Bispecific Molecules and Related Compositions and Methods

Abstract

Aspects of the present disclosure include bispecific molecules. The bispecific molecules include a cell targeting moiety and a glycan binding moiety. According to some embodiments, the cell targeting moiety is a cancer cell targeting moiety or an immune cell targeting moiety. In certain embodiments, the glycan binding moiety includes a sialoglycan binding domain of a lectin, a non-limiting example of which is sialic acid-binding immunoglobulin-like lectin (Siglec). The bispecific molecules may take a variety of forms, including heterodimeric molecules, fusion proteins, conjugates, and the like. Compositions, kits, and methods of using the bifunctional molecules, for example for therapeutic purposes, are also provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 170,297, filed April 2, 2021, which is incorporated herein by reference in its entirety.

[0002] Statement of government support This invention was made with Government support under Contracts CA226051, CA250324, and GM058867 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] Incorporation by reference of sequence listings provided as text files The Sequence Listing is provided herewith in a text file (S21-091_STAN-1838WO_SEQ_LIST_ST25) created on April 1, 2022 and having a file size of 425,000 bytes. The contents of the text file are incorporated herein by reference in their entirety.

[0004] Introduction Despite the remarkable benefits of cancer immunotherapy observed in selected cases, many tumors remain non-responsive to existing treatments. Thus, there is an unmet need for therapies that target additional immune checkpoints that drive cancer progression. Hypersialylation, or upregulation, of sialic acid monosaccharides on cell surfaces is an established hallmark of cancer that is associated with increased invasiveness and metastatic rates. Emerging evidence suggests that hypersialylation enables tumors to engage inhibitory glycan-binding receptors called Siglecs on immune cells. Siglec receptors are expressed by every immune cell class, and the intracellular domains of eight Siglec family members bear homology to the established PD-1 immune checkpoint. These studies establish Siglecs and their sialoglycan ligands as immune checkpoints that contribute to cancer progression.

[0005] The discovery and characterization of the Siglec-sialoglycan immune checkpoint has spurred interest in targeting Siglec receptors for checkpoint blockade. However, the lack of glycan-binding reagents with high affinity and selectivity has thus far prevented targeting of tumor-associated sialoglycan ligands for checkpoint blockade. The weak immunogenicity of mammalian glycan structures has historically hindered the development of anti-glycan antibodies. Even if glycan-binding antibodies were available, the identity of the sialoglycans used by tumors to engage Siglecs is not fully understood, precluding their use as targets. Soluble Siglec-Fc chimeras have been shown to maintain native sialoglycan binding specificity, but their binding affinity is too low to be used as decoy receptor therapeutics. Thus, effective agents are needed to target tumor-associated sialoglycans for checkpoint blockade. Summary of the Invention

[0006] Aspects of the present disclosure include bispecific molecules. The bispecific molecules include a cell targeting moiety and a glycan binding moiety. According to some embodiments, the cell targeting moiety is a cancer cell targeting moiety or an immune cell targeting moiety. In certain embodiments, the glycan binding moiety includes a sialoglycan binding domain of a lectin, a non-limiting example of which is sialic acid-binding immunoglobulin-like lectin (Siglec). The bispecific molecules may take a variety of forms, including heterodimeric molecules, fusion proteins, conjugates, and the like. Compositions, kits, and methods of using the bifunctional molecules, for example for therapeutic purposes, are also provided. [Brief description of the drawings]

[0007] [Figure 1a] FIG. 1 shows schematic and data demonstrating that antibody-lectin (AbLec) bispecific antibodies enable the use of lectin decoy receptors for checkpoint blockade. [Figure 1b]FIG. 1 shows schematic and data demonstrating that antibody-lectin (AbLec) bispecific antibodies enable the use of lectin decoy receptors for checkpoint blockade. [Figure 1c] FIG. 1 shows schematic and data demonstrating that antibody-lectin (AbLec) bispecific antibodies enable the use of lectin decoy receptors for checkpoint blockade. [Figure 1d] FIG. 1 shows schematic and data demonstrating that antibody-lectin (AbLec) bispecific antibodies enable the use of lectin decoy receptors for checkpoint blockade. [Figure 1e] FIG. 1 shows schematic and data demonstrating that antibody-lectin (AbLec) bispecific antibodies enable the use of lectin decoy receptors for checkpoint blockade. [Figure 2a] FIG. 1 shows a schematic and data demonstrating that AbLec blocks binding of targeted glycan-binding immune receptors. [Figure 2b] FIG. 1 shows a schematic and data demonstrating that AbLec blocks binding of targeted glycan-binding immune receptors. [Figure 2c] FIG. 1 shows a schematic and data demonstrating that AbLec blocks binding of targeted glycan-binding immune receptors. [Figure 2d] FIG. 1 shows a schematic and data demonstrating that AbLec blocks binding of targeted glycan-binding immune receptors. [Figure 2e] FIG. 1 shows a schematic and data demonstrating that AbLec blocks binding of targeted glycan-binding immune receptors. [Figure 2f] FIG. 1 shows a schematic and data demonstrating that AbLec blocks binding of targeted glycan-binding immune receptors. [Figure 3a] FIG. 1 shows schematics and data demonstrating that AbLec enhances antibody-dependent cellular phagocytosis and cytotoxicity in vitro. [Figure 3b] FIG. 1 shows schematics and data demonstrating that AbLec enhances antibody-dependent cellular phagocytosis and cytotoxicity in vitro. [Figure 3c]FIG. 1 shows schematics and data demonstrating that AbLec enhances antibody-dependent cellular phagocytosis and cytotoxicity in vitro. [Figure 3d] FIG. 1 shows schematics and data demonstrating that AbLec enhances antibody-dependent cellular phagocytosis and cytotoxicity in vitro. [Figure 3e] FIG. 1 shows schematics and data demonstrating that AbLec enhances antibody-dependent cellular phagocytosis and cytotoxicity in vitro. [Figure 4] Data demonstrating that AbLec enhancement of in vitro ADCP is dependent on expression of the target antigen (in this example, HER2, targeted by the trastuzumab arm). [Diagram 5] Data demonstrate that AbLec binds to human tumor cell lines and blocks Siglec receptor binding. [Figure 6a] FIG. 1 shows schematic and data demonstrating that AbLec is superior to combination immunotherapy through Siglec-dependent enhancement of anti-tumor immune responses in vitro. [Figure 6b] FIG. 1 shows schematic and data demonstrating that AbLec is superior to combination immunotherapy through Siglec-dependent enhancement of anti-tumor immune responses in vitro. [Figure 6c] FIG. 1 shows schematic and data demonstrating that AbLec is superior to combination immunotherapy through Siglec-dependent enhancement of anti-tumor immune responses in vitro. [Figure 6d] FIG. 1 shows schematic and data demonstrating that AbLec is superior to combination immunotherapy through Siglec-dependent enhancement of anti-tumor immune responses in vitro. [Figure 7a] FIG. 1 shows schematics and data demonstrating that the AbLec platform enables blockade of diverse glyco-immune checkpoint targets. [Figure 7b] FIG. 1 shows schematics and data demonstrating that the AbLec platform enables blockade of diverse glyco-immune checkpoint targets. [Figure 7c]FIG. 1 shows schematics and data demonstrating that the AbLec platform enables blockade of diverse glyco-immune checkpoint targets. [Figure 7d] FIG. 1 shows schematics and data demonstrating that the AbLec platform enables blockade of diverse glyco-immune checkpoint targets. [Figure 8] Data demonstrating that R7 AbLec enhances ADCC of CD20+Raji cells compared to rituximab. [Figure 9-1] FIG. 1 shows a schematic and data demonstrating the expression of diverse AbLec molecules. [Figure 9-2] FIG. 1 shows a schematic and data demonstrating the expression of diverse AbLec molecules. [Figure 9-3] FIG. 1 shows a schematic and data demonstrating the expression of diverse AbLec molecules. [Figure 9-4] FIG. 1 shows a schematic and data demonstrating the expression of diverse AbLec molecules. [Figure 9-5] FIG. 1 shows a schematic and data demonstrating the expression of diverse AbLec molecules. [Figure 10] FIG. 1 is a schematic diagram of AbLec as a modular agent for targeting the glycan immune checkpoint. [Figure 11] 1 is the amino acid sequence of an exemplary Rituximab-Siglec-7 AbLec heterodimer ("Ritux-Sig7 AbLec") containing a knobs-into-holes modified CH3 domain to promote heterodimer formation, as confirmed by mass spectrometry. [Figure 12] 1 is the amino acid sequence of an exemplary Rituximab-Siglec-9 AbLec heterodimer ("Ritux-Sig9 AbLec") containing a knobs-into-holes modified CH3 domain to promote heterodimer formation, as confirmed by mass spectrometry. [Figure 13] 1 is the amino acid sequence of an exemplary trastuzumab-Siglec-9 AbLec heterodimer ("Tras-Sig9 AbLec") containing a knobs-into-holes modified CH3 domain to promote heterodimer formation, as confirmed by mass spectrometry. [Figure 14] 1 is the amino acid sequence of an exemplary trastuzumab-Siglec-7 AbLec heterodimer ("Tras-Sig7 AbLec") containing a knobs-into-holes modified CH3 domain to promote heterodimer formation, as confirmed by mass spectrometry. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] Before the bispecific molecules, compositions, and methods of the present disclosure are described in more detail, it is to be understood that the bispecific molecules, compositions, and methods are not limited to the particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the bispecific molecules, compositions, and methods will be limited only by the appended claims.

[0009] When a range of values ​​is provided, it is understood that each intervening value between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the bispecific molecules, compositions, and methods to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the bispecific molecules, compositions, and methods, subject to any specifically excluded limits in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the bispecific molecules, compositions, and methods.

[0010] Certain ranges are presented herein with numerical values ​​preceded by the term "about." The term "about" is used herein to provide literal support for the exact number it precedes, as well as a number that is near or approximately the number it precedes. In determining whether a number is near or approximately a specifically recited number, the near or approximately unrecited number may be a number that, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0011] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the bispecific molecules, compositions and methods belong. Although any bispecific molecules, compositions and methods similar or equivalent to those described herein can also be used in the practice or testing of the bispecific molecules, compositions and methods, representative illustrative bispecific molecules, compositions and methods are described herein.

[0012] All publications and patents cited herein are incorporated by reference herein to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference herein to disclose and describe the materials and / or methods for which the publications are cited. The citation of any publication is for its disclosure prior to the filing date, and should not be construed as an admission that the bispecific molecules, compositions, and methods of the invention are not entitled to antedate such publication, since the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0013] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude any element. Thus, this statement is intended to serve as a predicate for the use of exclusive terminology, such as "solely," "only," or the use of a "negative" limitation in connection with the recitation of claim elements.

[0014] It is also understood that, for clarity, certain features of the bispecific molecules, compositions, and methods described in the context of separate embodiments may be provided in combination in a single embodiment. Conversely, for brevity, various features of the bispecific molecules, compositions, and methods described in the context of a single embodiment may also be provided separately or in any suitable subcombination. All combinations of the embodiments are specifically encompassed by the present disclosure and, to the extent such combinations encompass processes and / or compositions capable of carrying out the same, are disclosed herein as if each and every combination were individually and expressly disclosed. In addition, all subcombinations recited in the embodiments describing such variables are also specifically encompassed by the bispecific molecules, compositions, and methods of the present invention and are disclosed herein as if each and every such subcombination were individually and expressly disclosed herein.

[0015] As will be apparent to one of ordinary skill in the art after reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the method. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0016] bispecific molecules The present disclosure provides bispecific molecules. The bispecific molecules include a cell targeting moiety (e.g., a cancer cell targeting moiety or an immune cell targeting moiety) and a glycan binding moiety. In certain embodiments, the glycan binding moiety includes a sialoglycan binding domain of a lectin, a non-limiting example of which is sialic acid-binding immunoglobulin-like lectin (Siglec). The bispecific molecules may take a variety of forms, including heterodimeric molecules, fusion proteins, conjugates, and the like. As demonstrated herein, the bispecific molecules of the present disclosure that include a cancer cell targeting moiety and a glycan binding moiety are effective in enhancing anti-tumor immune responses, for example, by enhancing antibody-dependent cellular phagocytosis (ADCP) and / or cytotoxicity (ADCC). Furthermore, the enhancement requires a bispecific format, and the results demonstrate therapeutic synergy resulting from combining a tumor cell targeting arm and a glycan binding arm in a single bispecific molecule. As used herein, "synergy" or "synergistic effect," in reference to the effect produced by two or more individual components, refers to the phenomenon whereby the total effect produced by those components, when utilized in combination (where present in a single bispecific molecule), is greater than the sum of the individual effects of each component acting alone. Further details regarding bispecific molecules according to embodiments of the present disclosure are now provided.

[0017] Cell targeting moiety A variety of cell targeting moieties may be used in the bispecific molecules of the present disclosure. In certain embodiments, the cell targeting moiety is a cancer cell targeting moiety. By "cancer cell" is meant a cell that exhibits a neoplastic cell phenotype that may be characterized by one or more of the following exemplary properties: abnormal cell growth, abnormal cell proliferation, loss of density-dependent growth inhibition, anchorage-independent growth capacity, ability to promote tumor growth and / or development in immunocompromised non-human animal models, and / or any suitable indicator of cell transformation. "Cancer cell" may be used interchangeably herein with "tumor cell," "malignant cell," or "cancerous cell," and includes cancer cells of solid tumors, semi-solid tumors, hematological malignancies (e.g., leukemia cells, lymphoma cells, myeloma cells, etc.), primary tumors, metastatic tumors, and the like.

[0018] In certain embodiments, when the cell targeting moiety is a cancer cell targeting moiety, the cancer cell targeting moiety specifically binds to a molecule (eg, a protein) expressed on the surface of a cancer cell.Non-limiting examples of cancer cell surface molecules to which a cancer cell targeting moiety may specifically bind include 5T4, AXL receptor tyrosine kinase (AXL), B cell maturation antigen (BCMA), c-MET, C4.4a, carbonic anhydrase 6 (CA6), carbonic anhydrase 9 (CA9), cadherin-6, CD19, CD20, CD22, CD25, CD27L, CD30, CD33, CD37, CD44, CD44v6, CD56, CD70, CD74, CD79b, CD123, CD138, carcinoembryonic antigen (CEA), cKit, Criptotan. Protein, CS1, delta-like canonical notch ligand 3 (DLL3), endothelin receptor type B (EDNRB), ephrin A4 (EFNA4), epidermal growth factor receptor (EGFR), EGFRvIII, ectonucleotide pyrophosphatase / phosphodiesterase 3 (ENPP3), EPH receptor A2 (EPHA2), fibroblast growth factor receptor 2 (FGFR2), fibroblast growth factor receptor 3 (FGFR3), FMS-like tyrosine kinase 3 (FLT3), folate receptor 1 (FOLR1), GD2 ganglioside, glycoprotein nontransferase Translocation B (GPNMB), guanylate cyclase 2C (GUCY2C), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), integrin alpha, lysosome-associated membrane protein 1 (LAMP-1), Lewis Y, LIV-1, leucine-rich repeat containing 15 (LRRC15), mesothelin (MSLN), mucin 1 (MUC1), mucin 16 (MUC16), sodium-dependent phosphate transporter 2B (NaPi2b), nectin-4, NMB, NOTCH3, p-cadherin (p-CAD), program These include death receptor ligand 1 (PD-L1), programmed death receptor ligand 2 (PD-L2), prostate-specific membrane antigen (PSMA), protein tyrosine kinase 7 (PTK7), solute carrier family 44 member 4 (SLC44A4), SLIT-like family member 6 (SLITRK6), STEAP family member 1 (STEAP1), tissue factor (TF), T-cell immunoglobulin and mucin protein-1 (TIM-1), Tn antigen, trophoblast surface antigen (TROP-2), and Wilms' tumor 1 (WT1).

[0019] According to some embodiments, the cell targeting moiety is an immune cell targeting moiety. In certain embodiments, when the cell targeting moiety is an immune cell targeting moiety, the immune cell targeting moiety specifically binds to a molecule (e.g., a protein) expressed on the surface of an immune cell. The immune cell targeting moiety may be selected to target any desired immune cell, non-limiting examples of which include T cells, B cells, natural killer (NK) cells, macrophages, monocytes, neutrophils, dendritic cells, mast cells, basophils, and eosinophils. In some embodiments, the immune cell is a T cell. Exemplary T cell types include naive T cells (T N ), cytotoxic T cells (T CTL ), memory T cells (T MEM ), T memory stem cells (T SCM ), central memory T cells (T CM ), effector memory T cells (T EM ), tissue-resident memory T cells (T RM ), effector T cells (T EFF ), regulatory T cells (T REG ), helper T cells (T H , T H 1. T H 2. T H 17) CD4+ T cells, CD8+ T cells, virus-specific T cells, alpha-beta T cells (T αβ ), and gamma delta T cells (T γδ ) are mentioned.

[0020] Non-limiting examples of immune cell surface molecules to which an immune cell targeting moiety may specifically bind include PD-1, PD-L1, PD-L2, CLTA-4, VISTA, LAG-3, TIM-3, CD24, CD47, SIRP alpha, CD3, CD8, CD4, CD28, CD80, CD86, CD19, ICOS, OX40, OX40L, GD3 ganglioside, TIGIT, Siglec-2, Siglec-3, Siglec-7, Siglec-8. , Siglec-9, Siglec-10, Siglec-15, Galectin-9, B7-H3, B7-H4, CD40, CD40L, B7RP1, CD70, CD27, BTLA, HVEM, KIR, 4-1BB, 4-1BBL, CD226, CD155, CD112, GITR, GITRL, A2aR, CD137, CD137L, CD45, CD206, CD163, TRAIL, NKG2D, CD16, and TGF-beta.

[0021] According to some embodiments, the cell targeting moiety comprises a small molecule that binds to a cell surface molecule on the target cell. By "small molecule" is meant a compound having a molecular weight of 1000 atomic mass units (amu) or less. In certain embodiments, the small molecule is 750 amu or less, 500 amu or less, 400 amu or less, 300 amu or less, or 200 amu or less. In some embodiments, the small molecule is not made up of repeating molecular units such as those present in polymers. In certain embodiments, the target cell surface molecule is a receptor whose ligand is a small molecule, and the small molecule of the cell targeting moiety is a small molecule ligand (or a derivative thereof) of the receptor. Small molecules are known for use in targeting conjugates to target cells of interest. By way of example only, folic acid (FA) derivatives have been shown to effectively target certain cancer cells by binding to folate receptors, which are overexpressed in many epithelial tumors, for example. See, e.g., Vergote et al. (2015) Ther. Adv. Med. Oncol. 7(4):206-218. In another example, the small molecule sigma-2 has been proven effective in targeting cancer cells. See, for example, Hashim et al. (2014) Molecular Oncology 8(5):956-967. Sigma-2 is a small molecule ligand of the sigma-2 receptor, and is overexpressed in many proliferating tumor cells, including pancreatic cancer cells. In certain embodiments, the cell targeting portion of the bispecific molecule of the present disclosure comprises a small molecule, which has been demonstrated in the context of small molecule drug conjugates (SMDCs) to be effective in targeting the conjugate to a target cell of interest by binding to a cell surface molecule on the target cell.

[0022] According to certain embodiments, the cell targeting moiety comprises a ligand. As used herein, a "ligand" is a substance that forms a complex with a biomolecule to achieve a biological purpose. The ligand may be a substance selected from circulating factors, secreted factors, cytokines, growth factors, hormones, peptides, polypeptides, small molecules, and nucleic acids that form a complex with a cell surface molecule on the surface of the target cell. In certain embodiments, when the cell targeting moiety comprises a ligand, the ligand is modified so that a complex with the cell surface molecule occurs but the normal biological consequences of such complex formation do not occur. In certain aspects, the ligand is a ligand of a cell surface receptor present on the target cell.

[0023] In certain embodiments, the cell targeting moiety comprises an aptamer. "Aptamer" refers to a nucleic acid (e.g., an oligonucleotide) that has a specific binding affinity for a target cell surface molecule. Aptamers exhibit certain desirable properties for targeted delivery of bispecific molecules, such as ease of selection and synthesis, high binding affinity and specificity, low immunogenicity, and universal synthetic accessibility. Aptamers that bind to cell surface molecules are known, including, for example, TTA1 (a tumor-targeting aptamer for the extracellular matrix protein tenascin-C). Aptamers that find use in the bispecific molecules of the present disclosure include those described in Zhu et al. (2015) ChemMedChem 10(1):39-45; Sun et al. (2014) Mol. Ther. Nucleic Acids 3:e182; and Zhang et al. (2011) Curr. Med. Chem. 18(27):4185-4194.

[0024] According to some embodiments, the cell targeting moiety comprises a nanoparticle. As used herein, a "nanoparticle" is a particle having at least one dimension within the range of 1 nm to 1000 nm, 20 nm to 750 nm, 50 nm to 500 nm (including 100 nm to 300 nm, e.g., 120 to 200 nm). The nanoparticle may have any suitable shape, including, but not limited to, a sphere, a spheroid, a rod shape, a disk shape, a cone shape, a cube shape, a cylinder shape, a nanospiral shape, a nanospring shape, a nanoring shape, an arrow shape, a teardrop shape, a tetrapod shape, a prism shape, or any other suitable geometric or non-geometric shape. In certain embodiments, the nanoparticle comprises on its surface one or more of the other targeting moieties described herein, such as, for example, an antibody, a ligand, an aptamer, a small molecule, etc. Nanoparticles that find use with the bispecific molecules of the present disclosure include those described in Wang et al. (2010) Pharmacol. Res. 62(2):90-99; Rao et al. (2015) ACS Nano 9(6):5725-5740; and Byrne et al. (2008) Adv. Drug Deliv. Rev. 60(15):1615-1626.

[0025] In some embodiments, the cell targeting moiety specifically binds to a receptor expressed on the surface of a target cell. Such a cell targeting moiety may include, for example, an antigen binding domain of an antibody that specifically binds to the receptor, or a ligand for the receptor. Non-limiting examples of such cell surface receptors include stem cell receptors, immune cell receptors (e.g., T cell receptors, B cell receptors, etc.), growth factor receptors, cytokine receptors, hormone receptors, receptor tyrosine kinases, CD28, CD80, ICOS, CTLA4, PD1, PD-L1, BTLA, HVEM, CD27, 4-1BB, 4-1BBL, OX40, OX40L, DR3, GITR, CD30, SLAM, CD2, 2B4, TIM1, TIM2, TIM3, TIGIT, CD226 immune receptors such as CD160, LAG3, LAIR1, B7-1, B7-H1, and B7-H3; type I cytokine receptors such as interleukin-1 receptor, interleukin-2 receptor, interleukin-3 receptor, interleukin-4 receptor, interleukin-5 receptor, interleukin-6 receptor, interleukin-7 receptor, interleukin-9 receptor, interleukin-11 receptor, interleukin-12 receptor, interleukin-13 receptor, interleukin-15 receptor, interleukin-18 receptor, interleukin-21 receptor, interleukin-23 receptor, interleukin-27 receptor, erythropoietin receptor, GM-CSF receptor, G-CSF receptor, growth hormone receptor, prolactin receptor, leptin receptor, oncostatin M receptor, and leukemia inhibitory factor; interferon alpha / beta receptor, interferon gamma receptor, interferon type III receptor, interleukin-10 receptor, interferon Type II cytokine receptors such as interleukin-20 receptor, interleukin-22 receptor, and interleukin-28 receptor, tumor necrosis factor receptor 2(1B), tumor necrosis factor receptor 1, lymphotoxin beta receptor, OX40, CD40, Fas receptor, decoy receptor 3, CD27, CD30, 4-1BB, decoy receptor 2, decoy receptor 1, death receptor 5, death receptor 4, RANK, osteoprotegerin, TWEAK receptor, TACI, BAFF receptor, herpes virus entry mediator,Receptors of the tumor necrosis factor receptor superfamily, such as nerve growth factor receptor, B-cell maturation antigen, glucocorticoid-induced TNFR-related, TROY, death receptor 6, death receptor 3, and ectodysplasin A2 receptor; chemokine receptors, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, XCR1, ACKR1, ACKR2, ACKR3, ACKR4, and CCRL2; epidermal growth factor receptor (EGFR) family These include receptors of the fibroblast growth factor receptor (FGFR) family, receptors of the vascular endothelial growth factor receptor (VEGFR) family, receptors of the rearranged during transfection (RET) receptor family, receptors of the Eph receptor family, receptors capable of inducing cell differentiation (e.g., Notch receptors), adhesion receptors such as cell adhesion molecules (CAMs), integrin receptors, cadherins, selectins, and receptors of the discoidin domain receptor (DDR) family, transforming growth factor beta receptor 1, and transforming growth factor beta receptor 2. In some embodiments, such receptors are immune cell receptors selected from T cell receptors, B cell receptors, natural killer (NK) cell receptors, macrophage receptors, monocyte receptors, neutrophil receptors, dendritic cell receptors, mast cell receptors, basophil receptors, and eosinophil receptors.

[0026] In certain embodiments, the cell targeting moiety comprises an antigen-binding domain of an antibody. The term "antibody" may include antibodies or immunoglobulins of any isotype (e.g., IgG (e.g., IgG1, IgG2, IgG3, or IgG4), IgE, IgD, IgA, IgM, etc.), whole antibodies (e.g., antibodies composed of a tetramer, which in turn is composed of two dimers of a heavy chain polypeptide and a light chain polypeptide); fragments of antibodies (e.g., whole antibodies or single chain antibody fragments) that retain specific binding to a cell surface molecule of a target cell, including, but not limited to, single chain antibodies (e.g., scFv), single chain Fv (scFv), Fab, (Fab')2, (scFv')2, and bispecific antibodies, chimeric antibodies, monoclonal antibodies, human antibodies, humanized antibodies (e.g., humanized whole antibodies, humanized half antibodies, or humanized antibody fragments, e.g., humanized scFv), and fusion proteins comprising the antigen-binding portion of an antibody and a non-antibody protein. In certain embodiments, the antibody is selected from an IgG, Fv, single chain antibody, scFv, Fab, F(ab')2, or Fab'. The antibody may be detectably labeled, for example, with an in vivo imaging agent, a radioisotope, an enzyme that generates a detectable product, a fluorescent protein, etc. The antibody may be further conjugated to another moiety, such as a member of a specific binding pair, for example, biotin (a member of the biotin-avidin specific binding pair).

[0027] In some embodiments, where the cell targeting moiety comprises an antigen-binding domain of an antibody, the antigen-binding domain is of an antibody approved by the U.S. Food and Drug Administration and / or the European Medicines Agency (EMA) for use as a therapeutic antibody, e.g., to induce antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), etc. of specific disease-associated cells in a patient. Non-limiting examples of antigen-binding domains that may be used in the bispecific molecules of the present disclosure include adecatumumab, asclinbacumab, cixutumumab, conatumumab, daratumumab, drozitumab, durigotumab, durvalumab, dusigitumab, enfortumab, enoticumab, fizitumab, ganitumab, glembatumab, intetumumab, ipilimumab, iratumumab, icrucumab, lexatumumab, lucatumumab, mapatumumab, narunatumab, natumab ... tumumab, necitumumab, nesbacumab, ofatumumab, olaratumumab, panitumumab, patritumumab, pritumumab, radletumab, ramucirumab, rilotumumab, lobatumumab, seribantumab, talextumab, teprotumumab, tobetumab, vantictumab, besencumab, votumumab, zalutumumab, framvotumab, altumomab, anatumomab, arcitumomab, bectumomab, blinatumomab, detumomab, ibritumomab, minletumomab , mitumomab, moxetumomab, naptumomab, nofetumomab, pemtumomab, pintumomab, racotumomab, satumomab, solitomab, taplitumomab, tenatumomab, tositumomab, tremelimumab, abagovomab, igovomab, oregovomab, capromab, edrecolomab, nacolomab, amatuximab, bavituximab, brentuximab, cetuximab, dellotuximab, dinutuximab, ensituximab, futuximab, girentuximab , indatuximab, isatuximab, margetuximab, rituximab, siltuximab, ublituximab, ecromeximab, avituzumab, alemtuzumab, bevacizumab, bivatuzumab, brontixtuzumab, cantuzumab, cantuzumab, sitatuzumab, clivatuzumab, dacetuzumab, demcizumab, dalotuzumab, denintuzumab, elotuzumab, emactuzumab, emibetuzumab, enoblitzumab, etaracizumab, farletuzumab,Ficlatuzumab, gemtuzumab, imgatuzumab, inotuzumab, labetuzumab, rifastuzumab, lintuzumab, lorvotuzumab, lumuletuzumab, matuzumab, milatuzumab, nimotuzumab, obinutuzumab, ocaratuzumab, otlertuzumab, onartuzumab, opoltuzumab, palsatuzumab, pertuzumab, pinatuzumab, polatuzumab, sibrotuzumab, simtuzumab, tacituzumab and / or derived from an antibody selected from: tigatuzumab, trastuzumab, tucotuzumab, bundletuzumab, vanucizumab, veltuzumab, borsetuzumab, sofituzumab, catumaxomab, ertumaxomab, depatuxizumab, ontuxizumab, brontuzumab, tamtubetomab, or an antigen-binding variant thereof, such as a single chain version (e.g., scFv version).

[0028] In certain embodiments, the cell targeting moiety comprises an antibody heavy chain comprising a gamma, alpha, delta, epsilon, or mu antibody heavy chain or a fragment thereof. According to some embodiments, the antibody heavy chain or a fragment thereof is an IgG heavy chain or a fragment thereof, e.g., a human IgG1 heavy chain or a fragment thereof. In certain embodiments, the antibody heavy chain or a fragment thereof comprises a heavy chain variable region (V H Such an antibody heavy chain or fragment thereof may further comprise a heavy chain constant region or a fragment thereof. For example, when a heavy chain constant region or a fragment thereof is included in the cell targeting moiety, the heavy chain constant region of the antibody or a fragment thereof may comprise C H 1 domain, C H 2 domain, and / or C H According to some embodiments, the cell targeting moiety may comprise one or more of the V H、 C H 1 domain, C H 2 domain, and C H It is a full-length antibody heavy chain, which is an antibody heavy chain that comprises three domains.

[0029] In certain embodiments, the cell targeting moiety comprises an antibody light chain or a fragment thereof. According to some embodiments, the antibody light chain or a fragment thereof comprises a kappa (κ) light chain or a fragment thereof, or a lambda (λ) light chain or a fragment thereof. According to some embodiments, the antibody light chain or a fragment thereof comprises a light chain variable region (V L Such an antibody light chain or fragment thereof comprises an antibody light chain constant region (C L ) or a fragment thereof. In certain embodiments, the cell targeting moiety may further comprise a full-length antibody light chain, i.e., V L and C L is an antibody light chain comprising:

[0030] According to some embodiments, the cell targeting moiety comprises a variable heavy chain (V H ) domains of the antibody heavy and variable light chains (V L In certain embodiments, the cell targeting moiety comprises an antibody light chain comprising a C H 1 domain, hinge region, C H 2 domain, C H 3 domains, or any combination thereof. For example, the cell targeting moiety may comprise a C H 2 domain, C H The antibody may comprise an antibody heavy chain comprising one or more of the three domains, or both. Examples of such cell targeting moieties include those that comprise the Fragment Crystallizable (Fc) region.

[0031] The cell targeting moiety and / or glycan binding moiety of the bispecific molecules of the present disclosure may specifically bind to their respective targets. As used herein, a cell targeting moiety or glycan binding moiety "specifically binds" or "preferentially binds" to a target if it binds, e.g., with higher affinity, avidity, more readily, and / or for a longer period of time, than it binds to other substances in a sample and / or in vivo. In certain embodiments, a cell targeting moiety or glycan binding moiety may bind, e.g., with a ... 4 M -1An antibody "specifically binds" to a target if it binds or associates with the target with an affinity or Ka (i.e., the association rate constant of a particular binding interaction with units of 1 / M) that is equal to or greater than the equilibrium dissociation constant (KD) of a particular binding interaction with units of M (e.g., 10 -5 M~10 -13 In certain embodiments, specific binding may be defined as a binding ratio of the cell targeting moiety or the glycan binding moiety that is greater than or equal to about 10 -5 M or less, about 10 -6 M or less, about 10 -7 M or less, about 10 -8 M or less, or about 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 It means that the target is bound with a KD of equal to or less than M. The binding affinity of the cell targeting moiety or glycan binding moiety to the target can be readily determined using conventional techniques, such as by competitive ELISA (enzyme-linked immunosorbent assay), equilibrium dialysis, by using surface plasmon resonance (SPR) technology (e.g., BIAcore 2000 or BIAcore T200 instrument using the general procedures outlined by the manufacturer); by radioimmunoassay, etc.

[0032] Glycan-binding moiety A variety of glycan-binding moieties may be used in the bispecific molecules of the present disclosure. In certain embodiments, the glycan-binding moiety comprises a glycan-binding domain of a lectin. For example, the glycan-binding moiety may comprise a sialoglycan-binding domain of a sialoglycan-binding lectin. Non-limiting examples of sialoglycan-binding moieties include those that comprise the sialoglycan-binding domain of a sialic acid-binding immunoglobulin-like lectin (Siglec).

[0033] Siglecs are a family of immunomodulatory receptors whose function is regulated by glycan ligands. The Siglec family consists of 15 family members in humans that are expressed on a limited set of cells in the hematopoietic lineage, with known exceptions including Siglec-4 (MAG) on oligodendrocytes and Schwann cells and Siglec-6 on placental trophoblasts. Siglecs recognize sialic acid-containing glycan ligands on glycoproteins and glycolipids with unique but overlapping specificities via their outermost N-terminal V-set domains. Recognition of their ligands can affect cell signaling through immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in their cytoplasmic tails. In the majority of Siglecs, these ITIMs have the ability to recruit phosphatases, and therefore these members are referred to as inhibitory Siglecs. Exceptions include Siglec-1 and MAG, which lack such motifs, and activated Siglecs (Siglec-14 to -16), which associate with adaptor proteins that have immunoreceptor tyrosine-based activation motifs (ITAMs) via positively charged amino acids in their transmembrane domains.

[0034] Siglecs can be divided into two groups based on their genetic homology among mammalian species. The first group is present in all mammals and consists of Siglec-1 (sialoadhesin), Siglec-2 (CD22), Siglec-4, and Siglec-15. The second group consists of CD33-associated Siglecs, including Siglec-3 (CD33), Siglec-5, Siglec-6, Siglec-7, Siglec-8, Siglec-9, Siglec-10, Siglec-11, Siglec-14, and Siglec-16. Monocytes, monocyte-derived macrophages, and monocyte-derived dendritic cells have almost the same Siglec profile, i.e., high expression of Siglec-3, Siglec-7, and Siglec-9, low expression of Siglec-10, and expression of Siglec-1 upon stimulation with IFN-α. In contrast, macrophages have expression of mainly Siglec-1, Siglec-3, Siglec-8, Siglec-9, Siglec-11, Siglec-15, and Siglec-16, depending on their differentiation state. Conventional dendritic cells express Siglec-3, Siglec-7, and Siglec-9, similar to monocyte-derived dendritic cells, but in addition, they also express low levels of Siglec-2 and Siglec-15. Plasmacytoid dendritic cells express Siglec-1 and Siglec-5. Downregulation of Siglec-7 and Siglec-9 expression in monocyte-derived dendritic cells is observed after 48 h of stimulation with LPS, whereas in monocyte-derived macrophages, Siglec expression is unchanged upon stimulation with LPS. Siglecs are also present in other immune cells such as B cells, basophils, neutrophils, and NK cells.Further details regarding Siglecs can be found, for example, in Angata et al. (2015) Trends Pharmacol Sci. 36(10):645-660; Lubbers et al. (2018) Front. Immunol. 9:2807; Bochner et al. (2016) J Allergy Clin Immunol. 135(3):598-608; and Duan et al. (2020) Annu. Rev. Immunol. 38(1):365-395; the disclosures of which are incorporated by reference in their entireties for all purposes.

[0035] The glycan-binding moiety may comprise a sialoglycan-binding domain of any of the 15 human Siglec family members. In certain embodiments, the glycan-binding moiety comprises a sialoglycan-binding domain of a CD33-associated Siglec. According to some embodiments, the CD33-associated Siglec is Siglec-7 (UniProtKB-Q9Y286). In some embodiments, the glycan-binding moiety binds to a Siglec-7 ligand, and the glycan-binding moiety comprises an amino acid sequence set forth in SEQ ID NO: 15, or a Siglec-7 ligand-binding variant thereof comprising 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 15, or a fragment thereof that retains the ability to bind to a Siglec-7 ligand. In certain embodiments, the CD33-associated Siglec is Siglec-9 (UniProtKB-Q9Y336). In some embodiments, the glycan binding moiety binds to a Siglec-9 ligand, and the glycan binding moiety comprises an amino acid sequence set forth in SEQ ID NO:21, or a Siglec-9 ligand binding variant thereof comprising 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:21, or a fragment thereof that retains the ability to bind to a Siglec-9 ligand. According to some embodiments, the CD33-associated Siglec is Siglec-10 (UniProtKB-Q96LC7). In certain embodiments, the glycan binding moiety comprises the sialoglycan binding domain of Siglec-15 (UniProtKB-Q6ZMC9). According to some embodiments, the glycan binding moiety comprises the sialoglycan binding domain of a Siglec-like adhesin. See, e.g., Deng et al. (2014) PLoS Pathog 10(12):e1004540, and Bensing et al. (2018) Glycobiology 28(8):601-611, the disclosures of which are incorporated by reference in their entireties for all purposes.

[0036] In certain embodiments, the glycan-binding moiety comprises a glycan-binding domain of a C-type lectin. C-type lectins are a superfamily of proteins defined by the presence of at least one C-type lectin-like domain (CTLD), which recognize a broad repertoire of ligands and regulate a diverse range of physiological functions. Although most research attention has focused on the ability of C-type lectins to function in innate and adaptive antibacterial immune responses, it is increasingly recognized that these proteins have a major role in autoimmune diseases and contribute to many other aspects of multicellular existence. The term C-type lectin refers to proteins that are involved in the regulation of Ca 2+ Dependent carbohydrate-binding lectins and Ca 2+C-type lectins were introduced to distinguish them from CLR-independent carbohydrate-binding lectins. C-type lectins share at least one carbohydrate recognition domain, a compact structural module that contains conserved residue motifs and determines the carbohydrate specificity of CLRs. Of particular interest for their role in coupling both innate and adaptive immunity are the DECTIN-1 and DECTIN-2 family of genes, which are localized in the telomeric region of the natural killer cluster of genes. These two groups of C-type lectins are expressed primarily by cells of the myeloid lineage, such as monocytes, macrophages, dendritic cells (DCs), and neutrophils. C-type lectins not only function as antigen uptake receptors for internalization and presentation to T cells, but also trigger multiple signaling pathways that result in NF-κB, type I interferon (IFN), and / or inflammasome activation. This, in turn, leads to the production of pro- or anti-inflammatory cytokines and chemokines, which then fine-tune the adaptive immune response. Further details regarding C-type lectins can be found, for example, in Zelensky et al. (2005) FEBS J. 272:6179-6217; Geijtenbeek & Grinhuis (2009) Nature Reviews Immunology 9:465-479; Brown et al. (2018) Nature Reviews Immunology 18:374-389; Dambuza & Brown (2015) Curr. Opin. Immunol. 32:21-7; and Chiffoleau (2018) Front. Immunol. 9:227; the disclosures of which are incorporated by reference in their entireties for all purposes.According to some embodiments, the glycan-binding moiety comprises a glycan-binding domain of a C-type lectin selected from DECTIN-1, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), C-type lectin-like receptor-1 (CLEC-1), C-type lectin-like receptor 2 (CLEC-2), myeloid inhibitory C-type lectin-like receptor (MICL), CLEC9A, DC immunoreceptor (DCIR), DECTIN-2, blood DC antigen-2 (BDCA-2), macrophage-inducible C-type lectin (MINCLE), macrophage galactose lectin (MGL), and asialoglycoprotein receptor (ASGPR).

[0037] In certain embodiments, the glycan-binding moiety comprises a glycan-binding domain of a selectin. Selectins are C-type transmembrane lectins that mediate leukocyte trafficking and specific adhesive interactions between leukocytes, platelets, and endothelial cells and tumor cells. These lectins include SLe selectin, which is present on endothelial cells (E-selectin), leukocytes (L-selectin), and platelets (P-selectin) and is abundantly expressed in some tumor types. X and SLe AThey preferentially bind to glycans containing glycoepitopes. In the TME, selectins are functionally relevant in relation to leukocyte recruitment, tumor-promoting inflammation, and acquisition of metastatic potential. P-selectin (CD62P) mediates the interaction between activated platelets and cancer cells that contributes to tumorigenesis, and is therefore involved in tumor growth and metastasis. E-selectin (CD62E) also plays a major role in cancer cell adhesiveness at different events of the metastatic cascade, promoting tumor cell extravasation. Finally, L-selectin (CD62L), constitutively expressed on leukocytes, promotes cell adhesion and hematogenous metastasis by regulating tumor-leukocyte interactions and favoring embolization. Further details regarding selectins can be found, for example, in Cagnoni et al. (2016) Front Oncol. 6:109; Barthel et al. (2007) Expert Opin Ther Targets 11(11):1473-91; and Chen & Geng (2006) Arch Immunol Ther Exp 54(2):75-84; the disclosures of which are incorporated herein by reference in their entirety for all purposes. According to some embodiments, the glycan-binding moiety comprises a glycan-binding domain of a selectin selected from P-selectin (CD62P), E-selectin (CD62E), and L-selectin (CD62L).

[0038] According to some embodiments, the glycan-binding moiety comprises a glycan-binding domain of a galectin. Galectins are a family of highly conserved glycan-binding soluble lectins defined by a conserved carbohydrate recognition domain (CRD) and a common structural fold. Vasta GR (2012) Adv Exp Med Biol 946:21-36. Based on structural features, mammalian galectins have been classified into three types: prototype galectins (Gal-1, Gal-2, Gal-5, Gal-7, Gal-10, Gal-11, Gal-13, Gal-14, and Gal-15, which contain one CRD and exist as monomers or dimerize through non-covalent interactions), tandem repeat galectins (Gal-4, Gal-6, Gal-8, Gal-9, and Gal-12), which exist as bivalent galectins containing two different CRDs connected by a linker peptide, and finally, Gal-3, the only chimeric member of the galectin family. Galectins regulate different events in tumorigenesis and metastasis. Galectins contribute to immune tolerance and escape through the regulation of effector T cell apoptosis, clonal expansion, regulatory T cell (Treg) function, and cytokine secretion. The expression levels of several galectins are also altered during malignant transformation, confirming their role in cancer progression. Gal-1, abundantly secreted by almost all malignant tumor cells, has been characterized as a major promoter of an immunosuppressive pro-tumorigenic microenvironment. Another member of this family, Gal-3, has shown prominent pro-tumorigenic effects in numerous tumors. Similar to Gal-1, Gal-3 signaling contributes to tipping the balance towards an immunosuppressive TME by interacting with specific glycans and impairing anti-tumor responses. In this regard, Gal-3 has been shown to promote anergy of tumor-infiltrating lymphocytes (TILs).According to some embodiments, the glycan binding moiety comprises a glycan binding domain of a galectin selected from Gal-1, Gal-2, Gal-3, Gal-4, Gal-5, Gal-6, Gal-7, Gal-8, Gal-9, Gal-10, Gal-11, Gal-12, Gal-13, Gal-14, and Gal-15. In certain embodiments, the glycan binding moiety comprises the glycan binding domain of Gal-1. According to some embodiments, the glycan binding moiety comprises the glycan binding domain of Gal-3.

[0039] A "glycan-binding domain" or "sialoglycan-binding domain" of a lectin refers to a domain of a lectin or its glycan / sialoglycan-binding variant (e.g., glycan / sialoglycan-binding fragment) that is responsible for binding to the respective glycan. Siglecs, for example, contain an extracellular N-terminal V-set Ig (Ig-V) domain that is involved in binding sialoside ligands. In certain embodiments, a "variant" of any of the disclosed polypeptides or domains thereof contains one or more amino acid substitutions. According to some embodiments, the one or more amino acid substitutions are conservative substitutions. A "conservative substitution" is one in which an amino acid is replaced with another amino acid having similar properties such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic properties of the polypeptide to remain substantially unchanged. Modifications may be made in the polynucleotide and polypeptide structures contemplated in certain embodiments, including polypeptides that have at least approximately and still obtain functional molecules that encode variant or derivative polypeptides with desired characteristics. If it is desirable to alter the amino acid sequence of a polypeptide to produce an equivalent or even improved variant polypeptide, one skilled in the art can, for example, alter one or more of the codons in the encoding DNA sequence.

[0040] In addition to the glycan-binding domain of a lectin, the glycan-binding moiety may include one or more additional domains of the lectin or variants (e.g., fragments) thereof. By way of example, in the context of Siglecs, in addition to the sialoglycan-binding domain of a Siglec (e.g., Siglec-7, Siglec-9, Siglec-10, Siglec-15, etc.), the glycan-binding moiety may further include one or more (e.g., one, two, three, or more) Ig-like domains of a Siglec or fragments thereof. The amino acid sequences and domains (e.g., extracellular domains) of Siglecs and other lectins are known, and any such domains may be included in the glycan-binding moiety as desired and / or useful.

[0041] In certain embodiments, the glycan-binding moiety comprises an antibody heavy chain, including a gamma, alpha, delta, epsilon, or mu antibody heavy chain or a fragment thereof. According to some embodiments, the antibody heavy chain or a fragment thereof is an IgG heavy chain or a fragment thereof, such as a human IgG1 heavy chain or a fragment thereof. In certain embodiments, the antibody heavy chain or a fragment thereof comprises a heavy chain constant region or a fragment thereof. For example, when a heavy chain constant region or a fragment thereof is included in the glycan-binding moiety, the heavy chain constant region of the antibody or a fragment thereof is H 1 domain, C H 2 domain, and / or C H 3 domains, e.g., C H 2 domain and C H According to some embodiments, the glycan-binding moiety may comprise one or more of the C H 1 domain, C H 2 domain, and C H According to some embodiments, the glycan-binding moiety comprises a fragment crystallizable (Fc) region. According to some embodiments, each of the cell targeting moiety and the glycan-binding moiety comprises a C H 2 domain and / or C H and the bispecific molecule comprises a knobs-into-holes modified domain, e.g., a modified C HNon-limiting examples of such bispecific molecules of the present disclosure are shown diagrammatically (with amino acid sequences) in Figures 14-17.

[0042] A "knob-in-hole" strategy (non-limiting examples of which are described, for example, in WO 2006 / 028936) may be used to promote heterodimerization of portions of a bispecific molecule. Briefly, selected amino acids that form the interface of the CH3 domain in human IgG can be mutated at positions that affect CH3 domain interactions to promote heterodimer formation. An amino acid with a small side chain (hole) is introduced into the heavy chain of the portion that specifically binds to the first target, and an amino acid with a large side chain (knob) is introduced into the heavy chain of the portion that specifically binds to the second target. After co-expression of the two portions, a heterodimer is formed as a result of preferential interaction of the heavy chain with the "hole" with the heavy chain with the "knob". Exemplary CH3 substitution pairs that form knobs and holes are (expressed as modification position in first CH3 domain of first heavy chain / modification position in second CH3 domain of second heavy chain) T366Y7F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T3945 / Y407A, T366W / T394S, F405W / T394S, and T366W / T366S_L368A_Y407V.

[0043] Other strategies may be used such as promoting heavy chain heterodimerization using electrostatic interactions by substituting a positively charged residue at one CH3 surface and a negatively charged residue at the second CH3 surface as described in US2010 / 0015133; US2009 / 0182127; US2010 / 028637 or US2011 / 0123532. In other strategies, heterodimerization may be promoted by the following substitutions (expressed as modified position in the first CH3 domain of the first heavy chain / modified position in the second CH3 domain of the second heavy chain): L351 Y_F405A_Y407V T394W, T366I_K392M_T394W / F405A_Y407V, L351 Y_F405A_Y407V, T394W, T366I_K392M_T394W / F405A_Y407V ... Y_Y407A'T366A_K409F, L351Y_Y407A / T366V_K409F, Y407A / T366A_K409F or T350V_L351Y_F405A_Y407V / T350V_T366L_K392L_T394W.

[0044] According to some embodiments, the bispecific molecule of the present disclosure is a fusion protein comprising a cell targeting moiety fused to a glycan binding moiety. When the bispecific molecule is a fusion protein, the cell targeting moiety may be directly fused to the glycan binding moiety (e.g., at the N-terminus or C-terminus of the glycan binding moiety) or the cell targeting moiety may be indirectly fused to the glycan binding moiety via a linker. Any useful linker may be used, including, but not limited to, a serine-glycine linker. In certain embodiments, the linker is about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids in length, or any intervening length of amino acids. In some embodiments, the linker is 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, or more amino acids in length. Nucleic acids encoding the fusion proteins of the disclosure are also provided, as well as expression vectors comprising such nucleic acids, and host cells comprising such nucleic acids and / or expression vectors.

[0045] In certain embodiments, the bispecific molecule of the present disclosure is a conjugate comprising a cell targeting moiety conjugated to a glycan-binding moiety via a linker.Non-limiting examples of linkers that may be used in the conjugates of the present disclosure include ester linkers, amide linkers, maleimide or maleimide-based linkers; valine-citrulline linkers; hydrazone linkers; N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB) linkers; succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linkers; vinylsulfone-based linkers; polyethylene glycol (PEG), including but not limited to linkers comprising tetraethylene glycol; linkers comprising propanoic acid; linkers comprising caproic acid, and any combination thereof. In certain aspects, the linker is a chemically unstable linker, such as an acid-cleavable linker, that is stable at neutral pH (bloodstream pH 7.3-7.5) but undergoes hydrolysis upon internalization into the weakly acidic endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0) of target cells (e.g., cancer cells). Chemically unstable linkers include, but are not limited to, hydrazone-based linkers, oxime-based linkers, carbonate-based linkers, ester-based linkers, and the like. According to certain embodiments, the linker is an enzyme-labile linker, such as an enzyme-labile linker, that is stable in the bloodstream but undergoes enzymatic cleavage upon internalization into target cells, for example, by lysosomal proteases (e.g., cathepsin or plasmin) in the lysosomes of target cells (e.g., cancer cells). Enzyme-labile linkers include, but are not limited to, linkers containing a peptide bond, e.g., dipeptide-based linkers, such as valine-citrulline linkers, e.g., maleimidocaproyl-valine-citrulline-p-aminobenzyl (MC-vc-PAB) linker, valyl-alanyl-para-aminobenzyloxy (Val-Ala-PAB) linker, and the like.Chemically labile linkers, enzyme labile linkers, and non-cleavable linkers are known and are described, for example, in Ducry & Stump (2010) Bioconjugate Chem. 21:5-13.

[0046] In certain embodiments, the conjugates of the present disclosure include a linker that includes a valine-citrulline dipeptide, a valine-alanine dipeptide, or both. According to some embodiments, the linker is a valine-citrulline-paraaminobenzyloxy (Val-Cit-PAB) linker. In certain embodiments, the linker is a valylalanylparaaminobenzyloxy (Val-Ala-PAB) linker.

[0047] The cell targeting moiety may be conjugated to the glycan binding moiety using any convenient approach. For example, conjugating may include site-specifically conjugating the glycan binding moiety to a preselected amino acid of the cell targeting moiety (or vice versa). In certain aspects, the preselected amino acid is at the N-terminus or C-terminus of the cell targeting moiety. In other aspects, the preselected amino acid is internal to the cell targeting moiety, i.e., between the N-terminal amino acid and the C-terminal amino acid of the cell targeting moiety. In some embodiments, the preselected amino acid is an unnatural amino acid. Non-limiting examples of unnatural amino acids that may be provided on the cell targeting moiety (or the glycan binding moiety) to facilitate conjugation include those having a functional group selected from azide, alkyne, alkene, aminooxy, hydrazine, aldehyde (e.g., formylglycine, e.g., Catalent Pharma Solutions' SMARTag™ technology), nitrone, nitrile oxide, cyclopropene, norbornene, isocyanide, aryl halide, and boronic acid functional groups. Unnatural amino acids that may be incorporated and selected to provide desired functional groups are known and described, e.g., in Maza et al. (2015) Bioconjug. Chem. 26(9):1884-9; Patterson et al. (2014) ACS Chem. Biol. 9:592-605, Adumeau et al. (2016) Mol. Imaging Biol.(2):153-65, and elsewhere.

[0048] Numerous strategies are available for conjugating the cell targeting moiety and the glycan binding moiety through a linker. For example, the glycan binding moiety may be derivatized by covalently attaching a linker to the glycan binding moiety, the linker having a functional group capable of reacting with a "chemical handle" on the cell targeting moiety. Also by way of example, the cell targeting moiety may be derivatized by covalently attaching a linker to the cell targeting moiety, the linker having a functional group capable of reacting with a "chemical handle" on the glycan binding moiety. The functional group on the linker may be varied and may be selected based on compatibility with the chemical handle on the cell targeting moiety or the glycan binding moiety. According to one embodiment, the chemical handle is provided by incorporating a non-natural amino acid having a chemical handle into the cell targeting moiety or the glycan binding moiety. In some embodiments, conjugating the cell targeting moiety and the glycan binding moiety is by copper-free, strain-promoted cycloaddition, alkyne-azide cycloaddition, or the like.

[0049] Using the information provided herein, the cell targeting moieties and glycan binding moieties and fusion proteins of the present disclosure may be prepared using standard techniques known to those skilled in the art. For example, a nucleic acid sequence encoding the amino acid sequence of the cell targeting moiety and glycan binding moiety of the bispecific molecule of the present disclosure may be used to express the cell targeting moiety and glycan binding moiety. The nucleic acid sequence may be optimized to reflect the particular codon "choices" for various expression systems according to methods known to those skilled in the art. Using the sequence information provided, the nucleic acid may be synthesized according to several standard methods known to those skilled in the art.

[0050] Once the nucleic acid encoding the cell targeting moiety and / or glycan binding moiety of interest has been synthesized, it can be amplified and / or cloned according to standard methods. Molecular cloning techniques to achieve these ends are known in the art. A wide variety of cloning and in vitro amplification methods suitable for the construction of recombinant nucleic acids are known to those skilled in the art and are the subject of numerous textbooks and laboratory manuals.

[0051] Expression of a natural or synthetic nucleic acid encoding a cell targeting moiety and / or a glycan binding moiety of the present disclosure can be achieved by operably linking the nucleic acid encoding the cell targeting moiety and / or the glycan binding moiety to a promoter (either constitutive or inducible) and incorporating the construct into an expression vector to generate a recombinant expression vector. The vector may be suitable for replication and integration in prokaryotes, eukaryotes, or both. A typical cloning vector contains functionally appropriately oriented transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the nucleic acid encoding the cell targeting moiety and / or the glycan binding moiety. The vector optionally contains a generic expression cassette that includes at least one independent terminator sequence, e.g., a sequence that allows replication of the cassette in both eukaryotes and prokaryotes, as found in shuttle vectors, and a selection marker for both prokaryotic and eukaryotic systems.

[0052] To obtain high levels of expression of a cloned nucleic acid, it is common to construct an expression plasmid that typically contains a strong promoter to direct transcription, a ribosome binding site for translation initiation, and a transcription / translation terminator, each in a functional orientation relative to each other and to the protein coding sequence. Examples of regulatory regions suitable for this purpose in E. coli are the promoter and operator regions of the E. coli tryptophan biosynthetic pathway, phage lambda (P L), the left promoter of the ribosomal RNA (RIRNA) and the L-arabinose (araBAD) operon. It is also useful to include a selection marker in a DNA vector transformed in E. coli. Examples of such markers include genes specifying resistance to ampicillin, tetracycline, or chloramphenicol. Expression systems for expressing antibodies are available using, for example, E. coli, Bacillus, and Salmonella. E. coli systems can also be used.

[0053] The cell targeting and / or glycan binding moiety gene may be subcloned into an expression vector that allows for the addition of a tag (e.g., FLAG, hexahistidine, etc.) to the C-terminus or N-terminus of the cell targeting and / or glycan binding moiety to facilitate purification. Methods for transfecting and expressing genes in mammalian cells are known in the art. Transducing cells with a nucleic acid may include, for example, incubating lipid microparticles containing the nucleic acid with the cells or incubating a viral vector containing the nucleic acid with cells within the host range of the vector. Culturing cells used in the present disclosure, including cell lines and cultured cells from tissues (e.g., tumors) or blood samples, is known in the art.

[0054] Once the nucleic acid encoding the cell targeting moiety and / or glycan binding moiety of interest has been isolated and cloned, the nucleic acid can be expressed in a variety of recombinantly engineered cells known to those of skill in the art, including bacteria, yeast, filamentous fungi, insects (e.g., insects using baculovirus vectors), and mammalian cells.

[0055] Isolation and purification of the cell targeting and / or glycan binding moiety of interest can be achieved according to methods known in the art. For example, the protein can be isolated from the lysate of cells genetically modified to express the protein constitutively and / or upon induction, or from a synthetic reaction mixture, by immunoaffinity purification (or precipitation using protein L or A), washing to remove non-specifically bound material, and elution of the specifically bound cell targeting and / or glycan binding moiety. The isolated cell targeting and / or glycan binding moiety can be further purified by dialysis and other methods commonly used in protein purification methods. In one embodiment, the cell targeting and / or glycan binding moiety can be isolated using metal chelate chromatography methods. The cell targeting and / or glycan binding moiety of the present disclosure may contain modifications to facilitate isolation, as discussed above.

[0056] The cell targeting moiety and / or glycan binding moiety may be prepared in a substantially pure or isolated form (e.g., free of other polypeptides). The protein may be present in a composition in which the polypeptide is enriched relative to other components that may be present (e.g., other polypeptides or other host cell components). A purified cell targeting moiety and / or glycan binding moiety may be provided such that the cell targeting moiety and / or glycan binding moiety is present in a composition that is substantially free of other expressed proteins, e.g., less than 90%, usually less than 60%, more usually less than 50% of the composition is made up of other expressed proteins.

[0057] The cell targeting moiety and / or glycan binding moiety produced by a prokaryotic cell may require exposure to a chaotropic agent for proper folding. During purification from E. coli, for example, the expressed protein may be optionally denatured and then renatured. This can be achieved, for example, by solubilizing the bacterially produced cell targeting and / or glycan binding moiety in a chaotropic agent such as guanidine HCl. The cell targeting moiety and / or glycan binding moiety are then renatured by either slow dialysis or gel filtration. Alternatively, the nucleic acid encoding the cell targeting moiety and / or glycan binding moiety may be operably linked to a secretion signal sequence such as pelB so that the cell targeting moiety and / or glycan binding moiety are secreted into the periplasm in the correctly folded form.

[0058] Nucleic acids, expression vectors, and cells In view of the sections above relating to methods of producing the cell-targeting and / or glycan-binding moieties of the bispecific molecules of the present disclosure, it will be understood that the present disclosure also provides nucleic acids, expression vectors and cells.

[0059] In certain embodiments, nucleic acids are provided that encode any of the cell-targeting moieties of the bispecific molecules of the present disclosure, any of the glycan-binding moieties of the bispecific molecules of the present disclosure, or both. Non-limiting examples of nucleotide sequences encoding the cell-targeting and glycan-binding moieties of bispecific molecules according to embodiments of the present disclosure are provided in the Experimental Section below.

[0060] Also provided is an expression vector comprising any of the nucleic acids of the present disclosure. Expression of a natural or synthetic nucleic acid encoding a cell targeting moiety and / or a glycan binding moiety can be achieved by operably linking the nucleic acid encoding the cell targeting moiety and / or the glycan binding moiety to a promoter (either constitutive or inducible) and incorporating the construct into an expression vector to generate a recombinant expression vector. The vector may be suitable for replication and integration in prokaryotes, eukaryotes, or both. A typical cloning vector contains functionally appropriately oriented transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the nucleic acid encoding the cell targeting moiety and / or the glycan binding moiety. The vector optionally comprises a generic expression cassette that includes at least one independent terminator sequence, e.g., a sequence that allows replication of the cassette in both eukaryotes and prokaryotes, such as those found in shuttle vectors, and a selection marker for both prokaryotic and eukaryotic systems.

[0061] Cells comprising any of the nucleic acids and / or expression vectors of the present disclosure are also provided. According to some embodiments, the cells of the present disclosure comprise a nucleic acid encoding any of the cell-targeting moieties of the bispecific molecules of the present disclosure, any of the glycan-binding moieties of the bispecific molecules of the present disclosure, or both. In certain such embodiments, the bispecific molecule is a fusion protein (as described above), and the nucleic acid encodes the fusion protein. According to some embodiments, cells are provided that comprise a first nucleic acid encoding any of the cell-targeting moieties of the bispecific molecules of the present disclosure, and a second nucleic acid encoding any of the glycan-binding moieties of the bispecific molecules of the present disclosure. In certain embodiments, for example, the cells comprise a first expression vector comprising the first nucleic acid and a second expression vector comprising the second nucleic acid.

[0062] Also provided are methods of making a bispecific molecule of the disclosure, comprising culturing a cell of the disclosure under conditions suitable for the cell to express the cell targeting moiety and / or the glycan binding moiety, whereby the cell targeting moiety and / or the glycan binding moiety are produced. The conditions for culturing the cells such that the cell targeting moiety and / or the glycan binding moiety are expressed may vary. Such conditions may include culturing the cells in a suitable container (e.g., a cell culture plate or well thereof), in a suitable medium (e.g., cell culture medium such as DMEM, RPMI, MEM, IMDM, DMEM / F-12, etc.), at a suitable temperature (e.g., 32°C to 42°C (e.g., 37°C)) and pH (e.g., pH 7.0 to 7.7 (e.g., pH 7.4)), in an environment having a suitable percentage of CO2, e.g., 3% to 10% (e.g., 5%).

[0063] Further bispecific molecules Also provided by the present disclosure are bispecific molecules as described elsewhere herein, but where the second moiety binds to a target other than a glycan. That is, it will be understood, with the benefit of this disclosure, that the AbLec of the present disclosure provides proof of concept that the technology can be applied to contexts beyond the context of glycan binding. In certain embodiments, bispecific molecules are provided that include a cell targeting moiety (e.g., any of the cell targeting moieties described elsewhere herein) fused to an Fc region, and a moiety comprising a ligand binding domain of a receptor, which is also fused to an Fc region. In certain embodiments, the two moieties are heterodimerized via the Fc region. In some embodiments, the heterodimerization via the Fc region is via a knobs-in-holes strategy as described elsewhere herein.

[0064] In some embodiments, the portion comprising the ligand-binding domain of a receptor comprises a ligand-binding domain of a receptor that binds to a cell surface ligand. That is, the ligand-binding domain binds to a cell surface ligand. In certain embodiments, the cell surface ligand is present on the surface of a cell that also presents a target for the cell targeting moiety, such that the cell targeting moiety and the second portion (the portion comprising the ligand-binding domain of a receptor) bind to different types of molecules present on the surface of the same cell.

[0065] In certain embodiments, the portion of the receptor that comprises the ligand binding domain comprises a ligand binding domain of a stem cell receptor, an immune cell receptor, a growth factor receptor, a cytokine receptor, a hormone receptor, a receptor tyrosine kinase, a receptor of the epidermal growth factor receptor (EGFR) family (such as, for example, HER2 (human epidermal growth factor receptor 2)), a receptor of the fibroblast growth factor receptor (FGFR) family, a receptor of the vascular endothelial growth factor receptor (VEGFR) family, a receptor of the platelet-derived growth factor receptor (PDGFR) family, a receptor of the rearrangement during transfection (RET) receptor family, a receptor of the Eph receptor family, a receptor of the discoidin domain receptor (DDR) family, and a mucin protein (e.g., MUC1). In some embodiments, the portion of the receptor that comprises the ligand binding domain comprises the ligand binding domain of CD71 (transferrin receptor). In certain embodiments, the portion comprising the ligand binding domain of a receptor comprises the ligand binding domain of an immune cell receptor, non-limiting examples of which include a T cell receptor, a B cell receptor, a natural killer (NK) cell receptor, a macrophage receptor, a monocyte receptor, a neutrophil receptor, a dendritic cell receptor, a mast cell receptor, a basophil receptor, and an eosinophil receptor.

[0066] composition Aspects of the present disclosure further include compositions. According to some embodiments, the compositions of the present disclosure include the bispecific molecules of the present disclosure. For example, the bispecific molecule may be any of the bispecific molecules described in the bispecific molecules section above, the description of which is incorporated herein but will not be repeated for the sake of brevity.

[0067] In certain embodiments, the composition of the present disclosure comprises a bispecific molecule present in a liquid medium. The liquid medium may be an aqueous liquid medium, such as water, a buffer solution, etc. One or more additives may be present in such compositions, such as salts (e.g., NaCl, MgCl2, KCl, MgSO4), buffers (Tris buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 2-(N-morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.), solubilizers, detergents (e.g., non-ionic detergents such as Tween-20), nuclease inhibitors, protease inhibitors, glycerol, chelating agents, etc.

[0068] Aspects of the present disclosure further include pharmaceutical compositions. In some embodiments, a pharmaceutical composition of the present disclosure comprises a bispecific molecule of the present disclosure and a pharma- ceutically acceptable carrier.

[0069] The bispecific molecules can be incorporated into a variety of formulations for therapeutic administration. More specifically, the bispecific molecules can be formulated into pharmaceutical compositions by combination with suitable pharma- ceutically acceptable excipients or diluents, and may be formulated into solid, semi-solid, liquid or gaseous form preparations, such as tablets, capsules, powders, granules, ointments, solutions, injections, inhalants and aerosols.

[0070] Formulations of bispecific molecules for administration to an individual (e.g., suitable for human administration) are generally sterile and may further be free of detectable pyrogens or other contaminants that would contraindicate administration to a patient by a selected route of administration.

[0071] In pharmaceutical dosage forms, the bispecific molecules can be administered in the form of their pharma- ceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination with other pharma- ceutically active compounds. The following methods and carriers / excipients are merely examples and are in no way limiting.

[0072] For oral preparations, the bispecific molecules can be used alone or in combination with suitable excipients for making tablets, powders, granules, or capsules, e.g., conventional excipients such as lactose, mannitol, corn starch, or potato starch; binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch, or gelatin; disintegrating agents such as corn starch, potato starch, or sodium carboxymethylcellulose; lubricants such as talc or magnesium stearate; and diluents, buffers, wetting agents, preservatives, and flavorings, as appropriate.

[0073] The bispecific molecules can be formulated for parenteral (e.g., intravenous, intraarterial, intraosseous, intramuscular, intracerebral, intraventricular, intrathecal, subcutaneous, etc.) administration. In certain embodiments, the bispecific molecules are formulated for injection by dissolving, suspending, or emulsifying the bispecific molecule in an aqueous or non-aqueous solvent such as vegetable oil or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids, or propylene glycol, optionally formulated with conventional additives such as solubilizers, isotonicity agents, suspending agents, emulsifiers, stabilizers, and preservatives.

[0074] Pharmaceutical compositions comprising the bispecific molecules may be prepared by mixing the bispecific molecules having the desired purity with any physiologically acceptable carriers, excipients, stabilizers, surfactants, buffers, and / or isotonicity agents. Acceptable carriers, excipients, and / or stabilizers are non-toxic to recipients at the dosages and concentrations employed and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine, and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl paraben, benzalkonium chloride, or combinations thereof); arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, glycine, glycerol, glyceryl ester ... amino acids such as sucrose, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline, and combinations thereof; monosaccharides, disaccharides, and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid, and / or non-ionic surfactants such as Tween, Brij Pluronics, Triton-X, or polyethylene glycol (PEG).

[0075] The pharmaceutical composition may be in liquid form, lyophilized form, or liquid form reconstituted from lyophilized form, with the lyophilized preparation being reconstituted with a sterile solution prior to administration. The standard procedure for reconstituting a lyophilized composition is to add back a volume of purified water (typically equal to the volume removed during lyophilization), although solutions containing antimicrobial agents may be used to produce pharmaceutical compositions for parenteral administration.

[0076] Aqueous formulations of bispecific molecules may be prepared in pH buffer solutions, for example, at a pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5. Examples of buffers suitable for a pH within this range include phosphate buffers, histidine buffers, citrate buffers, succinate buffers, acetate buffers, and other organic acid buffers. The buffer concentration may be, for example, from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending on the buffer and the desired tonicity of the formulation.

[0077] An isotonicity agent may be included to adjust the tonicity of the formulation. Examples of isotonicity agents include sodium chloride, potassium chloride, glycerin, and any component from the group of amino acids, sugars, and combinations thereof. In some embodiments, the aqueous formulation is an isotonic solution, although hypertonic or hypotonic solutions may be suitable. The term "isotonic" refers to a solution that has the same isotonicity as some other solution to which it is compared, such as saline or serum. The isotonicity agent may be used in an amount of about 5 mM to about 350 mM, for example, in an amount of 100 mM to 350 mM.

[0078] Surfactants may also be added to the formulation to reduce aggregation in the formulation and / or minimize the formation of fine particles and / or reduce adsorption. Examples of surfactants include polyoxyethylene sorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenyl polyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymers (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). Examples of suitable polyoxyethylene sorbitan fatty acid esters are polysorbate 20 (sold under the trademark Tween 20™) and polysorbate 80 (sold under the trademark Tween 80™). Examples of suitable polyethylene-polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188™. Examples of suitable polyoxyethylene alkyl ethers are those sold under the trademark Brij™. Exemplary concentrations of the surfactant can range from about 0.001% to about 1% (w / v).

[0079] A lyoprotectant may be added to protect the bispecific molecule from destabilizing conditions during the lyophilization process. For example, known lyoprotectants include sugars (including glucose and sucrose), polyols (including mannitol, sorbitol, and glycerol), and amino acids (including alanine, glycine, and glutamic acid). For example, the lyoprotectant may be included in an amount of about 10 mM to 500 nM.

[0080] In some embodiments, the pharmaceutical compositions comprise a bispecific molecule and one or more of the components identified above (e.g., surfactants, buffers, stabilizers, tonicity agents), and are essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl paraben, benzalkonium chloride, and combinations thereof. In other embodiments, a preservative is included in the formulation, for example, at a concentration ranging from about 0.001 to about 2% (w / v).

[0081] kit Aspects of the present disclosure further include kits. In certain embodiments, the kits are used in practicing the methods of the present disclosure, such as methods comprising administering a pharmaceutical composition of the present disclosure to an individual to enhance anti-tumor immunity in the individual, administering a pharmaceutical composition of the present disclosure to an individual to enhance or suppress an immune response in the individual, etc.

[0082] Thus, in certain embodiments, the kits of the present disclosure comprise one or more unit doses of the pharmaceutical composition of the present disclosure, and instructions for administering the pharmaceutical composition to an individual in need thereof. The pharmaceutical composition included in the kit may comprise any of the bispecific molecules of the present disclosure, such as any of the bispecific molecules described herein above, which will not be repeated herein for the sake of brevity.

[0083] The kits of the present disclosure may include a quantity of the composition present in a unit dose, e.g., ampoule, or multiple dose format. Thus, in certain embodiments, the kits may include one or more (e.g., two or more) unit doses (e.g., ampoules) of a composition comprising a bispecific molecule of the present disclosure. The term "unit dose" as used herein refers to a physically discrete unit suitable as a unitary dose for human and animal subjects, each unit containing a predetermined amount of the composition calculated in an amount sufficient to produce a desired effect. The amount of a unit dose depends on various factors, such as the particular bispecific molecule used, the effect to be achieved, and the pharmacodynamics associated with the bispecific molecule in an individual. In yet other embodiments, the kits may include single or multiple doses of the composition.

[0084] In certain embodiments, the kits of the present disclosure include instructions for administering one or more unit doses of the pharmaceutical composition to an individual in need of enhanced anti-tumor immunity. According to some embodiments, the kits of the present disclosure include instructions for administering one or more unit doses of the pharmaceutical composition to an individual in need of an enhanced or suppressed immune response.

[0085] The instructions included in the kit (e.g., instructions for use (IFU)) may be recorded on a suitable recording medium. For example, the instructions can be printed on a substrate such as paper or plastic. As such, the instructions can be present in the kit as a package insert, on the label of the container of the kit or a component thereof (i.e., associated with the packaging or subpackaging), and the like. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer-readable storage medium, such as a portable flash drive, DVD, CD-ROM, diskette, and the like. In still other embodiments, the actual instructions are not present in the kit, but a means is provided for obtaining the instructions from a remote source, for example, via the Internet. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, the means for obtaining the instructions are recorded on a suitable substrate.

[0086] method Aspects of the disclosure include methods of using the bispecific molecules of the disclosure, which are useful in a variety of contexts, including in vitro and / or in vivo research and / or clinical applications.

[0087] In certain aspects, methods are provided for enhancing anti-tumor immunity in an individual in need thereof. Such methods include administering to the individual an effective amount of a pharmaceutical composition of the present disclosure, such as a pharmaceutical composition comprising a bispecific molecule of the present disclosure comprising a cancer cell targeting moiety and a glycan binding moiety. In certain embodiments, the method is for enhancing antibody-dependent cellular phagocytosis (ADCP) and / or cytotoxicity (ADCC) in the individual.

[0088] In certain aspects, methods are provided for enhancing or suppressing an immune response in an individual in need thereof, comprising administering to the individual an effective amount of a pharmaceutical composition of the present disclosure, e.g., a pharmaceutical composition comprising a bispecific molecule of the present disclosure comprising an immune cell targeting moiety and a glycan binding moiety.

[0089] An individual in need thereof may have a cell proliferative disorder. By "cell proliferative disorder" is meant a disorder in which unwanted cell proliferation of one or more subsets of cells of a multicellular organism occurs, resulting in harm to the organism (e.g., pain or reduced life expectancy). Cell proliferative disorders include, but are not limited to, cancer, precancer, benign tumors, vascular proliferative disorders (e.g., arthritis, restenosis, etc.), fibrotic disorders (e.g., hepatic cirrhosis, atherosclerosis, etc.), psoriasis, epidermal and dermoid cysts, lipomas, adenomas, capillary and cutaneous hemangiomas, lymphangiomas, nevi lesions, teratomas, nephromas, myofibromatosis, osteogenic tumors, dysplastic masses, mesangial cell proliferative disorders, and the like.

[0090] In some embodiments, the individual has cancer. The subject method can be used to treat a wide variety of cancers. As used herein, "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. Examples of cancers that can be treated using the subject method include, but are not limited to, carcinoma, lymphoma, blastoma, and sarcoma. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bile duct cancer, bladder cancer, liver cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancer. In certain embodiments, the individual has a cancer selected from a solid tumor, recurrent glioblastoma multiforme (GBM), non-small cell lung cancer, metastatic melanoma, melanoma, peritoneal cancer, epithelial ovarian cancer, glioblastoma multiforme (GBM), metastatic colorectal cancer, colorectal cancer, pancreatic ductal adenocarcinoma, squamous cell carcinoma, esophageal cancer, gastric cancer, neuroblastoma, fallopian tube carcinoma, bladder cancer, metastatic breast cancer, pancreatic cancer, soft tissue sarcoma, recurrent head and neck cancer squamous cell carcinoma, head and neck cancer, anaplastic astrocytoma, malignant pleural mesothelioma, breast cancer, squamous non-small cell lung cancer, rhabdomyosarcoma, metastatic renal cell carcinoma, basal cell carcinoma (basal cell epithelioma), and gliosarcoma. In certain aspects, the individual has a cancer selected from melanoma, Hodgkin's lymphoma, renal cell carcinoma (RCC), bladder cancer, non-small cell lung cancer (NSCLC), and head and neck squamous cell carcinoma (HNSCC).

[0091] The bispecific molecules of this disclosure may be administered via a route of administration selected from oral (e.g., in tablet form, capsule form, liquid form, etc.), parenteral (e.g., intravenous, intramuscular, subcutaneous, intramuscular, or epidural injection), topical, intranasal, or intratumoral administration.

[0092] The bispecific molecules of the present disclosure may be administered in a pharmaceutical composition in a therapeutically effective amount. By "therapeutically effective amount" is meant a dosage sufficient to produce a desired result, e.g., an amount sufficient to produce a beneficial or desired therapeutic (including prophylactic) result, such as the alleviation of symptoms of cancer and / or immune disorders, as compared to a control. With respect to cancer, in some embodiments, a therapeutically effective amount is sufficient to slow tumor growth, reduce tumor size, and / or the like. An effective amount can be administered in one or more administrations.

[0093] Aspects of the present disclosure include methods for treating cancer and / or immune disorders in individuals. Treatment means at least an improvement in one or more symptoms associated with cancer and / or immune disorders in an individual, and improvement is used broadly to refer to at least a decrease in the parameters associated with the cancer and / or immune disorder being treated, such as the magnitude of symptoms. Thus, treatment also includes the situation in which cancer and / or immune disorders or at least one or more symptoms associated therewith are completely inhibited, e.g., prevented from occurring, or stopped, e.g., discontinued, so that an individual no longer suffers from cancer and / or immune disorders or at least symptoms that characterize cancer and / or immune disorders.

[0094] The bispecific molecules of the present disclosure may be administered to an individual alone or in combination with a second agent. Second agents of interest include, but are not limited to, agents approved by the U.S. Food and Drug Administration and / or the European Medicines Agency (EMA) for use in the treatment of cancer. In some embodiments, the second agent is an immune checkpoint inhibitor. Immune checkpoint inhibitors of interest include, but are not limited to, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitors, programmed cell death-1 (PD-1) inhibitors, programmed cell death ligand-1 (PD-L1) inhibitors, lymphocyte activation gene-3 (LAG-3) inhibitors, T cell immunoglobulin domain and mucin domain 3 (TIM-3) inhibitors, indoleamine (2,3)-dioxygenase (IDO) inhibitors, T cell immunoreceptor with Ig and ITIM domain (TIGIT) inhibitors, V domain Ig suppressor of T cell activation (VISTA) inhibitors, B7-H3 inhibitors, and any combination thereof.

[0095] When the bispecific molecule of the present disclosure is administered together with a second agent, the bispecific molecule and the second agent may be administered to an individual according to any suitable administration regimen. According to certain embodiments, the bispecific molecule and the second agent are administered according to a dosing regimen approved for the individual's use. In some embodiments, administration of the bispecific molecule allows the second agent to be administered according to a dosing regimen with one or more lower and / or less frequent doses and / or a reduced number of cycles compared to those utilized when the second agent is administered without administration of the bispecific molecule. In certain aspects, administration of the second agent allows the bispecific molecule to be administered according to a dosing regimen with one or more lower and / or less frequent doses and / or a reduced number of cycles compared to those utilized when the bispecific molecule is administered without administration of the second agent.

[0096] In some embodiments, one or more doses of the bispecific molecule and the second agent are administered to an individual simultaneously, where "simultaneously" means either that the bispecific molecule and the second agent are present in the same pharmaceutical composition, or that the bispecific molecule and the second agent are administered within 1 hour or less, 30 minutes or less, or 15 minutes or less as separate pharmaceutical compositions.

[0097] In some embodiments, one or more doses of the bispecific molecule and the second agent are administered sequentially to the individual.

[0098] However, in some embodiments, the bispecific molecule and the second agent are administered to an individual in different compositions and / or at different times. For example, the bispecific molecule may be administered prior to administration of the second agent, e.g., in a particular cycle. Alternatively, the second agent may be administered prior to administration of the bispecific molecule, e.g., in a particular cycle. The second agent may be administered during a period beginning at least 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, or up to 5 days or more after administration of the first agent.

[0099] In one example, the second agent is administered to the individual for a desired period of time before the administration of the bispecific molecule.In certain embodiments, when the individual has cancer, such a regimen "primes" the cancer cells to enhance the anti-cancer effect of the bispecific molecule.Such a period of time that separates the step of administering the second agent from the step of administering the bispecific molecule is desirably long enough to allow the priming of the cancer cells so that the anti-cancer effect of the bispecific molecule is increased.

[0100] In some embodiments, the administration of one agent is specifically timed relative to the administration of another agent, for example, in some embodiments, the bispecific molecule is administered such that a particular effect is observed (e.g., observed or expected to be observed based on a population study showing a correlation between a given dosing regimen and a particular effect of interest).

[0101] In certain aspects, the desired relative dosing regimens of agents administered in combination may be evaluated or empirically determined, e.g., using ex vivo, in vivo, and / or in vitro models; in some embodiments, such evaluation or empirical determination is performed in vivo in a patient population (e.g., so that a correlation is established), or alternatively, in a particular individual of interest.

[0102] In some embodiments, the bispecific molecule and the second agent are administered according to an intermittent dosing regimen comprising at least two cycles. When two or more agents are administered in combination, each administered according to such an intermittent, cyclical regimen, the individual doses of the different agents may be interleaved with each other. In certain embodiments, one or more doses of the second agent are administered a period of time after a dose of the first agent. In some embodiments, each dose of the second agent is administered a period of time after a dose of the first agent. In certain embodiments, each dose of the first agent is followed by a dose of the second agent a period of time after each dose of the first agent. In some embodiments, two or more doses of the first agent are administered between at least a pair of doses of the second agent, and in certain embodiments, two or more doses of the second agent are administered between at least a pair of doses of the first agent. In some embodiments, the different doses of the same agent are separated by a common time interval, and in some embodiments, the time interval between the different doses of the same agent varies. In certain aspects, the different doses of the bispecific molecule and the second agent are separated from one another by a common time interval; in some embodiments, the different doses of the different agents are separated from one another by different time intervals.

[0103] One exemplary protocol for alternating two intermittent cyclic dosing regimens may include (a) a first dosing period during which a therapeutically effective amount of the bispecific molecule is administered to the individual; (b) a first rest period, (c) a second dosing period during which a therapeutically effective amount of a second agent is administered to the individual; and (d) a second rest period. A second exemplary protocol for alternating two intermittent cyclic dosing regimens may include (a) a first dosing period during which a therapeutically effective amount of a second agent is administered to the individual; (b) a first rest period, (c) a second dosing period during which a therapeutically effective amount of the bispecific molecule is administered to the individual; and (d) a second rest period.

[0104] In some embodiments, the first rest period and the second rest period may correspond to the same number of hours or days. Alternatively, in some embodiments, the first rest period and the second rest period are different, with the first rest period being longer than the second rest period, or vice versa. In some embodiments, each of the rest periods corresponds to 120 hours, 96 hours, 72 hours, 48 ​​hours, 24 hours, 12 hours, 6 hours, 30 hours, 1 hour, or less. In some embodiments, if the second rest period is longer than the first rest period, it can be defined as a number of days or weeks (e.g., 1 day, 3 days, 5 days, 1 week, 2 weeks, 4 weeks, or more) rather than hours.

[0105] Where the length of the first resting period is determined by the presence or occurrence of a particular biological or therapeutic event, the length of the second resting period may be determined based on different factors, either separately or in combination. Exemplary such factors may include the type and / or stage of the cancer to which the therapy is administered; the characteristics (e.g., pharmacokinetic characteristics) of the bispecific molecule, and / or one or more characteristics of the patient's response to therapy with the bispecific molecule. In some embodiments, the length of one or both resting periods may be adjusted in light of the pharmacokinetic characteristics (e.g., as assessed via plasma concentration levels) of one or the other of the administered agents. For example, the relevant resting period may be considered complete if the plasma concentration of the relevant agent falls below a predetermined level, optionally upon evaluation or other consideration of one or more characteristics of the individual's response.

[0106] In certain aspects, the number of cycles in which a particular agent is administered may be empirically determined, and in some embodiments, the exact regimen followed (e.g., number of doses, intervals between doses (e.g., relative to each other or to another event, such as administration of another therapy), amount of dose, etc.) may be different for one or more cycles as compared to one or more other cycles.

[0107] The bispecific molecule and the second agent may be administered together or independently via any suitable route of administration. The bispecific molecule and the second agent may be administered via a route of administration independently selected from oral, topical (e.g., by intravenous, intraarterial, subcutaneous, intramuscular, or epidural injection), or intranasal administration. According to certain embodiments, the bispecific molecule and the second agent are both administered orally (e.g., in tablet, capsule, liquid form, etc.) either simultaneously (in the same pharmaceutical composition or in separate pharmaceutical compositions) or sequentially.

[0108] Notwithstanding the scope of the appended claims, the present disclosure is also defined by the following embodiments: 1. A bispecific molecule comprising: a cell targeting moiety; and and a glycan-binding moiety.

[0109] 2. The bispecific molecule of embodiment 1, wherein the cell targeting moiety is a cancer cell targeting moiety.

[0110] 3. The cancer cell targeting moiety is selected from the group consisting of 5T4, AXL receptor tyrosine kinase (AXL), B cell maturation antigen (BCMA), c-MET, C4.4a, carbonic anhydrase 6 (CA6), carbonic anhydrase 9 (CA9), cadherin-6, CD19, CD20, CD22, CD25, CD27L, CD30, CD33, CD37, CD44, CD44v6, CD56, CD70, CD74, CD79b, CD123, CD138, carcinoembryonic antigen (CEA), cKit, Cripto protein, CS1, delta-like canonical notch ligand 3 (DLL3), and IL-1. 3), endothelin receptor type B (EDNRB), ephrin A4 (EFNA4), epidermal growth factor receptor (EGFR), EGFRvIII, ectonucleotide pyrophosphatase / phosphodiesterase 3 (ENPP3), EPH receptor A2 (EPHA2), fibroblast growth factor receptor 2 (FGFR2), fibroblast growth factor receptor 3 (FGFR3), FMS-like tyrosine kinase 3 (FLT3), folate receptor 1 (FOLR1), GD2 ganglioside, glycoprotein non-transferase B (GPNMB), guanylate cyclase 2C (GUCY2C) , human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), integrin alpha, lysosome-associated membrane protein 1 (LAMP-1), Lewis Y, LIV-1, leucine-rich repeat containing 15 (LRRC15), mesothelin (MSLN), mucin 1 (MUC1), mucin 16 (MUC16), sodium-dependent phosphate transport protein 2B (NaPi2b), nectin-4, NMB, NOTCH3, p-cadherin (p-CAD), programmed cell death receptor ligand 1 (PD-L1), programmed cell death receptor ligase 3. The bispecific molecule of embodiment 2, which binds to a cancer cell surface molecule selected from the group consisting of: prostate-specific ligand 2 (PD-L2), prostate-specific membrane antigen (PSMA), protein tyrosine kinase 7 (PTK7), solute carrier family 44 member 4 (SLC44A4), SLIT-like family member 6 (SLITRK6), STEAP family member 1 (STEAP1), tissue factor (TF), T-cell immunoglobulin and mucin protein-1 (TIM-1), Tn antigen, trophoblast surface antigen (TROP-2), and Wilms' tumor 1 (WT1).

[0111] 4. The bispecific molecule of embodiment 1, wherein the cell targeting moiety is an immune cell targeting moiety.

[0112] 5. The immune cell targeting moiety is selected from the group consisting of PD-1, PD-L1, PD-L2, CLTA-4, VISTA, LAG-3, TIM-3, CD24, CD47, SIRP alpha, CD3, CD8, CD4, CD28, CD80, CD86, CD19, ICOS, OX40, OX40L, GD3 ganglioside, TIGIT, Siglec-2, Siglec-3, Siglec-7, Siglec-8, Siglec-9, Siglec-10, Siglec-15, Ganglioside ... 5. The bispecific molecule of embodiment 4, which binds to an immune cell surface molecule selected from the group consisting of lectin-9, B7-H3, B7-H4, CD40, CD40L, B7RP1, CD70, CD27, BTLA, HVEM, KIR, 4-1BB, 4-1BBL, CD226, CD155, CD112, GITR, GITRL, A2aR, CD137, CD137L, CD45, CD206, CD163, TRAIL, NKG2D, CD16, and TGF-beta.

[0113] 6. The bispecific molecule of any one of embodiments 1-5, wherein the glycan binding moiety comprises a sialoglycan binding moiety.

[0114] 7. The bispecific molecule of embodiment 6, wherein the sialoglycan binding moiety comprises a sialoglycan binding domain of a lectin.

[0115] 8. The bispecific molecule of embodiment 7, wherein the lectin is a sialic acid-binding immunoglobulin-like lectin (Siglec).

[0116] 9. The bispecific molecule of embodiment 8, wherein the Siglec is a CD33-associated Siglec.

[0117] 10. The bispecific molecule of embodiment 9, wherein the CD33-associated Siglec is selected from the group consisting of Siglec-7, Siglec-9, and Siglec-10.

[0118] 11. The bispecific molecule of embodiment 10, wherein the CD33-associated Siglec is Siglec-7.

[0119] 12. The bispecific molecule of embodiment 10, wherein the CD33-associated Siglec is Siglec-9.

[0120] 13. The bispecific molecule of embodiment 8, wherein the Siglec is Siglec-15.

[0121] 14. The bispecific molecule of embodiment 6, wherein the sialoglycan binding moiety comprises the sialoglycan binding domain of a Siglec-like adhesin.

[0122] 15. The bispecific molecule of any one of embodiments 1-5, wherein the glycan-binding moiety comprises a glycan-binding domain of a C-type lectin.

[0123] 16. The bispecific molecule of embodiment 15, wherein the C-type lectin is DECTIN-1, lectin-like oxidized low density lipoprotein receptor-1 (LOX-1), C-type lectin-like receptor-1 (CLEC-1), C-type lectin-like receptor 2 (CLEC-2), myeloid inhibitory C-type lectin-like receptor (MICL), CLEC9A, DC immunoreceptor (DCIR), DECTIN-2, blood DC antigen-2 (BDCA-2), macrophage-inducible C-type lectin (MINCLE), macrophage galactose lectin (MGL), or asialoglycoprotein receptor (ASGPR).

[0124] 17. The bispecific molecule of any one of embodiments 1 to 5, wherein the glycan-binding moiety comprises a glycan-binding domain of a galectin.

[0125] 18. The bispecific molecule of embodiment 17, wherein the galectin is Gal-1, Gal-2, Gal-3, Gal-4, Gal-5, Gal-6, Gal-7, Gal-8, Gal-9, Gal-10, Gal-11, Gal-12, Gal-13, Gal-14, or Gal-15.

[0126] 19. The bispecific molecule of embodiment 17, wherein the galectin is Gal-1.

[0127] 20. The bispecific molecule of embodiment 17, wherein the galectin is Gal-3.

[0128] 21. The bispecific molecule of any one of embodiments 1-5, wherein the glycan-binding moiety comprises a glycan-binding domain of a selectin.

[0129] 22. The bispecific molecule of embodiment 21, wherein the selectin is P-selectin (CD62P), E-selectin (CD62E), or L-selectin (CD62L).

[0130] 23. The bispecific molecule of any one of embodiments 1-22, wherein the cell targeting moiety comprises a ligand for a receptor on the surface of the target cell, or a small molecule that binds to a cell surface molecule on the target cell.

[0131] 24. The bispecific molecule of any one of embodiments 1 to 22, wherein the cell targeting moiety comprises an antigen-binding domain of an antibody.

[0132] 25. The cell targeting moiety comprises a variable heavy chain (V H ) domains of the antibody heavy and variable light chains (V L 23. The bispecific molecule of any one of embodiments 1 to 22, comprising an antibody light chain comprising a CDR1-CDR2-GAL2 region.

[0133] 26. The bispecific molecule of embodiment 25, wherein the antibody heavy chain comprises a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or any combination thereof.

[0134] 27. The bispecific molecule of embodiment 25 or 26, wherein the antibody heavy chain comprises a CH2 domain, a CH3 domain, or both.

[0135] 28. The bispecific molecule of any one of embodiments 25 to 27, wherein the antibody heavy chain comprises a fragment crystallizable (Fc) region.

[0136] 29. The bispecific molecule of any one of embodiments 1-28, wherein the glycan-binding moiety comprises an antibody heavy chain domain.

[0137] 30. The bispecific molecule of embodiment 29, wherein the antibody heavy chain domain of the glycan binding moiety comprises a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or any combination thereof.

[0138] 31. The bispecific molecule of embodiment 29 or embodiment 30, wherein the antibody heavy chain domain of the glycan-binding moiety comprises a CH2 domain, a CH3 domain, or both.

[0139] 32. The bispecific molecule of any one of embodiments 29-31, wherein the antibody heavy chain domain of the glycan-binding moiety comprises a fragment crystallizable (Fc) region.

[0140] 33. The bispecific molecule of embodiment 31 or embodiment 32, wherein the cell targeting moiety comprises an antibody heavy chain comprising a CH3 domain and the bispecific molecule is a heterodimer comprising a knobs-into-holes modified CH3 domain.

[0141] 34. The bispecific molecule of any one of embodiments 1-32, wherein the bispecific molecule is a fusion protein comprising a cell targeting moiety fused to a glycan-binding moiety.

[0142] 35. The bispecific molecule of embodiment 34, wherein the cell targeting moiety is fused directly to the glycan-binding moiety.

[0143] 36. The bispecific molecule of embodiment 34, wherein the cell targeting moiety is indirectly fused to the glycan-binding moiety via a linker.

[0144] 37. The bispecific molecule of any one of embodiments 1-32, wherein the bispecific molecule is a conjugate comprising a cell targeting moiety conjugated to a glycan-binding moiety.

[0145] 38. A nucleic acid comprising: The cell targeting moiety of the bispecific molecule of any one of embodiments 1 to 36; The glycan-binding portion of the bispecific molecule according to any one of embodiments 1 to 36; or A nucleic acid that codes for both.

[0146] 39. An expression vector comprising the nucleic acid of embodiment 38.

[0147] 40. A pharmaceutical composition comprising: A bispecific molecule according to any one of embodiments 1 to 37, and a pharma- ceutically acceptable carrier.

[0148] 41. The pharmaceutical composition according to embodiment 40, wherein the cell targeting moiety is a cancer cell targeting moiety.

[0149] 42. The pharmaceutical composition of embodiment 40, wherein the cell targeting moiety is an immune cell targeting moiety.

[0150] 43. A kit comprising: One or more unit doses of the pharmaceutical composition according to any one of embodiments 40 to 42; and instructions for administering one or more unit doses of the pharmaceutical composition to an individual in need thereof.

[0151] 44. The kit according to embodiment 43, comprising two or more unit doses of the pharmaceutical composition.

[0152] 45. The kit of embodiment 43 or embodiment 44, wherein the cell targeting moiety is a cancer cell targeting moiety and the instructions include instructions for administering one or more unit doses of the pharmaceutical composition to an individual in need of enhanced anti-tumor immunity.

[0153] 46. ​​The kit of embodiment 43 or embodiment 44, wherein the cell targeting moiety is an immune cell targeting moiety and the instructions comprise instructions for administering one or more unit doses of the pharmaceutical composition to an individual in need of enhancing or suppressing an immune response.

[0154] 47. A method for enhancing anti-tumor immunity in an individual in need thereof, comprising: A method comprising administering to an individual an effective amount of the pharmaceutical composition of embodiment 41.

[0155] 48. A method for enhancing or suppressing an immune response in an individual in need thereof, comprising: A method comprising administering to an individual an effective amount of the pharmaceutical composition of embodiment 42.

[0156] 49. The method of embodiment 47 or embodiment 48, wherein administration is by parenteral administration.

[0157] 50. A bispecific molecule comprising: a cell targeting moiety fused to an Fc region; a portion comprising the ligand-binding domain of a receptor fused to an Fc region; A bispecific molecule in which a cell targeting moiety and a moiety comprising the ligand binding domain of a receptor are heterodimerized via the Fc region.

[0158] 51. The bispecific molecule according to embodiment 50, wherein the cell targeting moiety is as described in any one of embodiments 2 to 5.

[0159] 52. The bispecific molecule according to embodiment 50 or embodiment 51, wherein the portion comprising a ligand-binding domain of a receptor comprises a ligand-binding domain of a receptor that binds to a cell surface ligand.

[0160] 53. The bispecific molecule according to any one of embodiments 50 to 52, wherein the bispecific molecule is a heterodimer comprising a knobs-into-holes modified CH3 domain. EXAMPLES

[0161] The following examples are offered by way of illustration and not by way of limitation.

[0162] experiment Example 1 - Enhancement of anti-tumor immune responses using bispecific molecules containing a cancer cell targeting moiety and a glycan binding moiety Described herein are bispecific molecules comprising a cell targeting moiety and a glycan binding moiety, and the demonstration that such molecules are effective in enhancing anti-tumor immune responses, for example, by enhancing antibody-dependent cellular phagocytosis (ADCP) and / or cell-mediated cytotoxicity (ADCC).

[0163] As proof of principle, described herein is a new class of antibody-lectin bispecific molecules (sometimes referred to herein as "AbLec") that target tumor-associated sialoglycans for checkpoint blockade. In this approach, recombinant Siglec binding domains with sialoglycan-binding specificity are coupled to high affinity tumor-targeting antibody binding domains. AbLec is predicted to accumulate at high effective molar concentrations on the cancer cell surface, allowing otherwise low affinity recombinant Siglecs to bind to cancer cell surface sialoglycans at therapeutically relevant concentrations.

[0164] As an initial proof of concept, we used a knobs-in-holes approach to generate a panel of recombinant Siglec-7 and Siglec-9-Fc chimeras that dimerize with monovalent antibody chains derived from FDA-approved antibodies (trastuzumab and rituximab) targeting the common cancer antigens HER2 and CD20, respectively. These constructs are shown diagrammatically in Figures ​(Figure11-14), and their amino acid sequences are provided.

[0165] Schematics and data demonstrating that antibody-lectin (AbLec) bispecific antibodies enable the use of lectin decoy receptors for checkpoint blockade are provided in Figure 1a-e. (a) AbLec combines the beneficial properties of monoclonal antibodies (high affinity and selectivity for desired tumor, immune cell or tissue targets) with lectin decoy receptors (selectivity and specificity for cognate glycoconjugate ligands) while overcoming the limitations of each platform. (b) AbLec was engineered using a modified knobs-into-holes strategy using an IgG1 antibody framework. (c) Coexpression of trastuzumab heavy and light chains with Siglec-7-Fc or Siglec-9-Fc chains in Expi293 cells resulted in the expression of a single protein product. Reducing SDS-PAGE analysis showed that these products were composed of three disulfide-linked protein chains consistent with the molecular weights of the Siglec-Fc chain and the trastuzumab heavy and light chains. Western blotting against the HA and His6 tags on the Siglec-Fc and antibody heavy chains, respectively, further demonstrated that the full-length protein product was composed of both antibody and decoy receptor arms. Taken together, this evidence suggests that AbLec is a bifunctional heterodimer composed of both antibody and decoy receptor arms. (d) Dissociation constants K for Trastuzumab × Siglec-7 (T7) and Trastuzumab × Siglec-9 AbLec DThe K values ​​were measured by quantifying binding to SK-BR-3 cells at various concentrations via flow cytometry. SK-BR-3 cells express the HER2 antigen bound by trastuzumab, as well as the ligands Siglec-7 and Siglec-9 by flow cytometry. (e) The decoy receptor molecule (Siglec-7 / 9-Fc) binds only at low levels to SK-BR-3 cells, even at the highest concentration tested (200 nM), despite the fact that SK-BR-3 cells express Siglec-7 and -9 ligands (Figure 1d). However, by combining the decoy receptor arm with the high affinity trastuzumab antibody arm, AbLec is able to bind to SK-BR-3 cells at therapeutically relevant concentrations (left). AbLec exhibits a low nM K similar to the parent antibody, trastuzumab. D Values ​​are shown (right). Moreover, AbLec binding is cooperative. By mutating the conserved arginine residue in the Siglec-Fc binding site to alanine, T7A and T9A AbLec variants were generated that have Siglec-Fc arms with significantly reduced affinity for Siglec ligands, as previously reported. The mutant AbLec showed reduced binding to SK-BR-3 cells (middle) and an apparent K D This resulted in an approximately 2- to 3-fold increase in AbLec binding (right). This suggests that despite the low affinity of the Siglec-Fc decoy receptor molecule, it is able to bind to Siglec ligands when they accumulate at high local concentrations on the cell surface by high-affinity antibody-antigen interactions. The observed contribution of the Siglec-Fc arm to binding may explain why the AbLec dissociation constant is of the same order of magnitude as the bivalent parent antibody trastuzumab, despite having only one antibody arm.

[0166] Example 2 - AbLec blocks binding of targeted glycan-binding immune receptors In this example, it is demonstrated that AbLecs block the binding of targeted glycan-binding immune receptors. Schematics and data are shown in Figures 2a-f and 5. (a) It was hypothesized that if AbLecs, in addition to recruiting immune cells via antibody effector functions, could alleviate inhibitory signaling through the Siglec axis by blocking Siglec ligands on tumor cells, they would have the potential to enhance anti-tumor immune responses compared to parental monoclonal antibodies. (b) Dissociation constants (K D) values ​​were measured by quantifying binding to SK-BR-3 cells at various concentrations via flow cytometry. SK-BR-3 cells express the HER2 antigen bound by trastuzumab, as well as ligands for Siglec-7 and Siglec-9 by flow cytometry. The ability of T7 and T9 AbLec to compete with AF647-labeled Siglec-Fc reagents for binding to HER2+K562 cells expressing the target HER2 antigen and ligands for Siglec-7 and Siglec-9 was tested. Treatment of cells with increasing concentrations of T7 or T9 AbLec was observed by flow cytometry to enhance our ability to block binding of Siglec-7-Fc-AF647 (c) or Siglec-9-Fc-AF647 (d), respectively. AbLec reduced Siglec-Fc binding to near the level of the sialidase control, suggesting that they can block most of the sialic acid-dependent binding of Siglec receptors to cells. (e) AbLec also appears to block only the binding of targeted Siglecs. Treatment with T7 AbLec was able to block the binding of cognate Siglec-7-Fc to a greater extent than treatment with trastuzumab or the non-cognate T9 AbLec at all concentrations tested. (f) AbLec binds to and blocks the same epitope bound by the endogenous receptor. The ability of AbLec to compete with the anti-CD43 antibody MEM59 for binding to HER2+ K562 cells was tested. It has previously been shown that the main ligand for Siglec-7 expressed on K562 cells is the mucin glycoprotein CD43, and that MEM59 can block the binding of Siglec-7 to a sialylated epitope on CD43. We observed that the T7 AbLec was able to block MEM59 binding, suggesting that it binds to and blocks the same CD43 epitope bound by the endogenous Siglec-7 immune receptor. Furthermore, the T7 AbLec was able to block MEM59 binding to a greater extent than either trastuzumab or the non-cognate T9 AbLec.

[0167] FIG. 5 shows that trastuzumab hybrid AbLec binds to various HER2+ human tumor cell lines and blocks Siglec receptor binding. The plot on the left side of the figure shows that T7 AbLec binds to HCC-1954, SK-BR-3, BT-20, and ZR-75-1 cell lines expressing various levels of targeting HER2 antigen and ligand for Siglec-7. The graph on the right side of the figure shows that when we treated SK-BR-3 cells with increasing concentrations of T7 AbLec, our ability to block Siglec-7-Fc-AF647 binding to the cells was enhanced. T7 AbLec reduced Siglec-Fc binding to near the level of the sialidase control, suggesting that AbLec can block most of the sialic acid-dependent binding of Siglec receptors to cells.

[0168] Example 3 - AbLec enhances antibody-dependent cellular phagocytosis and cytotoxicity in vitro. In this example, it is demonstrated that AbLec enhances antibody-dependent cellular phagocytosis and cytotoxicity in vitro. Schematic diagrams and data are shown in Figures 3a-e and 4. (a) In vitro antibody-dependent cellular phagocytosis (ADCP) assays were performed using human macrophages isolated and differentiated from healthy donor peripheral blood. At the time of the assay, macrophages expressed Siglec-7 and Siglec-9. SK-BR-3 target cells were labeled with pHrodo red to allow quantification of ADCP by time-lapse fluorescence microscopy using an Incucyte instrument. (b) Images of macrophage / SK-BR-3 co-culture experiments after 5 hours of incubation show the level of red fluorescence as an indicator of phagocytosis. (c) Across n=3 unique donors, T7 and T9 AbLec significantly enhance ADCP of SK-BR-3 cells compared to trastuzumab or Siglec-Fc alone. (d) In vitro antibody-dependent cellular cytotoxicity (ADCC) assays were performed using human NK cells isolated from healthy donor peripheral blood. At the time of the assay, NK cells expressed Siglec-7. SK-BR-3 target cells were labeled with celltracker red and co-cultured with NK cells in the presence of Sytox green to allow assessment of ADCC activity via quantification of target cell death by flow cytometry. (e) Across n=3 unique donors, T7 AbLec significantly enhanced ADCC of SK-BR-3 cells compared to trastuzumab or Siglec-7-Fc alone.

[0169] Figure 4 shows that AbLec-mediated enhancement of ADCP is dependent on the expression of the target antigen (e.g., HER2). Across n=3 unique donors, T7 and T9 AbLec significantly enhanced ADCP of HER2+ K562 cells compared to trastuzumab or Siglec-7 / 9-Fc alone. However, for WT K562 cells that do not express HER2, we did not observe ADCP activity with either trastuzumab or AbLec. This suggests that AbLec immunotherapy can be specifically targeted to cells expressing the antigen of interest (e.g., tumor antigen, immune cell marker, etc.).

[0170] Example 4 - AbLec is superior to combination immunotherapy via Siglec-dependent enhancement of anti-tumor immune responses in vitro. This example demonstrates that AbLec is superior to combination immunotherapy through Siglec-dependent enhancement of antitumor immune responses in vitro. Schematic diagrams and data are shown in Figure 6a-6d. (a) T7 and T9 AbLec induced enhanced ADCP of HER2+ K562 cells compared to trastuzumab in combination with Siglec-7-Fc, Siglec-9-Fc, or V. cholerae sialidase across n=3 unique donors. T7 and T9 AbLec induced enhanced ADCP (b) and ADCC (c) of SK-BR-3 cells compared to trastuzumab in combination with Siglec-7 or Siglec-9 antagonist antibodies or V. cholerae sialidase across n=3 unique donors. (d) AbLec-mediated enhancement of ADCP and ADCC was Siglec-dependent. When macrophages (top) or NK cells (bottom) were incubated with Siglec-7 or Siglec-9 antagonist antibodies prior to co-culture with SK-BR-3 cells, which essentially function as Siglec-7 / 9 knockouts in immune cells, the ADCP or ADCC levels observed upon AbLec treatment were reduced to levels similar to those observed with trastuzumab treatment. However, AbLec enhanced in vitro ADCP and ADCC when the Siglec immunoreceptors were not blocked. This Siglec dependency was observed across n=3 unique donors, suggesting that the enhanced ADCP and ADCC observed with AbLec treatment is the result of blocking the targeted Siglec axis.

[0171] Example 5 - The AbLec platform enables blockade of diverse glyco-immune checkpoint targets. In this example, it is demonstrated that the AbLec platform enables the blockade of diverse glycoimmune checkpoint targets. Schematics and data are shown in Figures 7a-7d and 8. (a) Due to the modular structure of the knob-into-hole bispecific scaffold used to generate AbLec, the antibody and decoy receptor components can be easily exchanged for the production of additional checkpoint inhibitors. (b) SDS-PAGE characterization of additional AbLec candidates with diverse mechanisms of action. Magrolimab x Siglec-7 AbLec is engineered for dual checkpoint blockade of CD47 and Siglec-7 ligands on tumor cells. Magrolimab is an anti-CD47 antibody (Liu et al. 2015) currently in Phase III clinical trials for hematological cancers (Garcia-Manero et al. 2021). Pembrolizumab x Galectin-9 AbLec is engineered for dual checkpoint blockade of PD-1 and Galectin-9 ligands on exhausted T cells. Galectin-9 has been shown to contribute to immune evasion by binding to the TIM-3 checkpoint on T cells and contributing to T cell exhaustion (Yang et al. 2021). Trastuzumab × Siglec-10 AbLec was designed to simultaneously target HER2+ tumors and block the Siglec-10 immune checkpoint, which was recently shown to play a role in immune evasion in breast and ovarian cancer (Barkal et al. 2019). Functional characterization of rituximab × Siglec-7 (R7) and cetuximab × Siglec-7 (C7) AbLec demonstrates the utility of the AbLec platform for combined tumor targeting and glycoimmune checkpoint blockade in diverse tumor types. Across n=3 unique donors, R7(c) and C7(d) AbLec significantly enhance ADCP of CD20+ Ramos or EGFR+ K562 cells compared to parental antibody or Siglec-7-Fc alone.

[0172] Figure 8 shows that across n=3 unique donors, R7 AbLec also enhanced ADCC of CD20+Raji cells compared to rituximab (left). The enhancement of ADCC was a sialic acid-dependent effect, as there was no significant enhancement of ADCC by R7 AbLec compared to rituximab after treatment with sialidase (right).

[0173] Example 6 - Expression of diverse AbLec molecules Shown in Figure 9 are reducing and non-reducing SDS-PAGE and Western blot analysis of various AbLec molecules. Reducing SDS-PAGE demonstrated that each AbLec is composed of three disulfide-linked protein chains that match the molecular weights of the decoy receptor chain and the antibody heavy and light chains. Western blotting against HA and His6 tags on the decoy receptor and antibody heavy chains, respectively, further demonstrated that full-length AbLec is composed of both antibody and decoy receptor arms.

[0174] Materials and Methods Plasmid and protein sequences All AbLec plasmids were generated by Twist Bioscience. The DNA sequences are listed in Table 1 and the protein sequences are listed in Table 2. The AbLec sequences were inserted into the Twist Bioscience vector pTwist CMV BetaGlobin using the XhoI and NheI cleavage sites. Trastuzumab used in this specification was expressed from the pCDNA3.1 vector described in our previous work (Gray et al. 2020). Rituximab and cetuximab antibody variable sequences were generated by IDT and cloned into the variable regions of the VRC01 antibody plasmid vector (courtesy of Kim lab, Stanford) by using the In-Fusion cloning kit (Takara) according to the manufacturer's protocol.

[0175] Protein expression and purification Antibodies and cultured AbLecs were expressed in the Expi293F system (Thermo Fisher) and expressed according to established manufacturer protocols. For rituximab and cetuximab antibodies, a 1:1 weight ratio of heavy chain plasmid to light chain plasmid was used. For AbLecs, a 2:1:1 ratio of lectin:heavy chain:light chain was used. Trastuzumab antibody heavy and light chains were co-expressed from a single plasmid. After 7 days of expression, cells were pelleted at 300×g for 5 minutes, followed by a clarification spin at 3700×g for 40 minutes, and protein was collected from the supernatant by filtration through a 0.2 μm nylon filter (Fisher Scientific 0974025A). Antibodies were purified by manual gravity column using Protein A agarose (Fisher Scientific 20333) by loading the clarified supernatant onto the column 2x, sialidase treatment on the beads with 2μM ST sialidase for 0.5-2 hours at room temperature, then washing with 5x column volumes of PBS and eluting with 100mM glycine buffer pH 2.8 in 5mL increments into tubes pre-equilibrated with 150uL 1M Tris pH 8. Antibodies were buffer exchanged into PBS using a PD-10 column (GE). Cultured AbLec was purified by manual gravity column using Nickel-NTA agarose resin (Qiagen 30210). Briefly, AbLec supernatant was incubated with the resin (pre-equilibrated in PBS) for approximately 1 hour at 4C. The beads and supernatant were then loaded onto a chromatography column (BioRad 7321010) and the sialidase on the beads was treated with 2 μM ST sialidase for 2 h at room temperature, washed with 20× column volumes of PBS+20 mM imidazole, and eluted twice with 5× column volumes of PBS+250 mM imidazole. AbLec was buffer exchanged into PBS using a PD-10 desalting column.

[0176] Bioconjugation to generate Siglec-Fc-AF647 reagents Siglec-Fc DNA sequences were expressed in Expi293F cells co-expressing a stable human FGE protein for aldehyde tagging. Cells were expressed in the dark according to the Expi293 protocol from Thermo Fisher, then filtered through a 0.2 μm filter and loaded onto a column containing protein A agarose beads. Proteins were sialidase treated on the column for 2 hours (2 μM Salmonella Typhimurium sialidase, room temperature). Subsequently, they were washed with PBS and eluted in 100 mM glycine (pH 2.8), 10 mL buffered Tris pH 8 solution. Siglecs were buffer exchanged into an acidic buffer, concentrated, and conjugated with HIPS-azide according to the protocol from Gray, et al. (Gray et al. 2020). The Siglec-Fc-azide was then taken without further characterization, buffer exchanged into PBS, 100x molar equivalents of DBCO-AF647 (Click Chemistry Tools, 1302-1) in DMSO was added, and the reaction was mixed at room temperature at 500 rpm for 2 hours in the dark. The Siglecs were buffer exchanged by centrifugation six times on an Amicon column (30 kDa MWCO) in PBS, and AF647 addition was confirmed by using a NanoDrop spectrophotometer at 650 nm for the AF647 dye (extinction coefficient 239,000) and 280 nm for the proteins (using the extinction coefficients calculated for each Siglec by Expasy). 18

[0177] AbLec gel characterization For SDS-PAGE gels, 2 μg of protein with SDS dye alone (non-reducing conditions) or SDS dye + 1M beta-mercaptoethanol heated at 95C for 5 min (reducing conditions) was loaded onto Bis-Tris 4-12% Criterion™ XT Bis-Tris Protein Gel, 18-well (Bio-Rad 3450124) and run at 180V for 40 min with XT-MES buffer. Proteins were stained with Aquastain (Bulldog Bio AS001000) for 10 min followed by destaining in water for 10 min. For Western blotting of AbLec, 0.2 μg of protein was loaded onto SDS-PAGE gels and run as above, then the gels were transferred to nitrocellulose membranes using the Trans-Blot® Turbo™ RTA Midi Nitrocellulose Transfer Kit (Bio-Rad 1704271), 25V, 14 min. Membranes were blocked in blocking buffer (PBS+0.5% BSA) for 1 hour at room temperature, then stained with Invitrogen HA tag polyclonal antibody (SG77) (Thermo Fisher Scientific 71-5500) and purified anti-His Tag antibody (BioLegend 652502) in blocking buffer for 1 hour with shaking at room temperature. Membranes were washed three times in PBST (PBS+0.1% Tween) and subsequently stained with secondary antibodies IRDye® 800CW goat anti-mouse and IRDye® 680RD goat anti-rabbit (LI-COR) in PBST for 15 minutes at room temperature with shaking, followed by another three washes in PBST. All gels were imaged on an Odyssey® CLx Imaging System (LI-COR).

[0178] AbLec melting temperature characterization SYPRO orange dye (Thermo Fisher) was diluted to make a 25x stock and 5uL (final 5x concentration with antibody) was added to 20uL of AbLec, Siglec-Fc, or antibody at 0.2mg / mL in PBS to make 25uL per well in a 96-well qPCR plate. Protein denaturation was analyzed in the FRET fluorescence channel in qPCR by increasing the temperature from 25°C to 95°C by 0.5°C per minute.

[0179] AbLec mass spectrometry characterization For proteomic analysis, 4 micrograms of each AbLec dissolved in PBS were digested with trypsin. Samples were incubated with 5 mM dithiothreitol at 55°C for 18 min, followed by incubation with 15 mM iodoacetamide in the dark for 30 min at room temperature. Trypsin (Promega) was added at a 1:20 w:w ratio, and digestion was allowed to proceed overnight at room temperature. The following morning, samples were desalted by first quenching the digestion with formic acid to a final pH of approximately 2, followed by desalting on polystyrene-divinylbenzene solid-phase extraction (PS-DVB SPE) cartridges (Phenomenex, Torrance, CA). Samples were dried in a vacuum centrifuge after desalting and resuspended at 0.5 μg / μL in 0.2% aqueous formic acid.

[0180] Approximately 1 μg of peptides was injected per run and peptides were separated on a 25 cm EasySpray reversed-phase LC column (2 μm, 100 Å, 75 μm internal diameter packed with PepMap C18 particles, Thermo Fisher Scientific). The mobile phase (A: water with 0.2% formic acid and B: acetonitrile with 0.2% formic acid) was driven and controlled by a Dionex Ultimate 3000 RPLC nano system (Thermo Fisher Scientific). Gradient elution was performed at 300 nL / min. Mobile phase B was held at 0% for 6 min, followed by an increase to 5% at 7 min, 25% at 66 min, ramped to 90% B at 70 min, and washed at 90% B for 5 min. The flow rate was then returned to 0% B at 75.1 min and the column was re-equilibrated at 0% B for 15 min, giving a total run time of 90 min. Eluted peptides were analyzed on an Orbitrap Fusion Tribrid MS system (Thermo Fisher Scientific). Precursors were ionized using an EASY-Spray ionization source (Thermo Fisher Scientific) held at +2.2 kV relative to ground and the column was held at 40 °C. The inlet capillary temperature was held at 275 °C. Survey scans of peptide precursors were collected in the Orbitrap from 350 to 1350 m / z with an AGC target of 1,000,000, a maximum injection time of 50 ms, and a resolution of 60,000 at 200 m / z. Monoisotopic precursor selection was enabled for peptide isotope distributions, and precursors from z = 2 to 5 were selected for data-dependent MS / MS scans with cycle times of 2 s, dynamic exclusion was set to 30 s, and a ±10 ppm window was set around the precursor monoisotope. Precursor ions were selected in the quadrupole using an isolation window of 1 m / z. MS / MS scans were collected using HCD at 30 normalized collision energy (nce) with an AGC target of 100,000 and a maximum injection time of 54 ms. Mass spectrometry was performed in the Orbitrap with a resolution of 30,000 at 200 m / z and a scan range set to autocalc.

[0181] Raw data is then fed to Byonic 19The methionine oxidation (+15.994915) was calculated using the general 20 We set the nucleotide sequence of the nucleotide sequence of the nucleotides as 1 and 2 as variable modifications, identified protein N-terminal acetylation (+42.010565) and asparagine deamination (+0.984016) as rare 1 variable modifications, and set carbamidomethylation of cysteine ​​(+57.021464) as a fixed modification. Up to three common modifications and two rare modifications were allowed. A precursor ion search tolerance of 10 ppm and a product ion mass tolerance of 20 ppm were used for the search, allowing three missing cleavages for perfect trypsin specificity. Peptide spectral matches (PSMs) were created against a custom FASTA sequence file containing the appropriate combination of Siglec-7 and -9 holes and trastuzumab / rituximab knob and light chains. Peptides were filtered to a 1% false discovery rate (FDR) and a 1% protein FDR was applied according to the target-decoy method. 2 All peptide identifications were manually inspected and sequence coverage was calculated only from verified peptide identifications. The sequence coverage percentage is derived from the proportion of amino acids accounted for by peptide identifications relative to the total number of amino acids.

[0182] cell culture All cell lines were purchased from the American Type Culture Collection (ATCC). SK-BR-3, HCC-1954, K562, Raji, and Ramos were cultured in RPMI + 10% heat-inactivated fetal bovine serum (FBS) without antibiotic selection. Expi293F cells were a gift from the Kim lab at Stanford and cultured according to the Thermo Fisher Scientific user guide. Prepackaged lentiviral particles (G&P Biosciences) were used to transfect K562 with EGFR according to the manufacturer's protocol and selected for EGFR expression by culture in 1 μg / mL puromycin (InVivoGen). Prepackaged lentiviral particles (G&P Biosciences LTV-CD20) were used to transfect K562 with CD20 according to the manufacturer's protocol and sorted for CD20 expression using rituximab and a BV421-conjugated anti-human secondary antibody (Biolegend) as the staining reagent on the FACS instrument. HER2 WT was a gift from Mien-Chie Hung (Addgene plasmid #16257), and stable HER2 + Cell lines were generated according to their protocols, 21 Protein expression was verified by flow cytometry. Cell lines were not independently authenticated beyond the identity provided by ATCC. Cell lines were cultured in a humidified incubator at 5% CO2 and 37°C and tested negative for mycoplasma quarterly using a PCR-based assay.

[0183] AbLec K D Quantification HER2+ K562 and SK-BR-3 cells were isolated from cell culture supernatant or via dissociation with TrypLE (Gibco), respectively, washed with 1x PBS, and resuspended in blocking buffer. 60,000 cells were then distributed into wells of a 96-well V-bottom plate (Corning). Equal volumes of trastuzumab, Siglec-Fc, or AbLec at various concentrations (200-0.4 nM) were added to the cells and incubated with the cells for 1 h at 4 °C with periodic pipette mixing. Cells were washed three times in blocking buffer and pelleted by centrifugation at 300 g for 5 min at 4 °C between washes. Cells were resuspended in AF647 goat anti-human (BioLegend) in blocking buffer for 30 min at 4 °C. Cells were washed two more times, resuspended in blocking buffer, and analyzed for fluorescence by flow cytometry (BD LSR II). Gating was performed using FlowJo v.10.0 software (Tree Star) to remove debris and isolate single cells. The mean fluorescence intensity (MFI) of the cell population was normalized to the MFI of cells stained with 50 nM trastuzumab from each experimental replicate. GraphPad Prism 9 was used to fit MFI values ​​to a one-site total binding curve and the K was calculated as the antibody concentration required to achieve half-maximal binding. D values ​​were calculated.

[0184] Competitive binding with Siglec-Fc-AF647 reagent HER2+ K562 and SK-BR-3 cells were isolated from cell culture supernatant or via dissociation with TrypLE (Gibco), respectively, washed with 1x PBS, and resuspended in blocking buffer. Cells were dispensed for sialidase treatment with 100 nM V. cholerae sialidase for 30 min at 37 °C in blocking buffer. Then, 60,000 untreated or sialidase-treated cells were distributed into wells of a 96-well V-bottom plate (Corning). Various concentrations (100-0.4 nM) of trastuzumab or AbLec were added to the cells in equal volumes along with 200 nM Siglec-Fc-AF647 reagent and incubated with the cells for 2-3 h at 4 °C with regular pipette mixing. Cells were washed three times, resuspended in blocking buffer, pelleted by centrifugation at 300g for 5 min at 4°C between washes, and fluorescence analyzed by flow cytometry (BD LSR II). Gating was performed using FlowJo v.10.0 software (Tree Star) to remove debris and isolate single cells. The mean fluorescence intensity (MFI) of the cell population was normalized to the MFI of cells stained with 200 nM Siglec-Fc-AF 647 without antibody or AbLec from each experimental replicate. MFI values ​​were fitted to a one-site total binding curve using GraphPad Prism 9, and the K was calculated as the antibody concentration required to achieve half-maximal binding. D values ​​were calculated.

[0185] Isolation of donor NK cells PBMCs were isolated from the LRS chamber as described above and cultured at 2–4 × 10 in 90% heat-inactivated FBS + 10% DMSO. 7 Stocks were prepared with cells and stored in liquid nitrogen vapor until use. The day before use, stocks were thawed and NK cells were isolated using an EasySep NK Isolation Kit (StemCell Technologies 17955) and cells were cultured overnight with 0.5 μg / mL recombinant IL-2 (Biolegend 589106) in RPMI + 10% heat-inactivated FBS until use.

[0186] NK cell flow cytometry After activation with IL-2 for 24 hours, NK cells were collected from culture supernatants, washed with 1x PBS, and resuspended in blocking buffer. On the day of analysis, macrophages were stained with anti-CD16, Siglec-7, and isotype control in blocking buffer for 30 minutes at 4°C. After washing twice in PBS, NK cells were analyzed by flow cytometry (BD LSR II) and gated for CD16 positive cells using FlowJo v10. NK cells were >85% pure by flow cytometry.

[0187] NK cell killing assay Target cells were lifted and stained with celltracker deep red dye according to the manufacturer's protocol. NK cells and target cells were mixed at an effector to target (E / T) ratio of 4:1 and Sytox Green (Thermo) was added at 100 nM. Cell death was monitored by flow cytometry using red (FL4-A) staining. + ) Cells were selected and stained with Sytox Green + The percentage of death was analyzed by calculating as / total red blood cells. Replicates from three unique blood donors were plotted in Prism 9.0 (GraphPad Software, Inc).

[0188] Isolation and differentiation of donor macrophages LRS chambers were obtained from healthy anonymous blood bank donors and PBMCs were isolated using Ficoll-Paque (GE Healthcare Life Sciences) density gradient separation. Approximately 1 × 10 8 Monocytes were isolated by plating PBMCs for 1-2 h followed by rigorous washing 3 times with PBS+Ca+Mg to remove non-adherent cells. The medium was then replaced with IMDM containing 10% human AB serum (Gemini) and macrophages were allowed to differentiate for 7-9 days before being used for phagocytosis or flow cytometry experiments.

[0189] Macrophage flow cytometry Day 7-9 macrophages were lifted from the plates as above, then fixed with 4% formaldehyde (Thermo) in PBS for 15 min, washed 3 times with PBS, and stored at 4°C for 2-7 days until analysis. On the day of analysis, macrophages were stained with CD11b, CD14, Siglec-7, Siglec-9, Siglec-10, and isotype control in blocking buffer for 30 min at 4°C. After two washes in PBS, macrophages were analyzed by flow cytometry on an LSR II instrument and gated for CD11b and CD14 double positive cells using FlowJo v10. Macrophages were >85% pure by flow cytometry.

[0190] Phagocytosis assay Macrophages were washed with PBS and removed by incubation with 10 mL of TrypLE (Thermo) for 20 min at 37°C. RPMI + 10% HI FBS was added to an equal volume, and macrophages were pelleted by centrifugation at 300 x g for 5 min and resuspended in IncuCyte medium (RPMI + 10% HI FBS without phenol red). Macrophages (10,000 cells, 100 μL) were added to a 96-well flat-bottom plate (Corning) and incubated for 1 h at 37°C in a humidified incubator. Meanwhile, target cells were washed once with PBS and then treated with pHrodo red succinimydyl ester dye (Thermo Fisher) diluted 1:80,000 in PBS for 30 min at 37°C, washed once, and resuspended in IncuCyte medium. Finally, 10uL of 20x antibody or AbLec stock in PBS was added to the macrophages, followed by the addition of pHrodo Red stained target cells (90uL, 20,000 cells). Cells were plated by gentle centrifugation (50xg, 2 min). Two images were acquired per well at 1h intervals until maximum signal was reached (5h for breast cancer cell lines and K562 cells, 2h for Raji and Ramos cells). Quantification of pHrodo Red fluorescence was empirically optimized for phagocytosis of each cell line based on their background fluorescence and size. K562 was analyzed with a threshold of 0.8, edge sensitivity of -70, and areas were scaled from 100 to 2000μm. 2 Gating was performed on the 300–2000 RCU×μm 2 HCC-1954 was imaged with a threshold of 1.5, an edge sensitivity of -45, and a focus of 30-2000 μm. 2 The SK-BR-3 analysis had a threshold of 1, an edge sensitivity of -55, a minimum integrated intensity of 60, and a maximum area and eccentricity of 3000 and 0.96, respectively. Ramos and Raji gating had a threshold of 1.5, an edge sensitivity of -45, and a focus range of 100 to 2000 μm. 2 The total red object integrated intensity (RCU × μm 2A mean mean of 1000 cells / image was obtained for each image. For each experiment, maximum phagocytosis measured by pHrodo red was normalized to 1, and then triplicate technical well replicates were averaged for each biological replicate. Replicates from three unique blood donors were plotted in Prism 9.0 (GraphPad Software, Inc).

[0191] Acquiring IncuCyte Images For phagocytosis and toxicity assays, images were acquired over time using an Incucyte® S3 Live-Cell Analysis System (Essen BioScience) in a Thermo Fisher Scientific tissue culture incubator maintained at 37°C with 5% CO2. Data were acquired from a 10x objective with phase contrast, green fluorescent channel (e.g., 460±20, em: 524±20, acquisition time: 300ms), and red fluorescent channel (e.g., 585±20, em: 665±40, acquisition time: 400ms). Two images were acquired per well with time intervals. All cells were imaged using a 100μm radius, edge split on, hole fill: 0μm unless otherwise stated. 2 Analyzed by Top-Hat segmentation using

[0192] Cell growth and toxicity assays GFP + SK-BR-3, HER2 +K562 and HCC-1954 cells were lifted with 2 mL trypsin for 5 min at 37°C, rinsed with 8 mL normal growth medium, and cells were pelleted by centrifugation at 300 x g and resuspended in phenol red-free growth medium containing 50 nM Sytox Green cell death stain (Thermo Fisher) or 5 nM Sytox Red dead cell stain (Thermo Fisher) for the GFP-positive SK-BR-3 line to measure cytotoxicity. Cells were plated (10,000 cells / well, 95 uL) on flat-bottom 96-well plates, then 5 uL of AbLec, Siglec, or antibody in PBS was added, mixed, and then centrifuged at 30 x g for 1 min. Images were acquired every 2 hours for 3 days. SK-BR-3 cells were segmented using segmentation adjustments = 1 and 200 μm 2 Cell death was quantified by red fluorescence using a threshold of 0.3 RCU, edge sensitivity of -50 and area of ​​50-1000 square microns. HCC-1954 cells had a 0.9 segmentation adjustment, 300 square micron hole fill, and a minimum area of ​​200 square microns. Sytox green death events were detected with a threshold of 1, edge sensitivity of -45, and area of ​​100-3000 square microns. K562 phase segmentation adjustment was 0.2, with no hole fill and a minimum area of ​​60 microns. For the green fluorescence channel, the threshold was 2, edge sensitivity was -45, area was 50-800 microns, and eccentricity and integrated intensity were less than 0.95 and 40000, respectively.

[0193] statistical analysis Statistical analysis was performed in Prism (version 9). For binding curves, one-site specific binding curves were used to calculate the dissociation constants of antibodies and AbLec. For binding assays, NK cell cytotoxicity experiments, phagocytosis experiments and Siglec expression analysis, ordinary one-way ANOVA with Tukey's multiple comparison test was performed to compare treatment groups. In all cases, asterisks * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001,**** indicates p<0.0001.

[0194] Amino acid sequence The amino acid sequences of AbLec and antibodies used herein are shown in the table below. Amino acids in italics represent signal transduction sequences not present in the final purified molecule. HA and hexahistidine tags are underlined or bold, and the stop codon is denoted by an asterixis. * It is expressed as:

[0195] [Table 1-1]

[0196] [Table 1-2]

[0197] [Table 1-3]

[0198] [Table 1-4]

[0199] [Table 1-5]

[0200] [Table 1-6]

[0201] [Table 1-7]

[0202] [Table 1-8]

[0203] [Table 1-9]

[0204] Nucleotide sequence The nucleotide sequences encoding the AbLec and antibodies used herein are shown in the table below. The sequences include a transport signal peptide, peptide tags such as a hexahistidine tag and an HA tag, and a stop codon.

[0205] [Table 2-1]

[0206] [Table 2-2]

[0207] [Table 2-3]

[0208] [Table 2-4]

[0209] [Table 2-5]

[0210] [Table 2-6]

[0211] [Table 2-7]

[0212] [Table 2-8]

[0213]

Table 2-9

[0214]

Table 2-10

[0215]

Table 2-11

[0216]

Table 2-12

[0217]

Table 2-13

[0218]

Table 2-14

[0219]

Table 2-15

[0220]

Table 2-16

[0221]

Table 2-17

[0222]

Table 2-18

[0223]

Table 2-19

[0224]

Table 2-20

[0225]

Table 2-21

[0226]

Table 2-22

[0227] References Barkal,A.A.,R.E.Brewer,M.Markovic,M.Kowarsky,S.A.Barkal,B.W.Zaro,V.Krishnan,et al.2019.「CD24 Signalling through Macrophage Siglec-10 Is a Target for Cancer Immunotherapy.」Nature572:392-96. Garcia-Manero,Guillermo,Naval Guastad Daver,Jin Xu,Mark Chao,Trisha Chung,Anderson Tan,Vivian Wang,Andrew Wei,Paresh Vyas,and David Andrew Sallman.2021.「Magrolimab+Azacitidine versus Azacitidine+Placebo in Untreated Higher Risk(HR)Myelodysplastic Syndrome(MDS):The Phase 3,Randomized,ENHANCE Study.」Journal of Clinical Orthodontics:JCO39(15_suppl):TPS7055 TPS7055. Gray,M.A.,M.A.Stanczak,N.R.Mantuano,H.Xiao,J.F.A.Pijnenborg,S.A.Malaker,C.L.Miller,et al.2020.「Targeted Glycan Degradation Potentiates the Anticancer Immune Response in Vivo.」Nature Chemical Biology16:1376-1384. Ibarlucea-Benitez,Itziar,Polina Weitzenfeld,Patrick Smith,and Jeffrey V.Ravetch.2021.「Siglecs-7 / 9 Function as Inhibitory Immune Checkpoints in Vivo and Can Be Targeted to Enhance Therapeutic Antitumor Immunity.」Proceedings of the National Academy of Sciences of the United States of America118(26).https: / / doi.org / 10.1073 / pnas.2107424118. Liu,Jie,Lijuan Wang,Feifei Zhao,Serena Tseng,Cyndhavi Narayanan,Lei Shura,Stephen Willingham,et al.2015.「Pre-Clinical Development of a Humanized Anti-CD47 Antibody with Anti-Cancer Therapeutic Potential.」PloS One10(9):e0137345. Wisnovsky,Simon,Leonhard Mockl,Stacy A.Malaker,Kayvon Pedram,Gaelen T.Hess,Nicholas M.Riley,Melissa A.Gray,et al.2021.「Genome-Wide CRISPR Screens Reveal a Specific Ligand for the Glycan-Binding Immune Checkpoint Receptor Siglec-7.」Proceedings of the National Academy of Sciences of the United States of America118(5).https: / / doi.org / 10.1073 / pnas.2015024118. Yang,Riyao,Linlin Sun,Ching-Fei Li,Yu-Han Wang,Jun Yao,Hui Li,Meisi Yan,et al.2021.「Galectin-9 Interacts with PD-1 and TIM-3 to Regulate T Cell Death and Is a Target for Cancer Immunotherapy.」Nature Communications12(1):1-17.

[0228] Thus, the above merely illustrates the principles of the disclosure. It will be appreciated that those skilled in the art can devise various configurations that embody the principles of the present invention and are within the spirit and scope thereof, although not explicitly described or illustrated herein. Furthermore, all examples and conditional language recited herein are intended primarily to aid the reader in understanding the principles of the present invention and the concepts that the inventors have contributed to advancing the art, and should not be construed as being limited to such specifically recited examples and conditions. Furthermore, all statements herein that describe the principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Moreover, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Thus, the scope of the present invention is not limited to the exemplary embodiments shown and described herein.

Claims

1. 1. A bispecific molecule comprising: a cell targeting moiety that is a cancer cell targeting moiety; and and a glycan-binding moiety, Bispecific molecules.

2. The cancer cell targeting moiety is selected from the group consisting of 5T4, AXL receptor tyrosine kinase (AXL), B cell maturation antigen (BCMA), c-MET, C4.4a, carbonic anhydrase 6 (CA6), carbonic anhydrase 9 (CA9), cadherin-6, CD19, CD20, CD22, CD25, CD27L, CD30, CD33, CD37, CD44, CD44v6, CD56, CD70, CD74, CD79b, CD123, CD138, carcinoembryonic antigen (CEA), cKit, Cripto protein, CS1, delta-like canonical notch ligand 3 (DLL3), 3), endothelin receptor type B (EDNRB), ephrin A4 (EFNA4), epidermal growth factor receptor (EGFR), EGFRvIII, ectonucleotide pyrophosphatase / phosphodiesterase 3 (ENPP3), EPH receptor A2 (EPHA2), fibroblast growth factor receptor 2 (FGFR2), fibroblast growth factor receptor 3 (FGFR3), FMS-like tyrosine kinase 3 (FLT3), folate receptor 1 (FOLR1), GD2 ganglioside, glycoprotein nontransferase B (GPNMB), guanylate cyclase 2C (GUCY2C) , human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), integrin alpha, lysosome-associated membrane protein 1 (LAMP-1), Lewis Y, LIV-1, leucine-rich repeat-containing 15 (LRRC15), mesothelin (MSLN), mucin 1 (MUC1), mucin 16 (MUC16), sodium-dependent phosphate transport protein 2B (NaPi2b), nectin-4, NMB, NOTCH3, p-cadherin (p-CAD), programmed cell death receptor ligand 1 (PD-L1), programmed cell death receptor ligase 2. The bispecific molecule of claim 1, wherein the bispecific molecule binds to a cancer cell surface molecule selected from the group consisting of: prostate-specific membrane antigen (PSMA), protein tyrosine kinase 7 (PTK7), solute carrier family 44 member 4 (SLC44A4), SLIT-like family member 6 (SLITRK6), STEAP family member 1 (STEAP1), tissue factor (TF), T-cell immunoglobulin and mucin protein-1 (TIM-1), Tn antigen, trophoblast surface antigen (TROP-2), and Wilms' tumor 1 (WT1).

3. A bispecific molecule comprising: a cell targeting moiety; and and a glycan-binding moiety, Bispecific molecules.

4. The cell targeting moiety is selected from the group consisting of PD-1, PD-L1, PD-L2, CLTA-4, VISTA, LAG-3, TIM-3, CD24, CD47, SIRP alpha, CD3, CD8, CD4, CD28, CD80, CD86, CD19, ICOS, OX40, OX40L, GD3 ganglioside, TIGIT, Siglec-2, Siglec-3, Siglec-7, Siglec-8, Siglec-9, Siglec-10, Siglec-15, Galectin-9, 4. The bispecific molecule of claim 3, which is an immune cell targeting moiety that binds to an immune cell surface molecule selected from the group consisting of B7-H3, B7-H4, CD40, CD40L, B7RP1, CD70, CD27, BTLA, HVEM, KIR, 4-1BB, 4-1BBL, CD226, CD155, CD112, GITR, GITRL, A2aR, CD137, CD137L, CD45, CD206, CD163, TRAIL, NKG2D, CD16, and TGF-beta.

5. The bispecific molecule of any one of claims 1 to 4, wherein the glycan binding moiety comprises a sialoglycan binding moiety.

6. The bispecific molecule of claim 5 , wherein the sialoglycan binding moiety comprises a sialoglycan binding domain of a lectin.

7. The bispecific molecule of claim 6 , wherein the lectin is a sialic acid-binding immunoglobulin-like lectin (Siglec).

8. 8. The bispecific molecule of claim 7, wherein the Siglec is selected from the group consisting of Siglec-1, Siglec-2, Siglec-3, Siglec-4, Siglec-5, Siglec-6, Siglec-7, Siglec-8, Siglec-9, Siglec-10, Siglec-11, Siglec-14, Siglec-15, and Siglec-16.

9. The bispecific molecule of any one of claims 1 to 4, wherein the glycan-binding moiety comprises a glycan-binding domain of a protein selected from a C-type lectin, a galectin, a selectin, and a Siglec-like adhesin.

10. 5. The bispecific molecule of claim 1, wherein the cell targeting moiety comprises a ligand for a receptor on the surface of a target cell or a small molecule that binds to a cell surface molecule on a target cell.

11. The bispecific molecule of any one of claims 1 to 4, wherein the cell targeting moiety comprises an antigen-binding domain of an antibody.

12. The cell targeting moiety comprises a variable heavy chain (V Ｈ The antibody heavy and variable light chains (V Ｌ 5. The bispecific molecule of claim 1 , comprising an antibody light chain comprising a 2-aminobutyric acid (AAS) region.

13. The bispecific molecule of any one of claims 1 to 4, comprising a Fragment Crystallizable (Fc) domain.

14. The bispecific molecule of claim 13 , wherein the glycan binding moiety is fused to an Fc domain.

15. 15. The bispecific molecule of claim 14, wherein the cell targeting moiety is fused to an antibody heavy chain comprising a CH3 domain, and the bispecific molecule is a heterodimer comprising a knob-into-hole modified CH3 domain of an Fc region.

16. The bispecific molecule of any one of claims 1 to 4, wherein the bispecific molecule is a fusion protein comprising the cell targeting moiety fused to the glycan binding moiety.

17. A nucleic acid comprising: A cell targeting moiety of the bispecific molecule of any one of claims 1 to 4; The glycan-binding portion of the bispecific molecule of any one of claims 1 to 4; or A nucleic acid that codes for both.

18. An expression vector comprising the nucleic acid of claim 17.

19. 1. A pharmaceutical composition comprising: A bispecific molecule according to any one of claims 1 to 4, and a pharma- ceutically acceptable carrier.

20. A kit comprising:

20. One or more unit doses of the pharmaceutical composition of claim 19; and instructions for administering one or more unit doses of said pharmaceutical composition to an individual in need thereof.

21. 10. A pharmaceutical composition for use in a method for enhancing anti-tumor immunity in an individual in need thereof, said pharmaceutical composition comprising a bispecific molecule according to claim 1 or 2, The method comprises administering to the individual an effective amount of the pharmaceutical composition.

22. 5. A pharmaceutical composition for use in a method for enhancing or suppressing an immune response in an individual in need thereof, said pharmaceutical composition comprising a bispecific molecule according to claim 3 or 4, The method comprises administering to the individual an effective amount of the pharmaceutical composition.

Images