Oncolytic virus therapy with induced antitumor immunity

Abstract

The present invention provides an improved oncolytic virus that enhances collateral cell killing and induces anti-tumor immunity. The oncolytic virus comprises an oncolytic herpesvirus backbone genetically modified to encode a tumor cell-binding component and an immunoglobulin (Ig)-binding component. For example, the present invention provides an improved oncolytic virus comprising an oncolytic virus backbone genetically modified to encode a chimeric molecule comprising a tumor cell-binding component and an immunoglobulin (Ig)-aggregating component and to induce secretion of the chimeric molecule from infected cells, wherein the secreted chimeric molecule enhances collateral tumor cell killing and anti-tumor immunity in the presence of anti-viral antibodies or other Ig and innate immune cells.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates generally to methods and constructs for viral tumor therapy. [Background technology]

[0002] Background of the Invention Without limiting the scope of the invention, some background is provided in the context of existing oncolytic virus therapy. Oncolytic virus therapy for cancer relies on the use of oncolytic viruses, which are defined by their ability to selectively replicate in and destroy tumor cells while sparing normal cells.

[0003] Oncolytic viruses can kill cancer cells through a variety of methods, ranging from direct virus-mediated cell lysis to various cytotoxic immune effector mechanisms. However, current oncolytic virus therapies are limited by suboptimal oncolytic activity, susceptibility to suppression by innate or adaptive immune effectors, and a limited ability to induce tumor-specific immune responses, particularly against neoantigens.

[0004] Therefore, over the past decade, genetic engineering has been utilized to circumvent these problems and generate oncolytic virus strains with superior clinical activity. See, for example, Russell SJ, et al. Oncolytic virotherapy. Nat Biotechnol 30 (2012) 658-70. In 2015, Amgen obtained FDA approval of the first genetically engineered virus for the treatment of melanoma. Pol, J. et al. Oncoimmunology 5(1) (2016) e1115641. An injectable oncolytic virus called talimogene-laherparepvec or "T-Vec" originated from a primary isolate of human herpes simplex virus 1 (HSV-1) known as JSI (ECACC accession number 01010209), which demonstrated naturally enhanced oncolytic activity. JSI was attenuated by functionally deleting both copies of RL1 (encoding the neurovirulence factor ICP34.5) and US12 (encoding ICP47), and a cassette encoding human granulocyte-macrophage colony-stimulating factor (GM-CSF) under the control of the cytomegalovirus immediate-early promoter was inserted into the nonfunctional RL1 locus. The addition of GM-CSF favors the recruitment and activation of antigen-presenting cells, thereby promoting the initiation of tumor-targeting immune responses. T-Vec has demonstrated measurable, and in some cases durable, therapeutic efficacy in a relatively small percentage of melanoma patients. The therapeutic efficacy of this and other oncolytic viruses must and undoubtedly can be further improved. One improvement strategy is to enhance the ability of viral therapy to induce antitumor immunity. Several approaches have been reported in this effort, primarily by incorporating immunostimulatory genes into the viral genome to enhance tumor antigen presentation and induce T cell immunity. An alternative strategy is provided herein. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Russell SJ, et al. Oncolytic virotherapy. Nat Biotechnol 30 (2012) 658-70 [Non-patent document 2] Pol, J. et al. Oncoimmunology 5(1) (2016) e1115641 Summary of the Invention [Means for solving the problem]

[0006] Summary of the Invention Provided herein are improved oncolytic viruses that increase bystander cell killing and induce anti-tumor immunity, including methods for making and using such viral constructs in the treatment of cancer.

[0007] According to one aspect of the present disclosure, there is provided an improved oncolytic virus comprising an oncolytic virus backbone that encodes a chimeric molecule comprising a tumor cell-binding component and an immunoglobulin (Ig)-binding component and that is genetically modified to induce secretion of the chimeric molecule from infected cells, wherein the two secreted chimeric molecules enhance collateral cell killing and anti-tumor immunity in the presence of anti-tumor antibodies. In a specific aspect, the immunoglobulin-binding component comprises at least one Ig-binding "B" domain derived from peptostreptococcal protein L. In other embodiments, the immunoglobulin-binding component comprises at least four or five Ig-binding "B" domains derived from peptostreptococcal protein L.

[0008] In certain embodiments, the improved oncolytic viruses are constructed on a herpes simplex virus type 1 (HSV1) backbone, while in other embodiments, the oncolytic virus backbone is based on herpes simplex virus type 2 (HSV2). In certain embodiments, the HSV1 backbone contains at least one deletion of ICP34.5. In other embodiments, the HSV2 backbone contains an N-domain deletion of ICP10, which allows selective replication in tumor cells.

[0009] In certain embodiments, the tumor cell binding component of the improved oncolytic virus is an affibody that binds to a tumor antigen. In other embodiments, the tumor cell binding component is a ligand that binds to a tumor antigen in the form of a cell surface receptor expressed or overexpressed on tumor cells. In yet other embodiments, the tumor cell binding component is a single-chain antibody (scFv) or a single-domain antibody (nanobody). In certain embodiments, the tumor antigen is selected from human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), Erb-B2 receptor tyrosine kinase 3 (ErbB3), epithelial cell adhesion molecule (EpCAM), mesothelin (MSLN), MET proto-oncogene, insulin-like growth factor 1 receptor (IGF1R), ephrin receptor A3 (EphA3), TNF receptor apoptosis-inducing ligand receptor 1 (TRAIL-R1), TNF receptor apoptosis-inducing ligand receptor 2 (TRAIL-R2), vascular endothelial growth factor receptor (VEGFR), receptor activator of nuclear factor kappa beta ligand (RANKL), programmed death-ligand 1 (PD-L1), phosphatase of regenerating liver 3 (PRL-3), human melanoma antigen recognized by T cells, and carcinoembryonic antigen (CEA) gene family products. In certain embodiments, the human melanoma antigen recognized by T cells is selected from melanoma-associated antigen 1 (MAGE-A1); melanoma-associated antigen A3 (MAGE-A3); B melanoma antigen (BAGE); G antigen 2C (GAGE), melanoma antigen 1 recognized by T cells (MLANA), and premelanosome protein (PMEL).

[0010] In certain embodiments, the tumor cell binding component is a ligand that binds to a tumor antigen, for example, the extracellular domain of epidermal growth factor (EGF) that binds to the EGF receptor (EGFR) on tumor cells.

[0011] According to a further aspect, improved oncolytic viruses that increase collateral cell killing and induce anti-tumor immunity are provided, the viruses comprising an oncolytic herpesvirus backbone genetically modified to encode an affibody-Protein L (PL) cassette comprising an anti-HER2 affibody and multiple Protein L immunoglobulin binding domains fused in-frame to form an affibody-Protein L (PL) engineered for extracellular secretion by cells infected with the oncolytic virus. In certain embodiments, the affibody-PL cassette comprises a synthetic signal peptide (Sp), an anti-HER2 affibody (affibody), a linker (e.g., (GGGS)3, (Gly)8, (Gly)6, (EAAAK)3), multiple Protein L immunoglobulin binding domains (1-5, with 5 being optimal), and a growth hormone polyadenylation signal (polyA).

[0012] According to another aspect, an improved oncolytic virus that increases collateral cell killing and induces anti-tumor immunity is provided, the virus comprising an oncolytic herpesvirus backbone genetically modified to encode an extracellular domain of epidermal growth factor (EGF) that binds to the EGF receptor (EGFR) on tumor cells, fused in-frame to form EGF-PL, engineered for extracellular secretion by cells infected with the oncolytic virus, and multiple protein L domains.

[0013] An improved oncolytic virus that increases collateral cell killing and induces anti-tumor immunity, the virus comprising an oncolytic herpesvirus backbone genetically modified to encode an extracellular domain of epidermal growth factor (EGF) that binds to the EGF receptor (EGFR) on tumor cells, fused in frame to form EGF-PL, which is engineered for extracellular secretion by cells infected with the oncolytic virus, and multiple protein L domains.

[0014] Also provided is a method of treating cancer comprising administering a therapeutically effective amount of the improved oncolytic virus disclosed herein and a diluent or carrier.

[0015] For a more complete understanding of the present invention, including its features and advantages, reference is now made to the detailed description of the invention along with the accompanying drawings. [Brief explanation of the drawings]

[0016] [Figure 1] Figures 1A and 1B depict the design of Affibody-PL and its in vivo mechanism of action in the tumor microenvironment. Figure 1A depicts the gene cassette of the chimeric molecule Affibody-PL. Figure 1B depicts the predicted mechanism of action of Affibody-PL after delivery by oncolytic virus to HER2- or EGFR-expressing tumors.

[0017] [Figure 2-1] Figure 2A provides a schematic diagram of the stepwise assembly of FusOn-H2, and Figure 2B provides a schematic diagram of the stepwise assembly of FusOn-PL. [Figure 2-2] Figure 2C shows an alignment of the five domains of Finegoldia magna protein L as shown by Kastern (Kastern W et al. "Structure of Peptostreptococcal Protein L and Identification of a Repeated Immunoglobulin Light Chain-binding Domain" JBC 267 (18) (1992) 12820-12825).

[0018] [Figure 3] Figure 3 shows the results of efficient binding of affibody-PL to HER2 expressed on the surface of tumor cells. Tumor cells expressing various levels of HER2 (high in Skov3 cells, moderate in MCF7 cells, and completely negative in MDA-MB-231 cells) were sequentially incubated with 1) affibody-PL supernatant, 2) anti-HA antibody of mouse origin, and 3) FITC-conjugated goat anti-mouse IgG. The cells were then subjected to flow cytometry analysis.

[0019] [Figure 4] Figures 4A and 4B show the ability of affibody-PL to induce HER2-expressing tumor cell killing in PBMCs. PBMCs were prepared from normal human blood and mixed with SKOV3 tumor cells at a ratio of either 10 (R10) or 20 (R20) in the presence of 5 μg / ml IgG and either medium, supernatant collected from HEK293 cells transfected with a control vector (Ctl-Vec), or supernatant collected from HEK293 cells transfected with affibody-PL (Affibody-PL). After 24 hours, PBMCs and dead cells floating in the medium were removed, and remaining viable cells were stained with 0.1% crystal violet in ethanol. Figure 4A shows representative photomicrographs of each well from three different preparations. Figure 4B shows quantification of tumor cell killing. Stained tumor cells were lysed with 2% SDS, and the released dye was measured at a wavelength of 595 nM using a Spectramax 5 plate reader. The percentage of tumor cell killing was calculated by dividing the cell reading in wells without PBMCs (and others) by the reading in each of the triplicate wells. Asterisks indicate p<0.05 compared to medium and Ctl-Vec.

[0020] [Figure 5A] Figure 5A shows Western blot detection of affibody-PL expressed from FusOn-Affibody-PL. Supernatants were collected from Vero cells infected with either FusOn-H2 or FusOn-Affibody-PL. After filtration through a 0.1 μM filter, the supernatants were loaded onto gel electrophoresis and Western blotted using rabbit anti-HA tag IgG as the primary antibody. [Figure 5B] Figure 5B shows flow cytometry analysis of the binding of affibody-PL generated from FusOn-affibody-PL to HER2-expressing mouse tumor cells. The experiment was performed as in Figure 3, except that a mouse colon cancer cell line stably transduced with HER2 (CT26-HER2) was used. [Figure 5C] Figure 5C shows measurements of the ability of affibody-PL to induce HER2-expressing tumor cell killing in PBMCs. The experiment was performed as in Figure 4B, except that 1) the cells were of murine origin (CT26-HER2 and splenocytes harvested from Balb / c mice), 2) mouse Ig was used, and 3) supernatants were obtained from FusOn-H2 or FusOn-affibody-PL infected cells. Asterisks indicate p<0.05 compared to medium and FusOn-H2 supernatants.

[0021] [Figure 6] Figure 6A shows NK cell infiltration following viral treatment with FusOn-H2 and FusOn-Affibody-PL. Tumor tissues were harvested 3 days after treatment of mice with 1 × 10 pfu of either PBS, FusOn-H2, or FusOn-Affibody-PL. The harvested tumor tissues were divided in half. One half was embedded in paraffin, and tissue sections were used for immunohistological staining of NK cells. Positively stained NK cells are indicated by black arrows. The other half was used to prepare frozen sections, which were then immunohistochemically stained for both the NK marker (NCR1) and ki67 (Figure 6B). Positively stained NK cells are indicated by white arrowheads, and cells that stained positive for ki67 are indicated by white arrows.

[0022] [Figure 7]Figures 7A and 7B show the results of treatment evaluation and comparison of viral therapy with FusOn-H2 and FusOn-PL. CT26-HER2 tumor cells were implanted subcutaneously. When tumors reached a size of approximately 5 mm in diameter, mice were intratumorally treated with either 1 x 10 pfu of FusOn-PL or 1 x 10 pfu of FusOn-H2, or PBS as a negative control. Figure 7A shows the change in tumor size after treatment. Figure 7B shows the results of an IFN-γ ELISPO assay for class I neoantigen-specific antitumor immunity following viral therapy. The photographs show representative areas of wells from an ELISPO assay using the indicated neoantigen peptide or a no-peptide control. Asterisks indicate p<0.05 compared to either FusOn-H2 or PBS.

[0023] [Figure 8] Figure 8A shows the results of therapeutic evaluation of short-term viral therapy with FusOn-H2 and FusOn-PL. The experimental procedure was identical to that described in Figure 7A. Figure 8A shows the results of tumor size measurements taken continuously for 3 weeks before all mice were euthanized. Tumor growth rates were calculated by dividing the tumor volume measured at the indicated time by the tumor volume immediately before the start of treatment. Figure 8B shows the counting data from ELISPOT assays for the neoantigenic peptides and their mixtures listed in Table 2. Controls included no peptide or an irrelevant peptide (ovalbumin class II peptide (OVA323-339)). Asterisks indicate p<0.05 compared to FusOn-H2. A + indicates p<0.05 compared to PBS.

[0024] [Figure 9] Figure 9A shows a schematic of Synco-4 construction starting from Synco-2D, and Figure 9B shows the identification of Synco-47G transformants by green fluorescence.

[0025] [Figure 10]FIG. 10 shows the expression and secretion of EGF-PL by Synco-4 in 293 cells transfected with a plasmid containing an EGF-PL gene cassette (pCR-ul47-EGFPL) or infected with Synco-4.

[0026] [Figure 11] Figures 11A and 11B show that EGF-PL produced by Synco-4 binds to EGFR expressed on tumor cells with high efficiency and specificity. Figure 11A shows flow cytometry staining for EGFR expression on CT-26-EGFR cells, but not on other tumor cells. Figure 11B shows flow cytometry demonstrating that EGF-PL in the supernatant of Synco-4 infection can strongly bind to CD26-EGFR cells.

[0027] [Figure 12-1] Figures 12A-12G show the cytotoxic effect of Synco-4 on various tumor cell lines. Tumor cells were treated with Synco-4 at 0.003-3 MOI (multiplicity of infection). After 48 hours of incubation, viability was detected using an MTT assay kit. The data show that Synco-4 induced dose-dependent inhibition of tumor cell proliferation in Syrian hamster adenocarcinoma cells (HaP-T1) (Figure 12A), mouse breast cancer cells (4T-1) (Figure 12B), mouse colon carcinoma cells (CT-26) (Figure 12C), human glioblastoma cells (U87) (Figure 12D), human pharynx squamous carcinoma cells (FaDu) (Figure 12E), human hepatocellular carcinoma cells (HEPG2) (Figure 12F), and human osteosarcoma cells (MNNG-HOS1) (Figure 12G). [Figure 12-2] Same as above. [Figure 12-3] Same as above. [Figure 12-4] Same as above.

[0028] [Figure 13]FIG. 13 shows results demonstrating that Synco-4 had enhanced antitumor activity against CD26-EGFR tumors compared to the parent synco-2D. DETAILED DESCRIPTION OF THE INVENTION

[0029] [Table 3-1] [Table 3-2] Detailed Description of the Invention One of the major host defense mechanisms against viral infection is via innate immune cells, including NK cells and macrophages. These cells can rapidly eliminate introduced oncolytic viruses, presenting a major barrier to cancer virus therapy. Provided herein are constructs and methods for redirecting infiltrating innate immune cells to attack tumor cells instead. In exemplary embodiments disclosed herein, herpes simplex virus (HSV)-based oncolytic viruses, FsOn-H2 (SEQ ID NO: 36) or Synco-2D (SEQ ID NO: 35), are armed with a secreted chimeric molecule containing two separate components. In certain embodiments, component 1 is either an affibody (a peptide of approximately 58 aa) that binds to a tumor antigen such as HER2 or the extracellular domain of epidermal growth factor (EGF) receptor (EGFR) that binds to EGFR on tumor cells. The second component is protein L (PL), which can bind to various immunoglobulins (Ig), including antibodies against HSV. In vitro experiments disclosed herein demonstrate that the secreted chimeric molecule simultaneously binds to Fc receptors on innate immune cells (via engaged Ig) and HER2 or EGFR on tumor cells, actively engaging macrophages and NK cells with HER2- or EGFR-expressing tumor cells, resulting in efficient killing of the latter. Subsequent evaluation in mouse tumor models with limited tolerance to FusOn-H2 or Synco-2D demonstrates that arming viruses with this chimeric molecule can significantly enhance therapeutic activity. Furthermore, the data provided herein demonstrate that the combined killing action of engaged innate immune cells and oncolytic viruses provides the impetus to stimulate host antitumor immunity more efficiently than FusOn-H2 or Synco-2D viral therapy alone. Collectively, the data provided herein demonstrate that arming oncolytic viruses with this strategy represents a viable method for enhancing the oncolytic and immunotherapeutic effects of viral therapy.

[0030] While the making and use of various embodiments of the invention are discussed in detail below, it should be understood that the present invention provides many applicable inventive concepts that can be utilized in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.

[0031] Abbreviations: The following abbreviations are used throughout this application: [Table 4-1] [Table 4-2]

[0032] To facilitate understanding of the present invention and to avoid misunderstandings in interpreting the claims of the present invention, certain terms are defined below. Terms defined herein have meanings as commonly understood by one of ordinary skill in the art to which the present invention pertains. The terminology used to describe particular embodiments of the present invention does not delimit the invention except as outlined in the claims.

[0033] Terms such as "a," "an," and "the" are not intended to refer to the singular unless expressly defined as such, but include generic specific examples that may be used for illustration. The use of the terms "a" or "an," when used in conjunction with "comprising" in the claims and / or this specification, may mean "one," but may also coincide with "one or more," "at least one," and / or "one or more than one."

[0034] The use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer to the alternatives as mutually exclusive. Thus, unless otherwise stated, the term "or" in a group of alternatives means "any one or any combination" of the members of the group. Furthermore, unless expressly indicated to refer to the alternatives as mutually exclusive, the phrase "A, B and / or C" means an embodiment having element A alone, element B alone, element C alone, or any combination of A, B, and C taken together.

[0035] Similarly, for the avoidance of doubt, and unless otherwise expressly indicated to refer to alternatives as mutually exclusive, the phrase "at least one of," when combined with items in a list, means any single item in the list or any combination of items in the list. For example, unless otherwise defined, the phrase "at least one of A, B, and C" means "at least one of the group A, B, C, or any combination of A, B, and C." Thus, unless otherwise defined, the phrase requires one or more, and not necessarily all, of the listed items.

[0036] The terms "comprising" (and any of its forms, e.g., "comprise" and "comprises"), "having" (and any of its forms, e.g., "have" and "has"), "including" (and any of its forms, e.g., "includes" and "include") or "containing" (and any of its forms, e.g., "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0037] The term "effective," as used in the specification and claims, means adequate to bring about or achieve a desired, expected, or intended result.

[0038] The term "about" or "approximately" is defined as close to what would be understood by a person of ordinary skill in the art, and in one non-limiting embodiment, the term is defined as within 10%, within 5%, within 1%, and in certain aspects, within 0.5%.

[0039] To effectively infect and lyse tumor cells, oncolytic viruses must overcome host defense mechanisms that are normally initiated upon encountering an infectious pathogen. The innate immune system is the first line of defense, triggering an almost immediate response upon administration of an oncolytic virus. Thus, the innate immune response emerges as a critical barrier to cancer virotherapy. Key components of innate antiviral immunity include natural killer (NK) cells, macrophages, and interferons (IFNs). A study by Fulci et al. demonstrated that macrophage depletion during virotherapy significantly improved the therapeutic activity of oncolytic herpes simplex virus (HSV). See Fulci G, et al. Cyclophosphamide enhances glioma virotherapy by inhibiting innate immune responses. Proc Natl Acad Sci USA 103(34) (2006) 12873-8. Others have shown that oncolytic HSV recruits NK cells to the tumor site within hours of viral administration, resulting in rapid clearance of the introduced virus and therefore reduced therapeutic efficacy in a mouse glioblastoma model. These and other similar reports clearly highlight the importance of limiting innate antiviral immunity in cancer virotherapy.

[0040] On the other hand, NK cells and macrophages, two major cellular components of innate antiviral immunity, have the potential to kill malignant cells if properly activated or induced. Therefore, it seems promising to develop strategies for viral therapy that redirect infiltrating innate immune cells from eliminating oncolytic viruses and direct them to attack tumor cells instead.

[0041] Antibodies against oncolytic viruses can neutralize oncolytic viruses during viral propagation in tumor tissues, posing another major limiting factor for the effective therapeutic effect of viral therapy. Therefore, one approach is to exploit the widespread infiltration of these innate immune cells in viral therapy by converting them into tumor-targeting effector cells. One of the key activation mechanisms of NK cells and macrophages is antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC is triggered by the binding of the Fc portion of IgG, which becomes exposed when multiple IgG molecules aggregate into a multimeric form (e.g., in an immune complex), to Fc gamma receptors (FcγRs).

[0042] A potentially useful immunoglobulin agglutinating factor is Protein L ("PL"), an immunoglobulin (Ig)-binding protein derived from a cell wall protein produced by certain strains of the Gram-positive anaerobic bacterium Peptostreptococcus magnus (now known as Finegoldia magna). The PL gene product is a 76-106 kDa protein containing four or five highly homologous, continuous extracellular Ig-binding "B" domains, each 72-76 amino acid residues in length. See Kastern W et al. "Structure of Peptostreptococcal Protein L and Identification of a Repeated Immunoglobulin Light Chain-binding Domain" JBC 267 (18) (1992) 12820-12825. The amino acid sequence of the first kappa light chain-binding domain of Finegoldia magna Protein L is presented in SEQ ID NO:29. The amino acid sequence of the second kappa light chain binding domain of Finegoldia magna protein L is presented in SEQ ID NO: 30. The amino acid sequence of the third kappa light chain binding domain of Finegoldia magna protein L is presented in SEQ ID NO: 31. The amino acid sequence of the fourth kappa light chain binding domain of Finegoldia magna protein L is presented in SEQ ID NO: 32. The amino acid sequence of the fifth kappa light chain binding domain of Finegoldia magna protein L is presented in SEQ ID NO: 33. An exemplary codon-optimized sequence encoding the first kappa light chain binding domain of Finegoldia magna protein L is presented in SEQ ID NO: 1. An alignment of the five domains of Finegoldia magna protein L as set forth by Kastern (supra) is shown in Figure 2C.In certain embodiments, the immunoglobulin aggregating factor is formed by multiple kappa light chain binding domains of Finegoldia magna protein L, wherein each of the individual kappa light chain binding domains has at least 80% amino acid identity with the first kappa light chain binding domain of Finegoldia magna protein L set forth in SEQ ID NO: 29. In other embodiments, the immunoglobulin aggregating factor can be any combination of multiple kappa light chain binding domains of Finegoldia magna protein L set forth in any of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33.

[0043] Unlike Protein A and G, which bind to the Fc region of Ig, Protein L binds to the variable region of the kappa light chain of all classes of Ig, including IgG, IgM, IgA, IgE, and IgD. This allows the Fc region of the bound antibody to bind to FcγR on innate immune cells and initiate ADCC. Most importantly, like Protein A and G, Protein L also contains multiple copies (up to five) of the Ig-binding domain. This allows a single Protein L molecule to simultaneously bind to multiple units of Ig, creating multimeric Ig aggregates and potentially inducing Fc receptor oligomerization, thereby initiating ADCC. The amino acid sequence of Finegoldia magna Protein L containing five kappa light chain-binding domains is presented in SEQ ID NO: 34. An exemplary codon-optimized sequence encoding Finegoldia magna Protein L containing five kappa light chain-binding domains is presented in SEQ ID NO: 1.

[0044] In certain embodiments, the immunoglobulin aggregating factor comprises multiple kappa light chain binding domains, each of which has kappa light chain binding activity and has at least 80 percent amino acid sequence homology to the amino acid sequence of the first kappa light chain binding domain of Finegoldia magna protein L set forth in SEQ ID NO:29.

[0045] The strategy of using protein L to engage both Ig and innate immune cells and induce them to attack tumor cells could theoretically divert these two immune components from eliminating the oncolytic virus, thereby allowing sufficient viral replication and spread within tumor tissue, thus not only providing additional killing effects but also enhancing the oncolytic effects of the viral therapy itself.

[0046] In one embodiment provided herein, the above-described characteristics of the Protein L Ig-binding domain are utilized to enhance the therapeutic efficacy of oncolytic viruses. In this embodiment, the oncolytic viruses are engineered to redirect infiltrating innate immune cells to attack tumor cells instead of eliminating the introduced oncolytic virus. Specifically, an HSV-2-based oncolytic virus called "FusOn-H2" was constructed to express a soluble chimeric molecule consisting of a HER2-specific affibody that binds to Protein L. In another embodiment, an HSV-1-based oncolytic virus called "Synco-2D" was instead utilized. The binding of PL to EGF allows EGF-PL to target tumor cells overexpressing EGFR. This concept can be applied to other oncolytic virus constructs.

[0047] Anti-HER2 affibodies are short polypeptides with strong binding affinity for the HER2 tumor-associated antigen. In one embodiment, the anti-HER2 affibody has the sequence set forth in SEQ ID NO: 3. In vitro experiments described herein demonstrated that the chimeric molecule can actively engage macrophages and NK cells with HER2-expressing tumor cells in the presence of Ig, resulting in efficient killing of the latter. Subsequent evaluation in a mouse tumor model with limited tolerance to FusOn-H2 demonstrated that arming the virus with this chimeric molecule significantly enhanced therapeutic activity. Furthermore, the data presented herein demonstrate that the combined killing action of engaged innate immune cells and oncolytic viruses provides the impetus to stimulate host anti-tumor immunity more efficiently than FusOn-H2 viral therapy alone. Collectively, the data suggest that arming oncolytic viruses with this strategy represents a viable method for enhancing the oncolytic and immunotherapeutic effects of viral therapy.

[0048] The only oncolytic virus approved for clinical use by the U.S. Food and Drug Administration (FDA), Imlygic or T-VEC, has demonstrated measurable and, in some cases, durable therapeutic efficacy in a relatively small percentage of melanoma patients. The therapeutic efficacy of this and other oncolytic viruses must and undoubtedly can be further improved. One improvement strategy is to enhance the ability of viral therapy to induce antitumor immunity. Several approaches have been reported in this effort, primarily by incorporating immune-stimulating genes into the viral genome to enhance tumor antigen presentation and induce T cell immunity.

[0049] We present an alternative strategy designed to arm oncolytic viruses with chimeric molecules capable of inducing infiltrating innate immune cells to destroy tumor cells. This contrasts with other strategies that primarily focus on host adaptive immune cells, such as T cells. The chimeric element contains a tumor cell-specific targeting moiety and protein L as an Ig-binding domain. Because the lack of sufficient T cells in solid tumors is considered a major hurdle for cancer immunotherapy, such as checkpoint blockade, and other strategies designed to enhance effector T cells, one of the unique advantages of this strategy is that it exploits the widespread infiltration of these innate immune cells in viral therapy. Indeed, immunohistochemical staining of tumor tissues treated with FusOn-Affibody-PL revealed widespread infiltration of NK cells. The combined effects of Affibody-PL in infiltrating innate immune cells and inducing the local release of Affibody-PL or EGF-PL together result in additional tumor cell destruction, clearly contributing to the enhanced antitumor activity seen in in vivo studies. Indeed, half of the tumor-bearing mice treated with FusOn-Affibody-PL were completely tumor-free by the end of the experiment, and subsequent challenge of these mice with new tumor cells failed to initiate tumor growth in these mice, indicating that the administered viral therapy in these mice likely still conferred robust T cell-mediated antitumor immunity.

[0050] Tumor antigens present on tumor cells can be targeted by virus-encoded molecules that recognize and bind to tumor antigens. Examples of tumor antigens include human epidermal growth factor receptor 2 (also known as HER-2 and ErbB2, currently targeted in antibody-mediated therapy by trastuzumab (also known as HERCEPTIN®)), epidermal growth factor receptor (EGFR; also known as ErbB1), Erb-B2 receptor tyrosine kinase 3 (ErbB3), mesothelin, MET tyrosine kinase receptor, insulin-like growth factor 1 receptor (IGF1R), ephrin receptor A3 (EphA3), TNF receptor apoptosis-inducing ligand receptor 1 (TRAIL-R1), TNF receptor apoptosis-inducing ligand receptor 2 (TRAIL-R2), vascular endothelial growth factor receptor (VEGFR), receptor activator of nuclear factor kappa beta ligand (RANKL) and programmed death ligand 1 (PD-L1). New tumor antigens include the cancer-associated phosphatase PRL-3; human melanoma antigens recognized by T cells, including MAGE-1 (melanoma-associated antigen 1, also known as MAGEA1, OMIM entry 300016), MAGE-3 (also known as melanoma antigen, A3 family, OMIM entry 300174), BAGE (also known as B melanoma antigen, OMIM entry 605167), and the GAGE ​​family (also known as G antigen 2C, OMIM entry 300595), as well as MelanA (melanoma antigen 1 recognized by T cells, also known as MLANA, MART-1, OMIM entry 605513) and pre-melanosome protein (PMEL, also known as melanocyte protein 17, PMEL17, PMELGP100, gp100, OMIM entry 155550). Additional targets include certain gene products of the carcinoembryonic antigen (CEA) gene family, including OMIM entry 114890.

[0051] Thus, the targeting element of the chimeric molecule expressed by the virus specifically binds to a tumor antigen, such as those identified above. In certain embodiments, the targeting element expressed by the virus is an affibody, while in other embodiments, the targeting element is a single-chain variable fragment (scFv) or single-domain antibody directed against a cell surface tumor antigen. Non-limiting examples provided herein involve the use of a HER-2 affibody that binds to HER-2 on the tumor surface coupled with a PL. In other embodiments, the targeting element is a ligand for a cell surface receptor on tumor cells. Non-limiting examples provided herein involve the use of EGF that binds to EGFR on the tumor surface coupled with a PL.

[0052] In recent years, there has been increasing interest in utilizing neoantigens for cancer immunotherapy. Unlike tumor-associated tumor antigens, neoantigens originate from nonsynonymous mutations in the tumor cell genome and are therefore strictly tumor-specific. However, one of the challenges facing neoantigen-based immunotherapy is that neoepitopes are typically not shared among cancer patients. Current approaches to initially identify these neoantigens by exome sequencing followed by synthesis and delivery of antigenic epitopes to individual patients are cumbersome and can only be applied to cancer patients on a case-by-case basis. In principle, oncolytic virus therapy could provide a simple means of releasing these neoantigens in individual patients, ensuring efficient and timely presentation of neoantigens to the host immune system. However, such capabilities have not been previously explored. Studies on FusOn-affibody-PL-treated CT-26 tumor cells revealed that the armed virus was capable of inducing neoantigen-specific antitumor immunity against neoantigen peptides directed against both MHC class I and class II. The surprising data presented herein represent the first demonstration that viral therapy can induce measurable neoantigen-specific anti-tumor immunity.

[0053] Although the design of the affibody-PL limits its application to tumor cells overexpressing HER2, this strategy can be readily adapted to target other tumors by replacing the affibody with other binding moieties, such as ligands for various growth factor receptors or single-chain antibodies. Indeed, as provided herein, in one embodiment, the affibody in an affibody-PL chimeric construct was replaced with the binding domain of epidermal growth factor (EGF) to its receptor, and this construct was found to be capable of efficiently targeting tumor cells overexpressing EGFR (Figure 11A). In addition, we found that either EGF-PL (or the equivalent affibody-PL) could be incorporated into an HSV-1-based oncolytic virus, resulting in a similar potentiating effect (Figure 12). Thus, we expect that the disclosed strategy, including its potential for oncolytic viruses derived from other viruses, will be broadly applicable to the treatment of various malignancies.

[0054] The following examples are included for the purposes of complete disclosure, to illustrate methods of making the compositions and composites of the present invention, and to present certain properties of the compositions, and are not intended to limit the scope or teaching of the present disclosure in any way. [Example]

[0055] Example 1 Construction of the Disclosed Embodiments Figures 1A and 1B depict the design of an exemplary affibody-PL and its in vivo mechanism of action in the tumor microenvironment. Affibody molecules are short peptides, 58 amino acids in length, based on a three-alpha helix Z domain scaffold that can be selected from combinatorial libraries to bind to specific protein targets with strong affinity and specificity. See Feldwisch J, et al. "Engineering of affibody molecules for therapy and diagnostics" Methods Mol Biol. 899 (2012) 103-26. Examples of suitable affibodies selected for their strong binding affinity to HER2 are available. See Orlova A, et al. "Tumor imaging using a picomolar affinity HER2 binding affibody molecule," Cancer Res. 66(8) (2006) 4339-48; Steffen AC, et al. "Affibody-mediated tumor targeting of HER-2 expressing xenografts in mice," Eur J Nucl Med Mol Imaging 33(6) (2006) 631-8. The coding sequences for five immunoglobulin-binding domains (B1-B5) were selected from Peptostreptococcus magnus protein L as reported by Kastern et al. (supra). The anti-HER2 affibody and PL sequences were fused in-frame to construct affibody-PL. A signal peptide (Sp) was added to the N-terminus to render the chimeric molecule soluble. Figure 1A shows the gene cassette of affibody-PL, a chimeric molecule that can induce innate immune cells to attack tumor cells through a series of intermolecular engagements.From left to right, the sequence is a Rous sarcoma virus (RSV) long terminal repeat (LTR) (SEQ ID NO: 4), a synthetic signal peptide (Sp) (SEQ ID NO: 5) (as reported by Barash et al., "Human secretory signal peptide description by hidden Markov model and generation of a strong artificial signal peptide for secretory protein expression." Biochemical and Biophysical Research Communications 294 (2002) 835-842), an anti-HER2 affibody (affibody) (SEQ ID NO: 3), a linker-HA tag (HA) (SEQ ID NO: 6), the B1-B5 immunoglobulin binding domain of Protein L (SEQ ID NO: 2), and a bovine growth hormone polyadenylation signal (polyA) (SEQ ID NO: 7). As those skilled in the art will appreciate, other polyadenylation sites, such as SV40 polyA (SEQ ID NO: 28), can be used instead. The actual length of the coding sequence for each component is not proportional to the size of the depicted box.

[0056] The schematic diagram in Figure 1B illustrates the mechanism of action of affibody-PL when delivered to tumor tissue by oncolytic viruses. Local administration of the viral therapy assembles all components in the tumor microenvironment. The key to the illustrated intermolecular chain reaction is the soluble affibody-PL, which simultaneously binds to HER2-expressing tumor cells via the affibody and to immunoglobulins (Ig) via the PL. The Fc region of Ig subsequently binds and crosslinks to Fc receptors on the surface of NK cells and macrophages, resulting in the activation of these innate immune cells and tumor cell killing. This strategy is expected to enhance the overall antitumor effect of oncolytic viral therapy in two ways. First, it engages innate immune cells with tumor cells, thereby providing additional collateral antitumor activity. Second, it diverts two key components of antiviral immunity—neutralizing antibodies and innate immune cells, including NK cells and macrophages—from eliminating the introduced oncolytic virus, allowing viral therapy to exert its maximum oncolytic effect. Figure 1B depicts the predicted mechanism of action of affibody-PL after delivery by oncolytic viruses to HER2-expressing tumors, labeling each of the key components. The administered oncolytic viruses not only express affibody-PL in situ but also attract innate immune cells, such as NK cells and macrophages (Mφ), to the tumor site. The secreted affibody-PL engages infiltrating innate immune cells to tumor cells through a series of intermolecular binding events: affibody to HER2, PL to Ig, and Ig (via the Fc region) to NK cells or macrophages (via Fc receptors).

[0057] The composition of an embodiment of the chimeric virus is depicted in Figure 2A, which details the stepwise construction of FusOn-H2. The top image depicts the genome of wild-type HSV-2 (wtHSV-2, wild-type HSV-2 strain 186). The HSV genome is approximately 152 kb long and contains terminal repeats (TRs) and internal repeats (IRs), which are appropriately marked as well as the ICP10 and ICP47 genes. HSV-2 possesses several unique features, which have been previously exploited by the present inventors to convert it into an oncolytic agent. The HSV-2 ICP10 gene, the counterpart of ICP6 in HSV-1, contains a well-defined region (N domain) at its N-terminus that can bind and phosphorylate the GTPase-activating protein Ras-GAP, resulting in activation of the Ras / MEK / MAPK mitogenic pathway and induction and stabilization of c-Fos, both of which are essential for efficient HSV-2 replication. Deletion of this domain from the viral genome impairs viral growth in normal cells that normally have an inactive Ras signaling pathway. Because the Ras signaling pathway is a key regulator of normal cell growth, it is abnormally activated in most human tumors due to either mutations in the Ras gene itself or changes in upstream or downstream signaling components. We previously demonstrated that a mutant HSV-2 lacking the N domain of the ICP10 gene can selectively replicate in these tumor cells, while its replication is restricted in normal cells with an inactive Ras pathway. The mutant HSV-2 (FusOn-H2) was constructed by replacing the PK domain of the ICP10 gene with a DNA sequence encoding enhanced green fluorescent protein (EGFP).

[0058] The construction and use of FusOn-H2 is described in U.S. Patent No. 8,986,672, which is incorporated herein by reference in its entirety. In the construction of FusOn-H2, the N domain of the ICP10 gene was deleted and replaced with the EGFP gene under expression of a CMV (cytomegalovirus) immediate-early promoter, leaving the C-terminal ribonucleotide reductase (RR) domain intact, resulting in modified ICP10 (mICP10, SEQ ID NO: 8). Briefly, the HSV genomic region containing the ICP10 left flanking region (equivalent to nucleotides 85994-86999 of the HSV-2 genome) was amplified with primers 5'-TTGGTCTTCACCTACCGACA (SEQ ID NO:11), 3'-GACGCGATGAACGGAAAC (SEQ ID NO:12), while the RR domain and right flanking region (equivalent to nucleotides 88228-89347) was amplified with primers 5'-ACACGCCCTATCATCTGAGG (SEQ ID NO:13), 3'-AACATGATGAAGGGGCTTCC (SEQ ID NO:14). In other words, this is equivalent to a deletion of nucleotides 87000-87023, which is the ICP10 promoter region, and nucleotides 87024-88226, which are the first 1,204 nucleotides of the ICP10 domain (i.e., a deletion of amino acids 1-402 of the endogenous ICP10 polypeptide). The two PCR products were cloned into pNeb193 by EcoRI-NotI-XbaI ligation to generate pNeb-ICP10-deltaPK. A DNA sequence containing the CMV promoter-EGFP gene was then amplified by PCR from pSZ-EGFP with primers 5'-ATGGTGAGCAAGGGCGAG (SEQ ID NO: 15), 3'-CTTGTACAGCTCGTCCATGC (SEQ ID NO: 16). The PCR-amplified DNA was then cloned into the deleted PK locus of pNeb-ICP10-deltaPK by BglII and NotI ligation to generate pNeb-PKF-2.A PCR amplification strategy was designed to fuse the EGFP gene in frame with the remaining RR domain of the ICP10 gene, ensuring that the new protein product contained intact EGFP, facilitating the selection of recombinant viruses in subsequent experimental steps. The modified ICP10 gene was inserted into the virus by homologous recombination, in which pNeb-PKF-2 plasmid DNA was cotransfected with purified wt186 virion DNA into Vero cells using Lipofectamine (Invitrogen, Carlsbad, CA, USA). Recombinant viruses were screened and identified by selecting GFP-positive viral plaques. All GFP-positive plaques showed clear syncytia formation within infected cells, indicating that the modified virus was capable of inducing extensive cell membrane fusion. A total of six plaques were selected, including FusOn-H2, for further characterization.

[0059] Figure 2B depicts the stepwise construction of an embodiment of FusOn-Affibody-PL, which was constructed by first deleting the EGFP gene from FusOn-H2 and then inserting the Affibody-PL gene cassette (SEQ ID NO: 9) into the region next to the modified ICP10 gene on the HSV-2 genome.

[0060] Example 2 Affibody-PL engagement of innate immune cells We attempted to demonstrate that affibody-PL can actively engage innate immune cells to attack tumor cells when tested in vitro. First, we examined the ability of affibody-PL to selectively bind to tumor cells expressing HER2. 293 cells were transfected with a plasmid containing a gene cassette, pcDNA-affibody-PL constructed by inserting affibody-PL into the pcDNA3 plasmid, or a control plasmid (pcDNA3-EGFP from Addgene). After 24 hours (hr), the supernatant was harvested. 100 μl of the supernatant was added to three tumor cell lines (Skov3, MCF7, and MDA-MB-231, derived from serous cystadenocarcinoma) expressing different levels of HER2, allowing affibody-PL to initially bind to HER2 on the tumor cell surface. After washing, a FITC-conjugated anti-HA tag antibody was added. The stained cells were then analyzed by flow cytometry, and the results are shown in Figure 3. As shown, affibody-PL efficiently bound to Skov3 cells, which express high levels of HER2. More than 70% of the cells stained positive. In MCF7 cells, which express moderate levels of HER2, approximately half of the cells stained positive. In triple-negative MDA-MB-231 breast cancer cells, only 3% of the cells stained positive. These results demonstrate that the chimeric affibody construct allows the molecule to bind strongly to HER2-expressing tumor cells.

[0061] Next, we performed another in vitro experiment to examine whether affibody-PL could initiate the expected molecular interactions shown in Figure 1B and induce innate immune cells to kill HER2-expressing tumor cells. To more closely mimic the actual in vivo situation, peripheral blood mononuclear cells (PBMCs), containing NK cells and monocytes at 5-20% and 10-30%, respectively, were selected as the source of innate immune cells. SKOV3 cells were mixed with PBMCs in the presence of immunoglobulins (Sigma-Aldrich Ig) and affibody-PL-containing or control supernatants. After 20 hours, PBMCs were washed and stained with 0.1% crystal violet-ethanol solution, followed by direct visualization under a microscope to initially examine tumor cell viability (Figure 4A). The killing effect on tumor cells was further quantified by dissolving the cells in 2% SDS, and the released dye was measured using a Spectramax 5 plate reader at a wavelength of 595 nM (Figure 4B). The results show that the presence of affibody-PL resulted in significant tumor cell killing compared with mixtures without the chimeric molecule. This effect was particularly evident at a relatively low effector-to-target (E:T) ratio (10:1); wells with control supernatant showed very little tumor cell killing, while nearly 70% cell killing was detected in wells with affibody-PL. At an E:T ratio of 20:1, there was a notable increase in background killing in both wells with medium alone and control supernatant. However, tumor cell killing in wells with affibody-PL further increased to over 90 percent. Together, these results demonstrate the ability of affibody-PL to induce innate immune cells to kill tumor cells through a series of intermolecular engagements.

[0062] Example 3 Insertion of affibody-PL coding sequences into oncolytic HSV and in vitro characterization of the armed virus The affibody-PL coding sequence was then inserted into the genome of the HSV-2-based oncolytic virus FusOn-H2 by homologous recombination, as illustrated in Figures 2A and 2B, according to techniques previously described in certain of the inventors' publications. See Fu, X., et al. FusOn-H2 is a mutant herpes simplex virus type 2 in which the protein kinase domain of the ICP10 gene has been deleted, and is a potent oncolytic virus (Mol Ther. 13(5) (2006) 882-90; Fu X, et al. "Construction of an oncolytic herpes simplex virus that precisely targets hepatocellular carcinoma cells" Mol Ther. 20(2) (2012) 339-46); each of which is incorporated herein by reference. The full sequence of the resulting FusOn-affibody-PL is provided in SEQ ID NO: 17. Expression of affibody-PL from the new virus, FusOn-PL, was confirmed by Western blot analysis of supernatants collected from virus-infected cells, demonstrating that affibody-PL was abundantly expressed by FusOn-PL (Figure 5A). The properties of affibody-PL expressed from FusOn-PL were assessed by measuring its ability to bind to HER2-expressing tumor cells and induce innate immune cells to kill them, using the same methods as those described in Figures 3 and 4A-4B, respectively, but using cells of murine origin instead. The tumor cells used were the CT26 murine colon carcinoma cell line stably transduced with HER2. See Penichet ML, et al., "In vivo properties of three human HER2 / neu-expressing murine cell lines in immunocompetent mice," Lab Anim Sci. 49(2) (1999) 179-88. Splenocytes harvested from immunocompetent Balb / c mice were used as effector cells.Flow cytometry analysis of the binding of affibody-PL generated from FusOn-PL to HER2-expressing mouse tumor cells is shown in Figure 5B. The experiment was performed similarly to Figure 3, except that a mouse colon cancer cell line stably transduced with HER2 (CT26-HER2) was used. The results in Figure 5C, in which tumor cells were mixed with PBMCs, showed that, similar to those in Figure 4B, significant tumor cell killing was observed in wells to which supernatant containing affibody-PL was added, compared to the other two wells without this component. Collectively, these results demonstrate that affibody-PL can efficiently induce innate immune cells of mouse origin to kill mouse tumor cells when the tumor cells express the targeted tumor antigen.

[0063] Example 4 Treatment evaluation of FusOn-PL To evaluate the therapeutic effects of FusOn-PL and compare them with those of the parent FusOn-H2, we selected the murine CT26 colon tumor model, which is only marginally sensitive to the therapeutic effects of FusOn-H2. This allowed us to fully evaluate the therapeutic benefits of the incorporated affibody-PL. Specifically, a CT26 cell line stably transduced with the HER2 gene was used for this experiment. CT26-HER2 tumor cells were subcutaneously implanted into the right flank of syngeneic immunocompetent BALB / c mice. When tumors reached a size of approximately 5 mm in diameter, the mice were treated intratumorally with the same dose of either FusOn-PL or FusOn-H2. Three days after treatment, two mice from each group were euthanized, and tumor tissues were harvested to measure NK cell infiltration. The remaining animals were maintained for four weeks to evaluate the therapeutic effects by monitoring tumor size after treatment.

[0064] Immunohistochemical staining of harvested tumor tissues revealed that NK cells were rarely detected in PBS-treated control tumors, with NK cells detected at frequencies of approximately 1–3 per microscopic field within the tumor, a finding consistent with literature reports. As shown in Figure 6A, NK cells were readily detectable in tumors treated with both FusOn-H2 and FusOn-PL, consistent with reports that HSV-2 infection can induce significant NK cell infiltration at the infection site. Surprisingly, the presence of NK cells in tumor tissue treated with FusOn-PL was particularly prominent, significantly exceeding that observed with FusOn-H2 treatment.

[0065] To determine whether the increased presence of NK cells in FusOn-PL-treated tumors was due to enhanced local NK cell proliferation, tumor tissues were double-stained for both the NK cell marker (NCR1) and Ki67 protein. The results, presented in Figure 6B, showed that tumor cells stained strongly with Ki67 (particularly in PBS-treated tumor tissues), but there was no significant Ki67 staining for NK cells in tumor tissues treated with either FusOn-PL or FusOn-H2. This result therefore excludes the possibility of affibody-PL in stimulating NK cell proliferation and suggests that the increased presence of NK cells in FusOn-PL treatment is due to a positive feedback loop for their recruitment. Nevertheless, the increased presence of NK cells in FusOn-PL-treated tumor tissues allows secreted affibody-PL to efficiently act to direct them to tumor cell killing.

[0066] Figure 7A shows the results of an in vivo experiment designed to directly compare the therapeutic efficacy of FusOn-PL with that of the parent FusOn-H2 by treating HER2-positive tumor-bearing mice. The results show that administration of FusOn-H2 only slightly slowed tumor growth compared with the PBS control. In contrast, FusOn-PL treatment effectively prevented tumor growth for an extended period of time. Treated tumors were significantly smaller than those in the FusOn-H2-treated group. By the end of the experiment, 5 out of 10 mice in the FusOn-PL-treated group were tumor-free, whereas no tumor-free animals were detected in the other treatment groups. Collectively, these results demonstrate that incorporation of affibody-PL into oncolytic HSV can enhance the antitumor effects of viral therapy; this enhancement strategy may be particularly advantageous for patients whose tumors are relatively resistant to the direct oncolytic effects of the applied virus.

[0067] Subsequently, tumor-free mice in the FusOn-PL treatment group were challenged with fresh CT26-HER2 tumor cells implanted into the left flank. All three mice were completely protected from challenge for over 4 weeks, with no detectable tumor traces. Tumor challenge was not performed on mice in the other two treatment groups due to the large tumor burden, which required euthanasia of all mice. The challenged mice were maintained for an additional 4 weeks to monitor tumor growth. By the end of the experiment, no tumor formation was detected in any of the mice, indicating that FusOn-PL treatment had generated robust antitumor immunity and subsequently completely protected these mice from tumor challenge.

[0068] Tumor cells contain frequent point mutations that can lead to neoantigen formation. Inducing an immune response against these neoantigens is theoretically particularly attractive for cancer immunotherapy because they are strictly tumor-specific. The neoantigen profile of CT-26 cells was recently reported by Kreiter et al. (See Kreiter S, et al. "Mutant MHC class II epitopes drive therapeutic immune responses to cancer" Nature 520 (7549) (2015) 692-6). To determine whether FusOn-PL-mediated tumor cell killing could induce anti-neoantigen immunity, we first selected three mutant peptides (listed in Table 1) that predictedly contained MHC class I neoantigen epitopes and examined whether any cytotoxic T cells specific for these antigens could be detected in protected mice. Splenocytes harvested from the mice were stimulated with these peptides or a control peptide. The specificity of the T cell response was determined by ELISPOT assay. The results showed that splenocytes from one mouse responded to a single peptide in this assay (Fig. 7B), indicating that FusOn-PL virus treatment induced detectable induction of T cell responses against such class I neoantigenic epitopes.

[0069] [Table 1]

[0070] To further characterize neoantigen-specific immune responses to FusOn-PL and FusOn-H2 viral treatment, we essentially repeated the in vivo animal experiments shown in Figures 6A and 6B. The only difference was that, if all animals still survived, they were euthanized 21 days after viral treatment and their spleens were harvested for further analysis of anti-neoantigen immunity. Treatment data collected from this second in vivo experiment showed that, 21 days after the initiation of viral treatment, FusOn-PL was still significantly more effective than FusOn-H2 in preventing tumor growth, even with a relatively short treatment period (Figure 8A), and approximately 40% of the mice were tumor-free by the end of the experiment.

[0071] Studies by Kreiter et al. (supra) in three independent mouse tumor models, including CT-26, showed that the majority of the immunogenic mutanome was recognized by CD4+ T cells. To further characterize neoantigen-specific immunity, we selected a panel of MHC class II neoantigen peptides reported by Kreiter et al. (listed in Table 2) to measure antitumor immunity in these animals.

[0072] [Table 2]

[0073] Figure 8B shows the enumeration data from ELISPOT assays against these neoantigen peptides and their mixtures. Controls included no peptide or an irrelevant peptide (ovalbumin class II peptide (OVA323-339)). The results show that a significant increase in ELISPOT staining was detected for all four class II neoantigen peptides in animals treated with FusOn-PL, although the magnitude of the response varied among individual neoantigens (Figure 8B). Collectively, these data demonstrate that tumor disruption by FusOn-PL can induce neoantigen-specific antitumor immunity against both class I and class II MHC epitopes, whereas the parent FusOn-H2 was less efficient in inducing such immunity.

[0074] Example 5 HSV-1 constructs expressing PL We also achieved PL expression constructs of HSV-1 oncolytic viruses, engineered so that the tumor cell binding site was the epidermal growth factor receptor (EGFR). Alternative embodiments have affibodies against tumor cell antigens, such as HER2, which may be included in place of EGFR. Figure 9A shows a schematic diagram of Synco-4 construction starting from Synco-2D. As detailed in previous work by the present inventors and incorporated herein by reference, Synco-2D is an HSV-1-based oncolytic virus constructed by a deliberate process that can be summarized as follows: 1) deletion of both copies of the ICP34.5 gene (denoted as Δ34.5), 2) insertion of the high membrane fusion glycoprotein from gibbon ape leukemia virus (GALV.fus) into the Us3 gene (resulting in the inactivation of this gene), and 3) insertion of a bacterial artificial chromosome (BAC) into the UL46 and UL47 intergenic region. This virus is able to induce potent cell membrane fusion due to its intrinsic membrane fusion phenotype and the insertion of GALV.fus.

[0075] More specifically, Synco-2D was constructed by a process starting from fHSV-Δ-pac, a BAC-based construct containing a mutant HSV genome in which the diploid gene encoding γ34.5 and both copies of the HSV packaging signal were deleted. See Saeki, Y., et al. "Herpes simplex virus type 1 DNA amplified as bacterial artificial chromosome in Escherichia coli: rescue of replication-competent virus progeny and packaging of amplicon vectors." Hum. Gene Ther. 9 (1998) 2787-2794, 1998. Infectious HSV could not be generated from this construct unless an intact HSV packaging signal was provided in cis; otherwise, the virus would be conditional for replication due to the deletion of both copies of γ34.5. Therefore, construct "Baco-1," as previously described, was constructed by inserting a DNA sequence containing an HSV packaging signal and an enhanced GFP gene cassette into the unique PacI restriction site located in the BAC sequence of fHSV-Δ-pac. See Fu et al. "Expression of a Fusogenic Membrane Glycoprotein by an Oncolytic Herpes Simplex Virus Potentiates the Viral Antitumor Effect," Molec Ther 7 (6) (2003) 748-754. To generate Synco-2D, Baco-1 was first subjected to random mutagenesis. The syncytial phenotype was identified by screening the mutagenized virus on Vero cells. Circular viral DNA was then obtained from the new virus (Baco-F1) by extracting virion DNA from Vero cells immediately (1 hour) after virus infection.The viral DNA was then transformed into competent E. coli DH-10B cells by electroporation, and Baco-F1 DNA was purified from bacterial growth using a Qiagen kit. To insert the fusion-enhancing glycoprotein gene of gibbon ape leukemia virus (GALV.fus) into Baco-F1, the gene cassette encoding GFP in Baco-F1 was replaced with GALV.fus (driven by the HSV conditional UL38 promoter) using a forced ligation strategy. The ligation mixture was directly transfected into Vero cells using Lipofectamine (Life Technologies, Inc.) and incubated for 3–5 days to generate infectious virus. The resulting virus was subsequently plaque-purified. See Nakamori, M., et al., "Effective Therapy of metastatic ovarian cancer with an oncolytic herpes simplex virus incorporating two membrane-fusion mechanisms," Clinical Cancer Res. 9(7) (2003) 2727-2733, the entire contents of which are incorporated herein by reference. Subsequent characterization of Synco-2D led to the surprising finding that, despite its design, the GALV.fus gene cassette had inserted into the Us3 gene by unanticipated recombination.

[0076] To construct Synco-4, a gene cassette was first inserted into Synco-2D, replacing the BAC sequence contained in the viral genome by homologous recombination. This allowed the new virus, Synco-47G, to be easily identified under a microscope by its green color (shown in Figure 9B). A gene cassette containing an EGF-PL fusion gene (EGF ligand, SEQ ID NO: 10) linked to protein L (protein L construct SEQ ID NO: 2) for in-frame expression, driven by the RSV-LTR, and flanked by two LoxP sites (SEQ ID NO: 26 and SEQ ID NO: 27), was inserted into Synco-47G to replace the GFP gene, allowing Synco-4 to be selected as a "white" virus against a "green" background.

[0077] As shown in Figure 10, abundant expression and secretion of EGF-PL by Synco-4 was observed in 293 cells transfected with a plasmid (pCR-ul47-EGFPL) containing the EGF-PL gene cassette (shown in Figure 9A) or infected with Synco-4. Untransfected or uninfected 293 cells were used as a negative control. Forty-eight hours after transfection / infection, supernatants were collected, and secreted EGF-PL was detected by Western blot analysis. The results showed abundant EGF-PL in the supernatants of Synco-4-infected 293 cells, indicating that the transgene was efficiently expressed by the virus.

[0078] As shown in Figures 11A and 11B, EGF-PL produced by Synco-4 binds with high efficiency and specificity to EGFR expressed on tumor cells. The experiment in Figure 11A was performed by incubating the same supernatant collected from pCR-ul47-EGFPL-transfected cells as mentioned in

[0077] with either CT26 or CT26-EGFR, followed by reaction with PE-conjugated Ig. The results show flow cytometry staining for EGFR expression on CT-26-EGFR cells, but not on other tumor cells. The experiment in Figure 11B was performed by incubating the same supernatant collected from Synco-4-infected cells as mentioned in

[0077] with either CT26 or CT26-EGFR, followed by reaction with PE-conjugated Ig. The results showed flow cytometry demonstrating that EGF-PL in the supernatant of Synco-4 infection was able to bind strongly to CT26-EGFR cells, whereas the supernatant of parental FusOn-H2 did not show any binding.

[0079] As shown in Figures 12A–G, Synco-4 can induce dose-dependent inhibition of tumor cell proliferation in Syrian hamster adenocarcinoma cells (HaP-T1) (Figure 12A), mouse breast cancer cells (4T-1) (Figure 12B), mouse colon cancer cells (CT-26) (Figure 12C), human glioblastoma cells (U87) (Figure 12D), human pharyngeal squamous cell carcinoma cells (FaDu) (Figure 12E), human hepatocellular carcinoma cells (HEPG2) (Figure 12F), and human osteosarcoma cells (MNNG-HOS1) (Figure 12G). HaP-T1 cells were obtained from the European Collection of Authenticated Cell Cultures (ECACC). Other cell lines, including 4T-1, CT-26, U87, FaDu, HEPG2, and MNNG-HOS1, were purchased from ATCC. In the experiments, 5,000 cells / well were seeded into 96-well plates and cultured overnight. The culture medium was then removed and treated with 100 μl of serum-free medium containing various concentrations of Synco-4 at multiplicities of infection (MOI) ranging from 0.003 to 3. After 2 h of infection, the medium was removed and replaced with 100 μl of culture medium, followed by continued incubation for an additional 48 h. Cells were detected using an MTT assay kit (BioFloxx, Germany) according to the manufacturer's instructions. Briefly, 5 μl of MTT solution (5 mg / ml) was added and incubated in the dark for 4 h. The medium was then removed and 150 μl of DMSO was added. The mixture was then incubated at RT for 5 min with shaking, and the optical density (OD) was determined at a wavelength of 490 nm. Cell viability was calculated by normalizing to vehicle treatment and presented as percent inhibition. All experiments were performed in triplicate; n = 3–6.

[0080] As shown in Figure 13, Synco-4 exhibits enhanced antitumor activity against CT26-EGFR tumors compared to the parental Synco-2D. CT26-EGFR tumor cells were implanted subcutaneously into the right flank of Balb / c mice. When tumors reached a size of approximately 5 mm in diameter, mice were divided into three groups: 1) an equal volume (100 μl) of PBS; 2) 2×10 7 pfu Synco-2D, and 3) 2 × 10 7pfu of Synco-4. Tumor sizes were measured periodically and plotted. The results show that Synco-4 had enhanced antitumor activity against CT26-EGFR tumors compared with Synco-2D.

[0081] All publications, patents, and patent applications cited herein are hereby incorporated by reference as if set forth in their entirety herein. While the present invention has been described with reference to exemplary embodiments, this description is not intended to be construed in a limiting sense. Various modifications and combinations of the exemplary embodiments, as well as other embodiments of the present invention, will be apparent to those skilled in the art upon reference to this description. It is therefore intended that the appended claims encompass all such variations and enhancements. In certain embodiments, for example, the following items are provided: (Item 1) An improved oncolytic virus comprising an oncolytic virus backbone that encodes a chimeric molecule comprising a tumor cell binding component and an immunoglobulin (Ig) aggregation component and that has been genetically modified to induce secretion of the chimeric molecule from infected cells, wherein the secreted chimeric molecule, in the presence of anti-viral antibodies or other Ig and innate immune cells, increases collateral tumor cell killing and anti-tumor immunity. (Item 2) 2. The improved oncolytic virus of item 1, wherein the immunoglobulin aggregation component comprises at least one Ig-binding kappa light chain binding domain derived from peptostreptococcal protein L. (Item 3) 3. The improved oncolytic virus of item 2, wherein the immunoglobulin-binding component comprises at least four or five Ig-binding "B" domains derived from peptostreptococcal protein L. (Item 4) 2. The improved oncolytic virus of item 1, wherein the oncolytic virus backbone is based on herpes simplex virus type 1 (HSV1). (Item 5) 2. The improved oncolytic virus of item 1, wherein the oncolytic virus backbone is based on herpes simplex virus type 2 (HSV2). (Item 6) 2. The improved oncolytic virus of item 1, wherein the tumor cell-binding component is either an affibody, or a ligand, or a short peptide, or a single-chain antibody, or a single-domain antibody that binds to a tumor antigen. (Item 7) 7. The improved oncolytic virus according to item 6, wherein the tumor antigen is selected from human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), Erb-B2 receptor tyrosine kinase 3 (ErbB3), epithelial cell adhesion molecule (EpCAM), mesothelin (MSLN), MET proto-oncogene, insulin-like growth factor 1 receptor (IGF1R), ephrin receptor A3 (EphA3), TNF receptor apoptosis-inducing ligand receptor 1 (TRAIL-R1), TNF receptor apoptosis-inducing ligand receptor 2 (TRAIL-R2), vascular endothelial growth factor receptor (VEGFR), receptor activator of nuclear factor kappa beta ligand (RANKL), programmed death-ligand 1 (PD-L1), and hepatic regeneration phosphatase 3 (PRL-3). (Item 8) 2. The improved oncolytic virus of item 1, wherein the tumor cell-binding component is the extracellular domain of epidermal growth factor (EGF) that binds to EGF receptor (EGFR) on tumor cells. (Item 9) 5. The improved oncolytic virus of item 4, wherein the HSV1 backbone comprises at least one deletion of ICP34.5. (Item 10) 6. The improved oncolytic virus of item 5, wherein the HSV2 backbone comprises an N-domain deletion of ICP10 that allows selective replication in tumor cells. (Item 11) An improved oncolytic virus that increases collateral cell killing and induces anti-tumor immunity, the virus comprising an oncolytic herpesvirus backbone genetically modified to encode an affibody-Protein L (PL) cassette comprising an anti-HER2 affibody and multiple Protein L immunoglobulin binding domains fused in-frame to form an affibody-Protein L (PL) engineered for extracellular secretion by cells infected with the oncolytic virus. (Item 12) 13. The improved oncolytic virus of item 12, wherein the affibody-PL cassette comprises a synthetic signal peptide (Sp), the anti-HER2 affibody (affibody), a linker, the plurality of Protein L immunoglobulin binding domains, and a growth hormone polyadenylation signal (polyA). (Item 13) An improved oncolytic virus that increases collateral cell killing and induces anti-tumor immunity, the virus comprising an oncolytic herpesvirus backbone genetically modified to encode an extracellular domain of epidermal growth factor (EGF) that binds to the EGF receptor (EGFR) on tumor cells, fused in frame to form EGF-PL, which is engineered for extracellular secretion by cells infected with the oncolytic virus, and multiple protein L domains. (Item 15) 13. A method for treating cancer, comprising administering a therapeutically effective amount of the improved oncolytic virus of item 12 and a diluent or carrier. (Item 16) 16. The method of claim 15, wherein the improved oncolytic virus comprises SEQ ID NO: 17. (Item 15) 15. A method for treating cancer, comprising administering a therapeutically effective amount of the improved oncolytic virus of item 14 and a diluent or carrier. (Item 16) 16. The method of claim 15, wherein the improved oncolytic virus comprises SEQ ID NO: 25. (Item 17) A fusion protein comprising a tumor cell binding component and an immunoglobulin (Ig) aggregation component, wherein the immunoglobulin aggregation component comprises at least one Ig-binding kappa light chain binding domain derived from peptostreptococcal protein L. (Item 18) 18. The fusion protein of item 17, wherein the at least one Ig-binding kappa light chain binding domain derived from peptostreptococcal protein L has at least 80% amino acid identity with SEQ ID NO: 29. (Item 19) 16. The method of claim 15, wherein the improved oncolytic virus is administered to one or more of pancreatic cancer cells, breast cancer cells, colon cancer cells, glioma cancer cells, head and neck cancer cells, liver cancer cells, and bone cancer cells.

Claims

1. 1. An improved oncolytic virus comprising an oncolytic virus backbone that encodes a chimeric molecule comprising a tumor cell-binding component that is an affibody, a ligand, a single-chain antibody, or a single-domain antibody that binds to a tumor antigen, and an immunoglobulin (Ig) aggregation component that comprises five Ig-binding "B" domains derived from Peptostreptococcal Protein L, the chimeric molecule comprising, in that order, a first kappa light chain binding domain set forth in SEQ ID NO:29, a second kappa light chain binding domain set forth in SEQ ID NO:30, a third kappa light chain binding domain set forth in SEQ ID NO:31, a fourth kappa light chain binding domain set forth in SEQ ID NO:32, and a fifth kappa light chain binding domain set forth in SEQ ID NO:33, wherein the chimeric molecule is genetically modified to induce secretion of the chimeric molecule from infected cells.

2. 1. An improved oncolytic virus comprising an oncolytic virus backbone that encodes a chimeric molecule comprising a tumor cell-binding component and an immunoglobulin (Ig) aggregation component comprising five Ig-binding kappa light chain binding domains derived from Peptostreptococcal Protein L, the Ig-binding kappa light chain binding domains comprising, in that order, a first kappa light chain binding domain set forth in SEQ ID NO:29, a second kappa light chain binding domain set forth in SEQ ID NO:30, a third kappa light chain binding domain set forth in SEQ ID NO:31, a fourth kappa light chain binding domain set forth in SEQ ID NO:32, and a fifth kappa light chain binding domain set forth in SEQ ID NO:33, wherein the chimeric molecule is genetically modified to induce secretion of the chimeric molecule from infected cells, wherein the tumor cell-binding component is an epidermal growth factor (EGF) extracellular domain that binds to EGF receptor (EGFR) on tumor cells.

3. An improved oncolytic virus as described in claim 1 or 2, wherein the immunoglobulin (Ig) aggregation component comprises the sequence shown in SEQ ID NO:

34.

4. 3. The improved oncolytic virus of claim 1 or claim 2, wherein the oncolytic virus backbone is based on herpes simplex virus type 1 (HSV1).

5. 3. The improved oncolytic virus of claim 1 or claim 2, wherein the oncolytic virus backbone is based on herpes simplex virus type 2 (HSV2).

6. 2. The improved oncolytic virus of claim 1, wherein the tumor antigen is selected from human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), Erb-B2 receptor tyrosine kinase 3 (ErbB3), epithelial cell adhesion molecule (EpCAM), mesothelin (MSLN), MET proto-oncogene, insulin-like growth factor 1 receptor (IGF1R), ephrin receptor A3 (EphA3), TNF receptor apoptosis-inducing ligand receptor 1 (TRAIL-R1), TNF receptor apoptosis-inducing ligand receptor 2 (TRAIL-R2), vascular endothelial growth factor receptor (VEGFR), receptor activator of nuclear factor kappa beta ligand (RANKL), programmed death-ligand 1 (PD-L1), and hepatic regeneration phosphatase 3 (PRL-3).

7. 2. The improved oncolytic virus of claim 1, wherein the tumor cell binding component is the extracellular domain of epidermal growth factor (EGF) that binds to EGF receptor (EGFR) on tumor cells.

8. 5. The improved oncolytic virus of claim 4, wherein the HSV1 backbone comprises at least one deletion of ICP34.

5.

9. 6. The improved oncolytic virus of claim 5, wherein the HSV2 backbone comprises an N-domain deletion of ICP10 that allows selective replication in tumor cells.

10. 1. An improved oncolytic virus, the virus comprising an oncolytic herpesvirus backbone genetically modified to encode an affibody-Protein L (PL) cassette comprising an anti-HER2 affibody fused in-frame to form an affibody-PL fusion protein engineered for extracellular secretion by cells infected with the oncolytic virus, and a plurality of Protein L immunoglobulin binding domains, the plurality of Protein L immunoglobulin binding domains comprising five Ig binding "B" domains derived from Peptostreptococcal Protein L, the plurality of Protein L immunoglobulin binding domains comprising, in order: a first kappa light chain binding domain set forth in SEQ ID NO:29, a second kappa light chain binding domain set forth in SEQ ID NO:30, a third kappa light chain binding domain set forth in SEQ ID NO:31, a fourth kappa light chain binding domain set forth in SEQ ID NO:32, and a fifth kappa light chain binding domain set forth in SEQ ID NO:

33.

11. 11. The improved oncolytic virus of claim 10, wherein the affibody-PL cassette comprises a synthetic signal peptide (Sp), the anti-HER2 affibody (affibody), a linker, the plurality of Protein L immunoglobulin binding domains, and a growth hormone polyadenylation signal (polyA).

12. 1. An improved oncolytic virus comprising an oncolytic herpesvirus backbone genetically modified to encode an extracellular domain of epidermal growth factor (EGF) that binds to an EGF receptor (EGFR) on tumor cells, fused in frame to form EGF-PL engineered for extracellular secretion by cells infected with the oncolytic virus, and a plurality of Protein L immunoglobulin binding domains comprising, in that order, a first kappa light chain binding domain set forth in SEQ ID NO:29, a second kappa light chain binding domain set forth in SEQ ID NO:30, a third kappa light chain binding domain set forth in SEQ ID NO:31, a fourth kappa light chain binding domain set forth in SEQ ID NO:32, and a fifth kappa light chain binding domain set forth in SEQ ID NO:

33.

13. 11. A composition comprising the improved oncolytic virus of claim 10 and a diluent or carrier for treating cancer.

14. 14. The composition of claim 13, wherein the improved oncolytic virus comprises SEQ ID NO:

17.

15. 13. A composition comprising the improved oncolytic virus of claim 12 and a diluent or carrier for treating cancer.

16. 16. The composition of claim 15, wherein the improved oncolytic virus comprises SEQ ID NO:

25.

17. 1. A chimeric molecule comprising a tumor cell binding component that is an affibody, a ligand, a single-chain antibody, or a single-domain antibody that binds to a tumor antigen, and an immunoglobulin (Ig) aggregation component, wherein the immunoglobulin aggregation component comprises five Ig-binding "B" domains derived from Peptostreptococcal Protein L, the Ig-binding "B" domain comprising, in that order, a first kappa light chain binding domain set forth in SEQ ID NO:29, a second kappa light chain binding domain set forth in SEQ ID NO:30, a third kappa light chain binding domain set forth in SEQ ID NO:31, a fourth kappa light chain binding domain set forth in SEQ ID NO:32, and a fifth kappa light chain binding domain set forth in SEQ ID NO:

33.

18. The chimeric molecule of claim 17, wherein the tumor antigen is selected from human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), Erb-B2 receptor tyrosine kinase 3 (ErbB3), epithelial cell adhesion molecule (EpCAM), mesothelin (MSLN), MET proto-oncogene, insulin-like growth factor 1 receptor (IGF1R), ephrin receptor A3 (EphA3), TNF receptor apoptosis-inducing ligand receptor 1 (TRAIL-R1), TNF receptor apoptosis-inducing ligand receptor 2 (TRAIL-R2), vascular endothelial growth factor receptor (VEGFR), receptor activator of nuclear factor kappa beta ligand (RANKL), programmed death ligand 1 (PD-L1), and hepatic regeneration phosphatase 3 (PRL-3).

19. The chimeric molecule of claim 17, wherein the tumor antigen is selected from human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor (EGFR).

20. 19. The chimeric molecule of claim 17 or claim 18, wherein the five Ig-binding "B" domains derived from peptostreptococcal protein L comprise the sequence shown in SEQ ID NO:

34.

21. 16. The composition of claim 15, wherein the composition is administered to one or more of pancreatic cancer cells, breast cancer cells, colon cancer cells, glioma cancer cells, head and neck cancer cells, liver cancer cells, and bone cancer cells.

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