Antibodies to human sigle-7 and their use for immunotherapy

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 325,904 filed on March 31, 2022, U.S. Provisional Application No. 63 / 375,749 filed on September 15, 2022, and U.S. Provisional Application No. 63 / 490,156 filed on March 14, 2023, each of which is hereby incorporated by reference in its entirety. Background Art

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

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

[0005] Siglec 7 is primarily localized on human natural killer (NK) cells and monocytes. Siglec 7 regulates inhibitory signals on 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 participate in a special type of cell killing called antibody-dependent cell-mediated cytotoxicity (ADCC). Unlike T cells, which must be trained by antigen-presenting cells before they can recognize tumors, NK cells spontaneously lyse tumor cells without the need for antigen stimulation or pre-activation.

[0006] Virus-infected cells and tumor cells downregulate their expression of MHC-I. NK cell-mediated cytotoxicity is not restricted by MHC I molecules, but rather NK cells destroy cancer cells through perforin / granzyme-mediated lysis.

[0007] Siglec-7 has a preferential binding specificity for the Neu5Acα2,8Neu5Acα-(diSia) sequence, although certain branched α2,6-sialic acid residues (DSGb5, DSLc4) are also known to be preferential binding molecules.

[0008] There remains a need in the art for new cancer treatment methods that effectively treat cancer while minimizing negative side effects. The present invention meets this unmet need. Summary of the invention

[0009] In one embodiment, the invention relates to an antibody or fragment thereof that specifically binds to a sialic acid binding receptor. In one embodiment, the sialic acid binding receptor is Siglec-7.

[0010] In one embodiment, the antibody comprises a variable heavy chain sequence comprising the following CDR sequences: 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.

[0011] In one embodiment, the antibody comprises a variable heavy chain sequence comprising the following CDR sequences: 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.

[0012] 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.

[0013] 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.

[0014] In one embodiment, the antibody comprises a variable heavy chain sequence including a CDR sequence of SEQ ID NO: 1-3 and a variable light chain sequence including a CDR sequence of SEQ ID NO: 9-11. In one embodiment, the antibody comprises a variable heavy chain sequence including a CDR sequence of SEQ ID NO: 17-19 and a variable light chain sequence including a CDR sequence of SEQ ID NO: 25-27. In one embodiment, the antibody comprises a variable heavy chain sequence including a CDR sequence of SEQ ID NO: 33-35 and a variable light chain sequence including a CDR sequence of SEQ ID NO: 41-43. In one embodiment, the antibody comprises a variable heavy chain sequence including a CDR sequence of SEQ ID NO: 49-51 and a variable light chain sequence including a CDR sequence of SEQ ID NO: 57-59. In one embodiment, the antibody comprises a variable heavy chain sequence including a CDR sequence of SEQ ID NO: 65-67 and a variable light chain sequence including a CDR sequence of SEQ ID NO: 73-75. In one embodiment, the antibody comprises 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.

[0015] 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.

[0016] In one embodiment, the antibody comprises a sequence that is at least 95% identical to one or more of the variable heavy chain sequences of SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 36, SEQ ID NO: 52, SEQ ID NO: 68, or SEQ ID NO: 84. In one embodiment, the antibody comprises a sequence that is at least 95% identical to one or more of the variable light chain sequences of SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 44, SEQ ID NO: 60, SEQ ID NO: 76, or SEQ ID NO: 92. In one embodiment, the antibody comprises a fragment that includes 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 that comprises 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.

[0017] In one embodiment, the present invention relates to a nucleic acid molecule encoding an antibody or fragment thereof that specifically binds to a sialic acid binding receptor. In one embodiment, the sialic acid binding receptor is Siglec-7.

[0018] 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: a variable light chain sequence of SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 44, SEQ ID NO: 60, SEQ ID NO: 76, or SEQ ID NO: 92; a sequence having at least 95% identity to 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; a sequence having at least 95% identity to a variable light chain sequence of SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 44, SEQ ID NO: 60, SEQ ID NO: 76, 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 SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 44, SEQ ID NO: 60, SEQ ID NO: 76, or SEQ ID NO: 84. NO:28, SEQ ID NO:44, SEQ ID NO:60, SEQ ID NO:76, or SEQ ID NO:92, or a combination thereof.

[0019] 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.

[0020] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a 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 a 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; a nucleotide sequence having at least 95% identity to a 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 sequence having at least 95% identity to a 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; a sequence comprising 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; NO:56; SEQ ID NO:72; or 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.

[0021] In one embodiment, the present invention provides a combination of nucleic acid molecules, wherein the first 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, 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 first nucleic acid molecule and a second nucleic acid molecule, wherein the first nucleic acid molecule comprises a heavy chain coding sequence or a fragment or variant thereof of SEQ ID NO:8; SEQ ID NO:24; SEQ ID NO:40; SEQ ID NO:56; SEQ ID NO:72; or SEQ ID NO:88, and the second nucleic acid molecule comprises a light chain coding sequence or a fragment or variant thereof of SEQ ID NO:16; SEQ ID NO:32; SEQ ID NO:48; SEQ ID NO:64; SEQ ID NO:80; or SEQ ID NO:96.

[0022] In one embodiment, the present invention relates to a composition comprising a sialic acid binding receptor antibody. In one embodiment, the composition further comprises a pharmaceutically acceptable excipient. In one embodiment, the composition further comprises an adjuvant. In some embodiments, the composition further comprises a tumor antigen or a nucleotide sequence encoding a tumor antigen.

[0023] In one embodiment, the present invention relates to a composition comprising a nucleic acid molecule encoding a sialic acid binding receptor antibody. In one embodiment, the nucleic acid molecule comprises an expression vector. In one embodiment, the nucleic acid molecule is incorporated into a viral particle. In one embodiment, the composition further comprises a pharmaceutically acceptable excipient. In one embodiment, the composition further comprises an adjuvant. In some embodiments, the composition further comprises a nucleotide sequence encoding a tumor antigen.

[0024] In one embodiment, the present invention relates to a method for treating or preventing a disease or condition in a subject in need thereof, the method comprising administering a sialic acid binding receptor antibody, a nucleic acid molecule encoding a sialic acid binding receptor antibody, or a composition comprising a sialic acid binding receptor antibody or a nucleic acid molecule encoding a sialic acid binding receptor antibody. In one embodiment, the disease or condition is cancer, or a disease or condition associated with cancer. In one embodiment, the disease or condition is an infectious disease or condition.

[0025] 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.

[0026] In one embodiment, the invention relates to an immunogenic composition comprising an antibody or fragment thereof that specifically binds to a sialic acid binding receptor and a nucleic acid molecule encoding a tumor antigen.

[0027] In one embodiment, the disease or condition is cancer, or a disease or condition associated with cancer. In one embodiment, the cancer has increased sialic acid levels. In one embodiment, the cancer is ovarian cancer, melanoma, renal cell carcinoma, prostate cancer, colon cancer, breast cancer, head and neck squamous cell carcinoma, 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 is resistant to one or more of the drugs listed in Table 1.

[0028] In one embodiment, the invention relates to a method of increasing natural killer cell function in a subject in need thereof, the method comprising administering an immunogenic composition comprising an antibody or fragment thereof that specifically binds to a sialic acid binding receptor, and a nucleic acid molecule encoding a tumor antigen.

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

[0030] Figure 1 Binding of anti-Siglec-7 clones to human Siglec 7 is shown.

[0031] Figure 2 Shown is the expression of Siglec-7 by transduction.

[0032] Figure 3 Binding of anti-human Siglec 7 antibody is shown.

[0033] Figure 4 Depicted are exemplary experimental results demonstrating OVISE cell cytotoxicity by Siglec-7 antibody clones.

[0034] Figure 5 Depicted are exemplary experimental results demonstrating SKOV3 cell cytotoxicity by Siglec-7 antibody clones.

[0035] Figure 6 Depicted are exemplary experimental results demonstrating OVCAR10 cell cytotoxicity by Siglec-7 antibody clones.

[0036] Figure 7 Representative human melanoma expression of siglec-7 ligands is depicted. There is high expression of siglec-7 ligands on melanoma cells.

[0037] Figure 8 The cytotoxicity of Siglic-7 antibody against 451Lu BR human melanoma cell lines is depicted.

[0038] Fig. 9 The cytotoxicity of the Siglic-7 antibody against the WM1366 human melanoma cell line is depicted.

[0039] Fig.10 The 1205Lu human melanoma cell line cytotoxicity of the Siglic-7 antibody is depicted.

[0040] Fig.11 The cytotoxicity of the Siglic-7 antibody against the WM3311 human melanoma cell line is depicted.

[0041] Fig.12 The cytotoxicity of the Siglic-7 antibody against the WM983B BR human melanoma cell line is depicted.

[0042] Figures 13A to 13C depict the design and in vitro expression of Siglec-7 MAbs. (Figure 13A) Schematic diagram of a dual plasmid DNA construct encoding Siglec-7 MAbs. (Figure 13B) Expression analysis of Siglec-7 MAbs by Western blot. Numbers indicate molecular weight (kDa). (Figure 13C) Quantification of human IgG in the supernatant of anti-Siglec-7 transfected Expi293F by ELISA.

[0043] Figures 14A to 14E depict the binding properties of human Siglec-7 MAbs in vitro. (Figure 14A) Binding of Siglec-7 MAbs to recombinant human Siglec-7 analyzed by ELISA. Dose-dependent binding of DB7.1, DB7.2, and DB7.7 to human Siglec-7 was observed, however, there was no binding in the case of irrelevant controls. (Figure 14B) Binding of human Siglec7 MAbs to HEK293T cells expressing Siglec-7 analyzed by flow cytometry. No binding was observed in the case of secondary antibody controls or irrelevant MAbs only. Binding was present in 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 in DB7.2. (Figure 14C) Representative flow cytometry showing the binding of DB7.2 anti-Siglec-7 antibodies to human NK and T cells. DB7.2 binds primarily to NK cells. Positive (commercial fluorescent dye-conjugated mouse anti-human Siglec-7 antibody) and negative controls (0 μg / ml DB7.2 or no secondary antibody) are shown (Figure 14D). Summary of DB7.2 anti-Siglec-7 binding to different immune populations in PBMCs from multiple donors (n=4). (Figure 14E) Binding analysis of DB7.2 anti-Siglec-7 Ab to CD56dim and CD56bright NK cell subsets (n=4).

[0044] Fig.15 Describe the gating strategy for the flow cytometry analysis of Siglec-7 combination.Show representative examples of ND.First, select the most stable acquisition.Then, in order to ensure that only live single cells are analyzed from monocytes, forward scatter height (FSC-H)-with-forward scatter area (FSC-A) and side scatter area (SSC-A)-with-FSC-A graphs are used to exclude doublets and focus on single small lymphocytes.Dead cells are excluded by gating cells that are negative for viability marker Aqua Blue and positive for CD45.CD4+ and CD8+T lymphocytes are gated in CD3+ cells, and NK cells are identified by CD56 and CD16 expression in CD19-CD3- populations.

[0045] Figures 16A to 16D depict the 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 analysis. There was no binding in unrelated proteins in the case of pVax1. (Figure 16B) SPR analysis of DB7.2 on human Siglec-7 protein. Strong binding of DB7.2 was observed with a KD value of 44pM. No binding of (Figure 16C) unrelated ab to Siglec-7 or (Figure 16D) binding of DB7.2 to unrelated proteins was observed, indicating the specificity of DB7.2 for human Siglec-7.

[0046] Figure 17A to Figure 17I depict the in vitro cytotoxicity induced by human Siglec-7 MAb. In non-target (Figure 17A & B) HaCaT human keratinocytes and target OC cells, (Figure 17C & D) OVCAR10, (Figure 17E) OVISE, (Figure 17F) PEO-4 and (Figure 17G) TOV-21G cells induced by human Siglec-7 Mab in vitro cytotoxicity. The xCELLigence real-time cell analysis device (RTCA) of Agilent Technologies of the United States was used to measure in vitro cytotoxicity based on impedance. The conductivity was converted to a cell index (CI) by the xCELLigence device every 15 minutes. DB7.1, DB7.2 and DB7.7 did not induce killing in HaCaT cells. As shown in the images captured after 2 days of treatment, no killing was observed in the cells without Ab control and in the cells treated with DB7.1, DB7.2 and DB7.7, indicating that they do not have any off-target toxic effects. All three Siglec-7 MAbs effectively killed the mentioned group of human OC cells, including BRCA2 mutations and PARPi-resistant PEO-4 cells. DB7.2 Mab was found to exhibit the highest killing efficacy. The killing ability of DB7.2 was further confirmed in two additional OC cells (Figure 17H) CaOV3 cells and (Figure 17I) OVCAR3 cells. Arrows indicate the time points when antibodies and effector cells (human PBMCs) were added to 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) and yellow: DB7.7 (effector cells + target cells + DB7.7).

[0047] Figures 18A to 18C depict ablation of FcR binding of Siglec-7 Mab. (Figure 18A) Schematic diagram of FcR blocking on immune cells and its effect on Siglec-7 mediated killing. (Figure 18B) xCELLigence real-time analysis of killing of OVCAR3 cells by DB7.2 human Mab in the presence and absence of Fc blocking. (Figure 18C) Fc blocking maintains DB7.2 Siglec-7 MAb-mediated NK toxicity in OVISE cells. Images were captured two days after effector cells (E:T=5:1) and DB7.2 Mab were added to OVISE cells in the presence and absence of Fc blocking.

[0048] Figures 19A to 19I depict that ablation of FcR binding maintains the killing potential of Siglec-7 MAb, and the combination of Siglec-7 Mab and anti-PD1 leads to enhanced OC cell killing. (Figure 19A) Comparison of OVISE cell killing by DB7.2 and DB7.2_TM Mod. DB7.2_TM Mod is a variant of DB7.2 DMAb that contains a three-residue modification ("TM"; L234F / L235E / P331S) in the Fc domain to ablate FcR and C1q binding. The killing potency of DB7.2_TM Mod is 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). (FIG. 19B) Images after 24h of treatment with effector cells and DB7.2 / DB7.2_TMMod to target OVISE cells. Cell index and treatment dose curves for (FIG. 19C) DB7.2 and (FIG. 19D) DB7.2_TM Mod at 72h co-culture of OVISE cells and human PBMCs. Cell index and treatment dose curves for (FIG. 19E) DB7.2_TM Mod and (FIG. 19F) anti-PD1 (Pembrolizumab) at 72h co-culture of PEO4 cells and human PBMCs. Killing of target PEO4 cells by (FIG. 19G) DB7.2_TM Mod, (FIG. 19H) anti-PD1 (Pembrolizumab) and (FIG. 19I) DB7.2_TM Mod and anti-PD1 alone and in combination in the presence of human PBMCs; (E:T=5:1). The combination resulted in enhanced killing compared to CPI treatment alone.

[0049] Figures 20A to 20E depict the in vivo expression of Siglec-7 DNA-delivered MAbs in an ovarian cancer attack model and delay cancer progression. (Figure 20A) Schematic diagram of human Siglec-7 DNA-delivered MAbs administered to Balb / c mice. Anti-mouse CD4 and anti-mouse CD8 (both from InVivoMab) antibodies were first used to deplete the CD4 and CD8 T cells of mice. The mice were then electroporated with 50 μg of heavy chain + 50 μg of light chain human Siglec-7 DMAb. Mouse serum was collected at the specified time point. (Figure 20B) Human NK cells were stained with empty vector, DB7.1, DB7.2 and DB7.7 electroporated mouse serum or naive serum 14 days after DNA injection and electroporation, as well as unrelated controls and only secondary antibody controls. The 14th day serum of Siglec-7 MAb-immunized mice showed positive and strong NK cell staining, but no staining was observed in the case of empty vector controls, unrelated Mabs or naive serum. (FIG. 20C) Schematic diagram of electroporation of MAbs delivering Siglec-7 DNA to NSG-K mice challenged with OVISE cells. (FIG. 20D) Growth curves of OVISE tumors transplanted into NSG-K mice treated with DB7.2 or empty vector (n=5 mice per group). (FIG. 20E) Survival benefit in DB7.2 treated mice compared to empty vector controls. DB7.2 treated groups had significantly reduced tumor burden and enhanced survival compared to pVax1 control groups.

[0050] Figures 21A and 21B depict that Siglec-7 DMAb affects tumor growth in the OVISE challenge mouse model. (Figure 21A) Individual growth curves of OVISE tumors transplanted into NSG-K mice treated with DB7.2 DMAb or pVax1 empty vector (n = 5 mice per group). (Figure 21B) Representative images of mice bearing ovarian tumors treated with DB7.2 DMAb or pVax1 on day 37.

[0051] Figures 22A to 22K depict the generation, expression, binding, and specificity analysis of DB7.2xD2AP11:FSHR-targeted bispecific NK cell engagers. (Figure 22A) Schematic diagram of DNA constructs 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 in expi293F cells. (Figure 22C) Flow cytometry of Siglec-7 overexpressing HEK293T cells stained with secondary antibody only, DB7.2xD2AP11 NKCE, Siglec-7 antibody (F023-420 / DB7.2), and DB7.2xD2AP11 NKCE. (FIG. 22D) Flow cytometry of FSHR overexpressing K562 cells stained with secondary antibody only, non-FSHR targeted 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 antibody only was used as a negative control. High-resolution confocal images of fixed cells were captured using a Leica TCS SP8 WLL scanning laser confocal microscope and Leica LAS-X software (Leica Microsystems, Inc., Buffalo Grove, IL). Image post-processing included importing Huygens software for deconvolution (Scientific Volume Imaging, Laapersveld, Hilversum, The Netherlands). Fixed cell preparations were acquired using a 63X / 1.40 oil objective, 2X zoom, and a pinhole of 1AU. Cells were labeled with DAPI (nuclei), GFP (Siglec-7), and Texas Red (DB7.2xD2AP11 NKCE) and acquired sequentially with a HyD detector to maximize signal and minimize crosstalk. The scale bar corresponds to 20.0 microns. Specific 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 the xCELLigence real-time cell analyzer. No cell killing was observed in any of these non-FSHR expressing cells. Images show no induced cytotoxicity in HEK293T (G) and AGS (J) cells 3 days after the addition of effector cells (human PBMC; E: T = 10: 1) and DB7.2xD2AP11.Red: no Ab (effector cells + target cells only); purple: DB7.2xD2AP11 (effector cells + target cells + DB7.2xD2AP11 NKCE).

[0052] Figure 23A to Figure 23I depict NK cell engagers targeted by FSHR; DB7.2xD2AP11NKCE induces ovarian cancer cell toxicity in vitro. In vitro cytotoxicity induced by DB7.2xD2AP11NKCE in (Figure 23A & B) OVISE cells and (Figure 23C) OVCAR3-FSHR cells in the presence of human PBMCs. (Figure 23D & E) CaOV3, (Figure 23F) Kuramochi-FSHR and (Figure 23G) PEO-4 cells in the presence of human PBMCs. Real-time in vitro cytotoxicity analysis was completed by xCELLigence. E:T is 10:1 (A&B, F&G) and 5:1 (CE); Images (B&E) are captured 3 days after human PBMCs and NKCE are added to target cells. (FIG. 23H) Dose-dependent cell lysis induced by DB7.2xD2AP11 NKCE in FSHR expressing OVISE cells in the presence of human PBMCs (E:T=5:1). An IC50 value was obtained at 142.87 pM, indicating the potency of NKCE. (FIG. 23I) Cell index versus dose curve of DB7.2xD2AP11 NKCE in OVCAR3 cells. An IC50 value was obtained at 236.6 pM, confirming its high potency.

[0053] Figure 24A to Figure 24D depicts specific analysis of FSHR-targeted bispecific NK cell engagers. (Figure 24A) In vitro cytotoxicity caused by co-culture of NK cells and OVCAR3-FSHR cells in the presence of DB7.2xD2AP11 NKCE. (Figure 24B) Killing of AGS (FSHR-negative) cells by anti-Siglec-7Mab (DB7.2xD2AP11 NKCE does not induce killing of this cell line, indicating the specificity of the bispecific engager approach). Unrelated NKCE; DB7.2xIL13Rα2NKCE does not induce toxicity in (Figure 24C) FSHR overexpression OVCAR3 cells and (Figure 24D) OVISE cells in the presence of human PBMCs. Real-time in vitro cytotoxicity analysis was completed by xCELLigence; E:T=5:1. Arrows indicate the time of adding effector cells and NKCE.

[0054] Figures 25A to 25C depict dose-dependent cytotoxicity induced by DB7.2xD2AP11 NKCE in target FSHR overexpressing OVSE cells. DB7.2xD2AP11 NKCE induces dose-dependent killing in OVISE-FSHR cells in the presence of human PBMCs obtained from 3 different healthy human donors (Figure 25A) ND578, (Figure 25B) ND502, and (Figure 25C) ND609, analyzed at different concentrations; 120, 60, 30, 15, 7.5, 3.75, and 1.875 ng / ml. The red line indicates the absence of Ab cells (only effector cell target cells). In vitro cytotoxicity was measured based on impedance using an xCELLigence real-time cell analyzer. E:T=5:1; Arrows indicate the time when effector cells and NKCE were added to target OVISE-FSHR cells.

[0055] Fig.26 Depicted is the induction of dose-dependent cytotoxicity by DB7.2xD2AP11 NKCE in target OVCAR3 cells. DB7.2xD2AP11 NKCE induces dose-dependent killing in OVCAR3 cells in the presence of human PBMCs, analyzed at different concentrations; 120, 60, 30, 15, 7.5, 3.75 and 1.875 ng / ml. The red line indicates no Ab (effector cell target cells only). In vitro cytotoxicity was measured based on impedance using the xCELLigence real-time cell analysis device. E:T=10:1, arrows indicate the time when effector cells and NKCE were added to target OVCAR3 cells.

[0056] Figures 27A to 27E depict that DB7.2xD2AP11 NKCE leads to secretion of cytokines / cytotoxic molecules and affects 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 diagram of a tumor study evaluating the effect of DB7.2xD2AP11 NKCE on tumor progression in an OVCAR3-FSHR-challenged NSG-K mouse model. (Figure 27D) Average growth curves of OVCAR3-FSHR tumors transplanted into NSG-K mice treated with DB7.2xD2AP11 NKCE or empty vector (n = 5 mice per group). (Figure 27E) Survival benefit in DB7.2xD2AP11 NKCE mice compared to empty vector controls. Two-way ANOVA; *P<0.05, **P<0.01, ***P<0.001. DETAILED DESCRIPTION

[0057] The present invention relates to sialic acid receptor antibodies, fragments thereof, variants thereof, or nucleic acid molecules encoding the same, and methods for increasing natural killer cell activity in subjects in need thereof.

[0058] In one aspect, the present invention relates to a composition that can be used to increase or enhance an immune response, that is, to produce a more effective immune response by administering a sialic acid receptor antibody, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same. In one embodiment, the sialic acid receptor antibody is an antibody against Siglec-7.

[0059] In one aspect, the present invention relates to a sialic acid receptor antibody, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same in combination with a nucleic acid molecule encoding a tumor antigen.

[0060] In one aspect, the present invention relates to a method for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a sialic acid receptor antibody, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same. In one embodiment, the disease or condition is cancer. In one embodiment, the disease or condition is an infectious disease.

[0061] In one embodiment, the invention relates to a method of treating cancer or a disease or condition associated therewith in a subject in need thereof, the method comprising administering to the subject a sialic acid receptor antibody, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same in combination with a nucleic acid molecule encoding a tumor antigen. In one embodiment, the cancer has increased sialic acid levels.

[0062] definition

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice and testing of the present invention, exemplary methods and materials are described.

[0064] As used herein, the following terms each have the meaning associated with them in this section.

[0065] The articles "a" and "an" are used herein to refer to one or more than one (i.e., at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0066] As used herein, "about" when referring to a measurable value (e.g., an amount, a time interval, etc.) is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are suitable for performing the disclosed methods.

[0067] "Antibody" may refer to antibodies of the IgG, IgM, IgA, IgD or IgE class, or fragments, fragments or derivatives thereof, including Fab, F(ab')2, Fd and single-chain antibodies and derivatives thereof. The antibody may be an antibody isolated from a serum sample of a mammal, a polyclonal antibody, an affinity purified antibody or a mixture thereof, which exhibits sufficient binding specificity for the desired epitope or a sequence derived therefrom.

[0068] "Antigen" refers to a protein that has the ability to produce an immune response in a host. Antigens can be recognized and bound by antibodies. Antigens can originate from within the body or from the external environment.

[0069] "CDR" is defined as the complementary determining region amino acid sequence of an antibody, which is a hypervariable region of an immunoglobulin heavy chain and light chain. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, US Department of Health and Human Services, National Institutes of Health (1987). The variable portion of an immunoglobulin has three heavy chain CDRs (or CDR regions) and three light chain CDRs (or CDR regions). Therefore, "CDR" as used herein refers to all three heavy chain CDRs, or all three light chain CDRs (or all heavy chain CDRs and all light chain CDRs, if appropriate). The structure and protein folding of antibodies may mean that other residues are considered part of the antigen binding region, and the skilled person will understand this. See, e.g., Chothia et al., (1989) Conformations of immunoglobulin hypervariable regions; Nature 342, p 877-883.

[0070] "Antibody fragment" or "fragment of an antibody", used interchangeably herein, refers to a portion of an intact antibody including the antigen binding site or variable region. This portion does not include the constant heavy chain domains (i.e., CH2, CH3, or CH4, depending on the antibody isotype) of the Fc region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv) molecules, single-chain polypeptides containing only one light chain variable domain, single-chain polypeptides containing three CDRs of a light chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing three CDRs of a heavy chain variable region.

[0071] As used herein, "adjuvant" means any molecule added to the vaccines described herein to enhance the immunogenicity of the antigen.

[0072] As used herein, "coding sequence" or "encoding nucleic acid" may refer to a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence encoding an antibody as described herein. The coding sequence may also comprise a DNA sequence encoding an RNA sequence. The coding sequence may further include start and stop signals operably linked to regulatory elements, including a promoter and polyadenylation signal capable of directing expression in cells of an individual or mammal to which the nucleic acid is administered. The coding sequence may further include a sequence encoding a signal peptide.

[0073] As used herein, "complementary" or "complementary" may mean that a nucleic acid may have Watson-Crick (eg, AT / U and CG) or Hoogsteen base pairing between nucleotides or nucleotide analogs in the nucleic acid molecule.

[0074] "Disease" is a health condition in an animal in which the animal is unable to maintain homeostasis and in which the animal's health continues to deteriorate if the disease is not ameliorated.

[0075] In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Without treatment, the disorder does not necessarily cause a further decline in the animal's state of health.

[0076] A disease or condition is "relieved" if the signs or symptoms of the disease or condition are reduced in severity, if a patient experiences such signs or symptoms less frequently, or both.

[0077] "Encoding" refers to the inherent property of a specific nucleotide sequence in a polynucleotide (e.g., a gene, cDNA, or mRNA) as a template for synthesizing other polymers and macromolecules in biological processes having a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and the resulting biological properties. Thus, if the transcription and translation of the mRNA corresponding to a gene produces a protein in a cell or other biological system, the gene encodes the protein. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing) and the non-coding strand (used as a template for transcription of a gene or cDNA) may be referred to as encoding a protein or other product of the gene or cDNA.

[0078] An "effective amount" of a compound is an amount of the compound sufficient to provide an effect on the subject or system to which the compound is administered.

[0079] "Expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements for expression; other elements for expression may be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating recombinant polynucleotides.

[0080] As used herein, a "feedback mechanism" may refer to a process performed by software or hardware (or firmware) that receives the impedance of the desired tissue (before, during, and / or after energy pulse delivery) and compares it to a current value (preferably current) and adjusts the delivered energy pulse to achieve a preset value. The feedback mechanism may be performed by an analog closed-loop circuit.

[0081] "Fragment" may mean a polypeptide fragment of an antibody that is functional, i.e., can bind to a desired target and have the same intended effect as a full-length antibody. A fragment of an antibody may be 100% identical to the full length except for the deletion of at least one amino acid from the N and / or C terminus (with or without a signal peptide and / or methionine at position 1 in each case). A fragment may comprise more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% of the length of a particular full-length antibody, excluding any heterologous signal peptide added. The fragment may comprise a fragment of a polypeptide that is 95% or more, 96% or more, 97% or more, 98% or more identical to the antibody, and additionally comprises an N-terminal methionine or a heterologous signal peptide that is not included when calculating the percent identity. The fragment may further comprise an N-terminal methionine and / or a signal peptide, such as an immunoglobulin signal peptide, e.g., an IgE or IgG signal peptide. The N-terminal methionine and / or signal peptide may be attached to a fragment of the antibody.

[0082] A fragment of a nucleic acid sequence encoding an antibody may be 100% identical to the full length except for the deletion of at least one nucleotide from the 5' and / or 3' end (in each case with or without the sequence encoding a signal peptide and / or a methionine at position 1). A fragment may comprise more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% of the length of a particular full-length coding sequence, excluding any added heterologous signal peptide. The fragment may include a fragment encoding a polypeptide that is 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to an antibody, and optionally includes a sequence encoding an N-terminal methionine or a heterologous signal peptide that is not included when calculating percent identity. The fragment may further include a coding sequence for an N-terminal methionine and / or a signal peptide (such as an immunoglobulin signal peptide, e.g., an IgE or IgG signal peptide). The coding sequence encoding an N-terminal methionine and / or a signal peptide may be connected to a fragment of the coding sequence.

[0083] As used herein, "gene construct" refers to a DNA or RNA molecule containing a nucleotide sequence encoding a protein, such as an antibody. A gene construct may also refer to a DNA molecule that transcribes RNA. The coding sequence includes start and stop signals operably linked to regulatory elements, including promoters and polyadenylation signals that can direct expression in the cells of the individual to which the nucleic acid molecule is administered. As used herein, the term "expressible form" refers to a gene construct containing the necessary regulatory elements operably linked to the coding sequence encoding the protein, so that when present in the cells of the individual, the coding sequence will be expressed.

[0084] "Homologous" refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When one position in both of the two comparison sequences is occupied by the same base or amino acid monomer subunit, for example, if one position in each of the two DNA molecules is occupied by adenine, the two molecules are homologous at that position. The homology percentage between the two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in the two sequences are matched or homologous, the two sequences are 60% homologous. For example, the DNA sequences ATTGCC and TATGGC share 50% homology. Typically, comparison is performed when two sequences are aligned to provide maximum homology.

[0085] As used herein in the context of two or more nucleic acid or polypeptide sequences, "same" or "identity" means that the sequence has a specified percentage of identical residues in a specified region. The percentage can be calculated by: optimally aligning the two sequences, comparing the two sequences in a specified region, determining the number of positions where identical residues occur in the two sequences to derive the number of matching positions, dividing the number of matching positions by the total number of positions in the specified region, and multiplying the result by 100 to derive the percentage of sequence identity. In the case where the lengths of the two sequences are different or the alignment produces more than one staggered end and the specified comparison region includes only a single sequence, the residues of the single sequence are included in the denominator of the calculation but not in the numerator. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered to be equivalent. Identity can be performed manually or by using a computer sequence algorithm (e.g., BLAST or BLAST2.0).

[0086] "Isolated" means changed or removed from the natural state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated", but the same nucleic acid or peptide is "isolated" when it is partially or completely separated from the coexisting materials of its natural state. An isolated nucleic acid or protein can exist in a substantially purified form or can exist in a non-natural environment, such as a host cell.

[0087] In the context of the present invention, the following abbreviations for common nucleic acid bases are used: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0088] Unless otherwise indicated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The phrase "nucleotide sequence encoding a protein or RNA" may also include introns, to the extent that the nucleotide sequence encoding the protein contains introns (one or more) in some versions.

[0089] As used herein, “impedance” may be used when discussing a feedback mechanism and may be converted to a current value according to Ohm's law so that it can be compared with a preset current.

[0090] As used herein, "immune response" may refer to the activation of a host immune system (eg, a mammal's immune system) in response to the introduction of one or more nucleic acids and / or peptides. The immune response may be in the form of a cellular or humoral response, or both.

[0091] The terms "patient", "subject" and "individual" and the like are used interchangeably herein to refer to any animal or cell thereof (whether in vitro or in situ) suitable for the methods described herein. In some embodiments, the patient, subject or individual is a human.

[0092] "Parenteral" administration of the compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intradermal injection, or infusion techniques.

[0093] As used herein, "nucleic acid" or "oligonucleotide" or "polynucleotide" may mean at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Therefore, nucleic acid also encompasses the complementary strand of the depicted single strand. Many variants of nucleic acid can be used for the same purpose as a given nucleic acid. Therefore, nucleic acid also encompasses substantially identical nucleic acids and their complements. The single strand provides a probe that can hybridize with a target sequence under stringent hybridization conditions. Therefore, nucleic acid also encompasses probes that hybridize under stringent hybridization conditions.

[0094] Nucleic acids may be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. Nucleic acids may be DNA (both genomic and cDNA), RNA, or hybrids (wherein the nucleic acid may contain a combination of deoxyribonucleotides and ribonucleotides), and a combination of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acids may be obtained by chemical synthesis or recombinant methods.

[0095] As used herein, "operably linked" may mean that the expression of a gene is controlled by a promoter that is spatially linked thereto. A promoter may be located 5' (upstream) or 3' (downstream) of the gene it controls. The distance between the promoter and the gene may be about the same as the distance between the promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variations in this distance may be accommodated without loss of promoter function.

[0096] As used herein, "peptide," "protein," or "polypeptide" may mean a linked sequence of amino acids, and may be natural, synthetic, or a modification or combination of natural and synthetic.

[0097] As used herein, "promoter" means a synthetic or naturally derived molecule that can confer, activate or enhance the expression of nucleic acids in cells. A promoter may contain more than one specific transcriptional regulatory sequence to further enhance expression and / or change its spatial expression and / or temporal expression. A promoter may also contain distal enhancer or repressor elements, which may be located up to several thousand base pairs from the transcription start site. Promoters may be derived from sources including viruses, bacteria, fungi, plants, insects and animals. A promoter may constitutively or differentially regulate the expression of a genomic component with respect to the cell, tissue or organ where expression occurs, or with respect to the developmental stage where expression occurs, or in response to external stimuli (e.g., physiological stress, pathogens, metal ions or inducers). Representative examples of promoters include bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 late promoter and CMV IE promoter.

[0098] As used herein, the term "promoter / regulatory sequence" means a nucleic acid sequence required for the expression of a gene product that is operably linked to the promoter / regulatory sequence. In some cases, the sequence may be a core promoter sequence, while in other cases, the sequence may also include enhancer sequences and other regulatory elements required for the expression of the gene product. The promoter / regulatory sequence may be, for example, a sequence that expresses a gene product in a tissue-specific manner.

[0099] A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0100] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell substantially only when an inducer corresponding to the promoter is present in the cell.

[0101] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is of the tissue type corresponding to the promoter.

[0102] "Signal peptide" and "leader sequence" are used interchangeably herein and refer to an amino acid sequence that can be attached to the amino terminus of a protein described herein. The signal peptide / leader sequence generally directs the localization of the protein. The signal peptide / leader sequence used herein can promote the secretion of a protein from the cell in which it is produced. The signal peptide / leader sequence is generally cleaved from the remainder of the protein (generally referred to as the mature protein) when it is secreted from the cell. The signal peptide / leader sequence is attached to the N-terminus of the protein.

[0103] As used herein, "stringent hybridization conditions" may refer to conditions under which a first nucleic acid sequence (e.g., a probe) will hybridize to a second nucleic acid sequence (e.g., a target), for example in a complex nucleic acid mixture. Stringent conditions are sequence-dependent and will be different in different circumstances. Stringent conditions may be selected to be higher than the thermal melting point (T ) of a specific sequence at a defined ionic strength pH. m ) is about 5-10℃ lower. m The temperature at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (under defined ionic strength, pH and nucleic acid concentration) may be defined as the temperature at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (due to the presence of an excess of target sequence, the temperature at which 50% of the probes complementary to the target hybridize to the target sequence is defined as the temperature at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium). m Stringent conditions may be salt concentrations less than about 1.0 M sodium ions, such as about 0.01-1.0 M sodium ion concentrations (or other salts) at pH 7.0 to 8.3, and temperatures of at least about 30° C. for short probes (e.g., about 10-50 nucleotides) and at least about 60° C. for long probes (e.g., greater than about 50 nucleotides). Stringent conditions may also be achieved by the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal may be at least 2 to 10 times background hybridization. Exemplary stringent hybridization conditions include the following: 50% formamide, 5x SSC, and 1% SDS, incubated at 42° C., or 5xSSC, 1% SDS, incubated at 65° C., and washed in 0.2x SSC and 0.1% SDS at 65° C.

[0104] As used herein, "subject" and "patient" refer interchangeably to any vertebrate, including, but not limited to, mammals (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats and mice, non-human primates (e.g., monkeys, such as cynomolgus or rhesus monkeys, chimpanzees, etc.), and humans). In some embodiments, the subject may be human or non-human. The subject or patient may be receiving other forms of therapy.

[0105] As used herein, "substantially complementary" can mean that a first sequence is at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the complement of a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides or amino acids, or that the two sequences hybridize under stringent hybridization conditions.

[0106] As used herein, “substantially identical” may mean that the first and second sequences are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 80 97%, 98%, or 99% identical over a region of at least 10, 100, 200, 300, 400, 500, 600, 700, 8 ...

[0107] As used herein, a "synthetic antibody" refers to an antibody encoded by a recombinant nucleic acid sequence described herein and produced in a subject.

[0108] As used herein, "treat" or "treatment" may mean protecting a subject from a disease by means of preventing, inhibiting, suppressing, or completely eliminating the disease. Preventing a disease involves administering a vaccine of the present invention to a subject prior to the onset of the disease. Suppressing a disease involves administering a vaccine of the present invention to a subject after the induction of the disease but before its clinical appearance. Suppressing a disease involves administering a vaccine of the present invention to a subject after the clinical appearance of the disease.

[0109] "Therapeutic" treatment is treatment administered to a subject exhibiting signs or symptoms of a disease or condition, with the goal of reducing or eliminating the frequency or severity of those signs or symptoms.

[0110] As used herein, "treating a disease or condition" means reducing the frequency or severity, or both, of at least one sign or symptom of the disease or condition experienced by a patient.

[0111] As used herein, the phrase "therapeutically effective amount" refers to an amount sufficient or effective to prevent or treat (delay or prevent onset, prevent progression, inhibit, reduce or reverse) a disease or condition, including alleviating the signs and / or symptoms of such diseases and conditions.

[0112] As used herein, the term "treating" a disease or condition means reducing the frequency or severity of at least one sign or symptom of the disease or condition experienced by a subject.

[0113] As used herein, "variant" with respect to nucleic acids means (i) a portion or fragment of a reference nucleotide sequence; (ii) the complement of a reference nucleotide sequence or a portion thereof; (iii) a nucleic acid substantially identical to a reference nucleic acid or its complement; or (iv) a nucleic acid that hybridizes to a reference nucleic acid, its complement, or a sequence substantially identical thereto under stringent conditions.

[0114] Variants can be further defined as peptides or polypeptides whose amino acid sequences differ due to insertion, deletion or conservative substitution of amino acids but retain at least one biological activity. Representative examples of "biological activity" include the ability to be bound by a specific antibody or to promote an immune response. Variants can also refer to proteins having substantially the same amino acid sequence as a reference protein having an amino acid sequence retaining at least one biological activity. Conservative substitution of amino acids, i.e., replacing amino acids with different amino acids of similar properties (e.g., hydrophilicity, degree and distribution of charged regions), is considered in the art to generally involve minor changes. These minor changes can be identified in part by considering the hydropathic index of amino acids, as understood in the art. Kyte et al., J. Mol. Biol. 157: 105-132 (1982). The hydropathic index of amino acids is based on considerations of their hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids with a hydropathic index of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that will result in proteins retaining biological function. Considering the hydrophilicity of amino acids in the context of peptides allows calculation of the maximum local average hydrophilicity of the peptide, which is a useful measurement method that is reported to correlate well with antigenicity and immunogenicity. Substitution of amino acids with similar hydrophilicity values ​​can result in peptides retaining biological activity, such as immunogenicity, as understood in the art. Amino acids with hydrophilicity values ​​within ± 2 of each other can be substituted. Both the hydrophobicity index and the hydrophilicity value of amino acids are affected by the specific side chain of the amino acid. Consistent with this observation, amino acid substitutions compatible with biological function are understood to depend on the relative similarity of amino acids, particularly those side chains, as revealed by hydrophobicity, hydrophilicity, charge, size and other properties.

[0115] Variants can be substantially identical nucleic acid sequences over the full length of the entire gene sequence or its fragment. The nucleic acid sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical over the full length of the gene sequence or its fragment. Variants can be substantially identical amino acid sequences over the full length of the amino acid sequence or its fragment. The amino acid sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical over the full length of the amino acid sequence or its fragment.

[0116] "Vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Various vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ions or amphiphilic compounds, plasmids and viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that promote the transfer of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral vectors include but are not limited to adenoviral vectors, adeno-associated viral vectors, and retroviral vectors.

[0117] Range: Throughout this disclosure, various aspects of the invention may be presented in range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Therefore, the description of a range should be considered to have explicitly disclosed all possible sub-ranges as well as individual numerical values ​​within the range. For example, a description of a range such as from 1 to 6 should be considered to have explicitly disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within the range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0118] describe

[0119] The present invention provides 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, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same. The immunogenic composition of the present invention can be used to protect against diseases or conditions associated with changes in glycosylation, including but not limited to cancer and infectious diseases.

[0120] Therefore, in some embodiments, the present invention provides a composition comprising one or more Siglec-7 antibodies, fragments thereof, or variants thereof, or nucleic acid molecules encoding the same.

[0121] 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 of the present invention that specifically binds to Siglec-7, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same of the present invention.

[0122] In some embodiments, the present invention provides a method for treating or preventing cancer associated with increased Siglec-7, the method comprising administering to a subject an antibody or antibody-like molecule of the present invention that specifically binds to Siglec-7, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same.

[0123] Antibody composition

[0124] In one embodiment, the present invention relates to a composition comprising at least one Siglec-7 antibody, or a fragment thereof.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] In one embodiment, the antibody comprises a variable heavy chain sequence including a CDR sequence of SEQ ID NO: 1-3 and a variable light chain sequence including a CDR sequence of SEQ ID NO: 9-11. In one embodiment, the antibody comprises a variable heavy chain sequence including a CDR sequence of SEQ ID NO: 17-19 and a variable light chain sequence including a CDR sequence of SEQ ID NO: 25-27. In one embodiment, the antibody comprises a variable heavy chain sequence including a CDR sequence of SEQ ID NO: 33-35 and a variable light chain sequence including a CDR sequence of SEQ ID NO: 41-43. In one embodiment, the antibody comprises a variable heavy chain sequence including a CDR sequence of SEQ ID NO: 49-51 and a variable light chain sequence including a CDR sequence of Q ID NO: 57-59. In one embodiment, the antibody comprises a variable heavy chain sequence including a CDR sequence of SEQ ID NO: 65-67 and a variable light chain sequence including a CDR sequence of SEQ ID NO: 73-75. In one embodiment, the antibody comprises 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.

[0130] 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.

[0131] In some embodiments, variants of the amino acid sequences as described herein comprise at least about 60% identity, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over a specified region when compared to a defined amino acid sequence. In some embodiments, the variants of the amino acid sequences as described herein are over the full length of the variable heavy chain having the 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 having the amino acid 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: 87. 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over the full length of the variable light chain of one or more amino acid sequences in 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.

[0132] As used herein, the term "antibody" or "immunoglobulin" refers to proteins (including glycoproteins) of the immunoglobulin (Ig) superfamily of proteins. An antibody or immunoglobulin (Ig) molecule may be a tetramer comprising two identical light chain polypeptides and two identical heavy chain polypeptides. The two heavy chains are linked together by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. Each full-length Ig molecule contains at least two binding sites for a specific target or antigen.

[0133] Sialic acid binding receptor antibodies or antigen-binding fragments thereof include, but are not limited to, polyclonal antibodies, monoclonal fusion proteins, antibodies or fragments thereof, chimeric or chimeric fusion proteins, antibodies or fragments thereof, humanized fusion proteins, antibodies or fragments thereof, deimmunized humfusion proteins, antibodies or fragments thereof, fully humfusion proteins, antibodies or fragments thereof, single chain antibodies, single chain Fv fragments (scFv), Fv, Fd fragments, Fab fragments, Fab' fragments, F(ab')2 fragments, diabodies or antigen-binding fragments thereof, minibodies or antigen-binding fragments thereof, triabodies or antigen-binding fragments thereof, domain fusion proteins, antibodies or fragments thereof, camelid fusion proteins, antibodies or fragments thereof, dromedary fusion proteins, antibodies or fragments thereof, phage displayed fusion proteins, antibodies or fragments thereof, or antibodies or antigen-binding fragments thereof recognized by a repeating backbone array (e.g., repeating antigen display).

[0134] The immune system produces several different classes of Ig molecules (isotypes), including IgA, IgD, IgE, IgG, and IgM, each distinguished by the presence of a specific class of heavy chain polypeptides: alpha (a) present in IgA, delta (δ) present in IgD, epsilon (ε) present in IgE, gamma (γ) present in IgG, and mu (μ) present in IgM. There are at least five different gamma heavy chain polypeptides (isotypes) present in IgG. In contrast, there are only two light chain polypeptide isotypes, called kappa (κ) chains and lambda (λ) chains. The unique characteristics of an antibody isotype are defined by the sequence of the constant domain of the heavy chain.

[0135] IgG molecules contain two light chains (κ or λ forms) and two heavy chains (γ forms) held together by disulfide bonds. The κ and λ forms of the IgG light chain each contain a domain of relatively variable amino acid sequence, called the variable region (variably referred to as the "V L -","V κ -" or "Vλ -region) and a relatively conserved amino acid sequence domain, called the constant region (C L Similarly, each IgG heavy chain contains a variable region (V H -region) and one or more conserved regions: The complete IgG heavy chain contains three constant domains ("C H 1-", "C H 2-" and "C H 3-region”) and hinge region. At each V L - or V H -region, hypervariable regions (also called complementarity determining regions ("CDR")) are interspersed between relatively conserved framework regions ("FR"). Typically, the variable region of a light or heavy chain polypeptide contains four FRs and three CDRs, which are arranged along the polypeptide in the following order: NH2-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-COOH. CDRs and FRs together determine the three-dimensional structure of the IgG binding site, thereby determining the specific target protein or antigen to which the IgG molecule binds. Each IgG molecule is a dimer and can bind to two antigen molecules. Cleavage of dimeric IgG with the protease papain produces two identical antigen-binding fragments ("Fab") and an "Fc" fragment or Fc domain, so named because it is easy to crystallize.

[0136] As used throughout this disclosure, the term "antibody" also refers to a whole or complete antibody (e.g., IgM, IgG, IgA, IgD or IgE) molecule produced by any of the various methods known in the art and described herein. The term "antibody" includes polyclonal antibodies, monoclonal antibodies, chimeric or chimeric antibodies, humanized antibodies, deimmunized human antibodies and fully human antibodies. Antibodies can be made in any of a variety of species or derived from any of a variety of species, such as mammals such as humans, non-human primates (e.g., monkeys, baboons or chimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats and mice. The antibody can be a purified antibody or a recombinant antibody.

[0137] As used herein, the term "epitope" refers to a site on a protein to which an antibody binds."Overlapping epitopes" include at least one (eg, two, three, four, five or six) common amino acid residues.

[0138] In one embodiment, the antibody of the present invention specifically binds to a Siglec polypeptide. As used herein, the term "specific binding" or "specifically binds" refers to two molecules forming a relatively stable complex under physiological conditions. Generally, when the binding constant (K a ) is higher than 10 6 M -1Thus, an antibody can bind specifically to a target with a Ka of at least (or greater than) 10 6 (For example, at least or greater than 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 or 10 15 or higher)M -1 .

[0139] In one embodiment, the antibodies of the invention specifically bind to Siglec-7.

[0140] Methods for determining whether an antibody binds to a protein antigen and / or the affinity of an antibody for a protein antigen are known in the art. For example, the binding of an antibody to a protein antigen can be detected and / or quantified using various techniques, such as, but not limited to, Western blot, dot blot, surface plasmon resonance methods (e.g., BIAcore system; Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ) or enzyme-linked immunosorbent assay (ELISA). See, for example, Harlow and Lane (1988) "Antibodies: A Laboratory Manual" Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Benny KCLo (2004) "Antibody Engineering: Methods and Protocols," Humana Press (ISBN: 1588290921); Borrebaek (1992) "Antibody Engineering, A Practical Guide," WH Freeman and Co., NY; Borrebaek (1995) "Antibody Engineering," 2nd 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.

[0141] Immunoassays that can be used to analyze the immunospecific binding and cross-reactivity of antibodies include, but are not limited to, competitive and non-competitive assay systems using techniques such as Western blots, RIA, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays. Such assays are routine and well known in the art.

[0142] Antibodies can also be measured using any surface plasmon resonance (SPR)-based assay known in the art to characterize the kinetic parameters of the interaction of an antibody with its target or epitope. Any commercially available SPR instrument includes, but is not limited to, a BIAcore instrument (Biacore AB; Uppsala, Sweden); an IAsys instrument (Affinity Sensors; Franklin, Massachusetts); an IBIS system (Windsor Scientific Limited; Berks, UK), an SPR-CELLIA system (Nippon Laser and Electronics Lab; Hokkaido, Japan), and an SPR Detector Spreeta (Texas Instruments; Dallas, Texas) can be used for the methods described herein. See, e.g., Mullett et al. (2000) Methods 22:77-91; Dong et al. (2002) Reviews in Mol Biotech 82:303-323; Fivash et al. (1998) Curr Opin Biotechnol 9:97-101; and Rich et al. (2000) Curr Opin Biotechnol 11:54-61.

[0143] In some embodiments, antibodies and fragments thereof may be "chimeric". Chimeric antibodies and antigen-binding fragments thereof contain portions from two or more different species (e.g., mouse and human). Chimeric antibodies can be produced by splicing mouse variable regions of desired specificity onto human constant domain gene segments (see, e.g., U.S. Patent No. 4,816,567). In this way, non-human antibodies can be modified to make them more suitable for human clinical applications (e.g., methods for treating or preventing complement-associated diseases in human subjects).

[0144] The monoclonal antibodies disclosed herein include "humanized" forms of non-human (e.g., mouse) antibodies. Humanized or CDR-transplanted mAbs are particularly useful as therapeutic agents for humans because they are not rapidly cleared from the circulation like mouse antibodies and generally do not cause adverse immune responses. Methods for preparing humanized antibodies are generally well known in the art. For example, humanization can be performed substantially following the method of Winter and colleagues (see, e.g., Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; and Verhoeyen et al. (1988) Science 239:1534-1536)), by replacing the corresponding sequence of a human antibody with a rodent CDR or CDR sequence. See also, e.g., Staelens et al. (2006) Mol Immunol 43:1243-1257. In some embodiments, the humanized form of non-human (e.g., mouse) antibodies is a human antibody (receptor antibody), wherein the hypervariable (CDR) region residues of the receptor antibody are replaced by the hypervariable region residues of mice, rats, rabbits, or non-human primates from non-human species (donor antibodies), for example, with the desired specificity, affinity, and binding capacity. In some cases, the framework region residues of human immunoglobulins are also replaced by corresponding non-human residues (so-called "back mutations"). In addition, phage display libraries can be used to change the amino acid at the selected position in the antibody sequence. The characteristics of humanized antibodies are also affected by the selection of human frameworks. In addition, humanized and chimeric antibodies can be modified to be included in residues not present in the receptor antibody or the donor antibody, to further improve antibody properties, such as affinity or effector functions.

[0145] The disclosure also provides fully human antibodies.The term "human antibody" includes antibodies with variable regions and constant regions (if present) derived from human germline immunoglobulin sequences.Human antibodies can include amino acid residues not encoded by human germline immunoglobulin sequences (for example, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, the term "human antibody" does not include antibodies (i.e., humanized antibodies) in which the CDR sequences derived from germlines of other mammalian species such as mice have been transplanted to human framework sequences. Fully human antibodies or human antibodies can be derived from transgenic mice carrying human antibody genes (carrying variable (V), diversity (D), connection (J) and constant (C) exons) or from human cells. For example, transgenic animals (such as mice) can be produced now, which can produce complete human antibody repertoires in the absence of endogenous immunoglobulin production after immunity. (See, e.g., Jakobovits et al. (1993) Proc. Natl. Acad. Sci. USA 90:2551; Jakobovits et al. (1993) Nature 362:255-258; Bruggemann et al. (1993) Year in Immunol. 7:33; and Duchosal et al. (1992) Nature 355:258.) Transgenic mouse strains can be engineered to contain gene sequences from unrearranged human immunoglobulin genes. The human sequences can encode the heavy and light chains of human antibodies and will function correctly in mice, rearranging to provide a broad antibody repertoire similar to that of humans. Transgenic mice can be immunized with a target protein (to produce a diverse array of specific antibodies and their encoding RNAs. Nucleic acids encoding the antibody chain components of such antibodies can then be cloned from the animal into a display vector. Typically, separate populations of nucleic acids encoding heavy and light chain sequences are cloned and then inserted into a vector followed by recombination so that any given copy of the vector receives a random combination of heavy and light chains. The vector is designed to express the antibody chains so that they can be assembled and displayed on the outer surface of a display package containing the vector. For example, the antibody chains can be expressed as fusion proteins with a phage coat protein from the outer surface of a phage. Thereafter, the display package can be screened to display antibodies bound to the target.

[0146] Thus, in some embodiments, the present disclosure provides, for example, humanized, deimmunized, or primatized antibodies comprising one or more complementarity determining regions (CDRs) of the mouse monoclonal antibodies described herein that retain the ability of the mouse monoclonal antibody counterpart to bind to its antigen (e.g., at least 50, 60, 70, 80, 90, or 100%, or even greater than 100%).

[0147] In addition, human antibodies can be derived from phage display libraries (Hoogenboom et al. (1991) J. Mol. Biol. 227: 381; Marks et al. (1991) J. Mol. Biol, 222: 581-597; and Vaughan et al. (1996) Nature Biotech 14: 309 (1996)). Synthetic phage libraries can be created using random combinations of synthetic human antibody V regions. By selecting for antigen, fully human antibodies can be prepared in which the V regions are very similar to those of humans in nature. See, for example, U.S. Pat. Nos. 6,794,132, 6,680,209, 4,634,666 and Ostberg et al. (1983), Hybridoma 2: 361-367, the contents of each of which are hereby incorporated by reference in their entirety.

[0148] For the generation of human antibodies, see also Mendez et al. (1998) Nature Genetics 15: 146-156 and Green and Jakobovits (1998) J. Exp. Med. 188: 483-495, the disclosures of which are hereby incorporated by reference in their entireties. Human antibodies are further discussed and described in U.S. Patent Nos.: 5,939,598; 6,673,986; 6,1 14,598; 6,075,181; 6,162,963; 6,150,584; 6,713,610; and 6,657,103 and U.S. Patent Application Publication Nos. 2003-0229905A1, 2004-0010810A1, US 2004-0093622A1, 2006-0040363A1, 2005-0054055A1, 2005-0076395A1, and 2005-0287630A1. See also International Publication Nos. WO 94 / 02602, WO 96 / 34096 and WO 98 / 24893, and European Patent No. EP 0 463 151 Bl. The disclosures of each of the above patents, applications and references are hereby incorporated by reference in their entirety.

[0149] In another approach, other companies, including GenPharm International, Inc., have adopted the "minilocus" approach. In the minilocus approach, a foreign Ig locus is mimicked by including fragments (single genes) from the Ig locus. Thus, one or more VH genes, one or more DH genes, one or more JH genes, a mu constant region, and a second constant region (preferably a gamma constant region) form a construct for insertion into an animal. This method is described in, for example, U.S. Patent Nos.: 5,545,807; 5,545,806; 5,625,825; 5,625,126; 5,633,425; 5,661,016; 5,770,429; 5,789,650; and 5,814,318; 5,591,669; 5,612,205; 5,721,367; 5,789,215; 5,643,763; 5,569,825; 5,877,397; 6,300,129; 5,874,299; 6,255,458; and 7,041,871, the disclosures of which are hereby incorporated herein by reference. See also European Patent No. 0 546 073 Bl, International Patent Publication Nos. WO 92 / 03918, WO 92 / 22645, WO 92 / 22647, WO 92 / 22670, WO 93 / 12227, WO 94 / 00569, WO 94 / 25585, WO 96 / 14436, WO 97 / 13852 and WO 98 / 24884, the disclosures of each of which are hereby incorporated by reference in their entirety. See further Taylor et al. (1992) Nucleic Acids Res. 20:6287; Chen et al. (1993) Int. Immunol. 5:647; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90:3720-4; Choi et al. (1993) Nature Genetics 4:1 17; Lonberg et al. (1994) Nature 368:856-859; Taylor et al. (1994) International Immunology 6:579-591; Tuaillon et al. (1995) J. Immunol. 154:6453-65; Fishwild et al. (1996) Nature Biotechnology 14:845; and Tuaillon et al. (2000) Eur. J. Immunol. 10:2998-3005, the disclosures of each of which are hereby incorporated by reference in their entirety.

[0150] In some embodiments, a deimmunized antibody or antigen binding fragment thereof is provided. A deimmunized antibody or antigen binding fragment thereof is a modified antibody so that the antibody or its antigen binding fragment is non-immunogenic or less immunogenic to a given species (e.g., to humans). Deimmunization can be achieved by modifying fusion proteins, antibodies, or fragments thereof using any of a variety of techniques known to those skilled in the art (see, e.g., PCT Publication No. WO 04 / 108158 and WO 00 / 34317). For example, a fusion protein, an antibody, or its fragment can be deimmunized by identifying potential T cell epitopes and / or B cell epitopes in the amino acid sequence of a fusion protein, an antibody, or its fragment and removing more than one potential T cell epitope and / or B cell epitope from the fusion protein, an antibody, or its fragment (e.g., using recombinant technology). The modified antibody or its antigen binding fragment can then be optionally produced and tested to identify antibodies or its antigen binding fragments that retain more than one desired biological activity (e.g., such as binding affinity) but have reduced immunogenicity. Methods for identifying potential T cell epitopes and / or B cell epitopes can be performed using techniques known in the art, such as computational methods (see, e.g., PCT Publication No. WO 02 / 069232), in vitro or computer techniques, and biological assays or physical methods (e.g., such as determining the binding of a peptide to an MHC molecule, determining the binding of a peptide:MHC complex to a T cell receptor from a species that accepts a fusion protein, antibody, or fragment thereof, testing a protein or its peptide portion using a transgenic animal having an MHC molecule of a species that accepts an antibody or antigen-binding fragment thereof, or testing using a transgenic animal reconstituted with immune system cells from a species that accepts a fusion protein, antibody, or fragment thereof, etc.). In various embodiments, the deimmunized antibodies described herein include deimmunized antigen-binding fragments, Fab, Fv, scFv, Fab' and F(ab')2, monoclonal antibodies, murine antibodies, engineered antibodies (e.g., chimeric, single chain, CDR-grafted, humanized, fully human antibodies, and artificially selected antibodies), synthetic antibodies, and semisynthetic antibodies.

[0151] In some embodiments, the present disclosure also provides bispecific antibodies. Bispecific antibodies are monoclonal antibodies, preferably human antibodies or humanized antibodies with binding specificity to at least two different antigens. For example, in one embodiment, the bispecific antibody of the present invention comprises a domain with binding specificity to a Siglec protein or polypeptide, and a domain with binding specificity to an alternative protein or polypeptide. In one embodiment, the bispecific antibody of the present invention comprises a domain with binding specificity to a Siglec protein or polypeptide, and a domain with binding specificity to an alternative Siglec protein or polypeptide.

[0152] The method for preparing bispecific antibodies is within the knowledge of those skilled in the art. Traditionally, the recombinant production of bispecific antibodies is based on the coexpression of two heavy chain immunoglobulin / light chain pairs, wherein the two heavy chain / light chain pairs have different specificities (Milstein and Cuello (1983) Nature 305:537-539). The antibody variable domains (antibody-antigen binding sites) with the desired binding specificity can be fused to the immunoglobulin constant domain sequence. The fusion of the heavy chain variable region is preferably fused with the immunoglobulin heavy chain constant domain, including at least a portion of the hinge region, CH2 region and CH3 region. The DNA encoding the immunoglobulin heavy chain fusion and (if necessary) immunoglobulin light chain is inserted into a separate expression vector and cotransfected into a suitable host organism. For more details on exemplary currently known methods of producing bispecific antibodies, see Suresh et al. (1986) Methods in Enzymology 121:210; PCT Publication No. WO96 / 27011; Brennan et al. (1985) Science 229:81; Shalaby et al., J Exp Med (1992) 175:217-225; Kostelny et al. (1992) J Immunol 148(5):1547-1553; Hollinger et al. (1993) Proc Natl Acad Sci USA 90:6444-6448; Gruber et al. (1994) J Immunol 152:5368; and Tutt et al. (1991) J Immunol 147:60. Bispecific antibodies also include cross-linked antibodies or heteroconjugate antibodies. Heteroconjugate antibodies may be prepared using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in US Pat. No. 4,676,980, along with a number of cross-linking techniques.

[0153] Various techniques for preparing and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. See, for example, Kostelny et al. (1992) J Immunol 148 (5): 1547-1553. Leucine zipper peptides from Fos and Jun proteins can be connected to the Fab' portion of two different antibodies by gene fusion. Antibody homodimers can be reduced in the hinge region to form monomers and then oxidized to form antibody heterodimers. This method can also be used to produce antibody homodimers. The "double antibody" technology described by Hollinger et al. Proc Natl Acad Sci USA 90: 6444-6448 provides an alternative mechanism for preparing bispecific antibody fragments. The fragment comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) by a linker, and the linker is too short to allow pairing between the two domains on the same chain. Thus, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen binding sites. Another strategy for preparing bispecific antibody fragments by using single-chain Fv (scFv) dimers has also been reported. See, for example, Gruber et al. (1994) J Immunol 152:5368. Alternatively, the antibody may be a "linear antibody", such as described in Zapata et al. (1995) Protein Eng. 8 (10): 1057-1062. In short, these antibodies comprise a pair of tandem Fd fragments (VH-CH1-VH-CH1) that form a pair of antigen binding regions. Linear antibodies may be bispecific or monospecific.

[0154] 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.

[0155] The disclosure also encompasses variant forms of multispecific antibodies, such as Wu et al. (2007) Nat Biotechnol 25 (11): Double variable domain immunoglobulin (DVD-1g) molecules described in 1290-1297. DVD-1g molecules are designed so that two different light chain variable domains (VL) from two different parent antibodies are directly or via a short linker connected in series by recombinant DNA technology, followed by a light chain constant domain. Similarly, the heavy chain comprises two different heavy chain variable domains (VH) connected in series, followed by a constant domain CH1 and an Fc region. For example, PCT Publication No. WO 08 / 024188 and WO 07 / 024715 further describe methods for making DVD-Ig molecules from two parent antibodies.

[0156] The present disclosure also provides camelid or dromedary antibodies (e.g., antibodies derived from Bactrianus bactrianus, Calelus dromaderius, or lama paccos). Unlike typical two-chain (fragments) or four-chain (whole antibodies) antibodies from most mammals, such antibodies generally lack light chains. See U.S. Pat. No. 5,759,808; Stijlemans et al. (2004) J Biol Chem 279: 1256-1261; Dumoulin et al. (2003) Nature 424: 783-788; and Pleschberger et al. (2003) Bioconjugate Chem 14: 440-448.

[0157] Engineered libraries of camelid antibodies and antibody fragments are commercially available, for example, from Ablynx (Ghent, Belgium). Like other non-human derived antibodies, the amino acid sequence of camelid antibodies can be recombinantly altered to obtain a sequence that is more closely similar to the human sequence, i.e., nanobodies can be "humanized", thereby further reducing the potential immunogenicity of the antibody.

[0158] In some embodiments, the disclosure also provides antibodies or their antigen-binding fragments, which are variants of peptides, proteins or antibodies described herein. In some embodiments, such variant peptides, proteins or antibodies maintain the binding or inhibitory ability of parent peptides, proteins or antibodies. Methods for preparing variants of known proteins, peptides or antibodies are known in the art. In some embodiments, such variants include at least a single amino acid substitution, deletion, insertion or other modification. In some embodiments, fusion proteins, antibodies or their fragments described herein include more than two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acid modifications (e.g., amino acid substitutions, deletions or additions). In some embodiments, fusion proteins, antibodies or their fragments described herein do not contain amino acid modifications in CDR. In some embodiments, the fusion proteins, antibodies, or fragments thereof described herein contain more than one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acid modifications in the CDRs.

[0159] As used herein, the term "antibody fragment", "antigen-binding fragment", "antigen binding fragment" or similar terms refers to an antibody fragment that retains the ability to bind to an antigen, wherein the antigen binding fragment may optionally include other components that are not part of the original antibody (such as different framework regions or mutations) as well as fragments (more than one) from the original antibody. Examples include, but are not limited to, single-chain antibodies, single-chain Fv fragments (scFv), Fd fragments, Fab fragments, Fab' fragments, or F(ab')2 fragments. The scFv fragment is a single polypeptide chain that includes both the heavy chain and light chain variable regions of the antibody from which the scFv is derived. In addition, diabodies (Poljak (1994) Structure 2(12):1121-1123; Hudson et al. (1999) J. Immunol. Methods 23(1-2):177-189, the disclosures of each of which are hereby incorporated by reference in their entireties), minibodies, triabodies (Schoonooghe et al. (2009) BMC Biotechnol 9:70), and domain antibodies (also known as "heavy chain immunoglobulins" or camelids; Holt et al. (2003) Trends Biotechnol 21(11):484-490) that bind to complement component proteins (the disclosures of each of which are hereby incorporated by reference in their entireties) can be incorporated into the compositions described herein and used in the methods described herein. In some embodiments, any antigen-binding fragment described herein may be included in "antigen-binding fragment thereof" or equivalent terms, when referring to fragments associated with antibodies, whether or not such fragments are actually derived from antibodies, or are antigen-binding fragments that bind to the same epitope or overlapping epitopes or epitopes contained in antibody epitopes. Antigen-binding fragments thereof may include antigen-binding fragments that bind to the same or overlapping antigens as the original antibody, wherein the antigen-binding fragment includes a portion that is a fragment of the original antibody (e.g., more than one CDR, more than one variable region, etc.).

[0160] In some embodiments, the antibodies described herein comprise altered or mutated sequences that result in altered stability or half-life compared to the parent antibody. This includes, for example, increased stability or half-life to obtain higher affinity or longer in vitro or in vivo clearance time, or reduced stability or half-life to obtain lower affinity or faster clearance. In addition, the antibodies described herein may comprise more than one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) amino acid substitutions, deletions or insertions that result in altered post-translational modifications, including, for example, altered glycosylation patterns (e.g., adding more than one sugar component, losing more than one sugar component, or changing the composition of more than one sugar component).

[0161] In some embodiments, the antibodies described herein include reduced (e.g., or no) effector functions. The altered effector functions include, for example, modulation of one or more of the following activities: antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), apoptosis, binding to one or more Fc receptors, and proinflammatory responses. Modulation refers to an increase, decrease, or elimination of the effector function activity exhibited by the subject antibody containing the altered constant region compared to the activity of the unaltered form of the constant region. In specific embodiments, modulation includes situations where the activity is abolished or completely absent.

[0162] Antibodies with altered or no effector function can be produced by engineering or producing antibodies with variant constant regions, Fc regions, or heavy chain regions; antibodies with altered function and / or activity can be produced using recombinant DNA techniques and / or cell culture and expression conditions. For example, one or more amino acid substitutions, deletions, or insertions in regions (e.g., such as Fc regions or constant regions) that affect antibody function (including effector function) can be engineered using recombinant DNA techniques. Alternatively, changes in post-translational modifications (e.g., glycosylation patterns) can be achieved by manipulating cell culture and expression conditions that produce the antibody. Suitable methods for introducing one or more substitutions, additions or deletions into the Fc region of an antibody are well known in the art and include, for example, standard DNA mutagenesis techniques as described in Sambrook et al. (1989) "Molecular Cloning: A Laboratory Manual, 2nd Edition", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane (1988), supra; Borrebaek (1992), supra; Johne et al. (1993), supra; PCT Publication No. WO 06 / 53301; and U.S. Pat. No. 7,704,497.

[0163] Nucleic acid molecules

[0164] Provided herein are polynucleotides encoding Siglec-7 antibodies of the present invention, or fragments thereof. In some embodiments, the polynucleotides also include a sequence encoding a signal peptide operably linked to the 5' end of the coding sequence. In some embodiments, the polynucleotides also include a sequence encoding a linker sequence.

[0165] 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 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 that is at least 95% identical to one or more of the variable heavy chain sequences of SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, or SEQ ID NO:84; a sequence that is at least 95% identical to one or more of the variable light chain sequences of SEQ ID NO:12, SEQ ID NO:28, SEQ ID NO:44, SEQ ID NO:60, SEQ ID NO:76, or SEQ ID NO:92; ID NO:52, SEQ ID NO:68, or SEQ ID NO:84; or a fragment comprising at least 80% of the full length of a variable light chain sequence selected from SEQ ID NO:12, SEQ ID NO:28, SEQ ID NO:44, SEQ ID NO:60, SEQ ID NO:76, or SEQ ID NO:92, or a combination thereof.

[0166] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a 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.

[0167] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a 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.

[0168] In some embodiments, variants of the nucleotide sequences as described herein comprise at least about 60% identity, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over a specified region when compared to a defined nucleotide sequence. In some embodiments, variants of the nucleotide sequences as described herein comprise at least about 60% identity, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over the entire length of the nucleotide sequence. In some embodiments, a variant of SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, 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. Thus, in some embodiments, the variability is in regions outside of the CDR encoding regions.

[0169] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 8; SEQ ID NO: 24; SEQ ID NO: 40; SEQ ID NO: 56; SEQ ID NO: 72; or SEQ ID NO: 88. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 16; SEQ ID NO: 32; SEQ ID NO: 48; SEQ ID NO: 64; SEQ ID NO: 80; or SEQ ID NO: 96.

[0170] In some embodiments, a fragment of a nucleotide sequence as described herein comprises at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the full-length sequence of a defined nucleotide sequence. In some embodiments, a fragment of a nucleotide sequence as described herein comprises at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the full-length nucleotide sequence. In some embodiments, a fragment of SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, 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.

[0171] 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.

[0172] In one embodiment, the present invention provides a combination of nucleic acid molecules, wherein the first 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, 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 first nucleic acid molecule, or a fragment or variant thereof, comprising a heavy chain encoding 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, and a second nucleic acid molecule, or a fragment or variant thereof, comprising a light chain encoding 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.

[0173] The isolated nucleic acid can include any type of nucleic acid, including but not limited to DNA, cDNA and RNA. For example, in one embodiment, the composition includes an isolated DNA molecule encoding a protein inhibitor or a functional fragment thereof, including, for example, an isolated cDNA molecule. In one embodiment, the composition includes an isolated RNA molecule encoding a protein inhibitor or a functional fragment thereof.

[0174] The nucleic acid molecules of the present invention can be modified to improve their stability in serum or in a growth medium for cell culture. Modifications can be added to enhance the stability, functionality and / or specificity of the nucleic acid molecules of the present invention and to minimize their immunostimulatory properties. For example, in order to enhance stability, 3'-residues can be stabilized to prevent degradation, for example, they can be selected so that they consist of purine nucleotides (particularly adenosine or guanosine nucleotides). Alternatively, substitution of pyrimidine nucleotides by modified analogs, for example substitution of uridine by 2'-deoxythymidine is acceptable and does not affect the function of the molecule.

[0175] In one embodiment of the invention, the nucleic acid molecule may contain at least one modified nucleotide analog. For example, the termini may be stabilized by incorporating modified nucleotide analogs.

[0176] Non-limiting examples of nucleotide analogs include sugar- and / or backbone-modified ribonucleotides (i.e., including modifications to the phosphate-sugar backbone). For example, the phosphodiester bond of a natural RNA can be modified to include at least one nitrogen or sulfur heteroatom. In an exemplary backbone-modified ribonucleotide, the phosphate group connected to an adjacent ribonucleotide is replaced by a modified group (e.g., a phosphorothioate group).

[0177] Other examples of modifications are nucleobase-modified ribonucleotides, i.e., ribonucleotides containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. The base can be modified to block the activity of adenosine deaminase. Exemplary modified nucleobases include, but are not limited to, uridine and / or cytidine modified at the 5-position, such as 5-(2-amino)propyluridine, 5-bromouridine; adenosine and / or guanosine modified at the 8 position, such as 8-bromoguanosine; deazanucleotides, such as 7-deaza-adenosine; O- and N-alkylated nucleotides, such as N6-methyladenosine are suitable. The above modifications can be combined.

[0178] In some cases, the nucleic acid molecule comprises at least one of the following chemical modifications: 2'-H, 2'-O-methyl or 2'-OH modification of more than one nucleotide. In some embodiments, the nucleic acid molecule of the present invention may have enhanced resistance to nucleases. In order to increase nuclease resistance, the nucleic acid molecule may include, for example, 2'-modified ribose units and / or thiophosphate bonds. For example, 2'hydroxyl (OH) can be modified or replaced with many different "oxygen" or "deoxy" substituents. In order to increase nuclease resistance, the nucleic acid molecule of the present invention may include 2'-O-methyl, 2'-fluoro, 2'-O-methoxyethyl, 2'-O-aminopropyl, 2'-amino and / or thiophosphate bonds. The inclusion of locked nucleic acids (LNA), ethylene nucleic acids (ENA), such as 2'-4'-ethylene-bridged nucleic acids, and certain nucleobase modifications such as 2-amino-A, 2-thio (e.g., 2-thio-U), G-clamp modifications, may also increase binding affinity to the target.

[0179] In one embodiment, the nucleic acid molecule includes 2'-modified nucleotides, such as 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE) or 2'-ON-methylacetamido (2'-O-NMA). In one embodiment, the nucleic acid molecule includes at least one 2'-O-methyl modified nucleotide, and in some embodiments, all nucleotides of the nucleic acid molecule include a 2'-O-methyl modification.

[0180] The nucleic acid agents discussed herein include other unmodified RNA and DNA and RNA and DNA that have been modified (e.g., to improve efficacy), as well as polymers of nucleoside substitutes. Unmodified RNA refers to molecules in which the components of nucleic acid (i.e., sugar, base and phosphate moieties) are identical or substantially identical to those existing in nature (e.g., naturally occurring in the human body). Rare or rare but naturally occurring RNA has been referred to as modified RNA in the art, see, for example, Limbach et al. (Nucleic Acids Res., 1994, 22: 2183-2196). Such rare or rare RNA (often referred to as modified RNA) is usually the result of post-transcriptional modification and belongs to the term unmodified RNA used herein. As used herein, modified RNA refers to molecules in which one or more components of nucleic acid (i.e., sugar, base and phosphate moieties) are different from the components existing in nature (e.g., different from the components existing in the human body). Although they are referred to as "modified RNA", due to modification, they certainly include molecules that are not RNA in the strict sense. Nucleoside surrogates are molecules in which the ribose phosphate backbone is replaced with a non-ribose phosphate construct, which allows the bases to be presented in the correct spatial relationship such that hybridization is substantially similar to that seen when the ribose phosphate backbone is used, e.g., a non-charged mimetic of the ribose phosphate backbone.

[0181] Modifications of the nucleic acids of the invention may occur at one or more of the phosphate group, sugar group, backbone, N-terminus, C-terminus, or nucleobase.

[0182] The present invention also includes vectors into which the isolated nucleic acid of the present invention is inserted. The art is rich in suitable vectors that can be used in the present invention.

[0183] Therefore, in another aspect, the present invention relates to a vector comprising a nucleotide sequence of the present invention or a construct of the present invention. The selection of the vector will depend on the host cell to be subsequently introduced. In some embodiments, the vector of the present invention is an expression vector. Suitable host cells include a variety of prokaryotic and eukaryotic host cells. In a specific embodiment, the expression vector is selected from the group consisting of viral vectors, bacterial vectors and mammalian cell vectors. Systems based on prokaryotes and / or eukaryotic vectors can be used in the present invention to produce polynucleotides or their homologous polypeptides. Many such systems are commercially and widely available.

[0184] In some embodiments, expression of a synthetic nucleic acid encoding a protein is typically achieved by operably linking a nucleic acid encoding a protein or portion thereof to a promoter and incorporating the construct into an expression vector. The vector used is suitable for replication and optionally integration in eukaryotic cells. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters for regulating expression of the desired nucleic acid sequence.

[0185] The recombinant nucleic acid sequence construct may include more than one transcription termination region. The transcription termination region may be located downstream of the coding sequence to provide effective termination. The transcription termination region may be obtained from the same gene as the above-mentioned promoter, or may be obtained from more than one different gene.

[0186] The recombinant nucleic acid sequence construct may include more than one start codon. The start codon may be located upstream of the coding sequence. The start codon may be in frame with the coding sequence. The start codon may be associated with more than one signal required for effective translation initiation, such as, but not limited to, a ribosome binding site.

[0187] The recombinant nucleic acid sequence construct may include more than one termination or stop codon. The termination codon may be located downstream of the coding sequence. The termination codon may be in frame with the coding sequence. The termination codon may be associated with more than one signal required for effective translation termination.

[0188] The recombinant nucleic acid sequence construct may include more than one polyadenylation signal. The polyadenylation signal may include more than one signal required for efficient polyadenylation of the transcript. The polyadenylation signal may be located downstream of the coding sequence. The polyadenylation signal may be an SV40 polyadenylation signal, an LTR polyadenylation signal, a bovine growth hormone (bGH) polyadenylation signal, a human growth hormone (hGH) polyadenylation signal, or a human β-globin polyadenylation signal. The SV40 polyadenylation signal may be a polyadenylation signal from a pCEP4 plasmid (Invitrogen, San Diego, CA).

[0189] The recombinant nucleic acid sequence construct may include more than one leader sequence. The leader sequence may encode a signal peptide. The signal peptide may be an immunoglobulin (Ig) signal peptide, such as, but not limited to, an IgG signal peptide and an IgE signal peptide.

[0190] The vectors of the present invention can also be used for nucleic acid immunization using standard gene delivery protocols. Gene delivery methods are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are hereby incorporated by reference in their entirety.

[0191] The isolated nucleic acid of the present invention can be cloned into various types of vectors. For example, the nucleic acid can be cloned into vectors including but not limited to plasmids, phagemids, phage derivatives, animal viruses and cosmids. Particularly interesting vectors include expression vectors, replication vectors, probe generation vectors and sequencing vectors.

[0192] In addition, the vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described in, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. The virus that can be used as a vector includes but is not limited to retrovirus, adenovirus, adeno-associated virus, herpes virus and slow virus. In general, suitable vectors contain a replication origin, a promoter sequence, a convenient restriction endonuclease site and more than one selective marker (for example, WO 01 / 96584 with function in at least one organism; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0193] In addition, the expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described in, for example, Sambrook et al. (2012) and Ausubel et al. (1997) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and slow viruses. In general, suitable vectors contain a replication origin, a promoter sequence, a convenient restriction endonuclease site, and more than one selective marker that has a function in at least one organism. (See, for example, WO 01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193.

[0194] As an illustration, the vector into which the nucleic acid sequence is introduced may be a plasmid, which may or may not be integrated into the genome of the host cell when introduced into the cell. Exemplary, non-limiting examples of vectors into which the nucleotide sequence of the invention or the gene construct of the invention may be inserted include tet-on inducible vectors for expression in eukaryotic cells.

[0195] The vector can be obtained by conventional methods known to those skilled in the art (Sambrook et al., 2012). In a specific embodiment, the vector is a vector for transforming animal cells.

[0196] In one embodiment, the recombinant expression vector may also contain a nucleic acid molecule encoding a peptide or protein of the present invention as described elsewhere herein.

[0197] Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject's cells, either in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0198] For example, vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow long-term stable integration of transgenics and their propagation in daughter cells. Lentiviral vectors have additional advantages over vectors derived from oncogenic retroviruses such as murine leukemia viruses because they can transduce non-proliferating cells, such as hepatocytes. They also have the additional advantage of low immunogenicity. In one embodiment, the composition includes a vector derived from adeno-associated virus (AAV). Adeno-associated virus (AAV) vectors have become powerful gene delivery tools for treating various diseases. AAV vectors have many features that make them very suitable for gene therapy, including lack of pathogenicity, minimal immunogenicity, and the ability to transduce post-mitotic cells in a stable and effective manner. By selecting an appropriate combination of AAV serotypes, promoters, and delivery methods, the expression of specific genes contained in AAV vectors can be specific to more than one type of cell.

[0199] In some embodiments, the vector also includes conventional control elements that are operably linked to the transgene in a manner that allows the transgene to be transcribed, translated, and / or expressed in cells transfected with a plasmid vector or infected with a virus produced by the present invention. As used herein, "operably linked" sequences include expression control sequences adjacent to the gene of interest and expression control sequences that act in trans or remotely to control the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; effective RNA processing signals, such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and sequences that enhance secretion of the encoded product when desired. A large number of expression control sequences, including natural, constitutive, inducible, and / or tissue-specific promoters, are known in the art and can be used.

[0200] The promoter may be a promoter naturally associated with a gene or polynucleotide sequence, such as one that can be obtained by isolating a 5′ non-coding sequence upstream of a coding segment and / or exon. Such a promoter may be referred to as “endogenous”. Similarly, an enhancer may be an enhancer naturally associated with a polynucleotide sequence, located downstream or upstream of the sequence. Alternatively, certain advantages may be obtained by placing the coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not usually associated with a polynucleotide sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer that is not usually associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, as well as promoters or enhancers isolated from any other prokaryotic, viral or eukaryotic cell, and non-“naturally occurring” promoters or enhancers, i.e., different elements containing different transcriptional regulatory regions and / or mutations that alter expression. In addition to synthetically generating nucleic acid sequences of promoters and enhancers, sequences can also be generated using recombinant cloning and / or nucleic acid amplification techniques (including PCR) in conjunction with the compositions disclosed herein (U.S. Pat. No. 4,683,202, U.S. Pat. No. 5,928,906). In addition, it is believed that control sequences can also be utilized that direct the transcription and / or expression of sequences within non-nuclear organelles such as mitochondria and chloroplasts.

[0201] Of course, it is very important to utilize promoters and / or enhancers that effectively direct the expression of the DNA fragment in the cell type, organelle, and organism selected for expression. Those skilled in the art of molecular biology generally know how to use promoters, enhancers, and cell type combinations for protein expression, for example, see Sambrook et al. (2012). The promoter utilized can be constitutive, tissue-specific, inducible, and / or can be used to direct high-level expression of the introduced DNA fragment under appropriate conditions, so as to facilitate large-scale production of recombinant proteins and / or peptides. The promoter can be heterologous or endogenous.

[0202] The recombinant expression vector can also contain a selective marker gene, which helps select a transformed or transfected host cell. Suitable selective marker genes are genes encoding proteins such as G418 and hygromycin, which confer resistance to certain drugs, beta-galactosidase, chloramphenicol acetyltransferase, firefly luciferase or immunoglobulin or its part such as the Fc part of an immunoglobulin such as IgG. Selective markers can be directed to a carrier different from the nucleic acid of interest.

[0203] Additional promoter elements such as enhancers regulate the frequency of transcription initiation. Typically, they are located in the region of 30-110bp upstream of the start site, although recently many promoters have been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is usually flexible, so when elements are inverted or relatively moved to each other, promoter function is retained. In the thymidine kinase (tk) promoter, before activity begins to decline, the spacing between promoter elements can be increased to 50bp away. Depending on the promoter, it seems that each element can play a role synergistically or independently to activate transcription.

[0204] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive high-level expression of any polynucleotide sequence operably connected thereto. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, hemoglobin promoter and creatine kinase promoter. In addition, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also considered as part of the present invention. Use of an inducible promoter provides a molecular switch that can turn on the expression of a polynucleotide sequence to which it is operably linked when such expression is desired, or can turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0205] The enhancer sequence found on the vector also regulates the expression of the gene contained in the vector. Generally, the enhancer is combined with a protein factor to enhance the transcription of the gene. The enhancer can be located upstream or downstream of the gene it regulates. The enhancer can also be tissue-specific to enhance the transcription in a specific cell or tissue type. In one embodiment, the vector of the present invention comprises more than one enhancer to strengthen the transcription of the gene present in the vector.

[0206] In order to evaluate the expression of protein inhibitors, the expression vector to be introduced into the cell may also contain a selective marker gene or a reporter gene or both, so as to identify and select expressing cells from a cell population that is attempted to be transfected or infected by a viral vector. In other aspects, the selective marker may be carried on a separate DNA fragment and used for a co-transfection procedure. Both the selective marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in host cells. Useful selective markers include, for example, antibiotic resistance genes, such as neo, etc.

[0207] Reporter gene is used to identify potential transfected cells and to evaluate the function of regulatory sequences. Generally speaking, reporter gene is a gene that does not exist or is not expressed in a receptor organism or tissue, and the expression of its encoded polypeptide is shown as some properties that are easy to detect, such as enzyme activity. After DNA is introduced into the receptor cell, the expression of the reporter gene is detected at the appropriate time. Suitable reporter gene can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein gene (for example, Ui-Tei et al., 2000FEBS Letters 479:79-82). Suitable expression system is well-known, and known technology can be used to prepare or commercially obtain. Generally speaking, the construct with the minimum 5' flanking region showing the highest level reporter gene expression is identified as a promoter. This type of promoter region can be connected to a reporter gene, and is used to evaluate the ability of a reagent to regulate the promoter to drive transcription.

[0208] Methods for introducing genes into cells and expressing them are known in the art. In the context of expression vectors, vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical or biological means.

[0209] Physical methods for introducing peptides or proteins into host cells include calcium phosphate precipitation, lipofection, particle bombardment, and microinjection. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0210] Biological methods for introducing peptides or proteins of interest into host cells include the use of DNA and RNA vectors. Viral vectors, especially retroviral vectors, have become the most widely used methods for inserting genes into mammalian cells such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0211] Chemical means for introducing peptides or proteins into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0212] In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. It is contemplated that a lipid formulation is used to introduce nucleic acid into a host cell (in vitro, in vitro or in vivo). On the other hand, nucleic acid can be associated with a lipid. Nucleic acid associated with a lipid can be encapsulated in the aqueous interior of a liposome, dispersed in the lipid bilayer of the liposome, attached to the liposome via a linker associated with both a liposome and an oligonucleotide, embedded in the liposome, compounded with the liposome, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, included in lipids as a suspension, included or compounded with micelles, or otherwise associated with lipids. Lipid, lipid / DNA or lipid / expression vector related compositions are not limited to any specific structure in the solution. For example, they can exist with a double-layer structure, as micelles or using a "collapse" structure. They can also just be dispersed in the solution, and may form aggregates of uneven size or shape. Lipid is a fatty substance, which can be a naturally occurring or synthetic lipid. For example, lipids include naturally occurring fat droplets in the cytoplasm as well as the class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0213] Lipids suitable for use are available from commercial sources. For example, dimyristoylphosphatidylcholine ("DMPC") is available from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") is available from K&K Laboratories (Plainview, NY); cholesterol ("Choi") is available from Calbiochem-Behring; dimyristoylphosphatidylglycerol ("DMPG") and other lipids are available from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the only solvent because it evaporates more easily than methanol. "Liposome" is a general term covering various monolayer and multilayer lipid carriers formed by the generation of closed lipid bilayers or aggregates. Liposomes are characterized by having a vesicle structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. When phospholipids are suspended in an excess of aqueous solution, they form spontaneously. The lipid components rearrange themselves before forming a closed structure, and trap water and dissolved solutes between lipid bilayers (Ghosh et al., 1991 Glycobiology 5:505-10). However, compositions having structures different from normal vesicle structures in solution are also contemplated. For example, lipids may present micellar structures or exist only as non-uniform aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0214] ScFv Antibody

[0215] In one embodiment, the antibody fragment comprises a scFv fragment. In one embodiment, the ScFv antibody fragment relates to a Fab fragment without CH1 and CL regions. Therefore, in one embodiment, the scFv antibody fragment relates to a Fab fragment comprising VH and VL. In one embodiment, the scFv antibody fragment comprises a linker between VH and VL. In one embodiment, the scFv antibody fragment comprises VH, VL and CH2 and CH3 regions. In one embodiment, the scFv antibody fragment of the present invention has modified expression, stability, half-life, antigen binding, heavy chain-light chain pairing, tissue penetration or a combination thereof compared to the parent MAb.

[0216] In one embodiment, the scFv antibody fragment of the invention has at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 2.1-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6-fold, at least 2.7-fold, at least 2.8-fold, at least 2.9-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 8.5-fold, at least 9-fold, at least 9.5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, or more than 50-fold higher expression than the parent MAb.

[0217] In one embodiment, the scFv antibody fragment of the invention has at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 2.1-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6-fold, at least 2.7-fold, at least 2.8-fold, at least 2.9-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 8.5-fold, at least 9-fold, at least 9.5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, or more than 50-fold higher antigen binding than the parent MAb.

[0218] In one embodiment, the scFv antibody fragment of the invention has a half-life that is at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 2.1-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6-fold, at least 2.7-fold, at least 2.8-fold, at least 2.9-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 8.5-fold, at least 9-fold, at least 9.5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, or more than 50-fold longer than that of the parent MAb.

[0219] 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.

[0220] 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.

[0221] In one embodiment, the scFv antibody fragment of the invention has at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 2.1-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6-fold, at least 2.7-fold, at least 2.8-fold, at least 2.9-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 8.5-fold, at least 9-fold, at least 9.5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, or more than 50-fold greater heavy chain-light chain pairing than that of the parent MAb.

[0222] Delivery vehicle

[0223] In one embodiment, the invention provides a composition comprising a delivery vehicle comprising a sialic acid binding receptor antibody, a fragment thereof, or a nucleic acid molecule encoding the same, as described herein.

[0224] Exemplary delivery vehicles include, but are not limited to, microspheres, microparticles, nanoparticles, polymerosomes, liposomes, and micelles. For example, in some embodiments, the delivery vehicle is loaded with an anti-Siglec-7 polypeptide, a fragment thereof, or a nucleic acid molecule encoding it. In some embodiments, the delivery vehicle provides controlled release, delayed release, or sustained release of the cargo it carries. In some embodiments, the delivery vehicle includes a targeting portion that targets the delivery vehicle to a treatment site.

[0225] Pharmaceutical composition

[0226] The present invention also provides pharmaceutical compositions comprising more than one composition described herein. The preparations can be mixed with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier materials suitable for application to the treatment site. The pharmaceutical compositions can be sterilized and, if necessary, mixed with adjuvants such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure buffers, colorants and / or aromatic substances. They can also be combined with other active agents (e.g., other analgesics) as needed.

[0227] Administration of the compositions of the invention may be carried out, for example, by parenteral, intravenous, subcutaneous, intramuscular or intraperitoneal injection, or by infusion or by any other acceptable systemic method.

[0228] As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients; surfactants; dispersants; inert diluents; granulating agents and disintegrants; binders; lubricants; colorants; preservatives; physiologically degradable compositions, such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersants or wetting agents; emulsifiers, demulcents; buffers; salts; thickeners; fillers; emulsifiers; antioxidants; antibiotics; antifungal agents; stabilizers; and pharmaceutically acceptable polymers or hydrophobic materials. Other "additional ingredients" that may be included in the pharmaceutical compositions of the present invention are known in the art and are described, for example, in Genaro, ed. (1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA), which is incorporated herein by reference.

[0229] The composition of the present invention may contain about 0.005% to 2.0% preservatives, based on the total weight of the composition. Preservatives are used to prevent spoilage in the event of exposure to pollutants in the environment. Examples of preservatives useful according to the present invention include, but are not limited to, preservatives selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidazole lignocerae, and combinations thereof.

[0230] In one embodiment, the composition includes an antioxidant and a chelating agent that inhibits the degradation of one or more components of the composition. Exemplary antioxidants for some compounds are BHT, BHA, alpha-tocopherol, and ascorbic acid. Exemplary chelating agents include ethylenediaminetetraacetic acid salts (e.g., disodium edetate) and citric acid. Chelating agents can be used to chelate metal ions in the composition that may be harmful to the shelf life of the formulation. Although BHT and disodium edetate can be antioxidants and chelating agents for some compounds, respectively, other suitable and equivalent antioxidants and chelating agents can therefore be substituted as known to those skilled in the art.

[0231] Liquid suspensions can be prepared using conventional methods so that the compounds of the present invention or other compositions are suspended in aqueous or oily solvents. Aqueous solvents include, for example, water and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethanol, vegetable oils such as peanut oil, olive oil, sesame oil or coconut oil, fractionated vegetable oils and mineral oils such as liquid paraffin. Liquid suspensions may also include more than one additional component, including but not limited to suspending agents, dispersants or wetting agents, emulsifiers, mitigating agents, preservatives, buffers, salts, flavoring agents, coloring agents and sweeteners. Oily suspensions may also include thickeners. Known suspending agents include but are not limited to sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinyl pyrrolidone, tragacanth gum, gum arabic and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose. Known dispersants or wetting agents include, but are not limited to, naturally occurring phospholipids, such as lecithin, condensation products of alkylene oxides and fatty acids, condensation products with long-chain fatty alcohols, condensation products with partial esters derived from fatty acids and hexitol, or condensation products with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitol monooleate, respectively). Known emulsifiers include, but are not limited to lecithin and gum arabic. Known preservatives include, but are not limited to methyl, ethyl or n-propyl p-hydroxybenzoate, ascorbic acid and sorbic acid.

[0232] For oral use, tablets, dragees, liquids, drops, suppositories or capsules, caplets and gelcaps are particularly suitable. Other formulations suitable for oral administration include, but are not limited to, powder or granular formulations, aqueous or oily suspensions, aqueous or oily solutions, pastes, gels, toothpastes, mouthwashes, coatings, oral rinses, chewing gums, varnishes, sealants, oral and dental "dissolving strips" or emulsions. Compositions for oral use may be prepared according to any method known in the art, and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutical excipients suitable for making tablets. Such excipients include, for example, inert diluents such as lactose; granulating agents and disintegrants such as corn starch; binders such as starch; and lubricants such as magnesium stearate.

[0233] Tablets may be non-coated, or they may be coated using known methods to achieve delayed disintegration in the gastrointestinal tract of a subject, thereby providing sustained release and absorption of the active ingredient. For example, materials such as glyceryl monostearate or glyceryl distearate may be used to coat tablets. Further, for example, tablets may be coated using the methods described in U.S. Patent Nos. 4,256,108, 4,160,452, and 4,265,874 to form osmotic controlled release tablets. Tablets may also include sweeteners, flavoring agents, coloring agents, preservatives, or some combination of these to provide pharmaceutically elegant and palatable preparations.

[0234] Hard capsules containing the active ingredient can be made using physiologically degradable compositions such as gelatin. Such hard capsules contain the active ingredient and may also contain other ingredients including, for example, inert solid diluents such as calcium carbonate, calcium phosphate or kaolin.

[0235] Soft gelatin capsules containing the active ingredient can be made using physiologically degradable compositions such as gelatin. Such soft capsules contain the active ingredient, which can be mixed with water or an oily medium such as peanut oil, liquid paraffin or olive oil.

[0236] For oral administration, the compositions of the present invention may be in the form of tablets or capsules prepared by conventional means using pharmaceutically acceptable excipients such as binders; fillers; lubricants; disintegrants; or wetting agents. If desired, tablets may be coated using suitable methods and coating materials such as OPADRY available from Colorcon, West Point, Pa. TM Film coating systems (eg OPADRY TM OY type, OYC type, organic enteric-coated OY-P type, aqueous enteric-coated OY-A type, OY-PM type and OPADRY TM White, 32K18400) for coating.

[0237] Liquid preparations for oral administration may be in the form of solutions, syrups or suspensions. Liquid preparations can be prepared by conventional means using pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methylcellulose or hydrogenated edible fats); emulsifiers (e.g., lecithin or gum arabic); non-aqueous solvents (e.g., almond oil, oily esters or ethanol); and preservatives (e.g., methyl or propyl parabens or sorbic acid). Liquid preparations of the pharmaceutical compositions of the present invention suitable for oral administration can be prepared, packaged and sold in liquid form or in the form of dry products intended to be reconstituted with water or another suitable solvent before use.

[0238] Tablets containing active ingredients can be prepared, for example, by compressing or forming the active ingredient (optionally with one or more additional ingredients). Compressed tablets can be prepared by compressing a free-flowing form of the active ingredient, such as a powder or granular formulation (optionally mixed with one or more binders, lubricants, excipients, surfactants and dispersants) in a suitable device. Formed tablets can be prepared by forming a mixture of an active ingredient, a pharmaceutically acceptable carrier and a liquid at least sufficient to wet the mixture in a suitable device. Pharmaceutically acceptable excipients for making tablets include, but are not limited to, inert diluents, granulators and disintegrants, binders and lubricants. Known dispersants include, but are not limited to, potato starch and sodium starch glycolate. Known surfactants include, but are not limited to, sodium lauryl sulfate. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate and sodium phosphate. Known granulators and disintegrants include, but are not limited to, corn starch and alginic acid. Known binders include, but are not limited to, gelatin, gum arabic, pregelatinized corn starch, polyvinyl pyrrolidone and hydroxypropyl methylcellulose. Known lubricants include, but are not limited to, magnesium stearate, stearic acid, silicon dioxide, and talc.

[0239] The preparation of the pharmaceutical composition suitable for parenteral administration comprises an active ingredient combined with a pharmaceutically acceptable carrier (e.g., sterile water or sterile isotonic saline). Such preparations can be prepared, packaged or sold in a form suitable for bolus administration or continuous administration. Injectable preparations can be prepared, packaged or sold in a unit dose form such as an ampoule or a multi-dose container containing a preservative. Preparations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable preparations. Such preparations may also include more than one additional ingredient, including but not limited to suspending agents, stabilizers or dispersants. In one embodiment of a preparation for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granule) form for reconstitution with a suitable solvent (e.g., sterile pyrogen-free water) before parenteral administration of the reconstructed composition.

[0240] Pharmaceutical compositions can be prepared, packaged or sold in the form of sterile injectable aqueous or oily suspensions or solutions. The suspension or solution can be prepared according to known techniques and, in addition to the active ingredient, can also include additional ingredients such as dispersants, wetting agents or suspending agents as described herein. Such sterile injectable preparations can be prepared using nontoxic parenteral acceptable diluents or solvents (e.g., water or 1,3-butylene glycol). Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic monoglycerides or diglycerides. Other useful parenteral formulations include preparations including active ingredients including microcrystalline forms, liposome preparations, or as components of biodegradable polymer systems. Compositions for sustained release or implantation can include pharmaceutically acceptable polymers or hydrophobic materials, such as emulsions, ion exchange resins, poorly soluble polymers, or poorly soluble salts.

[0241] Excipients and other ingredients of the composition

[0242] Compositions can also include a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient can be a functional molecule, such as a solvent, adjuvant, carrier or diluent. A pharmaceutically acceptable excipient can be a transfection facilitator, which can include a surfactant, such as an immunostimulating complex (ISCOMS), Freund's incomplete adjuvant, LPS analogs (including monophosphoryl lipid A), muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations or nanoparticles, or other known transfection facilitators.

[0243] The transfection facilitator is a polyanion, a polycation (including poly-L-glutamic acid (LGS)), or a lipid. The transfection facilitator is poly-L-glutamic acid, and the concentration of the poly-L-glutamic acid in the composition may be less than 6 mg / ml. The transfection facilitator may also include a surfactant, such as an immunostimulating complex (ISCOMS), Freund's incomplete adjuvant, an LPS analog (including monophosphoryl lipid A), a muramyl peptide, a quinone analog, and a vesicle such as squalene and squalene, and hyaluronic acid, which may also be administered in combination with the composition. The composition may further include a transfection facilitating agent, such as a lipid, a liposome (including a phosphatidylcholine liposome or other liposomes known in the art, such as a DNA-liposome mixture (see, e.g., WO9324640), a calcium ion, a viral protein, a polyanion, a polycation or a nanoparticle, or other known transfection facilitating agents. The transfection facilitating agent is a polyanion, a polycation (including poly-L-glutamic acid (LGS)), or a lipid. The concentration of the transfection agent in the composition is less than 4 mg / ml, less than 2 mg / ml, less than 1 mg / ml, less than 0.750 mg / ml, less than 0.500 mg / ml, less than 0.250 mg / ml, less than 0.100 mg / ml, less than 0.050 mg / ml or less than 0.010 mg / ml.

[0244] Pharmaceutically acceptable excipients may be adjuvants other than the checkpoint inhibitor antibodies of the present invention. Additional adjuvants may be other genes expressed in alternative plasmids or delivered as proteins in combination with the above-mentioned plasmids in the composition. Adjuvants may be selected from the group consisting of: alpha-interferon (IFN-α), beta-interferon (IFN-β), gamma-interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), skin T cell attraction chemokine (CTACK), epithelial thymus expression chemokine (TECK), mucosa-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86, including IL-15 with deleted signal sequences and optionally including a signal peptide from IgE. The adjuvant can be IL-12, IL-15, IL-28, CTACK, TECK, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, PD-1, IL-10, IL-12, IL-18, or a combination thereof.

[0245] In addition to the antibodies of the present invention, other genes that can be used as adjuvants include genes encoding MCP-1, MIP-1a, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-C SF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, IL-22, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-1, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 and their functional fragments.

[0246] The composition may also include a genetic enhancer as described in US Serial No. 021,579, filed April 1, 1994, which is incorporated by reference in its entirety.

[0247] The composition may contain DNA in an amount of about 1 nanogram to 100 milligrams; about 1 microgram to about 10 milligrams; or preferably about 0.1 microgram to about 10 milligrams; or more preferably about 1 milligram to about 2 milligrams. In some preferred embodiments, the composition according to the present invention contains DNA in an amount of about 5 nanograms to about 1000 micrograms. In some preferred embodiments, the composition may contain DNA in an amount of about 10 nanograms to about 800 micrograms. In some preferred embodiments, the composition may contain DNA in an amount of about 0.1 to about 500 micrograms. In some preferred embodiments, the composition may contain DNA in an amount of about 1 to about 350 micrograms. In some preferred embodiments, the composition may contain about 25 to about 250 micrograms, about 100 to about 200 micrograms, about 1 nanogram to 100 milligrams; about 1 microgram to about 10 milligrams; about 0.1 microgram to about 10 milligrams; about 1 milligram to about 2 milligrams, about 5 nanograms to about 1000 micrograms, about 10 nanograms to about 800 micrograms, about 0.1 to about 500 micrograms, about 1 to about 350 micrograms, about 25 to about 250 micrograms, about 100 to about 200 micrograms of DNA.

[0248] The composition can be formulated according to the mode of administration to be used. The injectable pharmaceutical composition can be sterile, pyrogen-free and particle-free. Isotonic preparations or solutions can be used. Isotonic additives can include sodium chloride, dextrose, mannitol, sorbitol and lactose. The composition can include a vasoconstrictor. The isotonic solution can include phosphate buffered saline. The composition can also include stabilizers, including gelatin and albumin. Stabilizers can stabilize the formulation for a long time at room temperature or ambient temperature, including LGS or polycations or polyanions.

[0249] Application method

[0250] The present invention provides a method for increasing the function or activity of natural killer (NK) cells. This can be measured, for example, in a standard NK cell or T cell-based cytotoxicity assay, in which the ability of a therapeutic compound to stimulate Siglec-positive lymphocytes to kill sialic acid ligand-positive cells is measured. In one embodiment, the antibody preparation causes at least a 10% increase in the cytotoxicity of Siglec-restricted lymphocytes, optionally at least a 40% or 50% increase in lymphocyte cytotoxicity, or optionally at least a 70% increase in NK cell cytotoxicity, and reference is made to the cytotoxicity assay. In one embodiment, the antibody preparation causes at least a 10% increase in cytokine release from Siglec-restricted lymphocytes, optionally at least a 40% or 50% increase in cytokine release, or optionally at least a 70% increase in cytokine release, and reference is made to the cytotoxicity assay. In one embodiment, the antibody preparation results in at least a 10% increase in cell surface expression of cytotoxic markers (e.g., CD107 and / or CD137) of Siglec-restricted lymphocytes, optionally results in at least a 40% or 50% increase in cell surface expression of cytotoxic markers (e.g., CD107 and / or CD137), or optionally results in at least a 70% increase in cell surface expression of cytotoxic markers (e.g., CD107 and / or CD137).

[0251] The present invention also relates to a method of increasing an immune response in a subject. Increasing the immune response can be used to treat and / or prevent a disease in a subject. The method may include administering a vaccine disclosed herein to a subject. The subject administered the vaccine may have an increased or boosted immune response compared to a subject administered only the antigen. In some embodiments, the immune response may increase by about 0.5 times to about 15 times, about 0.5 times to about 10 times, or about 0.5 times to about 8 times. Alternatively, the immune response in a subject administered the vaccine may be increased by at least about 0.5-fold, at least about 1.0-fold, at least about 1.5-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 5.5-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 10.5-fold, at least about 11.0-fold, at least about 11.5-fold, at least about 12.0-fold, at least about 12.5-fold, at least about 13.0-fold, at least about 13.5-fold, at least about 14.0-fold, at least about 14.5-fold, or at least about 15.0-fold.

[0252] In still other alternative embodiments, the immune response in a subject administered the vaccine may be increased by about 50% to about 1500%, about 50% to about 1000%, or about 50% to about 800%. In other embodiments, the immune response in a subject administered the vaccine may be increased by at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, at least about 500%, at least about 550%, at least about 600%, at least about 650%, at least about 700%, at least about 750%, at least about 800%, at least about 850%, at least about 900%, at least about 950%, at least about 1000%, at least about 1050%, at least about 1100%, at least about 1150%, at least about 1200%, at least about 1250%, at least about 1300%, at least about 1350%, at least about 1450%, or at least about 1500%.

[0253] The vaccine dose may be between 1 μg and 10 mg of active ingredient / kg body weight / time, or may be between 20 μg and 10 mg of active ingredient / kg body weight / time. The vaccine may be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 days. The number of vaccine doses for effective treatment may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0254] Combination vaccines

[0255] In one embodiment, the present invention relates to the administration of the Siglec-7 antibody of the present invention, or a nucleic acid molecule encoding the same, in combination with a PD-(L)1 axis inhibitor. The immunogenic composition can be used to increase the immune response against a tumor.

[0256] In some embodiments, the Siglec-7 antibody of the present invention, or the nucleic acid molecule encoding the same, is administered simultaneously with administration of a PD-(L)1 axis inhibitor agent.

[0257] In some embodiments, the Siglec-7 antibody of the present invention, or the nucleic acid molecule encoding the same, is administered prior to administration of the PD-(L)1 axis inhibitor agent.

[0258] In some embodiments, the Siglec-7 antibody of the present invention, or the nucleic acid molecule encoding the same, is administered after administration of a PD-(L)1 axis inhibitor agent.

[0259] In some embodiments, the Siglec-7 antibody of the present invention, or the nucleic acid molecule encoding it, 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 before the administration of the PD-(L)1 axis inhibitor agent. Thus, in some embodiments, the PD-(L)1 axis inhibitor agent is administered to a subject who has previously been administered the Siglec-7 antibody of the present invention, or the nucleic acid molecule encoding it.

[0260] In some embodiments, the Siglec-7 antibody of the present invention, or the nucleic acid molecule encoding it, 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 the administration of the PD-(L)1 axis inhibitor agent. Therefore, in some embodiments, the Siglec-7 antibody of the present invention, or the nucleic acid molecule encoding it, is administered to a subject who has previously been administered a PD-(L)1 axis inhibitor agent.

[0261] In some embodiments, the combination of a Siglec-7 antibody of the invention, or a nucleic acid molecule encoding the same, and a PD-(L)1 axis inhibitor agent is administered prior to administration of one or more additional anti-cancer agents.

[0262] In some embodiments, the combination of a Siglec-7 antibody of the invention, or a nucleic acid molecule encoding the same, and a PD-(L)1 axis inhibitor agent is administered after administration of one or more additional anti-cancer agents.

[0263] In some embodiments, the combination of the Siglec-7 antibody of the invention, or the nucleic acid molecule encoding the same, and the PD-(L)1 axis inhibitor agent is administered concurrently with the administration of one or more additional anti-cancer agents.

[0264] The length of time between administration of the Siglec-7 antibody of the invention, or a nucleic acid molecule encoding the same, and the PD-(L)1 axis inhibitor agent or one or more additional anti-cancer agents can be a few minutes, such as about 1, 2, 5, 10, 30 or 60 minutes, or several 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.

[0265] One or more of the Siglec-7 antibodies of the present invention, or nucleic acid molecules encoding them, PD-(L)1 axis inhibitor agents, or additional anticancer agents can be administered in a pharmaceutically acceptable carrier. "Carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the antibodies of the present invention are administered. Such carriers can be liquids, such as water and oils, including oils of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. For example, 0.4% saline and 0.3% glycine can be used to formulate bispecific anti-EGFR / c-Met antibodies. 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 preparations, such as powders, capsules, and tablets, suitable carriers and additives include starch, sugars, diluents, granulating agents, lubricants, binders, and disintegrants. Solid oral preparations can also be coated with substances such as sugars or enteric coatings to adjust the main site of absorption. For parenteral administration, the carrier may comprise sterile water, and other excipients may be added to increase solubility or preservation. Injectable suspensions or solutions may also be prepared using aqueous carriers and appropriate additives. Suitable vehicles and formulations include other human proteins, such as, for example, human serum albumin as described in, 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, particularly see pp. 958-989.

[0266] The composition may contain pharmaceutically acceptable auxiliary substances as needed to approximate physiological conditions, such as pH adjustment and buffering agents, stabilizers, thickeners, lubricants and coloring agents, etc. 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 agent in the pharmaceutical preparation may vary from less than about 0.5% by weight, usually to at least about 1% to as much as 15%, 20%, 30%, 40% or 50%, and may be selected according to the specific mode of administration selected, mainly based on the required dose, liquid volume, viscosity, etc. Pharmaceutical compositions comprising solid forms may contain about 0.1 mg to about 2000 mg, such as 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.

[0267] The therapeutic compositions of the invention may possess desirable characteristics for effective treatment such as safety, so that the therapeutic composition itself does not cause illness or death; and provide ease of administration, few side effects, biological stability, and low cost per dose.

[0268] In one embodiment, the present invention relates to the administration of a Siglec-7 antibody encoded by a nucleic acid in combination with an immunogenic composition comprising a nucleic acid molecule encoding a tumor antigen. Therefore, 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 it, 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 the subject to which the vaccine is administered, thereby protecting against or treating cancer or related conditions.

[0269] The immunogenic composition can be a DNA vaccine, a peptide vaccine, or a combined DNA and peptide vaccine. A DNA vaccine can include a nucleic acid sequence encoding a tumor antigen. The nucleic acid sequence can be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The nucleic acid sequence can also include additional sequences encoding a linker, a leader, or a tag sequence that is linked to the tumor antigen via a peptide bond. A peptide vaccine can include an antigenic peptide, an antigenic protein, a variant thereof, a fragment thereof, or a combination thereof. A combined DNA and peptide vaccine can include the above-mentioned nucleic acid sequences encoding tumor antigens and antigenic peptides or proteins.

[0270] The vaccine can induce a humoral immune response in a subject to which the vaccine is administered. The induced humoral immune response can be specific to a tumor antigen. The induced humoral immune response can react with a tumor antigen. In a subject to which the vaccine is administered, the humoral immune response can induce about 1.5 times to about 16 times, about 2 times to about 12 times, or about 3 times to about 10 times. In a subject administered the vaccine, a humoral immune response can be induced by at least about 1.5 times, at least about 2.0 times, at least about 2.5 times, at least about 3.0 times, at least about 3.5 times, at least about 4.0 times, at least about 4.5 times, at least about 5.0 times, at least about 5.5 times, at least about 6.0 times, at least about 6.5 times, at least about 7.0 times, at least about 7.5 times, at least about 8.0 times, at least about 8.5 times, at least about 9.0 times, at least about 9.5 times, at least about 10.0 times, at least about 10.5 times, at least about 11.0 times, at least about 11.5 times, at least about 12.0 times, at least about 12.5 times, at least about 13.0 times, at least about 13.5 times, at least about 14.0 times, at least about 14.5 times, at least about 15.0 times, at least about 15.5 times, or at least about 16.0 times.

[0271] The humoral immune response induced by the vaccine may include an increased level of neutralizing antibodies associated with the subject to which the vaccine was administered, compared to a subject not administered the vaccine. The neutralizing antibodies may be specific to tumor antigens. The neutralizing antibodies may react with tumor antigens. The neutralizing antibodies may provide protection and / or treatment of cancer and its associated conditions in the subject to which the vaccine was administered.

[0272] The humoral immune response induced by the vaccine may include an increased level of IgG antibodies associated with the subject to which the vaccine is administered, compared to a subject to which the vaccine is not administered. These IgG antibodies may be specific to tumor antigens. These IgG antibodies may react with tumor antigens. Compared to a subject to which the vaccine is not administered, the level of IgG antibodies associated with the subject to which the vaccine is administered may increase by about 1.5 times to about 16 times, about 2 times to about 12 times, or about 3 times to about 10 times. The level of IgG antibodies associated with subjects administered the vaccine can be increased by at least about 1.5 times, at least about 2.0 times, at least about 2.5 times, at least about 3.0 times, at least about 3.5 times, at least about 4.0 times, at least about 4.5 times, at least about 5.0 times, at least about 5.5 times, at least about 6.0 times, at least about 6.5 times, at least about 7.0 times, at least about 7.5 times, at least about 8.0 times, at least about 8.5 times, at least about 9.0 times, at least about 9.5 times, at least about 10.0 times, at least about 10.5 times, at least about 11.0 times, at least about 11.5 times, at least about 12.0 times, at least about 12.5 times, at least about 13.0 times, at least about 13.5 times, at least about 14.0 times, at least about 14.5 times, at least about 15.0 times, at least about 15.5 times, or at least about 16.0 times compared to subjects not administered the vaccine.

[0273] The vaccine may induce a cellular immune response in a subject to which the vaccine is administered. The induced cellular immune response may be specific to a tumor antigen. The induced cellular immune response may be responsive to a tumor antigen. The induced cellular immune response may include eliciting CD8 + T cell response. Triggered CD8 + T cell responses can react to tumor antigens. + T cell responses can be multifunctional. The induced cellular immune response can include eliciting CD8 + T cell responses, including CD8 + T cells produce interferon-γ (IFN-γ), tumor necrosis factor α (TNF-α), interleukin-2 (IL-2), or a combination of IFN-γ and TNF-α.

[0274] The induced cellular immune response may include increased CD8 +T cell responses increased. CD8 + The T cell response can be increased by about 2-fold to about 30-fold, about 3-fold to about 25-fold, or about 4-fold to about 20-fold. The CD8 T cell response associated with the subject administered the vaccine is increased compared to the subject not administered the vaccine. + The T cell response can be increased by at least about 1.5 fold, at least about 2.0 fold, at least about 3.0 fold, at least about 4.0 fold, at least about 5.0 fold, at least about 6.0 fold, at least about 6.5 fold, at least about 7.0 fold, at least about 7.5 fold, at least about 8.0 fold, at least about 8.5 fold, at least about 9.0 fold, at least about 9.5 fold, at least about 10.0 fold, at least about 10.5 fold, at least about 11.0 fold, at least about 11.5 fold, at least about 12.0 fold, at least about 12.5 fold, at least about 13.0 fold, at least about 14.0 fold, at least about 15.0 fold, at least about 16.0 fold, at least about 17.5 fold, at least about 18.0 fold, at least about 19.0 fold, at least about 20.0 fold, at least about 21.0 fold, at least about 22.0 fold, at least about 23.0 fold, at least about 24.0 fold, at least about 25.0 fold, at least about 26.0 fold, at least about 27.0 fold, at least about 28.0 fold, at least about 29.0 fold, at least about 30.0 fold, at least about 31.0 fold, at least about 32.0 fold, at least about 33.0 fold, times, at least about 13.5 times, at least about 14.0 times, at least about 14.5 times, at least about 15.0 times, at least about 16.0 times, at least about 17.0 times, at least about 18.0 times, at least about 19.0 times, at least about 20.0 times, at least about 21.0 times, at least about 22.0 times, at least about 23.0 times, at least about 24.0 times, at least about 25.0 times, at least about 26.0 times, at least about 27.0 times, at least about 28.0 times, at least about 29.0 times, or at least about 30.0 times.

[0275] The induced cellular immune response may include CD3 producing IFNγ + CD8 + The frequency of T cells was increased in the subjects who received the vaccine compared to the subjects who did not receive the vaccine. + CD8 + IFN-γ + The frequency of T cells can be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold.

[0276] The induced cellular immune response may include CD3 producing TNF-α + CD8 + The frequency of T cells was increased in the subjects who received the vaccine compared to the subjects who did not receive the vaccine. + CD8 + TNF-α + The frequency of T cells can be increased at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, or 14-fold.

[0277] The induced cellular immune response may include CD3 producing IL-2+ CD8 + The frequency of T cells was increased in the subjects who received the vaccine compared to the subjects who did not receive the vaccine. + CD8 + IL-2 + The frequency of T cells may be increased by at least about 0.5-fold, 1.0-fold, 1.5-fold, 2.0-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, or 5.0-fold.

[0278] The induced cellular immune response may include CD3+ production of both IFN-γ and TNF-α + CD8 + The frequency of T cells was increased in the subjects who received the vaccine compared to the subjects who did not receive the vaccine. + CD8 + IFN-γ + TNF-α + The frequency of T cells can be increased by at least about 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 170-fold, or 180-fold.

[0279] Cellular immune responses induced by vaccines can include eliciting CD4 + T cell response. Initiation of CD4 + T cell responses can react to tumor antigens. + T cell responses can be multifunctional. The induced cellular immune response can include eliciting CD4 + T cell responses, including CD4 + T cells produce IFN-γ, TNF-α, IL-2, or a combination of IFN-γ and TNF-α.

[0280] The induced cellular immune response may include CD3 producing IFNγ + CD4 + The frequency of T cells was increased in the subjects who received the vaccine compared to the subjects who did not receive the vaccine. + CD4 + IFN-γ + The frequency of T cells can be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold.

[0281] The induced cellular immune response may include CD3 producing TNF-α + CD4 + The frequency of T cells was increased in the subjects who received the vaccine compared to the subjects who did not receive the vaccine. + CD4 + TNF-α + The frequency of T cells can be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, or 22-fold.

[0282] The induced cellular immune response may include CD3 producing IL-2 + CD4 + The frequency of T cells was increased in the subjects who received the vaccine compared to the subjects who did not receive the vaccine. + CD4 + IL-2 + The frequency of T cells can be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold, 39-fold, 40-fold, 45-fold, 50-fold, 55-fold, or 60-fold.

[0283] The induced cellular immune response may include CD3+ production of both IFN-γ and TNF-α + CD4 + The frequency of T cells was increased in the subjects who received the vaccine compared to the subjects who did not receive the vaccine. + CD4 + IFN-γ + TNF-α +The frequency of can be increased by at least about 2 times, 2.5 times, 3.0 times, 3.5 times, 4.0 times, 4.5 times, 5.0 times, 5.5 times, 6.0 times, 6.5 times, 7.0 times, 7.5 times, 8.0 times, 8.5 times, 9.0 times, 9.5 times, 10.0 times, 10.5 times, 11.0 times, 11.5 times, 12.0 times, 12.5 times, 13.0 times, 13.5 times, 1 4.0 times, 14.5 times, 15.0 times, 15.5 times, 16.0 times, 16.5 times, 17.0 times, 17.5 times, 18.0 times, 18.5 times, 19.0 times, 19.5 times, 20.0 times, 21 times, 22 times, 23 times, 24 times, 25 times, 26 times, 27 times, 28 times, 29 times, 30 times, 31 times, 32 times, 33 times, 34 times, or 35 times.

[0284] The vaccines of the present invention may have the characteristics desired for an effective vaccine, such as safety so that the vaccine itself does not cause disease or death; protection against disease caused by exposure to live pathogens such as viruses or bacteria; induction of neutralizing antibodies to protect cells; induction of protective T cells against intracellular pathogens; and providing ease of administration, few side effects, biological stability, and low cost per dose.

[0285] The immune response can be further induced when the vaccine is injected into different tissues such as muscle or skin.

[0286] Tumor Antigens

[0287] As described above, in some embodiments, the combination vaccine of the present invention comprises a combination of an anti-Siglec-9 antibody or a nucleic acid molecule encoding the same and a tumor antigen, a fragment thereof, a variant thereof, a nucleic acid molecule encoding the same, or a combination thereof.

[0288] Therefore, in some embodiments, the present invention includes a composition for enhancing an immune response to an antigen in a subject in need thereof, comprising a sialic acid binding receptor antibody of the present invention in combination with a synthetic antigen capable of generating an immune response in the subject, or a biologically functional fragment or variant thereof, or a nucleic acid molecule encoding the same.

[0289] In one embodiment, the antigen is a tumor-associated surface antigen. Illustrative examples of tumor-associated surface antigens are CD10, CD19, CD20, CD22, CD33, Fms-like tyrosine kinase 3 (FLT-3, CD135), chondroitin sulfate proteoglycan 4 (CSPG4, melanoma-associated chondroitin sulfate proteoglycan), epidermal growth factor receptor (EGFR), Her2neu, Her3, IGFR, CD133, IL3R, fibroblast activation protein (FAP), CDCP1, Derlin1, Tenascin, Frizzled protein 1-10, vascular antigen VEGFR2 (KDR / FLK1), VEGFR3 (FLT4, CD309), PDGFR-α (CD140a), PDGFR-β (CD140b), Endoglin, CLEC14, Tem1-8 and Tie2. Further examples may include A33, CAMPATH-1 (CDw52), carcinoembryonic antigen (CEA), carbonic anhydrase IX (MN / CA IX), CD21, CD25, CD30, CD34, CD37, CD44v6, CD45, CD133, de2-7EGFR, EGFRvIII, EpCAM, Ep-CAM, folate-binding protein, G250, Fms-like tyrosine kinase 3 (FLT-3, CD135), c-Kit (CD117), CSF1R (CD115), HLA-DR, IGFR, IL-2 receptor, IL3R, MCSP (melanoma-associated cell surface chondroitin sulfate proteoglycan), Muc-1, prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), prostate-specific antigen (PSA) and TAG-72. Examples of antigens expressed on the extracellular matrix of tumors are tenascin and fibroblast activation protein (FAP).

[0290] In the context of the present invention, a "tumor antigen" or "hyperproliferative disorder antigen" or "antigen associated with a hyperproliferative disorder" refers to an antigen that is common to a particular hyperproliferative disorder, such as cancer. The antigens discussed herein are included by way of example only. This list is not intended to be exclusive, and other examples will be apparent to those skilled in the art.

[0291] Tumor antigens are proteins produced by tumor cells that elicit an immune response, particularly a T cell mediated immune response.The choice of antigen binding portion of the invention will depend on the particular type of cancer to be treated. Tumor antigens are well known in the art and include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostate cancer-associated protein (prostein), PSMA, Her2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin.

[0292] In one embodiment, tumor antigens include more than one antigenic cancer epitope associated with a malignant tumor. Malignant tumors express many proteins that can be used as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens, such as MART-1, tyrosinase and GP 100 in melanoma and prostate acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the transformation-related molecule group, such as oncogene HER-2 / Neu / ErbB-2. Another group of target antigens is onco-fetal antigen, such as carcinoembryonic antigen (CEA). In B-cell lymphoma, tumor-specific idiotype immunoglobulin constitutes a true tumor-specific immunoglobulin antigen, which is unique to a single tumor. B cell differentiation antigens such as CD19, CD20 and CD37 are other candidates for target antigens in B-cell lymphoma. Some of these antigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targets for passive immunotherapy using monoclonal antibodies, but the success rate is limited.

[0293] The types of tumor antigens mentioned in the present invention may also be tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs). TSAs are specific to tumor cells and do not appear on other cells in the body. TAA-related antigens are not specific to tumor cells, but are also expressed on normal cells under conditions where a state of immune tolerance to the antigen cannot be induced. The expression of antigens on tumors can occur under conditions that enable the immune system to respond to the antigen. TAAs may be antigens expressed on normal cells during fetal development, when the immune system is immature and unable to respond, or they may be antigens that are usually present at very low levels on normal cells but expressed at much higher levels on tumor cells.

[0294] Non-limiting examples of TSA or TAA antigens include the following: differentiation antigens, such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multi-lineage antigens, such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens, such as CEA; overexpressed oncogenes and mutated tumor suppressor genes, such as p53, Ras, HER-2 / neu; unique tumor antigens caused by chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as Epstein Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA19-9, CA72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, β-HCG, BCA225, BTAA, CA125, CA 15-3\CA 27.29\BCAA, CA 195, CA242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP and TPS.

[0295] In one embodiment, the synthetic antigen is Muc-1.

[0296] Methods of delivering the composition

[0297] The present invention also relates to methods of delivering the composition to a subject in need thereof. The method of delivering may include administering the composition to a subject.

[0298] The mammal to which the composition is delivered can be a human, a primate, a non-human primate, a cow, a cattle, a sheep, a goat, an antelope, a bison, a buffalo, a bison, bovids, a deer, a hedgehog, an elephant, a camel, an alpaca, a mouse, a rat, and a chicken.

[0299] The composition can be administered by different routes, including oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, by inhalation, buccal administration, intrapleural, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal, intranasal, intrathecal and intraarticular or a combination thereof. For veterinary use, the composition can be administered in a suitable acceptable formulation in accordance with normal veterinary practice. The veterinarian can easily determine the dosage regimen and route of administration that is most suitable for a particular animal.

[0300] Treatment

[0301] In one embodiment, the invention provides a method of treating or preventing a disease or condition that would benefit from an increase in NK cell function or activity. Exemplary diseases and conditions that can be treated using the compositions and methods of the invention include, but are not limited to, cancer and infectious diseases.

[0302] The following are non-limiting examples of cancers that can be diagnosed or treated by the disclosed methods and compositions: acute lymphocytic leukemia, acute myeloid leukemia, adrenocortical carcinoma, appendix cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain and spinal cord tumors, brain stem glioma, brain tumors, breast cancer, bronchial tumors, Burkitt's lymphoma, carcinoid tumors, atypical teratoid / rhabdoid tumors of the central nervous system, embryonal tumors of the central nervous system, lymphomas of the central nervous system, cerebellar astrocytoma, cerebral astrocytoma / glioblast ... cell tumors, extrahepatic bile duct cancer, extrahepatic cancer, eye cancer, fungoides, gallbladder cancer, gastric cancer, gastrointestinal cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (gist), germ cell tumors, pregnancy cancer, gestational trophoblastic tumors, glioblastoma, glioma, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway gliomas, hypothalamic tumors, Intraocular (eye) cancer, intraocular melanoma, islet cell tumor, Kaposi's sarcoma, kidney (renal cell) cancer, Langerhans cell carcinoma, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer, lymphoma, macroglobulinemia, malignant fibrous histiocytoma and osteosarcoma of bone, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, mesothelioma, primary occult metastatic squamous neck cancer, mouth cancer (mouth cancer), multiple endocrine neoplasia syndrome, multiple myeloma, mycosis, myelodysplastic syndrome, myelodysplastic / myeloproliferative disease, myeloid leukemia, myeloid leukemia, myeloma, myeloproliferative disease, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oral cavity cancer, oropharyngeal cancercancer), osteosarcoma and malignant fibrous histiocytoma, osteosarcoma and malignant fibrous histiocytoma of bone, ovary, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low-grade malignant potential tumor, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, moderately differentiated pineal parenchymal tumor, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasma cell tumor, plasma cell tumor / multiple myeloma, pleuropulmonary blastoma, primary central nervous system cancer, primary central nervous system lymphoma, prostate cancer, colorectal cancer, renal cell (kidney) cancer, renal pelvis and ureter cancer, respiratory tract cancer involving the nut gene on chromosome 15, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sezary syndrome, skin cancer (melanoma), skin cancer (non-melanoma), skin cancer (skin carcinoma), small cell lung cancer, small intestine cancer, soft tissue cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer, gastric cancer, supratentorial primitive neuroectodermal tumor, supratentorial primitive neuroectodermal tumor and pineoblastoma, T-cell lymphoma, testicular cancer, laryngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumor, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, optic pathway and hypothalamic gliomas, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

[0303] In one embodiment, the cancer has a mutation in a cancer driver gene as described in Table 1. In one embodiment, the cancer is resistant to more than one drug listed in Table 1.

[0304] In one embodiment, the composition is used to treat cancers with high sialic acid levels. Cancers associated with high sialic acid levels include, but are not limited to, ovarian cancer, melanoma, renal cell carcinoma, prostate cancer, colon cancer, breast cancer, head and neck squamous cell carcinoma, skin cancer, and oral cancer.

[0305] Bacterial infection

[0306] In one embodiment, the infectious disease or condition is associated with bacteria. In some embodiments, the bacteria can be from any of the following phyla: Acidobacteria, Actinobacteria, Aquificae, Bacteroidetes, Caldiserica, Chlamydiae, Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus-Thermus, Dictyoglomi, Elusimi crobia, Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes, Lentisphaerae, Nitrospira, Planctomycetes, Proteobacteria, Spirochaetes, Synergistetes, Tenericutes, Thermodesulfobacteria, Thermotogae, and Verrucomicrobia.

[0307] The bacteria may be gram-positive or gram-negative. The bacteria may be aerobic or anaerobic. The bacteria may be autotrophic or heterotrophic. The bacteria may be mesophilic, neutrophile, extremophilic, acidophilic, alkaliphilic, thermophilic, psychrophilic, halophilic or osmophilic.

[0308] The bacteria may be anthrax bacteria, antibiotic-resistant bacteria, pathogenic bacteria, food poisoning bacteria, infectious bacteria, Salmonella bacterium, Staphylococcus bacterium, Streptococcus bacterium or tetanus bacterium. The bacteria may be mycobacteria, Clostridium tetani, Yersinia pestis, Bacillus anthracis, methicillin-resistant Staphylococcus aureus (MRSA), Clostridium difficile.

[0309] Viral infection

[0310] In one embodiment, the infectious disease or condition is associated with bacteria. In some embodiments, the virus is from one of the following families: Adenoviridae, Arenaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Papovaviridae, Paramyxoviridae, Parvoviridae, Picornaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, or Togaviridae. Viral antigens may be derived from human immunodeficiency virus (HIV), chikungunya virus (CHIKV), dengue virus, papillomaviruses such as human papillomavirus (HPV), poliovirus, hepatitis viruses such as hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV) and hepatitis E virus (HEV), smallpox virus (variola major and variola minor), vaccinia virus, influenza virus, rhinovirus, equine encephalitis virus, rubella virus, yellow fever virus, Norwalk virus (Norwalk virus), swine flu virus, erythritis virus, rubella virus, yellow fever virus, leukemia virus, leukopenia virus, leukemia ... virus), hepatitis A virus, human T-cell leukemia virus (HTLV-I), hairy cell leukemia virus (HTLV-II), California encephalitis virus, hantavirus (hemorrhagic fever), rabies virus, Ebola virus, Marburg virus, measles virus, mumps virus, respiratory syncytial virus (RSV), herpes simplex 1 (oral herpes), herpes simplex 2 (genital herpes), herpes zoster (varicella-zoster, also known as chickenpox), cytomegalovirus (CMV) such as human CMV, Epstein-Barr virus (EBV), flavivirus, foot-and-mouth disease virus, Lassa virus, arenavirus, severe acute respiratory syndrome-associated coronavirus (SARS), Middle East respiratory syndrome-associated coronavirus (MERS), severe acute respiratory syndrome-associated coronavirus 2 (SARS CoV2), or oncoviruses.

[0311] parasitic infection

[0312] In one embodiment, the infectious disease or condition is associated with a parasite. In some embodiments, the parasite may be a protozoan, a worm, or an ectoparasite. A worm (i.e., a flatworm (e.g., a fluke and a tapeworm), an acanthocephalic worm, or a round worm (e.g., a pinworm). An ectoparasite may be a louse, a flea, a tick, and a mite.

[0313] The parasite may be any parasite that causes any of the following diseases: Acanthamoeba keratitis, Amoebiasis, Ascariasis, Babesiosis, Balantidiasis, Baylisascariasis, Chagas disease disease), Clonorchiasis, Cochliomyia, Cryptosporidiosis, Diphyllobothriasis, Dracunculiasis, Echinococcosis, Elephantiasis, Enterobiasis, Fascioliasis, Fasciolopsiasis, Filariasis, Giardiasis, Gnathostomiasis, Hymenolepiasis, Isosporiasis, Katayama fever, Leishmaniasis, Lyme disease disease, Malaria, Metagonimiasis, Myiasis, Onchocerciasis, Pediculosis, Scabies, Schistosomiasis, Sleepingsickness, Strongyloidiasis, Taeniasis, Toxocariasis, Toxoplasmosis, Trichinosis, and Trichuriasis.

[0314] The parasites may be Acanthamoeba, Anisakis, Ascarislumbricoides, Botfly, Balantidium coli, Bedbug, Cestoda (tapeworms), Chiggers, Cochliomyia hominivorax, Entamoeba histolytica, Fasciola hepatica, Giardialamblia, Hookworm, Leishmania, Linguatula serrata, Liverfluke, Loa loa, Paragonimus-lung fluke, Pinworm, Plasmodium falciparum falciparum, Schistosoma, Strongyloides stercoralis, mites, tapeworms, Toxoplasma gondii, Trypanosoma, whipworms, or Wuchereria bancrofti.

[0315] Fungal infections

[0316] In one embodiment, the infectious disease or condition is associated with a fungus. In some embodiments, the fungus may be an Aspergillus species, Blastomyces dermatitidis, a Candida yeast (e.g., Candida albicans), Coccidioides, Cryptococcus neoformans, Cryptococcus gattii, a dermatophyte, a Fusarium species, Histoplasma capsulatum, Mucoromycotina, Pneumocystis jirovecii, Sporothrix schenckii, Exserohilum, or Cladosporium.

[0317] PD-(L)1 axis inhibitors

[0318] In some embodiments, the present invention provides a combination of an anti-Siglec-9 antibody of the present invention and a PD-(L)1 axis inhibitor. In various embodiments, the composition comprises an inhibitor of more than one gene or protein in the PD-(L)1 axis. In various embodiments, the present invention includes compositions and methods for reducing the level or activity of more than one gene or protein in the PD-(L)1 axis.

[0319] Based on the disclosure provided herein, those skilled in the art will understand that the reduction in the level or activity of one or more genes or proteins in the PD-(L)1 axis encompasses a reduction in the expression of a biomarker, including transcription, translation, or both. Once a skilled person in the art has mastered the teachings of the present invention, it will also be understood that the reduction in the level or activity of one or more genes or proteins in the PD-(L)1 axis includes a reduction in the amount of a polypeptide, a reduction in the amount of mRNA, a reduction in transcription, a reduction in translation, or a combination thereof; and it also includes reducing any activity of one or more genes or proteins in the PD-(L)1 axis.

[0320] 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, fragment of an antibody, fusion protein, aptamer, peptide and small molecule.

[0321] Based on the disclosure provided herein, one skilled in the art will appreciate that one method of reducing the mRNA and / or protein levels of more than one PD-(L)1 axis protein in a cell is to reduce or inhibit the expression of a nucleic acid encoding the PD-(L)1 axis protein. Therefore, molecules or compounds that inhibit or reduce gene expression (e.g., such as siRNA, antisense molecules, or ribozymes) can be used to reduce the protein level of the PD-(L)1 axis protein in a cell. However, the present invention should not be limited to these examples.

[0322] In one embodiment, RNAi is used to reduce the level or activity of PD-(L)1 axis protein. RNA interference (RNAi) is a phenomenon in which the introduction of double-stranded RNA (dsRNA) into a diverse range of organisms and cell types results in the degradation of complementary mRNA. In cells, long dsRNAs are cut into short 21-25 nucleotide small interfering RNAs or siRNAs by a ribonuclease called Dicer. The siRNA is then assembled with protein components into an RNA-induced silencing complex (RISC), unwinding in the process. The activated RISC then binds to the complementary transcript through base pairing interactions between the siRNA antisense strand and the mRNA. The bound mRNA is cut, and sequence-specific degradation of the mRNA leads to gene silencing. Chemical modification of siRNA can facilitate intravenous systemic delivery. Optimizing siRNA involves consideration of the overall G / C content, the C / T content at the end, the Tm, and the nucleotide content of the 3' overhang. Therefore, the present invention also includes a method for reducing the level of more than one PD-(L)1 axis protein using RNAi technology.

[0323] In some embodiments, the present invention includes an isolated nucleic acid encoding an inhibitor, such as a protein, antibody, siRNA, or antisense molecule, which is operably linked to a nucleic acid comprising a promoter / regulatory sequence such that the nucleic acid is preferably capable of directing the expression of the inhibitor encoded by the nucleic acid. Thus, the present invention encompasses expression vectors and methods for introducing exogenous DNA into cells and concomitantly expressing the exogenous DNA in cells.

[0324] In order to evaluate the expression of inhibitor, the expression vector to be introduced into the cell can also contain a selective marker gene or a reporter gene or both, so that from attempting to identify and select expressing cells by viral vector transfection or infected cell mass. In other embodiments, the selective marker can be carried on an independent DNA fragment and used for cotransfection procedures. Both sides of the selective marker and the reporter gene can have suitable regulatory sequences so that it can be expressed in host cells. Useful selective markers are known in the art and include, for example, antibiotic resistance genes, such as neomycin.

[0325] When the inhibitor of the present invention is a small molecule, standard methods known to those skilled in the art can be used to obtain small molecule antagonists. Such methods include chemical organic synthesis or biological means. Biological means include purification from biological sources, recombinant synthesis and in vitro translation systems using methods well known in the art.

[0326] Combinatorial libraries of molecularly diverse chemical compounds potentially useful for treating various diseases and conditions are well known in the art, as are methods for preparing the libraries. The methods can use various techniques well known to those skilled in the art, including solid phase synthesis, solution methods, parallel synthesis of single compounds, synthesis of chemical mixtures, rigid core structures, flexible linear sequences, deconvolution strategies, labeling techniques, and generation of unbiased molecular landscapes for lead discovery relative to biased structures for lead development.

[0327] In the general method of small library synthesis, the activated core molecule is condensed with many building blocks to obtain a combinatorial library of covalently linked core-building blocks. The shape and rigidity of the core determine the orientation of the building blocks in shape space. The library can be biased towards the biological structure characterized by targeting ("focused library") by changing the core, connection or building blocks, or a flexible core can be used to synthesize the library with less structural bias.

[0328] In another aspect of the invention, one or more proteins in the PD-(L)1 axis can be inhibited by inactivating and / or sequestering the protein(s). Thus, the effect of inhibiting one or more proteins in the PD-(L)1 axis can be achieved by using a trans-dominant negative mutant.

[0329] In one embodiment, antibodies specific for more than one protein in the PD-(L)1 axis can be used. As will be appreciated by those skilled in the art, any antibody that can recognize and bind to an antigen of interest can be used in the present invention. Methods for preparing and using antibodies are well known in the art. For example, polyclonal antibodies useful in the present invention are produced by immunizing rabbits according to standard immunological techniques well known in the art. Such techniques include immunizing animals with chimeric proteins comprising a portion of another protein, such as a maltose binding protein or glutathione (GSH) tag polypeptide portion, and / or a portion that renders the antigen protein of interest immunogenic (e.g., an antigen of interest conjugated to keyhole limpet hemocyanin (KLH)) and a portion comprising amino acid residues of the corresponding antigen protein. The chimeric protein is produced by cloning a suitable nucleic acid encoding the marker protein into a plasmid vector suitable for this purpose (e.g., but not limited to, pMAL-2 or pCMX).

[0330] Exemplary anti-PD-(L)1 axis antibodies include, but are not limited to, nivolumab Pembrolizumab sintilimab, cemiplimab tripolibamab, tislelizumab, spartalizumab, camrelizumab, dostralimab, genolimzumab, or cetrelimab, or an antibody that binds to PD-L1, such as envafolimab, atezolizumab, or Durvalumab Avelumab REGN2810, pidilizumab, MEDI0680, PDR001, PF-06801591, BGB-A317, TSR-042, and SHR-1210.

[0331] Cancer Therapy

[0332] In one embodiment, the invention provides a method for treating or preventing cancer, or a method for treating and preventing the growth or metastasis of a tumor. Related aspects of the invention provide methods for preventing, assisting in the prevention and / or reducing the metastasis of hyperplastic or tumor cells in an individual.

[0333] In one embodiment, the composition is used to treat cancers with high sialic acid levels, including but not limited to ovarian cancer, melanoma, renal cell carcinoma, prostate cancer, colon cancer, breast cancer, head and neck squamous cell carcinoma, and oral cancer.

[0334] One aspect of the invention provides a method of inhibiting metastasis in an individual in need thereof, the method comprising administering to the individual an effective amount of a nucleic acid molecule encoding a multivalent antibody of the invention, wherein the multivalent antibody is specific for the cancer to be treated. The invention also provides a method of inhibiting metastasis in an individual in need thereof, the method comprising administering to the individual an effective metastasis-inhibiting amount of a nucleic acid molecule encoding a multivalent antibody of the invention, wherein the multivalent antibody is specific for the cancer to be treated.

[0335] In some embodiments of treating or preventing cancer in an individual in need thereof or treating and preventing tumor metastasis in an individual in need thereof, a second agent, such as an anti-tumor agent, is administered to the individual. In some embodiments, the second agent comprises a second metastasis inhibitor, such as a plasminogen antagonist or an adenosine deaminase antagonist. In other embodiments, the second agent is an angiogenesis inhibitor.

[0336] The compositions of the present invention can be used to prevent, alleviate, minimize, control and / or reduce cancer in humans and animals. The compositions of the present invention can also be used to slow the growth rate of primary tumors. The compositions of the present invention can be used to prevent the spread of cancer cells when administered to a subject in need of treatment. Therefore, an effective amount of nucleic acid molecules encoding the multivalent antibodies of the present invention (wherein the multivalent antibodies are specific to the cancer to be treated) can be administered as part of a combination therapy with more than one drug or other agent. When used as part of a combination therapy, the reduction in metastasis and reduction in primary tumor growth provided by the compositions of the present invention allow for more effective and efficient use of any drug or drug therapy used to treat a patient. In addition, the control of metastasis by the compositions of the present invention provides a greater possibility for the subject to concentrate the disease in one location.

[0337] In one embodiment, the invention provides a method of treating cancer metastasis, the method comprising treating the subject with a complementary cancer therapy such as surgery, chemotherapy, chemotherapeutic agents, radiation therapy or hormonal therapy, or a combination thereof, prior to, concurrently with, or after treatment with a composition of the invention.

[0338] Chemotherapeutic agents include cytotoxic agents (e.g., 5-fluorouracil, cisplatin, carboplatin, methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, oxorubicin, carmustine (BCNU), lomustine (CCNU), cytarabine USP, cyclophosphamide, estramustine sodium phosphate, altretamine, hydroxyurea, ifosfamide, procarbazine, mitomycin, busulfan, cyclophosphamide, mitoxantrone, carboplatin, cisplatin, interferon alpha-2a recombinant, paclitaxel, teniposide, and streptozoci), cytotoxic alkylating agents (e.g., busulfan, chlorambucil, cyclophosphamide, melphalan, or ethionine), alkylating agents (e.g., asaley, AZQ, BCNU, busulfan, bisulphan, carboxyplatinum phthalate carboxyphthalatoplatinum, CBDCA, CCNU, CHIP, chlorambucil, chlorozotocin, cis-platinum, clomesone, cyanomorpholinodoxorubicin, cyclodisone, cyclophosphamide, dianhydrogalactitol, fluorodopan, hepsulfam, hydroxylamine lucanthone, ifosfamide, melphalan, methyl CCNU, mitomycin C, mitozolamide, nitrogen mustard, PCNU, piperazine, piperazine dione, pipobroman, porphyromycin, spirohydantoin mustard), streptozotocin, thiroxilon, tetraplatin, thiotepa, triethylene melamine, uracil nitrogen mustard, and Yoshi-864), antimitotic agents (e.g., allocolchicine, halichondrin M, colchicine, colchicine derivatives, Aplysia 10, maytansine, rhizoxin, paclitaxel derivatives, paclitaxel, thiocolchicine, tritylcysteine, vinblastine sulfate, and vincristine sulfate), plant biopharmaceuticals Bases (e.g., actinomycin D, bleomycin, L-asparaginase, idarubicin, vinblastine sulfate, vincristine sulfate, mitramycin, mitomycin, daunomycin, VP-16-213, VM-26, vinorelbine, and taxotere), biologics (e.g., interferon alpha, BCG, G-CSF, GM-CSF, and interleukin-2), topoisomerase I inhibitors (e.g., camptothecin, camptothecin derivatives, and morpholinodoxorubicin), topoisomerase II inhibitors (e.g.,mitoxantrone, aminafide, m-AMSA, anthrapyrazole derivatives, pyrazoloacridine, bisantrene hydrochloride, daunomycin, deoxydoxorubicin, menolide, N,N-dibenzyldaunomycin, oxanthrazole, rubidazone, VM-26 and VP-16), and synthetic preparations (e.g., hydroxyurea, procarbazine, o,p'-DDD, dacarbazine, CCNU, BCNU, cis-diaminedichloroplatinum, mitoxantrone, CBDCA, levamisole, hexamethylmelamine, all-trans retinoic acid, gliadel and porfimer sodium).

[0339] Antiproliferative agents are compounds that reduce the proliferation of cells. Antiproliferative agents include alkylating agents, antimetabolites, enzymes, biological response modifiers, miscellaneous agents, hormones and antagonists, androgen inhibitors (e.g., flutamide and leuprolide acetate), antiestrogens (e.g., tamoxifen citrate and its analogs, toremifene, droloxifene and raloxifene), and other examples of specific antiproliferative agents include, but are not limited to, levamisole, gallium nitrate, granisetron, sargramostim strontium-89 chloride, filgrastim, pilocarpine, dexrazoxane and ondansetron.

[0340] The compounds of the present invention can be administered alone or in combination with other anti-tumor agents, which include cytotoxic / antitumor agents and anti-angiogenic agents. Cytotoxic / antitumor agents are defined as agents that attack and kill cancer cells. Some cytotoxic / antitumor agents are alkylating agents that alkylate the genetic material in tumor cells, such as cisplatin, cyclophosphamide, nitrogen mustard, trimethylene thiophosphoramide, carmustine, busulfan, chlorambucil, belustine, uracil mustard, chlomaphazin and dacarbazine. Other cytotoxic / antitumor agents are antimetabolites for tumor cells, such as cytosine arabinoside, fluorouracil, methotrexate, mercaptopurine, thiopurine and methyl procarbazine. Other cytotoxic / antitumor drugs include antibiotics, such as doxorubicin, bleomycin, actinomycin, daunorubicin, mithramycin, mitomycin, mitomycin C and daunorubicin. There are many commercially available liposomal formulations of these compounds. Other cytotoxic / antitumor agents are mitotic inhibitors (vinca alkaloids). These include vincristine, vinblastine, and etoposide. Other cytotoxic / antitumor agents include paclitaxel and its derivatives, L-asparaginase, antitumor antibodies, dacarbazine, azacytidine, amsacrine, melphalan, VM-26, ifosfamide, mitoxantrone, and vindesine.

[0341] Anti-angiogenic agents are well known to those skilled in the art. Anti-angiogenic agents suitable for use in the methods and compositions of the present invention include anti-VEGF antibodies (including humanized and chimeric antibodies), anti-VEGF aptamers, and antisense oligonucleotides. Other known angiogenesis inhibitors include angiostatin, endostatin, interferon, interleukin 1 (including α and β), interleukin 12, retinoic acid, and tissue inhibitors of metalloproteinases-1 and -2 (TIMP-1 and -2). Small molecules, including topoisomerases, such as razoxane, a topoisomerase II inhibitor with anti-angiogenic activity, may also be used.

[0342] Other anticancer agents that can be used in combination with the compositions of the present invention include, but are not limited to: acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; binasafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride hydrochloride); decitabine; dexormaplatin;dezaguanine; dezaguanine mesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflornithine hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine sodium phosphate; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride hydrochloride); ifosfamide; ilmofosine; interleukin II (including recombinant interleukin II, or rIL2), interferon α-2a; interferon α-2b; interferon α-n1; interferon α-n3; interferon β-I a; interferon γ-I b; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate;liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride; megestrolacetate; melengestrol acetate acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid acid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium sodium); porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safingol;Safingol hydrochloride; semustine; simtrazene; sparrosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalansodium; tegafur; teloxantrone hydrochloride hydrochloride; temoporfin; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; hydrochloride; uracilmustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole;Zeniplatin; zinostatin; zorubicin hydrochloride. Other anticancer drugs include, but are not limited to: 20-epi-1,25-dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; acid; amrubicin; amsacrine; anagrelide; anastrozole; and andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen, prostate cancer; antiestrogens; antineoplastons; antisense oligonucleotides; aphidicolin glycine glycinate; apoptosis gene regulator; apoptosis regulator; apurinic nucleic acid (apurinic nucleic acid); ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR / ABL antagonists; benzochlorins; benzoylstaurosporine; β-lactam derivatives; β-alethine; betaclamycin B; betulinic acid acid); bFGF inhibitors; bicalutamide; bisantrene; bisaziridinylspermine;binafide; bistratene A; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN700; cartilage-derived inhibitor; carzelesin; casein kinase inhibitor (ICOS); castanospermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogs; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogs; conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox;diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenylspiromustine spiromustine; docetaxel; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride; estramustine analogs; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezlastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylenebisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat;imidazoacridones; imiquimod; immunostimulatory peptides; insulin-like growth factor-1 receptor inhibitors; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-Ntriacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate sulfate; leptolstatin; letrozole; leukemia inhibitory factor; interferon alpha; leuprolide + estrogen + progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogs; lipophilic disaccharide peptides; lipophilic platinum compounds; lissoclinamide 7 7); lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetiumtexaphyrin; lysofylline; cleaved peptide; maitansine; mannostatin A A); marimastat; masoprocol; maspin; matrixlysin inhibitor; matrix metalloproteinase inhibitor; menogaril; merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; mirimostim; mismatched double-stranded RNA; mitoguaone;Mitolactin; mitomycin analog; mitonafide; mitotoxinfibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotropin; monophosphoryl lipid A+ mycobacterial cell wall sk; mopidamol; multidrug resistance gene inhibitor; multiple tumor suppressor 1-based therapy; nitrogen mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyl narelin; N-substituted benzamides; nafarelin; nagrestip; naloxone + pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidants; nitrullyn; O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducers; ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogs; paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; sodium pentosan polysulfate; pentostatin; pentrozole; perflubron; perfosfamide;perillyl alcohol; phenazinomycin; phenyl acetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitors; platinum complexes; platinum compounds; platinum-triamine complexes; porfimer sodium; porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immunomodulators; protein kinase C inhibitors; protein kinase C inhibitors, microalgae; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugates hemoglobin polyoxyethylene conjugate; raf antagonist; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitor; ras inhibitor; ras-GAP inhibitor; retelliptine demethylated; rhenium etidronate Re186; rhizoxin; ribozyme; RII retinamide; rogletimide; rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetic; semustine; senescence-derived inhibitor 1; sense oligonucleotide; signal transduction inhibitor; signal transduction regulator; single-chain antigen binding protein; sizofiran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparsosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem cell division inhibitor; stipiamide; stromelysin inhibitor; sulfinosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycan; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetics; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene dichloride bichloride; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector systems, erythrocyte gene therapy; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin;Vorozole; zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer. In one embodiment, the anticancer drug is 5-fluorouracil, paclitaxel, or leucovorin. ;

[0343] Combination

[0344] In some embodiments, the present invention provides a method for treating, protecting against, and / or preventing cancer or a cancer-related disease or condition in a subject in need thereof, by administering a combination of a composition comprising a synthetic Siglec-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.

[0345] 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 so that the combination of anti-Siglec-7 and tumor antigen are both present in the subject. In one embodiment, the method can include administering a first composition of the invention comprising a synthetic Siglec-7 antibody, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same by any method described in detail elsewhere herein, and administering a second composition comprising a tumor antigen, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same less than 1 day, less than 2 days, less than 3 days, less than 4 days, less than 5 days, less than 6 days, less than 7 days, less than 8 days, less than 9 days, or less than 10 days after administration of the synthetic Siglec-7 antibody, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same. In one embodiment, the method may comprise administering a first composition of the present invention comprising a synthetic Siglec-7 antibody, fragment thereof, variant thereof, or nucleic acid molecules encoding the same by any of the methods described in detail above, and administering a second composition comprising a tumor antigen, fragment thereof, variant thereof, or nucleic acid molecules encoding the same more than 1 day, more than 2 days, more than 3 days, more than 4 days, more than 5 days, more than 6 days, less than 7 days, more than 8 days, more than 9 days, or more than 10 days after administration of the synthetic anti-Siglec-7 antibody, fragment thereof, variant thereof, or nucleic acid molecules encoding the 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 the same, and administering a second composition of the present invention comprising a synthetic anti-Siglec-7 antibody, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same by any of the methods described in detail above less than 1 day, less than 2 days, less than 3 days, less than 4 days, less than 5 days, less than 6 days, less than 7 days, less than 8 days, less than 9 days, or less than 10 days after administration of the composition comprising a tumor antigen, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same. In one embodiment, the method may comprise administering a first composition comprising a tumor antigen, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same, and administering a second composition of the present invention comprising a synthetic anti-Siglec-7 antibody, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same by any of the methods described in detail above more than 1 day, more than 2 days, more than 3 days, more than 4 days, more than 5 days, more than 6 days, less than 7 days, more than 8 days, more than 9 days, or more than 10 days after the administration of the composition comprising the tumor antigen, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same. In one embodiment, the method may comprise administering a first composition of the present invention comprising a synthetic anti-Siglec-7 antibody, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same, and a second composition comprising a tumor antigen, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same by any of the methods described in detail above.In one embodiment, the method may comprise administering a single composition comprising a synthetic anti-Siglec-7 antibody, fragment thereof, variant thereof, or nucleic acid molecule encoding the same of the present invention and a tumor antigen, fragment thereof, variant thereof, or nucleic acid molecule encoding the same.

[0346] The present invention is further illustrated in the following examples. It should be understood that these examples, although showing exemplary embodiments of the present invention, are given only by way of illustration. From the above discussion and these examples, those skilled in the art can determine the essential features of the present invention, and without departing from its spirit and scope, various changes and modifications can be made to the present invention to adapt it to various uses and conditions. Therefore, according to the above description, in addition to those shown and described herein, various modifications of the present invention will be apparent to those skilled in the art. Such modifications are also intended to fall within the scope of the appended claims.

[0347] Example

[0348] The present invention will be described in further detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise stated. Therefore, the present invention should never be interpreted as being limited to the following examples, but should be interpreted as covering any and all changes that become apparent due to the teachings provided herein.

[0349] Without further description, it is believed that one skilled in the art can use the foregoing description and the following illustrative examples to make and use the present invention and practice the claimed methods. Therefore, the following working examples should not be construed as limiting the remainder of the disclosure in any way.

[0350] Example 1: Generation of Siglec-7 Antibodies Using H2L2 Mice

[0351] Sialic acid-binding receptors such as Siglecs and selectins are readily exploited by cancer hypersialylation. Sialic acid (Sias) is a monosaccharide that is typically located at the outermost end of complex glycan chains that decorate the cell surface of all vertebrates. Sias are essential for embryonic development and mediate important intrinsic organismal functions. Seventy human genes are known to be involved in Sias biology, of which more than 10 have been shown to be human-specific. Most of these human-specific genetic changes occur in the Sia-recognizing Ig-like lectin (SIGLEC) gene, which is present in most types of white blood cells and is associated with the immune synapse. Upregulation of sialosides has long been associated with poor tumor prognosis and reduced immunogenicity.

[0352] The experiments described in this paper demonstrate the development and characterization of Siglec-7 antibodies ( Figure 1). In these experiments, the hybridoma supernatant showed specificity and binding to human Siglec-7. The Siglec7-pBMN-1-GFP plasmid was used to generate Siglec-7 stable 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 ).

[0353] The antibody was observed to exhibit cytotoxicity against OVISE cells ( Figure 4 ). OVISE cells are ovarian clear cell adenocarcinomas derived from metastatic sites: pelvis; hip bone of a 40-year-old Japanese woman. Experiments were designed to test the activity of the antibodies. Target cells: OVISE (20,000 cells / well); Effector cells: human PBMCs; E(effector):T(target)=5:1; Effector cells and antibodies (1:10) were added at time point 21.79 hours. Images were captured at 116 hours, whereby antibodies were observed to drive killing in OVISE cells. Killing was observed within 20 hours of incubation with antibodies and effector cells.

[0354] The antibody was also observed to exhibit cytotoxicity against SKOV3 cells ( Figure 5 ). SKOV3 is an ovarian serous cystadenocarcinoma cell derived from the metastatic site: ascites (female, 64 years old). Experiments were designed to test the activity of the antibody. Target cells: SKOV3 (10,000 cells / well); Effector cells: human PBMC E:T=5:1; Effector cells and antibody were added at 22.73 hrs. Images were captured at 90 hours, whereby antibody-driven killing of SKOV3 cells was observed. See Figure 5 .

[0355] It was also observed that the antibody exhibited cytotoxicity against OVCAR10 cells ( Figure 6 ). OVCAR10 is an ovarian human cancer cell line. Experiments were designed to test the activity of the antibodies. Target cells: OVCAR10 (10,000 cells / well); Effector cells: human PBMC E:T=5:1; Effector cells and antibodies were added at 22.73 hours. Images were captured at 90 hours, whereby antibody-driven killing of OVCAR10 cells was observed. See Figure 6 .

[0356] Example 2: Siglec-7 antibody treatment of melanoma

[0357] Skin cancer is one of the most common and dangerous human cancers. Among its different subtypes, melanoma arising from melanocytes represents a particularly severe disease. New FDA-approved immunotherapies with immune checkpoint blockade (CPI) have revolutionized melanoma therapy, improving outcomes. However, 40-50% of melanomas tend to be immune-cold, with poor T-cell infiltration, and respond poorly to CPIs.

[0358] In preliminary studies, several melanoma cell lines were observed to express high levels of Siglec-7 ligands, suggesting that NK cell inhibition through the Siglec-7 pathway is important in melanoma immunity. The results presented here show that anti-Siglec-7 antibodies can enhance NK cell killing activity against melanoma cancer cell lines ( Figure 8-12 ).

[0359] Melanoma cell lines 1205Lu, 451Lu MR, WM1366, WM3311, WM983BR, and WM3743 express high levels of Siglec 7 ligands and are amenable to treatment with Siglec 7 antibody therapy.

[0360] The WM983 cell line is a BRAFV600E mutant, wild type for PTEN, N-ras, c-KIT, and CDK4, and is a human metastatic melanoma cell line.

[0361] WM3743 is a primary post-chemotherapy melanoma cell line that exhibits mesenchymal morphology. The cell line expresses wild-type N-RAS, c-KIT, and CDK4 genes.

[0362] WM3311 is a human metastatic melanoma cell line. This cell line is established from the metastatic site from the patient. These cells are known to have mutations in GRIN2A, PRDM1, TERT, and are upregulated in Notch2. These cells are different from many melanoma cell lines in that they lack the common V600E (Val600Glu) mutation at codon 600 in the BRAF gene. This cell line is wild type for BRAF, N-RAS, c-KIT, and CDK4.

[0363] The results presented herein demonstrate that Siglec 7 antibodies can be used to selectively target Siglec 7-expressing melanomas (primary and metastatic and those resistant to therapy) as well as melanomas of many genotypes.

[0364] The parental Siglec-7 antibody is effective against melanoma resistant to Braf / Mek inhibitor treatment.

[0365] Example 3: Siglec-7 Glyco-Immune-Checkpoint MAb and NK Cell Engager Biologics Induce Targeting Ovarian Cancer Powerful anti-tumor immunity

[0366] Aggressive OC types such as HGSC and ovarian carcinosarcoma (OCS) are called "cold" tumors because they show low tissue-infiltrating lymphocyte (TIL) infiltration and are associated with poor therapeutic responses (Wu et al., 2021, Front Immunol 12, 672502, Hoogstad-van Evert et al., 2020, Gynecol Oncol 157, 810-816). Therefore, it is very important to participate in immune responses for cold tumors by additional means (Pugh-Toole et al., 2022, Curr Treat Options Oncol 23, 210-226). The innate immune effector system is of great significance because NK cells are the first line of defense for tumor immune surveillance (Wiernik et al., 2013, Clin Cancer Res 19, 3844-3855). Tumors avoid T cell responses by multiple mechanisms such as MHC downregulation and T cell exhaustion (exhaustion) program upregulation (Vinay et al., 2015, Semin Cancer Biol 35 Suppl, S185-S198). Similarly, cancer can bypass the surveillance function of NK cells by utilizing the host system through immune avoidance strategies, which include removing soluble ligands for NK activation receptors, the release of inhibitory cytokines, and the upregulation of HLA molecules, thus escaping NK immune function (Sabry et al., 2013, Front Immunol 4, 408), but NK anergy may also be mediated by other NK regulatory pathways. Recent studies have confirmed that many tumors show an increased level of sialic acid on their surface, which may act as a negative signal to NK cells, allowing tumors to avoid NK immune surveillance (Dobie et al., 2021, Br J Cancer 124, 76-90). One effort to strengthen NK activation against such highly sialylated tumors is the desialylation of target cells expressing Siglec-7 ligands (Jandus et al., 2014, J Clin Invest 124, 1810-1820). Since NK cells display Siglec-7 (receptor / sensor for different sialoglycan determinants), it can be used to inhibit NK-mediated targeted killing when it is co-engaged on the surface of human NK cells by tumors (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 can show regulatory negative signals through sialic acid polysaccharide tumor shield. Recent studies have shown that some Siglecs can act as sugar-immune checkpoint molecules that inhibit NK-mediated anti-tumor immunity in an MHC-independent manner (Hong et al., 2021, ACS Cent Sci 7, 1338-1346). In this study, the use of monoclonal antibodies and derivatives targeting Siglec-7 for tumor immunomodulation was examined to confirm and possibly overcome such sugar-CPI functions to improve the ability to affect tumor control.

[0367] Anti-tumor immune responses for the aggressive, difficult-to-treat phenotype of human OC (19) are the focus of these studies. The group developed several antibodies against Siglec-7 using transgenic humanized mouse models (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, three highly efficient Siglec-7 binding clones were focused on. These antibodies were engineered into human IgG1, and their binding to immune cells was studied. Strong staining of anti-Siglec-7 antibodies on NK cells was observed, including both bright and dim NK subsets, indicating its ability to bind to a large number of effector NK populations. In addition, expression can activate NK cells, and when these now activated cells are in the presence of several tumor lines, NK-mediated killing can occur against previously refractory OC targets. These human Siglec-7 antibodies drive NK immune function in the presence of different OC tumor target cells, promoting specific killing of target OC cells. Importantly, this approach should be relatively unaffected by tumor mutations. OC lines were tested with a variety of gene mutations including BRCA1, BRCA2, AKT2, TP53, SPOP, STAT3, MTOR, MEK1, MEK2, BRAF, etc. (Table 1), and it was observed that all mutations were activated by anti-Siglec-7 antibodies targeting NK cell targets.

[0368] Table 1: Ovarian cancer cell types studied, their driver mutations and drug resistance

[0369]

[0370]

[0371]

[0372]

[0373] These Mabs were studied in combination with PD-1CPI. It was found that dual therapy had a synergistic effect. However, it was observed that the efficacy of Siglec-7 antibodies was 10 times greater than that of the studied anti-PD1 antibody pembrolizumab, which indicates the potential value of this combined NK T cell anti-depletion group. Next, a humanized NSG mouse model in which human PBMCs were transplanted was used, and a single dose of DNA-encoded DB7.2Siglec-7 Mab was observed to demonstrate significant tumor control and an increase in median survival in mice with OVISE human ovarian tumors. Due to the limitation of the NSG model, it will develop graft-versus-host disease within a few months (Wunderlich et al., 2014, Blood 123, e134-144), so only a single dose of anti-Siglec-7 was administered, but it was still highly effective. This suggests that multiple administrations over time according to the current CPI (Hirsch et al., 2022, Nat Med 28, 2236-2237) may provide additional benefits. Notably, this is the first demonstration of the impact of Siglec-7-targeted MAbs alone and in combination with anti-PD1 (NK and T cell CPIs) for targeting OC or any human tumor, and suggests that more research is needed on the potential added value of such combinations, or that CPI-refractory tumors should be considered. These studies have important implications for tumor therapy, supporting the potential of novel non-T cell CPIs as a way to potentially enhance current immunotherapy strategies.

[0374] Next, the efficacy of Siglec-7 binding and activation of NK cells for targeted OC therapy was evaluated. Without being bound by theory, it is assumed that binding to Siglec-7 can directly engage NK cells and pull them to cell targets in an effective manner. New types of NK cell engagers are developed by creating a bispecific fusion between FSHR targeting MAbs and powerful anti-Siglec-7 Mabs. NKCE has become an interesting innate immune concept in the field of immuno-oncology. Currently, there is a bispecific that has entered the clinic; GTB-3550 engages CD16a-IL-15 / CD33 for AML and CD33+ malignancies (Gleason 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 being studied preclinically include NKG2D, NKp30, and NKp46. The phenotype of anti-Siglec-7 Mabs that show binding and activation of NK cells would otherwise be negatively affected by tumor sialic acid polysaccharide cloaks, indicating that Siglec-7 can provide a unique immune tool for specific tumor-NK activation and engagement. The bispecific developed here, DB7.2xD2AP1, shows powerful binding to both NK cells and ovarian cancer cells. It shows killing (pM levels) against different OC phenotypes. This is the first report of the development of a member of the Siglec family as an immune engager or the development of a Siglec-7 bispecific NK cell engager using such a method. This NKCE is specific to its target cells and is powerful in tumor cell killing, as evaluated by a panel of different human ovarian tumor lines. FSHR expression on the target is a prerequisite for NK killing activity because FSHR negative cells will not be killed. Interestingly, DB7.2xD2AP11 resulted in less production of cytokines and cytotoxic molecules, suggesting that it is important to investigate killing tools with potentially limited risk of cytokine release syndrome (CRS) (Pinto et al., 2022, Trends Immunol 43, 932-946).

[0375] During these studies, the focus was on OC cells expressing mutations that are particularly problematic for OC therapy. This includes PARPi-resistant OC cells and OCs displaying resistance against multiple drug targets, specifically HDACs (dagnostat, entinostat, belinstat), PI3Ks (daclis, bupanisib), mTORCs (omecilisib), Wee1 (CHEK1), TOP1 (butoconazole, mitoxantrone, irinotecan), DNA alkylating agents (oxaliplatin, cisplatin) and microtubule stabilizers (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., 2018, Biochim Biophys Acta Mol Cell Res 1865, 392-405; Ayestaran et al., 2020, Patterns (NY) 1, 100065). These studies included OC lines containing BRCA (BRCA1 & 2) mutations, as these mutations are the main risk in OC cancer patient groups. Approximately 50% of HGSCs exhibit an interrupted BRCA pathway via either germline or somatic mutations, or through epigenetic silencing of pathway members (Vaughan et al., 2011, Nat Rev Cancer 11, 719-725). PARP inhibition represents an important pathway for DNA damage repair, and therefore PARPi holds great promise in treating tumors with damaging mutations in BRCA1 / 2. However, resistance to PARPi has been reported to be a problem for treatment-resistant OC patients (Noordermeer et al., 2019, Trends Cell Biol 29, 820-834). The efficacy and consistency of killing FSHR+ tumors using Siglec-7 MAbs as NK CPIs in vitro and in vivo, as well as bispecific Siglec-7 NK engagers, are encouraging for the potential to bring additional tools to OC that respond poorly to treatment.

[0376] In summary, this study describes a potent Siglec-7 MAb that engages and activates NK cells and may further complement PD-1 immunotherapy for OC, representing a potential additional class of CPIs for NK cells in this setting. In addition, the data show that Siglec-7 can be used to build a novel NKCE and serve as a powerful tool for targeting receptor-positive OCs that drive significant anti-tumor responses in vitro and in vivo. While these studies are important for designing additional tools for OC, additional studies are needed to examine the usefulness of such biologics against additional difficult cancers.

[0377] Materials and methods are now described:

[0378] Cells and animals

[0379] 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 cancer, 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.

[0380] DNA-encoded MAb generation

[0381] Several humanized antibodies were generated for human Siglec-7. These MAbs bind to Siglec-7 and can stain NK. These hybridomas are sequenced here and used to develop Siglec-7 DNA-encoded monoclonal antibodies (DMAbs) as tools for in vitro expression (Patel et al., 2018, Cell Rep 25, 1982-1993e1984). As described, under the control of human cytomegalovirus (hCMV) promoter and bovine growth hormone (BGH) polyA signal, the final human IgG1 HC and LC are inserted into the pVax1 plasmid expression vector (Patel et al., 2018, Cell Rep 25, 1982-1993e1984). The plasmid is 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 evaluated by SDS-PAGE analysis.

[0382] Design of FSHRxSiglec-7NKCE

[0383] The FSHRxSiglec-7 natural killer cell engager (NKCE) was designed by encoding a codon-optimized scFv (DB-S7-2) of the Siglec-7 MAb, followed by an scFv (D2AP11) encoding the FSHR antibody and adding an enhanced optimized IgE leader sequence. Both constructs were subcloned into a modified pVax1 expression vector (Perales-Puchalt et al., 2019, JCI Insight 4). The FSHRxSiglec-7 NKCE was named DB7.2xD2AP11.

[0384] Flow cytometry

[0385] BD LSRII flow cytometer was used for staining of cells. BD FACS Aria cell sorter (BD Biosciences) was used for sorting of Siglec-7 / FSHR stably expressing cells. The anti-human antibodies used were directly fluorescent dye-conjugated. Antibodies used: anti-Siglec-7 (F023-420, BD Pharmingen), anti-Siglec 3 (6C5 / 2, R&D Systems). For non-conjugated primary antibodies, PE-secondary human (H+L) (Invitrogen) and PE / AF647-secondary human F(ab')2 (Jackson ImmunoResearch Laboratories Inc) were used. Live / Dead Violet Vitality Kit (Invitrogen) was used to exclude dead cells from the analysis. For fluorescent cell staining on 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 from BD Biosciences. PE-CF594(clone HIL-7RM21), CD25BUV737(clone 2A3), CD8 BUV496(clone RPA-T8), HLA-DR BV605(clone G46-6), CD38BUV661(clone HIT2), CD14 BV480(clone MP9), CD45RA BUV563(HI100), CD4BB790(cloneSK3), CD15 FITC(clone HI98), CD103 BB700(clone Ber-ACT8), CD161 APC(clone DX12).Briefly, cryopreserved PBMCs were thawed and left at 37C and 5% CO2 for 2 hours in complete R10 medium (RPMI supplemented with 10% FBS, 2mM L-glutamine, 100U / ml penicillin and 100mg / ml streptomycin) with 10U / ml DNAse I (Roche Life Sciences). The cells were washed with PBS and incubated for 20 minutes with 0, 1 or 10ug / ml DB7.2 Siglec-7 antibody diluted in fluorescence activated cell sorting (FACS) buffer. After washing with FACS buffer, the cells were incubated for 20 minutes with mouse anti-human IgG Fab secondary antibody PE (Invitrogen) diluted in FACS buffer. The cells were washed with FACS buffer, resuspended in PBS and prestained for 10 minutes at 37C 5% CO2 for chemokine receptors CCR7 and CXCR5. Cells were then stained for 10 minutes using Live / Dead Fixable Aqua (Invitrogen) for viability exclusion and then incubated for 20 minutes with a panel of directly conjugated monoclonal antibodies and Human Trustain FcX (Biolegend), diluted in equal parts of fluorescence activated cell sorting (FACS) buffer (PBS containing 0.1% sodium azide and 1% bovine serum albumin) and brilliant staining 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 analyzed by Flowjo software 10.8.1 (Tree Star Inc.).

[0386] Enzyme-linked immunosorbent assay (ELISA)

[0387] For quantification of human IgG in the serum of mice electroporated with Siglec-7 Mab and Siglec-7 DMAb, MaxiSorp plates were coated with 10 μg / mL of goat anti-human IgG Fc (Bethyl) at 4°C overnight. Plates were washed and blocked with 5% milk in PBS-T (0.05% Tween 20 in PBS) for 2 h at room temperature. Plates were washed and samples diluted in 1% NCS in PBS containing 0.2% Tween 20 were added and incubated at 37°C for 2 h. Plates were washed again and incubated with a 1:10,000 dilution of HRP-conjugated goat anti-human IgG (h+L) secondary antibody (Bethyl) for 1 h at room temperature. Plates were developed with SigmaFast OPD for 5-10 min and OD450 signals were measured.

[0388] In vitro cytotoxicity analysis using the xCELLigence real-time cell analyzer

[0389] The xCELLigence real-time cell analysis (RTCA) instrument from Agilent Technologies (USA) was used for impedance-based in vitro cytotoxicity assays. Impedance was expressed in arbitrary units, called cell index. Target cells were plated at 1x10 per well. 4 -2x10 4 The final cell concentration of cells was inoculated into a disposable sterile 96-well E-plate of the xCELLigence RTCA device. The instrument has been placed in a CO2 incubator during the experiment and is controlled by a cable connected to the control unit. The 96-well E-plate is placed in the xCELLigence RTCA device and incubated for 18-24 hours. Subsequently, effector cells (human PBMC / NK; E (effect): T (target) ratio = 5: 1 / 10: 1) and treatment agents (Siglec-7 MAb / NKCE) are added. Real-time analysis is performed for 3-7 days. The conductivity is converted to a unit-free cell index (CI) parameter by the xCELLigence device every 15 minutes, and images are captured at intervals of 1 hour. The generated data are standardized according to the time point when the effector cells and MAb / NKCE are added to the target cells and analyzed using RTCA / RTCA Pro software.

[0390] Western blot analysis

[0391] To verify the in vitro and in vivo expression of Siglec-7 MAb, the supernatant collected after transfection of Expi 293F cells with DNA encoding the antibody or the serum collected from mice immunized with Siglec-7 DMAb was heat inactivated, reduced, and loaded with Odyssey protein molecular weight (LI-COR). After electrophoresis, the sample was transferred to a polyvinylidene fluoride (PVDF) membrane via the iBlot-2 system (Thermo Fisher Scientific) and blocked with Odyssey blocking buffer (LI-COR). Heavy and light chains were detected using goat anti-human secondary antibodies (LI-COR). The expression of bispecific T and NK cell engagers was detected using goat anti-human IgG F (ab') 2 (Jackson ImmunoResearch Laboratories Inc) followed by donkey anti-goat antibodies (LICOR).

[0392] Immunofluorescence (IFA) analysis

[0393] HEK 293T cells transduced with Siglec-7 were inoculated in 2-well chamber slides and the cells were allowed to adhere overnight. The cells were permeabilized with 0.5% Triton 100 in PBS and then blocked with 5% goat serum. After that, bispecific NKCE was added and incubated overnight at 4°C. Subsequently, the slides were incubated with goat anti-human H+L (Texas Red-conjugated) secondary antibodies. Nuclear staining was completed with 4',6-diamino-2-phenylindole (DAPI). The samples were mounted on glass slides with the help of Fluoroshield mounting media (Invitrogen) and then observed using a Leica TCS SP8 WLL scanning laser confocal microscope.

[0394] Tumor attack

[0395] NOD / SCID-γ (NSG) mice were challenged with OVCAR3-FSHR cells. NSG mice were treated with 3x10 6 OVCAR3 or OVCAR3-FSHR cells expressing FSHR were injected subcutaneously in the right flank. After 3 days, when tumors became palpable, mice were inoculated with pVax1 (100 μg) or DDAP-NKCE (100 μg). On the same day of the expression vector administration, 10×10 6 PBMCs were injected intraperitoneally into each mouse. Mice were inoculated with DNA twice, 1 week apart, and tumor size was monitored regularly. Mice were euthanized when signs of graft-versus-host disease (GVHD) appeared. Tumor volume (V) was calculated according to the formula V = [(length x width 2 )] / 2; the width is the side with the smaller measurement. For Siglec-7 DMAb challenge, OVISE cells (0.8x10 6 ) challenged NOD / SCID-γ (NSG) mice. Ten days later, when tumors became palpable, mice were vaccinated with DB7.2 DMAb (50 μg + 50 μg; HC + LC) or pVax1 (100 μg), and then 10×10 6 PBMC were injected intraperitoneally into each mouse. On the same day of administration of the expression vector, 10x10 6 PBMCs were injected intraperitoneally into each mouse. The same procedure was followed thereafter. Animal experiments were approved by the Institutional Animal Care and Use Committee of the Wistar Institute.

[0396] Statistical analysis

[0397] All statistical analyses were performed using Graph Pad Prism. p-values ​​< 0.05 were considered statistically significant. Differences between the mean values ​​of the experimental groups were calculated using a two-tailed unpaired Student's t test or a one-way ANOVA where more than two quantitative variables were measured. Error bars represent the standard error of the mean. Comparisons between tumor sizes at each time point were performed using a two-way ANOVA with Fisher's least significant difference (LSD) test. The experimental results are now described.

[0398] In vitro expression and specificity evaluation of Siglec-7 MAb

[0399] To evaluate the potential of Siglec-7 blocking as a NK cell activation strategy, MAbs specific for human Siglec-7 were recently generated and their biological activity was studied in vitro. Here, codon- and RNA-optimized antibody expression cassettes of powerful Siglec-7 binders were generated and assembled into DNA vectors optimized for expression of human IgG1. These HC&LC individual combinations were named DB7.1 (DB-S7-1), DB7.2 (DB-S7-2), and DB7.7 (DB-S7-7) (Figure 13A). Expression was studied in vitro by transfecting expi293F cells with synthetic human Siglec-7 DNA vectors by Western blotting. These studies confirmed bands corresponding to the heavy and light antibody chains of the expressed antibodies in the transfected Expi293F supernatant, but not in the control wells transfected with the empty vector (Figure 13B). The in vitro expression of DB7.1, DB7.2, and DB7.7 was further confirmed by ELISA quantification (Figure 13C). By ELISA analysis, DB7.1, DB7.2 and DB7.7 Siglec-7 MAbs showed specific and dose-dependent binding to recombinant human Siglec-7 (Figure 14A). HEK293T cells were stably transduced for Siglec-7 expression, and these cells (HEK293T-Siglec7 cells) were used for evaluation of 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 from multiple donors (n=4) ( Fig.15). DB7.2 mainly binds to human NK cells (~90%), while lower levels of binding are observed in the case of CD8+T and CD4+T cells (Figure 14C&D). Binding of DB7.2 to innate lymphoid cells (ILC) was observed (~9%), which can be considered 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 >25% binding to mucosal-associated invariant T (MAIT) cells, which play an important role in immune defense against microbial infection in mucosal barriers and act as innate sensors of inflammation and viral infection (Parrot et al., 2020, Sci Immunol 5; Legoux et al., 2020, Immunity 53, 710-723). Additional studies of minor T cell populations expressing Siglec-7 are important for further research. Interestingly, DB7.2 MAb activated CD56 暗 and CD56 亮 NK cell subsets, indicating its ability to bind to a large portion of the human NK population (Figure 14E). DB7.2 also demonstrated strong binding to human Siglec-7 protein in Western blots (Figure 16A), and their binding was analyzed in surface plasmon resonance (SPR) analysis. The DB7.2 anti-Siglec-7 antibody showed a KD value of 44pM, as determined by the maximum point on the sensorgram using titration of a specified concentration of anti-Siglec-7 and representing saturation of the recombinant human Siglec-7 protein. In addition to the high affinity, DB7.2 (K off :10 -M ) with high specificity ( Figure 16B-D ).

[0400] Siglec-7 MAb induces potent and specific killing of target ovarian cancer cells in vitro

[0401] To evaluate the ability of Siglec-7 gene-optimized MAbs to activate human peripheral blood NK cells against human cancer lines, a panel of different human ovarian cancer cells (Table 1) (Bordoloi et al., 2022, JCI Insight 7) was assembled using HaCaT (human keratinocytes) as a Siglec-7 negative control. Importantly, DB7.1, DB7.2, and DB7.7 did not induce killing of HaCaT cells (Figure 17A & B). Next, the ability of MAbs to drive killing of determined human OC cells (OVISE, TOV-21G, OVCAR3, CaOV3, OVCAR10, and PEO-4) was examined with different disease phenotypes and mutations (Table 1) in the presence of human PBMC / NK cells. Although 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 showed enhanced killing induction against additional OC lines CaOV3 (Figure 17H) and OVCAR3 (Figure 17I) cells. In order to confirm that Siglec-7 MAb activity is independent of Fc receptor binding, it was found that (Figure 18A) DB7.2 potency was maintained in the presence of Fc blocking (Figure 18B & C). Next, the residue modification of the Fc of the antibody was performed to eliminate Fc binding and potential off-target effects. A variant of DB7.2 was designed, which contains multiple residue modifications in the Fc domain ("TM"; L234F / L235E / P331S) to remove 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), named DB7.2_TM Mod. The killing efficacy of DB7.2_TM Mod is maintained in OVISE cells (Figure 19A). In the presence of human PBMC, DB7.2_TM Mod was added to OVISE cells for 24h, resulting in powerful killing of target cells, but not against control (Figure 19B) cell targets. DB7.2 and DB7.2_TM Mod showed comparable IC50 values ​​of 82.67 and 192nM in OVISE cells (Figure 19C & D). In short, these studies illustrate the specificity of Siglec-7 Mab to activate immune killing and its ability to target OC cells. Combination of anti-PD-1 and DB7.2 MAb xCELLigence Assays demonstrate enhancement of OC killing

[0402] OC is one of the subgroups of cancers with a mild response to existing immune checkpoint blockade therapies, with an objective response rate (ORR) of ~8-9%. Rare durable responses have been reported in OC patients receiving checkpoint inhibitors (CPIs), and there are no FDA-approved CPIs for the management of OC to date (Farkkila et al., 2020, Nat Commun 11, 2543). However, there is evidence that a combination approach may prove important in optimizing the response of EOC patients to immunotherapy. Without being bound by theory, it is hypothesized that the combination of T cell activation of PD-1 and activation of NK cells by Siglec-7 Mab can more effectively synergistically target tumors. In order to test this hypothesis of simultaneously targeting PD-1 for T cells and engaging NK immune pathways; an antibody combination of DB7.2 and anti-PD1 (pembrolizumab) was studied in an xCELLigence assay targeting OC tumors. In an in vitro killing assay based on xCELLigence, DB7.2 showed a killing ability that simulated both BRCA2 mutation and PARPi-resistant PEO4 cells, EC50 68.87nM (Figure 19E&G), while anti-PD1-targeted PEO4 cells had a 10x lower EC50 of 680nM (Figure 19F&H). The combination of anti-PD-1 and DB7.2 showed further enhancement of PEO4 cell killing in the presence of human PBMCs (Figure 19I). These data illustrate that the combination of Siglec-7 Mab and anti-PD1 (NK and T cell CPI) enhances anti-tumor immunity.

[0403] In vivo expression of Siglec-7 MAb reduces tumor progression in an ovarian cancer challenge model

[0404] Next, the in vivo expression of Siglec-7 MAb clones was examined using DNA in vivo delivery. To this end, in a dual plasmid system of each anti-Siglec-7 clone (DB7.1, DB7.2 and DB7.7) and an empty vector control (pVax1), mice were injected with a DNA expression cassette of MAb (Patel et al., 2018, Cell Rep 25, 1982-1993e1984) at a dose of 50 μg + 50 μg (HC + LC), allowing the biological antibody production of mAb in vivo. As previously described, the injection was injected into the tibialis anterior muscle of mice on day 0 (Parzych et al. 2022, Nat Commun 13, 5886; Patel et al., 2018, Cell Rep25, 1982-1993e1984) (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, and no expression was detected in the serum of control animals or pre-bleed mice. To confirm the specificity of the antibodies generated in vivo, human NK cells were stained using the 14th day serum obtained from mice vaccinated with any of DB7.1, DB7.2 and DB7.7 and the serum from animals delivered with an empty vector control. The 14th day sera from all three groups positively stained human NK cells, while no staining was observed in the case of pVax1 or irrelevant antibody controls (Figure 20B). In order to examine the potential anti-tumor efficacy of Siglec-7 Mab delivery in animals, the NSG-K (NOD / SCID-γ-MHCI / II double knockout mutant) mouse tumor challenge model was used, in which OVISE human ovarian cancer cells (0.8x10 6 cells / mouse) and prospectively monitored tumor growth. 3 When the average size of , together with 10 million human PBMCs per mouse, 50μg+50μg (HC+LC) of DB7.2 or pVax1 vector control was delivered to the muscle as described (Parzych et al., 2022, Nat Commun 13, 5886; Patel et al., 2018, Cell Rep 25, 1982-1993e1984) (Figure 20C). Compared with the vector control, mice receiving DB7.2 Siglec-7 MAb showed a significant reduction in tumor burden / delayed tumor growth (Figure 20D, Figure 21). Significantly, Siglec-7 MAb significantly enhanced the median survival of tumor-bearing mice (Figure 20E). These data provide in vivo data for the first time, demonstrating the potential of Siglec-7 antibodies for direct treatment of human ovarian cancer.

[0405] Generation and expression of DB7.2xD2AP11 DNA-encoded bispecific NK cell engagers

[0406] NK engagers are an important new focus for targeting tumor cells (Gleason 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). As described above, based on the efficacy of anti-Siglec-7 MAbs to enhance NK activity against OC, the potential of this target is considered a new way to target NKCE. Without being bound by theory, it is hypothesized that this approach not only allows specific targeting of NK cells, but also prevents them from receiving negative TME signals, thereby generating a new type of NKCE. Therefore, in order to enhance tumor targeting using NK cell-specific Siglec-7 engagement, a NKCE was constructed in the form of two linked antibody binding fragments (scFVs), arranged to bind to the OC antigen FSHR as recently described (Bordoloi et al., 2022, JCI Insight 7) and connected to the anti-Siglec-7 FV form. A NKCE fusion sequence was designed that encodes the scFV of the DB7.2 anti-Siglec-7 antibody and the scFV of the anti-FSHR (clone D2AP11) (Bordoloi et al., 2022, JCI Insight 7) (Figure 22A), and incorporates a GS (glycine-serine) flexible linker (Perales-Puchalt et al., 2019, JCI Insight 4; Pratik et al., 2022, Molecular therapy Oncolytics). In vitro analysis showed that the DB7.2xD2AP11 NKCE was effectively expressed, as the production of the expected ~55KDa molecule was observed by transfection of Expi293F cells (Figure 22B). This NKCE was bound to two cellular targets by flow staining on Siglec-7 or FSHR overexpressing HEK293T (Figure 22C) or K562 (Figure 22D) cells, respectively. To visualize binding to human Siglec-7, high-resolution confocal imaging of 293T-Siglec-7 fixed cells was performed. Cells were labeled with DAPI (nuclei), GFP (Siglec-7), and Texas Red (DB7.2xD2AP11). DB7.2xD2AP11 NKCE demonstrated clear surface binding to GFP+ cells expressing human Siglec-7, but not in the secondary antibody control (Figure 22E).

[0407] A novel NK cell engager targeting the FSHR induces potent killing of multiple ovarian tumor lines in vivo and reduces Tumor burden

[0408] Next, the induction of in vitro tumor killing of DB7.2xD2AP11 NK cell engagers (NKCE) was evaluated using xCelligence RTCA assay format. Effector cells include direct PBMC and NK cell testing. NKCE was added 1 day after target OC cells were plated. As a control, 4 FSHR negative cell lines were studied, including HEK 293T 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 for these FSHR negative cells (Figure 22F-K) including unrelated cancer cells. As shown in Figure 22G&J, HEK293T and AGS cells were still healthy and adherent by being treated with DB7.2xD2AP11 NKCE for 3 days in the presence of human PMBC. In contrast, a variety of FSHR-expressing ovarian tumor lines OVISE, OVCAR3-FSHR, CaOV3, Kuramochi-FSHR, and PEO-4 (Figures 23A-G) were effectively killed by human PBMC 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-incubated with human NK cells also effectively killed OVCAR3-FSHR cells (Figure 24A). The specificity of this bispecific engagement mode was confirmed on FSHR-negative AGS cells; here, anti-Siglec-7 antibodies mediated the killing of AGS cells (Figure 24B), rather than DB7.2xD2AP11 NKCE (Figure 22I). As a further specificity control, non-FSHR-targeted NKCE (IL13Rα2-NKCE) was developed, and it was observed that it did not drive toxicity in FSHR-overexpressing OVCAR3 or against OVISE cells (Figure 24C & D). The DB7.2xD2AP11 NKCE induced concentration-dependent cytolysis in both OVISE-FSHR (in separate experiments using PBMCs from 3 different human donors) and OVCAR3 cells, showing EC50 values ​​of 142.87 pM and 236.6 pM, respectively, supporting the remarkable efficacy of this engager tool as a novel NKCE for targeting pathogenic cells (Figure 23H & I; Figure 25 and Fig.26). The cytokine / cytotoxic molecule secretion profile of OC-NKCE was examined. Co-culture of OVCAR3-FSHR cells and human PBMCs and DB7.2xD2AP11 NKCE (Figure 27A) generated enhanced production of soluble Fas (sFas) and granzyme A as part of specific immune activation, and reduced levels of IL-10 were observed (Figure 27B), which supports more effector-based activation. In order to directly evaluate the in vivo anti-tumor effect of DB7.2xD2AP11 NKCE, NSGK mice were challenged with OVCAR3-FSHR cells. NSG-K mice were administered 3 million OVCAR3-FSHR cells, and 7 days after tumor implantation, they were treated with nucleic acid-encoded DB7.2xD2AP11 (100 μg) or empty vector pVax1 (100 μg) for in vivo expression, twice every two weeks. At day 7, they were also inoculated with 10 million human PBMCs and tumor volumes were measured regularly (Figure 27C). Therapy with NKCE resulted in a significant reduction in tumor burden in OVCAR3-FSHR tumor-bearing mice, while no such effects were observed in the control treatment group (Figure 27D). In addition, DB7.2xD2AP11 NKCE treatment enhanced survival (Figure 27E), which supports further study of this approach.

[0409] Example 4: Sequence of Siglec 7 antibody clone (CDR sequence is underlined)

[0410] DB-S7-1 heavy chain

[0411] SEQ ID NO:4

[0412] QVQLVESGGGVVQPGRSLRLSCAAS GFTFRTDA MHWVRQAPGKGLEWVAV IWYDGSIK YHADSVKGRFTISRDNSKNTLYLEMNSLRAEDTAVYYC ARDRSNSLDH WGQGILVSVSS

[0413] DB-S7-1 heavy chain

[0414] SEQ ID NO:8

[0415] caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagcgtct ggattcaccttcaggaccgatgcc atgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagtt atttggtatgatggaagtattaaataccatgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctggaaatgaatagcctgagagccgaggacacggctgtatattattgt gcgagaga tcggtctaactctttggaccac tggggccagggaatcctggtcagcgtctcctcag

[0416] DB-S7-1 Light Chain

[0417] SEQ ID NO:12

[0418] EIVMTQSPATLSVSPGERATLSCRAS QSVSSN LAWYQQKPGQAPRLLIY GAS TRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYC QQYNNWPRT FGQGTKVEIK

[0419] DB-S7-1 Light Chain

[0420] SEQ ID NO:16

[0421] gaaatagtgatgacgcagtctccagccaccctgtctgtgtctccaggggaaagagccaccctctcctgcagggccagt cagagtgttagcagcaac ttagcctggtaccagcagaaacctggccaggctcccaggctcctcatctat ggtgcatcc accagggccactggtatcccagccaggttcagtggcagtgggtctgggacagagttcactctcaccatcagcagcctgcagtctgaagattttgcagtttattactgt cagcagtataataactggcctcggacg ttcggccaagggaccaaggtggaaatcaaac

[0422] DB-S7-2 Heavy Chain

[0423] SEQ ID NO:20

[0424] QVQLVESGGGVVQPGRSLRLSCAAS GFTFRTDA MHWVRQAPGKGLEWVAV IWYDGSIKYHADSVKGRFTISRDNSKNTLYLEMNSLRAEDTAVYYC ARDRSNSLDH WGQGILVSVSS

[0425] DB-S7-2 Heavy Chain

[0426] SEQ ID NO:24

[0427] caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagcgtct ggattcaccttcaggaccgatgcc atgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagtt atttggtatgatggaagtattaaa taccatgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctggaaatgaatagcctgagagccgaggacacggctgtatattattgt gcgagaga tcggtctaactctttggaccac tggggccagggaatcctggtcagcgtctcctcag

[0428] DB-S7-2 Light Chain

[0429] SEQ ID NO:28

[0430] DIVMTQSPLSLPVTPGEPASFSCRSS QSLLHSN GYNYLDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLKISRVEAADVGVYYC MQALQTPRT FGQGTKVEIK

[0431] DB-S7-2 Light Chain

[0432] SEQ ID NO:32

[0433] gatattgtgatgactcagtctccactctccctgcccgtcacccctggagagccggcctccttctcctgcaggtctagt cagagcctcctgcatagtaat ggatacaactatttggattggtacctgcagaagccagggcagtctccacagctcctgatctat ttgggttctaatcgggcctccggggtccctgacaggttcagtggcagtggatcaggcacagatttacactgaaaatcagcagagtggaggctgcggatgttggggtttactgc atgcaagctctacaaac tcctcggacg ttcggccaagggaccaaggtggaaatcaaac

[0434] DB-S7-3 heavy chain

[0435] SEQ ID NO:36

[0436] EVQLVESGGDLVQPGGSLRLSCAAS GFTFNSYW MSWVRQAPGKGLEWVAN IKHDGSEK YYVDSVKGRFTISRDSAKNSLYLQMNSLRAEDTAVYFC AREAVASSYFAY WGQGTLVTVSS

[0437] DB-S7-3 heavy chain

[0438] SEQ ID NO:40

[0439] gaggtgcagctggtggagtctggggagactttgtccagcctggggggcctgagactctctctgcagcctct ggattcacctttaatagctattgg atgagtgggtccgccaggctccaggggaagggctggagtgggtggccaac ataaaacatgatggaagtgaaaaa tacttgtggactctgtgaagggccgattcaccatctccagacagcgccaagaactcactgtatctgcaaatgaacagcctgagagccgaagacacggctgtgtatttctgt gcgagaga ggcagtggctagttcttactttgcctac tggggccagggaaccctggtcaccgtctcctcag

[0440] DB-S7-3 light chain

[0441] SEQ ID NO:44

[0442] EIVMTQSPATLSVSPGERATLSCRAS QSVSSN LAWYQQKPGQAPRLLIY GASTRAAGIPARFSGSGSGTEFTLTISSLQSEDFAVYYC QQYNNWPYT FGQGTKLEIK

[0443] DB-S7-3 light chain

[0444] SEQ ID NO:48

[0445] gaaatagtgatgacgcagtctccagccaccctgtctgtgtctccaggggaaagagccaccctctcctgcagggccagt cagagtgttagcagcaac ttagcctggtaccagcagaaacctggccaggctcccaggctcctcatctat ggtgcatcc accagggccgctggtatcccagccaggttcagtggcagtgggtctgggacagagttcactctcaccatcagcagcctgcagtctgaagattttgcagtttattactgt cagcagtataataactggccgtacact tttggccaggggaccaagctggagatcaaac

[0446] DB-S7-6 heavy chain

[0447] SEQ ID NO:52

[0448] QVQLVESGGGVVQPGRSLRLSCAAS GFPFSSYG MHWVRQAPGKGLEWVTF IWFDGNNK EYADSVKGRFIVSRDNSKNTLYLQMNSLRADDTAVYYC ARNGVYYGSGAYSDS WGQGTLVTISS

[0449] DB-S7-6 heavy chain

[0450] SEQ ID NO:56

[0451] CAGGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGCAGCCAGGCAGGAGCCTGAGACTGTCCTGCGCCGCCTCT GGCTTCCCCTTTAGCTCCTACGGA ATGCACTGGGTGAGGCAGGCACCTGGCAAGGGACTGGAGTGGGTGACCTTC ATCTGGTTTGACGGCAACAATAAGGAGTACGCCGATTCTGTGAAGGGCCGGTTCATCGTGAGCCGCGACAACTCCAAGAATACCCTGTATCTGCAGATGAACTCTCTGAGGGCCGACGATACAGCCGTGTACTATTGT GCCCGGAATGGC GTGTACTATGGCAGCGGCGCCTATTCCGATTCT TGGGGCCAGGGCACCCTGGTGACAATCTCTAGC

[0452] DB-S7-6 light chain

[0453] SEQ ID NO:60

[0454] DIVMTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGTDFTLKISRVAAEDVGVYYC MQALQTPYT FGQGTKLEIK

[0455] DB-S7-6 light chain

[0456] SEQ ID NO:64

[0457] gatattgtgatgactcagtctccactctccctgcccgtcacccctggagagccggcctccatctcctgcaggtctagt cagagcctcctgcatagtaat ggatacaactatttggattggtacctgcagaagccagggcagtctccacagctcctgatctat ttgggttct aatcgggcctccggggtccctgacaggttcagtggcagtggatcaggcacagattttacactgaaaatcagcagagtggcggctgaggatgttggggtttattactgc atgcaagctctacaaac tccgtacact tttggccaggggaccaagctggagatcaaac

[0458] DB-S7-7 heavy chain

[0459] SEQ ID NO:68

[0460] QVQLVESGGGVVQPGRSLRLSCAAS GFTFRTDA MHWVRQAPGKGLEWVAVIWYDGSIK YHADSVKGRFTISRDNSKNTLYLEMNSLRAEDTAVYYC ARDRSNSLDH WGQGILVSVSS

[0461] DB-S7-7 heavy chain

[0462] SEQ ID NO:72

[0463] caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagcgtct ggattcaccttcaggaccgatgcc atgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagtt atttggtatgatggaagtattaaa taccatgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctggaaatgaatagcctgagagccgaggacacggctgtatattattgt gcgagaga tcggtctaactctttggaccac tggggccagggaatcctggtcagcgtctcctcag

[0464] DB-S7-7 light chain

[0465] SEQ ID NO:76

[0466] EIVMTQSPATLSVSPGERATLSCRAS QSVSSN LAWYQQKPGQAPRLLIY GAS TRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYC QQYNNWPYT FGQGTRLEIK

[0467] DB-S7-7 light chain

[0468] SEQ ID NO:80

[0469] gaaatagtgatgacgcagtctccagccaccctgtctgtgtctccaggggaaagagccaccctctcctgcagggccagt cagagtgttagcagcaac ttagcctggtaccagcagaaacctggccaggctcccaggctcctcatctat ggtgcatccaccagggccactggtatcccagccaggttcagtggcagtgggtctgggacagagttcactctcaccatcagcagcctgcagtctgaagattttgcagtttattactgt cagcagtataataactggccgtacact tttggccaggggaccaggctggagatcaaac

[0470] DB-S7-8 heavy chain

[0471] SEQ ID NO:84

[0472] QVQLVESGGGVVQPGRSLRLSCAAS GFPFSSYG MHWVRQAPGKGLEWVTF IWFDGNNK DYADSVKGRFSVSRDNSKNTLYLQMNSLRADDTAVYYC ARNGVYYGSGAYVDY WGQGTLVTVSS

[0473] DB-S7-8 heavy chain

[0474] SEQ ID NO:88

[0475] gaggtgcaactggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagcgtct ggattcccattcagcagctatggc atgcactgggtccgccaggctccaggcaaggggctggagtgggtgacattt atatggtttgatggaaataataaa gactatgcagactccgtgaagggccgattcagcgtctccagagacaattccaagaacacgctgtatctgcaaatgaacagtctgagagccgatgacacggctgtttattactgt gcgagaaa tggggtttactatggttcgggggcttatgttgactac tggggccagggaaccctggtcaccgtctcctcag

[0476] DB-S7-8 light chain

[0477] SEQ ID NO:92

[0478] DIQMTQSPSTLSASVGDRVTITCRAS QSISYF LAWYQQKPGKAPKLLIY KASSLESGVPSRFSGSGSGTEFTLTISTLQPDDFATYYC QQYNSYSMYT FQGKLEIK

[0479] DB-S7-8 light chain

[0480] SEQ ID NO:96gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgccgggccagt cagagtattagttacttt ttggcctggtatcagcagaaaccagggaaagcccctaagctcctgatctat aaggcgtct agtttagaaagtggggtcccatcaaggttcagcggcagtggatctgggacagaattcactctcaccatcagcaccctgcagcctgatgattttgcaacttattactgc caacaatataatagttattc catgtacact tttggccaggggaccaagctggagatcaaac

[0481]

[0482]

[0483]

[0484] The disclosure of each and any patent, patent application, and publication cited herein is hereby incorporated by reference in its entirety. Although the present invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of the present invention may be designed by those skilled in the art without departing from the true spirit and scope of the present invention. The appended claims are intended to be interpreted as including all such embodiments and equivalent variations.

Claims

1. A Siglec-7 binding molecule comprising a Siglec-7 antibody, a Siglec-7 binding antibody fragment or a variant of a Siglec-7 antibody.

2. The Siglec-7 binding molecule according to claim 1, wherein the antibody comprises an amino acid sequence selected from the group consisting of: a) a variable heavy chain sequence comprising a CDR sequence selected from the group consisting of: SEQ ID NOs: 1-3; SEQ ID NOs: 17-19; SEQ ID NOs: 33-35; SEQ ID NOs: 49-51; SEQ ID NOs: 65-67; ​​and SEQ ID NOs: 81-83; b) a variable light chain sequence comprising a CDR sequence selected from the group consisting of: SEQ ID NOs: 9-11; SEQ ID NOs: 25-27; SEQ ID NOs: 41-43; SEQ ID NOs: 57-59; SEQ ID NOs: 73-75; and SEQ ID NOs: 89-91; c) a sequence that is at least 95% identical to one or more of the variable heavy chain sequences of SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:68, and SEQ ID NO:84; d) a sequence that is at least 95% identical to one or more of the variable light chain sequences of SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 44, SEQ ID NO: 60, SEQ ID NO: 76, 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.

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

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

92.

5. The Siglec-7 binding molecule or fragment thereof according to 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 multiple nucleic acid molecules, comprising one or more nucleotide sequences encoding the Siglec-7 binding molecule according to any one of claims 1 to 5.

7. The nucleic acid molecule or molecules according to claim 6, wherein the nucleic acid molecule or molecules comprises a) a nucleotide sequence encoding a variable heavy chain sequence, wherein the nucleotide sequence comprises a CDR encoding sequence selected from the group consisting of: SEQ ID NOs: 5-7; SEQ ID NOs: 21-23; SEQ ID NOs: 37-39; SEQ ID NOs: 53-55; SEQ ID NOs: 69-71; and SEQ ID NOs: 85-87; b) a nucleotide sequence encoding a variable light chain sequence, wherein the nucleotide sequence comprises a CDR encoding sequence selected from the group consisting of: SEQ ID NOs: 13-15; SEQ ID NOs: 29-31; SEQ ID NOs: 45-47; SEQ ID NOs: 61-63; SEQ ID NOs: 77-79; and SEQ ID NOs: 93-95; c) a nucleotide sequence having at least 95% identity to a variable heavy chain encoding sequence selected from the group consisting of: SEQ ID NO:8; SEQ ID NO:24; SEQ ID NO:40; SEQ ID NO:56; SEQ ID NO:72; and SEQ ID NO:88; d) a nucleotide sequence having at least 95% identity to a variable light chain encoding sequence selected from the group consisting of: SEQ ID NO: 16; SEQ ID NO: 32; SEQ ID NO: 48; SEQ ID NO: 64; SEQ ID NO: 80; and SEQ ID NO: 96; e) a fragment comprising at least 80% of the full-length sequence of a variable heavy chain encoding sequence selected from the group consisting of: SEQ ID NO: 8; SEQ ID NO: 24; SEQ ID NO: 40; SEQ ID NO: 56; SEQ ID NO: 72; and SEQ ID NO: 88; and f) a fragment comprising at least 80% of the full-length sequence of a variable light chain encoding sequence selected from the group consisting of: SEQ ID NO: 16; SEQ ID NO: 32; SEQ ID NO: 48; SEQ ID NO: 64; SEQ ID NO: 80; and SEQ ID NO:

96.

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

9. The nucleic acid molecule or molecules according to claim 7, wherein the nucleic acid molecule or molecules comprises a nucleotide sequence selected from the group consisting of: a) a first nucleotide sequence encoding a variable heavy chain sequence comprising SEQ ID NO: 8, and a second nucleotide sequence encoding a variable light chain sequence comprising SEQ ID NO: 16; b) a first nucleotide sequence encoding a variable heavy chain sequence comprising SEQ ID NO: 24, and a second nucleotide sequence encoding a variable light chain sequence comprising SEQ ID NO: 32; c) a first nucleotide sequence encoding a variable heavy chain sequence comprising SEQ ID NO: 40, and a second nucleotide sequence encoding a variable light chain sequence comprising SEQ ID NO: 48; d) a first nucleotide sequence encoding a variable heavy chain sequence comprising SEQ ID NO: 56, and a second nucleotide sequence encoding a variable light chain sequence comprising SEQ ID NO: 64; e) a first nucleotide sequence encoding a variable heavy chain sequence comprising SEQ ID NO: 72, and a second nucleotide sequence encoding a variable light chain sequence comprising SEQ ID NO: 80; and f) a first nucleotide sequence encoding a variable heavy chain sequence comprising SEQ ID NO: 88, and a second nucleotide sequence encoding a variable light chain sequence comprising SEQ ID NO:

96.

10. A composition comprising the Siglec-7 binding molecule according to any one of claims 1 to 5. The composition according to claim 10 , further comprising at least one selected from the group consisting of pharmaceutically acceptable excipients and adjuvants.

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

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

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

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

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

17. A composition comprising one or more nucleic acid molecules encoding Siglec-7 binding molecules according to any one of claims 6 to 9.

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

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

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

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

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

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

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

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

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

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

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

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

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

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