruboxil, safingol, santopine, SarCNU, sarcophytol A, sargramostim, Sdi1 mimetic, semustine, senescence derived inhibitor 1 1), sense oligonucleotide, signal transduction inhibitor, signal transduction modulator, single-chain antigen binding protein, schizofuran, sobuzoxane, sodium borocaptate, sodium phenylacetate, sorberol, somatomedin binding protein, sonermin, sparfosic acid, spicamycin D, spiromustine, splenopentin, spongiostatin 1, squalamine, stem cell inhibitor, stem cell division inhibitor, stipiamid, stromelysin inhibitor, sulfinosine, superactive vasoactive intestinal peptide antagonist, suragist, suramin, swainsonine, synthetic glycosaminoglycan, talimustine, tamoxifen methiodide, tauromustine, tazarotene, tecogalan sodium, tegafur, tellupyrium, telomerase inhibitor, temoporfin, temozolomide, teniposide, tetrachlorodecaoxide, tetrazomine, thaliblastine, thioco These include, but are not limited to, ralin, thrombopoietin, thrombopoietin mimetics, thymalfasin, thymopoietin receptor agonists, thymotrinan, thyrotropin, stannous etiopurpurin, tirapazamine, titanocene dichloride, topsentin, toremifene, totipotent stem cell factor, translation inhibitors, tretinoin, triacetyluridine, triciribine, trimetrexate, triptorelin, tropisetron, turosteride, tyrosine kinase inhibitors, tyrphostin, UBC inhibitors, ubenimex, urogenital sinus-derived growth inhibitor, urokinase receptor antagonists, vapreotide, variolin B, vector systems, red blood cell gene therapy, veraresol, veramine, verudine, verteporfin, vinorelbine, vinxartin, vitaxin, vorozole, zanoteron, zeniplatin, zilascorub, and zinostatin stimalamer.In one embodiment, the anticancer drug is 5-fluorouracil, taxol, or leucovorin.

[0284] Use in combination In some embodiments, the present invention provides methods for treating, protecting against, and / or preventing cancer or a cancer-related disease or disorder 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 same, and a composition comprising a tumor antigen, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding same.

[0285] 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 may be administered using any suitable method such that a combination of anti-Siglec-9 and tumor antigen is both present in the subject. In one embodiment, the method may comprise 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 invention by any of the methods described in detail 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 comprise administering a first composition comprising a synthetic Siglec-9 antibody of the present invention, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding same by any of the methods described in detail above, and administering a second composition comprising a tumor antigen, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding 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 administration of the synthetic anti-Siglec-9 antibody, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding same. In one embodiment, the method may comprise administering a first composition comprising the tumor antigen, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding same, and administering a second composition comprising the synthetic anti-Siglec-9 antibody of the invention, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding same by any of the methods described in detail 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 administration of the composition comprising the tumor antigen, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding same.In one embodiment, the method may comprise administering a first composition comprising the tumor antigen, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding same, and administering a second composition comprising the synthetic anti-Siglec-9 antibody of the invention, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding same, by any of the methods described in detail 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 administration of the composition comprising the tumor antigen, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding same. In one embodiment, the method may comprise simultaneously administering the first composition comprising the synthetic anti-Siglec-9 antibody of the invention, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding same, and the second composition comprising the tumor antigen, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding same, by any of the methods described in detail above. In one embodiment, the method may comprise administering a single composition comprising a synthetic anti-Siglec-9 antibody of the present invention, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding same and a tumor antigen, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding same.

[0286] The present invention is further illustrated in the following examples. It should be understood that these examples, while showing exemplary embodiments of the present invention, are provided for illustrative purposes only. From the above discussion and these examples, one skilled in the art can grasp the essential features of the present invention, and can make various changes and modifications to the present invention to adapt it to various applications and conditions without departing from the spirit and scope of the present invention. Thus, in addition to those shown and described herein, various modifications of the present invention will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. EXAMPLES

[0287] The present invention will be further described in 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 specified. Therefore, the present invention should not be interpreted as being limited to the following examples in any way, but should be interpreted as embracing any and all variations that become evident as a result of the teachings provided herein.

[0288] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the methods recited in the claims. Thus, the following examples are not to be construed as limiting in any way the remainder of the disclosure.

[0289] Example 1: Generation of Siglec-9 antibodies using H2L2 mice Sialic acid-binding receptors, such as Siglecs and selectins, are favorably situated to be explored due to hypersialylation in cancer. Sialic acid (Sia) is a monosaccharide typically found at the outermost end of complex glycan chains that decorate the cell surfaces of all vertebrates. Sialic acid is essential for embryonic development and mediates important and unique biological functions. Seventy human genes are known to be involved in sialic acid biology, with more than 10 documented to be unique to humans. Most of these human-specific genetic alterations are in sialic acid-recognizing Ig-like lectin (SIGLEC) genes, which are found in most types of white blood cells to the immune synapse. Upregulation of sialosides has long been correlated with poor tumor prognosis and reduced immunogenicity.

[0290] Siglec-9 is expressed on monocytes, macrophages, natural killer (NK) cells, dendritic cells, neutrophils, and a subset of T cells. Siglec-9 is an inhibitory receptor whose signals 1) inhibit the antitumor functions of NK cells and T cells, and 2) regulate the immune functions of myeloid cells.

[0291] The experiments described herein demonstrate the development and characterization of Siglec-9 antibodies (Figure 1). In these experiments, hybridoma supernatants demonstrated specificity and binding to human Siglec-9.

[0292] It was observed that the antibody exhibited cytotoxicity against OVISE cells (Figure 2). OVISE cells are ovarian clear cell adenocarcinoma arising from a metastatic site: pelvis (hipbone) of a 40-year-old Japanese woman. An experiment was designed to test the activity of the antibody. Target cells: OVISE (20,000 cells / well); Effector cells: human PBMC; E(effector):T(target)=5:1; Effector cells and antibody (1:10) were added at 20 and 56 hours. Images were taken after 100 hours, and it was observed that the Siglec-9 antibody caused the death of OVISE cells.

[0293] It was also observed that the antibody exhibited cytotoxicity against OVCAR10 cells (Figure 3). OVCAR10 is a human ovarian cancer cell line. An experiment was designed to test the activity of the antibody. Target cells: OVCAR10 (10,000 cells / well); Effector cells: human PBMCs E:T=5:1; effector cells and antibody were added at 28.67 hours. Images were taken after 53 hours and it was observed that the Siglec-9 antibody caused the death of OVCAR10 cells. See Figure 3.

[0294] The antibody showed no cytotoxicity in TC-1 cells, which were used as a control target. TC-1 is a mouse cell line that lacks Siglec-9 on its surface.

[0295] Example 2: Sequences - CDR sequences (underlined)

[0296] HB9-4G4(S9-DB-2) Heavy chain (SEQ ID NO:4) EVQLVESGGGLVKPGGSLRLSCAAS GFTFSNAW MSWVRQAPGKGLEWVGR IKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYC TTGWELQDYYYYGMDV WGQGTTVTVSS

[0297] HB9-4G4(S9-DB-2) Heavy chain (SEQ ID NO: 8) GAGGTGCAGCTGGTGGAGTCGGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTTAGACTCTCCTGTGCAGCCTCT GGATTCACTTTCAGTAACGCCTGG ATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCCGT ATTAAAAGCAAAACTGATGGTGGGACAACA GACTACGCTGCACCCGTGAAAGGCAGATTCACCATCTCAAGAGATGATTCAAAAAACACGCTGTATCTGCAAATGAACAGCCTGAAAACCGAGGACACAGCCGTGTATTACTGT ACCACAGGGTGGGAGCTACAGGACTACTACTACTACGGTATGGACGTC TGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

[0298] HB9-4G4(S9-DB-2) Light chain (SEQ ID NO: 12) IVMTQSPATLSVSPGERATLSCRAS QSVSSN LAWYQQKPGQAPRLLIY GAS TRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYC QQYNNWPLT FGGGTKVEIK

[0299] HB9-4G4(S9-DB-2) Light chain (SEQ ID NO: 16) ATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGT CAGAGTGTTAGCAGCAAC TTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT GGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGT CAGCAGTATAATAACTGGCCGCTCACT TTCGGCGGAGGGACCAAGGTGGAGATCAAA

[0300] HB9-6A8(S9-DB-3) Heavy chain (SEQ ID NO: 20) QVQLQQWGAGLLKPSETLSLTCAVY GGSFSGYY WSWIRQPPGKGLEWIGE INHSGST NYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYC ARGDCSGGSCPYWYFDL WGRGTLVTVSS

[0301] HB9-6A8(S9-DB-3) Heavy chain (SEQ ID NO: 24) CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTAT GGTGGGTCCTTCAGTGGTTACTACT GGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAA ATCAATCATAGTGGAAGCACC AACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGT GCGAGAGGTGATTGTAGTGGTGGTAGCTGTCCTTACTGGTACTTCGATCTC TGGGGCCGTGGCACCCTGGTCACTGTCTCCTCA

[0302] HB9-6A8(S9-DB-3) Light chain (SEQ ID NO: 28) DIVMTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC MQALQTPRT FGQGTKVEIK

[0303] HB9-6A8(S9-DB-3) Light chain (SEQ ID NO: 32) GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGGATACAACTAT TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTAT TTGGGTTCT AATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGCTCTACAAACTCCTCGGACG TTCGGCCAAGGGACCAAGGTGGAAATCAAA

[0304] HB9-6F3(S9-DB-4) Heavy chain (SEQ ID NO: 36) EVQLLESGGGLVQPGGSLRLSCEAS GFTFRNYA MSWVRQAPGKGLEWVSA IRGSGSRT YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC AKDEGFGDLLAHYVMDA WGQGASVTVSS

[0305] HB9-6F3(S9-DB-4) Heavy chain (SEQ ID NO: 40) GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGAAGCCTCA GGATTCACCTTTAGAAACTATGCC ATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCT ATTCGTGGTAGTGGTAGTAGAACATACTACGCAGACTCTGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGC GCGAAAGATGAGGGGTTCGGGGACTTATTAGCGCACTATGTTATGGATGCC TGGGGTCAAGGAGCTTCAGTCACTGTCTCCTCA

[0306] HB9-6F3(S9-DB-4) Light chain (SEQ ID NO: 44) EIVMTQSPATLSVSPGERATLSCRAS QSVSSN LAWYQQKPGQAPRLLIY GAS TRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYC QQYNNWPIT FGQGTRLEIK

[0307] HB9-6F3(S9-DB-4) Light chain (SEQ ID NO: 48) GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGT CAGAGTGTTAGCAGCAAC TTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT GGTGCATCC ACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGT CAGCAGTATAATAACTGGCCGATCACC TTCGGCCAAGGGACACGACTGGAGATTAAA

[0308] HB9-9B11(S9-DB-5) Heavy chain (SEQ ID NO: 52) EVQLLESGGGLVQPGGSLRLSCAAS GFTFSNYA MNWVRQAPGKGLEWVSA ISMSGGSTYYADSVKGRFTISRDNSKNTLYLQMNTLRAEDTAVYYC AKDEYSSGWYQFDY WGQGTLVTVSS

[0309] HB9-9B11(S9-DB-5) Heavy chain (SEQ ID NO: 56) GAGGTGCAGTTGTTGGAGTCTGGGGGGGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCT GGATTCACCTTTAGCAACTATGCC ATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCT ATTAGTATGAGTGGTGGTAGCACA TACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACGGCCGTATATTACTGT GCGAAAGACGAATATAGCAGTGGCTGGTACCAATTTGACTAT TGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0310] HB9-9B11(S9-DB-5) Light chain (SEQ ID NO: 60) DIQMTQSPSSLSASVGDRVTITCRAS QSISSY LNWYQQKPGKAPKLLIY AAS SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSYSTPLT FGGGTKVEIK

[0311] HB9-9B11(S9-DB-5) Light chain (SEQ ID NO: 64) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGT CAGAGCATTAGCAGCTAT TTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTAT GCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGT CAACAGAGTTACAGTACCCCGCTCACT TTCGGCGGAGGGACCAAGGTGGAGATCAAA

[0312] HB9-10B6(S9-DB-6) Heavy chain (SEQ ID NO: 68) QVQLVESGGGVVQPGRSLRLSCAAS GFTFSTYG MHWVRQAPGKGLEWVAV TWYDEYNK YYADSVKGRFTISRDNSKNMLYLQMNSLRAEDTAMYYC ARNVLRYFDWTLDY WGQGTLVTVSS

[0313] HB9-10B6(S9-DB-6) Heavy chain (SEQ ID NO: 72) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCT GGATTCACCTTCAGTACCTATGGC ATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTT ACATGGTATGATGAATATAATAAA TACTATGCAGACTCCGTGAAGGGCCGCTTCACCATCTCCAGAGACAATTCCAAGAACATGTTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTATGTATTACTGT GCGAGGAACGTATTACGATATTTTGACTGGACCCTTGACTAC TGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG

[0314] HB9-10B6(S9-DB-6) Light chain (SEQ ID NO: 76) IVLTQSPATLSLSPGERATLSCRAS QSVSSY LAWYQQKPGQAPRLLIY DASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQRSNWPPT FGQGTKVEIK

[0315] HB9-10B6(S9-DB-6) Light chain (SEQ ID NO: 80) ATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGT CAGAGTGTTAGCAGCTAC TTAGCCTGGTATCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT GATGCATCC AACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGT CAGCAGCGTAGCAACTGGCCTCCCACT TTTGGCCAGGGGACCAAGGTGGAGATCAAA

[0316] HB9-10E3(S9-DB-7) Heavy chain (SEQ ID NO: 84) EVQLLESGGGLVQPGGSLRLSCAAS GFTFSSYA MSWVRQAPGKGLEWVSA ISGSGGST YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC AKAGIAVAGGWYFDL WGRGTLVTVSS

[0317] HB9-10E3(S9-DB-7) Heavy chain (SEQ ID NO: 88) GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCT GGATTCACCTTTAGCAGCTATGCC ATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCT ATTAGTGGTAGTGGTGGTAGCACA TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGT GCGAAAGCCGGTATAGCAGTGGCTGGGGGATGGTACTTCGATCTC TGGGGCCGTGGCACCCTGGTCACTGTCTCCTCA

[0318] HB9-10E3(S9-DB-7) Light chain (SEQ ID NO: 92) IVLTQSPATLSLSPGERATLSCRAS QSVSSY LAWYQQKPGQAPRLLIY DAS NRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQRSNWPPT FGQGTKLEIK

[0319] HB9-10E3(S9-DB-7) Light chain (SEQ ID NO: 96) ATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGT CAGAGTGTTAGCAGCTAC TTAGCCTGGTATCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT GATGCATCC AACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGT CAGCAGCGTAGCAACTGGCCTCCCACT TTTGGCCAGGGGACCAAGCTGGAGATCAAA

[0320] HB9-11B10(S9-DB-8) Heavy chain (SEQ ID NO: 100) EVQLLESGGGLVQPGGSLRLSCAAS GFTFSNYA MNWVRQAPGKGLEWVSA ISMSGGST YYADSVKGRFTISRDNSKNTLYLQMNTLRAEDTAVYYC AKDEYSSGWY QFDYWGQGTLVTVSS

[0321] HB9-11B10(S9-DB-8) Heavy chain (SEQ ID NO: 104) GAGGTGCAGTTGTTGGAGTCTGGGGGGGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCT GGATTCACCTTTAGCAACTATGCC ATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCT ATTAGTATGAGTGGTGGTAGCACA TACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACGGCCGTATATTACTGT GCGAAAGACGAATATAGCAGTGGCTGGTAC CAATTTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0322] HB9-11B10(S9-DB-8) Light chain (SEQ ID NO: 108) EIVLTQSPATLSLSPGERATLSCRAS QSVNSY LAWYQQKPGQAPRLLIY DAS IRATGIPARFSGSGSGTDFTLTISSLEPEDFAFYYC QQRSNWPPT FGQGTKVEIK

[0323] HB9-11B10(S9-DB-8) Light chain (SEQ ID NO: 112) GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGT CAGAGTGTTAACAGCTAC TTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT GATGCATCC ATCAGGGCCACTGGCATCCCAGCCAGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCATTTTATTACTGT CAACAGCGTAGTAACTGGCCTCCGACG TTCGGCCAAGGGACCAAAGGTGGAAATCAAA

[0324] HB9-1F11 Heavy Chain: (SEQ ID NO:116) QVQLQQPGAELVKPGASVKLSCKAS GYTFTSYW MHWVKQRPGRGLEWIGR IDPNSGGT KYNEKFKSKATLTVDKPSSTAYMQLSSLTSEDSAVYYC ARYDYYGSSYFDY WGQGTTVTVSS

[0325] HB9-1F11 (SEQ ID NO: 120) CAAGTGCAGCTGCAGCAGCCTGGCGCCGAGCTGGTGAAACCTGGAGCCTCCGTGAAGCTGAGCTGTAAGGCCAGC GGCTACACCTTCACCAGCTACTGG ATGCACTGGGTCAAGCAGCGGCCAGGAAGAGGCCTGGAATGGATCGGCAGA ATCGACCCCAACAGCGGCGGCACA AAGTACAACGAGAAGTTCAAGAGCAAGGCTACACTGACAGTGGACAAGCCTAGCTCCACCGCCTACATGCAGCTGTCTAGCCTGACCAGCGAAGATAGCGCCGTGTACTACTGC GCCAGATACGACTACTACGGCAGCTCTTATTTTGATTAC TGGGGCCAGGGCACCACCGTGACCGTGTCCAGC

[0326] HB9-1F11 Light chain: (SEQ ID NO:124) QAVVTQESALTTSPGETVTLTCRSS TGAVTTSNY ANWVQEKPDHLFTGLIG GTN NRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFC ALWYSNHWV FGGGTKLTVL

[0327] HB9-1F11 (Sequence number 128) CAAGCCGTGGTGACACAGGAGAGCGCCCTGACCACATCCCCCGGCGAGACAGTGACCCTGACCTGTAGAAGCAGC ACAGGCGCTGTGACCACCTCTAACTAC GCCAACTGGGTCCAGGAAAAACCTGACCACCTGTTTACCGGCCTGATCGGCG GAACCAACA ACCGGGCCCCTGGAGTGCCAGCCAGATTCAGCGGCTCTCTGATCGGCGACAAGGCCGCTCTCACCATCACCGGCGCCCAGACAGAAGATGAGGCCATCTACTTCTGC GCCCTGTGGTACAGCAATCACTGGGTG TTCGGCGGAGGCACCAAGCTGACAGTGCTG

[0328] HB9-4C10 Heavy chain: (Sequence number 132) EVQLQQSGPELVKPGASVKISCKAS GYTFTDYY MNWVKQSHGKSLEWIGG INPNNGGT SYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYC ARNDGYRGYAMDY WGQGTSVTVSS

[0329] HB9-4C10 (Sequence number 136) GAGGTGCAGCTGCAGCAATCTGGACCTGAGCTGGTGAAGCCTGGCGCTTCTGTGAAGATCTCTTGTAAAGCCAGC GGCTACACCTTCACCGATTACTAC ATGAACTGGGTCAAGCAGAGCCACGGCAAAAGCCTGGAATGGATCGGCGGC ATCAACCCCAACAACGGCGGAACA AGCTACAACCAGAAGTTCAAGGGCAAGGCTACCCTGACCGTGGACAAGAGCAGCTCCACCGCCTACATGGAACTGCGGAGCCTGACAAGCGAGGACAGCGCCGTGTACTACTGC GCCAGAAATGATGGCTATAGAGGCTACGCCATGGACTAC TGGGGACAGGGCACCAGCGTGACAGTGTCCTCC

[0330] HB9-4C10 Light chain: (SEQ ID NO:140) DIVMSQSPSSLAVSVGEKVTMSCKSS QSLLYSSNQKNY LAWYQQKPGQSPKLLIY WAS TRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYCC QQYYSNPPLT FGAGTKLELK

[0331] HB9-4C10 (SEQ ID NO:144) GACATCGTGATGAGCCAGTCTCCATCTAGCCTGGCCGTGTCCGTGGGCGAGAAGGTGACCATGTCTTGTAAAAGCTCC CAGAGCCTGCTGTACAGCAGCAACCAGAAAAACTAC CTGGCCTGGTATCAGCAAAAGCCCGGCCAGAGCCCCAAGCTCCTGATCTAC TGGGCCTCC ACACGGGAAAGCGGAGTGCCTGATAGATTCACCGGCTCTGGCAGCGGAACCGACTTTACCCTGACCATCAGCAGCGTCAAGGCCGAGGACCTGGCTGTGTACTGCTGC CAGCAGTACTACAGCAATCCTCCTCTGACA TTCGGCGCCGGCACAAAGCTGGAACTGAAG

[0332] HB9-5B7 Heavy Chain: (SEQ ID NO:148) EVQLVESGGGLVQPGRSLRLSCAAS GFTFDDYA MHWVRQAPGKGLEWVSA ITWNSGHI DYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC AKVSYLSTASSLDY WGQGTLVTVSS

[0333] HB9-5B7 (SEQ ID NO:152) GAGGTGCAGCTGGTGGAAAGCGGAGGAGGCCTGGTTCAACCCGGCCGGAGCCTGAGACTGAGCTGTGCCGCTTCT GGCTTTACATTCGACGACTACGCC ATGCACTGGGTCAGACAGGCCCCTGGCAAGGGCCTCGAGTGGGTGTCCGCC ATCACCTGGAATAGCGGCCACATC GACTATGCTGATAGCGTGGAAGGCAGATTCACCATCAGCAGAGATAACGCCAAGAACTCCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGC GCCAAAGTGTCCTACCTGTCTACAGCCAGCAGCCTGGACTAC TGGGGCCAGGGCACCCTGGTGACCGTGTCTAGC

[0334] HB9-5B7 Light chain: (SEQ ID NO: 156) DIQMTQSPSSLSASVGDRVTITCRAS QGISNY LAWYQQKPGQVPKLLIY AAS ALQSGVPSRFSGSGSGTDFTLTITSLQPEDVATYYC HKYNSAPWT FGQGTEVEIK

[0335] HB9-5B7 (SEQ ID NO: 160) GACATCCAGATGACCCAGTCCCCTAGCTCTCTGAGCGCCAGCGTGGGCGACCGGGTGACCATCACCTGTAGAGCCTCT CAGGGCATCAGCAACTAC CTGGCCTGGTATCAGCAGAAACCCGGACAGGTGCCAAAGCTGCTGATCTAC GCCGCTTCT GCTCTGCAGAGCGGAGTCCCCAGCAGATTCAGCGGCTCCGGCAGCGGCACAGATTTTACCCTGACCATTACAAGCCTGCAACCTGAGGACGTGGCCACATACTACTGC CACAAGTACAACAGCGCCCCTTGGACC TTCGGCCAGGGCACCGAGGTGGAAATCAAG

[0336] HB9-7B8 Heavy chain: (SEQ ID NO:164) ELQLLESGGGLVQPGGSLRLSCAAS GFTFTNYA MNWVRQAPGKGLEWVSA ISGSGGRT YYADSVKGRFTISRDNSRNTLFLQMNSLRPEDTAVYYC AKDQTSGTTGYPYFAY WGQGTLVTVSS

[0337] HB9-7B8 (SEQ ID NO:168) GAGCTGCAGCTGCTGGAGTCTGGCGGCGGCCTGGTGCAGCCCGGCGGCTCCCTGAGGCTGAGCTGCGCCGCCTCC GGCTTTACATTCACAAATTACGCC ATGAATTGGGTGCGGCAGGCCCCAGGCAAGGGCCTGGAGTGGGTGTCCGCC ATCTCTGGCTCCGGCGGCAGAACC TATTACGCCGATTCCGTGAAGGGCAGATTCACCATCAGCAGAGACAACTCTCGCAATACCCTGTTTCTGCAGATGAATAGCCTGAGGCCCGAGGATACAGCCGTGTACTACTGC GCCAAGGATCAGACATCTGGCACAACAGGCTACCCTTATTTCGCCTAC TGGGGCCAGGGCACCCTGGTGACCGTGTCTTCC

[0338] HB9-7B8 Light chain: (SEQ ID NO:172) ETVMTQSPATLSVSPGERAILSCRAS QSVSSN LVWYQQKPGQAPRLFIY GAS TRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYC QQYNNWPRT FGQGTKVEIK

[0339] HB9-7B8 (SEQ ID NO:176) GAGACAGTGATGACCCAGAGCCCAGCCACCCTGTCCGTGTCTCCAGGCGAGAGGGCCATCCTGTCCTGTAGGGCCTCC CAGAGCGTGTCTTCCAAT CTGGTGTGGTACCAGCAGAAGCCAGGCCAGGCCCCTAGGCTGTTTATCTAC GGCGCCTCC ACAAGGGCCACCGGCATCCCTGCCAGATTCTCTGGCTCTGGCTCCGGCACAGAGTTCACCCTGACAATCTCTTCCCTGCAGTCTGAGGACTTTGCCGTGTACTATTGC CAGCAGTATAATAACTGGCCTAGGACC TTTGGCCAGGGCACAAAGGTGGAGATCAAGCA

[0340] HB9-14D4 Heavy chain: (SEQ ID NO: 180) QVQLVESGGGVVQPGRSLRLSCAAS GFAFSSYG MHWVRQAPGKGLEWVAV IWFDGTKK YYTDSVKGRFTISRDNSKNTLYLQMNTLRAEDTAVYYC ARDRGIGARRGPYYMDV WGKGTTVTVSS

[0341] HB9-14D4 (SEQ ID NO: 184) CAAGTGCAGCTGGTTGAGAGCGGCGGAGGCGTGGTGCAGCCCGGCAGAAGCCTGAGACTGTCTTGTGCTGCTTCT GGATTTGCCTTCAGCAGCTACGGC ATGCACTGGGTGCGGCAGGCCCCTGGCAAGGGCCTGGAATGGGTCGCCGTG ATCTGGTTCGACGGCACAAAGAAG TACTATACAGACAGCGTGAAGGGCAGATTCACCATCAGCAGAGATAATAGCAAGAACACCCTGTACCTGCAGATGAACACCCTGCGGGCCGAGGACACCGCCGTGTACTACTGC GCCAGAGATAGAGGCATCGGCGCCAGGCGGGGACCTTACTACATGGACGTG TGGGGCAAAGGCACCACAGTGACCGTGTCCTCC

[0342] HB9-14D4 Light chain: (SEQ ID NO: 188) DIQMTQSPSSLTASVGDRVTITCRAS QSISSY VNWYQQKPGKAPKVLIF AAS SLQSGVPSRFSGSGSGTDFTLTISSLQAEDVAVYYC QQYYSTPLT FGGGTKVEIK

[0343] HB9-14D4 (SEQ ID NO: 192) GATATCCAGATGACACAGAGCCCCAGCAGCCTGACCGCCTCTGTGGGCGACCGGGTGACCATCACCTGTAGAGCCAGC CAGTCCATCAGCTCCTAC GTGAACTGGTATCAGCAGAAACCTGGCAAGGCCCCTAAGGTGCTGATCTTT GCTGCCAGC AGCCTGCAGAGCGGAGTCCCATCTAGATTCAGCGGCTCTGGCAGCGGCACCGACTTCACCCTGACAATTAGCTCTCTGCAGGCCGAGGACGTGGCCGTGTACTACTGC CAGCAATACTACAGCACCCCTCTGACC TTCGGCGGCGGAACAAAGGTGGAAATCAAG

[0344] HB9-14H12 Heavy chain: (SEQ ID NO: 196) QVQLKESGPGLVAPSQSLSITCTVS GFLLISNG VHWVRQPPGKGLEWLGV IWAGGNTN YNSALMSRVSISKDNSKSQVFLKMKSLQTDDTAMYYC ARDFYDYDVFYYAMDY WGQGTSVTVSS

[0345] HB9-14H12 (SEQ ID NO: 200) CAAGTCCAGCTGAAGGAAAGCGGCCCAGGCCTGGTGGCCCCTAGCCAGAGCCTGAGCATCACATGTACCGTGTCC GGCTTCCTGCTGATCAGCAACGGC GTGCACTGGGTGCGGCAGCCTCCCGGCAAGGGCCTCGAGTGGCTGGGAGTG ATCTGGGCTGGCGGAAATACCAAC TACAACAGCGCCCTGATGTCTAGAGTGTCTATCTCTAAGGACAACAGCAAGTCCCAGGTGTTCCTGAAGATGAAAAGCCTGCAGACCGACGACACCGCCATGTACTACTGC GCCAGAGATTTCTACGATTATGACGTTTTTTACTACGCCATGGACTAC TGGGGCCAGGGCACCAGCGTGACAGTGTCCAGC

[0346] Light chain: HB9-14H12 (SEQ ID NO: 204) QAVVTQESALTTSPGETVPLTCRSS TGTVTTSNF ANWVQEKPDHLFTGLIG GTN NRAPGLPARFSGSLIGDKAALTITGAQTEDEAIFFC ALWYSNHFV FGGGSQLTVL

[0347] HB9-14H12 (SEQ ID NO: 208) CAAGCCGTGGTGACACAGGAATCCGCCCTCACAACCTCTCCAGGCGAGACAGTGCCTCTGACCTGTAGAAGCAGC ACCGGCACCGTGACCACCAGCAACTTC GCCAACTGGGTCCAGGAAAAGCCCGACCACCTGTTCACCGGCCTGATCGGC GGAACAAAC AACCGGGCCCCTGGCCTGCCTGCTAGATTCAGCGGCAGCCTGATCGGAGATAAGGCCGCTCTGACAATCACCGGCGCCCAGACCGAGGACGAGGCCATCTTTTTCTGC GCCCTGTGGTACAGCAATCACTTTGTG TTCGGCGGCGGATCTCAGCTGACCGTGCTG

[0348]

Table 1-1

Table 1-2

[0349] The disclosures of all patents, patent applications, and publications cited herein are incorporated herein by reference in their 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 may be devised by those skilled in the art without departing from the true spirit and scope of the present invention. It is intended that the appended claims be construed to include all such embodiments and equivalent variations.

Claims

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

2. The aforementioned antibody a) Variable heavy chain sequences containing CDR sequences selected from the group consisting of SEQ ID NOs: 1-3, 17-19, 33-35, 49-51, 65-67, 81-83, 97-99, 113-115, 129-131, 145-147, 161-163, 177-179, and 193-195, b) Variable light chain sequences containing CDR sequences selected from the group consisting of SEQ ID NOs: 9-11, 25-27, 41-43, 57-59, 73-75, 89-91, 105-107, 121-123, 137-139, 153-155, 169-171, 185-187, and 201-203, c) Sequences having at least 95% identity with one or more variable heavy chain sequences from among sequence numbers 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, and 196, d) Sequences having at least 95% identity with one or more variable light chain sequences from among sequence numbers 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, and 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 NOs: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, and 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 NOs: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, and 204. A Siglec-9 binding molecule according to claim 1, comprising an amino acid sequence selected from the group consisting of the following.

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

4. The aforementioned Sigrec-9 binding molecule, a) Variable heavy chain sequence of Sequence ID No. 4 and variable light chain sequence of Sequence ID No. 12, b) Variable heavy chain sequence of Sequence ID No. 20 and variable light chain sequence of Sequence ID No. 28, c) Variable heavy chain sequence of Sequence ID No. 36 and variable light chain sequence of Sequence ID No. 44 d) Variable heavy chain sequence of Sequence ID No. 52 and variable light chain sequence of Sequence ID No. 60, e) Variable heavy chain sequence of Sequence ID No. 68 and variable light chain sequence of Sequence ID No. 76, f) Variable heavy chain sequence of Sequence ID No. 84 and variable light chain sequence of Sequence ID No. 92, g) Variable heavy chain sequence of SEQ ID NO: 100 and variable light chain sequence of SEQ ID NO: 108, h) Variable heavy chain sequence of sequence number 116 and variable light chain sequence of sequence number 124, i) Variable heavy chain sequence of Sequence ID No. 132 and variable light chain sequence of Sequence ID No. 140, j) Variable heavy chain sequence of sequence number 148 and variable light chain sequence of sequence number 156, k) Variable heavy chain sequence of sequence number 164 and variable light chain sequence of sequence number 172, l) Variable heavy chain sequence of SEQ ID NO: 180 and variable light chain sequence of SEQ ID NO: 188, and m) Variable heavy chain sequence of SEQ ID NO: 196 and variable light chain sequence of SEQ ID NO: 204 A Siglec-9 binding molecule or fragment thereof according to claim 1, comprising an amino acid sequence selected from the group consisting of the following.

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

6. A nucleic acid molecule or combination of nucleic acid molecules comprising one or more nucleotide sequences encoding the Siglec-9 binding molecule described in claim 1.

7. The nucleic acid molecule a) A nucleotide sequence encoding a variable heavy chain sequence, comprising a CDR coding 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, comprising a CDR coding 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 with a variable heavy chain coding 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 with a variable light chain coding sequence selected from the group consisting of SEQ ID NOs: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, and 208, e) A fragment comprising at least 80% of the full-length sequence of a variable heavy-chain coding sequence selected from the group consisting of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, and 200, and f) A fragment comprising at least 80% of the full-length sequence of a variable light chain code sequence selected from the group consisting of SEQ ID NOs: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, and 208. A nucleic acid molecule according to claim 6, comprising an amino acid sequence selected from the group consisting of the following.

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

9. The nucleic acid molecule a) A first nucleotide sequence including sequence number 8 which encodes a variable heavy chain sequence, and a second nucleotide sequence including sequence number 16 which encodes a variable light chain sequence, b) A first nucleotide sequence including SEQ ID NO: 24 encoding a variable heavy chain sequence, and a second nucleotide sequence including SEQ ID NO: 32 encoding a variable light chain sequence, c) A first nucleotide sequence including sequence number 40 encoding a variable heavy chain sequence, and a second nucleotide sequence including sequence number 48 encoding a variable light chain sequence, d) A first nucleotide sequence including sequence number 56 encoding a variable heavy chain sequence, and a second nucleotide sequence including sequence number 64 encoding a variable light chain sequence, e) A first nucleotide sequence including SEQ ID NO: 72 encoding a variable heavy chain sequence, and a second nucleotide sequence including SEQ ID NO: 80 encoding a variable light chain sequence, f) A first nucleotide sequence including sequence number 88 encoding a variable heavy chain sequence, and a second nucleotide sequence including sequence number 96 encoding a variable light chain sequence, g) A first nucleotide sequence including sequence number 104 encoding a variable heavy chain sequence, and a second nucleotide sequence including sequence number 112 encoding a variable light chain sequence, h) A first nucleotide sequence including sequence number 120 encoding a variable heavy chain sequence, and a second nucleotide sequence including sequence number 128 encoding a variable light chain sequence, i) A first nucleotide sequence including sequence number 136 encoding a variable heavy chain sequence, and a second nucleotide sequence including sequence number 144 encoding a variable light chain sequence, j) A first nucleotide sequence including sequence number 152 encoding a variable heavy chain sequence, and a second nucleotide sequence including sequence number 160 encoding a variable light chain sequence, k) A first nucleotide sequence including sequence number 168 encoding a variable heavy chain sequence, and a second nucleotide sequence including sequence number 176 encoding a variable light chain sequence, l) A first nucleotide sequence including SEQ ID NO: 184 encoding a variable heavy chain sequence, and a second nucleotide sequence including SEQ ID NO: 192 encoding a variable light chain sequence, m) A first nucleotide sequence including SEQ ID NO: 200 encoding a variable heavy chain sequence, and a second nucleotide sequence including SEQ ID NO: 208 encoding a variable light chain sequence. The nucleic acid molecule according to claim 7, comprising a nucleotide sequence selected from the group consisting of the following.

10. A composition comprising the Siglec-9 binding molecule described in claim 1.

11. 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) monoaxial 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 according to claim 10, comprising a delivery vehicle containing the Siglec-7 binding molecule.

15. The composition according to claim 14, wherein the delivery vehicle is lipid nanoparticles.

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 the Siglec-9 binding molecule described in any one of claims 6 to 9.

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

19. The composition according to 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 according to claim 17, further comprising a PD-(L) monoaxial 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 according to claim 17, comprising a delivery vehicle containing the nucleic acid molecule.

23. The composition according to claim 22, wherein the delivery vehicle is lipid nanoparticles.

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

25. A pharmaceutical composition for treating or preventing a disease or disorder, comprising one or more nucleic acid molecules encoding the Siglec-9 binding molecule described in Claim 1 or the Siglec-9 binding molecule described in Claim 6.

26. The pharmaceutical composition according to claim 25, wherein the disease or disorder is cancer or a disease or disorder related to cancer.

27. The pharmaceutical composition according to claim 26, wherein the cancer has an increased level of sialic acid.

28. The pharmaceutical composition according to 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 pharmaceutical composition according to claim 25, wherein the disease or disorder is an infectious disease or disorder.

30. A pharmaceutical composition for increasing the function of natural killer cells, comprising one or more nucleic acid molecules encoding the Siglec-9 binding molecule described in Claim 1 or the Siglec-9 binding molecule described in Claim 6.

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