Recombinant human sialidase, sialidase fusion proteins and methods of use thereof

Abstract

The present invention generally relates to recombinant human sialidases and recombinant sialidase fusion proteins, wherein the sialidase optionally contains one or more mutations, such as at least one amino acid substitution, deletion, or addition, compared to wild-type human sialidase. The present invention also provides antibody conjugates comprising a sialidase and an antibody or a portion thereof. The present invention further relates to methods of using the sialidase fusion proteins or antibody conjugates to treat cancer.

Description

[Technical Field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 870,403, filed July 3, 2019, and U.S. Provisional Patent Application No. 62 / 957,011, filed January 3, 2020, the entire disclosures of each of which are incorporated herein by reference in their entirety.

[0002] FIELD OF THE INVENTION The present invention relates generally to recombinant human sialidases and recombinant sialidase fusion proteins and their use in the treatment of cancer. [Background technology]

[0003] background A growing body of evidence supports the role of glycans, and particularly sialoglycans, in various pathophysiological stages of tumor progression. Glycans regulate tumor growth, invasion, hematogenous metastasis, and angiogenesis (Fuster et al. (2005) NAT. REV. CANCER 5(7): 526-42). Sialylation of cell surface glycoconjugates is frequently altered in cancer, resulting in the expression of sialylated tumor-associated carbohydrate antigens. The expression of sialylated glycans by tumor cells is often associated with increased tumor aggressiveness and metastatic potential.

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

[0005] 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 advances 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 treat cancers associated with hypersialylated cancer cells. Summary of the Invention

[0006] Summary of the Invention The present invention is based, in part, on the discovery that it is possible to produce recombinant mutant forms of human sialidase enzymes and fusion proteins and / or antibody conjugates comprising such enzymes with suitable substrate specificity and activity that are useful 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.

[0007] Thus, in one aspect, the present invention provides recombinant mutant human sialidase enzymes, wherein the sialidase comprises: (a) a substitution of a proline residue at a position corresponding to position 5 of wild-type human Neu2 (P5); (b) a substitution of a lysine residue at a position corresponding to position 9 of wild-type human Neu2 (K9); (c) a substitution of a lysine residue at a position corresponding to position 44 of wild-type human Neu2 (K44); (d) a substitution of a lysine residue at a position corresponding to position 45 of wild-type human Neu2 (K45); (e) a substitution of a leucine residue at a position corresponding to position 54 of wild-type human Neu2 (L54). (f) substitution of a proline residue at position 62 of wild-type human Neu2 (P62); (g) substitution of a glutamine residue at position 69 of wild-type human Neu2 (Q69); (h) substitution of an arginine residue at position 78 of wild-type human Neu2 (R78); (i) substitution of an aspartic acid residue at position 80 of wild-type human Neu2 (D80); (j) substitution of an alanine residue at position 93 of wild-type human Neu2 (A93); (k) substitution of a glycine residue at position 107 of wild-type human Neu2 (l) substitution of a glutamine residue at position 108 of wild-type human Neu2 (Q108); (m) substitution of a glutamine residue at position 112 of wild-type human Neu2 (Q112); (n) substitution of a cysteine ​​residue at position 125 of wild-type human Neu2 (C125); (o) substitution of a glutamine residue at position 126 of wild-type human Neu2 (Q126); (p) substitution of an alanine residue at position 150 of wild-type human Neu2 (A150); (q) substitution of an alanine residue at position 16 of wild-type human Neu2 (r) substitution of an arginine residue at position 170 of wild-type human Neu2 (R170); (s) substitution of an alanine residue at position 171 of wild-type human Neu2 (A171); (t) substitution of a glutamine residue at position 188 of wild-type human Neu2 (Q188); (u) substitution of an arginine residue at position 189 of wild-type human Neu2 (R189); (v) substitution of an alanine residue at position 213 of wild-type human Neu2 (A213);(w) substitution of a leucine residue at position 217 of wild-type human Neu2 (L217); (x) substitution of a glutamic acid residue at position 225 of wild-type human Neu2 (E225); (y) substitution of a histidine residue at position 239 of wild-type human Neu2 (H239); (z) substitution of a leucine residue at position 240 of wild-type human Neu2 (L240); (aa) substitution of an arginine residue at position 241 of wild-type human Neu2 (R24 1); (bb) substitution of an alanine residue at position corresponding to position 242 of wild-type human Neu2 (A242); (cc) substitution of a valine residue at position corresponding to position 244 of wild-type human Neu2 (V244); (dd) substitution of a threonine residue at position corresponding to position 249 of wild-type human Neu2 (T249); (ee) substitution of an aspartic acid residue at position corresponding to position 251 of wild-type human Neu2 (D251); (ff) substitution of a glutamic acid residue at position corresponding to position 257 of wild-type human Neu2 (gg) substitution of a serine residue at position 258 of wild-type human Neu2 (S258); (hh) substitution of a leucine residue at position 260 of wild-type human Neu2 (L260); (ii) substitution of a valine residue at position 265 of wild-type human Neu2 (V265); (jj) substitution of a glutamine residue at position 270 of wild-type human Neu2 (Q270); (kk) substitution of a tryptophan residue at position 292 of wild-type human Neu2 (Ill) a substitution of a serine residue at position 301 of wild-type human Neu2 (S301); (mm) a substitution of a tryptophan residue at position 302 of wild-type human Neu2 (W302); (nn) a substitution of a valine residue at position 363 of wild-type human Neu2 (V363); or (oo) a substitution of a leucine residue at position 365 of wild-type human Neu2 (L365); or any combination of the foregoing substitutions. For example, the sialidase can include substitutions of K9, P62, A93, Q216, A242, Q270, S301, W302, V363, or L365, or any combination of the foregoing substitutions.

[0008] In some embodiments, in the sialidase: (a) the proline residue at the position corresponding to position 5 of wild-type human Neu2 is substituted with histidine (P5H); (b) the lysine residue at the position corresponding to position 9 of wild-type human Neu2 is substituted with aspartic acid (K9D); (c) the lysine residue at the position corresponding to position 44 of wild-type human Neu2 is substituted with arginine (K44R) or glutamic acid (K44E); (d) the lysine residue at the position corresponding to position 45 of wild-type human Neu2 is substituted with alanine (K45A), arginine (K45R), or glutamic acid (K45E). (e) the leucine residue at position 54 of wild-type human Neu2 is substituted with methionine (L54M); (f) the proline residue at position 62 of wild-type human Neu2 is substituted with asparagine (P62N), aspartic acid (P62D), histidine (P62H), glutamic acid (P62E), glycine (P62G), serine (P62S), or threonine (P62T); (g) the glutamine residue at position 69 of wild-type human Neu2 is substituted with histidine (Q69H); (h) the leucine residue at position 7 of wild-type human Neu2 is substituted with methionine (L54M); The arginine residue at position 8 was substituted with lysine (R78K); (i) the aspartic acid residue at position 80 of wild-type human Neu2 was substituted with proline (D80P); (j) the alanine residue at position 93 of wild-type human Neu2 was substituted with glutamic acid (A93E) or lysine (A93K); (k) the glycine residue at position 107 of wild-type human Neu2 was substituted with aspartic acid (G107D); (l) the glutamine residue at position 108 of wild-type human Neu2 was substituted with histidine (Q108H) (m) the glutamine residue at position corresponding to position 112 of wild-type human Neu2 is substituted with arginine (Q112R) or lysine (Q112K); (n) the cysteine ​​residue at position corresponding to position 125 of wild-type human Neu2 is substituted with leucine (C125L); (o) the glutamine residue at position corresponding to position 126 of wild-type human Neu2 is substituted with leucine (Q126L), glutamic acid (Q126E), phenylalanine (Q126F), histidine (Q126H), isoleucine (Q126I), or tyrosine (Q126Y);(p) the alanine residue at position 150 of wild-type human Neu2 is substituted with valine (A150V); (q) the cysteine ​​residue at position 164 of wild-type human Neu2 is substituted with glycine (C164G); (r) the arginine residue at position 170 of wild-type human Neu2 is substituted with proline (R170P); (s) the alanine residue at position 171 of wild-type human Neu2 is substituted with glycine (A171G); (t) the alanine residue at position 171 of wild-type human Neu2 is substituted with valine (A150V); (u) the glutamine residue at position 188 of wild-type human Neu2 is substituted with proline (Q188P); (v) the alanine residue at position 213 of wild-type human Neu2 is substituted with cysteine ​​(A213C), asparagine (A213N), serine (A213S), or threonine (A213T); (w) the leucine residue at position 217 of wild-type human Neu2 is substituted with proline (R189P); (x) the threonine residue at position 249 of wild-type human Neu2 is substituted with alanine (T249A); (y) the aspartic acid residue at position 251 of wild-type human Neu2 is substituted with glycine (D251G); (z) the glutamic acid residue at position 225 of wild-type human Neu2 is substituted with proline (E225P); (aa) the glutamic acid residue at position 239 of wild-type human Neu2 is substituted with alanine (T249A); the histidine residue is substituted with proline (H239P); (bb) the leucine residue at position corresponding to position 240 of wild-type human Neu2 is substituted with aspartic acid (L240D), asparagine (L240N), or tyrosine (L240Y); (cc) the arginine residue at position corresponding to position 241 of wild-type human Neu2 is substituted with alanine (R241A), aspartic acid (R241D), leucine (R241L), glutamine (R241Q), or tyrosine (R241Y);(dd) the alanine residue at position 242 of wild-type human Neu2 is substituted 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); (ee) wild-type human Neu2 (ff) the valine residue at position 244 of u2 is substituted with isoleucine (V244I), lysine (V244K), or proline (V244P); (ff) the glutamic acid residue at position 257 of wild-type human Neu2 is substituted with proline (E257P); (gg) the serine residue at position 258 is substituted with cysteine ​​(S258C); (hh) the leucine residue at position 260 of wild-type human Neu2 is substituted with aspartic acid (L260D), phenylalanine (L260F), glutamine (L260Q), or threonine. (ii) the valine residue at position 265 of wild-type human Neu2 is substituted with phenylalanine (V265F); (jj) the glutamine residue at position 270 of wild-type human Neu2 is substituted with alanine (Q270A), histidine (Q270H), phenylalanine (Q270F), proline (Q270P), serine (Q270S), or threonine (Q270T); (kk) the tryptophan residue at position 292 of wild-type human Neu2 is substituted with arginine (W292R); (ll) the serine residue at the position corresponding to position 301 of wild-type human Neu2 is replaced with alanine (S301A), aspartic acid (S301D), glutamic acid (S301E), phenylalanine (S301F), histidine (S301H), lysine (S301K), leucine (S301L), methionine (S301M), asparagine (S301N), proline (S301P), glutamine (S301Q), arginine (S301R), threonine (S301T), valine (S301V), tryptophan (S301W), or tyrosine (S301Y);(mm) The tryptophan residue at position 302 of wild-type human Neu2 is alanine (W302A), aspartic acid (W302D), phenylalanine (W302F), glycine (W302G), histidine (W302H), isoleucine (W302I), lysine (W302K), leucine (W302L), methionine (W302M), asparagine (W302N), proline (W302P), glutamine (W302Q), arginine (W302R), serine (W302S), threonine ( (nn) the valine residue at a position corresponding to position 363 of wild-type human Neu2 is substituted with arginine (V363R); or (oo) the leucine residue at a position corresponding to position 365 of wild-type human Neu2 is substituted with glutamine (L365Q), histidine (L365H), isoleucine (L365I), lysine (L365K), or serine (L365S); or the sialidase comprises any combination of the foregoing substitutions. For example, the sialidase can include a substitution selected from K9D, P62G, P62N, P62S, P62T, A93E, Q126Y, A242F, A242W, A242Y, Q270A, Q270T, S301A, S301R, W302K, W302R, V363R, and L365I, or any combination of the foregoing substitutions;

[0009] In some embodiments, the sialidase further comprises (a) a substitution or deletion of a methionine residue at a position corresponding to position 1 of wild-type human Neu2 (M1); (b) a substitution of a valine residue at a position corresponding to position 6 of wild-type human Neu2 (V6); (c) a substitution of an isoleucine residue at a position corresponding to position 187 of wild-type human Neu2 (I187); or (d) a substitution of a cysteine ​​residue at a position corresponding to position 332 of wild-type human Neu2 (C332); or any combination of the foregoing substitutions.

[0010] In some embodiments, in the sialidase, (a) the methionine residue at position corresponding to position 1 of wild-type human Neu2 is deleted (ΔM1), substituted with alanine (M1A), or substituted with aspartic acid (M1D); (b) the valine residue at position corresponding to position 6 of wild-type human Neu2 is substituted with tyrosine (V6Y); (c) the isoleucine residue at position corresponding to position 187 of wild-type human Neu2 is substituted with lysine (I187K); or (d) the cysteine ​​residue at position corresponding to position 332 of wild-type human Neu2 is substituted with alanine (C332A); or the sialidase includes any combination of the above substitutions.

[0011] In some embodiments, the sialidase has: (a) substitutions of M1D, V6Y, P62G, A93E, I187K, and C332A; (b) substitutions of M1D, V6Y, K9D, A93E, I187K, C332A, V363R, and L365I; (c) substitutions of M1D, V6Y, P62N, I187K, and C332A. (d) substitution of M1D, V6Y, I187K, Q270A, S301R, W302K, and C332A; (e) substitution of M1D, V6Y, P62S, I187K, Q270A, S301R, W302K, and C332A; (f) substitution of M1D, V6Y, P62T, I187K, Q270A, S301R, W30 (g) substitutions of M1D, V6Y, P62N, I187K, Q270A, S301R, W302K and C332A; (h) substitutions of M1D, V6Y, P62G, A93E, I187K, S301A, W302R and C332A; (i) substitutions of M1D, V6Y, P62G, A93E , Q126Y, I187K, Q270T and C332A substitutions; (j) M1D, V6Y, P62G, A93E, Q126Y, I187K and C332A substitutions; or (k) M1D, V6Y, P62G, A93E, Q126Y, I187K, A242F, Q270T and C332A substitutions.

[0012] In some embodiments, the sialidase has a different substrate specificity than the corresponding wild-type human sialidase. For example, in some embodiments, the sialidase can cleave α2,3, α2,6, and / or α2,8 linkages. In some embodiments, the sialidase can cleave α2,3 and α2,8 linkages.

[0013] In some embodiments, the sialidase comprises any of SEQ ID NOs: 48-54, 149, 154, 159, or 191, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 48-54, 149, 154, 159, or 191.

[0014] In another aspect, the present invention provides recombinant mutant human sialidases comprising a mutation or combination of mutations described in any of Tables 5-9, 11-13, or 15-30. In certain embodiments, the sialidase further comprises a mutation or combination of mutations described in any of Tables 1-4.

[0015] In another aspect, the present invention provides a fusion protein comprising (or consisting essentially of) (a) a recombinant mutant human sialidase disclosed herein; and (b) an immunoglobulin Fc domain and / or an immunoglobulin antigen-binding domain, wherein the sialidase and the Fc domain and / or the antigen-binding domain are linked by a peptide bond or an amino acid linker. In some embodiments, the fusion protein further comprises a linker, e.g., an amino acid linker, linking the sialidase enzyme and the Fc domain and / or the antigen-binding domain. In some embodiments, the immunoglobulin antigen-binding domain is linked (e.g., covalently or non-covalently) to a second immunoglobulin antigen-binding domain to generate an antigen-binding site.

[0016] In certain embodiments, the immunoglobulin Fc domain is derived from a human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, or IgM Fc domain, e.g., the immunoglobulin Fc domain is derived from a human IgG1, IgG2, IgG3, or IgG4 Fc domain, e.g., the immunoglobulin Fc domain is derived from a human IgG1 Fc domain.

[0017] In some embodiments, the immunoglobulin antigen binding domain is derived from an antibody selected from trastuzumab, daratumumab, girentuximab, ofatumumab, avelumab, and rituximab.

[0018] In another aspect, the present invention provides an antibody conjugate comprising any of the aforementioned fusion proteins. In some embodiments, the antibody conjugate comprises a single sialidase. In other embodiments, the antibody conjugate comprises two sialidases, which may be the same or different. In some embodiments, the antibody conjugate comprises two identical sialidases. In some embodiments, the antibody conjugate comprises a single antigen-binding site. In other embodiments, the antibody conjugate comprises two antigen-binding sites, which may be the same or different. In some embodiments, the antibody conjugate comprises two identical antigen-binding sites.

[0019] In certain embodiments, the antibody conjugate has a molecular weight of about 135 kDa to about 165 kDa, or the antibody conjugate has a molecular weight of about 215 kDa to about 245 kDa.

[0020] In some embodiments, the antibody conjugate comprises: (a) a first polypeptide comprising an immunoglobulin light chain; (b) a second polypeptide comprising an immunoglobulin heavy chain; and (c) a third polypeptide comprising an immunoglobulin Fc domain and a sialidase; wherein the first and second polypeptides are covalently linked together and the second and third polypeptides are covalently linked together, and wherein the first and second polypeptides together define an antigen-binding site. The third polypeptide can comprise, for example, in an N-terminal to C-terminal direction, a sialidase and an immunoglobulin Fc domain.

[0021] In some embodiments, the antibody conjugate comprises: (a) a first polypeptide comprising a first immunoglobulin light chain; (b) a second polypeptide comprising a first immunoglobulin heavy chain and a first sialidase; (c) a third polypeptide comprising a second immunoglobulin heavy chain and a second sialidase; and (d) a fourth polypeptide comprising a second immunoglobulin light chain; wherein the first and second polypeptides are covalently linked together, the third and fourth polypeptides are covalently linked together, and the second and third polypeptides are covalently linked together, wherein the first and second polypeptides together define a first antigen-binding site, and the third and fourth polypeptides together define a second antigen-binding site. The second and third polypeptides can comprise, for example, from N- to C-terminal, the first and second immunoglobulin heavy chains and the first and second sialidases, respectively.

[0022] In some embodiments, the antibody conjugate comprises: (a) a first polypeptide comprising a first sialidase, a first immunoglobulin Fc domain, and a first single-chain variable fragment (scFv); and (b) a second polypeptide comprising a second sialidase, a second immunoglobulin Fc domain, and an optional second single-chain variable fragment (scFv); wherein the first and second polypeptides are covalently linked together, and wherein the first scFv defines a first antigen-binding site and the second scFv, if present, defines a second antigen-binding site. The first polypeptide may comprise, e.g., from N- to C-terminus, the first sialidase, the first immunoglobulin Fc domain, and the first scFv. The second polypeptide may comprise, e.g., from N- to C-terminus, the second sialidase, the second immunoglobulin Fc domain, and an optional second scFv.

[0023] In some embodiments, the antibody conjugate comprises: (a) a first polypeptide comprising an immunoglobulin light chain; (b) a second polypeptide comprising an immunoglobulin heavy chain and a single-chain variable fragment (scFv); and (c) a third polypeptide comprising an immunoglobulin Fc domain and a sialidase, wherein the first and second polypeptides are covalently linked together, the second and third polypeptides are covalently linked together, the immunoglobulin light chain and the immunoglobulin heavy chain together define a first antigen-binding site, and the scFv defines a second antigen-binding site. The second polypeptide can comprise, for example, an immunoglobulin heavy chain and an scFv, from N- to C-terminus. The third polypeptide can comprise, for example, a sialidase and an immunoglobulin Fc domain, from N- to C-terminus.

[0024] In another aspect, the present invention provides an isolated nucleic acid comprising a nucleotide sequence encoding at least a portion of any of the aforementioned recombinant mutant human sialidases, any of the aforementioned fusion proteins, or any of the aforementioned antibody conjugates. In another aspect, the present invention provides an expression vector comprising any of the aforementioned nucleic acids. In another aspect, the present invention provides a host cell comprising any of the aforementioned expression vectors.

[0025] In another aspect, the present invention provides a pharmaceutical composition comprising any of the aforementioned recombinant mutant human sialidases, any of the aforementioned fusion proteins, or any of the aforementioned antibody conjugates.

[0026] In another aspect, the present invention provides a method for treating cancer in a subject in need thereof. The method comprises administering to the subject an effective amount of any of the aforementioned sialidases, any of the aforementioned fusion proteins, any of the aforementioned antibody conjugates, or any of the aforementioned pharmaceutical compositions. In some embodiments, the cancer is an epithelial cancer. 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, an adenocarcinoma, or a carcinoma. In some embodiments, the solid tumor is a tumor of the head and neck (e.g., pharynx), thyroid, lung (e.g., small cell or non-small cell lung cancer (NSCLC)), breast, lymphatic system, gastrointestinal (e.g., oral cavity, esophagus, stomach, liver, pancreas, small intestine, colon and rectum, anal canal), reproductive or genitourinary tract (e.g., kidney, urothelium, bladder, ovary, uterus, cervix, endometrium, prostate, testes), CNS (e.g., neural or glial cells, e.g., neuroblastoma or glioma), or skin (e.g., melanoma). 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 transformed CLL, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, hairy cell leukemia, myelodyplastic syndrome (MDS), lymphoma, Hodgkin's disease, malignant lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, or Rictor's syndrome (Richter's transformation). In some embodiments, the cancer is selected from endometrial cancer, ovarian cancer, cervical cancer, vulvar cancer, uterine cancer, fallopian tube cancer, breast cancer, prostate cancer, lung cancer, pancreatic cancer, urinary cancer, bladder cancer, head and neck cancer, oral cancer, and liver cancer.

[0027] In another aspect, the present invention provides a method for increasing expression of HLA-DR, CD86, CD83, IFNγ, IL-1b, IL-6, TNFα, IL-17A, IL-2, or IL-6 in a cell or tissue. The method comprises contacting the cell or tissue with an effective amount of any of the aforementioned sialidases, any of the aforementioned fusion proteins, any of the aforementioned antibody conjugates, or any of the aforementioned pharmaceutical compositions. In some embodiments, the cell is selected from a dendritic cell and a peripheral blood mononuclear cell (PBMC).

[0028] These and other aspects and features of the present invention are described in the following detailed description and claims. [Brief explanation of the drawings]

[0029] DESCRIPTION OF THE DRAWINGS The present invention can be more fully understood with reference to the following drawings. [Figure 1] Figure 1 shows an SDS-PAGE gel showing recombinant human Neu1, Neu2, Neu3 and Salmonella typhimurium (St-sialidase) under non-reducing and reducing conditions. Monomeric and dimeric species are indicated. [Figure 2] FIG. 2 is a bar graph showing the enzymatic activity of recombinant human Neu1, Neu2, and Neu3. [Figure 3] FIG. 3 is a line graph showing enzyme activity as a function of substrate concentration for recombinant human Neu2 and Neu3 at the indicated pH. [Figure 4] FIG. 4 shows a schematic representation of an exemplary sialic acid biotinylated probe that can be used in phage display or yeast display screening for Neu2 variants. [Figure 5] FIG. 5 shows an exemplary protocol that facilitates phage display screening of Neu2 variants. [Figure 6] FIG. 6 shows an exemplary protocol that facilitates yeast display screening of Neu2 variants. [Figure 7-1] Figure 7A shows an SDS-PAGE gel showing recombinant Neu2-Fc (wild-type) and Neu2-M106-Fc under non-reducing and reducing conditions. Figure 7B shows SEC-HPLC traces of Neu2-Fc (wild-type) and Neu2-M106-Fc. The monomer species has a retention time of 21 minutes. [Figure 7-2] Figure 7A shows an SDS-PAGE gel showing recombinant Neu2-Fc (wild-type) and Neu2-M106-Fc under non-reducing and reducing conditions. Figure 7B shows SEC-HPLC traces of Neu2-Fc (wild-type) and Neu2-M106-Fc. The monomer species has a retention time of 21 minutes. [Figure 8] FIG. 8 is a line graph showing the enzymatic activity of Neu2 variant M106. [Figure 9-1] 9A-9I show schematic representations of certain antibody conjugate constructs comprising a sialidase enzyme, e.g., a human sialidase enzyme, and an antigen-binding site. For each antibody conjugate construct comprising more than one (e.g., two) sialidases, the respective sialidases can be the same or different. For each antibody conjugate construct comprising more than one (e.g., two) antigen-binding site, the respective antigen-binding sites can be the same or different. For each antibody conjugate construct comprising an Fc domain, it is understood that the Fc domain can be a wild-type Fc domain or an engineered Fc domain. For example, the Fc domain can be engineered to include either a "knob" mutation, e.g., T366Y, or a "hole" mutation, e.g., Y407T, or both, to promote heterodimerization, or the Fc domain can be engineered to include one or more modifications, e.g., point mutations, to provide any other altered Fc domain functionality. [Figure 9-2]9A-9I show schematic representations of certain antibody conjugate constructs comprising a sialidase enzyme, e.g., a human sialidase enzyme, and an antigen-binding site. For each antibody conjugate construct comprising more than one (e.g., two) sialidases, the respective sialidases can be the same or different. For each antibody conjugate construct comprising more than one (e.g., two) antigen-binding site, the respective antigen-binding sites can be the same or different. For each antibody conjugate construct comprising an Fc domain, it is understood that the Fc domain can be a wild-type Fc domain or an engineered Fc domain. For example, the Fc domain can be engineered to include either a "knob" mutation, e.g., T366Y, or a "hole" mutation, e.g., Y407T, or both, to promote heterodimerization, or the Fc domain can be engineered to include one or more modifications, e.g., point mutations, to provide any other altered Fc domain functionality. [Figure 9-3] 9A-9I show schematic representations of certain antibody conjugate constructs comprising a sialidase enzyme, e.g., a human sialidase enzyme, and an antigen-binding site. For each antibody conjugate construct comprising more than one (e.g., two) sialidases, the respective sialidases can be the same or different. For each antibody conjugate construct comprising more than one (e.g., two) antigen-binding site, the respective antigen-binding sites can be the same or different. For each antibody conjugate construct comprising an Fc domain, it is understood that the Fc domain can be a wild-type Fc domain or an engineered Fc domain. For example, the Fc domain can be engineered to include either a "knob" mutation, e.g., T366Y, or a "hole" mutation, e.g., Y407T, or both, to promote heterodimerization, or the Fc domain can be engineered to include one or more modifications, e.g., point mutations, to provide any other altered Fc domain functionality. [Figure 10] Figure 10 shows a schematic representation of certain antibody conjugate constructs comprising a sialidase enzyme, e.g., a human sialidase enzyme, and an antigen-binding site. For each antibody conjugate construct comprising more than one (e.g., two) antigen-binding site, each antigen-binding site can be the same or different. For each antibody conjugate construct comprising an Fc domain, it is understood that the Fc domain can be a wild-type Fc domain or an engineered Fc domain. For example, the Fc domain can be engineered to include either a "knob" mutation, e.g., T366Y, or a "hole" mutation, e.g., Y407T, or both, to promote heterodimerization, or the Fc domain can be engineered to include one or more modifications, e.g., point mutations, to provide any other altered Fc domain functionality. [Figure 11-1] 11A-11D are schematic representations of exemplary fusion protein conjugates designated Raptor antibody-sialidase conjugate (FIG. 11A), Janus antibody-sialidase conjugate (FIG. 11B), Lobster antibody-sialidase conjugate (FIG. 11C), and Bunk antibody-sialidase conjugate (FIG. 11D). [Figure 11-2] 11A-11D are schematic representations of exemplary fusion protein conjugates designated Raptor antibody-sialidase conjugate (FIG. 11A), Janus antibody-sialidase conjugate (FIG. 11B), Lobster antibody-sialidase conjugate (FIG. 11C), and Bunk antibody-sialidase conjugate (FIG. 11D). [Figure 12] FIG. 12 shows an SDS-PAGE gel showing purified recombinant human Janus trastuzumab under non-reducing and reducing conditions. [Figure 13] FIG. 13 shows the SEC-HPLC trace of purified Janus trastuzumab, showing approximately 90% monomer purity. [Figure 14]FIG. 14 shows the enzymatic activity of Janus-trastuzumab assayed using 4-MU-Neu5Ac as a substrate. [Figure 15] Figure 15 shows binding to the HER2 antigen as determined by ForteBio Octet for Janus trastuzumab (top) and trastuzumab (bottom). The equilibrium dissociation constant (KD) is shown. [Figure 16-1] Figures 16A-D show testing of various configurations of antibody-sialidase conjugates in a mouse syngeneic tumor model using EMT6 mouse breast cancer cells genetically engineered to express human Her2. Mice were treated with intraperitoneal injection of 10 mg / kg of each test article on the days marked by black triangles, and tumor volumes (mm3) were recorded. Each line represents an individual mouse. Mice were treated with either trastuzumab (Figure 16A), Raptor (Figure 16B), Janus (Figure 16C), or Lobster (Figure 16D). [Figure 16-2] Figures 16A-D show testing of various configurations of antibody-sialidase conjugates in a mouse syngeneic tumor model using EMT6 mouse breast cancer cells genetically engineered to express human Her2. Mice were treated with intraperitoneal injection of 10 mg / kg of each test article on the days marked by black triangles, and tumor volumes (mm3) were recorded. Each line represents an individual mouse. Mice were treated with either trastuzumab (Figure 16A), Raptor (Figure 16B), Janus (Figure 16C), or Lobster (Figure 16D). [Figure 17-1]Figures 17A-D show testing of Janus antibody-sialidase conjugates in a mouse syngeneic tumor model using EMT6 mouse breast cancer cells genetically engineered to express human Her2. Mice were treated with 10 mg / kg Janus intraperitoneally on the days marked by black triangles, and tumor volume (mm) was recorded. Mice were also treated on the same days as Janus with either anti-mouse NK1.1 (10 mg / kg) to deplete natural killer cells (Figure 17A), clodronate liposomes (0.5 mg / mouse, three times a week for two weeks) to deplete macrophages (Figure 17B), or anti-mouse CD8α (10 mg / kg) to deplete CD8+ T cells (Figure 17C). Each line represents an individual mouse. Figure 17D shows the mean tumor volume with error bars for the indicated treatment groups from Example 5. [Figure 17-2] Figures 17A-D show testing of Janus antibody-sialidase conjugates in a mouse syngeneic tumor model using EMT6 mouse breast cancer cells genetically engineered to express human Her2. Mice were treated with 10 mg / kg Janus intraperitoneally on the days marked by black triangles, and tumor volume (mm) was recorded. Mice were also treated on the same days as Janus with either anti-mouse NK1.1 (10 mg / kg) to deplete natural killer cells (Figure 17A), clodronate liposomes (0.5 mg / mouse, three times a week for two weeks) to deplete macrophages (Figure 17B), or anti-mouse CD8α (10 mg / kg) to deplete CD8+ T cells (Figure 17C). Each line represents an individual mouse. Figure 17D shows the mean tumor volume with error bars for the indicated treatment groups from Example 5. [Figure 18]Figures 18A-B show testing of Janus antibody-sialidase conjugates in a mouse syngeneic orthotopic tumor model using a second source of EMT6 mouse breast cancer cells genetically engineered to express human Her2. Mice were treated with intraperitoneal injection of 10 mg / kg of each test article on the days indicated by the black triangles, and tumor volumes (mm) were recorded. Each line represents an individual mouse. Mice were treated with either vehicle, trastuzumab, Janus, or Janus loss of function (▼) (Figure 18A). Figure 18B shows a rechallenge experiment of either three Janus-treated mice (cured mice) from Figure 18A that had complete regression of the original EMT6-Her2 tumors or naive mice. Cured mice were inoculated with either EMT6-Her2 cells or parental EMT6 cells in the left and right lower flank regions. Naive mice were inoculated with EMT6-Her2 cells. [Figure 19] Figures 19A-B show testing of Janus antibody-sialidase conjugates in combination with anti-mouse PD1 in a mouse syngeneic orthotopic tumor model. Mice were treated by intraperitoneal injection of either 10 mg / kg anti-mouse PD1 alone (Figure 19A) or Janus and anti-mouse PD1 (each at 10 mg / kg, Figure 19B) on the days marked by black triangles (▼), and tumor volumes (mm3) were recorded. Each line represents an individual mouse. [Figure 20] Figure 20 shows the testing of various test articles in a mouse syngeneic tumor model injected with the B16 melanoma cell line expressing human Her2. Mice were treated with intraperitoneal injection of 10 mg / kg of either Janus, trastuzumab, or a combination of anti-mouse PD1 and anti-mouse CTLA4 (10 mg / kg each) on the days marked by black triangles (▼), and tumor volumes (mm3) were recorded. Each line represents an individual mouse. [Figure 21]Figure 21 shows the testing of Janus-trastuzumab in a mouse syngeneic tumor model using EMT6 mouse breast cancer cells genetically engineered to express human Her2. Each line represents an individual mouse. Tick marks indicate dosing frequency (a total of five doses over two weeks). Mice are treated with either Janus-trastuzumab or an isotype control. [Figure 22] Figure 22A shows an SDS-PAGE gel showing Neu2-M173-Fc under non-reducing and reducing conditions. Figure 22B shows an SEC-HPLC trace of Neu2-M173-Fc. The monomeric species has a retention time of 6.367 minutes. The monomeric species has a purity of approximately 90% after purification with Protein A and CHT chromatography. [Figure 23] FIG. 23 shows the enzymatic activity of Neu2-M173-Fc using 4-MU-Neu5Ac as substrate and fixed enzyme concentrations up to 2 μg / well. [Figure 24] Figure 24A shows an SDS-PAGE gel showing Neu2-M106 under non-reducing (NR) and reducing (R) conditions, and Figure 24B shows a schematic representation of the Neu2 structure with the location of the R243 cleavage site indicated. [Figure 25] FIG. 25 shows a reducing SDS-PAGE gel showing Neu2-M106 produced by large-scale or small-scale expression with (+) or without (-) trypsin treatment. [Figure 26] FIG. 26 shows an SDS-PAGE gel showing Neu2-M106 after incubation with trypsin and one of the protease inhibitors ferric citrate (Fe Cit), aprotinin, AEBSF, leupeptin, or E-64 at the concentrations indicated. [Figure 27] FIG. 27 is a table showing different mutations and combinations of mutations around the trypsin cleavage site in Neu2. [Figure 28-1]Figure 28A shows a reducing SDS-PAGE analysis of Neu2 variants with the indicated mutation at position A242 with or without trypsin treatment. Trypsin digestion was at 4°C for 5 minutes using 5,000% diluted trypsin. SDS was added to quench the digestion, and 2 μg of protein was loaded onto the gel. Figure 28B shows the enzymatic activity of Neu2 variants with the indicated mutation at position A242. Figure 28C shows the SEC-HPLC trace of Neu2 variants with the indicated mutation at position A242. Neu2-M106 (the mutation background in which the mutation at position A242 was tested) is shown as a control. [Figure 28-2] Figure 28A shows a reducing SDS-PAGE analysis of Neu2 variants with the indicated mutation at position A242 with or without trypsin treatment. Trypsin digestion was at 4°C for 5 minutes using 5,000% diluted trypsin. SDS was added to quench the digestion, and 2 μg of protein was loaded onto the gel. Figure 28B shows the enzymatic activity of Neu2 variants with the indicated mutation at position A242. Figure 28C shows the SEC-HPLC trace of Neu2 variants with the indicated mutation at position A242. Neu2-M106 (the mutation background in which the mutation at position A242 was tested) is shown as a control. [Figure 29] Figure 29 shows a reducing SDS-PAGE analysis of the indicated Neu2 variants with or without trypsin treatment. Neu2-M106 is shown as a control. For example, Neu2-M255 was shown to have more than 10-fold improved trypsin resistance relative to Neu2-M106. DETAILED DESCRIPTION OF THE INVENTION

[0030] Detailed Description Various features and aspects of the invention are described in further detail below.

[0031] The present invention relates to recombinant human sialidases that contain at least one mutation, such as at least one amino acid substitution, deletion, or addition (insertion), relative to wild-type human sialidase, which mutation, or a combination of mutations, can improve the expression, activity, or both expression and activity of the sialidase, and can improve its use in the diagnosis and / or treatment of cancer.

[0032] The present invention further relates to fusion proteins and / or antibody conjugates comprising a sialidase enzyme and an antibody or portion thereof, such as an immunoglobulin Fc domain and / or an antigen-binding domain. The sialidase enzyme portion of the fusion protein and / or antibody conjugate can contain at least one mutation relative to wild-type human sialidase.

[0033] The invention further relates to pharmaceutical compositions and methods of using the fusion proteins and / or antibody conjugates for treating cancer, such as solid tumors, soft tissue tumors, hematopoietic tumors, metastatic lesions or epithelial cell cancers.

[0034] I. Recombinant Human Sialidase As used herein, the term "sialidase" refers to any enzyme or functional fragment thereof that cleaves terminal sialic acid residues from a substrate, such as a glycoprotein or glycolipid. The term sialidase also 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 containing sialidase. Sialidase is also referred to as neuraminidase, and the two terms are used interchangeably herein unless otherwise indicated. As used herein, the term "functional fragment" of a sialidase refers to a fragment of a full-length sialidase that retains, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the enzymatic activity of the corresponding full-length naturally occurring sialidase. Sialidase enzyme activity can be assayed by any method known in the art, such as, for example, by measuring the release of sialic acid from the fluorogenic substrate 4-methylumbelliferyl-N-acetylneuraminic acid (4MU-NeuAc). In certain embodiments, the functional fragment comprises at least 100, 150, 200, 250, 300, 310, 320, 330, 340, 350, 360, or 370 contiguous amino acids present in a full-length, naturally occurring sialidase.

[0035] Four sialidases have also been found in the human genome, designated Neu1, Neu2, Neu3 and Neu4.

[0036] Human Neu1 is a lysosomal neuraminidase enzyme that functions in a complex with β-galactosidase and cathepsin A. The amino acid sequence of human Neu1 is shown in SEQ ID NO:7, and the nucleotide sequence encoding human Neu1 is shown in SEQ ID NO:23.

[0037] Human Neu2 is a cytosolic sialidase enzyme. The amino acid sequence of human Neu2 is set forth in SEQ ID NO:1, and the nucleotide sequence encoding human Neu2 is set forth in SEQ ID NO:24. Unless otherwise stated, as used herein, wild-type human Neu2 refers to human Neu2 having the amino acid sequence of SEQ ID NO:1.

[0038] Human Neu3 is a plasma membrane sialidase with ganglioside-specific activity. Human Neu3 has two isoforms: isoform 1 and isoform 2. The amino acid sequence of human Neu3, isoform 1 is set forth in SEQ ID NO:8, and the nucleotide sequence encoding human Neu3, isoform 1 is set forth in SEQ ID NO:25. The amino acid sequence of human Neu3, isoform 2 is set forth in SEQ ID NO:9, and the nucleotide sequence encoding human Neu3, isoform 2 is set forth in SEQ ID NO:34.

[0039] Human Neu4 has two isoforms; isoform 1 is a peripheral membrane protein and isoform 2 is localized to the lysosomal lumen. The amino acid sequence of human Neu4, isoform 1 is set forth in SEQ ID NO: 10, and the nucleotide sequence encoding human Neu4, isoform 1 is set forth in SEQ ID NO: 26. The amino acid sequence of human Neu4, isoform 2 is set forth in SEQ ID NO: 11, and the nucleotide sequence encoding human Neu4, isoform 2 is set forth in SEQ ID NO: 35.

[0040] Four sialidases have also been found in the mouse genome, designated Neu1, Neu2, Neu3, and Neu4. The amino acid sequence of mouse Neu1 is set forth in SEQ ID NO:38, and the nucleotide sequence encoding mouse Neu1 is set forth in SEQ ID NO:42. The amino acid sequence of mouse Neu2 is set forth in SEQ ID NO:39, and the nucleotide sequence encoding mouse Neu2 is set forth in SEQ ID NO:43. The amino acid sequence of mouse Neu3 is set forth in SEQ ID NO:40, and the nucleotide sequence encoding mouse Neu3 is set forth in SEQ ID NO:44. The amino acid sequence of mouse Neu4 is set forth in SEQ ID NO:41, and the nucleotide sequence encoding mouse Neu4 is set forth in SEQ ID NO:45.

[0041] Exemplary prokaryotic sialidases include sialidases from Salmonella typhimurium and Vibrio cholerae. The amino acid sequence of Salmonella typhimurium sialidase (St-sialidase) is set forth in SEQ ID NO:30, and the nucleotide sequence encoding the Salmonella typhimurium sialidase is set forth in SEQ ID NO:6. The amino acid sequence of Vibrio cholerae sialidase is set forth in SEQ ID NO:36, and the nucleotide sequence encoding the Vibrio cholerae sialidase is set forth in SEQ ID NO:37.

[0042] In some embodiments, the recombinant variant human sialidase has about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, e.g., about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% or more than 100% of the enzymatic activity of the corresponding (or template) wild-type human sialidase.

[0043] 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 a 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 linkages. In some embodiments, the sialidase can cleave α2,3 and α2,8 linkages.

[0044] In some embodiments, the expression yield 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), e.g., HEK293 cells, is greater than 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% of the expression yield of the corresponding wild-type human sialidase.

[0045] In certain embodiments, the recombinant variant human sialidase has about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% or greater than 100% of the enzymatic activity of the corresponding wild-type human sialidase, and The expression yield of the recombinant mutant human sialidase in animal cells, such as HEK293 cells, is 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% higher than the expression yield of the corresponding wild-type human sialidase.

[0046] In some embodiments, the amino acid sequence of the recombinant mutant human sialidase has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of the corresponding wild-type human sialidase.

[0047] a. Cysteine ​​residue substitution In some embodiments, the recombinant mutant human sialidase comprises a substitution of at least one cysteine ​​(cys, C) residue. It has been discovered that certain cysteine ​​residues in sialidases can inhibit the expression of functional proteins as a result of protein aggregation. Thus, in some embodiments, the recombinant mutant human sialidase comprises at least one mutation to remove a free cysteine ​​(e.g., for Neu1 (SEQ ID NO: 7), e.g., one or more mutations at C111, C117, C171, C183, C218, C240, C242, and C252; for Neu2 (SEQ ID NO: 1), e.g., one or more mutations at C125, C196, C219, C272, C332, and C352; for Neu3 (SEQ ID NO: 8), e.g., one or more mutations at 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), for example, one or more mutations at C88, C125, C126, C186, C191, C211, C223, C239, C276, C437, C453, C480, and C481). The free cysteine ​​may be substituted with any amino acid. In certain embodiments, the free cysteine ​​is substituted with serine (ser, S), isoleucine (iso, I), valine (val, V), phenylalanine (phe, F), leucine (leu, L), or alanine (ala, A). Exemplary cysteine ​​substitutions in Neu2 include C125A, C125I, C125S, C125V, C196A, C196L, C196V, C272S, C272V, C332A, C332S, C332V, C352L, and C352V.

[0048] In some embodiments, the recombinant mutant human sialidase comprises two or more cysteine ​​substitutions. Exemplary double or triple cysteine ​​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.

[0049] In some embodiments, the recombinant mutant human sialidase is a Neu2 sialidase and contains the substitutions C322A and C352L (SEQ ID NO: 5).

[0050] In some embodiments, the sialidase contains amino acid substitutions at 2, 3, 4, 5, or 6 cysteines typically present in human sialidases, such as Neu2 or Neu3.

[0051] In some embodiments, the recombinant mutant human sialidase comprises a substitution or combination of substitutions corresponding to a substitution or combination of substitutions listed in Table 1 (amino acid positions corresponding to wild-type human Neu2 (SEQ ID NO: 1)). [Table 1]

[0052] b. Substitution of residues to increase pI and / or decrease hydrophobicity The isoelectric point (pI) of a protein is the pH at which its net charge is zero. pI also generally indicates the pH at which a protein is least soluble, which may affect the ability to express and purify the protein. Generally, a protein has good solubility when its pI is two units higher than the pH of the solution. Human Neu2 has an expected pI of 7.5. Therefore, human Neu2 is minimally soluble around neutral pH, which is undesirable because expression and physiological systems are at neutral pH. In contrast, sialidase from Salmonella typhimurium (St-sialidase), which exhibits good solubility and recombinant expression, has a pI of 9.6. Therefore, to increase the expression of human Neu2 or other human sialidases, recombinant mutant human sialidases can be engineered to contain one or more amino acid substitution(s), where the substitution(s) increase the pI of the sialidase relative to the sialidase without the substitution(s). Furthermore, reducing the number of hydrophobic amino acids on the surface of a sialidase can improve expression of the sialidase, for example, by reducing aggregation. Thus, to increase expression of human Neu2 or other human sialidases, recombinant mutant human sialidases can be designed to contain one or more amino acid substitution(s), where the substitution(s) reduce the hydrophobicity of the surface of the sialidase relative to the sialidase without the substitution(s).

[0053] Thus, in some embodiments, the recombinant mutant human sialidase contains at least one amino acid substitution, wherein the substitution increases the isoelectric point (pI) of the sialidase and / or decreases the hydrophobicity of the sialidase relative to the sialidase without the substitution. This can be achieved by introducing one or more charged amino acids, e.g., positively or negatively charged amino acids, into the recombinant sialidase. In some embodiments, the amino acid substitution is for a charged amino acid, e.g., a positively charged amino acid, such as lysine (lys, K), histidine (his, H), or arginine (arg, R), or a negatively charged amino acid, such as aspartic acid (asp, D) or glutamic acid (glu, E). In some embodiments, the amino acid substitution is for 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.

[0054] In some embodiments, amino acid substitutions occur at surface-exposed D or E amino acids, in helices or loops, or at positions that have K or R at the corresponding position in St-sialidase. In some embodiments, amino acid substitutions occur at amino acids distant from or otherwise not involved in catalysis, amino acids that are not conserved by other human Neu proteins or St-sialidases or clostridial NanH, or amino acids that are not located within domains important for function (e.g., Asp-boxes or β-strands).

[0055] Exemplary amino acid substitutions in Neu2 that increase the isoelectric point (pI) of a sialidase and / or decrease the hydrophobicity of the sialidase relative to a sialidase not having the substitution include A2E, A2K, D215K, V325E, V325K, E257K, and E319K. In some embodiments, the recombinant mutant human sialidase contains 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.

[0056] In some embodiments, the recombinant mutant human sialidase comprises Table 2 (amino acid positions corresponding to substitutions or combinations of substitutions that correspond to substitutions or combinations of substitutions listed in wild-type human Neu2 (SEQ ID NO: 1)). [Table 2]

[0057] c. N-terminal peptide addition and N- or C-terminal substitution It has been discovered that adding a peptide sequence of two or more amino acids to the N-terminus of human sialidase can improve the expression and / or activity of the sialidase. In some embodiments, the peptide is at least 2 amino acids long, e.g., 2-20, 2-10, 2-5, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids long. In some embodiments, the peptide can form an α-helix or have a propensity to form an α-helix.

[0058] In mice, the Neu2 isoform (type B) found in the thymus contains six amino acids that are not present in the canonical isoform of Neu2 found in skeletal muscle. In certain embodiments herein, the N-terminal six amino acids of the mouse thymic Neu2 isoform, MEDLRP (SEQ ID NO: 4), or a variant thereof, can be added to human Neu, e.g., human Neu2. In certain embodiments, the recombinant mutant human sialidase comprises a peptide at least two amino acid residues long covalently linked to the N-terminal amino acid of the sialidase. In certain embodiments, the recombinant mutant human sialidase comprises the peptide MEDLRP (SEQ ID NO: 4) or EDLRP (SEQ ID NO: 3) covalently linked to the N-terminal amino acid of the sialidase. In certain embodiments, the sialidase can further comprise a cleavage site, e.g., a proteolytic cleavage site, located between the peptide, e.g., MEDLRP (SEQ ID NO: 4) or EDLRP (SEQ ID NO: 3), and the remainder of the sialidase. In some embodiments, the peptide, eg, MEDLRP (SEQ ID NO:4) or EDLRP (SEQ ID NO:3), can be post-translationally cleaved from the remainder of the sialidase.

[0059] Alternatively, or in combination with N-terminal addition, one to five amino acids of the 12 amino acid N-terminal region of the recombinant mutant human sialidase can be removed, for example, the N-terminal methionine can be removed. In some embodiments, when the recombinant mutant human sialidase is Neu2, the N-terminal methionine can be removed, the first five amino acids (MASLP; SEQ ID NO: 12) can be removed, or the second through fourth amino acids (ASLP; SEQ ID NO: 13) can be removed.

[0060] In some embodiments, 1 to 5 amino acids of the 12 amino acid N-terminal region of a recombinant mutant human sialidase are substituted with MEDLRP (SEQ ID NO: 4), EDLRP (SEQ ID NO: 3), or TVEKSVVF (SEQ ID NO: 14). For example, in some embodiments, when the recombinant mutant human sialidase is Neu2, amino acids MASLP (SEQ ID NO: 12), ASLP (SEQ ID NO: 13), or M are substituted with MEDLRP (SEQ ID NO: 4), EDLRP (SEQ ID NO: 3), or TVEKSVVF (SEQ ID NO: 14).

[0061] Human sialidases have a β-propeller structure characterized by a six-bladed β-sheet arranged toroidally around a central axis. Generally, hydrophobic interactions between the blades of the β-propeller, such as between the N- and C-terminal blades, enhance stability. Therefore, to increase the expression of human Neu2 or other human sialidases, recombinant mutant human sialidases can be engineered containing amino acid substitutions that increase hydrophobic interactions and / or hydrogen bonds between the N- and C-terminal β-propeller blades of the sialidase.

[0062] Thus, in some embodiments, the recombinant mutant human sialidase comprises a substitution of at least one wild-type amino acid residue, wherein the substitution increases hydrophobic interactions and / or hydrogen bonds between the N- and C-termini of the sialidase relative to the sialidase without the substitution. In some embodiments, the wild-type amino acid is substituted with asparagine (asn, N), lysine (lys, K), tyrosine (tyr, Y), phenylalanine (phe, F), or tryptophan (trp, W). Exemplary substitutions in Neu2 that increase hydrophobic interactions and / or hydrogen bonds 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.

[0063] In some embodiments, the recombinant mutant human sialidase comprises a combination of the above substitutions. For example, the recombinant mutant human Neu2 sialidase can comprise additional amino acids MEDLRP (SEQ ID NO: 4), EDLRP (SEQ ID NO: 3), or TVEKSVVF (SEQ ID NO: 14) at the N-terminus, in combination with at least one of L4N, L4K, V6Y, L7N, L4N and L7N, L4N and V6Y, and L7N, V12N, V12Y, V12L, V6Y, V6F, or V6W substitutions. 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 also contains at least one L4N, L4K, V6Y, L7N, L4N and L7N, L4N and V6Y, and L7N, V12N, V12Y, V12L, V6Y, V6F, or V6W substitution.

[0064] In some embodiments, the recombinant mutant human sialidase comprises a mutation or combination of mutations corresponding to a mutation or combination of mutations listed in Table 3 (amino acid positions corresponding to wild-type human Neu2 (SEQ ID NO: 1)). [Table 3]

[0065] Additionally, in some embodiments, the sialidase comprises a substitution or deletion of an N-terminal methionine at the N-terminus of the sialidase. For example, in some embodiments, the sialidase comprises a substitution of a methionine residue at a position corresponding to position 1 of wild-type human Neu2 (SEQ ID NO:1), e.g., the methionine at position 1 of wild-type human Neu2 is replaced with alanine (M1A) or aspartic acid (M1D). In other embodiments, the sialidase comprises a deletion (ΔM1) of a methionine residue at a position corresponding to position 1 of wild-type human Neu2 (SEQ ID NO:1).

[0066] In some embodiments, the recombinant mutant human sialidase comprises a substitution or combination of substitutions corresponding to a substitution or combination of substitutions listed in Table 4 (amino acid positions corresponding to wild-type human Neu2 (SEQ ID NO: 1)). [Table 4]

[0067] d. Substitution of residues to reduce proteolytic cleavage It has been discovered that certain sialidases (e.g., human Neu2) are susceptible to cleavage by proteases (e.g., trypsin). Consequently, proteolytic cleavage of the sialidase can occur during recombinant protein production, harvesting, purification, or formulation, during administration to a subject, or after administration to a subject. Thus, in some embodiments, the recombinant mutant human sialidase comprises a substitution of at least one wild-type amino acid residue, wherein the substitution reduces cleavage of the sialidase by a protease (e.g., trypsin) relative to a sialidase that does not have the substitution.

[0068] In some embodiments, incubation of a recombinant variant human sialidase with a protease (e.g., trypsin) results in 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 20%, about 1% to about 3 ...10%, about 1% to about 30%, about 1% to about 30%, about 1% to about 30%, about 1% to about 10%, about 1% to about 10%, about 1% to about 10%, or about 1% to about 10% of the proteolytic cleavage of the corresponding wild-type sialidase when incubated with the protease under the same conditions. In some embodiments, incubation of a recombinant mutant human sialidase with a protease (e.g., trypsin) results in less than 50%, 40%, 30%, 10%, 5%, 3%, 1%, or 0.5% of the proteolytic cleavage of the corresponding wild-type sialidase when incubated with the protease under the same conditions. Proteolytic cleavage can be assayed by any method known in the art, including, for example, by SDS-PAGE as described in Example 5 herein.

[0069] Exemplary substitutions that increase resistance to proteolytic cleavage include: (i) substitution of an alanine residue at a position corresponding to position 242 of wild-type human Neu2 (SEQ ID NO: 1), e.g., cysteine ​​(A242C), phenylalanine (A242F), glycine (A242G), histidine (A242H), isoleucine (A242I), lysine (A242K), leucine (A242L), methionine (A242M), asparagine (A242N), glutamine (A242Q), arginine (A242R), serine (A242S), valine (A242V), tryptophan (A242W). or tyrosine (A242Y); (ii) a substitution of an arginine residue at a position corresponding to position 243 of wild-type human Neu2 (SEQ ID NO: 1), such as with glutamic acid (R243E), histidine (R243H), asparagine (R243N), glutamine (R243Q), or lysine (R243K); (iii) a substitution of a valine residue at a position corresponding to position 244 of wild-type human Neu2 (SEQ ID NO: 1), such as with isoleucine (V244I), lysine (V244K), or proline (V244P); or (iv) any combination of the foregoing. 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 a substitution or combination of substitutions listed in Table 5 (amino acid positions corresponding to wild-type human Neu2 (SEQ ID NO: 1)). [Table 5]

[0070] Further exemplary substitutions that increase resistance to proteolytic cleavage (and / or increase expression yield and / or enzymatic activity) include: (i) substitution of a leucine residue at a position corresponding to position 240 of wild-type human Neu2 (SEQ ID NO: 1), for example, with aspartic acid (L240D), asparagine (L240N), or tyrosine (L240Y); (ii) substitution of an alanine residue at a position corresponding to position 213 of wild-type human Neu2 (SEQ ID NO: 1), for example, with cysteine ​​(A213C), asparagine (A213N), serine (A213S), or threonine (A213T); (iii) substitution of an arginine residue at a position corresponding to position 241 of wild-type human Neu2 (SEQ ID NO: 1), for example, with alanine (R241A), (iv) a substitution of a serine residue at a position corresponding to position 258 of wild-type human Neu2 (SEQ ID NO:1), such as with cysteine ​​(S258C); (v) a substitution of a leucine residue at a position corresponding to position 260 of wild-type human Neu2 (SEQ ID NO:1), such as with aspartic acid (L260D), phenylalanine (L260F), glutamine (L260Q), or threonine (L260T); (vi) a substitution of a valine residue at a position corresponding to position 265 of wild-type human Neu2 (SEQ ID NO:1), such as with phenylalanine (V265F); or (vii) a combination of any of the foregoing. In certain embodiments, it is contemplated that substitutions or combinations of substitutions at these positions may improve hydrophobic and / or aromatic interactions between secondary structural elements in the sialidase (e.g., between an α-helix and the nearest β-sheet), thereby stabilizing the structure and improving resistance to proteolytic cleavage.

[0071] 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 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 a combination of substitutions selected from: (i) A213C, A242F and S258C, (ii) A213C and A242F, (iii) A213T and A242F, (iv) R241Y and A242F, and (v) L240Y and A242F. In some embodiments, the recombinant mutant human sialidase comprises a substitution or combination of substitutions corresponding to a substitution or combination of substitutions listed in Table 6 (amino acid positions corresponding to wild-type human Neu2 (SEQ ID NO: 1)). [Table 6]

[0072] e. Other substitutions The present invention further provides recombinant mutant human sialidases comprising at least one of the following substitutions: I187K, A328E, K370N, or H210N. In some embodiments, the recombinant mutant human Neu2 comprises a substitution of the amino acid GDYDAPTHQVQW (SEQ ID NO: 15) with the amino acid SMDQGSTW (SEQ ID NO: 16) or STDGGKTW (SEQ ID NO: 17). In some embodiments, the recombinant mutant human Neu2 comprises a substitution of the amino acid PRPPAPEA (SEQ ID NO: 18) with the amino acid QTPLEAAC (SEQ ID NO: 19). In some embodiments, the recombinant mutant human Neu2 comprises a substitution of the amino acid NPRPPAPEA (SEQ ID NO: 20) with the amino acid SQNDGES (SEQ ID NO: 21).

[0073] The present invention further provides recombinant mutant human sialidases comprising at least one substitution at a position corresponding to V212, A213, Q214, D215, T216, L217, E218, C219, Q220, V221, A222, E223, V224, E225, or T225.

[0074] The present invention further provides recombinant mutant human sialidases comprising amino acid substitutions at positions identified in Table 7 (amino acid positions corresponding to wild-type human Neu2 (SEQ ID NO: 1). In some embodiments, the sialidase comprises an amino acid substitution identified in Table 7. In some embodiments, the sialidase comprises any combination of amino acid substitutions identified in Table 7. [Table 7-1] [Table 7-2] [Table 7-3]

[0075] For example, in some embodiments, the recombinant mutant human sialidase contains: (a) a substitution of a proline residue at a position corresponding to position 5 of wild-type human Neu2 (P5); (b) a substitution of a lysine residue at a position corresponding to position 9 of wild-type human Neu2 (K9); (c) a substitution of a lysine residue at a position corresponding to position 44 of wild-type human Neu2 (K44); (d) a substitution of a lysine residue at a position corresponding to position 45 of wild-type human Neu2 (K45); (e) a substitution of a leucine residue at a position corresponding to position 54 of wild-type human Neu2 (L54); (f) a substitution of a leucine residue at a position corresponding to position 62 of wild-type human Neu2 (L54). (g) substitution of a glutamine residue at position 69 of wild-type human Neu2 (Q69); (h) substitution of an arginine residue at position 78 of wild-type human Neu2 (R78); (i) substitution of an aspartic acid residue at position 80 of wild-type human Neu2 (D80); (j) substitution of an alanine residue at position 93 of wild-type human Neu2 (A93); (k) substitution of a glycine residue at position 107 of wild-type human Neu2 (G107); (l) substitution of an aspartic acid residue at position 108 of wild-type human Neu2 (D108); (m) substitution of a glutamine residue at position 112 of wild-type human Neu2 (Q112); (n) substitution of a cysteine ​​residue at position 125 of wild-type human Neu2 (C125); (o) substitution of a glutamine residue at position 126 of wild-type human Neu2 (Q126); (p) substitution of an alanine residue at position 150 of wild-type human Neu2 (A150); (q) substitution of a cysteine ​​residue at position 164 of wild-type human Neu2 (C164); (r) substitution of a cysteine ​​residue at position 164 of wild-type human Neu2 (C164); (s) substitution of an arginine residue at position 170 of wild-type human Neu2 (R170); (s) substitution of an alanine residue at position 171 of wild-type human Neu2 (A171); (t) substitution of a glutamine residue at position 188 of wild-type human Neu2 (Q188); (u) substitution of an arginine residue at position 189 of wild-type human Neu2 (R189); (v) substitution of an alanine residue at position 213 of wild-type human Neu2 (A213); (w) substitution of a leucine residue at position 217 of wild-type human Neu2 (L217);(x) substitution of a glutamic acid residue at position 225 of wild-type human Neu2 (E225); (y) substitution of a histidine residue at position 239 of wild-type human Neu2 (H239); (z) substitution of a leucine residue at position 240 of wild-type human Neu2 (L240); (aa) substitution of an arginine residue at position 241 of wild-type human Neu2 (R241); (bb) substitution of an arginine residue at position 242 of wild-type human Neu2 (R242). (cc) substitution of a valine residue at position 244 of wild-type human Neu2 (V244); (dd) substitution of a threonine residue at position 249 of wild-type human Neu2 (T249); (ee) substitution of an aspartic acid residue at position 251 of wild-type human Neu2 (D251); (ff) substitution of a glutamic acid residue at position 257 of wild-type human Neu2 (E257); (gg ...threonine residue at position 249 of wild-type human Neu2 (T249); (ee) substitution of an aspartic acid residue at position 251 of wild-type human Neu2 (D251); (ff) substitution of a glutamic acid residue at position 257 of wild-type human Neu2 (E257); (gg) substitution of a threonine residue at position 249 of wild-type human Neu2 (T249); (gg) substitution of a threonine residue at position 249 of wild-type human Neu2 (T249); (gg) substitution of a threonine residue at position 249 of wild-type human Neu2 (T249); (gg) substitution of a glutamic acid residue at position 257 of wild-type human Neu2 (E257); (gg) substitution of a glutamic acid residue at position 257 of wild-type human Neu2 (E257); (hh) substitution of a serine residue at position 258 of wild-type human Neu2 (S258); (ii) substitution of a leucine residue at position 260 of wild-type human Neu2 (L260); (iii) substitution of a valine residue at position 265 of wild-type human Neu2 (V265); (jj) substitution of a glutamine residue at position 270 of wild-type human Neu2 (Q270); (kk) substitution of a tryptophan residue at position 292 of wild-type human Neu2 (W2 (11) a substitution of a serine residue at position 301 of wild-type human Neu2 (S301); (mm) a substitution of a tryptophan residue at position 302 of wild-type human Neu2 (W302); (nn) a substitution of a valine residue at position 363 of wild-type human Neu2 (V363); or (oo) a substitution of a leucine residue at position 365 of wild-type human Neu2 (L365); or any combination of the foregoing substitutions. For example, the sialidase can include substitutions of K9, P62, A93, Q216, A242, Q270, S301, W302, V363, or L365, or any combination of the foregoing substitutions.

[0076] In some embodiments, in the sialidase: (a) the proline residue at the position corresponding to position 5 of wild-type human Neu2 is substituted with histidine (P5H); (b) the lysine residue at the position corresponding to position 9 of wild-type human Neu2 is substituted with aspartic acid (K9D); (c) the lysine residue at the position corresponding to position 44 of wild-type human Neu2 is substituted with arginine (K44R) or glutamic acid (K44E); (d) the lysine residue at the position corresponding to position 45 of wild-type human Neu2 is substituted with alanine (K45A), arginine (K45R), or glutamic acid (K45E). (e) the leucine residue at position 54 of wild-type human Neu2 is substituted with methionine (L54M); (f) the proline residue at position 62 of wild-type human Neu2 is substituted with asparagine (P62N), aspartic acid (P62D), histidine (P62H), glutamic acid (P62E), glycine (P62G), serine (P62S), or threonine (P62T); (g) the glutamine residue at position 69 of wild-type human Neu2 is substituted with histidine (Q69H); (h) the leucine residue at position 7 of wild-type human Neu2 is substituted with methionine (L54M); The arginine residue at position 8 was substituted with lysine (R78K); (i) the aspartic acid residue at position 80 of wild-type human Neu2 was substituted with proline (D80P); (j) the alanine residue at position 93 of wild-type human Neu2 was substituted with glutamic acid (A93E) or lysine (A93K); (k) the glycine residue at position 107 of wild-type human Neu2 was substituted with aspartic acid (G107D); (l) the glutamine residue at position 108 of wild-type human Neu2 was substituted with histidine (Q108H) (m) the glutamine residue at position corresponding to position 112 of wild-type human Neu2 is substituted with arginine (Q112R) or lysine (Q112K); (n) the cysteine ​​residue at position corresponding to position 125 of wild-type human Neu2 is substituted with leucine (C125L); (o) the glutamine residue at position corresponding to position 126 of wild-type human Neu2 is substituted with leucine (Q126L), glutamic acid (Q126E), phenylalanine (Q126F), histidine (Q126H), isoleucine (Q126I), or tyrosine (Q126Y);(p) the alanine residue at position 150 of wild-type human Neu2 is substituted with valine (A150V); (q) the cysteine ​​residue at position 164 of wild-type human Neu2 is substituted with glycine (C164G); (r) the arginine residue at position 170 of wild-type human Neu2 is substituted with proline (R170P); (s) the alanine residue at position 171 of wild-type human Neu2 is substituted with glycine (A171G); (t) the alanine residue at position 171 of wild-type human Neu2 is substituted with valine (A150V); (u) the glutamine residue at position 188 of wild-type human Neu2 is substituted with proline (Q188P); (v) the alanine residue at position 213 of wild-type human Neu2 is substituted with cysteine ​​(A213C), asparagine (A213N), serine (A213S), or threonine (A213T); (w) the leucine residue at position 217 of wild-type human Neu2 is substituted with proline (R189P); (x) the threonine residue at position 249 of wild-type human Neu2 is substituted with alanine (T249A); (y) the aspartic acid residue at position 251 of wild-type human Neu2 is substituted with glycine (D251G); (z) the glutamic acid residue at position 225 of wild-type human Neu2 is substituted with proline (E225P); (aa) the glutamic acid residue at position 239 of wild-type human Neu2 is substituted with alanine (T249A); the histidine residue is substituted with proline (H239P); (bb) the leucine residue at position corresponding to position 240 of wild-type human Neu2 is substituted with aspartic acid (L240D), asparagine (L240N), or tyrosine (L240Y); (cc) the arginine residue at position corresponding to position 241 of wild-type human Neu2 is substituted with alanine (R241A), aspartic acid (R241D), leucine (R241L), glutamine (R241Q), or tyrosine (R241Y);(dd) The alanine residues at the position corresponding to position 242 of wild-type human Neu2 are cysteine ​​(A242C), phenylalanine (A242F), glycine (A242G), histidine (A242H), isoleucine (A242I), lysine (A242K), leucine (A242L), methionine (A242M), asparagine (A242N), glutamine (A242Q), arginine (A242R), and serine (A (ee) the valine residue at the position corresponding to position 244 of wild-type human Neu2 is substituted with isoleucine (V244I), lysine (V244K) or proline (V244P); (ff) the glutamic acid residue at the position corresponding to position 257 of wild-type human Neu2 is substituted with proline (E257P); ( (gg) the serine residue at position 258 is substituted with a cysteine ​​(S258C); (hh) the leucine residue at position 260 of wild-type human Neu2 is substituted with an aspartic acid (L260D), phenylalanine (L260F), glutamine (L260Q), or threonine (L260T); (ii) the valine residue at position 265 of wild-type human Neu2 is substituted with a phenylalanine (V26 (jj) the glutamine residue at position corresponding to position 270 of wild-type human Neu2 is substituted with alanine (Q270A), histidine (Q270H), phenylalanine (Q270F), proline (Q270P), serine (Q270S), or threonine (Q270T); (kk) the tryptophan residue at position corresponding to position 292 of wild-type human Neu2 is substituted with arginine (W292R);(ll) The serine residue at position 301 of wild-type human Neu2 is alanine (S301A), aspartic acid (S301D), glutamic acid (S301E), phenylalanine (S301F), glycine (S301G), histidine (S301H), isoleucine (S301I), lysine (S301K), leucine (S301L), methionine (S301M), asparagine (S301N), and proline (S (mm) the tryptophan residue at position 302 of wild-type human Neu2 was replaced with alanine (W302A), aspartic acid (W302D), glutamic acid (W302E), or phenylalanine (W302F). , glycine (W302G), histidine (W302H), isoleucine (W302I), lysine (W302K), leucine (W302L), methionine (W302M), asparagine (W302N), proline (W302P), glutamine (W302Q), arginine (W302R), serine (W302S), threonine (W302T), valine (W302V), or tyrosine (W302Y); (n n) the valine residue at the position corresponding to position 363 of wild-type human Neu2 is substituted with arginine (V363R); or (oo) the leucine residue at the position corresponding to position 365 of wild-type human Neu2 is substituted with glutamine (L365Q), histidine (L365H), isoleucine (L365I), lysine (L365K), or serine (L365S); or the sialidase comprises any combination of the foregoing substitutions. For example, the sialidase can include a substitution selected from K9D, P62G, P62N, P62S, P62T, D80P, A93E, Q126H, Q126Y, R189P, H239P, A242T, Q270A, Q270S, Q270T, S301A, S301R, W302K, W302R, V363R, or L365I, or any combination of the foregoing substitutions;

[0077] In some embodiments, the recombinant mutant human sialidase comprises a deletion of a leucine residue at a position corresponding to position 184 of wild-type human Neu2 (ΔL184), a deletion of a histidine residue at a position corresponding to position 185 of wild-type human Neu2 (ΔH185), a deletion of a proline residue at a position corresponding to position 186 of wild-type human Neu2 (ΔP186), a deletion of an isoleucine residue at a position corresponding to position 187 of wild-type human Neu2 (ΔI187), and a deletion of a glutamine residue at a position corresponding to position 184 of wild-type human Neu2 (ΔQ188), or any combination of the foregoing deletions.

[0078] In some embodiments, the recombinant mutant human sialidase comprises an insertion, e.g., an insertion of an amino acid selected from S, T, Y, L, F, A, P, V, I, N, D, and H, between the threonine residue at the position corresponding to position 216 of wild-type human Neu2 and the leucine residue at the position corresponding to position 217 of wild-type human Neu2.

[0079] Further exemplary sialidase mutations and combinations of sialidase mutations are described in International (PCT) Patent Application No. PCT / US2019 / 012207, filed January 3, 2019, e.g., in the section entitled "I. Recombinant Human Sialidases" in the Detailed Description and in Examples 1, 2, 3, 4, 5, and 6 in the Examples.

[0080] f. Permutation combinations The present invention further provides recombinant mutant human sialidases comprising any combination of mutations contemplated herein. For example, a recombinant mutant sialidase enzyme can comprise a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more of the mutations contemplated herein. It is contemplated that a recombinant mutant sialidase enzyme can comprise 1-15, 1-10, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-15, 2-10, 2-7, 2-6, 2-5, 2-4, 2-3, 3-15, 3-10, 3-7, 3-6, 3-5, or 3-4 of the mutations contemplated herein.

[0081] For example, the recombinant mutant sialidase enzyme can include an M1 deletion (ΔM1), an M1A substitution, an M1D substitution, a V6Y substitution, a K9D substitution, a P62G substitution, a P62N substitution, a P62S substitution, a P62T substitution, an A93E substitution, an I187K substitution, a Q270A substitution, an S301R substitution, a W302K substitution, a C332A substitution, a V363R substitution, an L365I substitution, or any combination of the foregoing.

[0082] In some embodiments, the recombinant mutant sialidase enzyme comprises an M1 deletion (ΔM1), an M1A substitution, an M1D substitution, a V6Y substitution, an I187K substitution, a C332A substitution, or any combination of the foregoing. For example, the recombinant mutant sialidase enzyme can comprise: 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. A; M1D, V6Y and I187K; M1D, V6Y and C332A; M1D, I187K and C332A; ΔM1, V6Y and I187K; ΔM1, V6Y and C332A; ΔM1, I187K and C332A; V6Y, I187K and C332A; M1A, V6Y, I187K and C332A; M1D, V6Y, I187K and C332A; and ΔM1, V6Y, I187K and C332A.

[0083] In some embodiments, the recombinant mutant sialidase enzyme comprises (i) an amino acid substitution identified in Table 7 or any combination of amino acid substitutions identified in Table 7, and (ii) an M1 deletion (ΔM1), an M1A substitution, an M1D substitution, a V6Y substitution, an I187K substitution, a C332A substitution, or any combination of the foregoing. For example, recombinant mutant sialidase enzymes can include (i) an amino acid substitution identified in Table 7 or any combination of amino acid substitutions identified in Table 7, and (ii) M1A and V6Y; M1A and I187K; M1A and C332A; M1D and V6Y; M1D and I187K; M1D and C332A; ΔM1 and V6Y; ΔM1 and I187K; ΔM1 and C332A; V6Y and I187K; V6Y and C332A; I187K and C332A; M1A, V6Y and I187K; M1A, The mutations may include combinations of mutations selected from V6Y and C332A; M1A, I187K and C332A; M1D, V6Y and I187K; M1D, V6Y and C332A; M1D, I187K and C332A; ΔM1, V6Y and I187K; ΔM1, V6Y and C332A; ΔM1, I187K and C332A; V6Y, I187K and C332A; M1A, V6Y, I187K and C332A; M1D, V6Y, I187K and C332A; and ΔM1, V6Y, I187K and C332A.

[0084] In some embodiments, the recombinant mutant sialidase enzyme has: (a) substitutions of M1D, V6Y, P62G, A93E, I187K, and C332A; (b) substitutions of M1D, V6Y, K9D, A93E, I187K, C332A, V363R, and L365I; (c) substitutions of M1D, V6Y, P62N, I187K, and C (d) substitutions of M1D, V6Y, I187K, Q270A, S301R, W302K, and C332A; (e) substitutions of M1D, V6Y, P62S, I187K, Q270A, S301R, W302K, and C332A; (f) substitutions of M1D, V6Y, P62T, I187K, Q270A, S301R , W302K and C332A substitutions; (g) M1D, V6Y, P62N, I187K, Q270A, S301R, W302K and C332A substitutions; (h) M1D, V6Y, P62G, A93E, I187K, S301A, W302R and C332A substitutions; (i) M1D, V6Y, P62G, A9 (j) substitutions of M1D, V6Y, P62G, A93E, Q126Y, I187K, and C332A; or (k) substitutions of M1D, V6Y, P62G, A93E, Q126Y, I187K, A242F, Q270T, and C332A.

[0085] In some embodiments, the recombinant mutant human sialidase comprises a substitution of a serine residue at position 301 of wild-type human Neu2 (S301) combined with a substitution of a tryptophan residue at position 302 of wild-type human Neu2 (W302). For example, the recombinant mutant human sialidase can comprise a combination of substitutions corresponding to the combinations of substitutions listed in the column of Table 8 (amino acid positions corresponding to wild-type human Neu2 (SEQ ID NO: 1)). For example, the recombinant mutant human sialidase can comprise: a substitution of S301K and W302R; a substitution of S301K and W302K; or a substitution of S301A and W302S. [Table 8]

[0086] In some embodiments, the recombinant mutant human sialidase comprises a combination of substitutions corresponding to the combination of substitutions listed in the column of Table 9 (amino acid positions corresponding to wild-type human Neu2 (SEQ ID NO: 1)). [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4]

[0087] In some embodiments, the recombinant mutant human sialidase comprises the amino acid sequence of any of SEQ ID NOs: 48-54, 149, 154, 159, or 191, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 48-54, 149, 154, 159, or 191.

[0088] In some embodiments, the recombinant mutant human sialidase [Table 10-1] [Table 10-2] (SEQ ID NO: 47), wherein X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val, or absent, X2 is Ala or Lys, X3 is Asn or Leu, X4 is Pro or His, X5 is Phe, Trp, Tyr, or Val, X6 is Lys or Asp, X7 is Lys, Arg, or Glu, X8 is Lys, Ala, Arg, or Glu, X9 is Leu or Met, and X 10is Pro, Asn, Asp, His, Glu, Gly, Ser, or Thr, and X 11 is Gln or His, and X 12 is Arg or Lys, and X 13 is Ala, Glu, or Lys, and X 14 is Gly or Asp, and X 15 is Gln or His, and X 16 is Gln, Arg, or Lys, and X 17 is Ala, Cys, Ile, Ser, Val, or Leu, and X 18 is Gln or Leu, and X 19 is Ala or Val, and X 20 is Cys or Gly, and X 21 is Ala or Gly, and X 22 is Arg, Ile, or Lys, and X 23 is Ala, Cys, Leu, or Val, and X 24 is Leu, Ala, or Val, and X 25 is Thr or Ala, and X 26 is Asp or Gly, and X 27 is Glu or Lys, and X 28 is Gln, Ala, His, Phe or Pro, and X 29 is Cys or Val, and X 30 is Trp or Arg, and X 31 is Ser or Arg, and X 32 is Trp or Lys, and X 33 is Lys or Val, and X 34 is Ala, Cys, Ser or Val, and X 35 is Cys, Leu, or Val, and X 36 is Val or Arg, and X 37 is Leu, Gln, His, Ile, Lys, or Ser, and the sialidase comprises at least one mutation relative to wild-type human Neu2 (SEQ ID NO: 1).

[0089] In some embodiments, the recombinant mutant human sialidase [Table 11] (SEQ ID NO: 46), wherein X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val or absent, X2 is Phe, Trp, Tyr or Val, X3 is Lys or Asp, X4 is Pro, Asn, Asp, His, Glu, Gly, Ser or Thr, X5 is Ala, Glu or Lys, X6 is Arg, Ile or Lys, X7 is Gln, Ala, His, Phe or Pro, X8 is Ser or Arg, X9 is Trp or Lys, and X 10 is Ala, Cys, Ser or Val, and X 11 is Val or Arg, and X 12 is Leu, Gln, His, Ile, Lys, or Ser, and the sialidase comprises 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, and X 10 is Ala or Cys, and X 11 is Val or Arg, and X 12 is Leu or Ile.

[0090] In some embodiments, the recombinant mutant human sialidase [Table 12] (SEQ ID NO: 182), wherein X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val or absent, X2 is Ala or Lys, X3 is Asn or Leu, X4 is Pro or His, X5 is Phe, Trp, Tyr or Val, X6 is Lys or Asp, X7 is Lys, Arg or Glu, X8 is Lys, Ala, Arg or Glu, X9 is Leu or Met, and X 10 is Pro, Asn, Asp, His, Glu, Gly, Ser, or Thr, and X 11 is Gln or His, and X 12 is Arg or Lys, and X 13 is Asp or Pro, and X 14 is Ala, Glu, or Lys, and X 15 is Gly or Asp, and X 16 is Gln or His, and X 17 is Gln, Arg, or Lys, and X 18 is Ala, Cys, Ile, Ser, Val, or Leu, and X 19 is Gln, Leu, Glu, Phe, His, Ile, Leu, or Tyr, and X 20 is Ala or Val, and X 21 is Cys or Gly, and X 22 is Arg or Pro, and X 23 is Ala or Gly, and X 24 is Arg, Ile, or Lys, and X 25 is Gln or Pro, and X 26 is Arg or Pro, and X 27 is Ala, Cys, Leu, or Val, and X 28 is Ala, Cys, Asn, Ser or Thr, and X 29 is Leu, Ala, or Val, and X 30 is Glu or Pro, and X 31 is His or Pro, and X 32 is Leu, Asp, Asn, or Tyr, and X33 is Arg, Ala, Asp, Leu, Gln, or Tyr, and X 34 is Ala, Cys, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Val, Trp, or Tyr, and X 35 is Val, Ile, or Lys, and X 36 is Thr or Ala, and X 37 is Asp or Gly, and X 38 is Glu, Lys or Pro, and X 39 is Ser or Cys, and X 40 is Leu, Asp, Phe, Gln or Thr, and X 41 is Val or Phe, and X 42 is Gln, Ala, His, Phe, Pro, Ser, or Thr, and X 43 is Cys or Val, and X 44 is Trp or Arg, and X 45 is Ser, Arg, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Thr, Val, Trp, or Tyr, and X 46 is Trp, Lys, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Tyr, and X 47 is Lys or Val, and X 48 is Ala, Cys, Ser or Val, and X 49 is Cys, Leu, or Val, and X 50 is Val or Arg, and X 51 is Leu, Gln, His, Ile, Lys, or Ser, and the sialidase comprises at least one mutation relative to wild-type human Neu2 (SEQ ID NO: 1).

[0091] In some embodiments, the recombinant mutant human sialidase [Table 13] (SEQ ID NO: 183), wherein X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val or absent, X2 is Phe, Trp, Tyr or Val, X3 is Lys or Asp, X4 is Pro, Asn, Asp, His, Glu, Gly, Ser or Thr, and X5 is Ala, G 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, and X 10 is Ser, Arg, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Thr, Val, Trp, or Tyr, and X 11 is Trp, Lys, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Tyr, and X 12 is Ala, Cys, Ser or Val, and X 13 is Val or Arg, and X 14 is Leu, Gln, His, Ile, Lys, or Ser, and the sialidase comprises 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, and X 10 is Ser, Arg, or Ala, and X 11 is Trp, Lys or Arg, and X 12 is Ala or Cys, and X 13is Val or Arg, and X 14 is Leu or Ile.

[0092] In some embodiments, the recombinant mutant human sialidase contains conservative substitutions relative to the recombinant mutant human sialidase sequence disclosed herein. As used herein, the term "conservative substitution" refers to a substitution with a structurally similar amino acid. For example, conservative substitutions can include those 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. Conservative substitutions can 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).

[0093] Sequence identity can be determined by various methods within the skill of those skilled in the art, for example, by using publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. BLAST (Basic Local Alignment Search Tool) analysis (Karlin et al., (1990) PROC. NATL. ACAD. SCI. USA 87:2264-2268; Altschul, (1993) J. MOL. EVOL. 36, 290-300; Altschul et al., (1997) NUCLEIC ACIDS RES. 25:3389-3402, incorporated herein by reference) is adjusted for searching sequence similarity. For a discussion of basic issues in searching sequence databases, see Altschul et al., (1994) NATURE GENETICS 6:119-129, fully incorporated herein by reference. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Search parameters for histogram, description, alignment, expect (i.e., the statistical significance threshold for reporting matches to database sequences), cutoff, matrix, and filter are at default settings. The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et al., (1992) PROC. NATL. ACAD. SCI. USA 89:10915-10919, fully incorporated herein by reference).Four blastn parameters can be adjusted as follows: Q=10 (gap creation penalty); R=10 (gap extension penalty); wink=1 (generates word hits at every wink.sup.th position along the query); and gapw=16 (sets the window width over which gapped alignments are generated). Equivalent blastp parameter settings would be Q=9; R=2; wink=1; and gapw=32. Searches can also be performed using NCBI (National Center for Biotechnology Information) BLAST Advanced Option parameters (e.g.: -G, cost for an open gap [integer]: default = 5 for nucleotides / 11 for proteins; -E, cost for an extension gap [integer]: default = 2 for nucleotides / 1 for proteins; -q, penalty for a nucleotide mismatch [integer]: default = -3; -r, reward for a nucleotide match [integer]: default = 1; -e, expectation value [real number]: default = 10; -W, word size [integer]: default = 11 for nucleotides / 28 for megablast / 3 for proteins; -y, dropoff for blast extension in bits (X): default = 20 for blastn / 7 for others; -X, X dropoff value for gapped alignments (in bits): default = 15 for all programs but not applicable to blastn; and -Z, final X dropoff value for gapped alignments (in bits): 50 for blastn, 25 for others). ClustalW for pairwise protein alignments may also be used (default parameters may include, for example, a Blosum62 matrix and a Gap Opening Penalty=10 and a Gap Extension Penalty=0.1).Best-fit comparisons between sequences, available in the GCG package version 10.0, use DNA parameters GAP=50 (gap creation penalty) and LEN=3 (gap extension penalty). The equivalent settings for best-fit protein comparisons are GAP=8 and LEN=2.

[0094] II. Fusion Protein / Antibody Conjugates To promote the selective removal of sialic acid on hypersialylated cancer cells and / or in the tumor microenvironment, it may be useful to target a sialidase as described herein to such cells or the tumor microenvironment. Furthermore, to promote the removal of sialic acid by sialidase in a subject, it may be useful to extend the plasma half-life of the sialidase in the subject. This can be achieved by including the sialidase in a fusion protein and / or antibody conjugate (e.g., a chemically conjugated conjugate).

[0095] Thus, the present invention further provides a fusion protein comprising a sialidase enzyme, or a functional fragment thereof, and a portion or fragment of an antibody, such as an immunoglobulin Fc domain (also referred to herein as an Fc domain), or an immunoglobulin antigen-binding domain (also referred to herein as an antigen-binding domain). In some embodiments, the sialidase and the antibody, or portion thereof (e.g., the immunoglobulin Fc domain or antigen-binding domain), are linked by a peptide bond or an amino acid linker.

[0096] As used herein, unless otherwise indicated, the term "fusion protein" is understood to refer to a single polypeptide chain comprising amino acid sequences based on two or more separate proteins or polypeptide chains, where the two amino acid sequences may be fused together directly or via an intervening linker sequence, e.g., via an intervening amino acid linker. Nucleotide sequences encoding fusion proteins may be generated, for example, using conventional recombinant DNA techniques.

[0097] In some embodiments, the fusion protein comprises a tag, such as a Strep tag (e.g., a Strep II tag), a His tag (e.g., a 10x His tag), a myc tag, or a FLAG tag. The tag can be positioned at the C-terminus or N-terminus of the fusion protein. In some embodiments, the fusion protein comprises a sialidase moiety linked in the N-terminal to C-terminal direction to a polypeptide comprising an immunoglobulin heavy chain, wherein the sialidase moiety comprises an N-terminal addition of MEDLRP (SEQ ID NO: 4), and the Strep II tag is positioned at the C-terminus of the immunoglobulin heavy chain or the N-terminus of the sialidase moiety.

[0098] a. Sialidase moiety The sialidase portion of the fusion proteins described herein can be any sialidase, such as a fungal, bacterial, non-human mammalian, or human sialidase. In some embodiments, the sialidase portion is a recombinant human sialidase that contains at least one mutation relative to wild-type human sialidase, such as at least one amino acid substitution, deletion, or addition, as described above.

[0099] In some embodiments, the sialidase is any recombinant mutant human sialidase or functional fragment thereof disclosed herein.

[0100] In some embodiments, the sialidase portion comprises the 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 portion comprises an LSHSLST (SEQ ID NO: 22) peptide at the N-terminus. In some embodiments, the sialidase portion comprises an N-terminal addition and an A2K substitution of MEDLRP (SEQ ID NO: 4). In some embodiments, the sialidase portion comprises an N-terminal addition and a C332A substitution of MEDLRP (SEQ ID NO: 4). In some embodiments, the sialidase portion comprises an N-terminal addition, a C332A substitution, and a C352L substitution of MEDLRP (SEQ ID NO: 4).

[0101] In some embodiments, the sialidase portion comprises an M1 deletion (ΔM1), an M1A substitution, an M1D substitution, a V6Y substitution, a K9D substitution, a P62G substitution, a P62N substitution, a P62S substitution, a P62T substitution, an A93E substitution, a Q126Y substitution, an I187K substitution, an A242T substitution, a Q270A substitution, a Q270T substitution, an S301R substitution, an S301R substitution, a W302K substitution, a W302R substitution, a C332A substitution, a V363R substitution, an L365I substitution, or any combination of the foregoing.

[0102] In some embodiments, the sialidase portion comprises the amino acid sequence of any of SEQ ID NOs: 48-54, 149, 154, 159, or 191, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 48-54, 149, 154, 159, or 191.

[0103] b. Antibody part As used herein, unless otherwise indicated, the term "antibody" is understood to mean an intact antibody (e.g., an intact monoclonal antibody), or a fragment thereof, such as an Fc fragment of an antibody (e.g., an Fc fragment of a monoclonal antibody), or an antigen-binding fragment of an antibody (e.g., an antigen-binding fragment of a monoclonal antibody), such as an intact antibody, antigen-binding fragment, or Fc fragment that has been modified, engineered, or chemically conjugated. Examples of antigen-binding fragments include Fab, Fab', (Fab')2, Fv, single-chain antibodies (e.g., scFv), minibodies, and diabodies. Examples of modified or engineered antibodies include chimeric antibodies, humanized antibodies, and multispecific antibodies (e.g., bispecific antibodies). An example of a chemically conjugated antibody is an antibody conjugated to a toxin moiety.

[0104] In some embodiments, the fusion protein comprises an immunoglobulin Fc domain. As used herein, unless otherwise indicated, the term "immunoglobulin Fc domain" refers to a fragment of an immunoglobulin heavy chain constant region that can bind to an Fc receptor, either alone or in combination with a second immunoglobulin Fc domain. An immunoglobulin Fc domain can, for example, comprise an immunoglobulin CH2 and CH3 domain. An immunoglobulin Fc domain can, for example, comprise an immunoglobulin CH2 and CH3 domain and an immunoglobulin hinge region. The boundaries between the immunoglobulin hinge region, CH2 and CH3 domain are well known in the art and can be found, for example, in the PROSITE database (available on the World Wide Web at prosite.expasy.org).

[0105] In some embodiments, the immunoglobulin Fc domain is derived from a human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, or IgM Fc domain. A single amino acid substitution (S228P according to Kabat numbering; designated IgG4Pro) can be introduced to disrupt the heterogeneity observed in recombinant IgG4 antibodies. See Angal, S. et al. (1993) MOL. IMMUNOL. 30:105-108.

[0106] In some embodiments, the immunoglobulin Fc domain is derived from a human IgG1 isotype or another isotype that elicits 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:5).

[0107] In some embodiments, the immunoglobulin Fc domain is derived from the human IgG4 isotype or another isotype that elicits little or no antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-mediated cytotoxicity (CDC). In some embodiments, the immunoglobulin Fc domain is derived from the human IgG4 isotype.

[0108] In some embodiments, the immunoglobulin Fc domain comprises either a "knob" mutation, e.g., T366Y, or a "hole" mutation, e.g., Y407T (residue numbering according to EU numbering, Kabat, EA, et al. (1991) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, FIFTH EDITION, US Department of Health and Human Services, NIH Publication No. 91-3242), for heterodimerization with a second polypeptide.

[0109] In some embodiments, the fusion protein comprises an immunoglobulin antigen-binding domain. The inclusion of such a domain may improve targeting of the fusion protein to sialylated cancer cells and / or the tumor microenvironment. As used herein, unless otherwise indicated, the term "immunoglobulin antigen-binding domain" refers to a polypeptide that, alone or in combination with another immunoglobulin antigen-binding domain, defines an antigen-binding site. Exemplary immunoglobulin antigen-binding domains include, for example, an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, where the variable regions together define an antigen-binding site.

[0110] Immunoglobulin antigen-binding domains and / or antigen-binding sites include, for example, adecatumumab, ascrinvacumab, cixutumumab, conatumumab, daratumumab, drozitumab, durigotumab, durvalumab, dusigitumab, enfortumab, enoticumab, epratuxumab, figitumumab, ganitumab (ganitumab), glembatumumab, intetumumab, ipilimumab, iratumumab, icrucumab, lexatumumab, lucatumumab, mapatumumab, narnatumab, necitumumab, nesbacumab, ofatumumab, olaratumumab, panitumumab, patritumab, pritumumab, radretumab, ramucirumab, rilo Rilotumumab, robatumumab, seribantumab, tarextumab, teprotumumab, tovetumab, vantictumab, besencumab, votumumab, zalutumumab, flanvotumab, altumomab, anatumomab, arcitumomab mab), bectumomab, blinatumomab, detumomab, ibritumomab, minretumomab, mitumomab, moxetumomab, naptumomab, nofetumomab, pemtumomab, pintumomab, racotumomab, satumomab, solitomab, taplitumomab,Tenatumomab, tositumomab, tremelimumab, abagovomab, atezolizumab, durvalumab, avelumab, igovomab, oregovomab, capromab, edrecolomab, nacolomab, amatuximab, bavituximab, brentuximab, cetuximab, derlotuximab, dinutuximab, ensituximab, futuximab, girencki Cimab, indatuximab, isatuximab, margetuximab, rituximab, siltuximab, ublituximab, ecromeximab, abituzumab, alemtuzumab, bevacizumab, bivatuzumab, brontixituzumab, cantuzumab, cantuzumab, sitatuzumab, clivatuzumab, Dacetuzumab, demcizumab, dalotuzumab, denintuzumab, elotuzumab, emactuzumab, emibetuzumab, enoblituzumab, etaracizumab, farletuzumab, ficlatuzumab, gemtuzumab, imgatuzumab, inotuzumab, labetuzumab, rifastuzumab, lintuzumab, lirilumab, lorvotuzumab, lumletuzumab, matuzumab Mab, milatuzumab, moxetumomab, nimotuzumab, obinutuzumab, ocaratuzumab, otlertuzumab, onartuzumab, oportuzumab, parsatuzumab, pertuzumab, pidilizumab, pinatuzumab, polatuzumab, sibrotuzumab, simtuzumab, tacatuzumab, tigatuzumab,Trastuzumab, tucotuzumab, urelumab, vandortuzumab, vanucizumab, veltuzumab, borsetuzumab, sofituzumab, catumaxomab, ertumaxomab, depatuxizumab, ontuxizumab In some embodiments, the immunoglobulin antigen-binding domain may be derived from an antibody selected from trastuzumab, daratumumab, girentuximab, ofatumumab, and rituximab.

[0111] In some embodiments, the immunoglobulin antigen-binding domain is derived from trastuzumab. The trastuzumab heavy chain amino acid sequence is set forth in SEQ ID NO: 63, and the trastuzumab light chain amino acid sequence is set forth in SEQ ID NO: 64. The amino acid sequence of an exemplary scFv derived from trastuzumab is set forth in SEQ ID NO: 65.

[0112] Immunoglobulin antigen-binding domains and / or antigen-binding sites include, for example, adenosine A2a receptor (A2aR), A kinase anchoring protein 4 (AKAP4), B melanoma antigen (BAGE), brother of the regulator of imprinted sites (BORIS), breakpoint cluster region Abelson tyrosine kinase (BCR / ABL), CA125, CAIX, CD19, CD20, CD22, CD30, CD33, CD52, CD73, CD137, carcinoembryonic antigen (CEA), CS1, cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), estrogen receptor-binding site-associated antigen 9 (EBAG9), epidermal growth factor (EGF), epidermal growth factor receptor (EGFR), EGF-like module receptor 2 (EMR2), epithelial cell adhesion molecule (EpCAM). (17-1A), FR-α, G antigen (GAGE), disialoganglioside GD2 (GD2), glycoprotein 100 (gp100), human epidermal growth factor receptor 2 (Her2), hepatocyte growth factor (HGF), human papillomavirus 16 (HPV-16), heat shock protein 105 (HSP105), isocitrate dehydrogenase type 1 (IDH1), idiotype (NeuGcGM3), indoleamine-2,3-dioxygenase 1 (IDO1), IGF-1, IGF1R, IGG1K, killer cell immunoglobulin-like receptor (KIR), lymphocyte activation gene 3 (LAG-3), lymphocyte antigen 6 complex K (LY6K), matri Metalloproteinase 16 (MMP16), melanotransferrin (MFI2), melanoma antigen 3 (MAGE-A3), melanoma antigen C2 (MAGE-C2), melanoma antigen D4 (MAGE-D4), melanoma antigen recognized by T cells 1 (Melan-A / MART-1), N-methyl-N'-nitroso-guanidine human osteosarcoma transforming gene (MET), mucin 1 (MUC1), mucin 4 (MUC4), mucin 16 (MUC16), New York esophageal squamous cell carcinoma 1 (NY-ESO-1), prostaticacid phosphatase (PAP), programmed death receptor 1 (PD-1), programmed death receptor ligand 1 (PD-L1), phosphatidylserine, preferentially expressed antigen in melanoma (PRAME), prostate-specific antigen (PSA), protein tyrosine kinase 7 (PTK7, also known as colon cancer kinase 4 (CCK4)), receptor tyrosine kinase orphan receptor 1 (ROR1), scatter factor receptor kinase, sialyl-Tn, sperm-associated antigen 9 (SPAG-9), synovial sarcoma X chromosome breakpoints breakpoint 1 (SSX1), survivin, telomerase, T-cell immunoglobulin domain and mucin domain 3 (TIM-3), vascular endothelial growth factor (VEGF) (e.g., VEGF-A), vascular endothelial growth factor receptor 2 (VEGFR2), V-domain immunoglobulin-containing suppressor of T-cell activation (VISTA), Wilms' tumor 1 (WT1), X chromosome antigen 1b (XAGE-1b), 5T4, mesothelin, glypican 3 (GPC3), folate receptor alpha (FRα), prostate-specific membrane antigen (PSMA), cMET, CD38, B-cell maturation antigen (BCMA), CD123, CLDN6, CLDN9, LRRC15, PRLR (prolactin receptor), RING finger protein 43 (RNF43), Uroplakin-1 B (UPK1 B), tumor necrosis factor superfamily member 9 (TNFSF9), tumor necrosis factor receptor superfamily member 21 (TNFSRF21), bone morphogenetic protein receptor type 1B (BMPR1B), kringle domain-containing transmembrane protein 2 (KREMEN2), delta-like protein 3 (DLL3), Siglec7, and Siglec9. Further exemplary cancer antigens include those found on cancer stem cells, such as SSEA3, SSEA4, TRA-1-60, TRA-1-81, SSEA1, CD133 (AC133), CD90 (Thy-1), CD326 (EpCAM), Cripto-1 (TDGF1), PODXL-1 (podocalyxin-like protein 1), ABCG2, CD24, CD49f (integrin α6), Notch2, and CD146.(MCAM), CD10 (neprilysin), CD117 (c-KIT), CD26 (DPP-4), CXCR4, CD34, CD271, CD13 (alanine aminopeptidase), CD56 (NCAM), CD105 (endoglin), LGR5, CD114 (CSF3R), CD54 (ICAM-1), CXCR1,2, TIM-3 (HAVCR2), CD55 (DAF), DLL4 (delta-like ligand 4), CD20 (MS4A1), and CD96.

[0113] The present invention further provides antibody conjugates comprising one or more of the fusion proteins disclosed herein. As used herein, unless otherwise indicated, the term "antibody conjugate" is understood to refer to an antibody or functional fragment thereof comprising antigen-binding activity and / or Fc receptor-binding activity conjugated (e.g., covalently) to an additional functional moiety. In some embodiments, the antibody or functional antibody fragment is conjugated to a sialidase enzyme, such as a recombinant mutant human sialidase enzyme disclosed herein. In some embodiments, the antibody conjugate comprises a single polypeptide chain. In some embodiments, the antibody conjugate comprises two, three, four, or more polypeptide chains that are covalently or non-covalently linked together to form a multimeric complex, such as a dimeric, trimeric, or tetrameric complex.

[0114] Table 10 shows antibodies and antibody-drug conjugates suitable for use according to the present invention, the antigen bound by the antibody or antibody-drug conjugate, and, for a particular antibody, the type of cancer targeted by the antibody or antibody-drug conjugate. [Table 14-1] [Table 14-2] [Table 14-3]

[0115] c. Linker In some embodiments, the sialidase portion of the fusion protein can be directly linked or fused to the antibody portion of the fusion protein (e.g., the immunoglobulin Fc domain and / or the immunoglobulin antigen-binding domain). In other embodiments, the sialidase portion can be covalently linked to the antibody portion by a linker.

[0116] The linker can link one or more naturally occurring amino acids, the sialidase or functional fragment thereof, and the antibody moiety or fragment, where the amino acid (e.g., a cysteine ​​amino acid) can be introduced by site-directed mutagenesis. The linker can also include one or more unnatural amino acids. In certain situations, it is contemplated that a linker containing, for example, one or more sulfhydryl-reactive groups (e.g., maleimide) can be covalently bonded to a cysteine ​​in a sialidase moiety or antibody moiety that is a naturally occurring cysteine ​​residue or the product of site-directed mutagenesis.

[0117] The linker may be a cleavable linker or a non-cleavable linker. Optionally or additionally, the linker may be a flexible linker or a non-flexible linker.

[0118] The linker should be long enough to allow the sialidase and antibody moieties to be linked without steric hindrance from each other, and short enough to retain the intended activity of the fusion protein. The linker is preferably sufficiently hydrophilic to avoid or minimize instability of the fusion protein. The linker is preferably sufficiently hydrophilic to avoid or minimize insolubility of the fusion protein. The linker should be sufficiently stable in vivo (e.g., not cleaved by serum, enzymes, etc.) so that the fusion protein can be effective in vivo.

[0119] The linker can be from about 1 angstrom (Å) to about 150 Å in length, or from about 1 Å to about 120 Å in length, or from about 5 Å to about 110 Å in length, or from about 10 Å to about 100 Å in length. The linker can be 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 Å or more in length 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, 31 Å or less in length. Additionally, the linker can be about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110 and 120 Å in length.

[0120] In some embodiments, the linker comprises a polypeptide linker that links or fuses the sialidase portion of the fusion protein to the antibody portion of the fusion protein (e.g., the immunoglobulin Fc domain and / or the immunoglobulin antigen-binding domain). For example, it is contemplated that a gene encoding a sialidase portion linked directly or indirectly (e.g., via an amino acid-containing linker) to an antibody portion can be made and expressed using conventional recombinant DNA techniques. For example, the amino terminus of the sialidase portion can be linked to the carboxy terminus of either the light or heavy chain of the antibody portion. For example, for a Fab fragment, the amino or carboxy terminus of the sialidase portion can be linked to the first constant domain (CH1) of the antibody heavy chain. When a linker is used, the linker can 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 to 25 amino acid residues, 1 to 20 amino acid residues, 2 to 15 amino acid residues, 3 to 10 amino acid residues, 3 to 7 amino acid residues, 4 to 25 amino acid residues, 4 to 20 amino acid residues, 4 to 15 amino acid residues, 4 to 10 amino acid residues, 5 to 25 amino acid residues, 5 to 20 amino acid residues, 5 to 15 amino acid residues, or 5 to 10 amino acid residues. Exemplary linkers include glycine- and serine-rich linkers, such as (GlyGlyPro). n or (GlyGlyGlyGlySer) n wherein n is 1 to 5. In some embodiments, the linker comprises, consists of, or consists essentially of GGGGS (SEQ ID NO: 184). In some embodiments, the linker comprises, consists of, or consists essentially of GGGGSGGGGS (SEQ ID NO: 145). In some embodiments, the linker comprises, consists of, or consists essentially of EPKSS (SEQ ID NO: 146). Further exemplary linker sequences are disclosed, for example, in George et al. (2003) PROTEIN ENGINEERING 15:871-879, and U.S. Pat. Nos. 5,482,858 and 5,525,491.

[0121] In some embodiments, the fusion protein comprises the amino acid sequence of any of SEQ ID NOs: 66-85, 98-142, 150-153, 155-158, 160-163, 166-178, 185, 187, 189, or 192-197, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 66-85, 98-142, 150-153, 155-158, 160-163, 166-178, 185, 187, 189, or 192-197.

[0122] d. Antibody conjugates The present invention further provides an antibody conjugate comprising the fusion protein disclosed herein.The antibody conjugate may comprise a single polypeptide chain (i.e., the fusion protein disclosed herein), or the antibody conjugate may comprise additional polypeptide chains (e.g., one, two, or three additional polypeptide chains).For example, the antibody conjugate may comprise a first polypeptide (fusion protein) comprising a recombinant mutant human sialidase enzyme and an immunoglobulin heavy chain, and a second polypeptide comprising an immunoglobulin light chain, where, for example, the immunoglobulin heavy chain and light chain together define a single antigen-binding site.

[0123] In some embodiments, the antibody conjugate may comprise a single sialidase. In other embodiments, the antibody conjugate may comprise more than one (e.g., two) sialidases. When more than one sialidase is included, the sialidases may be the same or different. In some embodiments, the antibody conjugate may comprise a single antigen-binding site. In other embodiments, the antibody conjugate may comprise more than one (e.g., two) antigen-binding sites. When two antigen-binding sites are used, they may be the same or different. In some embodiments, the antibody conjugate comprises an immunoglobulin Fc fragment.

[0124] In some embodiments, the antibody conjugate comprises one or two immunoglobulin heavy chains, or functional fragments thereof. In some embodiments, the antibody conjugate comprises one or two immunoglobulin light chains, or functional fragments thereof. In some embodiments, the antibody conjugate comprises a sialidase fused to the N-terminus or C-terminus of an immunoglobulin heavy chain or an immunoglobulin light chain.

[0125] Figure 9 shows exemplary antibody conjugate constructs comprising one or more sialidase enzymes. For example, in Figure 9A, the first antigen-binding site is shown as 10, the second antigen-binding site is shown as 20, the sialidase is shown as 30, and the Fab is shown as 40. It is understood that in each of the constructs shown in Figures 9A-9I, the Fc may optionally be modified in some manner, for example, using knob-into-hole techniques, as shown at 50 in Figure 9B. Similar structures are shown with similar schematic representations throughout Figure 9.

[0126] Figure 9A shows an antibody conjugate construct comprising a first polypeptide comprising a first immunoglobulin light chain; a second polypeptide comprising a first immunoglobulin heavy chain; a third polypeptide comprising a second immunoglobulin heavy chain; and a fourth polypeptide comprising a second immunoglobulin light chain. The first and second polypeptides can be covalently linked together, the third and fourth polypeptides can be covalently linked together, and the second and third polypeptides can be covalently linked together. The covalent linkage can be a disulfide bond. In some embodiments, the first and second polypeptides together define a first antigen-binding site designated as 10, and the third and fourth polypeptides together define a second antigen-binding site designated as 20. A sialidase enzyme designated as 30 can be conjugated to the N- or C-terminus of the first and second immunoglobulin light chains or the first and second immunoglobulin heavy chains.

[0127] Figure 9B shows an antibody conjugate construct comprising a first polypeptide comprising a first immunoglobulin light chain; a second polypeptide comprising a first immunoglobulin heavy chain; a third polypeptide comprising a second immunoglobulin heavy chain; and a fourth polypeptide comprising a second immunoglobulin light chain. The first and second polypeptides may be covalently linked together, the third and fourth polypeptides may be covalently linked together, and the second and third polypeptides may be covalently linked together. The covalent linkage may be a disulfide bond. In some embodiments, the first and second polypeptides together define a first antigen-binding site, and the third and fourth polypeptides together define a second antigen-binding site. The sialidase enzyme may be conjugated to the N-terminus or C-terminus of the first immunoglobulin light chain or the first immunoglobulin heavy chain.

[0128] Figure 9C shows an antibody conjugate construct comprising a first polypeptide comprising an immunoglobulin light chain, a second polypeptide comprising an immunoglobulin heavy chain, and a third polypeptide comprising an immunoglobulin Fc domain. The first and second polypeptides can be covalently bonded together, and the second and third polypeptides can be covalently bonded together. The covalent bond can be a disulfide bond. In some embodiments, the first and second polypeptides together define an antigen-binding site. A sialidase enzyme can be conjugated to the N-terminus or C-terminus of the first immunoglobulin light chain or the first immunoglobulin heavy chain.

[0129] Figure 9D shows an antibody conjugate construct comprising a first polypeptide comprising an immunoglobulin light chain; a second polypeptide comprising an immunoglobulin heavy chain; and a third polypeptide comprising an immunoglobulin Fc domain and a first sialidase enzyme. The first and second polypeptides can be covalently linked together, and the second and third polypeptides can be covalently linked together. The covalent bond can be a disulfide bond. The third polypeptide comprises, from N- to C-terminus, a sialidase and an immunoglobulin Fc domain. In some embodiments, the first and second polypeptides together define an antigen-binding site. The optional second sialidase enzyme can be conjugated to the N- or C-terminus of the first immunoglobulin light chain or the first immunoglobulin heavy chain.

[0130] Figure 9E shows an antibody conjugate construct comprising a first polypeptide comprising an immunoglobulin light chain; a second polypeptide comprising an immunoglobulin heavy chain; and a third polypeptide comprising an immunoglobulin Fc domain and a first sialidase enzyme. The first and second polypeptides can be covalently linked together, and the second and third polypeptides can be covalently linked together. The covalent bond can be a disulfide bond. The third polypeptide comprises, from N- to C-terminus, an immunoglobulin Fc domain and a sialidase. In some embodiments, the first and second polypeptides together define an antigen-binding site. The optional second sialidase enzyme can be conjugated to the N- or C-terminus of the first immunoglobulin light chain or the first immunoglobulin heavy chain.

[0131] Figure 9F shows an antibody conjugate 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 can be covalently linked together. The covalent bond can be a disulfide bond. A sialidase enzyme can be conjugated to the N- or C-terminus of the first immunoglobulin Fc domain or the N- or C-terminus of the second immunoglobulin Fc domain. An optional second sialidase enzyme can be conjugated to the N- or C-terminus of the first immunoglobulin Fc domain or the N- or C-terminus of the second immunoglobulin Fc domain.

[0132] Figure 9G shows an antibody conjugate construct comprising a first polypeptide comprising an immunoglobulin light chain and a second polypeptide comprising an immunoglobulin heavy chain variable region. The first and second polypeptides can be covalently linked together. The covalent bond can be a disulfide bond. In some embodiments, the first and second polypeptides together define an antigen-binding site. A sialidase enzyme can be conjugated to the N-terminus or C-terminus of the immunoglobulin light chain or the immunoglobulin heavy chain variable region.

[0133] Figure 9H shows an antibody conjugate 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 can be covalently linked together. The covalent bond can be a disulfide bond. A sialidase enzyme can be conjugated to the N-terminus of the first immunoglobulin Fc domain or the second immunoglobulin Fc domain. An optional second sialidase enzyme can be conjugated to the N-terminus of the second immunoglobulin Fc domain or the first immunoglobulin Fc domain, respectively. A single-chain variable fragment (scFv) can be conjugated to the C-terminus of the first immunoglobulin Fc domain or the second immunoglobulin Fc domain, respectively. An optional second single-chain variable fragment (scFv) can be conjugated to the C-terminus of the first immunoglobulin Fc domain or the second immunoglobulin Fc domain, respectively.

[0134] Figure 9I shows an antibody conjugate construct similar to that shown in Figure 9H, except that each scFv is replaced with an immunoglobulin antigen-binding fragment, e.g., a Fab. For example, Figure 9I shows an antibody conjugate 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 can be covalently linked together. The covalent bond can be a disulfide bond. A sialidase enzyme can be conjugated to the N-terminus of the first immunoglobulin Fc domain or the second immunoglobulin Fc domain. An optional second sialidase enzyme can be conjugated to the N-terminus of the second immunoglobulin Fc domain or the first immunoglobulin Fc domain, respectively. An antibody fragment (Fab) can be conjugated or fused to the C-terminus of the first immunoglobulin Fc domain or the second immunoglobulin Fc domain. An optional second antibody fragment (Fab) can be conjugated or fused to the C-terminus of the second immunoglobulin Fc domain or the first immunoglobulin Fc domain, respectively. In the case of fusion, the C-terminus of the Fc domain is linked (either by a bond or an amino acid linker) to the first polypeptide chain defining the immunoglobulin antigen-binding fragment. In the case of an antibody having an antigen-binding site defined by a single variable region, this may be sufficient to confer binding affinity to the target antigen. In another example, for example, in the case of a human antibody, the first polypeptide chain defining the immunoglobulin antigen-binding fragment can be conjugated (e.g., covalently conjugated via a disulfide bond) to the second polypeptide chain defining the immunoglobulin antigen-binding fragment, where the two antigen-binding fragments together define an antigen-binding site for binding to the target antigen.

[0135] Figure 10 shows additional antibody conjugate constructs. For example, Figure 10 shows an antibody conjugate construct comprising a first polypeptide comprising an immunoglobulin light chain; a second polypeptide comprising an immunoglobulin heavy chain and an scFv; and a third polypeptide comprising an immunoglobulin Fc domain and a first sialidase enzyme. The first and second polypeptides can be covalently linked together, and the second and third polypeptides can be covalently linked together. The covalent linkage can be a disulfide bond. The second polypeptide comprises, from N- to C-terminal, a heavy chain and an scFv. The third polypeptide comprises, from N- to C-terminal, a sialidase and an immunoglobulin Fc domain. In some embodiments, the first and second polypeptides together define a first antigen-binding site. In some embodiments, the scFv defines a second antigen-binding site. Figure 10 shows a further antibody construct comprising a first polypeptide comprising an immunoglobulin light chain; a second polypeptide comprising an immunoglobulin heavy chain; and a third polypeptide comprising an immunoglobulin Fc domain and a first sialidase enzyme, wherein a Fab fragment is conjugated to the N-terminus of the immunoglobulin heavy chain. The first and second polypeptides can be covalently linked together, and the second and third polypeptides can be covalently linked together. The covalent bond can be a disulfide bond. The third polypeptide comprises, from N- to C-terminus, a sialidase and an immunoglobulin Fc domain. In some embodiments, the first and second polypeptides together define a first antigen-binding site. In some embodiments, the Fab fragment defines a second antigen-binding site. It is understood that in each of the constructs shown in Figure 10, the scFv, if present, can be replaced with the Fab fragment, or the Fab fragment, if present, can be replaced with the scFv. It is understood that in each of the constructs shown in Figure 10, the Fc may optionally be modified in several ways.

[0136] In some embodiments, the antibody conjugate comprises a first polypeptide comprising a first immunoglobulin light chain; a second polypeptide comprising a first immunoglobulin heavy chain and a first sialidase; a third polypeptide comprising a second immunoglobulin heavy chain and a second sialidase; and a fourth polypeptide comprising a second immunoglobulin light chain. An example of this embodiment is shown in FIG. 11A. The first and second polypeptides can be covalently linked together, the third and fourth polypeptides can be covalently linked together, and the second and third polypeptides can be covalently linked together. The covalent linkage can be a disulfide bond. In some embodiments, the first and second polypeptides together define a first antigen-binding site, and the third and fourth polypeptides together define a second antigen-binding site. In some embodiments, the second and third polypeptides comprise, from N- to C-terminal, the first and second immunoglobulin heavy chains and the first and second sialidases, respectively. In certain embodiments, the second and third polypeptides comprise, from N-terminal to C-terminal, a first and second sialidase and a first and second immunoglobulin heavy chain, respectively.

[0137] In some embodiments, the antibody conjugate comprises a first polypeptide comprising an immunoglobulin light chain; a second polypeptide comprising an immunoglobulin heavy chain; and a third polypeptide comprising an immunoglobulin Fc domain and sialidase. An example of this embodiment is shown in Figure 11B. The first and second polypeptides can be covalently bonded together, and the second and third polypeptides can be covalently bonded together. The covalent bond can be a disulfide bond. In some embodiments, the first and second polypeptides together define an antigen-binding site. In some embodiments, the third polypeptide comprises, from the N- to C-terminus, a sialidase and an immunoglobulin Fc domain, or from the N- to C-terminus, an immunoglobulin Fc domain and sialidase.

[0138] In certain embodiments, the first polypeptide comprises the amino acid sequence of SEQ ID NO:66, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:66. In certain embodiments, the second polypeptide comprises the amino acid sequence of SEQ ID NO:67 or 189, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:67 or 189. In certain embodiments, the third polypeptide comprises the amino acid sequence of any of SEQ ID NOs: 68-74, 98-112, 150, 151, 155, 156, 160, 161, 185, 187, 192, or 195, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 68-74, 98-112, 150, 151, 155, 156, 160, 161, 185, 187, 192, or 195.

[0139] In some embodiments, the third polypeptide is [Table 15] (SEQ ID NO: 76), wherein X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val or absent, X2 is Ala or Lys, X3 is Asn or Leu, X4 is Pro or His, X5 is Phe, Trp, Tyr or Val, X6 is Lys or Asp, X7 is Lys, Arg or Glu, X8 is Lys, Ala, Arg or Glu, X9 is Leu or Met, and X 10 is Pro, Asn, Asp, His, Glu, Gly, Ser, or Thr, and X 11 is Gln or His, and X 12 is Arg or Lys, and X 13 is Ala, Glu, or Lys, and X 14 is Gly or Asp, and X 15is Gln or His, and X 16 is Gln, Arg, or Lys, and X 17 is Ala, Cys, Ile, Ser, Val, or Leu, and X 18 is Gln or Leu, and X 19 is Ala or Val, and X 20 is Cys or Gly, and X 21 is Ala or Gly, and X 22 is Arg, Ile, or Lys, and X 23 is Ala, Cys, Leu, or Val, and X 24 is Leu, Ala, or Val, and X 25 is Thr or Ala, and X 26 is Asp or Gly, and X 27 is Glu or Lys, and X 28 is Gln, Ala, His, Phe or Pro, and X 29 is Cys or Val, and X 30 is Trp or Arg, and X 31 is Ser or Arg, and X 32 is Trp or Lys, and X 33 is Lys or Val, and X 34 is Ala, Cys, Ser or Val, and X 35 is Cys, Leu, or Val, and X 36 is Val or Arg, and X 37 is Leu, Gln, His, Ile, Lys, or Ser, and the sialidase comprises at least one mutation relative to wild-type human Neu2 (SEQ ID NO: 1).

[0140] In some embodiments, the third polypeptide is [Table 16-1] [Table 16-2] (SEQ ID NO: 75), wherein X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val or absent, X2 is Phe, Trp, Tyr or Val, X3 is Lys or Asp, X4 is Pro, Asn, Asp, His, Glu, Gly, Ser or Thr, X5 is Ala, Glu or Lys, X6 is Arg, Ile or Lys, X7 is Gln, Ala, His, Phe or Pro, X8 is Ser or Arg, X9 is Trp or Lys, and X 10 is Ala, Cys, Ser or Val, and X 11 is Val or Arg, and X 12 is Leu, Gln, His, Ile, Lys, or Ser, and the sialidase comprises 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, and X 10 is Ala or Cys, and X 11 is Val or Arg, and X 12 is Leu or Ile.

[0141] In some embodiments, the third polypeptide is [Table 17] (SEQ ID NO: 144), wherein X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val or absent, X2 is Ala or Lys, X3 is Asn or Leu, X4 is Pro or His, X5 is Phe, Trp, Tyr or Val, X6 is Lys or Asp, X7 is Lys, Arg or Glu, X8 is Lys, Ala, Arg or Glu, X9 is Leu or Met, and X 10 is Pro, Asn, Asp, His, Glu, Gly, Ser, or Thr, and X 11 is Gln or His, and X 12 is Arg or Lys, and X 13 is Ala, Glu, or Lys, and X 14 is Gly or Asp, and X 15 is Gln or His, and X 16 is Gln, Arg, or Lys, and X 17 is Ala, Cys, Ile, Ser, Val, or Leu, and X 18 is Gln or Leu, and X 19 is Ala or Val, and X 20 is Cys or Gly, and X 21 is Ala or Gly, and X 22 is Arg, Ile, or Lys, and X 23 is Ala, Cys, Leu, or Val, and X 24 is Leu, Ala, or Val, and X 25 is Thr or Ala, and X 26 is Asp or Gly, and X 27 is Glu or Lys, and X 28 is Gln, Ala, His, Phe or Pro, and X 29 is Cys or Val, and X 30 is Trp or Arg, and X 31 is Ser or Arg, and X 32 is Trp or Lys, and X 33 is Lys or Val, and X 34is Ala, Cys, Ser or Val, and X 35 is Cys, Leu, or Val, and X 36 is Val or Arg, and X 37 is Leu, Gln, His, Ile, Lys, or Ser, and X 38 is GGGGSGGGGS (SEQ ID NO: 145) or EPKSS (SEQ ID NO: 146), and the sialidase comprises at least one mutation relative to wild-type human Neu2 (SEQ ID NO: 1).

[0142] In some embodiments, the third polypeptide is [Table 18] (SEQ ID NO: 143), wherein X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val or absent, X2 is Phe, Trp, Tyr or Val, X3 is Lys or Asp, X4 is Pro, Asn, Asp, His, Glu, Gly, Ser or Thr, X5 is Ala, Glu or Lys, X6 is Arg, Ile or Lys, X7 is Gln, Ala, His, Phe or Pro, X8 is Ser or Arg, X9 is Trp or Lys, and X 10 is Ala, Cys, Ser or Val, and X 11 is Val or Arg, and X 12 is Leu, Gln, His, Ile, Lys, or Ser, and X 13is GGGGSGGGGS (SEQ ID NO: 145) or EPKSS (SEQ ID NO: 146), and the sialidase comprises 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, and X 10 is Ala or Cys, and X 11 is Val or Arg, and X 12 is Leu or Ile.

[0143] In some embodiments, the third polypeptide is [Table 19] (SEQ ID NO: 165), wherein X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val or absent, X2 is Ala or Lys, X3 is Asn or Leu, X4 is Pro or His, X5 is Phe, Trp, Tyr or Val, X6 is Lys or Asp, X7 is Lys, Arg or Glu, X8 is Lys, Ala, Arg or Glu, X9 is Leu or Met, and X 10 is Pro, Asn, Asp, His, Glu, Gly, Ser, or Thr, and X 11 is Gln or His, and X 12 is Arg or Lys, and X 13 is Asp or Pro, and X 14 is Ala, Glu, or Lys, and X 15 is Gly or Asp, and X 16 is Gln or His, and X 17 is Gln, Arg, or Lys, and X 18is Ala, Cys, Ile, Ser, Val, or Leu, and X 19 is Gln, Leu, Glu, Phe, His, Ile, Leu, or Tyr, and X 20 is Ala or Val, and X 21 is Cys or Gly, and X 22 is Arg or Pro, and X 23 is Ala or Gly, and X 24 is Arg, Ile, or Lys, and X 25 is Gln or Pro, and X 26 is Arg or Pro, and X 27 is Ala, Cys, Leu, or Val, and X 28 is Ala, Cys, Asn, Ser or Thr, and X 29 is Leu, Ala, or Val, and X 30 is Glu or Pro, and X 31 is His or Pro, and X 32 is Leu, Asp, Asn, or Tyr, and X 33 is Arg, Ala, Asp, Leu, Gln, or Tyr, and X 34 is Ala, Cys, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Val, Trp, or Tyr, and X 35 is Val, Ile, or Lys, and X 36 is Thr or Ala, and X 37 is Asp or Gly, and X 38 is Glu, Lys or Pro, and X 39 is Ser or Cys, and X 40 is Leu, Asp, Phe, Gln or Thr, and X 41 is Val or Phe, and X 42 is Gln, Ala, His, Phe, Pro, Ser, or Thr, and X 43 is Cys or Val, and X 44 is Trp or Arg, and X 45is Ser, Arg, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Thr, Val, Trp, or Tyr, and X 46 is Trp, Lys, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Tyr, and X 47 is Lys or Val, and X 48 is Ala, Cys, Ser or Val, and X 49 is Cys, Leu, or Val, and X 50 is Val or Arg, and X 51 is Leu, Gln, His, Ile, Lys, or Ser, and X 52 is GGGGS (SEQ ID NO: 184), GGGGSGGGGS (SEQ ID NO: 145) or EPKSS (SEQ ID NO: 146), and the sialidase comprises at least one mutation relative to wild-type human Neu2 (SEQ ID NO: 1).

[0144] In some embodiments, the third polypeptide is [Table 20] (SEQ ID NO: 164), wherein X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val or absent, X2 is Phe, Trp, Tyr or Val, X3 is Lys or Asp, X4 is Pro, Asn, Asp, His, Glu, Gly, Ser or Thr, and X5 is Ala, G 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, and X 10is Ser, Arg, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Thr, Val, Trp, or Tyr, and X 11 is Trp, Lys, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Tyr, and X 12 is Ala, Cys, Ser or Val, and X 13 is Val or Arg, and X 14 is Leu, Gln, His, Ile, Lys, or Ser, and X 15 is GGGGS (SEQ ID NO: 184), GGGGSGGGGS (SEQ ID NO: 145), or EPKSS (SEQ ID NO: 146), and the sialidase comprises 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, and X 10 is Ser, Arg, or Ala, and X 11 is Trp, Lys or Arg, and X 12 is Ala or Cys, and X 13 is Val or Arg, and X 14 is Leu or Ile.

[0145] In some embodiments, the first polypeptide comprises SEQ ID NO:66, the second polypeptide comprises SEQ ID NO:67, and the third polypeptide comprises SEQ ID NO:68. In some embodiments, the first polypeptide comprises SEQ ID NO:66, the second polypeptide comprises SEQ ID NO:67, and the third polypeptide comprises SEQ ID NO:69. In some embodiments, the first polypeptide comprises SEQ ID NO:66, the second polypeptide comprises SEQ ID NO:67, and the third polypeptide comprises SEQ ID NO:70. In some embodiments, the first polypeptide comprises SEQ ID NO:66, the second polypeptide comprises SEQ ID NO:67, and the third polypeptide comprises SEQ ID NO:71. In some embodiments, the first polypeptide comprises SEQ ID NO:66, the second polypeptide comprises SEQ ID NO:67, and the third polypeptide comprises SEQ ID NO:72. In some embodiments, the first polypeptide comprises SEQ ID NO:66, the second polypeptide comprises SEQ ID NO:67, and the third polypeptide comprises SEQ ID NO:73. In some embodiments, the first polypeptide comprises SEQ ID NO:66, the second polypeptide comprises SEQ ID NO:67, and the third polypeptide comprises SEQ ID NO:74. In some embodiments, the first polypeptide comprises SEQ ID NO:66, the second polypeptide comprises SEQ ID NO:67, and the third polypeptide comprises SEQ ID NO:98. In some embodiments, the first polypeptide comprises SEQ ID NO:66, the second polypeptide comprises SEQ ID NO:67, and the third polypeptide comprises SEQ ID NO:99. In some embodiments, the first polypeptide comprises SEQ ID NO:66, the second polypeptide comprises SEQ ID NO:67, and the third polypeptide comprises SEQ ID NO:100. In some embodiments, the first polypeptide comprises SEQ ID NO:66, the second polypeptide comprises SEQ ID NO:67, and the third polypeptide comprises SEQ ID NO:101. In some embodiments, the first polypeptide comprises SEQ ID NO:66, the second polypeptide comprises SEQ ID NO:67, and the third polypeptide comprises SEQ ID NO:102. In some embodiments, the first polypeptide comprises SEQ ID NO:66, the second polypeptide comprises SEQ ID NO:67, and the third polypeptide comprises SEQ ID NO:103.In some embodiments, the first polypeptide comprises SEQ ID NO:66, the second polypeptide comprises SEQ ID NO:67, and the third polypeptide comprises SEQ ID NO:104. In some embodiments, the first polypeptide comprises SEQ ID NO:66, the second polypeptide comprises SEQ ID NO:67, and the third polypeptide comprises SEQ ID NO:105. In some embodiments, the first polypeptide comprises SEQ ID NO:66, the second polypeptide comprises SEQ ID NO:67, and the third polypeptide comprises SEQ ID NO:106. In some embodiments, the first polypeptide comprises SEQ ID NO:66, the second polypeptide comprises SEQ ID NO:67, and the third polypeptide comprises SEQ ID NO:107. In some embodiments, the first polypeptide comprises SEQ ID NO:66, the second polypeptide comprises SEQ ID NO:67, and the third polypeptide comprises SEQ ID NO:108. In some embodiments, the first polypeptide comprises SEQ ID NO:66, the second polypeptide comprises SEQ ID NO:67, and the third polypeptide comprises SEQ ID NO:109. In some embodiments, the first polypeptide comprises SEQ ID NO:66, the second polypeptide comprises SEQ ID NO:67, and the third polypeptide comprises SEQ ID NO:110. In some embodiments, the first polypeptide comprises SEQ ID NO:66, the second polypeptide comprises SEQ ID NO:67, and the third polypeptide comprises SEQ ID NO:111. In some embodiments, the first polypeptide comprises SEQ ID NO:66, the second polypeptide comprises SEQ ID NO:67, and the third polypeptide comprises SEQ ID NO:112. In some embodiments, the first polypeptide comprises SEQ ID NO:66, the second polypeptide comprises SEQ ID NO:67, and the third polypeptide comprises SEQ ID NO:150. In some embodiments, the first polypeptide comprises SEQ ID NO:66, the second polypeptide comprises SEQ ID NO:67, and the third polypeptide comprises SEQ ID NO:151. In some embodiments, the first polypeptide comprises SEQ ID NO:66, the second polypeptide comprises SEQ ID NO:67, and the third polypeptide comprises SEQ ID NO:155. In some embodiments, the first polypeptide comprises SEQ ID NO:66, the second polypeptide comprises SEQ ID NO:67, and the third polypeptide comprises SEQ ID NO:156.In some embodiments, the first polypeptide comprises SEQ ID NO:66, the second polypeptide comprises SEQ ID NO:67, and the third polypeptide comprises SEQ ID NO:160. In some embodiments, the first polypeptide comprises SEQ ID NO:66, the second polypeptide comprises SEQ ID NO:67, and the third polypeptide comprises SEQ ID NO:161. In some embodiments, the first polypeptide comprises SEQ ID NO:66, the second polypeptide comprises SEQ ID NO:67, and the third polypeptide comprises SEQ ID NO:192. In some embodiments, the first polypeptide comprises SEQ ID NO:66, the second polypeptide comprises SEQ ID NO:67, and the third polypeptide comprises SEQ ID NO:195. In some embodiments, the first polypeptide comprises SEQ ID NO:66, the second polypeptide comprises SEQ ID NO:189, and the third polypeptide comprises SEQ ID NO:185. In some embodiments, the first polypeptide comprises SEQ ID NO:66, the second polypeptide comprises SEQ ID NO:189, and the third polypeptide comprises SEQ ID NO:187.

[0146] In some embodiments, the antibody conjugate comprises a first polypeptide comprising a first sialidase, a first immunoglobulin Fc domain, and a first single-chain variable fragment (scFv) (it is also understood that the scFv can be replaced by a first polypeptide chain of an immunoglobulin antigen-binding fragment, e.g., a Fab fragment); and a second polypeptide comprising a second sialidase, a second immunoglobulin Fc domain, and a second single-chain variable fragment (scFv) (it is also understood that the scFv can be replaced by a second polypeptide chain of an immunoglobulin antigen-binding fragment, e.g., a Fab fragment). An example of this embodiment is shown in FIG. 11C. The first and second polypeptides can be covalently linked together. The covalent bond can be a disulfide bond. In some embodiments, the first scFv defines a first antigen-binding site, and the second scFv defines a second antigen-binding site. In some embodiments, the first polypeptide comprises, from N- to C-terminal, a first sialidase, a first immunoglobulin Fc domain, and a first scFv. In some embodiments, the first polypeptide comprises, from N- to C-terminal, a first scFv, a first immunoglobulin Fc domain, and a first sialidase. In some embodiments, the second polypeptide comprises, from N- to C-terminal, a second sialidase, a second immunoglobulin Fc domain, and a second scFv. In some embodiments, the second polypeptide comprises, from N- to C-terminal, a second scFv, a second immunoglobulin Fc domain, and a second sialidase.

[0147] In certain embodiments, the first polypeptide comprises the amino acid sequence of any of SEQ ID NOs: 77 to 83, 166 to 178, 194, or 197, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 77 to 83, 166 to 178, 194, or 197. In certain embodiments, the second polypeptide comprises the amino acid sequence of any of SEQ ID NOs: 77 to 83, 166 to 178, 194, or 197, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 77 to 83, 166 to 178, 194, or 197.

[0148] In certain embodiments, the first and / or second polypeptide is [Table 21] (SEQ ID NO: 85), wherein X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val or absent, X2 is Ala or Lys, X3 is Asn or Leu, X4 is Pro or His, X5 is Phe, Trp, Tyr or Val, X6 is Lys or Asp, X7 is Lys, Arg or Glu, X8 is Lys, Ala, Arg or Glu, X9 is Leu or Met, and X 10 is Pro, Asn, Asp, His, Glu, Gly, Ser, or Thr, and X 11 is Gln or His, and X 12 is Arg or Lys, and X 13 is Ala, Glu, or Lys, and X 14 is Gly or Asp, and X 15 is Gln or His, and X 16 is Gln, Arg, or Lys, and X 17 is Ala, Cys, Ile, Ser, Val, or Leu, and X 18 is Gln or Leu, and X19 is Ala or Val, and X 20 is Cys or Gly, and X 21 is Ala or Gly, and X 22 is Arg, Ile, or Lys, and X 23 is Ala, Cys, Leu, or Val, and X 24 is Leu, Ala, or Val, and X 25 is Thr or Ala, and X 26 is Asp or Gly, and X 27 is Glu or Lys, and X 28 is Gln, Ala, His, Phe or Pro, and X 29 is Cys or Val, and X 30 is Trp or Arg, and X 31 is Ser or Arg, and X 32 is Trp or Lys, and X 33 is Lys or Val, and X 34 is Ala, Cys, Ser or Val, and X 35 is Cys, Leu, or Val, and X 36 is Val or Arg, and X 37 is Leu, Gln, His, Ile, Lys, or Ser, and the sialidase comprises at least one mutation relative to wild-type human Neu2 (SEQ ID NO: 1).

[0149] In certain embodiments, the first and / or second polypeptide is [Table 22-1] [Table 22-2] (SEQ ID NO: 84), wherein X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val or absent, X2 is Phe, Trp, Tyr or Val, X3 is Lys or Asp, X4 is Pro, Asn, Asp, His, Glu, Gly, Ser or Thr, X5 is Ala, Glu or Lys, X6 is Arg, Ile or Lys, X7 is Gln, Ala, His, Phe or Pro, X8 is Ser or Arg, X9 is Trp or Lys, and X 10 is Ala, Cys, Ser or Val, and X 11 is Val or Arg, and X 12 is Leu, Gln, His, Ile, Lys, or Ser, and the sialidase comprises 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, and X 10 is Ala or Cys, and X 11 is Val or Arg, and X 12 is Leu or Ile.

[0150] In certain embodiments, the first and / or second polypeptide is [Table 23] (SEQ ID NO: 180), wherein X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val or absent, X2 is Ala or Lys, X3 is Asn or Leu, X4 is Pro or His, X5 is Phe, Trp, Tyr or Val, X6 is Lys or Asp, X7 is Lys, Arg or Glu, X8 is Lys, Ala, Arg or Glu, X9 is Leu or Met, and X 10 is Pro, Asn, Asp, His, Glu, Gly, Ser, or Thr, and X 11 is Gln or His, and X 12 is Arg or Lys, and X 13 is Asp or Pro, and X 14 is Ala, Glu, or Lys, and X 15 is Gly or Asp, and X 16 is Gln or His, and X 17 is Gln, Arg, or Lys, and X 18 is Ala, Cys, Ile, Ser, Val, or Leu, and X 19 is Gln, Leu, Glu, Phe, His, Ile, Leu, or Tyr, and X 20 is Ala or Val, and X 21 is Cys or Gly, and X 22 is Arg or Pro, and X 23 is Ala or Gly, and X 24 is Arg, Ile, or Lys, and X 25 is Gln or Pro, and X 26 is Arg or Pro, and X 27 is Ala, Cys, Leu, or Val, and X 28 is Ala, Cys, Asn, Ser or Thr, and X 29 is Leu, Ala, or Val, and X 30 is Glu or Pro, and X 31 is His or Pro, and X 32 is Leu, Asp, Asn, or Tyr, and X33 is Arg, Ala, Asp, Leu, Gln, or Tyr, and X 34 is Ala, Cys, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Val, Trp, or Tyr, and X 35 is Val, Ile, or Lys, and X 36 is Thr or Ala, and X 37 is Asp or Gly, and X 38 is Glu, Lys or Pro, and X 39 is Ser or Cys, and X 40 is Leu, Asp, Phe, Gln or Thr, and X 41 is Val or Phe, and X 42 is Gln, Ala, His, Phe, Pro, Ser, or Thr, and X 43 is Cys or Val, and X 44 is Trp or Arg, and X 45 is Ser, Arg, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Thr, Val, Trp, or Tyr, and X 46 is Trp, Lys, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Tyr, and X 47 is Lys or Val, and X 48 is Ala, Cys, Ser or Val, and X 49 is Cys, Leu, or Val, and X 50 is Val or Arg, and X 51 is Leu, Gln, His, Ile, Lys, or Ser, and X 52 is GGGGS (SEQ ID NO: 184), GGGGSGGGGS (SEQ ID NO: 145) or EPKSS (SEQ ID NO: 146), and the sialidase comprises at least one mutation relative to wild-type human Neu2 (SEQ ID NO: 1).

[0151] In certain embodiments, the first and / or second polypeptide is [Table 24] (SEQ ID NO: 179), wherein X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val or absent, X2 is Phe, Trp, Tyr or Val, X3 is Lys or Asp, X4 is Pro, Asn, Asp, His, Glu, Gly, Ser or Thr, and X5 is Ala, G 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, and X 10 is Ser, Arg, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Thr, Val, Trp, or Tyr, and X 11 is Trp, Lys, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Tyr, and X 12 is Ala, Cys, Ser or Val, and X 13 is Val or Arg, and X 14 is Leu, Gln, His, Ile, Lys, or Ser, and X 15is GGGGS (SEQ ID NO: 184), GGGGSGGGGS (SEQ ID NO: 145), or EPKSS (SEQ ID NO: 146), and the sialidase comprises 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, and X 10 is Ser, Arg, or Ala, and X 11 is Trp, Lys or Arg, and X 12 is Ala or Cys, and X 13 is Val or Arg, and X 14 is Leu or Ile.

[0152] In some embodiments, the first and second polypeptides comprise SEQ ID NO:77. In some embodiments, the first and second polypeptides comprise SEQ ID NO:78. In some embodiments, the first and second polypeptides comprise SEQ ID NO:79. In some embodiments, the first and second polypeptides comprise SEQ ID NO:80. In some embodiments, the first and second polypeptides comprise SEQ ID NO:81. In some embodiments, the first and second polypeptides comprise SEQ ID NO:82. In some embodiments, the first and second polypeptides comprise SEQ ID NO:83. In some embodiments, the first and second polypeptides comprise SEQ ID NO:166. In some embodiments, the first and second polypeptides comprise SEQ ID NO:167. In some embodiments, the first and second polypeptides comprise SEQ ID NO:168. In some embodiments, the first and second polypeptides comprise SEQ ID NO:169. In some embodiments, the first and second polypeptides comprise SEQ ID NO:170. In some embodiments, the first and second polypeptides comprise SEQ ID NO:171. In some embodiments, the first and second polypeptides comprise SEQ ID NO:172. In some embodiments, the first and second polypeptides comprise SEQ ID NO: 173. In some embodiments, the first and second polypeptides comprise SEQ ID NO: 174. In some embodiments, the first and second polypeptides comprise SEQ ID NO: 175. In some embodiments, the first and second polypeptides comprise SEQ ID NO: 176. In some embodiments, the first and second polypeptides comprise SEQ ID NO: 177. In some embodiments, the first and second polypeptides comprise SEQ ID NO: 178. In some embodiments, the first and second polypeptides comprise SEQ ID NO: 194. In some embodiments, the first and second polypeptides comprise SEQ ID NO: 197.

[0153] In some embodiments, the antibody conjugate comprises: a first polypeptide comprising an immunoglobulin light chain; a second polypeptide comprising an immunoglobulin heavy chain and a single-chain variable fragment (scFv) (it is also understood that the scFv can be replaced by the first polypeptide chain of an immunoglobulin antigen-binding fragment, e.g., a Fab fragment); and a third polypeptide comprising an immunoglobulin Fc domain and a sialidase. An example of this embodiment is shown in FIG. 11D. The first and second polypeptides can be covalently linked together, and the second and third polypeptides can be covalently linked together. The covalent linkage can be a disulfide bond. In some embodiments, the first and second polypeptides together define a first antigen-binding site (i.e., the immunoglobulin light chain and the immunoglobulin heavy chain together define the first antigen-binding site). In some embodiments, the scFv defines a second antigen-binding site. In some embodiments, the second polypeptide comprises, from N- to C-terminal, an immunoglobulin heavy chain and an scFv, or an scFv and an immunoglobulin heavy chain, In some embodiments, the third polypeptide comprises, from N- to C-terminal, a sialidase and an immunoglobulin Fc domain, or a sialidase and an immunoglobulin Fc domain.

[0154] In some embodiments, the antibody conjugate has a molecular weight of about 135 kDa to about 165 kDa, e.g., about 140 kDa, hi other embodiments, the antibody conjugate has a molecular weight of about 215 kDa to about 245 kDa, e.g., about 230 kDa.

[0155] In some embodiments, an antibody conjugate comprises two polypeptides, each comprising an immunoglobulin Fc domain, wherein the first polypeptide has either a "knob" mutation, e.g., T366Y, or a "hole" mutation, e.g., Y407T, for heterodimerization with the second polypeptide, and the second polypeptide has either a "knob" mutation, e.g., T366Y, or a "hole" mutation, e.g., Y407T, respectively, for heterodimerization with the first polypeptide (residue numbers according to EU numbering; Kabat, EA, et al. (1991) supra). For example, in some embodiments, an antibody comprises two polypeptides, each comprising an immunoglobulin Fc domain derived from a human IgG1 Fc domain, wherein the first polypeptide comprises a Y407T mutation (e.g., the first polypeptide comprises SEQ ID NO:32 or SEQ ID NO:147), and the second polypeptide comprises a T366Y mutation (e.g., the second polypeptide comprises SEQ ID NO:33 or SEQ ID NO:148).

[0156] As used herein, the term "multispecific antibody" is understood to mean an antibody that specifically binds to at least two different antigens, i.e., an antibody that contains at least two antigen-binding sites that bind to at least two different antigens. As used herein, the term "bispecific antibody" is understood to mean an antibody that specifically binds to two different antigens, i.e., an antibody that contains two antigen-binding sites, each binding to a separate, different antigen. In other words, the first binding site binds to a first antigen, and the second binding site binds to a second, different antigen. Multispecific or bispecific antibodies can be, for example, human or humanized antibodies, and / or full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies).

[0157] The present invention encompasses antibody conjugates comprising antibody fragments, which may be produced by conventional means, such as enzymatic digestion, or by recombinant techniques. For a review of specific antibody fragments, see Hudson et al. (2003), supra.

[0158] In some embodiments, the antibody conjugate or fusion protein may be covalently or non-covalently linked to a biological modifier, which may be used to increase the solubility of the antibody, increase binding specificity, reduce immunogenicity or toxicity, or modify the pharmacokinetic profile of the antibody. For example, a biological modifier may be used to increase the molecular weight of an antibody to increase its circulating half-life.

[0159] It is contemplated that the antibody conjugate or fusion protein may be covalently linked to one or more (e.g., 2, 3, 4, 5, 6, 8, 9, 10, or more) biological modifiers, which may include linear or branched polymers. Exemplary biological modifiers may include various polymers, such as those described in U.S. Patent No. 7,842,789. Particularly useful are polyalkylene ethers, such as polyethylene glycol (PEG) and its derivatives (e.g., alkoxypolyethylene glycol, e.g., methoxypolyethylene glycol, ethoxypolyethylene glycol, etc.); block copolymers of polyoxyethylene and polyoxypropylene (Pluronics); polymethacrylates; carbomers; and branched or unbranched polysaccharides containing sugar monomers, such as D-mannose, D- and L-galactose, fucose, fructose, D-xylose, L-arabinose, and D-glucuronic acid.

[0160] In other embodiments, the biological modifier can be a hydrophilic polyvinyl polymer, such as polyvinyl alcohol and polyvinylpyrrolidone (PVP)-type polymer. The biological modifier can be a functionalized polyvinylpyrrolidone, e.g., carboxy- or amine-functionalized at one (or both) ends of the polymer (available from PolymerSource). Alternatively, the biological modifier can include poly N-(2-hydroxypropyl)methacrylamide (HPMA), or functionalized HPMA (amine, carboxy, etc.), poly(N-isopropylacrylamide), or functionalized poly(N-isopropylacrylamide). Alternatively, the biological modifier can include poly N-(2-hydroxypropyl)methacrylamide (HPMA), or functionalized HPMA (amine, carboxy, etc.), poly(N-isopropylacrylamide), or functionalized poly(N-isopropylacrylamide). The modifier prior to conjugation need not be, but preferably is, water-soluble; however, the final conjugate should be water-soluble.

[0161] Generally, biological modifiers are selected from the group consisting 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, about 5 kDa to about 60 kDa, and about 5kDa to about 70kDa, about 5kDa to about 80kDa, about 5kDa to about 90kDa, about 5kDa to about 100kDa, about 5kDa to about 150kDa, about 10kDa to about 20kDa, about 10kDa to about 30kDa, about 10kDa to about 40kDa, about 10kDa to about 50kDa, about 10 kDa ~ about 60kDa, about 10kDa - about 70kDa, about 10kDa - about 80kDa, about 10kDa - about 90kDa, about 10kDa - about 100kDa, about 10kDa - about 150kDa, about 20kDa - about 30kDa, about 20kDa - about 40kDa, about 20kDa - about 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 60k Da, approx. 30kDa ~ approx. 70kDa, approx. 30kDa ~ approx. 80kDa, approx. 30kDa ~ approx. 90kDa, approx. 30kDa ~ approx. 100kDa, approx. 30kDa ~ approx. 150kDa, approx. 40kDa ~ approx. 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 50k Da ~ about 150kDa, about 60kDa - about 70kDa, about 60kDa - about 80kDa, about 60kDa - about 90kDa, about 60kDa - about 100kDa, about 60kDa - about 150kDa, about 70kDa - about 80kDa, about 70kDa - about 90kDa, about 70kDa - about 100kDa,It may have a molecular weight of about 70 kDa to about 150 kDa, about 80 kDa to about 90 kDa, about 80 kDa to about 100 kDa, about 80 kDa to about 150 kDa, about 90 kDa to about 100 kDa, about 90 kDa to about 150 kDa, or about 100 kDa to about 150 kDa.

[0162] It is contemplated that the antibody conjugate or fusion protein is attached to about 10 or fewer polymer molecules (e.g., 9, 8, 7, 6, 5, 4, 3, 2, or 1), each polymer molecule having a molecular weight of at least about 20,000 D, or at least about 30,000 D, or at least about 40,000 D.

[0163] Although various polymers can be used as biological modifiers, it is contemplated that the antibody conjugates or fusion proteins described herein can be conjugated to polyethylene glycol (PEG) polymers. In one embodiment, the antibody conjugates or fusion proteins described herein are covalently linked to at least one PEG having an actual MW of at least about 20,000 D. In another embodiment, the antibody conjugates or fusion proteins described herein are covalently linked to at least one PEG having an actual MW of at least about 30,000 D. In another embodiment, the antibody conjugates or fusion proteins described herein are covalently linked to at least one PEG having an actual MW of at least about 40,000 D. In some embodiments, the PEG is methoxyPEG(5000)-succinimidyl propionate (mPEG-SPA), methoxyPEG(5000) succinimidyl succinate (mPEG-SS). Such PEGs are commercially available from Nektar Therapeutics or SunBiowest.

[0164] Attachment sites on antibody conjugates or fusion proteins for biological modifiers include the N-terminal amino group and the epsilon amino group found on lysine residues, as well as other amino, imino, carboxyl, sulfhydryl, hydroxyl, or other hydrophilic groups. Polymers can be directly covalently attached to antibody conjugates or fusion proteins using chemistries with or without the use of multifunctional (usually bifunctional) crosslinkers known in the art. For example, sulfhydryl groups can be derivatized by coupling to maleimide-substituted PEG (e.g., alkoxy-PEGamine plus sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, or PEG-maleimide, commercially available from Shearwater Polymers, Inc., Huntsville, Ala.).

[0165] III. Methods for Producing Recombinant Human Sialidases, Fusion Proteins, or Antibody Conjugates Methods for producing recombinant human sialidases, fusion proteins, such as those disclosed herein, antibodies, or antibody conjugates, such as those disclosed herein, are known in the art. For example, DNA molecules encoding light chain variable regions and / or heavy chain variable regions can be synthesized chemically or by recombinant DNA methodology. For example, the antibody sequences can be cloned from hybridomas by conventional hybridization or polymerase chain reaction (PCR) techniques using appropriate synthetic nucleic acid primers. The resulting DNA molecules encoding the desired variable regions can be ligated to other appropriate nucleotide sequences, such as constant region coding sequences and expression control sequences, to produce conventional gene expression constructs (i.e., expression vectors) encoding the desired antibodies. The production of a given gene construct is within the ordinary skill of those in the art.

[0166] Nucleic acids encoding the desired recombinant human sialidase, fusion protein, and / or antibody conjugate can be incorporated (ligated) into an expression vector, which can be introduced into host cells by conventional transfection or transformation techniques. Exemplary host cells are E. coli cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK 293) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells that do not otherwise produce IgG protein. Transformed host cells can be grown under conditions in which the host cells express genes encoding immunoglobulin light and / or heavy chain variable regions.

[0167] Specific expression and purification conditions vary depending on the expression system used.For example, when gene is expressed in E. coli, the gene 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.Expressed protein can be secreted.Expressed protein can accumulate in refractile or inclusion bodies, and can be recovered after cell disruption by French press or ultrasonic treatment.Refractile bodies can then be solubilized, and the protein can be refolded and / or cleaved by methods known in the art.

[0168] When a genetically engineered gene is expressed in a eukaryotic host cell, such as a CHO cell, the gene is first inserted into an expression vector containing a suitable eukaryotic promoter, secretion signal, polyA sequence, and stop codon. Optionally, the vector or gene construct may contain an enhancer and intron. In embodiments involving fusion proteins containing antibodies or portions thereof, the expression vector optionally contains a sequence encoding all or part of a constant region that allows the entire heavy or light chain to be expressed. The gene construct can be introduced into a eukaryotic host cell using conventional techniques.

[0169] The host cell is then incubated with a recombinant human sialidase or a V-type sialidase that can be linked to a moiety, each of which has a different function (e.g., cytotoxicity). L Or V H Fragment, V L -V H Heterodimer, V H -V L Or V L -V H They express fusion proteins and / or antibody conjugates comprising a single-chain polypeptide, a complete immunoglobulin heavy or light chain, or portions thereof. In some embodiments involving fusion proteins and / or antibody conjugates, host cells are transfected with a single vector expressing a polypeptide expressing a sialidase and all or part of a heavy chain (e.g., a heavy chain variable region), or a sialidase and a light chain (e.g., a light chain variable region), or a polypeptide expressing all or part of a heavy chain (e.g., a heavy chain variable region) or a light chain (e.g., a light chain variable region). In some embodiments, host cells are transfected with a single vector encoding (a) a polypeptide comprising a heavy chain variable region and a polypeptide comprising a light chain variable region, or (b) an entire immunoglobulin heavy chain and an entire immunoglobulin light chain, wherein in (a) or (b), the polypeptide may also comprise a sialidase. In some embodiments, the host cell is co-transfected with more than one expression vector (e.g., one expression vector expressing a polypeptide comprising all or part of a heavy chain or heavy chain variable region, optionally comprising a sialidase fused thereto, and another expression vector expressing a polypeptide comprising all or part of a light chain or light chain variable region, optionally comprising a sialidase fused thereto).

[0170] Polypeptides or fusion proteins containing sialidase, such as fusion proteins containing immunoglobulin heavy or light chain variable regions, can be produced by growing (culturing) host cells transfected with an expression vector encoding such variable regions under conditions that allow expression of the polypeptide. After expression, the polypeptide can be recovered and purified or isolated using techniques known in the art, for example, affinity tags such as glutathione-S-transferase (GST) or histidine tags.

[0171] In embodiments in which fusion proteins and / or antibody conjugates are produced, sialidase fused to a monoclonal antibody, its Fc domain, or antigen-binding domain can be produced by growing (culturing) host cells transfected with: (a) an expression vector encoding a complete or partial immunoglobulin heavy chain and another expression vector encoding a complete or partial immunoglobulin light chain; or (b) a single expression vector encoding both chains (e.g., complete or partial heavy and light chains) under conditions that allow expression of both chains. The sialidase is fused to one or more of the chains. The intact fusion protein and / or antibody conjugate can be recovered and purified or isolated using techniques known in the art, for example, affinity tags such as protein A, protein G, glutathione-S-transferase (GST), or histidine tags. It is within the ordinary skill in the art to express heavy and light chains from a single expression vector or two separate expression vectors.

[0172] In some embodiments, to express a protein, such as recombinant human sialidase, as a secreted protein, the native N-terminal signal sequence of the protein is replaced with, for example, MDMRVPAQLLGLLLLWLPGARC (SEQ ID NO: 28). In some embodiments, to express a protein, such as recombinant human sialidase, as a secreted protein, an N-terminal signal sequence, such as MDMRVPAQLLGLLLLWLPGARC (SEQ ID NO: 28), is added. Further exemplary N-terminal signal sequences include signal sequences derived from interleukin-2, CD-5, IgG kappa light chain, trypsinogen, serum albumin, and prolactin. In some embodiments, to express a protein, such as recombinant human sialidase, as a secreted protein, a C-terminal lysosomal signal motif, such as YGTL (SEQ ID NO: 29), is removed.

[0173] Methods for reducing or eliminating the antigenicity of antibodies and antibody fragments are known in the art. When the antibody is administered to humans, the antibody is preferably "humanized" to reduce or eliminate the antigenicity in humans. Preferably, each humanized antibody has the same or substantially the same affinity for the antigen as the non-humanized mouse antibody from which it was derived.

[0174] In one humanization approach, chimeric proteins are created in which mouse immunoglobulin constant regions are replaced with human immunoglobulin constant regions. See, e.g., Morrison et al., 1984, PROC. NAT. ACAD. SCI. 81:6851-6855; Neuberger et al., 1984, NATURE 312:604-608; U.S. Patent Nos. 6,893,625 (Robinson); 5,500,362 (Robinson); and 4,816,567 (Cabilly).

[0175] In the approach known as CDR grafting, the CDR of light chain and heavy chain variable region is grafted onto the framework of another species.For example, mouse CDR can be grafted onto human FR.In some embodiments, the CDR of light chain and heavy chain variable region of antibody is grafted onto human FR or consensus human FR.To make consensus human FR, FR from several human heavy chain or light chain amino acid sequence is aligned to identify consensus amino acid sequence. CDR porting is applicable to U.S. Patent No. 7,022,500 (Queen); U.S. Pat. ); No. 5,859,205 (Adair); No. 5,693,761 (Queen); No. 5,565,332 (Hoogenboom); No. 5,585,089 (Queen); No. 5,530,101 (Queen); Jones et al. (1986) NATURE 321: 522-525;Riechmann et al. (1988) NATURE 332: 323-327;Verhoeyen et al. (1988) SCIENCE 239: 1534-1536; and Winter (1998) FEBS LETT 430: 92-94.

[0176] "SUPERHUMANIZATION TM In an approach referred to as "selection of CDR sequences from human germline genes," human CDR sequences are selected from human germline genes based on the structural similarity of the human CDRs to the CDRs of the mouse antibody being humanized. See, e.g., U.S. Patent No. 6,881,557 (Foote); and Tan et al., 2002, J. IMMUNOL. 169:1119-1125.

[0177] Other methods for reducing immunogenicity include "reshaping," "hyperchimerization," and "veneering / resurfacing." See, e.g., Vaswami et al., 1998, ANNALS OF ALLERGY, ASTHMA, & IMMUNOL. 81:105; Roguska et al., 1996, PROT. ENGINEER 9:895-904; and U.S. Pat. No. 6,072,035 (Hardman). In the veneering / resurfacing approach, surface-accessible amino acid residues in a mouse antibody are replaced with amino acid residues more frequently found at the same positions in human antibodies. This type of antibody resurfacing is described, for example, in U.S. Pat. No. 5,639,641 (Pedersen).

[0178] Another approach to converting murine antibodies into a form suitable for medical use in humans is ACTIVMAB TM The technique is known as "human engineering" (Vaccinex, Inc., Rochester, NY), which involves vaccinia virus-based vectors for expressing antibodies in mammalian cells. High levels of combinatorial diversity of IgG heavy and light chains can be produced. See, e.g., U.S. Patent Nos. 6,706,477 (Zauderer); 6,800,442 (Zauderer); and 6,872,518 (Zauderer). Another approach to converting mouse antibodies into a form suitable for use in humans is the technique commercially practiced by KaloBios Pharmaceuticals, Inc. (Palo Alto, CA). This technique involves the use of a proprietary human "acceptor" library to create an "epitope-focused" library for antibody selection. Another approach to modifying mouse antibodies into a form suitable for medical use in humans is the HUMAN ENGINEERING TMtechnology, which is commercially practiced by XOMA (US) LLC. See, e.g., International (PCT) Publication No. WO 93 / 11794 and U.S. Patent Nos. 5,766,886 (Studnicka); 5,770,196 (Studnicka); 5,821,123 (Studnicka); and 5,869,619 (Studnicka).

[0179] Any suitable approach, including any of the approaches described above, may be used to reduce or eliminate human immunogenicity of an antibody.

[0180] It is also possible to produce fully human antibodies in mice. Fully human mAbs lacking any non-human sequences can be prepared from human immunoglobulin transgenic mice by techniques referenced, for example, in Lonberg et al., NATURE 368:856-859, 1994; Fishwild et al., NATURE BIOTECHNOLOGY 14:845-851, 1996; and Mendez et al., NATURE GENETICS 15:146-156, 1997. Fully human monoclonal antibodies can also be prepared and optimized from phage display libraries by techniques referenced, for example, in Knappik et al., J. MOL. BIOL. 296:57-86, 2000; and Krebs et al., J. IMMUNOL. METH. 254:67-84, 2001).

[0181] The present invention encompasses fusion proteins comprising antibody fragments, which may be produced by conventional means, such as enzymatic digestion or recombinant techniques. For a review of specific antibody fragments, see Hudson et al. (2003) NAT. MED. 9:129-134.

[0182] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were generated by proteolytic digestion of intact antibodies (see, e.g., Morimoto et al. (1992) JOURNAL OF BIOCHEMICAL AND BIOPHYSICAL METHODS 24:107-117; and Brennan et al. (1985) SCIENCE 229:81). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv, and ScFv antibody fragments can all be expressed in and secreted from E. coli, allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from antibody phage libraries. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically ligated to form F(ab')2 fragments (Carter et al. (1992) BIO / TECHNOLOGY 10:163-167). According to another approach, F(ab')2 fragments can be directly isolated from recombinant host cell culture. Fab and F(ab')2 fragments with increased in vivo half-lives containing salvage receptor binding epitope residues are described in U.S. Patent No. 5,869,046. Other techniques for producing antibody fragments will be apparent to those skilled in the art. In some embodiments, the antibody is a single-chain Fv fragment (scFv). See U.S. Patent Nos. 5,571,894 and 5,587,458.

[0183] Methods for producing bispecific antibodies are known in the art. See Milstein and Cuello (1983) NATURE 305:537, International (PCT) Publication No. WO93 / 08829, and Traunecker et al. (1991) EMBO J., 10:3655. For further details on producing bispecific antibodies, see, for example, Suresh et al. (1986) METHODS ENZYMOL. 121:210. Bispecific antibodies include cross-linked or "heteroconjugate" or "heterodimeric" antibodies. For example, one antibody in the heterodimer can be linked to avidin and the other to biotin. Heterodimeric antibodies can be produced using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, along with several cross-linking techniques.

[0184] Examples of heterodimeric or asymmetric IgG-like molecules include, but are not limited to, those obtained using the following technologies or formats: Triomab / Quadroma, Knobs-into-Holes, CrossMabs, Electrostatically Matched Antibodies, LUZ-Y, Strand Exchange Engineered Domain bodies, Biclonic and DuoBody.

[0185] Advantages of using antibody fragments (e.g., F(ab) and F(ab')2 fragments) include elimination of nonspecific binding between the Fc portion of the antibody and Fc receptors on cells (e.g., macrophages, dendritic cells, neutrophils, NK cells, and B cells). Fragments may also be able to penetrate tissues more efficiently due to their smaller size.

[0186] Heterodimeric or asymmetric antibodies allow for greater flexibility and novel formats for attaching various drugs to antibody arms. One common format for generating heterodimeric antibodies is the "knobs-into-holes" format. This format is specific to the heavy chain portion of the antibody constant region. The "knobs" are engineered by replacing small amino acids with larger ones that fit into the "holes," and the holes are engineered by replacing large amino acids with smaller ones. The "knobs" are connected to the "holes" by disulfide bonds between each chain. The "knobs-into-holes" configuration facilitates antibody-dependent cell-mediated cytotoxicity. Single-chain variable fragments (scFvs) are linked to the heavy and light chain variable domains via a short linker peptide. The linker is rich in glycines, which provide greater flexibility to the linker, and serines / threonines, which provide specificity. Two different scFv fragments can be joined together via a hinge region to either the heavy chain constant domain or the light chain constant domain. This confers bispecificity to the antibody, allowing it to bind to two different antigens. The "knobs-into-holes" format enhances heterodimer formation but does not suppress homodimer formation.

[0187] Several approaches to supporting heterodimerization are described, for example, in International (PCT) Publication Nos. WO96 / 27011, WO98 / 050431, WO2007 / 110205, WO2007 / 147901, WO2009 / 089004, WO2010 / 129304, WO2011 / 90754, WO2011 / 143545, WO2012 / 058768, WO2013 / 157954 and WO2013 / 096291, and European Patent Publication No. EP1870459. Typically, in approaches known in the art, the CH3 domains of a first heavy chain and a second heavy chain are both engineered in a complementary manner so that a heavy chain containing one engineered CH3 domain can no longer homodimerize with another heavy chain of the same structure (e.g., a CH3-engineered first heavy chain can no longer homodimerize with another CH3-engineered first heavy chain; a CH3-engineered second heavy chain can no longer homodimerize with another CH3-engineered second heavy chain). This forces a heavy chain containing one engineered CH3 domain to heterodimerize with another heavy chain containing a CH3 domain engineered in a complementary manner. Consequently, the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain are engineered in a complementary manner by amino acid substitutions such that the first and second heavy chains are forced to heterodimerize, while the first and second heavy chains can no longer homodimerize (e.g., for steric reasons).

[0188] IV. Pharmaceutical Compositions For therapeutic uses, the recombinant human sialidase or its fusion protein and / or antibody conjugate is preferably combined with a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable," as used herein, refers to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals, within the scope of sound medical judgment, without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0189] The term "pharmaceutically acceptable carrier," as used herein, refers to buffers, carriers, and excipients that are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers include any of the standard pharmaceutical carriers, such as phosphate-buffered saline solution, 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 Ed., Mack Publ. Co., Easton, PA

[1975] . Pharmaceutically acceptable carriers include buffers, solvents, dispersion media, coatings, isotonic and absorption delaying agents, etc., compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art.

[0190] In certain embodiments, pharmaceutical compositions may contain formulation materials to modify, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or permeability of the composition.In such embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); bulking agents; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colorants, flavoring agents, etc. agents) and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (such as glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancers (such as sucrose or sorbitol); isotonicity enhancers (such as alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, sorbitol); delivery vehicles: diluents; excipients and / or pharmaceutical adjuvants (Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990).

[0191] In some embodiments, the pharmaceutical composition can include nanoparticles, such as polymeric nanoparticles, liposomes, or micelles (see Anselmo et al. (2016) BIOENG. TRANSL. MED. 1: 10-29).

[0192] In some embodiments, the pharmaceutical composition may comprise a sustained- or controlled-delivery formulation. Techniques for formulating sustained- or controlled-delivery means, such as liposome carriers, bioerodible microparticles or porous beads, and depot injections, are also known to those skilled in the art. The sustained-release preparation may comprise, for example, porous polymeric microparticles or semipermeable polymer matrices in the form of shaped articles, such as films or microcapsules. The sustained-release matrix may comprise polyester, hydrogel, polylactide, copolymer of L-glutamic acid and gamma-ethyl-L-glutamic acid, poly(2-hydroxyethyl-inethacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. The sustained-release composition may also comprise liposomes, which may be prepared by any of several methods known in the art.

[0193] Pharmaceutical compositions comprising the recombinant human sialidase, recombinant human sialidase fusion protein, or antibody conjugate disclosed herein may be present in unit dosage form and may be prepared by any suitable method. Pharmaceutical compositions should be formulated to be compatible with their intended route of administration. Examples of routes of administration include intravenous (IV), intradermal, inhalation, transdermal, topical, transmucosal, intrathecal, and rectal administration. In some embodiments, the recombinant human sialidase, recombinant human sialidase fusion protein, or antibody conjugate disclosed herein is administered by IV infusion. In some embodiments, the recombinant human sialidase, recombinant human sialidase fusion protein, or antibody conjugate disclosed herein is administered by intratumoral injection. Useful formulations can be prepared by methods known in the pharmaceutical arts. See, for example, Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990). Formulation components suitable for parenteral administration include a sterile diluent, such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as EDTA; buffers, such as acetates, citrates or phosphates; and agents for the adjustment of tonicity, such as sodium chloride or dextrose.

[0194] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate-buffered saline (PBS). The carrier should be stable under the conditions of manufacture and storage and should be preserved against microorganisms. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

[0195] 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 (CaCl) or magnesium chloride (MgCl).

[0196] In certain 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 an amount of about 0.05 mM to about 4 mM, about 0.05 mM to about 3 mM, about 0.05 mM to about 2 mM, about 0.05 mM to about 1 mM, about 0.05 mM to about 0.5 mM, about 0.5 mM to about 4 mM, about 0.5 mM to about 3 mM, about 0.5 mM to about 2 mM, about 0.5 mM to about 1 mM, about 1 mM to about 4 mM, about 1 mM to about 3 mM, or about 1 mM to about 2 mM.

[0197] Preferably, the pharmaceutical preparation is sterile.Sterilization can be achieved by any suitable method, for example, by filtration through a sterile filtration membrane.When the composition is lyophilized, sterilization by filtration can be carried out before or after lyophilization and reconstitution.

[0198] The compositions described herein can be administered locally or systemically. Administration is generally parenteral. In a preferred embodiment, the pharmaceutical composition is administered subcutaneously, and in a more preferred embodiment, it is administered intravenously. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions.

[0199] Generally, a therapeutically effective amount of an active ingredient, such as a recombinant human sialidase or its fusion protein and / or antibody conjugate, ranges from 0.1 mg / kg to 100 mg / kg, e.g., 1 mg / kg to 100 mg / kg, e.g., 1 mg / kg to 10 mg / kg. The dosage depends on variables such as the type and severity of the disease or symptom being treated, the patient's overall health, the in vivo efficacy of the antibody, the pharmaceutical formulation, and the route of administration. To rapidly achieve the desired blood or tissue levels, the initial dose may be increased beyond the upper limit. Alternatively, the initial dose may be lower than optimal, and the daily dose may be gradually increased over the course of treatment. Human dosages may be optimized in a conventional Phase I dose-escalation study designed, for example, from 0.5 mg / kg to 20 mg / kg. The frequency of administration may vary depending on factors such as the route of administration, the dosage, the serum half-life of the recombinant human sialidase or its fusion protein and / or antibody conjugate, and the disease being treated. Exemplary administration frequencies are once a day, once a week, and once every two weeks. A preferred administration route is parenteral, for example, intravenous infusion. In some embodiments, recombinant human sialidase or its fusion protein and / or antibody conjugate is lyophilized and then reconstituted in buffered saline at the time of administration.

[0200] V. Therapeutic uses The compositions and methods disclosed herein can be used to treat various forms of cancer in a subject or to inhibit the growth of cancer in a subject. The present invention provides a method for treating cancer in a subject. The method includes administering to the subject an effective amount of a recombinant human sialidase or its fusion protein and / or antibody conjugate, such as a recombinant human sialidase, fusion protein, or antibody conjugate disclosed herein, alone or in combination with another therapeutic agent, to treat cancer in the subject. The term "effective amount," as used herein, refers to the amount of an active agent (e.g., a recombinant human sialidase or its fusion protein of the present invention) sufficient to produce a beneficial or desired result. An effective amount can be administered in one or more administrations, applications, or doses, and is not intended to be limited to a particular formulation or route of administration.

[0201] As used herein, "treat," "treating," and "treatment" refer to the treatment of a disease in a subject, e.g., a human. This includes (a) inhibiting the disease, i.e., halting its progression; and (b) alleviating the disease, i.e., causing regression of the disease state. As used herein, the terms "subject" and "patient" refer to an organism treated by the methods and compositions described herein. Preferably, such organisms include, but are not limited to, mammals (e.g., mice, monkeys, horses, cows, pigs, dogs, cats, etc.), and more preferably, humans.

[0202] Examples of cancer include solid tumors, soft tissue tumors, hematopoietic tumors and metastatic lesions.Examples of hematopoietic tumors include leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), B cell, T cell or FAB ALL, acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), for example, 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's lymphoma, multiple myeloma or Richter's syndrome (Richter's transformation). Examples of solid tumors include malignancies such as sarcomas, adenocarcinomas, and carcinomas of various organ systems, such as those affecting the head and neck (including pharynx), thyroid, lung (small cell or non-small cell lung carcinoma (NSCLC)), breast, lymphatic system, gastrointestinal (e.g., oral cavity, esophagus, stomach, liver, pancreas, small intestine, colon and rectum, anal canal), reproductive and genitourinary tract (e.g., kidney, urothelium, bladder, ovaries, uterus, cervix, endometrium, prostate, testes), CNS (e.g., neuronal or glial cells, e.g., neuroblastoma or glioma), or skin (e.g., melanoma).

[0203] In some embodiments, the cancer is an epithelial cancer, e.g., an epithelial cancer that upregulates the expression of sialylated glycans. Exemplary epithelial cancers include, but are not limited to, endometrial cancer, colon cancer, ovarian cancer, cervical cancer, vulvar cancer, uterine cancer or fallopian tube cancer, breast cancer, prostate cancer, lung cancer, pancreatic cancer, urinary tract cancer, bladder cancer, head and neck cancer, oral cancer, and liver cancer. Epithelial cancers also include carcinomas, such as lobular carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, adenocarcinoma (carcinoma adenomatosum), carcinoma of the adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchiolocarcinoma, bronchogenic carcinoma, cerebrioid carcinoma, cholangiocarcinoma, choriocarcinoma, colloid carcinoma, comedocarcinoma, corpus carcinoma, cribriform carcinoma, armor carcinoma, carcinoma cutaneum, and cylindrical carcinoma. carcinoma, cylindrical cell carcinoma, ductal carcinoma, carcinoma durum, embryonal carcinoma, encephalomoid carcinoma, epidermoid carcinoma, epithelial adenoid carcinoma, exophytic carcinoma, extraulcerative carcinoma, fibrous carcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, adenocarcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hürthle cell carcinoma, vitreous carcinomacarcinoma, hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, carcinoma in epidermis, carcinoma in situ (intraepithelial carcinoma), Krompecher's carcinoma, Kurticycky cell carcinoma, large cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullaris, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, myxomatodes, nasopharyngeal carcinoma, oat cell carcinoma, ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of the kidney, reserve cell carcinoma, sarcomatodes, Schneiderian carcinoma, scirrhous carcinoma, scrotal carcinoma, signet ring cell carcinoma, carcinoma simplex, small cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma carcinoma), spindle cell carcinoma, carcinoma spongiosum, squamous cell carcinoma, squamous cell carcinomaThese include carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, nodular carcinoma, tuberous carcinoma, verrucous carcinoma, and choriocarcinoma.

[0204] 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 a refractory cancer.

[0205] In some embodiments, the cancer is resistant or unresponsive to treatment with an antibody, eg, an antibody with ADCC activity, eg, trastuzumab.

[0206] The methods and compositions described herein can be used alone or in combination with other therapeutic agents and / or modalities. As used herein, the term "administered in combination" is understood to mean that two (or more) different therapies are delivered to a subject with a disorder so that the effects of the therapies on the patient overlap at points in time during the course of the subject's suffering. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so there is an overlap in the administration period. This is sometimes referred to herein as "simultaneous" or "concurrent delivery." In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments in either case, the treatments are more effective due to the combined administration. For example, the second treatment is more effective, e.g., a comparable effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent than would be seen if the second treatment were administered in the absence of the first treatment, or a comparable condition is seen with the first treatment. In some embodiments, delivery is such that the reduction in symptoms or other parameters associated with the disorder is greater than that observed when one treatment is delivered in the absence of the other treatment. The effects of the two treatments may be partially additive, wholly additive, or greater than additive. Delivery may be such that the effect of the first treatment delivered remains detectable when the second treatment is delivered.

[0207] In some embodiments, the methods or compositions described herein are administered in combination with one or more additional treatments, such as surgery, radiation therapy, or the administration of another therapeutic agent. In some embodiments, the additional treatment may include chemotherapy, such as a cytotoxic agent. In some embodiments, the additional treatment may include a targeted treatment, such as a tyrosine kinase inhibitor, a proteasome inhibitor, or a protease inhibitor. In some embodiments, the additional treatment may include an anti-inflammatory, anti-angiogenic, anti-fibrotic, or anti-proliferative compound, such as a steroid, a biological immunomodulator, a monoclonal antibody, an antibody fragment, an aptamer, an siRNA, an antisense molecule, a fusion protein, a cytokine, a cytokine receptor, a bronchodilator, a statin, an anti-inflammatory agent (e.g., methotrexate), or an NSAID. In some embodiments, the additional treatment may include a combination of different therapeutic agents.

[0208] In some embodiments, the methods or compositions described herein are administered in combination with a checkpoint inhibitor.The checkpoint inhibitor can be selected from, for example, a PD-1 antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, an adenosine A2A receptor antagonist, a B7-H3 antagonist, a B7-H4 antagonist, a BTLA antagonist, a KIR antagonist, a LAG3 antagonist, a TIM-3 antagonist, a VISTA antagonist, or a TIGIT antagonist.

[0209] 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 in the immune system, inhibiting or otherwise regulating T cell activity at the appropriate time to prevent an overactive immune response. However, cancer cells can use this checkpoint by expressing a ligand, such as PD-L1, that interacts with PD-1 on the surface of T cells to block or regulate T cell activity. Exemplary PD-1 / PD-L1-based immune checkpoint inhibitors include antibody-based therapeutic agents. Exemplary therapeutic methods using 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, e.g., in U.S. Patent Nos. 8,952,136, 8,779,105, 8,008,449, 8,741,295, 9,205,148, 9,181,342, 9,102,728, 9,102,727, 8,952,136, 8,927,697, 8,900,587, 8,735,553, and 7,488,802. Exemplary anti-PD-1 antibodies include, for example, nivolumab (Opdivo®, Bristol-Myers Squibb Co.), pembrolizumab (Keytruda®, Merck Sharp & Dohme Corp.), PDR001 (Novartis Pharmaceuticals), and pidilizumab (CT-011, Cure Tech). 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 (Tecentriq®, Genentech), duvalumab (AstraZeneca), MEDI4736, avelumab, and BMS 936559 (Bristol Myers Squibb Co.).

[0210] In some embodiments, methods or compositions described herein are administered in combination with CTLA-4 inhibitor.In CTLA-4 pathway, the interaction between CTLA-4 on T cell and its ligand (also known as CD80, B7-1 and CD86) on the surface of antigen-presenting cell (not cancer cell) causes T cell inhibition.Exemplary CTLA-4-based immune checkpoint inhibition methods are described in United States Patent (USP) No. 5,811,097, No. 5,855,887 and No. 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) Publication Nos. WO 98 / 42752, WO 00 / 37504, and WO 01 / 14424; and European Patent EP 1212422. B1. Exemplary CTLA-4 antibodies include ipilimumab or tremelimumab.

[0211] In certain embodiments, a method or composition described herein is administered in combination with (i) a PD-1 or PD-L1 inhibitor, such as a PD-1 or PD-L1 inhibitor disclosed herein, and (ii) a CTLA-4 inhibitor, such as a CTLA-4 inhibitor disclosed herein.

[0212] 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.

[0213] Exemplary cytotoxic agents that may be administered in combination with the methods or compositions described herein include, for example, antimicrotubule agents, topoisomerase inhibitors, antimetabolites, protein synthesis and degradation inhibitors, mitotic inhibitors, alkylating agents, platinizing agents, and the like. agents), inhibitors of nucleic acid synthesis, histone deacetylase inhibitors (HDAC inhibitors, e.g., vorinostat (SAHA, MK0683), entinostat (MS-275), panobinostat (LBH589), trichostatin A (TSA), mocetinostat (MGCD0103), belinostat (PXD101), romidepsin (FK228, depsipeptide)), DNA methyltransferase inhibitors, nitrogen mustards, nitrosoureas, ethylenimines, alkylsulfonates, triazenes, folic acid analogs, nucleoside analogs, ribonucleotide reductase inhibitors, vinca alkaloids, taxanes, epothilones, intercalating agents, drugs that can interfere with signal transduction pathways, drugs and radiation that promote apoptosis, or antibody molecule conjugates that bind to surface proteins to deliver toxic agents.In one embodiment, cytotoxic agents that may be administered with the methods or compositions described herein include platinum-based agents (e.g., cisplatin), cyclophosphamide, dacarbazine, methotrexate, fluorouracil, gemcitabine, capecitabine, hydroxyurea, topotecan, irinotecan, azacitidine, vorinostat, ixabepilone, bortezomib, taxanes (e.g., paclitaxel or docetaxel), cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, vinorelbine, colchicine, anthracyclines (e.g., doxorubicin or epirubicin). Daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, adriamycin, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, ricin, or maytansinoids.

[0214] The present invention also provides methods for increasing expression of HLA-DR, CD86, CD83, IFNγ, IL-1b, IL-6, TNFα, IL-17A, IL-2, or IL-6 in a cell, tissue, or subject. The methods include contacting the cell, tissue, or subject with an effective amount of a sialidase, fusion protein, and / or antibody conjugate, such as a sialidase, fusion protein, or antibody conjugate disclosed herein. In some embodiments, the cell is selected from a dendritic cell and a peripheral blood mononuclear cell (PBMC).

[0215] In certain embodiments, expression of HLA-DR, CD86, CD83, IFNγ, IL-1b, IL-6, TNFα, IL-17A, IL-2, or IL-6 in a cell, tissue, or 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% relative to similar or otherwise identical cells or tissues that have not been contacted with the sialidase, fusion protein, or antibody conjugate. Gene expression can be measured by any suitable method known in the art, for example, by ELISA or by Luminex multiplex assay.

[0216] The present invention also provides a method for promoting immune cell infiltration into tumors in a subject in need thereof. The method comprises administering to the subject an effective amount of a sialidase, fusion protein, and / or antibody conjugate, such as a sialidase, fusion protein, or antibody conjugate disclosed herein. In some embodiments, the immune cells are T cells, e.g., CD4+ and / or CD8+ T cells, e.g., CD69 + CD8 + and / or GzmB + CD8 + In some embodiments, the immune cell is a natural killer (NK) cell.

[0217] In certain embodiments, immune cell infiltration into a tumor in a subject is increased by at least about 10%, at least about 20%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1,000% relative to a similar or otherwise identical tumor and / or subject that has not been administered the sialidase, fusion protein, or antibody conjugate. Immune cell infiltration into a tumor can be measured by any suitable method known in the art, such as antibody staining.

[0218] The present invention also provides a method for increasing the number of circulating natural killer (NK) cells in a subject in need thereof, comprising administering to the subject an effective amount of a sialidase, fusion protein, and / or antibody conjugate, such as a sialidase, fusion protein, or antibody conjugate disclosed herein, to increase the number of circulating NK cells relative to before administration of the sialidase, fusion protein, or antibody conjugate.

[0219] In certain embodiments, the number of circulating NK cells in a subject is increased by at least about 10%, at least about 20%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1,000%, relative to a similar or otherwise identical subject that did not receive the sialidase, fusion protein, or antibody conjugate. Circulating NK cells in a subject can be measured by any suitable method known in the art, such as antibody staining.

[0220] The present invention also provides a method for increasing the number of T cells in a draining lymph node in a subject in need thereof. The method comprises administering to the subject an effective amount of a sialidase, fusion protein, and / or antibody conjugate, e.g., a sialidase, fusion protein, or antibody conjugate disclosed herein, to increase the number of T cells in the draining lymph node relative to before administration of the fusion protein, antibody conjugate, or pharmaceutical composition. In some embodiments, the immune cells are T cells, e.g., CD4+ and / or CD8+ T cells.

[0221] In certain embodiments, the number of T cells in the draining lymph nodes in a subject is increased by at least about 10%, at least about 20%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1,000%, relative to a similar or otherwise identical subject that did not receive the sialidase, fusion protein, or antibody conjugate. T cells in the draining lymph nodes in a subject can be measured by any suitable method known in the art, e.g., by antibody.

[0222] The present invention also provides methods for increasing expression of Cd3, Cd4, Cd8, Cd274, Ctla4, Icos, Pdcd1, Lag3, Il6, Il1b, Il2, Ifng, Ifna1, Mx1, Gzmb, Cxcl9, Cxcl12 and / or Ccl5 in a cell, tissue or subject. The method includes contacting a cell, tissue, or subject with an effective amount of a sialidase, fusion protein, and / or antibody conjugate, such as a sialidase, fusion protein, or antibody conjugate disclosed herein, to increase expression of Cd3, Cd4, Cd8, Cd274, Ctla4, Icos, Pdcd1, Lag3, Il6, Il1b, Il2, Ifng, Ifna1, Mx1, Gzmb, Cxcl9, Cxcl12, and / or Ccl5 relative to the cell, tissue, or subject prior to contact with the sialidase, fusion protein, or antibody conjugate.

[0223] In certain embodiments, expression of Cd3, Cd4, Cd8, Cd274, Ctla4, Icos, Pdcd1, Lag3, Il6, Il1b, Il2, Ifng, Ifna1, Mx1, Gzmb, Cxcl9, Cxcl12 and / or Ccl5 in a cell, tissue or 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% relative to a similar or otherwise identical cell, tissue or subject not contacted with the sialidase, fusion protein or antibody conjugate. Gene expression can be measured by any suitable method known in the art, for example, ELISA, Luminex multiplex assay or Nanostring technology.

[0224] The present invention also provides a method for removing sialic acid from cells or tissues, comprising contacting the cells or tissues with an effective amount of a sialidase, fusion protein, and / or antibody conjugate, such as a sialidase, fusion protein, or antibody conjugate disclosed herein. The present invention also provides a method for removing sialic acid from cells in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising a sialidase, fusion protein, and / or antibody conjugate, such as a sialidase, fusion protein, or antibody conjugate disclosed herein, thereby removing sialic acid from the cells.

[0225] 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, expression of MHC-II molecules in cells or tissues is increased by at least about 10%, at least about 20%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1,000% relative to similar or otherwise identical cells or tissues that have not been contacted with the sialidase, fusion protein, and / or antibody conjugate. 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.

[0226] The present invention also provides a method for increasing the phagocytosis of tumor cells, comprising contacting tumor cells with an effective amount of a sialidase, fusion protein, and / or antibody conjugate, such as a sialidase, fusion protein, or antibody conjugate disclosed herein, to remove sialic acid from the tumor cells, thereby increasing the phagocytosis of the tumor cells. In some embodiments, the present disclosure relates to a method for increasing the phagocytosis of tumor cells in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition, sialidase, fusion protein, and / or antibody conjugate, such as a sialidase, fusion protein, or antibody conjugate disclosed herein, to remove sialic acid from the tumor cells, thereby increasing the phagocytosis of the tumor cells.

[0227] In certain 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 a similar or otherwise identical tumor cell or population of tumor cells that has not been contacted with the sialidase, fusion protein, and / or antibody conjugate. Phagocytosis can be measured by any suitable method known in the art.

[0228] The present invention also provides a method for activating dendritic cells (DCs), comprising contacting DCs with tumor cells treated with a sialidase, a fusion protein, and / or an antibody conjugate, such as a sialidase, fusion protein, or antibody conjugate disclosed herein. In one embodiment, the present disclosure relates to a method for activating dendritic cells (DCs) or a population of DCs in a subject, comprising administering to the subject an amount of a pharmaceutical composition comprising a sialidase, a fusion protein, and / or an antibody conjugate, such as a sialidase, fusion protein, or antibody conjugate disclosed herein, effective to remove sialic acid from tumor cells in the subject, thereby activating DCs or a population of DCs in the subject.

[0229] In certain embodiments, activation of a DC or population of DCs is increased by at least about 10%, at least about 20%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1,000% relative to a similar or otherwise identical DC or population of DCs that has not been contacted with tumor cells treated with sialidase, fusion protein, and / or antibody conjugate. Activation can be measured by any suitable method known in the art.

[0230] The present disclosure also provides methods for reducing Siglec-15 binding activity, thereby increasing anti-tumor activity in a tumor microenvironment, comprising contacting T cells with a sialidase, a fusion protein, and / or an antibody conjugate, such as a sialidase, fusion protein, or antibody conjugate disclosed herein. In certain embodiments, the present disclosure relates to methods for reducing Siglec-15 binding activity, thereby increasing anti-tumor activity in a tumor microenvironment in a patient, comprising administering to the subject an effective amount of a pharmaceutical composition comprising a sialidase, a fusion protein, and / or an antibody conjugate, such as a sialidase, fusion protein, or antibody conjugate disclosed herein, thereby increasing anti-tumor activity (e.g., T cell activity) in the subject.

[0231] In certain 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, fusion protein, and / or antibody conjugate. Binding can be measured by any suitable method known in the art.

[0232] Throughout the description, when compositions are described as having, including, or comprising particular components, or when processes and methods are described as having, including, or comprising particular steps, it is further contemplated that there are compositions of the invention that consist essentially of or consist of the recited components, and that there are processes and methods of the invention that consist essentially of or consist of the recited process steps.

[0233] In this application, when an element or component is said to be included in and / or selected from a list of described elements or components, it is to be understood that the element or component can be any one of the described elements or components, or the element or component can be selected from a group consisting of two or more of the described elements or components.

[0234] Furthermore, it should be understood that the elements and / or features of the compositions or methods described herein, whether expressly or implicitly stated herein, can be combined in various ways without departing from the spirit and scope of the present invention. For example, when reference is made to a particular compound, the compound can be used in various embodiments of the compositions of the present invention and / or in the methods of the present invention, unless otherwise understood from the context. That is, although embodiments are described and illustrated herein in a manner that allows clear and concise application to be described and illustrated, it is intended and understood that the embodiments can be variously combined or separated without departing from the present teachings and invention(s). For example, it is understood that all features described and illustrated herein can be applicable to all aspects of the invention(s) described and illustrated herein.

[0235] The phrase "at least one" should be understood to include each of the listed items individually and various combinations of two or more of the listed items, unless otherwise understood from context and application. With respect to more than two listed items, the phrase "and / or" should be understood to have the same meaning, unless otherwise understood from context.

[0236] Use of the terms "include," "includes," "including," "have," "has," "having," "contain," "contains," or "containing," including grammatical equivalents thereof, is to be understood generally as open-ended and non-limiting, for example without excluding additional, unrecited elements or steps, unless the context specifically states or understands otherwise.

[0237] When the term "about" is used before a quantitative value, the invention also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term "about" refers to a ±10% variation from the nominal value, unless otherwise indicated or inferred.

[0238] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions may be performed simultaneously.

[0239] The use of any and all examples or exemplary terms herein, such as "such as" or "including," is intended merely to better describe the invention and does not pose a limitation on the scope of the invention unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. [Example]

[0240] Example The following examples are illustrative only and are not intended to limit the scope or content of the invention in any way.

[0241] Example 1 This example describes the construction of recombinant human sialidases (Neu1, Neu2, and Neu3).

[0242] Human sialidases Neu1, Neu2, Neu3 (isoform 1), and Neu4 (isoform 1) were expressed as secreted proteins with a 10xHis tag. To express Neu1 as a secreted protein, the native N-terminal signal peptide (MTGERPSTALPDRRWGPRILGFWGGCRVWVFAAIFLLLSLAASWSKA; SEQ ID NO: 27) was replaced with MDMRVPAQLLGLLLLWLPGARC (SEQ ID NO: 28), and the C-terminal lysosomal signal motif (YGTL; SEQ ID NO: 29) was removed. To express Neu2, Neu3, and Neu4 as secreted proteins, the N-terminal signal peptide MDMRVPAQLLGLLLLWLPGARC (SEQ ID NO: 28) was added to each.

[0243] Sialidase was expressed in 200 mL transfections of HEK293F human cells in 24-well plates using the pCEP4 mammalian expression vector with an N-terminal 6xHis tag. Sialidase was purified using a Ni-NTA column, quantified using a UV-Vis spectrometer (NanoDrop), and examined by SDS-PAGE as shown in Figure 1. Neu1 was well expressed with a yield of approximately 3 μg / mL and existed primarily in a monomeric form. Neu2 and Neu3 expression gave yields of approximately 0.15 μg / mL each, each existing primarily in a dimeric form. Neu4 had no detectable expression yield as measured by NanoDrop. Bacterial sialidase from Salmonella typhimurium (St-sialidase; SEQ ID NO: 30), used as a positive control for expression, gave a yield comparable to Neu1 and existed primarily in a monomeric form.

[0244] The activity of recombinantly expressed sialidases was assayed by measuring the release of sialic acid from the fluorogenic substrate 4-methylumbelliferyl-N-acetylneuraminic acid (4MU-NeuAc). As shown in Figure 2, Neu1 had no detectable activity above the no-enzyme control, consistent with previous reports showing that Neu1 is inactive unless complexed with β-galactosidase and protective protein / cathepsin A (PPCA). Neu2 and Neu3 were active. Enzyme kinetic assays were performed using Neu2 and Neu3. A fixed concentration of 1 nM enzyme was incubated with the fluorogenic substrate 4MU-NeuAc at concentrations ranging from 4000 μM to 7.8 μM. Assays were performed under both acidic (pH 5.6) and neutral (pH 7) conditions. As shown in Figure 3, both Neu2 and Neu3 were active in both acidic and neutral conditions and exhibited enzyme kinetics comparable to those previously reported.

[0245] Most of the recombinantly expressed sialidase migrated as aggregates or dimers on non-reducing SDS-PAGE gels. Subsequent treatment with the reducing agent dithiothreitol (DTT) yielded a monomeric form of the enzyme that migrated at 42 kDa on reducing SDS-PAGE gels (Figure 1).

[0246] Example 2 This example describes the construction of recombinant human sialidase with mutations that increase the expression and / or activity of the sialidase.

[0247] A. Rational Design Structural and sequence analysis identified residues A93 and P62 of Neu2 as candidates for substitutions that increase solubility and / or expression. In particular, comparison of homologous sialidase sequences indicated a preference for a D or E amino acid residue at the position corresponding to Neu2 position 93 and a preference for a G amino acid residue at the position corresponding to Neu2 position 62.

[0248] The β-propeller family of proteins is typically stabilized by extensive hydrogen-bonding interactions at the N- and C-termini of the protein. Structural analysis revealed that Neu2, a member of the β-propeller family, appears to lack these stabilizing interactions. In contrast, sialidases from Salmonella typhimurium and Micromonospora viridifaciens (the bacterial sialidases most homologous to human Neu2) have extensive hydrogen-bonding interactions at their N- and C-termini. Therefore, residues K9, V363, and L365 of Neu2 were mutated to promote hydrogen bonding between the N- and C-termini of Neu2.

[0249] B. Phage Display Neu2 was expressed in a phage display system to allow screening of Neu2 variants for both expression level and resistance to heat denaturation. Neu2 with V6Y and I187K substitutions was used as a template for library preparation. The designed phage display libraries 1, 2, and 3 are shown in Tables 11-13, respectively. Each library contained all possible combinations of the mutations shown. The fourth library contained random mutations generated by error-prone PCR. [Table 25] [Table 26] [Table 27]

[0250] The codon usage column in Tables 11-13 indicates the degenerate codon codes used in the design of the libraries, where the first, second, and third positions of a given codon encoding an amino acid are shown in Table 14 and as described in Mena et al. (2005) PROTEIN ENG DES SEL. 18(12):559-61. [Table 28]

[0251] Phage display libraries were screened for binding to conformation-specific antibodies and / or sialic acid biotinylated probes after heating to enrich for thermostability and expression. The sialic acid biotinylated probes and their synthesis are shown in Figure 4. An exemplary phage display screening procedure is shown in Figure 5. Briefly, a phage library expressing the desired Neu2 variants was generated. The phage were screened for binding to immobilized anti-Neu2 antibodies and / or sialic acid biotinylated probes. After washing to remove unbound phage, bound phage were eluted from the antibody or probe and analyzed, as appropriate.

[0252] C. Yeast Display Neu2 was also expressed in a yeast display system, allowing screening of Neu2 variants for both expression level and resistance to heat denaturation. Neu2 with V6Y and I187K substitutions was used as a template for library preparation. The designed yeast display libraries 1a, 1b, 1c, 1d, 2a, 2b, 2c, 3a, 3b, and 3c are shown in Tables 15-24, respectively. Each library contained all possible combinations of the mutations shown. Five additional sublibraries were generated by error-prone PCR with approximate average proportions of 1, 2, 3, 4, and 5 substitutions per enzyme. [Table 29] [Table 30] [Table 31] [Table 32] [Table 33] [Table 34] [Table 35] [Table 36] [Table 37] [Table 38]

[0253] The codon usage columns in Tables 15-24 indicate the degenerate codon codes used in the design of the libraries, where the first, second, and third positions of a given codon encoding an amino acid are shown herein above in Table 14 and as described in Mena et al. (2005) PROTEIN ENG DES SEL. 18(12):559-61.

[0254] Yeast display libraries were screened for binding to conformation-specific antibodies and / or sialic acid biotinylated probes after heating to enrich for thermal stability and expression. An exemplary yeast display screening procedure is shown in Figure 6. Briefly, a plasmid library encoding the desired Neu2 variants and yeast cells expressing the desired Neu2 variants on their surface were generated. The yeast cells were heat-shocked and then screened for binding to anti-Neu2 antibodies and / or sialic acid biotinylated probes on magnetic beads. The magnetic beads were isolated to remove unbound cells, and the bound cells were further analyzed for Neu2 affinity, activity, and stability as appropriate.

[0255] D. Results Mutant sialidases containing mutations identified using the rational design, phage display, and yeast display approaches described in this Example were expressed as secreted proteins with a C-terminal human Fc tag in Expi293F cells using the pCEP4 mammalian expression vector. Expression was assayed using a ForteBio Octet with an anti-human Fc sensor and Western blot, as described above, and enzyme activity was assayed using the fluorogenic substrate 4MU-NeuAc.

[0256] Expression and activity levels for the mutant sialidases are shown in Table 25. In Table 25, enzyme activity is indicated as "+++", indicating >2-fold higher activity than wild-type Neu2, "++", indicating activity equivalent to wild-type Neu2, "+", indicating lower activity than wild-type Neu2, or "-", indicating no detectable activity, and expression is indicated as "++++", indicating >15-fold higher expression than wild-type Neu2, "+++", indicating >6-fold higher expression than wild-type Neu2, "++", indicating 2-5-fold higher expression than wild-type Neu2, "+", indicating expression equivalent to wild-type Neu2, or "-", indicating no detectable expression. [Table 39-1] [Table 39-2]

[0257] To confirm these results, Neu2-M106 (having amino acid sequence SEQ ID NO:48, encoded by nucleotide sequence SEQ ID NO:89) was expressed and purified on a Protein A column. Figure 7A is an image of an SDS-PAGE gel showing recombinant wild-type human Neu2 and Neu2 variant M106 (each with a C-terminal human Fc tag) under non-reducing and reducing conditions. Figure 7B is an SEC-HPLC trace for recombinant wild-type human Neu2 and Neu2 variant M106 (each with a C-terminal human Fc tag). Neu2-Fc had a yield of 0.3 mg / liter and a monomer content of 7% as measured by SEC after Protein A purification, while Neu2-M106 had a yield of 20 mg / liter and a monomer content of 85%.

[0258] The enzymatic kinetics of Neu2-M106 was assayed by measuring the release of sialic acid from the fluorogenic substrate 4-methylumbelliferyl-N-acetylneuraminic acid (4MU-NeuAc) as described above. A fixed concentration of 2 μg / well of enzyme was incubated with concentrations of the fluorogenic substrate 4MU-NeuAc ranging from 4 mM to 0.03 μM. Figure 8 shows the enzymatic activity of the Neu2 variant M106. The enzymatic activity of Neu2-M106 was comparable to that of wild-type Neu2, with a K M was determined to be 230 μM.

[0259] Together, these results indicate that mutations identified by the rational design, phage display, and / or yeast display approaches described herein can increase sialidase stability and / or expression.

[0260] Example 3 This example describes the construction and expression of antibody-sialidase conjugates (ASCs), including antibody-sialidase genetic fusion proteins and fusion proteins with mutated human sialidase.

[0261] The configurations for four exemplary ASCs are shown in Figure 11. The first type of ASC, termed "Raptor," contains an antibody (with two heavy chains and two light chains), with a sialidase fused to the C-terminus of each heavy chain of the antibody (Figure 11A). The second type of ASC, termed "Janus," contains one antibody arm (with one heavy chain and one light chain) and one sialidase-Fc fusion, with a sialidase fused to the N-terminus of one arm of the Fc. Each Fc domain polypeptide in the Janus ASC contains either a "knob" (T366Y) or a "hole" (Y407T) mutation (residue numbers according to EU numbering; Kabat, EA, et al. (1991) supra) for heterodimerization (Figure 11B). The third type of ASC, termed "Lobster," contains two Fc domain polypeptides, each with a sialidase fused at the N-terminus of the Fc and an scFv fused at the C-terminus of the Fc (Figure 11C). The fourth type of ASC, termed "Bunk," contains one antibody arm (having one heavy chain and one light chain), an scFv fused at the C-terminus of one Fc arm, and one sialidase-Fc fusion with a sialidase fused at the N-terminus of the other Fc arm. Each Fc domain polypeptide in the Bunk ASC contains either a "knob" (T366Y) or a "hole" (Y407T) mutation (residue numbers according to EU numbering; see Kabat, EA, et al. (1991) supra) for heterodimerization (Figure 11D).

[0262] Janus ASCs and trastuzumab containing the Neu2 variants described in Example 2 were generated and tested for activity and expression. Expression was assayed using a ForteBio Octet with an anti-human Fc sensor and Western blot, as described above, and enzyme activity was assayed using the fluorogenic substrate 4MU-NeuAc. Expression and activity levels for Janus ASCs are shown in Table 26. In Table 26, enzyme activity is indicated as "+++," indicating >2-fold higher activity than wild-type Neu2, "++," indicating activity equivalent to wild-type Neu2, "+," indicating lower activity than wild-type Neu2, or "-," indicating no detectable activity. Expression is indicated as "++++," indicating >15-fold higher expression than wild-type Neu2, "+++," indicating >6-fold higher expression than wild-type Neu2, "++," indicating 2- to 5-fold higher expression than wild-type Neu2, "+," indicating expression equivalent to wild-type Neu2, or "-," indicating no detectable expression. [Table 40]

[0263] Additional Janus ASCs containing the Neu2 variants described in Example 2 and trastuzumab were generated and tested for activity and expression. Janus ASCs were expressed in Expi293F cells in 500 mL cultures and purified using protein A and ion exchange chromatography. Expression was assayed using a ForteBio Octet with an anti-human Fc sensor and Western blot, as described above, and enzyme activity was assayed using the fluorogenic substrate 4MU-NeuAc. Expression and activity levels for Janus ASCs are shown in Table 27. In Table 27, enzyme activity is indicated as "+++" indicating >2-fold higher activity than wild-type Neu2, "++" indicating activity equivalent to wild-type Neu2, "+" indicating lower activity than wild-type Neu2, or "-" indicating no detectable activity, and expression is indicated as "++++" indicating >15-fold higher expression than wild-type Neu2, "+++" indicating >6-fold higher expression than wild-type Neu2, "++" indicating 2-5-fold higher expression than wild-type Neu2, "+" indicating expression equivalent to wild-type Neu2, or "-" indicating no detectable expression. [Table 41]

[0264] Example 4 This example describes the construction of recombinant human sialidase with mutations that increase the expression and / or activity of the sialidase.

[0265] Unless otherwise indicated, the mutant sialidases of this example were expressed as secreted proteins with a C-terminal human Fc tag in Expi293F cells using the pCEP4 mammalian expression vector. Expression was assayed using a ForteBio Octet with an anti-human Fc sensor and Western blot, and enzyme activity was assayed using the fluorogenic substrate 4MU-NeuAc, as described above.

[0266] A mutant Neu2 sialidase containing a rationally designed substitution at position Q126 was constructed. Examination of the Neu2 crystal structure revealed that the Q126 mutation could increase interactions with neighboring amino acid residues.

[0267] Further mutant Neu2 sialidases were constructed containing rationally designed substitutions at position Q270. Examination of the Neu2 crystal structure revealed that mutations to specific amino acids at Q270 could stabilize the interaction with R237 and stabilize binding in the substrate pocket.

[0268] Additional mutant Neu2 sialidases were constructed that contain proline substitutions of amino acid residues in the β-turn (e.g., D80P, R189P, and / or H239P substitutions). Proline substitutions at these positions may stabilize the protein, for example, by affecting local protein folding.

[0269] The expression and activity levels for the resulting mutant sialidases are shown in Table 28. In Table 28, enzyme activity is indicated as "++", which indicates activity equivalent to wild-type Neu2, "+", which indicates activity less than wild-type Neu2, or "-", which indicates no detectable activity, and expression is indicated as "+++++", which indicates expression >40-fold higher than wild-type Neu2, "++++", which indicates expression >15-fold higher than wild-type Neu2, "+++", which indicates expression >6-fold higher than wild-type Neu2, "++", which indicates expression 2-5-fold higher than wild-type Neu2, "+", which indicates expression equivalent to wild-type Neu2, or "-", which indicates no detectable expression. [Table 42]

[0270] To confirm these results, Neu2-M173 (having amino acid sequence SEQ ID NO:159 encoded by nucleotide sequence SEQ ID NO:181) with a C-terminal human Fc tag was expressed and purified using Protein A and a ceramic hydroxyapatite (CHT) column. Figure 22A is an image of an SDS-PAGE gel showing Neu2-M173-Fc (with a C-terminal human Fc tag) under non-reducing and reducing conditions. Figure 22B is an SEC-HPLC trace for Neu2-M173-Fc (with a C-terminal human Fc tag). Neu2-M173-Fc had a yield of 120 mg / liter and a monomer content of 90%.

[0271] The enzymatic kinetics of Neu2-M173-Fc was assayed by measuring the release of sialic acid from the fluorogenic substrate 4-methylumbelliferyl-N-acetylneuraminic acid (4MU-NeuAc) as described above. A fixed concentration of 2 μg / well of enzyme was incubated with concentrations of the fluorogenic substrate 4MU-NeuAc ranging from 4 mM to 0.03 μM. Figure 23 shows the enzymatic activity of Neu2-M173-Fc. The enzymatic activity of Neu2-M173-Fc was comparable to that of wild-type Neu2, with a K M was determined to be 230 μM.

[0272] Additional mutant Neu2 sialidases were constructed containing rationally designed substitutions at positions S301 and / or W302. Mutations at S301 and / or W302 can affect interactions with neighboring amino acid residues and / or substrates.

[0273] Expression and activity levels for the mutant sialidases are shown in Table 29. In Table 29, enzyme activity is indicated as "++", which indicates activity equivalent to wild-type Neu2, "+", which indicates activity less than wild-type Neu2, or "-", which indicates no detectable activity, and expression is indicated as "+++++", which indicates expression >40-fold higher than wild-type Neu2, "++++", which indicates expression >15-fold higher than wild-type Neu2, "+++", which indicates expression >6-fold higher than wild-type Neu2, "++", which indicates expression 2-5-fold higher than wild-type Neu2, "+", which indicates expression equivalent to wild-type Neu2, or "-", which indicates no detectable expression. [Table 43-1] [Table 43-2]

[0274] Example 5 This example describes the construction of a recombinant human sialidase with mutations that reduce proteolytic cleavage.

[0275] Neu2-M106 (described in Example 2 and having the amino acid sequence SEQ ID NO:48) was expressed as an Fc-fusion single-chain protein using a CHO cell expression system in a large-scale (10 L) high-cell-density production run and purified using a protein A column. The resulting protein was analyzed by SDS-PAGE. The results are shown in Figure 24. Under reducing conditions, the protein contained a mixture of full-length (70 kDa, approximately 50%) and cleaved (40 kDa and 30 kDa, approximately 50%) fractions. However, under non-reducing conditions, there was no cleavage and the protein remained single-chain (Figure 24). Furthermore, when Neu2-M106 was expressed on a smaller scale (shorter duration of cell culture and lower cell density), there was no cleavage and the protein remained single-chain. Previous mass spectrometry analysis showed that the 40 kDa and 30 kDa molecular weight fractions observed under reducing conditions after large-scale production were the result of cleavage between amino acid residues R243 and V244 of the sialidase. The enzymatic activity of the cleaved Neu2-M106 was similar to that of the uncleaved Neu2-M106.

[0276] It was hypothesized that cleavage of Neu2-M106 could be due to the activity of intracellular proteases released as a result of cell lysis during protein production, recovery, and / or purification. To test this hypothesis, both cleaved Neu2-M106 (prepared using the large-scale production method described above, which results in cleavage) and uncleaved Neu2-M106 (prepared using the small-scale production method described above, which does not result in cleavage) were incubated with trypsin and analyzed by SDS-PAGE under reducing conditions (Figure 25). Briefly, trypsin digestion reactions were performed by incubating trypsin (5 μL, 0.005% solution in PBS) with Neu2-M106 (25 μL, 0.25 mg / mL in PBS pH 8.0) on ice for 5 minutes. The reactions were stopped by the addition of SDS gel loading buffer (5 μL) and run on a reducing SDS-PAGE gel to observe trypsin-mediated cleavage. SDS-PAGE analysis showed that incubation of uncleaved Neu2-M106 with trypsin produced a cleavage pattern identical to that of cleaved Neu2-M106. Furthermore, incubation of cleaved Neu2-M106 with trypsin produced an increase in the intensity of the bands corresponding to the cleavage products.

[0277] Neu2-M106 was also incubated with trypsin in the presence of various protease inhibitors. Briefly, trypsin digestion reactions were performed by incubating trypsin (0.005%) with Neu2-M106 (0.5 mg / mL) and protease inhibitors on ice for 5 minutes. The reactions were stopped by adding LDS gel loading buffer and run on a reducing SDS-PAGE gel to observe trypsin-mediated cleavage. The inhibitors used included ferric citrate (at 0.3 and 5 mM), aprotinin (at 5,000 and 20,000 U / mL), AEBSF (at 0.1 and 1 mM), leupeptin (at 1 and 10 μM), or E-64 (at 1 and 10 μM). As seen in Figure 26, protease inhibitors reduced the extent of trypsin cleavage.

[0278] Together, these results confirm that cleavage of Neu2-M106 after large-scale production is due to trypsin or a member of a similar class of proteases.

[0279] Next, we attempted to rationally design recombinant human sialidase with mutations that increase its resistance to trypsin cleavage.

[0280] Unless otherwise indicated, in the remainder of this example, mutant sialidases were expressed as secreted proteins with a C-terminal human Fc tag in Expi293F cells (at a 50 mL scale) using the pCEP4 mammalian expression vector. The resulting proteins were purified using a Protein A column. Expression was assayed using a ForteBio Octet with an anti-human Fc sensor and Western blot, as described above, and enzyme activity was assayed using the fluorogenic substrate 4MU-NeuAc. Protease cleavage was assayed by SDS-PAGE as described above.

[0281] First, R243 was mutated to amino acids of different polarity / charge, such as K, E, H, N, and Q. However, these mutations of R243 resulted in a complete loss of activity and a reduction in expression yield (R243 is also a conserved amino acid in similar sialidases).

[0282] Various amino acid residues surrounding the cleavage site were then mutated and tested for expression, activity, and resistance to trypsin cleavage. The substitutions and combinations of substitutions tested are shown in Figure 27. All mutations were tested in the Neu2-M106 background (i.e., including the following substitutions: M1D, V6Y, P62G, A93E, I187K, and C332A).

[0283] Although most of the mutant sialidases shown in Figure 27 expressed well, only two of the mutant sialidases (containing the V244I or A242C mutation) were active. The A242C mutation resulted in a more than 10-fold improved trypsin resistance and slightly lower activity (both relative to Neu2-M106). However, because having an unpaired cysteine ​​could potentially be a liability, A242 was mutated to all 19 other amino acids and assayed for activity and trypsin resistance. As seen in Figure 28, mutation of A242 to aromatic amino acids, such as F, W, and Y, resulted in a dramatic improvement in resistance to trypsin cleavage compared to Neu2-M106 (Figure 28A) and similar enzymatic activity to Neu2-M106 (Figure 28B). SEC analysis showed that proteins containing each of these mutations had a similar pattern to that of Neu2-M106 and a monomer content greater than 95% (Figure 28C).

[0284] Structural analysis indicated that replacing A242 with an aromatic amino acid could result in additional hydrophobic or stacking interactions with L260 and V265 (nonpolar amino acids located near A242). Therefore, L260 and V265 were also mutated to phenylalanine. These mutations, along with several other rationally designed mutations that could result in excess stability, for example, by increasing stacking interactions, were also tested for expression, activity, and protease resistance.

[0285] The selection results are shown in Figure 29. As shown in Figure 29, the combination of R241Y and A242F mutations (Neu2-M255) resulted in the highest resistance to trypsin cleavage (more than 10-fold improved trypsin resistance relative to Neu2-M106).

[0286] The expression, activity, and protease resistance levels for the mutant sialidases are shown in Table 30. In Table 30, enzyme activity is indicated as "++", which indicates activity equivalent to wild-type Neu2, "+", which indicates activity less than wild-type Neu2, or "-", which indicates no detectable activity, and expression is indicated as "+++++", which indicates expression >40-fold higher than wild-type Neu2, "++++", which indicates expression >15-fold higher than wild-type Neu2, "+++", which indicates expression >6-fold higher than wild-type Neu2, "++", which indicates expression 2-5-fold higher than wild-type Neu2, "+", which indicates expression equivalent to wild-type Neu2, or "-", which indicates no detectable expression. Protease / trypsin resistance is indicated as "+++" indicating >10-fold higher resistance than Neu2-M106, "++" indicating ≥5-fold higher resistance than Neu2-M106, "+" indicating equivalent resistance to Neu2-M106, or "-" indicating lower resistance than Neu2-M106. NT = not tested. [Table 44-1] [Table 44-2]

[0287] Example 6 This example describes the construction and expression of an antibody-sialidase genetic fusion protein with a mutated human sialidase, and an antibody-sialidase conjugate (ASC) containing the fusion protein.

[0288] Neu2 with M1D, V6Y, P62G, A93E, I187K, and C332A substitutions and trastuzumab were used to generate a Janus antibody-sialidase conjugate (ASC) referred to in this example as "Janus-trastuzumab." This Janus-trastuzumab (comprising a first polypeptide chain having the amino acid sequence SEQ ID NO:66 encoded by the nucleotide sequence SEQ ID NO:86, a second polypeptide chain having the amino acid sequence SEQ ID NO:67 encoded by the nucleotide sequence SEQ ID NO:87, and a third polypeptide chain having the amino acid sequence SEQ ID NO:68 encoded by the nucleotide sequence SEQ ID NO:88) was expressed and characterized for purity using SDS-PAGE and for enzymatic activity using 4MU-NeuAc, as described below.

[0289] Janus-trastuzumab was expressed in a 1 L transfection of Expi293 human cells using the pCEP4 mammalian expression vector. Janus-trastuzumab was purified using Protein A followed by cation exchange chromatography (Hitrap SP-HP, GE Lifesciences). The purified protein was analyzed by SDS-PAGE (Figure 12) and SEC-HPLC (Figure 13). The expression yield was 30 mg / L with a purity of 90% monomer as determined by SEC-HPLC.

[0290] The enzymatic activity of recombinantly expressed Janus-trastuzumab was assayed by measuring the release of sialic acid from the fluorogenic substrate 4-methylumbelliferyl-N-acetylneuraminic acid (4MU-NeuAc). Specifically, an enzyme kinetic assay was performed using a fixed concentration of enzyme at 2 μg / well, which was incubated with the fluorogenic substrate 4MU-NeuAc at concentrations ranging from 4000 μM to 7.8 μM. As shown in Figure 14, Janus-trastuzumab was enzymatically active with a Km of 0.48 mM.

[0291] Janus-trastuzumab was tested for antigen (Her2) binding using a ForteBio Octet with ASCs captured on an anti-Fc sensor soaked in serial dilutions of His-tagged Her2 (50–0.78 nM at 1:2 dilutions). Janus-trastuzumab bound to Her2 with binding affinity comparable to that of trastuzumab (Figure 15).

[0292] Example 7 This example describes the in vivo administration of an antibody-sialidase conjugate (ASC) containing bacterial sialidase.

[0293] The following ASCs were generated and tested in this example: (i) Janus ASC comprising Salmonella typhimurium sialidase (St-sialidase) and trastuzumab (comprising a first polypeptide chain having amino acid sequence SEQ ID NO:66 encoded by nucleotide sequence SEQ ID NO:86, a second polypeptide chain having amino acid sequence SEQ ID NO:67 encoded by nucleotide sequence SEQ ID NO:87, and a third polypeptide chain having amino acid sequence SEQ ID NO:90 encoded by nucleotide sequence SEQ ID NO:91); (ii) Raptor ASC comprising St-sialidase and trastuzumab (comprising first and fourth polypeptide chains having amino acid sequence SEQ ID NO:66 encoded by nucleotide sequence SEQ ID NO:86, and second and third polypeptide chains having amino acid sequence SEQ ID NO:92 encoded by nucleotide sequence SEQ ID NO:93); and (iii) Lobster ASC comprising St-sialidase and an scFv derived from trastuzumab (comprising first and second polypeptide chains having amino acid sequence SEQ ID NO:94 encoded by nucleotide sequence SEQ ID NO:95).

[0294] These ASCs were compared with trastuzumab in a mouse syngeneic tumor model injected with a mouse breast cancer cell line expressing human Her2 (EMT6-hHer2 cells). Female BALB / c mice, 6-8 weeks old, were injected with EMT6-Her2 tumor cells (5x10 cells) in 0.1 ml of PBS for tumor development.5 ) was inoculated subcutaneously into the right lower flank. 3 , average about 75~100mm 3 Upon reaching age 60, mice were randomly assigned to eight groups. Treatment groups are listed in Table 31, which shows the dosing schedule after randomization. Anti-mouse NK1.1 (clone: ​​PK136; BioXcell, 621717N1), anti-mouse CD8α (clone: ​​53-6.7; BioXcell, BE0004-1), and clodronate liposomes (FormuMax Scientific, Inc.) were included in the treatment groups as indicated. [Table 45]

[0295] The results of treatment with trastuzumab, and Raptor, Janus, and Lobster ASCs are shown in Figures 16A, 16B, 16C, and 16D, respectively. As can be seen, trastuzumab, when treated, did not produce a complete response in eight mice (defined as a regression below the limit of palpation at any point during the duration of the test, Figure 16A). This is in contrast to Raptor, in which two of eight animals showed a complete response (Figure 16B), Janus, in which three of eight animals showed a complete response (Figure 16C), and Lobster, in which two of eight animals showed a complete response (Figure 16D).

[0296] The results of administering Janus with NK depletion (anti-mouse NK1.1), macrophage depletion (clodronate liposomes), and CD8 T cell depletion (anti-mouse CD8α) are shown in Figure 17. As can be seen, compared to Janus treatment alone, which resulted in a complete response in 3 of 8 animals (Figure 16C), NK depletion reduced the number of complete responses to 1 of 8 animals (Figure 17A). Macrophage depletion also reduced the number of complete responses to 1 of 8 animals (Figure 17B). CD8 T cell depletion completely reversed the effect of Janus, with no animals showing a complete response (Figure 17C). Figure 17D shows the mean tumor volumes for vehicle, Janus alone, trastuzumab alone, and Janus with NK depletion, macrophage depletion, and CD8 T cell depletion. These results indicate that innate immunity (NK and macrophage dependent) and adaptive immunity (CD8 T cells) contribute to ASC activity in vivo.

[0297] Example 8 This example describes the in vivo administration of an antibody-sialidase conjugate (ASC) with bacterial sialidase.

[0298] In this example, the following ASCs were generated and tested: (i) a Janus ASC comprising Salmonella typhimurium sialidase (St-sialidase) and trastuzumab (comprising a first polypeptide chain having the amino acid sequence SEQ ID NO:66 encoded by the nucleotide sequence SEQ ID NO:86, a second polypeptide chain having the amino acid sequence SEQ ID NO:67 encoded by the nucleotide sequence SEQ ID NO:87, and a third polypeptide chain having the amino acid sequence SEQ ID NO:90 encoded by the nucleotide sequence SEQ ID NO:91); and (ii) a Janus ASC comprising St-sialidase with two loss-of-function mutations D100V and G231V and trastuzumab ("Janus-LOF", comprising a first polypeptide chain having the amino acid sequence SEQ ID NO:66 encoded by the nucleotide sequence SEQ ID NO:86, a second polypeptide chain having the amino acid sequence SEQ ID NO:67 encoded by the nucleotide sequence SEQ ID NO:87, and a third polypeptide chain having the amino acid sequence SEQ ID NO:96 encoded by the nucleotide sequence SEQ ID NO:97).

[0299] These ASCs were tested in a mouse syngeneic orthotopic tumor model injected with the human Her2-expressing, independent EMT6 cell line (EMT6-hHer2 cells, described in D'Amico et al. (2016) ANNALS OF ONCOLOGY, Volume 27, Issue suppl_8, 41P). Female BALB / c mice, 6-8 weeks old, were injected with EMT6-Her2 tumor cells (5x10 6 ) was inoculated by intramammary implantation. 3 Upon reaching age 6, mice were randomized into six groups. Treatment groups are listed in Table 32, which shows the dosing schedule after randomization. Anti-mouse PD1 was obtained from BioXcell (RMP1-14, catalog number 665418F1). [Table 46]

[0300] The results for Groups 1-4 (vehicle, trastuzumab, Janus, and Janus LOF) are shown in Figure 18A. As can be seen, 3 out of 6 animals treated with Janus had complete regression of tumor growth. Notably, both Janus LOF and trastuzumab were comparable to vehicle-treated animals.

[0301] Three mice with complete regression ("cured mice") were rechallenged with either the same EMT6-Her2 cells used initially or parental EMT6 cells (genetically engineered to lack human Her2 expression). For tumor development in all three cured mice, EMT6 and EMT6-Her2 cells (5x10) were added in 0.1 ml of PBS. 5 ) were inoculated subcutaneously into the lower right or lower left flank region, respectively. EMT6-Her2 cells were also inoculated subcutaneously into naive mice as a control. As can be seen in Figure 18B, neither EMT6-Her2 cells nor parental EMT6 cells produced tumor growth in cured mice, whereas EMT6-Her2 cells progressed to tumors in naive mice as expected. These results suggest that the antibody-sialidase conjugate of the present invention can induce long-term memory against tumors. Furthermore, the long-term memory is directed against tumor cells and is independent of the original targeted cancer antigen (in this case, Her2).

[0302] The results for groups 1, 5, and 6 (vehicle, anti-mouse PD1, and anti-mouse PD1 combined with Janus) are shown in Figures 19A and 19B. Anti-mouse PD1 had good activity, with 4 of 6 mice showing complete regression (similar to Janus alone, where 3 of 6 mice showed complete regression; see Figure 18A), while the combination of anti-mouse PD1 and Janus showed complete regression of tumor growth in all 6 mice (Figure 19B). There was no weight loss in any of the animals given this combination.

[0303] Example 9 This example describes the in vivo administration of an antibody-sialidase conjugate (ASC) with bacterial sialidase.

[0304] A Janus ASC (comprising a first polypeptide chain having amino acid sequence SEQ ID NO:66 encoded by nucleotide sequence SEQ ID NO:86, a second polypeptide chain having amino acid sequence SEQ ID NO:67 encoded by nucleotide sequence SEQ ID NO:87, and a third polypeptide chain having amino acid sequence SEQ ID NO:90 encoded by nucleotide sequence SEQ ID NO:91) comprising Salmonella typhimurium sialidase (St-sialidase) and trastuzumab was produced and tested in this example.

[0305] ASCs were tested in a mouse syngeneic tumor model injected with the B16 melanoma cell line expressing human Her2 (B16D5-Her2, Surana et al. CANCER IMMUNOL RES, 2(11): 1103-1112). Female C57BL / 6 mice, 6-8 weeks old, were injected with B16D5-Her2 tumor cells (5x10 5 ) was inoculated subcutaneously into the right lower flank area. 3 Upon reaching 100 mg / kg, mice were randomly assigned to three groups. Treatment groups are listed in Table 33, which shows the dosing schedule after randomization. Anti-mouse PD1 (RMP1-14, catalog number 665418F1) from BioXcell and anti-mouse CTLA4 (9D9, catalog number BE0164) from BioXcell were used in combination. [Table 47]

[0306] The B16 melanoma mouse model is considered a difficult tumor model to treat using immune-tumor approaches. Janus was compared to a combination of anti-mouse PD1 and anti-mouse CTLA4. The results are shown in Figure 20. While anti-mouse PD1 combined with anti-mouse CTLA4 had an effect on B16D5-Her2 tumor growth, this combination also resulted in significant weight loss in treated animals. By comparison, Janus demonstrated stronger antitumor activity without significant weight loss. Trastuzumab alone showed marginal activity in this model.

[0307] Example 10 This example describes the in vivo administration of an antibody-sialidase conjugate (ASC) containing human sialidase.

[0308] In this example, Janus trastuzumab as described in Example 3, comprising Neu2 with substitutions M1D, V6Y, P62G, A93E, I187K, and C332A and trastuzumab (comprising a first polypeptide chain having amino acid sequence SEQ ID NO:66 encoded by nucleotide sequence SEQ ID NO:86, a second polypeptide chain having amino acid sequence SEQ ID NO:67 encoded by nucleotide sequence SEQ ID NO:87, and a third polypeptide chain having amino acid sequence SEQ ID NO:68 encoded by nucleotide sequence SEQ ID NO:88) was made and tested.

[0309] Janus-trastuzumab was compared to an isotype control antibody in a mouse syngeneic tumor model injected with a mouse breast cancer cell line (EMT6-HER2) stably expressing human HER2. Female BALB / c mice, 6-8 weeks of age, were injected with EMT6-HER2 tumor cells (5x10 5 ) was inoculated subcutaneously into the right lower flank area. 3 , average about 75~100mm 3 When the mice reached 100 mg / kg, they were randomly assigned to groups of 8 animals each.

[0310] Mice were treated with an intraperitoneal injection of 10 mg / kg to increase tumor volume (mm 3 ) were recorded. Figure 21 shows the individual tumor growth for mice treated with Janus-trastuzumab or control. In this experiment, a significant tumor growth delay was observed after treatment with Janus-trastuzumab.

[0311] Incorporation by Reference The entire disclosures of each of the patent and scientific literature referenced herein are incorporated by reference for all purposes. Further, U.S. Provisional Patent Application No. 62 / 870,354, filed July 3, 2019, U.S. Provisional Patent Application No. 62 / 956,957, filed January 3, 2020, International (PCT) Patent Application No. PCT / US20 / 40815, filed July 33, 2020, U.S. Provisional Patent Application No. 62 / 870,348, filed July 3, 2019, U.S. Provisional Patent Application No. 62 / 956,977, filed January 3, 2020, No. 62 / 870,341, filed July 3, 2019; U.S. Provisional Patent Application No. 62 / 957,041, filed January 3, 2020; and International (PCT) Patent Application No. PCT / US20 / 40816, filed July 3, 2020, the entire disclosures of which are incorporated by reference for all purposes.

[0312] equivalent The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Therefore, the foregoing embodiments are to be considered in all respects as illustrative and not limiting of the invention described herein. The scope of the invention is, therefore, indicated by the appended claims rather than the foregoing description, and all changes that come within the meaning and range of equivalence of the claims are intended to be embraced therein. The present invention includes the following aspects. Item 1 Sialidase: (a) Substitution of a proline residue at the position corresponding to position 5 of wild-type human Neu2 (P5); (b) substitution of a lysine residue at the position corresponding to position 9 of wild-type human Neu2 (K9); (c) substitution of a lysine residue at the position corresponding to position 44 in wild-type human Neu2 (K44); (d) substitution of a lysine residue at the position corresponding to position 45 in wild-type human Neu2 (K45); (e) substitution of a leucine residue at the position corresponding to position 54 of wild-type human Neu2 (L54); (f) substitution of a proline residue at the position corresponding to position 62 of wild-type human Neu2 (P62); (g) substitution of a glutamine residue at the position corresponding to position 69 in wild-type human Neu2 (Q69); (h) substitution of an arginine residue at the position corresponding to position 78 of wild-type human Neu2 (R78); (i) substitution of an aspartic acid residue at the position corresponding to position 80 of wild-type human Neu2 (D80); (j) substitution of an alanine residue at the position corresponding to position 93 of wild-type human Neu2 (A93); (k) substitution of a glycine residue at the position corresponding to position 107 of wild-type human Neu2 (G107); (l) substitution of a glutamine residue at the position corresponding to position 108 of wild-type human Neu2 (Q108); (m) substitution of a glutamine residue at the position corresponding to position 112 of wild-type human Neu2 (Q112); (n) substitution of a cysteine ​​residue at the position corresponding to position 125 of wild-type human Neu2 (C125); (o) substitution of a glutamine residue at the position corresponding to position 126 in wild-type human Neu2 (Q126); (p) substitution of an alanine residue at the position corresponding to position 150 of wild-type human Neu2 (A150); (q) substitution of a cysteine ​​residue at the position corresponding to position 164 in wild-type human Neu2 (C164); (r) substitution of an arginine residue at the position corresponding to position 170 in wild-type human Neu2 (R170); (s) substitution of an alanine residue at the position corresponding to position 171 in wild-type human Neu2 (A171); (t) substitution of a glutamine residue at the position corresponding to position 188 in wild-type human Neu2 (Q188); (u) substitution of an arginine residue at the position corresponding to position 189 in wild-type human Neu2 (R189); (v) substitution of an alanine residue at the position corresponding to position 213 of wild-type human Neu2 (A213); (w) substitution of a leucine residue at the position corresponding to position 217 of wild-type human Neu2 (L217); (x) substitution of a glutamic acid residue at the position corresponding to position 225 of wild-type human Neu2 (E225); (y) substitution of a histidine residue at the position corresponding to position 239 in wild-type human Neu2 (H239); (z) substitution of a leucine residue at the position corresponding to position 240 in wild-type human Neu2 (L240); (aa) substitution of an arginine residue at the position corresponding to position 241 of wild-type human Neu2 (R241); (bb) substitution of an alanine residue at the position corresponding to position 242 of wild-type human Neu2 (A242); (cc) substitution of a valine residue at the position corresponding to position 244 in wild-type human Neu2 (V244); (dd) substitution of a threonine residue at the position corresponding to position 249 in wild-type human Neu2 (T249); (ee) substitution of an aspartic acid residue at the position corresponding to position 251 in wild-type human Neu2 (D251); (ff) substitution of a glutamic acid residue at the position corresponding to position 257 in wild-type human Neu2 (E257); (gg) substitution of a serine residue at the position corresponding to position 258 in wild-type human Neu2 (S258); (hh) substitution of a leucine residue at the position corresponding to position 260 in wild-type human Neu2 (L260); (ii) substitution of a valine residue at the position corresponding to position 265 of wild-type human Neu2 (V265); (jj) substitution of a glutamine residue at the position corresponding to position 270 in wild-type human Neu2 (Q270); (kk) substitution of a tryptophan residue at the position corresponding to position 292 in wild-type human Neu2 (W292); (ll) substitution of a serine residue at the position corresponding to position 301 in wild-type human Neu2 (S301); (mm) substitution of a tryptophan residue at the position corresponding to position 302 in wild-type human Neu2 (W302); (nn) substitution of a valine residue at a position corresponding to position 363 of wild-type human Neu2 (V363); or (oo) substitution of a leucine residue at the position corresponding to position 365 in wild-type human Neu2 (L365); or any combination of the above substitutions 1. A recombinant mutant human sialidase enzyme comprising: Section 2 (a) Substitution of a proline residue at the position corresponding to position 5 of wild-type human Neu2 (P5); (b) substitution of a lysine residue at the position corresponding to position 9 of wild-type human Neu2 (K9); (c) substitution of a lysine residue at the position corresponding to position 44 in wild-type human Neu2 (K44); (d) substitution of a lysine residue at the position corresponding to position 45 in wild-type human Neu2 (K45); (e) substitution of a leucine residue at the position corresponding to position 54 of wild-type human Neu2 (L54); (f) substitution of a proline residue at the position corresponding to position 62 of wild-type human Neu2 (P62); (g) substitution of a glutamine residue at the position corresponding to position 69 in wild-type human Neu2 (Q69); (h) substitution of an arginine residue at the position corresponding to position 78 of wild-type human Neu2 (R78); (i) substitution of an alanine residue at the position corresponding to position 93 of wild-type human Neu2 (A93); (j) substitution of a glycine residue at the position corresponding to position 107 of wild-type human Neu2 (G107); (k) substitution of a glutamine residue at the position corresponding to position 108 of wild-type human Neu2 (Q108); (l) substitution of a glutamine residue at the position corresponding to position 112 of wild-type human Neu2 (Q112); (m) substitution of a cysteine ​​residue at the position corresponding to position 125 of wild-type human Neu2 (C125); (n) substitution of a glutamine residue at the position corresponding to position 126 of wild-type human Neu2 (Q126); (o) substitution of an alanine residue at the position corresponding to position 150 of wild-type human Neu2 (A150); (p) substitution of a cysteine ​​residue at the position corresponding to position 164 in wild-type human Neu2 (C164); (q) substitution of an alanine residue at the position corresponding to position 171 of wild-type human Neu2 (A171); (r) substitution of a leucine residue at the position corresponding to position 217 in wild-type human Neu2 (L217); (s) substitution of a threonine residue at the position corresponding to position 249 in wild-type human Neu2 (T249); (t) substitution of an aspartic acid residue at the position corresponding to position 251 in wild-type human Neu2 (D251); (u) substitution of a glutamine residue at the position corresponding to position 270 in wild-type human Neu2 (Q270); (v) substitution of a tryptophan residue at the position corresponding to position 292 in wild-type human Neu2 (W292); (w) substitution of a serine residue at the position corresponding to position 301 in wild-type human Neu2 (S301); (x) substitution of a tryptophan residue at the position corresponding to position 302 of wild-type human Neu2 (W302); (y) substitution of a valine residue at the position corresponding to position 363 of wild-type human Neu2 (V363); or (z) substitution of a leucine residue at the position corresponding to position 365 of wild-type human Neu2 (L365); or any combination of the above substitutions Item 2. The sialidase according to Item 1, comprising: Section 3 3. The sialidase of paragraph 1 or 2, comprising a substitution of K9, P62, A93, Q216, A242, Q270, S301, W302, V363 or L365, or any combination of the foregoing substitutions. Section 4 Item 4. The sialidase of any one of Items 1 to 3, comprising a substitution of K9, P62, A93, Q270, S301, W302, V363, or L365, or any combination of the aforementioned substitutions. Section 5 In sialidase: (a) The proline residue at the position corresponding to position 5 of wild-type human Neu2 was replaced with a histidine (P5H); (b) the lysine residue at the position corresponding to position 9 of wild-type human Neu2 was replaced with aspartic acid (K9D); (c) the lysine residue at the position corresponding to position 44 of wild-type human Neu2 was substituted with arginine (K44R) or glutamic acid (K44E); (d) the lysine residue at the position corresponding to position 45 of wild-type human Neu2 is substituted with alanine (K45A), arginine (K45R), or glutamic acid (K45E); (e) the leucine residue at the position corresponding to position 54 of wild-type human Neu2 was replaced with a methionine (L54M); (f) the proline residue at the position corresponding to position 62 of wild-type human Neu2 is replaced with asparagine (P62N), aspartic acid (P62D), histidine (P62H), glutamic acid (P62E), glycine (P62G), serine (P62S), or threonine (P62T); (g) the glutamine residue at the position corresponding to position 69 of wild-type human Neu2 was replaced with a histidine (Q69H); (h) the arginine residue at the position corresponding to position 78 of wild-type human Neu2 was replaced with a lysine (R78K); (i) the aspartic acid residue at the position corresponding to position 80 of wild-type human Neu2 was replaced with a proline (D80P); (j) the alanine residue at the position corresponding to position 93 of wild-type human Neu2 is substituted with glutamic acid (A93E) or lysine (A93K); (k) the glycine residue at the position corresponding to position 107 of wild-type human Neu2 was replaced with aspartic acid (G107D); (l) the glutamine residue at the position corresponding to position 108 of wild-type human Neu2 was replaced with a histidine (Q108H); (m) the glutamine residue at the position corresponding to position 112 of wild-type human Neu2 is substituted with arginine (Q112R) or lysine (Q112K); (n) the cysteine ​​residue at the position corresponding to position 125 of wild-type human Neu2 was replaced with leucine (C125L); (o) a glutamine residue at a position corresponding to position 126 of wild-type human Neu2 is substituted with leucine (Q126L), glutamic acid (Q126E), phenylalanine (Q126F), histidine (Q126H), isoleucine (Q126I), or tyrosine (Q126Y); (p) the alanine residue at the position corresponding to position 150 of wild-type human Neu2 was replaced with valine (A150V); (q) the cysteine ​​residue at the position corresponding to position 164 of wild-type human Neu2 was replaced with a glycine (C164G); (r) the arginine residue at the position corresponding to position 170 of wild-type human Neu2 was replaced with proline (R170P); (s) the alanine residue at the position corresponding to position 171 of wild-type human Neu2 was replaced with glycine (A171G); (t) the glutamine residue at the position corresponding to position 188 of wild-type human Neu2 was replaced with a proline (Q188P); (u) the arginine residue at the position corresponding to position 189 of wild-type human Neu2 was replaced with a proline (R189P); (v) the alanine residue at the position corresponding to position 213 of wild-type human Neu2 is substituted with cysteine ​​(A213C), asparagine (A213N), serine (A213S), or threonine (A213T); (w) the leucine residue at the position corresponding to position 217 of wild-type human Neu2 is replaced with alanine (L217A) or valine (L217V); (x) the threonine residue at the position corresponding to position 249 of wild-type human Neu2 was replaced with alanine (T249A); (y) the aspartic acid residue at the position corresponding to position 251 of wild-type human Neu2 was replaced with a glycine (D251G); (z) the glutamic acid residue at the position corresponding to position 225 of wild-type human Neu2 was replaced with a proline (E225P); (aa) the histidine residue at the position corresponding to position 239 of wild-type human Neu2 was replaced with proline (H239P); (bb) the leucine residue at the position corresponding to position 240 of wild-type human Neu2 is replaced with aspartic acid (L240D), asparagine (L240N), or tyrosine (L240Y); (cc) the arginine residue at the position corresponding to position 241 of wild-type human Neu2 is replaced with alanine (R241A), aspartic acid (R241D), leucine (R241L), glutamine (R241Q), or tyrosine (R241Y); (dd) the alanine residue at the position corresponding to position 242 of wild-type human Neu2 is substituted 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); (ee) the valine residue at the position corresponding to position 244 of wild-type human Neu2 was replaced with isoleucine (V244I) or proline (V244P); (ff) the glutamic acid residue at the position corresponding to position 257 of wild-type human Neu2 was replaced with a proline (E257P); (gg) the serine residue at the position corresponding to position 258 is replaced with a cysteine ​​(S258C); (hh) the leucine residue at the position corresponding to position 260 of wild-type human Neu2 is replaced with aspartic acid (L260D), phenylalanine (L260F), glutamine (L260Q), or threonine (L260T); (ii) the valine residue at the position corresponding to position 265 of wild-type human Neu2 was replaced with phenylalanine (V265F); (jj) the glutamine residue at the position corresponding to position 270 of wild-type human Neu2 is substituted with alanine (Q270A), histidine (Q270H), phenylalanine (Q270F), proline (Q270P), serine (Q270S), or threonine (Q270T); (kk) the tryptophan residue at the position corresponding to position 292 of wild-type human Neu2 was replaced with arginine (W292R); (ll) the serine residue at the position corresponding to position 301 of wild-type human Neu2 is replaced with alanine (S301A), aspartic acid (S301D), glutamic acid (S301E), phenylalanine (S301F), histidine (S301H), lysine (S301K), leucine (S301L), methionine (S301M), asparagine (S301N), proline (S301P), glutamine (S301Q), arginine (S301R), threonine (S301T), valine (S301V), tryptophan (S301W), or tyrosine (S301Y); (mm) the tryptophan residue at the position corresponding to position 302 of wild-type human Neu2 is replaced with alanine (W302A), aspartic acid (W302D), phenylalanine (W302F), glycine (W302G), histidine (W302H), isoleucine (W302I), lysine (W302K), leucine (W302L), methionine (W302M), asparagine (W302N), proline (W302P), glutamine (W302Q), arginine (W302R), serine (W302S), threonine (W302T), valine (W302V), or tyrosine (W302Y); (nn) a valine residue at the position corresponding to position 363 of wild-type human Neu2 is substituted with arginine (V363R); or (oo) the leucine residue at the position corresponding to position 365 of wild-type human Neu2 is substituted with glutamine (L365Q), histidine (L365H), isoleucine (L365I), lysine (L365K), or serine (L365S); Alternatively, the sialidase comprises any combination of the foregoing substitutions. Item 5. The sialidase according to any one of Items 1 to 4. Section 6 In sialidase: (a) The proline residue at the position corresponding to position 5 of wild-type human Neu2 was replaced with a histidine (P5H); (b) the lysine residue at the position corresponding to position 9 of wild-type human Neu2 was replaced with aspartic acid (K9D); (c) the lysine residue at the position corresponding to position 44 of wild-type human Neu2 was substituted with arginine (K44R) or glutamic acid (K44E); (d) the lysine residue at the position corresponding to position 45 of wild-type human Neu2 is substituted with alanine (K45A), arginine (K45R), or glutamic acid (K45E); (e) the leucine residue at the position corresponding to position 54 of wild-type human Neu2 was replaced with a methionine (L54M); (f) the proline residue at the position corresponding to position 62 of wild-type human Neu2 is replaced with asparagine (P62N), aspartic acid (P62D), histidine (P62H), glutamic acid (P62E), glycine (P62G), serine (P62S), or threonine (P62T); (g) the glutamine residue at the position corresponding to position 69 of wild-type human Neu2 was replaced with a histidine (Q69H); (h) the arginine residue at the position corresponding to position 78 of wild-type human Neu2 was replaced with a lysine (R78K); (i) the alanine residue at the position corresponding to position 93 of wild-type human Neu2 is substituted with glutamic acid (A93E) or lysine (A93K); (j) the glycine residue at the position corresponding to position 107 of wild-type human Neu2 was replaced with aspartic acid (G107D); (k) the glutamine residue at the position corresponding to position 108 of wild-type human Neu2 was replaced with a histidine (Q108H); (l) the glutamine residue at the position corresponding to position 112 of wild-type human Neu2 was replaced with arginine (Q112R) or lysine (Q112K); (m) the cysteine ​​residue at the position corresponding to position 125 of wild-type human Neu2 was replaced with leucine (C125L); (n) the glutamine residue at the position corresponding to position 126 of wild-type human Neu2 was replaced with leucine (Q126L); (o) the alanine residue at the position corresponding to position 150 of wild-type human Neu2 was replaced with valine (A150V); (p) the cysteine ​​residue at the position corresponding to position 164 of wild-type human Neu2 was replaced with a glycine (C164G); (q) the alanine residue at the position corresponding to position 171 of wild-type human Neu2 was replaced with glycine (A171G); (r) the leucine residue at the position corresponding to position 217 of wild-type human Neu2 was replaced with alanine (L217A) or valine (L217V); (s) the threonine residue at the position corresponding to position 249 of wild-type human Neu2 was replaced with alanine (T249A); (t) the aspartic acid residue at the position corresponding to position 251 of wild-type human Neu2 was replaced with a glycine (D251G); (u) the glutamine residue at the position corresponding to position 270 of wild-type human Neu2 is substituted with alanine (Q270A), histidine (Q270H), phenylalanine (Q270F), or proline (Q270P); (v) the tryptophan residue at the position corresponding to position 292 of wild-type human Neu2 was replaced with arginine (W292R); (w) the serine residue at the position corresponding to position 301 of wild-type human Neu2 was replaced with arginine (S301R); (x) the tryptophan residue at the position corresponding to position 302 of wild-type human Neu2 was replaced with a lysine (W302K); (y) a valine residue at the position corresponding to position 363 of wild-type human Neu2 is substituted with arginine (V363R); or (z) the leucine residue at the position corresponding to position 365 of wild-type human Neu2 is substituted with glutamine (L365Q), histidine (L365H), isoleucine (L365I), lysine (L365K), or serine (L365S); Alternatively, the sialidase comprises any combination of the foregoing substitutions. Item 6. The sialidase according to any one of Items 1 to 5. Section 7 Item 7. The sialidase of any one of Items 1 to 6, comprising a substitution selected from K9D, P62G, P62N, P62S, P62T, A93E, Q126Y, A242F, A242W, A242Y, Q270A, Q270T, S301A, S301R, W302K, W302R, V363R and L365I, or any combination of the foregoing substitutions. Section 8 8. The sialidase of any one of items 1 to 7, comprising a substitution selected from the substitutions K9D, P62G, P62N, P62S, P62T, A93E, Q270A, S301R, W302K, V363R and L365I, or any combination of the foregoing substitutions. Section 9 (a) substitution or deletion of a methionine residue at the position corresponding to position 1 of wild-type human Neu2 (M1); (b) substitution of a valine residue at the position corresponding to position 6 of wild-type human Neu2 (V6); (c) substitution of an isoleucine residue at the position corresponding to position 187 of wild-type human Neu2 (I187); or (d) substitution of a cysteine ​​residue at the position corresponding to position 332 in wild-type human Neu2 (C332); or any combination of the above substitutions Item 9. The sialidase according to any one of Items 1 to 8, further comprising: Item 10 In sialidase: (a) The methionine residue at the position corresponding to position 1 of wild-type human Neu2 is deleted (ΔM1), substituted with alanine (M1A), or substituted with aspartic acid (M1D); (b) The valine residue at the position corresponding to position 6 of wild-type human Neu2 is replaced with a tyrosine (V6Y); (c) the isoleucine residue at the position corresponding to position 187 of wild-type human Neu2 is substituted with a lysine (I187K); or (d) the cysteine ​​residue at the position corresponding to position 332 of wild-type human Neu2 is substituted with alanine (C332A); Alternatively, the sialidase of paragraph 9, wherein the sialidase comprises any combination of the foregoing substitutions. Section 11 (a) M1D, V6Y, P62G, A93E, I187K, and C332A substitutions; (b) substitutions M1D, V6Y, K9D, A93E, I187K, C332A, V363R, and L365I; (c) M1D, V6Y, P62N, I187K, and C332A substitutions; (d) substitutions M1D, V6Y, I187K, Q270A, S301R, W302K, and C332A; (e) substitutions M1D, V6Y, P62S, I187K, Q270A, S301R, W302K, and C332A; (f) M1D, V6Y, P62T, I187K, Q270A, S301R, W302K, and C332A substitutions; (g) M1D, V6Y, P62N, I187K, Q270A, S301R, W302K, and C332A substitutions; (h) substitutions M1D, V6Y, P62G, A93E, I187K, S301A, W302R, and C332A; (i) M1D, V6Y, P62G, A93E, Q126Y, I187K, Q270T, and C332A substitutions; (j) substitutions M1D, V6Y, P62G, A93E, Q126Y, I187K, and C332A; or (k) Substitutions of M1D, V6Y, P62G, A93E, Q126Y, I187K, A242F, Q270T, and C332A Item 11. The sialidase according to Item 10, comprising: Item 12 (a) M1D, V6Y, P62G, A93E, I187K, and C332A substitutions; (b) substitutions M1D, V6Y, K9D, A93E, I187K, C332A, V363R, and L365I; (c) M1D, V6Y, P62N, I187K, and C332A substitutions; (d) substitutions M1D, V6Y, I187K, Q270A, S301R, W302K, and C332A; (e) substitutions M1D, V6Y, P62S, I187K, Q270A, S301R, W302K, and C332A; (f) substitutions of M1D, V6Y, P62T, I187K, Q270A, S301R, W302K, and C332A; or (g) Substitutions of M1D, V6Y, P62N, I187K, Q270A, S301R, W302K, and C332A Item 11. The sialidase according to Item 10, comprising: Section 13 Item 13. The sialidase according to any one of Items 1 to 12, which is selected from Neu1, Neu2, Neu3 and Neu4. Item 14 Item 14. The sialidase of Item 13, which is Neu2. Section 15 Item 15. The sialidase according to any one of Items 1 to 14, which has a substrate specificity different from that of the corresponding wild-type sialidase. Item 16 16. The sialidase of paragraph 15, which is capable of cleaving α2,3, α2,6 and / or α2,8 linkages. Item 17 17. The sialidase of item 15 or 16, which is capable of cleaving α2,3 and α2,8 linkages. Section 18 Item 18. The sialidase according to any one of Items 1 to 17, comprising any one of SEQ ID NOs: 48 to 54, 149, 154, 159, and 191. Section 19 Item 18. The sialidase according to any one of Items 1 to 17, comprising any one of SEQ ID NOs: 48 to 54. Section 20 A recombinant mutant human sialidase comprising a mutation or combination of mutations set forth in any one of Tables 5-9, 11-13 or 15-30, and optionally further comprising a mutation or combination of mutations set forth in any one of Tables 1-4. Section 21 (a) a recombinant mutant human sialidase according to any one of items 1 to 20; and (b) an immunoglobulin Fc domain and / or an immunoglobulin antigen-binding domain wherein the sialidase and the immunoglobulin Fc domain and / or the immunoglobulin antigen binding domain are linked by a peptide bond or an amino acid linker. Section 22 22. The fusion protein of paragraph 21, comprising an immunoglobulin Fc domain. Section 23 23. The fusion protein of paragraph 22, wherein the immunoglobulin Fc domain is derived from the Fc domain of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, or IgM. Section 24 24. The fusion protein of paragraph 23, wherein the immunoglobulin Fc domain is derived from the Fc domain of human IgG1, IgG2, IgG3, or IgG4. Section 25 25. The fusion protein of paragraph 24, wherein the immunoglobulin Fc domain is derived from a human IgG1 Fc domain. Section 26 26. The fusion protein according to any one of items 21 to 25, comprising an immunoglobulin antigen-binding domain. Section 27 27. The fusion protein of paragraph 26, wherein the immunoglobulin antigen-binding domain is linked to a second immunoglobulin antigen-binding domain to generate an antigen-binding site. Section 28 28. The fusion protein of paragraph 26 or 27, wherein the immunoglobulin antigen-binding domain is derived from an antibody selected from trastuzumab, daratumumab, girentuximab, ofatumumab, avelumab, and rituximab. Section 29 29. An antibody conjugate comprising the fusion protein according to any one of items 21 to 28. Item 30 30. The antibody conjugate of paragraph 29, comprising a single sialidase. Item 31 31. The antibody conjugate of paragraph 30, comprising two sialidases. Section 32 32. The antibody conjugate of paragraph 31, wherein the two sialidases are identical. Item 33 33. The antibody conjugate according to any one of items 29 to 32, comprising a single antigen-binding site. Section 34 34. The antibody conjugate of any one of items 29 to 33, comprising two antigen-binding sites. Section 35 35. The antibody conjugate of paragraph 34, wherein the two antigen-binding sites are identical. Section 36 Item 36. The antibody conjugate according to any one of Items 29 to 35, having a molecular weight of about 135 kDa to about 165 kDa. Section 37 Item 36. The antibody conjugate according to any one of Items 29 to 35, having a molecular weight of about 215 kDa to about 245 kDa. Section 38 The antibody conjugate: (a) a first polypeptide comprising an immunoglobulin light chain; (b) a second polypeptide comprising an immunoglobulin heavy chain; and (c) a third polypeptide comprising an immunoglobulin Fc domain and a sialidase. Includes; 38. The antibody conjugate of any one of items 29 to 37, wherein the first and second polypeptides are covalently linked together, the second and third polypeptides are conjugated together, and the first and second polypeptides together define an antigen-binding site. Section 39 39. The antibody conjugate of paragraph 38, wherein the third polypeptide comprises, from N-terminal to C-terminal, a sialidase and an immunoglobulin Fc domain. Section 40 The fusion protein: (a) a first polypeptide comprising a first immunoglobulin light chain; (b) a second polypeptide comprising a first immunoglobulin heavy chain and a first sialidase; (c) a third polypeptide comprising a second immunoglobulin heavy chain and a second sialidase; and (d) a fourth polypeptide comprising a second immunoglobulin light chain. Including, 38. The antibody conjugate of any of items 29 to 37, wherein the first and second polypeptides are covalently linked together, the third and fourth polypeptides are covalently linked together, the second and third polypeptides are covalently linked together, the first and second polypeptides together define a first antigen-binding site, and the third and fourth polypeptides together define a second antigen-binding site. Section 41 41. The antibody conjugate of paragraph 40, wherein the second and third polypeptides comprise, from N-terminal to C-terminal, a first and second immunoglobulin heavy chain and a first and second sialidase, respectively. Section 42 The fusion protein: (a) a first polypeptide comprising a first sialidase, a first immunoglobulin Fc domain, and a first single-chain variable fragment (scFv); and (b) a second polypeptide comprising a second sialidase, a second immunoglobulin Fc domain, and a second single-chain variable fragment (scFv). Including, 38. The antibody conjugate of any one of items 29 to 37, wherein the first and second polypeptides are covalently linked together, and the first scFv defines a first antigen-binding site and the second scFv defines a second antigen-binding site. Section 43 43. The antibody conjugate of paragraph 42, wherein the first polypeptide comprises, from N-terminal to C-terminal, a first sialidase, a first immunoglobulin Fc domain, and a first scFv, and the second polypeptide comprises, from N-terminal to C-terminal, a second sialidase, a second immunoglobulin Fc domain, and a second scFv. Section 44 The antibody conjugate: (a) a first polypeptide comprising an immunoglobulin light chain; (b) a second polypeptide comprising an immunoglobulin heavy chain and a single-chain variable fragment (scFv); and (c) a third polypeptide comprising an immunoglobulin Fc domain and a sialidase. Including, 38. The antibody conjugate of any of items 29 to 37, wherein the first and second polypeptides are covalently linked together, the second and third polypeptides are covalently linked together, the immunoglobulin light chain and immunoglobulin heavy chain together define a first antigen-binding site, and the scFv defines a second antigen-binding site. Section 45 45. The antibody conjugate of paragraph 44, wherein the second polypeptide comprises, from N-terminal to C-terminal, an immunoglobulin heavy chain and an scFv, and the third polypeptide comprises, from N-terminal to C-terminal, a sialidase and an immunoglobulin Fc domain. Section 46 An isolated nucleic acid comprising a nucleotide sequence encoding at least a portion of the recombinant mutant human sialidase of any one of items 1 to 20, the fusion protein of any one of items 21 to 28, or the antibody conjugate of any one of items 29 to 45. Section 47 47. An expression vector comprising the nucleic acid according to item 46. Section 48 A host cell comprising the expression vector according to item 47. Section 49 A pharmaceutical composition comprising the recombinant mutant human sialidase according to any one of Items 1 to 20, the fusion protein according to any one of Items 21 to 28, or the antibody conjugate according to any one of Items 29 to 45. Section 50 A method for treating cancer in a subject in need of cancer treatment, comprising administering to the subject in need of cancer treatment an effective amount of the recombinant mutant human sialidase of any one of Items 1 to 20, the fusion protein of any one of Items 21 to 28, the antibody conjugate of any one of Items 29 to 45, or the pharmaceutical composition of Item 49. Section 51 51. The method of paragraph 50, wherein the cancer is a solid tumor, a soft tissue tumor, a hematopoietic tumor, or a metastatic lesion. Section 52 52. The method of paragraph 51, wherein the solid tumor is a sarcoma, adenocarcinoma, or carcinoma. Section 53 53. The method of paragraph 51 or 52, wherein the solid tumor is a tumor of the head and neck (e.g., pharynx), thyroid, lung (e.g., small cell or non-small cell lung cancer (NSCLC)), breast, lymphatic system, gastrointestinal (e.g., oral cavity, esophagus, stomach, liver, pancreas, small intestine, colon and rectum, anal canal), reproductive or genitourinary tract (e.g., kidney, urothelium, bladder, ovary, uterus, cervix, endometrium, prostate, testes), CNS (e.g., neural or glial cells, e.g., neuroblastoma or glioma) or skin (e.g., melanoma). Section 54 52. The method of claim 51, wherein 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 transformed CLL, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, hairy cell leukemia, myelodyplastic syndrome (MDS), lymphoma, Hodgkin's disease, malignant lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, or Richter's syndrome (Richter transformation).

[0313] Sequence Listing [Table 48] [Table 49] [Table 50] [Table 51] [Table 52] Table 53 Table 54 Table 55 Table 56 Table 57 Table 58 Table 59 Table 60 Table 61 Table 62 Table 63 Table 64 Table 65 Table 66 Table 67 Table 68 Table 69 Table 70 Table 71 Table 72 Table 73 Table 74 Table 75 Table 76 Table 77 Table 78 Table 79 Table 80 Table 81 Table 82 Table 83 Table 84 Table 85 Table 86 Table 87 Table 88 Table 89 Table 90 Table 91 Table 92 Table 93 Table 94 Table 95 Table 96 Table 97 Table 98 Table 99 Table 100 Table 101 Table 102 Table 103 Table 104 Table 105 Table 106

Claims

1. comprising a substitution of a proline residue with a glycine (P62G) at a position corresponding to position 62 of wild-type human Neu2 (SEQ ID NO: 1); comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 159; and A recombinant mutant human Neu2 sialidase having sialidase enzyme activity.

2. (a) Substitution of an alanine residue (A93) at the position corresponding to position 93 of wild-type human Neu2; (b) substitution of a glutamine residue (Q126) at the position corresponding to position 126 in wild-type human Neu2; (c) a substitution of an alanine residue (A242) at a position corresponding to position 242 of wild-type human Neu2; or (d) Substitution of a glutamine residue (Q270) at the position corresponding to position 270 of wild-type human Neu2. or any combination of the above substitutions 2. The recombinant mutant human Neu2 sialidase of claim 1, further comprising:

3. A recombinant mutant human Neu2 sialidase as described in claim 2, comprising a substitution of A93.

4. A recombinant mutant human Neu2 sialidase as described in claim 2 or 3, comprising substitutions of A93, Q126, A242 and Q270.

5. In the sialidase: (a) the alanine residue at the position corresponding to position 93 of wild-type human Neu2 was substituted with glutamic acid (A93E) or lysine (A93K); (b) a glutamine residue at a position corresponding to position 126 of wild-type human Neu2 is substituted with leucine (Q126L), glutamic acid (Q126E), phenylalanine (Q126F), histidine (Q126H), isoleucine (Q126I), or tyrosine (Q126Y); (c) the alanine residue at the position corresponding to position 242 of wild-type human Neu2 is substituted 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); or (d) the glutamine residue at the position corresponding to position 270 of wild-type human Neu2 is substituted with alanine (Q270A), histidine (Q270H), phenylalanine (Q270F), proline (Q270P), serine (Q270S), or threonine (Q270T); Alternatively, the sialidase comprises any combination of the foregoing substitutions. The recombinant mutant human Neu2 sialidase of claim 4.

6. A recombinant mutant human Neu2 sialidase described in any one of claims 1 to 5, comprising an A93E substitution.

7. A recombinant mutant human Neu2 sialidase described in any one of claims 1 to 6, comprising A93E, Q126Y, A242F and Q270A substitutions.

8. (a) Substitution or deletion of a methionine residue at the position corresponding to position 1 of wild-type human Neu2 (M1); (b) substitution of a valine residue at the position corresponding to position 6 of wild-type human Neu2 (V6); (c) substitution of a cysteine ​​residue at the position corresponding to position 332 of wild-type human Neu2 (C332); or any combination of the above substitutions The recombinant mutant human Neu2 sialidase of any one of claims 1 to 7, further comprising:

9. In the sialidase: (a) The methionine residue at the position corresponding to position 1 of wild-type human Neu2 is deleted (ΔM1), substituted with alanine (M1A), or substituted with aspartic acid (M1D); (b) The valine residue at the position corresponding to position 6 of wild-type human Neu2 is replaced with a tyrosine (V6Y); (c) The cysteine ​​residue at the position corresponding to position 332 of wild-type human Neu2 is substituted with alanine (C332A); Alternatively, the sialidase comprises a combination of any of the foregoing substitutions.

10. The recombinant mutant human Neu2 sialidase of claim 9, wherein the sialidase comprises a V6Y substitution.

11. (a) substitutions of M1D, V6Y, P62G, A93E, and C332A; or (b) substitutions of M1D, V6Y, P62G, A93E, Q126Y, A242F, Q270T, and C332A; 11. The recombinant mutant human Neu2 sialidase of claim 9 or 10, comprising:

12. 12. The recombinant mutant human Neu2 sialidase of any one of claims 9 to 11, comprising the substitutions M1D, V6Y, P62G, A93E and C332A.

13. Replacement for M1D, V6Y, P62G, A93E, Q126Y, A242F, Q270T and C332A The recombinant mutant human Neu2 sialidase of any one of claims 9 to 11, comprising:

14. 14. The recombinant mutant human Neu2 sialidase of any one of claims 1 to 13, comprising SEQ ID NO:

48.

15. (a) a recombinant mutant human Neu2 sialidase according to any one of claims 1 to 14; and (b) an immunoglobulin Fc domain and / or an immunoglobulin antigen-binding domain wherein the sialidase and the immunoglobulin Fc domain and / or immunoglobulin antigen binding domain are linked by a peptide bond or an amino acid linker.

16. The fusion protein of claim 15, comprising an immunoglobulin Fc domain.

17. The fusion protein of claim 16, wherein the immunoglobulin Fc domain is derived from the Fc domain of human IgG1, IgG2, IgG3 or IgG4.

18. 18. The fusion protein of claim 17, wherein the immunoglobulin Fc domain is derived from a human IgG1 Fc domain.

19. The fusion protein of any one of claims 15 to 18, comprising an immunoglobulin antigen-binding domain.

20. 20. The fusion protein of claim 19, wherein the immunoglobulin antigen-binding domain is combined with a second immunoglobulin antigen-binding domain to generate an antigen-binding site.

21. 21. The fusion protein of claim 19 or 20, wherein the immunoglobulin antigen-binding domain is derived from an antibody selected from trastuzumab, daratumumab, girentuximab, ofatumumab, avelumab, and rituximab.

22. An antibody conjugate comprising the fusion protein of any one of claims 19 to 21.

23. The antibody conjugate: (a) a first polypeptide comprising an immunoglobulin light chain; (b) a second polypeptide comprising an immunoglobulin heavy chain; and (c) a third polypeptide comprising an immunoglobulin Fc domain and a sialidase. Including; 23. The antibody conjugate of claim 22, wherein the first and second polypeptides are covalently linked together, the second and third polypeptides are covalently linked together, and the first and second polypeptides together define an antigen-binding site.

24. 24. The antibody conjugate of claim 23, wherein the third polypeptide comprises, from N-terminal to C-terminal, the sialidase and an immunoglobulin Fc domain.

25. 26. An isolated nucleic acid comprising a nucleotide sequence encoding at least a portion of a recombinant mutant human Neu2 sialidase of any one of claims 1 to 14, a fusion protein of any one of claims 15 to 21, or an antibody conjugate of any one of claims 22 to 24.

26. 26. An expression vector comprising the nucleic acid of claim 25.

27. 27. A host cell comprising the expression vector of claim 26.

28. A pharmaceutical composition comprising a recombinant mutant human sialidase according to any one of claims 1 to 14, a fusion protein according to any one of claims 15 to 21, or an antibody conjugate according to any one of claims 22 to 24.

29. A composition for use in therapy, comprising a recombinant mutant human sialidase according to any one of claims 1 to 14, a fusion protein according to any one of claims 15 to 21 or an antibody conjugate according to any one of claims 22 to 24.

30. 29. A pharmaceutical composition according to claim 28 for use in therapy.

31. 31. The composition of claim 29 or the pharmaceutical composition of claim 30, wherein the treatment is treatment of cancer in a subject in need thereof.

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