Chalarase fusion molecules and related uses

Abstract

The present invention provides a fusion molecule comprising sialidase, and a bispecific molecule that engages an immune cell (e.g., a T cell or an NK cell) and a target cell underlying a disease. Related polynucleotides, vectors, and host cells are also described herein. The present invention further provides the therapeutic application of sialidase fusions in the treatment of cancer.

Description

Technical Field

[0001] Cross - Reference to Related Applications This patent application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 480,228, filed on January 17, 2023 (currently pending), and U.S. Provisional Patent Application No. 63 / 338,134, filed on May 4, 2022 (currently pending). The entire disclosure of the priority applications is hereby incorporated by reference in its entirety for all purposes.

[0002] Description of Government Support This invention was made with government support under Contract No. AI154138 and AI143884 awarded by the National Institutes of Health. The government has certain rights in this invention.

Background Art

[0003] A central theme in cancer immunotherapy is to activate the patient's own immune system for tumor control. Bispecific T - cell engagers (BiTEs) are off - the - shelf immunotherapeutic agents that recruit endogenous CD8+ and CD4+ T cells to eradicate tumor cells in a major histocompatibility complex (MHC) - independent manner. A BiTE molecule consists of two single - chain variable fragments (scFvs), one targeting a tumor - associated antigen and the other binding to CD3 on T cells. These two scFvs are covalently linked by a small linker peptide. Blinatumomab, which targets the CD19 antigen present on B cells, is the first BiTE approved by the U.S. Food and Drug Administration (FDA) for treating B - cell precursor acute lymphoblastic leukemia (ALL) in patients who still have a detectable level of cancer after chemotherapy.

[0004] However, similar to most T cell-based therapies, the promise of BiTEs for treating solid tumors is mainly hampered by limited penetration into tumor tissue and the immunosuppressive tumor microenvironment, where T cell suppression is regulated by the activity of tumor cells and adjacent stromal myeloid and lymphoid cells. In this unique microenvironment, changes in the cell surface epitopes of tumor cells and immune cells occur as a result of limited nutrient availability and accumulated metabolic waste products, which then alter the interaction between tumor cells and tumor-infiltrating T cells (TILs), ultimately leading to T cell exhaustion and poor tumor control. Therefore, activation approaches targeting the molecular and cellular components of the immunosuppressive tumor microenvironment can transform T cell-based cancer therapies, including those enabled by BiTEs.

Summary of the Invention

Problems to be Solved by the Invention

[0005] In the art, there is a need for means to enhance the effectiveness of immunotherapies based on bispecific immune cell engaging molecules such as BiTEs. The present invention aims to address these and other unmet needs.

Means for Solving the Problems

[0006] In one aspect, the present invention provides a fusion protein comprising (a) a bispecific molecule or bispecific antibody and (b) a sialidase or an enzymatic fragment thereof. The bispecific molecule in the fusion protein comprises two antibody fragments or moieties that bind to an immune cell and an antigen associated with or involved in a disease, respectively. In some embodiments, the bispecific molecule comprises, in tandem, a first scFv that targets an immune cell and a second scFv that targets a disease antigen. In some of these embodiments, the bispecific antibody is a bispecific T cell engager (BiTE), and the first scFv recognizes a T cell-specific molecule. In some of these fusion proteins, the target T cell-specific molecule is CD3. Some of the BiTE molecules used in the BiTE-sialidase fusion proteins of the present invention selectively engage γδ T cells (e.g., Vγ9Vδ2 T cells). In these embodiments, the target T cell-specific molecule is a TCR on the cell surface (e.g., Vγ9Vδ2 TCR). In some other embodiments, the bispecific antibody is a bispecific natural cell engager, and the first scFv recognizes a surface antigen on a natural immune cell. In some of these embodiments, the target natural immune cell is an NK cell or a macrophage. In some of these embodiments, the surface antigen on the natural immune cell is CD16A or NKp44. Some of the sialidase fusion proteins of the present invention target tumors. In these fusions, the second antibody fragment specifically binds to a tumor antigen. For example, some of the fusion proteins of the present invention comprise a bispecific molecule that engages an immune cell with a tumor cell expressing HER2 or PSMA.

[0007] In various embodiments, the sialidase in the fusion protein of the present invention is a human sialidase, a viral sialidase, or a bacterial sialidase. In some embodiments, the fusion protein uses human sialidase NEU1, NEU2, NEU3, NEU4, or an isoform thereof. In some embodiments, the fusion protein includes a bacterial sialidase, such as the sialidase of the human symbiotic bacterium Bifidobacterium longum subsp. infantis. In the fusion protein, the sialidase can be fused to either the C-terminus or the N-terminus of the bispecific molecule. In some embodiments, the sialidase is fused to the bispecific molecule via a GS linker. In some of these embodiments, the GS linker used can include the amino acid sequence (GmS)n, where m is an integer from 1 to 6 and n is an integer from 1 to 10. As a specific example, the linker used can be GGGSGGGS (SEQ ID NO: 2), GGGGSGGGGS (SEQ ID NO: 29), GGGGSGGGGSGGGGS (SEQ ID NO: 30), or GGGGSGGGGSGGGSGGGS (SEQ ID NO: 31). In some of the fusion proteins of the present invention, two antibody fragments or moieties (e.g., scFv) are also linked by a GS linker.

[0008] In some of the fusion proteins of the present invention, the bispecific molecule used includes an amino acid sequence that is at least 95% or 99% identical to the sequence shown in any one of SEQ ID NOs: 6, 10, 12, 14, 31, 32, and 40. In some embodiments, the bispecific molecule includes the amino acid sequence shown in any one of SEQ ID NOs: 6, 10, 12, 14, 31, 32, and 40, or a conservatively modified variant thereof. A portion of the sialidase fusion polypeptide of the present invention includes an amino acid sequence that is at least 95% or 99% identical to the sequence set forth in any one of SEQ ID NOs: 7, 8, 11, 13, 15, 23 - 28, and 41. In some embodiments, the sialidase fusion protein includes the amino acid sequence set forth in any one of SEQ ID NOs: 7, 8, 11, 13, 15, 23 - 28, and 41, or a conservatively modified variant thereof.

[0009] In some other embodiments, the present invention provides a polynucleotide molecule or sequence encoding the sialidase fusion protein or polypeptide described herein. Related vectors and host cells having such polynucleotide sequences are also encompassed by the present invention. In some related embodiments, the present invention provides a pharmaceutical composition containing a therapeutically effective amount of the sialidase fusion protein or coding polynucleotide sequence described herein and a pharmaceutically acceptable carrier. Some of the polynucleotide sequences of the present invention are directed to mRNA. Some of these embodiments of the present invention relate to lipid nanoparticles (LNPs) formulated with one mRNA molecule described herein. Therapeutic combinations or kits containing the sialidase fusion protein or polynucleotide are also provided by the present invention.

[0010] In another aspect, the present invention provides a method of treating or ameliorating the symptoms of a disease or disorder in a subject. The method includes administering to the subject a pharmaceutical composition containing the sialidase fusion polypeptide of the present invention. Some of the methods of the present invention specifically relate to treating tumors.

[0011] A further understanding of the nature and advantages of the present invention can be realized by reference to the remainder of the specification and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0012]

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Mode for Carrying Out the Invention

[0013] I. Overview Immunotherapies based on bispecific molecules that engage immune cells and target cells (e.g., BiTE) to activate a patient's immune system have gained momentum with the recent FDA approval of blinatumomab for treating B-cell malignancies. However, success in targeting solid tumors has been limited. The present invention is based, in part, on studies conducted by the inventors to develop fusion proteins comprising sialidase and a bispecific immune cell engager (e.g., BiTE), which enhance tumor cell sensitivity to bispecific molecule-mediated killing. The sialidase fusion bispecific molecules developed and investigated by the inventors include BiTE, as well as bispecific natural cell engagers such as bispecific killer cell engager (BiKE). As detailed herein, the inventors observed that a BiTE-sialidase fusion molecule specifically removes sialoglycans at the T cell-target tumor cell interface, promoting the lysis of T cell-dependent tumor cells. Enhanced tumor cell lysis was demonstrated to be independent of inhibitory sialoglycan-Siglec signaling but was due to stronger immune synapse formation induced by BiTE. As an example, a BiTE-sialidase fusion protein targeting human epidermal growth factor receptor 2 (Her2) and CD19 was shown to exhibit significantly better efficacy in killing tumor cells both in vitro and in vivo in a xenograft tumor model compared to BiTE alone. Enhanced cytolytic activity was also observed for sialidase-BiTE fusions targeting other tumor antigens, such as PSMA. Further studies conducted by the inventors using a syngeneic mouse model of melanoma demonstrated that the BiTE-sialidase fusion protein has treatment advantages over the parental BiTE.

[0014] In further studies, the inventors observed selective desialylation by sialidase fusion BIKEs targeting CD19 or EGFR. These sialidase fusion BiKEs also showed enhanced cytotoxicity compared to unfused NK cells. In a syngeneic mouse model, the in vivo efficacy of sialidase fusion BIKEs was also demonstrated using an EGFR-targeted BiKE-sialidase fusion protein. These results indicate that the sialidase bispecific molecular fusions described herein (e.g., BiTE-sialidase fusions and BiKE-sialidase fusions) can be used as next-generation bispecific immune cell-engaging molecules for cancer immunotherapy.

[0015] According to these studies, the present invention provides a fusion protein comprising a sialidase conjugated to a bispecific molecule or bispecific antibody that engages an immune cell (e.g., a T cell or NK cell) and a target antigen associated with a disease or disorder (e.g., cancer). Related polynucleotide sequences, expression vectors, and host cells, as well as their therapeutic uses, are also encompassed by the present invention.

[0016] The present invention, unless otherwise specified, can use conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology that are within the skill of those in the art. Such techniques are well described in the literature. (For example, Sambrook et al, ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook et al, ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D.N. Glover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984) Oligonucleotide Synthesis; Mullis et al. U.S. Pat. No. 4,683,195; Hames and Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984) Transcription And Translation; Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press) (1986); Perbal (1984) A Practical Guide To Molecular Cloning; the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Miller and Calos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); Wu et al, eds., Methods In Enzymology, Vols. 154 and 155; Mayer and Walker, eds.(1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London); Weir and Blackwell, eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986); see also Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

[0017] The general principles of antibody engineering are described in Borrebaeck, ed. (1995) Antibody Engineering (2nd ed.; Oxford Univ. Press). The general principles of protein engineering are described in Rickwood et al, eds. (1995) Protein Engineering, A Practical Approach (IRL Press at Oxford Univ. Press, Oxford, Eng.). The general principles of antibodies and antibody-hapten binding are described in Nisonoff (1984) Molecular Immunology (2nd ed.; Sinauer Associates, Sunderland, Mass.); and Steward (1984) Antibodies, Their Structure and Function (Chapman and Hall, New York, N.Y.). In addition, standard methods in immunology that are known in the art and not specifically described can be followed, such as Current Protocols in Immunology, John Wiley & Sons, New York; Stites et al, eds. (1994) Basic and Clinical Immunology (8th ed; Appleton & Lange, Norwalk, Conn.) and Mishell and Shiigi (eds)(1980) Selected Methods in Cellular Immunology (W.H. Freeman and Co., NY).

[0018] Standard references describing the general principles of immunology include Current Protocols in Immunology, John Wiley & Sons, New York; Klein (1982) J., Immunology: The Science of Self-Nonself Discrimination (John Wiley & Sons, NY); Kennett et al, eds. (1980) Monoclonal Antibodies, Hybridoma: A New Dimension in Biological Analyses (Plenum Press, NY); Campbell (1984) “Monoclonal Antibody Technology” in Laboratory Techniques in Biochemistry and Molecular Biology, ed. Burden et al, (Elsevier, Amsterdam); Goldsby et al, eds. (2000) Kuby Immunology (4th ed.; W.H. Freeman & Co.); Roitt et al. (2001) Immunology (6th ed.; London: Mosby); Abbas et al. (2005) Cellular and Molecular Immunology (5th ed.; Elsevier Health Sciences Division); Kontermann and Dubel (2001) Antibody Engineering (Springer Verlag); Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press); Lewin (2003) Genes VIII (Prentice Hall, 2003); Harlow and Lane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Press); Dieffenbach and Dveksler (2003) PCR Primer (Cold Spring Harbor Press).

[0019] II. Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide many common definitions of terms used in this invention to those of ordinary skill in the art: Academic Press Dictionary of Science and Technology, Morris (Ed.), Academic Press (1 st ed., 1992); Oxford Dictionary of Biochemistry and Molecular Biology, Smith et al. (Eds.), Oxford University Press (revised ed., 2000); Encyclopaedic Dictionary of Chemistry, Kumar (Ed.), Anmol Publications Pvt. Ltd. (2002); Dictionary of Microbiology and Molecular Biology, Singleton et al. (Eds.), John Wiley & Sons (3 rd ed., 2002); Dictionary of Chemistry, Hunt (Ed.), Routledge (1 st ed., 1999); Dictionary of Pharmaceutical Medicine, Nahler (Ed.), Springer-Verlag Telos (1994); Dictionary of Organic Chemistry, Kumar and Anandand (Eds.), Anmol Publications Pvt. Ltd. (2002); and A Dictionary of Biology (Oxford Paperback Reference), Martin and Hine (Eds.), Oxford University Press (4 th ed., 2000). Further, the following definitions are provided to assist the reader in the practice of this invention.

[0020] The term "antibody" or "antigen-binding fragment" refers to a (one or more) polypeptide chain(s) that exhibit strong monovalent, divalent or multivalent binding to a given antigen, one or more epitopes. Unless otherwise specified, the antibodies or antigen-binding fragments used in the present invention can have sequences derived from any vertebrate, camel, avian or fish species. They can be generated using any suitable technique, such as hybridoma technology, ribosome display, phage display, gene shuffling libraries, semi-synthetic or fully synthetic libraries or combinations thereof. Unless otherwise specified, the term "antibody" used in the present invention includes intact antibodies, antigen-binding polypeptide fragments and other designer antibodies described hereinafter or well-known in the art (see, for example, Serafini, J Nucl. Med. 34:533-6, 1993).

[0021] An intact "antibody" typically comprises at least two heavy (H) chains (about 50-70 kD) and two light (L) chains (about 25 kD) interconnected by disulfide bonds. The recognized immunoglobulin genes encoding the antibody chains include kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as numerous immunoglobulin variable region genes. The light chains are classified into either kappa or lambda. The heavy chains are classified into gamma, mu, alpha, delta, or epsilon, defining the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively.

[0022] Each heavy chain of an antibody consists of a heavy chain variable region (V H ) and a heavy chain constant region. The heavy chain constant region consists of three domains, C H1 , C H2 and C H3 . Each light chain consists of a light chain variable region (V L ) and a light chain constant region. The light chain constant region consists of one domain C LIt consists of. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors including various cells of the immune system and the first component (Clq) of the classical complement system.

[0023] The V H and V L regions can be further subdivided into hypervariable regions, also called complementarity-determining regions (CDRs), in which more conserved framework regions (FRs) are interspersed. Each V H and V L is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The positions and numbering systems of the CDR regions and FR regions are defined, for example, by Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, U.S. Government Printing Office (1987 and 1991).

[0024] Antibody fragments or antigen-binding fragments contain the antigen-binding portions of intact antibodies that retain the ability to bind to cognate antigens. Examples of such antibody fragments include: (i) Fab fragments, monovalent fragments consisting of V L , V H , C L and C H1 domains; (ii) F(ab’) 2 fragments, divalent fragments containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) Fd fragments consisting of V H domains and C H1 domains; (iv) Fv fragments consisting of the V L domains and V H domains of a single arm of an intact antibody; (v) disulfide-stabilized Fv (dsFv) having an interchain disulfide bond engineered between structurally conserved framework regions; (vi) V HSingle domain antibodies (dAbs) consisting of domains (see, e.g., Ward et al., Nature 341:544-546, 1989); and (vii) isolated complementarity determining regions (CDRs).

[0025] In some preferred embodiments, the antibody used to practice the invention is a single-chain antibody. The term "single-chain antibody" generally refers to a polypeptide linkage of V H domains and V L domains that are linked via a spacer peptide and may include additional domains or amino acid sequences at the amino and / or carboxyl termini. For example, a single-chain antibody may include a tether segment for linking to a coding polynucleotide. As an example, a single-chain variable region fragment (scFv) is a single-chain antibody. The V L domains and V H domains of an Fv fragment encoded by separate genes, in comparison, an scFv has two domains linked by a synthetic linker (e.g., via recombinant methods). This allows the V L regions and V H regions to be made as a single protein chain that pairs to form a monovalent molecule.

[0026] Antibodies or antigen-binding fragments for practicing the present invention can be produced by enzymatic or chemical modification of intact antibodies, or can be de novo synthesized using recombinant DNA methods, or can be identified using phage display libraries. All methods for making these antibodies or antigen-binding molecules are well known in the art. For example, single-chain antibodies can be identified using phage display libraries or ribosome display libraries, gene shuffling libraries (see, e.g., McCafferty et al., Nature 348:552-554, 1990; and U.S. Patent No. 4,946,778). In particular, scFv antibodies can be obtained using, for example, the methods described in Bird et al., Science 242:423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988. Fv antibody fragments can be generated as described in Skerra and Pluckthun, Science 240:1038-41, 1988. Disulfide-stabilized Fv fragments (dsFv) can be prepared using, for example, the methods described in Reiter et al., Int. J. Cancer 67:113-23, 1996. Similarly, single-domain antibodies (dAbs) can be produced by various methods described, for example, in Ward et al., Nature 341:544-546, 1989; and Cai and Garen, Proc. Natl. Acad. Sci. USA 93:6280-85, 1996. Camelid single-domain antibodies can be made using methods well known in the art, such as those described in Dumoulin et al., Nature Struct. Biol. 11:500-515, 2002; Ghahroudi et al., FEBS Letters 414:521-526, 1997; and Bond et al., J Mol Biol. 332:643-55, 2003. Other types of antigen-binding fragments (e.g., Fab, F(ab’) 2Alternatively, the (scFv or Fd fragment) can also be easily prepared by commonly practiced immunological methods. See, for example, Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1998. In some preferred embodiments, the scFv fragment used in the sialidase fusion of the present invention can be produced by recombinant expression.

[0027] Bispecific T cell engager antibodies, bispecific T cell engager molecules, or simply bispecific T cell engagers (BiTEs) are used interchangeably herein and refer to a group of bispecific antibodies that contain two single-chain variable fragments (scFvs) in tandem. One of the scFvs has binding specificity for the T cell receptor (TCR) complex, and the other recognizes an antigen on the target cell (e.g., a cell surface marker associated with or involved in a disease).

[0028] A "fusion" protein or polypeptide refers to a polypeptide composed of at least two polypeptides and a linker sequence or linker that operably links the two polypeptides into one continuous polypeptide. The two polypeptides linked in the fusion polypeptide typically originate from two independent sources, and thus, the fusion polypeptide contains two linked polypeptides that are not normally linked in nature.

[0029] "Linkage" refers to means for operably or functionally linking two biomolecules (e.g., a polypeptide or polynucleotide encoding two polypeptides), including but not limited to recombinant fusion, covalent bond, disulfide bond, ionic bond, hydrogen bond, and electrostatic bond. "Fusion" refers to linkage by covalent bond. "Linker" or "spacer" refers to a molecule or group of molecules that connects two biomolecules and helps to arrange the two molecules in a preferred orientation with minimal steric hindrance. Various linkages can be used in constructing the fusion molecules of the present invention. In some preferred embodiments, the polypeptide components of the sialidase fusion protein of the present invention are linked by peptide bonds.

[0030] When referring to nucleic acids, the term "operably linked" means the linkage of polynucleotide elements that are in a functional relationship. Nucleic acids are "operably linked" when they are in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. Being operably linked means that the linked DNA sequences are typically contiguous and, when it is necessary to link two protein-coding regions in the production of a fusion protein, are contiguous and within the reading frame.

[0031] As used herein, the terms "polynucleotide" or "nucleic acid" refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, that include purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural or derivatized nucleotide bases. The polynucleotides of embodiments of the invention can include sequences of deoxyribopolynucleotides (DNA), ribopolynucleotides (RNA), or DNA copies of ribopolynucleotides (cDNA) that can be isolated from natural sources, recombinantly produced, or artificially synthesized. Further examples of polynucleotides are polyamide polynucleotides (PNA). Polynucleotides and nucleic acids can exist as single-stranded or double-stranded. The backbone of a polynucleotide can include sugar and phosphate groups, as typically found in RNA or DNA, or modified or substituted sugars or phosphate groups. Polynucleotides can include modified nucleotides such as methylated nucleotides and nucleotide analogs. The nucleotide sequence can be interrupted by non-nucleotide components. Polymers made of nucleotides such as nucleic acids, polynucleotides, and polynucleotides can also be referred to herein as nucleotide polymers.

[0032] A polypeptide is a polymeric chain composed of amino acid residue monomers linked to each other via amide bonds (peptide bonds). Amino acids can be L - optical isomers or D - optical isomers. Generally, a polypeptide refers to a long polymer of amino acid residues, for example, consisting of at least 10, 20, 50, 100, 200, 500 or more amino acid residue monomers. However, unless otherwise specified, the term "polypeptide" as used herein typically includes short peptides containing two or more amino acid monomers, but usually includes 10, 15, or 20 or fewer amino acid monomers.

[0033] A protein is a long polymer of amino acids linked via peptide bonds and can be composed of two or more polypeptide chains. More specifically, the term "protein" refers to a molecule composed of one or more chains of amino acids in a specific order, for example, the order determined by the nucleotide base sequence in a gene encoding the protein. Proteins are essential for the structure, function, and regulation of the body's cells, tissues, and organs, and each protein has a unique function. Examples are hormones, enzymes, and antibodies. In some embodiments, the terms polypeptide and protein may be used interchangeably.

[0034] The enzyme sialidase or neuraminidase was first isolated from the bacterium Vibrio cholerae. This enzyme specifically cleaves the terminal sialic acid moiety from sialomucin and glycoproteins. Loss of PAS or alcian blue staining after sialidase treatment clearly indicates the presence of sialic acid in tissue test sections. When performing the combined alcian blue - PAS protocol after sialidase treatment, sialomucin, which is usually stained blue with alcian blue, is stained red with PAS. In addition to bacterial sialidase, enzymes with similar activity have been identified from viral species (e.g., influenza virus) and mammals (e.g., humans).

[0035] As used herein, the term "target molecule" or "target antigen" refers to the molecule of interest on the surface of a target cell (e.g., a tumor cell) that is specifically recognized by the bispecific molecule in the sialidase fusion protein of the present invention. Preferably, the target molecule for practicing the present invention is a polypeptide (e.g., a cell receptor or surface marker protein).

[0036] The term "conservatively modified variant" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, a conservatively modified variant refers to a nucleic acid that encodes the same or an essentially identical amino acid sequence or, when the nucleic acid does not encode an amino acid sequence, to an essentially identical sequence. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at any position where an alanine is specified by a codon, the codon can be modified to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent mutations" and represent one species of conservatively modified variation. Every nucleic acid sequence herein that encodes a polypeptide also represents every possible silent mutation of the nucleic acid. One of ordinary skill in the art will recognize that each codon in a nucleic acid (except for AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Thus, each silent mutation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

[0037] "Conservative substitution" with respect to a protein or polypeptide refers to the replacement of one amino acid with another amino acid having a similar side chain. Families of amino acid residues having side chains with similar charges are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Methods for identifying nucleotide and amino acid conservative substitutions that do not eliminate protein activity are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

[0038] The terms "identical" or "identity" percent in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are the same. When two sequences are compared and aligned to obtain maximum correspondence over a comparison window or designated region, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection, the two sequences have the same amino acid residues or nucleotides at a designated percentage (i.e., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identity may be mentioned over the designated region or, if not designated, over the entire sequence), the two sequences are "substantially identical". Identity may be present over a region of at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region of 100 - 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

[0039] Methods for aligning arrays for comparison are well known in the art. Optimal alignment of arrays for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482c, 1970, by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443, 1970, by the search for similar methods of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI), or by manual alignment and visual inspection (see, for example, Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003)). Two examples of algorithms suitable for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410, 1990, respectively.

[0040] The term "subject" refers to human and non-human animals (particularly non-human mammals). The term "subject" is used herein, for example, in connection with methods of treatment and methods of diagnosis, to refer to a human or animal subject. Animal subjects include animal models, for example, mammalian models of conditions or disorders associated with elevated Ebola virus expression such as CLL, ALL, mantle cell lymphoma, neuroblastoma, sarcoma, renal cell carcinoma, breast cancer, lung cancer, colon cancer, head and neck cancer, melanoma, and other cancers, but are not limited thereto. Other specific examples of non-human subjects include, for example, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys.

[0041] As used herein, the terms "treat", "treating", "treatment" and "therapeutically effective" do not necessarily mean 100% or complete treatment. Rather, there are various degrees of treatment that are recognized by those skilled in the art as having a potential benefit or therapeutic effect. In this regard, the treatment methods described herein can provide any amount of any level of treatment. Further, the treatment provided by the method can include treatment of one or more conditions or symptoms of the disease being treated.

[0042] A "vector" is a replicon, such as a plasmid, phage or cosmid, to which another polynucleotide segment can attach so as to bring about replication of the attached segment. A vector capable of directing the expression of a gene encoding one or more polypeptides is called an "expression vector".

[0043] III. Fusions Comprising Sialidase and Bispecific Immune Cell Engagers In one aspect, the present invention provides a fusion protein or fusion molecule comprising a sialidase (or an enzymatic fragment thereof) conjugated or linked to a bispecific immune cell engager molecule. As used herein, a bispecific immune cell engager molecule refers to any bispecific molecule (e.g., a bispecific antibody) that can specifically bind to both (1) a target antigen (e.g., a surface molecule or receptor) on a target cell and (2) an immune cell capable of exerting immune activity (e.g., cytotoxicity) against the target cell. Bispecific molecules suitable for the present invention can exist in various formats well-known in the art. See, e.g., Labrijn et al., Nat. Rev. Drug Discovery 18:585-608, 2019. In some embodiments, the immune cell engaged by the bispecific molecule is a T cell. In some of these embodiments, the bispecific immune cell engager used is a BiTE. In some other embodiments, the immune cell targeted by the bispecific molecule is a innate immune cell, e.g., an NK cell or a macrophage. In some of these embodiments, the bispecific immune cell engager used is a natural cell engager. In some other embodiments, the bispecific cell engaging molecule can comprise two antigen-binding arms linked via Fc-mediated heterodimerization, knob-into-hole, or other formats. Typically, the bispecific molecule binds to the immune cell via a surface marker antigen on the cell. For example, a BiTE suitable for the present invention can bind to an antigen in the TCR complex or a related protein, e.g., CD3. Similarly, a bispecific natural cell engager (e.g., a BiKE) that can be used in the present invention can target any specific surface marker on an innate immune cell, e.g., NKp44 or CD16 on an NK cell or a macrophage. Specific examples of bispecific natural cell engagers such as BiKE and their constructs are known in the art. See, e.g., Pinto et al., Trends Immunol. 43:933, 2022.

[0044] Some preferred embodiments of the present invention relate to BiTE - chalarase fusions. Examples of such fusions are shown, for example, in SEQ ID NOs: 7, 8, 11, 13, and 15. In some of these embodiments, one of the tandem scFvs in the BiTE recognizes the CD3 subunit of the T - cell receptor complex, and the other binds to an antigen on tumor cells. Many BiTE molecules and their use in cancer immunotherapy have been reported in the art. See, for example, Huehls et al., Immunol. Cell Biol. 93:290 - 296, 2015; Lejeune et al., Front. Immunol., Vol. 11, Article 762, 2020; Ross et al., PLoS One. 12:e0183390, 2017; Vafa et al., Front. Immunol., Vol. 10, Article 446, 2020; Haber et al., Sci. Rep. 11:14397, 2021; Ellerman, Methods 154:102 - 117, 2019; Lund et al., BMC Cancer 20:1214, 2020; and Einsele et al., Cancer 126:3192 - 3201, 2020. Using any of these known BiTEs and the BiTEs exemplified herein, BiTE - chalarase fusions can be constructed. BiTEs can be readily generated according to the description of the present invention or standard protocols routinely practiced in the art. By way of example, each scFv in the BiTE can be constructed by linking the heavy and light chains of each Fv with a serine - glycine linker sequence. As exemplified for the BiTE molecules herein, the linker can generally be constructed from two, three, or more SGGGG (SEQ ID NO: 34) repeats, making the peptide long and flexible enough to allow the heavy and light chains to associate in their normal conformation. Similar GS linkers can be used to link two scFvs, such as SEQ ID NOs: 1, 2, 29 - 31, and 34 - 37 exemplified herein. The length of this linker determines the flexibility of movement between the two scFvs and can be adjusted by including more or fewer repeats to optimize binding to both target cells.The entire BiTE molecule consists of one continuous polypeptide. In some embodiments, the complete BiTE molecule is approximately 55 kDa in size and approximately 11 nm in length.

[0045] In addition to BiTE, the sialidase fusion proteins of the present invention can also contain other types of immune cells that engage with the bispecific molecule. In some embodiments, a bispecific natural cell engager can be fused to the sialidase. By way of example, several bispecific molecules are described herein that engage NK cells (i.e., BiKE) via the CD16A surface marker and bind to CD19 or EFGR, respectively, on target cells. The sequences of the fusion proteins containing these BiKE and sialidase are shown in SEQ ID NOs: 23-28, respectively. As described herein, these BiKE-sialidase fusion molecules can selectively desialylate target cells and also exhibit enhanced cytotoxicity.

[0046] In some embodiments, the bispecific engagement molecule in the fusion proteins of the present invention is two antibody fragments (e.g., scFv or tandem V H -V L fragment) linked via two Fc arms each linked to an antibody fragment. In some of these fusion proteins, the two antibody fragments are linked via knob and hole mutations introduced into the two Fc arms, respectively. The use of "knob mutations" and "hole mutations" in Fc fusion dimerization is well known in the art. See, for example, Merchant et al., Nat. Biotechnol. 16, 677-681, 1998; Jendeberg et al., J. Immunol. Methods 201, 25-34, 1997; Ridgway et al., Protein Engineering 9:617, 1996; Rouet et al., Nat. Biotechnol. 32(2):136, 2014; and Xu et al., mAbs. 7(1):231-242, 2015. For example, the knob and hole mutations engineered for ligation are at the C of the Fc portion of the two antibody fragments. HIt may be the T366Y mutation and the Y407T mutation respectively introduced into the 3 regions.

[0047] In addition to the immune cell targeting functionality, the bispecific molecule in the fusion protein of the present invention also recognizes a target antigen associated with or involved in a disease or disorder (e.g., cancer). Typically, the target antigen is derived from a cell involved in or responsible for the onset of the disease. Any surface antigen on the cells that cause such a disease can be targeted with the bispecific molecule in the sialidase fusion. In some preferred embodiments, the target antigen is selectively or predominantly expressed on tumor cells. In some embodiments, the cell surface molecule targeted by the fusion protein of the present invention can be a receptor. The receptor can be an extracellular receptor. The receptor can be a cell surface receptor. By way of non-limiting example, the receptor can bind to a hormone, neurotransmitter, cytokine, growth factor, or cell recognition molecule. The receptor can be a transmembrane receptor. The receptor can be an enzyme-linked receptor. The receptor can be a G protein-coupled receptor (GPCR). The receptor can be a growth factor receptor. The cell surface molecule can be a non-receptor cell surface protein. The target molecule can be a surface antigen classification protein. By way of non-limiting example, the cell surface molecule can be selected from CD19, CD20, CD34, CD31, CD117, CD45, CD11b, CD15, CD24, CD114, CD182, CD14, CD11a, CD91, CD16, CD3, CD4, CD25, CD8, CD38, CD22, CD61, CD56, CD30, CD13, CLL1, CD33, CD123, or fragments or homologs thereof.

[0048] In addition to targeting the above cancer markers, the sialidase fusion of the present invention can also target antigens or neoantigens presented by MHC I or MHC II molecules on the surface of tumor cells. In some preferred embodiments, these antigens are presented only by tumor cells and never by normal ones. In some embodiments, the target antigen is a tumor-specific antigen (TSA), generally resulting from tumor-specific mutations. In some embodiments, the target antigen is an antigen presented by both tumor cells and normal cells, i.e., a tumor-associated antigen (TAA). In some embodiments, the target molecule on the tumor cell surface can be a peptide-free molecule. The cell surface molecule can contain lipids. The cell surface molecule can contain a lipid moiety or lipid group. The lipid moiety can contain sterols. The lipid moiety can contain fatty acids. The antigen can contain glycolipids. The cell surface molecule can contain carbohydrates.

[0049] Bispecific molecules that engage target cells with immune cells can be made by methods routinely practiced. As noted above, BiTEs or bispecific natural cell engagers targeting various tumor antigens or other disease-associated antigens have been reported in the art. These include various tumor cell surface markers such as Her2, CD19 or PSMA exemplified herein. Bispecific molecules specific for other cancer-targeting bispecific molecules can also be readily made. Suitable tumor cell surface targets for bispecific molecules include, for example, CD33, EGFR, EGFR vIII, CD66e, EphA2, MCSP (melanoma), EpCAM antigen (colon cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, and pancreatic cancer), CEA, and gp100 peptide (unresectable or metastatic uveal melanoma).

[0050] For the construction of the fusion protein of the present invention, any sialidase or its enzyme fragment can be used. Sialidase (neuraminidase) is a glycoside hydrolase enzyme that cleaves (cuts) the glycosidic bond of neuraminic acid. These enzymes are a large family and are found in various organisms. The most well-known neuraminidase is the viral neuraminidase, which is a drug target for preventing the spread of influenza infection. Viral neuraminidase is frequently used as an antigenic determinant found on the surface of the influenza virus. Some variants of influenza neuraminidase confer more pathogenicity to the virus than others. Other homologs with various functions are found in mammalian cells. As described below, at least seven mammalian sialidase homologs and isoforms are described in the human genome.

[0051] In some embodiments, the fusion proteins of the invention contain human sialidase. A number of human sialidases are known in the art. These include human sialidases NEU1, NEU2, NEU3, NEU4, and several isoforms. The sequences of these human enzymes (e.g., SEQ ID NOs: 16-22 herein), their functional characteristics and recombinant production have been reported in the literature. See, for example, Chavas et al., J. Biol. Chem. 280:469-75, 2005; Lipnicanova et al., Intl. J. Biol. Macromol. 148:857-868, 2020; Richards et al., Bioorg. Med. Chem., 26:5349-58, 2018; and U.S. Patent Application Publication No. 2020 / 0239512. In addition to wild-type human sialidase, variants or mutants of human sialidase, including conservatively modified variants, can also be used in the fusion proteins of the invention. These variants typically have enhanced or substantially the same enzyme activity as the wild-type sialidase. Additionally or alternatively, they can have other improved properties, such as biological or pharmaceutical properties. In some embodiments, the sialidase variant used is a recombinantly produced human sialidase mutant containing one or more amino acid substitutions described in International Publication No. 2021 / 003463.

[0052] In addition to human sialidase, sialidases obtained from other species and their modified variants can also be used for the construction of the fusion molecules of the present invention. These include, for example, viral neuraminidase and bacterial neuraminidase. Viral sialidases that have been widely studied are those derived from influenza virus (Orthomyxoviridae). Influenza sialidase has been widely studied and characterized functionally and structurally. Other examples of viral sialidases include, for example, sialidases derived from the Paramyxoviridae family. See, for example, Durrant et al., J. Phys. Chem. B. 120:8590 - 99, 2016; Vavricka et al., Nat. Commun. 4:e1491, 2013; Stelfox et al., Proc. Natl. Acad. Sci. USA 116:21514 - 20, 2019; and Villar et al., Glycoconj. J. 23:5 - 17, 2006. Known bacterial sialidases include, for example, S. typhimurium sialidase, V. cholerae sialidase, B. infantis sialidase, and B. bifidum sialidase. See, for example, Varghese et al., Proteins: Struct. Funct. Genet. 14:327 - 32, 1992; Fougerat et al., Mol. Metab., 12:76 - 88, 2018; Park et al., Biochim. Biophys. Acta, 1834:1510 - 1519, 2013; Nishiyama et al., mBio 8:e00928 - 17, 2017; Kaisar et al., mSphere 6:e01232 - 20, 2021; Prevato et al., PLoS One, 10(2015), Article e0135474; Cirillo et al., J. Biol. Chem., 291:10615 - 10624, 2016; and Crennell et al., Structure 2:535 - 544, 1994.

[0053] The method for producing the fusion protein of the present invention is not subject to any specific restrictions. The fusion protein of the present invention may be a fusion protein synthesized by chemical synthesis or a recombinant fusion protein produced by genetic engineering techniques. When chemically synthesizing the fusion protein of the present invention, for example, it can be synthesized by the Fmoc (fluorenylmethyloxycarbonyl) method or the tBoc (t-butyloxycarbonyl) method. Further, for example, peptide synthesizers available from Advanced ChemTech, PerkinElmer, Pharmacia, Protein Technology Instrument, Synthese-Vega, PerSeptive and Shimadzu Corporation can be used for chemical synthesis. When the fusion protein of the present invention is to be produced by genetic engineering techniques, the production can be carried out using conventional recombinant techniques routinely practiced in the art. Such techniques are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., (3 rd ed., 2000); and Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003). As described in detail below, the fusion protein can be produced by inserting a polynucleotide (e.g., DNA) encoding the fusion protein into an appropriate expression system.

[0054] In some preferred embodiments, the sialidase fusion protein of the present invention is produced according to recombinant techniques routinely practiced. Some specific exemplifications are described in detail in the following examples. As exemplified herein, the sialidase can be operably fused to either the N-terminus or the C-terminus of the bispecific molecule. Typically, the method involves removing the stop codon from a polynucleotide sequence (e.g., cDNA sequence) encoding one of the two fusion components (e.g., BiTE), and then adding in-frame a polynucleotide sequence (e.g., cDNA sequence) encoding the other component (e.g., sialidase) by ligation or overlap extension PCR. To ensure proper folding of the fusion partners and maintain biological activity, a linker peptide or spacer peptide can be used to link the GS-rich linker as shown in some of the sialidase fusions described in the examples herein between the two components of the fusion protein. The fusion protein of the present invention may further comprise a peptide sequence or tag for purification. Peptide sequences for purification that can be used are also known in the art. As exemplified herein, examples of peptide sequences for purification include a histidine tag sequence having an amino acid sequence of at least four, preferably at least six consecutive histidine residues, and the amino acid sequence of the glutathione binding domain in glutathione S-transferase.

[0055] IV. Polynucleotides, vectors and host cells for producing sialidase fusions In addition to the sialidase fusion proteins disclosed above, related embodiments of the present invention also include polynucleotide sequences encoding such fusions, expression constructs for expressing the fusion proteins, and host cells carrying the polynucleotides or expression constructs. The polynucleotides or nucleic acids of the present invention include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences. These include, but are not limited to, messenger RNA (mRNA), DNA / RNA hybrids, or synthetic nucleic acids. In some embodiments, the polynucleotides of the present invention include small nucleolar RNA (sno-RNA), microRNA (miRNA), small interfering RNA (siRNA), or Piwi-interacting RNA (piRNA). The nucleic acids of the present invention can be single-stranded, or partially or completely double-stranded (duplex). Double-stranded nucleic acids can be homoduplex or heteroduplex.

[0056] The recombinant construct or expression vector of the present invention has the polynucleotide sequence of the present invention encoding a sialidase fusion polypeptide. The recombinant construct of the present invention can be obtained by ligating (inserting) the polynucleotide (DNA) of the present invention into an appropriate vector. More specifically, the recombinant vector can be obtained by cleaving the purified polynucleotide (DNA) with an appropriate restriction enzyme, then inserting the cleaved polynucleotide into a restriction enzyme site or a multiple cloning site on an appropriate vector, and ligating the polynucleotide to the vector. The vector for inserting the polynucleotide sequence is not subject to any particular limitation as long as it can replicate in an appropriate host. The expression vector of the present invention is not subject to any particular limitation and can be, for example, a bacteriophage, plasmid, cosmid or phagemid. Examples of recombinant bacteriophage or phagemid vectors include those based on filamentous phages such as M13. Plasmid vectors include, for example, those based on plasmids derived from E. coli (e.g., pBR322, pBR325, pUC118 and pUC119), plasmids derived from Bacillus subtilis (e.g., pUB110 and pTP5), and plasmids derived from yeast (e.g., YEp13, YEp24 and YCp50). The expression vector can also include animal viruses such as retroviruses, vaccinia viruses and insect viruses (e.g., baculoviruses).

[0057] In the expression construct of the present invention, the polynucleotide encoding the sialidase fusion is generally ligated downstream of a promoter in an appropriate vector so as to be expressible. For example, when the host during transformation is an animal cell, preferred promoters include the promoter derived from SV40, retroviral promoters, metallothionein promoters, heat shock promoters, cytomegalovirus promoters, and SRα promoters. When the host is an Escherichia organism, preferred promoters include the tetracycline promoter, Trp promoter, T7 promoter, lac promoter, recA promoter, λ promoter, and lpp promoter. When the host is a Bacillus organism, preferred promoters include the SPO1 promoter, SPO2 promoter, and penP promoter. When the host is yeast, preferred promoters include the PHO5 promoter, PGK promoter, GAP promoter, ADH1 promoter, and GAL promoter. When the host is an insect cell, preferred promoters include the polyhedrin promoter and P10 promoter.

[0058] In addition to the above, the recombinant vector used in the present invention may optionally contain an enhancer, splicing signal, poly(A) addition signal, ribosome binding sequence (SD sequence), selection marker, etc. Examples of selection markers include the tetracycline resistance gene, carbenicillin resistance gene, dihydrofolate reductase gene, ampicillin resistance gene, and neomycin resistance gene. The recombinant vector of the present invention may further contain a polynucleotide having a nucleotide sequence encoding an amino acid sequence for enhancing translation and / or a polynucleotide having a nucleotide sequence encoding a peptide sequence for purification. For example, the vector can use a translation enhancer element (TEE) sequence (see, for example, Batten et al., FEBS Lett. 580:2591-7, 2006).

[0059] The present invention further provides a host cell expressing the sialidase fusion polypeptide described herein. The host cell is genetically engineered (transduced, transformed or transfected) with the recombinant construct or expression vector disclosed herein for the production of the fusion protein or the assay of its activity. To generate such cells, a recombinant construct carrying and expressing the sialidase fusion sequence can be introduced into a suitable host. The engineered host cell can be cultured in a conventional nutrient medium appropriately modified for the activation of the promoter, the selection of transformants or the amplification of specific genes such as the fusion gene encoding the sialidase fusion polypeptide. The culture conditions for the specific host cell selected for expression, such as temperature, pH, etc., will be readily apparent to those skilled in the art. In some embodiments, the sialidase fusion sequence is stably integrated into the chromosome of the host cell. In such host cells, the sialidase sequence and its expression are substantially maintained in successive generations of the cells. They are distinguished from host cells that transiently express the fusion polypeptide detailed herein.

[0060] Host cells for the production or expression of the constructs of the present invention can be, for example, higher eukaryotic cells such as mammalian cells, or lower eukaryotic cells such as yeast cells, or the host cells can be prokaryotic cells such as bacterial cells. The selection of a suitable host is within the scope of those skilled in the art and is also exemplified in the examples herein. Preferably, the host cells used are suitable for the expression of sialidase fusion by the cytidine deaminase polypeptide and for the induction of hypermutation in the target gene or polynucleotide. Representative examples of suitable host cells suitable for carrying out the present invention include, but are not limited to, bacterial cells such as E. coli, Streptomyces, Salmonella typhimurium; fungal cells such as yeast; insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS or 293 cells; adenovirus; plant cells, or any suitable cells already adapted or newly established for in vitro propagation. When the expression construct is a phage or phagemid vector, many suitable bacterial host cells can be used, for example, the E. coli ER2738 cell line as detailed in the following examples. Another example of such a host cell is the E. coli BW310 strain as exemplified in the following examples. This cell line is available from the Coli Genetic Stock Center (New Haven, CT) of Yale University. The BW310 cells do not have the ung gene (i.e., ung-genotype). The ung gene encodes uracil-DNA glycosylase, which prevents mutagenesis by removing uracil bases produced by cytosine deamination or misincorporation of dUMP residues from DNA molecules. This BW310 cell line is routinely used in the art for studying the expression and DNA mutator activity of AID or its orthologs (e.g., Ichikawa et al., J. Immunol. 177:355-361, 2006; and Hache et al., J. Biol. Chem. 280:10920-4, 2005).

[0061] The polynucleotides and related vectors of the present invention can be readily produced using standard molecular biology techniques or the protocols exemplified herein. For example, general protocols for cloning, transfection, transient gene expression, and obtaining stable transfected cell lines are described in the art and are available, for example, from Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., (3 rded., 2000); and Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003). Introduction of mutations into polynucleotide sequences by PCR is carried out, for example, as described in PCR Technology: Principles and Applications for DNA Amplification, H.A. Erlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, CA, 1990; Mattila et al., Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods and Applications 1:17, 1991. More specific teachings for preparing mRNA therapeutics and mRNA vaccines are also provided in the art.For example, detailed guidelines for producing therapeutic mRNA are described, for example, in U.S. Patent No. 9,464,124; U.S. Patent No. 9,447,164; U.S. Patent No. 9,428,535; U.S. Patent No. 9,334,328; U.S. Patent No. 9,303,079; U.S. Patent No. 9,301,993; U.S. Patent No. 9,295,689; U.S. Patent No. 9,283,287; U.S. Patent No. 9,271,996; U.S. Patent No. 9,255,129; U.S. Patent No. 9,254,311; U.S. Patent No. 9,233,141; U.S. Patent No. 9,221,891; U.S. Patent No. 9,220,792; U.S. Patent No. 9,220,755; U.S. Patent No. 9,216,205; U.S. Patent No. 9,192,651; U.S. Patent No. 9,186,372; U.S. Patent No. 9,181,319; U.S. Patent No. 9,149,506; U.S. Patent No. 9,114,113; U.S. Patent No. 9,107,886; U.S. Patent No. 9,095,552; U.S. Patent No. 9,089,604; U.S. Patent No. 9,061,059; U.S. Patent No. 9,050,297; U.S. Patent No. 8,999,380; U.S. Patent No. 8,980,864; U.S. Patent No. 8,822,663; U.S. Patent No. 8,754,062; U.S. Patent No. 8,710,200; U.S. Patent No. 8,680,069 and U.S. Patent No. 8,664,194.

[0062] Introduction of a vector or expression construct into a host cell can be carried out by various methods well-known to those skilled in the art, including, but not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, or electroporation (see, e.g., Brent et al. supra). Expression and, if desired, purification of the sialidase fusion polypeptide in the transfected or transformed host cell are carried out according to any of the methods routinely practiced in the art, e.g., as described in Sambrook et al., supra; and Brent et al., supra. Typically, to produce the fusion protein of the present invention, host cells having the expression vector are cultured under appropriate conditions that allow expression of the polynucleotide (DNA) encoding the fusion protein, thereby inducing formation and accumulation of the fusion polypeptide, and then the fusion polypeptide is isolated and purified. The fusion protein expressed in the host cell can be easily isolated and purified. Specifically, when the fusion protein of the present invention accumulates in the cultured bacteria or cultured cells, after completion of the culture, the bacteria or cells are disrupted using conventional techniques (e.g., sonication, lysozyme, freezing and thawing), and then an extract of the fusion protein of the present invention can be obtained by conventional methods such as centrifugation or filtration. When the sialidase fusion polypeptide accumulates in the periplasmic space, after completion of the culture, an extract containing the target protein may be obtained by a conventional method such as osmotic shock. When the fusion protein of the present invention accumulates in the culture broth, after completion of the culture, a culture supernatant containing the fusion protein of the present invention can be obtained using conventional methods such as centrifugation or filtration and separated from the bacteria or cells.

[0063] V. Therapeutic Uses The sialidase fusion molecules and related compositions described herein can be used to treat or ameliorate various tumor conditions that express antigens recognized by bispecific molecules in the fusion protein. The sialidase fusion molecules of the present invention can be administered directly to a subject under sterile conditions. The fusion protein can be administered alone or as an active ingredient of a pharmaceutical composition. The therapeutic compositions of the present invention can be used in combination with or in combination with other therapeutic agents. Various cancers can be treated by the methods of the present invention. These include cancers derived from any tissue such as, for example, tissues of the brain, esophagus, breast, colon, lung, glioma, ovary, uterus, testis, prostate, gastrointestinal tract, bladder, liver, thyroid, and skin. In some embodiments, the cancer to be treated is derived from bone. In some embodiments, the cancer to be treated is derived from blood. In these embodiments, the cancer can be derived from B cells, T cells, monocytes, platelets, leukocytes, neutrophils, eosinophils, basophils, lymphocytes, hematopoietic stem cells, or endothelial cell progenitor cells. In some embodiments, the cancer can be derived from CD19-positive B lymphocytes. In some embodiments, the cancer can be derived from stem cells. For example, the targeted cancer cells can be derived from pluripotent cells. In some embodiments, the cancer cells to be targeted can be derived from one or more endocrine glands. The endocrine gland can be a lymph gland, pituitary gland, thyroid gland, parathyroid gland, pancreas, gonad, or pineal gland.

[0064] Many tumors or cell proliferative disorders can be treated by the methods of the present invention. These include solid tumors, lymphomas, leukemias, and liposarcomas. The disorders to the treated condition can be acute, chronic, recurrent, refractory, accelerated, remission, stage I, stage II, stage III, stage IV, juvenile, or adult. Solid tumors that can be treated by the methods of the present invention include, for example, cancers from or derived from the brain, esophagus, breast, colon, lung, glioma, ovary, uterus, testis, prostate, gastrointestinal tract, bladder, liver, thyroid, and skin.

[0065] In some embodiments, the cancer to be treated is heterogeneous. In some embodiments, the cancer to be treated is a hematopoietic cell malignancy. For example, the cancer to be treated can be derived from myeloid cells or other blood cells. In these embodiments, the cancer can be derived from B cells, T cells, monocytes, platelets, white blood cells, neutrophils, eosinophils, basophils, lymphocytes, hematopoietic stem cells or endothelial cell progenitor cells. In some embodiments, the cancer can be derived from CD19-positive B lymphocytes. In some embodiments, the cancer can be derived from stem cells. For example, the targeted cancer cells can be derived from pluripotent cells. In some embodiments, the cancer cells to be targeted can be derived from one or more endocrine glands. The endocrine gland can be a lymph gland, pituitary gland, thyroid gland, parathyroid gland, pancreas, gonad or pineal gland.

[0066] In some embodiments, the cancer to be treated is a Her2-positive cancer. These include, for example, Her2-positive breast cancer and Her2-positive pancreatic cancer. In some embodiments, the cancer to be treated can be a PSMA-positive prostate cancer. In some embodiments, the cancer to be treated is a CD19-positive tumor or malignancy. In some of these embodiments, the cancer to be treated is a B cell cancer or B cell malignancy. B cell cancers or B cell malignancies include B cell lymphomas that account for the majority of non-Hodgkin lymphomas (NHL). Examples of these B cell cancers include, for example, diffuse large B cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (Waldenström macroglobulinemia), hairy cell leukemia (HCL), primary central nervous system (CNS) lymphoma, and primary intraocular lymphoma.

[0067] The sialidase fusion described in this specification can be used in combination with other known regimens for treating cancer. These include known anti-tumor drugs (antineoplastic drugs), tumor metastasis inhibitors, thrombus formation inhibitors, therapeutic agents for joint destruction, analgesics, anti-inflammatory drugs, immunomodulatory drugs (or immunoregulators) and / or immunosuppressive drugs, and these can be used without being limited to specific species as long as they work effectively or advantageously. Methods of co-administration with additional therapeutic agents are well known in the art (Hardman, et al. (eds.) (2001) Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New York, N.Y.; Poole and Peterson (eds.) (2001) Pharmacotherapeutics for Advanced Practice: A Practical Approach, Lippincott, Williams & Wilkins, Phila., Pa.; Chabner and Longo (eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams & Wilkins, Phila., Pa.).

[0068] VI. Pharmaceutical Compositions The present invention further provides a pharmaceutical composition comprising the sialidase fusion protein described herein and a pharmaceutically acceptable carrier. The pharmaceutical composition can be prepared from any of the sialidase fusion molecules described herein. The pharmaceutically acceptable carrier can be any suitable pharmaceutically acceptable carrier. It can be one or more compatible solid or liquid fillers, diluents, other excipients, or encapsulating substances (e.g., a physiologically acceptable carrier or a pharmacologically acceptable carrier) suitable for administration to a human or veterinary patient. The term "carrier" refers to a natural or synthetic organic or inorganic component with which the active ingredient is combined to facilitate the use of the active ingredient, e.g., the administration of the active ingredient to a subject. The pharmaceutically acceptable carrier can be mixed with one or more active ingredients, e.g., an adapter molecule, such that when one or more pharmaceutically acceptable carriers are present in the composition, the desired pharmaceutical effectiveness is not substantially impaired. Pharmaceutically acceptable materials can typically be administered to a subject, e.g., a patient, without causing significant undesirable physiological effects such as nausea, dizziness, rash, or stomach upset. For example, it is desirable that a composition containing a pharmaceutically acceptable carrier is not immunogenic when administered to a human patient for therapeutic purposes.

[0069] The pharmaceutical composition of the present invention can further contain a suitable buffer, for example, acetic acid in salt, citric acid in salt, boric acid in salt, and phosphoric acid in salt. The composition may also contain suitable preservatives such as benzalkonium chloride, chlorobutanol, parabens, and thimerosal. The pharmaceutical composition of the present invention can be provided in unit dosage form and can be prepared by any suitable method well known in the pharmaceutical art. Such methods include associating the antibody of the present invention with a carrier that constitutes one or more auxiliary components. Generally, the composition is prepared by uniformly and intimately associating the active agent with a liquid carrier, a micronized solid carrier, or both, and then shaping the product as necessary.

[0070] Compositions suitable for parenteral administration conveniently include a sterile aqueous preparation of the composition of the invention, which is preferably isotonic with the recipient's blood. This aqueous preparation can be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be used are water, Ringer's solution, and isotonic sodium chloride solution. Further, sterile fixed oils have conventionally been used as solvents or suspending media. For this purpose, any bland fixed oil such as synthetic monoglycerides or diglycerides can be used. Additionally, fatty acids such as oleic acid can be used in the preparation of injectables. Carrier formulations suitable for oral, subcutaneous, intravenous, intramuscular, etc. administration can be found in Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.

[0071] The pharmaceutical compositions of the present invention and their preparation for various routes of administration can be carried out according to methods well known in the art. For example, see Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20 th ed., 2000; and Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. Delivery systems useful in the context of the present invention include timed release delivery systems, delayed release delivery systems, and sustained release delivery systems such that delivery of the composition of the present invention occurs before sensitization of the site to be treated and over a time sufficient to cause sensitization. The compositions of the present invention can be used in combination with other therapeutic agents or treatments. Such systems can avoid repeated administration of the composition of the present invention, thereby enhancing convenience for the subject and the physician and may be particularly suitable for certain compositions of the present invention.

[0072] Many types of release delivery systems are available and are known to those of ordinary skill in the art. Suitable release delivery systems include polymer-based systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactone, polyesteramides, polyorthoesters, polyhydroxybutyrate, and polyanhydrides. Microcapsules of the aforementioned polymers containing a drug are described, for example, in U.S. Patent No. 5,075,109. Delivery systems include non-polymeric systems that are lipids containing sterols such as cholesterol, cholesterol esters, and fatty acids, or neutral fats such as mono-, di-, and triglycerides; hydrogel release systems; silastic systems; peptide-based systems; wax coatings; compressed tablets using conventional binders and excipients; and partially fused implants, among others. Specific examples include, but are not limited to, (a) erosion systems in which the active composition is included in matrix form as described in U.S. Patent Nos. 4,452,775, 4,667,014, 4,748,034, and 5,239,660, and (b) diffusion systems in which the active ingredient penetrates at a controlled rate from polymers as described in U.S. Patent Nos. 3,832,253 and 3,854,480. Additionally, pump-based hardware delivery systems can be used, some of which are implant-compatible.

[0073] Some embodiments of the present invention relate to pharmaceutical compositions or delivery formulations containing mRNA molecules or mRNA sequences encoding the sialidase fusion proteins described herein. The mRNA sequences can be used directly for therapeutic purposes as effective mRNA vaccines. The mRNA molecules may be designed with additional components to facilitate effective delivery of the mRNA in vivo, for example, complexed with polymer or lipid components. In some embodiments, the pharmaceutical composition of the present invention can contain (i) an effective amount of synthetic mRNA encoding the sialidase fusion protein described herein, (ii) a cell permeabilizer, and (iii) a pharmaceutically acceptable carrier. In these embodiments, the mRNA can include pseudouridine, 5'-methyl-cytidine, or a combination thereof. In some of these embodiments, the mRNA does not contain a substantial amount of one or more nucleotides selected from the group consisting of uridine, cytidine, and combinations of uridine and cytidine.

[0074] As exemplified herein, some embodiments of the present invention are directed to pharmaceutical compositions containing the mRNA molecules of the present invention formulated as lipid nanoparticle (LNP) formulations, including, for example, PEG lipids, which are used in many pharmaceutical compositions, cosmetic compositions, and drug delivery systems. In addition to the lipid materials exemplified herein, the LNPs described in US Patent Application Publication Nos. 20220047518 and 20200254086 can also be adapted and modified for delivery of the mRNA agents of the present invention. In some embodiments, the lipid nanoparticle formulations of the present invention contain ionizable lipids (such as ionizable cationic lipids), structural lipids, phospholipids, and lipids containing the mRNA agent. In some embodiments, the lipid nanoparticles include ionizable lipids, PEG-modified lipids, phospholipids, and structural lipids.

[0075] In some other embodiments, chemical modifications can be introduced into the mRNA sequence to promote certain desirable characteristics of the vaccine, for example, to reduce an unwanted innate immune response to the mRNA component and / or to promote a desirable level of protein expression. In various embodiments, chemical modification of the mRNA sequence includes the use of one or more chemically modified ribonucleosides or analogs. In some embodiments, the present invention provides a packaged pharmaceutical composition for treating a tumor, such as a kit or other container. Typically, the kit or container holds a therapeutically effective amount of the sialidase fusion protein or polynucleotide described herein. The kit may include instructions detailing the method of use of the fusion molecule for treating cancer.

[0076] [Example] The following examples are provided to further illustrate the invention but are not intended to limit its scope. Other variations of the invention will be readily apparent to those skilled in the art and are encompassed by the appended claims.

[0077] [Example 1] Removal of Tumor Cell Surface Sialic Acid Enhances BiTE-Mediated Tumor Cell Killing by T Cells To evaluate whether desialylation can enhance the sensitivity of tumor cells to BiTE-mediated cell killing by T cells, the inventors first constructed a BiTE molecule from the HER2-targeting scFv 4D5 and the human CD3-targeting scFv (4D5 BiTE). Next, the inventors treated HER2-positive SK-BR-3 human breast cancer cells with sialidase from Bifidobacterium longum subsp. infantis (B. infantis) to remove cell surface sialic acid. After this procedure, the treated cells were incubated with PBMCs from healthy human donors in the presence of 4D5 BiTE. Staining with FITC-Sambucus nigra lectin (SNA), which preferentially binds to sialic acid linked α-2,6 to terminal galactose, confirmed the success of cell surface desialylation. As shown in Figure 1a, desialylation significantly enhanced 4D5 BiTE-induced tumor cell killing by T cells compared to 4D5 BiTE alone. A similar trend was also observed when 4D5 BiTE treatment was combined with the sialylation inhibitor P-3Fax-Neu5Ac (Figure 1b). Next, the inventors treated SK-BR-3 and MCF-7 breast cancer cells with 4D5 BiTE and hPBMCs, respectively, in the presence or absence of B. infantis sialidase. Importantly, the addition of sialidase induced stronger killing of both cell lines compared to both controls of 4D5 BiTE+hPBMC and sialidase+hPBMC (Figure 1c). Enhanced cytotoxicity was also observed at different E:T ratios of 1:1, and the improvement of BiTE-induced killing was sialidase dose-dependent. To verify the advantage of sialidase treatment for BiTE-induced killing, another BiTE molecule targeting prostate-specific membrane antigen (PSMA) (PSMA BiTE) was constructed, and stronger enhancement of cytotoxicity was observed when sialidase and BiTE were added simultaneously (Figure 1d). Since BiTE engages only T cells to achieve tumor cell killing, the inventors repeated the killing assay with MCF-7 cells using purified T cells and confirmed that sialidase treatment actually resulted in better BiTE-mediated target cell killing. Furthermore, the addition of sialidase also significantly enhanced T cell activation and IFN-γ secretion (Figure 1, e and f).

[0078] [Example 2] Tumor desialylation promotes stronger immune synapse (IS) formation between T cells and tumor cells To elucidate the mechanism underlying the enhanced BiTE-induced cytotoxicity by desialylation, the inventors first examined whether the sialoglycan-Siglec (sialic acid-binding immunoglobulin-type lectin) inhibitory pathway is involved. Through their interactions with sialylated glycans abnormally expressed on tumor cells, immune cell-related Siglecs trigger signaling cascades that suppress immune cell activation and effector functions. Consistent with previous reports, T cells derived from PBMCs of healthy donors expressed negligible levels of Siglec-7 and Siglec-9 compared to their CD3-negative counterparts consisting mainly of B cells, NK cells, monocytes, and dendritic cells (Figure 2b). Nevertheless, the inventors observed a slight upregulation of both Siglec-9 and Siglec-7 after T cell activation. Compared to the expression of these Siglecs on newly isolated CD3-negative cells, the expression of Siglec-7 and -9 on activated T cells remained minimal. To further test whether the Siglec-9 inhibitory pathway plays a role in BiTE-induced T cell killing, a Siglec-9 blocking antibody was added together with 4D5 BiTE, and the level of target cell killing was analyzed. In contrast to sialidase addition, blocking of the Siglec-9 signal did not increase cytotoxicity, indicating a negligible role of Siglec-9 in BiTE-induced T cell killing (Figure 2c). To examine whether the enhanced BiTE-induced killing seen with desialylation is affected by CD28 co-stimulation, the high-affinity ligand for CD80, recombinant human CTLA-4, was added to determine whether the enhanced lysis by desialylation is attenuated. However, no change in enhanced lysis was detected even with the addition of high concentrations of CTLA-4 (Figure 2d).

[0079] The formation of BiTE-induced immune synapses (IS) between target cells and T cells is the essential mode of action of BiTE. The inventors hypothesized that the removal of cell surface sialosides could promote stronger BiTE-induced IS formation between target tumor cells and T cells, and thus better tumor cell killing. The accumulation of TCR-CD3 complex and F-actin in the synapse is a prominent feature of stable and functional cytolytic IS in T cells. To test whether desialylation can promote IS formation, the inventors imaged the IS formed between T cells and sialidase-treated and untreated SK-BR-3 cells by staining for F-actin and CD3ζ. The resulting immunofluorescence was imaged by confocal microscopy. As shown in Figure 2f, the inventors observed greater BiTE-induced IS formation between T cells and desialylated SK-BR-3 cells. To evaluate the stability of the formed IS, the inventors calculated the relative CD3 fluorescence intensity at the IS and the relative area of the IS. The IS formed by sialidase-treated tumor cells and T cells showed significantly stronger CD3 accumulation and a larger IS area compared to the IS formed by untreated tumor cells and T cells (Figure 2, g and h). The same trend was observed for BiTE-induced IS formation between SKOV-3 cells and T cells, and better IS was formed after sialidase treatment.

[0080] The interaction between CD2 and CD58 is known to play an important role in the formation of productive immune synapses. The inventors found that inhibition of this interaction with an anti-CD2 blocking antibody partially reversed the enhanced cytotoxicity from sialidase addition, strongly suggesting that desialylation causes stronger target cell killing by promoting closer interaction between target tumor cells and T cells (Figure 2e).

[0081] [Example 3] HER2-targeted BiTE sialidase fusion protein selectively desialylates HER2-positive cells Since it was confirmed that sialidase treatment enhanced the cell lysis of T cell-dependent tumor cells induced by BiTE, the present inventors next attempted to specifically induce sialidase at the tumor cell-T cell interface via BiTE conjugation. Restricting sialidase activity to the target cells enhances tumor cell killing and limits non-specific desialylation of cells of the immune system. Importantly, sialyl-Lewis X, a sialylated tetrasaccharide, is essential for the tethering and rolling of leukocytes in the pathway to inflammatory sites and tumor tissues. Non-specific desialylation would disrupt this glycan epitope on leukocytes and thereby interfere with their tumor homing and thus effective tumor control. For this purpose, the present inventors constructed 4D5 BiTE-B. infantis sialidase fusion proteins with sialidase introduced into the N-terminus (sialidase-4D5 BiTE) or C-terminus (4D5 BiTE-sialidase) of 4D5 BiTE, respectively (Figure 3a). To test whether the fusion protein could successfully remove sialic acid from the surface of tumor cells, SK-BR-3 (HER2+++) and SKOV-3, human ovarian adenocarcinoma cell lines (HER2+++), were treated with sialidase-4D5 BiTE or 4D5 BiTE-sialidase, respectively, and subsequently stained with α-2,6-sialic acid-binding lectin SNA. Desialylation was demonstrated by a decrease in SNA binding compared to untreated controls and was seen in both SK-BR-3 and SKOV-3 cells when treated with either fusion protein (Figure 3b). To determine whether the sialidase fusion protein could selectively desialylate HER2-positive cells in the presence of HER2-negative cells, the present inventors mixed SKOV-3 (HER2+++) cells and MDA-MB-468 (HER2-) cells and subsequently added 4D5 BiTE-sialidase. The present inventors observed that 4D5 BiTE-sialidase selectively desialylated HER2-positive SKOV-3 cells while sparing HER2-negative MDA-MB-468 cells at both 5 nM and 50 nM concentrations, thus confirming its selectivity for HER2-expressing cells (Figure 3c).

[0082] [Example 4] Anti-HER2 BiTE sialidase induces T cell-dependent in vitro cytotoxicity and T cell effector functions that are enhanced compared to HER2 BiTE Next, the inventors compared the T cell-dependent cytotoxicity mediated by both fusion proteins to that of the original 4D5 BiTE. At the same concentration of 4 nM, both fusion proteins induced a higher level of T cell-dependent cell lysis of SK-BR-3 and SKOV-3 cells compared to 4D5 BiTE (Figure 4, a and b). Specifically, in a dose-response assay, using SK-BR-3 and SKOV-3 cells as target cells, EC50 values that were 10-fold and 3-fold lower, respectively, for 4D5 BiTE sialidase compared to 4D5 BiTE were measured (4D5 BiTE EC50 = approximately 200 pM) (Figure 4, c and d). Consistent with these findings, 4D5 BiTE-sialidase induced the highest level of T cell activation as measured by the expression of the T cell activation markers CD25 and CD69 and the degranulation marker CD107a (Figure 4, e - g). Also, the strongest release of cytokines including IL-2, IFN-γ, and TNF-α was observed in T cells treated with 4D5 BiTE sialidase. In contrast, sialidase-4D5 BiTE unexpectedly decreased the production of pro-inflammatory cytokines by T cells. (Figure 4, h - j). A similar trend was seen for SKOV-3 cells, with 4D5 BiTE-sialidase inducing the strongest T cell activation. As a result, 4D5 BiTE-sialidase was selected for further study.

[0083] The above study showed that 4D5 BiTE sialidase-engaged T cells were activated better than 4D5 BiTE-engaged T cells. Therefore, it is interesting to determine whether the better T cell activation is derived from transcriptional changes induced by BiTE treatment. To systematically characterize the transcriptional changes in T cells engaged by the BiTE molecule, the inventors co-cultured 4D5 BiTE sialidase or 4D5 BiTE-treated CD3 with target MDA-MB-231 cells +Total transcriptome RNA sequencing (RNA-seq) analysis was performed on any of the T cells. Messenger RNA (mRNA) comparison of the volcano plots between 4D5 BiTE-sialidase and 4D5 BiTE-treated T showed that 1,191 transcripts were differentially expressed between these two groups (p<0.01, log2(fold change)>0.5) (Figure 4, k). The most highly expressed gene transcripts in 4D5 BiTE-sialidase-treated T cells encoded molecules important for T cell effector function, including cytolytic enzymes and cytokines (GZMB, LTA, LIF, IFNG), cytokine receptors (IL2RA), and transcriptional regulators (FOSB, BATF3). Notably, gene transcripts related to the memory phenotype (e.g., LEF1 and TCF7, etc.), inhibitory receptors (e.g., CD96 and PDCD4, etc.), and molecules involved in regulatory T cell generation (e.g., SMAD3) were strongly downregulated. Gene set enrichment analysis (GSEA) highlighted multiple important pathways upregulated in 4D5 BiTE-sialidase-treated T cells, including those related to the cell cycle, transcriptional activity, and cell metabolism. Importantly, the expression of transcripts involved in both oxidative phosphorylation and glycolysis was significantly increased. In contrast, the enrichment of downregulated genes was related to pathways associated with Wnt-β-catenin and TGF-β signaling. Consistently, cytokine signaling (Cytosig) analysis revealed that the growth-promoting and inflammatory cytokines, IL-2, IL-12, and IL-15, had the most clearly increased activity in 4D5 BiTE-sialidase-treated T cells, whereas the activity of the inhibitory cytokine TGF-β3 was downregulated. Collectively, compared to T cells treated with 4D5 BiTE, 4D5 BiTE-sialidase-treated T cells were in a more effector-differentiated state with higher oxidative phosphorylation, glycolytic activity, and effector function.

[0084] The inventors further tested BiTE-sialidase-mediated killing of cell lines with different cell surface HER2 expression levels: MDA-MB-231(+), MDA-MB-435(+) and MDA-MB-468(-). At a concentration of 4 nM compared to 4D5 BiTE, 4D5 BiTE-sialidase strongly enhanced the killing of cells with low levels of HER2 (HER2+), such as MDA-MB-231 and MDA-MB-435. Under these conditions, a stronger enhancement of killing was achieved than that measured for HER-high (HER2+++) cells (SK-BR-3 and SKOV-3 cells) (94 - 203% vs 22 - 24%) (Figure 4l). These observations suggest that desialylation can increase the sensitivity of cells that are normally relatively resistant to BiTE-mediated T cell killing. Importantly, 4D5 BiTE-sialidase did not induce killing of HER2-negative MDA-MB-468 cells or mouse melanoma B16-F10 cells expressing abundant sialoglycans, indicating exclusive specificity for HER2-positive cells (Figure 4, m).

[0085] [Example 5] BiTE-sialidase fusion proteins specific for CD19 and PSMA cause enhanced in vitro cytotoxicity and T cell activation To evaluate whether the BiTE - sialidase fusion format can be applied to improve the efficacy of BiTE molecules targeting other tumor - related antigens, the inventors designed and constructed two additional BiTE - sialidase molecules. The first was based on blinatumomab, an FDA - approved drug targeting CD19, a cell - surface marker on B cells and B - cell malignancies. The second was derived from a BiTE against prostate - specific membrane antigen (PSMA), a target for prostate cancer treatment. As shown in Figure 5a, compared to blinatumomab (CD19 BiTE), the sialidase fusion counterpart showed much stronger cytotoxicity against CD19 - positive Raji cells, with an EC50 of one - fifth (0.80 pM vs. 4.26 pM). Using the same concentration of 5 pM, CD19 BiTE - sialidase induced much higher T - cell activation and degranulation than blinatumomab (Figure 5, b - e). Consistent with better T - cell activation, CD19 BiTE - sialidase also induced stronger cytokine release (Figure 5, f - h). Similarly, much more potent killing of another CD19 - positive cell line, NALM - 6, was achieved by CD19 BiTE - sialidase. As the inventors observed for CD19 BiTE - sialidase, PSMA BiTE - sialidase also induced better killing of PSMA - positive PC3 cells and stronger T - cell activation compared to PSMA BiTE.

[0086] [Example 6] BiTE - sialidase enables better tumor control than BiTE in a xenograft model of immunodeficient mice Regarding inducing T - cell - dependent cell lysis of tumor cells in vitro, having demonstrated the superiority of the BiTE - sialidase fusion protein over the original BiTE molecule, the inventors sought to determine whether this enhanced efficacy could also be achieved in vivo. The inventors used NOD - Prkdc em26Cd52 IL2rg em26Cd22 / NjuCrl core isogenic (NCG) immunodeficient mice were used to select a human tumor mouse xenograft model and compare the anti-tumor immunity induced by 4D5 BiTE scilidase and 4D5 BiTE constructs (NCG; CRL572;. Charles River Laboratories). On day 0, 2.5 million SK-BR-3-luc cells were subcutaneously injected (s.c.) into NCG mice, and then 5 million hPBMCs were intraperitoneally administered (i.p.). On day 7, these NCG mice were divided into three groups and then received intravenous (i.v.) injections of PBS, 4D5 BiTE, or 4D5 BiTE-scilidase, respectively (Figure 6a). Five hours later, blood was collected from each mouse to measure serum IFN-γ levels. The 4D5 BiTE-scilidase group was found to have the highest level of serum IFN-γ, while the 4D5 BiTE treatment group had almost no increased IFN-γ levels compared to the PBS control group (Figure 6b). BiTE administration was continued twice a week until day 41. A second dose of 2 million hPBMCs per mouse was given on day 16. During this treatment process, tumor growth was monitored by longitudinal non-invasive bioluminescence imaging. As shown in Figures 6c and 6d, administration of 4D5 BiTE-scilidase significantly delayed in vivo tumor cell proliferation compared to 4D5 BiTE treatment and PBS control. Notably, by the end of the treatment regimen, the tumors of two mice treated with 4D5 BiTE-scilidase were completely eradicated. The inventors then used an orthotopic xenograft mouse leukemia model to examine the in vivo efficacy of the CD19 BiTE-scilidase fusion protein. In this model, CD19 +NALM-6 cells (800,000) and hPBMC (6,000,000) were intravenously (i.v.) injected into NCG mice on day 0 (Figure 6e). Recipient mice were divided into four groups on day 3 and received i.v. injections of PBS, 4D5 BiTE - sialidase, CD19 BiTE, or CD19 BiTE - sialidase, respectively. Significantly slower tumor progression was observed in the CD19 BiTE - sialidase - treated group compared to the CD19 BiTE - treated group, demonstrating a better in - vivo anti - tumor effect of the sialidase fusion protein (Figures 6, f and g). Notably, no obvious difference was detected between the PBS control group and the group receiving non - CD19 - targeted 4D5 BiTE - sialidase, indicating that the anti - tumor effect induced by the fusion protein depends on target engagement with tumor cells (Figure 6g).

[0087] [Example 7] In - vivo activity of BiTE - sialidase fusion in melanoma animal models The inventors further observed treatment advantages of BiTE-sialidase fusion proteins over parental BiTEs in a syngeneic mouse model of melanoma. Specifically, to evaluate the efficacy of BiTE-sialidase fusion proteins in an immunocompetent syngeneic mouse model, the inventors constructed a mouse CD3-engaging BiTE and the corresponding BiTE-sialidase from ScFv fragments derived from the anti-human EGFR antibody cetuximab and the anti-mouse CD3ε clone 17A2. The mouse melanoma cell line B16-EGFR5 (B16-E5) expressing chimeric mouse EGFR with six amino acid mutations that enable cetuximab binding was selected as the target cell. The fusion protein successfully induced desialylation of B16-E5 cells in vitro, as confirmed by SNA staining (Figure 7a). To compare the antitumor activities of EGFR BiTE and EGFR BiTE-sialidase in vivo, the inventors inoculated (s.c.) B16-E5 tumor cells into C57BL / 6J mice and subsequently administered EGFR BiTE or EGFR BiTE-sialidase intratumorally. Both groups conferred treatment advantages over the PBS control group, but EGFR BiTE-sialidase treatment significantly delayed tumor growth compared to the EGFR BiTE counterpart, in addition to providing a significant survival benefit to recipient mice (Figure 7, b and c). To profile the important effector cell types mediating tumor control by EGFR BiTE-sialidase treatment, the inventors depleted the CD8 + or CD4 + T cell populations in mice inoculated with B16-E5 tumor cells and treated with the different fusion proteins (Figure 14a). The results indicate that depletion of CD8 + T cells completely abrogated the antitumor benefit of EGFR BiTE-sialidase treatment, while depletion of CD4 + T cells had only a minimal effect (Figure 14b).

[0088] Next, the inventors investigated whether the BiTE sialidase fusion protein confers better tumor control by inducing changes in the immune cell composition in the tumor microenvironment. A single high dose of EGFR BiTE or EGFR BiTE sialidase was injected intratumorally on day 11 after tumor inoculation. Tumors and tumor-draining lymph nodes were harvested 3 days after treatment, at which time the fusion protein treatment group had a smaller tumor size compared to the BiTE treatment group. The inventors found that in the tumor-draining lymph nodes of both the EGFR BiTE and EGFR BiTE-sialidase treatment groups, the number of lymphocytes was significantly higher compared to the PBS control group, and the BiTE-sialidase treatment group had the highest CD8 + T cell count. Analysis of tumor-infiltrating immune cells revealed that compared to the EGFR BiTE treatment group, the BiTE-sialidase treatment group had a significantly higher frequency of CD8 + T cells and NK cells (CD45.2 + CD3 - NK1.1 + ), and a decreased frequency of myeloid cells CD45.2 + CD11b + NK1.1 - ). However, no obvious differences were observed in CD4 + T cells and dendritic cells (CD45.2 + CD11c + ).

[0089] Next, the inventors analyzed CD8 + T cells from different groups and found that the CD8 + T cells in the EGFR BiTE-sialidase treatment group were skewed towards a more effector-like phenotype. Collectively, these results demonstrate that the BiTE-sialidase fusion protein promotes the conversion of the immunosuppressive myeloid-rich and T cell-poor tumor microenvironment into a more immune-permissive tumor microenvironment that incorporates NK cells and CD8 + T cells, which results in a significant improvement in tumor control.

[0090] [Example 8] Desialylation efficiency of BiTE-sialidase fusion against free sialidase The inventors further compared the desialylation efficiency of both their 4D5 BiTE and sialidase fusion protein and free sialidase. The inventors measured the binding of two lectins PNA, which detect non-sialylated galactose residues, and MAL II, which is specific for α2-3-linked sialic acid, to target cells under three proteins of different concentrations. The results of the comparative study are shown in Figure 8. As can be seen from the figure, both fusion protein constructs showed better desialylation compared to free sialidase, demonstrating the advantage of targeting the tumor surface for stronger sialic acid removal. Furthermore, the C-terminal conjugate format 4D5 BiTE-sialidase showed better desialylation efficiency than the N-terminal conjugate version. This may explain the discrepancy in cytotoxicity and T cell activation observed between the two constructs.

[0091] [Example 9] BiTE sialidase fusion that selectively engages cytotoxic T cells BiTEs that redirect T cells via CD3 binding have demonstrated promising therapeutic potential as described above. Nevertheless, they were able to stimulate both cytolytic T cells and immunosuppressive regulatory T cells (Tregs) indiscriminately. Therefore, the inventors also constructed a bispecific gamma-delta T cell (γδT) cell engagerase fusion protein that selectively engages Vγ9Vδ2 T cells. Gamma-delta (γδ) T cells are a subset of T cells that promote lymphoid and myeloid inflammatory responses and are particularly essential for the initial inflammatory and immune responses. They contain a γδ T cell receptor (TCR) on their surface, in contrast to the αβ TCR on most T cells. For further information on general γδT cells and particularly Vγ9Vδ2 T cells, see, for example, Reis et al., Science 377:276-284, 2022; Kabelitz, Cells 9:2564, 2020; and Lin et al., Signal Transduct. Target Ther. 5:215, 2020. Thus, unlike conventional αβ T cells that engage molecules acting via CD3 binding, the involvement of γδ T cells is usually achieved by γδ TCR targeting.

[0092] An exemplary BiTE sialidase fusion that engages Vγ9Vδ2 T cells and the tumor marker Her2 is shown in SEQ ID NO: 41. The BiTE molecule in this fusion protein 4D5-7A5 (SEQ ID NO: 40) was constructed with a γδ TCR targeting scFv, 7A5 (SEQ ID NO: 39), and Her2 targeting scFv 4D5 (SEQ ID NO: 3). The BiTE sequence was then fused to B. infantis sialidase (SEQ ID NO: 5) using an appropriate GS linker, such as (GGGGS) 2 (SEQ ID NO: 29) exemplified herein. In addition to the exemplified linker, γδ T cells that engage the BiTE-sialidase fusion protein of the present invention may be other appropriate GS linkers described herein, such as (GGGGS) 3 (SEQ ID NO: 30) or (GGGGS) 4(SEQ ID NO: 31) can be easily used. These bispecific T cell engaging fusion molecules were able to have potent anti-tumor properties without a suppressive function. See, for example, Park et al., Exp Mol Med. 53:318-27, 2021.

[0093] [Example 10] In Vitro and In Vivo Activity of BiKE-Cialidase Fusion In addition to BiTE-Cialidase fusions, the inventors also constructed and examined the activity of a BiKE-Cialidase fusion protein targeting CD19 and EGFR. To identify the optimal design of the Cialidase fusion BiKE, as shown in Figure 9, B. infantis cialidase was fused to the N-terminus or C-terminus of BiKE having (G4S)n linkers of different lengths (n = 2, 3, or 4). Two tumor antigens (TA), CD19 and EGFR, were selected for targeting as a proof of concept. The nomenclature for each design of the Cialidase fusion BiKE is shown in the figure. All BiKE and Cialidase fusion BiKE (SEQ ID NOs: 23-28) were fused with a 6xhis tag at the C-terminus for purification and expressed in the Expi293f cell line.

[0094] To compare the cytotoxicity of anti-CD19 or anti-EGFR BiKE with different designs of Cialidase fusion BiKE, CD19+ NALM6 cells (Figure 10, left) and EGFR+ MDA-MB-231 cells (Figure 10, right) were used as target cells, and primary human NK cells were used as effector cells. Human NK cells were purified from PBMCs by magnetic negative selection and then left overnight in culture medium supplemented with 100 IU of IL2 before the killing assay. 1x10 4 target cells were seeded into 96-well plates, and BiKE, Cialidase fusion BiKE (10 pm for anti-CD19 engager and 100 pm for anti-EGFR engager) or PBS was added to a final volume of 100 μl, and the treatment was pre-incubated with the target cells at 37°C for 30 minutes. Next, 1x10 4Individual human NK cells were added at an effector-to-target (E / T) ratio of 1:1. The assay plates were incubated for 12 hours, and NK cell-mediated cytotoxicity was quantified by a luciferin reporter assay. Three independent experimental replicates were shown. Two-way analysis of variance (ANOVA). According to the results shown herein, the inventors selected the TAL4S design for future experiments. Unless otherwise specified, BIKE-CD19-Sia and BIKE-EGFR-Sia refer to the TAL4S format (SEQ ID NOs: 26 and 28).

[0095] The inventors compared the desialylation activity and selectivity of BiKE-CD19-Sia and BiKE-EGFR-Sia in co-culture assays containing CD19+ (Daudi) and EGFR+ (A549) cells, respectively. Daudi cells and A549 cells at a 1:1 ratio were treated with PBS, 10 nM BiKE-CD19-Sia, or 10 nM BiKE-EGFR-Sia in serum-free medium for 1 hour, followed by staining with SNA-biotin (targeting α2,6-linked sialoglycans) or MALII-biotin (targeting α2,3-linked sialoglycans) in HBSS buffer for 30 minutes. Next, the cell mixture was stained with streptavidin-APC and EGFR-Pc5.5. Representative flow cytometry dot plots from n = 3 independent experiments are shown in FIG. 11. Further data regarding the desialylation activity of the BiKE sialidase fusion molecules are shown in FIG. 13.

[0096] Next, the inventors compared the antitumor functions of BiKE-EGFR-Sia and BiKE-EGFR in vivo (FIG. 12). After subcutaneous inoculation of B15E5 cells into C57BL / 6 mice, when the tumor size reached 50 mm 2When this was reached, intraperitoneal (i.p.) treatment with PBS, 5 μg of BiKE-EGFR (low dose), 5 μg of BiKE-EGFR (high dose), and 9 μg of BiKE-EGFR-Sia was continued. Mice were injected i.p. with PBS or the engager every 2 days until the PBS-treated mice began to reach the tumor burden requiring euthanasia. Six independent experimental replicates were shown. Mean ± s.e.m.; **P < 0.01, ****P < 0.0001; two-way ANOVA.

[0097] [Example 11] BiTE mRNA Delivery Formulation The mRNA of BiTE-sialidase was produced using in vitro translation (IVT). Briefly, the coding fragment of each protein was prepared by cloning into the pCS2+MT vector with optimized 5’(3’) untranslated regions and polyA sequences. The IVT reaction followed a standard protocol but was performed using N1-methylpseudouridine-5’-triphosphate instead of typical uridine triphosphate. Finally, the mRNA was capped (Cap-1) using Vaccinia virus capping enzyme and 2’-O-methyltransferase New England Biolabs (NEB). The RNA-loaded LNP formulation was formed using the ethanol dilution method.

[0098] All lipids with the following specific molar ratios were used: 0.43 mg of ALC-0315 = (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), 0.05 mg of ALC-0159 = 2-[(polyethylene glycol)-2000]-N,N ditetradecylacetamide, 0.09 mg of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) 0.2 mg of cholesterol (46.3:9.4:42.7:1.6, molar lipid ratio, %); or SM-102 (heptadec-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate} PEG2000-DMG = 1-methoxypolyethylene glycol-2,3-dimyristylglycerol and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) cholesterol (50:10:38.5:1.5, molar lipid ratio, %).

[0099] All lipids having a specific molar ratio were dissolved in ethanol, and mRNA was dissolved in 10 mM citrate buffer (pH 4.0). The two solutions were rapidly mixed at a 3 / 1 aqueous to ethanol volume ratio (3 / 1 aqueous / ethanol, vol. / vol.) to achieve a final weight ratio of 40 / 1 (total lipid / mRNA), and then incubated at room temperature for 10 minutes. After LNP formation, the fresh LNP formulation was diluted with 1xPBS to 0.5 ng μl-1 mRNA (final ethanol concentration < 5%) for in vitro assays and size detection. For in vivo experiments, the formulation was dialyzed against 1xPBS for 2 hours and diluted with PBS for i.v. or s.c. injection.

[0100] [Example 12] Some exemplary methods and materials Cell lines and cell culture: SK-BR-3 cells, MCF7 cells, PC3 cells, Raji cells, SKOV-3 cells, MDA-MB-435 cells, MDA-MB-231 cells, MDA-MB-468 cells, NALM-6, NK92MI were obtained from ATCC and cultured as suggested. B16-E5 cells were kindly provided by Dr. Yangxin Fu. Expi293f cells were purchased from Thermo Fisher Scientific and cultured according to the protocol. For the culture of isolated human PBMC, AIM V (trademark) medium (Gibco (trademark) 12055091) supplemented with 10% FBS was used. All cells were cultured in an incubator at 37 °C supplemented with 5% CO2.

[0101] General gene cloning procedure: The protein sequences of ScFvs targeting human CD3, CD19, HER2, and PSMA were obtained from publicly available patents, reverse translated, and codon-optimized into DNA sequences. All ScFv sequences were synthesized from IDT. The sequences of EGFR and mouse CD3-binding ScFv were kindly provided by Dr. Yangxin Fu. The sequence of B. infantis sialidase was also kindly provided by George Peng Wang’s lab. For the molecular cloning process, NEBuilder HiFi DNA Assembly (New England BioLabs, E2621) was used to assemble the differential sequences. For BiTE molecules, two separate ScFv sequences were linked by a GS linker such as GGGS (SEQ ID NO: 1) or GGGGS (SEQ ID NO: 38). For BiTE and sialidase fusion proteins, the sialidase sequence was conjugated to the BiTE sequence via a 2xGGGGS linker (SEQ ID NO: 29).

[0102] Expression of BiTE, B. infantis sialidase, and BiTE-sialidase fusion proteins: All BiTE, sialidase, and BiTE-sialidase fusion proteins were fused with a 6xhis tag at the C-terminus for purification. For all BiTE and BiTE-sialidase fusion proteins, expression was performed in the Expi293f cell line (Thermo Fisher Scientific). Cell transfection and handling were performed according to the manufacturer's protocol. B. infantis sialidase was expressed in BL21 E. coli. For purification, all proteins were purified using Ni-NTA (nickel-nitrilo triacetic acid) resin from QIAGEN. After incubating the Expi293 cell supernatant with Ni-NTA resin, the nickel-loaded resin was washed with PBS and 20 mM imidazole. The protein was eluted with 250 mM imidazole, concentrated, and buffer-exchanged into PBS before use. The concentration of all proteins was determined by the Qubit protein quantification assay (Thermo Fisher Scientific, Q33211).

[0103] B. Infantis sialidase, 4D5 BiTE-sialidase fusion protein, and desialylation by P-3Fax-Neu5Ac: For the removal of sialic acid by B. Infantis sialidase or 4D5 BiTE-sialidase fusion protein, 500,000 cells were suspended in 100 mL of DMEM without serum. 1.5 mg of sialidase or fusion protein was added to each sample, and each sample was incubated at 37 °C for 1 hour. After incubation, the cells were washed twice with DPBS and then used for the killing experiment or staining. For desialylation by the inhibitor P-3Fax-Neu5Ac (R&D Systems, 117405-58-0), SK-BR-3 cells were cultured in a T25 flask supplemented with 100 mM P-3Fax-Neu5Ac for 3 days.

[0104] Detection of desialylation from SNA staining: For SNA staining, 500,000 cells with or without desialylation were suspended in 100 mL of HBSS buffer (Sigma-Aldrich, H6648) supplemented with 5 mM CaCl2 and MgCl2. SNA-FITC was added at 1:200 and DAPI was added at 1:2000 to each sample. The mixture was incubated on ice for 30 minutes and then washed twice with HBSS buffer. The samples were then analyzed by FACS. Desialylation was analyzed in the DAPI-negative live cell population using Flowjo.

[0105] Isolation of human PBMC and T cells: Human PBMC were collected from blood samples of multiple healthy donors. Briefly, blood samples were diluted with an equal volume of DPBS containing 2 mM EDTA. The mixture was then carefully layered onto Ficoll (Ficoll® Paque Plus, GE Healthcare, 17-1440-02) for gradient separation. After centrifugation at 650 g for 30 minutes with minimum acceleration and deceleration settings, the interphase was collected and washed twice with DPBS supplemented with 2 mM EDTA. Further T cell isolation from human PBMC was performed using the EasySep™ Human T Cell Isolation Kit (STEMCELL Technologies, 100-0695) according to the manufacturer's protocol.

[0106] Measurement of cytotoxicity by lactate dehydrogenase (LDH) release: T cell cytotoxicity induced by BiTE and BiTE-cyanidase fusion proteins was measured by lactate dehydrogenase (LDH) release using the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, G1780). 100 μL of medium containing tumor cells and hPBMC (tumor cells:hPBMC = 1:5) / well was exposed to different treatments and incubated at 37 °C in a 96-well plate (unless otherwise specified for different ratios elsewhere). After 24 hours of co-incubation, 50 μL of the culture supernatant from each well was transferred to a new flat-bottom 96-well plate, and LDH release was measured using the supplier's protocol. Specific killing was calculated using background subtraction and total lysis comparison as suggested by the supplier's protocol.

[0107] Cytokine Release and T Cell Surface Activation Marker Measurement: For the measurement of T cell cytokine release, similar to the cytotoxicity experiment, tumor cells and hPBMCs (tumor cells:hPBMCs = 1:5) were co-incubated per well in a 96-well plate with different treatments in 100 mL of medium at 37 °C for 24 hours. Then, 20 mL of the supernatant from each well was diluted with 100 mL of DPBS and used for the measurement of IFN-γ, IL-2, and TNFα. ELISA measurements were performed using the ELISA MAX (trademark) set. The IFN-γ, IL-2, and TNFα kits (BioLegend) and the experiments were conducted according to the manufacturer's protocol. The exact concentrations were calculated from the standard curves. For the measurement of cell surface activation markers, tumor cells and hPBMCs (tumor cells:hPBMCs = 1:5) were co-incubated per well in a 12-well plate with different treatments in 1 mL of medium at 37 °C for 24 hours. After incubation, the cells from each well were resuspended and stained with anti-CD3-PE, anti-CD69-FITC, anti-CD25-APC, or anti-CD107a-Pacific Blue (all from Biolegend, added at 1:200) at 4 °C for 30 minutes. Then, the cells were washed twice with FACS buffer (PBS containing 2.5% BSA) and analyzed using flow cytometry. Data analysis and calculation of the mean fluorescence intensity were performed by Flowjo. For transcriptome analysis, 1.2 million hPBMCs and 100,000 MDA-MB-231 cells were incubated together under the treatment of either 4 nM of 4D5 BiTE or 4 nM of 4D5 BiTE siaridase (3 replicates for each condition). After 48 hours of incubation, the mixture was stained with DAPI and CD3 to select the T cell population. The mRNA of T cells in each population was extracted using the Arcturus PicoPure RNA Isolation Kit (Thermo fisher). The mRNA samples were sent to Novogene for sequencing and initial analysis.

[0108] Flow cytometric analysis of Siglec-7 and Siglec-9 expression: Human PBMCs were collected from four healthy human donors. 500,000 freshly isolated human PBMCs were suspended in 100 mL of FACS buffer (PBS containing 2.5% BSA), and each sample was stained with anti-CD3-PE. Each sample was stained with either anti-Siglec-7-APC or anti-Siglec-9-APC (both from Biolegend, added at 1:200). After incubation at 4°C for 30 minutes, the cells were washed twice with FACS buffer and then analyzed using flow cytometry. The percentage of positive populations in both the Siglec-7 and Siglec-9 staining samples was analyzed by Flowjo. For T cells activated by BiTE, 80,000 tumor cells and 400,000 hPBMCs were co-incubated per well in 1 mL of medium in a 12-well plate at 37°C for 24 hours with or without BiTE and sialidase treatment. After incubation, the cells were resuspended and stained as described above for Siglec-7 and Siglec-9 expression analysis.

[0109] Staining of Human CD3ζ and Actin for Co-Focal Imaging: Briefly, 400,000 tumor cells were treated with 4 nM of 4D5 BiTE or 4 nM of 4D5 BiTE containing 15 mg / ML sialidase in 100 mL of serum-free DMEM at 37 °C for 1 hour. After incubation, all samples were washed twice with PBS and then incubated with 400,000 hPBMCs in 500 ml of PBS at 37 °C for 30 minutes. Next, all cells were transferred to coverslips in a 12-well plate in 1 ml of PBS and incubated at 37 °C for 30 minutes to attach the cells to the coverslips. 1 ml of 4% PFA was added to each well and incubated at room temperature (RT) for 20 minutes with shaking for cell fixation, and then each well was washed twice with ice-cold PBS. Washed at RT for 10 minutes with shaking. After fixation, 1 mL of 0.1% PBS-Triton 100 was added to each well at room temperature with shaking for 10 minutes to permeabilize the samples. PBST was used for washing three times, each time for 5 minutes at RT with shaking. Next, 1 mL of 2.5% FBS-PBST was used to block each sample at RT with shaking for 50 minutes. Then, the anti-CD247 (CD3ζ) antibody (Sigma-Aldrich, 12-35-22-00) was diluted 1:200 with FACs buffer, and the anti-actin antibody (Novus Biologicals, NBP267113) was diluted 1:500. 500 mL of each diluted antibody was added to the samples and incubated at room temperature for 1 hour with shaking. After using PBST for washing three times, the anti-rabbit 488 (Invitrogen, 35553) and anti-mouse 594 (Invitrogen, A-11005) secondary antibodies were diluted and used for staining at room temperature for 30 minutes with shaking. Finally, the samples were washed three times and each coverslip was transferred to a slide glass with mounting oil. The coverslip was sealed with nail oil. The samples were analyzed with a Zeiss LSM880 equipped with a 63x oil lens (NA 1.4). The relative mean fluorescence intensity (MFI) of CD3ζ accumulation and the relative contact area of IS were calculated by ImageJ.

[0110] Cluster formation analysis: Regarding the cluster formation experiment between SK-BR-3 cells and T cells. 500,000 SK-BR-3 cells and 1,000,000 hPMBCs were separately stained with CellTracker™ Green CMFDA (Thermo fisher) and PE anti-CD3. After washing, they were incubated at 37 °C for 2 hours under the treatment of 50 nM 4D5 BiTE with or without sialidase or 50 nM 4D5 BiTE-sialidase, and then the samples were analyzed by a FACS instrument. The cluster experiment of NALM-6 cells was performed in the same procedure and settings except that NALM-6 GL cells have GFP expression that does not require CellTracker staining. All results were analyzed by Flowjo.

[0111] RNA sequencing analysis: The quality of raw sequencing reads was verified using FastQC (FastQC: a quality control tool for high-throughput sequence data), which is available online. The reads were aligned to the genome, and gene reads were quantified using STAR version 2.7.0f (Dobin et al., Bioinformatics 29, 15 - 21, 2013) and the Ensembl version 101 GRCm38 genome and transcriptome annotation. Normalization, differential expression analysis, and principal component analysis were performed using the R package DESeq2 v1.35.0. Heatmaps were constructed using the R package ComplexHeatmap v2.12.0. R version 4.2.1 was used. Cytokine target expression analysis was performed using the python implementation of CytoSig (Jiang et al., Nature methods 18, 1181 - 1191, 2021). Gene set enrichment analysis was performed using GSEA (Subramanian et al., Proc. Natl. Acad. Sci. USA 102, 15545 - 15550, 2005).

[0112] Immunodeficient human tumor cell line xenograft mouse model: All animal experiments were approved by the TSRI Animal Care and Use Committee. Fifteen NCG (6-week-old male) mice (Charles Rivers Laboratories) were injected intraperitoneally with 5x10 6 human PBMC and subcutaneously with 2.5x10 6 SK-BR-3 cells on day 0. On day 6, the mice were imaged by BLI and grouped based on similar tumor burdens within each group. On day 7, three groups were treated intravenously (i.v.) with PBS, 6 mg of 4D5 BiTE, and 10 mg of 4D5 BiTE-sialidase, respectively. Blood was collected from each mouse 5 hours after BiTE administration, and serum IFN-γ levels were measured using ELISA MAX™ (Biologend). Drug treatment was continued twice a week, and the mice were administered a second dose of 2x10 6 human PBMC intraperitoneally on day 16, respectively. Throughout the test process, the tumor burden was imaged multiple times. For BLI imaging, 200 μL of 15 g / L D-luciferin, potassium salt (GoldBio) was injected intraperitoneally into each mouse, and the mice were imaged by an IVIS imaging system (PerkinElmer) 10 minutes later. For the NALM-6 model, twenty NCG (6-week-old male) mice (Charles Rivers Laboratories) were injected intravenously (i.v.) with 6x10 6 human PBMC and 0.8x10 6 NALM-6 cells on day 0. On day 3, all mice were imaged and divided into four groups. 1.5 mg of CD19 BiTE, 2.8 mg of CD19 BiTE-sialidase, 4D5 BiTE-sialidase, and PBS were injected into different groups, respectively. Tumor size was measured by BLI as described above until the death of the PBS control group.

[0113] B16-E5 syngeneic mouse model: For the B16-E5 syngeneic mouse model, 0.6x10 6Individual B16-E5 cells were subcutaneously injected on day 0. On day 8, tumor sizes were obtained by caliper measurement using the formula V = (W2xL) / 2, and the mice were divided into different groups. Intratumoral injections of 0.5 mg of EGFR BiTE, 0.93 mg of EGFR BiTE - sialidase, and PBS were performed on mice in different groups on days 8, 12, and 14. Tumor sizes were recorded every 2 days until the mice reached the endpoint of a tumor size of 1000 mm 3 . For tumor-infiltrating lymphocyte profiling, 15 C57BL / 6J mice (6-week-old males) were also subcutaneously injected with 0.6 x 10 6 individual B16-E5 cells on day 0. On day 11, tumor sizes were measured and the mice were divided into three groups. 1.5 mg of EGFR BiTE, 2.8 mg of EGFR BiTE - sialidase, and PBS were intratumorally injected into the tumors of different groups. On day 14, the tumors were harvested, and for profiling, tumor-infiltrating lymphocytes from each tumor of different groups were stained with multiple markers for different populations within CD45.2 lymphocytes.

[0114] Statistical analysis: Unless otherwise specified, results were shown using GraphPad Prism version 8.0.0 with the standard error of the mean (SEM) as error bars, and each dot represents a biological replicate. P-values were calculated using the built-in data analysis functions of Microsoft excel or GraphPad Prism8.

[0115] Some of the amino acid sequences exemplified herein Peptide linker: TIFF2025517636000002.tif791484D5 scFv targeting HER2 (SEQ ID NO: 3) TIFF2025517636000003.tif55153Anti-CD3 scFv (SEQ ID NO: 4) TIFF2025517636000004.tif55153Bifidobacterium longum subsp. infantis (B. infantis) sialidase (SEQ ID NO: 5) TIFF2025517636000005.tif871524D5 - CD3 BiTE (SEQ ID NO: 6) (underlined 4D5 scFv sequence; italicized anti - CD3 sequence) TIFF2025517636000006.tif1121534D5 - CD3 BiTE - sialidase fusion (SEQ ID NO: 7) (linker sequence connecting BiTE and sialidase in bold and italic) TIFF2025517636000007.tif198153Sialidase 4D5 - BiTE fusion (SEQ ID NO: 8) (linker sequence connecting sialidase and BiTE in bold and italic) TIFF2025517636000008.tif216153PSMA - targeted scFv (SEQ ID NO: 9) TIFF2025517636000009.tif56152PSMA - CD3 BiTE (SEQ ID NO: 10) (underlined anti - PSMA scFv; italicized anti - CD3; bold and italic GS linker) TIFF2025517636000010.tif110153PSMA - CD3 BiTE sialidase fusion (SEQ ID NO: 11) (linker sequence connecting BiTE and sialidase underlined and italicized) TIFF2025517636000011.tif208152Blinatumomab (CD19 BiTE) (SEQ ID NO: 12) TIFF2025517636000012.tif112152Blinatumomab - sialidase fusion (CD19 BiTE - sialidase) (SEQ ID NO: 13) TIFF2025517636000013.tif2071525E5 - BiTE (anti - Tn - MUC1) (SEQ ID NO: 14) TIFF2025517636000014.tif1131525E5 BiTE - sialidase fusion (SEQ ID NO: 15) TIFF2025517636000015.tif200151Human sialidase sequence (SEQ ID NOs: 16 - 22) Human NEU1 (SEQ ID NO: 16): TIFF2025517636000016.tif135151Human NEU 2 (Accession No. 17) TIFF2025517636000017.tif119152Human NEU 3, Isoform 1 (Accession No. 18) TIFF2025517636000018.tif136152Human NEU 3, Isoform 2 (Accession No. 19) TIFF2025517636000019.tif153152Human NEU 4, Isoform 1 (Accession No. 20) TIFF2025517636000020.tif159152Human NEU 4, Isoform 2 (Accession No. 21) TIFF2025517636000021.tif159152Human NEU 4, Isoform 3 (Accession No. 22) TIFF2025517636000022.tif160152CD19-CD16A BiKE (Accession No. 32) TIFF2025517636000023.tif110152EGFR-CD16A BiKE (Accession No. 33) TIFF2025517636000024.tif88152Anti-CD19 BiKE - Chalarase Fusion TAL2S (Accession No. 23) TIFF2025517636000025.tif208152Anti-EGFR BiKE - Chalarase Fusion TAL2S (Accession No. 24) TIFF2025517636000026.tif202152Additional BiKE - Chalarase Fusion Sequence (Underlined BiKE Sequence; Italic and Bold Linker): Anti-CD19 BiKE - Chalarase Fusion TAL3S (Accession No. 25) TIFF2025517636000027.tif209152Anti-CD19 BiKE - Chalarase Fusion TAL4S (Accession No. 26) TIFF2025517636000028.tif209152Anti-EGFR BiKE - Chalarase Fusion TAL3S (Accession No. 27) TIFF2025517636000029.tif209153 Anti-EGFR BiKE - Sialidase fusion TAL4S (SEQ ID NO: 28) TIFF2025517636000030.tif208153 7A5 scFv targeting Vγ9Vδ2 TCR (SEQ ID NO: 39): TIFF2025517636000031.tif571514 D5 - 7A5 BiTE (SEQ ID NO: 40) TIFF2025517636000032.tif1121524 D5 - 7A5 BiTE - Sialidase fusion (SEQ ID NO: 41): TIFF2025517636000033.tif208152 The foregoing invention has been described in some detail by way of illustration and example for the purpose of clarity of understanding, but it will be readily apparent to those skilled in the art that certain changes and modifications can be made without departing from the spirit or scope of the appended claims.

[0116] All publications, databases, GenBank sequences, patents, and patent applications cited herein are hereby incorporated by reference as if each were specifically and individually indicated to be incorporated by reference.

Claims

1. A fusion polypeptide comprising: (a) a bispecific molecule; and (b) a sialidase or an enzymatic fragment thereof, wherein the bispecific molecule comprises two antibody portions or antigen-binding fragments that bind to an immune cell and an antigen associated with a disease, respectively.

2. The fusion polypeptide of claim 1, wherein the bispecific molecule comprises in tandem a first scFv that binds to the immune cell and a second scFv that binds to the antigen associated with a disease.

3. The fusion polypeptide of claim 2, wherein the bispecific molecule is a bispecific T cell engager (BiTE) and the first scFv recognizes a T cell-specific molecule.

4. The fusion polypeptide of claim 3 , wherein the T cell specific molecule is CD3.

5. 4. The fusion polypeptide of claim 3, wherein the BiTE selectively engages gamma delta T (γδT) cells and the T cell-specific molecule is a Vγ9Vδ2 TCR.

6. The fusion polypeptide of claim 2, wherein the bispecific molecule is a bispecific innate cell engager and the first scFv recognizes a surface antigen on an innate immune cell.

7. The fusion polypeptide of claim 6 , wherein the innate immune cell is a NK cell or a macrophage.

8. The fusion polypeptide of claim 7, wherein the surface antigen is CD16A or NKp44.

9. The fusion polypeptide of claim 2 , wherein the disease is a tumor.

10. The fusion polypeptide of claim 9, wherein the second scFv binds to CD19, HER2 or PSMA.

11. The fusion polypeptide of claim 1 , wherein the sialidase is a human sialidase, a viral sialidase, or a bacterial sialidase.

12. The fusion polypeptide of claim 11 , wherein the human sialidase is NEU1, NEU2, NEU3, NEU4 or an isoform thereof.

13. 12. The fusion polypeptide of claim 11, wherein the bacterial sialidase is a B. infantis sialidase.

14. The fusion polypeptide of claim 1 , wherein the sialidase is fused to the C-terminus or N-terminus of the bispecific molecule.

15. The fusion polypeptide of claim 1, wherein the sialidase is fused to the bispecific molecule via a GS linker.

16. The fusion polypeptide of claim 15, wherein the GS linker comprises (GmS)n, where m is an integer from 1 to 6 and n is an integer from 1 to 10.

17. The GS linker is (GGGS) ２ (SEQ ID NO: 2), (GGGGS) ２ (SEQ ID NO: 29), (GGGGS) ３ (SEQ ID NO: 30) or (GGGGS) ４ The fusion polypeptide of claim 15 comprising: (SEQ ID NO: 31).

18. The fusion polypeptide of claim 1 , wherein the two antibody portions or antigen-binding fragments are linked by a GS linker.

19. The fusion polypeptide of claim 1, wherein the bispecific molecule comprises a sequence that is at least 95% or 99% identical to any one of SEQ ID NOs: 6, 10, 12, 14, 31, 32 and 40.

20. The fusion polypeptide of claim 1, wherein the bispecific molecule comprises a sequence set forth in any one of SEQ ID NOs: 6, 10, 12, 14, 31, 32 and 40, or a conservatively modified variant thereof.

21. The fusion polypeptide of claim 1, comprising a sequence at least 95% or 99% identical to any one of SEQ ID NOs: 7, 8, 11, 13, 15, 23-28 and 41.

22. 2. The fusion polypeptide of claim 1, comprising a sequence according to any one of SEQ ID NOs: 7, 8, 11, 13, 15, 23-28 and 41, or a conservatively modified variant thereof.

23. A pharmaceutical composition comprising a therapeutically effective amount of the fusion polypeptide of claim 1 and a pharma- ceutically acceptable carrier.

24. A kit comprising the fusion polypeptide of claim 1.

25. A method for treating or ameliorating a symptom of a disease or disorder in a subject, comprising administering to the subject a pharmaceutical composition comprising the fusion polypeptide of claim 1.

26. 26. The method of claim 25, wherein the disease is a tumor.

27. A polynucleotide encoding the fusion polypeptide of claim 1.

28. A vector harboring the polynucleotide of claim 27.

29. A lipid nanoparticle (LNP) formulated with the polynucleotide of claim 27.

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