Recombinant sialidases and methods of use thereof

Abstract

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

Description

[0001] Cross-reference with related applications

[0002] This application claims the benefit and priority of U.S. Provisional Patent Application Serial No. 62 / 870,336, filed July 3, 2019, and U.S. Provisional Patent Application Serial No. 62 / 957,027, filed January 3, 2020, the entire contents of each of which are incorporated herein by reference. Technical Field

[0003] The present invention generally relates to recombinant sialylase, methods and compositions for prolonging the serum half-life of recombinant sialylase, and their use in the treatment of sialic acid-related disorders. Background Technology

[0004] Increasing evidence supports the role of glycans, particularly sialylated glycans, in various pathophysiological steps of tumor progression. Glycans regulate tumor proliferation, invasion, hematogenous metastasis, and angiogenesis (Fuster et al., (2005) NAT.REV.CANCER 5(7):526-42). In cancer, the sialylation of cell surface glycoconjugates is frequently altered, leading to the expression of sialylated tumor-associated glycoantigens. The expression of sialylated glycans in tumor cells is often accompanied by increased tumor invasiveness and metastatic potential.

[0005] It has become increasingly apparent recently that the sialic acid-binding lectin family, Siglecs (sialic acid-binding immunoglobulin-like lectins), plays a role in cancer immunosuppression by binding to hypersialylated cancer cells and mediating the inhibition of signals from activated NK cell receptors, thereby inhibiting NK cell-mediated tumor cell killing (Jandus et al., (2014) J.CLIN.INVEST.124:1810-1820). =Jandus et al., (2014)PROC.NATL.ACAD.SCI.USA 111:14211-14216; Hudak et al., (2014)NAT.CHEM.BIOL.10:69-75). Similarly, the removal of sialic acid by sialidase can enhance NK cell-mediated tumor cell killing (Jandus, ibid.; Hudak, ibid.; Xiao et al., (2016)PROC.NATL.ACAD.SCI.USA 113(37):10304-9).

[0006] Cancer immunotherapy using immune checkpoint inhibitors, including 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 to treat cancers associated with hypersialylated cancer cells. Summary of the Invention

[0007] This invention is based in part on the discovery that it is possible to treat sialic acid-mediated disorders by administering sialic acid phosphatase or sialic acid phosphatase conjugated with a serum half-life enhancer. Surprisingly, it has been found that sialic acid phosphatase lacking a target component (e.g., an antibody-binding domain against a tumor antigen) or sialic acid phosphatase conjugated with a serum half-life enhancer can effectively treat sialic acid-mediated disorders (e.g., cancers, such as solid tumors) in vivo.

[0008] The present invention also relates to recombinant forms of sialidase, sialidase coupled with serum half-life enhancers, and pharmaceutical compositions thereof, which have suitable substrate specificity and activity for removing sialic acid and / or sialic acid-containing molecules from the surface of cancer cells and / or removing sialic acid and / or sialic acid-containing molecules from the tumor microenvironment and / or reducing the concentration of sialic acid and / or sialic acid-containing molecules in the tumor microenvironment.

[0009] Therefore, in some aspects, the present invention provides a pharmaceutical composition comprising, or substantially comprising, a sialidase coupled with a serum half-life enhancer that increases the serum half-life of sialidase when administered to a subject.

[0010] On the other hand, the present invention provides a method for treating sialic acid-related disorders in a subject in need. The method includes administering an effective amount of a pharmaceutical composition to the subject to treat the disorder, the pharmaceutical composition comprising sialic acid enzyme and a serum half-life enhancer that increases the serum half-life of the sialic acid enzyme upon administration to the subject.

[0011] In some embodiments, the sialidase is not coupled to a cancer antigen targeter that binds to cancer antigens associated with cancer cells.

[0012] In some embodiments, the sialidase is a functional fragment or variant of the full-length sialidase that exhibits at least 50% of the activity of the full-length sialidase.

[0013] In some embodiments, the sialidase and serum half-life enhancer are covalently linked together in the fusion protein or chemically coupled together.

[0014] In some embodiments, the serum half-life enhancer is selected from Fc domains, transferrin, albumin, XTEN, homo-amino acid polymers (HAP), proline-alanine-serine polymers (PAS), elastin-like peptides (ELP), albumin-binding domains, CTP fusions, GLK fusions, and polyethylene glycol.

[0015] In some embodiments, the serum half-life enhancer is an Fc domain.

[0016] In some embodiments, the serum half-life enhancer is not an Fc domain or polyethylene glycol.

[0017] In some embodiments, the sialidase contains one or more mutations relative to the template wild-type sialidase.

[0018] In some embodiments, the sialidase comprises: a substitution or deletion of a methionine residue (M1) at position 1 corresponding to wild-type human Neu2; a substitution of a valine residue (V6) at position 6 corresponding to wild-type human Neu2; a substitution of an isoleucine residue (I187) at position 187 corresponding to wild-type human Neu2; or a substitution of a cysteine ​​residue (C332) at position 332 corresponding to wild-type human Neu2; or any combination of the above substitutions. In some embodiments, in the sialidase: (a) the methionine residue at position 1 corresponding to wild-type Neu2 is deleted (ΔM1), replaced by alanine (M1A), or replaced by aspartic acid (M1D); (b) the valine residue at position 6 corresponding to wild-type Neu2 is replaced by tyrosine (V6Y); (c) the isoleucine residue at position 187 corresponding to wild-type Neu2 is replaced by lysine (I187K); or (d) the cysteine ​​residue at position 332 corresponding to wild-type Neu2 is replaced by alanine (C332A); or the sialidase comprises any combination of the above substitutions.

[0019] In some embodiments, the sialidase comprises: a substitution or deletion of a methionine residue (M1) at position 1 corresponding to wild-type Neu2; a substitution of a valine residue (V6) at position 6 corresponding to wild-type Neu2; a substitution of a proline residue (P62) at position 62 corresponding to wild-type Neu2; a substitution of an alanine residue (A93) at position 93 corresponding to wild-type Neu2; a substitution of an isoleucine residue (I187) at position 187 corresponding to wild-type Neu2; and a substitution of a valine residue (I187) at position 126 corresponding to wild-type Neu2. The substitution of a glutamine residue (Q126); the substitution of an alanine residue (A242) at position 242 corresponding to wild-type Neu2; the substitution of a glutamine residue (Q270) at position 270 corresponding to wild-type Neu2; the substitution of a serine residue (S301) at position 301 corresponding to wild-type Neu2; the substitution of a tryptophan residue (W302) at position 302 corresponding to wild-type Neu2; the substitution of a cysteine ​​residue (C332) at position 332 corresponding to wild-type Neu2; or any combination of the above substitutions.

[0020] In some embodiments, the sialidase comprises an alternative combination selected from the following:

[0021] (a)M1D, V6Y, P62G, A93E, I187K, C332A;

[0022] (b)M1D, V6Y, P62G, A93E, I187K, S301A, W302R, C332A;

[0023] (c)M1D, V6Y, P62G, A93E, Q126Y, I187K, A242F, Q270T, C332A;

[0024] (d)M1D,V6Y,P62G,A93E,Q126Y,I187K,C332A; and

[0025] (e)A93E, Q126Y, I187K, A242F, Q270T, C332A.

[0026] In some embodiments, the sialidase coupled with the serum half-life enhancer comprises an amino acid sequence selected from SEQ ID NO: 115, 152, 180, 184 and 188 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with an amino acid sequence selected from SEQ ID NO: 115, 152, 180, 184 and 188.

[0027] In some embodiments, the sialidase comprises: a substitution of a proline residue (P5) at position 5 corresponding to wild-type Neu2; a substitution of a lysine residue (K9) at position 9 corresponding to wild-type Neu2; a substitution of a lysine residue (K44) at position 44 corresponding to wild-type Neu2; a substitution of a lysine residue (K45) at position 45 corresponding to wild-type Neu2; a substitution of a leucine residue (L54) at position 54 corresponding to wild-type Neu2; a substitution of a proline residue (P62) at position 62 corresponding to wild-type Neu2; and a substitution of a glutamine residue at position 69 corresponding to wild-type Neu2. The substitutions are as follows: substitution of arginine residue (R78) at position 78 (corresponding to wild-type Neu2); substitution of aspartic acid residue (D80) at position 80 (corresponding to wild-type Neu2); substitution of alanine residue (A93) at position 93 (corresponding to wild-type Neu2); substitution of glycine residue (G107) at position 107 (corresponding to wild-type Neu2); substitution of glutamine residue (Q108) at position 108 (corresponding to wild-type Neu2); substitution of glutamine residue (Q112) at position 112 (corresponding to wild-type Neu2); and substitution of arginine residue (Q112) at position 125 (corresponding to wild-type Neu2). The substitution of cysteine ​​residue (C125) at position 126 (corresponding to wild-type Neu2); the substitution of glutamine residue (Q126) at position 126 (corresponding to wild-type Neu2); the substitution of alanine residue (A150) at position 150 (corresponding to wild-type Neu2); the substitution of cysteine ​​residue (C164) at position 164 (corresponding to wild-type Neu2); the substitution of arginine residue (R170) at position 170 (corresponding to wild-type Neu2); the substitution of alanine residue (A171) at position 171 (corresponding to wild-type Neu2); the substitution of glutamine residue (Q188) at position 188 (corresponding to wild-type Neu2); the substitution of arginine residue (R170) at position 171 (corresponding to wild-type Neu2); the substitution of arginine residue (R170) at position 170 (corresponding to wild-type Neu2); the substitution of arginine residue (Q188) at position 18 ... The substitution of an arginine residue (R189) at position 189 of Neu2; the substitution of an alanine residue (A213) at position 213 of Neu2 (corresponding to wild-type human Neu2); the substitution of a leucine residue (L217) at position 217 of Neu2 (corresponding to wild-type human Neu2); the substitution of a glutamic acid residue (E225) at position 225 of Neu2 (corresponding to wild-type human Neu2); the substitution of a histidine residue (H239) at position 239 of Neu2 (corresponding to wild-type human Neu2); the substitution of a leucine residue (L240) at position 240 of Neu2 (corresponding to wild-type human Neu2); and the substitution of an arginine residue (R241) at position 241 of Neu2 (corresponding to wild-type human Neu2).The substitutions are as follows: Alanine residue (A242) at position 242 (corresponding to wild-type Neu2); valine residue (V244) at position 244 (corresponding to wild-type Neu2); threonine residue (T249) at position 249 (corresponding to wild-type Neu2); aspartic acid residue (D251) at position 251 (corresponding to wild-type Neu2); glutamic acid residue (E257) at position 257 (corresponding to wild-type Neu2); serine residue (S258) at position 258 (corresponding to wild-type Neu2); leucine residue (L260) at position 260 (corresponding to wild-type Neu2); and valine residue (V244) at position 265 (corresponding to wild-type Neu2). The substitution of the following residues: (V265); (Q270) glutamine residue at position 270 of wild-type Neu2; (W292) tryptophan residue at position 292 of wild-type Neu2; (S301) serine residue at position 301 of wild-type Neu2; (W302) tryptophan residue at position 302 of wild-type Neu2; (C332) cysteine ​​residue at position 332 of wild-type Neu2; (V363) valine residue at position 363 of wild-type Neu2; or (L365) leucine residue at position 365 of wild-type Neu2; or any combination of the above substitutions.

[0028] In some embodiments, the sialidase is selected from bacterial sialidase, viral sialidase, and mammalian sialidase. In some embodiments, the sialidase is human sialidase. In some embodiments, the human sialidase is selected from neu1, neu2, neu3, and neu4. In some embodiments, the human sialidase is neu2.

[0029] In some embodiments, the pharmaceutical composition comprises about 0.01 mg / kg to about 100 mg / kg of the sialidase.

[0030] In some embodiments, the pharmaceutical composition comprises a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from anti-inflammatory agents, anti-angiogenic agents, anti-fibrotic agents, or anti-proliferative compounds (e.g., cytotoxic agents or checkpoint inhibitors).

[0031] In some embodiments, the pharmaceutical composition further comprises a stabilizing amount of a sialidase stabilizer. In some embodiments, the sialidase stabilizer is a cation. In some embodiments, the cation is selected from calcium and magnesium.

[0032] In some embodiments, the pharmaceutical composition is disposed in a sterile container (e.g., a bottle or tube). In some embodiments, the pharmaceutical composition is freeze-dried in the sterile container. In some embodiments, the pharmaceutical composition is present as a solution in the sterile container. In some embodiments, the sterile container is sealed with a diaphragm. In some embodiments, the sterile container has a label affixed thereto identifying the pharmaceutical composition contained therein.

[0033] On the other hand, this disclosure relates to a method of treating a sialic acid-related disorder in a subject in need, the method comprising administering to the subject a pharmaceutical composition comprising an effective amount of sialic acidase and a serum half-life enhancer that increases the serum half-life of the sialic acidase when administered to the subject, thereby treating the disorder.

[0034] In some embodiments, the sialic acid-related disorder is cancer. In some embodiments, the sialylase is not coupled to a cancer antigen targeter that binds to cancer antigens associated with cancer cells.

[0035] In some embodiments, the sialidase is a functional fragment of the full-length sialidase that exhibits at least 50% of the activity of the full-length sialidase. In some embodiments, the sialidase is a variant that exhibits at least 50% of the activity of the wild-type sialidase.

[0036] In some embodiments, the sialidase and the serum half-life enhancer are covalently linked together in the fusion protein. In some embodiments, the sialidase and the serum half-life enhancer are chemically coupled together.

[0037] In some embodiments, the serum half-life enhancer is selected from Fc domains, transferrin, albumin, XTEN, homopolymers of amino acids (HAP), proline-alanine-serine polymers (PAS), elastin-like peptides (ELP), and polyethylene glycol. In some embodiments, the serum half-life enhancer is an Fc domain. In some embodiments, the serum half-life enhancer is neither an Fc domain nor polyethylene glycol.

[0038] In some embodiments, the sialidase comprises one or more mutations relative to the template wild-type sialidase. In some embodiments, the sialidase comprises: a substitution or deletion of a methionine residue (M1) at position 1 corresponding to wild-type human Neu2; a substitution of a valine residue (V6) at position 6 corresponding to wild-type human Neu2; a substitution of an isoleucine residue (I187) at position 187 corresponding to wild-type human Neu2; or a substitution of a cysteine ​​residue (C332) at position 332 corresponding to wild-type human Neu2; or any combination of the above substitutions.

[0039] In some embodiments, in the sialidase: the methionine residue at position 1 corresponding to wild-type Neu2 is deleted (ΔM1), replaced by alanine (M1A), or replaced by aspartic acid (M1D); the valine residue at position 6 corresponding to wild-type Neu2 is replaced by tyrosine (V6Y); the isoleucine residue at position 187 corresponding to wild-type Neu2 is replaced by lysine (I187K); or the cysteine ​​residue at position 332 corresponding to wild-type Neu2 is replaced by alanine (C332A); or the sialidase comprises any combination of the above substitutions.

[0040] In some embodiments, the sialidase comprises: a substitution or deletion of a methionine residue (M1) at position 1 corresponding to wild-type Neu2; a substitution of a valine residue (V6) at position 6 corresponding to wild-type Neu2; a substitution of a proline residue (P62) at position 62 corresponding to wild-type Neu2; a substitution of an alanine residue (A93) at position 93 corresponding to wild-type Neu2; a substitution of an isoleucine residue (I187) at position 187 corresponding to wild-type Neu2; and a substitution of a valine residue (I187) at position 126 corresponding to wild-type Neu2. The substitution of a glutamine residue (Q126); the substitution of an alanine residue (A242) at position 242 corresponding to wild-type Neu2; the substitution of a glutamine residue (Q270) at position 270 corresponding to wild-type Neu2; the substitution of a serine residue (S301) at position 301 corresponding to wild-type Neu2; the substitution of a tryptophan residue (W302) at position 302 corresponding to wild-type Neu2; the substitution of a cysteine ​​residue (C332) at position 332 corresponding to wild-type Neu2; or any combination of the above substitutions.

[0041] In some embodiments, the sialidase comprises an alternative combination selected from the following:

[0042] (a) M1D, V6Y, P62G, A93E, I187K, C332A;

[0043] (b) M1D, V6Y, P62G, A93E, I187K, S301A, W302R, C332A;

[0044] (c) M1D, V6Y, P62G, A93E, Q126Y, I187K, A242F, Q270T, C332A;

[0045] (d) M1D, V6Y, P62G, A93E, Q126Y, I187K, C332A; and

[0046] (e)A93E, Q126Y, I187K, A242F, Q270T, C332A.

[0047] In some embodiments, the sialidase coupled with the serum half-life enhancer comprises an amino acid sequence selected from SEQ ID NO: 115, 152, 180, 184 and 188 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with an amino acid sequence selected from SEQ ID NO: 115, 152, 180, 184 and 188.

[0048] In some embodiments, the sialidase comprises: a substitution of a proline residue (P5) at position 5 corresponding to wild-type Neu2; a substitution of a lysine residue (K9) at position 9 corresponding to wild-type Neu2; a substitution of a lysine residue (K44) at position 44 corresponding to wild-type Neu2; a substitution of a lysine residue (K45) at position 45 corresponding to wild-type Neu2; a substitution of a leucine residue (L54) at position 54 corresponding to wild-type Neu2; a substitution of a proline residue (P62) at position 62 corresponding to wild-type Neu2; and a substitution of a glutamine residue at position 69 corresponding to wild-type Neu2. The substitutions are as follows: substitution of arginine residue (R78) at position 78 (corresponding to wild-type Neu2); substitution of aspartic acid residue (D80) at position 80 (corresponding to wild-type Neu2); substitution of alanine residue (A93) at position 93 (corresponding to wild-type Neu2); substitution of glycine residue (G107) at position 107 (corresponding to wild-type Neu2); substitution of glutamine residue (Q108) at position 108 (corresponding to wild-type Neu2); substitution of glutamine residue (Q112) at position 112 (corresponding to wild-type Neu2); and substitution of arginine residue (Q112) at position 125 (corresponding to wild-type Neu2). The substitution of cysteine ​​residue (C125) at position 126 (corresponding to wild-type Neu2); the substitution of glutamine residue (Q126) at position 126 (corresponding to wild-type Neu2); the substitution of alanine residue (A150) at position 150 (corresponding to wild-type Neu2); the substitution of cysteine ​​residue (C164) at position 164 (corresponding to wild-type Neu2); the substitution of arginine residue (R170) at position 170 (corresponding to wild-type Neu2); the substitution of alanine residue (A171) at position 171 (corresponding to wild-type Neu2); the substitution of glutamine residue (Q188) at position 188 (corresponding to wild-type Neu2); the substitution of arginine residue (R170) at position 171 (corresponding to wild-type Neu2); the substitution of arginine residue (R170) at position 170 (corresponding to wild-type Neu2); the substitution of arginine residue (Q188) at position 18 ... The substitution of an arginine residue (R189) at position 189 of Neu2; the substitution of an alanine residue (A213) at position 213 of Neu2 (corresponding to wild-type human Neu2); the substitution of a leucine residue (L217) at position 217 of Neu2 (corresponding to wild-type human Neu2); the substitution of a glutamic acid residue (E225) at position 225 of Neu2 (corresponding to wild-type human Neu2); the substitution of a histidine residue (H239) at position 239 of Neu2 (corresponding to wild-type human Neu2); the substitution of a leucine residue (L240) at position 240 of Neu2 (corresponding to wild-type human Neu2); and the substitution of an arginine residue (R241) at position 241 of Neu2 (corresponding to wild-type human Neu2).The substitutions are as follows: Alanine residue (A242) at position 242 (corresponding to wild-type Neu2); valine residue (V244) at position 244 (corresponding to wild-type Neu2); threonine residue (T249) at position 249 (corresponding to wild-type Neu2); aspartic acid residue (D251) at position 251 (corresponding to wild-type Neu2); glutamic acid residue (E257) at position 257 (corresponding to wild-type Neu2); serine residue (S258) at position 258 (corresponding to wild-type Neu2); leucine residue (L260) at position 260 (corresponding to wild-type Neu2); and valine residue (V244) at position 265 (corresponding to wild-type Neu2). The substitution of the following residues: (V265); (Q270) glutamine residue at position 270 of wild-type Neu2; (W292) tryptophan residue at position 292 of wild-type Neu2; (S301) serine residue at position 301 of wild-type Neu2; (W302) tryptophan residue at position 302 of wild-type Neu2; (C332) cysteine ​​residue at position 332 of wild-type Neu2; (V363) valine residue at position 363 of wild-type Neu2; or (L365) leucine residue at position 365 of wild-type Neu2; or any combination of the above substitutions.

[0049] In some embodiments, the sialidase is selected from bacterial sialidase, viral sialidase, and mammalian sialidase. In some embodiments, the mammalian sialidase is human sialidase. In some embodiments, the human sialidase is selected from neu1, neu2, neu3, and neu4. In some embodiments, the human sialidase is neu2.

[0050] In some embodiments, the sialidase is administered to the subject at a concentration of about 0.01 mg / kg to about 100 mg / kg.

[0051] In some embodiments, the cancer is a solid tumor, a soft tissue tumor, a hematopoietic tumor, or a metastatic lesion. In some embodiments, the solid tumor is a sarcoma, adenocarcinoma, or carcinoma. In some embodiments, the solid tumor is a tumor of the head and neck (e.g., pharynx), thyroid gland, lung (e.g., small cell or non-small cell lung cancer (NSCLC)), breast, lymph nodes, gastrointestinal tract (e.g., oral cavity, esophagus, stomach, liver, pancreas, small intestine, colon and rectum, anal canal), reproductive or genitourinary tract (e.g., kidney, urothelial, bladder, ovary, uterus, cervix, endometrium, prostate, testis), CNS (e.g., nerve or glial cell tumors, such as neuroblastoma or glioma), or skin (e.g., melanoma). In some embodiments, the cancer is breast cancer.

[0052] In some embodiments, the hematopoietic tumor is leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), B-cell, T-cell, or FAB ALL, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL) such as metastatic CLL, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, hairy cell leukemia, myelodysplastic syndrome (MDS), lymphoma, Hodgkin's disease, malignant lymphoma, non-Hodgkin's lymphoma, Burkitt lymphoma, multiple myeloma, or Richter's syndrome (Richter's transformation). In some embodiments, the cancer is lymphoma.

[0053] In some embodiments, administration of the pharmaceutical composition increases the expression of granzyme B, IFNγ, IL-10, IL-6, or IL-17A in the subject.

[0054] In some embodiments, the pharmaceutical composition is administered to the subject in combination with another therapeutic agent. In some embodiments, the therapeutic agent is selected from anti-inflammatory agents, anti-angiogenic agents, anti-fibrotic agents, or anti-proliferative compounds (e.g., cytotoxic agents or checkpoint inhibitors).

[0055] In some embodiments, the pharmaceutical composition further comprises a stabilizing amount of a sialidase stabilizer. In some embodiments, the sialidase stabilizer is a cation. In some embodiments, the cation is selected from calcium and magnesium.

[0056] In some embodiments, the pharmaceutical composition is placed in a sterile container (e.g., a bottle or tube) before administration.

[0057] In some embodiments, the method includes administering an effective amount of the pharmaceutical composition to the subject.

[0058] In some embodiments, this disclosure relates to a method for removing sialic acid from cells in a subject, the method comprising administering an effective amount of the pharmaceutical composition to the subject thereby removing sialic acid from the cells.

[0059] In some embodiments, the cells are tumor cells, dendritic cells (DCs), or monocytes. In some embodiments, the cells are monocytes, and the method results in increased expression of MHC-II molecules on the monocytes.

[0060] In some embodiments, this disclosure relates to a method for enhancing the phagocytic activity of tumor cells in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical composition in an amount that effectively removes sialic acid from the tumor cells, thereby enhancing the phagocytic activity of the tumor cells.

[0061] In some embodiments, this disclosure relates to a method for activating dendritic cells (DCs) in a subject, the method comprising administering the pharmaceutical composition to the subject in an amount that effectively removes sialic acid from tumor cells in the subject, thereby activating the DCs in the subject.

[0062] In some embodiments, this disclosure relates to a method for reducing Siglec-15 binding activity to enhance antitumor activity in the tumor microenvironment of a patient, the method comprising administering an effective amount of the pharmaceutical composition to the subject to enhance antitumor activity (e.g., T-cell activity) in the subject.

[0063] On the other hand, the present invention provides a method for expressing recombinant sialidase. The method may include: (a) providing cells containing nucleic acids encoding the recombinant sialidase; and (b) expressing the recombinant sialidase in the presence of a stabilizer. In some embodiments, the method further includes purifying the recombinant sialidase produced in step (b). The purification is performed in the presence of a stabilizer such as a cation (e.g., calcium or magnesium).

[0064] These and other aspects and features of the invention are described in the following detailed description and claims. Attached Figure Description

[0065] The invention can be more fully understood with reference to the following figures.

[0066] Figure 1Different configurations of sialidase-Fc fusion constructs are described. The 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 linked together, for example, by disulfide bonds. Figure 1 A shows a sialidase construct having two Fc domains and an N-terminus coupled to each Fc domain. Figure 1 B shows a sialidase construct having two Fc domains and a C-terminus coupled to the first Fc domain and an N-terminus coupled to the second Fc domain. Figure 1 C shows a sialidase construct having two Fc domains and an N-terminus coupled to the second Fc domain. Figure 1 D shows a sialidase construct having two Fc domains and a C-terminus coupled to the first Fc domain. Figure 1 E illustrates a sialidase construct having two Fc domains and a C-terminus coupled to each Fc domain. It should be understood that the Fc domains can be naturally occurring Fc domains or engineered Fc domains containing modifications, such as point mutations in each polypeptide chain that favor a mortar configuration or provide altered Fc domain function.

[0067] Figure 2 The depicted SDS-PAGE gels show recombinant human Neu1, Neu2, Neu3, and Salmonella typhimurium (St-sialidase) under non-reducing and reducing conditions. Monomers and dimers are labeled.

[0068] Figure 3 This is a bar graph showing the enzyme activities of recombinant human Neu1, Neu2, and Neu3.

[0069] Figure 4 This is a line graph showing the enzyme activities of recombinant human Neu2 and Neu3 as a function of substrate concentration at the indicated pH.

[0070] Figure 5A The depicted SDS-PAGE gels show recombinant wild-type human Neu2-Fc and Neu2-Fc variant M106 (“M106”) under non-reducing and reducing conditions. Figure 5B and 5C The SEC-HPLC trace of wild-type Neu2-Fc compared to M106 is shown, with the monomer having a retention time of 21 minutes.

[0071] Figure 6 This is a line graph showing the enzyme activity of M106 as a function of substrate concentration.

[0072] Figure 7 This is a bar graph showing the enzyme activity of Neu3-Fc in the supernatant (“supernatant”) or membrane-bound (“washed cells”) fraction of Expi293 cells.

[0073] Figure 8 It is the SEC-HPLC trace of Fc-ST sialidase, in which the monomeric substance has a retention time of 21 minutes.

[0074] Figure 9A -D is a series of line graphs showing tumor volume in a mouse A20 (lymphoma) syngeneic tumor model. Mice were treated twice weekly with a negative control (“isotype control”) at a dose of 10 mg / kg. Figure 9A ), Fc-ST sialidase ( Figure 9B ), acitumab (anti-mouse PD-L1 antibody), Figure 9C ) or a combination of Fc-ST sialidase and acimetidine ( Figure 9D The study lasted for 15 days, with tumor volume measured over time. Administration of FC-ST sialidase, alone or in combination with acitumab, reduced tumor volume.

[0075] Figure 10A -D is a series of line graphs showing tumor volume in a mouse syngeneic tumor model using EMT6 cells engineered to express human Her2. As indicated by the triangles, mice were administered isotype control (mediator control) twice weekly at a dose of 10 mg / kg. Figure 10A ), Fc-ST sialidase (FC-ST, Figure 10B ), Trastuzumab (anti-human Her2 antibody), Figure 10C ) or Fc human sialic acidase (M106, Figure 10D The study lasted for 15 days, with tumor volume measured over time. Fc human sialidase or Fc-ST sialidase reduced tumor volume.

[0076] Figure 11 The bar graph shows that by adding CaCl2, the activity of neuraminidase was stabilized for up to 14 days after incubation at 37°C.

[0077] Figure 12A The bar graph shows the neuraminidase activity in modulated culture medium of cells expressing the human neuraminidase Fc construct on the indicated day post-transfection, with or without 4 mM CaCl2. As shown, the presence of CaCl2 stabilizes the activity. Figure 12B The bar graph depicts the cell viability indicated by the number of days post-transfection in the presence or absence of 4 mM CaCl2.

[0078] Figure 13AThe bar graphs show how different concentrations of CaCl2 stabilized neuraminidase activity in modulated culture media of cells expressing the human neuraminidase Fc construct. The graphs also show the enzyme activity indicated on the days following transfection in the presence of 0, 0.05, 0.5, 1, 2, and 4 mM CaCl2. Figure 13B The total protein yield on day 6 is shown in the presence of 0, 0.05, 0.5, 1, 2 and 4 mM CaCl2.

[0079] The bar chart provided in Figure 14 depicts the differences in Hydra-3 (3) from different immune subsets. Figure 14A Hydra-7 Figure 14B ) and Hydra-9( Figure 14C Geometric mean fluorescence intensity (gMFI) produced by staining.

[0080] The bar chart provided in Figure 15 depicts the PNA (Personal Acids) from different immune subsets. Figure 15A MAL-II Figure 15B ) and SNA ( Figure 15C Geometric mean fluorescence intensity (gMFI) produced by staining.

[0081] The line graph provided in Figure 16 depicts the degree of desialylation of dendritic cells (DCs) induced by increasing the concentration of M106. Figure 16A The mean fluorescence intensity (MFI) was depicted, and Figure 16B The provided bar chart depicts the fold increase in desialylation compared to untreated DC.

[0082] Figure 17 provides a line graph depicting the degree of desialylation of BT-20 (breast cancer) tumor cells after treatment with gradually increasing concentrations of M106 (triangles) compared to the LOF control (squares), which is mediated by Hydra 9 binding (…). Figure 17A ) or PNA binding ( Figure 17B This is determined and measured using gMFI.

[0083] Figure 18 provides a line graph depicting the degree of desialylation of HT-29 tumor cells after treatment with gradually increasing concentrations of M106 (triangles) compared to the LOF control (squares), which is mediated by Hydra 9 binding (…). Figure 18A ) or PNA binding ( Figure 18B This is determined and measured using gMFI.

[0084] Figure 19 provides a line graph depicting the degree of desialylation of SK-BR-3 tumor cells after treatment with gradually increasing concentrations of M106 (triangles) compared to the LOF control (squares), which is mediated by Hydra 9 binding (…). Figure 19A MAL-II combination () Figure 19B ) or PNA binding ( Figure 19C This is determined and measured using gMFI.

[0085] Figure 20 provides a bar chart depicting the expression of CD83hi on DCs after incubation with SKBR3 tumor cells treated with or without M106, in the presence or absence of lipopolysaccharide (LPS) treatment (hollow bars versus solid bars). Figure 20A ) and CD86hi expression ( Figure 20B The percentage increase.

[0086] Figure 21 illustrates the dose-dependent enhancement of phagocytosis by M2-like macrophages on HT-29 tumor cells desialylated with M106 or LOF, as indicated. The tumor cells originated from two different healthy donors ( Figure 21A and Figure 21B Similar enhancements in the phagocytic activity of M2-like macrophages against desialylated BT20 and SKBR-3 tumor cells were described separately in Figure 21C and Figure 21D middle.

[0087] Figure 22 shows the dose-dependent enhancement of HLA-DR expression in monocytes after desialylation with M106 or LOF controls. The monocytes were obtained from two different healthy donors ( Figure 22A and Figure 22B ).

[0088] Figure 23 provides tumor growth curves depicting the in vivo activity of sialidase in a mouse MC38 syngeneic tumor model of this disclosure. For mice treated with isotype control (… Figure 23A Mice treated with M106 Figure 23B Mice treated with anti-PD-1 antibodies ( Figure 23C Mice treated with a combination of M106 and anti-PD-1 antibody () or mice Figure 23D For example, the tumor growth curve of a single mouse is shown. The triangles indicate the time of administration of the test substance.

[0089] Figure 24 provides tumor growth curves depicting the in vivo activity of sialidase of this disclosure in a mouse B16F10 syngeneic tumor model. For mice treated with isotype control (… Figure 24A Mice treated with M106 Figure 24B Mice treated with anti-PD-1 antibodies () or anti-PD-1 antibodies Figure 24C For example, the tumor growth curve of a single mouse is shown. Figure 24D It is a superposition of tumor growth curves from the same type control group and the M106 group. The triangle indicates the administration time of the test substance.

[0090] Figure 25 provides tumor growth curves depicting the in vivo activity of sialidase in a mouse EMT6 syngeneic tumor model of this disclosure. For mice treated with isotype control (… Figure 25A Mice treated with M106 or M106 Figure 25B For example, the tumor growth curve for each individual mouse is shown. The triangles indicate the timing of test substance administration.

[0091] Figure 26 The in vivo efficacy of M106, alone or in combination with avermab (“Ave”), at indicated doses was depicted in a mouse A20 syngeneic subcutaneous tumor model. Tumor growth curves were plotted for each mouse. Observed partial responses (PR) and complete responses (CR) were also labeled.

[0092] Figure 27 The in vivo efficacy of M106, alone or in combination with avermab, at indicated doses was depicted in a mouse A20 syngeneic subcutaneous tumor model. Tumor growth curves were plotted for each mouse. Triangles indicate dosing.

[0093] Figure 28 depicts the effects of ofumumab, the combination of ofumumab and Neu2-M106-Fc (“M106 FC”), and the isotype control on day 28 in an intravenous dissemination model of syngeneic EL4-CD20 lymphoma. Figure 28A ) or at the end of life on the 41st day ( Figure 28B The in vivo activity of the test substance is indicated by triangles representing the dosage of various test substances. The p-value is calculated using the Mantle-Cox test.

[0094] Figure 29 depicts the effects of treatment on CD4+ cells after untreated (“None”), treatment with defunctionalized sialidase (“LOF FC”), or treatment with sialidase (M106 (“M106 FC”) or BiNaNH2 (positive control)). Figure 29A ) and CD8+ cells ( Figure 29B Results of Siglec-15-Fc staining are shown. Allotype IgG1 staining as a negative control is also shown. As indicated, treatment of activated CD4 and CD8 cells with M106 or BiNaNH2 reduced Siglec-15-Fc staining compared to untreated or nonfunctional sialidase treatment. Bar charts showing fluorescence levels (gMFI) and underlying flow cytometry histogram data are provided in each figure.

[0095] Figure 30 depicts the use and Figure 30A CD4+ cells obtained using the same method in -B ( Figure 30A ) and CD8+ cells ( Figure 30BThe results of Siglec-15-Fc staining, in which PBMCs were derived from a second healthy donor.

[0096] Detailed description

[0097] This invention is based in part on the discovery that it is possible to treat sialic acid-mediated disorders by administering sialic acid phosphatase or sialic acid phosphatase conjugated with a serum half-life enhancer. Surprisingly, it has been found that sialic acid phosphatases lacking a target component (e.g., an antibody-binding domain against a tumor antigen) or sialic acid phosphatase conjugated with a serum half-life enhancer can effectively treat sialic acid-mediated disorders (e.g., cancer, such as solid tumors) in vivo. As a result, the constructs described herein can be used alone to treat sialic acid-mediated disorders such as cancer, or they can be combined with another agent, such as an anticancer agent, to treat said disorders such as cancer. For example, when used in combination with another anticancer agent, said constructs can enhance the activity of said anticancer agent, for example, by making said cancer more sensitive to treatment with said anticancer agent.

[0098] The present invention also relates to recombinant forms of sialidase, sialidase coupled with serum half-life enhancers, and pharmaceutical compositions thereof, which have suitable substrate specificity and activity for removing sialic acid and / or sialic acid-containing molecules from the surface of cancer cells and / or removing sialic acid and / or sialic acid-containing molecules from the tumor microenvironment and / or reducing the concentration of sialic acid and / or sialic acid-containing molecules in the tumor microenvironment.

[0099] The present invention also relates to pharmaceutical compositions and methods of treating cancers such as solid tumors, soft tissue tumors, hematopoietic tumors, metastatic lesions, or epithelial cell carcinomas using sialidase or sialidase coupled with a half-life extender.

[0100] The various features and aspects of the invention will now be discussed in more detail.

[0101] I. Recombinant sialidase

[0102] When used herein, the term "sialidase" refers to any enzyme or functional fragment or variant thereof that cleaves terminal sialic acid residues from a substrate such as a glycoprotein or glycolipid. The term sialidase includes variants having one or more amino acid substitutions, deletions, or insertions relative to the wild-type sialidase sequence and / or fusion proteins or conjugates comprising sialidase. Sialidase is also known as neuraminidase, and these two terms are used interchangeably herein unless otherwise specified. When used herein, the term "functional fragment" of sialidase refers to a fragment of the 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. The enzymatic activity of sialidase can be determined by any method known in the art, including, for example, by measuring the release of sialic acid from the fluorescent substrate 4-methylumbelliferyl ketone-N-acetylneuraminic acid (4MU-NeuAc). In some embodiments, the functional fragment comprises at least 100, 150, 200, 250, 300, 310, 320, 330, 340, 350, 360, or 370 consecutive amino acids present in full-length, naturally occurring sialidase.

[0103] The sialidase described herein can be any sialidase, such as viral, fungal, bacterial, non-human mammalian, or human sialidase. In some embodiments, the sialidase is a recombinant human sialidase as described above, which contains at least one mutation relative to wild-type human sialidase, such as the substitution, deletion, or addition of at least one amino acid.

[0104] In some embodiments, the sialidase is any recombinant mutant human sialidase or a functional fragment thereof disclosed herein.

[0105] In some embodiments, the sialidase comprises C332A and C352L mutations. In some embodiments, the sialidase comprises an N-terminal addition of MEDLRP (SEQ ID NO: 4) or EDLRP (SEQ ID NO: 3). In some embodiments, the sialidase comprises an LSHSLST (SEQ ID NO: 22) peptide at its N-terminus. In some embodiments, the sialidase comprises an N-terminal addition of MEDLRP (SEQ ID NO: 4) and an A2K substitution. In some embodiments, the sialidase comprises an N-terminal addition of MEDLRP (SEQ ID NO: 4) and a C332A substitution. In some embodiments, the sialidase comprises an N-terminal addition of MEDLRP (SEQ ID NO: 4), a C332A substitution, and a C352L substitution.

[0106] In some embodiments, the sialidase portion comprises M1 deletion (ΔM1), M1A substitution, M1D substitution, V6Y substitution, K9D substitution, P62G substitution, P62N substitution, P62S substitution, P62T substitution, A93E substitution, Q126Y substitution, I187K substitution, A242T substitution, Q270A substitution, Q270T substitution, S301R substitution, S301R substitution, W302K substitution, W302R substitution, C332A substitution, V363R substitution, L365I substitution, or any combination of the above deletions and substitutions.

[0107] In some embodiments, the sialidase comprises an amino acid sequence of any one of SEQ ID NO: 48-62, 169-171 or 196 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NO: 48-62, 169-171 or 196.

[0108] a. Viral sialidase

[0109] Exemplary viral sialidases include influenza A virus surface glycoprotein neuraminidase (e.g., NCBI accession number ACY01419.1, SEQ ID NO: 63), influenza B virus surface glycoprotein neuraminidase (e.g., NCBI accession number AIX94926.1, SEQ ID NO: 64), or influenza C virus surface glycoprotein neuraminidase, or variants or functional fragments thereof. Other exemplary viral sialidases include paramyxoviridae respiratory viruses type 1 and 3 parainfluenza viruses (e.g., NCBI accession number BAD89145.1, SEQ ID NO: 65), bovine parainfluenza virus type 3 (e.g., NCBI accession number ADQ43755, SEQ ID NO: 66), Sendai virus (e.g., UniProtKB accession number P04853.1, SEQ ID NO: 67), rubella virus, mumps virus, simian virus 5, and parainfluenza virus types 2 and 4a, 4b sialidase.

[0110] b. Prokaryotic sialidase

[0111] Exemplary prokaryotic sialidases include those derived from *Salmonella typhimurium* and *Vibrio cholera*. The amino acid sequence of *Salmonella typhimurium* sialidase (St-sialidase) is depicted in SEQ ID NO: 30, and the nucleotide sequence encoding *Salmonella typhimurium* sialidase is depicted in SEQ ID NO: 6. The amino acid sequence of *Vibrio cholera* sialidase is depicted in SEQ ID NO: 36, and the nucleotide sequence encoding *Vibrio cholera* sialidase is depicted in SEQ ID NO: 37.

[0112] Other exemplary sialidases include: sialidases from *Actinomyces viscosus* (Avis_NanH; Uniprot accession number AAA21932, SEQ ID NO: 68); *Arthrobacternicotianae* NA1 and NA2; sialidases from *Arthrobacter sialophilus*; *Arthrobacter ureafaciens* L, M1, M2, and S (GenBank accession number BAD66680, SEQ ID NO: 69); sialidases from *Bacteroides fragilis*; sialidases from *Clostridium chauvoei*; i A99 NanH (GenBank accession number CAA50436, SEQ ID NO: 70), NanI (GenBank accession number ABG83208, SEQ ID NO: 68); and sialidases from *Avis_NanH; Uniprot accession number AAA21932, SEQ ID NO: 68). NO: 71), NanJ (GenBank accession number ABG84247, SEQ ID NO: 72); sialidase from *Clostridium septicum* (e.g., GenBank accession number CAA44916.1, SEQ ID NO: 107); sialidase from *Clostridium sordellii*; sialidase from *Clostridium tertium* (e.g., GenBank accession number CAA69951, SEQ ID NO: 73); sialidase from *Corynebacterium diphtheriae* (e.g., GenBank accession number ACS34893, SEQ ID NO: 74); sialidase from *Haemophilus parasuis*; sialidase from *Micromonospora viridifaciens* (e.g., GenBank accession number BAA00852, SEQ ID NO: 72); NO: 75); Pasteurella multocida NanH (GenBank accession number AAG35310).1, SEQ ID NO: 76) and NanB (AAG35309, SEQ ID NO: 77); sialidases from *Pseudomonas aeruginosa* (e.g., GenBank accession AAG06182, SEQ ID NO: 78); sialidases from *Salmonella Typhimurium* (e.g., GenBank accession NP_459905, SEQ ID NO: 79); *Streptococcus pneumoniae* NanA (GenBank accession P62575, SEQ ID NO: 108), NanB (GenBank accession AAC44396, SEQ ID NO: 80), and NanC; sialidases from *Tannerella forsythia* (e.g., GenBank accession TF0035, SEQ ID NO: 81); and *Vibrio cholerae* (S. 1, SEQ ID NO: 76) and NanB (AAG35309, SEQ ID NO: 77); sialidases from *Vibrio cholerae* (S. 1, SEQ ID NO: 78); sialidases from *Salmonella Typhimurium* (e.g., GenBank accession NP_459905, SEQ ID NO: 79); *Streptococcus pneumoniae* NanA (GenBank accession P62575, SEQ ID NO: 108), NanB (GenBank accession AAC44396, SEQ ID NO: 80), and NanC; sialidases from *Tannerella forsythia* (e.g., GenBank accession TF0035, SEQ ID NO: 81); sialidases from *Vibrio cholerae* (S. 1, SEQ ID NO: 78); sialidases from *Salmonella forsythia* (S Sialidases from *Corynebacterium diphtheriae* (e.g., GenBank accession number YP_001217324, SEQ ID NO: 82); sialidases from *Corynebacterium diphtheriae* (*Corynebacterium diphtheriae* KCTC3075NanH, referred to as Cdip_NanH (GenBank accession number ACS34893, SEQ ID NO: 83) and their homologs; *Corynebacterium glutamicum* R hypothetical protein (Cglu_hypP; YP_001138502, SEQ ID NO: 84); *C. perfringens* NCTC 8239 sialidase I (Cper_NanI; ZP_02643014, SEQ ID NO: 82); NO: 85); Bacteroides fragilis YCH46 sialidase (Bfra_NanH; Uniprot accession number BAA05853, SEQ ID NO: 86); Micromonospora viridans sialidase (Mvir_NanH; Uniprot accession number BAA0085, SEQ ID NO: 87); Streptococcus pneumoniae (S.Sialidase of *Streptomyces coelicolor* A3(2) (Spne_NanA; P62575, SEQ ID NO: 88); Sialidase of *Streptomyces coelicolor* A3(2) (Scoe_NanH; NP_630638, SEQ ID NO: 89); Sialidase of *Streptomyces griseus* NBRC 13350 (Sgri_NanH; YP_001827941, SEQ ID NO: 90); Sialidase of *Propionibacterium acnes* SK137 (Pacn_NanH; ZP_03389398, SEQ ID NO: 91); Sialidase of *Macrobdella decora* (Mdec_NanL; AAC47263 ... NO: 92); Trypanosoma cruzi sialidase (Tcru_TS; GenBank accession number AAA99442, SEQ ID NO: 93); Akkermansia muciniphila (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: 92); NO: 95)(P31206); Bacteroides fragilis SBT3182; Bacteroides fragilis 4852; Bacteroides fragilis YM4000; Bacteroides thetaiotaomicron VPI-5482 sialidase (BtsA; BTSA; BT0455) (GenBank accession number Q8AAK9, SEQ ID NO: 96); Bacteroides vulgatus ATCC8482 / DSM1447 / NCTC 11154BVU_4143 (Uniprot accession number A6L7T1, SEQ ID NO: 97); Bifidobacterium bifidum JCM 1254 exonuclease-α-sialidase (SiaBb2; BBP_0054) (GenBank accession number BAK26854.1, SEQ ID NO: 96). NO: 98); Clostridium perfringens A99 “small” sialidase 1 (P10481, SEQ ID NO: 99); Clostridium perfringens (C.Clostridium perfringens ATCC 10543 sialidase 2 (NanH) (Uniprot accession number Q59311, SEQ ID NO: 100); Clostridium perfringens ATCC 13124 sialidase (CPF_0721) (Uniprot accession number Q0TT67, SEQ ID NO: 101); Clostridium perfringens str 13 exo-α-sialidase (NanI; CPSA; CPE0725) (Uniprot accession number Q8XMG4, SEQ ID NO: 102); Clostridium perfringens str 13 / ATCC 13124 exo-α-sialidase (NanJ; CPE0553 (Uniprot accession number Q8XMY5, SEQ ID NO: 103); Clostridium tertium ATCC Sialidase 14573 (NanH; SiaH) (Uniprot accession number P77848, SEQ ID NO: 104); *R. gnavus* ATCC 29149RgNanH (Uniprot accession number A7B557, SEQ ID NO: 105); *Salmonella typhimurium* TA262 / LT2 sialidase (NanH; STSA) (P29768, SEQ ID NO: 106).

[0113] Other exemplary sialidases include sialidases or neuraminidases derived from A. castellani, A. polyphaga, A. culbertsoni, A. astronyxis, A. hatchetti, A. palestinensis, A. rhysodes, E. tenella, E. maxima, E. necatrix, E. Spec, T. brucei, and T. rangeli.

[0114] c. Mouse sialidase

[0115] Four sialidases have also been identified in the mouse genome, designated Neu1, Neu2, Neu3, and Neu4. The amino acid sequence of mouse Neu1 is depicted in SEQ ID NO: 38, and the nucleotide sequence encoding mouse Neu1 is depicted in SEQ ID NO: 42. The amino acid sequence of mouse Neu2 is depicted in SEQ ID NO: 39, and the nucleotide sequence encoding mouse Neu2 is depicted in SEQ ID NO: 43. The amino acid sequence of mouse Neu3 is depicted in SEQ ID NO: 40, and the nucleotide sequence encoding mouse Neu3 is depicted in SEQ ID NO: 44. The amino acid sequence of mouse Neu4 is depicted in SEQ ID NO: 41, and the nucleotide sequence encoding mouse Neu4 is depicted in SEQ ID NO: 45.

[0116] d. Human sialidase

[0117] Four sialidases have been found in the human genome, known as Neu1, Neu2, Neu3, and Neu4.

[0118] Human Neu1 is a lysosomal neuraminidase that functions in a complex with β-galactosidase and cathepsin A. The amino acid sequence of human Neu1 is depicted in SEQ ID NO: 7, and the nucleotide sequence encoding human Neu1 is depicted in SEQ ID NO: 23.

[0119] Human Neu2 is a cytoplasmic sialylase. The amino acid sequence of human Neu2 is depicted in SEQ ID NO: 1, and the nucleotide sequence encoding human Neu2 is depicted in SEQ ID NO: 24.

[0120] Human Neu3 is a plasma membrane sialylase with specific activity against gangliosides. Human Neu3 has two isoforms: isoform 1 and isoform 2. The amino acid sequence of human Neu3 isoform 1 is depicted in SEQ ID NO: 8, and the nucleotide sequence encoding human Neu3 isoform 1 is depicted in SEQ ID NO: 25. The amino acid sequence of human Neu3 isoform 2 is depicted in SEQ ID NO: 9, and the nucleotide sequence encoding human Neu3 isoform 2 is depicted in SEQ ID NO: 34.

[0121] Human Neu4 has two isoforms: isoform 1 is a peripheral membrane protein, and isoform 2 is confined to the lysosomal lumen. The amino acid sequence of human Neu4 isoform 1 is described in SEQ ID NO: 10, and the nucleotide sequence encoding human Neu4 isoform 1 is described in SEQ ID NO: 26. The amino acid sequence of human Neu4 isoform 2 is described in SEQ ID NO: 11, and the nucleotide sequence encoding human Neu4 isoform 2 is described in SEQ ID NO: 35.

[0122] In some embodiments, the recombinant mutant human sialidase has about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more than 100% of the enzyme activity of the corresponding (or template) wild-type human sialidase.

[0123] In some embodiments, the recombinant mutant human sialidase has the same substrate specificity as the corresponding wild-type human sialidase. In other embodiments, the recombinant mutant human sialidase has different substrate specificity than the corresponding wild-type human sialidase. For example, in some embodiments, the recombinant mutant human sialidase can cleave α2,3, α2,6 and / or α2,8 bonds. In some embodiments, the sialidase can cleave α2,3 and α2,8 bonds.

[0124] In some embodiments, the expression level of the recombinant mutant human sialidase in mammalian cells such as HEK293 cells, CHO cells, mouse myeloma cells (NS0, Sp2 / 0) or human fibrosarcoma cells (HT-1080) such as HEK293 cells is higher than that of the corresponding wild-type human sialidase by about 10%, about 20%, about 50%, about 75%, about 100%, about 150%, about 200%, about 250%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1,000%.

[0125] In some embodiments, the recombinant mutant human sialidase has about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more than 100% of the enzyme activity of the corresponding wild-type human sialidase, and the... The expression levels of recombinant mutant human sialidase in mammalian cells, such as HEK293 cells, are approximately 10%, 20%, 50%, 75%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1,000% higher than those of the corresponding wild-type human sialidase.

[0126] In some embodiments, the amino acid sequence of the recombinant mutant human sialidase has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the corresponding wild-type human sialidase.

[0127] It should be understood that the sialidases described herein, such as human sialidase, can be modified to enhance one or more properties of the enzyme, such as increased expression, activity, or stability (e.g., increased resistance to protease degradation). Some of these properties apply to the various sialidases described herein, such as increased resistance to protease degradation.

[0128] i. Replacement of cysteine ​​residues

[0129] In some embodiments, the recombinant mutant human sialidase comprises a substitution of at least one cysteine ​​(cys, C) residue. It has been found that certain cysteine ​​residues in sialidase may inhibit the expression of functional proteins as a result of protein aggregation. Therefore, in some embodiments, the recombinant mutant human sialidase contains at least one mutation to remove free cysteine ​​(e.g., for Neu1 (SEQ ID NO: 7), one or more mutations of C111, C117, C171, C183, C218, C240, C242, and C252; for Neu2 (SEQ ID NO: 1), one or more mutations of C125, C196, C219, C272, C332, and C352; for Neu3 (SEQ ID NO: 7), one or more mutations of C125, C196, C219, C272, C332, and C352; for Neu3 (SEQ ID NO: 7), one or more mutations of C125, C196, C219, C272, C332, and C352). For NO: 8), mutations in one or more of 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), mutations in one or more of C88, C125, C126, C186, C191, C211, C223, C239, C276, C437, C453, C480, and C481. Free cysteine ​​can be replaced by any amino acid. In some embodiments, the free cysteine ​​is replaced by 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.

[0130] In some embodiments, the recombinant mutant human sialidase comprises two or more cysteine ​​substitutions. Exemplary double or triple substitutions in Neu2 include: C125S and C332S; C272V and C332A; C272V and C332S; C332A and C352L; C125S and C196L; C196L and C352L; C196L and C332A; C332A and C352L; and C196L, C332A, and C352L.

[0131] In some embodiments, the recombinant mutant human sialidase is Neu2 sialidase and includes replacements for C322A and C352L (SEQ ID NO: 5).

[0132] In some embodiments, the sialidase contains amino acid substitutions at 2, 3, 4, 5, or 6 cysteine ​​residues, which are typically present in human sialidases such as Neu2 or Neu3.

[0133] In some embodiments, the recombinant mutant human sialidase comprises substitutions or substitution combinations corresponding to the substitutions or substitution combinations listed in Table 1 (amino acid positions corresponding to wild-type human Neu2 (SEQ ID NO: 1)).

[0134] Table 1

[0135] replace C125A C125I C125S C125V C196A C196L C196V C272S C272V C332A C332S C332V C352L C352V C125S+C332S C272V+C332A C272V+C332S C332A+C352L C125S+C196L C196L+C352L C196L+C332A C196L+C332A+C352L

[0136] ii. Residue substitutions that increase pI and / or decrease hydrophobicity

[0137] 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 has the lowest solubility, affecting its ability to be expressed and purified. Generally, a protein has good solubility if its pI is more than 2 units higher than the pH of the solution. Human Neu2 has a predicted pI of 7.5. Therefore, human Neu2 has the lowest solubility near neutral pH, which is undesirable since expression and physiological systems are performed at neutral pH. Conversely, a sialidase (St-sialidase) from *Salmonella typhimurium*, exhibiting good solubility and recombinant expression, has a pI of 9.6. Therefore, to improve the expression of human Neu2 or other human sialidases, recombinant mutant human sialidases can be engineered to contain one or more amino acid substitutions, wherein these substitutions increase the pI of the sialidase relative to sialidase without said substitutions. Furthermore, reducing the number of hydrophobic amino acids on the sialidase surface may improve sialidase expression, for example, by reducing aggregation. Therefore, in order to enhance the expression of human Neu2 or other human sialidases, recombinant mutant human sialidases can be designed to contain one or more amino acid substitutions, wherein the substitutions reduce the hydrophobicity of the sialidase surface relative to sialidases without the substitutions.

[0138] Therefore, in some embodiments, the recombinant mutant human sialidase comprises at least one amino acid substitution, wherein the substitution increases the isoelectric point (pI) of the sialidase and / or decreases its hydrophobicity relative to sialidase without the substitution. This can be achieved by introducing one or more charged amino acids, such as positively or negatively charged amino acids, into the recombinant sialidase. In some embodiments, the amino acid substitution is a substitution with a charged amino acid, such as a positively charged amino acid like lysine (lys, K), histidine (his, H), or arginine (arg, R), or a negatively charged amino acid like aspartic acid (asp, D) or glutamic acid (glu, E). In some embodiments, the amino acid substitution is a substitution with a lysine residue. In some embodiments, 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.

[0139] In some embodiments, the amino acid substitution occurs at a surface-exposed D or E amino acid, in a helix or loop, or at a position having K or R at the corresponding site of St-sialidase. In some embodiments, the amino acid substitution occurs at an amino acid distant from the catalytic site or not involved in catalysis, an amino acid not conserved with other human Neu proteins or with St-sialidase or Clostridium NanH, or an amino acid not located in a functionally important domain (e.g., an Asp-box or β-chain).

[0140] Exemplary amino acid substitutions in Neu2 that increase the isoelectric point (pI) and / or decrease the hydrophobicity of the sialidase relative to the unsubstituted sialidase include A2E, A2K, D215K, V325E, V325K, E257K, and E319K. In some embodiments, the recombinant mutant human sialidase comprises two or more amino acid substitutions, including, for example, A2K and V325E, A2K and V325K, E257K and V325K, A2K and E257K, and E257K and A2K and V325K.

[0141] In some embodiments, the recombinant mutant human sialidase comprises substitutions or substitution combinations corresponding to the substitutions or substitution combinations listed in Table 2 (amino acid positions corresponding to wild-type human Neu2 (SEQ ID NO: 1)).

[0142] Table 2

[0143] replace A2K E72K D215K E257K V325K A2K+E257K A2K+V325E A2K+V325K E257K+V325K

[0144] iii. Addition of N-terminal peptides and replacement of N- or C-terminus

[0145] It has been found that adding a peptide sequence of two or more amino acids to the N-terminus of human sialidase can enhance the expression and / or activity of said sialidase. In some embodiments, the peptide is at least 2 amino acids long, for example, 2 to 20, 2 to 10, 2 to 5, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some embodiments, the peptide may form an α-helix or has a tendency to form an α-helix.

[0146] In mice, the Neu2 isoform (type B) found in the thymus contains six amino acids absent in the classic Neu2 isoform found in skeletal muscle. In some embodiments herein, the N-terminal six amino acids of the mouse thymic Neu2 isoform, MEDLRP (SEQ ID NO: 4) or a variant thereof, may be added to human Neu, such as human Neu2. In some embodiments, the recombinant mutant human sialidase comprises a peptide of at least two amino acid residues covalently bound to the N-terminal amino acid of the sialidase. In some embodiments, the recombinant mutant human sialidase comprises a peptide, MEDLRP (SEQ ID NO: 4) or EDLRP (SEQ ID NO: 3), covalently bound to the N-terminal amino acid of the sialidase. In some embodiments, the sialidase may further comprise a cleavage site, such as a proteolytic cleavage site, located between the peptide, such as MEDLRP (SEQ ID NO: 4) or EDLRP (SEQ ID NO: 3), and the remainder of the sialidase. In some embodiments, the peptide, such as MEDLRP (SEQ ID NO: 4) or EDLRP (SEQ ID NO: 3), may be cleaved from the remainder of the sialidase after translation.

[0147] Optionally or in combination with the N-terminal addition, 1-5 amino acids of the N-terminal region of the 12-amino acid recombinant mutant human sialidase may be removed, for example, the N-terminal methionine may be removed. In some embodiments, if the recombinant mutant human sialidase is Neu2, the N-terminal methionine may be removed, or the first 5 amino acids may be removed (MASLP; SEQ ID NO: 12), or the second to fourth amino acids may be removed (ASLP; SEQ ID NO: 13).

[0148] In some embodiments, 1-5 amino acids in the N-terminal region of the 12 amino acids of the recombinant mutant human sialidase are replaced with MEDLRP (SEQ ID NO: 4), EDLRP (SEQ ID NO: 3), or TVEKSVVF (SEQ ID NO: 14). For example, in some embodiments, if the recombinant mutant human sialidase is Neu2, then amino acids MASLP (SEQ ID NO: 12), ASLP (SEQ ID NO: 13), or M are replaced with MEDLRP (SEQ ID NO: 4), EDLRP (SEQ ID NO: 3), or TVEKSVVF (SEQ ID NO: 14).

[0149] Human sialidase possesses a β-propeller structure, characterized by six blade-shaped β-sheets arranged in a ring around a central axis. Typically, hydrophobic interactions between the blades of the β-propeller, including those between the N- and C-terminal blades, enhance stability. Therefore, to improve the expression of human Neu2 or other human sialidases, recombinant mutant human sialidases can be designed with amino acid substitutions that enhance hydrophobic interactions and / or hydrogen bonding between the N- and C-terminal β-propeller blades of the sialidase.

[0150] Therefore, in some embodiments, the recombinant mutant human sialidase comprises a substitution of at least one wild-type amino acid residue, wherein the substitution enhances the hydrophobic interaction and / or hydrogen bonding between the N- and C-termini of the sialidase relative to sialidase without the substitution. In some embodiments, the wild-type amino acid is replaced with asparagine (asn, N), lysine (lys, K), tyrosine (tyr, Y), phenylalanine (phe, F), or tryptophan (trp, W). Exemplary substitutions in Neu2 that enhance the hydrophobic interaction and / or hydrogen bonding between the N- and C-termini include L4N, L4K, V6Y, L7N, L4N and L7N, L4N and V6Y, and L7N, V12N, V12Y, V12L, V6Y, V6F, or V6W. In some embodiments, the sialidase comprises a V6Y substitution.

[0151] In some embodiments, the recombinant mutant human sialidase comprises a combination of the above-described substitutions. For example, the recombinant mutant human Neu2 sialidase may contain an added amino acid MEDLRP (SEQ ID NO: 4), EDLRP (SEQ ID NO: 3), or TVEKSVVF (SEQ ID NO: 14) at the N-terminus, and in combination, may contain at least one of the substitutions L4N, L4K, V6Y, L7N, L4N and L7N, L4N and V6Y, and L7N, V12N, V12Y, V12L, V6Y, V6F, or V6W. In some embodiments, the amino acids MASLP (SEQ ID NO: 12), ASLP (SEQ ID NO: 13), or M of the recombinant mutant human Neu2 sialidase are replaced with MEDLRP (SEQ ID NO: 4), EDLRP (SEQ ID NO: 3), or TVEKSVVF (SEQ ID NO: 14), and the recombinant mutant human Neu2 sialidase further comprises at least one of L4N, L4K, V6Y, L7N, L4N and L7N, L4N and V6Y, and L7N, V12N, V12Y, V12L, V6Y, V6F, or V6W.

[0152] In some embodiments, the recombinant mutant human sialidase comprises a mutation or combination of mutations corresponding to the mutations or combinations of mutations listed in Table 3 (the amino acid position corresponds to wild-type human Neu2 (SEQ ID NO: 1)).

[0153] Table 3

[0154]

[0155] Furthermore, in some embodiments, the sialidase includes a substitution or deletion of an N-terminal methionine residue at its N-terminus. For example, in some embodiments, the sialidase includes a substitution of a methionine residue at position 1 corresponding to wild-type human Neu2 (SEQ ID NO: 1), for example, replacing the methionine at position 1 corresponding to wild-type human Neu2 with alanine (M1A) or aspartic acid (M1D). In other embodiments, the sialidase includes a deletion (ΔM1) of a methionine residue at position 1 corresponding to wild-type human Neu2 (SEQ ID NO: 1).

[0156] In some embodiments, the recombinant mutant human sialidase comprises substitutions or substitution combinations corresponding to the substitutions or substitution combinations listed in Table 4 (amino acid positions corresponding to wild-type human Neu2 (SEQ ID NO: 1)).

[0157] Table 4

[0158] mutation The absence of M1, V6Y, I187K M1R, V6Y, I187K M1H, V6Y, I187K M1K, V6Y, I187K M1D, V6Y, I187K M1T, V6Y, I187K M1N, V6Y, I187K M1Q, V6Y, I187K M1G, V6Y, I187K M1A, V6Y, I187K M1V, V6Y, I187K M1L, V6Y, I187K M1F, V6Y, I187K M1Y, V6Y, I187K

[0159] d. Reduce the substitution of residues by protein hydrolysis cleavage

[0160] It has been found that certain sialidases (e.g., human Neu2) are readily cleaved by proteases (e.g., trypsin). Consequently, the proteolytic cleavage of these sialidases can occur during recombinant protein production, harvesting, purification, formulation, administration to a subject, or after administration to a subject, or any combination of these events. Therefore, in some embodiments, the recombinant mutant human sialidase comprises a substitution of at least one wild-type amino acid residue, wherein said substitution reduces the cleavage of the sialidase relative to sialidase without said substitution by proteases (e.g., trypsin).

[0161] In some embodiments, the recombinant mutant human sialidase is incubated with a protease (e.g., trypsin) to produce about 1% to about 50%, about 1% to about 40%, about 1% to about 30%, about 1% to about 20%, about 1% to about 10%, about 1% to about 5%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 5% to about 20%, about 5% to about 10%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%, about 30% to about 50%, about 30% to about 40%, or about 40% to about 50% of the corresponding wild-type sialidase when incubated with the protease under the same conditions. In some embodiments, the recombinant mutant human sialidase is incubated with a protease (e.g., trypsin) to produce 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 corresponding wild-type sialidase when incubated with the protease under the same conditions. The proteolytic cleavage can be measured by any method known in the art, including, for example, by SDS-PAGE as described in Example 5 herein.

[0162] Exemplary substitutions that enhance resistance to protein hydrolysis include: (i) a substitution of the alanine residue at position 242 corresponding to wild-type human Neu2 (SEQ ID NO: 1), for example, by substitution with cysteine ​​(A242C), phenylalanine (A242F), glycine (A242G), histidine (A242H), isoleucine (A242I), lysine (A242K), leucine (A242L), methionine (A242M), asparagine (A242N), glutamine (A242Q), arginine (A242R), serine (A242S), valine (A242V), tryptophan (A242W), or tyrosine (A242Y); (ii) a substitution corresponding to wild-type human Neu2 (SEQ ID NO: 1). (iii) A substitution of the arginine residue at position 243 of wild-type human Neu2 (SEQ ID NO: 1), for example, by glutamic acid (R243E), histidine (R243H), asparagine (R243N), glutamine (R243Q), or lysine (R243K); or (iv) a substitution of the valine residue at position 244 of wild-type human Neu2 (SEQ ID NO: 1), for example, by isoleucine (V244I), lysine (V244K), or proline (V244P); or (iv) any combination of the above substitutions. In some embodiments, the recombinant mutant human sialidase comprises a substitution selected from A242C, A242F, A242Y, and A242W. In some embodiments, the recombinant mutant human sialidase comprises a substitution or combination of substitutions corresponding to the substitutions or combinations of substitutions listed in Table 5 (amino acid positions corresponding to wild-type human Neu2 (SEQ ID NO: 1)).

[0163] Table 5

[0164]

[0165]

[0166] Additional exemplary substitutions that enhance resistance to proteolytic cleavage (and / or increase expression levels and / or enzyme activity) include: (i) a substitution of the leucine residue at position 240 corresponding to wild-type human Neu2 (SEQ ID NO: 1), for example, by substitution with aspartic acid (L240D), asparagine (L240N), or tyrosine (L240Y); (ii) a substitution of the alanine residue at position 213 corresponding to wild-type human Neu2 (SEQ ID NO: 1), for example, by substitution with cysteine ​​(A213C), asparagine (A213N), serine (A213S), or threonine (A213T); (iii) a substitution corresponding to wild-type human Neu2 (SEQ ID NO: 1). (iv) Substitution of the arginine residue at position 241 of wild-type human Neu2 (SEQ ID NO: 1), for example, by alanine (R241A), aspartic acid (R241D), leucine (R241L), glutamine (R241Q), or tyrosine (R241Y); (iv) Substitution of the serine residue at position 258 of wild-type human Neu2 (SEQ ID NO: 1), for example, by cysteine ​​(S258C); (v) Substitution of the leucine residue at position 260 of wild-type human Neu2 (SEQ ID NO: 1), for example, by aspartic acid (L260D), phenylalanine (L260F), glutamine (L260Q), or threonine (L260T); (vi) Substitution of the valine residue at position 265 of wild-type human Neu2 (SEQ ID NO: 1), for example, by phenylalanine (V265F); or (vii) Any combination of the above substitutions. In some embodiments, substitutions or combinations of substitutions at these locations are envisioned to improve hydrophobic and / or aromatic interactions between secondary structural elements in the sialidase (e.g., between the α-helix and the nearest β-sheet), thereby stabilizing the structure and enhancing resistance to proteolytic cleavage.

[0167] In some embodiments, the recombinant mutant sialidase comprises a mutation at position L240. In some embodiments, the recombinant mutant sialidase comprises a combination of mutations at the following 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; or (vii) L240 and A242. In some embodiments, the recombinant mutant human sialidase comprises an alternative combination selected from the following: (i) A213C, A242F, and S258C; (ii) A213C and A242F; (iii) A213T and A242F; (iv) R241Y and A242F; or (v) L240Y and A242F. In some embodiments, the recombinant mutant human sialidase comprises substitutions or substitution combinations corresponding to the substitutions or substitution combinations listed in Table 6 (amino acid positions corresponding to wild-type human Neu2 (SEQ ID NO: 1)).

[0168] Table 6

[0169] replace A242C, V244P A242R, V244R A242R, V244H A242Y, V244P A242T, V244P A242N, V244P A213C, A242F A213S, A242F A213T, A242F A213N, A242F A213C, A242F, S258C A242F, L260F A242F, V265F L240Y L240Y, L260F L240D, L260T L240N, L260T L240N, L260D L240N, L260Q L240Y, A242F R241A, A242F R241Y, A242F

[0170] iv. Other substitutions

[0171] The present invention also provides a recombinant mutant human sialidase comprising at least one of the following substitutions: I187K, A328E, K370N, or H210N. In some embodiments, the recombinant mutant human Neu2 comprises the amino acid GDYDAPTHQVQW (SEQ ID NO: 15) being replaced by the amino acid SMDQGSTW (SEQ ID NO: 16) or STDGGKTW (SEQ ID NO: 17). In some embodiments, the recombinant mutant human Neu2 comprises the amino acid PRPPAPEA (SEQ ID NO: 18) being replaced by the amino acid QTPLEAAC (SEQ ID NO: 19). In some embodiments, the recombinant mutant human Neu2 comprises the amino acid NPRPPAPEA (SEQ ID NO: 20) being replaced by the amino acid SQNDGES (SEQ ID NO: 21).

[0172] The present invention also provides a recombinant mutant human sialylase 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.

[0173] The present invention also provides a recombinant mutant human sialylase containing amino acid substitutions at the positions listed in Table 7 (amino acid positions corresponding to wild-type human Neu2 (SEQ ID NO: 1). In some embodiments, the sialylase contains the amino acid substitutions identified in Table 7. In some embodiments, the sialylase contains any combination of amino acid substitutions identified in Table 7.

[0174] Table 7

[0175]

[0176]

[0177]

[0178] For example, in some embodiments, the recombinant mutant human sialidase comprises: (a) a substitution of a proline residue (P5) at position 5 corresponding to wild-type human Neu2; (b) a substitution of a lysine residue (K9) at position 9 corresponding to wild-type human Neu2; (c) a substitution of a lysine residue (K44) at position 44 corresponding to wild-type human Neu2; (d) a substitution of a lysine residue (K45) at position 45 corresponding to wild-type human Neu2; (e) a substitution of a leucine residue (L54) at position 54 corresponding to wild-type human Neu2; and (f) a substitution of a proline residue (P5) at position 62 corresponding to wild-type human Neu2. (g) Substitution of glutamine residue (Q69) at position 69 of Neu2 in wild-type human; (h) Substitution of arginine residue (R78) at position 78 of Neu2 in wild-type human; (i) Substitution of aspartic acid residue (D80) at position 80 of Neu2 in wild-type human; (j) Substitution of alanine residue (A93) at position 93 of Neu2 in wild-type human; (k) Substitution of glycine residue (G107) at position 107 of Neu2 in wild-type human; (l) Substitution of glutamine residue (Q108) at position 108 of Neu2 in wild-type human. (m) Substitution of a glutamine residue (Q112) at position 112 corresponding to wild-type human Neu2; (n) Substitution of a cysteine ​​residue (C125) at position 125 corresponding to wild-type human Neu2; (o) Substitution of a glutamine residue (Q126) at position 126 corresponding to wild-type human Neu2; (p) Substitution of an alanine residue (A150) at position 150 corresponding to wild-type human Neu2; (q) Substitution of a cysteine ​​residue (C164) at position 164 corresponding to wild-type human Neu2; (r) Substitution of an arginine residue (R170) at position 170 corresponding to wild-type human Neu2. The substitutions are as follows: (s) substitution of an alanine residue (A171) at position 171 of wild-type human Neu2; (t) substitution of a glutamine residue (Q188) at position 188 of wild-type human Neu2; (u) substitution of an arginine residue (R189) at position 189 of wild-type human Neu2; (v) substitution of an alanine residue (A213) at position 213 of wild-type human Neu2; (w) substitution of a leucine residue (L217) at position 217 of wild-type human Neu2; and (x) substitution of a glutamic acid residue (E225) at position 225 of wild-type human Neu2.(y) Substitution of a histidine residue (H239) at position 239 corresponding to wild-type human Neu2; (z) Substitution of a leucine residue (L240) at position 240 corresponding to wild-type human Neu2; (aa) Substitution of an arginine residue (R241) at position 241 corresponding to wild-type human Neu2; (bb) Substitution of an alanine residue (A242) at position 242 corresponding to wild-type human Neu2; (cc) Substitution of a histidine residue (H239) at position 239 corresponding to wild-type human Neu2; (z) Substitution of a leucine residue (L240) at position 240 corresponding to wild-type human Neu2; (c) Substitution of a leucine residue (L240) at position 241 corresponding to wild-type human Neu2; (d) Substitution of a leucine residue (L240) at position 242 corresponding to wild-type human Neu2; (d) Substitution of a leucine residue (L240) at position 240 corresponding to wild-type human Neu2; (d) Substitution of a leucine residue (L240) at position 241 corresponding to wild-type human Neu2; (d) Substitution of a leucine residue (L241) at position 242 corresponding to wild-type human Neu2; (c) Substitution of a leucine residue (L242) at position 242 corresponding to wild-type human Neu2; (d) Substitution of a leucine residue (L240 ... (d) Substitution of a valine residue (V244) at position 244 of u2; (dd) Substitution of a threonine residue (T249) at position 249 of Neu2 (corresponding to wild-type human Neu2); (ee) Substitution of an aspartic acid residue (D251) at position 251 of Neu2 (corresponding to wild-type human Neu2); (ff) Substitution of a glutamic acid residue (E257) at position 257 of Neu2 (corresponding to wild-type human Neu2); (gg) Substitution of a silyl glutamic acid residue (E257) at position 258 of Neu2 (corresponding to wild-type human Neu2). Substitution of leucine residue (S258); (hh) substitution of leucine residue (L260) at position 260 corresponding to wild-type human Neu2; (ii) substitution of valine residue (V265) at position 265 corresponding to wild-type human Neu2; (jj) substitution of glutamine residue (Q270) at position 270 corresponding to wild-type human Neu2; (kk) substitution of tryptophan residue (W292) at position 292 corresponding to wild-type human Neu2. The substitutions may be: (11) a serine residue (S301) at position 301 corresponding to wild-type human Neu2; (2) a tryptophan residue (W302) at position 302 corresponding to wild-type human Neu2; (304) a valine residue (V363) at position 363 corresponding to wild-type human Neu2; or (405) a leucine residue (L365) at position 365 corresponding to wild-type human Neu2; or any combination of the above substitutions. For example, the sialidase may comprise substitutions of K9, P62, A93, Q216, A242, Q270, S301, W302, V363, or L365, or any combination of the above substitutions.

[0179] In some embodiments, in the sialidase: (a) the proline residue at position 5 corresponding to wild-type human Neu2 is replaced by histidine (P5H); (b) the lysine residue at position 9 corresponding to wild-type human Neu2 is replaced by aspartic acid (K9D); (c) the lysine residue at position 44 corresponding to wild-type human Neu2 is replaced by arginine (K44R) or glutamic acid (K44E); (d) the lysine residue at position 45 corresponding to wild-type human Neu2 is replaced by alanine (K45A), arginine (K45R), or glutamic acid (K45E); and (e) the leucine residue at position 54 corresponding to wild-type human Neu2... (f) The proline residue at position 62 of wild-type human Neu2 is replaced by asparagine (P62N), aspartic acid (P62D), histidine (P62H), glutamic acid (P62E), glycine (P62G), serine (P62S), or threonine (P62T); (g) The glutamine residue at position 69 of wild-type human Neu2 is replaced by histidine (Q69H); (h) The arginine residue at position 78 of wild-type human Neu2 is replaced by lysine (R78K); (i) The aspartic acid residue at position 80 of wild-type human Neu2 is replaced by proline (D). (j) The alanine residue at position 93 of wild-type human Neu2 is replaced by glutamic acid (A93E) or lysine (A93K); (k) The glycine residue at position 107 of wild-type human Neu2 is replaced by aspartic acid (G107D); (l) The glutamine residue at position 108 of wild-type human Neu2 is replaced by histidine (Q108H); (m) The glutamine residue at position 112 of wild-type human Neu2 is replaced by arginine (Q112R) or lysine (Q112K); (n) The cysteine ​​residue at position 125 of wild-type human Neu2 is replaced by leucine. (c125L); (o) The glutamine residue at position 126 of wild-type human Neu2 is replaced by leucine (Q126L), glutamic acid (Q126E), phenylalanine (Q126F), histidine (Q126H), isoleucine (Q126I), or tyrosine (Q126Y); (p) The alanine residue at position 150 of wild-type human Neu2 is replaced by valine (A150V); (q) The cysteine ​​residue at position 164 of wild-type human Neu2 is replaced by glycine (C164G); (r) The arginine residue at position 170 of wild-type human Neu2 is replaced by proline (R170P).(s) Alanine residue at position 171 of wild-type human Neu2 is replaced by glycine (A171G); (t) Glutamine residue at position 188 of wild-type human Neu2 is replaced by proline (Q188P); (u) Arginine residue at position 189 of wild-type human Neu2 is replaced by proline (R189P); (v) Alanine residue at position 213 of wild-type human Neu2 is replaced by cysteine ​​(A213C), asparagine (A213N), serine (A213S), or threonine (A213T); (w) Leucine residue at position 217 of wild-type human Neu2 is replaced by... The following residues were replaced: (x) threonine residue at position 249 of wild-type human Neu2 was replaced with alanine (T249A); (y) aspartic acid residue at position 251 of wild-type human Neu2 was replaced with glycine (D251G); (z) glutamic acid residue at position 225 of wild-type human Neu2 was replaced with proline (E225P); (aa) histidine residue at position 239 of wild-type human Neu2 was replaced with proline (H239P); (bb) leucine residue at position 240 of wild-type human Neu2 was replaced with aspartic acid (L217A). (L240D), asparagine (L240N), or tyrosine (L240Y); (cc) the arginine residue at position 241 of wild-type human Neu2 is replaced by 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 replaced by cysteine ​​(A242C), phenylalanine (A242F), glycine (A242G), histidine (A242H), isoleucine (A242I), lysine (A242K), leucine (A242L), or methionine (A242). M), asparagine (A242N), glutamine (A242Q), arginine (A242R), serine (A242S), valine (A242V), tryptophan (A242W), or tyrosine (A242Y) are replaced; (ee) the valine residue at position 244 of wild-type human Neu2 is replaced by isoleucine (V244I), lysine (V244K), or proline (V244P); (ff) the glutamate residue at position 257 of wild-type human Neu2 is replaced by proline (E257P); (gg) the serine residue at position 258 of wild-type human Neu2 is replaced by cysteine ​​(S258C);(hh) The leucine residue at position 260 of wild-type human Neu2 is replaced by aspartic acid (L260D), phenylalanine (L260F), glutamine (L260Q), or threonine (L260T); (ii) The valine residue at position 265 of wild-type human Neu2 is replaced by phenylalanine (V265F); (jj) The glutamine residue at position 270 of wild-type human Neu2 is replaced by alanine (Q270A), histidine (Q270H), phenylalanine (Q270F), proline (Q270P), serine (Q270S), or threonine (L260T). (Q270T) is replaced; (kk) corresponds to the tryptophan residue at position 292 of wild-type human Neu2 being replaced by arginine (W292R); (ll) corresponds to the serine residue at position 301 of wild-type human Neu2 being replaced by alanine (S301A), aspartic acid (S301D), glutamic acid (S301E), phenylalanine (S301F), glycine (S301G), histidine (S301H), isoleucine (S301I), lysine (S301K), leucine (S301L), methionine (S301M), asparagine (S301N), or proline (S301P). Glutamine (S301Q), arginine (S301R), threonine (S301T), valine (S301V), tryptophan (S301W), or tyrosine (S301Y) are replaced; (mm) the tryptophan residue at position 302 corresponding to wild-type human Neu2 is replaced by alanine (W302A), aspartic acid (W302D), glutamic acid (W302E), phenylalanine (W302F), glycine (W302G), histidine (W302H), isoleucine (W302I), lysine (W302K), leucine (W302L), methionine (W302M), or asparagine (W301Y). (02N), proline (W302P), glutamine (W302Q), arginine (W302R), serine (W302S), threonine (W302T), valine (W302V), or tyrosine (W302Y); (nn) the valine residue at position 363 corresponding to wild-type human Neu2 is replaced by arginine (V363R); or (oo) the leucine residue at position 365 corresponding to wild-type human Neu2 is replaced by glutamine (L365Q), histidine (L365H), isoleucine (L365I), lysine (L365K), or serine (L365S);Alternatively, the sialidase may comprise any combination of the above-described substitutions. For example, the sialidase may comprise 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 above-described substitutions.

[0180] In some embodiments, the recombinant mutant human sialidase comprises the deletion of a leucine residue at position 184 (ΔL184), a histidine residue at position 185 (ΔH185), a proline residue at position 186 (ΔP186), an isoleucine residue at position 187 (ΔI187), and a glutamine residue at position 184 (ΔQ188), or any combination of the above deletions.

[0181] In some embodiments, the recombinant mutant human sialidase includes an insertion between a threonine residue at position 216 corresponding to wild-type human Neu2 and a leucine residue at position 217 corresponding to wild-type human Neu2, for example, an insertion of an amino acid selected from S, T, Y, L, F, A, P, V, I, N, D, and H.

[0182] Other exemplary sialidase mutations and combinations thereof are described in International (PCT) Patent Application No. PCT / US2019 / 012207, filed January 3, 2019, including in the section entitled “I. Recombinant Human Sialidase” in the detailed description and in Examples 1, 2, 3, 4, 5 and 6 of the examples.

[0183] v. to substitute combinations

[0184] The present invention also provides a recombinant mutant human sialylase comprising any combination of mutations contemplated herein. For example, the recombinant mutant sialylase may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more combinations of mutations contemplated herein. It is contemplated that the recombinant mutant sialylase may comprise 1-15, 1-10, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-15, 2-10, 2-7, 2-6, 2-5, 2-4, 2-3, 3-15, 3-10, 3-7, 3-6, 3-5 or 3-4 mutations contemplated herein.

[0185] For example, the recombinant mutant sialidase may include M1 deletion (ΔM1), M1A substitution, M1D substitution, V6Y substitution, K9D substitution, P62G substitution, P62N substitution, P62S substitution, P62T substitution, A93E substitution, I187K substitution, Q270A substitution, S301R substitution, W302K substitution, C332A substitution, V363R substitution, L365I substitution, or any combination of the above deletions and substitutions.

[0186] In some embodiments, the recombinant mutant sialidase comprises M1 deletion (ΔM1), M1A substitution, M1D substitution, V6Y substitution, I187K substitution, C332A substitution, or any combination of the above deletions and substitutions. For example, the recombinant mutant sialidase may comprise a combination of mutations selected from: 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, I 187K 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.

[0187] In some embodiments, the recombinant mutant sialidase comprises: (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 of the above deletions and substitutions. For example, the recombinant mutant sialidase may comprise: (i) the amino acid substitutions identified in Table 8 or any combination of amino acid substitutions identified in Table 8, and (ii) a combination of mutations selected from the following: 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 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.

[0188] In some embodiments, the recombinant mutant sialidase comprises: (a) substitutions for M1D, V6Y, P62G, A93E, I187K, and C332A; (b) substitutions for M1D, V6Y, K9D, A93E, I187K, C332A, V363R, and L365I; (c) substitutions for M1D, V6Y, P62N, I187K, and C332A; (d) substitutions for M1D, V6Y, I187K, Q270A, S301R, W302K, and C332A; (e) substitutions for M1D, V6Y, P62S, I187K, Q270A, S301R, W302K, and C332A; and (f) substitutions for M1D, V6Y, P62T, I187K, Q270A, and S301R. (g) Replace M1D, V6Y, P62N, I187K, Q270A, S301R, W302K, and C332A; (h) Replace M1D, V6Y, P62G, A93E, I187K, S301A, W302R, and C332A; (i) Replace M1D, V6Y, P62G Replace with A93E, Q126Y, I187K, Q270T and C332A; or (j) replace with M1D, V6Y, P62G, A93E, Q126Y, I187K and C332A; or (k) replace with M1D, V6Y, P62G, A93E, Q126Y, I187K, A242F, Q270T and C332A.

[0189] In some embodiments, the recombinant mutant human sialidase comprises a combination of a substitution of a serine residue (S301) at position 301 corresponding to wild-type human Neu2 and a substitution of a tryptophan residue (W302) at position 302 corresponding to wild-type human Neu2. For example, the recombinant mutant human sialidase may comprise a combination of substitutions corresponding to the substitution combinations listed in the rows of Table 8 (amino acid positions corresponding to wild-type human Neu2 (SEQ ID NO: 1)). For example, the recombinant mutant human sialidase may comprise: S301K and W302R substitutions; S301K and W302K substitutions; or S301A and W302S substitutions.

[0190] Table 8

[0191]

[0192]

[0193] In some embodiments, the recombinant mutant human sialidase comprises a combination of substitutions corresponding to the substitution combinations listed in the rows of Table 9 (the amino acid position corresponds to wild-type human Neu2 (SEQ ID NO: 1)).

[0194] Table 9

[0195]

[0196]

[0197]

[0198]

[0199] In some embodiments, the recombinant mutant human sialidase comprises an amino acid sequence of any one of SEQ ID NO: 48-62, 169-171 or 196 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NO: 48-62, 169-171 or 196.

[0200] In some embodiments, the recombinant mutant human sialidase comprises the amino acid sequence X1X2SX3X4X5LQX6ESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASX7X8DEHAELIVX9RRGDYDAX 10 THQVQWX 11 AQEVVAQAX 12 LDGHRSMNPCPLYDX 13 QTGTLFLFFIAIPX 14 X 15 VTEX 16 QQLQTRANVTRLX 17 X 18 VTSTDHGRTWSSPRDLTDAAIGPX 19 YREWSTFAVGPGHX 20 LQLHDRX 21 RSLVVPAYAYRKLHPX 22 QRPIPSAFX 23 FLSHDHGRTWARGHFVAQDTX24ECQVAEVETGEQRVVTLNARSHLRARVQAQSX 25 NX 26 GLDFQX 27 SQLVKKLVEPPPX 28 GX 29 QGSVISFPSPRSGPGSPAQX 30 LLYTHPTHX 31 X 32 QRADLGAYLNPRPPAPEAWSEPX33 LLAKGSX 34 AYSDLQSMGTGPDGSPLFGX 35 LYEANDYEEIX 36 FX 37 MFTLKQAFPAEYLPQ (SEQ ID NO: 47), where X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val or not present; X2 is Ala or Lys; X3 is Asn or Leu; X4 is Pro or His; X5 is Phe, Trp, Tyr or Val; X6 is Lys or Asp; X7 is Lys, Arg or Glu; X8 is Lys, Ala, Arg or Glu; X9 is Leu or Met; X... 10 Is it Pro, Asn, Asp, His, Glu, Gly, Ser, or Thr, X 11 Is it Gln or His, X 12 Is it Arg or Lys, X 13 Is it Ala, Glu, or Lys, X? 14 Is it Gly or Asp, X 15 Is it Gln or His, X 16 Is it Gln, Arg, or Lys, X 17 Is it Ala, Cys, Ile, Ser, Val, or Leu, X? 18 Is it Gln or Leu, X 19 Is it Ala or Val, X? 20 Is it Cys or Gly, X 21 Is it Ala or Gly, X? 22 Is it Arg, Ile, or Lys, X 23 Is it Ala, Cys, Leu, or Val, X? 24 Is it Leu, Ala, or Val, X? 25 Is it Thr or Ala, X 26 Is it Asp or Gly, X 27 Is it Glu or Lys, X 28 Is it Gln, Ala, His, Phe, or Pro, X? 29 Is it Cys or Val, X 30 Is it Trp or Arg, X 31 Is it Ser or Arg, X 32 Is it Trp or Lys, X 33 Is it Lys or Val, X 34 Is it Ala, Cys, Ser, or Val, X? 35 Is it Cys, Leu, or Val, X?36 It is Val or Arg, and X 37 It is Leu, Gln, His, Ile, Lys, or Ser, and the sialidase contains at least one mutation relative to wild-type human Neu2 (SEQ ID NO: 1).

[0201] In some embodiments, the recombinant mutant human sialidase comprises the amino acid sequence X1ASLPX2LQX3ESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAX4THQVQWQAQEVVAQARLDGHRSMNPCPLYDX5QTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPX6QRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPX7GCQGSVISFPSPRSGPGSPAQWLLYTHPTHX8X9QRADLGAYLNPRPPAPEAWSEPVLLAKGSX 10 AYSDLQSMGTGPDGSPLFGCLYEANDYEEIX 11 FX 12 MFTLKQAFPAEYLPQ

[0202] (SEQ ID NO: 46), wherein X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val or not present; 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 it Ala, Cys, Ser, or Val, X? 11 It is Val or Arg, and X 12The sialidase is Leu, Gln, His, Ile, Lys, or Ser, and contains at least one mutation relative to wild-type human Neu2 (SEQ ID NO: 1). In some embodiments, X1 is Ala, Asp, Met, or absent; X2 is Tyr or Val; X3 is Lys or Asp; X4 is Pro, Asn, Gly, Ser, or Thr; X5 is Ala or Glu; X6 is Ile or Lys; X7 is Gln or Ala; X8 is Ser or Arg; X9 is Trp or Lys; X... 10 Is it Ala or Cys, X? 11 It is Val or Arg, and X 12 It is either Leu or Ile.

[0203] In some embodiments, the recombinant mutant human sialidase comprises the amino acid sequence X1X2SX3X4X5LQX6ESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASX7X8DEHAELIVX9RRGDYDAX 10 THQVQWX 11 AQEVVAQAX 12 LX 13 GHRSMNPCPLYDX 14 QTGTLFLFFIAIPX 15 X 16 VTEX 17 QQLQTRANVTRLX 18 X 19 VTSTDHGRTWSSPRDLTDAAIGPX 20 YREWSTFAVGPGHX 21 LQLHDX 22 X 23 RSLVVPAYAYRKLHPX 24 X 25 X 26 PIPSAFX 27 FLSHDHGRTWARGHFVX28QDTX 29 ECQVAEVX 30 TGEQRVVTLNARSX 31 X 32 X 33 X 34 RX 35 QAQSX 36 NX 37 GLDFQX 38 X 39 QX 40 VKKLX 41EPPPX 42 GX 43 QGSVISFPSPRSGPGSPAQX 44 LLYTHPTHX 45 X 46 QRADLGAYLNPRPPAPEAWSEPX 47 LLAKGSX 48 AYSDLQSMGTGPDGSPLFGX 49 LYEANDYEEIX 50 FX 51 MFTLKQAFPAEYLPQ

[0204] (SEQ ID NO: 172), wherein X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val or not present; X2 is Ala or Lys; X3 is Asn or Leu; X4 is Pro or His; X5 is Phe, Trp, Tyr or Val; X6 is Lys or Asp; X7 is Lys, Arg or Glu; X8 is Lys, Ala, Arg or Glu; X9 is Leu or Met; X 10 Is it Pro, Asn, Asp, His, Glu, Gly, Ser, or Thr, X 11 Is it Gln or His, X 12 Is it Arg or Lys, X 13 Is it Asp or Pro, X? 14 Is it Ala, Glu, or Lys, X? 15 Is it Gly or Asp, X 16 Is it Gln or His, X 17 Is it Gln, Arg, or Lys, X 18 Is it Ala, Cys, Ile, Ser, Val, or Leu, X? 19 Is it Gln, Leu, Glu, Phe, His, Ile, Leu, or Tyr, X 20 Is it Ala or Val, X? 21 Is it Cys or Gly, X 22 Is it Arg or Pro, X 23 Is it Ala or Gly, X? 24 Is it Arg, Ile, or Lys, X 25 Is it Gln or Pro, X 26 Is it Arg or Pro, X 27 Is it Ala, Cys, Leu, or Val, X? 28 Is it Ala, Cys, Asn, Ser, or Thr, X29 Is it Leu, Ala, or Val, X? 30 Is it Glu or Pro, X 31 Is it His or Pro, X? 32 Is it Leu, Asp, Asn, or Tyr, X 33 Is it Arg, Ala, Asp, Leu, Gln, or Tyr, X 34 Is it Ala, Cys, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Val, Trp, or Tyr, X 35 Is it Val, Ile, or Lys, X 36 Is it Thr or Ala, X 37 Is it Asp or Gly, X 38 Is it Glu, Lys, or Pro, X 39 Is it Ser or Cys, X 40 Is it Leu, Asp, Phe, Gln, or Thr, X 41 Is it Val or Phe, X 42 Is it Gln, Ala, His, Phe, Pro, Ser, or Thr, X 43 Is it Cys or Val, X 44 Is it Trp or Arg, X 45 Is it Ser, Arg, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Thr, Val, Trp, or Tyr, X 46 Is it Trp, Lys, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Tyr, X 47 Is it Lys or Val, X 48 Is it Ala, Cys, Ser, or Val, X? 49 Is it Cys, Leu, or Val, X? 50 It is Val or Arg, and X 51 It is Leu, Gln, His, Ile, Lys, or Ser, and the sialidase contains at least one mutation relative to wild-type human Neu2 (SEQ ID NO: 1).

[0205] In some embodiments, the recombinant mutant human sialidase comprises the amino acid sequence X1ASLPX2LQX3ESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAX4THQVQWQAQEVVAQARLDGHRSMNPCPLYDX5QTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCX6VTSTDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPX7QRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRX8RVQAQSTNDGLDFQESQLVKKLVEPPPX9GCQGSVISFPSPRSGPGSPAQWLLYTHPTHX 10 X 11 QRADLGAYLNPRPPAPEAWSEPVLLAKGSX 12 AYSDLQSMGTGPDGSPLFGCLYEANDYEEIX 13 FX 14 MFTLKQAFPAEYLPQ

[0206] (SEQ ID NO: 173), wherein X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val or not present; 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; X8 is Ala, Cys, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Val, Trp or Tyr; X9 is Gln, Ala, His, Phe, Pro, Ser or Thr; X 10 Is it Ser, Arg, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Thr, Val, Trp, or Tyr, X 11Is it Trp, Lys, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Tyr, X 12 Is it Ala, Cys, Ser, or Val, X? 13 It is Val or Arg, and X 14 The sialidase is Leu, Gln, His, Ile, Lys, or Ser, and contains at least one mutation relative to wild-type human Neu2 (SEQ ID NO: 1). In some embodiments, X1 is Ala, Asp, Met, or absent; X2 is Tyr or Val; X3 is Lys or Asp; X4 is Pro, Asn, Gly, Ser, or Thr; X5 is Ala or Glu; X6 is Gln or Tyr; X7 is Ile or Lys; X8 is Ala or Thr; X9 is Gln, Ala, or Thr; X... 10 Is it Ser, Arg, or Ala, X 11 Is it Trp, Lys, or Arg, X 12 Is it Ala or Cys, X? 13 It is Val or Arg, and X 14 It is either Leu or Ile.

[0207] In some embodiments, the recombinant mutant human sialidase contains conserved substitutions relative to the recombinant mutant human sialidase sequence disclosed herein. When used herein, the term "conserved substitution" refers to a substitution using structurally similar amino acids. For example, conserved substitutions may include substitutions within the following groups: Ser and Cys; Leu, Ile and Val; Glu and Asp; Lys and Arg; Phe, Tyr and Trp; and Gln, Asn, Glu, Asp, and His. Conserved substitutions may also be defined by the BLAST (Basic Local Alignment Search Tool) algorithm, a BLOSUM substitution matrix (e.g., a BLOSUM 62 matrix), or a PAM substitution:p matrix (e.g., a PAM 250 matrix).

[0208] Sequence identity can be determined in various ways within the technical scope of those skilled in the art, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. BLAST (Basic Local Alignment Search Tool) analysis using algorithms employed by programs blastp, blastn, blastx, tblastn, and 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, which are incorporated herein by reference) has been tailored for sequence similarity searches. For a discussion of the fundamental problems in searching sequence databases, see Altschul et al., (1994) NATURE GENETICS 6:119-129, the entirety of which is incorporated herein by reference. Those skilled in the art can determine suitable parameters for measuring alignment, including any algorithm required to achieve maximum alignment across the full length of the sequences being compared. Search parameters for histograms, descriptions, alignments, expected values ​​(i.e., the statistical significance thresholds used to report matches with database sequences), cutoff values, matrices, and filters are set to default. 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, the entirety of which is incorporated herein by reference). The four blastn parameters can be adjusted as follows: Q = 10 (gap generation penalty); R = 10 (gap extension penalty); wink = 1 (generate word hits at every wink-th position along the query sequence); and gapw = 16 (set the window width in which the aligned window with gaps is generated). The equivalent blastp parameter settings can be Q = 9; R = 2; wink = 1; and gapw = 32.Searches can also be performed using NCBI (National Center for Biotechnology Information). The BLAST advanced option parameters are used for information (e.g., -G, open gap cost [integer]: default = 5 for nucleotides / 11 for proteins; -E, extended gap cost [integer]: default = 2 for nucleotides / 1 for proteins; -q, nucleotide mismatch penalty [integer]: default = -3; -r, nucleotide match bonus [integer]: default = 1; -e, expected value [real number]: default = 10; -W, word length [integer]: default = 11 for nucleotides / 28 for megablast / 3 for proteins; -y, blast extension decay (X) in bytes: default = 20 for blastn / 7 for others; -X, X decay value for gapped alignment (in bytes): default = 15 for all programs, not for blastn; and -Z, final X decay value for gapped alignment (in bytes): 50 for blastn, 25 for others). ClustalW, used for pairwise protein alignment, can also be used (default parameters may include, for example, a Blosum62 matrix and a gap opening penalty of 10 and a gap extension penalty of 0.1). Bestfit comparisons between sequences available in GCG package version 10.0 use the DNA parameters GAP=50 (gap generation penalty) and LEN=3 (gap extension penalty). The equivalent settings in Bestfit protein comparisons are GAP=8 and LEN=2.

[0209] II. Serum half-life prolonging agents

[0210] When used herein, "serum half-life extender" refers to a component that can bind to sialidase to prolong its circulating half-life in the serum of a subject. In some embodiments, the serum half-life extender may be selected from the Fc domain (see, for example, Beck et al., (2011) MA). BS 4:1015-28), albumin (e.g., human serum albumin (HSA), see Weimer et al., (2013) "Recombinant albumin fusion proteins", in *Fusion protein technologies for biopharmaceuticals: applications and challenges*, edited by Schmidt S, Hoboken: Wiley; 2013, pp. 297-323), albumin-binding domains (e.g., HSA binders, see Walker et al., (2013) "Albumin-binding fusion proteins in the development of novel long-acting therapeutics ... Hoboken: Wiley; Hoboken: Wiley; 2013, pp. 297-323), albumin- Challenges), Hoboken: Wiley; 2013, p.325-43, transferrin (see Kim et al., (2010) J PHARMACOL EXP THER 334:682-92), XTEN (also known as recombinant PEG or "rPEG", see Schellenberger et al., (2009) NAT. BIOTECHNOL. 27:1186-90), amino acid homopolymer (HAP, see Schlapschy et al., (2007) PROTEIN ENG DES SEL. 20:273-84), proline-alanine-serine polymer (PAS, see Schlapschy et al., (2013) PROTEIN ENG DES SEL. 26:489-501), elastin-like peptide (ELP, see Floss et al., (2013) Fusion protein technologies for biopharmaceuticals: applications and challenges) and challenges), p.372-98), C-terminal peptide (CTP, Duijkers et al., (2002) HUM REPROD.17:1987-93), gelatin-like proteins (GLK, Huang et al., (2010) EUR J PHARM BIOPHARM 72:435-41) and polyethylene glycol (PEG).

[0211] Suitable serum half-life extenders also include a variety of different polymers, such as those described in U.S. Patent No. 7,842,789. For example, block polymers of polyethylene oxide and polypropylene oxide (Pluronics); polymethacrylates; carbomers; and branched or unbranched polysaccharides comprising sugar monomers such as D-mannose, D- and L-galactose, fucose, fructose, D-xylose, L-arabinose, and D-glucuronic acid can be used. In other embodiments, the serum half-life extender may be a hydrophilic polyvinyl polymer such as polyvinyl alcohol and polyvinylpyrrolidone (PVP) type polymers. The serum half-life extender may be a functionalized polyvinylpyrrolidone (available from PolymerSource) functionalized, for example, with carboxyl or amine groups at one (or both) ends of the polymer. Alternatively, the serum half-life extender may include poly(2-hydroxypropyl)methacrylamide (HPMA) or functionalized HPMA (amine, carboxyl, etc.), poly(N-isopropylacrylamide) or functionalized poly(N-isopropylacrylamide).

[0212] In one implementation, sialidase is covalently attached to a naturally occurring long-half-life polypeptide or protein, such as an Fc domain (Beck et al., ibid.), transferrin (Kim et al., ibid.), or albumin (Weimer et al., ibid.), by gene fusion (i.e., the generation of a recombinant fusion protein) or by chemical coupling, to form a fusion protein.

[0213] In another embodiment, sialidase is covalently attached to an inert polypeptide such as XTEN (also known as recombinant PEG or "rPEG," see Schellenberger, ibid.), amino acid homopolymer (HAP, see Schlapschy et al., (2007), ibid.), proline-alanine-serine polymer (PAS, see Schlapschy et al., (2013), ibid.), elastin-like peptide (ELP, see Floss et al., ibid.), or gelatin-like protein (GLK, Huang et al., ibid.) to form a fusion protein, among other functions. The inert polypeptide increases the size and hydrodynamic radius of the sialidase, thereby extending its half-life. In some embodiments, the XTEN polypeptide has about 25 amino acids to about 1500 amino acids, such as about 25 amino acids to about 100 amino acids, about 25 amino acids to about 250 amino acids, about 25 amino acids to about 500 amino acids, about 25 amino acids to about 750 amino acids, about 25 amino acids to about 1000 amino acids, about 25 amino acids to about 1250 amino acids, about 100 amino acids to about 250 amino acids, about 100 amino acids to about 500 amino acids, about 100 amino acids to about 750 amino acids, about 100 amino acids to about 1000 amino acids, about 100 amino acids to about 1250 amino acids, or about 100 amino acids to about 1500 amino acids. The length of 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 amino acids to 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.

[0214] In some embodiments, sialidase is chemically coupled to repeating chemical components such as PEG or hyaluronic acid (see Mero et al., (2013), CARB POLYMERS 92:2163-70), which increases the hydrodynamic radius of sialidase and thus prolongs its half-life.

[0215] In another embodiment, sialidase itself is polysialylated or covalently attached to a negatively charged, highly sialylated protein (e.g., the carboxyl-terminal peptide (CTP) of the β-chain of human chorionic gonadotropin (CG), see Duijkers et al., (2002) HUM REPROD 17:1987-93).

[0216] Methods for manufacturing and using the above-mentioned serum half-life extenders are known in the art. See also, for example, Strohl (2015) BIODRUGS 29:215-239.

[0217] In some embodiments, the sialidase is coupled to a serum half-life extender that is not an Fc domain and / or is not PEG.

[0218] It is envisioned that one or more sialidases can be covalently bound to one or more (e.g., 2, 3, 4, 5, 6, 8, 9, 10 or more) serum half-life extenders.

[0219] In some embodiments, the serum half-life of the sialidase coupled with the serum half-life enhancer is at least 24, 36, 48, or 60 hours.

[0220] Typically, the serum half-life extender may have a half-life of about 2 kDa to about 5 kDa, about 2 kDa to about 10 kDa, about 2 kDa to about 20 kDa, about 2 kDa to about 30 kDa, about 2 kDa to about 40 kDa, about 2 kDa to about 50 kDa, about 2 kDa to about 60 kDa, about 2 kDa to about 70 kDa, about 2 kDa to about 80 kDa, about 2 kDa to about 90 kDa, about 2 kDa to about 100 kDa, about 2 kDa to about 150 kDa, about 5 kDa to about 10 kDa, about 5 kDa to about 20 kDa, about 5 kDa to about 30 kDa, about 5 kDa to about 40 kDa, about 5 kDa to about 50 kDa, or about 5 kDa to about 60 kDa. 0 kDa, approximately 5 kDa to approximately 70 kDa, approximately 5 kDa to approximately 80 kDa, approximately 5 kDa to approximately 90 kDa, approximately 5 kDa to approximately 100 kDa, approximately 5 kDa to approximately 150 kDa, approximately 10 kDa to approximately 20 kDa, approximately 10 kDa to approximately 30 kDa, approximately 10 kDa to approximately 40 kDa, approximately 10 kDa to approximately 50 kDa, approximately 10 kDa to approximately 60 kDa, approximately 10 kDa to approximately 70 kDa, approximately 10 kDa to approximately 80 kDa, approximately 10 kDa to approximately 90 kDa, approximately 10 kDa to approximately 100 kDa, approximately 10 kDa to approximately 150 kDa, approximately 20 kDa to approximately 30 kDa, approximately 20 kDa to approximately 40 kDa, approximately 20 kDa a to approximately 50kDa, approximately 20kDa to approximately 60kDa, approximately 20kDa to approximately 70kDa, approximately 20kDa to approximately 80kDa, approximately 20kDa to approximately 90kDa, approximately 20kDa to approximately 100kDa, approximately 20kDa to approximately 150kDa, approximately 30kDa to approximately 40kDa, approximately 30kDa to approximately 50kDa, approximately 30kDa to approximately 60kDa, approximately 30kDa to approximately 70kDa, approximately 30kDa to approximately 80kDa, approximately 30kDa to approximately 90kDa, approximately 30kDa to approximately 100kDa, approximately 30kDa to approximately 150kDa, approximately 40kDa to approximately 50kDa, approximately 40kDa to approximately 60kDa, approximately 40kDa to approximately 70kDa kDa, approximately 40kDa to approximately 80kDa, approximately 40kDa to approximately 90kDa, approximately 40kDa to approximately 100kDa, approximately 40kDa to approximately 150kDa, approximately 50kDa to approximately 60kDa, approximately 50kDa to approximately 70kDa, approximately 50kDa to approximately 80kDa, approximately 50kDa to approximately 90kDa, approximately 50kDa to approximately 100kDa, approximately 50kDa to approximately 150kDa, approximately 60kDa to approximately 70kDa, approximately 60kDa to approximately 80kDa, approximately 60kDa to approximately 90kDa, approximately 60kDa to approximately 100kDa, approximately 60kDa to approximately 150kDa, approximately 70kDa to approximately 80kDa, approximately 70kDa to approximately 90kDaMolecules ranging from approximately 70 kDa to approximately 100 kDa, approximately 70 kDa to approximately 150 kDa, approximately 80 kDa to approximately 90 kDa, approximately 80 kDa to approximately 100 kDa, approximately 80 kDa to approximately 150 kDa, approximately 90 kDa to approximately 100 kDa, approximately 90 kDa to approximately 150 kDa, or approximately 100 kDa to approximately 150 kDa.

[0221] a.Fc structural domain

[0222] In some embodiments, the fusion protein comprises an immunoglobulin Fc domain. When used herein, unless otherwise specified, the terms "immunoglobulin Fc domain" or "Fc domain" or "Fc" refer to a segment of the constant region of the immunoglobulin heavy chain, which, alone or in combination with a second immunoglobulin Fc domain, or uncoupled or coupled to sialidase, is capable of binding to an Fc receptor. The immunoglobulin Fc domain may include, for example, immunoglobulin CH2 and CH3 domains. The immunoglobulin Fc domain may include, for example, immunoglobulin CH2 and CH3 domains and an immunoglobulin hinge region. The boundaries between the immunoglobulin hinge region, the CH2 and CH3 domains are well known in the art and can be found, for example, in the PROSITE database (available at prosite.expasy.org).

[0223] Figure 1 AE describes certain embodiments of a sialidase-Fc fusion construct comprising: a first polypeptide comprising a first immunoglobulin Fc domain and a second polypeptide comprising a second immunoglobulin Fc domain. The first and second polypeptides may be covalently linked together. The covalent bond may be a disulfide bond. A sialidase may be coupled to the N- or C-terminus of either the first or second immunoglobulin Fc domain. Optionally, a second sialidase may be coupled to either the N- or C-terminus of either the first or second immunoglobulin Fc domain.

[0224] Figure 1 A shows a sialidase construct having two Fc domains and an N-terminus coupled to each Fc domain. Figure 1 B shows a sialidase construct having two Fc domains and a C-terminus coupled to the first Fc domain and an N-terminus coupled to the second Fc domain. Figure 1 C shows a sialidase construct having two Fc domains and an N-terminus coupled to the second Fc domain. Figure 1 D shows a sialidase construct having two Fc domains and a C-terminus coupled to the first Fc domain. Figure 1E illustrates a sialidase construct having two Fc domains and a C-terminus coupled to each Fc domain. It should be understood that the Fc domains can be naturally occurring Fc domains or engineered Fc domains containing modifications, such as point mutations in each polypeptide chain that facilitate a mortise-and-tenon conformation or provide altered Fc domain function.

[0225] In some embodiments, the immunoglobulin Fc domain is derived from the Fc domains of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM. A single amino acid substitution (S228P according to the Kabat numbering system; referred to as IgG4Pro) can be introduced to eliminate the heterogeneity observed in recombinant IgG4 antibodies. See Angal, S et al., (1993) MOL. IMMUNOL. 30:105-108.

[0226] In some embodiments, 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 some embodiments, the immunoglobulin Fc domain is derived from a human IgG1 isotype (e.g., SEQ ID NO: 31 or SEQ ID NO: 69).

[0227] In some embodiments, the immunoglobulin Fc domain is derived from a human IgG4 isotype or another isotype that rarely or does not induce antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-mediated cytotoxicity (CDC). In some embodiments, the immunoglobulin Fc domain is derived from a human IgG4 isotype.

[0228] In some embodiments, the immunoglobulin Fc domain contains a "palm" mutation, such as T366Y, or a "mortar" mutation, such as Y407T, for heterodimerization with a second polypeptide (residue numbers according to the EU numbering system, Kabat, EA, et al., (1991) Sequences of Proteins of Immunological Interest, 5th Edition, Department of Health and Human Services, NIH Publication No. 91-3242). In some embodiments comprising a sialidase-Fc fusion having two Fc domains, the first Fc domain may contain a "palm" mutation (e.g., SEQ ID NO: 33 and SEQ ID NO: 148), and the second Fc domain may contain a "mortar" mutation (e.g., SEQ ID NO: 32 and SEQ ID NO: 147).

[0229] In some embodiments, the sialidase-Fc fusion protein comprises an amino acid sequence of any one of SEQ ID NO: 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 with any one of SEQ ID NO: 129-158, 177-192, and 197-200.

[0230] In some embodiments, the sialidase-Fc fusion protein comprises the amino acid sequence X1X2SX3X4X5LQX6ESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASX7X8DEHAELIVX9RRGDYDAX 10 THQVQWX 11 AQEVVAQAX 12 LDGHRSMNPCPLYDX 13 QTGTLFLFFIAIPX 14 X 15 VTEX 16 QQLQTRANVTRLX 17 X 18 VTSTDHGRTWSSPRDLTDAAIGPX 19 YREWSTFAVGPGHX 20 LQLHDRX 21 RSLVVPAYAYRKLHPX 22 QRPIPSAFX 23FLSHDHGRTWARGHFVAQDTX24ECQVAEVETGEQRVVTLNARSHLRARVQAQSX 25 NX 26 GLDFQX 27 SQLVKKLVEPPPX 28 GX 29 QGSVISFPSPRSGPGSPAQX 30 LLYTHPTHX 31 X 32 QRADLGAYLNPRPPAPEAWSEPX 33 LLAKGSX 34 AYSDLQSMGTGPDGSPLFGX 35 LYEANDYEEIX 36 FX 37 MFTLKQAFPAEYLPQGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0231] (SEQ ID NO: 159), wherein X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val or not present; X2 is Ala or Lys; X3 is Asn or Leu; X4 is Pro or His; X5 is Phe, Trp, Tyr or Val; X6 is Lys or Asp; X7 is Lys, Arg or Glu; X8 is Lys, Ala, Arg or Glu; X9 is Leu or Met; X 10 Is it Pro, Asn, Asp, His, Glu, Gly, Ser, or Thr, X 11 Is it Gln or His, X 12 Is it Arg or Lys, X 13 Is it Ala, Glu, or Lys, X? 14 Is it Gly or Asp, X 15 Is it Gln or His, X 16 Is it Gln, Arg, or Lys, X17 Is it Ala, Cys, Ile, Ser, Val, or Leu, X? 18 Is it Gln or Leu, X 19 Is it Ala or Val, X? 20 Is it Cys or Gly, X 21 Is it Ala or Gly, X? 22 Is it Arg, Ile, or Lys, X 23 Is it Ala, Cys, Leu, or Val, X? 24 Is it Leu, Ala, or Val, X? 25 Is it Thr or Ala, X 26 Is it Asp or Gly, X 27 Is it Glu or Lys, X 28 Is it Gln, Ala, His, Phe, or Pro, X? 29 Is it Cys or Val, X 30 Is it Trp or Arg, X 31 Is it Ser or Arg, X 32 Is it Trp or Lys, X 33 Is it Lys or Val, X 34 Is it Ala, Cys, Ser, or Val, X? 35 Is it Cys, Leu, or Val, X? 36 It is Val or Arg, and X 37 It is Leu, Gln, His, Ile, Lys, or Ser, and the sialidase contains at least one mutation relative to wild-type human Neu2 (SEQ ID NO: 1).

[0232] In some embodiments, the sialidase-Fc fusion protein comprises the amino acid sequence X1ASLPX2LQX3ESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAX4THQVQWQAQEVVAQARLDGHRSMNPCPLYDX5QTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPX6QRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPX7GCQGSVISFPSPRSGPGSPAQWLLYTHPTHX8X9QRADLGAYLNPRPPAPEAWSEPVLLAKGSX10 AYSDLQSMGTGPDGSPLFGCLYEANDYEEIX 11 FX 12 MFTLKQAFPAEYLPQGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0233] (SEQ ID NO: 160), wherein X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val or not present; 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 it Ala, Cys, Ser, or Val, X? 11 Is it Val or Arg, and X? 12 The sialidase is Leu, Gln, His, Ile, Lys, or Ser, and contains at least one mutation relative to wild-type human Neu2 (SEQ ID NO: 1). In some embodiments, X1 is Ala, Asp, Met, or absent; X2 is Tyr or Val; X3 is Lys or Asp; X4 is Pro, Asn, Gly, Ser, or Thr; X5 is Ala or Glu; X6 is Ile or Lys; X7 is Gln or Ala; X8 is Ser or Arg; X9 is Trp or Lys; X... 10 Is it Ala or Cys, X? 11 It is Val or Arg, and X 12 It is either Leu or Ile.

[0234] In some embodiments, the sialidase-Fc fusion protein comprises the amino acid sequence X1X2SX3X4X5LQX6ESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASX7X8DEHAELIVX9RRGDYDAX 10 THQVQWX 11 AQEVVAQAX 12 LDGHRSMNPCPLYDX 13 QTGTLFLFFIAIPX 14 X 15 VTEX 16 QQLQTRANVTRLX 17 X 18 VTSTDHGRTWSSPRDLTDAAIGPX 19 YREWSTFAVGPGHX 20 LQLHDRX 21 RSLVVPAYAYRKLHPX 22 QRPIPSAFX 23 FLSHDHGRTWARGHFVAQDTX24ECQVAEVETGEQRVVTLNARSHLRARVQAQSX 25 NX 26 GLDFQX 27 SQLVKKLVEPPPX 28 GX 29 QGSVISFPSPRSGPGSPAQX 30 LLYTHPTHX 31 X 32 QRADLGAYLNPRPPAPEAWSEPX 33 LLAKGSX 34 AYSDLQSMGTGPDGSPLFGX 35 LYEANDYEEIX 36 FX 37 MFTLKQAFPAEYLPQX 38DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 161), where X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val or does not exist; X2 is Ala or Lys; X3 is Asn or Leu; X4 is Pro or His; X5 is Phe, Trp, Tyr or Val; X6 is Lys or Asp; X7 is Lys, Arg or Glu; X8 is Lys, Ala, Arg or Glu; X9 is Leu or Met; X 10 Is it Pro, Asn, Asp, His, Glu, Gly, Ser, or Thr, X 11 Is it Gln or His, X 12 Is it Arg or Lys, X 13 Is it Ala, Glu, or Lys, X? 14 Is it Gly or Asp, X 15 Is it Gln or His, X 16 Is it Gln, Arg, or Lys, X 17 Is it Ala, Cys, Ile, Ser, Val, or Leu, X? 18 Is it Gln or Leu, X 19 Is it Ala or Val, X? 20 Is it Cys or Gly, X 21 Is it Ala or Gly, X? 22 Is it Arg, Ile, or Lys, X 23 Is it Ala, Cys, Leu, or Val, X? 24 Is it Leu, Ala, or Val, X? 25 Is it Thr or Ala, X 26 Is it Asp or Gly, X 27 Is it Glu or Lys, X 28 Is it Gln, Ala, His, Phe, or Pro, X? 29 Is it Cys or Val, X 30 Is it Trp or Arg, X 31 Is it Ser or Arg, X32 Is it Trp or Lys, X 33 Is it Lys or Val, X 34 Is it Ala, Cys, Ser, or Val, X? 35 Is it Cys, Leu, or Val, X? 36 Is it Val or Arg, X 37 Is it Leu, Gln, His, Ile, Lys, or Ser, X 38 It is GGGGSGGGGS (SEQ ID NO: 162) or EPKSS (SEQ ID NO: 163), and the sialidase contains at least one mutation relative to wild-type human Neu2 (SEQ ID NO: 1).

[0235] In some embodiments, the sialidase-Fc fusion protein comprises the amino acid sequence X1ASLPX2LQX3ESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAX4THQVQWQAQEVVAQARLDGHRSMNPCPLYDX5QTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPX6QRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPX7GCQGSVISFPSPRSGPGSPAQWLLYTHPTHX8X9QRADLGAYLNPRPPAPEAWSEPVLLAKGSX 10 AYSDLQSMGTGPDGSPLFGCLYEANDYEEIX 11 FX 12 MFTLKQAFPAEYLPQX 13 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0236] (SEQ ID NO: 164), where X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val or not present; 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 it Ala, Cys, Ser, or Val, X? 11 Is it Val or Arg, X 12 It is Leu, Gln, His, Ile, Lys, or Ser, and X 13 It is GGGGSGGGGS (SEQ ID NO: 162) or EPKSS (SEQ ID NO: 163), and the sialidase contains at least one mutation relative to wild-type human Neu2 (SEQ ID NO: 1). In some embodiments, X1 is Ala, Asp, Met, or absent; X2 is Tyr or Val; X3 is Lys or Asp; X4 is Pro, Asn, Gly, Ser, or Thr; X5 is Ala or Glu; X6 is Ile or Lys; X7 is Gln or Ala; X8 is Ser or Arg; X9 is Trp or Lys; X 10 Is it Ala or Cys, X? 11 It is Val or Arg, and X 12 It is either Leu or Ile.

[0237] In some embodiments, the sialidase-Fc fusion protein comprises the amino acid sequence X1X2SX3X4X5LQX6ESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASX7X8DEHAELIVX9RRGDYDAX 10 THQVQWX 11 AQEVVAQAX 12 LX13GHRSMNPCPLYDX 14 QTGTLFLFFIAIPX 15 X 16 VTEX 17 QQLQTRANVTRLX 18 X 19 VTSTDHGRTWSSPRDLTDAAIGPX 20 YREWSTFAVGPGHX21 LQLHDX 22 X 23 RSLVVPAYAYRKLHPX 24 X 25 X 26 PIPSAFX 27 FLSHDHGRTWARGHFVX 28 QDTX 29 ECQVAEVX 30 TGEQRVVTLNARSX 31 X 32 X 33 X 34 RX 35 QAQSX 36 NX 37 GLDFQX 38 X 39 QX 40 VKKLX 41 EPPPX 42 GX 43 QGSVISFPSPRSGPGSPAQX 44 LLYTHPTHX 45 X 46 QRADLGAYLNPRPPAPEAWSEPX 47 LLAKGSX 48 AYSDLQSMGTGPDGSPLFGX 49 LYEANDYEEIX 50 FX 51 MFTLKQAFPAEYLPQX 52 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0238] (SEQ ID NO: 165), wherein X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val or not present; X2 is Ala or Lys; X3 is Asn or Leu; X4 is Pro or His; X5 is Phe, Trp, Tyr or Val; X6 is Lys or Asp; X7 is Lys, Arg or Glu; X8 is Lys, Ala, Arg or Glu; X9 is Leu or Met; X 10 Is it Pro, Asn, Asp, His, Glu, Gly, Ser, or Thr, X 11 Is it Gln or His, X 12 Is it Arg or Lys, X 13 Is it Asp or Pro, X? 14 Is it Ala, Glu, or Lys, X? 15 Is it Gly or Asp, X 16 Is it Gln or His, X 17 Is it Gln, Arg, or Lys, X 18 Is it Ala, Cys, Ile, Ser, Val, or Leu, X? 19 Is it Gln, Leu, Glu, Phe, His, Ile, Leu, or Tyr, X 20 Is it Ala or Val, X? 21 Is it Cys or Gly, X 22 Is it Arg or Pro, X 23 Is it Ala or Gly, X? 24 Is it Arg, Ile, or Lys, X 25 Is it Gln or Pro, X 26 Is it Arg or Pro, X 27 Is it Ala, Cys, Leu, or Val, X? 28 Is it Ala, Cys, Asn, Ser, or Thr, X 29 Is it Leu, Ala, or Val, X? 30 Is it Glu or Pro, X 31 Is it His or Pro, X? 32 Is it Leu, Asp, Asn, or Tyr, X 33 Is it Arg, Ala, Asp, Leu, Gln, or Tyr, X 34 Is it Ala, Cys, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Val, Trp, or Tyr, X 35 Is it Val, Ile, or Lys, X 36 Is it Thr or Ala, X37 Is it Asp or Gly, X 38 Is it Glu, Lys, or Pro, X 39 Is it Ser or Cys, X 40 Is it Leu, Asp, Phe, Gln, or Thr, X 41 Is it Val or Phe, X 42 Is it Gln, Ala, His, Phe, Pro, Ser, or Thr, X 43 Is it Cys or Val, X 44 Is it Trp or Arg, X 45 Is it Ser, Arg, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Thr, Val, Trp, or Tyr, X 46 Is it Trp, Lys, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Tyr, X 47 Is it Lys or Val, X 48 Is it Ala, Cys, Ser, or Val, X? 49 Is it Cys, Leu, or Val, X? 50 Is it Val or Arg, X 51 Is it Leu, Gln, His, Ile, Lys, or Ser, X 52 It is GGGGS (SEQ ID NO: 174), GGGGSGGGGS (SEQ ID NO: 162), or EPKSS (SEQ ID NO: 163), and the sialidase contains at least one mutation relative to wild-type human Neu2 (SEQ ID NO: 1).

[0239] In certain embodiments, the sialidase-Fc fusion protein comprises the amino acid sequence X1ASLPX2LQX3ESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAX4THQVQWQAQEVVAQARLDGHRSMNPCPLYDX5QTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCX6VTSTDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPX7QRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRX8RVQAQSTNDGLDFQESQLVKKLVEPPPX9GCQGSVISFPSPRSGPGSPAQWLLYTHPTHX 10 X 11 QRADLGAYLNPRPPAPEAWSEPVLLAKGSX 12 AYSDLQSMGTGPDGSPLFGCLYEANDYEEIX 13 FX 14 MFTLKQAFPAEYLPQX15DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0240] (SEQ ID NO: 166), wherein X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val or not present; 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; X8 is Ala, Cys, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Val, Trp or Tyr; X9 is Gln, Ala, His, Phe, Pro, Ser or Thr; X 10 Is it Ser, Arg, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Thr, Val, Trp, or Tyr, X 11 Is it Trp, Lys, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Tyr, X 12 Is it Ala, Cys, Ser, or Val, X? 13 Is it Val or Arg, X 14 Is it Leu, Gln, His, Ile, Lys, or Ser, X 15 The enzyme is GGGGS (SEQ ID NO: 184), GGGGSGGGGS (SEQ ID NO: 162), or EPKSS (SEQ ID NO: 163), and the sialidase contains at least one mutation relative to wild-type human Neu2 (SEQ ID NO: 1). In some embodiments, X1 is Ala, Asp, Met, or absent; X2 is Tyr or Val; X3 is Lys or Asp; X4 is Pro, Asn, Gly, Ser, or Thr; X5 is Ala or Glu; X6 is Gln or Tyr; X7 is Ile or Lys; X8 is Ala or Thr; X9 is Gln, Ala, or Thr; X... 10 Is it Ser, Arg, or Ala, X 11 Is it Trp, Lys, or Arg, X 12 Is it Ala or Cys, X? 13 It is Val or Arg, and X 14 It is either Leu or Ile.

[0241] b. Polyethylene glycol (PEG)

[0242] In one embodiment, the serum half-life extender is polyethylene glycol (PEG) and its derivatives (e.g., alkoxy polyethylene glycol, such as methoxy polyethylene glycol, ethoxy polyethylene glycol, etc.). In one embodiment, the sialidase described herein is covalently attached to at least one PEG with an actual MW of at least about 20,000 D. In another embodiment, the sialidase is covalently attached to at least one PEG with an actual MW of at least about 30,000 D. In yet another embodiment, the sialidase is covalently attached to at least one PEG with an actual MW of at least about 40,000 D. In some embodiments, the PEG is methoxyPEG(5000)-succinimidylpropionate (mPEG-SPA) or methoxyPEG(5000)-succinimidylsuccinate (mPEG-SS). Such PEGs are commercially available from Nektar Therapeutics, SunBiowest, LaysanBio, or NOF. In one embodiment, the PEG may be branched or Y-shaped, as can be obtained from JenKemUSA or NOF, or comb-shaped, or synthesized by coupling two or more PEGs to a small molecule such as glutamic acid.

[0243] The ω-position of the PEG may include a hydroxyl or methoxy group, and the PEG may also contain an amino group at the ω-position. This amino group can then be coupled to various different reagents. In another embodiment of the invention, the biomodifier may be polyethylene glycol-modified poly-L-lysine or polyethylene glycol-modified poly-D-lysine.

[0244] The attachment sites on sialidase for PEG or its derivatives include the N-terminal amino and ε-amino groups present on lysine residues, as well as other amino, imino, carboxyl, thiol, hydroxyl, or other hydrophilic groups. PEG can be directly covalently bonded to sialidase with or without the use of chemically available and art-used multifunctional (typically bifunctional) crosslinking agents. For example, the PEG modifier can be coupled to the sialidase by using a thiol-reactive crosslinking agent and then reacting it with the thiol groups on the PEG. In some embodiments, the thiol group can be derived by coupling with a maleimide-substituted PEG (e.g., alkoxy-PEGamine plus 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfosuccinimide ester) or PEG-maleimide commercially available from Shearwater Polymers, Inc. (Huntsville, Ala).

[0245] c. Human serum albumin (HSA) and HSA binders

[0246] Human serum albumin (HSA) (molecular weight ~67 kDa) is the most abundant protein in blood plasma, present at approximately 50 mg / mL (600 μM), and has a half-life of about 20 days in humans. HSA is used to maintain plasma pH, contributes to colloid blood pressure, acts as a carrier for many metabolites and fatty acids, and serves as a major drug transport protein in plasma.

[0247] In some embodiments, the serum half-life extender is human serum albumin (HSA) or an HSA-binding peptide (see, for example, PCT Publications WO2013128027A1 and WO2014140358A1). Neonatal Fc receptors (FcRn) appear to be involved in prolonging the circulating lifetime of albumin (see Chaudhury et al., (2003) J.EXP.MED., 3:315-22). Albumin and IgG bind non-cooperatively to different sites on FcRn and form a trimolecular structure (see ibid.). The binding of human FcRn to HSA and human IgG is pH-dependent, stronger at acidic pH and weaker at neutral or physiological pH (see ibid.). This observation suggests that proteins and albumin-containing protein complexes are similar to IgG-containing (particularly Fc)-containing protein complexes, protected from degradation by pH-sensitive interactions with FcRn (see ibid.). The ability of a single HSA domain to bind immobilized soluble human FcRn was measured using surface plasmon resonance (SPR), showing that FcRn interacts with albumin via the D-III domain of albumin at sites different from the IgG binding site in a pH-dependent manner (see Chaudhury et al., (2006) BIOCHEM.45:4983-90 and PCT Publication No. WO2008068280A1).

[0248] Exemplary HSA-binding proteins are known in the art. For example, U.S. Patent Application Publication No. US20130316952A1 discloses a polypeptide that binds serum albumin having the amino acid sequence LKEAKEKAIEELKKAGITSDYYFDLINKAKTVEGVNALKDEILKA (SEQ ID NO: 109). Other exemplary polypeptides that bind HSA are described in the following literature: Dennis et al., (2002) JB IOL .C HEM .,277:35035-43; Jacobs et al., (2015)P ROTEIN E NG .D ES .S EL .,28:385-93; and Zorzi et al., (2017)NAT.COMMUN.,8:16092.

[0249] III. Connectors

[0250] In some embodiments, the sialidase may be directly linked to or fused to the serum half-life extender. In other embodiments, the sialidase may be covalently bound to the serum half-life extender via a linker.

[0251] The linker may be coupled to one or more natural amino acids, the sialidase or a functional fragment thereof, and the serum half-life extender, wherein the one or more natural amino acids (e.g., cysteine ​​amino acids) may be introduced by site-directed mutagenesis. The linker may include one or more non-natural amino acids. It is envisioned that, in some cases, a linker containing, for example, one or more thiol-reactive groups (e.g., maleimide) may covalently link cysteine ​​residues in the sialidase moiety or the serum half-life extender, which may be naturally occurring cysteine ​​residues or products of site-specific mutations.

[0252] The connector may be a cuttable connector or a non-cuttable connector. Optionally or additionally, the connector may be a flexible connector or a non-flexible connector.

[0253] The linker should be long enough to allow the sialidase and serum half-life extender to connect to each other without steric hindrance, and short enough to preserve the target activity of the fusion protein. The linker is preferably hydrophilic enough to avoid or minimize the instability of the fusion protein. The linker should be sufficiently stable in vivo (e.g., it is not cleaved by serum, enzymes, etc.) to allow for in vivo manipulation of the fusion protein.

[0254] The connector may have a diameter of approximately 1 angstrom. ) to approximately Length or approximately to approximately Length or approximately to approximately Length or approximately to approximately The connector may have a length greater than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 27, 30 angstroms or greater and / or less than about 110, 100, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32. Or even shorter lengths. Furthermore, the connectors can have lengths of approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, and... The length.

[0255] In some embodiments, the linker comprises a polypeptide linker that links or fuses the sialidase of the fusion protein to a serum half-life extender (e.g., an Fc domain). For example, a gene encoding a sialidase that is directly or indirectly (e.g., via an amino acid-containing linker) linked to a serum half-life extender is envisioned, which can be generated and expressed using conventional recombinant DNA techniques. For example, the amino terminus of the sialidase can be linked to the carboxyl terminus of the serum half-life extender. When a linker is used, the linker may contain hydrophilic amino acid residues such as Gln, Ser, Gly, Glu, Pro, His, and Arg. In some embodiments, the linker is a peptide containing 1-25 amino acid residues, 1-20 amino acid residues, 2-15 amino acid residues, 3-10 amino acid residues, 3-7 amino acid residues, 4-25 amino acid residues, 4-20 amino acid residues, 4-15 amino acid residues, 4-10 amino acid residues, 5-25 amino acid residues, 5-20 amino acid residues, 5-15 amino acid residues, or 5-10 amino acid residues. Exemplary linkers include glycine- and serine-rich linkers such as (GlyGlyPro). n (SEQ ID NO: 110) or (GlyGlyGlyGlySer) n (SEQ ID NO: 111), where n is 1-5. In some embodiments, the connector comprises, is composed of, or is substantially composed of GGGGS (SEQ ID NO: 174). In some embodiments, the connector comprises, is composed of, or is substantially composed of GGGGSGGGGS (SEQ ID NO: 162). In some embodiments, the connector comprises, is composed of, or is substantially composed of EPKSS (SEQ ID NO: 163). Other exemplary connector sequences are disclosed, for example, in George et al., (2003) PROTEIN ENGINEERING 15:871–879 and U.S. Patent Nos. 5,482,858 and 5,525,491.

[0256] IV. Methods for manufacturing sialidase and / or sialidase coupled with serum half-life enhancers.

[0257] Methods for producing sialidases, such as those disclosed herein, or sialidases conjugated with serum half-life enhancers, are known in the art. For example, DNA molecules encoding serum half-life enhancers (e.g., Fc domains) can be synthesized chemically or via recombinant DNA methods. For example, the sequence of said serum half-life enhancer can be cloned using conventional hybridization techniques or polymerase chain reaction (PCR) using suitable synthetic nucleic acid primers. The resulting DNA molecule encoding the protein of interest can be ligated with other suitable nucleotide sequences, including, for example, expression control sequences, to produce a conventional gene expression construct (i.e., an expression vector) encoding the desired serum half-life enhancer. The generation of the gene construct is determined to be within the conventional techniques of the art.

[0258] The nucleic acid encoding the required sialidase can be incorporated (ligated) into an expression vector, which can be introduced into host cells using conventional transfection or transformation techniques. Exemplary host cells include *E. coli* cells that do not normally produce IgG proteins, *C. hamster ovary* (CHO) cells, human embryonic kidney 293 (HEK 293) cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells. The transformed host cells can be grown under conditions that allow the host cells to express the sialidase.

[0259] Specific expression and purification conditions will vary depending on the expression system used. For example, if a gene is to be expressed in *E. coli*, it is first cloned into an expression vector by placing the engineered gene downstream of a suitable bacterial promoter, such as Trp or Tac, and a prokaryotic signal sequence. The expressed protein may be secreted. The expressed protein may accumulate in refractive bodies or inclusion bodies, which can be harvested after cell disruption by Freund's crusher or sonication. The refractive bodies are then dissolved, and the protein can be refolded and / or cleaved using methods known in the art.

[0260] If the engineered gene is to be expressed in a eukaryotic host cell, such as a CHO cell, it is first inserted into an expression vector containing a suitable eukaryotic promoter, secretion signal, poly A sequence, and stop codon. Optionally, the vector or gene construct may contain enhancers and introns. The gene construct can be introduced into the eukaryotic host cell using conventional techniques.

[0261] Peptides containing sialidase or fusion proteins, such as fusion proteins containing variable regions of the heavy or light chains of immunoglobulins, can be produced by growing (culturing) host cells transfected with an expression vector encoding such variable regions under conditions that allow expression of the peptide. After expression, the peptide can be harvested and purified or isolated using techniques known in the art, such as affinity tags like glutathione S-transferase (GST) or histidine tags.

[0262] In this embodiment, sialidase, or sialidase coupled to the Fc region, can be produced by growing (culturing) host cells under conditions that allow expression of two polypeptides, said host cells being transfected with expression vectors of: (a) an expression vector encoding one Fc polypeptide and a separate expression vector encoding another Fc polypeptide; or (b) a single expression vector encoding two Fc polypeptides. The sialidase will be fused into one or more of said strands. The complete sialidase-Fc domain fusion protein can be harvested and purified or isolated using techniques known in the art, such as protein A, protein G, affinity tags such as glutathione S-transferase (GST), or histidine tags.

[0263] In some embodiments, sialidase or sialidase coupled with a serum half-life extender is expressed and / or purified in the presence of a stabilizer. The stabilizer prevents one or more of the following from occurring during expression, purification, and / or storage: protein unfolding, protein misfolding, protein aggregation, protein inhibition, enzymatic loss, and / or protein degradation of the sialidase or sialidase coupled with a serum half-life extender: protein unfolding, protein misfolding, protein aggregation, protein inhibition, enzymatic loss, and / or protein degradation. In some embodiments, the stabilizer is a cation, such as a divalent cation. In some embodiments, 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).

[0264] In some embodiments, the stabilizer is present during expression and / or purification in amounts from about 0.05 mM to about 5 mM. For example, the stabilizer may be present in amounts from about 0.05 mM to about 4 mM, from about 0.05 mM to about 3 mM, from about 0.05 mM to about 2 mM, from about 0.05 mM to about 1 mM, from about 0.05 mM to about 0.5 mM, from about 0.5 mM to about 4 mM, from about 0.5 mM to about 3 mM, from about 0.5 mM to about 2 mM, from about 0.5 mM to about 1 mM, from about 1 mM to about 4 mM, from about 1 mM to about 3 mM, or from about 1 mM to about 2 mM.

[0265] In some embodiments, to express a protein such as sialidase as a secretory protein, the original N-terminal signal sequence of the protein is replaced with, for example, MDMRVPAQLLGLLLLWLPGARC (SEQ ID NO: 28). In some embodiments, to express a protein such as recombinant human sialidase as a secretory protein, an N-terminal signal sequence such as MDMRVPAQLLGLLLLWLPGARC (SEQ ID NO: 28) is added. Other exemplary N-terminal signal sequences include signal sequences derived from interleukin-2, CD-5, IgGκ light chain, trypsinogen, serum albumin, and prolactin. In some embodiments, to express a protein such as recombinant human sialidase as a secretory protein, a C-terminal lysosomal signal motif such as YGTL (SEQ ID NO: 29) is removed.

[0266] In some embodiments, when sialidase is chemically coupled to a serum half-life extender, the chemical coupling can be performed using methods known in the art. The attachment sites on the sialidase and / or the serum half-life extender include the N-terminal amino and ε-amino groups present on lysine residues, as well as other amino, imino, carboxyl, thiol, hydroxyl, or other hydrophilic groups. The serum half-life extender can be directly covalently bonded to the sialidase with or without the use of chemically available and known multifunctional (typically bifunctional) crosslinking agents used in the art. For example, in the case of PEG, the thiol group can be derived by coupling with a maleimide-substituted PEG (e.g., alkoxy-PEG amine plus 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfosuccinimide ester) or a PEG-maleimide commercially available from Shearwater Polymers, Inc. (Huntsville, Ala).

[0267] V. Pharmaceutical Composition

[0268] For therapeutic use, sialidase or sialidase conjugated with a half-life extender is preferably combined with a pharmaceutically acceptable carrier. When used herein, the term "pharmaceutical acceptable" means compounds, materials, compositions, and / or dosage forms that, to a reasonable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0269] When used herein, the term "medicinal carrier" refers to a buffer, carrier, or excipient suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio. Medicinal carriers include any standard pharmaceutical carrier such as phosphate-buffered saline solutions, water, emulsions (e.g., oil / water or water / oil emulsions), and various types of wetting agents. The compositions may also contain stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, for example, Martin, *Remington's Pharmaceutical Sciences*, 15th edition, Mack Publ. Co., Easton, PA

[1975] . Medicinal carriers include buffers, solvents, dispersion media, coatings, isotonic and absorption-delaying agents, etc., compatible with drug administration. The use of these media and reagents for the active pharmaceutical ingredient is known in the art.

[0270] In some embodiments, the pharmaceutical composition may contain formulation materials for modifying, maintaining, or preserving, for example, the pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeation of the composition. In these embodiments, suitable formulation materials include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine), antimicrobial agents, antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite), buffers (e.g., borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids), extenders (e.g., mannitol or glycine), chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)), complexing agents (e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin), fillers, monosaccharides, disaccharides, and other sugars (e.g., glucose, mannose, or dextrin), proteins (e.g., serum albumin, gelatin, or immunoglobulins), colorants, flavorings, and diluents, emulsifiers, hydrophilic polymers (e.g., polyvinylpyrrolidone), low molecular weight peptides, and salt-forming equilibrium ions. Ingredients include: sodium (e.g.), preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (e.g., glycerol, propylene glycol, or polyethylene glycol), sugar alcohols (e.g., mannitol or sorbitol), suspending agents, surfactants or wetting agents (e.g., Pluronic acid, PEG, dehydrated sorbitol esters, polysorbates such as polysorbate 20, polysorbate, triaton, tromethamine, lecithin, cholesterol, tetrabutylphenol), stability enhancers / stabilizers (e.g., sucrose, sorbitol, or cationic), swelling enhancers (e.g., alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, sorbitol), delivery media, diluents, excipients, and / or adjuvants (see Remington's Pharmaceuticals). Pharmaceutical Sciences, 18th ed., Mack Publishing Company, 1990).

[0271] In some embodiments, the pharmaceutical composition may contain a stabilizer. In some embodiments, the stabilizer is a cation, such as a divalent cation. In some embodiments, 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).

[0272] In some embodiments, the stabilizer is present in an amount of about 0.05 mM to about 5 mM. For example, the stabilizer may be present in amounts of about 0.05 mM to about 4 mM, about 0.05 mM to about 3 mM, about 0.05 mM to about 2 mM, about 0.05 mM to about 1 mM, about 0.05 mM to about 0.5 mM, about 0.5 mM to about 4 mM, about 0.5 mM to about 3 mM, about 0.5 mM to about 2 mM, about 0.5 mM to about 1 mM, about 1 mM to about 4 mM, about 1 mM to about 3 mM, or about 1 mM to about 2 mM.

[0273] In some embodiments, the pharmaceutical composition may contain nanoparticles such as polymer nanoparticles, liposomes or micelles (see Anselmo et al., (2016) BIOENG.TRANSL.MED.1:10-29).

[0274] In some embodiments, the pharmaceutical composition may contain a sustained or controlled delivery formulation. Techniques for formulating sustained or controlled delivery means, such as liposome carriers, bioerodible microparticles or porous beads, and reservoir injections, are also known to those skilled in the art. Sustained-release formulations may include, for example, porous polymer microparticles or semi-permeable polymer matrices in the form of molded articles such as films or microcapsules. Sustained-release matrices may include polyesters, hydrogels, polylactide, copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid, poly(2-hydroxyethyl methacrylate), ethylene-vinyl acetate, or poly(-)-3-hydroxybutyric acid. Sustained-release compositions may also include liposomes, which can be prepared by any of several methods known in the art.

[0275] Pharmaceutical compositions containing sialidase or sialidase conjugated with a half-life extender may be present in dose units and may be prepared by any suitable method. The pharmaceutical composition should be formulated to be compatible with its intended route of administration. Examples of routes of administration are intravenous (IV), intradermal, inhalation, transdermal, topical, transmucosal, intrathecal, and rectal 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 dosage forms may be prepared by methods known in the pharmaceutical field. See, for example, Remington's Pharmaceutical Sciences, 18th edition (Mack Publishing Company, 1990). Suitable dosage form components for parenteral administration include sterile diluents such as water for injection, saline solution, non-volatile oil, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents, antibacterial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as EDTA, buffers such as acetate, citrate or phosphate, and reagents for adjusting osmotic pressure such as sodium chloride or dextrose.

[0276] For intravenous administration, suitable carriers include physiological saline, antibacterial water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). The carrier should be stable under the conditions of manufacture and storage and should be antimicrobially preservative. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), or suitable mixtures thereof.

[0277] The pharmaceutical formulation is preferably sterile. Sterilization can be achieved by any suitable method, such as filtration through a sterile filter membrane. In the case of freeze-drying of the composition, sterilization by filtration can be performed before or after freeze-drying and reconstitution.

[0278] In some embodiments, the pharmaceutical composition is disposed in a sterile container (e.g., a bottle or tube). The pharmaceutical composition may be freeze-dried in the sterile container or present as a solution, for example. The sterile container may be sealed with a diaphragm and may have a label disposed thereon identifying the pharmaceutical composition contained therein.

[0279] The compositions described herein can be administered topically or systemically. Administration is typically parenteral. In a preferred embodiment, the pharmaceutical composition is administered subcutaneously, and in even more preferred embodiments, intravenously. Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.

[0280] Typically, the therapeutically effective dose of the active ingredient, such as sialidase or sialidase conjugated with 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 amount administered will depend on variables such as the type and severity of the disease or indication to be treated, the patient's overall health, the in vivo potency of the active ingredient, the drug formulation, and the route of administration. The initial dose may be increased above the upper limit to rapidly reach the desired blood or tissue levels. Alternatively, the initial dose may be below the optimal dose, and the daily dose may be gradually increased during treatment. Human doses can be optimized, for example, in a routine Phase I dose-escalation study designed to run at doses from 0.5 mg / kg to 20 mg / kg. Dosage frequency can vary with various factors, such as the route of administration, dose, the serum half-life of the sialidase or sialidase conjugated with a half-life extender, and the disease to be treated. Exemplary dosing frequencies are once daily, once weekly, and once every two weeks. The preferred route of administration is parenteral, such as intravenous infusion. In some embodiments, the sialidase or sialidase coupled with a half-life extender is freeze-dried and then reconstituted in buffered saline at the time of administration.

[0281] V. Therapeutic Uses

[0282] The compositions and methods disclosed herein can be used to treat various forms of cancer or to inhibit cancer growth in subjects. This invention provides a method for treating cancer in a subject. The method comprises administering, alone or in combination with another therapeutic agent, an effective amount of sialidase or sialidase coupled with a half-life extender to the subject to treat the cancer. When used herein, the term "effective amount" refers to an amount of active pharmaceutical agent (e.g., sialidase or sialidase coupled with a half-life extender according to the invention) sufficient to achieve a beneficial or desired result. Effective amounts may be administered once or multiple times, by application, or in doses, and are not intended to be limited to a particular formulation or route of administration.

[0283] When used herein, “treatment” means treating a disease in a subject, such as a human. This includes: (a) inhibiting the disease, i.e., stopping its development; and (b) alleviating the disease, i.e. causing a reduction in the disease state. When used herein, the terms “subject” and “patient” refer to an organism to be treated by the methods and compositions described herein. These organisms preferably include, but are not limited to, mammals (e.g., rodents, apes, equines, bovines, suidae, canines, felines, etc.), and more preferably include humans.

[0284] Examples of cancer include solid tumors, soft tissue tumors, hematologic malignancies, and metastatic lesions. Examples of hematologic malignancies include leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), B-cell, T-cell, or FAB ALL, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL) such as transformed CLL, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, hairy cell leukemia, myelodysplastic syndrome (MDS), lymphoma, Hodgkin's disease, malignant lymphoma, non-Hodgkin's lymphoma, Burkitt lymphoma, multiple myeloma, or Richter's syndrome (Richter's transformation). Examples of solid tumors include malignancies of various organ systems, such as sarcomas, adenocarcinomas, and malignant epithelial tumors, such as those affecting the head and neck (including the pharynx), thyroid gland, lungs (small cell or non-small cell lung cancer (NSCLC)), breast, lymphatic system, gastrointestinal tract (e.g., oral cavity, esophagus, stomach, liver, pancreas, small intestine, colon and rectum, anal canal), reproductive and genitourinary tract (e.g., kidneys, urothelial tract, bladder, ovaries, uterus, cervix, endometrium, prostate, testes), CNS (e.g., nerves or glial cells, such as neuroblastoma or glioma), or skin (e.g., melanoma).

[0285] In some embodiments, the cancer is an epithelial cancer, such as an epithelial cancer that upregulates the expression of sialylated glycans. Examples of epithelial cancers include, but are not limited to, endometrial cancer, colon cancer, ovarian cancer, cervical cancer, vulvar cancer, uterine or fallopian tube cancer, breast cancer, prostate cancer, lung cancer, pancreatic cancer, urinary system cancers, bladder cancer, head and neck cancer, oral cancer, and liver cancer. Epithelial carcinoma also includes malignant epithelial tumors, such as acinar carcinoma, adenocarcinoma, adenocystic carcinoma, adenoid cystic carcinoma, adenomatous carcinoma, adrenocortical carcinoma, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, basal carcinoid carcinoma, basal squamous cell carcinoma, bronchoalveolar carcinoma, bronchial carcinoma, bronchial carcinoma, cerebral carcinoma, cholangiocarcinoma, choriocarcinoma, colloid carcinoma, comedo carcinoma, uterine corpus cancer, cribriform carcinoma, skin cancer, columnar carcinoma, columnar cell carcinoma, ductal carcinoma, sclerosing carcinoma, embryonal carcinoma, cerebral carcinoma, epidermoid carcinoma, carcinoid epithelial carcinoma, explant carcinoma, ulcerative carcinoma, fibrous carcinoma, colloid carcinoma, giant cell carcinoma, adenocarcinoma, granular cell carcinoma, pilosula stromal carcinoma, polycythemia vera, hepatocellular carcinoma, eosinophilic carcinoma, hyalinoid carcinoma, adrenal carcinoma, infancy embryonal carcinoma, carcinoma in situ, intraepithelial carcinoma, intraepithelial carcinoma. Krompecher carcinoma, Kulchitzky cell carcinoma, large cell carcinoma, bean-shaped carcinoma, bean-shaped carcinoma, lipoma carcinoma, lymphoepithelial carcinoma, medullary carcinoma, medullary carcinoma, melanoma, soft carcinoma, mucinous carcinoma, mucinous cell carcinoma, mucinous epidermoid carcinoma, mucinous carcinoma, mucinous carcinoma, mucinous tumor-like carcinoma, nasopharyngeal carcinoma, oat cell carcinoma, ossifying carcinoma, osteoid carcinoma, papillary carcinoma, perihilar carcinoma, preinvasive carcinoma, acanthosis nigra, brain-like carcinoma, renal cell carcinoma, reserve cell carcinoma, sarcomatoid carcinoma, Schneider carcinoma, scleroderma, scrotal carcinoma, signet ring cell carcinoma, simple carcinoma, small cell carcinoma, potato-shaped carcinoma, spherical cell carcinoma, spindle cell carcinoma, cavernous carcinoma, squamous carcinoma, squamous cell carcinoma, string carcinoma, angiodilated carcinoma, angiodilated carcinoma, transitional cell carcinoma, nodular carcinoma, nodular carcinoma, verrucous carcinoma, and villous carcinoma.

[0286] In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is adenocarcinoma. In some embodiments, the cancer is metastatic cancer. In some embodiments, the cancer is refractory cancer.

[0287] In some embodiments, the cancer is resistant to or unresponsive to treatment with antibodies, such as trastuzumab, which has ADCC activity.

[0288] The methods and compositions described herein can be used alone or in combination with other therapeutic agents and / or methods. When used herein, the term “combined” administration is understood to mean delivering two (or more) different treatments to the subject during the course of the disorder, such that the therapeutic effects on the patient overlap at points in time. In some embodiments, the delivery of a second treatment begins while the delivery of one treatment is still in progress, resulting in overlap in administration. This is sometimes referred to herein as “simultaneous” or “parallel delivery.” In other embodiments, the delivery of one treatment ends before the delivery of another treatment begins. In some embodiments of either case, the treatment is more effective due to combined administration. For example, the second treatment is more effective, such as seeing an equivalent effect with less of the second treatment, or the second treatment alleviates symptoms to a greater extent than observed when the second treatment is administered in the absence of the first treatment, or a similar situation is observed when the first treatment is used. In some embodiments, the delivery results in a reduction in symptoms or other parameters associated with the disorder greater than the reduction observed when the other treatment is delivered in the absence of one treatment. The effects of the two treatments may be partially additive, fully additive, or greater than additive. The delivery method allows the effect of the first treatment to still be detectable when the second treatment is delivered.

[0289] In some embodiments, the methods or compositions described herein are administered in combination with one or more additional therapies, such as surgery, radiation therapy, or administration of another chemical agent. In some embodiments, the additional therapy may include chemotherapy, such as cytotoxic agents. In some embodiments, the additional therapy may include targeted therapies, such as tyrosine kinase inhibitors, proteasome inhibitors, or protease inhibitors. In some embodiments, the additional therapy may include anti-inflammatory, anti-angiogenic, anti-fibrotic, or anti-proliferative compounds, such as steroids, biological immunomodulators, monoclonal antibodies, antibody fragments, aptamers, siRNAs, antisense molecules, fusion proteins, cytokines, cytokine receptors, bronchodilators, statins, anti-inflammatory agents (e.g., methotrexate), or NSAIDs. In some embodiments, the additional therapy may include a combination of different classes of therapeutic agents.

[0290] In some embodiments, the methods or compositions described herein are administered in combination with a checkpoint inhibitor. The checkpoint inhibitor may be, for example, selected from 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.

[0291] In some embodiments, the checkpoint inhibitor is a PD-1 or PD-L1 inhibitor. PD-1 is a receptor present on the surface of T cells that acts as a checkpoint for the immune system, inhibiting or otherwise modulating T cell activity at appropriate times to prevent overactivation of the immune system. However, cancer cells can exploit this checkpoint by expressing ligands such as PD-L1, which interact with PD-1 on the surface of T cells, to shut down or modulate T cell activity. Exemplary PD-1 / PD-L1-based immune checkpoint inhibitors include antibody-based therapeutic agents. Exemplary treatment methods utilizing PD-1 / PD-L1-based immune checkpoint inhibition are described in U.S. Patent Nos. 8,728,474 and 9,073,994 and European Patent No. 1537878B1, and include, for example, the use of anti-PD-1 antibodies. Exemplary anti-PD-1 antibodies are described in, for example, U.S. Patent Nos. 8,952,136, 8,779,105, 8,008,449, 8,741,295, 9,205,148, 9,181,342, 9,102,728, 9,102,727, 8,952,136, 8,927,697, 8,900,587, 8,735,553, and 7,488,802. Exemplary anti-PD-1 antibodies include, for example, nivolumab (…). Bristol-Myers Squibb Co., pembrolizumab ( Merck Sharp & Dohme Corp., PDR001 (Novartis Pharmaceuticals), and pidilizumab (CT-011, Cure Tech). Exemplary 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. Exemplary anti-PD-L1 antibodies include, for example, atezolizumab (… Genentech, AstraZeneca (dvalumab), MEDI4736, acitumab, and BMS936559 (Bristol Myers Squibb Co.).

[0292] In some embodiments, the methods or compositions described herein are administered in combination with a CTLA-4 inhibitor. In the CTLA-4 pathway, the interaction of CTLA-4 on T cells with its ligands (such as CD80 and CD86, also known as B7-1) on the surface of antigen-presenting cells (rather than cancer cells) leads to T-cell suppression. Exemplary CTLA-4-based immune checkpoint inhibition methods are described in U.S. Patent Nos. 5,811,097, 5,855,887, and 6,051,227. Exemplary anti-CTLA-4 antibodies are described 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) Publications WO98 / 42752, WO00 / 37504, and WO01 / 14424, and European Patent No. EP 1212422 B1. Exemplary CTLA-4 antibodies include ipilimumab or trametumab.

[0293] In some embodiments, the methods or compositions described herein are administered in combination with (i) a PD-1 or PD-L1 inhibitor, such as the PD-1 or PD-L1 inhibitors disclosed herein and (ii) a CTLA-4 inhibitor, such as the CTLA-4 inhibitors disclosed herein.

[0294] In some embodiments, the methods or compositions described herein are administered in combination with a CD20 inhibitor. In some embodiments, the CD20 inhibitor is an anti-CD20 antibody. In some embodiments, the anti-CD20 antibody is selected from oflamb, rituximab, oligrizumab, iodine I 131 tosimomumab, oligrizumab, tiimumab, and hyaluronidase rituximab.

[0295] In some embodiments, the methods or compositions described herein are administered in combination with an IDO inhibitor. Exemplary IDO inhibitors include 1-methyl-D-tryptophan (known as indoximod), epacadostat (INCB24360), navoximod (GDC-0919), and BMS-986205.

[0296] Exemplary cytotoxic agents that can be administered in combination with the methods or compositions described herein include, for example, antimicrotubule agents, topoisomerase inhibitors, antimetabolites, protein synthesis and degradation inhibitors, mitosis inhibitors, alkylating agents, platinum-based agents, nucleic acid synthesis inhibitors, histone deacetylase inhibitors (HDAC inhibitors such as vorinostat (SAHA, MK0683), entenolol (MS-275), pabistal (LBH589), trichosmin A (TSA), mocetin ostat (MGCD0103), belistat (PXD101), romidesin (FK228, phenylethyl peptide)), DNA methyltransferase inhibitors, nitrogen mustard, nitrosourea, ethyleneimine, alkyl sulfonates, triazine, folic acid analogs, nucleoside analogs, ribonucleotide reductase inhibitors, vinca alkaloids, taxanes, epothilone, intercalating agents, agents that interfere with signal transduction pathways, agents that promote apoptosis, and antibody-drug conjugates that irradiate or bind to surface proteins to deliver toxic agents. In one embodiment, the cytotoxic agents that can be administered together with the methods or compositions described herein include platinum-based agents (e.g., cisplatin), cyclophosphamide, dacarbazine, methotrexate, fluorouracil, gemcitabine, capecitabine, hydroxyurea, topotecan, irinotecan, azacytidine, vorinostatin, ixaprilone, bortezomib, taxanes (e.g., paclitaxel or docetaxel), cytochalasin B, bacitracin D, ethidium bromide, emetine, mitomycin, etoposide, tenoporosisin, vincristine, vinblastine, colchicine, anthracyclines (e.g., doxorubicin or epirubicin), daunorubicin, dihydroxyanthraquinone dione, mitoxanthraquinone, photomycin, actinomycin D, doxorubicin, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, ricin, or maytansine compounds.

[0297] 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 includes contacting the cells, tissues, or subjects with an effective amount of sialidase or sialidase conjugated with a half-life extender to increase the expression of granzyme B, IL-1b, IL-2, IL-6, IL-10, IL-17A, HLA-DR, CD86, CD83, IFNγ, or TNFα in the cells, tissues, or subjects relative to the corresponding expression levels prior to contact with the sialidase or sialidase conjugated with a half-life extender. In some embodiments, the cells are selected from dendritic cells and peripheral blood mononuclear cells (PBMCs, e.g., monocytes).

[0298] In some embodiments, the expression of granzyme B, IL-1b, IL-2, IL-6, IL-10, IL-17A, HLA-DR, CD86, CD83, IFNγ, or TNFα in the 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% relative to similar or otherwise identical cells or tissues that have not been contacted with the sialidase or with a half-life extender.

[0299] The present invention also provides a method for removing sialic acid from cells or tissues. The method includes contacting the cells or tissues with an effective amount of sialic acid enzyme or sialic acid enzyme 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 sialic acid enzyme or sialic acid enzyme conjugated with a half-life extender, thereby removing sialic acid from the cells.

[0300] In some embodiments, the cells are tumor cells, dendritic cells (DCs), or monocytes. In some embodiments, the cells are monocytes, and the method results in increased expression of MHC-II molecules (e.g., HLA-DR) on the monocytes. In some embodiments, the expression of MHC-II molecules in the cells or tissues that are not yet in contact with the sialidase or a sialidase conjugated with a half-life extender 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%. Gene expression can be measured by any suitable method known in the art, such as by ELISA, by Luminex multiplex assay, or by flow cytometry as described in the embodiments herein.

[0301] The present invention also provides a method for enhancing the phagocytic activity of tumor cells. The method includes contacting the tumor cells with an amount of sialidase that effectively removes sialic acid from the tumor cells, or sialidase coupled with a half-life extender, thereby enhancing the phagocytic activity of the tumor cells. In some embodiments, this disclosure relates to a method for enhancing the phagocytic activity of tumor cells in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition containing an amount of sialidase that effectively removes sialic acid from the tumor cells, or sialidase coupled with a half-life extender, thereby enhancing the phagocytic activity of the tumor cells.

[0302] In some embodiments, phagocytosis is increased by at least about 10%, at least about 20%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1,000% relative to similar or otherwise identical tumor cells or populations of tumor cells that have not yet come into contact with the sialidase or a sialidase coupled with a half-life extender. Phagocytosis can be measured as described in Example 9 herein.

[0303] The present invention also provides a method for activating dendritic cells (DCs) or a population of DCs. The method includes contacting the DCs or population of DCs with tumor cells treated with sialidase or sialidase conjugated with a half-life extender. In some embodiments, this disclosure relates to a method for activating dendritic cells (DCs) or a population 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 effectively removes sialic acid from tumor cells in the subject, thereby activating the DCs or population of DCs in the subject.

[0304] In some embodiments, the activation of DCs or DC populations 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% relative to similar or otherwise identical DCs or DC populations that have not been contacted with tumor cells treated with the aforementioned sialidase or sialidase conjugated with a half-life extender. The activation can be measured as described in Example 8 herein.

[0305] The present invention also provides a method for reducing Siglec-15 binding activity to enhance antitumor activity in the tumor microenvironment, the method comprising contacting T cells with sialidase or sialidase conjugated with a half-life extender. In some embodiments, this disclosure relates to a method for reducing Siglec-15 binding activity in the tumor microenvironment of a patient to enhance antitumor activity, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising sialidase or sialidase conjugated with a half-life extender, thereby enhancing antitumor activity (e.g., T cell activity) in the subject.

[0306] In some embodiments, Siglec-15 binding activity is reduced by at least about 10%, at least about 20%, at least about 50%, at least about 75%, or about 100% relative to Siglec-15 that has not been contacted with the sialidase or with a half-life extender. The binding can be measured as described in Example 16 herein.

[0307] The present invention also provides a method for promoting the infiltration of immune cells into a tumor in a desired subject. The method includes administering to the subject an effective amount of sialidase or sialidase conjugated with a half-life extender, such as the sialidases disclosed herein or sialidase conjugated with a half-life extender. In some embodiments, the immune cells are T cells, such as CD4+ and / or CD8+ T cells, such as CD69. + CD8 + and / or GzmB + CD8 + T cells. In some embodiments, the immune cells are natural killer (NK) cells.

[0308] In some embodiments, relative to a similar or otherwise identical tumor and / or subject to which the sialidase or sialidase conjugated with a half-life extender has not been administered, the infiltration of immune cells into the tumor in the subject is increased by at least about 10%, at least about 20%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1,000%. The infiltration of immune cells into the tumor can be measured by any suitable method known in the art, such as antibody staining.

[0309] The present invention also provides a method for increasing the number of circulating natural killer (NK) cells in a subject in need. The method comprises administering to the subject an effective amount of sialidase or sialidase conjugated with a half-life extender, such as the sialidase disclosed herein or sialidase conjugated with a half-life extender, to increase the number of circulating NK cells relative to prior administration of the sialidase or sialidase conjugated with a half-life extender or the pharmaceutical composition.

[0310] In some embodiments, the number of circulating NK cells in a subject who has not been administered the sialidase or a similar or otherwise identical sialidase conjugated with a half-life extender 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%. The number of circulating NK cells in the subject can be measured by any suitable method known in the art, such as antibody staining.

[0311] The present invention also provides a method for increasing the number of T-cells in draining lymph nodes in a subject in need. The method includes administering to the subject an effective amount of sialidase or sialidase conjugated with a half-life extender, such as the sialidase disclosed herein or sialidase conjugated with a half-life extender, to increase the number of T-cells in the draining lymph nodes relative to prior administration of the sialidase or sialidase conjugated with a half-life extender or the pharmaceutical composition. In some embodiments, the immune cells are T-cells, such as CD4+ and / or CD8+ T-cells.

[0312] In some embodiments, relative to a subject who has not been administered the sialidase or a similar or otherwise identical sialidase conjugated with a half-life extender, the number of T cells in the draining lymph nodes of said subject is increased by at least about 10%, at least about 20%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1,000%. The number of T cells in the draining lymph nodes of the subject can be measured by any suitable method known in the art, such as an antibody.

[0313] The present invention also provides a method for increasing the expression of Cd3, Cd4, Cd8, Cd274, Ctla4, Icos, Pdcd1, Lag3, Il6, Il1b, Il2, Ifng, Ifna1, Mx1, Gzmb, Cxcl9, Cxcl12 and / or Ccl5 in cells, tissues or subjects. The method includes contacting the cells, tissues, or subjects with an effective amount of sialidase or sialidase conjugated with a half-life extender, such as the sialidase disclosed herein or sialidase conjugated with a half-life extender, to increase the expression of Cd3, Cd4, Cd8, Cd274, Ct1a4, Icos, Pdcd1, Lag3, Il6, Il1b, Il2, Ifng, Ifna1, Mx1, Gzmb, Cxcl9, Cxcl12, and / or Ccl5 relative to the cells, tissues, or subjects prior to contact with the sialidase or sialidase conjugated with a half-life extender or the pharmaceutical composition.

[0314] In some embodiments, the expression of Cd3, Cd4, Cd8, Cd274, Ctla4, Icos, Pdcd1, Lag3, Il6, Il1b, Il2, Ifng, Ifna1, Mx1, Gzmb, Cxcl9, Cxcl12 and / or Ccl5 is increased by at least about 10%, at least about 20%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1,000% in similar or otherwise identical cells, tissues, or subjects that have not been contacted with the sialidase or 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.

[0315] Throughout this specification, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that there may also be compositions of the invention that are substantially composed of or comprised of the described components, and processes and methods of the invention that are substantially composed of or comprised of the described process steps.

[0316] In this application, when an element or component is referred to as being included in and / or selected from the list of described elements or components, it should be understood that the element or component may be any one of the described elements or components, or the element or component may be selected from two or more of the described elements or components.

[0317] Furthermore, it should be understood that the elements and / or features of the compositions or methods described herein can be combined in various different ways without departing from the spirit and scope of the invention, whether express or implied herein. For example, when a particular compound is mentioned, that compound may be used in various different embodiments of the compositions of the invention and / or the methods of the invention, unless the context otherwise requires. In other words, in this application, embodiments are described and depicted in a manner that enables clear and concise writing and drawing, but it is intended and should be recognized that embodiments can be made in various different combinations or separations without departing from the teachings and inventions herein. For example, it should be recognized that all features described and depicted herein are applicable to all aspects of the invention described and depicted herein.

[0318] It should be understood that the expression “…at least one of…” includes the subject of each individual narrative following the expression, as well as various combinations of two or more of the subjects of the narratives, unless otherwise understood from the context and use. The expression “and / or” associated with the subject of three or more narratives should be understood to have the same meaning, unless otherwise understood from the context.

[0319] The use of the terms “including,” “having,” “meaning,” and their grammatical equivalents should generally be understood as open-ended and non-restrictive, for example, not excluding additional unstated elements or steps unless otherwise specifically stated or understood from the context.

[0320] Where the term "about" is used prior to a quantitative value, the invention also includes the specific quantitative value itself, unless otherwise specifically stated. When used herein, the term "about" means ±10% from the nominal value, unless otherwise indicated or speculated.

[0321] 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 operable. Furthermore, two or more steps or actions can be performed simultaneously.

[0322] The use of any and all instances or exemplary language, such as “e.g.” or “including,” herein is intended only to better illustrate the invention and does not constitute a limitation on the scope of the invention, unless otherwise claimed. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention. Example

[0323] The following examples are merely illustrative and are not intended to limit the scope or content of the invention in any way.

[0324] Example 1: Construction and expression of recombinant sialidase

[0325] This embodiment describes the construction of recombinant human sialidases (Neu1, Neu2, Neu3, and Neu4). Human sialidases Neu1, Neu2, Neu3 (isoform 1), and Neu4 (isoform 1) were expressed as secretory proteins with a 10×His tag.

[0326] To express Neu1 as a secretory protein, the original N-terminal signal peptide (MTGERPSTALPDRRWGPRILGFWGGCRVWVFA AIFLLLSLAASW SKA; 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 secretory proteins, the N-terminal signal peptide MDMRVPAQLLGLLLLWLPGARC (SEQ ID NO: 28) was added to each.

[0327] Sialidase was expressed in 200 mL of HEK293F human cells transfected with the pCEP4 mammalian expression vector in 24-well plates. Sialidase was purified using a Ni-NTA column, quantified using a UV-Vis spectrophotometer (NanoDrop), and as follows: Figure 2 The results were examined by SDS-PAGE. Neu1 was well expressed with a yield of ~3 μg / mL and was primarily present in monomeric form. Neu2 and Neu3 were each expressed with a yield of ~0.15 μg / mL and were primarily present in dimer form. Neu4 was not detected when measured by NanoDrop. Bacterial sialidase (bacterial sialidase; SEQ ID NO: 30) from Salmonella typhimurium was expressed in the same manner as Neu1-4 (above) and gave a yield comparable to Neu1, and was primarily present in monomeric form.

[0328] The activity of the recombinantly expressed sialidase was determined by measuring the release of sialic acid from the fluorescent substrate 4-methylumbelliferyl-N-acetylneuraminic acid (4MU-NeuAc). Figure 3As shown, Neu1 did not exhibit detectable activity higher than the enzyme-free control, consistent with previous reports indicating that Neu1 is inactive unless conjugated with β-galactosidase and the protective protein / cathetersin A (PPCA). Neu2 and Neu3 were active, as was bacterial sialidase. Enzyme kinetics were determined using Neu2 and Neu3. A fixed concentration of 1 nM of the enzyme was incubated with the fluorescent substrate 4MU-NeuAc at concentrations ranging from 4000 μM to 7.8 μM. Assays were performed under both acidic (pH 5.6) and neutral (pH 7) conditions. Figure 4 As shown, both Neu2 and Neu3 are active under acidic and neutral conditions and exhibit enzyme kinetics comparable to those previously reported.

[0329] Example 2: Construction and expression of recombinant sialidase-Fc fusion protein

[0330] This embodiment describes the construction of recombinant Fc sialidase genetic fusions, specifically Neu2-Fc, Neu3-Fc, and ST sialidase-Fc.

[0331] Fc-sialidases utilizing wild-type Neu2 (Neu2-Fc; SEQ ID NO: 113, encoded by SEQ ID NO: 114) and a variant Fc-sialidase named M106 (SEQ ID NO: 115, encoded by SEQ ID NO: 116) (M1D, V6Y, P62G, A93E, I187K, C332A, and human IgG1 with the Y407T mutation) were expressed, purified, and characterized. Neu2-Fc molecules were expressed in 1 L transfected 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 was obtained in yields of 0.3 mg / L, and M106 in yields of 20 mg / L.

[0332] Figure 5A The depicted SDS-PAGE gels show recombinant wild-type human Neu2-Fc and M106 under non-reducing and reducing conditions. Figure 5B The SEC-HPLC trace shown in -C compares wild-type Neu2-Fc with M106. The monomer has a retention time of 21 mins. Neu2-Fc ( Figure 5B M106 has a monomer purity of 7% SEC. Figure 5C It has 85% SEC monomer purity.

[0333] The activity of M106 was determined by measuring the release of sialic acid from the fluorescent substrate 4-methylumbelliferyl-N-acetylneuraminic acid (4MU-NeuAc). Enzyme kinetics assays were performed using a fixed concentration of enzyme (2 μg / well) and incubation with the fluorescent substrate 4MU-NeuAc at concentrations ranging from 4 mM to 0.03 μM. Figure 6 The enzyme activity of M106 was described.

[0334] Using the pCEP4 mammalian expression vector, the wild-type Neu3 FC sialidase (Neu3-Fc; SEQ ID NO: 117, encoded by SEQ ID NO: 118) was expressed in 100 ml of Expi293 human cells transfected. Activity was determined in both cell-modified medium (supernatant) and washed cell sediment using Neu3-Fc-expressing cells (N3-normal), Neu3-Fc-expressing cells treated with tunicamycin (N3-Tunic), and simulated transfection cells. Figure 7 Neu3-Fc activity was detected in cell deposits, representing surface-binding activity, while low levels of activity were detected in the supernatant, representing secreted Neu3-Fc. Treatment with tunicamycin, an inhibitor of S-acylation and N-glycosylation, did not alter either the surface-binding activity or the activity in the supernatant.

[0335] An Fc bacterial sialidase (Fc-ST sialidase) using Salmonella Typhimurium was constructed using a mortar-based Fc design. The Fc-ST sialidase comprises a dimer of two polypeptides: SEQ ID NO: 119 (pCEP-StSia-G4S2-hIgG1Fc-mortar, encoded by SEQ ID NO: 121) and SEQ ID NO: 120 (pCEP-StSia-G4S2-hIgG1Fc-mortar, encoded by SEQ ID NO: 122). The Fc-ST sialidase was expressed in 1 L of transfected Expi293 human cells using the pCEP4 mammalian expression vector. The Fc-ST sialidase was purified using protein A followed by cation exchange chromatography (Hitrap SP-HP, GE Lifesciences). Figure 8 The SEC-HPLC traces depicted showed that the expressed Fc-ST sialidase was a monomeric substance with a retention time of 21 minutes and SEC monomer purity of 75%.

[0336] The activity of Fc-ST sialidase was determined by measuring the release of sialic acid from the fluorescent substrate 4-methylumbelliferyl-N-acetylneuraminic acid (4MU-NeuAc). Enzyme kinetics assays were performed using a fixed concentration of enzyme (2 μg / well) and incubation with the fluorescent substrate 4MU-NeuAc at concentrations ranging from 4 mM to 0.03 μM. FCST exhibited approximately 3 x 10⁻⁶ ppm. 8 Activity of fluorescent AU.

[0337] Example 3: In vivo administration of Fc sialidase reduces tumor volume

[0338] This embodiment demonstrates how in vivo administration of the Fc sialidase of the present invention to a syngeneic mouse tumor model reduces tumor volume.

[0339] In a mouse syngeneic tumor model induced by injection of the murine lymphoma cancer cell line A20, the Fc-Salmonella typhimurium sialidase construct (Fc-ST sialidase) described in Example 2 was compared with avermab (anti-PD-L1 antibody). Six- to eight-week-old female BALB / c mice were injected with A20 tumor cells (5 x 10⁻⁶ cells) in 0.1 ml PBS in the right lower flank region. 5 Subcutaneous inoculation is used to treat tumor development. When the tumor reaches 50-100mm... 3 The average value is ~75-100mm 3 At that time, the mice were randomly assigned to 4 groups, with 8 animals in each group.

[0340] Mice were administered a negative control (“isotype control”) twice weekly at a dose of 10 mg / kg. Figure 9A ), Fc-ST sialidase ( Figure 9B ), acitumab (anti-mouse PD-L1 antibody), Figure 9C ) or a combination of Fc-ST sialidase and acimetidine ( Figure 9D The tumor was administered via intraperitoneal injection over 15 days, and tumor volume (mm²) was measured over time. 3 This embodiment demonstrates that the Fc sialidase of the present invention can reduce tumor volume in vivo.

[0341] The Fc-ST sialidase was evaluated in a second model using a mouse tumor cell line engineered to express human Her2 (EMT6-Her2 cells). In a mouse syngeneic tumor model injected with EMT6-Her2 cells, Fc-ST sialidase and the human Neu2-Fc construct M106 (described in Example 2) were compared with trastuzumab (an anti-HER2 antibody). Six- to eight-week-old female BALB / c mice were injected with EMT6-Her2 tumor cells (5 x 10⁻⁶ cells) in 0.1 ml PBS in the right lower flank region. 5Subcutaneous inoculation is used to treat tumor development. When the tumor reaches 50-100mm... 3 The average value is ~75-100mm 3 At that time, the mice were randomly assigned to 4 groups, with 8 animals in each group.

[0342] As indicated by the triangle, mice were administered 10 mg / kg of isotype control (medium control) twice weekly. Figure 10A ), Fc-ST sialidase (FC-ST, Figure 10B ), Trastuzumab (anti-human Her2 antibody), Figure 10C ) or Fc human sialic acidase (M106, Figure 10D The tumor was administered via intraperitoneal injection over 15 days, and tumor volume (mm²) was measured over time. 3 This embodiment demonstrates that the Fc sialidase of the present invention can reduce tumor volume in vivo.

[0343] Example 4: Divalent cations can stabilize the activity of sialidase.

[0344] This embodiment describes the ability of divalent cations, particularly calcium, to stabilize the activity of the sialidase of the present invention. Specifically, Fc-Neu2 sialidase (SEQ ID NO: 123) (M1D, V6Y, I187K, C332A) was expressed together with the heavy and light chains of trastuzumab (including a first polypeptide chain having an amino acid sequence SEQ ID NO: 124 encoded by a nucleotide sequence SEQ ID NO: 125, a second polypeptide chain having an amino acid sequence SEQ ID NO: 126 encoded by a nucleotide sequence SEQ ID NO: 127, and a third polypeptide chain having an amino acid sequence SEQ ID NO: 123 encoded by a nucleotide sequence SEQ ID NO: 128).

[0345] Purified proteins were incubated in PBS or PBS containing 4 mM CaCl2 at 37°C for up to 2 weeks. Samples containing approximately 2 μg of protein were determined by measuring the release of sialic acid from the fluorescent substrate 4-methylumbelliferyl ketone-N-acetylneuraminic acid (4MU-NeuAc). Measurements were performed at 37°C for 4 hours and on days 1, 3, 7, and 14. Results are shown in... Figure 11 As can be seen, adding CaCl2 to the enzyme preparation greatly stabilizes the enzyme activity.

[0346] To observe whether CaCl2 could stabilize enzyme activity during expression in mammalian cells, 4 mM CaCl2 was added to the expression medium of transiently transfected Expi293 cells starting 24 hours post-transfection. Figure 12A As shown, up to day 7, the addition of CaCl2 significantly increased the amount of secreted enzyme activity. However, as Figure 12B As shown, 4 mM CaCl2 leads to a decrease in cell viability.

[0347] To optimize the CaCl2 concentration that stabilizes enzyme activity while maintaining cell viability, five concentrations of CaCl2—0.05 mM, 0.5 mM, 1 mM, 2 mM, and 4 mM—were added on day 1 post-transfection. Figure 13A As shown, the conditioning medium was collected over a three-day period from day 4 to day 6 to determine enzyme activity (and therefore survival rate). Protein yield was also measured. Figure 13B It was found that 4 mM CaCl2 stabilized the activity and gave a moderate yield, but poor survival. It was found that using 0.5 mM CaCl2 to maintain sialidase activity under the tested conditions provided a higher protein yield and lower cytotoxicity.

[0348] Example 5: Distribution of sialyl polysaccharides in human PMBC subpopulations

[0349] This embodiment describes the distribution of sialyl glycans in different subsets of human peripheral blood mononuclear cells (PBMCs) obtained by flow cytometry. Sialoglycans, present on the surface of immune cells, play an important role in maintaining homeostasis. Imbalances in the distribution of sialyl glycans on immune cells have been reported in autoimmunity and immune surveillance escape mechanisms of tumor cells.

[0350] After isolating PBMCs using the Ficoll method, the cells were centrifuged at 350x g for 5 minutes using a benchtop centrifuge, washed twice with ice-cold PBS, and then subjected to Countess... TM The II automated cell counter (Thermo Fisher Scientific, Waltham, MA) was used to count cells, and aliquots of 250K cells were added to each well of a 96-well plate. Preparation was carried out in PBS containing Human Trustain FCX ​​(1 / 20 dilution) and LIVE / DEAD. TMFc blocking solution that can fix near-infrared dead cell dye (1 / 2000 dilution) was used, and cells were incubated on ice for 10 min. Cells were washed with ice-cold PBS (1% BSA) and centrifuged at 350 x g for 5 min. Cell surface sialylation was performed using Hydra and lectin reagents as shown in Table 10. Hydra-3, Hydra-7, and Hydra-9 are hexameric forms of the extracellular domains of human Siglec 3, Siglec 7, and Siglec 9, respectively (as described in International (PCT) Application Publication No. WO2019 / 237070). The lectins used included biotinylated elderberry (Sambucus Nigra) (SNA, Vector Laboratories, B-1305-2), biotinylated Machia Amurensis (MAL-II, Vector Laboratories, B-1265-1), and biotinylated peanut lectin (PNA, Vector Laboratories, B-1075-5). SNA is a lectin that preferentially binds to sialic acid attached to the terminal galactose residue via an α-2,6 linkage and, to a lesser extent, via an α-2,3 linkage. MAL-II is a lectin that binds to sialic acid attached via an α-2,3 linkage. PNA is a lectin that binds to the terminal galactose residue. Increased PNA staining can indicate the removal of terminal sialic acid by sialidase and the exposure of the underlying galactose.

[0351] Table 10

[0352] reagents Storage liquid Working concentration buffer solution Hydra-3 variable 250nM FACS staining buffer Hydra-7 variable 25nM FACS staining buffer Hydra-9 variable 75nM FACS staining buffer MAL-II 1mg / mL 2μg / mL PBS PNA 5mg / mL 1μg / mL FACS staining buffer SNA 2mg / mL 0.5 μg / mL FACS staining buffer

[0353] PBMCs were incubated on ice for 30 min with various Hydra and lectin reagents. Cells were washed in each well with 150 μL PBS (1% BSA) and centrifuged at 350 x g for 5 min. The solution in the plate was quickly decanted. AF-647 goat anti-mouse IgG antibody diluted 1 / 2000 in PBS was used as a second staining agent for Hydra reagents (Hydra-7 and Hydra-9). Streptavidin-conjugated Alexa Fluor 647 diluted 1 / 2000 in PBS was used as a second staining agent for lectin reagents (PNA, MAL-II, and SNA). Cells were incubated on ice for 15 min. Cell lineage-specific staining was performed using the specified antibodies as shown in Table 11. All antibodies except Live Dead staining reagent, which was purchased from Thermo Fisher Scientific (Waltham, MA), were purchased from [unspecified source]. (San Diego, CA)

[0354] Table 11

[0355]

[0356]

[0357] Prepare the master mixture (“staining mixture”) in FACS staining buffer using the reagents listed in Table 11, and add 30 μl aliquots of the staining mixture to each well / tube to achieve a final active antibody concentration of ~1 μg / ml. Incubate the cells on ice for 15 min. Prepare individual cell compensation controls. Wash the cells with PBS (1% BSA) and resuspend them in 4% paraformaldehyde at room temperature for 10 min. Wash the cells twice with PBS and resuspend the deposits in 150 μl of PBS. Perform flow cytometry using a BD FACSCelesta... TM (BD Biosciences) ran the sample.

[0358] Human PBMCs from two different healthy donors were stained with Hydra-3, Hydra-7, and Hydra-9, as depicted in Figure 14 (black and gray bars represent the two donors). As shown, monocyte and DC cell populations exhibited increased Hydra-9 staining compared to other cell populations. Figure 14A Compared to other cell populations, monocyte and dendritic cell populations showed increased Hydra-7 staining ( ). Figure 14B One donor showed increased Hydra-7 staining on CD4+ T cells. Monocyte and DC cell populations showed increased Hydra-3 staining compared to other cell populations. Figure 14C One donor showed increased Hydra-3 staining on CD4+ T cells.

[0359] The lectin staining (MAL-II, PNA, and SNA) of human PBMCs from healthy donors is depicted in Figure 15 (black and gray bars represent two independent donors). As shown, PNA staining is relatively low compared to Hydra-9 staining (see comparison of the Y-axis scale with Figure 14), but is specific for monocytes and dendritic cells (DCs). Figure 15A MAL-II staining showed that most immune cell populations ( Figure 15B Compared to other cell populations, T cells (CD4+ and CD8+) showed increased MAL-II staining. SNA staining was observed in most immune cell populations ( Figure 15C Among them, NK cells showed less SNA staining compared to other cell populations.

[0360] Example 6: Sialidase effectively desialylates dendritic cells (DCs).

[0361] This embodiment demonstrates the desialylation efficiency of the sialidase molecule of the present invention on dendritic cells (DCs) derived from human monocytes.

[0362] DCs are known to express high levels of Siglec (sialic acid-binding immunoglobulin-like lectins, such as Siglec-3, -7, and -9), which inhibit NK cell-mediated killing of tumor cells. Furthermore, DCs express large amounts of sialyl glycans, which, as demonstrated in the previous example, are ligands for Siglec molecules. The interaction of Siglecs on DCs with sialyl glycans on the same cell or on another interacting cell (e.g., cancer cells) regulates DC activation.

[0363] PBMCs were isolated from a single leukocyte sample (a blood sample rich in PBMCs) using the standard Ficoll density gradient method. After PBMC isolation, the cells were centrifuged at 350 x g for 5 minutes and then cooled with cold water. Wash twice with wash buffer (containing 5% BSA; Miltenyi Biotec). CD14+ monocytes were magnetically purified using CD14 microbeads (Miltenyi Biotec) and differentiated into dendritic cells. Specifically, CD14+ cells were purified using 0.8 x 10-1 microbeads. 6 Cells were resuspended at a concentration of 100 cells / mL in complete medium (RPMI medium containing 10% FBS) containing 50 ng / mL recombinant human GM-CSF and 50 ng / mL recombinant human IL-4. On day 0, cells were seeded in 6-well plates with 3 mL of cell suspension per well (2.4 x 10⁻⁶ cells / mL). 6 (cells / well). On days 3 and 6, remove half of the culture medium from each well, carefully without disturbing the loosely attached cells. Replenish each well with 1.5 mL of fresh culture medium containing 100 ng / mL rhGM-CSF and rhIL-4. On day 7, harvest differentiated DCs by gently rinsing them with culture medium, washing them once with complete culture medium, and then... 6 / mL resuspension.

[0364] For the desialylation assay, M106 (M1D, V6Y, P62G, A93E, I187K, C332A and human IgG1 Fc with the acetoside (Y407T) mutation and EPKSS (SEQ ID NO: 163) linker) (SEQ ID NO: 152, encoded by SEQ ID NO: 193) was used. This construct is as described in Example 2, but the EPKSS (SEQ ID NO: 163) linker is used instead of the GGGGSGGGGS (SEQ ID NO: 162) linker. The term "M106" as used herein refers to this construct. In addition, Neu2-FC variants known as LOF (M1D, V6Y, K9D, I187K, C332A, A93E, V363R, L365R, E218A, C219N, and human IgG1 Fc with the Y407T mutation (SEQ ID NO: 175, encoded by SEQ ID NO: 176)) were used as negative controls. 100,000 DCs were seeded per well in 96-well U-shaped plates, with 200 μl dispensed per well. LPS was used at the specified concentration of 0.3 ng / mL, and M106 and LOF constructs were used at the following concentrations (in μg / mL): 0, 6.25, 12.5, 25, 50, and 100. The DCs were incubated overnight (16 h) and then flow cytometry was performed for CD83, CD86, and MHCII (HLA-DR). Desialylation was measured by PNA staining as described in Example 5.

[0365] After incubation, the plate was centrifuged at 350x g for 4 minutes and the culture medium was removed. Cells were washed once with FACS staining buffer. Cells were then stained with 100 μl of human Trustain FcX (1 / 20 dilution) and LIVE / DEAD. TM Cells were immobilized and stained simultaneously with near-infrared dead cell staining agent (1 / 2000 dilution) in PBS solution and incubated on ice for 10 minutes. Cells were centrifuged and washed once with FACS buffer. 50 μL of PNA-Biotin (1 μg / mL, in FACS staining buffer) was added to each well and incubated on ice for 10 minutes. Cells were centrifuged and washed twice with FACS buffer. 50 μL of Alexa Fluor containing streptavidin was added to each well. TM The antibody mixture, including 647 (described in Table 12 below), was incubated on ice for 30 minutes. After incubation, the cells were washed twice with 150 μL of FACS buffer and resuspended in 125 μL of FACS buffer for flow cytometry acquisition. Flow cytometry data were analyzed on a BD FACSCelesta flow cytometer. TMData was acquired using the HTS (High-Throughput Sampler) option on the BD Biosciences (BDBiosciences) software. After acquisition, the signals were analyzed using FlowJo flow cytometry software (BD Biosciences).

[0366] Table 12

[0367]

[0368]

[0369] Figure 16 illustrates the degree of desialylation of DC by M106 based on PNA staining. Increased PNA staining indicates the removal of terminal sialic acid, exposing the underlying galactose residues recognized by PNA lectin. Figure 16A The results showed that as the concentration of M106 increased, the fluorescence (MFI) indicating PNA staining increased. Figure 16B The increase in PNA signal compared to untreated DC is shown as a fold. A significant dose-dependent increase in PNA signal was observed, indicating robust desialylation of DC.

[0370] Taken together, this embodiment demonstrates that M106 induces robust desialylation of DCs in a dose-dependent manner.

[0371] Example 7: Desialylation of tumor cell lines by sialidase

[0372] Sialotropic glycans play a role in maintaining tolerance and homeostasis under human physiological conditions. Overexpression of sialytropic glycans has been observed in tumor cell lines. This example demonstrates the ability of M106 to desialylate tumor cell lines BT-20, SKBR-3, and HT-29, as determined by Hydra-9 and lectin staining.

[0373] Use a suitable culture medium to grow BT-20 and HT-29 cells on a plate until 70-80% confluence. (Innovative Cell Technologies, Inc.) This enzyme mixture, containing proteolytic and collagenolytic enzymes, dissociates cells by incubating the plate at 37°C for 15 minutes. Upon cell dissociation, an equal volume of complete culture medium is added to neutralize the dissociation. Transfer the cell suspension and centrifuge at 300x g for 5 min. Discard the supernatant and wash the cells twice with cold PBS. Count the cells at 1x10⁻⁶. 6 Cells were resuspended at 100 cells / ml in culture medium. M106 and LOF were added to the cells at different dilutions. The cells were incubated at 37°C for 10 hours. After incubation, the cells were washed with PBS and transferred to 96-well round-bottom plates for staining. Staining was performed using Hydra-9 and PNA as described in Example 5.

[0374] Figure 17 illustrates the loss of Hydra 9 binding as measured by fluorescence (gMFI) after treatment with M106 (triangles) or the LOF control (squares). Figure 17A ) or increase in PNA staining ( Figure 17B The degree of desialylation of BT-20 cells was determined by M106. The IC50 for desialylation by M106 was 3.088 μg / mL for Hydra 9 and 58.75 μg / mL for SNA. Figure 18 depicts the loss of Hydra 9 binding (measured by fluorescence (gMFI)) after treatment with M106 (triangles) or LOF control (squares). Figure 18A ) or increase in PNA staining ( Figure 18B The degree of desialylation of BT-20 cells was determined by the Neu2-Fc variant M106. The IC50 for desialylation of Hydra 9 was 2.95 μg / mL, and for SNA it was 131.5 μg / mL.

[0375] Similar experiments were performed using SKBR-3 cells, in which cells were stained with MAL-II lectin in addition to Hydra 9 and PNA. For MAL-II staining, a final concentration of 2 μg / mL in PBS was used, and cells were stained at room temperature for 10 minutes. Figure 19 depicts the loss of Hydra 9 binding (measured by fluorescence) after treatment with M106 (triangles) or LOF control (circles). Figure 19A Loss of MAL-II staining Figure 19B ) or increase in PNA staining ( Figure 19C The degree of desialylation of SKBR-3 cells was determined by M106. The IC50 of desialylated SKBR-3 cells was 4.4 μg / mL for Hydra 9, approximately 120 μg / mL for MAL-II, and 22 μg / mL for SNA.

[0376] Taken together, this example demonstrates that M106 exhibits a dose-dependent removal of sialic acid from the cell surface of tumor cells. Loss of Hydra 9 staining is a more sensitive indicator than loss of MAL II staining or increase of PNA staining, with an EC50 of approximately 3 to 4 μg / mL for M106.

[0377] Example 8: Sialidase-induced desialylation of tumor cell lines enhances human dendritic cell activation.

[0378] Sialotropic glycans play a role in maintaining tolerance and homeostasis under human physiological conditions. Although overexpression of sialytropic glycans has been observed in tumor cell lines, as shown in the previous examples, the resulting sialytropic glycans can be removed using the sialylase of the present invention. This example demonstrates the effect of desialylation of tumor cell lines on dendritic cell activity.

[0379] In simple terms, dendritic cells (DCs) are generated from CD14+ monocytes isolated from PBMCs of healthy donors. The CD14+ cells are magnetically purified using the manufacturer's protocol (Miltenyi Cat#130-050-201). The purified cells are then cultured for 7 days in the presence of GM-CSF (R&D Systems Cat#7954-GM / CF) and IL-4 (R&D Systems Cat#6507-IL / CF) to produce immature DCs.

[0380] On the day of the experiment, using SKBR-3 tumor cells were harvested from T-75 culture flasks and washed twice with 10% FBS McCoy's 5A medium. The cells were then cultured at 5 x 10⁻⁶ cells / year. 6 The cells were resuspended in 10% FBS McCoy's 5A medium at 1 mL / mL. 100 μg / mL M106 was added to the sample, and the cells were incubated at 37°C for 4 hours. The untreated group underwent the same treatment except for the addition of M106. After 4 hours, the cells were washed twice with 10% FBS McCoy's 5A medium and incubated at 2 x 10 mL / mL. 6 Resuspend / mL in complete medium (10% FBS RPMI). Add 50 μl of suspension (100,000 DC) to the designated well.

[0381] Harvest the DCs, wash them in complete medium (10% FBS RPMI) and use 2x10 6 / ml resuspension. Add 50 μL of suspension (100,000 DC) to the specified well.

[0382] Add LPS (InvivoGen Cat#tlrl-pb5lps) to a final concentration of 0.3 ng / mL. Add complete culture medium (10% FBS RPMI) to a final volume of 200 μL per well. Incubate the assay plate overnight at 37°C. The next day, wash the cells with staining buffer and stain 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.

[0383] Figure 20 depicts the expression via CD83hi ( Figure 20A ) or CD86hi expression ( Figure 20B The effects of LPS on dendritic cell activation under various conditions were determined. Untreated DCs (“No Tx”) had low percentages of CD83hi and CD86hi. Addition of LPS to DCs strongly induced activation, as shown by the increased percentages of CD83h and CD86hi (“LPS”). When DCs were co-incubated with untreated SKBR-3 tumor cells, LPS-induced expression of both CD83 and CD86 was suppressed (see [link to study]). Figure 20A and 20B (Horizontal line in the image). Prior to co-incubation with DCs and LPS, the inhibition of DCs by SKBR-3 tumor cells was reversed after desialylation with M106 (“LPS+M106 FC”). Furthermore, sialidase treatment in the absence of LPS slightly enhanced DC activation (comparison of untreated and untreated SKBR-3 tumor cells with M106-treated SKBR-3 tumor cells (“M106FC”)).

[0384] This embodiment demonstrates that desialylation of tumor cells can reverse the immunosuppression of dendritic cells induced by sialyl polysaccharides, indicating that desialylation of tumor cells can elicit a stronger anti-tumor response.

[0385] Example 9 The effect of sialidase on the phagocytic activity of macrophages against tumor cells

[0386] Sialidin, present on the surface of immune cells, plays an important role in maintaining homeostasis. This embodiment demonstrates the effect of the sialidase of the present invention on the phagocytosis of HT-29 tumor cells by M2-like human macrophages.

[0387] PBMCs were isolated from whole blood of human volunteers using the Ficoll method. CD14+ monocytes were magnetically purified using CD14 microbeads. The CD14+ cells were then purified by scattering the cells at 1x10⁻⁶ microbeads. 6 The cells were resuspended at a concentration of 50 ng / mL in RPMI medium (10% FBS) containing recombinant human M-CSF, and monocytes were differentiated into M2-like macrophages. On day 0, cells were seeded at a volume of 20 mL in 150 mm tissue culture plates (20 x 10⁹ cells per plate). 6 (cells). On days 3 and 6, remove half of the culture medium from each well, carefully without disturbing the attached cells. Replenish M-CSF to a final concentration of 50 ng / mL. On day 7, collect the culture supernatant in a 50 mL tube and gently wash the plate with 20 mL of PBS. Add 20 mL of PBS. The plate was incubated for 20 minutes to detach the cells. The cells were then resuspended in complete RPMI medium supplemented with 10% FBS and non-essential amino acids (NEAA), sodium pyruvate, and HEPES containing 10 ng / ml M-CSF, and seeded in 100 μL of the medium in flat-bottomed 96-well plates at a rate of 50 cells / well / 100 μL.

[0388] use Harvest HT-29 cells from the culture flasks. Wash the cells with PBS. Dilute the cells with Cell Trace at a volume ratio of 1:1000. TM CFSE-labeled dye (FITC) conjugate (Thermo Fisher) was used to label cells (final concentration 10 μM). Cells were incubated at room temperature for 10 min, and the labeling reaction was quenched by adding an equal volume of chilled FBS. Cells were washed twice and inoculated with 1.2 x 10⁻⁶ cells / mL. 6 Cells / ml were resuspended in culture medium (McCoy's medium supplemented with 10% FBS). M106 and LOF were added at a maximum concentration of 100 μg / ml, followed by 2-fold dilutions. An untreated control group using untreated HT-29 cells was retained. Cells were incubated at 37°C for approximately 20 hours.

[0389] After incubation, the cells were centrifuged, washed with PBS, and then... 6 The cells were resuspended at a final cell density of 10 cells / mL in complete RPMI (10% FBS) medium. 100 μL of HT-29 cell suspension was added to suitable wells of M2-like macrophages at a macrophage:tumor cell ratio of 1:5 (E:T). The plates containing macrophages and tumor cells were incubated for 2 hours to allow phagocytosis. After 2 hours, the medium was gently removed using a multichannel pipette, and 200 μL of the medium was added to the plate. Incubate on ice for 45 minutes to dissociate both HT-29 cells and macrophages from the plate. Resuspend the cells and collect them in new 96-well plates. Centrifuge the plates, discard the supernatant, and wash the cell deposits in 200 μL of PBS.

[0390] The cell deposits were resuspended and blocked on ice for 5–7 minutes using human Trustain Fc inhibitor. After incubation, the cells were washed with PBS. Cells were stained with CD45 and CD14 fluorescent dyes as described in Table 13 below. Antibodies were purchased from [source missing]. .

[0391] Table 13

[0392] markers Fluorescent dyes clone Catalog Number Markers are used for CD14 BV421 MSE2 301830 macrophages CD45 APC 2D1 368512 macrophages

[0393] Prepare the master mixture in FACS staining buffer, adding staining antibodies at a 1:30 dilution. Add 30 μl of the master mixture to each well. Stain in parallel with a suitable compensation control (e.g., a single-color staining control for compensation according to standard flow cytometry practice for multicolor flow cytometry). Incubate cells on ice for 15 min, then wash with PBS and centrifuge at 350 g for 8 min. Then fix cells with 4% formaldehyde at room temperature for 10 min, then wash twice with PBS. Resuspend cells in 150 μL of PBS and run on a flow cytometer (BD FACSCelesta). TM It runs on (BD Biosciences).

[0394] The percentage of CFSE-positive, CD14+CD45+ macrophages was determined. CFSE-positive, CD14+CD45+ macrophages indicate the percentage of tumor cells phagocytosed by macrophages, because CFSE-positive tumor cells phagocytosed by CD14+CD45+ macrophages are CFSE-positive.

[0395] Figure 21 illustrates the dose-dependent enhancement of phagocytosis of desialylated HT-29 tumor cells by M2-like macrophages derived from two different healthy donors. Figure 21A and Figure 21B Pretreatment of HT-29 cells with sialidase at concentrations higher than 25 μg / mL showed reproducible enhancement in macrophage phagocytosis. Similar enhancements in the phagocytosis of desialylated BT20 and SKBR-3 tumor cells by M2-like macrophages were observed (respectively...). Figure 21C and Figure 21D ).

[0396] Therefore, treatment of tumor cells with the sialidase described herein leads to an increase in the phagocytic activity of macrophages on the tumor cells.

[0397] Example 10: Sialidase treatment enhances type II MHC expression on monocytes

[0398] This embodiment demonstrates the effect of the sialidase of the present invention on class II MHC (HLA-DR) expression on monocytes. MHC-II expression represents the antigen-presenting capacity of monocytes. Enhanced class II expression indicates an increased likelihood of antigen presentation to T cells to generate an effective immune response.

[0399] PBMCs were isolated from healthy volunteers using the Ficoll method and centrifuged at 350 x g for 10 minutes using a benchtop centrifuge. The cells were then washed twice with ice-cold PBS. The cells were resuspended in culture medium and washed with Countess... TM Count the cells using an automated cell counter (II). Adjust the final suspension to 2.5 x 10⁻⁶. 6Cells / L. Approximately 250,000 cells (100 μL) were seeded in 96-well round-bottom plates. Cells were incubated with M106 or LOF at a maximum concentration of 50 μg / mL, followed by a 2-fold dilution. An untreated group was included. Cells were incubated at 37°C for 18 hours. The plates were centrifuged at 350 x g for 10 minutes. Cell deposits were washed with cold PBS and blocked and stained using the FACS staining group described in Table 14. All antibodies except for LiveDead staining reagent purchased from Thermo Fisher were purchased from [unspecified source]. Sialotropic staining was performed using PNA lectin, confirmed by desialylation as described in Example 7.

[0400] Table 14

[0401]

[0402]

[0403] Figure 22 illustrates the dose-dependent enhancement of HLA-DR expression after M106 desialylation in monocytes from two different healthy donors compared to LOF. Figure 22A and Figure 22B ).

[0404] Therefore, this embodiment demonstrates that the desialylation of monocytes by the sialidase described herein leads to increased expression of class II MHC (HLA-DR) on monocytes. MHC-II expression represents the antigen-presenting capacity on monocytes; therefore, enhanced class II expression indicates enhanced antigen presentation to T cells, which can enhance the ability of T cells to generate an effective immune response.

[0405] Example 11: Sialidase treatment does not induce adverse cytokine release.

[0406] The stimulation of cytokine release by modulated medium derived from PBMCs incubated with M106 or LOF was measured. LPS (1 ng / mL) was used as a positive control. When measured via LEGENDplex... TM Human M1 / M2 macrophage group (10-plex); During measurements, in both independent donors, PBMC treatment with M106 (and LOF) at all treatment doses did not show an increase in TNF-α, IL-6, IL-1β, IL-1RA, or IL-10. Conversely, LPS showed significant cytokine induction. These results confirm that sialidase treatment of PBMCs does not induce adverse cytokine release.

[0407] Example 12: Sialidase treatment alone and in combination with anti-PD-1 antibody led to complete and partial reduction of tumor growth. Partial relief

[0408] This embodiment 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.

[0409] Sialidase treatment, both alone and in combination with other cancer therapies, was tested using an MC38 colon cancer cell model. Each mouse was treated with 5 x 10 μL of sialidase in 0.1 mL PBS in the right lower flank region. 5 Subcutaneous inoculation of tumor cells was performed to induce tumorigenesis. When the average tumor size reached approximately 50 mm... 3 Mice were randomly assigned to four study groups. Thirty-two mice were randomly assigned to four groups. Each mouse was administered M106, anti-mouse PD-1 antibody, a combination of Neu2-Fc variant M106 and anti-PD-1 antibody, or an isotype control at a dose of 10 mg / kg for five weeks. Figure 23 depicts the results in the isotype control group (…). Figure 23A M106 group ( Figure 23B ), anti-PD-1 antibody group ( Figure 23C ) or the combination of M106 and anti-PD-1 antibody ( Figure 23D Tumor growth in each mouse was measured. M106-treated mice showed complete remission (CR) of tumor growth in one animal, compared to no response in the isotype-treated group. The combination of M106 and anti-PD-1 antibody showed 1 CR and 1 partial response (PR), and a comprehensive reduction in tumor growth in all mice compared to the isotype control.

[0410] Next, sialidase treatment, both alone and in combination with other cancer therapies, was tested using a B16F10 melanoma cancer cell model. Each mouse was treated with 5 x 10 μL of sialidase in 0.1 mL PBS in the right lower flank region. 5 Subcutaneous inoculation of tumor cells was performed to induce tumorigenesis. When the average tumor size reached approximately 50 mm... 3 Mice were randomly assigned to three study groups. Twenty-four mice were randomly assigned to three groups. Mice were administered M106 (10 mg / kg), anti-mouse PD-1 antibody, or an isotype control twice weekly for a total of five doses. Figure 24 depicts the results in the isotype control group (…). Figure 24A M106 group ( Figure 24B or anti-PD-1 group ( Figure 24C Tumor growth in each mouse in the study. Figure 24D This is an overlay plot of the isotype control group on the M106 group, confirming the significant benefit of M106 in reducing tumor growth in a tumor model that is considered difficult to treat.

[0411] Next, sialidase treatment, both alone and in combination with other cancer therapies, was tested using the EMT6 cell line, a polyclonal cell line expressing human Her2. Each mouse was treated with 5 x 10 μL of sialidase in 0.1 mL PBS in the right lower flank region. 5 Subcutaneous inoculation of tumor cells was performed to induce tumorigenesis. When the average tumor size reached approximately 100 mm... 3 Mice were randomly assigned to two study groups. Sixteen mice were randomly assigned to two study groups. Mice were administered M106 at 10 mg / kg twice weekly for a total of five doses, or an isotype control. Figure 25 depicts the results in the isotype control group (…). Figure 25A ) or M106 FC group ( Figure 25B Tumor growth in each mouse in the M106 group was measured. Four out of eight mice treated with M106 showed complete remission (CR) of tumor growth, compared to only one out of eight mice in the same type of treatment group.

[0412] Therefore, as demonstrated in this embodiment, treatment with the sialidase disclosed herein leads to a reduction in cancer growth and, in some cases, complete remission in various different cancer types.

[0413] Example 13: Sialidase treatment, alone or in combination with anti-PD-L1 antibodies, leads to complete reduction of tumor growth. and partial relief

[0414] This example describes an in vivo assay of M106 and / or avermab (anti-PD-L1 antibody) in an A20 syngeneic mouse model. Mouse A20 cells express endogenous mouse PD-L1, which is bound by avermab. Five- to six-week-old female Balb / c mice were subcutaneously inoculated into the right lower flank region with mouse A20 B-cell lymphoma cells in matrix gel (1:1 volume ratio). When the tumor reached approximately 100 mm... 3 (The average tumor volume in each group was between 86 and 90 mm) 3 When the mice were within the range of [specific parameters], they were randomly assigned to groups of 8 mice. Table 15 describes the various arms of the study. Mice were treated intraperitoneally twice weekly with 5 or 10 mg / kg M106, avermab, and / or an antibody isotype control (as indicated) for a total of 5 doses. Tumor volume and body weight were recorded three times weekly.

[0415] Table 15

[0416]

[0417] Figure 26 Tumor growth was depicted for each mouse in each group. Complete responders (CR) and partial responders (PR) for each group are shown. As can be seen, M106, alone and in combination with acitumab (“Ave”), exhibited antitumor activity.

[0418] Mice with tumors that showed complete remission (CR) after M106 treatment (alone or in combination with acitumab) were re-irritated with mouse A20 cells (all approximately 12 weeks old) and compared with naive control mice injected with A20 cells at 6 or 12 weeks of age. Tumor volume and body weight were recorded three times weekly. As expected, tumors grew in both 6- and 12-week-old naive mice, but no tumor growth was observed in the re-irritated mice (data not shown).

[0419] Therefore, as demonstrated in this embodiment, in a B-cell lymphoma model, treatment with the sialidase disclosed herein resulted in a reduction in tumor growth and, in some cases, complete remission.

[0420] Example 14: Sialidase treatment alone or in combination with anti-PD-L1 antibodies leads to complete reduction of tumor growth and Partial relief

[0421] This example describes the in vivo testing of M106 and / or avermab (anti-PD-L1 antibody) in an A20 syngeneic mouse model. The experiment was performed as described in Example 13, except that six doses were administered (twice weekly for three weeks). Table 16 describes the various arms of the study. Mice were treated intraperitoneally twice weekly with 10 mg / kg M106, avermab, and / or an antibody isotype control for six weeks. Tumor volume and body weight were recorded three times weekly.

[0422] Table 16

[0423]

[0424] Figure 27 Tumor growth outcomes for each mouse in each group were depicted. ASCs based on avermab exhibited varying degrees of potency. As in Example 13, M106 exhibited the same activity as the combination of M106 and avermab.

[0425] Therefore, as demonstrated in this embodiment, in B-cell lymphoma models, treatment with the sialidase disclosed herein, alone or in combination with anti-PD-L1 antibodies, resulted in a reduction in tumor growth and, in some cases, complete remission.

[0426] Example 15: Sialidase treatment in combination with anti-CD20 antibody led to improved survival in mice with tumors. high

[0427] This example describes the in vivo administration of M106 in combination with an anti-CD20 antibody (oframumab) in a mouse syngeneic intravenous dispersal model using a mouse mammary cancer cell line expressing human CD20 (EL4 CD20 cells). Six- to eight-week-old female C57 / BL6 mice were IV-injected with 500,000 cells per mouse. Subsequently, mice were administered isotype control, oframumab, or a combination of oframumab and M106 as described in Table 17. Body weight and clinical observations were recorded daily.

[0428] Table 17

[0429]

[0430] Figure 28 depicts the survival curves of mice in each group. Figure 28A The survival rate up to day 28 was depicted. Figure 28B Overall survival (up to day 41) was depicted. Mice treated with ofamumumab showed survival migration compared to isotype controls, with the 50% survival point shifting from day 17 to day 24. Mice treated with a combination of ofamumumab and M106 showed even greater survival migration, shifting to day 30.

[0431] Therefore, this embodiment shows that in mice treated with anti-CD20 antibodies, treatment with the sialidase of the present invention leads to an increased survival rate.

[0432] Example 16: Sialidase treatment disrupts Siglec-15 activity on T cells

[0433] Siglec-15 is an important immunosuppressant. Under normal conditions, Siglec-15 is expressed only on certain myeloid cells, but it is widely upregulated in human cancer cells and tumor-infiltrating myeloid cells. Siglec-15 acts as a ligand and inhibits antigen-specific T cell responses in vitro and in vivo. Genetic ablation or antibody blockade of Siglec-15 enhances anti-tumor immunity in the tumor microenvironment (TME) and inhibits tumor growth in certain mouse models.

[0434] This embodiment demonstrates that neuraminidase treatment removes the Siglec-15 ligand, thereby disrupting Siglec-15 binding activity. It is believed that disruption of Siglec-15 binding activity in vivo will lead to increased antitumor immunity in the tumor microenvironment (TME) and inhibition of tumor growth.

[0435] Human PBMCs were thawed and stimulated with 1 μg / mL anti-CD3 (OKT3 clone) and anti-CD28 (clone CD28.2) antibodies (both from eBiosciences, Thermo Fisher Scientific) in complete RPMI medium (supplemented with 10% heat-inactivated FBS, non-essential amino acids, and sodium pyruvate) to a final concentration of 1 μg / mL. On day 2, floating cells were collected and replated in fresh complete RPMI medium, supplemented with 1 μg / mL anti-CD3 and anti-CD28 antibodies to continue cell stimulation. Three days later, cells were cultured at 10... 6 The inoculum was re-inoculated at a density of 1 / ml in 15 mL conical tubes and treated with the following different groups as described below: (1) no treatment; (2) a defunctionalized sialidase (“LOF FC”, as described in the preceding examples) at a final concentration of 50 μg / mL; (3) M106 at a final concentration of 50 μg / mL; and (4) BiNanH2 at a final concentration of 2 μg / mL. BiNanH2 is a potent sialidase derived from Bifidobacterium infantis and was used as a positive control.

[0436] Cells were added with anti-CD3 and anti-CD28 antibodies and incubated overnight at 37°C. The next day (approximately 14 hours later), the cells were centrifuged, the culture medium was removed, and then the cells were treated with human TruStain FcX Fc receptor blocker in PBS. ) and LIVE / DEAD TM Near-infrared staining agents can be used to block near-infrared dead cells. The cells are then blocked with heat-inactivated human serum (5%, in PBS).

[0437] Cells were stained with human Siglec-15-Fc (prepared by Palleon Pharmaceuticals; MW: ~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.

[0438] Next, the cells were stained with anti-human Fc-AF647 antibody in FACS staining buffer. The cells were incubated on ice for 5 minutes and then washed.

[0439] Then, as described in earlier embodiments, the cells were stained with CD4 and CD8 markers in FACS staining buffer. The cells were incubated on ice for 15 minutes and then washed. The cells were fixed and analyzed by flow cytometry (BDFACSCelesta). TM Run and analyze the data on (BD Biosciences).

[0440] Figure 29 depicts CD4+ cells after various treatments. Figure 29A ) and CD8+ cells ( Figure 29B Siglec-15-Fc staining results were shown. Allotype IgG1 staining as a control is also shown. As indicated, treatment with M106 FC or BiNaNH2 (positive control) reduced Siglec-15-Fc staining in activated CD4 and CD8 cells compared to untreated or LOF FC-treated cells. Figure 30 depicts CD4+ cells (CD4+ cells) using PBMCs from a second healthy donor. Figure 30A ) and CD8+ cells ( Figure 30B The results of Siglec-15-Fc staining were obtained. These results confirmed that the binding of Siglec-15 to activated T cells is sialic acid-dependent, and that this interaction is disrupted by the removal of sialic acid by neuraminidase.

[0441] Therefore, this embodiment demonstrates that neuraminidase treatment with the sialidase of the present invention removes the Siglec-15 ligand, thereby disrupting the binding activity of Siglec-15. It is believed that disruption of Siglec-15 binding activity in vivo will lead to increased antitumor immunity in the tumor microenvironment (TME) and inhibition of tumor growth.

[0442] By incorporating via reference

[0443] The full contents of every patent and scientific document mentioned in this article are incorporated herein by reference for all purposes.

[0444] Equivalence

[0445] This invention may be embodied in other specific ways without departing from its spirit or essential characteristics. Therefore, the above embodiments should be considered illustrative in all cases, and not as limiting of the invention described herein. Consequently, the scope of the invention is defined by the appended claims rather than by the foregoing description, and is intended to be encompassed by all variations within the equivalent meaning and scope of the claims.

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[0485]

[0486]

[0487]

[0488]

[0489]

[0490]

[0491]

Claims

1. A pharmaceutical composition comprising a sialidase conjugate, said sialidase conjugate comprising an Fc domain coupled to an sialidase that increases the serum half-life of the sialidase upon administration to a subject, wherein said sialidase is not linked to a target component, wherein said amino acid sequence of said sialidase is SEQ ID NO: 48, and The amino acid sequence of the sialidase conjugate is SEQ ID NO:

152.

2. The pharmaceutical composition of claim 1, wherein the sialidase and the Fc domain are covalently linked together in the fusion protein.

3. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition comprises a second therapeutic agent.

4. The pharmaceutical composition of claim 3, wherein the second therapeutic agent is selected from anti-inflammatory agents, anti-angiogenic agents, anti-fibrotic agents, and anti-proliferative compounds.

5. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition further comprises a stabilizing amount of sialidase stabilizer.

6. The pharmaceutical composition of claim 5, wherein the sialidase stabilizer is cationic.

7. The pharmaceutical composition of claim 6, wherein the cation is selected from calcium and magnesium.

8. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is disposed in a sterile container.

9. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition is freeze-dried in the sterile container.

10. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition is present as a solution in the sterile container.

11. The pharmaceutical composition of claim 8, wherein the sterile container is sealed with a diaphragm.

12. The pharmaceutical composition of claim 8, wherein the sterile container has a label disposed thereon identifying the pharmaceutical composition contained therein.

13. Use of sialidase in the preparation of a medicament for treating sialic acid-related disorders in a subject in need, wherein the sialidase is coupled to an Fc domain that increases the serum half-life of the sialidase upon administration to the subject to form a sialidase conjugate, wherein the sialidase is not linked to a target component, wherein the amino acid sequence of the sialidase is SEQ ID NO: 48, and The amino acid sequence of the sialidase conjugate is SEQ ID NO:

152. The sialic acid-related disorders mentioned above are B-cell lymphoma, melanoma, breast cancer, or colon cancer.

14. The use as claimed in claim 13, wherein the sialidase and the Fc domain are covalently linked together in the fusion protein.

15. The use as claimed in claim 13, wherein administration of the sialidase conjugate increases the expression of granzyme B, IFNγ, IL-10, IL-6, or IL-17A in the subject.

16. The use as described in claim 13, wherein the sialidase conjugate is administered to the subject in combination with another therapeutic agent.

17. The use as described in claim 16, wherein the therapeutic agent is selected from anti-inflammatory agents, anti-angiogenic agents, anti-fibrotic agents, and anti-proliferative compounds.

18. The use as described in claim 16, wherein the therapeutic agent is an anti-CD20 antibody.

19. The use as described in claim 18, wherein the anti-CD20 antibody is rituximab.

20. The use as claimed in claim 18, wherein the sialidase conjugate and the anti-CD20 antibody are simultaneously administered to the subject.

21. The use as described in claim 13, wherein the sialidase conjugate is formulated in a pharmaceutical composition further comprising a stabilizing amount of a sialidase stabilizer.

22. The use as claimed in claim 21, wherein the sialidase stabilizer is cationic.

23. The use as described in claim 22, wherein the cation is selected from calcium and magnesium.

24. The use as claimed in claim 21, wherein the pharmaceutical composition is placed in a sterile container prior to administration.

25. Use of the pharmaceutical composition according to any one of claims 1-12 in the preparation of a medicament for treating cancer in a subject. The cancers mentioned are B-cell lymphoma, melanoma, breast cancer, or colon cancer.

26. The use as claimed in claim 25, wherein the pharmaceutical composition is administered to the subject in combination with the anti-CD20 antibody.

27. The use as described in claim 26, wherein the anti-CD20 antibody is rituximab.

28. The use as claimed in claim 26, wherein the pharmaceutical composition and the anti-CD20 antibody are administered simultaneously to the subject.

29. The use as claimed in claim 26, wherein the pharmaceutical composition is administered after the subject has been given the anti-CD20 antibody, or the anti-CD20 antibody is administered after the subject has been given the pharmaceutical composition.

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