Recombinant human sialidases, sialidase fusion proteins, and methods of using the same
The invention relates generally to recombinant human sialidases and recombinant sialidase fusion proteins, wherein the sialidase optionally contains one or more mutations compared to wild-type human sialidase, e.g., a substitution, deletion, or addition of at least one amino acid. The invention also provides antibody conjugates including a sialidase and an antibody or a portion thereof. The invention further relates to methods of using the sialidase fusion proteins or antibody conjugates for treating cancer.
1 . A recombinant mutant human sialidase enzyme comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1; a substitution of the methionine residue at position 1 of SEQ ID NO: 1; and sialidase enzymatic activity.
2 . The recombinant mutant human sialidase enzyme of claim 1 , wherein the methionine residue at position 1 is substituted by arginine (M1R), histidine (M1H), lysine (M1K), aspartic acid (M1D), threonine (M1T), asparagine (M1N), glutamine (M1Q), glycine (M1G), alanine (M1A), valine (M1V), leucine (M1L), phenylalanine (M1F), or tyrosine (M1Y).
3 . The recombinant mutant human sialidase enzyme of claim 2 , wherein the methionine residue at position 1 is substituted by histidine (M1H).
4 . The recombinant mutant human sialidase enzyme of claim 2 , wherein the methionine residue at position 1 is substituted by leucine (M1L).
5 . The recombinant mutant human sialidase enzyme of claim 2 , wherein the methionine residue at position 1 is substituted by phenylalanine (M1F).
6 . The recombinant mutant human sialidase enzyme of claim 2 , wherein the methionine residue at position 1 is substituted by asparagine (M1N).
7 . The recombinant mutant human sialidase enzyme of claim 2 , wherein the methionine residue at position 1 is substituted by alanine (M1A).
8 . The recombinant mutant human sialidase enzyme of claim 2 , wherein the methionine residue at position 1 is substituted by threonine (M1T).
9 . The recombinant mutant human sialidase enzyme of claim 1 , further comprising one or more of (a) a substitution of the valine residue at position 6 of SEQ ID NO: 1; (b) a substitution of the isoleucine residue at position 187 of SEQ ID NO: 1 with a lysine (I187K); and (c) a substitution of the cysteine residue at position 332 of SEQ ID NO: 1.
10 . The recombinant mutant human sialidase enzyme of claim 9 , wherein the valine residue at position 6 is substituted by tyrosine (V6Y) and/or the cysteine residue at position 332 is substituted by alanine (C332A).
11 . A fusion protein comprising:
(a) the recombinant mutant human sialidase enzyme of claim 1 ; and
(b) an immunoglobulin Fc domain and/or an immunoglobulin antigen-binding domain;
wherein the recombinant mutant human sialidase enzyme and the immunoglobulin Fc domain and/or the immunoglobulin antigen-binding domain are linked by a peptide bond or an amino acid linker.
12 . An antibody conjugate comprising the fusion protein of claim 11 .
13 . The antibody conjugate of claim 12 , wherein 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 the immunoglobulin Fc domain and the recombinant mutant human sialidase enzyme;
wherein the first and second polypeptides are covalently linked together and the second and third polypeptides are linked together, and wherein the first polypeptide and the second polypeptide together define the immunoglobulin antigen-binding domain.
14 . An isolated nucleic acid comprising a nucleotide sequence encoding the recombinant mutant human sialidase enzyme of claim 1 .
15 . An expression vector comprising the nucleic acid of claim 14 .
16 . A host cell comprising the expression vector of claim 15 .
17 . A pharmaceutical composition comprising the recombinant mutant human sialidase enzyme of claim 1 .
18 . A method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of the recombinant mutant human sialidase enzyme of claim 1 .
19 . A method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of the antibody conjugate of claim 13 .
20 . A method of increasing expression of HLA-DR, CD86, CD83, IFNγ, IL-1b, IL-6, TNFα, IL-17A, IL-2, or IL-6 in a cell or tissue, the method comprising contacting the cell or tissue with an effective amount of the recombinant mutant human sialidase enzyme of claim 1 .