Anti-oncolytic virus antigen antibodies and methods of using the same

Abstract

To provide a method for targeting to cancer cells in an individual an antibody, fusion protein, or conjugate that specifically binds to an oncolytic virus (OV) antigen.SOLUTION: Provided is an antibody that specifically binds to Vaccinia Virus A56 antigen (VV A56) and, with respect to binding to VV A56, competes with an antibody comprising a variable heavy chain (VH) polypeptide including VHCDR1, VHCDR2, and VHCDR3 comprising specific amino acid sequences, and a variable light chain (VL) polypeptide including VLCDR1, VLCDR2, and VLCDR3 comprising specific amino acid sequences.SELECTED DRAWING: Figure 13B

Description

[Background technology]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 62 / 900,300, filed September 13, 2019. No. 3, filed on Oct. 1, 2003, which is incorporated herein by reference in its entirety.

[0002] Immunotherapy has become an effective treatment option for several malignancies. Oncolytic viruses (OV) selectively replicate in tumor tissue and lyse it. It is designed to simultaneously spare normal non-tumor host cells and restore anti-tumor immunity. This constitutes a next-generation immunotherapy approach for tumor treatment. The unique ability of OVs to target malignancies independent of their expression pattern makes them uniquely suited to other immune responses. Furthermore, OVs are an attractive alternative to immunotherapy approaches. ) and reprogram the immunosuppressive tumor microenvironment (TME), resulting in systemic anti-tumor effects. It can boost immunity.

[0003] Through genetic engineering, living, replicating viruses can be engineered to transduce cells through cell entry and transcriptional targeting. Not only is it highly tumor-selective, but it also allows for non-invasive monitoring of the pharmacokinetics of viral therapy. and for enhancing cytotoxic activity or immunogenic cell death, or immune modulators It is now possible to equip them with reporter genes. Currently, three OVs are being used for cancer treatment. These OVs are commercially available in Latvia, Georgia, and Armenia. Rigvir, approved in China; Oncorine H101, approved in the U.S.; Examples include talimogene laherparepvec (T-VEC), which has been approved for use in the treatment of rheumatoid arthritis. virus) is an unmodified enterocytopathic human orphan type 7 (ECHO-7) picornavirus Oncorine is a Chinese became the first oncolytic virus approved for clinical use and received regulatory approval This was the world's first recombinant oncolytic virus. , an attenuated serotype 5 adenoviral vector with four deletions in viral E3. corine remains the only adenovirus approved for cancer treatment. , and only when administered in combination with chemotherapy. mlygic™) was approved by the U.S. Food and Drug Administration (FDA) in 2015 as a treatment for non-resectable breast cancer. It was approved in the EU for the treatment of locally advanced or metastatic melanoma. The newest oncolytic virus approved for the treatment of cutaneous melanoma and approved by national regulatory authorities T-VEC is the first oncolytic virus approved in the United States. Recombinant human herpes simplex virus deleted for both copies of genotype 34.5 and viral ICP47. Virus type 1 (HSV1), which accelerates US11 expression, and cytomegalovirus (C Human granulocyte-macrophage colony-stimulating factor (GM-CSF) under the control of the MV promoter T-VEC encodes two copies of the T-VEC gene. It is currently administered intratumorally to high-grade melanoma lesions of the skin. It has been approved for the treatment of rheumatoid arthritis and has shown efficacy as a single agent for this indication.

[0004] OVs under clinical development include measles virus, Newcastle disease virus (NDV), and Budovirus, adenovirus, vaccinia virus (VV), herpes virus, cocci These include sacchiviruses, reoviruses, and retroviruses.

[0005] Measles virus is a negative-strand RNA paramyxovirus with high fusion and syncytial formation. Cell-cell fusion enhances bystander killing of tumor cells. These factors may induce immunogenic danger signals that may induce host-mediated cellular antitumor activity. sodium iodide symporter (MV-NIS) or soluble carcinoembryonic antigen (MV Recombinant Edmonston strain measles virus encoding β-CEA (C-CEA) has been shown to be effective in preventing multiple myeloma, ovarian cancer, and ovarian cancer. Relapsed or recurrent cancers, including follicular carcinoma, glioma, breast cancer, and mesothelioma It is currently undergoing Phase I / II clinical trials in patients with recurrent cancer.

[0006] NDV is an avian paramyxovirus and an oncolytic or oncolytic cancer vaccine NDV strains MTH-68 / H, HUJ, and PV701 have been tested as clinical Infection of avian cells but not mammalian cells was impaired and GM-CSF (Med Recombinant NDV-73T strain-based mesogen encoding immune (MEDI5395) NDV is in preclinical trials.

[0007] Rhabdoviruses are negative-sense RNA viruses. It is a lytic replication cycle of approximately 12 hours in permissive cells. The oncolytic rhabdovirus vesicular stomatitis virus (VSV) utilizes low-density lipoprotein (LDR) for cell entry. VSV uses the low-density lipoprotein (LDL) receptor to infect almost all cell types and produce a wide variety of It allows for the generation of a lytic infection in permissive cells.

[0008] Adenoviruses are icosahedral, non-enveloped, double-stranded DNA viruses, each capsid A long fibrous knob protrudes from the apex of the nucleus. Telomelysin in solid tumors Clinical data on CG0070 in bladder cancer and DNX-2401 in malignant brain tumors The data has been made public.

[0009] Vaccinia virus (VV) is a large endogenous virus with a linear genome approximately 190 kb in length. Enveloped double-stranded DNA viruses. Attenuation or tumor-specific targeting of these viruses. Modification has been achieved using a variety of deletion and insertion mutations, resulting in the loss of thymidine kinase function. This is a common feature of clinical oncolytic vaccinia viruses. TG6002 lacks thymidine kinase and viral ribonuclease. GL-ONC1 is a double deletion of its thymidine kinase (J2R), It has insertion mutations in the hemagglutinin HA (A56R) and F14.5L genes. Loss of ribonucleotides limits the ability of the virus to replicate in non-dividing cells and reduces viral ribonucleotide levels. Deletion of ductase further limits this ability. The two clinical vaccinia vectors used contain transfectants designed to improve tumor cell killing. JX-594 contains GM-CSF, just like T-VEC, and TG6 002 converts 5-fluorocytosine (5-FC) to 5-FU in infected cells. The nucleoside analogue conversion enzyme FCU1 is incorporated.

[0010] HSV1 is a large double-stranded DNA virus approximately 152 kb in length. The gamma 34.5 deletion virus currently approved by the FDA is T-VEC. , HSV1716 (Seprehvir), G207, and RP1. Attempts to enhance the anticancer effects of HSV in disease include the development of adaptive anticancer agents in treated patients. To simultaneously enhance anti-cancer and anti-viral immunity with the goal of developing tumor responses This includes including a therapeutic transgene for use in

[0011] Coxsackieviruses are single-stranded positive R-terminal fragments of approximately 7.4 kb enclosed in an icosahedral capsid. NA picornavirus. Oncolytic CVA21 (Viralytics, CAVA TAK) is derived from the Kuykendall strain and uses ICAM as the primary receptor for cell invasion. -1. Intratumoral CVA21 in combination with pembrolizumab or ipilimumab Phase I trials of viral injections are underway to enhance the overall effectiveness of these drugs. be.

[0012] Reoviruses are double-stranded RNA viruses that are not enveloped and have an outer and inner tag. It has an icosahedral capsid composed of a protein shell. It is being investigated in several Phase I clinical trials (Oncolytics) as an intratumoral or intravenous treatment. Biotech, Reolysin™).

[0013] Retroviral replicating vectors (Tocagen, Toca-511, vocimage) ne amiretrorepvec) converts the prodrug 5-FC into the anticancer drug 5-FU. It encodes a yeast cytosine deaminase (CD) that converts 5- Increases the local concentration of FU and reduces the overall systemic toxicity of the drug. Recurrent high-grade glioma In a Phase 1 trial of Toca-511 in patients, the overall survival was 13.6 months, The results were statistically significant compared with the control group. Summary of the Invention

[0014] Despite recent advances in OV-based therapy, there is still a lack of evidence that OVs are effective in treating, palliating, and / or preventing cancer. New and improved methods for the prevention of cancer and for improving survival of subjects with cancer Therefore, there is still a need for OV-based methods.

[0015] Binds specifically to vaccinia virus (VV or VACV) A56 or B5 antigens Antibodies are provided. Fusion proteins and conjugates comprising the antibodies are also provided. Pharmaceutical compositions and kits containing the fusion proteins and conjugates are also provided. Disclosed embodiments include, for example, antibodies, fusion proteins, and conjugates for therapeutic purposes. In certain embodiments, methods of administering to an individual with cancer an antibody of the present disclosure, the individual is infected with VV and administers the fusion protein or conjugate to the individual. and wherein the antibody, fusion protein or conjugate is capable of inhibiting the growth of the infected cancer cells. The virus is targeted to infected cancer cells by the VV antigens expressed on the surface of the cells. An embodiment of the present disclosure provides a method for detecting an oncolytic virus (OV) antigen. The antibody, fusion protein, or conjugate is targeted ( The method further includes a method for targeting. [Brief explanation of the drawings]

[0016] [Figure 1]Flow cytometry data of antibody binding to A549 and CaOV3 cells infected with VV Western Reserve VVdd(eGFP) are shown. [Figure 2] Flow cytometry data of antibody binding to A56 or B5 expressing HEK cells (Wyeth VV sequence) are shown. [Figure 3] Data for antibody binding to HEK-B5 cells are shown. The highest concentrations tested in flow cytometry assays (top panel) or ELISA (bottom panel) for binding to HEK-B5 are plotted, at 5 μg / ml for rabbit and mouse isotype controls (Rb IgG and mIgG isotypes, respectively), as well as the anti-B5 Immunetech antibody, IT anti-B5 IgG (identified as anti-B5 mIgG in Figure 3), and 1:1000 for the rabbit polyclonal antibody (c-VV). [Figure 4] Binding of the A048 antibody, Immunetech anti-B5 antibody, IT anti-B5 IgG (identified as IT anti-B5 mIgG in Fig. 4 ), and a human isotype control to VV Western Reserve VVdd(eGFP)-infected A549 cells (upper panel) and HT29 cells (lower panel) as measured by flow cytometry is shown. [Figure 5] Figure 1 shows antibody binding to VV-infected cells. Antibodies were tested for binding to VVdd(eGFP)-infected A549 cells by flow cytometry. All formats had similar EC50s, and no binding to uninfected cells was observed. [Figure 6] Immunofluorescence shows the binding of antibody A049 to virus-infected tumor tissue, specifically the binding of A049 anti-A56 hIgG1 to virus-infected colorectal tumor tissue. The top two panels are hematoxylin and eosin (H&E) stained. This is in comparison to a polyclonal anti-vaccinia virus antibody, which shows some nonspecific staining in uninfected tissue. [Figure 7]Figure 1 provides epitope binning data showing binding of biotinylated antibodies to VVdd(eGFP)-infected cells preincubated with non-biotinylated antibodies. Reduction of biotinylated antibody binding indicates a similar or overlapping epitope with the test antibody. Antibodies A047 and A049 appear to bind to different epitopes on A56. Antibodies A048 and A051 appear to bind to different epitopes on B5. [Figure 8] Data on antibody binding to virus particles are shown. Viral particles (VVdd(eGFP)) were coated onto plates and incubated with antibodies or the positive control NKp30. All antibodies bound to the virus except for antibody A054. [Figure 9] Data are provided showing neutralization of virus infection. Antibodies were added to A549 cells simultaneously with VVdd(eGFP) infection. Similar levels of infection were observed as compared to infection without antibody ("No antibody"). [Figure 10] Figure 1 shows antibody binding to mouse cell lines infected with VVCopenhagen (YFP). The percent YFP expression in each infected cell line indicates the proportion of cells infected with VVCopenhagen (YFP). Anti-A56-PE and anti-B5-PE antibodies bind to B16F10, CT26LacZ, and MC38 cells infected with VVCopenhagen (YFP). [Figure 11] Immunohistochemistry data are shown for the specific binding of antibodies to A56 or B5 antigens expressed on the surface of HEK cells or to U2OS cells infected with VVCopenhagen virus. Rabbit / mouse chimeric (antibodies A059 and A058) and full rabbit IgG format (antibodies A056 and A073) antibodies specific for A56 or B5 were able to specifically detect the target antigens on stably expressed HEK-A56 and HEK-B5 cells, respectively. Both anti-A56 and anti-B5 antibodies were able to detect protein expression on U2OS cells infected with VVCopenhagen virus. [Figure 12]1 shows detection of A56 and B5 from VV-treated tumors by immunohistochemistry. Data show detection of A56 and B5 proteins on the surface of tumors treated with vaccinia virus. [Figure 13] Panel A: Representative schematic of an example design of an anti-VV chimeric antigen receptor (CAR) construct. Panel B: Schematic of a therapeutic approach according to an embodiment of the present disclosure. As shown, OV infects cancer cells, which then present OV antigens (in this example, A56 or B5 OV antigens, which in some embodiments may be the OV's native A56 or B5 antigen) on their surface. Treating the infected cancer cells with CAR T cells containing a CAR specific for the presented OV antigen destroys the infected cancer cells. Adjacent cancer cells infected with OV released by the destroyed cancer cells can then be destroyed by CAR T cells containing a CAR specific for the presented OV antigen. [Figure 14] Flow cytometry data showing VV-CAR detection after lentiviral transduction are provided. The data show VV-CAR expression on transduced Jurkat cells. CAR downregulation, indicating specific target binding, is evident for all VV-CAR constructs tested. [Figure 15] Figure 1 shows the expression of CAR and eGFP on activated A56-CAR-06 positive Jurkat cells. Data demonstrates co-expression of A56-CAR-06 and eGFP on a transduced Jurkat cell line. After incubation with the target HEK A56 line, gating on the eGFP-positive population allows identification of the activated CAR population (CD69-positive) within the scFv-negative fraction. [Figure 16] We provide data demonstrating the specific activation of Jurkat cells expressing VV-CARs when co-cultured with target HEK cell lines. All VV-CARs tested demonstrate specific on-target activation as indicated by upregulation of CD69. Minimal cross-reactivity / intrinsic activation occurred when VV-CAR lines were co-cultured with negative / irrelevant target cell lines. [Figure 17]Transduction and enrichment of healthy donor primary T cells for A56-CAR-06 expression are shown. Data demonstrate stable expression of A56-CAR-06 on human primary T cells following standard clinical manufacturing protocols. The A56-CAR-06 enriched population was established by repeated cell sorting for CAR expression followed by clinical expansion. [Figure 18] Specific activation of human T cells expressing A56-CAR-06 when co-cultured with the target-expressing HEK-A56 line is shown. Human primary T cells expressing A56-CAR-06 showed significant upregulation of CD69 and CD137 after co-culture with the target HEK-A56 line. Minimal cross-reactivity was observed with negative / irrelevant target cell lines. [Figure 19] We provide data demonstrating that human primary T cells expressing A56-CAR-06 exhibit morphological signs of direct tumor killing 48 hours after co-culture with target HEK-A56 cells. [Figure 20] We provide data demonstrating that human T cells expressing B5-CAR-011 exhibit high percent specific cytotoxicity. Human primary T cells expressing B5-CAR-011 exhibited specific cytolysis at 24 hours, as determined by percent reduction in relative luminescence units (RLU). [Figure 21] Representative maximum intensity projection positron emission tomography (PET) images of 89Zr-DFO-A049 (anti-A56 antibody, DFO: deferoxamine) are shown for the same mouse at 1, 3, and 5 days post-injection. The tumor types: HEK-A56 (A56-expressing, left shoulder) and parental HEK (non-expressing, right shoulder), in addition to the spleen and liver, are indicated by arrows. Results are presented as the percentage of injected dose per gram of tissue (%ID / g). Clear preclinical A56-positive tumor PET imaging demonstrates efficient and specific accumulation of 89Zr-DFO-A049 in the positive tumor and the expected pharmacokinetics of the radioimmunoconjugate. DETAILED DESCRIPTION OF THE INVENTION

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

[0018] When a range of values is provided, the value between the upper and lower limits of that range (unless the context clearly indicates otherwise) Each intervening value (to the tenth of the lowest unit unless otherwise specified) and its stated range Any other stated or intervening values within the range are encompassed within the subject antibodies, compositions and methods. It is understood that the upper and lower limits of these smaller ranges may independently be Smaller ranges may be included, subject to any specifically excluded limitations within the stated ranges. If the range contains one or both of the limits, then either the upper or lower limit is included. The antibodies, compositions and methods also include those excluding both.

[0019] Certain ranges are presented herein with the numerical values preceded by the term "about." The term "is" is used to refer to the exact number it precedes, and to the number that the term precedes. Used herein to provide literal support for approximate numbers. In determining whether a number is close or approximate to an enumerated number, A number not listed is a number that is specifically listed in the context in which it is presented. It may be a substantially equivalent number.

[0020] Unless otherwise defined, all technical and scientific terms used herein are defined by the The same meaning as commonly understood by those skilled in the art to which the antibody, composition, and method pertains. Any antibodies, compositions and methods similar or equivalent to those described herein. Methods can also be used in practicing or testing such antibodies, compositions and methods. However, representative exemplary antibodies, compositions and methods are described herein.

[0021] All publications and patents cited herein are to be construed as being incorporated by reference in their entirety. All such references are incorporated herein by reference as if specifically and individually indicated to be incorporated by reference. The authors have disclosed materials and / or methods related to the work incorporated in this publication and to which the publication is cited. All publications cited herein are incorporated by reference to show and describe the invention. Any publication dates provided are for disclosures prior to the filing date and may need to be independently confirmed. The actual publication dates of the present invention may differ from those of the prior art, and the disclosures herein are hereby incorporated by reference. It should not be interpreted as an admission that there are no rights that precede things.

[0022] As used in this specification and the appended claims, the singular forms "a," "an," and "an" are used interchangeably. and "the" are intended to include plural referents unless the context clearly dictates otherwise. It is understood that the claims may be drafted to exclude any optional element. Further note: Thus, this statement does not necessarily imply a "solely" context for the enumeration of claim elements. "," "only," etc., or the use of "negative" limitations. It is intended to serve as a basis.

[0023] Identification of antibodies, compositions and methods described in the context of separate embodiments for clarity It will be appreciated that the above features may also be provided in combination in a single embodiment. In particular, for the sake of brevity, the antibodies, compositions and methods are described in the context of a single embodiment. The various features may be provided separately or in any suitable subcombination. All combinations of embodiments are specifically embraced by this disclosure, and any such combinations To the extent that the invention encompasses operable processes and / or compositions, Any and all combinations are disclosed herein just as if individually or explicitly disclosed. Furthermore, all sub-components listed in the embodiments describing such variables are Combinations are also specifically encompassed by the present antibodies, compositions and methods, and such The present invention is not to be construed as though all subcombinations were individually and expressly disclosed. Disclosed in the details.

[0024] As will be apparent to those skilled in the art upon reading this disclosure, the individual components described and illustrated herein may be Each of the embodiments may be combined with any of the other embodiments without departing from the scope or spirit of the disclosed methods. Individual features that can be easily separated from or combined with any of the features of the embodiments of Any recited method has elements and features. or in any other order which is logically possible.

[0025] antibody The present disclosure provides antibodies that specifically bind to vaccinia virus (VV or VACV) antigens. Vaccinia virus provides a method for viral replication independent of the host cell machinery. A double-stranded DNA genome of approximately 192 kb encodes numerous viral enzymes and factors that enable VV is a member of the poxvirus family, characterized by a cytoplasmic domain of up to 25 kb. They can stably harbor cloned exogenous DNA. Structurally, VVs are: The viral DNA and RNA polymerase and poly(A) polymerase are enclosed in a lipoprotein core membrane. It consists of various viral enzymes such as ribosomal reductase and a core region made up of The outer layer of the vesicle is composed of a double lipid membrane envelope. VV has the natural tropism of tumors, potent They have excellent lytic ability, a short life cycle with rapid cell-to-cell diffusion, and efficient gene expression. Attributes favorable for use in oncolytic virotherapy, such as expression and large cloning capacity VVs have many unique features that make them unique. VVs undergo a short cytoplasmic release of about 8 hours. Replication usually occurs approximately 2 days after infection. 3 hours later, at which point host cell nucleic acid synthesis ceases and cellular resources are directed toward viral replication. Cell lysis occurs between 12 and 48 hours, and packaged viral particles are released. VV does not depend on the host machinery for mRNA transcription and therefore Unlike other oncolytic viruses (OVs), VV does not require specific surface receptors for cell entry and therefore can infect a wide range of cells. This makes it possible.

[0026] The term "antibody" (also used interchangeably with "immunoglobulin") refers to a polyclonal antibody. antibody preparations, including clonal (e.g., rabbit polyclonal) and monoclonal antibody preparations. can be of any isotype (e.g., IgG (e.g., IgG1, IgG2, IgG3 or antibodies or immunoglobulins (e.g., IgG4, IgE, IgD, IgA, IgM, etc.), A full-length antibody (e.g., a tetramer composed of two dimers of heavy and light chain polypeptides) single chain antibodies (e.g., scFv); single chain Fv (scFv), Fab, (Fab to compounds including, but not limited to, (scFv')2, (scFv')2, and diabodies fragments of antibodies (e.g., fragments of full-length or single-chain antibodies) that retain specific binding of the antibody; Monoclonal antibodies, humanized antibodies, human antibodies; and antibody and non-antibody antibodies. In some embodiments, the antibody may be a fusion protein comprising the antigen-binding portion of an antibody. The antibodies include IgG, Fv, single chain antibodies, scFv, Fab, F(ab')2, and F(ab' The antibody may be selected from a member of a specific binding pair, such as biotin (biotin-antibody). can be further conjugated to other moieties such as members of a nucleotide-specific binding pair .

[0027] Immunoglobulin polypeptides include kappa and lambda light chains, and alpha, Gamma (IgG1, IgG2, IgG3, IgG4), Delta, Epsilon and Mu heavy chains of the human immunoglobulin family, or equivalents in other species. (usually about 25 kDa or about 214 amino acids) is separated by about 110 amino acids at the NH2-terminus and a kappa or lambda constant region at the COOH-terminus. The immunoglobulin "heavy chain" (of about 150 kDa or about 446 amino acids) of region (of about 116 amino acids) and one of the heavy chain constant regions mentioned above, e.g., gamma ( It contains approximately 330 amino acids.

[0028] Immunoglobulin light or heavy chain variable region (V L and V H ) is a "complementarity determination" It is a "framework" fragment interrupted by three hypervariable regions, also called "CDRs" or "CDRs." The framework regions and CDRs are defined. (E. Kabat et al., Sequences of proteins of immunological interest,4th ed. .Health and Human Services,Public Health Services, Bethesda, MD (1987); and Lefranc e t al.IMGT, the international ImMunoGeneTi cs information system.Nucl.Acids Res.,20 05,33,D593-D597). The arrangement of different light or heavy chain framework regions The sequences are relatively conserved within species. The interdigitated framework of the constituent light and heavy chains The framework regions of an antibody serve to position and align the CDRs. R is primarily responsible for binding to the epitope of the antigen. DR and Framework are defined according to Kabat, supra, unless otherwise stated. do.

[0029] Thus, an "antibody" may be any antibody that is genetically modified, for example, by immunoglobulin genes or fragments thereof. It is recognized that the term "protein" encompasses proteins having one or more polypeptides that can be encoded by the gene. The immunoglobulin genes involved are kappa, lambda, alpha, gamma, delta, and epsilon and mu constant region genes, as well as numerous immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, or alpha. They are classified as fa, delta, or epsilon, which in turn represent the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE are defined. 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.

[0030] A typical immunoglobulin (antibody) structural unit is known to comprise a tetramer. A dimer consists of two identical pairs of polypeptide chains, each pair containing one "light" chain (approximately 25 kJ / min). The N-terminus of each chain is primarily an antigen-binding domain. Defines a variable region of approximately 100-110 amino acids involved in antigen recognition. V L ) and variable heavy chain (V H ) refer to these light and heavy chains, respectively.

[0031] Antibodies include complete immunoglobulins and antibodies that can be genetically encoded or derived from various peptides. It includes a number of well-characterized fragments that can be produced by digestion with thiazides. Thus, for example, pepsin digests antibodies under disulfide bonds in the hinge region. F(ab)'2 (a light antibody fragment itself linked to VH-CHI by a disulfide bond) F(ab)'2 is produced by reducing the antibody under mild conditions. , cleaving the disulfide bond in the hinge region, thereby converting the (Fab')2 dimer into Fa The Fab' monomer essentially comprises 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). Various antibody fragments have been defined in terms of the digestion of intact antibodies. However, such Fab' fragments can be produced chemically or by utilizing recombinant DNA methodologies. Those skilled in the art will understand that the compounds can be synthesized de novo. As used herein, the term antibody refers to antibodies produced by modification of whole antibodies or Fab's. 2, IgG, IgM, IgA, scFv, dAb, nanobody, unibody, and dye De novo synthesized using recombinant DNA methodologies, including but not limited to abodies In certain embodiments, the antibodies of the present disclosure include IgG, Fv, single chain antibodies, s The antibody is selected from cFv, Fab, F(ab')2, and Fab'.

[0032] According to some embodiments, the antibodies of the present disclosure are monoclonal antibodies. "Clonal antibodies" refer to a population of antibodies that are substantially homogeneous (i.e., naturally occurring antibodies that may be present in trace amounts). a composition comprising one or more antibodies obtained from a population of antibodies (wherein the individual antibodies are identical except for mutations) Monoclonal antibodies are highly specific and target a single antigenic site, typically The modifier "monoclonal" refers to an antibody that is directed against a single epitope on an antigen. characterization of antibodies as being obtained from a substantially homogeneous population of antibodies, and It is not required that the antibody be produced by the method described above or that it be the only antibody in the composition.

[0033] In certain embodiments, an antibody of the present disclosure is a humanized antibody. Humanized antibodies are antibodies derived from non-human (e.g., rabbit, rodent, etc.) antibodies and are derived from human antibody fragments. recombinant DNA fragments modified to contain at least a portion of the framework and / or constant region. Humanized antibodies are also known as chimeric antibodies, where different regions may be derived from different species. Chimeric antibodies include antibodies that contain human constant regions (e.g., human The antibody may be an antibody comprising a variable region from any source linked to an Fc domain. Thus, in a chimeric antibody, the variable region can be non-human and the constant region is human. Ligated antibodies consist of a non-human "drug" linked to the framework regions of a human "recipient" antibody. For example, an antibody of the present disclosure that is in the form of an scFV may be an antibody that comprises CDRs from a "core" antibody. can be linked to a human constant region (e.g., an Fc domain) to form a human immunoglobulin .

[0034] Generally, a humanized antibody has a higher affinity for the antibody in a human host than a non-humanized version of the same antibody. The antibody may be modified, for example, by CDR grafting, veneering, or resurfacing. using a variety of techniques, including resurfacing, and chain shuffling. In certain embodiments, framework substitutions can be made to the CDRs and framework regions. Modeling framework residue interactions to identify framework residues important for antigen binding and sequence comparison to identify unique framework residues at specific positions. It is identified by:

[0035] Therefore, any of the antibodies described herein can be humanized using available methods. Rabbit or mouse CDRs can be replaced with human variable domain frameworks. By combining the human variable domain fragments, the correct spatial orientation of the fragments can be maintained. The framework is the same as or similar to the rabbit or mouse variable framework from which the CDRs are derived. This is because the CDRs are derived from rabbit or mouse variable framework amino acids. Human antibodies with framework sequences that show a high degree of sequence identity to the work domains This can be achieved by obtaining variable domains of the heavy and light chains. The framework regions can be derived from the same or different human antibody sequences. The sequence can be the sequence of a naturally occurring human antibody, or a consensus sequence of several human antibodies. It may be a sequence.

[0036] Rabbit or mouse donor immunoglobulin and appropriate human acceptor immunoglobulin Once the complementarity-determining regions of the antibody have been identified, the next step is to optimize the properties of the resulting humanized antibody. Determine which, if any, residues in these components should be substituted to achieve In general, substitution of human amino acid residues with rabbit or mouse residues is kept to a minimum. This is because the introduction of rabbit or mouse residues makes the antibody more human in humans. There is a risk of eliciting an anti-rabbit antibody (HARA) or human anti-mouse antibody (HAMA) response. Art-recognized methods for determining immune response in a particular patient are used. or can be performed to monitor HARA or HAMA responses during clinical trials Patients receiving humanized antibodies should be monitored at the start and throughout the duration of treatment. Immunogenicity assessment can be performed. HARA or HAMA responses can be assessed by, for example, These include methods known to those skilled in the art, such as BIACORE® and / or solid-phase ELISA assays. The method used to detect antibodies against the humanized therapeutic reagent in serum samples from patients In many embodiments, the subject humanized antibodies are measured in human subjects. and does not substantially induce a HARA response.

[0037] Specific amino acids from the human variable region framework residues are involved in the CDR conformation and / or Substitutions are selected based on their potential effect on the target site or antigen binding. The unnatural juxtaposition of human or mouse CDR regions with human variable framework regions creates an unnatural position. may result in structural constraints that can be corrected by substitution of specific amino acid residues. Unless the amino acid residues are properly selected, this will result in a loss of binding affinity. The three-dimensional structure of an immunoglobulin molecule can be determined by computer modeling. Computer hardware and software for generating images are well known in the art. Generally, starting from a resolved structure of an immunoglobulin chain or its domains, The chains being modeled are the chains or dopants of the solved three-dimensional structure. The chain or domain showing the greatest sequence similarity is compared for amino acid sequence similarity with the A sequence with at least 50% sequence identity is selected as the starting point for building a molecular model. Shared strands or domains are selected for modeling, preferably at least 60 Those sharing ≥ 70%, 80%, or 90% sequence identity were selected for modeling. The solved starting structure is the actual structure of the immunoglobulin chain or domain being modeled. The modified structure is then modified to allow for differences in amino acids between the original structure and the amino acids in the starting structure. The modified structures are assembled into composite immunoglobulins. Finally, energy minimization and All atoms are within the appropriate distance from each other and bond lengths and angles are within the chemically acceptable range. By checking that the model is within

[0038] For example, the framework residues defined by Kabat may be modified, for example, by Chothi a) in the structural loop residues defined by Amino acids adjacent to the CDR regions can be selected for substitution into the humanized antibody. The "interacting" residues include those that are directly associated with one or more of the CDRs in the primary sequence of the humanized immunoglobulin chain. Adjacent positions, e.g., CDRs as defined by Kabat or Chothia CDRs defined by (e.g., Chothia and Lesk JMB 19 6:901 (1987) These amino acids are particularly likely to interact with amino acids in the CDRs, if Selected from the acceptor, likely distorting the donor CDRs and reducing affinity Furthermore, these adjacent amino acids may directly interact with the antigen (Amit et al., Science, 233:747 (1986)), and these The amino acid selection is important to maintain all the antigen contacts that provide the affinity of the original antibody. It may be desirable to use the antibody to humanize any of the antibodies described herein. One approach that can be used is Williams, D., Matthews, D. & Jones,T.Humanising Antibodies by CDR Gr afting.Antibody Engineering 319-339(2010 ) doi:10.1007 / 978-3-642-01144-3_21;Kuram ochi, T., Igawa, T., Tsunoda, H. & Hattori, K.H. umanization and simultaneous optimization n of monoclonal antibody.Methods Mol.Bio l.1060,123-37(2014);Hwang,WY,Almagro,J .C., Buss, TN, Tan, P. & Foote, J. Use of hum an germline genes in a CDR homology-base d approach to antibody humanization.Meth ods 36,35-42(2005);Lo,BKAntibody human ization by CDR grafting.Methods Mol.Biol .248,135-59(2004); and Lefranc, M.-PP, Ehr enmann, F., Ginestoux, C., Giudicelli, V. & Du roux,P.Use of IMGT databases and tools f or antibody engineering and humanization .Methods Mol.Biol.907,3-37(2012) (their disclosures are , which are incorporated herein by reference in their entirety for all purposes. This includes, but is not limited to,

[0039] The antibodies of the present disclosure specifically bind to the VV A56 or VV B5 antigen. binds to other substances (e.g., in a sample) with greater affinity, avidity, and more readily and / or binds with a longer duration, it "specifically binds" or "binds" to the target. In certain embodiments, the antibody binds preferentially to, for example, about 10 4 M -1 More than affinity The affinity or Ka (i.e., the equilibrium association constant of a specific binding interaction in units of 1 / M) An antibody "specifically binds" to an antigen when it binds to or associates with the antigen. Alternatively, affinity can be determined by the ratio of M The equilibrium dissociation constant (KD) of a particular binding interaction in units (e.g., 10 -5 M~10 -1 3 In certain aspects, specific binding can be defined as a binding activity of an antibody that: about 10 -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 Binds to antigen with a KD of 0.1 M or less The binding affinity of an antibody to an antigen can be determined using conventional techniques, e.g., For example, competitive ELISA (enzyme-linked immunosorbent assay), equilibrium dialysis, and surface plasmon ion resonance (SPR) technique (e.g., BI using the general procedures outlined by the manufacturer) Acore 2000 or BIAcore T200 instrument) This can be readily determined by radioimmunoassay.

[0040] Whether an antibody of the present disclosure "competes" with a second antibody for binding to an antigen is determined by: This can be readily determined using competitive binding assays known in the art. Competing antibodies can be identified, for example, via antibody competition assays. A sample of one antibody can be bound to a solid support. A sample of the second antibody suspected of being the target of the antibody is added. One of the two antibodies is labeled. If labeled and unlabeled antibodies bind to separate, distinct sites on the antigen, they are suspected of competing. The labeled antibody binds to the same level regardless of whether the corresponding antibody is present. If the interaction sites are identical or overlapping, unlabeled antibodies will compete for and bind to the antigen. If there is an excess of unlabeled antibody, the labeled antibody will bind, e.g. Very little, if any, binding occurs.

[0041] For purposes of this disclosure, a competing antibody is one that inhibits binding of an antibody to an antigen by about 50% or more, about 60% or more. % or more, approximately 70% or more, approximately 80% or more, approximately 85% or more, approximately 90% or more, approximately 95% or more, or or by about 99% or more. Details of the antibody are known, e.g., Harlow and Lane, s,A Laboratory Manual,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,New York,1988,567-569,1988,ISBN 0-87969-314 Such assays can be quantitatively performed using purified antibodies. Establish a standard curve by titrating one antibody against itself. That is, the same antibody is used as both the label and the competitor. The ability of unlabeled competing antibodies to inhibit binding of the labeled antibody can be titrated. The results are plotted. and compare the concentrations required to achieve the desired degree of binding inhibition.

[0042] Anti-VV A56 antibody According to some embodiments, the antibody specifically binds to the VV A56 antigen (VV A56). The vaccinia virus A56 protein is originally a hemagglutinin protein. Although A56 has been characterized as a protein, it also has other functions. 2 of the vaccinia virus complement control protein (VCP) and the protease inhibitor (K2) They can bind to two viral proteins and anchor them to the surface of infected cells. Both proteins have biologically relevant functions at the cell surface, but neither can function by itself. The A56-K2 complex is unable to locate there. The A56-VCP complex reduces the amount of ATP and also prevents the formation of syncytia by infected cells. The deletion of the A56R gene protects vaccinia virus from complement attack. The gene encoding the A56 protein is not essential, which has various effects on pathogenicity. It may be used as an insertion point for foreign genes and is in clinical development as an oncolytic agent. It is deleted in some viruses.

[0043] In certain embodiments, the antibodies of the present disclosure specifically bind to VV A56, herein referred to as A56. Called 047 / A057, A049 / A059 / A056, A050 and A054 One, two, three, four, five, or all six of one or more of the following anti-VV A56 antibodies It competes for binding to VV A56 with an antibody having the complementarity-determining region (CDR) of According to some embodiments, the antibodies of the present disclosure specifically bind to VV A56 and A047 / A057, A049 / A059 / A056, A050 or A054 in the book One, two, three, four, five or all six CDRs of the anti-VV A56 antibody referred to Includes: A047 / A057, A049 / A059 / A056, A050 and A054 The variable heavy chain (V H ) polypeptide, variable light chain (V L ) polypeptide, and CDR The amino acid sequences are shown below in Table 1. All CDRs and fragments described throughout this disclosure are Framework regions are defined according to Kabat unless otherwise stated. [Table 1-1] [Table 1-2] [Table 1-3]

[0044] In certain embodiments, the antibodies of the present disclosure specifically bind to VV A56 and Regarding binding to V containing the amino acid sequence SSYWIC (SEQ ID NO: 3) H CDR1 and V containing the amino acid sequence CIYAGSGGSTYYATWAKG (SEQ ID NO: 4) H CD R2 and V containing the amino acid sequence AYSDRSGGYSFNL (SEQ ID NO: 5) H CDR3 and containing variable heavy chain (V H ) polypeptides, and V containing the amino acid sequence QASQSVDNNNYLA (SEQ ID NO: 6) L CDR1 and V containing the amino acid sequence SASSLAS (SEQ ID NO: 7) L CDR2 and V containing the amino acid sequence LGSYDCSDADCYA (SEQ ID NO: 8) L CDR3 and containing variable light chain (V L ) polypeptide, which competes with an antibody comprising the polypeptide.

[0045] In certain embodiments, such antibodies are selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, S It contains the six CDRs shown in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8. In some embodiments, the antibody has a sequence identity of 70% or more, 75% or more, with respect to the amino acid sequence set forth in SEQ ID NO: 1. , 80% or more, 85% or more, 90% or more, 95% or more, or 100% identity The variable heavy chain (V H ) polypeptide, the amino acid sequence set forth in SEQ ID NO:2 On the other hand, if it is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more The variable light chain (VLC) contains amino acid sequences with approximately 100% identity. L ) polypeptides, and includes both.

[0046] According to some embodiments, the antibodies of the present disclosure specifically bind to VV A56 and VV Regarding binding to A56, V containing the amino acid sequence DIYYIS (SEQ ID NO: 11) H CDR1 and V containing the amino acid sequence CTYAGSSGSTYYATWAKG (SEQ ID NO: 12) H C DR2 and V containing the amino acid sequence DRYPGTSGRVYGMDL (SEQ ID NO: 13) H CDR3 and a variable heavy chain (V H ) polypeptides, and V containing the amino acid sequence QASQSISDLLS (SEQ ID NO: 14)L CDR1 and V containing the amino acid sequence SASTLAS (SEQ ID NO: 15) L CDR2 and V containing the amino acid sequence QCNYYSPTYGNG (SEQ ID NO: 16) L CDR3 and containing variable light chain (V L ) polypeptide, which competes with an antibody comprising the polypeptide.

[0047] In certain embodiments, such antibodies are selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 , SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16. According to some embodiments, the antibody has a sequence identity of 70% or more to the amino acid sequence set forth in SEQ ID NO:9. , 75% or more, 80% or more, 85% or more, 90% or more, 95% or more or 100% The variable heavy chain (V H ) polypeptide, as set forth in SEQ ID NO: 10 70% or more, 75% or more, 80% or more, 85% or more, 90% or more of the amino acid sequence Variable light chains (V) containing amino acid sequences with 95% or more or 100% identity L ) Poly In certain embodiments, the antibody comprises an amino acid sequence set forth in SEQ ID NO:9, a peptide, or both. 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more of the acid sequence A variable heavy chain (V) containing an amino acid sequence with at least 100% identity H ) Polypeptide 70% or more, 75% or more, 80% or more of the amino acid sequence set forth in SEQ ID NO: 62, Amino acid sequences with 85% or more, 90% or more, 95% or more, or 100% identity containing variable light chain (V L ) polypeptide, or both. The antibody has a sequence similar to that of the amino acid sequence set forth in SEQ ID NO: 9, but is not specifically limited thereto. , 85% or more, 90% or more, 95% or more, or 100% identity A variable heavy chain (V H ) polypeptide, 70 to the amino acid sequence set forth in SEQ ID NO: 63 % or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more or 100 The variable light chain (V) contains an amino acid sequence with 100% identity to the L ) polypeptide, or both nothing.

[0048] In certain embodiments, the antibodies of the present disclosure specifically bind to VV A56 and Regarding binding to V containing the amino acid sequence SSYWLC (SEQ ID NO: 19) H CDR1 and V containing the amino acid sequence CIYNGDGSTHYASWAKG (SEQ ID NO: 20) H CD R2 and V containing the amino acid sequence DYTYNFYTYGFNL (SEQ ID NO: 21) H CDR3 and , including a variable heavy chain (V H ) polypeptides, and V containing the amino acid sequence QASQSVNIWAS (SEQ ID NO: 22) L CDR1 and V containing the amino acid sequence KASTLAS (SEQ ID NO: 23) L CDR2 and V containing the amino acid sequence QGGYPSSSSGWA (SEQ ID NO: 24) L CDR3 and containing variable light chain (V L ) polypeptide, which competes with an antibody comprising the polypeptide.

[0049] In certain embodiments, such antibodies are selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , and the six CDRs shown in SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24. According to some embodiments, the antibody has a sequence identity that is 70% or more similar to the amino acid sequence set forth in SEQ ID NO: 17. Above, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more or 100% Variable heavy chains (V) containing identical amino acid sequences H ) polypeptide, as set forth in SEQ ID NO: 18 70% or more, 75% or more, 80% or more, 85% or more, 90% or more of the amino acid sequence , a variable light chain (V) having an amino acid sequence with 95% or more or 100% identity L )Po peptide, or both.

[0050] According to some embodiments, the antibodies of the present disclosure specifically bind to VV A56 and VV Regarding binding to A56, V containing the amino acid sequence SSYWIC (SEQ ID NO: 27) H CDR1 and V containing the amino acid sequence CTYNGDGSTHYASWAKG (SEQ ID NO: 28) H CD R2 and V containing the amino acid sequence DYTDAFYTYGFNL (SEQ ID NO: 29) H CDR3 and A variable heavy chain (V H ) polypeptides, and V containing the amino acid sequence QASQSTSSYLA (SEQ ID NO: 30) L CDR1 and V containing the amino acid sequence RASSLAS (SEQ ID NO: 31) L CDR2 and V containing the amino acid sequence QTGFYGSSGHT (SEQ ID NO: 32) L CDR3 and Variable light chain (V L ) polypeptide, which competes with an antibody comprising the polypeptide.

[0051] In certain embodiments, such antibodies are selected from the group consisting of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 , and the six CDRs shown in SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32. According to some embodiments, the antibody has a sequence identity that is 70% or more similar to the amino acid sequence set forth in SEQ ID NO:25. Above, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more or 100% Variable heavy chains (V) containing identical amino acid sequences H ) polypeptide, as set forth in SEQ ID NO:26 70% or more, 75% or more, 80% or more, 85% or more, 90% or more of the amino acid sequence , a variable light chain (V) having an amino acid sequence with 95% or more or 100% identity L )Po peptide, or both.

[0052] The anti-VV A56 antibody of the present disclosure "competes" with a second antibody for binding to the VV A56 antigen. Suitable VV A56 antigens for determining whether they "match" are listed in Table 2 below. Wyeth, Western Reserve, and Cope, which have amino acid sequences An example is the nhagen A56 antigen. [Table 2]

[0053] Anti-VV B5 antibody According to some embodiments, an antibody that specifically binds to the VV B5 antigen (VV B5) The VV B5 protein contains four short contigs characteristic of complement regulatory proteins. A 42-kDa type I transmembrane domain with an extracellular domain composed of sensor repeats (SCRs) After the SCR, B5 has a stalk region and a short Both the SCR and CT bind B5 to the extracellular envelope. Although it is not essential for targeting to the EEV membrane, the latter is essential for targeting to the cell surface. B5 affects its transport to the endothelium and its recycling via endosomes. The mature virus (IMV) is wrapped to form the intracellular enveloped virus (IEV). It is necessary to

[0054] In certain embodiments, the antibodies of the present disclosure specifically bind to VV B5, herein referred to as A0 one or more of the anti-VV B5 antibodies designated 48 / A058 / A073 and A051 having one, two, three, four, five, or all six complementarity-determining regions (CDRs) of According to some embodiments, the antibody of the present disclosure competes with the antibody for binding to VV B5. The antibody specifically binds to VV B5 and is herein designated A048 / A058 / A073 or One, two, three, four, five or all six of the anti-VV B5 antibodies, called A051, The variable heavy chain (V) of the A048 / A058 / A073 and A051 antibodies contains all CDRs. H ) polypeptide, variable light chain (V L ) polypeptide, and the amino acid sequences of the CDRs are as follows: The results are shown in Table 3. [Table 3]

[0055] In certain embodiments, the antibodies of the present disclosure specifically bind to VV B5 and Regarding bonding, V containing the amino acid sequence SSYYMC (SEQ ID NO: 35) H CDR1 and V containing the amino acid sequence CIYTSSGSAYYANWAKG (SEQ ID NO: 36) H CD R2 and V containing the amino acid sequence NAVGSSYYLYL (SEQ ID NO: 37) H CDR3 and Variable heavy chain (V H ) polypeptides, and V containing the amino acid sequence QASQSVAGNNYLS (SEQ ID NO: 38) L CDR1 and V containing the amino acid sequence SVSTLAS (SEQ ID NO: 39) L CDR2 and V containing the amino acid sequence QGYYNDGIWA (SEQ ID NO: 40) L CDR3 and Variable light chain (V L ) polypeptide, competes with antibodies containing

[0056] In certain embodiments, such antibodies are selected from the group consisting of SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37 , SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40. According to some embodiments, the antibody has a sequence identity that is 70% or more similar to the amino acid sequence set forth in SEQ ID NO: 33. Above, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more or 100% Variable heavy chains (V) containing identical amino acid sequences H ) polypeptide, as set forth in SEQ ID NO: 34 70% or more, 75% or more, 80% or more, 85% or more, 90% or more of the amino acid sequence , a variable light chain (V) having an amino acid sequence with 95% or more or 100% identity L )Po peptide, or both.

[0057] According to some embodiments, the antibodies of the present disclosure specifically bind to VV B5 and VV Regarding binding to B5, V containing the amino acid sequence SYWMC (SEQ ID NO: 43) H CDR1 and V containing the amino acid sequence CIYGGSSGSTYYSNWAKG (SEQ ID NO: 44) H C DR2 and V containing the amino acid sequence DGSTWDYFRL (SEQ ID NO: 45) H CDR3 and Variable heavy chain (V H ) polypeptides, and V containing the amino acid sequence QASQSINTNYLS (SEQ ID NO: 46) L CDR1 and V containing the amino acid sequence QASTLES (SEQ ID NO: 47) L CDR2 and V containing the amino acid sequence QGYYTVENIGNP (SEQ ID NO: 48) L CDR3 and containing variable light chain (V L ) polypeptide, which competes with an antibody comprising the polypeptide.

[0058] In certain embodiments, such antibodies are selected from the group consisting of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45 , SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48. According to some embodiments, the antibody has a sequence identity that is 70% or more similar to the amino acid sequence set forth in SEQ ID NO:41. Above, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more or 100% Variable heavy chains (V) containing identical amino acid sequences H ) polypeptide, as set forth in SEQ ID NO:42 70% or more, 75% or more, 80% or more, 85% or more, 90% or more of the amino acid sequence , a variable light chain (V) having an amino acid sequence with 95% or more or 100% identity L )Po peptide, or both.

[0059] The anti-VV B5 antibodies of the present disclosure "compete" with a second antibody for binding to the VV B5 antigen. Suitable VV B5 antigens for determining whether a virus is "vaccinated" include those shown in Table 4 below. Wyeth, Western Reserve, and Copenhagen with amino acid sequences Gen B5 antigen. [Table 4]

[0060] bispecific antibody Bispecific antibodies are also provided. In certain embodiments, the bispecific antibodies of the present disclosure comprise any of the anti-VV A56 and anti-VV B5 antibodies of the disclosure, such as antibodies V H Polypeptide-V L a first antigen-binding domain comprising a pair of bispecific polypeptides; The antibody specifically binds to the VV antigen bound by the first antigen-binding domain. In certain embodiments, the bispecific antibody may comprise an antigen-binding domain of a first antigen-binding domain. A second antigen-binding domain that specifically binds to a VV antigen other than the VV antigen bound by the binding domain. Includes combined domains.

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

[0062] The bispecific antibodies of the present disclosure include antibodies having a full-length antibody structure and bispecific antibodies. As used herein, "full length" refers to two full length antibody heavy chains and a fragment thereof. Refers to an antibody having two full-length antibody light chains. A full-length antibody heavy chain (HC) is a well-known It consists of a chain variable domain and constant domains VH, CH1, CH2 and CH3. A full-length antibody light chain (LC) consists of the well-known light chain variable domain and the constant domain V A full-length antibody consists of one or both heavy chain C-terminal lysines. The term "Fab arm" refers to the antibody that specifically binds to an antigen. Refers to one heavy chain:light chain pair.

[0063] Full-length bispecific antibodies can be made, for example, by introducing substitutions at the heavy chain CH3 interface of each half molecule, In vitro or co-expression in a cell-free environment of two antibody half molecules with distinct specificities Two monospecific bivalent antibodies are used to promote heterodimer formation Fab arms can be generated using Fab arm exchange (or half molecule exchange) between The exchange reaction is the result of disulfide bond isomerization and dissociation-association of the CH3 domain. The heavy chain disulfide bonds in the hinge region of the parent monospecific antibody are reduced. A free cysteine from one of the heterologous antibodies is transferred to the cysteine of the second parent monospecific antibody molecule. Forms inter-heavy chain disulfide bonds with stearate residues, simultaneously dissociating the CH3 domain of the parent antibody -Association separates and reforms the CH3 domain of the Fab arm. The resulting products may also be designed to favor heterodimerization, each containing a distinct epitope. It is a bispecific antibody with two Fab arms or half molecules that bind to the same polypeptide.

[0064] "knob-in-hole" strategies (e.g., WO2006 / 028936) can be used to generate full-length bispecific antibodies. Then, the selected amino acids that form the interface of the CHS domain in human IgG are C Mutated at positions that affect H3 domain interactions and promote heterodimer formation The amino acid with a small side chain (hole) can specifically bind to the first antigen. The antibody heavy chain is introduced with an amino acid with a large side chain (knob) specific for the second antigen. After co-expression of the two antibodies, the heavy chain with the "hole" is introduced into the heavy chain of the antibody that binds to the antibody. Heterodimers are formed as a result of preferential interaction between the α- and β-heavy chains with the "knob" Exemplary CH3 substitution pairs that form knobs and holes are as follows (first helical group): Altered position in the first CH3 domain of the first heavy chain / alternative position in the second CH3 domain of the second heavy chain The alterations are expressed as: T366Y7F405A, T366W / F405W, F4 05W / Y407A, T394W / Y407T, T3945 / Y407A, T366W / T394S, F405W / T394S and T366W / T366S_L368A_Y4 07V.

[0065] US2010 / 0015133, US2009 / 0182127, US2010 / 02 8637 or US2011 / 0123532, on one CH3 surface. By substituting positively charged residues and negatively charged residues on the second CH3 surface, Other strategies may also be used, such as using electrostatic interactions to promote heavy chain heterodimerization. In another strategy, heterodimerization is achieved by modifying the first CH3 domain of the first heavy chain. The following substitutions (expressed as positions / altered positions in the second CH3 domain of the second heavy chain): May be promoted by: US2012 / 0149876 or US2013 / 01958 L351 Y_F405A_Y407V T394W, T366 as described in 49 I_K392M_T394W / F405A_Y407V, L351 Y_Y407A'T 366A_K409F, L351Y_Y407A / T366V_K409F, Y407A / T366A_K409F or T350V_L351Y_F405A_Y407V / T350V_T366L_K392L_T394W.

[0066] Single chain bispecific antibodies are also provided. In some embodiments, the single chain bispecific antibodies of the present disclosure The antibody is a bispecific scFv. For more information on bispecific scFvs, see, for example, Zh ou et al. (2017) J Cancer 8(18):3689-3696 can be found.

[0067] For producing multispecific (e.g., bispecific) antibodies from the antibodies described herein One possible approach is to look at Ellerman, D. (2019). Bispec ific T-cell engagers:Towards understandi ng variables influencing the in vitro po tency and tumor selectivity and their mo dulation to enhance their efficacy and s afety.”Methods 154:102-117;Brinkmann,Ua nd REKontermann (2017).”The making of bispecific antibodies.”mAbs 9(2):182-212 ; and Suurs, FV, et al. (2019).”A review of bispecific antibodies and antibody cons tructs in oncology and clinical challenges es." Pharmacol Ther 201:103-119 (the disclosure of which is incorporated herein by reference). (which are incorporated herein by reference in their entireties for all purposes), , but is not limited to this.

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

[0069] The chains of the anti-VV A56 or anti-VV B5 antibodies can be fused to any heterologous sequence of interest. The heterologous polypeptide of interest may be albumin, transferrin, XTEN, homoadenoids, or the like. amino acid polymers, proline-alanine-serine polymers, elastin-like peptides, or In certain embodiments, different The heterologous polypeptide is fused to the heterologous sequence when the antibody is administered to an individual in need thereof. Increases the stability and / or serum half-life of an antibody compared to the same antibody without the compound.

[0070] In certain embodiments, the fusion proteins of the present disclosure comprise single chain antibodies, e.g., antibodies as described above. any of the anti-VV A56 and anti-VVB5 antibodies of the present disclosure, including any of the antibodies of the present disclosure. Reka's V H Polypeptide-V L Single-chain antibodies (e.g., scFv) containing polypeptide pairs The scFv of the present disclosure includes scFvs containing the six CDRs of the scFvs listed in Table 5 below. In some embodiments, the scFv includes, but is not limited to, Fvs, and in some embodiments, scFvs are listed in Table 5. V of the scFv H 70% or more, 75% or more, 80% or more, 8% or more of the amino acid sequence Contains an amino acid sequence with 5% or more, 90% or more, 95% or more, or 100% identity Variable heavy chain (V H ) polypeptide and the V of the scFv listed in Table 5 L For the amino acid sequence 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more or 10 The variable light chain (V) contains an amino acid sequence with 0% identity L ) polypeptide. In 5, segments / domains of a polypeptide are indicated by alternating underlines, and the segments / domains The ID of the Inn is shown in the left column. [Table 5-1] [Table 5-2]

[0071] According to some embodiments, the fusion protein is a single chain antibody (e.g., a fusion protein comprising a single chain antibody, such as those described herein). scFv) of the present disclosure, The fusion protein comprises a single chain antibody, a transmembrane domain, and an intracellular signaling domain. It is a chimeric antigen receptor (CAR).

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

[0073] In some embodiments, the antigen binding domain of the CAR is followed by appropriate cell / cell contact, The antigen-binding domain is then transferred to an effector cell to allow antigen binding and / or activation. One or more spacers that distance the CAR from the cell surface (e.g., the surface of a T cell expressing the CAR). A spacer domain (as well as any other spacer domains described herein) follows. The nucleic acid sequence (e.g., a nucleic acid sequence, a nucleic acid fragment ... In certain embodiments, the spacer domain is an immunoglobulin. It is a portion of the ribonucleotide sequence that contains one or more heavy chain constant regions, e.g., CH2 and CH3. The spacer domain may be, but is not limited to, a naturally occurring immunoglobulin hinge domain. In one embodiment, the amino acid sequence of the region or modified immunoglobulin hinge region may be included. In the present invention, the spacer domain is CH2 and / or CH3 of IgG1, IgG4, or IgD. contains CH3. Exemplary spacer domains suitable for use in the CARs described herein include The hinges are derived from the extracellular domains of type 1 membrane proteins such as CD8α and CD4. and a region that is a wild-type hinge region derived from these molecules or mutants thereof. In certain embodiments, the hinge domain comprises a CD8α hinge region. In this embodiment, the hinge is a PD-1 hinge or a CD152 hinge.

[0074] The "transmembrane domain" (Tm domain) is the extracellular binding domain and the intracellular signaling domain. CARs that fuse with the CAR and anchor the CAR to the plasma membrane of a cell (e.g., an immune effector cell) The Tm domain is a portion of R. The Tm domain may be of any natural, synthetic, semi-synthetic, or recombinant origin. In some embodiments, the Tm domain can be derived from a T cell receptor, CD35, CD3ζ, CD3γ, CD3δ, CD4, CD5, CD8α, CD9, CD16, CD2 2, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD8 6, CD134, CD137, CD152, CD154 or PD-1 alpha or It is derived from a beta chain (eg, including at least the transmembrane domain or a functional portion thereof).

[0075] In one embodiment, the CAR comprises a Tm domain derived from CD8α. , CARs are composed of a Tm domain derived from CD8α and a length of, e.g., 1, 2, 3, 4, 5, a short oligo- or polypeptide linker of 6, 7, 8, 9 or 10 amino acids; This links the Tm domain of the CAR with the intracellular signaling domain. For example, A glycine-serine linker may be used as such a linker.

[0076] The "intracellular signaling" domain of CAR is responsible for the signal transduction from CAR that binds to the target molecule / antigen. Transmitting signals to the interior of immune effector cells to enhance effector cell function, e.g., activation activation, cytokine production, proliferation and / or cytotoxic activity (cell activation against CAR-bound target cells) Induced by release of cytotoxic factors or target molecule / antigen binding to the extracellular CAR domain It refers to the part of CAR that is involved in eliciting the cellular response (including other cellular responses elicited). Therefore, the term "intracellular signaling domain" refers to a domain that transmits effector function signals. refers to the part of a protein that instructs the cell to perform a specific function. When a truncated portion of the transduction domain is used, it is sufficient to transmit the effector function signal. Thus, such truncated portions can be used in place of the full-length intracellular signaling domain. The term intracellular signaling domain refers to a domain that is sufficient to transmit an effector function signal. This is meant to include any truncated portion of the intracellular signaling domain.

[0077] Signals generated by the T cell receptor (TCR) alone are not sufficient to fully activate T cells. Therefore, secondary or costimulatory signals are also required. Activation is mediated by two distinct classes of intracellular signaling domains: the TCR Primary signaling that initiates antigen-dependent primary activation via the TCR / CD3 complex (e.g., TCR / CD3 complex). a null transduction domain and a co-transduction domain that acts antigen-independently to provide a secondary or costimulatory signal Stimulatory signaling domains. Thus, the CARs of the present disclosure may contain one or more "co-stimulatory signaling domains." an intracellular signaling domain comprising a "transduction domain" and a "primary signaling domain"; It may include

[0078] The primary signaling domain mediates either stimulatory or inhibitory signaling of the TCR complex. Regulates primary activation. The primary signaling domain that acts stimulatory is the immunoreceptor tyrosine kinase (TIK). Signaling molecules known as syn-based activation motifs (or "ITAMs") The ITAM-containing primary signaling domain suitable for use in the CAR of the present disclosure may contain a Main, non-limiting examples include FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, These include those derived from CD3ζ, CD22, CD79α, CD79β, and CD66δ. In certain embodiments, a CAR comprises a CD3ζ primary signaling domain and one or more Contains the above costimulatory signaling domain. Intracellular primary signaling and costimulatory signaling The transduction domain is operably linked to the carboxyl terminus of the transmembrane domain.

[0079] In some embodiments, the CAR is expressed by an immune effector cell (e.g., and one or more costimulatory signaling domains to enhance the efficacy and proliferation of T cells. As used herein, a "costimulatory signaling domain" or "costimulatory domain" refers to a The term "domain" refers to the intracellular signaling domain of a costimulatory molecule or an active fragment thereof. Exemplary costimulatory molecules suitable for use in CARs contemplated in certain embodiments include: These include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, and T LR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54(ICAM), CD83, CD134(OX40), CD 137(4-1BB), CD278(ICOS), DAP10, LAT, KD2C, SL P76, TRIM, and ZAP70. In some embodiments, CAR is One or more selected from the group consisting of 4-1BB (CD137), CD28, and CD134 It comprises the CD3ζ costimulatory signaling domain and the CD3ζ primary signaling domain.

[0080] The CAR of the present disclosure may be any of a variety of suitable CARs, including, but not limited to, leader sequences. domain; hinge, spacer and / or linker domain; transmembrane domain; Stimulatory domains; signal transduction domains (e.g., CD3 ζ domain); ribosomal skipping domains restriction enzyme sequences; reporter protein domains; and / or similar Non-limiting examples of such domains that can be included in the CARs of the present disclosure include: Examples include those shown in Table 6 below. As will be appreciated by those skilled in the art, Table 6 The amino acid sequence of one or more of the indicated domains (e.g., linker, hinge, transmembrane , costimulation, signal transduction, ribosomal skip elements; restriction enzyme sequences; reporter Proteins, etc., may be modified as needed, for example, to improve the functionality of the CAR. It is possible. [Table 6-1] [Table 6-2] [Table 6-3]

[0081] In certain embodiments, the CAR of the present disclosure is directed to an antigen of interest (e.g., VV A56 antigen or V a single-chain antibody (e.g., any of the scFvs of the present disclosure) that binds to the V B5 antigen; D4, CD8α, CD154 and PD-1 transmembrane domain of 4-1BB (CD137), CD28, and CD134 one or more intracellular costimulatory signaling domains from a polypeptide selected from the group consisting of: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD7 Intracellular polypeptides selected from the group consisting of CD9α, CD79β and CD66δ Such CARs comprise an antigen-binding portion and a transmembrane domain. It may further comprise a spacer domain in between, for example a CD8 alpha hinge.

[0082] According to some embodiments, provided are those comprising, between the N-terminus and C-terminus, the amino acid sequence described herein. The variable heavy chain (V) of the antibody described in H ) polypeptide, linker, antibody variable light chain (V L ), CD8 hinge region (which in some embodiments is an extended CD8 hinge region), CD8 membrane Contains a transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain According to certain embodiments, provided is a CAR comprising, between the N-terminus and the C-terminus: , the variable light chain (V L ) Polypeptide, linker, variable heavy chain of antibody ( V H ), a CD8 hinge region (which in some embodiments is an extended CD8 hinge region), CD8 transmembrane domain, 4-1BB costimulatory domain, and CD3ζ signaling domain In certain embodiments, provided is a CAR comprising an N-terminal to C-terminal Between them, the variable heavy chain (V H ) Polypeptides, linkers, and variable light chains of antibodies Chain (V L ), CD28 hinge region, CD28 transmembrane domain, 4-1BB costimulatory domain and a CD3ζ signaling domain. For example, provided are those comprising, between the N-terminus and C-terminus, the variable light chain (VLC) of an antibody described herein. V L ) polypeptide, linker, antibody variable heavy chain (V H), CD28 hinge region, CD2 8 transmembrane domains, a 4-1BB costimulatory domain, and a CD3ζ signaling domain Any CAR of the present disclosure includes V H containing the N-terminal domain of the polypeptide For example, a leader sequence (e.g., a GM-CSFR leader sequence) may be used in the C It may be located at the N-terminus of the AR.

[0083] The amino acid sequences of exemplary anti-A56 CARs of the present disclosure are shown in Tables 7 and 8 below: The amino acid sequences of CARs in Table 7 are expressed as follows: an N-terminal leader sequence (here, N-terminal GM-CSFR The amino acid sequence of the CAR in Table 8 is the amino acid sequence of the leader sequence. It does not contain any amino acid sequence. It does not contain any desired leader sequence (e.g., the GM-CSFR leader sequence). As will be appreciated by those skilled in the art, the following sequence may be present at the N-terminus of such a CAR: One or more of the domains shown in Tables 7 and 8 (e.g., linker, hinge, transmembrane The amino acid sequences of the CARs (e.g., co-stimulatory, signal transduction, etc.) can be modified to improve, for example, the functionality of the CAR. In Tables 7 and 8, the polypeptide segments / Domains are indicated by alternating underlines, and the segment / domain ID is shown in the left column. . [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5]

[0084] The CARs of the present disclosure can optionally include one or more additional domains. Non-limiting examples of additional domains include ribosomal skipping elements, enzymatic domains, and the like. (e.g., a domain with nuclease activity, e.g., restriction endonuclease activity) , a domain that allows for detection of the CAR (e.g., a reporter protein domain (e.g., , fluorescent proteins (e.g., eGFP, mCherry, etc.), luminescent proteins, and / or or the like). For example, in certain embodiments, provided are , ribosomal skip elements, restriction enzyme domains and / or reporter proteins Exemplary CARs of the present disclosure having one or more of these characteristics are CARs containing a target domain. The amino acid sequence of a suitable anti-A56 CAR is shown in Table 9 below. As shown in Table 9, the amino acid sequence of one or more of the domains (e.g., Carrier, hinge, transmembrane, costimulatory, signal transduction, restriction enzyme sequences, reporter proteins, etc. etc.) can be modified as needed, for example, to improve CAR functionality, detection, etc. In Table 9, the CAR portion is shown in bold, and the segments / domains of the polypeptide are alternated. The segment / domain ID is shown in the left column. [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] [Table 9-5] [Table 9-6] [Table 9-7]

[0085] In certain embodiments, provided are CARs comprising one or more mouse CAR domains. Non-limiting examples of mouse CAR domains that can be included in the CARs of the present disclosure include: These include one or more of the murine CAR domains shown in Table 10 below. As will be understood, the amino acid sequence of one or more of the domains shown in Table 10 (e.g., For example, leader sequences, linkers, hinges, transmembrane sequences, costimulatory sequences, signal transduction sequences, ribosomal sequences, The following components (e.g., restriction enzyme sequences, reporter proteins, etc.) may be modified as necessary. obtain. [Table 10]

[0086] According to some embodiments, one or more mouse CAR domains (e.g., those listed in Table 1 above) A murine CAR is provided that comprises one or more of the domains described in any of the preceding paragraphs. Non-limiting examples of disclosed murine CARs include those shown in Table 11 below. As will be appreciated by those skilled in the art, one or more of the domains shown in Table 11 may be amino acid sequence (e.g., Leavire sequence, V H , Linker, V L , hinge, transmembrane, costimulatory, Signal transduction, ribosomal skipping elements, etc.) can be modified as needed. In Figure 11, the CAR portion is shown in bold. Each segment is underlined and the segment / domain ID is shown in the left column. [Table 11-1] [Table 11-2]

[0087] According to some embodiments, the CAR of the present disclosure is provided by a single polypeptide. In certain embodiments, the CAR of the present disclosure is provided by two or more polypeptides. When the CAR is provided by two or more polypeptides, the CAR may be provided by a biotin-linked polypeptide. Combined immunoreceptor (BBIR) format (e.g., Urbanska K, Powell DJ.Development of a novel universal imm une receptor for antigen targeting to in finity and beyond.Oncoimmunology.2012;1( 5):777-779.doi:10.4161 / onci.19730 and Urba nska K,Lanitis E,Poussin M,et al.A unive rsal strategy for adoptive immunotherapy of cancer through use of a novel T cell antigen receptor.2013;72(7):1844-1852.d See oi:10.1158 / 0008-5472.CAN-11-3890.A ); Switchable CAR format using the peptide NeoEpitope (PNE) (e.g., Kim et al. (2015) J Am Chem Soc. 2015 ;137(8):2832-2835;Ma et al.(2016)Proc Na tl Acad Sci 113(4):E450-8;Rodgers et al. (2016)Proc Natl Acad Sci.113(4):E459-E46 8;Viaud et al.(2018)Proc Natl Acad Sci 1 15(46):E10898-E10906); with leucine zipper SUPRA CAR format (e.g., Cho et al. (2108) Cell 173(6):1426-1438.e11); using FITC-folate CAR-T adapter molecule (CAM)-based formats (e.g., Lee et al. l.(2019)Cancer Res.79(2):387-396 and Lu et al. al. (2019) Front Oncol. 9:151); anti-FITC -Folate adapter format (e.g., Chu et al. (2018) Biosc i Trends.12(3):298-308); anti-FITC antibody adaptor TurCAR format (e.g., Tamada et al. (2012) Clin See Cancer Res. 18(23):6436-6445); Fc targeting (e.g., anti-CD16) CAR+anti-tumor antibody format (e.g., Kudo et al. (2014) Cancer Res. 74(1):93-103); Any useful multi-polypeptide containing the universal CAR format, including the CAR and similar. It may be provided in a peptide format.

[0088] Conjugates The present disclosure also provides conjugates. According to some embodiments, the conjugates of the present disclosure A conjugate is a compound that combines any of the antibodies or fusion proteins of the present disclosure with an antibody or fusion protein. and drugs conjugated to a conjugated protein. The term generally refers to either a covalent or non-covalent bond, usually a covalent bond, that refers to a chemical bond that proximally associates one molecule with a second molecule of interest. Drugs conjugated to antibodies or fusion proteins include chemotherapeutic agents, toxins, and radiation enhancers. Sensitizers, radioisotopes (e.g., therapeutic radioisotopes), detectable labels, and half-life extenders The extension part is selected from the

[0089] According to some embodiments, the agent is a therapeutic agent, for example, a chemotherapeutic agent. The therapeutic agent is delivered to the conjugate via specific binding of the antibody portion of the conjugate to the cell / tissue antigen. Contains agents that can affect the function of the cell / tissue to which the gate binds. If the function of the cells / tissues is pathological, drugs that reduce the function of the cells / tissues may be used. In embodiments, the conjugates of the present disclosure inhibit cell proliferation and / or kill cells / tissues. These drugs include drugs that reduce the function of target cells / tissues by , cytostatic and cytotoxic agents, e.g., targeting with or without internalization into target cells. The therapeutic agent may include an agent capable of killing target cells and tissue.

[0090] In certain embodiments, the therapeutic agent is an enediyne, a lexitropsin, a duocarmycin, a tandem oxamers, puromycins, dolastatins, maytansinoids, and vinca alkaloids In some embodiments, the cytotoxic agent is selected from the group consisting of peptides, Ritaxel, docetaxel, CC-1065, CPT-11 (SN-38), topotecan , doxorubicin, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin Sorubicin, dolastatin-10, echinomycin, combretastatin, calicheamicin Shin, Maytansin, Maytansin DM1, Maytansin DM4, DM-1, Aurista auristatin or other dolastatin derivatives, such as auristatin E or auristatin F Conductor, AEB (AEB-071), AEVB (5-benzoylvaleric acid-AE ester) ), AEFP (antibody-endostatin fusion protein), MMAE (monomethyl auric acid statin E), MMAF (monomethylauristatin F), pyrrolobenzodiazepine (PB D), eleutherobin, netrupson, or any combination thereof.

[0091] According to some embodiments, the agent is a hemiasterlin and hemiasterlin, such as HTI-286. Miasterin analogs (e.g., US Pat. No. 6,223,999, the entire disclosures of which are incorporated herein by reference) PN7,579,323, WO2004 / 026293, and USPN8,129,4 07), abrin, brucine, cyclotoxin, diphtheria toxin, batrachotoxin , Botulinum toxin, Shiga toxin, Endotoxin, Pseudomonas exotoxin, Pseudom onas endotoxin, tetanus toxin, pertussis toxin, anthrax toxin, cholera toxin, falcarinol, Fumonisin B1, fumonisin B2, aflatoxin, maurotoxin in), agitoxin, charybdotoxin, margatoxin, Slotoxin, scyllatoxin, heft calciceptin, taicatoxin, calcicludin, Geldanamycin, gelonin, lotaustralin, ochratoxin ) A, patulin, ricin, strychnine, trichothecenes, zearalenone (zearle none, and tetradotoxin Enzymatically active toxins and fragments thereof that can be used include diphtheria, exotoxin A chain, non-binding active fragment of diphtheria toxin, exotoxin A chain (Pseudomonas a eruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein Protein, dianthin protein, Phytolaca americana protein (P API, PAPII, and PAP-S), Momordica charantia Antibiotic, curcin, crotin, Sapaonaria officina LIS inhibitors, gelonin, mitogellin, restrictocin estrictocin), phenomycin, enomycin ( enomycin) and trichothecenes.

[0092] In certain embodiments, the agent is a radiosensitizer. 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 proteins include cisplatin, These include 5-fluorouracil (5-FU), AZD7762, and selumetinib. do.

[0093] In certain embodiments, the agent may be a therapeutic agent, e.g., a therapeutic agent and / or a detection agent (e.g., imaging agent). It is a radioisotope useful for immunohistochemistry (immunotherapy). It can be conjugated to an antibody or fusion protein. Non-limiting examples of radioisotopes 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, 1 65 Dy, 166 Dy, 169 Er, 18 F, 19 F, 52Fe 59 Fe 66 Ga 67 Ga 68 Ga 72 Ga 154-158 God 157 God 159 God 166 Ho 120 I 121 I 123 I 124 I 125 I 131 I 110 in 1 11 in 113m in 194 Ir 81m Cr 177 sun 51 Mn 52 Mn 99 For 13 N 15 N 15 Or 17 Or 32 Q 33 Q 211 Pb、 212 Pb 、 109 pd 149 PM 151 PM 142 Mr. 143 Mr. 191 PT. 193 m PT. 195m Pt. 223 Ra 142 Rb、 186 Re 188 Re 189 Re 、 105 Rh、 47 sc、 75 See 153 Sm、 117m Sn 121 Sn 83 Sr 、 89 Mr. 161 Tb 94 Tc99 Tc, 99m Tc, 227 Th, 201 Tl, 172 Tm, 127 Te, 90 Y, 169 Yb, 175 Yb, 133 X and 89 Zr These include, but are not limited to:

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

[0095] According to some embodiments, the radioisotope is a therapeutic radioisotope. In the form, the radioisotope may be an alpha-emitting radioisotope, e.g.,225 Ac, 211 A t, 212 Bi / 212 Pb, 213 Bi, 223 Ra, or 227 Th. Other In embodiments, the radioisotope is a beta-negative emitting radioisotope, e.g., 32 P, 3 3 P, 67 Cu, 90 Y, 131 I or 177 This is Lu.

[0096] According to some embodiments, the agent is a labeling agent. "Label" refers to the antibody or fusion protein that is intended for its intended use (e.g., in vitro and / or in vivo). The agent is an anti-cancer drug, so that it can be detected in in vivo studies and / or clinical applications. This means that the antibody or fusion protein is detectably labeled. The detectable label of interest is , a radioisotope (e.g., a gamma or positron emitter), an enzyme that produces a detectable product (e.g., horseradish peroxidase, alkaline phosphatase, lucifera In certain embodiments, antibodies or fusion proteins include: The protein is conjugated to a specific binding partner of the detectable label (e.g. , so that detection can occur via a detectable label, including avidin / streptavidin. , conjugated to biotin).

[0097] According to certain embodiments, the agent is capable of near-infrared (NIR) optical imaging, single photon emission Spectral computed tomography (SPECT) ± CT imaging, positron emission tomography (PE T) ± in vivo imaging such as CT imaging and nuclear magnetic resonance (NMR) spectroscopy Labeling agents that find use in such applications include fluorescent These include, but are not limited to, optical labels, radioisotopes, and the like. The imaging agent is a multi-modality imaging agent that allows in vivo imaging using two or more imaging modalities. and thrombin-modal in vivo imaging agents (e.g., Thorp-Greenwood, and Coogan(2011)Dalton Trans.40:6129-614 See 3).

[0098] In certain embodiments, the labeling agents find use in near-infrared (NIR) imaging applications. Such agents include Kodak X-SI GHT dye, Pz 247, DyLight750 and 800Fluors, Cy5. 5 and 7 Fluors, Alexa Fluor 680 and 750 Dyes, IRD Examples include, but are not limited to, ye680 and 800CW Fluors. According to some embodiments, the labeling agent finds use in SPECT imaging applications. Non-limiting examples of in vivo imaging agents include: 99m Tc, 111 In , 123 I, 201 T1, and 133 In certain embodiments, the labeling agent is suitable for PET imaging applications, e.g. 11 C. 13 N, 15 O. 18 F, 64 Cu, 6 2 Cu, 124 I, 76Br, 82 Rb, 68 In vivo applications have been found in Ga etc. It is a visualizing agent.

[0099] To extend half-life, the antibodies and fusion proteins of the present disclosure have improved pharmacokinetics. The drug may be conjugated to a drug that provides a specific profile (e.g., PEGylated, highly glycosylated, Modifications that can extend serum half-life are of interest. The antibody or fusion protein of Suitable methods for PEGylation of proteins include: Reagents are well known in the art and can be found, for example, in U.S. Pat. No. 5,849,860. PEG suitable for conjugation to proteins is generally water-soluble at room temperature. and has the general formula R(O-CH-CH) n OR, where R is hydrogen or alkane. R is a protecting group such as an alkyl group or an alkanol group, and n is an integer of 1 to 1000. When is a protecting group, it generally has 1 to 8 carbons. The PEG conjugated to the protein can be linear. The PEG conjugated to the substrate can also be branched. Branched PEG derivatives such as those described in No. 2002 / 002222, "star PEGs," and multi-arm PEGs Star PEGs have been described in the art, including, for example, U.S. Pat. No. 6,046,305. It is explained as follows.

[0100] If the antibody or fusion protein of interest is isolated from a source, the antibody or fusion protein The protein may be a member of a specific binding pair, such as biotin (biotin-avidin specific binding pair). members of the genus β-glucan, which may be conjugated to one or more moieties that facilitate purification, such as lectins. Antibodies can also be attached to polystyrene plates or beads, magnetic beads, test strips, membranes, etc. and the like. can.

[0101] If the antibody or fusion protein is detected in the assay, the antibody or fusion protein , a detectable label, such as a radioisotope (e.g., 89 Zr, 111 In, etc.), detection Enzymes that produce soluble products (e.g., luciferase, β-galactosidase, horseradish peroxidase, Dish peroxidase, alkaline phosphatase, etc.), fluorescent proteins, chromogenic Proteins, dyes (e.g., fluorescein isothiocyanate, rhodamine, phycoerythrin, etc.), Fluorescent compounds linked to proteins via metal chelating groups such as EDTA; Metals, e.g. 152 Eu or other lanthanides; chemiluminescent compounds, e.g., luminol , isoluminol, acridinium salts, etc.; bioluminescent compounds, e.g., luciferin; Indirect labels include antibodies specific to the protein of interest. Rarely, antibodies are coupled to secondary antibodies and members of specific binding pairs, e.g., biotin-avidin. can be detected via

[0102] Any of the above agents may be conjugated to an antibody or fusion protein via a linker. If present, the linker molecule may be a linker molecule between the antibody or fusion protein and the linking agent ( The antibody or fusion protein and the linked agent are The linker molecule may be, for example, about 6 to 100 nm long. The linker molecule may also be, for example, allylacetylene, 2-10 Ethylene glycol oligomers containing 1 monomer unit, diamines, diacids, amino acids, or can be a combination thereof.

[0103] When the linker is a peptide, the linker may be a peptide consisting of 1 amino acid (e.g., Gly) to 20 amino acids. amino acids or more, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids (4 amino acids to 10 Amino acids, 5-9 amino acids, 6-8 amino acids, or 7-8 amino acids The amino acids may be of any suitable length, such as 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, 26, 27, 28, 29, 30, 31, , 5, 6, or 7 amino acids.

[0104] The flexible linker is a glycine polymer (G) n , glycine-serine polymer, glycine alanine-alanine polymers, alanine-serine polymers, and those known in the art Other flexible linkers include glycine and glycine-serine polymers, which are relatively flexible. Unsynthesized amino acids may be used if desired, and neutral tethers between components may be used. Those skilled in the art will appreciate that antibodies or fusion tags conjugated to any of the above agents can function as The protein design may include a fully or partially flexible linker, thereby allowing the linker It is recognized that the may include a flexible linker and one or more moieties that impart a less flexible structure. You will recognize it.

[0105] According to some embodiments, the antibody or fusion protein comprises a non-cleavable linker. Contemplated non-cleavable linkers include, but are not limited to: However, examples of thioether linkers that can be used include: Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate A rate (SMCC) linker is included.

[0106] In certain embodiments, the antibody is conjugated to the agent via a cleavable linker. According to some embodiments, the linker is soluble in water at neutral pH (bloodstream pH 7.3-7.5). It is stable but does not penetrate the weakly acidic endosomes (pH 5.0-6) of target cells (e.g., cancer cells). 5) and undergoes acid cleavage upon internalization into lysosomes (pH 4.5-5.0) Chemically labile linkers, such as the linkers These include hydrazone linkers, oxime linkers, carbonate linkers, and ester linkers. In certain embodiments, the linker It is stable in the bloodstream, but can be released into the bloodstream, for example, by the lysosomes of target cells (e.g., cancer cells). Internalization into target cells by endosomal proteases (such as cathepsins or plasmin) The linker may be an enzyme-labile linker, such as an enzyme-labile linker that undergoes enzymatic cleavage upon administration. Enzyme-labile linkers include linkers containing peptide bonds, e.g., dipeptide-based linkers, such as valine-citrulline (VC) linkers, such as maleimidocaproy Lu-valine-citrulline-p-aminobenzyl (MC-vc-PAB) linker, valyl -alanyl-para-aminobenzyloxy (Val-Ala-PAB) linker. Examples include, but are not limited to, chemically labile linkers, enzyme-labile linkers and non-cleavable linkers are known, e.g., see Ducry & Stump (20 10)Bioconjugate Chem.21:5-13;Nolting,B.2 013)Methods Mol Biol.1045:71-100;Tsuchik ama and An(2018)Protein&Cell 9(1):33-46 It is described in detail in.

[0107] The agent is linked to the antibody or fusion protein directly or indirectly via a linker. Many strategies are available for this purpose. For example, drugs can be linked by covalently attaching a linker to the drug. The linker may be derivatized by adding a "chemical group" to the antibody or fusion protein. The functional groups on the linker can vary. The chemical handle may be selected based on its compatibility with the chemical handle on the antibody or fusion protein. According to embodiments, the chemical handle on the antibody or fusion protein comprises a chemical handle The amino acid sequence is provided by incorporating an unnatural amino acid having the desired structure into the antibody or fusion protein. Unnatural amino acids that find use in preparing the conjugates of the present disclosure include: Zides, alkynes, alkenes, aminooxys, hydrazines, aldehydes (e.g., formyl Glycine, e.g., SMART from Catalent Pharma Solutions ag™ technology), nitrones, nitrile oxides, cyclopropenes, norbornenes, having a functional group selected from a socianilide, an aryl halide, and a boronic acid functional group; Non-natural amino acids (such as methylamino acids) that can be incorporated into the antibodies of the conjugates of the present disclosure include: Non-natural amino acids (which may be selected to provide desired functional groups) are known, e.g. For example, Maza et al. (2015) Bioconjug. Chem. 26(9) :1884-9;Patterson et al.(2014)ACS Chem.B iol.9:592-605;Adumeau et al.(2016)Mol.Im Aging Biol. (2): 153-65. The amino acids can be synthesized, for example, via chemical synthesis or recombinant techniques, for example, by the synthesis of an antibody in a host cell. Orthogonal amino acids suitable for incorporating unnatural amino acids during translation of an enzyme or fusion protein Incorporating silyl-tRNA synthetase-tRNA pairs into antibodies or fusion proteins It can be done.

[0108] The functional groups of the unnatural amino acids present in the antibody or fusion protein may be azide, alkyne, , alkene, aminooxy, hydrazine, aldehyde, asaldehyde yde), nitrone, nitrile oxide, cyclopropene, norbornene, isocyanide , aryl halides, boronic acids, diazo, tetrazine, tetrazole, quadrocycline The compound may have a quadrocyclane, iodobenzene, or other suitable functional group. Often, a functional group on the linker is selected to react with a functional group on the unnatural amino acid (reverse As just one example, an azide-containing unnatural amino acid (e.g., 5- azido-L-norvaline) may be incorporated into the antibody or fusion protein, and the linker The linker portion of the drug moiety is such that the antibody or fusion protein and the linker-drug moiety are linked together. Containing an alkyne functionality, such that it can be covalently conjugated via dihydro-alkyne cycloaddition Conjugation can be achieved, for example, using a copper-catalyzed azide-alkyne cycloaddition reaction. This may be implemented using

[0109] In certain embodiments, the chemical handle on the antibody or fusion protein is a non-natural amino acid. Antibodies that do not contain unnatural amino acids may be prepared without reactive leaving groups or other electrophilic groups. as a nucleophile in a substitution reaction with a moiety having, for example, an antibody or fusion protein nucleophilic functional groups (e.g., the N-terminal amine or the primary amine of lysine, or any other By utilizing a nucleophilic amino acid residue, it can be conjugated to a drug. For example, a drug-linker moiety having an N-hydroxysuccinimidyl (NHS) ester and then prepared under aqueous conditions at high pH (approximately 10) or with N,N-diisopropylethylamine. In a polar organic solvent such as DMSO to which a non-nucleophilic base is added, the drug-linker moiety is Reacting with an antibody or fusion protein.

[0110] Linkers, specific molecules for linking drugs and / or antibodies or fusion proteins to each other Certain approaches involve the use of specific linkers, drugs and / or antibodies or fusion proteins, and functional groups selected and used to conjugate the various components to one another. It will be understood that this may vary depending on the application.

[0111] Methods for producing antibodies Using the information provided herein, the anti-VV A56 and anti-VV B5 antibodies of the present disclosure can be used to Antibodies and fusion proteins can be prepared using standard techniques well known to those skilled in the art. For example, a nucleic acid sequence encoding the amino acid sequence of an antibody or fusion protein of the disclosure can be used. The polypeptides provided herein can be used to express antibodies or fusion proteins. The peptide sequences (see, e.g., Tables 1, 3, 5-11) can be used to generate antibodies or fusion proteins. The appropriate nucleic acid sequence encoding the protein can then be determined, and the nucleic acid sequence can then be used to and administering one or more antibodies or fusion proteins specific for VV A56 or VV B5. The nucleic acid sequences can be expressed in a variety of expression systems according to standard methods well known to those skilled in the art. It can be optimized to reflect the particular codon "preferences" of the system. Using this information, nucleic acids can be synthesized according to several standard methods known to those skilled in the art. .

[0112] Once nucleic acid encoding the antibody of interest has been synthesized, it can be amplified and purified according to standard methods. Molecular cloning to achieve these goals can be used to identify and / or clone specific molecules. A wide variety of clones suitable for the construction of recombinant nucleic acids are known in the art. Cloning and in vitro amplification methods are known to those skilled in the art and are described in numerous textbooks and publications. This is the subject of the laboratory manual.

[0113] Expression of natural or synthetic nucleic acids encoding the antibodies and fusion proteins of the disclosure can be used to produce antibodies. Alternatively, the nucleic acid encoding the fusion protein may be placed under a promoter (either constitutive or inducible). ) and incorporating the construct into an expression vector to generate a recombinant expression vector. This can be achieved by using vectors that are prokaryotic, eukaryotic, or both. Typical cloning vectors can be suitable for replication and integration in mammals. and functionally oriented transcription and translation terminators useful for regulating the expression of nucleic acids that encode them. A vector contains at least one independent gene, an initiation sequence, and a promoter. termination sequences, such as those found in shuttle vectors, which are used in both eukaryotes and prokaryotes. It contains sequences that allow replication of the vector, and selectable markers for both prokaryotic and eukaryotic systems. Optionally, it contains a general expression cassette having the following structure:

[0114] To obtain high levels of expression of a cloned nucleic acid, a strong promoter that directs transcription is usually required. a suitable promoter, 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-encoding sequence. It is common to construct an expression plasmid containing the gene. Examples of suitable regulatory regions include the promoter and and operator region, the left-facing promoter of phage lambda (P L ), and L-ara The DNA used to transform E. coli is the araBAD operon. It is also useful to include selectable markers in the vector. Examples of such markers include: Genes specifying resistance to ampicillin, tetracycline, or chloramphenicol Expression systems for expressing antibodies include, for example, E. coli, Bacillus Us species and Salmonella are available. E. coli systems are also available. May be used.

[0115] Antibody genes may also be used to identify C- or N-terminal fragments of antibodies (e.g., IgG, Fab, scFv, etc.). Expression vectors that allow for the addition of tags at the termini (e.g., FLAG, hexahistidine, etc.) The gene can be transfected and subcloned into mammalian cells. Methods for expression are known in the art. For example, incubating nucleic acid-containing lipid microparticles with cells, or This may involve incubating the viral vector with cells within the vector's host range. Cell lines and cultured cells from tissue (e.g., tumor) or blood samples are included in the present disclosure. The cultivation of such cells is well known in the art.

[0116] Once nucleic acid encoding an antibody of interest has been isolated and cloned, it can be synthesized using techniques known to those skilled in the art. The nucleic acids can be expressed in a variety of recombinantly engineered cells. Examples of vectors include bacteria, yeast, filamentous fungi, insects (e.g., using baculovirus vectors), and mammalian cells.

[0117] Isolation and purification of the subject antibodies can be accomplished according to methods known in the art. For example, proteins can be expressed constitutively and / or upon induction. from lysates of cells genetically engineered to Purification (or precipitation using protein L or A) to remove non-specifically bound material The antibody can be isolated by washing to separate the bound antibody and eluting the specifically bound antibody. The isolated antibodies can be further purified by dialysis and other methods commonly used in protein purification methods. In one embodiment, the antibody can be purified to a high purity by metal chelate chromatography. The antibodies of the present disclosure may be isolated using a method to facilitate isolation, as discussed above. The invention may include specific modifications for the purpose of

[0118] Antibodies may be used in substantially pure or isolated form (e.g., free from other polypeptides). The protein may be prepared without other components that may be present (e.g., other a composition enriched in the polypeptide relative to other polypeptides or other host cell components Purified antibodies can be present in compositions in which the antibody is substantially free of other expressed proteins. a composition that does not contain, for example, less than 90%, usually less than 60%, more usually less than 50% of the composition Less than 100% of the total expressed protein may be provided.

[0119] Antibodies produced by prokaryotic cells require exposure to chaotropic agents for proper folding. For example, during purification from E. coli, the expressed protein The substance can be optionally denatured and then regenerated, e.g., by bacteria. By solubilizing the produced antibodies in a chaotropic agent such as guanidine HCl This can then be achieved by either slow dialysis or gel filtration. Alternatively, the nucleic acid encoding the antibody can be used to refold the antibody in its correctly folded form. operably linked to a secretion signal sequence such as pelB for secretion into the periplasm It can be done.

[0120] The present disclosure also provides cells that produce the antibodies of the present disclosure. Suitable cells are eukaryotic cells, such as For example, mammalian cells may be used to produce antibodies (e.g., monoclonal antibodies such as IgG) in vitro. hybrid cells or "hybridomas" capable of replicating the antibody For example, the present disclosure provides nucleotide sequences encoding the heavy and / or light chains of the antibodies of the present disclosure. A recombinant host cell (referred to herein as a "genetically modified" or "also known ... The present invention provides a host cell (also referred to as a "transformed host cell").

[0121] Recombinant DNA versions of the antigen-binding region of antibody molecules that bypass hybridoma generation Techniques for making such modifications are also contemplated herein. phage), yeast (e.g., Saccharomyces or Pichia ), insect or mammalian expression systems. The resulting antibody (e.g., Fab or scFv) is transported into the periplasmic space (the bacterial cell membrane and cell wall). Bacteriophages with leader sequences that allow the transport or secretion of Use a vector system to generate multiple functional fragments (e.g., For example, Fab or scFv) can be rapidly generated.

[0122] Antibodies that specifically bind to VV A56 and VV B5 are available from hybridoma, recombinant, Phage display technology, selected lymphocyte antibody method (SLAM) (1), or a combination of these can be prepared using a variety of techniques known in the art, including the use of combinations of For example, antibodies can be generated and isolated using phage display methods. Phage display can be used for high-throughput screening of protein interactions. Phages are used to generate repertoire or combinatorial antibody libraries. used to display antigen-binding domains expressed from a target cell (e.g., human or mouse). Phages expressing antigen-binding domains that bind to VV A56 or VV B5 can be used. VV A56 or VV B5 can be used to bind or bind to, for example, a solid surface or beads. can be selected or identified using captured labeled VV A56 or VV B5. The phage used in these methods typically contain phage gene III or Fab, Fv (light or heavy chain) recombinantly fused to either gene VIII protein individual Fv regions from phage) or disulfide-stabilized Fv antibody domains It is a filamentous phage containing the expressed fd and M13 binding domains. Combinatorial infection and in vivo recombination as a strategy for constructing DNA libraries Similarly, the production of high affinity human antibodies by chain shuffling is known. In terms of form, ribosome display has been used in conjunction with bacterioglobulin as a display platform. The cell surface library can be used to replace the phage. Such procedures may be used to isolate and subsequently screen monoclonal antibodies. This provides an alternative to traditional hybridoma techniques for cloning of

[0123] After phage selection, the antibody coding region from the phage is isolated and used to generate human antibodies or any desired antibody. can be used to generate whole antibodies, including antigen-binding fragments, of mammalian cells, insect cells, plant cells, The Fv, s and s sequences can be expressed in any desired host, including mammalian cells, yeast, and bacteria. Techniques for recombinant production of cFv, Fab, F(ab')2, and Fab' fragments are well known in the art. It can be applied using methods known in the art.

[0124] Nucleic acids, expression vectors and cells Considering the section above regarding methods for producing the antibodies and fusion proteins of the present disclosure, Thus, it will be understood that the present disclosure also provides nucleic acids, expression vectors and cells. .

[0125] In certain embodiments, provided are anti-VV A56 and anti-VV B5 antibodies of the present disclosure. Any of the antibodies of the present disclosure, including any of the antibodies described above, or the variable heavy chain (V H ) polypeptide, variable light chain (V L ) Polypeptide In some embodiments, the antibody is a nucleic acid encoding a single chain antibody. The nucleic acid encodes a single-chain antibody (e.g., scFv).

[0126] According to some embodiments, the anti-VV A56 antibodies A047 / A057, A04 9 / A059 / A056, A050 or A054 variable heavy chain (V H ) Polypeptide, Possible Variable light chain (V L Nucleic acids encoding the α- and β-glucan polypeptides, or both, are provided. Examples of suitable nucleotide sequences are shown below in Table 12. Framework Regions and CDRs The coding sequences are shown in upper and lower case, respectively. [Table 12-1] [Table 12-2]

[0127] According to some embodiments, the anti-VV B5 antibodies A048 / A058 / A07 The variable heavy chain (V H ) polypeptide, variable light chain (V L) Polypeptides, or Nucleic acids encoding either or both are provided. Examples of such nucleotide sequences are The sequences encoding the framework regions and CDRs are shown in Table 13. It is shown in lowercase and uppercase. [Table 13]

[0128] According to some embodiments, provided is a CAR of the disclosure, e.g., an antibody of the disclosure. VV A56 or anti-VV B5 antibodies H Polypeptides and V L Contains polypeptides Encoding a CAR comprising a single chain antibody, a transmembrane domain, and an intracellular signaling domain. Such single chain antibodies, transmembrane domains and intracellular signaling domains are nucleic acids. Examples of ins are described in detail above.

[0129] An exemplary nucleotide sequence encoding A56-CAR-01 described herein is Column number 155. An example of coding for A56-CAR-02 described herein. An exemplary nucleotide sequence is set forth in SEQ ID NO: 156. A56 as described herein -An exemplary nucleotide sequence encoding CAR-05 is set forth in SEQ ID NO: 157. An exemplary nucleotide sequence encoding A56-CAR-06 described herein is , set forth in SEQ ID NO: 158. The A56-CAR-07 gene encoding the A56-CAR-07 gene described herein An exemplary nucleotide sequence is set forth in SEQ ID NO: 159. An exemplary nucleotide sequence encoding 56-CAR-08 is set forth in SEQ ID NO: 160. Exemplary nucleotide sequences encoding A56-CAR-010 described herein The sequence is set forth in SEQ ID NO: 161. A56-CAR-020 as described herein An exemplary encoding nucleotide sequence is set forth in SEQ ID NO: 162. An exemplary nucleotide sequence encoding the described A56-CAR-021 is SEQ ID NO: 16 3. Exemplary nucleic acids encoding A56-CAR-027 described herein are The nucleotide sequence is set forth in SEQ ID NO: 164. A56-CAR as described herein An exemplary nucleotide sequence encoding -028 is set forth in SEQ ID NO:165. An exemplary nucleotide sequence encoding A56-CAR-029 described herein is Column number 166. Encoding A56-CAR-030 described herein An exemplary nucleotide sequence is set forth in SEQ ID NO: 167. B5 as described herein -An exemplary nucleotide sequence encoding CAR-03 is set forth in SEQ ID NO: 168. An exemplary nucleotide sequence encoding B5-CAR-04 described herein is: The sequence encoding B5-CAR-011 described herein is set forth in SEQ ID NO: 169. An exemplary nucleotide sequence is set forth in SEQ ID NO: 170. B5 as described herein - An exemplary nucleotide sequence encoding CAR-013 is set forth in SEQ ID NO: 171. Exemplary nucleotide sequences encoding B5-CAR-014 described herein is set forth in SEQ ID NO: 172. The B5-CAR-016 coding sequence described herein is An exemplary nucleotide sequence for this purpose is set forth in SEQ ID NO: 173. An exemplary nucleotide sequence encoding B5-CAR-019 is set forth in SEQ ID NO: 174. Exemplary nucleotide sequences encoding B5-CAR-022 described herein The sequence is set forth in SEQ ID NO: 175. Encoding the murine CAR shown in Table 11 Examples of nucleotide sequences are set forth in SEQ ID NOs: 176-179.

[0130] Also provided are expression vectors comprising any of the nucleic acids of the present disclosure. Expression of a natural or synthetic nucleic acid encoding a fusion protein results in the production of the antibody or fusion protein. The nucleic acid encoding the protein is operably linked to a promoter (either constitutive or inducible). and incorporating the construct into an expression vector to generate a recombinant expression vector. Vectors can be capable of replication and integration in prokaryotes, eukaryotes, or both. Typical cloning vectors may be suitable only for the expression of nucleic acids encoding antibodies. Functionally appropriate oriented transcription and translation terminators, initiation sequences, and and a promoter. The vector contains at least one independent termination sequence, e.g. Allows replication of cassettes in both eukaryotes and prokaryotes, as found in shuttle vectors A general expression vector containing sequences for the expression of ribosomal proteins and selectable markers for both prokaryotic and eukaryotic systems. Optionally contains a current cassette.

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

[0132] A method for producing an antibody or fusion protein of the disclosure, comprising: and culturing the cells of the present disclosure under conditions suitable for expressing the antigen-binding protein, wherein the antigen-binding protein is expressed. Also provided are methods by which antibodies or fusion proteins are produced. The conditions for culturing cells to induce expression may vary. In a container (e.g., a cell culture plate or well thereof), a suitable medium (e.g., DME Suitable for cell culture media such as M, RPMI, MEM, IMDM, DMEM / F-12, etc. 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 a suitable CO2 percentage, e.g. 3% to 10% ( The method may include culturing the cells in an environment having a pH of 1.5, such as 5%.

[0133] Chimeric antigen receptors that bind to oncolytic virus antigens Chimeric antigen receptors (CAs) of the present disclosure, as described in the Fusion Proteins section above, In addition to R), embodiments of the present disclosure further include anti-oncolytic viral antigen CARs. In embodiments, such CARs specifically bind to oncolytic virus (OV) antigens. It comprises an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. Such CARs find a variety of uses. For example, with the benefit of this disclosure, one may The CAR involves administering a pharmaceutical composition containing an anti-OV antigen CAR to an individual with cancer. The present invention finds use in methods for treating cancer in an individual, wherein cancer cells in the individual are infected with OV and express OV antigens on their surface. By expressing the CAR, the CAR can be targeted to cancer cells to treat cancer in individuals. It will be understood.

[0134] As used herein, an "oncolytic viral antigen" or "OV antigen" refers to an antigen that is present in a tumor An antigen encoded by the genome of a lytic virus (OV). In this case, the OV antigen is a native OV antigen, and "native" means that the antigen is found in the wild type of OV. This means that the protein is encoded by the type genome.

[0135] In some embodiments, the OV antigen is from the family Poxviridae, Herpesvir idae, Adenoviridae, Paramyxoviridae, Rha Family bdoviridae, family Reoviridae, family Picornaviridae, Wild-type viruses of the Parvoviridae or Coronaviridae families Such OV antigens are native to OV. The species may be heterologous or heterologous.

[0136] In particular embodiments, the OV antigens are viruses from the families listed in Table 14 below. The antigens encoded by the wild-type genome of the strain are listed in Table 14. Further non-limiting examples of viral species / strains, as well as non-limiting examples of such antigens, include: . [Table 14]

[0137] According to some embodiments, the OV antigen is Amsacta moorei entomopoietin. entomopoxvirus, Chironomus luridus, chicken smallpox virus, Melolontha melolontha entomopox virus, Molluscum contagiosum virus, mule deer pox virus, mucus Variola virus, Nile crocodile pox virus, orf virus, sheep pox virus, swinepox virus Virus, vaccinia virus, Yaba monkey tumor virus, Yokapox Virus, Acrobasis zelleri Entomopoxvirus, Adoxo phyes honmai entomopoxvirus, Aedes aegypti Tomopoxvirus, Anomala cupreaEntomopoxvirus, Ap hodius tasmaniae entomopoxvirus, Arphia cons persa entomopoxvirus, bovine papular stomatitis virus, camelpox virus, C Amputochironomus tentans entomopoxvirus, canary smallpox virus, Chironomus attenuatus entomopox virus, Chironomus plumosus entomopoxvirus, Choristo neura biennis entomopoxvirus, Choristoneura conflicta entomopoxvirus, Choristoneura dive rsuma entomopoxvirus, Choristoneura fumifera Entomopoxvirus, Choristoneura rosaceana Tomopoxvirus, Chorizagrotis auxiliaris entomopox Cowpox virus, cowpox virus, Demodex bonariensis entomopox Entomopoxvirus, Dermolepida albohirtum Rus, Diachasmimorpha entomopoxvirus, Ectromelia virus Rus, Figulus sublaevis Entomopoxvirus, Geotrup es sylvaticus entomopoxvirus, goatpox virus, Goeldi chironomus holoprasinus entomopoxvirus, wild rabbit Fibroma virus, Heliothis armigera entomopox virus, Juncopox virus, Locusta migratoria insect pox Lumpy skin disease virus, Melanoplus sanguine ipes entomopox virus, monkeypox virus, mynah pox) virus, Mythimna separata entomopox virus, O edaleus senegalensis entomopoxvirus, Operoph tera brumata Entomopoxvirus, New Zealand red deer Par apoxvirus, pigeonpox virus, pseudocowpox virus, Psittacinepox virus Virus, Pteropox virus, Quailpox virus, Rabbit line Fibromyalgia virus, raccoonpox virus, Schistocerca gregaria Ntomopoxvirus, skunkpox virus, sparrowpox virus, squirrel fibroma virus , squirrelpox virus, starlingpox virus, variola virus, varroapox virus ( Taterapox virus, turkeypox virus, variola virus, and haematopox virus Wild-type genomes of viruses in the Poxviridae family selected from ratpox virus Such OV antigens may be native or allogeneic to the OV. It can be a seed.

[0138] In a specific embodiment, the OV antigen is avian alphaherpesvirus type 1, Oum alphaherpesvirus. Alphaherpesvirus type 1, avian alphaherpesvirus type 2, geese and duck alphaherpesvirus rupesvirus type 1, pigeon alphaherpesvirus type 1, avian alphaherpesvirus alphaherpesvirus type 3, turkey alphaherpesvirus type 1, sea turtle alphaherpesvirus Herpesvirus type 5, human alphaherpesvirus type 1, spider monkey alphaherpesvirus type 1 , bovine alphaherpesvirus type 2, long-tailed macaque alphaherpesvirus type 2, human Alphaherpesvirus type 2, rabbit alphaherpesvirus type 4, and macacin cacine) Alphaherpesvirus type 1, Kangaroo (Macropod) Rufa herpesvirus type 1, Kangaroo alphaherpesvirus type 2, Panine Alphaherpesvirus type 3, baboon alphaherpesvirus type 2, squirrel monkey alphaherpesvirus Herpesvirus type 1, human beta-herpesvirus type 5, monkey beta-herpesvirus betaherpesvirus type 1, capuchin monkey betaherpesvirus type 1, African green monkey betaherpesvirus type 1 beta herpesvirus type 5, beta herpesvirus type 3, panine beta herpesvirus pesvirus type 2, baboon betaherpesvirus type 3, squirrel monkey betaherpesvirus 4, murine betaherpesvirus type 1, murine betaherpesvirus type 2, murine Betaherpesvirus type 8, human betaherpesvirus type 6A, human betaherpes Virus type 6B, human betaherpesvirus type 7, elephant betaherpesvirus type 1, Human gammaherpesvirus type 4, marmoset gammaherpesvirus type 3, long-tailed thrush Gamma herpesvirus type 14, gorilla gamma herpesvirus type 1, Macacin gamma Herpesvirus type 4, panin gamma herpesvirus type 1, hihi gamma herpesvirus gammaherpesvirus type 1, orangutan gammaherpesvirus type 2, squirrel monkey gammaherpesvirus 2, spider monkey gammaherpesvirus type 2, spider monkey gammaherpesvirus type 3, Cynomolgus monkey gammaherpesvirus type 4, Cricetida gammaherpesvirus type 2, human gamma Herpesvirus type 8, Macacine gamma herpesvirus type 5, murine gamma herpesvirus virus type 4, murine gammaherpesvirus type 7, hartebeest gammaherpesvirus 1, hartebeest gammaherpesvirus type 2, bovine gammaherpesvirus type 6 , Caprine gammaherpesvirus type 2, Bluebuck gammaherpesvirus type 1, Sheep Gammaherpesvirus type 2, porcine gammaherpesvirus type 3, porcine gammaherpesvirus Virus type 4, porcine gammaherpesvirus type 5, equine gammaherpesvirus type 2, equine Selected from gammaherpesvirus type 5 and weasel gammaherpesvirus type 1 Antigens encoded by the wild-type genome of viruses in the Herpesviridae family Such OV antigens can be native or heterologous to the OV.

[0139] According to some embodiments, the OV antigen is chicken avian adenovirus A, frogs Adenovirus A, human mastadenovirus C, ovine adenovirus D, sturgeon Ichtadenovirus A, bat mastadenovirus Rus A, bat mastadenovirus B, bat mastadenovirus C, bat Mastadenovirus D, bat mastadenovirus E, bat mastadenovirus Rus F, bat mastadenovirus G, bovine mastadenovirus D, bovine mastadenovirus Virus A, Bovine Mastadenovirus B, Bovine Mastadenovirus C, Canine Mastade Adenovirus A, Deer Adenovirus A, Deer Mast Adenovirus B, Dolphin Mast Adenovirus A, dolphin mastadenovirus B, duck adenovirus A, duck Avian adenovirus B, equine steatomatomatoma virus A, equine steatomatoma virus B, Yersinia aviadenovirus A, Chicken aviadenovirus B, Chicken aviadenoui Rus C, Chicken Avian Adenovirus D, Chicken Avian Adenovirus E, Goose Loon Adenovirus A, tit adenovirus A, human mast adenovirus A, human Tomato adenovirus B, human mastadenovirus D, human mastadenovirus E , Human mastadenovirus F, Human mastadenovirus G, Lizard atadenovirus A, Murine mastadenovirus A, Murine mastadenovirus B, Murine mastade Ovine mastadenovirus C, ovine mastadenovirus A, ovine mastadenovirus B, penguin Pedigree virus A, pigeon aviadenovirus A, pigeon aviadenovirus B, capuchin Monkey mastadenovirus A, porcine mastadenovirus A, porcine mastadenovirus B, porcine mastadenovirus C, opossum atadenovirus A, parrot atadenovirus Virus A, Parrot Avian Adenovirus B, Raptor Cyadenovirus A, Sea Lion Adenovirus Novirus A, simian adenovirus A, simian adenovirus B, simian adenovirus Adenovirus C, simian adenovirus D, simian adenovirus E, simian adenovirus Stored adenovirus F, simian adenovirus G, simian adenovirus H, Lumbar adenovirus I, Asian skua adenovirus A, Skunk bear adenovirus Virus A, Snake Tadenovirus A, Ristoceratopogon virus A, Tupaimastadenovirus A Novirus A, Turkey Avian Adenovirus B, Turkey Avian Adenovirus Virus C, turkey aviadenovirus D, and turkey aviadenovirus A Encoded by the wild-type genome of a virus of the family Adenoviridae selected from Such OV antigens can be native or heterologous to the OV. .

[0140] In certain embodiments, the OV antigen is Triabravirus 1, Hendra Henipavirus, Measles morbillivirus, mumps rubravirus, murine respirovirus, reptile pheovirus Lulavirus, salmon aquatic paramyxovirus, Achimota rubra Virus type 1, Achimotarbullavirus type 2, Avian avian avian virus type 10, Avian avian virus type 1 Avian arabulavirus type 11, avian arabulavirus type 12, avian arabulavirus type 13, Avian Triabravirus type 14, Avian Triabravirus type 15, Avian Triabravirus avian serotype 16, avian serotype 17, avian serotype 18, avian serotype 19, avian serotype 20, avian serotype 21, avian serotype 22, avian serotype 23, avian serotype 24, avian serotype 25, avian serotype 26, avian sero Avian tryabulavirus type 19, avian tryabulavirus type 2, avian tryabulavirus type 3, avian ...3, avian tryabulavirus type 3, avian try Triavirus type 4, Triavirus type 5, Triavirus type 6, Triavirus type 7 Type, Triabravirus type 8, Triabravirus type 9, Bat mumps rubravirus Bovine respirovirus type 3, canine morbillivirus, cedar henipavirus, cetamol Ammorbillivirus, feline morbillivirus, Ghanaian bat at) Henipavirus, Human Respirovirus Type 1, Human Respirovirus Type 3, Human Rubulavirus type 2, human rubulavirus type 4, mammalian rubulavirus type 5, Mapuera (M apuera rubulavirus, Menangle rubulavirus, Moji Mojiang henipavirus, Nipah henipavirus, seal Morbillivirus, porcine respirovirus type 1, porcine rabies virus, rinderpest virus (R inderpest morbillivirus), monkey rubra virus, small ruminant Small ruminant morbillivirus, Sosuga rubra Virus, Teviot rubulavirus, Tioman rubulavirus, Tuho Tuhoko rubulavirus type 1, Tuhoko rubulavirus type 2, and Tuhoko rubulavirus type 1 Wild-type genomes of Paramyxoviridae viruses selected from Paramyxoviridae type 3 Such OV antigens are native to OV. or may be heterologous.

[0141] According to some embodiments, the OV antigen is bovine fever ephemelovirus, Koisprivi Virus, Cryonopolis curiovi rus), Drosophila melanogaster sigma virus, Durham Tupa virus (D urham tupavirus), Flanders tupavirus, Indiana vesiculo Virus, Le Dantec virus, lettuce big vein-associated varicosaui varicosavirus, lettuce necrotic yellow tic yellows) Cytorhabdovirus, Niakha Sleep virus, Ran Potato mottle virus, Perch varicella virus, Potato yellow dwarf nucleola virus Puerto Almendras Almendra virus, rabies lyssavirus, Puerto Almendras virus, rabies virus, salmonid fish, Nobira grape Irus, Tibrogargantibrovirus, Adelaide River Ephemelovirus, Alagoas Vesiculovirus, Alfalfa dwarf cytorhabdovirus, Almpiwo piwar) sleep virus, American bat vesiculovirus, eel perhappus Virus, Aravan Lyssavirus, Arboretum Almendrau Virus, Australian bat virus, Balsa almendraui Rus, Barley yellow striate mosai c) Mosaic Cytorhabdovirus, Barule Redantevirus, Bas-Congo virus -Congo) Tibro virus, Beatrice Hill Tibro Virus, Berrimah Ephemeral Virus, Bokelo h) Bat lyssavirus, Broccoli necrotic yellow cytorhabdovirus, Karahas Carajas vesiculovirus, Chaco sleep virus, Chande Chandipura vesiculovirus, Coastal Plains al Plains) Tybrovirus, Cocal (Cocal) Vesiculovirus, Coffee Coffee ringspot dichorha virus virus), Colocasia bovonii-associated cytorhabdosigma virus, Kutbei Arme Coot Bay almendravirus, Datura yellow Leaf vein nucleorhabdovirus, Drosophila affinis sigma virus, Drosophila ananassae sigma virus, Drosophila i mmigrans sigma virus, Drosophila obscura sigma virus Drosophila tristis sigma virus, Duvenhaig nhage) Lyssavirus, Eggplant mottled dwarf arf) Nucleorhabdovirus, Ekpoma1 typhoid virus, Ekpoma2 typhoid virus rovirus, European bat 1 lyssavirus, European bat 2 lyssavirus Su, Festuca leaf streak cytorhabdovirus, Fikirini Redante virus, Fukuoka Redante virus, Gannoruwa ) Bat lyssavirus, Gray Lodge hapavirus, Har Hart Park Hapavirus, Hirame Nobirabudoyi Rus, Ikoma lyssavirus, Iriri cryovirus, I Irkut lyssavirus, Isfahan vesiculovirus Rus, Itacaiunas cryovirus, Joinjacaca oinjakaka Hapavirus, Jurona Vesiculovirus, Turtle Kamese Hapa virus, Kanyawara Redante virus , Kern Canyon Redante virus, Keu raliba) Redante virus, Khujand (Khujand) Lyssavirus, Kin Kimberley ephemerovirus, Klamath Tupau Virus, Kolente, Redante virus, Koolpin yah) Ephemeral virus, Kotonkan (Kotonkan) Ephemeral virus, Kumasi Redante virus, La Joya Hapa virus, Lago Lagos bat lyssavirus, Landjia hapavirus, Lettuce yellow mottle Cytorhabdovirus, Li Lleida bat lyssavirus, Maize Iranian mosaic virus ze Iranian mosaic) Nucleorhabdovirus, Maize stripe disease (Maize fine streak) Nucleorhabdovirus, Maize mosaic Nucleorhabdovirus, Malpais Springs Black virus, Manitoba hapavirus, Maraba virus Cyclovirus, Marco Hapavirus, Mokola Lissau virus, Morreton vesiculovirus, Mosqueirovirus ro) Hapavirus, Mossuril Hapavirus, Mount Elgon Bat Redantevirus, Moussa virus, Muscina sta bulans sigma virus, New Jersey vesiculovirus, Nga ingan) Hapavirus, Nishimuro (Nishimuro) Redantevirus, Nucol Nkolbisson Redante virus, Northern wheat mosaic disease Cytorhabdo Virus, Obodhiang ephemelovirus, Oita Redante virus, Ord River Hapa virus, Parry Creek (Parry Creek) Hapavirus, Perinet Vesiculovirus Rus, Pike fry sprivivirus, Pirivesiculovirus, Pi Piscine novirhabdovirus, Radi vesiculovirus, Rice yellow leaf nucleorhabdovirus, Rio Chico almendra virus Rochambeau cryovirus, Sea trout rout) Perhabdovirus, Sena Madureira Thripvirus, Shimoni bat lyssavirus, Snakehead virus Bererhabdovirus, Sonchus cytorhabdovirus 1, Sonchus ye Lawnnet nucleorhabdovirus, Sonchus yellow vein nucleorhabdovirus, Strawberry kudzu Wrinkle Cyt Rhabdovirus, Sweetwater Branch Tibrovirus, Taro Vein Taro vein chlorosis (nucleorhabdovirus), Tupaitupa Virus, West Caucasian bat lyssavirus, wheat American striatal mosaic cytoplasm Budovirus, Wongabel Hapavirus, Uhanredangtaeui Rus, Yata ephemeral virus, Yongjia redanteuil Selected from the vesiculoviruses Rus and Yug Bogdanovac Encoded by the wild-type genomes of selected Rhabdoviridae viruses Such OV antigens can be native or heterologous to the OV.

[0142] In a specific embodiment, the OV antigen is Aedes pseudoscutellaris Reovirus, Aquareovirus type A, Banna virus, Bluetongue (Bluetongue) virus, Colorado tick fever virus, Cypovirus 1, Er iocheir sinensis reovirus, Fiji disease virus, Idonoreoi Rus (Idnoreovirus) 1, mammalian orthoreovirus, Micromona pusilla reovirus, Mycoreovirus 1, Rice ragged stunt virus , rotavirus A, wound tumor virus, African horse sickness virus, aquareovirus B , Aquareovirus C, Aquareovirus D, Aquareovirus E, Aquareovirus Virus F, Aquareovirus G, Avian orthoreovirus, Baboon orthoreovirus, Changuinola virus, Chenuda virus, Chobar Golgi virus, Koripartawi Rus, Cypovirus 10, Cypovirus 11, Cypovirus 12, Cypovirus 13, Cypovirus 14, Cypovirus 15, Cypovirus 16, Cypovirus 2, Cypovirus 3, Cypovirus 4, Cypovirus 5, Cypovirus 6, Cypovirus Cypovirus 7, Cypovirus 8, Cypovirus 9, Echinochloa rugged Stunt virus, epizootic hemorrhagic disease virus, equine encephalopathy virus, eubenangi virus , Eyach virus, garlic dwarf virus, Great Island virus , Idonoreovirus 2, Idonoreovirus 3, Idonoreovirus 4, Idonoreovirus virus 5, Yeri virus, Kadipiro virus, Lebombo virus, Liaoning virus (Li ao ning virus, Mahlapitsi orthoreovirus, Maize rough dwarf virus, Rio Quarto disease (Mal de Rio Cuarto virus, Mycorreovirus 2, Mycorreovirus 3, Nelson virus Nelson Bay Orthoreovirus, Nilaparvata lugen Reovirus, Oat sterile dwarf virus, Orungo o) Virus, Palyam virus, Pangola stunt stunt) virus, Peruvian horse sickness virus, fish orthoreovirus, reptile orthoreovirus Reovirus, Rice black-streaked dwarf virus, Rice dwarf virus, Rice gall dwa rf virus, rotavirus B, rotavirus C, rotavirus D, rotavirus E, Rotavirus F, Rotavirus G, Rotavirus H, Rotavirus I, Southern rice black-streaked dwarf virus IRS, St. Croix River virus, Umatilla virus, Wadme Mite (Wad Medani) virus, Wallal virus, Vale Warrego virus, Wongorr virus, and Yunnan virus Wild viruses of the Reoviridae family selected from the Yunnan orbiviruses Such OV antigens are native to OV. The gene may be native or heterologous.

[0143] According to some embodiments, the OV antigen is selected from the group consisting of Aari virus A, Aichi virus A, Amphivirus A, Aquamavirus A, Abyssivirus A, Bopivirus Virus A, Kadisivirus A, Cardiovirus A, Cosavirus A, Blackfly virus Enterovirus B, Enterovirus C, Erbvirus A, Foot and Mouth Disease Virus, Gallivirus A, Hal Carcinoma virus A, Hepatovirus A, Fungivirus A, Kunsagivirus A, Limnipiu virus A, megrivirus A, micivirus A, mosavirus A, orivirus A, oshivirus A Virus A, Parechovirus A, Pacivirus A, Passerivirus A, Potamipivirus Su A, Labovirus A, Rosavirus A, Sacobuvirus A, Sarivirus A, Saperou Virus A, Senecavirus A, Shambavirus A, Cicinivirus A, Tessiovirus Teschovirus A, Torchivirus A, Tremovirus Virus A, Aichi virus B, Aichi virus C, Aichi virus D, Aichi virus E, Aichivirus F, Tori Sapelovirus, Abyssivirus B, Abyssivirus C, Bovine rhinitis A virus, bovine rhinitis B virus, cardiovirus B, cardiovirus C, Cosavirus B, Cosavirus D, Cosavirus E, Cosavirus F, Kurohivirus A, Enterovirus A, Enterovirus B, Enterovirus D, Enterovirus E, Enterovirus F, Enterovirus G, Enterovirus H, Enterovirus I, Enterovirus J, Enterovirus K, Enterovirus L, Equine rhinitis A virus, Hepatovirus B, Hepatovirus C, Hepatovirus D, Hepatovirus E, Hepatitis Hepatovirus F, Hepatovirus G, Hepatovirus H, Hepatovirus I, Kunsagi virus B, Kunsagi virus C, Limnipi virus B, Limnipi virus C, Megri virus B , Megrivirus C, Megrivirus D, Megrivirus E, Mishivirus B, Mishiwi Rus C, Parechovirus B, Parechovirus C, Parechovirus D, Rhinovirus A, Pico selected from rhinovirus B, rhinovirus C, and sapelovirus B It is an antigen encoded by the wild-type genome of a virus of the Rnaviridae family. OV antigens such as may be native or heterologous to the OV.

[0144] In a specific embodiment, the OV antigen is Adeno-associated Dependoparvovirus A, Carnivore Amdoparvovirus type 1, Decapod Hepandensovirus type 1, Decapod Penstildensovirus type 1, Diptera Brevidensovirus type 1, Galliformes Abeparvo Virus type 1, Lepidoptera ambidensovirus type 1, Lepidoptera iteradensovirus type 1, Spirit Long-legged erythroparvovirus type 1, primate tetraparvovirus type 1, rodent protoparvovirus Parvovirus type 1, ungulate bocaparvovirus type 1, ungulate copiparvovirus type 1, Adenovirus type 1 Dependent virus type B, Anseriformes dependent virus type 1, Aste roid) Ambidensovirus type 1, avian dependent parvovirus type 1, cockroach ( Blattodean Ambidensovirus type 1, cockroach (Blattodean) Ambidensovirus type 2, carnivore Amdoparvovirus type 2, carnivore Amdoparvo virus type 3, carnivore amdoparvovirus type 4, carnivore bocaparvovirus type 1, Carnivore bocaparvovirus type 2, Carnivore bocaparvovirus type 3, Carnivore bocaparvovirus Bocaparvovirus type 4, carnivore bocaparvovirus type 5, carnivore bocaparvovirus type 6, Carnivore protoparvovirus type 1, Chiroptera bocaparvovirus type 1, Chiroptera bocaparvo Virus type 2, Chiroptera Bocaparvovirus type 3, Chiroptera Bocaparvovirus type 4, Chiroptera Dependoparvovirus type 1, Chiroptera protoparvovirus type 1, Chiroptera tetraparvovirus Virus type 1, Decapod Ambidensovirus type 1, Decapod Ambidensovirus type 1, Bilateral Pterygota Ambidensovirus type 1, Diptera Brevidensovirus type 1, Euonymus Protopia Rubovirus type 1, Hemiptera Ambidensovirus type 1, Hemiptera Ambidensovirus type 2 , Hemiptera Ambidensovirus type 3, Hymenoptera Ambidensovirus type 1, , Orthoptera Densovirus Virus Bocaparvovirus type 1, Pinniped Bocaparvovirus type 1, Pinniped Bocaparvo Densoparvovirus type 2, pinniped Densoparvovirus type 1, primate Bocaparvovirus type 1, primate Bocaparvovirus type 2, primate erythroparvovirus type 2, primate erythroparvo erythroparvovirus type 3, primate erythroparvovirus type 4, primate protoparvovirus type 1, Protoparvovirus type 2 in long-legged mammals, protoparvovirus type 3 in primates, and erythroparvovirus type 2 in rodents parvovirus type 1, rodent protoparvovirus type 2, rodent protoparvovirus type 3, Squamate Dependoparvovirus type 1, Ungulate Bocaparvovirus type 2 type, ungulate bocaparvovirus type 3, ungulate bocaparvovirus type 4, ungulate bocaparvo Virus type 5, ungulates Bocaparvovirus type 6, ungulates Copiparvovirus type 2, ungulates Erythroparvovirus type 1, ungulate protoparvovirus type 1, ungulate protoparvo Virus type 2, ungulate tetraparvovirus type 3, and ungulate tetraparvovirus type 4 Encoded by the wild-type genome of a virus of the family Parvoviridae selected from Such OV antigens can be native or heterologous to the OV.

[0145] According to some embodiments, the OV antigen is alphacoronavirus type 1, avian coronavirus Virus, Bulbul coronavirus HKU11, Umatoro virus, Ma Mouse coronavirus, White bream virus, Balloon virus Python nid virus type 1, bat coronavirus CDPHE15, bat coronavirus virus HKU10, Dolphin coronavirus SW1, Betacoronavirus type 1, Bovine Nidovirus type 1, Bovine Torovirus, King Salmon Nidovirus type 1, Ban (Com mon moorhen) Coronavirus HKU21, Coronavirus HKU15, Fa Head minnow virus type 1, hedgehog coronavirus type 1, human coronavirus Rus 229E, Human coronavirus HKU1, Human coronavirus NL63, Human Toro virus, Middle East Respiratory Syndrome-associated coronavirus, Miniopterus bat coronavirus Virus type 1, Miniopterus bat coronavirus HKU8, mink coronavirus Virus type 1, Munia coronavirus type HKU13, Night heron ght heron) coronavirus HKU19, Pipistrellus bat ronavirus HKU5, porcine epidemic diarrhea virus, porcine torovirus, Rhinolop hus bat coronavirus HKU2, Rousettus bat coronavirus H KU9, SARS-CoV-2, Scotophilus bat coronavirus 512, Severe Acute Respiratory Syndrome-Associated Coronavirus, Thrush Coronavirus HKU12, Tylonycteris bat coronavirus HKU4, White Eye -eye) coronavirus HKU16, Wigeon coronavirus HK U20, and SARS-CoV-2 selected from the Coronaviridae family Such OV antigens are antigens encoded by the wild-type genome of the virus. It may be native or heterologous to the host.

[0146] In certain embodiments, the OV antigen is a vaccinia virus (e.g., antigens A33, A34 , A36, A56, B5, F12, F13, etc., for example, modified VV strains, JX-594, G L-ONC1, or Western Reserve, Wyeth, Lister, C openhagen, Temple of Heaven, Patwadangar, and modified vaccinia virus Ankara), adenovirus , HSV, reovirus, vesicular stomatitis virus, Newcastle disease virus, Seneca virus Rea virus, polio virus, measles virus, coxsackie virus, and marabou virus Such OV antigens are antigens encoded by the virus genome. It can be native or heterologous.

[0147] In certain embodiments, the OV antigens are modified versions of native OV antigens (also For example, in some embodiments, the OV antigen is a native antigen. , but which contain the same or substantially similar epitopes as those of the In certain embodiments, e.g., existing anti- The antibody (e.g., an antibody approved for therapeutic use) binds to the modified / engineered OV antigen. OV antigens have different epitopes compared to native antigens so that they do not bind to native antigens. modified / engineered to have a trope.

[0148] According to some embodiments, the genome of the OV is encoded by the wild-type genome of the OV. The recombinant vector may be modified to encode and express one or more proteins that are not otherwise required for the expression of the recombinant vector. Proteins are herein referred to as "foreign" to OV antigens or "not native" to the OV. The OV genome encodes and expresses heterologous OV antigens of interest. In certain embodiments, the heterologous OV antigen may be modified to express the antigen. Viral antigens that are heterologous to the antigen (e.g., of the viruses described elsewhere herein) In some embodiments, the antigen is an antigen encoded by either the wild-type genome. For example, heterologous OV antigens are tumor antigens, non-limiting examples of which include 5T4, AXL receptor tyrosine kinase, Axonase (AXL), B-cell maturation antigen (BCMA), c-MET, C4.4a, carbonate Anhydrase 6 (CA6), carbonic anhydrase 9 (CA9), cadherin-6, CD 19, CD20, CD22, CD25, CD27L, CD30, CD33, CD37, C D44v6, CD56, CD70, CD74, CD79b, CD123, and CD138 Carcinoembryonic antigen (CEA), cKit, Cripto protein, CS1, Delta-like canonica DLL3 (Lystagmus 3), endothelin receptor type B (EDNRB), Ephri Epidermal growth factor receptor (EGFR), EGFRvIII, ectonuclease A4 (EFNA4), epidermal growth factor receptor (EGFR), EGFRvIII, ectonuclease A4 Nucleotide pyrophosphatase / phosphodiesterase 3 (ENPP3), EPH receptor A2 (EPHA2), fibroblast growth factor receptor 2 (FGFR2), fibroblast growth factor receptor 3 (FGFR3), FMS-like tyrosine kinase 3 (FLT3), folate receptor 1 (F OLR1), glycoprotein non-transferase B (GPNMB), guanylate cyclase 2C (GU CY2C), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HE R3), integrin alpha, lysosome-associated membrane protein 1 (LAMP-1), Louis S-Y, LIV-1, leucine-rich repeat containing 15 (LRRC15), mesothelin (M SLN), mucin 1 (MUC1), mucin 16 (MUC16), sodium-dependent phosphate Transport protein 2B (NaPi2b), nectin-4, NMB, NOTCH3, p-cad Herin (p-CAD), prostate-specific membrane antigen (PSMA), protein tyrosine kinase 7 (PTK7), solute carrier family 44 member 4 (SLC44A4), SLIT-like protein Family Member 6 (SLITRK6), STEAP Family Member 1 (STEAP 1), tissue factor (TF), T-cell immunoglobulin and mucin protein-1 (TIM- 1), and trophoblast surface antigen (TROP-2).

[0149] Specific binding to tumor antigens that can be encoded and expressed by modifying the OV genome Antigen-binding domains that may be utilized in the anti-OV CAR or anti-OV conjugates of the present disclosure Main, non-limiting examples include the following antibodies: adecatumumab b), ascrinvacumab, cixutumumab umumab, conatumumab, daratumumab umumab, drozitumab, durigotumab otumab, durvalumab, dusidizumab gitumab), enfortumab, enoticumab ticumab, figitumumab, ganitumab tumab), Glembatumumab, Intetumumab ntetumumab, ipilimumab, iratumumab atumumab, Icrucumab, Lexatumumab tumumab, lucatumumab, mapatumumab tumumab, narnatumab, necitumumab umumab, nesvacumab, ofatumumab mumab, olaritumab, panitumumab umab, Patritumab, Pritumumab ab), Radretumab, Ramucirumab b), Rilotumumab, Robatumumab b), Seribantumab, Tarexuzumab mab), teprotumumab, tovetum ab), vantictumab, besencumab ab), Votumumab, Zalutumumab , Flanvotumab, Altumomab, Anatumomab, Arcitumomab, Bectumomab, Blinatumomab , Detumomab, Ibritumomab, Mi Minretumomab, Mitumomab, Moki Moxetumomab, Naptumomab, Nov Nofetumomab, Pemtumomab, Pimtumomab Pintumomab, Racotumomab, Satsumo Satumomab, Solitomab, Taplitumomab aplitumomab), tenatumomab, tositumomab Tositumomab, Tremelimumab, Abagoboma Abagovomab, Igovomab, Oregovomab govomab, capromab, edrecolomab omab, nacolomab, amatuximab b), Bavituximab, Brentuximab imab, cetuximab, dellotuximab ximab, dinutuximab, ensituximab Ituximab, Futuximab, Girentuximab entuximab, indatuximab, isatuximab (Isatuximab), Margetuximab, Rituximab Rituximab, Siltuximab, Ublituximab Ublituximab, Ecromeximab, Abi Abituzumab, Alemtuzumab, Beba Bevacizumab, Bivatuzumab, Bro Brontictuzumab, Cantuzumab b), Cantuzumab, Sitagumab ), clivatuzumab, dacetuzumab ab), Demcizumab, Dalotuzumab b), Denintuzumab, Elotuzumab ab), Emactuzumab, Emibetuzumab uzumab), Enoblituzumab, Etaracizumab Etaracizumab, Farletuzumab, Fix Ficlatuzumab, Gemtuzumab, Imgatuzumab, Inotuzumab, La Labetuzumab, rifastuzumab , Lintuzumab, Lorvotuzumab ), Lumretuzumab, Matuzumab , Milatuzumab, Nimotuzumab , obinutuzumab, ocaratuzumab mab, Otlertuzumab, Onartuzumab tuzumab), Oportuzumab, Parsatuzumab rsatuzumab), pertuzumab, pinatuzumab natuzumab), polatuzumab, sibrotuzumab ibrotuzumab), Simtuzumab, Tacatuzumab acatuzumab), tigatuzumab, trastuzumab Trastuzumab, Tucotuzumab, Bundletuzumab Vandortuzumab, Vanucizumab, Beltz Veltuzumab, borsetuzumab, sof Sofituzumab, Catumaxomab, Ertumaxomab, Depatuxizumab umab), Ontuxizumab, Brontuzumab ntuvetmab, the antigen-binding domain of tamtuvetmab or tumor antigen-binding variants thereof. As used herein, "variant" The antigen-binding domain is specific to a particular antigen (e.g., HER2 for trastuzumab). It binds heterologously but has fewer or more amino acids than the parent antibody (e.g., a fragment (e.g., scFv) of the antibody, which has one or more amino acid substitutions compared to the parent antibody It means having, or a combination thereof.

[0150] In some embodiments, the anti-OV antigen CAR or anti-OV antigen conjugate of the present disclosure The antigen-binding domain of the antibody may be used as a therapeutic antibody (e.g., to target specific disease-associated cells in a patient). United States Food and Drug Administration (U.S. Food and Drug Administration) and Drug Administration) and / or the European Medicines Agency Approved by the European Medicines Agency (EMA) or a fragment thereof that retains the ability to specifically bind to a target antigen (e.g., a fragment of an antibody cFv versions of such antibodies).

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

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

[0153] Nucleic acids encoding any of the anti-OV antigen CARs of the present disclosure, including such nucleic acids Expression vectors and cells containing such nucleic acids and expression vectors are also provided. .

[0154] According to some embodiments, provided are antibodies expressing an anti-OV antigen CAR on their surface. The target cells include, but are not limited to, immune cells. In certain embodiments, the immune cells are immune effector cells. Non-limiting examples of immune effector cells that can express a native CAR on their surface include T cells (i.e., the cells can be CAR T cells), NK cells, NKT cells, macrophages Also included are cytoplasmic cells of the present disclosure that express an anti-OV antigen CAR on their surface. Pharmaceutical compositions containing any of these are also provided. Such compositions include cells and pharmaceutically acceptable carriers. An acceptable carrier may be included. Examples of suitable pharmaceutically acceptable carriers are described in detail below. .

[0155] Conjugates that bind to oncolytic virus antigens The anti-VV A56 and anti-VV of the present disclosure described in the conjugates section above In addition to the B5 antibody and fusion protein conjugates, embodiments of the present disclosure also include anti-tumor lytic In certain embodiments, such conjugates further comprise a degradative viral antigen-antibody conjugate. Conjugates are antibodies that specifically bind to oncolytic virus (OV) antigens and their According to some embodiments, the drug is conjugated to an antibody. Gated agents include chemotherapeutic agents, toxins, radiosensitizers, and radioisotopes (e.g., The drug is selected from the group consisting of: The agent may be any of the drugs identified.

[0156] The anti-OV antibody conjugates of the present disclosure find a variety of uses. Advantageously, such conjugates may be used in pharmaceutical compositions comprising anti-OV antigen conjugates. The present invention finds use in methods comprising administering to an individual having cancer, The conjugate is then transferred to cells that are infected with OV and express OV antigens on their surface. It will be appreciated that the therapeutic agent may be targeted to cancer cells to treat cancer in an individual.

[0157] The antibody (or antibodies containing thereof) is added so that the conjugate specifically binds to the OV antigen of interest. fusion protein) can be selected. The OV antigen can be a native OV antigen. In embodiments, the antibody portion of the conjugate is a vaccinia virus (e.g., antigen A33 , A34, A36, A56, B5, F12, F13, etc., for example, JX-594, GL- ONC1, or Western Reserve, Wyeth, Lister, Cop enhagen, Temple of Heaven, Patwadangar, and Modified vaccinia virus (VV strains selected from Ankara, etc.), adenovirus, H SV, reovirus, vesicular stomatitis virus, Newcastle disease virus, Seneca Valley Viruses, poliovirus, measles virus, coxsackievirus, and Malabawi virus It is capable of specifically binding to a native antigen encoded by an OV selected from the virus.

[0158] In certain embodiments, the OV antigen is heterologous to the OV. For example, an antibody (or anti- The antibody portion of the conjugate is described in the CAR section above. The OV antigens can be selected to specifically bind to any of the heterologous OV antigens listed.

[0159] The antibody portion of the anti-OV antibody conjugate of the present disclosure is an antibody of the present disclosure, anti-VV A56 and and the sections above relating to anti-VV B5 antibodies and fusion protein conjugates. As described in The antibody may be provided in a single chain (e.g., scFv) format, or the like.

[0160] The anti-OV antibody conjugates of the present disclosure may be linked to a linker, e.g., the anti-VV A56 of the present disclosure. and the above sections relating to anti-VV B5 antibodies and fusion protein conjugates. The antibody is linked to the nucleotide sequence of the present invention via either a non-cleavable or cleavable linker as described in the section (or a fusion protein comprising the antibody).

[0161] Pharmaceutical compositions comprising any of the anti-OV antibody conjugates of the present disclosure are also provided. Such compositions may include the conjugate and a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers are described in detail below.

[0162] composition As summarized above, the present disclosure also provides compositions. According to some embodiments, the present disclosure The compositions described herein include antibodies, fusion proteins, or conjugates of the present disclosure. The antibodies, fusion proteins or conjugates are described in the antibody section above. It may be either an antibody, a fusion protein, or a conjugate, the description of which is incorporated herein by reference. are included but are not repeated here for brevity.

[0163] In certain aspects, the compositions of the present disclosure comprise an antibody, fusion protein or The liquid medium may be an aqueous liquid medium such as water, a buffer solution, or the like. one or more additives, such as salts (e.g., NaCl, MgCl, KCl, MgSO), Buffers (Tris buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanediol) sulfonic 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-amino dissolving agents, detergents (e.g., Tween-20 non-ionic detergents, nuclease inhibitors, protease inhibitors, glycerol, Chelating agents and the like may also be present in such compositions.

[0164] Aspects of the present disclosure further include pharmaceutical compositions. In some embodiments, the pharmaceutical compositions of the present disclosure The composition may be an anti-VV A56 antibody or an anti-VV B5 antibody (or a composition containing the same) of the present disclosure. The present invention also includes a pharmaceutically acceptable carrier, such as a pharmaceutically acceptable salt thereof, a soluble or soluble nucleotide ...

[0165] The antibodies, fusion proteins, or conjugates may be incorporated into a variety of formulations for therapeutic administration. More specifically, the antibody, fusion protein or conjugate may be suitably and formulated into a pharmaceutical composition by combining with a suitable pharmaceutically acceptable excipient or diluent. It can be used in tablets, capsules, powders, granules, ointments, solutions, injections, inhalants, aerosols, etc. The compositions can be formulated into solid, semi-solid, liquid or gaseous forms such as tablets.

[0166] Antibodies, fusion proteins or conjugates for administration to an individual (e.g., suitable for human administration) Preparations of adjugates are generally sterile and free of detectable pyrogens or selected The formulation may be free of other contaminants that contraindicate its administration to a patient by a different route of administration.

[0167] In pharmaceutical dosage forms, the antibody, fusion protein or conjugate may be administered in the form of a pharmaceutical They can be administered in the form of a therapeutically acceptable salt, or they can be administered alone or in combination with other pharmaceutically acceptable salts. The following methods and compositions can also be used in appropriate association and combination with other therapeutically active compounds. and carriers / excipients are merely examples and are in no way limiting.

[0168] For oral formulations, lactose, mannitol, corn starch, or potato Traditional additives such as starch, crystalline cellulose, cellulose derivatives, gum arabic, Binders such as starch or gelatin, corn starch, potato starch or or disintegrants such as sodium carboxymethylcellulose, talc, or stearic acid lubricants such as magnesium, and optionally diluents, wetting agents, preservatives and flavoring agents. , tablets, powders, granules or capsules, The conjugates can be used alone or in combination with suitable additives.

[0169] The antibody, fusion protein, or conjugate may be administered parenterally (e.g., intravenously, intraarterially, intravenously ... The compound can be formulated for administration (intramuscular, intracerebral, intraventricular, intrathecal, subcutaneous, etc.). In certain embodiments, the antibody, fusion protein, or conjugate is soluble in vegetable oil or other similar oils, synthetic fatty glycerides, esters of higher fatty acids, or propylene glycol, etc. Dissolve or suspend the antibody, fusion protein, or conjugate in an aqueous or non-aqueous solvent. or by emulsifying, and if necessary, by adding solubilizing agents, isotonicity agents, suspending agents, emulsifying agents The pharmaceutical composition can be formulated for injection using conventional additives such as antiseptics, stabilizers and antiseptics.

[0170] The pharmaceutical composition comprising the antibody, fusion protein or conjugate has a desired purity. The antibody, fusion protein, or conjugate may be mixed with any physiologically acceptable carrier, excipient, or , stabilizers, surfactants, buffers and / or tonicity agents. Acceptable carriers, excipients and / or stabilizers may be used depending on the dosage and and concentrations that are non-toxic to recipients and do not contain buffers, e.g., phosphate, citrate, and Other organic acids; ascorbic acid, glutathione, cysteine, methionine and citric acid Contains antioxidants, preservatives (e.g., ethanol, benzyl alcohol, phenol, m-chloride, Resole, p-chloro-m-cresol, methyl or propyl paraben, benzal chloride cornium, or a combination thereof), arginine, glycine, ornithine, lysine, Histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenyl thialanine, tyrosine, tryptophan, methionine, serine, proline, and Combinations of amino acids, monosaccharides, disaccharides, and other sugars, low molecular weight (approximately 10 residues or less) (less than 100%) polypeptides, proteins such as gelatin or serum albumin, and EDTA Chelating agents, trehalose, sucrose, lactose, glucose, mannose, maltose , galactose, fructose, sorbose, raffinose, glucosamine, N-methyl Sugars such as glucosamine, galactosamine, and neuraminic acid, and / or Tw een, Brij Pluronic, Triton-X, or polyethylene glycol These include non-ionic surfactants such as PEG-1.

[0171] The pharmaceutical composition may be in liquid form, lyophilized form, or in liquid form reconstituted from a lyophilized form. The lyophilized preparation may be in a sterile solution and will be reconstituted with a sterile solution prior to administration. The standard procedure for reconstituting the composition is to add a certain amount of purified water (typically the amount removed during lyophilization) to the The procedure for preparing a pharmaceutical composition for parenteral administration is to add back an amount equivalent to the volume of the original solution. A solution containing an antimicrobial agent may be used for this purpose.

[0172] Aqueous formulations of antibodies, fusion proteins, or conjugates may be used in aqueous solutions, for example, from about 4.0 to about 7. 0, or in the range of about 5.0 to about 6.0, or alternatively at a pH of about 5.5, in a pH buffer solution. Examples of buffers suitable for a pH in this range include phosphate buffers, buffers, histidine buffers, citrate buffers, succinate buffers, acetate buffers, and other The concentration of the buffer may vary depending, for example, on the buffer and the desired tonicity of the formulation. The concentration may be about 1 mM to about 100 mM, or about 5 mM to about 50 mM.

[0173] To adjust the tonicity of the formulation, a tonicity agent can be included in the formulation. The agents include sodium chloride, potassium chloride, glycerin, and amino acids and sugars. In some embodiments, the present invention may include any of the following components: The aqueous formulation is isotonic, although tonic or hypotonic solutions may be suitable. has the same tonicity as some other solution to which it is compared, such as saline or serum. The tonicity agent is added in an amount of about 5 mM to about 350 mM, for example, 100 mM It can be used in an amount of ∼350 mM.

[0174] To reduce aggregation and / or minimize the formation of particulates in the formulation Surfactants may be added to the formulation to reduce adsorption and / or surface active agents. Examples of the antibacterial agent include polyoxyethylene sorbitan fatty acid esters (Tween), poly Oxyethylene alkyl ether (Brij), alkylphenyl polyoxyethylene ether ether (Triton-X), polyoxyethylene-polyoxypropylene copolymer ( Poloxamer, Pluronic) and sodium dodecyl sulfate (SDS) are examples. Examples of suitable polyoxyethylene sorbitan fatty acid esters include polysorbate 20 (sold under the trademark Tween 20™), and Polysorbate 80 (Tween A suitable polyethylene-polypropylene copolymer is sold under the trademark n 80™. Examples of such compounds are Pluronic® F68 or Poloxamer 188 (trademark). Suitable polyoxyethylene alkyl ethers are those sold under the trade name An example is that sold under the trademark Brij™. Examples of surfactant concentrations are: It can range from about 0.001% to about 1% w / v.

[0175] Protecting antibodies and / or T cell activators from destabilizing conditions during the freeze-drying process For example, known cryoprotectants include sugars (glucose and sucrose), polyols (mannitol, sorbitol and glycerol Contains niacin, ... The cryoprotectant may be included in an amount of about 10 mM to 500 nM.

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

[0177] kit Aspects of the present disclosure further include kits. In certain embodiments, the kits include the methods of the present disclosure. For example, administering a pharmaceutical composition of the present disclosure to an individual to produce an antibody, fusion protein (e.g., a CAR ) or a conjugate thereof to oncolytic virus (e.g., VV)-infected cancer cells in the individual. The present invention finds use in carrying out methods that involve directing.

[0178] Thus, in certain embodiments, the kits of the present disclosure comprise any of the pharmaceutical compositions of the present disclosure. and instructions for administering the pharmaceutical composition to an individual in need thereof. The pharmaceutical compositions contained in the kit include antibodies, fusion proteins, and / or conjugates of the disclosure. 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 gates. The subject antibodies, fusion proteins, conjugates and containing any of the agents and features described above in the section relating to compositions. obtain.

[0179] The kits of the present disclosure may be present in unit dose, e.g., ampule, or multi-dose format. Thus, in certain embodiments, the kit may comprise a quantity of the composition of the present disclosure. one or more (e.g., A "unit dose" may contain two or more unit doses (e.g., ampoules). The term, as used herein, refers to a single dose suitable for human and animal subjects. refers to physically distinct units, each calculated to produce a quantity sufficient to produce a desired effect. The amount of a unit dose will vary depending on the particular antibody, fusion protein, etc. used. The substance and / or conjugate, the effect to be achieved, and the antibody, fusion The efficacy and safety of the drug will depend on various factors, including the pharmacodynamics associated with the protein and / or conjugate. In still other embodiments, the kit may include a single, multiple dose of the composition.

[0180] In certain embodiments, the kits of the present disclosure provide a method for administering a pharmaceutical composition to an individual, for example, by administering the pharmaceutical composition to an individual. The antibodies and fusion proteins present in the pharmaceutical composition are delivered to VV-infected cancer cells in individuals with cancer. targeting a protein or conjugate (e.g., to treat cancer in an individual) ), wherein the individual comprises cancer cells infected with VV, and the individual comprises antibodies, fusions, The protein or conjugate binds to VV antigens expressed on the surface of infected cancer cells. Thus, it is targeted to the infected cancer cells.

[0181] According to some embodiments, the kits of the present disclosure contain VV (e.g., JX-594, GL -ONC1, Western Reserve, Wyeth, Lister, Copenhagen, Tiantan, Patowada and modified vaccinia virus Ankara, etc. Such kits may include, for example, an antibody, fusion protein, or co-immunoglobulin of the present disclosure. an amount effective to infect cancer cells in the individual prior to administering the pharmaceutical composition containing the conjugate The pharmaceutical composition may further include instructions for administering the pharmaceutical composition containing the VV to an individual with cancer. .

[0182] Any instructions included with the kit (e.g., instructions for use (IFU)) should be included in the appropriate records. The instructions may be recorded on a medium. For example, the instructions may be printed on a substrate such as paper or plastic. As such, the instructions may be included in the kit as a package insert or as part of the kit. Present on the label of the container or its components (i.e., on the packaging or subpackaging) In other embodiments, the instructions are provided on a portable flash drive, DVD, CD, or - electronic storage residing on a suitable computer-readable storage medium such as a ROM, diskette, etc. In yet another embodiment, the actual instructions are present as a data file. It does not exist in the An example of this embodiment is a system that allows the user to view instructions and and / or a web address from which the instructions can be downloaded. Like the manifest, the means for obtaining the instructions is recorded on a suitable substrate.

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

[0184] In certain aspects, provided is an anti-VV A56 or anti-VV B5 antibody of the disclosure ( (including any fusion protein or conjugate containing such antibody) A method comprising administering an effective amount of a pharmaceutical composition containing any one of the above to an individual having cancer. The method includes administering to an individual an O antigen encoding a VV A56 or VV B5 antigen to which the antibody binds. VV-infected cancer cells (VV A56 or VV B5 antigens are negative for OV) the antibody, fusion protein or conjugate, which may be native or heterologous, Targeting of infected cancer cells by VV antigens expressed on the surface of infected cancer cells According to some embodiments, such methods comprise administering the pharmaceutical composition to an individual Before administering to the body, an effective amount of OV is administered to an individual to infect cancer cells. Such methods may find use, for example, in treating cancer in an individual. can be.

[0185] In certain embodiments, the pharmaceutical composition comprises an anti-VV A56 or anti-VV B5 antibody of the disclosure. For example, the pharmaceutical composition may comprise any of the anti-VV A56 or anti-VV A56 conjugates. The anti-VV B5 antibody is an in vivo imaging agent, and can be coupled to a detectable label or radioisotope. Such methods may include conjugating the conjugate to an in vivo and imaging the infected cancer cells in the individual using an imaging agent. The method of administering a conjugate containing a detectable label or a radioisotope to an individual includes For example, to isolate cancer cells from an individual for the purposes of diagnosis, prognosis, and / or monitoring anti-cancer therapy. The present invention finds use in imaging.

[0186] According to some embodiments, an antibody that specifically binds to an oncolytic virus (OV) antigen is Methods for targeting cancer cells in an individual are provided. Such methods involve targeting OV antigen-specific antibodies. a specific binding antibody (or a fusion protein or conjugate containing the antibody) The method comprises administering to an individual an effective amount of the pharmaceutical composition, wherein cancer cells in the individual become infected with OV and These express OV antigens on their surface. OV antigens are described elsewhere herein. In certain embodiments, the OV antigen is a native O For example, OV is a chimeric antigen that binds to an oncolytic virus antigen. Any of the viruses described in the section entitled "Receptors" or their engineered variants modified variants (e.g., OV modifications described below, e.g., for human GM-CSF) Transgenic, thymidine kinase gene deletion, and / or other The OV antigens may be native to the OV (an engineered variant having one or more antigens), and the OV antigens may be native to the OV. In some embodiments, the OV antigen is heterologous to the OV, e.g., heterologous to the OV. A viral antigen that is heterologous to the oncolytic virus antigen (e.g., a protein that binds to an oncolytic virus antigen, as described above) any of the viral OV antigens listed in the section entitled "Meta-antigen receptor" , tumor antigen, or any other heterologous OV antigen described elsewhere herein. According to some embodiments, such methods comprise, prior to administering the pharmaceutical composition to the individual: It further comprises infecting cancer cells by administering an effective amount of OV to an individual. Such methods find use, for example, in treating cancer in an individual.

[0187] Upon binding to the surface of the cancer cell, the antibody (or fusion protein) induces, e.g., antibody-dependent Through ADCC, complement is recruited in complement-dependent cytotoxicity (CDC) This allows epitope spreading via antibody-dependent cellular phagocytosis (ADCP). Cell injury through epitope spreading or by some other mechanism Antibodies can be modified in the Fc region to induce desired or enhanced effects. This can be achieved by introducing one or more amino acid substitutions into the Fc region of an antibody. Alternatively, this can be achieved by minimizing side effects or complications of the treatment. If it is desired to eliminate or reduce effector function to achieve the desired effect, certain other F The c region can be used.

[0188] In certain embodiments, the pharmaceutical composition comprises an anti-VV A56 or anti-VV B5 antibody. In vivo imaging of therapeutic agents, toxins, radiosensitizers, therapeutic radioisotopes, and antibodies The conjugate is a radioisotope that allows The drug may comprise a drug such as those described in the conjugates section above. The agent may be either

[0189] The antibody (or antibodies containing thereof) is added so that the conjugate specifically binds to the OV antigen of interest. fusion proteins) can be selected. OV antigens of interest include those described elsewhere herein. These include, but are not limited to, any of the native or heterologous OV antigens that have been identified. In certain embodiments, the OV antigen is a native OV antigen. V is a chimeric antigen receptor that binds to an oncolytic viral antigen, as described above in the section entitled "Chimeric Antigen Receptors That Bind Oncolytic Viral Antigens." any of the viruses described in the above, or engineered variants thereof (e.g., OV modifications described in, for example, transgenic for human GM-CSF, engineered genes with deletions of the ribozyme kinase gene and / or one or more of the following: In some embodiments, the OV antigen is native to the OV. The OV antigen is heterologous to the OV, e.g., a virus heterologous to the OV. Antigens (e.g., those described above in the section entitled "Chimeric Antigen Receptors that Bind Oncolytic Viral Antigens"). any of the viral OV antigens described in the section), tumor antigens, or In some embodiments, the antigen is any other heterologous OV antigen described elsewhere in the document. , an antigen is expressed exclusively under the control of the wild-type transcriptional regulatory elements of the OV for that antigen. In accordance with such an embodiment, the antigen is a native or heterologous OV antigen. The transcriptional regulatory elements of OV for the antigen are not altered compared to wild-type OV. In a specific embodiment, the antigen is a native OV antigen, the expression level of which is wild-type transcriptional regulatory elements by modification (e.g., substitution, duplication, etc.) of one or more wild-type transcriptional regulatory elements Altered (e.g., increased) compared to expression by OV. For example, the antigen may be Native OV antigen (e.g., native A56, native B5, or any desired antigen) The expression of the antigen can be regulated by the wild-type transcriptional regulatory elements of OV (or any other native OV antigen), one or more heterologous regulatory elements (e.g., human) that result in higher expression levels compared to The coding region of the native OV antigen is operably linked to a promoter (e.g., promoter). By combining the OV with the OV, the expression is increased compared to that of the wild-type OV. The OV may be a modified OV, for example, an OV containing one or more of the OV modifications described below. It is possible.

[0190] In certain embodiments, the OV antigen is heterologous to the OV. For example, a conjugate The antibody portion of the OV is directed against a heterologous OV antigen (e.g., the OV) as described elsewhere herein. Antibodies are engineered to specifically bind to one of the following antigens: heterologous viral antigens, tumor antigens, etc. can be selected.

[0191] The antibodies may be prepared in any desired format, as described in the previous section of this disclosure. , e.g., in a tetrameric format, a single chain (e.g., scFv) format, etc. According to some embodiments, the pharmaceutical composition comprises a detectable label or a radioisotope. In certain embodiments, the antibody is conjugated to a detectable label or a radioactive The isotope is an in vivo imaging agent. The methods described herein include, for example, for the purposes of diagnosis, prognosis and / or monitoring anti-cancer therapy. Furthermore, the present invention provides an in vivo imaging agent for imaging cancer cells in an individual in vivo. It may also include.

[0192] According to some embodiments, a CAR that specifically binds to an oncolytic virus (OV) antigen Methods for targeting OV antigens to cancer cells in an individual are provided. Such methods involve targeting OV antigens specifically to cancer cells in an individual. and administering to the individual an effective amount of a pharmaceutical composition comprising a CAR comprising an antigen-binding domain that specifically binds to the CAR. The individual's cancer cells are infected with OV and express OV antigens on their surface. CARs can be used to bind to cells, e.g., immune effector cells, e.g., T cells, NK cells, NK It can be expressed on the surface of immune cells such as T cells, macrophages, etc. For example, CARs may be present on the surface of T cells, in which case the method involves transmitting C to infected cancer cells in the individual. In some embodiments, such methods include targeting AR T cells. The method is performed by administering an effective amount of OV to the individual prior to administering the pharmaceutical composition to the individual. Such methods can be used, for example, to treat cancer in an individual. It finds use in

[0193] The antigen-binding domain of the CAR specifically binds to the OV antigen of interest. For example, any of the native or heterologous OV antigens described elsewhere herein In certain embodiments, the OV antigen is any of, but not limited to, For example, OV is an active OV antigen. any of the viruses listed in the section entitled "Antibody Antigen Receptor" or engineered variants of (e.g., OV modifications described below, e.g., human GM-CSF) Transgenics for thymidine kinase gene deletions and / or other tumors The OV antigen may be native to the OV (an engineered variant having one or more of the above). According to some embodiments, the OV antigen is heterologous to the OV, e.g., Viral antigens that are heterologous to the OV (e.g., binding to oncolytic viral antigens, see above) Any of the viral OV antigens described in the section entitled "Chimeric Antigen Receptors for ), tumor antigen, or any other heterologous OV antigen described elsewhere herein is.

[0194] Pharmaceutical compositions containing cells expressing a CAR on their surface can be prepared by a variety of methods. In some embodiments, the cells of the present disclosure can be transformed with a viral vector encoding a CAR. In some embodiments, the gene is produced by transfecting cells with a The cells are T cells, thereby providing a method for producing CAR T cells. In embodiments, such methods involve administering a population of T cells (e.g., a CAR T cell therapy). The goal is to activate T cells (T cells obtained from the individual being treated) and stimulate a population of T cells to proliferate. and transducing the T cells with a viral vector encoding the CAR. In some embodiments, a retroviral vector encoding a CAR, e.g., a cancer In some embodiments, the CAR is a maletroviral vector that transduces T cells. T cells are transduced with a lentiviral vector encoding

[0195] The cells of the present disclosure may be autologous / autogeneic (" self”) or non-autologous (“no self”) or non-autologous (“no n-self), for example, allogeneic, syngeneic or xenogeneic). "Autologous" as used herein refers to cells from the same individual. "Allogeneic" as used herein refers to cells of the same species that are genetically distinct from the cell in comparison. "Syngeneic," as used herein, refers to cells of a different individual that are genetically identical to the cell in comparison. In some embodiments, the cell is a T cell obtained from a mammal. In some embodiments, the mammal is a primate. In some embodiments, the individual is a human.

[0196] T cells are found in peripheral blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, and infected areas. tissue from the spleen, ascites, pleural effusion, splenic tissue, and tumors. T cells can be obtained from a number of sources. In certain embodiments, T cells are obtained by precipitation, e.g., F Blood collected from an individual using any number of known techniques, such as ICOLL™ separation It can be obtained from the liquid units.

[0197] In some embodiments, isolated or purified populations of T cells are used. In the embodiment, T CTL and T H Lymphocytes are purified from PBMCs. In embodiments, T CTL and T H Lymphocytes undergo activation, proliferation, and / or genetic modification. Either before or after the transformation, the naive N ), Memory (T MEM ), and Ephe Kutar (T EFF ) T cell subpopulations. A suitable approach for such selection is Techniques are known, including, for example, magnetic activated cell sorting (MACS), and TN is a CD45R A + 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 given by Wang et al. al.(2016)Blood 127(24):2980-90.

[0198] In some embodiments, the following markers are used: CD3, CD4, CD8, CD28, CD 45RA, CD45RO, CD62, CD127, and one or more of HLA-DR Specific subpopulations of T cells expressing IL-1 can be further isolated by positive or negative selection techniques. In some embodiments, CD62L, CCR7, CD28, CD27, CD122, CD127, CD197, or CD38 or CD62L, CD127, CD197 and CD38. Specific subpopulations are further isolated by positive or negative selection techniques. In this embodiment, the produced T cell composition is immunized with the following markers: CD57, CD244, CD16 do not express one or more of the following: PD-1, CTLA4, TI3, and LAG3. In some embodiments, the produced T cell compositions express the following markers: CD57, CD2 44, one or more of CD160, PD-1, CTLA4, TI3, and LAG3 Virtually not expressed.

[0199] To achieve a therapeutically effective dose of a T cell composition, T cells may be stimulated, activated, or immunized with T cells. T cells can be subjected to differentiation and / or expansion. No. 6,534,055, No. 6,905,680, No. 6,692,964, No. 5 ,858,358, No.6,887,466, No.6,905,681, No.7,144 ,575, No.7,067,318, No.7,172,869, No.7,232,566 No. 7,175,843, No. 5,883,223, No. 6,905,874, No. 6 Nos. 6,797,514 and 6,867,041, each of which is a (which is incorporated herein by reference in its entirety) In some embodiments, the T cells can be activated and expanded using The cells are activated and expanded for about 1 to 21 days, for example, about 5 to 21 days. In the T cell, a nucleic acid (e.g., an expression vector) encoding a CAR is introduced into the T cell. Before, about 1 to 4 days, about 1 to 3 days, about 1 to 2 days, about 2 to 3 days, about 2 to 3 days Activation and increase for 4 days, about 3 to 4 days, or about 1 day, about 2 days, about 3 days, or about 4 days It is propagated.

[0200] In some embodiments, the T cells are transfected with a nucleic acid (e.g., an expression vector) encoding a CAR. ) for about 6 hours, about 12 hours, about 18 hours, or about 24 hours before introducing it into T cells. In some embodiments, the T cells are transfected with a nucleic acid encoding a CAR (e.g., For example, an expression vector) is introduced into a T cell and simultaneously activated.

[0201] In some embodiments, suitable conditions for T cell culture include a suitable medium (e.g., a minimum of Essential medium or RPMI medium 1640 or X-vivo 15 (Lonza)), and serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), surin, IFN-γ, IL-4, IL-7, IL-21, GM-CSF, IL-10, IL-12, IL-15, TGFβ and TNF-α, or cells known to those skilled in the art and any other additives suitable for the growth of the plant, including, but not limited to, the nutrients necessary for growth and survival. Further illustrative examples of cell culture media include one or more essential factors such as amino acids, peptides, and the like. sodium benzoate and vitamins, and is serum-free or contains an adequate amount of blood serum (or plasma) or a defined set of hormones and / or T cell growth and RPMI 1640 supplemented with cytokines sufficient for proliferation, Click, AE VI-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15 and XV Including but not limited to ivo 20, Optimizer.

[0202] In some embodiments, the nucleic acid (e.g., expression vector) encoding the CAR is Clonal injection, transfection, lipofection, heat shock, enzyme electroporation, transduction, gene gun, microinjection, DEAE-de They are introduced into cells (e.g., T cells) by transfection, such as by xytosine-mediated transfer. In embodiments, the nucleic acid (e.g., expression vector) encoding the CAR is transduced using AAV. AAV vectors are introduced into cells (e.g., T cells) by I from AAV2. TR, and AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, and AAV7 , AAV8, AAV9 or AAV10. In some embodiments, the AAV vector comprises ITRs from AAV2 and ITRs from AAV6. In some embodiments, a nucleic acid encoding a CAR (e.g., an expression vector) is tter) by lentiviral or retroviral transduction of cells (e.g., T cells) The lentiviral vector backbone is compatible with HIV-1, HIV-2, and , Visna-Maedi virus (VMV), Caprine Arthritis-Encephalitis Virus (CAEV) ), Equine infectious anemia virus (EIAV), Feline immunodeficiency virus (FIV), Bovine immunodeficiency virus (BOV) may be derived from bacterial immunodeficiency virus (BIV) or simian immunodeficiency virus (SIV). Lentiviral vectors are integration competent. petent) or integrase-deficient lentiviral vector (TDLV) In one embodiment, an HIV-based vector backbone (i.e., an HIV system An IDLV vector containing the nucleotide sequence of the target gene (the nucleotide sequence of the target gene) is used.

[0203] In certain embodiments, a conjugate comprising an antibody that specifically binds to an oncolytic virus (OV) antigen is provided. Methods for targeting adjugates to cancer cells in an individual are provided. Such methods include administering to an individual an effective amount of a pharmaceutical composition comprising an antibody that specifically binds to a V antigen, Cancer cells in an individual become infected with OV and express OV antigens on their surface. According to an embodiment, such a method comprises administering an effective amount of OV to an individual prior to administering the pharmaceutical composition to the individual. Such methods further include administering the method to an individual to infect cancer cells. , for example, find use in treating cancer in an individual.

[0204] The antibody in the conjugate may be any of the OV antigens described elsewhere herein. Specific binding to an OV antigen of interest, including but not limited to: In some embodiments, the OV antigen is a native OV antigen. For example, the OV may be a native OV antigen, as described above in "Oncolytic of the virus described in the section entitled "Chimeric antigen receptors that bind to viral antigens" or engineered variants thereof (e.g., OV modifications described below, e.g., For example, transgenic for human GM-CSF, deletion of the thymidine kinase gene and / or other engineered variants) and OV antigens In some embodiments, the OV antigen is native to the OV. a viral antigen that is heterologous to the OV (e.g., a viral antigen that is heterologous to the OV (e.g., a viral antigen that is heterologous to the OV) ... Chimeric antigen receptors that bind to lytic viral antigens. any of the HIV 1, HIV 2, HIV 3, HIV 4, HIV 5, HIV 6, HIV 7, HIV 8, HIV 9, HIV 10, HIV 11, HIV 12, HIV 13, HIV 14, HIV 15, HIV 16, HIV 17, HIV 18, HIV 19, HIV 20, HIV 21, HIV 22, or any other heterologous OV antigen.

[0205] The OV administered to an individual in accordance with the present disclosure can be a wild-type OV or a modified OV. According to some embodiments, the OV is a modified OV, wherein the modified OV is a different virus. In some embodiments, the OV is a chimeric OV comprising two or more domains from the OV. is a modified OV(s) containing one or more modifications in one or more of the following functional categories: For example, non-chimeric or chimeric OVs): infection, replication, tropism, improved safety, immune reporter gene for imaging, inhibiting the host's antiviral immune response and promoting host antitumor In some embodiments, the OV is modified as follows: : A gene that encodes and expresses human GM-CSF and has a deletion of the thymidine kinase gene. (high levels of thymidine, typically found in cancer cells with mutated RAS or p53 genes) (which restricts or substantially restricts viral replication to cells having a reporter kinase), Ribonucleotides that encode and express genes (e.g., Lac Z, luciferase, etc.) The F2L gene alters one or more genes encoding nucleotide reductases (RR). Genes (maintaining the fidelity of DNA replication and providing precursors for TMP production by thymidylate synthase) Therapeutic proteins that disrupt the viral dUTPase (which encodes the enzyme responsible for both viral infection and viral damage) Quality (e.g., suicide genes - i.e., TK, cytosine deaminase, purine nucleosides) Phosphorylase, uracil phosphoribosyltransferase, thymidylate kinase, etc. Drug precursors can be converted into cytotoxic compounds, including but not limited to expressing immunostimulatory proteins (genes encoding proteins that stimulate immune responses); "Immunostimulatory proteins" are specific or specific antigen-binding proteins, including cytokines, chemokines, growth factors, etc. refers to proteins that have the ability to stimulate the immune system in a nonspecific manner), as well as Any of the above modified OVs may be used in combination as described elsewhere herein. Any of the OV antigens described may be encoded. For example, the modified OV may be As described above in the section entitled "Chimeric Antigen Receptors that Bind Oncolytic Viral Antigens" The OV antigens may be modified from any of the viruses described above, and the OV antigens may be native to the OV. In some embodiments, the OV antigen is heterologous to the OV, e.g., Viral antigens that are heterologous to the modified OV (e.g., the "Oncolytic Virus Antigens" section above) Among the viral OV antigens described in the section entitled "Chimeric Antigen Receptor Binding any), tumor antigen, or any other heterologous OV described elsewhere herein. It is an antigen.

[0206] In certain embodiments, administering a pharmaceutical composition comprising OV-infected cells (e.g., cancer cells) The pharmaceutical composition is provided as a method for treating a cancer cell, comprising administering to the patient a therapeutic agent for the treatment of a cancer cell. Furthermore, antibodies, conjugates or fusion proteins that specifically bind to the expressed OV antigens are The cells (e.g., cancer cells) may be removed from an individual during surgery. The cells (e.g., cancer cells) may be attacked by the immune system when administered to a patient. They may have been modified (and killed) in a laboratory to make them more likely to survive. The patient's immune system attacks these and similar cells remaining in the body. Conjugates or fusion proteins can be used according to this approach to Fc receptors on primary APCs promote the uptake of tumor particles / antigens, resulting in anti-tumor immunity. The response can be enhanced.

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

[0208] An individual in need thereof may have a cell proliferative disorder. "Harm" refers to the unwanted proliferation of one or more subsets of cells in a multicellular organism. It refers to a disorder that causes harm to an organism (e.g., pain or reduced life expectancy). Cell proliferative disorders include cancer, precancer, benign tumors, blood vessel 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, lesions Nevus (nevi lesions), teratomas, nephromas, myofibromatosis, bone forming tumors, dysplasia These include dysplastic masses and mesangial cell proliferative disorders. Examples include, but are not limited to:

[0209] In some embodiments, the individual has cancer. The subject methods include the treatment of a wide variety of cancers. As used herein, a "tumor" refers to a tumor, whether malignant or benign. not only the growth and proliferation of all neoplastic cells, but also all precancerous and cancerous The terms "cancer" and "cancerous" refer to cells and tissues that are typically unregulated. Refers to or describes a physiological condition in mammals characterized by cell proliferation / growth. Examples of cancers that can be treated using the methods include carcinoma, lymphoma, blastoma, and More specific examples of such cancers include, but are not limited to, cancers such as: 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 cancer or uterine cancer , salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver In certain embodiments, the individual has a solid tumor, tumor, recurrent glioblastoma multiforme (GBM), non-small cell lung cancer, metastatic melanoma, melanoma, Peritoneal cancer, epithelial ovarian cancer, glioblastoma multiforme (GBM), metastatic colorectal cancer, Colorectal cancer, pancreatic ductal adenocarcinoma, squamous cell carcinoma, esophageal cancer, gastric cancer, neuroblastoma, fallopian tube cancer Ductal carcinoma, bladder cancer, metastatic breast cancer, pancreatic cancer, soft tissue sarcoma, recurrent squamous cell carcinoma of the head and neck Cancer, 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 gliomas In certain embodiments, the individual has a cancer selected from melanoma, Hodgkin's lymphoma, Renal cell carcinoma (RCC), bladder cancer, non-small cell lung cancer (NSCLC), and squamous cell carcinoma of the head and neck have a cancer selected from the group consisting of: skin cancer (HNSCC);

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

[0211] The antibodies, fusion proteins and conjugates of the disclosure may be administered as pharmaceutical compositions in therapeutically effective amounts. A "therapeutically effective amount" is a dose sufficient to produce a desired result. means a beneficial or desired therapeutic outcome, such as a reduction in cancer symptoms, compared to a control. means an amount sufficient to produce a preventative effect (including a preventative effect). In embodiments, the therapeutically effective amount slows tumor growth, reduces tumor size, and An effective amount may be administered in one or more administrations. Cut.

[0212] As noted above, aspects of the present disclosure include methods for treating cancer in an individual. means an improvement in at least one or more symptoms associated with cancer in an individual, where improvement is achieved by a parameter broadly to refer to at least the reduction in the degree of symptoms associated with the cancer being treated, e.g. Therefore, treatment also involves determining whether an individual has cancer, or at least the characteristics that characterize cancer. To prevent further suffering from symptoms, The condition is completely inhibited, e.g., prevented from occurring, or stopped, e.g., This includes situations where the event is cancelled.

[0213] The antibodies, fusion proteins or conjugates of the disclosure may be used alone or in combination with a second agent. The second agent of interest may be a compound used in the treatment of cancer. approved by the U.S. Food and Drug Administration and / or the European Medicines Agency (EMA) for In some embodiments, the second agent includes, but is not limited to, an immunosuppressant. The immune checkpoint inhibitors of interest include cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitor, programmed cell death-1 (PD-1) inhibitor agents, programmed cell death ligand-1 (PD-L1) inhibitors, lymphocyte activation gene-3 ( LAG-3) inhibitor, T-cell immunoglobulin domain and mucin domain 3 (TIM- 3) Inhibitors, indoleamine (2,3)-dioxygenase (IDO) inhibitors, Ig and and T cell immunoreceptor containing ITIM domain (TIGIT) inhibitors, V of T cell activation Domain Ig suppressor (VISTA) inhibitors, B7-H3 inhibitors, and their applications Any combination of these is included, but is not limited to:

[0214] The antibody, fusion protein, or conjugate of the disclosure is administered in conjunction with a second agent In this case, the antibody, fusion protein or conjugate and the second agent may be administered in any suitable manner. According to certain embodiments, antibodies, fusion proteins, The compound or conjugate and second therapeutic agent are administered in a dosage regimen approved for their individual use. In some embodiments, the antibody, fusion protein, or conjugate is administered according to the method described above. Administration of a conjugate is in the absence of administration of an antibody, fusion protein, or conjugate, followed by administration of a second agent. one or more lower doses and / or more Second agent following a dosing regimen involving less frequent administration and / or a reduced number of cycles In certain embodiments, administering the second agent allows the second agent to be administered. and when the antibody, fusion protein, or conjugate is administered without In comparison, the antibody, fusion protein, or conjugate may be administered at one or more lower doses and and / or follow a dosing regimen with less frequent administration and / or a reduced number of cycles. This allows the drug to be administered in a controlled manner.

[0215] In some embodiments, one or more doses of the antibody, fusion protein, or conjugate The antibody and the second agent are administered to the individual simultaneously. The antibody or conjugate and the second agent may be present in the same pharmaceutical composition, or the antibody, fusion The combined protein or conjugate and the second agent are incubated for 1 hour or less, 30 minutes or less, or 15 minutes or less. This means that the two are administered as separate pharmaceutical compositions within minutes or less.

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

[0217] In some embodiments, the antibody, fusion protein or conjugate and a second The agents may be administered to an individual in different compositions and / or at different times. For example, antibodies, The fusion protein or conjugate may be administered prior to administration of a second therapeutic agent (e.g., a specific subunit). Alternatively, the second therapeutic agent may be administered as an antibody, a fusion protein, or It may be administered prior to administration of the conjugate (e.g., in a particular cycle). The second agent may be administered at least 1 hour, 3 hours, 6 hours, or 8 hours after administration of the first agent. After 12 hours, 24 hours, 48 hours, 72 hours, or up to 5 days or more It may be administered during the period in which the

[0218] In one example, the second agent may be administered as desired prior to administration of the antibody, fusion protein, or conjugate. In certain embodiments, such a regimen is administered to an individual for a period of time desired to induce cancer cells. "priming" to enhance the anti-cancer effect of antibodies, fusion proteins, or conjugates The step of administering the antibody, fusion protein, or conjugate is followed by the step of administering the second agent. Such a period of time separating the steps of administering the antibody, fusion protein or It allows priming of cancer cells, increasing the anti-cancer effect of the conjugate. It is long enough.

[0219] In some embodiments, the administration of one agent is timed specifically to the administration of another agent. For example, in some embodiments, the antibody, fusion protein, or conjugate The gate is the point at which a particular effect is observed (or, for example, a given dosing regimen and specific objectives are met). (expected to be observed based on population studies showing a correlation between the effects of do.

[0220] In certain embodiments, the desired relative dosing regimen of co-administered agents can be determined by, for example, ex vivo empirically assessed or determined using in vivo and / or in vitro models In some embodiments, such evaluation or empirical determination may be performed in vivo, in a patient. in a population (e.g., so that a correlation can be established), or alternatively in a specific individual of interest. It is held at.

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

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

[0223] In some embodiments, the first resting period and the second resting period occur at the same time or day. Alternatively, in some embodiments, the first and second pauses may correspond to a number. The periods are different, with the first rest period being longer than the second, or vice versa In some embodiments, each of the resting periods is 120 hours, 96 hours, 72 hours, , 48 hours, 24 hours, 12 hours, 6 hours, 30 hours, 1 hour or less. In this embodiment, the second rest period may be longer than the first rest period, which may be longer than several hours. Defined as days or weeks (e.g., 1 day, 3 days, 5 days, 1 week, 2 weeks, 4 weeks or more) It can be defined.

[0224] The length of the first resting period is determined by the presence or occurrence of a particular biological or therapeutic event. If so, the length of the second rest period may be based on different factors, either separately or in combination. Exemplary such factors include the type of cancer for which the treatment is administered and / or or stage of disease, characteristics of the antibody, fusion protein or conjugate (e.g., pharmacokinetic properties ), and / or patients to therapy with antibodies, fusion proteins or conjugates In some embodiments, one or both of the responses may be included. The length of the rest period may be assessed (e.g., via plasma concentration levels) of one or the other of the administered drugs. For example, if the plasma concentration of the relevant drug , optionally, below a predetermined level upon evaluation or other consideration of one or more characteristics of the individual's response. If the relevant suspension period is completed, the relevant suspension period may be considered to be completed.

[0225] In certain embodiments, the number of cycles for which a particular agent is administered can be empirically determined. In some embodiments, the exact regimen to be followed (e.g., number of doses, interval between doses) can be determined. The amounts of the doses (e.g., relative to each other or to another event such as the administration of another therapy) It may be different for one or more cycles compared to one or more other cycles.

[0226] The antibody, fusion protein or conjugate and second agent may be administered via any suitable route. The antibodies, fusion proteins or co-administrations may be administered together or separately via the same route. The conjugate and second agent may be administered orally (e.g., intravenously, intraarterially, subcutaneously, intramuscularly, or via an administration route independently selected from topical (by intravenous or epidural injection) or intranasal administration. According to certain embodiments, the antibody, fusion protein or conjugate may be administered. Both the first and second drugs are administered orally (e.g., in tablet, capsule, or liquid form). simultaneously (in the same pharmaceutical composition or in separate pharmaceutical compositions) or sequentially, such as It is administered either way.

[0227] Before administering the pharmaceutical composition to the individual, the OV is administered to the individual to infect cancer cells. When the method further comprises administering the compound to the cancer cells, any suitable administration regimen may be used. Poxvirus replication occurs in the cytoplasm, which is why the virus This is because the cytoplasm is sufficiently complex to have acquired all the functions necessary for genome replication. Once inside the core, gene expression is carried out by viral enzymes associated with the core. Divided into stages: early genes make up about 50% of the genome and are expressed before genome replication; Late genes are expressed after genome replication. The temporal regulation of expression is dependent on active DNA replication. Genome replication is provided by a self-priming late promoter. This leads to the formation of high molecular weight concatemers, which are then cleaved and It is thought that the virus is repaired and used to create the viral genome. This occurs in the nucleus, possibly interacting with cytoskeletal proteins (e.g., actin-binding proteins). This interaction occurs during the formation of inclusions in the cytoplasm, which then mature into virus particles. The virus is unique among DNA viruses because it replicates exclusively in the cytoplasm of the host cell Therefore, to encode various enzymes and proteins required for viral DNA replication. A large genome is required. During replication, vaccinia produces several infectious forms with different outer membranes. Forms: Intracellular mature virions (IMV), intracellular enveloped virions (IEV), cells associated enveloped virions (CEVs), and extracellular enveloped virions (EEVs).

[0228] To infect cancer cells with the OV, the OV is administered using a suitable route of administration. The route of administration may vary depending on the location and nature of the cancer, for example, intradermal, transdermal, parenteral, intravenous. intramuscular, intranasal, subcutaneous, local (e.g., near the tumor, especially in the vasculature or adjacent to the tumor) vascular system), percutaneous, intratracheal, intraperitoneal, intra-arterial, intravesical, intratumoral, inhalation, perfusion, lavage, and oral administration and formulations. Intratumoral injection or direct injection into the tumor vasculature. is specifically intended for discrete, solid, accessible tumors. l), regional or systemic administration may also be appropriate. For example, tumors may be contacted advantageously by administering multiple injections spaced approximately 1 cm apart. Continuous administration can also be used where appropriate, for example, by catheterization into the tumor or tumor vasculature. Such continuous perfusion can be applied by implanting a catheter. Over a period of about 1-2 hours, about 2-6 hours, about 6-12 hours, or about 12-24 hours Dosage regimens can vary and often depend on the type of tumor, tumor location, It depends on the progression of the disease and the health and age of the patient. Certain types of tumors require more aggressive treatment. At the same time, certain patients may be unable to tolerate more burdensome protocols. Clinicians are best placed to make such decisions.

[0229] Injection of the nucleic acid construct requires that the expression construct be able to pass through the specific gauge of needle required for injection. To the extent possible, it may be delivered by syringe or any other method used for injecting solutions. An exemplary needleless injection system that can be used to administer OVs is described in U.S. Pat. This system is exemplified in US Pat. No. 4,233, which includes an ampoule chamber for holding a solution. a nozzle defining a solution; and an energy device for forcing the solution through the nozzle to a delivery site. Another exemplary syringe system is capable of dispensing a precisely predetermined amount of solution at any depth. This allows for multiple injections (U.S. Patent No. 5,846,225). The mixture of nucleic acids encoding the same may be suitably combined with one or more excipients, carriers, or diluents. Dispersions may also be prepared in water, mixed with glycerol, liquid polyethylene glycol, It may also be prepared in water, and mixtures thereof, and in oils.

[0230] Physicians may administer OV at levels lower than those required to achieve the desired therapeutic effect. A dose of the vector may be prescribed and gradually increased until the desired effect is achieved. Alternatively, a physician may initiate a treatment regimen by administering a single dose of OV vector. and then until a therapeutic effect is achieved, e.g., a reduction in the volume or volume of one or more tumors. Progressively lower doses may be administered.

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

[0232] experiment Example 1 - Antibody Generation The A56 or B5 sequence from the Wyeth VV strain (SEQ ID NO: 180 and SEQ ID NO: 181, respectively) vaccinia virus A56 (CHO-A56) or B5 capsid (CHO-A56) was used to infect the We generated stable CHO cell lines expressing either the α- or β-side proteins (CHO-B5). Aluminum hydroxide (5 mg / injection) and CpG (Cat. No. ODN1826, 2 A mixture of CHO-A56 and CHO-B5 cells (initial 30 million per cell type in the first injection, and 45 million in subsequent injections) every six months Antibodies were raised in New Zealand White rabbits by subcutaneous immunization with 10 One week after the second injection, the rabbits were euthanized and the spleens were harvested (Cedarlane). B cells were cultured and isolated according to Babcook et al. (1). estern Reserve strain VVdd(eGFP)(Ottawa Hospita from Dr. John Bell of the Ohio Research Institute (OHRI) OVCAR3 cells (ATCC, catalog number 182 and 183) were infected with ATCC 182666. B cell supernatants were screened by flow cytometry for binding to the IgG antibody (HTB-161). Antibodies were identified by screening of cultured B cells (data not shown). To clone the antibody, thaw the frozen cells and use A56.hFc or B5.hFc solution. The human Fc fusion proteins were analyzed by serum plaque assay. As shown, the antibodies were generated in-house using the A56 or B5 sequences from the Wyeth VV strain. The isolated single B cells were lysed and antibody V genes were amplified by RT-PCR. PCR products corresponding to the heavy and light chains of the matched antibodies were cloned into the human IgG1 constant region and Cloned into Igκ constant region constructs (pTT5 / IgG1, pTT5 / Igk). To produce recombinant antibodies, the VH and VL chain plasmids were transfected with 293fectin (T Transfection of HEK293-6E cells was performed using a transfection kit (Hermo, Cat. No. 12347019). After 96 hours of secretion, the antibody-containing supernatant was centrifuged and sterile filtered. Cells were removed by filtration (0.22 μm). rap Mab Select SuRe column (GE, Catalog No.: 1100349 5) and a HiPrep 26 / 10 Desalting column (GE, catalog no. The antibody was purified using a 17508701 antibody. The purity of the antibody was confirmed by SDS-PAGE, UP The sequences of the generated antibodies are listed in Tables 1 and 3. Antibody IDs and formats are shown in Table 15 below. [Table 15]

[0233] Example 2 - Antibody binding to A56 and B5 antigens Lenti-x293T (Clontech, Catalog No. 632181) cells were used. A stable cell line was generated using the ELISA kit and the antigen A56 (Wyeth VV sequence) or HEK-A56 (GFP) and HEK-B5 expressing B5 (Wyeth VV sequence) (GFP) cells were generated using A549 (ATCC, Cat. No. CCL-185) and CaOv3 (ATCC, Cat. No. HTB-75) tumor cells were cultured in appropriate medium (A549 :F-12K Nutrient Mixture (Gibco, Catalog No. 2112 7-022); CaOV3: 10% FBS (Corning, Cat. No. 35-015 -CV) in DMEM (Gibco, Cat. No. 11995-040) Cells were grown to 80-90% confluence. Infected with VVdd(eGFP) at a multiplicity of infection of 0.5 in a 1000-well plate (Cat. No. A10491-01). The wells were then resuspended in growth medium 1:2 and incubated at 37°C in 5% CO2 for 6.5 hours. A549 cells were trypsinized and incubated overnight. Together with the cells, a 96-well V-bottom plate (Sarstedt, Cat. No. 82.158 3.001) approximately 1 x 10 per well5 The corresponding uninfected cells were plated. Stained A549 and CaOV3 cells, as well as HEK-A56 (GFP), HEK-B5 ( GFP) and HEK-WT cell lines were also cultured at 4 × 10 cells per well. 4 Plating at 100 cells Anti-vaccinia virus rabbit polyclonal antibody (CedarLane, catalogue number 1010001001001) was used. No. LS-C103289) was used as a positive control.

[0234] Anti-A56 antibody (A047, A049, A050 or A054), anti-B5 antibody (A04 8 or A051), and human IgG isotype control (Sigma, Cat. No. I 5154) was added to the cells and incubated on ice for 25 minutes. The cells were washed twice with PBS. The cells were washed, centrifuged at 450g for 4 minutes, and then diluted with 1:100 AlexaFluor 647 conjugate. dugate anti-rabbit IgG (Jackson, Cat. No. 115-605-046) or AlexaFluor 647-conjugated anti-human IgG (Jackson, Catalog) (Product ID: 109-605-098) and 1:1000 of the fixable viability dye eFluor 780 (eBioscience, Cat. No. 65-0865-14) and room temperature (RT). After washing twice with PBS, the cells were incubated in IC fixation buffer (eB Fix the cells in PBS (Bioscience, Cat. No. 00-8222-49) for 10 minutes and then in PBS. Washed twice, resuspended in 1% FBS in PBS, and then resuspended in BD LSRFortessa X- Analyze by flow cytometry on a 20 HTS system, FlowJo V 10 The data was processed using .5.3 software. The data was VV VVdd (eGFP) The binding of the antibody to infected cells is shown (Figure 1). The anti-A56 antibody was used to express HEK-A56 (GFP ) cells, and anti-B5 antibody specifically binds to HEK-B5(GFP) cells (Figure 2). Sex controls (HEK parental cells, uninfected A549 or CaOV3 cells, and also No binding was observed to an unrelated VV antigen (A33) expressed in HEK cells (data not shown). (Data not shown). The isotype control hIgG showed no binding to the cells.

[0235] Commercially available anti-B5 antibody (Immunetech, catalog number IT-012-009M1; (raised using VV Western Reserve immunogen) was used for binding to the B5 antigen. Anti-vaccinia rabbit polyclonal antibodies were detected by flow cytometry. A dilution series of Immunetech anti-B5 antibody (IT The results were compared with the isotype control (anti-B5 IgG) and the isotype control. For isotype control mouse antibody detection, goat anti-mouse A647 (Jackson, The IT anti-B5 IgG was obtained from HEK-B5 B5.hFc did not bind to cells (Figure 3, top panel). This antibody was generated based on the VV WyethB5 sequence by ELISA for In this assay, B5.hFc was used as a maxis The antibody was coated onto an ORP plate at 2 μg / ml and incubated overnight at 4°C. Following standard ELISA procedures, plates were washed, blocked with 5% nonfat dry milk, and incubated with primary antibodies. The bodies were incubated in block for 1 hour at RT and washed. Secondary detection was performed with goat anti-mIgG. Fc HRP (Jackson, catalog number 115-035-071) or anti-Rb IgG Fc HRP (Jackson ImmunoResearch, catalog no. No. 111-035-046) at RT for 1 hour, then washed and Add TMBK-Blue (Neogen, #308175) to the wells, then add 50 μl 1N HCl was added per well. By standard ELISA, IT anti-B5 IgG , and was negative for binding to B5.hFc soluble antigen (Fig. 3 , lower panel).

[0236] IT anti-B5 IgG was used to infect Western Reserve VVddeGFP. Binding to A549 and HT29 cells was also tested. Cells (ATCC Catalog No. HTB-38), McCoy's 5A Medium (Gibco #1660 0-082) + 10% FBS) were cultured until 80-90% confluence. Cells were infected with VVdd(eGFP) at a multiplicity of infection of 1 in serum-free RPMI. The cells were incubated at 37°C in 5% CO2 for 6.5 hours, supplemented with growth medium 1:2, and further The cells were trypsinized and plated in a 96-well V-bottom plate. Approximately 1 x 10 5 Anti-B5 antibody A048 hIgG1 and Human and IT anti-B5 IgG, as well as a human isotype control, were added to the cells and incubated on ice for 25 minutes. The cells were washed and incubated with AlexaFluor 647-conjugated Anti-human IgG and anti-mouse IgG (Jackson, #115-605-164), cont. Immunofluorescence assay using the fixable viability dye eFluor780 at 1:1000. After washing twice with 1% FBS in PBS, fixing, and resuspension, the samples were By flow cytometry on the LSRFortessa X-20 HTS system The data showed that the A048 antibody was more potent than IT anti-B5 IgG and We have shown that the antibody VVddeGFP binds to A549 and HT29 cells infected with VVddeGFP. Figure 4. Binding of A048 antibody to infected A549 and HT29 cells. The EC50 values are 6 nM and 1.1 nM, respectively. The EC50 values for IT anti-B5 IgG binding to 29 cells were 78 nM and 1 nM, respectively. It is 5.8nM.

[0237] Example 3 - Reformatting into mouse and rabbit antibodies Reformatting antibodies into full rabbit IgG or chimeric rabbit / mouse IgG2a Fc antibodies VVd was expressed in HEK293 cells and purified as described above. A549 cells infected with d(eGFP) were incubated with rabbit IgG, or mouse or human Binding by the antibodies as chimeras was tested. Anti-A56 antibodies (A056, A049, A059) and anti-B5 antibodies (A073, A048, A058), and rabbit IgG Isotype (Jackson, Cat. No. 011-000-003), human IgG Isotype (Sigma Aldrich, Cat. No. I5154) and mouse Ig G isotype (R&D Biosystems, catalog no. MAB004) The cells were washed and incubated with 1:100 Alex aFluor 647-conjugated anti-rabbit IgG (Jackson, catalog no. 115-605-046), or anti-human IgG (Jackson, Cat. No. 109 -605-098), or anti-mouse IgG (Jackson, Cat. No. 115-6 05-164), 1:1000 DAPI (BioLegend, Cat. No. 4228 01) and incubated at RT for 15 min. Following washing and fixation, the samples were Data were analyzed by cytometry. All formats showed similar EC50 values for VV V Confirm that it binds to Vdd(eGFP)-infected cells (Figure 5, Table 16). No binding was observed to the intact cells or with the isotype control. [Table 16]

[0238] Example 4 - Antibody binding to virus-infected tissue On day 0, BALB / c female mice were injected with 1 × 10 6 MC38 cells were transplanted. In some mice, VVdd(eGFP) was administered intravenously on days 11 and 13 after MC38 transplantation. 2.5 × 10 in 100 μl 7 pfu), and the mice were euthanized on day 16. 8 Tumor-bearing control mice were treated with PBS instead of VVdd(eGFP) and 1 day after implantation Mice were euthanized on day 2. Tumor tissue was collected in 10% neutral buffered formalin (NBF). Tissues were formalin-fixed, paraffin-embedded, sectioned, and stained. H&E staining images are shown below. Scan using the Aperio ScanScope AT2 digital slide scanner Tissues were deparaffinized and rehydrated according to standard protocols. Heat-induced epitope retrieval To do this, the tissue was placed in an antigen retriever (Sigma, Cat. No. C999-100ML). The slides were autoclaved at 121°C for 20 minutes. After cooling, the slides were rinsed twice with PBS. The sections were isolated using a PAP pen to create a hydrophobic barrier. The tissue was then placed in 200 μl of Hydrated with washing buffer (0.1% Tween-20, 0.1% BSA in PBS). Tissues were permeabilized using 0.2% Triton in S for 10 min at RT and washed three times. Sections were then blocked in blocking buffer (5% normal goat serum, 0.05% Tween-20 in PBS). After removing the blocking buffer, the antibody dilution was in a solution (1% normal goat serum, 0.05% Tween-20, 0.1% BSA in PBS). Primary antibodies were applied to the sections and incubated overnight at 4°C. ) hIgG1 was used at 12.5 μg / ml, and a positive control polyclonal rabbit anti-vaccine Nia virus antibody (CedarLane, catalog number LS-C103289) was 1:1 Human IgG1 (Sigma, Cat. No. I5154) and rabbit I gG (Jackson ImmunoResearch Laboratories, The isotype control (catalog number 011-000-003) was used at 25 μg / ml. On the day of staining, wash the tissue and prepare the secondary antibody (200 μl) in antibody diluent and incubate at room temperature in the dark for 1 h. Tissues stained with human primary antibodies were incubated for 5 hours. exaFluor 549-conjugated goat anti-human IgG (Jackson, Catalog) The primary rabbit antibody was detected at 10 μg / ml. AlexaFluor Plus 488 Goat Anti-Rabbit IgG (H+L) Antibody (T After incubation, the staining was detected using a ELISA kit (Hermo, Cat. No. A32731). The slides were washed three times for 5 minutes each. Hoechst stain (Sigma, Cat. No. B2 261) was added to the tissue at 1 μg / ml, incubated for 10 minutes, and then the excess was removed. The coverslip was coated with Fluoromont-G (Southern Biotech , catalog number 0100-01) and Leica SP5X Laser Scanner Imaging was performed using a ning confocal microscope. The data confirmed the binding of A049 to VV VVdd(eGFP)-infected tumor tissue and No binding to unvaccinated tumor tissue was observed (Figure 6). Near-virus antibodies exhibit non-specific binding to uninfected tumor tissue.

[0239] Example 5 - Antibody affinity to B5 and A56 proteins Antibody affinity was assessed using a Biacore T200. Series SCM On the 5 chip, A048 was immobilized on FC2, A051 was immobilized on FC3, and FC1 The blank was immobilized. The sensor surface was activated by injecting EDC and NHS. The ligand was immobilized as follows: 200 μL of sodium acetate (pH 4. 5) Prepare anti-B5 A048 hIgG1 (12 μg / mL) in 200 μl of acetic acid. A051 (12 μg / mL) was prepared in sodium chloride (pH 5.0). The chip was blocked by injecting ethanolamine. Finally, the chip was infused with 50 mM After amine-coupling approximately 1000 RU of each antibody, the chip was washed with buffer. All binding studies were performed in freshly prepared and filtered running buffer HBS The assay was performed in GE-EP+ (catalog no. BR100188). In the original (1.5 nM, 4.6 nM, 13.8 nM, 41.4 nM, and 124 nM) B5.hFc (MW: 150 kDa) was added as the analyte at 30 μl / min for 200 seconds. At the end of each cycle, a pH of 1.5, 10 ml of PBS was added. The IgG capture surface was regenerated by injecting M glycine at 30 μl / min for 60 seconds. Analyte-free running buffer was injected to perform double reference subtraction. Curves were fitted using the Furr coupling.

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

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

[0242] Example 6 - Epitope binning Antibodies were EZ-Link Sulfo-NHS-LC-Biotin (Thermo, Catalog number 21343) at a 20:1 ratio according to the manufacturer's protocol. A549 cells were infected with VVdd(eGFP) at an MOI of 0.5. On the day of the assay, the virus-infected A549 cells were incubated for 1 hour at 4°C for 1 hour and then fixed the day before the assay. Preincubated with 25 μg / mL of non-biotinylated antibody and incubated for 1 hour Next, biotinylated antibodies (10 μg / ml) were injected into the wells of a 96-well V-bottom plate. The final concentration was adjusted to 5 μg / ml and incubated for 1 hour. Visin-Alexa647 (Jackson, catalog number 016-600-084) Detection was performed using Intellicyt H to detect bound biotinylated antibodies. Samples were acquired on a TFC high-throughput flow cytometer. Anti-ovalbumin antibody (in-house product from the Center for Drug Research and Development (CDRD)) was used as an irrelevant control IgG. Addition of a second antibody to a VAVC-coated plate produced results similar to those of the first antibody tested. The data suggest that these anti-A56 antibodies target different epitopes on A56. Similarly, the data show that these anti-B5 antibodies appear to bind to B5. These results indicate that the antibodies appear to bind to different epitopes (Figure 7).

[0243] Example 7 - Antibody binding to virus particles A 96-well plate (Corning, Cat. No. 3368) was filled with sodium bicarbonate buffer (pH 9.6) 9×10 6 pfu / mL of VVdd(eGFP) was coated (50 After overnight incubation at 4°C, the coated wells were washed with PBS. After washing three times, the plate was incubated with fixation buffer for 10 minutes. The wells were washed four times with distilled water. The wells were blocked with 5% non-fat dry milk in PBS for 1 hour at RT. The antibodies (A049, A047, A050, A054, A048, and A051) were The antibody was titrated 1:4 in blocking buffer from a starting concentration of 0 μg / ml. The K cell cytotoxicity receptor NKp30 has been shown to bind to VV A56. As a positive control, NKp30-His (in-house) soluble protein was used at 20 μg / mL. hIgG1 was tested as a negative control at 1 μg / ml or 1 μg / ml. The wells were washed four times with water. The wells were then washed, 50 μl / well of antibody was added, and the wells were incubated for 1 hour at RT. The tube was washed with water and then incubated with 0.2 μg / mL of HRP-conjugated yam in blocking buffer. Add anti-hIgG Fc (Jackson, Cat. No. 109-035-098) The plates were then incubated at RT for 1 hour. After washing, 50 μl / well of TMB (Neogen Integrator) was added. , Cat. No. 308177) was added and the plate was incubated at RT for 30 minutes. The color development was stopped with 50 μl of 1 M HCl. Spectromax plate reader The absorbance at 450 nm was measured using SoftMaxPro and the data was processed using SoftMaxPro. The data show that all antibodies tested except A054 inhibited VVdd(eGFP) These results demonstrate that the IgG binds to virus particles (Figure 8).

[0244] Example 8 - Effect of antibodies on viral infection A549 cells were cultured in 96-well flat-bottom plates (Flacon, Cat. No. 353075). 5 x 10 cells per well 4 Cells were seeded at 1000 x 1000 cells per well and allowed to adhere overnight at 37°C and 5% CO2. MOI (1.5 x 10 6pfu / mL (MOI 1), 1.5 × 10 5 pfu / mL (MOI 0.1) and 1.5 × 10 4 pfu / mL (MOI 0.01)) VVd d(eGFP) was cultured in serum-free F-12K nutrient mixture (Gibco, Cat. No. 2112 7-022), an equal volume of 20 μg / mL of A047, A048, A049, and A050; A051, A054, hIgG (Sigma, Cat. No. I5154) or 1:10 Vaccinia virus polyclonal antibody (Cedarlane, catalog number LS-C Cells were incubated with VVdd(eGFP) / The cells were infected with the antibody mixture for 6.5 hours at 37°C and 5% CO2, and then cultured in 10% FBS (Corn F-12K Nutrient Mix Supplemented with ing 35-015-CV Lot 35015124) The mixture (Gibco, Cat. No. 21127-022) was added at a 1:2 dilution and incubated overnight. After incubation, the adherent cells were trypsinized and plated in a 96-well V-bottom plate ( and non-adherent cells transferred to the sham tubes (Sarstedt, Cat. No. 82.1583.001). The cells were centrifuged at 450g for 4 minutes and plated at 1:1000 fixable viable cells. Viability dye eFluor 780 (eBioscience, Catalog No. 65-0865 The cells were resuspended in PBS and incubated at room temperature for 15 minutes. , 1 in IC fixation buffer (eBioscience, Cat. No. 00-8222-49) Fix at RT for 10 min, wash with PBS, and finally count using CountBright absolute counting system. 6000 beads per well of PBS (Invitrogen, Cat. No. C36950) The cells were resuspended in 1x PBS with BD LSRFortessa X-20 HTS. Samples were analyzed by flow cytometry on the FlowJo V system, and data were analyzed. The data were processed using 10.5.3 Software. The data showed that the addition of these antibodies significantly increased the expression of tumor cells. These results demonstrate that the IL-16 expression vector does not inhibit or reduce VVdd(eGFP) infection (Figure 9).

[0245] Example 9 - Antibody Thermostability Antibody denaturation temperature (T m ) is Protein Thermo Shift Dye K It™ (ThermoFisher, Cat. No. 4461146) The results were determined by differential scanning fluorescence (DSF). Briefly, 31 μg / mL of antibody was added to each The reaction was performed using the Applied Biosystems B (ABI) kit with the recommended settings described in the kit manual. iosystems QuantStudio 7 Flex Real-Time P Antibody melting curves were generated using the CR System. er Protein Thermal Shift software (v.1.3) was used. and antibody T m (Table 18) were determined. m 1 was determined by DSF. [Table 18]

[0246] Example 10 - Antibody binding to vaccinia Copenhagen-infected mouse cell lines B16F10 (ATCC, Cat. No. CRL-6475), CT26LacZ (AT CC, Catalog No. CRL-2639) and MC38 (John Bell of OHRI) Tumor cells (gift from Dr. Ito) were cultured in 10 cm tissue culture plates (Thermo Scienti fic, Cat. No. 12-556-002, in the appropriate medium (B16F10:D MEM (Gibco, Cat. No. 11995-040), MC38:DMEM (Gib co, Catalog No. 11995-040), CT26LacZ:RPMI1640(Gi bco, Cat. No. 11875-093), 10% FBS (Gibco, Cat. No. Cells were grown to 80-90% confluence in serum-free medium (supplemented with 12483020). V at a multiplicity of infection of 0.5 in DMEM (Gibco, Cat. No. 11995-040) at VCopenhagen (YFP) (gift from Dr. John Bell (OHRI)) Cells were infected and incubated at 37°C in 5% CO for 6.5 hours. The growth medium was supplemented with 100 ml of PBS and further incubated overnight (approximately 16 hours). was trypsinized (TrypLE, Cat. No. 12604021) and placed in a 96-well U-bottom Approximately 1 x 1 per well in a plate (Sarstedt, Cat. No. 83.3925) 0 5 10 cells were plated alongside CT26LacZ and MC38 cells. Corresponding uninfected B16F10, CT26LacZ, and MC38 cells were also cultured in the wells. 1 x 10 5 Cells were plated at 1000 x 1000 cells per well. Cells were plated in PBS 3% FBS (FACs B The cells were washed twice with PBS and centrifuged at 1800 rpm for 3 minutes. and 1:3000 fixable viability dye Zombie NIR (Biolegend The FACS buffer was incubated with 100% Fibroblast Growth Factor (FGF) ... After washing twice with buffer, mouse Fc block (BD, Cat. No. 553141) was added. Resuspend the cells in FACS buffer and incubate at RT for 5 min. Without IgG, cells were incubated with 1:100 anti-A56-PE (A056, Rb IgG), 1:10 anti-B5-PE (A073, Rb IgG) at 1:0, or anti-vaccinia virus at 1:100 Rabbit polyclonal antibody (CedarLane, Cat. No. LS-C103289 ) (which served as a positive control) and incubated for 30 min at 4°C. The cells were washed twice with FACS buffer and resuspended. Anti-vaccinia virus primary antibody was used. The receiving wells were then treated with 1:100 anti-rabbit IgG-PE (Biolegend, Cat. The cells were stained with F (log no. 406421) and incubated for an additional 20 minutes at 4°C. After further washing with AC buffer, IC fixation buffer (eBioscience, catalog no. The cells were fixed in PBS (Product No. 00-8222-49) for 10 minutes, washed twice with PBS, and resuspended in FACS buffer. The cells were suspended and analyzed by flow cytometry on a Cytek Aurora flow cytometer. Data were processed using FlowJoV10.5.3 Software. Data show percent YFP expression in each infected cell line and are shown in Table 1. The percentage of cells infected with gen(YFP) is shown (Fig. 10A). Anti-B5-PE antibodies were positive for YFP and were associated with VVCopenhagen (YFP) infection. It was found to bind to B16F10, CT26LacZ, and MC38 cells, which had been previously reported. As expected, the anti-vaccinia virus rabbit polyclonal antibody Binding to uninfected negative control cells. was not observed.

[0247] Example 11 - Immunohistochemical detection of vaccinia A56 and B5 The HEK-B5(GFP) cell line was cultured on ten 15 cm tissue culture plates (Cornlin in the appropriate medium (10% FBS (Gibco, Cat. No. 353025)). HEK-DMEM (Gibco, catalog no. 12483020) supplemented with 100% ethanol. 11995-040)) to 90% confluence. OS cells (a gift from Dr. John Bell, OHRI) were cultured at 90% confluence. and infected with VVCopenhagen (YFP) at a multiplicity of infection of 0.025. The cells were incubated at 37°C in 5% CO for 72 hours. HEK-WT and U2OS cells were trypsinized (TrypLE, Cat. no. 12 604021), counted, 15 ml polypropylene tubes (FroggaBio, Catalog number TB15-500) in 2 x 10 8 Resuspend at a concentration of 10 cells / ml The cells were centrifuged at 1500 RPM for 5 minutes and then placed in 1 ml of molten Histogel (The Resuspend in 1000kJ / ml (RMO Scientific, Cat. No. HG-4000-012) The histogel was allowed to solidify on ice for 10 minutes. The histogel pellet was then removed by centrifugation in 10 ml of 10 mL of PBS. % neutral buffered formalin (Sigma, Cat. No. HT501128) and incubated at RT for 2 For immunohistochemistry (IHC), histogel sections (4 μm) were incubated at 37°C. Incubate overnight at RT, deparaffinize, and rehydrate using xylene and an ethanol gradient. Divide the cells in a Biocare decloaking chamber (110°C for 15 minutes). a DeCroaker reagent (Biocare, Cat. No. DV2004) was used to recover the antigen. The plates were then reassembled and loaded into the Intellipath FLX Autostainer. Brightfield IHC was performed at room temperature using Ocare reagent. Slides were then washed with Peroxide. ased-1 (Biocare, Cat. No. PX968) for 5 min, and Backg Treat with a round sniper (Biocare, Cat. No. BS966) for 10 minutes. Diluted with DaVinci Green (Biocare, Catalog No. PD900) After applying the primary antibody for 30 minutes, Mach2 HRP Polymer (primary antibody) either mouse or rabbit based on species) (Biocare, Cat. No. RHRP DAB Western Horseradish Perfume (520 and MHRP520) was applied for 30 minutes. Oxidase (Biocare, Cat. No. IPK5010G80) chromogenic substrate was added to the slide. CAT hematoxylin (Biocare, The stained slides were washed with water and then washed with PBS (Cat. No. CATHE) for 5 minutes at room temperature. Air dry and reassemble using Ecomount (Biocare, Cat. No. EM897L). The antibody dilutions were as follows: A058 anti-B5, Rb / Mu IgG2a:1 / 280(10μg / mL);A059 anti-A56, Rb / Mu IgG2 a:1 / 540(10μg / mL);A056 anti-A56, Rb IgG:1 / 840(1 0 μg / mL); A073 anti-B5, Rb IgG: 1 / 550 (10 μg / mL). A5 Antibodies specific for 6 or B5 were used to transfect stably transfected HEK-A56 cells and The target antigens on HEK-B5 cells and HEK-B6 cells were specifically detected (Figure 11). No cross-reactivity was observed with alternative antigens or HEK-WT cells. Both the anti-B5 antibody and the anti-B6 antibody could detect protein expression on infected U2OS cells. No background staining was detected on uninfected U2OS cells (Fig. 11). .

[0248] Example 12 - Immunohistochemical detection of A56 and B5 from VV-treated tumors C57BL / 6 wild-type mice (Jackson Laboratory, catalog no. 000664) in the right rear flank, 1 x 10 6 B16F10 (ATCC catalog number CRL-6475) tumor cells were subcutaneously implanted. Mice were grouped as follows: 1 dose of VVCopenhagen (YFP), 2 doses of VV Copenhagen (Y FP), PBS, and vesicular stomatitis virus (VSV) (Dr. John Bell (O All injections were 1 × 10 in a volume of 100 μl. 7 Dosage: PFU The tumor is 35-40 mm 2 The tumor was delivered into the tumor when it reached a size of 100 μg / cm. The mice were euthanized 1, 2, 3, or 7 days after injection. Tumors were collected and resuspended in 10% neutral buffered saline. The tumors were fixed in chloralmarin for 24 hours. After fixation, the tumors were embedded in paraffin wax. One to five 2 mm tumor punches were collected from each specimen. The tumor punches were arrayed for tumor tissue microarray analysis. A total of 100 TMA sections were prepared and sectioned onto glass slides. For IHC, TMA sections were prepared. (4 μm) was incubated overnight at 37°C, deparaffinized, and then purified with xylene and ethanol. Rehydrate using a gradient and place in a Biocare decloaking chamber (110 °C for 15 min) Diva DeKroker reagent (Biocare, catalog number DV2004) Antigen retrieval was performed using the Intellipath FLX Autostainer Brightfield IHC was performed at RT using Biocare reagents. xidazed-1 (Biocare, Cat. No. PX968) for 5 min, and Ba Ground Sniper (Biocare, Cat. No. BS966) for 10 minutes. Slides were processed with DaVinci Green (Biocare, Cat. No. Primary antibodies diluted in PD900 were applied for 30 minutes, followed by Mach2 HRP Poly mer (either mouse or rabbit based on the species of the primary antibody) (Biocare, Cat. DAB (catalog numbers RHRP520 and MHRP520) was applied for 30 minutes. Sladish peroxidase (Biocare, Cat. No. IPK5010G80 ) Chromogen substrate was added to the slides: CAT hematoxylin diluted 1 / 5 in dH2O (Biocare, Cat. No. CATHE) was added for 5 min at RT. The slides were washed with water, air dried and resuspended in Ecomount (Biocare, Cat. No. EM89 The slides were then coverslipped using anti-A56 (A056) or anti-B (A056). The cells were stained with 5(A073)Rb IgG and detected with anti-rabbit secondary antibody. The antibody dilutions were as follows: As follows: A056 anti-A56, Rb IgG: 1 / 840 (10 μg / mL); A0 73 Anti-B5, Rb IgG: 1 / 550 (10 μg / mL). DAB staining (dark gray) , showing positive protein detection (Figure 12). Antibodies against A56 or B5 The target antigen was specifically detected in the majority of cores taken from tumors with VVC. Increased protein detection was evident in tumors receiving multiple doses of openhagen (YFP). No cross-reactivity was observed against tumors treated with PBS or VSV. These findings support the role of virus-derived A56 and vaccinia virus-derived cytotoxic T cells in tumor cell proliferation after intratumoral delivery of vaccinia virus. The results demonstrate the specific detection and extent of B5 and B6 protein expression.

[0249] Example 13 - Design of VV chimeric antigen receptor (CAR) and its expression after lentiviral transduction detection All human VV-CAR constructs contain the GM-CSFRα leader sequence followed by A56 or B5scFv (derived from A56:A049, B5:A048), human CD8α hinge and transmembrane domain, human 4-1BB intracellular signaling domain, and human CD3 It contains the ζ intracellular signaling domain (Figure 13A) (3). Some CAR constructs contain , a reporter protein (e.g., eG) separated by a T2A ribosomal skip sequence The amino acid sequence of CAR is shown in Table 7.

[0250] A56-CAR-01, A56-CAR-02, B5-CAR-03, B5-CAR- The gene fragment encoding CAR-04, or A56-CAR-06, was obtained from Twist Biosciences. The gene fragment was then transferred to a second-generation transfer plasmid ( The clone was cloned into a donor (gift from Dr. Robert Holt, BC Cancer). Lentivirus encoding the V-CAR construct was transfected using standard calcium phosphate transfer assays. Introduction protocol (4) and second-generation packaging vectors (Rob Holt Each lentiviral transfection was generated using a donor (gift from Dr. BC Cancer). 500 μl of supernatant from the infection was used to infect Jurkat T cells (ATCC, Cataract). The Jurkat cells were then grown for a week and transduced with the IgG1 gene (log number TIB-152). 1×10 6 CAR-positive cells were cultured with goat anti-Rb IgG F(ab')2-Alexa Fluor 647 detection (Jackson Laboratories, Cat. No. 1 The enriched Jurkat CAR-positive population was selected based on the CAR-positive marker (11-605-047). The cells were grown for an additional week and then cultured with HEK-A56, HEK-B5, or HEK-WT cells. Cells were plated in a co-culture assay. The cells were cultured in a 1:1 effector:target (E :T) ratio(1×10 5 The cells were cultured overnight (16 hours) in PBS (total number of cells). and normal goat serum (Jackson Laboratories, Cat. No. 0 05-000-001) and blocked with CD45 v450 (BD, Cat. No. 560 367), CD69 PE-Cy7 (Biolegend, Cat. No. 310912) ) and stained with anti-Rb IgG Alexa Fluor 647. A56-CAR-01 A56-CAR-02 and A56-CAR-06 were cultured with the target HEK-A56 line. Such downregulation of cell surface CAR molecules was observed. The nodes show the activation of the CAR (Figure 14). Similarly, the B5-specific CAR, B5- CAR-03 and B5-CAR-04, when cultured with the target HEK-B5 line, exhibited surface C CAR-T cells were cultured with HEK WT or an irrelevant target antigen. These data support the conclusion that A56-specific or -specific downregulation of CAR was absent when the cells were cultured. Effector T cells transduced to express a B5-specific CAR are then injected with the appropriate antigen. These results suggest that the ATP-dependent activators are activated when they encounter target cells bearing the ATP-dependent activators A56 or B5, respectively. (as evidenced by downregulation of cell surface CAR expression).

[0251] Example 14 - CAR on activated A56-CAR-06 positive Jurkat T cells and eGFP expression The Jurkat A56-CAR-06 line, which co-expresses eGFP, was used in combination with the HEK-A56 line. The cells were cultured overnight (16 hours), and as expected from the results of Example 13, CAR expression was However, within the CAR "negative" population, eGF Furthermore, the eGFP-positive population was associated with a T cell activation marker. showed increased expression of CD69, whereas the eGFP-negative population showed minimal CD69 expression Upregulation of CD69 promotes CAR- These results show that the target with the appropriate antigen (in this case A56) After encountering target cells, CAR-T cells show downregulation of cell surface CAR expression, which However, they can still be distinguished by eGFP and CD69 expression, the latter of which can be targeted by CAR. This provides independent evidence of cell recognition.

[0252] Example 15 - Jurkat T cells expressing VV-CAR have the appropriate target antigen Specific activation when co-cultured with HEK cell lines A56-CAR-01, A56-CAR-02, B5-CAR-03, B5-CAR- Jurkat T cells expressing either A56-CAR-04 or A56-CAR-06 were 1:1 E:T ratio, 1 × 10 per condition 5 Total cell number of HEK-WT, HEK-A56 or co-cultured with HEK-B5 stable cells in triplicate. Cells were incubated overnight at 37°C in 5% CO2. The next day, the cells were washed with PBS, blocked with normal goat serum, and then incubated for 16 hours. Next, the CD45 Jurkat population was stained with CD6, CD69, and anti-RB IgG. 9 expression was assessed. All tested CARs were co-cultured with cells expressing the appropriate target antigen. The results showed increased CD69 expression, but not HEK WT cells or cells expressing an unrelated target antigen. When co-cultured with HEK cells expressing these genes, CD69 expression was minimal (Fig. 16). The findings suggest that CAR-T cells directed against A56 and B5 bind to the appropriate antigen (A56 and B5, respectively). It exhibits specific recognition of target cells expressing B6 and B5, with minimal cross-reactivity and / or It is demonstrated that it exhibits constitutive activity (tonic signaling).

[0253] Example 16 - Healthy donor primary transduced and enriched for A56-CAR-06 expression T cells CD4+ and CD8+ T cell populations were isolated from healthy donor PBMC samples. 10 5 Healthy donor T cells were injected into the Miltenyi TransAct™ (Mil Tenyi Biotec, Cat. No. 130-111-160) and activated with 3% human Serum (Sigma, Cat. No. H4522), gentamicin sulfate (Sandoz, DIN: 02268531), and human interleukin-7 (Miltenyi Biotec, Cat. No. 130-095-367) and interleukin-15 ( Miltenyi Biotec, Cat. No. 130-095-764)) (10 μg / ml) supplemented with Miltenyi TexMACS GMP (Miltenyi B iotec, Cat. No. 170-076-309) medium. 24 hours after activation. After 1 h, T cells were transduced with A56-CAR-06 lentivirus at an MOI of 0.25. Over the next 12 days, cells were plated at 1 x 10 per ml. 6 The cells were grown at subcellular density. After expansion, A56-CAR-06 transduced T cells were analyzed using a flow cytometer. The cytoplasmic fraction was sorted by eGFP positivity. 5 Highly purified cells were selected and The selected population was cultured as described above and plated in a single well of a round-bottom plate. Activation was performed at a cell density of 1 × 10 6 The cells were grown for a further 12 days, maintaining the concentration below 1 / ml. The final expanded and enriched population was >95% CAR-positive by flow cytometry. These findings support the conclusion that primary PBMC-derived human T cells express VV-CAR Can be easily transduced to express the construct and can be enriched and expanded after transduction Shows.

[0254] Example 17—Human T cells expressing A56-CAR-06 are derived from HEK2 T cells expressing A56 Specific activation is observed when co-cultured with 93T strain Human T cells expressing A56-CAR-06 were cultured using HEK-WT, HEK-A56, or HEK-B5 strain and 1 × 10 per condition at an E:T ratio of 1:1 5 Total cell count (1 The cells were co-cultured in triplicate for 6 hours. The next day, the cells were washed with PBS and blocked with normal goat serum. CD3 BV510 (Biolegend, Cat. No. 317332), CD4 5, CD69, CD137 BV650 (Biolegend, Cat. No. 56409 2) The CD3+eGFP+ T cell population was stained to detect the expression of anti-RB IgG. The expression of CD69 and CD137 was assessed by flow cytometry. T cells transduced with 56-CAR-06 were co-cultured with cells expressing the A56 target antigen. The expression of CD69 and CD137 was increased, but not in HEK-WT cells or in the presence of an unrelated antigen. When cocultured with HEK cells expressing CD69 and CD137, expression was minimal (Figure 1). 18) These findings were confirmed in primary T cells transduced to express A56-CAR-06. When cells are co-cultured with target cells expressing the A56 antigen on their surface, they undergo specific activation. Demonstrate what is shown.

[0255] Example 18 - Human T cells expressing A56-CAR-06 inhibit A56-expressing target cells induces morphological signs of cell death HEK-WT, HEK-A56, or HEK-B5 cells (2 × 10 each) 5 24 pieces) The cells were seeded onto well tissue culture plates and left overnight to adhere. The next day, 1 × 10 5 A56 pieces -CAR-06 T cells were added to each well and the cells were cultured for an additional 48 hours. A clear response was observed in the HEK-A56 target cell line when co-cultured with A56-CAR-06-positive T cells. They showed morphological changes, indicating direct tumor cell killing. T and HEK-B5 lines remained fully confluent without morphological changes. These findings were consistent with those of HEK-16 T cells after co-culture with A56-CAR-06 T cells (Figure 19). A56 cell monolayers were shown to be disrupted, providing evidence of a direct cytopathic effect. There are.

[0256] Example 19—Human T cells expressing B5-CAR-011 bind to B5-expressing target cells Cytotoxicity The mammalian expression vector pCCL-Luc encodes firefly luciferase. romycin (Dr. Jonathan Bramson, McMaster University) lentivirus was generated as described above in Example 13 using the ELISA kit (gift from the University of California, Santa Cruz, CA). The luciferase-encoding lentivirus was used to transfect HEK-WT, A56, and B5 The strain was transduced with puromycin (1.5 μg / ml, Sigma, Cat. No. P8 Resistance to puromycin (833) was selected for 2 weeks. The culture was grown and used as the target population for the VV-CAR killing assay. Target HEK lines expressing the enzyme were cultured in 96-well plates (Corning, Cat. no. 3 917) 2 x 10 per well 4 Cells were seeded in 100 μl and incubated overnight. Sorted primary human B5-CAR-011 T cells previously expanded were allowed to adhere. The resulting population was plated in triplicate in a 96-well plate (100 μl per well), Dividing into two-fold serial dilutions achieved an E:T ratio range of 10:1 to 0.625:1. The diluted T cell suspension was transferred to adherent HEK cells in a total volume of 200 μl. To determine the maximum and minimum relative luminescence units (RLU), three wells of target cells only were used. Three wells were plated with medium alone. The cells were cultured at 37°C in 5% CO2 for 24 hours. The next day, each well was filled with Xenolight™ D-luciferin (Perkin E) Add 22 μl of 10X stock of 10% ethanol (Imer, Cat. No. 122799) and store at room temperature in the dark. The plate was then incubated for 10 minutes. Elmer Wallac Envision 2104 Multilabel Re Figure 20A shows triplicate scans at each E:T ratio at 24 hours. The average RLU from triplicate wells is shown. Triplicate wells were averaged and percent specific cytotoxicity was calculated as follows: The percent specific cytotoxicity was determined by the formula: Percent specific cytotoxicity = 100 × (maximum luminescence RLU - test luminescence) RLU) / (minimum luminescence RLU-maximum luminescence RLU). B5-CAR-011 T cells The B5-expressing cells showed high percent specific cytotoxicity at several E:T ratios (Fig. 20B). B5-expressing target cells co-cultured with CAR-011 T cells showed clear cell death within 24 hours. Although they showed morphological signs, B5-CAR-011T cells did not show any significant differences with HEK293WT or HEK The compound showed no signs of cytotoxicity against 293A56 cells (Fig. 20C). 5-CAR-011 T cells efficiently and specifically killed HEK-B5 tumor cells. (Figure 20) By targeting OV antigens expressed on the surface of tumor cells, It can make cells more susceptible to destruction by OV-CAR.

[0257] Example 20 - Radiolabeled anti-A56 antibodies detect A56 in vivo by positron emission tomography Allows clear visualization of 6-expressing tumor cells Selected anti-A56 antibody (A049) for biodistribution studies and microPET imaging For zirconium-89( 89 This radiolabeled the cells in live mice. This allows for deep tissue imaging with high sensitivity and spatial resolution (5, 6). 89 Zr Its long half-life (78 hours) and favorable decay characteristics (6, 7) make it suitable for use with mAbs. Two milligrams of A049 was used at a 3:1 chelator:mAb molar ratio. p-SCN-Bn-deferoxamine (1-(4-isothiocyanatophenyl)-3-[ 6,17-dihydroxy-7,10,18,21-tetraoxo-27-(N-acetyl hydroxylamino)-6,11,17,22-tetraazahepteicosine]thiourea, It was conjugated with DFO (Macrocyclics, Plano, TZ, USA). The reaction was carried out in PBS pH 9 at 37°C for 1 hour at a final antibody concentration of 2 mg / mL. Next, the mixture was passed through a centrifugal filter unit (Amicon Ultracentrifuge) with a molecular weight cutoff of 50 kDa. Cardiac filter, Ultracel-50: regenerated cellulose, Millipore Cor (Billerica, MA, USA) to conjugate antibodies (DF O-A049) was purified from unconjugated deferoxamine and diluted in 0.25M sodium acetate. 5 mg / mL 2,5-dihydroxybenzoic acid (Sigma-Aldric) in HCl solution The purified immunoconjugate was washed once with PBS (Oakville, Ontario, Canada). The concentration of the blood filter solution was adjusted according to the manufacturer's recommendations (Sigma-Aldrich). Determined by Fold assay.

[0258] 89 Radiolabeling of immunoconjugates with Zr As previously mentioned (8), the ACSI (Richmond, Canada) TR-19 cyclotron Yttrium-89 disc (American Elements (USA, CA) By proton irradiation at 13 MeV onto a quartz crystal (Los Angeles, California), 89 Zr After irradiation, the disks were dissolved in HCl (10 mL, 2 M) and Triske (B The refinement was performed using a hydroxamate-based ZR resin supplied by Brütz, France. Briefly, the disk solution was loaded onto the resin and 10 mL of 2 M HCl was added. solution, followed by washing with 10 mL of water, and then with 0.5 mL of oxalic acid (0.05 M). I put it out.

[0259] For radiolabeling, immunoconjugate DFO-A049 (0.4 mg) and 89 Dissolve Zr solution (0.4 mL, pH 7, 232 MBq) in PBS (final volume: 1 mL, pH 7) and the resulting solution was incubated at room temperature for 1 hour. Radiochemical Yield (RCY) used 50 mM DTPA pH 7 (Sigma-Aldrich) as the solvent. Instant thin-layer chromatography silica gel (iTLC-SG, Agilent) determined using the NIRS Technologies (Santa Clara, CA, USA). Ta. 89 Zr-labeled antibody and free 89 The Rf values of Zr were 0 and 1, respectively. 96 Efficient labeling with % RCY was observed. 89 Zr-labeled antibody desalted with PD-10 column (GE Healthcare, London, UK). 89 Zr to min and filtered using a 50 kDa molecular weight cutoff filter (Millipore Corp.). It was concentrated using 89The specific activity of Zr-DFO-A049 was determined using a size-exclusion HPLC column ( BioSep-SEC-s3000, Phenomenex (California, USA) Torrance) using a Model 1200 Quaternary Pump and a Model 1200 UV Absorber. photometric detector, and Bioscan (Washington, DC, USA) NaI scintillation detector An Agilent HPLC system (Santa Clara, CA, USA) equipped with a detector was used. The HPLC buffer was 0.1M sodium phosphate monobasic dihydrate, 0. 1M sodium phosphate dibasic dodecahydrate, 0.1M sodium azide and 0.15M salt The gradient was an isocratic gradient of sodium chloride (pH 6.2-7.0). A specific radioactivity of 1000 / µg was obtained, sufficient for the in vivo characterization of the radioimmunoconjugates. The final radiochemical purity was determined using iTLC-SG as previously described and was >9 The rate was 9.9%.

[0260] Antibody immunoreactivity 89 The immunoreactive fraction of Zr-DFO-A049 was identified as A56 antigen (Wyeth VV sequence). HEK-293 cells stably modified to express IL-16 (HEK-A56) were used. Briefly, HEK-A56 cells were incubated with 1000 μg of ... 1.0~24.6×10 6 The cells were suspended in PBS (pH 7.4) at various concentrations of cells / mL. The remaining steps were performed as previously described (9). 89 Zr-DFO-A049 5 2% still showed efficient binding to the A56 protein.

[0261] Animal tumor models and antibody injections All animal experiments were conducted at the University of British Columbia. British Columbia Animal Care Commission mmittee (Vancouver, British Columbia, Canada) Institutional Guide The study was conducted at the BC Cancer Research Centre in accordance with the guidelines and under the supervision of accredited researchers. Animal Resources Research Centre The experiments were performed at the Center for Immunodeficiency Disease (NOD.Cg-Rag1tm1M) in 12-week-old female mice. om Il2rgtm1Wjl / SzJ(NRG) mice (obtained from an in-house breeding colony) HEK-A56 cells were injected subcutaneously into the left shoulder of each mouse, or non-A56-expressing HEK-293 parental cells were injected subcutaneously into the right shoulder of each mouse. (n=8 mice). For both cell lines, Matrigel (ratio 1:1, B 5 × 10 in D Bioscience (Mississauga, Ontario, Canada). 6 Individual cells Injected.

[0262] Positron Emission Tomography (PET) The tumor is about 100 mm 3 When the mice reached a normal blood pressure of 1000kJ / kg, they were placed in 2% i.m. oxygen. Anesthetized with soflurane 89 Zr-DFO-A049 (40.9±1.5μg, 4.1±0 The patient was given an intravenous injection of 0.2 MBq. Siemens (Knoxville, Tennessee, USA) Using the nveon microPET / CT scanner, we analyzed the data for one mouse injected PET images were acquired 1, 3, and 5 days after administration. Mice were also treated with C for decay correction. T scans were performed, followed by 20-minute static PET acquisitions on days 1 and 3, and on day 5. The eyes were scanned for 30 minutes. Three-dimensional conditional subset expectation maximization (3-di mensional ordered-subsets expectation ma simization (OSEM3D, 2 iterations) followed by a fast maximization pre-algorithm. Fast maximum a priori algorithm (Fa Images were reconstructed using a tMAP (18 iterations). Maximum intensity projection images were obtained using the mouse. in areas of high bone remodeling, especially in the epiphysis of long bones 89 Zr-DFO conjugate In addition to bone uptake due to the known demetallization of ATP, antibody processing and metabolism may also contribute to bone uptake. The study showed the expected accumulation in normal organs, such as the liver and spleen, which are involved in the administration of benzodiazepines (10). Notably, PET images showed that HEK-A56 tumors were significantly more abundant than negative control tumors (HEK-293). showed higher tumor uptake rates and tumor-to-background ratios for 89 This demonstrates specific and efficient tumor uptake of the Zr-DFO-A049 antibody (Figure 21). .

[0263] 89 Evaluation of Zr-DFO-A049 biodistribution On day 5, all mice were euthanized and biodistribution assays were performed as previously described ( 9 ). Organs of interest were collected for study. 89 The results for Zr-DFO-A049 are shown in Table 1. Shown in Figure 9. [Table 19]

[0264] Biodistribution results showed that the radioimmunoconjugate biodistribution was consistent with the expected distribution in normal organs. The PET imaging observations were confirmed with the intracellular distribution of the tumor. in A56-expressing HEK tumors compared with 1.44 ± 0.25% ID / g in tumors A high uptake of 7.71±4.52%ID / g was obtained (n=8, p<0.05). Therefore, these in vivo results (PET imaging and biodistribution studies) 89 Zr-DFO-A049 can be effectively and selectively delivered to A56-expressing tumors. This proves that:

[0265] Oncolytic virus-encoded proteins expressed on the surface of OV-infected tumor cells When antibodies targeting a target molecule are labeled with a radionuclide, they can be visualized by, for example, PET imaging. In vivo imaging of OV infection and radioimmunotherapy with therapeutic radioisotopes Local delivery to kill OV-infected cells and uninfected adjacent tumor cells It can be used to enable diagnostic, therapeutic, and theranostic applications (11 ,12).

[0266] References 1.Babcook, JS, Leslie, KB, Olsen, OA, Sa lmon, RA, Schrader, JW (1996) A novel str ategy for generating monoclonal antibodies es from single, isolated lymphocytes prod ucing antibodies of defined specificity s.Proc Natl Acad Sci USA 93(15):p.7843 -8. 2.Jarahian, M., Fiedler, M., Cohenen, A., Djand ji,D.,Hammerling,G.J.,Gati,C.,Cerwenka,A .,Turner,P.C.,Moyer,R.W.,Watzl,C.,Hengel ,H.,and Momburg,F.(2011)Modulation of NK p30- and NKp46-mediated natural killer c ell responses by poxviral hemagglutinin. PLoS Pathog 7,e1002195. 3.S.Guedan,H.Calderon,A.D.Posey,M.V Maus ,Engineering and Design of Chimeric Anti gen Receptors,Mol.Ther.Methods Clin.Dev. 12(2019)145-156.https: / / doi.org / 10.1016 / j.omtm.2018.12.009. 4.Molecular Cloning:A Laboratory Manual 3 rd edition(eds.Sambrook,J.& Russell,D.W .)(Cold Spring Harbor Laboratory Press,C old Spring Harbor,New York,USA,2001). 5.Kim JS.Combination Radioimmunotherapy Approaches and Quantification of Immuno- PET.Nuclear medicine and molecular imagi ng.2016;50(2):104-11. 6.van de Watering FC,Rijpkema M,Perk L,B rinkmann U,Oyen WJ,Boerman OC.Zirconium- 89 labeled antibodies:a new tool for mol ecular imaging in cancer patients.BioMed research international.2014;2014:203601 . 7.Zhang Y,Hong H,Cai W.PET tracers based on Zirconium-89.Current radiopharmaceut icals.2011;4(2):131-9. 8.Dias GM,Ramogida CF,Rousseau J,Zacchia NA,Hoehr C,Schaffer P,Lin K-S,Benard F. 89 Zr for antibody labeling and in vivo s tudies - A comparison between liquid and solid target production.Nucl Med Biol.2 018 Mar;58:1-7. 9.Rousseau J,Zhang Z,Wang X,Zhang C,Lau J,Rousseau E,et al.Synthesis and evaluat ion of bifunctional tetrahydroxamate che lators for labeling antibodies with(89)Z r for imaging with positron emission tom ography.Bioorg Med Chem Lett.2018;28(5): 899-905. 10.Rousseau J, Zhang Z, Dias GM, Zhang C, Co lpo N,Benard F,Lin KS.Design,synthesis and evaluation of novel bifunctional tet rahydroxamate chelators for PET imaging of 89 Zr-labeled antibodies.Bioorg Med Ch em Lett.2017;27(4):708-712. 11.Makvandi M.,et al.Alpha-Emitters and Targeted Alpha Therapy in Oncology:from Basic Science to Clinical Investigations .Target Oncol.2018 Apr;13(2):189-203. 12.Kraeber-Bodere F,Bodet-Milin C,Rousse au C, Eugene T, Pallardy A, Frampas E, et al. .Radioimmunoconjugates for the treatment of cancer.Seminars in oncology.2014;41( 5):613-22.

[0267] Thus, the foregoing merely illustrates the principles of the present disclosure. Although not described or illustrated herein, it embodies the principles of the present invention and is included within its spirit and scope. It will be appreciated that those skilled in the art will be able to devise various configurations for implementing the present invention. All examples and conditional language listed herein are based primarily on the principles of the present invention and the inventors' It is intended to aid the reader in understanding the concepts contributed to the advancement of the art. and should not be construed as a limitation to such specifically listed examples and conditions. The principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are also listed. All statements herein that relate to a compound or a derivative thereof are intended to encompass both structural and functional equivalents thereof. Additionally, such equivalents include both currently known equivalents and those developed in the future. This includes both the original and its equivalents, i.e., any development element that performs the same function regardless of structure. Accordingly, the scope of the present invention is intended to encompass all of the examples shown and described herein. It is not intended to be limited to the illustrated embodiments.

Claims

[Claim 1] The invention described in the specification and drawings of this application.

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