Anti-vaccinia virus antigen antibodies and related compositions and methods

Abstract

Antibodies that specifically bind to vaccinia virus B5 antigen (VV B5) are provided. In certain embodiments, the anti-VV B5 antibodies are humanized antibodies. Fusion proteins and conjugates comprising such antibodies are also provided. Pharmaceutical compositions comprising the antibodies, fusion proteins and conjugates of the present disclosure are also provided, as are methods of using such compositions, e.g., for treatment, in vivo imaging, etc. In certain aspects, methods are provided that include administering an antibody, fusion protein or conjugate of the present disclosure to an individual, the individual comprising cells infected with VV, and the antibody, fusion protein or conjugate is targeted to the infected cells by the VV B5 antigen expressed on the surface of the infected cells.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 195,536, filed June 1, 2021, and U.S. Provisional Patent Application No. 63 / 162,199, filed March 17, 2021, which are incorporated by reference in their entireties herein.

[0002] Introduction Immunotherapy has become an effective treatment option for multiple malignancies. Oncolytic viruses (OVs) can be engineered to selectively replicate and lyse in tumor tissues 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 engineer live replicating viruses that are not only highly tumor selective through cell entry and transcriptional targeting, but also equipped with reporter genes for non-invasive monitoring of the pharmacokinetics of virotherapy, and for the enhancement of cytotoxic activity or immunogenic cell death, or immune modulators.OVs in clinical development include measles virus, Newcastle disease virus (NDV), rhabdoviruses, adenoviruses, vaccinia virus (VV), herpesviruses, coxsackieviruses, reoviruses, and retroviruses.

[0004] 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 lacks 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.

[0005] 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. Summary of the Invention

[0006] Antibodies that specifically bind to vaccinia virus B5 antigen (VV B5) are provided. In certain embodiments, the anti-VV B5 antibodies are humanized antibodies. Fusion proteins and conjugates comprising such antibodies are also provided. Pharmaceutical compositions comprising the antibodies, fusion proteins and conjugates of the present disclosure are also provided, as are methods of using such compositions, e.g., for treatment, in vivo imaging, etc. In certain aspects, methods are provided that include administering an antibody, fusion protein or conjugate of the present disclosure to an individual, the individual comprising cells infected with VV, and the antibody, fusion protein or conjugate is targeted to the infected cells by the VV B5 antigen expressed on the surface of the infected cells. [Brief description of the drawings]

[0007] [Figure 1] Figure 1 shows binding of humanized antibodies to VV B5 protein. Antibodies were evaluated by ELISA for binding to VV B5 monomeric protein. Humanized anti-B5 hIgG1 antibodies were titrated and showed a similar pM EC50 compared to the A048 parent positive control. No binding was observed with the hIgG1 isotype control. [Figure 2-1] Figure 1 shows binding of humanized antibodies to VV infected cells. Antibodies were assessed for binding to VV infected cells by flow cytometry. HT29 cells were infected with VVddeGFP Western Reserve virus ("WR") or VV Copenhagen (YFP) virus ("Cop") and SKOV3 cells were infected with VVddeGFP Western Reserve virus ("WR"). Humanized anti-B5 hIgG1 antibodies were titrated and showed a similar EC50 in nM compared to A048 parental anti-B5 hIgG1 (positive control). The negative control was a human IgG1 isotype control. [Figure 2-2]Figure 1 shows binding of humanized antibodies to VV infected cells. Antibodies were assessed for binding to VV infected cells by flow cytometry. HT29 cells were infected with VVddeGFP Western Reserve virus ("WR") or VV Copenhagen (YFP) virus ("Cop") and SKOV3 cells were infected with VVddeGFP Western Reserve virus ("WR"). Humanized anti-B5 hIgG1 antibodies were titrated and showed a similar EC50 in nM compared to A048 parental anti-B5 hIgG1 (positive control). The negative control was a human IgG1 isotype control. [Figure 3-1] Figure 1 shows the binding levels of humanized antibodies over time to different cell lines infected with vaccinia virus. HT29 (A), SKOV3 (B), and OVCAR3 (C) cells were infected with vaccinia virus strains VVddeGFP Western Reserve ("WR") or VV Copenhagen (YFP) virus ("Cop") at an MOI of 1 or 0.1, or mock infected. Cells were harvested at the indicated time points to determine A048-H1L4 and A048-H3L2 binding levels in infected cells. Primary antibodies were conjugated to AlexaFluor-647. Data were acquired with a BD LSRFortessa X-20 and analyzed with FlowJo 10.8.1 software. Data represent three biological replicates. MOI, multiplicity of infection. [Figure 3-2] Figure 1 shows the binding levels of humanized antibodies over time to different cell lines infected with vaccinia virus. HT29 (A), SKOV3 (B), and OVCAR3 (C) cells were infected with vaccinia virus strains VVddeGFP Western Reserve ("WR") or VV Copenhagen (YFP) virus ("Cop") at an MOI of 1 or 0.1, or mock infected. Cells were harvested at the indicated time points to determine A048-H1L4 and A048-H3L2 binding levels in infected cells. Primary antibodies were conjugated to AlexaFluor-647. Data were acquired with a BD LSRFortessa X-20 and analyzed with FlowJo 10.8.1 software. Data represent three biological replicates. MOI, multiplicity of infection. [Figure 4-1] Schematic diagram of the design of humanized B5-CAR-050 or B5-CAR-051 chimeric antigen receptor (CAR) constructs (Figure 4A). Flow cytometry data showing humanized scFv CAR detection after lentiviral transduction and expansion of primary human T cells (Figure 4B). Specific activation of human T cells expressing B5-CAR-050 or B5-CAR-051 when co-cultured with target-expressing HT-29-B5, SKOV3-B5, and HEK293T-B5 lines (Figure 4C). Primary human T cells expressing B5-CAR-050 or B5-CAR-051 showed upregulation of CD137 after co-culture with target B5-expressing tumor lines. Minimal cross-reactivity observed with WT target cell lines. [Figure 4-2] Schematic diagram of the design of humanized B5-CAR-050 or B5-CAR-051 chimeric antigen receptor (CAR) constructs (Figure 4A). Flow cytometry data showing humanized scFv CAR detection after lentiviral transduction and expansion of primary human T cells (Figure 4B). Specific activation of human T cells expressing B5-CAR-050 or B5-CAR-051 when co-cultured with target-expressing HT-29-B5, SKOV3-B5, and HEK293T-B5 lines (Figure 4C). Primary human T cells expressing B5-CAR-050 or B5-CAR-051 showed upregulation of CD137 after co-culture with target B5-expressing tumor lines. Minimal cross-reactivity observed with WT target cell lines. [Figure 5-1] Data are presented demonstrating that human T cells expressing B5-CAR-050 or B5-CAR-051 exhibited a high rate of specific cytotoxicity (Figure 5A). Primary human T cells expressing B5-CAR-050 or B5-CAR-051 exhibited specific cytolysis at 24 hours as determined by the percentage reduction in relative luminescence units (RLU). Data are presented demonstrating that primary human T cells expressing B5-CAR-050 or B5-CAR-051 exhibited morphological signs of direct tumor killing after 24 hours of co-culture with the target B5-expressing tumor line (Figure 5B). [Figure 5-2]Data are presented demonstrating that human T cells expressing B5-CAR-050 or B5-CAR-051 exhibited a high rate of specific cytotoxicity (Figure 5A). Primary human T cells expressing B5-CAR-050 or B5-CAR-051 exhibited specific cytolysis at 24 hours as determined by the percentage reduction in relative luminescence units (RLU). Data are presented demonstrating that primary human T cells expressing B5-CAR-050 or B5-CAR-051 exhibited morphological signs of direct tumor killing after 24 hours of co-culture with the target B5-expressing tumor line (Figure 5B). [Figure 6-1] Data are presented demonstrating that human T cells expressing B5-CAR-050 or B5-CAR-051 exhibited a high percentage of specific cytotoxicity against vaccinia virus-infected target cell lines. Primary human T cells expressing B5-CAR-050 or B5-CAR-051 exhibited specific cell lysis at 24 hours as determined by the percentage reduction in relative luminescence units (RLU) against vaccinia virus-infected target cells (Figure 6A). In Figure 6A, B5-CAR-050, B5-CAR-051 and the parental B5-CAR-043 are identified as VV50_B5, VV51_B5 and VV43_B5_tEGFR, respectively. Data are presented demonstrating that primary human T cells expressing B5-CAR-050 or B5-CAR-051 exhibited morphological signs of direct tumor killing after 24 hours of co-culture with vaccinia virus-infected tumor lines (Figures 6B-D). [Figure 6-2]Data are presented demonstrating that human T cells expressing B5-CAR-050 or B5-CAR-051 exhibited a high percentage of specific cytotoxicity against vaccinia virus-infected target cell lines. Primary human T cells expressing B5-CAR-050 or B5-CAR-051 exhibited specific cell lysis at 24 hours as determined by the percentage reduction in relative luminescence units (RLU) against vaccinia virus-infected target cells (Figure 6A). In Figure 6A, B5-CAR-050, B5-CAR-051 and the parental B5-CAR-043 are identified as VV50_B5, VV51_B5 and VV43_B5_tEGFR, respectively. Data are presented demonstrating that primary human T cells expressing B5-CAR-050 or B5-CAR-051 exhibited morphological signs of direct tumor killing after 24 hours of co-culture with vaccinia virus-infected tumor lines (Figures 6B-D). [Figure 6-3] Data are presented demonstrating that human T cells expressing B5-CAR-050 or B5-CAR-051 exhibited a high percentage of specific cytotoxicity against vaccinia virus-infected target cell lines. Primary human T cells expressing B5-CAR-050 or B5-CAR-051 exhibited specific cell lysis at 24 hours as determined by the percentage reduction in relative luminescence units (RLU) against vaccinia virus-infected target cells (Figure 6A). In Figure 6A, B5-CAR-050, B5-CAR-051 and the parental B5-CAR-043 are identified as VV50_B5, VV51_B5 and VV43_B5_tEGFR, respectively. Data are presented demonstrating that primary human T cells expressing B5-CAR-050 or B5-CAR-051 exhibited morphological signs of direct tumor killing after 24 hours of co-culture with vaccinia virus-infected tumor lines (Figures 6B-D). [Figure 6-4]Data are presented demonstrating that human T cells expressing B5-CAR-050 or B5-CAR-051 exhibited a high percentage of specific cytotoxicity against vaccinia virus-infected target cell lines. Primary human T cells expressing B5-CAR-050 or B5-CAR-051 exhibited specific cell lysis at 24 hours as determined by the percentage reduction in relative luminescence units (RLU) against vaccinia virus-infected target cells (Figure 6A). In Figure 6A, B5-CAR-050, B5-CAR-051 and the parental B5-CAR-043 are identified as VV50_B5, VV51_B5 and VV43_B5_tEGFR, respectively. Data are presented demonstrating that primary human T cells expressing B5-CAR-050 or B5-CAR-051 exhibited morphological signs of direct tumor killing after 24 hours of co-culture with vaccinia virus-infected tumor lines (Figures 6B-D). [Figure 7] Predictive data are shown demonstrating that human T cells expressing humanized B5-CAR result in reduced tumor growth when administered in combination with vaccinia virus used to treat xenograft tumor models (Figure 7A). Predictive data demonstrating improved overall survival of combination therapy with humanized B5-CAR and vaccinia virus (Figure 7B). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] 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, as such 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 is not intended to be limiting, since the scope of the antibodies, compositions, and methods will be limited only by the appended claims.

[0009] When a range of values ​​is provided, it is understood that each intervening value between the upper and lower limit of that range, and any other stated or intervening value in that stated range, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, 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, and are also encompassed within the antibodies, compositions, and methods, subject to any specifically excluded limits in the stated range. When a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the antibodies, compositions, and methods.

[0010] Certain ranges are presented herein with numerical values ​​preceded by the term "about." The term "about" is used herein to provide literal support for the exact number it precedes, as well as a number that is near or approximately the number it precedes. In determining whether a number is near or approximately a specifically recited number, the near or approximately unrecited number may be a number that, in the context in which it is presented, provides a substantial equivalent to the specifically recited number.

[0011] 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 herein.

[0012] All publications and patents cited herein are incorporated by reference herein to the same extent 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 for 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 antibodies, compositions, and methods of the invention are not entitled to antedate such publication, since the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0013] 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 any element. Thus, this statement is intended to serve as a predicate for the use of exclusive terminology, such as "solely," "only," or the use of a "negative" limitation in connection with the recitation of claim elements.

[0014] It is also understood that, for clarity, certain features of the antibodies, compositions, and methods described in the context of separate embodiments may be provided in combination in a single embodiment. Conversely, for brevity, various features of the antibodies, compositions, and methods described in the context of a single embodiment may also be provided separately or in any suitable subcombination. All combinations of the embodiments are specifically encompassed by the present disclosure and, to the extent such combinations encompass operable processes and / or compositions, are disclosed herein as if each and every combination were individually and explicitly disclosed. In addition, all subcombinations recited in the embodiments describing such variables are also specifically encompassed by the antibodies, compositions, and methods of the present invention and are disclosed herein as if each and every such subcombination were individually and explicitly disclosed herein.

[0015] As will be apparent to one of ordinary skill in the art after reading this disclosure, each of the individual embodiments described and illustrated herein has distinct 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 method. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0016] Anti-VV B5 antibody Aspects of the present disclosure include antibodies that specifically bind to vaccinia virus (VV) B5 antigen (VV B5). In certain embodiments, the antibody is a humanized antibody that specifically binds to VV B5. Vaccinia virus is a member of the poxvirus family characterized by a double-stranded DNA genome of approximately 192 kb that encodes a number of 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 is composed of a core region made up 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 is composed of a double lipid membrane envelope. VV has unique characteristics that make it 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 genome integration. 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 are not dependent on the host machinery 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.

[0017] 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 preceding the transmembrane domain and a short cytoplasmic tail (CT). Both the SCR and CT are essential for targeting B5 to the extracellular enveloped virus (EEV) membrane, whereas the latter influences its trafficking to the cell surface and recycling via endosomes. B5 is required for wrapping of intracellular mature virus (IMV) to form intracellular enveloped virus (IEV).

[0018] The term "antibody" (also used interchangeably with "immunoglobulin") encompasses polyclonal (e.g., rabbit polyclonal) and monoclonal antibody preparations, where the antibody can be an antibody or immunoglobulin of any isotype (e.g., IgG (e.g., IgG1, IgG2, IgG3, or IgG4), IgE, IgD, IgA, IgM, etc.), whole antibodies (e.g., antibodies composed of a tetramer which in turn is composed of two dimers of a heavy and a light chain polypeptide), single chain antibodies (e.g., scFv), single chain Fv (scFv), Fab, (Fab')2, (scFv')2, and bispecific antibodies, fragments of antibodies (e.g., whole antibodies or single chain antibody fragments) that retain specific binding to a compound, including, but not limited to, chimeric antibodies, monoclonal antibodies, humanized antibodies, human antibodies, and fusion proteins comprising the antigen-binding portion of an antibody and a non-antibody protein. In some embodiments, the antibody is selected from an IgG, an Fv, a single chain antibody, a scFv, a Fab, a F(ab')2, and a F(ab'). The antibody may be further conjugated to another moiety, such as a member of a specific binding pair, e.g., biotin (a member of the biotin-avidin specific binding pair).

[0019] 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).

[0020] 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 framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, i.e., 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. The CDRs of the antibodies provided by this disclosure are defined according to Kabat above, unless otherwise indicated.

[0021] Thus, "antibody" encompasses a protein having one or more polypeptides genetically encodable, for example, by immunoglobulin genes or fragments of immunoglobulin genes. 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 comprise a human Fc domain.

[0022] 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 (VLC) is L ) and variable heavy chain (V H ) refer to these light and heavy chains respectively.

[0023] Antibodies encompass whole immunoglobulins and a number of well-characterized fragments that may 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 whole antibodies, one skilled in the art will understand that such Fab' fragments may 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 the 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 (e.g., scFv), Fab, F(ab')2, and Fab'.

[0024] According to some embodiments, the antibody of the present disclosure is a monoclonal antibody. "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" denotes 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.

[0025] As summarized above, according to some embodiments, the antibody of the present disclosure is a humanized antibody. In certain embodiments, an antibody is provided that is a humanized version of the parent rabbit antibody designated A048, described in International Application No. CA202 / 051230 (International Publication No. WO2021 / 046653), the disclosure of which is incorporated herein by reference in its entirety for all purposes. For example, the V of the A048 antibody is H V has been humanized against H , V of A048 antibody L V has been humanized against L or both.

[0026] 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 and CDR-grafted antibodies, where various regions may be derived from different species. A chimeric antibody may be an antibody that comprises variable regions 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 CDRs 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 an scFV may be linked to a human constant region (e.g., an Fc domain) to make it a human immunoglobulin.

[0027] Generally, humanized antibodies elicit a reduced immune response (exhibit reduced immunogenicity) 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, identifying framework residues important for antigen binding and sequence matching, and identifying unusual framework residues at specific positions.

[0028] When rabbit or mouse CDRs are replaced by a human variable domain framework, they can retain their correct spatial orientation, e.g., 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 whose framework sequences show a high degree of sequence identity with the rabbit or mouse variable framework domain from which the CDRs are derived. The heavy and light chain variable framework regions can be derived from the same or different human antibody sequences. The human antibody sequences can be the sequences of naturally occurring human antibodies or can be consensus sequences of several human antibodies.

[0029] Once the complementarity determining regions of a rabbit or mouse donor immunoglobulin and a suitable human acceptor immunoglobulin have been identified, the next step is to determine which, if any, residues of 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 of determining immune responses can be performed to monitor HARA or HAMA responses in a particular patient or during clinical trials. Patients administered a humanized antibody can be given an immunogenicity assessment at the start of and throughout administration of the therapy. HARA or HAMA responses are measured by detecting antibodies against the humanized therapeutic reagent in serum samples from the patient, 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.

[0030] Specific amino acids from the human variable region framework residues are selected for substitution based on their possible effect on CDR conformation and / or binding to antigen. Unnatural juxtaposition of rabbit or mouse CDR regions with human variable framework regions may result in unnatural conformational constraints, leading to loss of binding affinity unless corrected by substitution of specific amino acid residues. The 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, molecular models are created starting from a resolved structure of an immunoglobulin chain or domain thereof. The chains to be modeled are compared for amino acid sequence similarity with the chains or domains of the resolved three-dimensional structure, and the chains or domains showing the greatest sequence similarity are selected as the starting point for construction of the molecular model. Chains or domains sharing at least 50% sequence identity are selected for modeling, preferably those sharing 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 chains or modeled domains 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 ensuring that all atoms are within appropriate distances from each other and that bond lengths and angles are within chemically acceptable ranges.

[0031] 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 at positions 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 may interact specifically with amino acids in the CDRs and, if selected from the acceptor, may distort the donor CDRs and reduce affinity. Furthermore, 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 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.

[0032] The antibodies of the present disclosure specifically bind to the VV B5 antigen. An antibody "specifically binds" or "preferentially binds" to a target if it binds with higher affinity, avidity, more readily, and / or with a longer duration than it binds to other substances, e.g., samples. In certain embodiments, an antibody specifically binds to a target with a higher affinity, avidity, more readily, and / or with a longer duration than it binds to other substances, e.g., samples. 4 M-1 An antigen "specifically binds" 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) that is equal to or greater than 1 / M. Alternatively, affinity can be expressed as the equilibrium dissociation constant (KD) of the particular binding interaction in units of M (e.g., 10 -5 M~10 -13 In certain aspects, 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 general procedures outlined by the manufacturer), by radioimmunoassay, etc.

[0033] According to some embodiments, an antibody (non-humanized or humanized) is provided that competes with any of the antibodies (non-humanized or humanized) described elsewhere herein for binding to the VV B5 antigen. Whether an antibody of the present disclosure "competes" with a secondary antibody for binding to an antigen can be readily determined using competitive binding assays known in the art. Competing antibodies can be identified, for example, via antibody competition assays. For example, a sample of a primary antibody can be bound to a solid support. A sample of a secondary antibody suspected of being able to compete with such primary antibody is then added. One of the two antibodies is labeled. If the labeled and unlabeled antibodies 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 sites of interaction are identical or overlapping, the unlabeled antibody will compete and the amount of labeled antibody that binds to the antigen will be reduced. If unlabeled antibodies are present in excess, little, if any, of the labeled antibody will bind.

[0034] 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 quantitatively performed using purified antibodies. A standard curve can be established by titrating one antibody against itself; that is, the same antibody is used as 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.

[0035] In certain embodiments, the antibodies of the present disclosure are humanized versions of the parent rabbit antibody designated A048, which is described in International Application No. CA202 / 051230. H and V L The amino acid sequences of the polypeptides, and non-limiting examples of humanized versions of such VH and VL polypeptides (designated A048-H1 to H5 and A048-L1 to L5) are provided in Table 1 below (CDRs are underlined).

[0036] [Table 1-1]

[0037] [Table 1-2]

[0038] According to some embodiments, the antibodies of the disclosure comprise a variable heavy chain (V H ) polypeptide and the variable light chain (V L ) polypeptides. By way of example, the antibody of the present disclosure may be H and A048-L1 V L , A048-L2 V L , A048-L3V L , A048-L4V L , or A048-L5 V L Such antibodies may be referred to as A048-H1L1, A048-H1L2, A048-H1L3, A048-H1L4, or A048-H1L5, respectively. By way of further example, an antibody of the present disclosure may include a pair with A048-H2 V H and A048-L1 V L , A048-L2 V L , A048-L3V L , A048-L4V L , or A048-L5 V LSuch antibodies may be referred to as A048-H2L1, A048-H2L2, A048-H2L3, A048-H2L4, or A048-H2L5, respectively. As will be appreciated, the antibodies of the present disclosure may include any of the A048-H1 V H ~A048-H5 V H and A048-L1 V L ~A048-L5 V L may include any combination of.

[0039] In certain embodiments, an antibody of the present disclosure specifically binds to vaccinia virus B5 antigen (VV B5), the antibody being A variable heavy chain (VH) having 70% or more sequence identity to the amino acid sequence shown in SEQ ID NO:1. H ) polypeptide, V H The polypeptide is one or more mutations selected from the group consisting of Q1E, E2V, Q3T, E5L / K, G9P, G10V, K13Q, E15G / T, G16E, S17T, T19R, T21S, T23A, A41P, R44K, G45A, T74D, S75N / T, S76_T77insK, T77N / S, T78Q, V79L, T80Y / V, Q82T, T84N, R85S / N, L86M, T87R / D, A88P, A89E / V, T93V, F95Y, P112Q, and any combination thereof, numbered according to SEQ ID NO:1; V containing the amino acid sequence SSYYMC (SEQ ID NO: 13) H CDR1, V containing the amino acid sequence CIYTSSGSAYYA(N / D)(W / S)(A / V)KG (SEQ ID NO: 14) H CDR2, V containing the amino acid sequence NAVGSSYYLYL (SEQ ID NO: 17) H CDR3.

[0040] As used herein, a "mutation" refers to a mutation in the parent V of the A048 antibody shown in SEQ ID NO: 1 and SEQ ID NO: 7, respectively. H and V LThis includes amino acid substitutions, insertions of one or more amino acids, deletions of one or more amino acids, etc. of the polypeptide. According to some embodiments, the V H The polypeptide may include one of the mutations E2V, E5L / K, E15G / T, R44K, R85S / N, T87R / D, A89E / V, and P112Q, any combination thereof, or each of these. H The polypeptide may comprise each of the mutations E2V, E5L / K, E15G / T, R44K, R85S / N, T87R / D, A89E / V, P112Q. In certain embodiments, the E5L / K mutation is E5L, the E15G / T mutation is E15G, the R85S / N mutation is R85S, the T87R / D mutation is T87R, and / or the A89E / V mutation is A89E. According to some embodiments, the V of such an antibody is H The polypeptide comprises the mutations E5L, E15G, R85S, T87R, and A89E. H The polypeptide comprises one of the following mutations, any combination thereof, or each of Q1E, K13Q, T19R, T21S, T23A, T84N, T93V, and F95Y. H The polypeptide includes one of the following mutations, or any combination thereof: T74D, S75N / T, S76_T77insK, T77N / S, V79L, and T80Y / V. H The polypeptide is selected from the group consisting of parent A048 V H In certain embodiments, the T77N / S mutation is T77N. According to some embodiments, the T80Y / V mutation is T80Y. According to some embodiments, the V H The polypeptide comprises the amino acid sequence CIYTSSGSAYYADSVKG (SEQ ID NO: 16). H Includes CDR2.

[0041] According to some embodiments, the V of such antibodiesH The polypeptide may include one of the mutations Q3T, E5L / K, G9P, G10V, E15G / T, G16E, S17T, A41P, G45A, T74D, S75N / T, S76_T77insK, T77N / S, T78Q, T80Y / V, Q82T, R85S / N, L86M, T87R / D, A88P and A89E / V, or any combination thereof, or each of these. H The polypeptide may include each of the mutations Q3T, E5L / K, G9P, G10V, E15G / T, G16E, S17T, A41P, G45A, T74D, S75N / T, S76_T77insK, T77N / S, T78Q, T80Y / V, Q82T, R85S / N, L86M, T87R / D, A88P, and A89E / V. In certain embodiments, the E5L / K mutation is E5K, the E15G / T mutation is E15T, the S75N / T mutation is S75T, the T77N / S mutation is T77S, the T80Y / V mutation is T80V, the R85S / N mutation is R85N, the T87R / D mutation is T87D, and / or the A89E / V mutation is A89V. H The polypeptide includes the mutations E5K, E15T, R85N, T87D, and A89V.

[0042] In certain embodiments, the V of the humanized antibody of the present disclosure H The polypeptide comprises one or any desired combination of the above mutations and comprises 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the amino acid sequence set forth in one of SEQ ID NOs:2-6.

[0043] In certain embodiments, an antibody of the present disclosure specifically binds to VV B5, the antibody comprising: A variable light chain (V) having 70% or more sequence identity to the amino acid sequence shown in SEQ ID NO:7. L ) polypeptide, V L The polypeptide is one or more mutations selected from the group consisting of A1D / E, Q2I, V3Q, L4M, T7S, S9A, P10T / S, V11L, A13L, A14S, V15P, G17D / E, T18R, V19A, I21L, S22T, Q44K, P45A / V, N47K / R, V60I, S62A, K65S, Q72E / D, D79S, E81Q, C82P, D83E, A85F / V, T87V, G103Q, E106K, V107L, V108E, V109I, and any combination thereof, numbered according to SEQ ID NO:7; V containing the amino acid sequence QASQSVAGNNYLS (SEQ ID NO: 18) L CDR1, V containing the amino acid sequence SVSTLAS (SEQ ID NO: 19) L CDR2, V containing the amino acid sequence QGYYNDGIWA (SEQ ID NO: 20) L CDR3.

[0044] According to some embodiments, the V of such antibodies L The polypeptide may include one of, any combination of, or each of the mutations T7S, P10T / S, V11L, A14S, G17D / E, T18R, P45A / V, N47K / R, K65S, Q72E / D, D79S, C82P, A85F / V, G103Q, E106K, V108E, and V109I. L The polypeptide may include each of the mutations T7S, P10T / S, V11L, A14S, G17D / E, T18R, P45A / V, N47K / R, K65S, Q72E / D, D79S, C82P, A85F / V, G103Q, E106K, V108E, and V109I. L The polypeptide comprises one of the mutations G17D, S22T, Q44K, N47K, and E81Q, any combination thereof, or each. In certain embodiments, the P10T / S mutation is P10T, the P45A / V mutation is P45A, the Q72E / D mutation is Q72E, and / or the A85F / V mutation is A85F. The V of such an antibodyL The polypeptide may include one of, any combination of, or each of the mutations A1D, Q2I, V3Q, and L4M.

[0045] In certain embodiments, the V of the antibody of the present disclosure L The polypeptide comprises a mutation D83E. According to some embodiments, the P10T / S mutation is P10S, the P45A / V mutation is P45V, the Q72E / D mutation is Q72D, and / or the A85F / V mutation is A85V. In certain embodiments, L The polypeptide comprises one of, any combination of, or each of the mutations A1D, Q2I, V3Q, and L4M.

[0046] According to some embodiments, the V L The polypeptide may include one of, any combination of, or each of the mutations A1E, S9A, P10T, A13L, V15P, G17E, V19A, I21L, P45A, N47R, V60I, S62A, Q72D, D83E, A85F, T87V, and V107L. L The polypeptide can include each of the mutations A1E, S9A, P10T, A13L, V15P, G17E, V19A, I21L, P45A, N47R, V60I, S62A, Q72D, D83E, A85F, T87V, and V107L.

[0047] In certain embodiments, the V of the humanized antibody of the present disclosure L The polypeptide is V L It comprises one or any desired combination of mutations and has 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the amino acid sequence set forth in one of SEQ ID NOs:8-12.

[0048] Also provided by the present disclosure is an antibody (non-humanized or humanized) that specifically binds to VV B5, the antibody comprising: Variable heavy chain (V H ) a polypeptide comprising V containing the amino acid sequence SSYYMC (SEQ ID NO: 13) H CDR1, V containing the amino acid sequence CIYTSSGSAYYADSVKG (SEQ ID NO: 16) H CDR2, V containing the amino acid sequence NAVGSSYYLYL (SEQ ID NO: 17) H a variable heavy chain polypeptide comprising a CDR3; Variable light chain (V L ) a polypeptide comprising V containing the amino acid sequence QASQSVAGNNYLS (SEQ ID NO: 18) L CDR1, V containing the amino acid sequence SVSTLAS (SEQ ID NO: 19) L CDR2, V containing the amino acid sequence QGYYNDGIWA (SEQ ID NO: 20) L and a variable light chain polypeptide comprising:

[0049] The antibodies of the present disclosure specifically bind to the VV B5 antigen. Any of the antibodies can bind to the VV B5 antigen from one or more VV strains, non-limiting examples of which include the B5 antigen of the Wyeth, Western Reserve and / or Copenhagen strains. The amino acid sequences of the B5 antigens encoded by these strains are provided in Table 2 below.

[0050] [Table 2] Table 2 - B5 antigen sequences of Wyeth, Western Reserve and Copenhagen VVs

[0051] bispecific antibody Bispecific antibodies are also provided. In certain embodiments, the bispecific antibodies of the present disclosure comprise any of the VV B5 antibodies of the present disclosure, such as those described above. H Polypeptide-V LThe 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 the VV B5 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 B5 antigen bound by the first antigen-binding domain.

[0052] 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.

[0053] Bispecific antibodies of the present disclosure include antibodies having 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) consists of the known variable heavy chain and constant domains VH, CH1, CH2, and CH3. A full-length antibody light chain (LC) consists of the known variable light chain and constant domains VL and CL. A full-length antibody may lack the C-terminal lysine in either one or both heavy chains. The term "Fab arm" refers to one heavy:light chain pair that specifically binds to an antigen.

[0054] Full-length bispecific antibodies can be generated in vitro in a cell-free environment or using co-expression, for example, using Fab arm exchange (or half molecule exchange) between two monospecific bivalent antibodies, by introducing substitutions in the heavy chain CH3 interface in each half molecule to favor heterodimerization of two antibody half molecules with distinct specificities. The Fab arm exchange reaction is the result of a disulfide bond isomerization reaction and dissociation-association of the CH3 domains. The heavy chain disulfide bonds in the hinge region of the parent monospecific antibodies are reduced. The resulting free cysteine ​​of one of the parent monospecific antibodies forms an inter-heavy chain disulfide bond with a cysteine ​​residue of the second parent monospecific antibody molecule, and at the same time, the CH3 domain of the parent antibody is released and reformed by dissociation-association. The CH3 domain of the Fab arm can be engineered to favor heterodimerization over homodimerization. The resulting product is a bispecific antibody with two Fab arms or half molecules, each binding a different epitope.

[0055] A "knob-in-hole" strategy (see, for example, 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 (expressed as modification position in first CH3 domain of first heavy chain / modification position in second CH3 domain of second heavy chain) T366Y7F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T3945 / Y407A, T366W / T394S, F405W / T394S, and T366W / T366S_L368A_Y407V.

[0056] Other strategies may be used such as using electrostatic interactions to promote heavy chain heterodimerization by replacing positively charged residues on one CH3 surface with negatively charged residues on the second CH3 surface as described in U.S. Patent Application Publication Nos. 2010 / 0015133, 2009 / 0182127, 2010 / 028637, or 2011 / 0123532. In another strategy, heterodimerization can 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, T366L_K392M_T394W / F405A_Y407V, L351 Y_F405A_Y407V, T366L_K392M_T394W / ... Y_Y407A'T366A_K409F, L351Y_Y407A / T366V_K409F, Y407A / T366A_K409F or T350V_L351Y_F405A_Y407V / T350V_T366L_K392L_T394W.

[0057] Single chain bispecific antibodies are also provided. In some embodiments, the single chain bispecific antibodies of the present disclosure are bispecific scFv. Details regarding bispecific scFv can be found, for example, in Zhou et al. (2017) J Cancer 8 (18): 3689-3696.

[0058] 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.

[0059] Fusion proteins Aspects of the present disclosure further include fusion proteins. In certain embodiments, the fusion proteins of the present disclosure comprise any VV B5 antibody of the present disclosure fused to a heterologous amino acid sequence. H Polypeptide, V LThe heterologous amino acid sequence may be fused to the C-terminus of the antibody chain or to the N-terminus of the antibody chain. In certain embodiments, the fusion protein of the present disclosure includes a heterologous sequence at the C-terminus of the antibody chain and a heterologous sequence at the N-terminus of the antibody chain, where the heterologous sequences may be the same or different sequences. "Heterologous" as used in the context of a nucleic acid or polypeptide generally means that the nucleic acid or polypeptide is derived from a different source (e.g., a molecule of different sequence, a different species source, etc.) than the nucleic acid or polypeptide to which it is bound or linked, such that the nucleic acid or polypeptide is not found in nature. For example, in a fusion protein, the light chain polypeptide and the reporter polypeptide (e.g., GFP, red fluorescent protein (e.g., mCherry), luciferase, etc.) are said to be "heterologous" to each other. Similarly, CDRs from a rabbit antibody and a constant region from a human antibody are "heterologous" to each other.

[0060] V H Polypeptide and / or V L The polypeptide may be fused to any heterologous sequence of interest, including, but not limited to, albumin, transferrin, XTEN, homoamino acid polymers, proline-alanine-serine polymers, elastin-like peptides, or any combination thereof. In certain aspects, the heterologous polypeptide increases the stability and / or serum half-life of the anti-VV B5 antibody when the antibody is administered to an individual in need thereof, compared to the same antibody not fused to a heterologous sequence.

[0061] In certain embodiments, the fusion protein of the present disclosure comprises a single chain antibody, e.g., any VV B5 antibody of the present disclosure, such as an antibody as described above. H Polypeptide-V L and single chain antibodies (e.g., scFvs) comprising polypeptide pairs. Non-limiting example amino acid sequences of humanized scFvs according to embodiments of the present disclosure are provided in Table 3 below.

[0062] [Table 3]

[0063] As described in Table 3, V H and V L The orientation of is reversed, i.e., V L V H Also provided is an anti-VV B5scFv that is N-terminal to

[0064] 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.

[0065] 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 to accommodate 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 comprising the same light chain variable region and heavy chain variable region. 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: 26). In certain embodiments, 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 length of amino acids. 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.

[0066] 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 (and any other spacer domains, linkers, etc. described herein) can be derived from either natural, synthetic, semi-synthetic, or recombinant sources. In certain embodiments, the spacer domain is a portion of an immunoglobulin, including one or more heavy chain constant regions, such as, but not limited to, 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 may be wild-type hinge regions from these molecules or variants 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.

[0067] 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(s) or functional portion thereof) of the T cell receptor alpha or beta chain, CD35, CD3zeta, CD3gamma, CD3delta, CD4, CD5, CD8alpha, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, or PD-1.

[0068] In one embodiment, the CAR comprises a Tm domain derived from CD8α. In a particular embodiment, the CAR comprises a Tm domain derived from CD8α and a short oligo- or polypeptide linker, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids long, that connects the Tm domain of the CAR to the intracellular signaling domain. Such a linker may, for example, be a glycine-serine linker.

[0069] The "intracellular signaling" domain of a CAR refers to the portion of the CAR that is involved in transmitting a signal from CAR binding to a target molecule / antigen inside an immune effector cell to induce effector cell functions, such as activation, cytokine production, proliferative activity, and / or cytotoxic activity including release of cytotoxic factors to a CAR-bound target cell, or other cellular responses triggered by target molecule / antigen binding to the extracellular CAR domain. Thus, the term "intracellular signaling domain" refers to a portion of a protein that transmits an effector function signal and allows a cell to perform a specialized function. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion may be used in place of the full-length intracellular signaling domain, so long as it transmits an effector function signal. The term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transmit an effector function signal.

[0070] Signals generated by the T cell receptor (TCR) alone are insufficient to fully activate T cells; secondary or costimulatory signals are also required. Thus, T cell activation is mediated by two distinct classes of intracellular signaling domains: primary signaling domains, which initiate antigen-dependent primary activation through the TCR (e.g., the TCR / CD3 complex), and costimulatory signaling domains, which act antigen-independently to provide secondary or costimulatory signals. Thus, the CARs of the present disclosure can include intracellular signaling domains that include one or more "costimulatory signaling domains" and "primary signaling domains."

[0071] The primary signaling domain regulates the primary activation of the TCR complex in either a stimulatory or inhibitory manner. A primary signaling domain that acts in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif (or "ITAM"). 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 domain and the costimulatory signaling domain are operably linked to the carboxyl terminus of the transmembrane domain.

[0072] 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 CD3zeta primary signaling domain.

[0073] CARs of the present disclosure can include any of a variety of suitable domains, including, but not limited to, leader sequences, hinges, spacers, and / or linker domain(s), transmembrane domain(s), costimulatory domain(s), signal domain(s) (e.g., CD3 zeta domain(s)), ribosomal skip element(s), restriction enzyme sequence(s), reporter protein domain(s), etc. Non-limiting examples of such domains that can be included in CARs of the present disclosure include those shown in Table 4 below. As will be appreciated by one of skill in the art, the amino acid sequences of one or more of the domains shown in Table 4 (e.g., linker, hinge, transmembrane, costimulatory, signaling, ribosomal skip element, restriction enzyme sequence, reporter protein, etc.) can be modified as desired, such as for improved functionality of the CAR.

[0074] [Table 4-1]

[0075] [Table 4-2]

[0076] In certain aspects, a CAR of the disclosure comprises a single chain antibody that specifically binds to a VV B5 antigen (e.g., any of the scFvs of the disclosure), a transmembrane domain derived from a polypeptide selected from the group consisting of CD4, CD8α, CD154, and PD-1, one or more intracellular costimulatory signaling domains derived from a polypeptide selected from the group consisting of 4-1BB (CD137), CD28, and CD134, and an intracellular signaling domain derived 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 CD8α hinge, between the antigen binding portion and the transmembrane domain.

[0077] According to some embodiments, from N-terminus to C-terminus, the variable heavy chain (V H ) polypeptide, a linker, the variable light chain (V L ) polypeptide, 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, a CAR is provided that comprises, from N-terminus to C-terminus, a variable light chain (VLCNA) of an antibody described herein. L ) polypeptide, linker, variable heavy chain (V H In certain embodiments, a CAR is provided that comprises, from N-terminus to C-terminus, a variable heavy chain (VH) of an antibody described herein, 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. H ) polypeptide, a linker, the variable light chain (V L ), a CD28 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain. According to some embodiments, a CAR is provided that comprises, from N-terminus to C-terminus, a variable light chain (VLCNA) of an antibody described herein. L ) polypeptide, linker, variable heavy chain (V H ), a CD28 hinge region, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain. H The N-terminus of the polypeptide may include a domain. For example, a leader sequence (e.g., a GM-CSFRα leader sequence) may be present at the N-terminus of the CAR of the present disclosure.

[0078] Non-limiting example amino acid sequences of anti-VV B5 CARs according to embodiments of the present disclosure are provided in Table 5 below, where the amino acid sequences of the CARs in Table 5 include the amino acid sequence of an N-terminal leader sequence (here, an N-terminal GM-CSFRα leader sequence). Any desired leader sequence (e.g., a GM-CSFRα leader sequence) can be present at the N-terminus of such a CAR. As will be understood by those skilled in the art, the amino acid sequences of one or more of the domains (e.g., linker, hinge, transmembrane, costimulatory, signal transduction, etc.) shown in Table 5 can be modified as necessary, for example, to improve the functionality, etc., of the CAR. In Table 5, the segments / domains of the CAR are shown with alternating underlines, and the segment / domain IDs are shown in the left column.

[0079] [Table 5]

[0080] CARs of the present disclosure may optionally include one or more additional domains. Non-limiting examples of such additional domains include ribosomal skipping elements, enzyme domains (e.g., domains with nuclease activity, e.g., restriction endonuclease activity), domains that allow 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.

[0081] 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 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 with a 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 adaptor molecule (CAM)-based formats with FITC-folate (see, e.g., Lee et al.(2019) Cancer Res.79(2):387-396, and Lu et al.(2019) Proc Natl Acad Sci.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 other universal CAR formats.

[0082] 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 brings one molecule of interest into close association with a second molecule of interest. 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.

[0083] 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 an agent that reduces the function of the target cell / tissue by inhibiting cell proliferation and / or killing the cell / tissue. Such agents are varied 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.

[0084] In certain aspects, 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.

[0085] 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.

[0086] 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 an antibody or fusion protein include cisplatin, 5-fluorouracil (5-FU), AZD7762, selumetinib, and the like.

[0087] 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, 81mKr, 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.

[0088] 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 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.

[0089] 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.

[0090] According to some embodiments, the agent is a labeling agent. By "labeling agent" (or "detectable label") is meant an agent that detectably labels an 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 studies and / or clinical applications). Detectable labels of interest include radioisotopes (e.g., gamma-emitters 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 the detectable label, including avidin / streptavidin.

[0091] In 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).

[0092] 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, DyLight 750 and 800 Fluors, Cy 5.5 and 7 Fluors, Alexa Fluor 680 and 750 Dyes, IRDye 680 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 Tl, and 133 In certain embodiments, the labeling agent is an in vivo imaging agent that finds use in PET imaging applications, such as 11 C. 13 N, 15 O. 18 F, 64 Cu, 62 Cu, 124 I, 76 Br, 82 Rb, 68 Ga, etc.

[0093] 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" to contain one or more poly(ethylene glycol) (PEG) moieties. Methods and reagents suitable for PEGylation of proteins are well known in the art and may 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.

[0094] 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.

[0095] 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, such as a radioisotope (e.g., 89 Zr, 111 In, etc.), enzymes that produce a detectable product (e.g., luciferase, β-galactosidase, horseradish peroxidase, alkaline phosphatase, etc.), fluorescent proteins, chromogenic proteins, dyes (e.g., fluorescein isothiocyanate, rhodamine, phycoerythrin, etc.), fluorescent emitting metals attached to proteins through metal chelating groups such as EDTA, e.g. 152 These may include Eu, or other metals of the lanthanide series, 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, including members of a specific binding pair such as biotin-avidin.

[0096] 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.

[0097] 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.

[0098] As a flexible linker, 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 may be used when relatively unstructured amino acids are desired and may function as neutral tethers between components. Those skilled in the art will recognize that the design of an antibody or fusion protein conjugated to any of the above agents may include a fully or partially flexible linker, whereby the linker may include a flexible linker and one or more moieties that impart a less flexible structure.

[0099] According to some embodiments, the antibody or fusion protein is conjugated to the drug via a non-cleavable linker. Non-cleavable linkers of interest include, but are not limited to, thioether linkers. Examples of thioether linkers that can be used include succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker.

[0100] 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, that 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, that is stable in the bloodstream but undergoes enzymatic cleavage upon internalization into target cells, for example, by lysosomal proteases (e.g., cathepsin or plasmin) in the lysosomes of target cells (e.g., cancer cells). Enzyme-labile linkers include, but are not limited to, peptide bond-containing linkers, such as dipeptide-based linkers such as valine-citrulline (VC) linker, maleimidocaproyl-valine-citrulline-p-aminobenzyl (MC-vc-PAB) linker, and valyl-alanyl-para-aminobenzyloxy (Val-Ala-PAB) linker. 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, and elsewhere.

[0101] Numerous strategies are available for linking an agent directly to an antibody or fusion protein, or indirectly via a linker. For example, an agent may 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 may vary and may be 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 an unnatural amino acid having a chemical handle into the antibody or fusion protein. Unnatural amino acids that find use in preparing the conjugates of the present disclosure include unnatural amino acids having a functional group selected from azide, alkyne, alkene, amino-oxy, hydrazine, aldehyde (e.g., formylglycine, e.g., SMARTag™ technology from Catalent Pharma Solutions), 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 disclosure and that can be selected to provide a desired functional group are known and are 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, and elsewhere. Such unnatural amino acids can be incorporated into the antibody or fusion protein, 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.

[0102] The functional group of the unnatural amino acid present in the antibody or fusion protein may be an 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, for example, 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.

[0103] 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 that has 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 with the addition of a non-nucleophilic base such as N,N-diisopropylethylamine.

[0104] It is understood that the particular techniques for attaching the linker, agent, and / or antibody or fusion protein to one another can 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.

[0105] Methods for Producing Antibodies Using the information provided herein, the anti-VV B5 antibodies and fusion proteins of the present disclosure can be prepared using standard techniques 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, e.g., Tables 1, 3, 4, and 5) can be used to determine appropriate nucleic acid sequences encoding an antibody or fusion protein, and the nucleic acid sequences can then be used to express one or more antibodies or fusion proteins specific for 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.

[0106] Once the nucleic acid encoding the subject antibody or fusion protein has been synthesized, it can be amplified and / or cloned according to standard methods. Molecular cloning techniques to achieve these ends are known in the art. A wide variety of cloning and in vitro amplification methods suitable for the construction of recombinant nucleic acids are known to those of skill in the art and are the subject of numerous textbooks and laboratory manuals.

[0107] 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 generic expression cassette that includes at least one independent terminator sequence, a sequence that allows replication of the cassette in both eukaryotes and prokaryotes, such as those found in shuttle vectors, and a selection marker for both prokaryotic and eukaryotic systems.

[0108] To obtain high levels of expression of a cloned nucleic acid, it is common to construct an expression plasmid that typically contains a strong promoter to direct transcription, a ribosome binding site for translation initiation, and a transcription / translation terminator, each in a functional orientation relative to each other and to the protein coding sequence. Examples of regulatory regions suitable for this purpose in E. coli are the promoter and operator regions of the E. coli tryptophan biosynthetic pathway, phage lambda (P L ), the left promoter of the ribosomal RNA (RIRNA) and the L-arabinose (araBAD) operon. It is also useful to include a selection marker in a DNA vector 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, and Salmonella. E. coli systems can also be used.

[0109] The antibody gene(s) may also be subcloned into an expression vector that allows for the addition of tags (e.g., FLAG, hexahistidine, etc.) to the C-terminus or N-terminus of the antibody (e.g., IgG, Fab, scFv, etc.) to facilitate purification. Methods for transfecting and expressing genes in mammalian cells are known in the art. Transducing cells with a nucleic acid may include, for example, incubating lipid microparticles containing the nucleic acid with the cells, or incubating a viral vector containing the nucleic acid with cells within the host range of the vector. Culturing cells used in this disclosure, including cell lines and cultured cells from tissue (e.g., tumor) or blood samples, is well known in the art.

[0110] Once a nucleic acid encoding an antibody or fusion protein 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 fungi, insect (e.g., insects using baculovirus vectors), and mammalian cells.

[0111] Isolation and purification of the subject antibody or fusion protein can be accomplished according to methods known in the art. For example, the protein can be isolated from lysates of cells genetically modified to express the protein constitutively and / or upon induction, or from synthetic reaction mixtures by immunoaffinity purification (or precipitation using protein L or A), washing to remove non-specifically bound material, and elution of specifically bound antibody. The isolated antibody or fusion protein can be further purified by dialysis and other methods commonly used in protein purification methods. In one embodiment, the antibody can be isolated using metal chelate chromatography methods. The antibodies and fusion proteins of the present disclosure can include modifications to facilitate isolation, as discussed above.

[0112] Antibodies and fusion proteins may be prepared in a substantially pure or isolated form (e.g., free of other polypeptides). The protein may be present in a composition which is enriched for the polypeptide relative to other components which may be present (e.g., other polypeptides or other host cell components). Purified antibodies and fusion proteins may be provided such that the antibody or fusion protein is present in a composition which is substantially free of other expressed proteins, for example, such that less than 90%, usually less than 60%, and more usually less than 50% of the composition is made up of other expressed proteins.

[0113] Antibodies and fusion proteins produced by prokaryotic cells may require exposure to chaotropic agents for proper folding. During purification from E. coli, for example, the expressed proteins may be optionally denatured and then renatured. This can be accomplished, for example, by solubilizing bacterially produced antibodies and fusion proteins in a chaotropic agent such as guanidine HCl. The antibodies or fusion proteins are then renatured by either slow dialysis or gel filtration. Alternatively, the nucleic acids encoding the antibodies and fusion proteins may be operably linked to a secretion signal sequence such as pelB so that the antibodies and fusion proteins are secreted into the periplasm in a correctly folded form.

[0114] The present disclosure also provides cells that produce the antibodies and fusion proteins of the present disclosure, and suitable cells include eukaryotic cells, e.g., mammalian cells. For example, the present disclosure provides recombinant host cells (also referred to herein as "genetically modified host cells") that have been genetically modified with one or more nucleic acids comprising a nucleotide sequence encoding the heavy and / or light chains of an antibody of the present disclosure.

[0115] Techniques for making recombinant DNA versions of antigen-binding regions of antibody molecules that avoid the generation of hybridomas are also contemplated herein. The DNA is cloned, for example, into bacterial expression systems (e.g., bacteriophage), yeast expression systems (e.g., Saccharomyces or Pichia), insect expression systems, or mammalian expression systems. One suitable technique uses a bacteriophage lambda vector system with a leader sequence that directs the expressed antibody (e.g., Fab or scFv) to the periplasmic space (between the bacterial cell membrane and the cell wall) or to be secreted. Large numbers of functional fragments (e.g., Fab or scFv) can be rapidly generated for those that bind to the antigen of interest.

[0116] Antibodies and fusion proteins that specifically bind VV B5 can be prepared using a wide variety of techniques known in the art, including the use of recombinant, phage display technology, selected lymphocyte antibody method (SLAM), or a combination thereof. For example, antibodies can be generated and isolated using phage display methods. Phage display is used for high-throughput screening of protein interactions. Phages may be used to display antigen-binding domains expressed from repertoire or combinatorial antibody libraries (e.g., human or murine). Phages expressing antigen-binding domains that bind to VV B5 can be selected or identified using VV B5, for example, using labeled VV B5 bound or captured to a solid surface or bead. Phages used in these methods are typically filamentous phage that contain fd and M13 binding domains expressed from phages 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 also be screened for antibodies. Such procedures provide an alternative to traditional hybridoma technology for the isolation and subsequent cloning of monoclonal antibodies.

[0117] After phage selection, the antibody coding region from the phage can be isolated and used to produce whole antibodies or any desired antigenic fragment, and 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.

[0118] 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.

[0119] In certain embodiments, provided is an anti-VV B5 antibody or fusion protein of the disclosure, such as any of the antibodies and fusion proteins (e.g., CARs) described above, which comprises a variable heavy chain (V H ) polypeptide, variable light chain (V L In some embodiments, the antibody is a single chain antibody (e.g., scFv) and the nucleic acid encodes the single chain antibody. In some embodiments, a CAR of the disclosure, such as the V of the anti-VV B5 antibody of the disclosure, is provided. H Polypeptides and V L A nucleic acid encoding 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.

[0120] 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 anti-VV B5 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 comprises a generic expression cassette that includes at least one independent terminator sequence, e.g., 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.

[0121] 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, the variable heavy chain (VH) of the antibody of the present disclosure is H a first nucleic acid encoding a variable light chain (V L and 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.

[0122] Also provided is a method of making an anti-VV B5 antibody or fusion protein of the present disclosure, comprising culturing a cell of the present disclosure under conditions suitable for the cell to express the anti-VV B5 or fusion protein, where the antibody or fusion protein is produced. The conditions for culturing the cells such that the antibody or fusion protein is expressed can vary. Such conditions can 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)), and in an environment having a suitable percentage of CO2, e.g., 3% to 10% (e.g., 5%).

[0123] composition As summarized above, the present disclosure also provides compositions. According to some embodiments, the compositions of the present disclosure comprise the anti-VV B5 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 (e.g., CAR) or conjugates described in the above anti-VV B5 antibody section, the description of which is incorporated but will not be repeated here for brevity.

[0124] In certain aspects, the compositions of the present disclosure include an antibody, fusion protein, or conjugate present in a liquid medium. The liquid medium may be an aqueous liquid medium, such as water, a buffer, etc. One or more additives may be present in such compositions, 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.), solubilizers, detergents (e.g., non-ionic detergents such as Tween-20), nuclease inhibitors, protease inhibitors, glycerol, chelating agents, etc.

[0125] Aspects of the present disclosure further include pharmaceutical compositions. In some embodiments, the pharmaceutical compositions of the present disclosure comprise an anti-VV B5 antigen antibody (or a conjugate or fusion protein comprising same) of the present disclosure and a pharma- ceutical acceptable carrier.

[0126] Anti-VV B5 antibodies, fusion proteins (e.g., CARs) or conjugates can be incorporated into various formulations for therapeutic administration. More specifically, anti-VV B5 antibodies, fusion proteins or conjugates can be formulated into pharmaceutical compositions by combining with suitable pharma- ceutically acceptable excipients or diluents, and can be formulated into solid, semi-solid, liquid or gaseous formulations such as tablets, capsules, powders, granules, ointments, solutions, injections, inhalants, aerosols, etc.

[0127] 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 a selected route of administration.

[0128] 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.

[0129] For oral preparations, the antibody, fusion protein, or conjugate may be used alone or in combination with suitable additives for making tablets, powders, granules, or capsules, e.g., conventional additives such as lactose, mannitol, corn starch, or potato starch; binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch, or gelatin; disintegrating agents such as corn starch, potato starch, or sodium carboxymethylcellulose; lubricants such as talc or magnesium stearate; and, if necessary, diluents, buffers, wetting agents, preservatives, and flavorings.

[0130] 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 aspects, 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.

[0131] 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 (such as 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, glycine, glycerol ... amino acids such as sucrose, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline, and combinations thereof; monosaccharides, disaccharides, and other carbohydrates; 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 Pluronics, Triton-X, or polyethylene glycol (PEG).

[0132] The pharmaceutical composition may be in liquid form, lyophilized form, or liquid form reconstituted from lyophilized form, with the lyophilized preparation being reconstituted with a sterile solution prior to administration. The standard procedure for reconstituting a lyophilized composition is to add back a volume of purified water (typically equal to the volume removed during lyophilization), although solutions containing antimicrobial agents may be used to produce pharmaceutical compositions for parenteral administration.

[0133] 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. Buffer concentrations can be, for example, from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending on the buffer and the desired tonicity of the formulation.

[0134] An isotonicity agent may be included to adjust the tonicity of the formulation. Examples of 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 an isotonic solution, 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 may be used in an amount of about 5 mM to about 350 mM, for example, in an amount of 100 mM to 350 mM.

[0135] Surfactants may also be added to the formulation to reduce aggregation in the formulation and / or minimize the formation of fine particles 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 concentrations of the surfactant can range from about 0.001% to about 1% (w / v).

[0136] Lyoprotectants may also be added to protect the antibody and / or T cell activator from destabilizing conditions during the lyophilization process. For example, known lyoprotectants include sugars (including glucose and sucrose), polyols (including mannitol, sorbitol, and glycerol), and amino acids (including alanine, glycine, and glutamic acid). Lyoprotectants may be included in amounts of about 10 mM to 500 nM.

[0137] 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).

[0138] 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, e.g., methods comprising administering to an individual a pharmaceutical composition of the present disclosure to target an anti-VV B5 antibody, fusion protein (e.g., CAR) or conjugate to vaccinia virus-infected cells (including, but not limited to, cancer cells) of the individual.

[0139] Thus, in certain embodiments, the kits of the present disclosure include 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 anti-VV B5 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 appreciated, the kits of the present disclosure may include any of the agents and features described above in the sections related to the anti-VV B5 antibodies, fusion proteins, conjugates, and compositions of interest, which will not be repeated here for brevity.

[0140] 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 a composition comprising the anti-VV B5 antibody, fusion protein, and / or conjugate of the present disclosure. The term "unit dose" as used herein refers to a physically discrete unit suitable as a unit 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 a 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 or multiple dose of the composition.

[0141] In certain embodiments, the kits of the present disclosure include instructions for targeting the anti-VV B5 antibody, fusion protein, or conjugate present in the pharmaceutical composition to VV-infected cells in an individual. According to some embodiments, the infected cells are VV-infected cancer cells in an individual having cancer (e.g., for treating cancer in the individual), e.g., by administering the pharmaceutical composition to the individual, the individual comprising cancer cells infected with VV, and the anti-VV B5 antibody, fusion protein (e.g., CAR), or conjugate is targeted to the infected cancer cells by a VV antigen expressed on the surface of the infected cancer cells.

[0142] According to some embodiments, the kits of the present disclosure may further comprise 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 comprise instructions for administering to an individual with cancer a pharmaceutical composition comprising a VV in an amount effective to infect cells of the individual (e.g., infect cancer cells of an individual with cancer), e.g., prior to administering a pharmaceutical composition comprising an anti-VV B5 antibody, fusion protein, or conjugate of the present disclosure.

[0143] The instructions included in the kit (e.g., instructions for use (IFU)) may be recorded on a suitable recording medium. For example, the instructions may be printed on a substrate such as paper or plastic. As such, the instructions may be present within 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), or 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, or the like. In yet 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 from which the instructions can be downloaded. As with the instructions, the means for obtaining the instructions are recorded on a suitable substrate.

[0144] method Aspects of the present disclosure further include methods of using the anti-VV B5 antibodies, fusion proteins (e.g., CARs) and conjugates of the present disclosure, which are useful in a variety of contexts, including in vitro and / or in vivo research and / or clinical applications.

[0145] In certain aspects, provided are methods comprising administering to an individual an effective amount of a pharmaceutical composition comprising any of the disclosed anti-VV B5 antibodies (including any of the fusion proteins or conjugates comprising such antibodies), the individual comprising cells infected with a VV encoding a VV B5 antigen to which the antibody binds, and the anti-VV B5 antibody, fusion protein or conjugate is targeted to the infected cells by the VV B5 antigen expressed on the surface of the infected cells. According to some embodiments, such methods further comprise infecting the cells by administering an effective amount of a VV to the individual prior to administering the pharmaceutical composition to the individual.

[0146] Upon binding to an infected cell surface (e.g., an infected cancer cell surface), an anti-VV B5 antibody (or a fusion protein or conjugate comprising it) may induce cytotoxicity, for example, via antibody-dependent cellular cytotoxicity (ADCC), by recruiting complement in complement-dependent cytotoxicity (CDC), via antibody-dependent cellular cytotoxicity (ADCP), via epitope spreading, or by some other mechanism. The antibody may 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 may be used when it is desired to eliminate or reduce effector functions to minimize side effects or therapeutic complications.

[0147] According to some embodiments, provided is a method comprising administering to an individual an effective amount of a pharmaceutical composition comprising any of the anti-VV B5 antibodies (including any of the fusion proteins or conjugates comprising such antibodies) of the present disclosure, the individual comprising cancer cells infected with a VV encoding the VV B5 antigen to which the antibody binds, and the anti-VV B5 antibody, fusion protein or conjugate is targeted to the infected cancer cells by the VV B5 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 a VV to the individual prior to administering the pharmaceutical composition to the individual. Such methods find use, for example, in treating cancer in an individual.

[0148] In certain embodiments, the pharmaceutical composition comprises any of the anti-VV B5 antibody conjugates of the present disclosure. For example, the pharmaceutical composition may comprise a conjugate in which the 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 infected cells (e.g., infected cancer cells) of the individual using the in vivo imaging agent. The method of administering a conjugate comprising a detectable label or radioisotope to an individual finds use in imaging cells (e.g., cancer cells) of the individual, for example, for purposes of diagnosis, prognosis and / or monitoring treatment (e.g., anti-cancer therapy).

[0149] According to some embodiments, the pharmaceutical composition may comprise a conjugate of the present disclosure, wherein the 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 conjugates section above.

[0150] In certain embodiments, a method is provided for targeting a CAR that specifically binds to a VV B5 antigen to a VV-infected cell (e.g., a VV-infected cancer cell) of an individual. Such a method comprises administering to the individual an effective amount of a pharmaceutical composition comprising a CAR of the present disclosure, and the target cells of the individual are infected with VV and express the VV B5 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, etc. For example, the CAR can be present on the surface of a T cell, and the method is a method of targeting a CAR T cell to an infected cell (e.g., a cancer cell) of the individual. According to some embodiments, such a method further comprises infecting the cell (e.g., a cancer cell) 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.

[0151] 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 a CAR. In some embodiments, the cells are T cells, thereby providing a method of producing CAR T cells. In some embodiments, such a method includes 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 a CAR. In some embodiments, the T cells are transduced with a retroviral vector, e.g., a gamma retroviral vector, encoding a CAR. In some embodiments, the T cells are transduced with a lentiviral vector encoding a CAR.

[0152] 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 primate is a human.

[0153] T cells can be obtained from a number of 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.

[0154] 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 naive (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.

[0155] 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 markers selected from the group consisting of CD62L, CCR7, CD28, CD27, CD122, CD127, CD197, or CD38 or CD62L, CD127, CD197, and CD38 are further separated 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, TIM3, 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, TIM3, and LAG3.

[0156] 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, the disclosures of which are incorporated by reference in their entireties 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.

[0157] 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 simultaneously with the introduction of a nucleic acid (e.g., an expression vector) encoding a CAR into the T cells.

[0158] 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 amounts sufficient for the growth and proliferation of T cells.

[0159] In some embodiments, a nucleic acid (e.g., an expression vector) encoding a CAR is introduced into a cell (e.g., a T cell) by microinjection, transfection, lipofection, heat shock, electroporation, transduction, gene gun, microinjection, DEAE-dextran mediated transfer, etc. In some embodiments, a nucleic acid (e.g., an expression vector) encoding a CAR is introduced into a cell (e.g., a T cell) by AAV transduction. The AAV vector may comprise an ITR 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 comprises an ITR from AAV2 and a serotype from AAV6. In some embodiments, a nucleic acid (e.g., an expression vector) encoding a CAR is introduced into a cell (e.g., a T cell) by lentiviral or retroviral transduction. The lentiviral vector backbone may 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). The lentiviral vector may be integration-competent or an integrase-deficient lentiviral vector (TDLV). In one embodiment, an IDLV vector containing an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) is used.

[0160] In certain aspects, methods are provided for targeting a conjugate that specifically binds to the VV B5 antigen to VV-infected cells (e.g., VV-infected cancer cells) in an individual. Such methods include administering to the individual an effective amount of a pharmaceutical composition comprising a conjugate comprising an anti-VV B5 antibody, and the target cells (e.g., cancer cells) are infected with VV and express the VV B5 antigen on their surface. According to some embodiments, such methods further include infecting cells (e.g., cancer cells) by administering an effective amount of VV to the individual prior to administering the pharmaceutical composition to the individual. Such methods find use, for example, in treating cancer in an individual.

[0161] In certain aspects, a method is provided that includes administering a pharmaceutical composition comprising a cell (e.g., a cancer cell) infected with VV. The pharmaceutical composition may further include an anti-VV B5 antibody, conjugate, or fusion protein of the present disclosure that specifically binds to the VV B5 antigen expressed by the infected cell, e.g., the cancer cell. The cell (e.g., the cancer cell) may have been removed from the individual during surgery. The cell (e.g., the cancer cell) may have been modified (and killed) in the laboratory to make it more likely to be attacked by the immune system when administered to the patient. The patient's immune system then attacks the cell and similar cells that remain in the body. The antibody, conjugate, or fusion protein 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 and enhance the immune response against the tumor.

[0162] The pharmaceutical composition may be administered to any of a variety of individuals. In certain aspects, the individual is a "mammal" or "mammalian," terms used broadly to describe 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., a mouse model, a primate model, etc.) of a cell proliferative disorder, such as cancer.

[0163] 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.

[0164] 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 carcinoma, 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 aspects, 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).

[0165] The anti-VV B5 antibodies, fusion proteins and conjugates of the present disclosure may be administered via a route of administration selected from oral (e.g., in tablet form, capsule form, liquid form, etc.), parenteral (e.g., intravenous, intramuscular, subcutaneous, intramuscular, or epidural injection), topical, intranasal, or intratumoral administration.

[0166] The anti-VV B5 antibodies, fusion proteins and conjugates of the present disclosure can be administered as pharmaceutical compositions in a therapeutically effective amount. 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 prophylactic result), such as a reduction in symptoms, as compared to a control. With respect to 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.

[0167] As mentioned above, the present disclosure includes a method for treating an individual, e.g., cancer, in an individual. Treatment means the improvement of at least one or more symptoms associated with the disease state (e.g., cancer) of the individual, and improvement is used broadly to refer to the reduction of the parameters, e.g., the degree of symptoms associated with at least the disease state (e.g., cancer) being treated. Thus, treatment also includes the situation in which the disease state (e.g., cancer) or at least one or more symptoms associated therewith are completely inhibited, e.g., prevented from occurring, or stopped, e.g., discontinued, so that the individual no longer suffers from the disease state (e.g., cancer) or at least the symptoms that characterize the disease state (e.g., cancer).

[0168] The anti-VV B5 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.

[0169] 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 may 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 to an individual 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 that used 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 that used when the antibody, fusion protein or conjugate is administered without the second agent.

[0170] In some embodiments, one or more doses of the antibody, fusion protein or conjugate and the second agent are administered to an individual simultaneously, where "concurrently" means 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.

[0171] In some embodiments, one or more doses of the antibody, fusion protein or conjugate and the second agent are administered sequentially to the individual.

[0172] 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 particular cycle) before the administration of the second therapeutic agent. Alternatively, the second therapeutic agent may be administered (e.g., in a particular 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.

[0173] In one example, the second agent is administered to the individual for a desired period of time prior to administration of the antibody, fusion protein or conjugate. In certain aspects, such a regimen "primes" the cancer cells to enhance the anti-cancer effect of the antibody, fusion protein or conjugate. Such a period of time separating the step of administering the second agent from the step of administering the antibody, fusion protein or conjugate is desirably long enough to allow priming of the cancer cells such that the anti-cancer effect of the antibody, fusion protein or conjugate is increased.

[0174] 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, e.g., based on population studies showing a correlation between a given dosing regimen and a particular effect of interest).

[0175] In certain aspects, the desired relative dosing regimen of drugs administered in combination can be evaluated or empirically determined, e.g., using ex vivo, in vivo, and / or in vitro models, hi some embodiments, such evaluation or empirical determination is performed in vivo in a patient population (e.g., so that a correlation is established), or alternatively, in a particular individual of interest.

[0176] 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 administered in combination, each administered according to such an intermittent, cyclical schedule, the individual doses of the 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 one dose of the first agent. In some 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 by a dose of the second agent a period of time after each dose of the first agent. In some embodiments, two or more doses of the first agent are administered between at least a pair of doses of the second agent, and in certain aspects, two or more doses of the second agent are administered between at least a pair of doses of the first agent. In some embodiments, the 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.

[0177] One exemplary protocol for combining two intermittent cyclical dosing regimens may include (a) a first dosing period during which a therapeutically effective amount of an antibody, fusion protein, or conjugate is administered to the individual, (b) a first rest period, (c) a second dosing period during which a therapeutically effective amount of a second agent is administered to the individual, and (d) a second rest period. A second exemplary protocol for combining two intermittent cyclical dosing regimens may include (a) a first dosing period during which a therapeutically effective amount of a second agent is administered to the individual, (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 individual, and (d) a second rest period.

[0178] In some embodiments, the first rest period and the second rest period may correspond to the same number of hours or days. Alternatively, in some embodiments, the first rest period and the second rest period are different, with the first rest period being 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, it can be defined as a number of days or weeks (e.g., 1 day, 3 days, 5 days, 1 week, 2 weeks, 4 weeks, or more) rather than hours.

[0179] If the length of the first resting period is determined by the presence or occurrence of a particular biological or therapeutic event, the length of the second resting period may be determined based on different factors, separately or in combination. Exemplary such factors may 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 resting periods may 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 relevant resting period may be considered complete if the plasma concentration of the relevant agent falls below a predetermined level, optionally upon evaluation or other consideration of one or more characteristics of the individual's response.

[0180] In certain aspects, the number of cycles in which a particular agent is administered may be empirically determined, and in some embodiments, the exact regimen followed (e.g., number of doses, spacing of doses (e.g., relative to each other or to another event, such as administration of another therapy), amount of dose, etc.) may be different for one or more cycles as compared to one or more other cycles.

[0181] The antibody, fusion protein or conjugate and the second agent can be administered together or independently via any suitable route of administration. The antibody, fusion protein or conjugate and the second agent can be administered via an administration route independently selected from oral, topical (e.g., by 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, capsule, liquid form, etc.), either simultaneously (in the same pharmaceutical composition or in separate pharmaceutical compositions) or sequentially.

[0182] When the method further comprises infecting target cells (e.g., cancer cells) by administering the VV to an individual, any suitable administration regimen can be used to infect the cancer cells. In certain embodiments, such methods comprise simultaneously administering the VV and the pharmaceutical composition to the individual. The simultaneous administration can take various forms, including administering a first composition comprising the VV and a pharmaceutical composition comprising the antibody, fusion protein, or conjugate as separate compositions. According to some embodiments, the simultaneous administration comprises administering the VV and the antibody, fusion protein, or conjugate present in the same composition. In one non-limiting example, cells expressing a fusion protein (e.g., a CAR T cell of the present disclosure) are administered simultaneously with the VV, and the simultaneous administration comprises administering cells infected with the VV to the individual. As an example, a CAR T cell of the present disclosure infected with a VV can be administered to the individual to achieve simultaneous administration of the VV and the CAR T cell to the individual.

[0183] In certain embodiments, when the method further includes infecting a target cell (e.g., a cancer cell) by administering a VV to the individual, the target cell is infected by administering the VV to the individual prior to administering the pharmaceutical composition to the individual.

[0184] Any suitable approach can be used to infect target cells (e.g., cancer cells) in an individual. Poxvirus replication occurs in the cytoplasm because the virus is sufficiently complex to have acquired all the functions necessary for genome replication. Once in the cytoplasm, gene expression is carried out by viral enzymes associated with the core. Expression is divided into two stages: early genes, which represent about 50% of the genome, are expressed before genome replication, and late genes, which 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 self-prime, leading 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). Inclusions are formed in the cytoplasm that mature into viral particles. Vaccinia virus is unique among DNA viruses because it replicates exclusively in the cytoplasm of the host cell. Thus, a large genome is required to code for 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).

[0185] To infect target cells (e.g., cancer cells) with the VV, the VV is administered using a suitable administration route. The administration route 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, particularly the tumor vasculature or adjacent vasculature), transdermal, intratracheal, intraperitoneal, intraarterial, intravesical, intratumoral, inhalation, perfusion, lavage, and oral administration and formulations. Intratumoral injection, or direct injection into the 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.

[0186] 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 VV 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 a precisely predetermined amount of solution at any depth (U.S. Pat. No. 5,846,225). A mixture of VV 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.

[0187] A physician may begin by formulating a dose of the VV 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 VV vector, and then administer progressively lower doses until a therapeutic effect is achieved, for example, a reduction in the volume of one or more tumors.

[0188] Regardless of the scope of the appended claims, the present disclosure is also defined by the following embodiments.

[0189] 1. An antibody that specifically binds to vaccinia virus B5 antigen (VV B5), A variable heavy chain (VH) having 70% or more sequence identity to the amino acid sequence shown in SEQ ID NO:1. H ) a polypeptide comprising: H The polypeptide is one or more mutations selected from the group consisting of Q1E, E2V, Q3T, E5L / K, G9P, G10V, K13Q, E15G / T, G16E, S17T, T19R, T21S, T23A, A41P, R44K, G45A, T74D, S75N / T, S76_T77insK, T77N / S, T78Q, V79L, T80Y / V, Q82T, T84N, R85S / N, L86M, T87R / D, A88P, A89E / V, T93V, F95Y, P112Q, and any combination thereof, numbered according to SEQ ID NO:1; V containing the amino acid sequence SSYYMC (SEQ ID NO: 13) H CDR1, V containing the amino acid sequence CIYTSSGSAYYA(N / D)(W / S)(A / V)KG (SEQ ID NO: 14) H CDR2, V containing the amino acid sequence NAVGSSYYLYL (SEQ ID NO: 17) H a variable heavy chain polypeptide comprising a CDR3; A variable light chain (V) having 70% or more sequence identity to the amino acid sequence shown in SEQ ID NO:7. L ) a polypeptide comprising: L The polypeptide is one or more mutations selected from the group consisting of A1D / E, Q2I, V3Q, L4M, T7S, S9A, P10T / S, V11L, A13L, A14S, V15P, G17D / E, T18R, V19A, I21L, S22T, Q44K, P45A / V, N47K / R, V60I, S62A, K65S, Q72E / D, D79S, E81Q, C82P, D83E, A85F / V, T87V, G103Q, E106K, V107L, V108E, V109I, and any combination thereof, numbered according to SEQ ID NO:7; V containing the amino acid sequence QASQSVAGNNYLS (SEQ ID NO: 18) L CDR1, V containing the amino acid sequence SVSTLAS (SEQ ID NO: 19) L CDR2, V containing the amino acid sequence QGYYNDGIWA (SEQ ID NO: 20) LAn antibody comprising a variable light chain polypeptide comprising a CDR3.

[0190] 2.V H The antibody of embodiment 1, wherein the polypeptide comprises one of the following mutations: E2V, E5L / K, E15G / T, R44K, R85S / N, T87R / D, A89E / V, and P112Q, any combination thereof, or each.

[0191] 3.V H The antibody of embodiment 2, wherein the polypeptide comprises each of the mutations E2V, E5L / K, E15G / T, R44K, R85S / N, T87R / D, A89E / V, P112Q.

[0192] 4. The antibody of embodiment 2 or embodiment 3, wherein the E5L / K mutation is E5L.

[0193] 5. The antibody of any one of embodiments 2 to 4, wherein the E15G / T mutation is E15G.

[0194] 6. The antibody of any one of embodiments 2 to 5, wherein the R85S / N mutation is R85S.

[0195] 7. The antibody of any one of embodiments 2 to 6, wherein the T87R / D mutation is T87R.

[0196] 8. The antibody of any one of embodiments 2 to 7, wherein the A89E / V mutation is A89E.

[0197] 9.V H 9. The antibody of any one of embodiments 2 to 8, wherein the polypeptide comprises one of the following mutations: Q1E, K13Q, T19R, T21S, T23A, T84N, T93V, and F95Y, any combination thereof, or each of them.

[0198] 10. V H The polypeptide comprises the amino acid sequence CIYTSSGSAYYADSVKG (SEQ ID NO: 16). H10. The antibody of any one of embodiments 2 to 9, comprising CDR2.

[0199] 11. V H 10. The antibody of any one of embodiments 2 to 9, wherein the polypeptide comprises one of the following mutations: T74D, S75N / T, S76_T77insK, T77N / S, V79L, and T80Y / V, any combination thereof, or each.

[0200] 12. The antibody of embodiment 11, wherein the T77N / S mutation is T77N.

[0201] 13. The antibody of embodiment 11 or embodiment 12, wherein the T80Y / V mutation is T80Y.

[0202] 14. V H The polypeptide comprises the amino acid sequence CIYTSSGSAYYADSVKG (SEQ ID NO: 16). H 14. An antibody according to any one of embodiments 11 to 13, comprising CDR2.

[0203] 15. V H 4. The antibody of embodiment 2 or embodiment 3, wherein the polypeptide comprises one of the following mutations: Q3T, E5L / K, G9P, G10V, E15G / T, G16E, S17T, A41P, G45A, T74D, S75N / T, S76_T77insK, T77N / S, T78Q, T80Y / V, Q82T, R85S / N, L86M, T87R / D, A88P and A89E / V, any combination thereof, or each.

[0204] 16. The antibody of embodiment 15, wherein the E5L / K mutation is E5K.

[0205] 17. The antibody of embodiment 15 or embodiment 16, wherein the E15G / T mutation is E15T.

[0206] 18. The antibody of any one of embodiments 15 to 17, wherein the S75N / T mutation is S75T.

[0207] 19. The antibody of any one of embodiments 15 to 18, wherein the T77N / S mutation is T77S.

[0208] 20. The antibody of any one of embodiments 15 to 19, wherein the T80Y / V mutation is T80V.

[0209] 21. The antibody of any one of embodiments 15 to 20, wherein the R85S / N mutation is R85N.

[0210] 22. The antibody of any one of embodiments 15 to 21, wherein the T87R / D mutation is T87D.

[0211] 23. The antibody of any one of embodiments 15 to 22, wherein the A89E / V mutation is A89V.

[0212] 24. V H The antibody of any one of embodiments 1 to 3, wherein the polypeptide comprises 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the amino acid sequence set forth in one of SEQ ID NOs: 2 to 6.

[0213] 25. V H The antibody of any one of embodiments 2 to 9, wherein the polypeptide comprises 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:3.

[0214] 26. V H The antibody of embodiment 10, wherein the polypeptide comprises 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:5.

[0215] 27. V H14. The antibody of any one of embodiments 11 to 13, wherein the polypeptide comprises 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:2.

[0216] 28. V H The antibody of embodiment 14, wherein the polypeptide comprises 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:4.

[0217] 29. V H 24. The antibody of any one of embodiments 15 to 23, wherein the polypeptide comprises 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:6.

[0218] 30.V L 30. The antibody of any one of embodiments 1 to 29, wherein the polypeptide comprises one of the following mutations: T7S, P10T / S, V11L, A14S, G17D / E, T18R, P45A / V, N47K / R, K65S, Q72E / D, D79S, C82P, A85F / V, G103Q, E106K, V108E, and V109I, any combination thereof, or each.

[0219] 31. V L The antibody of embodiment 30, wherein the polypeptide comprises each of the mutations T7S, P10T / S, V11L, A14S, G17D / E, T18R, P45A / V, N47K / R, K65S, Q72E / D, D79S, C82P, A85F / V, G103Q, E106K, V108E, and V109I.

[0220] 32. V LThe antibody of embodiment 30 or embodiment 31, wherein the polypeptide comprises one of the following mutations: G17D, S22T, Q44K, N47K, and E81Q, any combination thereof, or each of them.

[0221] 33. An antibody according to any one of embodiments 30 to 32, wherein the P10T / S mutation is P10T.

[0222] 34. An antibody according to any one of embodiments 30 to 33, wherein the P45A / V mutation is P45A.

[0223] 35. The antibody of any one of embodiments 30 to 34, wherein the Q72E / D mutation is Q72E.

[0224] 36. The antibody of any one of embodiments 30 to 35, wherein the A85F / V mutation is A85F.

[0225] 37. V L 37. The antibody of any one of embodiments 33 to 36, wherein the polypeptide comprises one of the following mutations: A1D, Q2I, V3Q, and L4M, any combination thereof, or each of them.

[0226] 38. V L 33. An antibody according to any one of embodiments 30 to 32, wherein the polypeptide comprises the mutation D83E.

[0227] 39. An antibody according to any one of embodiments 30 to 32 or 38, wherein the P10T / S mutation is P10S.

[0228] 40. The antibody of any one of embodiments 30 to 32, 38, or 39, wherein the P45A / V mutation is P45V.

[0229] 41. The antibody of any one of embodiments 30 to 32 or 38 to 40, wherein the Q72E / D mutation is Q72D.

[0230] 42. An antibody according to any one of embodiments 30 to 32 or 38 to 41, wherein the A85F / V mutation is A85V.

[0231] 43. V L 43. The antibody of any one of embodiments 38 to 42, wherein the polypeptide comprises one of the mutations A1D, Q2I, V3Q, and L4M, any combination thereof, or each of them.

[0232] 44. V L 32. The antibody of embodiment 30 or embodiment 31, wherein the polypeptide comprises one of the following mutations: A1E, S9A, P10T, A13L, V15P, G17E, V19A, I21L, P45A, N47R, V60I, S62A, Q72D, D83E, A85F, T87V, and V107L, any combination thereof, or each of them.

[0233] 45. V L 45. The antibody of embodiment 44, wherein the polypeptide comprises each of the mutations A1E, S9A, P10T, A13L, V15P, G17E, V19A, I21L, P45A, N47R, V60I, S62A, Q72D, D83E, A85F, T87V, and V107L.

[0234] 46. ​​V L 32. The antibody of embodiment 1, 30 or 31, wherein the polypeptide comprises 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the amino acid sequence set forth in one of SEQ ID NOs:8 to 12.

[0235] 47. V L 37. The antibody of any one of embodiments 33 to 36, wherein the polypeptide comprises 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:9.

[0236] 48. V L38. The antibody of embodiment 37, wherein the polypeptide comprises 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:8.

[0237] 49. V L The antibody of any one of embodiments 38 to 42, wherein the polypeptide comprises 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:11.

[0238] 50.V L 44. The antibody of embodiment 43, wherein the polypeptide comprises 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:10.

[0239] 51. V L The antibody of embodiment 44 or embodiment 45, wherein the polypeptide comprises 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:12.

[0240] 52. An antibody that specifically binds to vaccinia virus B5 antigen (VV B5), Variable heavy chain (V H ) a polypeptide comprising V containing the amino acid sequence SSYYMC (SEQ ID NO: 13) H CDR1, V containing the amino acid sequence CIYTSSGSAYYADSVKG (SEQ ID NO: 16) H CDR2, V containing the amino acid sequence NAVGSSYYLYL (SEQ ID NO: 17) H a variable heavy chain polypeptide comprising a CDR3; Variable light chain (VL ) a polypeptide comprising V containing the amino acid sequence QASQSVAGNNYLS (SEQ ID NO: 18) L CDR1, V containing the amino acid sequence SVSTLAS (SEQ ID NO: 19) L CDR2, V containing the amino acid sequence QGYYNDGIWA (SEQ ID NO: 20) L An antibody comprising a variable light chain polypeptide comprising a CDR3.

[0241] 53. The antibody of embodiment 52, wherein the antibody is a humanized antibody.

[0242] 54. The antibody of any one of embodiments 1 to 53, wherein the antibody is an IgG.

[0243] 55. The antibody of embodiment 54, wherein the antibody comprises a human Fc domain.

[0244] 56. An antibody described in any one of embodiments 1 to 53, wherein the antibody is selected from the group consisting of Fab, F(ab')2, and F(ab').

[0245] 57. The antibody of any one of embodiments 1 to 53, wherein the antibody is a single-chain antibody.

[0246] 58. The antibody of embodiment 57, wherein the single chain antibody is an scFv.

[0247] 59. The antibody is a V as defined in any one of embodiments 1 to 53. H Polypeptide-V L The antibody of any one of embodiments 1 to 58, which is a bispecific antibody comprising a first antigen-binding domain comprising a polypeptide pair.

[0248] 60. The antibody of embodiment 59, wherein the bispecific antibody comprises a second antigen-binding domain that specifically binds to an antigen other than the vaccinia virus B5 antigen.

[0249] 61. The antibody of embodiment 60, wherein the antigen other than the vaccinia virus B5 antigen is an immune cell surface antigen.

[0250] 62. The antibody of embodiment 61, wherein the immune cell surface antigen is an immune effector cell surface antigen.

[0251] 63. The antibody of embodiment 62, wherein the immune cell surface antigen is a T cell surface antigen.

[0252] 64. The antibody of embodiment 63, wherein the antigen is a T cell stimulatory molecule.

[0253] 65. The antibody of embodiment 64, wherein the T cell stimulatory molecule is CD3 or CD28.

[0254] 66. The antibody of embodiment 62, wherein the immune cell surface antigen is a natural killer (NK) cell surface antigen.

[0255] 67. The antibody of embodiment 62, wherein the immune cell surface antigen is a macrophage cell surface antigen.

[0256] 68. A fusion protein comprising: A fusion protein comprising a chain of the antibody of any one of embodiments 1 to 53 fused to a heterologous sequence of amino acids.

[0257] 69. The fusion protein of embodiment 68, wherein the heterologous sequence of amino acids is fused to the C-terminus of the antibody chain.

[0258] 70. The fusion protein according to embodiment 68 or embodiment 69, wherein the antibody is a single-chain antibody according to embodiment 57 or 58.

[0259] 71. The fusion protein is A single chain antibody; A transmembrane domain; and an intracellular signaling domain.

[0260] 72. A conjugate comprising: An antibody according to any one of embodiments 1 to 67 or a fusion protein according to any one of embodiments 68 to 71; and a drug conjugated to an antibody or a fusion protein.

[0261] 73. The conjugate of embodiment 72, 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.

[0262] 74. The conjugate according to embodiment 73, wherein the radioisotope is a therapeutic radioisotope.

[0263] 75. The conjugate according to embodiment 73, wherein the detectable label is a radioactive label.

[0264] 76. The radioactive label is zirconium-89 ( 89 76. The conjugate of embodiment 75, wherein R is H, R is Zn.

[0265] 77. A conjugate according to any one of embodiments 72 to 76, wherein the drug is conjugated to the antibody or fusion protein via a non-cleavable linker.

[0266] 78. A conjugate according to any one of embodiments 72 to 76, wherein the drug is conjugated to the antibody or fusion protein via a cleavable linker.

[0267] 79. The conjugate according to embodiment 78, wherein the cleavable linker is an enzyme-cleavable linker.

[0268] 80. The conjugate according to embodiment 79, wherein the linker is cleavable by a lysosomal protease.

[0269] 81. The conjugate according to embodiment 80, wherein the linker is cleavable by cathepsin or plasmin.

[0270] 82. The variable heavy chain (VH) of the antibody according to any one of embodiments 1 to 67 H ) polypeptide, variable light chain (V L ) a nucleic acid encoding a polypeptide, or both.

[0271] 83. A nucleic acid encoding a fusion protein according to any one of embodiments 68 to 71.

[0272] 84. An expression vector comprising a nucleic acid according to embodiment 82 or embodiment 83.

[0273] 85. A cell comprising: A nucleic acid according to embodiment 82 or embodiment 83, or A cell comprising the expression vector described in embodiment 84.

[0274] 86. A cell according to embodiment 85, wherein the cell comprises a nucleic acid according to embodiment 83 or an expression vector comprising a nucleic acid according to embodiment 83.

[0275] 87. The cell of embodiment 86, wherein the nucleic acid encodes the CAR of embodiment 71, and the cell expresses the CAR on its surface.

[0276] 88. The cell of embodiment 87, wherein the cell is an immune cell.

[0277] 89. The cell of embodiment 88, wherein the cell is an immune effector cell.

[0278] 90. The cell of embodiment 89, wherein the cell is a T cell.

[0279] 91. The cell of embodiment 89, wherein the cell is a NK cell.

[0280] 92. The cell of embodiment 89, wherein the cell is a macrophage.

[0281] 93. A cell comprising: 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.

[0282] 94. a first expression vector comprising a first nucleic acid; and a second expression vector comprising the second nucleic acid.

[0283] 95. A pharmaceutical composition comprising a cell according to any one of embodiments 85 to 94.

[0284] 96. A pharmaceutical composition according to embodiment 95, comprising a cell according to any one of embodiments 87 to 92.

[0285] 97. The pharmaceutical composition according to embodiment 95 or embodiment 96, further comprising a pharma- ceutically acceptable carrier.

[0286] 98. A method for producing an antibody according to any one of embodiments 1 to 53, comprising culturing a cell according to any one of embodiments 85 to 94 under conditions suitable for the cell to express the antibody, thereby producing the antibody.

[0287] 99. A pharmaceutical composition comprising: An antibody according to any one of embodiments 1 to 67, and a pharma- ceutically acceptable carrier.

[0288] 100. A pharmaceutical composition comprising: A fusion protein according to any one of embodiments 68 to 71, and a pharma- ceutically acceptable carrier.

[0289] 101. A pharmaceutical composition comprising: A conjugate according to any one of embodiments 72 to 81, and a pharma- ceutically acceptable carrier.

[0290] 102. A kit comprising: A pharmaceutical composition according to any one of embodiments 95 to 97 or 99 to 101, and instructions for administering the pharmaceutical composition to an individual in need thereof.

[0291] 103. The kit according to embodiment 102, wherein the pharmaceutical composition is present in one or more unit doses.

[0292] 104. The kit according to embodiment 102, wherein the pharmaceutical composition is present in two or more unit doses.

[0293] 105. A method comprising: A method comprising administering a pharmaceutical composition according to any one of embodiments 95-97 or 99-101 to an individual having cancer, wherein the individual comprises cancer cells infected with vaccinia virus (VV), and wherein the antibody, fusion protein or conjugate is targeted to the infected cancer cells by the VV B5 antigen expressed on the surface of the infected cancer cells.

[0294] 106. The method of embodiment 105, further comprising infecting cancer cells by administering a VV to the individual prior to administering the pharmaceutical composition to the individual.

[0295] 107. The method of embodiment 105 or 106, wherein the method is a method for treating cancer in an individual.

[0296] 108. The method according to any one of embodiments 105 to 107, wherein the pharmaceutical composition according to embodiment 101 is administered to an individual, the conjugate comprises an antibody conjugated to a detectable label or a radioisotope that is an in vivo imaging agent, and the method comprises imaging infected cancer cells in the individual using the in vivo imaging agent. EXAMPLES

[0297] The following examples are offered by way of illustration and not by way of limitation.

[0298] experiment Example 1 - Design and expression of humanized V-region sequences This example describes the design and expression of humanized variable regions of rabbit anti-VV B5 antibody A048. The parent A048 antibody is described in International Application No. CA202 / 051230. Sequence-based CDR grafting was used to humanize the A048 variable regions (V regions). Using an in silico homology modeling tool (Schrodinger Bioluminate), the best-fitting candidate templates from the closest aligning human germline sequences for each variable (V) domain were analyzed, and rabbit CDRs were grafted into and compared with the non-humanized A048 parent rabbit antibody and germline antibody. Complementarity determining region (CDR) and framework (FW) sequences were considered taking into account a combination of Kabat, IMGT, Chothia, and in-house CDR annotations to define their involvement in antigen interaction. The start of FW1 was selected based on the rabbit antibody sequences in the IMGT database. Structural analysis of the homology models assessed conformational or sequence deviations that could affect the stability and conformation of the CDRs and identified suitable backmutations.

[0299] IGHV3_23 was found to be the best fit for A048 variable heavy chain (VH) domain sequence homology. The common germline IGHJ4_1 was found to be the best fit for A048 VH FW4 due to high sequence homology. IGKV1_5 was found to be the best fit for VL domain sequence homology. Additionally, IGKV1_27, which differs by five amino acids, was selected because several amino acid positions (IGKV1_5 to IGKV1_27, T10S, A45V, E72D, D83E, F85V) may affect the stability of the CDRs based on in silico modeling. IGKJ4_1 was selected for VL FW4 due to high sequence homology. In two VH candidates (A048_H3 and A048_H4), three amino acids were mutated to human sequences N63D, W64S, A65V (rabbit to human) at the extended end of CDR2, thereby making CDR2 more human. As this region is relatively distant from VH CDR3, it is unlikely to be involved in antigen binding, but has potential impact on immunogenicity due to exposure to solvent. In two VH candidates (A048_H2 and A048_H4), human-to-rabbit back mutations in FW3 (also known as HV4 loop) were introduced (human to rabbit: D74T, N75S, S76_T77delK, N77T, L79V, Y80T) to evaluate their potential impact on binding. In two VL candidates (A048_L2 and A048_L4), n-terminal exchanges in FW1 from human to rabbit were introduced (D1A, I2Q, Q3V, M4L) to assess the potential impact on binding.

[0300] The humanized VH domains (A048_H1, A048_H2, A048_H3, A048_H4) and humanized VL domains (A048_L1, A048_L2, A048_L3, A048_L4) were combined to generate 16 humanized IgGs for evaluation.

[0301] One additional humanized antibody was generated using structural homology modeling to initially identify the best template based on composite score and resolution using Schrodinger Bioluminate software. Structural analysis of the prototype V domains onto which the CDRs were grafted was checked for conformational and sequence deviations. The human Fv#3L7F template was selected for structural grafting of the rabbit CDRs of A048. The best matched human germline for 3L7F was IGHV2 for VH, while no predominant IGKV family was identified. No conformational or sequence deviations that could potentially affect the stability and conformation of the CDRs were observed in silico. Therefore, no back mutations were introduced after grafting the A048 rabbit CDRs onto #3L7F VH and VL, as described by A048_H5L5.

[0302] The sequences of the parental and humanized VH and VL domains are shown in Table 1.

[0303] Humanized VH and VL sequences were synthesized and cloned into ptt5 expression vectors (NRC) containing either the constant regions of human IgG1 heavy chain or human kappa light chain. To produce recombinant antibodies, VH and VL chain plasmids were transfected into HEK293 cells using FectoPro (Polyplus, Cat. No. 116-001). After 120 hours of secretion, cells were removed from the antibody-containing supernatant by centrifugation and sterile filtration (0.22 μm). Antibodies were purified using Protein A (Purolite Praesto Jetted A050) resin. Concentrations and yields of purified antibodies were determined by UV absorbance measurements at 280 nm (A280) using a biospectrophotometer. Concentration values ​​were calculated using the A280 extinction coefficients determined for the individual mAb sequences. Yields (mg / L conditioned medium "CM") determined by A280 measurements are shown in Table 9. The yields of humanized antibodies, 4-8 times higher than that of the parent chimeric antibody, suggest good manufacturability of the humanized antibodies. The purity of the antibodies was tested by SDS-PAGE. The percentage of complete antibodies was determined by HPLC-SEC. The antibodies (12 μL) were added to a glass vial insert in a standard HPLC vial. The samples (10 μL) were injected into an HPLC (Agilent) equipped with a WCL006 SEC column (Wyatt Technology, Cat. No.: WTC-010S5) and eluted with PBS, pH 7.4 at a flow rate of 0.5 mL / min for 40 min. The percentage of complete antibodies was determined by the peak area of ​​the mAb peak among the peak areas of other peaks (if present). The HPLC-SEC results are shown in Table 9.

[0304] Example 2 - Binding of humanized antibodies to VV B5 The humanized antibodies were evaluated by ELISA for binding to VV B5 protein. B5 protein was produced using the B5 sequence of Weiss vaccinia strain (Dr. John Bell OHRI). The B5 gene was synthesized in the ptt5 expression vector (NRC) with a C-terminal AVI tag for biotinylation and a HIS6 tag for purification and transfected into HEK293-6E cells (NRC) using 293fectin (Thermo, Cat. No. 12347019). After 96 hours of secretion, protein-containing supernatants were harvested and purified by IMAC and SEC using the AKTA Pure FPLC system. The protein was further purified by SEC using a Superdex 75 Increase 10 / 300 GL column (Cytiva, Catalog No.: 29148721) coupled with a Superdex 200 Increase 10 / 300 GL column (Cytiva, Catalog No.: 28990944) with PBS, pH 7.4. Fractions containing monomeric protein were combined and concentrated with Amicon Ultra Centrifugal Filter Devices, 10k MWCO.

[0305] For the binding ELISA, B5 monomer was coated onto 96-well ELISA plates (Greiner Bio-One High Bind) at 1 μg / mL in PBS and stored overnight at 4° C. After washing with PBS / Tween wash buffer, the plates were incubated in blocking buffer (1% BSA / PBS) for 1 hour at room temperature. Blocking buffer was removed and each anti-B5 hIgG1 antibody was added in serial titrations from 100 nm in blocking buffer. After 1 hour incubation at room temperature, the plates were washed 3 times and secondary antibody (rabbit anti-hIgG HRP) was added for 1 hour. Plates were washed and TMB was added followed by 2M H2SO4 stop solution. OD450 was read on a spectrophotometer. All humanized antibodies bound to B5 protein with similar EC50 as the A048 rabbit parent antibody, as shown in Table 6 below and Figure 1.

[0306] [Table 6]

[0307] Example 3 - Binding of humanized antibodies to vaccinia virus-infected cells Binding to vaccinia virus-infected cancer cells was determined by flow cytometry. HT29 (ATCC HTB-38) and SKOV3 (ATCC#HTB-77) cells were cultured at 27.3 × 10 in McCoy's 5A (Gibco, Cat. No. 16600-082) + 10% fetal bovine serum (Corning, Cat. No. 35-015-CV) in T175 tissue culture flasks (Corning, Cat. No. 353112). 6 Cells / well were seeded and allowed to adhere overnight at 37 °C, 5% CO. The next day, culture medium was aspirated and cells were resuspended in either VVdd (eGFP) or VVCopenhagen (YFP) (vaccinia virus strains Western Reserve and Copenhagen, respectively, both kindly provided by Dr. John Bell, OHRI) in 27.3 mL of serum-free McCoy's 5A (1 × 10 4Cells were infected at a multiplicity of infection (MOI) of 0.1 in 1000 mM NaCl / mL (100 mM Tris-HCl) at 37°C and 5% CO2 or mock infected. Two hours after infection, the infection medium was replaced with 27.3 mL of McCoy's 5A + 10% fetal bovine serum and incubated at 37°C and 5% CO2. At 48 hours post-infection, the culture medium of each flask was aspirated into a separate 50 mL Falcon tube. Cells were rinsed with 10 mL of phosphate-buffered saline (Gibco, Cat. No. 10010-023) and the wash was added to the corresponding tube. Cells were dissociated by incubating in 10 mL of cell dissociation buffer (Gibco, Cat. No. 13151014) / flask at 37°C for 60 min. Meanwhile, cells in the culture supernatant were pelleted by centrifuging the tubes at 400 x g for 5 min and the supernatant was discarded. After dissociation, cells were resuspended in 10 mL PBS, transferred to corresponding tubes, and pelleted by centrifugation at 400×g for 5 min. The supernatant was discarded and cells were stained for viability with LIVE / DEAD Fixable Violet Dead Cell Stain diluted 1:1000 in 27.3 mL PBS (Invitrogen, Cat. No. L34955) for 30 min at 4°C. After staining, cells were pelleted by centrifugation at 400×g for 5 min and resuspended in 27.3 mL staining buffer (phosphate buffered saline + 2 mM EDTA + 0.5% bovine serum albumin). Cells were transferred to 96-well V-bottom plates (Corning, Cat. No. 3894) at 50 μL / well (5×10 4The cells were seeded with 25 μL / well of human anti-B5 antibodies (A048-H1L1, A048-H1L2, A048-H1L3, A048-H1L4, A048-H2L1, A048-H2L2, A048-H2L3, A048-H2L4, A048-H3L5, A048-H4L6, A048-H5L7, A048-H6L8, A048-H7L9, A048-H8L10, A048-H9L11, A048-H10L12, A048-H10L21, A048-H10L31, A048-H10L4, A048-H10L5, A048-H10L6, A048-H10L7, A048-H10L8, A048-H10L9, A048-H20L13, A048-H20L22, A048-H20L31, A048-H20L4, A048-H30L5, A048-H30L6, A048-H30L7, A048-H30L8, A048-H30L9, A048-H30L10, A048-H30L11, A048-H30L12, A048-H30L13, A048-H30L14, A048-H30L15, A048-H30L16, A048-H30L17, A048-H30L18, A048-H30L19, A048-H30L21, A0 1, A048-H3L2, A048-H3L3, A048-H3L4, A048-H4L1, A048-H4L2, A048-H4L3, A048-H4L4, A048-H5L5, A048, or human IgG1k isotype control) and incubated at 4°C for 1 hour, after which 100 μL of staining buffer was added and centrifuged at 400 × g for 5 minutes.

[0308] Primary antibody binding was detected by incubating samples with 25 μL of 2 μg / mL AlexaFluor-647 conjugated goat anti-human IgG (Jackson ImmunoResearch, Cat. No. 109-605-098) in staining buffer for 30 minutes at 4° C. Cells were then fixed in 50 μL / well of 4% paraformaldehyde in PBS (Thermo Scientific, Cat. No. 19943-K2) for 15 minutes at room temperature, followed by washing with 100 μL of staining buffer. Samples were resuspended in 50 μL of staining buffer for data acquisition on an Intellicyte iQue Screener PLUS flow cytometer and analysis using Intellicyt ForeCyt software. All 17 humanized antibodies and the A048 parental anti-B5 antibody showed similar binding to vaccinia virus-infected cancer cells, as shown in Table 7 below and Figures 2A-C. Binding of the humanized antibodies to HT29 cells infected with the Western Reserve VV strain, HT29 cells infected with the Copenhagen VV strain, and SKOV3 cells infected with the Western Reserve VV strain are shown in Figures 2A, 2B, and 2C, respectively.

[0309] [Table 7]

[0310] Example 4 - Affinity of humanized antibodies to VV B5 The affinity of the humanized antibodies was evaluated using label-free biolayer interferometry on an OctetRed96E® biolayer interferometer according to standard protocols. Humanized anti-B5 antibodies were captured using an anti-human capture (AHC) biosensor (Sartorius) with 600 seconds of loading. The loaded sensor was then immersed in B5 monomer analyte (40 nM, titrated 1:2) for 600 seconds for association and 1800 seconds for dissociation. The sensor was regenerated for 30 seconds. A control biosensor without antibody capture was used for double reference subtraction. Data was analyzed using Octet Data Analysis software v11.1. The parent A048 antibody had a KD of 9.5 nM and all 17 humanized antibodies were similar, ranging from 3 to 12 nM KD (Table 8).

[0311] [Table 8]

[0312] Example 5 - Determination of self-association of humanized antibodies The tendency of antibodies to self-associate was determined from affinity capture self-interaction nanoparticle spectroscopy (AC-SINS) using gold nanoparticles (Au-NPs) (Ted Pella, Catalog No.: 15705) (1, 2). Briefly, goat IgG (Jackson, Catalog No.: 005-000-003) and goat anti-human Fc IgG (Jackson, Catalog No.: 109-005-098, 1:4 molar ratio) were used to coat the Au-NPs. The mixture was incubated at room temperature with rotation for 1 h. The reaction was quenched by adding a final concentration of poly(ethylene glycol) methyl ether thiol. The conjugated Au-NPs were then concentrated by centrifugation at 13,000×g for 5 min. 1 / 20 parts of the original volume of PBS was used to resuspend the Au-NPs. 100 μL of 50 μg / mL of each antibody was mixed with 10 μL of concentrated Au-NPs in quadruplicate on a 96-well plate (Thermo Scientific™ Nunc™ 96-well Polypropylene MicroWell™ Plate (Cat. No.: 12565369)). The mixture was incubated for 2 hours at room temperature. Wavelength scans were measured on a Synergy Neo2 plate reader. Maximum absorbance difference (Δλ max ) is the λ of each reaction max AC-SIN was calculated by subtracting that of PBS buffer. Data were analyzed with Excel's Linest function using quadratic polynomial fitting. Data are shown in Table 9. Cetuximab (DIN: 02271249, Lily), trastuzumab (DIN: 02240692, Roche) and infliximab (DIN: 02419475, Hospira) were used as controls. Infliximab has previously been reported to have a high AC-SIN (1), and based on the literature, <11 is used as the cutoff score.

[0313] Example 6 - Determination of polyreactivity of humanized antibodies The polyreactivity of humanized antibodies against negatively charged biomolecules was determined by ELISA (3). Briefly, ELISA plates (Nunc MaxiSorp™ flat bottom, Thermo, Cat. No. 44-2404-21) were coated overnight with 5 μg / mL human insulin (SigmaAlrich, Cat. No. I9278) and 10 μg / mL double-stranded DNA (SigmaAlrich, Cat. No. D1626-250MG). The plates were blocked with ELISA buffer (PBS, 1 mM EDTA, 0.05% Tween-20, pH 7.4). 10 μg / mL of test antibody was added in quadruplicates onto the plate and incubated for 2 h. 0.01 μg / mL of goat anti-human Fc conjugated with HRP was then added and the plate was incubated for 1 h. The signal was developed with TMB (Sigma, Cat. No. T0440-1L) and A was performed. 450 Absorbance was measured on a Synergy Neo2 plate reader. Signals were corrected for each antibody tested with the signal of uncoated wells. Data are shown in Table 9. Cetuximab (DIN: 02271249, Lily), trastuzumab (DIN: 02240692, Roche) and infliximab (DIN: 02419475, Hospira) were used as controls. These control antibodies have previously been reported to have low polyreactivity, and the humanized anti-VV B5 antibody is comparable.

[0314] Example 7 - Denaturation Temperature of Humanized Antibodies Antibody denaturation temperature (T m) was determined by 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 each antibody was then determined using ThermoFisher Protein Thermal Shift software (v.1.3). m Cetuximab (DIN: 02271249, Lily), trastuzumab (DIN: 02240692, Roche) and infliximab (DIN: 02419475, Hospira) were used as controls. Based on the literature, a T of ≥ 65°C was determined. m A value of 1 is used as the acceptable cutoff. The DSF of the humanized antibodies, as well as the HPLC-SEC, AC-SINS and polyreactivity data, are provided in Table 9.

[0315] [Table 9]

[0316] Example 8 - Binding of humanized antibodies to vaccinia virus-infected cells over time This example describes the determination of binding of an exemplary humanized anti-B5 antibody of the disclosure to vaccinia virus infected cells over time.

[0317] HT29, SKOV3, and OVCAR3 cells were seeded at 20,000 cells / well in clear flat-bottom 96-well tissue culture plates (Corning, Cat. No. 353072) in complete culture medium. For HT29 and SKOV3 cells, the complete culture medium was McCoy's 5A (Gibco, Cat. No. 16600-082) + 10% fetal bovine serum (Corning, Cat. No. 35-015-CV). For OVCAR3 cells, the complete culture medium was RPMI-1640 (Gibco, Cat. No. A10491-01) + 0.01 mg / mL bovine insulin (Sigma, Cat. No. I0516-5ML) + 20% fetal bovine serum and allowed to adhere overnight at 37°C, 5% CO2. The next day, culture medium was aspirated and cells were infected with either VVddeGFP Western Reserve ("WR") or VV Copenhagen (YFP) virus ("Cop") at a multiplicity of infection (MOI) of 0.1 or 1, or mock infected, in 100 μL of serum-free McCoy's 5A at 37° C. and 5% CO2.

[0318] Two hours after infection, the infection medium was replaced with 100 μL of the complete culture medium described above and incubated at 37° C., 5% CO2. At 2, 24, 48, 72, and 168 hours after infection, the culture supernatant was transferred to a V-bottom 96-well plate (Corning, Cat. No. 3894). The cells were then washed with 50 μL of phosphate-buffered saline (Gibco, Cat. No. L34955) and the wash was pooled with the supernatant. The cells were then dissociated by incubating in 25 μL of cell dissociation buffer (Gibco, Cat. No. 13151014) per well at 37° C. for 30 minutes. Meanwhile, the cells in the culture supernatant were pelleted by centrifuging the V-bottom plate at 400×g for 5 minutes and discarding the supernatant. After dissociation, the cells were resuspended in 100 μL / well of PBS and pooled with the collected supernatant in the V-bottom plate. Cells were centrifuged at 400×g for 5 min and resuspended in 50 μL / well LIVE / DEAD Fixable Violet Dead Cell Stain diluted 1:1000 in PBS (Invitrogen, Cat. No. L34955) for 30 min at 4° C. After staining, cells were washed with 100 μL / well staining buffer (phosphate buffered saline + 2 mM EDTA + 0.5% bovine serum albumin) and pelleted by centrifugation at 400×g for 5 min. Cells were resuspended in 50 μL / well humanized anti-vaccinia virus B5 antibody conjugated to AlexaFluor-647 (A048-H1L4 or A048-H3L2) at a concentration of 10 nM and incubated for 1 h at 4° C., after which 100 μL staining buffer was added and centrifuged at 400×g for 5 min. Cells were then fixed in 50 μL / well of 4% paraformaldehyde in PBS (Thermo Scientific, Cat. No. 19943-K2) for 15 min at room temperature, followed by washing with 100 μL of staining buffer. Samples were resuspended in 50 μL of staining buffer for data acquisition on a Beckton Dickson LSRFortessa X-20 and analysis using FlowJo 10.8.1 software.

[0319] The results show that both A048-H1L4 and A048-H3L2 bind similarly to vaccinia virus-infected cells (Figure 3A - HT29 cells, Figure 3B - SKOV3 cells, Figure 3C - OVCAR3 cells). The data demonstrate that both antibodies bind to multiple vaccinia virus-infected cell lines from different cancer indications and to cells infected with different vaccinia virus strains. Furthermore, the data show that the binding of A048-H1L4 and A048-H3L2 to vaccinia virus-infected cells can occur over a long period of time.

[0320] Example 9 - Design of humanized anti-VV B5 chimeric antigen receptors (CARs), detection, and activation after lentiviral transduction of human T cells In this example, a chimeric antigen receptor (CAR) is described that includes a humanized anti-VV B5 antibody described elsewhere herein. In this particular example, as shown in schematic form in FIG. 4A, the humanized anti-VV B5 CAR construct includes a GM-CSFRα leader sequence, a humanized B5 scFv (A048_H3 and A048_L2, referred to as B5-CAR-050 in this example, or A048_H1 and A048_L4, referred to as B5-CAR-051 in this example), a human CD8α hinge and transmembrane domain, a human 4-1BB intracellular signaling domain, and a human CD3ζ intracellular signaling domain. Some CAR constructs may also include a reporter protein (e.g., eGFP or truncated EGFR (tEGFR)) separated by a ribosomal skip sequence, e.g., a T2A ribosomal skip sequence. The amino acid sequence of the CAR is shown in Table 5.

[0321] Gene fragments encoding B5-CAR-050 or B5-CAR-051 were synthesized by Twist Biosciences. Gene fragments were cloned into second generation transfer plasmids (Twist Biosciences). Lentiviruses encoding VV-CAR constructs are generated using the Lipofectamine™ 3000 transfection reagent protocol (ThermoFisher Scientific, Cat. No. L300015) and second generation packaging vectors (AddGene). To optimize CAR transduction, CD4+ and CD8+ T cell populations were isolated from healthy donor PBMC samples. 1×10 6 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 ng / mL). 24 hours after activation, T cells were transduced with B5-CAR-050 or B5-CAR-051 lentivirus at an MOI of 1.0 to 5.0. Cells were cultured at 1 x 10 per mL. 6 The cells were grown for another 12 days at a cell density of less than 100 cells / ml. Figure 4B shows CD3+ and CAR expression data detected by soluble B5 protein bound to streptavidin-PE. In turn, CAR expression is absent on non-transduced T cells and T cells transduced with tEGFR (SEQ ID NO: 54) vector only. Anti-VV B5 CAR is detected on T cells transduced with lentivirus encoding the parental rabbit B5-CAR-043 CAR construct. However, CAR expression is highest in the two humanized CAR constructs (B5-CAR-050, B5-CAR-051).

[0322] A stable cell line (in this example, a cancer cell line) expressing VV B5 antigen (e.g., B5 antigen from Wyeth VV strain) was generated to allow characterization of the CAR. B5-CAR-050 or B5-CAR-051 CAR T cells were plated in co-culture assays with either HT-29-B5 and HT-29-WT (ATCC HTB-38), SKOV3-B5 and SKOV3-WT (ATCC HTB-77) cells, or HEK293T-B5 and HEK293T-WT (ATCC CRL-3216). Cells were cultured overnight (16 h) at an effector:target (E:T) ratio of 1:1 (total of 2 × 10 5 The next day, cells were washed with PBS, blocked with Human Trustain FcX (Biolegend, Cat. No. 422302), and stained for CD8 PerCP (Biolegend, Cat. No. 344708), CD3 BV750 (Biolegend, Cat. No. 344846), CD137 APC (BD, Cat. No. 561702), and CD4 AF700 (Biolegend, Cat. No. 344622). Data are shown in Figure 4C. B5-CAR-050 and B5-CAR-051 CAR T cells showed increased expression of CD137 when co-cultured with cells expressing the appropriate target antigen, but minimal expression of CD137 when co-cultured with WT cells, indicating target-specific activation. Both B5-CAR-050 and B5-CAR-051 CAR T cells showed enhanced expression of CD137 compared to the parental B5-CAR-043 CAR T cells. This analysis indicates that primary PBMC-derived human T cells can be readily transduced to express VV-CAR constructs and expanded following transduction. Furthermore, these data demonstrate that effector T cells transduced to express B5-CAR-050 or B5-CAR-051 CAR undergo activation upon encountering target cells bearing the VV B5 antigen.

[0323] Example 10 - Human T cells expressing B5-CAR-050 or B5-CAR-051 exhibit cytotoxicity against target cells expressing VV B5 The mammalian expression transfer plasmid pCCL Luc Puromycin encoding firefly luciferase was used to generate lentivirus as described in Example 9. HT-29, SKOV3, and HEK293T-WT 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. The expanded puromycin-resistant cell cultures were used as the target population for the VV-CAR killing assay. The target HT29, SKOV3, and HEK293T lines expressing luciferase were transduced at 2×10 in 100 μL per well of a 96-well plate (Corning, Cat. No. 3917). 4Cells were seeded at 100 μL / well and incubated overnight to allow for adhesion. Populations of expanded primary human B5-CAR-050, B5-CAR-051, parental B5-CAR-043, and vector control tEGFR T cells were seeded in triplicate in 96-well plates (100 μL / well) and diluted in a 2-fold dilution series to establish E:T ratios of 20:1 to 0.625:1 across CAR expression. The T cell suspension was then transferred to adherent tumor cells in a total volume of 200 μL. Additionally, triplicate wells of target cells alone and triplicate wells of media alone were plated to determine maximum and minimum relative luminescence units (RLU), respectively. Cells were cultured at 37°C in 5% CO2 for 24–36 h. After incubation, 22 μL of 10× stock of Xenolight™ D-luciferin (Perkin Elmer, Cat. No. 122799) was added to each well and incubated for 10 minutes at room temperature in the dark. The plate was then scanned on a luminescence plate reader (ThermoFisher Varioskan Lux plate reader). Triplicate wells were averaged and percent specific cytotoxicity was determined by the following formula: percent specific cytotoxicity=100×(maximum luminescence RLU−test luminescence RLU) / (maximum luminescence RLU−minimum luminescence RLU). As shown in FIG. 5A, percent specific cytotoxicity and relative luminescence for 1.25:1 E:T co-culture wells of B5-CAR-050, B5-CAR-051, parental B5-CAR-043, and vector control tEGFR showed high percent cytotoxicity specific to the B5 target cell line and cytotoxicity above background with the tEGFR vector control condition. Furthermore, B5-CAR-050 and B5-CAR-051 appeared to exhibit improved overall cytotoxicity compared to the parental B5-CAR-043. B5-expressing target cells co-cultured with B5-CAR-050 or B5-CAR-051 T cells at 10:1 E:T were also imaged for morphological signs of cell death at 24 hours and compared to B5 CARs cultured with WT tumor cells. B5-CAR-050 and B5-CAR-051 showed clear cell clustering and signs of cytotoxicity when cultured with tumor cell lines expressing the B5 target (Figure 5B).Moreover, the degree of CAR clustering appeared to be greater than that of the parental B5-CAR-043. These results indicate that B5-CAR-050 or B5-CAR-051 T cells efficiently and specifically kill tumor cells expressing B5. Furthermore, targeting OV antigens expressed on the surface of tumor cells can render the target cells susceptible to destruction by humanized VV-CAR.

[0324] Example 11 - Human T cells expressing B5-CAR-050 or B5-CAR-051 exhibit cytotoxicity against vaccinia virus-infected tumor cells Luciferase-expressing target HT29, SKOV3, and HEK293T WT lines were cultured at 2 × 10 per well of a 96-well plate (Corning, Cat. No. 3917). 4Tumor cells were seeded at 100 μL / well and incubated overnight to allow attachment. The next day, tumor cells were infected with vaccinia virus (non-limiting examples of which include Western Reserve strain, Copenhagen strain, etc.) at a multiplicity of infection ranging from 0.01 to 1.0 in 50 μL of serum-free DMEM (Gibco, Cat. No. 11995-040) per well and incubated for 24 hours at 37°C in 5% CO2. Corresponding mock uninfected wells are set as negative controls. Populations of expanded primary human B5-CAR-050, B5-CAR-051, parental B5-CAR-043, and vector control tEGFR T cells were plated in triplicate (50 μL / well) on vaccinia-infected plates to achieve an E:T ratio of 10:1 and a total volume of 200 μL per well. Additionally, triplicate wells containing only target cells (+ / -infected) and triplicate wells containing only T cells (+ / -infected) were plated and maximum and minimum relative luminescence units (RLU) were determined. Cells were cultured for an additional 24-48 hours at 37°C in 5% CO2. Each well received 22 μL of 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 on a luminescence plate reader (ThermoFisher Varioskan Lux plate reader). RLUs from triplicate wells were averaged and percent specific cytotoxicity was determined as described in Example 10. As shown in Figure 6A, percent specific cytotoxicity and relative luminescence for 10:1 E:T co-culture wells of B5-CAR-050, B5-CAR-051, parental B5-CAR-043, and vector control tEGFR demonstrated increased percent specific cytotoxicity to vaccinia virus infected target cell lines, cytotoxicity above background present in the tEGFR vector control condition. Vaccinia virus infected target cells co-cultured 10:1 E:T with B5-CAR-050 or B5-CAR-051 T cells were also imaged for morphological signs of cell death at 24 hours and compared to B5 CARs cultured with uninfected tumor cells (Figures 6B-6D).B5-CAR-050 and B5-CAR-051 showed clear signs of cell clustering and cytotoxicity when cultured with vaccinia virus-infected tumor cell lines. These findings demonstrate that B5-CAR-050 and B5-CAR-051 can specifically detect and kill vaccinia virus-infected tumor cells.

[0325] Example 12 - Antitumor Efficacy of Humanized B5-CAR on Vaccinia Virus-Infected Xenograft Tumor Models Mice bearing HT-29 tumors are treated with a combination of vaccinia virus and humanized B5-CAR. NSG mice are treated with 1×10 6 The cells are subcutaneously implanted and observed for tumor growth. When the tumor reaches a treatable size (approximately 30-40 mm 2 ), mice were given 1 × 10 7 PFU of vaccinia virus are injected intratumorally for a total of 1-3 times. Approximately 1-3 days after the last vaccinia virus injection, mice are injected with 5 × 10 6 B5-CAR positive T cells are given by tail vein injection. Tumor measurements are recorded every 2-3 days and animals are observed for survival. Control groups receive a combination of either intratumoral PBS injections and / or empty vector mock-transduced T cells delivered by tail vein injection. Figure 7A: Predicted tumor area in different treatment conditions. Figure 7B: Predicted overall survival in different treatment conditions.

[0326] References 1.Liu Y,Caffry I,Wu J,Geng SB,Jain T,Sun T,Reid F,Cao Y,Estep P,Yu Y,Vasquez M,Tessier PM,Xu Y.High-throughput screening for developability during early-stage antibody discovery using self-interaction nanoparticle spectroscopy.MAbs.2014 Mar-Apr;6(2):483-92.doi:10.4161 / mabs.27431.Epub 2013 Dec 6.PMID:24492294;PMCID:PMC3984336. 2.Shan L,Mody N,Sormani P,Rosenthal KL,Damschroder MM,Esfandiary R.Developability Assessment of Engineered Monoclonal Antibody Variants with a Complex Self-Association Behavior Using Complementary Analytical and in Silico Tools.Mol Pharm.2018 Dec 3;15(12):5697-5710.doi:10.1021 / acs.molpharmaceut.8b00867.Epub 2018 Nov 15.PMID:30395473. 3.Avery LB,Wade J,Wang M,Tam A,King A,Piche-Nicholas N,Kavosi MS,Penn S,Cirelli D,Kurz JC,Zhang M,Cunningham O,Jones R,Fennell BJ,McDonnell B,Sakorafas P,Apgar J,Finlay WJ,Lin L,Bloom L,O'Hara DM.Establishing in vitro in vivo correlations to screen monoclonal antibodies for physicochemical properties related to favorable human pharmacokinetics.MAbs.2018 Feb / Mar;10(2):244-255.doi:10.1080 / 19420862.2017.1417718.Epub 2018 Jan 29.PMID:29271699;PMCID:PMC5825195.

[0327] Thus, the above merely illustrates 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 present invention and are within the spirit and scope thereof, 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 present invention and the concepts that the inventors have contributed to advancing the art, and should not be construed as being limited to such specifically recited examples and conditions. Furthermore, all statements herein that describe the principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Moreover, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Thus, the scope of the present invention is not limited to the exemplary embodiments shown and described herein.

Claims

1. An antibody that specifically binds to vaccinia virus B5 antigen (VV B5), the antibody comprising: A variable heavy chain (VH) having 70% or more sequence identity to the amino acid sequence shown in SEQ ID NO:

1. H ) a polypeptide comprising one or more mutations selected from the group consisting of Q1E, E2V, Q3T, E5L / K, G9P, G10V, K13Q, E15G / T, G16E, S17T, T19R, T21S, T23A, A41P, R44K, G45A, T74D, S75N / T, S76_T77insK, T77N / S, T78Q, V79L, T80Y / V, Q82T, T84N, R85S / N, L86M, T87R / D, A88P, A89E / V, T93V, F95Y, P112Q, and any combination thereof, wherein the numbering is according to SEQ ID NO:1; V containing the amino acid sequence SSYYMC (SEQ ID NO: 13) H CDR1, V containing the amino acid sequence CIYTSSGSAYYA(N / D)(W / S)(A / V)KG (SEQ ID NO: 14) H CDR2, V containing the amino acid sequence NAVGSSYYLYL (SEQ ID NO: 17) H CDR3, Including, V H A polypeptide, and A variable light chain (V) having 70% or more sequence identity to the amino acid sequence shown in SEQ ID NO:

7. L ) a polypeptide comprising one or more mutations selected from the group consisting of A1D / E, Q2I, V3Q, L4M, T7S, S9A, P10T / S, V11L, A13L, A14S, V15P, G17D / E, T18R, V19A, I21L, S22T, Q44K, P45A / V, N47K / R, V60I, S62A, K65S, Q72E / D, D79S, E81Q, C82P, D83E, A85F / V, T87V, G103Q, E106K, V107L, V108E, V109I, and any combination thereof, wherein the numbering is according to SEQ ID NO:7; V containing the amino acid sequence QASQSVAGNNYLS (SEQ ID NO: 18) L CDR1, V containing the amino acid sequence SVSTLAS (SEQ ID NO: 19) L CDR2, V containing the amino acid sequence QGYYNDGIWA (SEQ ID NO: 20) L CDR3, Including, V L An antibody, including a polypeptide.

2. The V H 2. The antibody of claim 1, wherein the polypeptide comprises one of the following mutations: E2V, E5L / K, E15G / T, R44K, R85S / N, T87R / D, A89E / V, and P112Q, any combination thereof, or each.

3. (i) the E5L / K mutation is E5L; (ii) the E15G / T mutation is E15G; (iii) the R85S / N mutation is R85S; (iv) the T87R / D mutation is T87R, (v) the A89E / V mutation is A89E; and / or (vi) the V H 3. The antibody of claim 2, wherein the polypeptide comprises one of, any combination of, or each of the following mutations: Q1E, K13Q, T19R, T21S, T23A, T84N, T93V, and F95Y.

4. The V H The polypeptide comprises the amino acid sequence CIYTSSGSAYYANWAKG (SEQ ID NO: 15). H CDR2 or V comprising the amino acid sequence CIYTSSGSAYYADSVKG (SEQ ID NO: 16) H The antibody of claim 2 or 3, comprising CDR2.

5. The V H 4. The antibody of claim 2 or 3, wherein the polypeptide comprises one of the following mutations, any combination thereof, or each of: T74D, S75N / T, S76_T77insK, T77N / S, V79L, and T80Y / V.

6. (i) the T77N / S mutation is T77N; (ii) the T80Y / V mutation is T80Y; and / or (iii) the V H The polypeptide comprises the amino acid sequence CIYTSSGSAYYANWAKG (SEQ ID NO: 15). H CDR2 or V comprising the amino acid sequence CIYTSSGSAYYADSVKG (SEQ ID NO: 16) H The antibody of claim 5 comprising CDR2.

7. The V H 3. The antibody of claim 2, wherein the polypeptide comprises one of the following mutations, any combination thereof, or each of: Q3T, E5L / K, G9P, G10V, E15G / T, G16E, S17T, A41P, G45A, T74D, S75N / T, S76_T77insK, T77N / S, T78Q, T80Y / V, Q82T, R85S / N, L86M, T87R / D, A88P, and A89E / V.

8. (i) the E5L / K mutation is E5K; (ii) the E15G / T mutation is E15T; (iii) the S75N / T mutation is S75T; (iv) the T77N / S mutation is T77S, (v) the T80Y / V mutation is T80V; (vi) the R85S / N mutation is R85N; (vii) the T87R / D mutation is T87D, and / or (viii) The antibody of claim 7, wherein the A89E / V mutation is A89V.

9. The V H the polypeptide comprises 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to an amino acid sequence set forth in one of SEQ ID NOs:2-6; The V H the polypeptide comprises 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:2; The V H the polypeptide comprises 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:3; The V H the polypeptide comprises 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:5; The V H the polypeptide comprises 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:4; or The V H The antibody of claim 1, wherein the polypeptide comprises 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:

6.

10. The V L 10. The antibody of any one of claims 1 to 9, wherein the polypeptide comprises one of, any combination of, or each of the following mutations: T7S, P10T / S, V11L, A14S, G17D / E, T18R, P45A / V, N47K / R, K65S, Q72E / D, D79S, C82P, A85F / V, G103Q, E106K, V108E, and V109I.

11. (i) The V L the polypeptide comprises one of the following mutations, or any combination thereof: G17D, S22T, Q44K, N47K, and E81Q, (ii) the P10T / S mutation is P10T; (iii) the P45A / V mutation is P45A; (iv) the Q72E / D mutation is Q72E; and / or (v) The antibody of claim 10, wherein the A85F / V mutation is A85F.

12. the P10T / S mutation is P10T, the P45A / V mutation is P45A, the Q72E / D mutation is Q72E, the A85F / V mutation is A85F, and the V L The antibody of claim 10, wherein the polypeptide comprises one of, any combination of, or each of the mutations A1D, Q2I, V3Q, and L4M.

13. (i) The V L the polypeptide comprises the mutation D83E, (ii) the P10T / S mutation is P10S; (iii) the P45A / V mutation is P45V; (iv) the Q72E / D mutation is Q72D, (v) the A85F / V mutation is A85V; and / or (vi) the V L The antibody of claim 10, wherein the polypeptide comprises one of, any combination of, or each of the mutations A1D, Q2I, V3Q, and L4M.

14. The V L 11. The antibody of claim 10, wherein the polypeptide comprises one of, any combination of, or each of the following mutations: A1E, S9A, P10T, A13L, V15P, G17E, V19A, I21L, P45A, N47R, V60I, S62A, Q72D, D83E, A85F, T87V, and V107L.

15. The V L the polypeptide comprises 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to an amino acid sequence set forth in one of SEQ ID NOs:8-12; The V L the polypeptide comprises 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:9; The V L the polypeptide comprises 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:8; The V L the polypeptide comprises 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:11; The V L the polypeptide comprises 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:10; or The V L The antibody of claim 1, wherein the polypeptide comprises 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:

12.

16. An antibody that specifically binds to vaccinia virus B5 antigen (VV B5), the antibody comprising: Variable heavy chain (V H ) a polypeptide comprising V containing the amino acid sequence SSYYMC (SEQ ID NO: 13) H CDR1, V containing the amino acid sequence CIYTSSGSAYYADSVKG (SEQ ID NO: 16) H CDR2, V containing the amino acid sequence NAVGSSYYLYL (SEQ ID NO: 17) H CDR3, Including, V H A polypeptide, and Variable light chain (V L ) a polypeptide comprising V containing the amino acid sequence QASQSVAGNNYLS (SEQ ID NO: 18) L CDR1, V containing the amino acid sequence SVSTLAS (SEQ ID NO: 19) L CDR2, V containing the amino acid sequence QGYYNDGIWA (SEQ ID NO: 20) L CDR3, Including, V L Containing a polypeptide, An antibody, optionally wherein said antibody is a humanized antibody.

17. The antibody of any one of claims 1 to 16, wherein the antibody is an IgG or a single chain antibody.

18. The antibody of any one of claims 1 to 17, wherein the antibody is a bispecific antibody.

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 sequence of amino acids.

20. The antibody is a single chain antibody and the fusion protein is The single chain antibody; A transmembrane domain; and an intracellular signaling domain.

21. A conjugate comprising: An antibody according to any one of claims 1 to 18 or a fusion protein comprising said antibody, and a drug conjugated to the antibody or fusion protein.

22. 22. The conjugate of claim 21, 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.

23. The variable heavy chain (V) of the antibody according to any one of claims 1 to 18 H ) polypeptide, variable light chain (V L ) polypeptide, or both, or a nucleic acid encoding a fusion protein comprising said antibody.

24. 24. An expression vector comprising the nucleic acid of claim 23.

25. 24. A cell comprising the nucleic acid of claim 23 or an expression vector comprising said nucleic acid.

26. The cell of claim 25, wherein the nucleic acid encodes a CAR comprising the antibody, and the cell expresses the CAR on its surface.

27. The cell of claim 26 , wherein the cell is an immune cell.

28. 28. The cell of claim 27, wherein the cell is an immune effector cell, optionally wherein the cell is a T cell, a natural killer (NK) cell, or a macrophage.

29. A pharmaceutical composition comprising the cells of claim 28.

30. 20. A method of producing an antibody according to any one of claims 1 to 18, comprising culturing a cell comprising an expression vector encoding said antibody under conditions suitable for said cell to express said antibody, whereby said antibody is produced.

31. 1. A pharmaceutical composition comprising: An antibody according to any one of claims 1 to 18, a fusion protein comprising said antibody, or a conjugate comprising said antibody or fusion protein; and a pharma- ceutically acceptable carrier.

32. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 18, a fusion protein comprising said antibody, a conjugate comprising said antibody or the fusion protein, or a cell according to claim 28, the pharmaceutical composition is for use in a method comprising administering the pharmaceutical composition to an individual having cancer; The individual comprises cancer cells infected with vaccinia virus (VV), and the antibody, fusion protein, conjugate, or cell is targeted to the infected cancer cells by a VV B5 antigen expressed on the surface of the infected cancer cells.

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