Anti-oncolytic virus antigen antibody and method of use thereof

Abstract

Antibodies that specifically bind to vaccinia virus (VV) A56 or B5 antigens are provided. Fusion proteins and conjugates comprising the antibodies are also provided. Pharmaceutical compositions and kits comprising the antibodies, fusion proteins, and conjugates are also provided. Aspects of the present disclosure further include methods of using the antibodies, fusion proteins, and conjugates, e.g., for therapeutic purposes. In certain embodiments, methods are provided that include administering an antibody, fusion protein, or conjugate of the present disclosure to an individual having cancer, the individual comprising cancer cells infected with VV, and the antibody, fusion protein, or conjugate is targeted to the infected cancer cells by a VV antigen expressed on the surface of the infected cancer cells. Aspects of the present disclosure further include methods of targeting an antibody, fusion protein, or conjugate that specifically binds to an oncolytic virus (OV) antigen to cancer cells in an individual.

Description

[Background technology]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 900,303, filed Sep. 13, 2019, which is incorporated by reference in its entirety.

[0002] Immunotherapy has become an effective treatment option for multiple malignancies. Oncolytic viruses (OVs) can be engineered to selectively replicate in and lyse tumor tissue while sparing normal non-neoplastic host cells and simultaneously restore anti-tumor immunity, constituting a next-generation immunotherapeutic approach for tumor treatment. The unique ability of OVs to target malignancies independent of specific antigen expression patterns makes them an attractive alternative to other immunotherapeutic approaches. Moreover, OVs can promote the recruitment of tumor-infiltrating lymphocytes (TILs), reprogram the immunosuppressive tumor microenvironment (TME), and enhance systemic anti-tumor immunity.

[0003] Genetic engineering has made it possible to design live replicating viruses to be highly tumor selective through cell entry and transcriptional targeting, as well as to equip them with reporter genes for non-invasive monitoring of the pharmacokinetics of virotherapy, and for cytotoxic activity or immunogenic cell death, or for enhancement of immune modulators. Currently, three OVs are commercially available for the treatment of cancer. These OVs include Rigvir, approved in Latvia, Georgia, and Armenia; Oncorine H101, approved in China; and Talimogene laherparepvec (T-VEC), approved in the United States. Rigvir is an unmodified enterocytopathic human orphan type 7 (ECHO-7) picornavirus, with limited published data describing its efficacy. Oncorine became the first oncolytic virus approved for clinical use in China and the first recombinant oncolytic virus in the world to receive regulatory approval. It is an attenuated serotype 5 adenovirus vector with a deletion of viral E1B-55k and four deletions of viral E3. Oncorine remains the only adenovirus approved for cancer therapy, and only when administered in combination with chemotherapy. T-VEC (Imlygic™) was approved by the US Food and Drug Administration (FDA) in 2015 for the treatment of unresectable metastatic melanoma and subsequently in the EU for the treatment of locally advanced or metastatic cutaneous melanoma, making it the most recent oncolytic virus to gain national regulatory approval and the first to gain approval in the United States. T-VEC is a recombinant human herpes simplex virus type 1 (HSV1) with deletions of both copies of HSV1 gamma 34.5 and viral ICP47, accelerated expression of US11, and encodes two copies of human granulocyte-macrophage colony-stimulating factor (GM-CSF) under the cytomegalovirus (CMV) promoter. T-VEC is currently approved for intratumoral injection into cutaneous high-grade melanoma lesions and has demonstrated efficacy as a single agent for this indication.

[0004] OVs in clinical development include measles virus, Newcastle disease virus (NDV), rhabdovirus, adenovirus, vaccinia virus (VV), herpesvirus, coxsackievirus, reovirus, and retrovirus.

[0005] Measles virus is a negative-stranded RNA paramyxovirus that is highly fusogenic and induces the widespread cytopathic effect of syncytium formation. Cell-cell fusion increases bystander killing of tumor cells and induces immunogenic danger signals that may trigger host-mediated cellular antitumor activity. Recombinant Edmonston strain measles viruses encoding sodium iodide symporter (MV-NIS) or soluble carcinoembryonic antigen (MV-CEA) are in phase I / II clinical trials in patients with relapsed or recurrent cancers, including multiple myeloma, ovarian cancer, glioma, breast cancer, and mesothelioma.

[0006] NDV is an avian paramyxovirus that is being tested as an oncolytic or oncolytic cancer vaccine. NDV strains MTH-68 / H, HUJ and PV701 are being tested clinically. A recombinant NDV based on the mesogenic NDV-73T strain, which is impaired in infection of avian but not mammalian cells and encodes GM-CSF (Medimmune, MEDI5395), is in preclinical trials.

[0007] Rhabdoviruses are negative sense RNA viruses that exhibit a lytic replication cycle of approximately 12 hours in permissive cells. The best studied oncolytic rhabdovirus, vesicular stomatitis virus (VSV), uses low density lipoprotein (LDL) receptors for cell entry, allowing VSV to infect nearly all cell types and cause lytic infection in a wide variety of permissive cells.

[0008] Adenoviruses are icosahedral, non-enveloped, double-stranded DNA viruses with a long fibrous knob protruding from the apex of each capsid. Clinical data have been published for Telomelysin in solid tumors, CG0070 in bladder cancer, and DNX-2401 in malignant brain tumors.

[0009] Vaccinia viruses (VV) are large enveloped double-stranded DNA viruses with a linear genome of approximately 190 kb in length. Attenuation or tumor-specific targeting of these viruses has been achieved using a variety of deletion and insertion mutations, with loss of thymidine kinase function being a common feature of clinical oncolytic vaccinia viruses. JX-594 has a deletion of viral thymidine kinase, TG6002 has a double deletion of thymidine kinase and viral ribonucleotide reductase, and GL-ONC1 has insertion mutations in its thymidine kinase (J2R), hemagglutinin HA (A56R), and F14.5L genes. Loss of function of TK limits the virus's ability to replicate in non-dividing cells, and deletion of viral ribonucleotide reductase further limits this ability. Two clinical vaccinia vectors designed to enhance oncolytic effects contain transgenes designed to improve tumor cell killing. JX-594, like T-VEC, contains GM-CSF, and TG6002 incorporates the nucleoside analogue converting enzyme FCU1, which converts 5-fluorocytosine (5-FC) to 5-FU in infected cells.

[0010] HSV1 is a large double-stranded DNA virus approximately 152 kb in length. gamma34.5-deficient viruses being clinically evaluated include the currently FDA-approved T-VEC, HSV1716 (Seprehvir), G207, and RP1. Attempts to enhance the anticancer efficacy of HSV in metastatic disease include the inclusion of therapeutic transgenes used to simultaneously boost anticancer and antiviral immunity, with the goal of developing adaptive antitumor responses in treated patients.

[0011] Coxsackieviruses are single-stranded positive RNA picornaviruses of approximately 7.4 kb enclosed in an icosahedral capsid. Oncolytic CVA21 (Viralytics, CAVATAK) is derived from the Kuykendall strain and uses ICAM-1 as the primary receptor for cell entry. Phase I trials of intratumoral CVA21 virus injections in combination with pembrolizumab or ipilimumab are ongoing to enhance the overall efficacy of these agents.

[0012] Reovirus is a double-stranded RNA virus, non-enveloped, with an icosahedral capsid composed of an outer and inner protein shell. Reovirus as monotherapy has been investigated in several Phase I trials (Oncolytics Biotech, Reolysin™) as intratumoral or intravenous administration.

[0013] A retroviral replicating vector (Tocagen, Toca-511, vocimagene amiretrorepvec) encodes yeast cytosine deaminase (CD) that converts the prodrug 5-FC to the anticancer drug 5-FU, thereby increasing the local concentration of 5-FU in the tumor and reducing the overall systemic toxicity of the drug. A phase 1 trial of Toca-511 in patients with recurrent high-grade gliomas showed an overall survival of 13.6 months, a statistical improvement compared to external controls. Summary of the Invention

[0014] Despite recent advances in OV-based therapies, there remains a need for new and improved OV-based methods for the treatment, mitigation, and / or prevention of cancer, as well as for methods of improving survival of subjects with cancer.

[0015] Antibodies that specifically bind to vaccinia virus (VV or VACV) A56 or B5 antigens are provided. Fusion proteins and conjugates that include the antibodies are also provided. Pharmaceutical compositions and kits that include the antibodies, fusion proteins, and conjugates are also provided. Aspects of the present disclosure further include methods of using the antibodies, fusion proteins, and conjugates, for example, for therapeutic purposes. In certain embodiments, methods are provided that include administering an antibody, fusion protein, or conjugate of the present disclosure to an individual having cancer, the individual comprising cancer cells infected with VV, and the antibody, fusion protein, or conjugate is targeted to the infected cancer cells by a VV antigen expressed on the surface of the infected cancer cells. Aspects of the present disclosure further include methods of targeting an antibody, fusion protein, or conjugate that specifically binds to an oncolytic virus (OV) antigen to cancer cells of an individual. [Brief description of the drawings]

[0016] [Figure 1] Flow cytometry data of antibody binding to A549 and CaOV3 cells infected with VV Western Reserve VVdd(eGFP) are shown. [Diagram 2] Flow cytometry data of antibody binding to A56 or B5 expressing HEK cells (Wyeth VV sequence) is shown. [Diagram 3] Data for antibody binding to HEK-B5 cells are shown. The highest concentrations tested in flow cytometry assays (top panel) or ELISA (bottom panel) for binding to HEK-B5 are plotted, at 5 μg / ml for rabbit and mouse isotype controls (Rb IgG and mIgG isotypes, respectively), as well as the anti-B5 Immunetech antibody, IT anti-B5 IgG (identified as anti-B5 mIgG in FIG. 3), and 1:1000 for the rabbit polyclonal antibody (c-VV). [Figure 4]Binding of A048 antibody, Immunetech anti-B5 antibody, IT anti-B5 IgG (identified as IT anti-B5 mIgG in Fig. 4 ), and human isotype control to VV Western Reserve VVdd(eGFP)-infected A549 cells (upper panel) and HT29 cells (lower panel) as measured by flow cytometry is shown. [Diagram 5] Figure 1 shows antibody binding to VV-infected cells. Antibodies were tested for binding to VVdd(eGFP)-infected A549 cells by flow cytometry. All formats had similar EC50s and no binding was observed to uninfected cells. [Figure 6] Immunofluorescence antibody binding of antibody A049 to virus-infected tumor tissues, specifically binding of A049 anti-A56 hIgG1 to virus-infected colorectal tumor tissues. The top two panels are hematoxylin and eosin (H&E) stained. This is compared to a polyclonal anti-vaccinia virus antibody that shows some nonspecific staining on uninfected tissues. [Figure 7] Provides epitope binning data showing binding of biotinylated antibodies to VVdd(eGFP) infected cells pre-incubated with non-biotinylated antibodies. Reduction of biotinylated antibody binding indicates similar or overlapping epitopes with the test antibody. Antibodies A047 and A049 appear to bind different epitopes on A56. Antibodies A048 and A051 appear to bind different epitopes on B5. [Figure 8] Data on antibody binding to virus particles are shown. Viral particles (VVdd(eGFP)) were coated onto plates and incubated with antibodies or the positive control NKp30. All antibodies bound to the virus except for antibody A054. [Figure 9]

[0036] Figure 2 provides data showing neutralization of virus infection. Antibodies were added to A549 cells simultaneously with VVdd(eGFP) infection. Similar levels of infection were observed as compared to infection without antibody ("No Antibody"). [Figure 10]Antibody binding to mouse cell lines infected with VV Openhagen (YFP) is shown. Percent YFP expression in each infected cell line indicates the proportion of cells infected with VV Openhagen (YFP). Anti-A56-PE and anti-B5-PE antibodies bind to B16F10, CT26LacZ, and MC38 cells infected with VV Openhagen (YFP). [Figure 11] Immunohistochemistry data on specific binding of antibodies to A56 or B5 antigens expressed on the HEK cell surface or to U2OS cells infected with VV Openhagen virus are shown. Rabbit / mouse chimeric (antibodies A059 and A058) and full rabbit IgG format (antibodies A056 and A073) antibodies specific for A56 or B5 were able to specifically detect the target antigens on stably expressed HEK-A56 and HEK-B5 cells, respectively. Both anti-A56 and anti-B5 antibodies were able to detect protein expression on U2OS cells infected with VV Openhagen. [Figure 12] 1 shows detection of A56 and B5 from VV-treated tumors by immunohistochemistry. Data show detection of A56 and B5 proteins on the surface of tumors treated with vaccinia virus. [Figure 13] Panel A: Representative schematic of an example design of anti-VV chimeric antigen receptor (CAR) construct. Panel B: Schematic of a therapeutic approach according to an embodiment of the present disclosure. As shown, OV infects cancer cells, which then present OV antigens (in this example, A56 or B5 OV antigens, which in some embodiments may be the native A56 or B5 antigens of OV) on the surface of the cancer cells. Treating the infected cancer cells with CART cells containing a CAR specific for the presented OV antigen destroys the infected cancer cells. Adjacent cancer cells infected with OV released by the destroyed cancer cells can then be destroyed by CAR T cells containing a CAR specific for the presented OV antigen. [Figure 14]Flow cytometry data showing VV CAR detection after lentiviral transduction is provided. The data show VV-CAR expression on transduced Jurkat cells. Downregulation of CAR, indicating specific target binding, is evident for all VV-CAR constructs tested. [Figure 15] Figure 1 shows expression of CAR and eGFP on activated A56-CAR-06 positive Jurkat cells. Data shows co-expression of A56-CAR-06 and eGFP on transduced Jurkat cell lines. After culturing with the target HEK A56 line, an activated CAR population (CD69 positive) can be identified within the scFv negative fraction by gating on the eGFP positive population. [Figure 16] We provide data showing specific activation of Jurkat cells expressing VV-CARs when co-cultured with target HEK cell lines. All VV-CARs tested show specific on-target activation as indicated by upregulation of CD69. Minimal cross-reactivity / intrinsic activation occurred when VV-CAR lines were co-cultured with negative / irrelevant target cell lines. [Figure 17] Transduction and enrichment of A56-CAR-06 expression in healthy donor primary T cells. Data show stable expression of A56-CAR-06 on human primary T cells following standard clinical manufacturing protocols. A56-CAR-06 enriched populations were established by repeated cell sorting for CAR expression followed by clinical expansion. [Figure 18] Specific activation of human T cells expressing A56-CAR-06 when co-cultured with the target-expressing HEK-A56 line. Human primary T cells expressing A56-CAR-06 showed significant upregulation of CD69 and CD137 after co-culture with the target HEK-A56 line. Minimal cross-reactivity was observed with negative / irrelevant target cell lines. [Figure 19] We provide data demonstrating that human primary T cells expressing A56-CAR-06 show morphological signs of direct tumor killing 48 hours after co-culture with target HEK-A56 cells. [Figure 20]We provide data demonstrating that human T cells expressing B5-CAR-011 exhibit high percent specific cytotoxicity. Human primary T cells expressing B5-CAR-011 exhibited specific cytolysis at 24 hours as determined by percent reduction in relative luminescence units (RLU). [Figure 21] Representative maximum intensity projection positron emission tomography (PET) images of 89Zr-DFO-A049 (anti-A56 antibody, DFO: deferoxamine) at 1, 3, and 5 days post-injection for the same mouse are shown. The tumor types: HEK-A56 (expressing A56, left shoulder) and parental HEK (non-expressing, right shoulder) are indicated by arrows, in addition to the spleen and liver. Results are presented as percentage of injected dose per gram of tissue (%ID / g). Clear preclinical A56-positive tumor PET imaging is shown, with efficient and specific accumulation of 89Zr-DFO-A049 in positive tumors, and the expected pharmacokinetics of the radioimmunoconjugate. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Before describing the antibodies, compositions and methods of the present disclosure in more detail, it is to be understood that the antibodies, compositions and methods are not limited to particular embodiments described, which may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and that the scope of the antibodies, compositions and methods will be limited only by the appended claims, and that the terminology is not intended to be limiting.

[0018] When a range of values ​​is provided, it is understood that each intervening value between the upper and lower limit of that range (to one-tenth of the unit of the lower limit unless the context clearly dictates otherwise), and every other stated or intervening value within that stated range, is encompassed within the antibodies, compositions and methods. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, subject to any specifically excluded limits in the stated range. When one or both of the limits are included in a stated range, ranges excluding either or both of those included limits are also included in the antibodies, compositions and methods.

[0019] Certain ranges are presented herein with the term "about" preceding the numerical values. The term "about" is used herein to provide literal support for the exact number it precedes, as well as a number that is close to or approximately the number it precedes. In determining whether a number is close to or approximately a specific recited number, the close or approximate but unrecited number may be a number that is substantially equivalent to the specifically recited number in the context in which it is presented.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the antibodies, compositions and methods belong. Although any antibodies, compositions and methods similar or equivalent to those described herein can also be used in the practice or testing of the antibodies, compositions and methods, representative exemplary antibodies, compositions and methods are described here.

[0021] All publications and patents cited herein are incorporated by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference herein to disclose and describe the materials and / or methods in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date, and should not be construed as an admission that the present antibodies, compositions and methods are not entitled to antedate such publication, since the publication dates provided may be different from the actual publication dates which may need to be independently confirmed.

[0022] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude optional elements. As such, this statement is intended to serve as a literal basis for the use of exclusive terminology such as "solely," "only," and the like in connection with the recitation of claim elements or the use of a "negative" limitation.

[0023] It is recognized that certain features of the antibodies, compositions and methods that are described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the antibodies, compositions and methods that are described in the context of a single embodiment may be provided separately or in any suitable subcombination. All combinations of the embodiments are specifically encompassed by the present disclosure and are disclosed herein as if each and every combination were individually or explicitly disclosed, to the extent that such combinations encompass processes and / or compositions that can be performed. Furthermore, all subcombinations listed in the embodiments that describe such variables are also specifically encompassed by the present antibodies, compositions and methods and are disclosed herein as if each and every subcombination were individually and explicitly disclosed.

[0024] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has individual components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the methods of the present disclosure. Any recited method may be carried out in the order of events recited or in any other order which is logically possible.

[0025] antibody The present disclosure provides antibodies that specifically bind to vaccinia virus (VV or VACV) antigens. Vaccinia virus is a member of the poxvirus family characterized by a double-stranded DNA genome of approximately 192 kb that encodes numerous viral enzymes and factors that allow the virus to replicate independently of the host cell machinery. VV can stably accommodate up to 25 kb of cloned exogenous DNA. Structurally, VV consists of a core region composed of viral DNA and various viral enzymes, such as RNA polymerase and polyA polymerase, wrapped in a lipoprotein core membrane. The outer layer of the virus consists of a double lipid membrane envelope. VV has many unique features that endow it with attributes suitable for use in oncolytic virotherapy, such as natural tumor tropism, strong lytic ability, a short life cycle with rapid cell-to-cell spread, efficient gene expression, and large cloning capacity. VV has a short life cycle of approximately 8 hours that occurs in the cytoplasm, eliminating the risk of integration into the genome. Replication typically begins approximately 2 hours after infection, at which point host cell nucleic acid synthesis ceases and cellular resources are redirected toward viral replication. Cell lysis occurs between 12 and 48 hours, releasing packaged viral particles. VVs do not depend on host mechanisms for mRNA transcription, making them less susceptible to changes in host cell biology. Unlike other oncolytic viruses (OVs), VVs do not require specific surface receptors for cell entry, allowing them to infect a broad range of cells.

[0026] The term "antibody" (also used interchangeably with "immunoglobulin") encompasses polyclonal (e.g., rabbit polyclonal) and monoclonal antibody preparations, and the antibody may be an antibody or immunoglobulin of any isotype (e.g., IgG (e.g., IgG1, IgG2, IgG3 or IgG4), IgE, IgD, IgA, IgM, etc.), full-length antibody (e.g., an antibody composed of a tetramer composed of two dimers of heavy and light chain polypeptides); single chain antibody (e.g., scFv); single chain Fv (scFv), Fab, (Fab')2, (scFv')2, and diabody, fragments of an antibody (e.g., a full-length antibody or a single chain antibody fragment) that retain specific binding to a compound; chimeric antibody; monoclonal antibody, humanized antibody, human antibody; and fusion proteins comprising the antigen-binding portion of an antibody and a non-antibody protein. In some embodiments, the antibody is selected from IgG, Fv, single chain antibody, scFv, Fab, F(ab')2, and F(ab'). Antibodies can be further conjugated to other moieties, such as members of specific binding pairs, e.g., biotin (a member of the biotin-avidin specific binding pair).

[0027] Immunoglobulin polypeptides include kappa and lambda light chains, and alpha, gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu heavy chains, or equivalents in other species. A full-length immunoglobulin "light chain" (usually of about 25 kDa or about 214 amino acids) contains a variable region of about 110 amino acids at the NH2-terminus, and a kappa or lambda constant region at the COOH-terminus. A full-length immunoglobulin "heavy chain" (of about 150 kDa or about 446 amino acids) similarly contains a variable region (of about 116 amino acids) and one of the aforementioned heavy chain constant regions, e.g., gamma (of about 330 amino acids).

[0028] Immunoglobulin light or heavy chain variable regions (V L and V H) is composed of a "framework" region (FR) interrupted by three hypervariable regions, also called "complementarity determining regions" or "CDRs". The extent of the framework regions and CDRs have been defined (E. Kabat et al., Sequences of proteins of immunological interest, 4th ed. USDept. Health and Human Services, Public Health Services, Bethesda, MD (1987); and Lefranc et al. IMGT, the international ImMunoGeneTics information system. Nucl. Acids Res., 2005, 33, D593-D597). The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, being the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs. The CDRs are primarily responsible for binding to an epitope of an antigen. All CDRs and frameworks provided by this disclosure are defined according to Kabat above, unless otherwise stated.

[0029] Thus, an "antibody" encompasses a protein having one or more polypeptides genetically encodable, for example, by an immunoglobulin gene or fragments thereof. Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. In some embodiments, the antibodies of the present disclosure are IgG antibodies, e.g., IgG1 antibodies, such as human IgG1 antibodies. In some embodiments, the antibodies of the present disclosure include a human Fc domain.

[0030] A typical immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids that is primarily responsible for antigen recognition. The variable light chain (V L ) and variable heavy chain (V H ) refer to these light and heavy chains respectively.

[0031] Antibodies include complete immunoglobulins and a number of well-characterized fragments that can be genetically encoded or produced by digestion with various peptidases. Thus, for example, pepsin digests antibodies under the disulfide bond in the hinge region to produce F(ab)'2 (a dimer of Fab, itself a light chain bound to VH-CHI by a disulfide bond). F(ab)'2 can be reduced under mild conditions to cleave the disulfide bond in the hinge region, thereby converting the (Fab')2 dimer into a Fab' monomer. The Fab' monomer is essentially a Fab with a portion of the hinge region (for a more detailed description of other antibody fragments, see Fundamental Immunology, WE Paul, ed., Raven Press, NY (1993)). Although various antibody fragments have been defined with respect to digestion of complete antibodies, one skilled in the art will understand that such Fab' fragments can be synthesized de novo, either chemically or by utilizing recombinant DNA methodology. Thus, as used herein, the term antibody also includes antibody fragments produced by modification of whole antibodies or synthesized de novo using recombinant DNA methodologies, including, but not limited to, Fab'2, IgG, IgM, IgA, scFv, dAb, nanobodies, unibodies, and diabodies. In certain embodiments, the antibodies of the present disclosure are selected from IgG, Fv, single chain antibodies, scFv, Fab, F(ab')2, and Fab'.

[0032] According to some embodiments, the antibodies of the present disclosure are monoclonal antibodies. "Monoclonal antibody" refers to a composition comprising one or more antibodies obtained from a population of substantially homogeneous antibodies (i.e., a population in which the individual antibodies are identical except for naturally occurring mutations that may be present in minor amounts). Monoclonal antibodies are highly specific and directed against a single antigenic site, typically against a single epitope on the antigen. The modifier "monoclonal" indicates the nature of the antibody as being obtained from a substantially homogeneous population of antibodies and does not require that the antibody be produced by any particular method or be the only antibody in the composition.

[0033] In certain embodiments, the antibody of the present disclosure is a humanized antibody. As used herein, a humanized antibody is a recombinant polypeptide derived from a non-human (e.g., rabbit, rodent, etc.) antibody and modified to contain at least a portion of the framework and / or constant region of a human antibody. Humanized antibodies also encompass chimeric antibodies and CDR-grafted antibodies, where various regions may be derived from different species. A chimeric antibody may be an antibody that comprises a variable region from any source linked to a human constant region (e.g., a human Fc domain). Thus, in a chimeric antibody, the variable region may be non-human and the constant region is human. A CDR-grafted antibody is an antibody that comprises a CDR from a non-human "donor" antibody linked to the framework region of a human "recipient" antibody. For example, an antibody of the present disclosure in the form of a scFV may be linked to a human constant region (e.g., an Fc domain) to make it into a human immunoglobulin.

[0034] Generally, humanized antibodies induce a reduced immune response in a human host compared to a non-humanized version of the same antibody. Antibodies can be humanized using a variety of techniques, including, for example, CDR grafting, veneering or resurfacing, and chain shuffling. In certain embodiments, framework substitutions are identified by modeling the interactions of CDR and framework residues to identify framework residues important for antigen binding and by sequence comparison to identify unique framework residues at specific positions.

[0035] Therefore, any of the antibodies described herein can be humanized using available methods. When rabbit or mouse CDRs are replaced with human variable domain frameworks, they can retain their correct spatial orientation, for example, the human variable domain framework adopts the same or similar conformation as the rabbit or mouse variable framework from which the CDRs are derived. This can be achieved by obtaining the human variable domain from a human antibody that has a framework sequence that shows a high degree of sequence identity with the rabbit or mouse variable framework domain from which the CDRs are derived. The variable framework regions of the heavy and light chains can be derived from the same or different human antibody sequences. The human antibody sequence can be the sequence of a naturally occurring human antibody or can be a consensus sequence of several human antibodies.

[0036] Once the complementarity determining regions of rabbit or mouse donor immunoglobulin and suitable human acceptor immunoglobulin have been identified, the next step is to determine which, if any, residues in these components should be replaced to optimize the properties of the resulting humanized antibody. In general, rabbit or mouse replacement of human amino acid residues should be minimized, since the introduction of rabbit or mouse residues increases the risk that the antibody will elicit a human anti-rabbit antibody (HARA) or human anti-mouse antibody (HAMA) response in humans. Art-recognized methods for determining immune responses can be performed to monitor HARA or HAMA responses in specific patients or during clinical trials. Patients administered humanized antibodies can undergo immunogenicity assessments at the start of and throughout the administration of the therapy. HARA or HAMA responses are measured by detecting antibodies against the humanized therapeutic reagent in serum samples from patients, using methods known to those skilled in the art, including, for example, surface plasmon resonance technology (BIACORE) and / or solid-phase ELISA analysis. In many embodiments, the subject humanized antibodies do not substantially elicit a HARA response in human subjects.

[0037] Specific amino acids from human variable region framework residues are selected for substitution based on their potential effect on CDR conformation and / or antigen binding. Unnatural juxtaposition of rabbit or mouse CDR regions and human variable framework regions may result in unnatural conformational constraints, which will lead to loss of binding affinity unless corrected by substitution of specific amino acid residues. Selection of amino acid residues for substitution can be determined, in part, by computer modeling. Computer hardware and software for generating three-dimensional images of immunoglobulin molecules are known in the art. Generally, a molecular model is created starting from a resolved structure of an immunoglobulin chain or domain thereof. The chain to be modeled is compared for amino acid sequence similarity with the chain or domain of the resolved three-dimensional structure, and the chain or domain showing the greatest sequence similarity is selected as the starting point for construction of the molecular model. Chains or domains that share at least 50% sequence identity are selected for modeling, preferably those that share at least 60%, 70%, 80%, 90% or more sequence identity are selected for modeling. The solved starting structure is modified to allow for differences between the actual amino acids of the immunoglobulin chain or domain being modeled and those of the starting structure. The modified structures are then assembled into a composite immunoglobulin. Finally, the model is refined by energy minimization and by ensuring that all atoms are within appropriate distances from each other and that bond lengths and angles are within chemically acceptable ranges.

[0038] For example, if the framework residues defined by Kabat constitute the structural loop residues defined by Chothia, for example, amino acids present in rabbit or mouse antibodies can be selected for substitution into the humanized antibody. Residues "adjacent to the CDR region" include those amino acid residues that are located directly adjacent to one or more of the CDRs in the primary sequence of the humanized immunoglobulin chain, for example, the CDRs defined by Kabat or the CDRs defined by Chothia (see, for example, Chothia and Lesk JMB 196:901 (1987)). These amino acids are particularly likely to interact with amino acids in the CDRs, and if selected from the acceptor, are likely to distort the donor CDRs and reduce affinity. Furthermore, these adjacent amino acids may directly interact with the antigen (Amit et al., Science, 233:747 (1986)), and selecting these amino acids from the donor may be desirable to maintain all of the antigen contacts that provide the affinity of the original antibody.Approaches that can be used to humanize any of the antibodies described herein include those described in Williams, D., Matthews, D. & Jones, T. Humanising Antibodies by CDR Grafting. Antibody Engineering 319-339 (2010) doi:10.1007 / 978-3-642-01144-3_21; Kuramochi, T., Igawa, T., Tsunoda, H. & Hattori, K. Humanization and simultaneous optimization of monoclonal antibody. Methods Mol. Biol. 1060, 123-37 (2014); Hwang, WY, Almagro, JC, Buss, TN, Tan, P. & Foote, J. Use of human germline genes in a CDR homology-based approach to antibody humanization. Methods 36, 35-42 (2005); Lo, BC Antibody humanization by CDR grafting. Methods Mol. Biol. 248, 135-59 (2004); and Lefranc, M.-PP, Ehrenmann, F., Ginestoux, C., Giudicelli, V. & Duroux, P. Use of IMGT databases and tools for antibody engineering and humanization. Methods Mol. Biol. 907, 3-37 (2012), the disclosures of which are incorporated herein by reference in their entireties for all purposes.

[0039] The antibodies of the present disclosure specifically bind to VV A56 or VV B5 antigens. An antibody "specifically binds" or "preferentially binds" a target if it binds with higher affinity, avidity, more readily, and / or with longer duration than it binds to other substances (e.g., in a sample). In certain embodiments, an antibody specifically binds to a target with a ... 4 M -1 An antibody "specifically binds" an antigen if it binds or associates with the antigen with an affinity or Ka (i.e., the equilibrium association constant of the particular binding interaction in units of 1 / M) equal to or greater than 1 / M. Alternatively, affinity can be measured using the equilibrium dissociation constant (KD) of the particular binding interaction in units of M (e.g., 10 -5 M~10 -13 In certain embodiments, specific binding can be defined as a binding intensity of about 10 M or less. -5 M or less, about 10 -6 M or less, about 10 -7 M or less, about 10 -8 M or less, or about 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 This means that the antibody binds to the antigen with a KD of equal to or less than M. The binding affinity of an antibody to an antigen can be readily determined using conventional techniques, for example, by competitive ELISA (enzyme-linked immunosorbent assay), equilibrium dialysis, by using surface plasmon resonance (SPR) technology (e.g., BIAcore 2000 or BIAcore T200 instruments using the general procedures outlined by the manufacturer), by radioimmunoassay.

[0040] Whether an antibody of the present disclosure "competes" with a second antibody for binding to an antigen can be easily determined using competitive binding assays known in the art. Competing antibodies can be identified, for example, through antibody competition assays. For example, a sample of a first antibody can be bound to a solid support. A sample of a second antibody suspected of being capable of competing with such a first antibody is then added. One of the two antibodies is labeled. If the labeled antibody and the unlabeled antibody bind to separate and distinct sites on the antigen, the labeled antibody will bind to the same level regardless of whether the suspected competing antibody is present. However, if the interaction sites are identical or overlapping, the unlabeled antibody will compete and the amount of labeled antibody that binds to the antigen will decrease. If unlabeled antibody is present in excess, little, if any, labeled antibody will bind.

[0041] For the purposes of this disclosure, a competing antibody is one that reduces the binding of an antibody to an antigen by about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 99% or more. Details of the procedures for carrying out such competitive assays are known and can be found, for example, in Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988, 567-569, 1988, ISBN 0-87969-314-2. Such assays can be performed quantitatively using purified antibodies. A standard curve can be established by titrating one antibody against itself; that is, the same antibody is used for both the label and the competitor. The ability of an unlabeled competing antibody to inhibit the binding of the labeled antibody to the plate can be titrated. The results can be plotted and the concentrations required to achieve the desired degree of binding inhibition can be compared.

[0042] Anti-VV A56 antibody According to some embodiments, an antibody is provided that specifically binds to the VV A56 antigen (VV A56). Although the vaccinia virus A56 protein was originally characterized as a hemagglutinin protein, A56 has other functions. The A56 protein can bind two viral proteins, serine protease inhibitor (K2) and vaccinia virus complement control protein (VCP), and anchor them to the surface of infected cells. Both proteins have biologically relevant functions at the cell surface, but neither can locate there by itself. The A56-K2 complex reduces the amount of virus that superinfects infected cells and also prevents the formation of syncytia by infected cells. The A56-VCP complex can protect infected cells from complement attack. Deletion of the A56R gene has various effects on the pathogenicity of vaccinia viruses. The gene encoding the A56 protein is nonessential and may be used as an insertion point for foreign genes, and is deleted in some viruses in clinical development as oncolytic agents.

[0043] In certain embodiments, an antibody of the disclosure specifically binds to VV A56 and competes for binding to VV A56 with an antibody having one, two, three, four, five or all six complementarity determining regions (CDRs) of one or more of the anti-VV A56 antibodies referred to herein as A047 / A057, A049 / A059 / A056, A050 and A054. According to some embodiments, an antibody of the disclosure specifically binds to VV A56 and comprises one, two, three, four, five or all six CDRs of the anti-VV A56 antibodies referred to herein as A047 / A057, A049 / A059 / A056, A050 or A054. The variable heavy chain (V H ) polypeptide, variable light chain (V L ) polypeptide, and the amino acid sequences of the CDRs are shown below in Table 1. All CDR and framework regions described throughout this disclosure are defined according to Kabat unless otherwise stated. [Table 1-1] [Table 1-2] [Table 1-3]

[0044] In certain embodiments, the antibodies of the disclosure specifically bind to VV A56 and, for binding to VV A56, V containing the amino acid sequence SSYWIC (SEQ ID NO:3) H CDR1, V containing the amino acid sequence CIIAGSGGSTYYATWAKG (SEQ ID NO: 4) H CDR2, V containing the amino acid sequence AYSDRSGGYSFNL (SEQ ID NO:5) H CDR3 and a variable heavy chain (V H ) polypeptides, and V containing the amino acid sequence QASQSVDNNNYLA (SEQ ID NO:6) L CDR1, V containing the amino acid sequence SASSLAS (SEQ ID NO: 7) L CDR2, V containing the amino acid sequence LGSYDCSDADCYA (SEQ ID NO: 8) L CDR3 and a variable light chain (V L ) polypeptide, which competes with an antibody comprising the

[0045] In certain embodiments, such antibodies comprise the six CDRs set forth in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8. According to some embodiments, the antibodies comprise a variable heavy chain (VH) comprising an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more or 100% identity to the amino acid sequence set forth in SEQ ID NO:1. H ) polypeptide, a variable light chain (V) comprising an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100% identity to the amino acid sequence set forth in SEQ ID NO:2 L) polypeptides, or both.

[0046] According to some embodiments, the antibodies of the present disclosure specifically bind to VV A56 and have the following properties for binding to VV A56: V containing the amino acid sequence DIYYIS (SEQ ID NO: 11) H CDR1, V containing the amino acid sequence CTYAGSSGSTYYATWAKG (SEQ ID NO: 12) H CDR2, V containing the amino acid sequence DRYPGTSGRVYGMDL (SEQ ID NO: 13) H CDR3 and a variable heavy chain (V H ) polypeptides, and V containing the amino acid sequence QASQSISDLLS (SEQ ID NO: 14) L CDR1, V containing the amino acid sequence SASTLAS (SEQ ID NO: 15) L CDR2, V containing the amino acid sequence QCNYYSPTYGNG (SEQ ID NO: 16) L CDR3 and a variable light chain (V L ) polypeptide, which competes with an antibody comprising the

[0047] In certain embodiments, such antibodies comprise the six CDRs set forth in SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16. According to some embodiments, the antibodies comprise a variable heavy chain (VH) comprising an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more or 100% identity to the amino acid sequence set forth in SEQ ID NO:9. H ) polypeptide, a variable light chain (V) comprising an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100% identity to the amino acid sequence set forth in SEQ ID NO:10 LIn certain embodiments, the antibody comprises a variable heavy chain (VH) polypeptide comprising an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100% identity to the amino acid sequence set forth in SEQ ID NO:9. H ) polypeptide, a variable light chain (V) comprising an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100% identity to the amino acid sequence set forth in SEQ ID NO:62; L According to some embodiments, the antibody comprises a variable heavy chain (VH) polypeptide comprising an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100% identity to the amino acid sequence set forth in SEQ ID NO:9. H ) polypeptide, a variable light chain (V) comprising an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100% identity to the amino acid sequence set forth in SEQ ID NO:63 L ) polypeptides, or both.

[0048] In certain embodiments, the antibodies of the disclosure specifically bind to VV A56 and, for binding to VV A56, V containing the amino acid sequence SSYWLC (SEQ ID NO: 19) H CDR1, V containing the amino acid sequence CIYNGDGSTHYASWAKG (SEQ ID NO: 20) H CDR2, V containing the amino acid sequence DYTYNFYTYGFNL (SEQ ID NO: 21) H CDR3 and a variable heavy chain (V H ) polypeptides, and V containing the amino acid sequence QASQSVNIWAS (SEQ ID NO:22) L CDR1, V containing the amino acid sequence KASTLAS (SEQ ID NO:23) L CDR2, V containing the amino acid sequence QGGYPSSSSGWA (SEQ ID NO:24) L CDR3 and a variable light chain (V L) polypeptide, which competes with an antibody comprising the

[0049] In certain embodiments, such antibodies comprise the six CDRs set forth in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24. According to some embodiments, the antibodies comprise a variable heavy chain (VH) comprising an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more or 100% identity to the amino acid sequence set forth in SEQ ID NO: 17. H ) polypeptide, a variable light chain (V) comprising an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100% identity to the amino acid sequence set forth in SEQ ID NO:18. L ) polypeptides, or both.

[0050] According to some embodiments, the antibodies of the present disclosure specifically bind to VV A56 and have the following properties for binding to VV A56: V containing the amino acid sequence SSYWIC (SEQ ID NO:27) H CDR1, V containing the amino acid sequence CTYNGDGSTHYASWAKG (SEQ ID NO: 28) H CDR2, V containing the amino acid sequence DYTDAFYTYGFNL (SEQ ID NO: 29) H CDR3 and a variable heavy chain (V H ) polypeptides, and V containing the amino acid sequence QASQSTSSYLA (SEQ ID NO:30) L CDR1, V containing the amino acid sequence RASSLAS (SEQ ID NO:31) L CDR2, V containing the amino acid sequence QTGFYGSSGHT (SEQ ID NO:32) L CDR3 and a variable light chain (V L ) polypeptide, which competes with an antibody comprising the

[0051] In certain embodiments, such antibodies comprise the six CDRs set forth in SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32. According to some embodiments, the antibodies comprise a variable heavy chain (VH) comprising an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more or 100% identity to the amino acid sequence set forth in SEQ ID NO:25. H ) polypeptide, a variable light chain (V) comprising an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100% identity to the amino acid sequence set forth in SEQ ID NO:26; L ) polypeptides, or both.

[0052] Suitable VV A56 antigens for determining whether an anti-VV A56 antibody of the present disclosure "competes" with a second antibody for binding to the VV A56 antigen include the Wyeth, Western Reserve and Copenhagen A56 antigens having the amino acid sequences shown in Table 2 below. [Table 2]

[0053] Anti-VV B5 antibody According to some embodiments, an antibody is provided that specifically binds to the VV B5 antigen (VV B5). The VV B5 protein is a 42 kDa type I transmembrane glycoprotein with an extracellular domain composed of four short consensus repeats (SCRs) characteristic of complement control proteins. After the SCRs, B5 has a stalk region before the transmembrane domain and a short cytoplasmic tail (CT). Neither the SCR nor the CT is essential for targeting B5 to the extracellular enveloped virus (EEV) membrane, but the latter affects its trafficking to the cell surface and recycling through endosomes. B5 is required for intracellular mature virus (IMV) wrapping to form intracellular enveloped virus (IEV).

[0054] In certain embodiments, an antibody of the present disclosure specifically binds to VV B5 and competes for binding to VV B5 with an antibody having one, two, three, four, five or all six complementarity determining regions (CDRs) of one or more of the anti-VV B5 antibodies referred to herein as A048 / A058 / A073 and A051. According to some embodiments, an antibody of the present disclosure specifically binds to VV B5 and comprises one, two, three, four, five or all six CDRs of the anti-VV B5 antibody referred to herein as A048 / A058 / A073 or A051. The variable heavy chain (V H ) polypeptide, variable light chain (V L ) polypeptide, as well as the amino acid sequences of the CDRs, are shown in Table 3 below. [Table 3]

[0055] In certain embodiments, the antibodies of the disclosure specifically bind to VV B5 and, for binding to VV B5, V containing the amino acid sequence SSYYMC (SEQ ID NO:35) H CDR1, V containing the amino acid sequence CIYTSSGSAYYANWAKG (SEQ ID NO: 36) H CDR2, V containing the amino acid sequence NAVGSSYYLYL (SEQ ID NO:37) H CDR3 and a variable heavy chain (V H ) polypeptides, and V containing the amino acid sequence QASQSVAGNNYLS (SEQ ID NO:38) L CDR1, V containing the amino acid sequence SVSTLAS (SEQ ID NO:39) L CDR2, V containing the amino acid sequence QGYYNDGIWA (SEQ ID NO: 40) L CDR3 and a variable light chain (V L ) polypeptide, which competes with an antibody containing

[0056] In certain embodiments, such antibodies comprise the six CDRs set forth in SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:40. According to some embodiments, the antibodies comprise a variable heavy chain (VH) comprising an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more or 100% identity to the amino acid sequence set forth in SEQ ID NO:33. H ) polypeptide, a variable light chain (V) comprising an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100% identity to the amino acid sequence set forth in SEQ ID NO:34. L ) polypeptides, or both.

[0057] According to some embodiments, the antibodies of the present disclosure specifically bind to VV B5 and have the following properties for binding to VV B5: V containing the amino acid sequence SYWMC (SEQ ID NO: 43) H CDR1, V containing the amino acid sequence CIYGGSSGSTYYSNWAKG (SEQ ID NO: 44) H CDR2, V containing the amino acid sequence DGSTWDYFRL (SEQ ID NO: 45) H CDR3 and a variable heavy chain (V H ) polypeptides, and V containing the amino acid sequence QASQSINTNYLS (SEQ ID NO:46) L CDR1, V containing the amino acid sequence QASTLES (SEQ ID NO:47) L CDR2, V containing the amino acid sequence QGYYTVENIGNP (SEQ ID NO: 48) L CDR3 and a variable light chain (V L ) polypeptide, which competes with an antibody comprising the

[0058] In certain embodiments, such antibodies comprise the six CDRs set forth in SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48. According to some embodiments, the antibodies comprise a variable heavy chain (VH) comprising an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more or 100% identity to the amino acid sequence set forth in SEQ ID NO: 41. H ) polypeptide, a variable light chain (V) comprising an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100% identity to the amino acid sequence set forth in SEQ ID NO:42 L ) polypeptides, or both.

[0059] Suitable VV B5 antigens for determining whether an anti-VV B5 antibody of the present disclosure "competes" with a second antibody for binding to the VV B5 antigen include Wyeth, Western Reserve and Copenhagen B5 antigens having the amino acid sequences shown in Table 4 below. [Table 4]

[0060] bispecific antibody Bispecific antibodies are also provided. In certain embodiments, the bispecific antibodies of the present disclosure comprise either the V of any of the anti-VV A56 and anti-VV B5 antibodies of the present disclosure, such as those described above. H Polypeptide-V L The bispecific antibody comprises a first antigen-binding domain comprising a polypeptide pair. The bispecific antibody may comprise a second antigen-binding domain that specifically binds to a VV antigen bound by the first antigen-binding domain. In certain embodiments, the bispecific antibody comprises a second antigen-binding domain that specifically binds to a VV antigen other than the VV antigen bound by the first antigen-binding domain.

[0061] According to some embodiments, the bispecific antibody of the present disclosure comprises a second antigen-binding domain that specifically binds to an antigen other than a VV antigen. In certain embodiments, the antigen other than a VV antigen is an immune cell surface antigen. Non-limiting examples of immune cell surface antigens include immune effector cell surface antigens, such as T cell surface antigens, natural killer (NK) cell surface antigens, macrophage cell surface antigens, etc. Examples of T cell surface antigens that can be bound by the second antigen-binding domain include, but are not limited to, T cell stimulatory molecules, such as CD3, CD28, etc.

[0062] Bispecific antibodies of the present disclosure include antibodies with full-length antibody structures, and bispecific antibody fragments. As used herein, "full-length" refers to an antibody having two full-length antibody heavy chains and two full-length antibody light chains. A full-length antibody heavy chain (HC) is composed of the well-known heavy chain variable domain and constant domains VH, CH1, CH2, and CH3. A full-length antibody light chain (LC) is composed of the well-known light chain variable domain and constant domains VL and CL. A full-length antibody may lack the C-terminal lysine of one or both heavy chains. The term "Fab arm" refers to one heavy chain:light chain pair that specifically binds to an antigen.

[0063] Full-length bispecific antibodies can be generated, for example, using Fab arm exchange (or half molecule exchange) between two monospecific bivalent antibodies by introducing substitutions at the heavy chain CH3 interface of each half molecule to promote heterodimer formation in a cell-free environment in vitro or using co-expression of two antibody half molecules with distinct specificities. The Fab arm exchange reaction is the result of disulfide bond isomerization and dissociation-association of the CH3 domains. The heavy chain disulfide bonds in the hinge region of the parent monospecific antibodies are reduced. A free cysteine ​​from one of the parent monospecific antibodies forms an inter-heavy chain disulfide bond with a cysteine ​​residue of the second parent monospecific antibody molecule, while the CH3 domains of the parent antibodies are separated and reformed by dissociation-association. The CH3 domains of the Fab arms can be designed to favor heterodimerization over homodimerization. The resulting product is a bispecific antibody with two Fab arms or half molecules, each binding a distinct epitope.

[0064] A "knob-in-hole" strategy (see, e.g., WO2006 / 028936) can be used to generate full-length bispecific antibodies. Briefly, selected amino acids that form the interface of the CHS domain in human IgG can be mutated at positions that affect CH3 domain interactions to promote heterodimer formation. An amino acid with a small side chain (hole) is introduced into the heavy chain of an antibody that specifically binds to a first antigen, and an amino acid with a large side chain (knob) is introduced into the heavy chain of an antibody that specifically binds to a second antigen. After co-expression of the two antibodies, a heterodimer is formed as a result of preferential interaction of the heavy chain with the "hole" with the heavy chain with the "knob". Exemplary CH3 substitution pairs that form knobs and holes are as follows (represented as modified position in the first CH3 domain of the first heavy chain / modified position in the second CH3 domain of the second heavy chain): T366Y7F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T3945 / Y407A, T366W / T394S, F405W / T394S, and T366W / T366S_L368A_Y407V.

[0065] Other strategies may be used such as using electrostatic interactions to promote heavy chain heterodimerization by substituting a positively charged residue at one CH3 surface and a negatively charged residue at the second CH3 surface as described in US2010 / 0015133, US2009 / 0182127, US2010 / 028637 or US2011 / 0123532. In other strategies, heterodimerization may be promoted by the following substitutions (expressed as modified position in the first CH3 domain of the first heavy chain / modified position in the second CH3 domain of the second heavy chain): L351 Y_F405A_Y407V T394W, T366I_K392M_T394W / F405A_Y407V, L351 Y_F405A_Y407V, T394W, T366I_K392M_T394W / F405A_Y407V ... Y_Y407A'T366A_K409F, L351Y_Y407A / T366V_K409F, Y407A / T366A_K409F or T350V_L351Y_F405A_Y407V / T350V_T366L_K392L_T394W.

[0066] Single chain bispecific antibodies are also provided. In some embodiments, the single chain bispecific antibodies of the present disclosure are bispecific scFvs. Details regarding bispecific scFvs can be found, for example, in Zhou et al. (2017) J Cancer 8(18):3689-3696.

[0067] Approaches that may be used to generate multispecific (e.g., bispecific) antibodies from the antibodies described herein include, but are not limited to, Ellerman, D. (2019) "Bispecific T-cell engagers: Towards understanding variables influencing the in vitro potency and tumor selectivity and their modulation to enhance their efficacy and safety." Methods 154:102-117; Brinkmann, U. and RE Kontermann (2017) "The making of bispecific antibodies." mAbs 9(2):182-212; and Suurs, FV, et al. (2019) "A review of bispecific antibodies and antibody constructs in oncology and clinical challenges." Pharmacol Ther 201:103-119, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

[0068] Fusion proteins Fusion proteins are also provided. In certain embodiments, the fusion proteins of the present disclosure comprise a chain of any of the anti-VV A56 or anti-VV B5 antibodies of the present disclosure fused to a heterologous amino acid sequence. The heterologous amino acid sequence may be fused to the C-terminus of the antibody chain or the N-terminus of the antibody chain. In certain embodiments, the fusion proteins of the present disclosure comprise a heterologous sequence at the C-terminus of the antibody chain and a heterologous sequence at the N-terminus of the antibody chain, which may be the same or different sequences. When used in the context of a nucleic acid or polypeptide, "heterologous" generally means that the nucleic acid or polypeptide is derived from a different source (e.g., a molecule of different sequence, different species origin, etc.) than that to which it is associated or joined, such that it is not found in nature. For example, in a fusion protein, a light chain polypeptide and a reporter polypeptide (e.g., GFP, red fluorescent protein (e.g., mCherry), luciferase, etc.) are said to be "heterologous" to each other. Similarly, a CDR from a mouse antibody and a constant region from a human antibody are "heterologous" to each other.

[0069] The chains of anti-VV A56 or anti-VV B5 antibodies can be fused to any heterologous sequence of interest. Heterologous polypeptides of interest include, but are not limited to, albumin, transferrin, XTEN, homoamino acid polymers, proline-alanine-serine polymers, elastin-like peptides, or any combination thereof. In certain embodiments, the heterologous polypeptide increases the stability and / or serum half-life of the antibody when administered to an individual in need thereof, compared to the same antibody that is not fused to a heterologous sequence.

[0070] In certain embodiments, the fusion proteins of the present disclosure comprise single chain antibodies, such as any of the VV A56 and anti-VVB5 antibodies of the present disclosure, including any of the antibodies described above. H Polypeptide-V LThe scFvs of the present disclosure include, but are not limited to, scFvs that include the six CDRs of the scFvs listed in Table 5 below, and in some embodiments, the scFvs include the V and V CDRs of the scFvs listed in Table 5. H A variable heavy chain (VH) having an amino acid sequence that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100% identical to the amino acid sequence of H ) polypeptides and the V of scFvs listed in Table 5 L A variable light chain (V) having an amino acid sequence that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100% identical to the amino acid sequence of L In Table 5, segments / domains of the polypeptide are indicated with alternating underlines and the identity of the segment / domain is given in the left column. [Table 5-1] [Table 5-2]

[0071] According to some embodiments, when the fusion protein comprises a single chain antibody (e.g., any of the single chain antibodies of the present disclosure, including any of the scFvs described herein), the fusion protein is a chimeric antigen receptor (CAR) comprising the single chain antibody, a transmembrane domain, and an intracellular signaling domain.

[0072] The CAR of the present disclosure may include one or more linker sequences between the various domains. A "variable region linking sequence" is an amino acid sequence that connects the heavy chain variable region to the light chain variable region and provides a spacer function compatible with the interaction of the two sub-binding domains such that the resulting polypeptide retains the same specific binding affinity for the same target molecule as an antibody containing the same light and heavy chain variable regions. A non-limiting example of a variable region linking sequence is a serine-glycine linker, such as a serine-glycine linker comprising the amino acid sequence GGGGSGGGSGGGGS (G4S)3 (SEQ ID NO: 49). In certain aspects, the linker separates one or more heavy or light chain variable domains, hinge domains, transmembrane domains, costimulatory domains, and / or primary signaling domains. In certain embodiments, the CAR comprises one, two, three, four, or five or more linkers. In certain embodiments, the length of the linker is about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or any intervening amino acids in length. In some embodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids in length.

[0073] In some embodiments, the antigen binding domain of the CAR is followed by one or more spacer domains that distance the antigen binding domain from the effector cell surface (e.g., the surface of a T cell expressing the CAR) to allow for proper cell / cell contact, antigen binding and / or activation. The spacer domain (as well as any other spacer domains, linkers and / or the like described herein) can be derived from any source, natural, synthetic, semi-synthetic or recombinant. In certain embodiments, the spacer domain is a portion of an immunoglobulin, including, but not limited to, one or more heavy chain constant regions, such as CH2 and CH3. The spacer domain can comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region. In one embodiment, the spacer domain comprises the CH2 and / or CH3 of IgG1, IgG4 or IgD. Exemplary spacer domains suitable for use in the CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins such as CD8α and CD4, which can be wild-type hinge regions derived from these molecules or mutants thereof. In certain aspects, the hinge domain comprises a CD8α hinge region. In some embodiments, the hinge is a PD-1 hinge or a CD152 hinge.

[0074] The "transmembrane domain" (Tm domain) is the portion of the CAR that fuses the extracellular binding moiety with the intracellular signaling domain and anchors the CAR to the plasma membrane of a cell (e.g., an immune effector cell). The Tm domain can be derived from either natural, synthetic, semi-synthetic, or recombinant sources. In some embodiments, the Tm domain is derived from (e.g., including at least the transmembrane region or a functional portion thereof) the alpha or beta chain of the T cell receptor, CD35, CD3ζ, CD3γ, CD3δ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, or PD-1.

[0075] In one embodiment, CAR comprises the Tm domain derived from CD8α.In a particular aspect, CAR comprises the Tm domain derived from CD8α and a short oligo- or polypeptide linker, for example, between 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in length.This connects the Tm domain of CAR and the intracellular signaling domain.For example, glycine-serine linker can be used as such linker.

[0076] The "intracellular signaling" domain of a CAR refers to the portion of the CAR that is involved in transmitting a signal from the CAR that binds to a target molecule / antigen to the inside of an immune effector cell to induce effector cell functions, such as activation, cytokine production, proliferation and / or cytotoxic activity (including the release of cytotoxic factors to a CAR-bound target cell or other cellular responses elicited by target molecule / antigen binding to the extracellular CAR domain). Thus, the term "intracellular signaling domain" refers to the portion of a protein that transmits an effector function signal and instructs a cell to perform a specialized function. When a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used instead of the full-length intracellular signaling domain, so long as it transmits the effector function signal. The term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain that is sufficient to transmit an effector function signal.

[0077] Signals generated by the T cell receptor (TCR) alone are insufficient to fully activate T cells, and secondary or costimulatory signals are also required. Thus, T cell activation is mediated by two distinct classes of intracellular signaling domains: primary signaling domains that initiate antigen-dependent primary activation via the TCR (e.g., TCR / CD3 complex) and costimulatory signaling domains that act antigen-independently to provide secondary or costimulatory signals. Thus, the CAR of the present disclosure may include an intracellular signaling domain that includes one or more "costimulatory signaling domains" and "primary signaling domains."

[0078] The primary signaling domain regulates the primary activation of the TCR complex, either stimulatory or inhibitory. Primary signaling domains that act stimulatory may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs (or "ITAMs"). Non-limiting examples of ITAM-containing primary signaling domains suitable for use in the CARs of the present disclosure include those derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79α, CD79β, and CD66δ. In certain embodiments, the CAR comprises a CD3ζ primary signaling domain and one or more costimulatory signaling domains. The intracellular primary signaling and costimulatory signaling domains are operably linked to the carboxyl terminus of the transmembrane domain.

[0079] In some embodiments, the CAR comprises one or more costimulatory signaling domains to enhance the efficacy and proliferation of immune effector cells (e.g., T cells) expressing the CAR. As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to the intracellular signaling domain of a costimulatory molecule or an active fragment thereof. Exemplary costimulatory molecules suitable for use in the CARs contemplated in certain embodiments include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM and ZAP70. In some embodiments, the CAR comprises one or more costimulatory signaling domains selected from the group consisting of 4-1BB (CD137), CD28, and CD134, and a CD3ζ primary signaling domain.

[0080] The CAR of the present disclosure may include any of a variety of suitable domains, including, but not limited to, a leader sequence; hinge, spacer and / or linker domain; transmembrane domain; costimulatory domain; signal transduction domain (e.g., CD3ζ domain); ribosomal skip element; restriction enzyme sequence; reporter protein domain; and / or the like. Non-limiting examples of such domains that may be included in the CAR of the present disclosure include those shown in Table 6 below. As will be understood by those skilled in the art, the amino acid sequence of one or more of the domains shown in Table 6 (e.g., linker, hinge, transmembrane, costimulatory, signal transduction, ribosomal skip element; restriction enzyme sequence; reporter protein, etc.) may be modified as necessary, for example, to improve the functionality of the CAR, etc. [Table 6-1] [Table 6-2] [Table 6-3]

[0081] In certain embodiments, a CAR of the disclosure comprises a single chain antibody (e.g., any of the scFvs of the disclosure) that binds to an antigen of interest (e.g., VV A56 antigen or VV B5 antigen); a transmembrane domain from a polypeptide selected from the group consisting of CD4, CD8α, CD154, and PD-1; one or more intracellular costimulatory signaling domains from a polypeptide selected from the group consisting of 4-1BB (CD137), CD28, and CD134; and an intracellular signaling domain from a polypeptide selected from the group consisting of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79α, CD79β, and CD66δ. Such a CAR may further comprise a spacer domain, e.g., a CD8 alpha hinge, between the antigen binding portion and the transmembrane domain.

[0082] According to some embodiments, provided is a nucleotide sequence comprising, between the N-terminus and C-terminus, the variable heavy chain (VH) of an antibody described herein. H ) Polypeptides, linkers, and antibody variable light chains (V L ), a CD8 hinge region (which in some embodiments is an extended CD8 hinge region), a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain. According to certain embodiments, provided is a CAR comprising, between the N-terminus and C-terminus, a variable light chain (VLC) of an antibody described herein. L ) Polypeptides, linkers, and antibody variable heavy chains (V H ), a CD8 hinge region (which in some embodiments is an extended CD8 hinge region), a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain. In certain embodiments, provided is a CAR comprising, between the N-terminus and C-terminus, a variable heavy chain (VH) of an antibody described herein. H ) Polypeptides, linkers, and antibody variable light chains (V L ), a CD28 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain. According to some embodiments, provided is a CAR comprising, between the N-terminus and C-terminus, a variable light chain (VLC) of an antibody described herein. L ) Polypeptides, linkers, and antibody variable heavy chains (V H ), a CD28 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain. H The N-terminal domain of a polypeptide may be included. For example, a leader sequence (e.g., a GM-CSFR leader sequence) may be present at the N-terminus of the CAR of the present disclosure.

[0083] The amino acid sequences of exemplary anti-A56 CARs of the present disclosure are shown in Tables 7 and 8 below, where the amino acid sequences of the CARs in Table 7 include the amino acid sequence of an N-terminal leader sequence (here, an N-terminal GM-CSFR leader sequence) and the amino acid sequences of the CARs in Table 8 do not include the amino acid sequence of a leader sequence. Any desired leader sequence (e.g., a GM-CSFR leader sequence) can be present at the N-terminus of such a CAR. As will be understood by one of skill in the art, the amino acid sequences of one or more of the domains (e.g., linker, hinge, transmembrane, costimulatory, signaling, etc.) shown in Tables 7 and 8 can be modified as necessary, for example, to improve the functionality, etc., of the CAR. In Tables 7 and 8, segments / domains of the polypeptide are shown with alternating underlines, and the segment / domain IDs are shown in the left column. [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5]

[0084] CARs of the present disclosure may optionally include one or more additional domains. Non-limiting examples of such additional domains include ribosomal skipping elements, enzymatic domains (e.g., domains with nuclease activity, e.g., restriction endonuclease activity), domains that allow for detection of the CAR (e.g., reporter protein domains (e.g., fluorescent proteins (e.g., eGFP, mCherry, etc.), luminescent proteins, and / or the like)), and the like. For example, in certain embodiments, provided are CARs that include a ribosomal skipping element, a restriction enzyme domain, and / or a reporter protein domain. The amino acid sequences of exemplary anti-A56 CARs of the present disclosure having one or more of these features are shown in Table 9 below. As will be understood by one of skill in the art, the amino acid sequences of one or more of the domains shown in Table 9 (e.g., linker, hinge, transmembrane, costimulatory, signal transduction, restriction enzyme sequence, reporter protein, etc.) may be modified as needed, for example, to improve the functionality, detection, etc. of the CAR. In Table 9, the CAR portion is shown in bold and the polypeptide segments / domains are shown in alternating underlines. The segment / domain IDs are shown in the left column. [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] [Table 9-5] [Table 9-6] [Table 9-7]

[0085] In certain embodiments, the CAR provided comprises one or more mouse CAR domains.Non-limiting examples of mouse CAR domains that can be included in the CAR of the present disclosure include one or more of the mouse CAR domains shown in Table 10 below.As will be understood by those skilled in the art, the amino acid sequence of one or more of the domains shown in Table 10 (e.g., leader sequence, linker, hinge, transmembrane, costimulatory, signal transduction, ribosome skip element; restriction enzyme sequence; reporter protein, etc.) can be modified as necessary. [Table 10]

[0086] According to some embodiments, a murine CAR is provided that comprises one or more murine CAR domains (e.g., one or more of any of the domains set forth in Table 10 above). Non-limiting examples of murine CARs of the present disclosure include those shown in Table 11 below. As will be appreciated by those of skill in the art, the amino acid sequence of one or more of the domains set forth in Table 11 (e.g., the Leair sequence, the V H , Linker, V L The CAR moieties are shown in bold in Table 11. Segments / domains of the polypeptide are shown in alternating underlines, with the segment / domain identities shown in the left column. [Table 11-1] [Table 11-2]

[0087] According to some embodiments, the CAR of the present disclosure is provided by a single polypeptide. In certain embodiments, the CAR of the present disclosure is provided by two or more polypeptides. When the CAR is provided by two or more polypeptides, the CAR can be provided in a variety of formats, including biotin-binding immune receptor (BBIR) format (see, e.g., Urbanska K, Powell DJ. Development of a novel universal immune receptor for antigen targeting to infinity and beyond. Oncoimmunology. 2012;1(5):777-779. doi:10.4161 / onci.19730 and Urbanska K, Lanitis E, Poussin M, et al. A universal strategy for adoptive immunotherapy of cancer through use of a novel T cell antigen receptor. 2013;72(7):1844-1852. doi:10.1158 / 0008-5472. CAN-11-3890.A); a switchable CAR format using the peptide NeoEpitope (PNE) (see, e.g., Kim et al. (2015) J Am Chem Soc. 2015;137(8):2832-2835; Ma et al. (2016) Proc Natl Acad Sci 113(4):E450-8; Rodgers et al. (2016) Proc Natl Acad Sci. 113(4):E459-E468; Viaud et al. (2018) Proc Natl Acad Sci 115(46):E10898-E10906); SUPRA CAR formats with leucine zippers (see, e.g., Cho et al. (2108) Cell 173(6):1426-1438.e11); CAR-T adapter molecule (CAM)-based formats using FITC-folate (see, e.g., Lee et al. (2019) Cancer Res. 79(2):387-396 and Lu et al. (2019) Cancer Res. 113(4):E459-E468; Viaud et al. (2018) Proc Natl Acad Sci 115(46):E10898-E10906); al. (2019) Front Oncol.9:151); anti-FITC-folate adaptor format (see, e.g., Chu et al. (2018) Biosci Trends. 12(3):298-308); anti-FITC antibody adaptor CAR format (see, e.g., Tamada et al. (2012) Clin Cancer Res. 18(23):6436-6445); Fc-targeted (e.g., anti-CD16) CAR+anti-tumor antibody format (see, e.g., Kudo et al. (2014) Cancer Res. 74(1):93-103); and the like.

[0088] Conjugates The present disclosure also provides a conjugate. According to some embodiments, the conjugate of the present disclosure comprises any of the antibodies or fusion proteins of the present disclosure and a drug conjugated to the antibody or fusion protein. The term "conjugated" generally refers to a chemical bond, either covalent or non-covalent, usually covalent, that associates one molecule of interest with a second molecule of interest in close proximity. In certain embodiments, the drug conjugated to the antibody or fusion protein is selected from a chemotherapeutic agent, a toxin, a radiosensitizer, a radioisotope (e.g., a therapeutic radioisotope), a detectable label, and a half-life extending moiety.

[0089] According to some embodiments, the drug is a therapeutic agent, e.g., a chemotherapeutic agent. Therapeutic agents of interest include agents that can affect the function of the cell / tissue to which the conjugate is bound via specific binding of the antibody portion of the conjugate to the antigen of the cell / tissue. If the function of the cell / tissue is pathological, an agent that reduces the function of the cell / tissue may be used. In certain aspects, the conjugates of the present disclosure include agents that reduce the function of the target cell / tissue by inhibiting cell proliferation and / or killing the cell / tissue. Such agents are various and may include cytostatic agents and cytotoxic agents, e.g., agents that can kill the target cell tissue with or without internalization into the target cell.

[0090] In certain embodiments, the therapeutic agent is a cytotoxic agent selected from an enediyne, a lexitropsin, a duocarmycin, a taxane, a puromycin, a dolastatin, a maytansinoid, and a vinca alkaloid. In some embodiments, the cytotoxic agent is paclitaxel, docetaxel, CC-1065, CPT-11 (SN-38), topotecan, doxorubicin, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, dolastatin-10, echinomycin, combretastatin, calicheamicin, maytansine, maytansine DM1, maytansine DM4, DM-1, auristatin or other dolastatin derivatives such as auristatin E or auristatin F, AEB (AEB-071), AEVB (5-benzoylvaleric acid-AE ester), AEFP (antibody-endostatin fusion protein), MMAE (monomethylauristatin E), MMAF (monomethylauristatin F), pyrrolobenzodiazepines (PBDs), eleutherobin, netlopsin, or any combination thereof.

[0091] According to some embodiments, the agent is hemiasterlin and hemiasterlin analogs such as HTI-286 (see, e.g., USPN 7,579,323, WO 2004 / 026293, and USPN 8,129,407, the entire disclosures of which are incorporated herein by reference), abrin, brucine, cicutoxin, diphtheria toxin, batrachotoxin, botulinum toxin, shiga toxin, endotoxin, Pseudomonas exotoxin, Pseudomonas endotoxin, tetanus toxin, pertussis toxin, anthrax toxin, cholera toxin, falcarinol, fumonisin B1, fumonisin B2, aflatoxin, malatex, maltitol ... The protein toxin is selected from maurotoxin, agitoxin, charybdotoxin, margatoxin, slotoxin, scyllatoxin, hefutoxin, calciceptin, taicatoxin, calcicludin, geldanamycin, gelonin, lotaustralin, ochratoxin A, patulin, ricin, strychnine, trichothecene, zearalenone, and tetradotoxin. Enzymatically active toxins and fragments thereof which may be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the trichothecenes.

[0092] In certain embodiments, the agent is a radiosensitizer. As used herein, a "radiosensitizer" is an agent that enhances the ability of radiation to kill tumor cells. Non-limiting examples of radiosensitizers that can be conjugated to antibodies or fusion proteins include cisplatin, 5-fluorouracil (5-FU), AZD7762, selumetinib, etc.

[0093] In certain embodiments, the agent is a radioisotope, e.g., useful for therapy and / or detection (e.g., imaging). Non-limiting examples of radioisotopes that can be conjugated to an antibody or fusion protein include: 225 Ac, 111 Ag, 114 Ag, 71 As, 72 As, 77 As, 211 At, 198 Au, 199 Au, 212 Bi, 213 Bi, 75 Br, 76 Br, 11 C. 13 C. 55 Co, 62 Cu, 64 Cu, 67 Cu, 165 Dy, 166 Dy, 169 Er, 18 F, 19 F, 52 Fe, 59 Fe, 66 Ga, 67 Ga, 68 Ga, 72 Ga, 154-158 Gd, 157 Gd, 159 Gd, 166 Ho, 120 I, 121 I, 123 I, 124 I, 125 I, 131 I, 110 In, 111 In, 113m In, 194 Ir,81m Kr, 177 Lu, 51 Mn, 52 Mn, 99 Mo, 13 N, 15 N, 15 O. 17 O. 32 P, 33 P, 211 Pb, 212 Pb, 109 Pd, 149 Pm, 151 Pm, 142 Pr, 143 Pr, 191 P.T., 193m P.T., 195m Pt, 223 Ra, 142 Rb, 186 Re, 188 Re, 189 Re, 105 Rh, 47 Sc, 75 Se, 153 Sm, 117m Sn, 121 Sn, 83 Sr, 89 Sr, 161 Tb, 94 Tc, 99 Tc, 99m Tc, 227 Th, 201 Tl, 172 Tm, 127 Te, 90 Y, 169 Yb, 175 Yb, 133 X and 89 These include, but are not limited to, Zr.

[0094] In certain embodiments, the radioisotope is conjugated to the antibody or fusion protein via a chelator, for example, a bifunctional chelator. The bifunctional chelator may contain a metal chelating moiety that binds the radioisotope in a stable coordination complex and a reactive functional group that is covalently linked to a targeting moiety, such as any of the antibodies or fusion proteins of the present disclosure, so that the radioisotope can be appropriately directed to a desired molecular target in vivo. Non-limiting examples of bifunctional chelators that can be used to conjugate the antibodies or fusion proteins of the present disclosure to a radioisotope include p-SCN-Bn-DOTA and p-SCN-Bn-deferoxamine. Additional examples of bifunctional chelators that can be used to conjugate the antibodies or fusion proteins of the present disclosure to radioisotopes include those described in Price & Orvig (2014) Chem. Soc. Rev. 43:260; and Brechbiel (2008) QJ Nucl Med Mol Imaging 52(2):166-173.

[0095] According to some embodiments, the radioisotope is a therapeutic radioisotope. In certain embodiments, the radioisotope is an alpha-emitting radioisotope, e.g., 225 Ac, 211 At, 212 Bi / 212 Pb, 213 Bi, 223 Ra, or 227 In other embodiments, the radioisotope is a beta minus emitting radioisotope, e.g., 32 P, 33 P, 67 Cu, 90 Y, 131 I or 177 This is Lu.

[0096] According to some embodiments, the agent is a labeling agent. "Labeling agent" (or "detectable label") means that the agent detectably labels the antibody or fusion protein so that the antibody or fusion protein can be detected in the intended application (e.g., in vitro and / or in vivo research and / or clinical application). Detectable labels of interest include radioisotopes (e.g., gamma or positron emitters), enzymes that generate detectable products (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, etc.), fluorescent proteins, paramagnetic atoms, etc. In certain aspects, the antibody or fusion protein is conjugated to a specific binding partner of the detectable label (e.g., conjugated to biotin so that detection can occur via a detectable label that includes avidin / streptavidin).

[0097] According to certain embodiments, the agent is a labeling agent that finds use in in vivo imaging, such as near-infrared (NIR) optical imaging, single photon emission computed tomography (SPECT)±CT imaging, positron emission tomography (PET)±CT imaging, nuclear magnetic resonance (NMR) spectroscopy, etc. Labeling agents that find use in such applications include, but are not limited to, fluorescent labels, radioisotopes, etc. In certain aspects, the labeling agent is a multimodal in vivo imaging agent that allows in vivo imaging using two or more imaging techniques (see, for example, Thorp-Greenwood and Coogan (2011) Dalton Trans. 40:6129-6143).

[0098] In certain embodiments, the labeling agent is an in vivo imaging agent that finds use in near infrared (NIR) imaging applications.Such agents include, but are not limited to, Kodak X-SIGHT dyes, Pz 247, DyLight750 and 800Fluors, Cy5.5 and 7Fluors, Alexa Fluor680 and 750Dyes, IRDye680 and 800CW Fluors.According to some embodiments, the labeling agent is an in vivo imaging agent that finds use in SPECT imaging applications, non-limiting examples of which include: 99m Tc, 111 In, 123 I, 201 T1, and 133 In certain embodiments, the labeling agent is for PET imaging applications, e.g., 11 C. 13 N, 15 O. 18 F, 64 Cu, 62 Cu, 124 I, 76 Br, 82 Rb, 68 It is an in vivo imaging agent that finds use in Ga, etc.

[0099] To extend half-life, the antibodies and fusion proteins of the present disclosure may be conjugated to agents that provide improved pharmacokinetic profiles (e.g., by PEGylation, hyperglycosylation, etc.). Modifications that can extend serum half-life are of interest. The subject antibody or fusion protein may be "PEGylated" as containing one or more poly(ethylene glycol) (PEG) moieties. Methods and reagents suitable for PEGylation of proteins are well known in the art and can be found, for example, in U.S. Pat. No. 5,849,860. PEGs suitable for conjugation to proteins are generally water soluble at room temperature and have the general formula R(O-CH2-CH2) nThe PEG has the formula OR, where R is hydrogen or a protecting group such as an alkyl or alkanol group, and n is an integer between 1 and 1000. When R is a protecting group, it generally has 1 to 8 carbons. The PEG conjugated to the antibody or fusion protein of interest can be linear. The PEG conjugated to the antibody or fusion protein of interest can also be branched. Branched PEG derivatives, such as those described in U.S. Pat. No. 5,643,575, "star PEGs" and multi-arm PEGs. Star PEGs have been described in the art, including, for example, U.S. Pat. No. 6,046,305.

[0100] If the antibody or fusion protein of interest is isolated from a source, the antibody or fusion protein may be conjugated to one or more moieties that facilitate purification, such as members of a specific binding pair, e.g., biotin (a member of the biotin-avidin specific binding pair), lectins, etc. Antibodies can also be bound (e.g., immobilized) to a solid support, including, but not limited to, polystyrene plates or beads, magnetic beads, test strips, membranes, and the like.

[0101] Where an antibody or fusion protein is to be detected in an assay, the antibody or fusion protein may be tagged with a detectable label, e.g., a radioisotope (e.g., 89 Zr, 111 In, etc.), enzymes that produce detectable products (e.g., luciferase, β-galactosidase, horseradish peroxidase, alkaline phosphatase, etc.), fluorescent proteins, chromogenic proteins, dyes (e.g., fluorescein isothiocyanate, rhodamine, phycoerythrin, etc.); fluorescent metals attached to proteins via metal chelating groups such as EDTA, e.g. 152 Eu or other lanthanides; chemiluminescent compounds such as luminol, isoluminol, acridinium salts, etc.; bioluminescent compounds such as luciferin; fluorescent proteins, etc. Indirect labels include antibodies specific for the protein of interest, which can be detected via a secondary antibody and a member of a specific binding pair, such as biotin-avidin.

[0102] Any of the above agents may be conjugated to the antibody or fusion protein via a linker. If present, the linker molecule may be of sufficient length to allow the antibody or fusion protein and the linked agent some flexible movement between the antibody or fusion protein and the linked agent. The linker molecule may be, for example, about 6-50 atoms in length. The linker molecule may also be, for example, an aryl acetylene, an ethylene glycol oligomer containing 2-10 monomer units, a diamine, a diacid, an amino acid, or a combination thereof.

[0103] When the linker is a peptide, the linker can be of any suitable length, such as from 1 amino acid (e.g., Gly) to 20 or more amino acids, from 2 to 15 amino acids, from 3 to 12 amino acids (including from 4 to 10 amino acids, from 5 to 9 amino acids, from 6 to 8 amino acids, or from 7 to 8 amino acids), and can be 1, 2, 3, 4, 5, 6, or 7 amino acids in length.

[0104] The flexible linker is a glycine polymer (G) n , glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used when relatively unstructured amino acids are of interest and can function as neutral tethers between components. Those skilled in the art will recognize that the design of the antibody or fusion protein conjugated to any of the above agents can include a fully or partially flexible linker, whereby the linker can include a flexible linker and one or more moieties that impart a less flexible structure.

[0105] According to some embodiments, the antibody or fusion protein is conjugated to the drug via a non-cleavable linker.The non-cleavable linker of interest includes, but is not limited to, thioether linker.The example of the thioether linker that can be used includes succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker.

[0106] In certain embodiments, the antibody is conjugated to the drug via a cleavable linker. According to some embodiments, the linker is a chemically labile linker, such as an acid-cleavable linker, which is stable at neutral pH (bloodstream pH 7.3-7.5) but undergoes hydrolysis upon internalization into the weakly acidic endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0) of target cells (e.g., cancer cells). Chemically labile linkers include, but are not limited to, hydrazone-based linkers, oxime-based linkers, carbonate-based linkers, ester-based linkers, and the like. In certain embodiments, the linker is an enzyme-labile linker, such as an enzyme-labile linker, which is stable in the bloodstream but undergoes enzymatic cleavage upon internalization into target cells, for example, by lysosomal proteases (such as cathepsin or plasmin) in the lysosomes of target cells (e.g., cancer cells). The enzyme-labile linker includes, but is not limited to, a linker containing a peptide bond, for example, a dipeptide-based linker, for example, a valine-citrulline (VC) linker, for example, a maleimidocaproyl-valine-citrulline-p-aminobenzyl (MC-vc-PAB) linker, a valyl-alanyl-para-aminobenzyloxy (Val-Ala-PAB) linker, etc. Chemically unstable linkers, enzyme-labile linkers and non-cleavable linkers are known and are described in detail in, for example, Ducry & Stump (2010) Bioconjugate Chem. 21:5-13; Nolting, B. 2013) Methods Mol Biol. 1045:71-100; Tsusuchikama and An (2018) Protein & Cell 9 (1):33-46, etc.

[0107] Many strategies are available for linking an agent directly to an antibody or fusion protein, or indirectly through a linker. For example, an agent can be derivatized by covalently attaching a linker to the agent, the linker having a functional group capable of reacting with a "chemical handle" on the antibody or fusion protein. The functional group on the linker can be varied and selected based on compatibility with the chemical handle on the antibody or fusion protein. According to one embodiment, the chemical handle on the antibody or fusion protein is provided by incorporating a non-natural amino acid having a chemical handle into the antibody or fusion protein. The non-natural amino acids that find use for preparing the conjugates of the present disclosure include those having a functional group selected from azide, alkyne, alkene, aminooxy, hydrazine, aldehyde (e.g., formylglycine, e.g., Catalent Pharma Solutions' SMARTag™ technology), nitrone, nitrile oxide, cyclopropene, norbornene, isocyanide, aryl halide, and boronic acid functional groups. Unnatural amino acids that can be incorporated into the antibodies of the conjugates of the present disclosure (which unnatural amino acids can be selected to provide a desired functional group) are known and described, for example, in Maza et al. (2015) Bioconjug. Chem. 26(9):1884-9; Patterson et al. (2014) ACS Chem. Biol. 9:592-605; Adumeau et al. (2016) Mol. Imaging Biol. (2):153-65, etc. Such unnatural amino acids can be incorporated into the antibodies or fusion proteins, for example, via chemical synthesis or recombinant techniques, for example, using an orthogonal aminoacyl-tRNA synthetase-tRNA pair suitable for incorporating the unnatural amino acid during translation of the antibody or fusion protein in a host cell.

[0108] The functional group of the unnatural amino acid present in the antibody or fusion protein may be azide, alkyne, alkene, aminooxy, hydrazine, aldehyde, asaldehyde, nitrone, nitrile oxide, cyclopropene, norbornene, isocyanide, aryl halide, boronic acid, diazo, tetrazine, tetrazole, quadrocyclane, iodobenzene, or other suitable functional group, and the functional group on the linker is selected to react with the functional group of the unnatural amino acid (or vice versa). By way of example only, an azide-bearing unnatural amino acid (such as 5-azido-L-norvaline) may be incorporated into the antibody or fusion protein, and the linker portion of the linker-drug moiety may include an alkyne functional group such that the antibody or fusion protein and the linker-drug moiety are covalently conjugated by azide-alkyne cycloaddition. Conjugation may be carried out, for example, using a copper-catalyzed azide-alkyne cycloaddition reaction.

[0109] In certain embodiments, the chemical handle on the antibody or fusion protein does not include a non-natural amino acid. Antibodies that do not contain a non-natural amino acid can be conjugated to a drug by, for example, utilizing a nucleophilic functional group of the antibody or fusion protein (e.g., the N-terminal amine or the primary amine of lysine, or any other nucleophilic amino acid residue) as a nucleophile in a substitution reaction with a moiety that has a reactive leaving group or other electrophilic group. An example is to prepare a drug-linker moiety with an N-hydroxysuccinimidyl (NHS) ester and react the drug-linker moiety with the antibody or fusion protein under aqueous conditions at high pH (about 10) or in a polar organic solvent such as DMSO supplemented with a non-nucleophilic base such as N,N-diisopropylethylamine.

[0110] It will be understood that the particular approach for attaching the linker, agent and / or antibody or fusion protein to one another may vary depending on the particular linker, agent and / or antibody or fusion protein, as well as the functional groups selected and used to conjugate the various components to one another.

[0111] Methods for Producing Antibodies Using the information provided herein, the anti-VV A56 and anti-VV B5 antibodies and fusion proteins of the present disclosure can be prepared using standard techniques well known to those of skill in the art. For example, a nucleic acid sequence encoding the amino acid sequence of an antibody or fusion protein of the present disclosure can be used to express an antibody or fusion protein. The polypeptide sequences provided herein (see, for example, Tables 1, 3, 5-11) can be used to determine the appropriate nucleic acid sequence encoding an antibody or fusion protein, which can then be used to express one or more antibodies or fusion proteins specific for VV A56 or VV B5. Nucleic acid sequences can be optimized to reflect the particular codon "preferences" of various expression systems according to standard methods well known to those of skill in the art. Using the sequence information provided, nucleic acids can be synthesized according to several standard methods known to those of skill in the art.

[0112] Once the nucleic acid encoding the antibody of interest has been synthesized, it can be amplified and / or cloned according to standard methods. Molecular cloning techniques for achieving these ends are known in the art. A wide variety of cloning and in vitro amplification methods suitable for constructing recombinant nucleic acids are known to those skilled in the art and are the subject of numerous textbooks and laboratory manuals.

[0113] Expression of natural or synthetic nucleic acids encoding the antibodies and fusion proteins of the present disclosure can be achieved by operably linking the nucleic acid encoding the antibody or fusion protein to a promoter (either constitutive or inducible) and incorporating the construct into an expression vector to generate a recombinant expression vector. The vector may be suitable for replication and integration in prokaryotes, eukaryotes, or both. A typical cloning vector contains functionally appropriately oriented transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the nucleic acid encoding the antibody. The vector optionally contains a general expression cassette that contains at least one independent termination sequence, a sequence that allows the cassette to replicate in both eukaryotes and prokaryotes, such as those found in shuttle vectors, and a selection marker for both prokaryotic and eukaryotic systems.

[0114] To obtain high levels of expression of cloned nucleic acids, it is common to construct an expression plasmid that contains a strong promoter to direct transcription, a ribosome binding site for translation initiation, and a transcription / translation terminator, each operatively oriented relative to each other and to the protein-coding sequence. Examples of regulatory regions suitable for this purpose in E. coli include the promoter and operator regions of the E. coli tryptophan biosynthetic pathway, the leftward promoter of phage lambda (P L ), and the L-arabinose (araBAD) operon. It is also useful to include a selection marker in the DNA vector to be transformed in E. coli. Examples of such markers include genes specifying resistance to ampicillin, tetracycline, or chloramphenicol. Expression systems for expressing antibodies are available using, for example, E. coli, Bacillus species, and Salmonella. The E. coli system may also be used.

[0115] Antibody genes can also be subcloned into expression vectors that allow for the addition of tags (e.g., FLAG, hexahistidine, etc.) at the C-terminus or N-terminus of the antibody (e.g., IgG, Fab, scFv, etc.). Methods for transfecting and expressing genes in mammalian cells are known in the art. Transducing a cell with a nucleic acid can include, for example, incubating a lipid microparticle containing the nucleic acid with the cell, or incubating a viral vector containing the nucleic acid with a cell within the host range of the vector. The culture of cells used in the present disclosure, including cell lines and cultured cells from tissues (e.g., tumors) or blood samples, is well known in the art.

[0116] Once a nucleic acid encoding an antibody of interest has been isolated and cloned, it can be expressed in a variety of recombinantly engineered cells known to those of skill in the art, including bacterial, yeast, filamentous fungal, insect (e.g., using baculovirus vectors), and mammalian cells.

[0117] Isolation and purification of the subject antibodies can be accomplished according to methods known in the art. For example, the protein can be isolated from the lysate of cells genetically engineered to express the protein constitutively and / or upon induction, or from a synthesis reaction mixture, by immunoaffinity purification (or precipitation using protein L or A), washing to remove non-specifically bound materials, and elution of specifically bound antibodies. The isolated antibodies can be further purified by dialysis and other methods commonly used in protein purification methods. In one embodiment, the antibodies can be isolated using metal chelate chromatography methods. The antibodies of the present disclosure can include certain modifications to facilitate isolation, as discussed above.

[0118] The antibody may be prepared in a substantially pure or isolated form (e.g., free of other polypeptides). The protein may be present in a composition that is enriched for the polypeptide relative to other components that may be present (e.g., other polypeptides or other host cell components). A purified antibody may be provided in a composition in which the antibody is substantially free of other expressed proteins, e.g., less than 90%, usually less than 60%, and more usually less than 50% of the composition is made up of other expressed proteins.

[0119] Antibodies produced by prokaryotic cells may require exposure to chaotropic agents for proper folding. For example, during purification from E. coli, the expressed protein can be optionally denatured and then renatured. This can be accomplished, for example, by solubilizing bacterially produced antibodies in a chaotropic agent such as guanidine HCl. The antibody is then renatured by either slow dialysis or gel filtration. Alternatively, the nucleic acid encoding the antibody can be operably linked to a secretion signal sequence, such as pelB, so that the antibody is secreted into the periplasm in a correctly folded form.

[0120] The present disclosure also provides cells that produce the antibodies of the present disclosure, and suitable cells include eukaryotic cells, e.g., mammalian cells. The cells can be hybrid cells or "hybridomas" that can replicate antibodies (e.g., monoclonal antibodies such as IgG) in vitro. For example, the present disclosure provides recombinant host cells (also referred to herein as "genetically modified host cells") that are genetically modified with one or more nucleic acids that include nucleotide sequences encoding the heavy and / or light chains of the antibodies of the present disclosure.

[0121] Techniques for making recombinant DNA versions of antigen-binding regions of antibody molecules that bypass hybridoma generation are also contemplated herein. DNA is cloned into bacterial (such as bacteriophage), yeast (e.g., Saccharomyces or Pichia), insect or mammalian expression systems, etc. One suitable technique uses a bacteriophage lambda vector system with a leader sequence that allows the expressed antibody (e.g., Fab or scFv) to be translocated to the periplasmic space (between the bacterial cell membrane and the cell wall) or secreted. For those that bind to the antigen of interest, large numbers of functional fragments (e.g., Fab or scFv) can be rapidly generated.

[0122] Antibodies that specifically bind to VV A56 and VV B5 can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, phage display technology, selected lymphocyte antibody method (SLAM) (1), or a combination thereof. For example, antibodies can be generated and isolated using methods of phage display. Phage display is used for high-throughput screening of protein interactions. Phages can be utilized to display antigen-binding domains expressed from repertoire or combinatorial antibody libraries (e.g., human or mouse). Phages expressing antigen-binding domains that bind to VV A56 or VV B5 can be selected or identified using VV A56 or VV B5, for example, using labeled VV A56 or VV B5 bound or captured to a solid surface or bead. The phages used in these methods are typically filamentous phages containing fd and M13 binding domains expressed from the phage with Fab, Fv (individual Fv regions from light or heavy chains) or disulfide-stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII proteins. The production of high affinity human antibodies by chain shuffling is known, as is combinatorial infection and in vivo recombination as a strategy for constructing large phage libraries. In another embodiment, ribosome display can be used to replace bacteriophage as a display platform. Cell surface libraries can be screened for antibodies. Such procedures provide an alternative to traditional hybridoma techniques for the isolation and subsequent cloning of monoclonal antibodies.

[0123] After phage selection, the antibody coding region from the phage can be isolated and used to generate complete antibodies, including human antibodies or any desired antigen-binding fragment, and can be expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria. For example, techniques for recombinantly producing Fv, scFv, Fab, F(ab')2, and Fab' fragments can be used using methods known in the art.

[0124] Nucleic acids, expression vectors and cells In view of the sections above regarding methods of producing the antibodies and fusion proteins of the disclosure, it will be understood that the disclosure also provides nucleic acids, expression vectors and cells.

[0125] In certain embodiments, provided is a variable heavy chain (VH) antibody or fusion protein of the disclosure, including any of the anti-VV A56 and anti-VV B5 antibodies of the disclosure, e.g., any of the antibodies as described above. H ) polypeptide, variable light chain (V L ) polypeptide, or both. According to some embodiments, the antibody is a single chain antibody (e.g., scFv), and the nucleic acid encodes the single chain antibody.

[0126] According to some embodiments, the variable heavy chain (VH) of the anti-VV A56 antibody A047 / A057, A049 / A059 / A056, A050 or A054 H ) polypeptide, variable light chain (V L ) polypeptides, or both. Examples of such nucleotide sequences are shown in Table 12 below. Sequences encoding the framework regions and CDRs are shown in upper and lower case, respectively. [Table 12-1] [Table 12-2]

[0127] According to some embodiments, the variable heavy chain (VH) of the anti-VV B5 antibody A048 / A058 / A073 or A051 H ) polypeptide, variable light chain (V L Nucleic acids encoding the ribozyme, ribozyme, ribozyme, ribozyme-binding domain (Rds), ... [Table 13]

[0128] According to some embodiments, provided is a CAR of the disclosure, e.g., a VV of an anti-VV A56 or anti-VV B5 antibody of the disclosure. H Polypeptides and V L It is a nucleic acid that encodes a CAR comprising a single chain antibody comprising a polypeptide, a transmembrane domain, and an intracellular signaling domain. Examples of such single chain antibodies, transmembrane domains, and intracellular signaling domains are described in detail above.

[0129] An exemplary nucleotide sequence encoding A56-CAR-01 described herein is set forth in SEQ ID NO: 155. An exemplary nucleotide sequence encoding A56-CAR-02 described herein is set forth in SEQ ID NO: 156. An exemplary nucleotide sequence encoding A56-CAR-05 described herein is set forth in SEQ ID NO: 157. An exemplary nucleotide sequence encoding A56-CAR-06 described herein is set forth in SEQ ID NO: 158. An exemplary nucleotide sequence encoding A56-CAR-07 described herein is set forth in SEQ ID NO: 159. An exemplary nucleotide sequence encoding A56-CAR-08 described herein is set forth in SEQ ID NO: 160. An exemplary nucleotide sequence encoding A56-CAR-010 described herein is set forth in SEQ ID NO: 161. An exemplary nucleotide sequence encoding A56-CAR-020 described herein is set forth in SEQ ID NO: 162. An exemplary nucleotide sequence encoding A56-CAR-021 described herein is set forth in SEQ ID NO: 163. An exemplary nucleotide sequence encoding A56-CAR-027 described herein is set forth in SEQ ID NO: 164. An exemplary nucleotide sequence encoding A56-CAR-028 described herein is set forth in SEQ ID NO: 165. An exemplary nucleotide sequence encoding A56-CAR-029 described herein is set forth in SEQ ID NO: 166. An exemplary nucleotide sequence encoding A56-CAR-030 described herein is set forth in SEQ ID NO: 167. An exemplary nucleotide sequence encoding B5-CAR-03 described herein is set forth in SEQ ID NO: 168. An exemplary nucleotide sequence encoding B5-CAR-04 described herein is set forth in SEQ ID NO: 169. An exemplary nucleotide sequence encoding B5-CAR-011 described herein is set forth in SEQ ID NO: 170. An exemplary nucleotide sequence encoding B5-CAR-013 described herein is set forth in SEQ ID NO: 171. An exemplary nucleotide sequence encoding B5-CAR-014 described herein is set forth in SEQ ID NO: 172.An exemplary nucleotide sequence encoding B5-CAR-016 described herein is set forth in SEQ ID NO: 173. An exemplary nucleotide sequence encoding B5-CAR-019 described herein is set forth in SEQ ID NO: 174. An exemplary nucleotide sequence encoding B5-CAR-022 described herein is set forth in SEQ ID NO: 175. Examples of nucleotide sequences encoding murine CARs shown in Table 11 are set forth in SEQ ID NOs: 176-179.

[0130] Also provided is an expression vector comprising any of the nucleic acids of the present disclosure. Expression of natural or synthetic nucleic acids encoding the antibodies and fusion proteins of the present disclosure can be achieved by operably linking the nucleic acid encoding the antibody or fusion protein to a promoter (either constitutive or inducible) and incorporating the construct into an expression vector to generate a recombinant expression vector. The vector may be suitable for replication and integration in prokaryotes, eukaryotes, or both. A typical cloning vector contains functionally appropriately oriented transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the nucleic acid encoding the antibody. The vector optionally contains a general expression cassette that contains at least one independent termination sequence, a sequence that allows the cassette to replicate in both eukaryotes and prokaryotes, such as those found in shuttle vectors, and a selection marker for both prokaryotic and eukaryotic systems.

[0131] Also provided are cells comprising any of the nucleic acids and / or expression vectors of the present disclosure. According to some embodiments, the cells of the present disclosure comprise the V H V for Polypeptides and Antibodies L In certain such embodiments, the antibody is a single chain antibody (e.g., scFv) and the nucleic acid encodes the single chain antibody. According to some embodiments, provided is a variable heavy chain (VH) of the antibody of the present disclosure. H a first nucleic acid encoding a variable light chain (V Land a second nucleic acid encoding the polypeptide. In certain embodiments, for example, the cell comprises a first expression vector comprising the first nucleic acid and a second expression vector comprising the second nucleic acid.

[0132] Also provided is a method of making an antibody or fusion protein of the present disclosure, comprising culturing a cell of the present disclosure under suitable conditions for the cell to express the antibody or fusion protein, whereby the antibody or fusion protein is produced. The conditions for culturing the cells such that the antibody or fusion protein is expressed may vary. Such conditions may include culturing the cells in a suitable container (e.g., a cell culture plate or well thereof), in a suitable medium (e.g., cell culture medium such as DMEM, RPMI, MEM, IMDM, DMEM / F-12, etc.), at a suitable temperature (e.g., 32°C to 42°C (e.g., 37°C)) and pH (e.g., pH 7.0 to 7.7 (e.g., pH 7.4)), in an environment having a suitable percentage of CO2, e.g., 3% to 10% (e.g., 5%).

[0133] Chimeric antigen receptors that bind to oncolytic viral antigens In addition to the chimeric antigen receptor (CAR) of the present disclosure described in the fusion protein section above, aspects of the present disclosure further include anti-oncolytic virus antigen CARs. In certain embodiments, such CARs include an antigen binding domain that specifically binds to an oncolytic virus (OV) antigen, a transmembrane domain, and an intracellular signaling domain. Such CARs find various uses. For example, it will be understood that, with the benefit of the present disclosure, such CARs find use in a method that includes administering a pharmaceutical composition that includes an anti-OV antigen CAR to an individual with cancer, whereby the cancer cells in the individual are infected with OV and express OV antigens on their surface, thereby targeting the CAR to the cancer cells to treat the individual's cancer.

[0134] As used herein, an "oncolytic viral antigen" or "OV antigen" is an antigen encoded by the genome of an oncolytic virus (OV). In some embodiments, the OV antigen is a native OV antigen, where "native" means that the antigen is a protein encoded by the wild-type genome of OV.

[0135] In some embodiments, the OV antigen is an antigen encoded by the wild-type genome of a virus of the family Poxviridae, Herpesviridae, Adenoviridae, Paramyxoviridae, Rhabdoviridae, Reoviridae, Picornaviridae, Parvoviridae, or Coronaviridae. Such OV antigens may be native or heterologous to the OV.

[0136] In certain embodiments, the OV antigen is an antigen encoded by the wild-type genome of a virus from a family set forth in Table 14 below, which further includes non-limiting examples of viral species / strains within such families, as well as non-limiting examples of such antigens. [Table 14]

[0137] According to some embodiments, the OV antigen is selected from the group consisting of Amsacta moorei entomopoxvirus, Chironomus luridus entomopoxvirus, fowlpox virus, Melolontha melolontha entomopoxvirus, Molluscum contagiosum virus, mule deerpox virus, myxoma virus, Nile crocodilepox virus, orf virus, sheeppox virus, swinepox virus, vaccinia virus, Yaba monkey tumor virus, Yokapox virus, Acrobasis zelleri entomopoxvirus, Adoxophyes honmai entomopoxvirus, Aedes aegypti entomopoxvirus, Anomala cuprea entomopoxvirus, Aphodius tasmaniae entomopoxvirus, Arphia conspersa entomopoxvirus, bovine papular stomatitis virus, camelpox virus, Camptochironomus tentans entomopoxvirus, canarypox virus, Chironomus attenuatus entomopoxvirus, Chironomus plumosus entomopoxvirus, Choristoneura biennis entomopoxvirus, Choristoneura conflicta entomopoxvirus, Choristoneura diversuma entomopoxvirus, Choristoneura fumiferana entomopoxvirus, Choristoneura rosaceana entomopoxvirus, Chorizagrotis auxiliaris entomopoxvirus, cowpox virus, Demodema bonariensis entomopoxvirus, Dermolepida albohirtum entomopoxvirus, Diachasmimorpha entomopoxvirus, ectromelia virus, Figulus sublaevis entomopoxvirus, Geotrupes sylvaticus entomopoxvirus, goatpox virus, Goeldichironomusholoprasinus entomopoxvirus, Hare fibroma virus, Heliothis armigera entomopoxvirus, Juncopox virus, Locusta migratoria entomopoxvirus, Lumpy skin disease virus, Melanoplus sanguinipes entomopoxvirus, Monkeypox virus, Mynahpox virus, Mythimna separata entomopoxvirus, Oedaleus senegalensis entomopoxvirus, Operophtera brumata entomopoxvirus, New Zealand red deer Parapoxvirus, Pigeonpox virus, Pseudocowpox virus, Psittacinepox virus, Pteropox virus, Quailpox virus, Rabbit fibroma virus, Raccoonpox virus, Schistocerca The OV antigen is an antigen encoded by the wild-type genome of a virus of the family Poxviridae selected from: gregaria entomopoxvirus, skunkpoxvirus, sparrowpoxvirus, squirrel fibromavirus, squirrelpoxvirus, starlingpoxvirus, variolavirus, Tanapoxvirus, Greater Hadashian taterapoxvirus, turkeypoxvirus, variolavirus, and volepoxvirus. Such OV antigens may be native or heterologous to the OV.

[0138] In certain embodiments, the OV antigen is avian alphaherpesvirus type 1, parrot alphaherpesvirus type 1, avian alphaherpesvirus type 2, goose / duck alphaherpesvirus type 1, pigeon alphaherpesvirus type 1, avian alphaherpesvirus type 3, turkey alphaherpesvirus type 1, sea turtle alphaherpesvirus type 5, human alphaherpesvirus type 1, spider monkey alphaherpesvirus type 1, bovine alphaherpesvirus type 2, long-tailed monkey alphaherpesvirus type 2, human alphaherpesvirus type 1, orthopedic alphaherpesvirus type 2. betaherpesvirus 2, rabbit alphaherpesvirus 4, macaque alphaherpesvirus 1, kangaroo alphaherpesvirus 1, kangaroo alphaherpesvirus 2, panine alphaherpesvirus 3, baboon alphaherpesvirus 2, squirrel monkey alphaherpesvirus 1, human betaherpesvirus 5, owl monkey betaherpesvirus 1, capuchin monkey betaherpesvirus 1, African green monkey betaherpesvirus 5, macaque betaherpesvirus 1, leopard monkey betaherpesvirus 1, lemur ... betaherpesvirus 3, Panine betaherpesvirus 2, Baboon betaherpesvirus 3, Squirrel monkey betaherpesvirus 4, Murine betaherpesvirus 1, Murine betaherpesvirus 2, Murine betaherpesvirus 8, Human betaherpesvirus 6A, Human betaherpesvirus 6B, Human betaherpesvirus 7, Elephant betaherpesvirus 1, Human gammaherpesvirus 4, Marmoset gammaherpesvirus 3, Long-tailed macaque gammaherpesvirus 14, Goat Lyra gammaherpesvirus type 1, Macacain gammaherpesvirus type 4, Panin gammaherpesvirus type 1, Baboon gammaherpesvirus type 1, Orangutan gammaherpesvirus type 2, Squirrel monkey gammaherpesvirus type 2, Spider monkey gammaherpesvirus type 2, Spider monkey gammaherpesvirus type 3, Bovine gammaherpesvirus type 4, Cricetidae gammaherpesvirus type 2, Human gammaherpesvirus type 8, Macacain gammaherpesvirus type 5, Murine gammaherpesvirus type 4, Murine gammaherpesvirus type 7,and antigens encoded by the wild-type genome of a virus of the family Herpesviridae selected from hartebeest gammaherpesvirus type 1, hartebeest gammaherpesvirus type 2, bovine gammaherpesvirus type 6, caprine gammaherpesvirus type 2, bluebuck gammaherpesvirus type 1, ovine gammaherpesvirus type 2, porcine gammaherpesvirus type 3, porcine gammaherpesvirus type 4, porcine gammaherpesvirus type 5, equine gammaherpesvirus type 2, equine gammaherpesvirus type 5, and weasel gammaherpesvirus type 1. Such OV antigens may be native or heterologous to the OV.

[0139] According to some embodiments, the OV antigen is chicken avian adenovirus A, frog siadenovirus A, human mastadenovirus C, ovine tadenovirus D, sturgeon ichtadenovirus A, bat mastadenovirus A, bat mastadenovirus B, bat mastadenovirus C, bat mastadenovirus D, bat mastadenovirus E, bat mastadenovirus F, bat mastadenovirus G, bovine tadenovirus D, bovine mastadenovirus A, bovine Mast adenovirus B, Bovine mast adenovirus C, Dog mast adenovirus A, Deer atadenovirus A, Deer mast adenovirus B, Dolphin mast adenovirus A, Dolphin mast adenovirus B, Duck atadenovirus A, Duck aviadenovirus B, Horse mast adenovirus A, Horse mast adenovirus B, Peregrine falcon aviadenovirus A, Chicken aviadenovirus B, Chicken aviadenovirus C, Chicken aviadenovirus D, Chicken aviadenovirus E, Goose aviadenovirus A, Great tit Siadenovirus A, Human mastadenovirus A, Human mastadenovirus B, Human mastadenovirus D, Human mastadenovirus E, Human mastadenovirus F, Human mastadenovirus G, Lizard atadenovirus A, Murine mastadenovirus A, Murine mastadenovirus B, Murine mastadenovirus C, Ovine mastadenovirus A, Ovine mastadenovirus B, Penguin siadenovirus A, Pigeon aviadenovirus A, Pigeon aviadenovirus B, Capuchin mastadenovirus A, Pig mastadenovirus Virus A, porcine mastadenovirus B, porcine mastadenovirus C, opossum atadenovirus A, parrot atadenovirus A, parrot aviadenovirus B, raptor siaadenovirus A, sea lion mastadenovirus A, simian mastadenovirus A, simian mastadenovirus B, simian mastadenovirus C, simian mastadenovirus D, simian mastadenovirus E, simian mastadenovirus F, simian mastadenovirus G, simian mastadenovirus H, simian mastadenovirus I, skua siaadenovirus A,An antigen encoded by the wild-type genome of a virus of the Adenoviridae family selected from Skunk Bear Adenovirus A, Snake Adenovirus A, Squirrel Adenovirus A, Tree Shrew Adenovirus A, Turkey Aviadenovirus B, Turkey Aviadenovirus C, Turkey Aviadenovirus D, and Turkey Siadenovirus A. Such OV antigens can be native or heterologous to the OV.

[0140] In certain embodiments, the OV antigen is Triabularavirus 1, Hendra Henipavirus, Measles Morbillivirus, Mumps Rubulavirus, Murine Respirovirus, Reptile Ferulavirus, Salmon Aquaparamyxovirus, Achimota Rubulavirus 1, Achimota Rubulavirus 2, Avian Triabularavirus 10, Avian Triabularavirus 11, Avian Triabularavirus 12, Avian Triabularavirus 13, Avian Triabularavirus 14, Avian Triabularavirus 15, Avian Triabularavirus 16, Avian Triabularavirus 17, Avian Triabularavirus 19, Avian Triabularavirus 20, Avian Triabularavirus 21, Avian Triabularavirus 22, Avian Triabularavirus 23, Avian Triabularavirus 24, Avian Triabularavirus 25, Avian Triabularavirus 26, Avian Triabularavirus 27, Avian Triabularavirus 28, Avian Triabularavirus 29, Avian Triabularavirus 30, Avian Triabularavirus 31, Avian Triabularavirus 32, Avian Triabularavirus 33, Avian Triabularavirus 34, Avian Triabularavirus 35, Avian Triabularavirus 36, Avian Triabularavirus 37, Avian Triabularavirus 38, Avian Triabularavirus 39, Avian Triabularavirus 40, Avian Triabularavirus 41, Avian Triabularavirus 42, Avian Triabularavirus 43, Avian Triabularavirus 44, Avian Triabularavirus 45, Avian Triabularavirus 46, Avian Triabularavirus 47, Avian Triabularavirus 48, Avian Triabularavirus 4 rus 16, avian triabulus virus 17, avian triabulus virus 18, avian triabulus virus 19, avian triabulus virus 2, triabulus virus 3, triabulus virus 4, triabulus virus 5, triabulus virus 6, triabulus virus 7, triabulus virus 8, triabulus virus 9, bat mumps rubravirus, bovine respirovirus 3, canine morbillivirus, cedar henipavirus, cetacean morbillivirus, feline morbillivirus, Ghanaian free-tailed bat and an antigen encoded by a wild-type genome of a virus of the family Paramyxoviridae selected from among bat henipavirus, human respirovirus type 1, human respirovirus type 3, human rubulavirus type 2, human rubulavirus type 4, mammalian rubulavirus type 5, Mapuera rubulavirus, Menangle rubulavirus, Mojiang henipavirus, Nipah henipavirus, seal morbillivirus, porcine respirovirus type 1, butalubravirus, rinderpest morbillivirus, monkey rubulavirus, small ruminant morbillivirus, Susoga rubulavirus, Teviot rubulavirus, Tioman rubulavirus, Tuhoko rubulavirus type 1, Tuhoko rubulavirus type 2, and Tuhoko rubulavirus type 3. Such OV antigens may be native or heterologous to the OV.

[0141] According to some embodiments, the OV antigen is selected from the group consisting of bovine fever ephemerovirus, koisprivivirus, Curionopolis curiovirus, Drosophila melanogaster sigmavirus, Durham tupavirus, Flanders hapavirus, Indiana vesiculovirus, Le Dantec redantevirus, lettuce big vein associated varicosavirus, Lettuce necrotic yellows cytorhabdovirus, Niakha slipvirus, orchid necrotic spot virus, Perch perhabdovirus, Potato yellow dwarf nucleorhabdovirus, Puerto Almendras almendra virus, Rabies lyssavirus, Puerto Almendras virus, Rabies virus, Salmonid nobilhabdovirus, Tibrogargantibrovirus, Adelaide River ephemerovirus, Alagoas vesiculovirus, Alfalfa dwarf cytorhabdovirus, Almpiwar thrips virus, American bat vesiculovirus, Eel perhabdovirus, Aravan lyssavirus, Arboretum almendra virus, Australian bat sassavirus, Balsa almendra virus, Barley yellow striate mosaic cytorhabdovirus, Barleur redante virus, Bas-Congo tibrovirus, Beatrice hill virus, Hill tiblovirus, Berrimah ephemerovirus, Bokeloh bat lyssavirus, Broccoli necrotic yellow cytorhabdovirus, Carajas vesiculovirus, Chaco thripvirus, Chandipura vesiculovirus, Coastal Plains tiblovirus, Cocal vesiculovirus, Coffee ringspot dicolhavirus,dichorhavirus, Colocasia bobensis-associated cytorhabdovirus, Coot Bay almendravirus, Datura yellow vein nucleorhabdovirus, Drosophila affinis sigmavirus, Drosophila ananassae sigmavirus, Drosophila immigrans sigmavirus, Drosophila obscura sigmavirus, Drosophila tristis sigmavirus, Duvenhage lyssavirus, Eggplant mottled dwarf nucleorhabdovirus, Ekpoma 1 tibulovirus, Ekpoma 2 tibulovirus, European bat 1 lyssavirus, European bat 2 lyssavirus, Festuca leaf streak cytorhabdovirus, Fikirini redantevirus, Fukuoka redantevirus, Gannoruwa bat lyssavirus, Gray Lodge Lodge hapavirus, Hart Park hapavirus, Hirame novirhabdovirus, Ikoma lyssavirus, Iriri curiovirus, Irkut lyssavirus, Isfahan vesiculovirus, Itacaiunas curiovirus, Joinjakaka hapavirus, Jurona vesiculovirus, Kamese hapavirus, Kanyawara redantevirus, Kern Canyon Canyon Redantevirus, Keuralib Redantevirus, Khujand Lyssavirus, Kimberley Ephemerovirus, Klamath Tupavirus, Kolente Redantevirus, Koolpinyah Ephemerovirus, Kotonkan Ephemerovirus, Kumasi Redantevirus, La JollaJoya hapavirus, Lagos bat lyssavirus, Landjia hapavirus, Lettuce yellow mottle cytorhabdovirus, Lleida bat lyssavirus, Maize Iranian mosaic nucleorhabdovirus, Maize fine streak nucleorhabdovirus, Maize mosaic nucleorhabdovirus, Malpais Spring vesiculovirus, Manitoba hapavirus, Maraba vesiculovirus, Marco hapavirus, Mokola lyssavirus, Morreton vesiculovirus, Mosqueiro hapavirus, Mossuril hapavirus, Mount Elgon bat redantevirus, Moussa virus, Muscina stabulans sigmavirus, New Jersey vesiculovirus, Ngaingan hapavirus, Nishimuro redantevirus, Nkolbisson redantevirus, Wheat northern mosaic cytorhabdovirus, Obodhiang ephemerovirus, Oita redantevirus, Ord River hapavirus, Parry Creek hapavirus, Perinet vesiculovirus, Pike fry sprivivirus, Pirivesiculovirus, Piscine nobilhabdovirus, Radi vesiculovirus, Rice yellow leaf nucleorhabdovirus, Rio Chico almendravirus, Rochambeau curiovirus, Sea trout perhabdovirus, Sena madureiraThe OV antigens are antigens encoded by the wild-type genome of a virus of the family Rhabdoviridae selected from Madureira slipvirus, Shimoni bat lyssavirus, Snakehead novirhabdovirus, Sonchus cytorhabdovirus 1, Sonchus yellownet nucleorhabdovirus, Cornus yellow vein nucleorhabdovirus, Strawberry crinkle cytorhabdovirus, Sweetwater branch tibrovirus, Taro vein chlorosis nucleorhabdovirus, Tupaitupavirus, West Caucasian bat lyssavirus, Wheat American striatal mosaic cytorhabdovirus, Wongabel hapavirus, Wuhan redantevirus, Yata ephemerovirus, Yongjia redantevirus, and Yug Bogdanovac vesiculovirus. Such OV antigens may be native or heterologous to the OV.

[0142] In certain embodiments, the OV antigen is selected from the group consisting of Aedes pseudoscutellaris reovirus, Aquareovirus type A, Banna virus, Bluetongue virus, Colorado tick fever virus, Cypovirus 1, Eriocheir sinensis reovirus, Fiji disease virus, Idnoreovirus 1, Mammalian orthoreovirus, Micromonas pusilla reovirus, Mycoreovirus 1, Rice ragged stunt virus, Rotavirus A, Wound tumor virus, African horse sickness virus, Aquareovirus B, Aquareovirus C, Aquareovirus D, Aquareovirus E, Aquareovirus F, Aquareovirus G, Avian orthoreovirus, Baboon orthoreovirus, Changuinola virus, Chenuda virus, Chobar Golgi virus, Colipartavirus, Cypovirus 10, Cypovirus 11, Cypovirus 12, Cypovirus 13, Cypovirus 14, Cypovirus 15 , Cypovirus 16, Cypovirus 2, Cypovirus 3, Cypovirus 4, Cypovirus 5, Cypovirus 6, Cypovirus 7, Cypovirus 8, Cypovirus 9, Echinochloa ragged stunt virus, Epizootic hemorrhagic disease virus, Equine encephalopathy virus, Euvenangivirus, Eyach virus, Garlic dwarf virus, Great Island virus, Idonoreovirus 2, Idonoreovirus 3, Idonoreovirus 4, Idonoreovirus 5, Yeri virus, Kadipirovirus, Lebombovirus, Liaoning virus, ning virus, Mahlapitsi orthoreovirus, Maize rough dwarf virus, Mal de Rio Cuarto virus, Mycoreovirus 2, Mycoreovirus 3, Nelson Bay orthoreovirus, Nilaparvata lugens reovirus, Oat sterile dwarf virus, Orungo virus, Palyam virus, Pangola stunt virus,stunt virus, Peruvian horse sickness virus, fish orthoreovirus, reptile orthoreovirus, rice black-streaked dwarf virus, rice dwarf virus, Rice gall dwarf virus, Rotavirus B, Rotavirus C, Rotavirus D, Rotavirus E, Rotavirus F, Rotavirus G, Rotavirus H, Rotavirus I, Southern rice black-streaked dwarf virus, St. Croix River virus, Umatilla virus, Wad Medani virus, Wallal virus, Warrego virus, Wongorr virus, and Yunnan orbivirus. Such OV antigens may be native or heterologous to the OV.

[0143] According to some embodiments, the OV antigen is selected from the group consisting of Aarivirus A, Aichivirus A, Ampivirus A, Akuamavirus A, Abihepatovirus A, Abyssivirus A, Bopivirus A, Kadisivirus A, Cardiovirus A, Cosavirus A, Kurohivirus B, Enterovirus C, Erbovirus A, Foot and Mouth Disease Virus, Galivirus A, Harukavirus A, Hepatovirus A, Hunnivirus A, Kunsaguivirus A, Limnipivirus A, Meghrivirus A, Mishivirus A, Mosavirus A, Orivirus A, Oshivirus B, Oshivirus C, Oshivirus D, Oshivirus E, Oshivirus F, Oshivirus I ... Virus A, Parechovirus A, Pasivirus A, Passerivirus A, Potamipivirus A, Rabovirus A, Rosavirus A, Sacobuvirus A, Sarivirus A, Sapelovirus A, Senecavirus A, Shambavirus A, Citinivirus A, Teschovirus A, Torchivirus A, Tremovirus A, Aichivirus B, Aichivirus C, Aichivirus D, Aichivirus E, Aichivirus F, Torisapelovirus, Abyssivirus B, Abyssivirus C, U Bovine rhinitis A virus, Bovine rhinitis B virus, Cardiovirus B, Cardiovirus C, Cosavirus B, Cosavirus D, Cosavirus E, Cosavirus F, Kurohivirus A, Enterovirus A, Enterovirus B, Enterovirus D, Enterovirus E, Enterovirus F, Enterovirus G, Enterovirus H, Enterovirus I, Enterovirus J, Enterovirus K, Enterovirus L, Equine rhinitis A virus, Hepatovirus B, Hepatovirus C, Hepatovirus D, Hepatovirus E, Hepatovirus and antigens encoded by the wild-type genome of a virus of the Picornaviridae family selected from virus F, hepatovirus G, hepatovirus H, hepatovirus I, Kunsaguivirus B, Kunsaguivirus C, Limnipivirus B, Limnipivirus C, Meghrivirus B, Meghrivirus C, Meghrivirus D, Meghrivirus E, Micivirus B, Micivirus C, Parechovirus B, Parechovirus C, Parechovirus D, Rhinovirus A, Rhinovirus B, Rhinovirus C, and Sapelovirus B. Such OV antigens can be native or heterologous to the OV.

[0144] In certain embodiments, the OV antigen is Adeno-associated Dependoparvovirus A, Carnivore Amdoparvovirus Type 1, Decapod Hepandensovirus Type 1, Decapod Penstildensovirus Type 1, Dipteran Brevidensovirus Type 1, Galliform Abeparvovirus Type 1, Lepidoptera Ambidensovirus Type 1, Lepidoptera Iteradensovirus Type 1, Primate Erythroparvovirus Type 1, Primate Tetraparvovirus Type 1, Rodent Protoparvovirus Type 1, Ungulate Bocaparvovirus Type 1, Ungulate Copiparvovirus Type 1, Adeno-associated Dependoparvovirus Type 1, Virus type B, Anseriformes dependenvirus type 1, Asteroid ambidensovirus type 1, Avian dependenvovirus type 1, Blattodean ambidensovirus type 1, Blattodean ambidensovirus type 2, Carnivore amdoparvovirus type 2, Carnivore amdoparvovirus type 3, Carnivore amdoparvovirus type 4, Carnivore bocaparvovirus type 1, Carnivore bocaparvovirus type 2, Carnivore bocaparvovirus type 3, Carnivore bocapar ...1, Carnivore bocaparvovirus type 2, Carnivore bocaparvovirus type 3, Carnivore bocaparvovirus type 4, Carnivore bocaparvovirus type 1, Carnivore bocaparvovirus type 1, Carnivore bocaparvovirus type 2, Carnivore bocaparvovirus type 3, Carnivore bocaparvovirus type 4, Carnivore bocaparvovirus type 1, Carnivore bo Bovirus type 5, Carnivore bocaparvovirus type 6, Carnivore protoparvovirus type 1, Chiropteran bocaparvovirus type 1, Chiropteran bocaparvovirus type 2, Chiropteran bocaparvovirus type 3, Chiropteran bocaparvovirus type 4, Chiropteran dependoparvovirus type 1, Chiropteran protoparvovirus type 1, Chiropteran tetraparvovirus type 1, Decapod ambidensovirus type 1, Decapod ambidensovirus type 1, Dipteran ambidensovirus type 1, Dipteran brevidensovirus type 1, Euonymus protoparvovirus type 1, Hemiptera ambidensovirus type 1 type, Hemiptera ambidensovirus type 2, Hemiptera ambidensovirus type 3, Hymenoptera ambidensovirus type 1, Orthoptera densovirus bocaparvovirus type 1, Pinnipedia bocaparvovirus type 1, Pinnipedia bocaparvovirus type 2, Pinnipedia densoparvovirus type 1, Primate bocaparvovirus type 1, Primate bocaparvovirus type 2, Primate erythroparvovirus type 2, Primate erythroparvovirus type 3, Primate erythroparvovirus type 4, Primate protoparvovirus type 1, Primate protoparvovirus type 2, Primate protoparvovirus type 3,An antigen encoded by a wild-type genome of a virus of the family Parvoviridae selected from Rodent Erythroparvovirus Type 1, Rodent Protoparvovirus Type 2, Rodent Protoparvovirus Type 3, Squamate Dependoparvovirus Type 1, Ungulate Bocaparvovirus Type 2, Ungulate Bocaparvovirus Type 3, Ungulate Bocaparvovirus Type 4, Ungulate Bocaparvovirus Type 5, Ungulate Bocaparvovirus Type 6, Ungulate Copiparvovirus Type 2, Ungulate Erythroparvovirus Type 1, Ungulate Protoparvovirus Type 1, Ungulate Protoparvovirus Type 2, Ungulate Tetraparvovirus Type 3, and Ungulate Tetraparvovirus Type 4. Such OV antigens may be native or heterologous to the OV.

[0145] According to some embodiments, the OV antigen is selected from the group consisting of Alphacoronavirus type 1, Avian coronavirus, Bulbul coronavirus HKU11, Equine Torovirus, Murine coronavirus, White bream virus, Ball python nidovirus type 1, Bat coronavirus CDPHE15, Bat coronavirus HKU10, Dolphin coronavirus SW1, Betacoronavirus type 1, Bovine nidovirus type 1, Bovine Torovirus, King salmon nidovirus type 1, Common moorhen coronavirus HKU21, Coronavirus HKU15, Fathead minnow nidovirus type 1, Hedgehog coronavirus type 1, Human coronavirus 229E, Human coronavirus HKU1, Human coronavirus NL63, Human Torovirus, Middle East respiratory syndrome-associated coronavirus, Miniopterus bat coronavirus type 1, Miniopterus bat coronavirus HKU8, Mink coronavirus type 1, Munia coronavirus HKU13, Night heron coronavirus HKU14, Night heron coronavirus HKU15, Night heron coronavirus HKU16, Night heron coronavirus HKU17, Night heron coronavirus HKU18, Night heron coronavirus HKU19, Night heron coronavirus HKU20, Night heron coronavirus HKU21, Night heron coronavirus HKU22, Night heron coronavirus HKU23, Night heron coronavirus HKU24, Night heron coronavirus HKU25, Night heron coronavirus HKU26, Night heron coronavirus HKU27, Night heron coronavirus HKU28, Night heron coronavirus HKU29, Night heron coronavirus HKU30, Night heron coronavirus HKU31, Night heron coronavirus HKU32, Night heron coronavirus HKU33, Night heron coronavirus HKU34, Night heron coronavirus HKU35, Night heron coronavirus HKU36, Night heron coronavirus HKU37, Night heron coronavirus HKU38, Night heron coronavirus HKU39, Night heron coronavirus HKU39, Night heron coronavirus HKU39, Night heron coronavirus HKU39, Night heron coronavirus HKU39, Night heron coronavirus HKU39, Night heron coronavirus HKU19, Pipistrellus bat coronavirus HKU5, Porcine epidemic diarrhea virus, Porcine torovirus, Rhinolophus bat coronavirus HKU2, Rousettus bat coronavirus HKU9, SARS coronavirus type 2, Scotophilus bat coronavirus 512, Severe acute respiratory syndrome-associated coronavirus, Thrush coronavirus HKU12, Tylonycteris bat coronavirus HKU4, White-eye coronavirus HKU16, Wigeon coronavirus HKU20, and SARS-CoV-2. Such OV antigens can be native or heterologous to the OV.

[0146] In certain embodiments, the OV antigen is an antigen encoded by the genome of vaccinia virus (e.g., antigens A33, A34, A36, A56, B5, F12, F13, etc., such as modified VV strains JX-594, GL-ONC1, or VV strains selected from Western Reserve, Wyeth, Lister, Copenhagen, Temple of Heaven, Patwadangar, and modified vaccinia virus Ankara, etc.), adenovirus, HSV, reovirus, vesicular stomatitis virus, Newcastle disease virus, Seneca Valley virus, poliovirus, measles virus, coxsackievirus, and Maraba virus. Such OV antigens can be native or heterologous to the OV.

[0147] In certain embodiments, the OV antigen is a modified version (or derivative) of a native OV antigen. For example, according to some embodiments, the OV antigen contains the same or substantially similar epitopes of the native antigen, but is modified / engineered to confer a desired property, such as improved infectivity of the virus. In certain embodiments, the OV antigen is modified / engineered to have a different epitope compared to the native antigen, such that, for example, existing antibodies (e.g., antibodies approved for therapeutic use) bind to the modified / engineered OV antigen but not to the native antigen.

[0148] According to some embodiments, the genome of an OV may be modified to encode and express one or more proteins that are not encoded by the wild-type genome of the OV, and such proteins may be referred to herein as "heterologous" to the OV antigen or "non-native" to the OV. The genome of an OV may be modified to encode and express a heterologous OV antigen of interest. In certain embodiments, the heterologous OV antigen is a viral antigen that is heterologous to the OV expressing the antigen (e.g., an antigen encoded by the wild-type genome of any of the viruses described elsewhere herein).According to some embodiments, the heterologous OV antigen is a tumor antigen, non-limiting examples of which include 5T4, AXL receptor tyrosine kinase (AXL), B-cell maturation antigen (BCMA), c-MET, C4.4a, carbonic anhydrase 6 (CA6), carbonic anhydrase 9 (CA9), cadherin-6, CD19, CD20, CD22, CD25, CD27L, CD30, CD33, CD37, CD44v6, CD56, CD70, CD74, CD79b, CD123, CD138, carcinoembryonic antigen (CEAE), and / or VEGF. A), cKit, Cripto protein, CS1, delta-like canonical notch ligand 3 (DLL3), endothelin receptor type B (EDNRB), ephrin A4 (EFNA4), epidermal growth factor receptor (EGFR), EGFRvIII, ectonucleotide pyrophosphatase / phosphodiesterase 3 (ENPP3), EPH receptor A2 (EPHA2), fibroblast growth factor receptor 2 (FGFR2), fibroblast growth factor receptor 3 (FGFR3), FMS-like tyrosine kinase 3 (FL T3), folate receptor 1 (FOLR1), glycoprotein non-transferase B (GPNMB), guanylate cyclase 2C (GUCY2C), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), integrin alpha, lysosomal-associated membrane protein 1 (LAMP-1), Lewis Y, LIV-1, leucine-rich repeat-containing 15 (LRRC15), mesothelin (MSLN), mucin 1 (MUC1), mucin 16 (MUC16), sodium-dependent phosphate transport protein 2 These include B(NaPi2b), nectin-4, NMB, NOTCH3, p-cadherin (p-CAD), prostate-specific membrane antigen (PSMA), protein tyrosine kinase 7 (PTK7), solute carrier family 44 member 4 (SLC44A4), SLIT-like family member 6 (SLITRK6), STEAP family member 1 (STEAP1), tissue factor (TF), T-cell immunoglobulin and mucin protein-1 (TIM-1), and trophoblast surface antigen (TROP-2).

[0149] Non-limiting examples of antigen binding domains that specifically bind to tumor antigens that the OV genome can be engineered to encode and express and that may be utilized in the anti-OV CARs or anti-OV conjugates of the present disclosure include the following antibodies: Adecatumumab, Ascrinvacumab, Cixutumumab, Conatumumab, Daratumumab, Drozitumumab, Duligotumab, Durvalumab, Dusizitumab, and / or other antibodies that specifically bind to tumor antigens that the OV genome can be engineered to encode and express, and / or other antigen binding domains that may be utilized in the anti-OV CARs or anti-OV conjugates of the present disclosure include antibodies such as ... Durozitumab, Dusizitumab, and / or other antibodies that specifically bind to tumor antigens that the OV genome can be engineered to encode and express, and / or other antigen binding domains that may be utilized in the anti-OV CARs or anti-OV conjugates of the present disclosure. b), Enfortumab, Enoticumab, Figitumumab, Ganitumab, Glembatumumab, Intetumumab, Ipilimumab, Iratumumab, Icrucumab, Lexatumumab, Lucatumumab, Mapatumumab mab, Narnatumab, Necitumumab, Nesvacumab, Ofatumumab, Olaratumab, Panitumumab, Patritumumab, Pritumumab, Radretumab, Ramucirumab, Rilotumumab, Robatumumab , Seribantumab, Tarextumab, Teprotumumab, Tovetumab, Vantictumab, Vesencumab, Votumumab, Zalutumumab, Flanvotumab, Altumomab, Anatumomab, Arcitumomab,Bectumomab, Blinatumomab, Detumomab, Ibritumomab, Minretumomab, Mitumomab, Moxetumomab, Naptumomab, Nofetumomab, Pemtumomab, Pintumomab, Racotumomab, Satumomab umomab, Solitomab, Taplitumomab, Tenatumomab, Tositumomab, Tremelimumab, Abagovomab, Igovomab, Oregovomab, Capromab, Edrecolomab, Nacolomab, Amatuximab, Bavituximab Avituximab, Brentuximab, Cetuximab, Derlotuximab, Dinutuximab, Ensituximab, Futuximab, Girentuximab, Indatuximab, Isatuximab, Margetuximab, Rituximab, Siltuximab Siltuximab, Ublituximab, Ecromeximab, Abituzumab, Alemtuzumab, Bevacizumab, Bivatuzumab, Brontictuzumab, Cantuzumab, Citatuzumab, Crivatuzumab,Dacetuzumab, Demcizumab, Dalotuzumab, Denintuzumab, Elotuzumab, Emactuzumab, Emibetuzumab, Enoblituzumab, Etaracizumab, Farletuzumab, Ficlatuzumab, Gemtuzumab tuzumab, Imgatuzumab, Inotuzumab, Labetuzumab, Lifastuzumab, Lintuzumab, Lorvotuzumab, Lumretuzumab, Matuzumab, Milatuzumab, Nimotuzumab, Obinutuzumab, Ocaratuzumab uzumab, Otlertuzumab, Onartuzumab, Oportuzumab, Parsatuzumab, Pertuzumab, Pinatuzumab, Polatuzumab, Sibrotuzumab, Simtuzumab, Tacatuzumab, Tigatuzumab, Trastuzumab Tucotuzumab, Vandortuzumab, Vanucizumab, Veltuzumab, Borsetuzumab, Sofituzumab, Catumaxomab, Ertumaxomab, Depatuxizumab, Ontuxizumab, Brontuvetmab,The antigen-binding domain of Tamtuvetmab, or a tumor antigen-binding variant thereof. As used herein, "variant" means that the antigen-binding domain specifically binds to a particular antigen (e.g., HER2 for Trastuzumab), but has fewer or more amino acids than the parent antibody (e.g., is a fragment of the parent antibody (e.g., scFv)), has one or more amino acid substitutions compared to the parent antibody, or a combination thereof.

[0150] In some embodiments, the antigen binding domain of the anti-OV antigen CAR or anti-OV antigen conjugate of the present disclosure is derived from an antibody approved by the United States Food and Drug Administration and / or the European Medicines Agency (EMA) for use as a therapeutic antibody (e.g., for targeting specific disease-associated cells in a patient), or a fragment thereof that retains the ability to specifically bind to the target antigen (e.g., a single chain version of such an antibody, such as an scFv version of an antibody).

[0151] The antigen binding domain of the CAR of the present disclosure can be any suitable format, e.g., scFv, etc. Any suitable transmembrane and intracellular signaling domains can be used, including any of the transmembrane and intracellular signaling domains described in the fusion protein section above, as well as costimulatory domains, linker sequences / spacer domains, etc.

[0152] The CAR of the present disclosure can be provided by a single peptide or by two or more polypeptides. When the CAR is provided by two or more polypeptides, the CAR can be provided in any useful multi-polypeptide format, including those multi-peptide formats described above in the fusion protein section of the present disclosure.

[0153] Also provided are nucleic acids encoding any of the anti-OV antigen CARs of the present disclosure, expression vectors comprising such nucleic acids, and cells comprising such nucleic acids and expression vectors.

[0154] According to some embodiments, provided are cells expressing an anti-OV antigen CAR on its surface. Cells of interest include, but are not limited to, immune cells. In certain embodiments, the immune cells are immune effector cells. Non-limiting examples of immune effector cells that may express the anti-OV antigen CAR of the present disclosure on their surface include T cells (i.e., the cells may be CAR T cells), NK cells, NKT cells, macrophages, and the like. Also provided are pharmaceutical compositions comprising any of the cells of the present disclosure expressing an anti-OV antigen CAR on their surface. Such compositions may include cells and a pharmaceutically acceptable carrier. Examples of suitable pharmaceutically acceptable carriers are described in detail below.

[0155] Conjugates that bind to oncolytic virus antigens In addition to the anti-VV A56 and anti-VV B5 antibodies and fusion protein conjugates of the present disclosure described in the conjugate section above, aspects of the present disclosure further include anti-oncolytic virus antigen antibody conjugates. In certain embodiments, such conjugates include an antibody that specifically binds to an oncolytic virus (OV) antigen and a drug conjugated to the antibody. According to some embodiments, the drug conjugated to the antibody is selected from a chemotherapeutic agent, a toxin, a radiosensitizer, and a radioisotope (e.g., a therapeutic radioisotope). The drug can be any of such drugs described in the conjugate section above.

[0156] The anti-OV antibody conjugates of the present disclosure find a variety of uses. For example, it will be appreciated that, with the benefit of the present disclosure, such conjugates find use in methods comprising administering to an individual having cancer a pharmaceutical composition comprising an anti-OV antigen conjugate, whereby cancer cells in the individual become infected with OV and express OV antigens on their surface, thereby targeting the conjugate to the cancer cells to treat the individual's cancer.

[0157] The antibody (or fusion protein containing it) can be selected so that the conjugate specifically binds to the OV antigen of interest. The OV antigen can be a native OV antigen. In certain embodiments, the antibody portion of the conjugate can specifically bind to a native antigen encoded by an OV selected from vaccinia virus (e.g., antigens A33, A34, A36, A56, B5, F12, F13, etc., e.g., JX-594, GL-ONC1, or Western Reserve, Wyeth, Lister, Copenhagen, Temple of Heaven, Patwadangar, and modified vaccinia virus Ankara, etc.), adenovirus, HSV, reovirus, vesicular stomatitis virus, Newcastle disease virus, Seneca Valley virus, poliovirus, measles virus, coxsackievirus, and Maraba virus.

[0158] In certain embodiments, the OV antigen is heterologous to the OV. For example, an antibody (or a fusion protein comprising the antibody) can be selected such that the antibody portion of the conjugate specifically binds to any of the heterologous OV antigens described in the CAR section above.

[0159] The antibody portion of the anti-OV antibody conjugates of the disclosure may be provided in any desired format, e.g., a tetrameric format, a single chain (e.g., scFv) format, etc., as described in the sections above relating to the antibodies, anti-VV A56 and anti-VV B5 antibodies and fusion protein conjugates of the disclosure.

[0160] The anti-OV antibody conjugates of the present disclosure may include an agent conjugated to the antibody (or a fusion protein comprising the antibody) via a linker, e.g., any of the non-cleavable or cleavable linkers described in the sections above relating to the anti-VV A56 and anti-VV B5 antibody and fusion protein conjugates of the present disclosure.

[0161] Also provided is a pharmaceutical composition comprising any of the anti-OV antibody conjugates of the present disclosure. Such a composition may comprise a conjugate and a pharmaceutically acceptable carrier. Examples of suitable pharmaceutically acceptable carriers are described in detail below.

[0162] composition As summarized above, the present disclosure also provides compositions.According to some embodiments, the compositions of the present disclosure comprise the antibody, fusion protein or conjugate of the present disclosure.For example, the antibody, fusion protein or conjugate can be any of the antibodies, fusion proteins or conjugates described in the antibody section above, the description of which is incorporated but is not repeated here for the sake of brevity.

[0163] In certain aspects, the composition of the present disclosure comprises an antibody, fusion protein or conjugate present in a liquid medium. The liquid medium can be an aqueous liquid medium such as water, a buffer solution, etc. One or more additives, such as salts (e.g., NaCl, MgCl2, KCl, MgSO4), buffers (Tris buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 2-(N-morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.), dissolving agents, detergents (e.g., non-ionic detergents such as Tween-20), nuclease inhibitors, protease inhibitors, glycerol, chelating agents, etc., may be present in such compositions.

[0164] Aspects of the present disclosure further include pharmaceutical compositions. In some embodiments, the pharmaceutical compositions of the present disclosure include an anti-VV A56 antibody or an anti-VV B5 antibody (or a conjugate or fusion protein comprising the same) of the present disclosure and a pharma- ceutical acceptable carrier.

[0165] Antibody, fusion protein or conjugate can be incorporated into various preparations for therapeutic administration.More specifically, antibody, fusion protein or conjugate can be formulated into pharmaceutical composition by combining with suitable pharmaceutically acceptable excipient or diluent, and can be formulated into solid, semi-solid, liquid or gaseous form preparations such as tablets, capsules, powders, granules, ointments, solutions, injections, inhalants, aerosols, etc.

[0166] Formulations of antibodies, fusion proteins, or conjugates for administration to an individual (e.g., suitable for human administration) will generally be sterile and further free of detectable pyrogens or other contaminants that would contraindicate administration to a patient via the selected route of administration.

[0167] In pharmaceutical dosage forms, the antibodies, fusion proteins or conjugates can be administered in the form of their pharma- ceutically acceptable salts, or they can be used alone or in suitable association and combination with other pharma- ceutically active compounds. The following methods and carriers / excipients are merely examples and are in no way limiting.

[0168] For oral formulations, the antibody, fusion protein or conjugate can be used alone or in combination with suitable excipients to produce tablets, powders, granules or capsules using conventional excipients such as lactose, mannitol, corn starch or potato starch; binders such as microcrystalline cellulose, cellulose derivatives, gum arabic, corn starch or gelatin; disintegrating agents such as corn starch, potato starch or sodium carboxymethylcellulose; lubricants such as talc or magnesium stearate; diluents, wetting agents, preservatives and flavorings, as desired.

[0169] The antibody, fusion protein or conjugate can be formulated for parenteral (e.g., intravenous, intraarterial, intraosseous, intramuscular, intracerebral, intraventricular, intrathecal, subcutaneous, etc.) administration. In certain embodiments, the antibody, fusion protein or conjugate can be formulated for injection by dissolving, suspending or emulsifying the antibody, fusion protein or conjugate in an aqueous or non-aqueous solvent such as vegetable oil or other similar oils, synthetic aliphatic glycerides, esters of higher fatty acids, or propylene glycol, and, if necessary, with conventional additives such as solubilizers, isotonicity agents, suspending agents, emulsifiers, stabilizers and preservatives.

[0170] Pharmaceutical compositions containing the antibody, fusion protein or conjugate can be prepared by mixing the antibody, fusion protein or conjugate having the desired purity with any physiologically acceptable carrier, excipient, stabilizer, surfactant, buffer and / or isotonicity agent. Acceptable carriers, excipients and / or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers, such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid, preservatives (e.g., ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl paraben, benzalkonium chloride, or combinations thereof), arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, and the like. , amino acids such as leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline, and combinations thereof, monosaccharides, disaccharides, and other sugars, low molecular weight (less than about 10 residues) polypeptides, proteins such as gelatin or serum albumin, chelating agents such as EDTA, sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid, and / or non-ionic surfactants such as Tween, Brij Pluronic, Triton-X, or polyethylene glycol (PEG).

[0171] The pharmaceutical composition can be in liquid form, lyophilized form, or liquid form reconstituted from lyophilized form, and lyophilized preparations will be reconstituted with sterile solution before administration.The standard procedure for reconstituting lyophilized compositions is to add back a certain amount of pure water (typically equal to the volume removed during lyophilization), but a solution containing antibacterial agent may be used to prepare pharmaceutical compositions for parenteral administration.

[0172] Aqueous formulations of antibodies, fusion proteins or conjugates can be prepared in pH buffer solutions, for example, at a pH in the range of about 4.0 to about 7.0, or about 5.0 to about 6.0, or alternatively at about 5.5. Examples of buffers suitable for a pH in this range include phosphate buffers, histidine buffers, citrate buffers, succinate buffers, acetate buffers, and other organic acid buffers. The concentration of the buffer can be, for example, about 1 mM to about 100 mM, or about 5 mM to about 50 mM, depending on the buffer and the desired tonicity of the formulation.

[0173] To adjust the tonicity of the formulation, an isotonicity agent can be included in the formulation. Exemplary isotonicity agents include sodium chloride, potassium chloride, glycerin, and any component from the group of amino acids, sugars, and combinations thereof. In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable. The term "isotonic" refers to a solution that has the same tonicity as some other solution to which it is compared, such as physiological saline or serum. The isotonicity agent can be used in an amount of about 5 mM to about 350 mM, for example, in an amount of 100 mM to 350 mM.

[0174] Surfactants may be added to the formulation to reduce aggregation and / or minimize the formation of fine particles in the formulation and / or reduce adsorption. Examples of surfactants include polyoxyethylene sorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenyl polyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymers (Poloxamer, Pluronic) and sodium dodecyl sulfate (SDS). Examples of suitable polyoxyethylene sorbitan fatty acid esters are polysorbate 20 (sold under the trademark Tween 20™) and polysorbate 80 (sold under the trademark Tween 80™). Examples of suitable polyethylene-polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188™. Examples of suitable polyoxyethylene alkyl ethers are those sold under the trademark Brij™. Exemplary surfactant concentrations can range from about 0.001% to about 1% w / v.

[0175] Cryoprotectants may be added to protect the antibodies and / or T cell activators from destabilizing conditions during the lyophilization process. For example, known cryoprotectants include sugars (including glucose and sucrose), polyols (including mannitol, sorbitol and glycerol), and amino acids (including alanine, glycine and glutamic acid). Cryoprotectants can be included in amounts of about 10 mM to 500 nM.

[0176] In some embodiments, the pharmaceutical composition comprises an antibody, fusion protein or conjugate, and one or more of the components identified above (e.g., surfactants, buffers, stabilizers, tonicity agents), and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl paraben, benzalkonium chloride, and combinations thereof. In other embodiments, a preservative is included in the formulation, for example, at a concentration ranging from about 0.001 to about 2% (w / v).

[0177] kit Aspects of the present disclosure further include kits. In certain embodiments, the kits find use in carrying out the methods of the present disclosure, for example, methods comprising administering the pharmaceutical compositions of the present disclosure to an individual to target the antibody, fusion protein (e.g., CAR) or conjugate to oncolytic virus (e.g., VV)-infected cancer cells in the individual.

[0178] Thus, in certain embodiments, the kit of the present disclosure includes any of the pharmaceutical compositions of the present disclosure and instructions for administering the pharmaceutical composition to an individual in need thereof. The pharmaceutical composition included in the kit may include any of the antibodies, fusion proteins, and / or conjugates of the present disclosure, such as any of the antibodies, fusion proteins, and / or conjugates described above. As will be understood, the kit of the present disclosure may include any of the agents and features described above in the sections related to the subject antibodies, fusion proteins, conjugates, and compositions, which will not be repeated here for brevity.

[0179] The kit of the present disclosure may include a quantity of the composition present in a unit dose, e.g., ampoule, or multiple dose format. Thus, in certain embodiments, the kit may include one or more (e.g., two or more) unit doses (e.g., ampoules) of the composition comprising the antibody, fusion protein, and / or conjugate of the present disclosure. The term "unit dose" as used herein refers to a physically separate unit suitable as a single dose for human and animal subjects, each unit containing a predetermined amount of the composition calculated in an amount sufficient to produce a desired effect. The amount of the unit dose depends on various factors, such as the specific antibody, fusion protein, and / or conjugate used, the effect to be achieved, and the pharmacodynamics associated with the antibody, fusion protein, and / or conjugate in an individual. In yet other embodiments, the kit may include a single multiple dose of the composition.

[0180] In certain embodiments, the kits of the present disclosure include instructions for directing an antibody, fusion protein or conjugate present in the pharmaceutical composition to VV-infected cancer cells in an individual having cancer (e.g., to treat the individual's cancer), e.g., by administering the pharmaceutical composition to the individual, wherein the individual comprises cancer cells infected with VV and the antibody, fusion protein or conjugate is targeted to the infected cancer cells by a VV antigen expressed on the surface of the infected cancer cells.

[0181] According to some embodiments, the kits of the present disclosure include a pharmaceutical composition comprising a VV (e.g., a strain of VV selected from JX-594, GL-ONC1, Western Reserve, Wyeth, Lister, Copenhagen, Tiantan, Patwadangar, and modified vaccinia virus Ankara, etc.). Such kits may further include instructions for administering to an individual having cancer a pharmaceutical composition comprising a VV in an amount effective to infect cancer cells of the individual, e.g., prior to administering a pharmaceutical composition comprising an antibody, fusion protein, or conjugate of the present disclosure.

[0182] The instructions included in the kit (e.g., instructions for use (IFU)) can be recorded on a suitable recording medium. For example, the instructions can be printed on a substrate such as paper or plastic. As such, the instructions can be present in the kit as a package insert, on the label of the container of the kit or a component thereof (i.e., associated with the packaging or subpackaging), and the like. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer-readable storage medium, such as a portable flash drive, DVD, CD-ROM, diskette, and the like. In still other embodiments, the actual instructions are not present in the kit, but a means is provided for obtaining the instructions from a remote source, for example, via the Internet. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or downloaded. As with the instructions, the means for obtaining the instructions are recorded on a suitable substrate.

[0183] method Aspects of the present disclosure include methods of using the antibodies, fusion proteins (e.g., CARs) and conjugates of the present disclosure. The methods are useful in a variety of contexts, including in vitro and / or in vivo research and / or clinical applications.

[0184] In a particular aspect, provided is a method comprising administering an effective amount of a pharmaceutical composition comprising any of the anti-VV A56 or anti-VV B5 antibodies (including any of the fusion proteins or conjugates comprising such antibodies) of the present disclosure to an individual having cancer, wherein the individual comprises cancer cells infected with an OV encoding the VV A56 or VV B5 antigen to which the antibody binds (the VV A56 or VV B5 antigen can be native or heterologous to the OV), and the antibody, fusion protein or conjugate is targeted to the infected cancer cells by the VV antigen expressed on the surface of the infected cancer cells. According to some embodiments, such methods further comprise infecting the cancer cells by administering an effective amount of an OV to the individual prior to administering the pharmaceutical composition to the individual. Such methods find use, for example, in treating cancer in an individual.

[0185] In certain embodiments, the pharmaceutical composition comprises any of the anti-VV A56 or anti-VV B5 antibody conjugates of the present disclosure.For example, the pharmaceutical composition may comprise a conjugate in which the anti-VV A56 or anti-VV B5 antibody is conjugated to a detectable label or radioisotope that is an in vivo imaging agent.Such a method may further comprise imaging the infected cancer cells of the individual using the in vivo imaging agent.The method of administering the conjugate that comprises a detectable label or radioisotope to an individual finds use in imaging the cancer cells of the individual, for example, for the purpose of diagnosis, prognosis and / or monitoring anti-cancer therapy.

[0186] According to some aspects, a method is provided for targeting an antibody that specifically binds to an oncolytic virus (OV) antigen to cancer cells of an individual. Such a method comprises administering to an individual an effective amount of a pharmaceutical composition comprising an antibody that specifically binds to an OV antigen (or a fusion protein or conjugate comprising the antibody), wherein cancer cells in the individual are infected with the OV and express the OV antigen on their surface. The OV antigen can be any of the OV antigens described elsewhere herein. In certain embodiments, the OV antigen is a native OV antigen. For example, the OV can be any of the viruses described above in the section entitled "Chimeric antigen receptors that bind to oncolytic virus antigens," or an engineered variant thereof (e.g., an engineered variant having one or more of the OV modifications described below, e.g., transgenic for human GM-CSF, deletion of the thymidine kinase gene, and / or others), wherein the OV antigen is native to the OV. According to some embodiments, the OV antigen is heterologous to the OV, for example a viral antigen heterologous to the OV (e.g., any of the viral OV antigens described above in the section entitled "Chimeric Antigen Receptors Binding to Oncolytic Viral Antigens"), a tumor antigen, or any other heterologous OV antigen described elsewhere herein. According to some embodiments, such methods further comprise infecting cancer cells by administering an effective amount of OV to the individual prior to administering the pharmaceutical composition to the individual. Such methods find use, for example, in treating cancer in an individual.

[0187] Upon binding on the cancer cell surface, the antibody (or fusion protein) can induce cytotoxicity, for example, via antibody-dependent cellular cytotoxicity (ADCC), by recruiting complement in complement-dependent cytotoxicity (CDC), via antibody-dependent cellular phagocytosis (ADCP), via epitope spreading, or by some other mechanism. The antibody can be modified in the Fc region to provide desired or enhanced effector functions. This can be achieved by introducing one or more amino acid substitutions into the Fc region of the antibody. Alternatively, certain other Fc regions can be used when it is desirable to eliminate or reduce effector functions to minimize side effects or therapeutic complications.

[0188] In certain embodiments, the pharmaceutical composition may comprise a conjugate in which an anti-VV A56 or anti-VV B5 antibody is conjugated to an agent selected from a chemotherapeutic agent, a toxin, a radiosensitizer, a therapeutic radioisotope, and a radioisotope that allows in vivo imaging of the antibody. The agent may be any of the agents described in the conjugate section above.

[0189] The antibody (or fusion protein comprising it) can be selected so that the conjugate specifically binds to the OV antigen of interest. The OV antigen of interest includes, but is not limited to, any of the native or heterologous OV antigens described elsewhere herein. In certain embodiments, the OV antigen is a native OV antigen. For example, the OV can be any of the viruses described above in the section entitled "Chimeric Antigen Receptors Binding to Oncolytic Viral Antigens" or an engineered variant thereof (e.g., an engineered variant having one or more of the OV modifications described below, e.g., transgenic for human GM-CSF, deletion of the thymidine kinase gene, and / or others), and the OV antigen is native to the OV. According to some embodiments, the OV antigen is heterologous to the OV, e.g., a viral antigen heterologous to the OV (e.g., any of the viral OV antigens described above in the section entitled "Chimeric Antigen Receptors Binding to Oncolytic Viral Antigens"), a tumor antigen, or any other heterologous OV antigen described elsewhere herein. In some embodiments, the antigen is a native or heterologous OV antigen, the expression of which is exclusively under the control of the wild-type transcriptional regulatory elements of the OV for that antigen. That is, according to such embodiments, the transcriptional regulatory elements of the OV for the antigen are not modified compared to the wild-type OV. In certain embodiments, the antigen is a native OV antigen, the expression level of which is altered (e.g., increased) compared to the expression by the wild-type OV by modification (e.g., replacement, duplication, etc.) of one or more wild-type transcriptional regulatory elements of the OV. As an example, the antigen can be a native OV antigen (e.g., native A56, native B5, or any other native OV antigen of interest), the expression of which is increased compared to the expression by the wild-type OV by operably coupling the coding region of the native OV antigen to one or more heterologous regulatory elements (e.g., promoters such as human promoters) that result in a higher expression level compared to the wild-type transcriptional regulatory elements of the OV.Such an OV may be a modified OV, for example an OV that includes one or more of the OV modifications described below.

[0190] In certain embodiments, the OV antigen is heterologous to the OV. For example, an antibody can be selected such that the antibody portion of the conjugate specifically binds to any of the heterologous OV antigens described elsewhere herein (e.g., viral antigens, tumor antigens, etc. heterologous to the OV).

[0191] The antibody may be provided in any desired format, such as a tetrameric format, a single chain (e.g., scFv) format, etc., as described in the previous section of this disclosure. According to some embodiments, the pharmaceutical composition comprises an antibody conjugated to a detectable label or a radioisotope. In certain embodiments, the detectable label or radioisotope is an in vivo imaging agent. The method in which the antibody is conjugated to an in vivo imaging agent may further include detecting the in vivo imaging agent for imaging cancer cells of an individual in vivo, for example, for the purpose of diagnosis, prognosis and / or monitoring anti-cancer therapy.

[0192] According to some aspects, a method is provided for targeting a CAR that specifically binds to an oncolytic virus (OV) antigen to cancer cells of an individual. Such a method comprises administering to an individual an effective amount of a pharmaceutical composition comprising a CAR that comprises an antigen-binding domain that specifically binds to an OV antigen, and the cancer cells of the individual are infected with the OV and express the OV antigen on their surface. The CAR can be expressed on the surface of a cell, e.g., an immune cell, such as an immune effector cell, e.g., a T cell, a NK cell, a NKT cell, a macrophage, and the like. For example, the CAR can be present on the surface of a T cell, in which case the method is a method of targeting a CAR T cell to an infected cancer cell in an individual. According to some embodiments, such a method further comprises infecting the cancer cells by administering an effective amount of an OV to the individual prior to administering the pharmaceutical composition to the individual. Such a method finds use, for example, in treating cancer in an individual.

[0193] The antigen-binding domain of the CAR specifically binds to the OV antigen of interest. The OV antigen of interest includes, but is not limited to, any of the native or heterologous OV antigens described elsewhere herein. In certain embodiments, the OV antigen is a native OV antigen. For example, the OV can be any of the viruses described above in the section entitled "Chimeric Antigen Receptors Binding to Oncolytic Viral Antigens" or an engineered variant thereof (e.g., an engineered variant having one or more of the OV modifications described below, e.g., transgenic for human GM-CSF, deletion of the thymidine kinase gene, and / or others), and the OV antigen is native to the OV. According to some embodiments, the OV antigen is heterologous to the OV, e.g., a viral antigen heterologous to the OV (e.g., any of the viral OV antigens described above in the section entitled "Chimeric Antigen Receptors Binding to Oncolytic Viral Antigens"), a tumor antigen, or any other heterologous OV antigen described elsewhere herein.

[0194] Pharmaceutical compositions comprising cells expressing a CAR on their surface can be prepared by a variety of methods. In some embodiments, the cells of the present disclosure are produced by transfecting the cells with a viral vector encoding the CAR. In some embodiments, the cells are T cells, thereby providing a method of producing CAR T cells. In some embodiments, such methods include activating a population of T cells (e.g., T cells obtained from an individual to whom CAR T cell therapy is administered), stimulating the population of T cells to proliferate, and transducing the T cells with a viral vector encoding the CAR. In some embodiments, the T cells are transduced with a retroviral vector, e.g., a gamma retroviral vector, encoding the CAR. In some embodiments, the T cells are transduced with a lentiviral vector encoding the CAR.

[0195] Cells of the present disclosure can be autologous / autogeneic ("self") or non-autologous ("non-self", e.g., allogeneic, syngeneic or xenogeneic). As used herein, "autologous" refers to cells from the same individual. As used herein, "allogeneic" refers to cells of the same species that are genetically different from the comparative cell. As used herein, "syngeneic" refers to cells of a different individual that are genetically identical to the comparative cell. In some embodiments, the cell is a T cell obtained from a mammal. In some embodiments, the mammal is a primate. In some embodiments, the individual is a human.

[0196] T cells can be obtained from many sources, including, but not limited to, peripheral blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, splenic tissue, and tumors. In certain embodiments, T cells can be obtained from a unit of blood collected from an individual using any number of known techniques, such as sedimentation, e.g., FICOLL™ separation.

[0197] In some embodiments, an isolated or purified population of T cells is used. CTL and T H The lymphocytes are purified from PBMCs. CTL and T H Lymphocytes may be classified as naïve (T N ), Memory (T MEM ), and effector (T EFF ) T cell subpopulations. Suitable approaches for such selection are known and include, for example, magnetic activated cell sorting (MACS), in which TNs are CD45RA + CD62L + CD95 - and TSCM is CD45RA + CD62L + CD95 +and TCM is CD45RO + CD62L + CD95 + and TEM is CD45RO + CD62L - CD95 + An example of such a selection approach is described in Wang et al. (2016) Blood 127(24):2980-90.

[0198] In some embodiments, specific subpopulations of T cells expressing one or more of the following markers: CD3, CD4, CD8, CD28, CD45RA, CD45RO, CD62, CD127, and HLA-DR may be further isolated by positive or negative selection techniques. In some embodiments, specific subpopulations of T cells expressing one or more of the markers selected from the group consisting of CD62L, CCR7, CD28, CD27, CD122, CD127, CD197, or CD38 or CD62L, CD127, CD197, and CD38 are further isolated by positive or negative selection techniques. In some embodiments, the produced T cell composition does not express one or more of the following markers: CD57, CD244, CD160, PD-1, CTLA4, TI3, and LAG3. In some embodiments, the produced T cell compositions do not substantially express one or more of the following markers: CD57, CD244, CD160, PD-1, CTLA4, TI3, and LAG3.

[0199] To achieve a therapeutically effective dose of the T cell composition, the T cells may be subjected to one or more rounds of stimulation, activation and / or expansion. T cells can be activated and expanded generally using methods such as those described in, for example, U.S. Patent Nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905,681, 7,144,575, 7,067,318, 7,172,869, 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, and 6,867,041, each of which is incorporated by reference in its entirety for all purposes. In some embodiments, the T cells are activated and expanded for about 1 to 21 days, e.g., about 5 to 21 days. In some embodiments, the T cells are activated and expanded for about 1 to about 4 days, about 1 to about 3 days, about 1 to about 2 days, about 2 to about 3 days, about 2 to about 4 days, about 3 to about 4 days, or about 1, about 2, about 3, or about 4 days prior to introducing a nucleic acid (e.g., an expression vector) encoding a CAR into the T cells.

[0200] In some embodiments, the T cells are activated and expanded for about 6 hours, about 12 hours, about 18 hours, or about 24 hours prior to introducing a nucleic acid (e.g., an expression vector) encoding a CAR into the T cells. In some embodiments, the T cells are activated at the same time that a nucleic acid (e.g., an expression vector) encoding a CAR is introduced into the T cells.

[0201] In some embodiments, suitable conditions for T cell culture include an appropriate medium (e.g., Minimum Essential Medium or RPMI Medium 1640 or X-vivo 15 (Lonza)) and one or more factors necessary for proliferation and survival, including, but not limited to, serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, IL-21, GM-CSF, IL-10, IL-12, IL-15, TGFβ and TNF-α, or any other additives suitable for growing cells known to one of skill in the art. Further illustrative examples of cell culture media include, but are not limited to, RPMI 1640, Click, AEVI-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15 and X-Vivo 20, Optimizer, supplemented with amino acids, sodium pyruvate and vitamins, and either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or cytokines in sufficient amounts for T cell growth and proliferation.

[0202] In some embodiments, the nucleic acid (e.g., expression vector) encoding the CAR is introduced into the cell (e.g., T cell) by microinjection, transfection, lipofection, heat shock, electroporation, transduction, gene gun, microinjection, DEAE-dextran mediated transfer, etc. In some embodiments, the nucleic acid (e.g., expression vector) encoding the CAR is introduced into the cell (e.g., T cell) by AAV transduction. The AAV vector may include ITRs from AAV2 and a serotype from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In some embodiments, the AAV vector includes ITRs from AAV2 and a serotype from AAV6. In some embodiments, the nucleic acid (e.g., expression vector) encoding the CAR is introduced into the cell (e.g., T cell) by lentiviral or retroviral transduction. Lentiviral vector backbones can be derived from HIV-1, HIV-2, Visna-Maedi virus (VMV), Caprine Arthritis-Encephalitis Virus (CAEV), Equine Infectious Anemia Virus (EIAV), Feline Immunodeficiency Virus (FIV), Bovine Immunodeficiency Virus (BIV), or Simian Immunodeficiency Virus (SIV). Lentiviral vectors can be integration competent or integrase-deficient lentiviral vectors (TDLV). In one embodiment, an IDLV vector containing an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) is used.

[0203] In certain aspects, a method is provided for targeting a conjugate comprising an antibody that specifically binds to an oncolytic virus (OV) antigen to cancer cells of an individual. Such a method comprises administering to the individual an effective amount of a pharmaceutical composition comprising an antibody that specifically binds to an OV antigen, and the cancer cells of the individual are infected with the OV and express the OV antigen on their surface. According to some embodiments, such a method further comprises infecting the cancer cells by administering to the individual an effective amount of OV prior to administering the pharmaceutical composition to the individual. Such a method finds use, for example, in treating cancer in an individual.

[0204] The antibody of the conjugate specifically binds to the OV antigen of interest, including but not limited to any of the OV antigens described elsewhere herein. In certain embodiments, the OV antigen is a native OV antigen. For example, the OV can be any of the viruses described above in the section entitled "Chimeric Antigen Receptors Binding to Oncolytic Viral Antigens" or an engineered variant thereof (e.g., an engineered variant having one or more of the OV modifications described below, e.g., transgenic for human GM-CSF, deletion of the thymidine kinase gene, and / or others), and the OV antigen is native to the OV. According to some embodiments, the OV antigen is heterologous to the OV, e.g., a viral antigen heterologous to the OV (e.g., any of the viral OV antigens described above in the section entitled "Chimeric Antigen Receptors Binding to Oncolytic Viral Antigens"), a tumor antigen, or any other heterologous OV antigen described elsewhere herein.

[0205] The OV administered to an individual according to the present disclosure can be a wild-type OV or a modified OV. According to some embodiments, the OV is a modified OV, which is a chimeric OV that includes two or more domains from different viruses. In some embodiments, the OV is a modified OV (e.g., a non-chimeric or chimeric OV) that includes one or more modifications in one or more of the following functional categories: infection, replication, tropism, improved safety, a reporter gene for imaging, blocking the host's anti-viral immune response and enhancing the host's anti-tumor immune response, etc. According to some embodiments, the OV is modified to: encode and express human GM-CSF, have a deletion of the thymidine kinase gene (limiting or substantially limiting viral replication to cells with high levels of thymidine kinase, typically found in cancer cells with mutated RAS or p53 genes), a reporter gene (e.g., Lac The modified OVs may encode and express a viral protein (e.g., Z, luciferase, etc.), modify one or more genes encoding ribonucleotide reductase (RR), disrupt the F2L gene (encoding the viral dUTPase involved in both maintaining the fidelity of DNA replication and providing the precursor for TMP production by thymidylate synthase), express a therapeutic protein (e.g., a suicide gene - i.e., a gene encoding a protein capable of converting a precursor of a drug into a cytotoxic compound, including but not limited to TK, cytosine deaminase, purine nucleoside phosphorylase, uracil phosphoribosyltransferase, thymidylate kinase, etc.), express an immunostimulatory protein ("immunostimulatory protein" refers to a protein that has the ability to stimulate the immune system in a specific or non-specific manner, including cytokines, chemokines, growth factors, etc.), and any combination thereof. Any of the above modified OVs may encode any of the OV antigens described elsewhere herein. For example, the modified OV can be modified from any of the viruses described above in the section entitled "Chimeric Antigen Receptors that Bind Oncolytic Viral Antigens," where the OV antigen is native to the OV.According to some embodiments, the OV antigen is heterologous to the OV, e.g., a viral antigen heterologous to the modified OV (e.g., any of the viral OV antigens described above in the section entitled "Chimeric Antigen Receptors Binding to Oncolytic Viral Antigens"), a tumor antigen, or any other heterologous OV antigen described elsewhere herein.

[0206] In certain embodiments, methods are provided that include administering a pharmaceutical composition that includes a cell (e.g., a cancer cell) infected with an OV. The pharmaceutical composition may further include an antibody, conjugate, or fusion protein that specifically binds to an OV antigen expressed by the infected cell, e.g., a cancer cell. The cell (e.g., a cancer cell) may have been removed from an individual during surgery. The cell (e.g., a cancer cell) may have been modified (and killed) in a laboratory to make it more likely to be attacked by the immune system when administered to a patient. The patient's immune system then attacks the cell and similar cells that remain in the body. The antibodies, conjugates, or fusion proteins of the present disclosure can be used according to this approach to promote the uptake of tumor particles / antigens by Fc receptors on professional APCs, enhancing the immune response against the tumor.

[0207] The pharmaceutical composition can be administered to any of a variety of individuals. In certain aspects, the individual is a "mammal" or "mammalian", which terms are used broadly to refer to organisms within the class Mammalia, including carnivores (e.g., dogs and cats), rodents (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In some embodiments, the individual is a human. In certain aspects, the individual is an animal model (e.g., mouse model, primate model, etc.) of a cell proliferative disorder, such as cancer.

[0208] An individual in need thereof may have a cell proliferative disorder. By "cell proliferative disorder" is meant a disorder in which unwanted cell proliferation of one or more subsets of cells of a multicellular organism occurs, resulting in harm to the organism (e.g., pain or reduced life expectancy). Cell proliferative disorders include, but are not limited to, cancer, precancer, benign tumors, vascular proliferative disorders (e.g., arthritis, restenosis, etc.), fibrotic disorders (e.g., hepatic cirrhosis, atherosclerosis, etc.), psoriasis, epidermal and dermoid cysts, lipomas, adenomas, capillary and cutaneous hemangiomas, lymphangiomas, nevi lesions, teratomas, nephromas, myofibromatosis, osteogenic tumors, dysplastic masses, mesangial cell proliferative disorders, and the like.

[0209] In some embodiments, the individual has cancer. The subject method can be used to treat a wide variety of cancers. As used herein, "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. Examples of cancers that can be treated using the subject method include, but are not limited to, carcinoma, lymphoma, blastoma, and sarcoma. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bile duct cancer, bladder cancer, liver cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancer. In certain embodiments, the individual has a cancer selected from a solid tumor, recurrent glioblastoma multiforme (GBM), non-small cell lung cancer, metastatic melanoma, melanoma, peritoneal cancer, epithelial ovarian cancer, glioblastoma multiforme (GBM), metastatic colorectal cancer, colorectal cancer, pancreatic ductal adenocarcinoma, squamous cell carcinoma, esophageal cancer, gastric cancer, neuroblastoma, fallopian tube cancer, bladder cancer, metastatic breast cancer, pancreatic cancer, soft tissue sarcoma, recurrent head and neck cancer squamous cell carcinoma, head and neck cancer, anaplastic astrocytoma, malignant pleural mesothelioma, breast cancer, squamous non-small cell lung cancer, rhabdomyosarcoma, metastatic renal cell carcinoma, basal cell carcinoma (basal cell epithelioma), and gliosarcoma. In certain embodiments, the individual has a cancer selected from melanoma, Hodgkin's lymphoma, renal cell carcinoma (RCC), bladder cancer, non-small cell lung cancer (NSCLC), and head and neck squamous cell carcinoma (HNSCC).

[0210] The antibodies, fusion proteins and conjugates of the disclosure may be administered via a route of administration selected from oral (e.g., tablet form, capsule form, liquid form, etc.), parenteral (e.g., intravenous, intramuscular, subcutaneous, intramuscular, or epidural injection), topical, intranasal, or intratumoral administration.

[0211] The antibodies, fusion proteins and conjugates of the present disclosure can be administered as pharmaceutical compositions in therapeutically effective amounts. By "therapeutically effective amount" is meant a dose sufficient to produce a desired result, e.g., an amount sufficient to produce a beneficial or desired therapeutic result (including a preventative result), such as the alleviation of symptoms of cancer, as compared to a control. In the context of cancer, in some embodiments, a therapeutically effective amount is sufficient to slow tumor growth, reduce tumor size, and / or the like. An effective amount can be administered in one or more doses.

[0212] As mentioned above, the present disclosure includes a method for treating cancer in an individual. "Treatment" means improving at least one or more symptoms associated with cancer in an individual, and improvement is used in a broad sense to refer to the alleviation of parameters, for example, the severity of symptoms associated with at least the cancer being treated.Therefore, treatment also includes the situation in which cancer or at least one or more symptoms associated with it are completely inhibited, for example, prevented from occurring, or stopped, for example, discontinued, so that an individual no longer suffers from cancer or at least the symptoms that characterize cancer.

[0213] The antibody, fusion protein or conjugate of the present disclosure can be administered to an individual alone or in combination with a second agent. Second agents of interest include, but are not limited to, agents approved by the U.S. Food and Drug Administration and / or the European Medicines Agency (EMA) for use in the treatment of cancer. In some embodiments, the second agent is an immune checkpoint inhibitor. Immune checkpoint inhibitors of interest include, but are not limited to, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitors, programmed cell death-1 (PD-1) inhibitors, programmed cell death ligand-1 (PD-L1) inhibitors, lymphocyte activation gene-3 (LAG-3) inhibitors, T cell immunoglobulin domain and mucin domain 3 (TIM-3) inhibitors, indoleamine (2,3)-dioxygenase (IDO) inhibitors, T cell immunoreceptor with Ig and ITIM domain (TIGIT) inhibitors, V-domain Ig suppressor of T cell activation (VISTA) inhibitors, B7-H3 inhibitors, and any combination thereof.

[0214] When the antibody, fusion protein or conjugate of the present disclosure is administered together with a second agent, the antibody, fusion protein or conjugate and the second agent can be administered to an individual according to any suitable administration regimen. According to certain embodiments, the antibody, fusion protein or conjugate and the second therapeutic agent are administered according to a dosing regimen approved for the individual's use. In some embodiments, administration of the antibody, fusion protein or conjugate allows the second agent to be administered according to a dosing regimen with one or more lower doses and / or less frequent administrations, and / or a reduced number of cycles compared to when the second agent is administered without the antibody, fusion protein or conjugate. In certain aspects, administration of the second agent allows the antibody, fusion protein or conjugate to be administered according to a dosing regimen with one or more lower doses and / or less frequent administrations, and / or a reduced number of cycles compared to when the antibody, fusion protein or conjugate is administered without the second agent.

[0215] In some embodiments, one or more doses of the antibody, fusion protein or conjugate and the second agent are administered to an individual simultaneously. By "simultaneously" is meant that the antibody, fusion protein or conjugate and the second agent are present in the same pharmaceutical composition, or that the antibody, fusion protein or conjugate and the second agent are administered within 1 hour or less, 30 minutes or less, or 15 minutes or less as separate pharmaceutical compositions.

[0216] In some embodiments, one or more doses of the antibody, fusion protein or conjugate and the second agent are administered sequentially to the individual.

[0217] In some embodiments, the antibody, fusion protein or conjugate and the second agent are administered to an individual in different compositions and / or at different times.For example, the antibody, fusion protein or conjugate may be administered (e.g., in a certain cycle) before the administration of the second therapeutic agent.Alternatively, the second therapeutic agent may be administered (e.g., in a certain cycle) before the administration of the antibody, fusion protein or conjugate.The second agent may be administered in a period starting at least 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, or up to 5 days or more after the administration of the first agent may be administered.

[0218] In one example, the second agent is administered to an individual for a desired period of time prior to administration of the antibody, fusion protein or conjugate.In certain embodiments, such a regimen "primes" cancer cells to enhance the anti-cancer effect of the antibody, fusion protein or conjugate.Such a period of time that separates the step of administering the second agent from the step of administering the antibody, fusion protein or conjugate is desirably long enough to allow the priming of cancer cells so that the anti-cancer effect of the antibody, fusion protein or conjugate is increased.

[0219] In some embodiments, the administration of one agent is specifically timed relative to the administration of another agent. For example, in some embodiments, an antibody, fusion protein, or conjugate is administered such that a particular effect is observed (or is expected to be observed, for example, based on population studies showing a correlation between a given dosing regimen and a particular effect of interest).

[0220] In certain aspects, the desired relative dosing regimens of co-administered agents may be empirically evaluated or determined, e.g., using ex vivo, in vivo and / or in vitro models. In some embodiments, such evaluation or empirical determination is performed in vivo, in a patient population (e.g., so that correlations are established), or alternatively in a particular individual of interest.

[0221] In some embodiments, the antibody, fusion protein or conjugate and the second agent are administered according to an intermittent dosing regimen comprising at least two cycles. When two or more agents are combined and each is administered by such an intermittent cycling regimen, individual doses of different agents may be combined with each other. In certain aspects, one or more doses of the second agent are administered a period of time after a dose of the first agent. In certain embodiments, each dose of the second agent is administered a period of time after a dose of the first agent. In certain aspects, each dose of the first agent is followed a period of time by a dose of the second agent. In some embodiments, two or more doses of the first agent are administered between at least one pair of doses of the second agent, and in certain aspects, two or more doses of the second agent are administered between at least one pair of doses of the first agent. In some embodiments, different doses of the same agent are separated by a common time interval, and in some embodiments, the time interval between different doses of the same agent varies. In certain aspects, the different doses of the antibody, fusion protein or conjugate and the second agent are separated from each other by a common time interval, and in some embodiments, the different doses of the different agents are separated from each other by different time intervals.

[0222] One exemplary protocol for combining two intermittent cycling dosing regimens with each other can include (a) a first dosing period during which a therapeutically effective amount of an antibody, fusion protein or conjugate is administered to the patient, (b) a first rest period, (c) a second dosing period during which a therapeutically effective amount of a second agent is administered to the patient, and (d) a second rest period. A second exemplary protocol for combining two intermittent cycling dosing regimens with each other can include (a) a first dosing period during which a therapeutically effective amount of a second agent is administered to the patient, (b) a first rest period, (c) a second dosing period during which a therapeutically effective amount of an antibody, fusion protein or conjugate is administered to the patient, and (d) a second rest period.

[0223] In some embodiments, the first rest period and the second rest period may correspond to the same time or days. Alternatively, in some embodiments, the first rest period and the second rest period are different, and the first rest period is either longer than the second rest period or vice versa. In some embodiments, each of the rest periods corresponds to 120 hours, 96 hours, 72 hours, 48 ​​hours, 24 hours, 12 hours, 6 hours, 30 hours, 1 hour or less. In some embodiments, if the second rest period is longer than the first rest period, this may be defined as more than a few hours, days or weeks (e.g., 1 day, 3 days, 5 days, 1 week, 2 weeks, 4 weeks or more).

[0224] If the length of the first rest period is determined by the presence or occurrence of a particular biological or therapeutic event, the length of the second rest period can be determined based on different factors, either separately or in combination. Exemplary such factors can include one or more characteristics of the type and / or stage of the cancer to which the treatment is administered, the characteristics (e.g., pharmacokinetic characteristics) of the antibody, fusion protein or conjugate, and / or the patient's response to therapy with the antibody, fusion protein or conjugate. In some embodiments, the length of one or both rest periods can be adjusted in light of the pharmacokinetic characteristics (e.g., as assessed via plasma concentration levels) of one or the other of the administered agents. For example, the associated rest period can be considered complete when the plasma concentration of the associated agent falls below a predetermined level, optionally upon evaluation or other consideration of one or more characteristics of the individual's response.

[0225] In certain aspects, the number of cycles that a particular agent is administered may be empirically determined, and in some embodiments, the exact regimen followed (e.g., number of doses, intervals between doses (e.g., relative to each other or to another event, such as administration of another therapy), amount of doses, etc.) may be different for one or more cycles compared to one or more other cycles.

[0226] The antibody, fusion protein or conjugate and the second agent can be administered together or independently via any suitable administration route.The antibody, fusion protein or conjugate and the second agent can be administered via an administration route that is independently selected from oral, local (e.g., intravenous, intraarterial, subcutaneous, intramuscular, or epidural injection), or intranasal administration.According to certain embodiments, the antibody, fusion protein or conjugate and the second agent are both administered orally (e.g., in tablet form, capsule form, liquid form, etc.), either simultaneously (in the same pharmaceutical composition or in separate pharmaceutical compositions) or sequentially.

[0227] Where the method further comprises infecting cancer cells by administering an OV to the individual prior to administering the pharmaceutical composition to the individual, any suitable administration regimen can be used to infect the cancer cells. Poxvirus replication occurs in the cytoplasm because the virus is sufficiently complex to have acquired all the functions necessary for genome replication. Once inside the cytoplasm, gene expression is carried out by viral enzymes associated with the core. Expression is divided into two stages: early genes, which make up about 50% of the genome, are expressed before genome replication, and late genes are expressed after genome replication. Temporal control of expression is provided by late promoters that depend on active DNA replication. Genome replication is thought to involve self-priming, which leads to the formation of high molecular weight concatemers that are then cleaved and repaired to create the viral genome. Virus assembly occurs in the cytoskeleton, possibly involving interactions with cytoskeletal proteins (e.g., actin-binding proteins). Cytoplasmic inclusions form, which mature into viral particles. Vaccinia virus is unique among DNA viruses because it replicates exclusively in the cytoplasm of the host cell. Therefore, a large genome is required to encode the various enzymes and proteins required for viral DNA replication. During replication, vaccinia produces several infectious forms that differ in their outer membrane: intracellular mature virions (IMV), intracellular enveloped virions (IEV), cell-associated enveloped virions (CEV), and extracellular enveloped virions (EEV).

[0228] To infect cancer cells with OV, OV is administered using a suitable route of administration. The route of administration may vary depending on the location and nature of the cancer, and may include, for example, intradermal, transdermal, parenteral, intravenous, intramuscular, intranasal, subcutaneous, local (e.g., near the tumor, especially the tumor vasculature or adjacent vasculature), transdermal, intratracheal, intraperitoneal, intraarterial, intravesical, intratumoral, inhalation, perfusion, lavage, and oral administration and formulation. Intratumoral injection, or direct injection into tumor vasculature, is particularly contemplated for individual, solid, accessible tumors. Local, regional, or systemic administration may also be appropriate. The viral particles may be advantageously contacted by administering multiple injections into the tumor, for example, at intervals of about 1 cm. Sequential administration may also be applied, where appropriate, for example, by implanting a catheter into the tumor or tumor vasculature. Such continuous perfusion may occur, for example, over a period of about 1-2 hours, about 2-6 hours, about 6-12 hours, or about 12-24 hours after initiation of dosing. Dosing regimens may vary and often depend on the type of tumor, the location of the tumor, the progression of the disease, and the health and age of the patient. Certain types of tumors require more aggressive treatment, while at the same time, certain patients cannot tolerate more burdensome protocols. The clinician is best suited to make such decisions.

[0229] Injection of the nucleic acid construct can be delivered by a syringe or any other method used for injecting solutions, so long as the expression construct can pass through the particular gauge of the needle required for injection. An exemplary needle-free injection system that can be used to administer OVs is illustrated in U.S. Pat. No. 5,846,233. This system features a nozzle that defines an ampoule chamber for holding the solution, and an energy device for forcing the solution out of the nozzle to the delivery site. Another exemplary syringe system is one that allows multiple injections of precisely predetermined amounts of solution at any depth (U.S. Pat. No. 5,846,225). Mixtures of OV particles or nucleic acids encoding same may be prepared in water suitably mixed with one or more excipients, carriers, or diluents. Dispersions may be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as oils.

[0230] A physician may begin by prescribing a dose of the OV vector at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved. Alternatively, a physician may begin a treatment regimen by administering a single dose of the OV vector, and then administer progressively lower doses until a therapeutic effect is achieved, such as a reduction in the volume of one or more tumors.

[0231] The following examples are offered by way of illustration and not by way of limitation. EXAMPLES

[0232] experiment Example 1 - Generation of antibodies A56 or B5 sequences from the Wyeth VV strain (SEQ ID NO: 180 and SEQ ID NO: 181, respectively) were used to generate stable CHO cell lines expressing either vaccinia virus A56 (CHO-A56) or B5 capsid protein (CHO-B5). Antibodies were raised in New Zealand White rabbits by successive semi-monthly subcutaneous immunizations with a mixture of CHO-A56 and CHO-B5 cells (30 million per cell type in the first injection and 45 million in subsequent injections) mixed with aluminum hydroxide (5 mg / injection) and CpG (catalog no. ODN1826, 20 μg / injection). One week after the 10th injection, the rabbits were euthanized and the spleens were harvested (Cedarlane). B cells were cultured and isolated according to Babcook et al. (1). Antibodies were identified by screening B cell supernatants for binding by flow cytometry to OVCAR3 cells (ATCC, catalogue no. HTB-161) infected with VV Western Reserve strain VVdd(eGFP) (gift from Dr. John Bell, Ottawa Hospital Research Institute (OHRI), SEQ ID NOs: 182 and 183) (data not shown). To clone antibodies from cultured B cells, frozen cells were thawed and analysed by A56.hFc or B5.hFc hemolytic plaque assay. Human Fc fusion proteins were generated in-house using A56 or B5 sequences from Wyeth VV strains, as shown in Tables 2 and 4. Isolated single B cells were lysed and antibody V genes were amplified by RT-PCR. PCR products corresponding to the heavy and light chains of the matched antibodies were then cloned into human IgG1 constant region and Igκ constant region constructs (pTT5 / IgG1, pTT5 / Igk). To produce recombinant antibodies, VH and VL chain plasmids were transfected into HEK293-6E cells using 293fectin (Thermo, Cat. No. 12347019). After 96 hours of secretion, the antibody-containing supernatant was cleared of cells by centrifugation and sterile filtration (0.22 μm).Antibodies were purified using HiTrap Mab Select SuRe columns (GE, Catalog No. 11003495) and HiPrep 26 / 10 Desalting columns (GE, Catalog No. 17508701) on an AKTAExpress system. Antibody purity was tested by SDS-PAGE, UPLC-SEC and LC-MS. The sequences of the antibodies produced are shown in Tables 1 and 3. Antibody IDs and formats are shown in Table 15 below. [Table 15]

[0233] Example 2 - Binding of antibodies to A56 and B5 antigens Lenti-x293T (Clontech, catalog no. 632181) cells were used to generate stable cell lines, and HEK-A56(GFP) and HEK-B5(GFP) cells expressing antigens A56 (Wyeth VV sequence) or B5 (Wyeth VV sequence) on the cell surface. A549 (ATCC, catalog no. CCL-185) and CaOv3 (ATCC, catalog no. HTB-75) tumor cells were grown to 80-90% confluence in appropriate media (A549: F-12K Nutrient Mixture (Gibco, catalog no. 21127-022); CaOv3: DMEM (Gibco, catalog no. 11995-040) supplemented with 10% FBS (Corning, catalog no. 35-015-CV). Cells were infected with VVdd(eGFP) at a multiplicity of infection of 0.5 in serum-free RPMI (Gibco, Cat. No. A10491-01) and incubated for 6.5 h at 37°C in 5% CO2. Wells were supplemented with growth medium 1:2 and further incubated overnight. A549 cells were trypsinized and plated together with CaOV3 cells at approximately 1 × 10 per well in 96-well V-bottom plates (Sarstedt, Cat. No. 82.1583.001). 5 Corresponding uninfected A549 and CaOV3 cells, as well as HEK-A56(GFP), HEK-B5(GFP) and HEK-WT cell lines were also plated at 4 × 10 cells per well. 4Anti-vaccinia virus rabbit polyclonal antibody (CedarLane, catalog no. LS-C103289) was used as a positive control.

[0234] Anti-A56 antibody (A047, A049, A050 or A054), anti-B5 antibody (A048 or A051), and human IgG isotype control (Sigma, Cat. No. I5154) were added to the cells and incubated on ice for 25 min. Cells were washed twice with PBS, centrifuged at 450G for 4 min, and incubated with 1:100 AlexaFluor 647-conjugated anti-rabbit IgG (Jackson, Cat. No. 115-605-046) or AlexaFluor 647-conjugated anti-human IgG (Jackson, Cat. No. 109-605-098) and 1:1000 fixable viability dye eFluor 780 (eBioscience, Cat. No. 65-0865-14) for 15 min at room temperature (RT). After washing twice with PBS, cells were fixed for 10 min with IC fixation buffer (eBioscience, Cat. No. 00-8222-49), washed twice with PBS, resuspended in 1% FBS in PBS and analyzed by flow cytometry on a BD LSRFortessa X-20 HTS system and data processed with FlowJo V 10.5.3 Software. The data show binding of the antibodies to VV VVdd (eGFP) infected cells (Figure 1). Anti-A56 antibody specifically binds to HEK-A56 (GFP) cells and anti-B5 antibody to HEK-B5 (GFP) cells (Figure 2). No binding was observed to negative controls (HEK parental cells, uninfected A549 or CaOV3 cells and the irrelevant VV antigen A33 also expressed in HEK cells) (data not shown). The isotype control hIgG showed no binding to the cells.

[0235] A commercially available anti-B5 antibody (Immunetech, Cat. No. IT-012-009M1, raised using VV Western Reserve immunogen) was tested for binding to the B5 antigen. A dilution series of anti-vaccinia rabbit polyclonal antibody was compared by flow cytometry to the Immunetech anti-B5 antibody ("IT anti-B5 IgG") and isotype control under assay conditions similar to those described above. Goat anti-mouse A647 (Jackson, Cat. No. 115-005-146) was used for mouse antibody detection of IT anti-B5 IgG and mouse isotype control. IT anti-B5 IgG did not bind to HEK-B5 cells (Figure 3, top panel). An ELISA to assess binding to B5.hFc soluble antigen confirmed that this antibody does not bind to the B5 reagent made based on the VV WyethB5 sequence. For this assay, B5.hFc was coated at 2 μg / ml onto maxisorp plates and incubated overnight at 4°C. Following standard ELISA procedures, plates were washed, blocked with 5% nonfat dry milk, and primary antibodies were incubated in block for 1 h at RT and washed. Secondary detection was performed with goat anti-mIgG Fc HRP (Jackson, Cat. No. 115-035-071) or anti-Rb IgG Fc HRP (Jackson ImmunoResearch, Cat. No. 111-035-046) for 1 h at RT, followed by washing and addition of 50 μl / well TMBK-Blue (Neogen, #308175) followed by 50 μl / well 1N HCl. By standard ELISA, IT anti-B5 IgG was negative for binding to B5.hFc soluble antigen (Figure 3, lower panel).

[0236] IT anti-B5 IgG was also tested for binding to A549 and HT29 cells infected with Western Reserve VVddeGFP. A549 and HT29 cells (ATCC catalogue no. HTB-38, cultured in McCoy's 5A medium (Gibco #16600-082) + 10% FBS) were grown to 80-90% confluence. Cells were infected with VVdd(eGFP) at a multiplicity of infection of 1 in serum-free RPMI. Cells were incubated for 6.5 hours at 37°C in 5% CO2, replenished with growth medium 1:2 and further incubated overnight. Cells were trypsinized and plated at approximately 1 × 10 per well in 96-well V-bottom plates. 5 Cells were plated at 1000 x 1000 cells. Anti-B5 antibody A048 hIgG1 and IT anti-B5 IgG, as well as human isotype control, were added to the cells and incubated on ice for 25 min. Cells were washed and antibodies were detected as above using AlexaFluor 647-conjugated anti-human IgG and anti-mouse IgG (Jackson, #115-605-164) followed by 1:1000 fixable viability dye eFluor780. After two washes, fixation and resuspension in 1% FBS in PBS, samples were analyzed by flow cytometry on a BD LSRFortessa X-20 HTS system. The data show that the A048 antibody binds more strongly than IT anti-B5 IgG to A549 and HT29 cells infected with Western Reserve VVddeGFP (Figure 4). The EC50 for binding of the A048 antibody to infected A549 and HT29 cells is 6 nM and 1.1 nM, respectively. The EC50 for binding of IT anti-B5 IgG to infected A549 and HT29 cells is 78 nM and 15.8 nM, respectively.

[0237] Example 3 - Reformatting into Mouse and Rabbit Antibodies Antibodies were reformatted into full rabbit IgG or chimeric rabbit / mouse IgG2a Fc antibodies, expressed in HEK293 cells, and purified as described above. A549 cells infected with VVdd(eGFP) as described above were tested for binding by antibodies as rabbit IgG, or mouse or human chimeras. Anti-A56 antibodies (A056, A049, A059) and anti-B5 antibodies (A073, A048, A058), as well as rabbit IgG isotypes (Jackson, Cat. No. 011-000-003), human IgG isotypes (Sigma Aldrich, Cat. No. I5154), and mouse IgG isotypes (R&D Biosystems, Cat. No. MAB004) were added to infected cells and incubated on ice for 25 minutes. Cells were washed and incubated with 1:100 AlexaFluor 647-conjugated anti-rabbit IgG (Jackson, Cat. No. 115-605-046), or anti-human IgG (Jackson, Cat. No. 109-605-098), or anti-mouse IgG (Jackson, Cat. No. 115-605-164), 1:1000 DAPI (BioLegend, Cat. No. 422801) for 15 min at RT. Following washing and fixation, samples were analyzed by flow cytometry. The data confirm that all formats bind VV VVdd (eGFP) infected cells with similar EC50 (Figure 5, Table 16). No binding was observed to uninfected cells or with the isotype controls. [Table 16]

[0238] Example 4 - Antibody binding to virus-infected tissue On day 0, BALB / c female mice were 6 MC38 cells were transplanted. In some mice, VVdd(eGFP) was administered IT (2.5 × 10 in 100 μl) on days 11 and 13 after MC38 transplantation. 7pfu) and mice were euthanized on day 16. Control mice bearing MC38 tumors were treated with PBS instead of VVdd(eGFP) and euthanized on day 12 after implantation. Tumor tissues were collected in 10% neutral buffered formalin (NBF). Tissues were formalin fixed, paraffin embedded, sectioned, and stained. H&E stained images were scanned using an Aperio ScanScope AT2 digital slide scanner. Tissues were deparaffinized and rehydrated according to standard protocols. For heat-induced epitope retrieval, tissues were autoclaved at 121°C for 20 min in antigen retriever (Sigma, Cat. No. C999-100ML). After cooling, slides were rinsed twice with PBS and sections were isolated using a PAP pen to create a hydrophobic barrier. Tissues were hydrated with 200 μl of wash buffer (0.1% Tween-20, 0.1% BSA in PBS). Tissues were permeabilized for 10 min at RT using 0.2% Triton in PBS and washed three times. Sections were blocked for 60 min with blocking buffer (5% normal goat serum, 0.05% Tween-20, 0.1% BSA in PBS). After removing the blocking buffer, primary antibodies in antibody diluent (1% normal goat serum, 0.05% Tween-20, 0.1% BSA in PBS) were applied to the sections and incubated overnight at 4°C. A049 (Anti-A56) hIgG1 was used at 12.5 μg / ml and the positive control polyclonal rabbit anti-vaccinia virus antibody (CedarLane, catalog number LS-C103289) was used at 1:100. Human IgG1 (Sigma, Cat. No. I5154) and rabbit IgG (Jackson ImmunoResearch Laboratories, Cat. No. 011-000-003) isotype controls were used at 25 μg / ml. The following day, tissues were washed and secondary antibodies (200 μl) were prepared in antibody diluent and incubated for 1.5 hours at room temperature in the dark.Tissues stained with human primary antibodies were detected using AlexaFluor 549-conjugated goat anti-human IgG (Jackson, Cat. No. 109-585-003) at 1:100, and rabbit primary antibodies were detected using AlexaFluor Plus 488 goat anti-rabbit IgG (H+L) antibody (Thermo, Cat. No. A32731) at 10 μg / ml. After incubation, slides were washed three times for 5 min each. Hoechst stain (Sigma, Cat. No. B2261) was added to the tissues at 1 μg / ml and incubated for 10 min, after which the excess was removed. Fluoromont-G (Southern Biotech, Cat. No. 0100-01) was added to the coverslips and imaged using a Leica SP5X Laser Scanning Confocal Microscope. The data confirmed the binding of A049 to VV VVdd (eGFP)-infected tumor tissues, with no binding observed to uninfected tumor tissues (Figure 6). Polyclonal rabbit anti-vaccinia virus antibodies show non-specific binding to uninfected tumor tissue.

[0239] Example 5 - Antibody affinity to B5 and A56 proteins Antibody affinity was evaluated using a Biacore T200. On a Series SCM5 chip, A048 was immobilized on FC2, A051 on FC3, and FC1 was used as a blank immobilization. The sensor surface was activated by injecting EDC and NHS. Ligands were immobilized as follows: anti-B5 A048 hIgG1 (12 μg / mL) was prepared in 200 μL sodium acetate (pH 4.5) and A051 (12 μg / mL) was prepared in 200 μl sodium acetate (pH 5.0). Remaining activated groups were blocked by injecting ethanolamine. Finally, the chip was washed with 50 mM NaOH. After amine coupling of approximately 1000 RU of each antibody, the chip was equilibrated with buffer. All binding studies were performed in freshly prepared and filtered running buffer HBS-EP+ (GE, catalog no. BR100188). B5.hFc (MW: 150 kDa) was flowed as analyte at increasing concentrations of antigen (1.5 nM, 4.6 nM, 13.8 nM, 41.4 nM, and 124 nM) with 30 μl / min association for 200 s followed by one 30 min dissociation. At the end of each cycle, the IgG capture surface was regenerated by injecting 10 mM glycine, pH 1.5, at 30 μl / min for 60 s. Double reference subtraction was performed by injecting running buffer without analyte. Curves were fitted using 1:1 Langmuir coupling.

[0240] Similar experimental conditions were set for the A56 antibody, except that different concentrations of A56.hFc (0.6nM, 1.9nM, 5.6nM, 16.7nM and 50nM) were flowed over the sensor. Because bivalent antigens B5.hFc and A56.hFc are used, the interaction with the antibody may not be a simple 1:1 binding, so the apparent KD is listed (Table 17). The affinity of A051 could not be measured under these conditions, as it was at the detection limit of the Biacore T200. [Table 17]

[0241] Antibodies were assessed for affinity to A56.hFc or B5.hFc.

[0242] Example 6 - Epitope binning Antibodies were biotinylated using EZ-Link Sulfo-NHS-LC-Biotin (Thermo, Cat. No. 21343) at a ratio of 20:1 according to the manufacturer's protocol. A549 cells were infected with VVdd(eGFP) at an MOI of 0.5 and fixed the day before the assay. On the day of the assay, virus-infected A549 cells were preincubated with 25 μg / mL of non-biotinylated antibodies and incubated for 1 h. Then, biotinylated antibodies (10 μg / ml) were spiked into wells of a 96-well V-bottom plate to a final concentration of 5 μg / ml and incubated for 1 h. Finally, detection was performed using streptavidin-Alexa647 (Jackson, Cat. No. 016-600-084) to detect bound biotinylated antibodies. Samples were acquired on an Intellicyt HTFC high-throughput flow cytometer. An anti-ovalbumin antibody (in-house at the Center for Drug Research and Development (CDRD)) was used as an irrelevant control IgG. When a second antibody was added to the VAVC-coated plates, similar binding was observed as with the first antibody tested, so the data indicate that these anti-A56 antibodies appear to bind to a different epitope on A56. Similarly, the data indicate that these anti-B5 antibodies appear to bind to a different epitope on B5 (Figure 7).

[0243] Example 7 - Binding of antibodies to virus particles In a 96-well plate (Corning, Cat. No. 3368), 9 × 10 6VVdd(eGFP) was coated at pfu / mL (50 μl / well). After overnight incubation at 4°C, the coated wells were washed 3 times with PBS and fixing buffer was added and incubated for 10 min. The coated wells were then washed 4 times with distilled water. The wells were blocked with 5% nonfat dry milk in PBS for 1 h at RT. Antibodies (A049, A047, A050, A054, A048, A051) were diluted 1:4 and titrated in blocking buffer from a starting concentration of 20 μg / ml. NKp30, a cytotoxic receptor for NK cells, has been shown to bind VV A56 (2). NKp30-His (in-house) soluble protein was tested at 20 μg / mL or 1 μg / ml as a positive control and hIgG1 as a negative control. The wells were washed 4 times with water and 50 μl / well of antibody was added and incubated for 1 h at RT. Subsequently, wells were washed with water and 0.2 μg / mL HRP-conjugated goat anti-hIgG Fc (Jackson, Cat. No. 109-035-098) in blocking buffer was added and incubated for 1 h at RT. After washing, 50 μl / well TMB (Neogen, Cat. No. 308177) was added and the plate was incubated for 30 min at RT. Color development was stopped with 50 μl 1 M HCl. Absorbance at 450 nm was measured using a Spectromax plate reader and data was processed using SoftMaxPro. The data show that all antibodies tested except A054 bind to viral particles of VVdd (eGFP) (Figure 8).

[0244] Example 8 - Effect of antibodies on viral infection A549 cells were plated in 96-well flat-bottom plates (Flacon, Cat. No. 353075) at 5 × 10 cells per well. 4 The cells were seeded at 1.5 × 10 cells / well and allowed to adhere overnight at 37 °C and 5% CO2. 6 pfu / mL (MOI 1), 1.5 × 10 5 pfu / mL (MOI 0.1) and 1.5 × 10 4VVdd(eGFP) at 100 pfu / mL (MOI 0.01) was incubated with an equal volume of 20 μg / mL A047, A048, A049, A050, A051, A054, hIgG (Sigma, Cat. No. I5154) or 1:100 vaccinia virus polyclonal antibody (Cedarlane, Cat. No. LS-C103289) in serum-free F-12K nutrient mixture (Gibco, Cat. No. 21127-022) for 15 min at RT. Cells were infected with VVdd(eGFP) / antibody mixtures for 6.5 h at 37°C and 5% CO2, followed by addition of F-12K nutrient mixture (Gibco, Cat. No. 21127-022) supplemented with 10% FBS (Corning 35-015-CV lot 35015124) at a 1:2 dilution and cultured overnight. After incubation, the adherent cells were trypsinized and plated alongside the transferred non-adherent cells in 96-well V-bottom plates (Sarstedt, Cat. No. 82.1583.001). Cells were centrifuged at 450G for 4 min, resuspended in 1:1000 fixable viability dye eFluor 780 (eBioscience, Cat. No. 65-0865-14) and incubated at RT for 15 min. Cells were then washed with PBS, fixed with IC fixation buffer (eBioscience, Cat. No. 00-8222-49) for 10 min at RT, washed with PBS, and finally resuspended in 1x PBS with 6000 beads per well of CountBright absolute counting beads (Invitrogen, Cat. No. C36950). Samples were analyzed by flow cytometry on a BD LSRFortessa X-20 HTS system and data were processed with FlowJo V 10.5.3 Software. The data show that addition of these antibodies does not inhibit or reduce VVdd(eGFP) infection of tumor cells (Figure 9).

[0245] Example 9 - Antibody Thermostability Antibody denaturation temperature (T m) was determined from differential scanning fluorimetry (DSF) using the Protein Thermo Shift Dye Kit™ (ThermoFisher, Cat. No. 4461146). Briefly, 31 μg / mL of antibody was used in each reaction. Antibody melting curves were generated using an Applied Biosystems QuantStudio 7 Flex Real-Time PCR System with the recommended settings described in the kit manual. The T of the antibody was then determined using ThermoFisher Protein Thermal Shift software (v.1.3). m (Table 18) was determined. m 1 was determined by DSF. [Table 18]

[0246] Example 10 - Binding of antibodies to vaccinia Copenhagen-infected mouse cell lines B16F10 (ATCC, Cat. No. CRL-6475), CT26LacZ (ATCC, Cat. No. CRL-2639) and MC38 (kindly provided by Dr. John Bell at OHRI) tumor cells were grown in 10 cm tissue culture plates (Thermo Scientific, Cat. No. 12-556-002) to 80-90% confluence in the appropriate media (B16F10: DMEM (Gibco, Cat. No. 11995-040); MC38: DMEM (Gibco, Cat. No. 11995-040); CT26LacZ: RPMI1640 (Gibco, Cat. No. 11875-093), supplemented with 10% FBS (Gibco, Cat. No. 12483020)). Cells were infected with VVCopenhagen (YFP) (a gift from Dr. John Bell, OHRI) at a multiplicity of infection of 0.5 in serum-free DMEM (Gibco, Cat. No. 11995-040) and incubated at 37°C for 6.5 hours in 5% CO2. Plates were supplemented with growth medium at a 1:2 ratio and further incubated overnight (~16 hours). B16F10 cells were trypsinized (TrypLE, Cat. No. 12604021) and plated at approximately 1 × 10 per well in 96-well U-bottom plates (Sarstedt, Cat. No. 83.3925). 5 The corresponding uninfected B16F10, CT26LacZ and MC38 cells were plated at 1 × 10 per well. 5Cells were plated at 100x100 cells / well. Cells were washed twice with PBS 3% FBS (FACs Buffer) and centrifuged at 1800 rpm for 3 min. Cells were resuspended in PBS and incubated with fixable viability dye Zombie NIR (Biolegend, Cat. No. 423106) at 1:3000 for 15 min at RT. After washing twice with FACS buffer, cells were resuspended in FACS buffer containing mouse Fc block (BD, Cat. No. 553141) and incubated for 5 min at RT. Without further washing, cells were directly stained with either anti-A56-PE (A056, Rb IgG) at 1:100, anti-B5-PE (A073, Rb IgG) at 1:100, or anti-vaccinia virus rabbit polyclonal antibody (CedarLane, Cat. No. LS-C103289) at 1:100 (serving as positive control) and incubated at 4°C for 30 min. Cells were washed twice with FACS buffer and resuspended. Wells that received anti-vaccinia virus primary antibody were then stained with anti-rabbit IgG-PE (Biolegend, Cat. No. 406421) at 1:100 and incubated for an additional 20 minutes at 4°C. Cells were further washed with FACS buffer, then fixed with IC fixation buffer (eBioscience, Cat. No. 00-8222-49) for 10 minutes, washed twice with PBS, resuspended in FACS buffer, and analyzed by flow cytometry on a Cytek Aurora flow cytometer. Data were processed using FlowJoV10.5.3 Software. Data show percent YFP expression in each infected cell line, indicating the proportion of cells infected with VVCopenhagen (YFP) (Figure 10A). Anti-A56-PE and anti-B5-PE antibodies were found to bind to B16F10, CT26LacZ, and MC38 cells that were YFP-positive and therefore infected with VV Openhagen (YFP) (Figure 10B). As expected, anti-vaccinia virus rabbit polyclonal antibodies also bound to the infected (YFP-positive) cells. No binding was observed to uninfected negative control cells.

[0247] Example 11 - Immunohistochemical detection of vaccinia A56 and B5 The HEK-B5(GFP) cell line was grown to 90% confluence in ten 15 cm tissue culture plates (Corning, Cat. No. 353025) in the appropriate medium (HEK-DMEM (Gibco, Cat. No. 11995-040) supplemented with 10% FBS (Gibco, Cat. No. 12483020). In addition, U2OS cells (a gift from Dr. John Bell, OHRI) were grown to 90% confluence and infected with VVCopenhagen (YFP) at a multiplicity of infection of 0.025 and incubated for 72 hours at 37°C in 5% CO2. HEK-A56, HEK-B5, HEK-WT and U2OS cells were trypsinized (TrypLE, Cat. No. 12604021), counted and plated at 2 × 10 in 15 ml polypropylene tubes (FroggaBio, Cat. No. TB15-500). 8Cells were centrifuged at 1500 RPM for 5 minutes, resuspended in 1 ml of molten histogel (Thermo Scientific, Cat. No. HG-4000-012) and placed on ice for 10 minutes to allow the histogel to solidify. The histogel pellet was transferred to 10 ml of 10% neutral buffered formalin (Sigma, Cat. No. HT501128) and left at RT for 24 hours. For immunohistochemistry (IHC), histogel sections (4 μm) were incubated overnight at 37°C, deparaffinized, rehydrated using a xylene and ethanol gradient, antigen retrieved using Diva decloaking reagent (Biocare, Cat. No. DV2004) in a Biocare decloaking chamber (110°C for 15 minutes) and loaded onto an Intellipath FLX Autostainer. Brightfield IHC was performed at room temperature using Biocare reagents. Slides were treated with Peroxidased-1 (Biocare, Cat. No. PX968) for 5 min and Background Sniper (Biocare, Cat. No. BS966) for 10 min. Primary antibodies diluted in DaVinci Green (Biocare, Cat. No. PD900) were applied for 30 min, followed by Mach2 HRP Polymer (either mouse or rabbit based on the species of the primary antibody) (Biocare, Cat. No. RHRP520 and MHRP520) for 30 min. DAB Western Horseradish Peroxidase (Biocare, Cat. No. IPK5010G80) chromogen substrate was added to the slides. CAT Hematoxylin (Biocare, Cat. No. CATHE) diluted 1:5 in dH2O was added for 5 min at RT. Stained slides were washed with water, air-dried and coverslipped using Ecomount (Biocare, Cat. No. EM897L). The dilutions of the antibodies are as follows: A058 anti-B5, Rb / Mu IgG2a: 1 / 280 (10 μg / mL); A059 anti-A56, Rb / Mu IgG2a: 1 / 540 (10 μg / mL); A056 anti-A56, Rb IgG: 1 / 840 (10 μg / mL); A073 anti-B5, Rb IgG: 1 / 550 (10 μg / mL).Antibodies specific for A56 or B5 were able to specifically detect the target antigen on stably transfected HEK-A56 and HEK-B5 cells, respectively (Figure 11). No cross-reactivity was observed to alternative antigens or HEK-WT cells. Both anti-A56 and anti-B5 antibodies were able to detect protein expression on infected U2OS cells, and no background staining was detected on uninfected U2OS cells (Figure 11).

[0248] Example 12 - Immunohistochemical detection of A56 and B5 from VV-treated tumors C57BL / 6 wild-type mice (Jackson Laboratory, Cat. No. 000664) were injected with 1 × 10 6 B16F10 (ATCC, Cat. No. CRL-6475) tumor cells were implanted subcutaneously. Mice were grouped as follows: 1 dose of VV Copenhagen (YFP), 2 doses of VV Copenhagen (YFP), PBS, and Vesicular Stomatitis Virus (VSV) (a gift from Dr. John Bell, OHRI). All injections were 1 × 10 in a volume of 100 μl. 7 The dose was 100 PFU, and the tumor was 35-40 mm 2The tumors were delivered intratumorally when they reached a size of 10 μg / ml. Animals were euthanized 1, 2, 3, or 7 days after the first injection. Tumors were collected and fixed in 10% neutral buffered formalin for 24 h. After fixation, tumors were embedded in paraffin wax and one to five 2 mm tumor punches were taken from each resected tumor. Tumor punches were arrayed to create tumor tissue microarrays (TMA) and sectioned onto glass slides. For IHC, TMA sections (4 μm) were incubated overnight at 37 °C, deparaffinized, rehydrated using xylene and ethanol gradients, antigen retrieval was performed using Diva decloaking reagent (Biocare, catalog no. DV2004) in a Biocare decloaking chamber (110 °C for 15 min) and loaded onto an Intellipath FLX Autostainer. Brightfield IHC was performed at RT using Biocare reagents. Slides were treated with Peroxidazed-1 (Biocare, Cat. No. PX968) for 5 min and Background Sniper (Biocare, Cat. No. BS966) for 10 min. Primary antibodies diluted in DaVinci Green (Biocare, Cat. No. PD900) were applied for 30 min followed by Mach2 HRP Polymer (either mouse or rabbit based on the species of the primary antibody) (Biocare, Cat. No. RHRP520 and MHRP520) for 30 min. DAB Western Horseradish Peroxidase (Biocare, Cat. No. IPK5010G80) chromogen substrate was added to the slides. CAT Hematoxylin (Biocare, Cat. No. CATHE) diluted 1 / 5 in dH2O was added for 5 min at RT. Stained slides were washed with water, air-dried and coverslipped using Ecomount (Biocare, Cat. No. EM897L). Slides were stained with anti-A56 (A056) or anti-B5 (A073) Rb IgG and detected with anti-rabbit secondary antibody. The dilutions of the antibodies are as follows: A056 anti-A56, Rb IgG: 1 / 840 (10 μg / mL); A073 anti-B5, Rb IgG: 1 / 550 (10 μg / mL). DAB staining (dark grey) indicates positive protein detection (Figure 12).Antibodies against A56 or B5 were able to specifically detect the target antigen in the majority of cores taken from treated tumors. Increased protein detection was evident in tumors that received multiple doses of VVCopenhagen (YFP). No cross-reactivity was seen to tumors treated with PBS or VSV. These findings demonstrate the specific detection and extent of virus-derived A56 and B5 protein expression following intratumoral delivery of vaccinia virus.

[0249] Example 13 - Design of VV chimeric antigen receptor (CAR) and detection after lentiviral transduction All human VV-CAR constructs contain the GM-CSFRα leader sequence followed by the A56 or B5 scFv (A56:A049, derived from B5:A048), the human CD8α hinge and transmembrane domain, the human 4-1BB intracellular signaling domain, and the human CD3ζ intracellular signaling domain (Figure 13A) (3). Some CAR constructs contained a reporter protein (e.g., eGFP) separated by a T2A ribosomal skip sequence. The amino acid sequences of the CARs are shown in Table 7.

[0250] Gene fragments encoding A56-CAR-01, A56-CAR-02, B5-CAR-03, B5-CAR-04, or A56-CAR-06 were synthesized by Twist Biosciences. Gene fragments were cloned into second-generation transfer plasmids (gift from Dr. Robert Holt, BC Cancer). Lentiviruses encoding VV-CAR constructs were generated using a standard calcium phosphate transfection protocol (4) and second-generation packaging vectors (gift from Dr. Rob Holt, BC Cancer). 500 μl of supernatant from each lentiviral transfection was used to transduce Jurkat T cells (ATCC, catalog no. TIB-152). Jurkat cells were then expanded for 1 week and transduced with 1 × 10 6CAR-positive cells were sorted based on goat anti-Rb IgG F(ab')2-Alexa Fluor 647 detection (Jackson Laboratories, Cat. No. 111-605-047). The enriched Jurkat CAR-positive population was expanded for an additional week and plated in co-culture assays with HEK-A56, HEK-B5 or HEK-WT cells. Cells were cultured at a 1:1 effector:target (E:T) ratio (1 × 10 5 The cells were cultured overnight (16 hours) with 1000 total cells (1000 total cells). The next day, cells were washed with PBS, blocked with normal goat serum (Jackson Laboratories, Cat. No. 005-000-001), and stained with CD45 v450 (BD, Cat. No. 560367), CD69 PE-Cy7 (Biolegend, Cat. No. 310912), and anti-Rb IgG Alexa Fluor647. A56-CAR-01, A56-CAR-02, and A56-CAR-06 showed downregulation of cell surface CAR molecules when cultured with the target HEK-A56 line. Such downregulation of cell surface CAR molecules indicates CAR activation (Figure 14). Similarly, B5-specific CARs, B5-CAR-03 and B5-CAR-04, showed downregulation of surface CAR when cultured with the target HEK-B5 line. There was no downregulation of the CAR when CAR-T cells were cultured with the HEK WT line or with an irrelevant target antigen. These data indicate that effector T cells transduced to express A56- or B5-specific CARs undergo activation (as evidenced by downregulation of cell surface CAR expression) upon encounter with target cells bearing the appropriate antigen (A56 or B5, respectively).

[0251] Example 14 - Expression of CAR and eGFP on activated A56-CAR-06 positive Jurkat T cells Jurkat A56-CAR-06 line, which co-expresses eGFP, was cultured with HEK-A56 line overnight (16 hours), and as expected from the results of Example 13, CAR expression was observed to be reduced (Figure 15). However, within the CAR "negative" population, an eGFP-positive population was observed. Furthermore, the eGFP-positive population showed increased expression of CD69, a T cell activation marker, while the eGFP-negative population showed minimal CD69 expression. CD69 upregulation indicates CAR-T cell recognition of target cells bearing the appropriate antigen (in this case, A56). According to these results, after encounter with target cells bearing the appropriate antigen (in this case, A56), CAR-T cells show downregulation of cell surface CAR expression, but are still distinguishable by eGFP and CD69 expression, the latter being independent evidence of target cell recognition by CAR.

[0252] Example 15 - Jurkat T cells expressing VV-CAR show specific activation when co-cultured with HEK cell lines bearing appropriate target antigens Jurkat T cells expressing either A56-CAR-01, A56-CAR-02, B5-CAR-03, B5-CAR-04 or A56-CAR-06 were cultured at a 1:1 E:T ratio, 1 × 10 per condition. 5 Total cell numbers of 1000-1500 were co-cultured in triplicate with HEK-WT, HEK-A56, or HEK-B5 stable lines. Cells were cultured overnight (16 h) at 37 °C in 5% CO2. The next day, cells were washed with PBS, blocked with normal goat serum, and stained with CD45, CD69, and anti-RB IgG. CD69 expression in the CD45+ Jurkat population was then assessed. All CARs tested showed increased CD69 expression when co-cultured with cells expressing the appropriate target antigen, whereas CD69 expression was minimal when co-cultured with HEK WT cells or HEK cells expressing an unrelated target antigen (Figure 16). These findings demonstrate that CAR-T cells directed against A56 and B5 show specific recognition of target cells expressing the appropriate antigen (A56 and B5, respectively) with minimal cross-reactivity and / or constitutive activity (tonic signaling).

[0253] Example 16 - Healthy donor primary T cells transduced and enriched for A56-CAR-06 expression CD4+ and CD8+ T cell populations were isolated from healthy donor PBMC samples. 5 Healthy donor T cells were activated with Miltenyi TransAct™ (Miltenyi Biotec, Cat. No. 130-111-160) and grown in Miltenyi TexMACS GMP (Miltenyi Biotec, Cat. No. 170-076-309) medium supplemented with 3% human serum (Sigma, Cat. No. H4522), gentamicin sulfate (Sandoz, DIN:02268531), and human interleukin-7 (Miltenyi Biotec, Cat. No. 130-095-367) and interleukin-15 (Miltenyi Biotec, Cat. No. 130-095-764)) (10 μg / ml). 24 hours after activation, T cells were transduced with A56-CAR-06 lentivirus at an MOI of 0.25. Over the next 12 days, cells were transduced at 1×10 per ml. 6 After expansion, the A56-CAR-06 transduced T cell fraction was sorted by eGFP positivity using a flow cytometer. 5 Cells were sorted to high purity and plated in a single well of a 96-well round-bottom plate. The sorted population was activated as above and plated at a cell density of 1 × 10 6 The cells were then expanded for an additional 12 days, maintaining the total cell count at < 1 / ml. The final expanded and enriched population was > 95% CAR positive by flow cytometry (Figure 17). These findings indicate that primary PBMC-derived human T cells can be readily transduced to express VV-CAR constructs and can be enriched and expanded following transduction.

[0254] Example 17 - Human T cells expressing A56-CAR-06 show specific activation when co-cultured with HEK293T cells expressing A56 Human T cells expressing A56-CAR-06 were inoculated with HEK-WT, HEK-A56 or HEK-B5 lines at a 1:1 E:T ratio of 1 × 10 per condition. 5 A56-CAR-06 transduced T cells were co-cultured overnight (16 h) in triplicate at a total cell count of 1000x. The next day, cells were washed with PBS, blocked with normal goat serum, and stained to detect expression of CD3 BV510 (Biolegend, Cat. No. 317332), CD45, CD69, CD137 BV650 (Biolegend, Cat. No. 564092), and anti-RB IgG. CD3+eGFP+ T cell populations were assessed for expression of CD69 and CD137 by flow cytometry. A56-CAR-06 transduced T cells had increased expression of CD69 and CD137 when co-cultured with cells expressing the A56 target antigen, but had minimal expression of CD69 and CD137 when co-cultured with HEK-WT cells or HEK cells expressing an irrelevant antigen (Figure 18). These findings demonstrate that primary T cells transduced to express A56-CAR-06 exhibit specific activation when cultured with target cells expressing the A56 antigen on their surface.

[0255] Example 18 - Human T cells expressing A56-CAR-06 induce morphological signs of cell death in A56-expressing target cells HEK-WT, HEK-A56, or HEK-B5 cells (2 × 10 each) 5 1 × 10 5 A56-CAR-06 T cells were added to each well and the cells were cultured for an additional 48 hours. The images show clear morphological changes in the HEK-A56 target cell line when co-cultured with A56-CAR-06 positive T cells, indicating direct tumor cell killing. In contrast, the HEK-WT and HEK-B5 lines remained fully confluent without morphological changes (Figure 19). These findings indicate that the HEK-A56 cell monolayer is disrupted after co-culture with A56-CAR-06 T cells, providing evidence of a direct cytopathic effect.

[0256] Example 19 - Human T cells expressing B5-CAR-011 exhibit cytotoxicity against B5-expressing target cells The mammalian expression transfer plasmid pCCL Luc Puromycin (a gift from Dr. Jonathan Bramson, McMaster University) encoding firefly luciferase was used to generate lentivirus as described above in Example 13. HEK-WT, A56 and B5 lines were transduced with this luciferase-encoding lentivirus and selected for resistance to puromycin (1.5 μg / ml, Sigma, Cat. No. P8833) for 2 weeks. Puromycin-resistant cell cultures were expanded and used as the target population for the VV-CAR killing assay. The target HEK line expressing luciferase was cultured at 2×10 per well of a 96-well plate (Corning, Cat. No. 3917). 4Cells were seeded in 100 μl and incubated overnight to allow adhesion. Sorted populations of previously expanded primary human B5-CAR-011 T cells were plated in triplicate in 96-well plates (100 μl per well) and split into two-fold dilution series to achieve an E:T ratio range of 10:1 to 0.625:1. The diluted T cell suspension was then transferred to adherent HEK cells in a total volume of 200 μl. Additionally, 3 wells of target cells alone and 3 wells of media alone were plated to determine maximum and minimum relative luminescence units (RLU). Cells were cultured for 24 hours at 37°C in 5% CO2. The following day, each well received 22 μl of a 10X stock of Xenolight™ D-luciferin (Perkin Elmer, Cat. No. 122799) and incubated for 10 minutes at room temperature in the dark. Plates were then scanned with a luminescence plate reader (Perkin Elmer Wallac Envision 2104 Multilabel Reader). Figure 20A shows the average RLU from triplicate wells at each E:T ratio at 24 hours. Triplicate wells were averaged and percent specific cytotoxicity was determined by the following formula: percent specific cytotoxicity = 100 x (maximum luminescence RLU - test luminescence RLU) / (minimum luminescence RLU - maximum luminescence RLU). B5-CAR-011 T cells showed high percent specific cytotoxicity at multiple E:T ratios (Figure 20B). Furthermore, B5-expressing target cells co-cultured with B5-CAR-011 T cells showed clear morphological signs of cell death at 24 hours, whereas B5-CAR-011 T cells showed no signs of cytotoxicity against HEK293WT or HEK293A56 cells (Figure 20C). The results show that B5-CAR-011 T cells efficiently and specifically kill HEK-B5 tumor cells. (Figure 20) By targeting OV antigens expressed on the surface of tumor cells, the target cells can be made susceptible to destruction by OV-CAR.

[0257] Example 20 - Radiolabeled anti-A56 antibodies allow clear visualization of A56-expressing tumor cells in vivo by positron emission tomography We selected the anti-A56 antibody (A049) and immunofluorescence staining with zirconium-89 ( 89 We radioactively labeled the chromosome with 1,2-dichlorophenylalanine (Zr), which allows deep tissue imaging in living mice with high sensitivity and spatial resolution (5, 6). 89 Zr is well suited for use with mAbs due to its long half-life (78 h) and good decay characteristics (6, 7). Two milligrams of A049 were conjugated with p-SCN-Bn-deferoxamine (1-(4-isothiocyanatophenyl)-3-[6,17-dihydroxy-7,10,18,21-tetraoxo-27-(N-acetylhydroxylamino)-6,11,17,22-tetraazahepteicosine]thiourea, DFO (Macrocyclics, Plano, TZ, USA) at a molecular ratio of 3:1 chelator:mAb. The reaction was carried out for 1 h at 37 °C in PBS pH 9 with a final antibody concentration of 2 mg / mL. The antibody was then filtered through centrifugal filter units with a molecular weight cutoff of 50 kDa (Amicon ultracentrifugal filters, Ultracel-50: regenerated cellulose, Millipore Conjugated antibody (DFO-A049) was purified from unconjugated deferoxamine using a ELISA kit (Sigma-Aldrich, Billerica, MA, USA) and washed once with 5 mg / mL 2,5-dihydroxybenzoic acid (Sigma-Aldrich, Oakville, Ontario, Canada) in 0.25 M sodium acetate solution. The concentration of the purified immunoconjugate solution was determined by Bradford assay according to the manufacturer's recommendations (Sigma-Aldrich).

[0258] 89 Radiolabeling of immunoconjugates with Zr As previously described ( 8 ), proton irradiation at 13 MeV onto a yttrium-89 disk (American Elements, Los Angeles, CA, USA) in the ACSI (Richmond, Canada) TR-19 cyclotron was performed. 89Zr was generated. After irradiation, the disks were dissolved in HCl (10 mL, 2 M) and purified using a hydroxamate-based ZR resin supplied by Triske (Bruz, France). Briefly, the disk solution was loaded onto the resin and washed with 10 mL of 2 M HCl solution followed by 10 mL of water, then eluted with 0.5 mL of oxalic acid (0.05 M).

[0259] For radiolabeling, immunoconjugate DFO-A049 (0.4 mg) and 89 Zr solution (0.4 mL, pH 7, 232 MBq) was added to PBS (final volume: 1 mL, pH 7) and the resulting solution was incubated at room temperature for 1 h. Radiochemical yields (RCYs) were determined using instant thin layer chromatography silica gel (iTLC-SG, Agilent technologies, Santa Clara, CA, USA) with 50 mM DTPA pH 7 (Sigma-Aldrich) as the solvent. 89 Zr-labeled antibody and free 89 The Rf values ​​of Zr were 0 and 1, respectively. Efficient labeling with RCY of 96% was observed. Next, 89 Zr-labeled antibodies were desalted using a PD-10 desalting column (GE Healthcare, London, UK). 89 It was separated from Zr and concentrated using a 50 kDa molecular weight cutoff filter (Millipore Corp.). 89The specific activity of Zr-DFO-A049 was measured on an Agilent HPLC system (Santa Clara, CA, USA) equipped with a model 1200 quaternary pump, a model 1200 UV absorbance detector, and a Bioscan (Washington, DC, USA) NaI scintillation detector using a size-exclusion HPLC column (BioSep-SEC-s3000, Phenomenex (Torrance, CA, USA). The HPLC buffer was an isocratic gradient of 0.1 M sodium phosphate monobasic dihydrate, 0.1 M sodium phosphate dibasic dodecahydrate, 0.1 M sodium azide and 0.15 M sodium chloride (pH 6.2-7.0). A specific radioactivity of 0.1 MBq / µg was obtained, sufficient for the in vivo characterization of the radioimmunoconjugate. The final radiochemical purity was determined using iTLC-SG as previously described and was >99.9%.

[0260] Antibody immunoreactivity 89 The immunoreactive fraction of Zr-DFO-A049 was estimated according to the Lindmo cell-binding method using HEK-293 cells stably modified to express the A56 antigen (Wyeth VV sequence) (HEK-A56). Briefly, HEK-A56 cells were cultured at 1.0–24.6 × 10 6 The cells were suspended in PBS (pH 7.4) at various concentrations of cells / mL. The remaining procedures were carried out as previously described (9). The results were 89 It was shown that 52% of Zr-DFO-A049 still efficiently bound to the A56 protein.

[0261] Animal tumor models and antibody injections All animal experiments were performed at the Animal Resource Centre of the BC Cancer Research Centre, in accordance with the institutional guidelines of the Animal Care Committee of the University of British Columbia (Vancouver, BC, Canada) and under the supervision of certified researchers. 12-week-old female immunodeficient NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl / SzJ (NRG) mice (obtained from an in-house breeding colony) were subcutaneously injected with HEK-A56 cells in the left shoulder or A56 non-expressing HEK-293 parental cells in the right shoulder (n = 8 mice). For both cell lines, 5 × 10 cells were cultured in Matrigel (ratio 1:1, BD Bioscience, Mississauga, ON, Canada). 6 cells were injected.

[0262] Positron Emission Tomography (PET) The tumor is about 100 mm 3 ), mice were anesthetized with 2% isoflurane in oxygen. 89 Zr-DFO-A049 (40.9 ± 1.5 μg, 4.1 ± 0.2 MBq) was injected intravenously. PET images were acquired 1, 3, and 5 days after injection for one mouse using a Siemens (Knoxville, TN, USA) Inveon microPET / CT scanner. Mice underwent CT scans for attenuation correction, followed by 20-min static PET acquisitions on days 1 and 3, and a 30-min scan on day 5. Images were reconstructed using 3-dimensional ordered-subsets expectation maximization (OSEM3D), 2 iterations, followed by a fast maximum a priori algorithm (FastMAP), 18 iterations. Maximum intensity projection images were used to determine the maximal intensity of the periphery of the mouse, especially in high bone remodeling regions such as long bone epiphysis. 89In addition to bone uptake due to the known demetallation of the Zr-DFO conjugate, the results show the expected accumulation in normal organs such as the liver and spleen due to antibody processing and metabolism. (10) More importantly, PET images show higher tumor uptake and tumor-to-background ratios for HEK-A56 tumors compared to negative control tumors (HEK-293), 89 This demonstrates specific and efficient tumor uptake of the Zr-DFO-A049 antibody (Figure 21).

[0263] 89 Evaluation of Zr-DFO-A049 biodistribution On day 5, all mice were euthanized, and organs of interest were collected for biodistribution studies as previously described ( 9 ). 89 The results for Zr-DFO-A049 are shown in Table 19. [Table 19]

[0264] The biodistribution results confirm the PET imaging observations, with the expected biodistribution of the radioimmunoconjugate in normal organs. With respect to the tumors, a higher uptake of 7.71 ± 4.52% ID / g was obtained in A56-expressing HEK tumors compared to 1.44 ± 0.25% ID / g in A56-non-expressing HEK tumors (n = 8, p < 0.05). Thus, these in vivo results (PET imaging and biodistribution studies) are 89 We demonstrate that Zr-DFO-A049 can be effectively and selectively delivered to A56-expressing tumors.

[0265] Antibodies targeting oncolytic virus-encoded proteins expressed on the surface of OV-infected tumor cells, when labeled with radionuclides, can be used to enable diagnostic, therapeutic, and theranostic applications, for example, in in vivo imaging of OV infection by PET imaging, and in the local delivery of therapeutic radioisotopes to kill OV-infected cells and adjacent uninfected tumor cells by radioimmunotherapy (11, 12).

[0266] References 1. Babcook, JS, Leslie, KB, Olsen, OA, Salmon, RA, Schrader, JW (1996) A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities. Proc Natl Acad Sci USA 93(15):p.7843-8. 2.Jarahian, M., Fiedler, M., Cohnen, A., Djandji, D., Hammerling, GJ, Gati, C., Cerwenka, A., Turner, PC, Moyer, RW, Watzl, C., Hengel, H., and Momburg, F. (2011) Modulation of NKp30- and NKp46-mediated natural killer cell responses by poxviral hemagglutinin.PLoS Pathog 7,e1002195. 3.S.Guedan, H.Calderon,ADPosey,MV Maus,Engineering and Design of Chimeric Antigen Receptors,Mol.Ther.Methods Clin.Dev.12(2019)145-156.https: / / doi.org / 10.1016 / j.omtm.2018.12.009. 4.Molecular Cloning:A Laboratory Manual 3 rd edition(eds.Sambrook,J.& Russell,D.W.)(Cold Spring Harbor Laboratory Press,Cold Spring Harbor,New York,USA,2001). 5.Kim JS.Combination Radioimmunotherapy Approaches and Quantification of Immuno-PET.Nuclear medicine and molecular imaging.2016;50(2):104-11. 6.van de Watering FC,Rijpkema M,Perk L,Brinkmann U,Oyen WJ,Boerman OC.Zirconium-89 labeled antibodies:a new tool for molecular imaging in cancer patients.BioMed research international.2014;2014:203601. 7.Zhang Y,Hong H,Cai W.PET tracers based on Zirconium-89.Current radiopharmaceuticals.2011;4(2):131-9. 8.Dias GM,Ramogida CF,Rousseau J,Zacchia NA,Hoehr C,Schaffer P,Lin K-S,Benard F. 89 Zr for antibody labeling and in vivo studies - A comparison between liquid and solid target production.Nucl Med Biol.2018 Mar;58:1-7. 9.Rousseau J,Zhang Z,Wang X,Zhang C,Lau J,Rousseau E,et al.Synthesis and evaluation of bifunctional tetrahydroxamate chelators for labeling antibodies with(89)Zr for imaging with positron emission tomography.Bioorg Med Chem Lett.2018;28(5):899-905. 10.Rousseau J,Zhang Z,Dias GM,Zhang C,Colpo N,Benard F,Lin K-S.Design,synthesis and evaluation of novel bifunctional tetrahydroxamate chelators for PET imaging of 89 Zr-labeled antibodies.Bioorg Med Chem Lett.2017;27(4):708-712. 11.Makvandi M.,et al.Alpha-Emitters and Targeted Alpha Therapy in Oncology:from Basic Science to Clinical Investigations.Target Oncol.2018 Apr;13(2):189-203. 12.Kraeber-Bodere F,Bodet-Milin C,Rousseau C,Eugene T,Pallardy A,Frampas E,et al.Radioimmunoconjugates for the treatment of cancer.Seminars in oncology.2014;41(5):613-22.

[0267] The present disclosure includes the following embodiments. EMBODIMENT 1 It specifically binds to the vaccinia virus A56 antigen (VV A56), and V containing the amino acid sequence SSYWIC (SEQ ID NO:3) H CDR1, V containing the amino acid sequence CIIAGSGGSTYYATWAKG (SEQ ID NO: 4) H CDR2, V containing the amino acid sequence AYSDRSGGYSFNL (SEQ ID NO:5) H CDR3, and a variable heavy (VH) polypeptide comprising V containing the amino acid sequence QASQSVDNNNYLA (SEQ ID NO:6) L CDR1, V containing the amino acid sequence SASSLAS (SEQ ID NO: 7) L CDR2, V containing the amino acid sequence LGSYDCSDADCYA (SEQ ID NO: 8) L CDR3 and a variable light chain (V L ) polypeptides; V containing the amino acid sequence DIYYIS (SEQ ID NO: 11) H CDR1, V containing the amino acid sequence CTYAGSSGSTYYATWAKG (SEQ ID NO: 12) H CDR2, V containing the amino acid sequence DRYPGTSGRVYGMDL (SEQ ID NO: 13) H CDR3, and a variable heavy (VH) polypeptide comprising V containing the amino acid sequence QASQSISDLLS (SEQ ID NO: 14) L CDR1, V containing the amino acid sequence SASTLAS (SEQ ID NO: 15) L CDR2, V containing the amino acid sequence QCNYYSPTYGNG (SEQ ID NO: 16) L CDR3 and a variable light chain (V L ) polypeptides; V containing the amino acid sequence SSYWLC (SEQ ID NO: 19) H CDR1, V containing the amino acid sequence CIYNGDGSTHYASWAKG (SEQ ID NO: 20) H CDR2, V containing the amino acid sequence DYTYNFYTYGFNL (SEQ ID NO: 21) H CDR3 and a variable heavy chain (V H ) polypeptides, and V containing the amino acid sequence QASQSVNIWAS (SEQ ID NO:22) L CDR1, V containing the amino acid sequence KASTLAS (SEQ ID NO:23) L CDR2, V containing the amino acid sequence QGGYPSSSSGWA (SEQ ID NO:24) L CDR3 and a variable light chain (V L ) polypeptide; or V containing the amino acid sequence SSYWIC (SEQ ID NO:27) H CDR1, V containing the amino acid sequence CTYNGDGSTHYASWAKG (SEQ ID NO: 28) H CDR2, V containing the amino acid sequence DYTDAFYTYGFNL (SEQ ID NO: 29) H CDR3, and a variable heavy (VH) polypeptide comprising V containing the amino acid sequence QASQSTSSYLA (SEQ ID NO:30) L CDR1, V containing the amino acid sequence RASSLAS (SEQ ID NO:31) L CDR2, V containing the amino acid sequence QTGFYGSSGHT (SEQ ID NO:32) L CDR3 and a variable light chain (VL) polypeptide comprising An antibody that competes with an antibody comprising the EMBODIMENT 2 The antibody, V containing the amino acid sequence SSYWIC (SEQ ID NO:3) H CDR1, V containing the amino acid sequence CIIAGSGGSTYYATWAKG (SEQ ID NO: 4) H CDR2, V containing the amino acid sequence AYSDRSGGYSFNL (SEQ ID NO:5) H CDR3, and a variable heavy (VH) polypeptide comprising V containing the amino acid sequence QASQSVDNNNYLA (SEQ ID NO:6) L CDR1, V containing the amino acid sequence SASSLAS (SEQ ID NO: 7) L CDR2, V containing the amino acid sequence LGSYDCSDADCYA (SEQ ID NO: 8) L a variable light (VL) polypeptide comprising a CDR3; V containing the amino acid sequence DIYYIS (SEQ ID NO: 11) H CDR1, V containing the amino acid sequence CTYAGSSGSTYYATWAKG (SEQ ID NO: 12) H CDR2, V containing the amino acid sequence DRYPGTSGRVYGMDL (SEQ ID NO: 13) H CDR3, and a variable heavy (VH) polypeptide comprising V containing the amino acid sequence QASQSISDLLS (SEQ ID NO: 14) L CDR1, V containing the amino acid sequence SASTLAS (SEQ ID NO: 15) L CDR2, V containing the amino acid sequence QCNYYSPTYGNG (SEQ ID NO: 16) L a variable light (VL) polypeptide comprising a CDR3; V containing the amino acid sequence SSYWLC (SEQ ID NO: 19) H CDR1, V containing the amino acid sequence CIYNGDGSTHYASWAKG (SEQ ID NO: 20) H CDR2, V containing the amino acid sequence DYTYNFYTYGFNL (SEQ ID NO: 21) H CDR3 and a variable heavy chain (V H ) polypeptides, and V containing the amino acid sequence QASQSVNIWAS (SEQ ID NO:22) L CDR1, V containing the amino acid sequence KASTLAS (SEQ ID NO:23) L CDR2, V containing the amino acid sequence QGGYPSSSSGWA (SEQ ID NO:24) L a variable light (VL) chain polypeptide comprising: V containing the amino acid sequence SSYWIC (SEQ ID NO:27) H CDR1, V containing the amino acid sequence CTYNGDGSTHYASWAKG (SEQ ID NO: 28) H CDR2, V containing the amino acid sequence DYTDAFYTYGFNL (SEQ ID NO: 29) H CDR3, and a variable heavy (VH) polypeptide comprising V containing the amino acid sequence QASQSTSSYLA (SEQ ID NO:30) L CDR1, V containing the amino acid sequence RASSLAS (SEQ ID NO:31) L CDR2, V containing the amino acid sequence QTGFYGSSGHT (SEQ ID NO:32) L CDR3 and a variable light chain (VL) polypeptide comprising 2. The antibody of embodiment 1, comprising: EMBODIMENT 3 The antibody, A variable heavy chain (VH) having an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO:1. H ) polypeptides, and A variable light chain (VLC) having an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO:2. L 3. The antibody of embodiment 1 or 2, comprising a .DELTA.Asp-Asp-Csp-TgpAsp polypeptide. EMBODIMENT 4 The antibody, A variable heavy chain (VH) having an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO:9. H ) polypeptides, and A variable light chain (V) having an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO: 10. L 3. The antibody of embodiment 1 or 2, comprising a .DELTA.Asp-Asp-Csp-TgpAsp polypeptide. EMBODIMENT 5 The antibody, A variable heavy chain (VH) having an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO: 17. H ) polypeptides, and A variable light chain (VLC) having an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO: 18. L 3. The antibody of embodiment 1 or 2, comprising a .DELTA.Asp-Asp-Csp-TgpAsp polypeptide. EMBODIMENT 6 The antibody, A variable heavy chain (VH) comprising an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO:25. H ) polypeptides, and A variable light chain (V) comprising an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO:26. L 3. The antibody of embodiment 1 or 2, comprising a .DELTA.Asp-Asp-Csp-TgpAsp polypeptide. EMBODIMENT 7 The antibody, A variable heavy chain (VH) having an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO:9. H ) polypeptides, and A variable light chain (V) comprising an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO:62. L 3. The antibody of embodiment 1 or 2, comprising a .DELTA.Asp-Asp-Csp-TgpAsp polypeptide. EMBODIMENT 8 The antibody, A variable heavy chain (VH) having an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO:9. H ) polypeptides, and A variable light chain (V) comprising an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO:63. L 3. The antibody of embodiment 1 or 2, comprising a .DELTA.Asp-Asp-Csp-TgpAsp polypeptide. EMBODIMENT 9 It specifically binds to the vaccinia virus B5 antigen (VV B5), and V containing the amino acid sequence SSYYMC (SEQ ID NO:35) H CDR1, V containing the amino acid sequence CIYTSSGSAYYANWAKG (SEQ ID NO: 36) H CDR2, V containing the amino acid sequence NAVGSSYYLYL (SEQ ID NO:37) H CDR3, and a variable heavy (VH) polypeptide comprising V containing the amino acid sequence QASQSVAGNNYLS (SEQ ID NO:38) L CDR1, V containing the amino acid sequence SVSTLAS (SEQ ID NO:39) L CDR2, V containing the amino acid sequence QGYYNDGIWA (SEQ ID NO: 40) L CDR3 and a variable light chain (V L ) polypeptide; or V containing the amino acid sequence SYWMC (SEQ ID NO: 43) H CDR1, V containing the amino acid sequence CIYGGSSGSTYYSNWAKG (SEQ ID NO: 44) H CDR2, V containing the amino acid sequence DGSTWDYFRL (SEQ ID NO: 45) H CDR3, and a variable heavy (VH) polypeptide comprising V containing the amino acid sequence QASQSINTNYLS (SEQ ID NO:46) L CDR1, V containing the amino acid sequence QASTLES (SEQ ID NO:47) L CDR2, V containing the amino acid sequence QGYYTVENIGNP (SEQ ID NO: 48) L CDR3 and a variable light chain (VL) polypeptide comprising An antibody that competes with an antibody comprising the EMBODIMENT 10 The antibody, V containing the amino acid sequence SSYYMC (SEQ ID NO:35) H CDR1, V containing the amino acid sequence CIYTSSGSAYYANWAKG (SEQ ID NO: 36) H CDR2, V containing the amino acid sequence NAVGSSYYLYL (SEQ ID NO:37) H CDR3, and a variable heavy (VH) polypeptide comprising V containing the amino acid sequence QASQSVAGNNYLS (SEQ ID NO:38) L CDR1, V containing the amino acid sequence SVSTLAS (SEQ ID NO:39) L CDR2, V containing the amino acid sequence QGYYNDGIWA (SEQ ID NO: 40) L a variable light (VL) chain polypeptide comprising: V containing the amino acid sequence SYWMC (SEQ ID NO: 43) H CDR1, V containing the amino acid sequence CIYGGSSGSTYYSNWAKG (SEQ ID NO: 44) H CDR2, V containing the amino acid sequence DGSTWDYFRL (SEQ ID NO: 45) H CDR3, and a variable heavy (VH) polypeptide comprising V containing the amino acid sequence QASQSINTNYLS (SEQ ID NO:46) L CDR1, V containing the amino acid sequence QASTLES (SEQ ID NO:47) L CDR2, V containing the amino acid sequence QGYYTVENIGNP (SEQ ID NO: 48)L CDR3 and a variable light chain (VL) polypeptide comprising 10. The antibody of embodiment 9, comprising: EMBODIMENT 11 The antibody, A variable heavy chain (VH) having an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO: 33. H ) polypeptides, and A variable light chain (V) comprising an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO:34. L 11. The antibody of embodiment 9 or 10, comprising a ) polypeptide. EMBODIMENT 12 The antibody, A variable heavy chain (VH) having an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO:41. H ) polypeptides, and A variable light chain (V) comprising an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO:42. L 11. The antibody of embodiment 9 or 10, comprising a ) polypeptide. EMBODIMENT 13 The antibody according to any one of embodiments 1 to 12, wherein the antibody is a monoclonal antibody. EMBODIMENT 14 The antibody according to any one of embodiments 1 to 13, wherein the antibody is a humanized antibody. EMBODIMENT 15 The antibody according to any one of embodiments 1 to 14, wherein the antibody is an IgG. EMBODIMENT 16 The antibody of embodiment 15, wherein the antibody comprises a human Fc domain. EMBODIMENT 17 The antibody of embodiment 16, wherein the antibody is a human IgG1. EMBODIMENT 18 The antibody is Fab, F(ab') 2 15. The antibody according to any one of embodiments 1 to 14, which is selected from the group consisting of F(ab') and F(ab'). EMBODIMENT 19 The antibody of any one of embodiments 1 to 14, wherein the antibody is a single chain antibody. EMBODIMENT 20 The antibody of embodiment 19, wherein the single chain antibody is an scFv. EMBODIMENT 21 The antibody is a V H Polypeptide-V L The antibody of any one of embodiments 1 to 20, which is a bispecific antibody comprising a first antigen-binding domain comprising a polypeptide pair. EMBODIMENT 22 The antibody of embodiment 21, wherein the bispecific antibody comprises a second antigen-binding domain that specifically binds to an antigen other than a vaccinia virus antigen. EMBODIMENT 23 The antibody of embodiment 22, wherein the antigen other than a vaccinia virus antigen is an immune cell surface antigen. EMBODIMENT 24 The antibody of embodiment 23, wherein the immune cell surface antigen is an immune effector cell surface antigen. EMBODIMENT 25 The antibody of embodiment 24, wherein the immune cell surface antigen is a T cell surface antigen. EMBODIMENT 26 The antibody of embodiment 25, wherein the antigen is a T cell stimulatory molecule. EMBODIMENT 27 The antibody of embodiment 26, wherein the T cell stimulatory molecule is CD3 or CD28. EMBODIMENT 28 The antibody of embodiment 24, wherein the immune cell surface antigen is a natural killer (NK) cell surface antigen. EMBODIMENT 29 The antibody of embodiment 24, wherein the immune cell surface antigen is a macrophage cell surface antigen. EMBODIMENT 30 A fusion protein comprising: A fusion protein comprising a chain of the antibody of any one of embodiments 1 to 29 fused to a heterologous amino acid sequence. EMBODIMENT 31 The fusion protein of embodiment 30, wherein the heterologous amino acid sequence is fused to the C-terminus of the antibody chain. EMBODIMENT 32 The fusion protein of embodiment 30 or 31, wherein the antibody is a single chain antibody of embodiment 19 or 20. EMBODIMENT 33 The fusion protein is The single chain antibody; A transmembrane domain; Intracellular signaling domains and 33. The fusion protein of embodiment 32, which is a chimeric antigen receptor (CAR) comprising: EMBODIMENT 34 An antibody according to any one of embodiments 1 to 29 or a fusion protein according to any one of embodiments 30 to 33, a drug conjugated to said antibody or fusion protein; A conjugate comprising: EMBODIMENT 35 The conjugate of embodiment 34, wherein said agent is selected from the group consisting of a chemotherapeutic agent, a toxin, a radiosensitizer, a radioisotope, a detectable label, and a half-life extending moiety. EMBODIMENT 36 The conjugate of embodiment 35, wherein said radioisotope is a therapeutic radioisotope. EMBODIMENT 37 The conjugate according to embodiment 35, wherein said detectable label is a radioactive label. EMBODIMENT 38 The radioactive label is zirconium-89 ( 89 38. The conjugate of embodiment 37, wherein R is H, R is Zn. EMBODIMENT 39 The conjugate according to any one of embodiments 34 to 38, wherein said agent is conjugated to said antibody or fusion protein via a non-cleavable linker. EMBODIMENT 40 The conjugate according to any one of embodiments 34 to 38, wherein the agent is conjugated to the antibody or fusion protein via a cleavable linker. EMBODIMENT 41 The conjugate according to embodiment 40, wherein said cleavable linker is an enzyme-cleavable linker. EMBODIMENT 42 The conjugate of embodiment 41, wherein said linker is cleavable by a lysosomal protease. EMBODIMENT 43 The conjugate of embodiment 42, wherein said linker is cleavable by cathepsin or plasmin. EMBODIMENT 44 The variable heavy chain (VH) polypeptide and the variable light chain (VH) polypeptide of the antibody according to any one of embodiments 1 to 29 are L ) a nucleic acid encoding a polypeptide, or both. EMBODIMENT 45 A nucleic acid encoding the fusion protein according to any one of embodiments 30 to 33. EMBODIMENT 46 46. ​​An expression vector comprising a nucleic acid according to embodiment 44 or 45. EMBODIMENT 47 A nucleic acid according to embodiment 44 or 45, or The expression vector according to embodiment 46 , comprising a cell. EMBODIMENT 48 The variable heavy chain (V H a first nucleic acid encoding a polypeptide; The variable light chain (V L ) a second nucleic acid encoding a polypeptide; , comprising a cell. EMBODIMENT 49 a first expression vector comprising the first nucleic acid; a second expression vector comprising the second nucleic acid; 49. The cell of embodiment 48, comprising: EMBODIMENT 50 A method for producing an antibody according to any one of embodiments 1 to 28, comprising culturing a cell according to any one of embodiments 47 to 49 under conditions suitable for said cell to express said antibody, whereby said antibody is produced. EMBODIMENT 51 An antibody according to any one of embodiments 1 to 28, A pharma- ceutically acceptable carrier; 13. A pharmaceutical composition comprising: EMBODIMENT 52 A fusion protein according to any one of embodiments 30 to 33, A pharma- ceutically acceptable carrier; 13. A pharmaceutical composition comprising: EMBODIMENT 53 A conjugate according to any one of embodiments 34 to 43, A pharma- ceutically acceptable carrier; 13. A pharmaceutical composition comprising: EMBODIMENT 54 A pharmaceutical composition according to any one of embodiments 51 to 53, instructions for administering said pharmaceutical composition to an individual in need thereof; Including the kit. EMBODIMENT 55 The kit of embodiment 54, wherein the pharmaceutical composition is in one or more unit dosage forms. EMBODIMENT 56 The kit of embodiment 54, wherein the pharmaceutical composition is present in two or more unit dosage forms. EMBODIMENT 57 A method comprising administering a pharmaceutical composition according to any one of embodiments 51 to 53 to an individual having cancer, the individual comprising cancer cells infected with vaccinia virus (VV), and the antibody, fusion protein or conjugate being targeted to the infected cancer cells by a VV antigen expressed on the surface of the infected cancer cells. EMBODIMENT 58 The method of embodiment 57, further comprising infecting the cancer cells by administering a VV to the individual prior to administering the pharmaceutical composition to the individual. EMBODIMENT 59 The method of embodiment 57 or 58, wherein the method is a method of treating the cancer in the individual. EMBODIMENT 60 The method of any one of embodiments 57 to 59, wherein the pharmaceutical composition of embodiment 53 is administered to the individual, and the conjugate comprises the antibody conjugated to a detectable label or a radioisotope that is an in vivo imaging agent, and the method comprises imaging the infected cancer cells of the individual using the in vivo imaging agent. EMBODIMENT 61 1. A method for targeting an antibody that specifically binds to an oncolytic virus (OV) antigen to cancer cells in an individual, comprising: The method comprises administering to the individual a pharmaceutical composition comprising an antibody that specifically binds to the OV antigen, wherein the cancer cells in the individual are infected with OV and express the OV antigen on their surface. EMBODIMENT 62 The method of embodiment 61, further comprising infecting said cancer cells by administering said OV to said individual prior to administering said pharmaceutical composition to said individual. EMBODIMENT 63 The method of embodiment 61 or 62, wherein the OV antigen is native to the OV. EMBODIMENT 64 The method of embodiment 61 or 62, wherein the OV antigen is heterologous to the OV. EMBODIMENT 65 The method of any one of embodiments 61-64, wherein the OV is selected from the group consisting of vaccinia virus, adenovirus, herpes simplex virus (HSV), reovirus, vesicular stomatitis virus, Newcastle disease virus, Seneca Valley virus, poliovirus, measles virus, coxsackievirus, and Maraba virus. EMBODIMENT 66 The method of any one of embodiments 61-64, wherein the OV is a vaccinia virus (VV). EMBODIMENT 67 The method of embodiment 66, wherein the VV is JX-594 or GL-ONC1. EMBODIMENT 68 The method of embodiment 66, wherein the strain of VV is selected from the group consisting of Western Reserve, Wyeth, Lister, Copenhagen, Temple of Heaven, Patwadangar, and modified vaccinia virus Ankara. EMBODIMENT 69 The method of any one of embodiments 61 to 68, wherein the antibody is a monoclonal antibody. EMBODIMENT 70 The method of any one of embodiments 61 to 69, wherein the antibody is a humanized antibody. EMBODIMENT 71 The method of any one of embodiments 61 to 70, wherein the antibody is an IgG. EMBODIMENT 72 The method of embodiment 71, wherein the antibody is a human Fc domain. EMBODIMENT 73 73. The method of embodiment 72, wherein the antibody is human IgG1. EMBODIMENT 74 The antibody is Fab, F(ab') 2 The method of any one of embodiments 61-70, wherein the F(ab') is selected from the group consisting of F(ab'). EMBODIMENT 75 The method of any one of embodiments 61 to 70, wherein the antibody is a single chain antibody. EMBODIMENT 76 The method of embodiment 75, wherein the single chain antibody is an scFv. EMBODIMENT 77 The method of any one of embodiments 66-76, wherein the OV antigen is a VV A56 antigen. EMBODIMENT 78 The method of embodiment 77, wherein the pharmaceutical composition comprises an antibody of any one of embodiments 1 to 8. EMBODIMENT 79 The method of any one of embodiments 66 to 76, wherein the OV antigen is a VV B5 antigen. Embodiment 80 The method according to embodiment 79, wherein the pharmaceutical composition comprises an antibody according to any one of embodiments 9 to 12. Embodiment 81 The method of any one of embodiments 57 to 80, wherein said pharmaceutical composition comprises said antibody conjugated to a detectable label or a radioisotope. EMBODIMENT 82 82. The method of embodiment 81, wherein said detectable label or radioisotope is an in vivo imaging agent. EMBODIMENT 83 83. The method of embodiment 82, comprising detecting said in vivo imaging agent for imaging said cancer cells in vivo. EMBODIMENT 84 The method of any one of embodiments 61-83, wherein the method is a method of treating the cancer in the individual. EMBODIMENT 85 an antigen-binding domain that specifically binds to an oncolytic virus (OV) antigen; A transmembrane domain; Intracellular signaling domains and and a chimeric antigen receptor (CAR). EMBODIMENT 86 The CAR of embodiment 85, wherein the OV antigen is native to the OV. EMBODIMENT 87 The CAR of embodiment 85, wherein the OV antigen is heterologous to the OV. EMBODIMENT 88 The CAR of any one of embodiments 85 to 87, wherein the OV is selected from the group consisting of vaccinia virus, adenovirus, herpes simplex virus (HSV), reovirus, vesicular stomatitis virus, Newcastle disease virus, Seneca Valley virus, poliovirus, measles virus, and Maraba virus. EMBODIMENT 89 The CAR of any one of embodiments 85 to 87, wherein the OV antigen is an antigen encoded by vaccinia virus (VV). EMBODIMENT 90 The CAR of embodiment 89, wherein the VV is JX-594 or GL-ONC1. EMBODIMENT 91 The CAR of embodiment 89, wherein the strain of VV is selected from the group consisting of Western Reserve, Wyeth, Lister, Copenhagen, Temple of Heaven, Patwadangar, and modified vaccinia virus Ankara. EMBODIMENT 92 The CAR of embodiment 89, wherein the OV antigen is a VV A56 antigen. EMBODIMENT 93 The antigen-binding domain is V of the antibody according to any one of embodiments 1 to 8. H Polypeptide-V L The CAR of embodiment 92, comprising a polypeptide pair. EMBODIMENT 94 The CAR of embodiment 89, wherein the OV antigen is a VV B5 antigen. EMBODIMENT 95 The antigen-binding domain is V of the antibody according to any one of embodiments 9 to 12. H Polypeptide-V L The CAR of embodiment 94, comprising a polypeptide pair. EMBODIMENT 96 The CAR of any one of embodiments 85 to 95, wherein the antigen-binding domain comprises an scFv. EMBODIMENT 97 97. The CAR of any one of claims 85 to 96, wherein the CAR is provided by a single polypeptide. EMBODIMENT 98 97. The CAR of any one of claims 85 to 96, wherein the CAR is provided by two or more polypeptides. EMBODIMENT 99 A nucleic acid encoding a CAR described in any one of embodiments 85 to 98. EMBODIMENT 100 A cell comprising the nucleic acid of embodiment 99. EMBODIMENT 101 The cell of embodiment 100, wherein the cell expresses the CAR on its surface. EMBODIMENT 102 The cell of embodiment 100 or 101, wherein the cell is an immune cell. EMBODIMENT 103 The cell of embodiment 102, wherein the cell is an immune effector cell. EMBODIMENT 104 The cell of embodiment 103, wherein the cell is a T cell. EMBODIMENT 105 The cell of embodiment 103, wherein the cell is a NK cell. EMBODIMENT 106 The cell of embodiment 103, wherein the cell is a macrophage. EMBODIMENT 107 A pharmaceutical composition comprising a cell according to any one of embodiments 100 to 105. EMBODIMENT 108 1. A method of targeting a CAR that specifically binds to an oncolytic virus (OV) antigen to a cancer cell in an individual, comprising: A method comprising administering to the individual the pharmaceutical composition of embodiment 107, wherein the cancer cells of the individual are infected with OV and express the OV antigen on their surface. EMBODIMENT 109 The method of embodiment 108, further comprising infecting said cancer cells by administering said OV to said individual prior to administering said pharmaceutical composition to said individual. Embodiment 110 The method of embodiment 108 or 109, wherein the method is a method of treating the cancer in the individual. EMBODIMENT 111 A conjugate comprising: an antibody that specifically binds to an oncolytic virus (OV) antigen; a drug conjugated to the antibody; wherein said agent is selected from the group consisting of a chemotherapeutic agent, a toxin, a radiosensitizer, and a radioisotope. EMBODIMENT 112 The conjugate of embodiment 111, wherein said radioisotope is a therapeutic radioisotope. EMBODIMENT 113 The conjugate of embodiment 111 or 112, wherein the OV antigen is native to the OV. EMBODIMENT 114 The conjugate of embodiment 111 or 112, wherein the OV antigen is heterologous to the OV. EMBODIMENT 115 The conjugate according to any one of embodiments 111 to 114, wherein the OV antigen is an antigen encoded by an OV selected from the group consisting of vaccinia virus, adenovirus, HSV, reovirus, vesicular stomatitis virus, Newcastle disease virus, Seneca Valley virus, poliovirus, measles virus, coxsackievirus, and Maraba virus. EMBODIMENT 116 The conjugate according to any one of embodiments 111 to 114, wherein the OV antigen is an antigen encoded by vaccinia virus (VV). EMBODIMENT 117 The conjugate of embodiment 116, wherein said VV is JX-594 or GL-ONC1. EMBODIMENT 118 The conjugate of embodiment 116, wherein the strain of VV is selected from the group consisting of Western Reserve, Wyeth, Lister, Copenhagen, Temple of Heaven, Patwadangar, and modified vaccinia virus Ankara. EMBODIMENT 119 The conjugate of embodiment 116, wherein said OV antigen is a VV A56 antigen. Embodiment 120 The conjugate according to embodiment 119, wherein the antibody is an antibody according to any one of embodiments 1 to 8. EMBODIMENT 121 The conjugate of embodiment 116, wherein the OV antigen is a VV B5 antigen. EMBODIMENT 122 The conjugate according to embodiment 121, wherein the antibody is an antibody according to any one of embodiments 9 to 12. EMBODIMENT 123 The conjugate according to any one of embodiments 111 to 122, wherein the antibody is a monoclonal antibody. EMBODIMENT 124 The conjugate according to any one of embodiments 111 to 123, wherein the antibody is a humanized antibody. EMBODIMENT 125 The conjugate according to any one of embodiments 111 to 124, wherein the antibody is an IgG. EMBODIMENT 126 The conjugate of embodiment 125, wherein the antibody comprises a human Fc domain. EMBODIMENT 127 The conjugate of embodiment 126, wherein said antibody is human IgG1. EMBODIMENT 128 The antibody is Fab, F(ab') 2 and F(ab'). EMBODIMENT 129 The conjugate according to any one of embodiments 111 to 124, wherein the antibody is a single chain antibody. Embodiment 130 The conjugate of embodiment 129, wherein said single chain antibody is an scFv. EMBODIMENT 131 The conjugate according to any one of embodiments 111 to 130, wherein the drug is conjugated to the antibody via a non-cleavable linker. EMBODIMENT 132 The conjugate according to any one of embodiments 111 to 130, wherein the agent is conjugated to the antibody via a cleavable linker. EMBODIMENT 133 The conjugate according to embodiment 132, wherein said cleavable linker is an enzyme-cleavable linker. EMBODIMENT 134 The conjugate according to embodiment 133, wherein said linker is cleavable by a lysosomal protease. EMBODIMENT 135 The conjugate according to embodiment 134, wherein said linker is cleavable by cathepsin or plasmin. EMBODIMENT 136 A pharmaceutical composition comprising a conjugate according to any one of embodiments 111 to 135. EMBODIMENT 137 1. A method of targeting a conjugate comprising an antibody that specifically binds to an oncolytic virus (OV) antigen to cancer cells in an individual, comprising: A method comprising administering to the individual the pharmaceutical composition of embodiment 136, wherein the cancer cells of the individual are infected with OV and express the OV antigen on their surface. EMBODIMENT 138 The method of embodiment 137, further comprising infecting the cancer cells by administering the OV to the individual prior to administering the pharmaceutical composition to the individual. EMBODIMENT 139 The method of embodiment 137 or 138, wherein the method is a method of treating the cancer in the individual. Thus, the above merely describes the principles of the disclosure. It will be appreciated that those skilled in the art can devise various configurations that embody the principles of the invention and fall within its spirit and scope, although not explicitly described or illustrated herein. Furthermore, all examples and conditional language recited herein are intended primarily to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to advance the art, and should not be construed as limitations to such specifically recited examples and conditions. Furthermore, all descriptions herein reciting the principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. In addition, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any developed elements that perform the same function, regardless of structure. Thus, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein.

Claims

1. An antibody that specifically binds to vaccinia virus B5 antigen (VV B5), V comprising the amino acid sequence SSYYMC (SEQ ID NO:35) Ｈ CDR1, V containing the amino acid sequence CIYTSSGSAYYANWAKG (SEQ ID NO:36) Ｈ CDR2, V containing the amino acid sequence NAVGSSYYLYL (SEQ ID NO:37) Ｈ CDR3, and a variable heavy chain (V Ｈ ) polypeptides, and V containing the amino acid sequence QASQSVAGNNYLS (SEQ ID NO:38) Ｌ CDR1, V containing the amino acid sequence SVSTLAS (SEQ ID NO:39) Ｌ CDR2, V containing the amino acid sequence QGYYNDGIWA (SEQ ID NO: 40) Ｌ CDR3, and a variable light chain (V Ｌ ) polypeptide; or V containing the amino acid sequence SYWMC (SEQ ID NO:43) Ｈ CDR1, V containing the amino acid sequence CIYGGSSGSTYYSNWAKG (SEQ ID NO: 44) Ｈ CDR2, V containing the amino acid sequence DGSTWDYFRL (SEQ ID NO:45) Ｈ CDR3, and a variable heavy chain (V Ｈ ) polypeptides, and V containing the amino acid sequence QASQSINTNYLS (SEQ ID NO:46) Ｌ CDR1, V containing the amino acid sequence QASTLES (SEQ ID NO:47) Ｌ CDR2, V containing the amino acid sequence QGYYTVENIGNP (SEQ ID NO: 48) Ｌ CDR3, and a variable light chain (V Ｌ ) Polypeptide An antibody comprising:

2. The antibody that specifically binds to VV B5 is A variable heavy chain (VH) comprising an amino acid sequence having 75% or more identity to the amino acid sequence shown in SEQ ID NO:

33. Ｈ ) polypeptides, and A variable light chain (V) comprising an amino acid sequence having 75% or more identity to the amino acid sequence shown in SEQ ID NO:

34. Ｌ ) polypeptide, or A variable heavy chain (VH) comprising an amino acid sequence having 75% or more identity to the amino acid sequence shown in SEQ ID NO:

41. Ｈ ) polypeptides, and A variable light chain (V) comprising an amino acid sequence having 75% or more identity to the amino acid sequence shown in SEQ ID NO:

42. Ｌ ) polypeptides, The antibody described in claim 1.

3. The antibody of claim 1 or 2, wherein the antibody is a monoclonal antibody.

4. The antibody according to any one of claims 1 to 3, wherein the antibody is a humanized antibody.

5. The antibody according to any one of claims 1 to 4, wherein the antibody is an IgG.

6. The antibody of claim 5 , wherein the antibody comprises a human Fc domain.

7. The antibody is Fab, F(ab') ２ The antibody of any one of claims 1 to 4, which is selected from the group consisting of F(ab') and F(ab').

8. The antibody of any one of claims 1 to 4, wherein the antibody is a single chain antibody.

9. The antibody of claim 8 , wherein the single chain antibody is an scFv.

10. The antibody is a V Ｈ Polypeptide-V Ｌ The antibody of any one of claims 1 to 9, which is a bispecific antibody comprising a first antigen-binding domain comprising a polypeptide pair.

11. The antibody of claim 10 , wherein the bispecific antibody comprises a second antigen-binding domain that specifically binds to an antigen other than a vaccinia virus antigen.

12. The antibody of claim 11 , wherein the antigen other than a vaccinia virus antigen is an immune cell surface antigen.

13. The antibody of claim 12, wherein the immune cell surface antigen is an immune effector cell surface antigen.

14. The antibody of claim 13, wherein the immune cell surface antigen is a T cell surface antigen.

15. The antibody of claim 14, wherein the antigen is a T cell stimulatory molecule.

16. The antibody of claim 15, wherein the T cell stimulatory molecule is CD3 or CD28.

17. The antibody of claim 13, wherein the immune cell surface antigen is a natural killer (NK) cell surface antigen.

18. The antibody of claim 13, wherein the immune cell surface antigen is a macrophage cell surface antigen.

19. A fusion protein comprising: A fusion protein comprising a chain of an antibody according to any one of claims 1 to 18 fused to a heterologous amino acid sequence.

20. The fusion protein of claim 19 , wherein the heterologous amino acid sequence is fused to the C-terminus of the antibody chain.

21. The fusion protein of claim 19 or 20, wherein the antibody is a single chain antibody of claim 8 or 9.

22. The fusion protein is The single chain antibody; A transmembrane domain; Intracellular signaling domains and The fusion protein of claim 21, which is a chimeric antigen receptor (CAR) comprising:

23. An antibody according to any one of claims 1 to 18 or a fusion protein according to any one of claims 19 to 22, a drug conjugated to said antibody or fusion protein; A conjugate comprising:

24. 24. The conjugate of claim 23, wherein the agent is selected from the group consisting of a chemotherapeutic agent, a toxin, a radiosensitizer, a radioisotope, a detectable label, and a half-life extending moiety.

25. 25. The conjugate of claim 24, wherein the agent is a radioisotope or a detectable label, the radioisotope is a therapeutic radioisotope and the detectable label is a radiolabel.

26. The conjugate according to any one of claims 23 to 25, wherein the drug is conjugated to the antibody or fusion protein via a non-cleavable linker.

27. The conjugate according to any one of claims 23 to 25, wherein the agent is conjugated to the antibody or fusion protein via a cleavable linker.

28. The variable heavy chain (V) of the antibody according to any one of claims 1 to 18 Ｈ ) polypeptide and the variable light chain (V Ｌ ) a nucleic acid encoding a polypeptide.

29. A nucleic acid encoding a fusion protein according to any one of claims 19 to 22.

30. 30. An expression vector comprising the nucleic acid of claim 28 or 29.

31. 30. A nucleic acid according to claim 28 or 29, or The expression vector of claim 30. , comprising a cell.

32. The cell of claim 31, wherein the cell comprises a nucleic acid of claim 29, the nucleic acid encoding the fusion protein of claim 22, and the single chain antibody is an scFv.

33. The cell of claim 32 , wherein the cell expresses the fusion protein on its surface.

34. The cell of claim 32 or 33, wherein the cell is an immune cell.

35. 35. The cell of claim 34, wherein the cell is an immune effector cell.

36. The cell of claim 35 , wherein the cell is a T cell.

37. The cell of claim 35, wherein the cell is a natural killer (NK) cell.

38. The cell of claim 35 , wherein the cell is a macrophage.

39. The variable heavy chain (V Ｈ a first nucleic acid encoding a polypeptide; The variable light chain (V Ｌ ) a second nucleic acid encoding a polypeptide; , comprising a cell.

40. A first expression vector comprising the first nucleic acid; a second expression vector comprising the second nucleic acid; The cell of claim 39 .

41. 41. A method for producing an antibody according to any one of claims 1 to 18, comprising culturing a cell according to any one of claims 31, 39 and 40 under conditions suitable for said cell to express said antibody, whereby said antibody is produced.

42. An antibody according to any one of claims 1 to 18, A pharma- ceutically acceptable carrier; 13. A pharmaceutical composition comprising:

43. A fusion protein according to any one of claims 19 to 22, A pharma- ceutically acceptable carrier; 13. A pharmaceutical composition comprising:

44. A conjugate according to any one of claims 23 to 27, A pharma- ceutically acceptable carrier; 13. A pharmaceutical composition comprising:

45. A cell according to any one of claims 31 to 40, A pharma- ceutically acceptable carrier; 13. A pharmaceutical composition comprising:

46. 46. ​​The pharmaceutical composition of any one of claims 42 to 45, for use in a method comprising administering to an individual having cancer, wherein the individual comprises cancer cells infected with vaccinia virus (VV), and wherein the antibody, fusion protein, conjugate or cell is targeted to the infected cancer cells by a VV antigen expressed on the surface of the infected cancer cells.

47. 47. The pharmaceutical composition of claim 46, wherein the method further comprises infecting the cancer cells by administering a VV to the individual prior to administering the pharmaceutical composition to the individual.

48. 48. The pharmaceutical composition of claim 46 or 47, wherein the method is a method of treating the cancer in the individual.

49. The pharmaceutical composition according to any one of claims 46 to 48, comprising the fusion protein of claim 22 expressed on the surface of a T cell, a NK cell, or a macrophage.

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