Modified oncolytic virus

Abstract

To provide an oncolytic virus for use in improved treatment of cancer through improved direct oncolytic effects, viral replication and spread through tumors.SOLUTION: The present invention provides an oncolytic virus comprising: (i) a fusogenic protein-encoding gene; and (ii) an immune stimulatory molecule-encoding gene and also provides a pharmaceutical composition comprising a virus and a pharmaceutically acceptable carrier or diluent.SELECTED DRAWING: None

Description

[Technical Field] 【0001】 This invention relates to oncolytic immunotherapy agents and the use of oncolytic immunotherapy agents in the treatment of cancer. [Background technology] 【0002】 Viruses possess an inherent ability to invade cells with high efficiency. After entering a cell, viral genes are expressed and the virus replicates. This typically results in the death of the infected cell and, upon death, the release of the cell's antigenic components as the cell ruptures. As a result, virus-mediated cell death tends to trigger an immune response against these cellular components, including both those derived from the host cell and those encoded or incorporated by the virus itself. The immune response is also amplified by the host's recognition of so-called damage-associated molecular patterns (DAMPs), which assist in the activation of the immune response. 【0003】 Viruses also engage with various mediators of the innate immune response as part of the host response to recognition of viral infection through Toll-like receptors, cGAS / STING signaling, and recognition of pathogen-associated molecular patterns (PAMPs), which result in activation and inflammation of the interferon response, which is also an immunogenic signal to the host. These immune responses can provide immunogenic benefits to cancer patients, such as an immune response to tumor antigens providing a systemic overall benefit that leads to the treatment of non-virus-infected tumors, including micrometastatic disease, and providing vaccination against relapse. 【0004】 The direct ("oncolytic") effect of a combination of viruses, and the immune response to tumor antigens (including non-self "neoantigens," i.e., derived from specific mutant genes in individual tumors), is called "oncolytic immunotherapy." 【0005】 Viruses can also be used as delivery vehicles ("vectors") to express heterologous genes inserted into the viral genome in infected cells. These properties make viruses useful in a variety of biotechnological and medical applications. For example, viruses that express heterologous therapeutic genes can be used in gene therapy. In relation to oncolytic immunotherapy, the delivered genes may include genes encoding specific tumor antigens, genes intended to induce an immune response after viral replication and cell death or to increase the immunogenicity of released antigens, genes intended to form the generated immune response, genes to increase the general immune activation state of the tumor, or genes to increase the direct oncolytic properties (i.e., cytotoxic effects) of the virus. Importantly, viruses have the ability to deliver encoded molecules intended to help directly and selectively initiate, amplify, or form a systemic antitumor immune response in a tumor. This could have benefits such as reduced toxicity, or, compared to systemic administration of these same molecules targeting the same pathway or other molecules, the ability to concentrate beneficial effects on tumors (including those not infected with the virus) rather than off-target effects in normal (i.e., non-cancerous) tissues. 【0006】 For example, numerous viruses, such as herpes simplex virus (HSV), have been demonstrated to be useful in cancer oncolytic treatment. HSV for use in cancer oncolytic treatment must be inactivated so that it can invade and kill tumor cells, even though it is no longer pathogenic. Numerous inactivation mutations for HSV have been identified, including disruption of genes encoding ICP34.5, ICP6, and / or thymidine kinase. These do not prevent the virus from replicating in tumor tissue in culture or in vivo, but they do prevent significant replication in normal tissue. HSV with only the ICP34.5 gene disrupted replicates in many tumor cell types in vitro and, in mouse tumor models, selectively replicates in tumor tissue but not in surrounding tissue. Clinical trials of deleted ICP34.5, or deleted ICP34.5 and ICP6, HSV have also demonstrated safety and selective replication in human tumor tissue. 【0007】 As discussed above, oncolytic viruses such as HSV can also be used to deliver therapeutic genes in cancer treatment. A virus of this type, further deleting ICP47 and specifically ICP34.5 which encodes a heterologous gene for GM-CSF, has also been tested in clinical trials, including a Phase 3 trial in melanoma, demonstrating safety and efficacy in humans. Trial data showed that tumor responses were observed in injected tumors and to a lesser extent in uninjected tumors. Responses tended to be highly durable (several months to several years), and survival benefits appeared to be achieved in responding patients. Each of these demonstrated the involvement of the immune system in cancer treatment, in addition to direct oncolytic effects. However, this and other data regarding oncolytic viruses generally indicate that not all tumors respond to treatment, and not all patients achieve survival benefits. Consequently, improvements to this technique of oncolytic therapy are clearly needed. These could help increase the direct oncolytic effect of the therapy, the anti-tumor immunostimulatory effect of the treatment, or both of these effects together. 【0008】 In recent years, oncolytic immunotherapy has been shown to produce additive or synergistic therapeutic effects when combined with immune checkpoint blockade (i.e., inhibition or "antagonism" of immune checkpoint pathways), also known as immune co-inhibitory pathway blockade. Checkpoint (immunoinhibitory pathway) blockade is typically intended to block host immunosuppressive mechanisms that help prevent the development of autoimmunity. However, in cancer patients, these mechanisms may also help inhibit or block the induction of potentially beneficial effects of any immune response induced by the tumor. Alternatively, the immune response may not be fully enhanced due to a lack of activation or a lack of complete activation of the immune enhancement pathway. Therefore, drugs that mitigate these blockades (inhibit "immune co-inhibitory pathways") or stimulate immune enhancement pathways (i.e., activate or "agonize" "immune co-stimulatory pathways") are attractive for the testing and development of cancer treatments. Such approved or experimental drug targets include CTLA-4, PD-1, PD-L1, LAG-3, TIM-3, VISTA, CSF1R, IDO, CEACAM1, GITR, 4-1-BB, KIR, SLAMF7, OX40, CD40, ICOS, or CD47. 【0009】 Many of these approaches targeting or mitigating immune co-inhibitory pathways require a pre-existing immune response to the tumor for success; that is, the pre-existing immune response may be enhanced, or the blockade of the anti-tumor immune response may be mitigated. The presence of an inflammatory tumor microenvironment exhibiting such an ongoing response is also necessary. The pre-existing immune response to tumor neoantigens appears to be particularly important for the activity of immune co-inhibitory pathway blockade and associated drugs. Only some patients may have an ongoing immune response to tumor antigens, including neoantigens and / or an inflammatory tumor microenvironment, both of which are necessary for the optimal activity of these drugs. Therefore, oncolytics that can induce an immune response to tumor antigens, including neoantigens, and / or induce an inflammatory tumor microenvironment are attractive to use in combination with immune co-inhibitory pathway blockades and immunoenhancing agents. This is thought to explain the promising combined antitumor effects of oncolytics and immune co-inhibitory pathway blockade observed in mice and humans to date. 【0010】 The indoleamine 2,3-dioxygenase (IDO) pathway contributes to tumor-induced tolerance by creating a tolerogenic environment in tumors and tumor-discharging lymph nodes, through both direct suppression of T cells and enhancement of local regulatory T cell (Treg)-mediated immunosuppression. IDO catalyzes the rate-limiting step in tryptophan degradation along the kynurenine pathway, and both the decrease in local tryptophan concentration and the production of immunomodulatory tryptophan metabolites contribute to the immunosuppressive effect of IDO. IDO is chronically activated in many cancer patients with IDO activation, which correlates with a wider range of diseases. It can also function as an antagonist to other activators of antitumor immunity. Therefore, inhibitors of the IDO pathway are being developed as anticancer agents, particularly in combination with checkpoint blockers, such as those targeting CTLA-4, PD-1, or PDL-1. 【0011】 The above discussion demonstrates that there is still much room for improvement in oncolytic agents and cancer treatments that use them. [Overview of the Initiative] 【0012】 The present invention provides an oncolytic virus expressing a fusion-inducible protein and at least one immunostimulatory molecule. The oncolytic virus of the present invention provides an improved direct oncolytic effect, improved viral replication and transmission in tumors, mediated by the fusion-inducible protein, which (i) increases the amount of tumor antigens, including neoantigens, released for induction of an antitumor immune response, and (ii) enhances the expression of the immunostimulatory molecule encoded by the virus. The expression of the immunostimulatory molecule further increases and enhances the antitumor immune effect. The antitumor efficacy is improved when the oncolytic virus of the present invention is used as a single agent, and when the virus is used in combination with other anticancer modalities, such as chemotherapy, targeted drug treatment, radiation, immune checkpoint blockade and / or immunoenhancing agents. 【0013】 Accordingly, the present invention provides an oncolytic virus comprising (i) a gene encoding a fusion-inducible protein and (ii) a gene encoding an immunostimulatory molecule. The virus may encode one or more fusion-inducible proteins and / or one or more immunostimulatory molecules. 【0014】 The fusion-inducible protein is preferably a glycoprotein derived from gibbon ape leukemia virus (GALV) with a mutated or removed R transmembrane peptide (GALV-R-). The immunostimulatory molecule is preferably an agonist of an immunocostimulatory pathway such as GM-CSF and / or GITRL, 4-1-BBL, OX40L, ICOSL, or CD40L, or a modified version thereof. Examples of modified versions include agonists of the costimulatory pathway that are secreted rather than membrane-bound, and / or agonists modified to form protein polymers. The immunostimulatory molecule may be a protein capable of blocking CTLA-4-mediated signaling, such as an antibody or fragment thereof that binds to CTLA-4. 【0015】 The virus can be a modified clinical isolate, such as a modified clinical isolate of a virus, and the clinical isolate causes two or more tumor cell lines to die more rapidly and / or at lower doses in vitro than one or more reference clinical isolates of the same virus. 【0016】 The virus is preferably a herpes simplex virus (HSV), such as HSV1. HSV typically does not express functional ICP34.5 and / or functional ICP47, and / or expresses the US11 gene as a pre-early gene. 【0017】 The present invention also provides the following: - A pharmaceutical composition comprising the virus of the present invention and a pharmaceutically acceptable carrier or diluent, - The virus of the present invention for use in a method of treating the human or animal body by therapy, - The virus of the present invention for use in a method of treating cancer, the method optionally including administering a further anti-cancer agent, - A product comprising the virus of the present invention in a sterile vial, ampoule or syringe, - A method of treating cancer comprising administering a therapeutically effective amount of the virus or pharmaceutical composition of the present invention to a patient in need thereof, the method optionally including administering a further anti-cancer agent, wherein the anti-cancer agent is optionally an antagonist of an immune co-inhibitory pathway or an agonist of an immune co-stimulatory pathway, - Use of the virus of the present invention in the manufacture of a medicament for use in a method of treating cancer, the method optionally including administering a further anti-cancer agent, wherein the anti-cancer agent is optionally an antagonist of an immune co-inhibitory pathway or an agonist of an immune co-stimulatory pathway, - A method of treating cancer comprising administering a therapeutically effective amount of a oncolytic virus, an inhibitor of the indoleamine 2,3-dioxygenase (IDO) pathway and a further antagonist of an immune co-inhibitory pathway or an agonist of an immune co-stimulatory pathway to a patient in need thereof. BRIEF DESCRIPTION OF THE DRAWINGS 【0018】 [Figure 1] Figure 1 shows the structure of an exemplary virus of the present invention, including a gene encoding GALV-R- and a gene encoding GM-CSF inserted at the ICP34.5 locus, with the ICP47 gene deleted so that the US11 gene is under the regulation of the ICP47 pre-initial promoter. Figure 1 also shows similar viruses expressing only the gene encoding GALV-R (second panel) or only the gene encoding GM-CSF (third panel). Furthermore, viruses are shown in which the ICP34.5 and ICP47 genes are deleted but the inserted genes are not present. [Figure 2] This figure shows the structure of an exemplary virus of the present invention, which includes a gene encoding GALV-R-, a gene encoding GM-CSF, and a gene encoding CD40L. [Figure 3]Figure 3 shows the different capabilities of eight top-ranked HSV1 clinical isolates in killing Fadu, SK-mel-28, A549, HT1080, MIA-PA-CA-2, HT29, and MDA-MB-231 human tumor cell lines, as evaluated by crystal violet staining 24 or 48 hours after infection, with MOIs of 0.1, 0.01, or 0.001 as shown in the figure. The first and second ranked viral strains for each cell line are shown. Virus RH018A was ranked first for each of the Fadu, HT1080, MIA-PA-CA-2, and HT29 cell lines, and second for each of the SK-mel-28, A549, and MDA-MB-231 cell lines. RH004A was ranked 1st for both RH018A and RH015A for the HT29 cell line, 1st for both SK-mel-28 and A549 cell lines, and 2nd for the Fadu cell line. RH023A was ranked 1st for the MDA-MB-231 cell line and 2nd for the HT1080 cell line. RH031A was ranked 2nd for both the MIA-PA-CA-2 and HT29 cell lines. RH040A was ranked 2nd for both HT29 cell lines. [Figure 4] This figure compares the RH018A strain, the highest-ranked strain among all strains tested, with the "average" strain from screening (i.e., the RH065A strain). As shown by crystal violet staining 24 or 48 hours after infection with MOIs of 0.1, 0.01, and 0.001 in the SK-mel-28, HT1080, MDA-MB-231, Fadu, MIA-PA-CA-2, and A549 cell lines, the RH018A strain required approximately 10 times less cells to kill the same percentage of cells than the RH065A strain. [Figure 5-1]This figure shows the structure of an HSV1 virus modified by deletions of ICP34.5 and ICP47, in which the US11 gene is under the regulation of the ICP457 pre-initial promoter and a heterologous gene is included at the ICP34.5 locus. Unless otherwise noted in the figure, the virus was constructed using the RH018A strain. [Figure 5-2] This is a continuation of Figure 5-1. [Figure 5-3] This is a continuation of Figure 5-1. [Figure 5-4] This is a continuation of Figure 5-1. [Figure 5-5] This is a continuation of Figure 5-1. [Figure 5-6] This is a continuation of Figure 5-1. [Figure 5-7] This is a continuation of Figure 5-1. [Figure 5-8] This is a continuation of Figure 5-1. [Figure 5-9] This is a continuation of Figure 5-1. [Figure 5-10] This is a continuation of Figure 5-1. [Figure 5-11] This is a continuation of Figure 5-1. [Figure 6] This figure shows the results of an ELISA to detect the expression of human or mouse GM-CSF in the supernatant from BHK cells infected with virus 16 (mGM-CSF and GALVR-), virus 17 (hGM-CSF and GALVR-), and virus 19 (mGM-CSF). [Figure 7-1] This figure shows a comparison of the cell-killing abilities of the RH018A strain (virus 10), which lacks ICP34.5 and expresses GALVR- and GFP, and the virus expressing only GFP (virus 12), as determined by crystal violet staining in three cell lines at low magnification. [Figure 7-2] This is a continuation of Figure 7-1. [Figure 8-1] This figure shows a comparison of the cell-killing ability of the RH018A strain (virus 17), which is deleting ICP34.5 and ICP47 and expressing GALVR- and GM-CSF, and a conventional strain with the same modification, as determined by crystal violet staining in four cell lines. [Figure 8-2] This is a continuation of Figure 8-1. [Figure 9] This figure shows the efficacy of virus 16 (deleting ICP34.5 and ICP47 and expressing GALVR- and mGM-CSF) in treating mice with A20 lymphoma tumors on both sides. The virus or vehicle was injected into the tumor on the right side, and the effect on tumor size was observed for 30 days. The virus was effective against both injected and uninjected tumors. [Figure 10] This figure demonstrates the effects of virus 15 (deleting ICP34.5 and ICP47 and expressing GALVR- and GFP) and virus 24 (deleting ICP34.5 and ICP47 and expressing GFP) on rat 9L cells in vitro, as evaluated by crystal violet staining. The GALV-expressing virus (virus 15) showed increased rat 9L cell death in vitro compared to the GALV-non-expressing virus (virus 24). [Figure 11-1]This figure shows the antitumor effect of virus 16 in Balb / c mice with mouse CT26 tumors on both the left and right sides of the body. Next, groups of 10 mice were treated as follows: vehicle (injected into the right lateral tumor three times every other day), 5 × 10⁶ pfu of virus 16 (mRP1) injected into the right lateral tumor every other day, anti-mouse PD1 alone (10 mg / kg, intraperitoneal, every 3 days, BioXCell clone RMP1-14), anti-mouse CTLA-4 (3 mg / kg, intraperitoneal, every 3 days, BioXCell clone 9D9), virus 16 with anti-mouse PD1, virus 16 with anti-mouse CTLA4, 1-methyltryptophan (trypotophan) (I-MT, IDO inhibitor (5 mg / ml in drinking water)), 1-methyltryptophan with anti-mouse PD1, or 1-methyltryptophan and virus 16 with anti-mouse PD1. The effect on tumor size was observed for a further 30 days. Animals treated with a combination of virus and checkpoint blockade showed greater tumor reduction than single-treatment groups. Figure 11A shows that the combination of virus 16 and anti-PD1 has a superior antitumor effect compared to either anti-PD1 or virus alone. Figure 11B shows that the antitumor effect of virus 16 in combination with anti-CTLA-4 is better than the antitumor effect of either virus 16 or anti-CTLA-4 alone. Figure 11C shows that enhanced tumor reduction was observed using both anti-PD1 and IDO inhibition along with virus 16 compared to anti-PD1 and 1-MT inhibition in the absence of virus. [Figure 11-2] This is a continuation of Figure 11-1. [Figure 11-3] This is a continuation of Figure 11-1. [Figure 12-1] This figure shows the enhanced antitumor activity of virus 16 in combination with immune checkpoint blockade in mouse A20 tumors on both sides of Balb / c mice, compared to virus alone or checkpoint blockade alone (anti-PD1). [Figure 12-2] This is a continuation of Figure 12-1. [Figure 12-3] This is a continuation of Figure 12-1. [Figure 12-4] This is a continuation of Figure 12-1. [Figure 13] This figure shows the structures of ICP34.5 and ICP47 deletion viruses expressing GALVR-, GM-CSF, and codon-optimized anti-mouse or anti-human CTLA-4 antibody constructs (secreted scFv molecules linked to the human or mouse IgG1 Fc region). The scFv contains linked ([G4S]3) light and heavy variable chains derived from antibody 9D9 (US2011044953: mouse version) and ipilimumab (US20150283234, human version). The structure of the resulting CTLA-4 inhibitor is also shown. [Figure 14] This figure shows the antitumor effects of virus 16 and virus 19 in a human xenograft model (A549). Virus 16, virus 19, or the vehicle was injected three times over one week at three different dose levels (N=10 / group). The dose of the virus used is indicated. The antitumor effect of GALV-expressing virus 16 was better than that of GALV-nonexpressing virus 19. [Figure 15] This figure shows the effect of the virus of the present invention expressing GALVR- on 9L cells in the lateral surface of Fischer 344 rats. The following treatments were administered to groups of rats (10 rats per group) to one lateral surface of each rat only three times a week for three weeks: 50 μl vehicle, 50 μl of 107 pfu / ml of virus 19 (expressing mGM-CSF but not GALV R-), or 50 μl of 107 pfu / ml of virus 16 (expressing both mouse GM-CSF and GALV-R-). The effect on tumor growth was then observed for a further 30 days. Superior tumor control and reduction were observed with the virus expressing both GM-CSF and GALV-R- compared with the virus expressing GM-CSF alone. [Figure 16] This figure shows the antitumor effects of viruses expressing anti-mCTLA-4 (virus 27), mCD40L (virus 32), mOX4OL (virus 35), and m4-2BBL (virus 33), each of which also possesses mGM-CSF and GALV-R-, compared to virus 16 (which expresses GALV and mGM-CSF). [Modes for carrying out the invention] 【0019】 A brief explanation of sequence listings Sequence ID 1 is the nucleotide sequence of mouse GM-CSF. Sequence ID 2 is the nucleotide sequence of a codon-optimized version of mouse GM-CSF. Sequence ID 3 is the nucleotide sequence of human GM-CSF. Sequence ID 4 is the nucleotide sequence of a codon-optimized version of human GM-CSF. Sequence ID 5 is the amino acid sequence of mouse GM-CSF. Sequence ID 6 is the amino acid sequence of human GM-CSF. Sequence ID 7 is the nucleotide sequence of GALV-R-. Sequence ID 8 is the nucleotide sequence of a codon-optimized version of GALV-R- (the first three nucleotides are arbitrary). Sequence ID 9 is the amino acid sequence of GALV-R-. Sequence ID 10 is the nucleotide sequence of the codon-optimized version of the human membrane-bound version of CD40L. Sequence ID 11 is the amino acid sequence of the human membrane-bound version of CD40L. Sequence ID 12 is the nucleotide sequence of the codon-optimized version of the multimeric secreted version of human CD40L. Sequence ID 13 is the amino acid sequence of the multimeric secreted version of human CD40L. Sequence ID 14 is the nucleotide sequence of the codon-optimized version of the multimeric secreted version of mouse CD40L. Sequence ID No. 15 is the amino acid sequence of the multimeric secreted version of mouse CD40L. Sequence ID 16 is a codon-optimized version of the nucleotide sequence of wild-type human CD40L. Sequence ID No. 17 is the amino acid sequence of wild-type human CD40L. Sequence ID 18 is a codon-optimized version of the nucleotide sequence of wild-type mouse CD40L. Sequence ID 19 is the amino acid sequence of wild-type mouse CD40L. Sequence ID 20 is the nucleotide sequence of a codon-optimized version of mouse 4-1BBL. Sequence ID 21 is a codon-optimized version of the nucleotide sequence of human 4-1BBL. Sequence ID 22 is the nucleotide sequence of the codon-optimized version of the secretory mouse 4-1BBL. Sequence ID 23 is a nucleotide sequence of a codon-optimized version of human secreted 4-1BBL. Sequence ID 24 is the nucleotide sequence of a codon-optimized version of mouse GITRL. Sequence ID 25 is the nucleotide sequence of a codon-optimized version of human GITRL. Sequence ID 26 is the nucleotide sequence of a codon-optimized version of secreted mouse GITRL. Sequence ID 27 is the nucleotide sequence of a codon-optimized version of secreted human GITRL. Sequence ID 28 is the nucleotide sequence of the mouse OX40L gene, which is a codon-optimized version. Sequence ID 29 is the nucleotide sequence of a codon-optimized version of human OX40L. Sequence ID 30 is the nucleotide sequence of the codon-optimized version of the secretory mouse OX40L. Sequence ID 31 is the nucleotide sequence of a codon-optimized version of secreted human OX40L. Sequence ID 32 is the nucleotide sequence of a codon-optimized version of mouse ICOSL. Sequence ID 33 is a nucleotide sequence of a codon-optimized version of human ICOSL. Sequence ID 34 is the nucleotide sequence of the mouse scFv CTLA-4 antibody. The first 6 nucleotides and the last 8 nucleotides are restriction sites added for cloning purposes. Sequence ID 35 is the nucleotide sequence of the mouse scFv CTLA-4 antibody. The first 6 nucleotides and the last 8 nucleotides are restriction sites added for cloning purposes. Sequence ID 36 is the nucleotide sequence of the CMV promoter. Sequence ID 37 is the nucleotide sequence of the RSV promoter. Sequence ID 38 is the nucleotide sequence of BGH poly(A). Sequence ID 39 is the nucleotide sequence of late poly(A) in SV40. Sequence ID 40 is the nucleotide sequence of the SV40 enhancer promoter. Sequence ID 41 is the nucleotide sequence of rabbit β-globulin (RBG) polyA. Sequence ID 42 is the nucleotide sequence of GFP. Sequence ID 43 is the nucleotide sequence of the MoMuLV LTR promoter. Sequence ID 44 is the nucleotide sequence of the EF1a promoter. Sequence ID 45 is the nucleotide sequence of HGH poly(A). 【0020】 Detailed description of the invention Oncolytic viruses The virus of the present invention is oncolytic. Oncolytic viruses are viruses that infect and replicate in tumor cells, thereby killing them. Therefore, the virus of the present invention is replicable. Preferably, the virus is selectively replicable in tumor tissue. A virus is selectively replicable in tumor tissue if it replicates more effectively in tumor tissue than in non-tumor tissue. The ability of a virus to replicate in different tissue types can be determined using standard techniques in the art. 【0021】 The oncolytic effect depends on the virus replicating and killing the initially infected cells, and on the progeny virions that continue to infect and kill other tumor cells, resulting in proliferation within the tumor. Therefore, the ability of the virus of this invention to effectively kill tumor cells and proliferate within the tumor results in an optimal direct antitumor effect. Furthermore, the lysis of tumor cells accompanied by efficient proliferation and viral replication maximizes the amount of tumor antigen released, and therefore maximizes the potency of the induced antitumor immune response. 【0022】 The viruses of the present invention may be any virus having these characteristics, such as herpesviruses, poxviruses, adenoviruses, retroviruses, rhabdoviruses, paramyxoviruses, or reoviruses, or any species or strain within the larger group thereof. The viruses of the present invention may be wild-type (i.e., unmodified from the parent virus species) or may have gene disruption or gene addition. Which of these is the case depends on the virus species used. Preferably, the virus is a herpesvirus species, more preferably an HSV strain including HSV1 and HSV2 strains, and most preferably an HSV1 strain. In a particularly preferred embodiment, the viruses of the present invention are based on clinical isolates of the virus species used. Clinical isolates may be selected based on having characteristics that are particularly advantageous for cancer treatment. 【0023】 Clinical isolates may exhibit remarkably good antitumor effects compared to other strains of the same virus isolated from other patients, where the patient is the individual possessing the virus species under test. Virus strains used for comparison to identify useful viruses in this invention may be isolated from patients or otherwise healthy volunteers (i.e., those not possessing the virus species under test), preferably otherwise healthy volunteers. The HSV1 strain used to identify the virus in this invention is typically isolated from cold sores of individuals possessing HSV1, typically by swabbing using, for example, a Virocult (Sigma) brand swab / container containing a transport medium, and subsequently transported to a facility for further testing. 【0024】 After isolating the virus to be compared from an individual, a virus stock is typically prepared by growing the isolated virus, for example, on BHK cells or Vero cells. Preferably, this is done after three or fewer freeze-thaw cycles between taking the sample and growing it, for example, on BHK or Vero cells, in order to prepare a virus stock for further use. More preferably, the virus sample has undergone two or fewer freeze-thaw cycles, more preferably one freeze-thaw cycle, and most preferably no freeze-thaw cycles, before preparing a stock for further use. Lysates derived from cell lines infected with the virus thus prepared after isolation are typically compared by testing the virus's ability to kill tumor cell lines in vitro. Alternatively, the virus stock can be stored under appropriate conditions, for example, by freezing, before testing. The virus of the present invention may have remarkably good antitumor effects compared to other strains of the same virus isolated from other individuals, preferably from more than five individuals, more preferably from more than ten other individuals, and most preferably from more than 20 other individuals. 【0025】 A stock of clinical isolates identified as viruses for modification to produce the viruses of the present invention (i.e., having remarkably good properties for killing tumor cells compared to other viral strains with which they were compared) may be stored under appropriate conditions before and after modification and may be used to generate further stocks as needed. 【0026】 A clinical isolate is a strain of the virus species isolated from its natural host. The clinical isolate is preferably isolated for the purpose of testing and comparing it with other clinical isolates of the same virus species for desired properties, particularly its ability to kill human tumor cells. Clinical isolates that may be used for comparison may also include those present in a clinical repository, i.e., those previously recovered from clinical samples for clinical diagnosis or other purposes. In any case, the clinical isolates used for the comparison and identification of the viruses of the present invention are preferably subjected to minimal in vitro culture before being tested for desired properties, and preferably only cultured to produce sufficient stock for comparative testing purposes. Thus, the viruses used for comparison to identify the viruses of the present invention may also include deposited strains, where the deposited strain is isolated from a patient, preferably an HSV1 strain isolated from a patient's oral herpes. 【0027】 The virus may be a modified clinical isolate, where the clinical isolate kills two or more tumor cell lines more rapidly and / or at lower doses in vitro than one or more reference clinical isolates of the same type of virus. Typically, the clinical isolate kills two or more tumor cell lines within 72 hours, preferably within 48 hours, and more preferably within 24 hours of infection, with an infection multiplicity (MOI) of 0.1 or less, preferably 0.01 or less, and more preferably 0.001 or less. Preferably, the clinical isolate kills 2, 3, 4, 5, 6, 7, 8, 9, 10, or for example all, of a wide range of tumor cell lines, such as the following human tumor cell lines: U87MG (glioma), HT29 (colorectal), LNCaP (prostate), MDA-MB-231 (breast), SK-MEL-28 (melanoma), Fadu (squamous cell carcinoma), MCF7 (breast), A549 (lung), MIAPACA-2 (pancreas), CAPAN-1 (pancreas), and HT1080 (fibrosarcoma). 【0028】 Therefore, the virus of the present invention can kill cells from two or more different types of tumors, for example, three, four, five, six, or seven or more solid tumors, and the above cells include, but are not limited to, colorectal tumor cells, prostate tumor cells, breast tumor cells, ovarian tumor cells, melanoma cells, squamous cell carcinoma cells, lung tumor cells, pancreatic tumor cells, sarcoma cells and / or fibrosarcoma cells. 【0029】 The elimination of tumor cell lines can be determined by any suitable method. Typically, a sample is first isolated from a patient, preferably from herpes simplex in the case of HSV1, and used to infect another suitable cell line, such as BHK cells or Vero cells. Positive samples are typically identified by the presence of cytopathic effects (CPE) 24–72 hours post-infection, for example, 48 hours post-infection, and confirmed to be the target viral species by immunohistochemistry or PCR, for example. A viral stock is then generated from the positive samples. Samples from the viral stock are typically tested and compared to other samples similarly generated using swabs from different patients. Testing can be performed by determining a range of multiples of infection (MOI) and the level of CPE achieved at various time points post-infection. 【0030】 For example, an 80% confluent cell line can be infected with a viral sample at MOIs of 1, 0.1, 0.01, and 0.001, and the dual plates can be incubated for 24 and 48 hours at 37°C and 5% CO2, after which the degree of viral cell death can be determined. This can be determined, for example, by fixing the cells with glutaraldehyde and staining them with crystal violet using standard methods. The level of cell lysis can then be evaluated by standard methods such as macroscopic observation, microscopy (cell counting), and photography. This method can be repeated with incubated cells for shorter periods, e.g., 8, 12, or 16 hours, or longer periods, e.g., 72 hours, or with additional MOIs such as less than 0.0001, before cell death is determined. 【0031】 Growth curve experiments can also be performed to evaluate the ability of different clinical isolates to replicate in tumor cell lines in vitro. For example, 80% confluent cell lines are infected with viral samples at MOIs of 1, 0.1, 0.01, and 0.001, incubated at 37°C and 5% CO2, and the cells are lysed by freeze / thaw typically 0, 8, 16, 24, and 48 hours after infection, after which the degree of viral cell death is determined. This can be determined, for example, by assessing viral titer by a standard plaque assay. 【0032】 The clinical isolates of the present invention can kill infected tumor cell lines more rapidly and / or at a lower MOI than other clinical isolates it is compared with, preferably 2, 3, 4, 5, or 10 or more other clinical isolates of the same virus species. The clinical isolates of the present invention typically kill tumor cells present at a particular MOI and time point at a rate 10%, 25%, or 50% higher than at least one, preferably 2, 3, 4, 5, or 10 or more other clinical isolates of the same virus type compared at the same MOI and time point. The clinical isolates of the present invention typically kill the same or a greater proportion of tumor cells at an MOI that is half or less than half of the MOI at which one or more, preferably 2, 3, 4, 5, 10, or 15 or more other clinical isolates of the same virus species used for comparison at the same time point, typically 12, 24, and / or 48 hours, kill the same proportion of tumor cells. Preferably, the clinical isolates of the present invention typically kill the same or a greater proportion of tumor cells at an MOI 5 or 10 times lower than the MOI at which one or more other clinical isolates of the same viral species used for comparison at the same time point, typically 12, 24, and / or 48 hours, preferably 2, 3, 4, 5, 10, or 15 or more, kill the same proportion of tumor cells. The improved tumor cell killing ability of the viruses of the present invention is typically achieved compared to at least 50%, 75%, or 90% of other clinical isolates of the same viral species used for comparison. The viruses are preferably compared to at least four other viral strains of the same species, e.g., 7, 9, 19, 39, or 49 other viral strains. 【0033】 The isolated strains can be tested, for example, in batches of 4 to 8 virus strains at a time, on 4 to 8 tumor cell lines at a time. For each batch of the experiment, the degree of cell death achieved is ranked for each cell line from best (i.e., minimum viable cells / MOI at each time point) to worst (i.e., maximum viable cells / MOI at each time point) among the viruses compared in that experiment. The virus strain from each experiment that shows the best results across the entire range of tumor cell lines tested (i.e., consistently ranked as one of the best in killing cell lines) is then directly compared in further experiments using other clinical isolates and / or other tumor cell lines, and the best virus strain overall may be identified among more than 20 sampled virus strains. The virus ranked as the best overall is the virus of the present invention. 【0034】 In a preferred embodiment, the virus of the present invention is a strain selected from the following: RH018A shares with provisional accession number ECCAC16121904, RH004A shares with provisional accession number ECCAC16121902, RH031A shares with provisional accession number ECCAC16121907, RH040B shares with provisional accession number ECCAC16121908, RH015A shares with provisional accession number ECCAC16121903, RH021A shares with provisional accession number ECCAC16121905, RH023A strain with provisional accession number ECCAC16121906, and RH047A strain with provisional accession number ECCAC16121909. 【0035】 More preferably, the virus of the present invention is a strain selected from the following: RH018A shares with provisional accession number ECCAC16121904, RH004A shares with provisional accession number ECCAC16121902, RH031A shares with provisional accession number ECCAC16121907, RH040B shares with provisional accession number ECCAC16121908, and RH015A strain with provisional accession number ECCAC16121903. 【0036】 Most preferably, the virus of the present invention is the RH018A strain having accession number EACC16121904. 【0037】 The HSV of the present invention can selectively replicate in tumors, such as human tumors. Typically, the HSV replicates efficiently in target tumors but not efficiently in non-tumor tissues. This HSV may contain one or more mutations in one or more viral genes that inhibit replication in normal tissues but still allow replication in tumors. The mutations may be, for example, mutations that, when mediated by the HSV, inhibit the expression of functional ICP34.5, ICP6, and / or thymidine kinases. 【0038】 In one preferred embodiment, the gene encoding ICP34.5 is mutated to confer selective oncolytic activity to HSV. Mutations of the gene encoding ICP34.5 that prevent the expression of functional ICP34.5 are described by reference in Chou et al. (1990) Science 250:1262-1266, Maclean et al. (1991) J. Gen. Virol. 72:631-639 and Liu et al. (2003) Gene Therapy 10:292-303, which are incorporated herein by reference. The gene encoding ICP6 and / or the gene encoding thymidine kinase may also be inactivated, as with the other genes, as such inactivation does not interfere with viral infection or replication within the tumor. 【0039】 HSV may contain further mutations that enhance HSV replication in tumors. The enhancement resulting from viral replication in tumors not only leads to direct "oncolytic" tumor cell death by the virus, but also enhances the level of heterologous gene expression (i.e., genes inserted into the virus, which in the case of the virus of this invention encodes fusion-inducible proteins and immunomodulatory molecules), increasing the amount of tumor antigen released when tumor cells are killed. Both of these can also improve the immunogenic properties of the treatment for cancer. For example, in a preferred embodiment of the present invention, deletion of the gene encoding ICP47 in a manner that places the US11 gene under the regulation of a pre-initial promoter that normally regulates the expression of the gene encoding ICP47 results in enhanced replication in tumors (see Liu et al., 2003, incorporated herein by reference). 【0040】 Other mutations that place the sequence encoding US11, a late-stage HSV gene, under the control of a promoter independent of viral replication can also be introduced into the virus of the present invention. Such mutations enable the expression of US11 before HSV replication occurs, thereby enhancing viral replication in tumors. In particular, such mutations enhance replication in HSV lacking the gene encoding functional ICP34.5. 【0041】 Therefore, in one embodiment, the HSV of the present invention comprises a US11 gene operably linked to a promoter, and the promoter activity is independent of viral replication. The promoter may be a pre-early (IE) promoter or a non-HSV promoter that is active in mammalian, preferably human, tumor cells. The promoter may be a eukaryotic promoter, such as a promoter derived from a mammalian, preferably human, genome. The promoter may be a ubiquitous promoter (such as a β-actin or tubulin promoter) or a cell-specific promoter, such as a tumor-specific promoter. The promoter may be a viral promoter, such as a long-terminal repeat (MMLV LTR) promoter of Moloney mouse leukemia virus or a human or mouse cytomegalovirus (CMV) IE promoter. HSV pre-early (IE) promoters are well known in the art. The HSV IE promoter may be a promoter that drives the expression of ICP0, ICP4, ICP22, ICP27, or ICP47. 【0042】 The genes mentioned above, whose functional inactivation provides tumor-selective properties against viruses, can be made functionally inactive by any suitable method, for example, by deleting or substituting all or part of the gene and / or its regulatory sequence, or by inserting one or more nucleic acids into or in place of the gene and / or its regulatory sequence. For example, the viruses of the present invention can be produced using homologous recombination methods, which are standard in the art. Alternatively, a bacterial artificial chromosome (BAC)-based approach may be used. 【0043】 As used herein, the term “gene” is intended to mean a nucleotide sequence that codes for a protein, i.e., the coding sequence of a gene. The various genes referred to above can be rendered non-functional by mutation in the gene itself or in a regulatory sequence adjacent to the gene, such as a promoter sequence. Deletions can remove one or more parts of a gene, the entire gene, or the entire gene and all or part of the regulatory sequence. For example, a deletion of just one nucleotide in a gene can result in a frameshift. However, larger deletions can result, such as deletions of at least about 25%, more preferably at least about 50%, of the entire coding sequence and / or non-coding sequence. In one preferred embodiment, a gene is deleted that is in a functionally inactive state. For example, the entire gene and optionally a portion of the adjacent sequence can be removed from the virus. If two or more copies of a gene are present in the viral genome, both copies of the gene are rendered functionally inactive. 【0044】 A gene can be inactivated by substituting other sequences, for example, by substituting all or part of an endogenous gene with a heterologous gene and optionally a promoter sequence. If the promoter sequence is not substituted, the heterologous gene can be inserted so as to be regulated by the promoter of the inactivated gene. In the HSV of the present invention, the gene encoding ICP34.5 is preferably inactivated by inserting a heterologous gene, a promoter sequence operably linked thereto, and optionally other regulatory elements, such as a polyadenylation sequence, into each of the loci encoding ICP34.5. 【0045】 The virus of the present invention is used to express fusion-inducible proteins and immunostimulatory proteins in tumors. This is typically achieved by inserting heterologous genes encoding fusion-inducible proteins and heterologous genes encoding immunostimulatory proteins into the genome of a selectively replicating competent virus, where each gene is regulated by a promoter sequence. Because replication of such a virus occurs selectively in tumor tissue, the expression of fusion-inducible proteins and immunostimulatory proteins by the virus is also enhanced in tumor tissue compared to non-tumor tissue of the body. Enhanced expression occurs when expression is greater in the tumor compared to other tissues of the body. Thus, the present invention offers the benefit of selectively expressing both fusion-inducible proteins and immunostimulatory proteins in tumors, in combination with the antitumor effect provided by oncolytic viral replication. 【0046】 The virus of the present invention may include, in addition to the fusion-inducible protein and the immunostimulatory protein, one or more further heterologous genes, such as further fusion-inducible or immunostimulatory proteins. 【0047】 Fusion-inducible protein The virus of the present invention contains a gene encoding a fusion-inducing protein. The fusion-inducing protein may be any heterologous protein that can promote the fusion of a cell infected with the virus of the present invention into another cell. The fusion-inducing protein, preferably a wild-type or modified viral glycoprotein (i.e., one modified to enhance its fusion-inducing properties), is a protein that can induce intercellular fusion (syncitia formation) in the cell on which it is expressed. Examples of fusion-inducible glycoproteins include glycoproteins derived from VSV-G, syncytin-1 (derived from human endogenous retrovirus-W (HERV-W)) or syncytin-2 (derived from HERVFRDE1), paramyxovirus SV5-F, measles virus-H, measles virus-F, RSV-F, retroviruses or lentiviruses, such as gibbon ape leukemia virus (GALV), mouse leukemia virus (MLV), Mason-Pfizer monkey virus (MPMV), and equine infectious anemia virus (EIAV), in which the R transmembrane peptide has been removed (R-version). In a preferred embodiment, the fusion-inducible protein is derived from GALV and the R peptide has been removed (GALV-R-). 【0048】 The virus of the present invention may contain a gene encoding multiple copies of a fusion-inducible protein, preferably one or two copies. The virus may contain two or more different fusion-inducible proteins, including one of the fusion-inducible proteins listed above. 【0049】 The fusion-inducible protein expressed by the virus of the present invention may be identical to a naturally occurring protein, or it may be a modified protein. 【0050】 Genes encoding fusion-inducible proteins (fusion-inducible genes) may have naturally occurring nucleic acid sequences or modified sequences. The sequences of fusion-inducible genes may be modified, for example, to enhance the fusion-inducible properties of the encoded protein or to provide codon optimization, and therefore to increase the expression efficiency of the encoded protein. 【0051】 immunostimulatory molecules The virus of the present invention comprises one or more immunostimulatory molecules and / or one or more genes encoding immunostimulatory molecules. Immunostimulatory molecules include proteins that can assist in inducing an immune response, proteins that can mitigate inhibitory signals to the induction or efficacy of an immune response, and RNA molecules (e.g., shRNA, antisense RNA, RNAi, or microRNA) that inhibit the expression of immunosuppressive molecules. Examples of immunostimulatory molecules include IL-2, IL-12, IL-15, IL-18, IL-21, IL-24, CD40 ligand, GITR ligand, 4-1-BB ligand, OX40 ligand, ICOS ligand, flt3 ligand, type I interferons including interferon alpha and interferon beta, interferon gamma, type III interferons (IL-28, IL-29), other cytokines such as TNF alpha or GM-CSF, TGF beta, or immune checkpoint antagonists. Immune checkpoint antagonists include antibodies, single-strand antibodies, and RNA1 / siRNA / microRNA / antisense RNA knockdown approaches. Agonists in the immune-enhancing / costimulatory pathway include mutant or wild-type, soluble, secreted, and / or membrane-bound ligands, as well as agonist antibodies, including single-chain antibodies. With regard to targeting immune co-inhibitory or immune co-stimulatory pathways, proteins or other molecules (case-dependent agonists or antagonists) that target CTLA-4 (antagonist), PD-1 (antagonist), PD-L1 (antagonist), LAG-3 (antagonist), TIM-3 (antagonist), VISTA (antagonist), CSF1R (antagonist), IDO (antagonist), CEACAM1 (antagonist), GITR (agonist), 4-1-BB (agonist), KIR (antagonist), SLAMF7 (antagonist), OX40 (agonist), CD40 (agonist), ICOS (agonist), or CD47 (antagonist) are particularly preferred. Therefore, the virus of the present invention preferably encodes one or more of these molecules.More preferably, the virus of the present invention encodes GM-CSF and / or wild-type or modified versions of CD40L, ICOSL, 4-1-BBL, GITRL, or OX40L, most preferably GM-CSF. 【0052】 Inhibitors of co-inhibitory pathways may also be CTLA-4 inhibitors. CTLA-4 inhibitors are typically molecules such as peptides or proteins that bind to CTLA-4 and reduce or block CTLA-4 signaling, such as by reducing activation by B7. By reducing CTLA-4 signaling, inhibitors reduce or eliminate the blockade of the CTLA-4 immune stimulation pathway. 【0053】 CTLA-4 inhibitors are preferably antibodies or their antigen-binding fragments. The term “antibody” as used herein includes the whole antibody and any antigen-binding fragment (i.e., “antigen-binding moiety”) or their single chains. An antibody refers to a glycoprotein or its antigen-binding moiety comprising at least two heavy (H) chains and two light (kappa) (L) chains linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), which contain more conserved regions called framework regions (FRs). The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. 【0054】 The antibody is typically a monoclonal antibody. The antibody may also be a chimeric antibody. The antibody is preferably a humanized antibody, and more preferably a human antibody. 【0055】 The term “antigen-binding fragment” of an antibody refers to one or more fragments of an antibody that possess the ability to specifically bind to CTLA-4. The antigen-binding fragment also possesses the ability to inhibit CTLA-4 and therefore to reduce or eliminate CTLA-4 blockade in stimulative immune responses. Examples of suitable fragments include Fab fragments, F(ab')2 fragments, Fab' fragments, Fd fragments, Fv fragments, dAb fragments, and isolated complementarity-determining regions (CDRs). Single-chain antibodies such as scFv and heavy-chain antibodies such as VHH and camel antibodies are also intended to be included in the term “antigen-binding moiety” of an antibody. In a preferred embodiment, the antibody is scFv. Examples of suitable scFv molecules are disclosed, for example, in WO2007 / 123737 and WO2014 / 066532, which are incorporated herein by reference. scFv may be encoded by the nucleotide sequence shown in SEQ ID NO: 34 and SEQ ID NO: 35. 【0056】 The virus of the present invention may encode one or more immunostimulatory molecules, preferably one, two, three, or four immunostimulatory molecules, more preferably one or two immunostimulatory molecules. 【0057】 The sequences of genes encoding immunostimulatory molecules can be codon-optimized to increase the expression levels of each protein in target cells compared to when the unmodified sequences are used. 【0058】 Virus production The viruses of the present invention are constructed using methods well known in the art. For example, a plasmid encoding a packaged viral genome (for smaller viruses and single and multiple genomic component RNA viruses) or BAC (for larger DNA viruses, including herpesviruses) containing genes encoding fusion-inducible molecules and immunostimulatory molecules under appropriate regulatory conditions, constructed by standard molecular biology techniques, and transfected into tolerant cells from which recombinant viruses can be recovered. 【0059】 Alternatively, in a preferred embodiment, a plasmid containing a DNA region adjacent to the intended insertion site may be constructed and then co-transfected with viral genomic DNA into a suitable cell, thereby inducing homologous recombination between the region adjacent to the target insertion site in the plasmid and the same region in the parental virus. The recombinant virus can then be selected and purified by loss or addition of function inserted or deleted by the plasmid used for modification, such as by insertion or deletion of a marker gene such as GFP or lacZ from the parental virus at the intended insertion site. In the most preferred embodiment, the insertion site is the ICP34.5 locus of HSV, and therefore the plasmid used for the operation contains an HSV sequence adjacent to this insertion site, with an expression cassette encoding a fusion-inducible protein and an immunostimulatory molecule between them. In this case, the parental virus may contain a cassette encoding GFP instead of ICP34.5, and the recombinant viral plaque is selected by the loss of GFP expression. In the most preferred embodiment, the US11 gene of HSV is also expressed as an IE gene. This can be achieved via deletion of the ICP47 coding region or by other means. 【0060】 Sequences encoding fusion-inducible proteins and immunomodulatory molecules are inserted into the viral genome under appropriate regulatory conditions. This may be under the regulatory conditions of the native promoter of the viral species used, depending on the species and insertion site, or preferably under the regulation of a heterologous promoter. Suitable heterologous promoters include mammalian promoters such as the IEF2a promoter or the actin promoter. More preferably, potent viral promoters such as the CMV IE promoter, RSV LTR, MMLV LTR, other retroviral LTR promoters, or SV40-derived promoters are preferred. Preferably, each exogenous gene (i.e., encoding a fusion-inducible protein and an immunomodulatory molecule) is under the regulation of a separate promoter, but may also be expressed from a single RNA transcript, for example, by inserting an internal ribosome entry site (IRES) between protein-coding sequences. Each promoter-derived RNA is typically terminated with a polyadenylation sequence (e.g., mammalian sequences such as bovine growth hormone (BGH) poly-A sequence, synthetic polyadenylation sequence, rabbit betaglobin polyadenylation sequence, or viral sequences such as SV40 early or late polyadenylation sequence). 【0061】 The present invention also provides a poxvirus or HSV, preferably HSV1, that expresses at least three heterogenes, each of which is driven by a different promoter selected from the CMV promoter, RSV promoter, EF1a promoter, SV40 promoter, and retroviral LTR promoter. The virus may express, for example, four heterogenes, each of which is driven by a different promoter selected from the CMV promoter, RSV promoter, EF1a promoter, SV40 promoter, and retroviral LTR promoter. The retroviral LTR is preferably derived from MMLV (SEQ ID NO: 43), also known as MoMuLV. The heterogenes may be terminated by polyadenylation sequences. The polyadenylation sequences may be the same or different. Preferably, each heterogene is terminated by different polyadenylation sequences preferably selected from the BGH, SV40, HGH, and RBG polyadenylation sequences. 【0062】 The present invention also provides a poxvirus or HSV, preferably HSV1, that expresses at least three heterogenes, each of which is terminated by a different polyadenylation sequence selected from BGH, SV40, HGH, and RBG polyadenylation sequences. The virus may express, for example, four heterogenes, each terminated by one of the BGH, SV40, HGH, and RBG polyadenylation sequences. 【0063】 Pharmaceutical composition The present invention provides a pharmaceutical composition comprising a virus and a pharmaceutically acceptable carrier or diluent. Suitable carriers and diluents include isotonic salines, such as phosphate-buffered salines. The composition may further contain other components, such as sugars or proteins, to improve properties such as product stability. Alternatively, a lyophilized formulation that is reconstituted in a pharmaceutically acceptable carrier or diluent before use may be used. 【0064】 Where necessary, the choice of carrier is often a function of the delivery route of the composition. Within the scope of the present invention, compositions can be formulated for any suitable route and means of administration. Pharmaceutically acceptable carriers or diluents are used for compositions suitable for intratumoral administration, intravenous / intra-arterial administration, administration to the brain, or administration to body cavities (e.g., bladder, thoracic, or peritoneal administration). Compositions can be administered in any suitable form, preferably as a liquid. 【0065】 The present invention also provides a product containing the virus of the present invention in a sterile vial, ampoule, or syringe. 【0066】 Medical use / procedures The present invention provides a virus for use in therapeutic treatment of the human or animal body, particularly for use in methods of treating cancer. Cancer typically exists in mammals, preferably humans. This virus kills infected tumor cells by lysis and by fusing infected tumor cells together. The virus of the present invention also induces a systemic antitumor immune response enhanced by the expression of immunostimulatory molecules that also kill cancer cells. 【0067】 The present invention also provides a method for treating cancer, comprising administering a therapeutically effective amount of the virus of the present invention to an individual in need of it. 【0068】 The present invention further provides the use of the virus in the manufacture of pharmaceuticals for treating cancer. 【0069】 The virus of the present invention is particularly useful for the treatment of any solid tumor, including any adenocarcinoma, carcinoma, melanoma, or sarcoma. For example, the virus of the present invention is useful for the treatment of cancers of the head and neck, prostate, breast, ovary, lung, liver, endometrium, bladder, gallbladder, pancreas, colon, kidney, stomach / gastric, esophageal, or cervical cancers, mesothelioma, melanoma, or other skin cancers, lymphoma, glioma, or other cancers of the nervous system, or sarcomas such as soft tissue sarcoma. 【0070】 The virus of the present invention may be used to treat malignant tumors, including tumors that have metastasized from the site of the original tumor. In this embodiment, the virus may be administered to the primary tumor or one or more secondary tumors. 【0071】 The virus of the present invention can be administered in combination with other therapeutic agents, including chemotherapy, targeted therapy, immunotherapy (including one or more antagonists of the immune co-inhibitory pathway and / or one or more agonists of the immune costimulatory pathway), and / or in combination with radiotherapy, and / or in combination with any combination thereof. The therapeutic agent is preferably an anticancer agent. 【0072】 The virus of the present invention may be administered in combination with a second virus, such as a second oncolytic virus. 【0073】 For example, therapeutic agents may include immunogens (including recombinant or naturally occurring antigens, including such antigens or combinations of antigens delivered as the DNA or RNA on which they are encoded) to further stimulate an immune response against tumor cells, particularly neoantigens, such as cellular or humoral immune responses. The therapeutic agents may also include agents intended to increase or enhance the immune response, such as cytokines, agents intended to inhibit immune checkpoint pathways or stimulate immune enhancement pathways, or agents that inhibit the activity of regulatory T cells (Tregs) or myeloid-derived suppressor cells (MDSCs). 【0074】 The therapeutic agent may be a drug whose use in existing cancer treatment procedures is publicly known. The therapeutic agent may be a radiotherapy agent or a chemotherapeutic agent. The therapeutic agent may be selected from cyclophosphamide, alkylating agents, e.g., cisplatin or melphalan, plant alkaloids and terpenoids, e.g., vincristine or paclitaxel (Taxol), antimetabolites, e.g., 5-fluorouracil, topoisomerase inhibitors of type I or II, e.g., camptothecin or doxorubicin, cytotoxic antibiotics, e.g., actinomycin, anthracyclines, e.g., epirubicin, glucocorticoids, e.g., triamcinolone, inhibitors of protein, DNA and / or RNA synthesis, e.g., methotrexate and dacarbaxine, histone deacetylase (HDAC) inhibitors, or any other chemotherapeutic agent. 【0075】 The therapeutic agent may be one or a combination of the following: an immunotherapeutic agent or immunomodulator, e.g., a TLR agonist; an agent that downregulates T regulatory cells, e.g., cyclophosphamide; or an agent designed to block immune checkpoints or stimulate immune enhancement pathways, e.g., but not limited to monoclonal antibodies, e.g., CTLA-4 inhibitors, PD-1 inhibitors, PD-L1 inhibitors, LAG-3 inhibitors, TIM-3 inhibitors, VISTA inhibitors, CSF1R inhibitors, IDO inhibitors, CEACAM1 inhibitors, GITR agonists, 4-1-BB agonists, KIR inhibitors, SLAMF7 inhibitors, OX40 agonists, CD40 agonists, ICOS agonists, or CD47 inhibitors. In a preferred embodiment, the therapeutic agent is a CTLA-4 inhibitor, e.g., an anti-CTLA-4 antibody; a PD1 inhibitor, e.g., an anti-PD-1 antibody; or a PD-L1 inhibitor, e.g., an anti-PD-L1 antibody. Such inhibitors, agonists, and antibodies can be produced and tested by standard methods known in the art. 【0076】 Immunotherapy agents may also include bispecific antibodies, dendritic cell-based cell-based therapies, NK cells or engineered T cells, such as CAR-T cells or engineered T cells expressing T cell receptors. Immunotherapy agents may also include agents that target specific gene mutations occurring in tumors, agents intended to induce an immune response to specific tumor antigens or combinations of tumor antigens, such as neoantigens, and / or agents intended to activate the STING / cGAS pathway, TLR or other innate immune responses and / or inflammatory pathways, such as intratumor agents. 【0077】 For example, the virus of the present invention may be used in combination with dacarbazine, a BRAF inhibitor and / or a CTLA-4, PD1 or PD-L1 blocker to treat melanoma; in combination with Taxol, doxorubicin, vinorelbine, cyclophosphamide and / or gemcitabine to treat breast cancer; in combination with 5-fluorouracil and optionally leucovorin, irinotecan and / or oxaliplatin to treat colorectal cancer; in combination with Taxol, carboplatin, vinorelbine and / or gemcitabine, PD-1 or PD-L1 blocker to treat lung cancer; and in combination with cisplatin and / or radiotherapy to treat head and neck cancer. 【0078】 Therapeutic agents may include inhibitors of the indoleamine 2,3-dioxygenase (IDO) pathway. Examples of IDO inhibitors include epcadostat (INCB024360), 1-methyl-tryptophan, indoximod (1-methyl-D-tryptophan), GDC-0919, or F001287. 【0079】 The mechanism of action of IDO in suppressing antitumor immune responses can also suppress immune responses that arise after oncolytic virus therapy. IDO expression is induced by Toll-like receptor (TLR) activation and interferon-γ, both of which can result from oncolytic virus infection. One embodiment of the use of oncolytic virus therapy for cancer treatment involves a combination of an oncolytic virus, including a virus expressing an immunostimulatory protein and / or a fusion-inducible protein, an inhibitor of the IDO pathway, and optionally one or more further antagonists of immune co-inhibitory pathways, including those targeting CTLA-4, PD-1, and / or PD-L1, and / or one or more agonists of immune costimulatory pathways. 【0080】 The present invention also provides a method for treating cancer, comprising administering a therapeutically effective amount of oncolytic virus, an inhibitor of the indoleamine 2,3-dioxygenase (IDO) pathway, a further antagonist of an immunoco-inhibitory pathway, and / or an agonist of an immunoco-stimulatory pathway to a patient in need. 【0081】 The oncolytic virus is preferably a modified clinical isolate. The oncolytic virus is preferably a poxvirus, and more preferably an HSV, such as HSV1 and / or an HSV functionally inactive to ICP34.5 and / or ICP47. The oncolytic virus may express an immunostimulatory molecule, such as GM-CSF, and / or a molecule encoding a costimulatory pathway, such as CD4OL, GITRL, OX4OL, 4-I-BBL, or ICOSL, and / or an inhibitor of CTLA-4, and / or a fusion-inducible protein, such as a GALV fusion-inducible glycoprotein with a mutated or deleted R sequence. Further antagonists of the immune-costimulatory pathway are preferably an antagonist of CTLA-4, an antagonist of PD1, or an antagonist of PD-L1. For example, a further antagonist of the immune-coinhibitory pathway may be an inhibitor of the interaction between PD1 and PD-L1. 【0082】 When therapeutic agents and / or radiotherapy are used in conjunction with the virus of the present invention, the administration of the virus and therapeutic agents and / or radiotherapy may be performed simultaneously or separately over time. The compositions of the present invention may be administered before, simultaneously with, or after therapeutic agents or radiotherapy. A method of treating cancer may include multiple administrations of the virus and / or therapeutic agents and / or radiotherapy of the present invention. In preferred embodiments, when used in combination with immune checkpoint blockers or other immunostimulants, the virus of the present invention is administered once or more times before subsequent simultaneous administration of immune checkpoint blockers or other immunostimulants, or is administered simultaneously with the administration of immune checkpoint blockers or other immunostimulants without prior administration of the virus of the present invention. 【0083】 The virus of the present invention can be administered to a target by any suitable route. Typically, the virus of the present invention is administered by direct intratumor injection. Intratumor injections include direct injection into epidermal, subcutaneous, or nodular tumors, as well as image-guided injections (such as CT, MRI, or ultrasound) to localize the deposits more deeply or firmly to internal organs and other locations. The virus can be administered into a body cavity, for example, into the pleural cavity, bladder, or intraperitoneal cavity. The virus can be injected intravascularly, preferably into the blood vessel supplying the tumor. 【0084】 The therapeutic agents that can be combined with the virus of the present invention can be administered in vivo to human or animal subjects using a variety of known routes and techniques. For example, the composition may be provided as an injectable solution, suspension or emulsion and may be administered parenterally, subcutaneously, orally, epidermally, intradermally, intramuscularly, intra-arterially, intraperitoneally, or intravenously by conventional needles and syringes or by liquid-jet injection systems. The composition may be administered topically to skin or mucous membrane tissue such as the nose, trachea, intestine, sublingually, rectally, or vagina, or may be provided as finely divided sprays suitable for respiratory or pulmonary administration. In preferred embodiments, the composition may be administered intravenously, orally, or directly to tumors. 【0085】 The virus and / or therapeutic agent may be administered to a subject in an amount compatible with a therapeutically effective dose composition. The administration of the virus of the present invention is for the purpose of "treatment". As used herein, the terms "treatment" or "treatment" include, for its purpose, one or more of the following: prevention of any metastasis or further metastasis that may occur, reduction or elimination of symptoms, reduction or complete elimination of tumors or cancer, increase the time to progression of cancer in a patient, increase the time to recurrence after treatment, or increase survival time. 【0086】 Therapeutic treatment is administered for stage I, II, III, or IV cancer, preferably stage II, III, or IV, more preferably stage III or IV, before or after surgical intervention (i.e., after recurrence or incomplete removal of the tumor after surgery), preferably before any surgical intervention (for either primary or recurrent / metastatic disease resection), or after recurrence after surgery or incomplete surgical removal of the disease, i.e., while residual tumor remains. 【0087】 Therapeutic treatment can be performed after injecting the viral composition directly into a target tissue that may be a tumor, into a body cavity, or into a blood vessel. As a guideline, the amount of virus administered is 10 for HSV. 4 ~10 10 PFU, preferably 10 5 ~10 9 This is within the range of pfu for HSV. 4 ~10 7 pfu is administered to convert seronemically negative patients for HSV and to enhance immunity in seronemically positive patients, followed by higher doses (e.g., 10%). 6 ~10 9PFU may be administered thereafter. Typically, a pharmaceutical composition of up to 20 ml, essentially consisting of a virus and a suitable carrier or diluent that is pharmaceutically acceptable, may be used for direct injection into the tumor, or up to 50 ml (which may be subjected to further dilution in a suitable diluent before administration) may be used for administration into a body cavity or into the bloodstream. However, for some oncolytic therapeutic applications, larger or smaller volumes may also be used, depending on the tumor and the route and site of administration. 【0088】 The described routes of administration and dosages are intended as guidelines only, as a skilled physician can easily determine the optimal route of administration and dosage. Dosage may be determined according to various parameters, particularly the location and size of the tumor, the age, weight and condition of the patient being treated, and the route of administration. Preferably, the virus is administered by direct injection into the tumor. The virus may also be administered by intravascular or intracavitary injection. The optimal route of administration depends on the location and size of the tumor. Multiple doses may be required to achieve immunological or clinical effects, which are typically administered at intervals of 2 days to 12 weeks, preferably 3 days to 3 weeks, as needed. Repeated doses may be given for up to 5 years or more, preferably up to 1 month to 2 years, depending on the response rate of the type of tumor being treated and the response of a particular patient, as well as any combination therapy that may be given. Various embodiments of the present invention are shown below. 1. (i) A gene encoding a fusion-inducible protein, and (ii) an immunostimulatory molecule or an oncolytic virus containing a gene encoding an immunostimulatory molecule. 2. The virus described in 1 above, wherein the fusion-inducible protein is selected from the group consisting of glycoproteins derived from gibbon ape leukemia virus (GALV), mouse leukemia virus (MLV), Mason-Pfizer monkey virus (MPMV), or equine infectious anemia virus (EIAV) that are deficient in vesicular stomatitis virus (VSV) G protein, syncytin-1, syncytin-2, Simian virus 5 (SV5) F protein, measles virus (MV) H protein, MV F protein, respiratory syncytial virus (RSV) F protein, and R peptide. 3. The virus described in 1 or 2 above, wherein the immunostimulatory molecule is an antagonist of GM-CSF, IL-2, IL-12, IL-15, IL-18, IL-21, IL-24, type I interferon, interferon gamma, type III interferon, TNF-alpha, TGF-beta, an immune checkpoint antagonist, or an agonist of an immune enhancement pathway, such as CD40 ligand (CD40L), ICOS ligand, GITR ligand, 4-1-BB ligand, OX40 ligand, or flt3 ligand, or a modified version thereof. 4. (a) The fusion-inducible protein is a glycoprotein derived from gibbon leukemia virus (GALV) and has a mutated or removed R transmembrane peptide (GALV-R-), and / or (b) The virus described in any of items 1 to 3 above, wherein the immunostimulatory molecule is (i) GM-CSF or CD40L, GITR ligand, 4-1-BB ligand, OX40 ligand, ICOS ligand or a modified version thereof, or (ii) a CTLA-4 inhibitor. 5. Viruses according to any one of items 1 to 4 above, encoding more than 1 fusion-inducible proteins and / or more than 1 immunostimulatory molecules. 6. The virus described in item 5 above, wherein the immunostimulatory molecule is GM-CSF, and one or more of (i) CD40L, GITR ligand, 4-1-BB ligand, OX40 ligand, and ICOS ligand or a modified version thereof, and / or (ii) a CTLA-4 inhibitor. 7. The virus described in any of 4 to 6 above, wherein the CTLA-4 inhibitor is a CTLA-4 antibody or a fragment thereof. 8. A virus described in any of items 1-7 above, derived from a clinical isolate of the virus. 9. A modified clinical isolate of the virus described in any of items 1 to 8 above, which rapidly and / or at a lower dose in vitro kills two or more tumor cell lines than one or more reference clinical isolates of the same type of virus. 10. Clinical isolates RH018A shares with provisional accession number ECCAC16121904, RH004A shares with provisional accession number ECCAC16121902, RH031A shares with provisional accession number ECCAC16121907, RH040B shares with provisional accession number ECCAC16121908, RH015A shares with provisional accession number ECCAC16121903, RH021A shares with provisional accession number ECCAC16121905, RH023A shares with provisional accession number ECCAC16121906, or RH047A stock with provisional accession number ECCAC16121909 The virus described in item 8 or 9 above. 11. Any of the viruses described in 1 to 9 above, selected from the group consisting of herpesviruses, poxviruses, adenoviruses, retroviruses, rhabdoviruses, paramyxoviruses, and reoviruses. 12. Herpes simplex virus (HSV), one of the viruses listed in 1 to 11 above. 13. The virus described in item 12 above, which is HSV1. 14. HSV is, (a) Does not express functional ICP34.5, (b) Not expressing functional ICP47, and / or (c) Express the US11 gene as a pre-initial gene. Any of the viruses listed in items 10-13 above. 15. A virus according to any one of items 12 to 14 above, wherein a gene encoding a fusion-inducible protein and a gene encoding an immunostimulatory molecule are, respectively, inserted into the locus encoding ICP34.5 either by insertion or partial or complete deletion, either by means of insertion or partial or complete deletion, in an arbitrary and selective manner, in a back-to-back orientation relative to each other. 16. A virus according to any one of items 1 to 15 above, wherein the sequence of a gene encoding a fusion-inducible protein and / or the sequence of a gene encoding an immunostimulatory molecule are codon-optimized to increase the expression level in target cells. 17. A virus expressing three heterologous genes, each of which is driven by a different promoter selected from the CMV promoter, RSV promoter, SV40 promoter, and retroviral LTR promoter. 18. A virus according to any of items 1 to 17 above, which expresses three heterologous genes, each of which is driven by a different promoter selected from the CMV promoter, RSV promoter, SV40 promoter, and retroviral LTR promoter. 19. The virus described in 17 or 18 above, which expresses four heterologous genes, each driven by the CMV promoter, the RSV promoter, the SV40 promoter, and the retroviral LTR promoter, respectively. 20. A virus described in any of items 17-19 above, wherein the retrovirus LTR is derived from MMLV. 21. A virus expressing three heterologous genes, each of which is terminated by a different polyadenylation sequence selected from BGH, SV40, HGH, and RBG polyadenylation sequences. 22. A virus according to any one of items 1 to 21 above, wherein it expresses three heterologous genes, each of which is terminated by a different polyadenylation sequence selected from BGH, SV40, HGH, and RBG polyadenylation sequences. 23. The virus described in 21 or 22 above, which expresses four heterogeneous genes terminated by the BGH, SV40, HGH, and RBG polyadenylated sequences, respectively. 24. (a) HSV, (b) HSV1, or (c) poxvirus The virus described in any of the above items 17-23. 25. A pharmaceutical composition comprising the virus described in any of items 1 to 24 above and a carrier or diluent that is acceptable as a pharmaceutical. 26. Any of the viruses described in 1 to 24 above, for use in methods of treating the body of a human or animal by therapeutic means. 27. Any of the viruses described in 1 to 24 above for use in a method of treating cancer. 28. The virus for use described in 27 above, wherein the method comprises administering further anticancer agents. 29. Further anticancer agents selected from agents targeting immune co-inhibitory or immune co-stimulatory pathways, radiotherapy and / or chemotherapy, agents targeting specific gene mutations occurring in tumors, agents intended to induce an immune response to one or more tumor antigens or neoantigens, cell products derived from T cells or NK cells, STING, cGAS, TLR or other agents intended to stimulate innate immune responses and / or inflammatory pathways, second viruses, optionally oncolytic viruses, and combinations thereof, for use as described in 28 above. 30. The virus for use as described in 28 or 29 above, wherein the agent targeting the immune co-inhibitory pathway is a CTLA-4 inhibitor, PD-1 inhibitor, PD-L1 inhibitor, LAG-3 inhibitor, TIM-3 inhibitor, VISTA inhibitor, CSF1R inhibitor, IDO inhibitor, CEACAM1 inhibitor, KIR inhibitor, SLAMF7 or CD47 inhibitor, and / or the agent targeting the immune co-stimulatory pathway is a GITR agonist, 4-1-BB agonist, OX40 agonist, CD40 agonist or ICOS agonist. 31. A further anticancer agent is an antibody, which is a virus for use as described in any of paragraphs 28-30 above. 32. A method for the use of a virus according to any one of 28 to 31 above, comprising administering an inhibitor of the indoleamine 2,3-dioxygenase (IDO) pathway and a further antagonist of an immunoco-inhibitory pathway, or an agonist of an immunoco-stimulatory pathway. 33. Virus for use as described in any of 28-32 above, wherein the virus and further anticancer agents are administered separately. 34. A virus for use as described in any of the above 28-32, in which the virus and further anticancer drugs are administered simultaneously. 35. Virus for use as described in any of the above 28-34, wherein the cancer is a solid tumor. 36. A product containing any of the viruses described in items 1 to 35 above in a sterile vial, ampoule, or syringe. 37. A method for treating cancer, comprising administering a therapeutically effective amount of any of the viruses described in 1 to 24 above or the pharmaceutical composition described in 25 above to a patient in need thereof. 38. The method according to 37, further comprising administering a therapeutically effective dose of an additional anticancer drug to a patient in need. 39. The method according to 38, wherein the additional anticancer agent is selected from the group consisting of agents that target immune co-inhibitory or immune co-stimulatory pathways, radiotherapy and / or chemotherapy, agents that target specific gene mutations occurring in tumors, agents intended to induce an immune response to one or more tumor antigens or neoantigens, cell products derived from T cells or NK cells, STING, cGAS, TLR or other agents intended to stimulate innate immune responses and / or inflammatory pathways, a second virus, optionally oncolytic viruses, and combinations thereof. 40. The method according to 39 above, wherein the agent targeting the immune co-inhibitory pathway is a CTLA-4 inhibitor, PD-1 inhibitor, PD-L1 inhibitor, LAG-3 inhibitor, TIM-3 inhibitor, VISTA inhibitor, CSF1R inhibitor, IDO inhibitor, KIR inhibitor, SLAMF7 inhibitor, CEACAM1 inhibitor, or CD47 inhibitor, and / or the agent targeting the immune co-stimulatory pathway is a GITR agonist, 4-1-BB agonist, OX40 agonist, CD40 agonist, or ICOS agonist. 41. The method according to any one of the above 38-40, wherein the further anticancer agent includes an antibody. 42. The method according to any one of the above 38-41, wherein the virus and further anticancer drugs are administered separately. 43. The method according to any one of the above 38-41, wherein the virus and further anticancer drugs are administered simultaneously. 44. The method described in any of the above 38-43, wherein the cancer is a solid tumor. 45. Use of any of the viruses described in 1 to 24 above in the manufacture of a pharmaceutical product for use in a method of treating cancer. 46. ​​The use described in 45 above, wherein the method includes administering further anticancer drugs. 【0089】 The following examples illustrate the present invention. [Examples] 【0090】 [Example 1] Construction of the virus of the present invention The virus species used to illustrate the present invention is HSV, specifically HSV1. The HSV1 strains used for illustration are identified through the comparison of more than 20 primary clinical isolates of HSV1 for their ability to kill a panel of human tumor-derived cell lines, and the selection of the virus strains with the highest ability to kill a wide range of these cells quickly and at low doses. The tumor cell lines used for this comparison include U87MG (glioma), HT29 (colorectal), LNCaP (prostate), MDA-MB-231 (breast), SK-MEL-28 (melanoma), Fadu (squamous cell carcinoma), MCF7 (breast), A549 (lung), MIAPACA-2 (pancreas), CAPAN-1 (pancreas), and HT1080 (fibrosarcoma). Specifically, each primary clinical isolate of HSV is titrated against each cell line used for screening at MOIs such as 1, 0.1, 0.01, and 0.001, and the degree of cell death at time points such as 24 and 48 hours is evaluated for each dose. The degree of cell death can be assessed, for example, by microscopic evaluation of the percentage of viable cells at each time point, or by metabolic assays, such as MTT assays. 【0091】 Next, the exemplary virus of the present invention is constructed by deleting ICP47 from the viral genome using homologous recombination with a plasmid containing a region adjacent to HSV1 nucleotides 145300-145582 (HSV1 nucleotides 145300-145582 are the deleted sequence; HSV1 strain 17 sequence Genbank file NC 001806.2), which encodes GFP in between. GFP-expressing viral plaques are selected, and then GFP-removed and non-GFP-expressing plaques are selected by homologous recombination using empty adjacent regions. This results in an ICP47-deleted virus expressed as an IE protein, since US11 is under the regulation of the ICP47 promoter in this case. Next, ICP34.5 is deleted using homologous recombination with a plasmid containing a region adjacent to HSV1 nucleotides 124953-125727 (HSV1 nucleotides 124953-125727 are the deletion sequence; HSV1 strain 17 sequence Genbank file NC 001806.2), in which GFP is encoded. Viral plaques expressing GFP are selected again, and then GFP is removed by homologous recombination with the same adjacent region (however, in between, there is an expression cassette containing codon-optimized versions of the mouse GM-CSF sequence and the GALV R- sequence, respectively, driven by the CMV IE promoter and RSV promoter, oriented back-to-back), and viral plaques that do not express GFP are selected again. This viral construction is performed using methods standard in the art. 【0092】 The structure of the obtained virus is shown in Figure 1. The mGM-CSF and GALV-R- sequences are shown in SEQ ID NOs. 2 and 8, respectively. The structure of the obtained virus was confirmed by PCR, GM-CSF expression was confirmed by ELISA, and GALV-R- expression was confirmed by infection of human HT1080 tumor cells and observation of syncytial plaques. 【0093】 The viruses are also constructed using similar procedures, either without insertion into ICP34.5 or with only the mouse GM-CSF or GALV-R- gene inserted. The structures of these viruses are also shown in Figure 1. 【0094】 For human use, hGM-CSF is used, and its codon-optimized version sequence is shown in Sequence ID No. 4. 【0095】 [Example 2] Expression of two immune-stimulating molecules from a virus that expresses a fusion-inducible protein. Similar to the viruses expressing GALV-R- and mGM-CSF described above, a virus expressing a CD40L version was constructed. Here, instead of using a plasmid containing an expression cassette with the ICP34.5 flanking region and GM-CSF and GALV-R- driven by the CMV and RSV promoters, a plasmid containing an expression cassette with the ICP34.5 flanking region and GM-CSF, GALV, and CD40L driven by the CMV, RSV, and SV40 promoters was used for recombination using a virus containing GFP inserted into ICP34.5, and plaques that did not express GFP were selected again. 【0096】 [Example 3] Effects of combining the expression of oncolytic virus-derived fusion-inducible proteins and immunostimulatory molecules in a mouse tumor model. The GALV R-protein causes cell-cell fusion in human cells but not in mouse cells. This is because the PiT-1 receptor necessary to cause cell fusion has a sequence in mice that does not allow cell fusion to occur. As a result, mouse tumor cells expressing human PiT-1 are first prepared using standard methods in the art. Human PiT-1 is cloned into a lentiviral vector that also contains a selectable marker gene. The vector is transfected into target CT26 mouse colorectal cancer tumor cells, and clones resistant to the selectable marker are selected to generate CT26 / PiT-1 cells. PiT-1 expression is confirmed by Western blotting in untransfected cells and cells transfected with a lentivirus expressing PiT-1, and by transfection of a plasmid expressing GALV-R- and confirmation that cell fusion occurs. 【0097】 The utility of the present invention is demonstrated by administering CT26 / PiT-1 cells to both sides of Balb / c mice and growing CT26 / PiT-1 tumors to a diameter of about 0.5 cm. 【0098】 Next, the following treatments are administered to groups of mice (5 mice per group) 3 times per week for 2 weeks, each on one side of each mouse: - 50 μl of physiological saline (1 group), - 50 μl of 10 5 pfu / ml, 10 6 pfu, or 10 7 pfu / ml of HSV (3 groups), - 50 μl of 10 5 pfu / ml, 10 6 pfu / ml, or 10 7 pfu / ml of HSV in which only mouse GM-CSF is inserted (3 groups), - 50 μl of 10 5 pfu / ml, 10 6 pfu / ml, or 10 7pfu / ml virus (group 3), or - 50 μl of 10 mice containing both GM-CSF and GALV-R- 5 pfu / ml, 10 6 pfu / ml, or 10 7 pfu / ml viruses (group 3). 【0099】 Next, the effect on tumor growth is observed for up to one month. Compared to other groups, superior tumor control and reduction are observed, including through improved dose-response curves, in both tumors injected with GM-CSF and GALV-R- expressing viruses and tumors that are not injected. 【0100】 [Example 4] The effect of combined expression of oncolytic virus-derived fusion-inducible proteins and immunostimulatory molecules on the therapeutic effect of immune checkpoint blockade in a mouse tumor model. The experiment in Example 3 above is repeated, but the mice are given additional medication every two weeks via the intraperitoneal route using either an antibody targeting mouse PD-1 (10 mg / kg; Bioxcell RMP-1-14 on the same day as viral administration) or an antibody targeting mouse CTLA-4 (10 mg / kg; Bioxcell 9H10 on the same day as viral administration). An additional group of mice that do not receive antibody treatment is added. More specifically, the mouse groups receive (1) physiological saline, (2) HSV without gene insertion, (3) HSV with GM-CSF and GALV-R- insertion as in Example 3, (4) PD-1 antibody, (5) CTLA-4 antibody, (6) HSV without gene insertion + PD-1 antibody, (7) HSV without gene insertion + CTLA-4 antibody, (8) HSV with GM-CSF and GALV-R- and PD-1 antibody, or (9) HSV with GM-CSF and GALV-R- and CTLA-4 antibody. Compared to other groups, superior tumor control and reduction were observed, including through improved dose-response curves, in both injected and uninjected tumors using GM-CSF and GALV-R- expressing viruses, along with anti-PD-1 or anti-CTLA-4 antibodies. 【0101】 [Example 5] Recovery of clinical isolates The virus species used to illustrate this invention is HSV, specifically HSV1. To illustrate this invention, 181 volunteers with recurrent cold sores were recruited. These volunteers were given sample collection kits (including Sigma Virovult collection tubes) to swab their sores when they appeared, and these samples were then sent to Replimune (Oxford, UK). Swabs were received from 72 volunteers between June 2015 and February 2016. BHK cells were infected using each swab sample. Of these, 36 live virus samples were seeded and grown on BHK cells before being collected. These samples are detailed in Table 1. 【0102】 [Table 1] JPEG0007875832000002.jpg23489JPEG0007875832000003.jpg23689JPEG0007875832000004.jpg18689 The symbols A, B, C, etc. refer to multiple wipes originating from the same volunteer. 【0103】 [Example 6] Identification of clinical isolates with improved antitumor efficacy The ability of primary clinical isolates of HSV1 to kill a panel of human tumor-derived cell lines was tested. The tumor cell lines used for this comparison were HT29 (colorectal), MDA-MB-231 (breast), SK-MEL-28 (melanoma), Fadu (squamous cell carcinoma), MCF7 (breast), A549 (lung), MIAPACA-2 (pancreas), and HT1080 (fibrosarcoma). Using the cell lines, the CPE levels achieved within a range of MOI and time after infection were tested for each primary clinical isolate. 【0104】 The experiments were conducted in parallel using 5 to 8 new virus strains simultaneously. The virus strains were seeded in duplicate at a certain MOI range (0.001 to 1), and the degree of CPE after crystal violet staining was evaluated 24 and 48 hours after infection. The virus strains most effective in killing tumor cell lines were scored, and the two or three most effective strains were identified from each screening of 5 to 8 strains and compared in parallel in further experiments to identify the top strain for further development. 【0105】 The initial screening demonstrated substantial variability in the ability of different strains to kill various tumor cell lines. Of the first 29 strains tested, eight were identified in the initial screening for further comparison. These were strains RH004A, RH015A, RH018A, RH021A, RH023A, RH31A, RH040A, and RH047A. 【0106】 For further comparison, eight strains were tested in parallel against a panel of tumor cell lines, and their relative ability to kill these tumor cell lines was evaluated after observation for crystal violet staining and CPE. Figure 3 shows representative time points and MOIs for each virus on each cell line, demonstrating the different abilities of the viruses to kill the observed target tumor cell lines. 【0107】 There is substantial variation among the strains, and it has been found that certain strains may be particularly effective in killing one cell line, while others are not necessarily particularly effective in killing other cell lines, further demonstrating the degree of variability in the ability of HSV clinical strains to kill different types of tumor cells. 【0108】 Figure 3 also shows which virus strains are best and second best at killing each of the cell lines, allowing for ranking of the virus strains in terms of their overall relative ability to kill a panel of cell lines as a whole. This analysis demonstrated that strains RH004A, RH015A, RH018A, RH031A, and RH040A were relatively more effective than the others, and these five strains were selected for potential further development as oncolytic agents. Of these top five strains, the relative ranking based on their ability to kill an entire panel of cell lines was RH018A > RH004A > RH031A > RH040A > RH015A. 【0109】 More specifically, in these experiments, tumor cell lines were seeded into multi-well tissue culture plates to approximately 80% confluence on the day of infection. Representative wells from each tumor cell line were trypsin-treated to determine the cell count in the well. These cell counts were used to determine the volume of each clinical isolate required to achieve MOIs of 1, 0.1, 0.01, and 0.001. Separate wells of tumor cell lines were infected with clinical isolates at these MOIs. All infections were performed in quadruple incubation. Two wells were incubated for 24 hours, and two wells for 48 hours, both at 37°C and 5% CO2. The cells were then fixed with glutaraldehyde and stained with crystal violet. The level of cell lysis was then evaluated by macroscopic observation, microscopy (cell counting), and photography. 【0110】 The RH018A strain was the first to rank among all strains tested and was compared to the "average" strain from the screening (i.e., not among the top 8, but also not among the least effective strains that failed to kill the panel of tumor cell lines). This comparison showed that the RH018A strain was approximately 10 times more effective than this average strain (RH065A) in killing tumor cell lines (i.e., about one-tenth of the RH018A strain was needed to kill an equal proportion of cells compared to the RH065A strain). This is shown in Figure 4. 【0111】 [Example 7] Modification of clinical isolates In this example, the clinical isolate selected in Example 6 was modified by deleting ICP34.5 from the viral genome via homologous recombination using a plasmid containing a region adjacent to the gene encoding ICP34.5 (nucleotides 143680-145300 and 145,582-147,083; HSV1 strain 17 sequence Genbank file NC 001806.2), which in between encodes GFP and GALV-R-fusion-inducible glycoprotein. The structure of this virus (virus 10) is shown in Figure 5. 【0112】 Additionally, we constructed an additional virus based on the RH018A strain, which was deleting both ICP34.5 and ICP47 (using adjacent regions containing nucleotides 123464-124953 and 125727-126781; HSV1 strain 17 sequence Genbank file NC 001806.2) (placing US11 under the regulation of the ICP47 promoter). To construct these viruses, we first selected GFP-expressing viral plaques in which GFP was expressed instead of ICP47. Next, we removed GFP by homologous recombination using an empty adjacent region, selecting plaques that did not express GFP. This resulted in an ICP47-deleting virus, in which US11 is expressed as an IE protein, here under the regulation of the ICP47 promoter. Next, ICP34.5 was deleted by homologous recombination using a plasmid containing a region adjacent to HSV1 (nucleotides 143680-145300 and 145,582-147,083; HSV1 strain 17 sequence Genbank file NC 001806.2), which encodes GFP. Viral plaques expressing GFP were selected again, and then GFP was removed by homologous recombination using the same adjacent region (where, in this case, an expression cassette containing the inserted gene is present). The constructed viruses are shown in Figures 1 and 5. These contain, back-to-back, codon-optimized versions of the mouse GM-CSF sequence and the GALV R- sequence, respectively, driven by the CMV IE promoter and the RSV promoter, and again, viral plaques that do not express GFP were selected. This virus construction was performed using methods standard in the art. 【0113】 The mGM-CSF and GALV-R- sequences are shown in Sequence ID No. 2 and 8, respectively. The structure of the obtained virus was confirmed by PCR, GM-CSF expression was confirmed by ELISA, and GALV-R- expression was confirmed by infection of human HT1080 tumor cells and observation of syncytial plaques. 【0114】 hGM-CSF was used for human use, and the sequence of its codon-optimized version is shown in SEQ ID NO: 4. The structure of this virus is shown in Figure 5. Expression of mouse or human GM-CSF from viruses 16, 17, and 19 is shown in Figure 6. 【0115】 [Example 8] The virus of the present invention, modified for oncolytic use and expressing a fusion-inducible glycoprotein, exhibits increased tumor cell death in vitro compared to viruses that do not express the fusion-inducible glycoprotein. Virus 10 (see Figure 5), based on the clinical strain RH018A lacking ICP34.5 and expressing GALVR- and GFP, was compared to a virus expressing only GFP in vitro (Virus 12). As shown in Figure 7, Virus 10 showed increased cell death in a panel of human tumor cell lines compared to Virus 12. 【0116】 [Example 9] The virus of the present invention, modified for use in oncolysis, exhibits increased tumor cell death compared to similarly modified viruses not of the present invention. Virus 17 (see Figure 5), based on the clinical strain RH018A lacking ICP34.5 and ICP47 and expressing GALVR- and GM-CSF, was compared in vitro with a known virus that also lacks ICP34.5 and ICP47 but expresses only GM-CSF, and is not derived from the present strain. As shown in Figure 8, virus 17 showed increased cell death in a panel of human tumor cell lines compared to the previous virus. 【0117】 [Example 10] The virus of the present invention, modified for oncolytic use, effectively treats mouse tumors in vivo. Virus 16 was tested in mice with A20 lymphoma tumors on both sides of the body. One million tumor cells were initially transplanted into both sides of Balb / c mice, and the tumors were allowed to grow to a diameter of 0.5–0.7 cm. Next, the tumor on the right side was treated with 5 × 10⁶ virus 16 in a vehicle (10 mice). 6PFU (10 mice) was injected three times (every other day), and the effect on tumor size was observed for a further 30 days. This demonstrated that both injected and uninjected tumors were effectively treated with virus 16 (see Figure 9). 【0118】 [Example 11] Effect of combining the expression of a fusion-inducible protein from the oncolytic virus and an immunostimulatory molecule in a rat tumor model. The GALV R- protein induces cell fusion in human cells but not in mouse cells. However, GALV R- induces fusion in rat cells. 【0119】 The usefulness of the present invention was further demonstrated by administering 9L cells to the side of Fischer 344 rats, which allowed 9L tumors to grow to a diameter of approximately 0.5 cm. 【0120】 Next, the following treatment was administered to groups of rats (10 rats per group) three times a week for three weeks, to only one side of each rat: - 50 μl vehicle, - 10 of 50 μl 7 pfu / ml of Virus 19 (expressing mGM-CSF but not GALV R-), - 10 of 50 μl 7 Virus 16 with a pfu / ml concentration (expressing both mouse GM-CSF and GALV-R-). 【0121】 Next, the effect on tumor growth was observed for a further 30 days. This demonstrated superior tumor control and reduction by the GALV-R-expressing virus in both injected and uninjected tumors, demonstrating improved systemic effects. This is shown in Figure 15. Figure 10 shows that the GALV-expressing virus (virus 15) also showed increased death of rat 9L cells in vitro compared to the non-GALV-expressing virus (virus 24). 【0122】 [Example 12] The virus of the present invention, modified for use in oncolysis, is synergistic with immune checkpoint blockade in mouse tumor models. Virus 16 was tested in mice with CT26 tumors on both sides of their bodies. One million tumor cells were initially transplanted into each side of Balb / c mice, and the tumors were allowed to grow to a diameter of 0.5–0.6 cm. 【0123】 Next, a group of 10 mice was treated as follows: - Vehicle (3 injections into the tumor on the right side, every other day) - 5 x 10 injections into the tumor on the right side every other day 6 PFU virus 16, - Anti-mouse PD1 monotherapy (10 mg / kg ip, every 3 days, BioXCell clone RMP1-14), - Anti-mouse CTLA-4 (3 mg / kg ip, every 3 days, BioXCell clone 9D9), - Anti-mouse PD1 along with virus 16, - Anti-mouse CTLA4 along with virus 16, - 1-Methyltryptophan (IDO inhibitor (5 mg / ml in drinking water)), - 1-methyltryptophan along with anti-mouse PD1, - Anti-mouse PD1 along with 1-methyltryptophan and virus 16. 【0124】 The effect on tumor size was observed for a further 30 days. Greater tumor reduction was demonstrated in animals treated with a combination of virus and checkpoint blockade compared to animals treated with a single treatment (see Figure 11). Increased tumor reduction was also demonstrated with both anti-PD1 and IDO inhibition along with virus 16 compared to anti-PD1 alone along with virus 16 (see Figure 11). 【0125】 Furthermore, increased activity of virus 16 in combination with immune checkpoint blockade was observed in A20 tumors (Figure 12). 【0126】 [Example 13] Effect of expressing the oncolytic virus-derived fusion-inducible protein of the present invention in a human xenotransplantation model in immunodeficient mice. The GALV R protein induces cell fusion in human cells but not in mouse cells. However, the effect of GALV expression on antitumor activity can be evaluated using human xenograft tumors grown in immunodeficient mice. 【0127】 Therefore, the usefulness of the present invention was further demonstrated by administering A549 human lung cancer cells to the side of nude mice and allowing the tumors to grow to a diameter of approximately 0.5 cm. 【0128】 Next, the following treatment was administered to groups of mice (10 mice per group) three times over a week to the tumor-containing side of each mouse: - 50 μl vehicle, - 10 of 50 μl 7 pfu / ml of virus 16 (expressing both mouse GM-CSF and GALV-R-), - 10 of 50 μl 6 pfu / ml virus 16, - 10 of 50 μl 5 pfu / ml virus 16, - 10 of 50 μl 7 pfu / ml of virus 19 (expressing only mouse GM-CSF), - 10 of 50 μl 6 pfu / ml virus 19, - 10 of 50 μl 5 PFU / ML Virus 19 【0129】 Next, the effect on tumor growth was observed for a further 30 days. This experiment demonstrated superior tumor control and reduction by the GALV-R-expressing virus in both tumor models (see Figure 14). 【0130】 [Example 14] Expression of two immune-stimulating molecules from a virus that expresses a fusion-inducible protein. Similar to the viruses expressing GALV-R- and mGM-CSF described above (virus 16), viruses expressing mouse versions of CD40L (virus 32), ICOSL (virus 36), OX40L (virus 35), 4-1BBL (virus 33), and GITRL (virus 34) were further constructed. Here, instead of using plasmids containing expression cassettes with the ICP34.5 flanking region and GM-CSF and GALV-R- driven by CMV and RSV promoters, plasmids containing the ICP34.5 flanking region and expression cassettes with GM-CSF, GALV, and additional proteins driven by CMV, RSV, and MMLV promoters, respectively, were used for recombination with viruses containing GM-CSF, GALV, and GFP inserted into ICP34.5. Plaques that did not express GFP were selected again. Accurate insertion was confirmed by PCR, and expression of additional inserted genes was confirmed by Western blotting and / or ELISA. These viruses are shown in Figure 5. Similarly, viruses expressing anti-mouse and anti-human CTLA-4 in addition to GALV and mGM-CSF were also constructed (see viruses 27 and 31 in Figure 5, and also Figure 13). Figure 16 shows the effects of viruses expressing anti-mouse CTLA-4 (virus 27), mCD40L (virus 32), m4-1BBL (virus 33), or mOX40L (virus 35) in vivo, in addition to mGM-CSF and GALVR-. This showed enhanced activity in A20 tumors compared to virus 16 (expressing mGM-CSF and GALVR-). In these experiments, tumors were induced bilaterally in mice, and the virus or vehicle was injected only into the tumor on the right side. The dose of virus used was 5 × 10⁻⁶. 4 pfu (50 μl 1 × 10 in each case) 6 The dose was pfu / ml and administered three times over a week. This dose level of virus is sub-therapeutic for tumors not injected with virus 16, thereby clearly demonstrating the benefit of delivering additional molecules encoded by viruses 27, 32, 33, and 35. 【0131】 Deposit Information The following HSV1 strain was deposited by Replimune Limited with ECACC, Culture Collections, Public Health England, at Porton Down SP4 0JG, Salisbury, UK, on ​​December 19, 2016, and was assigned a designated provisional accession number. RH004A - Provisional Commission Number 16121902 RH015A - Provisional Commission Number 16121903 RH018A - Provisional Commission Number 16121904 RH021A - Provisional Commission Number 16121905 RH023A - Provisional Commission Number 16121906 RH031A - Provisional Contract Number 16121907 RH040B - Provisional Commission Number 16121908 RH047A - Provisional Contract Number 16121909 【0132】 SEQUENCE LISTING <110> Replimune Limited <120> Modified Oncolytic Virus <130> PA23-307 <150> GB1600380.8 <151> 2016-01-08 <150> GB1600381.6 <151> 2016-01-08 <150> GB1600382.4 <151> 2016-01-08 <160> 45 <170> PatentIn version 3.5 <210> 1 <211> 426 <212> DNA <213> Mus musculus <400> 1 atgtggctgc agaatttact tttcctgggc attgtggtct acagcctctc agcacccacc 60 cgctcaccca tcactgtcac ccggccttgg aagcatgtag aggccatcaa agaagccctg 120 aacctcctgg atgacatgcc tgtcacattg aatgaagagg tagaagtcgt ctctaacgag 180 ttctccttca agaagctaac atgtgtgcag acccgcctga agatattcga gcagggtcta 240 cggggcaatt tcaccaaact caagggcgcc ttgaacatga cagccagcta ctaccagaca 300 tactgcccc caactccgga aacggactgt gaaacacaag ttaccaccta tgcggatttc 360 atagacagcc ttaaaacctt tctgactgat atcccctttg aatgcaaaaa accagtccaa 420 aatga 426 <210> 2 <211> 426 <212> DNA <213> Mus musculus <400> 2 atgtggctcc agaacctcct cttctcggt atcgtcgtgt attcactctc cgcacctact 60 cgctcaccta tcactgtcac cagaccctgg aagcacgtgg aggccatcaa ggaggctctg 120 aacctgctgg acgatatgcc agtgaccctg aacgaggagg tggaggtggt gagcaacgag 180 ttctccttta agaagctgac ctgcgtgcag acaaggctga agatcttcga gcagggcctg 240 agaggaaact ttaccaagct gaagggcgcc ctgaacatga ccgcttctta ctaccagaca 300 tactgccccc ctacccccga gacagactgt gagacacagg tgaccacata cgccgacttc 360 attgatagcc tgaaaacatt cctgaccgac attccatttg agtgtaagaa gcccgtccag 420 aagtaa 426 <210> 3 <211> 435 <212> DNA <213> Homo sapiens <400> 3 atgtggctgc agagcctgct gctcttgggc actgtggcct gcagcatctc tgcacccgcc 60 cgctcgccca gccccagcac gcagccctgg gagcatgtga atgccatcca ggaggcccgg 120 cgtctcctga acctgagtag agacactgct gctgagatga atgaaacagt agaagtcatc 180 tcagaaatgt ttgacctcca ggagccgacc tgcctacaga cccgcctgga gctgtacaag 240 cagggcctgc ggggcagcct caccaagctc aagggcccct tgaccatgat ggccagccac 300 tacaagcagc actgccctcc aaccccggaa acttcctgtg caacccagat tatcacctttt 360 gaaagtttca aagagaacct gaaggacttt ctgcttgtca tcccctttga ctgctgggag 420 ccagtccagg agtga 435 <210> 4 <211> 435 <212> DNA <213> Homo sapiens <400> 4 atgtggctgc agtccctgct gctgctgggc accgtcgcct gttctatttc cgcacccgca 60 aggtcaccaa gtccatctac tcagccttgg gagcacgtga acgcaatcca ggaggcacgg 120 cggctgctga acctgagccg ggacaccgcc gccgagatga acgagacagt ggaagtgatc 180 agcgagatgt tcgatctgca ggagcccacc tgcctgcaga caaggctgga gctgtacaag 240 cagggcctgc gcggctctct gaccaagctg aagggcccac tgacaatgat ggccagccac 300 tataagcagc actgcccccc taccccccgag acaagctgtg ccacccagat catcacattc 360 gagtccttta aggagaacct gaaggatttt ctgctggtca ttccatttga ttgttgggag 420 cccgtccagg agtaa 435 <210> 5 <211> 141 <212> PRT <213> Mus musculus <400> 5 Met Trp Leu Gln Asn Leu Leu Phe Leu Gly Ile Val Val Tyr Ser Leu 1 5 10 15 Ser Ala Pro Thr Arg Ser Pro Ile Thr Val Thr Arg Pro Trp Lys His 20 25 30 Val Glu Ala Ile Lys Glu Ala Leu Asn Leu Leu Asp Asp Met Pro Val 35 40 45 Thr Leu Asn Glu Glu Val Glu Val Val Ser Asn Glu Phe Ser Phe Lys 50 55 60 Lys Leu Thr Cys Val Gln Thr Arg Leu Lys Ile Phe Glu Gln Gly Leu 65 70 75 80 Arg Gly Asn Phe Thr Lys Leu Lys Gly Ala Leu Asn Met Thr Ala Ser 85 90 95 Tyr Tyr Gln Thr Tyr Cys Pro Pro Thr Pro Glu Thr Asp Cys Glu Thr 100 105 110 Gln Val Thr Thr Tyr Ala Asp Phe Ile Asp Ser Leu Lys Thr Phe Leu 115 120 125 Thr Asp Ile Pro Phe Glu Cys Lys Lys Pro Val Gln Lys 130 135 140 <210> 6 <211> 144 <212> PRT <213> Homo sapiens <400> 6 Met Trp Leu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala Cys Ser Ile 1 5 10 15 Ser Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His 20 25 30 Val Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp 35 40 45 Thr Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu Met Phe 50 55 60 Asp Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys 65 70 75 80 Gln Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met 85 90 95 Met Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser 100 105 110 Cys Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys 115 120 125 Asp Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu 130 135 140 <210> 7 <211> 2010 <212> DNA <213> Gibbon leukemia virus <400> 7 atggtattgc tgcctgggtc catgcttctc acctcaaacc tgcaccacct tcggcaccag 60 atgagtcctg ggagctggaa aagactgatc atcctcttaa gctgcgtatt cggcggcggc 120 gggacgagtc tgcaaaataa gaacccccac cagcccatga ccctcacttg gcaggtactg 180 tcccaaactg gagacgttgt ctgggataca aaggcagtcc agcccccttg gacttggtgg 240 cccacactta aacctgatgt atgtgccttg gcggctagtc ttgagtcctg ggatatcccg 300 ggaaccgatg tctcgtcctc taaacgagtc agacctccgg actcagacta tactgccgct 360 tataagcaaa tcacctgggg agccataggg tgcagctacc ctcgggctag gactagaatg 420 gcaagctcta ccttctacgt atgtccccgg gatggccgga ccctttcaga agctagaagg 480 tgcggggggc tagaatccct atactgtaaa gaatgggatt gtgagaccac ggggaccggt 540 tattggctat ctaaatcctc aaaagacctc ataactgtaa aatgggacca aaatagcgaa 600 tggactcaaa aatttcaaca gtgtcaccag accggctggt gtaaccccct taaaatagat 660 ttcacagaca aagaaaatt atccaaggac tggataacgg gaaaaacctg gggattaaga 720 ttctatgtgt ctggacatcc aggcgtacag ttcaccattc gcttaaaaaat caccaacatg 780 ccagctgtgg footggtcc tgacctcgtc cttgtggaac areacctcc tagaacgtcc 840 ctcgctctcc cacctctct tcccccaagg gaagcgccac cgccatctct ccccgactct 900 aactccacag ccctggcgac tagtgcacaa actcccacgg tgagaaaaac aattgttacc 960 ctaaacactc cgcctcccac cacaggcgac agactttttg atcttgtgca gggggccttc 1020 ctaaccttaa atgctaccaa cccaggggcc actgagtctt gctggctttg tttggccatg 1080 ggcccccctt attatgaagc aatagcctca tcaggagagg tcgcctactc caccgacctt 1140 gaccggtgcc gctgggggac ccaaggaaag ctcaccctca ctgaggtctc aggacacggg 1200 ttgtgcatag gaaaggtgcc ctttacccat cagcatctct gcaatcagac cctatccatc 1260 aattcctccg gagaccatca gtatctgctc ccctccaacc atagctggtg ggcttgcagc 1320 actggcctca ccccttgcct ctccacctca gtttttaatc agactagaga tttctgtatc 1380 caggtccagc tgattcctcg catctattac tatcctgaag aagttttgtt acaggcctat 1440 gacaattctc accccaggac taaaagagag gctgtctcac ttaccctagc tgttttactg 1500 gggttgggaa tcacggcggg aataggtact ggttcaactg ccttaattaa aggacctata 1560 gacctccagc aaggcctgac aagcctccag atcgccatag atgctgacct ccgggccctc 1620 caagactcag tcagcaagtt agaggactca ctgacttccc tgtccgaggt agtgctccaa 1680 aataggagag gccttgactt gctgtttcta aaagaaggtg gcctctgtgc ggccctaaag 1740 gaagagtgct gtttttacat agaccactca ggtgcagtac gggactccat gaaaaaactc 1800 aaagaaaaac tggataaaag acagttagag cgccagaaaa gccaaaactg gtatgaagga 1860 tggttcaata actccccttg gttcactacc ctgctatcaa ccatcgctgg gcccctatta 1920 ctcctccttc tgttgctcat cctcgggcca tgcatcatca ataagttagt tcaattcatc 1980 aatgatagga taagtgcagt taaaatttaa 2010 <210> 8 <211> 2013 <212> DNA <213> Gibbon leukemia virus <400> 8 accatggtcc tgctgcctgg gtctatgctg ctgacttcta acctgcacca cctgcgacac 60 cagatgtctc ccggctcatg gaaacggctg atcatcctgc tgagctgcgt gttcggagga 120 ggaggcacct ccctgcagaa caagaatcct caccagccaa tgaccctgac atggcaggtg 180 ctgtcccaga caggcgacgt ggtgtgggat accaaggcag tgcagccacc ttggacatgg 240 tggcccaccc tgaagcctga cgtgtgcgcc ctggccgcct ccctggagtc ttgggacatc 300 cccggcacag acgtgagcag cagcaagagg gtgagaccac ccgactctga ttatacagcc 360 gcctacaagc agatcacctg gggcgccatc gggcttagct atcctcgggc ccgcacaagg 420 atggccagct ccacctttta cgtgtgccca cgcgacggaa ggaccctgtc tgaggcaagg 480 agatgtggcg gcctggagag cctgtattgc aaggagtggg attgtgagac cacaggcaca 540 ggctactggc tgtctaagtc tagcaaggac ctgatcaccg tgaagtggga tcaagaacagc 600 gagtggacac agaagttcca gcagtgccac cagaccggct ggtgtaatcc cctgaagaatc 660 gactttacag ataagggcaa gctgtccaag gactggatca ccggcaagac atggggcctg 720 agattctacg tgtctggcca ccctggcgtg cagtttacaa tccggctgaa gatcaccaac 780 atgccagcag tggcagtggg accagacctg gtgctggtgg agcagggacc tccacgcacc 840 tccctggccc tgccccctcc actgccccct agggaggccc caccccctag cctgcccgat 900 tctaacagca cagccctggc cacctccgcc cagaccccta cagtgcgcaa gaccatcgtg 960 acactgaata ccccaccccc taccacaggc gacaggctgt tcgatctggt gcagggcgcc 1020 tttctgacac tgaacgccac caatcctggc gcaaccgaga gctgctggct gtgcctggct 1080 atgggcccac cctactatga ggcaatcgcc tcctctggag aggtggcata ttccacagac 1140 ctggatagat gcagatgggg cacccagggc aagctgaccc tgacagaggt gtctggccac 1200 ggcctgtgca tcggcaaggt gccattcaca caccagcacc tgtgcaacca gaccctgagc 1260 atcaatagct ccggcgacca ccagtacctg ctgccaagca accactcctg gtgggcatgc 1320 tccacaggac tgaccccatg tctgagcacc agcgtgttca accagaccag agacttttgt 1380 atccaggtgc agctgatccc tcggatctac tattacccag aggaggtgct gctgcaggcc 1440 tatgataatt cccacccaag aacaaagagg gaggccgtgt ctctgaccct ggccgtgctg 1500 ctgggactgg gaatcacagc aggaatcggc acaggcagca ccgccctgat caagggacca 1560 atcgacctgc agcagggact gacctccctg cagatcgcca tcgacgccga tctgagagcc 1620 ctgcaggaca gcgtgtccaa gctggaggat tctctgacct ctctgagcga ggtggtgctg 1680 cagaacagga ggggcctgga cctgctgttc ctgaaggagg gaggactgtg cgccgccctg 1740 aaggaggagt gctgttttta tatcgaccac tctggcgccg tgcgggatag catgaagaag 1800 ctgaaggaga agctggataa gcgccagctg gagaggcaga agagccagaa ttggtacgag 1860 ggctggttca acaattcccc ctggtttacc acactgctgt ctaccatcgc aggacctctg 1920 ttattactgc tgctgctgct gatcctgggc ccatgtatca tcaacaagct ggtgcagttt 1980 atcaacgacc gaatctccgc agtgaaaatc taa 2013 <210> 9 <211> 669 <212> PRT <213> Gibbon leukemia virus <400> 9 Met Val Leu Leu Pro Gly Ser Met Leu Leu Thr Ser Asn Leu His His 1 5 10 15 Leu Arg His Gln Met Ser Pro Gly Ser Trp Lys Arg Leu Ile Ile Leu 20 25 30 Leu Ser Cys Val Phe Gly Gly Gly Gly Thr Ser Leu Gln Asn Lys Asn 35 40 45 Pro His Gln Pro Met Thr Leu Thr Trp Gln Val Leu Ser Gln Thr Gly 50 55 60 Asp Val Val Trp Asp Thr Lys Ala Val Gln Pro Pro Trp Thr Trp Trp 65 70 75 80 Pro Thr Leu Lys Pro Asp Val Cys Ala Leu Ala Ala Ser Leu Glu Ser 85 90 95 Trp Asp Ile Pro Gly Thr Asp Val Ser Ser Ser Lys Arg Val Arg Pro 100 105 110 Pro Asp Ser Asp Tyr Thr Ala Ala Tyr Lys Gln Ile Thr Trp Gly Ala 115 120 125 Ile Gly Cys Ser Tyr Pro Arg Ala Arg Thr Arg Met Ala Ser Ser Thr 130 135 140 Phe Tyr Val Cys Pro Arg Asp Gly Arg Thr Leu Ser Glu Ala Arg Arg 145 150 155 160 Cys Gly Gly Leu Glu Ser Leu Tyr Cys Lys Glu Trp Asp Cys Glu Thr 165 170 175 Thr Gly Thr Gly Tyr Trp Leu Ser Lys Ser Ser Lys Asp Leu Ile Thr 180 185 190 Val Lys Trp Asp Gln Asn Ser Glu Trp Thr Gln Lys Phe Gln Gln Cys 195 200 205 His Gln Thr Gly Trp Cys Asn Pro Leu Lys Ile Asp Phe Thr Asp Lys 210 215 220 Gly Lys Leu Ser Lys Asp Trp Ile Thr Gly Lys Thr Trp Gly Leu Arg 225 230 235 240 Phe Tyr Val Ser Gly His Pro Gly Val Gln Phe Thr Ile Arg Leu Lys 245 250 255 Ile Thr Asn Met Pro Ala Val Ala Val Gly Pro Asp Leu Val Leu Val 260 265 270 Glu Gln Gly Pro Pro Arg Thr Ser Leu Ala Leu Pro Pro Pro Leu Pro 275 280 285 Pro Arg Glu Ala Pro Pro Pro Ser Leu Pro Asp Ser Asn Ser Thr Ala 290 295 300 Leu Ala Thr Ser Ala Gln Thr Pro Thr Val Arg Lys Thr Ile Val Thr 305 310 315 320 Leu Asn Thr Pro Pro Pro Thr Thr Gly Asp Arg Leu Phe Asp Leu Val 325 330 335 Gln Gly Ala Phe Leu Thr Leu Asn Ala Thr Asn Pro Gly Ala Thr Glu 340 345 350 Ser Cys Trp Leu Cys Leu Ala Met Gly Pro Pro Tyr Tyr Glu Ala Ile 355 360 365 Ala Ser Ser Gly Glu Val Ala Tyr Ser Thr Asp Leu Asp Arg Cys Arg 370 375 380 Trp Gly Thr Gln Gly Lys Leu Thr Leu Thr Glu Val Ser Gly His Gly 385 390 395 400 Leu Cys Ile Gly Lys Val Pro Phe Thr His Gln His Leu Cys Asn Gln 405 410 415 Thr Leu Ser Ile Asn Ser Ser Gly Asp His Gln Tyr Leu Leu Pro Ser 420 425 430 Asn His Ser Trp Trp Ala Cys Ser Thr Gly Leu Thr Pro Cys Leu Ser 435 440 445 Thr Ser Val Phe Asn Gln Thr Arg Asp Phe Cys Ile Gln Val Gln Leu 450 455 460 Ile Pro Arg Ile Tyr Tyr Tyr Pro Glu Glu Val Leu Leu Gln Ala Tyr 465 470 475 480 Asp Asn Ser His Pro Arg Thr Lys Arg Glu Ala Val Ser Leu Thr Leu 485 490 495 Ala Val Leu Leu Gly Leu Gly Ile Thr Ala Gly Ile Gly Thr Gly Ser 500 505 510 Thr Ala Leu Ile Lys Gly Pro Ile Asp Leu Gln Gln Gly Leu Thr Ser 515 520 525 Leu Gln Ile Ala Ile Asp Ala Asp Leu Arg Ala Leu Gln Asp Ser Val 530 535 540 Ser Lys Leu Glu Asp Ser Leu Thr Ser Leu Ser Glu Val Val Leu Gln 545 550 555 560 Asn Arg Arg Gly Leu Asp Leu Leu Phe Leu Lys Glu Gly Gly Leu Cys 565 570 575 Ala Ala Leu Lys Glu Glu Cys Cys Phe Tyr Ile Asp His Ser Gly Ala 580 585 590 Val Arg Asp Ser Met Lys Lys Leu Lys Glu Lys Leu Asp Lys Arg Gln 595 600 605 Leu Glu Arg Gln Lys Ser Gln Asn Trp Tyr Glu Gly Trp Phe Asn Asn 610 615 620 Ser Pro Trp Phe Thr Thr Leu Leu Ser Thr Ile Ala Gly Pro Leu Leu 625 630 635 640 Leu Leu Leu Leu Leu Leu Ile Leu Gly Pro Cys Ile Ile Asn Lys Leu 645 650 655 Val Gln Phe Ile Asn Asp Arg Ile Ser Ala Val Lys Ile 660 665 <210> 10 <211> 759 <212> DNA <213> Artificial Sequence <220> <223> Homo sapiens <400> 10 atgatcgaga cctacaatca gacaagccca cggtccgccg caaccggact gcctatcagc 60 atgaagatct tcatgtacct gctgaccgtg tttctgatca cacagatgat cggctccgcc 120 ctgttcgccg tgtatctgca caggagactg gacaagatcg aggatgagcg caatctgcac 180 gaggacttcg tgtttatgaa gaccatccag cggtgcaaca caggcgagag gagcctgtct 240 ctgctgaatt gtgaggagat caagtcccag ttcgagggct ttgtgaagga tatcatgctg 300 aacaaggagg agacaaagaa ggacgaggat ccacagatcg cagcacacgt ggtgtccgag 360 gcaaactcta atgccgccag cgtgctgcag tgggccaaga agggctacta taccatgaag 420 tctaacctgg tgacactgga gaatggcaag cagctgaccg tgaagaggca gggcctgtac 480 tatatctatg cccaggtgac attctgctct aacagagagg caagctccca ggcacccttc 540 atcgtgggac tgtggctgaa gccctctagc ggcagcgaga ggatcctgct gaaggccgcc 600 aatacccact cctctagcca gctgtgcgag cagcagtcca tccacctggg aggcgtgttc 660 gagctgcagc ctggagccag cgtgttcgtg aacgtgacag acccatctca ggtgagccac 720 ggcaccggct tcacaagctt tggcctgctg aagctgtga 759 <210> 11 <211> 252 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens <400> 11 Met Ile Glu Thr Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly 1 5 10 15 Leu Pro Ile Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe Leu 20 25 30 Ile Thr Gln Met Ile Gly Ser Ala Leu Phe Ala Val Tyr Leu His Arg 35 40 45 Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp Phe Val 50 55 60 Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser Leu Ser 65 70 75 80 Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe Val Lys 85 90 95 Asp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Asp Glu Asp Pro Gln 100 105 110 Ile Ala Ala His Val Val Ser Glu Ala Asn Ser Asn Ala Ala Ser Val 115 120 125 Leu Gln Trp Ala Lys Lys Gly Tyr Tyr Thr Met Lys Ser Asn Leu Val 130 135 140 Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr 145 150 155 160 Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser 165 170 175 Gln Ala Pro Phe Ile Val Gly Leu Trp Leu Lys Pro Ser Ser Gly Ser 180 185 190 Glu Arg Ile Leu Leu Lys Ala Ala Asn Thr His Ser Ser Ser Gln Leu 195 200 205 Cys Glu Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro 210 215 220 Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His 225 230 235 240 Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu 245 250 <210> 12 <211> 1416 <212> DNA <213> Homo sapiens <400> 12 atgctgccct ttctgagcat gctggtgctg ctggtgcagc ctctgggaaa cctgggagcc 60 gagatgaaga gcctgtccca gagatctgtg cctaacacct gcacactggt catgtgcagc 120 cccaccgaga atggactgcc tggaagggac ggaagggatg gaagggaggg ccctcggggc 180 gagaagggcg acccaggact gcctggacca atgggactga gcggactgca gggaccaaca 240 ggacctgtgg gaccaaaggg agagaacgga tccgccggag agccaggccc taagggcgag 300 aggggcctgt ctggcccccc tggcctgcca ggcatcccag gccccgccgg caaggagggc 360 ccatccggca agcagggcaa tatcggcccc cagggcaagc ctggcccaaa gggcgaggca 420 ggaccaaagg gagaagtggg agcacctggc atgcagggat ccaccggagc aaagggatct acaggaccaa agggcgagcg cggcgcccca ggcgtgcagg gcgcccccgg caatgcagga gcagcaggac cagcaggacc tgcaggccca cagggcgccc ctggctctag gggcccaccc 600 ggcctgaagg gcgacagggg agtgcctggc gataggggca tcaaggaga gagcggactg 660 ccagattccg ccgccctgag gcagcagatg gaggccctga agggcaagct gcagaggctg gaggtggcct tctcccacta ccagaaggcc gccctgtttc cagacggcca caggagactg 780 gacaagatcg aggatgagcg caacctgcac gaggatttcg tgtttatgaa gaccatccag agatgcaaca caggcgagcg gtctctgagc ctgctgaatt gtgaggagat caagtctcag ttcgagggct ttgtgaagga catcatgctg aacaaggagg aacaagga ggagaatagc ttcgatgc agaagggcga tcagaatccc cagatcgcag cacacgtgat cagcgaggca agctccaaga ccacatccgt gctgcagtgg gccgagaagg gctactatac catgtccaac 1080 aatctggtga cactggagaa cggcaagcag ctgaccgtga agagacaggg cctgtactat 1140 atctatgccc aggtgacatt ctgctctaat cggggaggcct ctagccaggc cccttttatc 1200 gcctctctgt gcctgaagag cccaggcaga ttcgagcgga tcctgctgag ggccgccaac 1260 acccactcct ctgccaagcc atgcggacag cagagcatcc acctgggagg cgtgttcgag 1320 ctgcagccag gagcctccgt gtttgtgaat gtgacagacc catcccaggt gtctcacgga 1380 accggcttca catcctttgg cctgctgaag ctgtga 1416 <210> 13 <211> 471 <212> PRT <213> Homo sapiens <400> 13 Put Leu Pro Phe Leu Ser Put Leu Val Leu Leu Val Gln Pro Leu Gly 1 5 10 15 Donkey Leu Gly Ala Glu Met Lys Ser Leu Ser Gln Arg Ser Val Pro Donkey 20 25 30 Thr Cys Thr Leu Val Met Cys Ser Pro Thr Glu Asn Gly Leu Pro Gly 35 40 45 Arg Asp Gly Arg Asp Gly Arg Glu Gly Pro Arg Gly Glu Lys Gly Asp 50 55 60 Pro Gly Leu Pro Gly Pro Met Gly Leu Ser Gly Leu Gln Gly Pro Thr 65 70 75 80 Gly Pro Val Gly Pro Lys Gly Glu Asn Gly Ser Ala Gly Glu Pro Gly 85 90 95 Pro Lys Gly Glu Arg Gly Leu Ser Gly Pro Pro Gly Leu Pro Gly Ile 100 105 110 Pro Gly Pro Ala Gly Lys Glu Gly Pro Ser Gly Lys Gln Gly Asn Ile 115 120 125 Gly Pro Gln Gly Lys Pro Gly Pro Lys Gly Glu Ala Gly Pro Lys Gly 130 135 140 Glu Val Gly Ala Pro Gly Met Gln Gly Ser Thr Gly Ala Lys Gly Ser 145 150 155 160 Thr Gly Pro Lys Gly Glu Arg Gly Ala Pro Gly Val Gln Gly Ala Pro 165 170 175 Gly Asn Ala Gly Ala Ala Gly Pro Ala Gly Pro Ala Gly Pro Gln Gly 180 185 190 Ala Pro Gly Ser Arg Gly Pro Pro Gly Leu Lys Gly Asp Arg Gly Val 195 200 205 Pro Gly Asp Arg Gly Ile Lys Gly Glu Ser Gly Leu Pro Asp Ser Ala 210 215 220 Ala Leu Arg Gln Gln Met Glu Ala Leu Lys Gly Lys Leu Gln Arg Leu 225 230 235 240 Glu Val Ala Phe Ser His Tyr Gln Lys Ala Ala Leu Phe Pro Asp Gly 245 250 255 His Arg Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp 260 265 270 Phe Val Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser 275 280 285 Leu Ser Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe 290 295 300 Val Lys Asp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser 305 310 315 320 Phe Glu Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val 325 330 335 Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu 340 345 350 Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly 355 360 365 Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln 370 375 380 Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile 385 390 395 400 Only Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu 405 410 415 Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser 420 425 430 Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe 435 440 445 Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr 450 455 460 Ser Phe Gly Leu Leu Lys Leu 465,470 <210> 14 <211> 1412 <212> DNA <213> Muscles <400> 14 atgctgccct tcctgagcat gctggtgctg ctggtgcagc ctctgggcaa tctgggcgcc 60 gagatgaagt ccctgtctca gaggagcgtg ccaacacct gcacactggt catgtgctt 120 ccaaccgaga atggactgcc aggaagggac ggaagagatg gaaggggg accaagggg 180 gagaagggcg accctggact gcctggacca atgggactgt ccggactgca gggaccaaca 240 ggccctgtgg gaccaaaggg agagaatgga agcgccggag agccaggacc taagggagag 300 aggggcctgt ccggcccccc tggcctgcct ggcatcccag gccccgccgg caaggagggc 360 ccttctggca agcagggcaa catcggacca cagggcaagc ctggaccaaa gggagaggca 420 ggaccaaagg gagaagtggg agcaccccggc atgcagggca gcaccggagc aaagggatcc 480 accggcccta agggagagag aggagcacct ggagtgcagg gcgccccagg caatgcagga 540 gcagcaggac cagcaggacc tgcaggccca cagggcgccc caggcagccg gggcccacccc 600 ggcctgaagg gcgacagggg agtgccaggc gataggggca tcaagggaga gtccggactg 660 ccagactctg ccgccctgag gcagcagatg gaggccctga agggcaagct gcagaggctg 720 gaggtggcct tctccacta ccagaaggcc gccctgtttc cagaggaca caggagactg 780 gataaggtgg aggaggaggt gaacctgcac gaggatttcg tgttcatcaa gaagctgaag 840 aggtgcaaca agggcgaggg cagcctgtcc ctgctgaatt gtgaggagat gcggcgccag 900 ttcgaggacc tggtgaagga tatcaccctg aacaaggagg agaagaagga gaattcttttt 960 gagatgcaga ggggcgacga ggatcctcag atcgcagcac acgtggtgtc cgaggcaaac 1020 tctaatgccg ccagcgtgct gcagtgggcc aagaagggct actataccat gaagtctaac 1080 ctggtcatgc tggagaatgg caagcagctg acagtgaaga gagagggcct gtactacgtg 1140 tacacccagg tgacattctg cagcaacaga gagcccagct cccagcggcc tttatcgtg 1200 ggcctgtggc tgaagccctc tatcggaagc gagaggatcc tgctgaaggc agccaatacc 1260 cactctagct cccagctgtg cgagcagcag tccgtgcacc tgggaggcgt gttcgagctg 1320 caggcaggag caagcgtgtt cgtgaacgga cagaggccag ccaggtcatc cacagagtgg 1380 gcttctctag ctttggcctg ctgaagctgt ga 1412 <210> 15 <211> 470 <212> PRT <213> Mus musculus <400> 15 Met Leu Pro Phe Leu Ser Met Leu Val Leu Leu Val Gln Pro Leu Gly 1 5 10 15 Asn Leu Gly Ala Glu Met Lys Ser Leu Ser Gln Arg Ser Val Pro Asn 20 25 30 Thr Cys Thr Leu Val Met Cys Ser Pro Thr Glu Asn Gly Leu Pro Gly 35 40 45 Arg Asp Gly Arg Asp Gly Arg Glu Gly Pro Arg Gly Glu Lys Gly Asp 50 55 60 Pro Gly Leu Pro Gly Pro Met Gly Leu Ser Gly Leu Gln Gly Pro Thr 65 70 75 80 Gly Pro Val Gly Pro Lys Gly Glu Asn Gly Ser Ala Gly Glu Pro Gly 85 90 95 Pro Lys Gly Glu Arg Gly Leu Ser Gly Pro Pro Gly Leu Pro Gly Ile 100 105 110 Pro Gly Pro Ala Gly Lys Glu Gly Pro Ser Gly Lys Gln Gly Asn Ile 115 120 125 Gly Pro Gln Gly Lys Pro Gly Pro Lys Gly Glu Ala Gly Pro Lys Gly 130 135 140 Glu Val Gly Ala Pro Gly Met Gln Gly Ser Thr Gly Ala Lys Gly Ser 145 150 155 160 Thr Gly Pro Lys Gly Glu Arg Gly Ala Pro Gly Val Gln Gly Ala Pro 165 170 175 Gly Asn Ala Gly Ala Ala Gly Pro Ala Gly Pro Ala Gly Pro Gln Gly 180 185 190 Ala Pro Gly Ser Arg Gly Pro Pro Gly Leu Lys Gly Asp Arg Gly Val 195 200 205 Pro Gly Asp Arg Gly Ile Lys Gly Glu Ser Gly Leu Pro Asp Ser Ala 210 215 220 Ala Leu Arg Gln Gln Met Glu Ala Leu Lys Gly Lys Leu Gln Arg Leu 225 230 235 240 Glu Val Ala Phe Ser His Tyr Gln Lys Ala Ala Leu Phe Pro Asp Gly 245 250 255 His Arg Arg Leu Asp Lys Val Glu Glu Glu Val Asn Leu His Glu Asp 260 265 270 Phe Val Phe Ile Lys Lys Leu Lys Arg Cys Asn Lys Gly Glu Gly Ser 275 280 285 Leu Ser Leu Leu Asn Cys Glu Glu Met Arg Arg Gln Phe Glu Asp Leu 290 295 300 Val Lys Asp Ile Thr Leu Asn Lys Glu Glu Lys Lys Glu Asn Ser Phe 305 310 315 320 Glu Met Gln Arg Gly Asp Glu Asp Pro Gln Ile Ala Ala His Val Val 325 330 335 Ser Glu Ala Asn Ser Asn Ala Ala Ser Val Leu Gln Trp Ala Lys Lys 340 345 350 Gly Tyr Tyr Thr Met Lys Ser Asn Leu Val Met Leu Glu Asn Gly Lys 355 360 365 Gln Leu Thr Val Lys Arg Glu Gly Leu Tyr Tyr Val Tyr Thr Gln Val 370 375 380 Thr Phe Cys Ser Asn Arg Glu Pro Ser Ser Gln Arg Pro Phe Ile Val 385 390 395 400 Gly Leu Trp Leu Lys Pro Ser Ile Gly Ser Glu Arg Ile Leu Leu Lys 405 410 415 Ala Ala Asn Thr His Ser Ser Ser Gln Leu Cys Glu Gln Gln Ser Val 420 425 430 His Leu Gly Gly Val Phe Glu Leu Gln Ala Gly Ala Ser Val Phe Val 435 440 445 Asn Val Thr Glu Ala Ser Gln Val Ile His Arg Val Gly Phe Ser Ser 450 455 460 Phe Gly Leu Leu Lys Leu 465 470 <210> 16 <211> 786 <212> DNA <213> Homo sapiens <400> 16 atgatcgaaa catacaacca aacttctccc cgatctgcgg ccactggact gcccatcagc 60 atgaaaattt tttgtattt acttactgtt tttcttatca cccagatgat tgggtcagca 120 cttttgctg tgtatcttca tagaaggttg gacaagatag aagatgaaag gaatcttcat 180 gaagattttg tattcatgaa aacgatacag agatgcaaca caggagaaag atccttatcc 240 ttactgaact gtgaggagat taaaagccag tttgaaggct ttgtgaagga tataatgtta 300 aacaaagagg agacgaagaa agaaaacagc tttgaaatgc aaaaaggtga tcagaatcct 360 caaattgcgg cacatgtcat aagtgaggcc agcagtaaaa caacatctgt gttacagtgg 420 gctgaaaaag gatactacac catgagcaac aacttggtaa ccctggaaaa tgggaaacag 480 ctgaccgtta aaagacaagg actctattat atctatgccc aagtcacctt ctgttccaat 540 cgggaagctt cgagtcaagc tccatttata gccagcctct gcctaaagtc ccccggtaga 600 ttcgagagaa tcttactcag agctgcaaat acccacagtt ccgccaaacc ttgcgggcaa 660 caatccattc acttgggagg agtatttgaa ttgcaaccag gtgcttcggt gttgtcaat 720 gtgactgatc caagccaagt gagccatggc actggcttca cgtcctttgg cttactcaaa 780 ctctga 786 <210> 17 <211> 261 <212> PRT <213> Homo sapiens <400> 17 Met Ile Glu Thr Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly 1 5 10 15 Leu Pro Ile Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe Leu 20 25 30 Ile Thr Gln Met Ile Gly Ser Ala Leu Phe Ala Val Tyr Leu His Arg 35 40 45 Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp Phe Val 50 55 60 Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser Leu Ser 65 70 75 80 Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe Val Lys 85 90 95 Asp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser Phe Glu 100 105 110 Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val Ile Ser 115 120 125 Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly 130 135 140 Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln 145 150 155 160 Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr 165 170 175 Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser 180 185 190 Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala 195 200 205 Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His 210 215 220 Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn 225 230 235 240 Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe 245 250 255 Gly Leu Leu Lys Leu 260 <210> 18 <211> 783 <212> DNA <213> Mus musculus <400> 18 atgatagaaa catacagcca accttccccc agatccgtgg caactggact tccagcgagc 60 atgaagattt ttatgtattt acttactgtt ttccttatca cccaatgat tggatctgtg 120 ctttttgctg tgtatcttca tagagattg gataagtcg agaggaagt aaaccttcat 180 gagattttg tattcataa aaagctaag agatgcaca aaggagaagg atctttatcc 240 ttgctgaact gtgaggagat gagaaggcaa tttgagacc ttgtcagga tataacgtta 300 aaaaagaag agaaaaaaaacagcttt gaatgcaa gaggtgatga ggatcctcaa 360 attgcagcac acgttgtaag cgaagccaac agtaatgcag catccgttct acagtgggcc 420 aagaaaggat attackaccat gaaaagcaac ttggtaatgc ttgaaaatgg gaacagctg 480 acggttaaaa gagaggact ctattgtc tacactcag tcaccttg ctctaatcgg 540 gagccttcga gtcaaccccc attcatcgtc ggcctctggc tgaagcccag cagtggatct 600 gagagaatct tactcaggc ggcaatacc cacagttcct cccagctttg cgagcagcag 660 tctgttcact tgggcggagt gtttgaatta caagctggtg cttctgtgtt tgtcaacgtg 720 actgaagcaa gccaagtgat ccacagtt ggctctcat cttttggctt actcaactc 780 made 783 <210> 19 <211> 260 <212> PRT <213> Muscles <400> 19 With Glu Thr Tyr Ser Gln Pro Ser Pro Arg Ser Val Ala Thr Gly 1 5 10 15 Leu Pro Ala Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe Leu 20 25 30 Ile Thr Gln Met Ile Gly Ser Val Leu Phe Ala Val Tyr Leu His Arg 35 40 45 Arg Leu Asp Lys Val Glu Glu Glu Val Asn Leu His Glu Asp Phe Val 50 55 60 Phy Ile Lys Leu Lys Arg Cys Asn Lys Gly Glu Gly Ser Leu Ser 65 70 75 80 Leu Leu Asn Cys Glu Glu Met Arg Arg Gln Phe Glu Asp Leu Val Lys 85 90 95 Asp Ile Thr Leu Asn Lys Glu Glu Lys Lys Glu Asn Ser Phe Glu Met 100 105 110 Gln Arg Gly Asp Glu Asp Pro Gln Ile Ala Ala His Val Val Ser Glu 115 120 125 Ala Asn Ser Asn Ala Ala Ser Val Leu Gln Trp Ala Lys Lys Gly Tyr 130 135 140 Tyr Thr Met Lys Ser Asn Leu Val Met Leu Glu Asn Gly Lys Gln Leu 145 150 155 160 Thr Val Lys Arg Glu Gly Leu Tyr Tyr Val Tyr Thr Gln Val Thr Phe 165 170 175 Cys Ser Asn Arg Glu Pro Ser Ser Gln Arg Pro Phe Ile Val Gly Leu 180 185 190 Trp Leu Lys Pro Ser Ser Gly Ser Glu Arg Ile Leu Leu Lys Ala Ala 195 200 205 Asn Thr His Ser Ser Ser Gln Leu Cys Glu Gln Gln Ser Val His Leu 210 215 220 Gly Gly Val Phe Glu Leu Gln Ala Gly Ala Ser Val Phe Val Asn Val 225 230 235 240 Thr Glu Ala Ser Gln Val Ile His Arg Val Gly Phe Ser Ser Phe Gly 245 250 255 Leu Leu Lys Leu 260 <210> 20 <211> 930 <212> DNA <213> Mus musculus <400> 20 atggatcagc acacactgga cgtggaggat accgctgacg ctaggcaccc agctggcacc 60 tcctgccctt ctgatgccgc tctgctgcgc gacacaggac tgctggccga tgccgctctg 120 ctgtctgaca cagtgcggcc aaccaacgcc gctctgccaa ccgatgctgc ttaccctgct 180 gtgaacgtga gggacagaga ggctgcttgg ccacctgccc tgaacttctg cagccgccac 240 cctaagctgt acggcctggt ggccctggtg ctgctgctgc tgatcgctgc ttgcgtgcca 300 atctttaccc ggacagagcc acgccccgct ctgacaatca ccacatcccc caacctgggc 360 accagggaga acaacgccga tcaggtgaca ccagtgtctc acatcggctg ccccaacacc 420 acacagcagg gaagcccagt gttcgccaag ctgctggcta agaaccaggc cagcctgtgc 480 aacaccacac tgaactggca cagccaggac ggagctggaa gctcctacct gtcccagggc 540 ctgagatacg aggaggataa gaaggagctg gtggtggact cccctggact gtactacgtg 600 ttcctggagc tgaagctgtc tccaaccttt acaaacaccg gccacaaggt gcagggatgg 660 gtgtctctgg tgctgcaggc taagccccag gtggacgatt tcgataacct ggccctgacc 720 gtggagctgt ttccttgtag catggagaac aagctggtgg acaggtcttg gagccagctg 780 ctgctgctga aggctggcca caggctgtcc gtgggactga gagcctacct gcacggcgcc 840 caggatgctt acagagactg ggagctgagc taccctaaca ccacatcctt cggactgttt 900 ctggtgaagc ctgacaaccc atgggagtga 930 <210> 21 <211> 765 <212> DNA <213> Homo sapiens <400> 21 atggagtacg cctctgacgc cagcctggat ccagaggccc cttggccacc tgcaccaagg 60 gcccgcgcct gccgcgtgct gccctgggcc ctggtggccg gcctgttatt actgctgctg 120 ctggccgccg cctgcgccgt gttcctggca tgtccttggg ccgtgagcgg agccagagcc 180 tccccaggct ctgccgccag ccctcggctg agagagggac cagagctgtc cccagacgat 240 ccagcaggcc tgctggacct gaggcaggga atgtttgccc agctggtggc ccagaacgtg 300 ctgctgatcg acggccccct gtcctggtac tctgatcctg gcctggccgg cgtgtctctg 360 accggcggcc tgagctataa ggaggataca aaggagctgg tggtggccaa ggccggcgtg 420 tactacgtgt tcttccagct ggagctgagg agagtggtgg caggaggg ctctggaagc 480 gtgtccctgg ccctgcacct gcagcccctg cggagccgccg caggagccgc cgccctggcc 540 ctgaccgtgg acctgccacc agccagctcc attccgcctt cggctttcag 600 ggcagactgc tgcacctgtc tgccggacag aggctgggag tgcacctgca caccgaggcc 660 agggcccgcc acgcatggca gctgacccag ggagcaacag tgctgggcct gttccgcgtg 720 acacctgaga tcccagcagg cctgcctagc ccacggtccg to <210> 22 <211> 1389 <212> DNA <213> Mus musculus <400> 22 atgctgcctt tcctgtccat gctggtgctg ctggtgcagc cactgggcaa cctgggagcc 60 gagatgaagt ctctgagcca gcgcagcgtg cctaacacct gcacactggt catgtgctcc cctacagaga acggcctgcc aggaagggac ggaagagatg gaagggaggg accaagggga gagaagggcg accccggact gcctggacca atgggactga gcggcctgca gggaccaacc 240 ggccccgtgg gacctaaggg agagaacgga tccgctggag agccaggacc taagggagag 300 agaggactgt ctggaccacc tggactgcca ggaatcccag gaccagctgg caggaggga 360 ccatccggca agcagggaaa catcggacca cagggaaagc ctggaccaaa gggagaggct 420 ggacctaagg gagaagtggg cgccccagga atgcagggct ctacaggagc taagggcagc 480 accggaccaa agggagagag gggagccccc ggagtgcagg gagcccctgg caacgctgga 540 gccgctggcc cagccggacc cgctggccct cagggagccc ccggctctag gggaccacca 600 ggcctgaagg gagacagagg cgtgcccgga gatcggggca tcaagggga gagcggcctg 660 cctgactccg ccgctctgag acagcagatg gaggctctga agggcaagct gcagcggctg 720 gaggtggcct tctccacta ccagaaggcc gctctgtttc ctgacggaag gacagagccc 780 aggcctgctc tgaccatcac cacatctcca aacctgggca caagagagaa caacgccgat 840 caggtgaccc ccgtgtctca catcggatgc cctaacacca cacagcaggg cagccccgtg 900 tttgccaagc tgctggctaa gaaccaggcc agcctgtgca acaccacact gaactggcac 960 tcccaggatg gcgccggaag ctcctacctg tctcagggcc tgcggtacga ggaggacaag 1020 aaggagctgg tggtggatag cccaggcctg tactacgtgt tcctggagct gaagctgtcc 1080 cccaccttta caaacaccgg acacaaggtg cagggatggg tgagcctggt gctgcaggct 1140 aagccccagg tggacgattt cgacaacctg gccctgaccg tggagctgtt tccttgctct 1200 atggagaaca agctggtgga tagatcctgg agccagctgc tgctgctgaa ggctggacac 1260 cgcctgagcg tgggcctgag ggcttacctg cacggagctc aggacgctta cagggattgg 1320 gagctgtcct accctaacac cacatctttc ggcctgtttc tggtgaagcc agacaacccc 1380 tgggagtga 1389 <210> 23 <211> 1389 <212> DNA <213> Homo sapiens <400> 23 atgctgctgt tcctgctgtc cgccctggtg ctgctgaccc agcctctggg ctacctggag 60 gccgagatga agacctattc tcaccggaca atgccaagcg cctgcacact ggtcatgtgc 120 agcagcgtgg agtctggcct gccaggaagg gacggaaggg atggaaggga gggacctaga 180 ggcgagaagg gcgaccctgg cctgccagga gcagcaggac aggcaggaat gcccggccag 240 gccggccccg tgggacctaa gggcgacaac ggaagcgtgg gagagccagg accaaagggc 300 gataccggcc cttccggacc acctggacca ccaggcgtgc ctggcccagc cggcagggag 360 ggccctctgg gcaagcaggg caatatcggc ccacagggca agcccggccc taagggcgag 420 gccggcccca agggcgaagt gggcgcccct ggcatgcagg gaagcgccgg agcccgcggc 480 ctggccggac ctaagggcga gagaggcgtg cctggagaga ggggcgtgcc aggaaacaca 540 ggcgcagcag gatctgccgg agcaatggga ccccagggca gccctggcgc caggggccct 600 ccaggcctga agggcgacaa gggcatccca ggcgataagg gagcaaaggg agagagcggc 660 ctgccagatg tggcctccct gcgccagcag gtggaggccc tgcagggcca ggtgcagcac 720 ctgcaggccg ccttctctca gtacaagaag gtggagctgt ttccaaacgg cgcctgcccc 780 tgggccgtga gcggagcccg ggcctcccca ggctctgccg ccagccctag gctgcgcgag 840 ggaccagagc tgagcccaga cgatccagca ggcctgctgg acctgagaca gggaatgttc 900 gcccagctgg tggcccagaa tgtgctgctg atcgacggcc cactgtcctg gtactctgat 960 ccaggcctgg ccggcgtgtc cctgaccggc ggcctgtctt ataaggagga tacaaaggag 1020 ctggtggtgg ccaaggccgg cgtgtactac gtgttcttcc agctggagct gaggagagtg 1080 gtggcaggag agggatccgg atctgtgagc ctggccctgc acctgcagcc cctgcggtcc 1140 gccgcaggag ccgccgccct ggccctgacc gtggacctgc cacctgcctc tagcgaggca 1200 cgcaattccg ccttcggctt tcagggccgg ctgctgcacc tgtctgccgg acagagactg 1260 ggagtgcacc tgcacaccga ggcccgggcc agacacgcct ggcagctgac ccagggagca 1320 acagtgctgg gcctgtttag ggtgacacct gagatcccag ccggcctgcc aagcccccgc 1380 tccgagtga 1389 <210> 24 <211> 522 <212> DNA <213> Mus musculus <400> 24 atggaggaga tgcctctgag ggagagctcc ccacagaggg ccgagagatg caagaagagc 60 tggctgctgt gcatcgtggc tctgctgctg atgctgctgt gctctctggg caccctgatc 120 tacacaagcc tgaagccaac cgccatcgag tcctgtatgg tgaagttcga gctgtctagc 180 tccaagtggc acatgacatc ccccaagcct cactgcgtga acaccacatc tgacggaaag 240 ctgaagatcc tgcagagcgg cacctacctg atctacggac aggtcatccc cgtggacaag 300 aagtacatca aggataacgc ccctttcgtg gtgcagatct acaagaagaa cgacgtgctg 360 cagacactga tgaacgattt tcagatcctg cccatcggcg gagtgtacga gctgcacgct 420 ggcgacaaca tctacctgaa gttcaactcc aggatcaca tccagaagac caacacatac 480 tgggggaatca tcctgatgcc agatctgccc tttatctctt ga 522 <210> 25 <211> 600 <212> DNA <213> Homo sapiens <400> 25 atgaccctgc acccaagccc catcacatgc gagttcctgt tttctaccgc cctgatcagc 60 ccaaagatgt gcctgagcca cctggagaat atgcccctgt cccactctcg gacacaggga 120 gcccagagaa gctcctggaa gctctggctg ttctgctcta tcgtgatgct gctgttcctg 180 tgcagctttt cctggctgat cttcatcttt ctgcagctgg agacagccaa ggagccttgc 240 atggccaagt ttggccctct gccatccaag tggcagatgg cctctagcga gcccccttgc 300 gtgaacaagg tgagcgactg gaagctggag atcctgcaga acggcctgta cctgatctat 360 ggccaggtgg cccccaacgc caattacaac gacgtggccc cttcgaggt gcggctgtat 420 aagaacaagg atatgatcca gaccctgaca aataagtcta agatccagaa cgtgggcggc 480 acatacgagc tgcacgtggg cgacaccatc gacctgatct tcaacagcga gcaccaggtg 540 ctgaagaaca atacatattg gggcatcatc ctgctggcca acccccagtt tatctcctga 600 <210> 26 <211> 1164 <212> DNA <213> Mus musculus <400> 26 atgctgcctt tcctgtctat gctggtgctg ctggtgcagc cactgggcaa cctgggagcc 60 gagatgaaga gcctgtccca gagatccgtg cccaacacct gcacactggt catgtgctct 120 cctaccgaga acggcctgcc aggaagggac ggaagagatg gaagggaggg acctcgggga 180 gagaagggcg acccaggact gcctggacca atgggactga gcggcctgca gggaccaaca 240 ggccccgtgg gacctaaggg agagaacgga agcgccggag agccaggacc taagggagag 300 aggggactgt ccggaccacc tggactgcct ggaatcccag gaccagctgg caaggaggga 360 ccatccggca agcagggaaa catcggacca cagggaaagc ctggaccaaa gggagaggct 420 ggaccaaagg gagaagtggg cgctcctgga atgcagggct ccaccggagc caagggctct 480 acaggaccaa aaggagagag gggagctccc ggagtgcagg gagcccctgg caacgctgga 540 gccgctggcc cagccggacc cgctggccct cagggagccc caggcagcag gggaccaccc 600 ggcctgaagg gcgacagggg cgtgccagga gataggggca tcaagggaga gtctggcctg 660 ccagacagcg ccgctctgag acagcagatg gaggccctga agggcaagct gcagcggctg 720 gaggtggctt tctccacta ccagaaggcc gctctgtttc cagatggcag cctgaagccc 780 accgccatcg agtcctgcat ggtgaagttt gagctgagct cctctaagtg gcacatgaca 840 tctcccaagc ctcactgcgt gaacaccaca tctgacggca agctgaagat cctgcagagc 900 ggcacctacc tgatctacgg ccaggtcatc cccgtggaca agaagtacat caaggataac 960 gcccctttcg tggtgcagat ctacaagaag aacgacgtgc tgcagacact gatgaacgat 1020 tttcagatcc tgccaatcgg cggagtgtac gagctgcacg ctggcgacaa catctacctg 1080 aagttcaact ctaaggatca catccagaag accaacacat actggggcat catcctgatg 1140 ccagatctgc cctttatcag ctga 1164 <210> 27 <211> 1152 <212> DNA <213> Homo sapiens <400> 27 atgctgctgt tcctgctgtc tgccctggtg ctgctgaccc agccactggg ctacctggag 60 gccgagatga agacctattc ccaccgcaca atgccttctg cctgcacact ggtcatgtgc 120 agcagcgtgg agagcggcct gccaggaagg gacggaaag atggaaggga gggacccaga 180 ggcgagaagg gcgaccctgg cctgccagga gcagcagcac aggcaggaat gccaggccag 240 gccggccccg tgggccctaa gggcgacaat ggatccgtgg gagagccagg accaaagggc 300 gataccggcc cttctggacc acctggacca ccaggcgtgc ctggaccagc aggaagagag 360 ggacctctgg gcaagcaggg aaacatcgga ccacagggca agccaggccc taagggcgag 420 gccggcccca agggcgaagt gggcgcccct ggcatgcagg gatccgccgg agccaggggc 480 ctggccggac ctaagggcga gcgcggcgtg cctggagaga ggggcgtgcc aggaataca 540 ggcgcagcag gatctgccgg agcaatggga ccacagggca gccccggcgc cagaggccct 600 ccaggcctga agggcgacaa gggaatccct ggcgataagg gagcaaaggg agagagcggc 660 ctgccagacg tggcctccct gaggcagcag gtggaggccc tgcagggaca ggtgcagcac 720 ctgcaggccg ccttcagcca gtacaagaag gtggagctt ttccaatgg cgagacagcc 780 aaggagccct gcatggccaa gttcggccca ctgcccagca agtggcagat ggcctctagc 840 gagcccctt gcgtgaacaa ggtgagcgat tggaagctgg agatcctgca gaacggcctg 900 tacctgatct atggccaggt ggccccaac gccaattaca acgacgtggc cccttttgag 960 gtgcggctgt atagaacaa ggatatgatc cagaccctga caataagtc windowsccag 1020 aacgtgggag gcacctacga gctgcacgtg ggcgacacaa tcgacctgat cttcacagc 1080 gagcaccagg tgctgaagaa caatacatat tggggcatca tcctgctggc caacccccag 1140 tttatctcct ga 1152 <210> 28 <211> 597 <212> DNA <213> Muscles <400> 28 atggagggcg agggagtgca gccctggat gagaacctgg agaacggctc ccggcctcgc 60 ttcaagtgga agaagaccct gcggctggtg gtgtctgga tcaagggcgc cggaatgctg ctgtgcttta tctacgtgtg cctgcagctg agctcctctc ccgccaagga tccccctatc 180 cagaggctga gaggagctgt gaccaggtgc gaggacggac agctgttcat cagctcctac 300. aagaacgagt accagacaat ggaggtgcag aacaacagcg tggtcatcaa gtgtgatggc 360. ctgtacatca tctacctgaa gggatccttc tttcaggagg tgaagatcga cctgcacttt cgggaggatc acaacccaat ctctatcccc atgctgaacg acggcaggag aatcgtgttc 420 acagtggtgg ccagcctggc ttttaaggac aaggtgtacc tgaccgtgaa cgccccagat 480 acactgtgcg agcacctgca gatcaacgac ggagagctga tcgtggtgca gctgacccct ggctactgtg ctccagaggg atcttaccac agcacagtga accaggtgcc cctgtga 597 <210> 29 <211> 552 <212> DNA <213> Homo sapiens <400> 29 atggagagggg tgcagcccct ggaggagaac gtgggaatg ccgcccggcc tegttcgag 60 aggaacaagc tgctgctgt ggctctgtg atccaggcc tgggctgct gctgtgcttc 120 acctacatct gtctgcactt ttctgccctg caggtgagcc acagataccc ccgcatccag 180 agcatcagg tgcagttcac cgagtataag aaggaagg gctttatcct gatacccag 240 aaggaggacg agatcatgaa gtgcagac aattctgtga tcatcactg cgatggcttc 300 tacctgatct ccctgaaggg ctatttctct caggaagtga atcagcct gcactatcag 360 aaggacgagg agccactgtt tcagctgaag aaggtgcgga gcgtgaatttc cctgatggtg 420 gccagcctga cctacaagga caagtgtat ctgaacgtga ccacagataa tacatccctg 480 gacgatttcc acgtgaacgg cggcgagctg atcctgatcc accagaatcc cggcgagttt 540 tgcgtgctgt ga 552 <210> 30 <211> 1215 <212> DNA <213> Muscles <400> 30 atgctgccct tcctgtccat gctggtgctg ctggtgcagc ctctgggcaa cctgggagcc 60 gagatgaagt ctctgagcca gagatccgtg ccaaacacct gcaacactggt catgtgctct 120 cccaccgaga acggcctgcc tggaagggac ggagagatg gaagggaggg accccggga 180 gagaagggcg atcctggact gccaggacct atgggactga gcggcctgca gggaccaaca 240 ggccccgtgg gacctaaggg agagaacgga agcgccggag agccaggacc aaagggagag 300 aggggactgt ccggccccacc tggactgcct ggaatccctg gaccagctgg caaggaggga 360 ccttccggca agcagggaaa catcggacca cagggaaagc caggacctaa gggagaggct 420 ggaccaaagg gagaagtggg cgctcccgga atgcagggct ctaccggagc caagggcagc 480 agggaccta agggagagag gggagctcca ggagtgcagg gagccccccgg caacgctgga 540 gctgctggac cagctggacc agctggccct cagggagccc caggctctag gggaccacca 600 ggcctgaagg gcgacagggg cgtgccagga gataggggca tcaagggaga gagcggcctg 660 ccagattccg ccgctctgag acagcagatg gaggccctga agggcaagct gcagcggctg 720 gaggtggctt tcagccacta ccagaaggcc gctctgtttc ctgacggcag ctcctctcca 780 gccaaggatc ctccaatcca gcggctgcgc ggagctgtga ccaggtgcga ggatggccag 840 ctgttcatca gctcctacaa gaacgagtac cagacaatgg aggtgcagaa caactctgtg 900 gtcatcaagt gtgacggcct gtacatcatc tacctgaagg gcagcttctt tcaggaggtg 960 aagatcgacc tgcactttag agaggatcac aacccaatct ccatccccat gctgaacgac 1020 ggcaggagaa tcgtgttcac cgtggtggcc tctctggctt ttaaggacaa ggtgtacctg 1080 accgtgaacg cccccgatac actgtgcgag cacctgcaga tcaacgacgg cgagctgatc 1140 gtggtgcagc tgacccctgg atactgtgct ccagagggct cctaccactc tacagtgaac 1200 caggtgcctc tgtga 1215 <210> 31 <211> 1170 <212> DNA <213> Homo sapiens <400> 31 atgctgctgt tcctgctgag cgccctggtg ctgctgaccc agccactggg ctacctggag 60 gccgagatga agacctattc ccacagaaca atgccttctg cctgcacact ggtcatgtgc 120 agcagcgtgg agtccggcct gccaggaagg gacggcagag atggcaggga gggccccagg 180 ggcgagaagg gcgaccccgg cctgcctgga gcagcaggcc aggccggcat gccaggccag 240 gccggcccag tgggccccaa gggcgacaac ggcagcgtgg gcgagcccgg ccctaagggc 300 gataccggcc cctccggccc ccctggccca cccggcgtgc caggaccagc aggaagggag 360 ggaccactgg gcaagcaggg caatatcgga cctcagggca agcctggacc aaagggagag 420 gcaggaccaa agggagaagt gggcgcccct ggcatgcagg gatctgccgg agcccggggc 480 ctggccggcc ccaagggcga gagaggcgtg cccggcgaga ggggcgtgcc tggcaacaca 540 ggcgccgccg gctccgccgg cgccatggga cctcagggct ctccaggagc cagaggccct 600 ccaggcctga agggcgacaa gggaatccct ggcgataagg gagcaaaggg agagagcggc 660 ctgccagacg tggcctccct gcggcagcag gtggaggccc tgcagggcca ggtgcagcac 720 ctgcaggccg ccttcagcca gtacaagaag gtggagctgt ttcctaatgg cgtgtctcac 780 cgctacccac ggatccagag catcaaggtg cagttcaccg agtataagaa ggagaagggc 840 tttatcctga catctcagaa ggaggacgag atcatgaagg tgcagaacaa tagcgtgatc 900 atcaactgcg atggcttcta cctgatcagc ctgaagggct attttccca ggaagtgaat 960 atctctctgc actatcagaa ggatgaggag cctctgtttc agctgaagaa ggtgagatct 1020 gtgaacagcc tgatggtggc ctccctgacc tacaaggaca aggtgtatct gaacgtgacc 1080 acagataata catctctgga cgatttccac gtgaacggcg gcgagctgat cctgatccac 1140 cagaatcccg gcgagttttg cgtgctgtga 1170 <210> 32 <211> 969 <212> DNA <213> Mus musculus <400> 32 atgcagctga agtgtccatg cttcgtgtcc ctgggaacaa gacagcccgt ctggaagaaa 60 ctgcacgtga gctccggctt ctttagcggc ctggggctgt ttctgctgct gctgtctagt 120 ctgtgcgccg cttccgcaga gactgaagtc ggagccatgg tgggcagtaa cgtggtcctg 180 tcatgcatcg acccacaccg acggcatttc aacctgtctg gcctgtacgt gtattggcag 240 attgagaatc ccgaagtgtc agtcacctac tatctgcctt acaagagccc agggatcaac 300 gtggactcaa gctataaaaa tagggggcac ctgtccctgg attctatgaa gcagggaaac 360 ttcagcctgt acctgaaaaa tgtgacccct caggacacac aggagttcac ttgtcgcgtc 420 tttatgaaca ctgcaaccga actggtgaag attctggagg aagtggtccg gctgagagtc 480 gcagccaact ttagcactcc tgtgatctct accagtgatt cctctaatcc aggccaggag 540 cggacatata cttgcatgtc tagcgga taccccgac ctaatctgta ttggatcaac accacagaca attack tgataccgct ctgcagaaca attack cctgaacaag ctggggctgt atgacgtgat ctctactctg cggctgccat ggaccagtag aggagatgtg 720 ctgtgctgcg tggagaacgt ggccctgcac cagaatatca cctcaattag ccaggctgag tcctttaccg gcaacaatac aaagaatcct caggagacac aatacaatga actgaaagtg ctggtgccag tgctggccgt cctggctgca gcagctttcg tgtcttttat catctacaga900 aggacccgcc ctcaccgctc atacactgga cctaagaccg tgcagctgga actgacagac 960 catgcttga 969 <210> 33 <211> 909 <212> DNA <213> Homo sapiens <400> 33 atgcgtctgg gttcacctgg tctgctgtttt ctgctgtttt caagtctgcg tgctgatact caggagaagg aagtccgggc tatggtcgga agtgacgtgg agctgtcatg cgcttgtccc 120 gaagggtccc ggttcgacct gaacgatgtc tacgtgtatt ggcagacctc tgagagtaag 180 accgtggtca cataccacat ccctcagaac tccagcctgg aaaatgtgga ttcaaggtat 240 cggaacagag ccctgatgtc ccctgctggc atgctgcggg gagacttctc tctgagactg 300 tttaatgtga caccacagga tgagcagaaa ttccattgcc tggtcctgtc acagtccctg 360 ggatttcagg aggtgctgag tgtcgaagtg actctgcacg tcgccgctaa tttctccgtg 420 cctgtggtca gcgcaccaca tagcccctct caggacgagc tgacctttac atgtacttcc 480 atcaacggct acccccgccc taacgtgtac tggattaaca agactgacaa tagcctgctg 540 gatcaggcac tgcagaacga caccgtgttt ctgaatatgc gaggactgta cgatgtggtc 600 agcgtcctgc gtattgccag gaccccatct gtgaacatcg ggtgctgtat tgagaacgtc 660 ctgctgcagc agaatctgac agtggggagc cagactggta atgacatcgg cgagagggat 720 aagattaccg aaaaccccgt gagtacaggc gagaagaacg cagccacatg gtcaatcctg 780 gctgtgctgt gcctgctggt ggtcgtggct gtcgcaattg gctgggtgtg ccgcgatcgg 840 tgtctgcagc actcttatgc cggtgcttgg gcagtgagtc cagagactga actgaccggc 900 catgtctaa 909 <210> 34 <211> 1574 <212> DNA <213> Mus musculus <400> 34 cttaagatgg aaactgatac tctgctgctc tgggtgctgc tcctctgggt gcctggttca 60 actggggaca ttcgacgggc tgacattgtg atgacccaga ccacactgag cctgcccgtg 120 tccctgggcg accaggccag catctcctgc cggagctccc agtctatcgt gcacagcaac 180 ggaaacacat acctggagtg gtatctgcag aagcctggcc agtccccaaa gctgctgatc 240 tacaaggtgt ccaacaggtt cagcggcgtg cctgaccgct tttctggaag cggctccgga 300 acagatttca ccctgaagat cagcagggtg gaggctgagg acctgggcgt gtactactgc 360 ttccagggat cccacgtgcc ttacaccttt ggcggaggca caaagctgga gatcaagaga 420 gccgatgctg ctccaaccgt gtctggaagc ggaggcgggg gttctggagg cggtgggagc 480 ggtggcggag ggtctgaggc taagctgcag gagagcggcc ccgtgctggt gaagcctgga 540 gccagcgtga agatgtcctg taaggcttct ggatacacct tcacagacta ctacatgaac 600 tgggtgaagc agagccacgg caagtccctg gagtggatcg gagtgatcaa cccttacaac 660 ggcgacacct cttacaacca gaagtttaag ggcaaggcca ccctgacagt ggataagtct 720 agctccaccg cttacatgga gctgaacagc ctgacatccg aggattctgc cgtgtactac 780 tgtgctaggt actacggaag ctggttcgcc tactggggcc agggaacact gatcaccgtg 840 tccacagcca agaccacacc ccctagcgtg taccccctgg ctcctaggtc tagcagaggc 900 tgcaagccat gcatctgtac cgtgcccgag gtgagcagcg tgttcatctt tccacccaag 960 cccaaggacg tgctgaccat cacactgacc cctaaggtga catgcgtggt ggtggatatc 1020 agcaaggacg atccagaggt gcagttctcc tggtttgtgg acgatgtgga ggtgcacacc 1080 gcccagacac agccaaggga ggagcagttc aactccacct ttagatccgt gtctgagctg 1140 cccatcatgc accaggactg gctgaacgga aaggagttca agtgccgggt gaactccgcc 1200 gcttttcctg ctccaatcga gaagaccatc tctaagacaa agggccgccc aaaggctcca 1260 caggtgtaca ccatccctcc acccaaggag cagatggcta aggataaggt gagcctgacc 1320 tgtatgatca cagacttctt tcccgaggat atcacagtgg agtggcagtg gaacggacag 1380 cctgccgaga actacaagaa cacccagcca atcatggaca cagatggctc ttacttcgtg 1440 tacagcaagc tgaacgtgca gaagtctaac tgggaggctg gcaacacctt cacctgcagc 1500 gtgctgcacg aaggtctcca taatcaccac accgaaaaga gcctcagtca cagccctggg 1560 aaatgaggcg cgcc 1574 <210> 35 <211> 1484 <212> DNA <213> Homo sapiens <400> 35 cttaagatgg aaactgacac cctgctgctg tgggtcctgc tgctgtgggt gcctggatcc 60 accggcgata tcgtgctgac ccagtctcct ggcacactga gtctgtcacc aggggagcga 120 gcaacactgt cttgtagagc cagccagtct gtgggaagct cctacctggc ttggtatcag 180 cagaagccag gccaggcacc caggctgctg atctacggag ccttcagccg ggccactggc 240 attccagaca ggttctctgg aagtggctca gggaccgact tcaccctgac catcagccga 300 ctggagcccg aagacttcgc cgtgtactat tgccagcagt acggctctag tccttggact 360 tttggacagg gcaccaaagt ggagatcaag cgcggcgggg gaggctctgg gggaggcggg 420 agtggaggcg ggggatcaca ggtccagctg gtggaaagcg gcgggggagt ggtccagcca 480 ggccggagcc tgcggctgag ctgcgccgct tcaggattca cattttcaag ctataccatg 540 cactgggtcc ggcaggcacc agggaaggga ctggagtggg tgaccttcat cagctatgac 600 ggcaacaaca agtattacgc tgattccgtg aaagggaggt ttaccattag ccgcgacaac 660 tccaaaaata cactgtacct gcagatgaac agcctgcggg ccgaggatac tgctatctac 720 tattgcgcaa gaaccgggtg gctgggaccc ttcgactatt ggggccaggg gactctggtc 780 accgtgtcct ctgataagac acacacatgc cctccctgtc ctgcaccaga gctgctgggc 840 gggccatccg tgttcctgtt tccacccaag cctaaagaca ccctgatgat cagccggaca 900 cctgaagtca cttgcgtggt cgtggacgtg agtcacgagg atccagaagt caagtttaac 960 tggtacgtgg atggcgtcga ggtgcataat gccaagacca aacctcgcga ggaacagtac 1020 aatagcacat atcgagtcgt gtccgtcctg actgtgctgc atcaggattg gctgaacggc 1080 aaagagtata agtgcaaagt gagcaataag gcactgcctg ccccaatcga gaaaacaatt 1140 tccaaggcta aaggccagcc cagggaacct caggtgtaca ctctgcctcc aagtcgcgag 1200 gaaatgacca agaaccaggt gagcctgacc tgtctggtga aagggttcta tccatcagac 1260 attgcagtgg agtgggaaag caatggacag cccgaaaaca attacaagac cacaccccct 1320 gtgctggaca gcgatggctc cttctttctg tattctaagc tgactgtgga taaaagtcgc 1380 tggcagcagg ggaacgtctt tagctgttcc gtgatgcatg aggctctgca caatcattac 1440 acacagaagt ctctgagtct gtcacccggc aaatgaggcg cgcc 1484 <210> 36 <211> 632 <212> DNA <213> Artificial Sequence <220> <223> CMV promoter <400> 36 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 240 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat 420 agcggttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt 480 tttggcacca aaatcaacgg gactttccaa aatgcgtaa caactccgcc ccattgacgc 540 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta 600 gagaacccac tgcttactgg cttatcgaaa tt 632 <210> 37 <211> 394 <212> DNA <213> Artificial Sequence <220> <223> RSV promoter <400> 37 tgtacgggcc agatatacgc gtatctgagg ggactagggt gtgtttaggc gaaaagcggg 60 gcttcggttg tacgcggtta ggagtcccct caggatatag tagttcgct tttgcatagg 120 gagggggaaa tgtagtctta tgcaatacac ttgtagtctt gcaacatggt aacgatgagt 180 tagcaacatg ccttacaagg agagaaaaag caccgtgcat gccgattggt ggaagtaagg 240 tggtacgatc gtgccttatt aggaaggcaa cagacaggtc tgacatggat tggacgaacc 300 actgaattcc gcattgcaga gataattgta tttaagtgcc tagctcgata caataaacgc 360 catttgacca ttcaccacat tggtgtgcac ctcc 394 <210> 38 <211> 188 <212> DNA <213> Artificial Sequence <220> <223> BGH polyA <400> 38 ctgtgccttc tagttgccag ccatctgttg tttgccccctc cccgtgcct tccttgaccc 60 tggaaggtgc cactcccact gtccttttcct aataaaatga ggaaattgca tcgcattgtc 120 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 180 gggaagac 188 <210> 39 <211> 249 <212> DNA <213> Artificial Sequence <220> <223> SV40 late polyA <400> 39 gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa 60 tgctttatt gtgaaatttg tgatgctatt gctttatttg tgaaatttgt gatgctattg 120 ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt 180 ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca 240 aatgtggta 249 <210> 40 <211> 345 <212> DNA <213> Artificial Sequence <220> <223> SV40 enhancer promoter <400> 40 gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag 60 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc 120 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct 180 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg 240 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa 300 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagct 345 <210> 41 <211> 99 <212> DNA <213> Artificial Sequence <220> <223> Rabbit beta-globin polyA <400> 41 gacctctggc taataaagga aatttatttt cattgcaata gtgtgttgga attttttgtg 60 tctctcactc ggaaggacat atgggagggc aaatcattt 99 <210> 42 <211> 723 <212> DNA <213> Artificial Sequence <220> <223> GFP <400> 42 accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg 60 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc 120 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc 180 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg 240 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc 300 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc 360 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg 420 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag 480 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc 540 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac 600 cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg 660 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag 720 city ​​723 <210> 43 <211> 454 <212> DNA <213> Artificial Sequence <220> <223> MoMuLV LTR <400> 43 ttaattaagt aacgccattt tgcaaggcat ggaaaaatac ataactgaga atagagaagt 60 tcagatcaag gtcaggaaca gatggacag ctgaatatgg gccaaacagg atatctgtgg 120 taagcagttc ctgccccggc tcaggccaa gaacagatgg aacagctgaa tatgggccaa 180 acaggatatc tgtgtaagc agttcctgcc ccggctcagg gccagaaca gatggtcccc 240 agatgcggtc cagccctcag cagttcttag agaaccatca gatgttttcca gggtgcccca 300 aggacctgaa atgaccctgt gccttattg aactaccaa tcagttcgct tctcgcttct 360 gttcgcgcgc ttctgctccc cgagctcaat aaagagccc acaacccctc actcggggcg 420 ccagtcctcc gattgactga gtcgccccgct tag 454 <210> 44 <211> 1349 <212> DNA <213> Artificial Sequence <220> <223> The EF1alpha promoter <400> 44 tttaattaga gtaattcata CAAAggact cgcccctgcc ttggggaatc ccagggaccg 60 tcgttaaact cccactaacg tagaacccag agatcgctgc gttcccgccc cctcacccgc 120 ccgctctcgt catcactgag gtggagaaga gcatgcgtga ggctccggtg cccgtcagtg 180 ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgaac 240 cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg 300 cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct 360 ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc 420 tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacgc ccctggctgc 480 agtacgtgat tcttgatccc gagcttcggg ttggaagtgg gtgggagagt tcgaggcctt 540 gcggttaagg agccccttcg cctcgtgctt gagttgaggc ctggcttggg cgctggggcc 600 gccgcgtgcg aatctggtgg caccttcgcg cctgtctcgc tgctttcgat aagtctctag 660 ccatttaaaa tttttgatga cctgctgcga cgcttttttt ctggcaagat agtcttgtaa 720 atgcgggcca agatctgcac actggtattt cggtttttgg ggccgcgggc ggcgacgggg 780 cccgtgcgtc ccagcgcaca tgttcggcga ggcggggcct gcgagcgcgg ccaccgagaa 840 tcggacgggg gtagtctcaa gctggccggc ctgctctggt gcctggcctc gcgccgccgt 900 gtatcgcccc gccctgggcg gcaaggctgg cccggtcggc accagttgcg tgagcggaaa 960 gatggccgct tcccggccct gctgcaggga gctcaaaatg gaggacgcgg cgctcgggag 1020 agcgggcggg tgagtcaccc acacaaagga aaagggcctt tccgtcctca gccgtcgctt 1080 catgtgactc cacggagtac cgggcgccgt ccaggcacct cgattagttc tcgagctttt 1140 ggagtacgtc gtctttaggt tggggggagg ggttttatgc gatggagttt ccccacactg 1200 agtgggtgga gactgaagtt aggccagctt ggcacttgat gtaattctcc ttggaatttg 1260 cccttttga gtttggatct tggttcattc tcaagcctca gacagtggtt caaagtttttt 1320 ttcttccatt tcaggtgtcg tgacttaag 1349 <210> 45 <211> 481 <212> DNA <213> Artificial Sequence <220> <223> HGH polyA <400> 45 gacgggtggc atccctgtga cccctcccca gtgcctctcc tggccctgga agttgccact 60 ccagtgccca ccagccttgt cctaataaaa ttaagttgca tcatttgtc tgactaggtg 120 tccttctata atattatggg gtggagggg gtggtatgga gcaaggggca agttgggaag 180 acaacctgta gggcctgcgg ggtctattgg gaaccaagct ggagtgcagt ggcacaatct 240 tggctcactg caatctccgc ctcctgggtt caagcgattc tctgcctca gcctcccgag 300 ttgttgggat tccaggcatg catgaccagg ctcagctaat ttttgttttt ttggtagaga 360 cggggtttca ccatattggc caggctggtc tccaactcct aatctcaggt gatctaccca 420 ccttggcctc ccaaattgct gggattacag gcgtgaacca ctgctccctt ccctgtcctt 480 t 481

Claims

[Claim 1] A virus comprising a heterologous gene encoding a fusion-inducible protein and a gene encoding a CTLA-4 inhibitor, wherein the CTLA-4 inhibitor is a CTLA-4 antibody or a fragment thereof. [Claim 2] The virus according to claim 1, wherein the fusion-inducible protein is selected from the group consisting of glycoproteins derived from gibbon ape leukemia virus (GALV), mouse leukemia virus (MLV), Mason-Pfizer monkey virus (MPMV), or equine infectious anemia virus (EIAV) that are deficient in vesicular stomatitis virus (VSV) G protein, syncytin-1, syncytin-2, Simian virus 5 (SV5) F protein, measles virus (MV) H protein, MV F protein, respiratory syncytial virus (RSV) F protein, and R peptide. [Claim 3] The virus according to claim 1 or 2, wherein the fusion-inducible protein is a glycoprotein derived from gibbon leukemia virus (GALV), and the R transmembrane peptide is mutated or removed (GALV-R-). [Claim 4] The virus according to any one of claims 1 to 3, further comprising an immunostimulatory molecule. [Claim 5] Immune stimulating molecules, (a) IL-2, IL-12, IL-15, IL-18, IL-21, IL-24, type I interferon, interferon gamma, type III interferon, TNF-alpha, TGF-beta antagonists, immune checkpoint antagonists, or agonists of immune enhancement pathways; (b) Agonists of the immune-enhancing pathway that are agonists of CD40, ICOS, GITR, 4-1-BB, OX40, or flt3; (c) Agonists of the immunoenhancing pathway that are CD40 ligand (CD40L), ICOS ligand, GITR ligand, 4-1-BB ligand, OX40 ligand, or flt3 ligand; or (d) Immune checkpoint antagonists that are PD-1 inhibitors, PD-L1 inhibitors, LAG-3 inhibitors, TIM-3 inhibitors, VISTA inhibitors, CSF1R inhibitors, IDO inhibitors, CEACAM1 inhibitors, KIR antagonists, SLAMF7 inhibitors, or CD47 inhibitors. The virus according to claim 4. [Claim 6] The virus according to any one of claims 1 to 5, further comprising one or more immunostimulatory molecules. [Claim 7] A virus according to any one of claims 1 to 6, selected from the group consisting of herpesviruses, poxviruses, adenoviruses, retroviruses, rhabdoviruses, paramyxoviruses, and reoviruses. [Claim 8] The virus according to any one of claims 1 to 7, which is HSV1. [Claim 9] HSV1 RH018A strain with accession number ECACC16121904, RH004A strain with accession number ECACC16121902, RH031A strain with accession number ECACC16121907, RH040B strain with accession number ECACC16121908, RH015A strain with accession number ECACC16121903, RH021A strain with accession number ECACC16121905, RH023A strain with accession number ECACC16121906, or RH047A strain with accession number ECACC16121909 The virus according to claim 8, derived from the virus. [Claim 10] The virus according to claim 9, wherein HSV1 strain is RH018A strain having accession number ECACC16121904. [Claim 11] The virus, (i) Does not express functional ICP34.5, (ii) Not expressing functional ICP47, and / or (iii) Express the US11 gene as a pre-initial gene, The virus according to any one of claims 1 to 10, which is an HSV. [Claim 12] The virus according to any one of claims 1 to 11, wherein a gene encoding a fusion-inducible protein and a gene encoding a CTLA-4 inhibitor are, each under separate regulatory control, optionally inserted into the locus encoding ICP34.5 in a back-to-back orientation relative to each other, either by insertion or by partial or complete deletion. [Claim 13] (a) The sequence of the gene encoding the fusion-inducible protein and / or the sequence of the gene encoding the CTLA-4 inhibitor is codon-optimized to increase its expression level in target cells; and / or (b)(i) The virus expresses three heterologous genes, each of which is driven by a different promoter selected from the CMV promoter, RSV promoter, SV40 promoter and retroviral LTR promoter, and / or terminated by a different polyadenylation sequence selected from the BGH, SV40, HGH and RBG polyadenylation sequences; and / or (ii) The virus expresses four heterogeneous genes, each driven by the CMV promoter, RSV promoter, SV40 promoter, and retroviral LTR promoter, and / or terminated by different polyadenylation sequences selected from BGH, SV40, HGH, and RBG polyadenylation sequences. The virus according to any one of claims 1 to 12. [Claim 14] A pharmaceutical composition comprising the virus described in any one of claims 1 to 13 and a pharmaceutically acceptable carrier or diluent. [Claim 15] A composition comprising the virus according to any one of claims 1 to 13, for use in a method of treating the body of a human or animal by therapeutic means, or for use in a method of treating cancer. [Claim 16] Cancer, (a) Solid tumors; (b) adenocarcinoma, carcinoma or sarcoma; or (c) Cancers of the head and neck, prostate, breast, ovaries, lungs, liver, endometrium, bladder, gallbladder, pancreas, colon, kidneys, stomach / gastric, esophageal or cervical cancers, mesothelioma, melanoma, skin cancer, lymphoma, glioma, nervous system cancers, sarcomas or soft tissue sarcomas The composition according to claim 15. [Claim 17] The method is (a) By direct intratumor injection; (b)10 4 ~10 10 By direct intratumor injection of pfu in a range of doses; (c) By direct intratumor injection in multiple doses; or (d) Direct intratumor injection in multiple doses at intervals of 3 days to 3 weeks The composition according to claim 15 or 16, comprising administering an oncolytic virus. [Claim 18] The method is (a) Administering additional anticancer drugs simultaneously with or separately from the virus; (b) administering, concurrently with or separately from the virus, further anticancer agents selected from agents targeting immune co-inhibitory pathways, agents targeting immune costimulatory pathways, radiotherapy and / or chemotherapy, agents targeting specific gene mutations occurring in tumors, agents intended to induce an immune response to one or more tumor antigens or neoantigens, cell products derived from T cells or NK cells, STING, cGAS, TLR or other agents intended to stimulate innate immune responses and / or inflammatory pathways, a second virus, optionally oncolytic viruses, inhibitors of the indoleamine 2,3-dioxygenase (IDO) pathway, and combinations thereof; or (c) The composition according to any one of claims 15 to 17, comprising administering a further anticancer agent, which is an antibody, simultaneously with or separately from the virus. [Claim 19] (a) The drugs that target the immune co-inhibitory pathway are CTLA-4 inhibitors, PD-1 inhibitors, PD-L1 inhibitors, LAG-3 inhibitors, TIM-3 inhibitors, VISTA inhibitors, CSF1R inhibitors, IDO inhibitors, CEACAM1 inhibitors, KIR inhibitors, SLAMF7 inhibitors or CD47 inhibitors, and / or (b) The composition according to claim 18, wherein the agent targeting the immune costimulatory pathway is a GITR agonist, a 4-1-BB agonist, an OX40 agonist, a CD40 agonist, or an ICOS agonist.

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