Recombinant sialidase and its method of use

Abstract

In a method for treating cancer by desialylation of sialylated glycans expressed on the surface of cancer cells, the present invention provides a method for extending the serum half-life of the active ingredient, sialidase, and enhancing the therapeutic effect of said sialidase. [Solution] A pharmaceutical composition is provided that contains sialidase which, when administered to a subject, conjugates to a serum half-life enhancing factor that increases the serum half-life of sialidase.

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 sialoglycans, 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, and sia This leads to the expression of rilylated tumor-associated carbohydrate antigens. The expression of sialylated glycans by tumor cells is often associated with increased tumor aggressiveness and metastatic potential.

[0004] Recently, Siglec (sialic acid-binding immunoglobulin), a family of sialic acid-binding lectins, has been gaining attention. Phosphorus-like lectins bind to hypersialylated cancer cells and activate NK cell receptors, which are derived from signals. It has been shown that it plays a role in cancer immunosuppression by mediating the suppression of NK cells, 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, seared Enzymatic removal of sialic acid by treatment with ze 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 Initiative]

[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, the targeted portion (e.g., an anti-tumor antigen) Sialidases lacking a body-binding domain or conjugated to serum half-life enhancers It has been discovered that the allinase enzyme can effectively treat sialic acid-mediated disorders (e.g., cancer, e.g., solid tumors) in vivo.

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

[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, sialidase exhibits at least 50% of the activity of full-length sialidase. It is a functional fragment or variant of 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 comprises an 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 polymer. Selected from the group consisting of ethylene 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, sialidase has one or more mutations relative to the wild-type template sialidase. Includes differences.

[0017] In one embodiment, sialidase is located at the position corresponding to position 1 of wild-type human Neu2. Substitution or deletion of a valine residue (M1); valine residue at the position corresponding to position 6 in wild-type human Neu2. Substitution (V6); substitution of an isoleucine residue at the position corresponding to position 187 of wild-type human Neu2 (I187) ;or substitution of a cysteine ​​residue at the position corresponding to position 332 of wild-type human Neu2 (C332); or includes any combination of the substitutions described above. 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 wild The cysteine ​​residue at position 332 in type human Neu2 is substituted with alanine (C332A); or the sialidase contains one of the aforementioned combinations of substitutions.

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

[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, sialidase conjugated to serum half-life enhancer is an amino acid sequence or sequence number selected from the group consisting of SEQ ID NOs: 115, 152, 180, 184, and 188. Numbers: A small number of amino acid sequences selected from the group consisting of 115, 152, 180, 184, and 188. It contains amino acid sequences that make up at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

[0021] In one aspect, sialidase is prolyx at the position corresponding to position 5 of wild-type human Neu2. Substitution of lysine residue (P5); substitution of lysine residue at the position corresponding to position 9 of wild-type human Neu2 (K9); Lysine residue substitution at position 44 of biotype human Neu2 (K44); position 45 of wild-type human Neu2 Substitution of a lysine residue at the position corresponding to (K45); at the position corresponding to position 54 of wild-type human Neu2 Leucine residue substitution (L54); substitution of a proline residue at the position corresponding to position 62 of wild-type human Neu2. Substitution (P62); substitution of a glutamine residue at the position corresponding to position 69 of wild-type human Neu2 (Q69); substitution of an arginine residue at the position corresponding to position 78 of wild-type human Neu2 (R78); position 80 of wild-type human Neu2 Substitution of an aspartic acid residue at the corresponding position (D80); corresponding to position 93 of wild-type human Neu2. Substitution of an alanine residue at position (A93); substitution of a glycine residue at position 107 of wild-type human Neu2 (G107); substitution of a glutamine residue at position 108 of wild-type human Neu2 (Q108); substitution of a glutamine residue at position 112 of wild-type human Neu2 (Q112); substitution of a cysteine ​​residue at position 125 of wild-type human Neu2 (C125); substitution of a glutamine residue at position 126 of wild-type human Neu2 (Q126); position corresponding to position 150 of wild-type human Neu2 Substitution of an alanine residue at position (A150); cystamine at position 164 of wild-type human Neu2 Substitution of an arginine residue (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 the position corresponding to position 188 of wild-type human Neu2 (Q188); substitution of an arginine residue at the position corresponding to position 189 of wild-type human Neu2 (R189); substitution of an arginine residue at the position corresponding to position 213 of wild-type human Neu2 Alanine residue substitution at position (A213); leucine at position 217, corresponding to position 217 of wild-type human Neu2. Substitution of a residue (L217); substitution of a glutamate residue at the position corresponding to position 225 of wild-type human Neu2 (E225); substitution of a histidine residue at the position corresponding to position 239 of wild-type human Neu2 (H239); substitution of a leucine residue at the position corresponding to position 240 of wild-type human Neu2 (L240); substitution of an arginine residue at the position corresponding to position 241 of wild-type human Neu2 (R241); position corresponding to position 242 of wild-type human Neu2 Substitution of an alanine residue at position (A242); valine residue at position 244, corresponding to wild-type human Neu2. Substitution of a group (V244); substitution of a threonine residue at the position corresponding to position 249 of wild-type human Neu2 (T249); substitution of an aspartate residue at the position corresponding to position 251 of wild-type human Neu2 (D251); substitution of a glutamate residue at the position corresponding to position 257 of wild-type human Neu2 (E257); 2 Substitution of a serine residue at position 58 (S258); corresponding to position 260 in wild-type human Neu2 Leucine residue substitution at position (L260); valine residue at position 265 of wild-type human Neu2 Substitution of a group (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); corresponding to position 332 of wild-type human Neu2 Substitution of a cysteine ​​residue at position (C332); variant at position 363 of wild-type human Neu2. Substitution of a leucine residue (V363); or a leucine residue at the position corresponding to position 365 of wild-type human Neu2. Substitution of (L365); or any combination of the aforementioned 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 embodiment, the pharmaceutical composition 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 an anti-inflammatory agent, an anti-vasculitizing agent, an anti-fibrotic agent, or an antiproliferative compound (e.g., a cytotoxic agent or The group is selected from the group consisting of 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, sialidase exhibits at least 50% of the activity of full-length sialidase. This is a functional fragment of full-length sialidase. In one 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, serum half-life enhancers include Fc domains, transferrin, albumin, XTEN, homoamino acid polymers (HAP), proline-alanine-serine polymers (PAS), and elastin. Selected from the group consisting of stin-like peptides (ELPs) 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, sialidase has one or more mutations relative to the wild-type template sialidase. It contains differences. In one embodiment, sialidase is at the position corresponding to position 1 of wild-type human Neu2. Substitution or deletion of the methionine residue (M1); at the position corresponding to position 6 of wild-type human Neu2 Phosphorus residue substitution (V6); substitution of an isoleucine residue at the position corresponding to position 187 of wild-type human Neu2. This includes substitution (I187); 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, at the position corresponding to position 1 of wild-type human Neu2 The methionine residue is deleted (ΔM1), substituted with alanine (M1A), or replaced with asparagine. 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 is one of the above combinations of substitutions. Includes se.

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

[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, sialidase conjugated to serum half-life enhancer has an amino acid sequence selected from the group consisting of SEQ ID NOs: 115, 152, 180, 184, and 188, or a sequence Numbers: A small amount of amino acid sequences selected from the group consisting of 115, 152, 180, 184, and 188. It contains an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence.

[0037] In one aspect, sialidase is prolyx at the position corresponding to position 5 of wild-type human Neu2. Substitution of lysine residue (P5); substitution of lysine residue at the position corresponding to position 9 of wild-type human Neu2 (K9); Lysine residue substitution at position 44 of biotype human Neu2 (K44); position 45 of wild-type human Neu2 Substitution of a lysine residue at the position corresponding to (K45); at the position corresponding to position 54 of wild-type human Neu2 Leucine residue substitution (L54); substitution of a proline residue at the position corresponding to position 62 of wild-type human Neu2. Substitution (P62); substitution of a glutamine residue at the position corresponding to position 69 of wild-type human Neu2 (Q69); wild-type Arginine residue substitution at position 78 of human Neu2 (R78); position 80 of wild-type human Neu2 Substitution of an aspartic acid residue at the corresponding position (D80); corresponding to position 93 of wild-type human Neu2. Substitution of an alanine residue at position (A93); substitution of a glycine residue at position 107 of wild-type human Neu2 (G107); substitution of a glutamine residue at position 108 of wild-type human Neu2 (Q108); substitution of a glutamine residue at position 112 of wild-type human Neu2 (Q112); substitution of a cysteine ​​residue at position 125 of wild-type human Neu2 (C125); substitution of a glutamine residue at position 126 of wild-type human Neu2 (Q126); position corresponding to position 150 of wild-type human Neu2 Substitution of an alanine residue at position (A150); cystamine at position 164 of wild-type human Neu2 Substitution of an arginine residue (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 the position corresponding to position 188 of wild-type human Neu2 (Q188); substitution of an arginine residue at the position corresponding to position 189 of wild-type human Neu2 (R189); substitution of an arginine residue at the position corresponding to position 213 of wild-type human Neu2 Alanine residue substitution at position (A213); leucine at position 217, corresponding to position 217 of wild-type human Neu2. Substitution of a residue (L217); substitution of a glutamate residue at the position corresponding to position 225 of wild-type human Neu2 (E225); substitution of a histidine residue at the position corresponding to position 239 of wild-type human Neu2 (H239); substitution of a leucine residue at the position corresponding to position 240 of wild-type human Neu2 (L240); substitution of an arginine residue at the position corresponding to position 241 of wild-type human Neu2 (R241); position corresponding to position 242 of wild-type human Neu2 Substitution of an alanine residue at position (A242); valine residue at position 244, corresponding to wild-type human Neu2. Substitution of a group (V244); substitution of a threonine residue at the position corresponding to position 249 of wild-type human Neu2 (T249); substitution of an aspartate residue at the position corresponding to position 251 of wild-type human Neu2 (D251); substitution of a glutamate residue at the position corresponding to position 257 of wild-type human Neu2 (E257); substitution of a serine residue at the position corresponding to position 258 of wild-type human Neu2 (S258); position corresponding to position 260 of wild-type human Neu2 Leucine residue substitution at position (L260); valine residue at position 265 of wild-type human Neu2 Substitution of a group (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); corresponding to position 332 of wild-type human Neu2 Substitution of a cysteine ​​residue at position (C332); variant at position 363 of wild-type human Neu2. Substitution of a leucine residue (V363); or a leucine residue at the position corresponding to position 365 of wild-type human Neu2. Substitution of (L365); or any combination of the aforementioned 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 embodiments, cancer is a solid tumor, soft tissue tumor, hematopoietic malignancy, or metastatic lesion. In some embodiments, a solid tumor is a sarcoma, adenocarcinoma, or carcinoma. In some embodiments, 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 The urinary tract, lymphatic system, gastrointestinal tract (e.g., oral cavity, esophagus, stomach, liver, pancreas, small intestine, colon and rectum, anus) The anal canal, reproductive organs or urogenital tract (e.g., kidneys, urothelium, bladder, ovaries, uterus, cervix, endometrium, prostate, testes), CNS (e.g., nerves or glial cells, e.g., neuroblastoma) It is a tumor of the skin (e.g., melanoma) or a glioma. In one aspect, the cancer is breast cancer. That is the case.

[0041] In one aspect, hematopoietic malignancies include leukemia, acute leukemia, and acute lymphoblastic leukemia (ALL). B cells, T cells 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 It is either multiple myeloma or Richter syndrome (Richter transformation). In one aspect, the 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 an anti-inflammatory agent, an anti-vasculoforming agent, an anti-fibrotic agent, or an antiproliferative compound ( For example, it is selected from the group consisting of 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 reduces Siglec-15 binding activity, thereby reducing the tumor of a patient. The present invention relates to a method for increasing antitumor activity in a microenvironment, wherein the method involves administering an effective amount of a pharmaceutical composition to a subject, thereby increasing antitumor activity (e.g., T cell activity) in the subject. Includes the process.

[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 is performed by This can be done in the presence of a stabilizer such as thion (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 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, the sialidase or targeted moiety (e.g., tumor antigen) Sialider conjugated to serum half-life enhancer lacking an antibody-binding domain against It has been discovered that the enzyme 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 provides appropriate substrate specificity and sialic acid and / or from the surface of cancer cells. This invention relates to recombinant forms of sialidase enzymes having activity useful for the removal of sialic acid-containing molecules and / or sialic acid and / or sialic acid-containing molecules 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 refers to one or more amino acids relative to the wild-type sialidase sequence. Variants having acid substitution, deletion or insertion, and / or fusions including sialidase Contains proteins or conjugates. Sialidase is also called 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 enzymatic activity is, for example, that of the fluorescent substrate 4-methylumbelliferyl-N-acetylneutron. Known in the art, including measuring the release of sialic acid from lamic acid (4MU-NeuAc). It can be assayed by any method. 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, as described above, a sialidase having at least one mutation from wild-type human sialidase. For example, recombinant human sialidase containing at least one amino acid substitution, deletion, or addition. That is the case.

[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 includes N-terminal addition and C332A substitution of MEDLRP (SEQ ID NO: 4). In one embodiment, sialidase is N-terminal addition of MEDLRP (SEQ ID NO: 4), C332A substitution This includes substitution with C352L.

[0063] In one embodiment, the sialidase moiety is 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 replacement, A242T replacement, Q270A replacement, Q270T replacement, S301R replacement, S301R replacement, W302K replacement, W302R replacement This includes C332A substitution, V363R substitution, L365I substitution, or any combination of the above.

[0064] In one embodiment, sialidase is one of sequence numbers: 48-62, 169-171 or 196 At least one of the amino acid sequences, or one of 48-62, 169-171, or 196 It also includes amino acid sequences with 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity. .

[0065] a. Viral sialidase An example of viral sialidase is the surface glycoprotein of influenza A virus. Quality neuraminidase (e.g., NCBI accession number ACY01419.1, sequence number: 63), influenza B Viral surface glycoprotein neuraminidase (e.g., NCBI accession number AIX94926.1, SEQ ID NO: 1) Examples include :64) or influenza C virus surface glycoprotein neuraminidase or its variants or functional fragments. Other exemplary 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, sequence number: 66), Nydoid virus (e.g., UniProtKB entrusted P04853.1, sequence number: 67), Rubra virus, Mun Examples include Pusvirus, Simianvirus 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: The nucleotide sequence encoding Salmonella typhimurium sialidase, shown in SEQ ID NO: 30, is shown in SEQ ID NO: 6. The amino acid sequence of Vibrio cholerae sialidase is shown in SEQ ID NO: 36. The nucleotide sequence encoding Vibrio cholerae sialidase is shown in Sequence ID: 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; Arthrobacter sialophilus sialidase; and Arthrobacter ureafaciens L, M1, M2 and S (GenBank). Accession number BAD66680, Sequence ID: 69); sialidase derived from Bacteroides fragilis; Sialidase derived 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 derived from Mycobacterium malignantum (e.g., GenBank accession number CAA44916.1, SEQ ID NO: 107); Sialidase derived from Clostridium sordellii; Sialidase derived from Clostridium tertium (e.g., GenBank accession number CAA69951, SEQ ID NO: 73); Sialidase derived from Mycobacterium diphtheriae Haemophilus parasus (e.g., GenBank accession number ACS34893, sequence number: 74); Haemophilus parasusi Sialidase derived from Micromonospora viridifaciens (e.g., GenBank accession number BAA00852, sequence number: 75); Pastus Pasteurella multocida NanH (GenBank accession number AAG35310.1, SEQ ID NO: 76) and NanB (AAG35309, SEQ ID NO: 77); sialidase derived from Pseudomonas aeruginosa (e.g., GenBank accession number AAG06182, SEQ ID NO: 78); sialidase derived from Salmonella typhimurium (e.g., GenBank Accession number NP_459905, Sequence ID: 79); Streptococcus pneumoniae NanA (GenBank accession number P62575, Sequence ID: 108), NanB (GenBank accession number AAC44396, Sequence ID: 80), and NanC; Sialidase from Tannerella forsythia (e.g., GenBank accession number TF0035, Sequence ID: 81); Sialidase from Vibrio cholerae (e.g., GenBank accession number YP_001217324) (Sequence ID: 82), sialidase derived from *C. diphtheriae* (*Cdip_NanH*, specifically *C. diphtheriae KCTC3075 NanH (GenBank accession number ACS34893, Sequence ID: 83) and bisono homolog; Corynebacterium glutamicum (R) (tentative) Suggestion 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 (B. fragilis)YCH46 sialidase (Bfra_NanH; Uniprot accession number BAA05853, sequence number: 86); Micromonospora viridifaciens sialidase (Mvir_NanH) Uniprot accession number BAA0085, SEQ ID NO: 87); Streptococcus 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 decora) trans sialidase (Mdec_NanL; AAC47263, SEQ ID NO: 92); cruzi trypanosoma (T. cruzi) trans sialidase (Tcru_TS; GenBank accession number AAA99442, SEQ ID NO: 93); app Carmansia 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 Bacteroides thetaiotaomicron VPI-5482 sialidase (BtsA; BTSA; BT0455) (GenBank accession number Q8AAK9, SEQ ID NO: 96); Bacteroides vulgatus ATCC 8482 / DSM 1447 / NCTC 11154 BVU_4143 (Uniprot accession number A6L7T1, SEQ ID NO: 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, sequence number: 100); Clostridium perfringens ATCC 13124 sialidase (CPF_0721) (Uniprot accession number Q0TT67, sequence number: 101); Clostridium perfringens str 13 exo-α-sialidase Examples include 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 14573 sialidase (NanH;SiaH) (Uniprot accession number P77848, SEQ ID NO: 104); Ruminococcus gnavus ATCC 29149 RgNanH (Uniprot accession number A7B557, SEQ ID NO: 105); and Salmonella typhimurium TA262 / LT2 sialidase (NanH;STSA) (P29768, SEQ ID NO: 106).

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

[0069] c. Mouse sialidase Four sialidases have also been found in the mouse genome, including Neu1, Neu2, Neu3, and It is referred to as 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 sialydase Four sialidases have also been found in the human genome: Neu1, Neu2, Neu3, and Neu4 It is called that.

[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. Human Neu3, isoform 2 The amino acid sequence is shown in Sequence ID No. 9, and it encodes the human Neu3, isoform 2 nucleus. The Otid sequence is shown at sequence number 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. The nucleotide sequence encoding human Neu4, isoform 2 is shown in Sequence ID: 35.

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

[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 exhibits enzyme activity 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. The recombinant mutant human sialidase has sexual properties, and the expression yield of the recombinant mutant human sialidase in mammalian cells, 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.

[0079] In one aspect, the amino acid sequence of a recombinant mutant human sialidase is the same as the corresponding wild type. It has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the amino acid sequence of human sialyidase.

[0080] The sialidases described herein, such as human sialidase, are characterized by one or more properties of the enzyme. To enhance performance, for example, by improving expression, activity, and stability (for example, against protease degradation) It is understood that some of these properties may be modified to improve resistance to certain conditions. For example, improved resistance to protease degradation is 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 contribute to protein aggregation. As a result, it has been found that the expression of functional proteins can be inhibited. Therefore, in one embodiment, recombinant mutant human sialidase has at least one mutation to remove free cysteine ​​(for example, 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), C125, C196, C219 , one or more mutations in C272, C332 and C352; for Neu3 (SEQ ID NO: 8), one or more mutations in C7, C90, C99, C106, C127, C136, C189, C194, C226, C242, C250, C273, C279, C295, C356, C365, C368, C384, C383, C394 and C415; and for Neu4 (SEQ ID NO: 10) (Contains one or more mutations of C88, C125, C126, C186, C191, C211, C223, C239, C276, C437, C453, C480 and C481). Free cysteine ​​can be substituted with any amino acid. In this 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 and C332A; C272V and C332S; C332A and C352L; C125S and C196L; C196L and C352L Examples include C196L and C332A; C332A and C352L; and C196L, C332A and C352L. ru.

[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 is shown in Table 1 (wild-type human Neu2 (Sequence ID). :1) Includes the amino acid position corresponding to the substitution or combination of substitution listed in 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 least 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 the smallest possible pH around neutral. It is soluble, and this is undesirable as the expression and physiological systems are at a neutral pH. In contrast, sialidase derived 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 should have one or more amino acid substitutions (one or 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 sialidases without substitutions. Furthermore, a reduction in the number of hydrophobic amino acids on the surface of the 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) decrease the hydrophobicity of the surface of the sialidase compared to sialidases without substitutions.

[0087] Therefore, in one embodiment, recombinant mutant human sialidase has at least one This includes amino acid substitutions, where the substitution increases the isoelectric point (pI) of the sialidase and / or decreases the hydrophobicity of the sialidase compared to a sialidase without 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 a charged amino acid, For example, the amino acid substitution is for a positively charged amino acid, such as lysine (lys, K), histidine (his, H), or arginine (arg, R), or a negatively charged amino acid, such as 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, the amino acid substitution is a surface-exposed D or E amino acid in a helix or loop, or in a position having K or R at the corresponding position of St-sialidase. This occurs. In one embodiment, amino acid substitutions are amino acids that are separated 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 functionally important domains. This can occur, for example, with amino acids not located within the Asp-box or β-strand.

[0089] For sialidases without substitution, increasing the isoelectric point (pI) of the sialidase and / or Examples of amino acid substitutions in Neu2 that reduce the hydrophobicity of sialidase include A2E, A2K, D215K, V325E, V325K, E257K, and E319K. In one embodiment, recombinant The variant human sialyidase contains 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 is shown in Table 2 (wild-type human Neu2 (Sequence ID). :1) The substitution or combination of substitutions listed in the amino acid position corresponding to 1) This includes combinations of substitutions. [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, Peptides are at least two amino acid lengths, for example, 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 lengths. In this embodiment, the peptide may be capable of forming 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 is the same as the Neu2 isoform found in skeletal muscle. It contains six amino acids that are not present in the canonical isoform. In some aspects of this specification In this context, the N-terminal six amino acids of the mouse thymus Neu2 isoform, MEDLRP (SEQ ID NO: 4), or a variation thereof, can be added to human Neu, for example, human Neu2. In one embodiment, recombinant mutants 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 (Sequence No. 4) or EDLRP (Sequence No. 4) covalently bonded to the N-terminal amino acid of the sialidase. Includes (Sequence No. 3). In one embodiment, the sialidase may further include a cleavage site located between the peptide, e.g., MEDLRP (Sequence No. 4) or EDLRP (Sequence No. 3) and the rest of the sialidase, e.g., a proteolytic cleavage site. In one embodiment, the peptide, e.g., MEDLRP (Sequence No. 4) Column number: 4) or EDLRP (Sequence ID: 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 Can methionine be removed, or can the first five amino acids (MASLP; SEQ ID NO: 12) be removed? Alternatively, the second to fourth amino acids (ASLP; Sequence ID: 13) may be removed.

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

[0095] Human sialyidase has a toroidal arrangement of 6 blade-shaped β-septum cells around a central axis. It has a β-propeller structure characterized by [a specific feature]. Generally, hydrophobic interactions between β-propeller blades, such as between the N-terminal and C-terminal blades, enhance stability. Therefore, to increase the expression of human Neu2 or other human sialidases, amino acids that increase hydrophobic interactions and / or hydrogen bonds between the N-terminal and C-terminal β-propeller blades of the sialidase are used. Recombinant mutant human sialidases, including those involving substitution, can be designed.

[0096] Therefore, in one embodiment, recombinant mutant human sialidase has at least one This includes substitution of wild-type amino acid residues, where the substitution increases hydrophobic interactions and / or hydrogen bonding between the N-terminus and C-terminus of the sialidase compared to 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). An example in Neu2 is that it increases hydrophobic interactions and / or hydrogen bonding between the N-terminus and C-terminus. Examples of symbolic substitutions 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 contains the V6Y substitution.

[0097] In one embodiment, recombinant mutant human sialidase includes the above combination of 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, in combination with the above. The recombinant mutant may include 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 the 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 Allogeneic human Neu2 sialidase also contains at least one L4N, L4K, V6Y, L7N, L4N, and L7N This includes substitutions for L4N, V6Y, L7N, V12N, V12Y, V12L, V6Y, V6F, or V6W.

[0098] In one embodiment, recombinant mutant human sialidase is shown in Table 3 (wild-type human Neu2 (Sequence ID). :1) The mutations or combinations of mutations listed in the amino acid position corresponding to 1) This includes combinations of mutations. [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), 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 substitution of the N-terminal methionine at the position corresponding to position 1 of wild-type human Neu2 (SEQ ID NO: 1) This includes a deletion of a methionine residue (ΔM1) at the corresponding position.

[0100] In one embodiment, recombinant mutant human sialidase is shown in Table 4 (wild-type human Neu2 (Sequence ID). :1) The substitution or combination of substitutions listed in the amino acid position corresponding to 1) This includes combinations of substitutions. [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, the proteolytic cleavage of sialidases is This 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 sialyidase has at least one wild-type amino acid residue. This includes substitution, where the substitution is a protease (e.g., ) compared to an unsubstituted sialidase. It reduces the cleavage of sialidase by liposin.

[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%, 1% to 5%, 5% to 50%, 5% to 40%, 5% to 30%, 5% to 20%, 5% to 10%, 5% to 10%, 10% to 50%, 10% to 40%, 10% to 30%, and 10% to 30%. 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%, and This results in approximately 40% to 50% of the protein cleavage. 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 includes, for example, cleavage by SDS-PAGE as described in Example 5 of this specification. The assay can be performed by any method known in the art.

[0103] Exemplary substitutions that increase resistance to proteolytic cleavage include: (i) wild-type Substitutions of alanine residues at position 242 of Neu2 (Sequence ID: 1), such as 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), and tryptase. Substitution with fan (A242W) or tyrosine (A242Y); (ii) wild-type human Neu2 (Sequence ID: 1) (iii) Substitution of an arginine residue at the position corresponding to position 243, for example, by 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); also (iv) Any of the above combinations may be mentioned. In one embodiment, the recombinant mutant human sialidase includes substitutions selected from A242C, A242F, A242Y and A242W. In one embodiment, the recombinant mutant human sialidase corresponds to Table 5 (wild-type human Neu2 (SEQ ID NO: 1) The substitution or combination of substitutions corresponding to the substitution or combination of substitutions listed in the amino acid position. Includes. [Table 5]

[0104] Increases resistance to proteolytic cleavage (and / or expression yield and / or (This increases enzyme activity) Further exemplary substitutions include: (i) wild-type human Neu2 (SEQ ID NO: 1 Substitution of a leucine residue at the position corresponding to position 240 of ) for example, aspartic acid (L240D), as (ii) Substitution by paragine (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., cysteine ​​(A213C), asparagus (iii) Substitution of arginine residues at the position corresponding to position 241 of wild-type human Neu2 (SEQ ID NO: 1), e.g., substitution of alanine (R241A), aspartic acid (R241D), leucine (R241L), glutamine (R241Q), or tyrosine (R241Y); (iv) Substitution of serine residues at the position corresponding to position 258 of wild-type human Neu2 (SEQ ID NO: 1). Substance substitution, e.g., substitution 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), e.g., aspartic acid (L260D), phenyl Substitution with alanine (L260F), glutamine (L260Q), or threonine (L260T); (vi) wild (vii) substitution of a valine residue at the position corresponding to position 265 of human Neu2 (SEQ ID NO: 1), e.g., substitution with phenylalanine (V265F); or (vii) any combination of the above. In some embodiments, substitutions or combinations of substitutions at these positions may cause hydrophobic and / or aromatic interactions between secondary structure factors in the sialidase (e.g., between the α-helix and the nearest β-sheet). The aim is to improve the function, thereby stabilizing the structure and increasing resistance to proteolytic cleavage.

[0105] In certain embodiments, the recombinant mutant sialidase comprises a substitution at position L240. In certain embodiments, the recombinant mutant sialidase comprises a combination 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 certain embodiments, the recombinant mutant human sialidase comprises a combination 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 certain embodiments, the recombinant mutant human sialidase comprises a substitution or combination 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 a recombinant mutant human sialidase comprising at least one of the following substitutions: I187K, A328E, K370N or H210N. In certain embodiments, the 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 certain embodiments, the 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 certain embodiments, the 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 a recombinant variant human sialidase 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. A variant human sialidase is provided.

[0108] The present invention further provides a recombinant variant human sialidase comprising an amino acid substitution at an amino acid position corresponding to the position identified in Table 7 (corresponding to wild-type human Neu2 (SEQ ID NO: 1)). In certain embodiments, the sialidase comprises the amino acid substitutions identified in Table 7. In certain embodiments, the sialidase comprises any combination of the amino acid substitutions identified in Table 7.

Table 7-1

Table 7-2

Table 7-3

[0109] For example, in certain embodiments, the recombinant variant human sialidase is: (a) a substitution of a proline residue at a position corresponding to position 5 of wild-type human Neu2 (P5); (b) a substitution of a lysine residue at a position corresponding to position 9 of wild-type human Neu2 (K9); (c) a lysine residue at a position corresponding to position 44 of wild-type human Neu2 Substitutions (K44); (d) Lysine residue substitution at position 45 of wild-type human Neu2 (K45); (e) Leucine residue substitution at position 54 of wild-type human Neu2 (L54); (f) Proline residue substitution at position 62 of wild-type human Neu2 (P62); (g) Glutamine residue substitution at position 69 of wild-type human Neu2 (Q69); (h) Arginine residue substitution at position 78 of wild-type human Neu2 (R78); (i) Aspartate residue substitution at position 80 of wild-type human Neu2 (D80); (j) Alanine residue substitution at position 93 of wild-type human Neu2 (A93) (k) Substitution of a glycine residue at the position corresponding to position 107 of wild-type human Neu2 (G107); (l) wild-type (m) Substitution of a glutamine residue at the position corresponding to position 108 of human Neu2 (Q108); (n) Substitution of a glutamine residue at the position corresponding to position 112 of wild-type human Neu2 (Q112); (n) Substitution of a glutamine residue at the position corresponding to position 125 of wild-type human Neu2 (o) Substitution of a cysteine ​​residue at the corresponding position (C125); (p) 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) 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 the position corresponding to position 171 of wild-type human Neu2 (A171); (t) wild-type human (u) Substitution of a glutamine residue at position 188 of Neu2 (Q188); (u) Substitution of an arginine residue at position 189 of wild-type human Neu2 (R189); (v) Position 213 of wild-type human Neu2 (A213) Alanine residue substitution at the position corresponding to position 217 of wild-type human Neu2 (L217); (x) Glutamate residue substitution at the position corresponding to position 225 of wild-type human Neu2 (E225); (y) Histidine residue substitution at the position corresponding to position 239 of wild-type human Neu2 (H239); (z) Leucine residue substitution at the position corresponding to position 240 of wild-type human Neu2 (L240); (aa) Arginine residue substitution at the position corresponding to position 241 of wild-type human Neu2 (R241); (bb) Wild-type human Substitution of an alanine residue at position 242 of 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 (T249); (ee) at the position corresponding to position 251 of wild-type human Neu2 Substitution of aspartic acid residue (D251); (ff) Glucose at the position corresponding to position 257 of wild-type human Neu2 Substitution of tamic acid residue (E257); (gg) Serine residue at the position corresponding to position 258 of wild-type human Neu2 (S258); (hh) Substitution of a leucine residue at the position corresponding to position 260 of wild-type human Neu2 (L260); (ii) Substitution of a valine residue at the position corresponding to 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) wild-type human Neu2 Substitution of a tryptophan residue at position 292 (W292); (ll) Substitution of a serine residue at position 301 of wild-type human Neu2 (S301); (mm) Corresponding to position 302 of wild-type human Neu2 Substitution of a tryptophan residue at position (W302); (nn) corresponds to position 363 of wild-type human Neu2. Substitution of a valine residue at (V363); or (oo) at the position corresponding to position 365 of wild-type human Neu2 This includes leucine residue substitution (L365); or any combination of the aforementioned substitutions. For example, sialidase may contain substitutions of K9, P62, A93, Q216, A242, Q270, S301, W302, V363, or L365, or any combination of the aforementioned 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 position corresponding to position 9 of wild-type human Neu2 (c) The lysine residue in is replaced with aspartic acid (K9D); (d) The lysine residue at the position corresponding to position 44 in wild-type human Neu2 is replaced with arginine (K44R) or glutamic acid (K44E); (d) (e) The lysine residue at position 45 of biotype human Neu2 is substituted with alanine (K45A), arginine (K45R), or glutamate (K45E); (e) at position 54 of wild-type human Neu2 The leucine residue is substituted with methionine (L54M); (f) the position corresponding to position 62 of wild-type human Neu2. The proline residue in is replaced 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 in wild-type human Neu2 is replaced with histidine (Q69H). (h) The arginine residue at the position corresponding to position 78 of wild-type human Neu2 is lysine (R78K). Substitutions are made: (i) The aspartic acid residue at the position corresponding to position 80 of wild-type human Neu2 is replaced with proline (D80P); (j) The alanine residue at the position corresponding to position 93 of wild-type human Neu2 is replaced with glutamic acid (A93E) or lysine (A93K); (k) The glycine residue at the position corresponding to position 107 of wild-type human Neu2 is replaced with aspartic acid (G107D); (l) The glycine residue at position 108 The glutamine residue at the corresponding position is substituted 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 position 125 of wild-type human Neu2 is replaced with leucine (C125L); (o) The glutamine residue at position 126 of wild-type human Neu2 is replaced with leucine (Q126L), glutamic acid (Q126E), phenylalanine (Q126F), histidine (Q126H), isol (p) The alanine residue at position 150 of wild-type human Neu2 is replaced with valine (A150V); (q) the position corresponding to position 164 of wild-type human Neu2 The cysteine ​​residue at this position is replaced with glycine (C164G); (r) position 170 of wild-type human Neu2 (s) The arginine residue at the position corresponding to (t) is replaced with proline (R170P); (t) The alanine residue at the position corresponding to position 171 of wild-type human Neu2 is replaced with glycine (A171G); (t) The glutamine residue at the position corresponding to position 188 of wild-type human Neu2 is replaced with proline (Q188P); (u) The arginine residue at the position corresponding to position 189 of wild-type human Neu2 is replaced with proline (R189P); (v) The alanine residue at position 213 of biotype human Neu2 is substituted 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). Therefore; (x) The threonine residue at the position corresponding to position 249 in wild-type human Neu2 is alanine (T249A). (y) The aspartic acid residue at the position corresponding to position 251 of wild-type human Neu2 is replaced with glycine (D251G); (z) The glutamic acid residue at the position corresponding to position 225 of wild-type human Neu2 (aa) The histidine residue at the position corresponding to position 239 of wild-type human Neu2 is replaced with proline (H239P); (bb) The leucine residue at the position corresponding to position 240 of wild-type human Neu2 is replaced with aspartic acid (L240D), asparagine (L240N), or tyrosine (L240Y). Substituted; (cc) The arginine residue at 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 residue at position 242 of wild-type human Neu2 is cis Thein (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), Substituted with liptophan (A242W) or tyrosine (A242Y); (ee) position 244 of wild-type human Neu2 (ff) The valine residue at the position corresponding to (ff) is substituted with isoleucine (V244I), lysine (V244K), or proline (V244P); (gg) The glutamic acid residue at the position corresponding to position 257 of wild-type human Neu2 is substituted with proline (E257P); (gg) The serine residue at the position corresponding to position 258 is substituted with cysteine ​​(S258C); (hh) The leucine residue at the position corresponding to position 260 of wild-type human Neu2 is substituted with aspartic acid (L260D), phenylalanine (L260F), glutamine (L260Q), or threonine (L260T); (ii) The valine residue at the position corresponding to position 265 of wild-type human Neu2 is substituted with phenylalanine (L260F); Substituted with lanin (V265F); (jj) The glutamine residue at the position corresponding to position 270 of wild-type human Neu2 is substituted with alanine (Q270A), histidine (Q270H), phenylalanine (Q270F), proline (Q270P), serine (Q270S), or threonine (Q270T); (kk) Position 292 of wild-type human Neu2 The tryptophan residue at the corresponding position is substituted with arginine (W292R); (ll) The serine residue at the position corresponding to position 301 of wild-type human Neu2 is substituted with alanine (S301A), aspartic acid (S301D), glutamic acid (S301E), phenylalanine (S301F), glycine (S301G), histidine (S301H), isoleucine (S301I), lysine (S301K), leucine (S301L), methionine (S301M), asparagine (S301N), proline (S301P), glutamine (S301Q), arginine (S301R), threonine (S301T), valine (S301V), tryptophan (S301W), or tyrosine (S301Y). ;(mm)The tryptophan residues at position 302 in wild-type human Neu2 are alanine (W302A), aspartic acid (W302D), glutamic acid (W302E), phenylalanine (W302F), glycine (W302G), histidine (W302H), isoleucine (W302I), lysine (W302K), leucine (W302L), methionine (W302M), asparagine (W302N), proline (W302P), glutamine (W302Q), arginine (W302R), and serine (W302 (S) is substituted with threonine (W302T), valine (W302V), or tyrosine (W302Y); (nn) the valine residue at position 363 of wild-type human Neu2 is substituted with arginine (V363R); or (oo) the leucine residue at position 365 of wild-type human Neu2 is substituted with glutamine (L365Q), histidine (L365H), isoleucine (L365I), lysine (L365K), or serine (L365S); or the sialidase is any combination of the above substitutions. It includes. For example, sialidase may include 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 foregoing substitutions.

[0111] In certain embodiments, the recombinant mutant human sialidase corresponds to position 184 of wild-type human Neu2 Deletion of leucine residue (ΔL184) at the position corresponding to position 185 of wild-type human Neu2, histidine residue deletion (ΔH185) at the position corresponding to position 185 of wild-type human Neu2, proline residue deletion (ΔP186) at the position corresponding to position 186 of wild-type human Neu2, isoleucine residue deletion (ΔI187) at the position corresponding to position 187 of wild-type human Neu2 and glutamine residue deletion (ΔQ188) at the position corresponding to position 184 of wild-type human Neu2, or any combination of the foregoing deletions. at the position corresponding to position 186 of wild-type human Neu2, proline residue deletion (ΔP186), isoleucine residue deletion (ΔI187) at the position corresponding to position 187 of wild-type human Neu2 and glutamine residue deletion (ΔQ188) at the position corresponding to position 184 of wild-type human Neu2, or any combination of the foregoing deletions.

[0112] In certain embodiments, the recombinant mutant human sialidase corresponds to position 216 of wild-type human Neu2 Insertion between the threonine residue at the position corresponding to position 216 of wild-type human Neu2 and the leucine residue at the position corresponding to position 217 of wild-type human Neu2, for example, insertion of an amino acid selected from S, T, Y, L, F, A, P, V, I, N, D and H. including 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 in International (PCT) Patent Application No. PCT / US2019 / 012207 filed on January 3, 2019, for example, in the section entitled "I. Recombinant Human Sialidase" in the detailed description and in Examples 1, 2, 3, 4, 5 and 6 in the Examples. as described therein.

[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 It may include ~6, 2~5, 2~4, 2~3, 3~15, 3~10, 3~7, 3~6, ​​3~5, or 3~4. It is planned.

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

[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 C332A; M1D, V6Y and I187K; M1D, V6Y This may include combinations of mutations selected from 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.

[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) M1 deletion (ΔM1), M1A substitution, M1D substitution, V6Y substitution, I187K substitution, C332A substitution, or any combination thereof. 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 C This may include combinations of mutations selected from 332A;I187K and C332A;M1A, V6Y and I187K;M1A, 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 are: (a) M1D, V6Y, P62G, A93E, I18 (b) Replacement of 7K and C332A; (b) Placement of M1D, V6Y, K9D, A93E, I187K, C332A, V363R and L365I Replacement; (c) Replacement of M1D, V6Y, P62N, I187K and C332A; (d) Replacement of M1D, V6Y, I187K, Q270A, S301R (e) Replacement of W302K and C332A; (f) Replacement of M1D, V6Y, P62S, I187K, Q270A, S301R, W302K and C332A; (g) Replacement of M1D, V6Y, P62T, I187K, Q270A, S301R, W302K and C332A; (h) Replacement of M1D, V6Y, P62N, I187K, Q270A, S301R, W302K and C332A; (h) Replacement of M1D, V6Y, P62G, A93 Substitution of E, I187K, S301A, W302R and C332A; (i) substitution of M1D, V6Y, P62G, A93E, Q126Y, I187K, Q270T and C332A; or (j) 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 aspect, recombinant mutant human sialidase corresponds to position 302 of wild-type human Neu2. In combination with the substitution of a tryptophan residue at the position (W302) at position 301 of wild-type human Neu2, This includes a serine residue substitution at the corresponding position (S301). For example, recombinant mutant human sialidase may contain substitution combinations corresponding to the substitution combinations listed in the column of Table 8 (amino acid positions corresponding to wild-type human Neu2 (SEQ ID NO: 1)). For example, recombinant mutant human sialidase may contain: substitutions of S301K and W302R; substitutions of S301K and W302K; or substitutions of S301A and W302S. [Table 8]

[0120] In one embodiment, recombinant mutant human sialidase is shown in Table 9 (wild-type human Neu2 (Sequence ID). :1) The combination of substitutions corresponding to the amino acid position) listed in the column is include. [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 non-existent, and 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 Pro, Asn, Asp, His, Glu, Gly, Ser or Thr, and X 11 is Gln or His, and X 12 is 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 one embodiment, recombinant mutant human sialidase is [Table 11] (Sequence ID: 46) contains the amino acid sequence, where X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly X2 is His, Leu, Lys, Met, Phe, Thr, Val or is absent, 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 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 Ile or Lys, and X7 is Gln or Ala. X8 is Ser or Arg, X9 is Trp or Lys, X 10 is Ala or Cys, X 11 is Val or Arg, X 12 It is either Leu or Ile.

[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 is absent, and 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 Pro, Asn, Asp, His, Glu, Gly, Ser Or Thr, X 11 is Gln or His, X 12 is Arg or Lys, X 13 Asp also It is 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 ri, 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 31His 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 is 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 Yes, X 48 is Ala, Cys, Ser or Val, X 49 is Cys, Leu or Val, X 50 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, X5 is Ala, Glu, or Lys, X6 is Gln, Leu, Glu, Phe, His, Ile, Leu, or Tyr, X7 is Arg, Ile, or Lys, and X8 is Ala, Cys, Phe, Gly, His, Ile , Lys, Leu, Met, Asn, Gln, Arg, Ser, Val, Trp or Tyr, and 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 It is Leu, Gln, His, Ile, Lys or Ser, and the sialidase is wild-type human Neu2 (Sequence ID). :1) contains at least one mutation. 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, and X7 X is Ile or Lys, X8 is Ala or Thr, X9 is Gln, Ala or Thr, 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.

[0126] In one embodiment, a recombinant mutant human sialidase includes a conserved substitution in relation to the recombinant mutant human sialidase sequence 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. Existential substitutions can also be performed using 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 matrix). It can be defined by the 250 matrix.

[0127] Sequence identity can be verified by various methods within the scope of the art of the art, for example, by publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR). This can be determined using software. Programs such as blastp, blastn, blastx, tblastn and BLAST (Basic Local Alignment Search Tool) analysis using the algorithm employed by tblastx (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) is adapted for sequence similarity searches. For a discussion of fundamental issues in sequence database searches, see Altschul et al., (1994) NATURE GENETICS 6:119-129, as fully incorporated herein by reference. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm necessary to achieve the greatest alignment over the entire length of the sequences being compared. Histogram, description, alignment, expectation (that is) (Statistical significance threshold for reporting fit to database sequences), cutoff, The search parameters for tricks and filters are in 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, as fully incorporated herein by reference). The four blastn parameters can be adjusted as follows: Q=10 (gap generation penalty); R=10 (gap Gap extension penalty: wink=1 (generates a word hit at each position of wink.sup.th along the query); and gapw=16 (generates a gapped alignment). (Sets the window width to be used). Equivalent blastp parameter settings are Q=9;R=2;wink=1 ; and gapw=32. The search can also be performed using the NCBI (National Center for Biotechnology Information) BLAST Advanced Option parameters (e.g., -G, cost about open gap [integer]: default=5 / nucleotides / protein). Cost for nucleotides: 11;-E, cost for extension gap [integer]: default = nucleotide 2 for nucleotides / 1 for proteins; -q, Penalty for nucleotide mismatch [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 megablasts / 3 for proteins; -y, Blast extension in bits Drop-off for length (X): default = 20 for blastn / 7 for others; -X, X drop-off value (in bits) for gapped alignment: default = 15 for all programs but not applicable to blastn; and -Z, final X drop-off value (in bits) for gapped alignment: 50 for blastn, others Regarding other aspects (25), ClustalW is also used for pairwise protein alignment. (The default parameters are, for example, Blosum62 matrix and gap open pane.) (May include Gap Opening Penalty = 10 and Gap Extension Penalty = 0.1) In GCG package version 10.0, the optimal fit comparison between available sequences uses the DNA parameters GAP=50 (gap creation penalty) and LEN=3 (gap extension penalty). The equivalent settings for optimal protein synthesis 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, the serum half-life prolonger is an Fc domain (see, e.g., Beck et al. (2011) MABS 4:1015-28), albumin (For example, 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), Bumin-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), transferrin (see Kim et al. (2010) J PHARMACOL EXP THER 334:682-92), XTEN (also called recombinant PEG or "rPEG", see Schellenberger et al. (2009) NAT. BIOTECHNOL. 27:1186-90), homoamino acid poly Mar (HAP, see Schlapschy et al. (2007) PROTEIN ENG DES SEL. 20:273-84), Proli n-alanine-serine polymer (PAS, see Schlapschy et al. (2013) PROTEIN ENG DES SEL. 26:489-501), elastin-like peptide (ELP, see Floss et al. (2013) Fusion protein technologies for biopharmaceuticals: applications and challenges, p. 372-98), carboxy-terminal peptide (CTP, Duijkers et al. (2002) HUM REPROD. 17:1987-93), gelatin Chin-like protein (GLK, Huang et al. (2010) EUR J PHARM BIOPHARM 72:435-41) and Polyethylene glycol (PEG) may be selected.

[0129] Appropriate serum half-life prolongers also include species such as those described in U.S. Patent No. 7,842,789. This includes various polymers, such as block copolymers of polyoxyethylene and polyoxypropylene (Pluronics); polymethacrylates; carbomers; and D-mannose, D- and Branched or unbranched polysaccharides containing sugar monomers such as L-galactose, fucose, fructose, D-xylose, and L-arabinose, as well as D-glucuronic acid, may be used. In other embodiments, the serum half-life extender may be hydrophilic polyvinyl polymers such as polyvinyl alcohol and polyvinylpyrrolidone (PVP) type polymers. The serum half-life extender is Functionalized polyvinylpyrrolidone, for example, functionalized at one (or both) ends of the polymer It may be a carboxyl or amine (available from PolymerSource). Alternatively, Examples of serum half-life prolonging factors 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 has an Fc domain (Beck et al., above), and transfer Natural long half-life compounds such as nitrate (Kim et al., see above) or albumin (Weimer et al., see above) Covalently bonding to a polypeptide or protein during its development, it undergoes genetic fusion (i.e., recombination fusion). Fusion proteins are produced either by protein synthesis or chemical conjugation. To form.

[0131] In another embodiment, sialidase is XTEN (also called recombinant PEG or "rPEG", Schellenberger, see above), homoamino acid polymer (HAP, Schlapschy et al. (2007), above (See above), proline-alanine-serine polymer (PAS, Schlapschy et al., (2013), see above (See reference), covalently bond to inactive polypeptides such as elastin-like peptides (ELP, Floss et al., see above) or gelatin-like proteins (GLK, Huang et al., see above), thereby promoting genetic fusion. Fusion proteins are formed either by synthesis (i.e., the creation of recombinant fusion proteins) or chemical conjugate. Inactive polypeptides function to increase the size and hydrodynamic radius of sialidases in particular, thereby extending their half-life. In one embodiment, XTEN polypeptides range from approximately 25 amino acids to approximately 1500 amino acids (e.g., approximately 25 amino acids to approximately 100 amino acids, approximately 25 amino acids to approximately 250 amino acids, approximately 25 amino acids to approximately 500 amino acids, approximately 25 amino acids to approximately 750 amino acids, approximately 25 amino acids to approximately 1000 amino acids, approximately 25 amino acids to approximately 1250 amino acids, approximately 100 amino acids to approximately 250 amino acids, approximately 100 amino acids to approximately 250 amino acids, approximately 100 amino acids to approximately 500 amino acids). Mino acids, approximately 100 amino acids to approximately 750 amino acids, approximately 100 amino acids to approximately 1000 amino acids, approximately 100 amino acids to approximately 1250 amino acids, approximately 100 amino acids to approximately 1500 amino acids, approximately 250 amino acids to approximately 1250 amino acids, approximately 250 amino acids to approximately 1000 amino acids, approximately 250 amino acids to approximately 750 amino acids, approximately 250 amino acids to approximately 500 amino acids, approximately 500 amino acids to approximately 750 amino acids, approximately 500 amino acids to approximately 1000 amino acids, approximately 500 It has a length of approximately 1250 amino acids, approximately 500 amino acids to approximately 1500 amino acids, approximately 750 amino acids to approximately 1000 amino acids, approximately 750 amino acids to approximately 1250 amino acids, approximately 750 amino acids to approximately 1500 amino acids, approximately 1000 amino acids to approximately 1250 amino acids, approximately 1000 amino acids to approximately 1500 amino acids, or approximately 1250 amino acids to approximately 1500 amino acids.

[0132] In one embodiment, sialidase is converted into repeating chemical moieties such as PEG or hyaluronic acid. Scientifically conjugated (see Mero et al. (2013) CARB POLYMERS 92:2163-70), sial Increase the hydrodynamic radius of Dase, thereby extending the half-life.

[0133] In another embodiment, sialidase is polysialylated itself or a negatively charged, highly sialylated protein (e.g., the chorionic gonadotropin (CG) β-chain). It covalently binds to the ruboxy-terminal peptide (CTP), 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] One or more sialidases, one or more (e.g., 2, 3, 4, 5, 6, 8, 9, 10 or more) It is intended to be able to covalently bind to serum half-life prolonging factors.

[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, and 2kDa to 150kDa. Approximately 5kDa to 10kDa, approximately 5kDa to 20kDa, approximately 5kDa to 30kDa, approximately 5kDa to 40kDa, approximately 5kDa to 50kDa, approximately 5kDa to 60kDa, approximately 5kDa to 70kDa, approximately 5kDa to 80kDa, approximately 5kDa to 90kDa, approximately 5kDa ~100kDa, 5kDa~150kDa, 10kDa~20kDa, 10kDa~30kDa, 10kDa~40kDa, 10kDa~50kDa, 10kDa~60kDa, 10kDa~70kDa, 10kDa~80kDa, 10 kDa ~ approx. 90kDa, approx. 10kDa ~ approx. 100kDa, approx. 10kDa ~ approx. 150kDa, approx. 20kDa ~ approx. 30kDa, approx. 20kDa ~ Approximately 40kDa, approximately 20kDa to approximately 50kDa, approximately 20kDa to approximately 60kDa, approximately 20kDa to approximately 70kDa, approximately 20kDa to approximately 80kDa Approximately 20kDa to 90kDa, approximately 20kDa to 100kDa, approximately 20kDa to 150kDa, approximately 30kDa to 40kDa, approximately 30kDa to 50kDa, approximately 30kDa to 60kDa, approximately 30kDa to 70kDa, approximately 30kDa to 80kDa, approximately 30kDa to 90kDa, approximately 30kDa to 100kDa, approximately 30kDa to 150kDa, approximately 40kDa to 50kDa, approximately 40kDa to 60kDa, approximately 40kDa to 70kDa, approximately 40kDa to 80kDa, approximately 40 kDa ~ about 90kDa, about 40kDa - about 100kDa, about 40kDa - about 150kDa, about 50kDa - about 60kDa, about 50kDa - about 70kDa, about 50kDa - about 80kDa, about 50kDa - about 90kDa, about 50kDa - about 100k Da, about 50kDa to about 150kDa, about 60kDa to about 70kDa, about 60kDa to about 80kDa, about 60kDa to about 90kDa, about 60kDa to about 100kDa, about 60kDa to about 150kDa, about 70kDa to about 80kDa, about 70kDa It may have a molecular weight of approximately 90kDa, approximately 70kDa to approximately 100kDa, approximately 70kDa to approximately 150kDa, approximately 80kDa to approximately 90kDa, approximately 80kDa to approximately 100kDa, approximately 80kDa to approximately 150kDa, approximately 90kDa to approximately 100kDa, approximately 90kDa to approximately 150kDa, or approximately 100kDa to approximately 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 the Fc domain alone or in combination with a second immunoglobulin Fc domain. This refers to a fragment of the constant region of an immunoglobulin heavy chain that can bind to an Fc receptor either in combination or by being conjugated to or not 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 boundaries between the immunoglobulin hinge region and the CH2 and CH3 domains are 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 can be covalently bonded together. The covalent bond may be a disulfide bond. The sialidase enzyme is formed from the first immunoglobulin Fc domain... The N or C-terminus of the second immunoglobulin Fc domain or the N or C-terminus of the second immunoglobulin Fc domain is conjugated. It can be gated. Any second sialidase enzyme can also gate the N of the first immunoglobulin Fc domain. Alternatively, it can be conjugated to the C-terminus or the N-terminus or C-terminus of a 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 the structure with two Fc domains and a conjugate at the N-terminus of the second Fc domain. Figure 1D shows a construct containing a conjugated sialidase enzyme. Figure 1E shows a construct containing two Fc domains and a sialidase enzyme conjugated to the C-terminus of the first Fc domain. This 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, to 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) is the heterogeneity observed in recombinant IgG4 antibodies. It may be introduced to destroy. See Angal, S. et al. (1993) MOL. IMMUNOL. 30:105-108. Light.

[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 is heterodimeric with a second polypeptide. For identification, a "knob" mutation, e.g., T366Y, or a "hole" mutation, e.g., Y407T (EU numbering) Residue numbering by ing, Kabat, EA, et al. (1991) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, FIFTH EDITION, US Department of Health and Human Services, NIH It includes any of the following (Publication No. 91-3242). In one embodiment, which includes a sialidase-Fc fusion having two Fc domains, the first Fc domain is a "knob" mutation (e.g., SEQ ID NO: 33). The second Fc domain may include (and SEQ ID NO: 148), and the second Fc domain may include 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] (Sequence ID: 159) contains the amino acid sequence, where X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val or is absent, and 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 X10 is Pro, Asn, Asp, His, Glu, Gly, Ser or Thr, X11 is Gln or His, X12 is Arg or Lys, X13 is Ala, Glu or Lys, X14 is Gly or Asp, X15 is Gln or His, X16 is Gln, Arg or Lys, X17 is Ala, Cys, Ile, Ser, Val or Leu, X18 is Gln or Leu X19 is Ala or Val, X20 is Cys or Gly, and X21 is Ala or Gly. X22 is Arg, Ile, or Lys; X23 is Ala, Cys, Leu, or Val; X24 is Leu, Ala, or Val; X25 is Thr or Ala; X26 is Asp or Gly; and X27 is Glu. X28 is Lys, X28 is Gln, Ala, His, Phe or Pro, X29 is Cys or Val, X30 is Trp or Arg, X31 is Ser or Arg, X32 is Trp or Lys, X33 is Lys or Val, X34 is Ala, Cys, Ser or Val, and X35 is Cys, Leu or Val. Yes, X36 is Val or Arg, X37 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).

[0148] In certain embodiments, the sialidase-Fc fusion protein is

Table 15

[0149] In certain embodiments, the sialidase-Fc fusion protein is

Table 16

[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 Leu, Gln, His, Ile, Lys or Ser, X 13 This is GGGGSGGGGS (SEQ ID NO: 162) or EPKSS (SEQ ID NO: 163), and the sialidase has at least one mutation compared to wild-type human Neu2 (SEQ ID NO: 1). Includes. In one embodiment, X1 is Ala, Asp, Met or non-existent, 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 is Arg, X9 is Trp or Lys, 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 is absent, and 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 Pro, Asn, Asp, His, Glu, Gly, Ser Or Thr, X 11 is Gln or His, X 12 is Arg or Lys, X 13 Asp also It is 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 ri, 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 is 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 Yes, X 48 is Ala, Cys, Ser or Val, X 49 is Cys, Leu or Val, X 50 Val Or Arg, X 51 is Leu, Gln, His, Ile, Lys or Ser, X 52 is GGGGS (array number) (No.: 174), GGGGSGGGGS (Sequence ID: 162) or EPKSS (Sequence ID: 163), and Sealider Ze 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, X5 is Ala, Glu, or Lys, X6 is Gln, Leu, Glu, Phe, His, Ile, Leu, or Tyr, X7 is Arg, Ile, or Lys, and X8 is Ala, Cys, Phe, Gly, His, Ile , Lys, Leu, Met, Asn, Gln, Arg, Ser, Val, Trp or Tyr, and 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 is Leu, Gln, His, Ile, Lys or Ser, X 15 These are GGGGS (SEQ ID NO: 184), GGGGSGGGGS (SEQ ID NO: 162), or EPKSS (SEQ ID NO: 163), and the sialidase is wild-type human Neu2 (distributed The row number: 1) contains at least one mutation. 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, and X6 is Gln or Tyr. X7 is Ile or Lys, X8 is Ala or Thr, and X9 is Gln, Ala or Thr. , 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 prolonging factor is polyethylene glycol (PEG) and its inducer. Conductors (e.g., alkoxy polyethylene glycol, e.g., methoxy polyethylene glycol) These include ethanol, ethoxypolyethylene glycol, etc. In one embodiment, as described herein The sialidase is shared with at least one PEG having an actual MW of at least approximately 20,000D. They bind. In another embodiment, the sialidase has an actual MW of at least about 30,000 D. In another embodiment, the sialidase is covalently bonded to at least one PEG having an actual MW of at least about 40,000D. In one embodiment, the PEG is methoxyPEG(5000)-succinimidylpropionate (mPEG-SPA), methoxyPEG(5000)-succinimidylsuccinate (mPEG-SS). Such PEGs are commercially available from Nektar Therapeutics or SunBiowest or LaysanBio or NOF. In one embodiment, the PEG is It can be branched, Y-shaped, or comb-shaped so that it is available from JenKem USA or NOF, or it can 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 site for PEG or its derivatives on sialidase is located on a lysine residue. The N-terminal amino group and ε-amino group, as well as other amino, imino, carboxyl, and sul groups. Contains hydroxyl or other hydrophilic groups. PEG is produced using chemicals and Polyfunctional (usually bifunctional) crosslinking agents used in the art may be directly covalently bonded to sialidase with or without known use. For example, PEG variants can be thiol-reactive crosslinking linkers. By using this and then reacting it with the thiol group on PEG, the sialidase is converted to condyl It can be substituted. In one embodiment, the sulfhydryl group is a maleimide-substituted PEG (e.g., alkoxy-PEGamine + sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) or a commercially available product from Shearwater Polymers, Inc., Huntsville, Ala. It can be derivatized by coupling with PEG-maleimide.

[0156] c. Human serum albumin (HSA) and HSA binders Human serum albumin (HSA) (molecular mass approximately 67 kDa) is present in plasma at a concentration of approximately 50 mg / mL (600 μM). It is the most abundant protein in the blood and has a half-life of approximately 20 days in humans. HSA is found in blood. It works to maintain plasma pH, contributes to colloidal blood pressure, acts as a carrier for many metabolites and fatty acids, and serves 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, for example, PCT publication numbers WO2013128027A1 and WO2014140358A1). FcRn (FcRn) appears to be involved in extending 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 neutral or physiological pH. This is weaker (see above). This observation suggests that protein and albumin-containing protein complexes, as well as those containing IgG (especially Fc), are protected from degradation via pH-sensitive interactions with FcRn. This suggests protection (see above). Individual HSA domains are fixed in soluble human FcRn. Surface plasmon resonance (SPR) is used to measure the bonding ability of FcRn and albu It has been shown that albumin interacts with albumin via its D-III domain in a pH-dependent manner 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. Publication US20130316952A1 discloses a polypeptide that binds to serum albumin having the amino acid sequence LKEAKEKAIEELKKAGITSDYYFDLINKAKTVEGVNALKDEILKA (SEQ ID NO: 109). Further exemplary polypeptides that bind to HSA are disclosed in Dennis et al. (2002) J. BIOL. CHEM., 277: 35035-43; Jacobs et al. (2015) PROTEIN ENG. DES. SEL., 28: 385-93; and Zorzi This is described in et al. (2017) NAT. COMMUN., 8: 16092.

[0159] III. Linker In one embodiment, sialidase may be directly linked to or fused to the serum half-life prolonger. In another embodiment, sialidase may be covalently linked to the serum half-life prolonger by a linker.

[0160] Linkers consist of one or more natural amino acids, sialidase or its functional fragments, and blood. It can be coupled with a serum half-life extender, where one or more native amino acids (e.g., cysteine ​​amino acids) can be introduced by site-directed mutagenesis. The linker may contain one or more non-native amino acids. In some situations, a linker containing, for example, one or more sulfhydryl reactive groups (e.g., maleimide) may be introduced into the sialidase or serum half-life extender. The intention is for it to be able to covalently link with cysteine ​​residues present in or with cysteine ​​that are products of site-directed mutagenesis.

[0161] The linker may be a severable linker or a non-severable linker. Optionally or further, the linker may be a flexible linker or a non-flexible linker.

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

[0163] Linkers can be approximately 1 angstrom (Å) to 150 Å in length, or approximately 1 Å to 120 Å in length, or approximately 5 Å to 110 Å in length, or approximately 10 Å to 100 Å in length. Linkers can be approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 27, 30 or more. The angstrom length may be greater than the largest angstrom length 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 smaller angstrom lengths. Furthermore, the linker may 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 angstrom lengths.

[0164] In one embodiment, the linker comprises a polypeptide linker that links or fuses the sialidase to a serum half-life extension factor (e.g., the Fc domain) of the fusion protein. For example, a gene encoding a sialidase that is linked directly or indirectly (e.g., via amino acids containing the linker) to a serum half-life extension factor is generated and developed using conventional recombinant DNA technology. It is intended that this may be expressed. For example, the amino terminus of sialidase may be linked to the carboxyl terminus of a serum half-life prolonger. If a linker is used, the linker may include hydrophilic amino acid residues, such as Gln, Ser, Gly, Glu, Pro, His, and Arg. In some embodiments... And the linker consists of 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, and 4-15 amino acid residues. The peptide contains an acid residue, 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. An example linker is glycan. Syn and serine-rich linkers, e.g. (GlyGlyPro) n(Alternate number: 110) or (GlyGlyGlyGlySer) n (Sequence number: 111) is an example, where n is 1 to 5. In one embodiment, phosphorus The Kerr contains, consists of, or essentially consists of GGGGS (Sequence ID: 174). In one embodiment, the Linker contains, consists of, or also GGGGSGGGGS (Sequence ID: 162). It essentially consists of it. 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. Sialidase conjugated with sialidase and / or serum half-life enhancer How to make Methods for producing sialidase or sialidase conjugated with serum half-life enhancers, such as those disclosed herein, are known in the art. For example, DNA molecules encoding serum half-life enhancers (e.g., Fc domains) can be synthesized chemically or by recombinant DNA methodologies. For example, the sequences of serum half-life enhancers can be synthesized using appropriate synthetic nucleic acid primers by conventional hybridization techniques or polymerase chain reaction (PCR). It can be cloned using this technology. The resulting DNA molecule encoding the target protein is For example, it can be ligated to other suitable nucleotide sequences, including expression regulatory sequences, to produce a conventional gene expression construct (i.e., an expression vector) encoding a 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 the expression vector (ligation (This can be done), and then introduced into host cells by conventional transfection or transformation techniques. It can enter. Exemplary host cells include E. coli cells, Chinese hamster ovary (CHO) cells, Human embryonic kidney 293 (HEK 293) cells, HeLa cells, infant hamster kidney (BHK) cells, monkey kidney cells These are 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 sialidase.

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

[0168] When the reconstructed gene is expressed in a eukaryotic host cell, such as a CHO cell, the gene The gene first receives the appropriate eukaryotic promoter, secretory signal, poly(A) sequence, and stop code. It is inserted into an expression vector containing introns. Optionally, the vector or gene construct may contain enhancers and introns. The gene construct can be introduced into eukaryotic host cells 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, the sialidase or sialidase conjugated to the Fc region is: (a) an expression vector encoding one Fc polypeptide and another expression vector encoding another Fc polypeptide; or (b) a single expression vector encoding both Fc polypeptides. Sialidase can be produced by growing (culturing) lance-fected host cells under conditions that allow expression of both polypeptides. Sialidase is fused to one or more polypeptides. The intact sialidase-Fc domain fusion protein is obtained using techniques known in the art. For example, it can be recovered, purified, or isolated using 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 prolonger is expressed and / or purified in the presence of a stabilizer. The stabilizer is used for expression and purification. and / or during storage, conjugate to sialidase or serum half-life prolonging factor The sialidase prevents one or more of the following: protein unfolding, protein misfolding, protein aggregation, protein inhibition, enzymatic loss, and / or protein degradation. In one embodiment, the stabilizer is a cation, for example, a divalent cation. In one embodiment In this case, the cation is either 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 an amount 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, and about 0.05 mM to about 2 mM. , about 0.05mM to about 1mM, about 0.05mM to about 0.5mM, about 0.5mM to about 4mM, about 0.5mM to about 3mM, about 0.5mM to It is contained in quantities of approximately 2 mM, approximately 0.5 mM to approximately 1 mM, approximately 1 mM to approximately 4 mM, approximately 1 mM to approximately 3 mM, or (of) approximately 1 mM to approximately 2 mM. It is possible.

[0173] In one embodiment, in order to express a protein, such as sialidase, as a secreted protein, the native N-terminal signal sequence of the protein is replaced with, for example, MDMRVPAQLLGLLLLWLPGARC (SEQ ID NO: 28). In one embodiment, a protein, such as recombinant human sialidase, is expressed. To express the ligase 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 interleukin-2, CD-5, IgGκ light chain, trypsinogen, serum albumin, etc. Examples include signal sequences derived from prolactin. In one embodiment, in order to express a protein, such as recombinant human sialidase, as a secreted protein, the C-terminal lyso Remove somal signaling motifs, such as YGTL (SEQ ID NO: 29).

[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 extender 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. The serum half-life extender may be directly covalently bound to sialidase using chemical means 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 is Derivatization by coupling with 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.) It is possible.

[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 that, within the bounds of normal medical judgment, it is balanced by a reasonable benefit / risk ratio without excessive toxicity, irritation, allergic reactions or other problems or complications. Compounds, materials, compositions, and / or compounds suitable for use in contact with human and animal tissues. Alternatively, it refers to the dosage form.

[0176] The term "pharmaceutically acceptable carrier," when used herein, means a carrier that is balanced by a reasonable benefit / risk ratio without excessive toxicity, irritation, allergic response, or other problems or complications. This refers to buffers, carriers, and excipients suitable for use in contact with human and animal tissues. 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 contain 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] . 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 well 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, adsorption or penetration of the composition. In such embodiment, suitable formulation materials include, but are not limited to, amino acids (glycine, glutamine, asparagine, argy). Nin or lysine, etc.); antibacterial agents; antioxidants (ascorbic acid, sodium sulfite or sodium bisulfite, etc.); buffers (borates, bicarbonates, Tris-HCl, citrates, phosphates or other organic acids, etc.); bulking agents (mannitol or glycine, etc.); chelating agents (ethylenediaminetetraacetic acid (EDTA), etc.); complexing agents (caffeine, polyvinylpyrrolidone, β-cyclodextrin or hydroxypropyl-β-cyclodextrin, etc.); fillers; monosaccharides; disaccharides; and other carbohydrates (glucose, mannose or dextrin, etc.); proteins (serum albumin, gelatin or immunoglobulin, etc.); colorants, flavoring agents and diluents; emulsifiers; hydrophilic polymers (polyvinylpyrrolidone, etc.) Low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (benzalkonium chloride) (e.g., ammonium compounds, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (e.g., glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); suspending agents; surfactants or humectants (e.g., Pluronic acid, PEG-45). Polysorbates such as sorbitan ester, polysorbate 20, triton, tromethamine, lecithin, cholesterol, and tyloxapal. etc.); Stability enhancers / stabilizers (sucrose, sorbitol or cations, etc.); Tonicity enhancers (alkali metal halides, preferably sodium chloride or potassium chloride, man Nitol, sorbitol, etc.; delivery vehicle; diluent; excipient and / or pharmaceutical adjuvant; One example is Bant (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 approximately 0.05 mM to approximately 5 mM. For example, the stabilizer is It may exist in quantities of approximately 0.05 mM to 4 mM, approximately 0.05 mM to 3 mM, approximately 0.05 mM to 2 mM, approximately 0.05 mM to 1 mM, approximately 0.05 mM to 0.5 mM, approximately 0.5 mM to 4 mM, approximately 0.5 mM to 3 mM, approximately 0.5 mM to 2 mM, approximately 0.5 mM to 1 mM, approximately 1 mM to 4 mM, approximately 1 mM to 3 mM, or (of) approximately 1 mM to 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, the pharmaceutical composition may include sustained or controlled delivery formulations. Techniques for formulating sustained or controlled delivery means, such as liposome carriers, bio-erodible microparticles or porous beads, and depot injection formulations 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 semipermeable polymer matrices, such as films 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 secondary release composition may also include liposomes that can be prepared by any of several methods known in the art.

[0182] Pharmaceutical compositions containing sialidase or sialidase conjugated with a half-life extender may exist in unit dosage forms and may be prepared by any suitable method. Pharmaceutical compositions should be formulated to be compatible with their 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 field of medicine. See, for example, Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990). Formulation components suitable for parenteral administration are available. The formula includes bacterial diluents, such as sterile water for injection, saline solution, fixing 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 acetates, citrates, or phosphates; and agents for adjusting tonicity, such as sodium chloride or dextrose.

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

[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, for example, 1 mg / kg to 100 mg / kg or 1 mg / kg to 10 mg / kg. The dosage depends on the type and severity of the disease or symptom being treated. The effectiveness of the treatment depends on various variables, including the patient's overall health, the in vivo potency of the active components, the drug formulation, and the route of administration. To rapidly achieve the desired blood or tissue levels, the initial dose may be increased beyond the upper limit. 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 range, for example, from 0.5 mg / kg to 20 mg / kg. This can be optimized in conventional Phase I dose escalation studies designed to proceed up to a certain point. The dosage may vary depending on factors such as the route of administration, the dose, the serum half-life of the 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, the 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 prolonging factor, alone or in combination with another therapeutic agent, to treat cancer in the subject. The term “effective amount” as used herein means a sufficient amount of the active agent to produce a beneficial or desired result (e.g., the sialidase of the present invention). This refers to the amount of sialidase (or sialidase conjugated with a half-life prolonging factor). Effective dose It may be administered in one or more doses, applications, or dosages, and is not limited to specific formulations or routes of administration. It is not intended to be defined.

[0189] As used herein, “treat,” “treating,” and "Treatment" refers to the treatment of a disease in a subject, such as a human being. This means (a) inhibiting the disease, i.e., stopping its progression; and (b) alleviating the disease. , including, that is, causing a regression of the disease state. As used herein, the terms “subject” and “patient” mean an organism treated by the methods and compositions described herein. Preferably, such organisms are mammals (e.g., mice), but are not limited to these. Examples include monkeys, horses, cows, pigs, dogs, cats, etc., and humans are even more preferable. .

[0190] Examples of cancer 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), 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's syndrome (Richter's transformation). Examples of solid tumors include malignant diseases, e.g., sarcomas, adenocarcinomas, and carcinomas of various organ systems, e.g., head and neck (pharynx) (including), thyroid, lung (small cell or non-small cell lung cancer (NSCLC)), breast, lymphatic system, gastrointestinal tract (for example) (The oral cavity, esophagus, stomach, liver, pancreas, small intestine, colon and rectum, anal canal), reproductive organs and urogenital organs. Examples of affected organs include organs (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 some aspects, 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 adenomatosum, adrenal cortical carcinoma, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, baso squamous cell carcinoma, bronchioalveolar carcinoma, and tracheal carcinoma. bronchogenic carcinoma, cerebrum-like carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma ductal carcinoma, carcinoma durum, embryonic carcinoma, brain-like carcinoma, epidermoid carcinoma, carcinoma epitheliale adenoids, exophytic carcinoma, carcinoma ex ulcere), fibrous carcinoma (carcinoma fibrosum), gelatinous carcinoma (gelatinic carcinoma), colostomycosis Gelatinous carcinoma, giant cell carcinoma, giant cell carcinoma (carcinoma gigantocellulare), adenocarcinoma, granulosa cell carcinoma, hair matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hürthle cell carcinoma, hyaline carcinoma, hypomephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, carcinoma in epidermis , intraepithelial carcinoma, Krompecher's carcinoma Krutskyi cell carcinoma, large cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, mucinous adenocarcinoma, mucocellular carcinoma, mucinous epidermal carcinoma, mucinous carcinoma, mucosal carcinoma, myxomatous carcinoma, nasopharyngeal carcinoma, oat cell carcinoma, ossifying carcinoma Carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of the kidney, reserve cell carcinoma, sarcomatodes, Schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti, signet ring cell carcinoma, simple carcinoma, small cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes), transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma These include pyogenes carcinoma and choriocarcinoma (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 with other therapeutic agents and / or modal It may be used in combination with modality. As used herein, the term “combined” means that two (or more) different treatments are administered to a subject suffering from a disorder. This is understood to mean that the therapeutic effects on the patient overlap at points in time during the pain course. In some embodiments, there is an overlap in the duration of administration, as the delivery of one therapeutic agent is still taking place when the second delivery begins. This is sometimes referred to herein as "simultaneous" or "concurrent delivery." In another embodiment, the delivery of one treatment is completed before the delivery of another treatment is initiated. In some embodiments, the treatments are more effective due to the combined administration. For example, the second treatment is more effective, and a similar effect is observed with less of the second treatment, or the second treatment is more effective when the second treatment is administered in the absence of the first treatment. The symptoms are reduced to a greater extent than seen in the case of the first treatment, or the condition is equivalent to that seen in the first treatment. It can be observed. In some embodiments, the reduction of other parameters related to the symptoms or disorder is greater when one treatment is delivered in the absence of the other treatment than when the other treatment is delivered. It is such that the effects of the two treatments may be partially additive, and overall It can be additive or more than additive. Delivery is the effect of the first treatment delivered. This could be something that is still detectable when the second treatment is delivered.

[0195] In one embodiment, the method or composition described herein is one or more further curative treatments. It is administered in combination with other therapies, such as surgery, radiotherapy, or the administration of other therapeutic preparations. In some embodiments, further therapy may include chemotherapy, such as cytotoxic agents. In this context, further treatment may include targeted therapies, such as tyrosine kinase inhibitors, proteasome inhibitors, or protease inhibitors. In one embodiment, further treatment may include anti-inflammatory, anti-vasculitic, anti-fibrotic, or antiproliferative compounds, such as steroids, biological immunomodulators, monoclonal antibodies, antibody fragments, aptamers, siRNA, or antisense molecules. This may include fusion proteins, cytokines, cytokine receptors, bronchodilators, statins, anti-inflammatory agents (e.g., methotrexate), or NSAIDs. In one embodiment, further therapeutic Treatment may include combinations of different types of therapeutic drugs.

[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. To prevent an overactive immune response, inhibit T cell activity at the appropriate time or otherwise Otherwise, it is a receptor located on the surface of T cells that acts as a checkpoint in the immune system that regulates T cell activity. However, cancer cells interact with PD-1 on the surface of T cells, for example. The benefits of this checkpoint can be utilized by expressing ligands that block or modulate T cell activity, such as PD-L1. 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. Examples of exemplary anti-PD-1 antibodies include U.S. Patent Nos. 8,952,136, 8,779,105, and 8,008,449. These are described in publications No. 8,741,295, No. 9,205,148, No. 9,181,342, No. 9,102,728, No. 9,102,727, No. 8,952,136, No. 8,927,697, No. 8,900,587, No. 8,735,553, and No. 7,488,802. Examples of anti-PD-1 antibodies include, for example, nivolumab (Opdivo®, Bristol-Myers Squibb Co.) and pembrolizumab (Keytruda®, Merck Sharp & Dohme Corp.). PDR001 (Novartis Pharmaceuticals) and pidilizumab (CT-011, Cure Examples of anti-PD-L1 antibodies are described, for example, in U.S. Patent Nos. 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, there is an interaction between CTLA-4 on T cells and its ligands (also known as, for example, CD80, B7-1, and CD86) on the surface of antigen-presenting cells (not cancer cells). This causes T cell inhibition. An exemplary CTLA-4-based immune checkpoint inhibitor is It is listed 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 include (i) PD-1 or PD-L1 inhibitors, such as the PD-1 or PD-L1 inhibitors disclosed herein, and (ii) CTLA-4 inhibitors. For example, it is administered in combination with a CTLA-4 inhibitor as 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 ofatumumab, rituximab, ocrelizumab, iodine I131. The drug is selected from the group consisting of momab, obinutuzumab, ibritumomab, and hyaluronidaserituximab (ritixumab).

[0201] In one aspect, the methods or compositions described herein are combined with an IDO inhibitor. It is administered by rinsing. An example of an IDO inhibitor is 1-methyl-D-tryptophan (indoxy). Indoximod (commonly known as mod), epacadostat (INCB24360), Navox Examples include Mod (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, platinumating agents, nucleic acid synthesis inhibitors, histone deacetylase inhibitors (HDAC inhibitors), such as vorinostat (SAHA, MK0683), entinostat (MS-275), and panostat. Vinostat (LBH589), Trichostatin A (TSA), Mosetinostat (MGCD0103), Bellinostat (PXD101), Romidepsin (FK228, depsipeptide), DNA methyltransfer Examples include ribonucleotide reductase inhibitors, nitrogen mustard, nitrosourea, ethyleneimine, alkyl sulfonates, triazenes, folate analogs, nucleoside analogs, ribonucleotide reductase inhibitors, vinca alkaloids, taxanes, epotilone, 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, ixabepyrone, bortezomib, taxanes (e.g., paclitaxel or docetaxel), cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, vinorelbine, colchicine, anthracyclines (e.g., doxorubicin or epirubicin), daunorubicin, and di Hydroxyanthracinedione, mitoxantrone, mitramycin, actinomycin D, adriamycin, 1-dehydrotestosterone, glucocorticoid, procaine, These are 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 involves comparing the expression levels 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 with the corresponding expression levels before contact with sialidase or sialidase conjugated with a half-life extension factor. The process includes a step of contacting cells, tissues, or subjects with an effective amount of sialidase or sialidase conjugated with a half-life prolonger to increase the amount of sialidase. 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 related to sialidase. Or similar or so that sialidase conjugated with a half-life extension factor was not in contact with the sialidase. Otherwise, the same cells or tissue will be 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%. 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 performed on similar or otherwise identical tumor cells or populations of tumor cells that have not been in contact with sialidase or sialidase conjugated with a half-life prolonging factor, at least about 10%, at least about 20%, at least about 50%, and less Approximately 75%, at least approximately 100%, at least approximately 150%, at least approximately 200%, at least approximately 250%, at least approximately 300%, at least approximately 400%, at least approximately 500%, at least approximately 600%, and less The increase is at most about 700%, at least about 800%, at least about 900%, or at least about 1,000%. The phagocytic activity 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 prolonger. The activation is measured as described in Example 8 of this specification. It is possible.

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

[0212] In one embodiment, Siglec-15 binding activity conjugates sialidase or half-life prolongation factors. Compared to Siglec-15 that was not in contact with jugated sialidase, the reduction was at least approximately 10%, at least approximately 20%, at least approximately 50%, at least approximately 75%, or at least approximately 100%. The total can 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 sense, immune cells are These are natural killer (NK) cells.

[0214] In one embodiment, immune cell infiltration into tumors in subjects was at least about 10%, at least about 20%, compared to similar or otherwise identical tumors and / or subjects that were not administered sialidase or sialidase conjugated with a half-life prolonger. The increase is 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%. Immune cell infiltration into the tumor can be measured by any suitable method known in the art, for example, 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 at least about 10%, at least about 20%, at least about 50%, and at least in similar or otherwise identical subjects that were not administered sialidase or sialidase conjugated with a half-life prolonger. Approximately 75%, at least approximately 100%, at least approximately 150%, at least approximately 200%, at least approximately 250% The number of circulating NK cells in the subject may be measured by any suitable method known in the art, such as antibody staining.

[0217] The present invention also provides a method for increasing the number of T cells in draining lymph nodes in subjects who require an increase in the number of T cells in draining lymph nodes. This involves administering an effective amount of sialidase or sialidase conjugated with a half-life prolonging factor, for example, the sialidase or sialidase conjugated with a half-life prolonging factor disclosed herein, to a subject, and comparing the T cells in the inflowing lymph nodes with those prior to the administration of sialidase or sialidase conjugated with a serum half-life prolonging factor or the pharmaceutical composition. The process includes increasing the number of immune cells. 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 lymph nodes in the influx region of the subject is determined by sialidase Or, compared to similar or otherwise identical subjects who were not administered sialidase conjugated with a half-life prolonging factor, the number of T cells 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%. T cells in the influx region lymph nodes of the subjects are publicly known in the art. It can be measured by any suitable method of knowledge, such as by antibodies.

[0219] The present invention also relates to Cd3, Cd4, Cd8, Cd274, Ctla4, Icos in cells, tissues, or subjects. , Pdcd1, Lag3, Il6, Il1b, Il2, Ifng, Ifna1, Mx1, Gzmb, Cxcl9, Cxcl12 and / or This provides a method for increasing the expression of Ccl5. The method comprises contacting cells, tissues, or subjects with 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, 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 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, when a composition is described as having a specific component, including or comprising, or when a process and method has a specific step, including Where it is stated that a composition of the present invention is essentially composed of or comprises the described components, and where a process and method of the present invention is essentially composed of or comprises the described process steps.

[0222] In this application, if an element or component is to be said to be included in and / or selected from the list of elements or components described, then that element or component is described It may be any one of the elements or components listed, or the element or component An element may be selected from a group consisting of two or more elements or components that are listed. It should be understood.

[0223] Furthermore, elements and / or features of the compositions or methods described herein are subject to change. It should be understood that, whether explicitly or implicitly in the details, the invention can be combined in various ways without departing from the spirit and scope of the invention. For example, where a particular compound is referenced, that compound may be used in various embodiments of the composition of the invention and / or in the methods of the invention, unless otherwise understood from the context. In this application, the embodiments are described and shown in a manner that allows for clear and concise application to be described and illustrated, but the embodiments do not depart from the teachings and invention(s) (one or more) It is intended and understood that these features may be combined or separated from each other. For example, it is understood that all features described and shown herein may be applicable to all aspects of the invention(s) described and shown herein.

[0224] The expression "at least one" is used unless it is understood from the context and usage that it is not the case. Each of the items described after the expression, individually, and two or more of the items described. It should be understood that this includes combinations of the following. Regarding three or more items listed, the expression "And / or" have the same meaning unless the context suggests otherwise. It should be understood.

[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 They may be carried out simultaneously.

[0228] Any and all example or illustrative terms used herein, such as "such as" Alternatively, the use of "including" is simply intended to better illustrate the present invention. The scope of this invention is not limited unless otherwise claimed. Terms in this specification should not be construed as indicating any unclaimed element essential to the practice of this invention. [Examples]

[0229] Examples Example 1: Construction and expression of recombinant sialidase This example describes the construction of recombinant human sialyidases (Neu1, Neu2, Neu3, and Neu4). Human sialyidases Neu1, Neu2, Neu3 (isoform 1) and Neu4 (isoform 4) (1) was expressed as a secreted protein 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, N The terminal signal peptide MDMRVPAQLLGLLLLWLPGARC (SEQ ID NO: 28) was added to each of them.

[0231] Sialidase was expressed in 200 mL transfections of HEK293F human cells using the pCEP4 mammalian expression vector in a 24-well plate. Ni-NTA column was used to analyze the cells. Allidases were purified, 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 (bacterial sialidase; Sequence ID: 30) derived from Salmonella typhimurium was expressed in the same manner as Neu1-4 (described above), yielding a yield equivalent to Neu1, and was mainly present in monomeric form.

[0232] The activity of recombinant sialidase was assayed by measuring the release of sialic acid from the fluorescence-generating substrate 4-methylumbelliferyl-N-acetylneuraminic acid (4MU-NeuAc). (See Figure 3) As such, Neu1 does not have detectable activity above the enzyme-free control, unless Neu1 complexes with β-galactosidase and protective protein / cathepsin A (PPCA). This is consistent with previous reports indicating inactivity. Neu2 and Neu3 were active, similar to bacterial sialidases. Enzyme kinetic assays were performed using Neu2 and Neu3. 1 nM The enzyme at a fixed concentration was incubated with the fluorescence-generating substrate 4MU-NeuAc at concentrations ranging from 4000 μM to 7.8 μM. The assay was performed under both acidic (pH 5.6) and neutral (pH 7) conditions. The results are shown in Figure 4. Thus, both Neu2 and Neu3 were active under 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] Wild-type Neu2 (Neu2-Fc, coded by SEQ ID NO: 114; SEQ ID NO: 113) and M106 The variant Fc-sialidase (sequence number 115, encoded by sequence number 116) (human IgG1 Fc with M1D, V6Y, P62G, A93E, I187K, C332A and hole (Y407T) mutations) The Fc-sialidase used was expressed, purified, and characterized. The Neu2-Fc molecule was 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, while M106 yielded 20 mg / L. It had a yield of ttoles.

[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. The monomer species had 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). Fixed concentration: 2 μg / well The enzyme is incubated with the fluorescent substrate 4MU-NeuAc at concentrations ranging from 4 mM to 0.03 μM. Then, an enzyme kinetic assay was performed. Figure 6 shows the enzyme activity of M106.

[0237] Using the pCEP4 mammalian expression vector, 100 ml transfection of Expi293 human cells The wild-type Neu3 (Neu3-Fc, coded by sequence number 118; sequence number 117) is used within the system. FC sialidase was expressed. Neu3-Fc expressing cells (N3-normal), Neu3-Fc expressing cells treated with tunicamycin (N3-Tunic), and mock-transfected cells were cultured under both cell conditioning conditions. Activity was determined using the supernatant and washed cell pellet. Figure 7 shows that Neu3-Fc activity was detected in the cell pellet, indicating surface binding activity, and low levels of activity were detected in the supernatant, indicating secreted Neu3-Fc. S-acylation and N-glycosylation Treatment with tunicamycin, an inhibitor of this compounding process, did not alter the surface binding activity or the 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 contains 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). Therefore, Fc-ST sialidase was expressed in 1 L transfection of Expi293 human cells using the 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 The expressed Fc-ST sialidase has a retention time of 21 minutes and an SEC monomer purity of 75%. The SEC-HPLC trace shows that it was a monomer species.

[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). A fixed concentration of 2 μg / well of the enzyme was incubated with the fluorescent substrate 4MU-NeuAc at concentrations ranging from 4 mM to 0.03 μM. It was used as a reagent and an enzyme kinetic assay was performed. FC ST was 3 x 10 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] In a mouse syngeneic tumor model in which the Fc Salmonella sialidase construct (Fc-ST sialidase) described in Example 2 was injected with mouse lymphoma cancer cell line A20, avelumab (anti-PD-L1) was used. (Antibody) was compared. Female BALB / c mice, 6-8 weeks old, were subcutaneously placed in the right lower flank with 0.1 ml of PBS containing A20 tumor cells (5x10) for tumor development. 5 The drug was administered. The tumor was 50-100 mm. 3 , average about 75~100mm 3 Reach During this process, the mice were randomly assigned to four groups of eight animals each.

[0242] In mice, negative control ("isotype control", Figure 9A), Fc-ST sialidase (Figure 9B), and Abe Lumab (anti-mouse PD-L1 antibody, Figure 9C) or a combination of Fc-ST sialidase and avelumab (Figure 9D) was administered by intraperitoneal injection at a dose of 10 mg / kg twice a week for 15 days to reduce tumor volume (mm²). 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 injected into EMT6-Her2 cells. In a syngeneic tumor model, it was compared with trastuzumab (anti-HER2 antibody). Female BALB / c mice, 6-8 weeks old, were subcutaneously implanted in 0.1 ml of PBS containing EMT6-Her2 tumor cells (5x10) to induce 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 it reaches that point, move the mouse each The eight animals were randomly assigned to four groups.

[0244] As shown by the triangle, mice were given isotype control (vehicle control, Figure 10A), Fc-ST sialidase (FC-ST, Figure 10B), trastuzumab (anti-human Her2 antibody, Figure 10C), or Fc human sialidase. Alimidase (M106, Figure 10D) was administered intraperitoneally at a dose of 10 mg / kg twice a week for 15 days, 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. In particular, FcNeu2 sialidase (SEQ ID NO: 123) (M1D, V6Y, I187K , C332A) is used to form the heavy and light chains of trastuzumab (a first polypeptide chain having amino acid sequence sequence number 124 encoded by nucleotide sequence sequence number 125, and a second polypeptide chain having amino acid sequence sequence number 126 encoded by nucleotide sequence sequence number 127). It was expressed together with a third polypeptide chain having the amino acid sequence sequence number 123 encoded by the nucleotide sequence sequence number 128.

[0246] Incubate purified protein in PBS or PBS containing 4 mM CaCl2 at 37°C for up to 2 weeks. The samples were incubated. 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). The assay was 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. I made them do it.

[0247] To see if CaCl2 could stabilize its enzymatic activity during expression in mammalian cells Starting 24 hours after transfection, 4 mM CaCl2 was added to the transiently transfected Expi293 cell expression medium. As seen 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... Furthermore, 4 mM CaCl2 resulted in a decrease in cell viability.

[0248] To optimize the CaCl2 concentration that can stabilize enzyme activity while maintaining cell viability, five different concentrations of CaCl2—0.05 mM, 0.5 mM, 1 mM, 2 mM, and 4 mM—were used for transfection. The enzyme was added on day 1. On days 4-6, the conditioned medium was collected over a 3-day period, and the enzyme activity (and therefore viability) was determined as shown in Figure 13A. Protein yield The activity was also measured (Figure 13B). 4 mM CaCl2 stabilized the activity and yielded a moderate yield, but the yield was poor. It was found that this resulted in improved viability. Under the tested conditions, the use of 0.5 mM CaCl2 was found to maintain sialidase activity, provide higher protein yields, 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. TM Cells 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 Can be fixed An Fc blocking solution containing Nokin IR dead cell staining (1 / 2000 dilution) was prepared, and the cells were incubated on ice for 10 minutes. The cells were washed with ice-cold PBS (1% BSA) at 350 xg for 5 minutes. Cell surface siaroglycan staining using Hydra and lectin reagents as shown in Table 10. Color was used. Hydra-3, Hydra-7, and Hydra-9 are hexamer 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 has α-2,6 terminal galactose binding. It is a lectin that preferentially binds to sialic acid and binds more weakly to α-2,3 links. MAL-II is a lectin that binds to sialic acid in α-2,3 links. PNA is a terminal galactose residue It is a lectin that binds to the substrate. Increased PNA staining indicates the removal of terminal sialic acid by sialidase. Exposure to galactose below may be observed. [Table 20]

[0251] PBMCs were incubated on ice for 30 minutes with various hydra and lectin reagents. Wash each well with 150 μL of PBS (1% BSA) and centrifuge at 350 xg for 5 minutes. The sample was removed. The plate solution was 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. Avidin 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 those used for viable / dead staining were purchased from Biolegend® (San Diego, CA), which were purchased from Thermo Fisher Scientific (Waltham, MA). [Table 21]

[0252] Prepare a master mix ("stain mix") using the reagents listed in Table 11 in the FACS staining buffer, and add 30 μl of the stain mix to each well / tube for a final active antibody concentration of approximately 1 μg / ml. The cells were divided equally. The cells were incubated on ice for 15 minutes. Individual cellular compensation controls were also prepared. The cells were washed with PBS (1% BSA) and resuspended in 4% paraformaldehyde for 10 minutes at room temperature. The cells were washed twice with PBS and the pellet was resuspended in 150 μl of PBS. Flosa Itometer (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). Monocyte and DC cell populations show increased Hydra-9 staining compared to other cell populations (Figure 14A). Monocyte and DC cell populations also show increased Hydra-7 staining compared to other cell populations. This is shown (Figure 14B). One donor showed increased Hydra-7 staining against CD4+ T cells. Monocytes The DC cell population showed increased Hydra-3 staining compared to other cell populations (Figure 14C). One donor showed increased Hydra-3 staining for CD4+ T cells.

[0254] Figure 15 shows lectin staining (MAL-II, PNA, and SNA) of human PBMCs from healthy donors (black). (The gray bars indicate two independent donors.) As shown, PNA staining is Hydra-9 Although relatively low compared to staining (see the Y-axis scale in comparison with Figure 14), monocytes It is specific to 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 staining compared to other cell populations. It shows SNA staining.

[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, the DCs express many siaroglycans, which are ligands for the Siglec molecule, as shown in previous examples. The interaction between Siglec and siaroglycans on the 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, cold autoMACS (registered) was performed by centrifugation at 350 xg for 5 minutes. Cells were washed twice with a rinse solution (containing 5% BSA; Miltenyi Biotec). CD14+ monocytes were magnetically purified using CD14 microbeads (Miltenyi Biotec) and differentiated into dendritic cells. Specifically, CD14+ cells were treated with 50 ng / ml recombinant human GM-CSF and 50 ng / ml recombinant human IL-4. 0.8 cells x 10⁶ cells in complete medium (RPMI medium containing 10% FBS) 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 Thin 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 human IgG1 Fc with M106 (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. It has. In the following examples, the term "M106" refers to this structure. Human IgG1 Fc (Sequence ID: 176) possessing Neu2-FC variants (M1D, V6Y, K9D, I187K, C332A, A93E, V363R, L365R, E218A, C219N and hole (Y407T) mutations, referred to as LOF. The sample sequence number 175 was used as a negative control. 100,000 DC were used per well in 96 wells. Plate cultures were performed in a U-bottom format, with 200 μl distributed per well. M106 and LOF constructs were cultured using LPS at 0.3 ng / mL, as indicated, at the following concentrations (μg / mL): 0, 6.25, 12.5, and 25. Used with 50 and 100. Incubate the DC overnight (16 hours), then CD83, CD86. Flow analysis of MHCII (HLA-DR) was performed as described in Example 5. Desialization was measured.

[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 Includes 100 fixable near-IR dead cell stains (1 / 2000 dilution) Dead cells are stained by adding μl of the solution and incubating on ice for 10 minutes. The cells were centrifuged and washed once with FACS buffer. 50 μL of PNA-biotin (FACS staining) was then applied. 1 μg / mL of streptavidin Alexa Fluoride was added to each well in a color buffer and incubated on ice for 10 minutes. Cells were centrifuged and washed twice with FACS buffer. TM Add 50 μL of antibody cocktail containing 647 (listed in Table 12 below) to each well and leave on ice for 30 minutes. Incubation was performed. After incubation, the cells were washed twice with 150 μL of FACS buffer. The sample was resuspended in 125 μL of FACS buffer for flow cytometry acquisition. The flow cytometry data was obtained using the HTS (High Throughput Sampler) option. Itometer (BD FACSCelesta) TM The data was obtained from (BD Biosciences). After data acquisition, FlowJo The signals were analyzed using 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 acid and exposure of underlying galactose residues recognized by PNA lectins. This shows the increase in fluorescence (MFI) and PNA staining associated with the increase in M106 concentration. Figure 16B shows the multiplier increase in the PNA signal compared to untreated DCs. This demonstrates the robust desialylation of DCs. A clear dose-dependent increase in the PNA signal was observed.

[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. In this example, M106 was found in tumor cell lines BT-20, SKBR-3, and HT-29, as determined by Hydra-9 and lectin staining. It exhibits the ability to desialize.

[0263] BT-20 and HT-29 cells were placed on plates using appropriate media to reach 70-80% confluence. The cells were grown to this stage. Using Accutase® (Innovative Cell Technologies, Inc.), an enzyme mixture containing proteolytic and collagen-degrading enzyme activity, the cells were plated. The cells were dissociated by incubation at 37°C for 15 minutes. Once the cells were dissociated, an equivalent volume of complete medium was added to neutralize Accutase®. 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. The cells were counted to 1 x 10⁶ per ml. 6 The cells were resuspended in culture medium. M106 and LOF were added to the cells in various dilutions. The solution was added. The cells were incubated at 37°C for 10 hours. After incubation, the cells were transferred to PBS. The samples were washed 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 is related to Hydra 9. The level was 3.088 μg / mL, and for SNA it was 58.75 μg / mL. Figure 18 shows the results measured by fluorescence (gMFI). The degree of desialylation of BT-20 cells after treatment with M106 (triangle) or LOF control (square) is shown, determined by the disappearance of Hydra 9 binding (Figure 18A) or increased PNA staining (Figure 18B). The IC50 for desialylation with the Neu2-Fc variant M106 was 2.95 μg / mL for Hydra 9 and 131.5 μg / mL for PNA.

[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 M106 (triangle) 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. Alternatively, it shows the degree of desialylation of SKBR-3 cells after treatment with LOF control (round). Desaturation by M106 The IC50 for sialylation was 4.4 μg / ml using 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. ru 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) are generated from CD14+ monocytes isolated from PBMCs of healthy donors. CD14+ cells were magnetically purified using the manufacturer's protocol (Miltenyi, catalog number 130-050-201). The purified cells were then cultured for 7 days in the presence of GM-CSF (R&D Systems, catalog number 7954-GM / CF) and IL-4 (R&D Systems, catalog number 6507-IL / CF). Mature DCs were generated.

[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 5x10⁻¹⁴⁻¹ 6 The sample was resuspended in 10% FBS McCoy's 5A medium at a concentration of / mL. 100 μg / mL of M106 was added to the sample and incubated at 37°C for 4 hours. The untreated group was treated the same way except for the addition of M106 to the tube. After 4 hours, the fine Wash the cells twice with 10% FBS McCoy's 5A medium and then infuse them in complete medium (10% FBS RPMI) in 2x10 cells. 6 Resuspend with / mL The solution was cloudy. 50 μl (100,000 DC) of the supernatant was added to the designated well.

[0270] DCs are collected, washed in complete medium (10% FBS RPMI), and 2x10 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 then added to reach a final volume of 200 μL per well. The assay plate was 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. Increased percentages of CD83hi and CD86hi ("LPS") indicate... As shown, the addition of LPS to DCs strongly induces activation. 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"). Additionally, 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")). To raise.

[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 present invention in relation to the phagocytic activity of HT-29 tumor cells by M2-like human macrophages. This demonstrates the effect of sialidase.

[0275] PBMCs were isolated from the whole blood of human volunteers using the Ficol method. CD14+ monocytes were isolated from CD14 Monocytes were purified magnetically using microbeads. Monocytes were then combined with 50 ng / mL recombinant human M-CSF in a 1x10⁶ group. 6 CD14+ 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, the adhered cells were... Carefully, half of the culture medium was removed from each well to avoid disruption. 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 then placed in complete RPMI medium supplemented with 10% FBS and non-essential amino acids (NEAAs), sodium pyruvate, and HEPES, along with 10 ng / mL of M-CSF. The cells were resuspended and seeded in a flat-bottomed 96-well plate at a rate of 50K cells / well / 100μL.

[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). Cells were incubated at room temperature for 10 minutes. Then, an equal volume of chilled FBS was added to quench the labeling reaction. The cells were washed twice, and 1.2 x 10⁻⁶ cells were removed. 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 6 Cells / mL The cells were resuspended in complete RPMI (10% FBS) medium at final cell density. 100 μL of HT-29 cell suspension was added in a 1:5 ratio. M2-like macrophages were added to appropriate wells based on the macrophage:tumor cell ratio (E:T). Plates containing macrophages and tumor cells were incubated for 2 hours to observe phagocytic activity. The procedure was performed. 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] Resuspend the cell pellet and use human Trustin Fc blocking agent on ice for 5-7 minutes. The cells were then blocked. After incubation, the cells were washed with PBS. As shown in Table 13 below. The cells were then stained with CD45 and CD14 fluorescent dye markers. The 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 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. The cells were then washed twice with PBS. The 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 at concentrations exceeding 25 μg / mL showed reproducible phagocytosis by macrophages. An increase in activity was observed. Similar increases in phagocytosis of desialylated 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 efficacy of the sialidase of the present invention on MHC class II (HLA-DR) expression on monocytes. The results show that 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 Ficol method, PBMCs were isolated from healthy volunteers, and the cells were stored in 350 xg. Washed twice with ice-cold PBS using bench centrifugation for 10 minutes. Resuspended cells in culture medium and Countess TM II. Counted using an automated cell counter. The final suspension was 2.5 x 10⁻⁶. 6 The concentration was adjusted to cells / L. Approximately 250,000 cells (100 μL) were seeded into 96-well round-bottom plates. The cells were incubated with M106 or LOF at the top concentration of 50 μg / mL, at a 2-fold dilution. The untreated group was... The cells were incubated at 37°C for 18 hours. The plates were heated at 350 xg for 10 minutes. The cell pellet was washed with cold PBS and stained using the FACS staining panel described in Table 14. The samples were subjected to blocking and staining processes. All antibodies except for the Live Dead stain 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 leads to increased MHC class II (HLA-DR) expression on monocytes. Class I expression indicates antigen-presenting ability on monocytes. Therefore, enhanced class II expression indicates enhanced antigen presentation to T cells, which can increase the T cells' ability to produce an effective immune response. .

[0287] Example 11: Sialidase treatment does not produce harmful cytokine release. Regarding the stimulation of cytokine release, M106 or PBMCs incubated with LOF are used. Conditioned culture media were assayed. LPS (1 ng / mL) was used as a positive control. M106 (and LOF) treatment of PBMCs was performed using LEGENDplex. TM When measured using a human M1 / M2 macrophage panel (10plex; BioLegend®), TNF-α and IL-6 were observed in two independent donors. No increase was observed across all treatment doses of IL-1β, IL-1RA, or IL-10. In contrast, LPS showed clear cytokine induction. These results indicate that PBMC sialidase The principle demonstrates that it does not result in the release of harmful cytokines.

[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 Tumor cells were inoculated to induce tumor development. The average tumor size was approximately 50 mm. 3 When the mouse reaches The mice were randomized. 32 mice were randomly assigned to four test groups. The mice were given M106 anti-mouse PD-1, Neu2-Fc variant M106 and anti-PD-1 combination or isotype control Either drug was administered twice a week at five different doses, at 10 mg / kg for each drug. Figure 23 shows tumor growth in each mouse in one of the following groups: isotype control group (Figure 23A), M106 group (Figure 23B), anti-PD-1 group (Figure 23C), or combination of M106 and anti-PD-1 (Figure 23D). The M106-treated mice showed complete remission (CR) of tumor growth in one animal, compared to no mice that responded in the isotype-treated groups. The combination of M106 and anti-PD-1 showed complete remission (CR) in all mice compared to the isotype control. The results showed one complete response (CR), one partial response (PR), and an overall reduction in tumor growth.

[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 The mice were randomized when they reached a certain level. 24 mice were randomly assigned to three test groups. The mice were given M106 Either anti-mouse PD-1 or isotype control was administered at a dose of 10 mg / kg twice a week for 5 doses. Figure 24 shows the results for the isotype control group (Figure 24A), the M106 group (Figure 24B), or the anti-PD-1 group (Figure 24C). The tumor growth for each mouse group is shown. Figure 24D shows the isotype for the M106 group. This is a superposition of the control group and clearly demonstrates the benefit of M106 in reducing tumor growth in tumor models considered difficult to treat.

[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. Tumor size is approximately 100 mm 3 When this was reached, the mice were randomized. 16 mice were used in two trials. Mice were randomly assigned to groups. They were administered either M106 or an isotype control at a dose of 10 mg / kg twice a week for five doses. Figure 25 shows tumor growth in each mouse in either the isotype control group (Figure 25A) or the M106 FC group (Figure 25B). Isotype treatment group Compared to only 1 in 8 mice in the other group, 4 out of 8 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 completely eliminated tumor growth. and leads to partial remission This example describes the use of M106 and / or avelumab (anti-PD-L1) in the A20 syngeneic mouse model. This document describes an in vivo study. 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 reaches ( The average tumor volume in each group was 86-90 mm. 3 (The range was), mice were randomly divided into groups of 8 mice. Assigned to: Table 15 lists the arms of the various tests. Mice were given 5 or 10 mg / kg of M106, avelumab and / or antibody isotype control (as shown) twice a week. The patient was treated intraperitoneally with a total of five 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 was treated with monotherapy and avelumab. It showed antitumor activity when combined with "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 either 6 or 12-week-old A20 cells. Tumor volume and body weight were measured 3 times per week. The number of times recorded was as expected in both 6-week and 12-week naive mice. No tumor growth was observed in the proliferating and re-attacking mice (data not shown).

[0296] Therefore, as shown in this example, treatment with the sialidase disclosed herein reduces cancer growth in B-cell lymphoma models, and in some cases This leads to complete remission.

[0297] Example 14: Sialidase treatment alone and in combination with anti-PD-L1 antibody complete tumor growth Producing complete and partial remission This example describes the use of M106 and / or avelumab (anti-PD-L1) in the A20 syngeneic mouse model. The in vivo study is described below. The experiment was conducted in the same manner as in Example 13, except that six doses were administered (twice a week for three weeks). Table 16 describes the various arms of the study. Mice received 10 mg / kg of M106, avelumab and / or an antibody isotype control twice a week for a total of six doses. The procedure was performed intraperitoneally. 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 ASC They exhibited 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). 500,000 cells per mouse were administered intravenously to female C57 / BL6 mice aged 6-8 weeks. The mice were then injected. Subsequently, they were administered isotype controls, 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 the survival rate as of day 28. Figure 28B shows overall survival (as of day 41). Compared to isotype controls, the offer Mice treated with tumumab exhibited a survival shift, with the 50% survival point shifting from day 17 to day 24. This demonstrated that 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 it is widely upregulated on human cancer cells and tumor-infiltrating myeloid cells. Siglec-15 acts as a ligand to suppress antigens in vitro and in vivo. It suppresses the target T cell response. Genetic removal 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 mixed in complete RPMI medium (10% heat-inactivated FBS, non-essential amino acids, and pyruvate). Cells were stimulated with 1 μg / mL of anti-CD3 (OKT3 clone) and anti-CD28 (clone CD28.2) antibodies (both from eBiosciences, Thermo Fisher Scientific) in a sodium acid supplement. On day 2, suspended cells were harvested and re-cultured in fresh complete RPMI medium. The body was replenished with 1 μg / mL, and the cells were continuously stimulated. Three days later, the cells were 10 6The 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 next day (approximately 14 hours later), the cells were spun down to remove the culture medium, and then the cells were placed in PBS in a LIVE / DEAD state. TM Human TruStain FcX Fc receptor blocking agent (Bi) along with fixable near-IR dead cell staining. The cells were then blocked with olegend®. Next, the cells were treated with thermally inactivated human serum (5% in PBS). It was blocked.

[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 then placed on ice for 5 minutes. Incubate for 1 minute, then wash.

[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 Run it on (BD Biosciences) The data was analyzed.

[0310] Figure 29 shows the results of Siglec-15-Fc staining of CD4+ cells (Figure 29A) and CD8+ cells (Figure 29B) after various treatments. For control, isotype IgG1 staining is also shown. As shown, M106 FC also Treatment of activated CD4 and CD8 cells with BiNaNH2 (positive control) is compared to untreated cells or LOF (Low-of-Factor) cells. Compared to treatment with FC, Siglec-15-Fc staining was reduced. 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 sialic acid removal 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 parts 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 Sialidase exhibits at least 50% of the activity of full-length sialidase. A functional fragment, a pharmaceutical composition according to item 1 or 2. Section 4 The sialidase is a variant that exhibits at least 50% of the activity of the wild-type sialidase. or a pharmaceutical composition as described in any of items 1 to 3. 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 serum half-life enhancer is composed of an 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. A pharmaceutical composition selected from the group described in items 1 to 6. 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 sialidase contains one or more mutations compared to the wild-type sialidase template, items 1-9 The pharmaceutical composition described. 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 Sialidases that conjugate to serum half-life enhancers are sequence numbers: 115, 152, 180 The pharmaceutical composition according to claim 14, comprising an amino acid sequence selected from the group consisting of 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. Claim 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) 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) 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) Leucine residue substitution at 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: Section 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 containing approximately 0.01 mg / kg to approximately 100 mg / kg of sialidase, as described in any of items 1 to 20. thing. Section 22 A pharmaceutical composition according to any one of items 1 to 21, comprising a second therapeutic agent. Section 23 The pharmacopoeia described in paragraph 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). composition. 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 Sialidase is a conjugate for cancer antigen targeting agents that bind to cancer antigens associated with cancer cells. Do not use the method described in item 33. Item 35 Sialidase exhibits at least 50% of the activity of full-length sialidase. A functional fragment, as described in any of sections 32 to 34. Section 36 The sialidase is a variant that exhibits at least 50% of the activity of the wild-type sialidase. , or any of the methods described in items 32 to 35. 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. Section 39 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, as indicated in any of items 32-37. Method of loading. 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 sialidase contains one or more mutations compared to the wild-type sialidase template, item 32~ 41. Any of the methods described. 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 Sialidases that conjugate to serum half-life enhancers are sequence numbers: 115, 152, 180 The method according to claim 46, comprising an amino acid sequence selected from the group consisting of 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) 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) 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) Leucine residue substitution at 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. Section 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 Approximately 0.01 mg / kg to approximately 100 mg / kg of sialidase is administered to the subject, as per items 32-52. Or the method of description. 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 Solid tumors can occur in the head and neck (e.g., pharynx), thyroid, lungs (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, ovaries, uterus, cervix, endometrium, prostate, testes), CNS (e.g., nerve or glial cells, e.g., neuroblastoma or The method described in paragraph 54 or 55, which is a tumor of the nerve (glioma) or skin (e.g., melanoma). Section 57 Hematopoietic malignancies include leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), B cells, T cells, and 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, and pilocytic cell leukemia. Disease, myelodysplastic syndrome (MDS), lymphoma, Hodgkin's disease, malignant The method described in paragraph 56, which is 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 Therapeutic agents include anti-inflammatory agents, anti-vasculitizing agents, anti-fibrotic agents, or antiproliferative compounds (e.g., cytotoxic agents). The method described in item 61, selected from the group consisting of (sex 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. Item 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 item further comprises purifying the recombinant sialidase produced in step (b), item 74. Method of description. 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] Sequence 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

[Claim 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.

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