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

Abstract

To provide a recombinant human sialidase and a recombinant sialidase fusion protein useful for treating cancer.SOLUTION: The present invention relates generally to recombinant human sialidases and recombinant sialidase fusion proteins, wherein the sialidase optionally includes one or more mutations, e.g., a substitution, deletion or addition of at least one amino acid, as compared to a wild-type human sialidase. The present invention also provides antibody conjugates comprising a sialidase and an antibody or portion thereof. The invention further relates to methods of using sialidase fusion proteins or antibody conjugates to treat cancer.SELECTED DRAWING: None

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 a role for 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 increased sialoglycan activity. The expression of sialylated tumor-associated carbohydrate antigens by tumor cells is often associated with increased tumor aggressiveness and metastatic potential.

[0004] Recently, a family of sialic acid-binding lectins, Siglecs (sialic acid-binding immunoglobulins), has been identified. Phospholipase-like lectin (PLL) binds to hypersialylated cancer cells and transmits signals derived from activation of NK cell receptors. It has been shown to play a role in cancer immunosuppression by mediating the suppression of NK cells, thereby inhibiting NK cell-mediated tumor cell killing (Jandus et al. (2014) J. CLIN. INVEST. 124: 1810-1820; Laeubli et al. (2014) PROC. NATL. ACAD. SCI. USA 111: 14211-14216; Hudak et al. (2014) NAT. CHEM. BIOL. 10: 69-75). Enzymatic removal of sialic acid by treatment with enzymes 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, in part, involves removing sialic acid and / or sialic acid-containing molecules from the surface of cancer cells. and / or 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 tumor microenvironment and / or reducing the concentration of sialic acid and / or sialic acid-containing molecules in the tumor microenvironment. This invention is based in part on the discovery that it is possible to produce ducts.

[0007] Thus, in one aspect, the present invention provides a recombinant mutant human sialidase enzyme, wherein the sialidase comprises: (a) a substitution of a proline residue at a position corresponding to position 5 of wild-type human Neu2; (b) substitution of a lysine residue at the position corresponding to position 9 of wild-type human Neu2 (K9); (c) wild-type (d) substitution of a lysine residue at position 44 of human Neu2 (K44); (d) substitution of a lysine residue at position 45 of wild-type human Neu2 (K45); (e) substitution of a leucine residue at 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 an alanine residue at position 107 of wild-type human Neu2 (l) substitution of a glycine 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 a cysteine ​​residue at position 125 of wild-type human Neu2 (C125); (q) 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 170 of wild-type human Neu2 (C164). (s) substitution of an alanine residue at the corresponding position (R170); (s) substitution of an alanine residue at the position corresponding to position 171 of wild-type human Neu2 (A171); (t) substitution of a glutamine residue at the position corresponding to position 188 of wild-type human Neu2 (Q188); (u) substitution of an arginine residue at the position corresponding to position 189 of 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 leucine residue at the position corresponding to position 217 of wild-type human Neu2 (L217); (y) a substitution of a glutamic acid residue at position 225 of wild-type human Neu2 (E225); (y) a substitution of a histidine residue at position 239 of wild-type human Neu2 (H239); (z) a substitution of a histidine residue at position 240 of wild-type human Neu2 (H239). (aa) substitution of a leucine residue at the position corresponding to position 241 of wild-type human Neu2 (L240); (aa) substitution of a leucine residue at the position corresponding to position 241 of wild-type human Neu2 (L241); (bb) substitution of an arginine residue at position 242 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 the position corresponding to position 244 of wild-type human Neu2; (dd) substitution of a threonine residue at the position corresponding to position 249 of wild-type human Neu2 (T249) (ee) substitution of an aspartic acid residue at a position corresponding to position 251 of wild-type human Neu2 (D251); (ff) substitution of a glutamic acid residue at a position corresponding to position 257 of wild-type human Neu2 (E257); (gg) substitution of a serine residue at a position corresponding to position 258 of wild-type human Neu2 (S258); (hh) substitution of a leucine residue at a position corresponding to position 260 of wild-type human Neu2 (L260); (ii) substitution of a leucine residue at a position corresponding to position 265 of wild-type human Neu2 (L260). (jj) substitution of a valine residue at a position corresponding to position 270 of wild-type human Neu2 (V265); (kk) substitution of tryptophan at the position corresponding to position 292 of wild-type human Neu2; (11) substitution of a serine residue at the position corresponding to position 301 of wild-type human Neu2; (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 For example, the sialidase can include a substitution 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) a proline residue at a position corresponding to position 5 of wild-type human Neu2 is substituted with a histidine (P5H); and (b) a proline residue at a position corresponding to position 9 of wild-type human Neu2 is substituted with a histidine (P5H). (c) the lysine residue at position 44 of wild-type human Neu2 was substituted with an aspartic acid (K9D); (d) the lysine residue at position 44 of wild-type human Neu2 was substituted with an arginine (K44R) or a glutamic acid (K44E); The lysine residue at position 45 of wild-type human Neu2 was substituted with alanine (K45A), arginine (K45R), or glutamic acid (K45E); (e) the lysine residue at position 54 of wild-type human Neu2 was substituted with alanine (K45A), arginine (K45R), or glutamic acid (K45E); The leucine residue was replaced with a methionine (L54M); (f) the position corresponding to position 62 of wild-type human Neu2. (g) the proline residue at position 69 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 position 69 of wild-type human Neu2 is replaced with histidine (Q69H). (h) The arginine residue at position 78 of wild-type human Neu2 is replaced with a lysine (R78K). (i) the aspartic acid residue at position 80 of wild-type human Neu2 is substituted with proline (D80P); (j) the alanine residue at position 93 of wild-type human Neu2 is substituted with glutamic acid (A93E) or lysine (A93K); (k) the glycine residue at position 107 of wild-type human Neu2 is substituted with aspartic acid (G107D); (l) the glycine residue at position 108 of wild-type human Neu2 is substituted with glutamic acid (A93E) or lysine (A93K). (m) the glutamine residue at the position corresponding to position 112 of wild-type human Neu2 was replaced with arginine (Q112R) or lysine (Q112K). (n) the cysteine ​​residue at position 125 of wild-type human Neu2 is replaced with leucine (C125L); (o) the glutamine residue at position 126 of wild-type human Neu2 is replaced with leucine (Q126L), glutamic acid (Q126E), phenylalanine (Q126F), histidine (Q126H), or isopropyl alcohol (Q126L). (p) the alanine residue at position 150 of wild-type human Neu2 was replaced with valine (A150V); (q) the alanine residue at position 164 of wild-type human Neu2 was replaced with valine (A150V). The cysteine ​​residue at position 170 of wild-type human Neu2 was replaced with glycine (C164G); (s) an alanine residue at position 171 of wild-type human Neu2 is substituted with glycine (A171G); (t) a glutamine residue at position 188 of wild-type human Neu2 is substituted with proline (Q188P); (u) an arginine residue at position 189 of wild-type human Neu2 is substituted with proline (R189P); (v) an alanine residue at position 171 of wild-type human Neu2 is substituted with glycine (A171G); (v) a glutamine residue at position 188 of wild-type human Neu2 is substituted with proline (Q188P); (v) an arginine residue at position 189 of wild-type human Neu2 is substituted with proline (R189P); The alanine residue at position 213 of wild-type human Neu2 is replaced with cysteine ​​(A213C), asparagine (A213N), serine (A213S), or threonine (A213T); (w) the leucine residue at position 217 of wild-type human Neu2 is replaced with alanine (L217A) or valine (L217V). (x) The threonine residue at position 249 of wild-type human Neu2 is alanine (T249A). (y) the aspartic acid residue at position corresponding to position 251 of wild-type human Neu2 is replaced with glycine (D251G); (z) the glutamic acid residue at position corresponding to position 225 of wild-type human Neu2 is replaced with (aa) the histidine residue at position 239 of wild-type human Neu2 is substituted with proline (H239P); (bb) the leucine residue at position 240 of wild-type human Neu2 is substituted with aspartic acid (L240D), asparagine (L240N), or tyrosine (L240Y). (cc) the arginine residue at 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 position 242 of wild-type human Neu2 is replaced with cis- Leucine (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), thiamin (A242C), thiamin (A242F), thiamin (A242G), thiamin (A242H), thiamin (A242I), thiamin (A242K), thiamin (A242L), thiamin (A242M), thiamin (A242N), thiamin (A242Q), thiamin (A242R), thiamin (A242S), thiamin (A242V ... (ee) position 244 of wild-type human Neu2; (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 (L260T); (ii) the valine residue at position 265 of wild-type human Neu2 is substituted with phenylalanine (L260C). (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 glutamine residue at position 292 of wild-type human Neu2 is substituted with alanine (Q270A), histidine (Q270H), phenylalanine (Q270F), proline (Q270P), serine (Q270S), or threonine (Q270T). The tryptophan residue at the corresponding position was replaced with arginine (W292R); (ll) the serine residue at the position corresponding to position 301 of wild-type human Neu2 was 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 (S (mm) the tryptophan residue at position 302 of wild-type human Neu2 was 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), glycine (W302G), or tyrosine (S301Y); (nn) at a position corresponding to position 363 of wild-type human Neu2; or (oo) the valine 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 is and any combination of 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 the position corresponding to position 1 of wild-type human Neu2 is deleted (ΔM1), substituted with alanine (M1A), or asparagine. (b) the valine residue at position 6 of wild-type human Neu2 is replaced by tyrosine (M1D); (c) an isoleucine residue at the position corresponding to position 187 of wild-type human Neu2 (V6Y); the residue is substituted with lysine (I187K); or (d) at a position corresponding to position 332 of wild-type human Neu2. The cysteine ​​residue is replaced by alanine (C332A); alternatively, the sialidase is Any combination is included.

[0011] In some embodiments, the sialidase is: (a) M1D, V6Y, P62G, A93E, I187K, and C332A. (b) M1D, V6Y, K9D, A93E, I187K, C332A, V363R, and L365I substitutions; (c) M1D, V6Y (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; (g) substitutions of M1D, V6Y, P62N, I187K, Q270A, S301R, W302K, and C332A; (h) substitutions of M1D, V6Y, P62G, A93E, I187K, S301A , W302R and C332A substitutions; (i) 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 has a α2,3, α2,6, and / or In some embodiments, the sialidase can cleave α2,3 and α2,8 linkages. may be cut.

[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. include.

[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 method for the preparation of a recombinant mutant human sialidase, comprising: (a) administering to a mammalian subject the recombinant mutant human sialidase disclosed herein; and (b) an immunoglobulin Fc domain and / or an immunoglobulin antigen-binding domain. The present invention provides a fusion protein comprising (and consisting essentially of) a sialidase, wherein the sialidase and the Fc domain and / or antigen binding domain are linked by a peptide bond or an amino acid linker. In some embodiments, the fusion protein further comprises a linker, such as an amino acid, linking the sialidase enzyme and the Fc domain and / or the antigen binding domain. In some embodiments, the immunoglobulin antigen binding domain comprises a linker. The antibody is coupled (eg, covalently or non-covalently) to a brin 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 The antibody is derived from the 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 contains 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, wherein the first and second polypeptides together define an antigen-binding site. The third polypeptide comprises, e.g., from the N-terminus to the C-terminus, the sialidase and and an immunoglobulin Fc domain.

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

[0022] In some embodiments, the antibody conjugate comprises: (a) a first sialidase, a first immunoglobulin, (b) a first polypeptide comprising a phospho-Fc domain and a first single-chain variable fragment (scFv); and (b) a second sialidase, a second immunoglobulin Fc domain, and an optional second single-chain variable fragment. and a second polypeptide comprising an scFv; wherein the first and second polypeptides are covalently linked. together, where the first scFv defines a first antigen-binding site and the second scFv binds to any antigen-binding site present. If present, the first polypeptide defines a second antigen-binding site. The first polypeptide can comprise, for example, from N-terminus to C-terminus, a first sialidase, a first immunoglobulin Fc domain, and a first scFv. The second polypeptide may comprise, for example, from the N-terminus to the C-terminus, a second sialidase, a second It may comprise an 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 first polypeptide can comprise, for example, from N- to C-terminus, an immunoglobulin heavy chain and an scFv. The third polypeptide can comprise, for example, from N- to C-terminus, a sialidase and an immunoglobulin Fc domain.

[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 of 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 head and neck ( 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, ovaries, uterus, cervix, intrauterine In some embodiments, the hematopoietic tumor is a tumor of the thyroid gland, thyroid gland, kidney, liver, kidney, liver cirrhosis ... Myelodyplastic syndrome (MDS), lymphoma, Hodgkin's disease, malignant lymphoma, non- Hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, or Richter's syndrome (Richter's syndrome) In some embodiments, the cancer is endometrial cancer, ovarian cancer, cervical cancer, or vulvar cancer. 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 detecting HLA-DR, CD86, CD83, IFNγ, and / or IFN-γ in cells or tissues. The present invention provides a method for increasing expression of IL-1b, IL-6, TNFα, IL-17A, IL-2, or IL-6, the method comprising treating a cell or tissue with any of the aforementioned sialidases, any of the aforementioned fusion proteins, or In one embodiment, the method comprises contacting the cells with an effective amount of any of the aforementioned antibody conjugates or any of the aforementioned pharmaceutical compositions. In one embodiment, the cells are selected from dendritic cells and peripheral blood mononuclear cells (PBMCs).

[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 or both of a "knob" mutation, e.g., T366Y, or a "hole" mutation, e.g., Y407T, 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 or both of a "knob" mutation, e.g., T366Y, or a "hole" mutation, e.g., Y407T, 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 or both of a "knob" mutation, e.g., T366Y, or a "hole" mutation, e.g., Y407T, 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 enzyme portion can include at least one mutation relative to a wild-type human sialidase.

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

[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 refers to enzymes that contain one or more amino acid substitutions, deletions, or modifications relative to the wild-type sialidase sequence. variants with insertions, and / or fusion proteins or Sialidases are also referred to as neuraminidases, and the two terms are used interchangeably herein unless otherwise indicated. In this context, the term "functional fragment" of a sialidase refers to a fragment that retains, e.g., 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%, or at least a portion of the enzymatic activity of the corresponding full-length naturally occurring sialidase. A fragment of a full-length sialidase that retains at least 95% or 100% of the sialidase enzyme activity. The activity can be assayed by any method known in the art, such as by measuring the release of sialic acid from the fluorogenic substrate 4-methylumbelliferyl-N-acetylneuraminic acid (4MU-NeuAc). In some embodiments, the functional fragment contains at least 100, 150, 200, 250, 300, 310, 320, 330, 340, 350, 360, 400, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1100, 1110, 1200, 1210, 1220, 1300, 1310, 1320, 1330, 1400, 1410, 1420, 1430, 1440, 1500, 1510, 1 or 370 consecutive amino acids.

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

[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 shown in SEQ ID NO:1, and the nucleotide sequence encoding human Neu2 is shown in SEQ ID NO:24. Unless otherwise stated, as used herein, wild-type human Neu2 refers to the sequence of SEQ ID NO:1. It refers to human Neu2 having the amino acid sequence

[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 shown in SEQ ID NO:8, and the nucleotide sequence encoding human Neu3, isoform 1 is shown in SEQ ID NO:25. Human Neu3, isoform 2 The amino acid sequence of this is shown in SEQ ID NO: 9, and the nucleotide sequence encoding human Neu3, isoform 2 is shown in SEQ ID NO: 9. The octide sequence is shown 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 shown in SEQ ID NO: 10, and the nucleotide sequence encoding human Neu4, isoform 1 is shown in SEQ ID NO: 26. The amino acid sequence of human Neu4, isoform 2 is shown in SEQ ID NO: 11. and the nucleotide sequence encoding human Neu4, isoform 2 is shown in SEQ ID NO:35.

[0040] Four sialidases have also been found in the mouse genome: Neu1, Neu2, Neu3, and 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 those from Salmonella typhimurium and Vibrio cholerae. The amino acid sequence of Salmonella typhimurium sialidase (St-sialidase) is: The nucleotide sequence encoding the Salmonella typhimurium sialidase is set forth in SEQ ID NO: 30, and the amino acid sequence of the Vibrio cholerae sialidase is set forth in SEQ ID NO: 6. The nucleotide sequence encoding the Vibrio cholerae sialidase is shown in SEQ ID NO:37.

[0042] In some embodiments, the recombinant variant human sialidase exhibits 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%, or about 100% of the enzymatic activity of the corresponding (or template) wild-type human sialidase. It has about 100% or more than 100%.

[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 variant human sialidase in mammalian cells, e.g., HEK293 cells, is about 10%, about 20%, about 50%, or greater than the expression yield of the corresponding wild-type human sialidase. , 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.

[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 certain embodiments, the recombinant mutant human sialidase comprises a substitution of at least one cysteine ​​(cys, C) residue. Certain cysteine ​​residues in sialidases are involved in the prevention of protein aggregation. It has been discovered that this can result in inhibition of expression of a functional protein. Thus, in certain embodiments, the recombinant mutant human sialidase contains at least one mutation to remove a free cysteine ​​(e.g., for Neu1 (SEQ ID NO: 7), e.g., one or more of C111, C117, C171, C183, C218, C240, C242, and C252; for Neu2 (SEQ ID NO: 1), e.g., one or more of C125 , one or more mutations at C196, C219, C272, C332 and C352; for Neu3 (SEQ ID NO: 8), e.g. For example, 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), one or more mutations at C88, C125, C126, C186, C191, C211, C223, C239, C276, C437, C453, C480, and C481). In some embodiments, the free cysteine ​​can be replaced by serine (ser, S), isoleucine (iso, I), valine (val, V), phenylalanine (phe, F), leucine (leu, L), or alpha-amino acids. It is substituted with 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 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 is a mutant human sialidase as described in Table 1 (wild-type human Neu2 (SEQ ID NO: 1) corresponding amino acid positions) includes combinations of substitutions. [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 refers to the pH at which a protein is least soluble, which may affect the ability to express and purify the protein. Generally, a protein will dissolve well if its pI is 2 units higher than the pH of the solution. Human Neu2 has a predicted pI of 7.5. Therefore, human Neu2 is active around neutral pH. This 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 expression of human Neu2 or other human sialidases, recombinant mutant human sialidases can be engineered to contain one or more amino acids. The recombinant mutant human sialidase can be designed to contain one or more amino acid substitutions, where the substitution(s) increase the pI of the sialidase relative to a sialidase that does not have the substitution(s). Furthermore, reducing the number of hydrophobic amino acids on the surface of the 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 a sialidase that does not have the substitution(s).

[0053] Thus, in some embodiments, the recombinant mutant human sialidase comprises at least one The recombinant sialidase may include an 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, the amino acid substitution is at a surface-exposed D or E amino acid, in a helix or loop, or at a position that has a K or R at the corresponding position in St-sialidase. In some embodiments, amino acid substitutions occur in amino acids distant from or otherwise not involved in catalysis, amino acids not conserved by other human Neu proteins or St-sialidases or clostridial NanH, or in domains important for function (e.g., It occurs at amino acids that are not located within an Asp-box or a β-strand.

[0055] Increasing the isoelectric point (pI) of the sialidase relative to sialidase without the substitution and / or Exemplary amino acid substitutions in Neu2 that decrease the hydrophobicity of the sialidase include A2E, A2K, D215K, V325E, V325K, E257K, and E319K. Variant human sialidases contain 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 is a mutant human sialidase as described in Table 2 (wild-type human Neu2 (SEQ ID NO: :1) and amino acid positions corresponding to substitutions or combinations of substitutions that correspond to the substitutions or combinations of substitutions listed in 1). [Table 2]

[0057] c. N-terminal peptide addition and N- or C-terminal substitution It has been discovered that the addition of a peptide sequence of two or more amino acids to the N-terminus of a human sialidase can improve the expression and / or activity of the sialidase. The peptides are at least 2 amino acids in length, for example 2-20, 2-10, 2-5, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In embodiments, the peptide may be capable of forming an alpha helix or may have a propensity to form an alpha helix.

[0058] In mice, the Neu2 isoform (type B) found in the thymus is different from the Neu2 found in skeletal muscle. In one embodiment of the present invention, the N-terminal 6 amino acids of the mouse thymus Neu2 isoform, MEDLRP ( SEQ ID NO:4) or a variant thereof can be added to human Neu, e.g., human Neu2. In some embodiments, the recombinant mutant human sialidase comprises a covalently linked nucleotide sequence to the N-terminal amino acid of the sialidase. In one embodiment, the recombinant mutant human serotype comprises a peptide of at least two amino acid residues in length. The sialidase comprises the peptide MEDLRP (SEQ ID NO: 4) or EDLRP (SEQ ID NO: 3) covalently attached to the N-terminal amino acid of the sialidase. In some embodiments, the sialidase further comprises a peptide, e.g., a nucleotide sequence located between MEDLRP (SEQ ID NO: 4) or EDLRP (SEQ ID NO: 3) and the remainder of the sialidase. In some embodiments, the peptide, e.g., MEDLRP (SEQ ID NO: 4) or EDLRP (SEQ ID NO: 3), may contain a cleavage site, e.g., a proteolytic cleavage site, that cleaves the peptide from the remainder of the sialidase. It may be cut off after translation.

[0059] Alternatively, or in combination with the N-terminal addition, 1 to 5 amino acids in 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 The methionine can be removed, or the first five amino acids (MASLP; SEQ ID NO: 12) can be removed Alternatively, the second to fourth amino acids (ASLP; SEQ ID NO: 13) may be deleted.

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

[0061] Human sialidases have a β-propeller structure characterized by six-bladed β-sheets arranged toroidally around a central axis. Generally, the N- and C-terminal blades Hydrophobic interactions between the blades of the β-propeller, such as between the N-terminus of the sialidase, 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 C-terminal and C-terminal beta-propeller blades.

[0062] Thus, in some embodiments, the recombinant mutant human sialidase comprises at least one and a substitution of a wild-type amino acid residue, wherein the substitution increases hydrophobic interactions and / or hydrogen bonds between the N-terminus and C-terminus of the sialidase relative to a sialidase not having 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). Examples of amino acids in Neu2 that increase hydrophobic interactions and / or hydrogen bonds between the N- and C-termini include: Exemplary substitutions 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 the 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 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), resulting in a recombinant mutant human Neu2 sialidase. The variant human Neu2 sialidase also contains at least one of L4N, L4K, V6Y, L7N, L4N, and L7N. , including L4N and V6Y and L7N, V12N, V12Y, V12L, V6Y, V6F or V6W substitutions.

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

[0065] Furthermore, 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 an alanine (M1A) or an aspartic acid (M1D). In other embodiments, the sialidase comprises a substitution of a methionine residue at position 1 of wild-type human Neu2 (SEQ ID NO: 1) with an alanine (M1A) or an aspartic acid (M1D). It contains a deletion of a methionine residue (ΔM1) at the position corresponding to

[0066] In some embodiments, the recombinant mutant human sialidase is a mutant human sialidase as described in Table 4 (wild-type human Neu2 (SEQ ID NO: 1) corresponding amino acid positions) includes combinations of substitutions. [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 is The cleavage of sialidase by proteases (eg, trypsin) is reduced relative to the absence of sialidase.

[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 may be performed using methods such as SDS-PAGE, including, for example, as described in Example 5 herein. It may be assayed by any method known in the art.

[0069] Exemplary substitutions that increase resistance to proteolytic cleavage include: (i) wild-type human Substitution of an alanine residue at the position corresponding to position 242 of 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 (ii) wild-type human Neu2 (SEQ ID NO: 1) (iii) a substitution of an arginine residue at a position corresponding to position 243 of wild-type human Neu2 (SEQ ID NO: 1), for example, with glutamic acid (R243E), histidine (R243H), asparagine (R243N), glutamine (R243Q), or lysine (R243K); (iv) a substitution of a valine residue at a position corresponding to position 244 of wild-type human Neu2 (SEQ ID NO: 1); For example, substitution with isoleucine (V244I), lysine (V244K), or proline (V244P); and 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 selected from those listed in Table 5 (corresponding to wild-type human Neu2 (SEQ ID NO: 1)). Substitutions or combinations of substitutions corresponding to the substitutions or combinations of substitutions listed in (amino acid position) Includes: [Table 5]

[0070] Increased resistance to proteolytic cleavage (and / or expression yield and / or Further exemplary substitutions (which increase enzymatic activity) include: (i) substitution of a leucine residue at the position corresponding to position 240 of wild-type human Neu2 (SEQ ID NO: 1), e.g., aspartic acid (L240D), (ii) a substitution with alanine residue at the position corresponding to position 213 of wild-type human Neu2 (SEQ ID NO: 1), e.g., cysteine ​​(A213C), asparagine (L240N), or tyrosine (L240Y); (iii) a substitution of an arginine residue at a position corresponding to position 241 of wild-type human Neu2 (SEQ ID NO: 1) with an arginine (A213N), serine (A213S), or threonine (A213T); (iv) a substitution of a serine (R241A), aspartic acid (R241D), leucine (R241L), glutamine (R241Q), or tyrosine (R241Y) at a position corresponding to position 258 of wild-type human Neu2 (SEQ ID NO: 1). (v) substitution of a leucine residue at the position corresponding to position 260 of wild-type human Neu2 (SEQ ID NO: 1), e.g., aspartic acid (L260D), phenyl (vi) substitution with alanine (L260F), glutamine (L260Q), or threonine (L260T); (vii) a substitution of a valine residue at a position corresponding to position 265 of human Neu2 (SEQ ID NO: 1) with phenylalanine (V265F); or (vii) a combination of any of the foregoing. In some embodiments, substitutions or combinations of substitutions at these positions are used to improve hydrophobic and / or aromatic interactions between secondary structural elements in the sialidase (e.g., between an α-helix and the nearest β-sheet). It is contemplated that this may improve the activity of the hydroxybenzoates, 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 ... The 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 ... The present invention includes substitutions or combinations of substitutions corresponding to those listed in Table 1 (amino acid positions corresponding to type 1 human Neu2 (SEQ ID NO: 1)). [Table 6]

[0072] e. Other substitutions The present invention further provides a method for the treatment of rhesus malabsorption comprising administering to a subject therapies 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 amino acids GDYDAPTHQVQW (SEQ ID NO: 15) with amino acids SMDQGSTW (SEQ ID NO: 16) or STDGGKTW (SEQ ID NO: 17). In some embodiments, the recombinant mutant human Neu2 comprises a substitution of amino acids GDYDAPTHQVQW (SEQ ID NO: 15) with amino acids 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 variants comprising at least one substitution at a position corresponding to V212, A213, Q214, D215, T216, L217, E218, C219, Q220, V221, A222, E223, V224, E225, or T225. A variant human sialidase is provided.

[0074] The present invention further provides a method for the preparation of nucleic acids comprising the steps of: 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 comprises: (a) a substitution of a proline residue at the position corresponding to position 5 of wild-type human Neu2 (P5); (b) a substitution of a lysine residue at the position corresponding to position 9 of wild-type human Neu2 (K9); (c) a substitution of a lysine residue at the position corresponding to position 44 of wild-type human Neu2 (K9); (d) substitution of a lysine residue at position 45 of wild-type human Neu2 (K45); (e) substitution of a leucine residue at 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 (G107); (l) wild-type (m) a substitution of a glutamine residue at position 112 of wild-type human Neu2 (Q112); (n) a substitution of a glutamine residue at position 125 of wild-type human Neu2 (Q112); (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 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 an alanine residue at position 171 of wild-type human Neu2 (A171); (u) 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 arginine residue at position 213 of wild-type human Neu2 (R189). (w) substitution of an alanine residue at a position corresponding to position 217 of wild-type human Neu2 (L217); (x) substitution of a glutamic acid residue at a position corresponding to position 225 of wild-type human Neu2 (E225); (y) substitution of a histidine residue at a position corresponding to position 239 of wild-type human Neu2 (H239); (z) substitution of a leucine residue at a position corresponding to position 240 of wild-type human Neu2 (L240); (aa) substitution of an arginine residue at a position corresponding to position 241 of wild-type human Neu2 (R241); (bb) substitution of a glutamic acid residue at a position corresponding to position 225 of wild-type human Neu2 (E225); (y) substitution of a histidine residue at a position corresponding to position 239 of wild-type human Neu2 (H239); (z) substitution of a leucine residue at a position corresponding to position 240 of wild-type human Neu2 (L240); (aa) substitution of an arginine residue at a position corresponding to position 241 of wild-type human Neu2 (R241); (cc) substitution of an alanine residue at position 242 of wild-type human Neu2 (A242); (cc) substitution of a valine residue at position 244 of wild-type human Neu2 (V244); (dd) substitution of a valine residue at position 249 of wild-type human Neu2 (V249). (ee) substitution of a threonine residue at position 251 of wild-type human Neu2 (T249); Substitution of an aspartic acid residue (D251); (ff) a glutamic acid residue at the position corresponding to position 257 of wild-type human Neu2 Substitution of a hydroxyl residue (E257); (gg) a serine residue at the position corresponding to position 258 of wild-type human Neu2 (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 wild-type human Neu2 (ll) substitution of a tryptophan residue at position 292 of wild-type human Neu2 (W292); (ll) substitution of a serine residue at position 301 of wild-type human Neu2 (S301); (mm) substitution of a serine residue at position 302 of wild-type human Neu2 (S302). Substitution of a tryptophan residue at position (W302); (nn) at the position corresponding to position 363 in wild-type human Neu2 substitution of a valine residue at (V363); or (oo) at a position corresponding to position 365 of wild-type human Neu2 or a combination of any of the foregoing substitutions. For example, the sialidase can include a substitution of K9, P62, A93, Q216, A242, Q270, S301, W302, V363, or L365, or a combination of any of the foregoing substitutions.

[0076] In some embodiments, in the sialidase: (a) a proline residue at a position corresponding to position 5 of wild-type human Neu2 is substituted with a histidine (P5H); and (b) a proline residue at a position corresponding to position 9 of wild-type human Neu2 is substituted with a histidine (P5H). (c) the lysine residue at position 44 of wild-type human Neu2 was substituted with an aspartic acid (K9D); (d) the lysine residue at position 44 of wild-type human Neu2 was substituted with an arginine (K44R) or a glutamic acid (K44E); The lysine residue at position 45 of wild-type human Neu2 was substituted with alanine (K45A), arginine (K45R), or glutamic acid (K45E); (e) the lysine residue at position 54 of wild-type human Neu2 was substituted with alanine (K45A), arginine (K45R), or glutamic acid (K45E); The leucine residue was replaced with a methionine (L54M); (f) the position corresponding to position 62 of wild-type human Neu2. (g) the proline residue at position 69 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 position 69 of wild-type human Neu2 is replaced with histidine (Q69H). (h) The arginine residue at position 78 of wild-type human Neu2 is replaced with a lysine (R78K). (i) the aspartic acid residue at position 80 of wild-type human Neu2 is substituted with proline (D80P); (j) the alanine residue at position 93 of wild-type human Neu2 is substituted with glutamic acid (A93E) or lysine (A93K); (k) the glycine residue at position 107 of wild-type human Neu2 is substituted with aspartic acid (G107D); (l) the glycine residue at position 108 of wild-type human Neu2 is substituted with glutamic acid (A93E) or lysine (A93K). (m) the glutamine residue at the position corresponding to position 112 of wild-type human Neu2 was replaced with arginine (Q112R) or lysine (Q112K). (n) the cysteine ​​residue at position 125 of wild-type human Neu2 is replaced with leucine (C125L); (o) the glutamine residue at position 126 of wild-type human Neu2 is replaced with leucine (Q126L), glutamic acid (Q126E), phenylalanine (Q126F), histidine (Q126H), or isopropyl alcohol (Q126L). (p) the alanine residue at position 150 of wild-type human Neu2 was replaced with valine (A150V); (q) the alanine residue at position 164 of wild-type human Neu2 was replaced with valine (A150V). The cysteine ​​residue at position 170 of wild-type human Neu2 was replaced with glycine (C164G); (s) an alanine residue at position 171 of wild-type human Neu2 is substituted with glycine (A171G); (t) a glutamine residue at position 188 of wild-type human Neu2 is substituted with proline (Q188P); (u) an arginine residue at position 189 of wild-type human Neu2 is substituted with proline (R189P); (v) an alanine residue at position 171 of wild-type human Neu2 is substituted with glycine (A171G); (v) a glutamine residue at position 188 of wild-type human Neu2 is substituted with proline (Q188P); (v) an arginine residue at position 189 of wild-type human Neu2 is substituted with proline (R189P); The alanine residue at position 213 of wild-type human Neu2 is replaced with cysteine ​​(A213C), asparagine (A213N), serine (A213S), or threonine (A213T); (w) the leucine residue at position 217 of wild-type human Neu2 is replaced with alanine (L217A) or valine (L217V). (x) The threonine residue at position 249 of wild-type human Neu2 is alanine (T249A). (y) the aspartic acid residue at position corresponding to position 251 of wild-type human Neu2 is replaced with glycine (D251G); (z) the glutamic acid residue at position corresponding to position 225 of wild-type human Neu2 is replaced with (aa) the histidine residue at position 239 of wild-type human Neu2 is substituted with proline (H239P); (bb) the leucine residue at position 240 of wild-type human Neu2 is substituted with aspartic acid (L240D), asparagine (L240N), or tyrosine (L240Y). (cc) the arginine residue at 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 position 242 of wild-type human Neu2 is replaced with cis- Leucine (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), thiamin (A242C), thiamin (A242F), thiamin (A242G), thiamin (A242H), thiamin (A242I), thiamin (A242K), thiamin (A242L), thiamin (A242M), thiamin (A242N), thiamin (A242Q), thiamin (A242R), thiamin (A242S), thiamin (A242V ... (ee) position 244 of wild-type human Neu2; (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 (L260T); (ii) the valine residue at position 265 of wild-type human Neu2 is substituted with phenylalanine (L260C). (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 glutamine residue at position 292 of wild-type human Neu2 is substituted with alanine (Q270A), histidine (Q270H), phenylalanine (Q270F), proline (Q270P), serine (Q270S), or threonine (Q270T). the tryptophan residue at the corresponding position is 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), glycine (S301G), histidine (S301H), isoleucine (S301I), lysine (S301K), leucine (S301L), methionine (S301M), asparagine (S301N), proline (S301P), glutamine (S301Q), arginine (S301R), threonine (S301T), valine (S301V), tryptophan (S301W), or tyrosine (S301Y). (mm) The tryptophan residue at position 302 of wild-type human Neu2 was alanine (W302A), aspartic acid (W302D), glutamic acid (W302E), phenylalanine (W302F), glycine ( substituted with 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); ) 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 contains 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 corresponds to position 184 of wild-type human Neu2. deletion of a leucine residue (ΔL184) at the position corresponding to position 185 of wild-type human Neu2; Deletion of a histidine residue (ΔH185), a proline residue at the position corresponding to position 186 of wild-type human Neu2 The deletions include a deletion of a base (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 corresponds to position 216 of wild-type human Neu2. a threonine residue at the position corresponding to position 217 of wild-type human Neu2 and a leucine residue at the position corresponding to position 217 of wild-type human Neu2 and insertions between, for example, insertions of amino acids selected from S, T, Y, L, F, A, P, V, I, N, D and H.

[0079] Additional 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., under the heading "I. Recombinant Human Sialidase" section and Examples 1, 2, 3, 4, 5 and 6 in the Examples It is described in.

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

[0081] For example, recombinant mutant sialidase enzymes can be engineered to contain M1 deletion (ΔM1), M1A substitution, M1D substitution, V6Y substitution, substitution, K9D substitution, P62G substitution, P62N substitution, P62S substitution, P62T substitution, A93E substitution, I187K substitution, Q270A substitution, S301R substitution, W302K substitution, C332A substitution, V363R substitution, L365I substitution, or any of the above It may include any combination thereof.

[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. The recombinant mutant sialidase enzymes were: M1A and V6Y; M1A and I187K; M1A and C332A; M1D and V6Y; M1D and I187K; M1D and C332A; ΔM1 and V6Y; ΔM1 and I187K; ΔM1 and C332A; V6Y and I187K; V6Y and C332A; I187K and C332A; M1A, V6Y and I187K; M1A, V6Y and C332A; M1A, I187K and C332A; M1D, V6Y and I187K; M1D, V6Y and 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, V6 Y, I187K and C332A; and combinations of mutations selected from 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) M1D, V6Y, P62N, I187K, and C332A substitutions; (d) M1D, V6Y, I187K, Q270A, S301R , W302K and C332A substitutions; (e) M1D, V6Y, P62S, I187K, Q270A, S301R, W302K and C332A substitutions; (f) 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, A (i) substitutions of M1D, V6Y, P62G, A93E, Q126Y, I187K, Q270T and C332A; (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 corresponds to position 302 of wild-type human Neu2. at position 301 of wild-type human Neu2 combined with the substitution of a tryptophan residue at the corresponding position (W302). and a substitution of a serine residue (S301) at the corresponding position. For example, the recombinant mutant human sialidase can include a combination of substitutions corresponding to the combinations of substitutions listed in the columns of Table 8 (amino acid positions corresponding to wild-type human Neu2 (SEQ ID NO: 1)). For example, the recombinant mutant human sialidase can include: 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 is a mutant human sialidase as described in Table 9 (wild-type human Neu2 (SEQ ID NO: :1) the amino acid position corresponding to the substitution combinations listed in the column include. [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4]

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

[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, and X5 is Phe, Trp, Tyr or Val X6 is Lys or Asp, X7 is Lys, Arg or Glu, X8 is Lys, Ala, Arg or Glu, X9 is Leu or Met, and X10 is Pro, Asn, Asp, His, Glu, Gly, Ser, or Thr, 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 Ri, X 28 is Gln, Ala, His, Phe or Pro, and X 29 is Cys or Val, and X 30 Trp or Arg, and X 31 is Ser or Arg, and X 32 is Trp or Lys, and X 33 is Lys or Val, and X 34 is Ala, Cys, Ser or Val, and X 35 is Cys, Leu, or Val, and X 36 is Val or Arg, and X 37 is Leu, Gln, His, Ile, Lys, or Ser, and sialidase is It contains 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, and 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, and X8 is Ser or is 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, and 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 Ri, 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 32is 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 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 Yes, 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, and X4 is Pro, Asn, Asp, His, Glu, Gly, Ser or Thr. X5 is Ala, Glu, or Lys, X6 is Gln, Leu, Glu, Phe, His, Ile, Leu, or Tyr, X7 is Arg, Ile, or Lys, and X8 is Ala, Cys, Phe, Gly, His, Ile, Lys, L X is eu, Met, Asn, Gln, Arg, Ser, Val, Trp, or Tyr, X is Gln, Ala, His, Phe, Pro, Ser, or Thr, and X is 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 X1 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, and 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.

[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 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. Substitutions can also be identified using the BLAST (Basic Local Alignment Search Tool) algorithm, a BLOSUM substitution matrix (e.g., the BLOSUM 62 matrix) or a PAM substitution:p matrix (e.g., the PAM 250 matrix).

[0093] Sequence identity can be determined in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. This can be determined using the programs blastp, blastn, blastx, tblastn and BLAST (Basic Local Alignment Search Tool) analysis, using the algorithm used by tblastx (Karlin et al., (1990) PROC. NATL. ACAD. SCI. USA 87:2264-2268; Altschul, (1993) J. MOL. EVOL. 36, 290-300; Altschul et al., (1997) NUCLEIC ACIDS RES. 25:3389-3402, 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, incorporated herein by reference in its entirety. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment across the entire length of the sequences being compared. Histogram, Description, Alignment, Expect (i.e. (i.e., statistical significance threshold for reporting matches against database sequences), cutoffs, and match Search parameters for tricks and filters 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 creation penalty); wink=1 (generates word hits at every wink.sup.th position along the query); and gapw=16 (gapped alignments are generated). (Sets the window width used for the blastp test). The equivalent blastp parameter setting is 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 open gap [integer]: default = 5 for nucleotides / protein). -E, Cost for extension gap [integer]: default = nucleotide 2 for / 1 for protein; -q, penalty for nucleotide mismatch [integer]: default = -3; -r, reward for nucleotide match [integer]: default = 1; -e, expectation value [real number]: default = 10; -W, word size [integer]: default = nucle 11 for octides / 28 for megablasts / 3 for proteins; -y, blast extension in bits Length dropoff (X): default = 20 for blastn / 7 for others; -X, X dropoff value (in bits) for gapped alignments: default = 15 for all programs but not applicable to blastn; and -Z, Final X dropoff value (in bits) for gapped alignments: 50 for blastn, etc. 25) ClustalW for pairwise protein alignments is also used. (Default parameters are, for example, Blosum62 matrix and gap-open pairs.) Gap Opening Penalty = 10 and Gap Extension Penalty = 0.1 The best fit comparison between sequences available in the GCG package version 10.0 uses the DNA parameters GAP=50 (gap creation penalty) and LEN=3 (gap extension penalty). The equivalent settings for best-fit protein comparison are GAP=8 and LEN=2.

[0094] II. Fusion Protein / Antibody Conjugates Selective removal of sialic acid on hypersialylated cancer cells and / or in the tumor microenvironment To facilitate the removal of sialic acid by sialidase in a subject, it may be useful to target a sialidase as described herein to such cells or such a tumor microenvironment. Furthermore, to facilitate 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. These include fusion proteins and / or antibody conjugates. This can be achieved by including a sialidase in the conjugate (eg, a chemically conjugated conjugate).

[0095] Thus, the present invention further provides a method for the preparation of sialidase enzymes, or functional fragments thereof, and portions or fragments of antibodies, such as immunoglobulin Fc domains (also referred to herein as Fc domains). In some embodiments, fusion proteins comprising a sialidase and an immunoglobulin antigen binding domain (also referred to herein as an antigen binding domain) are provided. and the antibody or portion thereof (eg, 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" refers to a single protein comprising amino acid sequences based on two or more separate proteins or polypeptide chains. It is understood to refer to a polypeptide chain in which two amino acid sequences are connected, either directly or via an intervening linker. The nucleotide sequences encoding the fusion proteins can be generated, for example, using conventional recombinant DNA techniques. It is possible.

[0097] In some embodiments, the fusion protein contains a tag, such as a Strep tag (e.g., a Strep II tag). , His tag (e.g., 10x His tag), myc tag, or FLAG tag. In some embodiments, the fusion protein may be positioned at the C-terminus or N-terminus of the protein. a sialidase portion linked in a C-terminal direction to a polypeptide comprising an immunoglobulin heavy chain; wherein the sialidase portion comprises an N-terminal addition of MEDLRP (SEQ ID NO: 4), The II tag is placed 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, e.g., a fungal, bacterial, non-human mammalian, or human sialidase. In some embodiments, the sialidase portion contains at least one mutation, e.g., a sialidase-specific mutation, relative to a wild-type human sialidase. For example, a recombinant human serotype containing at least one amino acid substitution, deletion, or addition, as described above. It is alidase.

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

[0100] In some embodiments, the sialidase portion comprises the mutations C332A and C352L. In some embodiments, the sialidase comprises the N-terminal addition of MEDLRP (SEQ ID NO: 4) or EDLRP (SEQ ID NO: 3). In some embodiments, the sialidase portion comprises the peptide LSHSLST (SEQ ID NO: 22) 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, 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, or , K9D substitution, P62G substitution, P62N substitution, P62S substitution, P62T substitution, A93E substitution, Q126Y substitution, I187K substitution replacement, A242T replacement, Q270A replacement, Q270T replacement, S301R replacement, S301R replacement, W302K replacement, W302R replacement , a C332A substitution, a V363R substitution, a L365I substitution, or any combination of the foregoing.

[0102] In some embodiments, the sialidase portion has the amino acid sequence of any of SEQ ID NOs: 48-54, 149, 154, 159, or 191, or 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. It contains an amino acid sequence that

[0103] b. Antibody part As used herein, unless otherwise indicated, the term "antibody" refers to 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., a monoclonal antibody). antigen-binding fragments of monoclonal antibodies), e.g., modified or genetically engineered The term "antibody" is understood to mean an intact antibody, an antigen-binding fragment or an Fc fragment, either directly or chemically conjugated. Examples of antigen-binding fragments include Fab, Fab', (Fab')2, Fv, single chain antibodies ( Examples include scFv), minibodies and diabodies. Examples of 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 an immunoglobulin that binds to an Fc receptor, either alone or in combination with a second immunoglobulin Fc domain. "Fc" refers to a fragment of an immunoglobulin heavy chain constant region that can be obtained by cleaving an Fc domain. An immunoglobulin Fc domain can include, for example, immunoglobulin CH2 and CH3 domains. An immunoglobulin Fc domain can include, for example, immunoglobulin CH2 and CH3 domains and an immunoglobulin hinge region. The boundaries between the immunoglobulin hinge region, CH2 and CH3 domains 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) accounts for 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 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 is heterodimerized with a second polypeptide. For somatization, "knob" mutations, e.g., T366Y, or "hole" mutations, e.g., Y407T (EU numbering Residue numbers based on sequencing, 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).

[0109] In some embodiments, the fusion protein comprises an immunoglobulin antigen-binding domain. By including such a domain, the sialylated cancer cells and / or tumor microorganisms of the fusion protein are more likely to be sialylated. Targeting to the environment may be improved. 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, immunoglobulin heavy chain variable regions and immunoglobulin light chain variable regions, where the variable regions together define the antigen-binding site.

[0110] Immunoglobulin antigen-binding domains and / or antigen-binding sites can be, for example, Mab, ascrinvacumab, cixutumumab, conatumumab, daratumumab, drozitumab, durigotumab, durvalumab, dusigitumab, enfortumab, enoticumab, Epratuxumab, figitumumab, ganitumab , glembatumumab, intetumumab, ipilimumab, iratumumab, icrucumab, lexatumumab, lucatumumab, mapatumumab, narnatumab, necitumumab, nesbacumab, ofatumumab, olaratumumab, panitumumab, patritumumab, pritumumab, radretumab, ramucirumab, rilotumumab, lobatumumab, seribantumab, tarextumab, te Protumumab, tovetumab, vantictumab, besencumab, votumumab, zalutumumab, flanvotumab, altumomab, anatumomab, arcitumomab, bectumomab, blinatumomab, detumomab, ibritumomab , minretumomab, mitumomab, moxetumomab, naptumomab Mab, nofetumomab, pemtumomab, pintumomab, racotumomab, satumomab, solitomab, taplitumomab, tenatumomab, tositumomab, tremelli tumab, abagovomab, atezolizumab, durvalumab, avelumab, igovomab, oregovomab, capromab, edrecolomab, nacolomab, amatuximab, bavituximab, brentuximab, cetuximab , derlotuximab, dinutuximab, ensituximab , futuximab, girentuximab, indatuximab, isatuximab, margetuximab, rituximab, siltuximab, ublituximab, ecromeximab, abituzumab, alemtuzumab, bevacizumab, bivatuzumab, brontuximab ontictuzumab, 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, milatuzumab, moxetumomab, nimotuzumab, o Binutuzumab, ocaratuzumab, otlertuzumab, onartuzumab, oportuzumab, parsatuzumab, pertuzumab, pidilisumab pidilizumab, pinatuzumab, polatuzumab, sibrotuzumab Sibrotuzumab, simtuzumab, tacatuzumab, tigatuzumab, trastuzumab, tucotuzumab, urelumab, vandortuzumab, vanucizumab, veltuzumab, borsetuzumab Mab, sofituzumab, catumaxomab, ertumaxomab, depatuxizumab, ontuxizumab, brontuvetmab, tamtuvetmab, nivolumab, pembrolizumab, epratuzumab, MEDI9447, urelumab, utomilumab, hu3F8, hu14.18-IL-2, 3F8 / OKT3BsAb, lirilumab, BMS-986016, pidilizumab, AMP-224, AMP-514 In some embodiments, the immunoglobulin antigen-binding domain may be derived from an antibody selected from trastuzumab, daratumumab, girentuximab, ofatumumab, and rituximab.

[0111] In one embodiment, 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 sequence is shown in SEQ ID NO: 64. The amino acid sequence of an exemplary scFv derived from trastuzumab is the sequence Shown in number:65.

[0112] Immunoglobulin antigen-binding domains and / or antigen-binding sites may be selected from a wide variety of antigen-binding domains, including, for example, adenosine A2a receptor (A2aR), A kinase anchoring protein 4 (AKAP4), B melanoma antigen (BAGE), brother of the regulator of imprinted sites (BORIS), Brett et al. Breakpoint cluster region Abelson tyrosine kinase (BCR / ABL), CA125, CAIX, CD19, CD20, CD22, CD30, CD33, CD52, CD73, CD137, carcinoembryonic antigen Carcinoma of the genotype (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, Kira -cell immunoglobulin-like receptor (KIR), lymphocyte activation gene 3 (LAG-3), lymphocyte antigen 6 complex K (LY6K), matrix metalloproteinase 16 (MMP16), melanotransferrin (MFI2), melanoma antigen 3 (MAGE-A3), melanoma antigen C2 (MAGE-C2), melanoma antigen D4 (MAGE-D4), T cell Melanoma antigen 1 recognized by 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), prostatic acid 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), and smooth muscle. X chromosome 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 of T cell activation IgA-containing suppressor (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 ... Family member 21 (TNFSRF21), bone morphogenetic protein receptor type 1B (BMPR1B), kringle domain-containing transmembrane protein 2 (KREMEN2), Delta-like protein 3 (DLL3), Siglec7 and and Siglec9. Further exemplary cancer antigens These 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, 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), and CD55. (DAF), DLL4 (Delta-like ligand 4), CD20 (MS4A1) and CD96.

[0113] The present invention further provides antibody complexes comprising one or more of the fusion proteins disclosed herein. As used herein, unless otherwise indicated, the term "antibody conjugate" refers to an antibody conjugated (e.g., covalently coupled) to an additional functional moiety. The term "antibody conjugate" is understood to refer to an antibody or functional fragment thereof that comprises a sialidase enzyme, such as a recombinant mutant human sialidase enzyme disclosed herein, that binds to a sialidase enzyme, antigen binding activity, and / or Fc receptor binding activity. 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. contains further polypeptide chains.

[0114] Table 10 lists antibodies and antibody-drug conjugates suitable for use according to the present invention. The antigen bound by the antibody-drug conjugate, as well as the specific antibody, 1 shows the types of cancer targeted by the body or antibody-drug conjugates. [Table 14-1] [Table 14-2] [Table 14-3]

[0115] c. Linker In some embodiments, the sialidase portion of the fusion protein is directly attached 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 moiety can be attached, linked, or fused to the antigen by a linker. It can be covalently attached to the body moiety.

[0116] The linker may comprise one or more naturally occurring amino acids, a sialidase or a functional fragment thereof, and and an antibody portion or fragment, where an amino acid (e.g., a cysteine ​​amino acid) can be introduced by site-directed mutagenesis. The linker can also contain one or more unnatural amino acids. In certain circumstances, for example, a linker containing one or more sulfhydryl-reactive groups (e.g., maleimides) may be a naturally occurring cysteine ​​residue or a site-specific cysteine ​​residue. It is contemplated that the product of mutagenesis can be covalently bonded to a cysteine ​​in the sialidase portion or antibody portion.

[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., with respect to serum, enzymes, etc.) so that the fusion protein may be effective in vivo. (not cut off by

[0119] The linker can be about 1 angstrom (Å) to about 150 Å in length, or about 1 Å to about 120 Å in length, or about 5 Å to about 110 Å in length, or about 10 Å to about 100 Å in length. , about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 27, 30 Angstroms or more in length and / or 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 Angstroms or less in length. It can be length.

[0120] In some embodiments, the linker comprises a polypeptide linker that connects or fuses the sialidase portion of the fusion protein to the antibody portion of the fusion protein (e.g., an immunoglobulin Fc domain and / or an immunoglobulin antigen binding domain). For example, directly or indirectly It is contemplated that a gene encoding a sialidase moiety that is efficiently linked (e.g., via an amino acid-containing linker) to an antibody moiety can be made and expressed using conventional recombinant DNA techniques. For example, the amino terminus of the sialidase moiety can be linked to the carboxy terminus of either the light or heavy chain of the antibody moiety. For example, for a Fab fragment, the amino terminus of the sialidase The amino- or carboxy-terminus may be linked to the first constant domain (CH1) of the antibody heavy chain. When a linker is used, the linker may contain hydrophilic amino acid residues, such as Gln, Ser, Gly, Glu, Pro, His, and Arg. In some embodiments, the linker is 1 to 25 amino acid residues, 1 Up 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 Peptides containing 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 where 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 described, for example, in George et al. (2003) PROTEIN ENGINEERING 15:871-879, and and US Patent 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 antibody conjugates comprising the fusion proteins disclosed herein. The antibody conjugates comprise a single polypeptide chain (i.e., the fusion proteins disclosed herein). fusion protein), or the antibody conjugate may include additional polypeptide chains (e.g., 1, 2, or 3 additional polypeptide chains). For example, the antibody conjugate may include a first polypeptide comprising a recombinant mutant human sialidase enzyme and an immunoglobulin heavy chain. The fusion protein may comprise a second polypeptide comprising an immunoglobulin light chain, as well as a second polypeptide comprising an immunoglobulin light chain. , where, for example, the immunoglobulin heavy and light chains together define a single antigen-binding site.

[0123] In some embodiments, the antibody conjugate may include a single sialidase. In other embodiments, the antibody conjugate may include more than one (e.g., two) sialidases. When more than one sialidase is included, the sialidases can 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 an exemplary antibody conjugate construct 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 will be understood that in each of the constructs described, 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] FIG. 9A shows a first polypeptide comprising a first immunoglobulin light chain; a first immunoglobulin heavy chain; a second polypeptide comprising a second immunoglobulin heavy chain; a third polypeptide comprising a second immunoglobulin heavy chain; and and a fourth polypeptide comprising a second immunoglobulin light chain. The first and second polypeptides can be covalently bonded together, the third and fourth 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 polypeptide The first polypeptide and the second polypeptide together define a first antigen-binding site, denoted as 10, and the third polypeptide and the fourth polypeptide together define a second antigen-binding site, denoted as 20. The sialidase enzyme, designated as 30, can be conjugated to the N-terminus or C-terminus of the first and second immunoglobulin light chains or the first and second immunoglobulin heavy chains.

[0127] FIG. 9B shows a first polypeptide comprising a first immunoglobulin light chain; a first immunoglobulin heavy chain; a second polypeptide comprising a second immunoglobulin heavy chain; a third polypeptide comprising a second immunoglobulin heavy chain; and 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 one embodiment, the first polypeptide The peptide and the second polypeptide together define a first antigen-binding site, and the third polypeptide The sialidase enzyme binds to the N-terminus or the N-terminus of the first immunoglobulin light chain or the first immunoglobulin heavy chain, and the fourth polypeptide and the fourth polypeptide together define a second antigen-binding site. can be conjugated to the C-terminus.

[0128] FIG. 9C illustrates an antibody 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 present invention relates to a conjugate construct. 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. In some embodiments, the first and second polypeptides together define an antigen-binding site. The sialidase enzyme binds to the first immunoglobulin light chain or can be conjugated to the N-terminus or C-terminus of the first immunoglobulin heavy chain.

[0129] FIG. 9D shows a first polypeptide comprising an immunoglobulin light chain; a second polypeptide comprising an immunoglobulin heavy chain; and a second polypeptide comprising an immunoglobulin Fc domain and a first sialidase enzyme. 1 shows an antibody conjugate construct comprising a first and second polypeptide and a third polypeptide comprising the first and second polypeptides. The first and second polypeptides may be covalently linked together, and the second and third polypeptides may be covalently linked together. The covalent bond may be a disulfide bond. The third polypeptide may be covalently linked together from the N-terminus to the C-terminus. The first polypeptide and the second polypeptide together define an antigen-binding site. The optional second sialidase enzyme binds to the first immunoglobulin light chain or the first immunoglobulin heavy chain. The conjugated antibody may be conjugated to the N-terminus or C-terminus of

[0130] FIG. 9E shows a first polypeptide comprising an immunoglobulin light chain; a second polypeptide comprising an immunoglobulin heavy chain; and a second polypeptide comprising an immunoglobulin Fc domain and a first sialidase enzyme. 1 shows an antibody conjugate construct comprising a first and second polypeptide and a third polypeptide comprising the first and second polypeptides. The first and second polypeptides may be covalently linked together, and the second and third polypeptides may be covalently linked together. The covalent bond may be a disulfide bond. The third polypeptide may be covalently linked together from the N-terminus to the C-terminus. The first polypeptide and the second polypeptide together define an antigen-binding site. The optional second sialidase enzyme binds to the first immunoglobulin light chain or the first immunoglobulin heavy chain. The conjugated antibody may be conjugated to the N-terminus or C-terminus of

[0131] FIG. 9F illustrates a first polypeptide comprising a first immunoglobulin Fc domain and a second immunoglobulin Fc domain. 1 shows an antibody conjugate construct comprising a second polypeptide comprising a globulin Fc domain. The first and second polypeptides can be covalently linked together. The covalent bond can be a disulfide bond. The sialidase enzyme can be conjugated to the N-terminus or C-terminus of the first immunoglobulin Fc domain or the N-terminus or C-terminus of the second immunoglobulin Fc domain. An optional second sialidase enzyme can be conjugated to the N-terminus or C-terminus of the first immunoglobulin Fc domain. can be conjugated to the C-terminus, or to the N-terminus or C-terminus of the second immunoglobulin Fc domain.

[0132] FIG. 9G shows a first polypeptide comprising an immunoglobulin light chain; and a second polypeptide comprising an immunoglobulin heavy chain. 1 shows an antibody conjugate construct comprising a second polypeptide comprising a variable region. The polypeptides may be covalently bonded together. The covalent bond may be a disulfide bond. In some embodiments, the first polypeptide and the second polypeptide together define an antigen-binding site. The sialidase enzyme may be conjugated to the N-terminus or C-terminus of the immunoglobulin light chain or immunoglobulin heavy chain variable region.

[0133] FIG. 9H shows a first polypeptide comprising a first immunoglobulin Fc domain and a second immunoglobulin Fc domain. 1 shows an antibody conjugate construct comprising a second polypeptide comprising a globulin Fc domain. The first and second polypeptides can be covalently linked together. The covalent bond can be a disulfide bond. The 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. 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. The antibody may be conjugated to the C-terminus of each of the Fc domains.

[0134] FIG. 9I shows the results of a method in which each scFv is replaced with an immunoglobulin antigen-binding fragment, e.g., Fab. For example, Figure 9I shows an antibody conjugate construct similar to that shown in Figure 9H, except that it includes a first polypeptide comprising a first immunoglobulin Fc domain and a second immunoglobulin Fc domain. 1 shows an antibody conjugate construct comprising a first polypeptide and a second polypeptide comprising an Fc domain. The first and second polypeptides can be covalently linked together. The covalent bond can be a disulfide bond. The sialidase enzyme can be conjugated to the N-terminus of the first immunoglobulin Fc domain or the second immunoglobulin Fc domain. The antibody fragment (Fab) can be conjugated to the N-terminus of the first immunoglobulin Fc domain or the second immunoglobulin Fc domain, respectively. The optional second antibody fragment (Fab) can be conjugated or fused to the C-terminus of the immunoglobulin Fc domain of the first immunoglobulin. The Fc domain may be conjugated or fused to the C-terminus of each of the Fc domains. In the case of fusion, the C-terminus of the Fc domain is attached (either by a bond or an amino acid linker) to the first polypeptide chain that defines the immunoglobulin antigen-binding fragment. In the case of an antibody having a site, this may be sufficient to confer binding affinity to the target antigen. In other instances, for example in the case of a human antibody, the first site defining an immunoglobulin antigen-binding fragment may be sufficient to confer binding affinity to the target antigen. The polypeptide chain is linked to a second polypeptide chain that defines an immunoglobulin antigen-binding fragment. The two antigen-binding fragments may be conjugated (e.g., covalently conjugated, e.g., via a disulfide bond), where the two antigen-binding fragments together form an antigen-binding portion for binding to a target antigen. Determine the rank.

[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 bond can be a disulfide bond. The second polypeptide is linked, from the N-terminus to the C-terminus, to the heavy chain and the third polypeptide. The third polypeptide comprises, from N- to C-terminal, a sialidase and an immunoglobulin. In some embodiments, the first polypeptide and the second polypeptide together define a first antigen-binding site. In some embodiments, the scFv defines a second antigen-binding site. Figure 10 shows a first polypeptide comprising an immunoglobulin light chain; an immunoglobulin Fc domain; a second polypeptide comprising an immunoglobulin heavy chain; and a third polypeptide comprising an immunoglobulin Fc domain and a first sialidase enzyme, wherein the second polypeptide comprises a Fab fragment. The fragment is conjugated to the N-terminus of the immunoglobulin heavy chain. The first and second polypeptides may be covalently linked together, and the second and third polypeptides may be covalently linked together. The covalent bond may be a disulfide bond. The third polypeptide may be covalently linked together from the N-terminus to the C-terminus. In one embodiment, the first The polypeptide and the second polypeptide 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 can be optionally 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 and a fourth polypeptide comprising a second immunoglobulin light chain. An example of this embodiment is shown in Figure 11A. The first and second polypeptides can be covalently bonded together, the third and fourth 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 polypeptide The first polypeptide and the second polypeptide together define a first antigen-binding site, and the third polypeptide The first and fourth polypeptides together define a second antigen-binding site. In some embodiments, the second and third polypeptides comprise, from N- to C-terminal, a first and second immunoglobulin heavy chain and a first and second sialidase, respectively. In some embodiments, the second and third polypeptides comprise, from N- 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 an immunoglobulin Fc domain. and a third polypeptide comprising, from the N-terminus to the C-terminus, a sialidase and a 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-terminus to the C-terminus, a sialidase and a sialidase. and an immunoglobulin Fc domain, or from the N-terminus to the 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 has the amino acid sequence of any of SEQ ID NOs: 68 to 74, 98 to 112, 150, 151, 155, 156, 160, 161, 185, 187, 192, or 195, or a sequence at least similar to any of SEQ ID NOs: 68 to 74, 98 to 112, 150, 151, 155, 156, 160, 161, 185, 187, 192, or 195. amino acid sequences with at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity include.

[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, and 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 Ri, X 28 is Gln, Ala, His, Phe or Pro, and X 29 is Cys or Val, and X 30 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 sialidase is It contains 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, and 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 has at least one 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, and X8 is Ser or is 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, and 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 25is Thr or Ala, and X 26 is Asp or Gly, and X 27 is Glu or Lys Ri, X 28 is Gln, Ala, His, Phe or Pro, and X 29 is Cys or Val, and X 30 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 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, and 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 11is Val or Arg, and X 12 is Leu, Gln, His, Ile, Lys, or Ser, and X 13 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). In some embodiments, X1 is Ala, Asp, Met, or absent, X2 is Tyr or Val, and X3 is X1 is Lys or Asp, X2 is Pro, Asn, Gly, Ser or Thr, and X3 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, and 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 Ri, X 14is 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 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 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 Yes, 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 is wild-type human It contains at least one mutation relative to 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, and X4 is Pro, Asn, Asp, His, Glu, Gly, Ser or Thr. X5 is Ala, Glu, or Lys, X6 is Gln, Leu, Glu, Phe, His, Ile, Leu, or Tyr, X7 is Arg, Ile, or Lys, X8 is Ala, Cys, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Val, Trp, or Tyr, X9 is Gln, Ala, His, Phe, Pro, Ser, or Thr, 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 15 GGGGS (SEQ ID NO: 184), GGGGSGGGGS (SEQ ID NO: 185), :145) or EPKSS (SEQ ID NO:146), wherein 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 non- 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 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 one embodiment, 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 ... The peptide 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 the sequence In one embodiment, 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: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:68, the second polypeptide comprises SEQ ID NO:69, and the third polypeptide comprises SEQ ID NO:109. In one embodiment, 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 one embodiment, 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:150. In some embodiments, the first 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:155. In one embodiment, 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 another embodiment, 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 one embodiment, 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 one embodiment, 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 one embodiment, 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. wherein the first polypeptide comprises SEQ ID NO:66 and 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) (scFv is an immunoglobulin Fc domain). and a second sialidase, a second immunoglobulin Fc domain, and a second polypeptide chain of a second immunoglobulin antigen-binding fragment, e.g., a Fab fragment; a second polypeptide comprising a second single chain variable fragment (scFv) (scFv is an immunoglobulin antigen-binding fragment) It is also understood that the fragment may be replaced by a second polypeptide chain, e.g., a Fab fragment. An example of this embodiment is shown in Figure 11C. The first and second polypeptides are covalently linked together. The covalent bond can be a disulfide bond. In some embodiments, the first scFv is The first scFv defines a first antigen-binding site and the second scFv defines a second antigen-binding site. wherein the first polypeptide comprises, from the N-terminus to the C-terminus, a first sialidase, a first immune In some embodiments, the first polypeptide comprises, from N-terminal to C-terminal, a first scFv, a first immunoglobulin Fc domain, and a first scFv. In some embodiments, the second polypeptide comprises, from the N-terminus to the C-terminus: , a second sialidase, a second immunoglobulin Fc domain, and a second scFv. In such a manner, the second polypeptide comprises, from N-terminus to C-terminus, a second scFv, a second immunoglobulin, a It contains a globulin Fc domain and a second sialidase.

[0147] In one embodiment, the first polypeptide has the amino acid sequence of any one of SEQ ID NOs: 77 to 83, 166 to 178, 194, or 197, or At least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to either In one embodiment, the second polypeptide comprises an amino acid sequence having the amino acid sequence of any of SEQ ID NOs: 77 to 83, 166 to 178, 194, or 197, or a sequence that is at least 85%, 90%, 95%, 96%, 97%, or 98% identical to any of SEQ ID NOs: 77 to 83, 166 to 178, 194, or 197. or an amino acid sequence having 99% sequence identity.

[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, and 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 Ri, X 28 is Gln, Ala, His, Phe or Pro, and X29 is Cys or Val, and X 30 Trp or Arg, and X 31 is Ser or Arg, and X 32 is Trp or Lys, and X 33 is Lys or Val, and X 34 is Ala, Cys, Ser or Val, and X 35 is Cys, Leu, or Val, and X 36 is Val or Arg, and X 37 is Leu, Gln, His, Ile, Lys, or Ser, and sialidase is It contains 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, and 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 12is 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, and X8 is Ser or is 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, and 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 Ri, X 14 is Ala, Glu, or Lys, and X 15 is Gly or Asp, and X 16 is Gln or His, and X 17is 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 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 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 Yes, 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 is wild-type human It contains at least one mutation relative to 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, and X4 is Pro, Asn, Asp, His, Glu, Gly, Ser or Thr. X5 is Ala, Glu, or Lys, X6 is Gln, Leu, Glu, Phe, His, Ile, Leu, or Tyr, X7 is Arg, Ile, or Lys, X8 is Ala, Cys, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Val, Trp, or Tyr, X9 is Gln, Ala, His, Phe, Pro, Ser, or Thr, 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 Leu, Gl n, His, Ile, Lys, or Ser, and X 15 GGGGS (SEQ ID NO: 184), GGGGSGGGGS (SEQ ID NO: 185), :145) or EPKSS (SEQ ID NO:146), wherein 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 non- 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 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. and the second polypeptide comprises SEQ ID NO: 79. In some embodiments, the first and second polypeptides 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: 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: 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 one embodiment, the first and second polypeptides comprise SEQ ID NO: 178. The first and second polypeptides comprise SEQ ID NO: 194. In some embodiments, the first and second polypeptides The polypeptide comprises 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 second polypeptide comprising an immunoglobulin Fc domain and a sialidase. An example of this embodiment is shown in Figure 1 ID. 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 polypeptide and and the second polypeptide together define a first antigen-binding site (i.e., an immunoglobulin). In some embodiments, the immunoglobulin light chain and the immunoglobulin heavy chain together define a first antigen-binding site. In some embodiments, the scFv defines a second antigen-binding site. In some embodiments, the second polypeptide comprises, from N-terminal to C-terminal, an immunoglobulin heavy chain and an scFv, or, from N-terminal to C-terminal, an immunoglobulin heavy chain and an scFv. In some embodiments, the third polypeptide comprises , from the N-terminus to the C-terminus, sialidase and immunoglobulin Fc domain, or from the N-terminus to the C-terminus, From the C-terminus, it contains 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 certain embodiments, the antibody conjugate comprises two polypeptides, each comprising an immunoglobulin Fc domain, wherein the first polypeptide is heterodimerized with the second polypeptide. and the second polypeptide has either a "knob" mutation, e.g., T366Y, or a "hole" mutation, e.g., Y407T, for heterodimerization with the first polypeptide, respectively (EU number 1). Barring residue numbering, Kabat, EA, et al. (1991) supra). For example, in one embodiment, Thus, the 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" refers to an antibody that is specific for 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 at least two antigen-binding sites that bind to at least two different antigens. "Multispecific" is understood to mean an antibody that comprises two antigen-binding sites that bind to the same antigen. In other words, the first binding site binds to a first antigen and the second binding site binds to a second, different antigen. The specific or bispecific antibodies may be, for example, human or humanized antibodies, and / or full length antibodies. or an antibody fragment (for example, a F(ab')2 bispecific antibody).

[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 certain embodiments, the antibody conjugate or fusion protein may be covalently or non-covalently linked to a biological modifier, wherein the biological modifier Modifiers can be used to increase the solubility of antibodies, increase binding specificity, reduce immunogenicity or toxicity, or modify the pharmacokinetic profile of antibodies. For example, biological modifiers can 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 attached 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. Pat. No. 7,842,789. Polyalkylene ethers Particularly useful are polysaccharides such as polyethylene glycol (PEG) and its derivatives (e.g., alkoxypolyethylene glycols, 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 polymers. The polymer may be a functionalized polyvinylpyrrolidone, e.g., a polyvinylpyrrolidone having a functional group at one (or both) ends of the polymer. It can be end-functionalized with carboxy or amine groups (available from PolymerSource). The biological modifier may 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 may include poly N-(2-hydroxypropyl) methacrylamide (HPMA), or functionalized HPMA (amine, carboxy, etc.), poly(N-isopropylacrylamide), or functionalized poly(N-isopropylacrylamide). The modification prior to conjugation is water-soluble. It need not be soluble, but is preferably 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 5kDa to about 40kDa, about 5kDa to about 50kDa, about 5kDa to about 60kDa, 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 10kDa to about 60kDa, about 10kDa to about 70kDa, about 10kDa to about 80kDa, about 10kDa to Approximately 90kDa, approximately 10kDa to approximately 100kDa, approximately 10kDa to approximately 150kDa, approximately 20kDa to approximately 30kDa, approximately 20kDa to approximately 40kDa, approximately 20kDa to approximately 50kDa, approximately 20kDa to approximately 60kDa, approximately 20kDa to approximately 70kDa, approximately 20kDa to approximately 80kDa, approximately 20 kDa ~ approx. 90kDa, approx. 20kDa ~ approx. 100kDa, approx. 20kDa ~ approx. 150kDa, approx. 30kDa ~ approx. 40kDa, approx. 30kDa ~ approx. 50kDa, approx. Approximately 30kDa to approximately 100kDa, approximately 30kDa to approximately 150kDa, approximately 40kDa to approximately 50kDa, approximately 40kDa to approximately 60kDa, approximately 40kDa to approximately 70kDa, approximately 40kDa to approximately 80kDa, approximately 40kDa to approximately 90kDa, approximately 40kDa to approximately 100kDa, approximately 40kDa to approximately 15 0kDa, approx. 50kDa ~ approx. 60kDa, approx. 50kDa ~ approx. 70kDa, approx. 50kDa ~ approx. 80kDa, approx. 50kDa ~ approx. 90kDa, approx. 50kDa ~ approx. 100kDa, approx. ~ about 90kDa, about 60kDa to about 100kDa, about 60kDa to about 150kDa, about 70kDa to about 80kDa, about 70kDa to about 90kDa, about 70kDa to about 100kDa, about 70kDa to about 150kDa, about 80kDa to about 90kDa, about 80kDa to about 100kDa, about 80kDa to about 150kDa, about 90kDa to about 100kDa, about 90kDa to about 150kDa, or about 100kDa to about 150kDa The molecular weight of the compound may be 100 to 2000.

[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 a variety of polymers can be used as biological modifiers, the antibody conjugates or fusion proteins described herein are conjugated to polyethylene glycol (PEG) polymers. In one embodiment, the antibody conjugate or fusion protein described herein is attached to at least one PEG having an actual MW of at least about 20,000 D. In another embodiment, the antibody conjugate or fusion protein described herein is covalently linked to at least one PEG having an actual MW of at least about 30,000 D. In another embodiment, the antibody conjugate or fusion protein described herein is covalently attached to at least one PEG having an actual MW of at least about 40,000 D. In some embodiments, the PEG is methoxy PEG(5000)-succinimidyl propionate (mPEG-SPA), methoxy PEG(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 are the N-terminal amino group and the epsilon amino group found on lysine residues, as well as other amino, imidazole, and amide groups. The polymer may be directly covalently attached to the antibody conjugate or fusion protein using chemistries with or without the use of multifunctional (usually bifunctional) crosslinkers known in the art. For example, sulfhydryl groups can be linked to the antibody conjugate or fusion protein using maleimide-substituted PEG (e.g., alkoxy-PEGamine + sulfhydryl groups). phenylsuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate), or PEG-maleimide, available from Shearwater Polymers, Inc., Huntsville, Ala. It can be derivatized by coupling.

[0165] III. Methods for Producing Recombinant Human Sialidases, Fusion Proteins, or Antibody Conjugates law 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 the light chain variable region and / or heavy chain variable region can be synthesized chemically or by recombinant DNA methodology. For example, the sequence of the antibody is Using appropriate synthetic nucleic acid primers, the desired target gene can be cloned from the hybridoma by conventional hybridization techniques or polymerase chain reaction (PCR) techniques. The resulting DNA molecule encoding the variable region is then subjected to, for example, the addition of constant region coding sequences and expression control sequences. The gene expression constructs may be ligated to other appropriate nucleotide sequences, such as a control sequence, to generate a conventional gene expression construct (i.e., an expression vector) encoding the desired antibody. The generation of a given gene construct is within the ordinary skill in the art.

[0166] The desired recombinant human sialidase, fusion protein and / or antibody conjugate The encoding nucleic acid 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, and the like. Cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney (COS) cells, human hepatocellular carcinoma cells ( Transformed host cells are cells that encode immunoglobulin light and / or heavy chain variable regions (e.g., Hep G2) and myeloma cells that do not otherwise produce IgG proteins. The cells can be grown under conditions that express the gene encoding the desired gene.

[0167] Specific expression and purification conditions vary depending on the expression system used. For example, if a 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. The expressed protein can be secreted. The expressed protein can accumulate in refractile or inclusion bodies, and can be recovered after disruption of the cells by French press or sonication. The refractile bodies can then be solubilized, and the protein can be refolded and / or purified by methods known in the art. can be cut off.

[0168] The engineered gene is expressed in a eukaryotic host cell, e.g., a CHO cell. If desired, the gene is first inserted into a suitable eukaryotic promoter, secretion signal, polyA sequence and The gene construct is inserted into an expression vector containing a sequence encoding a constant region that includes a sequence encoding a heavy or light chain, or a stop codon. Optionally, the vector or gene construct may contain an enhancer and an 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 all or part of the 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 single-chain polypeptides, complete immunoglobulin heavy or light chains, or fusion proteins and / or antibody conjugates comprising portions thereof. In some embodiments, including conjugated proteins and / or antibody conjugates, the conjugates are transfected into host cells. The cells may contain 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 heavy chain (e.g., a heavy chain variable region). Polypeptides expressing all or part of a light chain (e.g., a light chain variable region) or a light chain (e.g., a light chain variable region) In some embodiments, the host cell is transfected with a single vector expressing (a) a polypeptide comprising a heavy chain variable region and a polypeptide comprising a light chain variable region. or (b) a single gene encoding an entire immunoglobulin heavy chain and an entire immunoglobulin light chain. In some embodiments, the host cell is transfected with more than one expression vector, wherein in (a) or (b), the polypeptide may also include a sialidase. vector (e.g., containing all or part of a heavy chain or heavy chain variable region, optionally fused thereto) one expression vector expressing a polypeptide comprising the selected sialidase, and a light chain or The vector is co-transfected with another expression vector expressing a polypeptide containing all or part of the light chain variable region, optionally containing 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 made, A sialidase fused to a monoclonal antibody, the Fc domain or antigen-binding domain of the antibody, can be prepared by: (a) an expression vector encoding a complete or partial immunoglobulin heavy chain, and (b) another expression vector encoding all or part of an immunoglobulin light chain; or (b) both Host cells transfected with a single expression vector encoding both chains (e.g., a complete or partial heavy and light chain) are grown (cultured) under conditions that allow expression of both chains. The sialidase is fused to one or more of the chains. Intact fusion proteins and / or antibody conjugates can be produced using techniques known in the art, e.g. For example, they can be recovered and purified or isolated using affinity tags such as protein A, protein G, glutathione-S-transferase (GST) or histidine tags. Expressing heavy and light chains from a single expression vector or from two separate expression vectors is well known in the art. It is within the ordinary skill of the art.

[0172] In some embodiments, to express a protein, e.g., recombinant human sialidase, as a secreted protein, the native N-terminal signal sequence of the protein is replaced with, e.g., MDMRVPAQLLGLLLLWLPGARC (SEQ ID NO: 28). In some embodiments, to express a protein, e.g., recombinant human sialidase, as a secreted protein, the N-terminal signal sequence, For example, MDMRVPAQLLGLLLLWLPGARC (SEQ ID NO: 28) is added. Further exemplary N-terminal signal sequences include those derived from interleukin 2, CD5, 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 The terminal lysosomal signal motif, eg, 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, mouse immunoglobulin constant regions are substituted with human immunoglobulins. Chimeric proteins are made in which the phospho-constant region is replaced by the phospho-constant region. 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 an approach known as CDR grafting, the CDRs of the light and heavy chain variable regions are grafted onto a framework from another species. For example, murine CDRs can be grafted onto human FRs. In this embodiment, the CDRs of the light and heavy chain variable regions of the antibody are grafted onto human FRs or consensus human FRs. To create the consensus human FRs, several human heavy or light chain antibodies are used. The FRs derived from the amino acid sequences are aligned to identify a consensus amino acid sequence. Patent No. 7,022,500 (Queen); Patent No. 6,982,321 (Winter); Patent No. 6,180,370 (Queen); Patent No. 6,054,297 (Carter); Patent No. 5,693,762 (Queen); Patent No. 5, No. 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 "human CDR sequences," the human CDR sequences are Selected from human germline genes based on structural similarity of the CDRs of the mouse antibody to be 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 to reduce immunogenicity include "reshaping," "over-texturing," Examples of approaches include "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 murine antibody are replaced with amino acid residues that are 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 known as the technology (Vaccinex, Inc., Rochester, NY), which is used to These include vaccinia virus-based vectors for expressing antibodies in IgG. 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 technology commercially practiced by KaloBios Pharmaceuticals, Inc. (Palo Alto, CA). This technology allows for the creation of "epitope-focused" libraries for antibody selection. Another approach to modifying mouse antibodies into a form suitable for medical use in humans involves the use of proprietary human "acceptor" libraries to engineer antibodies suitable for human medical use. TM Technique The technique is commercially available from XOMA (US) LLC. For example, International (PCT) Publication No. WO See 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 generate 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 generated 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) and can be prepared and optimized from phage display libraries.

[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 have all been expressed in Escherichia coli. Antibody fragments can be isolated from antibody phage libraries. Alternatively, Fab'-SH fragments can be cultured in E. coli and secreted from the culture, allowing the facile production of large amounts of these fragments. Fab and F(ab')2 fragments can be directly recovered from the host cell culture medium and chemically linked 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. Pat. No. 5,869,046. Other techniques for the production of 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 generating bispecific antibodies are known in the art. See Milstein and Cuello (1983) NATURE 305:537, International (PCT) Publication No. WO 93 / 08829, and Traunecker et al. (1991) EMBO J., 10:3655. For further details on generating bispecific antibodies, see See, e.g., Suresh et al. (1986) METHODS ENZYMOL. 121:210. Bispecific antibodies are crosslinked antibodies. Heteroconjugates or "heterodimeric" antibodies include antibodies in bridges or "heteroconjugates" or "heterodimers." For example, one of the antibodies in the heterodimer can be linked to avidin and the other to biotin. Heterodimeric antibodies The bodies may be made using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Patent 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, the following techniques: These include those obtained using or using the following formats: Triomab / Quadroma, Knobs-into-Holes, CrossMabs, Electrostatically Matched Antibodies, LUZ-Y, Strand Exchange Engineered Domain bodies, Biclonic and DuoBody.

[0185] The advantage of using antibody fragments (e.g., F(ab) and F(ab')2 fragments) is that they bind to the Fc portion of the antibody and Fc receptors on cells (e.g., macrophages, dendritic cells, neutrophils, NK cells, and B cells). These include the elimination of non-specific binding between fragments and fragments. 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 the antibody arms. One common format for creating heterodimeric antibodies is the "knobs-into-holes" format. This format utilizes the overlapping constant regions of the antibody. The "knobs-into-holes" configuration is specific to the chain portion. The "knobs" are engineered by replacing small amino acids with larger amino acids that fit into the "hole," and the holes are engineered by replacing large amino acids with smaller amino acids. The "knobs" are linked 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 glycine, which gives the linker greater flexibility, and serine / threonine, which gives the linker specificity. Two different scFv fragments can be linked together via a hinge region to 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" configuration The formula enhances heterodimer formation but does not suppress homodimer formation.

[0187] Some approaches that support 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, methods that are publicly known in the art are well known. In this approach, the CH3 domain of a first heavy chain and the CH3 domain of 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.) A heavy chain containing one engineered CH3 domain is thereby forced 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 use, recombinant human sialidase or its fusion protein and / or The antibody conjugates are preferably combined with a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable," as used herein, means, within the scope of sound medical judgment, a compound that can be administered to a subject with a reasonable degree of benefit / risk without undue toxicity, irritation, allergic response, or other problem or complication. "Human and animal contact lenses" refers to compounds, materials, compositions and / or dosage forms that are suitable for use in contact with human and animal tissues in proportion to the skin surface area.

[0189] The term "pharmaceutically acceptable carrier" as used herein means a compound that is capable of being administered orally and that is available without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers refer to buffers, carriers, and excipients suitable for use in contact with human and animal tissues. Pharmaceutically acceptable carriers include any of the standard pharmaceutical carriers, such as phosphate-buffered saline 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 include 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 (glycine, glutamine, asparagine, arginine, etc.), and the like. 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 and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone). low molecular weight polypeptides; salt-forming counterions (e.g., sodium); preservatives (e.g., benzalkonium chloride) benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (such as glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as Pluronic, PEG , sorbitan esters, polysorbates such as polysorbate 20, polysorbates, triton, tromethamine, lecithin, cholesterol, tyloxapal etc.); stability enhancers (such as sucrose or sorbitol); isotonicity enhancers (such as alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, sorbitol, etc.) delivery vehicle; diluent; excipient and / or pharmaceutical adjuvant (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. Sustained-release preparations may comprise, for example, porous polymeric microparticles or semipermeable polymer matrices in the form of shaped articles, such as films or microcapsules. Sustained-release matrices may comprise polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma-ethyl-L-glutamic acid, poly(2-hydroxyethyl-inethacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. Sustained release compositions may also include liposomes, which can 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. Carriers can be, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and and liquid polyethylene glycol), and their suitable It may be a mixture.

[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. It can be magnesium (MgCl2).

[0196] In some embodiments, the stabilizer is present in an amount of about 0.05 mM to about 5 mM. 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 component, e.g., 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 dosage may be increased beyond the upper limit. Alternatively, the initial dosage may be less than optimal, and the daily dosage may be gradually increased during the course of treatment. Human dosages range from, e.g., 0.5 mg / kg to 20 mg / kg. The frequency of administration can be optimized in a conventional Phase I dose-escalation study designed to be performed in a single dose. , route of administration, dosage, recombinant human sialidase or its fusion protein and / or The administration frequency may vary depending on factors such as the serum half-life of the antibody conjugate and the disease being treated. Exemplary administration frequencies are once daily, once weekly, and once every two weeks. A preferred route of administration is parenteral, e.g., intravenous infusion. In some embodiments, the recombinant human sialidase or its fusion protein and / or antibody conjugate is lyophilized and then At the time of administration, the drug is reconstituted in buffered saline.

[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 of treating cancer in a subject, comprising administering to the subject a recombinant human sialidase or a fusion protein and / or antibody conjugate thereof, such as those disclosed herein. The present invention includes administering an effective amount of a recombinant human sialidase, fusion protein, or antibody conjugate, alone or in combination with another therapeutic agent, to treat cancer in a subject. The term "effective amount," as used herein, refers to the amount of an active agent (e.g., a recombinant human sialidase or fusion protein thereof 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 may vary depending on the particular formulation or route of administration. is not intended to be limited to.

[0201] As used herein, "treat," "treating," and "Treatment" means the treatment of a disease in a subject, e.g., a human, by (a) inhibiting the disease, i.e., halting its progression; and (b) relieving the disease. As used herein, the terms "subject" and "patient" refer to an organism that is 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 myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), such as transformed CLL, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FLM), and leukemia. Examples of solid tumors include malignancies such as sarcomas, adenocarcinomas, and carcinomas of various organ systems, e.g., head and neck (including pharynx), thyroid, lung (small cell or non-small cell lung cancer (NSCLC)), breast, lymphoma, lymphoma, Hodgkin's disease, malignant lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, or Richter's syndrome (Richter's transformation). system, gastrointestinal (e.g., oral cavity, esophagus, stomach, liver, pancreas, small intestine, colon and rectum, anal canal), reproductive and genitourinary tract (e.g., kidneys, urothelium, bladder, ovaries, uterus, cervix, endometrium, prostate These include those affecting the glands, testes), the CNS (e.g., neurons or glial cells, such as neuroblastoma or glioma), or the skin (e.g., melanoma).

[0203] In some embodiments, the cancer is an epithelial cancer, such as 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, adenocarcinoma of the adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basoid carcinoma, basosquamous cell carcinoma, bronchioloalveolar carcinoma, bronchioalveolar carcinoma, bronchiolar ... Bronchogenic carcinoma, bronchogenic carcinoma, cerebrocarcinoma, cholangiocarcinoma, choriocarcinoma, colloid carcinoma, comedocarcinoma, corpus carcinoma, cribriform carcinoma carcinoma, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, ductal carcinoma, carcinoma durum, embryonic Stage cancer, epidermoid carcinoma, epidermoid carcinoma, epithelial adenoid carcinoma, exophytic carcinoma, extraulceral carcinoma, Fibrous carcinoma (carcinoma fibrosum), gelatinous carcinoma (gelatiniforni carcinoma), gelatinous gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, adenocarcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma , hepatocellular carcinoma, Hürthle cell carcinoma, vitreous carcinoma (hyaline carcinoma), adrenal gland-like carcinoma (hypemephroid carcinoma) carcinoma, infantile embryonal carcinoma, intraepithelial carcinoma, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Lutschitzky cell carcinoma, large cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, mucous cell carcinoma mucocellulare), mucinous epidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, nasopharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma carcinoma), prickle cell carcinoma, Pultaceous carcinoma, renal cell carcinoma of the kidney, reserve cell carcinoma, carcinoma sarcomatoides, Schneiderian carcinoma, scleroderma Cancer, scrotal carcinoma (carcinoma scroti), signet ring cell carcinoma, carcinoma simplex, small cell cancer, solanoid carcinoma, spheroidal cell carcinoma , spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, capillary Carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma These include verrucous carcinoma, verrucous carcinoma, and choriocarcinoma (carcinoma villosum).

[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 may be used alone or in combination with other therapeutic agents and / or modalities. As used herein, the term "administered in combination" means that two (or more) different therapies are administered to alleviate the suffering of a subject with a disorder. It is understood to mean that the treatments are delivered to a subject so that the effects of the treatments on the patient overlap at points in time during the course of pain. In some embodiments, the delivery of one treatment is still occurring when the delivery of a second treatment 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 is terminated before the delivery of the other treatment is initiated. In some embodiments in either case, the treatments are more effective due to their combined administration, e.g., the second treatment is more effective, e.g., the same effect is seen with less of the second treatment, or the second treatment is more effective than if the second treatment were administered in the absence of the first treatment. reduces symptoms to a greater extent than would be expected with the first treatment, or In some embodiments, delivery results in a reduction in symptoms or other parameters associated with the disorder that is greater than that observed with one treatment delivered in the absence of the other treatment. The effects of the two treatments may be partially additive or may be overall The delivery may be additive or greater than additive. The effect of the treatment may be such that it is still detectable when the second treatment is delivered.

[0207] In certain embodiments, the methods or compositions described herein may further comprise administering to a patient a therapeutically effective amount of one or more additional therapeutic agents. In some embodiments, the additional treatment may be administered in combination with a chemotherapy, such as surgery, radiation therapy, or 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 therapy, 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, Fusion protein, cytokine, cytokine receptor, bronchodialator In some embodiments, the anti-inflammatory agent may include a statin, an anti-inflammatory agent (e.g., methotrexate), or an NSAID. In this case, the additional treatment may involve a combination of therapeutic agents from different classes.

[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. inhibits or suppresses T cell activity at the appropriate time to prevent an overactive immune response. PD-1 is a receptor present on the surface of T cells that otherwise acts as a checkpoint in the immune system to regulate T cell activity. However, cancer cells can block this checkpoint by expressing ligands, such as PD-L1, that interact 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, for example, in U.S. Patent Nos. 8,952,136, 8,779,105, 8,008,449, 8,741,295, 9,205,148, 9,181,342, 9,102,728, 9,102,727, 8,952,136, 8,927,697, 8,900,587, 8,735,553, and 7,488,802. Examples of suitable anti-PD-1 antibodies include 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. Examples of anti-PD-L1 antibodies include atezolizumab (Tecentriq (registered trademark), Genentech), duplex (Duplex), and PD-L1 antibodies. These include Valmab (AstraZeneca), MEDI4736, avelumab, and BMS 936559 (Bristol Myers Squibb Co.).

[0210] In some embodiments, the methods or compositions described herein are administered in combination with a CTLA-4 inhibitor. The CTLA-4 pathway involves the interaction of CTLA-4 on T cells with its ligands (e.g., also known as CD80, B7-1, and CD86) on the surface of antigen-presenting cells (but not cancer cells). causes T cell inhibition. Exemplary CTLA-4-based immune checkpoint inhibition methods include: It is described in U.S. Patent Nos. 5,811,097, 5,855,887, and 6,051,227. Exemplary anti-CTLA-4 antibodies are described in U.S. Patent Nos. 6,984,720, 6,682,736, 7,311,910, 7,307,064, 7,109,003, 7,132,281, 6,207,156, 7,807,797, 7,824,679, 8,143,379, 8,263,073, 8,318,916, 8,017,114, 8,784,815, and 8,883,984; International (PCT) 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, the methods or compositions described herein comprise: (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, For example, in combination with 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 (indoxycycline), indoximod), epacadostat (INCB24360), naboximide (known as Modo (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, platinating agents, inhibitors of nucleic acid synthesis, histone deacetylase inhibitors (HDAC inhibitors, e.g., vorinostat (SAHA, MK0683), entinostat (MS-275), pancreatic cancer inhibitors, and the like. Binostat (LBH589), trichostatin A (TSA), mocetinostat (MGCD0103), belinostat (PXD101), romidepsin (FK228, depsipeptide), DNA methyltransferase enzyme inhibitors, nitrogen mustards, nitrosoureas, ethyleneimines, alkyl sulfur These include fonates, triazenes, folic acid analogs, nucleoside analogs, ribonucleotide reductase inhibitors, vinca alkaloids, taxanes, epothilones, intercalating agents, drugs that can interfere with signal transduction pathways, drugs that promote apoptosis and radiation, 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, diazepam, thiazolinone ... Hydroxyanthracinedione, mitoxantrone, mithramycin, actinomycin D, adriamycin, 1-dehydrotestosterone, glucocorticoids, procaine, Tetracaine, lidocaine, propranolol, puromycin, ricin, or maytansinoid.

[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. Cells, tissues or subjects with sialidase, fusion protein and / or antibody conjugates. In some embodiments, the cell is selected from a dendritic cell and a peripheral blood mononuclear cell (PBMC).

[0215] In some embodiments, the 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 determined by the expression of a sialidase, a fusion protein, or or antibody conjugate. Gene expression 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 tissue that have not been contacted with the antibody conjugate. Gene expression can be measured by any suitable method known in the art, for example, by ELISA, and can be measured by any suitable method known in the art, for example, by ELISA, or ... can be measured by Luminex multiplex assay.

[0216] The present invention also provides a method for promoting immune cell infiltration into a tumor in a subject in need thereof, the method comprising administering to the subject a sialidase, fusion protein and / or antibody conjugate, such as a sialidase disclosed herein. In one embodiment, the immune cells are T cells, e.g., CD4+ and / or CD8+ T cells, e.g., CD69 + CD8 + oh 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 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 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%, at least about 1000%, at least about 1500%, at least about 20 ... Infiltration of immune cells into tumors 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, the method comprising administering to the subject a sialidase, fusion protein and / or antibody conjugate, such as a sialidase disclosed herein. The method includes administering an effective amount of the sialidase, fusion protein, or antibody conjugate to the subject 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 a subject a sialidase, a fusion protein and / or an antibody conjugate, e.g. For example, administering an effective amount of 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. The cells are T cells, eg, CD4+ and / or CD8+ T cells.

[0221] In some embodiments, the number of T cells in the draining lymph nodes in a subject is determined by sialidase at least about 10%, at least about 20%, at least about 50%, or less than the fusion protein or antibody conjugate administered to a similar or otherwise identical subject who did not receive the fusion protein or antibody conjugate. 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%, The T cells in the draining lymph nodes in the subject are increased by at least about 700%, at least about 800%, at least about 900%, or at least about 1,000%. It can be measured by any suitable method, for example, by antibodies.

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

[0223] In some embodiments, the expression of Cd3, Cd4, Cd8, Cd274, Ctla4, Icos, Pdcd1, Lag3, Il6, Il1b, Il2, Ifng, Ifna1, Mx1, Gzmb, Cxcl9, Cxcl12, and / or Ccl5 in a cell, tissue, or subject is 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 Gene expression may be increased by 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1,000%. Gene expression may be measured by any suitable method known in the art, such as ELISA, Luminex multiplex assay, or Nanostring technology.

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

[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, the expression of MHC-II molecules in cells or tissues remains similar or different from cells that were not contacted with the sialidase, fusion protein, and / or antibody conjugate. The gene expression 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 an otherwise identical cell or tissue. Gene expression can be measured by any suitable method known in the art, for example, by ELISA, by Luminex multiplex assay, or by flow cytometry.

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

[0227] In some embodiments, phagocytosis is mediated by sialidase, fusion protein, and / or antibody complexes. at least about 10%, at least about 20%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 100%, at least about 200%, at least about 5 ... about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, Phagocytosis is increased by 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%. Phagocytosis can be measured by any suitable method known in the art.

[0228] The present invention also provides a method of activating dendritic cells (DCs) by combining DCs with sialidase, fusion proteins and / or antibody conjugates, such as those disclosed herein. In some embodiments, the present disclosure relates to a method of activating dendritic cells (DCs) or a population of DCs in a subject, the method comprising contacting the subject with a sialidase, fusion protein, and / or antibody conjugate, e.g., a sialidase effective to remove sialic acid from tumor cells in the subject, the sialidase, fusion protein, and / or antibody conjugate, e.g., a sialidase effective to remove sialic acid from tumor cells in the subject. For example, the method includes administering an amount of a pharmaceutical composition comprising a sialidase, fusion protein, or antibody conjugate disclosed herein, thereby activating DCs or a population of DCs in the subject.

[0229] In some embodiments, activation of DCs or populations of DCs is similar or different from that of DCs that have not been contacted with tumor cells treated with sialidase, fusion protein and / or antibody conjugates. Activation 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 an otherwise identical DC or population of DCs. Activation can be measured by any suitable method known in the art.

[0230] The present invention also provides a method for reducing Siglec-15 binding activity, thereby providing anti-tumor agents in the tumor microenvironment. The present invention provides a method for increasing tumor activity, the method comprising combining T cells with a sialidase, a fusion protein, and and / or antibody conjugates, e.g., sialidases, fusion proteins, In some embodiments, the present disclosure provides a method for reducing Siglec-15 binding activity, thereby increasing anti-tumor activity in the tumor microenvironment of a patient. The method further comprises administering to a subject a sialidase, a fusion protein, and / or The method includes administering an effective amount of a pharmaceutical composition comprising 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. It may be measured by any suitable method known in the art.

[0232] Throughout the description, when compositions are described as having, including, or comprising certain components, or when processes and methods have or include certain steps, When described as consisting of or comprising, 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, the element or component is included in the list. It may be any one of the elements or components described above, or An element may be selected from the group consisting of two or more of the listed elements or components. It should be understood.

[0234] Furthermore, elements and / or features of the compositions or methods described herein may be used in conjunction with the present specification. It should be understood that various combinations, whether express or implicit in the text, may be combined in various ways without departing from the spirit and scope of the invention. For example, when reference is made to a particular compound, the compound may be used in various embodiments of the compositions of the invention and / or methods of the invention, unless otherwise understood from the context. That is, Although the embodiments are described and illustrated herein in a manner that may describe and illustrate a clear and concise application, it is intended and understood that the embodiments may be variously combined or separated without parting from the present teachings and invention(s). For example, it is understood that all features described and illustrated herein may be applicable to all aspects of the invention(s) described and illustrated herein.

[0235] The expression "at least one" is used unless otherwise understood from the context and usage. Each of the items listed after the expression is treated individually and two or more of the items listed are treated differently. It should be understood that the expression "includes" includes combinations of three or more listed items. "And / or" shall have the same meaning unless otherwise understood from the context. It should be understood.

[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. They may be performed simultaneously.

[0239] Any and all examples or exemplary terms herein, e.g., "such as" The use of "including" is merely intended to better describe the invention, No limitation on the scope of the invention is imposed unless claimed, and 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] The pCEP4 mammalian expression vector with an N-terminal 6xHis tag was used in 24-well plates. Sialidase was expressed in a 200 mL transfection of HEK293F human cells. 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 each produced a yield of approximately 0.15 μg / mL. Neu1 and Neu2 were present primarily in dimeric form, respectively. Neu4 had no detectable expression yield as measured by NanoDrop. The bacterial sialidase from Salmonella typhimurium (St-sialidase; SEQ ID NO:30), used as a positive control for expression, gave yields comparable to Neu1 and was present primarily in monomeric form.

[0244] The activity of recombinantly expressed sialidase was assayed by measuring the release of sialic acid from the fluorogenic substrate 4-methylumbelliferyl-N-acetylneuraminic acid (4MU-NeuAc). As shown, Neu1 had no detectable activity above the no-enzyme control, indicating that Neu1 does not exhibit activity unless complexed with β-galactosidase and protective protein / cathepsin A (PPCA). This is consistent with previous reports showing that Neu2 and Neu3 are inactive. Enzyme kinetic assays were performed using Neu2 and Neu3. A fixed concentration of 1 nM of 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 under 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 includes recombinant sialidases containing mutations that increase sialidase expression and / or activity. The construction of a human sialidase is described.

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

[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. The fourth library contained random mutations generated by error-prone PCR. [Table 25] [Table 26] [Table 27]

[0250] The codon usage columns in Tables 11-13 represent the codons used in the design of the library. The degenerate codon codes used, where the first, second and third positions of a given codon encoding an amino acid, are shown in Table 14 and are listed in Mena et al. (2005) PROTEIN ENG DES SEL. 18(12):55 9-61. [Table 28]

[0251] Enrichment of binding and / or thermostability and expression for conformation-specific antibodies The phage display library was screened for sialic acid biotinylated probes after heating for the synthesis of sialic acid biotinylated probes. 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, the desired Neu2 A phage library expressing the variants was generated. Phages 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. 1, 2 per enzyme. Five additional substitutions were generated by error-prone PCR, with an approximate average proportion of 3, 4, and 5 substitutions. A sub-library was created. [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 represent the degenerate codons used in the design of the libraries. 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] Enrichment of binding and / or thermostability and expression for conformation-specific antibodies The yeast display library was screened for sialic acid biotinylated probes after heating to detect the sialic acid biotinylated probes. An exemplary yeast display screening procedure is shown in Figure 6. First, 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 probed 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 in pCEP4 mammalian expression vectors. The present vector was used to express a secreted protein with a C-terminal human Fc tag in Expi293F cells. 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. The activity was assayed.

[0256] The expression and activity levels for the mutant sialidases are shown in Table 25. In Table 25, the enzyme activity is ranked as follows: "+++" indicates >2-fold higher activity than wild-type Neu2, "+++" indicates activity equivalent to wild-type Neu2, and "+++" indicates activity equivalent to wild-type Neu2. Expression is shown as "++", indicating lower activity than wild-type Neu2, "+", indicating lower activity than wild-type Neu2, or "-", indicating no detectable activity; "++++", indicating >15-fold higher expression than wild-type Neu2. "+++" indicates expression >6-fold higher than wild-type Neu2; "++" indicates expression 2-5-fold higher than wild-type Neu2; Expression is indicated as "+" indicating expression equivalent to live-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 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. Regarding 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 after Protein A purification and a monomer content of 7% as determined by SEC, 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 the fluorogenic substrate 4MU-NeuAc at concentrations 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, and the K M was determined to be 230 μM.

[0259] Taken together, these results demonstrate that mutations identified by the rational design, phage display, and / or yeast display approaches described herein are sialida These results demonstrate that the stability and / or expression of the enzyme can be increased.

[0260] Example 3 This example includes antibody-sialidase genetic fusion proteins and mutant human sialidases. Construction of antibody-sialidase conjugates (ASCs) containing fusion proteins with sialidase and expression are described.

[0261] The configurations for four exemplary types of 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 in the Janus ASC The polypeptides contain 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). A third type of ASC, termed "Lobster," contains two Fc domain polypeptides, each of which is a nucleotide sequence. The ASCs comprise a single antibody arm (having one heavy chain and one light chain), with the sialidase fused at the N-terminus of the Fc and the scFv fused at the C-terminus of the Fc (Figure 11C). The fourth type of ASC, termed "Bunk," comprises one antibody arm (having one heavy chain and one light chain), with the scFv fused at the C-terminus of one arm of the Fc, and one sialidase-Fc fusion with the sialidase fused at the N-terminus of the other arm of the Fc. Each Fc domain polypeptide in the Bunk 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 11D).

[0262] Janus ASC and trastuzumab containing the Neu2 variants described in Example 2 were generated and tested for activity and expression. As described above, expression was assayed using ForteBio Octet with an anti-human Fc sensor and Western blot, and enzyme activity was assayed using the fluorogenic substrate 4MU-NeuAc. Expression and activity levels for Janus ASC are shown in Table 26. In Table 26, enzyme activity is graded as follows: "+++", indicating >2-fold higher activity than wild-type Neu2; "++" indicates activity equivalent to wild-type Neu2, "+" indicates activity lower than wild-type Neu2, or "+" indicates activity lower than wild-type Neu2. Expression is indicated as "-" showing no detectable activity, "++++" indicating expression >15-fold higher than wild-type Neu2, "+++" indicating expression >6-fold higher than wild-type Neu2, and "+++" indicating expression 2-5-fold higher than wild-type Neu2. "++" indicates high expression, "+" indicates expression equivalent to wild-type Neu2, or "detectable expression." It is shown as "-" when no [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 ForteBio Octet and Western blot with an anti-human Fc sensor, and enzyme activity was assayed using the fluorogenic substrate 4MU-NeuAc, as described above. The expression and activity levels for Janus ASC are shown in Table 27. In Table 27, the enzyme activity The activity was graded 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. The 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 includes recombinant sialidases containing mutations that increase sialidase expression and / or activity. The construction of a human sialidase is described.

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

[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 containing substitutions of amino acid residues in the β-turn with prolines (e.g., D80P, R189P, and / or H239P substitutions). Substitutions with may stabilize proteins, 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 "++", indicating activity equivalent to wild-type Neu2, "+", indicating activity less than wild-type Neu2, or "-", indicating no detectable activity, and expression is indicated as "++++++", indicating >40-fold higher expression than wild-type Neu2, "++++", indicating >15-fold higher expression than wild-type Neu2, "+++" indicates expression >6-fold higher than wild-type Neu2, and "+++" indicates expression 2-5-fold higher than wild-type Neu2. Denoted as "++", "+" indicating expression equivalent to wild-type Neu2, or "-" indicating no detectable expression. [Table 42]

[0270] To confirm these results, we used Neu2-M173 (nucleotide sequence: nucleotide sequence 1) with a C-terminal human Fc tag. expressing a vector encoding the amino acid sequence SEQ ID NO:159 (encoded by SEQ ID NO:181); Purification was performed 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 was purified with a yield of 120 mg / liter and a monomer content of 90%. Had the amount.

[0271] The enzymatic kinetics of Neu2-M173-Fc was investigated using the fluorogenic substrate 4-methylumbelliferyl-N as described above. Neu2-M173-Fc was assayed by measuring the release of sialic acid from 4-acetylneuraminic acid (4MU-NeuAc). A fixed concentration of 2 μg / well of enzyme was incubated with the fluorogenic substrate 4MU-NeuAc at concentrations 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] Further mutant Neu2 sialic acid mutants containing rationally designed substitutions at positions S301 and / or W302 Mutations at S301 and / or W302 may affect the interaction with adjacent amino acid residues and / or substrates.

[0273] The expression and activity levels for the mutant sialidases are shown in Table 29. In Table 29, enzyme activity is indicated as "++", indicating activity equivalent to wild-type Neu2, "+", indicating activity less than wild-type Neu2, or "-", indicating no detectable activity; expression was >40% higher than wild-type Neu2. "+++++" indicates 2-fold higher expression; "++++" indicates >15-fold higher expression than wild-type Neu2; "+++" indicates >6-fold higher expression than wild-type Neu2; "++" indicates 2- to 5-fold higher expression than wild-type Neu2; Denoted as "+" indicating expression equivalent to wild-type Neu2 or "-" indicating 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 synthesized at large scale (10 L). Expressed as an Fc-fused single chain protein using a CHO cell expression system for high cell density production 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 truncated (40 kDa and 30 kDa, approximately 50%) fractions. However, under non-reducing conditions, the truncated fractions were not present. No fragmentation occurred, and the protein remained single chain (Figure 24). When expressed in sialidase (with 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 Neu2-M106 was similar to that of the uncleaved Neu2-M106.

[0276] It was hypothesized that the 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 described above, which results in cleavage) and uncleaved Neu2-M106 (prepared using the small-scale production described above, which does not result in cleavage) were incubated with trypsin and analyzed by SDS-PAGE under reducing conditions (Figure 25). Briefly, trypsin (5 μL, 0.005% solution in PBS) and Neu2-M106 (25 μL, PBS pH 8.0) were incubated for 1 hour at 4°C. Trypsin digestion reactions were performed by incubation of Neu2-M106 (0.25 mg / mL in PBS) on ice for 5 min. The reactions were stopped by adding LDS gel loading buffer (5 μL) and run on a reducing SDS-PAGE gel to observe trypsin-mediated cleavage. SDS-PAGE analysis revealed uncleaved Neu2-M106 and trypsin-mediated cleavage. Incubation with syn resulted in a cleavage pattern identical to that of cleaved Neu2-M106. Furthermore, incubation of cleaved Neu2-M106 with trypsin resulted in This resulted in an increase in the intensity of the bands corresponding to the cleavage products.

[0277] Neu2-M106 was incubated with trypsin in the presence of various protease inhibitors. Briefly, trypsin (0.005%) and Neu2-M106 (0.5 mg / mL) with protease inhibitors were added to ice. Trypsin digestion reactions were performed by incubation for 5 minutes on a 5% COOH filter. Reactions were stopped by adding LDS gel loading buffer and run on a reducing SDS-PAGE gel to observe trypsin-mediated cleavage. 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 can be seen in Figure 26, protease inhibitors reduced the extent of trypsin cleavage.

[0278] Taken together, these results demonstrate that cleavage of Neu2-M106 after large-scale production is achieved by trypsin or for members 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, the 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 Protein A. The protein was purified using 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. I did.

[0281] First, R243 was mutated to amino acids of different polarities / charges, e.g., 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 substitution combinations tested are shown in Figure 27. All mutations were tested in the Neu2-M106 background (i.e., M1D, V6Y, P62G, A93E, I1 containing substitutions 87K and C332A).

[0283] Although most of the mutant sialidases shown in Figure 27 were well expressed, only two of the mutant sialidases (containing either the V244I or A242C mutation) were active. The A242C mutation resulted in more than 10-fold improved trypsin resistance and slightly lower activity (both relative to Neu2-M106). However, having an unpaired cysteine ​​can be a potential liability. Therefore, 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 increase 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 showed that replacing A242 with an aromatic amino acid resulted in the formation of L260 and V265 (the amino acid residues of A242). provide additional hydrophobic or stacking interactions to nearby nonpolar amino acids It was shown that L260 and V265 could be mutated to phenylalanine. Therefore, L260 and V265 were also mutated to phenylalanine. These mutations, along with several other rationally designed mutations that could confer extra 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 (10-fold more resistant than Neu2-M106). Highly improved trypsin resistance).

[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 "++", indicating activity equivalent to wild-type Neu2, "+", indicating activity less than wild-type Neu2, or "-", indicating no detectable activity, and expression is indicated as "++++++", indicating >40-fold higher expression than wild-type Neu2, "-", indicating >15-fold higher expression than wild-type Neu2, or "-", indicating no detectable activity. "++++" indicates expression >6-fold higher than wild-type Neu2, "+++" indicates expression 2-5-fold higher than wild-type Neu2 "++" indicates expression that is minimal, "+" indicates expression equivalent to wild-type Neu2, or 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 includes antibody-sialidase genetic fusion with a mutated human sialidase. and antibody-sialidase conjugates (ASCs) containing the fusion proteins. Construction and expression are described.

[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 (nucleotide sequence SEQ ID NO: 1) was synthesized using Neu2 with M1D, V6Y, P62G, A93E, I187K, and C332A substitutions and trastuzumab. a first polypeptide chain having the amino acid sequence SEQ ID NO:66 encoded by SEQ ID NO:86; A second antibody having the amino acid sequence SEQ ID NO:67 encoded by the 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 expressed and characterized for purity using SDS-PAGE and for enzymatic activity using 4MU-NeuAc as described below. I did it.

[0289] Janus-trastuzumab was expressed in a 1 L transfection of Expi293 human cells using the pCEP4 mammalian expression vector. Janus-trastuzumab was expressed in a 1 L transfection of Expi293 human cells using Protein A, followed by Purification was performed using 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 using the fluorogenic substrate 4-methylumbelliferin. The assay was performed by measuring the release of sialic acid from ferryl-N-acetylneuraminic acid (4MU-NeuAc). Specifically, an enzyme kinetic assay was performed using a fixed concentration of enzyme at 2 μg / well. This 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 reacts enzymatically with a Km of 0.48 mM. It was active in

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

[0292] Example 7 This example includes the preparation of an antibody-sialidase conjugate (ASC) containing bacterial sialidase. In vivo administration is described.

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

[0294] These ASCs were then cultured in mice injected with a mouse breast cancer cell line (EMT6-hHer2 cells) expressing human Her2. Female BALB / c mice, 6-8 weeks of age, were inoculated with EMT6-Her2 tumor cells (5x10) in 0.1 ml of PBS for tumor development. 5 ) was inoculated subcutaneously into the right lower flank. 3 , average about 75~100mm 3 When the mice reached 100 mg / kg, they were randomly divided into 8 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, where two out of eight animals had a complete response (Figure 16B), and Janus, where three out of eight animals had a complete response (Figure 16C). This is in contrast to the control group (Fig. 16C) and Lobster (Fig. 16D), which showed 2 out of 8 animals had a complete response.

[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, the 8 Compared to Janus treatment alone, which resulted in a complete response in 3 of 10 animals (Fig. 16C), NK depletion resulted in a complete response in 3 of 10 animals. Macrophage depletion also reduced the number of complete responses to 1 in 8 animals (Figure 17A). Macrophage depletion also reduced the number of complete responses to 1 in 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 effect of Janus on the immune response in mice treated with vehicle, Janus alone, trastuzumab, or trastuzumab. The mean tumor volumes for Janus alone and with NK, macrophage, and CD8 T cell depletion are shown. 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 includes an antibody-sialidase conjugate (ASC) with bacterial sialidase. In vivo administration of is described.

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

[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 to Once the mice reached the target age, they were randomly assigned to six groups. Treatment groups were assigned according to the dosing schedule after randomization. The antibodies are listed in Table 32. Anti-mouse PD1 was obtained from BioXcell (RMP1-14, catalog no. No. 665418F1). [Table 46]

[0300] The results for Groups 1 to 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. In contrast, 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 caused tumor growth in cured mice. In naive mice, EMT6-Her2 cells progressed to tumors as expected. The results suggest that the antibody-sialidase conjugates 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] Groups 1, 5, and 6 (vehicle, anti-mouse PD1, and anti-mouse PD1 combined with Janus) The results are shown in Figures 19A and 19B. Anti-mouse PD1 had good activity, inhibiting 4 of 6 mice. Janus alone showed complete regression in 3 of 6 mice (see Figure 18A). (Control), whereas the combination of anti-mouse PD1 and Janus resulted in complete regression of tumor growth in all six mice (Figure 19B). There was no weight loss in any of the animals given this combination.

[0303] Example 9 This example includes an antibody-sialidase conjugate (ASC) with bacterial sialidase. In vivo administration of is described.

[0304] Janus ASC (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:66 encoded by the nucleotide sequence SEQ ID NO:87) containing Salmonella typhimurium sialidase (St-sialidase) and trastuzumab a second polypeptide chain having the sequence SEQ ID NO:67, and a second polypeptide chain encoded by the nucleotide sequence SEQ ID NO:91; The third polypeptide chain (including the third polypeptide chain having the amino acid sequence SEQ ID NO:90) was prepared as described in this example. The cellulose acylate was prepared and tested in the following manner.

[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 When the mice reached 100 mg / kg, they were randomly assigned to one of three treatment groups. The dosing schedule after randomization is listed in Table 33. Anti-mouse PD1 (RMP1-14, Cat. No. 665418F1) obtained from BioXcell and anti-mouse CTLA4 (9D9, Catalog number BE0164) was used in combination. [Table 47]

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

[0307] Example 10 This example includes the illustration of an antibody-sialidase conjugate (ASC) containing human sialidase. In vivo administration is described.

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

[0309] A mouse syngeneic tumor model was injected with a mouse breast cancer cell line (EMT6-HER2) stably expressing human HER2. Janus trastuzumab was compared to an isotype control antibody in female BALB / c mice. Mice, 6–8 weeks of age, were injected with EMT6-HER2 tumor cells (5 × 10 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 study, significant tumor growth delay was observed after treatment with Janus trastuzumab. It was.

[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; U.S. Provisional Patent Application No. 62 / 956,957, filed July 33, 2020; International (PCT) Patent Application No. PCT / US20 / 40815, filed July 3, 2019; U.S. Provisional Patent Application No. 62 / 870,348, filed January 3, 2020; U.S. Provisional Patent Application No. 62 / 956,977, filed July 3, 2020 International (PCT) patent application number PCT / US20 / 40814 filed on July 3, 2019; U.S. provisional patent application number PCT / US20 / 40814 filed on July 3, 2019; The entire disclosure of each of U.S. Patent Application No. 62 / 870,341, 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 is 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 in 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 position 5 of wild-type human Neu2 was replaced with histidine (P5H). ; (b) The lysine residue at position 9 of wild-type human Neu2 was replaced with aspartic acid (K9D). Re; (c) The lysine residue at position 44 of wild-type human Neu2 is replaced by arginine (K44R) or arginine (K44R). substituted with 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 was replaced with methionine (L54M). Re; (f) The proline residue at position 62 of wild-type human Neu2 was replaced by asparagine (P62N), substituted with paragic acid (P62D), histidine (P62H), glutamic acid (P62E), glycine (P62G), serine (P62S), or threonine (P62T); (g) The glutamine residue at the position corresponding to position 69 of wild-type human Neu2 is replaced with a histidine (Q69H). be; (h) The arginine residue at position 78 of wild-type human Neu2 was replaced with a lysine (R78K). ; (i) The aspartic acid residue at position 80 of wild-type human Neu2 was replaced with proline (D80P). Converted; (j) The alanine residue at position 93 of wild-type human Neu2 is replaced by glutamic acid (A93E) or is substituted with lysine (A93K); (k) The glycine residue at position 107 of wild-type human Neu2 is an aspartic acid (G107D). Replaced; (l) The glutamine residue at position 108 of wild-type human Neu2 was replaced with a histidine (Q108H). Converted; (m) The glutamine residue at position 112 of wild-type human Neu2 is replaced by arginine (Q112R). substituted with lysine (Q112K); (n) The cysteine ​​residue at position 125 of wild-type human Neu2 is replaced with leucine (C125L). be; (o) The glutamine residue at position 126 of wild-type human Neu2 was replaced by leucine (Q126L), substituted with thiaminic acid (Q126E), phenylalanine (Q126F), histidine (Q126H), isoleucine (Q126I) or tyrosine (Q126Y); (p) The alanine residue at position 150 of wild-type human Neu2 was replaced with valine (A150V). ; (q) The cysteine ​​residue at position 164 of wild-type human Neu2 is replaced with glycine (C164G). be; (r) The arginine residue at position 170 of wild-type human Neu2 is replaced with proline (R170P). be; (s) The alanine residue at position 171 of wild-type human Neu2 was replaced with glycine (A171G). Re; (t) The glutamine residue at position 188 of wild-type human Neu2 is replaced with proline (Q188P). be; (u) The arginine residue at position 189 of wild-type human Neu2 is replaced with proline (R189P). be; (v) The alanine residue at position 213 of wild-type human Neu2 was replaced by cysteine ​​(A213C), aspartate (A213C). substituted with paragine (A213N), serine (A213S), or threonine (A213T); (w) The leucine residue at position 217 of wild-type human Neu2 is replaced by alanine (L217A) or substituted with valine (L217V); (x) The threonine residue at the position corresponding to position 249 of wild-type human Neu2 is replaced with alanine (T249A). be; (y) The aspartic acid residue at position 251 of wild-type human Neu2 is a glycine (D251G). Replaced; (z) The glutamic acid residue at position 225 of wild-type human Neu2 was replaced with a proline (E225P). Converted; (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 residues at position 242 of wild-type human Neu2 are cysteine ​​(A242C), phenylalanine (A242F), glycine (A242G), histidine (A242H), isoleucine (A242I), lysine (A242K), leucine (A242L), methionine (A242M), asparagine (A242N), glutamine (A242Q), arginine (A242R), serine (A242S), valine (A242V), and tryptophan (A242W). or substituted with 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 residues at position 301 of wild-type human Neu2 are 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), and valine (S301V). , substituted with 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 position 5 of wild-type human Neu2 is replaced with histidine (P5H); (b) the lysine residue at position 9 of wild-type human Neu2 is replaced with aspartic acid (K9D). Re; (c) The lysine residue at position 44 of wild-type human Neu2 is replaced by arginine (K44R) or arginine (K44R). substituted with 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 was replaced with methionine (L54M). Re; (f) The proline residue at position 62 of wild-type human Neu2 was replaced by asparagine (P62N), substituted with paragic acid (P62D), histidine (P62H), glutamic acid (P62E), glycine (P62G), serine (P62S), or threonine (P62T); (g) The glutamine residue at the position corresponding to position 69 of wild-type human Neu2 is replaced with a histidine (Q69H). be; (h) the arginine residue at the position corresponding to position 78 of wild-type human Neu2 is substituted with 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 substituted with lysine (A93K); (j) The glycine residue at position 107 of wild-type human Neu2 is an aspartic acid (G107D). Replaced; (k) The glutamine residue at position 108 of wild-type human Neu2 was replaced with a histidine (Q108H). Converted; (l) The glutamine residue at position 112 of wild-type human Neu2 is replaced by arginine (Q112R). substituted with lysine (Q112K); (m) The cysteine ​​residue at position 125 of wild-type human Neu2 is replaced with leucine (C125L). be; (n) The glutamine residue at position 126 of wild-type human Neu2 is replaced with leucine (Q126L). be; (o) The alanine residue at position 150 of wild-type human Neu2 is substituted with valine (A150V); (p) the cysteine ​​residue at position 164 of wild-type human Neu2 is substituted with glycine (C164G). be; (q) The alanine residue at position 171 of wild-type human Neu2 was replaced with glycine (A171G). Re; (r) The leucine residue at position 217 of wild-type human Neu2 is replaced by alanine (L217A) or substituted with valine (L217V); (s) The threonine residue at position 249 of wild-type human Neu2 is replaced with alanine (T249A). be; (t) The aspartic acid residue at position 251 of wild-type human Neu2 is a glycine (D251G). Replaced; (u) The glutamine residue at position 270 of wild-type human Neu2 was replaced by alanine (Q270A), and histidine was replaced by hydroxylase (H270A). substituted with thiamine (Q270H), phenylalanine (Q270F), or proline (Q270P); (v) The tryptophan residue at position 292 of wild-type human Neu2 is arginine (W292R). is replaced by; (w) The serine residue at position 301 of wild-type human Neu2 was replaced with arginine (S301R). Re; (x) The tryptophan residue at position 302 of wild-type human Neu2 is replaced with a lysine (W302K). Converted; (y) The valine residue at the position corresponding to position 363 of wild-type human Neu2 was replaced with arginine (V363R). or (z) The leucine residue at position 365 of wild-type human Neu2 was replaced by glutamine (L365Q), histidine (H365Q), and thiamin (H365Q). substituted with leucine (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 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 of the foregoing substitutions Item 7. The sialidase according to any one of Items 1 to 6, including any combination thereof. 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 position 1 of wild-type human Neu2 (M1); (b) substitution of a valine residue at 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: Section 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 position 187 of wild-type human Neu2 is replaced with lysine (I187K). to be; or (d) The cysteine ​​residue at position 332 of wild-type human Neu2 is replaced with alanine (C332A). be; 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: Item 13 Item 13. The sialidase according to any one of Items 1 to 12, which is selected from Neu1, Neu2, Neu3 and Neu4. Section 14 Item 14. The sialidase of Item 13, which is Neu2. Item 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. Section 17 17. The sialidase of item 15 or 16, which is capable of cleaving α2,3 and α2,8 linkages. Section 18 Any one of items 1 to 17, including any one of SEQ ID NOs: 48 to 54, 149, 154, 159, or 191. or the sialidase described. 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 comprising a mutation or combination of mutations set forth in any one of Tables 5-9, 11-13, or 15-30. , optionally further comprising a mutation or combination of mutations set forth in any one of Tables 1-4. Mutant human sialidase. 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 A fusion protein comprising a sialidase and an immunoglobulin Fc domain and / or or fusion proteins in which immunoglobulin antigen-binding domains are linked by peptide bonds or amino acid linkers. 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 The fusion protein according to paragraph 24, wherein the immunoglobulin Fc domain is derived from a human IgG1 Fc domain. Protein. Section 26 26. The fusion protein according to any one of items 21 to 25, comprising an immunoglobulin antigen-binding domain. Section 27 An immunoglobulin antigen-binding domain is bound to a second immunoglobulin antigen-binding domain 27. The fusion protein of claim 26, which produces 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. Item 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 chai...

Claims

[Claim 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) a 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 of wild-type human Neu2 (L365); or any combination of the above substitutions 1. A recombinant mutant human sialidase enzyme comprising:

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