Recombinant sialidase and its method of use

Abstract

The present invention relates generally to recombinant sialidases, methods and compositions for extending the serum half-life of recombinant sialidases, and their use in the treatment of sialic acid-related disorders.

Description

[Technical Field]

[0001] Other references regarding related applications This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 870,336, filed on 3 July 2019, and U.S. Provisional Patent Application No. 62 / 957,027, filed on 3 January 2020, the entire disclosures of said applications, which are incorporated herein by reference in their entirety.

[0002] Field of Invention The present invention generally relates to recombinant sialidase, methods and compositions for extending the serum half-life of recombinant sialidase, and their use in the treatment of sialic acid-related disorders. [Background technology]

[0003] background A growing body of evidence supports the role of glycans, and especially siaroglycans, in various pathophysiological stages of tumor progression. Glycans regulate tumor growth, invasion, hematogenous metastasis, and neovascularization (Fuster et al. (2005) NAT. REV. CANCER 5(7): 526-42). Sialylation of cell surface sugar conjugates is frequently altered in cancer, leading to the expression of sialylated tumor-associated carbohydrate antigens. Expression of sialylated glycans by tumor cells is often associated with increased tumor aggressiveness and metastatic capacity.

[0004] Recently, it has been revealed that Siglec (sialic acid-binding immunoglobulin-like lectin), a family of sialic acid-binding lectins, plays a role in cancer immunosuppression by binding to hypersialylated cancer cells and mediating the suppression of NK cell receptor activation-derived signals, thereby inhibiting NK cell-mediated tumor cell killing (Jandus et al. (2014) J. CLIN. INVEST. 124: 1810-1820; Laeubli et al. (2014) PROC. NATL. ACAD. SCI. USA 111: 14211-14216; Hudak et al. (2014) NAT. CHEM. BIOL. 10: 69-75). Similarly, enzymatic removal of sialic acid by sialidase treatment may enhance NK cell-mediated tumor cell killing (Jandus, see above; Hudak, see above; Xiao et al. (2016) PROC. NATL. ACAD. SCI. USA 113(37): 10304-9).

[0005] Cancer immunotherapy using immune checkpoint inhibitors containing antibodies that block the PD-1 / PD-L1 pathway has improved outcomes for many cancer patients. However, despite the progress made to date, many patients do not respond to currently available immune checkpoint inhibitors. Therefore, there remains a need for effective interventions to overcome the immunosuppressive tumor microenvironment and for treating cancers associated with hypersialylated cancer cells. [Overview of the project]

[0006] Summary of the Invention This invention is partly based on the discovery that sialic acid-mediated disorders can be treated by administering sialidase enzymes or sialidase enzymes conjugated with serum half-life enhancers. Surprisingly, it has been discovered that sialidase enzymes lacking a targeting moiety (e.g., an antibody-binding domain directed to a tumor antigen) or sialidase enzymes conjugated with serum half-life enhancers can effectively treat sialic acid-mediated disorders (e.g., cancer, e.g., solid tumors) in vivo.

[0007] The present invention further relates to a recombinant form of a sialidase enzyme, a sialidase enzyme conjugated to a serum half-life enhancer, and a pharmaceutical composition thereof, having appropriate substrate specificity and activity useful for removing sialic acid and / or sialic acid-containing molecules from the surface of cancer cells, and / or from the tumor microenvironment, and / or reducing the concentration of sialic acid and / or sialic acid-containing molecules in the tumor microenvironment.

[0008] Therefore, in a certain respect, the present invention provides a pharmaceutical composition comprising or essentially comprising sialidase conjugated with a serum half-life enhancing factor that increases the serum half-life of sialidase when administered to a subject.

[0009] In another aspect, the present invention provides a method for treating a sialic acid-related disorder in a subject requiring treatment for the disorder. The method comprises administering to the subject an effective amount of a pharmaceutical composition comprising or essentially comprising sialidase and a serum half-life enhancer that increases the serum half-life of sialidase when administered to the subject, thereby treating the disorder.

[0010] In one embodiment, the sialidase is not conjugated to a cancer antigen targeting agent that binds to cancer antigens associated with cancer cells.

[0011] In one embodiment, the sialidase is a functional fragment or variant of the full-length sialidase exhibiting at least 50% of the activity of the full-length sialidase.

[0012] In one embodiment, sialidase and serum half-life enhancer are covalently bound together or chemically conjugated together in a fusion protein.

[0013] In one embodiment, the serum half-life enhancer is selected from the group consisting of Fc domains, transferrin, albumin, XTEN, homoamino acid polymers (HAP), proline-alanine-serine polymers (PAS), elastin-like peptides (ELP), albumin-binding domains, CTP fusions, GLK fusions, and polyethylene glycol.

[0014] In one embodiment, the serum half-life enhancer is the Fc domain.

[0015] In one embodiment, the serum half-life enhancer is not an Fc domain or polyethylene glycol.

[0016] In one embodiment, the sialidase contains one or more mutations compared to the wild-type template sialidase.

[0017] In one embodiment, sialidase includes a substitution or deletion of a methionine residue at the position corresponding to position 1 of wild-type human Neu2 (M1); a substitution of a valine residue at the position corresponding to position 6 of wild-type human Neu2 (V6); a substitution of an isoleucine residue at the position corresponding to position 187 of wild-type human Neu2 (I187); or a substitution of a cysteine ​​residue at the position corresponding to position 332 of wild-type human Neu2 (C332); or any combination of the aforementioned substitutions. In one embodiment, in sialidase, (a) the methionine residue at the position corresponding to position 1 of wild-type human Neu2 is deleted (ΔM1), replaced with alanine (M1A), or replaced with aspartic acid (M1D); (b) the valine residue at the position corresponding to position 6 of wild-type human Neu2 is replaced with tyrosine (V6Y); (c) the isoleucine residue at the position corresponding to position 187 of wild-type human Neu2 is replaced with lysine (I187K); (d) or the cysteine ​​residue at the position corresponding to position 332 of wild-type human Neu2 is replaced with alanine (C332A); or the sialidase contains any combination of the above substitutions.

[0018] In one embodiment, sialidase is a result of substitution or deletion of a methionine residue at the position corresponding to position 1 of wild-type human Neu2 (M1); substitution of a valine residue at the position corresponding to position 6 of wild-type human Neu2 (V6); substitution of a proline residue at the position corresponding to position 62 of wild-type human Neu2 (P62); substitution of an alanine residue at the position corresponding to position 93 of wild-type human Neu2 (A93); substitution of an isoleucine residue at the position corresponding to position 187 of wild-type human Neu2 (I187); and a glutathione residue at the position corresponding to position 126 of wild-type human Neu2. This includes substitutions of a mine residue (Q126); substitutions of an alanine residue at the position corresponding to position 242 of wild-type human Neu2 (A242); substitutions of a glutamine residue at the position corresponding to position 270 of wild-type human Neu2 (Q270); substitutions of a serine residue at the position corresponding to position 301 of wild-type human Neu2 (S301); substitutions of a tryptophan residue at the position corresponding to position 302 of wild-type human Neu2 (W302); substitutions of a cysteine ​​residue at the position corresponding to position 332 of wild-type human Neu2 (C332); or any combination of the above substitutions.

[0019] In one aspect, sialidase is: (a)M1D, V6Y, P62G, A93E, I187K, C332A; (b)M1D, V6Y, P62G, A93E, I187K, S301A, W302R, C332A; (c)M1D, V6Y, P62G, A93E, Q126Y, I187K, A242F, Q270T, C332A; (d) M1D, V6Y, P62G, A93E, Q126Y, I187K, C332A; and (e)A93E, Q126Y, I187K, A242F, Q270T, C332A This includes combinations of permutations selected from the group consisting of the following.

[0020] In one embodiment, the sialidase conjugated to a serum half-life enhancer contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 115, 152, 180, 184, and 188, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% of an amino acid sequence selected from the group consisting of SEQ ID NOs: 115, 152, 180, 184, and 188.

[0021] In one embodiment, sialidase is a substitution of a proline residue at the position corresponding to position 5 of wild-type human Neu2 (P5); a substitution of a lysine residue at the position corresponding to position 9 of wild-type human Neu2 (K9); a substitution of a lysine residue at the position corresponding to position 44 of wild-type human Neu2 (K44); a substitution of a lysine residue at the position corresponding to position 45 of wild-type human Neu2 (K45); a substitution of a leucine residue at the position corresponding to position 54 of wild-type human Neu2 (L54); a substitution of a proline residue at the position corresponding to position 62 of wild-type human Neu2 (P62); and a substitution at position 69 of wild-type human Neu2 Substitution of a glutamine residue at the position corresponding to (Q69); substitution of an arginine residue at the position corresponding to position 78 of wild-type human Neu2 (R78); substitution of an aspartic acid residue at the position corresponding to position 80 of wild-type human Neu2 (D80); substitution of an alanine residue at the position corresponding to position 93 of wild-type human Neu2 (A93); substitution of a glycine residue at the position corresponding to position 107 of wild-type human Neu2 (G107); substitution of a glutamine residue at the position corresponding to position 108 of wild-type human Neu2 (Q108); substitution of glutamine at the position corresponding to position 112 of wild-type human Neu2 Substitution of a methyl residue (Q112); substitution of a cysteine ​​residue at the position corresponding to position 125 of wild-type human Neu2 (C125); substitution of a glutamine residue at the position corresponding to position 126 of wild-type human Neu2 (Q126); substitution of an alanine residue at the position corresponding to position 150 of wild-type human Neu2 (A150); substitution of a cysteine ​​residue at the position corresponding to position 164 of wild-type human Neu2 (C164); substitution of an arginine residue at the position corresponding to position 170 of wild-type human Neu2 (R170); substitution of an alanine residue at the position corresponding to position 171 of wild-type human Neu2 (A171); substitution of a glutamine residue at position 188 of wild-type human Neu2 (Q188); substitution of an arginine residue at position 189 of wild-type human Neu2 (R189); substitution of an alanine residue at position 213 of wild-type human Neu2 (A213); substitution of a leucine residue at position 217 of wild-type human Neu2 (L217); substitution of a glutamate residue at position 225 of wild-type human Neu2 (E225); substitution of a histidine residue at position 239 of wild-type human Neu2 (H239);Substitution of a leucine residue at position 240 of wild-type human Neu2 (L240); substitution of an arginine residue at position 241 of wild-type human Neu2 (R241); substitution of an alanine residue at position 242 of wild-type human Neu2 (A242); substitution of a valine residue at position 244 of wild-type human Neu2 (V244); substitution of a threonine residue at position 249 of wild-type human Neu2 (T249); substitution of an aspartic acid residue at position 251 of wild-type human Neu2 (D251); substitution of a glutamic acid residue at position 257 of wild-type human Neu2 (E257); substitution of a serine residue at position 258 of wild-type human Neu2 (S258); substitution of a leucine residue at position 260 of wild-type human Neu2 (L260); substitution of a valine residue at the position corresponding to position 265 of wild-type human Neu2 (V265); substitution of a glutamine residue at the position corresponding to position 270 of wild-type human Neu2 (Q270); substitution of a tryptophan residue at the position corresponding to position 292 of wild-type human Neu2 (W292); substitution of a serine residue at the position corresponding to position 301 of wild-type human Neu2 (S301); substitution of a tryptophan residue at the position corresponding to position 302 of wild-type human Neu2 (W302); substitution of a cysteine ​​residue at the position corresponding to position 332 of wild-type human Neu2 (C332); substitution of a valine residue at the position corresponding to position 363 of wild-type human Neu2 (V363); or substitution of a leucine residue at the position corresponding to position 365 of wild-type human Neu2 (L365); or any combination of the above substitutions.

[0022] In one embodiment, the sialidase is selected from the group consisting of bacterial sialidase, viral sialidase, and mammalian sialidase. In one embodiment, the sialidase is human sialidase. In one embodiment, the human sialidase is selected from the group consisting of neu1, neu2, neu3, and neu4. In one embodiment, the human sialidase is neu2.

[0023] In one aspect, pharmaceutical compositionIt contains approximately 0.01 mg / kg to approximately 100 mg / kg of sialidase.

[0024] In one embodiment, the pharmaceutical composition comprises a second therapeutic agent. In one embodiment, the second therapeutic agent is selected from the group consisting of anti-inflammatory agents, anti-vasoconducting agents, anti-fibrotic agents, or antiproliferative compounds (e.g., cytotoxic agents or checkpoint inhibitors).

[0025] In one embodiment, the pharmaceutical composition further comprises a stabilizing amount of a sialidase stabilizer. In one embodiment, the sialidase stabilizer is a cation. In one embodiment, the cation is selected from the group consisting of calcium and magnesium.

[0026] In one embodiment, the pharmaceutical composition is placed in a sterile container (e.g., a bottle or vial). In one embodiment, the pharmaceutical composition is freeze-dried in the sterile container. In one embodiment, the pharmaceutical composition exists as a solution in the sterile container. In one embodiment, the sterile container is sealed by a partition. In one embodiment, the sterile container has a label placed on it that identifies the pharmaceutical composition contained in the container.

[0027] In another aspect, the present disclosure relates to a method for treating a sialic acid-related disorder in a subject requiring treatment for the disorder, the method comprising administering to the subject a pharmaceutical composition comprising a sialidase and a serum half-life enhancer that increases the serum half-life of the sialidase when administered to the subject, thereby treating the disorder.

[0028] In one embodiment, sialic acid-related disorders are cancer. In another embodiment, sialidase is not conjugated to cancer antigen targeting agents that bind to cancer antigens associated with cancerous cells.

[0029] In one embodiment, the sialidase is a functional fragment of full-length sialidase exhibiting at least 50% of the activity of full-length sialidase. In another embodiment, the sialidase is a variant exhibiting at least 50% of the activity of wild-type sialidase.

[0030] In one embodiment, sialidase and serum half-life enhancer are covalently bound together in a fusion protein. In another embodiment, sialidase and serum half-life enhancer are chemically conjugated together.

[0031] In one embodiment, the serum half-life enhancer is selected from the group consisting of Fc domains, transferrin, albumin, XTEN, homoamino acid polymers (HAP), proline-alanine-serine polymers (PAS), elastin-like peptides (ELP), and polyethylene glycol. In one embodiment, the serum half-life enhancer is an Fc domain. In another embodiment, the serum half-life enhancer is neither an Fc domain nor polyethylene glycol.

[0032] In one embodiment, the sialidase contains one or more mutations from the wild-type sialidase template. In one embodiment, the sialidase contains a substitution or deletion of a methionine residue at the position corresponding to position 1 of wild-type human Neu2 (M1); a substitution of a valine residue at the position corresponding to position 6 of wild-type human Neu2 (V6); a substitution of an isoleucine residue at the position corresponding to position 187 of wild-type human Neu2 (I187); or a substitution of a cysteine ​​residue at the position corresponding to position 332 of wild-type human Neu2 (C332); or any combination of the aforementioned substitutions.

[0033] In one embodiment, in sialidase, the methionine residue at the position corresponding to position 1 of wild-type human Neu2 is deleted (ΔM1), replaced with alanine (M1A), or replaced with aspartic acid (M1D); the valine residue at the position corresponding to position 6 of wild-type human Neu2 is replaced with tyrosine (V6Y); the isoleucine residue at the position corresponding to position 187 of wild-type human Neu2 is replaced with lysine (I187K); or the cysteine ​​residue at the position corresponding to position 332 of wild-type human Neu2 is replaced with alanine (C332A); or the sialidase contains any combination of the aforementioned substitutions.

[0034] In one embodiment, sialidase is a result of substitution or deletion of a methionine residue at the position corresponding to position 1 of wild-type human Neu2 (M1); substitution of a valine residue at the position corresponding to position 6 of wild-type human Neu2 (V6); substitution of a proline residue at the position corresponding to position 62 of wild-type human Neu2 (P62); substitution of an alanine residue at the position corresponding to position 93 of wild-type human Neu2 (A93); substitution of an isoleucine residue at the position corresponding to position 187 of wild-type human Neu2 (I187); and a glutathione residue at the position corresponding to position 126 of wild-type human Neu2. This includes substitutions of a mine residue (Q126); substitutions of an alanine residue at the position corresponding to position 242 of wild-type human Neu2 (A242); substitutions of a glutamine residue at the position corresponding to position 270 of wild-type human Neu2 (Q270); substitutions of a serine residue at the position corresponding to position 301 of wild-type human Neu2 (S301); substitutions of a tryptophan residue at the position corresponding to position 302 of wild-type human Neu2 (W302); substitutions of a cysteine ​​residue at the position corresponding to position 332 of wild-type human Neu2 (C332); or any combination of the above substitutions.

[0035] In one aspect, sialidase is: (a)M1D, V6Y, P62G, A93E, I187K, C332A; (b)M1D, V6Y, P62G, A93E, I187K, S301A, W302R, C332A; (c)M1D, V6Y, P62G, A93E, Q126Y, I187K, A242F, Q270T, C332A; (d) M1D, V6Y, P62G, A93E, Q126Y, I187K, C332A; and (e)A93E, Q126Y, I187K, A242F, Q270T, C332A This includes combinations of permutations selected from the group consisting of the following.

[0036] In one embodiment, the sialidase conjugated to a serum half-life enhancer contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 115, 152, 180, 184, and 188, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% of an amino acid sequence selected from the group consisting of SEQ ID NOs: 115, 152, 180, 184, and 188.

[0037] In one embodiment, sialidase is a substitution of a proline residue at the position corresponding to position 5 of wild-type human Neu2 (P5); a substitution of a lysine residue at the position corresponding to position 9 of wild-type human Neu2 (K9); a substitution of a lysine residue at the position corresponding to position 44 of wild-type human Neu2 (K44); a substitution of a lysine residue at the position corresponding to position 45 of wild-type human Neu2 (K45); a substitution of a leucine residue at the position corresponding to position 54 of wild-type human Neu2 (L54); a substitution of a proline residue at the position corresponding to position 62 of wild-type human Neu2 (P62); and a substitution at position 69 of wild-type human Neu2 Substitution of a glutamine residue at the position corresponding to (Q69); substitution of an arginine residue at the position corresponding to position 78 of wild-type human Neu2 (R78); substitution of an aspartic acid residue at the position corresponding to position 80 of wild-type human Neu2 (D80); substitution of an alanine residue at the position corresponding to position 93 of wild-type human Neu2 (A93); substitution of a glycine residue at the position corresponding to position 107 of wild-type human Neu2 (G107); substitution of a glutamine residue at the position corresponding to position 108 of wild-type human Neu2 (Q108); substitution of glutamine at the position corresponding to position 112 of wild-type human Neu2 Substitution of a methyl residue (Q112); substitution of a cysteine ​​residue at the position corresponding to position 125 of wild-type human Neu2 (C125); substitution of a glutamine residue at the position corresponding to position 126 of wild-type human Neu2 (Q126); substitution of an alanine residue at the position corresponding to position 150 of wild-type human Neu2 (A150); substitution of a cysteine ​​residue at the position corresponding to position 164 of wild-type human Neu2 (C164); substitution of an arginine residue at the position corresponding to position 170 of wild-type human Neu2 (R170); substitution of an alanine residue at the position corresponding to position 171 of wild-type human Neu2 (A171); substitution of a glutamine residue at position 188 of wild-type human Neu2 (Q188); substitution of an arginine residue at position 189 of wild-type human Neu2 (R189); substitution of an alanine residue at position 213 of wild-type human Neu2 (A213); substitution of a leucine residue at position 217 of wild-type human Neu2 (L217); substitution of a glutamate residue at position 225 of wild-type human Neu2 (E225); substitution of a histidine residue at position 239 of wild-type human Neu2 (H239);Substitution of a leucine residue at position 240 of wild-type human Neu2 (L240); substitution of an arginine residue at position 241 of wild-type human Neu2 (R241); substitution of an alanine residue at position 242 of wild-type human Neu2 (A242); substitution of a valine residue at position 244 of wild-type human Neu2 (V244); substitution of a threonine residue at position 249 of wild-type human Neu2 (T249); substitution of an aspartic acid residue at position 251 of wild-type human Neu2 (D251); substitution of a glutamic acid residue at position 257 of wild-type human Neu2 (E257); substitution of a serine residue at position 258 of wild-type human Neu2 (S258); substitution of a leucine residue at position 260 of wild-type human Neu2 (L260); substitution of a valine residue at the position corresponding to position 265 of wild-type human Neu2 (V265); substitution of a glutamine residue at the position corresponding to position 270 of wild-type human Neu2 (Q270); substitution of a tryptophan residue at the position corresponding to position 292 of wild-type human Neu2 (W292); substitution of a serine residue at the position corresponding to position 301 of wild-type human Neu2 (S301); substitution of a tryptophan residue at the position corresponding to position 302 of wild-type human Neu2 (W302); substitution of a cysteine ​​residue at the position corresponding to position 332 of wild-type human Neu2 (C332); substitution of a valine residue at the position corresponding to position 363 of wild-type human Neu2 (V363); or substitution of a leucine residue at the position corresponding to position 365 of wild-type human Neu2 (L365); or any combination of the above substitutions.

[0038] In one embodiment, sialidase is selected from the group consisting of bacterial sialidase, viral sialidase, and mammalian sialidase. In one embodiment, mammalian sialidase is human sialidase. In one embodiment, human sialidase is selected from the group consisting of neu1, neu2, neu3, and neu4. In one embodiment, human sialidase is neu2.

[0039] In one embodiment, approximately 0.01 mg / kg to approximately 100 mg / kg of sialidase is administered to the subject.

[0040] In some aspects, cancer is a solid tumor, a soft tissue tumor, a hematopoietic malignancy, or a metastatic lesion. In some aspects, a solid tumor is a sarcoma, adenocarcinoma, or carcinoma. In some aspects, a solid tumor is a tumor of the head and neck (e.g., pharynx), thyroid gland, lung (e.g., small cell or non-small cell lung cancer (NSCLC)), breast, lymphatic system, gastrointestinal tract (e.g., oral cavity, esophagus, stomach, liver, pancreas, small intestine, colon and rectum, anal canal), genital or urogenital tract (e.g., kidney, urothelium, bladder, ovary, uterus, cervix, endometrium, prostate, testis), CNS (e.g., nerve or glial cells, e.g., neuroblastoma or glioma) or skin (e.g., melanoma). In some aspects, cancer is breast cancer.

[0041] In some embodiments, hematopoietic malignancies include leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), B-cell, T-cell or FAB ALL, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), e.g., transformed CLL, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, pilocytic cell leukemia, myelodysplastic syndrome (MDS), lymphoma, Hodgkin's disease, malignant lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, multiple myeloma, or Richter syndrome (Richter transformation). In some embodiments, cancer is lymphoma.

[0042] In one embodiment, administration of the pharmaceutical composition increases the expression of granzyme B, IFNγ, IL-10, IL-6, or IL-17A in the subject.

[0043] In one embodiment, the pharmaceutical composition is administered to a subject in combination with another therapeutic agent. In one embodiment, the therapeutic agent is selected from the group consisting of anti-inflammatory agents, anti-vasoconducting agents, anti-fibrotic agents, or antiproliferative compounds (e.g., cytotoxic agents or checkpoint inhibitors).

[0044] In one embodiment, the pharmaceutical composition further comprises a stabilizing amount of a sialidase stabilizer. In one embodiment, the sialidase stabilizer is a cation. In one embodiment, the cation is selected from the group consisting of calcium and magnesium.

[0045] In one embodiment, the pharmaceutical composition is placed in a sterile container (e.g., a bottle or vial) before administration.

[0046] In one embodiment, the method includes the step of administering an effective amount of a pharmaceutical composition to a subject.

[0047] In one embodiment, the present disclosure relates to a method for removing sialic acid from cells in a subject, the method comprising administering an effective amount of a pharmaceutical composition to the subject, thereby removing sialic acid from the cells.

[0048] In one embodiment, the cells are tumor cells, dendritic cells (DCs), or monocytes. In another embodiment, the cells are monocytes, and the method results in increased expression of MHC-II molecules on the monocytes.

[0049] In one embodiment, the present disclosure relates to a method for increasing the phagocytic activity of tumor cells in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition effective in removing sialic acid from tumor cells, thereby increasing the phagocytic activity of tumor cells.

[0050] In one embodiment, the present disclosure relates to a method for activating dendritic cells (DCs) in a subject, the method comprising administering to the subject an amount of a pharmaceutical composition effective in removing sialic acid from tumor cells in the subject, thereby activating DCs in the subject.

[0051] In one embodiment, the present disclosure relates to a method for reducing Siglec-15 binding activity to thereby increase antitumor activity in a patient's tumor microenvironment, the method comprising administering an effective amount of a pharmaceutical composition to a subject to thereby increase antitumor activity (e.g., T cell activity) in the subject.

[0052] In another aspect, the present invention provides a method for expressing recombinant sialidase. The method may include the steps of (a) providing cells containing nucleic acids encoding recombinant sialidase; and (b) expressing recombinant sialidase in the presence of a stabilizer. In one embodiment, the method further includes the step of purifying the recombinant sialidase produced in step (b). Purification may be carried out in the presence of a stabilizer such as a cation (e.g., calcium or magnesium).

[0053] These and other aspects and features of the present invention are described in the following detailed description and claims. [Brief explanation of the drawing]

[0054] Description of the drawing The present invention can be better understood by referring to the following drawings. [Figure 1]Figure 1 shows different configurations for sialidase-Fc fusion constructs. A sialidase-Fc fusion construct may comprise a first polypeptide containing a first immunoglobulin Fc domain ("Fc domain") and a second polypeptide containing a second immunoglobulin Fc domain. The first and second polypeptides may be covalently bonded together, for example, by one or more disulfide bonds. Figure 1A shows a construct having two Fc domains and sialidase enzymes conjugated to the N-terminus of each Fc domain. Figure 1B shows a construct having two Fc domains and sialidase enzymes conjugated to the C-terminus of the first Fc domain and the N-terminus of the second Fc domain. Figure 1C shows a construct having two Fc domains and sialidase enzymes conjugated to the N-terminus of the second Fc domain. Figure 1D shows a construct having two Fc domains and sialidase enzymes conjugated to the C-terminus of the first Fc domain. Figure 1E shows a construct having two Fc domains and a sialidase enzyme conjugated to the C-terminus of each Fc domain. It is understood that the Fc domains may be naturally occurring Fc domains or genetically engineered Fc domains, including modifications such as point mutations within each polypeptide chain that promote knob-into-hole construction or provide modified Fc domain function. [Figure 2] Figure 2 shows SDS-PAGE gels displaying recombinant human Neu1, Neu2, Neu3, and Salmonella typhimurium (ST-sialidase) under non-reducing and reducing conditions. Monomer and dimer species are shown. [Figure 3] Figure 3 is a bar graph showing the enzyme activity of recombinant human Neu1, Neu2, and Neu3. [Figure 4] Figure 4 is a line graph showing the enzyme activity as a function of substrate concentration for recombinant human Neu2 and Neu3 at the indicated pH. [Figure 5]Figure 5A shows SDS-PAGE gels displaying recombinant wild-type human Neu2-Fc and Neu2-Fc variant M106 ("M106") under non-reducing and reducing conditions. Figures 5B and 5C show SEC-HPLC traces comparing wild-type Neu2-Fc versus M106, where the monomer species have a retention time of 21 minutes. [Figure 6] Figure 6 is a line graph showing the enzyme activity of M106 as a function of substrate concentration. [Figure 7] Figure 7 is a bar graph showing the enzymatic activity of Neu3-Fc in supernatant ("supernatant") or membrane-bound ("washed cells") Expi293 cells. [Figure 8] Figure 8 shows the SEC-HPLC trace of Fc-ST sialidase, where the monomer species has a retention time of 21 minutes. [Figure 9] Figures 9A-D are a series of line graphs showing tumor volume in a mouse A20 (lymphoma) syngeneic tumor model. Mice were administered either a negative control ("isotype control," Figure 9A), Fc-ST sialidase (Figure 9B), avelumab (anti-mouse PD-L1 antibody, Figure 9C), or a combination of Fc-ST sialidase and avelumab (Figure 9D) at a dose of 10 mg / kg twice a week for 15 days, and tumor volume was measured over time. Administration of Fc-ST sialidase alone or in combination with avelumab reduced tumor volume. [Figure 10] Figures 10A–D are a series of line graphs showing tumor volume in a mouse syngeneic tumor model using EMT6 cells genetically engineered for human Her2 expression. Mice were administered 10 mg / kg twice a week for 15 days to either an isotype control (vehicle control, Figure 10A), Fc-ST sialidase (FC-ST, Figure 10B), trastuzumab (anti-human Her2 antibody, Figure 10C), or Fc human sialidase (M106, Figure 10D), as indicated by the triangles, and tumor volume was measured over time. Administration of Fc human sialidase or Fc-ST sialidase reduced tumor volume. [Figure 11]Figure 11 is a bar graph showing that neuraminidase activity after incubation at 37°C for 14 days is stabilized by the addition of CaCl2. [Figure 12] Figure 12A is a bar graph showing neuraminidase activity in conditioned medium of cells expressing the human neuraminidase Fc construct at indicated days after transfection in the presence or absence of 4 mM CaCl2. As shown, the presence of CaCl2 stabilizes the activity. Figure 12B is a bar graph showing cell viability at indicated days after transfection in the presence or absence of 4 mM CaCl2. [Figure 13] Figure 13A is a bar graph showing that neuraminidase activity is stabilized by different concentrations of CaCl2 in conditioned medium of cells expressing the human neuraminidase Fc construct. It shows the enzyme activity at the indicated days after transfection in the presence of 0, 0.05, 0.5, 1, 2, and 4 mM CaCl2. Figure 13B shows the total protein yield at day 6 in the presence of 0, 0.05, 0.5, 1, 2, and 4 mM CaCl2. [Figure 14] Figure 14 provides bar graphs showing the geometric mean fluorescence intensity (gMFI) obtained by staining with Hydra-3 (Figure 14A), Hydra-7 (Figure 14B), and Hydra-9 (Figure 14C) in different immunosubset populations. [Figure 15] Figure 15 provides bar graphs showing the geometric mean fluorescence intensity (gMFI) obtained by staining with PNA (Figure 15A), MAL-II (Figure 15B), and SNA (Figure 15C) in different immunosubset populations. [Figure 16] Figure 16 provides a line graph showing the degree of desialization of dendritic cells (DCs) by increasing concentrations of M106. Figure 16A shows the mean fluorescence intensity (MFI), and Figure 16B provides a bar graph showing the fold increase in desialization compared to untreated DCs. [Figure 17]Figure 17 provides a line graph showing the degree of desialylation of BT-20 (breast cancer) tumor cells after treatment with increased concentrations of M106 (triangle) compared to LOF control (square), as determined by Hydra 9 binding (Figure 17A) or PNA binding (Figure 17B) measured by gMFI. [Figure 18] Figure 18 provides a line graph showing the degree of desialylation of HT-29 tumor cells after treatment with increased concentrations of M106 (triangle), compared to the LOF control (square) when determined by Hydra 9 binding (Figure 18A) or PNA binding (Figure 18B) and measured as gMFI. [Figure 19] Figure 19 provides a line graph showing the degree of desialylation of SK-BR-3 tumor cells after treatment with increased concentrations of M106 (triangle), compared to an LOF control (square) determined by Hydra 9 binding (Figure 19A), MAL-II binding (Figure 19B), or PNA binding (Figure 19C) and measured as gMFI. [Figure 20] Figure 20 provides a bar graph showing the percentage increase in CD83hi expression (Figure 20A) and CD86hi expression (Figure 20B) on DCs after incubation with SKBR3 tumor cells treated with or without M106 in the presence or absence of lipopolysaccharide (LPS) treatment (white bars vs. colored bars). [Figure 21] Figure 21 shows dose-dependent enhancement of phagocytosis by M2-like macrophages in HT-29 tumor cells desialized by M106 or LOF, as shown. The tumor cells were derived from two different healthy donors (Figures 21A and 21B). Similar increases in phagocytosis of desialized BT20 and SKBR-3 tumor cells by M2-like macrophages are shown in Figures 21C and 21D, respectively. [Figure 22] Figure 22 provides a bar graph showing dose-dependent enhancement of HLA-DR expression after desialylation of monocytes under M106 or LOF control. Monocytes were obtained from two different healthy donors (Figures 22A and 22B). [Figure 23]Figure 23 provides tumor growth curves demonstrating the in vivo activity of the sialidase of this disclosure in a mouse MC38 syngeneic tumor model. Tumor growth curves for individual mice are shown for isotype control-treated mice (Figure 23A), M106-treated mice (Figure 23B), anti-PD-1-treated mice (Figure 23C), or mice treated with a combination of M106 and anti-PD-1 (Figure 23D). Triangles indicate the administration time of the test substance. [Figure 24] Figure 24 provides tumor growth curves demonstrating the in vivo activity of the sialidase of the present invention in a mouse B16F10 syngeneic tumor model. Tumor growth curves for individual mice are shown for isotype control-treated mice (Figure 24A), M106-treated mice (Figure 24B), or anti-PD-1-treated mice (Figure 24C). Figure 24D is a superposition of the tumor growth curves for the isotype control group and the M106 group. Triangles indicate the administration time of the test substance. [Figure 25] Figure 25 provides tumor growth curves demonstrating the in vivo activity of the sialidase of the present invention in a mouse EMT6 syngeneic tumor model. Tumor growth curves for each individual mouse are shown for isotype control-treated mice (Figure 25A) or M106-treated mice (Figure 25B). Triangles indicate administration time of the test substance. [Figure 26] Figure 26 shows the in vivo efficacy of M106 alone or in combination with avelumab ("Ave") at the indicated doses in a mouse A20 syngeneic subcutaneous tumor model. Tumor growth curves for each mouse are shown. Observed partial response (PR) and complete response (CR) are also shown. [Figure 27] Figure 27 shows the in vivo efficacy of M106 alone or in combination with avelumab at the indicated doses in a mouse A20 syngeneic subcutaneous tumor model. Tumor growth curves for each mouse are shown. Triangles indicate administration. [Figure 28]Figure 28 shows the in vivo activity of ofatumumab, ofatumumab combined with Neu2-M106-Fc ("M106 FC"), and isotype controls in a syngeneic EL4-CD20 lymphoma intravenously scattered model at survival end of day 28 (Figure 28A) or day 41 (Figure 28B). Triangles indicate administration of various test items. P-values ​​were calculated using the log-rank (Mantle-Cox) test. [Figure 29A] Figure 29 shows the results of Siglec-15-Fc staining of CD4+ cells (Figure 29A) and CD8+ cells (Figure 29B) after treatment with untreated ("none"), loss of function sialidase ("LOF FC"), or sialidase (M106 ("M106 FC") or BiNaNH2 (positive control)). Isotype IgG1 staining is also shown as a negative control. As shown, treatment of activated CD4 and CD8 cells with M106 or BiNaNH2 reduced Siglec-15-Fc staining compared to untreated or treatment with loss of function sialidase. Bar graphs showing fluorescence levels (gMFI) and flow cytometry histogram data are provided in each figure below. [Figure 29B] Figure 29 shows the results of Siglec-15-Fc staining of CD4+ cells (Figure 29A) and CD8+ cells (Figure 29B) after treatment with untreated ("none"), loss of function sialidase ("LOF FC"), or sialidase (M106 ("M106 FC") or BiNaNH2 (positive control)). Isotype IgG1 staining is also shown as a negative control. As shown, treatment of activated CD4 and CD8 cells with M106 or BiNaNH2 reduced Siglec-15-Fc staining compared to untreated or treatment with loss of function sialidase. Bar graphs showing fluorescence levels (gMFI) and flow cytometry histogram data are provided in each figure below. [Figure 30A] Figure 30 shows the results of Siglec-15-Fc staining of CD4+ cells (Figure 30A) and CD8+ cells (Figure 30B) using the same method as in Figures 29A-B, with PBMCs derived from a second healthy donor. [Figure 30B] Figure 30 shows the results of Siglec-15-Fc staining of CD4+ cells (Figure 30A) and CD8+ cells (Figure 30B) using the same method as in Figures 29A-B, with PBMCs derived from a second healthy donor. [Modes for carrying out the invention]

[0055] Detailed explanation The present invention is partly based on the discovery that sialic acid-mediated disorders can be treated by administering sialidase enzymes or sialidase enzymes conjugated to serum half-life enhancers. Surprisingly, it has been discovered that sialidase enzymes conjugated to sialidase or serum half-life enhancers lacking a targeting moiety (e.g., an antibody-binding domain against a tumor antigen) can effectively treat sialic acid-mediated disorders (e.g., cancer, e.g., solid tumors) in vivo. Consequently, the constructs described herein can be used alone to treat sialic acid-mediated disorders, e.g., cancer, or they can be used in combination with other agents, e.g., anticancer agents, to treat disorders, e.g., cancer. When used in combination with another anticancer agent, for example, the constructs can enhance the activity of the anticancer agent, for example, by making the cancer more sensitive to treatment with the anticancer agent.

[0056] The present invention further relates to recombinant forms of sialidase enzymes having appropriate substrate specificity and activity useful for removing sialic acid and / or sialic acid-containing molecules from the surface of cancer cells and / or from the tumor microenvironment and / or for reducing the concentration of sialic acid and / or sialic acid-containing molecules in the tumor microenvironment, sialidase enzymes conjugated to serum half-life enhancers and pharmaceutical compositions thereof.

[0057] The present invention further relates to pharmaceutical compositions and methods for using sialidase or sialidase conjugated with a half-life prolonging factor for the treatment of cancer, such as solid tumors, soft tissue tumors, hematopoietic tumors, metastatic lesions or epithelial cell carcinomas.

[0058] Various features and aspects of the present invention are described in more detail below.

[0059] I. Recombinant sialidase As used herein, the term “sialidase” means any enzyme or functional fragment or variant thereof that cleaves terminal sialic acid residues from a substrate, such as a glycoprotein or glycolipid. The term “sialidase” includes variants having one or more amino acid substitutions, deletions or insertions from a wild-type sialidase sequence, and / or fusion proteins or conjugates containing sialidase. Sialidase is also referred to as neuraminidase, and unless otherwise indicated, the two terms are used interchangeably herein. As used herein, the term “functional fragment” of sialidase means a fragment of full-length sialidase that retains, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the enzymatic activity of the corresponding full-length, naturally occurring sialidase. Sialidase enzyme activity can be assayed by any method known in the art, which includes, for example, measuring the release of sialic acid from the fluorescent substrate 4-methylumbelliferyl-N-acetylneuraminic acid (4MU-NeuAc). In one embodiment, the functional fragment contains at least 100, 150, 200, 250, 300, 310, 320, 330, 340, 350, 360, or 370 consecutive amino acids present in the full-length naturally occurring sialidase.

[0060] The sialidases described herein may be any sialidase, e.g., viral, fungal, bacterial, non-human mammalian, or human sialidase. In one embodiment, the sialidase is a recombinant human sialidase comprising at least one mutation from wild-type human sialidase, e.g., at least one amino acid substitution, deletion, or addition, as described above.

[0061] In one embodiment, the sialidase is any recombinant mutant human sialidase or a functional fragment thereof disclosed herein.

[0062] In one embodiment, the sialidase contains C332A and C352L mutations. In one embodiment, the sialidase contains an N-terminal addition of MEDLRP (SEQ ID NO: 4) or EDLRP (SEQ ID NO: 3). In one embodiment, the sialidase contains the LSHSLST (SEQ ID NO: 22) peptide at its N-terminus. In one embodiment, the sialidase contains an N-terminal addition of MEDLRP (SEQ ID NO: 4) and an A2K substitution. In one embodiment, the sialidase contains an N-terminal addition of MEDLRP (SEQ ID NO: 4) and a C332A substitution. In one embodiment, the sialidase contains an N-terminal addition of MEDLRP (SEQ ID NO: 4), a C332A substitution, and a C352L substitution.

[0063] In one embodiment, the sialidase moiety includes M1 deletion (ΔM1), M1A substitution, M1D substitution, V6Y substitution, K9D substitution, P62G substitution, P62N substitution, P62S substitution, P62T substitution, A93E substitution, Q126Y substitution, I187K substitution, A242T substitution, Q270A substitution, Q270T substitution, S301R substitution, S301R substitution, W302K substitution, W302R substitution, C332A substitution, V363R substitution, L365I substitution, or any combination of the above.

[0064] In one embodiment, the sialidase comprises an amino acid sequence of any of SEQ ID NOs: 48-62, 169-171, or 196, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of 48-62, 169-171, or 196.

[0065] a. Viral sialidase Examples of viral sialidases include influenza A virus surface glycoprotein neuraminidase (e.g., NCBI accession number ACY01419.1, SEQ ID NO: 63), influenza B virus surface glycoprotein neuraminidase (e.g., NCBI accession number AIX94926.1, SEQ ID NO: 64), or influenza C virus surface glycoprotein neuraminidase, or their variants or functional fragments. Other examples of viral sialidases include paramyxoviridae respirovirus parainfluenzavirus types 1 and 3 (e.g., NCBI accession number BAD89145.1, SEQ ID NO: 65), bovine parainfluenza virus type 3 (e.g., NCBI accession number ADQ43755, SEQ ID NO: 66), Sendai virus (e.g., UniProtKB accession number P04853.1, SEQ ID NO: 67), rubra virus, mumps virus, Simian virus type 5, and parainfluenza viruses types 2, 4a, and 4b.

[0066] b. Prokaryotic sialidases Examples of prokaryotic sialidases include those derived from Salmonella typhimurium and Vibrio cholerae. The amino acid sequence of Salmonella typhimurium sialidase (St-sialidase) is shown in SEQ ID NO: 30, and the nucleotide sequence encoding Salmonella typhimurium sialidase is shown in SEQ ID NO: 6. The amino acid sequence of Vibrio cholerae sialidase is shown in SEQ ID NO: 36, and the nucleotide sequence encoding Vibrio cholerae sialidase is shown in SEQ ID NO: 37.

[0067] Other exemplary prokaryotic sialidases include sialidase from Actinomyces viscosus (Avis_NanH; Uniprot accession number AAA21932, SEQ ID NO: 68); Arthrobacter nicotianae NA1 and NA2; sialidase from Arthrobacter sialophilus; Arthrobacter ureafaciens L, M1, M2 and S (GenBank accession number BAD66680, SEQ ID NO: 69); sialidase from Bacteroides fragilis; sialidase from Clostridium chauvoei; i A99 NanH (GenBank accession number CAA50436, SEQ ID NO: 70), NanI (GenBank accession number ABG83208, SEQ ID NO: 71), NanJ (GenBank accession number ABG84247, SEQ ID NO: 72); Sialidase from *Edema mercury* (e.g., GenBank accession number CAA44916.1, SEQ ID NO: 107); Sialidase from *Clostridium sordellii*; Sialidase from *Clostridium tertium* (e.g., GenBank accession number CAA69951, SEQ ID NO: 73); Sialidase from *Bacillus diphtheriae* (e.g., GenBank accession number ACS34893, SEQ ID NO: 74); Sialidase from *Haemophilus parasusis*; *Micromonospora viridifaciens* Sialidase derived from viridifaciens (e.g., GenBank accession number BAA00852, sequence number: 75); Pasteurella multocida NanH (GenBank accession number AAG35310).1. Sialidase from Pseudomonas aeruginosa (e.g., GenBank accession number AAG06182, Sialidase: 77); Sialidase from Salmonella typhimurium (e.g., GenBank accession number NP_459905, Sialidase: 79); Streptococcus pneumoniae NanA (GenBank accession number P62575, Sialidase: 108), NanB (GenBank accession number AAC44396, Sialidase: 80) and NanC; Sialidase from Tannerella forsythia (e.g., GenBank accession number TF0035, Sialidase: 81); Sialidase from Vibrio cholerae (e.g., GenBank accession number YP_001217324, Sialidase: 82), and Vibrio diphtheriae (C. Sialidase derived from *Bacillus diphtheriae* (designated as Cdip_NanH, *Bacillus diphtheriae* KCTC3075 NanH (GenBank accession number ACS34893, SEQ ID NO: 83) and its homolog; *Corynebacterium glutamicum* R virtual protein (Cglu_hypP; YP_001138502, SEQ ID NO: 84); *Clostridium perfringens* NCTC 8239 sialidase I (Cper_NanI; ZP_02643014, SEQ ID NO: 85); *Bacteroides fragilis* YCH46 sialidase (Bfra_NanH; Uniprot accession number BAA05853, SEQ ID NO: 86); *Micromonospora viridifaciens* (M. Streptococcus viridifaciens) sialidase (Mvir_NanH; Uniprot accession number BAA0085, sequence number: 87); Streptococcus pneumoniae (S.pneumoniae) NanA sialidase (Spne_NanA; P62575, SEQ ID NO: 88); Streptomyces coelicolor A3(2) sialidase (Scoe_NanH; NP_630638, SEQ ID NO: 89); Streptomyces griseus NBRC 13350 sialidase (Sgri_NanH; YP_001827941, SEQ ID NO: 90); Propionibacterium acnes SK137 sialidase (Pacn_NanH; ZP_03389398, SEQ ID NO: 91); Macrobdella decora trans sialidase (Mdec_NanL; AAC47263, SEQ ID NO: 92); Trypanosoma cruzi (T. cruzi) trans sialidase (Tcru_TS; GenBank accession number AAA99442, SEQ ID NO: 93); Ackermannsia muciniphylla (ATCC BAA-835 / DSM 22959) Amuc_0625 / Am0707 (Uniprot accession number B2UPI5, SEQ ID NO: 94); Bacteroides fragilis TAL2480 YCH46 sialidase (GenBank accession number BF1729, SEQ ID NO: 95) (P31206); Bacteroides fragilis SBT3182; Bacteroides fragilis 4852; Bacteroides fragilis YM4000; Bacteroides thetaiotaomicron VPI-5482 sialidase (BtsA; BTSA; BT0455) (GenBank accession number Q8AAK9, sequence number: 96); Bacteroides vulgatus ATCC 8482 / DSM 1447 / NCTC 11154 BVU_4143 (Uniprot accession number A6L7T1, sequence number: 97); Bifidobacterium bifidum JCM 1254 exo-α-sialidase (SiaBb2; BBP_0054) (GenBank accession number BAK26854.1, SEQ ID NO: 98); Clostridium perfringens A99 sialidase 1 "small" (P10481, SEQ ID NO: 99); Clostridium perfringens ATCC 10543 sialidase 2 (NanH) (Uniprot accession number Q59311, SEQ ID NO: 100); Clostridium perfringens ATCC 13124 sialidase (CPF_0721) (Uniprot accession number Q0TT67, SEQ ID NO: 101); Clostridium perfringens str 13 exo-α-sialidase (NanI;CPSA;CPE0725) (Uniprot accession number Q8XMG4, SEQ ID NO: 102); Clostridium perfringens str 13 / ATCC 13124 exo-α-sialidase (NanJ;CPE0553 (Uniprot accession number Q8XMY5, SEQ ID NO: 103); Clostridium tertium ATCC Examples include 14573 sialidase (NanH;SiaH) (Uniprot accession number P77848, SEQ ID NO: 104); Ruminococcus gnavus (R. gnavus) ATCC 29149 RgNanH (Uniprot accession number A7B557, SEQ ID NO: 105); and Salmonella typhimurium (S. typhimurium) TA262 / LT2 sialidase (NanH;STSA) (P29768, SEQ ID NO: 106).

[0068] Other exemplary sialidases include sialidases or neuraminidases derived from Acanthamoeba castellani, Acanthamoeba polyphaga, Acanthamoeba culbertsoni, Acanthamoeba astronyxis, Acanthamoeba hatchetti, Acanthamoeba palestinensis, Acanthamoeba rhysodes, chicken coccidia, Eimeria maxima, Eimeria necatrix, Eimeria spec, Trypanosoma brucei, and Trypanosoma rangeli.

[0069] c. Mouse sialidase Four sialidases have also been found in the mouse genome, designated Neu1, Neu2, Neu3, and Neu4. The amino acid sequence of mouse Neu1 is shown in SEQ ID NO: 38, and the nucleotide sequence encoding mouse Neu1 is shown in SEQ ID NO: 42. The amino acid sequence of mouse Neu2 is shown in SEQ ID NO: 39, and the nucleotide sequence encoding mouse Neu2 is shown in SEQ ID NO: 43. The amino acid sequence of mouse Neu3 is shown in SEQ ID NO: 40, and the nucleotide sequence encoding mouse Neu3 is shown in SEQ ID NO: 44. The amino acid sequence of mouse Neu4 is shown in SEQ ID NO: 41, and the nucleotide sequence encoding mouse Neu4 is shown in SEQ ID NO: 45.

[0070] d. Human sialydidase Four sialidases have also been discovered in the human genome, and are referred to as Neu1, Neu2, Neu3, and Neu4.

[0071] Human Neu1 is a lysosomal neuraminidase enzyme that functions in a complex with β-galactosidase and cathepsin A. The amino acid sequence of human Neu1 is shown in SEQ ID NO: 7, and the nucleotide sequence encoding human Neu1 is shown in SEQ ID NO: 23.

[0072] Human Neu2 is a cytosolic sialidase enzyme. The amino acid sequence of human Neu2 is shown in SEQ ID NO: 1, and the nucleotide sequence encoding human Neu2 is shown in SEQ ID NO: 24.

[0073] Human Neu3 is a plasma membrane sialidase with ganglioside-specific activity. Human Neu3 has two isoforms: isoform 1 and isoform 2. The amino acid sequence of human Neu3, isoform 1 is shown in SEQ ID NO: 8, and the nucleotide sequence encoding human Neu3, isoform 1 is shown in SEQ ID NO: 25. The amino acid sequence of human Neu3, isoform 2 is shown in SEQ ID NO: 9, and the nucleotide sequence encoding human Neu3, isoform 2 is shown in SEQ ID NO: 34.

[0074] Human Neu4 has two isoforms: isoform 1 is a superficial membrane protein, and isoform 2 is localized in the lysosome lumen. The amino acid sequence of human Neu4, isoform 1 is shown in SEQ ID NO: 10, and the nucleotide sequence encoding human Neu4, isoform 1 is shown in SEQ ID NO: 26. The amino acid sequence of human Neu4, isoform 2 is shown in SEQ ID NO: 11, and the nucleotide sequence encoding human Neu4, isoform 2 is shown in SEQ ID NO: 35.

[0075] In one embodiment, recombinant mutant human sialidase has approximately 5%, approximately 10%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, approximately 100%, or more than 100% of the enzymatic activity of the corresponding (or template) wild-type human sialidase.

[0076] In one embodiment, recombinant mutant human sialidase has the same substrate specificity as the corresponding wild-type human sialidase. In another embodiment, recombinant mutant human sialidase has different substrate specificity than the corresponding wild-type human sialidase. For example, in one embodiment, recombinant mutant human sialidase can cleave α2,3, α2,6 and / or α2,8 linkages. In one embodiment, sialidase can cleave α2,3 and α2,8 linkages.

[0077] In one aspect, the expression yield of recombinant mutant human sialidase in mammalian cells, e.g., HEK293 cells, CHO cells, mouse myeloma cells (NS0, Sp2 / 0), or human fibrosarcoma cells (HT-1080), e.g., HEK293 cells, is higher than approximately 10%, 20%, 50%, 75%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1,000% of the expression yield of the corresponding wild-type human sialidase.

[0078] In one embodiment, recombinant mutant human sialidase has enzyme activity that is approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or 100% higher than that of the corresponding wild-type human sialidase, and is found to be beneficial for mammalian cells. The expression yield of recombinant mutant human sialidase in cells, e.g., HEK293 cells, is approximately 10%, 20%, 50%, 75%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1,000% higher than the expression yield of the corresponding wild-type human sialidase.

[0079] In one embodiment, the amino acid sequence of a recombinant mutant human sialidase has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of the corresponding wild-type human sialidase.

[0080] It is understood that sialidases described herein, such as human sialidases, may be modified to enhance one or more of the enzyme's properties, for example, to improve expression, activity, or stability (e.g., to improve resistance to protease degradation). Some of these properties, such as improved resistance to protease degradation, are applicable to the various sialidases described herein.

[0081] i. Substitution of cysteine ​​residues In one embodiment, recombinant mutant human sialidase contains at least one cysteine ​​(cys, C) residue substitution. Certain cysteine ​​residues in sialidase have been found to inhibit the expression of functional proteins as a result of protein aggregation. Therefore, in one embodiment, recombinant mutant human sialidase contains at least one mutation to remove free cysteine ​​(e.g., for Neu1 (SEQ ID NO: 7), one or more mutations of C111, C117, C171, C183, C218, C240, C242 and C252; for Neu2 (SEQ ID NO: 1), one or more mutations of C125, C196, C219, C272, C332 and C352; for Neu3 (SEQ ID NO: 8), C7, C9 This includes one or more mutations of C0, C99, C106, C127, C136, C189, C194, C226, C242, C250, C273, C279, C295, C356, C365, C368, C384, C383, C394, and C415; as well as one or more mutations of C88, C125, C126, C186, C191, C211, C223, C239, C276, C437, C453, C480, and C481 for Neu4 (SEQ ID NO: 10). Free cysteine ​​may be substituted with any amino acid. In one embodiment, free cysteine ​​is substituted with serine (ser, S), isoleucine (iso, I), valine (val, V), phenylalanine (phe, F), leucine (leu, L), or alanine (ala, A). Exemplary cysteine ​​substitutions in Neu2 include C125A, C125I, C125S, C125V, C196A, C196L, C196V, C272S, C272V, C332A, C332S, C332V, C352L, and C352V.

[0082] In one embodiment, recombinant mutant human sialidase contains two or more cysteine ​​substitutions. Exemplary double or triple substitutions in Neu2 include: C125S and C332S; C272V and C332A; C272V and C332S; C332A and C352L; C125S and C196L; C196L and C352L; C196L and C332A; C332A and C352L; and C196L, C332A and C352L.

[0083] In one embodiment, the recombinant mutant human sialidase is Neu2 sialidase and contains substitutions C322A and C352L (SEQ ID NO: 5).

[0084] In one embodiment, the sialidase contains amino acid substitutions with 2, 3, 4, 5, or 6 cysteine ​​molecules, which are typically present in human sialidases such as Neu2 or Neu3.

[0085] In one embodiment, recombinant mutant human sialidase contains Table 1 (amino acid positions corresponding to substitutions or combinations of substitutions listed in wild-type human Neu2 (SEQ ID NO: 1)). [Table 1]

[0086] ii. Substitution of residues to increase pI and / or decrease hydrophobicity The isoelectric point (pI) of a protein is the pH at which its net charge is zero. pI also indicates the pH at which a protein is minimally soluble, affecting its ability to be expressed and purified. Generally, a protein has good solubility when its pI is 2 units higher than the pH of the solution. Human Neu2 has a predicted pI of 7.5. Therefore, human Neu2 has minimal solubility around neutral pH, which is undesirable since the expression and physiological systems are at neutral pH. In contrast, sialidase from Salmonella typhimurium (St-sialidase), which exhibits good solubility and recombinant expression, has a pI of 9.6. Therefore, to increase the expression of human Neu2 or other human sialidases, recombinant mutant human sialidases may be designed to contain one or more amino acid substitutions, where the substitution(s) increase the pI of the sialidase compared to sialidase without the substitution. Furthermore, reducing the number of hydrophobic amino acids on the surface of sialidase can improve sialidase expression, for example, by reducing aggregation. Therefore, to increase the expression of human Neu2 or other human sialidases, recombinant mutant human sialidases may be designed to contain one or more amino acid substitutions, where the substitution(s) reduce the hydrophobicity of the sialidase surface compared to sialidases without the substitution(s).

[0087] Therefore, in one embodiment, recombinant mutant human sialidase comprises at least one amino acid substitution, where the substitution increases the isoelectric point (pI) of the sialidase and / or decreases the hydrophobicity of the sialidase compared to sialidase without the substitution. This can be achieved by introducing one or more charged amino acids, such as positively or negatively charged amino acids, into the recombinant sialidase. In one embodiment, the amino acid substitution is for a charged amino acid, such as a positively charged amino acid, e.g., lysine (lys, K), histidine (his, H), or arginine (arg, R), or a negatively charged amino acid, e.g., aspartic acid (asp, D) or glutamic acid (glu, E). In one embodiment, the amino acid substitution is for a lysine residue. In one embodiment, the substitution increases the pI of the sialidase to about 7.75, about 8, about 8.25, about 8.5, about 8.75, about 9, about 9.25, about 9.5, or about 9.75.

[0088] In one embodiment, amino acid substitutions occur at surface-exposed D or E amino acids, within a helix or loop, or at positions where K or R is located at the corresponding position in the St-sialidase. In another embodiment, amino acid substitutions occur at amino acids that are far from the catalytic site or otherwise not included in the catalyst, amino acids that are not conserved by other human Neu proteins or St-sialidases or Clostridium NanH, or amino acids that are not located within functionally important domains (e.g., the Asp-box or β-strand).

[0089] Examples of amino acid substitutions in Neu2 that increase the isoelectric point (pI) of sialidase and / or decrease the hydrophobicity of sialidase compared to sialidase without substitutions include A2E, A2K, D215K, V325E, V325K, E257K, and E319K. In one embodiment, recombinant mutant human sialidases include two or more amino acid substitutions, such as A2K and V325E, A2K and V325K, E257K and V325K, A2K and E257K, and E257K and A2K and V325K.

[0090] In one embodiment, recombinant mutant human sialidase contains substitutions or combinations of substitutions corresponding to the substitutions or combinations of substitutions listed in Table 2 (amino acid positions corresponding to wild-type human Neu2 (SEQ ID NO: 1)). [Table 2]

[0091] iii. Addition of N-terminal peptides and N- or C-terminal substitutions It has been found that the addition of a peptide sequence of two or more amino acids to the N-terminus of human sialidase can improve sialidase expression and / or activity. In one embodiment, the peptide is at least two amino acid long, e.g., 2-20, 2-10, 2-5, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid long. In one embodiment, the peptide may be able to form an α-helix or may have a tendency to form an α-helix.

[0092] In mice, the Neu2 isoform (type B) found in the thymus contains six amino acids that are not present in the canonical isoform of Neu2 found in skeletal muscle. In one embodiment of this specification, the N-terminal six amino acids of the mouse thymic Neu2 isoform, MEDLRP (SEQ ID NO: 4), or a variation thereof, may be added to human Neu, e.g., human Neu2. In one embodiment, recombinant mutant human sialidase contains a peptide of at least two amino acid residues covalently bonded to the N-terminal amino acid of the sialidase. In one embodiment, recombinant mutant human sialidase contains the peptide MEDLRP (SEQ ID NO: 4) or EDLRP (SEQ ID NO: 3) covalently bonded to the N-terminal amino acid of the sialidase. In one embodiment, the sialidase may further contain a cleavage site located between the peptide, e.g., MEDLRP (SEQ ID NO: 4) or EDLRP (SEQ ID NO: 3), and the rest of the sialidase, e.g., a proteolytic cleavage site. In one embodiment, a peptide, such as MEDLRP (SEQ ID NO: 4) or EDLRP (SEQ ID NO: 3), can be post-translationally cleaved from the remainder of the sialidase.

[0093] Alternatively to, or in combination with, N-terminal addition, 1 to 5 amino acids may be removed from the 12-amino acid N-terminal region of recombinant mutant human sialidase, for example, the N-terminal methionine may be removed. In one embodiment, if the recombinant mutant human sialidase is Neu2, the N-terminal methionine may be removed, or the first 5 amino acids (MASLP; SEQ ID NO: 12) may be removed, or the 2nd to 4th amino acids (ASLP; SEQ ID NO: 13) may be removed.

[0094] In one embodiment, amino acids 1-5 of the 12-amino acid N-terminal region of recombinant mutant human sialidase are substituted with MEDLRP (SEQ ID NO: 4), EDLRP (SEQ ID NO: 3), or TVEKSVVF (SEQ ID NO: 14). For example, in one embodiment, if the recombinant mutant human sialidase is Neu2, the amino acids MASLP (SEQ ID NO: 12), ASLP (SEQ ID NO: 13), or M are substituted with MEDLRP (SEQ ID NO: 4), EDLRP (SEQ ID NO: 3), or TVEKSVVF (SEQ ID NO: 14).

[0095] Human sialidases have a β-propeller structure characterized by six blade-shaped β-sheets arranged toroidally around a central axis. Generally, hydrophobic interactions between β-propeller blades, such as between the N-terminal and C-terminal blades, enhance stability. Therefore, recombinant mutant human sialidases can be designed to increase the expression of human Neu2 or other human sialidases by including amino acid substitutions that increase hydrophobic interactions and / or hydrogen bonding between the N-terminal and C-terminal β-propeller blades of the sialidase.

[0096] Therefore, in one embodiment, the recombinant mutant human sialidase includes a substitution of at least one wild-type amino acid residue, where the substitution increases hydrophobic interactions and / or hydrogen bonding between the N-terminus and C-terminus of the sialidase compared to the sialidase without the substitution. In one embodiment, the wild-type amino acid is substituted with asparagine (asn, N), lysine (lys, K), tyrosine (tyr, Y), phenylalanine (phe, F), or tryptophan (trp, W). Exemplary substitutions in Neu2 that increase hydrophobic interactions and / or hydrogen bonding between the N-terminus and C-terminus include L4N, L4K, V6Y, L7N, L4N and L7N, L4N and V6Y and L7N, V12N, V12Y, V12L, V6Y, V6F, or V6W. In one embodiment, the sialidase includes the V6Y substitution.

[0097] In one embodiment, recombinant mutant human sialidases include combinations of the above substitutions. For example, recombinant mutant human Neu2 sialidase may contain an additional amino acid MEDLRP (SEQ ID NO: 4), EDLRP (SEQ ID NO: 3), or TVEKSVVF (SEQ ID NO: 14) at its N-terminus, which may be combined with at least one L4N, L4K, V6Y, L7N, L4N and L7N, L4N and V6Y and L7N, V12N, V12Y, V12L, V6Y, V6F, or V6W substitution. In one embodiment, the amino acid MASLP (SEQ ID NO: 12), ASLP (SEQ ID NO: 13), or M of recombinant mutant human Neu2 sialidase is replaced with MEDLRP (SEQ ID NO: 4), EDLRP (SEQ ID NO: 3), or TVEKSVVF (SEQ ID NO: 14), and the recombinant mutant human Neu2 sialidase also includes at least one L4N, L4K, V6Y, L7N, L4N and L7N, L4N and V6Y and L7N, V12N, V12Y, V12L, V6Y, V6F, or V6W substitution.

[0098] In one embodiment, recombinant mutant human sialidase contains mutations or combinations of mutations corresponding to the mutations or combinations of mutations listed in Table 3 (amino acid positions corresponding to wild-type human Neu2 (SEQ ID NO: 1)). [Table 3]

[0099] Furthermore, in one embodiment, the sialidase includes a substitution or deletion of the N-terminal methionine at the N-terminus of the sialidase. For example, in one embodiment, the sialidase includes a substitution of the methionine residue at the position corresponding to position 1 of wild-type human Neu2 (SEQ ID NO: 1), where, for example, the methionine at the position corresponding to position 1 of wild-type human Neu2 is substituted with alanine (M1A) or aspartic acid (M1D). In another embodiment, the sialidase includes a deletion (ΔM1) of the methionine residue at the position corresponding to position 1 of wild-type human Neu2 (SEQ ID NO: 1).

[0100] In one embodiment, recombinant mutant human sialidase contains substitutions or combinations of substitutions corresponding to the substitutions or combinations of substitutions listed in Table 4 (amino acid positions corresponding to wild-type human Neu2 (SEQ ID NO: 1)). [Table 4]

[0101] d. Substitution of residues to reduce proteolytic cleavage. Certain sialidases (e.g., human Neu2) have been found to be sensitive to cleavage by proteases (e.g., trypsin). Consequently, proteolytic cleavage of sialidase can occur during recombinant protein production, harvesting, purification, formulation, administration to subjects, or after administration to subjects, or any combination thereof. Therefore, in one embodiment, recombinant mutant human sialidase includes a substitution of at least one wild-type amino acid residue, where the substitution reduces cleavage of sialidase by proteases (e.g., trypsin) compared to sialidase without the substitution.

[0102] In one aspect, incubation of recombinant mutant human sialidase with a protease (e.g., trypsin) results in approximately 1% to 50%, 1% to 40%, 1% to 30%, 1% to 20%, 1% to 10%, and 1% to 1% of the proteolytic cleavage of the corresponding wild-type sialidase when incubated with the protease under the same conditions. This results in 5%, approximately 5% to approximately 50%, approximately 5% to approximately 40%, approximately 5% to approximately 30%, approximately 5% to approximately 20%, approximately 5% to approximately 10%, approximately 10% to approximately 50%, approximately 10% to approximately 40%, approximately 10% to approximately 30%, approximately 10% to approximately 20%, approximately 20% to approximately 50%, approximately 20% to approximately 40%, approximately 20% to approximately 30%, approximately 30% to approximately 50%, approximately 30% to approximately 40%, or approximately 40% to approximately 50%. In one embodiment, incubation of recombinant mutant human sialidase with a protease (e.g., trypsin) results in less than 50%, less than 40%, less than 30%, less than 10%, less than 5%, less than 3%, less than 1%, or less than 0.5% of the proteolytic cleavage of the corresponding wild-type sialidase when incubated with the protease under the same conditions. Proteolytic cleavage can be assayed by any method known in the art, including, for example, by SDS-PAGE as described in Example 5 of this specification.

[0103] Exemplary substitutions that increase resistance to proteolytic cleavage include: (i) substitutions of alanine residues at the position corresponding to position 242 of wild-type human Neu2 (SEQ ID NO: 1), e.g., cysteine ​​(A242C), phenylalanine (A242F), glycine (A242G), histidine (A242H), isoleucine (A242I), lysine (A242K), leucine (A242L), methionine (A242M), asparagine (A242N), glutamine (A242Q), arginine (A242R), serine (A242S), valine (A242V), tryptophan (A242W). (ii) substitution with tyrosine (A242Y); (ii) substitution of an arginine residue at the position corresponding to position 243 of wild-type human Neu2 (SEQ ID NO: 1), for example, substitution with glutamic acid (R243E), histidine (R243H), asparagine (R243N), glutamine (R243Q), or lysine (R243K); (iii) substitution of a valine residue at the position corresponding to position 244 of wild-type human Neu2 (SEQ ID NO: 1), for example, substitution with isoleucine (V244I), lysine (V244K), or proline (V244P); or (iv) any combination of the above. In one embodiment, recombinant mutant human sialidase includes substitutions selected from A242C, A242F, A242Y, and A242W. In one embodiment, recombinant mutant human sialidase contains substitutions or combinations of substitutions corresponding to the substitutions or combinations of substitutions listed in Table 5 (amino acid positions corresponding to wild-type human Neu2 (SEQ ID NO: 1)). [Table 5]

[0104] Further exemplary substitutions that increase resistance to proteolytic cleavage (and / or increase expression yield and / or enzyme activity) include: (i) substitution of a leucine residue at the position corresponding to position 240 of wild-type human Neu2 (SEQ ID NO: 1), e.g., substitution with aspartic acid (L240D), asparagine (L240N), or tyrosine (L240Y); (ii) substitution of an alanine residue at the position corresponding to position 213 of wild-type human Neu2 (SEQ ID NO: 1), e.g., substitution with cysteine ​​(A213C), asparagine (A213N), serine (A213S), or threonine (A213T); (iii) substitution of an arginine residue at the position corresponding to position 241 of wild-type human Neu2 (SEQ ID NO: 1), e.g., alanine (R241A), (iv) Substitution of a serine residue at the position corresponding to position 258 of wild-type human Neu2 (SEQ ID NO: 1), for example, by cysteine ​​(S258C); (v) Substitution of a leucine residue at the position corresponding to position 260 of wild-type human Neu2 (SEQ ID NO: 1), for example, by aspartic acid (L260D), phenylalanine (L260F), glutamine (L260Q), or threonine (L260T); (vi) Substitution of a valine residue at the position corresponding to position 265 of wild-type human Neu2 (SEQ ID NO: 1), for example, by phenylalanine (V265F); or (vii) Any combination of the above. In one embodiment, substitutions or combinations of substitutions at these positions may enhance hydrophobic and / or aromatic interactions between secondary structure factors in the sialidase (e.g., between the α-helix and the nearest β-sheet), thereby stabilizing the structure and improving resistance to proteolytic cleavage.

[0105] In one embodiment, recombinant mutant sialidase includes a substitution at position L240. In one embodiment, recombinant mutant sialidase includes combinations of mutations at positions (i) A213 and A242, (ii) A213, A242 and S258, (iii) L240 and L260, (iv) R241 and A242, (v) A242 and L260, (vi) A242 and V265, and (vii) L240 and A242. In one embodiment, recombinant mutant human sialidase includes combinations of substitutions selected from (i) A213C, A242F and S258C, (ii) A213C and A242F, (iii) A213T and A242F, (iv) R241Y and A242F, or (v) L240Y and A242F. In one embodiment, recombinant mutant human sialidase contains substitutions or combinations of substitutions corresponding to the substitutions or combinations of substitutions listed in Table 6 (amino acid positions corresponding to wild-type human Neu2 (SEQ ID NO: 1)). [Table 6]

[0106] iv. Other substitutions The present invention further provides recombinant mutant human sialidase comprising at least one of the following substitutions: I187K, A328E, K370N, or H210N. In one embodiment, recombinant mutant human Neu2 comprises a substitution of the amino acid GDYDAPTHQVQW (SEQ ID NO: 15) with the amino acid SMDQGSTW (SEQ ID NO: 16) or STDGGKTW (SEQ ID NO: 17). In one embodiment, recombinant mutant human Neu2 comprises a substitution of the amino acid PRPPAPEA (SEQ ID NO: 18) with the amino acid QTPLEAAC (SEQ ID NO: 19). In one embodiment, recombinant mutant human Neu2 comprises a substitution of the amino acid NPRPPAPEA (SEQ ID NO: 20) with the amino acid SQNDGES (SEQ ID NO: 21).

[0107] The present invention further provides recombinant mutant human sialidases comprising at least one substitution at a position corresponding to V212, A213, Q214, D215, T216, L217, E218, C219, Q220, V221, A222, E223, V224, E225, or T225.

[0108] The present invention further provides recombinant mutant human sialidases containing amino acid substitutions at amino acid positions corresponding to the positions identified in Table 7 (wild-type human Neu2 (SEQ ID NO: 1)). In one embodiment, the sialidase contains the amino acid substitutions identified in Table 7. In another embodiment, the sialidase contains any combination of amino acid substitutions identified in Table 7. [Table 7-1] [Table 7-2] [Table 7-3]

[0109] For example, in one embodiment, recombinant mutant human sialyidase has: (a) a proline residue substitution at the position corresponding to position 5 of wild-type human Neu2 (P5); (b) a lysine residue substitution at the position corresponding to position 9 of wild-type human Neu2 (K9); (c) a lysine residue substitution at the position corresponding to position 44 of wild-type human Neu2 (K44); (d) a lysine residue substitution at the position corresponding to position 45 of wild-type human Neu2 (K45); (e) a leucine residue substitution at the position corresponding to position 54 of wild-type human Neu2 (L54); (f) a position corresponding to position 62 of wild-type human Neu2 (g) Substitution of a proline residue at position 69 of wild-type human Neu2 (Q69); (h) Substitution of an arginine residue at position 78 of wild-type human Neu2 (R78); (i) Substitution of an aspartic acid residue at position 80 of wild-type human Neu2 (D80); (j) Substitution of an alanine residue at position 93 of wild-type human Neu2 (A93); (k) Substitution of a glycine residue at position 107 of wild-type human Neu2 (G107); (l) Substitution of a glycine residue at position 108 of wild-type human Neu2 (m) Substitution of a glutamine residue at the position corresponding to position 112 of wild-type human Neu2 (Q112); (n) Substitution of a cysteine ​​residue at the position corresponding to position 125 of wild-type human Neu2 (C125); (o) Substitution of a glutamine residue at the position corresponding to position 126 of wild-type human Neu2 (Q126); (p) Substitution of an alanine residue at the position corresponding to position 150 of wild-type human Neu2 (A150); (q) Substitution of a cysteine ​​residue at the position corresponding to position 164 of wild-type human Neu2 (C164); (r) Wild-type human N (s) Substitution of an arginine residue at position 170 of eu2 (R170); (t) Substitution of an alanine residue at position 171 of wild-type human Neu2 (A171); (u) Substitution of an arginine residue at position 189 of wild-type human Neu2 (R189); (v) Substitution of an alanine residue at position 213 of wild-type human Neu2 (A213); (w) Substitution of a leucine residue at position 217 of wild-type human Neu2 (L217);(x) Substitution of a glutamate residue at position 225 of wild-type human Neu2 (E225); (y) Substitution of a histidine residue at position 239 of wild-type human Neu2 (H239); (z) Substitution of a leucine residue at position 240 of wild-type human Neu2 (L240); (aa) Substitution of an arginine residue at position 241 of wild-type human Neu2 (R241); (bb) Substitution of an arginine residue at position 242 of wild-type human Neu2 Substitution of nin residue (A242); (cc) Substitution of valine residue at position 244 of wild-type human Neu2 (V244); (dd) Substitution of threonine residue at position 249 of wild-type human Neu2 (T249); (ee) Substitution of aspartate residue at position 251 of wild-type human Neu2 (D251); (ff) Substitution of glutamate residue at position 257 of wild-type human Neu2 (E257); (gg) Wild-type human Ne Substitution of a serine residue at position 258 of u2 (S258); (hh) Substitution of a leucine residue at position 260 of wild-type human Neu2 (L260); (ii) Substitution of a valine residue at position 265 of wild-type human Neu2 (V265); (jj) Substitution of a glutamine residue at position 270 of wild-type human Neu2 (Q270); (kk) Substitution of a tryptophan residue at position 292 of wild-type human Neu2 (W2 92);(ll) Substitution of a serine residue at the position corresponding to position 301 of wild-type human Neu2 (S301);(mm) Substitution of a tryptophan residue at the position corresponding to position 302 of wild-type human Neu2 (W302);(nn) Substitution of a valine residue at the position corresponding to position 363 of wild-type human Neu2 (V363); or(oo) Substitution of a leucine residue at the position corresponding to position 365 of wild-type human Neu2 (L365); or any combination of the above substitutions. For example, sialidase may contain substitutions of K9, P62, A93, Q216, A242, Q270, S301, W302, V363, or L365, or any combination of the above substitutions.

[0110] In one embodiment, in sialidase: (a) the proline residue at the position corresponding to position 5 of wild-type human Neu2 is replaced with histidine (P5H); (b) the lysine residue at the position corresponding to position 9 of wild-type human Neu2 is replaced with aspartic acid (K9D); (c) the lysine residue at the position corresponding to position 44 of wild-type human Neu2 is replaced with arginine (K44R) or glutamic acid (K44E); (d) the lysine residue at the position corresponding to position 45 of wild-type human Neu2 is replaced with alanine (K45A), arginine (K45R), or glutamic acid (K45A). E) is substituted with; (e) the leucine residue at the position corresponding to position 54 of wild-type human Neu2 is substituted with methionine (L54M); (f) the proline residue at the position corresponding to position 62 of wild-type human Neu2 is substituted with asparagine (P62N), aspartic acid (P62D), histidine (P62H), glutamic acid (P62E), glycine (P62G), serine (P62S), or threonine (P62T); (g) the glutamine residue at the position corresponding to position 69 of wild-type human Neu2 is substituted with histidine (Q69H); (h) the 7 (i) The arginine residue at position 8 is replaced with lysine (R78K); (j) The aspartic acid residue at position 80 of wild-type human Neu2 is replaced with proline (D80P); (k) The alanine residue at position 93 of wild-type human Neu2 is replaced with glutamic acid (A93E) or lysine (A93K); (g) The glycine residue at position 107 of wild-type human Neu2 is replaced with aspartic acid (G107D); (l) The glutamine residue at position 108 of wild-type human Neu2 is replaced with histidine (Q108H (m) The glutamine residue at the position corresponding to position 112 of wild-type human Neu2 is replaced with arginine (Q112R) or lysine (Q112K); (n) The cysteine ​​residue at the position corresponding to position 125 of wild-type human Neu2 is replaced with leucine (C125L); (o) The glutamine residue at the position corresponding to position 126 of wild-type human Neu2 is replaced with leucine (Q126L), glutamic acid (Q126E), phenylalanine (Q126F), histidine (Q126H), isoleucine (Q126I) or tyrosine (Q126Y);(p) The alanine residue at position 150 of wild-type human Neu2 is replaced with valine (A150V); (q) The cysteine ​​residue at position 164 of wild-type human Neu2 is replaced with glycine (C164G); (r) The arginine residue at position 170 of wild-type human Neu2 is replaced with proline (R170P); (s) The alanine residue at position 171 of wild-type human Neu2 is replaced with glycine (A171G); (t) The alanine residue at position 171 of wild-type human Neu2 is replaced with glycine (A171G); (u) The glutamine residue at position 188 is replaced with proline (Q188P); (v) The arginine residue at position 189 of wild-type human Neu2 is replaced with proline (R189P); (v) The alanine residue at position 213 of wild-type human Neu2 is replaced with cysteine ​​(A213C), asparagine (A213N), serine (A213S), or threonine (A213T); (w) The leucine residue at position 217 of wild-type human Neu2 is Substituted with alanine (L217A) or valine (L217V); (x) The threonine residue at the position corresponding to position 249 of wild-type human Neu2 is substituted with alanine (T249A); (y) The aspartic acid residue at the position corresponding to position 251 of wild-type human Neu2 is substituted with glycine (D251G); (z) The glutamic acid residue at the position corresponding to position 225 of wild-type human Neu2 is substituted with proline (E225P); (aa) The position corresponding to position 239 of wild-type human Neu2 (bb) The histidine residue is substituted with proline (H239P); (bb) The leucine residue at the position corresponding to position 240 of wild-type human Neu2 is substituted with aspartic acid (L240D), asparagine (L240N), or tyrosine (L240Y); (cc) The arginine residue at the position corresponding to position 241 of wild-type human Neu2 is substituted with alanine (R241A), aspartic acid (R241D), leucine (R241L), glutamine (R241Q), or tyrosine (R241Y);(dd) The alanine residues at position 242 of wild-type human Neu2 are cysteine ​​(A242C), phenylalanine (A242F), glycine (A242G), histidine (A242H), isoleucine (A242I), lysine (A242K), leucine (A242L), methionine (A242M), asparagine (A242N), glutamine (A242Q), arginine (A242R), and serine (A (242S), valine (A242V), tryptophan (A242W), or tyrosine (A242Y) is substituted; (ee) The valine residue at position 244 of wild-type human Neu2 is substituted with isoleucine (V244I), lysine (V244K), or proline (V244P); (ff) The glutamate residue at position 257 of wild-type human Neu2 is substituted with proline (E257P); ( (gg) The serine residue at position 258 is replaced with cysteine ​​(S258C); (hh) The leucine residue at position 260 of wild-type human Neu2 is replaced with aspartic acid (L260D), phenylalanine (L260F), glutamine (L260Q), or threonine (L260T); (ii) The valine residue at position 265 of wild-type human Neu2 is replaced with phenylalanine (V26 (5F) is substituted; (jj) the glutamine residue at position 270 of wild-type human Neu2 is substituted with alanine (Q270A), histidine (Q270H), phenylalanine (Q270F), proline (Q270P), serine (Q270S), or threonine (Q270T); (kk) the tryptophan residue at position 292 of wild-type human Neu2 is substituted with arginine (W292R);(ll) The serine residues at position 301 in wild-type human Neu2 are alanine (S301A), aspartic acid (S301D), glutamic acid (S301E), phenylalanine (S301F), glycine (S301G), histidine (S301H), isoleucine (S301I), lysine (S301K), leucine (S301L), methionine (S301M), asparagine (S301N), and proline (S (301P), glutamine (S301Q), arginine (S301R), threonine (S301T), valine (S301V), tryptophan (S301W), or tyrosine (S301Y) are substituted; (mm) The tryptophan residue at the position corresponding to position 302 in wild-type human Neu2 is alanine (W302A), aspartic acid (W302D), glutamic acid (W302E), or phenylalanine (W302F). , substituted with glycine (W302G), histidine (W302H), isoleucine (W302I), lysine (W302K), leucine (W302L), methionine (W302M), asparagine (W302N), proline (W302P), glutamine (W302Q), arginine (W302R), serine (W302S), threonine (W302T), valine (W302V), or tyrosine (W302Y); (n n) The valine residue at position 363 of wild-type human Neu2 is replaced with arginine (V363R); or (oo) The leucine residue at position 365 of wild-type human Neu2 is replaced with glutamine (L365Q), histidine (L365H), isoleucine (L365I), lysine (L365K), or serine (L365S); or the sialidase contains any combination of the aforementioned substitutions. For example, sialidase may contain substitutions selected from K9D, P62G, P62N, P62S, P62T, D80P, A93E, Q126H, Q126Y, R189P, H239P, A242T, Q270A, Q270S, Q270T, S301A, S301R, W302K, W302R, V363R, and L365I or any combination of the aforementioned substitutions.

[0111] In one embodiment, recombinant mutant human sialyidase includes a deletion of a leucine residue at position 184 of wild-type human Neu2 (ΔL184), a deletion of a histidine residue at position 185 of wild-type human Neu2 (ΔH185), a deletion of a proline residue at position 186 of wild-type human Neu2 (ΔP186), a deletion of an isoleucine residue at position 187 of wild-type human Neu2 (ΔI187), and a deletion of a glutamine residue at position 184 of wild-type human Neu2 (ΔQ188), or any combination of the aforementioned deletions.

[0112] In one embodiment, recombinant mutant human sialidase includes an insertion between a threonine residue at position 216 of wild-type human Neu2 and a leucine residue at position 217 of wild-type human Neu2, for example, an insertion of an amino acid selected from S, T, Y, L, F, A, P, V, I, N, D, and H.

[0113] Further exemplary sialidase mutations and combinations of sialidase mutations are described, for example, in the section titled "I. Recombinant Human Sialidase" in the Detailed Description and in Examples 1, 2, 3, 4, 5 and 6 in the Examples, in the International (PCT) Patent Application No. PCT / US2019 / 012207, filed on 3 January 2019.

[0114] v. Combinations of substitutions The present invention further provides recombinant mutant human sialidases comprising any combination of the mutations intended herein. For example, a recombinant mutant sialidase enzyme may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more combinations of the mutations intended herein. A recombinant mutant sialidase enzyme may comprise 1-15, 1-10, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-15, 2-10, 2-7, 2-6, 2-5, 2-4, 2-3, 3-15, 3-10, 3-7, 3-6, 3-5, or 3-4 of the mutations intended herein.

[0115] For example, recombinant mutant sialidase enzymes may include M1 deletion (ΔM1), M1A substitution, M1D substitution, V6Y substitution, K9D substitution, P62G substitution, P62N substitution, P62S substitution, P62T substitution, A93E substitution, I187K substitution, Q270A substitution, S301R substitution, W302K substitution, C332A substitution, V363R substitution, L365I substitution, or any combination of the above.

[0116] In one embodiment, recombinant mutant sialidase enzymes include M1 deletion (ΔM1), M1A substitution, M1D substitution, V6Y substitution, I187K substitution, C332A substitution, or any combination thereof. For example, recombinant mutant sialidase enzymes include: M1A and V6Y; M1A and I187K; M1A and C332A; M1D and V6Y; M1D and I187K; M1D and C332A; ΔM1 and V6Y; ΔM1 and I187K; ΔM1 and C332A; V6Y and I187K; V6Y and C332A; I187K and C332A; M1A, V6Y and I187K; M1A, V6Y and C332A; M1A, I187K and C332 A; M1D, V6Y and I187K; M1D, V6Y and C332A; M1D, I187K and C332A; ΔM1, V6Y and I187K; ΔM1, V6Y and C332A; ΔM1, I187K and C332A; V6Y, I187K and C332A; M1A, V6Y, I187K and C332A; M1D, V6Y, I187K and C332A; and combinations of mutations selected from ΔM1, V6Y, I187K and C332A.

[0117] In one embodiment, recombinant mutant sialidase enzymes include (i) amino acid substitutions identified in Table 8 or any combination of amino acid substitutions identified in Table 8 and (ii) )MThis includes 1 deletion (ΔM1), M1A substitution, M1D substitution, V6Y substitution, I187K substitution, C332A substitution, or any combination of the above. For example, recombinant mutant sialidase enzymes include (i) amino acid substitutions identified in Table 8 or any combination of amino acid substitutions identified in Table 8, and (ii) M1A and V6Y; M1A and I187K; M1A and C332A; M1D and V6Y; M1D and I187K; M1D and C332A; ΔM1 and V6Y; ΔM1 and I187K; ΔM1 and C332A; V6Y and I187K; V6Y and C332A; I187K and C332A; M1A, V6Y and I187K; M1A, This may include combinations of mutations selected from V6Y and C332A;M1A, I187K and C332A;M1D, V6Y and I187K;M1D, V6Y and C332A;M1D, I187K and C332A;ΔM1, V6Y and I187K;ΔM1, V6Y and C332A;ΔM1, I187K and C332A;V6Y, I187K and C332A;M1A, V6Y, I187K and C332A;M1D, V6Y, I187K and C332A; and ΔM1, V6Y, I187K and C332A.

[0118] In one embodiment, recombinant mutant sialidase enzymes include: (a) substitutions of M1D, V6Y, P62G, A93E, I187K and C332A; (b) substitutions of M1D, V6Y, K9D, A93E, I187K, C332A, V363R and L365I; (c) substitutions of M1D, V6Y, P62N, I187K and C3 (d) Replacement of 32A; (e) Replacement of M1D, V6Y, I187K, Q270A, S301R, W302K and C332A; (f) Replacement of M1D, V6Y, P62S, I187K, Q270A, S301R, W302K and C332A; (f) Replacement of M1D, V6Y, P62T, I187K, Q270A, S301R, (g) Replacement of W302K and C332A; (h) Replacement of M1D, V6Y, P62N, I187K, Q270A, S301R, W302K and C332A; (i) Replacement of M1D, V6Y, P62G, A93E, I187K, S301A, W302R and C332A; (i) Replacement of M1D, V6Y, P62G, A93E (j) Substitution of Q126Y, I187K, Q270T and C332A; or (k) Substitution of M1D, V6Y, P62G, A93E, Q126Y, I187K and C332A; or (k) Substitution of M1D, V6Y, P62G, A93E, Q126Y, I187K, A242F, Q270T and C332A.

[0119] In one embodiment, recombinant mutant human sialidase includes a serine residue substitution (S301) at the position corresponding to position 301 of wild-type human Neu2, combined with a tryptophan residue substitution (W302) at the position corresponding to position 302 of wild-type human Neu2. For example, recombinant mutant human sialidase may include combinations of substitutions corresponding to the combinations of substitutions listed in the columns of Table 8 (amino acid positions corresponding to wild-type human Neu2 (SEQ ID NO: 1)). For example, recombinant mutant human sialidase may include: the substitution of S301K and W302R; the substitution of S301K and W302K; or the substitution of S301A and W302S. [Table 8]

[0120] In one embodiment, recombinant mutant human sialidase contains combinations of substitutions corresponding to the combinations of substitutions listed in the column of Table 9 (amino acid positions corresponding to wild-type human Neu2 (SEQ ID NO: 1)). [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4]

[0121] In one embodiment, the recombinant mutant human sialyidase contains an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of the sequence numbers 48-62, 169-171, or 196.

[0122] In one embodiment, recombinant mutant human sialidase, [Table 10] (Sequence ID: 47) contains the amino acid sequence, where X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val or absent, X2 is Ala or Lys, X3 is Asn or Leu, X4 is Pro or His, X5 is Phe, Trp, Tyr, or Val, X6 is Lys or Asp. X7 is Lys, Arg, or Glu. X8 is Lys, Ala, Arg, or Glu, X9 is Leu or Met, and X 10 is Pro, Asn, Asp, His, Glu, Gly, Ser or Thr, X 11 is Gln or His, X 12is Arg or Lys, and X 13 is Ala, Glu or Lys, and X 14 is Gly or Asp, and X 15 is Gln or His, and X 16 is Gln, Arg or Lys, and X 17 is Ala, Cys, Ile, Ser, Val or Leu, and X 18 is Gln or Leu, and X 19 is Ala or Val, and X 20 is Cys or Gly, and X 21 is Ala or Gly, and X 22 is Arg, Ile or Lys, and X 23 is Ala, Cys, Leu or Val, and X 24 is Leu, Ala or Val, and X 25 is Thr or Ala, and X 26 is Asp or Gly, and X 27 is Glu or Lys, and X 28 is Gln, Ala, His, Phe or Pro, and X 29 is Cys or Val, and X 30 is Trp or Arg, and X 31 is Ser or Arg, and X 32 is Trp or Lys, and X 33 is Lys or Val, and X 34 is Ala, Cys, Ser or Val, and X 35 is Cys, Leu or Val, and X 36 is Val or Arg, and X 37 is Leu, Gln, His, Ile, Lys or Ser, and sialidase comprises at least one mutation relative to wild-type human Neu2 (SEQ ID NO: 1).

[0123] In certain embodiments, the recombinant mutant human sialidase is

Table 11

[0124] In one embodiment, recombinant mutant human sialidase, [Table 12] (Sequence ID: 172) contains the amino acid sequence, where X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val or absent, X2 is Ala or Lys, X3 is Asn or Leu, X4 is Pro or His, X5 is Phe, Trp, Tyr, or Val, X6 is Lys or Asp, X7 is Lys, Arg, or Glu, X8 is Lys, Ala, Arg, or Glu, X9 is Leu or Met, X 10 is Pro, Asn, Asp, His, Glu, Gly, Ser or Thr, X 11 is Gln or His, X 12 is Arg or Lys, X 13 It is Asp or Pro, X 14 is Ala, Glu or Lys, X 15 is Gly or Asp, X 16 is Gln or His, X 17 is Gln, Arg, or Lys, and X 18 is Ala, Cys, Ile, Ser, Val or Leu, X 19 is Gln, Leu, Glu, Phe, His, Ile, Leu or Tyr, X 20 is Ala or Val, X 21 is Cys or Gly, X 22 is Arg or Pro, X 23 is Ala or Gly, X 24 is Arg, Ile or Lys, and X 25 is Gln or Pro, X 26 is Arg or Pro, X 27 is Ala, Cys, Leu or Val, X 28 is Ala, Cys, Asn, Ser or Thr, X 29 is Leu, Ala, or Val, X 30 is Glu or Pro, X 31 His or Pro, X 32 is Leu, Asp, Asn, or Tyr, X33 is Arg, Ala, Asp, Leu, Gln or Tyr, X 34 is Ala, Cys, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Val, Trp or Tyr, X 35 is Val, Ile or Lys, X 36 is Thr or Ala, X 37 is Asp or Gly, X 38 is Glu, Lys or Pro, X 39 is Ser or Cys, and X 40 is Leu, Asp, Phe, Gln or Thr, X 41 is Val or Phe, X 42 is Gln, Ala, His, Phe, Pro, Ser or Thr, X 43 is Cys or Val, X 44 is Trp or Arg, X 45 is Ser, Arg, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Thr, Val, Trp or Tyr, X 46 is Trp, Lys, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Tyr, X 47 is Lys or Val, X 48 is Ala, Cys, Ser or Val, X 49 is Cys, Leu or Val, X 50 is Val or Arg, X 51 The mutants are Leu, Gln, His, Ile, Lys, or Ser, and the sialidase contains at least one mutation compared to wild-type human Neu2 (SEQ ID NO: 1).

[0125] In one embodiment, recombinant mutant human sialidase is [Table 13] The amino acid sequence (SEQ ID NO: 173) contains, where X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val or absent, X2 is Phe, Trp, Tyr, or Val, X3 is Lys or Asp, X4 is Pro, Asn, Asp, His, Glu, Gly, Ser, or Thr, and X5 is Ala X6 is Gln, Leu, Glu, Phe, His, Ile, Leu, or Tyr, X7 is Arg, Ile, or Lys, X8 is Ala, Cys, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Val, Trp, or Tyr, X9 is Gln, Ala, His, Phe, Pro, Ser, or Thr, X 10 is Ser, Arg, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Thr, Val, Trp or Tyr, X 11 is Trp, Lys, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Tyr, X 12 is Ala, Cys, Ser or Val, X 13 is Val or Arg, X 14 X1 is Leu, Gln, His, Ile, Lys, or Ser, and the sialidase contains at least one mutation compared to wild-type human Neu2 (SEQ ID NO: 1). In one embodiment, X1 is Ala, Asp, Met, or absent, X2 is Tyr or Val, X3 is Lys or Asp, X4 is Pro, Asn, Gly, Ser, or Thr, X5 is Ala or Glu, X6 is Gln or Tyr, X7 is Ile or Lys, X8 is Ala or Thr, X9 is Gln, Ala, or Thr, and X 10 is Ser, Arg or Ala, X 11 is Trp, Lys or Arg, and X 12 is Ala or Cys, X 13is Val or Arg, X 14 It is either Leu or Ile.

[0126] In one embodiment, recombinant mutant human sialidases include conserved substitutions to the recombinant mutant human sialidase sequences disclosed herein. As used herein, the term “conserved substitution” means a substitution of structurally similar amino acids. For example, conserved substitutions may include those within the following groups: Ser and Cys; Leu, Ile and Val; Glu and Asp; Lys and Arg; Phe, Tyr and Trp; and those within Gln, Asn, Glu, Asp and His. Conserved substitutions may also be defined by the BLAST (Basic Local Alignment Search Tool) algorithm, the BLOSUM substitution matrix (e.g., the BLOSUM 62 matrix), or the PAM substitution:p matrix (e.g., the PAM 250 matrix).

[0127] Sequence identity can be determined by various methods within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. BLAST (Basic Local Alignment Search Tool) analysis (Karlin et al., (1990) PROC. NATL. ACAD. SCI. USA 87:2264-2268; Altschul, (1993) J. MOL. EVOL. 36, 290-300; Altschul et al., (1997) NUCLEIC ACIDS RES. 25:3389-3402, as incorporated herein by reference) using algorithms employed by the programs blastp, blastn, blastx, tblastn, and tblastx is adapted for searching for sequence similarity. For a discussion of fundamental issues in searching sequence databases, see Altschul et al., (1994) NATURE GENETICS 6:119-129, which is fully referenced herein. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm necessary to achieve the greatest possible alignment over the entire length of the sequences being compared. Search parameters for histogram, description, alignment, expect (i.e., the statistical significance threshold for reporting the fit to the database sequences), cutoff, matrix, and filter are at their default settings. The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et al., (1992) PROC. NATL. ACAD. SCI. USA 89:10915-10919, which is fully referenced herein).The four blastn parameters can be adjusted as follows: Q=10 (gap generation penalty); R=10 (gap extension penalty); wink=1 (generates a word hit at each wink.sup.th position along the query); and gapw=16 (sets the window width at which a gapped alignment is generated). An equivalent blastp parameter setting could be Q=9; R=2; wink=1; and gapw=32. The search can also be performed using NCBI (National Center for Biotechnology Information) BLAST Advanced Option parameters (e.g., -G, Cost for open gaps [integer]: default=5 for nucleotides / 11 for proteins; -E, Cost for extension gaps [integer]: default=2 for nucleotides / 1 for proteins; -q, Penalty for nucleotide mismatches [integer]: default=-3; -r, Reward for nucleotide fit [integer]: default=1; -e, Predicted value [real number]: default=10; -W, Word size [integer]: default=11 for nucleotides / 28 for megablast / 3 for proteins; -y, Drop-off for blast extension in bits (X): default=20 for blastn / 7 for others; -X, X drop-off value for gapped alignments (in bits): default=15 for all programs but not applicable to blastn; and -Z, Final X drop-off value for gapped alignments (in bits): 50 for blastn, 25 for others). ClustalW can also be used for pairwise protein alignment (default parameters may include, for example, a Blosum62 matrix and a gap opening penalty of 10 and a gap elongation penalty of 0.1).In GCG package version 10.0, the optimal fit comparison between sequences uses DNA parameters GAP=50 (gap creation penalty) and LEN=3 (gap elongation penalty). The equivalent settings for optimal protein comparison are GAP=8 and LEN=2.

[0128] II. Serum half-life prolonging factors As used herein, “serum half-life prolonger” refers to a portion of the serum of a subject that can bind to sialidase to prolong its circulating half-life. In one embodiment, serum half-life prolonging factors include Fc domains (e.g., see Beck et al. (2011) MABS 4:1015-28), albumin (e.g., human serum albumin (HSA), see Weimer et al. (2013) Recombinant albumin fusion proteins. In: Schmidt S, editor. Fusion protein technologies for biopharmaceuticals: applications and challenges. Hoboken: Wiley; 2013, pp. 297-323), albumin-binding domains (e.g., HAS binders, see Walker et al. (2013) Albumin-binding fusion proteins in the development of novel long-acting therapeutics. In: Schmidt S, editor. Fusion protein technologies for biopharmaceuticals: applications and challenges. Hoboken: Wiley; 2013, pp. 325-43), and transferrin (Kim et al. (2010) J PHARMACOL EXP See THER 334:682-92), XTEN (also called recombinant PEG or "rPEG," see Schellenberger et al. (2009) NAT. BIOTECHNOL. 27:1186-90), homoamino acid polymer (HAP, see Schlapschy et al. (2007) PROTEIN ENG DES SEL. 20:273-84), proline-alanine-serine polymer (PAS, see Schlapschy et al. (2013) PROTEIN ENG DES SEL. 26:489-501), elastin-like peptide (ELP, see Floss et al.(See 2013) Fusion protein technologies for biopharmaceuticals: applications and challenges, pp. 372-98), carboxy-terminal peptides (CTP, Duijkers et al. (2002) HUM REPROD. 17:1987-93), gelatin-like proteins (GLK, Huang et al. (2010) EUR J PHARM BIOPHARM 72:435-41), and polyethylene glycol (PEG) can be selected.

[0129] Suitable serum half-life extenders also include a variety of polymers, such as those described in U.S. Patent No. 7,842,789. For example, block copolymers of polyoxyethylene and polyoxypropylene (Pluronics); polymethacrylates; carbomers; and branched or unbranched polysaccharides containing sugar monomers such as D-mannose, D- and L-galactose, fucose, fructose, D-xylose, L-arabinose, as well as D-glucuronic acid may be used. In other embodiments, serum half-life extenders may be hydrophilic polyvinyl polymers such as polyvinyl alcohol and polyvinylpyrrolidone (PVP) type polymers. Serum half-life extenders may be functionalized polyvinylpyrrolidones, for example, carboxylates or amines functionalized at one (or both) ends of the polymer (available from PolymerSource). Alternatively, serum half-life prolongers may include poly(N-(2-hydroxypropyl)methacrylamide (HPMA), or functionalized HPMA (amine, carboxy, etc.), poly(N-isopropylacrylamide), or functionalized poly(N-isopropylacrylamide).

[0130] In one embodiment, sialidase covalently binds to a naturally occurring long-half-life polypeptide or protein such as an Fc domain (Beck et al., see above), transferrin (Kim et al., see above), or albumin (Weimer et al., see above) to form a fusion protein by either genetic fusion (i.e., creation of a recombinant fusion protein) or chemical conjugation.

[0131] In another embodiment, sialidase covalently binds to inactive polypeptides such as XTEN (also referred to as recombinant PEG or "rPEG," see Schellenberger, above), homoamino acid polymers (HAP, see Schlapschy et al. (2007), above), proline-alanine-serine polymers (PAS, see Schlapschy et al., (2013), above), elastin-like peptides (ELP, see Floss et al., above), or gelatin-like proteins (GLK, see Huang et al., above) to form fusion proteins either by genetic fusion (i.e., creation of recombinant fusion proteins) or chemical conjugate. The inactive polypeptide functions to increase the size and hydrodynamic radius of the sialidase, thereby extending its half-life. In one aspect, XTEN polypeptide contains approximately 25 to 1500 amino acids (for example, approximately 25 to 100 amino acids, approximately 25 to 250 amino acids, approximately 25 to 500 amino acids, approximately 25 to 750 amino acids, approximately 25 to 1000 amino acids, approximately 25 to 1250 amino acids, approximately 100 to 250 amino acids, approximately 100 to 250 amino acids, approximately 100 to 500 amino acids, approximately 100 to 750 amino acids, approximately 100 to 1000 amino acids, approximately 100 to 1250 amino acids, and approximately 100 to 1500 amino acids). They have lengths of approximately 250 to 1250 amino acids, approximately 250 to 1000 amino acids, approximately 250 to 750 amino acids, approximately 250 to 500 amino acids, approximately 500 to 750 amino acids, approximately 500 to 1000 amino acids, approximately 500 to 1250 amino acids, approximately 500 to 1500 amino acids, approximately 750 to 1000 amino acids, approximately 750 to 1250 amino acids, approximately 750 to 1500 amino acids, approximately 1000 to 1250 amino acids, approximately 1000 to 1500 amino acids, or approximately 1250 to 1500 amino acids.

[0132] In one embodiment, sialidase chemically conjugates itself to repeating chemical moieties such as PEG or hyaluronic acid (see Mero et al. (2013) CARB POLYMERS 92:2163-70), increasing the hydrodynamic radius of the sialidase and thereby extending its half-life.

[0133] In another embodiment, sialidases are either polysialized themselves or covalently bonded to negatively charged, highly sialized proteins (e.g., carboxy-terminal peptides (CTPs) of chorionic gonadotropin (CG) β-chains, see Duijkers et al. (2002) HUM REPROD 17:1987-93).

[0134] Methods for producing and using the aforementioned serum half-life prolonging factors are known in the art. See, for example, Strohl (2015) BIODRUGS 29:215-239.

[0135] In one embodiment, sialidase is conjugated to a serum half-life prolonging factor that is not an Fc domain and / or PEG.

[0136] It is intended that one or more sialidases can covalently bind to one or more (e.g., 2, 3, 4, 5, 6, 8, 9, 10 or more) serum half-life prolongers.

[0137] In one embodiment, the serum half-life of the sialidase enzyme conjugated with a serum half-life enhancer is at least 24, 36, 48, or 60 hours.

[0138] Generally, serum half-life prolonging factors are approximately 2kDa to 5kDa, 2kDa to 10kDa, 2kDa to 20kDa, 2kDa to 30kDa, 2kDa to 40kDa, 2kDa to 50kDa, 2kDa to 60kDa, 2kDa to 70kDa, 2kDa to 80kDa, 2kDa to 90kDa, 2kDa to 100kDa, 2kDa to 150kDa, 5kDa to 10kDa, 5kDa to 20kDa, 5kDa to 30kDa, 5kDa to 40kDa, 5kDa to 50kDa, and 5kDa to 60kDa. kDa, about 5kDa to about 70kDa, about 5kDa to about 80kDa, about 5kDa to about 90kDa, about 5kDa to about 100kDa, about 5kDa to about 150kDa, about 10kDa to about 20kDa, about 10kDa to about 30kDa, about 10kDa to about 40kDa, about 10kDa to about 50 kDa, about 10kDa to about 60kDa, about 10kDa to about 70kDa, about 10kDa to about 80kDa, about 10kDa to about 90kDa, about 10kDa to about 100kDa, about 10kDa to about 150kDa, about 20kDa to about 30kDa, about 20kDa to about 40kDa, about 20kDa ~50kDa, 20kDa~60kDa, 20kDa~70kDa, 20kDa~80kDa, 20kDa~90kDa, 20kDa~100kDa, 20kDa~150kDa, 30kDa~40kDa, 30kDa~50kDa, approx. 30kDa to about 60kDa, about 30kDa to about 70kDa, about 30kDa to about 80kDa, about 30kDa to about 90kDa, about 30kDa to about 100kDa, about 30kDa to about 150kDa, about 40kDa to about 50kDa, about 40kDa to about 60kDa, about 40kDa to about 70k Da, approximately 40kDa to 80kDa, approximately 40kDa to 90kDa, approximately 40kDa to 100kDa, approximately 40kDa to 150kDa, approximately 50kDa to 60kDa, approximately 50kDa to 70kDa, approximately 50kDa to 80kDa, approximately 50kDa to 90kDa, approximately 50kDa ~100kDa, 50kDa~150kDa, 60kDa~70kDa, 60kDa~80kDa, 60kDa~90kDa, 60kDa~100kDa, 60kDa~150kDa, 70kDa~80kDa, 70kDa~90kDaIt may have a molecular weight of approximately 70kDa to 100kDa, approximately 70kDa to 150kDa, approximately 80kDa to 90kDa, approximately 80kDa to 100kDa, approximately 80kDa to 150kDa, approximately 90kDa to 100kDa, approximately 90kDa to 150kDa, or approximately 100kDa to 150kDa.

[0139] a. Fc domain In one embodiment, the fusion protein includes an immunoglobulin Fc domain. As used herein, unless otherwise indicated, the terms “immunoglobulin Fc domain,” “Fc domain,” or “Fc” refer to a fragment of the constant region of an immunoglobulin heavy chain that can bind to an Fc receptor, either alone or in combination with a second immunoglobulin Fc domain, or either conjugated to or without conjugated to a sialidase. An immunoglobulin Fc domain may include, for example, immunoglobulin CH2 and CH3 domains. An immunoglobulin Fc domain may include, for example, immunoglobulin CH2 and CH3 domains and an immunoglobulin hinge region. The boundary between the immunoglobulin hinge region, the CH2 and CH3 domains is well known in the art and can be found, for example, in the PROSITE database (prosite.expasy.org, available on the World Wide Web).

[0140] Figures 1A-E show an embodiment of a sialidase-Fc fusion construct comprising a first polypeptide containing a first immunoglobulin Fc domain and a second polypeptide containing a second immunoglobulin Fc domain. The first and second polypeptides may be covalently bonded to each other. The covalent bond may be a disulfide bond. The sialidase enzyme may be conjugated to the N or C terminus of the first immunoglobulin Fc domain or to the N or C terminus of the second immunoglobulin Fc domain. Any second sialidase enzyme may be conjugated to the N or C terminus of the first immunoglobulin Fc domain or to the N or C terminus of the second immunoglobulin Fc domain.

[0141] Figure 1A shows a construct having two Fc domains and sialidase enzymes conjugated to the N-terminus of each Fc domain. Figure 1B shows a construct having two Fc domains and sialidase enzymes conjugated to the C-terminus of the first Fc domain and the N-terminus of the second Fc domain. Figure 1C shows a construct having two Fc domains and sialidase enzymes conjugated to the N-terminus of the second Fc domain. Figure 1D shows a construct having two Fc domains and sialidase enzymes conjugated to the C-terminus of the first Fc domain. Figure 1E shows a construct having two Fc domains and sialidase enzymes conjugated to the C-terminus of each Fc domain. It is understood that the Fc domains may be naturally occurring Fc domains or genetically engineered Fc domains, including modifications such as point mutations within the respective polypeptide chain, that facilitate knob-in-hole construction or provide modified Fc domain functionality.

[0142] In one embodiment, the immunoglobulin Fc domain is derived from the human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM Fc domains. A single amino acid substitution (indicated as S228P;IgG4Pro according to Kabat numbering) may be introduced to disrupt the heterogeneity observed in recombinant IgG4 antibodies. See Angal, S. et al. (1993) MOL. IMMUNOL. 30:105-108.

[0143] In one embodiment, the immunoglobulin Fc domain is derived from a human IgG1 isotype or another isotype that induces antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-mediated cytotoxicity (CDC). In another embodiment, the immunoglobulin Fc domain is derived from a human IgG1 isotype (e.g., SEQ ID NO: 31 or SEQ ID NO: 69).

[0144] In one embodiment, the immunoglobulin Fc domain is derived from a human IgG4 isotype or another isotype that does little to no induce antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-mediated cytotoxicity (CDC). In one embodiment, the immunoglobulin Fc domain is derived from a human IgG4 isotype.

[0145] In one embodiment, the immunoglobulin Fc domain contains either a "knob" mutation, e.g., T366Y, or a "hole" mutation, e.g., Y407T (residue number according to EU numbering, Kabat, EA, et al. (1991) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, FIFTH EDITION, US Department of Health and Human Services, NIH Publication No. 91-3242) for heterodimerization with the second polypeptide. In one embodiment comprising a sialidase-Fc fusion having two Fc domains, the first Fc domain may contain a "knob" mutation (e.g., SEQ ID NO: 33 and SEQ ID NO: 148), and the second Fc domain may contain a "hole" mutation (e.g., SEQ ID NO: 32 and SEQ ID NO: 147).

[0146] In one embodiment, the sialidase-Fc fusion protein includes an amino acid sequence of any of SEQ ID NOs: 129-158, 177-192, and 197-200, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 129-158, 177-192, and 197-200.

[0147] In one embodiment, the sialidase-Fc fusion protein is [Table 14-1] [Table 14-2] Comprising the amino acid sequence of (sequence number: 159), where X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val or absent, X2 is Ala or Lys, X3 is Asn or Leu, X4 is Pro or His, X5 is Phe, Trp, Tyr or Val, X6 is Lys or Asp. X7 is Lys, Arg or Glu. X8 is Lys, Ala, Arg or Glu, X9 is Leu or Met, X 10 is Pro, Asn, Asp, His, Glu, Gly, Ser or Thr, X 11 is Gln or His, X 12 is Arg or Lys, X 13 is Ala, Glu or Lys, X 14 is Gly or Asp, X 15 is Gln or His, X 16 is Gln, Arg or Lys, X 17 is Ala, Cys, Ile, Ser, Val or Leu, X 18 is Gln or Leu, X 19 is Ala or Val, X 20 is Cys or Gly, X 21 is Ala or Gly, X 22 is Arg, Ile or Lys, X 23 is Ala, Cys, Leu or Val, X 24 is Leu, Ala or Val, X 25 is Thr or Ala, X 26 is Asp or Gly, X 27 is Glu or Lys, X 28 is Gln, Ala, His, Phe or Pro, X 29 is Cys or Val, X 30 is Trp or Arg, X 31 is Ser or Arg, X 32 is Trp or Lys, X 33is Lys or Val, and X 34 is Ala, Cys, Ser or Val, and X 35 is Cys, Leu or Val 、X 36 is Val or Arg, and X 37 is Leu, Gln, His, Ile, Lys or Ser, and the sialidase contains at least one mutation relative to wild-type human Neu2 (SEQ ID NO: 1).

[0148] In certain embodiments, the sialidase-Fc fusion protein is [Table 15] contains the amino acid sequence of (SEQ ID NO: 160), where X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val or is absent, X2 is Phe, Trp, Tyr or Val, X3 is Lys or Asp, X4 is Pro, Asn, Asp, His, Glu, Gly, Ser or Thr, X5 is Ala, Glu or Lys, X6 is Arg, Ile or Lys, X7 is Gln, Ala, His, Phe or Pro, X8 is Ser or Arg, X9 is Trp or Lys, and X 10 is Ala, Cys, Ser or Val, and X 11 is Val or Arg, and X 12 is Leu, Gln, His, Ile, Lys or Ser, and the sialidase contains at least one mutation relative to wild-type human Neu2 (SEQ ID NO: 1). In certain embodiments, X1 is Ala, Asp, Met or is absent, X2 is Tyr or Val, X3 is Lys or Asp, X4 is Pro, Asn, Gly, Ser or Thr, X5 is Ala or Glu, X6 is Ile or Lys, X7 is Gln or Ala, X8 is Ser or Arg, X9 is Trp or Lys, and X 10 is Ala or Cys, and X 11is Val or Arg, X 12 It is either Leu or Ile.

[0149] In one embodiment, the sialidase-Fc fusion protein is [Table 16] (Sequence ID: 161) contains the amino acid sequence, where X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val or absent, X2 is Ala or Lys, X3 is Asn or Leu, X4 is Pro or His, X5 is Phe, Trp, Tyr, or Val, X6 is Lys or Asp. X7 is Lys, Arg, or Glu. X8 is Lys, Ala, Arg, or Glu, X9 is Leu or Met, and X 10 is Pro, Asn, Asp, His, Glu, Gly, Ser or Thr, X 11 is Gln or His, X 12 is Arg or Lys, X 13 is Ala, Glu or Lys, X 14 is Gly or Asp, X 15 is Gln or His, X 16 is Gln, Arg, or Lys, and X 17 is Ala, Cys, Ile, Ser, Val or Leu, X 18 is Gln or Leu, X 19 is Ala or Val, X 20 is Cys or Gly, X 21 is Ala or Gly, X 22 is Arg, Ile or Lys, and X 23 is Ala, Cys, Leu or Val, X 24 is Leu, Ala, or Val, X 25 is Thr or Ala, X 26 is Asp or Gly, X 27 is Glu or Lys, X 28is Gln, Ala, His, Phe or Pro, X 29 is Cys or Val, X 30 is Trp or Arg, X 31 is either Ser or Arg, and X 32 is Trp or Lys, X 33 is Lys or Val, X 34 is Ala, Cys, Ser or Val, X 35 is Cys, Leu or Val, X 36 is Val or Arg, X 37 is Leu, Gln, His, Ile, Lys or Ser, X 38 The strain is either GGGGSGGGGS (SEQ ID NO: 162) or EPKSS (SEQ ID NO: 163), and the sialidase contains at least one mutation compared to wild-type human Neu2 (SEQ ID NO: 1).

[0150] In one embodiment, the sialidase-Fc fusion protein is [Table 17] (Sequence ID: 164) contains the amino acid sequence, where X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val or absent, X2 is Phe, Trp, Tyr, or Val, X3 is Lys or Asp, X4 is Pro, Asn, Asp, His, Glu, Gly, Ser, or Thr, X5 is Ala, Glu, or Lys, X6 is Arg, Ile, or Lys, X7 is Gln, Ala, His, Phe, or Pro, X8 is Ser, or Arg, X9 is Trp, or Lys, X 10 is Ala, Cys, Ser or Val, X 11 is Val or Arg, X 12 is Leu, Gln, His, Ile, Lys or Ser, X 13X is GGGGSGGGGS (SEQ ID NO: 162) or EPKSS (SEQ ID NO: 163), and the sialidase contains at least one mutation compared to wild-type human Neu2 (SEQ ID NO: 1). In one embodiment, X1 is Ala, Asp, Met or absent, X2 is Tyr or Val, X3 is Lys or Asp, X4 is Pro, Asn, Gly, Ser or Thr, X5 is Ala or Glu, X6 is Ile or Lys, X7 is Gln or Ala, X8 is Ser or Arg, X9 is Trp or Lys, and X 10 is Ala or Cys, X 11 is Val or Arg, X 12 It is either Leu or Ile.

[0151] In one embodiment, the sialidase-Fc fusion protein is [Table 18] (Sequence ID: 165) contains the amino acid sequence, where X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val or absent, X2 is Ala or Lys, X3 is Asn or Leu, X4 is Pro or His, X5 is Phe, Trp, Tyr, or Val, X6 is Lys or Asp, X7 is Lys, Arg, or Glu, X8 is Lys, Ala, Arg, or Glu, X9 is Leu or Met, X 10 is Pro, Asn, Asp, His, Glu, Gly, Ser or Thr, X 11 is Gln or His, X 12 is Arg or Lys, X 13 It is Asp or Pro, X 14 is Ala, Glu or Lys, X 15 is Gly or Asp, X 16 is Gln or His, X 17 is Gln, Arg, or Lys, and X 18is Ala, Cys, Ile, Ser, Val or Leu, X 19 is Gln, Leu, Glu, Phe, His, Ile, Leu or Tyr, X 20 is Ala or Val, X 21 is Cys or Gly, X 22 is Arg or Pro, X 23 is Ala or Gly, X 24 is Arg, Ile or Lys, and X 25 is Gln or Pro, X 26 is Arg or Pro, X 27 is Ala, Cys, Leu or Val, X 28 is Ala, Cys, Asn, Ser or Thr, X 29 is Leu, Ala, or Val, X 30 is Glu or Pro, X 31 His or Pro, X 32 is Leu, Asp, Asn, or Tyr, X 33 is Arg, Ala, Asp, Leu, Gln or Tyr, X 34 is Ala, Cys, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Val, Trp or Tyr, X 35 is Val, Ile or Lys, X 36 is Thr or Ala, X 37 is Asp or Gly, X 38 is Glu, Lys or Pro, X 39 is Ser or Cys, and X 40 is Leu, Asp, Phe, Gln or Thr, X 41 is Val or Phe, X 42 is Gln, Ala, His, Phe, Pro, Ser or Thr, X 43 is Cys or Val, X 44 is Trp or Arg, X 45is Ser, Arg, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Thr, Val, Trp or Tyr, X 46 is Trp, Lys, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Tyr, X 47 is Lys or Val, X 48 is Ala, Cys, Ser or Val, X 49 is Cys, Leu or Val, X 50 is Val or Arg, X 51 is Leu, Gln, His, Ile, Lys or Ser, X 52 These are GGGGS (SEQ ID NO: 174), GGGGSGGGGS (SEQ ID NO: 162), or EPKSS (SEQ ID NO: 163), and the sialidase contains at least one mutation compared to wild-type human Neu2 (SEQ ID NO: 1).

[0152] In one embodiment, the sialidase-Fc fusion protein is [Table 19] The amino acid sequence (SEQ ID NO: 166) contains, where X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val or absent, X2 is Phe, Trp, Tyr, or Val, X3 is Lys or Asp, X4 is Pro, Asn, Asp, His, Glu, Gly, Ser, or Thr, and X5 is Ala X6 is Gln, Leu, Glu, Phe, His, Ile, Leu, or Tyr, X7 is Arg, Ile, or Lys, X8 is Ala, Cys, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Val, Trp, or Tyr, X9 is Gln, Ala, His, Phe, Pro, Ser, or Thr, X 10is Ser, Arg, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Thr, Val, Trp or Tyr, X 11 is Trp, Lys, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Tyr, X 12 is Ala, Cys, Ser or Val, X 13 is Val or Arg, X 14 is Leu, Gln, His, Ile, Lys or Ser, X 15 X is GGGGS (SEQ ID NO: 184), GGGGSGGGGS (SEQ ID NO: 162), or EPKSS (SEQ ID NO: 163), and the sialidase contains at least one mutation compared to wild-type human Neu2 (SEQ ID NO: 1). In one embodiment, X1 is Ala, Asp, Met, or absent, X2 is Tyr or Val, X3 is Lys or Asp, X4 is Pro, Asn, Gly, Ser, or Thr, X5 is Ala or Glu, X6 is Gln or Tyr, X7 is Ile or Lys, X8 is Ala or Thr, X9 is Gln, Ala, or Thr, and X 10 is Ser, Arg or Ala, X 11 is Trp, Lys or Arg, and X 12 is Ala or Cys, X 13 is Val or Arg, X 14 It is either Leu or Ile.

[0153] b. Polyethylene glycol (PEG) In one embodiment, the serum half-life extender is polyethylene glycol (PEG) and its derivatives (e.g., alkoxy polyethylene glycol, e.g., methoxy polyethylene glycol, ethoxy polyethylene glycol, etc.). In one embodiment, the sialidase described herein is covalently bound to at least one PEG having an actual MW of at least about 20,000 D. In another embodiment, the sialidase is covalently bound to at least one PEG having an actual MW of at least about 30,000 D. In yet another embodiment, the sialidase is covalently bound to at least one PEG having an actual MW of at least about 40,000 D. In one embodiment, the PEG is methoxyPEG(5000)-succinimidylpropionate (mPEG-SPA) or methoxyPEG(5000)-succinimidyl succinate (mPEG-SS). Such PEGs are commercially available from Nektar Therapeutics or SunBiowest or LaysanBio or NOF. In one embodiment, PEG may be branched, Y-shaped, or comb-shaped so that it can be used from JenKem USA or NOF, or it may be synthesized by coupling two or more PEGs to a small molecule such as glutamic acid.

[0154] The ω-position of PEG may contain a hydroxyl group or a methoxy group, and PEG may also contain an amino group at the ω-position. Such amino groups can then be coupled to various drugs. In another embodiment of the present invention, the biological modifier may be pegylated poly-L-lysine or pegylated poly-D-lysine.

[0155] The binding sites on sialidases for PEG or its derivatives include the N-terminal amino group and ε-amino group found on lysine residues, as well as other amino, imino, carboxyl, sulfhydryl, hydroxyl, or other hydrophilic groups. PEG can be directly covalently bound to sialidases using chemistry and with or without known use of polyfunctional (usually bifunctional) crosslinking agents used in the art. For example, PEG variants can be conjugated to sialidases by using a thiol-reactive crosslinking linker and then reacting it with thiol groups on PEG. In some embodiments, sulfhydryl groups can be derivatized by coupling to maleimide-substituted PEG (e.g., alkoxy-PEGamine + sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) or PEG-maleimide commercially available from Shearwater Polymers, Inc., Huntsville, Ala.).

[0156] c. Human serum albumin (HSA) and HSA binders Human serum albumin (HSA) (molecular mass approximately 67 kDa) is the most abundant protein in plasma, present at approximately 50 mg / mL (600 μM), and has a half-life of approximately 20 days in humans. HSA plays a role in maintaining plasma pH, contributing to colloidal blood pressure, acting as a carrier for many metabolites and fatty acids, and serving as a major drug transport protein in plasma.

[0157] In one embodiment, the serum half-life prolonger is human serum albumin (HSA) or HSA-binding peptide (see, e.g., PCT publication numbers WO2013128027A1 and WO2014140358A1). Neonatal Fc receptors (FcRn) appear to be involved in prolonging the lifespan of circulating albumin (see Chaudhury et al. (2003) J. EXP. MED., 3: 315-22). Albumin and IgG bind non-cooperatively to other sites on FcRn to form a tri-molecular molecule (see above). The binding of human FcRn to HAS and human IgG is pH-dependent, being stronger at acidic pH and weaker at neutral or physiological pH (see above). This observation suggests that protein and albumin-containing protein complexes, as well as those containing IgG (particularly Fc), are protected from degradation via pH-sensitive interactions with FcRn (see above). Using surface plasmon resonance (SPR) to measure the ability of individual HSA domains to bind to immobilized soluble human FcRn, it was shown that FcRn and albumin interact in a pH-dependent manner via the D-III domain of albumin, at a site different from the IgG binding site (see Chaudhury et al. (2006) BIOCHEM. 45:4983-90 and PCT publication number WO2008068280A1).

[0158] Exemplary HSA-binding proteins are known in the art. For example, U.S. Patent Application Publication No. US20130316952A1 discloses a polypeptide that binds to serum albumin having the amino acid sequence LKEAKEKAIEELKKAGITSDYYFDLINKAKTVEGVNALKDEILKA (SEQ ID NO: 109). Further exemplary HSA-binding polypeptides are described in Dennis et al. (2002) J. BIOL. CHEM., 277: 35035-43; Jacobs et al. (2015) PROTEIN ENG. DES. SEL., 28: 385-93; and Zorzi et al. (2017) NAT. COMMUN., 8: 16092.

[0159] III. Linker In certain embodiments, sialidase can be directly linked or fused to a serum half-life extension factor. In other embodiments, sialidase can be covalently linked to a serum half-life extension factor via a linker.

[0160] The linker can couple to one or more natural amino acids, sialidase or a functional fragment thereof, and a serum half-life extension factor, where one or more natural amino acids (e.g., cysteine amino acids) can be introduced by site-directed mutagenesis. The linker can contain one or more unnatural amino acids. In certain situations, for example, a linker containing one or more sulfhydryl-reactive groups (e.g., maleimide) is contemplated to covalently link to a cysteine that is a naturally occurring cysteine residue in sialidase or a serum half-life extension factor or is the product of site-specific mutagenesis.

[0161] The linker can be a cleavable linker or a non-cleavable linker. Optionally or additionally, the linker can be a flexible linker or a non-flexible linker.

[0162] The linker should be long enough for the sialidase and the serum half-life extension factor to link to each other without steric hindrance and short enough to retain the intended activity of the fusion protein. The linker is preferably hydrophilic enough to avoid or minimize the instability of the fusion protein. The linker is preferably hydrophilic enough to avoid or minimize the insolubility of the fusion protein. The linker should be stable enough in vivo (e.g., it is not cleaved by serum, enzymes, etc.) to allow the fusion protein to be effective in vivo.

[0163] Linkers can be approximately 1 angstrom (Å) to approximately 150 Å in length, or approximately 1 Å to approximately 120 Å in length, or approximately 5 Å to approximately 110 Å in length, or approximately 10 Å to approximately 100 Å in length. Linkers can be greater than approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 27, 30 or greater angstrom lengths and / or less than approximately 110, 100, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31 or less Å lengths. Furthermore, the linker can be approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, and 120 Å in length.

[0164] In one embodiment, the linker comprises a polypeptide linker that links or fuses sialidase to a serum half-life extender (e.g., Fc domain) of the fusion protein. For example, a gene encoding sialidase linked directly or indirectly (e.g., via amino acids containing the linker) to a serum half-life extender is intended to be generated and expressed using conventional recombinant DNA technology. For example, the amino terminus of sialidase may be linked to the carboxy terminus of a serum half-life extender. When a linker is used, the linker may contain hydrophilic amino acid residues, e.g., Gln, Ser, Gly, Glu, Pro, His, and Arg. In one embodiment, the linker is a peptide containing 1-25 amino acid residues, 1-20 amino acid residues, 2-15 amino acid residues, 3-10 amino acid residues, 3-7 amino acid residues, 4-25 amino acid residues, 4-20 amino acid residues, 4-15 amino acid residues, 4-10 amino acid residues, 5-25 amino acid residues, 5-20 amino acid residues, 5-15 amino acid residues, or 5-10 amino acid residues. Examples of linkers include glycine and serine-rich linkers, such as (GlyGlyPro). n (Alternate number: 110) or (GlyGlyGlyGlySer) n(Sequence ID: 111) is an example, where n is 1 to 5. In one embodiment, the linker includes, consists of, or essentially consists of GGGGS (Sequence ID: 174). In one embodiment, the linker includes, consists of, or essentially consists of GGGGSGGGGS (Sequence ID: 162). In one embodiment, the linker includes, consists of, or essentially consists of EPKSS (Sequence ID: 163). Further exemplary linker sequences are disclosed, for example, in George et al. (2003) PROTEIN ENGINEERING 15:871-879 and U.S. Patents 5,482,858 and 5,525,491.

[0165] IV. Method for producing sialidase conjugated with sialidase and / or serum half-life enhancer. Methods for producing sialidase or sialidase conjugated with serum half-life enhancers, such as those disclosed herein, are known in the art. For example, a DNA molecule encoding a serum half-life enhancer (e.g., an Fc domain) can be synthesized chemically or by recombinant DNA methodologies. For example, the sequence of a serum half-life enhancer can be cloned by conventional hybridization techniques or polymerase chain reaction (PCR) techniques using appropriate synthetic nucleic acid primers. The resulting DNA molecule encoding the protein of interest can be ligated to other suitable nucleotide sequences, such as expression regulatory sequences, to produce a conventional gene expression construct (i.e., an expression vector) encoding the desired serum half-life enhancer. The production of the defined gene construct is within the scope of conventional techniques in the art.

[0166] The nucleic acid encoding the desired sialidase can be incorporated into an expression vector (ligated), which can then be introduced into host cells by conventional transfection or transformation techniques. Exemplary host cells include Escherichia coli cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK 293) cells, HeLa cells, infant hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells that do not otherwise produce IgG protein. Transformed host cells can be grown under conditions that allow the host cells to express the sialidase.

[0167] Specific expression and purification conditions vary depending on the expression system used. For example, if the gene is expressed in E. coli, it is first cloned into an expression vector by placing the genetically engineered gene downstream of a suitable bacterial promoter, such as Trp or Tac, and a prokaryotic signal sequence. The expressed protein can be secreted. The expressed protein can accumulate in refractiles or inclusion bodies and can be recovered after cell destruction by French press or sonication. The refractiles can then be solubilized, and the protein can be refolded and / or cleaved by methods known in the art.

[0168] When a reconstructed gene is to be expressed in a eukaryotic host cell, such as a CHO cell, the gene is first inserted into an expression vector containing an appropriate eukaryotic promoter, secretory signal, poly(A) sequence, and stop codon. Optionally, the vector or gene construct may contain enhancement factors and introns. The gene construct can be introduced into a eukaryotic host cell using conventional techniques.

[0169] Polypeptides containing sialidases or fusion proteins, such as fusion proteins containing immunoglobulin heavy chain variable regions or light chain variable regions, can be produced by growing (culturing) host cells transfected with an expression vector encoding such variable regions under conditions that enable polypeptide expression. After expression, the polypeptide can be recovered and purified or isolated using techniques known in the art, such as glutathione-S-transferase (GST) or affinity tags such as histidine tags.

[0170] In one embodiment, sialidase or sialidase conjugated to an Fc domain may be produced by: (a) an expression vector encoding one Fc polypeptide and another expression vector encoding another Fc polypeptide; or (b) growing (culturing) host cells transfected with a single expression vector encoding both Fc polypeptides under conditions that allow expression of both polypeptides. The sialidase is fused to one or more polypeptides. The intact sialidase-Fc domain fusion protein may be recovered and purified or isolated using techniques known in the art, such as affinity tags such as protein A, protein G, glutathione-S-transferase (GST), or histidine tags.

[0171] In one embodiment, sialidase or sialidase conjugated with a serum half-life extender is expressed and / or purified in the presence of a stabilizer. The stabilizer prevents one or more of the following from occurring during expression, purification and / or storage: protein unfolding, protein misfolding, protein aggregation, protein inhibition, enzymatic loss and / or protein degradation of the sialidase or sialidase conjugated with a serum half-life extender. In one embodiment, the stabilizer is a cation, for example, a divalent cation. In one embodiment, the cation is calcium or magnesium. The cation may be in the form of a salt, for example, calcium chloride (CaCl2) or magnesium chloride (MgCl2).

[0172] In one embodiment, the stabilizer is present in amounts of about 0.05 mM to about 5 mM during expression and / or purification. For example, the stabilizer may be present in amounts of about 0.05 mM to about 4 mM, about 0.05 mM to about 3 mM, about 0.05 mM to about 2 mM, about 0.05 mM to about 1 mM, about 0.05 mM to about 0.5 mM, about 0.5 mM to about 4 mM, about 0.5 mM to about 3 mM, about 0.5 mM to about 2 mM, about 0.5 mM to about 1 mM, about 1 mM to about 4 mM, about 1 mM to about 3 mM, or (of) about 1 mM to about 2 mM.

[0173] In one embodiment, to express a protein, such as sialidase, as a secreted protein, the protein's native N-terminal signal sequence is replaced with, for example, MDMRVPAQLLGLLLLWLPGARC (SEQ ID NO: 28). In another embodiment, to express a protein, such as recombinant human sialidase, as a secreted protein, an N-terminal signal sequence, such as MDMRVPAQLLGLLLLWLPGARC (SEQ ID NO: 28), is added. Further exemplary N-terminal signal sequences include those derived from interleukin-2, CD-5, IgGκ light chain, trypsinogen, serum albumin, and prolactin. In another embodiment, to express a protein, such as recombinant human sialidase, as a secreted protein, a C-terminal lysosomal signal motif, such as YGTL (SEQ ID NO: 29), is removed.

[0174] In one embodiment, when sialidase is chemically conjugated to a serum half-life extender, the chemical conjugation may be carried out using methods known in the art. Binding sites on sialidase and / or serum half-life extenders include the N-terminal amino group and the ε-amino group found on lysine residues, as well as other amino, imino, carboxyl, sulfhydryl, hydroxyl, or other hydrophilic groups. Serum half-life extenders may be directly covalently bound to sialidase using chemicals and with or without the known use of polyfunctional (usually bifunctional) crosslinking agents used in the art. For example, in the case of PEG, the sulfhydryl group can be derivatized by coupling to maleimide-substituted PEG (e.g., alkoxy-PEGamine + sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) or PEG-maleimide commercially available from Shearwater Polymers, Inc., Huntsville, Ala.).

[0175] V. Pharmaceutical Compositions For therapeutic use, sialidase or sialidase conjugated with a half-life extender is preferably combined with a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable," as used herein, means a compound, material, composition and / or dosage form that is suitable for use in contact with human and animal tissues, within the bounds of normal medical judgment, without excessive toxicity, irritation, allergic response or other problems or complications, and in proportion to a reasonable benefit / risk ratio.

[0176] The term “pharmaceutically acceptable carrier” as used herein means a buffer, carrier, and excipient suitable for use in contact with human and animal tissues, without excessive toxicity, irritation, allergic response, or other problems or complications, and in proportion to a reasonable benefit / risk ratio. Examples of pharmaceutically acceptable carriers include any of the standard pharmaceutical carriers, such as phosphate-buffered saline, water, emulsions (e.g., oil / water or water / oil emulsions), and various types of wetting agents. The composition may also include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA

[1975] . Examples of pharmaceutically acceptable carriers include buffers, solvents, dispersion media, coatings, isotonic agents, and absorption retarders suitable for pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art.

[0177] In one embodiment, the pharmaceutical composition may include formulation materials for modifying, maintaining, or preserving, for example, the pH, volumetric osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, or adsorption or penetration of the composition.In such embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulin); colorants, flavoring agents ( Agents) and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (such as glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as Pluronic acid, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal, etc.); stability enhancers / stabilizers (such as sucrose, sorbitol, or cations); tonicity enhancers (alkali metal halides, preferably sodium chloride or potassium chloride, mannitol) Examples include sorbitol, delivery vehicles, diluents, excipients, and / or pharmaceutical adjuvants (see Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990)).

[0178] In one embodiment, the pharmaceutical composition may contain a stabilizer. In one embodiment, the stabilizer is a cation, for example, a divalent cation. In one embodiment, the cation is calcium or magnesium. The cation may be in the form of a salt such as calcium chloride (CaCl2) or magnesium chloride (MgCl2).

[0179] In one embodiment, the stabilizer is present in an amount of about 0.05 mM to about 5 mM. For example, the stabilizer may be present in an amount of about 0.05 mM to about 4 mM, about 0.05 mM to about 3 mM, about 0.05 mM to about 2 mM, about 0.05 mM to about 1 mM, about 0.05 mM to about 0.5 mM, about 0.5 mM to about 4 mM, about 0.5 mM to about 3 mM, about 0.5 mM to about 2 mM, about 0.5 mM to about 1 mM, about 1 mM to about 4 mM, about 1 mM to about 3 mM, or (of) about 1 mM to about 2 mM.

[0180] In one embodiment, the pharmaceutical composition may include nanoparticles, such as polymer nanoparticles, liposomes, or micelles (see Anselmo et al. (2016) BIOENG. TRANSL. MED. 1: 10-29).

[0181] In some embodiments, a pharmaceutical composition may include a sustained-release or controlled-release formulation. Techniques for formulating sustained-release or controlled-release means, such as liposome carriers, bio-erodible microparticles or porous beads, and depot injections are also known to those skilled in the art. Sustained-release preparations may include, for example, porous polymeric microparticles in the form of formed articles or a semipermeable polymer matrix, such as a film or microcapsules. The sustained-release matrix may include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid, poly(2-hydroxyethyl methacrylate (inethacrylate)), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. The sustained-release composition may also include liposomes, which can be prepared by any of several methods known in the art.

[0182] Pharmaceutical compositions comprising sialidase or sialidase conjugated with a half-life extender may exist in unit dosage forms and may be prepared by any suitable method. The pharmaceutical composition should be formulated to be compatible with its intended route of administration. Examples of routes of administration include intravenous (IV), intradermal, inhalation, transdermal, topical, transmucosal, intrasacral, and transrectal administration. In some embodiments, sialidase or sialidase conjugated with a half-life extender is administered by IV infusion. In some embodiments, sialidase or sialidase conjugated with a half-life extender is administered by intratumoral injection. Useful formulations may be prepared by methods known in the pharmaceutical field. See, for example, Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990). Suitable formulation components for parenteral administration include sterile diluents, such as sterile water for injection, saline solution, fixative oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as EDTA; buffers, such as acetate, citrate, or phosphate; and agents for adjusting tonicity, such as sodium chloride or dextrose.

[0183] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). The carrier should be stable under manufacturing and storage conditions and protected from microorganisms. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol) and suitable mixtures thereof.

[0184] Preferably, the pharmaceutical preparation is sterilized. Sterilization can be achieved by any suitable method, such as filtration through a sterile filtration membrane. If the composition is freeze-dried, sterilization by filtration can be performed before or after freeze-drying and reconstitution.

[0185] In one embodiment, the pharmaceutical composition is placed in a sterile container (e.g., a bottle or vial). The pharmaceutical composition may, for example, be freeze-dried or exist as a solution in the sterile container. The sterile container may be sealed by a partition and may have a label placed thereon that identifies the pharmaceutical composition contained in the container.

[0186] The compositions described herein may be administered topically or systemically. Administration is generally parenteral. In a preferred embodiment, the pharmaceutical composition is administered subcutaneously, and in a more preferred embodiment, intravenously. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.

[0187] Generally, the therapeutically effective dose of sialidase conjugated with an active component, such as sialidase or a half-life extender, is in the range of 0.1 mg / kg to 100 mg / kg, e.g., 1 mg / kg to 100 mg / kg, or 1 mg / kg to 10 mg / kg. The dose depends on variables such as the type and severity of the disease or symptom being treated, the patient's overall health status, the in vivo potency of the active component, the pharmaceutical formulation, and the route of administration. To rapidly achieve the desired blood or tissue level, the initial dose may be increased beyond the above level. Alternatively, the initial dose may be lower than the optimal dose, and the daily dose may be gradually increased during the course of treatment. Human doses may be optimized in conventional Phase I dose escalation studies designed to be performed, for example, from 0.5 mg / kg to 20 mg / kg. The frequency of administration may vary depending on factors such as the route of administration, the dose, the serum half-life of sialidase or sialidase conjugated with a half-life extender, and the disease being treated. Exemplary administration frequencies are once daily, once weekly, and once every two weeks. The preferred route of administration is parenteral, for example, intravenous infusion. In one embodiment, sialidase or sialidase conjugated with a half-life extender is lyophilized and then reconstituted in buffered saline at the time of administration.

[0188] VI. Therapeutic uses The compositions and methods disclosed herein may be used to treat various forms of cancer in a subject or to inhibit the growth of cancer in a subject. The present invention provides a method for treating cancer in a subject. The method comprises administering to a subject an effective amount of sialidase or sialidase conjugated with a half-life extender, either alone or in combination with another therapeutic agent, to treat cancer in the subject. The term “effective amount” as used herein means an amount of the active agent (e.g., sialidase or sialidase conjugated with a half-life extender) sufficient to produce a beneficial or desired result. The effective amount may be administered in one or more doses, applications, or applications and is not intended to be limited to a particular formulation or route of administration.

[0189] As used herein, “treat,” “treating,” and “treatment” mean the treatment of a disease in a subject, such as a human. This includes (a) inhibiting the disease, i.e., stopping its progression; and (b) alleviating the disease, i.e., causing a regression of the disease state. As used herein, the terms “subject” and “patient” mean an organism to be treated by the methods and compositions described herein. Preferably, but not limited to, such organisms are mammals (e.g., mice, monkeys, horses, cattle, pigs, dogs, cats, etc.), and more preferably humans.

[0190] Examples of cancers include solid tumors, soft tissue tumors, hematopoietic malignancies, and metastatic lesions. Examples of hematopoietic malignancies include leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), B-cell, T-cell or FAB ALL, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), such as modified CLL, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, pilocytic cell leukemia, myelodysplastic syndrome (MDS), lymphoma, Hodgkin's disease, malignant lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, multiple myeloma, or Richter's syndrome (Richter's transformation). Examples of solid tumors include malignant diseases affecting various organ systems, such as sarcomas, adenocarcinomas and carcinomas of the head and neck (including the pharynx), thyroid gland, lungs (small cell or non-small cell lung cancer (NSCLC)), breasts, lymphatic system, gastrointestinal tract (e.g., oral cavity, esophagus, stomach, liver, pancreas, small intestine, colon and rectum, anal canal), reproductive and urogenital tracts (e.g., kidneys, urothelium, bladder, ovaries, uterus, cervix, endometrium, prostate, testes), CNS (e.g., nerve cells or glial cells, e.g., neuroblastoma or glioma), or skin (e.g., melanoma).

[0191] In one aspect, the cancer is an epithelial carcinoma, such as an epithelial carcinoma that upregulates the expression of sialylated glycans. Exemplary epithelial carcinomas include, but are not limited to, endometrial cancer, colon cancer, ovarian cancer, cervical cancer, vulvar cancer, uterine cancer or fallopian duct cancer, breast cancer, prostate cancer, lung cancer, pancreatic cancer, urinary tract cancer, bladder cancer, head and neck cancer, oral cancer, and liver cancer. Epithelial carcinomas also include carcinomas such as lobular carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, adenocarcinoma, adenocortical carcinoma, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, baso squamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebral carcinoma, cholangiocarcinoma, choriocarcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, armory carcinoma, and skin carcinoma. cutaneum), cylindrical carcinoma, cylindrical cell carcinoma, ductal carcinoma, carcinoma durum, embryonic carcinoma, encephaloid carcinoma, epidermoid carcinoma, carcinoma epitheliale adenoids, exophytic carcinoma, carcinoma ex ulcere, fibrous carcinoma fibrosum), gelatiniforni carcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, adenocarcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hürthle cell carcinoma, hyaline carcinomaCarcinoma, adrenal-like carcinoma (hypemephroid carcinoma), infantile embryonal carcinoma, carcinoma in situ, carcinoma in epidermis, carcinoma in situ (intraepithelial carcinoma), Krompecher's carcinoma, Krutskyi cell carcinoma, large cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, mucinous adenocarcinoma, mucinous cell carcinoma, mucinous epidermoid carcinoma, mucinous carcinoma (mucous) Carcinoma, myxomatous carcinoma, nasopharyngeal carcinoma, oat cell carcinoma, ossificans carcinoma, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of the kidney, reserve cell carcinoma, sarcomatodes carcinoma, Schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti, signet ring cell carcinoma, simple carcinoma, small cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma carcinoma), spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinomaExamples include carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, pyogenic carcinoma, and carcinoma villosum.

[0192] In some respects, cancer is breast cancer. In some respects, cancer is adenocarcinoma. In some respects, cancer is metastatic cancer. In some respects, cancer is refractory cancer.

[0193] In one embodiment, cancer is resistant to or unresponsive to treatment with antibodies, such as antibodies having ADCC activity, such as trastuzumab.

[0194] The methods and compositions described herein may be used alone or in combination with other therapeutic agents and / or modalities. As used herein, the term “administered in combination” is understood to mean that two (or more) different therapies are delivered to a subject such that the therapeutic effects on the patient overlap at a given point in time during the course of the distress of the subject with the disorder. In some embodiments, there is an overlap in the duration of administration, as the delivery of one therapy is still taking place when the delivery of the second therapy begins. This is sometimes referred to herein as “simultaneous” or “concurrent delivery.” In other embodiments, the delivery of one therapy ends before the delivery of the other therapy begins. In some embodiments of either case, the therapy is more effective due to the combined administration. For example, the second therapy is more effective, and a similar effect may be seen with, for example, less of the second therapy, or the second therapy reduces symptoms to a greater extent than would be seen if the second therapy were administered in the absence of the first therapy, or a similar situation may be seen with the first therapy. In one embodiment, the delivery is such that the reduction of other parameters related to the symptom or disorder is greater than that observed by the delivery of one treatment in the absence of the other. The effects of the two treatments may be partially additive, or entirely additive, or more than additive. The delivery may be such that the effect of the first treatment being delivered is still detectable when the second treatment is delivered.

[0195] In some embodiments, the methods or compositions described herein are administered in combination with one or more further treatments, such as surgery, radiotherapy, or the administration of another therapeutic preparation. In some embodiments, the further treatment may include chemotherapy, such as cytotoxic agents. In some embodiments, the further treatment may include targeted treatments, such as tyrosine kinase inhibitors, proteasome inhibitors, or protease inhibitors. In some embodiments, the further treatment may include anti-inflammatory, anti-vasculitizing, anti-fibrotic, or antiproliferative compounds, such as steroids, biological immunomodulators, monoclonal antibodies, antibody fragments, aptamers, siRNAs, antisense molecules, fusion proteins, cytokines, cytokine receptors, bronchodilators, statins, anti-inflammatory agents (such as methotrexate), or NSAIDs. In some embodiments, the further treatment may include combinations of different classes of therapeutic agents.

[0196] In one embodiment, the methods or compositions described herein are administered in combination with a checkpoint inhibitor. The checkpoint inhibitor may be selected from, for example, PD-1 antagonists, PD-L1 antagonists, CTLA-4 antagonists, adenosine A2A receptor antagonists, B7-H3 antagonists, B7-H4 antagonists, BTLA antagonists, KIR antagonists, LAG3 antagonists, TIM-3 antagonists, VISTA antagonists, or TIGIT antagonists.

[0197] In one embodiment, checkpoint inhibitors are PD-1 or PD-L1 inhibitors. PD-1 is a receptor present on the surface of T cells that acts as an immune system checkpoint, inhibiting or otherwise modulating T cell activity at the appropriate time to prevent an overactive immune response. However, cancer cells may exploit the benefits of this checkpoint by expressing ligands, such as PD-L1, that interact with PD-1 on the surface of T cells to block or modulate T cell activity. Exemplary PD-1 / PD-L1-based immune checkpoint inhibitors include antibody-based therapies. Exemplary therapeutic methods using PD-1 / PD-L1-based immune checkpoint inhibition are described in U.S. Patents 8,728,474 and 9,073,994, and European Patent No. 1537878B1, and include, for example, the use of anti-PD-1 antibodies. Exemplary anti-PD-1 antibodies are described, for example, in U.S. Patent Nos. 8,952,136, 8,779,105, 8,008,449, 8,741,295, 9,205,148, 9,181,342, 9,102,728, 9,102,727, 8,952,136, 8,927,697, 8,900,587, 8,735,553 and 7,488,802. Examples of anti-PD-1 antibodies include, for example, nivolumab (Opdivo®, Bristol-Myers Squibb Co.), pembrolizumab (Keytruda®, Merck Sharp & Dohme Corp.), PDR001 (Novartis Pharmaceuticals), and pidilizumab (CT-011, Cure Tech). Examples of anti-PD-L1 antibodies are described, for example, in U.S. Patents 9,273,135, 7,943,743, 9,175,082, 8,741,295, 8,552,154, and 8,217,149.Examples of anti-PD-L1 antibodies include, for example, atezolizumab (Tecentriq®, Genentech), durvalumab (AstraZeneca), MEDI4736, avelumab, and BMS 936559 (Bristol Myers Squibb Co.).

[0198] In one embodiment, the methods or compositions described herein are administered in combination with a CTLA-4 inhibitor. In the CTLA-4 pathway, the interaction between CTLA-4 on T cells and its ligands (also known as, e.g., CD80, B7-1, and CD86) on the surface of antigen-presenting cells (not cancer cells) results in T cell inhibition. Exemplary CTLA-4-based immune checkpoint inhibitory methods are described in U.S. Patents 5,811,097, 5,855,887, and 6,051,227. Exemplary anti-CTLA-4 antibodies are listed in U.S. Patent Nos. 6,984,720, 6,682,736, 7,311,910, 7,307,064, 7,109,003, 7,132,281, 6,207,156, 7,807,797, 7,824,679, 8,143,379, 8,263,073, 8,318,916, 8,017,114, 8,784,815 and 8,883,984, International (PCT) Publication Nos. WO98 / 42752, WO00 / 37504 and WO01 / 14424, and European Patent EP 1212422. This is described in B1. Examples of CTLA-4 antibodies include ipilimumab or tremelimumab.

[0199] In one embodiment, the methods or compositions described herein are administered in combination with (i) a PD-1 or PD-L1 inhibitor, for example, a PD-1 or PD-L1 inhibitor disclosed herein, and (ii) a CTLA-4 inhibitor, for example, a CTLA-4 inhibitor disclosed herein.

[0200] In one embodiment, the methods or compositions described herein are administered in combination with a CD20 inhibitor. In one embodiment, the CD20 inhibitor is an anti-CD20 antibody. In one embodiment, the anti-CD20 antibody is selected from the group consisting of ofatumumab, rituximab, ocrelizumab, iodine I131 tositumomab, obinutuzumab, ibritumomab, and hyaluronidase rituximab.

[0201] In some embodiments, the methods or compositions described herein are administered in combination with an IDO inhibitor. Examples of IDO inhibitors include 1-methyl-D-tryptophan (known as indoximod), epacadostat (INCB24360), napoximod (GDC-0919), and BMS-986205.

[0202] Examples of cytotoxic agents that may be administered in combination with the methods or compositions described herein include, for example, antimicrotubule agents, topoisomerase inhibitors, antimetabolites, protein synthesis and degradation inhibitors, mitotic inhibitors, alkylating agents, and platinum-platinating agents. Examples of drugs include nucleic acid synthesis inhibitors, histone deacetylase inhibitors (HDAC inhibitors, e.g., vorinostat (SAHA, MK0683), entinostat (MS-275), panobinostat (LBH589), trichostatin A (TSA), mosetinostat (MGCD0103), belinostat (PXD101), romidepsin (FK228, depsipeptide)), DNA methyltransferase inhibitors, nitrogen mustard, nitrosourea, ethyleneimine, alkyl sulfonates, triazenes, folate analogs, nucleoside analogs, ribonucleotide reductase inhibitors, vinca alkaloids, taxanes, epothirone, intercalating agents, drugs that can interfere with signaling pathways, drugs that promote apoptosis, and antibody molecule conjugates that bind to surface proteins to deliver radiation or toxic agents.In one embodiment, cytotoxic agents that may be administered together with the methods or compositions described herein include platinum-based drugs (e.g., cisplatin), cyclophosphamide, dacarbazine, methotrexate, fluorouracil, gemcitabine, capecitabine, hydroxyurea, topotecan, irinotecan, azacitidine, vorinostat, ixabépirone, bortezomib, taxanes (e.g., paclitaxel or docetaxel), cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, vinorelbine, colchicine, and anthracyclines (e.g., doxorubicin or epirubicin). These include daunorubicin, dihydroxyanthracine dione, mitoxantrone, mitramycin, actinomycin D, adriamycin, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, lysine, or mytansinoids.

[0203] The present invention also provides a method for increasing the expression of granzyme B, IL-1b, IL-2, IL-6, IL-10, IL-17A, HLA-DR, CD86, CD83, IFNγ, or TNFα in cells, tissues, or subjects. The method comprises contacting cells, tissues, or subjects with an effective amount of sialidase or sialidase conjugated with a half-life extender to increase the expression of granzyme B, IL-1b, IL-2, IL-6, IL-10, IL-17A, HLA-DR, CD86, CD83, IFNγ, or TNFα in cells, tissues, or subjects to the corresponding expression levels before contact with sialidase or sialidase conjugated with a half-life extender. In one embodiment, the cells are selected from dendritic cells and peripheral blood mononuclear cells (PBMCs, e.g., monocytes).

[0204] In one embodiment, the expression of granzyme B, IL-1b, IL-2, IL-6, IL-10, IL-17A, HLA-DR, CD86, CD83, IFNγ, or TNFα in cells, tissues, or subjects is increased by at least about 10%, at least about 20%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1,000% in cells, tissues, or subjects that were not exposed to similar or otherwise identical cells or tissues conjugated with sialidase or half-life extender. Gene expression can be measured by any suitable method known in the art, for example, by ELISA as described in the examples herein, by Luminex multiplex assay, or by flow cytometry.

[0205] The present invention also provides a method for removing sialic acid from cells or tissues. The method comprises contacting cells or tissues with an effective amount of sialidase or sialidase conjugated with a half-life extender. The present invention also provides a method for removing sialic acid from cells in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition containing sialidase or sialidase conjugated with a half-life extender, thereby removing sialic acid from cells.

[0206] In one embodiment, the cells are tumor cells, dendritic cells (DCs), or monocytes. In another embodiment, the cells are monocytes, and the method results in increased expression of MHC-II molecules (e.g., HLA-DR) on the monocytes. In another embodiment, the expression of MHC-II molecules in cells or tissues is increased by at least about 10%, at least about 20%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1,000% compared to similar or otherwise identical cells or tissues that have not been contacted with sialidase or sialidase conjugated with a half-life extender. Gene expression can be measured by any suitable method known in the art, for example, by ELISA as described in the examples herein, by Luminex multiplex assay, or by flow cytometry.

[0207] The present invention also provides a method for increasing the phagocytic activity of tumor cells. The method comprises contacting tumor cells with a sialidase or sialidase conjugated with a half-life extender effective in removing sialic acid from the tumor cells, thereby increasing the phagocytic activity of the tumor cells. In one embodiment, the present disclosure relates to a method for increasing the phagocytic activity of tumor cells in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition containing a sialidase or sialidase conjugated with a half-life extender effective in removing sialic acid from tumor cells, thereby increasing the phagocytic activity of the tumor cells.

[0208] In one embodiment, phagocytosis is increased by at least about 10%, at least about 20%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1,000% compared to similar or otherwise identical tumor cells or populations of tumor cells that have not been contacted with sialidase or sialidase conjugated with a half-life prolonger. Phagocytosis can be measured as described in Example 9 of this specification.

[0209] The present invention also provides a method for activating dendritic cells (DCs) or populations of DCs. The method comprises contacting DCs or populations of DCs with tumor cells treated with sialidase or sialidase conjugated with a half-life extender. In one embodiment, the disclosure relates to a method for activating dendritic cells (DCs) or populations of DCs in a subject, the method comprising administering to the subject an amount of a pharmaceutical composition containing sialidase or sialidase conjugated with a half-life extender that is effective in removing sialic acid from tumor cells in the subject, thereby activating DCs or populations of DCs in the subject.

[0210] In one embodiment, the activation of a DC or population of DCs is increased by at least about 10%, at least about 20%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1,000% compared to a similar or otherwise identical population of DCs or DCs that were not in contact with tumor cells treated with sialidase or sialidase conjugated with a half-life extender. The activation may be measured as described in Example 8 of this specification.

[0211] The present invention also provides a method for reducing Siglec-15 binding activity, thereby increasing antitumor activity in the tumor microenvironment, the method comprising contacting T cells with sialidase or sialidase conjugated with a half-life extender. In one embodiment, the present disclosure relates to a method for reducing Siglec-15 binding activity, thereby increasing antitumor activity in a patient's tumor microenvironment, the method comprising administering to a subject an effective amount of a pharmaceutical composition comprising sialidase or sialidase conjugated with a half-life extender, thereby increasing antitumor activity (e.g., T cell activity) in the subject.

[0212] In one embodiment, the Siglec-15 binding activity is reduced by at least about 10%, at least about 20%, at least about 50%, at least about 75%, or about 100% compared to Siglec-15 that has not been contacted with sialidase or sialidase conjugated with a half-life extender. Binding may be measured as described in Example 16 of this specification.

[0213] The present invention also provides a method for promoting the infiltration of immune cells into a tumor in subjects requiring the promotion of immune cell infiltration into a tumor. The method comprises administering to a subject an effective amount of sialidase or sialidase conjugated with a half-life extender, for example, sialidase or sialidase conjugated with a half-life extender as disclosed herein. In one embodiment, the immune cells are T cells, e.g., CD4+ and / or CD8+ T cells, e.g., CD69 + CD8 + and / or GzmB + CD8 + These are T cells. In one aspect, immune cells are natural killer (NK) cells.

[0214] In one embodiment, immune cell infiltration into tumors in subjects is increased by at least about 10%, at least about 20%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1,000% compared to similar or otherwise identical tumors and / or subjects that were not administered sialidase or sialidase conjugated with a half-life prolonger. Immune cell infiltration into tumors can be measured by any suitable method known in the art, such as antibody staining.

[0215] The present invention also provides a method for increasing the number of circulating natural killer (NK) cells in subjects requiring an increase in the number of circulating NK cells. The method comprises administering to a subject an effective amount of sialidase or sialidase conjugated with a half-life extender, for example, the sialidase or sialidase conjugated with a half-life extender disclosed herein, thereby increasing the number of circulating NK cells compared to before administration of the sialidase or sialidase conjugated with a serum half-life extender or pharmaceutical composition.

[0216] In one embodiment, the number of circulating NK cells in a subject is increased by at least about 10%, at least about 20%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1,000% compared to a similar or otherwise identical subject that was not administered sialidase or sialidase conjugated with a half-life prolonger. The number of circulating NK cells in a subject can be measured by any suitable method known in the art, for example, by antibody staining.

[0217] The present invention also provides a method for increasing the number of T cells in draining lymph nodes in subjects requiring an increase in the number of T cells in draining lymph nodes. The method comprises administering to a subject an effective amount of sialidase or sialidase conjugated with a half-life extender, for example, the sialidase or sialidase conjugated with a half-life extender disclosed herein, thereby increasing the number of T cells in draining lymph nodes compared to before administration of sialidase or sialidase conjugated with a serum half-life extender or a pharmaceutical composition. In one embodiment, the immune cells are T cells, for example, CD4+ and / or CD8+ T cells.

[0218] In one embodiment, the number of T cells in the influx region lymph nodes of a subject is increased by at least about 10%, at least about 20%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1,000% compared to a similar or otherwise identical subject that was not administered sialidase or sialidase conjugated with a half-life prolonger. The number of T cells in the influx region lymph nodes of a subject can be measured by any suitable method known in the art, for example, by an antibody.

[0219] The present invention also provides a method for increasing the expression of Cd3, Cd4, Cd8, Cd274, Ctla4, Icos, Pdcd1, Lag3, Il6, Il1b, Il2, Ifng, Ifna1, Mx1, Gzmb, Cxcl9, Cxcl12 and / or Ccl5 in cells, tissues or subjects. The method comprises the step of contacting cells, tissues, or subjects with an effective amount of sialidase or sialidase conjugated with a half-life extender, for example, sialidase or sialidase conjugated with a half-life extender disclosed herein, to increase the expression of Cd3, Cd4, Cd8, Cd274, Ctla4, Icos, Pdcd1, Lag3, Il6, Il1b, Il2, Ifng, Ifna1, Mx1, Gzmb, Cxcl9, Cxcl12, and / or Ccl5 in the cells, tissues, or subjects prior to contact with the sialidase or sialidase conjugated with a half-life extender or the pharmaceutical composition.

[0220] In one embodiment, the expression of Cd3, Cd4, Cd8, Cd274, Ctla4, Icos, Pdcd1, Lag3, Il6, Il1b, Il2, Ifng, Ifna1, Mx1, Gzmb, Cxcl9, Cxcl12 and / or Ccl5 in cells, tissues or subjects is increased by at least about 10%, at least about 20%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1,000% compared to similar or otherwise identical cells, tissues or subjects that have not been exposed to sialidase or sialidase conjugated with a half-life extender. Gene expression can be measured by any suitable method known in the art, such as ELISA, Luminex multiplex assay, or Nanostring technology.

[0221] Throughout the description, where a composition is described as having, including, or comprising certain components, or where a process and method is described as having, including, or comprising certain steps, it is intended that there are compositions of the present invention that are essentially composed of or consist of described components, and processes and methods of the present invention that are essentially composed of or consist of described process steps.

[0222] In this application, where it can be said that an element or component is included in and / or selected from a list of elements or components described, it should be understood that the element or component may be any one of the elements or components described, or may be selected from a group consisting of two or more of the elements or components described.

[0223] Furthermore, it should be understood that the elements and / or features of the compositions or methods described herein, whether expressly or implicitly, can be combined in various ways without departing from the spirit and scope of the invention. For example, where a particular compound is referenced, the compound can be used in various embodiments of the compositions and / or methods of the invention, unless otherwise understood from the context. That is, while embodiments in this application are described and shown in a manner that allows for clear and concise application to be described and illustrated, it is intended and understood that embodiments can be combined or separated in various ways without departing from the teachings and inventions(s). For example, it should be understood that all features described and shown herein may be applicable to all aspects of the inventions(s) described and shown herein.

[0224] The expression "at least one" should be understood to include each of the items listed after it individually and various combinations of two or more of the items listed, unless the context and usage otherwise suggest otherwise. With respect to three or more items listed, the expression "and / or" should be understood to have the same meaning, unless the context otherwise suggests otherwise.

[0225] The use of the terms "include," "includes," "including," "have," "has," "having," "contain," "contains," or "containing," including their grammatical equivalents, should generally be understood as open and unrestricted, without excluding, for example, further elements or processes not explicitly stated, unless the context specifically indicates otherwise or it is specifically understood from the context that they are not.

[0226] Where the term “about” precedes a quantitative value, the present invention also includes the specific quantitative value itself unless otherwise specifically stated. As used herein, the term “about” means a variation of ±10% from the nominal value unless otherwise indicated or inferred.

[0227] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains implementable. Furthermore, two or more steps or actions may be performed simultaneously.

[0228] The use of any and all example or illustrative terms herein, such as “such as” or “including,” is intended solely to better illustrate the invention and, unless otherwise claimed, does not limit the scope of the invention. Terms in this specification should not be construed as indicating any unclaimed element essential to the practice of the invention. [Examples]

[0229] Examples Example 1: Construction and expression of recombinant sialidase This example describes the construction of recombinant human sialidases (Neu1, Neu2, Neu3, and Neu4). Human sialidases Neu1, Neu2, Neu3 (isoform 1), and Neu4 (isoform 1) were expressed as secreted proteins with a 10xHis tag.

[0230] To express Neu1 as a secreted protein, the native N-terminal signal peptide (MTGERPSTALPDRRWGPRILGFWGGCRVWVFAAIFLLLSLAASWSKA; SEQ ID NO: 27) was replaced with MDMRVPAQLLGLLLLWLPGARC (SEQ ID NO: 28), and the C-terminal lysosomal signal motif (YGTL; SEQ ID NO: 29) was removed. To express Neu2, Neu3, and Neu4 as secreted proteins, the N-terminal signal peptide MDMRVPAQLLGLLLLWLPGARC (SEQ ID NO: 28) was added to each of them.

[0231] Sialidase was expressed in 24-well plates using the pCEP4 mammalian expression vector in 200 mL transfections of HEK293F human cells. Sialidase was purified using a Ni-NTA column, quantified using a UV-Vis spectrometer (NanoDrop), and tested by SDS-PAGE as shown in Figure 2. Neu1 was sufficiently expressed in a yield of approximately 3 μg / mL and was mainly present in monomeric form. Neu2 and Neu3 expression yielded yields of approximately 0.15 μg / mL each and were mainly present in dimeric form. Neu4 did not have a detectable expression yield when measured by NanoDrop. Bacterial sialidase derived from Salmonella typhimurium (bacterial sialidase; SEQ ID NO: 30) was expressed in the same manner as Neu1-4 (described above), yielding a yield similar to Neu1, and was mainly present in monomeric form.

[0232] The activity of recombinant sialidases was assayed by measuring the release of sialic acid from the fluorescence-generating substrate 4-methylumbelliferyl-N-acetylneuraminic acid (4MU-NeuAc). As shown in Figure 3, Neu1 did not exhibit detectable activity above the enzyme-free control, which is consistent with previous reports indicating that Neu1 is inactive unless complexed with β-galactosidase and protective protein / cathepsin A (PPCA). Neu2 and Neu3 were active, similar to bacterial sialidases. Enzyme kinetic assays were performed using Neu2 and Neu3. The enzymes at a fixed concentration of 1 nM were incubated with the fluorescence-generating substrate 4MU-NeuAc at concentrations ranging from 4000 μM to 7.8 μM. Assays were performed under both acidic (pH 5.6) and neutral (pH 7) conditions. As shown in Figure 4, both Neu2 and Neu3 were active under both acidic and neutral conditions and exhibited enzyme kinetics comparable to those previously reported.

[0233] Example 2: Construction and expression of recombinant sialidase-Fc fusion protein This example describes the construction of recombinant Fc sialidase gene fusions, particularly Neu2-Fc, Neu3-Fc, and ST sialidase-Fc.

[0234] Fc-sialidases were expressed, purified, and characterized using wild-type Neu2 (Neu2-Fc, encoded by SEQ ID NO: 114; SEQ ID NO: 113) and a variant Fc-sialidase called M106 (SEQ ID NO: 115, encoded by SEQ ID NO: 116) (human IgG1 Fc with M1D, V6Y, P62G, A93E, I187K, C332A, and hole (Y407T) mutations). Neu2-Fc molecules were expressed in 1 L transfections of Expi293 human cells using the pCEP4 mammalian expression vector. Neu2-Fc was purified using protein A, followed by cation exchange chromatography (Hitrap SP-HP, GE Lifesciences). Neu2-Fc yielded 0.3 mg / L, and M106 yielded 20 mg / L.

[0235] Figure 5A shows SDS-PAGE gels displaying recombinant wild-type human Neu2-Fc and M106 under non-reducing and reducing conditions. Figures 5B-C show SEC-HPLC traces comparing wild-type Neu2-Fc versus M106. Monomer species have a retention time of 21 minutes. Neu2-Fc (Figure 5B) had an SEC monomer purity of 7%, and M106 (Figure 5C) had an SEC monomer purity of 85%.

[0236] The activity of M106 was assayed by measuring the release of sialic acid from the fluorescent substrate 4-methylumbelliferyl-N-acetylneuraminic acid (4MU-NeuAc). Enzyme kinetic assays were performed using enzymes incubated with a fixed concentration of 2 μg / well with the fluorescent substrate 4MU-NeuAc at concentrations ranging from 4 mM to 0.03 μM. Figure 6 shows the enzyme activity of M106.

[0237] Using the pCEP4 mammalian expression vector, FC sialidase using wild-type Neu3 (Neu3-Fc encoded by SEQ ID NO: 118; SEQ ID NO: 117) was expressed in 100 ml transfections of Expi293 human cells. Neu3-Fc expressing cells (N3-normal), Neu3-Fc expressing cells treated with tunicamycin (N3-Tunic), and mock-transfected cells were used in both cell conditioning media (supernatant) and washed cell pellets to determine activity. Figure 7 shows that Neu3-Fc activity was detected in the cell pellet, indicating surface-bound activity, and low levels of activity were detected in the supernatant, indicating secreted Neu3-Fc. Treatment with tunicamycin, an inhibitor of S-acylation and N-glycosylation, did not alter surface-bound activity or activity in the supernatant.

[0238] Fc bacterial sialidase was constructed using Salmonella typhimurium (Fc-ST sialidase) with a knob-in-hole Fc design. Fc-ST sialidase contained dimers of two polypeptides: SEQ ID NO: 119 (pCEP-StSia-G4S2-hIgG1Fc-Hole, encoded by SEQ ID NO: 121) and SEQ ID NO: 120 (pCEP-StSia-G4S2-hIgG1Fc-Knob, encoded by SEQ ID NO: 122). Fc-ST sialidase was expressed in 1 L transfections of Expi293 human cells using a pCEP4 mammalian expression vector. Fc-ST sialidase was purified using protein A, followed by cation exchange chromatography (Hitrap SP-HP, GE Lifesciences). Figure 8 shows an SEC-HPLC trace indicating that the expressed Fc-ST sialidase was a monomer species with a retention time of 21 minutes and 75% SEC monomer purity.

[0239] The activity of Fc-ST sialidase was assayed by measuring the release of sialic acid from the fluorescent substrate 4-methylumbelliferyl-N-acetylneuraminic acid (4MU-NeuAc). Enzyme kinetic assays were performed using enzymes incubated with a fixed concentration of 2 μg / well with the fluorescent substrate 4MU-NeuAc at concentrations ranging from 4 mM to 0.03 μM. FC-ST was measured in 3x10⁻¹⁴⁻¹ 8 It exhibited activity that reached fluorescence AU.

[0240] Example 3: In vivo administration of Fc sialidase reduces tumor volume. This example demonstrates that in vivo administration of the Fc sialidase of the present invention reduces tumor volume in a syngeneic mouse tumor model.

[0241] The Fc Salmonella sialidase construct (Fc-ST sialidase) described in Example 2 was compared with avelumab (anti-PD-L1 antibody) in a mouse syngeneic tumor model injected with mouse lymphoma cell line A20. Female BALB / c mice, 6-8 weeks old, were subcutaneously implanted in 0.1 ml of PBS containing A20 tumor cells (5x10) to promote tumor development in the right lower flank. 5 The drug was administered. The tumor was 50-100 mm. 3 , average about 75~100mm 3 When this was reached, the mice were randomly assigned to four groups of eight animals each.

[0242] Mice were administered either a negative control ("isotype control," Figure 9A), Fc-ST sialidase (Figure 9B), avelumab (anti-mouse PD-L1 antibody, Figure 9C), or a combination of Fc-ST sialidase and avelumab (Figure 9D) twice a week for 15 days at a dose of 10 mg / kg intraperitoneally, and tumor volume (mm²) was measured. 3 The values ​​were measured over time. This example demonstrates that the Fc sialidase of the present invention can reduce tumor volume in vivo.

[0243] Fc-ST sialidase was evaluated in a second model using a mouse tumor cell line (EMT6-Her2 cells) genetically engineered to express human Her2. Fc-ST sialidase and the human Neu2 Fc construct M106 (described in Example 2) were compared to trastuzumab (anti-HER2 antibody) in a mouse syngeneic tumor model injected with EMT6-Her2 cells. Female BALB / c mice, 6-8 weeks old, were subcutaneously implanted in the right lower flank with EMT6-Her2 tumor cells (5x10) in 0.1 ml of PBS for tumor development. 5 The drug was administered. The tumor was 50-100 mm. 3 , average about 75~100mm 3 Upon reaching this point, the mice were randomly assigned to four groups of eight animals each.

[0244] As shown by the triangle, mice were administered either an isotype control (vehicle control, Figure 10A), Fc-ST sialidase (FC-ST, Figure 10B), trastuzumab (anti-human Her2 antibody, Figure 10C), or Fc human sialidase (M106, Figure 10D) twice a week for 15 days at a dose of 10 mg / kg via intraperitoneal injection, and tumor volume was measured over time. This example demonstrates that the Fc sialidase of the present invention can reduce tumor volume in vivo.

[0245] Example 4: Divalent cations can stabilize the activity of sialidase. This example describes the ability of divalent cations, particularly calcium, to stabilize the activity of the sialidase of the present invention. Specifically, FcNeu2 sialidase (SEQ ID NO: 123) (M1D, V6Y, I187K, C332A) was expressed together with the heavy and light chains of trastuzumab (including a first polypeptide chain having amino acid sequence sequence number 124 encoded by nucleotide sequence sequence number 125, a second polypeptide chain having amino acid sequence sequence number 126 encoded by nucleotide sequence sequence number 127, and a third polypeptide chain having amino acid sequence sequence number 123 encoded by nucleotide sequence sequence number 128).

[0246] Purified proteins were incubated in PBS or in PBS containing 4 mM CaCl2 at 37°C for up to 2 weeks. Samples containing approximately 2 μg of protein were assayed by measuring the release of sialic acid from the fluorescent substrate 4-methylumbelliferyl-N-acetylneuraminic acid (4MU-NeuAc). Assays were performed at 37°C at 4 hours and at 1, 3, 7, and 14 days. The results are shown in Figure 11. As can be seen, the addition of CaCl2 to the enzyme preparation significantly stabilized the enzyme activity.

[0247] To determine whether CaCl2 could stabilize enzyme activity during expression in mammalian cells, 4 mM CaCl2 was added to transiently transfected Expi293 cell expression medium, starting 24 hours after transfection. As shown in Figure 12A, the addition of CaCl2 significantly increased the amount of secreted enzyme activity up to day 7. However, as shown in Figure 12B, 4 mM CaCl2 resulted in a decrease in cell viability.

[0248] To optimize the CaCl2 concentration that could stabilize enzyme activity while maintaining cell viability, five concentrations of CaCl2—0.05 mM, 0.5 mM, 1 mM, 2 mM, and 4 mM—were added on day 1 post-transfection. Conditioned media over a 3-day time course were collected on days 4–6, and enzyme activity (and therefore viability) was determined as shown in Figure 13A. Protein yield was also measured (Figure 13B). It was found that 4 mM CaCl2 stabilized activity and yielded a moderate yield, but resulted in poor viability. Under the tested conditions, the use of 0.5 mM CaCl2 was found to maintain sialidase activity, provide a higher protein yield, and exhibit lower toxicity to cells.

[0249] Example 5: Siaroglycan profiles of a subset of human PBMCs This example describes the sialglycan profiles of different subsets of human peripheral blood mononuclear cells (PBMCs) using flow cytometry. Sialglycans present on the surface of immune cells play a crucial role in maintaining homeostasis. Imbalances in sialglycan profiles on immune cells are described in relation to autoimmunity and mechanisms of immune surveillance evasion by tumor cells.

[0250] After isolating PBMCs using the Ficol method, the cells were washed twice with ice-cold PBS using bench centrifugation at 350xg for 5 minutes, and then Countessed. TMCells were counted using the II Automated Cell Counter (Thermo Fisher Scientific, Waltham, MA), and 250K cells were equally divided into each well of a 96-well plate. Human Trustine FcX (1 / 20 dilution) and LIVE / DEAD were added to PBS. TM An Fc blocking solution containing fixable near-IR dead cell stain (1 / 2000 dilution) was prepared, and cells were incubated on ice for 10 minutes. Cells were washed with ice-cold PBS (1% BSA) at 350 xg for 5 minutes. Cell surface siaroglycan staining was performed using Hydra and lectin reagents as shown in Table 10. Hydra-3, Hydra-7, and Hydra-9 are hexamerized versions of the extracellular domains of human Siglec 3, Siglec 7, and Siglec 9, respectively (described in International (PCT) application publication number WO2019 / 237070). The lectins used included biotinylated elderberry (SNA, Vector Laboratories, B-1305-2), biotinylated Japanese pagoda tree (Machia Amurensis) (MAL-II, Vector Laboratories, B-1265-1), and biotinylated peanut glutinin (PNA, Vector Laboratories, B-1075-5). SNA is a lectin that preferentially binds to the sialic acid bound to the α-2,6 terminal galactose and binds more weakly to the α-2,3 linkage. MAL-II is a lectin that binds to the sialic acid in the α-2,3 linkage. PNA is a lectin that binds to the terminal galactose residue. Increased PNA staining may indicate removal of the terminal sialic acid by sialidase and exposure of the underlying galactose. [Table 20]

[0251] PBMCs were incubated on ice for 30 minutes with various Hydra and lectin reagents. Cells were washed with 150 μL of PBS (1% BSA) in each well and centrifuged at 350 xg for 5 minutes. Plate solutions were rapidly decanted. AF-647 goat anti-mouse IgG was used as a secondary stain for Hydra reagents (Hydra-7 and Hydra-9) at a 1 / 2000 dilution in PBS. Streptavidin conjugate Alexa Fluor 647 was used as a secondary stain for lectin reagents (PNA, MAL-II and SNA) at a 1 / 2000 dilution in PBS. Cells were incubated on ice for 15 minutes. Cell line-specific staining was performed using the antibodies shown, as shown in Table 11. All antibodies except for the viable-to-death staining antibodies were purchased from Biolegend® (San Diego, CA), purchased from Thermo Fisher Scientific (Waltham, MA). [Table 21]

[0252] A master mix ("staining mix") was prepared using the reagents listed in Table 11 in the FACS staining buffer, and 30 μl of the staining mix was divided equally into each well / tube for a final active antibody concentration of approximately 1 μg / ml. Cells were incubated on ice for 15 minutes. Individual cellular compensation controls were also prepared. Cells were washed with PBS (1% BSA) and resuspended in 4% paraformaldehyde for 10 minutes at room temperature. Cells were washed twice with PBS, and the pellet was resuspended in 150 μl of PBS. Flow cytometry (BD FACSCelesta) TM The sample was run using (BD Biosciences).

[0253] Human PBMCs from two different healthy donors were stained with Hydra-3, Hydra-7, and Hydra-9, as shown in Figure 14 (black and gray bars indicate the two donors). As shown, the monocyte and DC cell populations showed increased Hydra-9 staining compared to other cell populations (Figure 14A). The monocyte and DC cell populations showed increased Hydra-7 staining compared to other cell populations (Figure 14B). One donor showed increased Hydra-7 staining against CD4+ T cells. The monocyte and DC cell populations showed increased Hydra-3 staining compared to other cell populations (Figure 14C). One donor showed increased Hydra-3 staining against CD4+ T cells.

[0254] Figure 15 shows lectin staining (MAL-II, PNA, and SNA) of human PBMCs from healthy donors (black and gray bars represent two independent donors). As shown, PNA staining is relatively low compared to Hydra-9 staining (see scale on the Y axis in comparison to Figure 14) but is specific to monocytes and DCs (Figure 15A). MAL-II stains most immune cell populations (Figure 15B). T cells (CD4+ and CD8+) show increased MAL-II staining compared to other cell populations. SNA stains most immune cell populations (Figure 15C), and NK cells show lower SNA staining compared to other cell populations.

[0255] Example 6: Sialidase efficiently desializes dendritic cells (DCs). This example demonstrates the desialization efficiency of the sialidase molecule of the present invention against human monocyte-derived dendritic cells (DCs).

[0256] DCs are known to express high levels of Siglecs (sialic acid-binding immunoglobulin-like lectins, e.g., Siglec-3, -7, and -9), which inhibit NK cell-mediated killing of tumor cells. Furthermore, DCs express many sialoglycans, which are ligands for the Siglec molecule, as shown in previous examples. The interaction between Siglec and sialoglycans on DCs regulates DC activation, either on the same cell or on another interacting cell (e.g., cancer cells).

[0257] PBMCs were isolated from leukopak (blood sample enriched with PBMCs) using a standard Ficol density gradient method. After PBMC isolation, the cells were washed twice with cold autoMACS® rinse solution (containing 5% BSA; Miltenyi Biotec) by centrifugation at 350 xg for 5 minutes. CD14+ monocytes were magnetically purified using CD14 microbeads (Miltenyi Biotec) and differentiated into dendritic cells. Specifically, CD14+ cells were divided into 0.8 cells x 10⁶ cells in complete medium (RPMI medium containing 10% FBS) containing 50 ng / ml recombinant human GM-CSF and 50 ng / mL recombinant human IL-4. 6 The cells were resuspended at a concentration of / mL. On day 0, the cells were cultured in a 6-well plate with 3 ml of cell suspension per well (2.4 x 10). 6 Cells / well). On days 3 and 6, half of the medium was removed from each well, taking care not to disturb the loosely adhered cells. Each well was then refilled with 1.5 mL of fresh medium containing 100 ng / mL each of rhGM-CSF and rhIL-4. On day 7, differentiated DCs were harvested by gently flushing with medium, washed once with complete medium, and 2 x 10⁻⁶ cells were collected. 6 Resuspended in / mL.

[0258] For the desialylation assay, we used M106 (human IgG1 Fc with M1D, V6Y, P62G, A93E, I187K, C332A and hole (Y407T) mutations and the EPKSS (SEQ ID NO: 163) linker) (SEQ ID NO: 152, encoded by SEQ ID NO: 193). This is the construct described in Example 2, but with the EPKSS (SEQ ID NO: 163) linker instead of the GGGGSGGGGS (SEQ ID NO: 162) linker. In the following examples, the term "M106" refers to this construct. Furthermore, we used the Neu2-FC variant called LOF (human IgG1 with M1D, V6Y, K9D, I187K, C332A, A93E, V363R, L365R, E218A, C219N and hole (Y407T) mutations). Fc (Sequence ID: 175, coded by Sequence ID: 176) was used as a negative control. 100,000 DCs per well were cultured in a 96-well U-bottom format, with 200 μl distributed per well. M106 and LOF constructs were used at the following concentrations (μg / mL): 0, 6.25, 12.5, 25, 50, and 100, using LPS at 0.3 ng / mL where indicated. DCs were incubated overnight (16 hours), followed by flow analysis of CD83, CD86, and MHCII (HLA-DR). Desialylation was measured by PNA staining as described in Example 5.

[0259] After incubation, the plate was centrifuged at 350 xg for 4 minutes to remove the medium. The cells were washed once with FACS staining buffer. The cells were blocked and simultaneously stained with human Trustin FcX (1 / 20 dilution) and LIVE / DEAD in PBS. TM Dead cells were stained by adding 100 μl of a solution containing fixable near-IR dead cell stain (1 / 2000 dilution) and incubating on ice for 10 minutes. Cells were centrifuged and washed once with FACS buffer. 50 μl of PNA-biotin (1 μg / mL in FACS staining buffer) was added to each well and incubated on ice for 10 minutes. Cells were centrifuged and washed twice with FACS buffer. Streptoavidin Alexa FluorTM 50 μL of antibody cocktail containing 647 (listed in Table 12 below) was added to each well and incubated on ice for 30 minutes. After incubation, cells were washed twice with 150 μL of FACS buffer and resuspended in 125 μL of FACS buffer for flow cytometry acquisition. Flow cytometry data were obtained using the HTS (High Throughput Sampler) option on a flow cytometer (BD FACSCelesta). TM The data was acquired using BD Biosciences. After data acquisition, the signals were analyzed using FlowJo flow analysis software (BD Biosciences). [Table 22]

[0260] Figure 16 shows the degree of desialylation of DCs by M106 based on PNA staining. Increased PNA staining indicates removal of terminal sialic acids and exposure of underlying galactose residues recognized by PNA lectins. Figure 16A shows increased fluorescence (MFI) and PNA staining with increasing M106 concentration. Figure 16B shows a multiplier increase in the PNA signal compared to untreated DCs. A clear dose-dependent increase in the PNA signal was observed, indicating robust desialylation of DCs.

[0261] Together, this embodiment demonstrates that M106 induces robust desialization of DCs in a dose-dependent manner.

[0262] Example 7: Desialization of tumor cell lines by sialidase Siaroglycans play a role in maintaining tolerance and homeostasis under human physiological conditions. Overexpression of siaroglycans is observed in tumor cell lines. This example demonstrates that M106 has the ability to desialize tumor cell lines BT-20, SKBR-3, and HT-29, as determined by Hydra-9 and lectin staining.

[0263] BT-20 and HT-29 cells were grown on plates in appropriate media until they reached 70-80% confluence. Cells were dissociated by incubating the plates at 37°C for 15 minutes using Accutase® (Innovative Cell Technologies, Inc.), an enzyme mixture containing proteolytic and collagen-degrading enzyme activity. Once the cells were dissociated, Accutase® was neutralized by adding an equivalent volume of complete medium. The cell suspension was transferred and centrifuged at 300 xg for 5 minutes. The supernatant was discarded, and the cells were washed twice with cold PBS. Cells were counted to 1 x 10⁶ cells per ml. 6 The cells were resuspended in culture medium. M106 and LOF were added to the cells in various dilutions. The cells were incubated at 37°C for 10 hours. After incubation, the cells were washed with PBS and transferred to a 96-well round-bottom plate for staining. Staining was performed using Hydra-9 and PNA as in Example 5.

[0264] Figure 17 shows the degree of desialylation of BT-20 cells after treatment with M106 (triangle) or LOF control (square), as determined by the disappearance of Hydra 9 binding (Figure 17A) or the increase in PNA staining (Figure 17B), as measured by fluorescence (gMFI). The IC50 for desialylation by M106 was 3.088 μg / mL for Hydra 9 and 58.75 μg / mL for SNA. Figure 18 shows the degree of desialylation of BT-20 cells after treatment with M106 (triangle) or LOF control (square), as determined by the disappearance of Hydra 9 binding (Figure 18A) or the increase in PNA staining (Figure 18B), as measured by fluorescence (gMFI). The IC50 for desialylation by the Neu2-Fc variant M106 was 2.95 μg / mL for Hydra 9 and 131.5 μg / mL for SNA.

[0265] Similar experiments were performed using SKBR-3 cells, where the cells were stained with MAL-II lectin in addition to Hydra 9 and PNA. For MAL-II staining, cells were stained at room temperature for 10 minutes using a final concentration of 2 μg / mL in PBS. Figure 19 shows the degree of desialylation of SKBR-3 cells after treatment with M106 (triangle) or LOF control (circle), determined by the disappearance of Hydra 9 binding (Figure 19A), the disappearance of MAL-II staining (Figure 19B), or the increase in PNA staining (Figure 19C), as measured by fluorescence. The IC50 for desialylation by M106 was 4.4 μg / mL for Hydra 9, approximately 120 μg / mL for MAL-II, and 22 μg / mL for SNA.

[0266] Together, this example demonstrates that M106 exhibits dose-dependent removal of cell surface sialic acid from tumor cells. The disappearance of Hydra 9 staining is a more sensitive indicator compared to the disappearance of MAL II staining or the acquisition of PNA staining, with an EC50 of approximately 3-4 ug / mL for M106.

[0267] Example 8: Desialization of tumor cell lines by sialidase enhances human dendritic cell activation. Siaroglycans play a role in maintaining tolerance and homeostasis under human physiological conditions. Overexpression of siaroglycans is observed in tumor cell lines, but the resulting siaroglycans can be removed using the sialidase of the present invention, as shown in previous examples. This example demonstrates the effect of desialization of tumor cell lines on dendritic cell activity.

[0268] In short, dendritic cells (DCs) were generated from CD14+ monocytes isolated from healthy donor PBMCs. CD14+ cells were magnetically purified using the manufacturer's protocol (Miltenyi, catalog no. 130-050-201). The purified cells were then cultured for 7 days in the presence of GM-CSF (R&D Systems, catalog no. 7954-GM / CF) and IL-4 (R&D Systems, catalog no. 6507-IL / CF) to generate immature DCs.

[0269] On the day of the experiment, SKBR-3 tumor cells were harvested from a T-75 flask using Accutase® and washed twice with 10% FBS McCoy's 5A medium. The cells were then divided into 5x10⁻¹⁴ cells. 6 The cells were resuspended in 10% FBS McCoy's 5A medium at a concentration of / mL. 100 μg / mL of M106 was added to the samples, and they were incubated at 37°C for 4 hours. The untreated group was treated the same way except for the addition of M106 to the tubes. After 4 hours, the cells were washed twice with 10% FBS McCoy's 5A medium and rehydrated in complete medium (10% FBS RPMI) at a concentration of 2x10⁶. 6 The solution was resuspended in 1 / mL. 50 μl (100,000 DC) of the supernatant was added to the designated wells.

[0270] Collect DCs, wash them in complete medium (10% FBS RPMI), and prepare 2x10⁻¹⁴ cells. 6 The solution was resuspended in 1 / ml. 50 μL (100,000 DC) of the suspension was added to the designated wells.

[0271] LPS (InvivoGen, catalog number tlrl-pb5lps) was added to a final concentration of 0.3 ng / mL. Complete medium (10% FBS RPMI) was added to reach a final volume of 200 μL per well. The assay plates were incubated overnight at 37°C. The following day, the cells were washed with staining buffer and stained for DC markers (CD11c, CD209, CD1c, CD83, CD86, and HLA-DR). Desialylation of tumor cells was confirmed by staining with Hydra-9, as described in Example 6.

[0272] Figure 20 shows the effect of dendritic cell activation under various conditions, determined by CD83hi expression (Figure 20A) or CD86hi expression (Figure 20B). Untreated DCs ("no Tx") have low percentages of CD83hi and CD86hi. Addition of LPS to DCs strongly induces activation, as indicated by the increased percentages of CD83hi and CD86hi ("LPS"). LPS-induced expression of both CD83 and CD86 was inhibited when DCs were incubated with untreated SKBR-3 tumor cells (see horizontal lines in Figures 20A and 20B). Inhibition of DCs by SKBR-3 tumor cells is reversed after desialylation of SKBR-3 tumor cells with M106 prior to co-incubation with DCs and LPS ("LPS + M106 FC"). Furthermore, sialidase treatment slightly increases DC activation in the absence of LPS (comparing untreated and unprocessed SKBR-3 tumor cells with M106-treated SKBR-3 tumor cells ("M106 FC")).

[0273] This example demonstrates that desialylation of tumor cells can reverse siaroglycan-induced immunosuppression in DCs, suggesting that desialylation of tumor cells can induce a stronger antitumor response.

[0274] Example 9: Effect of sialidase on macrophage-mediated phagocytosis of tumor cells Siaroglycans present on the surface of immune cells play a crucial role in maintaining homeostasis. This example demonstrates the effect of the sialidase of the present invention on the phagocytic activity of HT-29 tumor cells by M2-like human macrophages.

[0275] PBMCs were isolated from the whole blood of human volunteers using the Ficol method. CD14+ monocytes were magnetically purified using CD14 microbeads. Monocytes were combined with 50 ng / mL recombinant human M-CSF at a rate of 1 x 10⁶ 6CD14+ cells were differentiated into M2-like macrophages by resuspending them in RPMI medium (10% FBS) at a concentration of 1 / mL. On day 0, the cells were cultured in 20 mL of 150 mm tissue culture plates (approximately 20 x 10 per plate). 6 Cells were seeded. On days 3 and 6, half of the culture medium was removed from each well, taking care not to disturb the adherent cells. M-CSF was replenished to a final concentration of 50 ng / mL. On day 7, the supernatant was collected in a 50 mL tube and the plate was gently washed with 20 mL of PBS. 20 mL of Accutase® was added and the plate was incubated for 20 minutes to detach the cells from the plate. The cells were resuspended in complete RPMI medium supplemented with 10% FBS and non-essential amino acids (NEAA), sodium pyruvate and HEPES, along with 10 ng / mL M-CSF, and seeded at a rate of 50K cells / well / 100 μL in a flat-bottomed 96-well plate.

[0276] HT-29 cells were collected from flasks using Accutase®. The cells were washed with PBS. The cells were then subjected to Cell Trace at a dilution of 1:1000 per volume (final concentration of 10 μM). TM Cells were labeled with CFSE-labeled dye (FITC) conjugate (Thermo Fisher). The cells were incubated at room temperature for 10 minutes, and the labeling reaction was quenched by adding an equal volume of chilled FBS. The cells were washed twice, resulting in 1.2 x 10⁶ cells. 6 Cells were resuspended in medium (McCoy's medium supplemented with 10% FBS) at a concentration of 100 μg / ml. M106 and LOF were added at the highest concentration of 100 μg / ml, followed by 2-fold dilutions. The untreated control group was stored together with untreated HT-29 cells. Cells were incubated at 37°C for approximately 20 hours.

[0277] After incubation, the cells were spun down, washed with PBS, and 2.5 x 10 6The cells were resuspended in complete RPMI (10% FBS) medium at a final cell density of cells / mL. 100 μL of HT-29 cell suspension was added to M2-like macrophages in appropriate wells at a macrophage:tumor cell ratio of 1:5 (E:T). The plate containing macrophages and tumor cells was incubated for 2 hours to allow phagocytosis. After 2 hours, the medium was slowly removed using a multichannel pipette, 200 μL of Accutase® was added to the plate, and the plate was incubated on ice for 45 minutes to detach both HT-29 and macrophages from the plate. The cells were resuspended and collected in a new 96-well bottom plate. The plate was spun down, the supernatant was discarded, and the cell pellet was washed in 200 μL of PBS.

[0278] The cell pellet was resuspended and blocked on ice for 5–7 minutes using human Trustin Fc blocking agent. After incubation, the cells were washed with PBS. The cells were stained for CD45 and CD14 fluorescent dye markers as shown in Table 13 below. Antibodies were purchased from Biolegend®. [Table 23]

[0279] A master mix was prepared in FACS staining buffer by adding the staining antibody at a 1:30 dilution. 30 μl of the master mix was added per well. Appropriate compensatory controls (e.g., monochromatic stained controls for compensation by standard flow cytometry for multichromatic flow cytometry) were stained concurrently. Cells were incubated on ice for 15 minutes, then washed with 350 g of PBS for 8 minutes. Cells were then fixed with 4% formaldehyde at room temperature for 10 minutes, followed by two washes with PBS. Cells were resuspended in 150 μl of PBS and subjected to flow cytometry (BD FACSCelesta). TM It was run on (BD Biosciences).

[0280] The percentage of CFSE-positive, CD14+CD45+ macrophages was determined. Since CFSE-positive tumor cells phagocytosed by CD14+CD45+ macrophages are CFSE-positive, CFSE-positive, CD14+CD45+ macrophages demonstrate a percentage of tumor cell phagocytosis by macrophages.

[0281] Figure 21 shows the dose-dependent increase in phagocytosis of desialized HT-29 tumor cells by M2-like macrophages derived from two different healthy donors (Figures 21A and 21B). HT-29 pretreated with sialidase concentrations above 25 μg / mL showed a reproducible increase in phagocytosis by macrophages. Similar increases in phagocytosis of desialized BT20 and SKBR-3 tumor cells by M2-like macrophages were observed (Figures 21C and 21D, respectively).

[0282] Therefore, treatment of tumor cells with the sialidase described herein resulted in increased phagocytosis of tumor cells by macrophages.

[0283] Example 10: Sialidase treatment enhances MHC class II expression on monocytes. This example demonstrates the effect of the sialidase of the present invention on MHC class II (HLA-DR) expression on monocytes. MHC-II expression indicates antigen-presenting ability on monocytes. Enhanced class II expression indicates enhanced antigen presentation to T cells for effective immune response.

[0284] Using the Ficoll method, PBMCs were isolated from healthy volunteers, and the cells were washed twice with ice-cold PBS using bench centrifugation at 350 xg for 10 minutes. The cells were resuspended in culture medium and Countessed. TM II. Counted using an automated cell counter. The final suspension was 2.5 x 10⁻⁶. 6The solution was adjusted to cells / L. Approximately 250,000 cells (100 μL) were seeded into 96-well round-bottom plates. Cells were incubated with M106 or LOF at the top concentration of 50 μg / mL, at a 2-fold dilution. An untreated group was included. Cells were incubated at 37°C for 18 hours. Plates were spun at 350 xg for 10 minutes. Cell pellets were washed with cold PBS and subjected to blocking and staining steps using the FACS staining panel listed in Table 14. All antibodies except for Live Dead stain, which was purchased from Thermo Fisher, were purchased from Biolegend®. Siaroglycan staining was performed using PNA lectin to confirm desialylation using the method described in Example 7. [Table 24]

[0285] Figure 22 shows dose-dependent enhancement of HLA-DR expression after M106 desialylation compared to LOF in monocytes from two different healthy donors (Figures 22A and 22B).

[0286] Therefore, this example demonstrates that desialylation of monocytes by the sialidase described herein results in increased MHC class II (HLA-DR) expression on monocytes. MHC-II expression indicates the antigen-presenting ability of monocytes. Thus, enhanced class II expression indicates enhanced antigen presentation to T cells, which can enhance the ability of T cells to produce an effective immune response.

[0287] Example 11: Sialidase treatment does not produce harmful cytokine release. For cytokine release stimulation, conditioned media derived from PBMCs incubated with M106 or LOF were assayed. LPS (1 ng / mL) was used as a positive control. M106 (and LOF) treatment of PBMCs was performed using LEGENDplex. TMWhen measured using a human M1 / M2 macrophage panel (10plex; BioLegend®), no increases were observed in any of the treatment doses for TNF-α, IL-6, IL-1β, IL-1RA, or IL-10 in two independent donors. In contrast, LPS showed clear cytokine induction. These results indicate that sialidase treatment of PBMCs does not result in harmful cytokine release.

[0288] Example 12: Sialidase treatment, alone and in combination with anti-PD-1 antibody, results in complete and partial remission of tumor growth. This example demonstrates that in vivo administration of the sialidase of the present invention can induce complete and partial remission of tumor growth in various mouse syngeneic tumor models.

[0289] Using an MC38 colon cancer cell model, sialidase treatment was tested both alone and in combination with other cancer therapies. 5x10 sialidase in 0.1 mL of PBS was administered subcutaneously to the right lower flank region of each mouse. 5 Tumor cells were inoculated to induce tumor development. The average tumor size was approximately 50 mm. 3 Mice were randomized when a certain threshold was reached. 32 mice were randomly assigned to four test groups. Mice were administered either M106, anti-mouse PD-1, a combination of Neu2-Fc variant M106 and anti-PD-1, or an isotype control, at five doses twice a week, at 10 mg / kg for each drug. Figure 23 shows tumor growth for each mouse in either the isotype control group (Figure 23A), the M106 group (Figure 23B), the anti-PD-1 group (Figure 23C), or the combination of M106 and anti-PD-1 (Figure 23D). M106-treated mice showed complete remission (CR) of tumor growth in one animal, compared to no responsive mice in the isotype-treated groups. The combination of M106 and anti-PD-1 showed one CR and one partial response (PR) as well as an overall reduction in tumor growth in all mice compared to the isotype control.

[0290] Next, sialidase treatment, both alone and in combination with other cancer therapies, was tested using a B16F10 melanoma cancer cell model. For tumor development, 5 x 10 units of sialidase in 0.1 mL of PBS were placed subcutaneously in the right lower flank region of each mouse. 5 Tumor cells were inoculated. The average tumor size was approximately 50 mm. 3 Mice were randomized when they reached a certain threshold. 24 mice were randomly assigned to three test groups. Mice were administered either M106, anti-mouse PD-1, or an isotype control at a dose of 10 mg / kg twice a week for five doses. Figure 24 shows tumor growth for each of the mice in the isotype control group (Figure 24A), the M106 group (Figure 24B), or the anti-PD-1 group (Figure 24C). Figure 24D is a superposition of the isotype control group to the M106 group, showing a clear benefit of M106 in reducing tumor growth in what is considered a difficult-to-treat tumor model.

[0291] Next, using the EMT6 cell line expressing human Her2 as a polyclonal cell line, sialidase treatment was tested both alone and in combination with other cancer therapies. For tumor development, 5 x 10¹⁶ sialidases were injected subcutaneously into the right lower flank region of each mouse in 0.1 mL of PBS. 5 Tumor cells were inoculated. The average tumor size was approximately 100 mm. 3 When the mice reached a certain level, they were randomized. Sixteen mice were randomly assigned to two test groups. The mice were administered either M106 or an isotype control at 10 mg / kg twice a week for five doses. Figure 25 shows tumor growth for each mouse in either the isotype control group (Figure 25A) or the M106 FC group (Figure 25B). Compared to only one in eight mice in the isotype treatment group, four out of eight M106-treated mice showed complete remission (CR) of tumor growth.

[0292] Therefore, as shown in this embodiment, treatment with the sialidases disclosed herein results in a reduction of cancer growth and, in some cases, complete remission in various cancer types.

[0293] Example 13: Sialidase treatment alone and in combination with anti-PD-L1 antibody results in complete and partial remission of tumor growth. This example describes an in vivo study of M106 and / or avelumab (anti-PD-L1) in an A20 syngeneic mouse model. Mouse A20 cells express endogenous mouse PD-L1, which is bound by avelumab. Female Balb / c mice, 5-6 weeks old, were inoculated subcutaneously into the right lower flank region with mouse A20 B-cell lymphoma cells in Matrigel (1:1 by volume). The tumor was approximately 100 mm. 3 When it reached this point (the average tumor volume for each group was 86-90 mm²), 3 (within the specified range), mice were randomly assigned to groups of 8 mice. Table 15 describes the arms of the various studies. Mice were treated intraperitoneally with 5 or 10 mg / kg of M106, avelumab and / or antibody isotype control (as shown) twice a week for a total of 5 doses. Tumor volume and body weight were recorded three times a week. [Table 25]

[0294] Figure 26 shows tumor growth in each mouse group. Complete response (CR) and partial response (PR) for each group are shown. As can be seen, M106 showed antitumor activity both alone and in combination with avelumab ("Ave").

[0295] Mice with tumors showing complete response (CR) derived from the M106 treatment group (either alone or in combination with avelumab) were divided into groups and re-attacked with mouse A20 cells (all approximately 12 weeks old) and compared to naive control mice injected with A20 cells at either 6 or 12 weeks old. Tumor volume and body weight were recorded three times per week. Tumors grew as expected in both 6-week and 12-week naive mice, and no tumor growth was observed in the re-attack mice (data not shown).

[0296] Therefore, as shown in this example, treatment with the sialidase disclosed herein results in a reduction of cancer growth and, in some cases, complete remission in B-cell lymphoma models.

[0297] Example 14: Sialidase treatment, alone and in combination with anti-PD-L1 antibodies, results in complete and partial remission of tumor growth. This example describes an in vivo study of M106 and / or avelumab (anti-PD-L1) in an A20 syngeneic mouse model. The experiment was conducted similarly to Example 13, except that six doses were administered (twice a week for three weeks). Table 16 describes the various arms of the study. Mice were intraperitoneally treated with 10 mg / kg of M106, avelumab, and / or an antibody isotype control twice a week for a total of six doses. Tumor volume and body weight were recorded three times a week. [Table 26]

[0298] Figure 27 shows the tumor growth results for each mouse in each group. Avelumab-based ASCs showed varying degrees of efficacy. Similar to Example 13, M106 showed activity similar to M106 combined with avelumab.

[0299] Therefore, as shown in this example, treatment with the sialidases disclosed herein, alone or in combination with anti-PD-L1 antibodies, results in reduced cancer growth and, in some cases, complete remission in B-cell lymphoma models.

[0300] Example 15: Sialidase treatment, in combination with an anti-CD20 antibody, resulted in improved survival in tumor-carrying mice. This example describes in vivo administration of M106 in combination with an anti-CD20 antibody (ofatumumab) in a mouse syngeneic intravenous dissemination model using a mouse mammary cancer cell line expressing human CD20 (EL4 CD20 cells). Female C57 / BL6 mice, 6-8 weeks old, were intravenously injected with 500,000 cells per mouse. Subsequently, the mice were administered isotype control, ofatumumab, or a combination of ofatumumab and M106 as described in Table 17. Body weight and clinical observations were recorded daily. [Table 27]

[0301] Figure 28 shows the survival curves for mice in each group. Figure 28A shows survival at day 28, and Figure 28B shows overall survival (at day 41). Compared to isotype controls, mice treated with ofatumumab showed a survival shift, with the 50% survival point shifting from day 17 to day 24. Mice treated with the combination of ofatumumab and M106 showed an even greater survival shift up to day 30.

[0302] Therefore, this example demonstrates that treatment with the sialidase of the present invention resulted in increased survival in mice treated with an anti-CD20 antibody.

[0303] Example 16: Sialidase treatment disrupts Siglec-15 activity on T cells. Siglec-15 is an important immunosuppressive factor. Under normal conditions, Siglec-15 is expressed only on certain myeloid cells, but is widely upregulated on human cancer cells and tumor-infiltrating myeloid cells. Siglec-15 acts as a ligand to suppress antigen-specific T cell responses in vitro and in vivo. Genetic depletion or antibody inhibition of Siglec-15 increases antitumor immunity in the tumor microenvironment (TME) and inhibits tumor growth in several mouse models.

[0304] This example demonstrates that neuraminidase treatment removes the Siglec-15 ligand, thereby disrupting Siglec-15 binding activity. Disruption of Siglec-15 binding activity in vivo is thought to induce increased antitumor immunity in TMEs, inhibiting tumor growth.

[0305] Human PBMCs were thawed and stimulated with 1 μg / mL of anti-CD3 (OKT3 clone) and anti-CD28 (clonal CD28.2) antibodies (both from eBiosciences, Thermo Fisher Scientific) in complete RPMI medium (supplemented with 10% heat-inactivated FBS, non-essential amino acids, and sodium pyruvate). On day 2, the suspended cells were harvested and re-cultured in fresh complete RPMI medium, and the cells were continuously stimulated by supplementing with 1 μg / mL of anti-CD3 and anti-CD28 antibodies. Three days later, the cells were 10 6 The seeds were re-seed into 15 mL conical tubes at a density of / ml and treated in the following different groups: (1) untreated; (2) loss of function sialidase ("LOF FC" as described in previous examples), final concentration 50 μg / mL; (3) M106, final concentration 50 μg / mL; and (4) BiNanH2 - final concentration 2 μg / mL. BiNanH2 is a potent sialidase derived from Bifidobacterium infantis, used as a positive control.

[0306] The cells were supplied with anti-CD3 anti-CD28 antibody and incubated overnight in a 37°C incubator. The following day (approximately 14 hours later), the cells were spun down to remove the medium, and then the cells were placed in PBS in a LIVE / DEAD state. TM Cells were blocked with human TruStain FcX Fc receptor blocking agent (Biolegend®) along with fixable near-IR dead cell staining. Subsequently, the cells were blocked with thermoactivated human serum (5% in PBS).

[0307] Cells were stained with human Siglec-15-Fc (prepared by Palleon Pharmaceuticals; MW: approximately 100 kDa) at a final concentration of 1 μM / 100 μg / mL. Cells were incubated on ice for 15 minutes and then washed with PBS.

[0308] Next, the cells were stained with anti-human Fc-AF647 antibody in FACS staining buffer. The cells were incubated on ice for 5 minutes and then washed.

[0309] Next, the cells were stained for CD4 and CD8 markers in FACS staining buffer as described in previous examples. The cells were incubated on ice for 15 minutes and then washed. The cells were fixed and scanned using a flow cytometer (BD FACSCelesta). TM We ran the simulation on BD Biosciences and analyzed the data.

[0310] Figure 29 shows the results of Siglec-15-Fc staining of CD4+ cells (Figure 29A) and CD8+ cells (Figure 29B) after various treatments. Isotype IgG1 staining is also shown as a control. As shown, treatment of activated CD4 and CD8 cells with M106 FC or BiNaNH2 (positive control) reduced Siglec-15-Fc staining compared to untreated cells or cells treated with LOF FC. Figure 30 shows the results of Siglec-15-Fc staining of CD4+ cells (Figure 30A) and CD8+ cells (Figure 30B) using PBMCs derived from a second healthy donor. These results indicate that Siglec-15 binding to activated T cells is sialic acid-dependent, and that removal of sialic acid by neuraminidase disrupts this interaction.

[0311] Therefore, this example demonstrates that neuraminidase treatment using the sialidase of the present invention removes the Siglec-15 ligand, thereby destroying Siglec-15 binding activity. Destruction of Siglec-15 binding activity in vivo is thought to lead to increased antitumor immunity in TMEs and inhibit tumor growth.

[0312] Reference The full disclosures of each patent and scientific document referred to herein are incorporated by reference for all purposes.

[0313] Equal portions The present invention may be realized in other specific forms without departing from its spirit or essential features. Therefore, the aforementioned embodiments are considered illustrative rather than limiting in all respects of the invention described herein. Accordingly, the scope of the invention is indicated not by the foregoing but by the appended claims, and all modifications within the meaning and scope of the equivalent claims are intended to be incorporated into the present invention. The following are examples of aspects of the present invention. Item 1 A pharmaceutical composition comprising a sialidase that, when administered to a subject, conjugates to a serum half-life enhancing factor that increases the serum half-life of sialidase. Section 2 The pharmaceutical composition according to claim 1, wherein the sialidase does not conjugate to a cancer antigen targeting agent that binds to cancer antigens associated with cancer cells. Section 3 The pharmaceutical composition according to claim 1 or 2, wherein the sialidase is a functional fragment of full-length sialidase exhibiting at least 50% of the activity of full-length sialidase. Section 4 A pharmaceutical composition according to any one of claims 1 to 3, wherein the sialidase is a variant exhibiting at least 50% of the activity of wild-type sialidase. Section 5 A pharmaceutical composition according to any one of items 1 to 4, wherein sialidase and serum half-life enhancer are covalently bonded together in a fusion protein. Section 6 A pharmaceutical composition according to any one of items 1 to 4, wherein sialidase and a serum half-life enhancer are chemically conjugated together. Section 7 The pharmaceutical composition according to claims 1 to 6, wherein the serum half-life enhancer is selected from the group consisting of Fc domain, transferrin, albumin, XTEN, homoamino acid polymer (HAP), proline-alanine-serine polymer (PAS), elastin-like peptide (ELP), albumin-binding domain, CTP fusion, GLK fusion, and polyethylene glycol. Section 8 A pharmaceutical composition according to items 1 to 7, wherein the serum half-life enhancer is an Fc domain. Section 9 The pharmaceutical composition according to items 1 to 7, wherein the serum half-life enhancer is not an Fc domain or polyethylene glycol. Item 10 The pharmaceutical composition according to claims 1 to 9, wherein the sialidase contains one or more mutations relative to the wild-type sialidase template. Section 11 Sialidase: (a) Substitution or deletion of a methionine residue at the position corresponding to position 1 of wild-type human Neu2 (M1); (b) Substitution of a valine residue at the position corresponding to position 6 of wild-type human Neu2 (V6); (c) Substitution of an isoleucine residue at the position corresponding to position 187 of wild-type human Neu2 (I187); or (d) Substitution of a cysteine ​​residue at position 332 of wild-type human Neu2 (C332); Or any combination of the aforementioned substitutions A pharmaceutical composition according to items 1 to 10, including the above. Section 12 In sialidase: (a) The methionine residue at the position corresponding to position 1 of wild-type human Neu2 is deleted (ΔM1), substituted with alanine (M1A), or substituted with aspartic acid (M1D); (b) The valine residue at position 6 of wild-type human Neu2 is replaced with tyrosine (V6Y); (c) The isoleucine residue at position 187 of wild-type human Neu2 is replaced with lysine (I187K); or (d) The cysteine ​​residue at position 332 of wild-type human Neu2 is replaced with alanine (C332A); Alternatively, the sialidase contains any of the aforementioned combinations of substitutions. The pharmaceutical composition described in item 11. Section 13 Sialidase: (a) Substitution or deletion of a methionine residue at the position corresponding to position 1 of wild-type human Neu2 (M1); (b) Substitution of a valine residue at the position corresponding to position 6 of wild-type human Neu2 (V6); (c) Substitution of a proline residue at position 62 of wild-type human Neu2 (P62); (d) Substitution of an alanine residue at position 93 of wild-type human Neu2 (A93); (e) Substitution of an isoleucine residue at position 187 of wild-type human Neu2 (I187); (f) Substitution of a glutamine residue at position 126 of wild-type human Neu2 (Q126); (g) Substitution of an alanine residue at position 242 of wild-type human Neu2 (A242); (h) Substitution of a glutamine residue at the position corresponding to position 270 of wild-type human Neu2 (Q270); (i) Substitution of a serine residue at position 301 of wild-type human Neu2 (S301); (j) Substitution of a tryptophan residue at position 302 of wild-type human Neu2 (W302); (k) Substitution of a cysteine ​​residue at position 332 of wild-type human Neu2 (C332); (l) or any combination of the above substitutions A pharmaceutical composition according to items 1 to 11, including the above. Section 14 Sialidase: (a)M1D, V6Y, P62G, A93E, I187K, C332A; (b)M1D, V6Y, P62G, A93E, I187K, S301A, W302R, C332A; (c)M1D, V6Y, P62G, A93E, Q126Y, I187K, A242F, Q270T, C332A; (d) M1D, V6Y, P62G, A93E, Q126Y, I187K, C332A; and (e)A93E, Q126Y, I187K, A242F, Q270T, C332A A pharmaceutical composition according to any of the preceding items, comprising a combination of substitutions selected from the group consisting of the following. Item 15 The pharmaceutical composition according to claim 14, wherein the sialidase conjugates to a serum half-life enhancer comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 115, 152, 180, 184, and 188, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% of an amino acid sequence selected from the group consisting of SEQ ID NOs: 115, 152, 180, 184, and 188. Item 16 Sialidase: (a) Substitution of a proline residue at the position corresponding to position 5 of wild-type human Neu2 (P5); (b) Substitution of a lysine residue at the position corresponding to position 9 of wild-type human Neu2 (K9); (c) Substitution of a lysine residue at position 44 of wild-type human Neu2 (K44); (d) Substitution of a lysine residue at the position corresponding to position 45 of wild-type human Neu2 (K45); (e) Substitution of a leucine residue at the position corresponding to position 54 of wild-type human Neu2 (L54); (f) Substitution of a proline residue at position 62 of wild-type human Neu2 (P62); (g) Substitution of a glutamine residue at position 69 of wild-type human Neu2 (Q69); (h) Arginine residue substitution (R78) at the position corresponding to position 78 of wild-type human Neu2; (i) Substitution of an aspartic acid residue at the position corresponding to position 80 of wild-type human Neu2 (D80); (j) Substitution of an alanine residue at position 93 of wild-type human Neu2 (A93); (k) Substitution of a glycine residue at position 107 of wild-type human Neu2 (G107); (l) Substitution of a glutamine residue at the position corresponding to position 108 of wild-type human Neu2 (Q108); (m) Substitution of a glutamine residue at position 112 of wild-type human Neu2 (Q112); (n) Substitution of a cysteine ​​residue at position 125 of wild-type human Neu2 (C125); (o) Substitution of a glutamine residue at position 126 of wild-type human Neu2 (Q126); (p) Substitution of an alanine residue at the position corresponding to position 150 of wild-type human Neu2 (A150); (q) Substitution of a cysteine ​​residue at position 164 of wild-type human Neu2 (C164); (r) Substitution of an arginine residue at the position corresponding to position 170 of wild-type human Neu2 (R170); (s) Substitution of an alanine residue at position 171 of wild-type human Neu2 (A171); (t) Substitution of a glutamine residue at position 188 of wild-type human Neu2 (Q188); (u) Arginine residue substitution (R189) at the position corresponding to position 189 of wild-type human Neu2; (v) Substitution of an alanine residue at position 213 of wild-type human Neu2 (A213); (w) Leucine residue substitution at position 217 of wild-type human Neu2 (L217); (x) Substitution of a glutamate residue at position 225 of wild-type human Neu2 (E225); (y) Substitution of a histidine residue at position 239 of wild-type human Neu2 (H239); (z) Substitution of a leucine residue at the position corresponding to position 240 of wild-type human Neu2 (L240); (aa) Arginine residue substitution (R241) at the position corresponding to position 241 of wild-type human Neu2; (bb) Substitution of an alanine residue at position 242 of wild-type human Neu2 (A242); (cc) Substitution of a valine residue at position 244 of wild-type human Neu2 (V244); (dd) Substitution of a threonine residue at position 249 of wild-type human Neu2 (T249); (ee) Substitution of an aspartic acid residue at position 251 of wild-type human Neu2 (D251); (ff) Substitution of a glutamate residue at position 257 of wild-type human Neu2 (E257); (gg) Substitution of a serine residue at position 258 of wild-type human Neu2 (S258); (hh) Leucine residue substitution at position 260 of wild-type human Neu2 (L260); (ii) Substitution of a valine residue at position 265 of wild-type human Neu2 (V265); (jj) Substitution of a glutamine residue at the position corresponding to position 270 of wild-type human Neu2 (Q270); (kk) Substitution of a tryptophan residue at position 292 of wild-type human Neu2 (W292); (ll) Substitution of a serine residue at position 301 of wild-type human Neu2 (S301); (mm) Substitution of a tryptophan residue at position 302 of wild-type human Neu2 (W302); (nn) Substitution of a cysteine ​​residue at position 332 of wild-type human Neu2 (C332); (oo) Substitution of a valine residue at position 363 of wild-type human Neu2 (V363); or (pp) Leucine residue substitution at position 365 of wild-type human Neu2 (L365); or any combination of the substitutions mentioned above A pharmaceutical composition according to any of the preceding items, including the following: Item 17 The pharmaceutical composition according to claims 1 to 16, wherein the sialidase is selected from the group consisting of bacterial sialidase, viral sialidase, and mammalian sialidase. Section 18 The pharmaceutical composition according to item 17, wherein the mammalian sialidase is human sialidase. Section 19 The pharmaceutical composition according to claim 18, wherein the human sialyidase is selected from the group consisting of neu1, neu2, neu3, and neu4. Section 20 The pharmaceutical composition according to item 19, wherein human sialyidase is neu2. Section 21 A pharmaceutical composition according to any one of items 1 to 20, containing approximately 0.01 mg / kg to approximately 100 mg / kg of sialidase. Section 22 A pharmaceutical composition according to any one of items 1 to 21, comprising a second therapeutic agent. Section 23 The pharmaceutical composition according to claim 22, wherein the second therapeutic agent is selected from the group consisting of anti-inflammatory agents, anti-vasoconducting agents, anti-fibrotic agents, or antiproliferative compounds (e.g., cytotoxic agents or checkpoint inhibitors). Section 24 A pharmaceutical composition according to any one of items 1 to 23, further comprising a stabilizing amount of a sialidase stabilizer. Section 25 The pharmaceutical composition according to item 24, wherein the sialidase stabilizer is a cation. Section 26 The pharmaceutical composition according to item 25, wherein the cation is selected from the group consisting of calcium and magnesium. Section 27 A pharmaceutical composition according to any one of items 1 to 26, which is placed in a sterile container (e.g., a bottle or vial). Section 28 The pharmaceutical composition according to item 27, which is freeze-dried in a sterile container. Section 29 The pharmaceutical composition described in item 28, which exists as a solution in a sterile container. Item 30 A pharmaceutical composition according to any one of items 27 to 29, wherein the sterile container is sealed with a partition. Section 31 A pharmaceutical composition according to any one of claims 27 to 30, wherein the sterile container has a label placed thereon for identifying the pharmaceutical composition contained in the container. Section 32 A method for treating a sialic acid-related disorder in a subject requiring treatment of the disorder, comprising the step of administering a pharmaceutical composition containing an effective amount of sialidase and a serum half-life enhancing factor that increases the serum half-life of sialidase when administered to the subject, thereby treating the disorder. Item 33 The method described in paragraph 32, in which sialic acid-related disorders are cancer. Section 34 The method according to paragraph 33, wherein the sialidase does not conjugate to a cancer antigen targeting agent that binds to cancer antigens associated with cancer cells. Section 35 The method according to any one of sections 32 to 34, wherein the sialidase is a functional fragment of full-length sialidase exhibiting at least 50% of the activity of full-length sialidase. Section 36 The method according to any one of sections 32 to 35, wherein the sialidase is a variant exhibiting at least 50% of the activity of wild-type sialidase. Section 37 A method described in any of sections 32-36, wherein sialidase and serum half-life enhancer are covalently bound together in a fusion protein. Section 38 A method described in any of sections 32-36, wherein sialidase and serum half-life enhancer are chemically conjugated together. Item 39 The method according to any one of claims 32 to 37, wherein the serum half-life enhancer is selected from the group consisting of Fc domain, transferrin, albumin, XTEN, homoamino acid polymer (HAP), proline-alanine-serine polymer (PAS), elastin-like peptide (ELP), and polyethylene glycol. Section 40 The method described in any of sections 32-39, wherein the serum half-life enhancer is an Fc domain. Section 41 The method described in any of sections 32-39, wherein the serum half-life enhancer is not an Fc domain or polyethylene glycol. Section 42 The method described in any of sections 32 to 41, wherein the sialidase contains one or more mutations compared to the wild-type sialidase template. Section 43 Sialidase: (a) Substitution or deletion of a methionine residue at the position corresponding to position 1 of wild-type human Neu2 (M1); (b) Substitution of a valine residue at the position corresponding to position 6 of wild-type human Neu2 (V6); (c) Substitution of an isoleucine residue at the position corresponding to position 187 of wild-type human Neu2 (I187); or (d) Substitution of a cysteine ​​residue at position 332 of wild-type human Neu2 (C332); Or any combination of the aforementioned substitutions A method described in any of paragraphs 32 to 42, including the method described in paragraphs 32 to 42. Section 44 In sialidase: (a) The methionine residue at the position corresponding to position 1 of wild-type human Neu2 is deleted (ΔM1), substituted with alanine (M1A), or substituted with aspartic acid (M1D); (b) The valine residue at position 6 of wild-type human Neu2 is replaced with tyrosine (V6Y); (c) The isoleucine residue at position 187 of wild-type human Neu2 is replaced with lysine (I187K); or (d) The cysteine ​​residue at position 332 of wild-type human Neu2 is replaced with alanine (C332A); Alternatively, the sialidase contains any of the aforementioned combinations of substitutions. The method described in item 43. Section 45 Sialidase: (a) Substitution or deletion of a methionine residue at the position corresponding to position 1 of wild-type human Neu2 (M1); (b) Substitution of a valine residue at the position corresponding to position 6 of wild-type human Neu2 (V6); (c) Substitution of a proline residue at position 62 of wild-type human Neu2 (P62); (d) Substitution of an alanine residue at position 93 of wild-type human Neu2 (A93); (e) Substitution of an isoleucine residue at position 187 of wild-type human Neu2 (I187); (f) Substitution of a glutamine residue at position 126 of wild-type human Neu2 (Q126); (g) Substitution of an alanine residue at position 242 of wild-type human Neu2 (A242); (h) Substitution of a glutamine residue at the position corresponding to position 270 of wild-type human Neu2 (Q270); (i) Substitution of a serine residue at position 301 of wild-type human Neu2 (S301); (j) Substitution of a tryptophan residue at position 302 of wild-type human Neu2 (W302); (k) Substitution of a cysteine ​​residue at position 332 of wild-type human Neu2 (C332); (l) or any combination of the above substitutions A method described in any of paragraphs 32 to 44, including the above. Section 46 Sialidase: (a)M1D, V6Y, P62G, A93E, I187K, C332A; (b)M1D, V6Y, P62G, A93E, I187K, S301A, W302R, C332A; (c)M1D, V6Y, P62G, A93E, Q126Y, I187K, A242F, Q270T, C332A; (d) M1D, V6Y, P62G, A93E, Q126Y, I187K, C332A; and (e)A93E, Q126Y, I187K, A242F, Q270T, C332A A method according to any one of items 32 to 45, including a combination of substitutions selected from the group consisting of the following. Section 47 The method according to claim 46, wherein the sialidase conjugates to a serum half-life enhancer comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 115, 152, 180, 184, and 188, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% of an amino acid sequence selected from the group consisting of SEQ ID NOs: 115, 152, 180, 184, and 188. Section 48 Sialidase: (a) Substitution of a proline residue at the position corresponding to position 5 of wild-type human Neu2 (P5); (b) Substitution of a lysine residue at the position corresponding to position 9 of wild-type human Neu2 (K9); (c) Substitution of a lysine residue at position 44 of wild-type human Neu2 (K44); (d) Substitution of a lysine residue at the position corresponding to position 45 of wild-type human Neu2 (K45); (e) Substitution of a leucine residue at the position corresponding to position 54 of wild-type human Neu2 (L54); (f) Substitution of a proline residue at position 62 of wild-type human Neu2 (P62); (g) Substitution of a glutamine residue at position 69 of wild-type human Neu2 (Q69); (h) Arginine residue substitution (R78) at the position corresponding to position 78 of wild-type human Neu2; (i) Substitution of an aspartic acid residue at the position corresponding to position 80 of wild-type human Neu2 (D80); (j) Substitution of an alanine residue at position 93 of wild-type human Neu2 (A93); (k) Substitution of a glycine residue at position 107 of wild-type human Neu2 (G107); (l) Substitution of a glutamine residue at the position corresponding to position 108 of wild-type human Neu2 (Q108); (m) Substitution of a glutamine residue at position 112 of wild-type human Neu2 (Q112); (n) Substitution of a cysteine ​​residue at position 125 of wild-type human Neu2 (C125); (o) Substitution of a glutamine residue at position 126 of wild-type human Neu2 (Q126); (p) Substitution of an alanine residue at the position corresponding to position 150 of wild-type human Neu2 (A150); (q) Substitution of a cysteine ​​residue at position 164 of wild-type human Neu2 (C164); (r) Substitution of an arginine residue at the position corresponding to position 170 of wild-type human Neu2 (R170); (s) Substitution of an alanine residue at position 171 of wild-type human Neu2 (A171); (t) Substitution of a glutamine residue at position 188 of wild-type human Neu2 (Q188); (u) Arginine residue substitution (R189) at the position corresponding to position 189 of wild-type human Neu2; (v) Substitution of an alanine residue at position 213 of wild-type human Neu2 (A213); (w) Leucine residue substitution at position 217 of wild-type human Neu2 (L217); (x) Substitution of a glutamate residue at position 225 of wild-type human Neu2 (E225); (y) Substitution of a histidine residue at position 239 of wild-type human Neu2 (H239); (z) Substitution of a leucine residue at the position corresponding to position 240 of wild-type human Neu2 (L240); (aa) Arginine residue substitution (R241) at the position corresponding to position 241 of wild-type human Neu2; (bb) Substitution of an alanine residue at position 242 of wild-type human Neu2 (A242); (cc) Substitution of a valine residue at position 244 of wild-type human Neu2 (V244); (dd) Substitution of a threonine residue at position 249 of wild-type human Neu2 (T249); (ee) Substitution of an aspartic acid residue at position 251 of wild-type human Neu2 (D251); (ff) Substitution of a glutamate residue at position 257 of wild-type human Neu2 (E257); (gg) Substitution of a serine residue at position 258 of wild-type human Neu2 (S258); (hh) Leucine residue substitution at position 260 of wild-type human Neu2 (L260); (ii) Substitution of a valine residue at position 265 of wild-type human Neu2 (V265); (jj) Substitution of a glutamine residue at the position corresponding to position 270 of wild-type human Neu2 (Q270); (kk) Substitution of a tryptophan residue at position 292 of wild-type human Neu2 (W292); (ll) Substitution of a serine residue at position 301 of wild-type human Neu2 (S301); (mm) Substitution of a tryptophan residue at position 302 of wild-type human Neu2 (W302); (nn) Substitution of a cysteine ​​residue at position 332 of wild-type human Neu2 (C332); (oo) Substitution of a valine residue at position 363 of wild-type human Neu2 (V363); or (pp) Leucine residue substitution at position 365 of wild-type human Neu2 (L365); or any combination of the substitutions mentioned above A method described in any of paragraphs 32 to 47, including the method described in paragraphs 32 to 47. Section 49 The method described in any of items 32 to 48, wherein the sialidase is selected from the group consisting of bacterial sialidase, viral sialidase, and mammalian sialidase. Item 50 The method described in item 49, wherein the mammalian sialidase is human sialidase. Section 51 The method according to item 50, wherein the human sialyidase is selected from the group consisting of neu1, neu2, neu3, and neu4. Section 52 The method described in item 51, wherein human sialyidase is neu2. Section 53 The method described in any of sections 32 to 52, wherein approximately 0.01 mg / kg to approximately 100 mg / kg of sialidase is administered to the subject. Section 54 The method described in any of sections 32 to 53, wherein the cancer is a solid tumor, soft tissue tumor, hematopoietic tumor, or metastatic lesion. Section 55 The method described in paragraph 54, wherein the solid tumor is a sarcoma, adenocarcinoma, or carcinoma. Section 56 The method according to paragraph 54 or 55, wherein the solid tumor is a tumor of the head and neck (e.g., pharynx), thyroid gland, lung (e.g., small cell or non-small cell lung cancer (NSCLC)), breast, lymphatic system, gastrointestinal tract (e.g., oral cavity, esophagus, stomach, liver, pancreas, small intestine, colon and rectum, anal canal), reproductive or urogenital tract (e.g., kidney, urothelium, bladder, ovary, uterus, cervix, endometrium, prostate, testis), CNS (e.g., nerve or glial cells, e.g., neuroblastoma or glioma) or skin (e.g., melanoma). Section 57 The method described in paragraph 56, wherein the hematopoietic malignancy is leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), B-cell, T-cell or FAB ALL, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), e.g., modified CLL, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, pilocytic cell leukemia, myelodysplastic syndrome (MDS), lymphoma, Hodgkin's disease, malignant lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, multiple myeloma, or Richter syndrome (Richter transformation). Section 58 The method described in paragraph 56, where the cancer is breast cancer. Section 59 The method described in paragraph 56, where the cancer is lymphoma. Section 60 The method according to any one of claims 32 to 59, wherein administration of a pharmaceutical composition increases the expression of granzyme B, IFNγ, IL-10, IL-6, or IL-17A in a subject. Section 61 The method according to any one of paragraphs 32 to 60, wherein a pharmaceutical composition is administered to a subject in combination with another therapeutic agent. Section 62 The method according to item 61, wherein the therapeutic agent is selected from the group consisting of anti-inflammatory agents, anti-vasoconducting agents, anti-fibrotic agents, or antiproliferative compounds (e.g., cytotoxic agents or checkpoint inhibitors). Section 63 The method according to any one of claims 32 to 62, wherein the pharmaceutical composition further comprises a stabilizing amount of a sialidase stabilizer. Section 64 The method according to item 63, wherein the sialidase stabilizer is a cation. Section 65 The method according to item 64, wherein the cation is selected from the group consisting of calcium and magnesium. Section 66 The method according to item 65, wherein the pharmaceutical composition is placed in a sterile container (e.g., a bottle or vial) before administration. Section 67 A method for treating cancer in a subject requiring cancer treatment, comprising the step of administering an effective amount of a pharmaceutical composition described in any of items 1 to 31 to the subject requiring cancer treatment. Section 68 A method for removing sialic acid from cells in a subject, comprising the step of administering an effective amount of a pharmaceutical composition described in any of items 1 to 31 to the subject, thereby removing sialic acid from the cells. Section 69 The method according to item 68, wherein the cells are tumor cells, dendritic cells (DCs), or monocytes. Section 70 The method according to item 69, wherein the cells are monocytes, and the method results in increased expression of MHC-II molecules on the monocytes. Section 71 A method for increasing the phagocytic activity of tumor cells in a subject, comprising the step of administering to a subject an effective amount of a pharmaceutical composition according to any one of items 1 to 31 in an amount effective for removing sialic acid from tumor cells, thereby increasing the phagocytic activity of tumor cells. Section 72 A method for activating dendritic cells (DCs) in a subject, comprising the step of administering to a subject an amount of a pharmaceutical composition described in any of items 1 to 31 that is effective in removing sialic acid from tumor cells in the subject, thereby activating dendritic cells (DCs) in the subject. Section 73 A method for reducing Siglec-15 binding activity and thereby increasing antitumor activity in the patient's tumor microenvironment, comprising the step of administering an effective amount of a pharmaceutical composition described in any of items 1 to 31 to a subject, thereby increasing antitumor activity (e.g., T cell activity) in the subject. Section 74 (a) A step of providing cells containing nucleic acids encoding recombinant sialidase; and (b) A step of expressing recombinant sialidase in the presence of a stabilizer. A method for expressing recombinant sialidase, including [specific ingredient / method]. Section 75 The method according to claim 74, further comprising purifying the recombinant sialidase produced in step (b). Section 76 The method described in item 75, wherein the purification is carried out in the presence of a stabilizer. Section 77 The method described in any of sections 74 to 76, wherein the stabilizer is a cation. Section 78 The method according to item 77, wherein the cation is selected from the group consisting of calcium and magnesium.

[0314] Match list Table 28 Table 29 Table 30 Table 31 Table 32 Table 33 Table 34 Table 35 Table 36 Table 37 Table 38 Table 39 Table 40 Table 41 Table 42 Table 43 Table 44 Table 45 Table 46 Table 47 Table 48 Table 49 Table 50 Table 51 Table 52 Table 53 Table 54 Table 55 Table 56 Table 57 Table 58 Table 59 Table 60 Table 61 Table 62 Table 63 Table 64 Table 65 Table 66 Table 67 Table 68 Table 69 Table 70 Table 71 Table 72 Table 73 Table 74 Table 75 Table 76 Table 77 Table 78 Table 79 Table 80 Table 81 Table 82 Table 83

Claims

1. A pharmaceutical composition for use in removing sialic acid from tumor cells or immune cells in a subject, comprising a sialidase conjugated to an Fc domain that increases the serum half-life of the sialidase when administered to a subject, wherein the sialidase is not linked to a targeting portion and the sialidase comprises the amino acid sequence of SEQ ID NO:

48.

2. The pharmaceutical composition according to claim 1, wherein the sialidase and the Fc domain are covalently bonded together or chemically conjugated together in the fusion protein.

3. The pharmaceutical composition according to claim 2, wherein the fusion protein containing sialidase conjugated to an Fc domain comprises the amino acid sequence of SEQ ID NO: 115 or 152.

4. The pharmaceutical composition according to any one of claims 1 to 3, further comprising a cation.

5. The pharmaceutical composition according to claim 4, wherein the cation is selected from the group consisting of calcium and magnesium.

6. The pharmaceutical composition according to any one of claims 1 to 5, for use in combination with an anti-CD20 antibody.

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