Changing viruses

Abstract

The present invention relates to oncolytic viruses encoding CTLA-4 inhibitors, such as anti-CTLA-4 antibodies or antigen-binding fragments thereof.

Description

Field of the Invention

[0001] The present invention relates to an oncolytic immunotherapeutic agent and use of the oncolytic immunotherapeutic agent in treating cancer. Background of the Invention

[0003] Viruses have the unique ability to enter cells with high efficiency. After entering the cell, the viral genes are expressed and the virus replicates. This usually leads to the death of infected cells and the release of antigenic components of the cell, because the cell ruptures when the cell dies. Therefore, virus-mediated cell death tends to lead to an immune response that is enhanced by host recognition of these cell components (including those components derived from host cells and those components encoded or incorporated by the virus itself) and due to so-called damage-associated molecular patterns (DAMPs) (which help activate immune responses).

[0004] Viruses also bind to various mediators of the innate immune response as part of the host response to recognition of viral infection (via, for example, Toll-like receptors and cGAS / STING signaling) and recognition of pathogen-associated molecular patterns (PAMPs), leading to activation of interferon responses and inflammation, which are also immunogenic signals to the host. These immune responses can have an immunogenic benefit to cancer patients, such that immune responses to tumor antigens provide systemic overall benefit, leading to treatment of tumors that are not infected with the virus (including micrometastatic disease), and providing vaccination against recurrence.

[0005] The combined direct ('oncolytic') effect of the virus and an immune response against tumour antigens (including non-self 'neoantigens', i.e. derived from specific mutated genes in an individual's tumour) is known as 'oncolytic immunotherapy'.

[0006] 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 for various biotechnological and medical applications. For example, viruses expressing heterologous therapeutic genes can be used for gene therapy. In the context of oncolytic immunotherapy, the genes delivered may include genes encoding specific tumor antigens, genes intended to induce an immune response or increase the immunogenicity of released antigens after viral replication and cell death, genes intended to shape the resulting immune response, genes that increase the general immune activation state of the tumor, or genes that increase the direct oncolytic properties (i.e., cytotoxic effects) of the virus. Importantly, viruses have the ability to deliver encoded molecules that are intended to help directly or selectively initiate, enhance, or shape a systemic anti-tumor immune response to a tumor, which may have benefits such as: reduced toxicity or focusing beneficial effects on tumors (including those not infected by the virus) rather than focusing off-target effects on normal (i.e., non-cancerous) tissues compared to systemic administration of these same molecules or systemic administration of other molecules targeting the same pathway.

[0007] It has been demonstrated that many viruses, including, for example, herpes simplex virus (HSV), can be used for oncolytic treatment of cancer. The HSV used for oncolytic treatment of cancer must be disabled so that it is no longer pathogenic, but can still enter tumor cells and kill them. Many disabling mutations for HSV have been identified, including the destruction of genes encoding ICP34.5, ICP6 and / or thymidine kinase, which can not prevent the virus from replicating in culture or in tumor tissue in vivo, but it prevents significant replication in normal tissue. Among them, only HSV with destroyed ICP34.5 gene replicates in many tumor cell types in vitro, and selectively replicates in tumor tissue in mouse tumor models, but not in surrounding tissues. Clinical trials of HSV with ICP34.5 or ICP34.5 and ICP6 deletions have also shown safety and selective replication in human tumor tissue.

[0008] As mentioned above, oncolytic viruses including HSV can also be used to deliver therapeutic genes in cancer treatment. This type of virus with ICP34.5 deletion, which has additionally deleted ICP47 and encodes a heterologous gene for GM-CSF, has also been tested in clinical trials, including Phase 3 trials conducted in melanoma, in which safety and effectiveness in humans are shown. GM-CSF is a proinflammatory cytokine that has multiple functions, including stimulating monocytes to leave the circulation and migrate into tissues, where they proliferate and mature into macrophages and dendritic cells. GM-CSF is important for the proliferation and maturation of antigen-presenting cells, and its activity is required for activating anti-tumor immune responses. Experimental data show that tumor responses can be seen in injected tumors, and tumor responses to a lesser extent can be seen in non-injected tumors. The response is often highly persistent (several months-several years), and survival benefits seem to be achieved in responding patients. In addition to direct oncolytic effects, each of these also indicates that the immune system is involved in cancer treatment. However, this and other data of oncolytic viruses generally indicate that not all tumors respond to treatment, and not all patients can gain a survival advantage. Therefore, there is a clear need for improvements in the field of oncolytic therapy.

[0009] It has recently been shown that oncolytic immunotherapy can lead to additive or synergistic therapeutic effects together with immune co-inhibitory pathway blockade (i.e., inhibition or 'antagonism' of immune checkpoint pathways, also known as immune co-inhibitory pathways). Immune co-inhibitory pathway blockade aims to block host immunosuppressive mechanisms, which are usually used to prevent the occurrence of autoimmunity. However, in cancer patients, these mechanisms can also be used to inhibit the induction of any immune response induced against the tumor or block the potential beneficial effects of the immune response.

[0010] The systemic blocking of these pathways by agents targeting cytotoxic T lymphocyte-associated molecule-4 (CTLA-4), PD-1 or PD-L1 has shown efficacy in many tumor types including melanoma and lung cancer. However, as expected, based on the mechanism of action, off-target toxicity may occur due to the induction of autoimmunity. Even so, these agents are sufficient to allow for considerable clinical utility. Other immune co-suppression pathways and related targets in development for agents thereof (mainly antibodies) include LAG-3, TIM-3, VISTA, CSF1R, IDO, CEACAM1, CD47. Due to the mechanism of action, the optimal clinical activity of these agents (such as PD1, PDL1, LAG-3, TIM-3, VISTA, CSF1R, IDO, CD47, CEACAM1) may require systemic administration or be present in all tumors, i.e., including the interface of targeted immune effector cells and tumors or other immunosuppressive mechanisms in / tumors. In certain cases, agents that are more localized, such as in only some tumors or in some lymph nodes, may also be most effective, such as agents that target CTLA-4.

[0011] An alternative method to increase the anti-tumor immune response of cancer patients is to target (activate) immune co-stimulatory pathways, i.e., to suppress immune co-suppression pathways in contrast. These pathways send activation signals to T cells and other immune cells, which are usually caused by the interaction of related ligands on antigen presenting cells (APC) and related receptors on T cells and other immune cells. Depending on ligand / receptor, these signals may cause activation of T cells and / or APC and / or NK cells and / or B cells (including specific subtypes) to increase, differentiation and proliferation of T cells to increase and / or differentiation and proliferation of APC and / or NK cells and / or B cells (including specific subtypes), or immunosuppressive T cells such as regulatory T cells to suppress activity. Therefore, it is expected that the activation of these pathways will lead to enhanced anti-tumor immune response, but it is also expected that the systemic activation of these pathways (i.e., general activation of immune response rather than specific or selective anti-tumor immune response) will cause considerable off-target toxicity in non-tumor tissues, and the degree of such off-target toxicity depends on the specific immune co-stimulatory pathways targeted. However, agents that target immune co-stimulatory pathways (primarily agonistic antibodies or, less commonly, soluble ligands to said receptors) (including agents targeting GITR, 4-1-BB, OX40, CD40, or ICOS) and are intended for systemic use (i.e., intravenous delivery) are in clinical development or have been proposed for clinical development.

[0012] In order for many of these methods targeting immune co-inhibition or co-inhibitory pathways to succeed, a pre-existing immune response to the tumor is required, that is, so that the pre-existing immune response can be enhanced or the blockade of the anti-tumor immune response can be delivered. It is also necessary to have an inflamed tumor microenvironment (which indicates this sustained response). The pre-existing immune response to tumor neoantigens seems to be particularly important for the activity of immune co-inhibitory pathway blockade and related drugs. Only some patients may have a sustained immune response to tumor antigens (including neoantigens and / or an inflamed tumor microenvironment, both of which are necessary for the optimal activity of these drugs). Therefore, oncolytic agents that can induce immune responses to tumor antigens (including neoantigens) and / or can induce an inflamed tumor microenvironment are attractive for use in combination with immune co-inhibitory pathway blockade and immune enhancement drugs. This may explain the promising combined anti-tumor effects of oncolytic agents and immune co-inhibitory pathway blockade in mice and humans observed so far.

[0013] The above discussion suggests that there is still much room for improving oncolytics as well as cancer therapies that exploit oncolytics, antitumor immune responses, and drugs that target immune co-inhibitory or co-stimulatory pathways. SUMMARY OF THE INVENTION

[0015] The present invention provides an oncolytic virus expressing an inhibitor of CTLA-4. The virus may also contain other immunomodulators. In particular, the virus may contain GM-CSF and / or at least one molecule targeting an immune co-stimulatory pathway. CTLA-4 inhibitors are used to block the co-inhibition pathway, i.e., interfere with the interaction between CTLA-4 and B7. GM-CSF helps to induce an inflammatory tumor microenvironment and stimulate the proliferation and maturation of antigen presenting cells (including dendritic cells), which helps to induce anti-tumor immune responses. These immune responses can be amplified by activating one or more immune co-stimulatory pathways using one or more immune co-stimulatory pathway activation molecules also delivered by an oncolytic virus.

[0016] Oncolytic viruses replicate within tumors, causing tumor cell lysis and release of tumor antigens, combined with local inflammation and activation of innate immune responses, all of which are beneficial for the activation of antitumor immune responses and the activity of inhibitors of CTLA-4 / B7 interactions.

[0017] The molecules that inhibit CTLA-4 / B7 interactions are delivered directly to the immune response-inducing tumor (including where it is expected to flow to the draining lymph nodes), and the immune enhancement produced by the inhibitor is concentrated on the tumor, thereby focusing on the tumor antigen present therein, reducing systemic toxicity and blocking the activation of regulatory T cells (Treg), and the activation of the regulatory T cells (Treg) can inhibit T cell activation in other ways at the immune response-inducing site. The molecules targeting CTLA-4 and the molecules of the optionally targeted immune co-stimulatory pathways are delivered to the tumor using an oncolytic virus to focus the amplification of the immune effect on the anti-tumor immune response, and reduce the amplification of the immune response for non-tumor antigens. Therefore, the immune cells in tumors and tumor-draining lymph nodes are generally affected by the molecules expressed by viruses rather than the selectivity of immune cells. This leads to an increase in the effectiveness of immune cell stimulation, and can also lead to a reduction in off-target toxicity. It is also important to focus the combined systemic immune co-inhibitory pathway blocking and immune co-stimulatory pathway activation on the tumor (i.e., so that the amplified immune response of the co-inhibitory blocking released therefrom is an anti-tumor immune response rather than a response for non-tumor antigens).

[0018] The present invention utilizes the following fact: when delivered by oncolytic virus, the action site of CTLA-4 blocking and optionally costimulatory pathway activation and GM-CSF expression is in tumor and / or tumor draining lymph nodes, but the result of this activation (enlarged systemic anti-tumor immune response) is systemic. This usually targets tumors, not just tumors to which oncolytic viruses have delivered one or more immunomodulatory molecules. Therefore, the oncolytic virus of the present invention provides improved cancer treatment by producing an improved immune response focused on tumors. The oncolytic virus of the present invention also provides improved anti-tumor immunostimulatory effects, so that immune-mediated effects on tumors (including micrometastatic diseases) that are not destroyed by oncolysis are enhanced, resulting in more effective destruction of these tumors and more effective long-term anti-tumor vaccination, to prevent future recurrence and improve overall survival rate.

[0019] The anti-tumor efficacy is improved when the oncolytic viruses of the present invention are used as a single agent and when the viruses are used in combination with other anti-cancer modalities, including chemotherapy, treatment with targeted agents, irradiation, and in preferred embodiments, immune checkpoint blockade drugs (i.e., antagonists of immune co-inhibitory pathways, such as antibodies to PD1 or PD-L1) and / or agonists of immune co-stimulatory pathways.

[0020] Therefore, the present invention provides an oncolytic virus encoding a CTLA-4 inhibitor. The CTLA-4 inhibitor is preferably an anti-CTLA-4 antibody or antibody-like molecule, or an antigen-binding fragment thereof.

[0021] The virus may further comprise: (i) a GM-CSF encoding gene; and / or (ii) an immune co-stimulatory pathway activating molecule or an immune co-stimulatory pathway activating molecule encoding gene. The virus may encode more than one immune co-stimulatory pathway activating molecule / gene.

[0022] The immune co-stimulatory pathway activating molecule is preferably GITRL, 4-1-BBL, OX40L, ICOSL or CD40L or any modified form thereof. Examples of modified forms include agonists of co-stimulatory pathways that are secreted rather than membrane-bound, and / or agonists that are modified to form multimers of the protein.

[0023] The virus can be a modified clinical isolate, such as a modified clinical isolate of a virus, wherein the clinical isolate kills two or more tumor cell lines in vitro more rapidly and / or at a lower dose than one or more reference clinical isolates of the same virus.

[0024] The virus is preferably a herpes simplex virus (HSV), such as HSV 1. HSV generally does not express a functional ICP34.5 and / or a functional ICP47 and / or expresses the US11 gene as an immediate early gene.

[0025] The present invention also provides:

[0026] - A pharmaceutical composition comprising the virus of the present invention and a pharmaceutically acceptable carrier or diluent;

[0027] - a virus according to the invention for use in a method of treatment of the human or animal body by therapy;

[0028] - a virus of the invention for use in a method of treating cancer, wherein the method optionally comprises the administration of an additional anti-cancer agent;

[0029] - a preparation comprising the virus of the invention in a sterile vial, ampoule or syringe;

[0030] - a method of treating cancer comprising administering to a patient in need thereof a therapeutically effective amount of a virus or pharmaceutical composition of the invention, wherein the method optionally comprises administering an additional anti-cancer agent;

[0031] - Use of a virus of the invention for the preparation of a medicament for use in a method for the treatment of cancer, wherein said method optionally comprises the administration of an additional anti-cancer agent. Sequence Listing <110> Replimune Limited <120> Changing viruses <130> N409771WO <140> <141> 2018-01-09 <150> GB 1700350.0 <151> 2017-01-09 <160> 46 <170> PatentIn version 3.5 <210> 1 <211> 108 <212> PRT <213> Homo sapiens <400> 1 Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu 1 5 10 15 Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Phe Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 2 <211> 215 <212> PRT <213> Homo sapiens <400> 2 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 3 <211> 118 <212> PRT <213> Human <400> 3 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 4 <211> 103 <212> PRT <213> Human <400> 4 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys 100 <210> 5 <211> 227 <212> PRT <213> Person <400> 5 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 6 <211> 448 <212> PRT <213> Human <400> 6 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leucine, Valine, Lysine, Glycine, Phenylalanine, Tyrosine, Proline, Serine, Aspartic acid, Isoleucine, Alanine, Valine, Glutamic acid, Tryptophan, Glutamic acid, Serine 370 375 380 Asparagine, Glycine, Glutamine, Proline, Glutamic acid, Asparagine, Asparagine, Tyrosine, Lysine, Threonine, Threonine, Proline, Proline, Valine, Leucine, Aspartic acid 385 390 395 400 Serine, Aspartic acid, Glycine, Serine, Phenylalanine, Phenylalanine, Leucine, Tyrosine, Serine, Lysine, Leucine, Threonine, Valine, Aspartic acid, Lysine, Serine 405 410 415 Arginine, Tryptophan, Glutamine, Glutamine, Glycine, Asparagine, Valine, Phenylalanine, Serine, Cysteine, Serine, Valine, Methionine, Histidine, Glutamic acid, Alanine 420 425 430 Leucine, Histidine, Asparagine, Histidine, Tyrosine, Threonine, Glutamine, Lysine, Serine, Leucine, Serine, Leucine, Serine, Proline, Glycine, Lysine 435 440 445 <210> 7 <211> 21 <212> PRT <213> Mouse (Mus musculus) <400> 7 Methionine, Glutamic acid, Threonine, Aspartic acid, Threonine, Leucine, Leucine, Leucine, Tryptophan, Valine, Leucine, Leucine, Leucine, Tryptophan, Valine, Proline 1 5 10 15 Glycine, Serine, Threonine, Glycine, Aspartic acid 20 <210> 8 <211> 15 <212> PRT <213> Artificial sequence <220> <223> (Gly4Ser)3 linker <400> 8 Glycine, Glycine, Glycine, Glycine, Serine, Glycine, Glycine, Glycine, Glycine, Serine, Glycine, Glycine, Glycine, Glycine, Serine 1 5 10 15 <210> 9 <211> 489 <212> PRT <213> Person <400> 9 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser 20 25 30 Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser 35 40 45 Val Gly Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala 50 55 60 Pro Arg Leu Leu Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Ile Pro 65 70 75 80 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 85 90 95 Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr 100 105 110 Gly Ser Ser Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 115 120 125 Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 145 150 155 160 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 165 170 175 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 180 185 190 Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val 195 200 205 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 210 215 220 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys 225 230 235 240 Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr 245 250 255 Leu Val Thr Val Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro 260 265 270 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 275 280 285 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 290 295 300 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 305 310 315 320 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 325 330 335 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 340 345 350 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 355 360 365 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 370 375 380 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 385 390 395 400 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 405 410 415 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 420 425 430 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 435 440 445 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 450 455 460 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 465 470 475 480 Lys Ser Leu Ser Leu Ser Pro Gly Lys 485 <210> 10 <211> 1470 <212> DNA <213> Human <400> 10 atggaaactg acaccctgct gctgtgggtc ctgctgctgt gggtgcctgg atccaccggc 60 gatatcgtgc tgacccagtc tcctggcaca ctgagtctgt caccagggga gcgagcaaca 120 ctgtcttgta gagccagcca gtctgtggga agctcctacc tggcttggta tcagcagaag 180 ccaggccagg cacccaggct gctgatctac ggagccttca gccgggccac tggcattcca 240 gacaggttct ctggaagtgg ctcagggacc gacttcaccc tgaccatcag ccgactggag 300 cccgaagact tcgccgtgta ctattgccag cagtacggct ctagtccttg gacttttgga 360 cagggcacca aagtggagat caagcgcggc gggggaggct ctgggggagg cgggagtgga 420 ggcgggggat cacaggtcca gctggtggaa agcggcgggg gagtggtcca gccaggccgg 480 agcctgcggc tgagctgcgc cgcttcagga ttcacatttt caagctatac catgcactgg 540 gtccggcagg caccagggaa gggactggag tgggtgacct tcatcagcta tgacggcaac 600 aacaagtatt acgctgattc cgtgaaaggg aggtttacca ttagccgcga caactccaaa 660 aatacactgt acctgcagat gaacagcctg cgggccgagg atactgctat ctactattgc 720 gcaagaaccg ggtggctggg acccttcgac tattggggcc aggggactct ggtcaccgtg 780 tcctctgata agacacacac atgccctccc tgtcctgcac cagagctgct gggcgggcca 840 tccgtgttcc tgtttccacc caagcctaaa gacaccctga tgatcagccg gacacctgaa 900 gtcacttgcg tggtcgtgga cgtgagtcac gaggatccag aagtcaagtt taactggtac 960 gtggatggcg tcgaggtgca taatgccaag accaaacctc gcgaggaaca gtacaatagc 1020 acatatcgag tcgtgtccgt cctgactgtg ctgcatcagg attggctgaa cggcaaagag 1080 tataagtgca aagtgagcaa taaggcactg cctgccccaa tcgagaaaac aatttccaag 1140 gctaaaggcc agcccaggga acctcaggtg tacactctgc ctccaagtcg cgaggaaatg 1200 accaagaacc aggtgagcct gacctgtctg gtgaaagggt tctatccatc agacattgca 1260 gtggagtggg aaagcaatgg acagcccgaa aacaattaca agaccacacc ccctgtgctg 1320 gacagcgatg gctccttctt tctgtattct aagctgactg tggataaaag tcgctggcag 1380 caggggaacg tctttagctg ttccgtgatg catgaggctc tgcacaatca ttacacacag 1440 aagtctctga gtctgtcacc cggcaaatga 1470 <210> 11 <211> 127 <212> PRT <213> Mouse <400> 11 Ile Arg Arg Ala Asp Ile Val Met Thr Gln Thr Thr Leu Ser Leu Pro 1 5 10 15 Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser 20 25 30 Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys 35 40 45 Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe 50 55 60 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 65 70 75 80 Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr 85 90 95 Cys Phe Gln Gly Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys 100 105 110 Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Gly Ser 115 120 125 <210> 12 <211> 134 <212> PRT <213> Mouse <400> 12 Glu Ala Lys Leu Gln Glu Ser Gly Pro Val Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Val Ile Asn Pro Tyr Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Gly Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Ile Thr Val Ser Thr Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu 115 120 125 Ala Pro Arg Ser Ser Arg 130 <210> 13 <211> 356 <212> PRT <213> Mouse <400> 13 Glu Ala Lys Leu Gln Glu Ser Gly Pro Val Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Val Ile Asn Pro Tyr Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Gly Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Ile Thr Val Ser Thr Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu 115 120 125 Ala Pro Arg Ser Ser Arg Gly Cys Lys Pro Cys Ile Cys Thr Val Pro 130 135 140 Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu 145 150 155 160 Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser 165 170 175 Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu 180 185 190 Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr 195 200 205 Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn 210 215 220 Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro 225 230 235 240 Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln 245 250 255 Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val 260 265 270 Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val 275 280 285 Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln 290 295 300 Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn 305 310 315 320 Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val 325 330 335 Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His 340 345 350 Ser Pro Gly Lys 355 <210> 14 <211> 519 <212> PRT <213> Mouse <400> 14 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ile Arg Arg Ala Asp Ile Val Met Thr Gln Thr 20 25 30 Thr Leu Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys 35 40 45 Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu 50 55 60 Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys 65 70 75 80 Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly 85 90 95 Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp 100 105 110 Leu Gly Val Tyr Tyr Cys Phe Gln Gly Ser His Val Pro Tyr Thr Phe 115 120 125 Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr 130 135 140 Val Ser Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 145 150 155 160 Gly Gly Ser Glu Ala Lys Leu Gln Glu Ser Gly Pro Val Leu Val Lys 165 170 175 Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe 180 185 190 Thr Asp Tyr Tyr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu 195 200 205 Glu Trp Ile Gly Val Ile Asn Pro Tyr Asn Gly Asp Thr Ser Tyr Asn 210 215 220 Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser 225 230 235 240 Thr Ala Tyr Met Glu Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val 245 250 255 Tyr Tyr Cys Ala Arg Tyr Tyr Gly Ser Trp Phe Ala Tyr Trp Gly Gln 260 265 270 Gly Thr Leu Ile Thr Val Ser Thr Ala Lys Thr Thr Pro Pro Ser Val 275 280 285 Tyr Pro Leu Ala Pro Arg Ser Ser Arg Gly Cys Lys Pro Cys Ile Cys 290 295 300 Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys 305 310 315 320 Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val 325 330 335 Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp 340 345 350 Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe 355 360 365 Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp 370 375 380 Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe 385 390 395 400 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys 405 410 415 Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys 420 425 430 Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp 435 440 445 Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys 450 455 460 Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser 465 470 475 480 Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr 485 490 495 Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser 500 505 510 Leu Ser His Ser Pro Gly Lys 515 <210> 15 <211> 1560 <212> DNA <213> Mouse <400> 15 atggaaactg atactctgct gctctgggtg ctgctcctct gggtgcctgg ttcaactggg 60 gacattcgac gggctgacat tgtgatgacc cagaccacac tgagcctgcc cgtgtccctg 120 ggcgaccagg ccagcatctc ctgccggagc tcccagtcta tcgtgcacag caacggaaac 180 acatacctgg agtggtatct gcagaagcct ggccagtccc caaagctgct gatctacaag 240 gtgtccaaca ggttcagcgg cgtgcctgac cgcttttctg gaagcggctc cggaacagat 300 ttcaccctga agatcagcag ggtggaggct gaggacctgg gcgtgtacta ctgcttccag 360 ggatcccacg tgccttacac ctttggcgga ggcacaaagc tggagatcaa gagagccgat 420 gctgctccaa ccgtgtctgg aagcggaggc gggggttctg gaggcggtgg gagcggtggc 480 ggagggtctg aggctaagct gcaggagagc ggccccgtgc tggtgaagcc tggagccagc 540 gtgaagatgt cctgtaaggc ttctggatac accttcacag actactacat gaactgggtg 600 aagcagagcc acggcaagtc cctggagtgg atcggagtga tcaaccctta caacggcgac 660 acctcttaca accagaagtt taagggcaag gccaccctga cagtggataa gtctagctcc 720 accgcttaca tggagctgaa cagcctgaca tccgaggatt ctgccgtgta ctactgtgct 780 aggtactacg gaagctggtt cgcctactgg ggccagggaa cactgatcac cgtgtccaca 840 gccaagacca caccccctag cgtgtacccc ctggctccta ggtctagcag aggctgcaag 900 ccatgcatct gtaccgtgcc cgaggtgagc agcgtgttca tctttccacc caagcccaag 960 gacgtgctga ccatcacact gacccctaag gtgacatgcg tggtggtgga tatcagcaag 1020 gacgatccag aggtgcagtt ctcctggttt gtggacgatg tggaggtgca caccgcccag 1080 acacagccaa gggaggagca gttcaactcc acctttagat ccgtgtctga gctgcccatc 1140 atgcaccagg actggctgaa cggaaaggag ttcaagtgcc gggtgaactc cgccgctttt 1200 cctgctccaa tcgagaagac catctctaag acaaagggcc gcccaaaggc tccacaggtg 1260 tacaccatcc ctccacccaa ggagcagatg gctaaggata aggtgagcct gacctgtatg 1320 atcacagact tctttcccga ggatatcaca gtggagtggc agtggaacgg acagcctgcc 1380 gagaactaca agaacaccca gccaatcatg gacacagatg gctcttactt cgtgtacagc 1440 aagctgaacg tgcagaagtc taactgggag gctggcaaca ccttcacctg cagcgtgctg 1500 cacgaaggtc tccataatca ccacaccgaa aagagcctca gtcacagccc tgggaaatga 1560 <210> 16 <211> 1574 <212> DNA <213> Mouse <400> 16 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> 17 <211> 1484 <212> DNA <213> Human <400> 17 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> 18 <211> 426 <212> DNA <213> Mouse <400> 18 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 tactgccccc caactccgga aacggactgt gaaacacaag ttaccaccta tgcggatttc 360 atagacagcc ttaaaacctt tctgactgat atcccctttg aatgcaaaaa accagtccaa 420 aaatga 426 <210> 19 <211> 426 <212> DNA <213> Mouse <400> 19 atgtggctcc agaacctcct cttcctcggt 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> 20 <211> 435 <212> DNA <213> Human <400> 20 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 tatcaccttt 360 gaaagtttca aagagaacct gaaggacttt ctgcttgtca tcccctttga ctgctgggag 420 ccagtccagg agtga 435 <210> 21 <211> 435 <212> DNA <213> Human <400> 21 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 tacccccgag acaagctgtg ccacccagat catcacattc 360 gagtccttta aggagaacct gaaggatttt ctgctggtca ttccatttga ttgttgggag 420 cccgtccagg agtaa 435 <210> 22 <211> 141 <212> PRT <213> Mouse <400> 22 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> 23 <211> 144 <212> PRT <213> Person <400> 23 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> 24 <211> 2010 <212> DNA <213> Gibbon ape leukemia virus <400> 24 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 tcgccctactc caccgacctt 1140 gaccggtgcc gctggggac ccaaggaaag ctcaccctca ctgaggtctc aggacacggg 1200 ttgtgcatag gaaaggtgcc ctttacccat cagcatctct gcaatcagac cctatccatc 1260 aattcctccg gagaccatca gtatctgctc ccctccaacc atagctggtg ggcttgcagc 1320 actggcctca ccccttgcct ctccacctca gtttttaatc agatagaga 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> 25 <211> 2013 <212> DNA <213> Gibbon ape leukemia virus <400> 25 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 atgccagcag tggcagtggg accagacctg gtgctggtgg agcaggacc tccacgcacc 840 tccctggccc tgccccctcc actgccccct agggaggcccc cacccctag cctgccccgat900 tctaacagca cagccctggc cacctccgcc cagaccccta cagtgcgca gaccatcgtg acactgaata ccccaccccc taccacaggc gacaggctgt tcgatctggt gcagggcgcc 1080. tttctgacac tgaacgccac caatcctggc gcaaccgaga gctgctggct gtgcctggct atgggcccac cctactatga ggcaatcgcc tcctctggag aggtggcata ttccacagac 1140 ctggatagat gcagatgggg cacccagggc aagctgaccc tgacagaggt gtctggccac ggcctgtgca tcggcaaggt gccattcaca caccagcacc tgtgcaacca gaccctgagc atcaatagct ccggcgacca ccagtacctg ctgccaagca accactcctg gtgggcatgc tccacaggac tgaccccatg tctgagcacc agcgtgttca accagaccag agacttttgt 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> 26 <211> 669 <212> PRT <213> Gibbon leukemia virus <400> 26 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> 27 <211> 759 <212> DNA <213> Artificial sequence <220> <223> Human-mouse hybrid <400> 27 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 atcgtgggac tgtggctgaa gccctctagc ggcagcgaga ggatcctgct gaaggccgcc 600 aatacccact cctctagcca gctgtgcgag cagcagtcca tccacctggg aggcgtgttc 660 aatacccact cctctagcca gctgtgcgag cagcagtcca tccacctggg aggcgtgttc 660 gagctgcagc ctggagccag cgtgttcgtg aacgtgacag acccatctca ggtgagccac 720 gagctgcagc ctggagccag cgtgttcgtg aacgtgacag acccatctca ggtgagccac 720 ggcaccggct tcacaagctt tggcctgctg aagctgtga 759 ggcaccggct tcacaagctt tggcctgctg aagctgtga 759 <210> 28<210> 28 <211> 252<211> 252 <212> PRT<212> PRT <213> Artificial Sequence<213> Artificial Sequence <220><220> <223> Human - Mouse Hybrid<223> Human - Mouse Hybrid <400> 28<400> 28 Met Ile Glu Thr Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly Met Ile Glu Thr Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly 1 5 10 15 1 5 10 15 Leu Pro Ile Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe Leu Leu Pro Ile Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe Leu 20 25 30 20 25 30 Ile Thr Gln Met Ile Gly Ser Ala Leu Phe Ala Val Tyr Leu His Arg Ile Thr Gln Met Ile Gly Ser Ala Leu Phe Ala Val Tyr Leu His Arg 35 40 45 35 40 45 Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp Phe Val Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp Phe Val 50 55 60 50 55 60 Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser Leu Ser Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser Leu Ser 65 70 75 80 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> 29 <211> 1416 <212> DNA <213> human <400> 29 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 480 acaggaccaa agggcgagcg cggcgcccca ggcgtgcagg gcgcccccgg caatgcagga 540 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 aatctggtga cactggaga cggcaagcag ctgaccgtga agagacaggg cctgtactat atctatgccc aggtgacatt ctgctctaat cgggaggcct 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> 30 <211> 471 <212> PRT <213> Human <400> 30 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 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 Ala 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> 31 <211> 1412 <212> DNA <213> Mouse <400> 31 atgctgccct tcctgagcat gctggtgctg ctggtgcagc ctctgggcaa tctgggcgcc 60 gagatgaagt ccctgtctca gaggagcgtg ccaaacacct gcacactggt catgtgctct 120 ccaaccgaga atggactgcc aggaagggac ggaagagatg gaagggaggg accaagggga 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 agcacccggc atgcagggca gcaccggagc aaagggatcc 480 accggcccta aggagagag aggagcacct ggagtgcagg gcgccccagg caatgcagga 540 gcagcaggac cagcaggacc tgcagccca cagggcgccc caggcagccg gggcccaccc 600 ggcctgaagg gcgacagggg agtgccaggc gataggggca tcaagggaga gtccggactg 660 ccagactctg ccgccctgag gcagcagatg gaggccctga agggcaagct gcagaggctg 720 gaggtggcct tctcccacta ccagaaggcc gccctgtttc cagacggaca 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 ttttatcgtg 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> 32 <211> 470 <212> PRT <213> Mouse <400> 32 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> 33 <211> 786 <212> DNA <213> Human <400> 33 atgatcgaaa catacaacca aacttctccc cgatctgcgg ccactggact gcccatcagc 60 atgaaaattt ttatgtattt acttactgtt tttcttatca cccagatgat tgggtcagca 120 ctttttgctg 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 gtttgtcaat 720 gtgactgatc caagccaagt gagccatggc actggcttca cgtcctttgg cttactcaaa 780 ctctga 786 <210> 34 <211> 261 <212> PRT <213> Human <400> 34 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> 35 <211> 783 <212> DNA <213> Mouse <400> 35 atgatagaaa catacagcca accttccccc agatccgtgg caactggact tccagcgagc 60 atgaagattt ttatgtattt acttactgtt ttccttatca cccaaatgat tggatctgtg 120 ctttttgctg tgtatcttca tagaagattg gataaggtcg aagaggaagt aaaccttcat 180 gaagattttg tattcataaa aaagctaaag agatgcaaca aaggagaagg atctttatcc 240 ttgctgaact gtgaggagat gagaaggcaa tttgaagacc ttgtcaagga tataacgtta 300 aacaaagaag agaaaaaaga aaacagcttt gaaatgcaaa gaggtgatga ggatcctcaa 360 attgcagcac acgttgtaag cgaagccaac agtaatgcag catccgttct acagtgggcc 420 aagaaaggat attataccat gaaaagcaac ttggtaatgc ttgaaaatgg gaaacagctg 480 acggttaaaa gagaaggact ctattatgtc tacactcaag tcaccttctg ctctaatcgg 540 gagccttcga gtcaacgccc attcatcgtc ggcctctggc tgaagcccag cagtggatct 600 gagagaatct tactcaaggc ggcaaatacc cacagttcct cccagctttg cgagcagcag 660 tctgttcact tgggcggagt gtttgaatta caagctggtg cttctgtgtt tgtcaacgtg 720 actgaagcaa gccaagtgat ccacagagtt ggcttctcat cttttggctt actcaaactc 780 tga 783 <210> 36 <211> 260 <212> PRT <213> Mouse <400> 36 Met Ile 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 Phe Ile Lys 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> 37 <211> 632 <212> DNA <213> Artificial sequence <220> <223> CMV promoter <400> 37 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 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt 480 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc 540 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta 600 gagaacccac tgcttactgg cttatcgaaa tt 632 <210> 38 <211> 394 <212> DNA <213> Artificial Sequence <220> <223> RSV Promoter <400> 38 tgtacgggcc agatatacgc gtatctgagg ggactagggt gtgtttaggc gaaaagcggg 60 gcttcggttg tacgcggtta ggagtcccct caggatatag tagtttcgct 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> 39 <211> 188 <212> DNA <213> Artificial sequence <220> <223> BGH polyA <400> 39 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 60 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 120 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 180 gggaagac 188 <210> 40 <211> 249 <212> DNA <213> Artificial sequence <220> <223> SV40 late polyA <400> 40 gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa 60 tgctttattt gtgaaatttg tgatgctatt gctttatttg tgaaatttgt gatgctattg 120 ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt 180 ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca 240 aatgtggta 249 <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 taa 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 gatggaacag ctgaatatgg gccaaacagg atatctgtgg 120 taagcagttc ctgccccggc tcagggccaa gaacagatgg aacagctgaa tatgggccaa 180 acaggatatc tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggtcccc 240 agatgcggtc cagccctcag cagtttctag agaaccatca gatgtttcca gggtgcccca 300 aggacctgaa atgaccctgt gccttatttg aactaaccaa tcagttcgct tctcgcttct 360 gttcgcgcgc ttctgctccc cgagctcaat aaaagagccc acaacccctc actcggggcg 420 ccagtcctcc gattgactga gtcgcccgct taag 454 <210> 44 <211> 1349 <212> DNA <213> Artificial sequence <220> <223> EF1alpha promoter <400> 44 ttaattaaga gtaattcata caaaaggact 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 ccctttttga gtttggatct tggttcattc tcaagcctca gacagtggtt caaagttttt 1320 ttcttccatt tcaggtgtcg tgacttaag 1349 <210> 45 <211> 345 <212> DNA <213> Artificial Sequence <220> <223> SV40 Enhancer Promoter <400> 45 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> 46 <211> 481 <212> DNA <213> Artificial Sequence <220> <223> HGH polyA <400> 46 gacgggtggc atccctgtga cccctcccca gtgcctctcc tggccctgga agttgccact 60 ccagtgccca ccagccttgt cctaataaaa ttaagttgca tcattttgtc tgactaggtg 120 tccttctata atattatggg gtggaggggg gtggtatgga gcaaggggca agttgggaag 180 acaacctgta gggcctgcgg ggtctattgg gaaccaagct ggagtgcagt ggcacaatct 240 tggctcactg caatctccgc ctcctgggtt caagcgattc tcctgcctca gcctcccgag 300 ttgttgggat tccaggcatg catgaccagg ctcagctaat ttttgttttt ttggtagaga 360 cggggtttca ccatattggc caggctggtc tccaactcct aatctcaggt gatctaccca 420 ccttggcctc ccaaattgct gggattacag gcgtgaacca ctgctccctt ccctgtcctt 480 t481 BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Fig. 1 depicts the structure of the virus for constructing an exemplary virus of the present invention, which comprises an anti-mouse or anti-human CTLA-4 construct, which is a codon-optimized secreted scFv molecule connected to a human or mouse IgG1 Fc region. The scFv contains variable light and heavy chains from 9D9 (initial mouse antibody for verifying CTLA-4; WO2007 / 123737: mouse form) or from ipilimumab (WO2014 / 066532; human form) connected by 15-mer [G4S] 3 (GGGGSGGGGSGGGGS). The virus is a modified form of strain HSV1RH018A (clinical strain 18). ICP34.5 and ICP47 genes are inactivated in the virus. The US11 gene is placed under the control of the ICP47 immediate early gene promoter by deleting the ICP47 promoter. The expression cassette is inserted into the ICP34.5 locus. In virus 17, the expression cassette contains the human GM-CSF gene under the control of the CMV promoter and the GALV gene under the control of the RSV promoter. Virus 16 is identical to virus 17 except that it contains human GM-CSF instead of mouse GM-CSF. Viruses 25 and 29 are identical to viruses 16 and 17, respectively, except that they each additionally contain a GFP gene under the control of the MMLV promoter in the expression cassette. Viruses 27 and 31 are identical to viruses 25 and 29, respectively, except that the GFP gene is replaced by mouse anti-CTLA4 and human anti-CTLA4, respectively.

[0034] FIG. 2 depicts the structure of plasmids used to construct exemplary viruses of the present invention.

[0035] Figure 3The structures of anti-mouse or human CTLA-4 constructs are shown, which are codon-optimized secreted scFv molecules linked to human or mouse IgG1 Fc regions. The scFv contains linked ([G4S]3) variable light and variable heavy chains from 9D9 (the initial mouse antibody originally used to validate CTLA-4; US2011044953: mouse form) or from ipilimumab (US20150283234; human form). The resulting structures of CTLA-4 inhibitors are also shown.

[0036] Figure 4 is a Western blot demonstrating that anti-mouse CTLA-4 is expressed by virus 27. The gel used is a reduced denatured PVDF membrane tris-glycine gel. Anti-CTLA-4 was detected using an alkaline phosphatase labeled anti-mouse IgG1 antibody. Lane 1: spectra broad range ladder; Lane 2: neat supernatant of virus 27; Lane 3: virus 27 supernatant diluted 1:2; Lane 4: virus 27 supernatant diluted 1:4; Lane 5: virus 27 supernatant diluted 1:8; Lane 6: virus 27 supernatant diluted 1:16; Lane 7: virus 27 supernatant diluted 1:32; Lane 8: negative control virus (neat supernatant). The expected size of anti-CTLA-4 (reduced) is 57 kDa.

[0037] Figure 5 The superior tumor control and shrinkage of uninjected tumors by a virus expressing anti-mCTLA-4 (virus 27) was shown compared to an otherwise identical virus not expressing CTLA-4 (virus 16). The virus dose used was 5x10 4 pfu (50ul of 1x10 6 pfu / ml), given three times in one week. For virus 16 uninjected tumors, this dose level of virus was subtherapeutic, allowing the benefit of delivery of additional molecules encoded by virus 27 to be clearly seen.

[0038] Figure 6 The superior tumor control and shrinkage in both injected and uninjected tumors of the virus expressing anti-mCTLA-4 (virus 27) compared to an otherwise identical virus not expressing CTLA-4 (virus 16) is shown. The dose of virus used in the right tumor of the virus expressing anti-mCTLA-4 (virus 27) was 5x10 4 pfu. Each line represents a different mouse.

[0039] Figure 7Shown are the effects of combined treatment of bilateral mouse A20 tumors using anti-PD1 and virus 27 expressing mGM-CSF, GALVR, and anti-mCTLA-4. The top panel shows the effect of anti-PD1 alone on injected (right) and uninjected (left) tumors. The middle panel shows the effect of virus 27 alone on injected (right) and uninjected (left) tumors. The bottom panel shows the excellent tumor control and shrinkage achieved when both anti-PD1 and virus 27 were injected into the right tumor. Improved anti-tumor effects of the combined treatment were observed in both injected (right) and uninjected (left) tumors. Each line represents a different mouse.

[0040] Figure 8 Shown in knock-in mice expressing human CTLA-4 in mouse MC38 tumors, superior tumor control and shrinkage effects of virus 31 expressing hGM-CSF, GALVR and anti-human CTLA-4 compared to virus 17 expressing only hGM-CSF and GALVR. The anti-tumor effect of virus 31 was observed when the virus was administered alone or in combination with anti-PD1. Superior tumor control and shrinkage of injected tumors were obtained using virus 31 expressing anti-human CTLA-4 compared to viruses that did not express anti-human CTLA-4 but were otherwise identical (left figure). This effect was further enhanced when treatment with the virus was combined with anti-PD1 treatment. Excellent tumor control and shrinkage were also observed in non-injected tumors (right figure) when treatment with either virus was combined with anti-PD1 treatment. This improvement was more pronounced for virus 31 expressing anti-CTLA-4 compared to virus 17 that did not express anti-CTLA-4. Each line represents a different mouse.

[0041] Brief description of sequence listing

[0042] SEQ ID NO: 1 is the amino acid sequence of the light chain variable region of the human CTLA-4 antibody used in the Examples.

[0043] SEQ ID NO: 2 is a complete light chain amino acid sequence, which comprises the light chain variable region amino acid sequence of the human CTLA-4 antibody used in the Examples.

[0044] SEQ ID NO: 3 is the amino acid sequence of the heavy chain variable region of the human CTLA-4 antibody used in the Examples.

[0045] SEQ ID NO: 4 is the heavy chain CH1 amino acid sequence of the human CTLA-4 antibody used in the Examples.

[0046] SEQ ID NO: 5 is the heavy chain CH2 / 3 amino acid sequence of the human CTLA-4 antibody used in the Examples.

[0047] SEQ ID NO: 6 is the complete heavy chain amino acid sequence of the human CTLA-4 antibody used in the Examples.

[0048] SEQ ID NO: 7 is the amino acid sequence of the signal peptide present in the CTLA-4 antibody of the Examples.

[0049] SEQ ID NO: 8 is the amino acid sequence of the linker between the light chain variable region and the heavy chain variable region present in the CTLA-4 antibody of the Examples.

[0050] SEQ ID NO: 9 is the amino acid sequence of the human scFv CTLA-4 antibody of the Example.

[0051] SEQ ID NO: 10 is the nucleotide sequence of the human scFv CTLA-4 antibody of the Example.

[0052] SEQ ID NO: 11 is the amino acid sequence of the light chain variable region of the murine CTLA-4 antibody used in the Examples.

[0053] SEQ ID NO: 12 is the amino acid sequence of the heavy chain variable region of the murine CTLA-4 antibody used in the Examples.

[0054] SEQ ID NO: 13 is the complete heavy chain amino acid sequence of the murine CTLA-4 antibody used in the Examples.

[0055] SEQ ID NO: 14 is the amino acid sequence of the murine scFv CTLA-4 antibody of the Example.

[0056] SEQ ID NO: 15 is the nucleotide sequence of the murine scFv CTLA-4 antibody of the Examples.

[0057] SEQ ID NO: 16 is the nucleotide sequence of an example murine scFv CTLA-4 antibody with insertion restriction sites at the N and C termini for cloning purposes, as present in an example virus. The restriction sites are the first six and last eight nucleotides of the sequence.

[0058] SEQ ID NO: 17 is the nucleotide sequence of an example human scFv CTLA-4 antibody with insertion restriction sites at the N and C termini for cloning purposes, as present in an exemplary virus. The restriction sites are the first six and last eight nucleotides of the sequence.

[0059] SEQ ID NO: 18 is the nucleotide sequence of mouse GM-CSF.

[0060] SEQ ID NO: 19 is the nucleotide sequence of a codon-optimized form of mouse GM-CSF.

[0061] SEQ ID NO: 20 is the nucleotide sequence of human GM-CSF.

[0062] SEQ ID NO:21 is the nucleotide sequence of a codon-optimized form of human GM-CSF.

[0063] SEQ ID NO:22 is the amino acid sequence of mouse GM-CSF.

[0064] SEQ ID NO: 23 is the amino acid sequence of human GM-CSF.

[0065] SEQ ID NO:24 is the nucleotide sequence of GALV-R-.

[0066] SEQ ID NO:25 is the nucleotide sequence of a codon-optimized form of GALV-R-.

[0067] SEQ ID NO:26 is the amino acid sequence of GALV-R-.

[0068] SEQ ID NO:27 is the nucleotide sequence of a codon-optimized form of the human / mouse hybrid membrane-bound form of CD40L.

[0069] SEQ ID NO:28 is the amino acid sequence of the human / mouse hybrid membrane-bound form of CD40L.

[0070] SEQ ID NO: 29 is the nucleotide sequence of a codon-optimized version of the multimeric secreted form of human CD40L.

[0071] SEQ ID NO: 30 is the amino acid sequence of the multimeric secreted form of human CD40L.

[0072] SEQ ID NO:31 is the nucleotide sequence of a codon-optimized version of the multimeric secreted form of mouse CD40L.

[0073] SEQ ID NO:32 is the amino acid sequence of the multimeric secreted form of mouse CD40L.

[0074] SEQ ID NO: 33 is the nucleotide sequence of wild-type human CD40L.

[0075] SEQ ID NO: 34 is the amino acid sequence of wild-type human CD40L.

[0076] SEQ ID NO:35 is the nucleotide sequence of wild-type mouse CD40L.

[0077] SEQ ID NO:36 is the amino acid sequence of wild-type mouse CD40L.

[0078] SEQ ID NO: 37 is the nucleotide sequence of CMV promoter.

[0079] SEQ ID NO:38 is the nucleotide sequence of RSV promoter.

[0080] SEQ ID NO:39 is the nucleotide sequence of BGH polyA.

[0081] SEQ ID NO:40 is the nucleotide sequence of SV40 late polyA.

[0082] SEQ ID NO:41 is the nucleotide sequence of rabbit β-globulin polyA.

[0083] SEQ ID NO:42 is the nucleotide sequence of GFP.

[0084] SEQ ID NO:43 is the nucleotide sequence of the retroviral LTR from MMLV.

[0085] SEQ ID NO:44 is the nucleotide sequence of EF1a promoter.

[0086] SEQ ID NO:45 is the nucleotide sequence of the SV40 promoter.

[0087] SEQ ID NO:46 is the nucleotide sequence of HGH polyA. DETAILED DESCRIPTION OF THE INVENTION

[0089] Oncolytic viruses

[0090] The virus of the present invention is oncolytic. Oncolytic viruses are viruses that infect tumor cells and replicate in tumor cells, thereby killing tumor cells. Therefore, the virus of the present invention has the ability to replicate. Preferably, the virus has the ability to selectively replicate in tumors. If the virus replicates more efficiently in tumor tissue than in non-tumor tissue, the virus has the ability to selectively replicate in tumor tissue. The ability of a virus to replicate in different tissue types can be determined using standard techniques in the art.

[0091] The virus of the present invention can be any virus with these characteristics, including herpes virus, pox virus, adenovirus, retrovirus, rhabdovirus, paramyxovirus or reovirus, or any species or strain in these larger groups. The virus of the present invention can be wild type (i.e. unchanged compared to the parent virus species), or have gene disruption or gene addition. As for which of these situations it belongs to, it will depend on the virus species to be used. Preferably, the virus is a species of herpes virus, more preferably a strain of HSV, including strains of HSV1 and HSV2, and most preferably a strain of HSV1. In a particularly preferred embodiment, the virus of the present invention is based on a clinical isolate of the virus species to be used. Clinical isolates can be selected based on its particularly advantageous properties for treating cancer.

[0092] The virus can be a modified clinical isolate, wherein the clinical isolate kills two or more tumor cell lines in vitro more quickly and / or with a lower dosage than one or more reference clinical isolates of the same virus species. Typically, the clinical isolate will kill two or more tumor cell lines within 48 hours, preferably within 24 hours, of an infection with a multiplicity of infection (MOI) less than or equal to 0.1. Preferably, the clinical isolate will kill a wide range of tumor cell lines, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 tumor cell lines or, for example, all cell lines in the following human tumor cell lines: U87MG (glioma), HT29 (colorectal cancer), LNCaP (prostate cancer), MDA-MB-231 (breast cancer), SK-MEL-28 (melanoma), Fadu (squamous cell carcinoma), MCF7 (breast cancer), A549 (lung cancer), MIAPACA-2 (pancreatic cancer), CAPAN-1 (pancreatic cancer), HT1080 (fibrosarcoma).

[0093] In a preferred embodiment, the virus of the present invention is a strain selected from the following:

[0094] strain RH018A, accession number ECCAC 16121904;

[0095] strain RH004A, accession number ECCAC 16121902;

[0096] strain RH031A, accession number ECCAC 16121907;

[0097] strain RH040B, accession number ECCAC 16121908;

[0098] strain RH015A, accession number ECCAC 16121903;

[0099] strain RH021A, accession number ECCAC 16121905;

[0100] strain RH023A, accession number ECCAC 16121906; and

[0101] strain RH047A, accession number ECCAC 16121909.

[0102] More preferably, the virus of the present invention is a strain selected from the following:

[0103] strain RH018A, accession number ECCAC 16121904;

[0104] strain RH004A, accession number ECCAC 16121902;

[0105] strain RH031A, accession number ECCAC 16121907;

[0106] strain RH040B, accession number ECCAC 16121908; and

[0107] strain RH015A, accession number ECCAC 16121903.

[0108] Most preferably, the virus of the invention is strain RH018A with accession number EACC 16121904. Any of the deposited virus strains may be modified as defined herein.

[0109] The HSV of the present invention is capable of selectively replicating in tumors (such as human tumors). Typically, HSV replicates efficiently in target tumors, but not in non-tumor tissues. The 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. For example, the mutation may be a mutation that prevents HSV from expressing functional ICP34.5, ICP6 and / or thymidine kinase.

[0110] In a preferred embodiment, the ICP34.5 encoding gene is mutated to confer selective oncolytic activity to HSV. Mutations of the ICP34.5 encoding gene that prevent functional ICP34.5 expression are described 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 ICP6 encoding gene and / or the thymidine kinase encoding gene may also be inactivated, as may other genes, as long as such inactivation does not prevent the virus from infecting the tumor or replicating therein.

[0111] HSV may contain additional mutations that enhance HSV replication in tumors. The resulting enhanced replication of the virus in tumors not only leads to enhanced direct "oncolytic" killing of tumor cells by the virus, but also increases the level of heterologous (i.e., genes inserted into the virus, in the case of the virus of the present invention, genes encoding CTLA-4 inhibitors, GM-CSF and / or immune co-stimulatory pathway activating molecules) gene expression and increases the amount of tumor antigens released when tumor cells die, both of which can also improve the immunogenic properties of the therapy for cancer treatment. For example, in a preferred embodiment of the present invention, the deletion of the ICP47 encoding gene in a manner that places the US11 gene under the control of the immediate early promoter that normally controls the expression of the ICP47 encoding gene leads to enhanced replication in tumors (see Liu et al., 2003, which is incorporated herein by reference).

[0112] Other mutations that place the US11 coding sequence (which is a HSV late gene) under the control of a promoter that is independent of viral replication can also be introduced into the virus of the present invention. Such mutations allow US11 to be expressed before HSV replication occurs and enhance viral replication in tumors. In particular, such mutations enhance the replication of HSV lacking a functional ICP34.5 coding gene.

[0113] Therefore, in one embodiment, the HSV of the present invention comprises a US11 gene operably linked to a promoter, wherein the activity of the promoter is independent of viral replication. The promoter may be an immediate early (IE) promoter or a non-HSV promoter active in mammalian (preferably human) tumor cells. The promoter may be, for example, a eukaryotic promoter, such as a promoter derived from a mammalian (preferably human) genome. The promoter may be a ubiquitous promoter (such as a promoter of β-actin or tubulin) or a cell-specific promoter, such as a tumor-specific promoter. The promoter may be a viral promoter, such as a Moloney murine leukemia virus long terminal repeat (MMLV LTR) promoter or a human or mouse cytomegalovirus (CMV) IE promoter. The HSV immediate early (IE) promoter is well known in the art. The HSV IE promoter may be a promoter that drives the expression of ICP0, ICP4, ICP22, ICP27 or ICP47.

[0114] The above genes (whose functional inactivation provides the virus with the property of tumor selectivity) can be functionally inactivated by any suitable method, for example, by deleting or replacing all or part of the gene and / or the control sequence of the gene, or by inserting or replacing one or more nucleic acids into or replacing the gene and / or the control sequence of the gene. For example, homologous recombination methods standard in the art can be used to produce the virus of the present invention. Alternatively, a method based on bacterial artificial chromosomes (BAC) can be used.

[0115] As used herein, the term "gene" is intended to mean a nucleotide sequence encoding a protein, i.e., the coding sequence of a gene. By mutating the gene itself or the control sequences flanking the gene, such as a promoter sequence, the various genes mentioned above can be rendered inoperative. Deletion can remove one or more parts of a gene, the entire gene, or the entire gene and all or some control sequences. For example, only one nucleotide in a gene can be deleted, resulting in a frameshift. However, larger deletions can be performed, such as at least about 25%, more preferably at least about 50% of the total coding sequence and / or non-coding sequence. In a preferred embodiment, deletion is performed on a gene to be functionally inactivated. For example, the entire gene and optionally some flanking sequences can be removed from a virus. When two or more gene copies are present in the viral genome, both copies of the gene are rendered functionally inactive.

[0116] Gene inactivation can be achieved by replacing other sequences, for example, by replacing all or part of an endogenous gene with a heterologous gene and optionally a promoter sequence. In the absence of a promoter sequence being replaced, a heterologous gene can be inserted so that it is controlled by the promoter of the gene to be functionally inactivated. In the HSV of the present invention, the ICP34.5 encoding gene is preferably inactivated by inserting one or more heterologous genes and one or more promoter sequences operably linked thereto or optionally other regulatory elements (such as a polyadenylation sequence) into the locus of each ICP34.5 encoding gene.

[0117] The virus of the present invention is used to express CTLA-4 inhibitors and optionally GM-CSF and / or immune co-stimulatory pathway activating molecules in tumors. This is usually achieved by inserting heterologous genes encoding CTLA-4 inhibitors and optionally heterologous genes encoding GM-CSF and / or heterologous genes encoding immune co-stimulatory pathway activating molecules into the genome of a virus with selective replication ability, wherein each gene is under the control of a promoter sequence. Since the replication of this virus will occur selectively in tumor tissue, the expression of the virus to CTLA-4 inhibitors and, if present, GM-CSF and / or immune co-stimulatory activating proteins is enhanced in tumor tissue compared to non-tumor tissues in vivo. In the case of higher expression in tumors compared to other tissues of the body, enhanced expression occurs. Proteins expressed by oncolytic viruses are also expected to be present in tumor-draining lymph nodes infected by oncolytic viruses, including due to the transport of expressed proteins and viruses from tumors to antigen presenting cells or thereon. Thus, the present invention provides the benefit of selective expression of a CTLA-4 inhibitor and any co-expressed GM-CSF and / or immune co-stimulatory pathway activating molecules in tumors and tumor draining lymph nodes in combination with the anti-tumor effects provided by oncolytic virus replication.

[0118] The virus of the present invention comprises a CTLA-4 inhibitor. A CTLA-4 inhibitor is a molecule that is usually a peptide or protein that binds to CTLA-4 and reduces or blocks signal transduction through CTLA-4. By reducing CTLA-4 signal transduction, the inhibitor reduces or removes the blockade of CTLA-4 on the immune stimulation pathway.

[0119] The CTLA-4 inhibitor is preferably an antibody or an antigen-binding fragment thereof.

[0120] The term "antibody" mentioned herein includes complete antibodies and any antigen-binding fragments thereof (i.e., "antigen-binding portions") or single chains. An antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (κ) (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. 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 antigens. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with 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 (Clq) of the classical complement system.

[0121] The antibody is usually a monoclonal antibody. The antibody may be a chimeric antibody. The antibody is preferably a humanized antibody, more preferably a human antibody.

[0122] The term "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retains the ability to specifically bind to CTLA-4. The antigen-binding fragment also retains the ability to inhibit CTLA-4, thereby reducing or eliminating the CTLA-4 blockade of the stimulatory immune response. Examples of suitable fragments include Fab fragments, F(ab')2 fragments, Fab' fragments, Fd fragments, Fv fragments, dAb fragments, and isolated complementary 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 portion" of an antibody. In a preferred embodiment, the antibody is a scFv. Examples of suitable scFv molecules are disclosed, for example, in WO2007 / 123737 and WO2014 / 066532 (which are incorporated herein by reference).

[0123] The antibody coding sequence generally encodes an antibody or antibody fragment having an N-terminal signal sequence. The signal sequence may have the amino acid sequence shown in SEQ ID NO: 7. For example, the signal sequence is contained in a scFv having an amino acid sequence shown in SEQ ID NO: 9 and encoded by the nucleotide sequence shown in SEQ ID NO: 10, and an scFv having an amino acid sequence shown in SEQ ID NO: 14 and encoded by the nucleotide sequence shown in SEQ ID NO: 15.

[0124] In an antibody or antibody fragment, the light chain and heavy chain sequences can be connected by an amino acid linker. The linker generally comprises about 15 to about 25 amino acids, such as about 18 or 20 amino acids. Any suitable linker can be used, such as a linker comprising glycine and serine residues (e.g., the amino acid sequence shown in SEQ ID NO:8). For example, the linker is included in an scFv with an amino acid sequence shown in SEQ ID NO:9 and encoded by the nucleotide sequence shown in SEQ ID NO:10, and an scFv with an amino acid sequence shown in SEQ ID NO:14 and encoded by the nucleotide sequence shown in SEQ ID NO:15. Both are preferred antibody fragments for use in the present invention.

[0125] Other antibody fragments with similar structures are also preferred. Therefore, the virus of the present invention can encode an antibody or fragment comprising or essentially consisting of a light chain variable region, a linker, a heavy chain variable region, a heavy chain CH1 domain, a heavy chain CH2 domain, and a heavy chain CH3 domain. The virus can also encode a signal sequence at the N-terminal end of the antibody.

[0126] The antibody or antibody fragment of the present invention may preferably comprise an Fc region, which is an IgG1, IgG2, IgG3 or IgG4 region, more preferably an IgG1 region. Preferably, the antibody is a scFv antibody in which the scFv is linked to the IgG heavy chain CH2 and CH3 domains.

[0127] Preferred CTLA-4 antibodies or fragments include the heavy chain variable region shown in SEQ ID NO:3 and / or the light chain variable region shown in SEQ ID NO:1 or the heavy chain variable region shown in SEQ ID NO:11 and / or the light chain variable region shown in SEQ ID NO:12. The antibody may include a heavy chain CH1 domain having the amino acid sequence shown in SEQ ID NO:4 and / or a CH2 / CH3 domain shown in SEQ ID NO:5. The antibody may include the light chain amino acid sequence shown in SEQ ID NO:2. The antibodies of the present invention may optionally include a variant of one of these heavy or light chain variable regions or CDR sequences. For example, the variant may be a substitution, deletion or addition variant of any of the above amino acid sequences.

[0128] Variant antibodies may comprise 1, 2, 3, 4, 5, up to 10, up to 20, up to 30 or more amino acid substitutions and / or deletions from the specific sequences and fragments discussed above, while maintaining the activity of the antibodies described herein. "Deletion" variants may comprise, for example, 1, 2, 3, 4 or 5 single amino acids or one or more small groups of amino acids (such as 2, 3, 4 or 5 amino acids). "Replacement" variants preferably include replacing one or more amino acids with the same number of amino acids and producing conservative amino acid substitutions. For example, amino acids may be replaced by alternative amino acids (e.g., another basic amino acid, another acidic amino acid, another neutral amino acid, another charged amino acid, another hydrophilic amino acid, another hydrophobic amino acid, another polar amino acid, another aromatic amino acid, or another aliphatic amino acid) having similar properties.

[0129] The virus of the present invention comprises one or more polynucleotide sequences encoding a CTLA-4 inhibitor. The polynucleotide sequence is under the control of a suitable promoter. The virus may comprise a first polynucleotide sequence encoding an antibody heavy chain variable region and a second polynucleotide encoding an antibody light chain variable region. The first polynucleotide may encode a full-length heavy chain and / or the second polynucleotide may encode a full-length light chain. The first and second polynucleotides may be under the control of a single promoter, optionally with an IRES, or may be under the control of two separate promoters. The separate promoters may be the same or different.

[0130] The first polynucleotide may comprise, consist essentially of, or consist of the heavy chain variable region encoding sequence shown in SEQ ID NO: 9, and / or the second polynucleotide may comprise, consist essentially of, or consist of the heavy chain variable region encoding sequence shown in SEQ ID NO: 10. The first polynucleotide may comprise, consist essentially of, or consist of the heavy chain variable region encoding sequence shown in SEQ ID NO: 19, and / or the second polynucleotide may comprise, consist essentially of, or consist of the heavy chain variable region encoding sequence shown in SEQ ID NO: 20.

[0131] The first and / or second polynucleotide sequence may be a variant of SEQ ID NO: 9, 10, 19 or 20. For example, the variant may be a substitution, deletion or addition variant of any of these nucleic acid sequences. The variant polynucleotide may comprise 1, 2, 3, 4, 5, up to 10, up to 20, up to 30, up to 40, up to 50, up to 75 or more nucleic acid substitutions and / or deletions from SEQ ID NO: 9, 10, 19 or 20.

[0132] Suitable variants can have at least 70% homology with any of the polynucleotides of the nucleic acid sequences disclosed herein, preferably at least 80% or 90%, more preferably at least 95%, 97% or 99% homology therewith. Preferably, at least for the coding region of the polynucleotide, there are homology and identity at these levels. The method of measuring homology is well known in the art, and it will be appreciated by those skilled in the art that in this specification, homology is calculated based on nucleic acid identity. This homology can be present in at least 15, preferably at least 30, for example at least 40, 60, 100, 200 or more continuous nucleotides. This homology can be present in the entire length of the unmodified polynucleotide sequence.

[0133] Methods for measuring polynucleotide homology or identity are known in the art. For example, the UWGCG package provides the BESTFIT program, which can be used to calculate homology (e.g., used under its default settings) (Devereux et al. (1984) Nucleic Acids Research 12, pp. 387-395).

[0134] The PILEUP and BLAST algorithms can also be used to calculate homology or align sequences (usually under their default settings), for example as described in Altschul SF (1993) J Mol Evol 36:290-300; Altschul, S, F et al. (1990) J Mol Biol 215:403-10.

[0135] Software for performing BLAST analysis can be publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy a certain positive threshold score T when aligned with a word of the same length in the database sequence. T is called the neighborhood wordscore threshold (Altschul et al., supra). These initial neighborhood word hits are used as seeds to initiate searches to find HSPs containing them. As long as the cumulative alignment score can be increased, the word hits are extended in both directions along each sequence. The extension of the word hits in each direction is stopped when: the cumulative alignment score becomes zero or lower due to the accumulation of one or more negative scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919), alignments (B) of 50, expectations (E) of 10, M=5, N=4, and a comparison of both strands.

[0136] The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787. One similarity measure provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences will occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in a comparison of a first sequence to a second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0137] In one embodiment, due to the redundancy of the genetic code, the variant sequence can be different from the specific sequence given in the sequence table. The DNA code has 4 main nucleic acid residues (A, T, C and G), and uses these residues to "spell" three-letter codons, which represent the amino acids in the proteins encoded in the genes of the organism. The linear sequence of the codons along the DNA molecule is translated into the linear sequence of amino acids in the proteins encoded by these genes. The code is highly degenerate, with 61 codons encoding 20 natural amino acids and 3 codons representing "stop" signals. Therefore, most amino acids are encoded by more than one codon-in fact, several amino acids are encoded by four or more different codons. Therefore, the variant polynucleotide of the present invention can encode the same polypeptide sequence as the polypeptide sequence encoded by another polynucleotide of the present invention, but because different codons are used to encode the same amino acid, different nucleic acid sequences can be used. Codons can be optimized to improve the expression level of the encoded protein in the target cell compared to using an unchanged sequence.

[0138] The virus of the present invention preferably comprises GM-CSF. The sequence of the gene encoding GM-CSF may be codon-optimized to increase the expression level of the corresponding protein in target cells compared to the case where the unaltered sequence is used.

[0139] The virus of the present invention preferably comprises one or more immune costimulatory pathway activating molecules and / or one or more genes encoding immune costimulatory pathway activating molecules. Immune costimulatory pathway activating molecules include proteins and nucleic acid molecules (e.g., aptamer sequences). Examples of immune costimulatory pathway activating molecules include CD40 ligands, GITR ligands, 4-1-BB ligands, OX40 ligands, ICOS ligands, flt3 ligands, TL1A, CD30 ligands, CD70 and single-chain antibodies targeting the respective receptors of these molecules (CD40, GITR, 4-1-BB, OX40, ICOS, flt3, DR3, CD30, CD27).

[0140] Activators of immune co-stimulatory pathways include mutant or wild-type, soluble, secreted and / or membrane-bound ligands, and agonistic antibodies including single-chain antibodies. The virus of the present invention preferably encodes one or more of CD40L, ICOSL, 4-1-BBL, GITRL or OX40L.

[0141] The virus of the present invention may encode one or more immune co-stimulatory pathway activating molecules, preferably 1, 2, 3 or 4 immune co-stimulatory pathway activating molecules, and more preferably 1 or 2 immune co-stimulatory pathway activating molecules.

[0142] The sequence of the gene encoding the immune co-stimulatory activating molecule can be codon-optimized to increase the expression level of the corresponding protein in the target cell compared to when the unaltered sequence is used.

[0143] In addition to CTLA-4 inhibitors and GM-CSF and / or immune co-stimulatory pathway activating molecules, the virus of the present invention may also include one or more additional heterologous genes. In a preferred embodiment, the virus may also include a fusogenic protein, such as GALVR-.

[0144] Fusogenic proteins can be any heterologous protein that can promote the fusion of cells infected by the virus of the present invention with another cell. Fusogenic proteins, preferably wild-type or modified viral glycoproteins (i.e., modified to increase their fusogenic properties), are proteins that can induce intercellular fusion (syncytium formation) of cells in which they are expressed. Examples of fusogenic glycoproteins include VSV-G, syncytin-1 (from human endogenous retrovirus-W (HERV-W)) or syncytin-2 (from HERVFRDE1), paramyxovirus SV5-F, measles virus-H, measles virus-F, RSV-F, glycoproteins from retroviruses or lentiviruses, such as gibbon ape leukemia virus (GALV), murine leukemia virus (MLV), Mason-Pfizer monkey virus (MPMV) and equine infectious anemia virus (EIAV) glycoproteins (wherein the R transmembrane peptide is removed (R-form)). In a preferred embodiment, the fusogenic protein is from GALV and the R-peptide has been removed (GALV-R-).

[0145] The viruses of the invention may optionally comprise multiple copies of a fusogenic protein encoding gene, preferably 1 or 2 copies.The viruses may comprise two or more different fusogenic proteins, including any of the fusogenic proteins listed above.

[0146] The fusogenic protein optionally expressed by the viruses of the invention can be the same as a naturally occurring protein, or can be a modified protein.

[0147] Fusogenic protein encoding gene (fusogenic gene) can have naturally occurring nucleic acid sequence or modified sequence.For example, the sequence of fusogenic gene can be modified to increase the fusogenic property of the encoded protein, or provide codon optimization, thereby improving the expression efficiency of the encoded protein.

[0148] The present invention also provides a virus expressing at least three heterologous genes, such as poxvirus or HSV, preferably HSV1, wherein each of the three heterologous genes is driven by a different promoter selected from the group consisting of CMV promoter, RSV promoter, EF1a promoter, SV40 promoter and retroviral LTR promoter. For example, the virus can express four heterologous genes, wherein each of the four heterologous genes is driven by a different promoter selected from the group consisting of CMV promoter, RSV promoter, EF1a promoter, SV40 promoter and retroviral LTR promoter. Retroviral LTR is preferably from MMLV. The heterologous genes can be terminated by a polyadenylation sequence. The polyadenylation sequences can be the same or different. Preferably, each heterologous gene is terminated by a different polyadenylation sequence, preferably selected from the group consisting of BGH, SV40, HGH and RBG polyadenylation sequences. The present invention also provides a virus, such as a poxvirus or HSV, preferably HSV1, which expresses at least three heterologous genes, wherein each of the three heterologous genes is terminated by a different polyadenylation sequence selected from the group consisting of BGH, SV40, HGH and RBG polyadenylation sequences. For example, the virus can express four heterologous genes terminated by each of the BGH, SV40, HGH and RBG polyadenylation sequences, respectively.

[0149] The at least three heterologous genes can be, for example, selected from CTLA-4 inhibitors, genes encoding GM-CSF, genes encoding immune co-stimulatory pathway activation molecules, and fusion-promoting genes. Examples of three heterologous genes are CTLA-4 inhibitors, genes encoding GM-CSF, and genes encoding immune co-stimulatory pathway activation molecules; CTLA-4 inhibitors, genes encoding GM-CSF, and fusion-promoting genes; and CTLA-4 inhibitors, genes encoding immune co-stimulatory pathway activation molecules, and fusion-promoting genes. For example, the four heterologous genes can be CTLA-4 inhibitors, genes encoding GM-CSF, genes encoding immune co-stimulatory pathway activation molecules, and fusion genes. The three or four heterologous genes may include, for example, two or more genes encoding immune co-stimulatory pathway activation molecules and / or two or more fusion-promoting genes.

[0150] In one embodiment, the promoters controlling the expression of the three heterologous genes are CMV, RSV and MMLV promoters. For example, a preferred virus may contain a GM-CSF gene under the control of a CMV promoter, a GALV gene under the control of a RSV promoter, and a CTLA-4 inhibitor under the control of a MMLV promoter.

[0151] In one embodiment, the polyadenylation sequences terminating the at least three heterologous genes are SV40, BGH and RBG polyadenylation sequences. Controlling the expression of the three heterologous genes are CMV, RSV and MMLV promoters. For example, a preferred virus may include a GM-CSF gene terminated by a BGH polyadenylation sequence, a GALV gene terminated by a SV40 polyadenylation sequence, and a CTLA-4 inhibitor terminated by a RGB polyadenylation sequence.

[0152] Any combination of various promoters and polyadenylation sequences can be used with any heterologous gene. For example, a preferred virus may contain the GM-CSF gene under the control of the CMV promoter and terminated by the BGH polyadenylation sequence, the GALV gene under the control of the RSV promoter and terminated by the SV40 polyadenylation sequence, and the CTLA-4 inhibitor under the control of the MMLV promoter and terminated by the RGB polyadenylation sequence.

[0153] Virus Generation

[0154] The virus of the present invention is constructed using methods disclosed in the art. For example, plasmids (for smaller viruses and single and multiple genome component RNA viruses) or BACs (for larger DNA viruses, including herpes viruses) encoding the viral genome to be packaged (including genes encoding fusogenic and immunostimulatory molecules under appropriate regulatory control) can be constructed by standard molecular biological techniques and transfected into permissive cells from which recombinant viruses can be recovered.

[0155] Alternatively, in a preferred embodiment, a plasmid containing a DNA region flanking the expected insertion site can be constructed and then co-transfected with viral genomic DNA into permissive cells so that homologous recombination occurs between the target insertion site flanking region in the plasmid and the same region in the parental virus. The recombinant virus can then be selected and purified by the loss or addition of function (insertion or deletion of a plasmid for modification, such as insertion or deletion of a marker gene such as GFP or lacZ from the parental virus at the expected insertion site). In a most preferred embodiment, the insertion site is the ICP34.5 locus of HSV, so the plasmid used for the operation contains HSV sequences flanking the insertion site, between which are expression cassettes encoding GM-CSF and immune co-stimulatory pathway activation molecules. In this case, the parental virus may contain a cassette encoding GFP that replaces ICP34.5, and recombinant virus plaques are selected by the loss of GFP expression. In a most preferred embodiment, the US11 gene of HSV is also expressed as an IE gene. This can be achieved by deleting the ICP47 coding region or by other means.

[0156] The CTLA-4 inhibitor and optionally the GM-CSF coding sequences as well as the immune co-stimulatory pathway activator coding sequences and / or additional protein coding sequences (such as sequences encoding fusogenic proteins such as GALVR-) are inserted into the viral genome under appropriate regulatory control. Depending on the species and insertion site, this may be under the regulatory control of the native promoter of the viral species of the invention used, or preferably under the control of a heterologous promoter. Suitable heterologous promoters include mammalian promoters such as the IEF2a promoter or the actin promoter. More preferred are strong viral promoters such as the CMV IE promoter, RSV LTR, MMLV LTR, other retroviral LTR promoters, or promoters derived from SV40. Preferably, each exogenous gene (e.g., encoding GM-CSF and immune co-stimulatory pathway activator) will be under the control of separate promoters, but may also be expressed from a single RNA transcript, for example by inserting an internal ribosome entry site (IRES) between the protein coding sequences. RNA derived from each promoter typically terminates with a polyadenylation sequence (e.g., a mammalian sequence such as the bovine or human growth hormone (BGH) poly A sequence, a synthetic polyadenylation sequence, the rabbit β-globin polyadenylation sequence, or a viral sequence such as the SV40 early or late polyadenylation sequence).

[0157] Each heterologous gene in the virus is usually under the control of a promoter. The promoters controlling the expression of heterologous genes can be the same or different. For example, anti-CTLA-4 and one or more of the genes encoding GM-CSF, fusogenic genes, and immune co-stimulatory pathway activating molecules can each be under the control of a CMV promoter, a RSV promoter, an EF1a promoter, a SV40 promoter, or a retroviral LTR promoter. Alternatively, for example, anti-CTLA-4 can be under the control of a retroviral LTR promoter such as an MMLV promoter, the GM-CSF gene can be under the control of a CMV promoter, and / or a fusogenic gene such as GALVR- can be under the control of an RSV promoter.

[0158] Pharmaceutical composition

[0159] The present invention provides a pharmaceutical composition comprising a virus and a pharmaceutically acceptable carrier or diluent. Suitable carriers and diluents include isotonic saline solutions, such as phosphate buffered saline. The composition may also include other ingredients, such as sugars or proteins, to improve properties, such as the stability of the product. Alternatively, a lyophilized formulation may be used, which is reconstituted in a pharmaceutically acceptable carrier or diluent before use.

[0160] If necessary, the choice of carrier is generally a function of the composition delivery route. In the present invention, the composition can be formulated for any suitable route and mode of administration. Pharmaceutically acceptable carriers or diluents are those that can be used for compositions suitable for intratumoral administration, intravenous / intraarterial administration, intracerebral administration, or administration in a body cavity (e.g., bladder, pleural cavity, or by intraperitoneal administration). The composition can be in any suitable form, preferably in liquid form.

[0161] The invention also provides an article of manufacture comprising the virus of the invention in a sterile vial, ampoule or syringe.

[0162] Medical uses / treatments

[0163] The present invention provides a virus of the present invention for use in treating a human or animal body by therapy, in particular for a method of treating cancer. Cancer is typically present in mammals, preferably in humans. The virus kills infected tumor cells by lysis and by fusing infected tumor cells to each other. The virus of the present invention also triggers a systemic anti-tumor immune response, enhanced by the expression of CTLA-4 inhibitors and optionally GM-CSF and immune co-stimulatory pathway activating molecules, which also kill cancer cells.

[0164] The invention also provides a method of treating cancer, comprising administering to an individual in need thereof a therapeutically effective amount of a virus of the invention.

[0165] The present invention further provides use of the virus of the present invention in preparing a medicament for treating cancer.

[0166] The viruses of the present invention are particularly suitable for treating any solid tumor, including any adenocarcinoma, carcinoma, melanoma or sarcoma. For example, the viruses of the present invention can be used to treat head and neck cancer, prostate cancer, breast cancer, ovarian cancer, lung cancer, liver cancer, endometrial cancer, bladder cancer, gallbladder cancer, pancreatic cancer, colon cancer, kidney cancer, stomach / gastric cancer, esophageal cancer or cervical cancer, mesothelioma, melanoma or other skin cancer, lymphoma, glioma or other cancers of the nervous system, or sarcoma such as soft tissue sarcoma.

[0167] The viruses of the invention can be used to treat malignant tumors, including tumors that have metastasized from the original tumor site. In this embodiment, the virus can be administered to the primary tumor or one or more secondary tumors.

[0168] The virus of the present invention can be administered in combination with other therapeutic agents (including chemotherapy, targeted therapy, immunotherapy (including immune checkpoint blockade, i.e., administration of one or more antagonists of immune co-inhibitory pathways, and / or one or more agonists of immune co-stimulatory pathways)), and / or in combination with radiotherapy and / or in combination with any combination of these therapies. The therapeutic agent is preferably an anticancer agent.

[0169] The viruses of the invention can be administered in combination with a second virus, such as a second oncolytic virus.

[0170] For example, the therapeutic agent may comprise an immunogen (including a recombinant or naturally occurring antigen, including such an antigen or a combination of antigens delivered with DNA or RNA encoding it / them) to further stimulate an immune response, such as a cellular or humoral immune response, against tumor cells, particularly tumor neoantigens. The therapeutic agent may be an agent intended to increase or enhance the immune response, such as a cytokine, an agent intended to inhibit an immune checkpoint pathway or stimulate an immune enhancement pathway, or an agent that inhibits the activity of regulatory T cells (Treg) or myeloid-derived suppressor cells (MDSC).

[0171] The therapeutic agent can be a medicament known for existing cancer treatment. The therapeutic agent can be radiotherapy or a chemotherapeutic agent. The therapeutic agent can be selected from cyclophosphamide, alkylating agents such as cisplatin or melphalan, plant alkaloids and terpenoids such as vincristine or paclitaxel (Taxol), antimetabolites such as 5-fluorouracil, topoisomerase inhibitors type I or II such as camptothecin or doxorubicin, cytotoxic antibiotics such as actinomycin, anthracyclines such as epirubicin, glucocorticoids such as triamcinolone, protein, inhibitors of DNA and / or RNA synthesis such as methotrexate and dacarbazine, histone deacetylase (HDAC) inhibitors or any other chemotherapeutic agents.

[0172] The therapeutic agent can be one or a combination of an immunotherapeutic agent or an immunomodulator, such as a TLR agonist; a drug that downregulates T-regulatory cells, such as cyclophosphamide; or an agent intended to block immune checkpoints or stimulate immune enhancement pathways, including but not limited to monoclonal antibodies, such as 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, such as an anti-CTLA-4 antibody, a PD1 inhibitor, such as an anti-PD-1 antibody or a PD-L1 inhibitor, such as an anti-PD-L1 antibody. Such inhibitors, agonists and antibodies can be produced and tested by standard methods known in the art.

[0173] Immunotherapeutics may also include bispecific antibodies, cell-based therapies based on dendritic cells, NK cells, or engineered T cells (such as CAR-T cells or T cells expressing engineered T cell receptors). Immunotherapeutics also include agents that target specific genetic mutations occurring in tumors, agents designed to induce immune responses against specific tumor antigens or combinations of tumor antigens (including new antigens), and / or agents designed to activate the STING / cGAS pathway, TLRs, or other innate immune responses and / or inflammatory pathways, including intratumoral agents.

[0174] For example, the viruses of the invention can be used in combination with dacarbazine, a BRAF inhibitor and / or a PD1 or PD-L1 blocker to treat melanoma; in combination with paclitaxel, 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 blockade to treat lung cancer; in combination with cisplatin and / or radiation therapy to treat head and neck cancer.

[0175] The therapeutic agent may be an inhibitor of the indoleamine 2,3-dioxygenase (IDO) pathway. Examples of IDO inhibitors include epacadostat (INCB024360), 1-methyl-tryptophan, indoximod (1-methyl-D-tryptophan), GDC-0919 or F001287.

[0176] The mechanism of action of IDO in suppressing anti-tumor immune response can also suppress the immune response produced after oncolytic virus treatment. IDO expression is induced by Toll-like receptor (TLR) activation and interferon-γ (both of which can be caused by oncolytic virus infection). An embodiment of using oncolytic virus therapy to treat cancer includes oncolytic virus, and optionally GM-CSF and / or immune costimulatory pathway activation molecules and / or one or more other protein coding sequences (such as sequences encoding fusion-promoting proteins such as GALVR-) (including viruses expressing CTLA-4 inhibitors) and inhibitors of IDO pathways and optionally another antagonist of immune co-inhibitory pathways and / or one or more agonists of immune costimulatory pathways (including those targeting PD-1 and / or PD-L1) combination.

[0177] When therapeutic agents and / or radiotherapy are used in combination with the virus of the present invention, the administration of the virus and therapeutic agents and / or radiotherapy can be simultaneous or separated by time. The composition of the present invention can be administered before, simultaneously with, or after the therapeutic agent or radiotherapy. The method for treating cancer may include multiple administrations of the virus and / or therapeutic agent and / or radiotherapy of the present invention. In a preferred embodiment, in combination with immune checkpoint blockade or other immunoenhancers, the virus of the present invention is administered once or multiple times, and then the immune checkpoint blocker is administered simultaneously or one or more other immunoenhancers are administered afterwards, or without prior administration of the virus of the present invention, the virus of the present invention is administered simultaneously with the immune checkpoint blocker or one or more other immunoenhancers.

[0178] The virus of the present invention can be administered to a subject by any suitable route. Typically, the virus of the present invention is administered by direct intratumoral injection. Intratumoral injection includes direct injection into superficial skin, subcutaneous or lymph node tumors, and imaging guidance (including CT, MRI or ultrasound) injection into deeper or more difficult to locate sites, including in internal organs and other places. The virus can be administered into a body cavity, for example, into the pleural cavity, in the bladder, or by intraperitoneal administration. The virus may be injected into a blood vessel (preferably a blood vessel supplying the tumor).

[0179] Can use multiple known approaches and technology to human or animal subjects in vivo administration can be with the therapeutic agent of the virus combination of the present invention.For example, composition can be provided as injection, suspension or emulsion, and use conventional needle and syringe or use liquid jet injection system by parenteral, subcutaneous, oral, epidermal, intradermal, intramuscular, intraarterial, intraperitoneal, intravenous injection administration.Composition can be topically applied to skin or mucosal tissue (such as nasal, intratracheal, intestinal, sublingual, rectal or vaginal), or provide with the finely dispersed spray suitable for breathing or pulmonary administration.In a preferred embodiment, composition is administered by intravenous infusion, oral or directly into tumor.

[0180] The virus and / or therapeutic agent can be administered to the subject in an amount compatible with the therapeutically effective dosage composition. The administration of the virus of the present invention is for "treatment" purposes. As used herein, the term "therapeutic" or "treatment" includes any one or more of the following as its goal: preventing any metastasis or further metastasis from occurring; alleviating or eliminating symptoms; reducing or completely eliminating tumors or cancers, increasing the time to reach patient cancer progression; increasing the time to recurrence after treatment; or prolonging survival time.

[0181] Therapeutic treatment may be administered to stage I, II, III or IV cancer, preferably stage II, III or IV, more preferably stage III or IV cancer, before or after surgical intervention (i.e., after tumor recurrence after surgery or incomplete resection thereof), preferably before any surgical intervention (to resect primary or recurrent / metastatic disease), or after surgery or after recurrence after incomplete surgical resection of the disease (i.e., when there is residual tumor).

[0182] Therapeutic treatment may be performed after direct injection of the viral composition into the target tissue, which may be a tumor, a body cavity or a blood vessel. As a guide, the amount of virus administered in the case of HSV is between 10 4 Up to 10 10 pfu, preferably 10 5 Up to 10 9 In the case of HSV, an initial lower dose (e.g., 10 4 Up to 10 7 pfu) to seroconvert patients seronegative for HSV and boost in those seropositive, followed by higher doses (e.g., 10 6 Up to 10 9 pfu). Typically, up to 20 ml of a pharmaceutical composition consisting essentially of the virus and a suitable pharmaceutically acceptable carrier or diluent may be used for direct injection into a tumor, or up to 50 ml may be used for administration into a body cavity (the composition may be further diluted into a suitable diluent prior to administration) or into the bloodstream. However, for some oncolytic therapy applications, larger or smaller volumes may also be used, depending on the tumor and the route and site of administration.

[0183] The administration route and dosage described are intended only as a guide, as a skilled practitioner will be able to easily determine the optimal administration route and dosage. The dosage can be determined according to various parameters, in particular according to the location of the tumor, the size of the tumor, the age, weight and condition of the patient to be treated, and the administration route. Preferably, the virus is administered by direct injection into the tumor or in a body cavity. The virus can also be administered by direct injection into a blood vessel. The optimal administration route will depend on the location and size of the tumor. Multiple doses may be required to achieve an immunological or clinical effect, and if necessary, the dosage is usually administered at intervals of 2 days to 12 weeks, preferably at intervals of 3 days to 3 weeks. Repeated doses of up to 5 years or more can be given, preferably up to one month to two years, depending on the response rate of the tumor type being treated and the response of the specific patient and any combination therapy that may also be given.

[0184] The following examples illustrate the invention.

[0185] Example 1. Construction of the virus of the present invention

[0186] The viral species used to exemplify the present invention is HSV, in particular HSV1.

[0187] A diagram of the plasmids used is shown in Figure 2. A diagram of the viruses is shown in Figure 1. All viruses were constructed using HSV1 strain RH018A. Plasmids used for virus construction were generated by a combination of gene synthesis and subcloning performed by Genscript Inc.

[0188] A virus expressing anti-mouse CTLA4 as well as mouse GM-CSF and GALV was constructed by co-transfecting plasmid 77 with virus 16 DNA to insert GFP into virus 16 (by selecting for GFP-expressing plaques) to produce virus 25. GFP was then knocked out of virus 25 by co-transfecting virus 25 DNA with plasmid 119. This produced virus 27.

[0189] A virus expressing anti-human CTLA4 as well as human GM-CSF and GALV was constructed by co-transfecting plasmid 78 with virus 17 DNA to insert GFP into virus 17 (by selecting for GFP-expressing plaques) to produce virus 29. GFP was then knocked out of virus 29 by co-transfecting virus 29 DNA with plasmid 122. This produced virus 31.

[0190] Viruses expressing anti-mouse CTLA-4 and co-stimulatory ligands as well as mouse GM-CSF and GALV were constructed by co-transfecting a plasmid encoding GFP driven by the SV40 promoter between the mouse GM-CSF coding sequence and the anti-mouse CTLA-4 coding sequence with virus 27. GFP was then knocked out from the resulting virus by replacing GFP with plasmids encoding each individual mouse co-stimulatory ligand.

[0191] Viruses expressing anti-human CTLA-4 and co-stimulatory ligands as well as human GM-CSF and GALV were constructed by co-transfecting a plasmid encoding GFP driven by the SV40 promoter between the human GM-CSF coding sequence and the anti-human CTLA-4 coding sequence with virus 31. GFP was then knocked out from the resulting virus by replacing GFP with plasmids encoding each individual human co-stimulatory ligand.

[0192] Figure 4 Western blot demonstrating expression of anti-mouse CTLA-4 from virus 27 is shown.

[0193] Example 2. Effect of combined expression of GALV, GM-CSF and anti-CTLA4 from oncolytic viruses

[0194] The utility of the present invention was demonstrated in the following manner: A20 cells were administered to both flanks of Balb / c mice and A20 tumors were allowed to grow to approximately 0.5 cm in diameter.

[0195] Groups of mice were then administered (3 times per week for 1 week into only one flank (right tumor) of each mouse) the following treatments:

[0196] -50 μl vehicle (1 group);

[0197] -50 μl of 10 6 pfu / ml of HSV inserted only into mouse GM-CSF and GALVR- (virus 16);

[0198] -50 μl of 10 6 pfu / ml of GALVR-, mouse GM-CSF, and anti-mouse CTLA-4 antibody-inserted HSV (virus 27);

[0199] The effect on tumor growth was then observed for up to one month. The viral dose used was 5x10 4 pfu (50ul, 1x10 6 pfu / ml), administered three times in one week. This viral dose level was subtherapeutic for virus 16-uninjected tumors, allowing for a clear benefit from the delivery of other molecules encoded by virus 27. Figure 5 and Figure 6 Superior tumor control and shrinkage in uninjected tumors with anti-CTLA-4 expressing virus compared to virus 16 not expressing CTLA-4 was shown.

[0200] Example 3. Effect of combined expression of GALV, GM-CSF and anti-CTLA4 and anti-PD-1 from oncolytic viruses

[0201] A20 cells were administered to both flanks of Balb / c mice, and A20 tumors were allowed to grow to approximately 0.5 cm in diameter.

[0202] Groups of mice (10 per group) were then administered (3 times per week, into one flank only of each mouse, for 1 week) the following treatments:

[0203] - 50 μl vehicle;

[0204] - intraperitoneal anti-mouse PD1 (Bioxcell RMP-1-14 10 mg / kg, once every three days);

[0205] -50 μl of 10 7 pfu / ml of GALVR-inserted HSV (virus 27), mouse GM-CSF, and anti-mouse CTLA-4 antibody

[0206] -50 μl of 10 7 pfu / ml of GALVR-, mouse GM-CSF and anti-mouse CTLA-4 antibody (Virus 27) and intraperitoneal anti-mouse PD1 (10 mg / kg every 3 days) HSV (Group 3).

[0207] The effects on tumor growth were then observed for up to 80 days. When the viral therapy was combined with anti-PD1 therapy, there was excellent tumor control and shrinkage in both injected and uninjected tumors. This data is shown in Figure 7 shown.

[0208] Example 4. Effects of combined expression of GALV, GM-CSF and anti-human CTLA4 from oncolytic viruses alone and in combination with anti-PD-1

[0209] MC38 cells were administered to the flanks of C57BL / 6 mice engineered by gene editing to express human CTLA-4 instead of mouse CTLA-4. This made the mice susceptible to anti-human CTLA-4 antibodies such as ipilimumab. MC38 tumors were grown to approximately 0.5 cm in diameter.

[0210] Groups of mice (10 per group) were then administered (3 times per week, into one flank only of each mouse, for 2 weeks) the following treatments:

[0211] - 50 μl vehicle;

[0212] -50 μl of 10 8 pfu / ml of virus 17 (i.e., expressing hGM-CSF and GALV);

[0213] -50 μl of 10 8pfu / ml of virus 31 (i.e., expressing hGM-CSF, GALV, and anti-human CTLA-4);

[0214] -50 μl of 10 8 pfu / ml of virus 17 and intraperitoneal anti-mouse PD1 (10 mg / kg every 3 days);

[0215] -50 μl of 10 8 pfu / ml of virus and intraperitoneal anti-mouse PD1 (10 mg / kg every 3 days).

[0216] The effects on tumor growth were then observed for up to 35 days. Excellent tumor control and shrinkage were observed in tumors injected with viruses expressing anti-human CTLA-4, an effect that was further enhanced when combined with anti-PD1. When treatment with either virus was combined with anti-PD1 treatment, excellent tumor control and shrinkage were observed in non-injected tumors. The improvement was even more pronounced with the virus expressing anti-CTLA4. This data is shown in Figure 8 middle.

[0217] Example 5. Effect of combined expression of GALV, GM-CSF and anti-CTLA4 and anti-PD-1 from oncolytic viruses

[0218] A20 cells were administered to both flanks of Balb / c mice, and A20 tumors were allowed to grow to approximately 0.5 cm in diameter.

[0219] Groups of mice (10 per group) were then administered (3 times per week, into one flank only of each mouse, for 2 weeks) the following treatments:

[0220] -50 μl vehicle (1 group);

[0221] - intraperitoneal anti-mouse PD1 (Bioxcell RMP-1-14 10 mg / kg, once every three days);

[0222] -50 μl of 10 5 pfu / ml, 10 6 pfu / ml or 10 7 pfu / ml of HSV inserted only into mouse GM-CSF and GALVR- (3 groups);

[0223] -50 μl of 10 5 pfu / ml, 10 6 pfu / ml or 10 7 pfu / ml of HSV inserted only into GALVR-, mouse GM-CSF, and anti-mouse CTLA-4 antibody, and intraperitoneal anti-mouse PD1 (10 mg / kg, every three days) (Group 3);

[0224] -50 μl of 10 5 pfu / ml, 10 6 pfu / ml or 10 7 pfu / ml of HSV inserted only into mouse GM-CSF and GALVR- and intraperitoneal anti-mouse PD1 (10 mg / kg, once every three days) (3 groups);

[0225] -50 μl of 10 5 pfu / ml, 10 6 pfu / ml or 10 7 pfu / ml of HSV inserted into GALVR-, mouse GM-CSF and anti-mouse CTLA-4 antibody and intraperitoneal anti-mouse PD1 (10 mg / kg, once every three days) (3 groups).

[0226] The effects on tumor growth were then observed for up to one month. Superior tumor control and shrinkage was observed in both injected and uninjected tumors with the anti-CTLA-4 expressing virus compared to the other group, including through an improved dose response curve, which was further enhanced when combined with anti-PD1 therapy.

[0227] Example 6. Effect of combined expression of GALV, GM-CSF and anti-human CTLA4 from oncolytic virus itself and in combination with anti-PD-1

[0228] MC38 cells were administered to the flanks of C57BL / 6 mice engineered by gene editing to express human CTLA-4 instead of mouse CTLA-4. This made the mice susceptible to anti-human CTLA-4 antibodies such as ipilimumab. MC38 tumors were grown to approximately 0.5 cm in diameter.

[0229] Groups of mice (10 per group) were then administered (3 times per week, into one flank only of each mouse, for 2 weeks) the following treatments:

[0230] -50 μl vehicle (1 group);

[0231] - intraperitoneal anti-mouse PD1 (Bioxcell RMP-1-14 10 mg / kg, once every three days);

[0232] -50 μl of 10 5 pfu / ml, 10 6 pfu / ml or 10 7 pfu / ml of HSV inserted only into mouse GM-CSF and GALVR- (3 groups);

[0233] -50μl 10 5pfu / ml, 10 6 pfu / ml or 10 7 pfu / ml of HSV inserted into GALVR-, mouse GM-CSF, and anti-mouse CTLA-4 antibody, and intraperitoneal anti-mouse PD1 (10 mg / kg, every three days) (3 groups);

[0234] -50 μl of 10 5 pfu / ml, 10 6 pfu / ml or 10 7 pfu / ml of HSV inserted only into mouse GM-CSF and GALVR- and intraperitoneal anti-mouse PD1 (10 mg / kg, once every three days) (3 groups);

[0235] -50μl 10 5 pfu / ml, 10 6 pfu / ml or 10 7 pfu / ml of HSV inserted into GALVR-, mouse GM-CSF and anti-mouse CTLA-4 antibody and intraperitoneal anti-mouse PD1 (10 mg / kg, once every three days) (3 groups).

[0236] The effects on tumor growth were then observed for up to one month. Superior tumor control and shrinkage was observed in both injected and uninjected tumors with the anti-CTLA-4 expressing virus compared to the other group, including through an improved dose response curve, which was further enhanced when combined with anti-PD1 therapy.

[0237] Example 7. Effect of combined expression of GALV, GM-CSF, anti-CTLA4 and immune co-stimulatory pathway activating molecules from oncolytic viruses

[0238] The experiment described in Example 3 above was repeated, but mice were administered viruses that additionally expressed ligands of immune co-stimulatory pathways as well as GALV, mGM-CSF, and anti-CTLA4.

[0239] More specifically, groups of mice received:

[0240] (1) Vehicle;

[0241] (2) intraperitoneal anti-mouse PD1;

[0242] (3) HSV with mGM-CSF, GALVR-, and anti-CTLA4 inserted as in Example 2;

[0243] (4) HSV inserted with mGM-CSF, GALVR-, anti-CTLA4, and mouse CD40L;

[0244] (5) HSV inserted with mGM-CSF, GALVR-, anti-CTLA4, and mouse 4-1BBL;

[0245] (6) HSV inserted with mGM-CSF, GALVR-, anti-CTLA4, and mouse GITRL;

[0246] (7) HSV inserted with mGM-CSF, GALVR-, anti-CTLA4, and mouse OX40L;

[0247] (8) HSV inserted with mGM-CSF, GALVR-, anti-CTLA4, and mouse ICOSL;

[0248] (9) inserting mGM-CSF, GALVR- and anti-CTLA4 HSV and intraperitoneal anti-PD1 as in Example 2;

[0249] (10) HSV inserted with mGM-CSF, GALVR-, anti-CTLA4, and mouse CD40L, and intraperitoneal anti-PD1;

[0250] (11) insertion of mGM-CSF, GALVR-, anti-CTLA4, and mouse 4-1BBL-containing HSV and intraperitoneal anti-PD1;

[0251] (12) HSV inserted into mGM-CSF, GALVR-, anti-CTLA4, and mouse GITRL, and intraperitoneal anti-PD1;

[0252] (13) HSV with mGM-CSF, GALVR-, anti-CTLA4 and mouse OX40L, and intraperitoneal anti-PD1; or

[0253] (14) inserted mGM-CSF, GALVR-, anti-CTLA4 and mouse ICOSL-containing HSV, and intraperitoneal anti-PD1.

[0254] Excellent tumor control was observed with viruses expressing immune co-stimulatory ligands.

[0255] Collection Information

[0256] The following HSV1 strains were deposited by Replimune Limited at ECACC, Culture Collections, Public Health England, Porton Down, Salisbury, SP4 0JG, United Kingdom on 19 December 2016 and were assigned the designated accession numbers:

[0257] RH004A – Accession No. 16121902

[0258] RH015A – Accession No. 16121903

[0259] RH018A – Accession No. 16121904

[0260] RH021A – Accession number 16121905

[0261] RH023A – Accession number 16121906

[0262] RH031A – Accession number 16121907

[0263] RH040B – Accession No. 16121908

[0264] RH047A – Accession number 16121909

Claims

1. An oncolytic herpes simplex virus type 1 (HSV1) expressing a CTLA-4 inhibitor, wherein the HSV1 is a modified HSV1 derived from strain RH018A with a deposit number of ECACC 16121904, wherein the CTLA-4 inhibitor is an anti-CTLA-4 antibody or an antigen-binding fragment thereof.

2. The virus of claim 1, wherein the fragment comprises a scFv molecule.

3. The virus of claim 1, wherein the fragment is a scFv molecule linked to one or more IgG1 constant regions.

4. The virus of claim 3, wherein the antibody or fragment comprises a light chain variable region sequence connected to an IgG heavy chain.

5. The virus of claim 4, wherein the antibody or fragment comprises (a) the light chain variable region sequence shown in SEQ ID NO: 1 and the heavy chain variable region sequence shown in SEQ ID NO: 3; or (b) the light chain variable region sequence shown in SEQ ID NO: 11 and the heavy chain variable region sequence shown in SEQ ID NO:

12.

6. The virus of claim 5, wherein the antibody or fragment comprises (a) the amino acid sequence of SEQ ID NO: 9; or (b) the amino acid sequence of SEQ ID NO:

14.

7. The virus of claim 6, wherein the antibody or fragment is encoded by the nucleotide sequence of SEQ ID NO:

10.

8. The virus of claim 6, wherein the antibody or fragment is encoded by the nucleotide sequence of SEQ ID NO:

15.

9. The virus of claim 1, wherein the virus further comprises an immune co-stimulatory pathway activating molecule or a gene encoding an immune co-stimulatory pathway activating molecule.

10. The virus of claim 9, wherein the immune co-stimulatory pathway activating molecule encoding gene encodes CD40 ligand (CD40L), ICOS ligand, GITR ligand, 4-1-BB ligand, OX40 ligand, TL1A, CD30 ligand, CD27 or flt3 ligand.

11. The virus of claim 10, wherein the gene encoding the immune co-stimulatory pathway activating molecule encodes a CD40 ligand, a GITR ligand, a 4-1-BB ligand, an OX40 ligand or an ICOS ligand.

12. The virus of claim 1, further comprising a gene encoding a fusogenic protein.

13. The virus of claim 12, wherein the fusogenic protein is selected from the group consisting of 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 a glycoprotein from gibbon ape leukemia virus (GALV), murine leukemia virus (MLV), Mason-Pfizer monkey virus (MPMV), or equine infectious anemia virus (EIAV) from which the R peptide has been deleted.

14. The virus of claim 13, wherein the fusogenic protein is a glycoprotein from Gibbon Ape Leukemia Virus (GALV) and the R transmembrane peptide has been mutated or removed (GALV-R-).

15. The virus of claim 9, encoding more than one immune co-stimulatory pathway activating molecule.

16. The virus of claim 1, wherein the HSV1 does not express functional ICP34.

5.

17. The virus of claim 1, wherein the HSV1 does not express functional ICP47.

18. The virus of claim 1, wherein the HSV1 expresses the US11 gene as an immediate early gene.

19. The virus of claim 1, wherein the anti-CTLA-4 inhibitory protein encoding gene has been inserted into the ICP34.5 encoding gene locus by insertion, partial deletion or complete deletion.

20. The virus of claim 19, wherein the anti-CTLA-4 inhibitory protein encoding gene is contained in a cassette that also contains one or more immunostimulatory genes and / or fusogenic protein encoding genes.

21. The virus of claim 20, wherein the immunostimulatory gene is a gene encoding an immune co-stimulatory pathway activating molecule.

22. The virus of claim 1, wherein the sequence encoding the CTLA-4 inhibitor is codon optimized to increase expression levels in target cells.

23. The virus of claim 22, wherein the virus further comprises a gene encoding an immune co-stimulatory pathway activating molecule and / or a fusogenic protein, and the sequence encoding the immune co-stimulatory pathway activating molecule and / or the sequence encoding the fusogenic protein is codon-optimized to increase the expression level in target cells.

24. The virus of claim 1, expressing three heterologous genes, wherein each of the three heterologous genes is driven by a different promoter selected from the group consisting of a CMV promoter, a RSV promoter, a SV40 promoter, and a retroviral LTR promoter.

25. The virus of claim 24, which expresses four heterologous genes driven by each of a CMV promoter, a RSV promoter, a SV40 promoter, and a retroviral LTR promoter, respectively.

26. The virus of claim 24, wherein the retroviral LTR promoter is from MMLV.

27. The virus of claim 25, wherein the retroviral LTR promoter is from MMLV.

28. The virus of claim 1, expressing three heterologous genes, wherein each of the three heterologous genes is terminated by a different polyadenylation sequence selected from the group consisting of BGH, SV40, HGH, and RBG polyadenylation sequences.

29. The virus of claim 28, which expresses four heterologous genes terminated by each of the BGH, SV40, HGH and RBG polyadenylation sequences, respectively.

30. A pharmaceutical composition comprising the virus according to any one of claims 1 to 29 and a pharmaceutically acceptable carrier or diluent.

31. Use of a virus according to any one of claims 1 to 29 for the preparation of a medicament for use in a method for the treatment of cancer.

32. The use of claim 31, wherein the method comprises administering an additional anti-cancer agent.

33. The use of claim 32, wherein the additional anti-cancer agent is selected from agents targeting immune co-inhibitory or immune co-stimulatory pathways, irradiation and / or chemotherapy, agents targeting specific genetic mutations occurring in tumors, agents designed to induce an immune response to one or more tumor antigens or neoantigens, cell products derived from T cells or NK cells, agents designed to stimulate STING, cGAS, TLR or other innate immune responses and / or inflammatory pathways, a second virus, and combinations thereof.

34. The use of claim 33, wherein the agent targeting the immune co-inhibitory pathway is a PD-1 inhibitor, a PD-L1 inhibitor, a LAG-3 inhibitor, a TIM-3 inhibitor, a VISTA inhibitor, a CSF1R inhibitor, an IDO inhibitor, a KIR inhibitor, a SLAMF7 inhibitor, a CEACAM1 inhibitor or a CD47 inhibitor, and / or the agent targeting the immune co-stimulatory pathway is a GITR agonist, a 4-1-BB agonist, an OX40 agonist, a CD40 agonist or an ICOS agonist.

35. The use of claim 33, wherein the second virus is an oncolytic virus.

36. The use of claim 32, wherein the additional anti-cancer agent comprises an antibody.

37. The use of any one of claims 32 to 36, wherein the virus and the additional anti-cancer agent are administered separately.

38. The use of any one of claims 32 to 36, wherein the virus and the additional anti-cancer agent are administered simultaneously.

39. The use of claim 31, wherein the cancer is a solid tumor.

40. The use of any one of claims 32-36, wherein the method comprises administering an inhibitor of the indoleamine 2,3-dioxygenase (IDO) pathway and another antagonist of an immune co-inhibitory pathway or an agonist of an immune co-stimulatory pathway.

41. Use of a virus according to any one of claims 1 to 29 in combination with an additional anti-cancer agent for the preparation of a medicament for use in a method of treating cancer.

42. The use of claim 41, wherein the additional anti-cancer agent is selected from agents targeting immune co-inhibitory or immune co-stimulatory pathways, irradiation and / or chemotherapy, agents targeting specific genetic mutations occurring in tumors, agents designed to induce an immune response to one or more tumor antigens or neoantigens, cell products derived from T cells or NK cells, agents designed to stimulate STING, cGAS, TLR or other innate immune responses and / or inflammatory pathways, a second virus, and combinations thereof.

43. The use of claim 42, wherein the second virus is an oncolytic virus.

44. The use of claim 42, wherein the agent targeting the immune co-inhibitory pathway is a PD-1 inhibitor, a PD-L1 inhibitor, a LAG-3 inhibitor, a TIM-3 inhibitor, a VISTA inhibitor, a CSF1R inhibitor, an IDO inhibitor, a KIR inhibitor, a SLAMF7 inhibitor, a CEACAM1 inhibitor or a CD47 inhibitor, and / or the agent targeting the immune co-stimulatory pathway is a GITR agonist, a 4-1-BB agonist, an OX40 agonist, a CD40 agonist or an ICOS agonist.

45. The use of claim 41, wherein the additional anti-cancer agent is an antibody.

46. ​​The use of claim 41, wherein the additional anticancer agent is an inhibitor of the indoleamine 2,3-dioxygenase (IDO) pathway and another antagonist of an immune co-inhibitory pathway or an agonist of an immune co-stimulatory pathway.

47. The use of claim 41, wherein the medicament is suitable for separate administration of the virus and the additional anti-cancer agent.

48. The use of claim 41, wherein the medicament is suitable for simultaneous administration of the virus and the additional anti-cancer agent.

49. The use of claim 41, wherein the cancer is a solid tumor.

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