Antibodies Against Human Siglec-7 and Their Use for Immunotherapy - Patent application

Abstract

The present invention provides compositions comprising anti-Siglec antibodies and nucleic acid molecules encoding same, as well as methods for treating or preventing diseases or disorders using same.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 325,904, filed March 31, 2022, U.S. Provisional Application No. 63 / 375,749, filed September 15, 2022, and U.S. Provisional Application No. 63 / 490,156, filed March 14, 2023, each of which is incorporated by reference in its entirety herein. [Background technology]

[0002] Cancers manipulate several immunological mechanisms to ensure a permissive local microenvironment that promotes tumor progression. Glycosylation is often cited as a hallmark of cancer. Altered surface glycosylation of tumor and infected cells is an important feature of cancer and various 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. The interaction of sialic acid-bound cancer and infected cells with Siglecs alters the immune cell phenotype, allowing them to evade the immune system.

[0003] Siglecs contain Sia-binding N-terminal V-set domains and C2-set Ig domains, which are thought to act as spacers or regulators of oligomerization. Most Siglecs function as transmembrane receptors in the immune system by recognizing Sia residues. Each type of Siglec recognizes a specific sialic acid structure.

[0004] Siglec7 is primarily localized in human natural killer (NK) cells and monocytes. Siglec7 controls the inhibitory signaling of NK cells. NK cells are essential innate immune cells that can directly kill unhealthy host cells, including virus-infected cells and tumor cells. NK cells are involved in a specialized form of cell killing known as antibody-dependent cell-mediated cytotoxicity (ADCC). Unlike T cells, which must be educated by antigen-presenting cells before they can recognize tumors, NK cells spontaneously lyse tumor cells without the need for antigen stimulation or prior activation.

[0005] Virus-infected and tumor cells down-regulate MHC-1 expression. NK cell-mediated cytotoxicity is not restricted by MHC-I molecules; rather, NK cells destroy cancer cells by perforin / granzyme-mediated lysis.

[0006] Siglec-7 has preferred binding specificity for the Neu5Acα2,8Neu5Acα-(diSia) sequence, but certain branched α2,6-sialyl residues (DSGb5, DSLc4) are also known to be preferred binding molecules.

[0007] There remains a need in the art for new cancer therapeutics that effectively treat cancer while minimizing adverse effects, and the present invention fulfills this unmet need. Summary of the Invention

[0008] In one embodiment, the present invention relates to an antibody or a fragment thereof that specifically binds to a sialic acid binding receptor. In one embodiment, the sialic acid binding receptor is Siglec-7.

[0009] In one embodiment, the antibody comprises a variable heavy chain sequence comprising the CDR sequences of SEQ ID NOs: 1-3; SEQ ID NOs: 17-19; SEQ ID NOs: 33-35; SEQ ID NOs: 49-51; SEQ ID NOs: 65-67; ​​or SEQ ID NOs: 81-83.

[0010] In one embodiment, the antibody comprises a variable heavy chain sequence comprising the CDR sequences of SEQ ID NOs: 9-11; SEQ ID NOs: 25-27; SEQ ID NOs: 41-43; SEQ ID NOs: 57-59; SEQ ID NOs: 73-75; or SEQ ID NOs: 89-91.

[0011] 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, or SEQ ID NO:84.

[0012] 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, or SEQ ID NO:92.

[0013] 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.

[0014] In one embodiment, the antibody comprises a variable heavy chain sequence of SEQ ID NO: 4 and a variable light chain sequence of SEQ ID NO: 12. In one embodiment, the antibody comprises a variable heavy chain sequence of SEQ ID NO: 20 and a variable light chain sequence of SEQ ID NO: 28. In one embodiment, the antibody comprises a variable heavy chain sequence of SEQ ID NO: 36 and a variable light chain sequence of SEQ ID NO: 44. In one embodiment, the antibody comprises a variable heavy chain sequence of SEQ ID NO: 52 and a variable light chain sequence of SEQ ID NO: 60. In one embodiment, the antibody comprises a variable heavy chain sequence of SEQ ID NO: 68 and a variable light chain sequence of SEQ ID NO: 76. In one embodiment, the antibody comprises a variable heavy chain sequence of SEQ ID NO: 84 and a variable light chain sequence of SEQ ID NO: 92.

[0015] 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, or SEQ ID NO:84. In one embodiment, the antibody comprises 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, or SEQ ID NO:92. In one embodiment, the antibody comprises 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, or SEQ ID NO:84. In one embodiment, the antibody comprises a fragment comprising at least 80% of the full length sequence 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, or SEQ ID NO:92.

[0016] 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. In one embodiment, the sialic acid binding receptor is Siglec-7.

[0017] In one embodiment, the nucleic acid molecule encodes an antibody comprising 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, or SEQ ID NO:84. In one embodiment, the nucleic acid molecule encodes an antibody comprising 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, or SEQ ID NO:92; a sequence having at least 95% identity to 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, or SEQ ID NO:84; a sequence having at least 95% identity to 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, or SEQ ID NO:92; 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, or SEQ ID NO:84; 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, or SEQ ID NO:92, or a combination thereof. In one embodiment, the nucleic acid molecule comprises a heavy chain CDR coding sequence of SEQ ID NO: 5-7; SEQ ID NO: 21-23; SEQ ID NO: 37-39; SEQ ID NO: 53-55; SEQ ID NO: 69-71; or SEQ ID NO: 85-87. In one embodiment, the 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; or SEQ ID NO: 93-95.

[0018] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the variable heavy chain sequence of SEQ ID NO:8; SEQ ID NO:24; SEQ ID NO:40; SEQ ID NO:56; SEQ ID NO:72; or SEQ ID NO:88; a nucleotide sequence encoding the variable light chain sequence of SEQ ID NO:16; SEQ ID NO:32; SEQ ID NO:48; SEQ ID NO:64; SEQ ID NO:80; or SEQ ID NO:96; SEQ ID NO:8; SEQ ID NO:24; SEQ ID NO:40; SEQ ID NO:56; SEQ ID NO:72; or a nucleotide sequence having at least 95% identity to the variable heavy chain sequence of SEQ ID NO:88; SEQ ID NO:16; SEQ ID NO:32; SEQ ID NO:48; SEQ ID NO:64; SEQ ID NO:80; or a sequence having at least 95% identity to the variable light chain sequence of SEQ ID NO:24; SEQ ID NO:40; SEQ ID NO:56; SEQ ID NO:72; or a fragment comprising at least 80% of the full length of the variable heavy chain sequence of SEQ ID NO:88; or a fragment comprising at least 80% of the full length of the variable light chain sequence of SEQ ID NO:16; SEQ ID NO:32; SEQ ID NO:48; SEQ ID NO:64; SEQ ID NO:80; or SEQ ID NO:96, or a combination thereof.

[0019] In one embodiment, the invention provides a combination of nucleic acid molecules, wherein a first nucleic acid molecule comprises a light chain CDR coding sequence of SEQ ID NO:5-7; SEQ ID NO:21-23; SEQ ID NO:37-39; SEQ ID NO:53-55; SEQ ID NO:69-71; or SEQ ID NO:85-87, and a 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; or SEQ ID NO:93-95. 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; or SEQ ID NO:88, 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; or SEQ ID NO:96, or a fragment or variant thereof.

[0020] 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 some embodiments, the composition further comprises an adjuvant. In some embodiments, the composition further comprises a tumor antigen or a nucleotide sequence encoding a tumor antigen.

[0021] 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.

[0022] In one embodiment, the present invention relates to a method of treating or preventing a disease or disorder in a subject in need thereof, the method comprising administering a sialic acid binding receptor antibody, a nucleic acid molecule encoding a sialic acid binding receptor antibody, or a composition comprising a sialic acid binding receptor antibody or a nucleic acid molecule encoding a sialic acid binding receptor antibody. In one embodiment, the disease or disorder is cancer or a cancer-related disease or disorder. In one embodiment, the disease or disorder is an infectious disease or disorder.

[0023] In one embodiment, the present invention relates to a method of increasing natural killer cell function in a subject in need thereof, the method comprising administering a sialic acid binding receptor antibody, a nucleic acid molecule encoding a sialic acid binding receptor antibody, or a composition comprising a sialic acid binding receptor antibody or a nucleic acid molecule encoding a sialic acid binding receptor antibody.

[0024] In one embodiment, the invention relates to an immunogenic composition comprising an antibody or a fragment thereof that specifically binds to a sialic acid-binding receptor and a nucleic acid molecule encoding a tumor antigen.

[0025] 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, skin cancer, or oral cancer. In one embodiment, the cancer has a mutation in a cancer driver gene as described in Table 1. In one embodiment, the cancer has drug resistance to one or more drugs described in Table 1.

[0026] In one embodiment, the present invention relates to a method for increasing natural killer cell function in a subject in need thereof, the method comprising administering an immunogenic composition comprising an antibody or fragment thereof that specifically binds to a sialic acid-binding receptor and a nucleic acid molecule encoding a tumor antigen.

[0027] In one embodiment, the present invention relates to a method for increasing natural killer cell function in a subject in need thereof, the method comprising administering an immunogenic composition comprising a nucleic acid molecule encoding an antibody or fragment thereof that specifically binds to a sialic acid-binding receptor and a nucleic acid molecule encoding a tumor antigen. [Brief description of the drawings]

[0028] [Figure 1] FIG. 1 shows binding of anti-Siglec-7 clones to human Siglec 7. [Diagram 2] FIG. 2 shows the expression of Siglec-7 by transduction. [Diagram 3] FIG. 3 shows binding of anti-human Siglec 7 antibody. [Figure 4] FIG. 4 shows exemplary experimental results showing cytotoxicity of OVISE cells by Siglec-7 antibody clones. [Diagram 5] FIG. 5 shows exemplary experimental results showing cytotoxicity of Siglec-7 antibody clones to SKOV3 cells. [Figure 6]FIG. 6 shows exemplary experimental results showing cytotoxicity of OVCAR10 cells by Siglec-7 antibody clones. [Figure 7] Figure 7 shows a representative example of siglec-7 ligand expression in human melanoma. Siglec-7 ligand is highly expressed in melanoma cells. [Figure 8] FIG. 8 shows the cytotoxicity of Siglic-7 antibody against 451Lu BR human melanoma cell line. [Figure 9] FIG. 9 shows the cytotoxicity of Siglic-7 antibody against WM1366 human melanoma cell line. [Figure 10] FIG. 10 shows the cytotoxicity of Siglic-7 antibody against 1205Lu human melanoma cell line. [Figure 11] FIG. 11 shows the cytotoxicity of Siglic-7 antibody against WM3311 human melanoma cell line. [Figure 12] FIG. 12 shows the cytotoxicity of Siglic-7 antibody against WM983B BR human melanoma cell line. [Figure 13] Figures 13A to 13C show the design and in vitro expression of Siglec-7 MAb. (Figure 13A) Schematic of the dual plasmid DNA construct encoding Siglec-7 MAb. (Figure 13B) Expression analysis of Siglec-7 MAb by Western blot. Numbers indicate molecular weight (kDa). (Figure 13C) Quantification of human IgG in anti-Siglec-7 transfected Expi293F supernatant by ELISA. [Figure 14]Figures 14A-E show the binding characteristics of human Siglec-7 MAbs in vitro (Figure 14A). Binding of Siglec-7 MAbs to recombinant human Siglec-7 by ELISA. Dose-dependent binding of DB7.1, DB7.2, and DB7.7 to human Siglec-7 was observed. (Figure 14B) Binding of human Siglec-7 MAbs to Siglec-7-expressing HEK293T cells was analyzed by flow cytometry. No binding was observed with secondary antibody control or irrelevant MAb alone. Binding was observed with the commercially available Siglec-7 Ab (F023-420, BD Pharmingen) and Siglec-7 MAbs DB7.1, DB7.2, and DB7.3, with the highest binding observed with DB7.2. (Figure 14C) Representative flow plots showing binding of DB7.2 anti-Siglec-7 antibody to human NK cells and T cells. DB7.2 binds primarily to NK cells. Positive control (commercially available fluorochrome-labeled mouse anti-human Siglec-7 antibody) and negative control (0 μg / ml DB7.2 or no secondary antibody) are shown (FIG. 14D). Summary plot of DB7.2 anti-Siglec-7 binding to different immune populations analyzed in PBMCs from multiple donors (n=4). (FIG. 14E) Binding analysis of DB7.2 anti-Siglec-7 Ab to CD56dim and CD56bright NK cell subsets (n=4). [Figure 15] Figure 15 shows the gating strategy used for flow cytometric analysis of Siglec-7 binding. A representative example of ND is shown. First, the most stable gating was selected. Second, to ensure that only live single cells were analyzed from mononuclear cells, doublets were excluded using forward scatter height (FSC-H)-forward scatter area (FSC-A) and side scatter area (SSC-A)-FSC-A plots, and singlet small lymphocytes were focused on. Dead cells were excluded by gating on cells negative for the viability marker Aqua Blue and positive for CD45. CD4+ and CD8+ T lymphocytes were gated within CD3+ cells, and NK cells were identified by CD56 and CD16 expression within the CD19-CD3- population. [Figure 16]Figures 16A to 16D show binding of Siglec-7 DMAb to human Siglec-7 protein. (Figure 16A) Binding of DB7.2 Siglec-7 DMAb to human Siglec-7 by Western blot. No binding was observed to unrelated proteins in the case of pVax1. (Figure 16B) SPR analysis of DB7.2 to human Siglec-7 protein. Strong binding of DB7.2 was observed, with a KD value of 44 pM. (Figure 16C) No binding was observed to Siglec-7 with an unrelated ab, or (Figure 16D) to an unrelated protein, demonstrating the specificity of DB7.2 for human Siglec-7. [Figure 17]Figure 17A to Figure 17I show the in vitro cytotoxicity induced by human Siglec-7 MAbs. In vitro cytotoxicity induced by human Siglec-7 MAbs in non-target (Figure 17A&B) HaCaT human keratinocyte cells and target OC cells; (Figure 17C&D) OVCAR10, (Figure 17E) OVISE, (Figure 17F) PEO-4, and (Figure 17G) TOV-21G cells. In vitro cytotoxicity was measured based on impedance using xCELLigence Real-Time Cell Analyzer (RTCA) from Agilent Technologies, USA. Electrical conductivity was converted to cell index (CI) by the xCELLigence instrument every 15 minutes. DB7.1, DB7.2, and DB7.7 did not induce killing in HaCaT cells. As shown in the images after 2 days of treatment, no killing was observed in DB7.1, DB7.2, and DB7.7 treated wells as well as in the no Ab control. All three Siglec-7 MAbs effectively killed human OC cells, including BRCA2 mutant and PARPi-resistant PEO-4 cells. DB7.2 MAb showed the highest killing ability. The killing ability of DB7.2 was further confirmed in two OC cell lines; (Fig. 17H) CaOV3 cells and (Fig. 17I) OVCAR3 cells. Arrows indicate the time points when the antibody and effector cells (human PBMCs) were added to the target cells; E:T=5:1 / 10:1. Red: no Ab (effector cells + target cells only); green: DB7.1 (effector cells + target cells + DB7.1); blue: DB7.2 (effector cells + target cells + DB7.2), yellow: DB7.7 (effector cells + target cells + DB7.7). [Figure 18]Figures 18A to 18C show inhibition of FcR binding of Siglec-7 MAbs. (Figure 18A) Schematic of FcR blockade on immune cells and its effect on killing by Siglec-7. (Figure 18B) xCELLigence real-time analysis of OVCAR3 cell killing by DB7.2 human MAb with and without Fc block. (Figure 18C) Fc block maintained DB7.2 Siglec-7 MAb-mediated NK toxicity in OVISE cells. Images were taken 2 days after effector cells (E:T=5:1) and DB7.2 MAb were added to OVISE cells with and without Fc block. [Figure 19] Figures 19A to 19I show that loss of FcR binding maintained the killing potency of Siglec-7 MAb, and the combination of Siglec-7 MAb and anti-PD1 enhanced the killing of OC cells. (Figure 19A) Comparison of killing of OVISE cells by DB7.2 and DB7.2 TM Mod. DB7.2 TM Mod is a mutant form of DB7.2 DMAb that contains a triple residue modification ("TM"; L234F / L235E / P331S) in the Fc domain that inhibits FcR and C1q binding. Killing potency of DB7.2 TM Mod was maintained in OVISE cells. Red: no Ab (effector cells + target cells only); dark blue: DB7.2 (effector cells + target cells + DB7.2); light blue (effector cells + target cells + DB7.2 TM Mod). (Figure 19B) Images after 24 hours of treatment with effector cells and DB7.2 / DB7.2 TM Mod to target OVISE cells. (Figure 19C) DB7.2 and (Figure 19D) DB7.2 TM Mod cell index vs. treatment dose curves for 72 hours of co-culture of OVISE cells with human PBMCs. (Figure 19E) DB7.2 TM Mod and (Figure 19F) anti-PD1 (pembrolizumab) cell index vs. treatment dose curves for 72 hours of co-culture of PEO4 cells with human PBMCs. (Figure 19G) DB7.2 TM Mod, (Figure 19H) anti-PD1 (pembrolizumab), and (Figure 19I) DB7.2 TM Mod and anti-PD1 alone and in combination in the presence of human PBMCs to kill target PEO4 cells; (E:T=5:1). This combination enhanced killing compared to individual CPI treatments. [Figure 20] Figures 20A-E show that Siglec-7 DNA delivery MAbs are expressed in vivo and slow cancer progression in an ovarian cancer challenge model. (Figure 20A) Schematic diagram of administration of human Siglec-7 DNA delivery MAbs to Balb / c mice. Mice were first depleted of CD4 and CD8 T cells using anti-mouse CD4 and anti-mouse CD8 antibodies (both from InVivoMab). Mice were then electroporated with human Siglec-7 DMAbs, 50 μg heavy chain + 50 μg light chain. Mouse serum was collected at the indicated time points. (Figure 20B) Staining of human NK cells with sera from mice electroporated with empty vector, DB7.1, DB7.2, and DB7.7, serum 14 days after DNA injection and electroporation, or naive serum alone along with irrelevant and secondary Ab controls. Day 14 sera from mice immunized with Siglec-7 MAbs showed positive and strong NK cell staining, whereas no staining was observed with empty vector control, irrelevant MAb, or naive serum. (Figure 20C) Schematic of Siglec-7 DNA delivery MAb electroporation into NSG-K mice challenged with OVISE cells. (Figure 20D) Growth curves of OVISE tumors implanted in NSG-K mice treated with DB7.2 or empty vector (n=5 mice per group). (Figure 20E) Survival benefit of DB7.2-treated mice compared to empty vector controls. DB7.2-treated groups had significantly reduced tumor burden and enhanced survival compared to pVax1 controls. [Figure 21] Figures 21A and 21B show that Siglec-7 DMAb affected tumor growth in an OVISE-challenged mouse model. (Figure 21A) Individual growth curves of OVISE tumors implanted in NSG-K mice treated with DB7.2 DMAb or pVax1 empty vector (n=5 mice per group). (Figure 21B) Representative images of ovarian tumor-bearing mice treated with DB7.2 DMAb or pVax1 on day 37. [Figure 22]Figures 22A to 22K show the generation, expression, binding and specificity analysis of DB7.2xD2AP11:FSHR-targeted bispecific NK cell engager. (Figure 22A) Schematic of DNA construct encoding DB7.2xD2AP11 NKCE; GS: glycine-serine, ScFV: single chain variable fragment. (Figure 22B) Western blot of in vitro expression of DB7.2xD2AP11 NKCE or pVax1 empty vector after transfection into expi293F cells. (Figure 22C) Flow cytometry plot of Siglec-7 overexpressing HEK293T cells stained with secondary antibody, DB7.2xD2AP11 NKCE, Siglec-7 antibody (F023-420 / DB7.2) and DB7.2xD2AP11 NKCE only. (FIG. 22D) Flow cytometry plots of FSHR-overexpressing K562 cells stained with secondary Ab only, non-FSHR-targeting T cell engager (IL13 receptor α2xCD3), D22AP11-TCE (FSHRxCD3) and DB7.2xD2AP11 NKCE. (FIG. 22E) IFA of DB7.2xD2AP11 NKCE in Siglec-7-overexpressing HEK293T cells. Staining with secondary Ab only served as a negative control. Leica TCS SP8 WLL scanning laser confocal microscope and Leica LAS-X software (Leica Microsystems, Inc.). Image post-processing included importing into Huygens software (Scientific Volume Imaging, Laapersveld, Hilversum, The Netherlands) for deconvolution. Fixed cell specimens were acquired with a 63X / 1.40 oil objective, 2x zoom, pinhole 1AU. Cells were labeled with DAPI (nuclei), GFP (Siglec-7) and Texas Red (DB7.2xD2AP11 NKCE) and acquired sequentially using the HyD detector to maximize signal and minimize crosstalk. Scale bar represents 20.0 microns. Specificity analysis of DB7.2xD2AP11 NKCE in FSHR negative (Figure 22F&G) HEK293T, (Figure 22H) GM05389, (Figure 22I&J) AGS and (Figure 22K) WM3743 cells using xCELLigence real-time cell analyzer.No cell death was observed in these non-FSHR expressing cells. Images showing that no cytotoxicity was induced in HEK293T (G) and AGS (J) cells after addition of effector cells (human PBMC; E:T=10:1) and DB7.2xD2AP11 for 3 days. Red: no Ab (effector cells + target cells only); Purple: DB7.2xD2AP11 (effector cells + target cells + DB7.2xD2AP11 NKCE). [Diagram 23] Figures 23A to 23I show that FSHR-targeted NK cell engager; DB7.2xD2AP11 NKCE induced ovarian cancer cytotoxicity in vitro. In vitro cytotoxicity induced by DB7.2xD2AP11 NKCE in the presence of human PBMC in Figures 23A and B) OVISE cells, and (Figure 23C) OVCAR3-FSHR cells. In the presence of human PBMC in (Figures 23D&E) CaOV3 cells, (Figure 23F) Kuramochi-FSHR cells, and (Figure 23G) PEO-4 cells. Real-time in vitro cytotoxicity analysis was performed with xCELLigence. E:T was 10:1 (A&B, F&G) and 5:1 (CE), and images shown in (B&E) were taken 3 days after addition of human PBMC and NKCE to target cells. (FIG. 23H) Dose-dependent cell lysis induced by DB7.2xD2AP11 NKCE in FSHR-expressing OVISE cells in the presence of human PBMC (E:T=5:1). The IC50 value was 142.87 pM, indicating the efficacy of NKCE. (FIG. 23I) Cell index vs. dose curve of DB7.2xD2AP11 NKCE in OVCAR3 cells. The IC50 value was 236.6 pM, confirming the efficacy of NKCE. [Figure 24]Figures 24A to 24D show specificity analysis of FSHR-targeted bispecific NK cell engagers. (Figure 24A) In vitro cytotoxicity by co-culture of NK cells with OVCAR3-FSHR cells in the presence of DB7.2xD2AP11 NKCE. (Figure 24B) Killing of AGS (FSHR-negative) cells by anti-Siglec-7 MAb (DB7.2xD2AP11 NKCE did not induce killing of this cell line, indicating the specificity of the bispecific engager approach). Irrelevant NKCE; DB7.2xIL13Rα2 NKCE did not induce toxicity in (Figure 24C) FSHR-overexpressing OVCAR3 cells and (Figure 24D) OVISE cells in the presence of human PBMC. Real-time in vitro cytotoxicity analysis was performed with xCELLigence; E:T=5:1. Arrows indicate the time of effector cell and NKCE addition. [Diagram 25] Figures 25A to 25C show the dose-dependent cytotoxicity induced by DB7.2xD2AP11 NKCE in target FSHR overexpressing OVISE cells. DB7.2xD2AP11 NKCE induced dose-dependent killing in OVISE-FSHR cells in the presence of human PBMCs (Figure 25A) ND578, (Figure 25B) ND502 and (Figure 25C) ND609 from three different healthy human donors and analyzed at different concentrations; 120, 60, 30, 15, 7.5, 3.75 and 1.875 ng / ml. Red line indicates no Ab (effector cells only target cells). In vitro cytotoxicity was measured based on impedance using xCELLigence real-time cell analyzer. E:T=5:1. Arrows indicate the time of addition of effector cells and NKCE to target OVISE-FSHR cells. [Figure 26]Figure 26 shows the dose-dependent cytotoxicity induced by DB7.2xD2AP11 NKCE in target OVCAR3 cells. DB7.2xD2AP11 NKCE induced dose-dependent killing in OVCAR3 cells in the presence of human PBMCs and was analyzed at different concentrations (120, 60, 30, 15, 7.5, 3.75 and 1.875 ng / ml). Red line indicates no Ab (effector cells only target cells). In vitro cytotoxicity was measured based on impedance using xCELLigence real-time cell analyzer. E:T=10:1, arrows indicate the time of addition of effector cells and NKCE to target OVCAR3 cells. [Figure 27] Figures 27A to 27E show that DB7.2xD2AP11 NKCE caused secretion of cytokines / cytotoxic molecules and influenced ovarian tumor growth in vivo. (Figure 27A) OVCAR3-FSHR cells were co-cultured with human PBMCs in the presence and absence of DB7.2xD2AP11 NKCE. (Figure 27B) Supernatants were collected after 72 hours and analyzed for cytokine / cytotoxic molecule secretion profiles. (Figure 27C) Schematic of tumor study to evaluate the effect of DB7.2xD2AP11 NKCE on tumor progression in OVCAR3-FSHR challenged NSG-K mouse model. (Figure 27D) Mean growth curves of OVCAR3-FSHR tumors implanted in NSG-K mice treated with DB7.2xD2AP11 NKCE or empty vector (n=5 mice per group). (Figure 27E) Survival benefit of DB7.2xD2AP11 NKCE mice compared to empty vector controls. Two-way ANOVA; *P<0.05, **P<0.01, ***P<0.001. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The present invention relates to sialic acid receptor antibodies, fragments, variants thereof, or nucleic acid molecules encoding same, and methods of use for enhancing natural killer cell activity in a subject in need thereof.

[0030] In one aspect, the invention relates to compositions that can be used to increase 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 the same. In one embodiment, the sialic acid receptor antibody is an antibody against Siglec-7.

[0031] In one aspect, the present invention relates to a combination of a sialic acid receptor antibody, a fragment or variant thereof, or a nucleic acid molecule encoding same, with a nucleic acid molecule encoding a tumor antigen.

[0032] 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.

[0033] 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 same, in combination with a nucleic acid molecule encoding a tumor antigen. In one embodiment, the cancer has increased levels of sialic acid.

[0034] 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.

[0035] As used herein, each of the following terms has the associated meaning in this section. The articles "a" and "an" are used herein to refer to one or to more than one (i.e., 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.

[0036] As used herein, "about" when referring to a measurable value, such as an amount, temporal duration, etc., is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the stated value, as appropriate variations for carrying out the disclosed methods. An "antibody" is an antibody of the IgG, IgM, IgA, IgD, or IgE class, or a fragment, fragment, or derivative thereof, including Fab, F(ab')2, Fd, 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, or a sequence derived therefrom.

[0037] An "antigen" is a protein that has the ability to provoke an immune response in a host. Antigens are recognized and bound by antibodies. Antigens can originate from within the body or from the external environment.

[0038] "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, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., US Department of Health and Human Services, National Institutes of Health (1987). There are three heavy and three light chain CDRs (or CDR regions) in the variable region of an immunoglobulin. Thus, as used herein, "CDR" refers to all three heavy chain CDRs or all three light chain CDRs (both all heavy chain CDRs and all light chain CDRs, if appropriate). The structure and protein folding of an antibody may mean that other residues are considered part of the antigen binding region, and will be understood as such by those skilled in the art. See, e.g., Chothia et al., (1989) Conformations of immunoglobulin hypervariable regions; Nature 342, p 877-883.

[0039] "Antibody fragment" or "fragment of an antibody", as used interchangeably herein, refers 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 containing only one light chain variable domain, single chain polypeptides containing the three CDRs of a light chain variable domain, single chain polypeptides containing only one heavy chain variable region, single chain polypeptides containing the three CDRs of a heavy chain variable region.

[0040] As used herein, "adjuvant" means a molecule added to the vaccines described herein to enhance the immunogenicity of an antigen.

[0041] 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 as defined herein. A coding sequence may also include a DNA sequence encoding an RNA sequence. A coding sequence may further include initiation and termination signals operably linked to regulatory elements including 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 include a sequence encoding a signal peptide.

[0042] As used herein, "complementarity" or "complementary" can mean that a nucleic acid can have Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairs between nucleotides or nucleotide analogs of a nucleic acid molecule.

[0043] A "disease" is a state of health in which an animal is unable to maintain homeostasis, and unless the disease is corrected, the animal's health will continue to deteriorate.

[0044] In contrast, an animal "disorder" is one in which the animal is able to maintain homeostasis, but the animal's health is less favorable than it would be in the absence of the disorder. Ignoring the disorder does not necessarily result in a further decline in the animal's health.

[0045] A disease or disorder is "alleviated" if the severity of a sign or symptom of the disease or disorder, the frequency with which such signs or symptoms are experienced by a patient, or both, are reduced.

[0046] "Encoding" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, mRNA, etc., to serve as a template for the synthesis of other polymers and macromolecules in biological processes, either having a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and the biological properties resulting therefrom. Thus, a gene encodes a protein when transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. 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 the gene or cDNA) can be said to encode the protein or other product of that gene or cDNA.

[0047] 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.

[0048] "Expression vector" refers to a vector containing a recombinant polynucleotide in which an expression control sequence is operably linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied in 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.

[0049] A "feedback mechanism" as used herein may refer to a process implemented by either software or hardware (or firmware) that receives the desired tissue impedance (before, during, and / or after delivery of a pulse of energy), compares it to a current value, preferably the current, and adjusts the pulse of energy delivered to achieve a preset value. The feedback mechanism may be implemented by an analog closed loop circuit.

[0050] By "fragment" is meant a polypeptide fragment of an antibody that is functional, i.e. binds to a desired target and exerts the same 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 either case with or without the signal peptide and / or 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, including 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 any added heterologous signal peptide). Fragments may be 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identical to an antibody and may further include fragments of a polypeptide including an N-terminal methionine or a heterologous signal peptide that are not included in calculating the 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 signal peptide may be linked to a fragment of an antibody. A fragment of a nucleic acid sequence encoding an antibody may be 100% identical to the full length except for at least one nucleotide deletion from the 5' and / or 3' end, in either case with or without sequences encoding a signal peptide and / or a methionine at position 1. Fragments can be 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, including 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 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 in calculating percent identity. Fragments may further include sequences encoding a signal peptide, such as an N-terminal methionine and / or an immunoglobulin signal peptide, e.g., an IgE or IgG signal peptide. The coding sequence encoding the N-terminal methionine and / or the signal peptide may be linked to the fragment of the coding sequence.

[0051] 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, including a promoter and a polyadenylation signal, that are capable of directing expression in the cells of an individual to whom 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.

[0052] "Homologous" refers to sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. If both positions of the two compared sequences are occupied by the same base or amino acid monomer subunit, e.g., if each position 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 times 100. For example, if 6 out of 10 positions of two sequences are matching or homologous, then the two sequences are 60% homologous. As an example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, the comparison is performed when the two sequences are aligned to give maximum homology.

[0053] "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. This 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 occur in both sequences to obtain the number of matched positions, dividing the number of matched 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 different in length, or if the alignment results in one or more offset ends, and if the specified region of comparison contains only a single sequence, the residues of the single 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. Identity can be determined manually or using computer sequence algorithms such as BLAST and BLAST 2.0.

[0054] "Isolated" means changed 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 it can exist in a non-native environment, such as a host cell.

[0055] In the context of the present invention, the following abbreviations are used for commonly occurring nucleobases: "A" for adenosine, "C" for cytosine, "G" for guanosine, "T" for thymidine, and "U" for uridine.

[0056] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. A nucleotide sequence that encodes a protein or RNA may also contain introns, to the extent that a nucleotide sequence that encodes a protein may, in some version, contain introns.

[0057] "Impedance" as used herein is sometimes 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.

[0058] As used herein, "immune response" may refer to activation of a host's immune system, e.g., a mammal's immune system, in response to the introduction of one or more nucleic acids and / or peptides. The immune response may be in the form of a cellular or humoral response, or both.

[0059] 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 are amenable to the methods described herein. In some embodiments, the patient, subject, or individual is a human.

[0060] "Parenteral" administration of the compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intradermal injection, or infusion techniques.

[0061] As used herein, "nucleic acid" or "oligonucleotide" or "polynucleotide" may refer to 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 can 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.

[0062] 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 deoxyribo- 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.

[0063] As used herein, "operably linked" may mean that the expression of a gene is under the control of a spatially linked promoter. 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, variations in this distance may be accommodated without impairing the function of the promoter.

[0064] As used herein, "peptide", "protein" or "polypeptide" refers to a linked sequence of amino acids, which may be natural, synthetic, or modified or combined natural and synthetic. As used herein, "promoter" may refer to a synthetic or naturally derived molecule capable of conferring, activating or enhancing expression of a nucleic acid in a cell. A promoter may contain one or more specific transcriptional regulatory sequences to further enhance expression and / or alter its spatial and / or temporal expression. A promoter may also contain distal enhancer or repressor elements, which may be as far as several thousand base pairs away from the transcription start site. Promoters may be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. Promoters may constitutively or differentially control expression of genetic components with respect to the cell, tissue or organ in which expression occurs, or the developmental stage in which expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, inducers, etc. 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.

[0065] 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 the core promoter sequence, and in other cases, this sequence may also include enhancer sequences and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may, for example, be one that confers tissue-specific expression of the gene product.

[0066] A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0067] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes that gene product to be substantially produced in a cell only if an inducer corresponding to that promoter is present in the cell.

[0068] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding a gene or specified by a gene, causes a gene product to be produced in a cell substantially only if that cell is a cell of the tissue type corresponding to the promoter.

[0069] "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 as defined 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. Upon secretion from the cell, the signal peptide / leader sequence is cleaved from the remainder of the protein, often referred to as the mature protein. The signal peptide / leader sequence is linked at the N-terminus of the protein.

[0070] As used herein, "stringent hybridization conditions" can 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 vary from situation to situation. Stringent conditions are those conditions that meet the thermal melting point (T) for a particular sequence at a pH of defined ionic strength. m) can be selected to be about 5 to 10°C lower than T m can be the temperature (at defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (because the target sequences are present in excess, T m (In this case, 50% of the probes are occupied at equilibrium). Stringent conditions are conditions in which the salt concentration is about 1.0 M sodium ion or less, e.g., 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., about 50 nucleotides or more). Stringent conditions can also be achieved by the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least 2 to 10 times higher than background hybridization. Exemplary stringent hybridization conditions are as follows: 50% formamide, 5xSSC, 1% SDS, incubate at 42° C., or 5xSSC, 1% SDS, incubate at 65° C., wash at 0.2xSSC, 0.1% SDS, 65° C.

[0071] "Subject" and "patient", as 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 such as cynomolgus or rhesus monkeys, chimpanzees), and humans. In some embodiments, a subject may be human or non-human. A subject or patient may be undergoing other forms of treatment.

[0072] As used herein, "substantially complementary" means that a first sequence is 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. "Hybrid" means that the sequence is at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence or that the two sequences hybridize under stringent hybridization conditions.

[0073] As used herein, "substantially identical" means that a first and 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, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 550, 560, 570, 580, 590, 610, 620, 630, 640, 650, 660, 670, 680, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790 , 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 or more regions, 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 over a region of the sequence.

[0074] As used herein, "synthetic antibody" refers to an antibody encoded by a recombinant nucleic acid sequence as described herein and produced in a subject.

[0075] As used herein, "treatment" or "treating" may mean protecting a subject from a disease by means of preventing, suppressing, inhibiting, or completely eliminating the disease. Preventing a disease involves administering a vaccine of the invention to a subject prior to the onset of the disease. Suppressing a disease involves administering a vaccine of the invention to a subject after the disease has been induced but prior to clinical appearance. Suppressing a disease involves administering a vaccine of the invention to a subject after the clinical appearance of the disease.

[0076] A "therapeutic" treatment is a treatment administered to a subject who exhibits signs or symptoms of a disease or disorder for the purpose of reducing or eliminating the frequency or severity of those signs or symptoms.

[0077] 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 a patient.

[0078] 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 the progression of, inhibit, reduce, or reverse) a disease or disorder, including alleviating the signs and / or symptoms of such diseases and disorders.

[0079] As used herein, "treating" a disease or disorder means reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject.

[0080] "Variant," as used herein with respect to a nucleic acid, means (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of the referenced nucleotide sequence or a portion thereof; (iii) a nucleic acid that is substantially identical to the referenced nucleic acid or its complement; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, its complement, or a sequence substantially identical thereto.

[0081] A variant can be 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, the ability to stimulate an immune response, and the like. 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 typically recognized in the art as involving minor changes. These minor changes can be identified, in part, by considering the hydrophilicity index of the amino acid, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydrophobicity index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids with similar hydrophobicity indexes can be substituted while retaining protein function. In one aspect, amino acids with hydrophobicity indexes of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that will result in a protein retaining biological function. By considering the hydrophilicity of the amino acids that make up a peptide, the maximum local average hydrophilicity of the peptide can be calculated. Substitution of amino acids with similar hydrophilicity values ​​can result in peptides that retain biological activity, such as immunogenicity, as understood in the art. Substitutions can be made with amino acids that have hydrophilicity values ​​within ±2 of each other. Both the hydrophobicity index and hydrophilicity value of an amino acid are influenced by the particular side chain of that amino acid. Consistent with that observation, it is understood that amino acid substitutions that are compatible with biological function depend on the relative similarity of the amino acids, particularly the side chains of those amino acids, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0082] 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.

[0083] A "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides bound to 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, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.

[0084] Ranges: Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity, and should not be construed as a flexible limitation of the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. For example, description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and individual numerical values ​​within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, 6. This applies regardless of the breadth of the range.

[0085] explanation Provided herein are antibodies or antibody-like molecules that specifically bind to Siglec-7. In one embodiment, the present invention provides an immunogenic composition comprising an antibody or antibody-like molecule that specifically binds to Siglec-7 of the present invention, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same. The immunogenic composition of the present invention can be used to protect against diseases or disorders associated with altered glycosylation, including, but not limited to, cancer and infectious diseases. Thus, in some embodiments, the present invention provides a composition comprising one or more antibodies against Siglec-7, a fragment thereof, or a variant thereof, or a nucleic acid molecule encoding the same.

[0086] 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-7, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding same of the present invention.

[0087] In some embodiments, the present invention provides a method for treating or preventing cancer associated with increased Siglec-7, comprising administering to a subject an antibody or antibody-like molecule that specifically binds to Siglec-7, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding same of the present invention.

[0088] antibody composition In one embodiment, the present invention relates to a composition comprising at least one Siglec-7 antibody or fragment thereof.

[0089] In one embodiment, the antibody comprises a variable heavy chain sequence comprising the CDR sequences of SEQ ID NOs: 1-3; SEQ ID NOs: 17-19; SEQ ID NOs: 33-35; SEQ ID NOs: 49-51; SEQ ID NOs: 65-67; ​​or SEQ ID NOs: 81-83.

[0090] In one embodiment, the antibody comprises a variable heavy chain sequence comprising the CDR sequences of SEQ ID NOs: 9-11; SEQ ID NOs: 25-27; SEQ ID NOs: 41-43; SEQ ID NOs: 57-59; SEQ ID NOs: 73-75; or SEQ ID NOs: 89-91.

[0091] 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, or SEQ ID NO:84, or a fragment or variant thereof.

[0092] 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, or SEQ ID NO: 92, or a fragment or variant thereof.

[0093] 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.

[0094] 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.

[0095] In some embodiments, variants of the amino acid sequences described herein include at least about 60% identity, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over a specified region when compared to the defined amino acid sequence. In some embodiments, variants of the amino acid sequences described herein include at least one or more of the following: a variable heavy chain having an amino acid sequence of SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, or SEQ ID NO:84, or a variable light chain having one or more of the amino acid sequences of SEQ ID NO:12, SEQ ID NO:28, SEQ ID NO:44, SEQ ID NO:60, SEQ ID NO:76, or SEQ ID NO:92. 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, or SEQ ID NO:84 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, or SEQ ID NO:84. 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, or SEQ ID NO:92 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, or SEQ ID NO:92.

[0096] As used herein, the term "antibody" or "immunoglobulin" refers to a protein (including glycoproteins) of the immunoglobulin (Ig) superfamily. 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 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.

[0097] Sialic acid binding receptor antibodies or antigen-binding fragments thereof include, but are not limited to, polyclonal antibodies, monoclonal fusion proteins, antibodies or fragments thereof, chimerized or chimeric fusion proteins, antibodies or fragments thereof, humanized fusion proteins, antibodies or fragments thereof, deimmunized human humfusion proteins, antibodies or fragments thereof, fully human fusion 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, camelized fusion proteins, antibodies or fragments thereof, dromedary fusion proteins, antibodies or fragments thereof, phage display fusion proteins, antibodies or fragments thereof, or antibodies or antigen-binding fragments thereof identified in repetitive backbone arrays (e.g., repetitive antigen display).

[0098] 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: α (a) found in IgA, δ (delta) found in IgD, ε (epsilon) found in IgE, γ (gamma) found in IgG, and μ (mu) found in IgM. There are at least five different gamma heavy chain polypeptides (isotypes) of IgG. In contrast, there are only two light chain polypeptide isotypes: kappa and lambda. The characteristics of an antibody isotype are defined by the sequence of the constant domain of the heavy chain.

[0099] IgG molecules consist of two light chains (κ or λ) and two heavy chains (γ) that are linked by disulfide bonds. The κ and λ types of IgG light chains each have a variable region (V L -," "V κ -" or "V λ The amino acid sequence of each protein is divided into two parts: a domain of relatively variable amino acid sequence called the constant region (C-region) and L Similarly, each IgG heavy chain contains domains of relatively conserved amino acid sequences called variable regions (V H -region) and one or more conserved regions. A complete IgG heavy chain contains three constant domains ("C H 1-Area", "C H 2-Area", "C H 3-region) and the hinge region. L - or V HWithin the -region, hypervariable regions, also known as complementarity determining regions (CDRs), are interspersed between relatively conserved framework regions (FRs). In general, the variable region of a light or heavy chain polypeptide contains 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, therefore, 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 domain, so named because it crystallizes easily.

[0100] 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 can be made in or derived from any of a variety of species, e.g., mammals such as humans, non-human primates (e.g., monkeys, baboons, chimpanzees), horses, cows, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, mice, etc. Antibodies can be purified or recombinant.

[0101] As used herein, the term "epitope" refers to a site on a protein that is bound by an antibody. "Overlapping epitopes" include at least one (e.g., two, three, four, five, or six) common amino acid residues.

[0102] In one embodiment, the antibody of the present invention specifically binds to a Siglec polypeptide. As used herein, the term "specific binding" or "specifically binds" refers to two molecules forming a complex that is relatively stable under physiological conditions. Binding is usually determined by an association constant (K a ) is 10 6 M -1 Therefore, an antibody should have at least (or more than) 10 6 (e.g., at least 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , or 10 15 above)M -1 It can specifically bind to a target having a Ka of

[0103] In one embodiment, the antibody of the invention specifically binds to Siglec-7.

[0104] Methods for determining whether an antibody binds to a protein antigen and / or the affinity of an antibody to a protein antigen are known in the art. For example, various techniques can be used to detect and / or quantify the binding of an antibody to a protein antigen, 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 KC Lo (2004) “Antibody Engineering: Methods and Protocols”, Humana Press (ISBN: 1588290921); Borrebaek (1992) “Antibody Engineering, A Practical Guide”, WH Freeman and Co., NY; Borrebaek (1995) "Antibody Engineering", 2nd Edition, Oxford University Press, NY, Oxford; Johne et al. (1993) J. Immunol. Meth. 160: 191-198; Jonsson et al. (1993) Ann. Biol. Clin. 51: 19- 26; and Jonsson et al. (1991) Biotechniques 11 :620-627. See also U.S. Patent No. 6,355,245.

[0105] Immunoassays that can be used to analyze antibody immunospecific binding and cross-reactivity include competitive and non-competitive assay systems using techniques such as, but not limited to, Western blots, RIAs, ELISAs (enzyme-linked immunosorbent assays), "sandwich" immunoprecipitation assays, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, etc. Such assays are routinely performed and well known in the art.

[0106] Antibodies can also be assayed using surface plasmon resonance (SPR)-based assays known in the art to characterize the kinetic parameters of the interaction of an antibody with its target or epitope. Any SPR instrument is commercially available, including but not limited to BIAcore Instruments (Biacore AB; Uppsala, Sweden); IAsys instruments (Affinity Sensors; Franklin, Massachusetts); IBIS system (Windsor Scientific Limited; Berks, UK); SPR-CELLIA systems (Nippon Laser and Electronics Lab; Hokkaido, Japan), and SPR Detector Spreeta (Texas Instruments; Dallas, Texas), and can be used in the methods described herein. See, e.g., Mullett et al. (2000) Methods 22: 77-91; Dong et al. (2002) Reviews in Mol Biotech 82: 303-323; Fivash et al. (1998) Curr Opin Biotechnol 9: 97-101; and Rich et al. (2000) Curr Opin Biotechnol 11:54-61.

[0107] Antibodies and fragments thereof can be "chimeric" in some embodiments. Chimeric antibodies and antigen-binding fragments thereof are composed of portions from two or more different species (e.g., mouse and human). Chimeric antibodies can be produced by splicing mouse variable regions with the desired specificity into 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 be suitable for human clinical applications (e.g., methods for treating or preventing complement-associated disorders in human subjects).

[0108] The monoclonal antibodies of the present disclosure include "humanized" versions of non-human (e.g., murine) antibodies. Humanized or CDR-grafted mAbs are particularly useful as therapeutic agents for humans because they are not cleared from the circulation as rapidly as murine antibodies and generally do not provoke adverse immune responses. Methods for preparing humanized antibodies are generally well known in the art. For example, humanization can be performed essentially by replacing rodent CDRs or CDR sequences with the corresponding sequences of a human antibody according to the method of Winter and coworkers (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, e.g., 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 with hypervariable region residues of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate that has the desired specificity, affinity, and binding capacity. In some cases, framework region residues of the human immunoglobulin are also replaced with corresponding non-human residues (so-called "backmutation"). Additionally, 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 backbone. Additionally, 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 and effector functions.

[0109] The disclosure also provides fully human antibodies. 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 mouse, have been grafted onto human framework sequences (i.e., humanized antibodies). Fully human or human antibodies can be derived from transgenic mice or human cells containing human antibody genes (having variable exons (V), divergent exons (D), joining exons (J), and constant exons (C)). For example, it is now possible to produce transgenic animals (e.g., mice) that are capable of producing a full repertoire of human antibodies upon immunization in the absence of endogenous immunoglobulin production (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 genetic sequences from unsequenced human immunoglobulin genes. The human genetic sequences can encode both human antibody heavy and light chains that function correctly in the mouse and, through rearrangement, provide a broad antibody repertoire similar to that of humans. Transgenic mice can be immunized with the target protein (to make a variety of specific antibodies and the RNA encoding them). Nucleic acids encoding the antibody chain components of such antibodies can be cloned from the animal into a display vector. Typically, separate populations of nucleic acids encoding heavy and light chain sequences are cloned, and the separate populations are recombined upon insertion into the vector.The vectors are designed to express antibody chains, which are assembled and displayed on the outer surface of a display package containing the vector. For example, antibody chains can be expressed from the outer surface of a phage as fusion proteins with a phage coat protein. The display packages can then be screened for display of an antibody that binds to a target.

[0110] Thus, in some embodiments, the disclosure provides humanized, deimmunized, or primatized antibodies that comprise one or more complementarity determining regions (CDRs) of, for example, a murine monoclonal antibody described herein and retain the ability (e.g., at least 50, 60, 70, 80, 90, or 100%, or even greater than 100%) of its murine monoclonal antibody counterpart to bind to its antigen.

[0111] Additionally, human antibodies can be derived from phage display libraries (Hoogenboom et al. (1991) J. Mol. Biol. 227:381; Marks et al. (1991) J. Mol. Biol, 222:581-597; and Vaughan et al. (1996) Nature Biotech 14:309 (1996)). Synthetic phage libraries can be generated using random combinations of synthetic human antibody V-regions. By selecting the antigen, fully human antibodies can be made in which the V-regions are highly human-like. 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.

[0112] For the production of human antibodies, see also the disclosures of Mendez et al. (1998) Nature Genetics 15: 146-156 and Green and Jakobovits (1998) J. Exp. Med. 188:483-495. Human antibodies are described in U.S. Patent 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, 2004-0010810, 2004-0093622, 2006-0040363, 2005-0054055, 2005-0076395, and 2005-0287630. See also International Publication Nos. WO 94 / 02602, WO 96 / 34096, and WO 98 / 24893, and European Patent No. EP 0463151 B1. The disclosures of each of the above cited patents, applications and references are incorporated herein by reference in their entirety.

[0113] As an alternative approach, in the GenPharm International, Inc. minilocus approach, an exogenous Ig locus is mimicked by including fragments (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 into 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; and 5,814,318; 5,591,669; 5,6 Nos. 12,205; 5,721,367; 5,789,215; 5,643,763; 5,569,825; 5,877,397; 6,300,129; 5,874,299; 6,255,458; and 7,041,871, the disclosures of which are incorporated herein by reference. See also European Patent No. 0546073B1, 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.Furthermore, Taylor et al. (1992) Nucleic Acids Res. 20: 6287; Chen et al. (1993) Int. Immunol. 5: 647; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90: 3720-4; Choi et al. (1993) Nature Genetics 4: 1 17; Lonberg et al. (1994) Nature 368: 856-859; Taylor et al. (1994) International Immunology 6: 579-591; Tuaillon et al. (1995) J. Immunol. 154: 6453- 65; Fishwild et al. (1996) Nature Biotechnology 14: 845; and Tuaillon et al. al. (2000) Eur. See J. Immunol. 10: 2998-3005, the disclosures of each of which are incorporated herein by reference in their entireties.

[0114] 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 to a given species (e.g., human). 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 can 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. 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, and have reduced immunogenicity. Methods for identifying potential T cell and / or B cell epitopes can be performed using techniques known in the art, such as computational methods (see, for example, PCT Publication No. WO 02 / 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 of the species that will receive the fusion protein, antibody or fragment thereof, testing of the protein or peptide portion thereof using transgenic animals bearing MHC molecules of the species that will receive the antibody or antigen-binding fragment thereof, or testing using transgenic animals reconstituted with immune system cells of the species that will receive 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, artificial antibodies (e.g., chimeric antibodies, single chain antibodies, CDR-grafted antibodies, humanized antibodies, fully human antibodies, artificially selected antibodies, etc.), synthetic antibodies and semi-synthetic antibodies.

[0115] 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 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 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.

[0116] Methods for producing bispecific antibodies are within the skill of the art. Traditionally, recombinant production of bispecific antibodies has been based on the co-expression of two immunoglobulin heavy / light chain pairs, where the two heavy / light chain pairs have 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 an immunoglobulin constant domain sequence. The heavy chain variable region fusion is preferably with an immunoglobulin heavy chain constant domain comprising at least part of the hinge, CH2 and CH3 regions. DNAs encoding the immunoglobulin heavy chain fusions and, optionally, 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 making 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 can 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.

[0117] Various techniques have also been described for producing and isolating bispecific antibody fragments directly from recombinant cell culture. For example, bispecific antibodies have been produced using leucine zippers. See, for example, Kostelny et al. (1992) J Immunol 148(5): 1547-1553. The leucine zipper peptides from the Fos and Jun proteins can be linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers are reduced at the hinge region to form monomers and then reoxidized to form the antibody heterodimers. This method can also be used for the production of antibody homodimers. The "diabody" technology described in Hollinger et al. (1993) Proc Natl Acad Sci USA 90:6444-6448 provided an alternative mechanism for making bispecific antibody fragments. These fragments consist of a heavy chain variable domain (VH) and a light chain variable domain (VL) joined 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 using single-chain Fv (scFv) dimers has also been reported. See, e.g., Gruber et al. (1994) J Immunol 152:5368. Alternatively, the antibody can be a "linear antibody" as described, e.g., in Zapata et al. (1995) Protein Eng. 8(10): 1057-1062. Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific.

[0118] 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.

[0119] The present disclosure also encompasses variants of multispecific antibodies such as dual variable domain immunoglobulin (DVD-lg) molecules described in Wu et al. (2007) Nat Biotechnol 25(11): 1290-1297. DVD-lg molecules are designed in which two different light chain variable domains (VL) from two different parent antibodies are linked in tandem, either directly or via a short linker by recombinant DNA techniques, followed by a light chain constant domain. Similarly, the heavy chain is linked in tandem with two different heavy chain variable domains (VH), followed by a constant domain CH1 and an Fc region. Methods for generating DVD-Ig molecules from two parent antibodies are further described, for example, in PCT Publication Nos. WO08 / 024188 and WO07 / 024715.

[0120] The present disclosure also provides camelid or dromedarian antibodies (e.g., antibodies from Camelus bactrianus, Calelus dromaderius, or lama paccos). Such antibodies are unlike the typical two-chain (fragment) or four-chain (whole antibody) 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.

[0121] 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 modified to obtain sequences that are closer to human sequences, i.e. nanobodies can be humanized, thereby further reducing the potential immunogenicity of the antibodies.

[0122] 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 maintain 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 comprise at least one amino acid substitution, deletion, insertion, or other modification. In some embodiments, the fusion proteins, antibodies, or fragments thereof described herein comprise 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 comprise 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.

[0123] As used herein, "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 which, like the fragment(s) from the original antibody, can optionally contain additional components not found in the original antibody (e.g., different framework regions or mutations). 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), domain antibodies (also known as "heavy chain immunoglobulins" or camelization; Holt et al. (2003) Trends Biotechnol 21(1 1):484-490), antibodies that bind to complement component proteins (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 "antigen-binding fragment thereof or equivalent term" when referring to a fragment related to an antibody, regardless of whether such fragment is actually derived from the antibody or is an antigen-binding fragment that binds to the same epitope or an overlapping epitope or an epitope contained within the epitope of the antibody. The antigen-binding fragment may include an antigen-binding fragment that binds to the same or overlapping antigen as the original antibody, and the antigen-binding fragment includes a portion that is a fragment of the original antibody (e.g., one or more CDRs, one or more variable regions, etc.).

[0124] In some embodiments, the antibodies described herein contain modified 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 more rapid clearance. Additionally, the antibodies described herein can 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., the addition of one or more sugar moieties, the loss of one or more sugar moieties, or a change in the composition of one or more sugar moieties).

[0125] In some embodiments, the antibodies described herein have reduced (e.g., or 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 inflammatory response. Modulation refers to an increase, decrease, or abolishment of an effector function activity exhibited by a subject antibody comprising an altered constant region compared to the activity of the unaltered constant region. In certain embodiments, modulation includes a situation in which activity is abolished or completely absent.

[0126] Antibodies with altered or unaltered effector functions can be produced by engineering or producing antibodies with variant constant, 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 can be used to engineer one or more amino acid substitutions, deletions, or insertions in regions (such as, for example, the Fc region or constant region) that affect antibody function, including effector function. Alternatively, alterations in post-translational modifications, such as, for example, glycosylation patterns, can be achieved by manipulating the cell culture and expression conditions under 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 standard DNA mutagenesis techniques described, for example, in Sambrook et al. (1989) "Molecular Cloning: A Laboratory Manual, 2nd Edition", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane (1988), supra; Borrebaek (1992), supra; Johne et al. (1993), supra; PCT publication no. WO 06 / 53301 ; and US patent no. 7,704,497.

[0127] nucleic acid molecule Provided herein are polynucleotides encoding the Siglec-7 antibodies or fragments thereof of the invention. In some embodiments, the polynucleotides also comprise a sequence encoding a signal peptide operably linked to the 5' end of the coding sequence. In some embodiments, the polynucleotides also comprise a sequence encoding a linker sequence.

[0128] In one embodiment, the nucleic acid molecule encodes an antibody comprising a variable heavy chain sequence selected from SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, or SEQ ID NO:84; a variable heavy chain sequence of SEQ ID NO:12, SEQ ID NO:28, SEQ ID NO:44, SEQ ID NO:60, SEQ ID NO:76, or SEQ ID NO:92; a sequence having at least 95% identity to one or more 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, or SEQ ID NO:84; a sequence having at least 95% identity to one or more 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, or SEQ ID NO:92; a fragment comprising at least 80% of the full length sequence of a variable heavy chain sequence of SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, or SEQ ID NO:84; or a fragment comprising at least 80% of the full length sequence of a 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, or SEQ ID NO:92, or a combination thereof.

[0129] 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, or SEQ ID NO:84. In one embodiment, the nucleic acid molecule comprises SEQ ID NO:5-7; SEQ ID NO:21-23; SEQ ID NO:37-39; SEQ ID NO:53-55; SEQ ID NO:69-71; or SEQ ID NO:85-87. 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; or SEQ ID NO:88. 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, or SEQ ID NO: 92. 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; or SEQ ID NO: 93-95. 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; or SEQ ID NO: 96.

[0130] 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%, 76%, 77%, 78%, 76%, 77%, 78%, 76%, 77%, 78%, 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 nucleic acid 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, or SEQ ID NO:84 comprises 100% identity to all three SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, or SEQ ID NO:84. 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, or SEQ ID NO:92 constitutes 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, or SEQ ID NO:92. Thus, in some embodiments, the variability is in regions other than the CDR coding regions.

[0131] 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; or SEQ ID NO:88. 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; or SEQ ID NO:96.

[0132] 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, or SEQ ID NO:84, 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, or SEQ ID NO:84. 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, or SEQ ID NO:92 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, or SEQ ID NO:92.

[0133] 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; or SEQ ID NO:88. 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; or SEQ ID NO:96.

[0134] In one embodiment, the invention provides a combination of nucleic acid molecules, wherein the first nucleic acid molecule consists of a light chain CDR coding sequence of SEQ ID NO:5-7; SEQ ID NO:21-23; SEQ ID NO:37-39; SEQ ID NO:53-55; SEQ ID NO:69-71; or SEQ ID NO:85-87, 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; or SEQ ID NO:93-95. In one embodiment, the combination of nucleic acid molecules comprises a light chain coding sequence of SEQ ID NO:8; SEQ ID NO:24; SEQ ID NO:40; SEQ ID NO:56; SEQ ID NO:72; or SEQ ID NO:88 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; or SEQ ID NO:96 or a fragment or variant thereof.

[0135] The isolated nucleic acid may be comprised of 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, including, for example, 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.

[0136] The nucleic acid molecules of the invention can be modified to improve stability in serum or in growth medium for cell culture. Modifications can be added to increase the stability, functionality, and / or specificity of the nucleic acid molecules of the invention and minimize immunostimulatory properties. For example, to increase stability, 3'-residues can be stabilized against degradation and can be selected to contain, for example, purine nucleotides, particularly adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides by modified analogs, for example, substitution of uridine by 2'-deoxythymidine, is tolerated and does not affect the function of the molecule.

[0137] 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 incorporating modified nucleotide analogues.

[0138] Non-limiting examples of nucleotide analogs include sugar- and / or backbone-modified ribonucleotides (i.e., containing modifications to the phosphate-sugar backbone). For example, the phosphodiester bond of natural RNA can be modified to include at least one nitrogen or sulfur heteroatom. In exemplary backbone-modified ribonucleotides, the phosphoester group that links adjacent ribonucleotides is replaced with a modified group, such as a phosphothioate group.

[0139] Another example of modification is nucleobase-modified ribonucleotide, i.e., ribonucleotide that contains at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. The base can be modified to inhibit the activity of adenosine deaminase. Exemplary modified nucleobases include 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. The above modifications may be combined.

[0140] In some cases, the nucleic acid molecule comprises at least one of the following chemical modifications: 2'-H, 2'-O-methyl, or 2'-OH modification of one or more nucleotides. In some embodiments, the nucleic acid molecule of the present invention may have enhanced resistance to nucleases. For increased nuclease resistance, the nucleic acid molecule may comprise, for example, 2'-modified ribose units and / or phosphorothioate linkages. For example, the 2' hydroxyl group (OH) can be modified or replaced with a number of different "oxy" or "deoxy" substituents. To enhance nuclease resistance, the nucleic acid molecule of the present invention may comprise 2'-O-methyl, 2'-fluoro, 2'-O-methoxyethyl, 2'-O-aminopropyl, 2'-amino, and / or phosphorothioate linkages. The 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), G-clamp modifications can also increase binding affinity to targets.

[0141] In one embodiment, the nucleic acid molecule comprises a 2'-modified nucleotide, such as a 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.

[0142] Nucleic acid agents contemplated herein include otherwise unmodified RNA and DNA, as well as RNA and DNA modified, for example to improve efficacy, and polymers of nucleoside surrogates. Unmodified RNA refers to molecules in which the components of nucleic acid, i.e., sugar, base, and phosphate moieties, are the same or essentially the same as those found in nature, e.g., as they occur naturally in the human body. In the art, rare or unusual, but naturally occurring, RNA has been referred to as modified RNA (see, e.g., Limbach et al. Nucleic Acids Res., 1994, 22:2183-2196). Such rare or unusual RNA, often referred to as modified RNA, is typically the result of post-transcriptional modifications and falls within the scope of the term unmodified RNA as used herein. Modified RNA, as used herein, refers to molecules in which one or more of the components of nucleic acid, i.e., sugar, base, and phosphate moieties, are different from those found in nature, e.g., in the human body. Although they are referred to as "modified RNA," they do, of course, include molecules that are not strictly speaking RNA, since they are modified. A nucleoside surrogate is a molecule in which the ribophosphate backbone is replaced with a non-ribophosphate backbone, which allows the bases to be presented in the correct spatial relationship such that hybridization is substantially similar to that seen with a ribophosphate backbone (e.g., an uncharged mimic of the ribophosphate backbone).

[0143] Modifications of the nucleic acids of the invention can be at one or more of the phosphate groups, sugar groups, backbone, N-terminus, C-terminus, or nucleobase.

[0144] The invention also includes vectors into which the isolated nucleic acid of the invention has been inserted. The art is replete with suitable vectors useful in the present invention.

[0145] 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 it is subsequently introduced. In some embodiments, the vector of the present invention is an expression vector. Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. In specific embodiments, the expression vector is selected from the group consisting of viral vectors, bacterial vectors and mammalian cell vectors. Systems based on prokaryotic and / or eukaryotic vectors can be employed for use with the present invention to produce polynucleotides, or their cognate polypeptides. Many such systems are widely available commercially.

[0146] 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 one suitable for replication and, optionally, integration in eukaryotic cells. Typical vectors include transcription and translation terminators, initiation sequences, and promoters useful for regulating expression of the desired nucleic acid sequence.

[0147] The recombinant nucleic acid sequence construct may contain one or more transcription termination regions. The transcription termination region may be 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.

[0148] The recombinant nucleic acid sequence construct may include one or more initiation codons. The initiation codon may be located upstream of the coding sequence. The initiation codon may be in frame with the coding sequence. The initiation codon may be associated with one or more signals necessary for efficient translation initiation, such as, but not limited to, a ribosome binding site.

[0149] A recombinant nucleic acid sequence construct may contain one or more termination or stop codons. The termination codon may be downstream of the coding sequence. The termination codon may be in frame with the coding sequence. The termination codon may be associated with one or more signals required for efficient translation termination.

[0150] 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 a pCEP4 plasmid (Invitrogen, San Diego, CA).

[0151] The recombinant nucleic acid construct may include one or more leader sequences. The leader sequence may encode a signal peptide. The signal peptide may be an immunoglobulin (Ig) signal peptide, such as, but not limited to, an IgG signal peptide and an IgE signal peptide. The vectors of the present invention may also be used for nucleic acid immunization using standard gene transfer protocols. Methods of gene transfer are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466.

[0152] 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, cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, sequencing vectors, and the like.

[0153] Furthermore, vectors can be provided to cells in the form of viral vectors.Viral vector technology is well known in the art and is 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 selectable markers (e.g., WO01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193).

[0154] 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), 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 selectable markers (see, for example, WO01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193).

[0155] By way of example, the vector into which the nucleic acid sequence is introduced may be a plasmid, which may or may not be integrated into the genome of the host cell when introduced into the cell. 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.

[0156] The vector can 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 transformation of animal cells.

[0157] In one embodiment, the recombinant expression vector can also include a nucleic acid molecule encoding a peptide or protein of the invention as described elsewhere herein.

[0158] Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene transfer systems. A gene of choice can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of a subject either in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used.

[0159] For example, vectors derived from retroviruses, such as lentiviruses, are suitable tools to achieve long-term gene transfer, as they allow long-term stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have the advantage over vectors derived from oncoretroviruses, such as murine leukemia viruses, that they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of being less immunogenic. In one embodiment, the composition comprises a vector derived from an adeno-associated virus (AAV). Adeno-associated virus (AAV) vectors have become a powerful gene transfer 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 transduce postmitotic cells in a stable and efficient manner. Expression of a particular gene contained within an AAV vector can be specifically targeted to one or more types of cells by selecting the appropriate combination of AAV serotype, promoter, and delivery method.

[0160] In some embodiments, the vector also contains conventional control elements operably linked to the transgene in a manner that allows its transcription, translation and / or expression in cells transfected with the plasmid vector or infected with the virus produced by the invention. As used herein, "operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance 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, if desired, sequences that enhance secretion of the encoded product. Numerous expression control sequences, including native, constitutive, inducible, and / or tissue-specific promoters, are known in the art and may be utilized.

[0161] A promoter may be one that is naturally associated with a gene or polynucleotide sequence, as obtained by isolating the 5' non-coding sequences located upstream of a coding segment and / or exon. Such a promoter is referred to as "endogenous". Similarly, an enhancer may be one that is naturally associated with a polynucleotide sequence and located either downstream or upstream of that sequence. Alternatively, certain advantages may be obtained by placing a coding polynucleotide segment under the control of a recombinant or heterologous promoter. 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, and "non-naturally occurring" promoters or enhancers, i.e., promoters or enhancers that contain different elements of different transcriptional regulatory regions and / or mutations that alter expression. In addition to synthetically producing promoter and enhancer nucleic acid sequences, the sequences may be produced using recombinant cloning and / or nucleic acid amplification techniques, including PCR, in conjunction with 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 within non-nuclear organelles, such as mitochondria, chloroplasts, and the like, may similarly be employed.

[0162] Naturally, it will be important to employ a promoter and / or enhancer that effectively induces 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 promoter, enhancer, and cell type combinations for protein expression. The promoter employed may be constitutive, tissue-specific, inducible, and / or useful under appropriate conditions to induce high-level expression of the introduced DNA segment, which is advantageous in large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

[0163] The recombinant expression vector may also contain a selectable marker gene to facilitate the selection of transformed or transfected host cells. Suitable selectable marker genes are G418 and hygromycin, which 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 selectable marker may be introduced on a vector separate from the nucleic acid of interest.

[0164] Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, these are located in the region 30-110 bp upstream of the start site, but recently, many promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements is often flexible, and promoter function is maintained even when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp before activity begins to decline. Depending on the promoter, individual elements appear to be able to function either cooperatively or independently to activate transcription.

[0165] 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 it. Another example of a suitable promoter is the elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences can also 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, as well as human gene promoters such as the acti 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 a polynucleotide sequence to which it is operably linked 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.

[0166] Enhancer sequences found in vectors also control the expression of genes contained therein. Enhancers usually bind protein factors to promote 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 invention contain one or more enhancers to enhance the transcription of genes present in the vector.

[0167] To evaluate the expression of protein inhibitors, the expression vector introduced into cells can also contain either a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from the cell population to be transfected or infected through the viral vector. In other embodiments, the selectable marker is carried on a separate DNA fragment and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to allow expression in the host cell. Useful selectable markers include antibiotic resistance genes, such as neo.

[0168] Reporter genes are used to identify potentially transfected cells or to evaluate the functionality of regulatory sequences. Generally, a reporter gene is a gene absent from or expressed by a recipient organism or tissue, and encodes a polypeptide whose expression is indicated by some easily detectable property, such as enzymatic activity. Expression of the reporter gene is assayed at an appropriate time after the DNA is introduced into the recipient cells. Suitable reporter genes include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and can 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 a promoter region can be linked to a reporter gene and used to evaluate drugs for their ability to modulate promoter-driven transcription. 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 the host cell by physical, chemical, or biological means.

[0169] Physical methods for introducing peptides or proteins into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0170] Biological methods for introducing peptides or proteins of interest into host cells include the use of DNA or RNA vectors. Viral vectors, particularly retroviral vectors, are the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors are derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, and the like. See, e.g., U.S. Pat. Nos. 5,350,674 and 5,585,362. 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, such as 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).

[0171] When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of 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 lipid-associated nucleic acid may be encapsulated in the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, or bound to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, encapsulated in a liposome, complexed with a liposome, dispersed in a solution containing lipids, mixed with lipids, bound to lipids, contained in a suspension in lipids, contained in micelles, complexed with micelles, or otherwise bound to lipids. The lipid, lipid / DNA, or lipid / expression vector associated compositions are not limited to a particular structure in solution. For example, they may exist as bilayer structures, micelles, or in "collapsed" structures. They may also simply be interspersed in the solution, or may form aggregates that are not uniform in size or shape. Lipids are fatty substances and may be naturally occurring or synthetic. For example, lipids include the lipid droplets that occur naturally in the cytoplasm and a class of compounds that contain long-chain aliphatic hydrocarbons, such as fatty acids, alcohols, amines, aminoalcohols, aldehydes, and their derivatives.

[0172] 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; dimyristyl phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, 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 are characterized by vesicular structures 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, incorporating water and dissolved solutes between the lipid bilayer (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have structures in solution other than the usual vesicular structures are also contemplated. For example, lipids may adopt micellar structures or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0173] 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 include the CH1 and CL regions. Thus, in one embodiment, the scFv antibody fragment relates to a Fab fragment that includes 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, a CH2 and a CH3 region. In one embodiment, the scFv antibody fragment of the invention has altered expression, stability, half-life, antigen binding, heavy-light chain pairing, tissue penetration or a combination thereof compared to the parent MAb.

[0174] In one embodiment, the scFv antibody fragment of the invention has expression 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 higher than the parent MAb.

[0175] In one embodiment, an scFv antibody fragment of the invention has antigen binding 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 higher than the parent MAb.

[0176] In one embodiment, the scFv antibody fragment of the invention has a half-life that is at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, at least 9.5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, or more than 50 fold longer than the parent MAB.

[0177] 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.

[0178] In one embodiment, the scFv antibody fragment of the invention has at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, at least 9.5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, or more than 50 fold greater tissue penetration than the parent MAB.

[0179] In one embodiment, the scFv antibody fragment of the invention has at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, at least 9.5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, or more than 50 fold greater heavy chain-light chain pairing than the parent MAb.

[0180] 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.

[0181] Exemplary delivery vehicles include, but are not limited to, microspheres, microparticles, nanoparticles, polymerosomes, liposomes, and micelles. For example, in some embodiments, the delivery vehicle carries an anti-Siglec-7 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 its loaded cargo. In some embodiments, the delivery vehicle includes a targeting moiety that targets the delivery vehicle to a treatment site.

[0182] Pharmaceutical Compositions The present invention also provides pharmaceutical compositions comprising one or more of the compositions described herein. The formulations can 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 can be mixed with auxiliary substances, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for affecting osmotic pressure buffers, coloring agents, and / or aromatic substances, if desired. They can also be combined with other active agents, such as other analgesics, if desired.

[0183] Administration of the compositions of the invention can be carried out, for example, by parenteral, intravenous, subcutaneous, intramuscular, or intraperitoneal injection, or by infusion, or by any other acceptable systemic method.

[0184] "Additional components" as used herein include, but are not limited to, one or more of the following: excipients; surfactants; dispersants; inert diluents; granulating and disintegrating agents; binders; lubricants; colorants; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, anti-inflammatory agents; buffers; salts; thickening agents; fillers; emulsifiers; antioxidants; antibiotics; antifungal agents; stabilizers; and pharmaceutically acceptable polymers or hydrophobic materials. Other "additional components" that can be included in the pharmaceutical composition 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.

[0185] The composition of the present invention may contain a preservative in an amount of about 0.005% to 2.0% by weight of the total 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 including benzyl alcohol, sorbic acid, parabens, imidurea, and combinations thereof.

[0186] In one embodiment, the composition comprises an antioxidant and a chelating agent that inhibits the degradation of one or more components of the composition. Exemplary antioxidants for some compounds are BHT, BHA, alpha-tocopherol and ascorbic acid. Exemplary chelating agents include 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, respectively, for some compounds, but other suitable and equivalent antioxidants and chelating agents may be substituted, as known to those skilled in the art.

[0187] Liquid suspensions can be prepared by using conventional methods to suspend the compound of the present invention or other compositions in aqueous or oily vehicles. Aqueous vehicles include, for example, water, isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis oil, olive oil, sesame oil, coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further include one or more additional components, including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, weight reducing agents, preservatives, buffers, salts, flavorings, colorings, and sweetening agents. Oily suspensions may further include 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 naturally occurring phosphatides such as lecithin, condensation products of alkylene oxides with fatty acids, with long chain aliphatic alcohols, with fatty acids with partial esters derived from hexitols, or with fatty acids with partial esters derived from hexitol anhydrides (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin, acacia, and the like. Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl parahydroxybenzoates, ascorbic acid, and sorbic acid.

[0188] Particularly suitable for oral administration are tablets, dragees, liquids, drops, suppositories, or capsules, caplets, gelcaps. Other formulations suitable for oral administration include, but are not limited to, powdered or granular formulations, aqueous or oily suspensions, aqueous or oily solutions, pastes, gels, dentifrices, mouthwashes, coatings, mouthwashes, chewing gums, varnishes, sealants, oral and dental "dissolving strips", or emulsions. Compositions intended for oral use can be prepared according to any method known in the art, and such compositions can contain one or more agents selected from the group comprising inert, non-toxic pharmaceutical excipients suitable for the manufacture of tablets. Such excipients include, for example, inert diluents such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricants such as magnesium stearate. Tablets may be uncoated or may be coated using known methods to achieve delayed disintegration in the subject's digestive tract, thereby providing sustained release and absorption of the active ingredient. For example, materials such as glyceryl monostearate or glyceryl distearate can be used to coat the tablets. For further example, the tablets can be coated to form osmotically controlled release tablets using the methods described in U.S. Patents 4,256,108; 4,160,452; and 4,265,874. The tablets can further include a sweetener, a flavoring agent, a coloring agent, a preservative, or some combination thereof to provide a pharma- ceutically elegant and palatable preparation.

[0189] Hard capsules containing the active ingredient can be manufactured using a physiologically degradable composition such as gelatin. Such hard capsules contain the active ingredient and can further contain additional ingredients including inert solid diluents such as calcium carbonate, calcium phosphate, kaolin, and the like.

[0190] Soft gelatin capsules containing the active ingredient can be prepared using physiologically degradable compositions such as gelatin. Such soft capsules consist of the active ingredient and can be mixed with water or an oil medium such as peanut oil, liquid paraffin, olive oil, etc. For oral administration, the compositions of the present invention can be in the form of tablets or capsules prepared by conventional means using pharma- ceutically acceptable excipients such as binders; fillers; lubricants; disintegrants; or wetting agents. If desired, tablets can be coated using suitable methods and coating materials such as OPADRY™ film coating systems available from Colorcon, West Point, Pa. (e.g., OPADRY™ OY type, OYC type, organic enteric OY-P type, aqueous enteric OY-A type, OY-PM type, and OPADRY™ White, 32K18400).

[0191] Liquid preparations for oral administration may be in the form of solution, syrup or suspension. Liquid preparations can be prepared by conventional means using pharma- ceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methylcellulose or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxybenzoates or sorbic acid). Liquid preparations of the pharmaceutical compositions of the present invention suitable for oral administration can be prepared, packaged, and sold either 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.

[0192] Tablets containing the active ingredient can be prepared, for example, by compressing or molding the active ingredient, optionally mixed with one or more additional ingredients. Compressed tablets can be prepared by mixing the active ingredient in a free-flowing form, such as a powder or granular formulation, with one or more of the following: binders, lubricants, excipients, surface active agents, and dispersants, and compressing in a suitable device. Molded tablets can be prepared 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. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, sodium phosphate, and the like. 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, hydroxypropyl methylcellulose, and the like. Known lubricants include, but are not limited to, magnesium stearate, stearic acid, silica, talc, and the like.

[0193] 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 can be prepared, packaged, or sold in a form suitable for bolus or continuous administration. Injectable formulations can be prepared, packaged, or sold in unit dosage form, such as ampoules or 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, stabilizing agents, or 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.

[0194] The pharmaceutical compositions can be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. The suspension or solution can be formulated according to known techniques and can contain, in addition to the active ingredient, additional ingredients such as dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable preparations can be prepared using a non-toxic parenterally acceptable diluent or solvent, such as 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 administrable 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 can be composed of pharma-ceutically acceptable polymeric or hydrophobic materials, such as emulsions, ion exchange resins, sparingly soluble polymers, or sparingly soluble salts.

[0195] 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-facilitating agent, which may include immunostimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogs including monophosphoryl lipid A, muramyl peptides, quinone analogs, squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or surface active agents such as nanoparticles, or other known transfection-facilitating agents.

[0196] The transfection facilitating agent is a polyanion, polycation, or lipid, including poly-L-glutamic acid (LGS). 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 used administered with the composition. The composition may also include transfection facilitating agents such as lipids, liposomes including lecithin liposomes as DNA-liposome mixtures (see, e.g., WO9324640) or other liposomes known in the art, calcium ions, viral proteins, polyanions, polycations, or nanoparticles. The transfection facilitating agent is a polyanion, polycation, or lipid, including poly-L-glutamic acid (LGS), and the concentration of the transfection facilitating 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.

[0197] The pharma- ceutically acceptable excipient may be an adjuvant in addition to the checkpoint inhibitor antibody of the present invention. Additional adjuvants may be other genes expressed in alternative plasmids or delivered as proteins in combination with the above plasmids in the composition. The adjuvant may be selected from the group including: 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 a deleted signal sequence, and optionally including a signal peptide from IgE). The adjuvant can be IL-12, IL-15, IL-28, CTACK, TECK, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, PD-1, IL-10, IL-12, IL-18, or a combination thereof.

[0198] Other genes useful as adjuvants in addition to the antibodies of the invention include those encoding: MCP-1, MIP-1a, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant IL-18, CD4 0, CD40L, vascular growth factor, fibroblast growth factor, IL-7, IL-22, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF , DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-1, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 and their functional fragments.

[0199] The composition may further include a gene enhancer such as those described in US Serial No. 021,579, filed April 1, 1994, which is incorporated by reference in its entirety.

[0200] The composition may comprise DNA in an amount of about 1 nanogram to about 100 milligrams; about 1 microgram to about 10 milligrams; or preferably about 0.1 microgram to about 10 milligrams; or more preferably about 1 milligram to about 2 milligrams. In some preferred embodiments, the composition according to the present invention comprises about 5 nanograms to about 1000 micrograms of DNA. In some preferred embodiments, the composition may comprise about 10 nanograms to about 800 micrograms of DNA. In some preferred embodiments, the composition may contain about 0.1 to about 500 micrograms of DNA. In some preferred embodiments, the composition may contain about 1 to about 350 micrograms of DNA. In some preferred embodiments, the compositions can comprise about 25 to about 250 micrograms, about 100 to about 200 micrograms, about 1 nanogram to about 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 micrograms to about 500 micrograms; about 1 micrograms to about 350 micrograms; about 25 micrograms to about 250 micrograms; or about 100 micrograms to about 200 micrograms of DNA.

[0201] The composition can be formulated according to the mode 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 can include sodium chloride, dextrose, mannitol, sorbitol, and lactose. The composition can include a vasoconstrictor. The isotonic solution can include phosphate buffered saline. The composition can further include a stabilizer, including gelatin and albumin. The stabilizer can allow the formulation to be stable for an extended period of time at room or ambient temperature, including LGS or a polycation or polyanion.

[0202] 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 described cytotoxicity assays. 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 described cytotoxicity assays. In one embodiment, the antibody preparation causes at least a 10% increase, 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) by Siglec-restricted lymphocytes.

[0203] The present invention is also directed to a method of increasing an immune response in a subject. Increasing the immune response can be used to treat and / or prevent disease in a subject. The method can include administering a vaccine disclosed herein to a subject. A subject administered the vaccine can have an increased or boosted 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 administered the vaccine may be increased by at least about 0.5-fold, at least about 1.0-fold, at least about 1.5-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 5.5-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 10.5-fold, at least about 11.0-fold, at least about 11.5-fold, at least about 12.0-fold, at least about 12.5-fold, at least about 13.0-fold, at least about 13.5-fold, at least about 14.0-fold, at least about 14.5-fold, or at least about 15.0-fold.

[0204] In yet other alternative embodiments, the immune response in a subject administered the vaccine can be increased by about 50% to about 1500%, about 50% to about 1000%, or about 50% to about 800%. In other embodiments, the immune response in a subject administered the vaccine may be increased by at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, at least about 500%, at least about 550%, at least about 600%, at least about 650%, at least about 700%, at least about 750%, at least about 800%, at least about 850%, at least about 900%, at least about 950%, at least about 1000%, at least about 1050%, at least about 1100%, at least about 1150%, at least about 1200%, at least about 1250%, at least about 1300%, at least about 1350%, at least about 1450%, or at least about 1500%.

[0205] The vaccine dosage can be 1 μg to 10 mg of active ingredient / kg body weight / dose, and can be 20 μg to 10 mg of ingredient / kg body weight / dose. The vaccine can 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 can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0206] Combination vaccine In one embodiment, the invention relates to the administration of a Siglec-7 antibody of the invention or a nucleic acid molecule encoding same in combination with a PD-(L)1 axis inhibitor. This immunogenic composition can be used to increase the immune response against tumors.

[0207] In some embodiments, a Siglec-7 antibody of the invention, or a nucleic acid molecule encoding same, is administered simultaneously with administration of a PD-(L)1 axis inhibitor.

[0208] In some embodiments, a Siglec-7 antibody of the invention, or a nucleic acid molecule encoding same, is administered prior to administration of a PD-(L)1 axis inhibitor.

[0209] In some embodiments, the Siglec-7 antibody of the invention, or a nucleic acid molecule encoding same, is administered following administration of a PD-(L)1 axis inhibitor.

[0210] In some embodiments, a Siglec-7 antibody of the invention, or a nucleic acid molecule encoding same, is administered about 1, 2, 5, 10, 30 or 60 minutes or hours, such as about 2, 4, 6, 10, 12, 24 or 36 hours, or such as about 2, 4, 7, 14, 21, 28, 35, 42, 49, 56 days or more, prior to administration of a PD-(L)1 axis inhibitor. Thus, in some embodiments, a PD-(L)1 axis inhibitor is administered to a subject who has previously been administered a Siglec-7 antibody of the invention, or a nucleic acid molecule encoding same.

[0211] In some embodiments, the Siglec-7 antibody of the invention, or a nucleic acid molecule encoding same, is administered about 1, 2, 5, 10, 30 or 60 minutes or hours, such as about 2, 4, 6, 10, 12, 24 or 36 hours, or such as about 2, 4, 7, 14, 21, 28, 35, 42, 49, 56 days or more, after administration of a PD-(L)1 axis inhibitor. Thus, in some embodiments, the Siglec-7 antibody of the invention, or a nucleic acid molecule encoding same, is administered to a subject who has previously been administered a PD-(L)1 axis inhibitor.

[0212] In some embodiments, the combination of a Siglec-7 antibody or a nucleic acid molecule encoding same of the invention and a PD-(L)1 axis inhibitor is administered prior to the administration of one or more additional anti-cancer agents.

[0213] In some embodiments, the combination of an aSiglec-7 antibody of the invention, or a nucleic acid molecule encoding same, and a PD-(L)1 axis inhibitor is administered following administration of one or more additional anti-cancer agents.

[0214] In some embodiments, the combination of a Siglec-7 antibody, or a nucleic acid molecule encoding same, of the invention and a PD-(L)1 axis inhibitor is administered simultaneously with the administration of one or more additional anti-cancer agents.

[0215] The length of time between administration of a Siglec-7 antibody of the invention, or a nucleic acid molecule encoding same, and a PD-(L)1 axis inhibitor or one or more additional anti-cancer agents can be a few minutes, e.g., about 1, 2, 5, 10, 30 or 60 minutes, or a few hours, e.g., about 2, 4, 6, 10, 12, 24 or 36 hours, or about 2, 4, 7, 14, 21, 28, 35, 42, 49, 56 days or more.

[0216] The Siglec-7 antibody of the invention, or the nucleic acid molecule encoding same, the PD-(L)1 axis inhibitor, or one or more of the additional anti-cancer agents, can be administered in a pharma- ceutically acceptable carrier. "Carrier" refers to a diluent, adjuvant, excipient, or vehicle in which the antibody of the invention is administered. Such vehicles can be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. For example, 0.4% saline and 0.3% glycine can be used to formulate the bispecific anti-EGFR / c-Met antibody. These solutions are sterile and generally free of particulate matter. They can be sterilized by conventional, well-known sterilization techniques (e.g., filtration). For solid oral formulations, such as powder capsules and tablets, suitable carriers and excipients include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrants, and the like. Solid oral preparations may be coated with substances such as sugars or enteric coated to adjust the primary site of absorption. For parenteral administration, the carrier consists of sterile water, and other excipients may be added to enhance solubility and preservation. Injectable suspensions or solutions may also be prepared using aqueous carriers with appropriate additives. For suitable vehicles and formulations containing other human proteins, such as human serum albumin, see, for example, Remington: The Science and Practice of Pharmacy, 21st Edition, Troy, DB ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing pp 691-1092, especially pages 958-989.

[0217] The composition may contain pharma- ceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusting and buffering agents, stabilizers, thickeners, lubricants, and colorants. The concentration of the Siglec-7 antibody of the present invention, or the nucleic acid molecule encoding it, and the PD-(L)1 axis inhibitor in the pharmaceutical formulation may vary from less than about 0.5%, usually at least about 1% to 15%, 20%, 30%, 40% or 50% by weight, and may be selected primarily based on the required amount, liquid volume, viscosity, etc., according to the particular mode of administration selected. Pharmaceutical compositions including solid forms may contain about 0.1 mg to about 2000 mg, for example, about 1 mg, about 5 mg, about 10 mg, about 25 mg, about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 300 mg, about 500 mg, about 600 mg or about 1000 mg of active ingredient.

[0218] The therapeutic compositions of the present invention can have the characteristics required for an effective therapeutic agent, such as safety so that the therapeutic composition itself does not cause disease or death, ease of administration, few side effects, biological stability, and low cost per dose.

[0219] In one embodiment, the present invention relates to the administration of a nucleic acid-encoded Siglec-7 antibody in combination with an immunogenic composition comprising a nucleic acid molecule encoding a tumor antigen. Thus, in some embodiments, the present invention relates to an immunogenic composition, such as a vaccine, comprising a tumor antigen, a fragment thereof, or a variant thereof, a nucleic acid molecule encoding the same, or a combination thereof. The immunogenic composition can be used to increase the immune response against the tumor antigen. The combination vaccine can significantly induce an immune response in a subject administered the vaccine, thereby preventing or treating cancer or a related condition.

[0220] The immunogenic composition can be a DNA vaccine, a peptide vaccine, or a combination of DNA and peptide vaccines. The DNA vaccine can include a nucleic acid sequence encoding a tumor antigen. The nucleic acid sequence can be DNA, RNA, cDNA, variants thereof, fragments thereof, or a combination thereof. The nucleic acid sequence can also include additional sequences encoding linker, leader, or tag sequences linked to the tumor antigen by peptide bonds. The peptide vaccine can include antigenic peptides, antigenic proteins, variants thereof, fragments thereof, or a combination thereof. The combination of DNA and peptide vaccine can include the above nucleic acid sequences encoding the tumor antigen and antigenic peptide or protein.

[0221] The vaccine can induce a humoral immune response in a subject administered with the vaccine. The induced humoral immune response can be specific to a tumor antigen. The induced humoral immune response can be reactive to a tumor 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 administered with the vaccine. The humoral immune response may 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 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 in a subject administered the vaccine.

[0222] The humoral immune response induced by the vaccine can include increased levels of neutralizing antibodies associated with vaccinated subjects compared to non-vaccinated subjects. The neutralizing antibodies can be specific to tumor antigens. The neutralizing antibodies can be reactive with tumor antigens. The neutralizing antibodies can provide protection and / or treatment against cancer and related conditions in vaccinated subjects.

[0223] The humoral immune response induced by the vaccine can include increased levels of IgG antibodies associated with vaccinated subjects compared to non-vaccinated subjects. These IgG antibodies can be specific to tumor antigens. These IgG antibodies can be reactive with tumor antigens. The level of IgG antibodies associated with vaccinated subjects 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 non-vaccinated subjects. The level of IgG antibodies associated with a subject receiving the vaccine may 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 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.

[0224] The vaccine can induce a cellular immune response in a subject administered the vaccine. The induced cellular immune response can be specific to a tumor antigen. The induced cellular immune response can be reactive to a tumor antigen. The induced cellular immune response can be CD8 +The method includes inducing a CD8 T cell response. + The T cell response may be reactive to tumor antigens. + T cell responses can be polyfunctional. The induced cellular immune response is + This can include inducing a T cell response, in this case CD8 + 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.

[0225] The induced cellular immune response was higher in vaccinated subjects compared to non-vaccinated subjects, with CD8 + This may include an increase in CD8 T cell responses associated with the vaccinated subject. + 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 compared to a subject that has not received the vaccine. + The T cell response can be at least about 1.5 fold, at least about 2.0 fold, at least about 3.0 fold, at least about 4.0 fold, at least about 5.0 fold, at least about 6.0 fold, at least about 6.5 fold, at least about 7.0 fold, at least about 7.5 fold, at least about 8.0 fold, at least about 8.5 fold, at least about 9.0 fold, at least about 9.5 fold, at least about 10.0 fold, at least about 10.5 fold, at least about 11.0 fold, at least about 11.5 fold, at least about 12.0 fold, at least about 12.5 fold, at least about 13.0 fold, The increase may be 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.

[0226] The induced cellular immune response was characterized by CD3 + CD8 + The vaccine may include increasing the frequency of CD3 T cells associated with the vaccine recipient. + CD8 + IFN-γ + The frequency of T cells may be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold compared to subjects who do not receive the vaccine.

[0227] The induced cellular immune response was characterized by CD3 + CD8 + The increased frequency of CD3 T cells associated with the vaccinated subject may include + CD8 TNF-α ++ The frequency of T cells may be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, or 14-fold compared to subjects who do not receive the vaccine.

[0228] The induced cellular immune response was characterized by CD3 + CD8 + The increased frequency of CD3 T cells associated with the vaccinated subject may include + CD8 + IL-2 + The frequency of T cells may be increased by at least about 0.5-fold, 1.0-fold, 1.5-fold, 2.0-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, or 5.0-fold compared to subjects who do not receive the vaccine.

[0229] The induced cellular immune response was characterized by CD3 + CD8 + The vaccine may include increasing the frequency of CD3 T cells associated with the vaccine recipient. + CD8 + IFN-γ TNF-α ++The frequency of T cells may 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.

[0230] Vaccine-induced cellular immune responses are highly conserved in the CD4 + The method includes inducing a CD4 T cell response. + T cell responses can react with tumor antigens. + T cell responses can be polyfunctional. The induced cellular immune response is + This may include inducing a T cell response, in this case CD4 + The T cells produce IFN-γ, TNF-α, IL-2, or a combination of IFN-γ and TNF-α.

[0231] The induced cellular immune response was characterized by CD3 + CD4 + The vaccine may include increasing the frequency of CD3 T cells associated with the vaccine recipient. + CD4 + IFN-γ + The frequency of T cells may be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold compared to subjects who do not receive the vaccine.

[0232] The induced cellular immune response was characterized by CD3 + CD4 + The increased frequency of CD3 T cells associated with the vaccinated subject may include + CD4 + TNF-α + The frequency of T cells is 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.

[0233] The induced cellular immune response was characterized by CD3 + CD4 + The vaccine may include increasing the frequency of CD3 T cells associated with the vaccine recipient. + CD4 + IL-2 + The frequency of T cells is at least about 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, 20x, 21x, 22x, 23x, 24x, 25x, 26x, 27x, 28x, 29x, 30x, 31x, 32x, 33x, 34x, 35x, 36x, 37x, 38x, 39x, 40x, 45x, 50x, 55x, or 60x.

[0234] The induced cellular immune response was characterized by CD3 + CD4 + The vaccine may include increasing the frequency of CD3 T cells associated with the vaccine recipient. + CD4 + IFN-γ TNF-α ++ is at least about 2x, 2.5x, 3.0x, 3.5x, 4.0x, 4.5x, 5.0x, 5.5x, 6.0x, 6.5x, 7.0x, 7.5x, 8.0x, 8.5x, 9.0x, 9.5x, 10.0x, 10.5x, 11.0x, 11.5x, 12.0x, 12.5x, 13.0x, 13.5x, 14.0x, 15.0x, 16.0x, 17.0x, 18.0x, 19.0x, 20.0x, 21.0x, 22.0x, 23.0x, 24.0x, 25.0x, 26.0x, 27.0x, 28.0x, 29.0x, 30.0x, 31.0x, 32.0x, 33.0x, 34.0x, 35.0x, 36.0x, 37.0x, 38.0x, 39.0x, 40.0x, 41.0x, 42.0x, 43.0x, 44.0x, 45.0x, 46.0x, 47.0x, 48.0x, 49.0x, 50.0x, 51.0x, 52.0x, 53.0x, 54.0x, 55.0x, 56.0x, 57.0x, 58.0x, 59.0x, 60.0x, 61.0x, 62.0x, 63.0x, 64.0x, fold, 14.5-fold, 15.0-fold, 15.5-fold, 16.0-fold, 16.5-fold, 17.0-fold, 17.5-fold, 18.0-fold, 18.5-fold, 19.0-fold, 19.5-fold, 20.0-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, or 35-fold increase.

[0235] The vaccine of the present invention can have the characteristics required for an effective vaccine, such as being safe so that the vaccine itself does not cause disease or death, being protective against disease resulting from exposure to live pathogens such as viruses or bacteria, inducing neutralizing antibodies to prevent cellular invasion, inducing protective T cells against intracellular pathogens, ease of administration, few side effects, biological stability, low cost per dose, etc. The vaccine can be administered to different tissues such as muscle or skin to further induce an immune response.

[0236] Tumor antigens As described above, in some embodiments, the combination vaccine of the present invention comprises an anti-Siglec-7 antibody or a nucleic acid molecule encoding same and a tumor antigen, a fragment thereof, a variant thereof, a nucleic acid molecule encoding same, or a combination thereof.

[0237] 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 the same, capable of generating an immune response in a subject.

[0238] In one embodiment, the antigen is a tumor-associated surface antigen. Exemplary 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-alpha (CD140a), PDGFR-beta (CD140b), endoglin, CLEC14, Tem1-8, and Tie2. Further examples may include A33, CAMPATH-1 (CDw52), calcynoembrionic antigen (CEA), carboanhydrase IX (MN / CA IX), 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), TAG-72. Examples of antigens expressed in the extracellular matrix of tumors include tenascin and fibroblast activation protein (FAP).

[0239] In the context of the present invention, "tumor antigen" or "hyperproliferative disorder antigen" or "antigen associated with hyperproliferative disorder" refers to an antigen common to a particular hyperproliferative disorder, such as cancer. The antigens discussed herein are included merely as examples. This list is not exhaustive, and further examples will be readily apparent to one of skill in the art.

[0240] Tumor antigens are proteins produced by tumor cells that provoke an immune response, particularly an immune response mediated by T cells. The choice of antigen-binding moiety of the invention depends on the particular type of cancer to be treated. Tumor antigens are well known in the art and include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase mut hsp70-2, M-CSF, prostase, prostate specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivin, 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.

[0241] 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 target antigen is an oncofetal antigen such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific aho-type immunoglobulins constitute truly tumor-specific immunoglobulin antigens specific for individual tumors. B-cell differentiation antigens such as CD19, CD20, and CD37 are also candidates for target antigens in B-cell lymphomas. Some of these antigens (CEA, HER-2, CD19, CD20, idiotypes) have been used as targets for passive immunotherapy using monoclonal antibodies, with limited success. The type of tumor antigen referred to in the present invention may be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). TSAs are unique to tumor cells and are not present on other cells in the body. TAA-associated antigens are not unique to tumor cells and are also expressed on normal cells under conditions that do not allow induction of a state of immunological 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. TAA may be an antigen expressed on normal cells during fetal development, when the immune system is too immature to respond, or it may be an antigen that is usually present at very low levels on normal cells, but is expressed at much higher levels on tumor cells.

[0242] 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, tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15, overexpressed embryonic antigens such as CEA; overexpressed oncogenes or 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, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 / CA 27.29 / BCAA, CA 195, CA 242, 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, TAAL6, TAG72, TLP, and TPS.

[0243] In one embodiment, the synthetic antigen is Muc-1.

[0244] Methods for delivering the composition The present invention also relates to a method of delivering the composition to a subject in need thereof. The delivery method may include administering the composition to the subject. 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 bovine, a deer, a hedgehog, an elephant, a llama, an alpaca, a mouse, a rat, and a chicken.

[0245] The composition can be administered by different routes, including orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, via inhalation, via buccal administration, intrathoracically, intravenously, intraarterially, intraperitoneally, subcutaneously, intramuscularly, intranasally, intrathecally, and intraarticularly, or combinations thereof. For veterinary use, the composition can be administered in a suitably acceptable formulation according to normal veterinary practice. A veterinarian can easily determine the most appropriate administration regimen and route for a particular animal.

[0246] Treatment method In one embodiment, the present invention provides a method for the treatment or prevention of 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.

[0247] The following are non-limiting examples of cancers that can be diagnosed or treated by the disclosed methods and compositions: acute lymphoblastic leukemia, acute myeloid leukemia, adrenal cortical 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 teratoma / rhabdoid tumor, central nervous system embryonal tumor, central nervous system lymphoma, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, cerebral astrocytoma / malignant glioma, cervical cancer, pediatric visual pathway tumor, chordoma, chronic lymphocytic leukemia, chronic bone Myeloid leukemia, chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, skin cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymomas, ependymomas, esophageal cancer, Ewing family tumors, extracranial cancer, extramaxillary germ cell tumors, extrahepatic bile duct cancer, extrahepatic cancer, eye cancer, mycosis fungoides, gallbladder cancer, gastric cancer, gastrointestinal cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (gist), germ cell tumors, pregnancy cancer, gestational trophoblastic tumors, glioblastoma, glioma, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, hypothalamic tumors of the eye, intraocular (eyeball) cancer, intraocular melanoma, islet cell tumors, Kaposi's sarcoma, kidney (renal cell) cancer, Langerhans cell carcinoma, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer, lymphoma, macroglobulinemia, malignant fibrous histiocytoma and osteosarcoma of bone, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell neck cancer of unknown primary, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis, myelodysplastic syndrome, myelodysplastic / myeloproliferative disorders, myeloid leukemia, myelogenous leukemia, myeloma, myeloproliferative disorders, nasal and Paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cavity cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer, ovarian germinoma, ovarian low malignant potential tumor, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, moderately differentiated pineal parenchymal tumor, pineoblastoma and upper ventricular 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 involving the Nat gene on chromosome 15, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sézary 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, potential primary neuroectodermal tumor, potential primary neuroectodermal tumor and pineoblastoma, T-cell lymphoma, testicular cancer, pharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma, renal pelvis and ureter transitional cell carcinoma, choriocarcinoma, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

[0248] In one embodiment, the cancer has a mutation in a cancer driver gene listed in Table 1. In one embodiment, the cancer has drug resistance to one or more drugs listed in Table 1.

[0249] 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.

[0250] bacterial infection In one embodiment, the infectious disease or disorder is associated with bacteria. In some embodiments, the bacteria can be from any one of the following phyla: Acidobacteria, Actinobacteria, Aquificae, Bacteroidetes, Caldiserica, Chlamydiae, Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus-Thermus, Dictyoglomi, Elusimicrobia, Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes, Lentisphaerae, Nitrospira, Planctomycetes, Proteobacteria, Spirochaetes, Synergistetes, Tenericutes, Thermodesulfobacteria, Thermotogae, and Verrucomicrobia.

[0251] 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 neutrophilic, neutrophilic, extreme neutrophilic, acidophilic, alkaliphilic, thermophilic, psychophilic, alkaliphilic, or osmophilic.

[0252] The bacteria may be Bacillus anthracis, antibiotic resistant bacteria, disease causing bacteria, food poisoning bacteria, infectious bacteria, Salmonella, Staphylococcus, Streptococcus, or Clostridium tetani. The bacteria may be Mycobacteria, Clostridium tetani, Yersinia pestis, Bacillus anthracis, Methicillin-resistant Staphylococcus aureus (MRSA), or Clostridium difficile.

[0253] Viral infection In one embodiment, the infectious disease or disorder is associated with bacteria. In some embodiments, the virus is from one of the following families: Adenoviridae, Arenaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Papoviridae, Paramyxoviridae, Parvoviridae, Picornaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, or Togaviridae. Viral antigens include human immunodeficiency virus (HIV), chikungunya virus (CHIKV), dengue virus, papilloma virus, e.g., human papilloma virus (HPV), poliovirus, hepatitis virus, e.g., hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), hepatitis E virus (HEV), smallpox virus (variola major and smallpox), 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 (HTLV-I), and the like. -II), California encephalitis virus, Hantavirus (hemorrhagic fever), Rabies virus, Ebola virus, Marburg virus, Measles virus, Mumps virus, Respiratory syncytial virus (RSV), Herpes simplex 1 (oral herpes), Herpes simplex 2 (genital herpes), Shingles (varicella zoster, also known as chickenpox), Cytomegalovirus (CMV), e.g. human CMV, Epstein-Barr virus (EBV), Flaviviruses, Foot and mouth disease virus, Lassa virus, Arenaviruses, Severe acute respiratory syndrome-related coronavirus (SARS), Middle East respiratory syndrome-related coronavirus (MERS), Severe acute respiratory syndrome-related coronavirus 2 (SARS CoV 2), or cancer-causing viruses.

[0254] Parasitic infection In one embodiment, the infectious disease or disorder is associated with a parasite. In some embodiments, the parasite may be a protozoan, a helminth, or an ectoparasite. The helminth (i.e., earthworm) may be a flatworm (e.g., fuluquim and tapeworm), a tapeworm, or a roundworm (e.g., pinworm). The ectoparasite may be a louse, a flea, a tick, a mite, etc.

[0255] The parasite can be any parasite that causes any of the following diseases: acanthamoeba keratitis, amebiasis, ascariasis, babesiosis, balantidiosis, baylisascariasis, Chagas disease, clonorchiasis, encephalopathy, cryptosporidiosis, diphyllobothriasis, dracunculiasis, echinococcosis, elephantiasis, intestinal worm disease, fascioliasis, fascioliasis, filariasis, giardiasis, gnathostomiasis, hymenobiosis, isosporiosis, Katayama fever, leishmaniasis, Lyme disease, malaria, metagonimiasis, myiasis, onchocerciasis, pediculosis, scabies, schistosomiasis, sleeping sickness, strongyliasis, taeniasis, toxocariasis, toxoplasmosis, trichinosis, and trichuriasis.

[0256] Parasites include Acanthamoeba, Anisakis, Ascaris lumbricoides, Botfly, Balantidium coli, Bedbug, Cestoda, 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, or Wuchereria bancrofti.

[0257] fungal infection In one embodiment, the infectious disease or disorder is associated with a fungus. In some embodiments, the fungus can be an Aspergillus species, Blastomyces dermatitidis, a Candida yeast (e.g., Coccidioides, Cryptococcus neoformans, Cryptococcus gattii, a dermatophyte, a Fusarium species, Histoplasma capsulatum, a Mucoromycotina, Pneumocystis jirovecii, Sporothrix schenckii, Exserohilum, or Cladosporium.

[0258] PD-(L)1 axis inhibitors In some embodiments, the invention provides a combination of a Siglec-7 binding molecule 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 of 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.

[0259] It will be understood by one of skill in the art based on the disclosure provided herein that a decrease in the level or activity of one or more genes or proteins in the PD-(L)1 axis encompasses a decrease in expression of a biomarker, including transcription, translation, or both. It will also be understood by one of skill in the art that, in accordance with the teachings of the present invention, 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, and also includes a decrease in the activity of one or more genes or proteins in the PD-(L)1 axis.

[0260] Exemplary inhibitors of the PD-(L)1 axis include, but are not limited to, small interfering RNA (siRNA), microRNA, antisense nucleic acid, ribozyme, expression vector encoding a transdominant negative mutant, antibody, antibody fragment, fusion protein, aptamer, peptide, small molecule.

[0261] 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 to reduce or inhibit 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.

[0262] 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 variety 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 called Dicer. The siRNAs are then assembled into RNA-induced silencing complexes (RISCs) with protein components, unwinding in the process. Activated RISCs bind to complementary transcripts 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 modifications of siRNAs allow for intravenous systemic administration. Optimization of siRNAs requires consideration of 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.

[0263] In some embodiments, the invention includes an isolated nucleic acid encoding an inhibitor, such as a protein, antibody, siRNA, or antisense molecule, operably linked to a nucleic acid comprising a promoter / regulatory sequence, preferably capable of directing expression of the inhibitor encoded by the nucleic acid. Thus, the invention encompasses methods for introducing an expression vector and exogenous DNA into a cell and simultaneously expressing the exogenous DNA in the cell.

[0264] To assess the expression of the inhibitor, the expression vector introduced into the cells may also contain either a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from the cell population to be transfected or infected through the viral vector. In other embodiments, the selectable marker is carried on a separate DNA fragment and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to allow expression in the host cell. Useful selectable markers are known in the art and include, for example, antibiotic resistance genes such as neomycin.

[0265] When the inhibitors of the present invention are small molecules, the small molecule antagonists can 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.

[0266] Combinatorial libraries of molecularly diverse chemical compounds that may be useful in the treatment of various diseases and conditions are well known in the art, as are methods for generating the libraries, which may use a variety of techniques well known to those skilled in the art, including solid-phase synthesis, solution methods, parallel synthesis of single compounds, synthesis of chemical mixtures, rigid core structures, flexible linear sequences, deconvolution strategies, tagging techniques, and the generation of unbiased molecular landscapes for lead discovery and biased structures for lead development.

[0267] A common approach to small molecule library synthesis involves condensing an activated core molecule with multiple building blocks to yield a combinatorial library of covalently linked core-building block ensembles. The shape and rigidity of the core determine the orientation of the building blocks in shape space. Libraries can be biased to target distinct biological structures by varying the core, linkages, and building blocks ("focused libraries"), or can be synthesized with less structural bias using flexible cores.

[0268] In another aspect of the invention, one or more proteins of the PD-(L)1 axis can be inhibited by methods that inactivate and / or sequester the proteins. Thus, inhibiting the effect of one or more proteins of the PD-(L)1 axis can be achieved by using transdominant negative mutants. In one embodiment, antibodies specific for one or more proteins of the PD-(L)1 axis can be used. As will be appreciated by those of skill in the art, any antibody capable of recognizing and binding to 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 made by immunizing rabbits according to standard immunological techniques well known in the art. Such techniques include immunizing animals with chimeric proteins that contain a portion of another protein, such as a maltose binding protein or glutathione (GSH) tag polypeptide portion, and / or a portion that renders the antigenic protein of interest immunogenic (e.g., keyhole limpet hemocyanin, an antigen of interest conjugated with KLH), and a portion that contains amino acid residues of the respective antigenic protein. Chimeric proteins are produced by cloning an appropriate nucleic acid encoding the marker protein into a plasmid vector suitable for this purpose, such as, but not limited to, pMAL-2 or pCMX.

[0269] Exemplary anti-PD-(L)1 axis antibodies include, but are not limited to, nivolumab (OPDIVO®), pembrolimumab (KEYTRUDA®), sintilimumab, semipilimumab (LIBTAYO®), tripolimab, tisulizumab, spartalizumab, camrelizumab, dostralimumab, genolimuzumab, or cetrelimumab, or antibodies that bind to PD-L1, e.g., PD-L1 antibodies, include envafolimab, atezolizumab (TECENTRIQ®), durvalumab (IMFINZI®), avelumab (BAVENCIO®), REGN2810, pidilizumab, MEDI0680, PDR001, PF-06801591, BGB-A317, TSR-042, and SHR-1210.

[0270] Cancer Treatment In one embodiment, the invention provides a method for treating or preventing cancer, or treating and preventing tumor growth or metastasis. In a related aspect, an exemplary embodiment of the invention provides a method for preventing, aiding in the prevention, and / or reducing metastasis of hyperplastic or neoplastic cells in an individual.

[0271] In one embodiment, the composition is used to treat cancers with high levels of sialic acid, including but not limited to ovarian cancer, melanoma, renal cell carcinoma, prostate cancer, colon cancer, breast cancer, head and neck squamous cell carcinoma, and oral cancer.One aspect of the present 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 present invention, wherein the multivalent antibody is specific for the cancer to be treated.The present invention further 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 present invention, wherein the multivalent antibody is specific for the cancer to be treated.

[0272] In some embodiments of the treatment or prevention of cancer or the treatment and prevention of 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.

[0273] The compositions of the present invention can be used to prevent, alleviate, minimize, control, and / or reduce cancer in humans and animals. The compositions of the present invention can also be used to slow the growth rate of primary tumors. The compositions of the present invention can be used to block the spread of cancer cells when administered to a subject in need of treatment. As such, an effective amount of a nucleic acid molecule encoding a multivalent antibody of the present invention (wherein the multivalent antibody is specific for the cancer to be treated) can 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 primary tumor growth provided by the compositions of the present invention allows for more effective and efficient use of the medicines or drug therapies used to treat the patient. Furthermore, the control of metastasis by the compositions of the present invention gives the subject a greater ability to focus the disease in one place.

[0274] In one embodiment, the invention provides a method of treating cancer metastasis comprising treating the subject with a complementary therapy for the cancer, such as surgery, chemotherapy, chemotherapeutic agents, radiation therapy, or hormonal therapy, or a combination thereof, prior to, simultaneously with, or after treatment with a composition of the invention.

[0275] Chemotherapeutic agents include cytotoxic agents (e.g., 5-fluorouracil, cisplatin, carboplatin, methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, oxorubicin, carmustine (BCNU), lomustine (CCNU), cytarabine USP, cyclophosphamide, estramucin sodium phosphate, altretamine, hydroxyurea, ifosfamide, procarbazine, mitomycin, busulfan, cyclophosphamide, mitoxantrone, carboplatin, cisplatin, interferon alpha -2a recombinant, paclitaxel, teniposide, and streptozocytose), cytotoxic alkylating agents (e.g., busulfan, chlorambucil, cyclophosphamide, melphalan, or ethylsulfonic acid), alkylating agents (e.g., asarray, AZQ, BCNU, busulfan, bisulfan, carboxyphthalatoplatinum, CBDCA, CCNU, CHIP, chlorambucil, chlorozotocin, cisplatinum, clomesone, cyanomorpholinodoxorubicin, cyclodizone, cyclophosphamide, dianhydrogalactitol, fluoro Dopan, hepsulfam, 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, Yoshi-864), antirheumatic drugs (e.g., allocolchicine, halichondrin M, colchicine, colchicine derivatives, dolastatin 10, mei tansin, rhizoxin, paclitaxel derivatives, paclitaxel, thiocolchicine, tritylcysteine, vinblastine sulfate, 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),These include 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-diaminedichloroplatinum, mitoxantrone, CBDCA, levamisole, hexamethylmelamine, all-trans retinoic acid, gliadel, and porfimer sodium).

[0276] Antiproliferative agents are compounds that reduce cell proliferation. Antiproliferative agents include alkylating agents, antimetabolites, enzymes, biological response modifiers, miscellaneous agents, hormones and antagonists, androgen inhibitors (e.g., flutamide and leuprolide acetate), antiestrogens (e.g., tamoxifen citrate and its analogs, toremifene, droloxifene and roloxifene), and other examples. 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.

[0277] The compounds of the present invention can be administered alone or in combination with other anti-tumor agents, including cytotoxic / antineoplastic agents and anti-angiogenic agents. Cytotoxic / antineoplastic agents are defined as agents that attack and kill cancer cells. Cytotoxic / antineoplastic agents include alkylating agents that alkylate the genetic material of tumor cells, such as cisplatin, cyclophosphamide, nitrogen mustard, trimethylene thiophosphoramide, carmustine, busulfan, chlorambucil, verstine, uracil mustard, chromafazine, and dacabanidine. Other cytotoxic / antineoplastic agents are antimetabolites against tumor cells, such as cytosine arabinoside, fluorouracil, methotrexate, mercaptopyridine, azathioprime, and procarbazine. Other cytotoxic / antineoplastic agents are antibiotics, such as doxorubicin, bleomycin, dactinomycin, daunorubicin, mithramycin, mitomycin, mitomycin C, and daunomycin. Numerous liposomal formulations of these compounds are commercially available. Further cytotoxic / antineoplastic agents are the mitotic inhibitors (vinca alkaloids). These include vincristine, vinblastine, and etoposide. Other cytotoxic / antineoplastic agents include taxol and its derivatives, L-asparaginase, antitumor antibodies, dacarbazine, azacitidine, amsacrine, melphalan, VM-26, ifosfamide, mitoxantrone, and vindesine.

[0278] Antiangiogenic agents are well known to those skilled in the art. Antiangiogenic agents suitable for use in the methods and compositions of the present invention include anti-VEGF antibodies, including humanized and chimeric antibodies, anti-VEGF aptamers and antisense oligonucleotides. Other known angiogenesis inhibitors include angiostatin, endostatin, interferon, interleukin 1 (including alpha and beta), interleukin 12, retinoic acid, tissue inhibitors of metalloproteinase-1 and -2 (TIMP-1 and -2), and the like. Small molecule compounds that contain topoisomerase, such as razoxane, a topoisomerase II inhibitor with angiogenesis inhibitory activity, can also be used.

[0279] Other anti-cancer drugs that can be used in combination with the compositions of the present invention include, but are not limited to, acivicin; aclarubicin; acodazole hydrochloride; acronain; adzelesin; aldesleukin; altretamine; ammomycin; amethanthrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizeresin ;Bleomycin sulfate;Brequinar sodium;Bropirimine;Busulfan;Cactinomycin;Calsterone;Caracemide;Carbetimer;Carboplatin;Carmustine;Carubicin hydrochloride;Carzelesin;Cedefingol;Chlorambucil;Silormycin;Cisplatin;Cladribine;Crisnatol mesylate;Cyclophosphamide;Cytarabine;Dacarbazine;Dactinomycin;Daunorubicin hydrochloride;Decitabine;Dexormaplatin;Desaguanine;Desaguanine mesylate;Diazicon;Docetaxel;Doxorubicin;Doxol Bicine hydrochloride;Droloxifene citrate;Dromostanolone propionate;Duazomycin;Edatrexate;Eflunitine hydrochloride;Elsamitrucin;Enloplatin;Enpromate;Epipropizine;Epirubicin hydrochloride;Elburozole;Esorubicin hydrochloride;Estramustine;Estramustine sodium phosphate;Etanidazole;Etoposide;Etoposide phosphate;Etoprine;Fadrozole hydrochloride;Fazarabine;Fenretinide;Floxuridine;Fludarabine phosphate fluorouracil;Fluorocytabine ;Fosquidone;Fostriecin sodium;Gemcitabine;Gemcitabine hydrochloride;Hydroxyurea;Idarubicin hydrochloride;Ifosfamide;Irmofosine;Interleukin II (including recombinant interleukin II or rIL2), interferon alpha-2a;Interferon alpha-2b;Interferon alpha-n1;Interferon alpha-n3;Interferon beta-Ia;Interferon gamma-Ib;Iproplatin;Irinotecan hydrochloride;Lanreotide letrozole acetate;Leuprolide acetate;Liriozole hydrochloride;Lometrexol sodium; Lomustine; Rosoxantrone hydrochloride; Masoprocol; Maytansine; Mechlorethamine hydrochloride; Megestrol acetate; Melengestrol acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate sodium; Metoprine; Methodepa; Mitindomide; Mitocalcine; Mitochromine; Mitogillin; Mitomarcin; Mitomycin; Mitospar; Mitotane mitoxantrone hydrochloride; Mycophenolic acid; Nono Codazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pegaspagase; Periomycin; Pentamustine; Peplomycin sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone hydrochloride; Plicamycin; Promestane; Porfimer sodium; Porfiromycin; Prednimustine; Procarbazine hydrochloride; Puromycin; Puromycin hydrochloride; Pirazofurin; Ribopurin; Rogletimide; Safingo ru;safingol hydrochloride;semustine;simtrazene;sparphosate sodium;sparsomycin;spirogermanium hydrochloride;spiromustine;spiroplatin;streptonigrin;streptozocin;sulofenur;tallysomycin tecogalan sodium;tegafur;teroxantrone hydrochloride;temoporfin;teniposide;teroxylon;testolactone;thiamiprine;thioguanine;thiotepa;tiazofurin;tirapazamine;toremifenic acid citrate ;Triciribine phosphate;Trimetrexate;Trimetrexate glucuronate;Triptorelin;Tubrozole hydrochloride;Uracil mustard;Uredep;Vapreotide;Verteporfin;Vinblastine sulfate;Vincristine sulfate;Vindesine sulfate;Binepidine sulfate;Biricine sulfate;Benzyl sulfate;Vinorelbine tartrate;Vinorcidine sulfate;Vorozole;Zeniplatin;Zinostatin;Zorubicin hydrochloride.Other anticancer drugs include, but are not limited to:20-epi-1,25 dihydroxyvitamin D3;5-ethynyluracil;Abiraterone;Aclarubicin;Acylfulvene;Adesipenol;Adozelesin;Aldesleukin;ALL-TK antagonists;Altretamine;Ambamustine;Amidox;Amifostine;Aminolevulinic acid;Amrubicin;Amsacrine;Anagrelide;Anastrozole;Andrographolide;Angiogenesis inhibitors;Antagonist D;Antagonist G;Antarelix;Anti-dorsalized morphogenetic protein-1;Anti-androgen, prostate cancer;Anti-estrogen;Antineoplaston;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 antagonists;Benzochlorins;Benzoylstaurosporine;Beta-lactam derivatives;Beta-arrestins;Betaclamicin B;Betulinic acid;bFGF inhibitors;Bicalutamide Bisantrene;Bisaziridinylspermine;Bisnafide;Bistratin A;Bizurecin;Brefurate;Bropirimine;Budotitanium;Buthionine sulfoximine;Calcipotriol;Calophostin C;Camptothecin derivatives;Canarypox IL-2;Capecitabine;Carboxamido-aminotriazole Carboxamidotriazole;CaRest M3;CARN700;cartilage-derived inhibitor;carzelesin;casein kinase inhibitor (ICOS);castanospermine;cecropin B;cetrorelix;chlorin;chloroquinoxaline sulfonamide;cicaprost;cis-porphyrin gradribine;clomiphene analogs;clotrimazole;collismycin A;collismycin B;combretastatin A4;combretastatin analogs;conagenin;crambesidin 816;crisnatol;cryptophy Syn 8;Cryptophycin A derivatives;Cracin A;Cyclopentane thraquinones cycloplatam;Sipemycin;Cytarabine ocosfate;Cytolytic factors;Cytostatin;Dacliximab;Decitabine;Dehydrodemnin B;Deslorelin;Dexamethasone;Dexyphosphamide;Dexrazoxane;Dexverapamil;Diazicon;Didemnin B;didox.Diethylnorspermine;Dihydro-5-azacytidine;Dihydrotaxol, 9-;Dioxamycin;Diphenylspiromustine;Docetaxel;Docosanol;Dolasetron;Doxifluridine;Droloxifene;Dronabinol;Duocarmycin SA;Ebselen;Ecomustine;Edelfosine;ElecolomabEfurnitine;Elemene;Emiteflu;Epirubicin;Epristeride;Estramustine analogs;Estrogen agonists;Estrogen antagonists;Etanidazole;Etoposide phosphate;Exemestane;Fadrozole;Fazarabine;Fenretinide;Filgrastim;Finasteride Flavopiridol;Freseras tin;fluasterone;fludarabine;fluorodaunornithine hydrochloride;forphenimex;formestane;fostriecin;fotemustine;gadolinium texaphyrin;gallium nitrate;galocitabine;ganirelix;gelatinase inhibitor gemcitabine;glutathione inhibitors;hepsulfame;heregulin;hexamethylene bisacetamide;hypericin;ibandronate;idarubicin;idoxifene;idramantone;ilmofosine;ilomastat;imidazoacridone;imiquimod;immunostimulating peptide insulin-like growth factor-1 Receptor inhibitors;Interferon agonists;Interferons;Interleukins;Iobenguane;Iododoxsorbicin;Ipomeanol, 4-;Ilopract;Irsogladine;Isobengazole;Isohomohalichondrin B;Itasetron;Jasplakinolide;Kahalide F. Lamellarin-N triacetate;Lanreotide;Leinamycin;Lenograstim;Lentinan sulfate;Leptolstatin;Letrozole;Leukemia inhibitory factor;Leukocyte alpha interferon;Leuprolide + estrogen + progesterone;Leuprorelin ;Levamisole;Lyazol linear polyamine analogs;Lipophilic disaccharide peptides;Lipophilic platinum compounds;Lysoclinamide 7;Lovaplatin;Lombricine;Lometrexol;Lonidamine;Losoxantrone;Lovastatin;Loxoribine;Lultotecan;Lutetium texaphyrin;Rhizophyllin lytic peptides;Maytansine;Mannostatin A;Marimastat;Masoprocol;Maspin;Matrilysin inhibitors;Matrix metalloproteinase inhibitors;Menogaril;Melbarone;Meterelin;Methioninase;Metoclopramide;MIF inhibitors;Mifepristone;miltefosine;mirimostim;mismatched double-stranded RNA;mitoguazone;mitolactol;mitomycin analogues;mitonafide;mitotoxin fibroblast growth factor-saporin;mitoxantrone;mofalotene;molgramostim;monoclonal antibodies, human chorionic gonadotrophin;monophosphoryl lipid A+myobacterial cell wall sk;mopidamol;multidrug resistance gene inhibitors;multiple tumor suppressor 1-based therapy;mustard antisputum;mycaperoxide B;mycobacterial cell wall extract;myriaporone;N-acetyl Dinaline;N-substituted benzamides;Nafarelin;Nagressip;Naloxone + pentazocine;Napavine;Naphthalpine;Nartograstim;Nedaplatin;Nemorubicin;Neridronic acid;Neutral endopeptidase;Nilutamide;Nisamycin;Nitric oxide regulators;Nitric oxide antioxidants;Nitrulline;O6-benzylguanine;Octreotide;Oxenone;Oligonucleotides;Onapristone;Ondansetron;Oracin;Oral cytokine inducers;Ormaplatin;Osateron;Oxaliplatin;Oxaunomycin;Paclitaxel Ritaxel analogues;Paclitaxel derivatives;Palauamine;Palmitoyl rhizoxin;Pamidronic acid;Panaxytriol;Panomyphen;Parabactin;Pazeliptin;Pegaspargase;Perdecin;Pentosan polysulfate sodium;Pentostatin;Pentrozole;Perflubron perphosphamide;Perillyl alcohol;Phenazinomycin;Phenylacetate;Phosphatase inhibitors;Picibanil;Pilocarpine hydrochloride;Pirarubicin;Piritrexim;Plasetin A;Plasetin B;Plasminogen activator inhibitors; Platinum complexes;Platinum compounds;Platinum-triamine complexes;Porfimer sodium;Porfiromycin;Prednisone;Propyl bis-acridone;Prostaglandin J2;Proteasome inhibitors;Protein A-based immunomodulators;Protein kinase C inhibitors;Protein kinase C inhibitors, microalgae;Protein tyrosine phosphatase inhibitors;Purine nucleoside phosphorylase inhibitors;Purpurins;Pyrazoloacridines;Pyridoxylated hemoglobin polyoxyethylene conjugates;raf antagonists;Raltitrexed;Ramosetron;ras farnesyl protein transferase inhibitors;ras inhibitors;ras-GAP inhibitors;demethylated reterliptin;rhenium Re 186 etidronate;rhizoxin;ribozyme;RII retinamide;logretimide;rohitukin;romurtide;roquinimex rubiginone B1;ruboxil;safingol;santopine;SarCNU;sarcophytol; A;Sargramostim;Sdi 1 mimetics;Semustine;Senogenesis-derived inhibitor 1;Sense oligonucleotides;Signal transduction inhibitors;Signal transduction modulators;Single-chain antigen-binding proteins;Schizofuran;Sobuzoxane;Sodium borocaptate;Sodium phenylacetate;Sorberol;Somatomedin-binding proteins;Sonamine;Sparphostinic acid;Spicamycin D;Spiromustine;Splenopentin;Spongistatin 1;Squalamine;Stem cell inhibitors;Stem cell division inhibitors;Stipiamide;Stromelysin inhibitors;Sulfinosine;Superactive vasoactive intestinal peptide antagonists;Sladista;Suramin;Swainsonine;Synthetic glycosaminoglycans;Talimustine tamoxifen methiodide;Tauromustine;Tazarotene;Tecogalan sodium;Tegafur;Terlapyrylium;Telomerase inhibitors;Temoporfin;Temozolomide;Teniposide;Tetrachlorodecaoxide ;Tetrazomine;Taliblastine;Thiocoraline;Thrombopoietin;Thrombopoietin mimetics;Thymalfasin;Thymopoietin receptor agonists;Thymotrin;Thyroid-stimulating hormone;Tin ethyl etiopurpurin;Tirapazamine;Titanocene bichloride;Topsentin;Toremifene;Totipotent stem cell factor;Translation inhibitors tretinoin;Triacetyluridine;Triciribine;Trimetrexate;Triptorelin;Tropisetron;Turosteride;Tyrosine kinase inhibitors;Tyrphostin;UBC inhibitors;Ubenimex;Urogenitourinary sinus-derived growth inhibitor;Urokinase receptor antagonist vapreotide;Variolin B;Vector systems, red blood cell gene therapy;Veraresol;Veramine;Vergin;Verteporfin;Vinorelbine;Vinxartin;Vitaxin;Vorozole;Zanotherone;Zeniplatin;Zilascorub;And zinostatin styramer. In one embodiment, the anticancer drug is 5-fluorouracil, taxol, or leucovorin.

[0280] 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-7 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.

[0281] In one embodiment, a composition comprising a synthetic anti-Siglec-7 antibody, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same, and a composition comprising a tumor antigen, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same, can be administered using any suitable method such that the combination of anti-Siglec-7 and tumor antigen are both present in the subject. In one embodiment, the method comprises administering a first composition comprising a synthetic Siglec-7 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, where the second composition comprising a tumor antigen, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same of the invention is administered 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-7 antibody, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same. In one embodiment, the method may comprise administration of a first composition comprising a synthetic Siglec-7 antibody, fragment or variant thereof or a nucleic acid molecule encoding same of the present invention by any of the methods detailed above, and administration of a second composition comprising a tumor antigen, fragment or variant thereof or a nucleic acid molecule encoding same, where the second composition comprising a tumor antigen, fragment or variant thereof or a nucleic acid molecule encoding same of the present invention is administered 1 or more days, 2 or more days, 3 or more days, 4 or more days, 5 or more days, 6 or more days, 7 or more days, 8 or more days, 9 or more days, or 10 or more days after administration of the synthetic anti-Siglec-7 antibody, fragment or variant thereof or a nucleic acid molecule encoding same.In one embodiment, the method may comprise administration of a first composition comprising a tumor antigen, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding same, and administration of a second composition comprising a synthetic anti-Siglec-7 antibody, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding same, the second composition comprising a synthetic anti-Siglec-7 antibody of the present invention, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding same is administered 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 a tumor antigen, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding same, and administering a second composition comprising a synthetic anti-Siglec-7 antibody, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding same, wherein one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more days after administration of the composition comprising the tumor antigen, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding same, the second composition comprising the synthetic anti-Siglec-7 antibody of the invention, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding same, is administered by any of the methods detailed above. In one embodiment, the method may comprise simultaneously administering a first composition comprising the synthetic anti-Siglec-7 antibody of the invention, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding same, by any of the methods detailed above, and a second 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 single composition comprising a synthetic anti-Siglec-7 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.

[0282] The present invention will be further described in the following examples. It should be understood that these examples, while showing exemplary embodiments of the present invention, are given for illustrative purposes only. From the above discussion and these examples, those skilled in the art can grasp the essential features of the present invention, and can make various changes and modifications to adapt the present invention to various applications and conditions without departing from the spirit and scope thereof. 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 be included within the scope of the appended claims. EXAMPLES

[0283] The present invention will be described in more 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 construed as being limited in any way to the following examples, but rather as embracing any variations that become evident as a result of the teachings provided herein. Without further explanation, it is believed that one skilled in the art can use the preceding description and the following illustrative examples to make and use the present invention and carry out the claimed method. Therefore, the following examples should not be construed as limiting the remainder of the disclosure in any way.

[0284] Example 1: Generation of Siglec-7 antibody using H2L2 mice Sialic acid-binding receptors, such as Siglecs and selectins, are in a position to be exploited by cancer hypersialylation. Sialic acids (Sias) are monosaccharides typically found at the outermost ends of complex glycans that decorate the cell surfaces of all vertebrates. Sialic acids are essential for embryonic development and mediate important functions inherent to the organism. Seventy human genes are known to be involved in sialic acid biology, and more than 10 genes have been demonstrated to be human-specific. Most of these human-specific genetic alterations are Sialocyanine-Recognizing Ig-Like Lectin to Immunological Synapse (SIGLEC) genes. Sialoside upregulation has long been correlated with poor tumor prognosis and reduced immunogenicity.

[0285] The experiments described here demonstrate the development and characterization of Siglec-7 antibodies (Figure 1). In these experiments, hybridoma supernatants showed specificity and binding to human Siglec-7. Siglec7-pBMN-1-GFP plasmid was used to generate stable Siglec-7 expressing cells by retroviral transduction (Figure 2). Anti-human Siglec 7 antibodies bound to human NK cells from different donors at concentrations ranging from 10-50 ng / ml (Figure 3).

[0286] As a result, the antibody showed cytotoxicity against OVISE cells (Figure 4). OVISE cells are ovarian clear cell adenocarcinoma derived from metastasis: OVISE cells are ovarian clear cell adenocarcinoma metastasized to the pelvis; sternum of a 40-year-old Japanese woman. The experiment was designed to examine the activity of the antibody. Target cells OVISE (20,000 cells / well); Effector cells: Effector cells and antibody (1:10) were added at 21.79 hours. Images were taken after 116 hours, and it was observed that the antibody killed OVISE cells. Killing was observed within 20 hours of incubation of the antibody with effector cells.

[0287] It was also observed that the antibody exhibited cytotoxicity against SKOV3 cells (Figure 5). SKOV3 are ovarian serous cystadenocarcinoma cells derived from a metastatic site: The experiment was designed to examine the activity of the antibody. Target cells SKOV3 (10,000 cells / well); Effector cells: Effector cells and antibodies were added at 22.73 hours. It was observed that the antibody was killing the SKOV3 cells. See Figure 5.

[0288] It was also observed that the antibody exhibited cytotoxicity against OVCAR10 cells (Figure 6). OVCAR10 is an ovarian human cancer cell line. The experiment was designed to investigate the activity of the antibody. Target cells OVCAR10 (10,000 cells / well); effector cells: human PBMC E:T=5:1; effector cells and antibody were added at 22.73 hours. It was observed that the antibody killed OVCAR10 cells. See Figure 6.

[0289] Example 2: Treatment of melanoma with Siglec-7 antibody Skin cancer is one of the most common and dangerous human cancers. Among its various subtypes, melanoma, which arises from melanocytes, is a particularly serious disease. Immune checkpoint inhibition (CPI), a new immunotherapy approved by the FDA, has revolutionized melanoma treatment and improved outcomes. However, 40-50% of melanomas tend to be immunologically cold with poor T cell infiltration, and therefore have low responsiveness to CPIs.

[0290] In early studies, it was observed that several melanoma cell lines express high levels of Siglec-7 ligands, suggesting that suppression of NK cells by the Siglec-7 pathway is important in melanoma immunity. The results presented here show that anti-Siglec-7 antibodies enhance the killing activity of NK cells against melanoma cell lines (Figures 8-12). The melanoma cell lines 1205Lu, 451Lu MR, WM1366, WM3311, WM983BR, and WM3743 express high levels of Siglec7 ligands and are amenable to treatment with Siglec7 antibodies. The WM983 cell line is a BRAFV600E mutant line, with wild-type PTEN, N-ras, c-KIT, and CDK4, and is a human metastatic melanoma cell line.

[0291] WM3743 is a post-chemotherapy primary melanoma cell line that displays mesenchymal morphology. The cell line expresses wild-type N-RAS, c-KIT, and CDK4 genes. WM3311 is a human metastatic melanoma cell line. The cell line was established from a metastatic site in a patient. These cells are known to harbor mutations in GRIN2A, PRDM1, and TERT, and to have upregulated Notch2. Unlike many melanoma cell lines, these cells do not harbor the common V600E (Val600Glu) mutation at codon 600 of the BRAF gene. The cell line is wild-type for BRAF, N-RAS, c-KIT, and CDK4.

[0292] The results presented here demonstrate that Siglec 7 antibodies can selectively target Siglec 7-expressing melanomas (primary, metastatic, and treatment-resistant) and across multiple genotypes of melanoma.

[0293] Parental Siglec-7 antibodies are effective in melanomas resistant to Braf / Mek inhibitor treatment.

[0294] Example 3: Siglec-7 glycan immune checkpoint MAbs and NK cell engager biologics induce potent antitumor immunity against ovarian cancer Aggressive OC types such as HGSC and ovarian carcinosarcoma (OCS) are referred to as “cold” tumors due to their low tissue infiltrating lymphocyte (TIL) infiltration and poor therapeutic response (Wu et al., 2021, Front Immunol 12, 672502, Hoogstad-van Evert et al., 2020, Gynecol Oncol 157, 810-816). Thus, engaging the immune response by additional means is crucial for immune targeting of cold tumors (Pugh-Toole et al., 2022, Curr Treat Options Oncol 23, 210-226). Considering the innate immune effector system is important, as NK cells serve as the first line of defense in tumor immune surveillance (Wiernik et al., 2013, Clin Cancer Res 19, 3844-3855). Tumors evade T cell responses through multiple mechanisms, including MHC downmodulation and upregulation of T cell exhaustion programs (Vinay et al., 2015, Semin Cancer Biol 35 Suppl, S185-S198). Similarly, cancers can exploit host systems to evade NK cell surveillance and escape NK immune functions through immune evasion strategies, such as shedding soluble ligands of NK activating receptors, releasing inhibitory cytokines, and upregulating HLA molecules (Sabry et al., 2013, Front Immunol 4, 408), but NK unresponsiveness may also be mediated by additional NK regulatory pathways. Recent studies have identified that many tumors have elevated levels of sialic acid on their surface, which likely acts as a negative signal for NK cells, allowing tumors to evade NK immune surveillance (Dobie et al., 2021, Br J Cancer 124, 76-90). One approach to enhance NK activation against such hypersialylated tumors is the desialylation of target cells expressing ligands for Siglec-7 (Jandus et al., 2014, J Clin Invest 124, 1810-1820).NK cells express Siglec-7, a receptor / sensor for distinct sialoglycan determinants, which, when engaged on the human NK cell surface through tumor association, plays a role in inhibiting NK-mediated target killing (Prescher et al., 2017, J Med Chem 60, 941-956; Fong et al., 2018, Proc Natl Acad Sci USA 115, 10410-10415; Meril et al., 2020, Mol Carcinog 59, 713-723). These may represent a regulatory negative signal by the sialoglycan tumor shield. Very recent studies have suggested that some Siglecs may function as glycan immune checkpoint molecules and inhibit NK-mediated antitumor immunity in an MHC-independent manner (Hong et al., 2021, ACS Cent Sci 7, 1338-1346). In this study, we aimed to use monoclonal antibodies and their derivatives targeting Siglec-7 for tumor immunomodulation to confirm and possibly overcome such glycan CPI functions, improving their ability to affect tumor control.

[0295] Antitumor immune responses against the difficult-to-treat aggressive phenotype of human OC (19) were the focus of these studies. Using transgenic humanized mouse models, the research group developed several antibodies against Siglec-7 (Bordoloi et al., 2022, JCI Insight 7; Perales-Puchalt et al., 2019, JCI Insight 4; Bordoloi et al., 2021, Genes Cancer 12, 51-64). In this study, we focused on three potent clones that bind Siglec-7. These antibodies were generated as human IgG1 and their binding to immune cells was studied. Strong staining of anti-Siglec-7 antibodies on NK cells was observed, including both light and dim NK subsets, suggesting their ability to bind to bulk effector NK populations. In addition, expression can activate NK cells, and when these activated cells are in the presence of some tumor lines, NK-mediated killing can occur against previously refractory OC targets. These human Siglec-7 antibodies promote NK immune function in the presence of diverse OC tumor target cells and facilitate specific killing of targeted OC cells. Importantly, this approach is relatively agnostic to tumor mutations. OC lines were tested for multiple genetic mutations, including BRCA1, BRCA2, AKT2, TP53, SPOP, STAT3, MTOR, MEK1, MEK2, BRAF, and others (Table 1), and all mutations were observed to be amenable to activation of NK cell targets by anti-Siglec-7 antibodies.

[0296] [Table 1A]

[0297] [Table 1B]

[0298] These MAbs were studied in combination with PD-1 CPIs. The dual therapy was found to be synergistic. However, the potency of the Siglec-7 antibody was observed to be 10-fold higher than the anti-PD1 antibody Pembrolizumab examined, suggesting the potential value of this combined NK T cell anti-excretory panel. Next, a humanized NSG mouse model engrafted with human PBMCs was utilized, and a single dose of DNA-encoded DB7.2 Siglec-7 MAb was observed to significantly control tumors and increase median survival in mice bearing OVISE human ovarian tumors. Due to the limitation of the NSG model, which is that it takes several months for graft-versus-host disease (Wunderlich et al., 2014, Blood 123, e134-144) to develop, anti-Siglec-7 was only administered in a single dose, but was still highly effective. This suggests that multiple doses over time, as current CPIs (Hirsch et al., 2022, Nat Med 28, 2236-2237) are utilized, would likely provide additional benefit. Notably, this is the first demonstration of the impact of a Siglec-7-targeting MAb alone, as well as in combination with anti-PD1 (NK and T cell CPIs) targeting OC or any human tumor, suggesting that further investigation of the potential added value to such combinations, or in CPI-refractory tumors, is warranted. These studies have important implications for tumor therapy supporting the potential of novel non-T cell CPIs to augment current immunotherapy strategies.

[0299] Next, the efficacy of Siglec-7 binding to and activation of NK cells in targeted OC therapy was evaluated. Without being bound by theory, it was hypothesized that binding to Siglec-7 could directly attract NK cells and efficiently recruit them to target cells. By creating a bispecific fusion of a MAb targeting FSHR and a potent anti-Siglec-7 MAb, a new type of NK cell engager was developed. NKCE has emerged as an interesting innate immune concept in the field of immuno-oncology. Currently, one such bispecific antibody has been applied clinically. GTB- 3550, which engages CD16a-IL-15 / CD33, is effective against AML and CD33+ malignancies (Gleason et al., et al., 2014, Blood 123, 3016-3026; Ibarlucea-Benitez et al., 2021, Proc Natl Acad Sci USA 118; Demaria et al., 2021, Eur J Immunol 51, 1934-1942). Other NK targets that have been preclinically studied include NKG2D, NKp30, and NKp46. The phenotype demonstrated with anti-Siglec-7 antibodies is the binding and activation of NK cells that are adversely affected by tumor sialoglycan cloaks, suggesting that Siglec-7 could be a unique immune tool to specifically activate and engage tumors with NK. The bispecific developed here, DB7.2xD2AP1, potently bound to ovarian cancer cells as well as NK cells. It showed killing potency (pM level) against diverse ovarian cancer phenotypes. This is the first report of developing a member of the Siglec family as an immune engager and the first report of using such an approach to develop a Siglec-7 bispecific NK cell engager. This NKCE was specific for the target cells and potent in killing tumor cells as assessed with various human ovarian tumor lines. FSHR expression on the target was a prerequisite for NK killing activity, as FSHR-negative cells were not killed. Interestingly, DB7.2xD2AP11 caused less production of cytokines and cytotoxic molecules.This suggests that research into killing tools that limit the potential risk of cytokine release syndrome (CRS) is important (Pinto et al., 2022, Trends Immunol 43, 932-946).

[0300] These studies focused on OC cells expressing mutations that are particularly problematic for OC therapy. This includes PARPi-resistant OC cells and OCs resistant to a variety of drug targets, including HDAC (dacinostat, entinostat, belinostat), PI3K (dactolisib, bupallisib), mTORC (omipallisib), Wee1 (CHEK1), TOP1 (galiviscoquinazole, mitoxantrone, irinotecan), DNA alkylating agents (oxaliplatin, cisplatin), and microtubule stabilizing agents (docetaxel) (Table 1) (Ai et al., 2021, Oncogene 40, 2496-2508; Diaz Osterman et al., 2019, Elife 8; Estep et al., 2007, PLoS One 2, e1279; Kapoor et al., 2019, PLoS One 2, e1279; al., 2018, Biochim Biophys Acta Mol Cell Res 1865, 392-405; Ayestaran et al., 2020, Patterns (NY) 1, 100065). These studies included OC lines with BRCA (BRCA1 and 2) mutations, which are the major risk in OC cancer patient cohorts. Approximately 50% of HGSCs show BRCA pathway disruption due to germline or somatic mutations or epigenetic silencing of pathway members (Vaughan et al., 2011, Nat Rev Cancer 11, 719-725). PARP inhibition is a key pathway for DNA damage repair, therefore PARPi shows great promise for the treatment of tumors with disruptive mutations in BRCA1 / 2. However, resistance to PARPi has been reported to be a problem in treating patients with resistant OC (Noordermeer et al., 2019, Trends Cell Biol 29, 820-834).The efficacy and consistency of killing FSHR+ tumors in vitro and in vivo using both Siglec-7 MAb as an NK CPI and bispecific Siglec-7 NK engagers may provide a new tool for treating poorly responsive OC.

[0301] In summary, this study describes potent Siglec-7 MAbs that engage and activate NK cells, in this case as a potential additional class of CPIs for NK cells that can further complement PD-1 immunotherapy for OC. Furthermore, Siglec-7 was shown to construct a novel NKCE and serve as a powerful tool to target receptor-positive OC with significant antitumor effects in vitro and in vivo. Although these studies are important for designing new tools for OC, other studies are justified to explore the utility of such biologics for other challenging cancers.

[0302] Materials and methods are described below:

[0303] Cells and animals Cell lines used in the experiments included OVCAR3, CaOV3, TOV-21G, OVISE, OVCAR10, PEO-4, Kuramochi cells, HaCaT human keratinocytes, WM3743 melanoma cells, human embryonic kidney 293T, Expi293F, AGS gastric carcinoma, and GM05389 human fibroblasts. 293T cells were retrovirally transduced to express Siglec-7. OVCAR3, OVISE, and Kuramochi cells were retrovirally transduced with human FSHR to express FSHR as previously described (46). K562 cells were purchased from ATCC and retrovirally transduced to express FSHR.

[0304] Generation of DNA-encoded MAbs Several humanized antibodies against human Siglec-7 have been generated. These MAbs were able to bind to Siglec-7 and stain NK cells. Here, these hybridomas were sequenced and used to develop Siglec-7 DNA-encoded monoclonal antibodies (DMAbs) as a tool for in vitro expression (Patel et al., 2018, Cell Rep 25, 1982-1993 e1984). The final human IgG1 HC and LC were inserted into the pVax1 plasmid expression vector under the control of the human cytomegalovirus (hCMV) promoter and the bovine growth hormone (BGH) polyA signal as described (Patel et al., 2018, Cell Rep 25, 1982-1993 e1984). The plasmids were then transfected into Expi293F cells using the Expifectamine 293 Expression Kit (Thermo Fisher Scientific) to produce recombinant antibodies. The purity and apparent molecular weight of the recombinant antibodies were assessed by SDS-PAGE analysis.

[0305] Design of FSHRx Siglec-7 NKCE FSHRxSiglec-7 natural killer cell engager (NKCE) was designed by encoding a codon-optimized scFv of Siglec-7 MAb (DB-S7-2) followed by an scFv of FSHR antibody (D2AP11) with an enhancement-optimized IgE leader sequence. Both constructs were subcloned into a modified pVax1 expression vector (Perales-Puchalt et al., 2019, JCI Insight 4). FSHRxSiglec-7 NKCE was designated DB7.2xD2AP11.

[0306] Flow cytometry A BD LSRII flow cytometer was used for cell staining. A BD FACS Aria cell sorter (BD Biosciences) was used for sorting Siglec-7 / FSHR stable expressing cells. The anti-human antibodies used were directly fluorescently labeled. Antibodies used were: anti-Siglec-7 (F023-420, BD Pharmingen), anti-Siglec 3 (6C5 / 2, R&D Systems). The unconjugated primary antibodies used were PE-secondary anti-human (H+L) (Invitrogen) and PE / AF647-secondary anti-human F(ab')2 (Jackson ImmunoResearch Laboratories Inc). A Live / Dead Violet Viability Kit (Invitrogen) was used to exclude dead cells from the analysis. For fluorescent cytometry staining of PBMCs, the following antibodies were used: CD69 PE-Cy5 (clone FN50), PD-1 BV421 (clone EH12.2H7), CCR7 APC-Cy7 (G043H7), CD19 BV785 (clone HIB19), CD27 BV650 (clone 0323), CD56 BV570 (clone HCD56), CD16 BV711 (clone 3G8), CD21 PE-Cy7 (clone BU32) and Siglec-7 (clone 6-434) from Biolegend; CD11c BUV395 (clone B-ly6), CXCR5 BV750 (clone RF8B2), CD3 BUV805 (UCHT1), CD45 AF700 (clone HI30), CD127 PE-CF594 (clone HIL-7RM21), CD25 BUV737 (clone 2A3), CD8 BUV496 (clone RPA-T8), HLA-DR BV605 (clone G46-6), CD38 BUV661 (clone HIT2), CD14 BV480 (clone MP9), CD45RA BUV563 (HI100), CD4 BB790 (clone SK3), CD15 FITC (clone HI98), CD103 BB700 (clone Ber-ACT8), CD161 APC (clone DX12) were obtained from BD Biosciences.Briefly, cryopreserved PBMCs were thawed and incubated for 2 h at 37 °C, 5% CO2 in complete R10 medium (RPMI supplemented with 10% FBS, 2 mM Lglutamine, 100 U / ml penicillin, 100 mg / ml streptomycin) supplemented with 10 U / ml DNAse I (Roche Life Sciences). Cells were washed with PBS and incubated for 20 min with DB7.2 Siglec-7 antibody at 0, 1 or 10 μg / ml diluted in fluorescence-activated cell sorting (FACS) buffer. After washing with FACS buffer, cells were incubated for 20 min with mouse anti-human IgG Fab secondary antibody PE (Invitrogen) diluted in FACS buffer. Cells were washed with FACS buffer and resuspended in PBS before prestaining for chemokine receptors CCR7 and CXCR5 for 10 min at 37 °C, 5% CO2. Cells were then incubated for 20 min with a panel of direct-conjugated monoclonal antibodies and human Trustai FcX (Biolegend) diluted at 1:1 in fluorescence-activated cell sorting (FACS) buffer (PBS containing 0.1% sodium azide and 1% bovine serum albumin) and Brilliant Stain buffer (BD Biosciences). Stained cells were washed and fixed in PBS containing 1% paraformaldehyde (Sigma-Aldrich). Samples were acquired using a FACS Symphony A5 cytometer and analysis was performed with Flowjo software 10.8.1 (Tree Star Inc.).

[0307] Enzyme-linked immunosorbent assay (ELISA) To quantify human IgG in Siglec-7 MAbs and Siglec-7 DMAb electroporated mouse serum, MaxiSorp plates were coated with 10 μg / mL goat-human IgG Fc (Bethyl) overnight at 4°C. Plates were washed and blocked with 5% milk in PBS-T (0.05% Tween® 20 in PBS) for 2 h at RT. Plates were washed and samples diluted in 1% NCS in PBS with 0.2% Tween® 20 were added and incubated for 2 h at 37°C. Plates were washed again and incubated with 1:10,000 diluted HRP-conjugated goat anti-human IgG (H+L) secondary antibody (Bethyl) for 1 h at RT. Plates were developed in SigmaFast OPD for 5-10 min and OD450 signals were measured.

[0308] In vitro cytotoxicity analysis using the xCELLigence Real-time Cell Analyzer In vitro cytotoxicity assays were performed based on impedance using the xCELLigence Real-Time Cell Analyzer (RTCA) from Agilent Technologies, USA. Impedance is expressed in arbitrary units called cell index. Target cells were plated in a disposable sterile 96-well E-plate of the xCELLigence RTCA instrument at a final cell concentration of 1x10 per well. 4 ~2x10 4 Cells were seeded as 100 μg / well plates. During the experiment, the device was placed in a CO2 incubator and controlled by a cable connected to a control unit. The 96-well E-Plates were placed in the xCELLigence RTCA device and incubated for 18–24 h. Afterwards, effector cells (human PBMC / NK; E(effector):T(target) ratio = 5:1 / 10:1) and treatment (Siglec-7 MAbs / NKCE) were added. Real-time analysis was performed for 3–7 days. Electrical conductance was converted to a unitless cell index (CI) parameter by the xCELLigence device every 15 min, and images were taken at 1-h intervals. The generated data were normalized according to the time points when effector cells and MAbs / NKCE were added to the target cells and analyzed using RTCA / RTCA Pro software.

[0309] Western blot analysis To verify the in vitro and in vivo expression of Siglec-7 MAbs, supernatants collected after transfection of Expi 293F cells with DNA encoding the antibodies or sera collected from mice after immunization with Siglec-7 MAbs were heat inactivated, reduced, and loaded with Odyssey Protein Molecule Weight (LI-COR). After electrophoresis, samples were transferred to polyvinylidene difluoride (PVDF) membranes via the iBlot-2 system (Thermo Fisher Scientific) and blocked with Odyssey Blocking Buffer (LI-COR). Heavy and light chains were detected with goat anti-human secondary antibodies (LI-COR). Expression of bispecific T cell and NK cell engagers was detected with goat anti-human IgG F(ab')2 (Jackson ImmunoResearch Laboratories Inc) followed by donkey anti-goat antibodies (LICOR).

[0310] Immunofluorescence (IFA) analysis Siglec-7-transduced HEK 293T cells were seeded in 2-well chamber slides and allowed to adhere overnight. Cells were permeabilized with 0.5% Triton® 100 in PBS and blocked with 5% goat serum. Bispecific NKCE was then added and incubated overnight at 4°C. Slides were then incubated with goat anti-human H+L (Texas Red conjugated) secondary antibody. Nuclear staining was performed with 4',6-diamidino-2-phenylindole (DAPI). Samples were mounted on glass slides using Fluoroshield mounting medium (Invitrogen) and viewed using a Leica TCS SP8 WLL scanning laser confocal microscope.

[0311] Tumor challenge NOD / SCID-γ (NSG) mice were challenged with OVCAR3-FSHR cells. NSG mice were challenged with 3x10 6FSHR-expressing OVCAR3 or OVCAR3-FSHR cells were injected subcutaneously into the right flank. Three days later, when tumors were palpable, mice were inoculated with pVax1 (100 μg) or DDAP-NKCE (100 μg). On the same day as expression vector administration, 10 × 10 6 PBMCs were injected intraperitoneally into each mouse. Mice were inoculated with DNA twice at a weekly interval and tumor size was monitored periodically. Mice were euthanized when signs of graft-versus-host disease (GVHD) appeared. Tumor volume (V) was calculated as V = [(length × width) × 100] × 100]. 2 For Siglec-7 DMAb challenge, NOD / SCID-γ (NSG) mice were inoculated with OVISE cells (0.8 × 10 6 Ten days later, when tumors were palpable, mice were inoculated with DB7.2 DMAb (50 μg + 50 μg; HC + LC), or pVax1 (100 μg), and then each mouse was challenged with 10 × 10 6 PBMCs were injected intraperitoneally. The same procedure was then followed. Animal experiments were approved by the Institutional Animal Care and Use Committee of The Wistar Institute.

[0312] statistical analysis All statistical analyses were performed using Graph Pad Prism. A p-value <0.05 was considered statistically significant. Differences between the means of experimental groups were calculated using two-tailed unpaired Student's t-test or one-way ANOVA when more than two quantitative variables were measured. Error bars represent the standard error of the mean. Comparisons of tumor size at each time point were performed using two-way ANOVA with Fisher's least significant difference (LSD) test. The experimental results are described below.

[0313] In vitro expression and specificity evaluation of Siglec-7 MAbs To evaluate the potential of Siglec-7 blockade as a strategy for NK cell activation, MAbs specific for human Siglec-7 were recently generated and their biological activity studied in vitro. Here, codon- and RNA-optimized antibody expression cassettes of potent Siglec-7 binders were generated and incorporated into DNA vectors optimized for the expression of human IgG1. These individual combinations of HC and LC were named DB7.1 (DB-S7-1), DB7.2 (DB-S7-2), and DB7.7 (DB-S7-7), respectively (Figure 13A). In vitro expression was examined by Western blot analysis by transfecting expi293F cells with synthetic human Siglec-7 DNA vectors. These studies revealed bands corresponding to the heavy and light antibody chains of the expressed antibody in the supernatants of transfected Expi293F cells but not in the control wells transfected with empty vector (Figure 13B). In vitro expression of DB7.1, DB7.2 and DB7.7 was further confirmed by ELISA quantification (Figure 13C). DB7.1, DB7.2 and DB7.7 Siglec-7 MAbs showed specific and dose-dependent binding to recombinant human Siglec-7 analyzed by ELISA (Figure 14A). HEK293T cells were stably expressed with Siglec-7 and these cells (HEK293T-Siglec7 cells) were used to evaluate binding by Siglec-7 MAbs. As shown in Figure 14B, DB7.1, DB7.2 and DB7.7 showed specific binding to HEK293T-Siglec-7 cells. The most potent binder, DB7.2, was further analyzed for binding in PBMCs of multiple donors (n=4) (Figure 15). DB7.2 bound primarily to human NK cells (~90%), whereas lower levels of binding were observed in the case of CD8+ and CD4+ T cells (Figure 14C and D). Binding of DB7.2 was observed to innate lymphoid cells (ILCs) (~9%), which are considered to be the innate counterparts of CD4+ T helper 1 (TH1), TH2, and TH17 cells (Eberl et al., 2015, Science 348, aaa6566). In addition, DB7.2 showed over 25% binding to mucosal-associated invariant T (MAIT) cells.MAIT cells play a key role in immune defense against microbial infections at mucosal barriers and function as innate sensors of inflammation and viral infections (Parrot et al., 2020, Sci Immunol 5; Legoux et al., 2020, Immunity 53, 710-723). Further study of the minor T cell population expressing Siglec-7 is important. Interestingly, DB7.2 MAb stained both CD56dim and CD56bright NK cell subsets, suggesting its ability to bind to the majority of the human NK population (Figure 14E). DB7.2 also showed strong binding to human Siglec-7 protein by Western blot (Figure 16A) and binding was also analyzed by surface plasmon resonance (SPR) analysis. DB7.2 anti-Siglec-7 antibody showed a KD value of 44 pM, which was determined by titrating the concentration of anti-Siglec-7 antibody, and the maximum point on the sensorgram indicates saturation of recombinant human Siglec-7 protein. In addition to the high affinity, high specificity (Koff: 10-M) of DB7.2 was also observed in surface plasmon resonance (SPR) assays (Figures 16B-D).

[0314] Siglec-7 MAbs potently and specifically killed target ovarian cancer cells in vitro To evaluate the ability of Siglec-7 gene-optimized MAbs to activate human peripheral blood NK cells against human cancer lines, a diverse panel of human ovarian cancer cells was assembled (Table 1) with HaCaT (human keratinocyte cells) as a Siglec-7 negative control (Bordoloi et al., 2022, JCI Insight). Importantly, DB7.1, DB7.2 and DB7.7 did not induce killing of HaCaT cells (Figure 17A and B). Next, the ability of MAbs to induce killing of defined human OC cells (OVISE, TOV-21G, OVCAR3, CaOV3, OVCAR10 and PEO-4) was examined in the presence of human PBMC / NK cells with different disease phenotypes and mutations (Table 1). While all three clones induced killing of all four OC targets (Figure 17C-G), clone DB7.2 showed the highest potency in these assays. DB7.2 further showed enhanced killing induction against two OC lines, CaOV3 (Figure 17H) and OVCAR3 (Figure 17I) cells. To confirm the Siglec-7 MAb activity independent of Fc receptor binding (Figure 18A), we found that the potency of DB7.2 was maintained in the presence of Fc blockade (Figures 18B and C). Next, we performed residue modifications in the Fc of the antibody to eliminate Fc binding and potential off-target effects. We designed a variant of DB7.2 containing multiple residue modifications ("TM"; L234F / L235E / P331S) in the Fc domain to abolish FcR and complement (C1q) binding (Oganesyan et al., 2008, Acta Crystallogr D Biol Crystallogr 64, 700-704; Parzych et al., 2022, Nat Commun 13, 5886), and DB7.2 I named it TM Mod. DB7.2 The killing efficacy of TM Mod was maintained in OVISE cells (Figure 19A). Addition of TM Mod after 24 hours potently killed target cells, but not control (Figure 19B) cell targets. TM Mod showed comparable IC50 values ​​of 82.67 nM and 192 nM, respectively, in OVISE cells (Figures 19C and D). These studies demonstrate the specificity of Siglec-7 MAbs for immune killing activation and their ability to target OC cells.

[0315] Combination of anti-PD-1 antibody and DB7.2 MAbs shows enhanced OC killing in xCELLigence assay OC is one of the subsets of cancers that responds more slowly to existing immune checkpoint inhibitor therapies, with an objective response rate (ORR) of 8–9%. OC patients treated with checkpoint inhibitors (CPIs) report less frequent durable responses, and no CPIs have been FDA-approved for the management of OC to date (Farkkila et al., 2020, Nat Commun 11, 2543). However, evidence indicates that combinatorial approaches are important to optimize EOC patients' response to immunotherapy. Without being bound by theory, it was hypothesized that combining T cell-activating PD-1 with NK cell activation by Siglec-7 MAb might synergistically target tumors more efficiently. To test this hypothesis, the simultaneous targeting of PD-1 on T cells and engagement in the NK immune pathway was examined in an xCELLigence assay with the antibody combination DB7.2 and anti-PD1 (Pembrolizumab) targeted to OC tumors. DB7.2 showed simulated killing ability against both BRCA2 mutant and PARPi resistant PEO4 cells with EC50 68.87 nM (Figure 19E and G), whereas anti-PD1 targeted PEO4 cells with 10-fold lower EC50 680 nM (Figure 19F and H) in an xCELLigence-based in vitro killing assay. Combination of anti-PD-1 and DB7.2 showed further enhanced killing of PEO4 cells in the presence of human PBMC (Figure 19I). These data indicate that the combination of Siglec-7 MAb and anti-PD1 (NK and T cell CPI) enhances anti-tumor immunity.

[0316] In vivo expression of Siglec-7 MAbs suppresses tumor progression in an ovarian cancer challenge model Next, the in vivo expression of the Siglec-7 MAb clones was investigated using a DNA delivery in vivo approach. For that purpose, mice were injected with DNA expression cassettes of the MAbs at a dose of 50 μg + 50 μg (HC + LC) in a two-plasmid system for each anti-Siglec-7 clone (DB7.1, DB7.2 and DB7.7) together with an empty vector control (pVax1) that allows biological antibody production of the mAbs in vivo (Patel et al., 2018, Cell Rep 25, 1982-1993 e1984). Injections were performed in the tibialis anterior muscle of mice on day 0 as previously described (Parzych et al., 2022, Nat Commun 13, 5886; Patel et al., 2018, Cell Rep 25, 1982-1993 e1984) (Figure 20A). The presence of human IgG was observed in the serum of mice injected with DB7.1, DB7.2, and DB7.7 Siglec-7 MAbs', with no expression detected in the serum of control animals or pre-bleed mice. To confirm the specificity of the in vivo generated antibodies, human NK cells were stained with day 14 sera from mice inoculated with DB7.1, DB7.2, and DB7.7, as well as serum from empty vector control animals. Day 14 sera from all three groups positively stained human NK cells, whereas no staining was observed with pVax1 or irrelevant antibody control (Figure 20B). To examine the potential antitumor efficacy of Siglec-7 MAb delivery in animals, an NSG-K (NOD / SCID-γ-MHC I / II double knockout mutant) mouse tumor challenge model was used to inject OVISE human ovarian cancer cells (0.8x10 6 Each patient was injected with 100 μg / mL of 10 ... 3When tumors reached an average size of >100 μg, 50 μg + 50 μg (HC + LC) of DB7.2 or pVax1 vector control were delivered intramuscularly as described (Parzych et al., 2022, Nat Commun 13, 5886; Patel et al., 2018, Cell Rep 25, 1982-1993 e1984) along with 10 million human PBMCs per mouse (Figure 20C). Mice administered DB7.2 Siglec-7 MAb showed a significant reduction in tumor burden / tumor growth delay compared to vector control (Figure 20D, Figure 21). Of note, Siglec-7 MAb significantly extended the median survival of tumor-bearing mice (Figure 20E). These data are the first in vivo data demonstrating the potential of Siglec-7 antibodies in the direct treatment of human ovarian cancer.

[0317] Generation and expression of bispecific NK cell engager encoded by DB7.2xD2AP11 DNA NK engagers are an important new focus for targeting tumor cells (Gleason et al., et al., 2014, Blood 123, 3016-3026; Ibarlucea-Benitez et al., 2021, Proc Natl Acad Sci USA 118; Demaria et al., 2021, Eur J Immunol 51, 1934-1942). Based on the potency of anti-Siglec-7 MAbs to enhance NK activity against OC as described above, the potential of this target was explored as a new targeted NKCE approach. Without being bound by theory, it was hypothesized that this approach would not only allow for specific targeting of NK cells but would also prevent NK cells from receiving negative TME signals, resulting in novel NKCE. Therefore, to enhance tumor targeting using NK cell-specific Siglec-7 binding, we constructed an NKCE in the form of an anti-Siglec-7 FV linked to two linked antibody binding fragments (scFVs) arranged to bind the OC antigen FSHR as described recently (Bordoloi et al., 2022, JCI Insight 7). An NKCE fusion sequence encoding the scFV of the DB7.2 anti-Siglec-7 antibody and the scFV of anti-FSHR (clone D2AP11) (Bordoloi et al., 2022, JCI Insight 7) (Figure 22A) was designed incorporating a GS (glycine-serine) flexible linker (PPerales-Puchalt et al., 2019, JCI Insight 4; Pratik et al., 2022, Molecular therapy Oncolytics). In vitro analysis demonstrated that DB7.2xD2AP11 NKCE was efficiently expressed, as transfection into Expi293F cells produced the expected ∼55 KDa molecule (Figure 22B), which bound to both cellular targets upon flow staining in HEK293T cells (Figure 22C) or K562 cells (Figure 22D) overexpressing Siglec-7 or FSHR, respectively.To visualize binding to human Siglec-7, we performed high-resolution confocal imaging of 293T-Siglec-7 fixed cells. Cells were labeled with DAPI (nuclei), GFP (Siglec-7), and Texas Red (DB7.2xD2AP11). DB7.2xD2AP11 NKCE clearly bound to GFP+ cells expressing human Siglec-7, but not to the secondary antibody control (Figure 22E).

[0318] A novel NK cell engager targeting FSHR induces potent killing in multiple ovarian tumor lines and reduces tumor burden in vivo Next, DB7.2xD2AP11 NK cell engagers (NKCEs) were evaluated for induction of tumor killing in vitro using the xCelligence RTCA assay format. Effector cells included direct NK cells as well as PBMCs tested. NKCEs were added the day after plating target OC cells. As controls, four FSHR-negative cell lines were studied, including HEK293T cells (Urbanska et al., 2015, Cancer Immunol Res 3, 1130-1137), AGS human gastric cancer cells, GM05389 human fibroblasts, and WM3743 human melanoma cells. No off-target killing was observed against these FSHR-negative cells, including unrelated cancer cells (Figure 22F-K). As shown in Figures 22G and J, HEK293T and AGS cells remained healthy and adherent after treatment with DB7.2xD2AP11 NKCE for 3 days in the presence of human PMBCs. In contrast, multiple FSHR-expressing ovarian tumor lines OVISE, OVCAR3-FSHR, CaOV3, Kuramochi-FSHR and PEO-4 (Figures 23A-G) were efficiently killed by human PBMCs in the presence of DB7.2xD2AP11 NKCE. As an additional specificity control, purified human NK cells were used directly as effector cells. DB7.2xD2AP11 NKCE co-cultured with human NK cells also effectively killed OVCAR3-FSHR cells (Figure 24A). Anti-Siglec-7 antibodies killed AGS cells (Figure 24B), but DB7.2xD2AP11 NKCE did not (Figure 22I). As an additional specificity control, NKCE not targeting FSHR (IL13Rα2-NKCE) was developed and observed to show no toxicity against FSHR-overexpressing OVCAR3 or OVISE cells (Figure 24C&D). DB7.2xD2AP11 NKCE induced concentration-dependent cell lysis in both OVISE-FSHR (separate experiments with PBMCs from three different human donors) and OVCAR3 cells, with EC50 values ​​of 142.87 pM and 236.6 pM, respectively, supporting the significant potency of this engagement tool to target pathogenic cells as a novel NKCE (Figure 23H&I; Figure 25 and Figure 26).The secretion profile of cytokines / cytotoxic molecules of OC-NKCE was examined. Co-culture of DB7.2xD2AP11 NKCE with OVCAR3-FSHR cells and human PBMC (Figure 27A) resulted in enhanced production of soluble Fas (sFas) and granzyme A as part of specific immune activation, and IL-10 levels were observed to be decreased compared to no Ab control or empty vector control groups (Figure 27B), supporting more effector-based activation. To directly evaluate the in vivo antitumor effect of DB7.2xD2AP11 NKCE, NSGK mice were challenged with OVCAR3-FSHR cells. NSG-K mice were administered 3 million OVCAR3-FSHR cells and were allowed to express in vivo nucleic acid-encoded DB7.2xD2AP11 (100 μg) or empty vector; pVax1 (100 μg) twice at 2-week intervals 7 days after tumor implantation. We also inoculated 10 million human PBMCs on day 7 and measured tumor volume periodically (Figure 27C). Treatment with NKCE significantly reduced tumor burden in mice bearing OVCAR3-FSHR tumors, whereas no such effect was observed in the control treatment group (Figure 27D). Furthermore, DB7.2xD2AP11 NKCE therapy improved survival (Figure 27E), supporting further study of this approach.

[0319] Example 4: Sequences of Siglec7 antibody clones (underlined CDR sequences) DB-S7-1 heavy chain SEQ ID NO:4 QVQLVESGGGVVQPGRSLRLSCAAS GFTFRTDA MHWVRQAPGKGLEWVAV IWYDGSIK YHADSVKGRFTISRDNSKNTLYLEMNSLRAEDTAVYYC ARDRSNSLDH WGQGILVSVSS DB-S7-1 heavy chain SEQ ID NO:8 caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagcgtct ggattcaccttcaggaccgatgcc atgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagtt atttggtatgatggaagtattaaa taccatgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctggaaatgaatagcctgagagccgaggacacggctgtatattattgt gcgagagatcggtctaactctttggaccac tggggccagggaatcctggtcagcgtctcctcag DB-S7-1 light chain sequence number 12 EIVMTQSPATLSVSPGERATLSCRAS QSVSSN LAWYQQKPGQAPRLLIY GAS TRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYC QQYNNWPRT FGQGTKVEIK DB-S7-1 light chain sequence number 16 gaaatagtgatgacgcagtctccagccaccctgtctgtgtctccagggggaaagagccaccctctcctgcagggccagt cagagtgttagcagcaac ttagcctggtaccagcagaaacctggccaggctcccaggctcctcatctat ggtgcatcc accagggccactggtatcccagccaggttcagtggcagtgggtctgggacagagttcactctcaccatcagcagcctgcagtctgaagattttgcagtttattactgt cagcagtataataactggcctcggacg ttcggccaagggaccaaggtggaaatcaaac DB-S7-2 heavy chain sequence number 20 QVQLVESGGGVVQPGRSLRLSCAAS GFTFRTDA MHWVRQAPGKGLEWVAV IWYDGSIK YHADSVKGRFTISRDNSKNTLYLEMNSLRAEDTAVYC ARDRSNSLDH WGQGILVSVSS DB-S7-2 heavy chain sequence number 24 caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagcgtct ggattcaccttcaggaccgatgcc atgcactgggtccgccaggctccaggcaaggggctggagatgggtggcagtt atttggtatgatggaagtattaaa taccatgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctggaaatgaatagcctgagagccgaggacacggctgtatattattgt gcgagagatcggtctaactctttggaccac tggggccagggaatcctggtcagcgtctcctcag DB-S7-2 light chain sequence no. 28 DIVMTQSPLSLPVTPGEPASFSCRSS QSLLHSN GYNYLDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLKISRVEAADVGVYYC MQALQTPRT FGQGTKVEIK DB-S7-2 light chain sequence number 32 gatatgtgatgactcagtctccactctccctgcccgtcacccctggagagccggcctccttctcctgcaggtctagt cagagcctcctgcatagtaat ggatacaactatttggattggtacctgcagaagccagggcagtctccacagctcctgatctat ttggggttct aatcgggcctccggggtccctgacaggttcagtggcagtggatcaggcacagatttacactgaaaatcagcagagtggaggctgcggatgttggggtttattactgc atgcaagctctacaaactcctcggacg ttcggccaagggaccaaggtggaaatcaaac DB-S7-3 heavy chain sequence no. 36 EVQLVESGGDLVQPGGSLRLSCAAS GFTFNSYW MSWVRQAPGKGLEWVAN IKHDGSEK YYVDSVKGRFTISRDSAKNSLYLQMNSLRAEDTAVYFC AREAVASSYFAY WGQGTLVTVSS DB-S7-3 heavy chain Sequence number 40 gaggtgcagctggtggagtctgggggagacttggtccagcctggggggtccctgagactctcctgtgcagcctct ggattcacctttaatagctattgg atgagttgggtccgccaggctccagggaagggtctggagtgggtggccaac ataaaacatgatggaagtgaaaaa tactatgtggactctgtgaagggccgattcaccatctccagagacagcgccaagaactcactgtatctgcaaatgaacagcctgagagccgaagacacggctgtgtatttctgt gcgagagaggcagtggctagttcttactttgcctac tggggccagggaaccctggtcaccgtctcctcag DB-S7-3 light chain Sequence number 44 EIVMTQSPATLSVSPGERATLSCRAS QSVSSN LAWYQQKPGQAPRLLIY GAS TRAAGIPARFSGSGSGTEFTLTISSLQSEDFAVYYC QQYNNWPYT FGQGTKLEIK DB-S7-3 light chain Sequence number 48 gaaatagtgatgacgcagtctccagccaccctgtctgtgtctccaggggaaagagccaccctctcctgcagggccagt cagagtgttagcagcaac ttagcctggtaccagcagaaacctggccaggctcccaggctcctcatctat ggtgcatcc accagggccgctggtatcccagccaggttcagtggcagtgggtctgggacagagttcactctcaccatcagcagcctgcagtctgaagattttgcagtttattactgt cagcagtataataactggccgtacact tttggccaggggaccaagctggagatcaaac DB-S7-6 heavy chain SEQ ID NO:52 QVQLVESGGGVVQPGRSLRLSCAAS GFPFSSYG MHWVRQAPGKGLEWVTF IWFDGNNK EYADSVKGRFIVSRDNSKNTLYLQMNSLRADDDTAVYYC ARNGVYYGSGAYSDS WGQGTLVTISS DB-S7-6 heavy chain SEQ ID NO:56 CAGGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGCAGCCAGGCAGGAGCCTGAGACTGTCCTGCGCCGCCTCT GGCTCCCCTTTAGCTCCTACGGA ATGCACTGGGTGAGGCAGGCACCTGGCAAGGGACTGGAGTGGGTGACCTTC ATCTGGTTTGACGGCAACAATAAG GAGTACGCCGATTCTGTGAAGGGCCGGTTCATCGTGAGCCGCGACAACTCCAAGAATACCCTGTATCTGCAGATGAACTCTCTGAGGCCGACGATACAGCCGTGTACTATTGT GCCGGAATGGCGTGTACTATGGCAGCGGCGCCTATTCCGATTCT TGGGGCCAGGGCACCCTGGTGACAATCTCTAGC DB-S7-6 light chain SEQ ID NO:60 DIVMTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLKISRVAAEDVGVYYC MQALQTPYT FGQGTKLEIK DB-S7-6 light chain SEQ ID NO:64 gatattgtgatgactcagtctccactctccctgcccgtcacccctggagagccggcctccatctcctgcaggtctagt cagagcctcctgcatagtaat ggatacaactatttggattggtacctgcagaagccagggcagtctccacagctcctgatctat ttggggttctaatcgggcctccggggtccctgacaggttcagtggcagtggatcaggcacagatttacactgaaaatcagcagagtggcggctgaggatgttggggtttattactgc atgcaagctctacaaactccgtacact tttggccagggggaccaagctggagatcaaac DB-S7-7 heavy chain sequence number 68 QVQLVESGGGVVQPGRSLRLSCAAS GFTFRTDA MHWVRQAPGKGLEWVAV IWYDGSIK YHADSVKGRFTISRDNSKNTLYLEMNSLRAEDTAVYC ARDRSNSLDH WGQGILVSVSS DB-S7-7 heavy chain sequence no. 72 caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagcgtct ggattcaccttcaggaccgatgcc atgcactgggtccgccaggctccaggcaaggggctggagatgggtggcagtt atttggtatgatggaagtattaaa taccatgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctggaaatgaatagcctgagagccgaggacacggctgtatattattgt gcgagagatcggtctaactctttggaccac tggggccagggaatcctggtcagcgtctcctcag DB-S7-7 light chain sequence no. 76 EIVMTQSPATLSVSPGERATLSCRAS QSVSSN LAWYQQKPGQAPRLLIY GAS TRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYC QQYNNWPYT FGQGTRLEIK DB-S7-7 light chain sequence no.80 gaaatagtgatgacgcagtctccagccaccctgtctgtgtctccagggggaaagagccaccctctcctgcagggccagt cagagtgttagcagcaac ttagcctggtaccagcagaaacctggccaggctcccaggctcctcatctat ggtgcatcc accagggccactggtatcccagccaggttcagtggcagtgggtctgggacagagttcactctcaccatcagcagcctgcagtctgaagattttgcagtttattactgt cagcagtataataactggccgtacact tttggccagggggaccaggctggagatcaaac DB-S7-8 heavy chain sequence number 84 QVQLVESGGGVVQPGRSLRLSCAAS GFPFSSYG MHWVRQAPGKGLEWVTF IWFDGNNK DYADSVKGRFSVSRDNSKNTLYLQMNSLRADDTAVYYC ARNGVYYGSGAYVDY WGQGTLVTVSS DB-S7-8 heavy chain sequence number88 gaggtgcaactggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagcgtct ggattcccattcagcagctatggc atgcactgggtccgccaggctccaggcaaggggctggagtgggtgacattt atatggtttgatggaaataataaa gactatgcagactccgtgaagggccgattcagcgtctccagagacaattccaagaacacgctgtatctgcaaatgaacagtctgagagccgatgacacggctgtttattactgt gcgagaaatggggtttactatggttcgggggcttatgttgactac tggggccagggaaccctggtcaccgtctcctcag DB-S7-8 light chain sequence number 92 DIQMTQSPSTLSASVGDRVTITCRAS QSISYF LAWYQQKPGKAPKLLIY KASSLESGVPSRFSGSGSGTEFTLTISTLQPDDFATYYC QQYNSYSMYT FGQGTKLEIK DB-S7-8 light chain SEQ ID NO:96 gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgccgggccagt cagagtattagttacttt ttggcctggtatcagcagaaaccagggaaagcccctaagctcctgatctat aaggcgtct agtttagaaagtggggtcccatcaaggttcagcggcagtggatctgggacagaattcactctcaccatcagcaccctgcagcctgatgattttgcaacttattactgc caacaatataatagttattccatgtacact tttggccaggggaccaagctggagatcaaac

[0320] [Table 2]

[0321] The disclosures of each patent, patent application, and publication cited herein are incorporated herein by reference in their entirety. Although the present invention has been disclosed with reference to specific embodiments, it will be apparent to those skilled in the art that other embodiments and modifications of the present invention may be devised 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-7 binding molecule comprising a Siglec-7 antibody, a Siglec-7 binding antibody fragment or a variant of a Siglec-7 antibody.

2. The antibody is: a) a variable heavy chain sequence comprising CDR sequences selected from the group comprising: SEQ ID NOs: 1-3; SEQ ID NOs: 17-19; SEQ ID NOs: 33-35; SEQ ID NOs: 49-51; SEQ ID NOs: 65-67; ​​and SEQ ID NOs: 81-83; b) a variable light chain sequence comprising CDR sequences selected from the group comprising SEQ ID NOs: 9-11; SEQ ID NOs: 25-27; SEQ ID NOs: 41-43; SEQ ID NOs: 57-59; SEQ ID NOs: 73-75; and SEQ ID NOs: 89-91; c) 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, and SEQ ID NO:84; d) 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, and SEQ ID NO:92; e) a fragment comprising at least 80% of the full length sequence of a variable heavy chain sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, and SEQ ID NO:84; and f) A fragment comprising at least 80% of the full length sequence of a variable light chain sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 44, SEQ ID NO: 60, SEQ ID NO: 76, and SEQ ID NO:

92. The Siglec-7 binding molecule of claim 1, comprising an amino acid sequence selected from the group comprising:

3. The Siglec-7 binding molecule comprises: a) a variable heavy chain sequence comprising the CDR sequences of SEQ ID NOs: 1-3, and a variable light chain sequence comprising the CDR sequences of SEQ ID NOs: 9-11; b) a variable heavy chain sequence comprising the CDR sequences of SEQ ID NOs: 17-19, and a variable light chain sequence comprising the CDR sequences of SEQ ID NOs: 25-27; c) a variable heavy chain sequence comprising the CDR sequences of SEQ ID NOs: 33-35, and a variable light chain sequence comprising the CDR sequences of SEQ ID NOs: 41-43; d) a variable heavy chain sequence comprising the CDR sequences of SEQ ID NOs: 49-51, and a variable light chain sequence comprising the CDR sequences of SEQ ID NOs: 57-59; e) a variable heavy chain sequence comprising the CDR sequences of SEQ ID NOs: 65-67, and a variable light chain sequence comprising the CDR sequences of SEQ ID NOs: 73-75; and f) a variable heavy chain sequence comprising the CDR sequences of SEQ ID NOs: 81-83, and a variable light chain sequence comprising the CDR sequences of SEQ ID NOs: 89-91; The Siglec-7 binding molecule or fragment thereof of claim 1, comprising an amino acid sequence selected from the group comprising:

4. The Siglec-7 binding molecule comprises: a) a variable heavy chain sequence of SEQ ID NO: 4 and a variable light chain sequence of SEQ ID NO: 12; b) the variable heavy chain sequence of SEQ ID NO:20 and the variable light chain sequence of SEQ ID NO:28; c) the variable heavy chain sequence of SEQ ID NO: 36 and the variable light chain sequence of SEQ ID NO: 44; d) the variable heavy chain sequence of SEQ ID NO:52 and the variable light chain sequence of SEQ ID NO:60; e) the variable heavy chain sequence of SEQ ID NO:68 and the variable light chain sequence of SEQ ID NO:76; and f) the variable heavy chain sequence of SEQ ID NO: 84 and the variable light chain sequence of SEQ ID NO: 92 The Siglec-7 binding molecule or fragment thereof of claim 1, comprising an amino acid sequence selected from the group comprising:

5. The Siglec-7 binding molecule or a fragment thereof of claim 1, wherein the Siglec-7 binding molecule comprises a fusion protein comprising a Siglec-7 antibody, a Siglec-7 binding antibody fragment, or a variant of a Siglec-7 antibody.

6. A nucleic acid molecule, or a combination of nucleic acid molecules, comprising one or more nucleotide sequences encoding a Siglec-7 binding molecule according to any one of claims 1 to 5.

7. The nucleic acid molecule(s) comprises: a) a nucleotide sequence encoding a variable heavy chain sequence, said nucleotide sequence comprising sequences encoding CDRs selected from the group comprising 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; and SEQ ID NOs: 85-87; b) a nucleotide sequence encoding a variable light chain sequence, said nucleotide sequence comprising sequences encoding CDRs selected from the group comprising 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; and SEQ ID NOs: 93-95; c) a nucleotide sequence having at least 95% identity to 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; and SEQ ID NO:88; d) a nucleotide sequence having at least 95% identity to a variable light chain coding sequence selected from the group consisting of SEQ ID NO:16; SEQ ID NO:32; SEQ ID NO:48; SEQ ID NO:64; SEQ ID NO:80; and SEQ ID NO:96; 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 NO:8; SEQ ID NO:24; SEQ ID NO:40; SEQ ID NO:56; SEQ ID NO:72; and SEQ ID NO:88; and f) A fragment comprising at least 80% of the full length sequence of a variable light chain coding sequence selected from the group consisting of SEQ ID NO: 16; SEQ ID NO: 32; SEQ ID NO: 48; SEQ ID NO: 64; SEQ ID NO: 80; and SEQ ID NO:

96. The nucleic acid molecule(s) according to claim 6, comprising:

8. The nucleic acid molecule(s) comprises: a first nucleotide sequence comprising SEQ ID NOs:5-7 encoding a variable heavy chain sequence, and a second nucleotide sequence comprising SEQ ID NOs:13-15 encoding a variable light chain sequence; a first nucleotide sequence comprising SEQ ID NOs:21-23 encoding a variable heavy chain sequence, and a second nucleotide sequence comprising SEQ ID NOs:29-31 encoding a variable light chain sequence; a first nucleotide sequence comprising SEQ ID NOs: 37-39 encoding a variable heavy chain sequence, and a second nucleotide sequence comprising SEQ ID NOs: 45-47 encoding a variable light chain sequence; a first nucleotide sequence comprising SEQ ID NOs:53-55 encoding a variable heavy chain sequence, and a second nucleotide sequence comprising SEQ ID NOs:61-63 encoding a variable light chain sequence; a first nucleotide sequence comprising SEQ ID NOs:69-71 encoding a variable heavy chain sequence, and a second nucleotide sequence comprising SEQ ID NOs:77-79 encoding a variable light chain sequence; and A first nucleotide sequence comprising SEQ ID NOs: 85-87 encoding a variable heavy chain sequence, and a second nucleotide sequence comprising SEQ ID NOs: 93-95 encoding a variable light chain sequence. The nucleic acid molecule(s) of claim 7, comprising a nucleotide sequence selected from the group comprising:

9. The nucleic acid molecule(s) comprises: a first nucleotide sequence comprising SEQ ID NO:8 encoding a variable heavy chain sequence, and a second nucleotide sequence comprising SEQ ID NO:16 encoding a variable light chain sequence; a first nucleotide sequence comprising SEQ ID NO:24 encoding a variable heavy chain sequence, and a second nucleotide sequence comprising SEQ ID NO:32 encoding a variable light chain sequence; a first nucleotide sequence comprising SEQ ID NO:40 encoding a variable heavy chain sequence, and a second nucleotide sequence comprising SEQ ID NO:48 encoding a variable light chain sequence; a first nucleotide sequence comprising SEQ ID NO:56 encoding a variable heavy chain sequence, and a second nucleotide sequence comprising SEQ ID NO:64 encoding a variable light chain sequence; a first nucleotide sequence comprising SEQ ID NO:72 encoding a variable heavy chain sequence, and a second nucleotide sequence comprising SEQ ID NO:80 encoding a variable light chain sequence; and A first nucleotide sequence comprising SEQ ID NO:88 encoding a variable heavy chain sequence, and a second nucleotide sequence comprising SEQ ID NO:96 encoding a variable light chain sequence. The nucleic acid molecule(s) of claim 7, comprising a nucleotide sequence selected from the group comprising:

10. A composition comprising the Siglec-7 binding molecule of any one of claims 1 to 5.

11. The composition of claim 10, further comprising at least one selected from the group consisting of pharma- ceutically acceptable excipients and adjuvants.

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

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

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

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

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

17. A composition comprising one or more nucleic acid molecule(s) encoding the Siglec-7 binding molecule of any one of claims 6 to 9.

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

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

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

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

22. 20. The composition of claim 17, comprising a delivery vehicle comprising the nucleic acid molecule(s).

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

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

25. A method for treating or preventing a disease or disorder in a subject in need thereof, comprising administering to the subject a Siglec-7 binding molecule according to any one of claims 1 to 5, one or more nucleic acid molecules encoding a Siglec-7 binding molecule according to any one of claims 6 to 9, or a composition according to any one of claims 10 to 24.

26. 26. The method of claim 25, wherein the disease or disorder is cancer or a cancer-related disease or disorder.

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

28. 28. The method of claim 27, wherein the cancer is selected from the group including 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.

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

30. A method of increasing natural killer cell function in a subject in need thereof, comprising administering to the subject a Siglec-7 binding molecule according to any one of claims 1 to 5, one or more nucleic acid molecules encoding a Siglec-7 binding molecule according to any one of claims 6 to 9, or a composition according to any one of claims 10 to 24.

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