SMAGP fusion molecules and methods of use thereof

Abstract

Provided herein are fusion molecules comprising the extracellular domain of SMAGP that can modulate leukocyte activity. Also provided are polynucleotides, vectors, and host cells encoding these fusion molecules, as well as methods for making and using these fusion molecules.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 510,290, filed June 26, 2023, U.S. Provisional Application No. 63 / 504,348, filed May 25, 2023, U.S. Provisional Application No. 63 / 449,846, filed March 3, 2023, and U.S. Provisional Application No. 63 / 478,836, filed January 6, 2023, the contents of each of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing The contents of the electronically submitted Sequence Listing XML (Name: 206873_SL.xml, Size: 73,597 bytes, Created: January 2, 2024) are incorporated herein by reference in their entirety.

[0003] The present disclosure relates to fusion molecules comprising the extracellular domain of SMAGP, methods for making these fusion molecules, and methods for using these fusion molecules to modulate leukocyte activity in a subject. [Background technology]

[0004] Phagocytes are immune cells that play a key role in both the early and late stages of the immune response. Their primary role is to circulate and travel through tissues, ingesting and destroying both microorganisms and cellular debris. However, activation of phagocytes can also contribute to the pathology of autoimmune diseases such as lupus, rheumatoid arthritis, multiple sclerosis, hemophagocytic lymphohistiocytosis (HLH), and immune thrombocytopenia (ITP).

[0005] Thus, there is a need for novel compositions and methods capable of inhibiting phagocyte activation for use in the treatment of autoimmune diseases and other inflammatory disorders. Summary of the Invention

[0006] The present disclosure provides novel fusion molecules containing the SMAGP extracellular domain that can regulate leukocyte activity. In particular, these fusion molecules can inhibit the phagocytic and inflammatory activity of phagocytes, both of which can contribute to the pathology of autoimmune diseases. Also provided are polynucleotides, vectors, and host cells encoding these fusion molecules, as well as methods for producing and using these fusion molecules.

[0007] In one aspect, the disclosure provides a fusion molecule comprising the extracellular domain (ECD) of SMAGP and a half-life extending moiety.

[0008] In some embodiments, the SMAGP ECD is wild-type human, mouse, or cynomolgus SMAGP ECD.

[0009] In some embodiments, the SMAGP ECD comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3.

[0010] In some embodiments, the SMAGP ECD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3, or a variant thereof comprising 1-5 amino acid changes.

[0011] In some embodiments, the SMAGP ECD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3.

[0012] In some embodiments, the half-life extending moiety is an Fc region.

[0013] In some embodiments, the Fc region is a human IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 Fc region.

[0014] In some embodiments, the Fc region is a human IgG1 Fc region.

[0015] In some embodiments, the Fc region is a human IgG1 Fc region containing D265A and N297A mutations numbered according to the EU numbering system.

[0016] In some embodiments, the Fc region is a human IgG1 Fc region comprising L234A, L235A, and P329G mutations numbered according to the EU numbering system.

[0017] In some embodiments, the Fc region is a human IgG1 Fc region containing S239D and I332E mutations numbered according to the EU numbering system.

[0018] In some embodiments, the Fc region is a human IgG4 Fc region.

[0019] In some embodiments, the Fc region is a human IgG4 Fc region containing the S228P mutation numbered according to the EU numbering system.

[0020] In some embodiments, the Fc region is a murine IgG 2a The Fc region of

[0021] In some embodiments, the Fc region comprises a murine IgG1A polypeptide comprising the L234A, L235A, and P329G mutations numbered according to the EU numbering system. 2a The Fc region of

[0022] In some embodiments, the Fc region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-18 and 50-57.

[0023] In some embodiments, the half-life extending moiety is selected from the group consisting of human serum albumin, a serum albumin binding moiety, and polyethylene glycol (PEG).

[0024] In some embodiments, the serum albumin binding moiety is an antibody or an antigen-binding portion thereof.

[0025] In some embodiments, the serum albumin binding moiety is a VH or a VHH.

[0026] In some embodiments, the SMAGP ECD is covalently linked to the half-life extending moiety via a linker.

[0027] In some embodiments, the linker is a peptide linker.

[0028] In some embodiments, the peptide linker is an Fc hinge region or a portion thereof.

[0029] In some embodiments, the Fc hinge region is a human Fc hinge region or a portion thereof.

[0030] In some embodiments, the human Fc hinge region is a human IgG1, IgG2, IgG3, or IgG4 hinge region or portion thereof.

[0031] In some embodiments, the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 35-49, and 58-61, or a variant thereof comprising 1 to 5 amino acid changes.

[0032] In some embodiments, the half-life extending moiety is linked to the C-terminus of the SMAGP ECD.

[0033] In some embodiments, the half-life extending moiety is linked to the N-terminus of the SMAGP ECD.

[0034] In some embodiments, the fusion molecule comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-34.

[0035] In some embodiments, the fusion molecule comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-34.

[0036] In some embodiments, the fusion molecule comprises a dimer of two polypeptides, each polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-34.

[0037] In some embodiments, the fusion molecule further comprises a targeting moiety that binds to a target molecule on a cell.

[0038] In some embodiments, the cell is selected from the group consisting of a diseased cell, a senescent cell, a cancer cell, a B cell, a T cell, and a dendritic cell.

[0039] In some embodiments, the target molecule is selected from the group consisting of DEP1, NTAL, EBP50, STX4, VAMP3, ARMCX3, B2MG, LANCL1, PLD3, VPS26A, DPP4, SCAMP4, MICA / B, TNFRSF10D / CD264, NOTCH1, NOTCH3, CD36, oxidized vimentin, ICAM-1, uPAR, DEP1 / PTPRJ / CD148, CD264, TNFRSF10D, TRAILR4, and CD26.

[0040] In some embodiments, the cells are selected from diseased cells and senescent cells.

[0041] In some embodiments, the target molecule is ADAM9, B7-H3 / CD276, BCMA, CA6, CA9, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD70, CD79b, CD123, CD138, CD157 / BST1, P-cadherin CDH3, CEACAM5, CEACAM6, CLDN6, CLDN18.2, DLL3, EGFR, EGFRvIII, ENPP3, ENTPD2, EpCAM, FGR3, FLT 3, FOLR1, GPA33, GPC3, GPNMB, GPRC5D, GUCY2C, Her2, HHLA2, LAMP1, SLC39A6 / Liv-1, mesothelin, MUC16 / CA125, MUC17, SLC34A1 / NaPi2a, Nectin-4, CD274 / PD-L1, PSCA, PSMA / FOLH1, PVR, PVRIg, ROR1, SLITRK6, SSTR2, STEAP1, TROP2, and TMEM97.

[0042] In some embodiments, the cells are cancer cells.

[0043] In some embodiments, the target molecule is a B cell surface antigen.

[0044] In some embodiments, the target molecule is CD20.

[0045] In some embodiments, the targeting moiety is an antibody or antigen-binding fragment thereof.

[0046] In some embodiments, the targeting moiety is rituximab or an antigen-binding fragment thereof.

[0047] In some embodiments, the fusion molecule further comprises a payload molecule.

[0048] In some embodiments, the payload molecule is selected from the group consisting of mRNA, miRNA, circular RNA (cRNA), tRNA, siRNA, sgRNA, antisense oligonucleotide, peptide, virus, viral RNA genome, viral DNA genome, vector, plasmid, DNA, and drug.

[0049] In some embodiments, the payload molecule is encapsulated or attached to the nanoparticle.

[0050] In some embodiments, the virus is an adeno-associated virus or a lentivirus.

[0051] In some embodiments, the vector is a DNA vector.

[0052] In another aspect, the present disclosure provides a fusion molecule comprising the extracellular domain (ECD) of SMAGP and a payload molecule.

[0053] In some embodiments, the SMAGP ECD is wild-type human, mouse, or cynomolgus SMAGP ECD.

[0054] In some embodiments, the SMAGP ECD comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3.

[0055] In some embodiments, the SMAGP ECD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3, or a variant thereof comprising 1-5 amino acid changes.

[0056] In some embodiments, the SMAGP ECD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3.

[0057] In some embodiments, the payload molecule is selected from the group consisting of mRNA, miRNA, cRNA, tRNA, siRNA, sgRNA, antisense oligonucleotide, peptide, virus, viral RNA genome, viral DNA genome, vector, plasmid, DNA, radionuclide, and drug.In some embodiments, the virus is an adeno-associated virus or lentivirus.In some embodiments, the vector is a DNA vector.

[0058] In some embodiments, the payload molecule modulates the activity of cells expressing CLEC10A or CD177.

[0059] In some embodiments, the payload molecule is designed or selected to modulate the activity of neutrophils.

[0060] In some embodiments, the payload molecule is selected from the group consisting of a small molecule drug that inhibits the NADPH oxidase complex involved in ROS production, a degranulation inhibitor, and a promoter of neutrophil apoptosis. In some embodiments, the small molecule drug that inhibits the NADPH oxidase complex involved in ROS production is selected from the group consisting of diphenyleneiodonium and stelazine. In some embodiments, the degranulation inhibitor is nexinhib-20.

[0061] In some embodiments, the payload molecule is a nucleic acid molecule encoding a chimeric antigen receptor for neutrophils.

[0062] In some embodiments, the payload molecule is encapsulated or attached to a nanoparticle, hi some embodiments, the nanoparticle is a lipid nanoparticle.

[0063] In some embodiments, the SMAGP ECD is covalently attached to the payload molecule via a linker.

[0064] In some embodiments, the SMAGP ECD is covalently attached to the nanoparticle via a linker, and the payload molecule is encapsulated or attached to the nanoparticle.

[0065] In some embodiments, the nanoparticle is a lipid nanoparticle and the linker is linked to a lipid component of the lipid nanoparticle.

[0066] In some embodiments, the lipid component of the lipid nanoparticle to which the linker is attached is selected from the group consisting of an ionizable lipid, a PEGylated lipid, a helper lipid, and cholesterol.

[0067] In some embodiments, the helper lipid is selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-dibenzocyclooctyl (DBCO PE).

[0068] In some embodiments, the helper lipid is DBCO PE, the SMAGP ECD comprises an N-terminal azidohomoalanine, and the linker comprises a clickable DARPin that is linked to the DBCO PE and the N-terminal azidohomoalanine.

[0069] In some embodiments, the pegylated lipid is selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG(2000) maleimide), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-5000] (DSPE-PEG(5000) maleimide), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG(2000)), and 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

[0070] In some embodiments, the pegylated lipid is DSPE-PEG(2000)maleimide or DSPE-PEG(5000)maleimide, and the linker comprises a cysteine ​​fused to the C-terminus of the SMAGP ECD that binds the pegylated lipid.

[0071] In some embodiments, the linker is a peptide linker.

[0072] In some embodiments, the peptide linker is an Fc hinge region or a portion thereof.

[0073] In some embodiments, the Fc hinge region is a human Fc hinge region or a portion thereof.

[0074] In some embodiments, the human Fc hinge region is a human IgG1, IgG2, IgG3, or IgG4 hinge region or portion thereof.

[0075] In some embodiments, the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 35-49, and 58-61, or a variant thereof comprising 1 to 5 amino acid changes.

[0076] In some embodiments, the payload molecule is linked to the C-terminus of the SMAGP ECD.

[0077] In some embodiments, the payload molecule is linked to the N-terminus of the SMAGP ECD.

[0078] In another aspect, the disclosure provides a fusion molecule comprising the extracellular domain (ECD) of SMAGP and a targeting moiety that binds to a target molecule on a cell.

[0079] In some embodiments, the SMAGP ECD is wild-type human, mouse, or cynomolgus SMAGP ECD.

[0080] In some embodiments, the SMAGP ECD comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3.

[0081] In some embodiments, the SMAGP ECD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3, or a variant thereof comprising 1-5 amino acid changes.

[0082] In some embodiments, the SMAGP ECD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3.

[0083] In some embodiments, the cell is selected from the group consisting of a diseased cell, a senescent cell, a cancer cell, a B cell, a T cell, and a dendritic cell.

[0084] In some embodiments, the target molecule is selected from the group consisting of DEP1, NTAL, EBP50, STX4, VAMP3, ARMCX3, B2MG, LANCL1, PLD3, VPS26A, DPP4, SCAMP4, MICA / B, TNFRSF10D / CD264, NOTCH1, NOTCH3, CD36, oxidized vimentin, ICAM-1, uPAR, DEP1 / PTPRJ / CD148, CD264, TNFRSF10D, TRAILR4, and CD26.

[0085] In some embodiments, the cells are selected from diseased cells and senescent cells.

[0086] In some embodiments, the target molecule is ADAM9, B7-H3 / CD276, BCMA, CA6, CA9, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD70, CD79b, CD123, CD138, CD157 / BST1, P-cadherin CDH3, CEACAM5, CEACAM6, CLDN6, CLDN18.2, DLL3, EGFR, EGFRvIII, ENPP3, ENTPD2, EpCAM, FGR3, FLT 3, FOLR1, GPA33, GPC3, GPNMB, GPRC5D, GUCY2C, Her2, HHLA2, LAMP1, SLC39A6 / Liv-1, mesothelin, MUC16 / CA125, MUC17, SLC34A1 / NaPi2a, Nectin-4, CD274 / PD-L1, PSCA, PSMA / FOLH1, PVR, PVRIg, ROR1, SLITRK6, SSTR2, STEAP1, TROP2, and TMEM97.

[0087] In some embodiments, the cells are cancer cells.

[0088] In some embodiments, the target molecule is a B cell surface antigen.

[0089] In some embodiments, the target molecule is CD20.

[0090] In some embodiments, the targeting moiety is an antibody or antigen-binding fragment thereof.

[0091] In some embodiments, the targeting moiety is rituximab or an antigen-binding fragment thereof.

[0092] In some embodiments, the targeting moiety is linked to the C-terminus of the SMAGP ECD.

[0093] In some embodiments, the targeting moiety is linked to the N-terminus of the SMAGP ECD.

[0094] In some embodiments, the fusion molecule binds to a SMAGP ECD receptor on a cell.

[0095] In some embodiments, the SMAGP ECD is glycosylated.

[0096] In some embodiments, the SMAGP ECD is aglycosylated.

[0097] In some embodiments, the fusion molecule further comprises a detectable label, optionally, the detectable label is selected from the group consisting of an enzyme, a fluorescent label, and a radioisotope.

[0098] In another aspect, the present disclosure provides a fusion molecule comprising an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-34.

[0099] In some embodiments, the fusion molecule comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-34.

[0100] In some embodiments, the fusion molecule comprises a dimer of two polypeptides, each polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-34.

[0101] In another aspect, the disclosure provides a polynucleotide comprising a nucleotide sequence encoding any of the fusion molecules described above and provided herein.

[0102] In another aspect, the disclosure provides an expression vector comprising any of the polynucleotides described above and herein.

[0103] In another aspect, the present disclosure provides a recombinant host cell comprising any of the polynucleotides or expression vectors described above and herein.

[0104] In another aspect, the present disclosure provides a nanoparticle comprising any of the fusion molecules described above and herein, any of the polynucleotides described above and herein, or any of the expression vectors described above and herein.

[0105] In some embodiments, the nanoparticle is a lipid nanoparticle and the polynucleotide is mRNA.

[0106] In another aspect, the present disclosure provides a method of producing any of the fusion molecules described above and herein, comprising culturing any of the host cells described above and herein under conditions such that the fusion molecule is produced.

[0107] In another aspect, the present disclosure provides a pharmaceutical composition comprising any of the fusion proteins described above and herein, or any of the polynucleotides described above and herein, or any of the expression vectors described above and herein, and a pharmaceutically acceptable carrier or excipient.

[0108] In another aspect, the disclosure provides a method of modifying the activity of a leukocyte, comprising contacting the leukocyte with any of the fusion proteins described above and herein.

[0109] In some embodiments, the leukocytes are selected from the group consisting of myeloid cells, macrophages, Kupffer cells, histiocytes, microglia, osteoclasts, dendritic cells, mast cells, neutrophils, regulatory T cells, tumor-infiltrating regulatory T cells, and granulocytes.

[0110] In some embodiments, the leukocyte is a macrophage, hi some embodiments, the macrophage is selected from the group consisting of an M0 macrophage, an M1 macrophage, an M2 macrophage, an M2a macrophage, an M2b macrophage, an M2c macrophage, and an M2d macrophage.

[0111] In some embodiments, the activity comprises one or more of phagocytosis, cytokine production, chemokine production, antigen presentation, growth factor production, and protease production.

[0112] In some embodiments, the activity comprises phagocytosis of a population of cells or cell-like structures, in some embodiments, the population of cells or cell-like structures is selected from the group consisting of cancer cells, immune cells, neurons, red blood cells, and platelets.

[0113] In some embodiments, the activity of white blood cells is increased.

[0114] In some embodiments, the activity of white blood cells is reduced.

[0115] In another aspect, the disclosure provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the fusion molecules described above and herein, any of the polynucleotides described above and herein, any of the expression vectors described above and herein, or any of the pharmaceutical compositions described above and herein.

[0116] In some embodiments, the disease or disorder is selected from the group consisting of an autoimmune disorder, an autoimmune disorder characterized by excessive phagocytic activity, atherosclerosis, cancer, an inflammatory disease or disorder, a lymphoproliferative disorder, an infectious disease, macrophage activation syndrome (MAS), a cytokine-associated disorder, a central nervous system (CNS) disease, a disease of overactivated microglia, a disease of overactivated osteoclasts, osteoporosis, cancer-associated bone degradation and / or metastasis, multiple myeloma, a bone disease, systemic juvenile idiopathic arthritis, an allergy, a cancer with low PD-L1 expression, a cancer resistant to PD-1 and / or PD-L1 inhibitors, a disease or disorder characterized by multinucleated giant cells, and an atopic disease.

[0117] In some embodiments, the cancer-associated bone degradation and / or metastasis is due to a disease or disorder selected from multiple myeloma, breast cancer, and prostate cancer.

[0118] In some embodiments, the autoimmune disorder is lupus, rheumatoid arthritis, multiple sclerosis, hemophagocytic lymphohistiocytosis (HLH), or immune thrombocytopenia (ITP).

[0119] In some embodiments, the CNS disease is Alzheimer's disease, schizophrenia, or Huntington's disease.

[0120] In some embodiments, the disease or disorder is a lymphoproliferative disorder and the fusion molecule comprises a targeting moiety comprising rituximab or an antigen-binding fragment thereof.

[0121] In some embodiments, the disease or disorder is selected from the group consisting of atherosclerosis, cancer, an inflammatory disease or disorder, and an infectious disease.

[0122] In some embodiments, the disease or disorder is selected from the group consisting of H. pylori infection, COVID, severe COVID, IgA nephropathy, and ovarian cancer.

[0123] In some embodiments, the disease or disorder characterized by multinucleated giant cells is selected from the group consisting of Langerhans cell histiocytosis and granuloma.

[0124] In some embodiments, the disease or disorder is selected from the group consisting of inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, acute-on-chronic liver failure (ACLF), COVID, severe COVID, antineutrophil cytoplasmic antibody-associated vasculitis (AAV), autoimmune vasculitis, asthma, acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), diseases in which regulatory T cells express CD177, Kawasaki disease, septic shock, renal cell carcinoma, hepatocellular carcinoma, breast cancer, lung cancer, and colorectal cancer.

[0125] In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of a chemotherapeutic agent, hi some embodiments, the chemotherapeutic agent is paclitaxel.

[0126] In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of an immune checkpoint modulator, hi some embodiments, the immune checkpoint modulator is an anti-PD-1 agent or an anti-PD-L1 agent.

[0127] In some embodiments, the method further comprises administering to the subject therapeutically effective amounts of a chemotherapeutic agent and an immune checkpoint modulator, hi some embodiments, the chemotherapeutic agent is paclitaxel and the immune checkpoint modulator is an anti-PD-1 agent or an anti-PD-L1 agent.

[0128] In some embodiments, the disease or disorder is ovarian cancer.

[0129] In another aspect, the present disclosure provides a method for identifying a modulator of the interaction between SMAGP and CLEC10A, the method comprising measuring the binding of SMAGP to CLEC10A in the presence and absence of a test compound, wherein the test compound is identified as a modulator of the interaction between SMAGP and CLEC10A based on an increase or decrease in the amount of binding of SMAGP to CLEC10A in the presence of the test compound relative to the amount of binding of SMAGP to CLEC10A in the absence of the test compound.

[0130] In another aspect, the present disclosure provides a method for identifying a promoter of the interaction between SMAGP and CLEC10A, the method comprising measuring the binding of SMAGP to CLEC10A in the presence and absence of a test compound, wherein the test compound is identified as a promoter of the interaction between SMAGP and CLEC10A based on an increase in the amount of binding of SMAGP to CLEC10A in the presence of the test compound relative to the amount of binding of SMAGP to CLEC10A in the absence of the test compound.

[0131] In another aspect, the present disclosure provides a method for identifying an inhibitor of the interaction between SMAGP and CLEC10A, the method comprising measuring the binding of SMAGP to CLEC10A in the presence and absence of a test compound, wherein the test compound is identified as an inhibitor of the interaction between SMAGP and CLEC10A by a decrease in the amount of binding of SMAGP to CLEC10A in the presence of the test compound relative to the amount of binding of SMAGP to CLEC10A in the absence of the test compound.

[0132] In some embodiments, SMAGP and / or CLEC10A are expressed on the surface of the cell.

[0133] In some embodiments, SMAGP and / or CLEC10A are recombinant proteins. In some embodiments, the recombinant proteins are fusion molecules comprising an Fc region.

[0134] In another aspect, the present disclosure provides a method for identifying a modulator of the interaction between SMAGP and CD177, the method comprising measuring the binding of SMAGP to CD177 in the presence and absence of a test compound, wherein the test compound is identified as a modulator of the interaction between SMAGP and CD177 based on an increase or decrease in the amount of binding of SMAGP to CD177 in the presence of the test compound relative to the amount of binding of SMAGP to CD177 in the absence of the test compound.

[0135] In another aspect, the present disclosure provides a method for identifying a promoter of the interaction between SMAGP and CD177, the method comprising measuring the binding of SMAGP to CD177 in the presence and absence of a test compound, wherein the test compound is identified as a promoter of the interaction between SMAGP and CD177 based on an increase in the amount of SMAGP binding to CD177 in the presence of the test compound relative to the amount of SMAGP binding to CD177 in the absence of the test compound.

[0136] In another aspect, the present disclosure provides a method for identifying an inhibitor of the interaction between SMAGP and CD177, the method comprising measuring the binding of SMAGP to CD177 in the presence and absence of a test compound, wherein the test compound is identified as an inhibitor of the interaction between SMAGP and CD177 by a decrease in the amount of binding of SMAGP to CD177 in the presence of the test compound relative to the amount of binding of SMAGP to CD177 in the absence of the test compound.

[0137] In some embodiments, SMAGP and / or CD177 are expressed on the surface of the cell.

[0138] In some embodiments, SMAGP and / or CD177 are recombinant proteins. In some embodiments, the recombinant protein is a fusion molecule comprising an Fc region.

[0139] In some embodiments, the test compound is selected from the group consisting of a small molecule, a polypeptide, or a nucleic acid. In some embodiments, the polypeptide is an antibody. In some embodiments, the nucleic acid is a DNA or RNA aptamer.

[0140] In some embodiments, the amount of binding is measured using enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), or flow cytometry.

[0141] In another aspect, the disclosure provides a method of treating a subject having a tumor with a high expression level of CLEC10A compared to a reference level of CLEC10A, the method comprising administering to the subject a therapeutically effective amount of a compound that antagonizes the binding of CLEC10A to SMAGP. In some embodiments, the compound is any of the fusion molecules described above and herein, any of the polynucleotides described above and herein, any of the expression vectors described above and herein, or any of the pharmaceutical compositions described above and herein.

[0142] In another aspect, the present disclosure provides a method for identifying a subject who would benefit from treatment with a fusion molecule that antagonizes the binding of CLEC10A to SMAGP, comprising determining the expression level of CLEC10A in a biological sample from the subject relative to a reference level of CLEC10A, wherein a higher expression level of CLEC10A in the biological sample compared to the reference level indicates that the subject would benefit from the treatment. In some embodiments, the biological sample is a tumor sample. In some embodiments, the expression level of CLEC10A in the biological sample is determined by immunohistochemistry, fluorescent in situ hybridization (FISH), or chromogenic in situ hybridization (CISH). In some embodiments, CLEC10A is expressed on the surface of myeloid cells and SMAGP is expressed on the surface of tumor cells.

[0143] In another aspect, the present disclosure provides a method for identifying a subject who will benefit from treatment with a fusion molecule comprising the extracellular domain (ECD) of SMAGP, comprising measuring the expression level of CD177 in a biological sample from the subject and comparing it to a reference level of CD177, wherein a higher expression level of CD177 compared to the reference level indicates that the subject will benefit from the treatment. In some embodiments, the biological sample is a tumor sample. In some embodiments, the expression level of CD177 in the biological sample is determined by immunohistochemistry, fluorescent in situ hybridization (FISH), or chromogenic in situ hybridization (CISH). In some embodiments, CD177 is expressed on the surface of neutrophils or regulatory T cells, and SMAGP is expressed on the surface of tumor cells.

[0144] In some embodiments, the method further comprises administering to a subject a therapeutically effective amount of any of the fusion molecules described above and herein, any of the polynucleotides described above and herein, any of the expression vectors described above and herein, or any of the pharmaceutical compositions described above and herein.

[0145] In another aspect, the present disclosure provides a fusion molecule as described above and herein, a polynucleotide as described above and herein, an expression vector as described above and herein, a nanoparticle as described above and herein, or a composition as described above and herein, for use in modifying the activity of a leukocyte.

[0146] In another aspect, the present disclosure provides a fusion molecule as described above and herein, a polynucleotide as described above and herein, an expression vector as described above and herein, a nanoparticle as described above and herein, or a composition as described above and herein for use in medicine.

[0147] In another aspect, the present disclosure provides a fusion molecule as described above and herein, a polynucleotide as described above and herein, an expression vector as described above and herein, a nanoparticle as described above and herein, or a composition as described above and herein, for use in treating a disease or disorder in a subject in need thereof.

[0148] In another aspect, the present disclosure provides the use of a fusion molecule as described above and herein, a polynucleotide as described above and herein, an expression vector as described above and herein, a nanoparticle as described above and herein, or a composition as described above and herein in the manufacture of a medicament for the treatment of a disease or disorder in a subject in need thereof. [Brief explanation of the drawings]

[0149] [Figure 1] [Figure 1A] Schematic diagram of the structure of the SMAGP extracellular domain (ECD)-IgG1 Fc fusion molecule. The SMAGP ECD is shown in light gray, and the Fc is shown in dark gray. [Figure 1B] HPLC-SEC chromatogram of purified human SMAGP ECD-hIgG1 Fc (hSMAGP ECD-hIgG1 Fc) and accompanying data table. [Figure 2][Figure 2A] Flow cytometry histograms showing the binding of hIgG1 Fc control, hSMAGP ECD-hIgG1 Fc, and cynomolgus monkey SMAGP ECD-hIgG1 Fc (cSMAGP ECD-hIgG1 Fc) to human macrophages from two different donors. [Figure 2B] Flow cytometry histograms showing the binding of hIgG1 Fc control, hSMAGP ECD-hIgG1 Fc, and cynomolgus monkey SMAGP ECD-hIgG1 Fc (cSMAGP ECD-hIgG1 Fc) to human macrophages from two different donors. [Figure 2C] Flow cytometry histograms showing the binding of hIgG1 Fc control and hSMAGP ECD-hIgG1 Fc to human neutrophils (Figure 2C) and human monocytes (Figure 2D). [FIG. 2D] Flow cytometry histograms showing binding of hIgG1 Fc control and hSMAGP ECD-hIgG1 Fc to human neutrophils (FIG. 2C) and human monocytes (FIG. 2D). [Figure 3] (A and B) are bar graphs showing the extent of phagocytosis of Ramos tumor cells by macrophages from two different donors in the presence of the indicated concentrations of anti-CD47 antibody and hSMAGP ECD-hIgG1 Fc or hIgG1 Fc control. Additional controls include macrophages cocultured with Ramos SMAGP-overexpressing cells in the presence of anti-CD47 antibody and macrophages cocultured with the Ramos parental cell line in the absence of anti-CD47 antibody. [Figure 4] A and B are bar graphs showing the extent of recovery of Ramos tumor cells after inhibition of the phagocytosis assay shown in Figures 3A and 3B. [Figure 5][Figure 5A] A bar graph showing the extent of phagocytosis of A375 tumor cells by macrophages from three different donors in the presence of the indicated concentrations of anti-CD47 antibody and hSMAGP ECD-hIgG1 Fc or hIgG1 Fc control, quantified at 4 hours. [Figure 5B] A graph showing the time course of the extent of phagocytosis of A375 tumor cells by macrophages from a representative donor in the presence of the indicated concentrations of anti-CD47 antibody and hSMAGP ECD-hIgG1 Fc or hIgG1 Fc control. [Figure 6] [Figure 6A] Bar graph showing the extent of phagocytosis of Ramos tumor cells by macrophages from three different donors in the presence of the indicated concentrations of rituximab and hIgG1 Fc control, hSMAGP ECD-hIgG1 Fc, CD24 ECD-hIgG1 Fc, or CD47 ECD-hIgG1 Fc, quantified at 3 hours. [Figure 6B] The extent of recovery of Ramos tumor cells after inhibition of phagocytosis is shown. [Figure 7] 1 is a bar graph showing the extent of phagocytosis of Ramos tumor cells by macrophages from three different donors in the presence of the indicated concentrations of rituximab and hIgG1-Fc, hSMAGP ECD-hIgG1 Fc, hIgG1 LALA PG, hSMAGP ECD-hIgG1 LALA PG Fc, hIgG4, and hSMAGP ECD-hIgG4. [Figure 8][Figure 8A] Bar graph showing the extent of phagocytosis of Ramos tumor cells by macrophages treated with hSMAGP ECD-hIgG1 Fc or hIgG1 Fc control in the presence of MCSF plus the indicated polarity stimulus (M0, no polarity stimulus; M1, induced by LPS and IFN-γ; M2, induced by IL-10; M2, induced by TGF-β; M2, induced by TGF-β and IL-10; and M2, induced by TGF-β, IL-10, and IL-4), quantified at 2 hours, as indicated. [Figure 8B] Graph showing the time course of the extent of phagocytosis of Ramos cells by M1-polarized macrophages (induced by LPS and IFN-γ) treated with hSMAGP ECD-hIgG1 Fc or hIgG1 Fc control, quantified over 21 hours, as indicated. [Figure 9] [Figure 9A] A graph showing the time course of the extent of phagocytosis of red blood cells (RBCs) by macrophages from a representative donor in the presence of the indicated concentrations of anti-RBC antibody and hIgG1 Fc control, hSMAGP ECD-hIgG1 Fc, or medium alone. [Figure 9B] A bar graph showing the extent of phagocytosis of RBCs by macrophages from three donors in the presence of anti-RBC antibody and hIgG1 Fc control, hSMAGP ECD-hIgG1 Fc, or medium alone at 10 hours. [Figure 9C] A set of three graphs showing dose-response curves for each of three donors, corresponding to the area under the curve of the total area of ​​red objects over time plotted against the indicated opsonizing antibody concentrations. [Figure 10] 1 is a set of three graphs showing the percentage of viable RBCs remaining after phagocytosis by macrophages from three donors in the presence of either hIgG1 Fc control or hSMAGP ECD-hIgG1 Fc at the indicated concentrations. [Figure 11] A, B, and C are bar graphs showing the extent of TNFα (FIG. 11A), IL-12p70 (FIG. 11B), and IL-6 (FIG. 11C) secretion after overnight treatment with medium alone, hIgG1 Fc control, or hSMAGP ECD-hIgG1 Fc, plus or minus LPS. [Figure 12] (A-C) are bar graphs showing the extent of binding of each of a panel of lectins to hSMAGP ECD-hIgG1 Fc (FIG. 12A), cSMAGP ECD-hIgG1 Fc (FIG. 12B), and buffer (FIG. 12C) plus streptavidin-Cy3 in the presence of cations. The identities of the lectins tested are not shown, except for CLEC10A / MGL. NC = immobilization buffer, PC1 = positive control (consisting of amine-PEG-biotin), PC2 = human IgG, PC3 = mouse IgG, M = marker. [Figure 13] (A-C) Bar graphs showing the extent of binding of each of a panel of lectins to hSMAGP ECD-hIgG1 Fc (FIG. 13A), cSMAGP ECD-hIgG1 Fc (FIG. 13B), and buffer (FIG. 13C) plus streptavidin-Cy3 in the absence of cations. The identities of the lectins tested are not shown, except for CLEC10A / MGL. NC = immobilization buffer, PC1 = positive control (consisting of amine-PEG-biotin), PC2 = human IgG, PC3 = mouse IgG, M = marker. [Figure 14] 1 is a flow cytometry histogram showing binding of hSMAGP ECD-hIgG1 Fc to CLEC10A-overexpressing or control cells. [Figure 15] 1 is a bar graph showing the extent of binding of hSMAGP ECD-hIgG1 Fc to recombinant CLEC10A ECD or a negative control in an ELISA assay. [Figure 16] A and B are images of slides of membrane proteins expressed in HEK293 cells and probed with hSMAGP ECD-hIgG1 Fc (FIG. 16A) or CTLA4-hFc (control) (FIG. 16B). [Figure 17] 1 is a flow cytometry histogram showing binding of hSMAGP ECD-hIgG1 Fc to CD177-overexpressing or control cells. [Figure 18] 1 is a bar graph showing the extent of binding of hSMAGP ECD-hIgG1 Fc to recombinant CD177 ECD or a negative control in an ELISA assay. [Figure 19] 1 is a bar graph showing the extent of binding of aglycosylated scrambled hSMAGP ECD peptide, aglycosylated hSMAGP ECD peptide, and aglycosylated hSMAGP ECD peptide to recombinant CD177 ECD or a negative control in the presence of anti-CD177 antibody in an ELISA assay. [Figure 20] A and B are a set of flow cytometry histograms showing representative expression of CD177 (FIG. 20A) and CLEC10A (FIG. 20B) on M0 and M2a / c human macrophages. [Figure 21] [Figure 21A] A set of flow cytometry histograms showing representative expression of CD177 (Figure 21A) and CLEC10A (Figure 21B) on neutrophils, monocytes, B cells, T cells, NK cells, and NKT cells. [Figure 21B] A set of flow cytometry histograms showing representative expression of CD177 (Figure 21A) and CLEC10A (Figure 21B) on neutrophils, monocytes, B cells, T cells, NK cells, and NKT cells. DETAILED DESCRIPTION OF THE INVENTION

[0150] The present disclosure provides novel fusion molecules containing the SMAGP extracellular domain that can regulate leukocyte activity. In particular, these fusion molecules can inhibit the phagocytic and inflammatory activity of phagocytes, both of which can contribute to the pathology of autoimmune diseases. Also provided are polynucleotides, vectors, and host cells encoding these fusion molecules, as well as methods for producing and using these fusion molecules.

[0151] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are merely exemplary and explanatory and are not intended to limit any claimed subject matter. In this application, the use of the singular includes the plural unless specifically stated otherwise. 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. In this application, the use of "or" means "and / or" unless stated otherwise. Furthermore, the use of "including," as well as other forms such as "include," "includes," and "included," is not limiting. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0152] As used herein, the term "SMAGP" refers to a small cell adhesion glycoprotein. An exemplary human SMAGP is set forth in NCBI reference sequence NM_001033873.1. An exemplary human SMAGP extracellular domain (ECD) is set forth in SEQ ID NO: 1.

[0153] As used herein, the term "half-life extending moiety" refers to any molecule that, when linked to SMAGP ECD, is capable of extending the serum half-life of SMAGP ECD.

[0154] As used herein, the term "Fc region" refers to the portion of an immunoglobulin formed by the Fc domains of its two heavy chains. In some embodiments, the Fc region is formed from an IgG1 heavy chain constant region. In some embodiments, residues 356 and 358, respectively, numbered according to the EU numbering system, are D and L. In some embodiments, residues 356 and 358, respectively, numbered according to the EU numbering system, are E and M. In some embodiments, the IgG1 heavy chain constant region comprises a G1m1(a), G1m2(x), G1m3(f), or G1m17(z) allotype. See, e.g., Jefferis and Lefranc (2009) mAbs 1(4):332-338 and de Taeye et al. (2020) Front Immunol. 11:740 (incorporated herein by reference in their entireties).

[0155] As used herein, the term "variant Fc region" refers to a variant of an Fc region that has one or more modifications relative to a native Fc region. Modifications may include amino acid substitutions, additions and / or deletions, attachment of additional moieties, and / or modifications of native glycans. The term encompasses heterodimeric Fc regions in which each of the constituent Fc domains is different. The term also encompasses single-chain Fc regions in which the constituent Fc domains are linked together by linker moieties.

[0156] As used herein, the term "Fc domain" refers to the portion of a single antibody heavy chain that contains both the CH2 and CH3 domains of the antibody. In some embodiments, the Fc domain includes at least a portion of the hinge region (e.g., the amino acid sequence EPKSX (SEQ ID NO: 4), where X is cysteine ​​or serine), the CH2 domain, and the CH3 domain.

[0157] As used herein, the term "hinge region" refers to the portion of an antibody heavy chain molecule that connects the CH1 domain to the CH2 domain. In some embodiments, the hinge region is derived from an IgG1 antibody. In some embodiments, the hinge region comprises an upper hinge region, a core hinge region, and / or a lower hinge region, e.g., as described in Chiu et al. (2019) Antibodies (Basel) 8(4):55 (incorporated herein by reference in its entirety). In some embodiments, the hinge region comprises the amino acid sequence EPKSX (SEQ ID NO: 4) (wherein X is cysteine ​​or serine). In some embodiments, the hinge region comprises the amino acid sequence EPKSXDKTHT (SEQ ID NO: 58), where X is cysteine ​​or serine. In some embodiments, the hinge region comprises the amino acid sequence EPKSXDKTHTCPPCP (SEQ ID NO: 59), where X is cysteine ​​or serine. In some embodiments, the hinge region comprises the amino acid sequence EPKSXDKTHTCPPCPAPELL (SEQ ID NO: 60), where X is cysteine ​​or serine. In some embodiments, the hinge region comprises the amino acid sequence EPKSXDKTHTCPPCPAPELLGGP (SEQ ID NO: 61), where X is cysteine ​​or serine.

[0158] As used herein, the term "CH2 domain" generally refers to the portion of an antibody heavy chain corresponding to residues 231-340 of human IgG1 numbered according to the EU numbering system. In some cases, the CH2 domain may begin after residue 231 of human IgG1 numbered according to the EU numbering system (e.g., spanning residues 236-340, 237-340, or 239-340).

[0159] As used herein, the term "CH3 domain" refers to the portion of an antibody heavy chain corresponding to residues 341-446 or 341-447 of human IgG1, numbered according to the EU numbering system.

[0160] As used herein, the term "serum albumin-binding moiety" refers to any molecule that specifically binds to serum albumin (e.g., human serum albumin). Examples of serum albumin-binding moieties include antibodies, such as Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFvs), single-chain Fvs (scFvs), CDRs, VH domains, VL domains, single-domain antibodies (sdAbs), VHH domains, camelid antibodies, and polypeptides derived from any of the above antigen-binding fragments. The term also encompasses synthetic antigen-binding proteins or antibody-mimetic proteins, such as affibodies, lipocalins, and DARPins.

[0161] As used herein, the terms "antibody" and "antibodies" include full-length antibodies, antigen-binding fragments of full-length antibodies, and molecules comprising an antibody CDR, VH domain, or VL domain. Examples of antibodies include monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, heteroconjugate antibodies, antibody-drug conjugates, single domain antibodies (sdAbs), monovalent antibodies, single chain antibodies or single chain Fvs (scFvs), camelid antibodies, single domain antibodies (sdAbs), humanized antibodies, VHH domains, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), and antigen-binding fragments of any of the above. Antibodies can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or species (e.g., murine IgG 2a or IgG 2b ) can be.

[0162] As used herein, the terms "VH" and "VH domain" are used interchangeably and refer to the heavy chain variable region of an antibody.

[0163] As used herein, the terms "VL" and "VL domain" are used interchangeably and refer to the light chain variable region of an antibody.

[0164] As used herein, the terms "VHH" and "VHH domain" are used interchangeably to refer to the variable region of a heavy chain antibody (e.g., a camelid heavy chain antibody) or a humanized variant thereof.

[0165] The determination of " percent identity " between two sequences (for example, amino acid sequence or nucleic acid sequence) can be achieved using mathematical algorithm.A specific non-limiting example of the mathematical algorithm utilized for comparing two sequences is the algorithm of Karlin S&Altschul SF, (1990) PNAS 87:2264-2268 (each of which is incorporated herein by reference in its entirety), modified as in Karlin S&Altschul SF, (1993) PNAS 90:5873-5877.Such algorithm is incorporated into the NBLAST and XBLAST programs of Altschul SF et al., (1990) J Mol Biol 215:403 (each of which is incorporated herein by reference in its entirety).To obtain the nucleotide sequence homologous to the nucleic acid molecules described herein, BLAST nucleotide search can be carried out using, for example, NBLAST nucleotide program parameters set, such as score=100, word length=12, etc. To obtain amino acid sequences homologous to the protein molecules described herein, BLAST protein searches can be performed using XBLAST program parameters set, for example, score=50, word length=3, etc. To obtain gapped alignments for comparison purposes, GappedBLAST can be used as described in Altschul SF et al., (1997) Nucleic Acids Res 25:3389-3402, the entire contents of which are incorporated herein by reference. Alternatively, PSI BLAST can be used to perform an iterated search to detect distant relationships between molecules. When using the above-mentioned BLAST, GappedBLAST, and PSI BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used (see, for example, the National Center for Biotechnology Information (NCBI) on the worldwide web, ncbi.nlm.nih.gov).Another specific, non-limiting example of a mathematical algorithm utilized for comparing sequences is the algorithm of Myers and Miller, (1988), CABIOS 4:11-17, which is incorporated herein by reference in its entirety. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically, only exact matches are counted.

[0166] As used herein, the term "payload molecule" refers to any molecule to which a fusion molecule can be linked (e.g., for delivery to a cell by binding the fusion molecule to the cell).

[0167] As used herein, the term "polynucleotide" refers to a polymer of DNA or RNA. Polynucleotide sequences may be single-stranded or double-stranded, contain natural, non-natural, or modified nucleotides, and may include natural, non-natural, or modified internucleotide linkages, such as phosphoramidate or phosphorothioate linkages, instead of the phosphodiester bonds found between nucleotides in unmodified polynucleotide sequences. Polynucleotide sequences include, but are not limited to, all polynucleotide sequences obtained by any means available in the art, including, but not limited to, recombinant means, such as cloning polynucleotide sequences from recombinant libraries or cell genomes, using conventional cloning techniques and the polymerase chain reaction, and synthetic means.

[0168] As used herein, the term "linked" refers to a physical connection (e.g., directly or indirectly connected) between amino acid sequences (e.g., different segments, regions, or domains). Linked regions, domains, and segments of the fusion molecules of the present disclosure can be contiguous or non-contiguous (e.g., connected to each other via a linker). In some embodiments, the linkage is a covalent bond. In some embodiments, the linkage is a non-covalent bond.

[0169] As used herein, the term "EU numbering system" refers to the EU numbering convention for antibody constant regions as described in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) and Kabat et al., Sequences of Proteins of Immunological Interest, USDept. Health and Human Services, 5th edition, 1991, each of which is incorporated herein by reference in its entirety.

[0170] As used herein, the terms "treat," "treating," and "treatment" refer to therapeutic or prophylactic measures as described herein. In some embodiments, the method of "treatment" employs administration of a fusion protein to a subject having a disease or disorder or predisposed to having such a disease or disorder to prevent, cure, delay, reduce the severity of, or ameliorate a disease or disorder, or one or more symptoms of a recurrent disease or disorder, or to extend the subject's survival beyond that expected in the absence of such treatment.

[0171] As used herein, in the context of administering therapy, the term "effective amount" refers to the amount of therapy that achieves a desired prophylactic or therapeutic effect.

[0172] As used herein, the term "subject" includes any human or non-human animal. In one embodiment, the subject is a human or non-human mammal. In one embodiment, the subject is a human.

[0173] fusion molecule The present disclosure provides fusion molecules comprising the SMAGP extracellular domain (ECD) and one or more moieties selected from the group consisting of a half-life extending moiety, a targeting moiety, and a payload molecule. In some embodiments, the present disclosure provides fusion molecules comprising the SMAGP ECD and a half-life extending moiety. In some embodiments, the present disclosure provides fusion molecules comprising the SMAGP ECD and a targeting moiety. In some embodiments, the present disclosure provides fusion molecules comprising the SMAGP ECD and a payload molecule.

[0174] SMAGP extracellular domain The present disclosure provides fusion molecules comprising the SMAGP extracellular domain (ECD). The SMAGP ECD disclosed herein can be derived from any mammalian species, including, for example, humans, rodents (e.g., mice, rats, rabbits, guinea pigs), or non-human primates (e.g., chimpanzees, macaques). In some embodiments, the SMAGP ECD is wild-type human, mouse, or cynomolgus monkey SMAGP ECD. In some embodiments, the SMAGP ECD is a variant of wild-type human, mouse, or cynomolgus monkey SMAGP ECD. Exemplary SMAGP ECDs are shown in Table 1 below. [Table 1]

[0175] In some embodiments, the disclosure provides a fusion molecule comprising a SMAGP ECD, wherein the fusion molecule comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3. In some embodiments, the SMAGP ECD comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3.

[0176] In some embodiments, the disclosure provides a fusion molecule comprising a SMAGP ECD, the fusion molecule comprising a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3, comprising 1, 2, 3, 4, or 5 amino acid changes (e.g., substitutions, insertions, and / or deletions). In some embodiments, the SMAGP ECD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3. In some embodiments, the amino acid sequence of the SMAGP ECD consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3.

[0177] In some embodiments, the disclosure provides a fusion molecule comprising a SMAGP ECD, wherein the fusion molecule comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3, and wherein one or more amino acids from the N-terminus are absent. In some embodiments, the SMAGP ECD comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3, and wherein the N-terminal methionine is absent. In some embodiments, the SMAGP ECD comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3, and wherein one or more amino acids from the C-terminus are absent.

[0178] Half-life extension moiety In some embodiments, the present disclosure provides a fusion molecule comprising a SMAGP ECD and a half-life extending moiety. The half-life extending moiety can be linked to the N-terminus or N-terminus of the SMAGP ECD.

[0179] In some embodiments, the half-life extending moiety comprises an Fc region or a derivative thereof. Any Fc region can be used in the fusion molecules disclosed herein. In certain embodiments, the Fc region is a human immunoglobulin (Ig) of any isotype of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), any class of immunoglobulin molecule (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass of immunoglobulin molecule (e.g., IgG2a and IgG2b). In one embodiment, the Fc region is an IgG Fc region (e.g., a human IgG region). In one embodiment, the Fc region is an IgG1 Fc region (e.g., a human IgG1 region). In one embodiment, the Fc region is a chimeric Fc region comprising portions of several different Fc regions. Suitable examples of chimeric Fc regions are described in US2011 / 0243966A1, which is incorporated herein by reference in its entirety. A variety of Fc region gene sequences (eg, human constant region gene sequences) are available in the form of public deposits.

[0180] In some embodiments, the present disclosure provides a fusion molecule comprising a SMAGP ECD and an Fc region, wherein the Fc region is that of human IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2. The Fc region can be of any allotype or isoallotype. In some embodiments, the present disclosure provides a fusion molecule comprising a SMAGP ECD and an Fc region, wherein the Fc region is that of an IgG1 allotype selected from the group consisting of G1m1(a), G1m2(x), G1m3(f), and G1m17(z). See, e.g., Jefferis and Lefranc (2009) mAbs 1(4):332-338. In some embodiments, the present disclosure provides a fusion molecule comprising a SMAGP ECD and an Fc region, wherein the Fc region is that of an IgG2 allotype or isoallotype. In some embodiments, the Fc region is an IgG2 Fc region of the G2m23(n) allotype. (Id.) In some embodiments, the present disclosure provides a fusion molecule comprising an SMAGP ECD and an Fc region, wherein the Fc region is an IgG3 Fc region of any allotype or isoallotype. In some embodiments, the IgG3 Fc region of the allotype is selected from the group consisting of G3m21(g1), G3m28(g5), G3m11(b0), G3m5(b1), G3m13(b3), G3m14(b4), G3m10(b5), G3m15(s), G3m16(t), G3m6(c3), G3m24(c5), G3m26(u), and G3m27(v). (Id.) In some embodiments, the present disclosure provides a fusion molecule comprising a SMAGP ECD and an Fc region, wherein the Fc region is an Fc region of an IgA of any allotype or isoallotype. In some embodiments, an Fc region of an IgA of an allotype selected from the group consisting of A2m1 and A2m2. (Id.)

[0181] In some embodiments, the Fc region is a variant of a wild-type Fc region. In some embodiments, the variant Fc region differs from the Fc region of any native immunoglobulin. In some embodiments, the native immunoglobulin is a human immunoglobulin. In some embodiments, the immunoglobulin is IgA, IgD, IgE, or IgG. In some embodiments, the immunoglobulin is IgG. In some embodiments, the immunoglobulin is human IgA, human IgD, human IgE, or human IgG. In some embodiments, the immunoglobulin is human IgG. In some embodiments, the IgG is IgG1, IgG2, IgG3, or IgG4. In some embodiments, the human IgG is human IgG1, human IgG2, human IgG3, or human IgG4. In some embodiments, the variant Fc region differs from the Fc region of human IgG1. In some embodiments, the Fc region of human IgG1 comprises the G1m1(a), G1m2(x), G1m3(f), or G1m17(z) allotype. In some embodiments, the native immunoglobulin is a mouse immunoglobulin. In some embodiments, the mouse IgG is mouse IgG1, mouse IgG2a, mouse IgG2b, or mouse IgG3. In some embodiments, the variant Fc region is different from the Fc region of mouse IgG2a.

[0182] In some embodiments, the present disclosure provides a fusion molecule comprising a SMAGP ECD and an Fc region, wherein the Fc region comprises a CH2 and CH3 domain and at least a portion of a hinge region. In certain embodiments, the SMAGP ECD is directly linked to the hinge region. In more specific embodiments, the hinge region comprises the amino acid sequence EPKSX (SEQ ID NO: 4), where X is cysteine ​​or serine.

[0183] The Fc region can be linked to the N-terminus of the SMAGP ECD or the C-terminus of the SMAGP ECD. The Fc region can be linked directly to the SMAGP ECD or can be linked to the SMAGP ECD via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In some embodiments, the peptide linker comprises or consists of 1, 2, 3, 4, or 5 amino acids. In some embodiments, the peptide comprises or consists of 4 or 5 amino acids. Any peptide linker can be used. Exemplary peptide linkers are described herein.

[0184] In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of a fusion molecule described herein to alter one or more functional properties of the fusion molecule, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. Mutations can be introduced into the CH2 domain, CH3 domain, and / or hinge region.

[0185] In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the hinge region so that the number of cysteine ​​residues in the hinge region is altered (e.g., increased or decreased), for example, as described in U.S. Patent No. 5,677,425 (incorporated herein by reference in its entirety). The number of cysteine ​​residues in the hinge region can be altered, for example, to alter (e.g., increase or decrease) the stability of the fusion molecule.

[0186] In some embodiments, one or more amino acid mutations (e.g., substitutions, insertions, or deletions) are introduced into the Fc region to modify (e.g., decrease or increase) the half-life of the fusion molecule in vivo. For examples of mutations that modify (e.g., decrease or increase) the half-life of an antibody in vivo, see, for example, WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631, and U.S. Patent Nos. 5,869,046, 6,121,022, 6,277,375, and 6,165,745 (all of which are incorporated herein by reference in their entirety). In some embodiments, one or more amino acid mutations (e.g., substitutions, insertions, or deletions) are introduced into the Fc region to increase the half-life of the fusion molecule in vivo.

[0187] In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the Fc region to increase or decrease the affinity of the fusion molecule to the Fc receptor (e.g., activating Fc receptor) on the surface of effector cells.The mutations in the Fc region of fusion molecules that increase or decrease the affinity of the fusion molecule to the Fc receptor, and the techniques for introducing such mutations into the Fc receptor or its fragments, are known to those skilled in the art.Examples of mutations in the Fc receptor of an antibody that can be made to modify the affinity of the antibody to the Fc receptor are described, for example, in Smith P et al., (2012) PNAS 109:6181-6186, U.S. Patent No. 6,737,056, and WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631, all of which are incorporated herein by reference in their entirety.

[0188] In a further embodiment, one or more amino acid substitutions are introduced into the IgG constant domain Fc region to alter the effector function(s) of the fusion molecule. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 239, 243, 267, 292, 297, 300, 318, 320, 322, 328, 330, 332, and 396, numbered according to the EU numbering system, can be replaced with a different amino acid residue, resulting in a fusion molecule with altered affinity for an effector ligand while retaining the functionality of the parent molecule. The effector ligand for which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, each of which is incorporated herein by reference in its entirety. In certain embodiments, deletion or inactivation of the Fc region (by point mutation or other means) can reduce Fc receptor binding of circulating fusion molecules, thereby increasing tumor localization. For example, see U.S. Patent Nos. 5,585,097 and 8,591,886, each of which is incorporated herein by reference in its entirety, for a description of mutations that delete or inactivate constant domains, thereby increasing tumor localization. In certain embodiments, one or more amino acid substitutions can be introduced into the Fc region of the fusion molecules described herein to remove potential glycosylation sites on the Fc region, which may reduce Fc receptor binding (see, e.g., Shields RL et al., (2001) J Biol Chem 276:6591-604, incorporated herein by reference in its entirety).

[0189] In various embodiments, one or more of the following mutations may be made in the Fc region of the fusion molecules described herein: an N297A substitution, an N297Q substitution, and an S228P substitution, an L234A substitution, an L234F substitution, an L235A substitution, an L235F substitution, an L235V substitution, an L237A substitution, an S239D substitution, an E233P substitution, an L234V substitution, an L235A substitution, a C236 deletion, a P238A substitution, an S239D substitution, an F243L substitution, a D265A substitution, an S267E substitution, an L328F substitution, an R292P substitution, a Y300L substitution, an A327Q substitution, a P329A substitution, a P329G substitution, an A332L substitution, an I332E substitution, or a P396L substitution, numbered according to the EU numbering system.

[0190] In a specific embodiment, the fusion molecule described herein comprises an IgG1 Fc region with an N297Q or N297A amino acid substitution, numbered according to the EU numbering system. In one embodiment, the fusion molecule described herein comprises an IgG1 Fc region with a mutation selected from the group consisting of D265A, N297A, and combinations thereof (DANA), numbered according to the EU numbering system. In one embodiment, the fusion molecule described herein comprises an IgG1 Fc region with a mutation selected from the group consisting of S239D, I332E, and combinations thereof (DE), numbered according to the EU numbering system. In one embodiment, the fusion molecule described herein comprises an IgG1 Fc region with a mutation selected from the group consisting of D265A, P329A, and combinations thereof, numbered according to the EU numbering system. In another embodiment, the fusion molecule described herein comprises an IgG1 Fc region with a mutation selected from the group consisting of L234A, L235A, and combinations thereof (LALA), numbered according to the EU numbering system. In another embodiment, a fusion molecule described herein comprises an IgG1 Fc region having a mutation selected from the group consisting of L234A, L235A, P329G, and combinations thereof (LALAPG), numbered according to the EU numbering system. In another embodiment, a fusion molecule described herein comprises an IgG2a Fc region having a mutation selected from the group consisting of L234A, L235A, P329G, and combinations thereof (LALAPG), numbered according to the EU numbering system. In another embodiment, a fusion molecule described herein comprises an IgG1 Fc region having a mutation selected from the group consisting of L234F, L235F, N297A, and combinations thereof, numbered according to the EU numbering system. In a specific embodiment, the amino acid residues in the Fc region of a fusion molecule described herein at positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain, numbered according to the EU numbering system, are not L, L, and D, respectively.This approach is described in detail in WO 14 / 108483, which is incorporated herein by reference in its entirety. In certain embodiments, the amino acids corresponding to positions L234, L235, and D265 in the Fc region of human IgG1, numbered according to the EU numbering system, are F, E, and A, or A, A, and A, respectively. In certain embodiments, the amino acids corresponding to positions L234, L235, and P329 in the Fc region of human IgG1, numbered according to the EU numbering system, are A, A, and G, respectively. In certain embodiments, the amino acids corresponding to positions D265 and N297 in the Fc region of human IgG1, numbered according to the EU numbering system, are A and A, respectively. In certain embodiments, the amino acids corresponding to positions S239 and I332 in the Fc region of human IgG1, numbered according to the EU numbering system, are D and E, respectively.

[0191] In certain embodiments, one or more amino acids selected from amino acid residues 329, 331, and 322, numbered according to the EU numbering system, in the Fc region of a fusion molecule described herein can be replaced with a different amino acid residue, thereby resulting in the molecule having altered C1q binding and / or reduced or abolished complement-dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Pat. No. 6,194,551 (Idusogie et al.), incorporated herein by reference in its entirety. In certain embodiments, one or more amino acid residues within amino acid positions 231-238, numbered according to the EU numbering system, in the N-terminal region of the CH2 domain of an antibody described herein are altered, thereby altering the molecule's ability to fix complement. This approach is further described in WO 94 / 29351, incorporated herein by reference in its entirety. In certain embodiments, the Fc region of the fusion molecules described herein may be modified to increase the ability of the molecule to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to modify any of the following positions, numbered according to the EU numbering system: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 358, 360, 361, 362, 363, 36 The fusion molecule may be modified to increase its affinity for an Fcγ receptor by mutating (e.g., introducing an amino acid substitution) one or more amino acids at positions 05, 307, 309, 312, 315, 320, 322, 324, 326, 327, 328, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439. This approach is further described in WO 00 / 42072, which is incorporated by reference in its entirety.

[0192] In certain embodiments, the Fc region of the fusion molecules described herein may have a mutation selected from the group consisting of D265A, P329A, and combinations thereof, numbered according to the EU numbering system. In certain embodiments, the Fc region of the fusion molecules described herein may have a mutation selected from the group consisting of L235A, L237A, and combinations thereof, numbered according to the EU numbering system. In certain embodiments, the Fc region of the fusion molecules described herein may have a mutation selected from the group consisting of S267E, L328F, and combinations thereof, numbered according to the EU numbering system. In certain embodiments, the Fc region of the fusion molecules described herein may have a mutation selected from the group consisting of S239D, I332E, optionally A330L, and combinations thereof, numbered according to the EU numbering system. In certain embodiments, the Fc region of the fusion molecules described herein may contain a mutation selected from the group consisting of L235V, F243L, R292P, Y300L, P396L, and combinations thereof, numbered according to the EU numbering system. In certain embodiments, the Fc region of the fusion molecules described herein may contain a mutation selected from the group consisting of S267E, L328F, and combinations thereof, numbered according to the EU numbering system.

[0193] In some embodiments, the Fc region of the fusion molecules described herein comprises a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256, numbered according to the EU numbering system. See U.S. Patent No. 7,658,921 (incorporated herein by reference in its entirety). This type of mutant IgG, termed "YTE mutant," has been shown to exhibit a four-fold increased half-life compared to the wild-type version of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281:23514-24, incorporated herein by reference in its entirety). In certain embodiments, the fusion molecule comprises an IgG Fc region comprising one or more amino acid substitutions at amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, numbered according to the EU numbering system.

[0194] In a specific embodiment, a fusion molecule described herein comprises an Fc region of human IgG4, in which the serine at amino acid residue 228, numbered according to the EU numbering system, is substituted with a proline (S228P).

[0195] In some embodiments, the Fc region is human IgG1 Fc or a derivative thereof. In some embodiments, the human IgG Fc or human IgG1 Fc comprises an amino acid sequence that is at least 85, 90, 95, 96, 97, 98, or 99% identical to the amino acid sequence of human IgG1 Fc. In some embodiments, the human IgG Fc or IgG1 Fc comprises an amino acid sequence that is at least 85, 90, 95, 96, 97, 98, 99, or 100% identical to the amino acid sequence provided in Table 2 below.

[0196] In some embodiments, the Fc region is human IgG4 Fc or a derivative thereof. In some embodiments, the human IgG Fc or human IgG4 Fc comprises an amino acid sequence that is at least 85, 90, 95, 96, 97, 98, or 99% identical to the amino acid sequence of human IgG4 Fc. In some embodiments, the human IgG Fc or IgG4 Fc comprises an amino acid sequence that is at least 85, 90, 95, 96, 97, 98, 99, or 100% identical to the amino acid sequence provided in Table 2 below.

[0197] In some embodiments, the Fc region is murine IgG2a Fc or a derivative thereof. In some embodiments, the murine IgG Fc or murine IgG2a Fc comprises an amino acid sequence that is at least 85, 90, 95, 96, 97, 98, or 99% identical to the amino acid sequence of murine IgG2a Fc. In some embodiments, the murine IgG Fc or IgG2a Fc comprises an amino acid sequence that is at least 85, 90, 95, 96, 97, 98, 99, or 100% identical to the amino acid sequence provided in Table 2 below. [Table 2-1] [Table 2-2] [Table 2-3]

[0198] In some embodiments, any IgG Fc or derivative thereof, with or without a linker, can be linked to the N- or C-terminus of any of the SMAGP ECDs listed in Table 1 above. In some embodiments, human IgG1 Fc or derivative thereof, with or without a linker, can be linked to the N- or C-terminus of any of the SMAGP ECDs listed in Table 1 above. In some embodiments, the amino acid sequence of the human IgG1 Fc comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, and 50-57. In some embodiments, a derivative of human IgG1 Fc comprises an amino acid sequence at least 85, 90, 95, 96, 97, 98, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, and 50-57.

[0199] In some embodiments, human IgG1 Fc or derivative thereof comprising DANA mutations can be linked, with or without a linker, to the N- or C-terminus of any of the SMAGP ECDs listed above in Table 1. In some embodiments, the amino acid sequence of human IgG1 Fc comprising DANA mutations comprises or consists of the amino acid sequence of SEQ ID NO: 7 or 8. In some embodiments, derivatives of human IgG1 Fc comprising DANA mutations comprise an amino acid sequence that is at least 85, 90, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 7 or 8.

[0200] In some embodiments, the human IgG1 Fc or derivative thereof comprising the LALAPG mutation can be linked, with or without a linker, to the N- or C-terminus of any of the SMAGP ECDs listed above in Table 1. In some embodiments, the amino acid sequence of the human IgG1 Fc comprising the LALAPG mutation comprises or consists of the amino acid sequence of SEQ ID NO: 9 or 10. In some embodiments, the derivative of the human IgG1 Fc comprising the LALAPG mutation comprises an amino acid sequence that is at least 85, 90, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 9 or 10.

[0201] In some embodiments, human IgG1 Fc or derivative thereof comprising DE mutations can be linked, with or without a linker, to the N- or C-terminus of any of the SMAGP ECDs listed above in Table 1. In some embodiments, the amino acid sequence of human IgG1 Fc comprising DE mutations comprises or consists of the amino acid sequence of SEQ ID NO: 11 or 12. In some embodiments, derivatives of human IgG1 Fc comprising DE mutations comprise an amino acid sequence that is at least 85, 90, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 11 or 12.

[0202] In some embodiments, human IgG4 Fc or derivative thereof comprising the S228P mutation can be linked, with or without a linker, to the N- or C-terminus of any of the SMAGP ECDs listed above in Table 1. In some embodiments, the amino acid sequence of human IgG4 Fc comprising the S228P mutation comprises or consists of the amino acid sequence of SEQ ID NO: 13 or 14. In some embodiments, the derivative of human IgG4 Fc comprising the S228P mutation comprises an amino acid sequence that is at least 85, 90, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 13 or 14.

[0203] In some embodiments, murine IgG2a Fc or a derivative thereof can be linked, with or without a linker, to the N- or C-terminus of any of the SMAGP ECDs listed above in Table 1. In some embodiments, the amino acid sequence of murine IgG2a Fc comprises or consists of the amino acid sequence of SEQ ID NO: 15 or 16. In some embodiments, a derivative of murine IgG2a Fc comprises an amino acid sequence that is at least 85, 90, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 15 or 16.

[0204] In some embodiments, murine IgG2a Fc or a derivative thereof comprising the LALAPG mutation can be linked, with or without a linker, to the N- or C-terminus of any of the SMAGP ECDs listed above in Table 1. In some embodiments, the amino acid sequence of the murine IgG2a Fc comprising the LALAPG mutation comprises or consists of the amino acid sequence of SEQ ID NO: 17 or 18. In some embodiments, the derivative of the murine IgG2a Fc comprising the LALAPG mutation comprises an amino acid sequence that is at least 85, 90, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 17 or 18.

[0205] In some embodiments, the fusion molecule comprises or consists of an amino acid sequence shown in Table 3 below. [Table 3-1] [Table 3-2] [Table 3-3]

[0206] In some embodiments, the present disclosure provides a fusion molecule comprising an amino acid sequence at least 85, 90, 95, 96, 97, 98, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 19-34. In some embodiments, the present disclosure provides a fusion molecule comprising the amino acid sequence of any one of SEQ ID NOs: 19-34. In some embodiments, the present disclosure provides a fusion molecule consisting of the amino acid sequence of any one of SEQ ID NOs: 19-34.

[0207] In some embodiments, the present disclosure provides a fusion molecule comprising a dimer of two polypeptides, wherein each polypeptide comprises an amino acid sequence at least 85, 90, 95, 96, 97, 98, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 19-34. In some embodiments, the present disclosure provides a fusion molecule comprising a dimer of two polypeptides, wherein each polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 19-34. In some embodiments, the present disclosure provides a fusion molecule comprising a dimer of two polypeptides, wherein each polypeptide consists of the amino acid sequence of any one of SEQ ID NOs: 19-34. In some embodiments, the present disclosure provides a fusion molecule comprising a dimer of two polypeptides, wherein each polypeptide consists of the amino acid sequence of any one of SEQ ID NOs: 19-34.

[0208] In some embodiments, the half-life extending moiety is serum albumin (e.g., human serum albumin) or a derivative thereof.

[0209] In some embodiments, the half-life extending moiety is a serum albumin binding moiety. Any moiety that can specifically bind to serum albumin (e.g., human serum albumin) in a physiological setting can be used in the fusion molecules described herein. Suitable serum albumin binding moieties include, but are not limited to, antibodies or antigen-binding portions thereof, as well as non-antibody binding molecules such as avimers, DARPins, affibodies, and lipocalins. In some embodiments, the albumin binding moiety is a VH or VHH that specifically binds to human serum albumin.

[0210] In some embodiments, the half-life extending moiety is a non-proteinaceous molecule, such as a polymer. In some embodiments, the non-proteinaceous molecule is linked to the fusion molecule in place of the Fc region. In some embodiments, the non-proteinaceous molecule is linked to the fusion molecule in addition to the Fc region.

[0211] Examples of non-proteinaceous half-life extending moieties include polymer molecules selected from the group consisting of polyalkylene glycols (PAGs), such as polyethylene glycol (PEG) and polypropylene glycol (PPG), branched PEGs, hydroxyalkyl starches (HASs), e.g., hydroxyethyl starch (HES), polysialic acid (PSA), polyvinyl alcohol (PVA), polycarboxylates, poly(vinylpyrrolidone), polyethylene-co-maleic anhydride, polystyrene-co-maleic anhydride, dextrans, including carboxymethyl-dextran, or polyalkylene oxides (PAOs), including any other biopolymer suitable for reducing immunogenicity and / or increasing functional in vivo half-life and / or serum half-life. Another example of a polymer molecule is human serum albumin or another abundant plasma protein. In general, polyalkylene glycol-derived polymers are biocompatible, non-toxic, non-antigenic, non-immunogenic, have various water-soluble properties, and are easily excreted from the body.

[0212] PEG has the advantage of having only a few crosslinkable reactive groups compared to polysaccharides such as dextran. In particular, monofunctional PEG, such as methoxypolyethylene glycol (mPEG), is interesting because its coupling chemistry is relatively simple (only one reactive group is available for conjugation with the linking group on the polypeptide). Therefore, as the risk of crosslinking is eliminated, the resulting conjugated fusion molecules described herein are more homogeneous, and the reaction between the polymer molecule and the variant polypeptide is easier to control.

[0213] To effect covalent attachment of a polymer molecule(s) to the fusion molecules described herein, the hydroxyl end groups of the polymer molecules must be provided in activated form, i.e., with reactive functional groups (examples of which include primary amino groups, hydrazide (HZ), thiol, succinate (SUC), succinimidyl succinate (SS), succinimidyl succinamide (SSA), succinimidyl propionate (SPA), succinimidyl butyrate (SBA), succinimidyl carboxymethylate (SCM), benzotriazole carbonate (BTC), N-hydroxysuccinimide (NHS), aldehyde, nitrophenyl carbonate (NPC), and tresylate (TRES)). Suitable activated polymer molecules are commercially available, for example, from Shearwater Polymers, Inc., Huntsville, Ala., USA, or PolyMASC Pharmaceuticals plc, UK.

[0214] Alternatively, the polymer molecules can be activated by conventional methods known in the art, such as those disclosed in WO 90 / 13540. Specific examples of activated linear or branched polymer molecules for use herein are described in the Shearwater Polymers, Inc. 1997 and 2000 Catalogs (Functionalized Biocompatible Polymers for Research and Pharmaceuticals, Polyethylene Glycol and Derivatives, incorporated herein by reference). Specific examples of activated PEG polymers include the following linear PEGs: NHS-PEG (e.g., SPA-PEG, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG, SG-PEG, and SCM-PEG), and branched PEGs such as NOR-PEG, BTC-PEG, EPOXPEG, NCO-PEG, NPC-PEG, CDI-PEG, ALD-PEG, TRES-PEG, VS-PEG, IODO-PEG, and MAL-PEG, and PEG2-NHS, as well as those disclosed in U.S. Patent Nos. 5,932,462 and 5,643,575, both of which are incorporated herein by reference. Additionally, the following publications disclose useful polymer molecules and / or PEGylation chemistries:U.S. Patent No. 5,824,778, U.S. Patent No. 5,476,653, WO97 / 32607, EP229,108, EP402,378, U.S. Patent No. 4,902,502, U.S. Patent No. 5,281,698, U.S. Patent No. 5,122,614, U.S. Patent No. 5,219,564, WO92 / 16555, WO94 / 04193, WO94 / 14758, WO94 / 17039, WO94 / 18247, WO94 / 28024, WO95 / 00162, WO95 / 1192 4, WO95 / 13090, WO95 / 33490, WO96 / 00080, WO97 / 18832, WO98 / 41562, WO98 / 48837, WO9 9 / 32134, WO99 / 32139, WO99 / 32140, WO96 / 40791, WO98 / 32466, WO95 / 06058, EP439508, WO97 / 03106, WO96 / 21469, WO95 / 13312, EP921131, U.S. Pat. No. 5,736,625, WO98 / 05363, E P809996, U.S. Patent No. 5,629,384, WO96 / 41813, WO96 / 07670, U.S. Patent No. 5,473,034, U.S. Patent No. 5,516,673, EP605963, U.S. Patent No. 5,382,657, EP510356, EP400472, EP183503, and EP154316.

[0215] Specific examples of activated PEG polymers that are particularly preferred for coupling to cysteine ​​residues include the following linear PEGs: vinylsulfone-PEG (VS-PEG), preferably vinylsulfone-mPEG (VS-mPEG), maleimide-PEG (MAL-PEG), preferably maleimide-mPEG (MAL-mPEG), and orthopyridyl-disulfide-PEG (OPSS-PEG), preferably orthopyridyl-disulfide-mPEG (OPSS-mPEG). Typically, such PEG or mPEG polymers have a size of about 5 kDa, about 10 kDa, about 12 kDa, or about 20 kDa.

[0216] Conjugation of the fusion molecules and activated polymer molecules described herein can be carried out using any conventional method, for example, as described in the following references, which also describe suitable methods for activating the polymer molecules: Harris and Zalipsky, eds., Poly(ethylene glycol) Chemistry and Biological Applications, AZC Washington; RF Taylor, (1991), "Protein immobilization. Fundamental and applications," Marcel Dekker, NY; S.S. Wong, (1992), "Chemistry of Protein Conjugation and Crosslinking," CRC Press, Boca Raton; G.T. Hermanson et al., (1993), "Immobilized Affinity Ligand Techniques," Academic Press, NY;

[0217] Those skilled in the art will recognize that the activation method and / or conjugation chemistry used will depend on the linking group(s) of the fusion protein (examples of which are further provided above), as well as the functional groups of the polymer (e.g., amine, hydroxyl, carboxyl, aldehyde, sulfhydryl, succinimidyl, maleimide, vinylsulfone, or haloacetate). PEGylation can be directed to conjugation to all available linking groups on the fusion molecule (i.e., those linking groups exposed on the surface of the polypeptide), or can be directed to one or more specific linking groups, such as the N-terminal amino group or cysteine ​​residues described in U.S. Pat. No. 5,985,265. Furthermore, conjugation can be achieved in one step or in a stepwise manner (e.g., as described in WO 99 / 55377).

[0218] For PEGylation to cysteine ​​residues (see above), the fusion protein is typically treated with a reducing agent, such as dithiothreitol (DDT), prior to PEGylation. The reducing agent is then removed by any conventional method, such as desalting. Conjugation of PEG to cysteine ​​residues is typically carried out in a suitable buffer solution, pH 6-9, at temperatures ranging from 4°C to 25°C for up to 16 hours.

[0219] It will be understood that PEGylation will be designed to produce an optimal molecule with respect to the number of PEG molecules attached, the size and morphology of such molecules (e.g., whether they are linear or branched), and the attachment site(s) on the fusion molecule. The molecular weight of the polymer used can be selected, for example, based on the desired effect to be achieved.

[0220] In connection with conjugation to only a single linking group on the fusion molecule (e.g., the N-terminal amino group), it may be advantageous for the polymer molecule, which may be linear or branched, to have a high molecular weight, preferably about 10-25 kDa, e.g., about 15-25 kDa, e.g., about 20 kDa.

[0221] Typically, polymer conjugation is carried out under conditions designed to allow as many available polymer binding groups as possible to react with the polymer molecule. This is achieved by using a suitable molar excess of polymer relative to the polypeptide. Typically, the molar ratio of activated polymer molecules to polypeptide is up to about 1000-1, for example, up to about 200-1, or up to about 100-1. However, in some cases, the ratio may be slightly lower, such as up to about 50-1, 10-1, 5-1, 2-1, or 1-1, to achieve optimal reaction.

[0222] It is also contemplated that the polymer molecule may be coupled to the fusion molecule via a linker. Suitable linkers are well known to those skilled in the art. A preferred example is cyanuric chloride (Abuchowski et al., (1977), J. Biol. Chem., 252, 3578-3581; U.S. Patent No. 4,179,337; Shafer et al., (1986), J. Polym. Sci. Polym. Chem. Ed., 24, 375-378).

[0223] Following conjugation, any remaining activated polymer molecules are blocked according to methods known in the art, for example, by adding a primary amine to the reaction mixture, and the resulting unactivated polymer molecules are removed by an appropriate method.

[0224] It will be understood that varying degrees of PEGylation can be obtained, depending on circumstances such as the amino acid sequence of the fusion molecule, the nature of the activated PEG compound used, and the specific PEGylation conditions, including the molar ratio of PEG to polypeptide, with higher degrees of PEGylation generally being obtained with higher ratios of PEG to fusion molecule. However, the PEGylated fusion protein resulting from any given PEGylation process will usually contain a stochastic distribution of conjugated fusion proteins having slightly different degrees of PEGylation.

[0225] To improve the biological half-life of the fusion proteins described herein, chemical modifications such as PEGylation or HESylation can be applied.

[0226] HAS and HES non-proteinaceous polymers and methods for producing HAS or HES conjugates are disclosed, for example, in WO02 / 080979, WO03 / 070772, WO057092391, and WO057092390.

[0227] Polysialylation is another technique that uses the natural polymer polysialic acid (PSA) to extend the half-life and improve the stability of therapeutic peptides and proteins. PSA is a polymer of sialic acid (a sugar). When used for protein and therapeutic peptide drug delivery, polysialic acid provides a protective microenvironment upon conjugation. This increases the active lifespan of the fusion molecule in the circulation and prevents it from being recognized by the immune system. PSA polymers are naturally found in the human body. They were adopted by certain bacteria that evolved over millions of years to coat their walls. These naturally polysialylated bacteria were then able to evade the body's defenses thanks to molecular mimicry. PSA, the ultimate in natural stealth technology, can be easily produced from such bacteria in large quantities with defined physical properties. Bacterial PSA, even when conjugated to proteins, is completely non-immunogenic because it is chemically identical to human PSA.

[0228] targeting part The present disclosure provides a fusion molecule comprising the SMAGP extracellular domain (ECD) and a targeting moiety that binds to a target molecule on a cell. Those skilled in the art will recognize suitable targeting moieties, such as proteins, peptides, antibodies, nucleic acids, nucleic acid analogs, carbohydrates, and small molecules. Other non-limiting examples of targeting moieties include sugars and polymers. The targeting moiety is selected based on the target cell type, tissue, or organ to enable sufficiently specific delivery of the fusion molecule to the desired target.

[0229] In some embodiments, the targeting moiety binds to a target molecule on a cell selected from the group consisting of a diseased cell, a senescent cell, a cancer cell, a B cell, a T cell, and a dendritic cell.

[0230] In some embodiments, the targeting moiety binds to a target molecule on a diseased or senescent cell, and the target molecule is selected from the group consisting of DEP1, NTAL, EBP50, STX4, VAMP3, ARMCX3, B2MG, LANCL1, PLD3, VPS26A, DPP4, SCAMP4, MICA / B, TNFRSF10D / CD264, NOTCH1, NOTCH3, CD36, oxidized vimentin, ICAM-1, uPAR, DEP1 / PTPRJ / CD148, CD264, TNFRSF10D, TRAILR4, and CD26.

[0231] In some embodiments, the targeting moiety binds to a target molecule on a cancer cell, and the target molecule is ADAM9, B7-H3 / CD276, BCMA, CA6, CA9, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD70, CD79b, CD123, CD138, CD157 / BST1, P-cadherin CDH3, CEACAM5, CEACAM6, CLDN6, CLDN18.2, DLL3, EGFR, EGFRvIII, ENPP3, ENTPD2, EpC AM, FGR3, FLT3, FOLR1, GPA33, GPC3, GPNMB, GPRC5D, GUCY2C, Her2, HHLA2, LAMP1, SLC39A6 / Liv-1, mesothelin, MUC16 / CA125, MUC17, SLC34A1 / NaPi2a, Nectin4, CD274 / PD-L1, PSCA, PSMA / FOLH1, PVR, PVRIg, ROR1, SLITRK6, SSTR2, STEAP1, TROP2, and TMEM97.

[0232] In some embodiments, the targeting moiety binds to a target molecule on a cancer cell, and the target molecule is selected from the group consisting of ADAM9, B7-H3 / CD276, CEACAM6, CLDN6, DLL3, ENTPD2, FOLR1, GPA33, GPC3, mesothelin, nectin4, PSMA / FOLH1, PVR, and SLITRK6.

[0233] In some embodiments, the targeting moiety binds to a target molecule on a cancer cell from a liquid tumor, and the target molecule is selected from the group consisting of BCMA, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD70, CD79b, CD123, CD138, CD157 / BST1, FGR3, and FLT3.

[0234] In some embodiments, the targeting moiety binds to a target molecule on a cancer cell from a solid tumor, and the target molecule is selected from the group consisting of ADAM9, B7-H3 / CD276, CA6, CA9, P-cadherin CDH3, CEACAM5, CEACAM6, CLDN6, CLDN18.2, DLL3, EGFR, EGFRvIII, ENPP3, ENTPD2, EpCAM, FOLR1, GPA33, GPC3, GPNMB, GPRC5D, GUCY2C, Her2, HHLA2, LAMP1, SLC39A6 / Liv-1, mesothelin, MUC16 / CA125, MUC17, SLC34A1 / NaPi2a, Nectin4, CD274 / PD-L1, PSCA, PSMA / FOLH1, PVR, PVRIg, ROR1, SLITRK6, SSTR2, STEAP1, TROP2, and TMEM97.

[0235] In some embodiments, the targeting moiety binds to a target molecule on a B cell, such as a B cell surface antigen. In some embodiments, the B cell surface antigen is CD20. In some embodiments, the targeting moiety that binds to the target molecule is an antibody or an antigen-binding fragment thereof. In some embodiments, the targeting moiety is rituximab or an antigen-binding fragment thereof. Rituximab-containing fusion molecules are particularly useful in the treatment of lymphoproliferative disorders such as lymphoma or rheumatoid arthritis (RA).

[0236] Payload molecules The present disclosure provides fusion molecules comprising a SMAGP extracellular domain (ECD), eg, a glycosylated or aglycosylated SMAGP ECD, and a payload molecule.

[0237] In some embodiments, the payload molecule is selected from the group consisting of mRNA, miRNA, cRNA, tRNA, siRNA, sgRNA, antisense oligonucleotide, peptide, virus, viral RNA genome, viral DNA genome, vector, plasmid, DNA, radionuclide, and drug (e.g., small molecule drug). In some embodiments, the payload molecule is encapsulated or attached to the nanoparticle. In some embodiments, the payload molecule is a virus selected from an adeno-associated virus or a lentivirus. In some embodiments, the payload molecule is a DNA vector. In some embodiments, the drug is a cytotoxic drug.

[0238] In some embodiments, the payload molecule modulates the activity of cells expressing CLEC10A (also known as C-type lectin domain family 10 member A, MGL, MGL1, or MMGL; see, e.g., UniProtKB Reference Sequence Q8IUN9 (e.g., Q8IUN9-1, updated 2003-03-01) and Q8IUN9-2) in which exemplary human CLEC10A is described), or CD177 (also known as Cluster of Differentiation 177; see, e.g., UniProtKB Reference Sequence Q8N6Q3 (e.g., Q8N6Q3-1, updated 2006-04-04) in which exemplary human CD177 is described). In some embodiments, the payload molecule is designed or selected to modulate (e.g., promote or inhibit) the activity of neutrophils, e.g., neutrophils expressing CD177, as described in Voels et al. (2022) Front Immunol. 13:1003871 (incorporated herein by reference in its entirety), for treating, e.g., a disease or disorder in which neutrophils play a role, e.g., atherosclerosis, cancer (e.g., as described in Astarita et al. (2021) PLoS One 16(12):e0260800 and Kim et al. (2021) Nat Commun. 12(1):5764, each of which is incorporated herein by reference in its entirety), an inflammatory disease or disorder (e.g., as described in Filep (2022) Front Immunol. 13:866747, which is incorporated herein by reference in its entirety), or an infectious disease. For example, the payload molecule can be selected from the group consisting of small molecule drugs that inhibit the NADPH oxidase complex involved in the production of ROS (e.g., diphenyleneiodonium or stelazine), degranulation inhibitors (e.g., nexin hib-20), and promoters of neutrophil apoptosis (e.g., roscovitine).In some embodiments, the payload molecule is a nucleic acid molecule, e.g., mRNA, miRNA, cRNA, tRNA, siRNA, sgRNA, antisense oligonucleotide, viral RNA genome, viral DNA genome, vector, plasmid, or DNA, for delivery to neutrophils (e.g., CD177-expressing neutrophils or another CD177-expressing cell), e.g., for generating engineered chimeric antigen receptor neutrophils (CAR-N), e.g., as described in Chang et al. (2022) Cell Rep. 40(3):111128 (incorporated herein by reference in its entirety). In some embodiments, the payload molecule is directly conjugated to, e.g., the SMAGP ECD, or the payload molecule is encapsulated or attached to a nanoparticle (e.g., a lipid nanoparticle). In some embodiments, the payload molecule is attached to a mutant or variant SMAGP ECD. In some embodiments, the payload molecule is attached to an aglycosylated SMAGP ECD.

[0239] In some embodiments, the nanoparticles are lipid nanoparticles, and the linker is linked to a lipid component of the lipid nanoparticle. In some embodiments, the lipid component of the lipid nanoparticle to which the linker is linked is selected from the group consisting of an ionizable lipid, a PEGylated lipid, a helper lipid, and cholesterol. In some embodiments, the helper lipid is selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-dibenzocyclooctyl (DBCO PE). In some embodiments, the helper lipid is DBCO PE, the SMAGP ECD comprises an N-terminal azidohomoalanine, and the linker comprises a clickable DARPin bound to DBCO PE and the N-terminal azidohomoalanine. In some embodiments, the pegylated lipid is selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG(2000) maleimide), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-5000] (DSPE-PEG(5000) maleimide), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG(2000)), and 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159). In some embodiments, the pegylated lipid is DSPE-PEG(2000)maleimide or DSPE-PEG(5000)maleimide, and the linker comprises a cysteine ​​fused to the C-terminus of the SMAGP ECD that binds the pegylated lipid.

[0240] Linker The present disclosure provides a fusion molecule comprising a SMAGP ECD linked to one or more moieties selected from the group consisting of a half-life extending moiety, a targeting moiety, and a payload molecule. The half-life extending moiety, the targeting moiety, or the payload molecule can be linked to the N-terminus or C-terminus of the SMAGP ECD. Alternatively, the half-life extending moiety, the targeting moiety, or the payload molecule can be linked at a position other than the N-terminus or C-terminus of the SMAGP ECD.

[0241] In some embodiments, the half-life extending moiety, targeting moiety, or payload molecule can be non-covalently attached to the SMAGP ECD. In some embodiments, the half-life extending moiety, targeting moiety, or payload molecule can be covalently attached to the SMAGP ECD.

[0242] In some embodiments, the half-life extending moiety, targeting moiety, or payload molecule may be directly linked (e.g., fused) to the N-terminus, C-terminus, or other region of the SMAGP ECD. In some embodiments, the half-life extending moiety, targeting moiety, or payload molecule is linked to the N-terminus, C-terminus, or other region of the SMAGP ECD via a linker. In some embodiments, the linker is a non-cleavable linker. As used herein, the term "non-cleavable linker" refers to a linker that is not easily cleaved by one or more of a given enzyme, chemical agent, or light irradiation. In some embodiments, the enzyme is a protease.

[0243] In some embodiments, the linker is a synthetic compound linker, such as a chemical crosslinker. Non-limiting examples of suitable commercially available crosslinkers include N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS3), dithiobis(succinimidyl propionate) (DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES).

[0244] As described above, the Fc region disclosed herein can comprise a hinge region or a portion thereof. Thus, a half-life extending moiety, targeting moiety, or payload molecule can be linked to the N-terminus or C-terminus of the SMAGP ECD via this hinge region. In some embodiments, one or more amino acids are included between the SMAGP ECD and the half-life extending moiety, targeting moiety, or payload molecule. In some embodiments, the one or more amino acids included between the SMAGP ECD and the half-life extending moiety, targeting moiety, or payload molecule are amino acids of a natural hinge region. In some embodiments, the SMAGP ECD is fused to a half-life extending moiety, targeting moiety, or payload molecule via the hinge region or a portion thereof. In some embodiments, the hinge region is an IgG hinge region, for example, a human IgG hinge region. In some embodiments, the peptide linker is a portion of a hinge region comprising the amino acid sequence EPKSX (SEQ ID NO: 4), where X is C or S.

[0245] In some embodiments, the linker is a peptide linker. Examples of peptide linkers are well known, and one skilled in the art can select a suitable peptide linker for use in linking the SMAGP ECD to a half-life extending moiety, a targeting moiety, or a payload molecule.

[0246] Peptide linkers can be of any length. In some embodiments, the length and amino acid composition of the linker peptide sequence can be optimized to vary the orientation and / or proximity of moieties to one another to achieve a desired activity. In some embodiments, the peptide linker is about 1 to about 100 amino acids in length, about 8 to about 40 amino acids in length, or about 15 to about 25 amino acids in length. In some embodiments, the peptide linker is 1 to 100 amino acids in length, 8 to 40 amino acids in length, or 15 to 25 amino acids in length. In some embodiments, the peptide linker is about 8 amino acids in length, about 9 amino acids in length, about 10 amino acids in length, about 11 amino acids in length, about 12 amino acids in length, about 13 amino acids in length, about 14 amino acids in length, about 15 amino acids in length, about 16 amino acids in length, about 17 amino acids in length, about 18 amino acids in length, about 19 amino acids in length, about 20 amino acids in length, about 21 amino acids in length, about 22 amino acids in length, about 23 amino acids in length, about 24 amino acids in length, about 25 amino acids in length, about 26 amino acids in length, about 27 amino acids in length, about 28 amino acids in length, about 29 amino acids in length, about 30 amino acids in length, about 31 amino acids in length, about 32 amino acids in length, about 33 amino acids in length, about 34 amino acids in length, about 35 amino acids in length, about 36 amino acids in length, about 37 amino acids in length, about 38 amino acids in length, about 39 amino acids in length, or about 40 amino acids in length. In some embodiments, the peptide linker is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids in length.

[0247] In some embodiments, the peptide linker contains only glycine and / or serine residues (e.g., a glycine-serine linker or a GS linker). Examples of such peptide linkers include Gly(x)Ser (where x is 0-6) or SerGly(x) (where x is 0-6), (GlyGlyGlySer)n (SEQ ID NO: 35) (where n is an integer equal to or greater than 1), and (SerGlyGlyGly)n (SEQ ID NO: 36) (where n is an integer equal to or greater than 1). In some embodiments, the linker peptide is modified so that the amino acid sequence GSG (which occurs at the junction of a traditional Gly / Ser linker peptide repeat) is absent. For example, in some embodiments, the peptide linker comprises an amino acid sequence selected from the group consisting of (GGGXX)nGGGGS (SEQ ID NO: 37) and GGGGS(XGGGS)n (SEQ ID NO: 38), where X is any amino acid that can be inserted into the sequence and does not result in a polypeptide containing the sequence GSG, and n is 0-4. In some embodiments, the sequence of the linker peptide is (GGGX1X2)nGGGGS (SEQ ID NO: 39), where X1 is P, X2 is S, and n is 0 to 4. In some other embodiments, the sequence of the linker peptide is (GGGX1X2)nGGGGS (SEQ ID NO: 40), where X1 is G, X2 is Q, and n is 0 to 4. In some other embodiments, the sequence of the linker peptide is (GGGX1X2)nGGGGS (SEQ ID NO: 41), where X1 is G, X2 is A, and n is 0 to 4. In still other embodiments, the sequence of the linker peptide is GGGGS(XGGGS)n (SEQ ID NO: 42), where X is P, and n is 0 to 4. In some embodiments, a linker peptide of the present disclosure comprises or consists of the amino acid sequence (GGGGA)2GGGGS (SEQ ID NO: 43). In some embodiments, the linker peptide comprises or consists of the amino acid sequence (GGGGQ)2GGGGS (SEQ ID NO: 44). In other embodiments, the linker peptide comprises or consists of the amino acid sequence (GGGPS)2GGGGS (SEQ ID NO: 45).In another embodiment, the linker peptide comprises or consists of the amino acid sequence GGGGS(PGGGS)2 (SEQ ID NO:46). In yet another embodiment, the linker peptide comprises or consists of the amino acid sequence GSGGS (SEQ ID NO:47) or SGGSGS (SEQ ID NO:48). In another embodiment, the linker peptide comprises or consists of the amino acid sequence GGGGGSGGGGSGGGSGGGGS (SEQ ID NO:49). In some embodiments, the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 35-49, 58-61, or a variant thereof containing 1 to 5 amino acid changes.

[0248] In some embodiments, the peptide linker is a GS linker about 20 amino acids in length. In some embodiments, the peptide linker is a GS linker about 20 amino acids in length.

[0249] Polynucleotides, vectors, and production methods The present disclosure also provides polynucleotides encoding any of the fusion molecules or portions thereof described herein. In some embodiments, the polynucleotide encodes the SMAGP ECD of the present disclosure. In some embodiments, the polynucleotide encodes the Fc region of the present disclosure. In some embodiments, the polynucleotide encodes one or more of a half-life extending moiety, a targeting moiety, and a payload molecule. In some embodiments, the polynucleotide encodes the SMAGP ECD and the Fc region, and optionally a linker. In some embodiments, the polynucleotide encodes the SMAGP ECD and one or more of a half-life extending moiety, a targeting moiety, and a payload molecule, and optionally a linker. In some embodiments, the polynucleotides described herein are DNA molecules. In some embodiments, the polynucleotides described herein are RNA molecules.

[0250] In some embodiments, the polynucleotide comprises a nucleotide sequence that encodes a fusion molecule comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 19-34. In some embodiments, the polynucleotide comprises a nucleotide sequence that encodes a fusion molecule comprising the amino acid sequence of any one of SEQ ID NOs: 19-34. In some embodiments, the polynucleotide comprises a nucleotide sequence that encodes a fusion molecule consisting of the amino acid sequence of any one of SEQ ID NOs: 19-34.

[0251] In some embodiments, the polynucleotide comprises a first nucleotide sequence encoding a SMAGP ECD and a second nucleotide sequence encoding a half-life extending moiety, a targeting moiety, or a payload molecule. In some embodiments, the polynucleotide comprises a first nucleotide sequence encoding a SMAGP ECD and an Fc region. In some embodiments, the first nucleotide sequence encodes a SMAGP ECD consisting of an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 1-3. In some embodiments, the second nucleotide sequence encodes an Fc domain consisting of an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 5-18 and 50-57. In some embodiments, the first nucleotide sequence encodes a SMAGP ECD comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 1-3. In some embodiments, the second nucleotide sequence encodes an Fc domain comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 5-18 and 50-57. In some embodiments, the first nucleotide sequence encodes a SMAGP ECD comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 1-3, and the second nucleotide sequence encodes an Fc domain comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 5-18 and 50-57.

[0252] In some embodiments, the first or second nucleotide sequence also encodes a linker. In some embodiments, the first nucleotide sequence encodes the SMAGP ECD and linker, and the second nucleotide sequence encodes the Fc region. In some embodiments, the first nucleotide sequence encodes the SMAGP ECD, and the second nucleotide sequence encodes the Fc region and linker. The linker can be any linker described herein, including a portion of a hinge region, a peptide linker, or a linker comprising the amino acid sequence of any one of SEQ ID NOs: 4, 35-49, or 58-61. In some embodiments, the linker consists of the amino acid sequence of any one of SEQ ID NOs: 4, 35-49, or 58-61.

[0253] Also provided herein is a polynucleotide encoding the above-provided fusion molecule or a portion thereof, which is optimized, for example, by codon / RNA optimization, replacement with a heterologous signal sequence, and elimination of mRNA instability elements. Thus, a method for generating an optimized nucleic acid for recombinant expression by introducing codon changes in mRNA and / or elimination of inhibitory regions can be carried out by adapting the optimization methods described in, for example, U.S. Patent Nos. 5,965,726, 6,174,666, 6,291,664, 6,414,132, and 6,794,498, all of which are incorporated herein by reference in their entirety. For example, potential splice sites and instability elements (e.g., A / T or A / U-rich elements) in RNA can be mutated without altering the amino acids coded by the nucleic acid sequence to increase the stability of the RNA for recombinant expression. Modifications take advantage of the degeneracy of the genetic code, for example, by using alternative codons for the same amino acid. In one embodiment, it may be desirable to modify one or more codons to encode a conservative variation, e.g., a similar amino acid having a similar chemical structure and properties and / or function as the native amino acid.

[0254] Polynucleotides can be obtained by any method known in the art, and the nucleotide sequence of a polynucleotide can be determined by any method known in the art. The nucleotide sequences encoding the fusion molecules or portions thereof described herein, and modified versions of these fusion molecules, can be determined using methods well known in the art, i.e., nucleotide codons known to encode specific amino acids are assembled to generate nucleic acids encoding the fusion molecules or portions thereof. Polynucleotides encoding such proteins can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier G et al., (1994), BioTechniques 17:242-6 (incorporated herein in its entirety)). Briefly, this involves synthesizing overlapping oligonucleotides containing portions of the sequence encoding the fusion molecules or portions thereof, annealing and ligating the oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

[0255] Alternatively, polynucleotides encoding the fusion molecules or portions thereof described herein can be generated from nucleic acids from a suitable source using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells producing the polypeptide of interest. Such PCR amplification methods can be used to obtain nucleic acids containing sequences encoding the fusion molecules or portions thereof. The amplified nucleic acids can be cloned into vectors for expression in host cells and further cloning.

[0256] If a clone containing nucleic acid encoding a particular polypeptide is not available, but the sequence of the polypeptide is known, the nucleic acid encoding the polypeptide can be chemically synthesized or obtained from a suitable source (e.g., a cDNA library generated from, or nucleic acid isolated therefrom, any tissue or cell that expresses a polypeptide described herein, preferably polyA+ RNA) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific for the particular gene sequence to identify a cDNA clone from a cDNA library that encodes the polypeptide, for example. Amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using any method well known in the art.

[0257] DNA encoding the fusion molecules described herein, or portions thereof, can be readily isolated and sequenced using conventional procedures. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells (e.g., CHO cells from the CHO GS System™ (Lonza)), or myeloma cells that do not otherwise produce the molecules described herein.

[0258] Also provided are polynucleotides that hybridize under high stringency, medium or low stringency hybridization conditions to polynucleotides encoding the fusion molecules or portions thereof described herein.

[0259] Hybridization conditions are described in the art and are known to those skilled in the art. For example, hybridization under stringent conditions can include hybridization to filter-bound DNA in 6x sodium chloride / sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2x SSC / 0.1% SDS at about 50-65°C, and hybridization under highly stringent conditions can include hybridization to filter-bound nucleic acid in 6x SSC at about 45°C, followed by one or more washes in 0.1x SSC / 0.2% SDS at about 68°C. Hybridization under other stringent hybridization conditions is known to those skilled in the art and has been described, e.g., Ausubel FM et al., eds., (1989) Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York, pages 6.3.1-6.3.6 and 2.10.3, which is incorporated herein by reference in its entirety.

[0260] In one aspect, provided herein are cells (e.g., host cells) that (e.g., recombinantly) express the fusion molecules, or portions thereof, described herein, and associated polynucleotides and expression vectors. Provided herein are vectors (e.g., expression vectors) for recombinant expression in host cells, preferably mammalian cells (e.g., CHO cells), comprising a polynucleotide that includes a nucleotide sequence encoding a fusion molecule, or portion thereof, described herein. Also provided herein are host cells for recombinant expression of the fusion molecules, or portions thereof, described herein, comprising such vectors. Provided herein are methods for producing the fusion molecules, or portions thereof, described herein, comprising expressing the fusion molecules, or portions thereof, from a host cell.

[0261] Recombinant expression of the proteins described herein generally involves the construction of an expression vector containing a polynucleotide encoding the polypeptide. Once a polynucleotide encoding a polypeptide described herein is obtained, a vector for producing the polypeptide can be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a fusion molecule or a portion thereof by expressing a polynucleotide containing a polypeptide-encoding nucleotide sequence are described herein. Methods well known to those skilled in the art can be used to construct an expression vector containing a polypeptide-encoding sequence and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also provided are replicable vectors containing a nucleotide sequence encoding a fusion molecule or a portion thereof operably linked to a promoter. Such vectors can include, for example, a nucleotide sequence encoding the Fc region of a polypeptide (see, for example, WO 86 / 05807 and WO 89 / 01036, and U.S. Pat. No. 5,122,464, which are incorporated herein by reference in their entireties).

[0262] The expression vector can be transferred to a cell (e.g., a host cell) by conventional techniques, and the resulting cell can then be cultured by conventional techniques to produce a fusion molecule or portion thereof described herein. Thus, provided herein are host cells comprising a polynucleotide encoding or comprising a fusion molecule or portion thereof described herein.

[0263] In some embodiments, the vector is a non-viral vector. Exemplary non-viral vectors include, but are not limited to, plasmid DNA, transposons, episomal plasmids, minicircles, ministrings, and oligonucleotides (e.g., mRNA, naked DNA). In some embodiments, the vector is a DNA plasmid vector.

[0264] In some embodiments, the vector is a viral vector. The viral vector may be replication-competent or replication-defective. The viral vector may be integrating or non-integrating. Several virus-based systems have been developed for gene transfer into mammalian cells, and a suitable viral vector can be selected by one skilled in the art. Exemplary viral vectors include, but are not limited to, adenoviral vectors (e.g., adenovirus 5), adeno-associated viral (AAV) vectors (e.g., AAV2, 3, 5, 6, 8, 9), retroviral vectors (MMSV, MSCV), lentiviral vectors (e.g., HIV-1, HIV-2), gamma retroviral vectors, herpesvirus vectors (e.g., HSV1, HSV2), alphavirus vectors (e.g., SFV, SIN, VEE, M1), flavivirus (e.g., Kunjin, West Nile, Dengue virus), rhabdovirus vectors (e.g., rabies virus, VSV), measles virus vectors (e.g., MV-Edm), Newcastle disease virus vectors, poxvirus vectors (e.g., VV), measles virus, and picornavirus vectors (e.g., coxsackievirus).

[0265] In some embodiments, a vector or expression cassette comprises one or more additional elements, including but not limited to, a promoter, an enhancer, a polyadenylation (polyA) sequence, and a selection gene.

[0266] In some embodiments, the vector comprises a polynucleotide sequence that encodes an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence listed in any of Tables 1-3. In some embodiments, the vector comprises a polynucleotide sequence that encodes an amino acid sequence listed in any of Tables 1-3.

[0267] Pharmaceutical Compositions The present disclosure provides compositions (e.g., pharmaceutical compositions) comprising the fusion molecules described herein, nucleic acid molecules (e.g., expression vectors) encoding the fusion molecules, or host cells expressing the fusion molecules. The pharmaceutical compositions described herein are formulated with suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerability, etc. Many suitable formulations can be found in formularies known to all pharmacists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (e.g., LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowax (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998) J Pharm Sci Technol 52:238-311.

[0268] The dosage of the fusion molecules described herein, nucleic acid molecules (e.g., expression vectors) encoding the fusion molecules, or host cells expressing the fusion molecules administered to a patient can vary depending on the patient's age and size, the target disease, condition, route of administration, etc. Preferred dosages are typically calculated by body weight or body surface area. The frequency and duration of treatment can be adjusted depending on the severity of the condition. Effective dosages and schedules for administering the fusion proteins or component peptides, or nucleic acid molecules, or expression vectors encoding them described herein can be determined empirically, for example, by monitoring the patient's progress through periodic evaluations and adjusting the dosage accordingly. Furthermore, interspecies scaling of dosages can be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0269] Various delivery systems (e.g., liposomes, microparticles, encapsulation in microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis) are known and can be used to administer the pharmaceutical compositions disclosed herein (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Introduction methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active agents. Administration can be systemic or local.

[0270] Any pharmaceutical composition described herein can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, for subcutaneous delivery, a pen delivery device facilitates application when delivering the pharmaceutical composition disclosed herein. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In disposable pen delivery devices, there is no replaceable cartridge. Rather, disposable pen delivery devices are pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is empty of the pharmaceutical composition, the entire device is discarded.

[0271] In certain circumstances, pharmaceutical compositions can be delivered in a sustained-release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, a polymeric material can be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Press, Boca Raton, Florida. In yet another embodiment, a sustained-release system can be placed near the target of the composition, thereby requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other sustained-release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0272] Injectable preparations may include dosage forms for intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, drip infusion, etc. These injectable preparations may be prepared by publicly known methods. For example, injectable preparations may be prepared by dissolving, suspending, or emulsifying any of the fusion molecules described herein in a sterile aqueous or oily medium conventionally used for injections. Aqueous media for injections include, for example, saline, isotonic solutions containing glucose, and other adjuvants, which may be used in combination with suitable solubilizers such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)). Oily media include, for example, sesame oil and soybean oil, which may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The injection solution prepared in this manner is preferably filled into an appropriate ampule.

[0273] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms with unit doses suitable for the dosage of active ingredients.Such dosage forms in unit doses include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0274] Treatment method The present disclosure provides methods comprising administering to a subject in need thereof a composition (e.g., a pharmaceutical composition described herein) comprising a fusion molecule described herein, a nucleic acid molecule (e.g., an expression vector) encoding the fusion molecule, or a composition comprising a host cell expressing the fusion molecule. In certain embodiments, the present disclosure provides a fusion molecule described herein, a polynucleotide encoding the fusion molecule, an expression vector comprising a polynucleotide encoding the fusion molecule, a nanoparticle comprising the fusion molecule, or a composition comprising the fusion molecule (e.g., a pharmaceutical composition described herein) for use in medicine.

[0275] In some embodiments, the present disclosure provides a method for modifying the activity of a leukocyte, the method comprising contacting the leukocyte with an effective amount of a fusion molecule described herein, a nucleic acid molecule (e.g., an expression vector) encoding the fusion molecule, or a host cell expressing the fusion molecule. In some embodiments, the leukocyte is selected from the group consisting of a bone marrow cell, a macrophage (e.g., an M0 macrophage, an M1 macrophage, an M2 macrophage, an M2a macrophage, an M2b macrophage, an M2c macrophage, or an M2d macrophage), a Kupffer cell, a histiocyte, a microglia, an osteoclast, a dendritic cell, a mast cell, a neutrophil, a regulatory T cell, a tumor-infiltrating regulatory T cell, and a granulocyte. In some embodiments, the activity of the leukocyte comprises one or more of phagocytosis, cytokine production, chemokine production, antigen presentation, growth factor production, and protease production. In some embodiments, the activity comprises phagocytosis of a population of cells or cell-like structures. In some embodiments, the population of cells or cell-like structures is selected from the group consisting of cancer cells, immune cells, neurons, red blood cells, and platelets. In some embodiments, leukocyte activity is increased. In some embodiments, leukocyte activity is decreased. In some embodiments, leukocyte activity is increased or decreased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, or at least about 200% of activity.

[0276] The present disclosure also provides a method for treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a fusion molecule, a nucleic acid molecule (e.g., an expression vector) encoding the fusion molecule, or a host cell expressing the fusion molecule, described herein. Non-limiting examples of diseases or disorders include, for example, autoimmune disorders (e.g., autoimmune disorders characterized by excessive phagocytic activity), lymphoproliferative disorders, macrophage activation syndrome (MAS), cytokine-associated disorders, central nervous system (CNS) diseases, diseases of overactivated microglia, diseases of overactivated osteoclasts, osteoporosis, cancer-related bone degradation and / or metastasis, multiple myeloma, bone diseases, systemic juvenile idiopathic arthritis, allergies, cancer, cancer with low PD-L1 expression, cancer resistant to PD-1 and / or PD-L1 inhibitors, diseases or disorders characterized by multinucleated giant cells, and atopic diseases (e.g., atopic dermatitis). As used herein, a "cancer with low PD-L1 expression" is a cancer having tumors that express PD-L1 with a tumor proportionality score (TPS) or combined positive score (CPS) of ≥ 1%, ≥ 10%, or ≥ 50%, as determined by an FDA-approved test. See, e.g., https: / / www.keytrudahcp.com / biomarker-testing / pd-l1 / .

[0277] In some embodiments, the autoimmune disorder is selected from the group consisting of allogeneic islet graft rejection, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, Alzheimer's disease, antineutrophil cytoplasmic autoantibodies (ANCA), autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune myocarditis, autoimmune neutropenia, autoimmune oophoritis and orchitis, immune thrombocytopenia (ITP or idiopathic thrombocytopenic purpura, idiopathic thrombocytopenic purpura, immune-mediated thrombocytopenia, or primary immune thrombocytopenia), autoimmune urticaria, Behcet's disease, bullous pemphigoid ( BP), cardiomyopathy, Castleman syndrome, celiac sprue dermatitis, chronic fatigue and immune deficiency syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, dilated cardiomyopathy, discoid lupus, epidermolysis bullosa acquisita, essential mixed cryoglobulinemia, factor VIII deficiency, fibromyalgia and fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Goodpasture's syndrome, graft-versus-host disease (GVHD), Hashimoto's thyroiditis, hemophagocytic lymphohistiocytosis (HLH), hemophilia A, Idiopathic inflammatory myopathy (IIM), idiopathic membranous nephropathy, idiopathic pulmonary fibrosis, IgA neuropathy, IgM polyneuropathy, immune thrombocytopenia (ITP), immune-mediated necrotizing myopathy (IMNM), juvenile arthritis, Kawasaki disease, lichen planus, lichen sclerosus, lupus erythematosus, lupus nephritis, Meniere's disease, mixed connective tissue disease, mucous membrane pemphigoid, multiple sclerosis, type 1 diabetes, multifocal motor neuropathy (MMN), myasthenia gravis (MG), generalized myasthenia gravis (gMG), myositis, paraneoplastic bullous pemphigoid, pemphigoid gestationis, pemphigus vulgaris (PV), pemphigus foliaceus (P) F), pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis, dermatomyositis (DM), necrotizing autoimmune myopathy (NAM), antisynthetase syndrome (AsyS), primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, relapsing polychondritis, Raynaud's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, solid organ transplant rejection, stiff-body syndrome, systemic lupus erythematosus, Takayasu's arteritis, toxic epidermal necrolysis (TEN), Stevens-Johnson syndrome (SJS),The disease is selected from the group consisting of temporal arteritis / giant cell arteritis, thrombotic thrombocytopenic purpura, ulcerative colitis, uveitis, dermatitis herpetiformis vasculitis, antineutrophil cytoplasmic antibody-associated vasculitis, vitiligo, and Wegener's granulomatosis.

[0278] In some embodiments, the lymphoproliferative disorder is selected from the group consisting of chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia, large granular lymphocyte disorder, lymphocytosis, natural killer cell leukemia, prolymphocytic leukemia, follicular lymphoma, hemophagocytic lymphohistiocytosis (HLH), B-cell lymphoma, T-cell lymphoma, and multiple myeloma. Fusion molecules comprising a targeting moiety comprising rituximab or an antigen-binding fragment thereof are particularly suitable for treating lymphoproliferative disorders.

[0279] In some embodiments, the CNS disease is Alzheimer's disease, schizophrenia, or Huntington's disease.

[0280] In some embodiments, the cancer-associated bone degradation and / or metastasis is due to a disease or disorder selected from multiple myeloma, breast cancer, and prostate cancer.

[0281] Non-limiting examples of cancers that can be treated with the fusion molecules or compositions disclosed herein include solid tumors, blood cancers, leukemia, lymphoma, osteosarcoma, rhabdomyosarcoma, neuroblastoma, kidney cancer, renal transitional cell carcinoma, bladder cancer, Wilms' carcinoma, ovarian cancer, pancreatic cancer, breast cancer (e.g., characterized by mutations in BRCA1 and / or BRCA2 or Her2+), prostate cancer, bone cancer, lung cancer (e.g., non-small cell lung cancer or small cell lung cancer), gastric cancer, colon cancer, cervical cancer, synovial sarcoma, head and neck cancer, and ovarian cancer. Cancers include, but are not limited to, cervical cancer, squamous cell carcinoma, multiple myeloma, renal cell carcinoma, retinoblastoma, hepatoblastoma, hepatocellular carcinoma, melanoma, renal rhabdoid tumor, Ewing's sarcoma, chondrosarcoma, brain cancer, glioblastoma, meningioma, pituitary adenoma, vestibular schwannoma, primitive neuroectodermal tumor, medulloblastoma, astrocytoma, anaplastic astrocytoma, oligodendroglioma, ependymoma, choroid plexus papilloma, polycythemia vera, thrombocythemia, idiopathic myelofibrosis, soft tissue sarcoma, thyroid cancer, endometrial cancer, carcinoid cancer, liver cancer, epithelial cancer, and peritoneal cancer. In certain embodiments, the cancer is a metastatic cancer, such as the types of metastatic cancer described above.

[0282] In certain embodiments, the cancer is a solid tumor, a hematological cancer (e.g., leukemia, lymphoma, myeloma), and metastatic lesions thereof. In one embodiment, the cancer is a solid tumor. Examples of solid tumors include malignant tumors, such as sarcomas and carcinomas (e.g., adenocarcinomas) of various organ systems affecting the lung, breast, lymphatic, gastrointestinal or colon, genital and genitourinary tract (e.g., kidney, urothelium, bladder cells), pharynx, CNS (e.g., brain, neural or glial cells), skin (e.g., melanoma), head and neck (e.g., head and neck squamous cell carcinoma (HNCC)), and pancreas. For example, melanoma, colon cancer, gastric cancer, rectal cancer, renal cell carcinoma, breast cancer (e.g., breast cancer that does not express one, two, or all of estrogen receptors, progesterone receptors, or Her2 / neu, e.g., triple-negative breast cancer; or Her2+ breast cancer), liver cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC), e.g., NSCLC with squamous and / or non-squamous histology), or small cell lung cancer), prostate cancer, head and neck cancer (e.g., HPV+ squamous cell carcinoma), small intestine cancer, and esophageal cancer.

[0283] In one embodiment, the cancer is a blood cancer, such as leukemia, lymphoma, or myeloma. In one embodiment, the cancer is a leukemia, such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute myeloblastic leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), chronic lymphocytic leukemia (CLL), or hairy cell leukemia. In one embodiment, the cancer is a lymphoma, e.g., B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), activated B-cell-like (ABC) diffuse large B-cell lymphoma, germinal center B-cell (GCB) diffuse large B-cell lymphoma, mantle cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, relapsed non-Hodgkin's lymphoma, refractory non-Hodgkin's lymphoma, relapsed follicular non-Hodgkin's lymphoma, Burkitt's lymphoma, small lymphocytic lymphoma, follicular lymphoma, lymphoplasmacytic lymphoma, or extranodal marginal zone lymphoma. In one embodiment, the cancer is a myeloma, e.g., multiple myeloma.

[0284] In some embodiments, the disease or disorder is selected from the group consisting of H. pylori infection, COVID-19, severe COVID-19, IgA nephropathy, ovarian cancer, inflammatory bowel disease (IBD), acute-on-chronic liver failure (ACLF), antineutrophil cytoplasmic antibody-associated vasculitis (AAV), autoimmune vasculitis, asthma, acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), diseases in which regulatory T cells express CD177, Kawasaki disease, septic shock, renal cell carcinoma, hepatocellular carcinoma, breast cancer, lung cancer, and colorectal cancer.

[0285] The administration of the composition according to the method described herein can result in the reduction of the severity, signs, symptoms or markers of disease or disorder in patients with disease or disorder.In this context, " reduction " means a statistically significant reduction in such level.The reduction (absolute reduction or reduction in the difference between the elevated level and normal level in a subject) can be, for example, at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or less than the detection level of the assay used.

[0286] In one embodiment, the fusion molecule or composition thereof is administered to a subject simultaneously or sequentially with an additional therapeutic agent. In one embodiment, the additional therapeutic agent is an anti-inflammatory agent. In one embodiment, the additional therapeutic agent is a corticosteroid. In one embodiment, the additional therapeutic agent is rituximab, daclizumab, basiliximab, muronomab-cd3, infliximab, adalimumab, omalizumab, efalizumab, natalizumab, tocilizumab, eculizumab, golimumab, canakinumab, ustekinumab, or belimumab. In one embodiment, the additional therapeutic agent is a leukocyte-depleting agent.

[0287] In one embodiment, the additional therapeutic agent is a B cell depleting agent. In one embodiment, the B cell depleting agent is an antibody. In one embodiment, the B cell depleting antibody is an antibody that specifically binds to CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD53, CD70, CD72, CD74, CD75, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85, or CD86.

[0288] In some embodiments, the additional therapeutic agent is a chemotherapeutic agent, hi one embodiment, the chemotherapeutic agent is paclitaxel.

[0289] In one embodiment, the additional therapeutic agent is an immune checkpoint modulator. In one embodiment, the immune checkpoint modulator is an anti-PD-1 agent or an anti-PD-L1 agent. In one embodiment, the immune checkpoint modulator is an anti-PD-1 antibody or an anti-PD-L1 antibody. In one embodiment, the additional therapeutic agent is a combination of a chemotherapeutic agent and an immune checkpoint modulator. In one embodiment, the chemotherapeutic agent is paclitaxel and the immune checkpoint modulator is an anti-PD-1 agent or an anti-PD-L1 agent. In one embodiment, the disease or disorder is ovarian cancer and the additional therapeutic agent is a combination of paclitaxel and an anti-PD-1 agent or an anti-PD-L1 agent.

[0290] The fusion molecules of the present disclosure can be further linked to or co-expressed with another functional molecule, e.g., another peptide or protein. For example, the fusion molecules can be operatively linked (e.g., by chemical bonding, genetic fusion, non-covalent bonding, or other methods) to one or more other molecular entities, such as antibodies or antibody fragments, to produce bispecific or multispecific binding molecules with second or additional binding specificities.

[0291] The following examples demonstrate that CLEC10A and CD177 are receptors for SMAGP. Therefore, the fusion molecules described herein can also be used to assay CLEC10A or CD177 (e.g., human CLEC10A or CD177) protein levels. In some embodiments, CLEC10A or CD177 (e.g., human CLEC10A or CD177) protein levels can be measured in biological samples using classical immunohistological methods known to those skilled in the art, including immunoassays such as enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, or Western blotting. Suitable assay labels are known in the art and include enzyme labels such as glucose oxidase, iodine ( 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 121In), and technetium ( 99 These labels include radioisotopes such as Tc), luminescent labels such as luminol, and fluorescent labels such as fluorescein and rhodamine, as well as biotin. Such labels can be used to label the fusion molecules described herein. Alternatively, antibodies that recognize the fusion molecules described herein can be labeled and used in combination with the fusion molecules to detect SMAGP (e.g., human SMAGP) protein levels. Thus, in certain embodiments, the present disclosure relates to the use of the fusion molecules of the present disclosure for in vitro detection of any of SMAGP, CLEC10A, or CD177 (e.g., human SMAGP, CLEC10A, or CD177) proteins in biological samples. In further embodiments, the present disclosure relates to the use of a fusion molecule of the present disclosure for assaying and / or detecting SMAGP, CLEC10A, or CD177 (e.g., human SMAGP, CLEC10A, or CD177) protein levels in a biological sample in vitro, optionally wherein the fusion molecule is conjugated to a radionuclide or detectable label and / or carries a label as described herein, and / or wherein immunohistological methods are used.

[0292] Assaying the expression level of SMAGP, CLEC10A, or CD177 (e.g., human SMAGP, CLEC10A, or CD177) protein is intended to include qualitatively or quantitatively measuring or estimating the level of SMAGP, CLEC10A, or CD177 (e.g., human SMAGP, CLEC10A, or CD177) protein in a first biological sample, either directly (e.g., by determining or estimating absolute protein levels) or relatively (e.g., by comparing with disease-related protein levels in a second biological sample). The expression level of SMAGP, CLEC10A, or CD177 (e.g., human SMAGP, CLEC10A, or CD177) polypeptide in a first biological sample can be measured or estimated and compared with a standard or reference SMAGP, CLEC10A, or CD177 (e.g., human SMAGP, CLEC10A, or CD177) protein level. The standard may be, for example, taken from a second biological sample obtained from an individual without the disorder, or determined by averaging levels from a population of individuals without the disorder. As is understood in the art, once a "standard" SMAGP, CLEC10A, or CD177 (e.g., human SMAGP, CLEC10A, or CD177) polypeptide level is known, it can be repeatedly used as a standard for comparison. Accordingly, in further embodiments, the present disclosure relates to an in vitro method for assaying and / or detecting SMAGP, CLEC10A, or CD177 protein levels (e.g., human SMAGP, CLEC10A, or CD177 protein levels) in a biological sample, comprising qualitatively or quantitatively measuring or estimating SMAGP, CLEC10A, or CD177 protein (e.g., human SMAGP, CLEC10A, or CD177 protein) in the biological sample by immunohistological methods. In certain embodiments, the binding between SMAGP and CLEC10A or CD177 is measured using enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), or flow cytometry.

[0293] As used herein, the term "biological sample" refers to any biological sample obtained from a subject, cell line, tissue, or other cell source that potentially expresses SMAGP, CLEC10A, or CD177 (e.g., human SMAGP, CLEC10A, or CD177). Methods for obtaining tissue biopsies and body fluids from animals (e.g., humans or cynomolgus monkeys) are well known in the art. Biological samples include peripheral blood mononuclear cells (PBMCs).

[0294] The fusion molecules described herein are well known and standard to those skilled in the art and can be used for prognostic, diagnostic, monitoring, and screening applications, including in vitro and in vivo applications based on the present description. Prognostic, diagnostic, monitoring, and screening assays and kits for in vitro evaluation and assessment can be used to predict, diagnose, and monitor patient samples to determine a patient's suitability for drug clinical trials or the administration of specific chemotherapeutic agents, radiotherapeutic agents, or antibodies (including combinations thereof) against different drugs or antibodies. Thus, in certain embodiments, the present disclosure relates to fusion molecules and / or compositions of the present disclosure for use as diagnostics. In certain embodiments, the present disclosure relates to fusion molecules and / or compositions of the present disclosure for use in methods for predicting, diagnosing, and / or monitoring a subject. In another embodiment, the present disclosure relates to the use of fusion molecules of the present disclosure for predicting, diagnosing, and / or monitoring a subject by assaying and / or detecting human SMAGP, CLEC10A, or CD177 protein levels in a subject's biological sample in vitro.

[0295] kit Any of the compositions described herein can be included in a kit. In a non-limiting example, the kit includes one or more of a fusion molecule described herein, a nucleic acid molecule (e.g., an expression vector) encoding the fusion molecule, or a host cell expressing the fusion molecule.

[0296] The kit may further include reagents or instructions for using in a subject a fusion molecule described herein, a nucleic acid molecule (e.g., an expression vector) encoding the fusion molecule, or a host cell expressing the fusion molecule. It may also include one or more buffers.

[0297] The components of the kit may be packaged either in aqueous media or in lyophilized form. The container means of the kit will generally include at least one vial, test tube, flask, bottle, syringe, or other container means into which the components may be placed, and preferably, suitably aliquoted. Where there are more than two components in the kit (such as when the labeling reagent and label are packaged together), the kit will also generally include a second, third, or other additional container into which the additional components may be placed separately. The kit may also include a second container means for containing a sterile, pharmaceutically acceptable buffer and / or other diluent. However, various combinations of components may also be included in the vial. The kits of the present disclosure will also typically include a means for containing the fusion molecules described herein, nucleic acid molecules (e.g., expression vectors) encoding the fusion molecules, or host cells expressing the fusion molecules, and any other reagent containers in close confinement for commercial sale.

[0298] When the components of the kit are provided in one and / or more liquid solutions, the liquid solution is an aqueous solution, and a sterile aqueous solution is particularly preferred.However, the components of the kit can also be provided as dry powder(s).When reagents and / or components are provided as dry powder, the powder can be reconstituted by adding a suitable solvent.It is also envisioned that the solvent can be provided in a separate container. [Example]

[0299] The presently disclosed embodiments are provided by way of illustration and description and are not intended to limit the scope of the present disclosure.

[0300] Example 1 Generation and characterization of SMAGP ECD-hIgG1 Fc fusion molecules The extracellular domains of human SMAGP (Met1-Leu36) and cynomolgus SMAGP (Met1-Leu35) were separately fused to the wild-type Fc domain of human IgG1, including the hinge, CH2, and CH3 domains. To avoid an unpaired cysteine, the Cys residue in the upper hinge region was mutated to Ser (C220S) in each molecule. The fusion molecules are referred to herein as hSMAGP ECD-hIgG1 Fc and cSMAGP ECD-hIgG1 Fc, respectively. A schematic diagram of these fusion molecules is shown in Figure 1A.

[0301] Protein expression vectors encoding each of these fusion molecules, along with their N-terminal leader sequences, were transfected into HEK293 cells. The fusion proteins were captured using Protein A resin, and the buffer was exchanged into PBS buffer at pH 7.0 after elution. The resulting purified proteins were quantified by OD280. The purity of each protein was determined using a Perkin Elmer GXII capillary electrophoresis system, and the degree of protein aggregation was determined by HPLC-SEC. As shown in Figure 1B, hSMAGP ECD-hIgG1 Fc was approximately 98% pure and showed minimal aggregation. The estimated MW of hSMAGP ECD-hIgG1 Fc (103.25 kDa) was significantly higher than the calculated MW (59.7 kDa), suggesting that the protein was glycosylated. Similar results were obtained for cSMAGP ECD-hIgG1 Fc.

[0302] Example 2 Binding of SMAGP ECD-hIgG1 Fc to human macrophages, neutrophils, and monocytes Human macrophages were generated from monocytes isolated from fresh peripheral blood leukapheresis products from two healthy human donors. Briefly, CD14 +Monocytes were sorted via negative selection using a stem cell monocyte enrichment kit. Monocytes were then cultured in RPMI containing Glutamax + heat-inactivated fetal bovine serum and MCSF (40 ng / ml) in 10 cm tissue culture-treated plates for 6 days. Macrophages were detached using Accutase enzyme digestion, washed, and plated in 96-well plates for 5 e 4 Cells were seeded at 200 μg / well. Cells were then Fc-R blocked with antibodies against CD32, CD16, and CD64 (25 μg / ml) in FACS buffer for 30 minutes at 4°C. After washing, cells were then stained with hIgG1 Fc control, hSMAGP ECD-hIgG1 Fc, or cSMAGP ECD-hIgG1 Fc in FACS buffer for 30 minutes, followed by secondary staining with anti-hIgG-647 for 30 minutes at 4°C. After washing, macrophages were resuspended in FACS buffer plus NucBlue (2 drops / ml) and read on a flow cytometer.

[0303] As shown in Figures 2A and 2B, binding of both hSMAGP ECD-hIgG1 Fc and cSMAGP ECD-hIgG1 Fc to primary human macrophages relative to the hIgG1 Fc control was demonstrated. This study was repeated with similar results.

[0304] In further experiments, whole blood was obtained from healthy human donors. Red blood cells were lysed, and samples were then Fc-R blocked with antibodies against CD32, CD16, and CD64 (25 μg / ml) in FACS buffer for 30 minutes at 4°C. After washing, cells were then stained with viability dye, lineage markers (CD3, CD56, CD14, CD19, and CD15), and hIgG1 Fc control or hSMAGP ECD-hIgG1 Fc in FACS buffer for 30 minutes, followed by secondary staining with anti-hIgG-647 for 30 minutes at 4°C. After washing, cells were resuspended in FACS buffer and read on a flow cytometer.

[0305] As shown in Figures 2C and 2D, binding of hSMAGP ECD-hIgG1 Fc relative to the hIgG1 Fc control was demonstrated on both neutrophils (CD15+, Figure 2C) and monocytes (CD14+, CD15-, Figure 2D). This study was repeated with similar results.

[0306] Example 3 Inhibition of human macrophage phagocytosis of Ramos cells by hSMAGP ECD-hIgG1 Fc Human macrophages were generated as described in Example 2. Following the monocyte culture step, macrophages were detached using Accutase enzyme digestion, washed, and plated in 96-well plates for 3 e 5 After removing the medium and washing the cells, they were incubated with hSMAGP ECD-hIgG1 Fc (0.63, 1.25, 2.5, 5, 10, 20, or 40 μg / ml; the 0.63 μg / ml condition was tested on only one donor) or hIgG1 Fc control (20 or 40 μg / ml; the 20 μg / ml condition was tested on only one donor) at 37°C for 30–45 min. Ramos tumor cells were stained with 100 nM pHrodo-Red-SE in dPBS for 30 min, quenched, washed, and then incubated with anti-CD47 antibody (10 μg / ml) (clone B6H12) for 6 e 5 100 μg / well of IgG1 Fc was added to the cultures. Images were acquired via Incucyte after 1 hour of incubation to assess the total area of ​​red objects corresponding to tumor cell phagocytosis. Additional controls included macrophages co-cultured with Ramos SMAGP-overexpressing cells in the presence of anti-CD47 antibody and macrophages co-cultured with the Ramos parental cell line in the absence of anti-CD47 antibody. Remaining tumor cells at the end of the assay were assessed by flow cytometry and normalized to the 40 μg / ml IgG1 Fc control hIgG1-Fc group.

[0307] As shown in Figures 3A and 3B, hSMAGP ECD-hIgG1 Fc dose-dependently inhibited anti-CD47 blockade-induced phagocytosis of Ramos tumor cells by human macrophages from two separate donors measured at 1 hour relative to the IgG1 Fc control. As expected, Ramos SMAGP-overexpressing tumor cells and Ramos cells without anti-CD47 were not phagocytosed efficiently by human macrophages. For donor 23-derived macrophages (Figure 3A), statistical significance was observed for the 40 μg / ml hSMAGP ECD-hIgG1 Fc, Ramos SMAGP-overexpressing cells, and non-anti-CD47 conditions, respectively, relative to the 40 μg / ml IgG1 Fc control (p<0.001 (***) using one-way ANOVA). For donor 27-derived macrophages (Figure 3B), statistical significance was observed for the 20 μg / ml hSMAGP ECD-hIgG1 Fc, 40 μg / ml hSMAGP ECD-hIgG1 Fc, Ramos SMAGP-overexpressing cells, and non-anti-CD47 conditions relative to the 40 μg / ml IgG1 Fc control (p<0.033 (*), p<0.001 (***) using one-way ANOVA).

[0308] As shown in Figures 4A and 4B, incubation with hSMAGP ECD-hIgG1 Fc resulted in increased tumor cell recovery relative to incubation with the IgG1 Fc control in a dose-dependent manner, consistent with the inhibition of phagocytosis shown in Figures 3A and 3B. As expected, Ramos SMAGP-overexpressing tumor cells and Ramos cells without anti-CD47 were not phagocytosed as efficiently by human macrophages, resulting in increased tumor cell recovery. For donor 23-derived macrophages (Figure 4A), statistical significance was observed for 10, 20, and 40 μg / ml hSMAGP ECD-hIgG1 Fc, Ramos SMAGP-overexpressing cells, and the non-anti-CD47 condition, respectively, relative to the 40 μg / ml IgG1 Fc control (p<0.033 (*), p<0.002 (**), and p<0.0001 (****) using one-way ANOVA). For macrophages from donor 27 (Figure 4B), statistical significance was observed for 5, 10, 20, and 40 μg / ml hSMAGP ECD-hIgG1 Fc, Ramos SMAGP-overexpressing cells, and non-anti-CD47 conditions, respectively, versus the 40 μg / ml IgG1 Fc control (p<0.033 (*), p<0.002 (**), p<0.001 (***) using one-way ANOVA).

[0309] Example 4 Inhibition of human macrophage phagocytosis by hSMAGP ECD-hIgG1 Fc in A375 tumor cells Depending on the donor, cells were isolated from fresh peripheral blood leukapheresis products from healthy human donors using negative selection or CD14 +Human macrophages were generated from isolated pan-monocytes using selection. Monocytes were differentiated into macrophages by culturing for 7 days in RPMI medium supplemented with 10% FBS in the presence of recombinant human MCSF. On day 6, macrophages were detached, replated in 96-well plates in MCSF-containing medium, and allowed to re-adhere overnight. On day 7, A375 tumor cells were detached, washed with PBS, and stained with the pH-sensitive dye pHrodo-Red-SE. Macrophages were stimulated with hIgG1-Fc (Bio-X-Cell) or hSMAGP ECD-hIgG1-Fc for approximately 45 minutes. After this preincubation, 10 μg / ml of anti-CD47 (magrolimab biosimilar, R&D Systems) was added to all macrophage wells, and labeled tumor cells were added at a 1:1 effector:target ratio. Images were acquired via Incucyte at 25 hours after culture, and the total area of ​​red matter corresponding to tumor cell phagocytosis was assessed. The total area of ​​red matter corresponding to tumor cell phagocytosis was quantified at 4 hours for three donors. This demonstrated significant inhibition of phagocytosis of hSMAGP ECD-hIgG1 Fc relative to the control for each donor (Figure 5A). This observation was confirmed over a 25-hour phagocytosis time course for a representative donor (Figure 5B). In Figure 5A, one-way ANOVA was used to assess statistical significance (p<0.05 (*), p<0.01 (**), p<0.001 (***), p<0.0001 (****)).

[0310] Because A375 tumor cells do not express Fc receptors, the results of this experiment indicate that expression of Fc receptors on tumor cells is not required for the inhibition of phagocytosis by hSMAGP ECD-hIgG1 Fc. Although hSMAGP ECD-hIgG1 Fc can bind to macrophages and other myeloid cells via both the SMAGP ECD and hIgG1 Fc domains, either in cis or trans, we speculate that hSMAGP ECD-hIgG1 Fc may also bind to tumor cells that express Fc receptors via the hIgG1 Fc domain, thus "studding" tumor cells with hSMAGP ECD-hIgG1 Fc and mimicking overexpressing tumor cells.

[0311] Example 5 Inhibition of human macrophage phagocytosis by hSMAGP ECD-hIgG1 Fc in the presence of rituximab Human macrophages were generated from pan-monocytes isolated using negative selection from fresh peripheral blood leukapheresis products from healthy human donors. Monocytes were differentiated into macrophages by culturing for 7 days in RPMI medium supplemented with 10% FBS in the presence of recombinant human MCSF. On day 6, macrophages were detached, replated in MCSF medium in 96-well plates, and allowed to re-adhere overnight. On day 7, Ramos tumor cells were harvested and stained with the pH-sensitive dye pHrodo-Red-SE. Macrophages were stimulated for approximately 45 minutes with hIgG1-Fc (Bio-X-Cell), hSMAGP ECD-hIgG1-Fc, CD24 ECD-hIgG1-Fc (R&D Systems), or CD47 ECD-hIgG1-Fc (R&D Systems). After this pre-incubation, 0.5 μg / ml (Figure 6A) or 1 μg / ml (Figure 6B) of anti-CD20 (rituximab biosimilar) was added to all macrophage wells, and labeled tumor cells were added at an effector:target ratio of 1:2. Images were acquired via Incucyte after 3 hours of culture, and the total area of ​​red objects corresponding to tumor cell phagocytosis was assessed (Figure 6A). Phagocytosis was allowed to proceed for 24 hours, after which all cells were detached, stained with anti-CD11b antibody, resuspended in FACS buffer, and 100 μl of this solution was acquired on an Attune flow cytometer. Live CD11b in 100 μl - The amount of cells was determined via analysis in FlowJo (Figure 6B). In both Figures 6A and 6B, one-way ANOVA was used to assess statistical significance (p<0.05 (*), p<0.01 (**), p<0.001 (***), p<0.0001 (****)).

[0312] As shown in Figures 6A and 6B, hSMAGP ECD-hIgG1 Fc inhibited anti-CD20-induced phagocytosis of Ramos cells by human macrophages compared with the isotype control, as indicated by a decrease in the red area at 3 hours (Figure 6A) and an increase in the total number of remaining tumor cells at 24 hours (Figure 6B). In contrast, CD24-hIgG1-Fc and CD47-hIgG1-Fc significantly enhanced phagocytosis at 3 hours (Figure 6A) and tended to reduce the amount of remaining tumor cells at 24 hours (Figure 6B). These results demonstrate that hSMAGP ECD-hIgG1 Fc reduces phagocytosis by two different measures and that this is not an inherent property of human Fc fusion proteins. Unlike hSMAGP ECD-hIgG1 Fc, other "Don't Eat Me" signaling protein ECDs derived from CD24 and CD47, each fused to hIgG1-Fc, enhanced phagocytosis. These data highlight the surprising nature of the potent inhibition of phagocytosis exhibited by hSMAGP ECD-hIgG1 Fc.

[0313] Example 6 Inhibition of human macrophage phagocytosis by hSMAGP ECD-hIgG1 Fc in the presence of rituximab Human macrophages were generated and seeded as in Example 5. On day 7, Ramos tumor cells were harvested and stained with the pH-sensitive dye pHrodo-Red-SE. Macrophages were stimulated for approximately 45 minutes with 20 μg / ml of hIgG1-Fc (Bio-X-Cell), hSMAGP ECD-hIgG1 Fc, hIgG1 LALA PG (Bio-X-Cell), hIgG4 (Ichor Bio), hSMAGP ECD-hIgG1 LALA PG Fc, and hSMAGP ECD-hIgG4. After this preincubation, 0.5 μg / ml of anti-CD20 (rituximab biosimilar) was added to all macrophage wells, and labeled tumor cells were added at an effector:target ratio of 1:2. Images were acquired via Incucyte after 3 hours of culture, and the total area of ​​red objects, corresponding to tumor cell phagocytosis, was assessed (Figure 7). One-way ANOVA was used to assess statistical significance (p<0.05(*), p<0.01(**), p<0.001(***), p<0.0001(****)).

[0314] Interestingly, only hSMAGP ECD-hIgG1 Fc, which contains a functional Fc domain, reduced phagocytosis compared to its isotype-matched control. The Fc-dead (hSMAGP-hIgG1 LALA PG) or hIgG4-fusion variant of hSMAGP ECD did not reduce phagocytosis. Based on these data, binding of the Fc domain to Fc receptors may be important for hSMAGP-mediated inhibition of phagocytosis. Comparison with the isotype-matched control, as well as the data in Example 5, indicates that inhibition of phagocytosis is not solely due to the Fc domain, as hSMAGP ECD-hIgG1 Fc inhibits phagocytosis compared to its isotype-matched control, whereas other DEM signals fused to hIgG1 do not. Collectively, these data highlight the uniqueness and potential therapeutic importance of hSMAGP ECD-hIgG1 Fc.

[0315] Example 7 Inhibition of human macrophage phagocytosis of Ramos cells by hSMAGP ECD-hIgG1 Fc across macrophage polarization states Human macrophages were generated from pan-monocytes isolated using negative selection from fresh peripheral blood leukapheresis products from healthy human donors. Monocytes were differentiated into macrophages by culturing for 7 days in RPMI medium supplemented with 10% FBS in the presence of recombinant human MCSF. On day 6, macrophages were detached, replated in 96-well plates in MCSF medium, and allowed to re-adhere overnight in the presence of MCSF plus polarizing stimuli (IFN-γ, LPS, TGF-β, IL-10, IL-4), as indicated. LPS was purchased from Invivogen; all other polarizing stimuli were purchased from R&D Systems. On day 7, Ramos tumor cells were harvested and stained with the pH-sensitive dye pHrodo-Red-SE. Macrophages were stimulated with hIgG1-Fc or hSMAGP ECD-hIgG1-Fc for approximately 1 hour. hIgG1-Fc was purchased from Bio-X-Cell. After this preincubation, 10 μg / ml of anti-CD47 antibody (B6H12, Bio-X-Cell) was added to all macrophage wells, followed by the addition of labeled tumor cells (effector:target ratio of 1:2). Images were acquired via Incucyte at 21 h. For each polarity condition (M0, no polarity stimulus; M1, induced by LPS and IFN-γ; M2, induced by IL-10; M2, induced by TGF-β; M2, induced by TGF-β and IL-10; and M2, induced by TGF-β, IL-10, and IL-4) treated with hSMAGP ECD-hIgG1 Fc or hIgG1 Fc control, the total area of ​​red objects corresponding to tumor cell phagocytosis was determined 2 h after coculture (Figure 8A). Statistical significance was assessed using multiple unpaired t-tests corrected for multiple comparisons (p<0.01 (**), p<0.001 (***)). The time course of phagocytosis of Ramos cells by M1-polarized macrophages (induced by LPS and IFN-γ) treated with hSMAGP ECD-hIgG1 Fc or hIgG1 Fc control in the presence of anti-CD47 antibody (B6H12) was determined over 21 hours (Figure 8B).

[0316] As shown in Figure 8A, hSMAGP ECD-hIgG1 Fc inhibited anti-CD47-induced phagocytosis of Ramos cells by human macrophages compared to the isotype control, as indicated by the significantly reduced red area across all macrophage polarization conditions tested. Strong inhibition of phagocytosis by inflammatory M1 macrophages was observed over the entire time course of the assay (Figure 8B). These results demonstrate that hSMAGP ECD-hIgG1 Fc can broadly inhibit phagocytosis by macrophages across various polarization states / stimulation conditions. In particular, the inhibition of phagocytosis in inflammatory M1 macrophages suggests that hSMAGP ECD-hIgG1 Fc could be used to inhibit phagocytosis under inflammatory conditions (e.g., diseases or disorders such as autoimmune disorders that involve excessive phagocytosis causing pathology).

[0317] Example 8 Inhibition of human macrophage phagocytosis of opsonized erythrocytes by SMAGP ECD-hIgG1 Fc Human macrophages were generated from pan-monocytes isolated using negative selection from fresh peripheral blood leukapheresis products from healthy human donors. Monocytes were differentiated into macrophages by culturing for 7 days in RPMI medium supplemented with 10% FBS in the presence of recombinant human MCSF. On day 6, macrophages were detached, replated in MCSF medium in 96-well plates, and allowed to re-adhere overnight. On day 7, red blood cells (RBCs) from pooled human donors (Rockland Immunochemicals) were washed and stained with the pH-sensitive dye pHrodo-Red-SE. RBCs were then opsonized for 30 minutes at 37°C with 1.25, 2.5, 5, 10, 20, or 40 μg / mL of polyclonal anti-RBC antibody (Rockland Immunochemicals) or no anti-RBC antibody. While RBCs were opsonizing, macrophages were stimulated with 20 μg / mL hIgG1 Fc (Bio-X-Cell) or SMAGP ECD-hIgG1 Fc, or medium alone, for 30 minutes. Opsonized RBCs were washed and added to the macrophages at a 1:5 E:T ratio. Images were acquired via Incucyte every 1–2 hours after incubation, and the total area of ​​red matter corresponding to RBC phagocytosis was assessed for each time point. A time course for a representative donor with a 20 μg / mL opsonizing antibody concentration is shown in Figure 9A. Figure 9B shows the total area of ​​red matter at 10 hours with a 20 μg / mL opsonizing antibody concentration for all three donors tested. Statistical significance was determined using a multiple unpaired t-test (p<0.05 (*), p<0.01 (**), p<0.001 (***), p<0.0001 (****)). Total phagocytosis for each concentration of opsonizing antibody, with or without hSMAGP ECD-hIgG1 Fc or hIgG1 Fc, was quantified by calculating the area under the curve of the total area of ​​red objects over time and plotted against the opsonizing antibody concentration for each donor in Figure 9C.

[0318] Human macrophages were then generated as described in the preceding paragraph of this example. On day 7, RBCs from pooled human donors (Rockland Immunochemicals) were washed and stained with the pH-sensitive dyes pHrodo-Red-SE and Cytopainter green. The RBCs were then opsonized with 10 μg / mL of polyclonal anti-RBC antibody or isotype control (Rockland Immunochemicals) for 30 minutes at 37°C. While the RBCs were opsonized, macrophages were stimulated with either 2.5, 5, 10, or 20 μg / mL of hIgG1 Fc (Bio-X-Cell) or hSMAGP ECD-hIgG1 Fc, or medium alone, for 30 minutes. The opsonized RBCs were washed and added to the macrophages at a 1:5 E:T ratio and incubated overnight. All cells were then detached and resuspended in FACS buffer, and 100 μL of this solution was analyzed on an Attune flow cytometer. Macrophages were distinguished from RBCs by size. The amount of remaining live RBCs in 100 μL was determined via analysis in FlowJo. The amount of remaining RBCs was normalized to the amount of remaining RBCs for RBCs opsonized with isotype control antibody in the presence of macrophages from each donor. The percentage of remaining RBCs versus the concentration of hSMAGP ECD-hIgG1 Fc or hIgG1 Fc is plotted in Figure 10.

[0319] The results demonstrated that hSMAGP ECD-hIgG1 Fc was able to suppress RBC phagocytosis by human macrophages induced by anti-RBC antibodies compared to the isotype hIgG1 Fc control, as indicated by both a reduced area of ​​red objects (Figures 9A-9C) as well as a higher percentage of surviving RBCs (Figure 10).

[0320] Example 9 Macrophage cytokine expression in the presence and absence of LPS stimulation Human macrophages were generated from monocytes isolated from fresh peripheral blood leukapheresis products from healthy human donors. Briefly, CD14 + Monocytes were sorted via negative selection using a stem cell monocyte enrichment kit. They were then cultured in RPMI containing Glutamax®, heat-inactivated fetal bovine serum, and MCSF in tissue culture-treated plates for 5 days. Cells were detached and replated in 96-well plates in MCSF (MO) for an additional 2 days. Cells were treated overnight with either no Fc, hIgG1 Fc control (20 μg / ml), or hSMAGP ECD-hIgG1 Fc (20 μg / ml) plus or minus LPS (100 ng / ml). The next day, plates were spun, and supernatants were collected. The amount of released cytokines was analyzed in multiplex using the U-PLEX cytokine assay (Meso Scale Discovery) according to the manufacturer's instructions.

[0321] As shown in Figures 11A-11C, hSMAGP ECD-hIgG1 Fc did not significantly modulate the release of TNFα (Figure 11A), IL-12p70 (Figure 11B), or IL-6 (Figure 11C) either at steady state or in the presence of 100 ng / ml LPS. Three representative donors are shown in Figure 11. This study was repeated with similar results. These data suggest that hSMAGP ECD-hIgG1 Fc does not induce an enhanced general inflammatory effect in macrophages under the conditions tested.

[0322] Example 10 Identification and characterization of CLEC10A as a binding partner of SMAGP To identify cognate cell surface receptors for SMAGP that may potentially mediate signaling that reduces macrophage phagocytosis of tumor cells, we evaluated the binding interactions of hSMAGP ECD-hIgG1 Fc and cSMAGP ECD-hIgG1 Fc with a library of human lectins. Lectins are a broad category of surface-expressed proteins that mediate various protein-protein and protein-carbohydrate interactions. A set of 37 recombinant human lectins was selected based on their reported expression and potential role in immune cells. Human recombinant lectin microarrays were created by positioning the lectins at specific locations and immobilizing them on microarray slides coated with polyvalent N-hydroxysuccinimide (NHS). Custom microarrays were fabricated in a 16-subarray format. Each subarray contained four replicates of each human lectin, and also included negative and positive controls and markers.

[0323] The recombinant human lectin microarray assay utilized the accumulation of fluorescent signals at designated spots to identify specific lectins to which the probe reagent could bind. Recombinant SMAGP-Fc fusions served as probes and were first labeled with biotin, then hybridized to the array, and subsequently prepared for assay with fluorescent streptavidin. Specifically, hSMAGP ECD-hIgG1 Fc and cSMAGP ECD-hIgG1 Fc were labeled with EZ-link NHS-LC-LC-biotin, followed by overnight dialysis (in PBS, 10 kDa cutoff) and concentration (30 kDa cutoff). The biotinylated probes were then ready to hybridize to the microarray.

[0324] To determine whether hSMAGP ECD-hIgG1 Fc or cSMAGP ECD-hIgG1 Fc interacts with either lectin, biotinylated versions were analyzed using custom microarrays. Microarrays were pretreated with buffer TBS-T (supplemented with 2 mM CaCl2 and MgCl2) for 30 minutes at room temperature. hSMAGP ECD-hIgG1 Fc or cSMAGP ECD-hIgG1 Fc (5 μg / ml) was then added to the microarray and incubated for 1 hour at room temperature. After incubation, the microarray was washed, and streptavidin-Cy3 (0.5 μg / ml) was added to the microarray. After incubation for 1 hour at room temperature, the microarray was washed and scanned at 532 nm using high laser intensity (1 PMT). Specific binding of CLEC10A only was observed with hSMAGP ECD-hIgG1 Fc (Figure 12A) and cSMAGP ECD-hIgG1 Fc (Figure 12B), but not with the negative control consisting of buffer alone (Figure 12C). As expected, PC1 (consisting of amine-PEG-biotin) showed a positive signal (Figures 12A-12C).

[0325] CLEC10A is a C-type lectin that requires calcium for efficient substrate binding. To test binding specificity, microarray binding assays were performed in the absence of 2 mM CaCl2 and MgCl2. As expected, CLEC10A binding of hSMAGP ECD-hIgG1 Fc and cSMAGP ECD-hIgG1 Fc was lost in the absence of cations (Figures 13A-13C). Thus, CLEC10A binding of hSMAGP ECD-hIgG1 Fc and cSMAGP ECD-hIgG1 Fc is cation-dependent. Considering that lectins generally lose carbohydrate-binding activity when cations are removed, these data suggest that the observed binding is likely due to glycan-receptor interactions. Thus, the glycans on hSMAGP ECD-hIgG1 Fc and cSMAGP ECD-hIgG1 Fc may generate a multivalent scaffold for CLEC10A binding.

[0326] To test the binding of hSMAGP ECD-hIgG1 Fc to CLEC10A, HEK293 cells were transfected with expression vectors encoding ZsGreen1 alone (control) or ZsGreen1 and CLEC10A. Live cell transfectants, including control-only transfected cells, were incubated with 5 μg / mL hSMAGP ECD-hIgG1 Fc and PBS + Ca2+. + / Mg2 + The cells were incubated in assay buffer. Cells were washed, and binding was detected by flow cytometry using an AF647-labeled anti-human Fc secondary antibody. A 3.3-fold change in median fluorescence intensity averaged across two replicates was observed in CLEC10A-expressing cells compared to the control ( FIG. 14 ).

[0327] Binding of recombinant hSMAGP ECD-hIgG1 Fc to recombinant CLEC10A ECD was assessed by enzyme-linked immunosorbent assay (ELISA). Human CLEC10A (His-tagged extracellular domain) or human IgG1 Fc (control) protein was incubated in PBS + 2 mM Ca2+. + / Mg2 + The antibody was immobilized in buffer on a Corning 96-well clear flat-bottom polystyrene high-binding microplate overnight at 4°C. The plate was blocked with 5% skim milk, and then biotinylated hSMAGP ECD-hIgG1 Fc protein (10 μg / ml) was added to the wells. Binding of biotinylated hSMAGP ECD-hIgG1 Fc to the immobilized protein was detected using HRP-conjugated streptavidin. The signal was developed using TMB substrate. The assay was performed in triplicate. Binding of hSMAGP ECD-hIgG1 Fc to CLEC10A ECD was observed, as shown in Figure 15.

[0328] In conclusion, lectin library surveys identified CLEC10A as a specific binding partner of hSMAGP ECD-hIgG1 Fc and cSMAGP ECD-hIgG1 Fc, with cation-dependent binding. Binding of hSMAGP ECD-hIgG1 Fc to CLEC10A was confirmed both in CLEC10A-overexpressing cells and in ELISA assays using recombinant CLEC10A ECD.

[0329] Example 11 Identification and characterization of CD177 as a binding partner of SMAGP To identify one or more additional cognate cell surface receptors for SMAGP in addition to CLEC10A, hits from a library screen of over 6,000 full-length human plasma membrane proteins were identified, expressed in HEK293 cells, and probed with 5 μg / mL hSMAGP ECD-hIgG1 Fc or 1 μg / mL CTLA4-hFc (control). All expression vectors encoding membrane proteins were spotted in duplicate with a fluorescent expression control. Binding was detected using the AF647 anti-hIgG Fc secondary antibody. Comparison of hits with SMAGP-hFc (Figure 16A) and CTLA4-hFc (Figure 16B) identified CD177 as a SMAGP-specific binding protein.

[0330] The binding of hSMAGP ECD-hIgG1 Fc to CD177 was reassessed in a live cell assay using flow cytometry. HEK293 cells were transfected with expression vectors encoding ZsGreen1 alone (control) or ZsGreen1 and CD177. Live cell transfectants, including control-only transfected cells, were incubated with 5 μg / mL of hSMAGP ECD-hIgG1 Fc. Cells were washed, and binding was detected by flow cytometry using an AF647-labeled anti-human Fc secondary antibody. Assays were performed in duplicate. A 157-fold change in median fluorescence intensity was observed in CD177-expressing cells compared to the control (FIG. 17).

[0331] Binding of recombinant hSMAGP ECD-hIgG1 Fc to recombinant CD177 ECD was assessed by enzyme-linked immunosorbent assay (ELISA). Human CD177 (His-tagged extracellular domain) or human IgG1 Fc (control) protein was incubated in PBS + 2 mM Ca2+. + / Mg2 + The antibody was immobilized in buffer on a Corning 96-well clear flat-bottom polystyrene high-binding microplate overnight at 4°C. The plate was blocked with 5% skim milk, and then biotinylated hSMAGP ECD-hIgG1 Fc protein (10 μg / ml) was added to the wells. Binding of biotinylated hSMAGP ECD-hIgG1 Fc to the immobilized protein was detected using HRP-conjugated streptavidin. The signal was developed using TMB substrate. The assay was performed in triplicate. Binding of hSMAGP ECD-hIgG1 Fc to CD177 ECD was observed, as shown in Figure 18.

[0332] To evaluate the binding of aglycosylated hSMAGP ECD to CD177, the hSMAGP ECD peptide (H2N-MTSLLTTPSPREELMTTPILQPTEALSPEDGASTALK-biotin-amide (SEQ ID NO: 62)) was chemically synthesized. An additional lysine residue was added to the C-terminus to facilitate biotinylation. Another peptide with a scrambled hSMAGP ECD sequence (H2N-TRTTLIMESTTPALMLLEPLGSPQDAPTSTEEAPSLK-biotin-amide (SEQ ID NO: 63)) was synthesized as a control. Peptide binding to recombinant CD177 ECD-Fc (CD177 extracellular domain with an Fc tag) or hIgG1-Fc (IgG1 domain with an Fc tag) negative control was evaluated by enzyme-linked immunosorbent assay (ELISA). CD177 ECD-Fc and hIgG1-Fc were immobilized onto separate Corning 96-well clear flat-bottom polystyrene high-binding microplates in PBS overnight at 4°C. Plates were blocked using 5% skim milk. Biotinylated hSMAGP ECD peptide or a scrambled control (10 μg / ml) was preincubated with HRP-conjugated streptavidin (5 μg / ml) and added to the plate. In the control assay, anti-CD177 antibody (Biolegend catalog no. 315802; 10 μg / ml) was premixed with the biotinylated SMAGP ECD peptide. Signals were developed using TMB substrate. Assays were performed in triplicate.

[0333] As shown in Figure 19, the hSMAGP ECD peptide showed binding to CD177-Fc, whereas the scrambled hSMAGP ECD peptide and the hSMAGP ECD peptide plus anti-CD177 antibody showed no binding. Importantly, this result revealed that the interaction between the hSMAGP ECD and CD177 ECD does not require glycosylation of the hSMAGP ECD. Therefore, recombinant expression of the hSMAGP ECD is not required to bind to CD177 ECD.

[0334] In conclusion, a survey of a human plasma membrane protein library identified CD177 as a specific binding partner of hSMAGP ECD-hIgG1 Fc. Binding of hSMAGP ECD-hIgG1 Fc to CD177 was confirmed both in CD177-overexpressing cells and in an ELISA assay using recombinant CD177 ECD, and binding of aglycosylated hSMAGP ECD peptide to CD177 was confirmed in a separate ELISA assay using recombinant CD177 ECD.

[0335] Example 12 CD177 and CLEC10A expression on various cell types Human macrophages were generated from monocytes isolated from fresh peripheral blood leukapheresis products from healthy human donors. Briefly, CD14 + Monocytes were sorted via negative selection using a stem cell monocyte enrichment kit. They were then cultured in RPMI containing Glutamax plus heat-inactivated fetal bovine serum and MCSF in tissue culture-treated plates for 5 days. Cells were detached and replated in either MCSF (M0) or MCSF plus IL-4, IL-10, and TGF-β (M2a / c) for an additional 2 days. Cells were Fc-blocked and then stained with anti-CD177, anti-CLEC10A, or isotype control and viability dye. Cells were washed and run on a flow cytometer.

[0336] As shown in Figure 20A, CD177 expression was not detected in either M0 or M2a / c human macrophages. As shown in Figure 20B, CLEC10A expression was detected in both M0 and M2a / c human macrophages. Three representative donors are shown.

[0337] In further experiments, whole blood was obtained from healthy human donors. Red blood cells were lysed, and then the cells were Fc-blocked. Peripheral blood cells were stained with lineage markers (CD3, CD56, CD14, CD19, and CD15), viability dyes, and either anti-CD177, anti-CLEC10A, or isotype controls. Cells were washed and run on a flow cytometer.

[0338] As shown in Figure 21A, peripheral blood neutrophils (CD15+) were found to strongly express CD177. CD177 expression was not detected on peripheral blood monocytes (CD14+, CD15-), B cells (CD19+), T cells (CD3+CD56-), NK cells (CD56+), or NKT cells (CD56+CD3+). As shown in Figure 21B, CLEC10A expression was not detected on any of the peripheral blood cell types tested.

[0339] Example 13 Evaluation of SMAGP ECD-Fc fusion molecules in a mouse model of ITP Immune thrombocytopenia (ITP) is an autoimmune disorder manifested by a decrease in the number of platelets in the blood due to autoantibodies against platelet membrane antigens, leading to splenic sequestration and phagocytosis by macrophages. ITP is associated with bleeding disorders ranging from mild to fatal. ITP is usually treated with corticosteroids, intravenous immunoglobulins, anti-D immunoglobulins, or immunosuppressants. The benefits of these treatments may be transient, and some patients may require platelet transfusions or splenectomy. Therefore, novel therapies, particularly those that can reduce phagocytic activity, are needed for ITP patients.

[0340] SMAGP ECD mouse Fc fusion molecules, including wild-type and mutant variants (including variants with mutations in the Fc domain), are evaluated in a field-recognized mouse model of ITP (see, e.g., Samuelsson et al. (2001) Science 291(5503):484-486 and Huang et al. (2010) Blood 116(23):5002-5009, each of which is incorporated herein by reference in its entirety). ITP is induced by intravenous administration of 0.1 mg / kg body weight of anti-platelet (PLT) monoclonal antibody (mAb, rat anti-mouse integrin αIIb / CD41Ig, clone MWReg30, BD Biosciences). To analyze PLT counts, whole blood samples (50–100 μL) from mice were collected from the retro-orbital venous plexus and mixed with anticoagulant ACD solution (38 mM citric acid, 75 mM sodium citrate, 100 mM dextrose) in Eppendorf tubes. PLT counts were then measured 0, 2, 4, and 24 h after anti-CD41 Ig MWReg30 treatment using a hematology analyzer (KX-21N; Sysmex). To investigate the effect of the SMAGP ECD-Fc fusion molecule on ITP, mice were treated intravenously or intraperitoneally with 1–50 mg / kg of SMAGP ECD-Fc fusion molecule or vehicle 10–120 min before ITP induction. IVIg (1 g / kg) was used as a positive control to reduce the deleterious effects of CD41 Ig on PLT counts.

[0341] Example 14 Evaluation of SMAGP ECD-Fc fusion molecules in a mouse model of HLH Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome resulting from overwhelming immune activation, most frequently affecting infants and young children. The cause of HLH can be primary or related to genetic mutations in specific genes, such as the perforin 1 (PRF1) gene. Macrophage activation syndrome (MAS) is a term used to describe a type of secondary HLH that occurs in people with autoimmune or autoinflammatory diseases. The diseases most commonly associated with MAS are juvenile systemic arthritis, adult-onset Still's disease, and systemic lupus erythematosus. HLH is characterized by overt macrophage activation, cytokine production, and phagocytosis, leading to excessive inflammation and tissue destruction. HLH is typically treated with steroids and chemotherapy. Patients with HLH require treatments that suppress macrophage activity and are more tolerable.

[0342] SMAGP ECD mouse Fc fusion molecules, including wild-type and mutant variants (including variants with mutations in the Fc domain), were evaluated in field-recognized mouse models of primary HLH (see, e.g., Jordan et al. (2004) Blood 104(3):735-743 and Huang et al. (2017) Haematologica 102(11):1956-1968, each of which is incorporated herein by reference in its entirety). Perforin 1 (PRF1)-deficient mice exhibit all hallmarks of HLH after infection with lymphocytic choriomeningitis virus (LCMV). After LCMV infection, perforin-deficient mice develop fever, splenomegaly, pancytopenia, hypertriglyceridemia, hypofibrinogenemia, and elevated levels of multiple serum cytokines, with hemophagocytosis evident in many tissues. C57BL / 6-Prf1tm1Sdz(pfp- / -) mice were infected with LCMV for 5e 1 ~10e 4Particle-forming units (PFU) of the SMAP-ECD-Fc fusion molecule are injected intravenously or intraperitoneally. Mice are sacrificed and analyzed 8–14 days after inoculation. Spleen weight, hemoglobin (Hb), platelets (PLT), and neutrophils in the blood, as well as CD80 and CD80 expression on splenic macrophages, are evaluated as HLH symptoms. Survival of mice from day 20 onward is also assessed. To investigate the effect of the SMAP ECD-Fc fusion molecule on HLH pathology, mice are treated intravenously or intraperitoneally with 1–50 mg / kg SMAP fusion protein or vehicle every 2–3 days before and after LCMV injection.

[0343] Repeated CpG treatment of mice is a model of secondary HLH (see, e.g., Behrens et al. (2011) J Clin Invest. 121(6):2264-2277, Das et al. (2016) Blood 127(13):1666-1675, and Huang et al. (2017) Haematologica 102(11):1956-1968, each of which is incorporated herein by reference in its entirety). C57BL / 6 mice are injected with 50-75 μg of CpG DNA on days 0, 2, 4, 6, and 8 as described. Mice are euthanized, and organs are harvested on day 9 for analysis of spleen size and cellularity, blood hemoglobin, and platelet count. The effect of the SMAGP ECD-Fc fusion molecule on secondary HLH pathology will be assessed by intravenous or intraperitoneal treatment with 1-50 mg / kg of the SMAGP ECD-Fc fusion molecule or vehicle every 2 days before and during CpG administration.

[0344] Example 15 SMAGP ECD-lipid nanoparticle fusions SMAGP ECD, e.g., glycosylated or aglycosylated SMAGP ECD, can bind to leukocytes, e.g., myeloid cells, macrophages (e.g., M0 macrophages, M1 macrophages, M2 macrophages, M2a macrophages, M2b macrophages, M2c macrophages, or M2d macrophages), neutrophils, Kupffer cells, histiocytes, microglia, osteoclasts, dendritic cells, mast cells, regulatory T cells, tumor-infiltrating regulatory T cells, and granulocytes. Conjugation of SMAGP ECD to nanoparticles encapsulating a payload (e.g., mRNA, circular RNA (cRNA), tRNA, miRNA, siRNA, sgRNA, antisense oligonucleotides, peptides, viruses, viral DNA genomes, viral RNA genomes, vectors, DNA constructs, plasmids, or drugs) can facilitate delivery of the payload to leukocytes, thereby modulating the function of such leukocytes.

[0345] Because the cell plasma membrane is not permeable to highly hydrophilic molecules, delivery of nucleic acids (e.g., RNA or DNA) is particularly challenging. Nanoparticles, including lipid nanoparticle (LNP) formulations, have been shown to have a very high capacity for delivering nucleic acids into cells. One such application is the delivery of siRNA (e.g., Onpattro® (patisiran), Alnylam; see, e.g., Akinc et al. (2019) Nat Nanotechnol. 14(12):1084-1087, incorporated herein by reference in its entirety) or mRNA (e.g., encoding the spike protein of SARS-CoV-2, BioNtech / Pfizer and Moderna). Lipid nanoparticles generally contain ionized lipids that enable endosomal escape, pegylated lipids to improve half-life in circulation (e.g., 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG(2000)), or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159)) (e.g., from Avanti Polar Lipids), helper lipids (e.g., 1,2-distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-dibenzocyclooctyl (DBCO-PE)) (e.g., from Avanti Polar Lipids). It consists of four components: lipids, and thiols (or their derivatives).These individual components can be chemically conjugated to the SMAGP ECD to selectively deliver payloads to leukocytes, including macrophages and dendritic cells, to enhance antigen presentation of vaccines against infectious diseases or cancer (e.g., SARS-CoV-2 vaccines or cancer vaccines) (see, e.g., Sahin et al. (2020) Nature 585(7823):107-112), or to enhance the tumor-killing activity of macrophages against tumor cells (e.g., by expressing IRF5 mRNA) (see, e.g., Zhang et al. (2019) Nature Communications 10:3974, incorporated herein by reference in its entirety) or tolerogenic activity for the treatment of autoimmune diseases (see, e.g., Krienke et al. (2021) Science 371(6525):145-153, incorporated herein by reference in its entirety).

[0346] Conjugation of the SMAGP ECD to the helper lipids DSPE or PEG lipids can occur at the amino or carboxy terminus of the SMAGP protein using bioorthogonal / click chemistry (see, e.g., Simon et al. (2012) Bioconjugate Chem. 23(2):279-286, incorporated herein by reference in its entirety).

[0347] Click chemistry can be performed in either direction, as both the unnatural Met analogs azidohomoalanine (Aha) and homopropargylglycine (Hpg) can be efficiently introduced simply by changing the growth medium immediately prior to induction and by using a methionine-auxotrophic E. coli strain. The resulting "clickable" SMAGP ECD conjugates can be expressed in large quantities and biorthogonally coupled to "clickable" substrates containing the corresponding azides or alkynes for Cu(I)-dependent or Cu(I)-independent click chemistry, respectively, for substituted cyclooctynes.

[0348] For N-terminal coupling, e.g., to the N-terminal azidohomoalanine of SMAGP ECD, the clickable DARPin Aha-Ec1 is reacted with a 2-fold molar excess of DBCO-PE at 4° C. Aliquots are taken over time, snap frozen, and stored at −20° C. The SMAGP ECD conjugated to the DBCO helper lipid can then be used for nanoprecipitation of the payload LNP components.

[0349] Because human SMAGP ECD lacks any cysteines, a single C-terminal cysteine ​​can be introduced for maleimide coupling from Avanti Polar lipid to maleimide 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG(2000)) or its PEG5000 analog to generate PEG-conjugated SMAGP ECD. SMAGP engineered with a C-terminal cysteine ​​is mixed with a 2-fold molar excess of DSPE-PEG(2000) or (5000) from a 5 mM stock in PBS and vortexed. The click reaction is carried out at 4°C for 72 hours in PBS, pH 7.2, without stirring, and the PEGylated SMAGP is separated from free DSPE-PEG and unconjugated SMAGP using anion exchange chromatography. Conjugation can occur on the DSPE-PEG prior to nanoprecipitation of the payload and LNP components or after the LNP payload has been formulated. ***

[0350] The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to be within the scope of the appended claims.

[0351] All references (e.g., publications or patents or patent applications) cited in this specification are incorporated by reference herein in their entirety for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0352] Other embodiments are within the scope of the following claims.

Claims

1. A fusion molecule comprising the SMAGP extracellular domain (ECD) and a half-life extending moiety.

2. The fusion molecule of claim 1 , wherein the SMAGP ECD is a wild-type human, mouse, or cynomolgus monkey SMAGP ECD.

3. 3. The fusion molecule of claim 1, wherein the SMAGP ECD comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3.

4. 3. The fusion molecule of claim 1, wherein the SMAGP ECD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3, or a variant thereof comprising 1 to 5 amino acid changes.

5. The fusion molecule of any one of claims 1 to 4, wherein the SMAGP ECD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3.

6. The fusion molecule of any one of claims 1 to 5, wherein the half-life extending moiety is an Fc region.

7. The Fc region is human IgG １ , IgG ２ , IgG ３ , IgG ４ , IgA １ , or IgA ２ The fusion molecule of claim 6, wherein the Fc region is

8. The Fc region is human IgG １ The fusion molecule of claim 6, wherein the Fc region is

9. the Fc region comprising the D265A and N297A mutations numbered according to the EU numbering system. １ The fusion molecule of claim 6, wherein the Fc region is

10. a human IgG Fc region comprising L234A, L235A, and P329G mutations numbered according to the EU numbering system; １ The fusion molecule of claim 6, wherein the Fc region is

11. the Fc region comprising the S239D and I332E mutations numbered according to the EU numbering system. １ The fusion molecule of claim 6, wherein the Fc region is

12. The Fc region is human IgG ４ The fusion molecule of claim 6, wherein the Fc region is

13. a human IgG Fc region comprising the S228P mutation numbered according to the EU numbering system; ４ The fusion molecule of claim 6, wherein the Fc region is

14. The Fc region is mouse IgG ２ａ The fusion molecule of claim 6, wherein the Fc region is

15. a murine IgG Fc region comprising L234A, L235A, and P329G mutations numbered according to the EU numbering system; ２ａ The fusion molecule of claim 6, wherein the Fc region is

16. The fusion molecule of claim 6, wherein the Fc region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-18 and 50-57.

17. The fusion molecule of any one of claims 1 to 5, wherein the half-life extending moiety is selected from the group consisting of human serum albumin, a serum albumin binding moiety, and polyethylene glycol (PEG).

18. 18. The fusion molecule of claim 17, wherein the serum albumin binding moiety is an antibody or an antigen-binding portion thereof.

19. The fusion molecule of claim 17, wherein the serum albumin binding portion is a VH or a VHH.

20. 20. The fusion molecule of any one of claims 1 to 19, wherein the SMAGP ECD is covalently attached to the half-life extending moiety via a linker.

21. The fusion molecule of claim 20 , wherein the linker is a peptide linker.

22. 22. The fusion molecule of claim 21, wherein the peptide linker is an Fc hinge region or a portion thereof.

23. 23. The fusion molecule of claim 22, wherein the Fc hinge region is a human Fc hinge region or a portion thereof.

24. The human Fc hinge region is selected from the group consisting of human IgG １ , IgG ２ , IgG ３ , or IgG ４ 24. The fusion molecule of claim 23, wherein the hinge region of

25. 25. The fusion molecule of any one of claims 21 to 24, wherein the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 35-49, 58-61, or a variant thereof comprising 1 to 5 amino acid changes.

26. 26. The fusion molecule of any one of claims 1 to 25, wherein the half-life extending moiety is linked to the C-terminus of the SMAGP ECD.

27. 26. The fusion molecule of any one of claims 1 to 25, wherein the half-life extending moiety is linked to the N-terminus of the SMAGP ECD.

28. The fusion molecule of any one of claims 1 to 26, comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 19 to 34.

29. The fusion molecule of any one of claims 1 to 26, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19 to 34.

30. 2. The fusion molecule of claim 1, comprising a dimer of two polypeptides, each polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-34.

31. The fusion molecule of any one of claims 1 to 30, further comprising a targeting moiety that binds to a target molecule on a cell.

32. 32. The fusion molecule of claim 31 , wherein the cell is selected from the group consisting of a diseased cell, a senescent cell, a cancer cell, a B cell, a T cell, and a dendritic cell.

33. The fusion molecule of claim 31, wherein the target molecule is selected from the group consisting of DEP1, NTAL, EBP50, STX4, VAMP3, ARMCX3, B2MG, LANCL1, PLD3, VPS26A, DPP4, SCAMP4, MICA / B, TNFRSF10D / CD264, NOTCH1, NOTCH3, CD36, oxidized vimentin, ICAM-1, uPAR, DEP1 / PTPRJ / CD148, CD264, TNFRSF10D, TRAILR4, and CD26.

34. The fusion molecule of claim 33, wherein the cell is selected from a diseased cell and a senescent cell.

35. The target molecule is ADAM9, B7-H3 / CD276, BCMA, CA6, CA9, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD70, CD79b, CD123, CD138, CD157 / B ST1, P-cadherin CDH3, CEACAM5, CEACAM6, CLDN6, CLDN18.2, DLL3, EGFR, EGFRvIII, ENPP3, ENTPD2, EpCAM, FGR3, FLT3, FOLR1, GPA3 32. The fusion molecule of claim 31, wherein the fusion molecule is selected from the group consisting of: 3, GPC3, GPNMB, GPRC5D, GUCY2C, Her2, HHLA2, LAMP1, SLC39A6 / Liv-1, mesothelin, MUC16 / CA125, MUC17, SLC34A1 / NaPi2a, Nectin-4, CD274 / PD-L1, PSCA, PSMA / FOLH1, PVR, PVRIg, ROR1, SLITRK6, SSTR2, STEAP1, TROP2, and TMEM97.

36. The fusion molecule of claim 35, wherein the cell is a cancer cell.

37. 32. The fusion molecule of claim 31 , wherein the target molecule is a B cell surface antigen.

38. The fusion molecule of any one of claims 35 to 37, wherein the target molecule is CD20.

39. The fusion molecule of any one of claims 31 to 38, wherein the targeting moiety is an antibody or an antigen-binding fragment thereof.

40. 40. The fusion molecule of any one of claims 35 to 39, wherein the targeting moiety is rituximab or an antigen-binding fragment thereof.

41. The fusion molecule of any one of claims 1 to 40, further comprising a payload molecule.

42. 42. The fusion molecule of claim 41, wherein the payload molecule is selected from the group consisting of mRNA, miRNA, circular RNA (cRNA), tRNA, siRNA, sgRNA, antisense oligonucleotide, peptide, virus, viral RNA genome, viral DNA genome, vector, plasmid, DNA, radionuclide, and drug.

43. The fusion molecule of claim 42, wherein the payload molecule is encapsulated or attached to a nanoparticle.

44. The fusion molecule of claim 42, wherein the virus is an adeno-associated virus or a lentivirus.

45. The fusion molecule of claim 42, wherein the vector is a DNA vector.

46. A fusion molecule comprising an SMAGP extracellular domain (ECD) and a payload molecule.

47. 47. The fusion molecule of claim 46, wherein the SMAGP ECD is a wild-type human, mouse, or cynomolgus monkey SMAGP ECD.

48. 48. The fusion molecule of claim 46 or 47, wherein the SMAGP ECD comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3.

49. 48. The fusion molecule of claim 46 or 47, wherein the SMAGP ECD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3, or a variant thereof comprising 1 to 5 amino acid changes.

50. 50. The fusion molecule of any one of claims 46-49, wherein the SMAGP ECD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3.

51. The fusion molecule of any one of claims 46 to 50, wherein the payload molecule is selected from the group consisting of mRNA, miRNA, cRNA, tRNA, siRNA, sgRNA, antisense oligonucleotide, peptide, virus, viral RNA genome, viral DNA genome, vector, plasmid, DNA, radionuclide, and drug.

52. The fusion molecule of any one of claims 46 to 51, wherein the payload molecule modulates the activity of a cell expressing CLEC10A or CD177.

53. The fusion molecule of any one of claims 46 to 51, wherein the payload molecule is designed or selected to modulate the activity of neutrophils.

54. The fusion molecule of any one of claims 46 to 52, wherein the payload molecule is selected from the group consisting of small molecule drugs that inhibit the NADPH oxidase complex involved in the production of ROS, degranulation inhibitors, and promoters of neutrophil apoptosis.

55. 55. The fusion molecule of claim 54, wherein the small molecule drug that inhibits the NADPH oxidase complex involved in the production of ROS is selected from the group consisting of diphenyleneiodonium and stelazine.

56. The fusion molecule of claim 54, wherein the degranulation inhibitor is nexin hib-20.

57. 55. The fusion molecule of claim 54, wherein the promoter of neutrophil apoptosis is roscovitine.

58. The fusion molecule of any one of claims 46 to 53, wherein the payload molecule is a nucleic acid molecule encoding a chimeric antigen receptor for neutrophils.

59. The fusion molecule of any one of claims 51 to 58, wherein the payload molecule is encapsulated or attached to a nanoparticle.

60. The fusion molecule of claim 59, wherein the nanoparticle is a lipid nanoparticle.

61. 52. The fusion molecule of claim 51, wherein the virus is an adeno-associated virus or a lentivirus.

62. The fusion molecule of claim 51 , wherein the vector is a DNA vector.

63. The fusion molecule of any one of claims 46 to 62, wherein the SMAGP ECD is covalently attached to the payload molecule via a linker.

64. The fusion molecule of any one of claims 46 to 63, wherein the SMAGP ECD is covalently attached to a nanoparticle via a linker, and the payload molecule is encapsulated or attached to the nanoparticle.

65. The fusion molecule of claim 64, wherein the nanoparticle is a lipid nanoparticle and the linker is linked to a lipid component of the lipid nanoparticle.

66. 66. The fusion molecule of claim 65, wherein the lipid component of the lipid nanoparticle to which the linker is attached is selected from the group consisting of an ionizable lipid, a pegylated lipid, a helper lipid, and cholesterol.

67. 67. The fusion molecule of claim 66, wherein the helper lipid is selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-dibenzocyclooctyl (DBCO PE).

68. 68. The fusion molecule of claim 67, wherein the helper lipid is DBCO PE, the SMAGP ECD comprises an N-terminal azidohomoalanine, and the linker comprises a clickable DARPin that binds to the DBCO PE and the N-terminal azidohomoalanine.

69. 67. The fusion molecule of claim 66, wherein the pegylated lipid is selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG(2000)maleimide), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-5000] (DSPE-PEG(5000)maleimide), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG(2000)), and 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

70. 70. The fusion molecule of claim 69, wherein the pegylated lipid is DSPE-PEG(2000)maleimide or DSPE-PEG(5000)maleimide, and the linker comprises a cysteine ​​fused to the C-terminus of the SMAGP ECD that binds the pegylated lipid.

71. 64. The fusion molecule of claim 63, wherein the linker is a peptide linker.

72. 72. The fusion molecule of claim 71, wherein the peptide linker is an Fc hinge region or a portion thereof.

73. 73. The fusion molecule of claim 72, wherein the Fc hinge region is a human Fc hinge region or a portion thereof.

74. The human Fc hinge region is selected from the group consisting of human IgG １ , IgG ２ , IgG ３ , or IgG ４ 74. The fusion molecule of claim 73, wherein the hinge region of

75. 75. The fusion molecule of any one of claims 71 to 74, wherein the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 35-49, 58-61, or a variant thereof comprising 1 to 5 amino acid changes.

76. The fusion molecule of any one of claims 46 to 75, wherein the payload molecule is linked to the C-terminus of the SMAGP ECD.

77. The fusion molecule of any one of claims 46 to 75, wherein the payload molecule is linked to the N-terminus of the SMAGP ECD.

78. A fusion molecule comprising the SMAGP extracellular domain (ECD) and a targeting moiety that binds to a target molecule on a cell.

79. 79. The fusion molecule of claim 78, wherein the SMAGP ECD is a wild-type human, mouse, or cynomolgus monkey SMAGP ECD.

80. 80. The fusion molecule of claim 78 or 79, wherein the SMAGP ECD comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3.

81. 80. The fusion molecule of claim 78 or 79, wherein the SMAGP ECD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3, or a variant thereof comprising 1 to 5 amino acid changes.

82. 82. The fusion molecule of any one of claims 78-81, wherein the SMAGP ECD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3.

83. The fusion molecule of any one of claims 78 to 82, wherein the cell is selected from the group consisting of a diseased cell, a senescent cell, a cancer cell, a B cell, a T cell, and a dendritic cell.

84. The fusion molecule of any one of claims 78 to 82, wherein the target molecule is selected from the group consisting of DEP1, NTAL, EBP50, STX4, VAMP3, ARMCX3, B2MG, LANCL1, PLD3, VPS26A, DPP4, SCAMP4, MICA / B, TNFRSF10D / CD264, NOTCH1, NOTCH3, CD36, oxidized vimentin, ICAM-1, uPAR, DEP1 / PTPRJ / CD148, CD264, TNFRSF10D, TRAILR4, and CD26.

85. 85. The fusion molecule of claim 84, wherein the cell is a diseased cell or a senescent cell.

86. The target molecule is ADAM9, B7-H3 / CD276, BCMA, CA6, CA9, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD70, CD79b, CD123, CD138, CD157 / BST1 , P-cadherin CDH3, CEACAM5, CEACAM6, CLDN6, CLDN18.2, DLL3, EGFR, EGFRvIII, ENPP3, ENTPD2, EpCAM, FGR3, FLT3, FOLR1, GPA33, GPC 3, GPNMB, GPRC5D, GUCY2C, Her2, HHLA2, LAMP1, SLC39A6 / Liv-1, mesothelin, MUC16 / CA125, MUC17, SLC34A1 / NaPi2a, Nectin-4, CD274 / PD-L1, PSCA, PSMA / FOLH1, PVR, PVRIg, ROR1, SLITRK6, SSTR2, STEAP1, TROP2, and TMEM97.

87. The fusion molecule of claim 86, wherein the cell is a cancer cell.

88. The fusion molecule of any one of claims 78 to 82, wherein the target molecule is a B cell surface antigen.

89. The fusion molecule of any one of claims 86 to 88, wherein the target molecule is CD20.

90. 90. The fusion molecule of any one of claims 78 to 89, wherein the targeting moiety is an antibody or an antigen-binding fragment thereof.

91. 91. The fusion molecule of any one of claims 86 to 90, wherein the targeting moiety is rituximab or an antigen-binding fragment thereof.

92. 92. The fusion molecule of any one of claims 78-91, wherein the targeting moiety is linked to the C-terminus of the SMAGP ECD.

93. 92. The fusion molecule of any one of claims 78-91, wherein the targeting moiety is linked to the N-terminus of the SMAGP ECD.

94. 94. The fusion molecule of any one of claims 1 to 93, wherein the fusion molecule binds to a SMAGP ECD receptor on a cell.

95. The fusion molecule of any one of claims 1 to 94, wherein the SMAGP ECD is glycosylated.

96. The fusion molecule of any one of claims 1 to 94, wherein the SMAGP ECD is aglycosylated.

97. 97. The fusion molecule of any one of claims 1 to 96, further comprising a detectable label, optionally wherein the detectable label is selected from the group consisting of an enzyme, a fluorescent label, and a radioisotope.

98. A fusion molecule comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-34.

99. 99. The fusion molecule of claim 98, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-34.

100. 100. The fusion molecule of claim 98 or 99, comprising a dimer of two polypeptides, each polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-34.

101. A polynucleotide comprising a nucleotide sequence encoding the fusion molecule of any one of claims 1 to 100.

102. An expression vector comprising the polynucleotide of claim 101.

103. 103. A recombinant host cell comprising the polynucleotide of claim 101 or the expression vector of claim 102.

104. A nanoparticle comprising a fusion molecule according to any one of claims 1 to 100, a polynucleotide according to claim 101, or an expression vector according to claim 102.

105. The nanoparticle of claim 104, wherein the nanoparticle is a lipid nanoparticle and the polynucleotide of claim 101 is mRNA.

106. 102. A method for producing a fusion molecule according to any one of claims 1 to 100, comprising culturing a recombinant host cell according to claim 103 under conditions such that said fusion molecule is produced.

107. A composition comprising a fusion molecule according to any one of claims 1 to 100, a polynucleotide according to claim 101, an expression vector according to claim 102, or a nanoparticle according to claim 104 or 105, and a pharmaceutically acceptable carrier or excipient.

108. 101. A method of modifying the activity of leukocytes, comprising contacting said leukocytes with a fusion molecule according to any one of claims 1 to 100.

109. 109. The method of claim 108, wherein the leukocytes are selected from the group consisting of myeloid cells, macrophages, Kupffer cells, histiocytes, microglia, osteoclasts, dendritic cells, mast cells, neutrophils, regulatory T cells, tumor-infiltrating regulatory T cells, and granulocytes.

110. 110. The method of claim 109, wherein the leukocyte is a macrophage.

111. 111. The method of claim 110, wherein the macrophage is selected from the group consisting of M0 macrophage, M1 macrophage, M2 macrophage, M2a macrophage, M2b macrophage, M2c macrophage, and M2d macrophage.

112. 112. The method of any one of claims 108-111, wherein the activity comprises one or more of phagocytosis, cytokine production, chemokine production, antigen presentation, growth factor production, and protease production.

113. 113. The method of any one of claims 108 to 112, wherein the activity comprises phagocytosis of a population of cells or cell-like structures.

114. 114. The method of claim 113, wherein the population of cells or cell-like structures is selected from the group consisting of cancer cells, immune cells, neurons, red blood cells, and platelets.

115. 115. The method of any one of claims 108 to 114, wherein the activity of the leukocytes is increased.

116. 115. The method of any one of claims 108 to 114, wherein the activity of the leukocytes is reduced.

117. 107. A method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the fusion molecule of any one of claims 1 to 100, the polynucleotide of claim 101, the expression vector of claim 102, the nanoparticle of claim 104 or 105, or the composition of claim 107.

118. 118. The method of claim 117, wherein the disease or disorder is selected from the group consisting of an autoimmune disorder, an autoimmune disorder characterized by excessive phagocytic activity, atherosclerosis, cancer, an inflammatory disease or disorder, a lymphoproliferative disorder, an infectious disease, macrophage activation syndrome (MAS), a cytokine-associated disorder, a central nervous system (CNS) disease, a disease of overactivated microglia, a disease of overactivated osteoclasts, osteoporosis, cancer-associated bone degradation and / or metastasis, multiple myeloma, a bone disease, systemic juvenile idiopathic arthritis, allergies, cancers with low PD-L1 expression, cancers resistant to PD-1 and / or PD-L1 inhibitors, a disease or disorder characterized by multinucleated giant cells, and an atopic disease.

119. 119. The method of claim 118, wherein the cancer-associated bone degradation and / or metastasis is caused by a disease or disorder selected from multiple myeloma, breast cancer, and prostate cancer.

120. 119. The method of claim 118, wherein the autoimmune disorder is lupus, rheumatoid arthritis, multiple sclerosis, hemophagocytic lymphohistiocytosis (HLH), or immune thrombocytopenia (ITP).

121. 119. The method of claim 118, wherein the CNS disease is Alzheimer's disease, schizophrenia, or Huntington's disease.

122. 119. The method of claim 118, wherein the disease or disorder is a lymphoproliferative disorder and the fusion molecule comprises a targeting moiety comprising rituximab or an antigen-binding fragment thereof.

123. 118. The method of claim 117, wherein the disease or disorder is selected from the group consisting of atherosclerosis, cancer, an inflammatory disease or disorder, and an infectious disease.

124. 118. The method of claim 117, wherein the disease or disorder is selected from the group consisting of H. pylori infection, COVID, severe COVID, IgA nephropathy, and ovarian cancer.

125. 119. The method of claim 118, wherein the disease or disorder characterized by multinucleated giant cells is selected from the group consisting of Langerhans cell histiocytosis and granuloma.

126. The method of claim 117, wherein the disease or disorder is selected from the group consisting of inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, acute exacerbation of chronic liver failure (ACLF), COVID, severe COVID, antineutrophil cytoplasmic antibody-associated vasculitis (AAV), autoimmune vasculitis, asthma, acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), Kawasaki disease, septic shock, diseases in which regulatory T cells express CD177, renal cell carcinoma, hepatocellular carcinoma, breast cancer, lung cancer, and colorectal cancer.

127. 127. The method of any one of claims 117 to 126, further comprising administering to the subject a therapeutically effective amount of a chemotherapeutic agent.

128. 128. The method of claim 127, wherein the chemotherapeutic agent is paclitaxel.

129. 127. The method of any one of claims 117-126, further comprising administering to said subject a therapeutically effective amount of an immune checkpoint modulator.

130. 130. The method of claim 129, wherein the immune checkpoint modulator is an anti-PD-1 agent or an anti-PD-L1 agent.

131. 127. The method of any one of claims 117-126, further comprising administering to the subject therapeutically effective amounts of a chemotherapeutic agent and an immune checkpoint modulator.

132. 132. The method of claim 131, wherein the chemotherapeutic agent is paclitaxel and the immune checkpoint modulator is an anti-PD-1 agent or an anti-PD-L1 agent.

133. 133. The method of any one of claims 117 to 132, wherein the disease or disorder is ovarian cancer.

134. A method for identifying a modulator of the interaction between SMAGP and CLEC10A, the method comprising measuring the binding of SMAGP to CLEC10A in the presence and absence of a test compound, wherein the test compound is identified as a modulator of the interaction between SMAGP and CLEC10A by an increase or decrease in the amount of binding of SMAGP to CLEC10A in the presence of the test compound relative to the amount of binding of SMAGP to CLEC10A in the absence of the test compound.

135. A method for identifying an agent that promotes the interaction between SMAGP and CLEC10A, the method comprising measuring the binding of SMAGP to CLEC10A in the presence and absence of a test compound, wherein the test compound is identified as an agent that promotes the interaction between SMAGP and CLEC10A based on an increase in the amount of binding of SMAGP to CLEC10A in the presence of the test compound relative to the amount of binding of SMAGP to CLEC10A in the absence of the test compound.

136. A method for identifying an inhibitor of the interaction between SMAGP and CLEC10A, the method comprising measuring the binding of SMAGP to CLEC10A in the presence and absence of a test compound, wherein the test compound is identified as an inhibitor of the interaction between SMAGP and CLEC10A by a decrease in the amount of binding of SMAGP to CLEC10A in the presence of the test compound relative to the amount of binding of SMAGP to CLEC10A in the absence of the test compound.

137. 137. The method of any one of claims 134 to 136, wherein the SMAGP and / or CLEC10A is expressed on the surface of a cell.

138. 137. The method of any one of claims 134 to 136, wherein the SMAGP and / or CLEC10A are recombinant proteins.

139. 139. The method of claim 138, wherein the recombinant protein is a fusion molecule comprising an Fc region.

140. A method for identifying a modulator of the interaction between SMAGP and CD177, the method comprising measuring the binding of SMAGP to CD177 in the presence and absence of a test compound, wherein an increase or decrease in the amount of binding of SMAGP to CD177 in the presence of the test compound relative to the amount of binding of SMAGP to CD177 in the absence of the test compound identifies the test compound as a modulator of the interaction between SMAGP and CD177.

141. A method for identifying an agent that promotes the interaction between SMAGP and CD177, the method comprising measuring the binding of SMAGP to CD177 in the presence and absence of a test compound, wherein the test compound is identified as a promoter of the interaction between SMAGP and CD177 based on an increase in the amount of SMAGP binding to CD177 in the presence of the test compound relative to the amount of SMAGP binding to CD177 in the absence of the test compound.

142. A method for identifying an inhibitor of the interaction between SMAGP and CD177, the method comprising measuring the binding of SMAGP to CD177 in the presence and absence of a test compound, wherein the test compound is identified as an inhibitor of the interaction between SMAGP and CD177 by a decrease in the amount of binding of SMAGP to CD177 in the presence of the test compound relative to the amount of binding of SMAGP to CD177 in the absence of the test compound.

143. A method according to any one of claims 140 to 142, wherein the SMAGP and / or CD177 are expressed on the surface of a cell.

144. 144. The method of any one of claims 140 to 143, wherein the SMAGP and / or CD177 are recombinant proteins.

145. The method of claim 144, wherein the recombinant protein is a fusion molecule comprising an Fc region.

146. 146. The method of any one of claims 134 to 145, wherein the test compound is selected from the group consisting of a small molecule, a polypeptide, or a nucleic acid.

147. 147. The method of claim 146, wherein the polypeptide is an antibody.

148. 147. The method of claim 146, wherein the nucleic acid is a DNA or RNA aptamer.

149. 149. The method of any one of claims 134 to 148, wherein the amount of binding is measured using enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), or flow cytometry.

150. A method for treating a subject having a tumor with a high expression level of CLEC10A compared to a reference level of CLEC10A, the method comprising administering to the subject a therapeutically effective amount of a compound that antagonizes the binding of CLEC10A to SMAGP.

151. The method of claim 150, wherein the compound is a fusion molecule according to any one of claims 1 to 100, a polynucleotide according to claim 101, an expression vector according to claim 102, a nanoparticle according to claim 104 or 105, or a composition according to claim 107.

152. A method for identifying a subject who would benefit from treatment with a fusion molecule that antagonizes the binding of CLEC10A to SMAGP, the method comprising determining the expression level of CLEC10A in a biological sample derived from the subject relative to a reference level of CLEC10A, wherein a higher expression level of CLEC10A in the biological sample compared to the reference level indicates that the subject would benefit from the treatment.

153. 153. The method of claim 152, wherein the biological sample is a tumor sample.

154. The method of claim 152 or 153, wherein the expression level of CLEC10A in the biological sample is determined by immunohistochemistry, fluorescent in situ hybridization (FISH), or chromogenic in situ hybridization (CISH).

155. The method of any one of claims 152 to 154, wherein the CLEC10A is expressed on the surface of a myeloid cell and the SMAGP is expressed on the surface of a tumor cell.

156. A method for identifying a subject who would benefit from treatment with a fusion molecule comprising the SMAGP extracellular domain (ECD), comprising measuring the expression level of CD177 in a biological sample from the subject and comparing it with a reference level of CD177, wherein a higher expression level of CD177 compared to the reference level indicates that the subject would benefit from the treatment.

157. 157. The method of claim 156, wherein the biological sample is a tumor sample.

158. The method of claim 156 or 157, wherein the expression level of CD177 in the biological sample is determined by immunohistochemistry, fluorescent in situ hybridization (FISH), or chromogenic in situ hybridization (CISH).

159. The method of any one of claims 156 to 158, wherein the CD177 is expressed on the surface of a neutrophil or a regulatory T cell, and the SMAGP is expressed on the surface of a tumor cell.

160. 160. The method of any one of claims 152 to 159, further comprising administering to the subject a therapeutically effective amount of a fusion molecule of any one of claims 1 to 97, a polynucleotide of claim 98, an expression vector of claim 99, a nanoparticle of claim 101 or 102, or a composition of claim 104.

161. A fusion molecule according to any one of claims 1 to 100, a polynucleotide according to claim 101, an expression vector according to claim 102, a nanoparticle according to claim 104 or 105, or a composition according to claim 107 for use in modifying the activity of leukocytes.

162. A fusion molecule according to any one of claims 1 to 100, a polynucleotide according to claim 101, an expression vector according to claim 102, a nanoparticle according to claim 104 or 105, or a composition according to claim 107 for use in medicine.

163. 107. A fusion molecule according to any one of claims 1 to 100, a polynucleotide according to claim 101, an expression vector according to claim 102, a nanoparticle according to claim 104 or 105, or a composition according to claim 107 for use in treating a disease or disorder in a subject in need thereof.

164. 107. Use of a fusion molecule according to any one of claims 1 to 100, a polynucleotide according to claim 101, an expression vector according to claim 102, a nanoparticle according to claim 104 or 105, or a composition according to claim 107 in the manufacture of a medicament for the treatment of a disease or disorder in a subject in need thereof.

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