Bispecific natural killer engagers targeting Siglec-7

Abstract

The present invention provides natural killer engagers specific for activating natural killer cells via binding to Siglec-7, and nucleic acid molecules encoding same, as well as methods of using same to treat or prevent a disease or disorder.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 325,887, filed March 31, 2022, U.S. Provisional Application No. 63 / 375,784, filed September 15, 2022, and U.S. Provisional Application No. 63 / 490,156, filed March 14, 2023, each of which is incorporated by reference in its entirety. [Background technology]

[0002] Ovarian cancer (OC) is the most lethal gynecologic malignancy. It is the fifth leading cause of cancer deaths in women and the most common cause of deaths from cancers of the female reproductive system. According to the American Cancer Society, an estimated 21,410 women will be newly diagnosed with ovarian cancer in 2021, and 13,770 women will die due to OC (Kurnit et al., 2021, Obstet Gynecol 137: 108-21; www_cancer_org). OC is a highly heterogeneous cancer, with 90% of tumors being of epithelial origin. The most common subtype of epithelial ovarian cancer (EOC) is high-grade serous carcinoma, which accounts for approximately 70-80% of cases. On the other hand, low-grade serous (<5%), endometrioid (10%), clear cell (10%), and mucinous (3%) are less common subtypes (Barnes et al., 2021, Genome Med 13: 140).

[0003] Surgery and chemotherapy are the main treatments for OC (Yang et al., 2020, Front Immunol 11: 577869). Unfortunately, these treatments are only partially successful, with many patients developing chemotherapy resistance within a few years after the first treatment and thus facing disease recurrence (Yang et al., 2020, Front Immunol 11: 577869). The high mortality rate of OC is also associated with a low rate of early detection, often due to subjective symptoms and a paucity of less invasive techniques for primary detection. Therefore, OC remains an area in need of improved and new therapeutic approaches (Banno et al., 2014, Biomed Res Int 2014: 232817). Due to the close interaction between ovarian tumor cells and the tumor microenvironment, it is important to develop therapies that can not only target tumor cells but also maintain antitumor functions in this microenvironment (CSSOCR, 2016). An expanding area of ​​research is immune-based therapies for OC. Such research includes immune checkpoint inhibitors (ICIs), chimeric antigen receptors (CARs), and T cell receptor (TCR)-engineered T cells (Yang et al., 2020, Front Immunol 11: 577869). In particular, a major obstacle in the development of CAR therapy is finding targets with specific expression restricted to the surface of tumor cells, not outside the tumor tissue (Perales-Puchalt et al., 2017, Clin Cancer Res 23: 441-53). The follicle-stimulating hormone receptor (FSHR) is one such target that has been reported to be selectively expressed in ovarian granulosa cells and with lower levels of expression in the ovarian endothelium. FSHR is expressed in 50-70% of serous ovarian cancer cases, making it an important potential target for immunotherapy (Perales-Puchalt et al., 2017, Clin Cancer Res 23: 441-53).

[0004] mAbs are important tools in the diagnosis, classification, treatment, and monitoring of certain cancers. Examples include various forms of anti-HER2 antibodies for classification and treatment of breast cancer (Hayes et al., 2007, N Engl J Med 357: 1496-506; Pegram et al., 1998, J Clin Oncol 16: 2659-71), anti-CD20 antibodies for the treatment of lymphoma (Maloney et al., 1997, Blood 90: 2188-95), anti-CA125 antibodies for follow-up of OC (Bast et al., 1983, N Engl J Med 309: 883-7), and anti-PSA antibodies for the detection of prostate cancer (Siddall et al., 1986, Clin Chem 32: 2040-3). A recent important area in the field of antibody therapeutics is the study of bispecific T cell engagers (Perales-Puchalt et al., 2019, Mol Ther 27: 314-25). These are a new class of immunotherapeutics with the ability to simultaneously bind both T cells and tumor cells and promote the cytolytic function of T cells against specific tumor cells (Hipp et al., 2017, Leukemia 31: 2278).

[0005] In the case of OC, there are limited therapeutic options, especially for highly aggressive ovarian tumors such as high-grade serous ovarian cancer (HGSOC) and ovarian carcinosarcoma (OCS). These tumors respond poorly to existing ICI therapies and are often referred to as immunologically “cold” tumors (Wu et al., 2021, Front Immunol 12: 672502). Thus, T cell approaches alone may not be effective enough to treat challenging OC cases. It may be important to engage additional effector components of the immune system. In this regard, there are limited studies linking the effects of innate mechanisms, such as NK, with OC tumors, and no specific targeting methods for OC have been reported (Hoogstad-van Evert et al., 2020, Gynecol Oncol 157: 810-6).

[0006] Skin cancer is one of the most common and dangerous human cancers. Among its various subtypes, melanoma arising from melanocytes is a particularly serious disease. Immune checkpoint inhibition (CPI), a new FDA-approved immunotherapy, has revolutionized the treatment of melanoma and improved treatment outcomes. However, 40-50% of melanomas are immunologically cold, have poor T-cell infiltration, and tend to respond poorly to CPIs. For this reason, additional approaches are needed.

[0007] There remains a need in the art for immunotherapies that can effectively treat cancer, autoimmune diseases, and infectious diseases while minimizing adverse effects. The present invention fulfills this unmet need. Summary of the Invention

[0008] In one embodiment, the present invention provides a bispecific natural killer engager (NKCE) or fragment thereof comprising an antibody or fragment thereof that specifically binds to Siglec-7 and an antibody or fragment thereof that specifically binds to an antigen.

[0009] In one embodiment, the antigen is a tumor antigen. In one embodiment, the tumor antigen is follicle stimulating hormone receptor (FSHR) or interleukin 13 receptor subunit alpha 2 (IL-13R alpha 2).

[0010] In one embodiment, the Siglec-7 binding arm of the bispecific NKCE comprises a variable heavy chain sequence comprising the CDR sequences of SEQ ID NOs: 1-3, 17-19, 33-35, 49-51, 65-67, or 81-83.

[0011] In one embodiment, the Siglec-7 binding arm of the bispecific NKCE comprises a variable heavy chain sequence comprising the CDR sequences of SEQ ID NOs: 9-11, 25-27, 41-43, 57-59, 73-75, or 89-91.

[0012] In one embodiment, the Siglec-7 binding arm of the bispecific NKCE 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 Siglec-7 binding arm of the bispecific NKCE 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 bispecific NKCE comprises the amino acid sequence of SEQ ID NO:98, SEQ ID NO:100, or SEQ ID NO:102.

[0015] In one embodiment, the present invention relates to a nucleic acid molecule encoding a bispecific natural killer engager (NKCE) or a fragment thereof comprising an antibody or a fragment thereof that specifically binds to Siglec-7 and an antibody or a fragment thereof that specifically binds to an antigen.

[0016] In one embodiment, the nucleic acid molecule comprises a heavy chain CDR encoding the sequence of SEQ ID NO:5 to 7, SEQ ID NO:21 to 23, SEQ ID NO:37 to 39, SEQ ID NO:53 to 55, SEQ ID NO:69 to 71, or SEQ ID NO:85 to 87. In one embodiment, the nucleic acid molecule comprises a light chain CDR encoding the sequence of SEQ ID NO:13 to 15, SEQ ID NO:29 to 31, SEQ ID NO:45 to 47, SEQ ID NO:61 to 63, SEQ ID NO:77 to 79, or SEQ ID NO:93 to 95.

[0017] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the variable heavy chain sequence of SEQ ID NO:8, SEQ ID NO:24, SEQ ID NO:40, SEQ ID NO:56, SEQ ID NO:72, or SEQ ID NO:88; a nucleotide sequence encoding the variable light chain sequence of SEQ ID NO:16, SEQ ID NO:32, SEQ ID NO:48, SEQ ID NO:64, SEQ ID NO:80, or SEQ ID NO:96; a nucleotide sequence having at least 95% identity to the variable heavy chain sequence of SEQ ID NO:8, SEQ ID NO:24, SEQ ID NO:40, SEQ ID NO:56, SEQ ID NO:72, or SEQ ID NO:88; a sequence having at least 95% identity to 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; a fragment comprising at least 80% of the full length sequence of the variable heavy chain sequence of SEQ ID NO:8, SEQ ID NO:24, SEQ ID NO:40, SEQ ID NO:56, SEQ ID NO:72, or SEQ ID NO:88; or a fragment comprising at least 80% of the full length sequence 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.

[0018] In one embodiment, the nucleic acid molecule encoding the bispecific NKCE comprises the nucleotide sequence of SEQ ID NO:97, SEQ ID NO:99, or SEQ ID NO:101.

[0019] In one embodiment, the invention provides a method of treating or preventing a disease or disorder in a subject in need thereof comprising administering to the subject a nucleic acid molecule of the invention.

[0020] In one embodiment, the disease or disorder is selected from the group consisting of a disease or disorder associated with a bacterial infection, a disease or disorder associated with a viral infection, an autoimmune disease or disorder, cancer, or a disease or disorder associated with cancer.

[0021] In one embodiment, the cancer is selected from the group consisting of ovarian cancer, breast cancer, prostate cancer, renal cancer, colorectal cancer, gastric cancer, lung cancer, testicular cancer, skin cancer, and endometrial cancer.

[0022] In one embodiment, the invention provides a method of increasing natural killer cell function in a subject in need thereof comprising administering to the subject a nucleic acid molecule of the invention.

[0023] In one embodiment, the invention provides a method of directing natural killer cells to a target cell or particle in a subject in need thereof, comprising administering to the subject a nucleic acid molecule of the invention. [Brief description of the drawings]

[0024] [Fig. 1A-1I] Figure 1A-I show a series of images showing the generation of anti-human FSHR antibodies. (Figure 1A) Depiction of FSHR structure. (Figure 1B) Cloning strategy into pBMN-I-GFP expression vector. (Figure 1C) Mouse immunization scheme. (Figure 1D) cAMP response of K562 and K562-FSHR to different doses of FSH hormone. (Figure 1E) Western blot of phospho-phospho-p44 / 42 (Erk1 / 2) and p44 / 42 (Erk1 / 2) 20 min after stimulation of K562 and K562-FSHR cells with 1 μg / ml FSH. Analysis of variance. ***p<0.001. (Figure 1F) Flow cytometry plots of CaOV3, OVCAR3, and TOV-21G stained with DDAP (the most potent down-selected antibody clone) or with secondary APC-labeled antibody without primary antibody. (Figure 1G) Flow cytometry plots of TOV-21G parental or FSHR CRISPR stained with DDAP. (Figure 1H) Flow cytometry plots of K562, K562-FSHR, and K562-LHCGR21G stained with DDAP or with secondary APC-conjugated antibody without primary antibody. (Figure 1I) Flow cytometry plots of A20(GFP-) / A20-Fhsr(GFP+) and ID8-Defb29 / Vegf-a vs ID8-Defb29 / Vegf-a-Fshr cells stained with DDAP (both cell lines were transfected with mouse FSHR). [Fig. 2A-2I]Figures 2A-I are a series of immunohistochemical and immunocytochemical images showing that DDAP binds to FSHR and induces antibody-dependent cellular cytotoxicity. (Figure 2A) Immunohistochemical images of frozen sections of tumors from K562, K562-FSHR, OVCAR3, and TOV-21G cell lines stained with DDAP. 40x magnification, scale bar 50 μm. (Figure 2B) Immunofluorescence images of 293T cells transfected with human FSHR and stained with mouse anti-human FSHR or DDAP antibodies, followed by secondary anti-mouse IgG. (Figure 2C) Immunofluorescence images of 293T cells transfected with mouse FSHR and stained with mouse anti-mouse FSHR or DDAP antibodies, followed by secondary anti-mouse IgG. (Figure 2D) Immunofluorescence images of 293T cells transfected with pVax1 empty vector and stained with DDAP antibodies, followed by secondary anti-mouse IgG. B-D: Scale bar 10 μm. (Fig. 2E) Absorbance values ​​of isotype ELISA performed with DDAP antibody. (Fig. 2F) Cytotoxicity by ADCC of DDAP or irrelevant mouse IgG2a (C1.18.4) against K562-FSHR. (Fig. 2G) Cytotoxicity by ADCC of DDAP or irrelevant mouse IgG2a (C1.18.4) against K562. (Fig. 2H) Cytotoxicity by ADCC of DDAP or irrelevant mouse IgG2a (C1.18.4) against OVCAR3 cells. t-test, ANOVA. ***p<0.001, ns=not significant. (Fig. 2I) In vitro cytotoxicity obtained by co-culturing PBMCs and OVCAR3-FSHR cells with different concentrations (1000ng / ml, 500ng / ml, 250ng / ml, 31.25ng / ml) of DDAP anti-FSHR antibody or medium alone. Moderate dose-dependent killing was observed, but bactericidal efficacy was completely lost at 31.25 ng / ml. [Figures 3A-3I]Figure 3A-I show a series of images showing the generation, expression, and antitumor activity of FSHR TCE. (Figure 3A) Diagram of TCE engaging FSHR and T cell receptor (TCR). GS, glycine-serine; VH, heavy chain variable region; VL, light chain variable region. (Figure 3B) Schematic of DNA construct encoding DDAP-TCE. (Figure 3C) Western blot of in vitro expression of DDAP-TCE or pVax1 empty vector after transfection into Expi293F cells. (Figure 3D) Binding specificity of DDAP-TCE was demonstrated using K562 cells that lack natural expression of FSHR. No binding of DDAP-TCE was observed in K562 cells that do not express FSHR. (Figure 3E) Binding of DDAP-TCE to K562 cells overexpressing FSHR. (Figure 3F) Binding of DDAP-TCE to FSHR demonstrated using an additional FSHR expressing cell, CaOV3. The shift in the peak of DDAP-TCE compared to pVax1 and secondary antibody alone indicates that it binds to FSHR. (Figure 3G) Binding of DDAP-TCE to FSHR demonstrated using OVCAR3 cells transduced with pBMN-I-GFP plasmid encoding FSHR for overexpression of FSHR. There is a significant shift in the peak in OVCAR3 cells overexpressing FSHR compared to empty vector and secondary antibody alone controls. (Figure 3H) Flow staining of primary human T cells with DDAP-TCE and empty vector control. (Figure 3I) In vitro cytotoxicity resulting from co-culture of PBMCs (effector cells, E) with OVCAR3-FSHR cells (target cells, T) in the presence of two different concentrations (31.25 ng / ml and 7.81 ng / ml) of in vitro generated DDAP-TCE or medium alone. We observed very potent killing induced by DDAP-TCE, whereas FSHR antibody alone was unable to induce killing of OVCAR3-FSHR cells at this concentration, indicating enhanced potency of the designed TCE. [Figure 4A-4L]Figure 4A-L show a series of images showing that anti-Siglec7 antibody clones induce specific killing of target ovarian cancer cells. Evaluation of the cytotoxicity effects of DB-S7-1, DB-S7-2, and DB-S7-7 anti-Siglec7 clones in non-target human cells, (Figure 4A and Figure 4B) A549 lung adenocarcinoma cells, (Figure 4C and Figure 4D) HaCaT human keratinocytes, (Figure 4E and Figure 4F) GMO5389 human fibroblasts, and target human ovarian cancer cells, (Figure 4G and Figure 4H) OVISE cells, (Figure 4I and Figure 4J) OVCAR8 cells, (Figure 4K and Figure 4L) SKOV3 cells. In vitro cytotoxicity was measured based on impedance using xCELLigence real-time cell analyzer (RTCA) from Agilent Technologies, USA. Electrical conductivity is converted to a unitless cell index (CI) parameter by the xCELLigence device every 15 min and images are captured at 1 h intervals. The data generated is normalized to the time point when effector (E) cells (PBMC) and antibodies were added to target (T) cells (all E:T = 5:1). Data was analyzed using RTCA / RTCAPro software. No nonspecific killing was obtained in A549, HaCaT, and GMO5389 cells, whereas potent killing was observed in OVISE, OVCAR8, and SKOV3 target OC cells. The specific killing activity of DB-S7-2 was found to be the highest. Arrows indicate the time point when antibodies and effector cells were added to target cells. Images shown show killing 3 days after addition of effector cells and antibodies. [Figure 5A-5J]Figure 5A-5J show a series of in vitro human Siglec7 DMAb design, expression, and functionality. (Figure 5A) Schematic of the double plasmid DNA construct encoding Siglec7 DMAb. (Figure 5B) Western blot analysis of Siglec7 DMAb expressed in Expi293F cells. Numbers indicate molecular weight (kDa). (Figure 5C) Quantification of human IgG in Expi293F supernatants transfected with human Siglec7 DMAb by ELISA. (Figures 5D and 5E) In vitro cytotoxicity induced by fully human Siglec7 recombinant antibodies in OVCAR8 cells, (Figure 5F) TOV-21G cells, (Figure 5G) OVISE cells, and (Figure 5H) PEO-4 cells. All recombinant antibodies effectively killed the panel of human ovarian cancer cells mentioned above, including BRCA2 mutant cells and PARPi resistant PEO-4 cells. It can be seen that DB-S7-2 recombinant antibody exhibits the highest killing activity. The killing ability of DB-S7-2 was further confirmed in two ovarian cancer cells [CaOV3 cells (Figure 5I) and OVCAR3 cells (Figure 5J)]. The arrows indicate the time points when the antibody and effector cells (human PBMCs) were added to the target cells (E:T=5:1). The images shown in (Figure 5D) show the killing of OVCAR8 target OC cells after addition of human PBMCs and anti-Siglec7 antibodies for 3 days. [Figures 6A-6G]Figure 6A-G shows a series of images showing that Siglec7 DMAb is expressed in vivo and delays cancer progression in an ovarian cancer challenge model, and that inhibition of FcγR increases Siglec7-mediated NK cytotoxicity. (Figure 6A) Schematic of human Siglec7 DMAb administration to Balb / c mice. Mice were first depleted of CD4 and CD8 T cells using anti-mouse CD4 and anti-mouse CD8 antibodies (both from InVivoMab). Mice were then electroporated with 50 μg heavy chain + 50 μg light chain human Siglec7 DMAb. Mouse serum was collected at the indicated time points. (Figure 6B) Western blot of human IgG from mice serum electroporated with DB-S7-1, DB-S7-2, and DB-S7-7 DMAb or drug-naive serum 14 days after DNA injection and electroporation. (FIG. 6C) Human NK cells were stained using mouse sera electroporated with empty vector, DB-S7-1, DB-S7-2, and DB-S7-7 DMAb, or treatment-naïve sera, along with irrelevant and secondary antibody controls only, 14 days after DNA injection and electroporation. Day 14 sera from mice immunized with Siglec7 DMAb showed positive and strong NK cell staining, whereas no staining was observed with empty vector control, irrelevant antibody control, or treatment-naïve sera. (FIG. 6D) Schematic of Siglec7 DMAb electroporation into NSG mice challenged with OVISEOC cells (FIG. 6E). Growth curves of OVISE tumors implanted in NSG mice treated with Siglec7 DMAb or empty vector (n=5 mice per group). (FIG. 6F) xCELLigence real-time analysis of OVCAR3 cell killing by DB-S7-2 human antibody in the presence or absence of Fc block (FIG. 6G). Fc blockade enhanced Siglec7-mediated NK toxicity in OVISE cells. Images were captured 2 days after addition of effector cells (E:T=5:1) and DB-S7-2 antibody to OVISE cells in the presence or absence of Fc blockade. [Figures 7A-7H]Figures 7A-H show a series of images depicting the generation and expression of DDAP-NKCE, and the cytokine secretion profile and specificity analysis of FSHR-targeted bispecific T cell and NK cell engagers. (Figure 7A) Schematic of the DNA construct encoding DDAP-NKCE. GS: glycine-serine, ScFV: single chain variable fragment. (Figure 7B) Western blot of in vitro expression of DDAP-NKCE or pVax1 empty vector after transfection into expi293F cells. (Figure 7C) Flow cytometry plots of Siglec7-overexpressing HEK293T cells stained with secondary antibody only, DDAP-TCE, commercial Siglec7 antibody, DB-S7-2 anti-Siglec7 antibody, and DDAP-NKCE. (Figure 7D) Flow cytometry plots of FSHR-overexpressing K562 cells stained with secondary antibody only, non-FSHR-targeted TCE (IL13 receptor alpha 2xCD3), and DDAP-NKCE. (Figure 7E) IFA analysis of DDAP-NKCE in HEK293T cells overexpressing Siglec7. Staining with secondary antibody alone served as a negative control. High-resolution confocal images of fixed cells were acquired using a Leica TCS SP8 WLL scanning laser confocal microscope and Leica LAS-X software (Leica Microsystems, Inc., BuffaloGrove, IL). Post-processing of images included importing into Huygens software (Scientific Volume Imaging, Laapersveld, Hilversum, The Netherlands) for deconvolution. Fixed cell preparations were acquired using a 63× / 1.40 oil immersion objective, 2× zoom, and a 1 AU pinhole. Cells were labeled with DAPI (nuclei), GFP (Siglec7), and Texas Red (DDAP-NKCE) and acquired sequentially on a HyD detector to maximize signal and minimize crosstalk. Scale bar corresponds to 20.0 microns. (FIG. 7F) Secretion profile of cytokines (sFas and granulysin) in the presence of DDAP-NKCE in co-cultures of OVCAR3-FSHR and human PBMCS (E:T=5:1).Supernatants used for analysis of cytokine secretion profiles were collected 48 hours after addition of effector cells and NKCE targeting OVCAR3-FSHR cells. 2-way ANOVA; *P<0.05, **P<0.01, ***P<0.001. (Figure 7G) Specificity analysis of FSHR targeting T and NK cell engagers in FSHR-negative HEK293T cells. (Figure 7H) Images showing no cytotoxicity induced in HEK293T cells over 3 days after addition of effector cells (human PBMC; E:T=5:1) and DDAP-NKCE / DDAP-TCE. [Figure 8A-8J]Figures 8A-8J show a series of images showing that FSHR-targeted bispecific T and NK cell engagers induced ovarian cancer cytotoxicity in vitro and in vivo. In vitro cytotoxicity resulting from co-culture of PBMCs with (Figures 8A and 8B) OVISE cells, (Figures 8C and 8D) CaOV3 cells, (Figure 8E) OVCAR3-FSHR cells, (Figure 8F) PEO-4 cells, and (Figure 8G) Kuramochi-FSHR cells in the presence and absence of DDAP-TCE / DDAP-NKCE. Real-time in vitro cytotoxicity analysis was performed by xCELLigence. E:T is 10:1 (Figures 8A-8D) and 5:1 (Figures 8E-8G). Images shown (Figures 8B and 8D) were captured 3 days after addition of human PBMCs and bispecific antibodies to target cells. (FIG. 8H) Comparison of killing efficiency of DDAP-NKCE in the presence and absence of anti-Fas antibody. In the presence of anti-Fas antibody, DDAP-NKCE reduced the killing of target OVCAR3-FSHR cells. Red line: E+T (only tumor cells and PBMCs as effector cells), gray line: tumor cells, PBMCs, and anti-Fas antibody, magenta line: tumor cells, PBMCs, and DDAP-NKCE, and purple line: tumor cells, PBMCs, and DDAP-NKCE in the presence of anti-Fas antibody. (FIG. 8I) Schematic of tumor study to evaluate the effect of bispecific antibodies on tumor progression in NSG mouse model challenged with OVCAR3-FSHR. (FIG. 8J) Mean growth curves of OVCAR3-FSHR tumors implanted in NSG mice treated with DDAP-NKCE / DDAP-TCE or empty vector (n=5 mice per group). Two-way ANOVA; *P<0.05, **P<0.01, ***P<0.001. [Figure 9A-9D]Figure 9A-9D show a series of images showing the screening of anti-human FSHR antibodies. (Figure 9A) Schematic of flow cytometry plots showing potential outcomes in the screening process using K562 and K562-FSHR. (Figure 9B) Flow cytometry plots of K562(GFP-) / K562-Fhsr(GFP+) cells stained with serum (1:1000 dilution) of mice immunized with human FSHR or empty vector and anti-mouse IgG APC. (Figure 9C) Representative example of flow cytometry screening output strategy to detect FSHR binding antibodies from hybridomas as flow plot and fold mean fluorescence intensity of K562-FSHR / K562. (Figure 9D) Waterfall plot showing binding of hybridoma supernatants to FSHR measured as fold MFIK562-FSHR / K562. After the first screening experiment, the top 20 clones (left side of red bar) were used to proceed further. [Figure 10A-10C]Figures 10A-10C show a series of images showing that anti-Siglec7 antibodies efficiently bind to human Siglec7. (Figure 10A) Flow cytometry plots of Siglec7-overexpressing HEK293T cells stained with DB-S7-1, DB-S7-2, and DB-S7-7 anti-Siglec7 clones and isotype control (anti-Siglec3). (Figure 10B) Flow cytometry plots of Siglec9-overexpressing HEK293T cells stained with DB-S7-1, DB-S7-2, and DB-S7-7 anti-Siglec7 clones and anti-Siglec9 Ab (KB, Biolegend). (Figure 10C) IFA analysis of DB-S7-1, DB-S7-2, and DB-S7-7 anti-Siglec7 clones in Siglec7-overexpressing HEK293T cells. 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). Post-processing of images included importing into Huygens software (Scientific Volume Imaging, Laapersveld, Hilversum, The Netherlands) for deconvolution. Fixed cell preparations were acquired using a 63× / 1.40 oil immersion objective, 2× zoom, and 1 AU pinhole. Cells were labeled with DAPI (nuclei), GFP (Siglec7), and Texas Red (anti-Siglec7) and acquired sequentially on a HyD detector to maximize signal and minimize crosstalk. Scale bar corresponds to 20.0 microns. [Figures 11A-11C]Figures 11A-11C show a series of images showing quantification of human IgG in serum of mice immunized with Siglec7 DMAb. Balb / c mice (depleted of CD4 and CD8 T cells) were electroporated with 50 μg heavy chain + 50 μg light chain human Siglec7 DMAb. Mouse serum was collected at various time points. Expression levels of human IgG quantified by ELISA from mouse serum electroporated with (Figure 11A) DB-S7-1 DMAb, (Figure 11B) DB-S7-2 DMAb, and (Figure 11C) DB-S7-7 DMAb (n=5 mice per group). [Figure 12] FIG. 12 is a schematic diagram of FcγR blocking on immune cells and its effect on Siglec7-mediated toxicity. [Figure 13A-13D] Figures 13A-D show a series of images showing that FSHR-targeted bispecific T cell and NK cell engagers induce ovarian cancer cytotoxicity in the presence of purified immune cells, but no cell killing by FSHR-nontargeted NKCE. (Figure 13A) In vitro cytotoxicity resulting from co-culture of T cells and OVCAR3-FSHR cells in the presence of DDAP-TCE, (Figure 13B) NK cells and OVCAR3-FSHR cells in the presence of DDAP-NKCE. Real-time in vitro cytotoxicity analysis was performed by xCELLigence; E:T=5:1 (Figure 13B). No toxicity was induced in FSHR-overexpressing OVCAR3 cells by (Figure 13C) FSHR-nontargeted TCE (IL13Rα2-TCE) and (Figure 13D) FSHR-nontargeted NKCE (IL13Rα2-NKCE) in the presence of human PBMC. Arrows indicate the time when effector cells and TCE / NKCE were added. [Figure 14]Figure 14 is an image showing a combination study to engage both T cells and NK cells to target Kuramochi-FSHR cells. DDAP-NKCE and DDAP-TCE were combined at suboptimal doses to evaluate the combination effect. This combination showed synergistic effects on FSHR-expressing Kuramochi cells; green line: both DDAP-TCE and DDAP-NKCE were added. PBMCs were added as effector cells; E:T=5:1. Arrows indicate the time points when effector cells and DDAP-TCE / NKCE / both were added. [Figure 15] FIG. 15 is an image showing the development of a T cell engager that targets IL-13Ra2. [Figure 16] FIG. 16 is an image showing treatment of GBM with a T cell engager targeting IL-13Ra2. [Figure 17] FIG. 17 is an image showing the design of a bispecific NK cell engager targeting IL13Ra2 and Siglec7. [Figure 18] FIG. 18 is an image showing that IL13Ra2-NKCE did not induce killing of OVCAR and OVISE cells (ovarian cancer cells that do not express IL13Ra2). [Figure 19] FIG. 19 is an image showing expression of IL-13Ra2 on human melanoma cell lines. [Figure 20] FIG. 20 is an image showing cytotoxicity of melanoma cells by IL13Ra2-NKCE. [Figures 21A-21C] Figures 21A-C show the design and in vitro expression of Siglec-7 MAb. (Figure 21A) Schematic of the dual plasmid DNA construct encoding Siglec-7 MAb. (Figure 21B) Expression analysis of Siglec-7 MAb by Western blot. Numbers indicate molecular weight (kDa). (Figure 21C) Quantification of human IgG in anti-Siglec7-transfected Expi293F supernatants by ELISA. [Fig. 22A-22E]Figures 22A-E show in vitro binding characterization of human Siglec7 MAbs. (Figure 22A) Binding of Siglec7 MAbs to recombinant human Siglec7 analyzed by ELISA. Dose-dependent binding of DB7.1, DB7.2, and DB7.7 to human Siglec7 was observed, whereas no binding was observed with an irrelevant control. (Figure 22B) Binding of human Siglec7 MAbs to Siglec7-expressing HEK293T cells analyzed by flow cytometry. No binding was observed with the secondary antibody control alone or with an irrelevant MAb. Binding was observed with the commercial Siglec-7 Ab (F023-420, BD Pharmingen) and the Siglec-7 MAbs DB7.1, DB7.2, and DB7.3, with the highest binding observed with DB7.2. (FIG. 22C) Representative flow plot showing binding of DB7.2 anti-Siglec-7 antibody to human NK cells and T cells. DB7.2 binds primarily to NK cells. Positive control (commercially available fluorochrome-conjugated mouse anti-human Siglec-7 antibody) and negative controls (DB7.2 at 0 μg / ml or no secondary antibody) are shown (FIG. 22D). Summary plot of binding of DB7.2 anti-Siglec-7 to various immune populations analyzed in PBMCs of multiple donors (n=4). (FIG. 22E) Binding analysis of DB7.2 anti-Siglec-7 Ab to CD56dim and CD56bright NK cell subsets (n=4). [Diagram 23] Figure 23 shows the gating strategy used for flow cytometric analysis of Siglec-7 binding. A representative example of ND is shown. First the most stable acquisition was selected. Then, to ensure that only live single cells from the mononuclear cells were analyzed, doublets were excluded and singlet small lymphocytes were focused on using forward scatter height (FSC-H) vs. forward scatter area (FSC-A) plots and side scatter area (SSC-A) vs. FSC-A plots. Dead cells were excluded by gating on cells negative for the viability marker Aqua Blue and positive for CD45. CD4+ and CD8+ T lymphocytes were gated within CD3+ cells, and NK cells were identified by CD56 and CD16 expression within the CD19-CD3- population. [Fig. 24A-24D]Figures 24A-D show binding of Siglec-7 DMAb to human Siglec-7 protein. (Figure 24A) Binding of DB7.2 Siglec-7 DMAb to human Siglec-7 analyzed by Western blot. In the case of pVax1, there is no binding to an unrelated protein. (Figure 24B) SPR analysis of DB7.2 to human Siglec-7 protein. Strong binding of DB7.2 was observed with a K value of 44 pM. (Figure 24C) No binding of the unrelated ab to Siglec-7 or (Figure 24D) binding of DB7.2 to an unrelated protein was observed, indicating that DB7.2 is specific for human Siglec-7. [Fig. 25A-25I]Figures 25A-I show in vitro cytotoxicity induced by human Siglec-7 MAbs. Figures 25A and B show in vitro cytotoxicity induced by human Siglec-7 MAbs in non-target (Figures 25A and B) HaCaT human keratinocytes and target OC cells [(Figures 25C and D) OVCAR10, (Figure 25E) OVISE, (Figure 25F) PEO-4, and (Figure 25G) TOV-21G cells]. In vitro cytotoxicity was measured based on impedance using an xCELLigence real-time cell analyzer (RTCA) (Agilent Technologies, USA). Electrical conductivity is converted to cell index (CI) by the xCELLigence device every 15 minutes. DB7.1, DB7.2, and DB7.7 did not induce killing of HaCaT cells. As shown in images captured 2 days after treatment, no killing was observed in the no antibody control and DB7.1, DB7.2, and DB7.7 treated wells, indicating the absence of off-target toxic effects. All three Siglec-7 MAbs effectively killed the aforementioned panel of human OC cells, including BRCA2 mutant and PARPi resistant PEO-4 cells. DB7.2 MAb was found to exert the highest killing potential. The killing ability of DB7.2 was further confirmed in two additional OC cell types; (Figure 25H) CaOV3 cells and (Figure 25I) OVCAR3 cells. Arrows indicate the time points when antibody and effector cells (human PBMCs) were added to the target cells; E:T=5:1 / 10:1. Red: no antibody (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). [Figures 26A-26C]Figures 26A-C show ablation of FcR binding of Siglec-7 MAb. (Figure 26A) Schematic of FcR blocking on immune cells and its effect on Siglec-7 mediated cell killing. (Figure 26B) xCELLigence real-time analysis of OVCAR3 cell killing by DB7.2 human MAb in the presence or absence of Fc block. (Figure 26C) Fc block maintained DB7.2 Siglec-7 MAb mediated NK toxicity in OVISE cells. Images were captured 2 days after addition of effector cells (E:T=5:1) and DB7.2 MAb to OVISE cells in the presence or absence of Fc block. [Fig. 27A-27I]Figures 27A-I show that removal of FcR binding maintained the killing potency of Siglec-7 MAb, and the combination of Siglec-7 MAb and anti-PD1 enhanced OC cell killing. (Figure 27A) Comparison of OVISE cell killing by DB7.2 and DB7.2_TM Mod. DB7.2_TM Mod is a variant of DB7.2DMAb that contains a triple residue modification ("TM"; L234F / L235E / P331S) in the Fc domain that removes FcR and C1q binding. Killing potency of DB7.2_TM Mod was maintained in OVISE cells. Red: no Ab (effector cells + target cells only), dark blue: DB7.2 (effector cells + target cells + DB7.2), light blue (effector cells + target cells + DB7.2_TM Mod). (Figure 27B) Images after 24 hours of treatment with effector cells and DB7.2 / DB7.2_TM Mod targeting OVISE cells. (Figure 27C) DB7.2 and (Figure 27D) DB7.2_TM Mod cell index vs. treatment dose curves after 72 hours of co-culture of OVISE cells with human PBMCs. (Figure 27E) DB7.2_TM Mod and (Figure 27F) anti-PD1 (pembrolizumab) cell index vs. treatment dose curves after 72 hours of co-culture of PEO4 cells with human PBMCs. Killing of target PEO4 cells by (Figure 27G) DB7.2_TM Mod, (Figure 27H) anti-PD1 (pembrolizumab), and (Figure 27I) individual treatments and combination of DB7.2_TM Mod and anti-PD1 (E:T=5:1) in the presence of human PBMCs. The combination enhanced killing compared to individual CPI treatments. [Fig. 28A-28E]Figures 28A-E show that Siglec-7 DNA delivery MAb is expressed in vivo and slows cancer progression in an ovarian cancer challenge model. (Figure 28A) Schematic of human Siglec-7 DNA delivery MAb administration to Balb / c mice. Mice were first depleted of CD4 and CD8 T cells using anti-mouse CD4 and anti-mouse CD8 (both from InVivoMab) antibodies. Mice were then electroporated with 50 μg heavy chain + 50 μg light chain human Siglec-7 DMAb. Mouse serum was collected at the indicated time points. (Figure 28B) 14 days after DNA injection and electroporation, human NK cells were stained with mouse serum electroporated with empty vector, DB7.1, DB7.2, and DB7.7, or with drug-naive serum, along with irrelevant and secondary Ab controls only. Day 14 sera from mice immunized with Siglec-7 MAb showed positive and strong NK cell staining, whereas no staining was observed with empty vector control, irrelevant MAb, or drug naive serum. (Figure 28C) Schematic of Siglec-7 DNA delivery MAb electroporation into NSG-K mice challenged with OVISE cells. (Figure 28D) Growth curves of OVISE tumors implanted in NSG-K mice treated with DB7.2 or empty vector (n=5 mice per group). (Figure 28E) Survival advantage of DB7.2-treated mice compared to empty vector control. DB7.2-treated groups showed significantly reduced tumor burden and improved survival compared to pVax1 control groups. [Figure 29A-29B] Figures 29A and 29B show that Siglec-7 DMAb affected tumor growth in an OVISE challenge mouse model. (Figure 29A) Individual growth curves of OVISE tumors implanted in NSG-K mice treated with DB7.2DMAb or pVax1 empty vector (n=5 mice per group). (Figure 29B) Representative images of ovarian tumor-bearing mice treated with DB7.2DMAb or pVax1 at day 37. [Fig. 30A-30K]Figures 30A-K show the generation, expression, binding, and specificity analysis of DB7.2xD2AP11:FSHR-targeted bispecific NK cell engager. (Figure 30A) Schematic of DNA construct encoding DB7.2xD2AP11 NKCE; GS: glycine-serine, ScFV: single chain variable fragment. (Figure 30B) Western blot of in vitro expression of DB7.2xD2AP11 NKCE or pVax1 empty vector after transfection into expi293F cells. (Figure 30C) Flow cytometry plot 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. 30D) Flow cytometry plots of FSHR-overexpressing K562 cells stained with secondary antibody only, non-FSHR-targeting T cell engager (IL13 receptor alpha 2xCD3), D22AP11-TCE (FSHRxCD3), and DB7.2xD2AP11 NKCE. (FIG. 30E) IFA of DB7.2xD2AP11 NKCE in Siglec-7-overexpressing HEK293T cells. Staining with secondary antibody only served 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). Post-processing of images included importing into Huygens software (Scientific Volume Imaging, Laapersveld, Hilversum, The Netherlands) for deconvolution. Fixed cell preparations were acquired using a 63X / 1.40 oil immersion objective, 2x zoom, and a 1AU pinhole. Cells were labeled with DAPI (nuclei), GFP (Siglec-7), and Texas Red (DB7.2xD2AP11 NKCE) and acquired sequentially on a HyD detector to maximize signal and minimize crosstalk. Scale bar corresponds to 20.0 microns.Specificity analysis of DB7.2xD2AP11 NKCE in FSHR-negative (Figure 30F and G) HEK293T, (Figure 30H) GM05389, (Figure 30I and J) AGS, and (Figure 30K) WM3743 cells using xCELLigence real-time cell analyzer. No cell killing was observed in any of these FSHR non-expressing cells. Images show no cytotoxicity induced in HEK293T (G) and AGS (J) cells after addition of effector cells (human PBMC; E:T=10:1) and DB7.2xD2AP11 for 3 days. Red: no antibody (effector cells + target cells only); Purple: DB7.2xD2AP11 (effector cells + target cells + DB7.2xD2AP11 NKCE). [Fig. 31A-31I] Figures 31A-I show FSHR-targeted NK cell engager; DB7.2xD2AP11 NKCE induced ovarian cancer cytotoxicity in vitro. In vitro cytotoxicity induced by DB7.2xD2AP11 NKCE in the presence of human PBMCs in (Figures 31A and B) OVISE cells, (Figure 31C) OVCAR3-FSHR cells, (Figures 31D and E) CaOV3 cells, (Figure 31F) Kuramochi-FSHR cells, and (Figure 31G) PEO-4 cells. Real-time in vitro cytotoxicity analysis was performed by xCELLigence. E:T is 10:1 (A and B, F and G) and 5:1 (C-E). Images shown (B and E) were captured 3 days after addition of human PBMCs and NKCE to target cells. (Figure 31H) Dose-dependent cell lysis (E:T=5:1) induced by DB7.2xD2AP11 NKCE in FSHR-expressing OVISE cells in the presence of human PBMCs. IC50 value was obtained at 142.87 pM, indicating the efficacy of NKCE. (Figure 31I) Cell index vs. dose curve of DB7.2xD2AP11 NKCE in OVCAR3 cells. IC50 value was obtained at 236.6 pM, confirming its high potency. [Fig. 32A-32D]Figures 32A-D show specificity analysis of FSHR-targeted bispecific NK cell engagers. (Figure 32A) In vitro cytotoxicity resulting from co-culture of NK cells with OVCAR3-FSHR cells in the presence of DB7.2xD2AP11 NKCE. (Figure 32B) Killing of AGS (FSHR-negative) cells by anti-Siglec-7 MAb (DB7.2xD2AP11 NKCE did not induce killing of this cell line, demonstrating the specificity of the bispecific engager approach). Irrelevant NKCE (DB7.2xIL13Rα2NKCE) did not induce toxicity in (Figure 32C) FSHR-overexpressing OVCAR3 cells and (Figure 32D) OVISE cells in the presence of human PBMC. Real-time in vitro cytotoxicity analysis was performed by xCELLigence; E:T=5:1. Arrows indicate the time when effector cells and NKCE were added. [Fig. 33A-33C] Figures 33A-C show that DB7.2xD2AP11 NKCE induced dose-dependent cytotoxicity in OVISE cells overexpressing the target FSHR. DB7.2xD2AP11 NKCE induced dose-dependent killing in OVISE-FSHR cells when analyzed at different concentrations (120, 60, 30, 15, 7.5, 3.75, and 1.875 ng / ml) in the presence of human PBMCs obtained from three different healthy human donors (Figure 33A) ND578, (Figure 33B) ND502, (Figure 33C) ND609. The red line indicates no antibody (only effector and target cells). In vitro cytotoxicity was measured based on impedance using a xCELLigence real-time cell analyzer. E:T=5:1; arrows indicate the time when effector cells and NKCE were added to the target OVISE-FSHR cells. [Diagram 34]Figure 34 shows that DB7.2xD2AP11 NKCE induced dose-dependent cytotoxicity in target OVCAR3 cells. DB7.2xD2AP11 NKCE induced dose-dependent cell death in OVCAR3 cells when analyzed at different concentrations (120, 60, 30, 15, 7.5, 3.75, and 1.875 ng / ml) in the presence of human PBMCs. The red line indicates no antibody (only effector and target cells). In vitro cytotoxicity was measured based on impedance using a xCELLigence real-time cell analyzer. E:T=10:1, arrows indicate the time when effector cells and NKCE were added to target OVCAR3 cells. [Fig. 35A-35E] Figures 35A-E show that DB7.2xD2AP11 NKCE caused secretion of cytokines / cytotoxic molecules and influenced ovarian tumor growth in vivo. (Figure 35A) OVCAR3-FSHR cells were co-cultured with human PBMCs in the presence and absence of DB7.2xD2AP11 NKCE. (Figure 35B) Supernatants were collected after 72 hours and the secretion profile of cytokines / cytotoxic molecules was analyzed. (Figure 35C) Schematic of tumor study to evaluate the effect of DB7.2xD2AP11 NKCE on tumor progression in NSG-K mouse model challenged with OVCAR3-FSHR. (Figure 35D) Mean growth curves of OVCAR3-FSHR tumors implanted in NSG-K mice treated with DB7.2xD2AP11 NKCE or empty vector (n=5 mice per group). (FIG. 35E) Survival advantage of DB7.2xD2AP11 NKCE mice compared to empty vector controls. Two-way ANOVA; *P<0.05, **P<0.01, ***P<0.001. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Detailed Description The present invention relates to the development of a new class of bispecific NK cell engagers (NKCEs) that simultaneously target both Siglec7 and FSHR (DDAP NKCEs). In another embodiment, the bispecific NKCEs simultaneously target both Siglec7 and IL13Ra2.

[0026] In one aspect, the present invention relates to compositions that can be used to increase or enhance an immune response, i.e., generate a more effective immune response, by administering NKCE, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding same. In one embodiment, NKCE targets Siglec-7.

[0027] In one aspect, the invention relates to an NKCE comprising a combination of a sialic acid receptor antibody, or a fragment thereof, or a variant thereof, and an antibody, or a fragment thereof, or a variant thereof, specific for binding to a tumor antigen. In some embodiments, the invention relates to a nucleic acid molecule encoding an NKCE comprising a combination of a sialic acid receptor antibody, or a fragment thereof, or a variant thereof, and an antibody, or a fragment thereof, or a variant thereof, specific for binding to a tumor antigen.

[0028] In one embodiment, the present invention relates to a method for treating a disease or disorder in a subject in need thereof, comprising administering to the subject NKCE, a fragment thereof, a variant thereof, or a nucleic acid molecule encoding the same. In one embodiment, the disease or disorder is cancer. In one embodiment, the disease or disorder is an infectious disease.

[0029] In one embodiment, the present invention relates to a method for treating cancer or a related disease or disorder in a subject in need of such treatment, comprising administering to the subject an NKCE comprising a combination of a sialic acid receptor antibody, or a fragment thereof, or a variant thereof, and an antibody specific for binding to a tumor antigen, a viral glycoprotein, an MHC-binding antibody fragment, or a fragment thereof, or a variant thereof, or a nucleic acid molecule encoding the same.

[0030] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described.

[0031] As used herein, each of the following terms has the meaning associated with it in this section.

[0032] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0033] As used herein, "about" when referring to a measurable value, such as an amount, duration in time, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as appropriate variations for performing the disclosed methods.

[0034] "Antibody" means an antibody of the IgG, IgM, IgA, IgD, or IgE class, or a fragment or derivative thereof, including Fab, F(ab')2, Fd, and single chain antibodies, and derivatives thereof. The antibody may be an antibody isolated from a mammalian serum sample, a polyclonal antibody, an affinity purified antibody, or a mixture thereof that exhibits sufficient binding specificity for the desired epitope, or sequences derived therefrom.

[0035] "Antigen" refers to a protein that has the ability to generate an immune response in a host. An antigen can be recognized and bound by an antibody. Antigens can originate from within the body or from the external environment.

[0036] "CDR" is defined as the complementarity determining region amino acid sequences of an antibody, which are the hypervariable regions of the immunoglobulin heavy and light chains. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., US Department of Health and Human Services, National Institutes of Health (1987). There are three heavy chain CDRs (or CDR regions) and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. Thus, as used herein, "CDR" refers to all three heavy chain CDRs or all three light chain CDRs (or both all heavy chain CDRs and all light chain CDRs, as appropriate). Depending on the structure and protein folding of the antibody, other residues may be considered to be part of the antigen binding region and may be understood as such by the skilled artisan. See, e.g., Chothia et al., (1989) Conformations of immunoglobulin hypervariable regions; Nature 342, p 877-883.

[0037] "Antibody fragment" or "fragment of an antibody", as used interchangeably herein, refers to a portion of an intact antibody that contains the antigen binding site or variable region. This portion does not include the constant heavy chain domains (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 that contain only one light chain variable domain, single-chain polypeptides that contain three CDRs of a light chain variable domain, single-chain polypeptides that contain only one heavy chain variable region, and single-chain polypeptides that contain three CDRs of a heavy chain variable region.

[0038] As used herein, "adjuvant" means any molecule added to the vaccines described herein to increase the immunogenicity of an antigen.

[0039] As used herein, a "coding sequence" or "encoding nucleic acid" may refer to a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence encoding an antibody described herein. A coding sequence may also comprise a DNA sequence encoding an RNA sequence. A coding sequence may further comprise initiation and termination signals operably linked to regulatory elements comprising a promoter and a polyadenylation signal capable of directing expression in cells of an individual or mammal to which the nucleic acid is administered. A coding sequence may further comprise a sequence encoding a signal peptide.

[0040] As used herein, "complement" or "complementary" can mean that a nucleic acid has Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairing between nucleotides or nucleotide analogs of a nucleic acid molecule.

[0041] A "disease" is a condition in the health of an animal where the animal is unable to maintain homeostasis and where the animal's health continues to deteriorate if the disease is not ameliorated.

[0042] In contrast, a "disorder" in an animal is a health state in which the animal is able to maintain homeostasis, but in which the animal's health state is less favorable than it would be in the absence of the disorder. Leaving the disorder untreated does not necessarily result in an animal's health state becoming further deteriorated.

[0043] A disease or disorder is "alleviated" if the severity of a sign or symptom of the disease or disorder, the frequency with which a patient experiences such signs or symptoms, or both, are reduced.

[0044] "Encoding" refers to the inherent property of a particular sequence of a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers or macromolecules in biological processes that have a particular sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a particular sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if the protein is produced in a cell or other biological system by transcription and translation of the mRNA corresponding to the gene. 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, which is used as a template for transcription of the gene or cDNA, are said to code for the protein or other product of that gene or cDNA.

[0045] An "effective amount" of a compound is an amount of the compound sufficient to confer a benefit on a subject or system to which the compound is administered.

[0046] "Expression vector" refers to a vector that contains a recombinant polynucleotide that includes an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression. Other elements for expression can be supplied by the host cell or an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate a recombinant polynucleotide.

[0047] As used herein, a "feedback mechanism" may refer to a process implemented by either software or hardware (or firmware) that receives the desired tissue impedance (before, during, and / or after delivery of an energy pulse), compares it to a current value (preferably the current), and adjusts the delivered energy pulse to achieve a preset value. The feedback mechanism may be implemented by an analog closed loop circuit.

[0048] "Fragment" may refer to a polypeptide fragment of an antibody that is functional, i.e., capable of binding to a desired target and having the same intended effect as the full-length antibody. An antibody fragment is 100% identical to the full-length except for the absence of at least one amino acid from the N-terminus and / or C-terminus, in either case with or without a signal peptide and / or methionine at position 1. A fragment may comprise a percentage of 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more of the length of a particular full-length antibody, excluding the heterologous signal peptide. Fragments include fragments of polypeptides that are 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to an antibody, and further include an N-terminal methionine or a heterologous signal peptide, which are not included in the percent identity calculation. Fragments may further include an N-terminal methionine and / or a signal peptide, such as an immunoglobulin signal peptide, such as an IgE or IgG signal peptide. The N-terminal methionine and / or signal peptide may be linked to a fragment of an antibody.

[0049] Fragments of a nucleic acid sequence encoding an antibody are 100% identical to the full length except for the absence of at least one nucleotide from the 5' and / or 3' end, in either case with or without a signal peptide and / or methionine at position 1. Fragments can include 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more of the length of a particular full length coding sequence, excluding any added heterologous signal peptide. Fragments can include fragments that encode a polypeptide that is 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the antibody, and optionally include sequences encoding an N-terminal methionine or a heterologous signal peptide that are not included in the percent identity calculation. Fragments can further include a coding sequence for an N-terminal methionine and / or a signal peptide (e.g., an immunoglobulin signal peptide, such as an IgE or IgG signal peptide). The coding sequence encoding an N-terminal methionine and / or a signal peptide may be linked to the fragment of the coding sequence.

[0050] As used herein, a "genetic construct" refers to a DNA or RNA molecule that contains a nucleotide sequence that codes for a protein, such as an antibody. A genetic 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 a promoter and a polyadenylation signal, that can induce expression in the cells of an individual to which the nucleic acid molecule is administered. As used herein, the term "expressible form" refers to a genetic construct that includes the necessary regulatory elements operably linked to a coding sequence that encodes a protein, such that the coding sequence is expressed when present in the cells of an individual.

[0051] "Homologous" refers to sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. If both positions of two sequences being compared are occupied by the same base or amino acid monomer subunit, for example, if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions being compared, multiplied by 100. For example, if 6 out of 10 positions of two sequences are matching or homologous, then the two sequences are 60% homologous. As an example, the DNA sequences ATTGCC and TATGGC share 50% homology. Usually, the comparison is performed when the two sequences are aligned to give maximum homology.

[0052] "Identical" or "identity" as used herein in the context of two or more nucleic acid or polypeptide sequences means that the sequences have the same percentage of residues over a specified region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over a specified region, calculating the number of positions where identical residues occur in both sequences to calculate the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to calculate the percentage of sequence identity. If the two sequences are of different length, or if the alignment produces one or more staggered ends, and the identified comparison region contains only one sequence, the residues of that one 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 equivalent. Identity can be performed manually or using computer sequence algorithms such as BLAST and BLAST 2.0.

[0053] "Isolated" means altered or removed from its 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" if 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-native environment, such as, for example, a host cell.

[0054] In the context of the present invention, the following abbreviations are used for commonly occurring nucleobases: "A" for adenosine, "C" for cytosine, "G" for guanosine, "T" for thymidine, and "U" for uridine.

[0055] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA may also include introns, to the extent that a nucleotide sequence encoding a protein may, in some version, contain introns.

[0056] As used herein, "impedance" is used when describing a feedback mechanism and can be converted to a current value according to Ohm's law, allowing comparison to a preset current.

[0057] As used herein, "immune response" means activation of a host's immune system (e.g., a mammalian immune system) in response to the introduction of one or more nucleic acids and / or peptides. The immune response can take the form of a cellular or humoral response, or both.

[0058] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal or cells thereof, in vitro or in situ, amenable to the methods described herein. In some embodiments, the patient, subject, or individual is a human.

[0059] "Parenteral" administration of the compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intradermal injection, or infusion techniques.

[0060] As used herein, "nucleic acid" or "oligonucleotide" or "polynucleotide" may mean at least two nucleotides covalently linked. A description of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also includes the complementary strand of a described single strand. Many variants of a nucleic acid can be used for the same purpose as a specific nucleic acid. Thus, a nucleic acid also includes substantially identical nucleic acids and their complements. A single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also includes a probe that hybridizes under stringent hybridization conditions.

[0061] Nucleic acids can be single-stranded or double-stranded, or can contain portions of both double-stranded and single-stranded sequences. Nucleic acids can be DNA (both genomic and cDNA), RNA, or hybrids, and can contain combinations of deoxyribonucleotides and ribonucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acids can be obtained by chemical synthesis or recombinant methods.

[0062] As used herein, "operably linked" may mean that the expression of a gene is under the control of a promoter to which the gene is spatially connected. The promoter may be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene is approximately the same as the distance between the promoter and the gene it controls in the gene from which it is derived. As is known in the art, changes in this distance can be accommodated without loss of promoter function.

[0063] As used herein, "peptide," "protein," or "polypeptide" refers to a linked sequence of amino acids, which may be natural, synthetic, or a modified or combination of natural and synthetic.

[0064] A "promoter" as used herein may refer to a synthetic or naturally derived molecule capable of conferring, activating, or enhancing expression of a nucleic acid in a cell. A promoter may contain one or more specific transcriptional regulatory sequences to further enhance expression and / or alter the spatial and / or temporal expression of expression. A promoter may also contain distal enhancer or repressor elements located as far away as several thousand base pairs from the start site of transcription. Promoters may be derived from sources such as viruses, bacteria, fungi, plants, insects, animals, etc. A promoter may constitutively or differentially control the expression of genetic components with respect to the cell, tissue, or organ in which expression occurs, or with respect to the developmental stage in which expression occurs, or in response to an external stimulus such as physiological stress, a pathogen, a metal ion, or an inducer. Representative examples of promoters include a bacteriophage T7 promoter, a bacteriophage T3 promoter, an SP6 promoter, a lac operator promoter, a tac promoter, an SV40 late promoter, an SV40 early promoter, an RSV-LTR promoter, a CMVIE promoter, an SV40 early promoter, or an SV40 late promoter, and a CMVIE promoter.

[0065] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to the promoter / regulatory sequence. In some cases, this sequence is the core promoter sequence, and in other cases, this sequence also includes enhancer sequences and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may, for example, be one that expresses the gene product in a tissue-specific manner.

[0066] 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.

[0067] An "inducible" 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 substantially only when an inducer corresponding to the promoter is present in the cell.

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

[0069] "Signal peptide" and "leader sequence" are used interchangeably herein and refer to an amino acid sequence that can be linked to the amino terminus of a protein described herein. A signal peptide / leader sequence typically directs the localization of a protein. As used herein, a signal peptide / leader sequence may facilitate secretion of a protein from the cell in which it is produced. A signal peptide / leader sequence is often cleaved from the remainder of the protein (often referred to as the mature protein) upon secretion from the cell. A signal peptide / leader sequence is linked to the N-terminus of a protein.

[0070] As used herein, "stringent hybridization conditions" may refer to conditions under which a first nucleic acid sequence (e.g., a probe) hybridizes to a second nucleic acid sequence (e.g., a target), such as in a complex mixture of nucleic acids. Stringent conditions are sequence-dependent and will vary from situation to situation. Stringent conditions may be selected to be about 5-10°C lower than the thermal melting point (Tm) of a particular sequence at a defined ionic strength pH. Tm may be the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (the target sequence is present in excess, so at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions are conditions with a pH of 7.0-8.3, a salt concentration of less than about 1.0 M sodium ion, for example, about 0.01-1.0 M sodium ion concentration (or other salt), and a temperature 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., more than about 50 nucleotides). Stringent conditions can also be achieved by adding a destabilizing agent such as formamide. For selective or specific hybridization, a positive signal will be at least 2-10 times higher than background hybridization. Exemplary stringent hybridization conditions include: 50% formamide, 5xSSC, and 1% SDS, incubated at 42° C., or 5xSSC, 1% SDS, incubated at 65° C., washed with 0.2xSSC, and 0.1% SDS at 65° C.

[0071] 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, mice, non-human primates (e.g., monkeys such as cynomolgus monkeys, rhesus monkeys, chimpanzees, etc.), and humans). In some embodiments, the subject can be human or non-human. The subject or patient may be undergoing other forms of therapy.

[0072] As used herein, "substantially complementary" means that a first sequence is substantially complementary to a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more nucleotides or amino acids. It can mean that a 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 the sequence, or that the two sequences hybridize under stringent hybridization conditions.

[0073] As used herein, "substantially identical" means 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, 800, 900, 1000, 1100, Over a region of 1 or more nucleotides or amino acids, or with respect to nucleic acids, when a first sequence is substantially complementary to the complement of a second sequence, it can mean at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.

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

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

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

[0077] As used herein, "treating a disease or disorder" means reducing the frequency or severity, or both, of at least one sign or symptom of the disease or disorder experienced by the patient.

[0078] As used herein, the phrase "therapeutically effective amount" refers to an amount sufficient or effective to prevent or treat (delay or prevent the onset of, prevent, inhibit, reduce, or reverse) a disease or disorder, including alleviating the signs and / or symptoms of such diseases and disorders.

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

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

[0081] A variant can be further defined as a peptide or polypeptide that differs in amino acid sequence by insertion, deletion, or conservative substitution of amino acids, but retains at least one biological activity. Representative examples of "biological activity" include the ability to bind to a specific antibody or to stimulate an immune response. A variant can also refer to a protein having an amino acid sequence substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity. Conservative substitutions of amino acids, i.e., replacing one amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree and distribution of charged regions), are recognized in the art as usually involving minor changes. These minor changes can be identified in part by considering the hydropathic index of the amino acid, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one embodiment, amino acids with a hydropathic index of ±2 are substituted. The hydrophilicity of amino acids can also be used to identify substitutions that will result in a protein retaining biological function. Considering the hydrophilicity of amino acids in the context of a peptide, the maximum local average hydrophilicity of the peptide can be calculated, which is a useful measure that has been reported to correlate well with antigenicity and immunogenicity. Substitution of amino acids with similar hydrophilicity values ​​may result in peptides that retain biological activity, such as immunogenicity, as understood in the art. Substitutions can be made with amino acids with hydrophilicity values ​​within ±2 of each other. Both the hydrophobic index and hydrophilicity value of an amino acid are influenced by the particular side chain of that amino acid. Consistent with this observation, it is understood that amino acid substitutions that are compatible with biological function depend on the relative similarity of amino acids, particularly the similarity of the side chains of these amino acids as revealed by their hydrophobicity, hydrophilicity, charge, size, and other properties.

[0082] A variant may be a nucleic acid sequence that is substantially identical over the entire length of the complete gene sequence or a fragment thereof. The nucleic acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the entire length of the gene sequence or a fragment thereof. A variant may be an amino acid sequence that is substantially identical over the entire length of the amino acid sequence or a fragment thereof. The amino acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the entire length of the amino acid sequence or a fragment thereof.

[0083] A "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the inside of a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, etc.

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

[0085] explanation Provided herein is an NKCE comprising a domain that specifically binds to a sialic acid-binding receptor, a fragment thereof, or a variant thereof, and further a domain that specifically binds to an antigen expressed by a target cell of interest, and a nucleic acid molecule encoding the same. In one embodiment, the sialic acid-binding receptor is a sialic acid-binding immunoglobulin-type lectin (Siglec) polypeptide or a selectin polypeptide. In one embodiment, the NKCE is specific for binding to Siglec-7, which is directly associated with NK cells, and can direct the killing and elimination of pathogenic cells.

[0086] In one embodiment, the present invention provides an immunogenic composition comprising the NKCE of the present invention or a nucleic acid molecule encoding the same. The immunogenic composition of the present invention can be used to protect against diseases or disorders, including, but not limited to, cancer and infectious diseases. In some embodiments, the immunogenic composition of the present invention can be used for cell-specific targeting of glycoproteins on cancer cells, autoimmune cells, or infected target cells.

[0087] Thus, in some embodiments, the present invention provides compositions comprising nucleic acid molecules encoding one or more NKCEs comprising a domain that specifically binds to a sialic acid binding receptor, a fragment thereof, or a variant thereof, and further comprising a domain that specifically binds to an antigen expressed by a target cell of interest.

[0088] In some embodiments, the present invention provides a method for treating or preventing a disease or disorder comprising administering to a subject a bispecific sialic acid binding receptor antibody of the present invention or a nucleic acid molecule encoding same.

[0089] In some embodiments, the present invention provides a method for treating or preventing cancer comprising administering to a subject a bispecific sialic acid binding receptor antibody, a fragment thereof, or a variant thereof, or a nucleic acid molecule encoding same, comprising a domain that specifically binds to a sialic acid binding receptor, a fragment thereof, or a variant thereof, and further comprising a domain that specifically binds to a cancer antigen or a nucleic acid molecule encoding same.

[0090] antibody composition In some embodiments, the present invention relates to a composition comprising at least one NKCE comprising a domain specific for binding to a sialic acid-binding receptor. In one embodiment, the sialic acid-binding receptor is a Siglec polypeptide or a selectin polypeptide. In one embodiment, the Siglec is Siglec7.

[0091] In one embodiment, the present invention relates to a composition comprising an NKCE comprising at least one Siglec7 binding domain or a fragment thereof. In one embodiment, the Silgec-7 binding domain or a fragment thereof of the bispecific NKCE comprises a variable heavy chain sequence of SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 36, SEQ ID NO: 52, SEQ ID NO: 68, or SEQ ID NO: 84. In one embodiment, the Silgec-7 binding domain or a fragment thereof of the bispecific NKCE comprises a variable light chain sequence of SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 44, SEQ ID NO: 60, SEQ ID NO: 76, or SEQ ID NO: 92. In one embodiment, the bispecific NKCE or a fragment thereof comprises a sequence of SEQ ID NO: 98, SEQ ID NO: 100, or SEQ ID NO: 102.

[0092] In some embodiments, variants of the amino acid sequences described herein include at least about 60% identity, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over a designated region when compared to the defined amino acid sequence. In some embodiments, variants of the amino acid sequences described herein have at least about 60% repeat sequence identity over the entire length of at least one of the amino acid sequences of SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:36, SEQ ID NO:44, SEQ ID NO:52, SEQ ID NO:60, SEQ ID NO:68, SEQ ID NO:76, SEQ ID NO:84, SEQ ID NO:92, SEQ ID NO:98, SEQ ID NO:100, or SEQ ID NO:102. 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 higher identity.

[0093] In some embodiments, fragments of the amino acid sequences described herein comprise at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the full length sequence of the defined amino acid sequence. In some embodiments, a fragment of an amino acid sequence described herein comprises at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of at least one full length sequence of SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:36, SEQ ID NO:44, SEQ ID NO:52, SEQ ID NO:60, SEQ ID NO:68, SEQ ID NO:76, SEQ ID NO:84, SEQ ID NO:92, SEQ ID NO:98, SEQ ID NO:100, or SEQ ID NO:102.

[0094] The term "antibody" or "immunoglobulin" as used herein refers to a protein (including glycoproteins) of the immunoglobulin (Ig) superfamily. An antibody or immunoglobulin (Ig) molecule may be a tetramer containing two identical light chain polypeptides and two identical heavy chain polypeptides. The two heavy chains are linked to each other 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.

[0095] Sialic acid binding receptor antibodies, or antigen-binding fragments thereof, include, but are not limited to, polyclonal antibodies, monoclonal fusion proteins, antibodies or fragments thereof, chimerized or chimeric fusion proteins, antibodies or fragments thereof, humanized fusion proteins, antibodies or fragments thereof, deimmunized humanized fusion proteins, antibodies or fragments thereof, fully human fusion proteins, antibodies or fragments thereof, single chain antibodies, single chain Fv fragments (scFv), Fv, Fd fragments, Fab fragments, Fab' fragments, F(ab')2 fragments, diabodies or antigen-binding fragments thereof, minibodies or antigen-binding fragments thereof, triabodies or antigen-binding fragments thereof, domain fusion proteins, antibodies or fragments thereof, camel 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 identified in a repetitive backbone array (e.g., repetitive antigen display).

[0096] The immune system produces several different classes of Ig molecules (isotypes), including IgA, IgD, IgE, IgG, and IgM, each distinguished by the particular class of heavy chain polypeptide present: alpha (a) for IgA, delta (δ) for IgD, epsilon (ε) for IgE, gamma (γ) for IgG, and mu (μ) for IgM. There are at least five different gamma heavy chain polypeptides (isotypes) for IgG. In contrast, light chain polypeptides are of only two isotypes, called kappa (κ) and lambda (λ) chains. The unique characteristics of an antibody isotype are defined by the sequence of the constant domain of the heavy chain.

[0097] An IgG molecule contains two light chains (κ or λ) and two heavy chains (γ) that are linked by disulfide bonds. The κ and λ IgG light chains each contain a variable region (V L Area”, “V κ Area" or "V λ Each IgG heavy chain contains a domain of relatively variable amino acid sequence called the constant region (CL region) and a domain of relatively conserved amino acid sequence called the variable region (V region).H A complete IgG heavy chain contains three constant domains ("C H 1 area”, “C H 2 area" and "C H Each V L Area or V H Within the region, hypervariable regions, also called complementarity determining regions ("CDRs"), are interspersed between relatively conserved framework regions ("FRs"). Generally, the variable region of a light or heavy chain polypeptide contains four FRs and three CDRs arranged in the following order along the polypeptide: NH2-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-COOH. Together, the CDRs and FRs determine the three-dimensional structure of the IgG binding site and therefore the specific target protein or antigen to which the IgG molecule binds. Each IgG molecule is a dimer and can bind two antigen molecules. Cleavage of the dimeric IgG with the protease papain produces two identical antigen-binding fragments ("Fab") and an "Fc" fragment or Fc domain. It is so named because it is easily crystallized.

[0098] The term "antibody" as used throughout this disclosure further refers to whole or intact antibody (e.g., IgM, IgG, IgA, IgD, or IgE) molecules produced by any of a variety of methods known in the art and described herein. The term "antibody" includes polyclonal antibodies, monoclonal antibodies, chimerized or chimeric antibodies, humanized antibodies, deimmunized human antibodies, and fully human antibodies. Antibodies can be generated or derived from a variety of species, including mammals such as humans, non-human primates (e.g., monkeys, baboons, chimpanzees), horses, cows, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. Antibodies can be purified antibodies or recombinant antibodies.

[0099] As used herein, the term "epitope" refers to a site on a protein to which an antibody binds. "Overlapping epitopes" include at least one (e.g., two, three, four, five, or six) common amino acid residues.

[0100] In one embodiment, the antibody of the present invention specifically binds to a Siglec polypeptide. As used herein, the term "specific binding" or "specifically binds" refers to two molecules that form a complex that is relatively stable under physiological conditions. Typically, the binding constant (K a ) is 10 6 M -1 If the binding is higher, it is considered specific. Thus, an antibody should have a binding affinity of at least 10 6 M -1 (or more) (e.g., at least 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , or 10 15 M -1 , or more (or more) K a can specifically bind to the target.

[0101] In one embodiment, the NKCE of the present invention comprises a domain that specifically binds to Siglec-7.

[0102] 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, binding of an antibody to a protein antigen can be detected and / or quantified using a variety of techniques, including, but not limited to, Western blot, dot blot, surface plasmon resonance (e.g., BIAcore system; Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ), or enzyme-linked immunosorbent assay (ELISA). For example, Harlow and Lane (1988) "Antibodies: A Laboratory Manual" Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY;Benny KC Lo (2004) "Antibody Engineering: Methods and Protocols," Humana Press (ISBN: 1588290921);Borrebaek (1992) "Antibody Engineering, A Practical Guide," WH Freeman and Co., NY;Borrebaek (1995) "Antibody Engineering," 2nd Edition, Oxford University Press, NY, Oxford; Johne et al. (1993) J. Immunol. Meth. 160: 191-198; Jonsson et al. (1993) Ann. Biol. Clin. 51: 19- 26; and Jonsson et al. (1991) Biotechniques 11 :620-627). See also U.S. Patent No. 6,355,245.

[0103] Immunoassays that can be used to analyze immunospecific binding and cross-reactivity of antibodies include, but are not limited to, competitive and noncompetitive assays using techniques such as Western blots, RIAs, ELISAs (enzyme-linked immunosorbent assays), "sandwich" immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays. Such assays are routine and well known in the art.

[0104] The antibodies can also be assayed using any surface plasmon resonance (SPR)-based assay known in the art to characterize the kinetic parameters of the interaction of the antibody with its target or epitope. Any commercially available SPR instrument can be used in the methods described herein, including, but not limited to, the BIAcore instrument (Biacore AB; Uppsala, Sweden); the lAsys instrument (Affinity Sensors; Franklin, Massachusetts); the IBIS system (Windsor Scientific Limited; Berks, UK), the SPR-CELLIA system (Nippon Laser and Electronics Lab; Hokkaido, Japan), and the SPR detector Spreeta (Texas Instruments; Dallas, Texas). See, e.g., Mullett et al. (2000) Methods 22: 77-91; Dong et al. (2002) Reviews in Mol Biotech 82: 303-323; Fivash et al. (1998) Curr Opin Biotechnol 9: 97-101; and Rich et al. (2000) Curr Opin Biotechnol 11:54-61.

[0105] Antibodies and fragments thereof may be "chimeric" in some embodiments. Chimeric antibodies and antigen-binding fragments thereof contain portions derived from two or more different species (e.g., mouse and human). Chimeric antibodies can be produced by splicing mouse variable regions with the desired specificity into human constant domain gene segments (see, e.g., U.S. Pat. No. 4,816,567). In this way, non-human antibodies can be modified to make them more suitable for human clinical applications (e.g., methods for treating or preventing complement-related diseases in human subjects).

[0106] The monoclonal antibodies of the present disclosure include "humanized" forms of non-human (e.g., murine) antibodies. Humanized or CDR-grafted mAbs are particularly useful as human therapeutics because they are not cleared from the circulation as rapidly as murine antibodies and do not usually induce adverse immune responses. Methods for preparing humanized antibodies are generally well known in the art. For example, humanization can be performed by replacing rodent CDRs or CDR sequences with the corresponding sequences of a human antibody, essentially following the method of Winter and coworkers (see, e.g., Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; and Verhoeyen et al. (1988) Science 239: 1534-1536). See also, e.g., Staelens et al. (2006) Mol Immunol 43:1243-1257. In some embodiments, humanized forms of non-human (e.g., murine) antibodies are human antibodies (recipient antibodies) in which hypervariable (CDR) region residues of the recipient antibody are replaced with hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and binding capacity. In some cases, framework region residues of the human immunoglobulin are also replaced with corresponding non-human residues (so-called "backmutation"). Furthermore, phage display libraries can be used to vary amino acids at selected positions within the antibody sequence. The properties of the humanized antibody are also influenced by the choice of human framework. Furthermore, humanized and chimeric antibodies can be modified to include residues that are not found in the recipient antibody or donor antibody to further improve antibody properties, such as affinity or effector function.

[0107] Fully human antibodies are also provided in the present disclosure. The term "human antibody" includes antibodies having variable and constant regions (if present) derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (i.e., humanized antibodies). Fully human or human antibodies may be obtained from transgenic mice having human antibody genes (having variable (V), diversity (D), joining (J), and constant (C) exons) or from human cells. For example, it is now possible to generate transgenic animals (e.g., mice) that, upon immunization, are capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production (see, e.g., Jakobovits et al. (1993) Proc. Natl. Acad. Sci. USA 90:2551: Jakobovits et al. (1993) Nature 362:255-258; Bruggemann et al. (1993) Year in Immunol. 7:33; and Duchosal et al. (1992) Nature 355:258). Transgenic mouse strains can be engineered to contain genetic sequences of unrearranged human immunoglobulin genes. The human sequences encode both heavy and light chains of human antibodies, function correctly in the mouse, and are rearranged to provide a broad antibody repertoire similar to that of humans. The transgenic mice are immunized with target proteins (to generate a variety of specific antibodies and the RNA encoding them). 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 the separate populations are combined upon insertion into a vector, so that any copy of the vector receives a random combination of heavy and light chains. The vector is designed to express an antibody chain, so that the antibody chain can be assembled and displayed on the outer surface of a display package containing the vector. For example, the antibody chain can be expressed as a fusion protein with a phage coat protein from the outer surface of a phage. The display package can then be screened for display of an antibody that binds to a target.

[0108] Thus, in some embodiments, the disclosure provides, e.g., humanized, deimmunized, or primatized antibodies that comprise one or more of the complementarity determining regions (CDRs) of a murine monoclonal antibody described herein, and retain the ability (e.g., at least 50, 60, 70, 80, 90, or 100%, or greater than 100%) of the murine monoclonal antibody counterpart to bind to its antigen.

[0109] Additionally, human antibodies can be obtained from phage display libraries (Hoogenboom et al. (1991) J. Mol. Biol. 227:381; Marks et al. (1991) J. Mol. Biol, 222:581-597; and Vaughan et al. (1996) Nature Biotech 14:309 (1996)). Synthetic phage libraries can be generated that use random combinations of synthetic human antibody V-regions. Depending on the selection of antigens, fully human antibodies can be generated in which the V-regions are essentially very similar to human. See, e.g., U.S. Pat. Nos. 6,794,132, 6,680,209, 4,634,666, and Ostberg et al. (1983), Hybridoma 2:361- 367. The contents of each of these references are incorporated herein by reference in their entirety.

[0110] For the generation of human antibodies, see also Mendez et al. (1998) Nature Genetics 15: 146-156 and Green and Jakobovits (1998) J. Exp. Med. 188:483-495, the disclosures of which are incorporated herein by reference in their entireties. Human antibodies are further discussed and described in U.S. Patent Nos. 5,939,598, 6,673,986, 6,114,598, 6,075,181, 6,162,963, 6,150,584, 6,713,610, and 6,657,103, as well as U.S. Patent Application Publication Nos. 2003-0229905A1, 2004-0010810A1, US2004-0093622A1, 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 0463151 B1. The disclosures of each of the above patents, applications, and references are incorporated herein by reference in their entirety.

[0111] As an alternative approach, other companies, including GenPharm International, Inc., have utilized a "minilocus" approach. In the minilocus approach, portions (individual genes) of the Ig locus are included to mimic the exogenous Ig locus. Thus, one or more VH genes, one or more DH genes, one or more JH genes, a μ constant region, and a second constant region (preferably a γ constant region) are formed into a construct for insertion into an animal. This approach is described, for example, in U.S. Patent Nos. 5,545,807, 5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, 5,789,650, and 5,814,318, 5,591,669, 5,612, 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 incorporated herein by reference. See also European Patent No. 0546073Bl, 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 incorporated herein by reference in their entirety.Furthermore, Taylor et al. (1992) Nucleic Acids Res. 20: 6287;Chen et al. (1993) Int. Immunol. 5: 647;Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90: 3720-4;Choi et al. (1993) Nature Genetics 4: 1 17;Lonberg et al. (1994) Nature 368: 856-859;Taylor et al. (1994) International Immunology 6: 579-591;Tuaillon et al. (1995) J. Immunol. 154: 6453- 65;Fishwild et al. Tuaillon et al. (2000) Eur. J. Immunol. 10: 2998-3005, the disclosures of each of which are incorporated herein by reference in their entireties.

[0112] In some embodiments, a deimmunized antibody or antigen-binding fragment thereof is provided. A deimmunized antibody or antigen-binding fragment thereof is an antibody that has been modified so that the antibody or antigen-binding fragment thereof is non-immunogenic or less immunogenic to a particular species (e.g., human). Deimmunization can be achieved by modifying the fusion protein, antibody, or fragment thereof using any of a variety of techniques known to those of skill in the art (see, e.g., PCT Publication Nos. WO04 / 108158 and WO00 / 34317). For example, a fusion protein, antibody, or fragment thereof can be deimmunized by identifying potential T-cell and / or B-cell epitopes within the amino acid sequence of the fusion protein, antibody, or fragment thereof, and removing one or more of the potential T-cell and / or B-cell epitopes from the fusion protein, antibody, or fragment thereof, e.g., using recombinant techniques. The modified antibody or antigen-binding fragment thereof can then be optionally generated and tested to identify an antibody or antigen-binding fragment thereof that retains one or more desired biological activities, e.g., binding affinity, but has reduced immunogenicity. Methods for identifying potential T cell epitopes and / or B cell epitopes can be carried out using techniques known in the art, such as computational methods (see, e.g., PCT Publication No. WO 02 / 069232), in vitro or in silico techniques, and biological or physical methods (e.g., determining the binding of the peptide to an MHC molecule, determining the binding of a peptide:MHC complex to a T cell receptor of the species that will receive the fusion protein, antibody, or fragment thereof, testing the protein or a peptide portion thereof using transgenic animals bearing MHC molecules of the species that will receive the antibody or antigen-binding fragment thereof, or testing using transgenic animals reconstituted with immune system cells of the species that will receive the fusion protein, antibody, or fragment thereof, etc.). In various embodiments, the deimmunized antibodies described herein include deimmunized antigen-binding fragments, Fab, Fv, scFv, Fab', and F(ab')2, monoclonal antibodies, murine antibodies, engineered antibodies (such as chimeric antibodies, single chain antibodies, CDR-grafted antibodies, humanized antibodies, fully human antibodies, and artificially selected antibodies), synthetic antibodies, and semi-synthetic antibodies.

[0113] In some embodiments, the present disclosure also provides bispecific antibodies. Bispecific antibodies are monoclonal antibodies, preferably human or humanized antibodies, that have binding specificities for at least two different antigens. For example, in one embodiment, the NKCE of the present invention comprises one domain that has binding specificity for a Siglec protein or polypeptide and one domain that has binding specificity for an alternative protein or polypeptide. In one embodiment, the NKCE of the present invention comprises one domain that has binding specificity for a Siglec protein or polypeptide and one domain that has binding specificity for an alternative Siglec protein or polypeptide.

[0114] Methods for producing NKCE are within the knowledge of those skilled in the art. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy / light chain pairs, where the two heavy / light chain pairs have different specificities (Milstein and Cuello (1983) Nature 305:537- 539). Antibody variable domains with the desired binding specificities (antibody antigen-binding sites) can be fused to immunoglobulin constant domain sequences. The fusion of the heavy chain variable region is preferably with an immunoglobulin heavy chain constant domain comprising at least a portion of the hinge, CH2, and CH3 regions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into a suitable host organism. For details of exemplary methods currently known for generating bispecific antibodies, see, e.g., Suresh et al. (1986) Methods in Enzymology 121:210; PCT Publication No. WO 96 / 27011; Brennan et al. (1985) Science 229:81; Shalaby et al, J Exp Med (1992) 175:217-225; Kostelny et al. (1992) J Immunol 148(5): 1547-1553; Hollinger et al. (1993) Proc Natl Acad Sci USA 90:6444-6448; Gruber et al. (1994) J Immunol 152:5368; and Tutt et al. (1991) J Immunol 147:60. Bispecific antibodies also include cross-linked or heteroconjugate antibodies. Heteroconjugate antibodies can be made using any convenient cross-linking method. Suitable cross-linking agents are known in the art, and are disclosed in U.S. Patent No. 4,676,980, along with a number of cross-linking techniques.

[0115] Various techniques have also been described for producing and isolating bispecific antibody fragments directly from recombinant cell culture. For example, bispecific antibodies have been produced using leucine zippers. See, for example, Kostelny et al. (1992) J Immunol 148(5): 1547-1553. The leucine zipper peptides from the Fos and Jun proteins can be linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers can be reduced at the hinge region to form monomers and then reoxidized to form antibody heterodimers. This method can also be used to produce antibody homodimers. The "diabody" technology described in Hollinger et al. (1993) Proc Natl Acad Sci USA 90:6444-6448 provides an alternative mechanism for making bispecific antibody fragments. The fragments contain a heavy chain variable domain (VH) connected to a light chain variable domain (VL) by a linker that is too short to allow pairing between the two domains on the same chain. Thus, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thus forming two antigen-binding sites. Another strategy for generating bispecific antibody fragments using single-chain Fv (scFv) dimers has also been reported. See, e.g., Gruber et al. (1994) J Immunol 152:5368. Alternatively, the antibodies may be "linear antibodies" as described, e.g., in Zapata et al. (1995) Protein Eng. 8(10): 1057-1062. Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions. Linear antibodies may be bispecific or monospecific.

[0116] Antibodies with more than three valencies (eg, trispecific antibodies) are also contemplated and are described, for example, in Tutt et al. (1991) J Immunol 147:60.

[0117] The present disclosure also includes variants of multispecific antibodies, such as dual variable domain immunoglobulin (DVD-lg) molecules, described in Wu et al. (2007) Nat Biotechnol 25(11): 1290-1297. DVD-lg molecules are designed such that two different light chain variable domains (VL) from two different parent antibodies are linked in tandem by recombinant DNA technology, either directly or via a short linker, followed by a light chain constant domain. Similarly, the heavy chain comprises two different heavy chain variable domains (VH) linked in tandem, followed by a constant domain CH1 and an Fc region. Methods for generating DVD-Ig molecules from two parent antibodies are further described, for example, in PCT Publication Nos. WO08 / 024188 and WO07 / 024715.

[0118] The present disclosure also provides camelid or dromedary antibodies (e.g., antibodies from Camelus bactrianus, Calelus dromaderius, or lama paccoss). Such antibodies differ from the typical two-chain (fragment) or four-chain (whole antibody) antibodies from most mammals and generally lack light chains. See U.S. Patent 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.

[0119] Engineered libraries of camelid antibodies and antibody fragments are commercially available, for example from Ablynx (Ghent, Belgium). As with other antibodies of non-human origin, the amino acid sequences of camelid antibodies can be recombinantly modified to obtain sequences that more closely resemble human sequences, i.e. nanobodies can be "humanized" to further reduce potential immunogenicity of the antibodies.

[0120] In some embodiments, the present disclosure also provides antibodies or antigen-binding fragments thereof that are variants of the peptides, proteins, or antibodies described herein. In some embodiments, such mutant peptides, proteins, or antibodies maintain the binding or inhibitory ability of the parent peptide, protein, or antibody. Methods for preparing variants of known proteins, peptides, or antibodies are known in the art. In some embodiments, such variants include at least a single amino acid substitution, deletion, insertion, or other modification. In some embodiments, the fusion proteins, antibodies, or fragments thereof described herein include two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) amino acid modifications (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the fusion proteins, antibodies, or fragments thereof described herein do not include amino acid modifications in the CDRs. In some embodiments, the fusion proteins, antibodies, or fragments thereof described herein comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acid modifications in a CDR.

[0121] As used herein, the term "antibody fragment," "antigen-binding fragment," "antigen-binding fragment," or similar terms refer to a fragment of an antibody that retains the ability to bind to an antigen, where the antigen-binding fragment may optionally include additional compositions not part of the original antibody (e.g., different framework regions or mutations) and fragments from the original antibody. Examples include, but are not limited to, single chain antibodies, single chain Fv fragments (scFv), Fd fragments, Fab fragments, Fab' fragments, or F(ab')2 fragments. An scFv fragment is a single polypeptide chain that contains both the heavy and light chain variable regions of the antibody from which the scFv is derived. Additionally, diabodies (Poljak (1994) Structure 2(12): 1121-1123; Hudson et al. (1999) J. Immunol. Methods 23(1-2): 177-189, the disclosures of each of which are incorporated herein by reference in their entireties), minibodies, triabodies (Schoonooghe et al. (2009) BMC Biotechnol 9:70), and domain antibodies (also called "heavy chain immunoglobulins" or camelids, Holt et al. (2003) Trends Biotechnol 21(1 1):484-490), which bind to complement component proteins (the disclosures of each of which are incorporated herein by reference in their entireties), can be incorporated into the compositions and used in the methods described herein. In some embodiments, any of the antigen-binding fragments described herein may be included under the term "antigen-binding fragment thereof" or equivalent terms when referring to a fragment related to an antibody, regardless of whether such fragment is actually derived from the antibody or is an antigen-binding fragment that binds to the same epitope or an overlapping epitope or an epitope included in the epitope of the antibody. The antigen-binding fragment includes antigen-binding fragments that bind to the same or overlapping antigen as the original antibody, where the antigen-binding fragment includes a portion that is a fragment of the original antibody (e.g., one or more CDRs, one or more variable regions, etc.).

[0122] In some embodiments, the antibodies described herein contain altered or mutated sequences that have altered stability or half-life compared to the parent antibody. This includes, for example, increased stability or half-life due to improved affinity or extended clearance time in vitro or in vivo, or decreased stability or half-life due to decreased affinity or more rapid clearance. Additionally, the antibodies described herein can contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acid substitutions, deletions, or insertions that result in altered post-translational modifications, including, for example, altered glycosylation patterns (e.g., addition of one or more sugar moieties, loss of one or more sugar moieties, or a change in the composition of one or more sugar moieties).

[0123] In some embodiments, the antibodies described herein comprise reduced (e.g., or no) effector function. Altered effector function includes, for example, modulation of one or more of the following activities: antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), apoptosis, binding to one or more Fc receptors, and proinflammatory response. Modulation refers to the effector function activity exhibited by the subject antibody comprising the altered constant region being increased, decreased, or eliminated compared to the activity of the unaltered constant region. In certain embodiments, modulation includes situations in which the activity is eliminated or completely absent.

[0124] Antibodies with altered or no effector functions can be generated by engineering or producing antibodies with variant constant regions, Fc, or heavy chain regions, and recombinant DNA technology and / or cell culture and expression conditions can be used to produce antibodies with altered function and / or activity. For example, recombinant DNA technology can be used to incorporate one or more amino acid substitutions, deletions, or insertions in regions that affect antibody function, including effector function, such as the Fc region or constant region. Alternatively, alterations in post-translational modifications, such as glycosylation patterns, can be achieved by manipulating the cell culture and expression conditions in which the antibody is produced. Suitable methods for introducing one or more substitutions, additions, or deletions into an Fc region of an antibody are known in the art and include standard DNA mutagenesis techniques as described, for example, in Sambrook et al. (1989) "Molecular Cloning: A Laboratory Manual, 2nd Edition," Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane (1988), supra; Borrebaek (1992), supra; Johne et al. (1993), supra; PCT Publication No. WO06 / 53301, and U.S. Pat. No. 7,704,497.

[0125] nucleic acid molecule Provided herein is a polynucleotide encoding the NKCE antibody or fragment thereof of the present invention. In some embodiments, the polynucleotide also comprises a sequence encoding a signal peptide operably linked to the 5' end of the coding sequence. In some embodiments, the polynucleotide also comprises a sequence encoding a linker sequence.

[0126] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding an NKCE comprising a Siglec-7 binding arm comprising at least one of SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:36, SEQ ID NO:44, SEQ ID NO:52, SEQ ID NO:60, SEQ ID NO:68, SEQ ID NO:76, SEQ ID NO:84, or SEQ ID NO:92.

[0127] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the NKCE of SEQ ID NO:98, SEQ ID NO:100, or SEQ ID NO:102.

[0128] In one embodiment, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:24, SEQ ID NO:32, SEQ ID NO:40, SEQ ID NO:48, SEQ ID NO:56, SEQ ID NO:64, SEQ ID NO:72, SEQ ID NO:80, SEQ ID NO:88, or SEQ ID NO:96 encoding an NKCE comprising a Siglec-7 binding arm.

[0129] In one embodiment, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:97, SEQ ID NO:99, or SEQ ID NO:101 which encodes a bispecific NKCE.

[0130] In one embodiment, the nucleic acid molecule comprises an RNA molecule corresponding to at least one of the nucleotide sequences of SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:24, SEQ ID NO:32, SEQ ID NO:40, SEQ ID NO:48, SEQ ID NO:56, SEQ ID NO:64, SEQ ID NO:72, SEQ ID NO:80, SEQ ID NO:88, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:99, or SEQ ID NO:101.

[0131] In one embodiment, the nucleic acid molecule comprises a DNA molecule corresponding to at least one of the nucleotide sequences of SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:24, SEQ ID NO:32, SEQ ID NO:40, SEQ ID NO:48, SEQ ID NO:56, SEQ ID NO:64, SEQ ID NO:72, SEQ ID NO:80, SEQ ID NO:88, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:99, or SEQ ID NO:101.

[0132] In some embodiments, variants of the nucleotide sequences described herein comprise at least about 60% identity, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over a designated region when compared to a defined nucleotide sequence. In some embodiments, variants of the nucleotide sequences described herein have at least about 6 to 10 nucleotides across the entire length of at least one of the nucleotide sequences SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:24, SEQ ID NO:32, SEQ ID NO:40, SEQ ID NO:48, SEQ ID NO:56, SEQ ID NO:64, SEQ ID NO:72, SEQ ID NO:80, SEQ ID NO:88, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:99, or SEQ ID NO:101. including 0% 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.

[0133] In some embodiments, fragments of the nucleotide sequences described herein comprise at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the full length sequence of the defined nucleotide sequence. In some embodiments, a fragment of a nucleotide sequence described herein has at least about 60% identity, 61%, or more identity to the full length sequence of SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:24, SEQ ID NO:32, SEQ ID NO:40, SEQ ID NO:48, SEQ ID NO:56, SEQ ID NO:64, SEQ ID NO:72, SEQ ID NO:80, SEQ ID NO:88, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:99, or SEQ ID NO:101. Includes 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.

[0134] The isolated nucleic acid can comprise any type of nucleic acid, including, but not limited to, DNA, cDNA, and RNA. In one embodiment, the composition comprises an isolated RNA molecule encoding NKCE or a functional fragment thereof.

[0135] The nucleic acid molecules of the present invention can be modified to improve stability. Modifications can be added to increase the stability, functionality, and / or specificity of the nucleic acid molecules of the present invention, and to minimize the immunostimulatory properties. For example, to increase stability, the 3' residues can be stabilized against degradation, for example, they may be selected to 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 tolerated and does not affect the function of the molecule.

[0136] In one embodiment of the invention, the nucleic acid molecule may comprise at least one modified nucleotide analogue. For example, the termini may be stabilized by incorporating modified nucleotide analogues.

[0137] Non-limiting examples of nucleotide analogs include sugar-modified and / or backbone-modified backbone ribonucleotides (i.e., containing modifications to the phosphate-sugar backbone). For example, the phosphodiester bond of natural RNA can be modified to include at least one nitrogen or sulfur heteroatom. In exemplary backbone-modified ribonucleotides, the phosphate ester group attached to the adjacent ribonucleotide is replaced by a modified group, for example, a phosphothioate group.

[0138] Other examples of modifications are nucleobase-modified ribonucleotides, i.e., ribonucleotides that contain at least one non-natural nucleobase instead of a natural nucleobase. The base may be modified to inhibit the activity of adenosine deaminase. Exemplary modified nucleobases include, but are not limited to, uridine and / or cytidine modified at the 5-position, such as 5-(2-amino)propyluridine, 5-bromouridine; adenosine and / or guanosine modified at the 8-position, such as 8-bromoguanosine; deazanucleotides, such as 7-deazaadenosine; O-alkylated and N-alkylated nucleotides, such as N6-methyladenosine. The above modifications can also be combined.

[0139] In some examples, the nucleic acid molecule comprises at least one of the following chemical modifications: 2'-H, 2'-O-methyl, or 2'-OH modification of one or more nucleotides. In some embodiments, the nucleic acid molecule of the present invention may have enhanced resistance to nucleases. To increase nuclease resistance, the nucleic acid molecule can comprise, for example, 2'-modified ribose units and / or phosphorothioate linkages. For example, the 2' hydroxyl group (OH) can be modified or replaced with several different "oxy" or "deoxy" substituents. To increase nuclease resistance, the nucleic acid molecule of the present invention can comprise 2'-O-methyl, 2'-fluorine, 2'-O-methoxyethyl, 2'-O-aminopropyl, 2'-amino, and / or phosphorothioate linkages. Inclusion of locked nucleic acids (LNA), ethylene nucleic acids (ENA), 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, can also increase binding affinity to targets.

[0140] In one embodiment, the nucleic acid molecule comprises a 2'-modified nucleotide, such as 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA). In one embodiment, the nucleic acid molecule comprises at least one 2'-O-methyl modified nucleotide, and in some embodiments, all of the nucleotides of the nucleic acid molecule comprise a 2'-O-methyl modification.

[0141] Nucleic acid agents discussed herein include RNA and DNA that are not modified in nature, as well as RNA and DNA that are modified, for example to improve efficacy, and polymers of nucleoside substitutes. Unmodified RNA refers to molecules in which the components of the nucleic acid, i.e., sugar, base, and phosphate moieties, are the same or essentially the same as those found in nature, e.g., in the human body. Rare or unusual but naturally occurring RNAs are referred to in the art as modified RNAs, see, for example, Limbach et al. (Nucleic Acids Res., 1994, 22:2183-2196). Such rare or unusual RNAs are often referred to as modified RNAs, and are usually the result of post-transcriptional modifications, and are within the scope of the term "unmodified RNA" as used herein. Modified RNAs, as used herein, refer to molecules in which one or more of the components of the nucleic acid, i.e., sugar, base, and phosphate moieties, are different from those found in nature, e.g., in the human body. Although these are referred to as "modified RNAs," they are of course also intended to include molecules that are not strictly speaking RNAs because they have been modified. Nucleoside surrogates are molecules in which the ribophosphate backbone is replaced with a non-ribophosphate construct such that the bases are presented in the correct spatial relationship and hybridization is substantially similar to that seen with the ribophosphate backbone (e.g., an uncharged mimic of the ribophosphate backbone).

[0142] Modifications of the nucleic acids of the invention can be at one or more of the phosphate groups, sugar groups, backbone, N-terminus, C-terminus, or nucleobases.

[0143] The invention also includes vectors into which the isolated nucleic acids of the invention are inserted. The art is replete with suitable vectors useful in the present invention.

[0144] Thus, in another aspect, the present invention relates to a vector comprising the nucleotide sequence of the present invention or the construct of the present invention. The choice of vector depends on the host cell into which it is subsequently introduced. In some embodiments, the vector of the present invention is an expression vector. Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. In certain embodiments, the expression vector is selected from the group consisting of viral vectors, bacterial vectors, and mammalian cell vectors. Prokaryotic and / or eukaryotic vector-based systems can be used in the present invention to produce polynucleotides or their cognate polypeptides. Many such systems are commercially available and widely available.

[0145] In some embodiments, expression of synthetic nucleic acids encoding proteins is usually achieved by operably linking the nucleic acid encoding the protein or a portion thereof to a promoter and incorporating the construct into an expression vector. The vector used is suitable for replication and, optionally, integration into eukaryotic cells. Typical vectors include transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence.

[0146] The recombinant nucleic acid sequence construct may include one or more transcription termination regions. The transcription termination region may be downstream of the coding sequence to provide efficient termination. The transcription termination region may be obtained from the same gene as the promoter or from one or more different genes.

[0147] The recombinant nucleic acid construct may include one or more initiation codons. The initiation codon may be upstream of the coding sequence. The initiation codon may be in frame with the coding sequence. The initiation codon may be associated with one or more signals required for efficient translation initiation, such as, but not limited to, a ribosome binding site.

[0148] A recombinant nucleic acid sequence construct can include one or more termination or stop codons. The termination codon can be downstream of the coding sequence. The termination codon can be in frame with the coding sequence. The termination codon can be associated with one or more signals required for efficient translation termination.

[0149] The recombinant nucleic acid sequence construct may include one or more polyadenylation signals. The polyadenylation signal may include one or more signals required for efficient polyadenylation of the transcript. The polyadenylation signal may be located downstream of the coding sequence. The polyadenylation signal may be an SV40 polyadenylation signal, an LTR polyadenylation signal, a bovine growth hormone (bGH) polyadenylation signal, a human growth hormone (hGH) polyadenylation signal, or a human β-globin polyadenylation signal. The SV40 polyadenylation signal may be a polyadenylation signal from a pCEP4 plasmid (Invitrogen, San Diego, CA).

[0150] The recombinant nucleic acid construct can include one or more leader sequences. The leader sequence can encode a signal peptide. The signal peptide can be an immunoglobulin (Ig) signal peptide, such as, but not limited to, an IgG signal peptide and an IgE signal peptide.

[0151] The vector of the present invention can also be used for nucleic acid immunization using standard gene delivery protocols.The method of gene delivery is known in the art.See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, the entirety of which is incorporated herein by reference.

[0152] The isolated nucleic acids of the present invention can be cloned into many types of vectors. For example, the nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0153] Furthermore, the vector may be provided to the cell in the form of a viral vector. Viral vector technology is known in the art and described, for example, in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector comprises an origin of replication functional in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers (e.g., WO01 / 96584, WO01 / 29058, and U.S. Pat. No. 6,326,193).

[0154] Furthermore, the expression vector may be provided to the cell in the form of a viral vector.Viral vector technology is known in the art and described, for example, in Sambrook et al. (2012), and in Ausubel et al. (1997), and in other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, suitable vectors include an origin of replication that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers (see, for example, WO01 / 96584, WO01 / 29058, and U.S. Patent No. 6,326,193).

[0155] By way of example, the vector into which the nucleic acid sequence is introduced may be a plasmid, which when introduced into a cell may or may not be integrated into the genome of the host cell. Illustrative, non-limiting examples of vectors into which the nucleotide sequence of the invention or the genetic construct of the invention may be inserted include tet-on inducible vectors for expression in eukaryotic cells.

[0156] The vector can be obtained by conventional methods known to those skilled in the art (Sambrooke et al., 2012). In a particular embodiment, the vector is a vector useful for transforming animal cells.

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

[0158] Numerous virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. Recombinant viruses can then be isolated and delivered to target cells in vivo or in vitro. Numerous retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Numerous adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

[0159] For example, vectors derived from retroviruses, such as lentiviruses, are suitable tools to achieve long-term gene transfer, as they allow long-term stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from oncoretroviruses, such as murine leukemia viruses, that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of being less immunogenic. In one embodiment, the composition includes a vector derived from an adeno-associated virus (AAV). Adeno-associated virus (AAV) vectors have become a powerful gene delivery tool for the treatment of various diseases. AAV vectors have many characteristics that make them ideal for gene therapy, including non-pathogenicity, minimal immunogenicity, and the ability to stably and efficiently transduce post-mitotic cells. Expression of a particular gene contained in an AAV vector can be specifically targeted to one or more cell types by selecting the appropriate combination of AAV serotype, promoter, and delivery method.

[0160] In some embodiments, the vector also includes conventional control elements operably linked to the transgene in a manner that allows transcription, translation, and / or expression in cells transfected with the plasmid vector or infected with the virus produced by the present invention. As used herein, "operably linked" sequences include both expression control sequences adjacent to the gene of interest and expression control sequences acting in trans or at a distance to control the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing signals and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that increase translation efficiency (i.e., Kozak consensus sequences); sequences that increase protein stability; and, if desired, sequences that increase secretion of the encoded product. Numerous expression control sequences, including native, constitutive, inducible, and / or tissue-specific promoters, are known in the art and can be utilized.

[0161] A promoter is one that is naturally associated with a gene or polynucleotide sequence and can be obtained by isolating 5' non-coding sequences located upstream of the coding segment and / or exon. Such promoters are sometimes referred to as "endogenous". Similarly, an enhancer is one that is naturally associated with a polynucleotide sequence and is located downstream or upstream of that sequence. Alternatively, certain advantages are obtained by placing a coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a polynucleotide sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer that is not normally associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers can include promoters or enhancers of other genes, promoters or enhancers isolated from other prokaryotes, viruses, or eukaryotic cells, and promoters or enhancers that are not "naturally occurring", i.e., different elements of different transcriptional regulatory regions and / or mutations that alter expression. In addition to producing promoter and enhancer nucleic acid sequences synthetically, the sequences can also be produced in conjunction with the compositions disclosed herein using recombinant cloning and / or nucleic acid amplification techniques, including PCR (U.S. Patent Nos. 4,683,202 and 5,928,906).Furthermore, it is contemplated that control sequences that direct transcription and / or expression of sequences in non-nuclear organelles, such as mitochondria, chloroplasts, etc., can also be used.

[0162] Naturally, it will be important to use promoters and / or enhancers that effectively direct the expression of the DNA segment in the cell type, organ, and organism selected for expression. Those skilled in the art of molecular biology generally know how to use combinations of promoters, enhancers, and cell types for protein expression. See, for example, Sambrook et al. (2012). The promoters used can be constitutive, tissue-specific, inducible, and / or useful for inducing high-level expression of the introduced DNA segment under appropriate conditions, which is advantageous for large-scale production of recombinant proteins and / or peptides. The promoters can be heterologous or endogenous.

[0163] The recombinant expression vector may also contain a selection marker gene that facilitates the selection of transformed or transfected host cells. Suitable selection marker genes are proteins such as G418 and hygromycin that confer resistance to certain drugs, β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or genes encoding immunoglobulins or portions thereof, such as the Fc portion of an immunoglobulin, such as IgG. The selection marker may be introduced into a vector separate from the nucleic acid of interest.

[0164] Additional promoter elements, e.g. enhancers, regulate the frequency of transcription initiation. Usually, these are located in the region 30-110 bp upstream of the start site, but recently, some promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements is often flexible, so that promoter function is preserved even if elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, activity begins to decrease when the spacing between promoter elements is increased to 50 bp. In some promoters, individual elements appear to be able to function cooperatively or independently to activate transcription.

[0165] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive high levels of expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is the elongation growth factor-1 alpha (EF-1 alpha). However, other constitutive promoter sequences can also be used, including but not limited to the Simian Virus 40 (SV40) early promoter, the mouse mammary tumor virus (MMTV) promoter, the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr virus immediate early promoter, the Rous sarcoma virus promoter, and human gene promoters, including but not limited to the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also considered as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on expression of an operably linked polynucleotide sequence when such expression is desired, and turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, tetracycline promoters, and the like.

[0166] Enhancer sequences present on vectors also control the expression of genes contained therein. Enhancers usually bind protein factors to promote the transcription of genes. Enhancers can be located upstream or downstream of the gene they control. Enhancers can also be tissue-specific to promote transcription in certain cell or tissue types. In one embodiment, the vectors of the present invention contain one or more enhancers that promote the transcription of genes present in the vector.

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

[0168] Reporter genes are used to identify potentially transfected cells and evaluate the function of regulatory sequences. Generally, reporter genes are genes that encode a polypeptide that is not present or expressed by the recipient organism or tissue and whose expression is manifested by an easily detectable property, such as an enzymatic activity. Expression of the reporter gene is measured at an appropriate time after the DNA is introduced into the recipient cell. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and can be prepared using known techniques or are commercially available. Generally, the construct with the minimal 5' flanking region that results in the highest expression level of the reporter gene is identified as the promoter. Such promoter regions are linked to the reporter gene and can be used to evaluate drugs for their ability to modulate promoter-driven transcription.

[0169] Methods for introducing and expressing genes into cells are known in the art. In the context of an expression vector, the vector can be easily introduced into a host cell, such as a mammalian, bacterial, yeast, or insect cell, by any method in the art. For example, the expression vector can be transferred into the host cell by physical, chemical, or biological means.

[0170] Physical methods for introducing peptides or proteins into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, e.g., Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0171] 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, are the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be obtained from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0172] 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).

[0173] When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for introducing nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another embodiment, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained as a suspension in lipids, contained in micelles or lipid nanoparticles, or otherwise associated with lipids. The lipid, lipid / DNA, or lipid / expression vector associated compositions are not limited to a particular structure in solution. For example, they may exist in a bilayer structure, as micelles, or in a "collapsed" structure. They may also simply be interspersed in the solution, or may form aggregates that are not uniform in size or shape. Lipids are fatty substances and can be natural or synthetic. For example, lipids include the lipid droplets that occur naturally in the cytoplasm, as well as a class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0174] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("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; dimyristyl phosphatidylglycerol ("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 that includes a variety of unilamellar and multilamellar lipid vesicles formed by the formation of sealed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before forming a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have structures in solution that differ from the usual vesicular structure are also included. For example, lipids may adopt micellar structures or exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0175] ScFv antibody In one embodiment, the antibody fragment comprises an scFv fragment. In one embodiment, the ScFv antibody fragment relates to a Fab fragment lacking the CH1 and CL regions. Thus, in one embodiment, the scFv antibody fragment relates to a Fab fragment comprising a VH and a VL. In one embodiment, the scFv antibody fragment comprises a linker between the VH and VL. In one embodiment, the scFv antibody fragment comprises a VH, a VL, and a CH2 and a CH3 region. In one embodiment, the scFv antibody fragment of the invention has modified expression, stability, half-life, antigen binding, heavy-light chain pairing, tissue penetration, or a combination thereof, compared to the parent MAb.

[0176] 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.

[0177] In one embodiment, an scFv antibody fragment of the invention has an antigen binding affinity that is at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, at least 9.5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, or more than 50 fold greater than the parent MAb.

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

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

[0180] In one embodiment, an scFv antibody fragment of the invention has at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, at least 9.5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, or more than 50 fold, tissue penetration than the parent MAb.

[0181] In one embodiment, an scFv antibody fragment of the invention has at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, at least 9.5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, or more than 50 fold, greater heavy chain-light chain pairing than the parent MAb.

[0182] Delivery Vehicle In one embodiment, the present invention provides a composition comprising a delivery vehicle comprising NKCE, a fragment thereof, or a nucleic acid molecule encoding same, as described herein. In one embodiment, the nucleic acid molecule encoding NKCE comprises an mRNA molecule.

[0183] Exemplary delivery vehicles include, but are not limited to, microspheres, microparticles, nanoparticles, polymersomes, liposomes, and micelles.For example, in some embodiments, the delivery vehicle is a lipid nanoparticle loaded with a nucleic acid molecule encoding the NKCE of the present invention or a fragment thereof.In one embodiment, the nucleic acid molecule encoding the NKCE comprises an mRNA molecule.

[0184] In some embodiments, the delivery vehicle provides for controlled, delayed, or continuous release of its cargo, hi some embodiments, the delivery vehicle includes a targeting moiety that targets the delivery vehicle to a treatment site.

[0185] In some cases, delivering coding mRNA to express proteins has many advantages over methods using proteins, plasmid DNA, or viral vectors. During mRNA transfection, the coding sequence of the desired protein is the only material delivered to the cell, thus avoiding all side effects associated with the plasmid backbone, viral genes, and viral proteins. More importantly, unlike DNA- and viral-based vectors, mRNA does not risk being integrated into the genome, and protein production begins immediately after mRNA delivery. For example, high levels of circulating protein have been measured within 15-30 minutes after in vivo injection of coding mRNA. In certain embodiments, using mRNA rather than protein also has many advantages. Proteins often have short half-lives in the circulation, necessitating frequent administration for protein therapy, whereas mRNA provides a template for continuous protein production over several days. Protein purification is problematic and may contain aggregates and other impurities that cause adverse effects (Kromminga and Schellekens, 2005, Ann NY Acad Sci 1050:257-265).

[0186] A variety of assays can be performed to confirm the presence of an mRNA sequence in a host cell, including "molecular biological" assays known to those skilled in the art, such as Northern blotting and RT-PCR, and "biochemical" assays, such as detecting the presence or absence of specific peptides by immunogenic means (ELISA and Western blot), or by the assays described herein to identify agents within the scope of the invention.

[0187] CAR molecule In one embodiment, the present invention provides a chimeric antigen receptor (CAR) comprising a binding domain comprising the NKCE of the present invention. In one embodiment, the CAR comprises an antigen binding domain. In one embodiment, the antigen binding domain is a targeting domain, where the targeting domain directs cells expressing the CAR to cells or particles expressing a sialic acid binding receptor.

[0188] In various embodiments, a CAR can be a "first generation," "second generation," "third generation," "fourth generation," or "fifth generation" CAR (see, e.g., Sadelain et al., Cancer Discov. 3(4):388-398 (2013); Jensen et al., Immunol. Rev. 257:127-133 (2014); Sharpe et al., Dis. Model Mech. 8(4):337-350 (2015); Brentjens et al., Clin. Cancer Res. 13:5426-5435 (2007); Gade et al., Cancer Res. 65:9080-9088 (2005); Maher et al., Nat. Biotechnol. 20:70-75 (2002); Kershaw et al., J. Immunol. 173:2143-2150 (2002)). (2004); see Sadelain et al., Curr. Opin. Immunol. (2009); Hollyman et al., J. Immunother. 32:169-180 (2009)).

[0189] The "first generation" CARs used in the present invention comprise an antigen-binding domain, e.g., a single chain variable fragment (scFv) fused to a transmembrane domain fused to the cytoplasmic / intracellular domain of a T cell receptor chain. "First generation" CARs usually have the intracellular domain of the CD3 zeta chain, which is the main transmitter of signals from the endogenous T cell receptor (TCR). "First generation" CARs provide novel antigen recognition and can activate both CD4+ and CD8+ T cells via the CD3 zeta chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation.

[0190] The "second generation" CARs used in the present invention contain an antigen-binding domain, e.g., a single chain variable fragment (scFv) fused to an intracellular signaling domain capable of activating T cells and a costimulatory domain designed to increase T cell potency and persistence (Cancer Discov. 3:388-398 (2013)). Thus, the CAR design can combine antigen recognition and signaling, two functions performed by two physiologically separate complexes, the TCR heterodimer and the CD3 complex. "Second generation" CARs contain intracellular domains of various costimulatory molecules, such as CD28, 4-1BB, ICOS, and OX40, in the cytoplasmic tail of the CAR to provide additional signals to the cell.

[0191] "Second generation" CARs provide both costimulation, e.g., by CD28 or 4-1BB domains, and activation, e.g., by CD3ζ signaling domains. Preclinical studies have shown that "second generation" CARs can improve the antitumor activity of T cells. For example, the potent efficacy of "second generation" CAR-modified T cells was demonstrated in clinical trials targeting the CD19 molecule in patients with chronic lymphoblastic leukemia (CLL) and acute lymphoblastic leukemia (ALL) (Davila et al., Oncoimmunol. 1(9):1577-1583 (2012)).

[0192] "Third generation" CARs provide multiple costimulation by including both CD28 and 4-1BB domains, and activation by including a CD3zeta activation domain.

[0193] "Fourth generation" CARs provide costimulation, e.g., by CD28 or 4-1BB domains, and activation, e.g., by a CD3ζ signaling domain, in addition to constitutive or inducible chemokine components.

[0194] "5th generation" CARs provide costimulation, e.g., by CD28 or 4-1BB domains, and activation, e.g., by CD3ζ signaling domains, constitutive or inducible chemokine components, and intracellular domains of cytokine receptors (e.g., IL-2Rβ).

[0195] In various embodiments, the CAR can be included in a multivalent CAR system (e.g., a dual CAR or "tandem CAR" system). Multivalent CAR systems include systems or cells that include multiple CARs, and systems or cells that include bivalent / bispecific CARs that target multiple antigens.

[0196] In the embodiments disclosed herein, the CAR generally comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain, as described above. In certain non-limiting embodiments, the antigen-binding domain is a bispecific sialic acid-binding receptor antibody or variant thereof specific for binding to a sialic acid-binding receptor.

[0197] substrate In one embodiment, the present invention provides a scaffold, substrate, or device comprising NKCE, a fragment thereof, or a nucleic acid molecule encoding the same. For example, in some embodiments, the present invention provides a tissue engineering scaffold (including, but not limited to, hydrogel, electrospun scaffold, polymer matrix, etc.) comprising a modulator. In certain embodiments, NKCE, a fragment thereof, or a nucleic acid molecule encoding the same may be coated along the surface of the scaffold, substrate, or device. In certain embodiments, NKCE, a fragment thereof, or a nucleic acid molecule encoding the same is encapsulated within the scaffold, substrate, or device.

[0198] Pharmaceutical Compositions The present invention also provides pharmaceutical compositions comprising one or more of the compositions described herein. The formulations can be used in admixture with conventional excipients, i.e., pharma-ceutically acceptable organic or inorganic carrier substances suitable for administration to the treatment site. The pharmaceutical compositions are sterilized and can be mixed with auxiliary substances, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic buffers, coloring agents, and / or aromatic substances, if necessary. They can also be combined with other active agents, such as other analgesics, if necessary.

[0199] Administration of the compositions of the invention can be by, for example, parenteral, intravenous, subcutaneous, intramuscular, or intraperitoneal injection, or infusion, or other acceptable systemic methods.

[0200] As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients, surfactants. Dispersing agents, inert diluents, granulating and disintegrating agents, binders, lubricants, coloring agents, preservatives, physiologically degradable compositions such as gelatin, aqueous vehicles and solvents, oily vehicles and solvents, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, buffers, salts, thickening agents, fillers, emulsifiers, antioxidants, antibiotics, antifungal agents, stabilizers, and pharma-ceutically acceptable 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.

[0201] The composition of the present invention may contain a preservative in an amount of about 0.005% to 2.0% by weight of the total composition. Preservatives are used to prevent spoilage when exposed to pollutants in the environment. Examples of preservatives useful according to the present invention include, but are not limited to, those selected from the group of benzyl alcohol, sorbic acid, parabens, imidurea, and combinations thereof.

[0202] In one embodiment, the composition comprises an antioxidant and a chelating agent that inhibits the decomposition of one or more components of the composition. Examples of antioxidants for some compounds include BHT, BHA, alpha tocopherol, and ascorbic acid. Examples of chelating agents include edetate (e.g., disodium edetate) and citric acid. Chelating agents are useful for chelating metal ions in the composition that may adversely affect the shelf life of the formulation. Although BHT and disodium edetate can be antioxidants and chelating agents, respectively, for some compounds, other suitable and equivalent antioxidants and chelating agents may be used instead, as known to those skilled in the art.

[0203] Liquid suspensions can be prepared using conventional methods to achieve the suspension of the compounds of the present invention or other compositions in aqueous or oily vehicles. Aqueous vehicles include, for example, water and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils (e.g., peanut oil, olive oil, sesame oil, or coconut oil, fractionated vegetable oils), and mineral oils (e.g., liquid paraffin). Liquid suspensions can further include one or more additional ingredients, including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavoring agents, coloring agents, and sweeteners. Oily suspensions can further include a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats and oils, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose. Known dispersing or wetting agents include, but are not limited to, natural phospholipids such as lecithin, condensation products of alkylene oxides with fatty acids, with long chain aliphatic alcohols, with partial esters derived from fatty acids and hexitols, or with partial esters derived from fatty acids and hexitol anhydrides (polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin and acacia. Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl parahydroxybenzoates, ascorbic acid, and sorbic acid.

[0204] For oral administration, tablets, dragees, liquids, drops, suppositories, or capsules, caplets, and gel capsules are particularly suitable. Other formulations suitable for oral administration include, but are not limited to, powder or granular formulations, aqueous or oily suspensions, aqueous or oily solutions, pastes, gels, toothpastes, mouthwashes, coatings, mouthwashes, chewing gums, varnishes, sealants, oral and dental "dissolving strips", or emulsions. Compositions intended for oral use can 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 the manufacture of tablets. Such excipients include, for example, inert diluents such as lactose, granulating and disintegrating agents such as corn starch, binding agents such as starch, and lubricants such as magnesium stearate.

[0205] The tablets may be uncoated or may be coated using known methods to delay disintegration in the gastrointestinal tract of a subject, thereby achieving sustained release and absorption of the active ingredient. For example, the tablets may be coated using materials such as glyceryl monostearate or glyceryl distearate. For further example, the tablets may be coated using methods described in U.S. Patent Nos. 4,256,108, 4,160,452, and 4,265,874 to form osmotically controlled release tablets. The tablets may further comprise a sweetener, a flavoring agent, a coloring agent, a preservative, or a combination thereof to provide a medicamentously elegant and palatable formulation.

[0206] Hard capsules containing the active ingredient can be manufactured using a physiologically degradable composition such as gelatin. Such hard capsules contain the active ingredient and can further contain additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin.

[0207] Soft gelatin capsules containing the active ingredient can be prepared using a physiologically degradable composition such as gelatin. Such soft capsules contain the active ingredient, which can be mixed with water or an oil medium (e.g., peanut oil, liquid paraffin, or olive oil).

[0208] For oral administration, the compositions of the present invention may be in the form of tablets or capsules prepared by conventional means with pharma- ceutically acceptable excipients, such as binders, fillers, lubricants, disintegrants, or wetting agents. If desired, tablets can be coated using suitable methods and coating materials, such as OPADRY™ film coating systems available from Colorcon, West Point, Pa. (e.g., OPADRY™ OY type, OYC type, organic enteric OY-P type, aqueous enteric OY-A type, OY-PM type, and OPADRY™ White, 32K18400).

[0209] Liquid preparations for oral administration can be in the form of solution, syrup, or suspension. Liquid preparations can be prepared in a conventional manner using pharma- ceutically acceptable additives, such as suspending agents (e.g., sorbitol syrup, methylcellulose, or hydrogenated edible fats and oils), emulsifying agents (e.g., lecithin, acacia), non-aqueous vehicles (e.g., almond oil, oily esters, or ethyl alcohol), and preservatives (e.g., methyl or propyl p-hydroxybenzoate or sorbic acid). Liquid 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 a dry product that is intended to be reconstituted with water or other suitable vehicle before use.

[0210] Tablets containing the active ingredient may be prepared, for example, by compressing or molding the active ingredient, optionally with one or more additional ingredients. Compressed tablets may be prepared by mixing the active ingredient in a free-flowing form, such as a powder or granular formulation, with one or more of an optional binder, lubricant, excipient, surfactant, and dispersant, and compressing in a suitable device. Molded tablets may be prepared by molding in a suitable device a mixture of the active ingredient, a pharma- ceutically acceptable carrier, and at least sufficient liquid to moisten the mixture. Pharmaceutically acceptable excipients used in the manufacture of tablets include, but are not limited to, inert diluents, granulating and disintegrating agents, binders, and lubricants. Known dispersing agents include, but are not limited to, potato starch and sodium starch glycolate. Known surfactants include, but are not limited to, sodium lauryl sulfate. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate. Known granulating and disintegrating agents include, but are not limited to, corn starch and alginic acid. Known binding agents include, but are not limited to, gelatin, acacia, pregelatinized maize starch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Known lubricants include, but are not limited to, magnesium stearate, stearic acid, silica, and talc.

[0211] A pharmaceutical composition formulation suitable for parenteral administration is a combination of the active ingredient and a pharma- ceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations can be prepared, packaged, or sold in a form suitable for bolus administration or continuous administration. Injectable formulations can be prepared, packaged, or sold in unit dosage form, such as ampoules or multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further include one or more additional components, including, but not limited to, suspending agents, stabilizing agents, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granule) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0212] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. The suspension or solution may be prepared according to known techniques and may contain, in addition to the active ingredient, additional ingredients such as dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable preparations may be prepared using a non-toxic parenterally acceptable diluent or solvent, for example, water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or diglycerides. Other parenterally administrable formulations that are useful include those that contain the active ingredient in microcrystalline form, in a liposomal formulation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may include pharma-ceutically acceptable polymers or hydrophobic materials, such as emulsions, ion exchange resins, sparingly soluble polymers, or sparingly soluble salts.

[0213] Excipients and other components of the composition The composition may further comprise a pharma- ceutically acceptable excipient. The pharma-ceutically acceptable excipient may be a functional molecule such as a vehicle, an adjuvant, a carrier, or a diluent. The pharma-ceutically acceptable excipient may be a transfection-facilitating agent, which may comprise a surfactant, which may comprise, for example, immune stimulating complexes (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-facilitating agents.

[0214] The transfection facilitating agent is a polyanion, a polycation (including poly-L-glutamic acid (LGS)), or a lipid. The transfection facilitating agent is poly-L-glutamic acid, which may be present in the composition at a concentration of less than 6 mg / ml. The transfection facilitating agent may also include surfactants, such as immune stimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogs including monophosphoryl lipid A, muramyl peptides, quinone analogs, squalene and vesicles such as squalene, and hyaluronic acid may also be administered in combination with the composition. The composition may also include a transfection facilitating agent, such as lipids, liposomes (including lecithin liposomes or other liposomes known in the art) as a DNA-liposome mixture (see, for example, WO9324640), calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents. The transfection facilitating agent is a polyanion, a polycation (including poly-L-glutamic acid (LGS)), or a lipid. The concentration of the transfection agent in the composition is less than 4 mg / ml, less than 2 mg / ml, less than 1 mg / ml, less than 0.750 mg / ml, less than 0.500 mg / ml, less than 0.250 mg / ml, less than 0.100 mg / ml, less than 0.050 mg / ml, or less than 0.010 mg / ml.

[0215] The pharma- ceutically acceptable excipient may be an adjuvant in addition to the checkpoint inhibitor antibody of the present invention. Additional adjuvants may be other genes expressed in alternative plasmids or delivered as proteins in combination with the above plasmids in the composition. The adjuvant may be selected from the group 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), cutaneous T-cell-attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosa-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86 (including IL-15 with the signal sequence deleted 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.

[0216] Other genes that may be useful as adjuvants in addition to the antibodies of the invention include those encoding MCP-1, MIP-1a, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, 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, SAPK, SAP-1, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK ligand, Ox40, Ox40 ligand, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2, and functional fragments thereof.

[0217] The composition may further comprise a gene enhancer as described in U.S. Patent Application Serial No. 021,579, filed April 1, 1994, which is incorporated by reference in its entirety.

[0218] The composition may contain DNA in an amount of about 1 nanogram to about 100 milligrams, about 1 microgram to about 10 milligrams, or preferably about 0.1 microgram to about 10 milligrams, or more preferably about 1 milligram to about 2 milligrams. In some preferred embodiments, the composition according to the present invention contains about 5 nanograms to about 1000 micrograms of DNA. In some preferred embodiments, the composition can contain about 10 nanograms to about 800 micrograms of DNA. In some preferred embodiments, the composition can contain about 0.1 micrograms to about 500 micrograms of DNA. In some preferred embodiments, the composition can contain about 1 microgram to about 350 micrograms of DNA. In some preferred embodiments, the composition comprises about 25 to about 250 micrograms, about 100 to about 200 micrograms, about 1 nanogram to 100 milligrams, about 1 microgram to about 10 milligrams, about 0.1 micrograms to about 10 milligrams, about 1 milligram to about 2 milligrams, about 5 nanograms to about 1000 micrograms, about 10 nanograms to about 800 micrograms, about 0.1 to about 500 micrograms, about 1 to about 350 micrograms, about 25 to about 250 micrograms, or about 100 to about 200 micrograms of DNA.

[0219] The composition can be prepared according to the method of administration used. The injectable pharmaceutical composition can be sterile, pyrogen-free, and particulate-free. An isotonic formulation or solution can be used. Additives for isotonicity include sodium chloride, dextrose, mannitol, sorbitol, and lactose. The composition can include a vasoconstrictor. Isotonic solutions include phosphate buffered saline. The composition can further include a stabilizer, including gelatin and albumin. The stabilizer can make the formulation containing LGS or a polycation or polyanion stable for an extended period of time at room or ambient temperature.

[0220] Delivery Methods Using Engineered Immune Cells In one embodiment, the present invention provides a method for delivering a bispecific sialic acid binding receptor antibody to a target cell, providing an engineered immune cell expressing the bispecific sialic acid binding receptor antibody. In one embodiment, the immune cell is engineered for endogenous secretion of the bispecific sialic acid binding receptor antibody of the present invention.

[0221] In various embodiments, the present invention relates to compositions comprising immune cells engineered for the expression or endogenous secretion of bispecific anti-sialic acid binding receptor antibodies targeting tumor cells. Examples of immune cells that can be engineered for the expression or secretion of the bispecific sialic acid binding receptor antibodies of the present invention include, but are not limited to, T cells, B cells, natural killer (NK) cells, or macrophages. In some embodiments, the immune cells further comprise a chimeric antigen receptor (CAR). Thus, in some embodiments, the present invention relates to the use of CAR T cells for the expression or delivery of the bispecific sialic acid binding receptor antibodies of the present invention.

[0222] Method of administration The present invention provides a method for increasing the function or activity of natural killer (NK) cells. This can be measured, for example, in standard NK cell or T cell-based cytotoxicity assays, in which the ability of a therapeutic compound to stimulate the killing of sialic acid ligand-positive cells by Siglec-positive lymphocytes is measured. In one embodiment, the antibody preparation causes at least a 10% enhancement in cytotoxicity of Siglec-restricted lymphocytes, optionally at least a 40% or 50% enhancement in lymphocyte cytotoxicity, or optionally at least a 70% enhancement in NK cytotoxicity and for the cytotoxicity assays described. In one embodiment, the antibody preparation causes at least a 10% enhancement in cytokine release by Siglec-restricted lymphocytes, optionally at least a 40% or 50% enhancement in cytokine release, or optionally at least a 70% enhancement in cytokine release and for the cytotoxicity assays described. In one embodiment, the antibody preparation causes at least a 10% enhancement in cell surface expression of cytotoxicity markers (e.g., CD107 and / or CD137) by Siglec-restricted lymphocytes, optionally at least a 40% or 50% enhancement, or optionally at least a 70% enhancement in cell surface expression of cytotoxicity markers (e.g., CD107 and / or CD137).

[0223] The present invention also relates to a method of increasing an immune response in a subject. The increased immune response can be used to treat and / or prevent disease in a subject. The method can include administering to the subject a vaccine as disclosed herein. A subject administered the vaccine can have an increased or enhanced immune response compared to a subject administered only the antigen. In some embodiments, the immune response can be increased by about 0.5 to about 15 times, about 0.5 to about 10 times, or about 0.5 to about 8 times. Alternatively, the immune response in a subject administered the vaccine can be increased by at least about 0.5-fold, at least about 1.0-fold, at least about 1.5-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 5.5-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 10.5-fold, at least about 11.0-fold, at least about 11.5-fold, at least about 12.0-fold, at least about 12.5-fold, at least about 13.0-fold, at least about 13.5-fold, at least about 14.0-fold, at least about 14.5-fold, or at least about 15.0-fold.

[0224] In yet 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 another embodiment, 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%.

[0225] The vaccine dosage can be 1 μg-10 mg active ingredient / kg body weight / dose, and 20 μg-10 mg ingredient / kg body weight / dose. The vaccine can be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. The number of vaccine doses for effective treatment can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0226] vaccine In one embodiment, the invention relates to the administration of a bispecific antibody comprising a combination of a sialic acid receptor antibody, or a fragment thereof, or a variant thereof, and an antibody specific for binding to a tumor antigen, or a nucleic acid molecule encoding a bispecific antibody comprising a combination of a sialic acid receptor antibody, or a fragment thereof, or a variant thereof, and an antibody specific for binding to a tumor antigen. The immunogenic composition can be used to increase killing of target cells expressing the tumor antigen.

[0227] The immunogenic composition may be a DNA vaccine, a peptide vaccine, or a combination of DNA and peptide vaccines. The DNA vaccine may comprise a nucleic acid sequence encoding a tumor antigen. The nucleic acid sequence may be DNA, RNA, cDNA, variants thereof, fragments thereof, or combinations thereof. The nucleic acid sequence may also comprise additional sequences encoding linker, leader, or tag sequences that are linked by peptide bonds to the sequence encoding the bispecific antibody of the present invention.

[0228] Vaccine-induced tumor cell killing includes an increase in the level of killing of cells expressing a target tumor antigen in a subject administered the vaccine compared to a subject not administered the vaccine. The level of tumor cell killing in a subject administered the vaccine can be increased by about 1.5-fold to about 16-fold, about 2-fold to about 12-fold, or about 3-fold to about 10-fold compared to a subject not administered the vaccine. The level of tumor cell killing in a subject administered the vaccine can be increased by at least about 1.5-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 5.5-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8-fold, or at least about 10-fold compared to a subject not administered the vaccine. The increase may be at least about 1.0 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.

[0229] The vaccines of the present invention can have the characteristics required for an effective vaccine, such as being safe so that the vaccine itself does not cause illness or death, being protective against illness caused by the presence of cells expressing the target antigen, being easy to administer, having few side effects, being biologically stable, and having a low cost per dose.

[0230] In some embodiments, the NKCE is directed to a pathogen-associated antigen or a viral antigen, which can be used to target NK cells to cells infected with a pathogen or virus. In some embodiments, the antigen includes, but is not limited to, an antigen of coronavirus (e.g., SARS-CoV-2), influenza virus, Zika virus, Ebola virus, Japanese encephalitis virus, mumps virus, measles virus, rabies virus, varicella zoster virus, Epstein-Barr virus (HHV-4), cytomegalovirus, herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2), human immunodeficiency virus type 1 (HIV-1), JC virus, arbovirus, enterovirus, West Nile virus, dengue virus, poliovirus, and varicella zoster virus. In some embodiments, the antigen comprises a bacterial antigen, including but not limited to, Streptococcus pneumoniae, Neisseria meningitides, Streptococcus agalactiae, and Escherichia coli antigens. In some embodiments, the antigen comprises a fungal or protozoan antigen, including but not limited to, candidiasis, aspergillosis, cryptococcosis, and Toxoplasma gondii antigens.

[0231] autoantigen The NKCE of the present invention can be specific for binding to an autoantigen. In some embodiments, the autoantigen is an antigen associated with an autoimmune disease or disorder. In some embodiments, the autoantigen is a tumor antigen.

[0232] Thus, in some embodiments, the present invention includes compositions for inducing natural killer cells to tumor cells. In some embodiments, the tumor cells express the antigen targeted by the NKCE of the present invention. As a non-limiting example, in one embodiment, the present invention provides a bispecific FSHR-Siglec7NKCE that induces natural killer cells to tumor cells expressing FSHR. Examples of tumor cells expressing FSHR include, but are not limited to, ovarian, breast, prostate, renal, colorectal, gastric, lung, testicular, endometrial, and thyroid cancer tumor cells.

[0233] In one embodiment, the antigen targeted by the NKCE of the present invention is a tumor-associated surface antigen. Specific examples of tumor-associated surface antigens are CD10, CD19, CD20, CD22, CD33, Fms-like tyrosine kinase 3 (FLT-3, CD135), chondroitin sulfate proteoglycan 4 (CSPG4, melanoma-associated chondroitin sulfate proteoglycan), epidermal growth factor receptor (EGFR), Her2neu, Her3, IGFR, CD133, IL3R, fibroblast activation protein (FAP), CDCP1, Derlin1, tenascin, frizzled 1-10, vascular antigen VEGFR2 (KDR / FLK1), VEGFR3 (FLT4, CD309), PDGFR-alpha (CD140a), PDGFR-beta, (CD140b) endoglin, CLEC14, Tem1-8, and Tie2. Further examples may include A33, CAMPATH-1 (CDw52), carcinoembryonic antigen (CEA), carboanhydrase IX (MN / CAIX), 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.

[0234] In the context of the present invention, "tumor antigen" or "hyperproliferative disease antigen" or "antigen associated with hyperproliferative disease" refers to an antigen common to a particular hyperproliferative disease, such as cancer. The antigens discussed herein are provided merely as examples. This list is not intended to be limiting, and further examples will be readily apparent to one skilled in the art.

[0235] Tumor antigens are proteins produced by tumor cells that can be targeted by the NKCE of the present invention. The choice of antigen-binding portion of the NKCE of the present invention depends on the particular type of cancer to be treated. Tumor antigens are 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, muthsp70-2, M-CSF, prostase, prostate specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.

[0236] In one embodiment, the tumor antigen comprises one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express a large number of proteins that can serve as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and GP100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-associated molecules, such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens are carcinoembryonic antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotypic immunoglobulins constitute truly tumor-specific immunoglobulin antigens that are unique to each individual tumor. B-cell differentiation antigens, such as CD19, CD20, and CD37, are other candidates for target antigens in B-cell lymphomas. Some of these antigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targets for passive immunotherapy with monoclonal antibodies, but with limited success.

[0237] The type of tumor antigen referred to in the present invention can be a tumor specific antigen (TSA) or a tumor associated antigen (TAA). TSA is specific to tumor cells and is not present in other cells in the body. TAA associated antigens are not specific to tumor cells and are also expressed in normal cells under conditions that cannot induce a state of immunological tolerance to the antigen. Expression of antigens in tumors can occur under conditions that allow the immune system to respond to the antigen. TAA can be an antigen that is expressed in normal cells during fetal development, when the immune system is too immature to respond, or an antigen that is usually present at very low levels in normal cells but is expressed at very high levels in tumor cells.

[0238] Non-limiting examples of TSA or TAA antigens include: differentiation antigens, such as MART-1 / MelanA (MART-I), gp100 (Pmel17), tyrosinase, TRP-1, TRP-2, and tumor-specific multilineage antigens, such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens, such as CEA; overexpressed oncogenes and mutated tumor suppressor genes, such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations, such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA19-9, CA72-4, CAM17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA125, CA15-3\CA27.29\BCAA, CA195, CA242, CA-50, CAM43, CD68\P1, and CO-029. These include FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0239] In one embodiment, the invention provides anti-Siglec7 anti-FSHRNKCE. In one embodiment, the invention provides an anti-Siglec7 anti-IL13Ra2NKCE.

[0240] Combination vaccines In one embodiment, the present invention relates to administering the NKCE of the present invention or a nucleic acid molecule encoding same in combination with a PD-(L)1 axis inhibitor. Immunogenic compositions can be used to increase the immune response against tumors.

[0241] In some embodiments, the NKCE of the present invention or a nucleic acid molecule encoding same is administered simultaneously with administration of a PD-(L)1 axis inhibitor.

[0242] In some embodiments, the NKCE of the present invention or a nucleic acid molecule encoding same is administered prior to administration of a PD-(L)1 axis inhibitor.

[0243] In some embodiments, the NKCE of the present invention or a nucleic acid molecule encoding same is administered following administration of a PD-(L)1 axis inhibitor.

[0244] In some embodiments, the NKCE of the present invention or a nucleic acid molecule encoding same is administered about 1, 2, 5, 10, 30, or 60 minutes or hours, such as about 2, 4, 6, 10, 12, 24, or 36 hours, or such as about 2, 4, 7, 14, 21, 28, 35, 42, 49, 56 days or more, prior to administration of the PD-(L)1 axis inhibitor. Thus, in some embodiments, the PD-(L)1 axis inhibitor is administered to a subject who has previously been administered the NKCE of the present invention or a nucleic acid molecule encoding same.

[0245] In some embodiments, the NKCE of the present invention or a nucleic acid molecule encoding same is administered about 1, 2, 5, 10, 30, or 60 minutes or hours, e.g., about 2, 4, 6, 10, 12, 24, or 36 hours, or, e.g., about 2, 4, 7, 14, 21, 28, 35, 42, 49, 56 days or more, after administration of a PD-(L)1 axis inhibitor. Thus, in some embodiments, the Siglec-7 antibody or a nucleic acid molecule encoding same is administered to a subject who has previously been administered a PD-(L)1 axis inhibitor.

[0246] In some embodiments, the combination of the NKCE or nucleic acid molecule encoding same of the present invention and a PD-(L)1 axis inhibitor is administered prior to the administration of one or more additional anti-cancer agents.

[0247] In some embodiments, the combination of the NKCE or nucleic acid molecule encoding same of the present invention and a PD-(L)1 axis inhibitor is administered following administration of one or more additional anti-cancer agents.

[0248] In some embodiments, the combination of the NKCE or nucleic acid molecule encoding same of the present invention and a PD-(L)1 axis inhibitor is administered simultaneously with the administration of one or more additional anti-cancer agents.

[0249] The length of time between administration of the NKCE or nucleic acid molecule encoding same of the present invention and the PD-(L)1 axis inhibitor or one or more additional anti-cancer agents may be a few minutes, e.g., about 1, 2, 5, 10, 30, or 60 minutes, or a few hours, e.g., about 2, 4, 6, 10, 12, 24, or 36 hours, or e.g., about 2, 4, 7, 14, 21, 28, 35, 42, 49, 56 days, or more.

[0250] One or more NKCEs, or nucleic acid molecules encoding same, PD-(L)1 axis inhibitors, or additional anti-cancer agents of the present invention can be administered in a pharma- ceutically acceptable carrier. A "carrier" refers to a diluent, adjuvant, excipient, or vehicle administered with the antibody of the present invention. Such vehicles can be liquids, such as water and oils, including oils of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. For example, a bispecific anti-EGFR / c-Met antibody can be prepared using 0.4% saline and 0.3% glycine. 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, tablets, and the like, suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrants, and the like. Solid oral preparations can also be coated with substances such as sugars to regulate the primary site of absorption, or enteric coated. For parenteral administration, the carrier comprises sterile water, and other excipients may be added to increase solubility or preservation. Injectable suspensions or solutions may also be prepared using aqueous carriers with appropriate additives. Suitable vehicles and formulations, including other human proteins such as human serum albumin, are described, for example, in 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, See especially pp. 958-989.

[0251] The composition may contain pharma- ceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusters, buffers, stabilizers, thickeners, lubricants, colorants, etc. The concentration of the NKCE of the present invention, or the nucleic acid molecule encoding it, and the PD-(L)1 axis inhibitor in the pharmaceutical formulation may vary from less than about 0.5% by weight, usually at least about 1% by weight up to 15%, 20%, 30%, 40%, or 50% by weight, and may be selected primarily based on the required dose, fluid volume, viscosity, etc., depending on the particular method of administration selected. Pharmaceutical compositions, including solid forms, may contain about 0.1 mg to about 2000 mg, e.g., 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.

[0252] Therapeutic compositions of the invention can have characteristics desired for an effective therapeutic agent, such as safety so that the therapeutic composition itself does not cause illness or death, ease of administration, few side effects, biological stability, and low cost per dose.

[0253] PD-(L)1 axis inhibitors In some embodiments, the present invention provides a combination of a bispecific NKCE of the present invention with a PD-(L)1 axis inhibitor. In various embodiments, the composition comprises an inhibitor of one or more genes or proteins in the PD-(L)1 axis. In various embodiments, the present invention includes compositions and methods for reducing the level or activity of one or more genes or proteins in the PD-(L)1 axis.

[0254] One of skill in the art will understand, based on the disclosure provided herein, that reducing the level or activity of one or more genes or proteins in the PD-(L)1 axis includes reducing expression of a biomarker, including transcription, translation, or both. One of skill in the art will also understand, upon comprehending the teachings of the present invention, that reducing the level or activity of one or more genes or proteins in the PD-(L)1 axis includes reducing the amount of polypeptide, reducing the amount of mRNA, reducing transcription, reducing translation, or a combination thereof, including reducing any activity of one or more genes or proteins in the PD-(L)1 axis.

[0255] Exemplary inhibitors of the PD-(L)1 axis include, but are not limited to, small interfering RNA (siRNA), microRNA, antisense nucleic acids, ribozymes, expression vectors encoding transdominant negative mutants, antibodies, antibody fragments, fusion proteins, aptamers, peptides, and small molecules.

[0256] One of ordinary skill in the art will understand, based on the disclosure provided herein, that one way to reduce the mRNA and / or protein levels of one or more PD-(L)1 axis proteins in a cell is to reduce or inhibit expression of a nucleic acid encoding the PD-(L)1 axis protein. Thus, the protein levels of a PD-(L)1 axis protein in a cell can be reduced using molecules or compounds that inhibit or reduce gene expression, such as, for example, siRNAs, antisense molecules, or ribozymes. However, the present invention is not limited to these examples.

[0257] In one embodiment, RNAi is used to reduce the level or activity of PD-(L)1 axis proteins. RNA interference (RNAi) is a phenomenon in which the introduction of double-stranded RNA (dsRNA) into a variety of organisms and cell types results in the degradation of complementary mRNA. In cells, long dsRNA is cleaved into short, 21-25 nucleotide small interfering RNAs (i.e., siRNAs) by a ribonuclease known as Dicer. The siRNAs are then assembled into RNA-induced silencing complexes (RISCs) with protein components, unwinding in the process. The activated RISC then binds to the complementary transcripts through base pairing interactions between the siRNA antisense strand and the mRNA. The bound mRNA is cleaved, and gene silencing occurs through sequence-specific degradation of the mRNA. Chemical modification of siRNAs aids in intravenous systemic delivery. To optimize siRNAs, the overall G / C content, terminal C / T content, Tm, and nucleotide content of the 3' overhangs must be considered. Thus, the present invention also includes methods of reducing the levels of one or more PD-(L)1 axis proteins using RNAi technology.

[0258] In some embodiments, the invention includes an isolated nucleic acid encoding an inhibitor, such as a protein, an antibody, an siRNA, or an antisense molecule, operably linked to a nucleic acid comprising a promoter / regulatory sequence, such that the nucleic acid is preferably capable of directing expression of the inhibitor encoded by the nucleic acid. Thus, the invention encompasses expression vectors and methods for introducing exogenous DNA into a cell and simultaneously expressing the exogenous DNA in the cell.

[0259] The expression vector introduced into the cells to evaluate the expression of the inhibitor can also contain either a selection marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from the cell population to be transfected or infected via the viral vector. In other embodiments, the selection marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selection marker and the reporter gene may be flanked by appropriate regulatory sequences to allow expression in the host cell. Useful selection markers are known in the art and include, for example, antibiotic resistance genes such as neomycin.

[0260] When the inhibitors of the present invention are small molecules, standard methods known to those skilled in the art can be used to obtain small molecule antagonists, including chemical organic synthesis or biological means, including purification from biological sources, recombinant synthesis, and in vitro translation systems, using methods well known in the art.

[0261] Combinatorial libraries of molecularly diverse compounds potentially useful for the treatment of various diseases and conditions are well known in the art, as are methods for generating the libraries, which can use a variety of techniques well known to those of skill in the art, including solid-phase synthesis, solution methods, parallel synthesis of single compounds, synthesis of chemical mixtures, rigid core structures, flexible linear arrays, deconvolution strategies, tagging techniques, and the generation of unbiased molecular landscapes for lead discovery and biased structures for lead development.

[0262] In a common method for small-scale library synthesis, an activated core molecule is condensed with a number of building blocks to yield a combinatorial library of covalently linked core-building block ensembles. The shape and rigidity of the core dictate the orientation of the building blocks in shape space. Libraries can be biased by modifying the core, bonds, or building blocks to target characterized biological structures ("focused libraries"), or can be synthesized with less structural bias using flexible cores.

[0263] In another embodiment of the invention, one or more proteins of the PD-(L)1 axis can be inhibited by inactivating and / or sequestering the proteins. Thus, inhibiting the effect of one or more proteins of the PD-(L)1 axis can be achieved by using transdominant negative mutants.

[0264] In one embodiment, antibodies specific for one or more proteins of the PD-(L)1 axis can be used. As will be appreciated by one of skill in the art, any antibody capable of recognizing and binding to an antigen of interest is useful in the present invention. Methods for making and using antibodies are well known in the art. For example, polyclonal antibodies useful in the present invention are generated by immunizing rabbits according to standard immunological techniques well known in the art. Such techniques include immunizing animals with chimeric proteins that contain a portion of another protein, such as a maltose binding protein or glutathione (GSH) tag polypeptide portion, and / or a portion that renders the antigen protein of interest immunogenic (e.g., an antigen of interest conjugated to keyhole limpet hemocyanin (KLH)), and a portion that contains the respective antigenic protein amino acid residues. Chimeric proteins are generated by cloning appropriate nucleic acids encoding the marker proteins into a plasmid vector suitable for this purpose, such as, but not limited to, pMAL-2 or pCMX.

[0265] Examples of anti-PD-(L)1 axis antibodies include, but are not limited to, nivolumab (OPDIVO®), pembrolimumab (KEYTRUDA®), sintilimab, cemiplimab (LIBTAYO®), tripolibamab, tislelizumab, spartalizumab, camrelizumab, dostralimab, genolimuzumab, or cetrelimab; or PD Antibodies that bind to PD-L1 (PD-L1 antibodies) are embafolimab, atezolizumab (TECENTRIQ®), durvalumab (IMFINZI®), avelumab (BAVENCIO®), REGN2810, pidilizumab, MEDI0680, PDR001, PF-06801591, BGB-A317, TSR-042, SHR-1210).

[0266] Methods for delivering the composition The present invention also relates to a method of delivering the composition to a subject in need thereof. The delivery method includes administering the composition to the subject. In some embodiments, the present invention relates to the administration of the NKCE of the present invention, or a fragment thereof, or a nucleic acid molecule encoding the same. In some embodiments, the nucleic acid molecule is a DNA molecule. In some embodiments, the nucleic acid molecule is an RNA molecule. In some embodiments, the nucleic acid molecule is an mRNA molecule.

[0267] Administration includes, but is not limited to, intravenous delivery of antibodies, DNA injection, liposome-mediated delivery, and nanoparticle-enhanced delivery.

[0268] The mammal to which the composition is delivered can be a human, a primate, a non-human primate, a cow, a beef cattle, a sheep, a goat, an antelope, a bison, a buffalo, a bison, a bovine, a deer, a hedgehog, an elephant, a llama, an alpaca, a mouse, a rat, and a chicken.

[0269] The compositions can be administered by a variety of routes, including orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, by inhalation, buccal administration, intrapleurally, intravenously, intraarterially, intraperitoneally, subcutaneously, intramuscularly, intranasally, intrathecally, intraarticularly, or combinations thereof. For veterinary use, the compositions can be administered in an appropriately acceptable formulation in accordance with normal veterinary practice. A veterinarian can readily determine the administration schedule and route most suitable for a particular animal.

[0270] Treatment method In one embodiment, the present invention provides a method for the treatment or prevention of a disease or disorder that would benefit from increased NK cell function or activity. Exemplary diseases and disorders that can be treated using the compositions and methods of the present invention include, but are not limited to, cancer and infectious diseases.

[0271] The following are non-limiting examples of cancers that can be diagnosed or treated by the disclosed methods and compositions: acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, appendix cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain and spinal cord tumors, brain stem glioma, brain tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, central nervous system atypical teratoid / rhabdomyosarcoma-like tumor, central nervous system embryonal tumor, central nervous system pulmonary tumor, central nervous system pulmonary tumor, central nervous system pulmonary fibrosis ... Systemic lymphoma, cerebellar astrocytoma, brain astrocytoma / malignant glioma, brain astrocytoma / malignant glioma, cervical cancer, childhood visual pathway tumors, chordoma, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorder, colon cancer, colorectal cancer, craniopharyngioma, skin cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, Ewing's family of tumors, extracranial cancer, extragonadal germ cell tumor, extrahepatic bile duct cancer, extrahepatic cancer, eye cancer, fungoides tumors, gallbladder cancer, stomach cancer, gastrointestinal cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), germ cell tumors, gestational cancer, gestational trophoblastic tumors, glioblastoma, glioma, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway gliomas, hypothalamic tumors, intraocular (eye) cancer, intraocular malignant melanoma, pancreatic islet cell tumors, Kaposi's sarcoma, kidney (renal cell) cancer, Langerhans cell carcinoma, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer, lymphoma, macroglobulinemia, malignant fibrous histiocytoma and osteosarcoma of bone, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell neck cancer of unknown primary, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis, myelodysplastic syndrome, myelodysplastic / myeloproliferative disorders, myelogenous leukemia, myeloid leukemia, myeloma, myeloproliferative disorders, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cancer, oral cavity cancercancer), oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma, osteosarcoma and malignant fibrous histiocytoma of bone, ovary, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal parenchymal tumor of intermediate differentiation, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasma cell neoplasm, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, primary central nervous system cancer, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter cancer, respiratory cancer related to the nut gene on chromosome 15 , retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, sarcoma, Sezary syndrome, skin cancer (melanoma), skin cancer (non-melanoma), skin cancer, small cell lung cancer, small intestine cancer, soft tissue cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell cervical cancer, gastric cancer, supratentorial primitive neuroectodermal tumor, supratentorial primitive neuroectodermal tumor and pineoblastoma, T-cell lymphoma, testicular cancer, pharyngeal cancer, 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, visual pathway and hypothalamic glioma, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

[0272] In one embodiment, the composition is used to treat cancers with high levels of sialic acid. Cancers associated with high levels of sialic acid include, but are not limited to, ovarian cancer, melanoma, renal cell carcinoma, prostate cancer, colon cancer, breast cancer, squamous cell carcinoma of the head and neck, and oral cancer.

[0273] cancer treatment In one embodiment, the invention provides a method for treating or preventing cancer, or treating and preventing tumor growth or metastasis. Related exemplified aspects of the invention provide a method for preventing, assisting in the prevention, and / or reducing metastasis of hyperplastic or tumor cells in an individual.

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

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

[0276] In some embodiments of the treatment or prevention of cancer or the treatment and prevention of tumor metastasis in an individual in need of treatment, a second agent such as an antitumor agent is administered to the individual.In some embodiments, the second agent comprises a second metastasis inhibitor such as a plasminogen antagonist or an adenosine deaminase antagonist.In other embodiments, the second agent is an angiogenesis inhibitor.

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

[0278] In one embodiment, the invention provides a method of treating cancer metastasis comprising treating a subject with a complementary therapy for cancer, such as surgery, chemotherapy, chemotherapeutic agents, radiation therapy, or hormonal therapy, or a combination thereof, prior to, concurrently with, or following treatment with a composition of the invention.

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

[0280] Antiproliferative agents are compounds that reduce cell proliferation. Antiproliferative agents include alkylating agents, antimetabolites, enzymes, biological response modifiers, miscellaneous drugs, hormones and antagonists, androgen inhibitors (e.g., flutamide and leuprolide acetate), antiestrogens (e.g., tamoxifen citrate and its analogs, toremifene, droloxifene, and roloxifene). Further examples of specific antiproliferative agents include, but are not limited to, levamisole, gallium nitrate, granisetron, sargramostim strontium-89 chloride, filgrastim, pilocarpine, dexrazoxane, and ondansetron.

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

[0282] Antiangiogenic agents are well known to those skilled in the art. Antiangiogenic agents suitable for use in the methods and compositions of the present invention include anti-VEGF antibodies, including humanized and chimeric antibodies, anti-VEGF aptamers, and antisense oligonucleotides. Other known inhibitors of angiogenesis include angiostatin, endostatin, interferon, interleukin 1 (including alpha and beta), interleukin 12, retinoic acid, and tissue inhibitors of metalloproteinases-1 and -2 (TIMP-1 and -2). Small molecules that contain topoisomerase, such as razoxane, a topoisomerase II inhibitor with antiangiogenic activity, can also be used.

[0283] Other anti-cancer drugs that can be used in combination with the compositions of the present invention include, but are not limited to, acivicin, aclarubicin, acodazole hydrochloride, acronine, adozelesin, aldesleukin, altretamine, ambomycin, amethanthrone acetate, aminoglutethimide, amsacrine, anastrozole, anthramycin, asparaginase, asperlin, azacytidine, azetepa, azotomycin, batimastat, benzodepa, bicalutamide, bisantrene hydrochloride, bisnafide dimesylate, bizeresin, and burenesulfate. Leomycin, brequinar sodium, bropirimine, busulfan, cactinomycin, calstron, caracemide, carbetimer, carboplatin, carmustine, carubicin hydrochloride, carzelesin, cedefingol, chlorambucil, ciloremycin, cisplatin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin hydrochloride, decitabine, dexormaplatin, desaguanine, desaguanine mesylate, diazicon, docetaxel, doxorubicin, doxorubicin hydrochloride , droloxifene, droloxifene citrate, dromostanolone propionate, duazomycin, edatrexate, eflornithine hydrochloride, elsamitrucin, enloplatin, enpromate, epipropidine, epirubicin hydrochloride, elbrozole, esorubicin hydrochloride, estramustine, estramustine sodium phosphate, etanidazole, etoposide, etoposide phosphate, etoprine, fadrozole hydrochloride, fazarabine, fenretinide, floxuridine, fludarabine phosphate, fluorouracil, fluorocitabine foskidone, fostriecin sodium, gemcitabine, gemcitabine hydrochloride, hydroxyurea, idarubicin hydrochloride, ifosfamide, irmofosine, interleukin II (including recombinant interleukin II or rIL2), interferon alpha 2a, interferon alpha 2b, interferon alpha n1, interferon alpha n3, interferon beta-Ia, interferon gamma-Ib, iproplatin, irinotecan hydrochloride, lanreotide acetate, letrozole, leuprolide acetate, liarozole hydrochloride,lometrexol sodium, lomustine, losoxantrone hydrochloride, masoprocol, maytansine, mechlorethamine hydrochloride, megestrol acetate, melengestrol acetate, melphalan, menogaril, mercaptopurine, methotrexate, methotrexate sodium, metoprine, meturedepa, mitindomide, mitocalcine, mitochromine, mitogillin, mitomarcine, mitomycin, mitospar, mitotane, mitoxantrone hydrochloride, mycophenolic acid, noco Dazol, nogalamycin, ormaplatin, oxisuran, paclitaxel, pegaspargase, periomycin, pentamustine, peplomycin sulfate, perfosfamide, pipobroman, piposulfan, piroxantrone hydrochloride, plicamycin, promestane, porfimer sodium, porfiromycin, prednimustine, procarbazine hydrochloride, puromycin, puromycin hydrochloride, pyrazofurin, ribopurin, rogletimide, safingol, hydrochloride Safingol, semustine, simtrazene, sparfosate sodium, sparsomycin, spirogermanium hydrochloride, spiromustine, spiroplatin, streptonigrin, streptozocin, sulofenur, tallysomycin, tecogalan sodium, tegafur, teroxantrone hydrochloride, temoporfin, teniposide, teroxylon, testolactone, thiamiprine, thioguanine, thiotepa, tiazofurin, tirapazamine, toremifene citrate, and toresodium acetate Thoron, triciribine phosphate, trimetrexate, trimetrexate glucuronate, triptorelin, tuburozole hydrochloride, uracil mustard, uredepa, vapreotide, verteporfin, vinblastine sulfate, vincristine sulfate, vindesine, vindesine sulfate, vinepidine sulfate, vinglisine sulfate, vinleurosine sulfate, vinorelbine tartrate, vinrocidine 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, adecipenol, adzelesin, aldesleukin, ALL-TK antagonists, altretamine, ambamustine, amidox,Amifostine, aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole, andrographolide, angiogenesis inhibitor, antagonist D, antagonist G, antarelix, anti-dorsal morphogenetic protein-1, antiandrogen, prostate cancer, antiestrogen, antineoplastic agent, antisense oligonucleotide, aphidicolin glycinate, apoptotic gene regulator, apoptotic control agent, apurinic acid, ara-CDP-DL-PTBA, arginine deaminase, asulaculin, atamestane, atrimustine, axinastatin, axinastatin-1 tin 2, axinastatin 3, azasetron, azatoxin, azatyrosine, baccatin III derivatives, balanol, batimastat, BCR / ABL antagonists, benzochlorine, benzoylstaurosporine, beta-lactam derivatives, beta-arretin, beta-clamycin B, betulinic acid, bFGF inhibitors, bicalutamide, bisantrene, bisaziridinylspermine, bisnafide, bisstraten A, bizelesin, brefullate, bropirimine, budotitane, buthionine sulfoximine, calcipotriol, calphostin C, camptothecin derivatives, camptothecin derivatives, Nariapox IL-2, capecitabine, carboxamide aminotriazole, carboxyamidotriazole, CaRestM3, CARN700, cartilage derived inhibitor, carzelesin, casein kinase inhibitor (ICOS), castanospermine, cecropin B, cetrorelix, chlorin, chloroquinoxaline sulfonamide, cicaprost, cis-porphyrin, cladribine, clomiphene analogue, clotrimazole, collismycin A, collismycin B, combretastatin A4, combretastatin analogue, conagenin, clambecidin 816, clamiphene analogue ... Lisnatol, cryptophycin 8, cryptophycin A derivative, curacin A, cyclopentaquinone, cycloplatam, sipemycin, cytarabine ocphosphate, cytolytic factor, cytostatin, dacliximab, decitabine, dehydrodidemnin B, deslorelin, dexamethasone, dexphosphamide, dexrazoxane, dexverapamil, diazicon, didemnin B, didox, diethylnorspermine, dihydro-5-azacytidine, dihydrotaxol (9-), dioxamycin, diphenylspiromustine, docetaxel,Docosanol, dolasetron, doxifluridine, droloxifene, dronabinol, duocarmycin SA, ebselen, ecomustine, edelfosine, edrecolomab, eflornithine, elemene, emiteflu, epirubicin, epristeride, estramustine analogues, estrogen agonists, estrogen antagonists, etanidazole, etoposide phosphate, exemestane, fadrozole, fazarabine, fenretinide, filgrastim, finasteride, flavopiridol, frezelastine, fluasterone, fludarabine, fluorouracil hydrochloride Daunorhysine, forfenimex, formestane, fostriecin, fotemustine, gadolinium texaphyrin, gallium nitrate, gallocitabine, ganirelix, gelatinase inhibitors, gemcitabine, glutathione inhibitors, hepsulfame, heregulin, hexamethylene bisacetamide, hypericin, ibandronate, idarubicin, idoxifene, idramantone, ilmofosine, ilomastat, imidazoacridone, imiquimod, immunostimulating peptides, insulin-like growth factor-1 receptor inhibitors, interferon agonists, interferon , interleukin, iobenguane, iododoxorubicin, ipomeanol (4-), ilopract, irsogladine, isobengazole, isohomohalichondrin B, itasetron, jasplakinolide, kahalalide F, lamellarin-N triacetate, lanreotide, leinamycin, lenograstim, lentinan sulfate, leptolstatin, letrozole, leukemia inhibitory factor, leukocyte alpha interferon, leuprolide + estrogen + progesterone, leuprorelin, levamisole, liarozole, linear polyamine analogs, lipophilic disaccharide peptides tides, lipophilic platinum compounds, lysoclinamide 7, lobaplatin, lombricine, lometrexol, lonidamine, losoxantrone, lovastatin, loxoribine, lurtotecan, lutetium texaphyrin, lysofylline, lytic peptides, maytansine, mannostatin A, marimastat, masoprocol, maspin, matrilysin inhibitors, matrix metalloproteinase inhibitors, menogaril, mervalone, meterelin, methioninase, metoclopramide, MIF inhibitors, mifepristone, miltefosine, millimostim, mismatched double-stranded RNA,Mitoguazone, Mitolactol, Mitomycin analogues, Mitonafide, Mitotoxin fibroblast growth factor saporin, Mitoxantrone, Mofalotene, Molgramostim, Monoclonal antibodies, Human chorionic gonadotropin, Monophosphoryl lipid A+ Myobacterium cell wall sk, Mopidamol, Multidrug resistance gene inhibitors, Multiple tumor suppressor 1-based therapy, Mustard anticancer drugs, Mycaperoxide B, Mycobacterium tuberculosis cell wall extract, Myriaporone, N-acetylglutaminone, N-substituted benzamides, Nafarelin, Nagressip, Naloxone+Pentazo Syn, napavine, naphterpin, nartograstim, nedaplatin, nemorubicin, neridronic acid, neutral endopeptidase, nilutamide, nisamycin, nitric oxide regulators, nitroxide antioxidants, nitrulline, O6-benzylguanine, octreotide, oxenone, oligonucleotides, onapristone, ondansetron, oracin, oral cytokine inducers, ormaplatin, osaterone, oxaliplatin, oxaunomycin, paclitaxel, paclitaxel analogues, paclitaxel derivatives, palladium, palmitoyl rhizoxin , pamidronate, panaxytriol, panomyphen, parabactin, pazelliptin, pegaspargase, perdecin, pentosan polysulfate sodium, pentostatin, pentrozole, perflubron, perphosphamide, perillyl alcohol, phenazinomycin, phenylacetic acid, phosphatase inhibitors, picibanil, pilocarpine hydrochloride, pirarubicin, piritrexim, prasetin A, prasetin B, plasminogen activator inhibitors, platinum complexes, platinum compounds, platinum triamine complexes, porfimer sodium, porfiromycin, pre Donisone, propyl bisacridone, prostaglandin J2, proteasome inhibitors, protein A-based immunomodulators, protein kinase C inhibitors, microalgae-derived protein kinase C inhibitors, protein tyrosine phosphatase inhibitors, purine nucleoside phosphorylase inhibitors, purpurins, pyrazoloacridines, pyridoxylated hemoglobin polyoxyethylene complexes, raf antagonists, raltitrexed, ramosetron, ras farnesyl protein transferase inhibitors, ras inhibitors, ras-GAP inhibitors, demethylated reteliptin,Rhenium Re186 etidronic acid, rhizoxin, ribozyme, RII retinamide, logretimide, rohitucine, romurtide, roquinimex, rubidinone B1, ruboxil, safingol, santopine, SarCNU, sarcophytol A, sargramostim, Sdi1 mimetic, semustine, senescence induction inhibitor 1, sense oligonucleo, Otides, signal transduction inhibitors, signal transduction modulators, single chain antigen binding proteins, schizofuran, sobuzoxane, sodium borocaptate, sodium phenylacetate, sorberol, somatomedin binding proteins, sonermin, sparfosic acid, spicamycin D, spiromustine, splenopentin, spongiostatin 1, squalamine, stem cell inhibitors, stem cell division inhibitors, stipiamid, stromelysin inhibitors, sulfinosine, superactive vasoactive intestinal peptide antagonists, slajista, suramin, swainsonine, synthetic glycosaminoglycans, talimustine, tamoxifen methiodide, tauromustine, tazarotene, tecogalan sodium, tegafur, tellupilium, telomerase inhibitors, temoporfin, temozolomide, teniposide, tetrachlorodecaoxide, tetrazomine, talibustine, thio Coraline, thrombopoietin, thrombopoietin mimetics, thymalfasin, thymopoietin receptor agonist, thymotrinan, thyroid stimulating hormone, tin ethyl etiopurpurin, tirapazamine, titanocene dichloride, topsentin, toremifene, totipotent stem cell factor, translation inhibitors, tretinoin, triacetyluridine, triciribine, trimetrexate, triptorelin, tropisetron, turosteride, tyrosine kinase inhibitors, tyrphostin, UBC inhibitors, ubenimex, urogenital sinus-derived growth inhibitor, urokinase receptor antagonists, vapreotide, variolin B, vector systems, red blood cell gene therapy, veraresol, veramine, verudine, verteporfin, vinorelbine, vinxartin, vitaxin, vorozole, zanoteron, zeniplatin, zilascorub, and zinostatin stimalamer. In one embodiment, the anticancer agent is 5-fluorouracil, taxol, or leucovorin.

[0284] The present invention will be further described in the following examples. It should be understood that these examples, which show exemplary embodiments of the present invention, are for illustrative purposes only. From the above description and these examples, one skilled in the art can ascertain the essential features of the present invention, and can make various changes and modifications to the present invention in order to adapt the present invention to various applications and conditions without departing from the spirit and scope of the present invention. Therefore, in addition to those shown and described herein, various modifications of the present invention will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

[0285] Experimental Example The present invention will be described in more detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, the present invention should not be construed as being limited to the following examples in any way, but rather as embracing any variations that become evident as a result of the teachings provided herein.

[0286] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods, and therefore the following examples are not to be construed as limiting in any way the remainder of the disclosure.

[0287] Example 1: Immunotherapy of ovarian cancer targeting FSHR via innate and adaptive immunity Monoclonal antibodies against FSHR were developed and experiments were planned to focus on the most potent of these reagents. Anti-FSHR antibodies bound to ovarian serous and clear cell adenocarcinoma cells expressing multiple FSHRs, establishing their specificity. Utilizing this as a tool, experiments were planned to develop a bispecific T cell engager (DDAP TCE) targeting FSHR and evaluate this bispecific engager in a therapeutic model for the treatment of ovarian cancer (OC). DDAP TCE in the presence of human PBMCs was highly specific for killing FSHR-positive ovarian tumor lines. It is believed that engaging the innate components of the immune system may potentially enhance tumor control. As increasing evidence suggests that OC is susceptible to attack by natural killer (NK) cells, antibodies against human Siglec7, an inhibitory receptor present on human NK cells, were developed and their binding to NK cells was demonstrated. Experiments were planned to use these to create a new class of bispecific NK cell engagers (NKCEs) that simultaneously target both Siglec7 and FSHR (DDAP NKCEs). This NKCE potently killed FSHR-positive OC targets in in vitro and in vivo assays. These studies demonstrate for the first time the utility of targeting FSHR against major subsets of OC, the high immunopotency of bispecific tools focusing on FSHR and CD3, and the in vivo impact of Siglec7 antibodies against OC or human tumors, as well as new targets of NK cell activation by bispecific engagers against diverse OC populations. Combination studies involving both T cells and NK cells may be interesting as new tools for the treatment of OC.

[0288] Methods and materials are described here.

[0289] Cell lines and animals ID8-Defb29 / Vegf-a-Fshr, ID8-Defb29 / Vegf-a, OVCAR3, CaOV3, TOV-21G, and SKOV3 cells were kindly provided by JR Conejo-Garcia (Department of Immunology, Moffitt Cancer Center, Tampa, Florida). OVISE, OVCAR8, OVCAR10, PEO-4, and Kuramochi cells were kindly provided by R. Zhang (The Wistar Institute). HaCaT human keratinocytes were kindly provided by M. Herlyn (The Wistar Institute). Human embryonic kidney 293T, Expi293F, and mouse myeloma cell line Sp2.0 / 0 were obtained from ATCC. 293T cells were retrovirally transduced to express Siglec7. OVCAR3 and Kuramochi cells were retrovirally transduced with human FSHR to express FSHR as previously described (Perales-Puchalt et al., 2019, JCI Insight 4). K562 and A20 were also purchased from ATCC and retrovirally transduced to express human and mouse FSHR, respectively. The expression vector pBMN-I-GFP (Nolan Lab) was purchased from Addgene.

[0290] Transgenic H2L2 mice were obtained from Harbor Biomed. Balb / c mice were purchased from The Jackson Laboratory. NSG mice were purchased from The Wistar Institute Animal Facility.

[0291] Immunization of mice, generation of hybridomas, and generation of DNA-encoded mAbs Human FSHR (Uniprot P23945) was RNA and codon optimized for expression in mice and cloned into a modified pVax1 vector (Genscript). For generation of FSHR hybridomas, Balb / c mice were immunized by injection into the tibialis anterior muscle with 25 μg of DNA resuspended in 30 μl of water, followed by electroporation with a CELLECTRA device (Inovio Pharmaceuticals). Hybridomas were generated by fusing the mouse myeloma cell line Sp2.0 / 0 with spleen cells using 50% polyethylene glycol 1500 (Roche).

[0292] Several humanized antibodies against human Siglec7 were generated in H2L2 mice. These mAbs were able to bind Siglec7 and stain NK cells. These hybridomas were sequenced and used to develop Siglec7 DNA-encoded monoclonal antibodies (DMAbs) as a tool for in vitro expression (Patel et al., 2018, Cell Rep 25: 1982-93 e4). The final human IgG1 HC and LC were inserted into the pVax1 plasmid expression vector under the control of the human cytomegalovirus (hCMV) promoter and the bovine growth hormone (BGH) polyA signal as described (Patel et al., 2018, Cell Rep 25: 1982-93 e4). The plasmids were then transfected into Expi293F cells using the Expifectamine 293 Expression Kit (Thermo Fisher Scientific) to generate recombinant antibodies. The purity and apparent molecular weight of the recombinant antibodies were assessed by SDS-PAGE analysis.

[0293] Design of FSHRxCD3 TCE and FSHRxSiglec7 NKCE Experiments were set up to design a FSHRxCD3 DNA-encoded bispecific T cell engager (TCE) by encoding a codon-optimized scFv of FSHR MAb (DDAP) followed by a scFv of modified UCHT1 anti-human CD3 antibody with the addition of an IgE leader sequence. FSHRxSiglec7 natural killer cell engager (NKCE) was designed by encoding a codon-optimized scFv of Siglec7 MAb (DB-S7-2) followed by a scFv of FSHR antibody (DDAP) with the addition of an enhanced optimized IgE leader sequence. Both constructs were subcloned into a modified pVax1 expression vector (Perales-Puchalt et al., 2019, Mol Ther 27: 314-25). FSHRxCD3 TCE and FSHRxSiglec7 NKCE are hereafter referred to as DDAP-TCE and DDAP-NKCE, respectively.

[0294] Flow cytometry A BD LSRII flow cytometer was used for cell staining. A BD FACS Aria cell sorter (BD Biosciences) was used for sorting cells stably expressing Siglec7 / FSHR. The anti-human antibodies used were directly fluorochrome conjugated. The antibodies used were anti-Siglec7 (F023-420, BD Pharmingen), anti-Siglec3 (6C5 / 2, R&D Systems), and anti-Siglec9 (KB, Biolegend). The primary antibodies used were PE-secondary anti-human (H+L) (Invitrogen), PE / AF647-secondary anti-human F(ab')2 (Jackson ImmunoResearch Laboratories Inc), and APC-secondary anti-mouse IgG (Poly4053, BioLegend). The Live / Dead Violet Viability Kit (Invitrogen) was used to exclude dead cells from the analysis.

[0295] Enzyme-linked immunosorbent assay (ELISA) For DDAP isotyping, ELISA plates were coated with DDAP in PBS overnight, then the plates were blocked and the following HRP-conjugated antibodies were added: anti-mouse IgA, anti-mouse IgM, anti-mouse IgG1, anti-mouse IgG2a, anti-mouse IgG2b, anti-mouse IgG3, and anti-mouse kappa light chain (all from Bethyl).

[0296] For quantification of human IgG in mouse serum from expi293F transfected with Siglec7 DMAb and electroporated with Siglec7 DMAb, MaxiSorp plates were coated with 10 μg / mL goat anti-human IgG Fc (Bethyl) overnight at 4°C. Plates were washed and blocked with 5% milk in PBS-T (PBS with 0.05% Tween 20) for 2 h at room temperature. Plates were washed and samples diluted in PBS containing 0.2% Tween 20 with 1% NCS were added and incubated for 2 h at 37°C. 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.

[0297] Measurement of cyclic AMP 25,000 K562 or K562-hFSHR cells were seeded in 96-well plates. Cells were washed twice with warm PBS and then resuspended in 100 μl serum-free RPMI containing 0.5 mM IBMX (Cayman chemicals) with or without DDAP antibody. After 30 min incubation at 37°C, FSH (50 ng / ml or 1 μg / ml) or PBS was added. After 1 h, cells were washed with ice-cold PBS, lysed, and cyclic AMP measurements were performed according to the manufacturer's instructions (Cell Signaling).

[0298] Immunoblotting Protein extraction, denaturation, and Western blotting were performed as previously described (Tesone et al., 2016). Membranes were blotted with the following antibodies: anti-phospho-p44 / 42 MAPK (Erk1 / 2) (Thr202 / Tyr204) (#9101, Cell Signaling), anti-p44 / 42 MAPK (Erk1 / 2) (clone 137F5, Cell Signaling), anti-LHCGR (clone 8G9A2, Abcam), and anti-β-actin (A5441, Sigma-Aldrich). Images were captured with an ImageQuantLAS 4000 (GE Healthcare Life Sciences).

[0299] In vitro cytotoxicity assay by measurement of luciferase expression 10,000 OVCAR3 cells per well were seeded in 96-well plates and, after 18 h, primary peripheral blood mononuclear cells (PBMCs) were added. After 4 h of co-incubation, cells were stained with 7AAD (Invitrogen), Annexin V (Biolegend), and anti-human CD45 (Biolegend), followed by flow cytometry-based cytotoxicity assays as previously described (Perales-Puchalt et al., 2017, Clin Cancer Res 23: 441-53). Experiments were performed to stably transfect K562 and K562-FSHR with firefly luciferase. 20,000 K562 and K562-FSHR expressing luciferase were seeded in 96-well plates and co-incubated with PBMCs for 5 h. After incubation, cells were lysed and luciferase expression was measured using CytoTox Glo (Promega) as previously described (Zhang et al., 2009, Cancer Res 69: 6506-14). Cytotoxicity was calculated as (max viability control - individual well) / (max viability control - max mortality control) x 100 and calculated as a percentage or relative to the control (PBMCs containing mouse IgG2a isotype control C1.18.4).

[0300] In vitro cytotoxicity analysis using the xCELLigence Real-time Cell Analyzer In vitro cytotoxicity assays were performed based on impedance using the xCELLigence Real-Time Cell Analysis Instrument (RTCA) (Agilent Technologies, USA). Impedance is expressed in arbitrary units called cell index (Durdagi et al., 2021, Mol Ther 30:963-974). Target cells were plated at 1 × 10 per well in disposable sterile 96-well E-plates in the xCELLigence RTCA instrument. 4 ~2×10 4 Cells were seeded at a final cell concentration of 100 mM NaCl. During the experiment, the device was placed in a CO2 incubator and controlled by a cable connected to a control unit. The 96-well E-plate was placed in the xCELLigence RTCA device and incubated for 18–24 h. Effector cells (human PBMCs; E(effector):T(target) ratio = 5:1 / 10:1) and treatments (antibody / TCE / NKCE) were then added. Real-time analysis was performed for 3–7 days. Electrical conductivity was converted to a unitless cell index (CI) parameter by the xCELLigence device every 15 min, and images were captured at 1-h intervals. The data generated were normalized based on the time point of effector cell and antibody / TCE / NKCE addition to target cells and analyzed using RTCA / RTCA Pro software.

[0301] Immunohistochemistry and immunocytochemistry Mouse tumors were frozen in OCT (TissueTek) and frozen sections were cut. 293T were grown on poly-L-lysine-coated cover slides (Sigma) and transfected with human or mouse FSHR expression vectors. Slides were then fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 in PBS. Sections were blocked using 5% normal goat serum and stained with DDAP antibody followed by AF647-conjugated secondary antibodies specific for human or mouse IgG (Invitrogen). Slides were viewed using a Leica TCS SP-5 confocal microscope and Leica LAS-X software (immunohistochemistry) or a Nikon ECLIPSE 80i microscope and NIS-Element Imaging (immunohistochemistry).

[0302] Western blot analysis To demonstrate in vitro and in vivo expression of Siglec7 DMAb, supernatants collected after transfection of DNA encoding the antibody into Expi293F cells or serum collected from mice immunized with Siglec7 DMAb were heat inactivated, reduced, and loaded with Odyssey Protein Molecular Weight (LI-COR). After electrophoresis, samples were transferred to polyvinylidene difluoride (PVDF) membranes via the iBlot-2 system (Thermo Fisher Scientific) and blocked using Odyssey Blocking Buffer (LI-COR). Heavy and light chains were detected using goat anti-human secondary antibodies (LI-COR). Expression of bispecific T cell and NK cell engagers was detected using goat anti-human IgGF(ab')2 (Jackson ImmunoResearch Laboratories Inc) followed by donkey anti-goat antibodies (LICOR).

[0303] Immunofluorescence (IFA) analysis: Siglec7-transduced HEK293T cells were seeded in 2-well chamber slides and cells were allowed to adhere overnight. Cells were permeabilized using 0.5% Triton 100 in PBS followed by blocking using 5% goat serum. Primary antibodies (bispecific) were then added and incubated overnight at 4°C. Slides were then incubated with goat anti-human H+L (Texas Red conjugated) secondary antibody. Nuclear staining was performed using 4',6-diamidino-2-phenylindole (DAPI). Samples were mounted on glass slides using Fluoroshield mounting medium (Invitrogen) and observed using a Leica TCS SP8 WLL scanning laser confocal microscope.

[0304] Cytokine secretion profile analysis: OVCAR3-FSHR (target) cells 1 × 10 4 Cells were seeded at a density of 1000 cells / well. After overnight incubation, PBMCs (effector cells; E:T=5:1) and DDAP-NKCE or pVax1 were added to the target cells. After 48 hours, supernatants were collected and secreted cytokines were analyzed with the LEGENDplex™ Human CD8 / NKPanel (13-plex) multiplex bead-based assay (Biolegend) according to the manufacturer's protocol.

[0305] Tumor challenge: NOD / SCID-γ (NSG) mice were challenged with OVISE and OVCAR3-FSHR cells. 6 OVISE cells (1:1 in PBS and Matrigel) were injected subcutaneously into the right flank. Seven days after tumor implantation, when tumors were palpable, mice were treated with pVax1 (100 μg) or Siglec7 DMAb (50 μg HC + 50 μg LC). On the same day that expression vectors were administered, 7 × 10 6 PBMCs were injected intraperitoneally into each mouse. Mice were inoculated three times, one week apart. Tumor size was monitored periodically by caliper measurement. Mice were euthanized when signs of graft-versus-host disease (GVHD) appeared. Tumor volume (V) was calculated as V = [(length × width) 2)] / 2. The width is the smaller of the measured values.

[0306] In the OVCAR3-FSHR challenge model, NSG mice were inoculated with 3 × 10 FSHR-expressing OVCAR3-FSHR cells in the right flank. 6 OVCAR3 or OVCAR3-FSHR cells were subcutaneously injected. Three days after tumors became palpable, mice were inoculated with pVax1 (100 μg), DDAP-TCE (100 μg), or DDAP-NKCE (100 μg). On the same day that expression vectors were administered, 10 × 10 6 PBMCs were injected intraperitoneally into each mouse. Mice were inoculated twice with a 1-week interval, and the same procedures were followed thereafter. Animal experiments were approved by the Institutional Animal Care and Use Committee at The Wistar Institute. Human PBMCs from healthy donors were provided by the Human Immunology Core at the University of Pennsylvania.

[0307] statistical analysis All statistical analyses were performed using Graph Pad Prism. A p-value <0.05 was considered statistically significant. Differences between the means of experimental groups were calculated using unpaired two-tailed Student's t-test or one-way analysis of variance where three or more quantitative variables were measured. Error bars represent the standard error of the mean. Comparisons of tumor size at each time point were performed using two-way analysis of variance with Fisher's least significant difference (LSD) test.

[0308] Here, the results will be explained.

[0309] Generation of anti-human FSHR antibodies and flow cytometric screening Follicle-stimulating hormone receptor (FSHR) is a tumor-associated antigen present in ovarian cancer (Perales-Puchalt et al., 2017, Clin Cancer Res 23: 441-53), prostate cancer (Mariani et al., 2006, J Urol 175: 2072-7), and neovascularization in 80% of cancers (Radu et al., 2010, N Engl J Med 363: 1621-30). FSHR is a G protein-coupled receptor with seven transmembrane domains (Fan and Hendrickson, 2005, Nature 433: 269-77). This complex structure poses a challenge for traditional antigen-protein approaches. We generated a codon-optimized sequence of human FSHR for direct in vivo immunization (Figure 1A). FSHR cDNA was subcloned into the pBMN-I-GFP expression vector (Figure 1B) and inoculated into mice by DNA injection followed by in vivo electroporation. Mice were immunized every 2 weeks, and serum was collected 1 week after each immunization for analysis of antibody levels (Figure 1C).

[0310] To detect anti-FSHR antibodies that bind to native FSHR expressed on the cell membrane, stably transduced K562 cells were generated to overexpress human FSHR (K562-FSHR). To demonstrate correct folding and function of the recombinant FSHR, experiments were performed to test the response of K562-FSHR cells to follicle-stimulating hormone (FSH). K562-FSHR increased cyclic AMP production and ERK phosphorylation upon FSH stimulation, whereas parental K562 did not (Figure 1D and Figure 1E).

[0311] To track the binding ability of immune serum to FSHR, K562 (GFP-) and K562-FSHR (GFP+) cells were mixed in equal ratios, serum diluted to 1:1000 was added, followed by anti-mouse IgG APC-conjugated secondary antibody (Figures 9A and 9B), and the mean fluorescence intensity (MFI) fold of K562-FSHR compared to wild-type K562 was measured. If the 1:1000 diluted serum exceeded 20-fold the MFI, a final immunization was performed by boosting with FSHR-overexpressing A20 cells 3 weeks after the previous immunization. The boosted mice were sacrificed 4 days later for hybridoma generation, as described (Bordoloi et al., 2021, ACS Pharmacol Transl Sci 4: 1349-61; Choi et al., 2020, Hum Vaccin Immunother 16: 907-18).

[0312] Two weeks after fusion, supernatants from 15 96-well plates were screened by flow cytometry to analyze potential hybridomas (Figure 9C). The top 20 clones based on MFI fold were expanded for further analysis (Figure 9D). Highly potent clones were selected based on high binding specificity.

[0313] Anti-FSHR antibodies bind to FSHR with high specificity DDAP binds to ovarian cancer cell lines that spontaneously express FSHR (Perales-Puchalt et al., 2017, Clin Cancer Res 23: 441-53; Zhang et al., 2009, Cancer Res 69: 6506-14). All cell lines (CAOV3, OVCAR3, and TOV-21G) showed the expected expression of FSHR by DDAP staining (Figure 1F). To further confirm that the signal induced by DDAP corresponds to FSHR, CRISPR-mediated deletion of FSHR in TOV-21G cell line was performed. Flow cytometry staining with DDAP antibody showed that there was no binding to TOV-21G cell line after FSHR knockout (Figure 1G). The clone was highly specific for FSHR, since K562 cells transfected with the FSHR homologous protein LHCGR (sequence homology is approximately 46% in the ECD and 72% in the 7TMD) (Ulloa-Aguirre et al., 2018, Front Endocrinol (Lausanne) 9: 707) showed no cross-reactivity in flow cytometry analysis (Figure 1H). Since mouse disease models are important for studying the in vivo efficacy and safety of new therapeutics, experiments were performed to further test whether DDAP could also bind to mouse FSHR. Mouse FSHR was expressed in mouse tumor lines A20 and ID8-Defb29 / Vegf-a, and the binding of DDAP to transfected and non-transfected cells was again tested by flow cytometry. DDAP was observed to bind well to mouse FSHR as well as human FSHR (Figure 1I).

[0314] FSHR + Anti-FSHR antibodies to detect tumor cells Immunohistochemical detection of proteins from biological samples is a common method to determine protein expression from tumors and other specimens to better classify them for prognostic or therapeutic purposes. To examine whether DDAP detects FSHR by immunohistochemistry, solid tumors were generated in NSG immunodeficient mice. To generate tumors, 5 million K562, K562-FSHR, OVCAR-3, or TOV-21G were injected into the axilla of NSG mice in 50% PBS / Matrigel (Corning). DDAP detected FHSR from frozen tumor sections (Figure 2A). Furthermore, this antibody is useful for staining FSHR in immunocytochemical analysis. Human or mouse FSHR-transduced 293T cells were stained with DDAP. DDAP was able to bind both human and mouse FSHR similarly to polyclonal anti-human antibodies, but was unable to bind to mock-transfected 293T cells confirming this activity (Figure 2B-D).

[0315] Anti-FSHR antibodies induce antibody-dependent cellular cytotoxicity (ADCC) To determine the isotype of DDAP, ELISA was performed and DDAP was found to be IgG2a (Figure 2E), an isotype capable of inducing ADCC (Akiyama et al., 1984, Cancer Res 44: 5127-31). ADCC capacity was tested using K562 with or without FSHR. DDAP was able to increase PBMC cytotoxicity against K562-FSHR, but not against K562 (Figures 2F and G). Unmodified FSHR + To examine its ability to induce ADCC against ovarian cancer cell lines, OVCAR3 cells were co-cultured with PBMCs in the presence of DDAP or an irrelevant IgG2a antibody. We found that physiological expression levels of FSHR in ovarian cancer cells were sufficient to target DDAP-mediated cytotoxicity with increasing doses of the antibody (Figure 2H). The killing activity of OVCAR3-FSHR cells was also assessed by xCelligence assay and was found to induce dose-dependent killing of FSHR-overexpressing OVCAR3 cells (Figure 2I). Thus, DDAP inhibits the target FSHR +It can be used to induce selective cytotoxicity in cells with moderate activity.

[0316] DDAP-TCE generation, expression, and cytotoxicity Bispecific T cell engagers represent a recent important advancement in the field of monoclonal technology. Because the DDAP anti-FSHR antibody showed initial levels of ADCC, experiments were designed to improve this potential. A TCE targeting FSHR (DDAP-TCE) was designed (Gary et al., 2021, iScience 24: 102699; Patel et al., 2018, Cell Rep 25: 1982-93 e4; Perales-Puchalt et al., 2019, Mol Ther 27: 314-25). Experiments were designed to genetically optimize the scFv of the FSHR mAb and fuse it with an optimized sequence scFv encoding anti-CD3 (modified UCHT1) (Figure 3A and B). DDAP-TCE was efficiently expressed in vitro upon DNA transfection into Expi293F cells (Figure 3C). This new bispecific antibody did not show non-specific binding to K562 cells that do not have native FSHR expression (Figure 3D) and maintained binding to K562-FSHR cells (Figure 3E). Binding to FSHR was further confirmed with CaOV3 (Figure 3F) and OVCAR3-FSHR cells (Figure 3G). CD3 binding of the DDAP-TCE bispecific antibody was confirmed using human primary T cells (Figure 3H). To determine the functionality of this new bispecific antibody expressed in vitro, DDAP-TCE supernatant or medium alone containing OVCAR3-FSHROC cells were co-cultured with human PBMCs in vitro and quantitatively analyzed using a real-time xCELLigence-based killing assay for tumor cell clearance. Incubation with immune cells + DDAP-TCE resulted in a very strong cytotoxic effect on OVCAR3-FSHR cells. No cytotoxicity was observed in the absence of DDAP-TCE or effector cells (Figure 3I).

[0317] Generation and screening of anti-human Siglec7 antibodies We generated and screened hybridomas that produce mAbs that react specifically with human Siglec7 expressed in cell lines. Three mAbs targeting Siglec7 were sequenced and further characterized. DB-S7-1, DB-S7-2, and DB-S7-7 showed strong cell binding, whereas anti-Siglec3 (6C5 / 2, R&D Systems) did not (Figure 10A). To demonstrate the specificity of these three anti-Siglec7 antibodies, we confirmed their binding to 293T cells overexpressing Siglec9 (293T-Siglec9). Siglec-9 also belongs to the Siglec-3 / CD-33-related Siglecs and shows homology (84% similarity) with Siglec7 (Angata and Varki, 2000, J Biol Chem 275: 22127-35). A commercially available anti-Siglec9 antibody (clone K8, Biolegend) showed binding to 293T-Siglec9 cells as expected, whereas DB-S7-1, DB-S7-2, and DB-S7-7 anti-Siglec7 antibody clones showed no binding, confirming the specificity of the anti-Siglec7 antibody (Figure 10B). To further visualize the binding of anti-Siglec7 antibodies to human Siglec7, high-resolution confocal imaging of 293T-Siglec7 fixed cells was performed. Cells were labeled with DAPI (nuclei), GFP (Siglec7), and Texas Red (anti-Siglec7). As shown in Figure 10C, DB-S7-2 bound strongly to Siglec7; DB-S7-1 and DB-S7-7 also showed binding to human Siglec7, whereas no binding was observed with the secondary antibody only control.

[0318] Cytotoxicity test of human anti-Siglec7 antibody To confirm the ability of anti-Siglec7 antibodies to induce cytotoxicity through NK cell activation, impedance-based in vitro cytotoxicity assays were performed using the xCELLigence real-time cell analyzer. Target cells (A549, HaCaT, GMO5389, OVISE, OVCAR8, and SKOV3) were placed in the xCELLigence RTCA device and incubated for 18–24 h before adding human PBMCs and anti-Siglec7 antibodies. Real-time analysis of A549 (lung adenocarcinoma cells), HaCaT (human keratinocytes), and GMO5389 (human fibroblasts) showed that no cytotoxicity was induced by the presence of anti-Siglec7 antibodies DB-S7-1, DB-S7-2, and DB-S7-7 when cultured up to 3 days after the addition of effector cells and antibodies (Figures 4A, 4C, and 4E). Images captured 3 days after adding effector cells and antibodies to target cells show no killing in the presence of antibodies compared to control wells without antibodies (Figures 4B, 4D, and 4F). When cytotoxicity is assessed in various human OC cells, DB-S7-2 is able to induce significant tumor cell killing. For OVISE and SKOV3 cells, killing was observed within 24 hours of treatment with antibodies and effector cells, whereas for OVCAR8 cells, killing by DB-S7-2 appears after approximately 30 hours. DB-S7-1 and DB-S7-7 were also able to induce killing of OVISE, OVCAR8, and SKOV3 human ovarian cancer cells, although the killing effect of these reagents was not as strong as DB-S7-2 (Figures 4G, 4I, and 4K). As shown in the images, no attached tumor cells were observed in the treated wells 3 days after adding effector cells and treating with DB-S7-2. Similar killing was observed for clones DB-S7-1 and DB-S7-7 compared to control wells (Figures 4H, 4J, and 4L).

[0319] In vitro expression and tumor cell killing of Siglec7 DMAb To rapidly evaluate the anti-Siglec7 DMAb in vivo, we developed expression cassettes as DNA vectors for direct delivery. Experiments were designed to encode the codon and RNA-optimized sequences of the heavy and light chains of DB-S7-1, DB-S7-2, and DB-S7-7 in the pVax1 plasmid expression vector as a dual plasmid (Figure 5A). Antibody expression was tested in vitro by transfecting the synthetic human Siglec7 DNA vector into expi293F cells and Western blot analysis 5 days after transfection. Bands corresponding to the heavy and light chain antibodies were clearly identifiable in the supernatants of Expi293F transfected with human Siglec7 DMAb, but not in the empty factor-transfected control wells (Figure 5B). DB-S7-1, DB-S7-2, and DB-S7-7 were expressed in vitro at 7.5 μg / ml, 4.297 μg / ml, and 7.824 μg / ml, respectively (Figure 5C). The generated fully human recombinant antibodies were then further evaluated for their ability to induce killing in OC cells (OVCAR10 (Figures 5D and E), TOV-21G cells (Figure 5F), OVISE cells (Figure 5G), and PEO-4 cells (Figure 5H) cells). Although all three clones induced OC cell killing, clone DB-S7-2 showed the highest potency. Cytotoxicity induced by DB-S7-2 was also evaluated in two additional OC lines (CaOV3 cells (Figure 5I) and OVCAR3 cells (Figure 5J)). DB-S7-2 retained effective killing ability in both OC cells, indicating the high potency of this anti-Siglec7 mAb clone.

[0320] In vivo expression of Siglec7 DNA-delivered MAb and its effect on tumor progression in an ovarian cancer challenge model After confirming in vitro expression, we tested the in vivo expression of DMAb clones DB-S7-1, DB-S7-2, and DB-S7-7. In these studies, 50 μg + 50 μg (HC + LC) of each anti-Siglec7 clone was injected as described in (Duperret et al., 2018, Cancer Res 78: 6363-70; Patel et al., 2018, Cell Rep 25: 1982-93 e4) using EP to directly inject DNA, increasing transfection efficiency into mouse tibialis anterior muscle (Figure 6A). The presence of human IgG in the serum of mice injected with DB-S7-1, DB-S7-2, and DB-S7-7 Siglec7 DMAbs was significant, but not in the serum of controls (data not shown) or prebled mice (Figure 6B, Figure 11). DB-S7-1 DMAb showed the highest in vivo expression, with levels as high as 50 μg / ml in mouse serum (Figure 11). Furthermore, human NK cells were stained with day 14 sera from mice immunized with DB-S7-1, DB-S7-2, and DB-S7-7 DMAb (DMAb showed highest expression on day 14) and the empty vector control. Day 14 sera from all three groups positively stained human NK cells, whereas no staining was observed with pVax1 or the irrelevant antibody control (Figure 6C).

[0321] To determine the antitumor effect of Siglec7 DNA delivery in vivo, NOD / SCID-γ (NSG) mice were then transfected with the OVISE human ovarian cancer cell line (0.8 × 10 6 The tumors were challenged with 1000 cells / mouse. The tumors were 50 mm in size on average. 3 When the ovarian tumors exceeded 100% in vivo, 50 μg + 50 μg (HC + LC) of DB-S7-1 or DB-S7-2 DMAb or pVax1 empty vector were delivered intramuscularly by electroporation (Figure 6D). Notably, the groups receiving DB-S7-1 and DB-S7-2 Siglec7 (Figure 6E) DMAb showed a significant reduction in tumor burden / delay in tumor growth compared to the empty vector control, indicating that Siglec7 antibodies can affect ovarian tumor growth in vivo.

[0322] Fc blocking enhanced the killing efficiency of anti-Siglec7 antibodies Fc gamma receptors (FcγRs) are expressed primarily on the surface of lymphoid and myeloid cells, such as granulocytes, macrophages, and NK cells. However, they are uniquely assigned to each cell type; FcγRIIb is expressed exclusively by B cells and FcγRIIIa is expressed exclusively by NK cells, whereas different combinations of FcγRs are expressed by various other immune cells, allowing for a balanced antibody-mediated cellular response (Bournazos et al., 2020, Nat Rev Immunol 20: 633-43; Romain et al., 2014, Blood 124: 3241-9; van der Poel and Carroll, 2017, Nat Immunol 18: 874-5). Similar to native human mAbs, DB-S7-2 anti-Siglec7 antibody expresses a variable domain at one end that binds to Siglec7 present on immune cells, thereby potentially inhibiting interactions between antigens and other glycoproteins. However, the other end of the antibody has a constant Fc region that binds to various Fc receptors and engages with the unique arm of the immune system (Sanseviero, 2019, J Clin Med 8). If anti-Siglec7 antibodies bind to immune cells through their Fc region and not just their Fab region by ligation with FcγR, Siglec7-mediated cytotoxicity may be prevented and one immune cell may be retargeted to another immune cell (Figure 12). Experiments were designed to preliminarily evaluate this issue by blocking FcγR present on immune cells using an Fc blocker (Biolegend). As shown in Figure 6F, enhanced killing was observed after blocking FcγR on immune cells compared to when FcγR was not blocked. With FcγR blockade, killing of OVCAR3 cells was observed to occur at an earlier time point. Images shown in Figure 6G show that blocking FcγR on OVISE cells enhanced killing. FcγR blocking appears to improve both the quality and quantity of immune cell-mediated antibody-dependent cytotoxicity by allowing more immune cells to participate in cytotoxicity targeting OC cells via Siglec7-mediated signaling.

[0323] Generation and expression of DDAP-NKCE DNA-encoded bispecific NK engagers NK cell engagers (NKCEs) have emerged as a promising innate immune modality in the field of immuno-oncology. Currently, the development of single-chain variable fragment (scFv) recombinant reagents with the ability to specifically target CD16 on NK cells and tumor antigens of interest is under evaluation for clinical use (Gleason et al., 2014, Blood 123: 3016-26; Ibarlucea-Benitez et al., 2021, Proc Natl Acad Sci USA 118). Based on the above preliminary data, the potential engagement of dual-activating NKCEs targeting Siglec7 may provide an additional immune tool for tumor targeting and thus may be a promising therapeutic strategy in OC.

[0324] Therefore, experiments were designed to develop a Siglec7-based bispecific NKCE to eliminate potential Fc-mediated effects and engage NK cells more directly, improving targeting potential. It was constructed as two linked antibody binding fragments (scFvs), thus one engaging the target tumor antigen FSHR, while the second engaging the innate immune system via binding to Siglec7, which is highly expressed on NK cells. The optimized sequence encoding the scFv of DB-S7-2 anti-Siglec7 antibody was fused to the optimized sequence encoding the scFv of anti-FSHR (clone DDAP) (Figure 7A) using a GS (glycine-serine) flexible linker (Perales-Puchalt et al., 2019, Mol Ther 27: 314-25). This DDAP-NKCE was efficiently expressed in vitro as a molecule of approximately 55 KDa, as tested by transfection of Expi293F cells (Figure 7B). Binding of DDAP-NKCE was confirmed to bind both cellular targets by flow staining of HEK293T (Figure 7C) or K562 (Figure 7D) cells overexpressing Siglec7 or FSHR, respectively. To visualize the binding of this new bispecific NKCE to human Siglec7, high-resolution confocal imaging of 293T-Siglec7 fixed cells was performed. Cells were labeled with DAPI (nuclei), GFP (Siglec7), and Texas Red (DDAP-NKCE). DDAP-NKCE is observed to show clear surface binding to GFP+ cells expressing human Siglec7 (Figure 7E).

[0325] Cytokine secretion profile of a novel NK cell engager targeting FSHR Cytokines are involved in promoting lymphocyte proliferation, survival, differentiation, and activation (Romain et al., 2014, Blood 124: 3241-9). Because cytokine production is a critical component of NK cell function (Gleason et al., 2012, Mol Cancer Ther 11: 2674-84), we designed experiments to investigate the cytokine secretion profile of our new DDAP-NKCE. A recent study conducted by Gauthier's group reported that CD20-NK cell engagers, which target two other receptors present on human NK cells, NKp46 and CD16, elicited barely detectable cytokine release but exhibited high antitumor potential, demonstrating the critical profile of such NKCEs (Gauthier et al., 2019, Cell 177: 1701-13 e16). Increased production of sFas and Granulysin was observed in DDAP-NKCE treated groups compared to the empty vector control group (Figure 7F). DDAP-NKCE, which engages FSHR and primarily NK immune cells, shows reduced induction of proinflammatory cytokines, which may be important in clinical development, and these immune profiles, distinct from other NKCEs, confirm the uniqueness of the Siglec7NK bispecific engager.

[0326] A novel bispecific T cell and NK cell engager targeting FSHR elicited potent killing in multiple ovarian tumor lines and reduced tumor burden in vivo To determine the functionality of DDAP-NKCE, xCELLigence real-time killing assays were performed to evaluate the potential of DDAP-NKCE to induce killing of a panel of human ovarian tumor lines. DDAP-NKCE delivered in vivo was compared to DDAP-TCE for inducing tumor killing in vitro. Effector cells (PBMC / T / NK cells) and TCE / NKCE were added 1 day after seeding of target OC cells. HEK293T cells were used as a control (FSHR-negative cell line) (Urbanska et al., 2015, Cancer Immunol Res 3: 1130-7). Of note, no nonspecific off-target killing in FSHR-negative 293T cells was observed (Figure 7G and H). DDAP-TCE and DDAP-NKCE effectively killed OVCAR3-FSHR cells in the presence of only purified human T cells and NK cells, respectively (Figure 13A and 13B). Further evaluation of various ovarian tumor cells expressing FSHR demonstrated that DDAP-NKCE was highly efficient in killing OVISE (Figure 8A and B), CaOV3 (Figure 8C and D), OVCAR3-FSHR (Figure 8E), PEO-4 cells (Figure 8F), and Kuramochi-FSHR cells (Figure 8G) in the presence of human PBMCs as a source of T and NK cells. Importantly, Kuramochi and PEO-4 harbor BRCA2 mutations, the latter also showing resistance to PARP inhibitors (Sakai et al., 2009, Cancer Res 69: 6381-6), yet are not spared from killing by DDAP-NKCE. Similarly, DDAP-TCE was found to exert potent killing activity as a DDAP-NKCE bispecific antibody; however, the efficacy of both is altered in PEO-4 cells. These differences suggest important implications, potentially complementary, that require further development and investigation. Furthermore, NKCE (IL13Rα2-NKCE) and TCE (IL13Rα2-TCE) that do not target FSHR did not induce toxicity in FSHR-expressing OVCAR3 cells (Figures 13C and 13D). To further demonstrate the sFas-mediated killing of DDAP-NKCE, the killing effect of this novel NKCE was compared in the presence and absence of anti-Fas antibody.Fas (CD95, apoptosis antigen 1) is a member of the death receptor subfamily of the TNF receptor superfamily. Fas / FasL binding is known to induce the extrinsic apoptosis pathway. We observed that in the presence of anti-Fas antibodies, the killing of OVCAR3-FSHR cells by DDAP-NKCE was reduced. This suggests a novel killing mechanism of DDAP-NKCE through the involvement of Fas.

[0327] To further evaluate the in vivo antitumor effects of DDAP-NKCE and DDAP-TCE, NSG mice were challenged with OVCAR3-FSHR cells. NSG mice were administered OVCAR3-FSHR cells and 4 days after tumor implantation, DDAP-TCE, DDAP-NKCE, or empty vector were administered twice, 1 week apart. On day 4, mice were also inoculated with human PBMCs and tumor volumes were measured periodically (Figure 8H). Treatment with both bispecific antibodies significantly reduced tumor burden in OVCAR3-FSHR tumor-bearing mice, but not in the control-treated group (Figure 8I), supporting the potential synergistic effect of this approach (Figure 14).

[0328] Engineering FSHR to engage innate or adaptive immunity for ovarian cancer immunotherapy Although important advances have been made in the field of OC treatment, recurrent OC remains a harbinger of extremely poor prognosis and highly lethal cancer types (Izar et al., 2020, Nat Med 26: 1271-9; Kurnit et al., 2021, Obstet Gynecol 137: 108-21; Hamanishi et al., 2016, Int Immunol 28: 339-48). There are several reasons to believe that OC may respond well to immunotherapy treatment, as ovarian cancer cells express cancer-specific antigens, which can induce antitumor immune responses after immunotherapy, but immunotherapy response rates in OC patients remain rather modest (Coleman, 2016, Nat Rev Clin Oncol 13: 71-2). In particular, a major obstacle in the development of targeted therapies is finding targets with specific expression restricted to the surface of tumor cells and not healthy tissues (Perales-Puchalt et al., 2017, Clin Cancer Res 23: 441-53). FSHR is one such target that is selectively expressed in ovarian granulosa cells (Perales-Puchalt et al., 2017, Clin Cancer Res 23: 441-53), so experiments were designed to study its potential target in OC. Follicle-stimulating hormone is an important ovarian epithelial cell proliferation inducer that functions through binding with FSHR. Overexpression of FSHR is responsible for upregulation of oncogenic pathways and increased EOC proliferation. Thus, FSHR may be utilized as an important therapeutic target to induce T cells against OC (Perales-Puchalt et al., 2017, Clin Cancer Res 23: 441-53). Recently, the tumor impact, tolerability, and potential safety of immune targeting of FSHR in an immune-competent mouse model have been reported (Perales-Puchalt et al., 2019, JCI Insight 4).Injection of the optimized DNA sequence, followed by electroporation, confers overexpression of the protein in its native conformation that can induce strong immune cellular and humoral responses (Tebas et al., 2017, N Engl J Med; Yan et al., 2013, Cancer Immunol Res 1: 179-89). Here, we extend our experiments to generate an anti-FSHR monoclonal antibody and use this reagent to develop a new biologic. The results presented here demonstrate the generation and characterization of a potent anti-FSHR antibody and its application as an immunotherapeutic tool for OC. Several individual clones (including clone DDAP) were selected for additive testing based on their potency and reactivity. The MAb supports detection of FSHR expression in samples by multiple methods, including flow cytometry, ELISA, and immunocytochemistry. This could extend its potential impact to the clinic and help determine FSHR status in patient samples for personalized medicine approaches.

[0329] A recent important tool in the field of antibody technology for cancer therapy is the bispecific T cell engager approach. Most of these are in preclinical and early clinical research, but there is an approved cancer bispecific product, Blincynto (blinatumomab), which is used as a treatment for acute lymphoblastic leukemia (ALL) that targets CD19 on B cells and engages T cells via linked anti-CD3 binding (Sheridan, 2021, Nat Biotechnol 39: 251-4). Bispecific T cell engagers can redirect both CD4 and CD8 T cells to kill tumor cells, independent of endogenous antigen-specific TCR recognition by T cells (Dao et al., 2015, Nat Biotechnol 33: 1079-86). Despite showing high antitumor potency, the progress rate of these tools has been slower than expected due to several factors (including tumor antigen specificity, extratumoral activity, specific cytokine release issues, and short processing half-life) (Perales-Puchalt et al., 2019, Mol Ther 27: 314-25). Based on the specificity of the DDAP anti-FSHR clone, it was thought that this could be an important target for research. To rapidly evaluate these new potential tools in the OC model, direct in vivo delivery of DNA was used. DDAP-TCE binds to both FSHR and CD3 termini, and potent killing activity was observed at nanogram levels (7.81ng / ml) in the presence of human PBMCs. This DDAP-TCE had significantly better specific tumor killing than the DDAP parent antibody.

[0330] Some aggressive OC types, such as HGSOC and OCS, are referred to as "cold" tumors, with limited therapeutic approaches and poor responsiveness due to low TIL infiltration, leading to high mortality rates (Wu et al., 2021, Front Immunol 12: 672502). Therefore, T cell approaches and natural effector cells such as NK may be important tools for research (Hoogstad-van Evert et al., 2020, Gynecol Oncol 157: 810-6). NK cells serve as the first line of defense in tumor immune surveillance (Wiernik et al., 2013, Clin Cancer Res 19: 3844-55). Recent limited data suggest that OC may respond to NK cell attack (Hoogstad-van Evert et al., 2020, Gynecol Oncol 157: 810-6). Early NK bispecific studies have focused on NK cell-associated receptors. NK cells can find immune targets through engagement of NKG2D and natural cytotoxicity receptors, which are involved in the regulation of natural cytotoxicity. In addition, the activating receptor CD16 (FcγRIII), which strongly binds certain Ig isotypes, promotes NK-driven ADCC as a result of antibody trafficking to the local tumor environment (Wiernik et al., 2013, Clin Cancer Res 19: 3844-55). Thus, immune responses elicited by NK cells depend on interactions between receptors present on NK cells, direct recognition of tumors at the site, or a humoral response of the host to target cell ligands that engage NK cells (Wiernik et al., 2013, Clin Cancer Res 19: 3844-55).

[0331] As mentioned above, NK cells play a role in tumor immune surveillance as they can lyse tumor cells, but tumor cells often utilize immune evasion strategies to escape this immune function. A recent important area related to immune evasion is through tumors that exhibit increased surface sialic acid glycosylation as an immunostatic decoy against NK attack. NK cells display Siglec7, which, when engaged on the surface of human NK cells via tumor association, can inhibit NK-mediated target killing (Prescher et al., 2017, J Med Chem 60: 941-56). Siglecs have recently been designated as glycoimmune checkpoints, although much of this research is in its infancy. Through interactions with sialylated glycan ligands overexpressed on cancer cells, inhibitory Siglecs may affect protective antitumor immunity (Hong et al., 2021, ACS Cent Sci 7: 1338-46). Therefore, targeting Siglec7 is a promising therapeutic strategy to enhance antitumor immune responses in OC (Ibarlucea-Benitez et al., 2021, Proc Natl Acad Sci USA 118). To achieve this, several human-like antibodies against Siglec7 using transgenic humanized mice have been developed (Bordoloi et al., 2021, ACS Pharmacol Transl Sci 4: 1349-61; Perales-Puchalt et al., 2019, Mol Ther 27: 314-25), which offer the advantages of research and rapid application due to the V regions derived from the human V gene lineage (Tu and Zheng, 2016, Methods Mol Biol 1371: 157-76). Several key clones were developed and further experiments were planned with three highly potent clones (DB-S7-1, DB-S7-2, and DB-S7-7) that had high binding affinity to recombinant human Siglec7 (Siglec7 is expressed on OC cells and human NK cells). It was observed that the antibody was able to drive NK cell function in the presence of tumor target cells promoting specific killing of target OC cells without nonspecifically killing cellular targets.These are important tools for further evaluation.

[0332] Here, we present further characterization of DDAP-NKCE. This is believed to be the first report of a Siglec7-based bispecific NK cell engager. Using the sequence of DB-S7-2, which showed the highest potency, we designed this new bispecific human NKCE and associated it with anti-FSHR. This DDAP-NKCE was highly potent in tumor-specific cell killing, as assessed using a panel of human ovarian tumor cells. Targets included cell lines with mutations in the BRCA gene and cell lines resistant to chemotherapy or poly ADP-ribose polymerase inhibitors (PARPi). Despite showing impressive activity in cases of sporadic high-grade serous OC and BRCA-associated OC, unfortunately, as with classical chemotherapy, many patients have been reported to eventually acquire resistance to PARPi treatment (Franzese et al., 2019, Cancer Treat Rev 73: 1-9; Liu et al., 2014, Gynecol Oncol 133: 362-9; McMullen et al., 2020, Cancers (Basel) 12; Yang et al., 2020, Front Immunol 11: 577869), supporting the importance of novel approaches to target such OC tumors. The high efficacy shown by both DDAP-TCE and DDAP-NKCE (bispecific T and NK cell engagers) using FSHR targeting is promising. Notably, both NK and T cell engagers targeting FSHR were found to be equally potent in reducing in vivo tumor burden / tumor progression in an ovarian tumor-bearing mouse model.

[0333] Differences were observed for both FSHR-targeting bispecific antibodies, DDAP-TCE and DDAP-NKCE. Both are very powerful approaches to affect tumors in vitro and in vivo. DDAP-NKCE exerts strong killing power in the presence of a reduced cytokine-producing phenotype. These studies demonstrate for the first time the utility of targeting FSHR against a major subset of OC, that bispecific tools focused on FSHR and CD3 are highly immunopotent, that Siglec7mAb engages and activates NK cells, and finally that Siglec7 NKCE is a powerful tool to target OC and may affect tumor growth in vivo, and that combining these two approaches may provide additional benefits. The results presented here show for the first time a new target for NK cell activation by bispecific engagers, and diverse OC populations are considered potential targets for these therapeutic approaches. These can be directly applied to OC research and diagnosis. Further efforts are needed to further explore these important tools for progress in the application of these approaches to ovarian and other cancers expressing FSHR. SiglecNKCE needs to be further investigated in the context of other solid and liquid tumor targets.

[0334] Example 2: Designed bispecific NK cell engager targeting IL13Ra2 and Siglec7 (IL13Ra2-NKCE) Figure 17 shows a schematic diagram of the designed IL13Ra2 targeting bispecific NK cell engager and targeting Siglec7 (IL13Ra2-NKCE). IL13Ra2-NKCE showed potent activity in killing antigen-positive melanoma cells (Figure 20).

[0335] Example 3: Siglec-7 glycoimmune checkpoint MAb and NK cell engager biologics induce potent antitumor immunity against ovarian cancer Aggressive OCs, such as HGSC and ovarian carcinosarcoma (OCS), are referred to as “cold” tumors due to their low tissue infiltrating lymphocyte (TIL) infiltration and poor therapeutic response (Wu et al., 2021, Front Immunol 12, 672502, Hoogstad-van Evert et al., 2020, Gynecol Oncol 157, 810-816). Therefore, engaging the immune response with additional means is crucial for immune targeting of cold tumors (Pugh-Toole et al., 2022, Curr Treat Options Oncol 23, 210-226). Considering the innate immune effector system is important, as NK cells serve as the first line of defense for tumor immune surveillance (Wiernik et al., 2013, Clin Cancer Res 19, 3844-3855). Tumors evade T cell responses through multiple mechanisms, including MHC downmodulation and upregulation of T cell exhaustion programs (Vinay et al., 2015, Semin Cancer Biol 35 Suppl, S185-S198). Similarly, cancers can exploit host systems to circumvent NK cell surveillance and thus escape NK immune functions through immune evasion strategies, including release of soluble ligands for NK activating receptors, release of inhibitory cytokines, and upregulation of HLA molecules (Sabry et al., 2013, Front Immunol 4, 408). However, NK unresponsiveness may also be mediated by additional NK regulatory pathways. Recent studies have identified that many tumors exhibit increased levels of sialic acid on their surface, which likely acts as a negative signal to NK cells, allowing tumors to evade NK immune surveillance (Dobie et al., 2021, Br J Cancer 124, 76-90). One approach to enhance NK activation against such hypersialylated tumors is desialylation of target cells expressing ligands for Siglec-7 (Jandus et al., 2014, J Clin Invest 124, 1810-1820).NK cells display a receptor / sensor for distinct sialoglycan determinants, Siglec-7, which, when engaged on the surface of human NK cells via tumor association, can inhibit NK-mediated target killing (Prescher et al., 2017, J Med Chem 60, 941-956; Fong et al., 2018, Proc Natl Acad Sci USA 115, 10410-10415; Meril et al., 2020, Mol Carcinog 59, 713-723). These may represent a regulatory negative signal by the sialoglycan tumor shield. Very recent studies suggest that some Siglecs may function as glycoimmune checkpoint molecules that inhibit NK-mediated antitumor immunity in an MHC-independent manner (Hong et al., 2021, ACS Cent Sci 7, 1338-1346). This study explored the use of monoclonal antibodies and derivatives targeting Siglec7 for tumor immunomodulation to confirm and possibly overcome such glyco-CPI functions, improving their ability to impact tumor control.

[0336] The focus of these studies was the antitumor immune response against human OC (19), a phenotype that is difficult to treat aggressively. Using transgenic humanized mouse models, the group developed several antibodies against Siglec-7 (Bordoloi et al., 2022, JCI Insight 7; Perales-Puchalt et al., 2019, JCI Insight 4; Bordoloi et al., 2021, Genes Cancer 12, 51-64). In this study, we focused on three highly potent Siglec-7 binding clones. These antibodies were engineered as human IgG1 and tested for binding to immune cells. Strong staining of anti-Siglec-7 antibodies against NK cells was observed, including both light and dim NK subsets, suggesting the ability to bind to bulk effector NK populations. Furthermore, expression activates NK cells, and if these activated cells are present in some tumor lines, NK-mediated killing may occur against previously refractory OC targets. These human Siglec-7 antibodies drive NK immune function in the presence of diverse OC tumor target cells, promoting specific killing of targeted OC cells. Importantly, this approach should be relatively tolerant of tumor mutations. OC lines were tested with multiple gene mutations, including BRCA1, BRCA2, AKT2, TP53, SPOP, STAT3, MTOR, MEK1, MEK2, and BRAF, among others (Table 1), and all mutations were observed to be targets for anti-Siglec-7 antibody activation of NK cell targets. [Table 1-1] [Table 1-2]

[0337] These Mabs were tested in combination with PD-1 CPIs. This dual treatment was found to be synergistic. However, the potency of the Siglec-7 antibody was observed to be 10-fold higher than the tested anti-PD1 antibody pembrolizumab, suggesting the potential value of this combined NK T cell anti-depletion panel. Next, a humanized NSG mouse model engrafted with human PBMCs was used, and a single dose of DNA-encoded DB7.2 Siglec-7 MAb was observed to significantly suppress tumors and increase median survival in mice bearing OVISE human ovarian tumors. Due to the limitation of the NSG model of developing graft-versus-host disease over several months (Wunderlich et al., 2014, Blood 123, e134-144), anti-Siglec-7 was only administered in a single dose, but was still highly effective. This suggests that multiple dosing over an extended period of time, as current CPIs (Hirsch et al., 2022, Nat Med 28, 2236-2237) are utilized, will likely provide additional benefit. Of note, this is the first demonstration of the impact of Siglec-7-targeting MAbs alone and in combination with anti-PD1 (NK and T cell CPIs) targeting OC or any human tumor, suggesting that the potential added value of such combinations should be further studied or considered in CPI-refractory tumors. These studies have important implications for tumor therapy supporting the possibility of novel non-T cell CPI approaches that may augment current immunotherapy strategies.

[0338] We next evaluated the efficacy of Siglec-7 binding and activation of NK cells for targeted OC therapy. Without wishing to be bound by theory, we hypothesized that binding to Siglec-7 may directly engage NK cells and efficiently attract NK cells to cellular targets. By creating a bispecific fusion of a MAb targeting FSHR with a potent anti-Siglec-7 MAb, we developed a new type of NK cell engager. NKCE has emerged as an intriguing innate immune concept in the field of immuno-oncology. Currently, there is one bispecific antibody being introduced into the clinic; GTB-3550, which engages CD16a-IL-15 / CD33 against AML and CD33+ malignancies (Gleason et al., et al., 2014, Blood 123, 3016-3026; Ibarlucea-Benitez et al., 2021, Proc Natl Acad Sci USA 118; Demaria et al., 2021, Eur J Immunol 51, 1934-1942). Other NK targets being investigated preclinically include NKG2D, NKp30, and NKp46. The phenotype demonstrated for anti-Siglec-7 MAbs of NK cell binding and activation that would otherwise be adversely affected by tumor sialoglycan cloaks suggests that Siglec-7 may be a unique immune tool for specific tumor-NK activation and engagement. The bispecific antibody DB7.2xD2AP1 developed here showed potent binding to both NK cells and ovarian cancer cells. It exhibits killing activity (pM level) against diverse OC phenotypes. This is the first report of the development of a member of the Siglec family as an immune engager or the use of such an approach to develop a Siglec-7 bispecific NK cell engager. This NKCE was target cell specific and potent in tumor cell killing, as assessed using a panel of different human ovarian tumor lines. FSHR expression in the target was a prerequisite for NK killing activity, as FSHR-negative cells were not killed.Interestingly, DB7.2×D2AP11 reduced the production of cytokines and cytotoxic molecules, suggesting that it is important to investigate killing tools with limited potential risk of cytokine release syndrome (CRS) (Pinto et al., 2022, Trends Immunol 43, 932-946).

[0339] During these studies, attention was drawn to OC cells expressing mutations that are particularly problematic for OC therapy. This includes PARPi-resistant OC cells and OCs that exhibit resistance to a variety of drug targets, including HDACs (dacinostat, entinostat, belinostat), PI3K (dactolisib, buparlisib), mTORC (omipalisib), Wee1 (CHEK1), TOP1 (galibiscoquinazole, mitoxantrone, irinotecan), DNA alkylating agents (oxaliplatin, cisplatin), and microtubule stabilizing agents (docetaxel) (Table 1) (Ai et al., 2021, Oncogene 40, 2496-2508; Diaz Osterman et al., 2019, Elife 8; Estep et al., 2007, PLoS One 2, e1279; Kapoor et al., 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 and 2) mutations, which are a major risk in OC cancer patient cohorts. Approximately 50% of HGSCs exhibit a disrupted BRCA pathway, either through germline or somatic mutations or epigenetic silencing of pathway members (Vaughan et al., 2011, Nat Rev Cancer 11, 719-725). Because PARP inhibition is a key pathway for DNA damage repair, PARPi shows great promise for treating tumors with disruptive mutations in BRCA1 / 2. However, resistance to PARPi has been reported to be problematic in treating resistant OC patients (Noordermeer et al., 2019, Trends Cell Biol 29, 820-834). The efficacy and consistency of killing FSHR+ tumors in vitro and in vivo using Siglec-7 MAbs as NKCPIs and bispecific Siglec-7NK engagers suggests that they may provide additional tools for treating poorly responsive OC.

[0340] In summary, this study describes a potent Siglec-7 MAb that can engage and activate NK cells to further complement PD-1 immunotherapy for OC, potentially representing an additional class of CPI, in this case for NK cells. Furthermore, the data show that Siglec-7 can be used to construct new NKCEs and serve as a powerful tool to target receptor-positive OC eliciting remarkable antitumor responses in vitro and in vivo. Although these studies are important for designing additional tools for OC, other studies are needed to explore the utility of such biologics for additional challenging cancers.

[0341] The materials and methods are described below.

[0342] Cells and animals Cell lines used in the experiments included OVCAR3, CaOV3, TOV-21G, OVISE, OVCAR10, PEO-4, Kuramochi cells, HaCaT human keratinocytes, WM3743 melanoma cells, human embryonic kidney 293T, Expi293F, AGS gastric carcinoma, and GM05389 human fibroblasts. 293T cells were retrovirally transduced to express Siglec-7. OVCAR3, OVISE, and Kuramochi cells were retrovirally transduced to express human FSHR as previously described (46). K562 cells were purchased from ATCC and retrovirally transduced to express FSHR.

[0343] Generation of DNA-encoded MAbs Several humanized antibodies against human Siglec-7 were generated. These MAbs were able to bind to Siglec-7 and stain NK cells. Here, these hybridomas were sequenced and used to develop Siglec-7 DNA-encoded monoclonal antibodies (DMAbs) as a tool for in vitro expression (Patel et al., 2018, Cell Rep 25, 1982-1993 e1984). The final human IgG1 HC and LC were inserted into the pVax1 plasmid expression vector under the control of the human cytomegalovirus (hCMV) promoter and the bovine growth hormone (BGH) polyA signal as described (Patel et al., 2018, Cell Rep 25, 1982-1993 e1984). The plasmids were then transfected into Expi293F cells using the Expifectamine 293 Expression Kit (Thermo Fisher Scientific) to generate the recombinant antibodies. The purity and apparent molecular weight of the recombinant antibodies were assessed by SDS-PAGE analysis.

[0344] Design of FSHRxSiglec7 NKCE FSHRxSiglec7 natural killer cell engager (NKCE) was designed encoding a codon-optimized scFv of Siglec-7 MAb (DB-S7-2) followed by an scFv of FSHR antibody (D2AP11) supplemented with an enhanced optimized IgE leader sequence. Both constructs were subcloned into a modified pVax1 expression vector (Perales-Puchalt et al., 2019, JCI Insight 4). FSHRxSiglec7 NKCE is designated DB7.2×D2AP11.

[0345] Flow cytometry A BD LSRII flow cytometer was used for cell staining. A BD FACS Aria cell sorter (BD Biosciences) was used for sorting cells stably expressing Siglec-7 / FSHR. The anti-human antibodies used were directly fluorochrome-conjugated. Antibodies used: anti-Siglec-7 (F023-420, BD Pharmingen), anti-Siglec3 (6C5 / 2, R&D Systems). For unconjugated primary antibodies, PE-secondary anti-human (H+L) (Invitrogen) and PE / AF647-secondary anti-human F(ab')2 (Jackson ImmunoResearch Laboratories Inc) were used. To exclude dead cells from the analysis, a Live / Dead Violet viability kit (Invitrogen) was used. The following antibodies were used for fluorescent cytometry staining of PBMCs: 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 BUV395 (clone B-ly6), CXCR5 BV750 (clone RF8B2), CD3 BUV805 (UCHT1), CD45 AF700 (clone HI30), and ... PE-CF594 (clone HIL-7RM21), CD25 BUV737 (clone 2A3), CD8 BUV496 (clone RPA-T8), HLA-DR BV605 (clone G46-6), CD38 BUV661 (clone HIT2), CD14 BV480 (clone MP9), CD45RA BUV563 (HI100), CD4 BB790 (clone SK3), CD15 FITC (clone HI98), CD103 BB700 (clone Ber-ACT8), CD161 APC (clone DX12).Briefly, cryopreserved PBMCs were thawed and incubated in complete R10 medium (RPMI supplemented with 10% FBS, 2 mM L-glutamine, 100 U / ml penicillin, 100 mg / ml streptomycin) containing 10 U / ml DNAseI (Roche Life Sciences) for 2 h at 37° C., 5% CO2. Cells were washed with PBS and incubated for 20 min with 0, 1, or 10 μg / ml DB7.2 Siglec-7 antibody diluted in fluorescence-activated cell sorting (FACS) buffer. After washing with FACS buffer, cells were incubated for 20 min with mouse anti-human IgGFab secondary antibody PE (Invitrogen) diluted in FACS buffer. Cells were washed with FACS buffer, resuspended in PBS, and prestained for 10 min with chemokine receptors CCR7 and CXCR5 at 37° C., 5% CO2. Cells were then stained for 10 min using Live / Dead Fixable Aqua (Invitrogen) and then incubated for 20 min with a panel of directly conjugated monoclonal antibodies and Human Trustain FcX (Biolegend) diluted in equal volumes of fluorescence-activated cell sorting (FACS) buffer (PBS containing 0.1% sodium azide and 1% bovine serum albumin) and Brilliant Stain Buffer (BD Biosciences). Stained cells were washed and fixed with 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.).

[0346] Enzyme-linked immunosorbent assay (ELISA) For quantification of human IgG in mouse serum electroporated with Siglec-7 MAb and Siglec-7 DMAb, MaxiSorp plates were coated with 10 μg / mL goat anti-human IgG Fc (Bethyl) overnight at 4 °C. Plates were washed and blocked with 5% milk in PBS-T (0.05% Tween 20 in PBS) for 2 h at room temperature. Plates were washed and samples diluted in 1% NCS in PBS with 0.2% Tween 20 were added and incubated for 2 h at 37 °C. Plates were washed again and incubated with 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 signal was measured.

[0347] In vitro cytotoxicity analysis using the xCELLigence Real-time Cell Analyzer In vitro cytotoxicity measurements were performed based on impedance using the xCELLigence Real-Time Cell Analyzer (RTCA) (Agilent Technologies, USA). Impedance is expressed in arbitrary units called cell index. Target cells were plated at 1 × 10 per well in disposable sterile 96-well E-plates of the xCELLigence RTCA device. 4 ~2×10 4 Cells were seeded at a final cell concentration of 1000 x 1000 cells. During the experiment, the device was placed in a CO2 incubator and controlled by a cable connected to a control unit. The 96-well E-plate was placed in the xCELLigence RTCA device and incubated for 18–24 h. Then, effector cells (human PBMC / NK, E(effector):T(target) ratio = 5:1 / 10:1) and treatment (Siglec-7 MAb / NKCE) were added. Real-time analysis was performed for 3–7 days. Electrical conductivity was converted to a unitless cell index (CI) parameter by the xCELLigence device every 15 min, and images were captured at 1-h intervals. The generated data were normalized based on the time point when effector cells and MAb / NKCE were added to the target cells and analyzed using the RTCA / RTCA Pro software.

[0348] Western blot analysis To demonstrate in vitro and in vivo expression of Siglec-7 MAbs, supernatants collected after transfection of Expi293F cells with DNA encoding the antibodies, or serum collected from mice immunized with Siglec-7 MAbs, were heat inactivated, reduced, and loaded on an Odyssey Protein Molecular Weight Analyzer (LI-COR). After electrophoresis, samples were transferred to polyvinylidene difluoride (PVDF) membranes via the iBlot-2 system (Thermo Fisher Scientific) and blocked using Odyssey blocking buffer (LI-COR). Heavy and light chains were detected using a goat anti-human secondary antibody (LI-COR). Expression of bispecific T cell and NK cell engagers was detected using a goat anti-human IgG F(ab')2 (Jackson ImmunoResearch Laboratories Inc) followed by a donkey anti-goat antibody (LICOR).

[0349] Immunofluorescence (IFA) analysis Siglec-7 transduced HEK293T cells were seeded in 2-well chamber slides and cells were allowed to adhere overnight. Cells were permeabilized using 0.5% Triton 100 in PBS followed by blocking using 5% goat serum. Bispecific NKCE was then added and incubated overnight at 4°C. Slides were then incubated with goat anti-human H+L (Texas Red conjugated) secondary antibody. Nuclear staining was performed using 4',6-diamidino-2-phenylindole (DAPI). Samples were mounted on glass slides using Fluoroshield mounting medium (Invitrogen) and observed using a Leica TCS SP8 WLL scanning laser confocal microscope.

[0350] Tumor challenge NOD / SCID-γ (NSG) mice were challenged with OVCAR3-FSHR cells. NSG mice were challenged with 3 × 10 6FSHR-expressing OVCAR3 cells or OVCAR3-FSHR cells were subcutaneously injected. Three days later, when tumors were palpable, mice were inoculated with pVax1 (100 μg) or DDAP-NKCE (100 μg). On the same day as expression vector administration, 10 × 10 6 PBMCs were injected intraperitoneally into each mouse. Mice were inoculated twice with DNA, one week apart, and tumor size was followed periodically. Mice were euthanized when signs of graft-versus-host disease (GVHD) appeared. Tumor volume (V) was calculated using the formula V = [(length × width 2 For Siglec-7 DMAb challenge, NOD / SCID-γ (NSG) mice were injected with OVISE cells (0.8 × 10 6 Ten days later, when tumors were palpable, mice were challenged with DB7.2 DMAb (50 μg + 50 μg; HC + LC) or pVax1 (100 μg), followed by inoculation of each mouse with 10 × 10 6 PBMCs were intraperitoneally injected. The same procedures were then performed. Animal experiments were approved by the Institutional Animal Care and Use Committee at The Wistar Institute.

[0351] statistical analysis All statistical analyses were performed using Graph Pad Prism. A p-value <0.05 was considered statistically significant. Differences between the means of experimental groups were calculated using unpaired two-tailed Student's t-test or one-way analysis of variance where three or more quantitative variables were measured. Error bars represent the standard error of the mean. Comparison of tumor size at each time point was performed using two-way analysis of variance with Fisher's least significant difference (LSD) test. Next, the experimental results are described.

[0352] In vitro expression and specificity evaluation of Siglec-7 MAbs To evaluate the potential of Siglec-7 inhibition as a strategy for NK cell activation, MAbs specific for human Siglec-7 were recently generated and their biological activity studied in vitro. Here, codon- and RNA-optimized antibody expression cassettes for potent Siglec-7 binders were generated and assembled into DNA vectors optimized for the expression of human IgG1. These individual combinations of HC a...

Claims

1. A bispecific natural killer cell engager (NKCE) comprising an antibody or fragment thereof that specifically binds to a target cell of interest, and an antibody or fragment thereof that specifically binds to Siglec7, linked to the antibody or fragment thereof.

2. The NKCE according to claim 1, wherein the target cells for the purpose are tumor cells.

3. The NKCE according to claim 2, comprising an antibody or fragment thereof that specifically binds to a sialic acid-binding receptor, which is linked to an antibody or fragment thereof that specifically binds to a tumor antigen.

4. The NKCE according to claim 2, wherein the tumor antigen is follicle-stimulating hormone receptor (FSHR) or interleukin-13 receptor subunit alpha-2 (IL13Ra2).

5. The NKCE according to claim 1, wherein the antibody or fragment thereof that specifically binds to Siglec7 comprises an amino acid sequence selected from the group consisting of the following: a) Variable heavy chain sequences containing CDR sequences selected from the group consisting of SEQ ID NOs: 1-3, 17-19, 33-35, 49-51, 65-67, and 81-83; b) Variable light chain sequences containing CDR sequences selected from the group consisting of SEQ ID NOs: 9-11, 25-27, 41-43, 57-59, 73-75, and 89-91; c) Sequences having at least 95% identity with one or more variable heavy chain sequences from sequence number 4, sequence number 20, sequence number 36, sequence number 52, sequence number 68, and sequence number 84; d) Sequences having at least 95% identity with one or more variable light chain sequences from sequence number 12, sequence number 28, sequence number 44, sequence number 60, sequence number 76, and sequence number 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 NOs: 4, 20, 36, 52, 68, and 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 NOs: 12, 28, 44, 60, 76, and 92.

6. The NKCE according to claim 1, wherein the antibody or fragment thereof that specifically binds to Siglec7 comprises an amino acid sequence selected from the group consisting of the following: a) Variable heavy chain sequences containing the CDR sequences of SEQ ID NOs. 1-3 and variable light chain sequences containing the CDR sequences of SEQ ID NOs. 9-11; b) Variable heavy chain sequences containing the CDR sequences of SEQ ID NOs. 17-19 and variable light chain sequences containing the CDR sequences of SEQ ID NOs. 25-27; c) Variable heavy chain sequences containing the CDR sequences of SEQ ID NOs. 33-35 and variable light chain sequences containing the CDR sequences of SEQ ID NOs. 41-43; d) Variable heavy chain sequences containing the CDR sequences of SEQ ID NOs. 49-51 and variable light chain sequences containing the CDR sequences of SEQ ID NOs. 57-59; e) Variable heavy chain sequences containing the CDR sequences of SEQ ID NOs. 65-67 and variable light chain sequences containing the CDR sequences of SEQ ID NOs. 73-75; and f) Variable heavy chain sequences containing CDR sequences of SEQ ID NOs. 81-83 and variable light chain sequences containing CDR sequences of SEQ ID NOs. 89-91.

7. The NKCE according to claim 1, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 98, SEQ ID NO: 100, and SEQ ID NO:

102.

8. A composition comprising NKCE as described in any one of claims 1 to 7.

9. The composition according to claim 8, further comprising at least one selected from the group consisting of pharmaceutically acceptable excipients and adjuvants.

10. The composition according to claim 8, further comprising a PD-(L) monoaxial inhibitor.

11. The composition according to claim 8, comprising a delivery vehicle containing the aforementioned NKCE.

12. The composition according to claim 11, wherein the delivery vehicle is lipid nanoparticles.

13. A nucleic acid molecule or combination of nucleic acid molecules comprising one or more nucleotide sequences encoding an NKCE or a fragment thereof as described in any one of claims 1 to 7.

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

15. The nucleic acid molecule or combination according to claim 13, comprising a combination of at least two nucleic acid molecules encoding the aforementioned NKCE.

16. The nucleic acid molecule or combination according to claim 13, comprising a combination of four nucleic acid molecules encoding the aforementioned NKCE.

17. The nucleic acid molecule or combination according to claim 13, comprising a single nucleic acid molecule encoding the aforementioned NKCE.

18. A nucleic acid molecule or combination according to claim 17, comprising a nucleotide sequence encoding an NKCE selected from the group consisting of SEQ ID NO: 98, SEQ ID NO: 100, and SEQ ID NO:

102.

19. The nucleic acid molecule or combination according to claim 18, wherein the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 97, SEQ ID NO: 99, and SEQ ID NO:

101.

20. The nucleic acid molecule or combination according to claim 13, wherein the nucleic acid molecule is selected from the group consisting of RNA molecules and DNA molecules.

21. A composition comprising the nucleic acid molecule or combination described in claim 13.

22. The composition according to claim 21, further comprising a PD-(L) monoaxial inhibitor.

23. The composition according to claim 21, comprising a delivery vehicle containing the nucleic acid molecule or combination thereof.

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

25. The composition according to claim 21, further comprising at least one selected from the group consisting of pharmaceutically acceptable excipients and adjuvants.

26. The composition according to claim 8 for treating or preventing a disease or disorder in a subject requiring it.

27. The composition according to claim 26, wherein the disease or disorder is selected from the group consisting of a disease or disorder related to bacterial infection, a disease or disorder related to viral infection, an autoimmune disease or disorder, cancer, or a disease or disorder related to cancer.

28. The composition according to claim 27, wherein the cancer is selected from the group consisting of ovarian cancer, breast cancer, prostate cancer, kidney cancer, colorectal cancer, gastric cancer, lung cancer, testicular cancer, skin cancer, and endometrial cancer.

29. The composition according to claim 8 for increasing the natural killer cell function of a subject that requires it.

30. The composition according to claim 8 for inducing natural killer cells in target cells or particles of a subject that require it.

31. The composition according to claim 30, wherein the target cells are selected from the group consisting of tumor cells, pathogen cells or particles, bacterial cells, virus-infected cells, and cells that express antigens related to autoimmune diseases or disorders.

32. The composition according to claim 31, wherein the tumor cells are derived from cancer selected from the group consisting of ovarian cancer, breast cancer, prostate cancer, kidney cancer, colorectal cancer, gastric cancer, lung cancer, testicular cancer, skin cancer, and endometrial cancer.

33. The composition according to claim 21 for treating or preventing a disease or disorder in a subject requiring it.

34. The composition according to claim 33, wherein the disease or disorder is selected from the group consisting of a disease or disorder related to bacterial infection, a disease or disorder related to viral infection, an autoimmune disease or disorder, cancer, or a disease or disorder related to cancer.

35. The composition according to claim 34, wherein the cancer is selected from the group consisting of ovarian cancer, breast cancer, prostate cancer, kidney cancer, colorectal cancer, gastric cancer, lung cancer, testicular cancer, and endometrial cancer.

36. The composition according to claim 21 for increasing natural killer cell function in subjects that require it.

37. The composition according to claim 21 for inducing natural killer cells in target cells or particles of a subject that require it.

38. The composition according to claim 37, wherein the target cells are selected from the group consisting of tumor cells, pathogen cells or particles, bacterial cells, virus-infected cells, and cells that express antigens associated with autoimmune diseases or disorders.

39. The composition according to claim 37, wherein the tumor cells are derived from cancer selected from the group consisting of ovarian cancer, breast cancer, prostate cancer, kidney cancer, colorectal cancer, gastric cancer, lung cancer, testicular cancer, skin cancer, and endometrial cancer.

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