Sialidase-PD-1-Antibody Fusion Protein and Methods of Use Thereof

Abstract

The present invention generally relates to a fusion protein of a recombinant sialidase and an anti-PD-1 immunoglobulin antigen-binding domain. The present invention also provides an antibody conjugate comprising a sialidase and an anti-PD-1 antibody or a portion thereof. The present invention further relates to a method of using the sialidase fusion protein or antibody conjugate to treat cancer.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 134,415, filed January 6, 2021, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0002] The present invention relates generally to recombinant sialidase fusion proteins and antibody conjugates and their use in the treatment of cancer. [Background technology]

[0003] A growing body of evidence supports the role of glycans, and particularly sialoglycans, in various pathophysiological stages of tumor progression. Glycans regulate tumor growth, invasion, hematogenous metastasis, and angiogenesis (Fuster et al. (2005) NAT. REV. CANCER 5(7):526-42). Sialylation of cell surface glycoconjugates is frequently altered in cancer, resulting in the expression of sialylated tumor-associated carbohydrate antigens. The expression of sialylated glycans by tumor cells is often associated with increased tumor aggressiveness and metastatic potential (Julien S., Delannoy P. (2015) Sialic Acid and Cancer. In: Taniguchi N., Endo T., Hart G., Seeberger P., Wong CH. (eds) Glycoscience: Biology and Medicine. Springer, Tokyo. https: / / doi.org / 10.1007 / 978-4-431-54841-6_193).

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

[0005] PD-1 is a receptor present on the surface of T cells that acts as an immune system checkpoint, suppressing or otherwise regulating T cell activity at appropriate times to prevent an excessive immune response. However, cancer cells can exploit this checkpoint by expressing ligands (e.g., PD-L1) that interact with PD-1 on the surface of T cells and reduce T cell activity. Many anti-PD-1 antibodies have been developed for use in cancer immunotherapy. For example, nivolumab is approved in the United States for use in the treatment of, e.g., certain melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), mesothelioma, renal cell carcinoma (RCC), Hodgkin's lymphoma, squamous cell carcinoma of the head and neck, urothelial carcinoma, colorectal cancer, hepatocellular carcinoma, and esophageal squamous cell carcinoma, and pembrolizumab is approved in the United States for use in the treatment of, e.g., certain melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), squamous cell carcinoma of the head and neck, Hodgkin's lymphoma, primary mediastinal large B-cell lymphoma, urothelial carcinoma, gastric cancer, esophageal cancer, cervical cancer, hepatocellular carcinoma, Merkel cell carcinoma, renal cell carcinoma (RCC), endometrial cancer, cutaneous squamous cell carcinoma, and triple-negative breast cancer.

[0006] Cancer immunotherapy using immune checkpoint inhibitors, including antibodies that block the PD-1 / PD-L1 pathway, has improved outcomes for many cancer patients. However, despite the advances made to date, many patients do not respond to currently available immune checkpoint inhibitors. Therefore, there remains a need for effective interventions to overcome the immunosuppressive tumor microenvironment and treat cancers associated with hypersialylated cells. Summary of the Invention

[0007] The present invention is based, in part, on the discovery that it is possible to generate fusion proteins comprising a sialidase enzyme and an anti-PD-1 immunoglobulin, or a portion thereof (e.g., an antigen-binding domain and / or an immunoglobulin Fc domain), and / or antibody conjugates comprising a sialidase enzyme and an anti-PD-1 antibody, or a portion thereof (e.g., an antigen-binding domain and / or an immunoglobulin Fc domain). The sialidase enzyme portion of the fusion protein and / or antibody conjugate can comprise at least one mutation compared to wild-type sialidase. Such mutations, or combinations of such mutations, can improve the expression, activity, or both expression and activity of the sialidase, thereby improving its use in the diagnosis and / or treatment of cancer. The fusion proteins and / or antibody conjugates have suitable substrate specificity and activity that are useful for removing sialic acid and / or sialic acid-containing molecules from the surface of cells (e.g., PD-1-expressing cells) and / or removing sialic acid and / or sialic acid-containing molecules from the tumor microenvironment and / or reducing the concentration of sialic acid and / or sialic acid-containing molecules in the tumor microenvironment.

[0008] Accordingly, in one aspect, the invention provides a fusion protein comprising (or consisting essentially of) (a) a sialidase enzyme, and (b) an anti-PD-1 immunoglobulin antigen-binding domain.

[0009] In certain embodiments, the sialidase is a human sialidase (eg, a recombinant mutant human sialidase). In certain embodiments, the sialidase comprises (a) a substitution or deletion of a methionine residue (M1) at a position corresponding to position 1 of wild-type human Neu2, (b) a substitution of a valine residue (V6) at a position corresponding to position 6 of wild-type human Neu2, (c) a substitution of a lysine residue (K9) at a position corresponding to position 9 of wild-type human Neu2, (d) a substitution of an alanine residue (A42) at a position corresponding to position 42 of wild-type human Neu2, (e) a substitution of a proline residue (P62) at a position corresponding to position 62 of wild-type human Neu2, (f) a substitution of an alanine residue (A93) at a position corresponding to position 93 of wild-type human Neu2, (g) a substitution of a glutamine residue (Q126) at a position corresponding to position 126 of wild-type human Neu2, or (h) an isoleucine residue at a position corresponding to position 187 of wild-type human Neu2. (i) a substitution of an alanine residue (A242) at a position corresponding to position 242 of wild-type human Neu2; (j) a substitution of a glutamine residue (Q270) at a position corresponding to position 270 of wild-type human Neu2; (k) a substitution of a serine residue (S301) at a position corresponding to position 301 of wild-type human Neu2; (l) a substitution of a tryptophan residue (W302) at a position corresponding to position 302 of wild-type human Neu2; (m) a substitution of a cysteine ​​residue (C332) at a position corresponding to position 332 of wild-type human Neu2; (n) a substitution of a valine residue (V363) at a position corresponding to position 363 of wild-type human Neu2; or (o) a substitution of a leucine residue (L365) at a position corresponding to position 365 of wild-type human Neu2, or any combination of the foregoing substitutions.

[0010] In certain embodiments, in the sialidase, (a) the methionine residue at the position corresponding to position 1 of wild-type human Neu2 is deleted (ΔM1), substituted with alanine (M1A), or substituted with aspartic acid (M1D), (b) the valine residue at the position corresponding to position 6 of wild-type human Neu2 is substituted with tyrosine (V6Y), (c) the lysine residue at the position corresponding to position 9 of wild-type human Neu2 is substituted with aspartic acid (K9D), and (d) the alanine residue at the position corresponding to position 42 of wild-type human Neu2 is substituted with arginine (K9D). (e) the proline residue at position 62 of wild-type human Neu2 is replaced by asparagine (P62N), aspartic acid (P62D), histidine (P62H), glutamic acid (P62E), glycine (P62G), serine (P62S), or threonine (P62T); (f) the alanine residue at position 93 of wild-type human Neu2 is replaced by glutamic acid (A93E) or lysine (A93K); (g) the alanine residue at position 126 of wild-type human Neu2 is replaced by glutamic acid (A93E) or lysine (A93K). (h) the isoleucine residue at position 187 of wild-type human Neu2 is substituted by lysine (I187K); and (i) the alanine residue at position 242 of wild-type human Neu2 is substituted by cysteine ​​(A242C), phenylalanine (A242F), glycine (A242G), histidine (A242H), isoleucine (Q126I), or tyrosine (Q126Y). (j) the glutamine residue at position 270 of wild-type human Neu2 is replaced by alanine (Q270A), histidine (Q270H), phenylalanine (Q270F), proline (Q270P), serine (Q270S), or tyrosine (A242Y).or threonine (Q270T), and (k) the serine residue at position 301 of wild-type human Neu2 is replaced by alanine (S301A), aspartic acid (S301D), glutamic acid (S301E), phenylalanine (S301F), histidine (S301H), lysine (S301K), leucine (S301L), methionine (S301M), asparagine (S301N), proline (S301P), glutamine (S30 (1) the tryptophan residue at position 302 of wild-type human Neu2 is replaced by alanine (W302A), aspartic acid (W302D), phenylalanine (W302F), glycine (W302G), histidine (W302H), isoleucine (W302I), lysine (W302K), leucine (W302L), methionine (W302M), asparagine (W302N), proline (W302P), glutamine (W302Q), arginine (W302R), serine (W302S), threonine (W302T), valine (W302V), or tyrosine (W302Y), and (m) the cysteine ​​residue at position 332 of wild-type human Neu2 is replaced by alanine (C332A). (n) the valine residue at the position corresponding to position 363 of wild-type human Neu2 is substituted with arginine (V363R); or (o) the leucine residue at the position corresponding to position 365 of wild-type human Neu2 is substituted with glutamine (L365Q), histidine (L365H), isoleucine (L365I), lysine (L365K), or serine (L365S); or the sialidase comprises any combination of the foregoing substitutions. For example, the sialidase can include a modification selected from ΔM1, M1A, M1D, V6Y, K9D, A42R, P62G, P62N, P62S, P62T, A93E, Q126Y, I187K, A242F, A242W, A242Y, Q270A, Q270T, S301A, S301R, W302K, W302R, C332A, V363R, and L365I, or any combination of the foregoing modifications.

[0011] In certain embodiments, the sialidase has (a) an M1D substitution, a V6Y substitution, a P62G substitution, an A93E substitution, an I187K substitution, and a C332A substitution; (b) an M1D substitution, a V6Y substitution, a K9D substitution, an A93E substitution, an I187K substitution, a C332A substitution, a V363R substitution, and a L365I substitution; (c) an M1D substitution, a V6Y substitution, a P62N substitution, an I187K substitution, and a C332A substitution; (d) an M1D substitution, a V6Y substitution, an I187 K substitution, Q270A substitution, S301R substitution, W302K substitution, and C332A substitution, (e) M1D substitution, V6Y substitution, P62S substitution, I187K substitution, Q270A substitution, S301R substitution, W302K substitution, and C332A substitution, (f) M1D substitution, V6Y substitution, P62T substitution, I187K substitution, Q270A substitution, S301R substitution, W302K substitution, and C332A substitution, (g) M1D substitution, V6Y substitution, P62N substitution, I (h) M1D substitution, V6Y substitution, P62G substitution, A93E substitution, I187K substitution, S301A substitution, W302R substitution, and C332A substitution; (i) M1D substitution, V6Y substitution, P62G substitution, A93E substitution, Q126Y substitution, I187K substitution, Q270T substitution, and C332A substitution; (j) M1D substitution, V6Y substitution, P62G substitution , A93E substitution, Q126Y substitution, I187K substitution, and C332A substitution; (k) M1D substitution, V6Y substitution, P62G substitution, A93E substitution, Q126Y substitution, I187K substitution, A242F substitution, Q270T substitution, and C332A substitution; or (l) M1D mutation, V6Y mutation, A42R mutation, P62G mutation, A93E mutation, Q126Y mutation, I187K mutation, A242F mutation, Q270T mutation, and C332A mutation.

[0012] In certain embodiments, the sialidase is selected from Neu1, Neu2, Neu3, and Neu4, for example, the sialidase is Neu2.

[0013] In some embodiments, the sialidase has a different substrate specificity than the corresponding wild-type human sialidase. For example, in some embodiments, the sialidase can cleave α2,3, α2,6, and / or α2,8 linkages. In some embodiments, the sialidase can cleave α2,3 and α2,8 linkages.

[0014] In some embodiments, the sialidase comprises any of SEQ ID NOs: 48-63, 94, 97, 100, or 126, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 48-63, 94, 97, 100, or 126.

[0015] In certain embodiments, the sialidase comprises a mutation or combination of mutations shown in any one of Tables 1-9.

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

[0017] In certain embodiments, an anti-PD-1 immunoglobulin antigen-binding domain is associated (e.g., covalently or non-covalently) with a second anti-PD-1 immunoglobulin antigen-binding domain to provide an anti-PD-1 antigen-binding site.

[0018] In certain embodiments, the anti-PD-1 immunoglobulin antigen-binding domain is derived from an antibody selected from nivolumab, pembrolizumab, cemiplimab, spartalizumab (PDR001), TX-4014, camrelizumab (SHR1210), sintilimab (IBI308), tislelizumab (BGB-A317), toripalimab (JS001), dostallimab (TSR-042, WBP-285), INCMGA00012 (MGA012), AMP-514, and pidilizumab, e.g., the anti-PD-1 immunoglobulin antigen-binding domain is derived from nivolumab or pembrolizumab.

[0019] In certain embodiments, the sialidase and the immunoglobulin Fc domain and / or the anti-PD-1 immunoglobulin antigen binding domain are linked by a peptide bond or an amino acid linker.

[0020] In certain embodiments, the fusion protein comprises any one of SEQ ID NOs: 67-73, 78, 81-87, 95, 96, 98, 99, 101, 102, 105, 106, 108, 111, 112, 115, 122, 123, 125, 127, 128, 130, 132, 134, or 145.

[0021] In another embodiment, the present invention provides an antibody conjugate comprising any of the aforementioned fusion proteins. In some embodiments, the antibody conjugate comprises a single sialidase. In other embodiments, the antibody conjugate comprises two sialidases, which may be the same or different. In some embodiments, the antibody conjugate comprises two identical sialidases. In some embodiments, the antibody conjugate comprises a single anti-PD-1 antigen-binding site. In other embodiments, the antibody conjugate comprises two anti-PD-1 antigen-binding sites, which may be the same or different. In some embodiments, the antibody conjugate comprises two identical anti-PD-1 antigen-binding sites.

[0022] In certain embodiments, the antibody conjugate has a molecular weight of about 135 kDa to about 165 kDa, or the antibody conjugate has a molecular weight of about 215 kDa to about 245 kDa.

[0023] In some embodiments, the antibody conjugate comprises: (a) a first polypeptide comprising an immunoglobulin light chain; (b) a second polypeptide comprising an immunoglobulin heavy chain; and (c) a third polypeptide comprising an immunoglobulin Fc domain and a sialidase; wherein the first and second polypeptides are covalently linked together and the second and third polypeptides are covalently linked together, and wherein the first and second polypeptides together define an anti-PD-1 antigen-binding site. The third polypeptide can comprise, for example, in N-terminal to C-terminal direction, a sialidase and an immunoglobulin Fc domain. The first polypeptide may, for example, comprise SEQ ID NO: 77, the second polypeptide may, for example, comprise SEQ ID NO: 105, and / or the third polypeptide may, for example, comprise any one of SEQ ID NOs: 67-73, 78, 81-87, 95, 96, 98, 99, 101, 102, 106, 108, 111, 112, 115, 122, 123, 125, 127, or 128.

[0024] In some embodiments, the antibody conjugate comprises: (a) a first polypeptide comprising a first immunoglobulin light chain; (b) a second polypeptide comprising a first immunoglobulin heavy chain and a first sialidase; (c) a third polypeptide comprising a second immunoglobulin heavy chain and a second sialidase; and (d) a fourth polypeptide comprising a second immunoglobulin light chain; wherein the first and second polypeptides are covalently linked together, the third and fourth polypeptides are covalently linked together, and the second and third polypeptides are covalently linked together, wherein the first and second polypeptides together define a first anti-PD-1 antigen-binding site, and the third and fourth polypeptides together define a second anti-PD-1 antigen-binding site. The second and third polypeptides can comprise, e.g., from N-terminus to C-terminus, the first and second immunoglobulin heavy chains and the first and second sialidases, respectively. The first polypeptide and the fourth polypeptide can comprise, for example, SEQ ID NO: 77. The second polypeptide and the third polypeptide can comprise, for example, SEQ ID NO: 145.

[0025] In some embodiments, the antibody conjugate comprises: (a) a first polypeptide comprising a first sialidase, a first immunoglobulin Fc domain, and a first single-chain variable fragment (scFv); and (b) a second polypeptide comprising a second sialidase, a second immunoglobulin Fc domain, and an optional second single-chain variable fragment (scFv); wherein the first and second polypeptides are covalently linked together, and wherein the first scFv defines a first anti-PD-1 antigen-binding site and the second scFv, if present, defines a second anti-PD-1 antigen-binding site. The first polypeptide can comprise, e.g., from N-terminus to C-terminus, the first sialidase, the first immunoglobulin Fc domain, and the first scFv. The second polypeptide can comprise, e.g., from N-terminus to C-terminus, the second sialidase, the second immunoglobulin Fc domain, and an optional second scFv.

[0026] In some embodiments, the antibody conjugate comprises: (a) a first polypeptide comprising an immunoglobulin light chain; (b) a second polypeptide comprising an immunoglobulin heavy chain and a single-chain variable fragment (scFv); and (c) a third polypeptide comprising an immunoglobulin Fc domain and a sialidase, wherein the first and second polypeptides are covalently linked together, the second and third polypeptides are covalently linked together, the immunoglobulin light chain and the immunoglobulin heavy chain together define a first anti-PD-1 antigen-binding site, and the scFv defines a second anti-PD-1 antigen-binding site. The second polypeptide can comprise, e.g., from N- to C-terminus, an immunoglobulin heavy chain and an scFv. The third polypeptide can comprise, e.g., from N- to C-terminus, a sialidase and an immunoglobulin Fc domain.

[0027] In another aspect, the invention provides an isolated nucleic acid comprising a nucleotide sequence encoding at least a portion of any of the aforementioned fusion proteins or any of the aforementioned antibody conjugates. In another aspect, the invention provides an expression vector comprising any of the aforementioned nucleic acids. In another aspect, the invention provides a host cell comprising any of the aforementioned expression vectors.

[0028] In another aspect, the present invention provides a pharmaceutical composition comprising any of the aforementioned fusion proteins or any of the aforementioned antibody conjugates.

[0029] In another aspect, the present invention provides a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of any of the aforementioned fusion proteins, any of the aforementioned antibody conjugates, or any of the aforementioned pharmaceutical compositions.

[0030] In certain embodiments, the cancer is selected from melanoma, bladder cancer, breast cancer, cervical cancer, esophageal cancer, gastric cancer, renal cancer, lung cancer (e.g., NSCLC), ovarian cancer, metastatic Merkel cell carcinoma (MCC), metastatic urothelial carcinoma (UC), pancreatic cancer, colon cancer, gastric cancer, AML, and multiple myeloma. For example, in certain embodiments, the cancer is NSCLC or melanoma.

[0031] These and other aspects and features of the present invention are described in the following detailed description and claims.

[0032] The present invention can be more fully understood with reference to the following drawings. [Brief explanation of the drawings]

[0033] [Figure 1]

[0033] Figure 1 shows an SDS-PAGE gel showing recombinant human Neu1, Neu2, Neu3 and Salmonella typhimurium (St-sialidase) under non-reducing and reducing conditions. Monomeric and dimeric species are indicated. [Figure 2] 1 is a bar graph showing the enzymatic activity of recombinant human Neu1, Neu2, and Neu3. [Figure 3] 1 is a line graph showing enzyme activity as a function of substrate concentration for recombinant human Neu2 and Neu3 at the indicated pH. [Figure 4A]Schematic representations of certain antibody conjugate constructs comprising a sialidase enzyme, e.g., a human sialidase enzyme, and an anti-PD-1 antigen-binding site are shown. For each antibody conjugate construct comprising more than one (e.g., two) sialidases, the respective sialidases can be the same or different. For each antibody conjugate construct comprising more than one (e.g., two) anti-PD-1 antigen-binding site, the respective anti-PD-1 antigen-binding sites can be the same or different. For each antibody conjugate construct comprising an Fc domain, it is understood that the Fc domain can be a wild-type Fc domain or an engineered Fc domain. For example, the Fc domain can be engineered to include either or both of a "knob" mutation, e.g., T366Y, or a "hole" mutation, e.g., Y407T, to promote heterodimerization, or the Fc domain can be engineered to include one or more modifications, e.g., point mutations, to provide any other altered Fc domain functionality. [Figure 4B] Schematic representations of certain antibody conjugate constructs comprising a sialidase enzyme, e.g., a human sialidase enzyme, and an anti-PD-1 antigen-binding site are shown. For each antibody conjugate construct comprising more than one (e.g., two) sialidases, the respective sialidases can be the same or different. For each antibody conjugate construct comprising more than one (e.g., two) anti-PD-1 antigen-binding site, the respective anti-PD-1 antigen-binding sites can be the same or different. For each antibody conjugate construct comprising an Fc domain, it is understood that the Fc domain can be a wild-type Fc domain or an engineered Fc domain. For example, the Fc domain can be engineered to include either or both of a "knob" mutation, e.g., T366Y, or a "hole" mutation, e.g., Y407T, to promote heterodimerization, or the Fc domain can be engineered to include one or more modifications, e.g., point mutations, to provide any other altered Fc domain functionality. [Figure 4C] Schematic representations of certain antibody conjugate constructs comprising a sialidase enzyme, e.g., a human sialidase enzyme, and an anti-PD-1 antigen-binding site are shown. For each antibody conjugate construct comprising more than one (e.g., two) sialidases, the respective sialidases can be the same or different. For each antibody conjugate construct comprising more than one (e.g., two) anti-PD-1 antigen-binding site, the respective anti-PD-1 antigen-binding sites can be the same or different. For each antibody conjugate construct comprising an Fc domain, it is understood that the Fc domain can be a wild-type Fc domain or an engineered Fc domain. For example, the Fc domain can be engineered to include either or both of a "knob" mutation, e.g., T366Y, or a "hole" mutation, e.g., Y407T, to promote heterodimerization, or the Fc domain can be engineered to include one or more modifications, e.g., point mutations, to provide any other altered Fc domain functionality. [Figure 4D]Schematic representations of certain antibody conjugate constructs comprising a sialidase enzyme, e.g., a human sialidase enzyme, and an anti-PD-1 antigen-binding site are shown. For each antibody conjugate construct comprising more than one (e.g., two) sialidases, the respective sialidases can be the same or different. For each antibody conjugate construct comprising more than one (e.g., two) anti-PD-1 antigen-binding site, the respective anti-PD-1 antigen-binding sites can be the same or different. For each antibody conjugate construct comprising an Fc domain, it is understood that the Fc domain can be a wild-type Fc domain or an engineered Fc domain. For example, the Fc domain can be engineered to include either or both of a "knob" mutation, e.g., T366Y, or a "hole" mutation, e.g., Y407T, to promote heterodimerization, or the Fc domain can be engineered to include one or more modifications, e.g., point mutations, to provide any other altered Fc domain functionality. [Figure 4E] Schematic representations of certain antibody conjugate constructs comprising a sialidase enzyme, e.g., a human sialidase enzyme, and an anti-PD-1 antigen-binding site are shown. For each antibody conjugate construct comprising more than one (e.g., two) sialidases, the respective sialidases can be the same or different. For each antibody conjugate construct comprising more than one (e.g., two) anti-PD-1 antigen-binding site, the respective anti-PD-1 antigen-binding sites can be the same or different. For each antibody conjugate construct comprising an Fc domain, it is understood that the Fc domain can be a wild-type Fc domain or an engineered Fc domain. For example, the Fc domain can be engineered to include either or both of a "knob" mutation, e.g., T366Y, or a "hole" mutation, e.g., Y407T, to promote heterodimerization, or the Fc domain can be engineered to include one or more modifications, e.g., point mutations, to provide any other altered Fc domain functionality. [Figure 4F] Schematic representations of certain antibody conjugate constructs comprising a sialidase enzyme, e.g., a human sialidase enzyme, and an anti-PD-1 antigen-binding site are shown. For each antibody conjugate construct comprising more than one (e.g., two) sialidases, the respective sialidases can be the same or different. For each antibody conjugate construct comprising more than one (e.g., two) anti-PD-1 antigen-binding site, the respective anti-PD-1 antigen-binding sites can be the same or different. For each antibody conjugate construct comprising an Fc domain, it is understood that the Fc domain can be a wild-type Fc domain or an engineered Fc domain. For example, the Fc domain can be engineered to include either or both of a "knob" mutation, e.g., T366Y, or a "hole" mutation, e.g., Y407T, to promote heterodimerization, or the Fc domain can be engineered to include one or more modifications, e.g., point mutations, to provide any other altered Fc domain functionality. [Figure 4G]Schematic representations of certain antibody conjugate constructs comprising a sialidase enzyme, e.g., a human sialidase enzyme, and an anti-PD-1 antigen-binding site are shown. For each antibody conjugate construct comprising more than one (e.g., two) sialidases, the respective sialidases can be the same or different. For each antibody conjugate construct comprising more than one (e.g., two) anti-PD-1 antigen-binding site, the respective anti-PD-1 antigen-binding sites can be the same or different. For each antibody conjugate construct comprising an Fc domain, it is understood that the Fc domain can be a wild-type Fc domain or an engineered Fc domain. For example, the Fc domain can be engineered to include either or both of a "knob" mutation, e.g., T366Y, or a "hole" mutation, e.g., Y407T, to promote heterodimerization, or the Fc domain can be engineered to include one or more modifications, e.g., point mutations, to provide any other altered Fc domain functionality. [Figure 4H] Schematic representations of certain antibody conjugate constructs comprising a sialidase enzyme, e.g., a human sialidase enzyme, and an anti-PD-1 antigen-binding site are shown. For each antibody conjugate construct comprising more than one (e.g., two) sialidases, the respective sialidases can be the same or different. For each antibody conjugate construct comprising more than one (e.g., two) anti-PD-1 antigen-binding site, the respective anti-PD-1 antigen-binding sites can be the same or different. For each antibody conjugate construct comprising an Fc domain, it is understood that the Fc domain can be a wild-type Fc domain or an engineered Fc domain. For example, the Fc domain can be engineered to include either or both of a "knob" mutation, e.g., T366Y, or a "hole" mutation, e.g., Y407T, to promote heterodimerization, or the Fc domain can be engineered to include one or more modifications, e.g., point mutations, to provide any other altered Fc domain functionality. [Figure 4I] Schematic representations of certain antibody conjugate constructs comprising a sialidase enzyme, e.g., a human sialidase enzyme, and an anti-PD-1 antigen-binding site are shown. For each antibody conjugate construct comprising more than one (e.g., two) sialidases, the respective sialidases can be the same or different. For each antibody conjugate construct comprising more than one (e.g., two) anti-PD-1 antigen-binding site, the respective anti-PD-1 antigen-binding sites can be the same or different. For each antibody conjugate construct comprising an Fc domain, it is understood that the Fc domain can be a wild-type Fc domain or an engineered Fc domain. For example, the Fc domain can be engineered to include either or both of a "knob" mutation, e.g., T366Y, or a "hole" mutation, e.g., Y407T, to promote heterodimerization, or the Fc domain can be engineered to include one or more modifications, e.g., point mutations, to provide any other altered Fc domain functionality. [Figure 5] Schematic representations of certain antibody conjugate constructs comprising a sialidase enzyme, e.g., a human sialidase enzyme, and an antigen-binding site are shown. For each antibody conjugate construct comprising more than one (e.g., two) antigen-binding site, each antigen-binding site can be the same or different. For each antibody conjugate construct comprising an Fc domain, it is understood that the Fc domain can be a wild-type Fc domain or an engineered Fc domain. For example, the Fc domain can be engineered to include either a "knob" mutation, e.g., T366Y, or a "hole" mutation, e.g., Y407T, or both, to promote heterodimerization, or the Fc domain can be engineered to include one or more modifications, e.g., point mutations, to provide any other altered Fc domain functionality. [Figure 6A]Schematic representation of exemplary fusion protein conjugates designated Raptor antibody sialidase conjugate (A), Janus antibody sialidase conjugate (B), Lobster antibody sialidase conjugate (C), Bunk antibody sialidase conjugate (D), and Lobster-Fab antibody sialidase conjugate (E). [Figure 6B] Schematic representation of exemplary fusion protein conjugates designated Raptor antibody sialidase conjugate (A), Janus antibody sialidase conjugate (B), Lobster antibody sialidase conjugate (C), Bunk antibody sialidase conjugate (D), and Lobster-Fab antibody sialidase conjugate (E). [Figure 6C] Schematic representation of exemplary fusion protein conjugates designated Raptor antibody sialidase conjugate (A), Janus antibody sialidase conjugate (B), Lobster antibody sialidase conjugate (C), Bunk antibody sialidase conjugate (D), and Lobster-Fab antibody sialidase conjugate (E). [Figure 6D] Schematic representation of exemplary fusion protein conjugates designated Raptor antibody sialidase conjugate (A), Janus antibody sialidase conjugate (B), Lobster antibody sialidase conjugate (C), Bunk antibody sialidase conjugate (D), and Lobster-Fab antibody sialidase conjugate (E). [Figure 6E] Schematic representation of exemplary fusion protein conjugates designated Raptor antibody sialidase conjugate (A), Janus antibody sialidase conjugate (B), Lobster antibody sialidase conjugate (C), Bunk antibody sialidase conjugate (D), and Lobster-Fab antibody sialidase conjugate (E). [Figure 7]A shows a size-exclusion chromatography (SEC) trace of ASC#3. B shows a size-exclusion chromatography (SEC) trace of ASC#6. Peaks (triangles) and corresponding elution times are indicated. [Figure 8] Blockade of the interaction between PD-L1 and PD-1 by the indicated ASCs is shown, as measured by fold induction of a PD-1 / PD-L1-linked NFAT-inducible luciferase reporter. Pembrolizumab was included as a control. ASC#3-1, ASC#3-2, and ASC#3-3 represent ASC#3 derived from three different preparations. [Figure 9] Blockade of the interaction between human PD-L1-Fc and human PD-1 by the indicated ASCs, as measured by ELISA. Pembrolizumab was included as a control. [Figure 10A] Figure 1 shows the in vivo efficacy of anti-PD-1 antibody sialidase conjugate ASC#3 in the MC38 mouse syngeneic subcutaneous tumor model. Pembrolizumab and isotype antibodies were included as controls. In A, mean tumor volumes ± SEM over 18 days are shown for the indicated treatments. Triangles indicate drug administration. In B, individual tumor volumes at day 18 are shown. *adjusted p<0.05, ***adjusted p<0.001, ns not significant (determined by one-way ANOVA). [Figure 10B] Figure 1 shows the in vivo efficacy of anti-PD-1 antibody sialidase conjugate ASC#3 in the MC38 mouse syngeneic subcutaneous tumor model. Pembrolizumab and isotype antibodies were included as controls. In A, mean tumor volumes ± SEM over 18 days are shown for the indicated treatments. Triangles indicate drug administration. In B, individual tumor volumes at day 18 are shown. *adjusted p<0.05, ***adjusted p<0.001, ns not significant (determined by one-way ANOVA). [Figure 11A]Figure 1 shows the in vivo efficacy of the anti-PD-1 antibody sialidase conjugate ASC#3 in the CT26 mouse syngeneic subcutaneous tumor model (female BALB / c-hPD-1 / hPD-L1 mice bearing CT26-hPD-L1 tumors). Pembrolizumab and isotype antibody were included as controls. In A, mean tumor volumes ± SEM over 18 days are shown for the indicated treatments. Triangles indicate drug administration. In B, individual tumor volumes at day 18 are shown. ****Adjusted p<0.001 (determined by one-way ANOVA) (applies to both the isotype vs. pembrolizumab and isotype vs. ASC#3 comparisons). [Figure 11B] Figure 1 shows the in vivo efficacy of the anti-PD-1 antibody sialidase conjugate ASC#3 in the CT26 mouse syngeneic subcutaneous tumor model (female BALB / c-hPD-1 / hPD-L1 mice bearing CT26-hPD-L1 tumors). Pembrolizumab and isotype antibody were included as controls. In A, mean tumor volumes ± SEM over 18 days are shown for the indicated treatments. Triangles indicate drug administration. In B, individual tumor volumes at day 18 are shown. ****Adjusted p<0.001 (determined by one-way ANOVA) (applies to both the isotype vs. pembrolizumab and isotype vs. ASC#3 comparisons). DETAILED DESCRIPTION OF THE INVENTION

[0034] The present invention is based, in part, on the discovery that it is possible to generate fusion proteins comprising a sialidase enzyme and an anti-PD-1 immunoglobulin, or portion thereof (e.g., the antigen-binding domain and / or the immunoglobulin Fc domain), and / or antibody conjugates comprising a sialidase enzyme and an anti-PD-1 antibody, or portion thereof (e.g., the antigen-binding domain and / or the immunoglobulin Fc domain). The sialidase enzyme portion of the fusion protein and / or antibody conjugate can comprise at least one mutation compared to wild-type sialidase. Such mutations, or combinations of such mutations, can improve the expression, activity, or both expression and activity of the sialidase, thereby improving its use in the diagnosis and / or treatment of cancer. The fusion proteins and / or antibody conjugates have suitable substrate specificity and activity useful for removing sialic acid and / or sialic acid-containing molecules from the surface of cells (e.g., PD-1-expressing cells) and / or removing sialic acid and / or sialic acid-containing molecules from the tumor microenvironment and / or reducing the concentration of sialic acid and / or sialic acid-containing molecules in the tumor microenvironment.

[0035] The present invention further relates to pharmaceutical compositions and methods of using the fusion proteins and / or antibody conjugates for treating cancer.

[0036] I. Sialidase-anti-PD-1 fusion protein To facilitate the selective removal of sialic acid on cells (e.g., PD-1-expressing cells) and / or in the tumor microenvironment, it may be useful to target a sialidase as described herein to such cells or the tumor microenvironment. Furthermore, to facilitate the removal of sialic acid by the sialidase in a subject, it may be useful to extend the plasma half-life of the sialidase in the subject. This can be achieved by including the sialidase in a fusion protein and / or antibody conjugate (e.g., a chemically conjugated conjugate).

[0037] Accordingly, the present invention further provides a fusion protein comprising a sialidase enzyme, or a functional fragment thereof, and a portion or fragment of an anti-PD-1 antibody, e.g., an immunoglobulin Fc domain (also referred to herein as an Fc domain), or an immunoglobulin antigen-binding domain (also referred to herein as an antigen-binding domain). In some embodiments, the sialidase and the anti-PD-1 antibody, or portion thereof (e.g., the immunoglobulin Fc domain or antigen-binding domain), are linked by a peptide bond or an amino acid linker.

[0038] As used herein, unless otherwise indicated, the term "fusion protein" is understood to refer to a single polypeptide chain comprising amino acid sequences based on two or more separate proteins or polypeptide chains, where the two amino acid sequences may be fused together directly or via an intervening linker sequence, e.g., via an intervening amino acid linker. Nucleotide sequences encoding such fusion proteins may be produced, for example, using conventional recombinant DNA techniques.

[0039] In certain embodiments, the fusion protein comprises a tag, such as a Strep tag (e.g., a Strep II tag), a His tag (e.g., a 10x His tag), a myc tag, or a FLAG tag. The tag can be located at the C-terminus or N-terminus of the fusion protein.

[0040] a. sialidase portion As used herein, the term "sialidase" refers to any enzyme or functional fragment thereof that cleaves terminal sialic acid residues from a substrate, such as a glycoprotein or glycolipid. The term sialidase also includes variants having one or more amino acid substitutions, deletions, or insertions relative to the wild-type sialidase sequence, and / or fusion proteins or conjugates comprising a sialidase. Sialidases are also referred to as neuraminidases, and the two terms are used interchangeably herein unless otherwise indicated. As used herein, the term "functional fragment" of a sialidase refers to a fragment of a full-length sialidase that retains, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the enzymatic activity of the corresponding full-length naturally occurring sialidase. Sialidase enzyme activity can be assayed by any method known in the art, such as, for example, by measuring the release of sialic acid from the fluorogenic substrate 4-methylumbelliferyl-N-acetylneuraminic acid (4MU-NeuAc). In certain embodiments, the functional fragment comprises at least 100, 150, 200, 250, 300, 310, 320, 330, 340, 350, 360, or 370 contiguous amino acids present in a full-length, naturally occurring sialidase.

[0041] In certain embodiments, the sialidase portion of the sialidase-anti-PD-1 fusion protein is derived from a eukaryotic sialidase, eg, a mammalian sialidase, eg, a human or mouse sialidase.

[0042] Four sialidases (Neu1, Neu2, Neu3, and Neu4) are encoded in the human genome. Human Neu1 is a lysosomal neuraminidase enzyme that functions in a complex with β-galactosidase and cathepsin A. The amino acid sequence of human Neu1 is shown in SEQ ID NO:7, and the nucleotide sequence encoding human Neu1 is shown in SEQ ID NO:23.

[0043] Human Neu2 is a cytosolic sialidase enzyme. The amino acid sequence of human Neu2 is set forth in SEQ ID NO:1, and the nucleotide sequence encoding human Neu2 is set forth in SEQ ID NO:24. Unless otherwise stated, as used herein, wild-type human Neu2 refers to human Neu2 having the amino acid sequence of SEQ ID NO:1.

[0044] Human Neu3 is a plasma membrane sialidase with ganglioside-specific activity. Human Neu3 has two isoforms: isoform 1 and isoform 2. The amino acid sequence of human Neu3, isoform 1 is set forth in SEQ ID NO:8, and the nucleotide sequence encoding human Neu3, isoform 1 is set forth in SEQ ID NO:25. The amino acid sequence of human Neu3, isoform 2 is set forth in SEQ ID NO:9, and the nucleotide sequence encoding human Neu3, isoform 2 is set forth in SEQ ID NO:34.

[0045] Human Neu4 has two isoforms; isoform 1 is a peripheral membrane protein and isoform 2 is localized to the lysosomal lumen. The amino acid sequence of human Neu4, isoform 1 is set forth in SEQ ID NO: 10, and the nucleotide sequence encoding human Neu4, isoform 1 is set forth in SEQ ID NO: 26. The amino acid sequence of human Neu4, isoform 2 is set forth in SEQ ID NO: 11, and the nucleotide sequence encoding human Neu4, isoform 2 is set forth in SEQ ID NO: 35.

[0046] Four sialidases have also been found in the mouse genome, designated Neu1, Neu2, Neu3, and Neu4. The amino acid sequence of mouse Neu1 is set forth in SEQ ID NO:38, and the nucleotide sequence encoding mouse Neu1 is set forth in SEQ ID NO:42. The amino acid sequence of mouse Neu2 is set forth in SEQ ID NO:39, and the nucleotide sequence encoding mouse Neu2 is set forth in SEQ ID NO:43. The amino acid sequence of mouse Neu3 is set forth in SEQ ID NO:40, and the nucleotide sequence encoding mouse Neu3 is set forth in SEQ ID NO:44. The amino acid sequence of mouse Neu4 is set forth in SEQ ID NO:41, and the nucleotide sequence encoding mouse Neu4 is set forth in SEQ ID NO:45.

[0047] In certain embodiments, the sialidase portion of the sialidase-anti-PD-1 fusion protein is derived from a prokaryotic sialidase. Exemplary prokaryotic sialidases include sialidases from Salmonella typhimurium and Vibrio cholerae. The amino acid sequence of Salmonella typhimurium sialidase (St-sialidase) is set forth in SEQ ID NO:30, and the nucleotide sequence encoding the S. typhimurium sialidase is set forth in SEQ ID NO:6. The amino acid sequence of Vibrio cholerae sialidase is set forth in SEQ ID NO:36, and the nucleotide sequence encoding the Vibrio cholerae sialidase is set forth in SEQ ID NO:37.

[0048] In certain embodiments, the sialidase portion of the sialidase-anti-PD-1 fusion protein is a mutant sialidase (e.g., a recombinant mutant human sialidase). In certain embodiments, the recombinant mutant human sialidase has about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more than 100% of the enzymatic activity of the corresponding (or template) wild-type human sialidase.

[0049] In certain embodiments, the recombinant mutant human sialidase has the same substrate specificity as the corresponding wild-type human sialidase. In other embodiments, the recombinant mutant human sialidase has a different substrate specificity than the corresponding wild-type human sialidase. For example, in certain embodiments, the recombinant mutant human sialidase can cleave α2,3, α2,6, and / or α2,8 linkages. In certain embodiments, the sialidase can cleave α2,3 and α2,8 linkages.

[0050] In some embodiments, the expression yield of the recombinant mutant human sialidase in mammalian cells, such as HEK293 cells, CHO cells, mouse myeloma cells (NS0, Sp2 / 0), or human fibrosarcoma cells (HT-1080), e.g., HEK293 cells, is greater than about 10%, about 20%, about 50%, about 75%, about 100%, about 150%, about 200%, about 250%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1,000% of the expression yield of the corresponding wild-type human sialidase.

[0051] In certain embodiments, the recombinant variant human sialidase has about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% or greater than 100% of the enzymatic activity of the corresponding wild-type human sialidase, and The expression yield of the recombinant mutant human sialidase in animal cells, such as HEK293 cells, is about 10%, about 20%, about 50%, about 75%, about 100%, about 150%, about 200%, about 250%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1,000% higher than the expression yield of the corresponding wild-type human sialidase.

[0052] In some embodiments, the amino acid sequence of the recombinant mutant human sialidase has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of the corresponding wild-type human sialidase.

[0053] 1. Substitution of cysteine ​​residues In some embodiments, the recombinant mutant human sialidase comprises a substitution of at least one cysteine ​​(cys, C) residue. It has been discovered that certain cysteine ​​residues in sialidases can inhibit the expression of functional proteins as a result of protein aggregation. Thus, in some embodiments, the recombinant mutant human sialidase comprises at least one mutation to remove a free cysteine ​​(e.g., for Neu1 (SEQ ID NO: 7), e.g., one or more mutations at C111, C117, C171, C183, C218, C240, C242, and C252; for Neu2 (SEQ ID NO: 1), e.g., one or more mutations at C125, C196, C219, C272, C332, and C352; for Neu3 (SEQ ID NO: 8), e.g., one or more mutations at C7 , C90, C99, C106, C127, C136, C189, C194, C226, C242, C250, C273, C279, C295, C356, C365, C368, C384, C383, C394, and C415; and for Neu4 (SEQ ID NO: 10), for example, one or more mutations at C88, C125, C126, C186, C191, C211, C223, C239, C276, C437, C453, C480, and C481). The free cysteine ​​may be substituted with any amino acid. In certain embodiments, the free cysteine ​​is substituted with serine (ser, S), isoleucine (iso, I), valine (val, V), phenylalanine (phe, F), leucine (leu, L), or alanine (ala, A). Exemplary cysteine ​​substitutions in Neu2 include C125A, C125I, C125S, C125V, C196A, C196L, C196V, C272S, C272V, C332A, C332S, C332V, C352L, and C352V.

[0054] In some embodiments, the recombinant mutant human sialidase comprises two or more cysteine ​​substitutions. Exemplary double or triple cysteine ​​substitutions in Neu2 include: C125S and C332S; C272V and C332A; C272V and C332S; C332A and C352L; C125S and C196L; C196L and C352L; C196L and C332A; C332A and C352L; and C196L, C332A and C352L.

[0055] In some embodiments, the recombinant mutant human sialidase is a Neu2 sialidase and comprises the substitutions C322A and C352L (SEQ ID NO: 5).

[0056] In some embodiments, the sialidase contains amino acid substitutions at 2, 3, 4, 5, or 6 cysteines typically present in human sialidases, such as Neu2 or Neu3.

[0057] In some embodiments, the recombinant mutant human sialidase comprises a substitution or combination of substitutions corresponding to a substitution or combination of substitutions listed in Table 1 (amino acid positions corresponding to wild-type human Neu2 (SEQ ID NO: 1)). [Table 1]

[0058] 2. Substitution of residues to increase pI and / or decrease hydrophobicity The isoelectric point (pI) of a protein is the pH at which its net charge is zero. pI also generally indicates the pH at which a protein is least soluble, which may affect the ability to express and purify the protein. Generally, a protein has good solubility when its pI is two units higher than the pH of the solution. Human Neu2 has an expected pI of 7.5. Therefore, human Neu2 is minimally soluble around neutral pH, which is undesirable because expression and physiological systems are at neutral pH. In contrast, sialidase from Salmonella typhimurium (St-sialidase), which exhibits good solubility and recombinant expression, has a pI of 9.6. Therefore, to increase the expression of human Neu2 or other human sialidases, recombinant mutant human sialidases can be engineered to contain one or more amino acid substitution(s), where the substitution(s) increase the pI of the sialidase relative to the sialidase without the substitution(s). Furthermore, reducing the number of hydrophobic amino acids on the surface of a sialidase can improve expression of the sialidase, for example, by reducing aggregation. Thus, to increase expression of human Neu2 or other human sialidases, recombinant mutant human sialidases can be designed to contain one or more amino acid substitution(s), where the substitution(s) reduce the hydrophobicity of the surface of the sialidase relative to the sialidase without the substitution(s).

[0059] Thus, in some embodiments, the recombinant mutant human sialidase contains at least one amino acid substitution, wherein the substitution increases the isoelectric point (pI) of the sialidase and / or decreases the hydrophobicity of the sialidase relative to the sialidase without the substitution. This can be achieved by introducing one or more charged amino acids, e.g., positively or negatively charged amino acids, into the recombinant sialidase. In some embodiments, the amino acid substitution is for a charged amino acid, e.g., a positively charged amino acid, such as lysine (lys, K), histidine (his, H), or arginine (arg, R), or a negatively charged amino acid, such as aspartic acid (asp, D) or glutamic acid (glu, E). In some embodiments, the amino acid substitution is for a lysine residue. In some embodiments, the substitution increases the pI of the sialidase to about 7.75, about 8, about 8.25, about 8.5, about 8.75, about 9, about 9.25, about 9.5, or about 9.75.

[0060] In some embodiments, amino acid substitutions occur at surface-exposed D or E amino acids, in helices or loops, or at positions that have K or R at the corresponding position in St-sialidase. In some embodiments, amino acid substitutions occur at amino acids distant from or otherwise not involved in catalysis, amino acids that are not conserved by other human Neu proteins or St-sialidases or clostridial NanH, or amino acids that are not located within domains important for function (e.g., Asp-boxes or β-strands).

[0061] Exemplary amino acid substitutions in Neu2 that increase the isoelectric point (pI) of a sialidase and / or decrease the hydrophobicity of the sialidase relative to a sialidase not having the substitution include A2E, A2K, D215K, V325E, V325K, E257K, and E319K. In some embodiments, the recombinant mutant human sialidase contains two or more amino acid substitutions, including, for example, A2K and V325E, A2K and V325K, E257K and V325K, A2K and E257K, and E257K and A2K and V325K.

[0062] In certain embodiments, the recombinant mutant human sialidase comprises substitutions or combinations of substitutions corresponding to the substitutions or combinations of substitutions listed in Table 2 (amino acid positions corresponding to wild-type human Neu2 (SEQ ID NO: 1)). [Table 2]

[0063] 3. N-terminal peptide addition and N- or C-terminal substitution It has been discovered that the addition of a peptide sequence of two or more amino acids to the N-terminus of a human sialidase can improve the expression and / or activity of the sialidase. In some embodiments, the peptide is at least two amino acids long, e.g., 2-20, 2-10, 2-5, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids long. In some embodiments, the peptide can form an α-helix or have a propensity to form an α-helix.

[0064] In mice, the Neu2 isoform (type B) found in the thymus contains six amino acids that are not present in the canonical isoform of Neu2 found in skeletal muscle. In certain embodiments herein, the N-terminal six amino acids of the mouse thymic Neu2 isoform, MEDLRP (SEQ ID NO: 4), or a variant thereof, can be added to human Neu, e.g., human Neu2. In certain embodiments, the recombinant mutant human sialidase comprises a peptide at least two amino acid residues long covalently linked to the N-terminal amino acid of the sialidase. In certain embodiments, the recombinant mutant human sialidase comprises the peptide MEDLRP (SEQ ID NO: 4) or EDLRP (SEQ ID NO: 3) covalently linked to the N-terminal amino acid of the sialidase. In certain embodiments, the sialidase can further comprise a cleavage site, e.g., a proteolytic cleavage site, located between the peptide, e.g., MEDLRP (SEQ ID NO: 4) or EDLRP (SEQ ID NO: 3), and the remainder of the sialidase. In some embodiments, the peptide, eg, MEDLRP (SEQ ID NO:4) or EDLRP (SEQ ID NO:3), can be post-translationally cleaved from the remainder of the sialidase.

[0065] Alternatively, or in combination with N-terminal addition, one to five amino acids of the 12 amino acid N-terminal region of the recombinant mutant human sialidase can be removed, for example, the N-terminal methionine can be removed. In some embodiments, when the recombinant mutant human sialidase is Neu2, the N-terminal methionine can be removed, the first five amino acids (MASLP; SEQ ID NO: 12) can be removed, or the second through fourth amino acids (ASLP; SEQ ID NO: 13) can be removed.

[0066] In some embodiments, 1 to 5 amino acids of the 12 amino acid N-terminal region of a recombinant mutant human sialidase are substituted with MEDLRP (SEQ ID NO: 4), EDLRP (SEQ ID NO: 3), or TVEKSVVF (SEQ ID NO: 14). For example, in some embodiments, when the recombinant mutant human sialidase is Neu2, amino acids MASLP (SEQ ID NO: 12), ASLP (SEQ ID NO: 13), or M are substituted with MEDLRP (SEQ ID NO: 4), EDLRP (SEQ ID NO: 3), or TVEKSVVF (SEQ ID NO: 14).

[0067] Human sialidases have a β-propeller structure characterized by a six-bladed β-sheet arranged toroidally around a central axis. Generally, hydrophobic interactions between the blades of the β-propeller, such as between the N- and C-terminal blades, enhance stability. Therefore, to increase the expression of human Neu2 or other human sialidases, recombinant mutant human sialidases can be engineered containing amino acid substitutions that increase hydrophobic interactions and / or hydrogen bonds between the N- and C-terminal β-propeller blades of the sialidase.

[0068] Thus, in some embodiments, the recombinant mutant human sialidase comprises a substitution of at least one wild-type amino acid residue, wherein the substitution increases hydrophobic interactions and / or hydrogen bonding between the N- and C-termini of the sialidase relative to a sialidase without the substitution. In some embodiments, the wild-type amino acid is substituted with asparagine (asn, N), lysine (lys, K), tyrosine (tyr, Y), phenylalanine (phe, F), or tryptophan (trp, W). Exemplary substitutions in Neu2 that increase hydrophobic interactions and / or hydrogen bonding between the N- and C-termini include L4N, L4K, V6Y, L7N, L4N and L7N, L4N and V6Y and L7N, V12N, V12Y, V12L, V6Y, V6F, or V6W. In some embodiments, the sialidase comprises a V6Y substitution.

[0069] In some embodiments, the recombinant mutant human sialidase comprises a combination of the above substitutions. For example, the recombinant mutant human Neu2 sialidase can comprise the additional amino acids MEDLRP (SEQ ID NO: 4), EDLRP (SEQ ID NO: 3), or TVEKSVVF (SEQ ID NO: 14) at the N-terminus, in combination with at least one of L4N, L4K, V6Y, L7N, L4N and L7N, L4N and V6Y and L7N, V12N, V12Y, V12L, V6Y, V6F, or V6W substitutions. In some embodiments, the amino acids MASLP (SEQ ID NO: 12), ASLP (SEQ ID NO: 13), or M of the recombinant mutant human Neu2 sialidase are replaced with MEDLRP (SEQ ID NO: 4), EDLRP (SEQ ID NO: 3), or TVEKSVVF (SEQ ID NO: 14), and the recombinant mutant human Neu2 sialidase also comprises at least one L4N, L4K, V6Y, L7N, L4N and L7N, L4N and V6Y and L7N, V12N, V12Y, V12L, V6Y, V6F, or V6W substitution.

[0070] In some embodiments, the recombinant mutant human sialidase comprises a mutation or combination of mutations corresponding to a mutation or combination of mutations listed in Table 3 (amino acid positions corresponding to wild-type human Neu2 (SEQ ID NO: 1)). [Table 3]

[0071] Furthermore, in some embodiments, the sialidase comprises a substitution or deletion of an N-terminal methionine at the N-terminus of the sialidase. For example, in some embodiments, the sialidase comprises a substitution of a methionine residue at a position corresponding to position 1 of wild-type human Neu2 (SEQ ID NO:1), e.g., the methionine at position 1 of wild-type human Neu2 is replaced with alanine (M1A) or aspartic acid (M1D). In other embodiments, the sialidase comprises a deletion (ΔM1) of a methionine residue at a position corresponding to position 1 of wild-type human Neu2 (SEQ ID NO:1).

[0072] In certain embodiments, the recombinant mutant human sialidase comprises a substitution or combination of substitutions corresponding to a substitution or combination of substitutions listed in Table 4 (amino acid positions corresponding to wild-type human Neu2 (SEQ ID NO: 1)). [Table 4]

[0073] 4. Substitution of residues to reduce proteolytic cleavage It has been discovered that certain sialidases (e.g., human Neu2) are susceptible to cleavage by proteases (e.g., trypsin). Consequently, proteolytic cleavage of the sialidase can occur during recombinant protein production, harvesting, purification, or formulation, during administration to a subject, or after administration to a subject. Thus, in certain embodiments, the recombinant mutant human sialidase comprises a substitution of at least one wild-type amino acid residue, which reduces cleavage of the sialidase by a protease (e.g., trypsin) compared to a sialidase without such substitution.

[0074] In certain embodiments, incubation of a recombinant mutant human sialidase with a protease (e.g., trypsin) results in proteolytic cleavage of about 1% to about 50%, about 1% to about 40%, about 1% to about 30%, about 1% to about 20%, about 1% to about 3 ... about 10%, about 1% to about 5%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 5% to about 20%, about 5% to about 10%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%, about 30% to about 50%, about 30% to about 40%, or about 40% to about 50%. In certain embodiments, incubation of a recombinant variant human sialidase with a protease (e.g., trypsin) results in less than 50%, less than 40%, less than 30%, less than 10%, less than 5%, less than 3%, less than 1%, or less than 0.5% proteolytic cleavage compared to the proteolytic cleavage of the corresponding wild-type sialidase when incubated with the protease under the same conditions. Proteolytic cleavage may be assayed by any method known in the art, including, for example, by SDS-PAGE as described in Example 4 herein.

[0075] Exemplary substitutions that increase resistance to proteolytic cleavage include: (i) substitution of an alanine residue at a position corresponding to position 242 of wild-type human Neu2 (SEQ ID NO: 1), e.g., cysteine ​​(A242C), phenylalanine (A242F), glycine (A242G), histidine (A242H), isoleucine (A242I), lysine (A242K), leucine (A242L), methionine (A242M), asparagine (A242N), glutamine (A242Q), arginine (A242R), serine (A242S), valine (A242V), tryptophan (A242W). or tyrosine (A242Y); (ii) a substitution of an arginine residue at a position corresponding to position 243 of wild-type human Neu2 (SEQ ID NO: 1), such as with glutamic acid (R243E), histidine (R243H), asparagine (R243N), glutamine (R243Q), or lysine (R243K); (iii) a substitution of a valine residue at a position corresponding to position 244 of wild-type human Neu2 (SEQ ID NO: 1), such as with isoleucine (V244I), lysine (V244K), or proline (V244P); or (iv) any combination of the foregoing. In certain embodiments, the recombinant mutant human sialidase comprises a substitution selected from A242C, A242F, A242Y, and A242W. In certain embodiments, the recombinant mutant human sialidase comprises a substitution or combination of substitutions corresponding to the substitutions or combinations of substitutions set forth in Table 5 (amino acid positions correspond to wild-type human Neu2 (SEQ ID NO: 1)). [Table 5]

[0076] Further exemplary substitutions that increase resistance to proteolytic cleavage (and / or increase expression yield and / or enzymatic activity) include: (i) substitution of a leucine residue at a position corresponding to position 240 of wild-type human Neu2 (SEQ ID NO: 1), e.g., with aspartic acid (L240D), asparagine (L240N), or tyrosine (L240Y); (ii) substitution of an alanine residue at a position corresponding to position 213 of wild-type human Neu2 (SEQ ID NO: 1), e.g., with cysteine ​​(A213C), asparagine (A213N), serine (A213S), or threonine (A213T); (iii) substitution of an arginine residue at a position corresponding to position 241 of wild-type human Neu2 (SEQ ID NO: 1), e.g., with alanine (R241A), arginine (R241B), or arginine (R241C). (iv) substitution of a serine residue at a position corresponding to position 258 of wild-type human Neu2 (SEQ ID NO: 1), for example with cysteine ​​(S258C); (v) substitution of a leucine residue at a position corresponding to position 260 of wild-type human Neu2 (SEQ ID NO: 1), for example with aspartic acid (L260D), phenylalanine (L260F), glutamine (L260Q) or threonine (L260T); (vi) substitution of a valine residue at a position corresponding to position 265 of wild-type human Neu2 (SEQ ID NO: 1), for example with phenylalanine (V265F); or (vii) any combination of the foregoing. In certain embodiments, it is contemplated that substitutions or combinations of substitutions at these positions may improve hydrophobic and / or aromatic interactions between secondary structural elements in the sialidase (e.g., between an α-helix and the nearest β-sheet), thereby stabilizing the structure and improving resistance to proteolytic cleavage.

[0077] In certain embodiments, the recombinant mutant sialidase comprises a mutation at position L240. In some aspects, the recombinant mutant sialidase comprises a combination of mutations at positions (i) A213 and A242, (ii) A213, A242 and S258, (iii) L240 and L260, (iv) R241 and A242, (v) A242 and L260, (vi) A242 and V265, or (vii) L240 and A242. In some aspects, the recombinant mutant human sialidase comprises a combination of substitutions selected from (i) A213C, A242F and S258C, (ii) A213C and A242F, (iii) A213T and A242F, (iv) R241Y and A242F, and (v) L240Y and A242F. In certain embodiments, the recombinant mutant human sialidase comprises a substitution or combination of substitutions corresponding to those listed in Table 6 (amino acid positions corresponding to wild-type human Neu2 (SEQ ID NO: 1)). [Table 6]

[0078] 5. Other substitutions In certain embodiments, the recombinant mutant human sialidase comprises at least one of the following substitutions: I187K, A328E, K370N, or H210N. In certain embodiments, the recombinant mutant human Neu2 comprises a substitution of the amino acid GDYDAPTHQVQW (SEQ ID NO: 15) with the amino acid SMDQGSTW (SEQ ID NO: 16) or STDGGKTW (SEQ ID NO: 17). In certain embodiments, the recombinant mutant human Neu2 comprises a substitution of the amino acid PRPPAPEA (SEQ ID NO: 18) with the amino acid QTPLEAAC (SEQ ID NO: 19). In certain embodiments, the recombinant mutant human Neu2 comprises a substitution of the amino acid NPRPPAPEA (SEQ ID NO: 20) with the amino acid SQNDGES (SEQ ID NO: 21).

[0079] In certain embodiments, the recombinant mutant human sialidase comprises at least one substitution at a position corresponding to V212, A213, Q214, D215, T216, L217, E218, C219, Q220, V221, A222, E223, V224, E225, or T225.

[0080] In certain embodiments, the recombinant mutant human sialidase comprises an amino acid substitution at a position set forth in Table 7 (an amino acid position corresponding to wild-type human Neu2 (SEQ ID NO: 1)). In some embodiments, the sialidase comprises an amino acid substitution identified in Table 7. In some embodiments, the sialidase comprises any combination of amino acid substitutions identified in Table 7. [Table 7-1] [Table 7-2] [Table 7-3]

[0081] For example, in some embodiments, the recombinant mutant human sialidase contains: (a) a substitution of a proline residue at a position corresponding to position 5 of wild-type human Neu2 (P5); (b) a substitution of a lysine residue at a position corresponding to position 9 of wild-type human Neu2 (K9); (c) a substitution of an alanine residue at a position corresponding to position 42 of wild-type human Neu2 (A42); (d) a substitution of a lysine residue at a position corresponding to position 44 of wild-type human Neu2 (K44); (e) a substitution of a lysine residue at a position corresponding to position 45 of wild-type human Neu2 (K45); (f) a substitution of a lysine residue at a position corresponding to position 54 of wild-type human Neu2 (K56). (g) substitution of a leucine residue at position 62 of wild-type human Neu2 (P62); (h) substitution of a glutamine residue at position 69 of wild-type human Neu2 (Q69); (i) substitution of an arginine residue at position 78 of wild-type human Neu2 (R78); (j) substitution of an aspartic acid residue at position 80 of wild-type human Neu2 (D80); (k) substitution of an alanine residue at position 93 of wild-type human Neu2 (A93); (l) substitution of an alanine residue at position 107 of wild-type human Neu2 (m) substitution of a glutamine residue at position 108 of wild-type human Neu2 (Q108); (n) substitution of a glutamine residue at position 112 of wild-type human Neu2 (Q112); (o) substitution of a cysteine ​​residue at position 125 of wild-type human Neu2 (C125); (p) substitution of a glutamine residue at position 126 of wild-type human Neu2 (Q126); (q) substitution of an alanine residue at position 150 of wild-type human Neu2 (A150); (r) substitution of a cysteine ​​residue at position 125 of wild-type human Neu2 (C125); (p) substitution of a glutamine residue at position 126 of wild-type human Neu2 (Q126); (q) substitution of an alanine residue at position 150 of wild-type human Neu2 (A150); (s) substitution of an arginine residue at position 170 of wild-type human Neu2 (R170); (t) substitution of an alanine residue at position 171 of wild-type human Neu2 (A171); (u) substitution of a glutamine residue at position 188 of wild-type human Neu2 (Q188); (v) substitution of an arginine residue at position 189 of wild-type human Neu2 (R189); (w) substitution of an alanine residue at position 213 of wild-type human Neu2 (A213);(x) substitution of a leucine residue at position 217 of wild-type human Neu2 (L217); (y) substitution of a glutamic acid residue at position 225 of wild-type human Neu2 (E225); (z) substitution of a histidine residue at position 239 of wild-type human Neu2 (H239); (aa) substitution of a leucine residue at position 240 of wild-type human Neu2 (L240); (bb) substitution of an arginine residue at position 241 of wild-type human Neu2 (R2 41); (cc) substitution of an alanine residue at position corresponding to position 242 of wild-type human Neu2 (A242); (dd) substitution of a valine residue at position corresponding to position 244 of wild-type human Neu2 (V244); (ee) substitution of a threonine residue at position corresponding to position 249 of wild-type human Neu2 (T249); (ff) substitution of an aspartic acid residue at position corresponding to position 251 of wild-type human Neu2 (D251); (gg) substitution of a glutamic acid residue at position corresponding to position 257 of wild-type human Neu2 (hh) substitution of a serine residue at position 258 of wild-type human Neu2 (S258); (ii) substitution of a leucine residue at position 260 of wild-type human Neu2 (L260); (jj) substitution of a valine residue at position 265 of wild-type human Neu2 (V265); (kk) substitution of a glutamine residue at position 270 of wild-type human Neu2 (Q270); (ll) substitution of a tryptophan residue at position 292 of wild-type human Neu2 (mm) a substitution of a serine residue at position 301 of wild-type human Neu2 (S301); (nn) a substitution of a tryptophan residue at position 302 of wild-type human Neu2 (W302); (oo) a substitution of a valine residue at position 363 of wild-type human Neu2 (V363); or (pp) a substitution of a leucine residue at position 365 of wild-type human Neu2 (L365); or any combination of the foregoing substitutions. For example, the sialidase can include substitutions of K9, A42, P62, A93, Q216, A242, Q270, S301, W302, V363, or L365, or any combination of the foregoing substitutions.

[0082] In some embodiments, in the sialidase: (a) the proline residue at the position corresponding to position 5 of wild-type human Neu2 is substituted with histidine (P5H); (b) the lysine residue at the position corresponding to position 9 of wild-type human Neu2 is substituted with aspartic acid (K9D); (c) the alanine residue at the position corresponding to position 42 of wild-type human Neu2 is substituted with arginine (A42R) or aspartic acid (A42D); (d) the lysine residue at the position corresponding to position 44 of wild-type human Neu2 is substituted with arginine (K44R) or glutamine. (e) the lysine residue at position 45 of wild-type human Neu2 is substituted with alanine (K45A), arginine (K45R), or glutamic acid (K45E); (f) the leucine residue at position 54 of wild-type human Neu2 is substituted with methionine (L54M); (g) the proline residue at position 62 of wild-type human Neu2 is substituted with asparagine (P62N), aspartic acid (P62D), histidine (P62H), glutamic acid (P62E), glycine (P62G), (h) the glutamine residue at position 69 of wild-type human Neu2 is substituted with histidine (Q69H); (i) the arginine residue at position 78 of wild-type human Neu2 is substituted with lysine (R78K); (j) the aspartic acid residue at position 80 of wild-type human Neu2 is substituted with proline (D80P); (k) the alanine residue at position 93 of wild-type human Neu2 is substituted with glutamic acid (A93E) or lysine (A93F). (l) the glycine residue at position 107 of wild-type human Neu2 is substituted with aspartic acid (G107D); (m) the glutamine residue at position 108 of wild-type human Neu2 is substituted with histidine (Q108H); (n) the glutamine residue at position 112 of wild-type human Neu2 is substituted with arginine (Q112R) or lysine (Q112K); (o) the cysteine ​​residue at position 125 of wild-type human Neu2 is substituted with leucine (C125L);(p) the glutamine residue at position 126 of wild-type human Neu2 is substituted with leucine (Q126L), glutamic acid (Q126E), phenylalanine (Q126F), histidine (Q126H), isoleucine (Q126I), or tyrosine (Q126Y); (q) the alanine residue at position 150 of wild-type human Neu2 is substituted with valine (A150V); (r) the cysteine ​​residue at position 164 of wild-type human Neu2 is substituted with glycine (C164G); (s) the alanine residue at position 170 of wild-type human Neu2 is substituted with valine (A150V). (t) the alanine residue at position 171 of wild-type human Neu2 is substituted with glycine (A171G); (u) the glutamine residue at position 188 of wild-type human Neu2 is substituted with proline (Q188P); (v) the arginine residue at position 189 of wild-type human Neu2 is substituted with proline (R189P); (w) the alanine residue at position 213 of wild-type human Neu2 is substituted with cysteine ​​(A213C), asparagine (A213N), or serine (A213C). (x) the leucine residue at position 217 of wild-type human Neu2 is substituted with alanine (L217A) or valine (L217V); (y) the threonine residue at position 249 of wild-type human Neu2 is substituted with alanine (T249A); (z) the aspartic acid residue at position 251 of wild-type human Neu2 is substituted with glycine (D251G); (aa) the glutamic acid residue at position 225 of wild-type human Neu2 is substituted with proline (E225P). (bb) the histidine residue at position corresponding to position 239 of wild-type human Neu2 is substituted with proline (H239P); (cc) the leucine residue at position corresponding to position 240 of wild-type human Neu2 is substituted with aspartic acid (L240D), asparagine (L240N), or tyrosine (L240Y); (dd) the arginine residue at position corresponding to position 241 of wild-type human Neu2 is substituted with alanine (R241A), aspartic acid (R241D), leucine (R241L), glutamine (R241Q), or tyrosine (R241Y);(ee) The alanine residues at the position corresponding to position 242 of wild-type human Neu2 are cysteine ​​(A242C), phenylalanine (A242F), glycine (A242G), histidine (A242H), isoleucine (A242I), lysine (A242K), leucine (A242L), methionine (A242M), asparagine (A242N), glutamine (A242Q), arginine (A242R), and serine (A (ff) the valine residue at position corresponding to position 244 of wild-type human Neu2 is substituted with isoleucine (V244I), lysine (V244K) or proline (V244P); (gg) the glutamic acid residue at position corresponding to position 257 of wild-type human Neu2 is substituted with proline (E257P); (h (h) the serine residue at the position corresponding to position 258 is substituted with a cysteine ​​(S258C); (ii) the leucine residue at the position corresponding to position 260 of wild-type human Neu2 is substituted with an aspartic acid (L260D), phenylalanine (L260F), glutamine (L260Q), or threonine (L260T); (jj) the valine residue at the position corresponding to position 265 of wild-type human Neu2 is substituted with a phenylalanine (V265 (kk) the glutamine residue at position corresponding to position 270 of wild-type human Neu2 is substituted with alanine (Q270A), histidine (Q270H), phenylalanine (Q270F), proline (Q270P), serine (Q270S), or threonine (Q270T); (ll) the tryptophan residue at position corresponding to position 292 of wild-type human Neu2 is substituted with arginine (W292R);(mm) The serine residue at the position corresponding to position 301 of wild-type human Neu2 is alanine (S301A), aspartic acid (S301D), glutamic acid (S301E), phenylalanine (S301F), glycine (S301G), histidine (S301H), isoleucine (S301I), lysine (S301K), leucine (S301L), methionine (S301M), asparagine (S301N), proline ( (nn) the tryptophan residue at position 302 of wild-type human Neu2 is replaced with alanine (W302A), aspartic acid (W302D), glutamic acid (W302E), or phenylalanine (W302F). , glycine (W302G), histidine (W302H), isoleucine (W302I), lysine (W302K), leucine (W302L), methionine (W302M), asparagine (W302N), proline (W302P), glutamine (W302Q), arginine (W302R), serine (W302S), threonine (W302T), valine (W302V) or tyrosine (W302Y); ) the valine residue at the position corresponding to position 363 of wild-type human Neu2 is substituted with arginine (V363R); or (pp) the leucine residue at the position corresponding to position 365 of wild-type human Neu2 is substituted with glutamine (L365Q), histidine (L365H), isoleucine (L365I), lysine (L365K), or serine (L365S); or the sialidase comprises any combination of the foregoing substitutions. For example, the sialidase can include a substitution selected from K9D, A42R, P62G, P62N, P62S, P62T, D80P, A93E, Q126H, Q126Y, R189P, H239P, A242T, Q270A, Q270S, Q270T, S301A, S301R, W302K, W302R, V363R, and L365I, or any combination of the foregoing substitutions;

[0083] In some embodiments, the recombinant mutant human sialidase comprises a deletion of a leucine residue at a position corresponding to position 184 of wild-type human Neu2 (ΔL184), a deletion of a histidine residue at a position corresponding to position 185 of wild-type human Neu2 (ΔH185), a deletion of a proline residue at a position corresponding to position 186 of wild-type human Neu2 (ΔP186), a deletion of an isoleucine residue at a position corresponding to position 187 of wild-type human Neu2 (ΔI187), and a deletion of a glutamine residue at a position corresponding to position 184 of wild-type human Neu2 (ΔQ188), or any combination of the foregoing deletions.

[0084] In some embodiments, the recombinant mutant human sialidase comprises an insertion, e.g., an insertion of an amino acid selected from S, T, Y, L, F, A, P, V, I, N, D, and H, between the threonine residue at position corresponding to position 216 of wild-type human Neu2 and the leucine residue at position corresponding to position 217 of wild-type human Neu2.

[0085] Additional examples of sialidase mutations and combinations of sialidase mutations are described in International (PCT) Patent Application Publication No. WO2019 / 136167 (including those in the detailed description in the section entitled "I. Recombinant Human Sialidases" and in the examples of Examples 1, 2, 3, 4, 5, and 6) and International (PCT) Patent Application Publication No. WO2021 / 003469 (including those in the detailed description in the section entitled "I. Recombinant Human Sialidases" and in the examples of Examples 2, 3, 4, and 5), and International (PCT) Patent Application No. PCT / US2021 / 040240, filed July 2, 2021 (including those in the detailed description in the section entitled "I. Recombinant Human Sialidases" and in the examples of Examples 2, 3, 4, and 5).

[0086] 6. Combining Permutations In certain embodiments, the recombinant mutant human sialidase comprises any combination of the mutations contemplated herein. For example, the recombinant mutant sialidase enzyme can comprise a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more of the mutations contemplated herein. It is contemplated that the recombinant mutant sialidase enzyme can comprise 1-15, 1-10, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-15, 2-10, 2-7, 2-6, 2-5, 2-4, 2-3, 3-15, 3-10, 3-7, 3-6, 3-5, or 3-4 of the mutations contemplated herein.

[0087] For example, the recombinant mutant sialidase enzyme can include an M1 deletion (ΔM1), an M1A substitution, an M1D substitution, a V6Y substitution, a K9D substitution, a P62G substitution, a P62N substitution, a P62S substitution, a P62T substitution, an A93E substitution, an I187K substitution, a Q270A substitution, an S301R substitution, a W302K substitution, a C332A substitution, a V363R substitution, an L365I substitution, or any combination of the foregoing.

[0088] In some embodiments, the recombinant mutant sialidase enzyme comprises an M1 deletion (ΔM1), an M1A substitution, an M1D substitution, a V6Y substitution, an I187K substitution, a C332A substitution, or any combination of the foregoing. For example, the recombinant mutant sialidase enzyme can include: M1A and V6Y; M1A and I187K; M1A and C332A; M1D and V6Y; M1D and I187K; M1D and C332A; ΔM1 and V6Y; ΔM1 and I187K; ΔM1 and C332A; V6Y and I187K; V6Y and C332A; I187K and C332A; M1A, V6Y and I187K; M1A, V6Y and C332A; M1A, I187K and C332A; The mutations may include combinations of mutations selected from M1D, V6Y and I187K; M1D, V6Y and C332A; M1D, I187K and C332A; ΔM1, V6Y and I187K; ΔM1, V6Y and C332A; ΔM1, I187K and C332A; V6Y, I187K and C332A; M1A, V6Y, I187K and C332A; M1D, V6Y, I187K and C332A; and ΔM1, V6Y, I187K and C332A.

[0089] In some embodiments, the recombinant mutant sialidase enzyme comprises (i) an amino acid substitution identified in Table 7 or any combination of amino acid substitutions identified in Table 7, and (ii) an M1 deletion (ΔM1), an M1A substitution, an M1D substitution, a V6Y substitution, an I187K substitution, a C332A substitution, or any combination of the foregoing. For example, recombinant mutant sialidase enzymes can include (i) an amino acid substitution identified in Table 7 or any combination of amino acid substitutions identified in Table 7, and (ii) M1A and V6Y; M1A and I187K; M1A and C332A; M1D and V6Y; M1D and I187K; M1D and C332A; ΔM1 and V6Y; ΔM1 and I187K; ΔM1 and C332A; V6Y and I187K; V6Y and C332A; I187K and C332A; M1A, V6Y and I187K; M1A, V and C332A; M1A, I187K and C332A; M1D, V6Y and I187K; M1D, V6Y and C332A; M1D, I187K and C332A; ΔM1, V6Y and I187K; ΔM1, V6Y and C332A; ΔM1, I187K and C332A; V6Y, I187K and C332A; M1A, V6Y, I187K and C332A; M1D, V6Y, I187K and C332A; and ΔM1, V6Y, I187K and C332A.

[0090] In some embodiments, the recombinant mutant sialidase enzyme has: (a) substitutions of M1D, V6Y, P62G, A93E, I187K, and C332A; (b) substitutions of M1D, V6Y, K9D, A93E, I187K, C332A, V363R, and L365I; (c) substitutions of M1D, V6Y, P62N, I187K, and C332A; (d) substitutions of M1D, V6Y, I (e) substitutions of M1D, V6Y, P62S, I187K, Q270A, S301R, W302K, and C332A; (f) substitutions of M1D, V6Y, P62T, I187K, Q270A, S301R, W302K, and C332A; (g) substitutions of M1D, V6Y, P62N, I187K, (h) substitutions of Q270A, S301R, W302K, and C332A; (i) substitutions of M1D, V6Y, P62G, A93E, I187K, S301A, W302R, and C332A; (j) substitutions of M1D, V6Y, P62G, A93E, Q126Y, I187K, Q270T, and C332A. (k) substitutions of M1D, V6Y, P62G, A93E, Q126Y, I187K, A242F, Q270T, and C332A; or (l) mutations M1D, V6Y, A42R, P62G, A93E, Q126Y, I187K, A242F, Q270T, and C332A.

[0091] In some embodiments, the recombinant mutant human sialidase comprises a substitution of a serine residue at position 301 of wild-type human Neu2 (S301) combined with a substitution of a tryptophan residue at position 302 of wild-type human Neu2 (W302). For example, the recombinant mutant human sialidase can comprise a combination of substitutions corresponding to the combinations of substitutions listed in the columns of Table 8 (amino acid positions corresponding to wild-type human Neu2 (SEQ ID NO: 1)). For example, the recombinant mutant human sialidase can comprise: a substitution of S301K and W302R; a substitution of S301K and W302K; or a substitution of S301A and W302S. [Table 8]

[0092] In some embodiments, the recombinant mutant human sialidase comprises a combination of substitutions corresponding to the combination of substitutions listed in the column of Table 9 (amino acid positions corresponding to wild-type human Neu2 (SEQ ID NO: 1)). [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4]

[0093] In certain embodiments, the recombinant mutant human sialidase comprises the amino acid sequence of any one of SEQ ID NOs: 48-63, 94, 97, 100, or 126, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 48-63, 94, 97, 100, or 126.

[0094] In certain embodiments, the recombinant mutant human sialidase is X1X2SX3X4X5LQX6ESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRX7SX8X9DEHAELIVX 10 RRGDYDAX 11 THQVQWX 12 AQEVVAQAX 13 LX 14 GHRSMNPCPLYDX 15 QTGTLFLFFIAIPX 16 X 17 VTEX 18 QQLQTRANVTRLX 19 X 20 VTSTDHGRTWSSPRDLTDAAIGPX 21 YREWSTFAVGPGHX 22LQLHDX 23 X 24 RSLVVPAYAYRKLHPX 25 X 26 X 27 PIPSAFX 28 FLSHDHGRTWARGHFVX 29 QDTX 30 ECQVAEVX 31 TGEQRVVTLNARSX 32 X 33 X 34 X 35 RX 36 QAQSX 37 NX 38 GLDFQX 39 X 40 QX 41 VKKLX 42 EPPPX 43 GX 44 QGSVISFPSPRSGPGSPAQX 45 LLYTHPTHX 46 X 47 QRADLGAYLNPRPPAPEAWSEPX 48 LLAKGSX 49 AYSDLQSMGTGPDGSPLFGX 50 LYEANDYEEIX 51 FX 52 MFTLKQAFPAEYLPQ (SEQ ID NO: 64), In the sequence, X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val, or absent; X2 is Ala or Lys; X3 is Asn or Leu; X4 is Pro or His; X5 is Phe, Trp, Tyr, or Val; X6 is Lys or Asp; X7 is Ala or Arg; X8 is Lys, Arg, or Glu; X9 is Lys, Ala, Arg, or Glu; 10 is Leu or Met, and X 11 is Pro, Asn, Asp, His, Glu, Gly, Ser, or Thr, and X 12 is Gln or His, and X 13is Arg or Lys, and X 14 is Asp or Pro, and X 15 is Ala, Glu or Lys, and X 16 is Gly or Asp, and X 17 is Gln or His, and X 18 is Gln, Arg, or Lys, and X 19 is Ala, Cys, Ile, Ser, Val, or Leu, and X 20 is Gln, Leu, Glu, Phe, His, Ile, Leu, or Tyr, and X 21 is Ala or Val, and X 22 is Cys or Gly, and X 23 is Arg or Pro, and X 24 is Ala or Gly, and X 25 is Arg, Ile, or Lys, and X 26 is Gln or Pro, and X 27 is Arg or Pro, and X 28 is Ala, Cys, Leu, or Val, and X 29 is Ala, Cys, Asn, Ser, or Thr, and X 30 is Leu, Ala, or Val, and X 31 is Glu or Pro, and X 32 is His or Pro, and X 33 is Leu, Asp, Asn, or Tyr, and X 34 is Arg, Ala, Asp, Leu, Gln, or Tyr, and X 35 is Ala, Cys, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Val, Trp, or Tyr, and X 36 is Val, Ile, or Lys, and X 37 is Thr or Ala, and X 38 is Asp or Gly, and X 39 is Glu, Lys, or Pro, and X 40 is Ser or Cys, and X 41is Leu, Asp, Phe, Gln, or Thr, and X 42 is Val or Phe, and X 43 is Gln, Ala, His, Phe, Pro, Ser, or Thr, and X 44 is Cys or Val, and X 45 is Trp or Arg, and X 46 is Ser, Arg, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Thr, Val, Trp, or Tyr, and X 47 is Trp, Lys, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Tyr, and X 48 is Lys or Val, and X 49 is Ala, Cys, Ser, or Val, and X 50 is Cys, Leu, or Val, and X 51 is Val or Arg, and X 52 is Leu, Gln, His, Ile, Lys, or Ser, and the sialidase comprises at least one mutation compared to wild-type human Neu2 (SEQ ID NO: 1).

[0095] In certain embodiments, the recombinant mutant human sialidase is X1ASLPX2LQX3ESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRX4SKKDEHAELIVLRRGDYDAX5THQVQWQAQEVVAQARLDGHRSMNPCPLYDX6QTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCX7VTSTDH GRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPX8QRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRX9RVQAQSTNDGLDFQESQLVKKLVEPPPX 10 GCQGSVISFPSPRSGPGSPAQWLLYTHPTHX 11X 12 QRADLGAYLNPRPPAPEAWSEPVLLAKGSX 13 AYSDLQSMGTGPDGSPLFGCLYEANDYEEIX 14 FX 15 MFTLKQAFPAEYLPQ (SEQ ID NO: 65), In the sequence, X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val, or absent; X2 is Phe, Trp, Tyr, or Val; X3 is Lys or Asp; X4 is Arg or Ala; X5 is Pro, Asn, Asp, His, Glu, Gly, Ser, or Thr; X6 is Ala, Glu, or Lys; X7 is Gln, Leu, Glu, Phe, His, Ile, Leu, or Tyr; X8 is Arg, Ile, or Lys; X9 is Ala, Cys, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Val, Trp, or Tyr; 10 is Gln, Ala, His, Phe, Pro, Ser, or Thr, and X 11 is Ser, Arg, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Thr, Val, Trp, or Tyr, and X 12 is Trp, Lys, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Tyr, and X 13 is Ala, Cys, Ser, or Val, and X 14 is Val or Arg, and X 15is Leu, Gln, His, Ile, Lys, or Ser, and the sialidase comprises at least one mutation compared to wild-type human Neu2 (SEQ ID NO: 1). In certain embodiments, X1 is Ala, Asp, Met, or absent, X2 is Tyr or Val, X3 is Lys or Asp, X4 is Arg or Ala, X5 is Pro, Asn, Gly, Ser, or Thr, X6 is Ala or Glu, X7 is Gln or Tyr, X8 is Ile or Lys, X9 is Ala or Thr, and X 10 is Gln, Ala, or Thr, and X 11 is Ser, Arg, or Ala, and X 12 is Trp, Lys, or Arg, and X 13 is Ala or Cys, and X 14 is Val or Arg, and X 15 is Leu or Ile.

[0096] In some embodiments, the recombinant variant human sialidase comprises a conservative substitution relative to the recombinant variant human sialidase sequence disclosed herein. As used herein, the term "conservative substitution" refers to a substitution with a structurally similar amino acid. For example, conservative substitutions can include those within the following groups: Ser and Cys; Leu, Ile, and Val; Glu and Asp; Lys and Arg; Phe, Tyr, and Trp; and Gln, Asn, Glu, Asp, and His. Conservative substitutions can also be defined by the BLAST (Basic Local Alignment Search Tool) algorithm, a BLOSUM substitution matrix (e.g., a BLOSUM 62 matrix), or a PAM substitution:p matrix (e.g., a PAM 250 matrix).

[0097] Sequence identity can be determined in a variety of ways within the skill of one in the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. BLAST (Basic Local Alignment Search Tool) analysis, using the algorithm used by the programs blastp, blastn, blastx, tblastn, and tblastx (Karlin et al., (1990) PROC. NATL. ACAD. SCI. USA 87:2264-2268; Altschul, (1993) J. MOL. EVOL. 36:290-300; Altschul et al., (1997) NUCLEIC ACIDS RES. 25:3389-3402 (incorporated herein by reference), is tailored to searching for sequence similarity. For a discussion of basic issues in searching sequence databases, see Altschul et al., (1994) NATURE GENETICS 6:119-129, fully incorporated herein by reference. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Search parameters for histogram, description, alignment, expect (i.e., the statistical significance threshold for reporting matches against database sequences), cutoff, matrix, and filter are at default settings. The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et al., (1992) PROC. NATL. ACAD. SCI. USA 89:10915-10919, fully incorporated herein by reference).Four blastn parameters can be adjusted as follows: Q=10 (gap creation penalty); R=10 (gap extension penalty); wink=1 (generates word hits at every wink.sup.th position along the query); and gapw=16 (sets the window width over which gapped alignments are generated). Equivalent blastp parameter settings would be Q=9; R=2; wink=1; and gapw=32. Searches can also be performed using NCBI (National Center for Biotechnology Information) BLAST Advanced Option parameters (e.g.: -G, cost for an open gap [integer]: default = 5 for nucleotides / 11 for proteins; -E, cost for an extension gap [integer]: default = 2 for nucleotides / 1 for proteins; -q, penalty for a nucleotide mismatch [integer]: default = -3; -r, reward for a nucleotide match [integer]: default = 1; -e, expectation value [real number]: default = 10; -W, word size [integer]: default = 11 for nucleotides / 28 for megablast / 3 for proteins; -y, dropoff for blast extension in bits (X): default = 20 for blastn / 7 for others; -X, X dropoff value for gapped alignments (in bits): default = 15 for all programs but not applicable to blastn; and -Z, final X dropoff value for gapped alignments (in bits): 50 for blastn, 25 for others). ClustalW for pairwise protein alignments may also be used (default parameters may include, for example, a Blosum62 matrix and a Gap Opening Penalty=10 and a Gap Extension Penalty=0.1).The best-fit comparison between sequences available in the GCG package version 10.0 uses DNA parameters GAP=50 (gap creation penalty) and LEN=3 (gap extension penalty). The equivalent settings for best-fit protein comparisons are GAP=8 and LEN=2.

[0098] b.Antibody part Unless otherwise specified, the term "antibody," as used herein, will be understood to mean an intact antibody (e.g., an intact monoclonal antibody) or a fragment thereof, such as an antigen-binding fragment of an antibody (e.g., an antigen-binding fragment of a monoclonal antibody) or an Fc fragment of an antibody (e.g., an Fc fragment of a monoclonal antibody), including modified, engineered, or chemically conjugated intact antibodies, antigen-binding fragments, or Fc fragments. Examples of antigen-binding fragments include Fab, Fab', (Fab')2, Fv, single-chain antibodies (e.g., scFv), minibodies, and diabodies. Examples of modified or engineered antibodies include chimeric antibodies, humanized antibodies, and multispecific antibodies (e.g., bispecific antibodies). An example of a chemically conjugated antibody is an antibody conjugated to a toxin moiety.

[0099] In some embodiments, the fusion protein comprises an immunoglobulin Fc domain. As used herein, unless otherwise indicated, the term "immunoglobulin Fc domain" refers to a fragment of an immunoglobulin heavy chain constant region that can bind to an Fc receptor, either alone or in combination with a second immunoglobulin Fc domain. An immunoglobulin Fc domain can include, for example, immunoglobulin CH2 and CH3 domains. An immunoglobulin Fc domain can include, for example, immunoglobulin CH2 and CH3 domains and an immunoglobulin hinge region. The boundaries between the immunoglobulin hinge region, CH2 and CH3 domains are well known in the art and can be found, for example, in the PROSITE database (available on the World Wide Web at prosite.expasy.org).

[0100] In some embodiments, the immunoglobulin Fc domain is derived from a human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, or IgM Fc domain. A single amino acid substitution (S228P according to Kabat numbering; designated IgG4Pro) can be introduced to disrupt the heterogeneity observed in recombinant IgG4 antibodies. See Angal, S. et al. (1993) MOL. IMMUNOL. 30:105-108.

[0101] In some embodiments, the immunoglobulin Fc domain is derived from a human IgG1 isotype or another isotype that elicits antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-mediated cytotoxicity (CDC). In some embodiments, the immunoglobulin Fc domain is derived from a human IgG1 isotype (e.g., SEQ ID NO:31, SEQ ID NO:5, or SEQ ID NO:139).

[0102] In some embodiments, the immunoglobulin Fc domain is derived from the human IgG4 isotype or another isotype that elicits little or no antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-mediated cytotoxicity (CDC). In some embodiments, the immunoglobulin Fc domain is derived from the human IgG4 isotype.

[0103] In certain embodiments, the immunoglobulin Fc domain comprises either a "knob" mutation, e.g., T366Y, or a "hole" mutation, e.g., Y407T (residue numbers according to EU numbering; Kabat, EA, et al. (1991) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, FIFTH EDITION, USDapartment of Health and Human Services, NIH Publication No. 91-3242), for heterodimerization with a second polypeptide. For example, in certain embodiments, the immunoglobulin Fc domain is derived from a human IgG1 Fc domain and comprises a Y407T mutation (e.g., the fusion protein comprises SEQ ID NO:32, SEQ ID NO:92, SEQ ID NO:141, or SEQ ID NO:143). In certain embodiments, the immunoglobulin Fc domain is derived from a human IgG1 Fc domain and comprises a T366Y mutation (e.g., the fusion protein comprises SEQ ID NO:33, SEQ ID NO:93, SEQ ID NO:142, or SEQ ID NO:144).

[0104] In certain embodiments, the immunoglobulin Fc domain is modified to prevent glycosylation of the Fc domain. For example, in certain embodiments, the immunoglobulin Fc domain is derived from a human IgG1 Fc domain and contains a mutation at position N297 (e.g., an N297A mutation or an N297G mutation) (residue numbers are according to EU numbering (Kabat, EA, et al., supra)). For example, in certain embodiments, the fusion protein comprises SEQ ID NO: 140, SEQ ID NO: 143, or SEQ ID NO: 144.

[0105] In some embodiments, the fusion protein comprises an immunoglobulin antigen-binding domain. The inclusion of such a domain may enhance targeting of the fusion protein to sialylated cells (e.g., PD-1-expressing cells) and / or the tumor microenvironment. As used herein, unless otherwise indicated, the term "immunoglobulin antigen-binding domain" refers to a polypeptide that, alone or in combination with another immunoglobulin antigen-binding domain, defines an antigen-binding site. Exemplary immunoglobulin antigen-binding domains include, for example, an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, where the variable regions together define an antigen-binding site (e.g., an anti-PD-1 antigen-binding site).

[0106] In certain embodiments, the immunoglobulin antigen-binding domain is derived from an anti-PD-1 antibody. Examples of anti-PD-1 antibodies are described in, for example, U.S. Patent Nos. 8,952,136, 8,779,105, 8,008,449, 8,741,295, 9,205,148, 9,181,342, 9,102,728, 9,102,727, 8,952,136, 8,927,697, 8,900,587, 8,735,553, and 7,488,802. Examples of anti-PD-1 antibodies include, for example, nivolumab (Opdivo®, Bristol-Myers Squibb Co.), pembrolizumab (Keytruda®, Merck Sharp & Dohme Corp.), spartalizumab (PDR001, Novartis Pharmaceuticals), pidilizumab (CT-011, Cure Tech), cemiplimab, TX-4014, camrelizumab (SHR1210), sintilimab (IBI308), tislelizumab (BGB-A317), toripalimab (JS001), dostallimab (TSR-042, WBP-285), INCMGA00012 (MGA012), and AMP-514.

[0107] In certain embodiments, the immunoglobulin antigen-binding domain is derived from pembrolizumab. The pembrolizumab heavy chain amino acid sequence is set forth in SEQ ID NO: 136, and the pembrolizumab light chain amino acid sequence is set forth in SEQ ID NO: 77. The heavy chain variable region derived from pembrolizumab is set forth in SEQ ID NO: 137, and the light chain variable region derived from pembrolizumab is set forth in SEQ ID NO: 138.

[0108] c. linker In some embodiments, the sialidase portion of the fusion protein can be directly linked or fused to the anti-PD-1 antibody portion of the fusion protein (e.g., the immunoglobulin Fc domain and / or the immunoglobulin antigen binding domain). In other embodiments, the sialidase portion can be covalently linked to the anti-PD-1 antibody portion by a linker.

[0109] The linker can link one or more naturally occurring amino acids, the sialidase or functional fragment thereof, and the antibody moiety or fragment, where the amino acid (e.g., a cysteine ​​amino acid) can be introduced by site-directed mutagenesis. The linker can also include one or more unnatural amino acids. In certain circumstances, it is contemplated that a linker containing, for example, one or more sulfhydryl-reactive groups (e.g., maleimide) can be covalently bonded to a cysteine ​​in a sialidase moiety or antibody moiety that is a naturally occurring cysteine ​​residue or the product of site-directed mutagenesis.

[0110] The linker may be a cleavable linker or a non-cleavable linker. Optionally or additionally, the linker may be a flexible linker or a non-flexible linker.

[0111] The linker should be long enough to allow the sialidase and antibody moieties to be linked without steric hindrance from each other, and short enough to retain the intended activity of the fusion protein. The linker is preferably sufficiently hydrophilic to avoid or minimize instability of the fusion protein. The linker is preferably sufficiently hydrophilic to avoid or minimize insolubility of the fusion protein. The linker should be sufficiently stable in vivo (e.g., not cleaved by serum, enzymes, etc.) so that the fusion protein can be effective in vivo.

[0112] The linker can be about 1 angstrom (Å) to about 150 Å in length, or about 1 Å to about 120 Å in length, or about 5 Å to about 110 Å in length, or about 10 Å to about 100 Å in length. The linker can be about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 27, 30 Å or more in length and / or less than about 110, 100, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31 Å or less in length. Additionally, the linker can be about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110 and 120 Å in length.

[0113] In some embodiments, the linker comprises a polypeptide linker that links or fuses the sialidase portion of the fusion protein to the anti-PD-1 antibody portion of the fusion protein (e.g., the immunoglobulin Fc domain and / or the immunoglobulin antigen-binding domain). For example, it is contemplated that a gene encoding a sialidase portion linked directly or indirectly (e.g., via an amino acid-containing linker) to an antibody portion can be made and expressed using conventional recombinant DNA techniques. For example, the amino terminus of the sialidase portion can be linked to the carboxy terminus of either the light chain or the heavy chain of the antibody portion. For example, for a Fab fragment, the amino or carboxy terminus of the sialidase portion can be linked to the first constant domain (CH1) of the antibody heavy chain. When a linker is used, the linker can contain hydrophilic amino acid residues, such as Gln, Ser, Gly, Glu, Pro, His, and Arg. In some embodiments, the linker is a peptide containing 1 to 25 amino acid residues, 1 to 20 amino acid residues, 2 to 15 amino acid residues, 3 to 10 amino acid residues, 3 to 7 amino acid residues, 4 to 25 amino acid residues, 4 to 20 amino acid residues, 4 to 15 amino acid residues, 4 to 10 amino acid residues, 5 to 25 amino acid residues, 5 to 20 amino acid residues, 5 to 15 amino acid residues, or 5 to 10 amino acid residues. Exemplary linkers include glycine and serine-rich linkers, such as (GlyGlyPro). n or (GlyGlyGlyGlySer) n wherein n is 1 to 5. In some embodiments, the linker comprises, consists of, or consists essentially of GGGGS (SEQ ID NO: 121). In some embodiments, the linker comprises, consists of, or consists essentially of GGGGSGGGGS (SEQ ID NO: 90). In some embodiments, the linker comprises, consists of, or consists essentially of EPKSS (SEQ ID NO: 91). Further exemplary linker sequences are disclosed, for example, in George et al. (2003) PROTEIN ENGINEERING 15:871-879, and U.S. Pat. Nos. 5,482,858 and 5,525,491.

[0114] In certain embodiments, the fusion protein comprises the amino acid sequence of any one of SEQ ID NOs: 67-73, 78, 81-87, 95, 96, 98, 99, 101, 102, 105, 106, 108, 111, 112, 115, 122, 123, 125, 127, 128, 130, 132, 134, or 145, or SEQ ID NOs: 67-73 , 78, 81-87, 95, 96, 98, 99, 101, 102, 105, 106, 108, 111, 112, 115, 122, 123, 125, 127, 128, 130, 132, 134, or 145, and having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of

[0115] d. Antibody conjugates The present invention further provides antibody conjugates comprising one or more of the fusion proteins disclosed herein. As used herein, unless otherwise indicated, the term "antibody conjugate" is understood to refer to an antibody or functional fragment thereof comprising antigen-binding activity (e.g., anti-PD-1 antigen-binding activity) and / or Fc receptor-binding activity conjugated (e.g., covalently) to an additional functional moiety. In certain embodiments, the antibody conjugate comprises a single polypeptide chain. In certain embodiments, the antibody conjugate comprises two, three, four, or more polypeptide chains that are covalently or non-covalently linked together to form a multimeric complex, e.g., a dimeric, trimeric, or tetrameric complex. For example, an antibody conjugate can comprise a first polypeptide (fusion protein) comprising a recombinant mutant human sialidase enzyme and an immunoglobulin heavy chain, and a second polypeptide comprising an immunoglobulin light chain, where, for example, the immunoglobulin heavy and light chains together define a single antigen-binding site (e.g., an anti-PD-1 antigen-binding site).

[0116] In some embodiments, the antibody conjugate may comprise a single sialidase. In other embodiments, the antibody conjugate may comprise more than one (e.g., two) sialidases. When more than one sialidase is included, the sialidases may be the same or different. In some embodiments, the antibody conjugate may comprise a single anti-PD-1 antigen-binding site. In other embodiments, the antibody conjugate may comprise more than one (e.g., two) anti-PD-1 antigen-binding sites. When two antigen-binding sites are used, they may be the same or different. In some embodiments, the antibody conjugate comprises an immunoglobulin Fc fragment.

[0117] In some embodiments, the antibody conjugate comprises one or two immunoglobulin heavy chains, or functional fragments thereof. In some embodiments, the antibody conjugate comprises one or two immunoglobulin light chains, or functional fragments thereof. In some embodiments, the antibody conjugate comprises a sialidase fused to the N-terminus or C-terminus of an immunoglobulin heavy chain or an immunoglobulin light chain.

[0118] Figure 4 shows exemplary antibody-conjugate constructs comprising one or more sialidase enzymes. For example, in Figure 4A, a first anti-PD-1 antigen-binding site (e.g., V H Domain and V L The first anti-PD-1 antigen binding site (defined by the domain) is shown as 10, the second anti-PD-1 antigen binding site is shown as 20, the sialidase is shown as 30, and the Fc is shown as 40. It will be understood that in each of the constructs shown in Figures 4A-4I, the Fc may optionally be modified in several ways, for example, using knob-into-hole techniques as shown at 50 in Figure 4B. Similar structures are shown with similar schematic representations throughout Figure 4.

[0119] Figure 4A shows an antibody conjugate construct comprising a first polypeptide comprising a first immunoglobulin light chain; a second polypeptide comprising a first immunoglobulin heavy chain; a third polypeptide comprising a second immunoglobulin heavy chain; and a fourth polypeptide comprising a second immunoglobulin light chain. The first and second polypeptides can be covalently linked together, the third and fourth polypeptides can be covalently linked together, and the second and third polypeptides can be covalently linked together. The covalent linkage can be a disulfide bond. In some embodiments, the first and second polypeptides together define a first anti-PD-1 antigen-binding site designated as 10, and the third and fourth polypeptides together define a second anti-PD-1 antigen-binding site designated as 20. A sialidase enzyme designated as 30 can be conjugated to the N- or C-terminus of the first and second immunoglobulin light chains or the first and second immunoglobulin heavy chains.

[0120] Figure 4B shows an antibody conjugate construct comprising a first polypeptide comprising a first immunoglobulin light chain; a second polypeptide comprising a first immunoglobulin heavy chain; a third polypeptide comprising a second immunoglobulin heavy chain; and a fourth polypeptide comprising a second immunoglobulin light chain. The first and second polypeptides can be covalently linked together, the third and fourth polypeptides can be covalently linked together, and the second and third polypeptides can be covalently linked together. The covalent linkage can be a disulfide bond. In some embodiments, the first and second polypeptides together define a first anti-PD-1 antigen-binding site, and the third and fourth polypeptides together define a second anti-PD-1 antigen-binding site. The sialidase enzyme can be conjugated to the N-terminus or C-terminus of the first immunoglobulin light chain or the first immunoglobulin heavy chain.

[0121] Figure 4C shows an antibody conjugate construct comprising a first polypeptide comprising an immunoglobulin light chain; a second polypeptide comprising an immunoglobulin heavy chain; and a third polypeptide comprising an immunoglobulin Fc domain. The first and second polypeptides can be covalently linked together, and the second and third polypeptides can be covalently linked together. The covalent linkage can be a disulfide bond. In some embodiments, the first and second polypeptides together define an anti-PD-1 antigen binding site. The sialidase enzyme can be conjugated to the N-terminus or C-terminus of the first immunoglobulin light chain or the first immunoglobulin heavy chain.

[0122] Figure 4D shows an antibody conjugate construct comprising a first polypeptide comprising an immunoglobulin light chain; a second polypeptide comprising an immunoglobulin heavy chain; and a third polypeptide comprising an immunoglobulin Fc domain and a first sialidase enzyme. The first and second polypeptides can be covalently linked together, and the second and third polypeptides can be covalently linked together. The covalent linkage can be a disulfide bond. The third polypeptide comprises, from N- to C-terminal, a sialidase and an immunoglobulin Fc domain. In some embodiments, the first and second polypeptides together define an anti-PD-1 antigen-binding site. The optional second sialidase enzyme can be conjugated to the N- or C-terminus of the first immunoglobulin light chain or the first immunoglobulin heavy chain.

[0123] Figure 4E shows an antibody conjugate construct comprising a first polypeptide comprising an immunoglobulin light chain; a second polypeptide comprising an immunoglobulin heavy chain; and a third polypeptide comprising an immunoglobulin Fc domain and a first sialidase enzyme. The first and second polypeptides can be covalently linked together, and the second and third polypeptides can be covalently linked together. The covalent linkage can be a disulfide bond. The third polypeptide comprises, from N- to C-terminal, an immunoglobulin Fc domain and a sialidase. In some embodiments, the first and second polypeptides together define an anti-PD-1 antigen-binding site. The optional second sialidase enzyme can be conjugated to the N- or C-terminus of the first immunoglobulin light chain or the first immunoglobulin heavy chain.

[0124] Figure 4F shows an antibody conjugate construct comprising a first polypeptide comprising a first immunoglobulin Fc domain and a second polypeptide comprising a second immunoglobulin Fc domain. The first and second polypeptides can be covalently linked together. The covalent bond can be a disulfide bond. A sialidase enzyme can be conjugated to the N-terminus or C-terminus of the first immunoglobulin Fc domain or the N-terminus or C-terminus of the second immunoglobulin Fc domain. An optional second sialidase enzyme can be conjugated to the N-terminus or C-terminus of the first immunoglobulin Fc domain or the N-terminus or C-terminus of the second immunoglobulin Fc domain.

[0125] Figure 4G shows an antibody conjugate construct comprising a first polypeptide comprising an immunoglobulin light chain; and a second polypeptide comprising an immunoglobulin heavy chain variable region. The first and second polypeptides can be covalently linked together. The covalent bond can be a disulfide bond. In some embodiments, the first and second polypeptides together define an anti-PD-1 antigen binding site. A sialidase enzyme can be conjugated to the N-terminus or C-terminus of the immunoglobulin light chain or the immunoglobulin heavy chain variable region.

[0126] Figure 4H shows an antibody conjugate construct comprising a first polypeptide comprising a first immunoglobulin Fc domain and a second polypeptide comprising a second immunoglobulin Fc domain. The first and second polypeptides can be covalently linked together. The covalent bond can be a disulfide bond. A sialidase enzyme can be conjugated to the N-terminus of the first immunoglobulin Fc domain or the second immunoglobulin Fc domain. An optional second sialidase enzyme can be conjugated to the N-terminus of the second immunoglobulin Fc domain or the first immunoglobulin Fc domain, respectively. A single-chain variable fragment (scFv) can be conjugated to the C-terminus of the first immunoglobulin Fc domain or the second immunoglobulin Fc domain, respectively. An optional second single-chain variable fragment (scFv) can be conjugated to the C-terminus of the first immunoglobulin Fc domain or the second immunoglobulin Fc domain, respectively.

[0127] Figure 4I shows an antibody conjugate construct similar to that shown in Figure 4H, except that each scFv is replaced with an immunoglobulin antigen-binding fragment, e.g., a Fab. For example, Figure 4I shows an antibody conjugate construct comprising a first polypeptide comprising a first immunoglobulin Fc domain and a second polypeptide comprising a second immunoglobulin Fc domain. The first and second polypeptides can be covalently linked together. The covalent bond can be a disulfide bond. A sialidase enzyme can be conjugated to the N-terminus of the first immunoglobulin Fc domain or the second immunoglobulin Fc domain. An optional second sialidase enzyme can be conjugated to the N-terminus of the second immunoglobulin Fc domain or the first immunoglobulin Fc domain, respectively. An antibody fragment (Fab) can be conjugated or fused to the C-terminus of the first immunoglobulin Fc domain or the second immunoglobulin Fc domain. An optional second antibody fragment (Fab) can be conjugated or fused to the C-terminus of the second immunoglobulin Fc domain or the first immunoglobulin Fc domain, respectively. In the case of fusion, the C-terminus of the Fc domain is linked (either by a bond or an amino acid linker) to a first polypeptide chain defining an anti-PD-1 immunoglobulin antigen-binding fragment. In the case of an antibody having an antigen-binding site defined by a single variable region, this may be sufficient to confer binding affinity to a target antigen (e.g., PD-1). In another example, for example, in the case of a human antibody, a first polypeptide chain defining an immunoglobulin antigen-binding fragment can be conjugated (e.g., covalently conjugated, e.g., via a disulfide bond) to a second polypeptide chain defining an immunoglobulin antigen-binding fragment, where the two antigen-binding fragments together define an antigen-binding site for binding to a target antigen (e.g., PD-1).

[0128] Figure 5 shows additional antibody conjugate constructs. For example, Figure 5 shows an antibody conjugate construct comprising a first polypeptide comprising an immunoglobulin light chain; a second polypeptide comprising an immunoglobulin heavy chain and an scFv; and a third polypeptide comprising an immunoglobulin Fc domain and a first sialidase enzyme. The first and second polypeptides can be covalently linked together, and the second and third polypeptides can be covalently linked together. The covalent linkage can be a disulfide bond. The second polypeptide comprises, from N- to C-terminal, the heavy chain and the scFv. The third polypeptide comprises, from N- to C-terminal, the sialidase and the immunoglobulin Fc domain. In some embodiments, the first and second polypeptides together define a first antigen-binding site. In some embodiments, the scFv defines a second antigen-binding site. Figure 5 shows additional antibody constructs comprising a first polypeptide comprising an immunoglobulin light chain; a second polypeptide comprising an immunoglobulin heavy chain; and a third polypeptide comprising an immunoglobulin Fc domain and a first sialidase enzyme, wherein a Fab fragment is conjugated to the N-terminus of the immunoglobulin heavy chain. The first and second polypeptides can be covalently linked together, and the second and third polypeptides can be covalently linked together. The covalent linkage can be a disulfide bond. The third polypeptide comprises, from N- to C-terminus, a sialidase and an immunoglobulin Fc domain. In some embodiments, the first and second polypeptides together define a first antigen-binding site. In some embodiments, the Fab fragment defines a second antigen-binding site. It is understood that in each of the constructs shown in Figure 5, the scFv, if present, can be replaced with the Fab fragment, or the Fab fragment, if present, can be replaced with the scFv. It is understood that in each of the constructs shown in Figure 5, the Fc can optionally be modified in several ways.

[0129] In some embodiments, the antibody conjugate comprises a first polypeptide comprising a first immunoglobulin light chain; a second polypeptide comprising a first immunoglobulin heavy chain and a first sialidase; a third polypeptide comprising a second immunoglobulin heavy chain and a second sialidase; and a fourth polypeptide comprising a second immunoglobulin light chain. An example of this embodiment is shown in Figure 6A. The first and second polypeptides can be covalently linked together, the third and fourth polypeptides can be covalently linked together, and the second and third polypeptides can be covalently linked together. The covalent linkage can be a disulfide bond. In some embodiments, the first and second polypeptides together define a first anti-PD-1 antigen-binding site, and the third and fourth polypeptides together define a second anti-PD-1 antigen-binding site. In some embodiments, the second and third polypeptides comprise, from N- to C-terminal, first and second immunoglobulin heavy chains and first and second sialidases, respectively. In certain embodiments, the second and third polypeptides comprise, from N-terminal to C-terminal, a first and second sialidase and a first and second immunoglobulin heavy chain, respectively.

[0130] In certain embodiments, the first polypeptide and the fourth polypeptide comprise the amino acid sequence of SEQ ID NO: 77, or comprise an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 77. In certain embodiments, the second polypeptide and the third polypeptide comprise the amino acid sequence of SEQ ID NO: 145, or comprise an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 145.

[0131] In some embodiments, the antibody conjugate comprises a first polypeptide comprising an immunoglobulin light chain; a second polypeptide comprising an immunoglobulin heavy chain; and a third polypeptide comprising an immunoglobulin Fc domain and a sialidase. An example of this embodiment is shown in Figure 6B. The first and second polypeptides can be covalently linked together, and the second and third polypeptides can be covalently linked together. The covalent linkage can be a disulfide bond. In some embodiments, the first and second polypeptides together define an anti-PD-1 antigen-binding site. In some embodiments, the third polypeptide comprises, from N-terminal to C-terminal, a sialidase and an immunoglobulin Fc domain, or, from N-terminal to C-terminal, an immunoglobulin Fc domain and a sialidase.

[0132] In certain embodiments, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 77, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 77. In certain embodiments, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 105, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 105. In certain embodiments, the third polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 67-73, 78, 81-87, 95, 96, 98, 99, 101, 102, 106, 108, 111, 112, 115, 122, 123, 125, 127, or 128, or comprises an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 67-73, 78, 81-87, 95, 96, 98, 99, 101, 102, 106, 108, 111, 112, 115, 122, 123, 125, 127, or 128.

[0133] In certain embodiments, the third polypeptide is X1X2SX3X4X5LQX6ESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRX7SX8X9DEHAELIVX 10 RRGDYDAX 11 THQVQWX 12 AQEVVAQAX 13 LX 14 GHRSMNPCPLYDX 15 QTGTLFLFFIAIPX 16 X 17 VTEX 18 QQLQTRANVTRLX 19 X 20 VTSTDHGRTWSSPRDLTDAAIGPX 21 YREWSTFAVGPGHX 22 LQLHDX 23 X 24 RSLVVPAYAYRKLHPX 25 X 26 X 27 PIPSAFX 28 FLSHDHGRTWARGHFVX 29 QDTX 30 ECQVAEVX 31 TGEQRVVTLNARSX 32 X 33 X 34 X 35 RX 36 QAQSX 37 NX 38 GLDFQX 39 X 40 QX 41 VKKLX 42 EPPPX 43 GX 44 QGSVISFPSPRSGPGSPAQX 45 LLYTHPTHX 46 X 47 QRADLGAYLNPRPPAPEAWSEPX 48 LLAKGSX 49 AYSDLQSMGTGPDGSPLFGX 50 LYEANDYEEIX 51 FX 52 MFTLKQAFPAEYLPQX 53DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 66), In the sequence, X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val, or absent; X2 is Ala or Lys; X3 is Asn or Leu; X4 is Pro or His; X5 is Phe, Trp, Tyr, or Val; X6 is Lys or Asp; X7 is Ala or Arg; X8 is Lys, Arg, or Glu; X9 is Lys, Ala, Arg, or Glu; 10 is Leu or Met, and X 11 is Pro, Asn, Asp, His, Glu, Gly, Ser, or Thr, and X 12 is Gln or His, and X 13 is Arg or Lys, and X 14 is Asp or Pro, and X 15 is Ala, Glu, or Lys, and X 16 is Gly or Asp, and X 17 is Gln or His, and X 18 is Gln, Arg, or Lys, and X 19 is Ala, Cys, Ile, Ser, Val, or Leu, and X 20 is Gln, Leu, Glu, Phe, His, Ile, Leu, or Tyr, and X 21 is Ala or Val, and X 22 is Cys or Gly, and X 23 is Arg or Pro, and X 24is Ala or Gly, and X 25 is Arg, Ile, or Lys, and X 26 is Gln or Pro, and X 27 is Arg or Pro, and X 28 is Ala, Cys, Leu, or Val, and X 29 is Ala, Cys, Asn, Ser, or Thr, and X 30 is Leu, Ala, or Val, and X 31 is Glu or Pro, and X 32 is His or Pro, and X 33 is Leu, Asp, Asn, or Tyr, and X 34 is Arg, Ala, Asp, Leu, Gln, or Tyr, and X 35 is Ala, Cys, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Val, Trp, or Tyr, and X 36 is Val, Ile, or Lys, and X 37 is Thr or Ala, and X 38 is Asp or Gly, and X 39 is Glu, Lys, or Pro, and X 40 is Ser or Cys, and X 41 is Leu, Asp, Phe, Gln, or Thr, and X 42 is Val or Phe, and X 43 is Gln, Ala, His, Phe, Pro, Ser, or Thr, and X 44 is Cys or Val, and X 45 is Trp or Arg, and X 46 is Ser, Arg, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Thr, Val, Trp, or Tyr, and X 47 is Trp, Lys, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Tyr, and X 48is Lys or Val, and X 49 is Ala, Cys, Ser, or Val, and X 50 is Cys, Leu, or Val, and X 51 is Val or Arg, and X 52 is Leu, Gln, His, Ile, Lys, or Ser, and X 53 is GGGGS (SEQ ID NO: 121), GGGGSGGGGS (SEQ ID NO: 90), or EPKSS (SEQ ID NO: 91), and the sialidase comprises at least one mutation compared to wild-type human Neu2 (SEQ ID NO: 1).

[0134] In certain embodiments, the third polypeptide is X1ASLPX2LQX3ESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRX4SKKDEHAELIVLRRGDYDAX5THQVQWQAQEVVAQARLDGHRSMNPCPLYDX6QTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCX7VTSTDH GRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPX8QRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRX9RVQAQSTNDGLDFQESQLVKKLVEPPPX 10 GCQGSVISFPSPRSGPGSPAQWLLYTHPTHX 11 X 12 QRADLGAYLNPRPPAPEAWSEPVLLAKGSX 13 AYSDLQSMGTGPDGSPLFGCLYEANDYEEIX 14 FX 15 MFTLKQAFPAEYLPQX 16DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 76), In the sequence, X1 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Thr, Val, or absent; X2 is Phe, Trp, Tyr, or Val; X3 is Lys or Asp; X4 is Arg or Ala; X5 is Pro, Asn, Asp, His, Glu, Gly, Ser, or Thr; X6 is Ala, Glu, or Lys; X7 is Gln, Leu, Glu, Phe, His, Ile, Leu, or Tyr; X8 is Arg, Ile, or Lys; X9 is Ala, Cys, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Val, Trp, or Tyr; 10 is Gln, Ala, His, Phe, Pro, Ser, or Thr, and X 11 is Ser, Arg, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Thr, Val, Trp, or Tyr, and X 12 is Trp, Lys, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Tyr, and X 13 is Ala, Cys, Ser, or Val, and X 14 is Val or Arg, and X 15 is Leu, Gln, His, Ile, Lys, or Ser, and X 16is GGGGS (SEQ ID NO: 121), GGGGSGGGGS (SEQ ID NO: 90), or EPKSS (SEQ ID NO: 91), and the sialidase comprises at least one mutation compared to wild-type human Neu2 (SEQ ID NO: 1). In certain embodiments, X1 is Ala, Asp, Met, or absent, X2 is Tyr or Val, X3 is Lys or Asp, X4 is Arg or Ala, X5 is Pro, Asn, Gly, Ser, or Thr, X6 is Ala or Glu, X7 is Gln or Tyr, X8 is Ile or Lys, X9 is Ala or Thr, and X 10 is Gln, Ala, or Thr, and X 11 is Ser, Arg, or Ala, and X 12 is Trp, Lys, or Arg, and X 13 is Ala or Cys, and X 14 is Val or Arg, and X 15 is Leu or Ile.

[0135] In certain embodiments, the first polypeptide comprises SEQ ID NO:77, the second polypeptide comprises SEQ ID NO:105, and the third polypeptide comprises SEQ ID NO:111. In certain embodiments, the first polypeptide comprises SEQ ID NO:77, the second polypeptide comprises SEQ ID NO:105, and the third polypeptide comprises SEQ ID NO:115. In certain embodiments, the first polypeptide comprises SEQ ID NO:77, the second polypeptide comprises SEQ ID NO:105, and the third polypeptide comprises SEQ ID NO:125.

[0136] In some embodiments, the antibody conjugate comprises a first polypeptide comprising a first sialidase, a first immunoglobulin Fc domain, and a first single-chain variable fragment (scFv) (it is also understood that the scFv can be replaced by a first polypeptide chain of an immunoglobulin antigen-binding fragment, e.g., a Fab fragment); and a second polypeptide comprising a second sialidase, a second immunoglobulin Fc domain, and a second single-chain variable fragment (scFv) (it is also understood that the scFv can be replaced by a second polypeptide chain of an immunoglobulin antigen-binding fragment, e.g., a Fab fragment). An example of this embodiment is shown in Figure 6C (it will be understood that in the construct shown in Figure 6C, the scFv, if present, can be replaced by a Fab fragment, or the Fab fragment, if present, can be replaced by the scFv). The first and second polypeptides can be covalently linked together. The covalent bond can be a disulfide bond. In some embodiments, the first scFv defines a first anti-PD-1 antigen-binding site, and the second scFv defines a second anti-PD-1 antigen-binding site. In some embodiments, the first polypeptide comprises, from N-terminal to C-terminal, a first sialidase, a first immunoglobulin Fc domain, and a first scFv. In some embodiments, the first polypeptide comprises, from N-terminal to C-terminal, a first scFv, a first immunoglobulin Fc domain, and a first sialidase. In some embodiments, the second polypeptide comprises, from N-terminal to C-terminal, a second sialidase, a second immunoglobulin Fc domain, and a second scFv. In some embodiments, the second polypeptide comprises, from N-terminal to C-terminal, a second scFv, a second immunoglobulin Fc domain, and a second sialidase.

[0137] In some embodiments, the antibody conjugate comprises: a first polypeptide comprising an immunoglobulin light chain; a second polypeptide comprising an immunoglobulin heavy chain and a single-chain variable fragment (scFv) (it is also understood that the scFv can be replaced by the first polypeptide chain of an immunoglobulin antigen-binding fragment, e.g., a Fab fragment); and a third polypeptide comprising an immunoglobulin Fc domain and a sialidase. An example of this embodiment is shown in Figure 6D. The first and second polypeptides can be covalently linked together, and the second and third polypeptides can be covalently linked together. The covalent linkage can be a disulfide bond. In some embodiments, the first and second polypeptides together define a first anti-PD-1 antigen-binding site (i.e., the immunoglobulin light chain and the immunoglobulin heavy chain together define the first anti-PD-1 antigen-binding site). In some embodiments, the scFv defines a second anti-PD-1 antigen-binding site. In some embodiments, the second polypeptide comprises, from N- to C-terminal, an immunoglobulin heavy chain and an scFv, or an scFv and an immunoglobulin heavy chain. In some embodiments, the third polypeptide comprises, from N- to C-terminal, a sialidase and an immunoglobulin Fc domain, or a sialidase and an immunoglobulin Fc domain.

[0138] In certain embodiments, the antibody conjugate comprises a first polypeptide comprising a first immunoglobulin light chain, a second polypeptide comprising a first sialidase, a first immunoglobulin Fc domain, and a first immunoglobulin heavy chain variable region, a third polypeptide comprising a second sialidase, a second immunoglobulin Fc domain, and a second immunoglobulin heavy chain variable region, and a fourth polypeptide comprising a second immunoglobulin light chain. It will also be understood that an immunoglobulin heavy chain variable region can replace an immunoglobulin light chain, and an immunoglobulin light chain can replace an immunoglobulin heavy chain variable region (e.g., an antibody conjugate can comprise a first polypeptide comprising a first immunoglobulin heavy chain variable region; a second polypeptide comprising a first sialidase, a first immunoglobulin Fc domain, and a first immunoglobulin light chain; a third polypeptide comprising a second sialidase, a second immunoglobulin Fc domain, and a second immunoglobulin light chain; and a fourth polypeptide comprising a second immunoglobulin heavy chain variable region). An example of this embodiment is shown in Figure 6E. The second and third polypeptides can be covalently linked to each other. The covalent bond can be a disulfide bond. In certain embodiments, the first and second polypeptides define a first anti-PD-1 antigen-binding site, and the third and fourth polypeptides define a second anti-PD-1 antigen-binding site. In certain embodiments, the second polypeptide comprises, from N-terminal to C-terminal, a first sialidase, a first immunoglobulin Fc domain, and a first immunoglobulin heavy chain variable region, hi certain embodiments, the third polypeptide comprises, from N-terminal to C-terminal, a second sialidase, a second immunoglobulin Fc domain, and a second immunoglobulin heavy chain variable region.

[0139] In some embodiments, the antibody conjugate has a molecular weight of about 135 kDa to about 165 kDa, e.g., about 140 kDa, hi other embodiments, the antibody conjugate has a molecular weight of about 215 kDa to about 245 kDa, e.g., about 230 kDa.

[0140] In some embodiments, an antibody conjugate comprises two polypeptides, each comprising an immunoglobulin Fc domain, wherein the first polypeptide has either a "knob" mutation, e.g., T366Y, or a "hole" mutation, e.g., Y407T, for heterodimerization with the second polypeptide, and the second polypeptide has either a "knob" mutation, e.g., T366Y, or a "hole" mutation, e.g., Y407T, for heterodimerization with the first polypeptide (residue numbers according to EU numbering; Kabat, EA, et al. (1991) supra). For example, in some embodiments, an antibody comprises two polypeptides, each comprising an immunoglobulin Fc domain derived from a human IgG1 Fc domain, wherein the first polypeptide comprises a Y407T mutation (e.g., the first polypeptide comprises SEQ ID NO: 32 or SEQ ID NO: 92), and the second polypeptide comprises a T366Y mutation (e.g., the second polypeptide comprises SEQ ID NO: 33 or SEQ ID NO: 93).

[0141] As used herein, the term "multispecific antibody" is understood to mean an antibody that specifically binds to at least two different antigens, i.e., an antibody that comprises at least two antigen-binding sites that bind to at least two different antigens. As used herein, the term "bispecific antibody" is understood to mean an antibody that specifically binds to two different antigens, i.e., an antibody that comprises two antigen-binding sites, each binding to a separate, different antigen. In other words, the first binding site binds to a first antigen and the second binding site binds to a second, different antigen. Multispecific or bispecific antibodies can be, for example, human or humanized antibodies, and / or full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies).

[0142] The present invention encompasses antibody conjugates comprising antibody fragments, which may be produced by conventional means, such as enzymatic digestion, or by recombinant techniques. For a review of specific antibody fragments, see Hudson et al. (2003), supra.

[0143] In some embodiments, the antibody conjugate or fusion protein may be covalently or non-covalently linked to a biological modifier, which may be used to increase the solubility of the antibody, increase binding specificity, reduce immunogenicity or toxicity, or modify the pharmacokinetic profile of the antibody. For example, a biological modifier may be used to increase the molecular weight of an antibody to increase its circulating half-life.

[0144] It is contemplated that the antibody conjugate or fusion protein may be covalently attached to one or more (e.g., 2, 3, 4, 5, 6, 8, 9, 10, or more) biological modifiers, which may include linear or branched polymers. Exemplary biological modifiers may include various polymers, such as those described in U.S. Pat. No. 7,842,789. Particularly useful are polyalkylene ethers, such as polyethylene glycol (PEG) and its derivatives (e.g., alkoxypolyethylene glycol, e.g., methoxypolyethylene glycol, ethoxypolyethylene glycol, etc.); block copolymers of polyoxyethylene and polyoxypropylene (Pluronics); polymethacrylates; carbomers; and branched or unbranched polysaccharides containing sugar monomers, such as D-mannose, D- and L-galactose, fucose, fructose, D-xylose, L-arabinose, and D-glucuronic acid.

[0145] In other embodiments, the biological modifier can be a hydrophilic polyvinyl polymer, such as polyvinyl alcohol and polyvinylpyrrolidone (PVP)-type polymer. The biological modifier can be a functionalized polyvinylpyrrolidone, e.g., carboxy- or amine-functionalized at one (or both) ends of the polymer (available from PolymerSource). Alternatively, the biological modifier can include poly-N-(2-hydroxypropyl) methacrylamide (HPMA), or functionalized HPMA (amine, carboxy, etc.), poly(N-isopropylacrylamide), or functionalized poly(N-isopropylacrylamide). Alternatively, the biological modifier can include poly-N-(2-hydroxypropyl) methacrylamide (HPMA), or functionalized HPMA (amine, carboxy, etc.), poly(N-isopropylacrylamide), or functionalized poly(N-isopropylacrylamide). The modifier prior to conjugation need not be, but preferably is, water-soluble; however, the final conjugate should be water-soluble.

[0146] Generally, biological modifiers are from about 2 kDa to about 5 kDa, from about 2 kDa to about 10 kDa, from about 2 kDa to about 20 kDa, from about 2 kDa to about 30 kDa, from about 2 kDa to about 40 kDa, from about 2 kDa to about 50 kDa, from about 2 kDa to about 60 kDa, from about 2 kDa to about 70 kDa, from about 2 kDa to about 80 kDa, from about 2 kDa to about 90 kDa, from about 2 kDa to about 100 kDa, from about 2 kDa to about 150 kDa, from about 5 kDa to about 10 kDa, from about 5 kDa to about 20 kDa, from about 5 kDa to about 30 kDa, from about 5 kDa to about 40 kDa, from about 5 kDa to about 50 kDa, from about 5 kDa to about 60 kDa, 5kDa to about 70kDa, about 5kDa to about 80kDa, about 5kDa to about 90kDa, about 5kDa to about 100kDa, about 5kDa to about 150kDa, about 10kDa to about 20kDa, about 10kDa to about 30kDa, about 10kDa to about 40kDa, about 10kDa to about 50kDa, about 10 kDa ~ approx. 60kDa, approx. 10kDa ~ approx. 70kDa, approx. 10kDa ~ approx. 80kDa, approx. 10kDa ~ approx. 90kDa, approx. 10kDa ~ approx. 100kDa, approx. Approximately 20kDa to approximately 60kDa, approximately 20kDa to approximately 70kDa, approximately 20kDa to approximately 80kDa, approximately 20kDa to approximately 90kDa, approximately 20kDa to approximately 100kDa, approximately 20kDa to approximately 150kDa, approximately 30kDa to approximately 40kDa, approximately 30kDa to approximately 50kDa, approximately 30kDa to approximately 60k Da, approx. 30kDa ~ approx. 70kDa, approx. 30kDa ~ approx. 80kDa, approx. 30kDa ~ approx. 90kDa, approx. 30kDa ~ approx. 100kDa, approx. 30kDa ~ approx. 150kDa, approx. 40kDa ~ approx. 80kDa, approximately 40kDa to approximately 90kDa, approximately 40kDa to approximately 100kDa, approximately 40kDa to approximately 150kDa, approximately 50kDa to approximately 60kDa, approximately 50kDa to approximately 70kDa, approximately 50kDa to approximately 80kDa, approximately 50kDa to approximately 90kDa, approximately 50kDa to approximately 100kDa, approximately 50k Da ~ about 150kDa, about 60kDa - about 70kDa, about 60kDa - about 80kDa, about 60kDa - about 90kDa, about 60kDa - about 100kDa, about 60kDa - about 150kDa, about 70kDa - about 80kDa, about 70kDa - about 90kDa, about 70kDa - about 100kDa,It may have a molecular weight of about 70 kDa to about 150 kDa, about 80 kDa to about 90 kDa, about 80 kDa to about 100 kDa, about 80 kDa to about 150 kDa, about 90 kDa to about 100 kDa, about 90 kDa to about 150 kDa, or about 100 kDa to about 150 kDa.

[0147] It is contemplated that the antibody conjugate or fusion protein is attached to about 10 or fewer polymer molecules (e.g., 9, 8, 7, 6, 5, 4, 3, 2, or 1), each polymer molecule having a molecular weight of at least about 20,000 D, or at least about 30,000 D, or at least about 40,000 D.

[0148] Although various polymers can be used as biological modifiers, it is contemplated that the antibody conjugates or fusion proteins described herein can be conjugated to polyethylene glycol (PEG) polymers. In one embodiment, the antibody conjugates or fusion proteins described herein are covalently linked to at least one PEG having an actual MW of at least about 20,000 D. In another embodiment, the antibody conjugates or fusion proteins described herein are covalently linked to at least one PEG having an actual MW of at least about 30,000 D. In another embodiment, the antibody conjugates or fusion proteins described herein are covalently linked to at least one PEG having an actual MW of at least about 40,000 D. In some embodiments, the PEG is methoxyPEG(5000)-succinimidyl propionate (mPEG-SPA), methoxyPEG(5000) succinimidyl succinate (mPEG-SS). Such PEGs are commercially available from Nektar Therapeutics or SunBiowest.

[0149] Attachment sites on antibody conjugates or fusion proteins for biological modifiers include the N-terminal amino group and the epsilon amino group found on lysine residues, as well as other amino, imino, carboxyl, sulfhydryl, hydroxyl, or other hydrophilic groups. Polymers can be directly covalently attached to antibody conjugates or fusion proteins using chemistries with or without the use of multifunctional (usually bifunctional) crosslinkers known in the art. For example, sulfhydryl groups can be derivatized by coupling to maleimide-substituted PEG (e.g., alkoxy-PEGamine plus sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, or PEG-maleimide, commercially available from Shearwater Polymers, Inc., Huntsville, Ala.).

[0150] II. Methods of Making Fusion Proteins or Antibody Conjugates Methods for producing fusion proteins (e.g., those disclosed herein), antibodies, or antibody conjugates (e.g., those disclosed herein) are known in the art. For example, DNA molecules encoding the light chain variable region and / or heavy chain variable region can be synthesized chemically or by recombinant DNA methodology. For example, the antibody sequence can be cloned from a hybridoma by conventional hybridization or polymerase chain reaction (PCR) techniques using appropriate synthetic nucleic acid primers. The resulting DNA molecule encoding the variable region of interest can be ligated to other appropriate nucleotide sequences, such as constant region coding sequences and expression control sequences, to produce a conventional gene expression construct (i.e., expression vector) encoding the desired antibody. The production of a given gene construct is within the ordinary skill of those in the art.

[0151] Nucleic acids encoding the desired fusion proteins and / or antibody conjugates can be incorporated (ligated) into expression vectors, which can be introduced into host cells by conventional transfection or transformation techniques. Exemplary host cells are E. coli cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK 293) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells that do not otherwise produce IgG protein. Transformed host cells can be grown under conditions in which the host cells express genes encoding immunoglobulin light and / or heavy chain variable regions.

[0152] Specific expression and purification conditions vary depending on the expression system used. For example, if a gene is expressed in E. coli, the gene is first cloned into an expression vector by placing the engineered gene downstream of a suitable bacterial promoter, such as Trp or Tac, and a prokaryotic signal sequence. The expressed protein can be secreted. The expressed protein can accumulate in refractile or inclusion bodies and can be recovered after disruption of the cells by French press or sonication. The refractile bodies can then be solubilized, and the protein can be refolded and / or cleaved by methods known in the art.

[0153] When a genetically engineered gene is to be expressed in a eukaryotic host cell, such as a CHO cell, the gene is first inserted into an expression vector containing a suitable eukaryotic promoter, secretion signal, polyA sequence, and stop codon. Optionally, the vector or gene construct may contain an enhancer and intron. In embodiments involving fusion proteins containing antibodies or portions thereof, the expression vector optionally contains sequences encoding all or part of a constant region that allows the entire heavy or light chain to be expressed. The gene construct can be introduced into a eukaryotic host cell using conventional techniques.

[0154] The host cell is then exposed to a sialidase and a V, each of which may be conjugated to a moiety with another function (e.g., cytotoxicity). L Or V H Fragment, V L -V H Heterodimer, V H -V L Or V L -V H They express fusion proteins and / or antibody conjugates comprising single-chain polypeptides, complete immunoglobulin heavy or light chains, or portions thereof. In some embodiments, including fusion proteins and / or antibody conjugates, host cells are transfected with a single vector expressing a polypeptide expressing a sialidase and all or part of a heavy chain (e.g., a heavy chain variable region), or a sialidase and a light chain (e.g., a light chain variable region), or a polypeptide expressing all or part of a heavy chain (e.g., a heavy chain variable region) or a light chain (e.g., a light chain variable region). In some embodiments, host cells are transfected with a single vector encoding (a) a polypeptide comprising a heavy chain variable region and a polypeptide comprising a light chain variable region, or (b) an entire immunoglobulin heavy chain and an entire immunoglobulin light chain, where in (a) or (b), the polypeptide may also include a sialidase. In some embodiments, the host cell is co-transfected with more than one expression vector (e.g., one expression vector expressing a polypeptide comprising all or part of a heavy chain or heavy chain variable region, optionally comprising a sialidase fused thereto, and another expression vector expressing a polypeptide comprising all or part of a light chain or light chain variable region, optionally comprising a sialidase fused thereto).

[0155] Polypeptides containing fusion proteins (e.g., fusion proteins containing immunoglobulin heavy or light chain variable regions) can be produced by growing (culturing) host cells transfected with an expression vector encoding such variable regions under conditions that allow expression of the polypeptide. After expression, the polypeptide can be recovered and purified or isolated using techniques known in the art, for example, affinity tags such as glutathione-S-transferase (GST) or histidine tags.

[0156] In embodiments in which fusion proteins and / or antibody conjugates are produced, sialidase fused to a monoclonal antibody, Fc domain, or antigen-binding domain of an antibody can be produced by growing (culturing) host cells transfected with: (a) an expression vector encoding a complete or partial immunoglobulin heavy chain and another expression vector encoding a complete or partial immunoglobulin light chain; or (b) a single expression vector encoding both chains (e.g., complete or partial heavy and light chains) under conditions that allow expression of both chains. The sialidase will be fused to one or more of the chains. The intact fusion protein and / or antibody conjugate can be recovered and purified or isolated using techniques known in the art, for example, affinity tags such as protein A, protein G, glutathione-S-transferase (GST), or histidine tags. It is within the ordinary skill in the art to express the heavy and light chains from a single expression vector or from two separate expression vectors.

[0157] In some embodiments, to express a protein, e.g., a fusion protein, as a secreted protein, the native N-terminal signal sequence of the protein is replaced with, e.g., MDMRVPAQLLGLLLLWLPGARC (SEQ ID NO: 28). In some embodiments, to express a protein, e.g., a fusion protein, as a secreted protein, an N-terminal signal sequence, e.g., MDMRVPAQLLGLLLLWLPGARC (SEQ ID NO: 28), is added. Further exemplary N-terminal signal sequences include signal sequences from interleukin-2, CD-5, IgG kappa light chain, trypsinogen, serum albumin, and prolactin. In some embodiments, to express a protein, e.g., a fusion protein, as a secreted protein, a C-terminal lysosomal signal motif, e.g., YGTL (SEQ ID NO: 29), is removed.

[0158] Methods for reducing or removing the antigenicity of antibodies and antibody fragments are known in the art. When the antibodies are administered to humans, they are preferably "humanized" to reduce or remove their antigenicity in humans. Preferably, each humanized antibody has the same or substantially the same affinity for the antigen as the non-humanized murine antibody from which it was derived.

[0159] In one humanization approach, chimeric proteins are generated in which mouse immunoglobulin constant regions are replaced with human immunoglobulin constant regions (see, e.g., Morrison et al., 1984, PROC. NAT. ACAD. SCI. 81:6851-6855; Neuberger et al., 1984, NATURE 312:604-608; U.S. Patent Nos. 6,893,625 (Robinson); 5,500,362 (Robinson); and 4,816,567 (Cabilly).

[0160] In an approach known as CDR grafting, the CDRs of the light and heavy chain variable regions are grafted onto a framework derived from another species. For example, mouse CDRs can be grafted onto human FRs. In some embodiments, the CDRs of the light and heavy chain variable regions of an antibody are grafted onto human FRs or consensus human FRs. To create consensus human FRs, FRs from several human heavy or light chain amino acid sequences are aligned to identify a consensus amino acid sequence. CDR porting is applicable to U.S. Patent No. 7,022,500 (Queen); U.S. Pat. ); No. 5,859,205 (Adair); No. 5,693,761 (Queen); No. 5,565,332 (Hoogenboom); No. 5,585,089 (Queen); No. 5,530,101 (Queen); Jones et al.(1986)NATURE 321:522-525;Riechmann et al.(1988)NATURE 332:323-327;Verhoeyen et al.(1988)SCIENCE 239:1534-1536; and Winter (1998) FEBS LETT 430:92-94.

[0161] In an approach referred to as "SUPERHUMANIZATION™," human CDR sequences are selected from human germline genes based on the structural similarity of the human CDRs to the CDRs of the mouse antibody being humanized. See, e.g., U.S. Patent No. 6,881,557 (Foote); and Tan et al., 2002, J. IMMUNOL. 169:1119-1125.

[0162] Other methods for reducing immunogenicity include "reshaping," "hyperchimerization," and "veneering / resurfacing." See, e.g., Vaswami et al., 1998, ANNALS OF ALLERGY, ASTHMA, & IMMUNOL. 81:105; Roguska et al., 1996, PROT. ENGINEER 9:895-904; and U.S. Pat. No. 6,072,035 (Hardman). In the veneering / resurfacing approach, surface-accessible amino acid residues in a murine antibody are replaced with amino acid residues that are more frequently found at the same positions in human antibodies. This type of antibody resurfacing is described, for example, in U.S. Pat. No. 5,639,641 (Pedersen).

[0163] Another approach to converting murine antibodies into a form suitable for medical use in humans is ACTIVMAB TM The technique is known as "human engineering" (Vaccinex, Inc., Rochester, NY), which involves vaccinia virus-based vectors for expressing antibodies in mammalian cells. High levels of combinatorial diversity of IgG heavy and light chains can be produced. See, e.g., U.S. Patent Nos. 6,706,477 (Zauderer); 6,800,442 (Zauderer); and 6,872,518 (Zauderer). Another approach to converting mouse antibodies into a form suitable for use in humans is the technique commercially practiced by KaloBios Pharmaceuticals, Inc. (Palo Alto, CA). This technique involves the use of a proprietary human "acceptor" library to create an "epitope-focused" library for antibody selection. Another approach to modifying mouse antibodies into a form suitable for medical use in humans is the HUMAN ENGINEERING TMtechnology, which is commercially practiced by XOMA (US) LLC. See, e.g., International (PCT) Publication No. WO 93 / 11794 and U.S. Patent Nos. 5,766,886 (Studnicka); 5,770,196 (Studnicka); 5,821,123 (Studnicka); and 5,869,619 (Studnicka).

[0164] Any suitable approach, including any of the approaches described above, may be used to reduce or eliminate human immunogenicity of an antibody.

[0165] It is also possible to generate fully human antibodies in mice. Fully human mAbs, lacking any non-human sequences, can be prepared from human immunoglobulin transgenic mice by techniques referenced, for example, in Lonberg et al., NATURE 368:856-859, 1994; Fishwild et al., NATURE BIOTECHNOLOGY 14:845-851, 1996; and Mendez et al., NATURE GENETICS 15:146-156, 1997. Fully human monoclonal antibodies can also be prepared and optimized from phage display libraries by techniques referenced, for example, in Knappik et al., J. MOL. BIOL. 296:57-86, 2000; and Krebs et al., J. IMMUNOL. METH. 254:67-84, 2001).

[0166] The present invention encompasses fusion proteins comprising antibody fragments, which may be produced by conventional means, such as enzymatic digestion or recombinant techniques. For a review of specific antibody fragments, see Hudson et al. (2003) NAT. MED. 9:129-134.

[0167] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were generated by proteolytic digestion of intact antibodies (see, e.g., Morimoto et al. (1992) JOURNAL OF BIOCHEMICAL AND BIOPHYSICAL METHODS 24:107-117; and Brennan et al. (1985) SCIENCE 229:81). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv, and ScFv antibody fragments can all be expressed in and secreted from E. coli, allowing the facile production of large amounts of these fragments. Antibody fragments can also be isolated from antibody phage libraries. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically ligated to form F(ab')2 fragments (Carter et al. (1992) BIO / TECHNOLOGY 10:163-167). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab')2 fragments with increased in vivo half-lives containing salvage receptor binding epitope residues are described in U.S. Patent No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to those skilled in the art. In some embodiments, the antibody is a single-chain Fv fragment (scFv). See U.S. Patent Nos. 5,571,894 and 5,587,458.

[0168] Methods for producing bispecific antibodies are known in the art. See Milstein and Cuello (1983) NATURE 305:537, International (PCT) Publication No. WO 93 / 08829, and Traunecker et al. (1991) EMBO J., 10:3655. For further details on producing bispecific antibodies, see, for example, Suresh et al. (1986) METHODS ENZYMOL. 121:210. Bispecific antibodies include cross-linked or "heteroconjugate" or "heterodimeric" antibodies. For example, one antibody in the heterodimer can be linked to avidin and the other to biotin. Heterodimeric antibodies can be produced using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, along with several cross-linking techniques.

[0169] Examples of heterodimeric or asymmetric IgG-like molecules include, but are not limited to, those obtained using the following technologies or formats: Triomab / Quadroma, Knobs-into-Holes, CrossMabs, Electrostatically Matched Antibodies, LUZ-Y, Strand Exchange Engineered Domain bodies, Biclonic and DuoBody.

[0170] Advantages of using antibody fragments (e.g., F(ab) and F(ab')2 fragments) include elimination of non-specific binding between the Fc portion of the antibody and Fc receptors on cells (e.g., macrophages, dendritic cells, neutrophils, NK cells, and B cells). Fragments may also be able to penetrate tissues more efficiently due to their smaller size.

[0171] Heterodimeric or asymmetric antibodies allow for greater flexibility and novel formats for attaching various drugs to antibody arms. One common format for generating heterodimeric antibodies is the "knobs-into-holes" format. This format is specific to the heavy chain portion of the antibody constant region. The "knobs" are engineered by replacing small amino acids with larger ones that fit into the "holes," and the holes are engineered by replacing large amino acids with smaller ones. The "knobs" are connected to the "holes" by disulfide bonds between each chain. The "knobs-into-holes" configuration facilitates antibody-dependent cell-mediated cytotoxicity. Single-chain variable fragments (scFvs) are linked to the heavy and light chain variable domains via a short linker peptide. The linker is rich in glycines, which provide greater flexibility to the linker, and serine / threonines, which provide specificity. Two different scFv fragments can be joined together via a hinge region to either the heavy chain constant domain or the light chain constant domain. This confers bispecificity to the antibody, allowing it to bind to two different antigens. The "knobs-into-holes" format enhances heterodimer formation but does not suppress homodimer formation.

[0172] Several approaches to supporting heterodimerization are described, for example, in International (PCT) Publication Nos. WO96 / 27011, WO98 / 050431, WO2007 / 110205, WO2007 / 147901, WO2009 / 089004, WO2010 / 129304, WO2011 / 90754, WO2011 / 143545, WO2012 / 058768, WO2013 / 157954 and WO2013 / 096291, and European Patent Publication No. EP1870459. Typically, in approaches known in the art, the CH3 domain of a first heavy chain and the CH3 domain of a second heavy chain are both engineered in a complementary manner so that a heavy chain containing one engineered CH3 domain can no longer homodimerize with another heavy chain of the same structure (e.g., a CH3-engineered first heavy chain can no longer homodimerize with another CH3-engineered first heavy chain; a CH3-engineered second heavy chain can no longer homodimerize with another CH3-engineered second heavy chain), thereby forcing a heavy chain containing one engineered CH3 domain to heterodimerize with another heavy chain containing a CH3 domain engineered in a complementary manner. Consequently, the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain are engineered in a complementary manner by amino acid substitutions such that the first and second heavy chains are forced to heterodimerize, while the first and second heavy chains can no longer homodimerize (e.g., for steric reasons).

[0173] III. Pharmaceutical Compositions For therapeutic uses, the fusion protein and / or antibody conjugate is preferably combined with a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable," as used herein, refers to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0174] The term "pharmaceutically acceptable carrier," as used herein, refers to buffers, carriers, and excipients that are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers include any of the standard pharmaceutical carriers, such as phosphate-buffered saline solution, water, emulsions (e.g., oil / water or water / oil emulsions), and various types of wetting agents. The compositions may also contain stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA

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

[0175] In certain embodiments, pharmaceutical compositions may contain formulation materials to modify, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or permeability of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); bulking agents; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colorants, flavoring agents, etc. agents) and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (such as glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancers (such as sucrose or sorbitol); isotonicity enhancers (such as alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, sorbitol, etc.); delivery vehicles: diluents; excipients and / or pharmaceutical adjuvants (Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990).

[0176] In some embodiments, the pharmaceutical composition can include nanoparticles, such as polymeric nanoparticles, liposomes, or micelles (see Anselmo et al. (2016) BIOENG.TRANSL.MED. 1:10-29).

[0177] In some embodiments, the pharmaceutical composition may comprise a sustained- or controlled-delivery formulation. Techniques for formulating sustained- or controlled-delivery means, such as liposome carriers, bioerodible microparticles or porous beads, and depot injections, are also known to those skilled in the art. Sustained-release preparations may comprise, for example, porous polymeric microparticles or semipermeable polymer matrices in the form of shaped articles, such as films or microcapsules. Sustained-release matrices may include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma-ethyl-L-glutamic acid, poly(2-hydroxyethyl-inethacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. Sustained-release compositions may also comprise liposomes, which may be prepared by any of several methods known in the art.

[0178] Pharmaceutical compositions comprising the sialidase fusion proteins or antibody conjugates disclosed herein may be present in unit dosage form and may be prepared by any suitable method. Pharmaceutical compositions should be formulated to be compatible with their intended route of administration. Examples of routes of administration include intravenous (IV), intradermal, inhalation, transdermal, topical, transmucosal, intrathecal, and rectal administration. In some embodiments, the sialidase fusion proteins or antibody conjugates disclosed herein are administered by IV infusion. In some embodiments, the sialidase fusion proteins or antibody conjugates disclosed herein are administered by intratumoral injection. Useful formulations can be prepared by methods known in the pharmaceutical arts. See, e.g., Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990). Formulation components suitable for parenteral administration include sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.

[0179] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). The carrier should be stable under the conditions of manufacture and storage and preserved against microorganisms. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

[0180] In some embodiments, the pharmaceutical composition may contain a stabilizer. In some embodiments, the stabilizer is a cation, such as a divalent cation. In some embodiments, the cation is calcium or magnesium. The cation may be in the form of a salt, such as calcium chloride (CaCl) or magnesium chloride (MgCl).

[0181] In certain embodiments, the stabilizer is present in an amount of about 0.05 mM to about 5 mM. For example, the stabilizer may be present in an amount of about 0.05 mM to about 4 mM, about 0.05 mM to about 3 mM, about 0.05 mM to about 2 mM, about 0.05 mM to about 1 mM, about 0.05 mM to about 0.5 mM, about 0.5 mM to about 4 mM, about 0.5 mM to about 3 mM, about 0.5 mM to about 2 mM, about 0.5 mM to about 1 mM, about 1 mM to about 4 mM, about 1 mM to about 3 mM, or about 1 mM to about 2 mM.

[0182] Preferably, the pharmaceutical preparation is sterile. Sterilization can be achieved by any suitable method, for example, filtration through a sterile filtration membrane. If the composition is lyophilized, sterilization by filtration can be performed before or after lyophilization and reconstitution.

[0183] The compositions described herein can be administered locally or systemically. Administration is generally parenteral. In a preferred embodiment, the pharmaceutical compositions are administered subcutaneously, and in an even more preferred embodiment, intravenously. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.

[0184] Generally, a therapeutically effective amount of an active component, e.g., a fusion protein and / or antibody conjugate, ranges from 0.1 mg / kg to 100 mg / kg, e.g., 1 mg / kg to 100 mg / kg, e.g., 1 mg / kg to 10 mg / kg. The dosage depends on variables such as the type and severity of the disease or symptom being treated, the patient's overall health, the in vivo efficacy of the antibody, the pharmaceutical formulation, and the route of administration. To rapidly achieve the desired blood or tissue levels, the initial dose may be increased beyond the upper limit. Alternatively, the initial dose may be lower than optimal, and the daily dose may be gradually increased over the course of treatment. Human dosages may be optimized in a conventional Phase I dose-escalation study designed, for example, from 0.5 mg / kg to 20 mg / kg. The dosing frequency may vary depending on factors such as the route of administration, dosage, serum half-life, and the disease being treated of the fusion protein and / or antibody conjugate. Exemplary dosing frequencies are once daily, once weekly, and once every two weeks. A preferred route of administration is parenteral, e.g., intravenous infusion. In certain embodiments, the fusion protein and / or antibody conjugate is lyophilized and then reconstituted in buffered saline at the time of administration.

[0185] IV. Therapeutic uses The compositions and methods disclosed herein can be used to treat various forms of cancer in a subject or to inhibit the growth of cancer in a subject. The present invention provides a method of treating cancer in a subject. The method includes administering to the subject an effective amount of a sialidase-anti-PD-1 fusion protein and / or antibody conjugate, such as a fusion protein or antibody conjugate disclosed herein, alone or in combination with another therapeutic agent, to treat cancer in the subject. The term "effective amount," as used herein, refers to the amount of an active agent (e.g., a fusion protein of the invention) sufficient to produce a beneficial or desired result. An effective amount may be administered in one or more administrations, applications, or doses, and is not intended to be limited to a particular formulation or route of administration.

[0186] As used herein, "treat," "treating," and "treatment" refer to the treatment of a disease in a subject, e.g., a human. This includes (a) inhibiting the disease, i.e., halting its progression; and (b) palliating the disease, i.e., causing regression of the disease state. As used herein, the terms "subject" and "patient" refer to an organism treated by the methods and compositions described herein. Preferably, such organisms include, but are not limited to, mammals (e.g., mice, monkeys, horses, cows, pigs, dogs, cats, etc.), and more preferably, humans.

[0187] Examples of cancer include solid tumors, soft tissue tumors, hematopoietic tumors, and metastatic lesions. Examples of hematopoietic tumors include leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), B-cell, T-cell, or FAB ALL, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), such as transformed CLL, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, hairy cell leukemia, myelodyplastic syndrome (MDS), lymphoma, Hodgkin's disease, malignant lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, and Richter's syndrome (Richter's transformation). Examples of solid tumors include malignancies such as sarcomas, adenocarcinomas, and carcinomas of various organ systems, such as those affecting the head and neck (including pharynx), thyroid, lung (small cell or non-small cell lung carcinoma (NSCLC)), breast, lymphatic system, gastrointestinal (e.g., oral cavity, esophagus, stomach, liver, pancreas, small intestine, colon and rectum, anal canal), reproductive and genitourinary tract (e.g., kidney, urothelium, bladder, ovaries, uterus, cervix, endometrium, prostate, testes), CNS (e.g., neuronal or glial cells, such as neuroblastoma or glioma), or skin (e.g., melanoma and metastatic Merkel cell carcinoma (MCC)).

[0188] In some embodiments, the cancer is an epithelial cancer, e.g., an epithelial cancer that upregulates expression of sialylated glycans. Exemplary epithelial cancers include, but are not limited to, endometrial cancer, colon cancer, ovarian cancer, cervical cancer, vulvar cancer, uterine cancer or fallopian tube cancer, breast cancer, prostate cancer, lung cancer, pancreatic cancer, urinary tract cancer, bladder cancer, head and neck cancer, oral cancer, and liver cancer. Epithelial cancers also include carcinomas, such as lobular carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, adenocarcinoma (carcinoma adenomatosum), carcinoma of the adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchiolocarcinoma, bronchogenic carcinoma, cerebrioid carcinoma, cholangiocarcinoma, choriocarcinoma, colloid carcinoma, comedocarcinoma, corpus carcinoma, cribriform carcinoma, armor carcinoma, carcinoma cutaneum, and cylindrical carcinoma. carcinoma, cylindrical cell carcinoma, ductal carcinoma, carcinoma durum, embryonal carcinoma, encephalomoid carcinoma, epidermoid carcinoma, epithelial adenoid carcinoma, exophytic carcinoma, extraulcerative carcinoma, fibrous carcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, adenocarcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hürthle cell carcinoma, vitreous carcinomacarcinoma, hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, carcinoma in epidermis, carcinoma in situ (intraepithelial carcinoma), Krompecher's carcinoma, Kurticycky cell carcinoma, large cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullaris, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, myxomatodes, nasopharyngeal carcinoma, oat cell carcinoma, ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of the kidney, reserve cell carcinoma, sarcomatodes, Schneiderian carcinoma, scirrhous carcinoma, scrotal carcinoma, signet ring cell carcinoma, carcinoma simplex, small cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma carcinoma), spindle cell carcinoma, carcinoma spongiosum, squamous cell carcinoma, squamous cell carcinomaThese include carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, nodular carcinoma, tuberous carcinoma, verrucous carcinoma, and choriocarcinoma.

[0189] In certain embodiments, the cancer is selected from lung bronchioloalveolar carcinoma (BAC), bladder cancer, female reproductive tract malignancies (e.g., uterine serous adenocarcinoma, endometrial carcinoma, vulvar squamous cell carcinoma, and uterine sarcoma), ovarian surface epithelial carcinoma (e.g., ovarian clear cell carcinoma, epithelial ovarian carcinoma, fallopian tube carcinoma, and primary peritoneal carcinoma), breast cancer, non-small cell lung cancer (NSCLC), male reproductive tract malignancies (e.g., testicular cancer), retroperitoneal or peritoneal carcinoma, gastroesophageal adenocarcinoma, esophagogastric junction carcinoma, liver hepatocellular carcinoma, esophageal and esophagogastric junction carcinoma, and esophageal and esophagogastric junction carcinoma. Selected from synovial carcinoma, cervical cancer, bile duct carcinoma, pancreatic adenocarcinoma, extrahepatic bile duct adenocarcinoma, small intestinal malignancies, gastric adenocarcinoma, carcinoma of unknown primary (CUP), colorectal adenocarcinoma, esophageal carcinoma, prostate adenocarcinoma, renal carcinoma, head and neck squamous cell carcinoma, thymic carcinoma, non-melanoma skin cancer, thyroid cancer (e.g., papillary carcinoma), head and neck cancer, anal cancer, non-epithelial ovarian cancer (non-EOC), metastatic urothelial carcinoma (UC), uveal melanoma, malignant pleural mesothelioma, small cell lung cancer (SCLC), central nervous system cancer, neuroendocrine tumors, and soft tissue tumors.

[0190] In some embodiments, the cancer is melanoma, non-small cell lung cancer, colon cancer, breast cancer, bladder cancer, or renal cancer.

[0191] In some embodiments, the cancer is an adenocarcinoma. In some embodiments, the cancer is a metastatic cancer. In some embodiments, the cancer is a refractory cancer.

[0192] In some embodiments, the cancer is resistant or non-responsive to treatment with an antibody, e.g., pembrolizumab.

[0193] In certain embodiments, the cancer is associated with or otherwise mediated by PD-1, hi certain embodiments, the cancer is a PD-L1-expressing cancer, e.g., the cancer comprises cells that express PD-L1.

[0194] The methods and compositions described herein can be used alone or in combination with other therapeutic agents and / or modalities. As used herein, the term "administered in combination" is understood to mean that two (or more) different therapies are delivered to a subject with a disorder such that the effects of the therapies on the patient overlap at points in time during the course of the subject's affliction. In some embodiments, the delivery of one treatment is still occurring when the delivery of a second treatment begins, so there is an overlap in the administration period. This is sometimes referred to herein as "simultaneous" or "concurrent delivery." In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some cases, the treatments are more effective due to their combined administration. For example, the second treatment is more effective, e.g., a comparable effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent than would be seen if the second treatment were administered in the absence of the first treatment, or a comparable condition is seen with the first treatment. In some embodiments, delivery is such that the reduction in symptoms or other parameters associated with the disorder is greater than that observed when one treatment is delivered in the absence of the other treatment. The effects of the two treatments may be partially additive, wholly additive, or greater than additive. Delivery may be such that the effect of the first treatment delivered remains detectable when the second treatment is delivered.

[0195] In some embodiments, the methods or compositions described herein are administered in combination with one or more additional treatments, such as surgery, radiation therapy, or the administration of another therapeutic preparation. In some embodiments, the additional treatment may include chemotherapy, such as a cytotoxic agent. In some embodiments, the additional treatment may include targeted treatment, such as a tyrosine kinase inhibitor, a proteasome inhibitor, or a protease inhibitor. In some embodiments, the additional treatment may include an anti-inflammatory, anti-angiogenic, anti-fibrotic, or anti-proliferative compound, such as a steroid, a biological immunomodulator, a monoclonal antibody, an antibody fragment, an aptamer, an siRNA, an antisense molecule, a fusion protein, a cytokine, a cytokine receptor, a bronchodilator, a statin, an anti-inflammatory agent (e.g., methotrexate), or an NSAID. In some embodiments, the additional treatment may include a combination of different therapeutic agents.

[0196] In some embodiments, the methods or compositions described herein are administered in combination with a second checkpoint inhibitor, which may be selected from, for example, a second PD-1 antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, an adenosine A2A receptor antagonist, a B7-H3 antagonist, a B7-H4 antagonist, a BTLA antagonist, a KIR antagonist, a LAG3 antagonist, a TIM-3 antagonist, a VISTA antagonist, or a TIGIT antagonist.

[0197] In some embodiments, the checkpoint inhibitor is a second PD-1 inhibitor or PD-L1 inhibitor. PD-1 is a receptor present on the surface of T cells that acts as a checkpoint in the immune system, inhibiting or otherwise regulating T cell activity at the appropriate time to prevent an overactive immune response. However, cancer cells can exploit this checkpoint by expressing a ligand, e.g., PD-L1, that interacts with PD-1 on the surface of T cells to block or regulate T cell activity. Exemplary PD-1 / PD-L1-based immune checkpoint inhibitors include antibody-based therapeutic agents. Exemplary therapeutic methods using PD-1 / PD-L1-based immune checkpoint inhibition are described in U.S. Patent Nos. 8,728,474 and 9,073,994 and European Patent No. 1537878B1, and include, for example, the use of anti-PD-1 antibodies. Exemplary anti-PD-1 antibodies are described, e.g., in U.S. Patent Nos. 8,952,136, 8,779,105, 8,008,449, 8,741,295, 9,205,148, 9,181,342, 9,102,728, 9,102,727, 8,952,136, 8,927,697, 8,900,587, 8,735,553, and 7,488,802. Exemplary anti-PD-1 antibodies include, for example, nivolumab (Opdivo®, Bristol-Myers Squibb Co.), pembrolizumab (Keytruda®, Merck Sharp & Dohme Corp.), PDR001 (Novartis Pharmaceuticals), and pidilizumab (CT-011, Cure Tech). Exemplary anti-PD-L1 antibodies are described, for example, in U.S. Patent Nos. 9,273,135, 7,943,743, 9,175,082, 8,741,295, 8,552,154, and 8,217,149. Exemplary anti-PD-L1 antibodies include atezolizumab (Tecentriq®, Genentech), duvalumab (AstraZeneca), MEDI4736, avelumab, and BMS 936559 (Bristol Myers Squibb Co.).

[0198] In some embodiments, the methods or compositions described herein are administered in combination with a CTLA-4 inhibitor. In the CTLA-4 pathway, the interaction of CTLA-4 on T cells with its ligands (e.g., also known as CD80, B7-1, and CD86) on the surface of antigen-presenting cells (not cancer cells) leads to T cell inhibition. Exemplary CTLA-4-based immune checkpoint inhibition methods are described in U.S. Patent Nos. 5,811,097, 5,855,887, and 6,051,227. Exemplary anti-CTLA-4 antibodies are described in U.S. Patent Nos. 6,984,720, 6,682,736, 7,311,910, 7,307,064, 7,109,003, 7,132,281, 6,207,156, 7,807,797, 7,824,679, 8,143,379, 8,263,073, 8,318,916, 8,017,114, 8,784,815, and 8,883,984; International (PCT) Publication Nos. WO 98 / 42752, WO 00 / 37504, and WO 01 / 14424; and European Patent EP 1212422. B1. Exemplary CTLA-4 antibodies include ipilimumab or tremelimumab.

[0199] In certain embodiments, the methods or compositions described herein are administered in combination with a CTLA-4 inhibitor, such as a CTLA-4 inhibitor disclosed herein.

[0200] In some embodiments, the methods or compositions described herein are administered in combination with an IDO inhibitor. Exemplary IDO inhibitors include 1-methyl-D-tryptophan (known as indoximod), epacadostat (INCB24360), navoximod (GDC-0919), and BMS-986205.

[0201] Exemplary cytotoxic agents that may be administered in combination with the methods or compositions described herein include, for example, antimicrotubule agents, topoisomerase inhibitors, antimetabolites, protein synthesis and degradation inhibitors, mitotic inhibitors, alkylating agents, platinizing agents, and the like. agents), inhibitors of nucleic acid synthesis, histone deacetylase inhibitors (HDAC inhibitors, e.g., vorinostat (SAHA, MK0683), entinostat (MS-275), panobinostat (LBH589), trichostatin A (TSA), mocetinostat (MGCD0103), belinostat (PXD101), romidepsin (FK228, depsipeptide)), DNA methyltransferase inhibitors, nitrogen mustards, nitrosoureas, ethylenimines, alkylsulfonates, triazenes, folic acid analogs, nucleoside analogs, ribonucleotide reductase inhibitors, vinca alkaloids, taxanes, epothilones, intercalating agents, drugs that can interfere with signal transduction pathways, drugs and radiation that promote apoptosis, or antibody molecule conjugates that bind to surface proteins to deliver toxic agents.In one embodiment, cytotoxic agents that may be administered with the methods or compositions described herein include platinum-based agents (e.g., cisplatin), cyclophosphamide, dacarbazine, methotrexate, fluorouracil, gemcitabine, capecitabine, hydroxyurea, topotecan, irinotecan, azacitidine, vorinostat, ixabepilone, bortezomib, taxanes (e.g., paclitaxel or docetaxel), cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, thiazolinone ... The active ingredient is benzocaine, etoposide, tenoposide, vincristine, vinblastine, vinorelbine, colchicine, anthracyclines (e.g., doxorubicin or epirubicin), daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, adriamycin, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, ricin, or maytansinoids.

[0202] The present invention also provides methods for increasing expression of HLA-DR, CD86, CD83, IFNγ, IL-1b, IL-6, TNFα, IL-17A, IL-2, or IL-6 in a cell, tissue, or subject. The methods comprise contacting the cell, tissue, or subject with an effective amount of a fusion protein and / or antibody conjugate, e.g., a fusion protein or antibody conjugate disclosed herein. In one embodiment, the cell is selected from a dendritic cell and a peripheral blood mononuclear cell (PBMC).

[0203] In certain embodiments, expression of HLA-DR, CD86, CD83, IFNγ, IL-1b, IL-6, TNFα, IL-17A, IL-2, or IL-6 in a cell, tissue, or subject is increased by at least about 10%, at least about 20%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1,000%, relative to similar or otherwise identical cells or tissues not contacted with the fusion protein or antibody conjugate. Gene expression can be measured by any suitable method known in the art, for example, by ELISA or by Luminex multiplex assay.

[0204] The present invention also provides a method for promoting immune cell infiltration into a tumor in a subject in need thereof, comprising administering to the subject an effective amount of a fusion protein and / or antibody conjugate, e.g., a fusion protein or antibody conjugate disclosed herein. In one embodiment, the immune cells are T cells, e.g., CD4+ and / or CD8+ T cells, e.g., CD69 + CD8 + and / or GzmB + CD8 + In some embodiments, the immune cell is a natural killer (NK) cell.

[0205] In certain embodiments, immune cell infiltration into a tumor in a subject is increased by at least about 10%, at least about 20%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1,000%, relative to a similar or otherwise identical tumor and / or subject that has not received the fusion protein or antibody conjugate. Immune cell infiltration into a tumor can be measured by any suitable method known in the art, such as antibody staining.

[0206] The present invention also provides a method for increasing the amount of circulating natural killer (NK) cells in a subject in need thereof, comprising administering to the subject an effective amount of a fusion protein and / or antibody conjugate, such as a fusion protein or antibody conjugate disclosed herein, to increase the number of circulating NK cells relative to before administration of the fusion protein, antibody conjugate, or pharmaceutical composition.

[0207] In certain embodiments, the number of circulating NK cells in a subject is increased by at least about 10%, at least about 20%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1,000%, relative to a similar or otherwise identical subject that did not receive the fusion protein or antibody conjugate. Circulating NK cells in a subject can be measured by any suitable method known in the art, such as antibody staining.

[0208] The present invention also provides a method for increasing the amount of T cells in a draining lymph node in a subject in need thereof. The method comprises administering to the subject an effective amount of a fusion protein, antibody conjugate, and / or pharmaceutical composition, e.g., a fusion protein, antibody conjugate, and / or pharmaceutical composition disclosed herein, to increase the number of T cells in the draining lymph node relative to before administration of the fusion protein, antibody conjugate, or pharmaceutical composition. In some embodiments, the immune cells are T cells, e.g., CD4+ and / or CD8+ T cells.

[0209] In certain embodiments, the number of T cells in the draining lymph nodes in a subject is increased by at least about 10%, at least about 20%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1,000%, relative to a similar or otherwise identical subject that has not been administered the fusion protein, antibody conjugate, or pharmaceutical composition. T cells in the draining lymph nodes in a subject can be measured by any suitable method known in the art, such as antibody staining.

[0210] The present invention also provides methods for increasing expression of Cd3, Cd4, Cd8, Cd274, Ctla4, Icos, Pdcd1, Lag3, Il6, Il1b, Il2, Ifng, Ifna1, Mx1, Gzmb, Cxcl9, Cxcl12 and / or Ccl5 in a cell, tissue or subject. The method includes contacting a cell, tissue, or subject with an effective amount of a fusion protein, antibody conjugate, and / or pharmaceutical composition, such as a fusion protein, antibody conjugate, and / or pharmaceutical composition disclosed herein, to increase expression of Cd3, Cd4, Cd8, Cd274, Ctla4, Icos, Pdcd1, Lag3, Il6, Il1b, Il2, Ifng, Ifna1, Mx1, Gzmb, Cxcl9, Cxcl12, and / or Ccl5 relative to the cell, tissue, or subject prior to contact with the fusion protein, antibody conjugate, or pharmaceutical composition.

[0211] In certain embodiments, expression of Cd3, Cd4, Cd8, Cd274, Ctla4, Icos, Pdcd1, Lag3, Il6, Il1b, Il2, Ifng, Ifna1, Mx1, Gzmb, Cxcl9, Cxcl12 and / or Ccl5 in a cell, tissue or subject is increased by at least about 10%, at least about 20%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1,000% relative to a similar or otherwise identical cell, tissue or subject not contacted with the fusion protein, antibody conjugate, or pharmaceutical composition. Gene expression can be measured by any suitable method known in the art, for example, ELISA, Luminex multiplex assay or Nanostring technology.

[0212] The present invention also provides a method for removing sialic acid from cells or tissues, comprising contacting the cells or tissues with an effective amount of a fusion protein and / or antibody conjugate, such as a fusion protein or antibody conjugate disclosed herein. The present invention also provides a method for removing sialic acid from cells in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising the fusion protein and / or antibody conjugate, such as a fusion protein or antibody conjugate disclosed herein, thereby removing sialic acid from the cells.

[0213] In some embodiments, the cells are tumor cells, dendritic cells (DCs), or monocytes. In some embodiments, the cells are monocytes, and the method results in increased expression of MHC-II molecules (e.g., HLA-DR) on the monocytes. In some embodiments, expression of MHC-II molecules in cells or tissues is increased by at least about 10%, at least about 20%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1,000% relative to similar or otherwise identical cells or tissues that have not been contacted with the fusion protein and / or antibody conjugate. Gene expression can be measured by any suitable method known in the art, for example, by ELISA, by Luminex multiplex assay, or by flow cytometry.

[0214] The present disclosure also provides a method for increasing the phagocytosis of tumor cells. The method comprises contacting tumor cells with an effective amount of a fusion protein and / or antibody conjugate, such as a fusion protein or antibody conjugate disclosed herein, to remove sialic acid from the tumor cells, thereby increasing the phagocytosis of the tumor cells. In one embodiment, the present disclosure relates to a method for increasing the phagocytosis of tumor cells in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition, a fusion protein and / or an antibody conjugate (e.g., a fusion protein or antibody conjugate disclosed herein) in an amount effective to remove sialic acid from the tumor cells, thereby increasing the phagocytosis of the tumor cells.

[0215] In certain embodiments, phagocytosis is increased by at least about 10%, at least about 20%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1,000% relative to a similar or otherwise identical tumor cell or population of tumor cells that has not been contacted with the fusion protein and / or antibody conjugate. Phagocytosis can be measured by any suitable method known in the art.

[0216] The present disclosure also provides methods for activating dendritic cells (DCs), comprising contacting DCs with tumor cells treated with a fusion protein and / or antibody conjugate, e.g., a fusion protein or antibody conjugate disclosed herein. In one embodiment, the present disclosure relates to a method of activating dendritic cells (DCs) or a population of DCs in a subject, comprising administering to the subject an amount of a pharmaceutical composition comprising a fusion protein and / or antibody conjugate, e.g., a fusion protein or antibody conjugate disclosed herein, effective to remove sialic acid from tumor cells in the subject, thereby activating DCs or a population of DCs in the subject.

[0217] In certain embodiments, activation of a DC or population of DCs is increased by at least about 10%, at least about 20%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1,000% relative to a similar or otherwise identical DC or population of DCs that has not been contacted with tumor cells treated with the fusion protein and / or antibody conjugate. Activation can be measured by any suitable method known in the art.

[0218] The present disclosure also provides methods for reducing Siglec-15 binding activity, thereby increasing anti-tumor activity in a tumor microenvironment, comprising contacting T cells with a fusion protein and / or antibody conjugate, e.g., a fusion protein or antibody conjugate disclosed herein. In certain embodiments, the present disclosure relates to methods for reducing Siglec-15 binding activity, thereby increasing anti-tumor activity in a tumor microenvironment of a patient, comprising administering to the subject an effective amount of a pharmaceutical composition comprising the fusion protein and / or antibody conjugate, e.g., a fusion protein or antibody conjugate disclosed herein, thereby increasing anti-tumor activity (e.g., T cell activity) in the subject.

[0219] In certain embodiments, Siglec-15 binding activity is reduced by at least about 10%, at least about 20%, at least about 50%, at least about 75%, or about 100% relative to Siglec-15 that has not been contacted with the fusion protein and / or antibody conjugate. Binding can be measured by any suitable method known in the art.

[0220] Throughout the description, when compositions are described as having, including, or comprising particular components, or when processes and methods are described as having, including, or comprising particular steps, it is further contemplated that there are compositions of the invention that consist essentially of or consist of the recited components, and that there are processes and methods of the invention that consist essentially of or consist of the recited process steps.

[0221] In this application, when an element or component is said to be included in and / or selected from a list of described elements or components, it should be understood that the element or component can be any one of the described elements or components, or the element or component can be selected from a group consisting of two or more of the described elements or components.

[0222] Furthermore, it should be understood that elements and / or features of the compositions or methods described herein, whether expressly or implicitly stated herein, may be combined in various ways without departing from the spirit and scope of the invention. For example, when reference is made to a particular compound, the compound may be used in various embodiments of the compositions of the invention and / or methods of the invention, unless otherwise understood from the context. That is, although embodiments are described and illustrated herein in a manner that may describe and illustrate a clear and concise application, it is intended and understood that the embodiments may be variously combined or separated without departing from the present teachings and invention(s). For example, it is understood that all features described and illustrated herein may be applicable to all aspects of the invention(s) described and illustrated herein.

[0223] The phrase "at least one" should be understood to include each of the listed items individually and various combinations of two or more of the listed items, unless otherwise understood from context and application. With respect to more than two listed items, the phrase "and / or" should be understood to have the same meaning, unless otherwise understood from context.

[0224] Use of the terms "include," "includes," "including," "have," "has," "having," "contain," "contains," or "containing," including grammatical equivalents thereof, is to be understood generally as open-ended and non-limiting, for example without excluding additional, unrecited elements or steps, unless the context specifically states or understands otherwise.

[0225] When the term "about" is used before a quantitative value, the invention also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term "about" refers to a ±10% variation from the nominal value, unless otherwise indicated or inferred.

[0226] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions may be performed simultaneously.

[0227] The use of any and all examples or exemplary terms herein, such as "such as" or "including," is intended merely to better describe the invention and does not pose a limitation on the scope of the invention unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. [Example]

[0228] The following examples are illustrative only and are not intended to limit the scope or content of the invention in any way.

[0229] Example 1 This example describes the construction of recombinant human sialidases (Neu1, Neu2, and Neu3).

[0230] Human sialidases Neu1, Neu2, Neu3 (isoform 1), and Neu4 (isoform 1) were expressed as secreted proteins with a 10xHis tag. To express Neu1 as a secreted protein, the native N-terminal signal peptide (MTGERPSTALPDRRWGPRILGFWGGCRVWVFAAIFLLLSLAASWSKA; SEQ ID NO: 27) was replaced with MDMRVPAQLLGLLLLWLPGARC (SEQ ID NO: 28), and the C-terminal lysosomal signal motif (YGTL; SEQ ID NO: 29) was removed. To express Neu2, Neu3, and Neu4 as secreted proteins, the N-terminal signal peptide MDMRVPAQLLGLLLLWLPGARC (SEQ ID NO: 28) was added to each.

[0231] Sialidase was expressed in 200 mL transfections of HEK293F human cells in 24-well plates using the pCEP4 mammalian expression vector with an N-terminal 6xHis tag. Sialidase was purified using a Ni-NTA column, quantified using a UV-Vis spectrometer (NanoDrop), and examined by SDS-PAGE as shown in Figure 1. Neu1 was expressed well, with a yield of approximately 3 μg / mL, and was present primarily in the monomeric form. Neu2 and Neu3 expression gave yields of approximately 0.15 μg / mL, each of which was present primarily in the dimeric form. Neu4 had no detectable expression yield as measured by NanoDrop. Bacterial sialidase from Salmonella typhimurium (St-sialidase; SEQ ID NO: 30), used as a positive control for expression, gave a yield comparable to Neu1 and was present primarily in the monomeric form.

[0232] The activity of recombinantly expressed sialidases was assayed by measuring the release of sialic acid from the fluorogenic substrate 4-methylumbelliferyl-N-acetylneuraminic acid (4MU-NeuAc). As shown in Figure 2, Neu1 had no detectable activity above the no-enzyme control, consistent with previous reports showing that Neu1 is inactive unless complexed with β-galactosidase and protective protein / cathepsin A (PPCA). Neu2 and Neu3 were active. Enzyme kinetic assays were performed using Neu2 and Neu3. A fixed concentration of 1 nM enzyme was incubated with concentrations of the fluorogenic substrate 4MU-NeuAc ranging from 4000 μM to 7.8 μM. Assays were performed under both acidic (pH 5.6) and neutral (pH 7) conditions. As shown in Figure 3, both Neu2 and Neu3 were active under both acidic and neutral conditions and exhibited enzyme kinetics comparable to those previously reported.

[0233] Most of the recombinantly expressed sialidase migrated as aggregates or dimers on non-reducing SDS-PAGE gels. Subsequent treatment with the reducing agent dithiothreitol (DTT) yielded a monomeric form of the enzyme that migrated at 42 kDa on reducing SDS-PAGE gels (Figure 1).

[0234] Example 2 This example describes the construction of an anti-PD-1 antibody sialidase conjugate (ASC).

[0235] An example of an anti-PD-1 antibody ASC is called "Janus" and contains one antibody arm (comprising one heavy chain and one light chain) and one sialidase-Fc fusion in which sialidase is fused to the N-terminus of one Fc arm. Each Fc domain polypeptide in the Janus ASC contains either a "knob" (T366Y) or a "hole" (Y407T) mutation for heterodimerization (residue numbers are according to EU numbering (Kabat, EA, et al. (1991) supra)) (see, e.g., Figure 6B).

[0236] A first Janus ASC was constructed using Neu2 containing the M1D, V6Y, P62G, A93E, I187K, and C332A mutations and pembrolizumab (an anti-PD-1 antibody). This ASC (designated ASC#1, which comprises a first polypeptide chain having the amino acid sequence of SEQ ID NO:77 encoded by the nucleotide sequence of SEQ ID NO:104, a second polypeptide chain having the amino acid sequence of SEQ ID NO:105 encoded by the nucleotide sequence of SEQ ID NO:110, and a third polypeptide chain having the amino acid sequence of SEQ ID NO:111 encoded by the nucleotide sequence of SEQ ID NO:114) was expressed and subjected to purity characterization using SDS-PAGE and enzymatic activity characterization using 4MU-NeuAc, as described below.

[0237] ASC#1 was expressed in 1000 mL of Expi293 human cells transfected with the pCEP4 mammalian expression vector, purified using Protein A, subjected to ceramic hydroxyapatite chromatography, quantified with a UV-Vis spectrophotometer (NanoDrop), and examined by SDS-PAGE.

[0238] The activity of ASC#1 is assayed by measuring the release of sialic acid from the fluorogenic substrate 4-methylmethylumbelliferyl-N-acetylneuraminic acid (4MU-NeuAc). Specifically, an enzyme kinetic assay is performed by incubating a fixed concentration (1 nM) of the enzyme with concentrations of the fluorogenic substrate 4MU-NeuAc ranging from 4000 μM to 7.8 μM. Active enzyme fluoresces upon liberating sialic acid. The assay is performed at pH 5.6.

[0239] A second Janus ASC is constructed using Neu2 containing the M1D, V6Y, P62G, A93E, Q126Y, I187K, A242F, Q270T, and C332A mutations and pembrolizumab. This ASC (designated ASC#2, which comprises a first polypeptide chain having the amino acid sequence of SEQ ID NO:77 encoded by the nucleotide sequence of SEQ ID NO:104, a second polypeptide chain having the amino acid sequence of SEQ ID NO:105 encoded by the nucleotide sequence of SEQ ID NO:110, and a third polypeptide chain having the amino acid sequence of SEQ ID NO:115 encoded by the nucleotide sequence of SEQ ID NO:124) is expressed and characterized for purity using SDS-PAGE and enzymatic activity using 4MU-NeuAc as described above.

[0240] An additional example ASC construct (termed "Lobster-Fab") comprises two Fc domain polypeptides, each of which comprises a sialidase fused to the N-terminus of the Fc and a Fab fused to the C-terminus of the Fc (Figure 6E).

[0241] A first Lobster-Fab ASC was constructed using Neu2 containing the M1D, V6Y, A42R, P62G, A93E, Q126Y, I187K, A242F, Q270T, and C332A mutations and pembrolizumab. This ASC (designated ASC#4) comprises first and fourth polypeptide chains having the amino acid sequence of SEQ ID NO:77 encoded by the nucleotide sequence of SEQ ID NO:104, and second and third polypeptide chains having the amino acid sequence of SEQ ID NO:130 encoded by the nucleotide sequence of SEQ ID NO:131. A second Lobster-Fab ASC is constructed using Neu2 containing the M1D, V6Y, A42R, P62G, A93E, Q126Y, I187K, A242F, Q270T, and C332A mutations and pembrolizumab. This ASC (designated ASC#5) comprises first and fourth polypeptide chains having the amino acid sequence of SEQ ID NO: 132 encoded by the nucleotide sequence of SEQ ID NO: 133, and second and third polypeptide chains having the amino acid sequence of SEQ ID NO: 134 encoded by the nucleotide sequence of SEQ ID NO: 135. ASC#4 and ASC#5 are expressed and subjected to characterization of purity using SDS-PAGE and enzymatic activity using 4MU-NeuAc as described above.

[0242] An additional example ASC construct (termed "Raptor") comprises an antibody (comprising two heavy chains and two light chains) that comprises a sialidase fused to the C-terminus of each heavy chain of the antibody (see, e.g., Figure 6A).

[0243] Example 3 This example describes the construction and characterization of an anti-PD-1 antibody sialidase conjugate (ASC).

[0244] A Janus ASC was constructed using Neu2 containing the M1D, V6Y, A42R, P62G, A93E, Q126Y, I187K, A242F, Q270T, and C332A mutations, the variable region of pembrolizumab, and a human IgG1 Fc domain containing the N297A mutation. This ASC (designated ASC#3) comprises a first polypeptide chain having the amino acid sequence of SEQ ID NO:77 encoded by the nucleotide sequence of SEQ ID NO:104, a second polypeptide chain having the amino acid sequence of SEQ ID NO:105 encoded by the nucleotide sequence of SEQ ID NO:110, and a third polypeptide chain having the amino acid sequence of SEQ ID NO:125 encoded by the nucleotide sequence of SEQ ID NO:129. A Raptor ASC was constructed using Neu2 containing the M1D, V6Y, P62G, A93E, Q126Y, I187K, A242F, Q270T, and C332A mutations, the variable region of pembrolizumab, and a human IgG1 Fc domain containing the N297A mutation. This ASC (designated ASC#6) comprises a first polypeptide chain having the amino acid sequence of SEQ ID NO:77 encoded by the nucleotide sequence of SEQ ID NO:104, and a second polypeptide chain having the amino acid sequence of SEQ ID NO:145 encoded by the nucleotide sequence of SEQ ID NO:146.

[0245] ASC#3 and ASC#6 were expressed in 1,000 mL of Expi293 human cells transfected with the pCEP4 mammalian expression vector. These ASCs were purified using Protein A, then subjected to ceramic hydroxyapatite chromatography, quantified with a UV-Vis spectrophotometer (NanoDrop), and examined by SDS-PAGE and size-exclusion chromatography (SEC). Figure 7 shows the SEC traces of ASC#3 and ASC#6, demonstrating that 91.6% and 95.6%, respectively, were present in monomeric form, with no detectable endotoxin.

[0246] The activity of ASC#3 and ASC#6 was assayed by measuring the release of sialic acid from the fluorogenic substrate 4-methylmethylumbelliferyl-N-acetylneuraminic acid (4MU-NeuAc). Enzyme kinetic assays were performed by incubating a fixed concentration (1 nM) of enzyme with concentrations of the fluorogenic substrate 4MU-NeuAc ranging from 4,000 μM to 7.8 μM. Active enzymes emit fluorescence upon liberation of sialic acid. Assays were performed at pH 5.6. 1 / 2 (the concentration of ASC required for half the conversion of the substrate) was 2.54 μg / mL, and the A 1 / 2 was 86.84 μg / mL.

[0247] ASC#3 and ASC#6 were tested for their ability to block the interaction between PD-1 and PD-L1. They were incubated with (i) engineered CHO-K1 cells expressing human PD-L1 and a TCR activation protein, and (ii) Jurkat T cells expressing a luciferase reporter driven by human PD-1, TCR, and an NFAT response element. In the absence of intervention, PD-L1 interacts with PD-1, inhibiting TCR-mediated luminescence, while blocking the PD-L1 / PD-1 interaction results in a luminescent signal. After 6 hours of incubation, luciferase substrate was added, and luminescence was measured. Relative luminescence units (RLU) were calculated by subtracting the background (substrate and medium only) from the assay wells. Fold induction was calculated by dividing the RLU of induced cells minus the background RLU by the RLU of the no-antibody control minus the background RLU (Fold induction = RLU(induction - background) / RLU(no-antibody control - background)). Pembrolizumab was used as a positive control, and the fold induction obtained in the assay was 4-5 over the range of 0.1-10 μg / mL, with an EC 50The EC of ASC#3 and ASC#6 was 0.92 nM. As shown in Figure 8, ASC#3 and ASC#6 increased luminescence in a dose-dependent manner, indicating that these ASCs have the ability to disrupt PD-L1 / PD-1 interaction. 50 were 7.23 nM and 0.53 nM, respectively, and the EC 50 Two other ASC#3 preparations also had similar EC 50 The values ​​shown are: EC 50 It is contemplated that the difference in values ​​is a result of different ASC formats (e.g., ASC#3 has only one PD-1-binding antibody arm, while ASC#6 has two PD-1-binding antibody arms).

[0248] ASC#3 and ASC#6 were also tested for their ability to block the binding of biotinylated human PD-L1 Fc fusion (hPD-L1-Fc) to human PD-1 (hPD-1). Three-fold serial dilutions of ASC and pembrolizumab were prepared and mixed with hPD-L1-Fc to a final concentration of 1 μg / mL. The mixtures of antibody and hPD-L1-Fc were loaded onto hPD-1-coated binding ELISA wells. ASC or antibodies that bind to the hPD-L1-binding epitope on hPD-1 compete for binding and reduce the hPD-L1-Fc binding signal. Residual binding of hPD-L1-Fc to hPD-1 was detected using HRP-conjugated streptavidin. The plate was developed with TMB, stop buffer was added to the plate, and the absorbance was read at 450 nm using a SpectraMax plate reader. A450 absorbance curve and IC using GraphPad Prism software 50 As shown in Figure 9, ASC#3 blocked hPD-L1 binding to hPD-1 at single-digit nanomolar concentrations, and ASC#6 showed subnanomolar blockade. These results are consistent with those obtained from the cell-based assays described above.

[0249] Binding of purified ASC#3 and ASC#6 to recombinant human hPD-1 was measured using a ForteBio octet. ASC was captured on an AHC (anti-human IgG-Capture) biosensor. The hPD-1 analyte was varied in concentration from 100 nM in a two-fold dilution series. A buffer reference was used to subtract the signal and baseline the signal. KD, Kon, and Koff values ​​were obtained using a 1:1 fitting model. The KD for ASC#3 and ASC#6 were determined to be 10.3 nM and 10.6 nM, respectively.

[0250] Table 10 provides a summary of some of the characteristics of ASC#3 and ASC#6. [Table 10]

[0251] Example 4 This example describes the in vivo administration of an anti-PD-1 antibody sialidase conjugate (ASC) containing human sialidase.

[0252] The MC38 mouse tumor line, engineered to express human PD-L1, was propagated as a syngeneic subcutaneous tumor in C57BL6 transgenic mice (Biocytogen Inc.) engineered to express human PD-L1 and human PD-1, in which mouse PD-L1 and mouse PD-1 had been disrupted. Tumors were allowed to develop in 6- to 8-week-old mice by subcutaneous inoculation of tumor cells into the right lower flank. Tumors ranged from 90 to 136 mm. 3 At this point, mice were randomly assigned to three groups of eight animals each, with a group mean of 109 mm 3 It was decided.

[0253] Mice were treated with intraperitoneal injection of ASC#3 (described above in Example 3; 10 mg / kg), pembrolizumab (5 mg / kg), or isotype control (30 mg / kg), and tumor volume (mm 3) were recorded. Figure 10A shows individual tumor volumes, and Figure 10B shows the mean tumor volume on day 18. As shown in Figure 10B, administration of ASC#3 significantly reduced tumor growth, and this reduction was comparable to the response produced by the positive control pembrolizumab.

[0254] The CT26 mouse tumor line, engineered to express human PD-L1, was propagated as a syngeneic subcutaneous tumor in transgenic BALB / c mice (Gempharmatech Inc.) engineered to express human PD-L1 and human PD-1, in which mouse PD-L1 and mouse PD-1 had been disrupted. Tumors were allowed to develop in 6- to 8-week-old mice by subcutaneous inoculation of tumor cells into the right lower flank. Tumors were 90-120 mm 3 Once the mice reached this size, they were randomly assigned to three groups of six animals each, with a group mean of approximately 104.06–104.36 mm. 3 It was decided.

[0255] Mice were treated with intraperitoneal injection of ASC#3 (described above in Example 3; 10 mg / kg), pembrolizumab (5 mg / kg), or isotype control (10 mg / kg), and tumor volume (mm 3 ) were recorded. Figure 11A shows individual tumor volumes, and Figure 11B shows the mean tumor volume on day 18. As shown in Figure 11B, administration of ASC#3 significantly reduced tumor growth, and this reduction was comparable to the response produced by the positive control pembrolizumab.

[0256] Incorporation by Reference The entire disclosure of each of the patent and scientific literatures referred to herein is incorporated by reference for all purposes.

[0257] equivalent The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Therefore, the foregoing embodiments are to be considered in all respects as illustrative and not limiting of the invention described herein. The scope of the invention is, therefore, indicated by the appended claims, rather than the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

[0258] Sequence Listing SEQ ID NO:1: MASLPVLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAPTHQVQWQAQEVVAQARLDGHRSMNPCPLYDAQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPIQRP IPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPQGCQGSVISFPSPRSGPGSPAQWLLYTHPTHSWQRADLGAYLNPRPPAPEAWSEPVLLAKGSCAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQ

[0259] SEQ ID NO:2: MEDLRPMATCPPVLQKETLFRTGVHAYRIPALLYLKKQKTLLAFAEKRASKTDEHAELIVLRRGSYNEATNRVKWQPEEVVTQAQLEGRSMNPCPLYDKQTKTLFLFFIAVPGRVSEHHQLHTKVNVTRLCCVSSTDHGRTWSPIQDLTETTIGSTHQEWATFAVGPGHCLQLRNPAGSLLVPAYAYRKLHP AQKPTPFAFCFISLDHGHTWKLGNFVAENSLECQVAEVGTGAQRMVYLNARSFLGARVQAQSPNDGLDFQDNRVVSKLVEPPHGCHGSVVAFHNPISKPHALDTWLLYTHPTDSRNRTNLGVYLNQMPLDPTAWSEPTLLAMGICAYSDLQNMGQGPDGSPQFGCLYESGNYEEIIFLIFTLKQAFPTVFDAQ

[0260] SEQ ID NO:3: EDLRP

[0261] SEQ ID NO:4: MEDLRP

[0262] SEQ ID NO:5: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0263] SEQ ID NO:6:

[0264] SEQ ID NO:7: ENDFGLVQPLVTMEQLLWVSGRQIGSVDTFRIPLITATPRGTLLAFAEARKMSSSDEGAKFIALRRSMDQGSTWSPTAFIVNDGDVPDGLNLGAVSDVETGVVFLFYSLCAHKAGCQVASTMLVWSKDDGVSWSTPRNLSLDIGTEVFAPGPGSGIQKQREPRKGRLIVCGHGTLERDVF CLLSDDHGASWRYGSGVSGIPYGQPKQENDFNPDECQPYELPDGSVVINARNQNNYHCHCRIVLRSYDACDTLRPRDVTFDPELVDPVVAAGAVVTSSGIVFFSNPAHPEFRVNLTLRWSFSNGTSWRKETVQLWPGPSGYSSLATLEGSMDGEEQAPQLYVLYEKGRNHYTESISVAKISV

[0265] SEQ ID NO:8: MEEVTTCSFNSPLFRQEDDRGITYRIPALLYIPPTHTFLAFAEKRSTRRDEDALHLVLRRGLRIGQLVQWGPLKPLMEATLPGHRTMNPCPVWEQKSGCVFLFFICVRGHVTERQQIVSGRNAARLCFIYSQDAGCSWSEVRDLTEEVIGSELKHWATFAVGPGHGIQLQSGRLVIPAYTYYIPSWFFCFQLPCKTRPHSLMIYSDDLGVTWHH GRLIRPMVTVECEVAEVTGRAGHPVLYCSARTPNRCRAEALSTDHGEGFQRLALSRQLCEPPHGCQGSVVSFRPLEIPHRCQDSSSKDAPTIQQSSPGSSLRLEEEA GTPSESWLLYSHPTSRKQRVDLGIYLNQTPLEAACWSRPWILHCGPCGYSDLAALEEEGLFGCLFECGTKQECEQIAFRLFTHREILSHLQGDCTSPGRNPSQFKSN

[0266] SEQ ID NO:9: MRPADLPPRPMEESPASSSAPTETEEPGSSAEVMEEVTTCSFNSPLFRQEDDRGITYRIPALLYIPPTHTFLAFAEKRSTRRDEDALHLVLRRGLRIGQLVQWGPLKPLMEATLP GHRTMNPCPVWEQKSGCVFLFFICVRGHVTERQQIVSGRNAARLCFIYSQDAGCSWSEVRDLTEEVIGSELKHWATFAVGPGHGIQLQSGRLVIPAYTYYIPSWFFCFQLPCKTR PHSLMIYSDDLGVTWHHGRLIRPMVTVECEVAEVTGRAGHPVLYCSARTPNRCRAEALSTDHGEGFQRLALSRQLCEPPHGCQGSVVSFRPLEIPHRCQDSSSKDAPTIQQSSPG SSLRLEEEAGTPSESWLLYSHPTSRKQRVDLGIYLNQTPLEAACWSRPWILHCGPCGYSDLAALEEEGLFGCLFECGTKQECEQIAFRLFTHREILSHLQGDCTSPGRNPSQFKSN

[0267] SEQ ID NO:10: MGVPRTPSRTVLFERERTGLTYRVPSLLPVPPGPTLLAFVEQRLSPDDSHAHRLVLRRGTLAGGSVRWGALHVLGTAALAEHRSMNPCPPVHDAGTGTVFLFFIAVLGHTPEAVQIATGRNA ARLCCVASRDAGLSWGSARDLTEEAIGGAVQDWATFAVGPGHGVQLPSGRLLVPAYTYRVDRRECFGKICRTSPHSFAFYSDDHGRTWRCGGLVPNLRSGECQLAAVDGGQAGSFLYCNAR SPLGSRVQALSTDEGTSFLPAERVASLPETAWGCQGSIVGFPAPAPNRPRDDSWSVGPGSPLQPPLLPGVHEPPEEAAVDPRGGQVPGGPFSRLQPRGDGPRQPGPRPGVSGDVGSWTLA LPMPFAAPPQSPTWLLYSHPVGRRARLHMGIRLSQSPLDPRSWTEPWVIYEGPSGYSDLASIGPAPEGGLVFACLYESGARTSYDEISFCTFSLREVLENVPASPKPPNLGDKPRGCCWPS

[0268] SEQ ID NO:11: MMSSAAFPRWLSMGVPRTPSRTVLFERERTGLTYRVPSLLPVPPGPTLLAFVEQRLSPDDSHAHRLVLRRGTLAGGSVRWGALHVLGTAALAEHRSMNPCPPVHDAGTGTVFLFFIAVLGHTPEA VQIATGRNAARLCCVASRDAGLSWGSARDLTEEAIGGAVQDWATFAVGPGHGVQLPSGRLLVPAYTYRVDRRECFGKICRTSPHSFAFYSDDHGRTWRCGGLVPNLRSGECQLAAVDGGQAGSF LYCNARSPLGSRVQALSTDEGTSFLPAERVASLPETAWGCQGSIVGFPAPAPNRPRDDSWSVGPGSPLQPPLLPGVHEPPEEAAVDPRGGQVPGGPFSRLQPRGDGPRQPGPRPGVSGDVGSW TLALPMPFAAPPQSPTWLLYSHPVGRRARLHMGIRLSQSPLDPRSWTEPWVIYEGPSGYSDLASIGPAPEGGLVFACLYESGARTSYDEISFCTFSLREVLENVPASPKPPNLGDKPRGCCWPS

[0269] SEQ ID NO:12: MASLP

[0270] SEQ ID NO:13: ASLP

[0271] SEQ ID NO:14: TVEKSVVF

[0272] SEQ ID NO:15: GDYDAPTHQVQW

[0273] SEQ ID NO:16: SMDQGSTW

[0274] SEQ ID NO:17: STDGGKTW

[0275] SEQ ID NO:18: PRPPAPEA

[0276] SEQ ID NO:19: QTPLEAAC

[0277] SEQ ID NO:20: NPRPPAPEA

[0278] SEQ ID NO:21: SQNDGES

[0279] SEQ ID NO:22: LSHSLST

[0280] SEQ ID NO:23:

[0281] SEQ ID NO:24:

[0282] SEQ ID NO:25:

[0283] SEQ ID NO:26:

[0284] SEQ ID NO:27: MTGERPSTALPDRRWGPRILGFWGGCRVWVFAAIFLLLSLAASWSKA

[0285] SEQ ID NO:28: MDMRVPAQLLGLLLLWLPGARC

[0286] SEQ ID NO:29: YGTL SEQ ID NO:30: MTVEKSVVFKAEGEHFTDQKGNTIVGSGSGGTTKYFRIPAMCTTSKGTIVVFADARHNTASDQSFIDTAAARSTDGGKTWNKKIAIYNDRVNSKLSRVMDPTCIVANIQGRETILVMVGKWNNNDKTWGAYRDKAPDTDWDLVLYKSTDGVTFSKVETNIHDIVTKNGTISAMLGGVGSGLQLNDGKLVF PVQMVRTKNITTVLNTSFIYSTDGITWSLPSGYCEGFGSENNIIEFNASLVNNIRNSGLRRSFETKDFGKTWTEFPPMDKKVDNRNHGVQGSTITIPSGNKLVAAHSSAQNKNNDYTRSDISLYAHNLYSGEVKLIDDFYPKVGNASGAGYSCLSYRKNVDKETLYVVYEANGSIEFQDLSRHLPVIKSYN

[0287] SEQ ID NO:31: EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0288] SEQ ID NO:32: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0289] SEQ ID NO:33: EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPSREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0290] SEQ ID NO:34:

[0291] SEQ ID NO:35:

[0292] SEQ ID NO:36: MRFKNVKKTALMLAMFGMATSSNAALFDYNATGDTEFDSPAKQGWMQDNTNNGSGVLTNADGMPAWLVQGIGGRAQWTYSLSTNQHAQASSFGWRMTTEMKVLSGGMITNYYANGTQRVLPIISLDSSGNLVVEFEGQTGRTVLATGTAATEYHKFELVFLPGSNPSASFYFDGKLIRDNIQPTASKQNMIVWGN GSSNTDGVAAYRDIKFEIQGDVIFRGPDRIPSIVASSVTPGVVTAFAEKRVGGGDPGALSNTNDIITRTSRDGGITWDTELNLTEQINVSDEFDFSDPRPIYDPSSNTVLVSYARWPTDAAQNGDRIKPWMPNGIFYSVYDVASGNWQAPIDVTDQVKERSFQIAGWGGSELYRRNTSLNSQQDWQSNAKIRIVD GAANQIQVADGSRKYVVTLSIDESGGLVANLNGVSAPIILQSEHAKVHSFHDYELQYSALNHTTTLFVDGQQITTWAGEVSQENNIQFGNADAQIDGRLHVQKIVLTQQGHNLVEFDAFYLAQQTPEVEKDLEKLGWTKIKTGNTMSLYGNASVNPGPGHGITLTRQQNISGSQNGRLIYPAIVLDRFFLNVMSI YSDDGGSNWQTGSTLPIPFRWKSSSILETLEPSEADMVELQNGDLLLTARLDFNQIVNGVNYSPRQQFLSKDGGITWSLLEANNANVFSNISTGTVDASITRFEQSDGSHFLLFTNPQGNPAGTNGRQNLGLWFSFDEVTWKGPIQLVNGASAYSDIYQLDSENAIVIVETDNSNMRILRMPITLLKQKLTLSQN

[0293] SEQ ID NO:37:

[0294] SEQ ID NO:38: MVGADPTRPRGPLSYWAGRRGQGLAAIFLLLVSAAESEARAEDDFSLVQPLVTMEQLLWVSGKQIGSVDTFRIPLITATPRGTLLAFAEARKKSASDEGAKFIAMRRSTDQGSTWSSTAFIVDDGEASDGLNLGAVVNDVDTGIVFLIYTLCAHKVNCQVASTMLVWSKDDGISWSPRPRNLSVDIGTEMFAPGPGSGIQKQREP GKGRLIVCGHGTLERDVFCLLSDDHGASWHYGTGVSGIPFGQPKHDHDFNPDECQPYELPDGSVIINARNQNNYHCRCRIVLRSYDACDTLRPRDVTFDPELVDPVVAAGALATSSGIVFFSNPAHPEFRVNLTLRWSFSNGTSWLKERVQVWPGPSGYSSLTALENSTDGKKQPPQLFVLYEKGLNRYTESISMVKISVYGTL

[0295] SEQ ID NO:39: MTVQPSPWFSDLRPMATCPVLQKETLFRTGVHAYRIPALLYLKKQKTLLAFAEKRASKTDEHAELIVLRRGSYNEATNRVKWQPEEVVTQAQLEGRSMNPCPLYDKQTKTLFLFFIAVPGRVSEHHQLHTKVNVTRLCCVSSTDHGRTWSPIQDLTETTIGSTHQEWATFAVGPGHCLQLRNPAGSLLVPAYAYR KLHPAQKPTPFAFCFISLDHGHTWKLGNFVAENSLECQVAEVGTGAQRMVYLNARSFLGARVQAQSPNDGLDFQDNRVVSKLVEPPHGCHGSVVAFHNPISKPHALDTWLLYTHPTDSRNRTNLGVYLNQMPLDPTAWSEPTLLAMGICAYSDLQNMGQGPDGSPQFGCLYESGNYEEIIFLIFTLKQAFPTVFDAQ

[0296] SEQ ID NO:40: MEEVPPYSLSSTLFQQEEQSGVTYRIPALLYLPPTHTFLAFAEKRTSVRDEDAACLVLRRGLMKGRSVQWGPQRLLMEATLPGHRTMNPCPVWEKNTGRVYLFFICVRGHVTERCQIVWGKNAARLCFLCSEDAGCSWGEVKDLTEEVIGSEVKRWATFAVGPGHGIQLHSGRLIIPAYAYYVSRWFLCFACSVKPHSLMIYSDDFGVTWHHGKFIEPQVTGECQVAEVAGTAGNPVLYCSARTPSRFRAEAFSTDSGGCFQKPTLNPQLHEPRTGCQGSVVSFRPLKMPNTYQDSIGKGAPATQKCPLLDSPLEVEKGAETPSATWLLYSHPTSKRKRINLGIYYNRNPLEVNCWSRPWILNRGPSGYSDLAVVEEQDLVACLFECGEKNEYERIDFCLFSDHEVLSCEDCTSPSSD

[0297] Sequence number: 41: METAGAPFCFHVDSLVPCSYWKVMGPTRVPRRTVLFQRERTGLTYRVPALLCVPPRPTLLAFAEQRLSPDDSHAHRLVLRRGTLTRGSVRWGTLSVLETAVLEEHRSMNPCPVLDEHSGTIFLFFIAVLGHTPEAVQIATGKNAARLCCVTSCDAGLTWGSVRDLTEEAIGAALQDWATFAVGPGHGVQLRSGRLLVPAYTYHVDRRECFGKICWTSPHSLAFYSDDHGISWHCGGLVPNLRSGECQLAAVDGDFLYCNARSPLGNRVQALSADEGTSFLPGELVPTLAETARGCQGSIVGFLAPPSIEPQDDRWTGSPRNTPHSPCFNLRVQESSGEGARGLLERWMPRLPLCYPQSRSPENHGLEPGSDGDKTSWTPECPMSSDSMLQSPTWLLYSHPAGRRARLHMGIYLSRSPLDPHSWTEPWVIYEGPSGYSDLAFLGPMPGASLVFACLFESGTRTSYEDISFCLFSLADVLENVPTGLEMLSLRDKAQGHCWPS

[0298] SEQ ID NO:42:

[0299] SEQ ID NO:43:

[0300] SEQ ID NO:44:

[0301] SEQ ID NO:45:

[0302] SEQ ID NO:46: X1ASLPX2LQX3ESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAX4THQVQWQAQEVVAQARLDGHRSMNPCPLYDX5QTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCL QLHDRARSLVVPAYAYRKLHPX6QRPIPSAFCFLSHHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPX7GCQGSVISFPSPRSGPGSPAQWLLYTHPTHX8X9QRADLGAYLNPRPPAPEAWSEPVLLAKGSX 10 AYSDLQSMGTGPDGSPLFGCLYEANDYEEIX 11 FX 12 MFTLKQAFPAEYLPQ

[0303] SEQ ID NO:47: X1X2SX3X4X5LQX6ESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASX7X8DEHAELIVX9RRGDYDAX 10 THQVQWX 11 AQEVVAQAX 12 LDGHRSMNPCPLYDX 13 QTGTLFLFFIAIPX 14 X 15 VTEX 16 QQLQTRANVTRLX 17 X 18 VTSTDHGRTWSSPRDLTDAAIGPX 19 YREWSTFAVGPGHX 20 LQLHDRX 21 RSLVVPAYAYRKLHPX 22 QRPIPSAFX 23 FLSHDHGRTWARGHFVAQDTX 24 ECQVAEVETGEQRVVTLNARSHLRARVQAQSX25 NX 26 GLDFQX 27 SQLVKKLVEPPPX 28 GX 29 QGSVISFPSPRSGPGSPAQX 30 LLYTHPTHX 31 X 32 QRADLGAYLNPRPPAPEAWSEPX 33 LLAKGSX 34 AYSDLQSMGTGPDGSPLFGX 35 LYEANDYEEIX 36 FX 37 MFTLKQAFPAEYLPQ

[0304] SEQ ID NO:48: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAGTHQVQWQAQEVVAQARLDGHRSMNPCPLYDEQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP IPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPQGCQGSVISFPSPRSGPGSPAQWLLYTHPTHSWQRADLGAYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQ

[0305] SEQ ID NO:49: DASLPYLQDESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAPTHQVQWQAQEVVAQARLDGHRSMNPCPLYDEQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP IPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPQGCQGSVISFPSPRSGPGSPAQWLLYTHPTHSWQRADLGAYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIRFIMFTLKQAFPAEYLPQ

[0306] SEQ ID NO:50: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDANTHQVQWQAQEVVAQARLDGHRSMNPCPLYDAQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP IPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPQGCQGSVISFPSPRSGPGSPAQWLLYTHPTHSWQRADLGAYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQ

[0307] SEQ ID NO:51: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAPTHQVQWQAQEVVAQARLDGHRSMNPCPLYDAQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP IPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPAGCQGSVISFPSPRSGPGSPAQWLLYTHPTHRKQRADLGAYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQ

[0308] SEQ ID NO:52: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDASTHQVQWQAQEVVAQARLDGHRSMNPCPLYDAQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP IPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPAGCQGSVISFPSPRSGPGSPAQWLLYTHPTHRKQRADLGAYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQ

[0309] SEQ ID NO:53: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDATTHQVQWQAQEVVAQARLDGHRSMNPCPLYDAQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP IPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPAGCQGSVISFPSPRSGPGSPAQWLLYTHPTHRKQRADLGAYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQ

[0310] SEQ ID NO:54: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDANTHQVQWQAQEVVAQARLDGHRSMNPCPLYDAQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP IPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPAGCQGSVISFPSPRSGPGSPAQWLLYTHPTHRKQRADLGAYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQ

[0311] SEQ ID NO:55: ASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAPTHQVQWQAQEVVAQARLDGHRSMNPCPLYDAQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP IPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPQGCQGSVISFPSPRSGPGSPAQWLLYTHPTHSWQRADLGAYLNPRPPAPEAWSEPVLLAKGSCAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQ

[0312] SEQ ID NO:56: MASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAPTHQVQWQAQEVVAQARLDGHRSMNPCPLYDAQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP IPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPQGCQGSVISFPSPRSGPGSPAQWLLYTHPTHSWQRADLGAYLNPRPPAPEAWSEPVLLAKGSCAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQ

[0313] SEQ ID NO:57: ASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAPTHQVQWQAQEVVAQARLDGHRSMNPCPLYDAQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP IPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPQGCQGSVISFPSPRSGPGSPAQWLLYTHPTHSWQRADLGAYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQ

[0314] SEQ ID NO:58: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAPTHQVQWQAQEVVAQARLDGHRSMNPCPLYDAQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP IPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPQGCQGSVISFPSPRSGPGSPAQWLLYTHPTHSWQRADLGAYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQ

[0315] SEQ ID NO:59: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAPTHQVQWQAQEVVAQARLDGHRSMNPCPLYDAQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP IPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPQGCQGSVISFPSPRSGPGSPAQWLLYTHPTHSWQRADLGAYLNPRPPAPEAWSEPVLLAKGSCAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQ

[0316] SEQ ID NO:60: AASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAPTHQVQWQAQEVVAQARLDGHRSMNPCPLYDAQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP IPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPQGCQGSVISFPSPRSGPGSPAQWLLYTHPTHSWQRADLGAYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQ

[0317] SEQ ID NO:61: MASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAPTHQVQWQAQEVVAQARLDGHRSMNPCPLYDAQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP IPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPQGCQGSVISFPSPRSGPGSPAQWLLYTHPTHSWQRADLGAYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQ

[0318] SEQ ID NO:62: AASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAPTHQVQWQAQEVVAQARLDGHRSMNPCPLYDAQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP IPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPQGCQGSVISFPSPRSGPGSPAQWLLYTHPTHSWQRADLGAYLNPRPPAPEAWSEPVLLAKGSCAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQ

[0319] SEQ ID NO:63: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRRSKKDEHAELIVLRRGDYDAGTHQVQWQAQEVVAQARLDGHRSMNPCPLYDEQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCYVTSTDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRFRVQAQSTNDGLDFQESQLVKKLVEPPPTGCQGSVISFPSPRSGPGSPAQWLLYTHPTHSWQRADLGAYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQ

[0320] Accession number: 64: X1X2SX3X4X5LQX6ESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRX7SX8X9DEHAELIVX 10 RRGDYDAX 11 THQVQWX 12 AQEVVAQAX 13 LX 14 GHRSMNPCPLYDX 15 QTGTLFLFFIAIPX 16 X 17 VTEX 18 QQLQTRANVTRLX 19 X 20 VTSTDHGRTWSSPRDLTDAAIGPX 21 YREWSTFAVGPGHX 22 LQLHDX 23 X 24 [[ID=,36]]RSLVVPAYAYRKLHPX 25 X 26 X 27 PIPSAFX 28 FLSHDHGRTWARGHFVX 29 QDTX 30 ECQVAEVX 31 TGEQRVVTLNARSX 32 X 33X 34 X 35 RX 36 QAQSX 37 NX 38 GLDFQX 39 X 40 QX 41 VKKLX 42 EPPPX 43 GX 44 QGSVISFPSPRSGPGSPAQX 45 LLYTHPTHX 46 X 47 QRADLGAYLNPRPPAPEAWSEPX 48 LLAKGSX 49 AYSDLQSMGTGPDGSPLFGX 50 LYEANDYEEIX 51 FX 52 MFTLKQAFPAEYLPQ

[0321] SEQ ID NO:65: X1ASLPX2LQX3ESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRX4SKKDEHAELIVLRRGDYDAX5THQVQWQAQEVVAQARLDGHRSMNPCPLYDX6QTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCX7VTSTDH GRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPX8QRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRX9RVQAQSTNDGLDFQESQLVKKLVEPPPX 10 GCQGSVISFPSPRSGPGSPAQWLLYTHPTHX 11 X 12 QRADLGAYLNPRPPAPEAWSEPVLLAKGSX 13 AYSDLQSMGTGPDGSPLFGCLYEANDYEEIX 14 FX 15 MFTLKQAFPAEYLPQ

[0322] SEQ ID NO:66: X1X2SX3X4X5LQX6ESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRX7SX8X9DEHAELIVX 10 RRGDYDAX 11 THQVQWX 12 AQEVVAQAX 13 LX 14 GHRSMNPCPLYDX 15 QTGTLFLFFIAIPX 16 X 17 VTEX 18 QQLQTRANVTRLX 19 X 20 VTSTDHGRTWSSPRDLTDAAIGPX 21 YREWSTFAVGPGHX 22 LQLHDX 23 X 24 RSLVVPAYAYRKLHPX 25 X 26 X 27 PIPSAFX 28 FLSHDHGRTWARGHFVX 29 QDTX 30 ECQVAEVX 31 TGEQRVVTLNARSX 32 X 33 X 34 X 35 RX 36 QAQSX 37 NX 38 GLDFQX 39 X 40 QX 41 VKKLX 42 EPPPX 43 GX 44 QGSVISFPSPRSGPGSPAQX 45 LLYTHPTHX 46 X 47 QRADLGAYLNPRPPAPEAWSEPX 48 LLAKGSX 49 AYSDLQSMGTGPDGSPLFGX 50 LYEANDYEEIX 51 FX 52 MFTLKQAFPAEYLPQX 53DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0323] SEQ ID NO:67: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAGTHQVQWQAQEVVAQARLDGHRSMNPCPLYDEQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYREW STFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPQGCQGSVISFPSPRSGPGSPAQWLLYTHPTHSWQRADLG AYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0324] SEQ ID NO:68: DASLPYLQDESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAPTHQVQWQAQEVVAQARLDGHRSMNPCPLYDEQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYREW STFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPQGCQGSVISFPSPRSGPGSPAQWLLYTHPTHSWQRADLG AYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIRFIMFTLKQAFPAEYLPQGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0325] SEQ ID NO:69: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDANTHQVQWQAQEVVAQARLDGHRSMNPCPLYDAQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYREW STFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPQGCQGSVISFPSPRSGPGSPAQWLLYTHPTHSWQRADLG AYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0326] SEQ ID NO:70: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAPTHQVQWQAQEVVAQARLDGHRSMNPCPLYDAQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYREW STFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPAGCQGSVISFPSPRSGPGSPAQWLLYTHPTHRKQRADLG AYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0327] SEQ ID NO:71: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDASTHQVQWQAQEVVAQARLDGHRSMNPCPLYDAQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYREW STFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPAGCQGSVISFPSPRSGPGSPAQWLLYTHPTHRKQRADLG AYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0328] SEQ ID NO:72: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDATTHQVQWQAQEVVAQARLDGHRSMNPCPLYDAQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYREW STFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPAGCQGSVISFPSPRSGPGSPAQWLLYTHPTHRKQRADLG AYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0329] SEQ ID NO:73: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDANTHQVQWQAQEVVAQARLDGHRSMNPCPLYDAQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYREW STFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPAGCQGSVISFPSPRSGPGSPAQWLLYTHPTHRKQRADLG AYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0330] SEQ ID NO:74: X1ASLPX2LQX3ESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAX4THQVQWQAQEVVAQARLDGHRSMNPCPLYDX5QTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPX6QRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPX7GCQGSVISFPSPRSGPGSPAQWLLYTHPTHX8X9QRADLGAYLNPRPPAPEAWSEPVLLAKGSX 10 AYSDLQSMGTGPDGSPLFGCLYEANDYEEIX 11 FX 12 MFTLKQAFPAEYLPQGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0331] Sequence number: 75: X1X2SX3X4X5LQX6ESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASX7X8DEHAELIVX9RRGDYDAX 10 THQVQWX 11 AQEVVAQAX 12 LDGHRSMNPCPLYDX 13 QTGTLFLFFIAIPX 14 X 15 VTEX 16 QQLQTRANVTRLX 17 X 18 VTSTDHGRTWSSPRDLTDAAIGPX19 YREWSTFAVGPGHX 20 LQLHDRX 21 RSLVVPAYAYRKLHPX 22 QRPIPSAFX 23 FLSHDHGRTWARGHFVAQDTX 24 ECQVAEVETGEQRVVTLNARSHLRARVQAQSX 25 NX 26 GLDFQX 27 SQLVKKLVEPPPX 28 GX 29 QGSVISFPSPRSGPGSPAQX 30 LLYTHPTHX 31 X 32 QRADLGAYLNPRPPAPEAWSEPX 33 LLAKGSX 34 AYSDLQSMGTGPDGSPLFGX 35 LYEANDYEEIX 36 FX 37 MFTLKQAFPAEYLPQGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0332] Sequence number: 76: X1ASLPX2LQX3ESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRX4SKKDEHAELIVLRRGDYDAX5THQVQWQAQEVVAQARLDGHRSMNPCPLYDX6QTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCX7VTSTDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPX8QRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRX9RVQAQSTNDGLDFQESQLVKKLVEPPPX 10 GCQGSVISFPSPRSGPGSPAQWLLYTHPTHX 11 X 12 QRADLGAYLNPRPPAPEAWSEPVLLAKGSX 13 AYSDLQSMGTGPDGSPLFGCLYEANDYEEIX 14 FX 15 MFTLKQAFPAEYLPQX 16 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0333] Sequence number: 77: EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0334] SEQ ID NO:78: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAGTHQVQWQAQEVVAQARLDGHRSMNPCPLYDEQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYRE WSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPQGCQGSVISFPSPRSGPGSPAQWLLYTHPTTHSWQRAD LGAYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0335] SEQ ID NO:79:

[0336] SEQ ID NO:80:

[0337] SEQ ID NO:81: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAGTHQVQWQAQEVVAQARLDGHRSMNPCPLYDEQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYRE WSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPQGCQGSVISFPSPRSGPGSPAQWLLYTHPTTHSWQRAD LGAYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0338] SEQ ID NO:82: DASLPYLQDESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAPTHQVQWQAQEVVAQARLDGHRSMNPCPLYDEQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYRE WSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPQGCQGSVISFPSPRSGPGSPAQWLLYTHPTTHSWQRAD LGAYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIRFIMFTLKQAFPAEYLPQEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0339] SEQ ID NO:83: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDANTHQVQWQAQEVVAQARLDGHRSMNPCPLYDAQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYRE WSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPQGCQGSVISFPSPRSGPGSPAQWLLYTHPTTHSWQRAD LGAYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0340] SEQ ID NO:84: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAPTHQVQWQAQEVVAQARLDGHRSMNPCPLYDAQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYRE WSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPAGCQGSVISFPSPRSGPGSPAQWLLYTHPTHRKQRAD LGAYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0341] SEQ ID NO:85: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDASTHQVQWQAQEVVAQARLDGHRSMNPCPLYDAQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYRE WSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPAGCQGSVISFPSPRSGPGSPAQWLLYTHPTHRKQRAD LGAYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0342] SEQ ID NO:86: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDATTHQVQWQAQEVVAQARLDGHRSMNPCPLYDAQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYRE WSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPAGCQGSVISFPSPRSGPGSPAQWLLYTHPTHRKQRAD LGAYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0343] SEQ ID NO:87: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDANTHQVQWQAQEVVAQARLDGHRSMNPCPLYDAQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYRE WSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPAGCQGSVISFPSPRSGPGSPAQWLLYTHPTHRKQRAD LGAYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0344] SEQ ID NO:88: X1ASLPX2LQX3ESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAX4THQVQWQAQEVVAQARLDGHRSMNPCPLYDX5QTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPX6QRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPX7GCQGSVISFPSPRSGPGSPAQWLLYTHPTHX8X9QRADLGAYLNPRPPAPEAWSEPVLLAKGSX 10 AYSDLQSMGTGPDGSPLFGCLYEANDYEEIX 11 FX 12 MFTLKQAFPAEYLPQX 13 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0345] Sequence number: 89: X1X2SX3X4X5LQX6ESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASX7X8DEHAELIVX9RRGDYDAX 10 THQVQWX 11 AQEVVAQAX 12 LDGHRSMNPCPLYDX 13 QTGTLFLFFIAIPX 14 X 15 VTEX 16 QQLQTRANVTRLX 17 X 18VTSTDHGRTWSSPRDLTDAAIGPX 19 YREWSTFAVGPGHX 20 LQLHDRX 21 RSLVVPAYAYRKLHPX 22 QRPIPSAFX 23 FLSHDHGRTWARGHFVAQDTX 24 ECQVAEVETGEQRVVTLNARSHLRARVQAQSX 25 NX 26 GLDFQX 27 SQLVKKLVEPPPX 28 GX 29 QGSVISFPSPRSGPGSPAQX 30 LLYTHPTHX 31 X 32 QRADLGAYLNPRPPAPEAWSEPX 33 LLAKGSX 34 AYSDLQSMGTGPDGSPLFGX 35 LYEANDYEEIX 36 FX 37 MFTLKQAFPAEYLPQX 38 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0346] SEQ ID NO:90: GGGGSGGGGS

[0347] SEQ ID NO:91: EPKSS

[0348] SEQ ID NO:92: EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0349] SEQ ID NO:93: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPSREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0350] SEQ ID NO:94: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAGTHQVQWQAQEVVAQARLDGHRSMNPCPLYDEQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP IPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPQGCQGSVISFPSPRSGPGSPAQWLLYTHPTHARQRADLGAYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQ

[0351] SEQ ID NO:95: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAGTHQVQWQAQEVVAQARLDGHRSMNPCPLYDEQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYREW STFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPQGCQGSVISFPSPRSGPGSPAQWLLYTHPTHARQRADLG AYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0352] SEQ ID NO:96: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAGTHQVQWQAQEVVAQARLDGHRSMNPCPLYDEQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYRE WSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPQGCQGSVISFPSPRSGPGSPAQWLLYTHPTHARQRAD LGAYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0353] SEQ ID NO:97: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAGTHQVQWQAQEVVAQARLDGHRSMNPCPLYDEQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCYVTSTDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP IPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPTGCQGSVISFPSPRSGPGSPAQWLLYTHPTHSWQRADLGAYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQ

[0354] SEQ ID NO:98: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAGTHQVQWQAQEVVAQARLDGHRSMNPCPLYDEQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCYVTSTDHGRTWSSPRDLTDAAIGPAYREW STFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPTGCQGSVISFPSPRSGPGSPAQWLLYTHPTHSWQRADLG AYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0355] SEQ ID NO:99: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAGTHQVQWQAQEVVAQARLDGHRSMNPCPLYDEQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCYVTSTDHGRTWSSPRDLTDAAIGPAYRE WSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPTGCQGSVISFPSPRSGPGSPAQWLLYTHPTTHSWQRAD LGAYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0356] SEQ ID NO:100: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAGTHQVQWQAQEVVAQARLDGHRSMNPCPLYDEQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCYVTSTDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP IPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRFRVQAQSTNDGLDFQESQLVKKLVEPPPTGCQGSVISFPSPRSGPGSPAQWLLYTHPTHSWQRADLGAYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQ

[0357] SEQ ID NO:101: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAGTHQVQWQAQEVVAQARLDGHRSMNPCPLYDEQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCYVTSTDHGRTWSSPRDLTDAAIGPAYREW STFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRFRVQAQSTNDGLDFQESQLVKKLVEPPPTGCQGSVISFPSPRSGPGSPAQWLLYTHPTHSWQRADLG AYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0358] SEQ ID NO:102: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAGTHQVQWQAQEVVAQARLDGHRSMNPCPLYDEQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCYVTSTDHGRTWSSPRDLTDAAIGPAYRE WSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRFRVQAQSTNDGLDFQESQLVKKLVEPPPTGCQGSVISFPSPRSGPGSPAQWLLYTHPTTHSWQRAD LGAYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0359] SEQ ID NO:103:

[0360] sequence number:104: GAGATCGTGCTGACACAGAGCCCTGCCACACTGTCACTGTCTCCAGGCGAGAGAGCCACACTGAGCTGTAGAGCTTCTAAGGGCGTGTCACCAGCGGCTACTCTTACCTGCACTGGTATCAGCAGAAGCCCGGCCAGGCTCCTAGACTGCTGATCTACCTGG CCAGCTACCTGGAAAGCGGAGTGCCTGCTAGATTCAGCGGCTCTGGCTCTGGCACCGACTTCACCCTGGACAATCAGCAGCCTGGAACCTGAGGACTTCGCCGTGTACTACTGCCAGCACAGCAGAGATCTGCCCCTGACATTTGGCGGAGGCACCAAGGTGGAA ATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCCAGAGAGGCCAAAGTACAGTGGAAGGTGATAACGCCCTCCAATCGGGTAACTCCCAGGAGGTTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCTCGCCCGTCACAAAGAGCTTCACAGGGGAGAGTGT

[0361] sequence number:105: QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQ GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0362] SEQ ID NO:106: TVEKSVVFKAEGEHFTDQKGNTIVGSGSGGTTKYFRIPAMCTTSKGTIVVFADARHNTASDQSFIDTAAARSTDGGKTWNKKIAIYNDRVNSKLSRVMDPTCIVANIQGRETILVMVGKWNNNDKTWGAYRDKAPDTDWDLVLYKSTDGVTFSKVET NIHDIVTKNGTISAMLGGVGSGLQLNDGKLVFPVQMVRTKNITTVLNTSFIYSTDGITWSLPSGYCEGFGSENNIIEFNASLVNNIRNSGLRRSFETKDFGKTWTEFPPMDKKVDNRNHGVQGSTITIPSGNKLVAAHSSAQNKNNDYTRSDISLYAH NLYSGEVKLIDDFYPKVGNASGAGYSCLSYRKNVDKETLYVVYEANGSIEFQDLSRHLPVIKSYNGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGGGSHHHHHHHH

[0363] SEQ ID NO:107:

[0364] SEQ ID NO:108: TVEKSVVFKAEGEHFTDQKGNTIVGSGSGGTTKYFRIPAMCTTSKGTIVVFADARHNTASDQSFIDTAAARSTDGGKTWNKKIAIYNDRVNSKLSRVMDPTCIVANIQGRETILVMVGKWNNNDKTWGAYRDKAPDTDWDLVLYKSTDGVTFSKVET NIHDIVTKNGTISAMLGGVGSGLQLNDGKLVFPVQMVRTKNITTVLNTSFIYSTDGITWSLPSGYCEGFGSENNIIEFNASLVNNIRNSGLRRSFETKDFGKTWTEFPPMDKKVDNRNHGVQGSTITIPSGNKLVAAHSSAQNKNNDYTRSDISLYAH NLYSGEVKLIDDFYPKVGNASGAGYSCLSYRKNVDKETLYVVYEANGSIEFQDLSRHLPVIKSYNGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGGGSHHHHHHHH

[0365] SEQ ID NO:109:

[0366] SEQ ID NO:110:

[0367] SEQ ID NO:111: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAGTHQVQWQAQEVVAQARLDGHRSMNPCPLYDEQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYRE WSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPQGCQGSVISFPSPRSGPGSPAQWLLYTHPTTHSWQRAD LGAYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0368] SEQ ID NO:112: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAGTHQVQWQAQEVVAQARLDGHRSMNPCPLYDEQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYREW STFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPQGCQGSVISFPSPRSGPGSPAQWLLYTHPTHSWQRADLG AYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0369] SEQ ID NO:113:

[0370] SEQ ID NO:114:

[0371] SEQ ID NO:115: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAGTHQVQWQAQEVVAQARLDGHRSMNPCPLYDEQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCYVTSTDHGRTWSSPRDLTDAAIGPAYRE WSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRFRVQAQSTNDGLDFQESQLVKKLVEPPPTGCQGSVISFPSPRSGPGSPAQWLLYTHPTTHSWQRAD LGAYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0372] SEQ ID NO:116: X1ASLPX2LQX3ESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAX4THQVQWQAQEVVAQARLDGHRSMNPCPLYDX5QTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCX6VTSTDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPX7QRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRX8RVQAQSTNDGLDFQESQLVKKLVEPPPX9GCQGSVISFPSPRSGPGSPAQWLLYTHPTHX 10 X 11 QRADLGAYLNPRPPAPEAWSEPVLLAKGSX 12 AYSDLQSMGTGPDGSPLFGCLYEANDYEEIX 13 FX 14 MFTLKQAFPAEYLPQX 15 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0373] Sequence number: 117: X1X2SX3X4X5LQX6ESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASX7X8DEHAELIVX9RRGDYDAX 10 THQVQWX 11 AQEVVAQAX 12 LX 13 GHRSMNPCPLYDX 14 QTGTLFLFFIAIPX 15 X 16 VTEX 17 QQLQTRANVTRLX18 X 19 VTSTDHGRTWSSPRDLTDAAIGPX 20 YREWSTFAVGPGHX 21 LQLHDX 22 X 23 RSLVVPAYAYRKLHPX 24 X 25 X 26 PIPSAFX 27 FLSHDHGRTWARGHFVX 28 QDTX 29 ECQVAEVX 30 TGEQRVVTLNARSX 31 X 32 X 33 X 34 RX 35 QAQSX 36 NX 37 GLDFQX 38 X 39 QX 40 VKKL X 41 EPPPX 42 GX 43 QGSVISFPSPRSGPGSPAQX 44 LLYTHPTHX 45 X 46 QRADLGAYLNPRPPAPEAWSEPX 47 LLAKGSX 48 AYSDLQSMGTGPDGSPLFGX 49 LYEANDYEEIX 50 FX 51 MFTLKQAFPAEYLPQX 52 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0374] Sequence number: 118:

[0375] Array number: 119: X1X2SX3X4X5LQX6ESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASX7X8DEHAELIVX9RRGDYDAX 10 THQVQWX 11 AQEVVAQAX 12 LX 13 GHRSMNPCPLYDX 14 QTGTLFLFFIAIPX 15 X 16 VTEX 17 QQLQTRANVTRLX 18 X 19 VTSTDHGRTWSSPRDLTDAAIGPX 20 YREWSTFAVGPGHX 21 LQLHDX 22 X 23 RSLVVPAYAYRKLHPX 24 X 25 X 26 PIPSAFX 27 FLSHDHGRTWARGHFVX 28 QDTX 29 ECQVAEVX 30 TGEQRVVTLNARSX 31 X 32 X 33 X 34 RX 35 QAQSX 36 NX 37 GLDFQX 38 X 39 QX 40 VKKL X 41 EPPPX 42 GX 43 QGSVISFPSPRSGPGSPAQX 44 LLYTHPTHX 45 X 46 QRADLGAYLNPRPPAPEAWSEPX 47 LLAKGSX 48 AYSDLQSMGTGPDGSPLFGX 49 LYEANDYEEIX 50 FX 51 MFTLKQAFPAEYLPQ

[0376] SEQ ID NO:120: X1ASLPX2LQX3ESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAX4THQVQWQAQEVVAQARLDGHRSMNPCPLYDX5QTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCX6VSTDHHGRTWSSPRDLTDAAIGP AYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPX7QRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRX8RVQAQSTNDGLDFQESQLVKKLVEPPPX9GCQGSVISFPSPRSGPGSPAQWLLYTHPTHX 10 X 11 QRADLGAYLNPRPPAPEAWSEPVLLAKGSX 12 AYSDLQSMGTGPDGSPLFGCLYEANDYEEIX 13 FX 14 MFTLKQAFPAEYLPQ

[0377] SEQ ID NO:121: GGGGS

[0378] SEQ ID NO:122: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAGTHQVQWQAQEVVAQARLDGHRSMNPCPLYDEQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCYVTSTDHGRTWSSPRDLTDAAIGPAYRE WSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPTGCQGSVISFPSPRSGPGSPAQWLLYTHPTTHSWQRAD LGAYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0379] SEQ ID NO:123: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAGTHQVQWQAQEVVAQARLDGHRSMNPCPLYDEQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCYVTSTDHGRTWSSPRDLTDAAIGPAYRE WSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRFRVQAQSTNDGLDFQESQLVKKLVEPPPTGCQGSVISFPSPRSGPGSPAQWLLYTHPTTHSWQRAD LGAYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0380] SEQ ID NO:124:

[0381] SEQ ID NO:125: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRRSKKDEHAELIVLRRGDYDAGTHQVQWQAQEVVAQARLDGHRSMNPCPLYDEQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCYVTSTDHGRTWSSPRDLTDAAIGPAYRE WSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRFRVQAQSTNDGLDFQESQLVKKLVEPPPTGCQGSVISFPSPRSGPGSPAQWLLYTHPTTHSWQRAD LGAYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0382] SEQ ID NO:126: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAGTHQVQWQAQEVVAQARLDGHRSMNPCPLYDEQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCYVTSTDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRP IPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPQGCQGSVISFPSPRSGPGSPAQWLLYTHPTHSWQRADLGAYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQ

[0383] SEQ ID NO:127: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAGTHQVQWQAQEVVAQARLDGHRSMNPCPLYDEQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCYVTSTDHGRTWSSPRDLTDAAIGPAYRE WSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPQGCQGSVISFPSPRSGPGSPAQWLLYTHPTTHSWQRAD LGAYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0384] SEQ ID NO:128: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAGTHQVQWQAQEVVAQARLDGHRSMNPCPLYDEQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCYVTSTDHGRTWSSPRDLTDAAIGPAYREW STFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPQGCQGSVISFPSPRSGPGSPAQWLLYTHPTHSWQRADLG AYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0385] SEQ ID NO:129:

[0386] SEQ ID NO:130: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRRSKKDEHAELIVLRRGDYDAGTHQVQWQAQEVVAQARLDGHRSMNPCPLYDEQTGTLFLFFIAIP GQVTEQQQLQTRANVTRLCYVSTDHHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRPIPSAFCFLSHDHGRTWARGHFVA QDTLECQVAEVETGEQRVVTLNARSHLRFRVQAQSTNDGLDFQESQLVKKLVEPPPTGCQGSVISFPSPRSGPGSPAQWLLYTHPTHSWQRADLGAYLNPRPPAPEA WSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLVQSGVEVKK PGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTS

[0387] SEQ ID NO:131:

[0388] SEQ ID NO:132: QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTS

[0389] SEQ ID NO:133: CAGGTTCAGCTGGTGCAGTCTGGCGTGGAAGTGAAGAAACCTGGGGCCAGCGTGAAGGTGTCCTGTAAAGCCAGCGGCTACACCTTTACCAACTACTACATGTACTGGGTCCGACAGGCCCCAGGCCAAGGACTTGAATGGATGGGCGGCATCAACCCCAGCAACGGCGG CACCAACTTCAACGAGAAGTTCAAGAACAGAGTGACCCTGACCACCGACAGCAGCACCACAGCTACATGGAACTGAAGTCCCTGCAGTTCGACGACACCGCCGTGTACTACTGCGCCAGACGGGACTACAGATTCGACATGGGCTTCGACTATTGGGGCCAGGGCA CCACCGTGACAGTCTCTTCTGCtagcACCAAGGGCCCATCcGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCcTGGAACTCAGGCGCtCTGACCAGCGGCTG CACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAAACTagc

[0390] sequence number:134: DASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRRSKKDEHAELIVLRRGDYDAGTHQVQWQAQEVVAQARLDGHRSMNPCPLYDEQTGTLFLFFIAI PGQVTEQQQLQTRANVTRLCYVTSTDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPKQRPIPSAFCFLSHDHGRTWARGHF VAQDTLECQVAEVETGEQRVVTLNARSHLRFRVQAQSTNDGLDFQESQLVKKLVEPPPTGCQGSVISFPSPRSGPGSPAQWLLYTHPTHSWQRADLGAYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSEIVLTQ SPATLLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGGTKVEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0391] SEQ ID NO:135:

[0392] SEQ ID NO:136: QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWG QGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0393] SEQ ID NO:137: QVQLVQSVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS

[0394] SEQ ID NO:138: EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGGTKVEIK

[0395] SEQ ID NO:139: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0396] SEQ ID NO:140: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0397] SEQ ID NO:141: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0398] SEQ ID NO:142: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0399] SEQ ID NO:143: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0400] SEQ ID NO:144: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0401] SEQ ID NO:145: MGWSCIILFLVATATGVHSQVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFD DTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSDASLPYLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRR GDYDAGTHQVQWQAQEVVAQARLDGHRSMNPCPLYDEQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCYVTSTDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHC LQLHDRARSLVVPAYAYRKLHPKQRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRFRVQAQSTNDGLDFQESQLVKKLVEPPPTGC QGSVISFPSPRSGPGSPAQWLLYTHPTHSWQRADLGAYLNPRPPAPEAWSEPVLLAKGSAAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQ

[0402] SEQ ID NO:146:

Claims

1. (a) a recombinant mutant human sialidase; (b) an anti-PD-1 immunoglobulin antigen-binding domain; and 1. A fusion protein comprising: (i) substitution of an alanine residue at a position corresponding to position 42 of wild-type human Neu2 (A42); (ii) substitution of a proline residue at a position corresponding to position 62 of wild-type human Neu2 (P62); (iii) substitution of an alanine residue at a position corresponding to position 93 of wild-type human Neu2 (A93); (iv) substitution of a glutamine residue at a position corresponding to position 126 of wild-type human Neu2 (Q126); (v) substitution of an alanine residue at a position corresponding to position 242 of wild-type human Neu2 (A242); and / or (vi) Substitution of a glutamine residue at a position corresponding to position 270 of wild-type human Neu2 (Q270). A fusion protein comprising:

2. The sialidase is (a) substitution or deletion of a methionine residue (M1) at a position corresponding to position 1 of wild-type human Neu2; (b) substitution of a valine residue (V6) at the position corresponding to position 6 of wild-type human Neu2; (c) substitution of a lysine residue (K9) at a position corresponding to position 9 of wild-type human Neu2; (d) substitution of a serine residue at a position corresponding to position 301 of wild-type human Neu2 (S301); (e) substitution of a tryptophan residue at a position corresponding to position 302 of wild-type human Neu2 (W302); (f) substitution of a cysteine ​​residue at a position corresponding to position 332 of wild-type human Neu2 (C332); (g) a substitution of a valine residue at a position corresponding to position 363 of wild-type human Neu2 (V363); or (h) substitution of a leucine residue at a position corresponding to position 365 of wild-type human Neu2 (L365); or any combination of the foregoing substitutions The fusion protein of claim 1, further comprising:

3. In the sialidase, (a) a methionine residue at the position corresponding to position 1 of wild-type human Neu2 is either deleted (ΔM1), replaced by alanine (M1A), or replaced by aspartic acid (M1D); (b) a valine residue at a position corresponding to position 6 of wild-type human Neu2 is replaced by a tyrosine (V6Y); (c) an alanine residue at a position corresponding to position 42 of wild-type human Neu2 is replaced by arginine (A42R); (d) the lysine residue at the position corresponding to position 9 of wild-type human Neu2 is replaced by an aspartic acid (K9D); (e) the proline residue at a position corresponding to position 62 of wild-type human Neu2 is replaced by asparagine (P62N), aspartic acid (P62D), histidine (P62H), glutamic acid (P62E), glycine (P62G), serine (P62S), or threonine (P62T); (f) an alanine residue at a position corresponding to position 93 of wild-type human Neu2 is replaced by glutamic acid (A93E) or lysine (A93K); (g) a glutamine residue at a position corresponding to position 126 of wild-type human Neu2 is replaced by leucine (Q126L), glutamic acid (Q126E), phenylalanine (Q126F), histidine (Q126H), isoleucine (Q126I), or tyrosine (Q126Y); (h) the alanine residue at a position corresponding to position 242 of wild-type human Neu2 is replaced by cysteine ​​(A242C), phenylalanine (A242F), glycine (A242G), histidine (A242H), isoleucine (A242I), lysine (A242K), leucine (A242L), methionine (A242M), asparagine (A242N), glutamine (A242Q), arginine (A242R), serine (A242S), valine (A242V), tryptophan (A242W), or tyrosine (A242Y); (i) the glutamine residue at a position corresponding to position 270 of wild-type human Neu2 is replaced by alanine (Q270A), histidine (Q270H), phenylalanine (Q270F), proline (Q270P), serine (Q270S), or threonine (Q270T); (j) the serine residue at a position corresponding to position 301 of wild-type human Neu2 is replaced by alanine (S301A), aspartic acid (S301D), glutamic acid (S301E), phenylalanine (S301F), histidine (S301H), lysine (S301K), leucine (S301L), methionine (S301M), asparagine (S301N), proline (S301P), glutamine (S301Q), arginine (S301R), threonine (S301T), valine (S301V), tryptophan (S301W), or tyrosine (S301Y); (k) the tryptophan residue at a position corresponding to position 302 of wild-type human Neu2 is replaced by alanine (W302A), aspartic acid (W302D), phenylalanine (W302F), glycine (W302G), histidine (W302H), isoleucine (W302I), lysine (W302K), leucine (W302L), methionine (W302M), asparagine (W302N), proline (W302P), glutamine (W302Q), arginine (W302R), serine (W302S), threonine (W302T), valine (W302V), or tyrosine (W302Y); (l) the cysteine ​​residue at the position corresponding to position 332 of wild-type human Neu2 is replaced by alanine (C332A); (m) a valine residue at a position corresponding to position 363 of wild-type human Neu2 is replaced by an arginine (V363R); or (n) the leucine residue at the position corresponding to position 365 of wild-type human Neu2 is replaced by glutamine (L365Q), histidine (L365H), isoleucine (L365I), lysine (L365K), or serine (L365S); Alternatively, the sialidase comprises any combination of the above substitutions. A fusion protein according to claim 1 or 2.

4. 4. The fusion protein of claim 3, wherein the sialidase comprises a substitution selected from: ΔM1, M1A, M1D, V6Y, K9D, A42R, P62G, P62N, P62S, P62T, A93E, Q126Y, A242F, A242W, A242Y, Q270A, Q270T, S301A, S301R, W302K, W302R, C332A, V363R, and L365I, or any combination of said substitutions.

5. The sialidase is (a) an M1D substitution, a V6Y substitution, a P62G substitution, an A93E substitution, and a C332A substitution; (b) an M1D substitution, a V6Y substitution, a K9D substitution, an A93E substitution, a C332A substitution, a V363R substitution, and an L365I substitution; (c) an M1D substitution, a V6Y substitution, a P62N substitution, and a C332A substitution; (d) an M1D substitution, a V6Y substitution, a Q270A substitution, a S301R substitution, a W302K substitution, and a C332A substitution; (e) an M1D substitution, a V6Y substitution, a P62S substitution, a Q270A substitution, a S301R substitution, a W302K substitution, and a C332A substitution; (f) an M1D substitution, a V6Y substitution, a P62T substitution, a Q270A substitution, a S301R substitution, a W302K substitution, and a C332A substitution; (g) an M1D substitution, a V6Y substitution, a P62N substitution, a Q270A substitution, a S301R substitution, a W302K substitution, and a C332A substitution; (h) an M1D substitution, a V6Y substitution, a P62G substitution, an A93E substitution, a S301A substitution, a W302R substitution, and a C332A substitution; (i) an M1D substitution, a V6Y substitution, a P62G substitution, an A93E substitution, a Q126Y substitution, a Q270T substitution, and a C332A substitution; (j) an M1D substitution, a V6Y substitution, a P62G substitution, an A93E substitution, a Q126Y substitution, and a C332A substitution; (k) an M1D substitution, a V6Y substitution, a P62G substitution, an A93E substitution, a Q126Y substitution, an A242F substitution, a Q270T substitution, and a C332A substitution, or (l) M1D mutation, V6Y mutation, A42R mutation, P62G mutation, A93E mutation, Q126Y mutation, A242F mutation, Q270T mutation, and C332A mutation. The fusion protein of claim 4 .

6. The fusion protein of any one of claims 1 to 5, wherein the sialidase is Neu2.

7. The fusion protein of any one of claims 1 to 6, wherein the sialidase comprises any one of SEQ ID NOs: 48-63, 94, 97, 100, or 126.

8. The fusion protein according to any one of claims 1 to 7, further comprising an immunoglobulin Fc domain.

9. 9. The fusion protein of claim 8, wherein the immunoglobulin Fc domain is derived from a human IgG1, IgG2, IgG3 or IgG4 Fc domain, optionally wherein the immunoglobulin Fc domain is derived from a human IgG1 Fc domain.

10. The fusion protein of any one of claims 1 to 9, wherein the anti-PD-1 immunoglobulin antigen-binding domain is associated with a second anti-PD-1 immunoglobulin antigen-binding domain to provide an anti-PD-1 antigen-binding site.

11. The fusion protein of any one of claims 1 to 10, wherein the anti-PD-1 immunoglobulin antigen-binding domain is derived from an antibody selected from nivolumab, pembrolizumab, cemiplimab, PDR001, and pidilizumab, and optionally, the anti-PD-1 immunoglobulin antigen-binding domain is derived from pembrolizumab.

12. The fusion protein of any one of claims 1 to 11, wherein the sialidase and the immunoglobulin Fc domain and / or the anti-PD-1 immunoglobulin antigen binding domain are linked by a peptide bond or an amino acid linker.

13. The fusion protein of any one of claims 1 to 12, wherein the fusion protein comprises any one of SEQ ID NOs: 67-73, 78, 81-87, 95, 96, 98, 99, 101, 102, 105, 106, 108, 111, 112, 115, 122, 123, 125, 127, 128, 130, 132, or 134.

14. An antibody conjugate comprising the fusion protein according to any one of claims 1 to 13.

15. and / or 15. The antibody conjugate of claim 14, which comprises a single anti-PD-1 antigen binding site.

16. The antibody conjugate: (a) a first polypeptide comprising an immunoglobulin light chain, optionally, said first polypeptide comprising SEQ ID NO:77; (b) a second polypeptide comprising an immunoglobulin heavy chain, optionally wherein the second polypeptide comprises SEQ ID NO: 105; and (c) a third polypeptide comprising an immunoglobulin Fc domain and a sialidase, optionally wherein said third polypeptide comprises, from N-terminal to C-terminal direction, said sialidase and said immunoglobulin Fc domain, and / or said third polypeptide comprises any one of SEQ ID NOs: 67-73, 78, 81-87, 95, 96, 98, 99, 101, 102, 106, 108, 111, 112, 115, 122, 123, 125, 127, or 128. Including; 16. The antibody conjugate of claim 14 or 15, wherein said first and second polypeptides are covalently linked together, said second and third polypeptides are covalently linked together, and said first and second polypeptides together define an anti-PD-1 antigen binding site.

17. An isolated nucleic acid comprising a nucleotide sequence encoding at least a part of the fusion protein of any one of claims 1 to 13 or the antibody conjugate of any one of claims 14 to 16.

18. An expression vector comprising the nucleic acid of claim 17.

19. A host cell comprising the expression vector of claim 18.

20. A pharmaceutical composition comprising a fusion protein according to any one of claims 1 to 13 or an antibody conjugate according to any one of claims 14 to 16.

21. A pharmaceutical composition according to claim 20 for use in treatment.

22. A composition for use in therapy comprising a fusion protein according to any one of claims 1 to 13 or an antibody conjugate according to any one of claims 14 to 16.

23. The pharmaceutical composition of claim 21 or the composition of claim 22, wherein the treatment is treatment of cancer in a subject in need of such treatment.

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