Genetically engineered oncolytic herpes simplex viruses that deliver chemokines and tumor-associated / specific antigens

Abstract

Disclosed is a genetically modified oncolytic herpes simplex virus (oHSV) encoding a truncated non-signaling variant of at least one tumor-associated / specific antigen and at least one chemokine. The expression of the truncated non-signaling variant and the chemokine is controlled by an immediate early gene promoter of HSV, and upon replication of the oHSV in tumor cells, the truncated non-signaling variant is expressed and presented as a biomarker on the surface of the tumor cells, and the chemokine is expressed and released to induce chemotaxis of immune cells to tumor cells. The genetically modified oHSV can be used in combination with CAR-T, ADC and / or BiTE therapy.

Description

[Technical Field]

[0001] The present disclosure relates to oncolytic herpes simplex viruses genetically engineered to carry a gene encoding at least one chemokine and / or at least one tumor-associated / specific antigen, and their use in combination with tumor-targeted therapeutics, including CAR-T, BiTE, and ADC, for the treatment of various tumors. [Background technology]

[0002] Adoptive transfer of CAR-T cells into patients with hematological malignancies has shown remarkable results. However, this approach has been largely ineffective in patients with solid tumors. It appears unlikely that CAR-T cell therapy alone will be sufficient to induce a complete response in most cancers. Combining CAR-T cells with other cancer treatments that have different mechanisms of action and may synergize with T cells may reduce tumor escape and increase the success rate of CAR-T cell therapy.

[0003] Oncolytic virotherapy is a cancer treatment method that uses native or genetically modified viruses that selectively replicate in cancer cells. Following the FDA approval of Talimogene laherparepvec (T-VEC), an oncolytic herpes simplex virus type 1 (HSV-1) modified to express GM-CSF, the field of oncolytic virotherapy has attracted renewed attention. Furthermore, oncolytic viruses (OVs) can be further modified to selectively deliver therapeutic transgenes to the tumor microenvironment, thereby improving their antitumor efficacy or promoting antitumor immune responses. Preclinical studies using CAR-T cells in combination with oncolytic viruses equipped with cytokines, chemokines, BiTEs, or immune checkpoint inhibitors have demonstrated improved therapeutic efficacy. For example, oncolytic adenoviruses modified to express IL-15 and RANTES or IL-2 and TNF-α have been shown to increase the accumulation and survival of CAR-T cells in the tumor microenvironment. Vaccinia virus expressing the CXCR3 ligand CXCL11 was used to attract effector cells after transduction to improve intratumoral delivery of CAR-T cells. Another report demonstrated that expression of an oncolytic adenovirus BiTE targeting a second tumor antigen can address heterogeneity in antigen expression. As expected, all of these combinations of CAR-T cells and armed OVs resulted in improved tumor control and prolonged survival compared with monotherapy with each agent.

[0004] Recent studies have engineered oncolytic viruses to express a non-signaling truncated CD19 (CD19t) protein for tumor-selective delivery, enabling targeting of CD19-CAR T cells. Infection of tumor cells with an oncolytic vaccinia virus encoding CD19t (OV19t) resulted in de novo production of CD19 on the cell surface prior to virus-mediated tumor lysis. Cocultured CD19-CAR T cells secreted cytokines and demonstrated effective cytolytic activity against infected tumors. Using several mouse tumor models, delivery of OV19t promoted tumor control after administration of CD19-CAR T cells. OV19t induced local immunity characterized by tumor infiltration of endogenous and adoptively transferred T cells. CAR T-cell-mediated tumor killing also induced virus release from dying tumor cells, thereby promoting CD19t expression in tumors. In the study, more than 50% of mice treated with this combination therapy were cured, although some mice only responded temporarily or did not respond at all (Park et al., Sci.Transl.Med.12,eaaz1863(2020)).

[0005] US20190233536A1 discloses a modified adenovirus, specifically Enadenotucirev (EnAd), equipped with at least two bispecific T cell engagers (BiTEs), each containing at least two binding domains, at least one of which is specific for a surface antigen on a target immune cell, e.g., a target T cell. By equipping an adenovirus with BiTE molecules, the bispecific antibody fragment molecules can "piggyback" on the adenovirus's ability to selectively infect cancer cells, thereby enabling targeted delivery of the BiTE to tumor cells. Upon adenovirus infection, the BiTE molecules are synthesized and secreted by tumor cells, acting locally and spreading beyond the virus's direct footprint. This therefore allows the BiTE to spread beyond the immediate site of infection, but limits viral spread far beyond the infected tumor cell nest. This minimizes the risk of undesired off-target effects. Summary of the Invention

[0006] A first aspect of the present disclosure relates to a genetically modified oncolytic herpes simplex virus (oHSV), the genome of which incorporates a polynucleotide encoding (a) a truncated, non-signaling variant of at least one tumor-associated / specific antigen and (b) at least one chemokine, wherein expression of the truncated, non-signaling variant and at least one chemokine is controlled by an immediate-early gene promoter of HSV, and during replication of the oHSV in tumor cells, the truncated, non-signaling variant is expressed and presented on the surface of the tumor cells as a biomarker, and at least one chemokine is expressed and released to induce chemotaxis of immune cells to the tumor cells.

[0007] Another aspect of the present disclosure relates to a genetically modified oncolytic herpes simplex virus (oHSV), wherein the genome of the oHSV incorporates a polynucleotide encoding a truncated, non-signaling variant of at least one tumor-associated / specific antigen, the expression of the truncated, non-signaling variant is controlled by an immediate early gene promoter of the HSV, and upon replication of the oHSV in tumor cells, the truncated, non-signaling variant is expressed and presented as a biomarker on the surface of the tumor cells.

[0008] Another aspect of the present disclosure relates to a pharmaceutical kit for the treatment of cancer comprising, separately, a genetically modified oncolytic herpes simplex virus (oHSV) as described herein and a tumor-targeted therapeutic agent, the tumor-targeted therapeutic agent comprising a targeting moiety specific for a truncated, non-signaling mutant of at least one tumor-associated / specific antigen encoded by a polynucleotide, and an effector moiety for killing or inhibiting the growth of cancer cells.

[0009] Another aspect of the present disclosure relates to a method for treating cancer in a subject, comprising simultaneously or sequentially administering to the subject a pharmaceutically effective amount of a genetically modified oncolytic herpes simplex virus (oHSV) and a tumor-targeted therapeutic agent, wherein the genome of the oHSV incorporates a polynucleotide encoding (a) a truncated, non-signaling mutant of at least one tumor-associated / specific antigen and preferably (b) at least one chemokine, the expression of which is controlled by an immediate-early gene promoter of an HSV, and the tumor-targeted therapeutic agent comprises a targeting moiety specific for the truncated, non-signaling mutant of at least one tumor-associated / specific antigen encoded by the polynucleotide, and an effector moiety for killing or inhibiting the growth of cancer cells.

[0010] Further aspects of the present disclosure may be gleaned from the detailed description set forth below with reference to the drawings. [Brief explanation of the drawings]

[0011] [Figure 1] Figure 1 shows schematic diagrams of the oHSV backbones of T7201, T7202, T7203, T7204, T7011, T7012, and T7013 (collectively referred to as the "T7 series"). (A) Schematic diagram of T3011, a genetically modified oHSV encoding hPD-1 antibody (hPD-1-Ab) (an immune checkpoint inhibitor) and the cytokine hIL-12, in which the internal inverted repeat regions (b'a' and a'c') are replaced with a polynucleotide encoding hIL-12, and an expression cassette for hPD-1-Ab is introduced between the UL3 and UL4 genes of the UL fragment. A more detailed description of T3011 is available in WO2017 / 181420 (IMMV503), the disclosure of which is incorporated herein by reference in its entirety. (B) Schematic diagram of exemplary T7 series viruses described herein. These genetically modified oHSVs encode hPD-1-Ab, hIL-12, a tumor-associated antigen, and a chemokine, with the internal inverted repeat regions (b'a' and a'c') replaced by a polynucleotide encoding hIL-12. An expression cassette for hPD-1-Ab is inserted between genes UL3 and UL4 of the UL fragment, and a TAA + chemokine expression cassette is inserted between genes UL37 and UL38 of the UL fragment. (C) Schematic diagram of exemplary genetically modified oHSVs T7201, T7202, T7203, and T7204. In inset B, the TAA + chemokine expression cassette is embodied as one TAA (a truncated, non-signaling variant of a tumor-associated antigen) and one chemokine. (D) Schematic diagram of exemplary genetically modified oHSVs T7011, T7012, and T7013. In inset B, the TAA+chemokine expression cassette is embodied as two different TAAs plus one chemokine, designated TAA1+TAA2+chemokine. The HSV-IE (immediate early) promoter and PolyA tail are located upstream and downstream of the expression cassette, respectively. [Figure 2]2 shows a flow chart of the construction of T7 series oHSVs (i.e., T7011 to T7013 and T7201 to T7204 described above). The construction involves several steps of cloning using the bacterial artificial chromosome (BAC) system. [Figure 3] Figure 3 shows the release of CCL5 after T7011 infection in 293T, HEp-2, and Tca8113 cells. CCL5 expression and release were rapid and robust. Secreted CCL5 was detected as early as 4 hours postinfection, reaching a maximum of 5,000 pg / mL. After T7011 virus infection, secretion was stable and maintained for at least 4 days, demonstrating that CCL5 is secreted. [Figure 4] Figure 4 shows the expression of cleaved CD19, BCMA, Trop-2, and HER2 on the cell surface. Different cleaved antigens, encoded by T7011, T7012, and T7013, respectively, were simultaneously expressed on the tumor cell surface. [Figure 5] Figure 5 shows the antitumor effects of T7 series (T7011, T7012, and T7013) oHSV viruses. The IC values of the T7 series oHSV viruses were comparable to that of T3011, indicating that the T7 series viruses possess similar broad antitumor activity compared to T3011. [Figure 6] Figure 6 shows that T7 series (T7011, T7012, and T7013) oHSV viruses have no infectious activity in either CAR-T cells or normal T cells. [Figure 7] Figure 7 shows that T7 series (T7011, T7012, and T7013) oHSV viruses have no cell killing activity in either CAR-TCD19 cells or normal T cells. [Figure 8] Figure 8 shows that the combined treatment of T7011 and CAR-TCD19 significantly improved the antitumor effect. [Figure 9] Figure 9 shows that T7011 virus infection can specifically synergize with the antitumor activity of CAR-TCD19. [Figure 10]Figure 10 shows that the combined treatment of T7012 and CAR-TCD19 significantly improved the antitumor effect. [Figure 11] Figure 11 shows that T7012 virus infection can specifically synergize with the antitumor activity of CAR-TCD19. [Figure 12] Figure 12 shows that the combined treatment of T7013 and CAR-TCD19 significantly improved the anti-tumor effect. [Figure 13] Figure 13 shows that T7013 virus infection can specifically synergize with the antitumor activity of CAR-TCD19. [Figure 14] Figure 14 shows that T7 series (T7011, T7012, and T7013) oHSV viruses lack cell killing ability in either CAR-NKCD19 or NK cells. [Figure 15] Figure 15 shows viral replication of HSV-1(F) and T7011 in CAR-NKCD19 and NK cells. [Figure 16] Figure 16 shows that T7011 does not negatively affect the proliferation of CAR-NKCD19 cells. *p<0.05, ***p<0.001. [Figure 17] Figure 17 shows that T7011 has no negative effect on NK cell proliferation. *p<0.05, **p<0.01, ***p<0.001. [Figure 18] Figure 18 shows that the combined treatment of T7011 and CAR-NKCD19 significantly improved the anti-tumor effect. [Figure 19] Figure 19 shows that T7011 viral infection can specifically synergize with the antitumor activity of CAR-NKCD19.

[0012] "Detailed Description of the Invention" Definition: It should be noted that the term "one" or "an" entity refers to one or more of that entity. For example, "one truncated, non-signaling variant" is understood to refer to one or more truncated, non-signaling variants. Thus, the terms "one or more" and "at least one" can be used interchangeably herein.

[0013] As used herein, the term "antibody fragment" or "antigen-binding fragment" refers to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. The term "antibody fragment" includes aptamers, spiegelmers, and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

[0014] Antibodies, antigen-binding polypeptides, variants, or derivatives thereof of the present disclosure include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, or chimeric antibodies, single-chain antibodies, epitope-binding fragments such as Fab, Fab', and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv)), fragments comprising the VK or VH domains, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies against the LIGHT antibodies disclosed herein). Immunoglobulin or antibody molecules of the present disclosure may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule. For example, an anti-PD-1 antibody may refer to an antigen-binding fragment thereof, such as a Fab fragment or an scFv thereof.

[0015] "Specifically binds," "specific for," or "having specificity for" generally mean that an antibody binds to an epitope via its antigen-binding domain and that such binding involves some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope if it binds to the epitope via its antigen-binding domain more readily than it would to a random, unrelated epitope. The term "specificity" is used herein to quantify the relative affinity of a particular antibody for binding to a particular epitope. For example, antibody "A" may be considered to have higher specificity for a given epitope than antibody "B," or antibody "A" may be said to bind epitope "C" with greater specificity than to the related epitope "D."

[0016] As used herein, "cancer" or "tumor," used interchangeably herein, refer to a group of diseases involving abnormal cell growth that can invade or spread to other parts of the body and that can be treated according to the present disclosure. Not all tumors are cancerous; benign tumors do not spread to other parts of the body. Possible signs and symptoms include a new lump, abnormal bleeding, persistent cough, unexplained weight loss, changes in bowel movements, etc. There are over 100 known types of cancer that affect humans. As used herein, "cancer" includes, but is not limited to, solid cancers (e.g., tumors) and hematologic malignancies. "Hematologic malignancies," also known as blood cancers, are cancers that originate in blood-forming tissues, such as the bone marrow or other cells of the immune system. Hematological malignancies include leukemias (e.g., acute myeloid leukemia (AML), acute promyelocytic leukemia, acute lymphocytic leukemia (ALL), acute mixed lineage leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia (CLL), hairy cell leukemia, and large granular lymphocytic leukemia), myelodysplastic syndromes (MDS), myeloproliferative disorders (polycythemia vera, essential thrombocythemia, primary myelofibrosis, and chronic myeloid leukemia). ), lymphoma, multiple myeloma, MGUS and similar disorders, Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), primary mediastinal large B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, transformed follicular lymphoma, splenic marginal zone lymphoma, lymphocytic lymphoma, T-cell lymphoma, and other B-cell malignancies. "Solid tumors" include, but are not limited to, bone cancer, pancreatic cancer, skin cancer, cancer of the head and neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, prostate cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood tumors, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, tumors of the spinal axis, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, and environmentally induced cancers, including asbestos-induced cancer.

[0017] As used herein, the term "treatment" or "treatment" refers to both therapeutic treatment and prophylactic measures, the purpose of which is to prevent or slow (alleviate) an undesirable physiological change or disorder, such as, for example, the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of the disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, improvement or palliation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Subjects in need of treatment include those already with the condition or disorder as well as those susceptible to the condition or disorder, or those in whom the condition or disorder is to be prevented.

[0018] "Subject" or "individual" or "animal" or "patient" or "mammal" means any subject for whom diagnosis, prognosis, or treatment is desired, particularly a mammalian subject. Mammalian subjects include humans, livestock, farm and zoo animals, sport animals, or pets, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, dairy cows, etc.

[0019] As used herein, phrases such as "patient in need of treatment" or "subject in need of treatment" include subjects, e.g., mammalian subjects, who would benefit from the administration of oHSV-1 or compositions of the disclosure, e.g., for detection, diagnostic procedures, and / or treatment.

[0020] It will also be understood by those skilled in the art that the modified genomes disclosed herein may be modified such that they differ in nucleotide sequence from the modified polynucleotide from which they were derived. For example, a polynucleotide or nucleotide sequence derived from a designated DNA sequence may be similar to the starting sequence, e.g., have a certain percent identity to the starting sequence, e.g., it may be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the starting sequence.

[0021] Additionally, nucleotide or amino acid substitutions, deletions, or insertions may be made that result in conservative substitutions or changes in "non-essential" amino acid regions. For example, a polypeptide or amino acid sequence derived from a designated protein may be identical to the starting sequence except for one or more distinct amino acid substitutions, insertions, or deletions, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more distinct amino acid substitutions, insertions, or deletions. In certain embodiments, a polypeptide or amino acid sequence derived from a designated protein has 1 to 5, 1 to 10, 1 to 15, or 1 to 20 distinct amino acid substitutions, insertions, or deletions relative to the starting sequence.

[0022] A "therapeutically effective amount" or "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount may vary depending on factors such as, for example, the individual's disease state, age, sex, and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, an improvement in the patient's health status, a reduction in tumor burden, a halt or slowing of tumor growth, and / or a lack of metastasis of cancer cells to other sites in the body.

[0023] CAR-T cells are T cells that express a chimeric antigen receptor. T cells expressing a CAR molecule may be helper T cells, cytotoxic T cells, virus-specific cytotoxic T cells, memory T cells, or gamma delta (γδ) T cells. A chimeric antigen receptor (CAR) is a recombinant fusion protein that includes 1) an extracellular ligand-binding domain, i.e., an antigen-recognition domain, 2) a transmembrane domain, and 3) a signaling domain. The extracellular ligand-binding domain is an oligo- or polypeptide capable of binding to a ligand. Preferably, the extracellular ligand-binding domain can interact with a cell surface molecule, which may be an antigen, receptor, peptide ligand, target protein ligand, or target polypeptide. In the present disclosure, the extracellular ligand-binding domain can interact with a truncated, non-signaling mutant of a tumor-associated antigen or tumor-specific antigen.

[0024] Typically, the extracellular ligand-binding domain is linked to the signaling domain of the chimeric antigen receptor (CAR) by the transmembrane domain I. The transmembrane domain traverses the cell membrane, anchors the CAR to the surface of the T cell, connects the extracellular ligand-binding domain to the signaling domain, and influences the expression of the CAR on the surface of the T cell. The transmembrane domain may further comprise a hinge region between the extracellular ligand-binding domain and the transmembrane domain. The term "hinge region" generally refers to any oligo- or polypeptide that functions to connect the transmembrane domain to the extracellular ligand-binding domain. In particular, the hinge region is used to provide greater flexibility and accessibility to the extracellular ligand-binding domain. The hinge region may comprise up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids. The hinge region may be derived from all or a portion of a naturally occurring molecule, such as CD28, 4-1BB (CD137), OX-40 (CD134), CD3ζ, T-cell receptor α or β chain, CD45, CD4, CD5, CD8, CD8α, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, ICOS, CD154, or from all or a portion of an antibody constant region. Alternatively, the hinge region may be a synthetic sequence that corresponds to a naturally occurring hinge sequence, or the hinge region may be an entirely synthetic hinge sequence.

[0025] Chimeric antigen receptors (CARs) further comprise a signaling domain or intracellular signaling domain of the CAR, responsible for intracellular signaling following binding of the extracellular ligand-binding domain to a target, resulting in immune cell activation and an immune response. In other words, the signaling domain is involved in activating at least one of the normal effector functions of the immune cell in which the CAR is expressed. For example, the effector function of a T cell may be cytolytic activity, including cytokine secretion, or helper T cell activity. Thus, the term "signaling domain" refers to a portion of a protein that transmits an effector signal and instructs the cell to perform a specialized function. Examples of signaling domains for use in CARs include the cytoplasmic sequences of T cell receptors and coreceptors that act in concert to initiate signaling following antigen receptor binding, any derivatives or variants of these sequences, and any synthetic sequences with the same function. Signaling domains include two different classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation and those that act antigen-independently to provide secondary or costimulatory signals. Primary cytoplasmic signaling sequences may contain signaling motifs known as ITAMs, immunoreceptor tyrosine-based activation motifs. ITAMs are well-defined signaling motifs found in the cytoplasmic tails of various receptors that function as binding sites for the syk / zap70 class of tyrosine kinases. Non-limiting examples of ITAMs that can be used in the present disclosure may include those derived from TCRζ, FcRγ, FcRβ, FcRε, CD3γ, CD3δ, CD3ε, CDS, CD22, CD79a, CD79b, and CD66d. In one embodiment, the signaling domain of the CAR may comprise a CD3ζ signaling domain comprising an amino acid sequence of at least 80%, 90%, 95%, 97%, or 99% sequence identity. The present disclosure contemplates, without limitation, the combined use of any CAR-T with the engineered oHSV described herein.

[0026] A typical antibody-drug conjugate (ADC) contains a monoclonal antibody that can bind to a specific surface antigen on cancer cells. These antibodies include several proteins found on the surface of immune system B and T cells, such as CD20, CD22, human epidermal growth factor receptor 2 (Her2), and prostate-specific membrane antigen (PSMA). These antibodies are connected to a highly toxic drug by a cleavable linker unit. The drug is designed to trigger apoptosis of cancer cells by inducing irreversible DNA damage or disrupting cell division. ADC contains a monoclonal antibody that can bind to a specific surface antigen on cancer cells. These antibodies include several proteins found on the surface of immune system B and T cells, such as CD20, CD22, human epidermal growth factor receptor 2 (Her2), and prostate-specific membrane antigen (PSMA). These antibodies are connected to a highly toxic drug by a cleavable linker unit. The drug is designed to trigger apoptosis of cancer cells by inducing irreversible DNA damage or disrupting cell division.

[0027] The mechanism of antibody-drug conjugates (ADCs) is that antibodies recognize and bind to specific antigens, triggering a series of reactions that then lead to endocytosis and entry into the cytoplasm, where the highly toxic drug is released from lysosomal enzymes, killing the cancer cells. Compared to traditional chemotherapy, which indiscriminately damages both cancer cells and normal tissues, targeted drug delivery allows the drug to act directly on cancer cells, reducing damage to normal cells. A typical antibody-drug conjugate consists of three parts: a drug, a linker unit, and an antibody. The selection of the specific antibody and drug depends on the specific disease and has a significant impact on the safety and efficacy of the conjugate. The stability of the linker unit and the mode of attachment to the antibody play a crucial role in the development of ADC drugs. Factors that determine the efficacy of an antibody-drug conjugate include the stability and cleavage susceptibility of the linker unit, cell surface activation, internalization, transport, and cytotoxin release. The present disclosure contemplates, without limitation, the combined use of any ADC with the T7 series oHSV described herein.

[0028] Bispecific T cell engagers (BiTEs) are relatively simple bispecific molecules that target the CD3E subunit of the TCR complex on T cells and target an antigen of interest, such as a cancer antigen. Because BiTEs are specific for the TCR complex, they can activate resident T cells to kill cells expressing a specific target antigen on their surface, such as cancer cells. A key feature of BiTEs is their ability to enable CD4+ and resting CD8+ T cells to target cancer cells. In other words, BiTE-activated T cells can kill cells regardless of MHC expression on the cell surface. This is important because some tumor cells downregulate MHC, conferring resistance to drugs such as CAR-T cells and immTAC. Unfortunately, compared with full-length antibodies, BiTEs have poor circulatory dynamics. This means that when administered to patients, the majority of BiTEs do not reach target cells. Furthermore, the use of a high-affinity anti-CD3 ScFv as part of a BiTE can lead to strong binding to T cells in the blood, which also hinders delivery to tumors. As a result, BiTEs cannot be effectively delivered to tumor cells and therefore do not reach their full potential as anti-cancer therapies. The present disclosure contemplates, without limitation, the combined use of any BiTE with an oHSV described herein.

[0029] As used herein, the term "tumor-associated / specific antigen" refers to a tumor-associated antigen, a tumor-specific antigen, or both. For example, the term "at least one tumor-associated / specific antigen" refers to at least one tumor-associated antigen or at least one tumor-specific antigen, and may include pairs of tumor-associated and tumor-specific antigens. For example, the term "a truncated, non-signaling variant of at least one tumor-associated / specific antigen" is intended to include a truncated, non-signaling variant of one tumor-associated antigen, a truncated, non-signaling variant of two or more tumor-associated antigens, a truncated, non-signaling variant of one tumor-specific antigen, a truncated, non-signaling variant of two or more tumor-specific antigens, a truncated, non-signaling variant of one tumor-associated antigen and two or more tumor-specific antigens, and a truncated, non-signaling variant of two or more tumor-associated antigens and one tumor-specific antigen. For example, the term "two tumor-associated / specific antigens" may include two tumor-associated antigens, two tumor-specific antigens, and a combination of one tumor-associated antigen and one tumor-specific antigen.

[0030] As used herein, a "biomarker," "truncated non-signaling variant," "truncated variant," or "non-signaling variant" of a particular tumor-associated or tumor-specific antigen refers to a variant of a tumor-associated or tumor-specific antigen that has been mutated, deleted, or otherwise modified to abolish signaling of its wild-type counterpart in a signaling pathway. The variant exposes at least a portion of an epitope on the antigen, allowing it to be bound by an antibody or its antigen-binding fragment (e.g., scFv) specifically directed against the wild-type antigen. A commonly known non-signaling variant of a tumor-associated or tumor-specific antigen is, when the antigen is a transmembrane protein, the extracellular domain of the antigen, the extracellular-transmembrane domain of the antigen, or an equivalent having at least 90% amino acid sequence identity to the extracellular domain or the extracellular-transmembrane domain. The equivalent is unable to transmit a signal but exposes at least a portion of an epitope on the extracellular domain of the antigen.

[0031] For example, a non-signaling variant of CD19 (also referred to herein as non-signaling CD19) is the 323 amino acid extracellular-transmembrane domain of wild-type CD19 (SEQ ID NO: 14). Non-signaling BCMA is the 77 amino acid extracellular-transmembrane domain of wild-type BCMA (SEQ ID NO: 15). Non-signaling HER2 is the 675 amino acid extracellular-transmembrane domain of wild-type HER2 (SEQ ID NO: 16). Non-signaling Trop-2 is the 297 amino acid extracellular-transmembrane domain of wild-type Trop-2 (SEQ ID NO: 17).

[0032] The extracellular and transmembrane domains can be easily obtained by routine practice in the art. The amino acid sequences of the extracellular / transmembrane domains of various tumor-associated or tumor-specific antigens are available from public resources, including NCBI (https: / / www.ncbi.nlm.nih.gov / protein). It should be noted that, when expressed on the surface of tumor cells, non-signaling mutants are recognized and bound by antibodies or antigen-binding fragments of antibodies specific for the tumor-associated or tumor-specific antigen. The antigen-binding fragments may be part of CAR-immune cells (e.g., CAR-T cells or CAR-NK cells) or BiTEs. The antibodies may be combined with chemotherapeutic drugs to form ADCs. However, non-signaling mutants do not induce signaling pathways like their wild-type counterparts. Those skilled in the art would easily be able to test and verify whether a mutant of a tumor-associated or tumor-specific antigen is non-signaling. For example, this may be determined by detecting the levels of downstream proteins in the normal signaling pathway known for the wild-type antigen.

[0033] Genetically engineered oncolytic herpes simplex virus (oHSV): The present disclosure provides genetically modified oncolytic herpes simplex viruses (oHSVs). The genetically modified oHSVs are engineered to express a truncated, non-signaling variant of at least one tumor-associated or tumor-specific antigen during replication in susceptible cells, such as solid tumor cells. The inventors have successfully demonstrated the expression of different truncated tumor-associated or tumor-specific antigens on the cell surface after infection and replication of the genetically modified oHSV in tumor cells. When the non-signaling tumor-associated or tumor-specific antigen is expressed and subsequently presented on the surface of tumor cells, the tumor cells can be targeted for various antigen-directed therapies, such as CAR-T therapy. In some embodiments, the genetically modified oHSVs of the present disclosure are further engineered to express at least one chemokine during replication in susceptible cells, such as solid tumor cells. The inventors have demonstrated that secreted chemokines were detected as early as 4 hours after infection, and this was maintained for at least 4 days after oHSV infection. The expression and release of chemokines induces chemotaxis of immune cells, such as T cells or CAR T cells, towards susceptible cells, which promotes the trafficking and infiltration of immune cells into the tumor mass.

[0034] In some embodiments, a genetically modified oncolytic herpes simplex virus (oHSV) is provided, wherein the genome of the oHSV incorporates a polynucleotide encoding a truncated, non-signaling variant of at least one tumor-associated / specific antigen, and expression of the truncated, non-signaling variant is controlled by an immediate early gene promoter of the HSV.

[0035] In some embodiments, a genetically modified oncolytic herpes simplex virus (oHSV) is provided, wherein the genome of the oHSV incorporates a polynucleotide encoding (a) a truncated, non-signaling variant of at least one tumor-associated / specific antigen, and (b) at least one chemokine, wherein expression of the truncated, non-signaling variant and the at least one chemokine is controlled by an immediate-early gene promoter of the HSV.

[0036] In some embodiments, a genetically modified oncolytic herpes simplex virus (oHSV) is provided, wherein the genome of the oHSV incorporates a polynucleotide encoding (a) a truncated, non-signaling mutant of one tumor-associated antigen or one tumor-specific antigen, and (b) a chemokine, wherein expression of the truncated, non-signaling mutant and the chemokine is controlled by an immediate-early gene promoter of the HSV.

[0037] In some embodiments, a genetically modified oncolytic herpes simplex virus (oHSV) is provided, wherein the genome of the oHSV incorporates a polynucleotide encoding (a) truncated, non-signaling variants of two tumor-associated / specific antigens and (b) a chemokine, and expression of the truncated, non-signaling variants and the chemokine is controlled by an immediate-early gene promoter of an HSV. In some embodiments, the two tumor-associated / specific antigens comprise two of the same or different tumor-associated antigens. In some embodiments, the two tumor-associated / specific antigens comprise two of the same or different tumor-specific antigens. In some embodiments, the two tumor-associated / specific antigens comprise one tumor-associated antigen and one tumor-specific antigen.

[0038] In some embodiments, a genetically modified oncolytic herpes simplex virus (oHSV) is provided, the genome of which incorporates a polynucleotide encoding (a) a truncated, non-signaling variant of at least one tumor-associated / specific antigen, and (b) two chemokines, wherein expression of the truncated, non-signaling variant and the chemokines is controlled by an immediate-early gene promoter of an HSV. In some embodiments, the two chemokines are the same. In some embodiments, the two chemokines are different.

[0039] In some embodiments, a genetically modified oncolytic herpes simplex virus (oHSV) is provided, the genome of which incorporates a polynucleotide encoding (a) truncated, non-signaling variants of two tumor-associated / specific antigens and (b) two chemokines, wherein expression of the truncated, non-signaling variants and the chemokines is controlled by an HSV immediate-early gene promoter. In some embodiments, the two chemokines are the same. In some embodiments, the two chemokines are different. In some embodiments, the two tumor-associated / specific antigens comprise two of the same or different tumor-associated antigens. In some embodiments, the two tumor-associated / specific antigens comprise two of the same or different tumor-specific antigens. In some embodiments, the two tumor-associated / specific antigens comprise one tumor-associated antigen and one tumor-specific antigen.

[0040] Thus, in some embodiments of the present disclosure, a genetically modified oHSV has integrated into its genome a first polynucleotide encoding a truncated, non-signaling variant of at least one tumor-associated / specific antigen and a second polynucleotide encoding at least one chemokine, and expression of the truncated, non-signaling variant and the at least one chemokine is controlled by an immediate-early gene promoter of an HSV.

[0041] In some embodiments, the genetically modified oHSV has integrated into its genome a first polynucleotide encoding a truncated, non-signaling variant of a first tumor-associated / specific antigen, a second polynucleotide encoding a truncated, non-signaling variant of a second tumor-associated / specific antigen, and a third polynucleotide encoding a chemokine, and expression of the truncated, non-signaling variant and the chemokine is controlled by an immediate early gene promoter of an HSV.

[0042] In some embodiments, the tumor-associated / specific antigen, the first or second tumor-associated / specific antigen is HER2, PSMA, BCMA, CD20, CD33, CD19, CD22, CD123, CD30, GPC-3, CEA, Claudin 18.2, EpCAM, GD2, MSLN, EGFR, MUC1, EGF VIII, CD38, Trop-2, c-MET, Nectin-4, CD79b, CCK4, GPA33, HLA-A2, CLEC12A, p-cadherin, TDO2, MART-1, Pmel 17, MAGE-1, AFP, CA125, TRP-1, TRP-2, NY-ESO, PSA, CDK4, BCA225, CA125, MG7-Ag, NY-CO-1, RCAS1, SDCCAG16, TAAL6, and TAG72. In some embodiments, the tumor-associated / specific antigen is selected from the group consisting of HER2, Trop-2, BCMA, and CD19.

[0043] In some embodiments, the chemokine is selected from the group consisting of CXCL1-CXCL17, CCL1-CCL28, XCL1, XCL2, and CX3CL1. In preferred embodiments, the chemokine is selected from the group consisting of CXCL9, CXCL10, CXCL11, CXCL12, CCL3, CCL4, CCL5, CCL19, and CCL21. In preferred embodiments, the chemokine is CCL5.

[0044] In some embodiments, the HSV immediate-early gene promoter is an HSV-1 or HSV-2 immediate-early gene promoter. In some embodiments, the HSV immediate-early gene promoter is selected from the group consisting of IE1 (ICP0 promoter), IE2 (ICP27 promoter), IE3 (ICP4 promoter), and IE4 / 5 (ICP22 and ICP47 promoters) of HSV-1. In a preferred embodiment, the HSV immediate-early gene promoter is the HSV-1 immediate-early gene promoter IE4 / 5.

[0045] In some embodiments, the truncated, non-signaling variant is the extracellular-transmembrane domain of a tumor-associated / specific antigen. For example, a truncated, non-signaling variant of CD19 is the extracellular-transmembrane domain of CD19. For example, a truncated, non-signaling variant of BCMA is the extracellular-transmembrane domain of BCMA. For example, a truncated, non-signaling variant of HER2 is the extracellular-transmembrane domain of HER2. For example, a truncated, non-signaling variant of Trop-2 is the extracellular-transmembrane domain of Trop-2. In some embodiments, the truncated, non-signaling variant is the extracellular domain of a tumor-associated / specific antigen. In some embodiments, the truncated, non-signaling variant is the extracellular domain linked to part of the transmembrane domain of a tumor-associated / specific antigen. In some embodiments, the truncated, non-signaling variant is a variant of a wild-type tumor-associated / specific antigen lacking part or all of the signaling domain.

[0046] In a preferred embodiment, the genetically modified oHSV originates from HSV type 1 (HSV-1) or HSV type 2 (HSV-2). In a preferred embodiment, the genetically modified oHSV originates from the F strain of HSV-1.

[0047] In preferred embodiments, the polynucleotides described herein encode (i) a truncated, non-signaling mutant of CD19 and (ii) CCL5. In preferred embodiments, the polynucleotides encode (i) a truncated, non-signaling mutant of Trop-2 and (ii) CCL5. In preferred embodiments, the polynucleotides encode (i) a truncated, non-signaling mutant of HER2 and (ii) CCL5. In preferred embodiments, the polynucleotides encode (i) a truncated, non-signaling mutant of BCMA and (ii) CCL5.

[0048] In preferred embodiments, the polynucleotides described herein encode (i) a truncated, non-signaling mutant of CD19, (ii) a truncated, non-signaling mutant of BCMA, and (iii) CCL5. In preferred embodiments, the polynucleotides encode (i) a truncated, non-signaling mutant of CD19, (ii) a truncated, non-signaling mutant of Trop-2, and (iii) CCL5. In preferred embodiments, the polynucleotides encode (i) a truncated, non-signaling mutant of CD19, (ii) a truncated, non-signaling mutant of HER2, and (iii) CCL5.

[0049] In some embodiments, the tumor cells are solid tumor cells. In some embodiments, the tumor cells do not express the tumor-associated or tumor-specific antigen encoded by the polynucleotide. In some embodiments, the tumor cells express the tumor-associated or tumor-specific antigen encoded by the polynucleotide.

[0050] In some embodiments, such genetically modified oHSVs are further modified to delete a nucleic acid fragment from the genome of the oHSV, thereby reducing or eliminating a particular undesirable characteristic of the oHSV for its intended use. In one embodiment, the genetically modified oHSV has deleted an internal inverted repeat region, a fragment encoding a viral gene, or both. In one embodiment, the deleted nucleic acid fragment of the oHSV is positions 117005-132096 of the P prototype genome of the HSV-1 F strain. In one embodiment, the viral gene-encoding fragment is a nucleic acid fragment encoding γ34.5. In one embodiment, both copies of the gene γ34.5 are deleted.

[0051] In some embodiments, such genetically modified oHSV are further modified to encode an immunostimulatory agent, an immunotherapeutic agent, or both. In one embodiment, the immunostimulatory agent is selected from the group consisting of GM-CSF, IL-2, IL-12, IL-15, IL-24, and IL-27. In one embodiment, the immunotherapeutic agent is an anti-PD-1 antibody, an anti-CTLA4 antibody, or an antigen-binding fragment thereof. In one embodiment, the genetically modified oHSV encodes IL-12. In one embodiment, the genetically modified oHSV encodes an anti-PD-1 antibody or an antigen-binding fragment thereof. In one embodiment, the genetically modified oHSV encodes IL-12 and an anti-PD-1 antibody or an antigen-binding fragment thereof.

[0052] It should be noted that the expression of the truncated, non-signaling variant of the tumor-associated / specific antigen and the chemokine must be controlled by an immediate-early gene promoter of HSV, such as the IE4 / 5 promoter, so that the tumor-associated / specific antigen is expressed immediately after viral infection and before tumor cell lysis due to viral replication. The polynucleotide encoding the truncated, non-signaling variant and the polynucleotide encoding the chemokine may be operably linked to the same immediate-early promoter. In another embodiment, the polynucleotide encoding the truncated, non-signaling variant and the polynucleotide encoding the chemokine may be operably linked to different immediate-early promoters. When the oHSV is further equipped with an immunostimulatory agent, an immunotherapeutic agent, or both, such as IL-12 or an anti-PD-1 antibody, the expression of the immunostimulatory agent and / or the immunotherapeutic agent is not necessarily controlled by an immediate-early promoter, but is preferably controlled by a different and relatively late promoter, such as the CMV promoter or the Egr-1 promoter. In one embodiment, the polynucleotide encoding IL-12 is operably linked to the Egr-1 promoter. In another embodiment, the polynucleotide encoding the scFv-anti-hPD1 is operably linked to a CMV promoter.

[0053] In one embodiment, the genetically modified oHSV has integrated into its genome a first polynucleotide encoding a truncated, non-signaling CD19 and a second polynucleotide encoding CCL5, and expression of the truncated, non-signaling CD19 and the CCL5 is controlled by the HSV-1 IE4 / 5 promoter.

[0054] In one embodiment, the genetically modified oHSV has integrated into its genome a first polynucleotide encoding a truncated, non-signaling BCMA and a second polynucleotide encoding CCL5, and expression of the truncated, non-signaling BCMA and the CCL5 is controlled by the HSV-1 IE4 / 5 promoter.

[0055] In one embodiment, the genetically modified oHSV has integrated into its genome a first polynucleotide encoding a truncated, non-signaling Trop-2 and a second polynucleotide encoding CCL5, and expression of the truncated, non-signaling Trop-2 and the CCL5 is controlled by the HSV-1 IE4 / 5 promoter.

[0056] In one embodiment, the genetically modified oHSV has integrated into its genome a first polynucleotide encoding a truncated, non-signaling HER2 and a second polynucleotide encoding CCL5, and expression of the truncated, non-signaling HER2 and the CCL5 is controlled by the HSV-1 IE4 / 5 promoter.

[0057] In one embodiment, the genetically modified oHSV has integrated into its genome a first polynucleotide encoding a truncated, non-signaling CD19, a second polynucleotide encoding a truncated, non-signaling BCMA9, and a third polynucleotide encoding CCL5, and expression of the truncated, non-signaling CD19, the truncated, non-signaling BCMA9, and the CCL5 is controlled by the HSV-1 IE4 / 5 promoter.

[0058] In one embodiment, the genetically modified oHSV has integrated into its genome a first polynucleotide encoding a truncated, non-signaling CD19, a second polynucleotide encoding a truncated, non-signaling Trop-2, and a third polynucleotide encoding CCL5, and expression of the truncated, non-signaling CD19, the truncated, non-signaling Trop-2, and the CCL5 is controlled by the HSV-1 IE4 / 5 promoter.

[0059] In one embodiment, the genetically modified oHSV has integrated into its genome a first polynucleotide encoding a truncated, non-signaling CD19, a second polynucleotide encoding a truncated, non-signaling HER2, and a third polynucleotide encoding CCL5, and expression of the truncated, non-signaling CD19, the truncated, non-signaling HER2, and the CCL5 is controlled by the HSV-1 IE4 / 5 promoter.

[0060] In one embodiment, the genetically modified oHSV has integrated into its genome a first polynucleotide encoding a truncated, non-signaling mutant of any one selected from the group consisting of CD19, BCMA, Trop-2, and HER2, a second polynucleotide encoding CCL5, a third polynucleotide encoding an anti-PD-1 antibody, and a fourth polynucleotide encoding IL-12, wherein expression of the truncated, non-signaling mutant CD19 and the CCL5 is controlled by the HSV-1 IE4 / 5 promoter, and the internal inverted repeat region of the oHSV is deleted.

[0061] In one embodiment, the genetically modified oHSV has integrated into its genome a first polynucleotide encoding a truncated, non-signaling CD19, a second polynucleotide encoding a truncated, non-signaling mutant of any one selected from the group consisting of BCMA, Trop-2, and HER2, a third polynucleotide encoding CCL5, a fourth polynucleotide encoding an anti-PD-1 antibody, and a fifth polynucleotide encoding IL-12, and the expression of the truncated, non-signaling CD19 and the CCL5 is controlled by the HSV-1 IE4 / 5 promoter.

[0062] In one embodiment, the genetically modified oHSV has integrated into its genome a first polynucleotide encoding a truncated, non-signaling CD19, a second polynucleotide encoding a truncated, non-signaling mutant of any one selected from the group consisting of BCMA, Trop-2, and HER2, a third polynucleotide encoding CCL5, a fourth polynucleotide encoding an anti-PD-1 antibody, and a fifth polynucleotide encoding IL-12, wherein expression of the truncated, non-signaling CD19 and the CCL5 is controlled by the HSV-1 IE4 / 5 promoter, and the internal inverted repeat region of the oHSV is deleted.

[0063] In one embodiment, the genetically modified oHSV comprises a first polynucleotide encoding a truncated, non-signaling CD19, a second polynucleotide encoding a truncated, non-signaling variant of any one selected from the group consisting of BCMA, Trop-2, and HER2, a third polynucleotide encoding CCL5, a fourth polynucleotide encoding an anti-PD-1 antibody, and a fifth polynucleotide encoding IL-12 integrated into its genome, wherein expression of the truncated, non-signaling CD19 and the CCL5 is controlled by the HSV-1 IE4 / 5 promoter, the internal inverted repeat region of the oHSV is deleted, and all single-copy genes of the oHSV are retained.

[0064] In one embodiment, the genetically modified oHSV comprises a first polynucleotide encoding a truncated, non-signaling CD19, a second polynucleotide encoding a truncated, non-signaling variant of any one selected from the group consisting of BCMA, Trop-2, and HER2, a third polynucleotide encoding CCL5, a fourth polynucleotide encoding an anti-PD-1 antibody, and a fifth polynucleotide encoding IL-12 integrated into its genome, wherein expression of the truncated, non-signaling CD19 and the CCL5 is controlled by the HSV-1 IE4 / 5 promoter, wherein both copies of the internal inverted repeat and γ34.5 of the oHSV are deleted, and all single-copy genes of the oHSV are retained.

[0065] In one embodiment, the PolyA tail is located downstream of the polynucleotides encoding the truncated antigen and chemokine, for example, the polynucleotides encoding the truncated non-signaling mutant and chemokine are arranged as 5'-CD19-CCL5-PolyA-3', 5'-BCMA-CCL5-PolyA-3', 5'-HER2-CCL5-PolyA-3', 5'-CD19-BCMA-CCL5-PolyA-3', 5'-CD19-Trop-2-CCL5-PolyA-3', or 5'-CD19-HER2-CCL5-PolyA-3'.

[0066] In one embodiment, the integration of polynucleotides encoding the truncated non-signaling mutant, immunotherapeutic agent, and chemokine into the oHSV genome does not disrupt the function of any viral genes. For example, a polynucleotide encoding an anti-PD-1 antibody or antigen-binding fragment thereof is introduced between the UL3 and UL4 genes of the virus, and a polynucleotide encoding the truncated non-signaling mutant and chemokine is introduced between the UL37 and UL38 genes of the virus. Also, in one embodiment, a polynucleotide encoding IL2 replaces the internal inverted repeat region of the viral genome.

[0067] In the present disclosure, the tumor-associated / specific antigen encoded by the oHSV may be heterologous or homologous to the tumor cells infected with the oHSV. In one embodiment, the tumor cells express a tumor-associated / specific antigen that is different from the tumor-associated / specific antigen encoded by the oHSV. For example, the tumor cells overexpress CD22, while the genetically modified oHSV of the present disclosure expresses CD19, HER2, or both. In another embodiment, the tumor cells express the same tumor-associated / specific antigen as the tumor-associated / specific antigen encoded by the oHSV. For example, the tumor cells express HER2 at low levels, while the genetically modified oHSV of the present disclosure expresses HER2. In another embodiment, the tumor cells lack detectable known tumor-associated / specific antigens.

[0068] The tumor cells infected with the genetically modified oHSV of the present disclosure are hematological tumor cells or solid tumor cells.

[0069] Advantageously, presentation of a non-signaling tumor-associated / specific antigen on the surface of tumor cells converts tumor cells from negative to positive for that particular tumor-associated / specific antigen, thereby rendering the tumor responsive to therapies targeting that particular tumor-associated / specific antigen. Expression of the heterologous polynucleotide is controlled by an immediate-early gene promoter, such as HSV-1 IE4 / 5, which produces a translation product at a very early stage of HSV entry and replication into tumor cells. For example, oHSV may be engineered to express cleaved, non-signaling CD19, a transmembrane protein specifically expressed on normal and most tumor B cells. The cleaved, non-signaling CD19 is then presented on the surface of tumor cells prior to oHSV infection-induced cell lysis and serves as a target for CD19-directed CAR-T therapy, such as Kymriah®, Yescarta®, or Tecartus®. In other words, expression of non-signaling CD19 converts tumor cells that are generally insensitive to CD19-directed CAR-T therapy due to the lack of CD19 antigen on the tumor cells into tumor cells that are susceptible to CD19-directed CAR-T therapy, and in this case, the tumor is likely to respond to CD19-directed CAR-T therapy.

[0070] A key advantage of genetically engineered oHSVs encoding one or more non-signaling tumor-associated / specific antigens is that they provide a versatile tool for combined use with different tumor antigen-targeted therapies, eliminating the need to repeatedly design, test, and manufacture an oHSV for each tumor antigen-targeted therapy. It has been shown that when two different non-signaling tumor-associated / specific antigens are encoded by the same oHSV, they can be simultaneously expressed and presented on the tumor cell surface before viral infection lyses the tumor cell. Presentation of two or more different non-signaling tumor-associated / specific antigens converts tumor cells into double- or even triple-positive tumor cells, allowing different tumor-targeted therapies to be effective against the tumor cells. This helps improve the specificity and efficacy of corresponding tumor-targeted therapies, such as CAR-T cell therapy.

[0071] An additional advantage of some of the genetically modified oHSVs disclosed herein is that, in addition to tumor-associated / specific antigens, they encode at least one chemokine, whose expression and release further aids in the trafficking and infiltration of immune cells into tumor cells. Thus, CAR-T, CAR-NK, and the like are particularly advantageous when used in combination with the oHSVs described herein. However, without being bound by any theory, the genetically modified oHSVs disclosed herein can be used independently, and the secretion of chemokines induces the body's immune cells, such as T cells, to chemotaxis toward tumors, killing tumor cells in conjunction with the anti-tumor effects of the virus.

[0072] Combining oHSV with targeted tumor therapy: Another aspect of the present disclosure relates to the use of any of the above-described genetically modified oHSVs in combination with tumor-targeting therapeutic agents for the treatment of various cancers. As described above, the genetically modified oHSVs disclosed herein express non-signaling tumor-associated / specific antigens, which are then presented on the surface of tumor cells. This provides an opportunity for therapeutic agents designed to target tumor-associated / specific antigens to target oHSV-infected tumor cells.

[0073] In the present disclosure, tumor-targeting therapeutic agents comprise a targeting moiety specific for a truncated, non-signaling variant of at least one tumor-associated / specific antigen encoded by oHSV, and an effector moiety for killing or inhibiting the growth of cancer cells. The targeting moiety has specificity for a non-signaling tumor-associated / specific antigen expressed on the surface of tumor cells upon oHSV entry and replication into tumor cells. For example, the targeting moiety can be an antigen-binding domain of an antibody against the tumor-associated / specific antigen, such as an antibody, scFv, Fab, or chimeric antigen receptor moiety of a CAR-T cell. The effector moiety is useful for killing or inhibiting the growth of cancer cells. For example, the effector moiety can be immune cells, including T cells and natural killer cells, a portion of a BiTE capable of binding to T cells, or the drug moiety of an antibody-drug conjugate.

[0074] In some embodiments, the tumor-targeted therapeutic is selected from the group consisting of chimeric antigen receptor T (CAR-T) cells, chimeric antigen receptor NK (CAR-NK) cells, bispecific T-cell engagers (BiTEs), and antibody-drug conjugates (ADCs). In some embodiments, the tumor-targeted therapeutic is a CAR-T cell. In some embodiments, the tumor-targeted therapeutic is a CAR-NK cell. In some embodiments, the tumor-targeted therapeutic is a BiTE. In some embodiments, the tumor-targeted therapeutic is an ADC.

[0075] In some embodiments, the tumor-targeting therapeutic agent is a CD19-targeting CAR-T cell. In some embodiments, the tumor-targeting therapeutic agent is a CD19- or EpCAM-targeting BiTE. In some embodiments, the tumor-targeting therapeutic agent is a HER2-, Trop-2-, Nectin-4-, BCMA-, CD33-, CD30-, CD22-, or CD79b-targeting ADC.

[0076] In some embodiments, the tumor-targeting therapeutic is a CD19-targeting CAR-T cell. In some embodiments, the tumor-targeted therapeutic is selected from the group consisting of Tecartus®, Kymriah®, Yescarta®, JWCAR-029, IM19CAR-T, CNCT19, BZ019, HD CD19 CAR-T, pCAR-19B, CD19-CART, CT032, iPD1 CD19 eCAR-T, LCAR-B38M, CT103A, CAR-BCMA T, AU-101, 4SCAR-PSMA, PSMA-CART, P-PSMA-101, C-CAR066, MB-CART20.1, PBCAR20A, LB1095, LB1901, PRGN-3006, AMG553, CT041, CD30.CAR-T, and CAR-GPC3 T.

[0077] In some embodiments, the tumor-targeted therapeutic is a CD19- or EpCAM-targeted BiTE, hi some embodiments, the tumor-targeted therapeutic is selected from the group consisting of Blincyto®, AMG420, PF-3135, and GBR1302.

[0078] In some embodiments, the tumor-targeting therapeutic is a HER2-, Trop-2-, Nectin-4-, BCMA-, CD33-, CD30-, CD22-, or CD79b-targeting ADC. In some embodiments, the tumor-targeted therapeutic agent is selected from the group consisting of Kadcyla®, Enhertu®, SHR-A1811, TAA013, RC-48, BAT8001, ARX788, A166, Trodelvy®, BAT8003, DAC-002, DS-1062, SKB264, RC-108, TR1801-ADC, Padccv®, Polivy®, Adcetris®, Mylotarg®, Blenrep®, PSMA ADC, ADCT-402, PTK7-ADC, and TRS005.

[0079] In a preferred embodiment, the genetically modified oHSV for use in combination with any of the above-described tumor-targeting therapeutic agents is a genetically modified oHSV having incorporated into its genome a polynucleotide encoding (a) truncated, non-signaling mutants of two tumor-associated / specific antigens and (b) a chemokine, wherein expression of the truncated, non-signaling mutants and the chemokine is controlled by an HSV immediate-early gene promoter. In some embodiments, the two tumor-associated / specific antigens comprise two of the same or different tumor-associated antigens. In some embodiments, the two tumor-associated / specific antigens comprise two of the same or different tumor-specific antigens. In some embodiments, the two tumor-associated / specific antigens comprise one tumor-associated antigen and one tumor-specific antigen.

[0080] In a preferred embodiment, the genetically modified oHSV for use in combination with a tumor-targeted therapeutic agent is a genetically modified oHSV that expresses truncated, non-signaling mutants of both CD19 and BCMA, and CCL5, and said tumor-targeted therapeutic agent is a CD19-targeted CAR-T cell, e.g., Kymriah®, Yescarta®, or Tecartus®, a CD19-targeted BiTE, e.g., Blinatumomab, a BCMA-targeted ADC, e.g., Blenrep®, or any combination thereof.

[0081] In a preferred embodiment, the genetically modified oHSV for use in combination with a tumor-targeted therapeutic agent is a genetically modified oHSV that expresses truncated, non-signaling mutants of both CD19 and HER2, and CCL5, and said tumor-targeted therapeutic agent is a CD19-targeted CAR-T cell, e.g., Kymriah®, Yescarta®, or Tecartus®, a CD19-targeted BiTE, e.g., Blinatumomab, a HER2-targeted ADC, e.g., Kadcyla® or Enhertu®, or any combination thereof.

[0082] In a preferred embodiment, the genetically modified oHSV for use in combination with a tumor-targeted therapeutic agent is a genetically modified oHSV that expresses truncated, non-signaling mutants of both CD19 and Trop-2, and CCL5, and said tumor-targeted therapeutic agent is a CD19-targeted CAR-T cell, e.g., Kymriah®, Yescarta®, or Tecartus®, a CD19-targeted BiTE, e.g., Blinatumomab, a Trop-2-targeted ADC, e.g., Trodelvy®, or any combination thereof.

[0083] The combination of oHSV and tumor-targeted therapy may be embodied, for example, as a pharmaceutical kit. Thus, in one aspect, a pharmaceutical kit for treating cancer is provided that separately comprises a genetically modified oncolytic herpes simplex virus (oHSV) described herein and a tumor-targeted therapeutic agent, the tumor-targeted therapeutic agent comprising a targeting moiety specific for a truncated, non-signaling mutant of at least one tumor-associated / specific antigen encoded by a polynucleotide, and an effector moiety for killing or inhibiting the growth of cancer cells.

[0084] In some embodiments, a pharmaceutical kit for the treatment of cancer comprises a genetically modified oncolytic herpes simplex virus (oHSV) encoding (i) a truncated, non-signaling mutant of CD19, (ii) a truncated, non-signaling mutant of BCMA, and (iii) CCL5, and separately a CD19- or BCMA-targeted CAR-T, ADC, or BiTE. In some embodiments, the CD19- or BCMA-targeted CAR-T, ADC, or BiTE is selected from the group consisting of Tecartus®, Kymriah®, Yescarta®, ADCT-402 (ADC Therapeutics), Blinatumomab, JNJ-68284528 (JNJ-4528, Legend Biotech), Blenrep (or GSK2857916), AMG420 (Amgen), and PF-3135 (Pfizer).

[0085] In some embodiments, a pharmaceutical kit for treating cancer comprises a genetically modified oncolytic herpes simplex virus (oHSV) encoding (i) a truncated, non-signaling mutant of CD19, (ii) a truncated, non-signaling mutant of Trop-2, and (iii) CCL5, and separately a CD19- or Trop-2-targeted CAR-T, ADC, or BiTE. In some embodiments, the CD19- or Trop-2-targeted CAR-T, ADC, or BiTE is selected from the group consisting of Tecartus®, Kymriah®, Yescarta®, ADCT-402 (ADC Therapeutics), Blinatumomab, and Trodelvy® (Immunomedics).

[0086] In some embodiments, a pharmaceutical kit for treating cancer comprises a genetically modified oncolytic herpes simplex virus (oHSV) encoding (i) a truncated, non-signaling mutant of CD19, (ii) a truncated, non-signaling mutant of HER2, and (iii) CCL5, and separately a CD19- or HER2-targeted CAR-T, ADC, or BiTE. In some embodiments, the CD19- or HER2-targeted CAR-T, ADC, or BiTE is selected from the group consisting of Tecartus®, Kymriah®, Yescarta®, ADCT-402 (ADC Therapeutics), Blinatumomab, AU-101 (Aurora Biopharma), Kadcyla® (Roche), Enhertu® (Daiichi Sankyo), and GBR1302 (Ichnos Sciences SA).

[0087] Treatment method: Another aspect of the present disclosure relates to a method of treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a genetically modified oHSV described herein and a tumor-targeted therapeutic agent described herein, wherein the administration of both the oHSV and the tumor-targeted therapeutic agent occurs simultaneously or sequentially.

[0088] In some embodiments, a subject is first administered a therapeutically effective amount of a genetically modified oHSV described herein, followed by a tumor-targeted therapeutic agent described herein. In such embodiments, the interval between administrations is within a range of 0.5 to 12 hours, e.g., 0.5 to 9 hours, 0.5 to 8 hours, 0.5 to 7 hours, 0.5 to 6 hours, 0.5 to 5 hours, 0.5 to 4 hours, 0.5 to 3 hours, 0.5 to 2 hours, 0.5 to 2.5 hours, 0.5 to 1.5 hours, or 0.5 to 1 hour. For example, the oHSV is administered 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, 9, 10, 11, or 12 hours before the administration of the tumor-targeted therapeutic agent.

[0089] In some embodiments, the methods comprise administering to the subject therapeutically effective amounts of a genetically modified oncolytic herpes simplex virus (oHSV) encoding (i) a truncated, non-signaling mutant of CD19, (ii) a truncated, non-signaling mutant of BCMA, and (iii) CCL5, and a CD19- or BCMA-targeted CAR-T, ADC, or BiTE. In some embodiments, the CD19- or BCMA-targeted CAR-T, ADC, or BiTE is selected from the group consisting of Tecartus®, Kymriah®, Yescarta®, ADCT-402 (ADC Therapeutics), Blinatumomab, JNJ-68284528 (JNJ-4528, Legend Biotech), Blenrep (or GSK2857916), AMG420 (Amgen), and PF-3135 (Pfizer).

[0090] In some embodiments, the method comprises administering to the subject therapeutically effective amounts of a genetically modified oncolytic herpes simplex virus (oHSV) encoding (i) a truncated, non-signaling mutant of CD19, (ii) a truncated, non-signaling mutant of Trop-2, and (iii) CCL5, and a CD19- or Trop-2-targeted CAR-T, ADC, or BiTE. In some embodiments, the CD19- or Trop-2-targeted CAR-T, ADC, or BiTE is selected from the group consisting of Tecartus®, Kymriah®, Yescarta®, ADCT-402 (ADC Therapeutics), Blinatumomab, and Trodelvy® (Immunomedics).

[0091] In some embodiments, the method comprises administering to the subject therapeutically effective amounts of a genetically modified oncolytic herpes simplex virus (oHSV) encoding (i) a truncated, non-signaling mutant of CD19, (ii) a truncated, non-signaling mutant of HER2, and (iii) CCL5, and a CD19- or HER2-targeted CAR-T, ADC, or BiTE. In some embodiments, the CD19- or HER2-targeted CAR-T, ADC, or BiTE is selected from the group consisting of Tecartus®, Kymriah®, Yescarta®, ADCT-402 (ADC Therapeutics), Blinatumomab, AU-101 (Aurora Biopharma), Kadcyla® (Roche), Enhertu® (Daiichi Sankyo), and GBR1302 (Ichnos Sciences SA).

[0092] Combination of the oHSV described herein with various tumor antigen-directed CAR-T cells, ADCs, or BiTEs provides significantly improved antitumor efficacy against various tumors. oHSV directly disrupts barriers and manipulates the tumor microenvironment through direct tumor cell lysis. oHSV equipped with payloads such as chemokines and cytokines further improves T cell trafficking and infiltration into tumor mass. Furthermore, highly tumor-specific antigens (e.g., CD19, BCMA) delivered by oHSV on the surface of solid tumor cells improve the specificity and safety of tumor-targeted therapies, such as CAR-T therapy, by reducing on-target off-tumor toxicity. The present disclosure relates, for example, to the following: [1] A genetically modified oncolytic herpes simplex virus (oHSV), comprising: The genome of the oHSV contains (a) a truncated, non-signaling mutant of at least one tumor-associated / specific antigen, and (b) incorporating a polynucleotide encoding at least one chemokine; expression of the truncated non-signaling mutant and the at least one chemokine is controlled by an immediate early gene promoter of HSV; and A genetically modified oHSV in which, upon replication of the oHSV in tumor cells, the truncated, non-signaling mutant is expressed and presented as a biomarker on the surface of the tumor cells, and the at least one chemokine is expressed and released to induce chemotaxis of immune cells toward the tumor cells. [2] The at least one tumor-associated / specific antigen is selected from the group consisting of HER2, PSMA, BCMA, CD20, CD33, CD19, CD22, CD123, CD30, GPC-3, CEA, Claudin 18.2, EpCAM, GD2, MSLN, EGFR, MUC1, EGF-R VIII, CD38, Trop-2, c-MET, Nectin-4, CD79b, CCK4, GPA33, HLA-A2, CLEC12A, p-cadherin, TDO2, MART-1, Pmel 17, MAGE-1, AFP, CA125, TRP-1, TRP-2, NY-ESO, PSA, CDK4, BCA225, CA125, MG7-Ag, NY-CO-1, RCAS1, SDCCAG16, TAAL6, and TAG72. [3] The genetically modified oHSV according to [1] or [2], wherein the at least one chemokine is selected from the group consisting of CXCL1 to CXCL17, CCL1 to CCL28, XCL1, XCL2, and CX3CL1. [4] The genetically modified oHSV according to any one of [1] to [3], wherein the truncated non-signaling mutant is an extracellular domain, an extracellular-transmembrane domain, or an equivalent having at least 90% amino acid sequence identity to the extracellular domain or the extracellular-transmembrane domain. [5] The genetically modified oHSV according to any one of [1] to [4], wherein the HSV immediate early gene promoter is selected from the group consisting of HSV-1 IE1 (ICP0 promoter), IE2 (ICP27 promoter), IE3 (ICP4 promoter), and IE4 / 5 (ICP22 and ICP47 promoters). [6] The genetically modified oHSV according to any one of [1] to [5], wherein the polynucleotide encodes truncated, non-signaling mutants of two tumor-associated / specific antigens and at least one chemokine. [7] The genetically modified oHSV according to any one of [1] to [6] above, wherein the at least one chemokine includes CCL5. [8] The genetically modified oHSV according to any one of [1] to [7] above, wherein the polynucleotide is inserted between UL37 and UL38. [9] the polynucleotide is (a) a truncated, non-signaling mutant of CD19, a truncated, non-signaling mutant of BCMA, and CCL5; (b) a truncated, non-signaling mutant of CD19, a truncated, non-signaling mutant of Trop-2, and CCL5; (c) a truncated, non-signaling mutant of CD19, a truncated, non-signaling mutant of HER2, and CCL5; (d) a truncated, non-signaling mutant of CD19 and CCL5; (e) truncated, non-signaling mutants of Trop-2 and CCL5; (f) a truncated, non-signaling mutant of BCMA and CCL5, or (g) The genetically modified oHSV according to any one of [1] to [8] above, which encodes a truncated, non-signaling mutant of HER2 and CCL5.

[10] The genetically modified oHSV according to any one of [1] to [9] above, wherein the HSV immediate-early gene promoter is the HSV-1 immediate-early gene promoter IE4 / 5.

[11] The genetically modified oHSV according to any one of [1] to

[10] above, wherein the PolyA tail is located downstream of the polynucleotide encoding the cleaved antigen and chemokine.

[12] The genetically modified oHSV according to any one of [1] to

[11] above, wherein the tumor cells are solid tumor cells.

[13] The genetically modified oHSV according to any one of [1] to

[12] above, wherein the tumor cells do not express the tumor-associated / specific antigen encoded by the polynucleotide.

[14] The genetically modified oHSV according to any one of [1] to

[13] above, wherein the oHSV is further modified so that a nucleic acid fragment of the oHSV is deleted.

[15] The genetically modified oHSV according to any one of [1] to

[14] , wherein the fragment of the nucleic acid of the oHSV is an internal inverted repeat region of the oHSV, a fragment encoding a viral gene, or both.

[16] The genetically modified oHSV according to

[14] , wherein the fragment of the oHSV nucleic acid is located at positions 117005 to 132096 in the P prototype genome of the F strain.

[17] The genetically modified oHSV according to any one of [1] to

[16] above, wherein the oHSV is further modified to encode an immunostimulatory agent, an immunotherapeutic agent, or both.

[18] The genetically modified oHSV according to

[17] , wherein the immunostimulant is selected from the group consisting of GM-CSF, IL-2, IL-12, IL-15, IL-24, and IL-27.

[19] The genetically modified oHSV according to

[17] or

[18] , wherein the immunotherapeutic agent is an anti-PD-1 antibody, an anti-CTLA4 antibody, or an antigen-binding fragment thereof.

[20] The genetically modified oHSV according to any one of

[17] to

[19] , wherein the immunostimulant is IL-12 and the immunotherapeutic agent is an anti-PD-1 antibody or an antigen-binding fragment thereof.

[21] A pharmaceutical kit for treating cancer, comprising separately the oHSV described in any one of [1] to

[20] above and a tumor-targeting therapeutic agent, wherein the tumor-targeting therapeutic agent comprises a targeting moiety specific for a truncated, non-signal-transducing mutant of the at least one tumor-associated / specific antigen encoded by the polynucleotide, and an effector moiety for killing cancer cells or inhibiting their proliferation.

[22] The pharmaceutical kit according to

[21] , wherein the tumor-targeting therapeutic agent is selected from the group consisting of CAR-T cells, CAR-NK cells, BiTEs, and ADCs.

[23] The pharmaceutical kit according to

[22] , wherein the tumor-targeting therapeutic agent is selected from the group consisting of CD19-targeting CAR-T cells, CD19-targeting CAR-NK cells, and CD19- or EpCAM-targeting BiTEs.

[24] The pharmaceutical kit according to

[22] , wherein the tumor-targeting therapeutic agent is an ADC targeting HER2, Trop-2, Nectin-4, BCMA, CD33, CD30, CD22, or CD79b.

[25] 22. The pharmaceutical kit according to claim 21, wherein the tumor-targeting therapeutic agent is selected from the group consisting of Tecartus®, Kymriah®, Yescarta®, JWCAR-029, IM19CAR-T, CNCT19, BZ019, HD CD19 CAR-T, pCAR-19B, CD19-CART, CT032, iPD1 CD19 eCAR-T, LCAR-B38M, CT103A, CAR-BCMA T, AU-101, 4SCAR-PSMA, PSMA-CART, P-PSMA-101, C-CAR066, MB-CART20.1, PBCAR20A, LB1095, LB1901, PRGN-3006, AMG553, CT041, CD30.CAR-T, and CAR-GPC3 T.

[26] The pharmaceutical kit according to

[22] , wherein the tumor-targeting therapeutic agent is selected from the group consisting of Blincyto (registered trademark), AMG420, PF-3135, and GBR1302.

[27] The pharmaceutical kit according to

[22] , wherein the tumor-targeting therapeutic agent is selected from the group consisting of Kadcyla®, Enhertu®, SHR-A1811, TAA013, RC-48, BAT8001, ARX788, A166, Trodelvy®, BAT8003, DAC-002, DS-1062, SKB264, RC-108, TR1801-ADC, Padcetris®, Polivy®, Adcetris®, Mylotarg®, Blenrep®, PSMA ADC, ADCT-402, PTK7-ADC, and TRS005.

[28] A genetically modified oncolytic herpes simplex virus (oHSV), comprising: A genetically modified oHSV in which a polynucleotide encoding a truncated, non-signaling mutant of at least one tumor-associated / specific antigen is incorporated into the genome of the oHSV, the expression of the truncated, non-signaling mutant being controlled by an immediate early gene promoter of the HSV, and the truncated, non-signaling mutant being expressed and presented as a biomarker on the surface of the tumor cell during replication of the oHSV in the tumor cell.

[29] A pharmaceutical kit for treating cancer, comprising separately the oHSV described in

[28] and a tumor-targeting therapeutic agent, wherein the tumor-targeting therapeutic agent comprises a targeting moiety specific for a truncated, non-signal-transducing mutant of the at least one tumor-associated / specific antigen encoded by the polynucleotide, and an effector moiety for killing cancer cells or inhibiting their proliferation.

[0093] array: The amino acid or nucleic acid sequences described in this disclosure are provided in Table 1 below. [Table 1] DETAILED DESCRIPTION OF THE INVENTION

[0094] (Example) Construction of oHSV-1 T7201, T7202, T7203, T7204, T7011, T7012, and T7013: Oncolytic herpes simplex viruses (oHSV-1) T7201, T7202, T7203, and T7204 carry coding sequences for IL-12, anti-PD-1 antibody, CCL5, and a truncated, non-signaling variant of one tumor-associated antigen (TAA) that functions as a biomarker. The truncated, non-signaling variants expressed by T7201, T7202, T7203, and T7204 as biomarkers are CD19, BCMA, Trop-2, and HER2, respectively. Figure 1C shows a schematic diagram of the viral backbone of T7201 to T7204.

[0095] Oncolytic herpes simplex viruses (oHSV-1) T7011, T7012, and T7013 carry coding sequences for IL-12, anti-PD-1 antibody, CCL5, and two truncated, non-signaling variants of tumor-associated antigens (TAAs) that function as biomarkers. The truncated, non-signaling variants expressed by T7011, T7012, and T7013 as biomarkers are CD19 plus BCMA, CD19 plus Trop-2, and CD19 plus HER2, respectively. Figure 1D shows a schematic diagram of the viral backbone of T7011, T7012, and T7013. Figure 2 shows a flowchart of the construction of T7-series oHSVs.

[0096] The coding sequences of the two biomarkers and CCL5, linked by a T2A self-cleaving peptide sequence (SEQ ID NO: 1), are translated within a single open reading frame driven by the HSV-1 immediate early gene promoter (IE4 / 5 promoter). The expression cassette is inserted between the UL37 and UL38 genes.

[0097] Additionally, T7201, T7202, T7203, T7204, T7011, T7012, and T7013 contain an anti-human PD-1 antibody expression cassette inserted between UL3 and UL4, and a modified internal repeat (IR) region replaced by an IL-12 expression cassette. Recombinant viruses were constructed in several steps using the bacterial artificial chromosome (BAC) system. Details of virus construction are described below.

[0098] An IL-12 expression cassette flanked by nucleotide 117005 upstream and nucleotide 132096 downstream of the wild-type genome, which was PCR-amplified from the HSV-1 viral genome using two sets of primers (SEQ ID NOs: 2, 3 and 4, 5), respectively, was inserted into the gene replacement plasmid pKO5 to generate pKO1407. pKO1407 was then transfected into E. coli harboring the wild-type BAC by electroporation to generate BAC-T2010. Next, a CMV promoter cassette driving an anti-PD-1 antibody gene flanked by nucleotide 11658 upstream and nucleotide 11659 downstream of the wild-type genome, which was PCR-amplified from the HSV-1 viral genome using two sets of primers (SEQ ID NOs: 6, 7 and 8, 9), respectively, was ligated into pKO5 at the BglII and PacI sites to generate the pKOE1002 plasmid. The pKOE1002 plasmid was then transfected by electroporation into E. coli containing BAC-T2010 to generate BAC-T3011. The expression cassette containing one or two biomarkers (i.e., tumor-associated / specific antigens) and the CCL5 gene was flanked by nucleotide 84220 upstream and nucleotide 84221 downstream in the wild-type genome. The upstream and downstream flanking sequences were PCR-amplified from the HSV-1 viral genome using two sets of primers (SEQ ID NOs: 10 and 11) and (SEQ ID NOs: 12 and 13), respectively. DNA fragments containing the biomarkers, CCL5, and flanking sequences were ligated into pKO5 at the XbaI and PacI sites to generate the pKO7201, pKO7202, pKO7203, pKO7204, pKO7011, pKO7012, and pKO7013 plasmids. Next, the pKO7201, pKO7202, pKO7203, pKO7204, pKO7011, pKO7012, and pKO7013 plasmids were transfected into E. coli containing BAC-T3011 by electroporation to generate BAC-T7201, BAC-T7202, BAC-T7203, BAC-T7204, BAC-T7011, BAC-T7012, and BAC-T7013, respectively.T7201, T7202, T7203, T7204, T7011, T7012 and T7013 viruses were obtained by transfection of the corresponding BAC plasmids followed by several steps of plaque purification and amplification in Vero cells.

[0099] Virus titration: The virus titers were measured by plaque formation assay. Briefly, the virus stock solution was serially diluted and then inoculated onto a monolayer of Vero cells in a T25 flask. After 2 hours of absorption, the medium was replaced with DMEM medium supplemented with 1% FBS plus 0.05% (wt / vol) human pooled immunoglobulin for 72 hours. The cells were fixed with absolute methanol for 5 minutes, rinsed with distilled water, and stained with crystal violet. Plaques were counted, and the titers of infectious virus particles were calculated. The titers of T7011, T7012, and T7013 are shown in Table 2 below, and the titers of T7201, T7202, T7203, and T7204 are shown in Table 4. [Table 2]

[0100] Detection of CCL5 secretion after viral infection: 1×10 human embryonic kidney 293T, human laryngeal carcinoma Hep-2, and human tongue squamous cell carcinoma Tca8113 cells 6T25 flasks were inoculated with T7011 at a density of 100 cells / flask. After overnight incubation, cells were either mock infected or infected with T7011 at 1 PFU / cell. After 2 hours of incubation, the inoculum was replaced with fresh medium. Cell supernatants were collected at 0, 4, 8, 12, 24, 48, 72, and 96 hours postinfection in preparation for ELISA assays to quantify CCL5 secretion. Figure 3 shows the release of CCL5 after T7011 infection in 293T, Hep-2, and Tca8113 cells. CCL5 expression and release were rapid and robust. Secreted CCL5 was detected as early as 4 hours postinfection, reaching a maximum of 5000 pg / mL. Secretion was stable and maintained for at least 4 days after T7011 virus infection, demonstrating that CCL5 is secreted.

[0101] Expression of IL-12, anti-PD-1 antibody and CCL5 by ELISA assay: 6 × 10 Vero cells 6 The cells were seeded into T150 flasks at a density of 100 cells / flask. After overnight incubation, the cells were infected with T7201, T7202, T7203, T7204, T7011, T7012, and T7013 at 0.01 PFU / cell. Cell supernatants collected 48 hours postinfection were used in ELISA assays to detect the expression levels of IL-12, anti-PD-1 antibody, and CCL5. The results are shown in Tables 3 and 4. As shown in Table 3, CCL5 expression was high and very stable in the tested viruses. IL-12 and PD-1 Ab expression were virtually identical between T7011 and T7013, while T7012 showed the highest IL-12 expression and the lowest PD-1 Ab expression. [Table 3] [Table 4]

[0102] Expression and presentation of cleaved CD19, BCMA, Trop-2, and HER2 on the cell surface by immunofluorescence assay: Hep-2 cells (4 x 10 5 ) were inoculated onto cover slips in individual wells of 6-well plates and incubated for 24 hours to allow cells to adhere. Cells were then mock infected or exposed to T7011, T7012, and T7013 viruses at 5 PFU / cell for 1 hour. The inoculum was replaced with fresh medium. Cells were rinsed with PBS and fixed with 4% paraformaldehyde for 10 minutes at room temperature for the indicated times, followed by blocking with 5% skim milk. Cells infected with T7011 were co-stained with antibodies against CD19 (catalog no. 302204, Biolegend) and BCMA (catalog no. NBP1-97637, Novus), cells infected with T7012 were co-stained with antibodies against CD19 (catalog no. 302204, Biolegend) and Trop-2 (catalog no. PA5-47030, Invitrogen), and cells infected with T7013 were stained overnight at 4°C with antibodies against CD19 (catalog no. 302204, Biolegend) and primary HER2 antibody (catalog no. MAB1129-100, R&D systems). The cells were then incubated with Alexa Fluor 488-conjugated anti-mouse (Cat. No. A32766, Invitrogen), Alexa Fluor 568-conjugated anti-rabbit (Cat. No. A11036, Invitrogen), and Alexa Fluor 568-conjugated anti-goat (Cat. No. A11057, Invitrogen) secondary antibodies at room temperature for 1 hour. The cells were then washed with PBS and embedded in mounting medium (Cat. No. 8961S, Cell Signaling Technology). Images were acquired and processed using a Nikon confocal laser scanning microscope (HD25, 120x magnification) and are shown in Figure 4. As can be seen from Figure 4, the different truncated antigens encoded by T7011, T7012, and T7013, respectively, were simultaneously expressed on the tumor cell surface.

[0103] Neurotoxicity studies: Six-week-old female BALB / c mice were anesthetized and then intracranially injected with 50 μL of 10-fold serial dilutions of HSV-1(F), T3011, T7011, T7012, or T7013 virus in groups of eight mice. Mock-treated controls were inoculated with the same volume of DPBS containing 10% glycerol. Mice were monitored for 14 days, and the 50% lethal dose (LD) was calculated from the mortality data according to the Reed and Muench method. 50 ) was calculated.

[0104] As shown in Table 5 below, the LD of T7011, T7012, T7013 and T3011 50 The values were 158-, 316-, 100-, and 268-fold higher than HSV-1(F), respectively, indicating that, like the T3011 virus, the neurovirulence of T7011, T7012, and T7013 was significantly attenuated compared to HSV-1(F). [Table 5]

[0105] Antitumor activity of T7 series oHSV viruses: Tumor cells were seeded into 96-well plates at a density of 10,000 cells / well. After overnight incubation, cells were infected with T3011, T7011, T7012, and T7013 at 0.01, 0.1, 1, 5, 10, 33.33, and 100 PFU / cell in triplicates. At 48 hours post-infection (pi), cell viability was measured by CellTiter-Glo. Percent cell growth inhibition was calculated according to the manufacturer's instructions. The concentration resulting in 50% cell growth inhibition by virus infection (IC) was determined by fitting the data to a dose-response curve using GraphPad Prism software. 50 ) (PFU / cell) was calculated.

[0106] As shown in Fig. 5, the IC of T7 series viruses 50The IC values were comparable to those of T3011, indicating that the T7 series viruses possess similar broad antitumor activity compared to T3011. On the other hand, the IC values in HCT116, Hep-2, PC-3, MDA-MB-231, and A375 cells were significantly higher than those in T3011. 50 The values were slightly higher than those of other tumor cell lines, indicating that these cell lines were relatively resistant to infection with T7 series viruses and were selected for further combo studies.

[0107] Infectious activity of T7 series oHSV viruses: Hep-2 cells, non-transduced normal T cells, and CD19 CAR-T (CAR-T) cells were cultured at 5 × 10 5 Cells were seeded into 12-well plates at a density of 100 cells / well and infected with HSV-1(F), T7011, T7012, and T7013 at 1 PFU / cell. Cell pellets were collected at 24 and 48 hours (h) post-infection and then washed with PBS. The cell pellets were then resuspended in DPBS + 10% glycerol and subjected to three freeze-thaw cycles. Virus progeny were titrated on Vero cells.

[0108] As shown in Figure 6, the virus yields in Hep-2 cells infected with all viruses were significantly higher than those in normal T cells and CAR-T cells. In particular, the virus yields in both normal T cells and CAR-T cells were 10% or higher at 24 or 48 hours after infection. 3 These results indicate that wild-type HSV-1(F) virus has low infectious activity in CAR-T or normal T cells, whereas the attenuated T7011, T7012, and T7013 viruses have no infectious activity.

[0109] Cytotoxic activity of T7 series oHSV viruses: CD19 CAR-T CD19 ) cells and 4 × 10 untransduced normal T cells 4Cells were seeded at 0.01, 0.1, 1, and 10 PFU / cell in 96-well plates and infected with T7011, T7012, and T7013 in triplicate. Cell viability was measured by CellTiter-Glo at 24 and 48 hours post-infection (pi). Relative cell viability was calculated as a percentage of untreated cells.

[0110] As shown in Figure 7, cell viability was not reduced upon infection with the T7 series viruses, demonstrating that the T7 series viruses had no cell-killing activity against either CAR-T or normal T cells.

[0111] T7011 and CAR-T CD19 Antitumor effect of the combination: 1×10 human laryngeal cancer cells Hep-2, human melanoma cells A375, and human prostate cancer cells PC-3 4 The cells were seeded into 96-well plates at a density of 10 cells / well. After overnight incubation, the cells were infected with or without T7011 at 0.01, 0.03, 0.1, 0.3, and 1 PFU / cell in triplicate. At 24 h postinfection, 4 × 10 cells were seeded at an effector-to-target (E:T) ratio of 4:1 for coculture with tumor cells. 4 CAR-T cells / well CD19 Or T cells were added. Non-transduced normal T cells served as a control. After 24 hours of co-culture, cell viability was measured by CellTiter-Glo. Relative cell viability was calculated as a percentage of untreated cells.

[0112] As shown in Figure 8, T7011 and CAR-T CD19 The combination of T7011 and CAR-T showed more than 60% higher efficacy than the single agents. This result shows that the antitumor effect was greater in the T7011 and CAR-T groups than in the T7011 and CAR-T groups alone. CD19 In contrast, the combination of T7011 and normal T cells showed only a slight antitumor effect.

[0113] Furthermore, human laryngeal cancer cells Hep-2, human melanoma cells A375, and human prostate cancer cells PC-3 were cultured at 1 × 10 4 Cells were seeded into 96-well plates at a density of 1 × 10 cells / well. After overnight incubation, cells were infected with or without T7011 or T3011 at 1 PFU / cell in triplicate. At 24 h post-infection, 4 × 10 cells were infected at an effector to target (E:T) ratio of 4:1 for an additional 24 h of co-culture. 4 CAR-T cells / well CD19 Cells were added. Untreated cells served as the untreated control. Cell viability was measured by CellTiter-Glo. Relative cell viability was calculated as a percentage of untreated cells.

[0114] As shown in Figure 9, the T7011 and CAR-T combination treatment groups showed significantly higher efficacy compared to the single treatment group and the T3011 and CAR-T combination treatment group. CD19 Only the combination treatment of T3011 and CAR-T significantly reduced cell viability. CD19 All of these results suggest that T7011 virus infection is a major risk factor for CAR-T CD19 This indicates that it can specifically synergize with the antitumor activity of

[0115] T7012 and CAR-T CD19 Antitumor effect of the combination: 1×10 human laryngeal cancer cells Hep-2, human melanoma cells A375, and human prostate cancer cells PC-3 4 The cells were seeded into 96-well plates at a density of 10 cells / well. After overnight incubation, the cells were infected with or without T7012 at 0.01, 0.03, 0.1, 0.3, and 1 PFU / cell in triplicate. At 24 h postinfection, 4 × 10 cells were seeded at an effector-to-target (E:T) ratio of 4:1 for coculture with tumor cells. 4 CAR-T cells / well CD19 Or T cells were added. Non-transduced normal T cells served as a control. After 24 hours of co-culture, cell viability was measured by CellTiter-Glo. Relative cell viability was calculated as a percentage of untreated cells.

[0116] As shown in Figure 10, T7012 and CAR-T CD19 The combination of T7012 and CAR-T showed more than 50% higher efficacy than the single agents. This result shows that the antitumor effect was greater in the T7012 and CAR-T groups than in the T7012 and CAR-T groups alone. CD19 The combination of T7012 and normal T cells demonstrated a significant improvement in tumor response. In contrast, the combination of T7012 and normal T cells showed only a slight antitumor effect.

[0117] Furthermore, human laryngeal cancer cells Hep-2, human melanoma cells A375, and human prostate cancer cells PC-3 were cultured at 1 × 10 4 Cells were seeded into 96-well plates at a density of 10 cells / well. After overnight incubation, cells were infected with or without T7012 or T3011 at 1 PFU / cell in triplicate. At 24 h post-infection, 4 × 10 cells were added at an effector to target (E:T) ratio of 4:1 for an additional 24 h of co-culture. 4 CAR-T cells / well CD19 Cells were added. Untreated cells served as the untreated control. Cell viability was measured by CellTiter-Glo. Relative cell viability was calculated as a percentage of untreated cells.

[0118] As shown in Figure 11, T7012 and CAR-T treatment significantly improved the survival rate compared to the single treatment group and the T3011 and CAR-T combination treatment group. CD19 Only the combination treatment of T3011 and CAR-T significantly reduced cell viability. CD19 All of these results suggest that T7012 virus infection is a major risk factor for CAR-T CD19 This indicates that it can specifically synergize with the antitumor activity of

[0119] T7013 and CAR-T CD19 Antitumor effect of the combination: 1×10 human laryngeal cancer cells Hep-2, human melanoma cells A375, and human prostate cancer cells PC-3 4The cells were seeded into 96-well plates at a density of 10 cells / well. After overnight incubation, the cells were infected with or without T7013 at 0.01, 0.03, 0.1, 0.3, and 1 PFU / cell in triplicate. At 24 h postinfection, 4 × 10 cells were seeded at an effector-to-target (E:T) ratio of 4:1 for coculture with tumor cells. 4 CAR-T cells / well CD19 Or T cells were added. Non-transduced normal T cells served as a control. After 24 hours of co-culture, cell viability was measured by CellTiter-Glo. Relative cell viability was calculated as a percentage of untreated cells.

[0120] As shown in Figure 12, T7013 and CAR-T CD19 The combination of T7013 and CAR-T showed more than 60% higher efficacy than the single agents. This result shows that the antitumor effect was greater in the T7013 and CAR-T groups than in the T7013 and CAR-T groups alone. CD19 The combination of T7013 and normal T cells demonstrated a significant improvement in tumor response. In contrast, the combination of T7013 and normal T cells showed only a slight antitumor effect.

[0121] Furthermore, human laryngeal cancer cells Hep-2, human melanoma cells A375, and human prostate cancer cells PC-3 were cultured at 1 × 10 4 Cells were seeded into 96-well plates at a density of 10 cells / well. After overnight incubation, cells were infected with or without T7013 or T3011 at 1 PFU / cell in triplicate. At 24 h post-infection, 4 × 10 cells were added at an effector to target (E:T) ratio of 4:1 for an additional 24 h of co-culture. 4 CAR-T cells / well CD19 Cells were added. Untreated cells served as the untreated control. Cell viability was measured by CellTiter-Glo. Relative cell viability was calculated as a percentage of untreated cells.

[0122] As shown in Figure 13, the T7013 and CAR-T combination treatment groups showed a significant improvement compared to the single treatment group and the T3011 and CAR-T combination treatment group. CD19 Only the combination treatment of T3011 and CAR-T significantly reduced cell viability.CD19 All of these results suggest that T7013 virus infection is a major risk factor for CAR-T CD19 This indicates that it can specifically synergize with the antitumor activity of

[0123] CAR-NK CD19 Cytotoxic activity of T7 series (T7011, T7012, and T7013) oHSV viruses in human and NK cells: NK cells were isolated from PBMCs according to methods known in the art. CD19 CAR-NK (CAR-NK CD19 ) cells and 4 × 10 normal untransduced NK cells 4 Cells were seeded into 96-well plates at 0.01, 0.1, 1, and 10 PFU / cell with HSV-1(F), T7011, T7012, and T7013 in triplicate. Cell viability was measured by CellTiter-Glo at 24, 48, and 72 hours postinfection (pi). Relative cell viability was calculated as a percentage of untreated cells.

[0124] As shown in Figure 14, cell viability was not reduced upon infection with the T7 series viruses, demonstrating that the T7 series viruses had no cell-killing activity against either CAR-NK cells or normal NK cells.

[0125] CAR-NK CD19 Viral replication of HSV-1(F) and T7011 in cells and NK cells: CAR-NK CD19 5 × 10 cells and NK cells 5 Cells were seeded into 12-well plates at a density of 10 cells / well and infected with HSV-1(F) and T7011 at 1 PFU / cell in the presence (+IL-2) or absence of IL-2. Cell pellets were collected at 2, 24, 48, and 72 hours postinfection. The cell pellets were then washed with PBS, resuspended in DPBS + 10% glycerol, and freeze-thawed three times. Virus progeny were titrated on Vero cells.

[0126] As shown in Figure 15, the T7011 virus inhibited CAR-NK CD19 It has no infectious activity in cells or normal NK cells.

[0127] CAR-NK CD19 Effect of T7011 on cell proliferation: CAR-NK CD19 5 x 10 cells 5 Cells were seeded into 12-well plates at a density of 10 cells / well and infected with or without HSV-1(F) and T7011 at 0.1 or 1 PFU / cell in the presence (+IL-2) or absence of IL-2. Cell pellets were collected at 24, 48, and 72 hours postinfection, and viable cells were counted by trypan blue staining.

[0128] As shown in Figure 16, T7011 is a CAR-NK CD19 It does not have a negative effect on cell proliferation.

[0129] Effect of T7011 on CAR-NK cell proliferation: 5 × 10 NK cells 5 Cells were seeded into 12-well plates at a density of 10 cells / well and infected with or without HSV-1(F) and T7011 at 0.1 or 1 PFU / cell in the presence (+IL-2) or absence of IL-2. Cell pellets were collected at 24, 48, and 72 hours postinfection, and viable cells were counted by trypan blue staining.

[0130] As shown in Figure 17, T7011 does not have a negative effect on NK cell proliferation.

[0131] T7011 and CAR-NK CD19 Cell killing effect of the combination: Human laryngeal carcinoma Hep-2, human melanoma A375, and human prostate cancer PC-3 cells were seeded in a 96-well plate. After overnight incubation, the cells were infected with T7011 at 0.01, 0.1, and 1 PFU / cell in triplicate. At 24 hours postinfection, CAR-NK cells were co-cultured with tumor cells at an effector-to-target (E:T) ratio of 2:1. CD19 Cells were added. After an additional 24 or 48 hours, cell viability was measured by CellTiter-Glo. Relative cell viability was calculated as a percentage of untreated cells.

[0132] As shown in Figure 18, T7011 and CAR-NK CD19 The combination was more effective than the single agents.

[0133] Furthermore, human melanoma A375 cells were cultured at 1 × 10 4 The cells were seeded into a 96-well plate at a density of 1 PFU / cell. After overnight incubation, NK, CAR-NK, and CAR-NK cells were transfected at 1 PFU / cell. CD19 Twenty-four hours after infection, cells were infected with CAR-NK at an effector to target (E:T) ratio of 2:1 for an additional 24 hours of co-culture. CD19 Cells or NK cells were added. Cell viability was measured by CellTiter-Glo. Relative cell viability was calculated as a percentage of untreated cells.

[0134] As shown in Figure 19, the T7011 and CAR-NK treatment groups showed significantly higher IL-1 expression than the single treatment groups and the T7011 and NK combination treatment groups. CD19 The combined treatment significantly reduced cell viability. All of these results suggest that T7011 virus infection may be a contributing factor to the survival of CAR-T cells. CD19 This indicates that it can specifically synergize with the antitumor activity of

Claims

1. A genetically modified oncolytic herpes simplex virus type 1 (oHSV-1), comprising: The genome of the oHSV contains (a) a truncated, non-signaling mutant of at least one tumor-associated / specific antigen, and (b) at least one chemokine a polynucleotide encoding expression of the truncated non-signaling mutant and the at least one chemokine is controlled by an immediate early gene promoter of HSV; and Upon replication of the oHSV-1 in tumor cells, the truncated, non-signaling mutant is expressed and presented as a biomarker on the surface of the tumor cells, and the at least one chemokine is expressed and released to induce chemotaxis of immune cells to the tumor cells; the oHSV-1 is further modified such that a fragment of nucleic acid of the oHSV-1 is deleted, the fragment of nucleic acid of the oHSV-1 being from positions 117005 to 132096 of the P prototype genome of the F strain; the oHSV-1 is further modified to encode IL-12 and an anti-PD-1 antibody or antigen-binding fragment thereof; the polynucleotide is (i) a truncated, non-signaling mutant of CD19, a truncated, non-signaling mutant of BCMA, and CCL5; (ii) a truncated, non-signaling mutant of CD19, a truncated, non-signaling mutant of Trop-2, and CCL5; (iii) a truncated, non-signaling mutant of CD19, a truncated, non-signaling mutant of HER2, and CCL5; (iv) truncated, non-signaling mutants of CD19 and CCL5; (v) a truncated, non-signaling mutant of Trop-2 and CCL5; (vi) a truncated, non-signaling mutant of BCMA and CCL5, or (vii) encoding a truncated, non-signaling mutant of HER2 and CCL5; and The genetically modified oHSV-1, wherein the truncated non-signaling mutant is the extracellular domain or the extracellular-transmembrane domain.

2. The genetically modified oHSV-1 of claim 1, wherein the HSV immediate early gene promoter is selected from the group consisting of HSV-1 IE1 (ICP0 promoter), IE2 (ICP27 promoter), IE3 (ICP4 promoter), and IE4 / 5 (ICP22 and ICP47 promoters).

3. The genetically modified oHSV-1 of claim 1, wherein the polynucleotide is inserted between UL37 and UL38.

4. The genetically modified oHSV-1 according to claim 1, wherein the immediate early gene promoter of HSV is the immediate early gene promoter IE4 / 5 of HSV-1.

5. 2. The genetically modified oHSV-1 of claim 1, wherein a PolyA tail is located downstream of the polynucleotide encoding the cleaved antigen and chemokine.

6. The genetically modified oHSV-1 of claim 1, wherein the tumor cell is a solid tumor cell.

7. The genetically modified oHSV-1 of claim 1, wherein the tumor cells do not express the tumor-associated / specific antigen encoded by the polynucleotide.

8. 1. A pharmaceutical kit for the treatment of cancer, comprising separately the oHSV-1 of claim 1 and a tumor-targeting therapeutic agent, the tumor-targeting therapeutic agent comprising a targeting moiety specific for a truncated, non-signaling mutant of the at least one tumor-associated / specific antigen encoded by the polynucleotide, and an effector moiety for killing or inhibiting the proliferation of cancer cells, The pharmaceutical kit, wherein the tumor-targeting therapeutic agent is selected from the group consisting of CAR-T cells, CAR-NK cells, BiTEs, and ADCs.

9. the tumor-targeted therapeutic agent is selected from the group consisting of CD19-targeted CAR-T cells, CD19-targeted CAR-NK cells, and CD19-targeted BiTEs; or The pharmaceutical kit of claim 8, wherein the tumor-targeting therapeutic agent is a HER2-, Trop-2-, or BCMA-targeting ADC.

10. 9. The pharmaceutical kit of claim 8, wherein the tumor-targeting therapeutic agent is selected from the group consisting of Tecartus®, Kymriah®, Yescarta®, Blincyto®, Kadcyla®, Enhertu®, Trodelvy®, and Blenrep®.

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