WO2022147481A1

WO2022147481A1 - Combination therapy of an oncolytic virus delivering a foreign antigen and an engineered immune cell expressing a chimeric receptor targeting the foreign antigen

Info

Publication number: WO2022147481A1
Application number: PCT/US2021/073204
Authority: WO
Inventors: Nancy Chang
Original assignee: Ansun Biopharma Inc.
Priority date: 2020-12-30
Filing date: 2021-12-30
Publication date: 2022-07-07
Also published as: WO2022147480A1; TW202241479A

Abstract

The present application provides methods and compositions for treating cancers (such as solid tumors) using a recombinant oncolytic virus encoding a foreign antigen (e.g., a bacterial sialidase) and an engineered immune cell (e.g., a CAR-NK cell) expressing a chimeric receptor specifically recognizing the foreign antigen.

Description

COMBINATION THERAPY OF AN ONCOLYTIC VIRUS DELIVERING A FOREIGN ANTIGEN AND AN ENGINEERED IMMUNE CELL EXPRESSING A CHIMERIC RECEPTOR TARGETING THE FOREIGN ANTIGEN

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority benefit of U.S. Provisional Application 63/132,420 filed December 30, 2020, the contents of which are incorporated herein by reference in their entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

[0002] The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 208712001340SEQLIST.TXT, date recorded: December 28, 2021, size: 308,635 bytes).

FIELD

[0003] The present application relates to methods and compositions for treating cancer with an oncolytic virus (e.g., vaccinia virus) encoding a foreign antigen and an engineered immune cell expressing a chimeric receptor specifically recognizing the foreign antigen.

BACKGROUND

[0004] Cancer is the second leading cause of death in the United States. In recent years, great progress has been made in cancer immunotherapy, including immune checkpoint inhibitors, T cells with chimeric antigen receptors, and oncolytic viruses.

[0005] Oncolytic viruses are naturally occurring or genetically modified viruses that infect, replicate in, and eventually kill cancer cells while leaving healthy cells unharmed. A recently completed Phase III clinical trial of the oncolytic herpes simplex virus T-VEC in 436 patients with unresectable stage IIIB, IIIC or IV melanoma was reported to meet its primary end point, with a durable response rate of 16.3% in patients receiving T-VEC compared to 2.1% in patients receiving GM-CSF. Based on the results from this trial, FDA approved T-VEC in 2015.

[0006] Oncolytic virus constructs from at least eight different species have been tested in various phases of clinical trials, including adenovirus, herpes simplex virus- 1, Newcastle disease virus, reovirus, measles virus, coxsackievirus, Seneca Valley virus, and vaccinia virus. It has become clear that oncolytic viruses are well tolerated in patients with cancer. The clinical benefits of oncolytic viruses as stand-alone treatments, however, remain limited. Due to concerns on the safety of oncolytic viruses, only highly attenuated oncolytic viruses (either naturally avirulent or attenuated through genetic engineering) have been used in both preclinical and clinical studies. Since the safety of oncolytic viruses has now been well established it is time to design and test oncolytic viruses with maximal anti-tumor potency. Oncolytic viruses with a robust oncolytic effect will release abundant tumor antigens to prime or activate immune cells including T and NK cells, resulting in a strong immunotherapeutic effect.

BRIEF SUMMARY

[0007] The present application provides methods and compositions for treating cancer using an oncolytic virus and an engineered immune cell expressing a chimeric receptor.

[0008] One aspect of the present application provides a method of treating a cancer in an individual in need thereof comprising administering to the individual: (a) an effective amount of a recombinant oncolytic virus comprising a nucleotide sequence encoding a foreign antigen; and (b) an effective amount of an engineered immune cell expressing a chimeric receptor specifically recognizing said foreign antigen. In some embodiments, the foreign antigen comprises an anchoring domain. In some embodiments, the anchoring domain is positively charged at physiologic pH. In some embodiments, the anchoring domain is a glycosaminoglycan (GAG)-binding domain. In some embodiments, the foreign antigen comprises a transmembrane domain. In some embodiments, the anchoring domain or the transmembrane domain is located at the carboxy terminus of the foreign antigen. In some embodiments, the foreign antigen comprises a stabilization domain. In some embodiments, the stabilization domain is an Fc domain. In some embodiments, the foreign antigen comprises a domain (e.g., an Fc domain) that induces ADCC effects by the engineered immune cell.

[0009] In some embodiments according to any one of the methods described above, the nucleotide sequence encoding the foreign antigen is operably linked to a promotor. In some embodiments, the promotor is a viral promoter that can be an early promoter, an intermediate promoter, or a late promoter or an early/late hybrid promoter. In some embodiments, the oncolytic virus is a poxvirus and the promoter is a poxvirus early promoter, a late promoter or a hybrid early/late promoter. In some embodiments, the promotor is a viral late promoter. In some embodiments, the promoter is an F17R late promoter (e.g., SEQ ID NO: 61). In some embodiments, the promoter is a hybrid early-late promoter. In some embodiments, the promoter comprises a partial or complete nucleotide sequence of a human promoter. In some embodiments, the human promoter is a tissue or tumor-specific promoter. In some embodiments, the promoter is a synthetic promoter.

[0010] In some embodiments according to any one of the methods described above, the foreign antigen is a viral protein or fragment thereof. In some embodiments, the foreign antigen is a bacterial protein or fragment thereof.

[0011] In some embodiments according to any one of the methods described above, the foreign antigen is a sialidase. In some embodiments, the sialidase is a protein having exo- sialidase activity (Enzyme Commission EC 3.2.1.18) including bacterial, fungal, viral sialidase and derivatives thereof. In some embodiments, the sialidase is an anhydrosialidase as defined by Enzyme Commission EC 4.2.2.15. In some embodiments, the sialidase is a Neu5Ac alpha(2,6)-Gal sialidase, a Neu5Ac alpha(2,3)-Gal sialidase, or a Neu5Ac alpha(2,8)-Gal sialidase.

[0012] In some embodiments according to any one of the methods described above, the foreign antigen is a bacterial sialidase. In some embodiments, the bacterial sialidase is selected from the group consisting of: Clostridium pe/fringens sialidase, Actinomyces viscosus sialidase, and Arthrobacter ureafaciens sialidase, Salmonella typhimurium sialidase and Vibrio cholera sialidase. In some embodiments, the bacterial sialidase is Actinomyces viscosus sialidase (“avSial”).

[0013] In some embodiments according to any one of the methods described above, wherein the foreign antigen is a sialidase, the sialidase is a naturally occurring sialidase. In some embodiments, the sialidase is an engineered protein comprising a sialidase catalytic domain. In some embodiments, the sialidase comprises an anchoring domain. In some embodiments, the sialidase is a fusion protein comprising a sialidase catalytic domain fused to an anchoring domain. In some embodiments, the anchoring domain is positively charged at physiologic pH. In some embodiments, the anchoring domain is a glycosaminoglycan (GAG)-binding domain. In some embodiments, the sialidase comprises a transmembrane domain. In some embodiments, the sialidase comprises an anchoring domain or a transmembrane domain located at the carboxy terminus of the sialidase. In some embodiments, the sialidase comprises from the N-terminus to the C-terminus: a sialidase catalytic domain, a hinge region, and a transmembrane domain.

[0014] In some embodiments according to any one of the methods described above, wherein the foreign antigen is a sialidase, the sialidase comprises an amino acid sequence having at least about 80% (e.g., at least about any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-33 and 53-54. In some embodiments, the sialidase comprises an amino acid sequence having at least about 80% (e.g., at least about any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 2.

[0015] In some embodiments according to any one of the methods described above, the foreign antigen is DAS181 or a derivative thereof. In some embodiments, the chimeric receptor comprises an anti-DAS181 antibody moiety that is not cross-reactive with human native amphiregulin or neuraminidase.

[0016] In some embodiments according to any one of the methods described above, the chimeric receptor is a Chimeric Antigen Receptor (CAR). In some embodiments, the CAR comprises an anti-sialidase antibody moiety, a transmembrane domain, and an intracellular domain. In some embodiments, the intracellular domain comprising a CD28 intracellular signaling sequence and an intracellular signaling sequence of CD3^. In some embodiments, the anti-sialidase antibody moiety comprises an antibody heavy chain variable domain (VH) comprising a heavy chain complementarity determining region (CDR-H) 1 comprising the amino acid sequence of SEQ ID NO: 111, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 112, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 113; and an antibody light chain variable domain (VL) comprising a light chain complementarity determining region (CDR-L) 1 comprising the amino acid sequence of SEQ ID NO: 114, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 115, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 116. In some embodiments, the anti-sialidase antibody moiety comprises a VH comprising an amino acid sequence having at least about 80% e.g., at least about any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 117, and a VL comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 118. In some embodiments, the anti-sialidase antibody moiety is a scFv. In some embodiments, the anti-sialidase antibody moiety comprises an amino acid sequence having at least about 80% (e.g., at least about any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 119. [0017] In some embodiments according to any one of the methods described above, the engineered immune cell is selected from the group consisting of T cell, Natural Killer (NK) cell, natural killer T (NKT) cell, macrophage and combinations thereof. In some embodiments, the engineered immune cell is NK cell. In some embodiments, the engineered immune cell is a T cell, such as y8T cell. In some embodiments, the engineered immune cell is a macrophage. In some embodiments, the engineered immune cell is NKT cell.

[0018] In some embodiments according to any one of the methods described above, the oncolytic virus is a virus selected from the group consisting of: vaccinia virus, reovirus, Seneca Valley virus (SVV), vesicular stomatitis virus (VSV), Newcastle disease virus (NDV), herpes simplex virus (HSV), morbillivirus virus, retrovirus, influenza virus, Sinbis virus, poxvirus, measles virus, cytomegalovirus (CMV), lentivirus, adenovirus, and derivatives thereof. In some embodiments, the virus is Talimogene Laherparepvec. In some embodiments, the virus is a reovirus. In some embodiments, the virus is an adenovirus having an E1ACR2 deletion.

[0019] In some embodiments according to any one of the methods described above, the oncolytic virus is a poxvirus. In some embodiments, the poxvirus is a vaccinia virus. In some embodiments, the vaccinia virus is of a strain selected from the group consisting of Dryvax, Lister, M63, LIVP, Tian Tan, Modified Vaccinia Ankara, New York City Board of Health (NYCBOH), Dairen, Ikeda, LC16M8, Tashkent, IHD-J, Brighton, Dairen I, Connaught, Elstree, Wyeth, Copenhagen, Western Reserve, Elstree, CL, Lederle-Chorioallantoic, AS, and derivatives thereof. In some embodiments, the virus is vaccinia virus Western Reserve.

[0020] In some embodiments according to any one of the methods described above, the recombinant oncolytic virus comprises one or more mutations that reduce immunogenicity of the virus compared to a corresponding wild-type strain. In some embodiments, the virus is a vaccinia virus, and the one or more mutations are in one or more proteins selected from the group consisting of A14, A17, A13, LI, H3, D8, A33, B5, A56, F13, A28, and A27. In some embodiments, the one or more mutations are in one or more proteins selected from the group consisting of A27L, H3L, D8L and L1R.

[0021] In some embodiments according to any one of the methods described above, the virus is a vaccinia virus, and the virus comprises one or more proteins selected from the group consisting of: (a) a variant vaccinia virus (VV) H3L protein that comprises an amino acid sequence having at least 90% amino acid sequence identity to any one of SEQ ID NOS: 66- 69; (b) a variant vaccinia virus (VV) D8L protein that comprises an amino acid sequence having at least 90% amino acid sequence identity to any one of SEQ ID NOS: 70-72 or 85; (c) a variant vaccinia virus (VV) A27L protein that comprises an amino acid sequence having at least 90% amino acid sequence identity to SEQ ID NO: 73; and (d) a variant vaccinia virus (VV) L1R protein that comprises an amino acid sequence having at least 90% amino acid sequence identity to SEQ ID NO: 74.

[0022] In some embodiments according to any one of the methods described above, the oncolytic virus further comprises a second nucleotide sequence encoding a heterologous protein or nucleic acid. In some embodiments, the second nucleotide sequence encodes a heterologous protein. In some embodiments, the heterologous protein is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA-4, PD-1, PD-L1, TIGIT, LAG3, TIM-3, VISTA, B7-H4, or HLA-G. In some embodiments, the immune checkpoint inhibitor is an antibody. In some embodiments, the heterologous protein is an inhibitor of an immune suppressive receptor. In some embodiments, the immune suppressive receptor is LILRB, TYRO3, AXL, or MERTK. In some embodiments, the inhibitor of an immune suppressive receptor is an anti-LILRB antibody. In some embodiments, the heterologous protein is a multi-specific immune cell engager. In some embodiments, the heterologous protein is a bispecific T cell engager (BiTE). In some embodiments, the heterologous protein is selected from the group consisting of cytokines, costimulatory molecules, tumor antigen presenting proteins, anti-angiogenic factors, tumor-associated antigens, foreign antigens, and matrix metalloproteases (MMP). In some embodiments, the heterologous protein is IL-15, IL-12, IL2, modified IL-2 with reduced toxicity or better function, IL18, modified IL-18 with less or no binding to the IL-18 binding protein, Flt3L, CCL5, CXCL10, or CCL4 and any modified forms of such cytokines that still have the antitumor immunity, or an inhibitor of any binding proteins that can block and neutralize these cytokine function and activities.

[0023] In some embodiments according to any one of the methods described above, the virus comprises two or more additional nucleotide sequences, wherein each nucleotide sequence encodes a heterologous protein.

[0024] In some embodiments according to any one of the methods described above, the engineered immune cell further comprises a heterologous nucleotide sequence encoding a cytokine. In some embodiments, the cytokine is 11-15.

[0025] In some embodiments according to any one of the methods described above, the engineered immune cell and the recombinant oncolytic virus are administered simultaneously. In some embodiments, the recombinant oncolytic virus is administered prior to administration of the engineered immune cell.

[0026] In some embodiments according to any one of the methods described above, the recombinant oncolytic virus is administered via a carrier cell (e.g., an immune cell or a stem cell, such as a mesenchymal stem cell).

[0027] In some embodiments according to any one of the methods described above, the recombinant oncolytic virus is administered as a naked virus. In some embodiments, the recombinant oncolytic virus is administered via direct intratumoral injection. In some embodiments, the method further comprises administering to the individual an effective amount of an immunotherapeutic agent. In some embodiments, the immunotherapeutic agent is selected from the group consisting of chemotherapy, radiotherapy, radioimmunotherapy, a mono or multi- specific antibody, a cell therapy, a cancer vaccine (e.g., a dendritic cell-based cancer vaccine), a cytokine, PI3Kgamma inhibitor, a TLR9 ligand, an HDAC inhibitor, a LILRB2 inhibitor, a MARCO inhibitor, and an immune checkpoint inhibitor.

[0028] In some embodiments according to any one of the methods described above, the cancer is a solid cancer.

[0029] One aspect of the present application provides a pharmaceutical composition comprising: (a) a recombinant oncolytic virus comprising a nucleotide sequence encoding a foreign antigen; (b) an engineered immune cell expressing a chimeric receptor specifically recognizing said foreign antigen; and (c) a pharmaceutically acceptable carrier. In some embodiments, the foreign antigen is a bacterial sialidase, such as avSial. In some embodiments, the foreign antigen is DAS181 or a derivative thereof. In some embodiments, the engineered immune cell is a NK cell, a T cell e.g., y8 T cell), a NKT cell, or a macrophage. In some embodiments, the engineered immune cell is a NK cell. In some embodiments, the chimeric receptor is a CAR.

[0030] Another aspect of the present application provides an isolated antibody or antigenbinding fragment thereof that specifically binds Actinomyces viscosus sialidase, comprising a VH comprising an CDR-H1 comprising the amino acid sequence of SEQ ID NO: 111, a CDR- H2 comprising the amino acid sequence of SEQ ID NO: 112, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 113; and a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 114, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 115, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 116. [0031] In some aspects, provided herein is a recombinant oncolytic virus comprising a first nucleotide sequence encoding a sialidase and a second nucleotide sequence encoding a multispecific immune cell engager, wherein the first nucleotide sequence and the second nucleotide sequence are operably linked to one or more promoters.

[0032] In some embodiments, the multispecific immune cell engager is a bispecific immune cell engager. In some embodiments, the multispecific immune cell engager comprises a first antigen-binding domain capable of specifically recognizing a tumor antigen and a second antigen-binding domain capable of specifically recognizing a cell surface molecule of an immune effector cell. In some embodiments, the tumor antigen is selected from the group consisting of fibroblast activation protein (FAP), epithelial cellular adhesion molecule (EpCAM), and epidermal growth factor receptor (EGFR). In some embodiments, the tumor antigen is FAP. In some embodiments, the cell surface molecule on the effector cell is CD3 or 41-BB. In some embodiments, the cell surface marker on the effector cell is CD3s.

[0033] In some embodiments according to any of the recombinant oncolytic viruses described above, the first antigen-binding domain is an scFv, and/or the second antigen binding domain is an scFv. In some embodiments, the tumor antigen is FAP and the first antigenbinding domain comprises: (i) a first light chain complementarity-determining region (CDR- Ll) having the amino acid sequence of SEQ ID NO: 86, (i) a second light chain complementarity-determining region (CDR-L2) having the amino acid sequence of SEQ ID NO: 87, (iii), a third light chain complementarity-determining region (CDR-L3) having the amino acid sequence of SEQ ID NO: 88, (iv) a first heavy chain complementarity-determining region (CDR-H1) having the amino acid sequence of SEQ ID NO: 89, (v) a second heavy chain complementarity-determining region (CDR-H2) having the amino acid sequence of SEQ ID NO: 90, and (vi) a third heavy chain complementarity-determining region (CDR-H3) having the amino acid sequence of SEQ ID NO: 91. In some embodiments, the tumor antigen is FAP and the first antigen-binding domain comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 98. In some embodiments, the first antigenbinding domain comprises the amino acid sequence of SEQ ID NO: 98.

[0034] In some embodiments according to any of the recombinant oncolytic viruses described above, the cell surface molecule on the effector cell is CD3, and the second antigenbinding domain comprises: (i) a first light chain complementarity-determining region (CDR- Ll) having the amino acid sequence of SEQ ID NO: 92, (ii) a second light chain complementarity-determining region (CDR-L2) having the amino acid sequence of SEQ ID NO: 93, (iii), a third light chain complementarity-determining region (CDR-L3) having the amino acid sequence of SEQ ID NO: 94, (iv) a first heavy chain complementarity-determining region (CDR-H1) having the amino acid sequence of SEQ ID NO: 95, (v) a second heavy chain complementarity-determining region (CDR-H2) having the amino acid sequence of SEQ ID NO: 96, and (vi) a third heavy chain complementarity-determining region (CDR-H3) having the amino acid sequence of SEQ ID NO: 97. In some embodiments, the cell surface molecule on the effector cell is CD 3 and the second antigen-binding domain comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 99. In some embodiments, the cell surface molecule on the effector cell is CD3 and the second antigenbinding domain comprises the amino acid sequence of SEQ ID NO: 99.

[0035] In some embodiments according to any of the recombinant oncolytic viruses described above, the multispecific immune cell engager comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% identity to the amino acid sequence of SEQ ID N O: 100. In some embodiments according to any of the recombinant oncolytic viruses described above, the multispecific immune cell engager comprises the amino acid sequence of SEQ ID N O: 100.

[0036] In some embodiments, the recombinant oncolytic virus is a virus selected from the group consisting of: vaccinia virus, reovirus, Seneca Valley virus (SVV), vesicular stomatitis virus (VSV), Newcastle disease virus (NDV), herpes simplex virus (HSV), morbillivirus virus, retrovirus, influenza virus, Sinbis virus, poxvirus, measles virus, cytomegalovirus (CMV), lentivirus, adenovirus, and derivatives thereof. In some embodiments, the virus is Talimogene Laherparepvec. In some embodiments, the virus is a reovirus. In some embodiments, the virus is an adenovirus having an E1ACR2 deletion.

[0037] In some embodiments according to any one of the recombinant oncolytic viruses described above, the recombinant oncolytic virus is a poxvirus. In some embodiments, the poxvirus is a vaccinia virus. In some embodiments, the vaccinia virus is of a strain selected from the group consisting of Dryvax, Lister, M63, LIVP, Tian Tan, Modified Vaccinia Ankara, New York City Board of Health (NYCBOH), Dairen, Ikeda, LC16M8, Tashkent, IHD-J, Brighton, Dairen I, Connaught, Elstree, Wyeth, Copenhagen, Western Reserve, Elstree, CL, Lederle-Chorioallantoic, AS, and derivatives thereof. In some embodiments, the virus is vaccinia virus Western Reserve.

[0038] In some embodiments according to any one of the recombinant oncolytic viruses described above, the recombinant oncolytic virus comprises one or more mutations that reduce immunogenicity of the virus compared to a corresponding wild-type strain. In some embodiments, the virus is a vaccinia virus, and the one or more mutations are in one or more proteins selected from the group consisting of A14, A17, A13, LI, H3, D8, A33, B5, A56, F13, A28, and A27. In some embodiments, the one or more mutations are in one or more proteins selected from the group consisting of A27L, H3L, D8L and L1R.

[0039] In some embodiments, the virus is a vaccinia virus, and the virus comprises one or more proteins selected from the group consisting of: (a) a variant vaccinia virus (VV) H3L protein that comprises an amino acid sequence having at least 90% amino acid sequence identity to any one of SEQ ID NOS: 66-69; (b) a variant vaccinia virus (VV) D8L protein that comprises an amino acid seqOuence having at least 90% amino acid sequence identity to any one of SEQ ID NOS: 70-72 or 85; (c) a variant vaccinia virus (VV) A27L protein that comprises an amino acid sequence having at least 90% amino acid sequence identity to SEQ ID NO: 73; and (d) a variant vaccinia virus (VV) L1R protein that comprises an amino acid sequence having at least 90% amino acid sequence identity to SEQ ID NO: 74.

[0040] In some embodiments according to any of the recombinant oncolytic viruses described above, the recombinant oncolytic virus is a vaccinia virus, and the recombinant oncolytic virus comprises a disruption of a thymidine kinase (TK) gene. In some embodiments, the first and second nucleotide sequences (encoding the sialidase and the multispecific immune cell engager, respectively) are inserted into the TK gene. In some embodiments, the recombinant oncolytic virus comprises a disruption of a vaccinia growth factor (VGF) gene.

[0041] In some embodiments according to any one of the recombinant oncolytic viruses described above, the sialidase is a Neu5Ac alpha(2,6)-Gal sialidase, a Neu5Ac alpha(2,3)-Gal sialidase, or a Neu5Ac alpha(2,8)-Gal sialidase.

[0042] In some embodiments according to any one of the recombinant oncolytic viruses described above, the sialidase is any protein having exo-sialidase activity (Enzyme Commission EC 3.2.1.18) including bacterial, human, fungal, viral sialidase and derivatives thereof. In some embodiments, the bacterial sialidase is selected from the group consisting of: Clostridium perfringens sialidase, Actinomyces viscosus sialidase, and Arthrobacter ureafaciens sialidase, Salmonella typhimurium sialidase and Vibrio cholera sialidase.

[0043] In some embodiments according to any one of the recombinant oncolytic viruses described above, the sialidase is a human sialidase or a derivative thereof. In some embodiments, the sialidase is NEU1, NEU2, NEU3, or NEU4.

[0044] In some embodiments according to any one of the recombinant oncolytic viruses described above, the sialidase is a naturally occurring sialidase.

[0045] In some embodiments according to any one of the recombinant oncolytic viruses described above, the sialidase comprises an anchoring domain. In some embodiments, the sialidase is a fusion protein comprising a sialidase catalytic domain fused to an anchoring domain. In some embodiments, the anchoring domain is positively charged at physiologic pH. In some embodiments, the anchoring domain is a glycosaminoglycan (GAG)-binding domain. [0046] In some embodiments according to any one of the recombinant oncolytic viruses described above, the sialidase is a protein having exo-sialidase activity as defined by Enzyme Comission EC 3.2.1.18.

[0047] In some embodiments according to any one of the recombinant oncolytic viruses described above, the sialidase is an anhydrosialidase as defined by Enzyme Commission EC 4.2.2.15.

[0048] In some embodiments according to any one of the recombinant oncolytic viruses described above, the sialidase comprises an amino acid sequence having at least about 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-33, 53-54, and 105. In some embodiments, the sialidase comprises an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the sialidase is DAS181 or a derivative thereof.

[0049] In some embodiments according to any one of the recombinant oncolytic viruses described above, the nucleotide sequence encoding the sialidase further encodes a secretion sequence operably linked to the sialidase. In some embodiments, the secretion sequence comprises the amino acid sequence of any one of SEQ ID NOs: 40, 101 and 102.

[0050] In some embodiments according to any one of the recombinant oncolytic viruses described above, the sialidase comprises a transmembrane domain. In some embodiments, the sialidase comprises from the N-terminus to the C-terminus: a sialidase catalytic domain, a hinge region, and a transmembrane domain. In some embodiments, the sialidase comprises from the N-terminus to the C-terminus: a sialidase catalytic domain, a hinge region, an IgG Fc region, and a transmembrane domain. In some embodiments, the hinge region is an IgGl hinge region. In some embodiments, the IgG Fc domain comprises the amino acid sequence of SEQ ID NO: 104.

[0051] In some embodiments according to any one of the recombinant oncolytic viruses described above, the sialidase comprises an immunoglobulin G (IgG) Fc (fragment, crystallizable) domain. In some embodiments, the sialidase comprises from the N-terminus to the C-terminus: a sialidase catalytic domain, an IgG Fc domain, and a transmembrane domain. In some embodiments according to any one of the recombinant oncolytic viruses described above, the IgG Fc domain comprises the amino acid sequence of SEQ ID NO: 104. [0052] In some embodiments according to any one of the recombinant oncolytic viruses described above, the sialidase comprises an anchoring domain or a transmembrane domain located at the carboxy terminus of the sialidase.

[0053] In some embodiments according to any one of the recombinant oncolytic viruses described above, the one or more promotors comprise a viral promoter that can be an early promoter, an intermediate promoter, or a late promoter, or an early/late hybrid promoter. In some embodiments, the recombinant oncolytic virus is a poxvirus and the promoter is a poxvirus early promoter, a late promoter or a hybrid early/late promoter.

[0054] In some embodiments according to any one of the recombinant oncolytic viruses described above, the one or more promoters comprise a viral late promoter. In some embodiments, the promoter is an F17R late promoter (SEQ ID NO: 61).

[0055] In some embodiments according to any one of the recombinant oncolytic viruses described above, the one or more promoters comprise a hybrid early-late promoter.

[0056] In some embodiments according to any one of the recombinant oncolytic viruses described above, the one or more promoters comprise a promoter comprising the nucleotide sequence of SEQ ID NO: 107.

[0057] In some embodiments according to any one of the recombinant oncolytic viruses described above, the promoter comprises a partial or complete nucleotide sequence of a human promoter. In some embodiments, the human promoter is a tissue or tumor-specific promoter.

[0058] In some embodiments according to any one of the recombinant oncolytic viruses described above, the one or more promotors comprise a first promoter that is operably linked to the first nucleotide sequence and a second promoter that is operably linked to the second nucleotide sequence. In some embodiments, the first promoter is an F17R promoter and the second promoter is a pE/L promoter. In some embodiments, the F17R promoter comprised the nucleic acid sequence of SEQ ID NO: 61. In some embodiments, the pE/L promoter comprises the nucleic acid sequence of SEQ ID NO: 107.

[0059] In some embodiments according to any one of the recombinant oncolytic viruses described above, the recombinant oncolytic virus further comprises an additional nucleotide sequence encoding a heterologous protein or nucleic acid. In some embodiments, the additional nucleotide sequence encodes a heterologous protein.

[0060] In some embodiments according to any one of the recombinant oncolytic viruses described above, the heterologous protein is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA-4, PD-1, PD-L1, TIGIT, LAG3, TIM-3, VISTA, B7-H4, or HLA-G. In some embodiments, the immune checkpoint inhibitor is an antibody.

[0061] In some embodiments according to any one of the recombinant oncolytic viruses described above, the heterologous protein is an inhibitor of an immune suppressive receptor. In some embodiments, the immune suppressive receptor is LILRB, TYRO3, AXL, or MERTK. In some embodiments, the inhibitor of an immune suppressive receptor is an anti-LILRB antibody.

[0062] In some embodiments according to any one of the recombinant oncolytic viruses described above, the heterologous protein is a multi-specific immune cell engager. In some embodiments, the heterologous protein is a bispecific T cell engager (BiTE).

[0063] In some embodiments according to any one of the recombinant oncolytic viruses described above, the heterologous protein is selected from the group consisting of cytokines, costimulatory molecules, tumor antigen presenting proteins, anti-angiogenic factors, tumor- associated antigens, foreign antigens, and matrix metalloproteases (MMP).

[0064] In some embodiments according to any one of the recombinant oncolytic viruses described above, the heterologous protein is an inhibitor of CD55 or CD59.

[0065] In some embodiments according to any one of the recombinant oncolytic viruses described above, the heterologous protein is IL- 15, IL- 12, IL2, modified IL-2 with reduced toxicity or better function, IL18, modified IL- 18 with less or no binding to the IL- 18 binding protein, Flt3L, CCL5, CXCL10, or CCL4 and any modified forms of such cytokines that still have the anti-tumor immunity, or an inhibitor of any binding proteins that can block and neutralize these cytokine function and activities.

[0066] In some embodiments according to any one of the recombinant oncolytic viruses described above, the heterologous protein is a bacterial polypeptide.

[0067] In some embodiments according to any one of the recombinant oncolytic viruses described above, the heterologous protein is a tumor-associated antigen selected from the group consisting of carcinoembryonic antigen, alphafetoprotein, MUC16, survivin, glypican-3, B7 family members, LILRB, CD19, BCMA, NY-ESO-1, CD20, CD22, CD33, CD38, CEA, EGFR (such as EGFRvIII), GD2, HER2, IGF1R, mesothelin, PSMA, ROR1, WT1, NY-ESO- 1, Fibulin-3, CDH17, and other tumor antigens with clinical significance

[0068] In some embodiments according to any one of the recombinant oncolytic viruses described above, the virus comprises two or more additional nucleotide sequences, wherein each nucleotide sequence encodes a heterologous protein. [0069] In some aspects, provided herein is a recombinant vaccinia virus of a Western Reserve strain comprising a first nucleotide sequence encoding a sialidase having the amino acid sequence of SEQ ID NO: 105, and a second nucleotide sequence encoding a bispecific immune cell engager having the amino acid sequence of SEQ ID NO: 100; wherein the first nucleotide sequence and the second nucleotide sequence are operably linked to one or more promoters; and wherein the recombinant vaccinia virus comprises a disruption or deletion of a thymidine kinase (TK) gene and a disruption or deletion of a vaccinia growth factor (VGF) gene.

[0070] One aspect of the present application provides a pharmaceutical composition comprising the recombinant oncolytic virus of any one of the preceding claims and a pharmaceutically acceptable carrier.

[0071] One aspect of the present application provides a carrier cell comprising any one of the recombinant oncolytic viruses described above. In some embodiments, the carrier cell is an engineered immune cell or a stem cell (e.g., a mesenchymal stem cell) or B cells or leukocytes. In some embodiments, the engineered immune cell is a Chimeric Antigen Receptor (CAR)-T cell (including CAR-yST cell), CAR-NK, CAR-NKT, or CAR-macrophage.

[0072] One aspect of the present application provides a method of treating a cancer in an individual in need thereof, comprising administering to the individual an effective amount of any one of the recombinant oncolytic viruses, pharmaceutical compositions, or carrier cells described above.

[0073] In some embodiments, the method comprises administering to the individual an effective amount of any one of the recombinant oncolytic viruses described above. In some embodiments, the recombinant oncolytic virus is administered via a carrier cell (e.g., an immune cell or a stem cell, such as a mesenchymal stem cell).

[0074] In some embodiments, the cancer is an FAP positive cancer. In some embodiments, the cancer is selected from the group consisting of lung cancer, colon cancer, and breast cancer. In some embodiments, administering the recombinant oncolytic virus activates and/or expands CD4⁺ and/or CD8⁺ T-cells in the individual. In some embodiments, administering the recombinant oncolytic virus increases tumor-infiltrating lymphocytes in the individual.

[0075] In some embodiments, the recombinant oncolytic virus is administered as a naked virus. In some embodiments, the recombinant oncolytic virus is administered via direct intratumoral injection. In some embodiments, the method further comprises administering to the individual an effective amount of an immunotherapeutic agent. In some embodiments, the immunotherapeutic agent is selected from the group consisting of chemotherapy, radiotherapy, radioimmunotherapy, a mono or multi-specific antibody, a cell therapy, a cancer vaccine (e.g., a dendritic cell-based cancer vaccine), a cytokine, PI3Kgamma inhibitor, a TLR9 ligand, an HDAC inhibitor, a LILRB2 inhibitor, a MARCO inhibitor, and an immune checkpoint inhibitor.

[0076] Also provided are compositions, kits and articles of manufacture for use in any one the methods described above.

BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Fig. 1: Detection of 2,6 sialic acid (by FITC-SNA) on A549 and MCF cells by fluorescence microscopy. A549 and MCF cells were fixed and incubated with FITC-SNA for one hour at 37°C before imaged under fluorescence microscope to show the FITC-SNA labeled cells (left) and overlay with brightfield cells (right)

[0078] Fig. 2: Effective removal of 2,6 sialic acid, 2,3 sialic acid, and exposure of galactose on A549 cells by DAS181 treatment. A549 were treated with DAS 181 for two hours at 37°C and incubated with staining reagents one hour before imaged under fluorescence microscope to show effective removal of sialic acids on tumor cells.

[0079] Fig. 3: Effective removal of 2,6 sialic acid on A549 cells by DAS181 but not DAS185 treatment. A549 were treated with DAS181 for 30 minutes or two hours at 37°C and incubated with FITC-SNA for one hour before examined using flow cytometry to show effective removal of 2,6 sialic acids on tumor cells.

[0080] Fig. 4: Effective removal of 2,3 sialic acid on A549 cells by DAS181 but not DAS185 treatment. A549 were treated with DAS181 for 30 minutes or two hours at 37°C and incubated with FITC-MALII for one hour before examined using flow cytometry to show effective removal of 2,3 sialic acids on tumor cells

[0081] Fig. 5: Effective exposure of galactose on A549 cells by DAS181 but not DAS185 treatment. A549 were treated with DAS181 for 30 minutes or two hours at 37°C and incubated with FITC-PNA for one hour before examined using flow cytometry to show effective exposure of galactose on tumor cells

[0082] Fig. 6: DAS 181 treatment and PBMC stimulation regimen do not affect A549-red cell proliferation. A549-Red cells were seeded at 2k/well overnight, followed by replacement of medium containing reagents listed on the left. Scan by IncuCyte was initiated immediately after the reagents were added (0 hr) and scheduled for every 3 hr. A549-red cell proliferation is monitored by analyzing the nuclear (red) counts. Kinetic readouts reveal no effect on A549 cell proliferation by vehicle, DAS181, or various stimulation reagents, without the presence of PBMCs.

[0083] Fig. 7: Detection of cytotoxicity in A549-red cells following co-culturing with PBMCs from Donor 1 with or without DAS181 treatment. These results showed that DAS181 treatment significantly boost anti-tumor cytotoxicity by PBMCs from Donor 1. A549-Red cells were seeded at 2k/well overnight, followed by co-culturing with lOOK/well Donor- 1 PBMCs (E:T=50:l) in the presence of medium (no activation), CD3+CD28+IL-2 (T cell activation), or CD3+CD29+IL-2+IL-15+IL-21 (T and NK cell activation). Representative images were taken by IncuCyte at 0 hr and 72 hrs post adding PBMCs.

[0084] Fig. 8: Detection of cytotoxicity in A549-red cells following co-culturing with PBMCs from Donor 2 with or without DAS 181 treatment. These results showed that DAS 181 treatment significantly boost anti-tumor cytotoxicity by PBMCs from Donor 2. A549-Red cells were seeded at 2k/well overnight, followed by co-culturing with lOOk/well Donor-1 PBMCs (E:T=50:l) in the presence of medium (no activation), CD3+CD28+IL-2 (T cell activation), or CD3+CD29+IL-2+IL-15+IL-21 (T and NK cell activation). Representative images were taken by IncuCyte at 0 hr and 72 hrs post adding PBMCs.

[0085] Figs. 9A-9C: Detection of cytotoxicity in A549-red cells following co-culturing with PBMCs from Donor 1 with or without DAS181 treatment. These results showed that DAS181 treatment significantly boost anti-tumor cytotoxicity by PBMCs from Donor 1. A549-red tumor cells were seeded at 2k cells/well in 96-well plate. After overnight incubation, PBMCs from Donor 1 mixed with (A) medium (B) CD3/CD28/IL-2, or (C) CD3/CD28/IL-2/IL-15/IL- 21 were added into each well as indicated E:T ratio. At mean time, DAS 181 (100 nM) was added. Plates were scanned by IncuCyte every 3hr for total 72hrs. Proliferation is monitored by analyzing RFP cell counts.

[0086] Figs. 10A-10C: Detection of cytotoxicity in A549-red cells following co-culturing with PBMCs from Donor 2 with or without DAS181 treatment. These results showed that DAS 181 treatment significantly boost anti-tumor cytotoxicity by PBMCs from Donor 2. A549- red tumor cells were seeded at 2k cells/well in 96-well plate. After overnight incubation, PBMCs from Donor 2 mixed with (A) medium, (B) CD3/CD28/IL-2, or (C) CD3/CD28/IL- 2/IL-15/IL-21 were added into each well as indicated E:T ratio. At mean time, DAS 181 (100 nM) was added. Plates were scanned by IncuCyte every 3hr for total 72hrs. Proliferation is monitored by analyzing RFP cell counts. [0087] Fig. 11: DAS 181 enhances NK-mediated tumor lysis by vaccinia virus, measured by MTS assay. ® =T-test P value <0.05, suggesting that DAS181 alone boosts NK cell-mediated U87 tumor killing in vitro, compared to enzyme-dead DAS185. * = T-Test P value <0.05.

[0088] Fig. 12: DAS181 increases NK-mediated tumor killing by vaccinia virus as measured by MTS assay. * = T-test P value <0.05, suggesting that DAS181 increases NK cell-mediated killing of U87 cells by VV in vitro.

[0089] Fig. 13: DAS181 significantly enhanced expression of maturation markers (CD80, CD86, HLA-Dr, HLA-ABC) in human DC cells that were cultured alone or exposed to VV- infected tumor cells. * = T-test P value <0.05.

[0090] Fig. 14: DAS181 significantly enhanced TNF-alpha production by THP-1 derived macrophages. * = T-test P value <0.05

[0091] Fig. 15: DAS181 treatment promotes oncolytic adenovirus-mediated tumor cell killing and growth prohibition. A549-red tumor cells were seeded at 2K cells/well in 96-well plates. After overnight incubation, DAS181 vehicle, oncolytic adenovirus, and DAS 181 were added as indicated. CD3/CD28/IL-2 were also added into each well with the amount described previously. Graph showed that DAS181 plus oncolytic adenovirus effectively reduced tumor cell proliferation.

[0092] Figs. 16A-16B: DAS181 treatment enhances PBMC-mediated tumor cell killing by oncolytic virus. A549-red tumor cells were seeded at 2K cells/well in 96-well plate. After overnight incubation, fresh PBMCs were added at densities of lOK/well (A) or 40K/well (B). CD3, CD28, IL-2, DAS181, and oncolytic adenovirus were added as indicated in the graph following with the timed scans by IncuCyte. Graph showed that DAS 181 plus oncolytic adenovirus dramatically enhanced human PBMC-mediated tumor cell eradication.

[0093] Fig. 17: Schematic of a portion of a vaccinia virus construct encoding a sialidase.

[0094] Figs. 18A-18B: DAS 181 expressed by Sialidase- VV has in vitro activity towards sialic acid-containing substrates. (A) Standard curve of DAS181 activity at 0.5 nM, 1 nM, and 2 nM. (B) IxlO⁶ cells infected with Sialidase-VV express DAS181 equivalent to 0.78nM - 1.21 nM DAS 181 in 1ml medium in vitro.

[0095] Fig. 19: Sialidase-VV enhances Dendritic cell maturation. GM-CSF/IL4 derived human DC were cultured with Sial-VV or VV infected U87 tumor cell lysate for 24 hours. LPS was used as control. DC were collected and stained with antibodies against CD80, CD86, HLA- DR, and HLA-ABC. The expression of DC maturation markers was determined by flow analysis. The results suggested that Sial-VV enhanced DC maturation. * = T-test P value <0.05 [0096] Fig. 20: Sialidase-VV induced IFN-gamma and IL2 expression by T cells. CD3 antibody-activated human T cells were co-cultured with A594 tumor cells in the presence of Sial-VV- or VV-infected tumor cells lysate for 24 hours, and cytokine IFNy or IL-2 expression was measured by ELISA. The results suggested that Sial- VV-infected tumor cell lysate induced IFNy and IL2 expression by human T cells. * = T-test P value <0.05

[0097] Fig. 21: Sialidase-VV enhances T cell-mediated tumor cell lytic activity. CD3 Ab activated human T cells were co-cultured with Sial-VV- or VV-infected A594 tumor cells for 24 hours, and tumor cell viability was determined by MTS assay. The results suggested that Sial-VV infection of tumor cells resulted in enhanced tumor killing. * = T-test P value <0.05. [0098] FIGS. 22A-22C: Impact of DAS181 and secreted sialidase Constructs 1, 2, and 3 on cell surface a2,3 sialic acid (FIG. 22A); a2,6 sialic acid (FIG. 22B) and galactose (FIG. 22C). FIG. 22A: A549-red cells were transfected by Construct- 1, 2 or 3. After overnight incubation, transfected cells were lifted and re-seeded in 24-well plate. After additional 24hrs, 48hrs and 72hrs, cells were fixed and stained with MALII-FITC for Ihr before performing flow. Treat non-transfected cells with lOOnM DAS181 for 2hrs before fixed. Vehicle prepared for DAS181 was used to treat another set of non-transfected cells as control. FIG. 22B : A549-red cells were transfected by Construct- 1, 2 and 3. After overnight incubation, transfected cells were lifted and re-seeded in 24-well plate. After additional 24hrs, 48hrs and 72hrs, cells were fixed and stained with SNA-FITC for Ihr before performing flow. Treat non-transfected cells with lOOnM DAS181 for 2hrs before fixed. Vehicle prepared for DAS181 was used to treat another set of non-transfected cells as control. FIG. 22C: A549-red cells were transfected by Construct- 1, 2 and 3. After overnight incubation, transfected cells were lifted and re-seeded in 24-well plate. After additional 24hrs, 48hrs and 72hrs, cells were fixed and stained with PNA-FITC for Ihr before performing flow. Treat non-transfected cells with lOOnM DAS181 for 2hrs before fixed. Vehicle prepared for DAS181 was used to treat another set of non-transfected cells as control.

[0099] FIGS. 23A-23C: Impact of DAS181 and transmembrane sialidase Constructs 1, 4, 5 and 6 on cell surface a2,3 sialic acid (FIG. 23A); a2,6 sialic acid (FIG. 23B); and galactose (FIG. 23C). FIG. 23A: A549-red cells were transfected by Construct-1, 4, 5, and 6. After overnight incubation, transfected cells were lifted and re-seeded in 24-well plate. After additional 24hrs, 48hrs and 72hrs, cells were fixed and stained with MALII-Biotinylated for Ihr followed by FITC-streptavidin for an additional Ihr. The 2, 3-sialic acid level was detected by flow cytometry. FIG. 23B: A549-red cells were transfected by Construct- 1, 4, 5, and 6. After overnight incubation, transfected cells were lifted and re-seeded in 24-well plate. In additional 24hrs, 48hrs and 72hrs, cells were fixed and stained with SNA-FITC for Ihr. The 2, 6-sialic acid level was detected by flow cytometry. FIG. 23C: A549-red cells were transfected by Construct- 1, 4, 5, and 6. After overnight incubation, transfected cells were lifted and reseeded in 24-well plate. After additional 24hrs, 48hrs and 72hrs, cells were fixed and stained with PNA-FITC for Ihr. The galactose level was detected by flow cytometry.

[0100] FIG. 24: Stable expression of Construct 1 increases oncolytic virus and PBMC- mediated A549 cell killing. Freshly isolated PBMCs were incubated with A549-red parental cells only or with cells stable expressing Construct- 1 or cells stable expressing Construct- 1 with 1MOI or 5MOI on two separated plates (Plate 2 and 4).

[0101] FIG. 25: Stable expression of Construct 4 increases oncolytic virus and PBMC- mediated A549 cell killing. Fresh isolated PBMCs were activated and incubated with A549- red cells only or with cells stable expressing Construct-4 or cells stable expressing Construct- 4 with 1MOI or 5MOI OL in two separated plates (Plate 2 and 4).

[0102] FIG. 26: Design of exemplary sialidase expression constructs for recombination into the TK gene of Western Reserve VV to generate oncolytic virus encoding a sialidase. Exemplary constructs are shown for endocellular sialidase, secreted sialidase with an anchoring domain, and cell surface expressed sialidase with a transmembrane domain.

[0103] FIG. 27: PCR detection of Sialidase expression: CV-1 cells were infected with Sialidase- VV at an MOI of 0.2. After 48 hours, CV-1 cells were collected, and DNA were extracted using Wizard® SV Genomic DNA Purification System and used as template for Sialidase PCR amplification. PCR was conducted using standard PCR protocol. Expected PCR product size is 125 Ibp.

[0104] FIG. 28: U87 or CV-1 cells were infected with control VV, SP-, Endo- or TM-Sial- VVs at MOI 1. The cells were collected at 24, 48, 72, or 96 hours. Virus titers were determined by plaque assay.

[0105] FIG. 29: U87 tumor cells were infected with control VV, SP-, Endo- or TM-Sial- VVs at MOI 0.1, 1, or 5. Tumor killing was measured by MTS assay.

[0106] FIG. 30: The expression of DC maturation marker HLA-ABC is enhanced by culture with oncolytic virus encoding secreted or transmembrane sialidase.

[0107] FIG. 31: The expression of DC maturation marker HLA-DR is enhanced by culture with oncolytic virus encoding secreted or transmembrane sialidase.

[0108] FIG. 32: The expression of DC maturation marker CD80 is enhanced by culture with oncolytic virus encoding secreted or transmembrane sialidase. [0109] FIG. 33: The expression of DC maturation marker CD86 is enhanced by culture with oncolytic virus encoding secreted or transmembrane sialidase.

[0110] FIG. 34: Sial-VV enhances NK-mediated tumor lysis in vitro. Negative selected human NK cells (Astarte, WA) and VV-U87 cells (ATCC, VA) were co-cultured, and tumor killing efficacy was measured by LDH assay (Abeam, MA). The results suggested that Sial- VVs enhanced NK cell-mediated U87 tumor killing in vitro. (* P value, the Sial-VV vs Mock VV in U87 and NK culture).

[0111] FIG. 35: Results indicate that TM-sial-VV significantly inhibited tumor growth compared to control VV in vivo (tumor cells inoculated in right flank of mouse).

[0112] FIG. 36 Results indicate that TM-sial-VV significantly inhibited tumor growth compared to control VV in vivo (tumor cells inoculated in left flank of mouse).

[0113] FIG. 37: Mouse body weight was unaffected by treatment with Sial-VV or VV The results didn’t show the difference on the mouse body weight.

[0114] FIGS. 38A-38B: Sialidase armed oncolytic vaccinia virus significantly enhanced CD8+ and CD4+ T cell infiltration within tumor. * p value: treatment group vs control VV group. FIG. 38 A shows quantification of the results. FIG. 38B shows the FACS plots.

[0115] FIG. 39: TM-Sial-VV decreased the ratio of Treg/CD4+ T cells within the tumor, compared to control VV. * p value: treatment group vs control VV group.

[0116] FIG. 40: Sialidase armed oncolytic vaccinia virus significantly enhanced NK and NKT cell infiltration within tumor. * p value: treatment group vs control VV group.

[0117] FIG. 41: TM-Sial-VV significantly increased PD-L1 expression within tumor cells (p <0.05).

[0118] FIGS. 42A-42B: Results indicating that an exemplary oncolytic virus comprising a nucleotide sequence encoding a sialidase and a bispecific immune cell engager (vvDD-Sial- FAP/CD3) reduced the level of a-2,6-sialic acid linkages on cell surface.

[0119] FIG. 43: Results indicating that vvDD-Sial-FAP/CD3 induced antibody-dependent cellular cytotoxicity (ADCC) for A549 in the presence of Jurkat effector T cells.

[0120] FIGS. 44A-44B: Conditioned media from A549 cells infected with vvDD-Sial- FAP/CD3 induced T-cell activation in the presence of Jurkat cells for FAP-positive COLO829 colon cancer cells (FIG 44A) compared to FAP-negative A549 cells (FIG. 44B). [0121] FIG. 45: Infection of HCT116 human colon cancer cells mixed with FAP- expressing normal human dermal fibroblasts with vvDD-Sial-FAP/CD3 resulted in significantly higher LDH compared to mock infected or vvDD infected cells in the presence of PBMCs. [0122] FIGS. 46A-46C: A549 human lung adenocarcinoma cells were co-cultured with normal human dermal fibroblasts (NhDF) were mock-infected or infected with vvDD or vvDD-Sial-FAP/CD3 at 0.1 pfu/cell. The next day, peripheral blood mononuclear cells (PBMCs) were added at 10:1 (Effector:Target). One day later, cells were harvested and analyzed for the expression of CD25 activation marker on CD4+ (FIG. 46A) and CD8+ (FIG. 46B) T cells using flow cytometry. Supernatants were analyzed for granzyme B release using ELISA (FIG. 46C).

[0123] FIGS. 47A-47B: HCT116 human colon cancer cells co-cultured with normal human dermal fibroblasts (NhDF) or HCC1143 human breast cancer cells were mock- infected or infected with vvDD or vvDD-Sial-FAP/CD3 at 0.1 pfu/cell. The next day, peripheral blood mononuclear cells (PBMCs) were added at 10:1 (Effector:Target). Two days later, cells were harvested and analyzed for the expression of activation markers, CD69 and CD25, on CD4+ (FIG. 47A) and CD8+ (FIG. 47B) T cells using flow cytometry [0124] FIG. 48: A549 human lung adenocarcinoma cells co-cultured with cancer- associated fibroblasts (CAFs) were mock-infected or infected with vvDD or vvDD-Sial- FAP/CD3 expressing green or yellow fluorescent protein, respectively, at 0.3 pfu/cell. The expression of either GFP or YFP was monitor by imaging. Increase in the intensity of the fluorescent proteins indicates the spread of the virus within tumor spheroids.

[0125] FIG. 49: A549 human lung adenocarcinoma cells co-cultured with cancer- associated fibroblasts (CAFs) were mock-infected or infected with vvDD or vvDD-Sial- FAP/CD3 at 0.3 pfu/cell. The next day, peripheral blood mononuclear cells (PBMCs) labeled by CellTracker DeepRed were added at 10:1 (Effector:Target). Images taken two days after addition of PBMCs are shown. Infection with vvDD-Sial-FAP/CD3 resulted in the increase of red fluorescence in the core of the tumor spheroid, indicating enhanced tumor-infiltrating lymphocytes.

[0126] FIG. 50A shows schematics of avSial CAR (also referred herein as “DAS181 CAR”) and CD 19 CAR constructs.

[0127] FIG. SOB shows binding of DAS181-biotin protein to avSial CAR (D004) comprising various scFv clones in 293T cells.

[0128] FIGS. 50C-50D show transduction efficiencies of various avSial CAR (D004) to 293T cells as determined by anti-mouse Fab staining (FIG. 50C) and protein L staining (FIG. 50D).

[0129] FIG. 51A shows transduction rates of NT, CD19 CAR and avSial CAR (D004) NK cells. [0130] FIG. 51B shows total cell counts in NT NK, CD 19 CAR NK and avSial CAR (D004) NK samples.

[0131] FIG. 51C shows transduction rates of NT, CD19 CAR and avSial CAR (D004) NK cells.

[0132] FIG. 52 shows avSial CAR NK cells had enhanced anti-tumor effects against A549 tumor cells expressing Tm-Fc-sialidase than against A549 tumor cells expressing Tm- sialidase.

[0133] FIG. 53 shows avSial CAR NK cells had enhanced anti-tumor effects against A375 tumor cells expressing Tm-Fc-sialidase than against A375 tumor cells expressing Tm- sialidase.

[0134] FIG. 54 shows avSial CAR NK cells exhibited enhanced anti-tumor effects against A549 and A375 tumor cells expressing membrane sialidase than CAR19 CAR NK cells and NT NK cells in a second tumor re -challenge test.

[0135] FIG. 55 shows avSial CAR NK cells exhibited enhanced anti-tumor effects against A375 tumor cells expressing membrane sialidase than CAR19 CAR NK cells and NT NK cells in a second tumor re-challenge test.

[0136] FIG. 56 shows at different NK doses, avSial CAR NK cells were more efficacious in controlling tumor than NT and CD 19 CAR NK cells.

[0137] FIG. 57 shows avSial CAR NK cells persisted longer in vitro than non-transduced (NT) NK cells. In this experiment, cell cultures were maintained regularly in complete NK medium except without IL 15 cytokine support.

[0138] FIG. 58 shows that tumor cells expressing membrane sialidase were not stable.

[0139] FIG. 59 shows membrane sialidase expression levels increased after passages in A375 tumor cells.

[0140] FIG. 60 shows membrane sialidase expression levels increased after passages in A549 tumor cells.

[0141] FIG. 61 shows desialyation of A375 and A549 tumor cells expressing membrane sialidase.

DETAILED DESCRIPTION

[0142] The present application provides compositions and methods for treating cancers with an oncolytic virus (e.g., vaccinia virus) encoding a foreign antigen and an engineered immune cell (e.g., NK cell) expressing a chimeric receptor (e.g., CAR) specifically recognizing the foreign antigen. In some embodiments, the foreign antigen is a bacterial sialidase such as DAS181 or a derivative thereof. The recombinant oncolytic viruses described herein are capable of delivering a foreign antigen such as sialidase to tumor cells and/or the tumor cell environment, which is specifically recognized by the engineered immune cell, thereby enhancing immune response against the tumor cells. In some embodiments, the delivered sialidase can reduce sialic acid present on tumor cells or immune cells and render the tumor cells more vulnerable to killing by immune cells whose effectiveness may otherwise be diminished by hypersialylation of cancer cells.

I. Definitions

[0143] Terms are used herein as generally used in the art, unless otherwise defined as follows. [0144] As used herein, “treatment” or “treating” is an approach for obtaining beneficial or desired results including clinical results. For purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: decreasing one more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread of the disease, preventing or delaying the occurrence or recurrence of the disease, delay or slowing the progression of the disease, ameliorating the disease state, providing a remission (whether partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing the quality of life, and/or prolonging survival. Also encompassed by “treatment” is a reduction of pathological consequence of the disease. The methods of the present application contemplate any one or more of these aspects of treatment.

[0145] The terms “individual,” “subject” and “patient” are used interchangeably herein to describe a mammal, including humans. In some embodiments, the individual is human. In some embodiments, an individual suffers from a cancer. In some embodiments, the individual is in need of treatment.

[0146] As is understood in the art, an “effective amount” refers to an amount of a composition sufficient to produce a desired therapeutic outcome (e.g., reducing the severity or duration of, stabilizing the severity of, or eliminating one or more symptoms of cancer). For therapeutic use, beneficial or desired results include, e.g., decreasing one or more symptoms resulting from the disease (biochemical, histologic and/or behavioral), including its complications and intermediate pathological phenotypes presented during development of the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing effect of another medication, delaying the progression of the disease, and/or prolonging survival of patients. In some embodiments, an effective amount of the therapeutic agent may extend survival (including overall survival and progression free survival); result in an objective response (including a complete response or a partial response); relieve to some extent one or more signs or symptoms of the disease or condition; and/or improve the quality of life of the subject.

[0147] As used herein the term ‘‘wild type” is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms.

[0148] The terms “non-naturally occurring” or “engineered” are used interchangeably and indicate the involvement of the hand of man. The terms, when referring to nucleic acid molecules or polypeptides mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and as found in nature.

[0149] As used herein, “sialidase” refers to a naturally occurring or engineered sialidase that is capable of catalyzing the cleavage of terminal sialic acids from carbohydrates on glycoproteins or glycolipids. As used herein, “sialidase” can refer to a domain of a naturally occurring or non-naturally occurring sialidase that is capable of catalyzing cleavage of terminal sialic acids from carbohydrates on glycoproteins or glycolipids. The term “sialidase” also encompasses fusion proteins comprising a naturally occurring or non-naturally occurring sialidase protein or an enzymatically active fragment or domain thereof and another polypeptide, fragment or domain thereof, e.g., an anchoring domain or a transmembrane domain.

[0150] The term “sialidase” as used herein encompasses sialidase catalytic domain proteins. A "sialidase catalytic domain protein" is a protein that comprises the catalytic domain of a sialidase, or an amino acid sequence that is substantially homologous to the catalytic domain of a sialidase, but does not comprise the entire amino acid sequence of the sialidase. The catalytic domain is derived from, wherein the sialidase catalytic domain protein retains substantially the functional activity as the intact sialidase the catalytic domain is derived from. A sialidase catalytic domain protein can comprise amino acid sequences that are not derived from a sialidase. A sialidase catalytic domain protein can comprise amino acid sequences that are derived from or substantially homologous to amino acid sequences of one or more other known proteins, or can comprise one or more amino acids that are not derived from or substantially homologous to amino acid sequences of other known proteins. [0151] As used herein, “expression” refers to the process by which a polynucleotide is transcribed from a DNA template (such as into an mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as “gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0152] The term “antibody” is used in its broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies, etc.), humanized antibodies, chimeric antibodies, full-length antibodies and antigen-binding fragments, single chain Fv, nanobodies, Fc fusion proteins, thereof, so long as they exhibit the desired antigenbinding activity. Antibodies and/or antibody fragments may be derived from murine antibodies, rabbit antibodies, chicken antibodies, human antibodies, fully humanized antibodies, camelid antibody variable domains and humanized versions, shark antibody variable domains and humanized versions, and camelized antibody variable domains.

[0153] The term “hypervariable region” or “HVR,” as used herein, refers to each of the regions of an antibody variable domain, which are hypervariable in sequence. HVRs may form structurally defined loops (“hypervariable loops”). Generally, native four-chain antibodies comprise six HVRs; three in the VH (Hl, H2, H3), and three in the VL (LI, L2, L3). HVRs are interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three HVRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, HVR1, FR2, HVR2, FR3, HVR3, FR4. HVRs generally comprise amino acid residues from the hypervariable loops and/or from the “complementarity determining regions” (CDRs), CDRs being of highest sequence variability and/or involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32 (LI), 50-52 (L2), 91-96 (L3), 26-32 (Hl), 53-55 (H2), and 96-101 (H3). (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acid residues 24-34 of LI, 50-56 of L2, 89-97 of L3, 31-35B of Hl, 50-65 of H2, and 95-102 of H3 (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)). With the exception of CDR1 in VH, CDRs generally comprise the amino acid residues that form the hypervariable loops. CDRs also comprise “specificity determining residues,” or “SDRs,” which are residues that contact antigen. SDRs are contained within regions of the CDRs called abbreviated-CDRs, or a-CDRs. Exemplary a-CDRs (a- CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-Hl, a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31-34 of LI, 50-55 of L2, 89-96 of L3, 31-35B of Hl, 50-58 of H2, and 95-102 of H3 (Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)).

[0154] Table 1 below provides exemplary CDR definitions according to various algorithms known in the art.

Table 1. CDR Definitions

[0155] The term "recombinant" when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified.

[0156] The terms “virus” or “virus particle” are used according to its plain ordinary meaning within Virology and refers to a virion including the viral genome (e.g. DNA, RNA, single strand, double strand), viral capsid and associated proteins, and in the case of enveloped viruses (e.g. herpesvirus, poxvirus), an envelope including lipids and optionally components of host cell membranes, and/or viral proteins.

[0157] As used herein, “oncolytic viruses” refer to viruses that selectively replicate in and selectively kill tumor cells in subjects having a tumor. These include viruses that naturally preferentially replicate and accumulate in tumor cells, such as poxviruses, and viruses that have been engineered to do so. Some oncolytic viruses can kill a tumor cell following infection of the tumor cell. For example, an oncolytic virus can cause death of the tumor cell by lysing the tumor cell or inducing cell death of the tumor cell. Exemplary oncolytic viruses include, but are not limited to, poxviruses, herpesviruses, adenoviruses, adeno-associated viruses, lentiviruses, retroviruses, rhabdoviruses, papillomaviruses, vesicular stomatitis virus, measles virus, Newcastle disease virus, picomavirus, Sindbis virus, papillomavirus, parvovirus, reovirus, and coxsackievirus.

[0158] The term “poxvirus” is used according to its plain ordinary meaning within Virology and refers to a member of Poxviridae family capable of infecting vertebrates and invertebrates which replicate in the cytoplasm of their host. In embodiments, poxvirus virions have a size of about 200 nm in diameter and about 300 nm in length and possess a genome in a single, linear, double-stranded segment of DNA, typically 130-375 kilobase. The term poxvirus includes, without limitation, all genera of poxviridae (e.g., betaentomopoxvirus, yatapoxvirus, cervidpoxvirus, gammaentomopoxvirus, leporipoxvirus, suipoxvirus, molluscipoxvirus, crocodylidpoxvirus, alphaentomopoxvirus, capripoxvirus, orthopoxvirus, avipoxvirus, and parapoxvirus). In embodiments, the poxvirus is an orthopoxvirus (e.g., smallpox virus, vaccinia virus, cowpox virus, monkeypox virus), parapoxvirus (e.g., orf virus, pseudocowpox virus, bovine popular stomatitis virus), yatapoxvirus (e.g., tanapox virus, yaba monkey tumor virus) or molluscipoxvirus (e.g., molluscum contagiosum virus). In embodiments, the poxvirus is an orthopoxvirus (e.g., cowpox virus strain Brighton, raccoonpox virus strain Herman, rabbitpox virus strain Utrecht, vaccinia virus strain WR, vaccinia virus strain IHD, vaccinia virus strain Elstree, vaccinia virus strain CL, vaccinia virus strain Lederle-Chorioallantoic, or vaccinia virus strain AS). In embodiments, the poxvirus is a parapoxvirus (e.g., orf virus strain NZ2 or pseudocowpox virus strain TJS).

[0159] As used herein, a “modified virus” or a “recombinant virus” refers to a virus that is altered in its genome compared to a parental strain of the virus. Typically modified viruses have one or more truncations, substitutions (replacement), mutations, insertions (addition) or deletions (truncation) of nucleotides in the genome of a parental strain of virus. A modified virus can have one or more endogenous viral genes modified and/or one or more intergenic regions modified. Exemplary modified viruses can have one or more heterologous nucleotide sequences inserted into the genome of the virus. Modified viruses can contain one or more heterologous nucleotide sequences in the form of a gene expression cassette for the expression of a heterologous gene. Modifications can be made using any method known to one of skill in the art, including as provided herein, such as genetic engineering and recombinant DNA methods.

[0160] “Percent (%) amino acid sequence identity” with respect to the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. 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. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program MUSCLE (Edgar, R.C., Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, R.C., BMC Bioinformatics 5(1):113, 2004, each of which are incorporated herein by reference in their entirety for all purposes).

[0161] The term “epitope” as used herein refers to the specific group of atoms or amino acids on an antigen to which an antibody or diabody binds. Two antibodies or antibody moieties may bind the same epitope within an antigen if they exhibit competitive binding for the antigen.

[0162] The terms “polypeptide” or “peptide” are used herein to encompass all kinds of naturally occurring and synthetic proteins, including protein fragments of all lengths, fusion proteins and modified proteins, including without limitation, glycoproteins, as well as all other types of modified proteins (e.g., proteins resulting from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP-ribosylation, pegylation, biotinylation, etc. ).

[0163] As use herein, the terms “specifically binds,” “specifically recognizing,” and “is specific for” refer to measurable and reproducible interactions, such as binding between a target and an antibody (such as a diabody). In certain embodiments, specific binding is determinative of the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules (e.g., cell surface receptors). For example, an antibody that specifically recognizes a target (which can be an epitope) is an antibody (such as a diabody) that binds this target with greater affinity, avidity, more readily, and/or with greater duration than its bindings to other molecules. In some embodiments, the extent of binding of an antibody to an unrelated molecule is less than about 10% of the binding of the antibody to the target as measured, e.g., by a radioimmunoassay (RIA). In some embodiments, an antibody that specifically binds a target has a dissociation constant (KD) of <10^-5 M, <10^-6 M, <10^-7 M, <10" ⁸ M, <10^-9 M, <1O ¹⁰ M, <10^-11 M, or <10 ¹² M. In some embodiments, an antibody specifically binds an epitope on a protein that is conserved among the protein from different species. In some embodiments, specific binding can include, but does not require exclusive binding. Binding specificity of the antibody or antigen-binding domain can be determined experimentally by methods known in the art. Such methods comprise, but are not limited to Western blots, ELISA, RIA, ECL, IRMA, EIA, BIACORETM and peptide scans.

[0164] The term “simultaneous administration,” as used herein, means that a first therapy and second therapy in a combination therapy are administered with a time separation of no more than about 15 minutes, such as no more than about any of 10, 5, or 1 minutes. When the first and second therapies are administered simultaneously, the first and second therapies may be contained in the same composition (e.g., a composition comprising both a first and second therapy) or in separate compositions e.g., a first therapy in one composition and a second therapy is contained in another composition).

[0165] As used herein, the term “sequential administration” means that the first therapy and second therapy in a combination therapy are administered with a time separation of more than about 15 minutes, such as more than about any of 20, 30, 40, 50, 60, or more minutes. Either the first therapy or the second therapy may be administered first. The first and second therapies are contained in separate compositions, which may be contained in the same or different packages or kits.

[0166] As used herein, the term “concurrent administration” means that the administration of the first therapy and that of a second therapy in a combination therapy overlap with each other.

[0167] The term “pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

[0168] A “pharmaceutically acceptable carrier” refers to one or more ingredients in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, cryoprotectant, tonicity agent, preservative, and combinations thereof. Pharmaceutically acceptable carriers or excipients have preferably met the required standards of toxicological and manufacturing testing and/or are included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug administration or other state/federal government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.

[0169] The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.

[0170] An “article of manufacture” is any manufacture (e.g., a package or container) or kit comprising at least one reagent, e.g. , a medicament for treatment of a disease or condition (e.g. , cancer), or a probe for specifically detecting a biomarker described herein. In certain embodiments, the manufacture or kit is promoted, distributed, or sold as a unit for performing the methods described herein.

[0171] It is understood that embodiments of the invention described herein include “consisting” and/or “consisting essentially of’ embodiments.

[0172] Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”.

[0173] As used herein, reference to “not” a value or parameter generally means and describes "other than" a value or parameter. For example, the method is not used to treat disease of type X means the method is used to treat disease of types other than X.

[0174] The term “about X-Y” used herein has the same meaning as “about X to about Y.” [0175] As used herein and in the appended claims, the singular forms “a,” “an,” or “the” include plural referents unless the context clearly dictates otherwise.

[0176] The term “and/or” as used herein a phrase such as “A and/or B” is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term “and/or” as used herein a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

II. Methods of treatment

[0177] The present application provides methods of treating a cancer (e.g., solid tumor) in an individual in need thereof, comprising administering to the individual an effective amount of a recombinant oncolytic virus and an engineered immune cell (or a composition comprising engineered immune cells). In some embodiments, the engineered immune cell expresses a chimeric receptor that targets a heterologous protein expressed by the recombinant oncolytic virus. In some embodiments, the heterologous protein is a sialidase (e.g., DAS181 or a derivative thereof, such as a membrane-bound form of DAS181), and the chimeric receptor specifically recognizes the sialidase. In some embodiments, the sialidase is DAS181 or a derivative thereof, and wherein the chimeric receptor comprises an anti-DAS181 antibody that is not cross-reactive with human native amphiregulin or any other human antigen.

[0178] In some embodiments, there is provided a method of treating a cancer in an individual in need thereof comprising administering to the individual: (a) an effective amount of a recombinant oncolytic virus comprising a nucleotide sequence encoding a foreign antigen; and (b) an effective amount of engineered immune cells expressing a chimeric receptor specifically recognizing said foreign antigen. In some embodiments, the foreign antigen is a non-human protein (e.g., a bacterial protein). In some embodiments, the oncolytic virus is a vaccinia virus. In some embodiments, the engineered immune cell further expresses a heterologous nucleotide sequence encoding a cytokine. In some embodiments, the oncolytic virus further comprises a heterologous nucleic acid sequence encoding a multi-specific immune cell engager, such as a bispecific antibody that specifically binds FAP and CD3E. In some embodiments, the cancer is a solid cancer.

[0179] In some embodiments, there is provided a method of treating a cancer in an individual in need thereof comprising administering to the individual: (a) an effective amount of a recombinant oncolytic virus comprising a nucleotide sequence encoding a foreign antigen; and (b) an effective amount of engineered immune cells expressing a CAR specifically recognizing said foreign antigen. In some embodiments, the foreign antigen is a non-human protein (e.g., a bacterial protein). In some embodiments, the oncolytic virus is a vaccinia virus. In some embodiments, the engineered immune cell further expresses a heterologous nucleotide sequence encoding a cytokine. In some embodiments, the oncolytic virus further comprises a heterologous nucleic acid sequence encoding a multi-specific immune cell engager, such as a bispecific antibody that specifically binds FAP and CD3E. In some embodiments, the cancer is a solid cancer.

[0180] In some embodiments, there is provided a method of treating a cancer in an individual in need thereof comprising administering to the individual: (a) an effective amount of a recombinant oncolytic virus comprising a nucleotide sequence encoding a foreign antigen; and (b) an effective amount of engineered NK cells expressing a CAR specifically recognizing said foreign antigen. In some embodiments, the foreign antigen is a non-human protein (e.g., a bacterial protein). In some embodiments, the oncolytic virus is a vaccinia virus. In some embodiments, the engineered immune cell further expresses a heterologous nucleotide sequence encoding a cytokine, such as IL- 15. In some embodiments, the oncolytic virus further comprises a heterologous nucleic acid sequence encoding a multi-specific immune cell engager, such as a bispecific antibody that specifically binds FAP and CD3E. In some embodiments, the chimeric receptor is a CAR. In some embodiments, the cancer is a solid cancer.

[0181] In some embodiments, there is provided a method of treating a cancer in an individual in need thereof, comprising administering to the individual: (a) an effective amount of a recombinant oncolytic virus comprising a nucleotide sequence encoding a sialidase, or an effective amount of carrier cells comprising the recombinant oncolytic virus; and (b) an effective amount of engineered immune cells expressing a chimeric receptor specifically recognizing the sialidase. In some embodiments, the sialidase is a bacterial sialidase (e.g., Clostridium perfringens sialidase, Actinomyces viscosus sialidase, Arthrobacter ureafaciens sialidase, Salmonella typhimurium sialidase or Vibrio cholera sialidase). In some embodiments, the sialidase comprises an anchoring domain. In some embodiments, the anchoring domain is a GAG-binding protein domain, e.g., the epithelial anchoring domain of human amphiregulin. In some embodiments, the anchoring domain is positively charged at physiologic pH. In some embodiments, the anchoring domain is a GPI linker. In some embodiments, the sialidase is DAS181. In some embodiments, the sialidase comprises a transmembrane domain. In some embodiments, the chimeric receptor specifically recognizes the sialidase e.g., DAS 181) and is not cross-reactive with human native amphiregulin or any other human antigen. In some embodiments, the engineered immune cells are T cells or NK cells. In some embodiments, the chimeric receptor is a CAR. In some embodiments, the engineered immune cell further expresses a heterologous nucleotide sequence encoding a cytokine, such as IL-15. In some embodiments, the oncolytic virus further comprises a heterologous nucleic acid sequence encoding a multi-specific immune cell engager, such as a bispecific antibody that specifically binds FAP and CD3E. In some embodiments, the cancer is a solid cancer.

[0182] In some embodiments, there is provided a method of treating a cancer in an individual, comprising administering to the individual: (a) an effective amount of a recombinant oncolytic virus comprising a nucleotide sequence encoding a sialidase; and (b) an effective amount of an engineered natural killer (NK) cell comprising a chimeric antigen receptor (CAR) specifically recognizing the sialidase. In some embodiments, the sialidase is a bacterial sialidase (e.g., Clostridium perfringens sialidase, Actinomyces viscosus sialidase, Arthrobacter ureafaciens sialidase, Salmonella typhimurium sialidase or Vibrio cholera sialidase). In some embodiments, the sialidase comprises an anchoring domain. In some embodiments, the anchoring domain is a GAG-binding protein domain, e.g., the epithelial anchoring domain of human amphiregulin. In some embodiments, the anchoring domain is positively charged at physiologic pH. In some embodiments, the anchoring domain is a GPI linker. In some embodiments, the sialidase is DAS181. In some embodiments, the sialidase comprises a transmembrane domain. In some embodiments, the chimeric receptor specifically recognizes the sialidase (e.g., DAS 181) and is not cross-reactive with human native amphiregulin or any other human antigen. In some embodiments, the engineered immune cell further expresses a heterologous nucleotide sequence encoding a cytokine, such as IL- 15. In some embodiments, the oncolytic virus further comprises a heterologous nucleic acid sequence encoding a multispecific immune cell engager, such as a bispecific antibody that specifically binds FAP and CD3E. In some embodiments, the cancer is a solid cancer.

[0183] In some embodiments, there is provided a method of treating a cancer e.g., a solid cancer) in an individual, comprising administering to the individual: (a) an effective amount of a recombinant oncolytic virus comprising a first nucleotide sequence encoding a sialidase (e.g. , a DAS181 or a derivative thereof), and a second nucleotide sequence encoding a bispecific antibody that specifically binds FAP and CD3E; and (b) an effective amount of NK cells expressing a CAR and IL- 15, wherein the CAR comprises an antigen-binding domain that specifically binds to the sialidase, a transmembrane domain and an intracellular domain. In some embodiments, the oncolytic virus is a vaccinia virus comprising a disruption or deletion of a thymidine kinase (TK) gene and a vaccinia growth factor (VGF) gene. In some embodiments, the sialidase comprises from the N-terminus to the C-terminus: a sialidase domain, an Fc domain and a transmembrane domain. In some embodiments, the sialidase comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 105 or 106. In some embodiments, the bispecific antibody comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 100. In some embodiments, the CAR comprises an anti-sialidase scFv comprising an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 119. In some embodiments, the CAR comprises from the N-terminus to the C-terminus: an anti-sialidase scFv, a CD8 hinge region, a CD8 transmembrane domain, a co-stimulatory domain of CD28, and an intracellular domain of CD3^. In some embodiments, the CAR comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the IL-15 comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 121.

[0184] In some embodiments, there is provided a method of delivering a foreign antigen to cancer cells in an individual, comprising administering to the individual an effective amount of a recombinant oncolytic virus comprising a nucleotide sequence encoding a foreign antigen. In some embodiments, the foreign antigen is a bacterial protein. In some embodiments, the foreign antigen is a sialidase. In some embodiments, the foreign antigen is a bacterial sialidase (e.g., Clostridium pe/fringens sialidase, Actinomyces viscosus sialidase, Arthrobacter ureafaciens sialidase, Salmonella typhimurium sialidase or Vibrio cholera sialidase). In some embodiments, the sialidase is a sialidase catalytic domain of DAS181. In some embodiments, the method further comprises administering engineered immune cells. In some embodiments, the engineered immune cells express a chimeric receptor specifically recognizing the foreign antigen. In some embodiments, the engineered immune cells are NK cells. In some embodiments, the chimeric receptor is a CAR. In some embodiments, the engineered immune cell further expresses a heterologous nucleotide sequence encoding a cytokine, such as IL-15. In some embodiments, the oncolytic virus further comprises a heterologous nucleic acid sequence encoding a multi-specific immune cell engager, such as a bispecific antibody that specifically binds FAP and CD3E. In some embodiments, the chimeric receptor is a CAR.

[0185] In some embodiments, there is provided a method of treating a cancer in an individual in need thereof, comprising administering to the individual an effective amount of engineered immune cells, wherein the immune cells express a recombinant oncolytic virus encoding a foreign antigen. In some embodiments, the immune cells express a chimeric receptor that specifically recognizes the foreign antigen. In some embodiments, the foreign antigen is a sialidase, such as a bacterial sialidase (e.g., Clostridium perfringens sialidase, Actinomyces viscosus sialidase, Arthrobacter ureafaciens sialidase, Salmonella typhimurium sialidase or Vibrio cholera sialidase). In some embodiments, the sialidase comprises an anchoring domain. In some embodiments, the anchoring domain is a GAG-binding protein domain, e.g., the epithelial anchoring domain of human amphiregulin. In some embodiments, the anchoring domain is positively charged at physiologic pH. In some embodiments, the anchoring domain is a GPI linker. In some embodiments, the sialidase is DAS181. In some embodiments, the sialidase comprises a transmembrane domain. In some embodiments, the chimeric receptor specifically recognizes the sialidase (e. g. , DAS 181) and is not cross-reactive with human native amphiregulin or any other human antigen. In some embodiments, the engineered immune cells are NK cells. In some embodiments, the chimeric receptor is a CAR. In some embodiments, the engineered immune cell further expresses a heterologous nucleotide sequence encoding a cytokine, such as IL-15. In some embodiments, the oncolytic virus further comprises a heterologous nucleic acid sequence encoding a multi-specific immune cell engager, such as a bispecific antibody that specifically binds FAP and CD3E. In some embodiments, the chimeric receptor is a CAR. In some embodiments, the cancer is a solid cancer.

[0186] In some embodiments, there is provided a method of treating a cancer in an individual in need thereof, comprising administering to the individual an effective amount of engineered immune cells, wherein the immune cells express a recombinant oncolytic virus encoding a sialidase. In some embodiments, the immune cells express a chimeric receptor that specifically recognizes the sialidase encoded by the virus. In some embodiments, the oncolytic virus is a vaccinia virus. In some embodiments, the immune cells are NK cells. In some embodiments, the engineered immune cell further expresses a heterologous nucleotide sequence encoding a cytokine, such as IL-15. In some embodiments, the oncolytic virus further comprises a heterologous nucleic acid sequence encoding a multi-specific immune cell engager, such as a bispecific antibody that specifically binds FAP and CD3E. In some embodiments, the chimeric receptor is a CAR. In some embodiments, the cancer is a solid cancer.

[0187] The methods described herein comprise administration of a recombinant oncolytic virus. In some embodiments, the oncolytic virus is a vaccinia virus (also referred herein as “VV”). Suitable oncolytic viruses and derivatives thereof are described in the “Oncolytic Viruses” subsection below.

[0188] In some embodiments, the method comprises administering to the individual an effective amount of a recombinant oncolytic virus comprising a nucleotide sequence encoding a sialidase, wherein the nucleotide sequence encoding the heterologous protein is operably linked to a promoter. In some embodiments, the oncolytic virus is a vaccinia virus, reovirus, Seneca Valley virus (SVV), vesicular stomatitis virus (VSV), Newcastle disease virus (NDV), herpes simplex virus (HSV), morbillivirus virus, retrovirus, influenza virus, Sinbis virus, poxvirus, measles virus, cytomegalovirus (CMV), lentivirus, adenovirus, or coxsackievirus, or a derivative thereof. In some embodiments, the oncolytic virus is Talimogene Laherparepvec. In some embodiments, the oncolytic virus is a reovirus. In some embodiments, the oncolytic virus is an adenovirus (e.g., an adenovirus having an E1ACR2 deletion). [0189] In some embodiments, the oncolytic virus is a poxvirus. In some embodiments, the poxvirus is a vaccinia virus. In some embodiments, the vaccinia virus is of a strain such as Dryvax, Lister, M63, LIVP, Tian Tan, Modified Vaccinia Ankara, New York City Board of Health (NYCBOH), Dairen, Ikeda, LC16M8, Tashkent, IHD-J, Brighton, Dairen I, Connaught, Elstree, Wyeth, Copenhagen, Western Reserve, Elstree, CL, Lederle-Chorioallantoic, or AS, or a derivative thereof. In some embodiments, the virus is vaccinia virus Western Reserve.

[0190] In some embodiments, the recombinant oncolytic virus is administered via a carrier cell (e.g., an immune cell or stem cell, such as a mesenchymal stem cell). In some embodiments, the recombinant oncolytic virus is administered as a naked virus. In some embodiments, the recombinant oncolytic virus is administered via intratumoral injection.

[0191] In some embodiments, the recombinant oncolytic virus described herein comprises a nucleotide sequence encoding a foreign antigen, such as a sialidase. In some embodiments, the foreign antigen is membrane-bound. In some embodiments, the foreign antigen comprises a transmembrane domain. In some embodiments, the foreign antigen comprises an anchoring moiety. In some embodiments, the foreign antigen comprises a stabilization domain, such as an Fc domain. In some embodiments, the recombinant oncolytic virus encodes one or more additional heterologous proteins, such as additional immunotherapeutic agents (e.g., a bispecific T cell engager such as an anti-FAP anti-CD3 bispecific antibody). In some embodiments, the recombinant oncolytic virus comprises a nucleotide sequence encoding an immune checkpoint inhibitor. Exemplary foreign antigens, including sialidase constructs, and other heterologous proteins encoded by the oncolytic virus include, but are not limited to those described in the subsection B “Foreign antigen and other heterologous proteins” below.

[0192] In some embodiments, the method comprises administering a recombinant oncolytic virus comprising a nucleotide sequence encoding a sialidase, wherein the nucleotide sequence encoding the heterologous protein is operably linked to a promoter, and wherein the recombinant oncolytic virus comprises one or more mutations that reduce immunogenicity of the virus compared to a corresponding wild-type strain. In some embodiments, the virus is a vaccinia virus e.g., a vaccinia virus Western Reserve), and the one or more mutations are in one or more proteins selected from the group consisting of A27L, H3L, D8L and L1R or other immunogenic proteins (e.g., A14, A17, A13, LI, H3, D8, A33, B5, A56, F13, A28, and A27). In some embodiments, the one or more mutations are in one or more proteins selected from the group consisting of A27L, H3L, D8L and L1R. In some embodiments, the virus comprises one or more proteins selected from the group consisting of: (a) a variant vaccinia virus (VV) H3L protein that comprises an amino acid sequence having at least 90% amino acid sequence identity to any one of SEQ ID NOS: 66-69; (b) a variant vaccinia virus (VV) D8L protein that comprises an amino acid sequence having at least 90% amino acid sequence identity to any one of SEQ ID NOS: 70-72 or 85; (c) a variant vaccinia virus (VV) A27L protein that comprises an amino acid sequence having at least 90% amino acid sequence identity to SEQ ID NO: 73; and (d) a variant vaccinia virus (VV) L1R protein that comprises an amino acid sequence having at least 90% amino acid sequence identity to SEQ ID NO: 74.

[0193] The recombinant oncolytic viruses (e.g., vaccinia virus) described herein encodes a foreign antigen. In some embodiments, the foreign antigen is a sialidase. In some embodiments, the sialidase is a bacterial sialidase. In some embodiments, the sialidase is a secreted sialidase. In some embodiments, the sialidase comprises a membrane anchoring moiety or a transmembrane domain. Suitable sialidases and derivatives or variants thereof are described in the “Sialidase” subsection below. In some embodiments, the recombinant oncolytic virus encodes one or more heterologous proteins or nucleic acids that promote an immune response or inhibit an immune suppressive protein, as described in the “Multispecific immune cell engager” and “Other heterologous proteins or nucleic acids” subsection below.

[0194] In some embodiments, the method comprises administering a recombinant oncolytic virus comprising a nucleotide sequence encoding a sialidase, wherein the sialidase is operably linked to a promoter. In some embodiments, the sialidase is a Neu5Ac alpha(2,6)-Gal sialidase or a Neu5Ac alpha(2,3)-Gal sialidase. In some embodiments, the sialidase is a bacterial sialidase (e.g., a Clostridium pe /fringe ns sialidase, Actinomyces viscosus sialidase, and Arthrobacter ureafaciens sialidase, Salmonella typhimurium sialidase or Vibrio cholera sialidase) or a derivative thereof.

[0195] In some embodiments, the sialidase comprises all or a portion of the amino acid sequence of a large bacterial sialidase or can comprise amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to all or a portion of the amino acid sequence of a large bacterial sialidase. In some embodiments, the sialidase domain comprises SEQ ID NO: 2 or 27, or a sialidase sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 12. In some embodiments, a sialidase domain comprises the catalytic domain of the Actinomyces viscosus sialidase extending from amino acids 274-666 of SEQ ID NO: 26, having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to amino acids 274-666 of SEQ ID NO: 26.

[0196] In some embodiments, the sialidase is a naturally occurring sialidase. In some embodiments, the sialidase is a fusion protein comprising a sialidase catalytic domain. [0197] In some embodiments, the sialidase comprises an anchoring moiety. In some embodiments, the sialidase is a fusion protein comprising a sialidase catalytic domain fused to an anchoring domain. In some embodiments, the anchoring domain is positively charged at physiologic pH. In some embodiments, the anchoring domain is a glycosaminoglycan (GAG)- binding domain.

[0198] In some embodiments, the sialidase comprises an amino acid sequence having at least about 80% (e.g. , at least about 85%, 90%, or 95%) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-33 or 53-54. In some embodiments, the sialidase comprises an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, or 95%) sequence identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the sialidase is DAS181.

[0199] In some embodiments, the sialidase comprises a transmembrane domain. In some embodiments, the anchoring domain or the transmembrane domain is located at the carboxy terminus of the sialidase.

[0200] The nucleotide sequence encoding the foreign antigen (e.g., sialidase protein) and/or heterologous protein is operably linked to a promoter. In some embodiments, the promoter is a viral promoter, such as an early, late, or early/late viral promoter. In some embodiments, the promoter is a hybrid promoter. In some embodiments, the promoter is comprises a promoter sequence of a human promoter (e.g., a tissue- or tumor- specific promoter). Suitable promoters are described in the “Promoters for expression of heterologous proteins or nucleic acids” subsection below.

[0201] The methods described herein comprise administering an effective amount of engineered immune cells expressing a chimeric receptor, such as any one of the engineered immune cells described in the “Engineered immune cells” section below. In non-limiting examples, the engineered immune cells can be T-cells (e.g., oc[3T cells or y8T cells), natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells (DC), cytokine-induced killer (CIK) cells, cytokine-induced natural killer (CINK) cells, lymphokine-activated killer (LAK) cells, tumor-infiltrating lymphocytes (TILs), macrophages, peripheral blood mononuclear cells (PBMCs) or combinations thereof. In some embodiments, the cell therapy comprises PBMC cells that have been stimulated with various cytokine and antibody combinations to activate effector T cells (CD3, CD38 and IL- 2) or, in some cases, T cells and NK cells (CD3, CD28, IL-15 and IL-21). In some embodiments, the engineered immune cells are CAR-T, CAR-NK, CAR-macrophage or CAR-NKT cells. [0202] In some embodiments, the engineered immune cells express a chimeric receptor that recognizes a foreign antigen expressed by tumor cells, such as a heterologous protein delivered to the tumor cells via any one of the recombinant oncolytic viruses provided herein. In some embodiments, the foreign antigen delivered by the recombinant oncolytic virus is a bacterial peptide or a bacterial sialidase, e.g., DAS181 (SEQ ID NO: 2). In some embodiments, the foreign antigen is a sialidase comprising a transmembrane domain. In some embodiments, the foreign antigen is DAS 181 without an AR tag and fused to a C-terminal transmembrane domain (e.g., SEQ ID NO: 31).

[0203] In some embodiments, the method further comprises administering to the individual an effective amount of an additional immunotherapy. In some embodiments, the recombinant oncolytic virus is administered before, after, or simultaneously with the additional immunotherapy. In some embodiments, the additional immunotherapy is a multi-specific immune cell engager e.g., a bispecific molecule such as a BiTE), a cell therapy, a cancer vaccine (e.g., a dendritic cell (DC) cancer vaccine), a cytokine (e.g., IL- 15, IL- 12, modified IL-2 having no or reduced binding to the alpha receptor, modified IL- 18 with no or reduced binding to IL-18 BP, CXCL10, or CCL4), an immune checkpoint inhibitor (e.g., an inhibitor of CTLA-4, PD-1, PD-L1, B7-H4, TIGIT, LAG3, TIM3 or HLA), a master switch anti-LILRB, and bispecific anti-LILRB-4-lBB, Anti-FAP-CD3, a PI3Kgamma inhibitor, a TLR9 ligand, an HD AC inhibitor, a LILRB2 inhibitor, a MARCO inhibitor, etc. Suitable immune cell engagers and immune checkpoint inhibitors are described in the “Other heterologous proteins or nucleic acids” subsection below.

[0204] In some embodiments, administering the recombinant oncolytic virus increases tumor cell killing by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% compared to the engineered immune cell and/or additional immunotherapy alone.

[0205] One aspect of the present application provides methods of reducing sialylation of cancer cells in an individual, comprising administering to the individual an effective amount of any one of the recombinant oncolytic viruses and the engineered immune cells described herein. In some embodiments, the sialidase reduces surface sialic acid on tumor cells. In some embodiments the sialidase reduces surface sialic acid on tumor cells by at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, or 90%. In some embodiments, the sialidase cleaves both a2,3 and a2,6 sialic acids from the cell surface of tumor cells. In some embodiments, the sialidase increases cleavage of both a2,3 and a2,6 sialic acids by at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, or 90%. [0206] In some embodiments, administration of the recombinant oncolytic virus encoding a sialidase increases killing of tumor cells in an individual by the engineered immune cells. In some embodiments, administration of the recombinant oncolytic virus encoding a sialidase increases killing of tumor cells by NK cells. In some embodiments, administration of the recombinant oncolytic virus encoding a sialidase increases killing by NK cells by at least at least 5%, 10%, 15%, 20%, 30%, 40%, or 50%. In some embodiments, the recombinant oncolytic virus encoding a sialidase increases killing by NK cells by at least at least 5%, 10%, 15%, 20%, 30%, 40%, or 50% compared to recombinant oncolytic virus lacking sialidase. In some embodiments, administration of the recombinant oncolytic virus encoding a sialidase increases killing of tumor cells by T cells. In some embodiments, the recombinant oncolytic virus encoding a sialidase increases killing of tumor cells by T cells by at least at least 5%, 10%, 15%, 20%, 30%, 40%, or 50% . In some embodiments, administration of the recombinant oncolytic virus encoding a sialidase increases killing of tumor cells by T cells by at least at least 5%, 10%, 15%, 20%, 30%, 40%, or 50% compared to recombinant oncolytic virus lacking sialidase. In some embodiments, administration of the recombinant oncolytic virus encoding a sialidase increases killing of tumor cells by PBMCs. In some embodiments, the recombinant oncolytic virus encoding a sialidase increases killing of tumor cells by PBMCs by at least at least 5%, 10%, 15%, 20%, 30%, 40%, or 50% compared to recombinant oncolytic virus lacking sialidase.

[0207] In some embodiments, administration of the recombinant oncolytic virus encoding a sialidase enhances cytokine production in an individual. In some embodiments, administration of the recombinant oncolytic virus encoding a sialidase enhances cytokine production by T- lymphocytes. In some embodiments, administration of the recombinant oncolytic virus encoding a sialidase enhances T-lymphocyte mediated cytokine production locally in a tumor microenvironment of the individual. In some embodiments, the cytokines include IL2 and IFN- gamma. In some embodiments, administering recombinant oncolytic virus encoding a sialidase increases cytokine production by at least at least 5%, 10%, 20%, 30%, 40%, or 50% compared to administering an oncolytic virus lacking sialidase. In some embodiments, administering recombinant oncolytic virus encoding a sialidase increases IL2 production by at least 2.5-fold, at least 3-fold, or at least 4- fold compared to administering an oncolytic virus lacking sialidase. In some embodiments, administering recombinant oncolytic virus encoding a sialidase increases IFN-gamma production by at least 5%, 10%, 20%, 30%, 40%, or 50% compared to administering an oncolytic virus lacking sialidase. [0208] The methods described herein are suitable for treating a variety of cancers. As used herein, cancer is a term for diseases caused by or characterized by any type of malignant tumor or hematological malignancy, including metastatic cancers, solid tumors, lymphatic tumors, and blood cancers.

[0209] Cancers include leukemias, lymphomas (Hodgkins and non-Hodgkins), sarcomas, melanomas, adenomas, carcinomas of solid tissue including breast cancer and pancreatic cancer, hypoxic tumors, squamous cell carcinomas of the mouth, throat, larynx, and lung, genitourinary cancers such as cervical and bladder cancer, hematopoietic cancers, head and neck cancers, and nervous system cancers, such as gliomas, astrocytomas, meningiomas, etc., benign lesions such as papillomas, and the like.

[0210] Many cancer cells are hypersialylated. The recombinant oncolytic viruses described herein are capable of delivering sialidase to tumor cells and the tumor microenvironment. Within the tumor microenvironment the sialidase can remove terminal sialic acid residues on cancer cells, thereby reducing the barrier for entry of immune cells or immunotherapy reagents and promote cellular immunity against cancer cells. For instance, a group of receptors called Siglect (Sialic acid-binding immunoglobulin like lectins) on immune cells will bind its inhibitory receptor ligands, which are sialylated glycoconjugates on tumor cells. In some embodiments, the removal of sialic acid prevents binding of such ligands to Siglect on immune cells and thus abolishes the suppression of immunity against tumor cells.

[0211] In some embodiments, delivery of a sialidase by the recombinant oncolytic virus can reduce sialic acid present on tumor cells and render the tumor cells more vulnerable to killing by immune cells, immune cell-based therapies and other therapeutic agents whose effectiveness is diminished by hyper sialylation of cancer cells.

[0212] In some embodiments, the cancer comprises a solid tumor. In some embodiments of any of the methods provided herein, the cancer is an adenocarcinoma, a metastatic cancer and/or is a refractory cancer. In certain embodiments of any of the foregoing methods, the cancer is a breast, colon or colorectal, lung, ovarian, pancreatic, prostate, cervical, endometrial, head and neck, liver, renal, skin, stomach, testicular, thyroid or urothelial cancer. In certain embodiments of any of the foregoing methods, the cancer is an epithelial cancer, e.g., an endometrial cancer, ovarian cancer, cervical cancer, vulvar cancer, uterine cancer, fallopian tube cancer, breast cancer, prostate cancer, lung cancer, pancreatic cancer, urinary cancer, bladder cancer, head and neck cancer, oral cancer or liver cancer. In some embodiments, the cancer is selected from human alveolar basal epithelial adenocarcinoma, human mamillary epithelial adenocarcinoma, and glioblastoma. In some embodiments, the cancer is an FAP positive cancer (i.e., a cancer that expresses FAP) and/or a cancer associated with FAP positive stromal cells (e.g., tumor-associated fibroblasts). In some embodiments, the cancer is selected from the group consisting of lung cancer, colon cancer, and breast cancer.

[0213] In some embodiments, the engineered immune cells and the recombinant oncolytic virus are administered separately (e.g., as monotherapy) or together simultaneously (e.g., in the same or separate formulations) as combination therapy. In some embodiments, the recombinant oncolytic virus is administered prior to administration of the engineered immune cells. In nonlimiting examples, the recombinant oncolytic virus can be administered 1 or more, 2 or more, 4 or more, 6 or more, 8 or more, 10 or more, 12 or more, 24 or more, or 48 or more hours prior to the engineered immune cells comprising the chimeric receptor. In some embodiments, a population of carrier cells e.g., engineered immune cells) expressing the recombinant oncolytic virus is administered prior to a population of engineered immune cells expressing a chimeric antigen receptor targeting a heterologous protein expressed by the recombinant oncolytic virus. In non-limiting examples, the carrier cells (e.g., engineered immune cells) comprising the recombinant oncolytic virus can be administered 1 or more, 2 or more, 4 or more, 6 or more, 8 or more, 10 or more, 12 or more, 24 or more, or 48 or more hours prior to the engineered immune cells comprising the chimeric receptor targeting a heterologous protein expressed by the recombinant oncolytic virus. In some embodiments, the time period between administration of the recombinant oncolytic virus (e.g., in a pharmaceutical composition or a carrier cell comprising the recombinant oncolytic virus) and administration of the engineered immune cells expressing the chimeric receptor is sufficient to allow the virus to express the heterologous protein or nucleic acid in the tumor cells.

[0214] The recombinant oncolytic virus and the engineered immune cells comprising the chimeric receptor and in some embodiments, additional immunotherapeutic agent(s) may be administered using any suitable routes of administration and suitable dosages. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary artisan. Animal experiments provide reliable guidance for the determination of effective doses for human therapy. Interspecies scaling of effective doses can be performed following the principles laid down by Mordenti, J. and Chappell, W. “The Use of Interspecies Scaling in Toxicokinetics,” In Toxicokinetics and New Drug Development, Yacobi et al., Eds, Pergamon Press, New York 1989, pp. 42-46.

[0215] In some embodiments, the recombinant oncolytic virus, the engineered immune cells comprising the chimeric receptor and in some embodiments, additional immunotherapeutic agent(s) are administered sequentially (e.g., the recombinant oncolytic virus can be administered prior to the engineered immune cells, and/or prior to other therapeutic agents such as bi-specific antibody of FAP/CD3, bi-specific or trispecific antibody of LILRB-4-1BB, PD-1 antibody, etc. as described above). In some embodiments, the recombinant oncolytic virus, the engineered immune cells and/or additional immunotherapeutic agent(s) are administered simultaneously or concurrently. In some embodiments, the recombinant oncolytic virus, the engineered immune cells and/or additional immunotherapeutic agent(s) are administered in a single formulation. In some embodiments, the recombinant oncolytic virus, the engineered immune cells and/or additional immunotherapeutic agent(s) are administered as separate formulations.

[0216] The methods of the present invention may be combined with conventional chemotherapeutic, radiologic and/or surgical methods of cancer treatment.

A. Oncolytic Viruses

[0217] The present application provides recombinant oncolytic viruses for use in treating a cancer, comprising at least one nucleotide sequence encoding a heterologous protein. In some embodiments, the heterologous protein is operably linked to a promoter. In some embodiments, the heterologous protein is a foreign antigen. In some embodiments, the heterologous protein is a sialidase such as bacterial sialidase.

[0218] In some embodiments, the present application provides a recombinant oncolytic virus comprising a nucleotide sequence encoding a sialidase. In some embodiments, the nucleotide sequence encoding the sialidase is operably linked to a promoter. In some embodiments, the recombinant oncolytic virus further comprises a second nucleotide sequence encoding a heterologous protein or nucleic acid.

[0219] In some embodiments, the present application provides a recombinant oncolytic virus comprising a first nucleotide sequence encoding a sialidase and a second nucleotide sequence encoding a heterologous protein or nucleic acid, wherein the first nucleotide sequence is operably linked to a promoter and the second nucleotide sequence is operably linked to a promoter. In some embodiments, the first nucleotide sequence and the second nucleotide sequence are operably linked to the same promoter. In some embodiments, the first nucleotide sequence and the second nucleotide sequence are operably linked to different promoters. In some embodiments, the recombinant oncolytic virus comprises two or more nucleotide sequences, wherein each nucleotide sequence encodes a heterologous protein or nucleic acid. In some embodiments, the second nucleotide sequence encodes a heterologous protein selected from the group consisting of immune checkpoint inhibitors, inhibitors of immune suppressive receptors, multi-specific immune cell engager (e.g., a BiTE), cytokines, costimulatory molecules, tumor antigen presenting proteins, anti-angiogenic factors, tumor-associated antigens, foreign antigens, and matrix metalloproteases (MMP), Regulatory molecules of Macrophage or monocyte functions (antibodies to LILRBs), antibodies to folate receptor beta, tumor cell specific antigens (CD19, CDH17, etc.) or antibodies to tumor scaffold (FAP, fibulin-3, etc.).

[0220] In some embodiments, there is provided a recombinant vaccinia virus comprising a first nucleotide sequence encoding a sialidase, wherein the first nucleotide sequence is operably linked to a promoter. In some embodiments, the vaccinia virus further comprises a second nucleotide encoding a heterologous protein, e.g., an immune checkpoint inhibitor, an inhibitor of an immune suppressive receptor, a cytokine, a costimulatory molecule, a tumor antigen presenting protein, an anti-angiogenic factor, a tumor-associated antigen, a foreign antigen, or a matrix metalloprotease (MMP), Regulatory molecules of Macrophage or monocyte functions (antibodies to LILRBs), antibodies to folate receptor beta, tumor cell specific antigens (CD19, CDH17, etc.) or antibodies to tumor scaffold (FAP, fibulin-3, etc.) wherein the second nucleotide sequence is operably linked to the same or a different promoter. In some embodiments, the virus is vaccinia virus Western Reserve. In some embodiments, the virus is a vaccinia virus, and the one or more mutations are in one or more proteins selected from the group consisting of A14, A17, A13, LI, H3, D8, A33, B5, A56, F13, A28, and A27. In some embodiments, the one or more mutations are in one or more proteins selected from the group consisting of A27L, H3L, D8L and L1R.

[0221] In some embodiments, there is provided a recombinant vaccinia virus comprising a first nucleotide sequence encoding a sialidase, wherein the first nucleotide sequence is operably linked to a promoter. In some embodiments, the vaccinia virus further comprises a second nucleotide encoding a heterologous protein, wherein the heterologous protein is a membranebound complement activation modulator such as CD55, CD59, CD46, CD35, factor H, C4- binding protein, or other identified complement activation modulators, and wherein the second nucleotide sequence is operably linked to the same or a different promoter. In some embodiments, the virus is vaccinia virus Western Reserve. In some embodiments, the virus is a vaccinia virus, and the one or more mutations are in one or more proteins selected from the group consisting of A14, A17, A13, LI, H3, D8, A33, B5, A56, F13, A28, and A27. In some embodiments, the one or more mutations are in one or more proteins selected from the group consisting of A27L, H3L, D8L and L1R. [0222] In some embodiments, there is provided a recombinant oncolytic viruses (e.g., vaccinia virus) comprising a first nucleotide sequence encoding a Actinomyces viscosus sialidase or a derivative thereof, wherein the first nucleotide sequence is operably linked to a promoter. In some embodiments, the oncolytic virus further comprises a second nucleotide sequence encoding a heterologous protein (e.g., an immune checkpoint inhibitor, an inhibitor of an immune suppressive receptor, a cytokine, a costimulatory molecule, a tumor antigen presenting protein, an anti-angiogenic factor, a tumor-associated antigen, a foreign antigen, or a matrix metalloprotease (MMP)), wherein the second nucleotide sequence is operably linked to the same or a different promoter. In some embodiments, the recombinant oncolytic virus is an enveloped virus e.g., vaccinia virus) and the heterologous protein is a membrane-bound complement activation modulator such as CD55, CD59, CD46, CD35, factor H, C4-binding protein, or other identified complement activation modulators. In some embodiments, the sialidase comprises an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 1 or 26.

[0223] In some embodiments, there is provided a recombinant oncolytic viruses (e.g., vaccinia virus) encoding a sialidase comprising an anchoring domain (e.g., DAS181). In some embodiments, the oncolytic virus further comprises a second nucleotide sequence encoding a heterologous protein or nucleic acid. In some embodiments, the anchoring domain is a glycosaminoglycan (GAG)-binding domain. In some embodiments, the anchoring domain is positively charged at physiologic pH. In some embodiments, the anchoring domain is located at the carboxy terminus of the sialidase. In some embodiments, the sialidase is derived from an Actinomyces viscosus sialidase. In some embodiments, the sialidase is DAS 181. In some embodiments, the nucleotide sequence encoding the sialidase further encodes a secretion sequence operably linked to the sialidase. In some embodiments, the secretion sequence is operably linked to the amino terminus of the sialidase.

[0224] In some embodiments, there is provided a recombinant oncolytic viruses (e.g., vaccinia virus) encoding a sialidase comprising a transmembrane domain. In some embodiments, the transmembrane domain comprises an amino acid sequence selected from SEQ ID NOs: 45-52. In some embodiments, the oncolytic virus further comprises a second nucleotide sequence encoding a heterologous protein or nucleic acid. In some embodiments, the sialidase is derived from an Actinomyces viscosus sialidase. In some embodiments, the nucleotide sequence encoding the sialidase further encodes a secretion sequence operably linked to the sialidase. [0225] Numerous oncolytic viruses, including Vaccinia virus, Coxsackie virus, Adenovirus, Measles, Newcastle disease virus, Seneca Valley virus, Coxsackie A21, Vesicular stomatitis virus, Parvovirus Hl, Reovirus, Herpes virus, Lentivirus, and Poliovirus, and Parvovirus. Vaccinia Virus Western Reserve, GLV-lh68, ACAM2000, and OncoVEX GFP, are available. The genomes of these oncolytic virus can be genetically modified to insert a nucleotide sequence encoding a protein that includes all or a catalytic portion of a sialidase. The nucleotide sequence encoding a protein that includes all or a catalytically active portion of a sialidase is placed under the control of a viral expression cassette so that the sialidase is expressed by infected cells.

[0226] Oncolytic viruses (OVs) have the ability to preferentially accumulate in and replicate in and kill tumor cells, relative to normal cells. This ability can be a native feature of the virus (e.g., pox virus, reovirus, Newcastle disease virus and mumps virus), or the viruses can be modified or selected for this property. Viruses can be genetically attenuated or modified so that they can circumvent antiviral immune and other defenses in the subject (e.g., vesicular stomatitis virus, herpes simplex virus, adenovirus) so that they preferentially accumulate in tumor cells or the tumor microenvironment, and/or the preference for tumor cells can be selected for or engineered into the virus using, for example, tumor-specific cell surface molecules, transcription factors and tissue-specific microRNAs (see, e.g., Cattaneo el al, Nat. Rev. Microbiol., 6(7):529-540 (2008); Dorer et al, Adv. Drug Deliv. Rev., 61(7- 8):554-571 (2009); Kelly et al., Mol. Ther., 17(3):409-416 (2009); andNaiket al., Expert Opin. Biol. Ther., 9(9): 1163-1176 (2009)).

[0227] Other unmodified oncolytic viruses include any known to those of skill in the art, including those selected from among viruses designated GLV-lh68, JX-594, JX-954, Colo Adi, MV-CEA, MV-NIS, ONYX-015, B18R, H101, OncoVEX GM-CSF, Reolysin, NTX-010, CCTG-102, Cavatak, Oncorine, and TNFerade.

[0228] Suitable oncolytic viruses have been described, for example, in W02020097269, which is incorporated herein by reference in its entirety. Oncolytic viruses described herein include for example, vesicular stomatitis virus, see, e.g., U.S. Patent Nos. 7,731,974, 7,153,510, 6,653,103 and U.S. Pat. Pub. Nos. 2010/0178684, 2010/0172877, 2010/0113567, 2007/0098743, 20050260601, 20050220818 and EP Pat. Nos. 1385466, 1606411 and 1520175; herpes simplex virus, see, e.g., U.S. Patent Nos. 7,897,146, 7731,952, 7,550,296, 7,537,924, 6,723,316, 6,428,968 and U.S. Pat. Pub. Nos. 2011/0177032, 2011/0158948, 2010/0092515, 2009/0274728, 2009/0285860, 2009/0215147, 2009/0010889, 2007/0110720, 2006/0039894 and 20040009604; retroviruses, see, e.g., U.S. Patent Nos. 6,689,871, 6,635,472, 6,639,139, 5,851,529, 5,716,826, 5,716,613 and U.S. Pat. Pub. No. 20110212530; and adeno-associated viruses, see, e.g. , U.S. Patent Nos. 8,007,780, 7,968,340, 7,943,374, 7,906,111, 7,927,585, 7,811,814, 7,662,627, 7,241,447, 7,238,526, 7,172,893, 7,033,826, 7,001,765, 6,897,045, and 6,632,670.

[0229] In some embodiments, the oncolytic virus is a vesicular stomatitis virus (VSV). VSV has been used in multiple oncolytic virus applications. In addition, VSV has been engineered to express an antigenic protein of human papilloma virus (HPV) as a method to treat HPV positive cervical cancers via vaccination (REF 18337377, 29998190) and to express pro- inflammatory factors to increase the immune reaction to tumors (REF 12885903). Various methods for engineering VSV to encode an additional gene have been described (REF 7753828). Briefly, the VSV RNA genome is reverse transcribed to a complementary, doubled stranded-DNA with an upstream T7 RNA polymerase promoter and an appropriate location within the VSV genome for gene insertion is identified e.g., within the noncoding 5’ or 3’ regions flanking VSV glycoprotein (G) (REF 12885903). Restriction enzyme digestion can be accomplished, e.g., with Mlu I and Nhe I, yielding a linearized DNA molecule. An insert consisting of a DNA molecule encoding the gene of interest flanked by appropriate restriction sites can be ligated into the linearized VSV genomic DNA. The resulting DNA can be transcribed with T7 polymerase, yielding a complete VSV genomic RNA containing the inserted gene of interest. Introduction of this RNA molecule to a mammalian cell, e.g., via transfection and incubation results in viral progeny expressing the protein encoded by the gene of interest.

[0230] In some embodiments, the recombinant oncolytic virus is an adenovirus. In some embodiments, the adenovirus is an adenovirus serotype 5 virus (Ad5). Ad5 contains a human E2F-1 promoter, which is a retinoblastoma (Rb) pathway-defective tumor specific transcription regulatory element that drives expression of the essential Ela viral genes, restricting viral replication and cytotoxicity to Rb pathway-defective tumor cells (REF 16397056). A hallmark of tumor cells is Rb pathway defects. Engineering a gene of interest into Ad5 is accomplished through ligation into Ad5 genome. A plasmid containing the gene of interest is generated via and digested, e.g., with AsiSI and Pack An Ad5 DNA plasmid, e.g., PSF-AD5 (REF Sigma OGS268) is digested with AsiSI and Pad and ligated with recombinant bacterial ligase or co-transformed with RE digested gene of interest into permissive E.coli as has been reported for the generation of human granulocyte macrophage colony stimulating factor (GM-CSF) expressing Ad5 (REF 16397056). Recovery of the DNA and transfection into a permissive host, e.g., human embryonic kidney cells (HEK293) or HeLa yields virus encoding the gene of interest.

[0231] In some embodiments, the recombinant oncolytic virus is a modified oncolytic virus (e.g., a derivative of any one of the viruses described herein). In some embodiments, the recombinant oncolytic virus comprises one or more mutations that reduce immunogenicity of the virus compared to a corresponding wild-type strain.

[0232] Delivery of oncolytic viruses can be achieved via direct intratumoral injection. While direct intratumoral delivery can minimize the exposure of normal cells to the virus, there often are limitations due to, e.g., inaccessibility of the tumor site e.g., brain tumors) or for tumors that are in the form of several small nodules spread out over a large area or for metastatic disease. Viruses can be delivered via systemic or local delivery, such as by intravenous administration, or intraperitoneal administration, and other such routes. Systemic delivery can deliver virus not only to the primary tumor site, but also to disseminated metastases.

Vaccinia virus (W)

[0233] In some embodiments, the recombinant oncolytic virus is a vaccinia virus (VV). Various strains of VV have been used as templates for OV therapeutics; the unifying feature is deletion of the viral thymidine kinase (TK) gene, rendering a virus dependent upon actively replicating cells, i.e. neoplastic cells, for productive replication and thus these VVs have preferential infectivity of cancer cells exemplified by the Western Reserve (WR) strain of VV (REF 25876464). Production of VV’s with a gene of interest inserted in the genome may be accomplished with homologous recombination utilizing lox sites.

[0234] In some embodiments, the virus is a modified vaccinia virus. In some embodiments, the virus is a modified vaccinia virus comprising one or more mutations. In some embodiments, the one or more mutations are in one or more proteins such as Al 4, A17, A13, LI, H3, D8, A33, B5, A56, F13, A28, and A27. In some embodiments, the one or more mutations are in one or more proteins selected from the group consisting of A27L, H3L, D8L and L1R. Exemplary mutations have been described, for example, in international patent publication W02020086423, which is incorporated herein by reference in its entirety.

[0235] A limiting factor in the use of VVs as cancer treatment delivery vectors is the strong neutralizing antibody (Nab) response induced by the injection of VV into the bloodstream that limits the ability of the virus to persist and spread and prevents vector re-dosing. The NAbs recognize and bind viral glycoproteins embedded in the VV envelope, thus preventing virus interaction with host cell receptors. A number of VV glycoproteins involved in host cell receptor recognition have been identified. Among them, proteins H3L, L1R, A27L, D8L, A33R, and B5R have been shown to be targeted by NAbs, with A27L, H3L, D8L and L1R being the main NAb antigens presented on the surface of mature viral particles. A27L, H3L, and D8L are the adhesion molecules that bind to host glycosaminoglycans (GAGs) heparan sulfate (HS) (A27L and H3L) and chondroitin sulfate (CS) (D8L) and mediate endocytosis of the virus into the host cell. L1R protein is involved in virus maturation. Modified vaccinia viruses comprising mutations in one or more of these proteins have been described in international patent publication W02020086423, which is herein incorporated by reference in its entirety.

[0236] In some embodiments, the modified vaccinia virus comprises one or more proteins selected from the group consisting of: (a) a variant vaccinia virus (VV) H3L protein that comprises an amino acid sequence having at least 90% (e.g., at least 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) amino acid sequence identity to any one of SEQ ID NOS: 66-69; (b) a variant vaccinia virus (VV) D8L protein that comprises an amino acid sequence having at least 90% (e.g., at least 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) amino acid sequence identity to any one of SEQ ID NOS: 70-72 or 85; (c) a variant vaccinia virus (VV) A27L protein that comprises an amino acid sequence having at least 90% (e.g., at least 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) amino acid sequence identity to SEQ ID NO: 73; and (d) a variant vaccinia virus (VV) L1R protein that comprises an amino acid sequence having at least 90% (e.g., at least 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) amino acid sequence identity to SEQ ID NO: 74.

[0237] In some embodiments, the variant VV H3L protein comprises amino acid substitution or deletion at one or more of the following amino acid residues: 14, 15, 16, 33, 34, 35, 38, 40, 44, 45, 52, 131, 134, 135, 136, 137, 154, 155, 156, 161, 166, 167, 168, 198, 227, 250, 253, 254, 255, and 256, wherein the amino acid numbering is based on SEQ ID NO: 66. In some embodiments, the variant VV H3L comprises one or more amino acid mutations selected from the group consisting of I14A, D15A, R16A, K38A, P44A, E45A, V52A, E131A, T134A, L136A, R137A, R154A, E155A, I156A, M168A, I198A, E250A, K253A, P254A, N255A, and F256A, wherein the amino acid numbering is based on SEQ ID NO: 66.

[0238] In some embodiments, the variant VV D8L protein comprises amino acid substitution or deletion at one or more of the following amino acid residues: 44, 48, 98, 108, 117, and 220, wherein the amino acid numbering is based on SEQ ID NO: 70. In some embodiments, the variant VV D8L construct comprises one or more amino acid mutations selected from the group consisting of R44A, K48A, K98A, K108A, K117A, and R220A, wherein the amino acid numbering is based on SEQ ID NO: 70.

[0239] In some embodiments, the variant VV A27L protein comprises amino acid substitution or deletion at one or more of the following amino acid residues: 27, 30, 32, 33, 34, 35, 36, 37, 39, 40, 107, 108, and 109, wherein the amino acid numbering is based on SEQ ID NO: 73. In some embodiments, the variant A27L construct comprises one or more amino acid mutations selected from the group consisting of K27A, A30D, R32A, E33A, A34D, I35A, V36A, K37A, D39A, E40A, R107A, P108A, and Y109A, wherein the amino acid numbering is based on SEQ ID NO: 73.

[0240] In some embodiments, the variant VV L1R protein comprises amino acid substitution or deletion at one or more of the following amino acid residues: 25, 27, 31, 32, 33, 35, 58, 60, 62, 125, and 127, wherein the amino acid numbering is based on SEQ ID NO: 74. In some embodiments, the variant L1R construct comprises one or more amino acid mutations selected from the group consisting of E25A, N27A, Q31A, T32A, K33A, D35A, S58A, D60A, D62A, K125A, and K127A, wherein the amino acid numbering is based on SEQ ID NO: 74.

[0241] In some embodiments, the variant VV H3L protein comprises amino acid substitution or deletion at one or more of the following amino acid residues: 14, 15, 16, 33, 34, 35, 38, 40, 44, 45, 52, 131, 132, 134, 135, 136, 137, 154, 155, 156, 161, 166, 167, 168, 195, 198, 199, 227, 250, 251, 252, 253, 254, 255, 256, 258, 262, 264, 266, 268, 272, 273, 275, and 277, wherein the amino acid numbering is based on SEQ ID NO: 68. In some embodiments, the variant H3L construct comprises one or more amino acid mutations selected from the group consisting of I14A, D15A, R16A, K33A, F34A, D35A, K38A, N40A, P44A, E45A, V52A, E131A, D132A, T134A, F135A, L136A, R137A, R154A, E155A, I156A, K161A, L166A, VI 67 A, M168A, E195A, I198A, V199A, R227A, E250A, N251A, M252A, K253A, P254A, N255A, F256A, S258A, T262P, A264T, K266I, Y268C, M272K, Y273N, F275N, and T277A, wherein the amino acid numbering is based on SEQ ID NO: 68.

[0242] In some embodiments, the variant VV D8L protein comprises amino acid substitution or deletion at one or more of the following amino acid residues: 43, 44, 48, 53, 54, 55, 98, 108, 109, 144, 168, 177, 196, 199, 203, 207, 212, 218, 220, 222, and 227, wherein the amino acid numbering is based on SEQ ID NO: 72. In some embodiments, the variant VV D8L construct comprises one or more amino acid mutations selected from the group consisting of V43A, R44A, K48A, S53A, G54A, G55A, K98A, K108A, K109A, A144G, T168A, S177A, L196A, F199A, L203A, N207A, P212A, N218A, R220A, P222A, and D227A, wherein the amino acid numbering is based on SEQ ID NO: 72. [0243] In some embodiments, there is provided a composition comprising a recombinant vaccinia virus of a Western Reserve strain comprising a nucleic acid sequence having at least any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% identity to the nucleic acid sequence of SEQ ID NO: 108. In some embodiments, the first nucleotide sequence and the second nucleotide sequence are operably linked to one or more promoters. In some embodiments, the recombinant vaccinia virus comprises a disruption or deletion of a thymidine kinase (TK) gene and a disruption or deletion of a vaccinia growth factor (VGF) gene. In some embodiments, the nucleic acid sequence having at least any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% identity to the nucleic acid sequence of SEQ ID NO: 108 is integrated into the TK gene of the vaccinia virus.

B. Foreign antigen and other heterologous proteins

[0244] The oncolytic viruses described herein comprises a nucleotide sequence encoding a foreign antigen. In some embodiments, the foreign antigen is a sialidase, such as bacterial sialidase. In some embodiments, the oncolytic virus further comprises one or more nucleotide sequences encoding heterologous protein(s) or nucleic acid(s). In some embodiments, the oncolytic virus further comprises a nucleotide sequence encoding a multispecific immune cell engager as described in the section “Multispecific immune cell engager” below, e.g., a bispecific antibody that specifically binds FAP and CD3. In some embodiments, the oncolytic virus further comprises a nucleotide sequence encoding a heterologous protein or nucleic acid as described in the section “Other heterologous proteins or nucleotide sequences” below, e.g., an immune checkpoint inhibitor.

[0245] In some embodiments, the foreign antigen is a non-human protein. In some embodiments, the foreign antigen is a viral polypeptide, including a viral protein or a peptide fragment thereof. In some embodiments, the foreign antigen is a bacterial polypeptide, including a bacterial protein or a peptide fragment thereof. In some embodiments, the foreign antigen is a naturally occurring protein or an antigenic fragment (i.e., a peptide fragment that can be specifically recognized by the engineered immune cells comprising a chimeric receptor) thereof. In some embodiments, the foreign antigen is a synthetic polypeptide. In some embodiments, the foreign antigen is a fusion protein comprising an antigenic peptide fused to one or more polypeptide sequences that contribute to the antigenic activity (i.e., the ability to be specifically recognized by the engineered immune cells comprising a chimeric receptor) of the antigenic peptide. For example, the foreign antigen can include an anchoring domain that promotes interaction between the foreign antigen and cell surface of tumor cells. For example, in a sialidase construct, the anchoring domain and sialidase domain can be arranged in any appropriate way that allows the protein to bind at or near a target cell membrane such that the therapeutic sialidase can exhibit an extracellular activity that removes sialic acid residues. The foreign antigen can have more than one anchoring domains. In cases in which the foreign antigen has more than one anchoring domain, the anchoring domains can be the same or different. The foreign antigen can comprise one or more transmembrane domains (e.g., one or more transmembrane alpha helices). The foreign antigen can have more than one antigen peptides, such as more than one sialidase domain. In cases in which a foreign antigen has more than one sialidase domain, the sialidase domains can be the same or different. Where the foreign antigen comprises multiple anchoring domains, the anchoring domains can be arranged in tandem (with or without linkers) or on alternate sides of other domains, such as antigen peptides e.g., sialidase domains). Where a foreign antigen comprises multiple antigen peptides (e.g., sialidase domains), the antigen peptides (e.g., sialidase domains) can be arranged in tandem (with or without linkers) or on alternate sides of other domains. In some embodiments, the foreign antigen comprises a stabilization domain, such as an Fc domain. In some embodiments, the foreign antigen comprises a domain (e.g., an Fc domain) that induces ADCC effects by the engineered immune cell.

1. Sialidase

[0246] In some embodiments, the recombinant oncolytic virus encodes a heterologous protein that includes all or a catalytic portion of a sialidase that is capable of removing sialic acid (N- acetylneuraminic acid (Neu5Ac)), e.g., from a glycan on a human cell. In general, Neu5Ac is linked via an alpha 2,3, an alpha 2,6 or alpha 2,8 linkage to the penultimate sugar in glycan on a protein by any of a variety of sialyl transferases.

[0247] The heterologous protein, in addition to a naturally occurring sialidase or catalytic portion thereof can, optionally, include peptide or protein sequences that contribute to the therapeutic activity of the protein. For example, the protein can include an anchoring domain that promotes interaction between the protein and a cell surface. The anchoring domain and sialidase domain can be arranged in any appropriate way that allows the protein to bind at or near a target cell membrane such that the therapeutic sialidase can exhibit an extracellular activity that removes sialic acid residues. The protein can have more than one anchoring domains. In cases in which the polypeptide has more than one anchoring domain, the anchoring domains can be the same or different. The protein can comprise one or more transmembrane domains (e.g. , one or more transmembrane alpha helices). The protein can have more than one sialidase domain. In cases in which a compound has more than one sialidase domain, the sialidase domains can be the same or different. Where the protein comprises multiple anchoring domains, the anchoring domains can be arranged in tandem (with or without linkers) or on alternate sides of other domains, such as sialidase domains. Where a compound comprises multiple sialidase domains, the sialidase domains can be arranged in tandem (with or without linkers) or on alternate sides of other domains.

Sialidase catalytic activity

[0248] In some embodiments, the sialidase has exo-sialidase activity as defined by Enzyme Commission EC 3.2.1.18. In some embodiments, the sialidase is an anhydrosialidase as defined by Enzyme Commission EC 4.2.2.15.

[0249] In some embodiments, the sialidase expressed by the oncolytic virus can be specific for Neu5Ac linked via alpha 2,3 linkage, specific for Neu5Ac linked via an alpha 2,6 specific for Neu5Ac linked via alpha 2,8 linkage, or can cleave Neu5Ac linked via an alpha 2,3 linkage or an alpha 2,6 linkage. In some embodiments, the sialidase can cleave Neu5Ac linked via an alpha 2,3 linkage, an alpha 2,6 linkage, or an alpha 2,8 linkage. A variety of sialidases are described in Tables 2-5.

[0250] A sialidase that can cleave more than one type of linkage between a sialic acid residue and the remainder of a substrate molecule, in particular, a sialidase that can cleave both alpha(2,6)-Gal and alpha(2,3)-Gal linkages or both alpha(2,6)-Gal and alpha(2,3)-Gal linkages and alpha(2,8)-Gal linkages can be used in the compounds of the disclosure. Sialidases included are the large bacterial sialidases that can degrade the receptor sialic acids Neu5Ac alpha(2,6)-Gal and Neu5Ac alpha(2,3)-Gal. For example, the bacterial sialidase enzymes from Clostridium perfringens (Genbank Accession Number X87369), Actinomyces viscosus (GenBankX62276), Arthrobacter ureafaciens GenBank (AY934539), or Micromonospora viridifaciens (Genbank Accession Number DO 1045) can be used.

[0251] In some embodiments, the sialidase comprises all or a portion of the amino acid sequence of a large bacterial sialidase or can comprise amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to all or a portion of the amino acid sequence of a large bacterial sialidase. In some embodiments, the sialidase domain comprises SEQ ID NO: 2 or 27, or a sialidase sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 12. In some embodiments, a sialidase domain comprises the catalytic domain of the Actinomyces viscosus sialidase extending from amino acids 274-666 of SEQ ID NO: 26, having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to amino acids 274-666 of SEQ ID NO: 26.

[0252] Additional sialidases include the human sialidases such as those encoded by the genes NEU2 (SEQ ID NO: 4; Genbank Accession Number Y16535; Monti, E, Preti, Rossi, E., Ballabio, A andBorsaniG. (1999) Genomics 57:137-143) and NEU4 (SEQ ID NO: 6; Genbank Accession Number NM080741; Monti et al. (2002) Neurochem Res 27:646-663). Sialidase domains of compounds of the present disclosure can comprise all or a portion of the amino acid sequences of a sialidase or can comprise amino acid sequences having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to all or a portion of the amino acid sequences of a sialidase. In some embodiments, where a sialidase domain comprises a portion of the amino acid sequences of a naturally occurring sialidase, or sequences having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to a portion of the amino acid sequences of a naturally occurring sialidase, the portion comprises essentially the same activity as the intact sialidase. In some embodiments, the sialidase expressed by the recombinant oncolytic virus is a sialidase catalytic domain protein. As used herein a “sialidase catalytic domain protein” comprises a catalytic domain of a sialidase but does not comprise the entire amino acid sequence of the sialidase from which the catalytic domain is derived. A “sialidase catalytic domain protein” has sialidase activity, and the term as used herein is interchangeable with a “sialidase”. In some embodiments, a sialidase catalytic domain protein comprises at least 10%, at least 20%, at least 50%, at least 70% of the activity of the sialidase from which the catalytic domain sequence is derived. In some embodiments, a sialidase catalytic domain protein comprises at least 90% of the activity of the sialidase from which the catalytic domain sequence is derived.

[0253] A sialidase catalytic domain protein can include other amino acid sequences, such as but not limited to additional sialidase sequences, sequences derived from other proteins, or sequences that are not derived from sequences of naturally occurring proteins. Additional amino acid sequences can perform any of a number of functions, including contributing other activities to the catalytic domain protein, enhancing the expression, processing, folding, or stability of the sialidase catalytic domain protein, or even providing a desirable size or spacing of the protein.

[0254] In some embodiments, the sialidase catalytic domain protein is a protein that comprises the catalytic domain of the A. viscosus sialidase. In some embodiments, an A. viscosus sialidase catalytic domain protein comprises amino acids 270-666 of the A. viscosus sialidase sequence (SEQ ID NO: 26; GenBank WP_003789074). In some embodiments, an A. Viscosus sialidase catalytic domain protein comprises an amino acid sequence that begins at any of the amino acids from amino acid 270 to amino acid 290 of the A. viscosus sialidase sequence (SEQ ID NO: 26) and ends at any of the amino acids from amino acid 665 to amino acid 901 of said A. viscosus sialidase sequence (SEQ ID NO: 26), and lacks any A. viscosus sialidase protein sequence extending from amino acid 1 to amino acid 269.

[0255] In some embodiments, an A. viscosus sialidase catalytic domain protein comprises amino acids 274-681 of the A. viscosus sialidase sequence (SEQ ID NO: 26) and lacks other A. viscosus sialidase sequence. In some embodiments, an A. viscosus sialidase catalytic domain protein comprises amino acids 274-666 of the A. viscosus sialidase sequence (SEQ ID NO: 26) and lacks any other A. viscosus sialidase sequence. In some embodiments, an A. viscosus sialidase catalytic domain protein comprises amino acids 290-666 of the A. viscosus sialidase sequence (SEQ ID NO: 26) and lacks any other A. viscosus sialidase sequence. In yet other embodiments, an A. viscosus sialidase catalytic domain protein comprises amino acids 290- 681 of the A. viscosus sialidase sequence (SEQ ID NO: 26) and lacks any other A. viscosus sialidase sequence.

[0256] In some embodiments, useful sialidase polypeptides for expression by an oncolytic virus include polypeptides comprising a sequence that is 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 27 or comprises 375, 376, 377, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, or 392 contiguous amino acids of SEQ ID NO: 27.

[0257] In some embodiments, the sialidase is DAS181, a functional derivative thereof (e.g., a fragment thereof), or a biosimilar thereof. In some embodiments, the sialidase comprises an amino acid sequence that is at least about 80% (e.g., at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identical to SEQ ID NO: 2. In some embodiments, the sialidase comprises 414, 413, 412, 411, or 410 contiguous amino acids of SEQ ID NO: 2. In some embodiments, the sialidase comprises a fragment of DAS181 without the anchoring domain (AR domain). In some embodiments, the sialidase comprises an amino acid sequence that is at least about 80% (e.g., at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identical to SEQ ID NO: 27.

[0258] DAS 181 is a recombinant sialidase fusion protein with a heparin- binding anchoring domain. DAS181 and methods for preparing and formulating DAS181 are described in US 7,645,448; US 9,700,602 and US 10,351,828, each of which is herein incorporated by reference in their entirety for any and all purposes. [0259] In some embodiments, the sialidase is a secreted form of DAS 181, a functional derivative thereof, or a biosimilar thereof. In some embodiments, the nucleotide sequence encoding a secreted form of DAS 181 encodes a secretion sequence operably linked to DAS 181, wherein the secretion sequence enables secretion of the protein from eukaryotic cells. In some embodiments, the sialidase comprises an amino acid sequence that is at least about 80% (e.g., at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identical to SEQ ID NO: 28. In some embodiments, the sialidase comprises an amino acid sequence that is at least about 80% (e.g., at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identical to SEQ ID NO: 28. In some embodiments, the sialidase comprises 414, 413, 412, 411, or 410 contiguous amino acids of SEQ ID NO: 28.

[0260] In some embodiments, the sialidase is a transmembrane form of DAS 181 , a functional derivative thereof, or a biosimilar thereof. In some embodiments, the sialidase comprises an amino acid sequence that is at least about 80% e.g., at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identical to SEQ ID NO: 31. In some embodiments, the sialidase comprises an amino acid sequence that is at least about 80% (e.g., at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or 100% identical to SEQ ID NO: 31. In some embodiments, the sialidase comprises 414, 413, 412, 411, or 410 contiguous amino acids of SEQ ID NO: 31.

Table 2: Engineered Sialidases

Table 3: Human Sialidases

Table 4: Sialidases in organisms that are largely commensal with humans

Table 5: Additional sialidases

Anchoring Domain

[0261] In some embodiments, the foreign antigen (e.g., sialidase) comprises an anchoring domain (also referred herein as an “anchoring moiety”). As used herein, an “extracellular anchoring domain” or “anchoring domain” is any moiety that interacts with an entity that is at or on the exterior surface of a target cell or is in close proximity to the exterior surface of a target cell. An anchoring domain can serve to retain a foreign antigen (e.g., sialidase) of the present disclosure at or near the external surface of a target cell. An extracellular anchoring domain may bind 1) a molecule expressed on the surface of a cancer cell, or a moiety, domain, or epitope of a molecule expressed on the surface of a cancer cell, 2) a chemical entity attached to a molecule expressed on the surface of a cancer cell, or 3) a molecule of the extracellular matrix surrounding a cancer cell.

[0262] An exemplary anchoring domain binds to heparin/sulfate, a type of GAG that is ubiquitously present on cell membranes. Many proteins specifically bind to heparin/heparan sulfate, and the GAG-binding sequences in these proteins have been identified (Meyer, F A, King, M and Gelman, R A. (1975) Biochimica et Biophysica Acta 392: 223-232; Schauer, S. ed., pp 233. Sialic Acids Chemistry, Metabolism and Function. Springer-Verlag, 1982). For example, the GAG-binding sequences of human platelet factor 4 (PF4) (SEQ ID NO:77), human interleukin 8 (IL8) (SEQ ID NO:78), human antithrombin III (AT III) (SEQ ID NO:80), human apoprotein E (ApoE) (SEQ ID NO: 80), human angio-associated migratory cell protein (AAMP) (SEQ ID NO:81), or human amphiregulin (SEQ ID NO:82) have been shown to have very high affinity to heparin.

[0263] In some embodiments, the anchoring domain is a non-protein anchoring moiety, such as a phosphatidylinositol (GPI) linker.

Linkers

[0264] A foreign antigen may comprise one or more polypeptide linkers disposed between different domains. For example, a protein that includes a sialidase or a catalytic domain thereof can optionally include one or more polypeptide linkers that can join various domains of the sialidase. Linkers can be used to provide optimal spacing or folding of the domains of a protein. The domains of a protein joined by linkers can be sialidase domains, anchoring domains, transmembrane domains, or any other domains or moieties of the foreign antigen that provide additional functions such as enhancing protein stability, facilitating purification, etc. Some preferred linkers include the amino acid glycine. For example, linkers having the sequence: (GGGGS (SEQ ID NO: 55))n, where n is 1-20. In some embodiments, the linker is a hinge region of an immunoglobulin. Any hinge or linker sequence capable of keeping the catalytic domain free of steric hindrance can be used to link a domain of a sialidase to another domain (e.g., a transmembrane domain or an anchoring domain). In some embodiments, the linker is a hinge domain comprising the sequence of SEQ ID NO: 62. Secretion sequence

[0265] In some embodiments, the nucleotide sequence encoding the foreign antigen (e.g., sialidase) further encodes a secretion sequence (e.g., a signal sequence or signal peptide) operably linked to the foreign antigen e.g., sialidase). The terms “secretion sequence,” “signal sequence,” and “signal peptide” are used interchangeably. In some embodiments, the secretion sequence is a signal peptide operably linked to the N-terminus of the protein. In some embodiments, the length of the secretion sequence ranges between 10 and 30 amino acids (e.g. , between 15 and 25 amino acids, between 15 and 22 amino acids, or between 20 and 25 amino acids). In some embodiments, the secretion sequence enables secretion of the protein from eukaryotic cells. During translocation across the endoplasmic reticulum membrane, the secretion sequence is usually cleaved off and the protein enters the secretory pathway. In some embodiments, the nucleotide sequence encodes, from N-terminus to C-terminus, a secretion sequence, a sialidase, and a transmembrane domain, wherein the sialidase is operably linked to the secretion sequence and the transmembrane domain. In some embodiments, the N-terminal secretion sequence is cleaved resulting in a protein with an N-terminal extracellular domain. An exemplary secretion sequence is provided in SEQ ID NO: 40.

Transmembrane domain

[0266] In some embodiments, the foreign antigen (e.g., sialidase) comprises a transmembrane domain. In some embodiments, the antigenic peptide (e.g., sialidase domain) can be joined to a mammalian (preferably human) transmembrane (TM) domain. This arrangement permits the foreign antigen (e.g., sialidase) to be expressed on the cell surface. Suitable transmembrane domain include, but are not limited to a sequence comprising human CD28 TM domain (NM_006139; FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 46), human CD4 TM domain (M35160; MALIVLGGVAGLLLHGLGIFF (SEQ ID NO: 47); human CD8 TM1 domain (NM_001768; IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 48); human CD8 TM2 domain (NM_001768; IYIWAPLAGTCGVLLLSLVITLY (SEQ ID NO: 49); human CD8 TM3 domain (NM_001768; IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 50); human 41BB TM domain (NM_001561; IISFFLALTSTALLFLLFF LTLRFSVV (SEQ ID NO: 51); human PDGFR TM1 domain (VVISAILA LVVLTIISLIILI; SEQ ID NO:52); and human PDGFR TM2 domain

NAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR; SEQ ID NO: 45) [0267] In some embodiments, the nucleotide sequence encoding a sialidase encodes a protein comprising, from amino terminus to carboxy terminus, a secretion sequence (e.g. , SEQ ID NO: 40), a sialidase (e.g., a sialidase comprising an amino acid sequence selected from SEQ ID NOs: 1-27, and a transmembrane domain (e.g., a transmembrane domain selected from SEQ ID NOs: 45-52). However, any suitable secretion sequence, sialidase domain sequence, or transmembrane domain may be used. In some embodiments, the nucleotide sequence encoding a sialidase encodes a protein comprising, from amino terminus to carboxy terminus, a secretion sequence (e.g., SEQ ID NO: 40), the sialidase of SEQ ID NO: 27, and a transmembrane domain (e.g., a transmembrane domain selected from SEQ ID NOs: 45-52).

[0268] In some embodiments, the sialidase has at least 50%, at least 60%, at least 65%, 80% (e.g., at least about any one of 85%, 86%, 87%, 88%, 89%) or at least 90% (e.g., at least about any one of 91%, 92%, 94%, 96%, 98%, or 99%) sequence identity to a sequence selected from SEQ ID NOs: 31. In some embodiments, the sialidase comprises a sequence selected from SEQ ID NOs: 31. In some embodiments, the sialidase comprises the amino acid sequence of SEQ ID NO: 31.

Stabilization domain

[0269] In some embodiments, the foreign antigen (e.g., sialidase) comprises a stabilization domain. The stabilizing domain can be any suitable domain that stabilizes the inhibitory polypeptide. In some embodiments, the stabilizing domain extends the half-life of the inhibitory polypeptide in vivo. In some embodiments, the stabilizing domain is an Fc domain. In some embodiments, the stabilizing domain is an albumin domain.

[0270] In some embodiments, the stabilization domain is an immunoglobulin G (IgG) Fc (fragment, crystallizable) domain. In some embodiments, the Fc domain is selected from the group consisting of Fc fragments of IgG, IgA, IgD, IgE, IgM, and combinations and hybrids thereof. In some embodiments, the Fc domain is derived from a human IgG. In some embodiments, the Fc domain comprises the Fc domain of human IgGl, IgG2, IgG3, IgG4, or a combination or hybrid IgG. In some embodiments, the IgG Fc domain comprises an amino acid sequence having at least any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 99% identity to the amino acid sequence of SEQ ID NO: 104. In some embodiments, the IgG Fc domain comprises the amino acid sequence of SEQ ID NO: 104.

[0271] In some embodiments, the Fc domain induces or enhances Antibody-dependent cellular cytotoxicity (ADCC) effects by the engineered immune cell. In some embodiments, the Fc domain in the foreign antigen (e.g., sialidase) binds to CD 16 on NK cells. [0272] In some embodiments, the foreign antigen (e.g., sialidase) comprises from the N- terminus to the C-terminus: an antigenic peptide (e.g., a sialidase catalytic domain), an IgG Fc domain, and a transmembrane domain. In some embodiments, the sialidase comprises an amino acid sequence having at least any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 99% identity to the amino acid sequence of SEQ ID NO: 105 or 106. In some embodiments, the sialidase comprises the amino acid sequence of SEQ ID NO: 105 or 106.

[0273] In some embodiments, the sialidase comprises a transmembrane domain. In some embodiments, the sialidase comprises from the N-terminus to the C-terminus: a sialidase catalytic domain, a hinge region, and a transmembrane domain. In some embodiments, the sialidase comprises from the N-terminus to the C-terminus: a sialidase catalytic domain, a hinge region, an IgG Fc region, and a transmembrane domain. In some embodiments, the hinge region is an IgGl hinge region. In some embodiments, the IgG Fc domain comprises the amino acid sequence of SEQ ID NO: 104.

2. Multispecific immune cell engager

[0274] In some embodiments, the recombinant oncolytic virus further comprises a nucleotide sequence encoding a multispecific immune cell engager. In some embodiments, the multispecific immune cell engager is a bispecific immune cell engager. In some embodiments, the heterologous protein is a bispecific T cell engager (BiTE). Exemplary bispecific immune cell engagers have been described, for example, in international patent publication WO2018049261, herein incorporated by reference in its entirety. In some embodiments, the bispecific immune cell engager comprises a first antigen-binding domain (such as scFv) specifically recognizing a tumor antigen (such as EpCAM, FAP, or EGFR, etc.) and a second antigen-binding domain (such as scFv) specifically recognizing a cell surface molecule on an effector cell (such as CD3 or 4-1BB on T lymphocytes). Tumor antigens can be a tumor- associated antigen (TAA) or a tumor-specific antigen (TSA). In some embodiments, TAA or TSA is expressed on a cell of a solid tumor. Tumor antigens include, but are not limited to, EpCAM, FAP, EphA2, HER2, GD2, EGFR, VEGFR2, and Glypican-3 (GPC3), CDH17, Fibulin-3, HHLA2, Folate receptors, etc. In some embodiments, the tumor antigen is EpCAM. In some embodiments, the tumor antigen is FAP. In some embodiments, the tumor antigen is EGFR.

[0275] As described above, effector cells include, but are not limited to T lymphocyte, B lymphocyte, natural killer (NK) cell, dendritic cell (DC), macrophage, monocyte, neutrophil, NKT-cell, or the like. In some embodiments, the effector cell is a T lymphocyte. In some embodiments, the effector cell is a cytotoxic T lymphocyte. Cell surface molecules on an effector cell include, but are not limited to CD3, CD4, CD5, CD8, CD 16, CD28, CD40, CD64, CD89, CD134, CD137, NKp46, NKG2D, or the like. In some embodiments, the cell surface molecule is CD3.

[0276] A cell surface molecule on an effector cell of the present application is a molecule found on the external cell wall or plasma membrane of a specific cell type or a limited number of cell types. Examples of cell surface molecules include, but are not limited to, membrane proteins such as receptors, transporters, ion channels, proton pumps, and G protein-coupled receptors; extracellular matrix molecules such as adhesion molecules (e.g., integrins, cadherins, selectins, or NCAMS); see, e.g., U.S. Pat. No. 7,556,928, which is incorporated herein by reference in its entirety. Cell surface molecules on an effector cell include but not limited to CD3, CD4, CD5, CD8, CD16, CD27, CD28, CD38, CD64, CD89, CD134, CD137, CD154, CD226, CD278, NKp46, NKp44, NKp30, NKG2D, and an invariant TCR.

[0277] The cell surface molecule-binding domain of an engager molecule can provide activation to immune effector cells. The skilled artisan recognizes that immune cells have different cell surface molecules. For example CD3 is a cell surface molecule on T-cells, whereas CD 16, NKG2D, or NKp30 are cell surface molecules on NK cells, and CD3 or an invariant TCR are the cell surface molecules on NKT-cells. Engager molecules that activate T-cells may therefore have a different cell surface molecule-binding domain than engager molecules that activate NK cells. In some embodiments, e.g., wherein the immune cell is a T- cell, the activation molecule is one or more of CD3, e.g., CD3y, CD35 or CD3s; or CD27, CD28, CD40, CD134, CD137, and CD278. In other some embodiments, e.g., wherein the immune cell is a NK cell, the cell surface molecule is CD 16, NKG2D, or NKp30, or wherein the immune cell is a NKT-cell, the cell surface molecule is CD3 or an invariant TCR.

[0278] CD3 comprises three different polypeptide chains (a, 5 and y chains), is an antigen expressed by T cells. The three CD3 polypeptide chains associate with the T-cell receptor (TCR) and the (J-chain to form the TCR complex, which has the function of activating signaling cascades in T cells. Currently, many therapeutic strategies target the TCR signal transduction to treat diseases using anti-human CD3 monoclonal antibodies. The CD3 specific antibody OKT3 is the first monoclonal antibody approved for human therapeutic use, and is clinically used as an immunomodulator for the treatment of allogenic transplant rejections. [0279] In some embodiments, the tumor antigen is FAP. In some embodiments, the cell surface molecule on the effector cell is CD3 or 41-BB. In some embodiments, the cell surface marker on the effector cell is CD3s. In some embodiments according to any of the recombinant oncolytic viruses described above, the first antigen-binding domain is a scFv, and the second antigen binding domain is a scFv. In some embodiments, the multispecific immune cell engager comprises a first scFv that recognizes FAP, and a second scFv that recognizes CD3/ CD3s.

[0280] In some embodiments, the tumor antigen is FAP and the first antigen-binding domain comprises: (i) a first light chain complementarity-determining region (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 86, (ii) a second light chain complementaritydetermining region (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 87, (iii), a third light chain complementarity-determining region (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 88, (iv) a first heavy chain complementarity-determining region (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 89, (v) a second heavy chain complementarity-determining region (CDR-H2) comprising g the amino acid sequence of SEQ ID NO: 90, and (vi) a third heavy chain complementarity-determining region (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 91. In some embodiments, the tumor antigen is FAP and the first antigen-binding domain comprises an amino acid sequence having at least any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% identity to the amino acid sequence of SEQ ID NO: 98. In some embodiments, the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 98.

[0281] In some embodiments, the cell surface molecule on the effector cell is CD3, and the second antigen-binding domain comprises: (i) a first light chain complementarity-determining region (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 92, (ii) a second light chain complementarity-determining region (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 93, (iii), a third light chain complementarity-determining region (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 94, (iv) a first heavy chain complementarity-determining region (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 95, (v) a second heavy chain complementarity-determining region (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 96, and (vi) a third heavy chain complementarity-determining region (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 97. In some embodiments, the cell surface molecule on the effector cell is CD3 and the second antigen-binding domain comprises an amino acid sequence having at least any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% identity to the amino acid sequence of SEQ ID NO: 99. In some embodiments, the cell surface molecule on the effector cell is CD3 and the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 99. [0282] In some embodiments, recombinant oncolytic virus comprises a second nucleotide sequence encoding a multispecific immune cell engager comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% identity to the amino acid sequence of SEQ ID NO: 100. In some embodiments according to any of the recombinant oncolytic viruses described above, the multispecific immune cell engager comprises the amino acid sequence of SEQ ID NO: 100.

[0283] In some cases the amino acids that differ from those from the reference sequences described above (e.g., SEQ ID NO: 98 for the first scFv, SEQ ID NO: 99 for the second scFv, or SEQ ID NO: 100 for the multispecific immune cell engager) are conservative substitutions or highly conservative substitutions. Conservative substitutions and highly conservative substitutions can be as defined in the “Sialidase” section above.

[0284] In some embodiments, the second nucleotide sequence further encodes a signal peptide sequence operably linked to the multispecific immune cell engager. In some embodiments the signal peptide sequence comprises the amino acid sequence of SEQ ID NO: 103. In some embodiments, the second nucleotide sequence encodes an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% identity to SEQ ID NO: 101. In some embodiments, the second nucleotide sequence encodes the amino acid sequence of SEQ ID NO: 101. In some embodiments, the second nucleotide sequence comprises a nucleic acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% identity to the nucleic acid sequence of SEQ ID NO: 109. In some embodiments, the second nucleotide sequence comprises the nucleic acid sequence of SEQ ID NO: 109.

3. Other heterologous proteins or nucleotide sequences

[0285] In some embodiments according to any one of the recombinant oncolytic viruses described above, the oncolytic virus further comprises a second nucleotide sequence encoding a heterologous protein or nucleic acid. In some embodiments, the second nucleotide sequence encodes a heterologous protein.

[0286] In some embodiments according to any one of the recombinant oncolytic viruses described above, the heterologous protein is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA-4, PD-1, PD-L1, TIGIT, LAG3, TIM-3, VISTA, B7-H4, or HLA-G. In some embodiments, the immune checkpoint inhibitor is an antibody. In some embodiments, the immune checkpoint modulator is an immune checkpoint inhibitor, such as an inhibitor or an antagonist antibody or a decoy ligand of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CD160, CD73, CTLA-4, B7-H4, TIGIT, VISTA, or 2B4. In some embodiments, the immune checkpoint modulator is an inhibitor of PD-1. In some embodiments, the immune checkpoint inhibitor is an antibody against an immune checkpoint molecule, such as an anti-PD-1 antibody. In some embodiments, the immune checkpoint inhibitor is a ligand that binds to the immune checkpoint molecule, such as soluble or free PD-L1/PD-L2. In some embodiments, the immune checkpoint inhibitor is an extracellular domain of PD- 1 fused to an Fc fragment of an immunoglobulin (such as IgG4 Fc)) that can block PDL-1 on tumor cell surface binding to the immune check point PD-1 on immune cells. In some embodiments, the immune checkpoint inhibitor is a ligand that binds to HHLA2. In some embodiments, the immune checkpoint inhibitor is an extracellular domain of TMIGD2 fused to an Fc fragment of an immunoglobulin, such as IgG4 Fc. In some embodiments, the immune checkpoint inhibitor is a ligand that binds to at least two different inhibitory immune checkpoint molecules (e.g. bispecific), such as a ligand that binds to both CD47 and CXCR4. In some embodiments, the immune checkpoint inhibitor comprises an extracellular domain of SIRPa and a CXCL12 fragment fused to an Fc fragment of an immunoglobulin, such as IgG4 Fc. These molecules can bind to CD47 on cancer cell, thus stopping its interaction with SIRPalpha to block the “don’t eat me” signal to macrophages and dendritic cells.

[0287] In some embodiments, the heterologous protein is an inhibitor of an immune suppressive receptor. The immune suppressive receptor can be any receptor expressed by an immune effector cell that inhibits or reduces an immune response to tumor cells. Exemplary effector cell includes without limitation a T lymphocyte, a B lymphocyte, a natural killer (NK) cell, a dendritic cell (DC), a macrophage, a monocyte, a neutrophil, an NKT-cell, or the like. In some embodiments, the immune suppressive receptor is LILRB, TYRO3, AXL, Folate receptor beta or MERTK. In some embodiments, the inhibitor of an immune suppressive receptor is an anti-LILRB antibody.

[0288] In some embodiments, the heterologous protein reduces neutralization of the recombinant oncolytic virus by the immune system of the individual. In some embodiments, the recombinant oncolytic virus is an enveloped virus (e.g., vaccinia virus), and the heterologous protein is a complement activation modulator (e.g. , CD55 or CD59). Complement is a key component of the innate immune system, targeting the virus for neutralization and clearance from the circulatory system. Complement activation results in cleavage and activation of C3 and deposition of opsonic C3 fragments on surfaces. Subsequent cleavage of C5 leads to assembly of the membrane attack complex (C5b, 6, 7, 8, 9), which disrupts lipid bilayers.

[0289] In some embodiments, recombinant oncolytic virus is an enveloped virus (e.g., vaccinia virus), and the heterologous protein is a complement activation modulator such as CD55, CD59, CD46, CD35, factor H, C4-binding protein, or other identified complement activation modulators. Without wishing to be bound by theory, expression of the complement activation modulators on the virus envelope surface (e.g., the vaccinia virus envelope) results in a virus having the ability to modulate complement activation and reduce complement- mediated virus neutralization as compared to the wild-type virus. In some embodiments, the heterologous nucleotide sequence encodes a domain of human CD55, CD59, CD46, CD35, factor H, C4-binding protein, or other identified complement activation modulators. In another embodiment, the heterologous nucleic acid encodes a CD55 protein that comprises an amino acid sequence having the sequence of SEQ ID NO: 58. In view of the disclosure presented herein, one of ordinary skill in the art would readily employ other complement activation modulators (e.g. CD59, CD46, CD35, factor H, C4-binding protein, etc.) in any one of the enveloped recombinant oncolytic viruses (e.g., vaccinia virus) presented herein.

[0290] In some embodiments, the heterologous protein is a cytokine. In some embodiments, the heterologous protein is IL-15, IL-12, IL-2, IL-18, CXCL10, or CCL4, or a modified protein (e.g., a fusion protein) derived from of any of the aforementioned proteins. In some embodiments, the heterologous protein is a derivative of IL-2 that is modified to have reduced side effects. In some embodiments, the heterologous protein is modified IL- 18 that lacks binding to IL18-BP. In some embodiments, the heterologous protein is a fusion protein comprising an inflammatory cytokine and a stabilizing domain. The stabilizing domain can be any suitable domain that stabilizes the inhibitory polypeptide. In some embodiments, the stabilizing domain extends the half-life of the inhibitory polypeptide in vivo. In some embodiments, the stabilizing domain is an Fc domain. In some embodiments, the stabilizing domain is an albumin domain.

[0291] In some embodiments, the Fc domain is selected from the group consisting of Fc fragments of IgG, IgA, IgD, IgE, IgM, and combinations and hybrids thereof. In some embodiments, the Fc domain is derived from a human IgG. In some embodiments, the Fc domain comprises the Fc domain of human IgGl, IgG2, IgG3, IgG4, or a combination or hybrid IgG. In some embodiments e.g., a fusion protein derived from IL- 12 or IL-2), the Fc domain has a reduced effector function as compared to corresponding wildtype Fc domain (such as at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95% reduced effector function as measured by the level of antibody-dependent cellular cytotoxicity (ADCC)).

[0292] In some embodiments, the inflammatory cytokine and the stabilization domain are fused to each other via a linker, such as a peptide linker. A peptide linker may have a naturally occurring sequence, or a non-naturally occurring sequence. For example, a sequence derived from the hinge region of heavy chain only antibodies may be used as the linker. The peptide linker can be of any suitable length. In some embodiments, the peptide linker tends not to adopt a rigid three-dimensional structure, but rather provide flexibility to a polypeptide. In some embodiments, the peptide linker is a flexible linker. Exemplary flexible linkers include glycine polymers, glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art.

[0293] In some embodiments, the heterologous protein is a bacterial or a viral polypeptide. In some embodiments the heterologous protein is a tumor-associated antigen selected from carcinoembryonic antigen, alphafetoprotein, MUC16, survivin, glypican-3, B7 family members, LILRB, CD19, BCMA, NY-ESO-1, CD20, CD22, CD33, CD38, CEA, EGFR (such as EGFRvIII), GD2, HER2, IGF1R, mesothelin, PSMA, ROR1, WT1, NY-ESO-1, CDH17, and other tumor antigens with clinical significance.

[0294] In some embodiments, the recombinant oncolytic virus comprises two or more additional nucleotide sequences, wherein each nucleotide sequence encodes any one of the heterologous proteins or nucleic acids described herein.

Antagonists or inhibitors

[0295] Antagonist, as used herein, is interchangeable with inhibitor. In some embodiments, the heterologous protein is an inhibitor (i.e., an antagonist) of a target protein, wherein the target protein is an immune suppressive protein (e.g., a checkpoint inhibitor or other inhibitor of immune cell activation). In some embodiments, the target protein is an immune checkpoint protein. In some embodiments, the target protein is PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CD160, CD73, CTLA-4, B7-H4, TIGIT, VISTA, or 2B4. In some embodiments, the target protein is CTLA-4, PD-1, PD-L1, B7-H4, or HLA-G. In some embodiments, the target protein is an immune suppressive receptor selected from LILRB, TYRO3, AXL, or MERTK.

[0296] The antagonist inhibits the expression and/or activity of the target protein (e.g., an immune suppressive receptor or an immune checkpoint protein). In some embodiments, the antagonist inhibits expression of the target protein (e.g., mRNA or protein level) by at least about any one of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more. Expression levels of a target protein can be determined using known methods in the art, including, for example, quantitative Polymerase Chain Reaction (qPCR), microarray, and RNA sequencing for determining RNA levels; and Western blots and enzyme-linked immunosorbent assays (ELISA) for determining protein levels.

[0297] In some embodiments, the antagonist inhibits activity (e.g., binding to a ligand or receptor of the target protein, or enzymatic activity) of the target protein by at least about any one of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more. Binding can be assessed using known methods in the art, including, for example, Surface Plasmon Resonance (SPR) assays, and gel shift assays.

[0298] The antagonist may be of any suitable molecular modalities, including, but are not limited to, small molecule inhibitors, oligopeptides, peptidomimetics, RNAi molecules (e.g., small interfering RNAs (siRNA), short hairpin RNAs (shRNA), microRNAs (miRNA)), antisense oligonucleotides, ribozymes, proteins (e.g., antibodies, inhibitory polypeptides, fusion proteins, etc.), and gene editing systems. i. Antibodies

[0299] In some embodiments, the antagonist inhibits binding of the target protein (e.g., an immune checkpoint protein or immune suppressive protein) to a ligand or a receptor. In some embodiments, the antagonist is an antibody that specifically binds to the target protein (e.g., CTLA-4, PD-1, PD-L1, B7-H4, HLA-G, LILRB, TYRO3, AXL, or MERTK, Folate receptor beta, etc.), or an antigen-binding fragment thereof. In some embodiments, the antagonist is a polyclonal antibody. In some embodiments, the antagonist is a monoclonal antibody. In some embodiments, the antagonist is a full-length antibody, or an immunoglobulin derivative. In some embodiments, the antagonist is an antigen-binding fragment. Exemplary antigen-binding fragments include, but are not limited to, a single-chain Fv (scFv), a Fab, a Fab’, a F(ab’)2, a Fv, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a single-domain antibody e.g., VHH), a Fv-Fc fusion, a scFv-Fc fusion, a scFv-Fv fusion, a diabody, a tribody, and a tetrabody. In some embodiments, the antagonist is a scFv. In some embodiments, the antagonist is a Fab or Fab’. In some embodiments, the antagonist is a chimeric, human, partially humanized, fully humanized, or semi-synthetic antibody. Antibodies and/or antibody fragments may be derived from murine antibodies, rabbit antibodies, human antibodies, fully humanized antibodies, camelid antibody variable domains and humanized versions, shark antibody variable domains and humanized versions, and camelized antibody variable domains. In some embodiments, the antagonist is a bi-specific molecule (e.g., a bi-specific antibody or bi-specific Fab, bi-specific scFv, antibody-Fc fusion protein Fv, etc.) or a tri-specific molecule (e.g., a tri-specific antibody comprised of Fab, scFv, VH or Fc fusion proteins etc.).

[0300] In some embodiments, the antibody comprises one or more antibody constant regions, such as human antibody constant regions. In some embodiments, the heavy chain constant region is of an isotype selected from IgA, IgG, IgD, IgE, and IgM. In some embodiments, the human light chain constant region is of an isotype selected from K and X. In some embodiments, the antibody comprises an IgG constant region, such as a human IgGl, IgG2, IgG3, or IgG4 constant region. In some embodiments, when effector function is desirable, an antibody comprising a human IgGl heavy chain constant region or a human IgG3 heavy chain constant region may be selected. In some embodiments, when effector function is not desirable, an antibody comprising a human IgG4 or IgG2 heavy chain constant region, or IgGl heavy chain with mutations, such as N297A/Q, negatively impacting FcyR binding may be selected. In some embodiments, the antibody comprises a human IgG4 heavy chain constant region. In some embodiments, the antibody comprises an S241P mutation in the human IgG4 constant region.

[0301] In some embodiments, the antibody comprises an Fc domain. The term “Fc region,” “Fc domain” or “Fc” refers to a C-terminal non-antigen binding region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native Fc regions and variant Fc regions. In some embodiments, a human IgG heavy chain Fc region extends from Cys226 to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present, without affecting the structure or stability of the Fc region. Unless otherwise specified herein, numbering of amino acid residues in the IgG or Fc region is according to the EU numbering system for antibodies, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. In some embodiments, the antibody comprises a variant Fc region has at least one amino acid substitution compared to the Fc region of a wild type IgG or a wild-type antibody.

[0302] In some embodiments, the antibody is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed. [0303] Antibodies that specifically bind to a target protein can be obtained using methods known in the art, such as by immunizing a non-human mammal and obtaining hybridomas therefrom, or by cloning a library of antibodies using molecular biology techniques known in the art and subsequence selection or by using phage display. ii. Nucleic acid agents

[0304] In some embodiments, the heterologous nucleic acid is a nucleic acid agent that downregulates the target protein. In some embodiments, the antagonist inhibits expression (e.g., mRNA or protein expression) of the target protein. In some embodiments, the antagonist is a siRNA, a shRNA, a miRNA, an antisense oligonucleotide, or a gene editing system.

[0305] In some embodiments, the antagonist is an RNAi molecule. In some embodiments, the antagonist is a siRNA. In some embodiments, the antagonist is a shRNA. In some embodiments, the antagonist is a miRNA.

[0306] A skilled in the art may could readily design an RNAi molecule or a nucleic acid encoding an RNAi molecule to downregulate the target protein. The term “RNAi” or “RNA interference” as used herein refers to biological process in which RNA molecules inhibit gene expression or translation by specific binding to a target mRNA molecule. See for example Zamore et al., 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428-429; Elbashir et al., 2001, Nature, 411, 494-498; and Kreutzer et al., International PCT Publication No. WO 00/44895; Zernicka-Goetz et al., International PCT Publication No. WO 01/36646; Fire, International PCT Publication No. WO 99/32619; Plaetinck et al., International PCT Publication No. WO00/01846; Mello and Fire, International PCT Publication No. WO 01/29058; Deschamps- Depaillette, International PCT Publication No. WO 99/07409; and Li et al., International PCT Publication No. WO 00/44914; Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237; Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al., 2002, RNA, 8, 842-850; Reinhart et al., 2002, Gene & Dev., 16, 1616-1626; and Reinhart & Bartel, 2002, Science, 297, 1831). Exemplary RNAi molecules include siRNA, miRNA and shRNA.

[0307] A siRNA can be a double-stranded polynucleotide molecule comprising self- complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleotide sequence or a portion thereof. In some embodiments, the siRNA comprises one or more hairpin or asymmetric hairpin secondary structures. In some embodiments, the siRNA may be constructed in a scaffold of a naturally occurring miRNA. The siRNA molecules need not be limited to those molecules containing only RNA, but further encompasses chemically modified nucleotides and non-nucleotides.

[0308] RNAi may be designed using known methods in the art. For example, siRNA may be designed by classifying RNAi sequences, for example 1000 sequences, based on functionality, with a functional group being classified as having greater than 85% knockdown activity and a non-functional group with less than 85% knockdown activity. The distribution of base composition was calculated for entire the entire RNAi target sequence for both the functional group and the non-functional group. The ratio of base distribution of functional and nonfunctional group may then be used to build a score matrix for each position of RNAi sequence. For a given target sequence, the base for each position is scored, and then the log ratio of the multiplication of all the positions is taken as a final score. Using this score system, a very strong correlation may be found of the functional knockdown activity and the log ratio score. Once the target sequence is selected, it may be filtered through both fast NCBI blast and slow Smith Waterman algorithm search against the Unigene database to identify the gene-specific RNAi or siRNA. Sequences with at least one mismatch in the last 12 bases may be selected.

[0309] In some embodiments, the antagonist is an antisense oligonucleotide, e.g., antisense RNA, DNA or PNA. In some embodiments, the antagonist is a ribozyme. An “antisense” nucleic acid refers to a nucleotide sequence complementary to a “sense” nucleic acid encoding a target protein or fragment (e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence). The antisense nucleic acid can be complementary to an entire coding strand, or to a portion thereof or a substantially identical sequence thereof. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of the mRNA, e.g., between the -10 and +10 regions of the target gene nucleotide sequence of interest. In some embodiments, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence. An antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length. An antisense nucleic acid can be constructed using chemical synthesis or enzyme ligation reactions using standard procedures. For example, an antisense nucleic acid e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids (e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used). Antisense nucleic acid also can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation.

[0310] An antisense nucleic acid is a ribozyme in some embodiments. A ribozyme having specificity for a target nucleotide sequence can include one or more sequences complementary to such a nucleotide sequence, and a sequence having a known catalytic region responsible for mRNA cleavage (e.g., U.S. Pat. No. 5,093,246 or Haselhoff and Gerlach, Nature 334: 585-591 (1988)). For example, a derivative of a Tetrahymena L-19 IVS RNA is sometimes utilized in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an mRNA (e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742). Target mRNA sequences may be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules (e.g., Bartel & Szostak, Science 261: 1411-1418 (1993)).

[0311] In some embodiments, the antagonist is a gene -editing system, such as a CRISPR/Cas gene editing system, Transcription activator-like effector nuclease or TALEN gene editing system, Zinc-finger gene editing system, etc. In some embodiments, the antagonist is a geneediting system that knocks-down a target protein, e.g., in a tissue-specific manner. In some embodiments, the antagonist is a gene-editing system that silences expression of the target protein.

[0312] In some embodiments, the gene-editing system comprises a guided nuclease such as an engineered (e.g., programmable or targetable) nuclease to induce gene editing of a target sequence (e.g., DNA sequence or RNA sequence) encoding the target protein. Any suitable guided nucleases can be used including, but not limited to, CRISPR-associated protein (Cas) nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, other endo- or exo-nucleases, variants thereof, fragments thereof, and combinations thereof. In some embodiments, the gene-editing system comprises a guided nuclease fused to a transcription suppressor. In some embodiments, the gene-editing system further comprises an engineered nucleic acid that hybridizes to a target sequence encoding the target protein. In some embodiments, the gene-editing system is a CRISPR-Cas system comprising a Cas nuclease (e.g., Cas9) and a guide RNA (i.e., gRNA).

4. Promoters for expression of heterologous proteins or nucleic acids

[0313] The nucleotide sequences encoding heterologous proteins (e.g., foreign antigen such as sialidase) or nucleic acids described herein can be operably linked to a promoter. In some embodiments, at least a first nucleotide sequence encoding the foreign antigen e.g., sialidase) and a second nucleotide sequence encoding an additional heterologous protein or nucleic acid are operably linked to the same promoter. In some embodiments, all of the nucleic acids encoding the heterologous proteins or nucleic acids are operably linked to the same promoter. In some embodiments, all of the nucleic acids encoding the heterologous proteins or nucleic acids are operably linked to different promoters.

[0314] In some embodiments, the promoter is a viral promoter. Viral promoters can include, but are not limited to, VV promoter, poxvirus promoter, adenovirus late promoter, Cowpox ATI promoter, or T7 promoter. The promoter may be a vaccinia virus promoter, a synthetic promoter, a promoter that directs transcription during at least the early phase of infection, a promoter that directs transcription during at least the intermediate phase of infection, a promoter that directs transcription during early/late phase of infection, or a promoter that directs transcription during at least the late phase of infection.

[0315] In some embodiments, the promoter described herein is a constitutive promoter. In some embodiments, the promoter described herein is an inducible promoter.

[0316] Promoters suitable for constitutive expression in mammalian cells include but are not limited to the cytomegalovirus (CMV) immediate early promoter (US 5,168,062), the RSV promoter, the adenovirus major late promoter, the phosphoglycerate kinase (PGK) promoter (Adra et al., 1987, Gene 60: 65-74), the thymidine kinase (TK) promoter of herpes simplex virus (HSV)-l and the T7 polymerase promoter (W098/10088). Vaccinia virus promoters are particularly adapted for expression in oncolytic poxviruses. Representative examples include without limitation the vaccinia 7.5K, H5R, 11K7.5 (Erbs et al. , 2008, Cancer Gene Ther. 15(1): 18-28), TK, p28, pll, pB2R, pA35R and K1L promoters, as well as synthetic promoters such as those described in Chakrabarti et al. (1997, Biotechniques 23: 1094-7; Hammond et al, 1997, J. Virol Methods 66: 135-8; and Kumar and Boyle, 1990, Virology 179: 151-8) as well as early/late chimeric promoters. Promoters suitable for oncolytic measles viruses include without limitation any promoter directing expression of measles transcription units (Brandler and Tangy, 2008, CIMID 31: 271).

[0317] Inducible promoters belong to the category of regulated promoters. The inducible promoter can be induced by one or more conditions, such as a physical condition, microenvironment of the host cell, or the physiological state of the host cell, an inducer (i.e., an inducing agent), or a combination thereof.

[0318] Appropriate promoters for expression can be tested in vitro {e.g. in a suitable cultured cell line) or in vivo {e.g. in a suitable animal model or in the subject). When the encoded immune checkpoint modulator(s) comprise(s) an antibody and especially a mAh, examples of suitable promoters for expressing the heavy component of said immune checkpoint modulator comprise CMV, SV and vaccinia virus pH5R, F17R and pllK7.5 promoters; examples of suitable promoters for expressing the light component of said immune checkpoint modulator comprise PGK, beta-actin and vaccinia virus p7.5K, F17R and pA35R promoters.

[0319] Promoters can be replaced by stronger or weaker promoters, where replacement results in a change in the attenuation of the virus. As used herein, replacement of a promoter with a stronger promoter refers to removing a promoter from a genome and replacing it with a promoter that effects an increased the level of transcription initiation relative to the promoter that is replaced. Typically, a stronger promoter has an improved ability to bind polymerase complexes relative to the promoter that is replaced. As a result, an open reading frame that is operably linked to the stronger promoter has a higher level of gene expression. Similarly, replacement of a promoter with a weaker promoter refers to removing a promoter from a genome and replacing it with a promoter that decreases the level of transcription initiation relative to the promoter that is replaced. Typically, a weaker promoter has a lessened ability to bind polymerase complexes relative to the promoter that is replaced. As a result, an open reading frame that is operably linked to the weaker promoter has a lower level of gene expression. The viruses can exhibit differences in characteristics, such as attenuation, as a result of using a stronger promoter versus a weaker promoter. For example, in vaccinia virus, synthetic early/late and late promoters are relatively strong promoters, whereas vaccinia synthetic early, P7.5k early/late, P7.5k early, and P28 late promoters are relatively weaker promoters (see e.g., Chakrabarti et al. (1997) BioTechniques 23 (6) 1094-1097). In some embodiments, the promoter described herein is a weak promoter. In some embodiments, the promoter described herein is a strong promoter.

[0320] In some embodiments, the promoter is a viral promoter of the oncolytic virus. In some embodiments, the promoter is an early viral promoter, a late viral promoter, an intermediate viral promoter, or an early/late viral promoter. In some embodiments, the promoter is a synthetic viral promoter, such as a synthetic early, early/late, or late viral promoter.

[0321] In some embodiments, the promoter is a vaccinia virus promoter. Exemplary vaccinia viral promoters for use in the present invention can include, but are not limited to, P7.sk, Piik, PSE, PSEL, PSL, H5R, TK, P28, C11R, G8R, F17R, I3L, I8R, AIL, A2L, A3L, H1L, H3L, H5L, H6R, H8R, DIR, D4R, D5R, D9R, DHL, D12L, D13L, MIL, N2L, P4b or KI promoters. [0322] Exemplary vaccinia early, intermediate and late stage promoters include, for example, vaccinia P7.sk early/late promoter, vaccinia PEL early/late promoter, vaccinia P13 early promoter, vaccinia Pnk late promoter and vaccinia promoters listed elsewhere herein. Exemplary synthetic promoters include, for example, PSE synthetic early promoter, PSEL synthetic early/late promoter, PSL synthetic late promoter, vaccinia synthetic promoters listed elsewhere herein (Patel et al., Proc. Natl. Acad. Sci. USA 85: 9431-9435 (1988); Davison and Moss, J Mol Biol 210: 749-769 (1989); Davison et al., Nucleic Acids Res. 18: 4285-4286 (1990); Chakrabarti et al. , BioTechniques 23: 1094-1097 (1997)). Combinations of different promoters can be used to express different gene products in the same virus or two different viruses.

[0323] In some embodiments, the promoter directs transcription during at least the late phase of infection (such as F17R promoter, shown in SEQ ID NO: 61) is employed. In some embodiments, the late promoter is selected from the group consisting of F17R, I2L late promoter, L4R late promoter, P7.sk early/late promoter, PEL early/late promoter, Pi ik late promoter, PSEL synthetic early/late promoter, and PSL synthetic late promoter. The late vaccinia viral promoter F17R is only activated after VV infection in tumor cells, thus tumor selective expression of the heterologous protein or nucleic acid from VV will be further enhanced by the use of F17R promoter. In some embodiments, the late expression of a heterologous protein or nucleic acid of the present invention allows for sufficient viral replication before T-cell activation and mediated tumor lysis.

[0324] In some embodiments, the promoter is a hybrid promoter. In some embodiments, the hybrid promoter is a synthetic early/late viral promoter. In some embodiments, the promoter comprises a partial or complete nucleotide sequence of a human promoter. In some embodiments, the human promoter is a tissue or tumor-specific promoter. In some embodiments, the tumor-specific promoter can be a promoter that drives enhanced expression in tumor cells, or that drives expression specifically in tumor cells (e.g., a promoter that drives expression of a tumor tumor-associated antigen (TAA) or a tumor- specific antigen (TSA)). In some embodiments, the hybrid promoter comprises a partial or complete nucleotide sequence of a tissue or tumor- specific promoter and a nucleotide sequence (e.g., a CMV promoter sequence) that increase the strength of the hybrid promoter relative to the tissue- or tumorspecific promoter. Non-limiting examples of hybrid promoters comprising tissue- or tumorspecific promoters include hTERT and CMV hybrid promoters or AFP and CMV hybrid promoters.

[0325] In some embodiments according to any one of the recombinant oncolytic viruses described herein, the one or more promotors comprise a first promoter that is operably linked to the first nucleotide sequence and a second promoter that is operably linked to the second nucleotide sequence. In some embodiments, the first promoter is an F17R promoter and the second promoter is a pE/L promoter. In some embodiments, the F17R promoter comprised the nucleic acid sequence of SEQ ID NO: 61. In some embodiments, the pE/L promoter comprises the nucleic acid sequence of SEQ ID NO: 107.

C. Engineered immune cells

[0326] The present application further provides engineered immune cells for treatment of a cancer in an individual in need thereof. In some embodiments, the engineered immune cells comprise chimeric receptors that specifically recognize a foreign antigen (e.g., a bacterial sialidase) encoded by any one of the recombinant oncolytic viruses described herein.

[0327] In some aspects of the present application, provided are engineered immune cells expressing a chimeric receptor. In some embodiments, the immune cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a suppressor T cell, an NK cell, an NK- T cell and a macrophage. In some embodiments, the engineered immune cell is an NK cell. In some embodiments, the engineered immune cell is a T cell. In some embodiments, the engineered immune cell is a y8T cell. In some embodiments, the engineered immune cell is an NKT cell. In some embodiments, the engineered immune cell is a macrophage. In some embodiments, the engineered immune cell is a mixture of two or more types of immune cells, such as T cells and NK cells. In some embodiments, the engineered immune cell is PBMC.

[0328] Some embodiments of the engineered immune cells described herein comprise one or more engineered chimeric receptors, which are capable of activating an immune cell (e.g., T cell or NK cell) directly or indirectly against a tumor cell expressing a target antigen. Exemplary engineered receptors include, but are not limited to, chimeric antigen receptor (CAR), engineered T cell receptor, and TCR fusion protein.

[0329] In some embodiments, the engineered immune cells are autologous cells (cells obtained from the subject to be treated). In some embodiments, the engineered immune cells are allogeneic cells, which can include a variety of readily isolable and/or commercially available cells/cell lines.

[0330] In some embodiments, the engineered immune cells express a chimeric receptor that specifically recognizes a foreign antigen encoded by the recombinant oncolytic virus. In some embodiments, the engineered immune cells express a chimeric receptor that specifically recognizes a sialidase encoded by the recombinant oncolytic virus. [0331] In some embodiments, the engineered immune cell further comprises a heterologous nucleotide sequence encoding a co-stimulatory ligand. In some embodiments, the costimulatory ligand is a cytokine. Exemplary co-stimulatory ligands include, without limitation, tumor necrosis factor (TNF) ligands, cytokines (such as IL-2, IL-12, IL-15 or IL21), and immunoglobulin (Ig) superfamily ligands. See, for example, US10117897B2, the contents of which are incorporated herein by reference. In some embodiments, the engineered immune cell is an NK cell comprising a heterologous nucleotide sequence encoding IL- 15, such as human IL-15. An exemplary sequence of human IL-15 is SEQ ID NO: 121.

[0332] In some embodiments, the engineered immune cell comprises a vector comprising a first nucleotide sequence encoding a chimeric receptor (e.g., CAR) and a second nucleotide sequence encoding the co-stimulatory ligand (e.g., cytokine such as IL- 15), wherein the first nucleotide sequence and the second nucleotide sequence are linked to each other via a third nucleotide sequence encoding a self-splicing peptide, such as a 2 A peptide, e.g., T2A. An exemplary construct is shown in FIG. 50A.

1. Chimeric antigen receptor (CAR)

[0333] “Chimeric antigen receptor” or “CAR” as used herein refers to an engineered receptor that can be used to graft one or more target-binding specificities onto an immune cell, such as T cells or NK cells. In some embodiments, the chimeric antigen receptor comprises an extracellular target binding domain, a transmembrane domain, and an intracellular signaling domain of a T cell receptor and/or other receptors.

[0334] Some embodiments of the engineered immune cells described herein comprise a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises an antigenbinding moiety and an effector protein or fragment thereof comprising a primary immune cell signaling molecule or a primary immune cell signaling domain that activates the immune cell expressing the CAR directly or indirectly. In some embodiments, the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. Also provided an engineered immune cells (e.g., T cell or NK cell) comprising the CAR. The antigen-binding moiety and the effector protein or fragment thereof may be present in one or more polypeptide chains. Exemplary CAR constructs have been described, for example, in US9765342B2, W02002/077029, and WO2015/142675, which are hereby incorporated by reference. Any one of the known CAR constructs may be used in the present application.

[0335] In some embodiments, the primary immune cell signaling molecule or primary immune cell signaling domain comprises an intracellular domain of a molecule selected from the group consisting of CD3 , FcRy, FcR(3, CD3y, CD35, CD3a, CD5, CD22, CD79a, CD79b, and CD66d. In some embodiments, the intracellular signaling domain consists of or consists essentially of a primary immune cell signaling domain. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain of CD3^. In some embodiments, the CAR further comprises a costimulatory molecule or fragment thereof. In some embodiments, the costimulatory molecule or fragment thereof is derived from a molecule selected from the group consisting of CD27, CD28, 4-1BB, 0X40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds CD83. In some embodiments, the intracellular signaling domain further comprises a co-stimulatory domain comprising a CD28 intracellular signaling sequence. In some embodiments, the intracellular signaling domain comprises a CD28 intracellular signaling sequence and an intracellular signaling sequence of CD3^.

[0336] The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Transmembrane regions of particular use in this invention may be derived from (i.e. comprise at least the transmembrane region(s) of) the CD28, CD3s. CD3^, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD 137, or CD 154. In some embodiments, the CAR is a CD- 19 CAR comprising including CD19 scFv from clone FMC63 (Nicholson IC, et al. Mol Immunol. 1997), a CH2-CH3 spacer, a CD28-TM, 41BB, and CD3^. In some embodiments, the transmembrane domain may be synthetic, in which case it may comprise predominantly hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan and valine may be found at each end of a synthetic transmembrane domain. In some embodiments, a short oligo- or polypeptide linker, having a length of, for example, between about 2 and about 10 (such as about any of 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids in length may form the linkage between the transmembrane domain and the intracellular signaling domain. In some embodiments, the linker is a glycine-serine doublet.

[0337] In some embodiments, the transmembrane domain that is naturally associated with one of the sequences in the intracellular domain is used (e.g., if an intracellular domain comprises a CD28 co-stimulatory sequence, the transmembrane domain is derived from the CD28 transmembrane domain). In some embodiments, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex. [0338] In some embodiments, the CAR comprises a transmembrane domain of CD8. In some embodiments, the CAR comprises a transmembrane domain of CD28.

[0339] In some embodiments, the CAR further comprises a hinge region disposed between the antigen-binding domain and the transmembrane domain. In some embodiments, the hinge region is derived from CD 8.

[0340] The intracellular signaling domain of the CAR is responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been placed in. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. Thus, the term “intracellular signaling domain” refers to the portion of a protein, which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term “intracellular signaling sequence” is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[0341] Examples of intracellular signaling domains for use in the CAR of the present application include the cytoplasmic sequences of the TCR and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability.

[0342] It is known that signals generated through the TCR alone may be insufficient for full activation of the T cell and that a secondary or co-stimulatory signal may also be required. Thus, T cell activation can be said to be mediated by two distinct classes of intracellular signaling sequence: those that initiate antigen-dependent primary activation through the TCR (primary signaling sequences) and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (co-stimulatory signaling sequences).

[0343] Primary signaling sequences regulate primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary signaling sequences that act in a stimulatory manner may contain signaling motifs, which are known as immunoreceptor tyrosine-based activation motifs or IT AMs. The CAR constructs in some embodiments comprise one or more IT AMs. Examples of IT AM containing primary signaling sequences that are of particular use in the invention include those derived from CD3^, FcRy, FcRp, CD3y, CD35, CD3s. CD5, CD22, CD79a, CD79b, and CD66d. [0344] In some embodiments, the CAR comprises a primary signaling sequence derived from CD3^. For example, the intracellular signaling domain of the CAR can comprise the CD3^ intracellular signaling sequence by itself or combined with any other desired intracellular signaling sequence(s) useful in the context of the CAR described herein. For example, the intracellular domain of the CAR can comprise a CD3^ intracellular signaling sequence and a costimulatory signaling sequence. The costimulatory signaling sequence can be a portion of the intracellular domain of a costimulatory molecule including, for example, CD27, CD28, 4- 1BB (CD137), 0X40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA- 1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, and the like.

[0345] In some embodiments, the intracellular signaling domain of the CAR comprises the intracellular signaling sequence of CD3^ and the intracellular signaling sequence of CD28. In some embodiments, the intracellular signaling domain of the CAR comprises the intracellular signaling sequence of CD3^ and the intracellular signaling sequence of 4-1BB. In some embodiments, the intracellular signaling domain of the CAR comprises the intracellular signaling sequence of CD3^ and the intracellular signaling sequences of CD28 and 4-1BB. [0346] In some embodiments, the antigen-binding domain is an antibody fragment. In some embodiments, the antigen binding moiety comprises a scFv or a Fab. In some embodiments, the antigen binding moiety is directed to a foreign antigen that is delivered to tumor cells (e.g., by a recombinant oncolytic virus). In some embodiments, the foreign antigen is DAS181 or its derivatives (e.g. a transmembrane form of the sialidase domain of DAS 181 without anchoring domain, as described in Examples 11 and 15).

[0347] In some embodiments, the sialidase domain (e.g., a non-human sialidase or a derivative thereof, such as a sialidase domain of DAS 181) delivered to tumor cells using an oncolytic virus functions both to remove sialic acid from the surface of tumor cells and as a foreign antigen that enhances immune cell-mediated killing of tumor cells. In some embodiments, the sialidase-armed oncolytic virus is combined with an engineered immune cell that specifically targets the sialidase domain (e.g., DAS181), thereby enhancing killing of tumor cells infected by the oncolytic virus.

[0348] In some embodiments, the antigen-binding domain specifically binds to a sialidase, such as avSial. In some embodiments, the antigen-binding domain specifically binds to DAS181 or a derivative thereof. In some embodiments, the antigen- binding domain is an antibody fragment derived from any one of the anti-sialidase antibodies derived in Section III “Anti-sialidase antibodies” below. In some embodiments, the antigen-binding domain is a scFv of D004.

[0349] In some embodiments, the CAR comprises an antigen-binding domain that specifically binds Actinomyces viscosus sialidase (e.g., DAS181 or a derivative thereof), wherein the antigen-binding domain comprises a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 111, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 112, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 113; and a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 114, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 115, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 116. In some embodiments, the antigen-binding domain comprises a VH comprising an amino acid sequence having at least any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% identity to the amino acid sequence of SEQ ID NO: 117, and a VL comprising an amino acid sequence having at least any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% identity to the amino acid sequence of SEQ ID NO: 118. In some embodiments, the antigen-binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 117, and a VL comprising the amino acid sequence of SEQ ID NO: 118. In some embodiments, the antigen-binding domain comprises a scFv comprising an amino acid sequence having at least any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% identity to the amino acid sequence of SEQ ID NO: 119. In some embodiments, the antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 119.

[0350] In some embodiments, there is provided a CAR comprising from the N-terminus to the C-terminus: an anti-sialidase scFv (such as a D004 scFv), a CD8 hinge region, a CD8 transmembrane domain, a co-stimulatory domain of CD28, and an intracellular signaling domain of CD3^. An exemplary CAR construct is shown in FIG. 50A. In some embodiments, the CAR comprises an amino acid sequence having at least any one of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% identity to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 120.

[0351] Also provided herein are engineered immune cells (such as lymphocytes, e.g., T cells, NK cells, or combinations thereof) expressing any one of the CARs described herein. Also provided is a method of producing an engineered immune cell expressing any one of the CARs described herein, the method comprising introducing a vector comprising a nucleic acid encoding the CAR into the immune cell. In some embodiments, introducing the vector into the immune cell comprises transducing the immune cell with the vector. In some embodiments, introducing the vector into the immune cell comprises transfecting the immune cell with the vector. Transduction or transfection of the vector into the immune cell can be carried about using any method known in the art.

2. Engineered T cell receptor

[0352] In some embodiments, the chimeric receptor is a T cell receptor. In some embodiments, wherein the engineered immune cell is a T cell, the T cell receptor is an endogenous T cell receptor. In some embodiments, the engineered immune cell with the TCR is pre-selected. In some embodiments, the T cell receptor is a recombinant TCR. In some embodiments, the TCR is specific for the foreign antigen (e.g., sialidase) encoded by the oncolytic virus. In some embodiments, the TCR has an enhanced affinity to the foreign antigen. Exemplary TCRs and methods for introducing the TCRs to immune cells have been described, for example, in US5830755, and Kessels et al. Immunotherapy through TCR gene transfer. Nat. Immunol. 2, 957-961 (2001). In some embodiments, the engineered immune cell is a TCR-T cell.

3. TCR fusion protein (TFP)

[0353] In some embodiments, the engineered immune cell comprises a TCR fusion protein (TFP). “TCR fusion protein” or “TFP” as used herein refers to an engineered receptor comprising an extracellular target-binding domain fused to a subunit of the TCR-CD3 complex or a portion thereof, including TCRoc chain, TCR|3 chain, TCRy chain, TCR8 chain, CD3E. CD38, or CD3y. The subunit of the TCR-CD3 complex or portion thereof comprise a transmembrane domain and at least a portion of the intracellular domain of the naturally occurring TCR-CD3 subunit. The TFP comprises the extracellular domain of the TCR-CD3 subunit or a portion thereof.

[0354] Exemplary TFP constructs comprising an antibody fragment as the target-binding moiety have been described, for example, in WO2016187349 and WO2018098365, which are hereby incorporated by reference.

4. Targeting Engineered Immune Cells to Foreign Antigens.

[0355] The engineered immune cells described herein can be targeted to a foreign antigen. In some embodiments, engineered immune cells can be targeted to a foreign antigen (e.g., a bacterial peptide or a bacterial sialidase) that is delivered to tumor cells using a recombinant oncolytic virus. [0356] Engineered immune cells can be delivered to the patient in any way known in the art for delivering engineered immune cells (e.g. , CART-T, CAR-NK, or CAR-NKT cells). In some embodiments, sialidase expressed on the surface of or secreted by sialidase expressing engineered immune cells may remove sialic acids from sialoglycans expressed on immune cells and/or tumor cells. The removal of the sialic acid on tumor cell can further activate the Dendritic cells, macrophages, T and NK cell that are no longer engaged with the inihibitory signals of the tumor cells via Siglecs/sialic acid axis and other Selectins interactions. These interactions can further enhance immune activation against cancer and change the tumor microenvironment (TME). With respect to tumor cells, as they are desialylated, they become exposed to attack by activated NK cells and T cells and other immune cells, resulting in reduction in tumor size.

[0357] In some embodiments, the engineered immune cells set forth herein can be engineered to express sialidase, such as, without limitation, sialidase domain of DAS181 fused to a transmembrane domain, on the immune cell surface membrane, such that the sialidase is membrane bound. In some embodiments, the sialidase can be fused to a transmembrane domain. [0358] Without being bound by any theory or hypothesis, membrane bound sialidases will not be freely circulating and will only come into contact with the tumor cells expressing the oncolytic virus. In this way, the sialidases will not desialylate non-targeted cells, such as erythrocytes, but will instead eliminate sialic acid primarily only from tumor cells.

5. Engineered immune cell compositions

[0359] The present application also provides a composition comprising engineered immune cells expressing a chimeric receptor that specifically recognizes a foreign antigen encoded by the recombinant oncolytic virus. In some embodiments, there is provide a composition comprising engineered immune cells expressing a chimeric receptor that specifically recognizes a sialidase, such as Actinomyces viscosus sialidase, e.g., DAS 181 or a derivative thereof. In some embodiments, there is provided a composition comprising NK cells expressing a chimeric receptor that specifically recognizes a sialidase, such as Actinomyces viscosus sialidase, e.g., DAS181 or a derivative thereof. In some embodiments, there is provided a composition comprising NK cells expressing a CAR comprising an anti-DAS181 scFv (e.g., D004 scFv), a transmembrane domain and an intracellular domain e.g., a co-stimulatory domain of CD28 and an intracellular signaling domain of CD3( .

[0360] In some embodiments, there is provided a method of preparing a composition of engineered NK cells expressing a CAR, comprising: (a) activating peripheral blood cells; (b) transducing the activated peripheral blood cells with a vector encoding the CAR; and (c) incubating the transduced cells in NK MACS medium.

D. Oncolytic Virus and Carrier Cell

[0361] In some embodiments, the present application provides a carrier cell comprising any one of the recombinant oncolytic viruses described herein. In some embodiments, the carrier cell is an immune cell or a stem cell (e.g., a mesenchymal stem cell). In some embodiments, the carrier cell is a B cell. In some embodiments, the carrier cell is a leukocyte. In some embodiments, the engineered immune cell is a Chimeric Antigen Receptor (CAR)-T, CAR- NK, CAR-NKT, or CAR-macrophage cell. In some embodiments, the immune cell is an engineered immune cell, such as any of the engineered immune cells described in subsection C above.

[0362] In some embodiments, there is provided a composition comprising an engineered immune cell comprising a recombinant oncolytic virus encoding a sialidase. In some embodiments, the recombinant oncolytic virus is a vaccinia virus. In some embodiments, the vaccinia virus is a Western Reserve strain. In some embodiments, the vaccinia virus is a modified vaccinia virus (e.g., a vaccinia virus comprising one or more mutations, wherein the mutations are in one or more proteins such as A14, A17, A13, LI, H3, D8, A33, B5, A56, F13, or A28). In some embodiments, the sialidase is derived from an Actinomyces viscosus sialidase. In some embodiments, the sialidase is DAS181. In some embodiments, the nucleotide sequence encoding the sialidase further encodes a secretion sequence operably linked to the sialidase. In some embodiments, the sialidase further comprises a transmembrane domain. In some embodiments, the engineered immune cell encodes a chimeric receptor. In some embodiments, the chimeric receptor is a chimeric antigen receptor. In some embodiments, the CAR specifically recognizes the sialidase encoded by the oncolytic virus. In some embodiments, the engineered immune cell is a cytotoxic T cell, a helper T cell, a suppressor T cell, an NK cell, and an NK-T cell. In some embodiments, the engineered immune cell is an autologous cell of a patient or an allogeneic cell.

[0363] The present application further provides immune cells comprising any one of the recombinant oncolytic viruses provided herein. In some embodiments, the immune cells comprising a recombinant oncolytic virus are prepared by incubating the immune cells with the recombinant oncolytic virus. In some embodiments, the immune cells comprising a recombinant oncolytic virus are prepared by engineering a nucleotide sequence encoding the recombinant oncolytic virus into the cells (e.g., by transducing or transfecting the cells with the construct).

[0364] The population of carrier cells (e.g., immune cells or stem cells) can be infected with the oncolytic virus. The sialidase containing virus may be administered in any appropriate physiologically acceptable cell carrier. The multiplicity of infection will generally be in the range of about 0.001 to 1000, e.g., in the range of 0.001 to 100. The virus-containing cells may be administered one or more times. Alternatively, viral DNA may be used to transfect the effector cells, employing liposomes, general transfection methods that are well known in the art (such as calcium phosphate precipitation and electroporation), etc. Due to the high efficiency of transfection of viruses, one can achieve a high level of modified cells. In some embodiments, the engineered immune cell comprising the recombinant oncolytic virus can be prepared by incubating the immune cell with the virus for a period of time. In some embodiments, the immune cell can be incubated with the virus for a time sufficient for infection of the cell with virus, and expression of the one or more virally encoded heterologous protein(s) (e.g., sialidase and/or any of the immunomodulatory proteins described herein).

[0365] The population of carrier cells (e.g., immune cells or stem cells) comprising the recombinant oncolytic virus may be injected into the recipient. Determination of suitability of administering cells of the invention will depend, inter alia, on assessable clinical parameters such as serological indications and histological examination of tissue biopsies. Generally, a pharmaceutical composition is administered. Routes of administration include systemic injection, e.g. intravascular, subcutaneous, or intraperitoneal injection, intratumor injection, etc.

III. Anti-sialidase antibodies

[0366] The present application provides anti-sialidase antibodies and antigen-binding fragments thereof. In some embodiments, the anti-sialidase antibodies and antigen-binding fragments thereof described herein specifically bind Actinomyces viscosus sialidase, also referred herein as “avSial” or “avSialidase.” In some embodiments, the present application provides anti-DAS181 antibodies and antigen-binding fragments thereof.

[0367] In some embodiments, the anti-sialidase antibody or antigen-binding fragment comprises one, two, three, four, five, or all six of the CDRs shown for any of an exemplary anti-avSial antibody D004 (also referred herein as “illustrative anti-sialidase antibody” or “parental anti-sialidase antibody”) described in Table A, below. [0368] In some embodiments, the anti-sialidase antibody or antigen-binding fragment comprises one, two, three, four, five, or six CDRs of antibody D004 as shown in Table A. In some embodiments, the anti-sialidase antibody or antigen-binding fragment comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 117. In certain embodiments, a VH sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 117, but retains the ability to bind avSial as the antibody comprising SEQ ID NO: 117. In certain embodiments, a total of 1 to 13 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13) amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 117. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In a particular embodiment, the VH comprises one, two or three CDRs selected from the group consisting of: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 111, or a variant thereof comprising up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, (b) a CDR- H2 comprising the amino acid sequence of SEQ ID NO: 112, or a variant thereof comprising up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 113, or a variant thereof comprising up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions.

[0369] In some embodiments, the anti-sialidase antibody or antigen-binding fragment comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 118. In certain embodiments, a VL sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the amino acid sequence of SEQ ID NO: 118, but retains the ability to bind avSial as the antibody comprising SEQ ID NO: 118. In certain embodiments, a total of 1 to 11 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 118. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In a particular embodiment, the VL comprises one, two or three CDRs selected from the group consisting of (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 114, or a variant thereof comprising up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; (b) a CDR- L2 comprising the amino acid sequence of SEQ ID NO: 115, or a variant thereof comprising up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 116, or a variant thereof comprising up to 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions. [0370] In one embodiment, the anti-sialidase antibody or antigen-binding fragment comprises a VH comprising the amino acid sequence of SEQ ID NO: 117, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 117; and a VL comprising the amino acid sequence of SEQ ID NO: 118, or an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 118.

[0371] In some embodiments, the anti-sialidase antibody or antigen-binding fragment comprises a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 111, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 112, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 113; and a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 114, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 115, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 116.

[0372] In some embodiments, the anti-sialidase antibody or antigen-binding fragment comprises a CDR-H1, a CDR-H2, and a CDR-H3 of a VH having the sequence set forth in SEQ ID NO: 117; and a CDR-L1, a CDR-L2, and a CDR-L3 of a VL having the sequence set forth in SEQ ID NO: 118.

Table A. anti-avSial antibody sequences

[0373] In some embodiments, the anti-sialidase antibody is a full-length antibody. In some embodiments, the anti-sialidase antibody comprises an Fc region of an immunoglobulin, such as a human IgGl, IgG2, IgG3 or IgG4. In some embodiments, the anti-sialidase antibody is an antigen-binding fragment.

[0374] In some embodiments, the anti-sialidase antigen-binding fragment is a scFv comprising from the N-terminus to the C-terminus, a VH, a peptide linker, and a VL. In some embodiments, the anti-sialidase antigen-binding fragment is a scFv comprising from the N- terminus to the C-terminus, a VL, a peptide linker, and a VH.

[0375] In some embodiments, the anti-sialidase antigen-binding fragment comprises an amino acid sequence having at least 80% (e.g. , at least 85%, 90%, 95%, 98%, or 99%; or 100%) sequence identity with SEQ ID NO: 119. In some embodiments, the anti-sialidase antigenbinding fragment comprises the amino acid sequence of SEQ ID NO: 119.

[0376] The anti-sialidase antibodies of the present disclosure may be produced using any techniques known in the art, including conventional monoclonal antibody methodology e.g., a standard somatic cell hybridization technique (see e.g., Kohler and Milstein, Nature 256:495 (1975)), viral or oncogenic transformation of B lymphocytes, or recombinant antibody technologies.

[0377] Hybridoma production is a very well-established procedure. The common animal system for preparing hybridomas is the murine system. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known. One well-known method that may be used for making human antibodies provided by the present disclosure involves the use of a XenoMouse™ animal system. XenoMouse™ mice are engineered mouse strains that comprise large fragments of human immunoglobulin heavy chain and light chain loci and are deficient in mouse antibody production (see e.g., Green et al., (1994) Nature Genetics 7:13-21; W02003/040170). Immunization of animals can be carried out by any method known in the art (see e.g., Harlow and Lane, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Press, 1990). Methods for immunizing non-human animals such as mice, rats, sheep, goats, pigs, cattle and horses are well known in the art (see e.g., Harlow and Lane, supra, and U.S. Pat. No. 5,994,619). The sialidase antigen may be administered with an adjuvant to stimulate the immune response. Exemplary adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptides) or ISCOM (immunostimulating complexes). After immunization of an animal with a sialidase antigen, antibody-producing immortalized cell lines are prepared from cells isolated from the immunized animal. After immunization, the animal is sacrificed and lymph node and/or splenic B cells are immortalized. Methods of immortalizing cells include, but are not limited to, transferring them with oncogenes, inflecting them with the oncogenic virus cultivating them under conditions that select for immortalized cells, subjecting them to carcinogenic or mutating compounds, fusing them with an immortalized cell, e.g., a myeloma cell, and inactivating a tumor suppressor gene (see e.g., Harlow and Lane, supra). If fusion with myeloma cells is used, the myeloma cells preferably do not secrete immunoglobulin polypeptides (a non-secretory cell line). Immortalized cells are screened using sialidase, a portion thereof, or a cell expressing sialidase. Anti-sialidase antibody-producing cells, e.g., hybridomas, are selected, cloned and further screened for desirable characteristics, including robust growth, high antibody production and desirable antibody characteristics, as discussed further below. Hybridomas can be expanded in vivo in syngeneic animals, in animals that lack an immune system, e.g., nude mice, or in cell culture in vitro. Methods of selecting, cloning and expanding hybridomas are well known to those of ordinary skill in the art.

[0378] Anti-sialidase antibodies of the present disclosure may also be prepared using phage display or yeast display methods. Such display methods for isolating human antibodies are established in the art (see e.g., Knappik, et al. (2000) J. Mol. Biol. 296, 57-86; Feldhaus et al. (2003) Nat Biotechnol 21:163-170.

[0379] In some further embodiments, the present disclosure provides derivatives of any of the anti-sialidase antibodies described herein. In some embodiments, the anti-sialidase antibody derivative is derived from modifications of the amino acid sequences of an illustrative anti-sialidase antibody of the present disclosure while conserving the overall molecular structure of the parental antibody amino acid sequence. Amino acid sequences of any regions of the parental antibody chains may be modified, such as framework regions, CDR regions, or constant regions. Types of modifications include substitutions, insertions, deletions, or combinations thereof, of one or more amino acids of the parental antibody.

[0380] Amino acid substitutions encompass both conservative substitutions and nonconservative substitutions. The term “conservative amino acid substitution” means a replacement of one amino acid with another amino acid where the two amino acids have similarity in certain physico-chemical properties such as polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, substitutions typically may be made within each of the following groups: (a) nonpolar (hydrophobic) amino acids, such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; (b) polar neutral amino acids, such as glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; (c) positively charged (basic) amino acids, such as arginine, lysine, and histidine; and (d) negatively charged (acidic) amino acids, such as aspartic acid and glutamic acid.

[0381] The modifications may be made in any positions of the amino acid sequences of the anti-sialidase antibody, including the CDRs, framework regions, or constant regions. In one embodiment, the present disclosure provides an anti-sialidase antibody derivative that contains the Vn and VL CDR sequences of an illustrative anti-sialidase antibody of this disclosure, yet contains framework sequences different from those of the illustrative anti-sialidase antibody. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the Genbank database or in the “VBase” human germline sequence database (Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 (1991); Tomlinson et al., J. Mol. Biol. 227:776-798 (1992); and Cox et al., Eur. J. Immunol. 24:827-836 (1994)). Framework sequences that may be used in constructing an antibody derivative include those that are structurally similar to the framework sequences used by illustrative antibodies of the disclosure For example, the CDR- Hl, CDR-H2, and CDR-H3 sequences, and the CDR-E1, CDR-E2, and CDR-E3 sequences of an illustrative antibody can be grafted onto framework regions that have the identical sequence as that found in the germline immunoglobulin gene from which the framework sequence derive, or the CDR sequences can be grafted onto framework regions that contain one or more mutations as compared to the germline sequences.

[0382] In some embodiments, the anti-sialidase antibody derivative is a chimeric anti- sialidase antibody which comprises an amino acid sequence of an illustrative antibody of the disclosure. In one example, one or more CDRs from one or more illustrative anti-sialidase antibodies are combined with CDRs from an anti-sialidase antibody from a non-human animal, such as mouse or rat. In another example, all of the CDRs of the chimeric anti-sialidase antibody are derived from one or more illustrative anti-sialidase antibodies. In some particular embodiments, the chimeric anti-sialidase antibody comprises one, two, or three CDRs from the heavy chain variable region and/or one, two, or three CDRs from the light chain variable region of an illustrative anti-sialidase antibody. Chimeric antibodies can be generated using conventional methods known in the art.

[0383] Another type of modification is to mutate amino acid residues within the CDR regions of the VH and/or VL chain. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation(s) and the effect on antibody binding, or other functional property of interest, can be evaluated in in vitro or in vivo assays known in the art. Typically, conservative substitutions are introduced. The mutations may be amino acid additions and/or deletions. Moreover, typically no more than one, two, three, four or five residues within a CDR region are altered. In some embodiments, the anti-sialidase antibody derivative comprises 1, 2, 3, or 4 amino acid substitutions in the heavy chain CDRs and/or in the light chain CDRs. In another embodiment, the amino acid substitution is to change one or more cysteines in an anti- sialidase antibody to another residue, such as, without limitation, alanine or serine. The cysteine may be a canonical or non-canonical cysteine. In one embodiment, the anti-sialidase antibody derivative has 1, 2, 3, or 4 conservative amino acid substitutions in the heavy chain HVR regions relative to the amino acid sequences of an illustrative anti-sialidase antibody.

[0384] Modifications may also be made to the framework residues within the VH and/or VL regions. Typically, such framework variants are made to decrease the immunogenicity of the antibody. One approach is to “back mutate” one or more framework residues to the corresponding germline sequence. An antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived. To return the framework region sequences to their germline configuration, the somatic mutations can be “back mutated” to the germline sequence by, for example, site-directed mutagenesis or PCR-mediated mutagenesis.

IV. Pharmaceutical compositions, kits and articles of manufacture

[0385] Further provided by the present application are pharmaceutical compositions comprising any one of the recombinant oncolytic viruses, carrier cells comprising a recombinant oncolytic virus, and/or engineered immune cells (s) described herein, and a pharmaceutically acceptable carrier.

[0386] In some embodiments, there is provided a composition (e.g., a pharmaceutical composition) comprising (a) a recombinant oncolytic virus comprising a nucleotide sequence encoding a foreign antigen; and (b) an engineered immune cell expressing a chimeric receptor specifically recognizing said foreign antigen. In some embodiments, the foreign antigen is a bacterial antigen. In some embodiments, the foreign antigen is a sialidase such as DAS181.

[0387] In some embodiments, the present application provides a pharmaceutical composition comprising an oncolytic virus (such as VV) comprising a first nucleotide sequence encoding a sialidase and optionally any one or more of the other heterologous proteins or nucleic acids described herein, and an engineered immune cell expressing a chimeric receptor (e.g., a CAR- T, CAR-NK, or CAR-NKT cell) that specifically binds the sialidase. In some embodiments, the one or more heterologous protein or nucleic acid can modulate and enhance immune cell function such as anti LILRB, Anti-folate receptor beta, bi-specific antibody such as anti- LILRB/4-1BB, etc.

[0388] In some embodiments, the present application provides a first pharmaceutical composition comprising a recombinant oncolytic virus (such as VV) comprising a first nucleotide sequence encoding a sialidase and optionally any one or more of the other heterologous proteins or nucleic acids described herein, and optionally a pharmaceutically acceptable carrier; and a second pharmaceutical composition comprising an engineered immune cell expressing a chimeric receptor (e.g., a CAR-T, CAR-NK, or CAR-NKT cell) that specifically binds the sialidase, and optionally a pharmaceutically acceptable carrier.

[0389] In some embodiments, there is provided a pharmaceutical composition comprising: (a) a recombinant oncolytic virus comprising a nucleotide sequence encoding a sialidase (e.g., DAS181 or a derivative thereof); (b) NK cells expressing a CAR specifically recognizing the sialidase; and (c) a pharmaceutically acceptable carrier. In some embodiments, the oncolytic virus is a vaccinia virus. In some embodiments, the oncolytic virus further comprises a nucleotide sequence encoding a multispecific immune cell engager, such as a bispecific antibody that specifically binds FAP and CD3E. In some embodiments, the NK cell further expresses IL- 15.

[0390] Pharmaceutical compositions can be prepared by mixing the recombinant oncolytic viruses and/or engineered immune cells described herein having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers, antioxidants including ascorbic acid, methionine, Vitamin E, sodium metabisulfite; preservatives, isotonicifiers (e.g. sodium chloride), stabilizers, metal complexes (e.g. Zn-protein complexes); chelating agents such as EDTA and/or non-ionic surfactants.

[0391] The formulation can include a carrier. The carrier is a macromolecule which is soluble in the circulatory system and which is physiologically acceptable where physiological acceptance means that those of skill in the art would accept injection of said carrier into a patient as part of a therapeutic regime. The carrier preferably is relatively stable in the circulatory system with an acceptable plasma half-life for clearance. Such macromolecules include but are not limited to soy lecithin, oleic acid and sorbitan trioleate.

[0392] The formulations can also include other agents useful for pH maintenance, solution stabilization, or for the regulation of osmotic pressure. Examples of the agents include but are not limited to salts, such as sodium chloride, or potassium chloride, and carbohydrates, such as glucose, galactose or mannose, and the like.

[0393] In some embodiments, the pharmaceutical composition is contained in a single -use vial, such as a single-use sealed vial. In some embodiments, the pharmaceutical composition is contained in a multi-use vial. In some embodiments, the pharmaceutical composition is contained in bulk in a container. In some embodiments, the pharmaceutical composition is cryopreserved.

[0394] In some embodiments, the systems provided herein can be stably and indefinitely stored under cryopreservation conditions, such as, for example, at -80 °C, and can be thawed as needed or desired prior to administration. For example, the systems provided herein can be stored at a preserving temperature, such as - 20 °C or -80 °C, for at least or between about a few hours,. 1, 2, 3, 4 or 5 hours, or days, including at least or between about a few years, such as, but not limited to, 1 , 2, 3 or more years, for example for at least or about 1, 2, 3, 4 or 5 hours to at least or about 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 or 72 hours or 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 days or 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5 or 12 months or 1, 2, 3, 4 or 5 or more years prior to thawing for administration. The systems provided herein also stably can be stored under refrigeration conditions such as, at 4 °C and/or transported on ice to the site of administration for treatment. For example, the systems provided herein can be stored at 4 °C or on ice for at least or between about a few hours, such as, but not limited to, 1 , 2, 3, 4 or 5 hours, to at least or about 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 or more hours prior to administration for treatment.

[0395] The present application further provides kits and articles of manufacture for use in any embodiment of the treatment methods described herein. The kits and articles of manufacture may comprise any one of the formulations and pharmaceutical compositions described herein.

[0396] In some embodiments, there is provided a kit comprising one or more nucleic acid constructs for expression any one of the recombinant oncolytic viruses described herein, and instructions for producing the recombinant oncolytic virus. In some embodiments, the kit further comprises instructions for treating a cancer.

[0397] In some embodiments, there is provided a kit comprising any one of the recombinant oncolytic viruses described herein and any one of the engineered immune cells expressing a chimeric receptor (e.g. , CAR-NK cells) described herein, and instructions for treating a cancer. In some embodiments, the kit further comprises an additional immunotherapeutic agent (e.g., an immune checkpoint inhibitor). In some embodiments, the kit further comprises one or more additional therapeutic agents for treating the cancer. In some embodiments, the antagonist, the recombinant oncolytic virus and the engineered immune cells and optionally the additional immunotherapeutic agent(s) are in a single composition (e.g., a composition comprising the engineered immune cells and a recombinant oncolytic virus). In some embodiments, the recombinant oncolytic virus and the engineered immune cells and optionally the additional immunotherapeutic agent(s) for treating the cancer are in separate compositions.

[0398] The kits of the invention are in suitable packaging. Suitable packaging includes, but is not limited to, vzals, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Kits may optionally provide additional components such as buffers and interpretative information. The present application thus also provides articles of manufacture, which include vials (such as sealed vials), bottles, jars, flexible packaging, and the like.

[0399] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

EXAMPLES

[0400] The examples below are intended to be purely exemplary of the invention and should therefore not be considered to limit the invention in any way. The following examples and detailed description are offered by way of illustration and not by way of limitation.

Example 1: DAS181 Treatment Reduces Surface Sialic Acid on Tumor Cells

[0401] In this study the impact of DAS181 on the sialic acid burden of certain tumor cells was examined. Briefly, FACs and image-based quantitation of a-2,3 and a-2,6 sialic acid modifications on A549 (human alveolar basal epithelial adenocarcinoma) and MCF (human mamillary epithelial adenocarcinoma) tumor cells were conducted. Galactose exposure after sialic acid removal in A549 and MCF7 cells was detected by PNA-FITC using flow cytometry analysis and imaging approaches. As discussed above, there are two sialic acid is most often attached to the penultimate sugar by an a-2,3 linkage or an a-2,6 linkage, which can that can be detected by Maackia Amurensis Lectin II (MAL II) and Sambucus Nigra Lectin (SNA), respectively. In addition, surface galactose (e.g., galactose exposed after sialic acid removal) can be detected using Peanut Agglutinin (PNA).

[0402] FIG 1 depicts the detection of a-2,6 sialic acid by FITC-SNA on A549 and MCF cells by fluorescence imaging. [0403] A549 cells were treated with various concentrations of DAS181 and them stained to image 2,6 linked sialic acid (FITC-SNA), a-2,3 linked sialic acid (FITC-MALII) or galactose (FITC-PNA). As can be seen in FIG 2, DAS181 effectively removed both 2,3 and 2,6 linked sialic acid and exposed galactose.

[0404] In contrast, DAS 185, a variant of DAS181 lacking sialidase activity due to Y348F mutation, was not able to remove a-2,6 linked sialic acid or a-2,3 linked sialic acid. As shown in FIG 3, incubation of A549 cells with DAS 185 had essentially no impact on surface a-2,3 linked sialic acid, while DAS181 reduced surface a-2,3 linked sialic acid in a concentration dependent manner (cells stained with FITC-MALII; results shown in FIG. 3). Similarly, incubation of A549 cells with DAS 185 had essentially no impact on surface a2,6 linked sialic acid, while DAS181 reduced surface a-2,6 linked sialic acid in a concentration dependent manner (cells stained with FITC-SNA; results shown in FIG 4). Consistent with these results, incubation of A549 cells with DAS 185 had essentially no impact on surface galactose, while DAS181 increased surface galactose in a concentration dependent manner (cells stained with FITC-PNA; results shown in FIG. 5).

Example 2: DAS181 Treatment Increases PBMC-Mediated Tumor Cell Killing

[0405] Example 1 demonstrated that DAS181 effectively reduces the sialic acid burden of tumor cells with broad specificity (e.g., cleaving both a-2,3 vs. a-2,6 linkages). Example 2 demonstrates that treatment of tumor cells with DAS 181 significantly enhances PBMC- mediated killing of the treated tumor cells compared to untreated tumor cells.

[0406] Briefly, FACs and image-based quantitation of a-2,3 and a-2,6 sialic acid

[0407] A549 cells were genetically labelled with a red fluorescent protein (A549-red). Fresh human PBMCs were harvested and stimulated with various cytokine and antibody combinations to activate effector T cells (CD3, CD38 and IL-2) or, in some cases, T cells and NK cells (CD3, CD28, IL-15 and IL-21). Activated PBMCs were then co-cultured with A549- red cells that had been exposed to DAS181 (100 nM). Tumor cell killing by PBMCs was monitored by live cell imaging and quantification with IncuCyte. The cell culture medium was collected and analyzed by ELISA to assess cytokine production by PBMCs.

[0408] FIG. 6 shows that neither the treatments used to stimulate PBMC nor DAS181 in combination with treatment used to stimulate PBMC impact A549-red cell proliferation.

[0409] FIG. 7 shows that DAS181 significantly increases tumor cell toxicity mediated by PBMC (Donor 1), both T cell mediated and NK cell mediated, compared to a vehicle only control. Similar results were observed using PBMC from a different donor (Donor 2; FIG. 8). FIGS. 9A-C presents a quantification of the data presented in FIG. 7. FIG. 9A shows quantification of A549-red cells following treatment with PBMCs with or without DAS181 at the indicated effector cell : tumor cell ratios. FIG. 9B shows quantification of A549-red cells following treatment with PBMCs stimulated with CD3, CD38 and IL-2 to activate effector T cells with or without DAS181 at the indicated effector cell : tumor cell ratios. FIG. 9C shows quantification of A549-red cells following treatment with PBMCs stimulated with CD3, CD28, IL- 15 and IL-21 to activate effector T and NK cells with or without DAS181 at the indicated effector cell : tumor cell ratios. FIGS. 10A-10C show the same quantifications, respectively, using PBMCs from a different donor (Donor 2).

Example 3: NK Cell Mediated Killing of Tumor Cells by Oncolytic Vaccinia Virus and DAS181

[0410] In this study the impact of an oncolytic vaccinia virus (Western Reserve, VV) and DAS181 on NK cell-mediated killing was examined. DAS185, a variant protein lacking sialidase activity was used as a control. This Example demonstrates that exposure to DAS181 increases tumor cell killing by an oncolytic virus.

[0411] Briefly, tumor cells (U87-GFP) were plated in a 96-well tissue culture plate at 5x10⁴ cells per well (lOOul) in DMEM and incubated overnight at 37°C. On Day 2 the cells were infected with VV at MOI 0.5, 1, or 2 in fetal bovine serum-free medium for 2 hours and then exposed to InM DAS181 or 1 mM DAS185. Tumor cells were then mixed with purified NK cells at Effector:Tumor (E:T) = 1:1, 5:1, 10:1. The cells were cultured in medium supplemented with 2% FBS in order to decrease neuraminidase/sialidase background. After 24 hrs, tumor killing were measured by MTS assay (96 well plate), and cell culture medium was collected. Expression of IFN gamma were measured by ELISA. The results of this study are shown in FIG. 11 and FIG. 12 where it can be seen the DAS181, but not inactive DAS 185, increased tumor cell killing by oncolytic vaccinia virus.

Example 4: Impact of DAS181 on DC Maturation and Macrophage activity in the Presence of Tumor Cells

[0412] In this study, the impact of DAS181 on monocyte-derived dendritic cells or macrophages was examined. DAS185, a variant protein lacking sialidase activity was used as a control.

[0413] Briefly, monocyte-derived dendritic cells (DC) were prepared by resuspending 5xl0⁶ adherent PBMC in 3 ml of medium supplemented with 100 ng/ml of GM-CSF and 50 ng/ml of IL-4. After 48 hrs, 2 ml of fresh medium supplemented with 100 ng/ml of GM-CSF and 50 ng/ml of IL-4 was added to each well. After another 72 hrs, tumor cells (U87-GFP) were plated in 24-well plates in DMEM. The tumor cells were infected with VV at various MOI in FBS free medium for 2 hours. DC cultured in the presence of InM DAS181 or DAS185 were mixed with tumor cells at 1:1 tumor cell:DC ratio. Dendritic cell maturation (expression of CD86, CD80, MHC-II, MHC-I).

[0414] In addition, THP-1 cells were cultured in RPMI 1640 medium (Invitrogen) containing 10% heat-inactivated FBS. THP-1 cells in a 6-well plate (3xl0e6 cells/well) were stimulated with PMA (20 ng/ml) in the absence and in the presence of InM of Sialidase DAS181 or DAS185. Cell culture medium volume was 2ml. On day 5, tumor cells (U87- GFP, DMEM cell culture medium) were plated in a 24-well tissue culture plate. Tumor were infected with VV at various MOI (i.e. 0.5, 1, 2) in FBS free medium for 2 hours. For THP-1 cell culture, 1.5 ml cell culture medium was removed by pipette. The differentiated THP-1 cells were further stimulated for 12 h by ionomycin (lug/ml) and PMA (20 ng/ml) also in the absence and in the presence of InM of Sialidase DAS181 or DAS185 and tumor cells- VV at tumor macrophage ratio of 1:1. The THP-1 cells were cultured in medium supplemented with 2% FBS in order to decrease neuraminidase background. On day 6, the concentration of cytokine in the culture medium was measured by ELISA array.

[0415] As can be seen in FIG 13, DAS181 significant enhanced expression of dendritic cell maturation markers whether the cells were cultured alone or with vaccinia virus infected tumor cells.

[0416] Additionally, the results of this study demonstrate that exposure to DAS 181 increased and increased TNF-alpha secretion by THP-1 derived macrophage (FIG 14).

Example 5: DAS181 Increases Oncolytic Adenovirus Tumor Cell Killing in the Absence of Immune Cells

[0417] This Example provides unexpected results demonstrating that treatment with DAS181 increases oncolytic virus tumor cell killing, even in the absence of immune cells.

[0418] A549 cells were genetically labelled with red fluorescent protein (A549-red). Tumor cell proliferation and killing by oncolytic adenovirus (Ad5) in the presence or absence of DAS181 was monitored by live cell imaging and quantification with IncuCyte. The cell culture medium was collected for ELISA measurement of cytokine production by PBMCs. As shown in FIG 15, DAS 181 increased oncolytic adenovirus-mediated tumor cell killing and growth inhibition. Example 6: DAS181 Increases Oncolytic Adenovirus Tumor Cell Killing in the Presence of PBMC

[0419] As shown in Example 5, treatment with DAS181 increases killing of tumor cells by an oncolytic virus in the absence of immune cells. Example 6 provides results demonstrating that treatment with DAS 181 also increases tumor cell killing when present together with oncolytic virus in the presence of PBMC

[0420] A549 cells were genetically labelled by a red fluorescent protein (A549-red). Fresh human PBMCs were harvested and stimulated with proper cytokine and antibody combinations to activate effector T cells. Activated PBMCs were then co-cultured with A549-red cells that have been treated with DAS181 with or without the oncolytic adenovirus (Ad5). Tumor cell killing by PBMCs was monitored by live cell imaging and quantification with IncuCyte. The cell culture medium was collected for ELISA measurement of cytokine production by PBMCs. As shown in FIG 16, DAS 181 significantly increased tumor cell killing when present together with oncolytic adenovirus in the presence of PBMC.

Example 7: Construction and Characterization of an Oncolytic Virus Expressing DAS181

[0421] A construct designed for expression of DAS 181 is depicted schematically in FIG 17. [0422] To generate a recombinant VV expressing DAS181, a pSEM-1 vector was modified to include a sequence encoding DAS181 as well as two loxP sites (loxP site sequences are shown in SEQ ID NO: 62) with the same orientation flanking the sequence encoding the GFP protein (the GFP coding sequence is shown in SEQ ID NO: 63). (pSEM-l-TK-DAS181-GFP). DAS181 expression is under the transcriptional control of the F17R late promoter in order to limit the expression within tumor tissue. The sequence of a portion of an exemplary construct is shown in SEQ ID NO: 65.

[0423] Western Reserve VV was used as the parental virus. VV expressing DAS181 was generated by recombination with pSEM-l-TK-DAS181-GFP into the TK gene of Western Reserve VV to generated VV-DAS181.

[0424] Recombinant virus can be generated as follows.

Transfection:

[0425] Seed CV-1 cells in 6-well plate at 5xl0⁵ cells/2 ml DMEM-10% FBS/well and grow overnight. Prepare parent VV virus (1 ml/well) by diluting a virus stock in DMEM/2% FBS at MOI 0.05. Remove medium from CV-1 wells and immediately add VV, and culture for 1-2 hours. CV-1 cells should be 60-80% confluent at this point. Transfection mix in 1.5 ml tubes. For each Transfection, dilute 9 pl Genejuice in 91 ul serum-free DMEM and incubate at room temperature for 5 min. Add 3ug pSEM-l-TK-DAS181-GFP DNA gently by pipetting up and down two or three times. Leave at room temperature for 15 min. Aspirate VV virus from the CV-1 well and wash the cells once with 2 ml serum-free DMEM. Add 2 ml DMEM-2% FBS and add the DNA-Genejuice solution drop-by-drop. Incubate at 37°C for 48-72 hr or until all the cells round up. Harvest the cells by pipetting repeatedly. Release the virus from cells by repeated freeze-thawing of the harvested cells by first placing them in dry-ice/ethanol bath and then thawing them in a 37 °C water bath and vortexing. Repeat the freeze-thaw cycling three times. The cell lysate can be stored at -80°C.

Plaque Isolation:

[0426] Seed CV-1 cells in 6-well plates at 5xl0⁵ cells/2ml DMEM-10% FBS/well and grow overnight. CV-1 cells should be 60-80% confluent when receiving cell lysate. Sonicate the cell lysate on ice using sonic dismembrator with an ultrasonic convertor probe for 4 cycles of 30s until the material in the suspension is dispersed. Make 10-fold serial dilutions of the cell lysate in DMEM-2% FBS. Add 1 ml of the cell lysate-medium per well at dilutions 10"², 10"³, 10"⁴, incubate at 37°C. Pick well-separated GFP+ plaques using pipet tip. Rock the pipet tip slightly to scrape and detach cells in the plaque. Gently transfer to a microcentrifuge tube containing 0.5 ml DMEM medium. Freeze-thaw three times and sonicate. Repeat the same process of plaque isolation 3-5 times.

Virus amplification:

[0427] Seed CV-1 cells 5xl0⁵ cells/2ml DMEM-10% FBS/well and grow overnight in 6- well plate. CV-1 should be confluent when starting the experiment. Infect 1 well with 250 ul of plaque lysate/lml DMEM-2% FBS, and incubate at 37°C for 2 h. Remove the plaque lysate and add 2 ml fresh DMEM-2% FBS, and incubate for 48-72 hr until cells round up. Collect the cells by repeatedly pipetting, freeze-thaw 3 times and sonicate. Add half of the cell lysate in 4ml DMEM-2%FBS and infect CV-1 cells in 75-CM2 flask, after 2 h, remove virus and add 12 ml DMEM-2%FBS and culture 48-72 h (until cell round up). Harvest the cells, spin down 5 min at 1800 G, and discard supernatant and resuspend in 1 ml DMEM-2.5% FBS.

Virus titration:

[0428] Seed CV-1 cells 5xl0⁵ cells/2ml DMEM-10% FBS/well and grow overnight in 6- well plate. Dilute virus in DMEM-2% FBS, 50 ul virus/4950 ul DMEM-2% FBS (A, IO ²), 500ul A/4500ul medium (B, 10³), and 500 ul B/4500 ul medium (C, HF⁴). IO’⁷ to IO ¹⁰ for virus stock. Remove medium and wash lx with PBS, and cells were infected with 1ml virus dilution in duplicate. Incubate the cells for 1 h, rock the plate every 10 min. 1 h later, remove the virus and add 2 ml DMEM-10% FBS and incubate 48 h. Remove the medium, add 1 ml of 0.1% crystal violet in 20% ethanol for 15 min at room temperature. Remove the medium and allow to dry at room temperature for 24 hr. Count the plaque and express as plaque forming units (pfu) per ml.

Detection of DAS181 Expression by VV-DAS181 :

[0429] CV-1 cells were infected with VV-DAS181 at MOI 0.2. 48 hours later, CV-1 cells were collected. DNA was extracted using Wizard SV Genomic DAN Purification System and used as template for DAS181 PCR amplification. PCR was conducted using standard PCR protocol and primer sequences (SialF:

GGCGACCACCCACAGGCAACACCAGCACCTGCCCCA (SEQ ID NO: 56) and SialR: CCGGTTGCGCCTATTCTTGCCGTTCTTGCCGCC (SEQ ID NO: 57)). The expected PCR product (1251 bp) was found.

Example 8: DAS181 Expressed by Vaccinia Virus is Active In Vitro

[0430] Example 8 provides results demonstrating that delivery of DAS 181 to cells using an oncolytic virus results in sialidase activity equivalent to treatment with approximately 0.78nM- 1.21 nM of purified DAS181 in 1 ml medium.

[0431] CV-1 cells were plated in six well plate. The cells were transduced with Sialidase - VV or control VV at MOI 0.1 or MOI 1. After 24 hrs, transfected cells were collected, and single cell suspension were made in PBS at 3xl0⁶/500 pl. Cell lysate was prepared using Sigma’s Mammalian cell lysis kit for protein extraction (Sigma, MCL1-1KT), and supernatant was collected. The sialidase (DAS 181) activity was measured using Neuraminidase Assay Kit (Abeam, ab 138888) according to manufacturer’s instruction. 1 nM, 2 nM, and 10 nM DAS181 was added to the VV-cell lysate as control and generated the standard curve. IxlO⁶ cells infected with Sialidase-VV express DAS181 equivalent to 0.78nM-1.21 nM of DAS181 in 1 ml medium. As shown in FIG 18, the DAS 181 has sialidase activity in vitro.

Example 9: Vaccinia Virus-Sialidase Promotes Dendritic Cell Maturation

[0432] Example 9 provides results demonstrating that an oncolytic virus encoding a sialidase promotes dendritic cell maturation compared to an oncolytic virus without a sialidase.

[0433] To determine if Sialidase-VV can promote DC activation and maturation, adherent human PBMC were re-suspend at 5xl0⁶ cells in 3 ml medium supplemented with 100 ng/ml of GM-CSF and 50 ng/ml of IL-4 then cultured in 6-well plates with 2ml per well of fresh medium supplemented with same concentrations of GM-CSF and IL-4. 6 days post cell culture, the cells were cultured in the presence of Sialidase-VV infected tumor cell lysate, VV- infected tumor cell lysate, VV-infected tumor cell lysate plus synthetic DAS181 protein, or LPS (positive control). After another 24 hrs, expression of CD86, CD80, MHC-II, MHC-I were determined by flow cytometry. As shown in FIG 19, Sialidase-VV promotes the expression of markers indicative of dendritic cell activation and maturation compared to treatment with VV alone.

Example 10: Sialidase-VV enhances T lymphocyte-mediated cytokine production and oncolytic activity

[0434] To assess whether DAS181 can activate human T cells by inducing IFN-gamma (IFNr) and IL-2 expressing, human PBMCs were activated by adding CD3 antibody at 10 pg/ml, proliferation was further stimulated by adding IL-2 by every 48 hrs. On day 15, tumor cells (A549) were infected with VVs at MOI 0.5, 1, or 2 in 2.5% FBS medium for 2 hours. Activated T cells were added to the culture at effector:target ratio of 5: 1 or 10: 1 in the presence of CD3 antibody at 1 ug/ml. After another 24 hrs, tumor cytotoxicity was measured, and cell culture medium was collected for cytokine array. As can be seen in FIG. 20, Sialidase-VV induces a significantly greater IL-2 and IFN-gamma expression by CD3 activated T cells than does VV. In addition, as can be seen in FIG. 21, Sialidase-VV elicits stronger anti-tumor response than VV at an E:T of 5:1.

Example 11: Generation of expression constructs for secreted and transmembrane DAS181

[0435] Secreted and transmembrane forms of DAS181 were created to examine impact on sialidase activity. As a negative control, secreted and transmembrane forms of a point mutant that very substantially reduces sialidase activity were also created. Finally, secreted and transmembrane forms of Neu2, an alternative sialidase, were also constructed.

[0436] To facilitate the secretion of DAS 181 from cells, a DNA sequence encoding the signal peptide of the mouse Immunoglobulin kappa chain was added to the N-terminus of DAS181 sequence by gene synthesis and then together cloned into a mammalian expression vector pcDNA3.4. To restrict the DAS181 sialidase activity on the cell surface, a DNA sequence encoding the DAS181 catalytic domain was synthesized and cloned in-frame with the human PDGFR beta transmembrane domain in a mammalian expression vector pDisplay. For controls, DNA sequences encoding secreted and transmembrane versions of DAS185, a mutant protein lacking sialidase activity, were similarly synthesized and cloned into pcDNA3.4 and pDisplay vectors, respectively. In addition, constructs expressing secreted and transmembrane versions of human Neu2 sialidases were generated in the same manner. The sequences for the following constructs were shown: construct 1 (secreted DAS 181; SEQ ID NO: 34), construct 4 (transmembrane DAS181; SEQ ID NO: 37), construct 2 (secreted DAS 185; SEQ ID NO: 35), construct 5 (transmembrane DAS 185; SEQ ID NO: 38), construct 3 (secreted human Neu2; SEQ ID NO: 36) and construct 6 (transmembrane human Neu2; SEQ ID NO: 39).

Example 12: Enzymatic activity of secreted and transmembrane sialidases

[0437] For ectopic expression, mammalian expression vectors (detailed in Example 11) were transfected into HEK293 cells using jetPRIME transfection reagent (Polyplus Transfection #114-15) following the manufacturer’s protocol. Briefly, Human embryonic kidney cells (HEK293) were plated at ~ 2 x 10⁵ live cells per well in 6-well tissue culture plates and grown to confluency by incubation at 37°C, 5% CO2, and 95% relative humidity (typically overnight). Two microliters equivalent to 2 micrograms of DNA was diluted into 200 microliters jetPRIME Buffer followed by 4 microliters of jetPRIME reagent. Tubes were vortexed, briefly centrifuged at 1,000 x g (~10 seconds) and incubated for 10 minutes at room temperature. During the incubation, the media on all wells was replenished with fresh culture media (MEM + 10% FBS). Transfections were added to individual wells and the plate returned to the incubator for 24 hours. Following incubation, supernatants were reserved. Single cell suspensions were created using non-enzymatic cell dissociation reagent Versene (Gibco #15040-066). Monolayers were washed 1 time with DPBS and 500 microliters Versene was added the plate incubated until cells dissociated from vessel surface; 500 microliters complete media was added and the cells were centrifuged for 5 minutes at 300xg. The supernatant was aspirated and cells were suspended in 300 microliters compete media for enzymatic assay.

[0438] For each of the resulting transfection cultures, supernatant and resuspended cells were evaluated for activity utilizing the ability of the sialidase to enzymatically cleave the Anorogenic substrate, 2'-(4-Methylumbelliferyl)-a-D-N-acetylneuraminic acid sodium salt hydrate (MuNaNa) to release the Auorescent molecule 4-methylumbelliferone (4-Mu). The resulting free 4-Mu is excited at 365 nM and the emission is read at 445 nm using a Auorescent plate reader. BrieAy, 100 pl of each sample was plated into a black, non-treated 96-well plate. The plate was incubated in a water bath at 37°C for approximately 30 minutes and subsequently mixed with pre-incubated (37°C, 30 minutes) 100 pM MuNaNa. The Auorescence was kinetically measured at 30 second intervals for 60 minutes using a Molecular Devices SpectraMax M5e multi-mode plate reader. The amount of 4-Mu generated by cleavage was quantified by comparison to a standard curve of pure 4-Mu, ranging from 100-5 pM. Reaction rates were determined for each sample by dividing the amount of 4-Mu produced (< 20 pM) by the time (seconds) required to do so. The observed reaction rates were compared to determine the approximate relative activity of each sample solution (Table 6). It was shown that the supernatant from the secreted DAS181 transfection and the resuspended cells from the transmembrane DAS181 transfection were the most active and approximately equal. All DAS185 and Neu2 sample solutions showed negligible activity compared to the DAS181 sample solutions. The Neu2 sample solutions were equivalent to the background. Furthermore, the observed reaction rates were compared to a standard curve of known concentrations of DAS181, ranging from 1000-60 pM. The supernatant from the secreted DAS181 transfection and the resuspended cells from the transmembrane DAS181 transfection were extrapolated to be approximately equivalent to 4000 pM DAS181. All other samples were observed to be approximately equivalent to or less than 90 pM DAS181.

Table 6

*Conditioned Media samples were spun down to remove any debris and tested neat.

**Cells were harvested, spun down and resuspended in 300 pL media.

All values are adjusted to remove background activity from the media.

All values determined describe the specific sample tested. Values between samples cannot be directly compared because the enzyme concentrations will vary.

Example 13: Secreted DAS181 and transmembrane DAS181 reduce surface sialic acids on tumor cells

[0439] The effect of secreted and transmembrane sialidases on cell surface sialic acid removal and galactose exposure were examined by imaging and flow cytometry after transient transfection of various expression constructs into A549-red cells using Fugene HD (Promega) following the instructions provided by the manufactures. Briefly, A549-Red cell were plated at 2 x 10⁵ cells per well in 2 ml of A549-Red complete growth medium in 6-well plates. For each well of cells to be transfected, 3 pg of plasmid DNA and 9 pl of Fugene HD were diluted into 150 pl of Opti-MEM® I Reduced Serum Medium, mixed gently and incubated for 5 minutes at room temperature to form DNA-Fugene HD complexes. The above DNA-Fugene HD complexes directly to each well containing cells and the cells were incubated at 37°C in a CO2 incubator overnight before further experiments.

[0440] For imaging experiments, transfected cells were re-seeded as 8,000 cells per well in 96-well plates. Then cells were fixed and stained for a2,3-sialic acid; a2,6-sialic acid; and galactose following cell culture for 24 hr, 48 hr or 72 hr. Cells were incubated with SNA-FITC at 40pg/ml, PNA-FITC at 20pg/ml for Ih at room temperature to stain a2,6-sialic acid, and galactose, separately. For a2,3-sialic acid, cells were incubated with Biotinylated MA II at 40pg/ml for Ihr, followed by FITC-Streptavidin for an additional Ihr. To detect HA-tag expression, cells are incubated with HA-Tag rabbit mAh (1:200) for Ihr at room temperature, followed by Donkey anti-rabbit- Alexa Fluor647 for an additional Ihr. The images are taken by Keyence Fluorescent Microscopy.

[0441] Images taken 24 hr post transfection showed that, similar to recombinant DAS181 treatment, secreted DAS181 (Construct 1) and transmembrane DAS 181 (Construct 4) transfection removed both a2,3 and a2,6 sialic acids from cell surface with a concomitant increased galactose staining. Cell transfected with enzyme-inactive DAS 185 (Constructs 2, 5) or human Neu2 (Constructs 3, 6) showed similar staining pattern as vehicle control cells, consistent with the enzyme activity results.

[0442] Images taken 72 hr post transfection more evidently demonstrated that only secreted and transmembrane DAS 181 transfections were capable of efficiently removing tumor cell surface sialic acids. However, it is possible that human Neu2 was not expressed well by the cells as staining of HA tag present in the transmembrane constructs was only positive in the cells transfected with the DAS181 and DAS 185 constructs.

[0443] For flow cytometry analysis, transfected cells were re- seeded at IxlO⁵ cells per well in 24-well plates. Then cells are fixed and stained for a2,3-sialic acid, a2,6-sialic acid, and galactose following cell culture for 24 hr, 48 hr or 72 hr. Results were analyzed using Acea Flow cytometer system. The results of secreted construct transfections, with recombinant DAS181 treatment as control, are shown in FIGS. 22A-22C for a2,3 (FIG. 22A) and a2,6 (FIG. 22B) sialic acids, and galactose (FIG. 22C). The results of transmembrane construct transfections, with secreted DAS181 transfection as control, are shown in FIGS. 23A-23C for a2,3 (FIG. 23A) and a2,6 (FIG. 23B) sialic acids, and galactose (FIG. 23C). Consistent with the imaging study results, secreted DAS181 and transmembrane DAS181 transfections led to removal of cell surface a2,3 and a2,6 sialic acids, and exposure of galactose, whereas transfections with secreted and transmembrane DAS 185 or human Neu2 had little effect.

Example 14: Secreted DAS181 and transmembrane DAS181 increase tumor cell killing mediated by PBMC and oncolytic virus

[0444] Because secreted DAS181 and transmembrane DAS181 were shown to remove cell surface sialic acid efficiently, their effect on PBMC and oncolytic virus-mediated tumor cell killing were evaluated with cells transfected with secreted and transmembrane DAS181. Because transient transfection can have deleterious effect on cell growth, stable pool cells for secreted and transmembrane DAS181 were generated by culturing the transfected A549-red cells in the presence of 1 mg/ml G418 for 3 weeks until the control non-transfected cells were completely killed off. Stable pool transfected A549-red cells with DAS181 were seeded into 96-well plate at density of 2000 cells per well. A549-red parental cells were seeded as controls. The next day, the complete growth medium was removed and replaced with 50 ul of medium with or without oncolytic virus. Freshly isolated PBMC were counted and resuspended at 200,000/ml in A549 complete medium with anti-CD3/anti-CD28/IL2, then 50pl freshly PBMC were added to the cells. The cell growth was monitored by Essen Incucyte up to 5 days based on the counted red objects. As shown in FIG. 24, secreted DAS181 expression sensitized activated PBMC-mediated tumor cells killing and increased oncolytic virus associated PBMC- mediated cell killing at both MOI of 1 and 5. As shown in FIG. 25, transmembrane DAS181 expression significantly sensitized A549-red cells to activated PBMC killing. A far greater effect was virus was observed at MOI of 5, than at MOI of 1. It is possible that the potency of sialidase activity and oncolytic virus as single agent could be masking the additive effect when they were combined together under certain experimental conditions.

Example 15: Generation of Sialidase-Armed Oncolytic Vaccinia Virus

[0445] This Example demonstrates generation of exemplary oncolytic virus constructs encoding sialidase. Constructs were successfully generated for Endo-Sial-VV, SP-Sial-VV, and TM-Sial-VV.

1.1. Design of pSEM-l-Sidalidase-GFP/RFP.

[0446] To generate the recombinant VV expressing Sialidase, pSEM-1 vectors were created using gene synthesis. The construct comprises of the gene encoding Sialidase, the gene encoding GFP or RFP, and two loxP sites with the same orientation flanking GFP/RFP (pSEM- 1-Sialidase-GFP/RFP). The inserted Sialidase is under the transcriptional control of the F17R late promoter in order to limit the Sialidase expression within tumor tissue. The simplified design of the plasmids is as show in FIG. 26.

1.2. Generation of SP-Sial-VV and TM-Sial-VV.

[0447] Vaccinia virus (VV) strain WR was used as the parental virus for recombination with Sialidase to create VVs that expresses Sialidase in three different isoforms: i) constrained to the intracellular compartment (Endo-Sial-VV); ii) secreted to the extracellular environment (SP-Sial-VV); or iii) localized at the cell surface (TM-Sial-VV).

[0448] Sialidase- VVs were generated by insertion of pSEM-l-TK-Sialidase-GFP, pSEM-1- TK-SP-Sialidase-RFP or pSEM-l-TK-TM-Sialidase-GFP into the TK gene of VV through homologous recombination. All the viruses were produced and quantified by titration on CV- 1 cells.

1.2.1. VV. endo-Sial-VV, SP-Sial-VV and TM-Sial-VV quantification by titration

[0449] After obtaining the recombinant viruses and having their stocks amplified, infectious particles were titrated by plaque assay. Briefly, CV-1 cells seeded in a 12-well plate were infected with serial dilutions of VV, endo-Sial-VV, SP-Sial-VV or TM-Sial-VV. After 48 h of infection, cells were fixed and stained with 20% Ethanol/ 0.1% Crystal Violet and virus plaques were counted. We prepared aliquots of 10⁶ of each virus stock in 100 pl of 10 mM Tris-HCl pH 9.0 for shipping. Therefore, all viruses are at 10⁷ pfu/ml.

1.2.2 Detection of virus recombination by PCR

[0450] In order to confirm that Sialidase isoforms were successfully inserted into VV genome, PCR was performed according to standard protocols to amplify the constructs using each virus stock as the template DNA. To do so, PCR primers were designed to specifically bind to the regions shown in Figure 2. These primers will be able to confirm that: i) the constructs were successfully inserted into VV genome; ii) the constructs maintained their respective modifications during recombination (i.e. secretion and transmembrane domains). The primer sequences used were the following:

Sial-fwd: 5’ - GGCCACACTGCTCGCCCAGCCAGTTCATG (SEQ ID NO: 56)

Sial-rev: 5’ - ATGCCTCCACCGAGCTGCCAGCAAGCATG (SEQ ID NO: 57) SP-Sial-rev: 5’ - TCCTGTCTTGCATTGCACTAAGTCTTG (SEQ ID NO: 83) TM-Sial-fwd: 5’ - TCATCACTAACGTGGCTTCTTCTGCCAAAGCATG (SEQ ID NO: 84) [0451] A band of the predicted size of Sialidase was detected in all three isoforms, demonstrating successful generation of the sialidase VV constructs (FIG. 27). When sialFWD + SPsialREV primer pair was used, only SP-Sial-VV and TM-Sial-VV showed bands of the expected size for SP-Sial, which confirms that these viruses have the secretion signal. Finally, when TM-sial-fwd + SP-Sial-rev primer was used, only TM-sial-VV showed a strong band of the predicted size of TM-Sialidase. This data confirms that VV recombinants were successfully generated and that the constructs for the three isoforms are intact within the virus genome.

Example 16: Sialidase-VVs’ are able to infect, replicate in, and lyse tumor cells in vitro.

[0452] This Example provides results demonstrating that Endo-Sial-VV, SP-Sial-VV, and TM-Sial-VV have comparable infectivity and replication activity in CV-1 and U87 cells, and comparable lytic activity in U87 and A549 cells to parental vaccinia virus, indicating the transgene didn’t impair the VV’s infectivity, replication, and lytic ability. Tumor cells were infected with Sialidase-VV, or parental VV at increasing MOIs. At various time points (24, 48, 72 or 96 hours) post infection, the cells were harvested and subjected to plaque assay and MTS assays to determine virus replication.

[0453] As shown in FIG. 28, the replication ability of the virus was not affected by modification with sialidase. CV-1 or U87 cells were plated in 12-well tissue culture plate and infected with Sialidase-VVs or VV at MOIs 0. 1 in 2.5% FBS medium for 2 hours followed by culturing in complete medium. At various time points post infection (24, 48, 72, or 96 hours), the cells were harvested and virus replication was determined by plaque assay using CV-1 cells. [0454] Furthermore, as shown in FIG. 29, the lytic activity of the modified vaccinia viruses was comparable to that of parental vaccinia virus in U87 and A549 cells, as shown in FIG. 29 and Tables 7-9 below.

Table 8. Percent (%) A549 cell survival

Example 17: Sialidase-VVs enhance Dendritic Cell maturation in vitro

[0455] This Example provides results demonstrating: SP-, & TM-Sial-VV activated human DC by enhancing the its expression of maturation markers. Both SP-Sial-VV and TM-Sial-VV induced activation of DC effectively in vitro.

[0456] The effect of oncolytic viruses encoding a sialidase on maturation of DCs was evaluated. GM-CSF/IL4 derived human DC (Astarte, WA) were cultured with VV-U87 tumor cells (ATCC, VA) for 24 hours. DC were collected and stained with antibodies against DC maturation markers CD86, CD80, HLA-ABC, HLA-Dr on DCs were determined by flow cytometry. Cell were collected and stained with HLA-Dr-FITC (abl93620, Abeam, MA) and HEA-ABC-PE (abl55381, Abeam, MA), or CD80-FITC (abl8279, Abeam, MA) and CD86- PE (ab234226, Abeam, MA) antibodies and subjected to flow analysis (Sony SA3800).

[0457] FIGS. 30-33 show expression of DC maturation markers HLA-ABC, HLA-DR, CD80, and CD86, respectively. Culturing DCs together with U87 tumor cells infected with SP- Sial-VV or TM-Sial-VV enhanced expression of DC maturation markers compared to that of DC cells cultures with U87 infected with VV or U87 alone.

Example 18: Sialidase-VVs enhance NK-mediated tumor cell killing in vitro

[0458] This Example provides results demonstrating that Sial-VVs enhance NK-mediated cytotoxicity. VV-infected tumor cells were co-cultured with NK, and specific lysis of the tumor cells was determined.

[0459] Protocol: Negative selected human NK cells (Astarte, WA) and VV-U87 cells (ATCC, VA) were co-cultured, and tumor killing efficacy was measured by LDH assay (Abeam, MA). As shown in FIG. 34, the results suggested that Sial-VVs enhanced NK cell- mediated U87 tumor killing in vitro. (* P value, the Sial-VV vs Mock VV in U87 and NK culture)

Specific lysis was calculated as: experimental target cell release - target cells spontaneous release

% = 100 % x - target cells maximum release - target cells spontaneous release

*P-value: T.Test were used with 1 tail and type 1 analysis.

Example 19: Sialidase-VVs inhibit tumor growth in vivo

[0460] The Examples above demonstrate the surprising beneficial effects of Sialidase-VVs in vitro in promoting immune cell activation and cytotoxicity. Example 19 provides results demonstrating that Sialidase-VVs significantly inhibit tumor growth in vitro compared to control VV.

[0461] To test the effect of Sialidase-VVs on tumor growth in vivo, 2xl0⁵ and 2xl0⁴ B16- F10 tumor cells were inoculated on the right or left flank of C57 mice. When the tumor size on the right or left flank reached 100mm (14 days), 4xl0⁷ pfu VVs were injected intratumorally into the tumor on the right or left site every other day for 3 doses. Tumor size was measured. FIG. 35 shows the tumor size on the right flank. The results indicated that TM-sial-VV significantly inhibited tumor growth compared to control VV. SP-sial VV inhibited tumor growth, albeit to a lesser extent. FIG. 36 shows the tumor size on the left flank. The results indicated that TM-sial-VV significantly inhibited tumor growth compared to control VV.

[0462] FIG. 37 shows that there was no significant difference in mouse body weight for mice treated with the various VVs or PBS control. 2xl0⁵ and 2xl0⁴ B16-F10 tumor cells were inoculated on the right or left flank of C57 mice. When the right tumor size reached 100mm (14 days), 4xl0⁷ pfu VVs were injected intratumorally every other day for 3 doses. Sialidase armed oncolytic vaccinia virus significantly enhances CD8+ and CD4+ T cell infiltration within tumor

[0463] Tumor cells were inoculated on the right flank of C57 mice, and the resulting tumors were intratumorally injected with VVs as described above (every other day for 3 doses). 7 days after the first VV treatment, tumor tissues (n=6) were collected and subjected to flow analysis to analyze CD8+ and CD4+ T cell infiltration within the tumor. * p value: treatment group vs control VV group. FIG. 38A shows quantification of the results and p values demonstrating significant enhancement of CD8+ and CD4+ T cell infiltration by sialidase armed oncolytic vaccinia virus. FIG. 38B shows the FACS plots. The results demonstrated that sialidase armed oncolytic vaccinia virus significantly enhanced CD8+ and CD4+ T cell infiltration within tumor compared to control vaccinia virus. Sialidase armed oncolytic vaccinia virus significantly decreased the ratio of Treg/CD4+ T cells within the tumor

[0464] Tumor cells were inoculated on the right flank of C57 mice, and the resulting tumors were intratumorally injected with VVs as described above (every other day for 3 doses). 7 days after the first VV treatment, tumor tissues (n=6) were collected and subjected to flow analysis to determine the ratio of Treg/CD4+ T cells within the tumor. As shown in FIG. 39, TM-Sial-VV decreased the ratio of Treg/CD4+ T cells within the tumor, compared to control VV. * p value: treatment group vs control VV group.

Sialidase armed oncolytic vaccinia virus significantly enhances NK and NKT cell infiltration within tumor

[0465] Tumor cells were inoculated on the right flank of C57 mice, and the resulting tumors were intratumorally injected with VVs as described above (every other day for 3 doses). 7 days after the first VV treatment, tumor tissues (n=6) were collected and subjected to flow analysis to determine the number of NK1.1+ NK cells. As shown in FIG. 40, sialidase armed oncolytic vaccinia virus significantly enhanced NK and NKT cell infiltration within tumor. * p value: treatment group vs control VV group.

[0466] Tumor cells were inoculated on the right flank of C57 mice, and the resulting tumors were intratumorally injected with VVs as described above (every other day for 3 doses). 7 days after the first VV treatment, tumor tissues (n=6) were collected and subjected to flow analysis to determine expression of PD-L1. As shown in FIG. 41, transmembrane bound sialidase armed oncolytic virus significantly increased PD-L1 expression within tumor cells (p<0.05, TM-Sial-VV vs Control VV).

Example 20: Glycoimmune checkpoint and tumor stroma-targeted oncolytic vaccinia virus

[0467] This example describes the generation of a vvDD-Sial-FAP/CD3, an oncolytic vaccinia virus expressing a membrane-bound sialidase to remove sialic acids from the cell surface glycans, and fibroblast activation protein (FAP)-targeted T cell engager to eliminate glycoimmune checkpoint and tumor stroma, respectively. A vvDD-Sial-FAP/CD3 was designed as an engineered vaccinia virus of Western Reserve (WR) strain with: (1) an insertional disruption of the viral thymidine kinase (TK) gene with the sialidase and FAP/CD3 transgenes. TK is an essential enzyme for the pyrimidine synthesis pathway; viral TK gene deletion thus results in selective replication of virus in rapidly dividing cancerous cells with high intracellular nucleotide pools, and (2) a deletion of the vaccinia growth factor (VGF) gene for greater dependence on the cell cycling status of the cancer cells. The vvDD virus has been described (see McCart JA, et al. Systemic cancer therapy with a tumor- selective vaccinia virus mutant lacking thymidine kinase and vaccinia growth factor genes. Cancer Res 2001;61:8751-8757, the content of which is herein incorporated by reference in its entirety). The nucleic acid construct used to integrate the sialidase and FAP/CD3 T cell engager into the TK gene of vvDD comprised the nucleic acid sequence shown in SEQ ID NO: 108.

[0468] Results showed that sialidase expressed from vvDD-Sial-FAP/CD3 efficiently cleaves the sialic acids from the cell surface and the fusion of Fc on sialidase induced antibody-dependent cell-mediated cytotoxicity (ADCC) using an ADCC reporter assay.

Sialidase expressed from vvDD-Sial-FAP/CD3 efficiently cleaves sialic acids from cells. Human lung adenocarcinoma cells were mock-infected or infected with either vvDD or Sial- FAP/CD3-vvDD at 0.05 pfu/cell. About 20 hrs after infection, cells were harvested and incubated with allophycocyanin (APC)-labeled Sambucus Nigra Lectin (SNA) to detect a- 2,6-sialic acid linkages on cell surface. As shown in FIG. 42A, A decrease in the APC signals indicates the removal of a-2,6-sialic acid linkages by virus-expressed sialidase. As shown in FIG. 42B, virus-expressed sialidase reduced the level of a-2,6-sialic acid linkages to -40% compared to the control.

ADCC reporter assays demonstrate efficacy of vvDD-Sial-FAP/CD3

[0469] A549 human lung adenocarcinoma cells were mock-infected or infected with either vvDD or vvDD-Sial-FAP/CD3 at the indicated pfu/cell. The next day, effector cells expressing a reporter (Jurkat cells stably expressing FcyRIIIa receptor and a nuclear factor of activated T cells (NF AT) response element driving expression of firefly luciferase) were added to the infected A549 cells. The NF AT response element-driven luciferase expression acts as an early reporter of ADCC. Six hours later, luciferase substrate was added to wells and luminescence in each well was measured to quantify the luciferase activity in effector cells. As shown in FIG. 43, mock or vvDD infected A549 cells did not result in luciferase expression (the lines for mock infection and vvDD infection are flat overlapping lines in the graph and cannot be distinguished). The A549 cells infected with vvDD-Sial-FAP/CD3 did induce luciferase expression in the Jurkat reporter effector cells, indicating activation of the ADCC pathway.

[0470] In another experiment, A549 human lung adenocarcinoma cells were mock-infected or infected with either vvDD or vvDD-Sial-FAP/CD3 at 0.05 pfu/cell. About 20 hrs after infection, supernatants were collected and passed through 0.2 mm filters to generate Conditioned Media. The indicated amounts of Conditioned Media were added to COLO829 (FAP-positive) or A549 (FAP-negative) cells, followed by addition of the T cell receptor (TCR)/CD3 Effector cells, Jurkat cells that express a luciferase reporter driven by Nuclear Factor of Activated T Cells (NF AT) response element. Five hours later, luciferase substrate was added to wells and luminescence in each well was measured to quantify the luciferase activity in effector cells. As shown in FIGS. 44A-44B, the conditioned media from cells infected with vvDD-Sial-FAP/CD3 resulted in luciferase expression indicative of ADCC in the FAP-positive, COLO829 cells in a concentration-dependent manner, but resulted in much lower luciferase expression in the FAP-negative A549 cell line, indicating the specific effect of the FAP/CD3 bispecific T cell engager. Conditioned media from mock infected cells or cells infected with vvDD did not result in luciferase expression.

[0471] In vitro efficacy studies were also conducted using HCT116 human colon cancer cells mixed with FAP-expressing normal human dermal fibroblasts or FAP-positive HCC1143 human breast cancer cells in the presence of human peripheral blood mononuclear cells. HCT116 cells co-cultured with normal human dermal fibroblasts (NhDF) were mock- infected or infected with vvDD or vvDD-Sial-FAP/CD3 at 0.1 pfu/cell. The next day, peripheral blood mononuclear cells (PBMCs) were added at a ratio of 10: 1 (Effector:Target). Two days later, cell culture supernatants were collected for lactate dehydrogenase (LDH) assay to measure cytotoxicity. As shown in FIG. 45, infection of HCT116 human colon cancer cells mixed with FAP-expressing normal human dermal fibroblasts with vvDD-Sial- FAP/CD3 resulted in significantly higher LDH compared to mock infected or vvDD infected cells in the presence of PBMCs. vvDD-Sial-FAP/CD3 infected cells induced activation of T cells in vitro

[0472] To assess activation of T cells by cells infected with vvDD-Sial-FAP/CD3, A549 human lung adenocarcinoma cells co-cultured with normal human dermal fibroblasts (NhDF) were mock-infected or infected with vvDD or vvDD-Sial-FAP/CD3 at 0.1 pfu/cell. The next day, peripheral blood mononuclear cells (PBMCs) were added at 10:1 (Effector:Target). One day later, cells were harvested and analyzed for the expression of CD25 activation marker on CD4+ and CD8+ T cells using flow cytometry. Infection with vvDD-Sial-FAP/CD3 significantly increased the % of CD25⁺ cells in the population of CD4⁺ T cells (FIG. 46A) and in the population of CD8⁺ T cells (FIG. 46B) compared to mock-infected or vvDD infected cells. Supernatants were analyzed for granzyme B release using ELISA. As shown in FIG. 46C, infection with vvDD-Sial-FAP/CD3 resulted in significantly more granzyme B release compared to mock-infected or vvDD-infected controls.

[0473] To assess T cell activation by vvDD-Sial-FAP/CD3 infected cells of other cancer types, HCT116 human colon cancer cells co-cultured with normal human dermal fibroblasts (NhDF) or HCC1143 human breast cancer cells were similarly mock-infected or infected with vvDD or vvDD-Sial-FAP/CD3 at 0.1 pfu/cell. The next day, peripheral blood mononuclear cells (PBMCs) were added at 10:1 (Effector:Target). Two days later, cells were harvested and analyzed for the expression of activation markers, CD69 and CD25, on CD4+ and CD8+ T cells using flow cytometry. As shown in FIG. 47A, infection with vvDD-Sial- FAP/CD3 significantly increased the % of CD69⁺ cells in the population of CD4⁺ T cells compared to mock-infected or vvDD infected cells for both the HCT116/NhDF and HCC1143 cells. Similarly, as shown in FIG. 47B, infection with vvDD-Sial-FAP/CD3 significantly increased the % of CD69⁺ cells in the population of CD8⁺ T cells compared to mock-infected or vvDD infected cells for both the HCT116/NhDF and HCC1143 cells.

[0474] Thus, in both the colon cancer and breast cancer tumor models, vvDD-Sial- FAP/CD3 induced activation of both CD4⁺ and CD8⁺ T cells, as measured by the upregulation of CD69 and CD25 markers and increased granzyme B release, which resulted in enhanced cell killing. vvDD-Sial-FAP/CD3 enhanced lymphocyte infiltration in tumor spheroids

[0475] Using A549 co-cultured with CAFs microtissues, we showed that vvDD-Sial- FAP/CD3 spread efficiently within the tumor spheroid. In addition, vvDD-Sial-FAP/CD3 increased tumor-infiltrated lymphocytes, leading to enhanced cell killing.

[0476] A549 human lung adenocarcinoma cells co-cultured with cancer-associated fibroblasts (CAFs) were mock-infected or infected with vvDD or vvDD-Sial-FAP/CD3 expressing green or yellow fluorescent protein, respectively, at 0.3 pfu/cell. The expression of either GFP or YFP was monitor by imaging. Increase in the intensity of the fluorescent proteins indicates the spread of the virus within tumor spheroids. As shown in FIG. 48, vvDD-Sial-FAP/CD3 spread efficiently within the tumor spheroid. [0477] To assay the effect of vvDD-Sial-FAP/CD3 on lymphocyte infiltration in tumor spheroids, A549 human lung adenocarcinoma cells co-cultured with cancer-associated fibroblasts (CAFs) were mock-infected or infected with vvDD or vvDD-Sial-FAP/CD3 at 0.3 pfu/cell. The next day, peripheral blood mononuclear cells (PBMCs) labeled by CellTracker DeepRed were added at 10:1 (Effector:Target). Images taken two days after addition of PBMCs are shown. Infection with vvDD-Sial-FAP/CD3 resulted in the increase of red fluorescence (Cy5-PBMC) in the core of the tumor spheroid, indicating enhanced tumorinfiltrating lymphocytes (FIG. 49).

[0478] Taken together, the results provided in this example demonstrated potent anti-tumor and -stroma effects induced by vvDD-Sial-FAP/CD3 in multiple cancer models. In vivo efficacy of vvDD-Sial-FAP/CD3, either alone or in combination with checkpoint inhibitor or NK cell therapy, is being evaluated.

Example 21: Combination therapy of engineered chimeric antigen receptor natural killer (CAR-NK) cells targeting avSialidase and avSialidase-armed oncolytic vaccinia virus for treating solid tumors

[0479] Chimeric antigen receptor (CAR) - engineered natural killer (NK) cell therapy has emerged as a promising platform for adoptive immunotherapy for cancer. However, CAR- NK or CAR-T therapy so far has achieved limited efficacy in solid tumors compared with hematologic malignancies. One of the challenges is the lack of prevalent tumor-specific surface antigen target in solid tumors. Hence, we developed a novel oncolytic vaccinia virus (VV) to deliver a dual functional glycol-immune checkpoint inhibitor (see Example 20) and universal tumor cell marker combined with CAR-NK cell therapy to increase anti-tumor efficacy. Using the vaccinia virus deleted in both viral TK and VGF genes (vvDD) to drive the expression of a membrane-bound sialidase derived from Actinomyces viscosus (avSial) under the viral late promoter, we observed efficient desialylation of both VV-infected and non-infected tumor cells, which would alleviate the suppression of sialic acid on NK and other immune cells within TME. Further, the surface bound avSial on VV-infected tumor cells can also serve as a universal target for avSial-CAR NK cells, with less concern for cross-reactivity to normal human tissues and antigen loss. Methods

Retroviral and Plasmid Constructs

[0480] Retroviral vectors were constructed using a synthetic DNA approach (Genewiz). Briefly, CAR containing DAS181-specific scFv, CD8 transmembrane domain, CD28 and CD3 cytoplasmic region (SEQ ID NO: 120) in a SFG retroviral backbone were synthesized and linked together using HiFi assembly (New England Biolabs). The vectors also contained an IE-15 gene (SEQ ID NO: 121) separated by a Thosea asigna virus 2A (T2A) peptide bond-skipping polypeptide. D004 scFv sequence targeting DAS181 was derived from antibodies originally generated through mice immunization studies and screened via ELISA (Genescript).

DAS181 binding affinity assay

[0481] The binding affinity of scFvs to DAS181 were tested in 293T cells. Briefly, a variety of avSial CAR (also referred herein as “DAS 181 CAR”) constructs containing different scFvs were transfected into 293T cells using Genejuice following manufacture’s protocol. After 48 hours, 293T cells were harvested and stained with DAS181 biotin (Ansun), followed by streptavidin APC (Biolegend), and analyzed by fluorescence-activated cell sorting (FACS) to identify DAS181 positive binding cells. Flow cytometry was performed on a Novocyte 3000 (ACEA Biosciences) and analyzed with ACEA NovoExpress software. avSial CAR NK cells generation

[0482] Retroviral supernatants were produced by transient transfection of a packaging cell line (Biovec). Peripheral blood cells from healthy donors were obtained by leukapheresis, activated and then subject to retroviral transduction. Cells were maintained in NK MACS medium (Miltenyi Biotec). At day 12 and day 20 after activation, a portion of the cells were harvested and subjected to FACS analysis for transduction efficiency as well as NK and T cells composition via staining of anti-mouse Fab PE (Jackson Immunoresearch), anti-CD56 APCcy7, anti-CD3 BV510 (Biolegend).

Membrane sialidase expressing tumor cell lines

[0483] To test the function of avSial CAR NK cells, tumor cell lines expressing membrane sialidase were generated. Retro viral vectors containing Tm. sialidase (i.e., sialidase with a transmembrane domain) or Tm.Fc.sialidase (i.e., sialidase with a transmembrane domain and an Fc domain) were generated via PCR-based methods. A549 and A375 tumor cells lines were double transduced with a retroviral vector containing Tm.sialidase or Tm.Fc.sialidase and a second retroviral vector containing an enhanced green fluorescent protein (eGFP). Double positive transduced cells were flow sorted using a Sony SH800 sorter. Flow cytometry confirmed over 90% of A375 and over 86% of A549 cells were double positive with eGFP and membrane sialidase initially and over 95% A375 and over 93% A549 cells were double positive at late passages. Desialylations of tumor cell surface were also accessed via flow cytometry analysis of the binding of SNA (Sambucus nigra agglutinin, lectin binds a2,6-linked sialic acid), MAA (Maackia amurensis agglutinin; lectin that mainly recognizes a2-3 linked sialic acid), and PNA (Peanut agglutinin; binding terminal galactose residues, binding increased after sialic acids removing).

Assessing cytotoxicity of NK cells

[0484] Co-culture assays were performed with unmodified and transduced NK cells against an eGFP modified A549 or A375 tumor cells expressing a membrane sialidase at various E:T ratios. For the E:T ratio 4:1 and 2:1, a second dose of fresh tumor cells were added at 72 hrs for tumor re-challenge. Tumor cells (green) proliferation were monitored by real-time fluorescent microscopy (IncuCyte; Essen Biosciences) for 6 to 7 days. The total green object integrated intensity (GCU x um²/well) metric was used to quantify green fluorescence. Each condition was performed at least in duplicates. The whole well was imaged at 4x objective. [0485] For 3D spheroid assays, 5000 tumor cells (A375 or A549 expressing eGFP and membrane sialidase) were seeded in U bottom plates for 4 days to allow spheroid formation. Unmodified, CD 19 CAR transduced, or avSial CAR transduced NK cells were then added at the dose of 50k, 25k or 12.5k cells per well. Tumor spheroid growth was monitored using IncuCyte spheroid module for 6 to 7 days.

Results

[0486] We developed anti-avSial antibodies through mice immunization study. NK cells expressing CAR constructs based on selected single chain variable fragments (scFv) specific for DAS 181 and containing CD28 co-stimulatory domain and CD3^ signaling domain were generated. The CAR NK cells further express human IL- 15 gene to improve NK persistence and function. The binding affinity of anti-avSial scFv to avSial idase were assessed in CAR constructs-transfected 293T cells. The selected anti-avSial scFv CAR were packaged into gamma retroviral vectors to transduce activated and expanded NK cells derived from peripheral blood of healthy donors. For proof-of-concept, target tumor cell lines A375 and A549 expressing transmembrane sialidase and GFP were also generated. Compared with CD 19 CAR NK and none transduced (NT) NK cells, avSial CAR NK had markedly increased cytotoxicity against avSial-expressing tumor cells in co-culture assays at low E:T ratios <= 1:2. Upon tumor cell re-challenge, avSial CAR NK also controlled the tumor growth significantly better than CD 19 CAR NK or non-transduced (NT) NK cells. Furthermore, avSial CAR NK cells completely eliminated A375 tumor spheroids expressing transmembrane sialidase whereas CD 19 CAR NK or NT NK cells only transiently controlled the tumor spheroids growth. Taken together, we have developed an effective avSial targeting CAR IL 15 NK that can be used to demonstrate enhanced efficacy and versatility in combination with avSial-armed VV for treatment of solid tumors.

DAS181 recombinant protein binds 293 T cells expressing CAR specific to DAS181

[0487] avSial CAR constructs (FIG.50A) were made with different sequences of DAS181 scFv and D004 was identified to have the highest percentage of cells binding to DAS181 biotinylated recombinant protein. The CDR sequences of the scFv corresponding to the D004 CAR construct are shown in Table A. CD 19 specific CAR was also made as a control construct. The transduction efficiency was also determined by anti-mouse Fab (FIG. 50C) or protein L staining (FIG. SOD).

NK cells successfully engineered to express CAR constructs on cell surface

[0488] Peripheral blood cells from two healthy donors were used to generate CAR NK cells. On Day 12 and Day 20, transduction efficiencies were around 40%. Protein L staining showed a lower rate compared with anti-mouse Fab staining in CD 19 CAR NK (FIG. 51A). Anti-mouse Fab staining revealed comparable staining of CAR expression between CD 19 CAR and avSial CAR (FIGS. 51A and SIC). On Day 5, 1 million cells were transfected. After expansion, On Day 20, 20 to 40 million cells grew (FIG. SIB). avSial CAR NK killed tumor cells expressing membrane sialidase

[0489] To test the cytotoxicity of avSial CAR NK cells (D004), A375 cells expressing Tm.sialidase or Tm.Fc. sialidase were co-cultured with none transduced (NT), CD19 CAR NK, or avSial CAR NK. As shown in FIG. 52 and FIG. 53, avSial CAR NK consistently killed both A375 and A549 cells expressing membrane sialidase better than NT or CD19 CAR NK cells. Interestingly, avSial CAR NK killed tumor cells expressing Tm.Fc.sialidase better than Tm.sialidase.

[0490] In a tumor re-challenge assay, NK cells were co-cultured with tumor cells expressing membrane sialidase for 72 hrs at the E:T ratio of 4 to 1. There was no difference among the tumor killing effects by NT, CD 19 CAR NK or avSial CAR NK cells initially. However, after a second round of fresh tumor cells were added at 72 hours, avSial CAR NK cells demonstrated markedly enhanced killing effects against A375 or A549 tumor cells expressing membrane sialidase (FIGS. 54-55).

[0491] avSial CAR NK function was further tested in a 3D tumor spheroid assay. NT NK cells temporarily reduced tumor spheroid size. However, after 2 days, tumor grew back. CD 19 CAR NK cells containing IL- 15 transgene were able to control tumor spheroid growth without rebound. Only avSial CAR NK completely eradicated tumor spheroids. FIG. 56 showed that at different NK doses, avSial CAR NK cells were more efficacious in controlling tumor than NT and CD 19 CAR NK cells.

[0492] To test avSial CAR NK persistence in vitro, avSial CAR NK cells and NT NK cells at day 20, were cultured for 6 weeks without IL- 15 cytokine support. FIG. 57 showed live cell counts of NK cells prepared from two different donors over 6 weeks. NT NK rapidly died after one week, whereas avSial CAR NK maintained a higher lever for 3 to 4 weeks before the live cell counts declined. A significant portion of avSial CAR NK cells persisted for the entire 6 weeks. The data demonstrate durable cytotoxicity of avSial CAR NK cells compared to NT control.

Tumor cells expressing membrane sialidase were not stable

[0493] We noticed that after one- month passage, A375 or A549 tumor cells expressing membrane sialidase (Tm.sialidase or Tm.Fc. sialidase) were prone to cell death (FIG. 58). We assessed membrane sialidase expression using flow cytometry. The late passages had higher membrane sialidase expression compared to early passages (FIGS. 59-60). These results show that sialidase renders tumor cells vulnerable. As the tumor cells became desialyated (FIG. 61 and Table 9), tumor cell surface proteins were revealed and make the cells more sensitive to environmental stress such as trypsinization.

Table 9. Quantification of data in FIG. 53.

EXEMPLARY SEQUENCES

SEQ ID NO: 3 Human Neul sialidase

MTGERPSTALPDRRWGPRILGFWGGCRVWVFAAIFLLLSLAASWSKAENDFGLVQP

LVTMEQLLWVSGRQIGSVDTFRIPLITATPRGTLLAFAEARKMSSSDEGAKFIALRRS

MDQGSTWSPTAFIVNDGDVPDGLNLGAVVSDVETGVVFLFYSLCAHKAGCQVAST

MLVWSKDDGVSWSTPRNLSLDIGTEVFAPGPGSGIQKQREPRKGRLIVCGHGTLERD

GVFCLLSDDHGASWRYGSGVSGIPYGQPKQENDFNPDECQPYELPDGSVVINARNQ

NNYHCHCRIVLRSYDACDTLRPRDVTFDPELVDPVVAAGAVVTSSGIVFFSNPAHPE

FRVNLTLRWSFSNGTSWRKETVQLWPGPSGYSSLATLEGSMDGEEQAPQLYVLYEK

GRNHYTESISVAKISVYGTL

SEQ ID NO: 4 Human Neu2 sialidase

MASLPVLQKESVFQSGAHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRG

DYDAPTHQVQWQAQEVVAQARLDGHRSMNPCPLYDAQTGTLFLFFIAIPGQVTEQQ

QLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHD

RARSLVVPAYAYRKLHPIQRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVET

GEQRVVTLNARSHLRARVQAQSTNDGLDFQESQLVKKLVEPPPQGCQGSVISFPSPR

SGPGSPAQWLLYTHPTHSWQRADLGAYLNPRPPAPEAWSEPVLLAKGSCAYSDLQS

MGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAFPAEYLPQ

SEQ ID NO: 5 Human Neu3 sialidase

MEEVTTCSFNSPLFRQEDDRGITYRIPALLYIPPTHTFLAFAEKRSTRRDEDALHLVLR

RGLRIGQLVQWGPLKPLMEATLPGHRTMNPCPVWEQKSGCVFLFFICVRGHVTERQ

QIVSGRNAARLCFIYSQDAGCSWSEVRDLTEEVIGSELKHWATFAVGPGHGIQLQSG

RLVIPAYTYYIPSWFFCFQLPCKTRPHSLMIYSDDLGVTWHHGRLIRPMVTVECEVAE

VTGRAGHPVLYCSARTPNRCRAEALSTDHGEGFQRLALSRQLCEPPHGCQGSVVSFR

PLEIPHRCQDSSSKDAPTIQQSSPGSSLRLEEEAGTPSESWLLYSHPTSRKQRVDLGIY

LNQTPLEAACWSRPWILHCGPCGYSDLAALEEEGLFGCLFECGTKQECEQIAFRLFT

HREILSHLQGDCTSPGRNPSQFKSN

SEQ ID NO: 6 Human Neu4 sialidase

MGVPRTPSRTVLFERERTGLTYRVPSLLPVPPGPTLLAFVEQRLSPDDSHAHRLVLRR

GTLAGGSVRWGALHVLGTAALAEHRSMNPCPVHDAGTGTVFLFFIAVLGHTPEAVQ

IATGRNAARLCCVASRDAGLSWGSARDLTEEAIGGAVQDWATFAVGPGHGVQLPS

GRLLVPAYTYRVDRRECFGKICRTSPHSFAFYSDDHGRTWRCGGLVPNLRSGECQLA

AVDGGQAGSFLYCNARSPLGSRVQALSTDEGTSFLPAERVASLPETAWGCQGSIVGF

PAPAPNRPRDDSWSVGPGSPLQPPLLGPGVHEPPEEAAVDPRGGQVPGGPFSRLQPR

GDGPRQPGPRPGVSGDVGSWTLALPMPFAAPPQSPTWLLYSHPVGRRARLHMGIRL

SQSPLDPRSWTEPWVIYEGPSGYSDLASIGPAPEGGLVFACLYESGARTSYDEISFCTF

SLREVLENVPASPKPPNLGDKPRGCCWPS

SEQ ID NO: 7 Human Neu4 isoform 2 sialidase

MMSSAAFPRWLSMGVPRTPSRTVLFERERTGLTYRVPSLLPVPPGPTLLAFVEQRLSP

DDSHAHRLVLRRGTLAGGSVRWGALHVLGTAALAEHRSMNPCPVHDAGTGTVFLF

FIAVLGHTPEAVQIATGRNAARLCCVASRDAGLSWGSARDLTEEAIGGAVQDWATF

AVGPGHGVQLPSGRLLVPAYTYRVDRRECFGKICRTSPHSFAFYSDDHGRTWRCGG

LVPNLRSGECQLAAVDGGQAGSFLYCNARSPLGSRVQALSTDEGTSFLPAERVASLP

ETAWGCQGSIVGFPAPAPNRPRDDSWSVGPGSPLQPPLLGPGVHEPPEEAAVDPRGG

QVPGGPFSRLQPRGDGPRQPGPRPGVSGDVGSWTLALPMPFAAPPQSPTWLLYSHPV GRRARLHMGIRLSQSPLDPRSWTEPWVIYEGPSGYSDLASIGPAPEGGLVFACLYESG

ARTSYDEISFCTFSLREVLENVPASPKPPNLGDKPRGCCWPS

SEQ ID NO: 8 Human Neu4 isoform 3 sialidase

MMSSAAFPRWLQSMGVPRTPSRTVLFERERTGLTYRVPSLLPVPPGPTLLAFVEQRL

SPDDSHAHRLVLRRGTLAGGSVRWGALHVLGTAALAEHRSMNPCPVHDAGTGTVF

LFFIAVLGHTPEAVQIATGRNAARLCCVASRDAGLSWGSARDLTEEAIGGAVQDWA

TFAVGPGHGVQLPSGRLLVPAYTYRVDRRECFGKICRTSPHSFAFYSDDHGRTWRCG

GLVPNLRSGECQLAAVDGGQAGSFLYCNARSPLGSRVQALSTDEGTSFLPAERVASL

PETAWGCQGSIVGFPAPAPNRPRDDSWSVGPGSPLQPPLLGPGVHEPPEEAAVDPRG

GQVPGGPFSRLQPRGDGPRQPGPRPGVSGDVGSWTLALPMPFAAPPQSPTWLLYSHP

VGRRARLHMGIRLSQSPLDPRSWTEPWVIYEGPSGYSDLASIGPAPEGGLVFACLYES

GARTSYDEISFCTFSLREVLENVPASPKPPNLGDKPRGCCWPS

SEQ ID NO: 9 A. viscosus nanH sialidase

MTSHSPFSRRRLPALLGSLPLAATGLIAAAPPAHAVPTSDGLADVTITQVNAPADGLY

SVGDVMTFNITLTNTSGEAHSYAPASTNLSGNVSKCRWRNVPAGTTKTDCTGLATH

TVTAEDLKAGGFTPQIAYEVKAVEYAGKALSTPETIKGATSPVKANSLRVESITPSSS

QENYKLGDTVSYTVRVRSVSDKTINVAATESSFDDLGRQCHWGGLKPGKGAVYNC

KPLTHTITQADVDAGRWTPSITLTATGTDGATLQTLTATGNPINVVGDHPQATPAPA

PDASTELPASMSQAQHLAANTATDNYRIPAIPPPPMGTCSSPTTSARRTTATAAATTP

NPNHIVQRRSTDGGKTWSAPTYIHQGTETGKKVGYSDPSYVVDHQTGTIFNFHVKSY

DQGWGGSRGGTDPENRGIIQAEVSTSTDNGWTWTHRTITADITKDKPWTARFAASG

QGIQIQHGPHAGRLVQQYTIRTAGGPVQAVSVYSDDHGKTWQAGTPIGTGMDENKV

VELSDGSLMLNSRASDGSGFRKVAHSTDGGQTWSEPVSDKNLPDSVDNAQIIRAFPN

AAPDDPRAKVLLLSHSPNPRPWCRDRGTISMSCDDGASWTTSKVFHEPFVGYTTIAV

QSDGSIGLLSEDAHNGADYGGIWYRNFTMNWLGEQCGQKPAEPSPGRRRRRHPQRH

RRRSRPRRPRRALSPRRHRHHPPRPSRALRPSRAGPGAGAHDRSEHGAHTGSCAQSA

PEQTDGPTAAPAPETSSAPAAEPTQAPTVAPSVEPTQAPGAQPSSAPKPGATGRAPSV

VNPKATGAATEPGTPSSSASPAPSRNAAPTPKPGMEPDEIDRPSDGTMAQPTGAPAR

RVPRRRRRRRPAAGCLARDQRAADPGPCGCRGCRRVPAAAGSPFEELNTRRAGHPA

LSTD

SEQ ID NO: 10 A. viscosus nanA sialidase

MTTTKSSALRRLSALAGSLALAVTGIIAAAPPAHATPTSDGLADVTITQTHAPADGIY

AVGDVMTFDITLTNTSGQARSFAPASTNLSGNVLKCRWSNVAAGATKTDCTGLATH

TVTAEDLKAGGFTPQIAYEVKAVGYKGEALNKPEPVTGPTSQIKPASLKVESFTLASP

KETYTVGDVVSYTVRIRSLSDQTINVAATDSSFDDLARQCHWGNLKPGQGAVYNCK

PLTHTITQADADHGTWTPSITLAATGTDGAALQTLAATGEPLSVVVERPKADPAPAP

DASTELPASMSDAQHLAENTATDNYRIPAITTAPNGDLLVSYDERPRDNGNNGGDSP

NPNHIVQRRSTDGGKTWSAPSYIHQGVETGRKVGYSDPSYVVDNQTGTIFNFHVKSF

DQGWGHSQAGTDPEDRSVIQAEVSTSTDNGWSWTHRTITADITRDNPWTARFAASG

QGIQIHQGPHAGRLVQQYTIRTADGVVQAVSVYSDDHGQTWQAGTPTGTGMDENK

VVELSDGSLMLNSRASDGTGFRKVATSTDGGQTWSEPVPDKNLPDSVDNAQIIRPFP

NAAPSDPRAKVLLLSHSPNPRPWSRDRGTISMSCDNGASWVTGRVFNEKFVGYTTIA

VQSDGSIGLLSEDGNYGGIWYRNFTMGWVGDQCSQPRPEPSPSPTPSAAPSAEPTSEP

TTAPAPEPTTAPSSEPSVSPEPSSSAIPAPSQSSSATSGPSTEPDEIDRPSDGAMAQPTGG

AGRPSTSVTGATSRNGLSRTGTNALLVLGVAAAAAAGGYLVLRIRRARTE

SEQ ID NO: 11 S. oralis nanA sialidase MNYKSLDRKQRYGIRKFAVGAASVVIGTVVFGANPVLAQEQANAAGANTETVEPG

QGLSELPKEASSGDLAHLDKDLAGKLAAAQDNGVEVDQDHLKKNESAESETPSSTE

TPAEEANKEEESEDQGAIPRDYYSRDLKNANPVLEKEDVETNAANGQRVDLSNELD

KLKQLKNATVHMEFKPDASAPRFYNLFSVSSDTKENEYFTMSVLDNTALIEGRGAN

GEQFYDKYTDAPLKVRPGQWNSVTFTVEQPTTELPHGRVRLYVNGVLSRTSLKSGN

FIKDMPDVNQAQLGATKRGNKTVWASNLQVRNLTVYDRALSPDEVQTRSQLFERG

ELEQKLPEGAKVTEKEDVFEGGRNNQPNKDGIKSYRIPALLKTDKGTLIAGTDERRL

HHSDWGDIGMVVRRSSDNGKTWGDRIVISNPRDNEHAKHADWPSPVNIDMALVQD

PETKRIFAIYDMFLESKAVFSLPGQAPKAYEQVGDKVYQVLYKQGESGRYTIRENGE

VFDPQNRKTDYRVVVDPKKPAYSDKGDLYKGNELIGNIYFEYSEKNIFRVSNTNYL

WMSYSDDDGKTWSAPKDITHGIRKDWMHFLGTGPGTGIALRTGPHKGRLVIPVYTT

NNVSYLSGSQSSRVIYSDDHGETWQAGEAVNDNRPVGNQTIHSSTMNNPGAQNTES

TVVQLNNGDLKLFMRGLTGDLQVATSHDGGATWDKEIKRYPQVKDVYVQMSAIHT

MHEGKEYILLSNAGGPGRNNGLVHLARVEENGELTWLKHNPIQSGKFAYNSLQELG

NGEYGLLYEHADGNQNDYTLSYKKFNWDFLSRDRISPKEAKVKYAIQKWPGIIAME

FDSEVLVNKAPTLQLANGKTATFMTQYDTKTLLFTIDPEDMGQRITGLAEGAIESMH

NLPVSLAGSKLSDGINGSEAAIHEVPEFTGGVNAEEAAVAEIPEYTGPLATVGEEVAP

TVEKPEFTGGVNAEEAPVAEMPEYTGPLSTVGEEVAPTVEKPEFTGGVNAVEAAVH

ELPEFKGGVNAVLAASNELPEYRGGANFVLAASNDLPEYIGGVNGAEAAVHELPEY

KGDTNLVLAAADNKLSLGQDVTYQAPAAKQAGLPNTGSKETHSLISLGLAGVLLSL FAFGKKRKE

SEQ ID NO: 12 .S', oralis nanH sialidase

MSDLKKYEGVIPAFYACYDDQGEVSPERTRALVQYFIDKGVQGLYVNGSSGECIYQS

VEDRKLILEEVMAVAKGKLTIIAHVACNNTKDSMELARHAESLGVDAIATIPPIYFRL

PEYSVAKYWNDISAAAPNTDYVIYNIPQLAGVALTPSLYTEMLKNPRVIGVKNSSMP

VQDIQTFVSLGGEDHIVFNGPDEQFLGGRLMGAKAGIGGTYGAMPELFLKLNQLIAE

KDLETARELQYAINAIIGKLTSAHGNMYGVIKEVLKINEGLNIGSVRSPLTPVTEEDRP VVEAAAQLIRETKERFL

SEQ ID NO: 13 S. mitis nanA sialidase

MNQRHFDRKQRYGIRKFTVGAASVVIGAVVFGVAPALAQEAPSTNGETAGQSLPEL

PKEVETGNLTNLDKELADKLSTATDKGTEVNREELQANPGSEKAAETEASNETPATE

SEDEKEDGNIPRDFYARELENVNTVVEKEDVETNPSNGQRVDMKEELDKLKKLQNA

TIHMEFKPDASAPRFYNLFSVSSDTKVNEYFTMAILDNTAIVEGRDANGNQFYGDYK

TAPLKIKPGEWNSVTFTVERPNADQPKGQVRVYVNGVLSRTSPQSGRFIKDMPDVN

QVQIGTTKRTGKNFWGSNLKVRNLTVYDRALSPEEVKKRSQLFERGELEKKLPEGA

KVTDKLDVFQGGENRKPNKDGIASYRIPALLKTDKGTLIAGADERRLHHSDWGDIG

MVVRRSDDKGKTWGDRIVISNPRDNENARRAHAGSPVNIDMALVQDPKTKRIFSIFD

MFVEGEAVRDLPGKAPQAYEQIGNKVYQVLYKKGEAGHYTIRENGEVFDPENRKTE YRVVVDPKKPAYSDKGDLYKGEELIGNVYFDYSDKNIFRVSNTNYLWMSYSDDDG KTWSAPKDITYGIRKDWMHFLGTGPGTGIALHSGPHKGRLVIPAYTTNNVSYLGGSQ SSRVIYSDDHGETWHAGEAVNDNRPIGNQTIHSSTMNNPGAQNTESTVVQLNNGDL

KLFMRGLTGDLQVATSKDGGATWEKDVKRYADVKDVYVQMSAIHTVQEGKEYIIL

SNAGGPGRYNGLVHVARVEANGDLTWIKHNPIQSGKFAYNSLQDLGNGEFGLLYEH

ATATQNEYTLSYKKFNWDFLSKDGVAPTKATVKNAVEMSKNVIALEFDSEVLVNQP

PVLKLANGNFATFLTQYDSKTLLFAASKEDIGQEITEIIDGAIESMHNLPVSLEGAGVP

GGKNGAKAAIHEVPEFTGAVNGEGTVHEDPAFEGGINGEEAAVHDVPDFSGGVNGE VAAIHEVPEFTGGINGEEAAKLELPSYEGGANAVEAAKSELPSYEGGANAVEAAKLE LPSYESGAHEVQPASSNLPTLADSVNKAEAAVHKGKEYKANQSTAVQAMAQEHTY QAPAAQQHLLPKTGSEDKSSLAIVGFVGMFLGLLMIGKKRE

SEQ ID NO: 14 S. mitis nanA_l sialidase

MNQSSLNRKNRYGIRKFTIGVASVAIGSVLFGITPALAQETTTNIDVSKVETSLESGAP VSEPVTEVVSGDLNHLDKDLADKLALATNQGVDVNKHNLKEETSKPEGNSEHLPVE SNTGSEESIEHHPAKIEGADDAVVPPRDFFARELTNVKTVFEREDLATNTGNGQRVD LAEELDQLKQLQNATIHMEFKPDANAPQFYNLFSVSSDKKKDEYFSMSVNKGTAMV EARGADGSHFYGSYSDAPLKIKPGQWNSVTFTVERPKADQPNGQVRLYVNGVLSRT NTKSGRFIKDMPDVNKVQIGATRRANQTMWGSNLQIRNLTVYNRALTIEEVKKRSH LFERNDLEKKLPEGAEVTEKKDIFESGRNNQPNGEGINSYRIPALLKTDKGTLIAGGD ERRLHHFDYGDIGMVIRRSQDNGKTWGDKLTISNLRDNPEATDKTATSPLNIDMVLV QDPTTKRIFSIYDMFPEGRAVFGMPNQPEKAYEEIGDKTYQVLYKQGETERYTLRDN GEIFNSQNKKTEYRVVVNPTEAGFRDKGDLYKNQELIGNIYFKQSDKNPFRV ANTSY LWMSYSDDDGKTWSAPKDITPGIRQDWMKFLGTGPGTGIVLRTGAHKGRILVPAYT TNNISHLGGSQSSRLIYSDDHGQTWHAGESPNDNRPVGNSVIHSSNMNKSSAQNTES TVLQLNNGDVKLFMRGLTGDLQVATSKDGGVTWEKTIKRYPEVKDAYVQMSAIHT MHDGKEYILLSNAAGPGRERKNGLVHLARVEENGELTWLKHNPIQNGEFAYNSLQE LGGGEYGLLYEHRENGQNYYTLSYKKFNWDFVSKDLISPTEAKVSQAYEMGKGVF GLEFDSEVLVNRAPILRLANGRTAVFMTQYDSKTLLFAVDKKDIGQEITGIVDGSIES MHNLTVNLAGAGIPGGMNAAESVEHYTEEYTGVLGTSGVEGVPTISVPEYEGGVNS

ELALVSEKEDYRGGVNSASSVVTEVLEYTGPLSTVGSEDAPTVSVLEYEGGVNIDSP EVTEAPEYKEPIGTSGYELAPTVDKPAYTGTIEPLEKEENSGAIIEEGNVSYITENNNK PLENNNVTTSSIISESSKLKHTLKNATGSVQIHASEEVLKNVKDVKIQEVKVSSLSSLN YKAYDIQLNDASGKAVQPKGTVIVTFAAEQSVENVYYVDSKGNLHTLEFLQKDGEV TFETNHFSIYAMTFQLSLDNVVLDNHREDKNGEVNSASPKLLSINGHSQSSQLENKV SNNEQSKLPNTGEDKSISTVLLGFVGVILGAMIFYRRKDSEG

SEQ ID NO: 15 S. mitis nanA_2 sialidase

MDKKKIILTSLASVAVLGAALAASQPSLVKAEEQPTASQPAGETGTKSEVTSPEIKQA EADAKAAEAKVTEAQAKVDTTTPVADEAAKKLETEKKEADEADAAKTKAEEAKKT ADDELAAAKEKAAEADAKAKEEAKKEEDAKKEEADSKEALTEALKQLPDNELLDK KAKEDLLKAVEAGDLKASDILAELADDDKKAEANKETEKKLRNKDQANEANVATT PAEEAKSKDQLPADIKAGIDKAEKADAARPASEKLQDKADDLGENVDELKKEADAL KAEEDKKAETLKKQEDTLXEAKEALKSAKDNGFGEDITAPLEKAVTAIEKERDAAQ NAFDQAASDTKAVADELNKLTDEYNKTLEEVKAAKEKEANEPAKPVEEEPAKPAEK TEAEKAAEAKTEADAKVAELQKKADEAKTKADEATAKATKEAEDVKAAEKAKEE ADKAKTDAEAELAKAKEEAEKAKAKVEELKKEEKDNLEALKAALDQLEKDIDADA TITNKEEAKKALGKEDILAAVEKGDLTAGDVLKELENQNATAEATKDQDPQADEIG ATKQEGKPLSELPAADKEKLDAAYNKEASKPIVKKLQDIADDLVEKIEKLTKVADKD KADATEKAKAVEEKNAALDKQKETLDKAKAALETAKKNQADQAIQDGLQDAVTK LEASFASAKTAADEAQAKFDEVNEVVKAYKAAIDELTDDYNATLGHIENLKEVPKG EEPKDFSGGVNDDEAPSSTPNTNEFTGGANDADAPTAPNANEFAGGVNDEEAPTTE NKPEFNGGVNDEEAPTVPNKPEGEAPKPTGENAKDAPVVKLPEFGANNPEIKKILDEI AKVKEQIKDGEENGSEDYYVEGLKERLADLEEAFDTLSKNLPAVNKVPEYTGPVTPE NGQTQPAVNTPGGQQGGSSQQTPAVQQGGSGQQAPAVQQGGSNQQVPAVQQTNTP

AVAGTSQDNTYQAPAAKEEDKKELPNTGGQESAALASVGFLGLLLGALPFVKRKN

SEQ ID NO: 16 S. mitis nanA_3 sialidase MKYRDFDRKRRYGIRKFAVGAASVVIGTVVFGANPVLAQEQANAAGANTETVEPG QGLSELPKEASSGDLAHLDKDLAGKLAAAQDNGVEVDQDHLKKNESAESETPSSTE TPAEGTNKEEESEDQGAIPRDYYSRDLKNANPVLEKEDVETNAANGQRVDLSNELD KLKQLKNATVHMEFKPDASAPRFYNLFSVSSDTKENEYFTISVLDNTALIEGRGANG EQFYDKYTDAPLKVRPGQWNSVTFTVEQPTTELPHGRVRLYVNGVLSRTSLKSGNFI KDMPDVNQAQLGATKRGNKTVWASNLQVRNLTVYDRALSPDEVQTRSQLFERGEL EQKLPEGAKVTEKEDVFEGGRNNQPNKDGIKSYRIPALLKTDKGTLIAGTDERRLHH SDWGDIGMVVRRSSDNGKTWGDRIVISNPRDNEHAKHADWPSPVNIDMALVQDPE TKRIFAIYDMFLESKAVFSLPGQAPKAYEQVGDKVYQVLYKQGESGRYTIRENGEVF DPQNRKTDYRVVVDPKKPAYSDKGDLYKGNELIGNIYFEYSEKNIFRVSNTNYLWM SYSDDDGKTWSAPKDITHGIRKDWMHFLGTGPGTGIALRTGPHKGRLVIPVYTTNN VSYLSGSQSSRVIYSDDHGETWQAGEAVNDNRPVGNQTIHSSTMNNPGAQNTESTV VQLNNGDLKLFMRGLTGDLQVATSHDGGATWDKEIKRYPQVKDVYVQMSAIHTM HEGKEYILLSNAGGPGRNNGLVHLARVEENGELTWLKHNPIQSGKFAYNSLQDLGN GEYGLLYEHADGNQNDYTLSYKKFNWDFLTKDWISPKEAKVKYAIEKWPGILAMEF DSEVLVNKAPTLQLANGKTARFMTQYDTKTLLFTVDSEDMGQKVTGLAEGAIESM HNLPVSVAGTKLSNGMNGSEAAVHEVPEYTGPLGTAGEEPAPTVEKPEFTGGVNGE

EAAVHEVPEYTGPLGTSGEEPAPTVEKPEFTGGVNAVEAAAHEVPEYTGPLGTSGKE PAPTVEKPEYTGGVNAVEAAVHEVPEYTGPLATVGEEAAPKVDKPEFTGGVNAVEA AVHELPEYTGGVNAADAAVHEIAEYKGADSLVTLAAEDYTYKAPLAQQTLPDTGN KESSLLASLGLTAFFLGLFAMGKKREK

SEQ ID NO: 17 S. mitis nanA_4 sialidase

MEKIWREKSCRYSIRKLTVGTASVLLGAVFLASHTVSADTIKVKQNESTLEKTTAKT DTVTKTTESTEHTQPSEAIDHSKQVLANNSSSESKPTEAKVASATTNQASTEAIVKPN ENKETEKQELPVTEQSNYQLNYDRPTAPSYDGWEKQALPVGNGEMGAKVFGLIGEE RIQYNEKTLWSGGPRPDSTDYNGGNYRERYKILAEIRKALEDGDRQKAKRLAEQNL VGPNNAQYGRYLAFGDIFMVFNNQKKGLDTVTDYHRGLDITEATTTTSYTQDGTTF KRETFSSYPDDVTVTHLTQKGDKKLDFTVWNSLTEDLLANGDYSAEYSNYKSGHVT TDPNGILLKGTVKDNGLQFASYLGIKTDGKVTVHEDSLTITGASYATLLLSAKTNFA QNPKTNYRKDIDLEKTVKGIVEAAQGKYYETLKRNHIKDYQSLFNRVKLNLGGSNIA QTTKEALQTYNPTKGQKLEELFFQYGRYLLISSSRDRTDALPANLQGVWNAVDNPP WNADYHLNVNLQMNYWPAYMSNLAETAKPMINYIDDMRYYGRIAAKEYAGIESK DGQENGWLVHTQATPFGWTTPGWNYYWGWSPAANAWMMQNVYDYYKFTKDET YLKEKIYPMLKETAKFWNSFLHYDQASDRWVSSPSYSPEHGTITIGNTFDQSLVWQL FHDYMEVANHLNVDKDLVTEVKAKFDKLKPLHINKEGRIKEWYEEDSPQFTNEGIE NNHRHVSHLVGLFPGTLFSKDQAEYLEAARATLNHRGDGGTGWSKANKINLWARL LDGNRAHRLLAEQLKYSTLENLWDTHAPFQIDGNFGATSGIAEMLLQSHTGYIAPLP ALPDAWKDGQVSGLVARGNFEVSMQWKDKNLQSLSFLSNVGGDLVVDYPNIEASQ VKVNGKPVKATVLKDGRIQLATQKGDVITFEHFSGRVTSLTAVRQNGVTAELTFNQ

VEGATHYVIQRQVKDESGQTSATREFVTNQTHFIDRSLDPQLAYTYTVKAMLGNVS TQVSEKANVETYNQLMDDRDSRIQYGSAFGNWADSELFGGTEKFADLSLGNYTDK DATATIPFNGVGIEIYGLKSSQLGIAEVKIDGKSVGELDFYTAGATEKGSLIGRFTGLS DGAHVMTITVKQEHKHRGSERSKISLDYFKVLPGQGTTIEKMDDRDSRIQYGSQFKD WSDTELYKSTEKYADINNSDPSTASEAQATIPFTGTGIRIYGLKTSALGKALVTLDGK EMPSLDFYTAGATQKATLIGEFTNLTDGNHILTLKVDPNSPAGRKKISLDSFDVIKSP AVSLDSPSIAPLKKGDKNISLTLPAGDWEAIAVTFPGIKDPLVLRRIDDNHLVTTGDQ TVLSIQDNQVQIPIPDETNRKIGNAIEAYSIQGNTTSSPVVAVFTKKDEKKVENQQPTT SKGDDPAPIVEIPEYTKPIGTAGLEQPPTVSIPEYTQPIGTAGLEQAPTVSIPEYTKPVG TAGIEQAPTVSIPEYTKPIGTAGLEQAPTVSIPEYTQPIGTAGLEQPPTVSIPEYTKSIGT AGLEQPPVVNVPEYTQPIGTAGIEQPPTVSIPEYTKPIGTAGQEQALTVSIPEYTKPIGT

AGQEQAPTVSVPEYKLRVLKDERTGVEIIGGATDLEGISHISSRRVLAQELFGKTYDA

YDLHLKNSTDQSLQPKGSVLVRLPISSAVENVYYLTPSKELQALDFTIREGMAEFTTS

HFSTYAVVYQANGASTTAEQKPSETDIKPLANSSEQVSSSPDLVQSTNDSPKEQLPAT

GETSNPLLFLSGLSLVLTATFLLKSKKDESN

SEQ ID NO: 18 S. mitis nanA_5 sialidase

MKQYFLEKGRIFSIRKLTVGVASVAVGLTFFASGNVAASELVTEPKLEVDGQSKEVA

DVKHEKEEAVKEEAVKEEVTEKTELTAEKATEEAKTAEVAGDVLPEEIPDRAYPDTP

VKKVDTAAIVSEQESPQVETKSILKPTEVAPTEGEKENRAVINGGQDLKRINYEGQPA

TSAAMVYTIFSSPLAGGGSQRYLNSGSGIFVAPNIMLTVAHNFLVKDADTNAGSIRG

GDTTKFYYNVGSNTAKNNSLPTSGNTVLFKEKDIHFWNKEKFGEGIKNDLALVVAP

VPLSIASPNKAATFTPLAEHREYKAGEPVSTIGYPTDSTSPELKEPIVPGQLYKADGVV

KGTEKLDDKGAVGITYRLTSVSGLSGGGIINGDGKVIGIHQHGTVDNMNIAEKDRFG

GGLVLSPEQLAWVKEIIDKYGVKGWYQGDNGNRYYFTPEGEMIRNKTAVIGKNKYS

FDQNGIATLLEGVDYGRVVVEHLDQKDNPVKENDTFVEKTEVGTQFDYNYKTEIEK

TDFYKKNKEKYEIVSIDGKAVNKQLKDTWGEDYSVVSKAPAGTRVIKVVYKVNKG

SFDLRYRLKGTDQELAPATVDNNDGKEYEVSFVHRFQAKEITGYRAVNASQEATIQ

HKGVNQVIFEYEKIEDPKPATPATPVVDPKDEETEIGNYGPLPSKAQLDYHKEELAAF

IHYGMNTYTNSEWGNGRENPQNFNPTNLDTDQWIKTLKDAGFKRTIMVVKHHDGF

VIYPSQYTKHTVAASPWKDGKGDLLEEISKSATKYDMNMGVYLSPWDANNPKYHV

STEKEYNEYYLNQLKEILGNPKYGNKGKFIEVWMDGARGSGAQKVTYTFDEWFKYI

KKAEGDIAIFSAQPTSVRWIGNERGIAGDPVWHKVKKAKITDDVKNEYLNHGDPEG

DMYSVGEADVSIRSGWFYHDNQQPKSIKDLMDIYFKSVGRGTPLLLNIPPNKEGKFA

DADVARLKEFRATLDQMYATDFAKGATVTASSTRKNHLYQASNLTDGKDDTSWAL

SNDAKTGEFrVDLGQKRRFDVVELKEDIAKGQRISGFKVEVELNGRWVPYGEGSTV

GYRRLVQGQPVEAQKIRVTITNSQATPILTNFSVYKTPSSIEKTDGYPLGLDYHSNTT

ADKANTTWYDESEGIRGTSMWTNKKDASVTYRFNGTKAYVVSTVDPNHGEMSVY

VDGQKVADVQTNNAARKRSQMVYETDDLAPGEHTIKLVNKTGKAIATEGIYTLNN

AGKGMFELKETTYEVQKGQPVTVTIKRVGGSKGAATVHVVTEPGTGVHGKVYKDT

TADLTFQDGETEKTLTIPTIDFTEQADSIFDFKVKMTSASDNALLGFASEATVRVMKA

DLLQKDQVSHDDQASQLDYSPGWHHETNSAGKYQNTESWASFGRLNEEQKKNASV

TAYFYGTGLEIKGFVDPGHGIYKVTLDGKELEYQDGQGNATDVNGKKYFSGTATTR

QGDQTLVRLTGLEEGWHAVTLQLDPKRNDTSRNIGIQVDKFITRGEDSALYTKEELV

QAMKNWKDELAKFDQTSLKNTPEARQAFKSNLDKLSEQLSASPANAQEILKIATAL

QAILDKEENYGKEDTPTSEQPEEPNYDKAMASLSEAIQNKSKELSSDKEAKKKLVEL

SEQALTAIQEAKTQDAVDKALQAALTSINQLQATPKEEVKPSQPEEPNYDKAMASLA

EAIQNKSKELGSDKESKKKLVELSEQALTAIQEAKTQDAVDKALQAALTSINQLQAT

PKEEAKPSQPEEPNYDKAMASLAEAIQNKSKELGSDKEAKKKLVELSEQALTAIQEA

KTQDAVDKALQAALTSINQLQATPKEEVKHSIVPTDGDKELVQPQPSLEVVEKVINF

KKVKQEDSSLPKGETRVTQVGRAGKERILTEVAPDGSRTIKLREVVEVAQDEIVLVG

TKKEESGKIASSVHEVPEFTGGVIDSEATIHNLPEFTGGVTDSEAAIHNLPEFTGGVTD

SEAAIHNLPEFTGGMTDSEAAIHNLPEFTGGMTDSEGVAHGVSNVEEGVPSGEATSH

QESGFTSDVTDSETTMNEIVYKNDEKSYVVPPMLEDKTYQAPANRQEVLPKTGSED

GSAFASVGIIGMFLGMIGIVKRKKD

SEQ ID NO: 19 S. mitis nanH sialidase MSGLKKYEGVIPAFYACYDDAGEVSPERTRALVQYFIDKGVQGLYVNGSSGECIYQS

VEDRKLILEEVMAVAKGKLTIIAHVACNNTKDSIELARHAESLGVDAIATIPPIYFRLP

EYSVAKYWNDISAAAPNTDYVIYNIPQLAGVALTPSLYTEMLKNPRVIGVKNSSMPV

QDIQTFVSLGGDDHIVFNGPDEQFLGGRLMGAKAGIGGTYGAMPELFLKLNQLIADK

DLETARELQYAINAIIGKLTAAHGNMYCVIKEVLKINEGLNIGSVRSPLTPVTEEDRPV

VEAAAQLIRESKERFL

SEQ ID NO: 20 P. gingivalis sialidase

MANNTLLAKTRRYVCLVVFCCLMAMMHLSGQEVTMWGDSHGVAPNQVRRTLVK

VALSESLPPGAKQIRIGFSLPKETEEKVTALYLLVSDSLAVRDLPDYKGRVSYDSFPIS

KEDRTTALSADSVAGRCFFYLAADIGPVASFSRSDTLTARVEELAVDGRPLPLKELSP

ASRRLYREYEALFVPGDGGSRNYRIPSILKTANGTLIAMADRRKYNQTDLPEDIDIVM

RRSTDGGKSWSDPRIIVQGEGRNHGFGDVALVQTQAGKLLMIFVGGVGLWQSTPDR

PQRTYISESRDEGLTWSPPRDITHFIFGKDCADPGRSRWLASFCASGQGLVLPSGRVM

FVAAIRESGQEYVLNNYVLYSDDEGGTWQLSDCAYHRGDEAKLSLMPDGRVLMSV

RNQGRQESRQRFFALSSDDGLTWERAKQFEGIHDPGCNGAMLQVKRNGRNQMLHS

LPLGPDGRRDGAVYLFDHVSGRWSAPVVVNSGSSAYSDMTLLADGTIGYFVEEDDE

ISLVFIRFVLDDLFDARQ

SEQ ID NO: 21 T. forsythia siaHI sialidase

MTKKSSISRRSFLKSTALAGAAGMVGTGGAATLLTSCGGGASSNENANAANKPLKE

PGTYYVPELPDMAADGKELKAGIIGCGGRGSGAAMNFLAAANGVSIVALGDTFQDR

VDSLAQKLKDEKNIDIPADKRFVGLDAYKQVIDSDVDVVIVATPPNFRPIHFQYAVE

KSKHCFLEKPICVDAVGYRTIMATAKQAQAKNLCVITGTQRHHQRSYIASYQQIMN

GAIGEITGGTVYWNQSMLWYRERQAGWSDCEWMIRDWVNWKWLSGDHIVEQHV

HNIDVFTWFSGLKPVKAVGFGSRQRRITGDQYDNFSIDFTMENGIHLHSMCRQIDGC

ANNVSEFIQGTKGSWNSTDMGIKDLAGNVIWKYDVEAEKASFKQNDPYTLEHVNWI

NTIRAGKSIDQASETAVSNMAAIMGRESAYTGEETTWEAMTAAALDYTPADLNLGK

MDMKPFVVPVPGKPLEKK

SEQ ID NO: 22 T. forsythia nanH sialidase

MKKFFWIIGLFISMLTTRAADSVYVQNPQIPILIDRTDNVLFRIRIPDATKGDVLNRLTI

RFGNEDKLSEVKAVRLFYAGTEAGTKGRSRFAPVTYVSSHNIRNTRSANPSYSVRQD

EVTTVANTLTLKTRQPMVKGINYFWVSVEMDRNTSLLSKLTPTVTEAVINDKPAVIA

GEQAAVRRMGIGVRHAGDDGSASFRIPGLVTTNEGTLLGVYDVRYNNSVDLQEHID

VGLSRSTDKGQTWEPMRIAMSFGETDGLPSGQNGVGDPSILVDERTNTVWVVAAW

THGMGNARAWTNSMPGMTPDETAQLMMVKSTDDGRTWSEPINITSQVKDPSWCFL

LQGPGRGITMRDGTLVFPIQFIDSLRVPHAGIMYSKDRGETWHIHQPARTNTTEAQV

AEVEPGVLMLNMRDNRGGSRAVSITRDLGKSWTEHSSNRSALPESICMASLISVKAK

DNIIGKDLLFFSNPNTTEGRHHITIKASLDGGVTWLPAHQVLLDEEDGWGYSCLSMID

RETVGIFYESSVAHMTFQAVKIKDLIR

SEQ ID NO: 23 A. muciniphila sialidase

MTWLLCGRGKWNKVKRMMNSVFKCLMSAVCAVALPAFGQEEKTGFPTDRAVTVF

SAGEGNPYASIRIPALLSIGKGQLLAFAEGRYKNTDQGENDIIMSVSKNGGKTWSRPR

AIAKAHGATFNNPCPVYDAKTRTVTVVFQRYPAGVKERQPNIPDGWDDEKCIRNFM

IQSRNGGSSWTKPQEITKTTKRPSGVDIMASGPNAGTQLKSGAHKGRLVIPMNEGPF

GKWVISCIYSDDGGKSWKLGQPTANMKGMVNETSIAETDNGGVVMVARHWGAGN

CRRIAWSQDGGETWGQVEDAPELFCDSTQNSLMTYSLSDQPAYGGKSRILFSGPSAG RRIKGQVAMSYDNGKTWPVKKLLGEGGFAYSSLAMVEPGIVGVLYEENQEHIKKLK

FVPITMEWLTDGEDTGLAPGKKAPVLK

SEQ ID NO: 24 A. muciniphila sialidase

MGLGLLCALGLSIPSVLGKESFEQARRGKFTTLSTKYGLMSCRNGVAEIGGGGKSGE

ASLRMFGGQDAELKLDLKDTPSREVRLSAWAERWTGQAPFEFSIVAIGPNGEKKIYD

GKDIRTGGFHTRIEASVPAGTRSLVFRLTSPENKGMKLDDLFLVPCIPMKVNPQVEM

ASSAYPVMVRIPCSPVLSLNVRTDGCLNPQFLTAVNLDFTGTTKLSDIESVAVIRGEE

APIIHHGEEPFPKDSSQVLGTVKLAGSARPQISVKGKMELEPGDNYLWACVTMKEGA

SLDGRVVVRPASVVAGNKPVRVANAAPVAQRIGVAVVRHGDFKSKFYRIPGLARSR

KGTLLAVYDIRYNHSGDLPANIDVGVSRSTDGGRTWSDVKIAIDDSKIDPSLGATRG

VGDPAILVDEKTGRIWVAAIWSHRHSIWGSKSGDNSPEACGQLVLAYSDDDGLTWS

SPINITEQTKNKDWRILFNGPGNGICMKDGTLVFAAQYWDGKGVPWSTIVYSKDRG

KTWHCGTGVNQQTTEAQVIELEDGSVMINARCNWGGSRIVGVTKDLGQTWEKHPT

NRTAQLKEPVCQGSLLAVDGVPGAGRVVLFSNPNTTSGRSHMTLKASTNDAGSWPE

DKWLLYDARKGWGYSCLAPVDKNHVGVLYESQGALNFLKIPYKDVLNAKNAR

SEQ ID NO: 25 B. thetaiotaomicron sialidase

MKRNHYLFTLILLLGCSIFVKASDTVFVHQTQIPILIERQDNVLFYFRLDAKESRMMD

EIVLDFGKSVNLSDVQAVKLYYGGTEALQDKGKKRFAPVDYISSHRPGNTLAAIPSY

SIKCAEALQPSAKVVLKSHYKLFPGINFFWISLQMKPETSLFTKISSELQSVKIDGKEAI

CEERSPKDIIHRMAVGVRHAGDDGSASFRIPGLVTSNKGTLLGVYDVRYNSSVDLQE

YVDVGLSRSTDGGKTWEKMRLPLSFGEYDGLPAAQNGVGDPSILVDTQTNTIWVVA

AWTHGMGNQRAWWSSHPGMDLYQTAQLVMAKSTDDGKTWSKPINITEQVKDPSW

YFLLQGPGRGITMSDGTLVFPTQFIDSTRVPNAGIMYSKDRGKTWKMHNMARTNTT

EAQVVETEPGVLMLNMRDNRGGSRAVAITKDLGKTWTEHPSSRKALQEPVCMASLI

HVEAEDNVLDKDILLFSNPNTTRGRNHITIKASLDDGLTWLPEHQLMLDEGEGWGYS

CLTMIDRETIGILYESSAAHMTFQAVKLKDLIR

SEQ ID NO: 26 A. viscosus sialidase

MTSHSPFSRRHLPALLGSLPLAATGLIAAAPPAHAVPTSDGLADVTITQVNAPADGLY

SVGDVMTFNITLTNTSGEAHSYAPASTNLSGNVSKCRWRNVPAGTTKTDCTGLATH

TVTAEDLKAGGFTPQIAYEVKAVEYAGKALSTPETIKGATSPVKANSLRVESITPSSS

KEYYKLGDTVTYTVRVRSVSDKTINVAATESSFDDLGRQCHWGGLKPGKGAVYNC

KPLTHTITQADVDAGRWTPSITLTATGTDGTALQTLTATGNPINVVGDHPQATPAPA

PDASTELPASMSQAQHVAPNTATDNYRIPAITTAPNGDLLISYDERPKDNGNGGSDA

PNPNHIVQRRSTDGGKTWSAPTYIHQGTETGKKVGYSDPSYVVDHQTGTIFNFHVKS

YDHGWGNSQAGTDPENRGIIQAEVSTSTDNGWTWTHRTITADITKDNPWTARFAAS

GQGIQIQHGPHAGRLVQQYTIRTAGGAVQAVSVYSDDHGKTWQAGTPVGTGMDEN

KVVELSDGSLMLNSRASDSSGFRKVAHSTDGGQTWSEPVSDKNLPDSVDNAQIIRAF

PNAAPDDPRAKVLLLSHSPNPKPWSRDRGTISMSCDDGASWTTSKVFHEPFVGYTTI

AVQSDGSIGLLSEDAHDGANYGGIWYRNFTMNWLGEQCGQKPAEPSPAPSPTAAPS

AAPSEQPAPSAAPSTEPTQAPAPSSAPEPSAVPEPSSAPAPEPTTAPSTEPTPTPAPSSAP

EPSAGPTAAPAPETSSAPAAEPTQAPTVAPSAEPTQVPGAQPSAAPSEKPGAQPSSAP

KPDATGRAPSVVNPKATAAPSGKASSSASPAPSRSATATSKPGMEPDEIDRPSDGAM

AQPTGGASAPSAAPTQAAKAGSRLSRTGTNALLVLGLAGVAVVGGYLLLRARRSKN

SEQ ID NO: 27 DAS181 without initial Met and without anchoring domain

GDHPQATPAPAPDASTELPASMSQAQHLAANTATDNYRIPAITTAPNGDLLISYDERP

KDNGNGGSDAPNPNHIVQRRSTDGGKTWSAPTYIHQGTETGKKVGYSDPSYVVDH QTGTIFNFHVKSYDQGWGGSRGGTDPENRGIIQAEVSTSTDNGWTWTHRTITADITK DKPWTARFAASGQGIQIQHGPHAGRLVQQYTIRTAGGAVQAVSVYSDDHGKTWQA GTPIGTGMDENKVVELSDGSLMLNSRASDGSGFRKVAHSTDGGQTWSEPVSDKNLP DSVDNAQIIRAFPNAAPDDPRAKVLLLSHSPNPRPWSRDRGTISMSCDDGASWTTSK VFHEPFVGYTTIAVQSDGSIGLLSEDAHNGADYGGIWYRNFTMNWLGEQCGQKPA

SEQ ID NO: 28 Construct 1: mIg-K_DAS181 Protein sequence

METDTLLLWVLLLWVPGSTGDGDHPOATPAPAPDASTELPASMSOAQHLAANTAT DNYRIPAITTAPNGDLLISYDERPKDNGNGGSDAPNPNHIVQRRSTDGGKTWSAPTYI HQGTETGKKVGYSDPSYVVDHQTGTIFNFHVKSYDQGWGGSRGGTDPENRGIIQAE VSTSTDNGWTWTHRTITADITKDKPWTARFAASGQGIQIQHGPHAGRLVQQYTIRTA GGAVQAVSVYSDDHGKTWQAGTPIGTGMDENKVVELSDGSLMLNSRASDGSGFRK VAHSTDGGQTWSEPVSDKNLPDSVDNAQIIRAFPNAAPDDPRAKVLLLSHSPNPRPW SRDRGTISMSCDDGASWTTSKVFHEPFVGYTTIAVQSDGSIGLLSEDAHNGADYGGI WYRNFTMNWLGEQCGQKPAKRKKKGGKNGKNRRNRKKKNP

SEQ ID NO: 29 Construct 2: mIg-K_DAS185 Protein sequence

METDTLLLWVLLLWVPGSTGDGDHPOATPAPAPDASTELPASMSOAQHLAANTAT DNYRIPAITTAPNGDLLISYDERPKDNGNGGSDAPNPNHIVQRRSTDGGKTWSAPTYI HQGTETGKKVGYSDPSYVVDHQTGTIFNFHVKSYDQGWGGSRGGTDPENRGIIQAE VSTSTDNGWTWTHRTITADITKDKPWTARFAASGQGIQIQHGPHAGRLVQQYTIRTA GGAVQAVSVYSDDHGKTWQAGTPIGTGMDENKVVELSDGSLMLNSRASDGSGFRK VAHSTDGGQTWSEPVSDKNLPDSVDNAQIIRAFPNAAPDDPRAKVLLLSHSPNPRPW SRDRGTISMSCDDGASWTTSKVFHEPFVGFTTIAVQSDGSIGLLSEDAHNGADYGGI WYRNFTMNWLGEQCGQKPAKRKKKGGKNGKNRRNRKKKNP

SEQ ID NO: 30 Construct 3: mIg-K_Neu2-AR Protein sequence

METDTLLLWVLLLWVPGSTGDMASLPVLOKESVFQSGAHAYRIPALLYLPGOQSLL AFAEQRASKKDEHAELIVLRRGDYDAPTHQVQWQAQEVVAQARLDGHRSMNPCPL YDAQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTSTDHGRTWSSPRDLTDAAI GPAYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLHPIQRPIPSAFCFLSHDHGR TWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRARVQAQSTNDGLDFQESQ LVKKLVEPPPQGCQGSVISFPSPRSGPGSPAQWLLYTHPTHSWQRADLGAYLNPRPP APEAWSEPVLLAKGSCAYSDLQSMGTGPDGSPLFGCLYEANDYEEIVFLMFTLKQAF

PAEYLPQKRKKKGGKNGKNRRNRKKKNP

SEQ ID NO: 31 Construct 4: DAS181(-AR)_TM Protein Sequence

METDTLLLWVLLLWVPGSTGDYPYDVPDYAGATPARSPGMGDHPQATPAPAPDAS TELPASMSQAQHLAANTATDNYRIPAITTAPNGDLLISYDERPKDNGNGGSDAPNPN HIVQRRSTDGGKTWSAPTYIHQGTETGKKVGYSDPSYVVDHQTGTIFNFHVKSYDQ GWGGSRGGTDPENRGIIQAEVSTSTDNGWTWTHRTITADITKDKPWTARFAASGQGI QIQHGPHAGRLVQQYTIRTAGGAVQAVSVYSDDHGKTWQAGTPIGTGMDENKVVE LSDGSLMLNSRASDGSGFRKVAHSTDGGQTWSEPVSDKNLPDSVDNAQIIRAFPNAA PDDPRAKVLLLSHSPNPRPWSRDRGTISMSCDDGASWTTSKVFHEPFVGFTTIAVQS DGSIGLLSEDAHNGADYGGIWYRNFTMNWLGEOCGOKPAVDEQKLISEEDLNAVG QDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR

SEQ ID NO: 32 Construct 5: DAS185(-AR)_TM Protein Sequence

METDTLLLWVLLLWVPGSTGDYPYDVPDYAGATPARSPGMGDHPQATPAPAPDAS TELPASMSQAQHLAANTATDNYRIPAITTAPNGDLLISYDERPKDNGNGGSDAPNPN HIVQRRSTDGGKTWSAPTYIHQGTETGKKVGYSDPSYVVDHQTGTIFNFHVKSYDQ

GWGGSRGGTDPENRGIIQAEVSTSTDNGWTWTHRTITADITKDKPWTARFAASGQGI

QIQHGPHAGRLVQQYTIRTAGGAVQAVSVYSDDHGKTWQAGTPIGTGMDENKVVE

LSDGSLMLNSRASDGSGFRKVAHSTDGGQTWSEPVSDKNLPDSVDNAQIIRAFPNAA

PDDPRAKVLLLSHSPNPRPWSRDRGTISMSCDDGASWTTSKVFHEPFVGFTTIAVQS

DGSIGLLSEDAHNGADYGGIWYRNFTMNWLGEOCGOKPAVDEQKLISEEDLNAVG

QDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR

SEQ ID NO: 33 Construct 6: Neu2_TM Protein Sequence

METDTLLLWVLLLWVPGSTGDYPYDVPDYAGATPARSPGMASLPVLOKESVFOSG

AHAYRIPALLYLPGQQSLLAFAEQRASKKDEHAELIVLRRGDYDAPTHQVQWQAQE

VVAQARLDGHRSMNPCPLYDAQTGTLFLFFIAIPGQVTEQQQLQTRANVTRLCQVTS

TDHGRTWSSPRDLTDAAIGPAYREWSTFAVGPGHCLQLHDRARSLVVPAYAYRKLH

PIQRPIPSAFCFLSHDHGRTWARGHFVAQDTLECQVAEVETGEQRVVTLNARSHLRA

RVQAQSTNDGLDFQESQLVKKLVEPPPQGCQGSVISFPSPRSGPGSPAQWLLYTHPT

HSWQRADLGAYLNPRPPAPEAWSEPVLLAKGSCAYSDLQSMGTGPDGSPLFGCLYE

ANDYEEIVFLMFTLKOAFPAEYLPOVDEOKLISEEDLNAVGODTQEVIVVPHSLPFKV

VVISAILALVVLTIISLIILIMLWQKKPR

Not underlined = Sialidase Domain

Key to Underlined Sequences:

N-Terminal Portion

METDTLLLWVLLLWVPGSTGD = Signal

YPYDVPDYA = HA Tag

GATPARSPG = Cloning Site

C-Terminal Portion

VD = Cloning Site

EQKLISEEDL = Myc Tag

NAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR = TM Domain

SEQ ID NO: 34 Construct 1: mIg-K_DAS181 Nucleotide sequence

ATGgagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtgacGGCGACCACCCACAG

GCAACACCAGCACCTGCCCCAGATGCCTCCACCGAGCTGCCAGCAAGCATGTCC

CAGGCACAGCACCTGGCAGCAAATACCGCAACAGACAACTACAGAATCCCCGCC

ATCACCACAGCCCCAAATGGCGATCTGCTGATCAGCTATGACGAGCGCCCCAAG

GATAACGGAAATGGAGGCTCCGACGCACCAAACCCTAATCACATCGTGCAGCGG

AGATCTACCGATGGCGGCAAGACATGGAGCGCCCCTACCTACATCCACCAGGGC

ACCGAGACAGGCAAGAAGGTCGGCTACTCTGACCCAAGCTATGTGGTGGATCAC

CAGACCGGCACAATCTTCAACTTTCACGTGAAGTCCTATGACCAGGGATGGGGA

GGCTCTAGGGGCGGCACCGATCCTGAGAATCGCGGCATCATCCAGGCCGAGGTG

TCTACCAGCACAGACAACGGCTGGACCTGGACACACCGGACCATCACAGCCGAC

ATCACAAAGGATAAGCCCTGGACCGCAAGATTCGCAGCAAGCGGACAGGGCATC

CAGATCCAGCACGGACCTCACGCAGGCCGGCTGGTGCAGCAGTACACCATCAGA

ACAGCAGGAGGAGCAGTGCAGGCCGTGTCCGTGTATTCTGACGATCACGGCAAG

ACCTGGCAGGCAGGCACCCCAATCGGCACAGGCATGGACGAGAATAAGGTGGTG

GAGCTGAGCGATGGCTCCCTGATGCTGAACTCTAGGGCCAGCGACGGCTCCGGC

TTCCGCAAGGTGGCACACTCTACAGACGGAGGACAGACCTGGTCCGAGCCCGTG

TCTGATAAGAATCTGCCTGACAGCGTGGATAACGCCCAGATCATCCGGGCCTTTC

CTAATGCCGCCCCAGACGATCCCAGAGCCAAGGTGCTGCTGCTGTCCCACTCTCC

AAACCCAAGGCCTTGGAGCCGGGACAGAGGCACAATCAGCATGTCCTGCGACGA

TGGCGCCAGCTGGACCACATCCAAGGTGTTCCACGAGCCATTTGTGGGCTACACC ACAATCGCCGTGCAGTCTGATGGCAGCATCGGACTGCTGAGCGAGGACGCACAC

AATGGCGCCGATTACGGCGGCATCTGGTATCGGAACTTCACCATGAACTGGCTG

GGCGAGCAGTGTGGCCAGAAGCCAGCCAAGCGGAAGAAGAAGGGCGGCAAGAA

CGGCAAGAATAGGCGCAACCGGAAGAAGAAGAACCCCTGATGA

SEQ ID NO: 35 Construct 2: mIg-K_DAS185 Nucleotide sequence

ATGgagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtgacGGCGACCACCCACAG

GCAACACCAGCACCTGCCCCAGATGCCTCCACCGAGCTGCCAGCAAGCATGTCC

CAGGCACAGCACCTGGCAGCAAATACCGCAACAGACAACTACAGAATCCCCGCC

ATCACCACAGCCCCAAATGGCGATCTGCTGATCAGCTATGACGAGCGCCCCAAG

GATAACGGAAATGGAGGCTCCGACGCACCAAACCCTAATCACATCGTGCAGCGG

AGATCTACCGATGGCGGCAAGACATGGAGCGCCCCTACCTACATCCACCAGGGC

ACCGAGACAGGCAAGAAGGTCGGCTACTCTGACCCAAGCTATGTGGTGGATCAC

CAGACCGGCACAATCTTCAACTTTCACGTGAAGTCCTATGACCAGGGATGGGGA

GGCTCTAGGGGCGGCACCGATCCTGAGAATCGCGGCATCATCCAGGCCGAGGTG

TCTACCAGCACAGACAACGGCTGGACCTGGACACACCGGACCATCACAGCCGAC

ATCACAAAGGATAAGCCCTGGACCGCAAGATTCGCAGCAAGCGGACAGGGCATC

CAGATCCAGCACGGACCTCACGCAGGCCGGCTGGTGCAGCAGTACACCATCAGA

ACAGCAGGAGGAGCAGTGCAGGCCGTGTCCGTGTATTCTGACGATCACGGCAAG

ACCTGGCAGGCAGGCACCCCAATCGGCACAGGCATGGACGAGAATAAGGTGGTG

GAGCTGAGCGATGGCTCCCTGATGCTGAACTCTAGGGCCAGCGACGGCTCCGGC

TTCCGCAAGGTGGCACACTCTACAGACGGAGGACAGACCTGGTCCGAGCCCGTG

TCTGATAAGAATCTGCCTGACAGCGTGGATAACGCCCAGATCATCCGGGCCTTTC

CTAATGCCGCCCCAGACGATCCCAGAGCCAAGGTGCTGCTGCTGTCCCACTCTCC

AAACCCAAGGCCTTGGAGCCGGGACAGAGGCACAATCAGCATGTCCTGCGACGA

TGGCGCCAGCTGGACCACATCCAAGGTGTTCCACGAGCCATTTGTGGGCTTCACC

ACAATCGCCGTGCAGTCTGATGGCAGCATCGGACTGCTGAGCGAGGACGCACAC

AATGGCGCCGATTACGGCGGCATCTGGTATCGGAACTTCACCATGAACTGGCTG

GGCGAGCAGTGTGGCCAGAAGCCAGCCAAGCGGAAGAAGAAGGGCGGCAAGAA

CGGCAAGAATAGGCGCAACCGGAAGAAGAAGAACCCCTGATGA

SEQ ID NO: 36 Construct 3: mIg-K_Neu2-AR Nucleotide Sequence

ATGgagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtgacATGGCCAGCCTGCCT

GTGCTGCAGAAGGAGAGCGTGTTCCAGTCCGGCGCCCACGCATACAGAATCCCC

GCCCTGCTGTATCTGCCTGGCCAGCAGTCCCTGCTGGCCTTTGCCGAGCAGAGAG

CCTCTAAGAAGGACGAGCACGCAGAGCTGATCGTGCTGAGGAGGGGCGACTACG

ATGCACCAACCCACCAGGTGCAGTGGCAGGCACAGGAGGTGGTGGCACAGGCA

AGGCTGGACGGACACCGCAGCATGAATCCATGCCCCCTGTATGATGCCCAGACC

GGCACACTGTTCCTGTTCTTTATCGCAATCCCCGGCCAGGTGACCGAGCAGCAGC

AGCTGCAGACCAGAGCCAACGTGACAAGACTGTGCCAGGTGACCTCCACAGACC

ACGGCAGGACCTGGAGCAGCCCTCGCGACCTGACAGATGCAGCAATCGGACCAG

CATACAGGGAGTGGTCTACATTCGCCGTGGGCCCTGGCCACTGCCTGCAGCTGCA

CGATCGGGCCAGAAGCCTGGTGGTGCCAGCCTACGCCTATCGGAAGCTGCACCC

CATCCAGAGACCTATCCCATCTGCCTTCTGCTTTCTGAGCCACGACCACGGCAGA

ACTTGGGCCAGAGGCCACTTTGTGGCCCAGGATACACTGGAGTGTCAGGTGGCA

GAGGTGGAGACCGGAGAGCAGAGGGTGGTGACACTGAATGCACGCAGCCACCT

GAGGGCCCGCGTGCAGGCCCAGTCCACCAACGACGGCCTGGATTTCCAGGAGTC

TCAGCTGGTGAAGAAGCTGGTGGAGCCACCTCCACAGGGATGTCAGGGCTCTGT

GATCAGCTTTCCCTCCCCTCGGTCTGGCCCAGGCAGCCCAGCACAGTGGCTGCTG

TACACCCACCCCACACACTCCTGGCAGAGGGCAGACCTGGGAGCATATCTGAAT CCAAGACCCCCTGCACCAGAGGCCTGGTCCGAGCCTGTGCTGCTGGCCAAGGGC TCTTGCGCCTACAGCGACCTGCAGAGCATGGGCACCGGACCTGATGGCTCTCCAC TGTTCGGCTGTCTGTACGAGGCCAACGATTATGAGGAGATCGTGTTCCTGATGTT

TACACTGAAGCAGGCCTTTCCTGCCGAGTATCTGCCACAGAAGCGGAAGAAGAA GGGCGGCAAGAACGGCAAGAATCGGAGAAACCGGAAGAAGAAGAACCCTTGAT GA

SEQ ID NO: 37 Construct 4: DAS181(-AR)_TM Nucleotide sequence atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtgacTATCCA

TATGATGTTCCAGATTATGCTGGGGCCACGCCGGCCAGATCTCCCGGGATGGGCG

ACCACCCACAGGCAACACCAGCACCTGCCCCAGATGCCTCCACCGAGCTGCCAG

CAAGCATGTCCCAGGCACAGCACCTGGCAGCAAATACCGCAACAGACAACTACA

GAATCCCCGCCATCACCACAGCCCCAAATGGCGATCTGCTGATCAGCTATGACG

AGCGCCCCAAGGATAACGGAAATGGAGGCTCCGACGCACCAAACCCTAATCACA

TCGTGCAGCGGAGATCTACCGATGGCGGCAAGACATGGAGCGCCCCTACCTACA

TCCACCAGGGCACCGAGACAGGCAAGAAGGTCGGCTACTCTGACCCAAGCTATG

TGGTGGATCACCAGACCGGCACAATCTTCAACTTTCACGTGAAGTCCTATGACCA

GGGATGGGGAGGCTCTAGGGGCGGCACCGATCCTGAGAATCGCGGCATCATCCA

GGCCGAGGTGTCTACCAGCACAGACAACGGCTGGACCTGGACACACCGGACCAT

CACAGCCGACATCACAAAGGATAAGCCCTGGACCGCAAGATTCGCAGCAAGCGG

ACAGGGCATCCAGATCCAGCACGGACCTCACGCAGGCCGGCTGGTGCAGCAGTA

CACCATCAGAACAGCAGGAGGAGCAGTGCAGGCCGTGTCCGTGTATTCTGACGA TCACGGCAAGACCTGGCAGGCAGGCACCCCAATCGGCACAGGCATGGACGAGA ATAAGGTGGTGGAGCTGAGCGATGGCTCCCTGATGCTGAACTCTAGGGCCAGCG

ACGGCTCCGGCTTCCGCAAGGTGGCACACTCTACAGACGGAGGACAGACCTGGT

CCGAGCCCGTGTCTGATAAGAATCTGCCTGACAGCGTGGATAACGCCCAGATCA

TCCGGGCCTTTCCTAATGCCGCCCCAGACGATCCCAGAGCCAAGGTGCTGCTGCT

GTCCCACTCTCCAAACCCAAGGCCTTGGAGCCGGGACAGAGGCACAATCAGCAT GTCCTGCGACGATGGCGCCAGCTGGACCACATCCAAGGTGTTCCACGAGCCATTT GTGGGCTACACCACAATCGCCGTGCAGTCTGATGGCAGCATCGGACTGCTGAGC

GAGGACGCACACAATGGCGCCGATTACGGCGGCATCTGGTATCGGAACTTCACC

ATGAACTGGCTGGGCGAGCAGTGTGGCCAGAAGCCAGCCGTCGACGAACAAAA ACTCATCTCAGAAGAG

GATCTGaatgctgtgggccaggacacgcaggaggtcatcgtggtgccacactccttgccctttaaggtggtggtgatctcagcca tcctggccctggtggtgctcaccatcatctcccttatcatcctcatcatgctttggcagaagaagccacgt

SEQ ID NO: 38 Construct 5: DAS185(-AR)_TM Nucleotide sequence atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtgacTATCCATATGATGTTCC

AGATTATGCTGGGGCCACGCCGGCCAGATCTCCCGGGATGGGCGACCACCCACA

GGCAACACCAGCACCTGCCCCAGATGCCTCCACCGAGCTGCCAGCAAGCATGTC CCAGGCACAGCACCTGGCAGCAAATACCGCAACAGACAACTACAGAATCCCCGC CATCACCACAGCCCCAAATGGCGATCTGCTGATCAGCTATGACGAGCGCCCCAA

GGATAACGGAAATGGAGGCTCCGACGCACCAAACCCTAATCACATCGTGCAGCG

GAGATCTACCGATGGCGGCAAGACATGGAGCGCCCCTACCTACATCCACCAGGG

CACCGAGACAGGCAAGAAGGTCGGCTACTCTGACCCAAGCTATGTGGTGGATCA

CCAGACCGGCACAATCTTCAACTTTCACGTGAAGTCCTATGACCAGGGATGGGG

AGGCTCTAGGGGCGGCACCGATCCTGAGAATCGCGGCATCATCCAGGCCGAGGT

GTCTACCAGCACAGACAACGGCTGGACCTGGACACACCGGACCATCACAGCCGA CATCACAAAGGATAAGCCCTGGACCGCAAGATTCGCAGCAAGCGGACAGGGCAT CCAGATCCAGCACGGACCTCACGCAGGCCGGCTGGTGCAGCAGTACACCATCAG AACAGCAGGAGGAGCAGTGCAGGCCGTGTCCGTGTATTCTGACGATCACGGCAA

GACCTGGCAGGCAGGCACCCCAATCGGCACAGGCATGGACGAGAATAAGGTGGT

GGAGCTGAGCGATGGCTCCCTGATGCTGAACTCTAGGGCCAGCGACGGCTCCGG

CTTCCGCAAGGTGGCACACTCTACAGACGGAGGACAGACCTGGTCCGAGCCCGT

GTCTGATAAGAATCTGCCTGACAGCGTGGATAACGCCCAGATCATCCGGGCCTTT

CCTAATGCCGCCCCAGACGATCCCAGAGCCAAGGTGCTGCTGCTGTCCCACTCTC

CAAACCCAAGGCCTTGGAGCCGGGACAGAGGCACAATCAGCATGTCCTGCGACG

ATGGCGCCAGCTGGACCACATCCAAGGTGTTCCACGAGCCATTTGTGGGCTTCAC

CACAATCGCCGTGCAGTCTGATGGCAGCATCGGACTGCTGAGCGAGGACGCACA

CAATGGCGCCGATTACGGCGGCATCTGGTATCGGAACTTCACCATGAACTGGCTG

GGCGAGCAGTGTGGCCAGAAGCCAGCCGTCGACGAACAAAAACTCATCTCAGAA

GAGGATCTGaatgctgtgggccaggacacgcaggaggtcatcgtggtgccacactccttgccctttaaggtggtggtgatctc agccatcctggccctggtggtgctcaccatcatctcccttatcatcctcatcatgctttggcagaagaagccacgt

SEQ ID NO: 39 Construct 6: Neu2_TM Nucleotide sequence atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtgacTATCCATATGATGTTCC

AGATTATGCTGGGGCCACGCCGGCCAGATCTCCCGGGATGGCCAGCCTGCCTGT

GCTGCAGAAGGAGAGCGTGTTCCAGTCCGGCGCCCACGCATACAGAATCCCCGC

CCTGCTGTATCTGCCTGGCCAGCAGTCCCTGCTGGCCTTTGCCGAGCAGAGAGCC

TCTAAGAAGGACGAGCACGCAGAGCTGATCGTGCTGAGGAGGGGCGACTACGAT

GCACCAACCCACCAGGTGCAGTGGCAGGCACAGGAGGTGGTGGCACAGGCAAG

GCTGGACGGACACCGCAGCATGAATCCATGCCCCCTGTATGATGCCCAGACCGG

CACACTGTTCCTGTTCTTTATCGCAATCCCCGGCCAGGTGACCGAGCAGCAGCAG

CTGCAGACCAGAGCCAACGTGACAAGACTGTGCCAGGTGACCTCCACAGACCAC

GGCAGGACCTGGAGCAGCCCTCGCGACCTGACAGATGCAGCAATCGGACCAGCA

TACAGGGAGTGGTCTACATTCGCCGTGGGCCCTGGCCACTGCCTGCAGCTGCACG

ATCGGGCCAGAAGCCTGGTGGTGCCAGCCTACGCCTATCGGAAGCTGCACCCCA

TCCAGAGACCTATCCCATCTGCCTTCTGCTTTCTGAGCCACGACCACGGCAGAAC

TTGGGCCAGAGGCCACTTTGTGGCCCAGGATACACTGGAGTGTCAGGTGGCAGA

GGTGGAGACCGGAGAGCAGAGGGTGGTGACACTGAATGCACGCAGCCACCTGA

GGGCCCGCGTGCAGGCCCAGTCCACCAACGACGGCCTGGATTTCCAGGAGTCTC

AGCTGGTGAAGAAGCTGGTGGAGCCACCTCCACAGGGATGTCAGGGCTCTGTGA

TCAGCTTTCCCTCCCCTCGGTCTGGCCCAGGCAGCCCAGCACAGTGGCTGCTGTA

CACCCACCCCACACACTCCTGGCAGAGGGCAGACCTGGGAGCATATCTGAATCC

AAGACCCCCTGCACCAGAGGCCTGGTCCGAGCCTGTGCTGCTGGCCAAGGGCTC

TTGCGCCTACAGCGACCTGCAGAGCATGGGCACCGGACCTGATGGCTCTCCACTG

TTCGGCTGTCTGTACGAGGCCAACGATTATGAGGAGATCGTGTTCCTGATGTTTA

CACTGAAGCAGGCCTTTCCTGCCGAGTATCTGCCACAGGTC

GACGAACAAAAACTCATCTCAGAAGAGGATCTGaatgctgtgggccaggacacgcaggaggtcatc gtggtgccacactccttgccctttaaggtggtggtgatctcagccatcctggccctggtggtgctcaccatcatctcccttatcatcctcat catgctttggcagaagaagccacgt

SEQ ID NO : 40 Exemplary amino acid secretion sequence

METDTLLLWVLLLWVPGSTGD

SEQ ID NO: 41 HA tag amino acid sequence

YPYDVPDYA

SEQ ID NO: 42 N-terminal cloning site amino acid sequence GATPARSPG SEQ ID NO: 43 C-terminal cloning site amino acid sequence VD

SEQ ID NO: 44 Myc Tag amino acid sequence

EQKLISEEDL

SEQ ID NO: 53 Salmonella typhimurium sialidase

TVEKSVVFKAEGEHFTDQKGNTIVGSGSGGTTKYFRIPAMCTTSKGTIVVFADARHN

TASDQSFIDTAAARSTDGGKTWNKKIAIYNDRVNSKLSRVMDPTCIVANIQGRETILV

MVGKWNNNDKTWGAYRDKAPDTDWDLVLYKSTDDGVTFSKVETNIHDIVTKNGTI

SAMLGGVGSGLQLNDGKLVFPVQMVRTKNITTVLNTSFIYSTDGITWSLPSGYCEGF

GSENNIIEFNASLVNNIRNSGLRRSFETKDFGKTWTEFPPMDKKVDNRNHGVQGSTIT

IPSGNKLVAAHSSAQNKNNDYTRSDISLYAHNLYSGEVKLIDDFYPKVGNASGAGYS

CLSYRKNVDKETLYVVYEANGSIEFQDLSRHLPVIKSYN

SEQ ID NO: 54 Vibrio cholera sialidase

MRFKNVKKTALMLAMFGMATSSNAALFDYNATGDTEFDSPAKQGWMQDNTNNGS

GVETNADGMPAWEVQGIGGRAQWTYSESTNQHAQASSFGWRMTTEMKVESGGMI

TNYYANGTQRVLPIISLDSSGNLVVEFEGQTGRTVLATGTAATEYHKFELVFLPGSNP

SASFYFDGKLIRDNIQPTASKQNMIVWGNGSSNTDGVAAYRDIKFEIQGDVIFRGPDR

IPSIVASSVTPGVVTAFAEKRVGGGDPGALSNTNDIITRTSRDGGITWDTELNLTEQIN

VSDEFDFSDPRPIYDPSSNTVLVSYARWPTDAAQNGDRIKPWMPNGIFYSVYDVASG

NWQAPIDVTDQVKERSFQIAGWGGSELYRRNTSLNSQQDWQSNAKIRIVDGAANQI

QVADGSRKYVVTLSIDESGGLVANLNGVSAPIILQSEHAKVHSFHDYELQYSALNHT

TTLFVDGQQITTWAGEVSQENNIQFGNADAQIDGRLHVQKIVLTQQGHNLVEFDAFY

LAQQTPEVEKDLEKLGWTKIKTGNTMSLYGNASVNPGPGHGITLTRQQNISGSQNGR

LIYPAIVLDRFFLNVMSIYSDDGGSNWQTGSTLPIPFRWKSSSILETLEPSEADMVELQ

NGDLLLTARLDFNQIVNGVNYSPRQQFLSKDGGITWSLLEANNANVFSNISTGTVDA

SITRFEQSDGSHFLLFTNPQGNPAGTNGRQNLGLWFSFDEGVTWKGPIQLVNGASAY

SDIYQLDSENAIVIVETDNSNMRILRMPITLLKQKLTLSQN

SEQ ID NO: 55 Lv-CD19-CAR Plasmid DNA sequence

ATGGAGTTTGGACTGAGCTGGCTGTTTCTCGTGGCCATTCTGAAGGGCGTCCAGT

GCAGCAGAGACATCCAGATGACCCAGACAACCAGCTCTCTGAGCGCTAGCCTCG

GAGATAGAGTGACCATTAGCTGTAGAGCCTCCCAAGACATTTCCAAGTACCTCA

ACTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCA

GCAGACTGCACTCCGGAGTGCCCTCTAGGTTTTCCGGATCCGGCAGCGGCACAG

ACTACTCTCTGACCATCTCCAATCTGGAGCAAGAGGACATCGCCACCTACTTCTG

CCAGCAAGGCAACACACTGCCTTACACATTCGGCGGCGGAACAAAGCTCGAACT

GAAAAGAGGCGGCGGCGGAAGCGGAGGAGGAGGATCCGGAGGCGGAGGATCCG

GCGGAGGAGGCTCCGAAGTCCAGCTGCAACAAAGCGGACCCGGACTGGTGGCTC

CCAGCCAATCTCTGAGCGTGACATGCACAGTGTCCGGCGTCTCTCTGCCCGACTA

CGGAGTCAGCTGGATTAGACAGCCTCCTAGAAAGGGACTGGAGTGGCTGGGAGT

CATCTGGGGCAGCGAGACCACCTACTATAACTCCGCCCTCAAGTCTAGGCTCACC

ATCATCAAAGACAACAGCAAGAGCCAAGTGTTCCTCAAGATGAACAGCCTCCAG

ACCGACGACACCGCCATCTACTACTGCGCCAAACACTACTACTACGGAGGCAGC

TACGCTATGGATTACTGGGGCCAAGGCACCACAGTCACAGTGAGCAGCTATGTG

ACCGTGAGCAGCCAAGACCCCGCCAAAGATCCCAAGTTCTGGGTGCTGGTCGTG

GTGGGAGGCGTGCTGGCTTGTTATTCTCTGCTGGTGACCGTGGCCTTCATCATCTT

CTGGGTGAGGAGCAAGAGATCCAGACTGCTGCACAGCGACTACATGAACATGAC ACCTAGAAGGCCCGGCCCCACAAGGAAACATTACCAGCCCTACGCCCCCCCTAG AGACTTCGCTGCCTATAGATCCAAGAGAGGAAGAAAAAAGCTGCTCTACATCTT

CAAGCAGCCCTTCATGAGGCCCGTGCAAACAACACAAGAGGAGGACGGATGTAG CTGTAGATTCCCCGAGGAGGAAGAGGGAGGATGCGAGCTGAGAGTGAAGTTCTC TAGGAGCGCCGATGCTCCCGCTTATCAGCAAGGCCAGAACCAGCTGTACAATGA GCTGAATCTGGGAAGAAGGGAAGAATACGACGTGCTGGATAAGAGGAGGGGAA GAGACCCCGAGATGGGAGGCAAGCCTAGAAGGAAGAACCCCCAAGAGGGACTG

TACAACGAGCTCCAAAAGGACAAGATGGCTGAAGCCTACAGCGAGATCGGAATG AAGGGAGAGAGAAGGAGGGGCAAGGGCCACGATGGACTCTACCAAGGCCTCAG CACAGCCACCAAGGACACCTACGACGCTCTGCACATGCAAGCTCTGCCCCCAGA TGATGA

SEQ ID NO: 56 Lv-CD19-CAR Translated amino acid sequence

MEFGLSWLFLVAILKGVQCSRDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWY QQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLP YTFGGGTKLELKRGGGGSGGGGSGGGGSGGGGSEVQLQQSGPGLVAPSQSLSVTCT VSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFL

KMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTTVTVSSYVTVSSQDPAKDPKF WVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPY APPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKF SRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPDD

SEQ ID NO: 57 CD19-scFv amino acid sequence

MEFGLSWLFLVAILKGVQCSRDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWY QQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLP YTFGGGTKLELKRGGGGSGGGGSGGGGSGGGGSEVQLQQSGPGLVAPSQSLSVTCT

VSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFL KMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTTVTVS

SEQ ID NO: 58 CD55-A27 amino acid sequence

MDCGLPPDVPNAQPALEGRTSFPEDTVITYKCEESFVKIPGEKDSVICLKGSQWSDIE EFCNRSCEVPTRLNSASLKQPYITQNYFPVGTVVEYECRPGYRREPSLSPKLTCLQNL KWSTAVEFCKKKSCPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGS SVQWSDPLPECREIYCPAPPQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYC

TVNNDEGEWSGPPPECRGGGGSGGGGSGGGGSDGTLFPGDDDLAIPATEFFSTKAA KAPEDKAADAAAAAADDNEETLKQRLTNLEKKITNVTTKFEQIEKCCKRNDEVLFR LENHAETLRAAMISLAKKIDVQTGRAAAE

TK-left (SEQ ID NO: 59) agttgataatcggccccatgttttcaggtaaaagtacagaattaattagacgagttagacgttatcaaatagctcaatataaatgcgtgact ataaaatattctaacgataatagatacggaacgggactatggacgcatgataagaataattttgaagcattggaagcaactaaactatgt gatgtcttggaatcaattacagatttctccgtgataggtatcgatgaaggacagttctttccagacattgttgaatt

Sialidase (reverse complement): (SEQ ID NO: 60) tcatcaggggttcttcttcttccggttgcgcctattcttgccgttcttgccgcccttcttcttccgcttggctggcttctggccacactgctcgc ccagccagttcatggtgaagttccgataccagatgccgccgtaatcggcgccattgtgtgcgtcctcgctcagcagtccgatgctgcca tcagactgcacggcgattgtggtgtagcccacaaatggctcgtggaacaccttggatgtggtccagctggcgccatcgtcgcaggac atgctgattgtgcctctgtcccggctccaaggccttgggtttggagagtgggacagcagcagcaccttggctctgggatcgtctgggg cggcattaggaaaggcccggatgatctgggcgttatccacgctgtcaggcagattcttatcagacacgggctcggaccaggtctgtcc tccgtctgtagagtgtgccaccttgcggaagccggagccgtcgctggccctagagttcagcatcagggagccatcgctcagctccac caccttattctcgtccatgcctgtgccgattggggtgcctgcctgccaggtcttgccgtgatcgtcagaatacacggacacggcctgca ctgctcctcctgctgttctgatggtgtactgctgcaccagccggcctgcgtgaggtccgtgctggatctggatgccctgtccgcttgctg cgaatcttgcggtccagggcttatcctttgtgatgtcggctgtgatggtccggtgtgtccaggtccagccgttgtctgtgctggtagacac ctcggcctggatgatgccgcgattctcaggatcggtgccgcccctagagcctccccatccctggtcataggacttcacgtgaaagttga agattgtgccggtctggtgatccaccacatagcttgggtcagagtagccgaccttcttgcctgtctcggtgccctggtggatgtaggtag gggcgctccatgtcttgccgccatcggtagatctccgctgcacgatgtgattagggtttggtgcgtcggagcctccatttccgttatcctt ggggcgctcgtcatagctgatcagcagatcgccatttggggctgtggtgatggcggggattctgtagttgtctgttgcggtatttgctgc caggtgctgtgcctgggacatgcttgctggcagctcggtggaggcatctggggcaggtgctggtgttgcctgtgggtggtcgcccat

F17R: (SEQ ID NO: 61) gaatttcattttgtttttttctatgctataa

LoxP: (SEQ ID NO: 62) ataacttcgtataatgtatgctatacgaagttat

GFP: (SEQ ID NO: 63)

Atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttca gcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgcc ctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaa gtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagt tcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctgga gtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacat cgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaacca ctacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccg ggatcactctcggcatggacgagctgtacaag

TK-right: (SEQ ID NO: 64) aattctgtgagcgtatggcaaacgaaggaaaaatagttatagtagccgcactcgatgggacatttcaacgtaaaccgtttaataatatttt gaatcttattccattatctgaaatggtggtaaaactaactgctgtgtgtatgaaatgctttaaggaggcttccttttctaaacgattgggtgag gaaaccgagatagaaataa

SEQ ID NO: 65 Sequence of a portion of a vaccinia virus construct for expressing a sialidase (DAS181). atgaacggcggacatattcagttgataatcggccccatgttttcaggtaaaagtacagaattaattagacgagttagacgttatcaaatag ctcaatataaatgcgtgactataaaatattctaacgataatagatacggaacgggactatggacgcatgataagaataattttgaagcatt ggaagcaactaaactatgtgatgtcttggaatcaattacagatttctccgtgataggtatcgatgaaggacagttctttccagacattgttg aattagatcgataaaaattaattaattacccgggtaccacatttgtagaggttttacttgctttaaaaaacctcccacacctccccctgaacc tgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaat aaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgctcgaagcggccggcctcatcagggg ttcttcttcttccggttgcgcctattcttgccgttcttgccgcccttcttcttccgcttggctggcttctggccacactgctcgcccagccagtt catggtgaagttccgataccagatgccgccgtaatcggcgccattgtgtgcgtcctcgctcagcagtccgatgctgccatcagactgca cggcgattgtggtgtagcccacaaatggctcgtggaacaccttggatgtggtccagctggcgccatcgtcgcaggacatgctgattgt gcctctgtcccggctccaaggccttgggtttggagagtgggacagcagcagcaccttggctctgggatcgtctggggcggcattagg aaaggcccggatgatctgggcgttatccacgctgtcaggcagattcttatcagacacgggctcggaccaggtctgtcctccgtctgtag agtgtgccaccttgcggaagccggagccgtcgctggccctagagttcagcatcagggagccatcgctcagctccaccaccttattctc gtccatgcctgtgccgattggggtgcctgcctgccaggtcttgccgtgatcgtcagaatacacggacacggcctgcactgctcctcctg ctgttctgatggtgtactgctgcaccagccggcctgcgtgaggtccgtgctggatctggatgccctgtccgcttgctgcgaatcttgcgg tccagggcttatcctttgtgatgtcggctgtgatggtccggtgtgtccaggtccagccgttgtctgtgctggtagacacctcggcctggat gatgccgcgattctcaggatcggtgccgcccctagagcctccccatccctggtcataggacttcacgtgaaagttgaagattgtgccgg tctggtgatccaccacatagcttgggtcagagtagccgaccttcttgcctgtctcggtgccctggtggatgtaggtaggggcgctccatg tcttgccgccatcggtagatctccgctgcacgatgtgattagggtttggtgcgtcggagcctccatttccgttatccttggggcgctcgtc atagctgatcagcagatcgccatttggggctgtggtgatggcggggattctgtagttgtctgttgcggtatttgctgccaggtgctgtgcc tgggacatgcttgctggcagctcggtggaggcatctggggcaggtgctggtgttgcctgtgggtggtcgcccatttatagcatagaaaa aaacaaaatgaaattcaagctttcactaattccaaacccacccgctttttatagtaagtttttcacccataaataataaatacaataattaattt ctcgtaaaagtagaaaatatattctaatttattgcacggtaaggaagtagatcataactcgagataacttcgtataatgtatgctatacgaag ttatctagcgctaccggtcgccaccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacgg cgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgca ccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccac atgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactac aagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggc aacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggt gaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggc cccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcct gctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaatagactagcgctcaataacttcgtataatgt atgctatacgaagttatgcggccgcttcctcgctcactgacgctagcgccctatagtgagtcgtattacagatccaattctgtgagcgtat ggcaaacgaaggaaaaatagttatagtagccgcactcgatgggacatttcaacgtaaaccgtttaataatattttgaatcttattccattatc tgaaatggtggtaaaactaactgctgtgtgtatgaaatgctttaaggaggcttccttttctaaacgattgggtgaggaaaccgagatagaa ataataggaggtaatgatatgtatcaatcggtgtgtagaaagtgttacatcgactcata

SEQ ID NO: 66 mutant vaccinia virus (VV) H3L protein

MAAAKTPVIVVPVAAALPSETFPNVHEHINDQAAADVADAEVMAAKRNVVVAKDD PDHYKDYAFIQWTGGNIRNDDKYTHFFSGFCNTMCTEETKRNIARHEAEWDSNFFT EEENKKVEYVVIVENDNVIAAIAFEAPVEKAMHDKKIDIEQMAEAITGNAVKTEAAA DKNHAIFTYTGGYDVSESAYIIRVTTAENIADEIIKSGGESSGFYFEIARIENEMKINAQ IEDNAAKYVEHDPREVAEHRFANMAAAAWSRIGTAATKRYPGVMYAFTTPEISFFG EFDINVIGEIVIEFIMFMEIFNVKSKEEWFETGTFVTAFI

SEQ ID NO: 67 mutant vaccinia virus (VV) H3E protein

MAAAKTPVIVVPVIDREPSETFPNVHEHINDQKFDDVKDNEVMAEKRNVVVVKDDP DHYKDYAFIQWTGGNIRNDDKYTHFFSGFCNTMCTEETKRNIARHEAEWDSNFFTE EENKKVEYVVIVENDNVIEDITFERPVEKAMHDKKIDIEQMREIITGNKVKTEEVMD KNHAIFTYTGGYDVSESAYIIRVTTAENIVDEIIKSGGESSGFYFEIARIENEMKINRQIE DNAAKYVEHDPREVAEHRFGWMKPNFWFRIGPATVIRCPGVKNANTAPEISFFGEFD INVIGEIVIEFIMFMEIFNVKSKEEWFETGTFVTAFI

SEQ ID NO: 68 mutant vaccinia virus (VV) H3E protein

MAAAKTPVIVVPVAAAEPSETFPNVHEHINDQAAADVADAEVMAAKRNVVVAKDD PDHYKDYAFIQWTGGNIRNDDKYTHFFSGFCNTMCTEETKRNIARHEAEWDSNFFT EEENKKVEYVVIVENDNVIAAIAAAAPVEKAMHDKKIDIEQMAAAITGNAVKTEAA ADKNHAIFTYTGGYDVSESAYIIRVTTAENAADEIIKSGGESSGFYFEIARIENEMKIN AQIEDNAAKYVEHDPREVAEHRFAAAAAAAWARIGPATTIRCPGVKNANTAPEISFF GEFDINVIGEIVIEFIMFMEIFNVKSKEEWFETGTFVTAFI

SEQ ID NO: 69 mutant vaccinia virus (VV) H3E protein

MAAAKTPVIVVPVIDREPSETFPNVHEHINDQKFDDVKDNEVMAEKRNVVVVKDDP DHYKDYAFIQWTGGNIRNDDKYTHFFSGFCNTMCTEETKRNIARHEAEWDSNFFTE EENKKVEYVVIVENDNVIEDITFERPVEKAMHDKKIDIEQMREIITGNKVKTEEVMD KNHAIFTYTGGYDVSESAYIIRVTTAENIVDEIIKSGGESSGFYFEIARIENEMKINRQIE DNAAKYVEHDPRLVAEHRFGWMKPNFWFRIGPATVIRCPGVKNANTAPLISFFGLFD INVIGLIVILFIMFMLIFNVKSKLLWFLTGTFVTAFI

SEQ ID NO: 70 mutant vaccinia virus (VV) D8L protein

MPQQLSPINIETKKAISNARLKPLDIHYNESKPTTIQNTGALVAINFAGGYISGGFLPNE YVLSSLHIYWGKEDDYGSNHLIDVYKYSGEINLVHWNAKKYSSYEEAAKHDDGLIII SIFLQVLDHKNVYFQKIVNQLDSIRSANTSAPFDSVFYLDNLLPSKLDYFTYLGTTINH SADAVWIIFPTPINIHSDQLSKFRTLLSSSNHDGKPHYITENYANPYKLNDDTQVYYS

GEIIRAATTSPARENYFMRWLSDLRETCFSYYQKYIEENKTFAIIAIVFVFILTAILFFM SRRYSREKQN

SEQ ID NO: 71 mutant vaccinia virus (VV) D8L protein

MPQQLSPINIETKKAISNARLKPLDIHYNESKPTTIQNTGKLFWINFKGGYISGWFLPN

EYVLSSLHIYWGKEDDYGSNHLIDVYKYSGEINLVHWNKKKYSSYEEAKKHDDGLII

ISIFLQVLDHKNVYFQKIVNQLDSIRSTNTSAPFDSVFYLDNLLPSKLDYFSYLGTTIN

HYADAVWIIFPTPINIHSDQLSKYRTLSSSSNHDGKTHYITECYRNLYKLNGDTQVYY

SGEIIRAATTSPARENYFMRWLSDLRETCFSYYQKYIEENKTFAIIAIVFVFILTAILFF MSRRYSREKQN

SEQ ID NO: 72 mutant vaccinia virus (VV) D8L protein

MPQQLSPINIETKKAISNARLKPLDIHYNESKPTTIQNTGKLAAINFAGGYIAAAFLPN

EYVLSSLHIYWGKEDDYGSNHLIDVYKYSGEINLVHWNAKKYSSYEEAAAHDDGLII

ISIFLQVLDHKNVYFQKIVNQLDSIRSGNTSAPFDSVFYLDNLLPSKLDYFAYLGTTIN

HAADAVWIIFPTPINIHSDQASKARTLASSSAHDGKAHYITEAYANAYKLNADTQVY YSGEIIRAATTSPARENYFMRWLSDLRETCFSYYQKYIEENKTFAIIAIVFVFILTAILFF MSRRYSREKQN

SEQ ID NO: 73 mutant vaccinia virus (VV) A27L protein

MDGTLFPGDDDLAIPATEFFSTKAAKAPEDKAADAAAAAADDNEETLKQRLTNLEK

KITNVTTKFEQIEKCCKRNDEVLFRLENHAETLRAAMISLAKKIDVQTGRAAAE

SEQ ID NO: 74 mutant vaccinia virus (VV) L1R protein

MGAAASIQTTVNTLSERISSKLEQAAAASAAAACAIEIGNFYIRQNHGCNLTVKNMC

AAAAAAQLDAVLSAATETYSGLTPEQKAYVPAMFTAALNIQTSVNTVVRDFENYVK

QTCNSSAVVDNALAIQNVIIDECYGAPGSPTNLEFINTGSSKGNCAIKALMQLTTKAT

TQIAPKQVAGTGVQFYMIVIGVIILAALFMYYAKRMLFTSTNDKIKLILANKENVHW TTYMDTFFRTSPMVIATTDMQN

SEQ ID NO: 75 SialF primer

GGCGACCACCCACAGGCAACACCAGCACCTGCCCCA

SEQ ID NO: 76 SialR primer

CCGGTTGCGCCTATTCTTGCCGTTCTTGCCGCC

SEQ ID NO: 77 Human Platelet Factor 4 (PF4)

NGRRICLDLQAPLYKKIIKKLLES

SEQ ID NO: 78 Human Interleukin 8 (IL8) GRELCLDPKENWVQRVVEKFLKRAENS SEQ ID NO: 79 Human Antithrombin III (AT-III)

QIHFFFAKLNCRLYRKANKSSKLVSANRLFGDKS

SEQ ID NO: 80 Human Apoprotein E (ApoE)

ELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAG

SEQ ID NO: 81 Human Angio- Associated Migratory Cell Protein (AAMP) RRLRRMESESES

SEQ ID NO: 82 Human Amphiregulin (AR)

KRKKKGGKNGKNTTNTKKKNP

SEQ ID NO: 83 SP-Sial-rev

TCCTGTCTTGCATTGCACTAAGTCTTG

SEQ ID NO: 84 TM-Sial-fwd

TCATCACTAACGTGGCTTCTTCTGCCAAAGCATG

SEQ ID NO: 85 mutant vaccinia virus (VV) D8L protein

MPQQLSPINIETKKAISNARLKPLDIHYNESKPTTIQNTGKLLWINFKGGYISGWFLPN

EYVLSSLHIYWGKEDDYGSNHLIDVYKYSGEINLVHWNKKKYSSYEEAKKHDDGLII

ISIFLQVLDHKNVYFQKIVNQLDSIRSTNTSAPFDSVFYLDNLLPSKLDYFSYLGTTIN

HYADAVWIIFPTPINIHSDQLSKYRTLSSSSNHDGKTHYITECYRNLYKLNGDTQVYY

SGEIIRAATTSPARENYFMRWLSDLRETCFSYYQKYIEENKTFAIIAIVFVFILTAILFF

MSRRYSREKQN

SEQ ID NO: 86 FAP scFv CDR-L1

RASKSVSTSAYSYMH

SEQ ID NO: 87 FAP CDR-L2

LASNLES

SEQ ID NO: 88 FAP CDR-L3:

QHSRELPYT

SEQ ID NO: 89 FAP CDR-H1

ENIIH

SEQ ID NO: 90 FAP CDR-H2

WFHPGSGSIKYNEKFKD

SEQ ID NO: 91 FAP CDR-H3

HGGTGRGAMDY

SEQ ID NO: 92 CD3a scFv CDR-L1

RASSSVSYMN

SEQ ID NO: 93 CD3a CDR-L2

DTSKVAS

SEQ ID NO: 94 CD3a CDR-L3: QQWSSNPLT

SEQ ID NO: 95 CD3a CDR-H1

RYTMH

SEQ ID NO: 96 CD3a CDR-H2

YINPSRGYTNYNQKFKD

SEQ ID NO: 97 CD3e CDR-H3

YYDDHYCLDY

SEQ ID NO: 98 FAP scFv

MDWIWRILFLVGAATGAHSQVQLKQSGAELVKPGASVKLSCKTSGYTFTENIIHWV KQRSGQGLEWIGWFHPGSGSIKYNEKFKDKATLTADKSSSTVYMELSRLTSEDSAVY FCARHGGTGRGAMDYWGQGTSVTVSSGGGGSGGGGSGGSAQILMTQSPASSVVSL GQRATISCRASKSVSTSAYSYMHWYQQKPGQPPKLLIYLASNLESGVPPRFSGSGSGT DFTLNIHPVEEEDAATYYCQHSRELPYTFGGGTKLEIKRAGS

SEQ ID NO: 99 CD3e scFv

VDDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPS RGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYW GQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYM NWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQ QWSSNPLTFGAGTKLELKS

SEQ ID NO: 100 CD3s/HAP bispecific immune cell engager (without signal sequence) QVQLKQSGAELVKPGASVKLSCKTSGYTFTENIIHWVKQRSGQGLEWIGWFHPGSG SIKYNEKFKDKATLTADKSSSTVYMELSRLTSEDSAVYFCARHGGTGRGAMDYWG QGTSVTVSSGGGGSGGGGSGGSAQILMTQSPASSVVSLGQRATISCRASKSVSTSAYS YMHWYQQKPGQPPKLLIYLASNLESGVPPRFSGSGSGTDFTLNIHPVEEEDAATYYC QHSRELPYTFGGGTKLEIKRAGSGGGGSGGGGSGGGGSVDDIKLQQSGAELARPGAS VKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTT DKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGG GSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYD TSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK S

SEQ ID NO: 101 CD3s/HAP bispecific immune cell engager (with signal sequence) MDWIWRILFLVGAATGAHSQVQLKQSGAELVKPGASVKLSCKTSGYTFTENIIHWV KQRSGQGLEWIGWFHPGSGSIKYNEKFKDKATLTADKSSSTVYMELSRLTSEDSAVY FCARHGGTGRGAMDYWGQGTSVTVSSGGGGSGGGGSGGSAQILMTQSPASSVVSL GQRATISCRASKSVSTSAYSYMHWYQQKPGQPPKLLIYLASNLESGVPPRFSGSGSGT DFTLNIHPVEEEDAATYYCQHSRELPYTFGGGTKLEIKRAGSGGGGSGGGGSGGGGS VDDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPS RGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYW GQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYM NWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQ QWSSNPLTFGAGTKLELKS

SEQ ID NO: 102 exemplary signal peptide sequence MYRMQLLSCIALSLALVTNS

SEQ ID NO: 103 exemplary signal peptide sequence MDWIWRILFLVGAATGAHS

SEQ ID NO: 104 IgG Fc domain amino acid sequence

SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 105 Sialidase-IgG Fc-transmembrane domain (without signal peptide sequence) MGDHPQATPAPAPDASTELPASMSQAQHLAANTATDNYRIPAITTAPNGDLLISYDE RPKDNGNGGSDAPNPNHIVQRRSTDGGKTWSAPTYIHQGTETGKKVGYSDPSYVVD HQTGTIFNFHVKSYDQGWGGSRGGTDPENRGIIQAEVSTSTDNGWTWTHRTITADIT KDKPWTARFAASGQGIQIQHGPHAGRLVQQYTIRTAGGAVQAVSVYSDDHGKTWQ AGTPIGTGMDENKVVELSDGSLMLNSRASDGSGFRKVAHSTDGGQTWSEPVSDKNL PDSVDNAQIIRAFPNAAPDDPRAKVLLLSHSPNPRPWSRDRGTISMSCDDGASWTTS KVFHEPFVGYTTIAVQSDGSIGLLSEDAHNGADYGGIWYRNFTMNWLGEQCGQKPA EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGKAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR

SEQ ID NO: 106 Sialidase-IgG Fc-transmembrane domain (with signal peptide sequence) MYRMQLLSCIALSLALVTNSMGDHPQATPAPAPDASTELPASMSQAQHLAANTATD NYRIPAITTAPNGDLLISYDERPKDNGNGGSDAPNPNHIVQRRSTDGGKTWSAPTYIH QGTETGKKVGYSDPSYVVDHQTGTIFNFHVKSYDQGWGGSRGGTDPENRGIIQAEV STSTDNGWTWTHRTITADITKDKPWTARFAASGQGIQIQHGPHAGRLVQQYTIRTAG GAVQAVSVYSDDHGKTWQAGTPIGTGMDENKVVELSDGSLMLNSRASDGSGFRKV AHSTDGGQTWSEPVSDKNLPDSVDNAQIIRAFPNAAPDDPRAKVLLLSHSPNPRPWS RDRGTISMSCDDGASWTTSKVFHEPFVGYTTIAVQSDGSIGLLSEDAHNGADYGGIW YRNFTMNWLGEQCGQKPAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGKAVGQDTQEVIVVPHSLPFKVVVISAILALVVLT IISLIILIMLWQKKPR

SEQ ID NO: 107 pE/L promoter

TATTTATATTCCAAAAAAAAAAAATAAAATTTCAATTTTT

SEQ ID NO: 108: Sial-FAP/CD3 nucleic acid construct

GGCCGGCCTCATCATGATTTCAGTTCCAGTTTAGTCCCAGCCCCGAAGGTCAGGG GATTAGAGGACCACTGCTGACAATAGTAGGTTGCAGCATCTTCGGCCTCCATGCT TGAAATGGTCAGGCTATATGAAGTCCCACTGCCAGATCCGGAGAACCGGTAGGG GACTCCACTAGCCACCTTAGAGGTGTCATAGATCCATCGCTTTGGAGAAGTCCCG GATTTCTGCTGGTACCAGTTCATATAGCTGACACTAGAGGAGGCCCGGCATGTCA TGGTCACTTTTTCGCCAGGGGAAGCTGACATAATGGCGGGAGACTGAGTCAGCT GGATGTCAGAGCCTCCTCCGCCGGAGCCGCCTCCTCCGCTTCCTCCTCCTCCGCTT

GACACTGTCAGAGTTGTCCCCTGTCCCCAATAATCCAGACAATAATGATCGTCAT

AGTAGCGAGCGCAATAGTACACGGCGCTGTCCTCTGATGTCAGACTAGACAGCT

GCATGTATGCGGTGGAGCTTGACTTATCGGTAGTCAGAGTAGCCTTGTCTTTAAA

CTTCTGGTTGTAGTTGGTGTATCCCCTGCTTGGATTAATGTACCCGATCCATTCCA

GTCCCTGCCCAGGTCGCTGCTTCACCCAGTGCATTGTGTAGCGAGTGAATGTATA

GCCGGAGGTCTTACAGCTCATTTTCACTGATGCTCCTGGTCTAGCCAGCTCTGCTC

CAGACTGCTGCAGCTTGATATCGTCGACTGAGCCTCCCCCGCCACTTCCTCCTCCT

CCAGATCCTCCTCCTCCGGATCCCGCCCGTTTTATTTCCAGCTTGGTCCCCCCTCC

GAACGTGTACGGAAGCTCCCTACTGTGCTGACAGTAATAGGTTGCAGCATCCTCC

TCCTCCACAGGGTGGATGTTGAGGGTGAAGTCTGTCCCAGACCCACTGCCACTGA

ACCTGGGAGGGACCCCAGATTCTAGGTTGGATGCAAGATAGATGAGGAGTTTGG

GTGGCTGTCCTGGTTTCTGTTGGTACCAGTGCATATAACTATAGGCAGATGTACT

GACACTTTTGCTGGCCCTGCATGAGATGGTGGCCCTCTGCCCCAGAGATACAACT

GAGGAAGCAGGAGACTGGGTCATCAGAATTTGTGCACTACCGCCAGAGCCACCT

CCGCCTGAACCGCCTCCACCACTCGAGACGGTGACTGAGGTTCCTTGACCCCAGT

AGTCCATAGCTCCTCGCCCAGTTCCTCCGTGTCTTGCACAGAAATAGACCGCAGA

GTCTTCAGATGTCAATCTACTAAGCTCCATATAGACTGTGCTGGAGGATTTGTCC

GCAGTCAATGTGGCCTTGTCCTTGAATTTCTCATTGTACTTTATACTACCACTTCC

AGGGTGAAACCACCCAATCCACTCAAGACCCTGCCCAGACCTCTGCTTTACCCAG

TGTATAATATTTTCAGTGAAGGTGTAGCCAGAAGTCTTGCAGGACAGCTTCACTG

ATGCCCCGGGTTTCACCAGTTCAGCTCCAGACTGCTTCAGCTGCACCTGAGAATG

AGCGCCGGTAGCAGCGCCGACGAGGAAGAGGATGCGCCAGATCCAGTCCATTAT

TTATATTCCAAAAAAAAAAAATAAAATTTCAATTTTTCATATGTCATCACTAACG

TGGCTTCTTCTGCCAAAGCATGATGAGGATGATAAGGGAGATGATGGTGAGCAC

CACCAGGGCCAGGATGGCTGAGATCACCACCACCTTAAAGGGCAAGGAGTGTGG

CACCACGATGACCTCCTGCGTGTCCTGGCCCACAGCTTTGCCGGGGCTCAGAGAC

AGAGATTTCTGGGTGTAGTGGTTGTGCAGAGCCTCGTGCATGACGGAGCAGCTG

AACACATTGCCTTGTTGCCATCTAGACTTATCCACGGTCAGCTTGGAGTACAGAA

AGAAGCTGCCGTCGGAATCGAGCACGGGGGGTGTGGTCTTGTAGTTGTTCTCGG

GCTGTCCGTTGCTTTCCCATTCGACGGCGATGTCGCTAGGGTAGAAGCCTTTCAC

GAGGCATGTGAGGGACACTTGATTCTTGGTCATCTCCTCTCTAGAGGGAGGGAG

GGTATACACTTGAGGTTCCCTAGGCTGGCCCTTGGCCTTGCTGATGGTCTTCTCA

ATGGGGGCGGGCAGAGCCTTGTTGCTGACCTTGCATTTATACTCTTTGCCGTTCA

GCCAGTCTTGGTGCAGCACTGTCAGCACGGACACGACTCTATAGGTGCTGTTATA

CTGTTCCTCTCTAGGCTTGGTCTTAGCGTTATGCACCTCCACGCCGTCCACGTACC

AGTTGAACTTGACCTCGGGGTCTTCATGGGACACATCCACCACCACGCATGTCAC

CTCGGGTGTTCTAGAAATCATGAGGGTGTCCTTGGGTTTGGGGGGAAACAGAAA

CACGGAGGGTCCGCCGAGCAGCTCGGGAGCGGGACAAGGAGGGCATGTGTGGG

TCTTATCGCAGCTCTTGGGCTCGGCTGGCTTCTGGCCACACTGCTCGCCCAGCCA

GTTCATGGTGAAGTTCCGATACCAGATGCCGCCGTAATCGGCGCCATTGTGTGCG

TCCTCGCTCAGCAGTCCGATGCTGCCATCAGACTGCACGGCGATTGTGGTGTAGC

CCACAAATGGCTCGTGGAACACCTTGGATGTGGTCCAGCTGGCGCCATCGTCGCA

GGACATGCTGATTGTGCCTCTGTCCCGGCTCCAAGGCCTTGGGTTTGGAGAGTGG

GACAGCAGCAGCACCTTGGCTCTGGGATCGTCTGGGGCGGCATTAGGAAAGGCC

CGGATGATCTGGGCGTTATCCACGCTGTCAGGCAGATTCTTATCAGACACGGGCT

CGGACCAGGTCTGTCCTCCGTCTGTAGAGTGTGCCACCTTGCGGAAGCCGGAGCC

GTCGCTGGCCCTAGAGTTCAGCATCAGGGAGCCATCGCTCAGCTCCACCACCTTA

TTCTCGTCCATGCCTGTGCCGATTGGGGTGCCTGCCTGCCAGGTCTTGCCGTGATC

GTCAGAATACACGGACACGGCCTGCACTGCTCCTCCTGCTGTTCTGATGGTGTAC TGCTGCACCAGCCGGCCTGCGTGAGGTCCGTGCTGGATCTGGATGCCCTGTCCGC TTGCTGCGAATCTTGCGGTCCAGGGCTTATCCTTTGTGATGTCGGCTGTGATGGTC CGGTGTGTCCAGGTCCAGCCGTTGTCTGTGCTGGTAGACACCTCGGCCTGGATGA TGCCGCGATTCTCAGGATCGGTGCCGCCCCTAGAGCCTCCCCATCCCTGGTCATA GGACTTCACGTGAAAGTTGAAGATTGTGCCGGTCTGGTGATCCACCACATAGCTT GGGTCAGAGTAGCCGACCTTCTTGCCTGTCTCGGTGCCCTGGTGGATGTAGGTAG GGGCGCTCCATGTCTTGCCGCCATCGGTAGATCTCCGCTGCACGATGTGATTAGG GTTTGGTGCGTCGGAGCCTCCATTTCCGTTATCCTTGGGGCGCTCGTCATAGCTG ATCAGCAGATCGCCATTTGGGGCTGTGGTGATGGCGGGGATTCTGTAGTTGTCTG TTGCGGTATTTGCTGCCAGGTGCTGTGCCTGGGACATGCTTGCTGGCAGCTCGGT GGAGGCATCTGGGGCAGGTGCTGGTGTTGCCTGTGGGTGGTCGCCCATACTGTTT GTGACAAGTGCAAGACTTAGTGCAATGCAAGACAGGAGTTGCATCCTGTACAT7T ATAGCATAGAAAAAAACAAAATGAAATTCAAGCTT

SEQ ID NO: 109 FAP/CD3 immune cell engager nucleotide sequence

TGATTTCAGTTCCAGTTTAGTCCCAGCCCCGAAGGTCAGGGGATTAGAGGACCAC TGCTGACAATAGTAGGTTGCAGCATCTTCGGCCTCCATGCTTGAAATGGTCAGGC TATATGAAGTCCCACTGCCAGATCCGGAGAACCGGTAGGGGACTCCACTAGCCA CCTTAGAGGTGTCATAGATCCATCGCTTTGGAGAAGTCCCGGATTTCTGCTGGTA CCAGTTCATATAGCTGACACTAGAGGAGGCCCGGCATGTCATGGTCACTTTTTCG CCAGGGGAAGCTGACATAATGGCGGGAGACTGAGTCAGCTGGATGTCAGAGCCT CCTCCGCCGGAGCCGCCTCCTCCGCTTCCTCCTCCTCCGCTTGACACTGTCAGAGT TGTCCCCTGTCCCCAATAATCCAGACAATAATGATCGTCATAGTAGCGAGCGCAA TAGTACACGGCGCTGTCCTCTGATGTCAGACTAGACAGCTGCATGTATGCGGTGG AGCTTGACTTATCGGTAGTCAGAGTAGCCTTGTCTTTAAACTTCTGGTTGTAGTTG GTGTATCCCCTGCTTGGATTAATGTACCCGATCCATTCCAGTCCCTGCCCAGGTC GCTGCTTCACCCAGTGCATTGTGTAGCGAGTGAATGTATAGCCGGAGGTCTTACA GCTCATTTTCACTGATGCTCCTGGTCTAGCCAGCTCTGCTCCAGACTGCTGCAGCT TGATATCGTCGACTGAGCCTCCCCCGCCACTTCCTCCTCCTCCAGATCCTCCTCCT CCGGATCCCGCCCGTTTTATTTCCAGCTTGGTCCCCCCTCCGAACGTGTACGGAA GCTCCCTACTGTGCTGACAGTAATAGGTTGCAGCATCCTCCTCCTCCACAGGGTG GATGTTGAGGGTGAAGTCTGTCCCAGACCCACTGCCACTGAACCTGGGAGGGAC CCCAGATTCTAGGTTGGATGCAAGATAGATGAGGAGTTTGGGTGGCTGTCCTGGT TTCTGTTGGTACCAGTGCATATAACTATAGGCAGATGTACTGACACTTTTGCTGG CCCTGCATGAGATGGTGGCCCTCTGCCCCAGAGATACAACTGAGGAAGCAGGAG ACTGGGTCATCAGAATTTGTGCACTACCGCCAGAGCCACCTCCGCCTGAACCGCC TCCACCACTCGAGACGGTGACTGAGGTTCCTTGACCCCAGTAGTCCATAGCTCCT CGCCCAGTTCCTCCGTGTCTTGCACAGAAATAGACCGCAGAGTCTTCAGATGTCA ATCTACTAAGCTCCATATAGACTGTGCTGGAGGATTTGTCCGCAGTCAATGTGGC CTTGTCCTTGAATTTCTCATTGTACTTTATACTACCACTTCCAGGGTGAAACCACC CAATCCACTCAAGACCCTGCCCAGACCTCTGCTTTACCCAGTGTATAATATTTTC AGTGAAGGTGTAGCCAGAAGTCTTGCAGGACAGCTTCACTGATGCCCCGGGTTTC ACCAGTTCAGCTCCAGACTGCTTCAGCTGCACCTGAGAATGAGCGCCGGTAGCA GCGCCGACGAGGAAGAGGATGCGCCAGATCCAGTCCAT

SEQ ID NO: 110 nucleotide sequence encoding Sial-IgG Fc-transmembrane

CTAACGTGGCTTCTTCTGCCAAAGCATGATGAGGATGATAAGGGAGATGATGGT GAGCACCACCAGGGCCAGGATGGCTGAGATCACCACCACCTTAAAGGGCAAGGA GTGTGGCACCACGATGACCTCCTGCGTGTCCTGGCCCACAGCTTTGCCGGGGCTC AGAGACAGAGATTTCTGGGTGTAGTGGTTGTGCAGAGCCTCGTGCATGACGGAG CAGCTGAACACATTGCCTTGTTGCCATCTAGACTTATCCACGGTCAGCTTGGAGT

ACAGAAAGAAGCTGCCGTCGGAATCGAGCACGGGGGGTGTGGTCTTGTAGTTGT

TCTCGGGCTGTCCGTTGCTTTCCCATTCGACGGCGATGTCGCTAGGGTAGAAGCC

TTTCACGAGGCATGTGAGGGACACTTGATTCTTGGTCATCTCCTCTCTAGAGGGA

GGGAGGGTATACACTTGAGGTTCCCTAGGCTGGCCCTTGGCCTTGCTGATGGTCT

TCTCAATGGGGGCGGGCAGAGCCTTGTTGCTGACCTTGCATTTATACTCTTTGCC

GTTCAGCCAGTCTTGGTGCAGCACTGTCAGCACGGACACGACTCTATAGGTGCTG

TTATACTGTTCCTCTCTAGGCTTGGTCTTAGCGTTATGCACCTCCACGCCGTCCAC

GTACCAGTTGAACTTGACCTCGGGGTCTTCATGGGACACATCCACCACCACGCAT GTCACCTCGGGTGTTCTAGAAATCATGAGGGTGTCCTTGGGTTTGGGGGGAAACA GAAACACGGAGGGTCCGCCGAGCAGCTCGGGAGCGGGACAAGGAGGGCATGTG

TGGGTCTTATCGCAGCTCTTGGGCTCGGCTGGCTTCTGGCCACACTGCTCGCCCA

GCCAGTTCATGGTGAAGTTCCGATACCAGATGCCGCCGTAATCGGCGCCATTGTG

TGCGTCCTCGCTCAGCAGTCCGATGCTGCCATCAGACTGCACGGCGATTGTGGTG

TAGCCCACAAATGGCTCGTGGAACACCTTGGATGTGGTCCAGCTGGCGCCATCGT

CGCAGGACATGCTGATTGTGCCTCTGTCCCGGCTCCAAGGCCTTGGGTTTGGAGA

GTGGGACAGCAGCAGCACCTTGGCTCTGGGATCGTCTGGGGCGGCATTAGGAAA

GGCCCGGATGATCTGGGCGTTATCCACGCTGTCAGGCAGATTCTTATCAGACACG

GGCTCGGACCAGGTCTGTCCTCCGTCTGTAGAGTGTGCCACCTTGCGGAAGCCGG

AGCCGTCGCTGGCCCTAGAGTTCAGCATCAGGGAGCCATCGCTCAGCTCCACCAC

CTTATTCTCGTCCATGCCTGTGCCGATTGGGGTGCCTGCCTGCCAGGTCTTGCCGT

GATCGTCAGAATACACGGACACGGCCTGCACTGCTCCTCCTGCTGTTCTGATGGT

GTACTGCTGCACCAGCCGGCCTGCGTGAGGTCCGTGCTGGATCTGGATGCCCTGT

CCGCTTGCTGCGAATCTTGCGGTCCAGGGCTTATCCTTTGTGATGTCGGCTGTGAT

GGTCCGGTGTGTCCAGGTCCAGCCGTTGTCTGTGCTGGTAGACACCTCGGCCTGG

ATGATGCCGCGATTCTCAGGATCGGTGCCGCCCCTAGAGCCTCCCCATCCCTGGT CATAGGACTTCACGTGAAAGTTGAAGATTGTGCCGGTCTGGTGATCCACCACATA GCTTGGGTCAGAGTAGCCGACCTTCTTGCCTGTCTCGGTGCCCTGGTGGATGTAG

GTAGGGGCGCTCCATGTCTTGCCGCCATCGGTAGATCTCCGCTGCACGATGTGAT

TAGGGTTTGGTGCGTCGGAGCCTCCATTTCCGTTATCCTTGGGGCGCTCGTCATA

GCTGATCAGCAGATCGCCATTTGGGGCTGTGGTGATGGCGGGGATTCTGTAGTTG

TCTGTTGCGGTATTTGCTGCCAGGTGCTGTGCCTGGGACATGCTTGCTGGCAGCT CGGTGGAGGCATCTGGGGCAGGTGCTGGTGTTGCCTGTGGGTGGTCGCCCATACT GTTTGTGACAAGTGCAAGACTTAGTGCAATGCAAGACAGGAGTTGCATCCTGTA

CAT

SEQ ID NO: 117 D004 VH (CDR sequences are underlined)

EVOLOQSGPELVKPGASLKIPCRASGYTFTDYNMDWVKOSHGKSLEWIGDINPNNG GTISNQKFKGKATLTVDKSSSTASMELRSLTSEDTAVYYCAIRRYYGNHWYFDVWG TGTSVTVSS

SEQ ID NO: 118 D004 VL (CDR sequences are underlined)

DIRMTOSPSSMYASLGERVTIACKASODINIYLSWFOQKPGTSPKTLIYRANRLMDGV PSRFSASGSGODYSLTISSLEYEDMGIYYCLQYDEFPPTFGGGTKVEIK

SEQ ID NO: 119 D004 scFv

MYRMQEESCIAESEAEVTNSDIRMTQSPSSMYASEGERVTIACKASQDINIYESWFQQ

KPGTSPKTEIYRANREMDGVPSRFSASGSGQDYSETISSEEYEDMGIYYCEQYDEFPP

TFGGGTKVEIKGGGGSGGGGSGGGGSEVQEQQSGPEEVKPGASEKIPCRASGYTFTD YNMDWVKQSHGKSLEWIGDINPNNGGTISNQKFKGKATLTVDKSSSTASMELRSLT

SEDTAVYYCAIRRYYGNHWYFDVWGTGTSVTVSS

SEQ ID NO: 120 D004 CAR MYRMQLLSCIALSLALVTNSDIRMTQSPSSMYASLGERVTIACKASQDINIYLSWFQQ KPGTSPKTLIYRANRLMDGVPSRFSASGSGQDYSLTISSLEYEDMGIYYCLQYDEFPP TFGGGTKVEIKGGGGSGGGGSGGGGSEVQLQQSGPELVKPGASLKIPCRASGYTFTD YNMDWVKQSHGKSLEWIGDINPNNGGTISNQKFKGKATLTVDKSSSTASMELRSLT SEDTAVYYCAIRRYYGNHWYFDVWGTGTSVTVSSTRTTTPAPRPPTPAPTIASQPLSL RPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHS DYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSGSRVKFSRSADAPAYQQGQNQLYN ELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID NO: 121 IL15 MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKI EDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILAN NSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINT

While certain embodiments have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS What is claimed is:

1. A method of treating a cancer in an individual in need thereof comprising administering to the individual: (a) an effective amount of a recombinant oncolytic virus comprising a nucleotide sequence encoding a foreign antigen; and (b) an effective amount of an engineered immune cell expressing a chimeric receptor specifically recognizing said foreign antigen.

2. The method of claim 1, wherein the foreign antigen comprises an anchoring domain.

3. The method of claim 2, wherein the anchoring domain is positively charged at physiologic pH.

4. The method of claim 2 or 3, wherein the anchoring domain is a glycosaminoglycan (GAG)-binding domain.

5. The method of any one of claims 1-4, wherein the foreign antigen comprises a transmembrane domain.

6. The method of any one of claims 2-5, wherein the anchoring domain or the transmembrane domain is located at the carboxy terminus of the foreign antigen.

7. The method of any one of claims 1-6, wherein the foreign antigen comprises a stabilization domain.

8. The method of claim 7, wherein the stabilization domain is an Fc domain.

9. The method of any one of claims 1-8, wherein the nucleotide sequence encoding the foreign antigen is operably linked to a promotor.

10. The method of claim 9, wherein the promoter is a viral late promoter.

11. The method of claim 10, wherein the promoter is an F17R late promoter.

12. The method of any one of claims 1-11, wherein the foreign antigen is a bacterial protein.

13. The method of any one of claims 1-12, wherein the foreign antigen is a sialidase.

14. The method of claim 13, wherein the sialidase is a protein having exo-sialidase activity.

15. The method of claim 13 or 14, wherein the sialidase is selected from the group consisting of: Clostridium perfringens sialidase, Actinomyces viscosus sialidase, and Arthrobacter ureafaciens sialidase, Salmonella typhimurium sialidase and Vibrio cholera sialidase.

16. The method of any one of claims 13-15, wherein the sialidase comprises an amino acid sequence having at least about 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-28, 31, and 53-54.

17. The method of claim 16, wherein the sialidase comprises an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 2.

18. The method of claim 17, wherein the sialidase is DAS181 or a derivative thereof.

19. The method of claim 18, wherein the chimeric receptor comprises an anti-DAS181 antibody moiety that is not cross-reactive with human native amphiregulin or neuraminidase.

20. The method of one of claims 1-19, wherein the chimeric receptor is a Chimeric Antigen Receptor (CAR).

21. The method of claim 20, wherein the CAR comprises an anti-sialidase antibody moiety, a transmembrane domain, and an intracellular domain.

22. The method of claim 21, wherein the anti-sialidase antibody moiety is a scFv.

23. The method of claim 21 or 22, wherein the anti-sialidase antibody moiety comprises an antibody heavy chain variable domain (VH) comprising a heavy chain complementarity determining region (CDR-H) 1 comprising the amino acid sequence of SEQ ID NO: 111, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 112, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 113; and an antibody light chain variable domain (VL) comprising a light chain complementarity determining region (CDR-L) 1 comprising the amino acid sequence of SEQ ID NO: 114, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 115, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 116.

24. The method of any one of claims 21-23, wherein the intracellular domain comprising a CD28 intracellular signaling sequence and an intracellular signaling sequence of CD3^.

25. The method of any one of claims 1-24, wherein the engineered immune cell is selected from the group consisting of T cell, natural killer (NK) cell, natural killer T (NKT) cell, macrophage and combinations thereof.

26. The method of claim 25, wherein the engineered immune cell is NK cell.

27. The method of any one of claims 1-26, wherein the oncolytic virus is a virus selected from the group consisting of: vaccinia virus, reovirus, Seneca Valley virus (SVV), vesicular stomatitis virus (VSV), Newcastle disease virus (NDV), herpes simplex virus (HSV), morbillivirus virus, retrovirus, influenza virus, Sinbis virus, poxvirus, measles virus, cytomegalovirus (CMV), lentivirus, adenovirus, coxsackievirus, and derivatives thereof.

28. The method of claim 27, wherein the oncolytic virus is a poxvirus. 147

29. The method of claim 28, wherein the poxvirus is a vaccinia virus.

30. The method of claim 29, wherein the vaccinia virus is of a strain selected from the group consisting of Dryvax, Lister, M63, LIVP, Tian Tan, Modified Vaccinia Ankara, New York City Board of Health (NYCBOH), Dairen, Ikeda, LC16M8, Tashkent, IHD-J, Brighton, Dairen I, Connaught, Wyeth, Copenhagen, Western Reserve, Elstree, CL, Lederle- Chorioallantoic, AS, and derivatives thereof.

31. The method of claim 30, wherein the virus is vaccinia virus Western Reserve.

32. The method of any one of claims 1-31, wherein the recombinant oncolytic virus further comprises a second nucleotide sequence encoding a heterologous protein.

33. The method of claim 32, wherein the heterologous protein is an immune checkpoint inhibitor.

34. The method of claim 32, wherein the heterologous protein is a multi-specific immune cell engager.

35. The method of any one of claims 1-34, wherein the engineered immune cell further comprises a heterologous nucleotide sequence encoding a cytokine.

36. The method of claim 35, wherein the engineered immune cell is a NK cell, and wherein the cytokine is IL- 15.

37. The method of any one of claims 1-36, wherein the engineered immune cell and the recombinant oncolytic virus are administered simultaneously.

38. The method of any one of claims 1-36, wherein the recombinant oncolytic virus is administered prior to administration of the engineered immune cell.

39. The method of any one of claims 1-38, wherein the recombinant oncolytic virus is delivered via a carrier cell.

40. The method of any one of claims 1-39, wherein the cancer is a solid cancer.

41. A pharmaceutical composition comprising: (a) a recombinant oncolytic virus comprising a nucleotide sequence encoding a foreign antigen; (b) an engineered immune cell expressing a chimeric receptor specifically recognizing said foreign antigen; and (c) a pharmaceutically acceptable carrier.

42. The pharmaceutical composition of claim 41, wherein the foreign antigen is a bacterial sialidase.

43. The pharmaceutical composition of claim 41 or 42, wherein the engineered immune cell is a NK cell.

44. A kit comprising: (a) a recombinant oncolytic virus comprising a nucleotide sequence encoding a foreign antigen; and (b) an engineered immune cell expressing a chimeric receptor specifically recognizing said foreign antigen.

45. An engineered NK cell comprising a CAR, wherein the CAR comprises an anti- sialidase antibody moiety, a transmembrane domain, and an intracellular domain.

46. An isolated antibody or antigen-binding fragment thereof that specifically binds Actinomyces viscosus sialidase, comprising a VH comprising an CDR-H1 comprising the amino acid sequence of SEQ ID NO: 111, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 112, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 113; and a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 114, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 115, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 116.