oncolytic virus, pharmaceutical composition, manufacturing product, method for treating cancer and u

专利摘要:
The present invention relates to an oncolytic virus encoding a ctla-4 inhibitor such as an anti-ctla-4 antibody or antigen binding fragment thereof.
公开号:BR112019013215A2
申请号:R112019013215
申请日:2018-01-09
公开日:2019-12-10
发明作者:Stuart Coffin Robert
申请人:Replimune Ltd；
IPC主号:

专利说明:

“ONCOLYTIC VIRUS, PHARMACEUTICAL COMPOSITION, MANUFACTURING PRODUCT, METHOD FOR TREATING CANCER AND USE”
Field of the Invention [001] The invention relates to an oncolytic immunotherapeutic agent and the use of the oncolytic immunotherapeutic agent in the treatment of cancer.
Background of the Invention [002] Viruses have a unique ability to enter cells with high efficiency. After entering cells, viral genes are expressed and the virus replicates. This usually results in the death of the infected cell and the release of the cell's antigenic components as the cell ruptures when it dies. As a result, virus-mediated cell death tends to result in an immune response to these cell components, including both those derived from the host cell and those encoded or incorporated into the virus itself, and enhanced due to the host's recognition of so-called molecular patterns associated with damage (DAMPs), which helps activate the immune response.
[003] Viruses also engage with various mediators of the innate immune response as part of the host's response to the recognition of a viral infection, for example, through tolllike receptors and cGAS / STING signaling and the recognition of molecular patterns associated with pathogens (PAMPs), resulting in the activation of responses to interferon and inflammation, which are also immunogenic signs for the host. These immune responses can result in the immunogenic benefit for cancer patients, so immune responses to tumor antigens provide an overall systemic benefit, resulting in the treatment of tumors that have not been infected with the virus, including micrometastatic disease, and provide vaccination against relapse .
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2/52 [004] The direct combined (oncolytic) effects of the virus, and immune responses against tumor antigens (including non-autoantigen neo-antigens, that is, derived from the specific mutated genes in individual tumors) are called “oncolytic immunotherapy” .
[005] 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 a variety of medical and biotechnological applications. For example, viruses that express heterologous therapeutic genes can be used for gene therapy. In the context of oncolytic immunotherapy, distributed genes may include those that encode specific tumor antigens, genes designed to induce immune responses or increase the immunogenicity of antigens released after viral replication and cell death, genes designed to shape the immune response that is generated, genes for increase the state of general immune activation of the tumor or genes to increase the direct oncolytic properties (i.e., cytotoxic effects) of the virus. Importantly, viruses have the ability to deliver encoded molecules that are designed to help initiate, improve or shape systemic anti-tumor immune response directly and selectively to tumors, which can have benefits, for example, of reduced toxicity or beneficial effects of concentration on tumors (including those not infected by the virus) rather than off-target effects on normal tissues (ie, non-cancerous), compared to systemic administration of those same molecules or systemic administration of other molecules targeting the same pathways.
[006] A number of viruses, including, for example, herpes simplex virus (HSV), have been shown to be useful in the oncolytic treatment of cancer. HSV for use in cancer treatment for cancer must be inactivated so that it is no longer pathogenic, but can still enter and kill cells
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3/52 tumor. A series of inactivation mutations for HSV, including disruption of the genes encoding ICP34.5, ICP6 and / or thymidine kinase, have been identified, which do not prevent the virus from replicating in culture or in tumor tissue in vivo, but prevent significant replication in normal tissue. HSVs in which only the ICP34.5 genes have been disrupted replicate in many types of tumor cells in vitro, and selectively replicate in tumor tissue, but not in the surrounding tissues, in tumor models in mice. Clinical trials of deleted ICP34.5 or deleted ICP34.5 and ICP6, HSV also showed safety and selective replication in tumor tissue in humans.
[007] As discussed above, an oncolytic virus, including HSV, can also be used to deliver a therapeutic gene in the treatment of cancer. A deleted ICP34.5 virus of this type additionally deleted for ICP47 and encoding a heterologous gene for GM-CSF has also been tested in clinical trials, including a Phase 3 melanoma trial, in which safety and efficacy in humans have been shown. GM-CSF is a proinflammatory cytokine that has multiple functions, including the stimulation of monocytes to leave the circulation and migrate to the tissue where they proliferate and mature to macrophages and dendritic cells. GM-CSF is important for the proliferation and maturation of antigen presenting cells, the activity of which is necessary for the activation of an antitumor immune response. The trial data demonstrated that tumor responses could be seen in injected tumors, and to a lesser extent in uninjected tumors. Responses tend to be highly durable (months to years), and a survival benefit appeared to be obtained in responder patients. Each of these indicated involvement of the immune system in the treatment of cancer, in addition to the direct oncolytic effect. However, these and other data with oncolytic viruses have shown that, in general, all tumors that do not respond to treatment, and not all patients obtain a survival advantage. Of that
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4/52 way, improvements to the oncolytic therapy technique are clearly needed.
[008] Recently, it has been shown that oncolytic immunotherapy can result in additive or synergistic therapeutic effects in conjunction with the blocking of the immune co-inhibitory pathway (ie, inhibition or “antagonism” of immunological checkpoint pathways, also called co-inhibitory pathways). Blocking the immune co-inhibitory pathway is intended to block the host's immune inhibitory mechanisms that normally serve to prevent the occurrence of autoimmunity. However, in cancer patients these mechanisms can also serve to inhibit induction or block the potentially beneficial effects of any immune response induced to tumors.
[009] Systemic blocking of these pathways by agents that target molecule associated with cytotoxic T-lymphocyte -4 (CTLA-4), PD-1 or PDL1 have shown efficacy of a number of tumor types, including melanoma and lung cancer. However, unsurprisingly, based on the mechanism of action, off-target toxicity can occur due to the induction of autoimmunity. Even so, these agents are tolerable enough to provide great clinical utility. Another immune co-inhibitory pathway and related targets for whose agents (mainly antibodies) are under development include LAG-3, TIM-3, VISTA, CSF1 R, IDO, CEACAM1, CD47. The optimal clinical activity of these agents, for example, PD1, PDL1, LAG-3, TIM-3, VISTA, CSF1R, IDO, CD47, CEACAM1, may require systemic administration or presence in all tumors, due to the mechanism of action , including targeting the interface of immune effector cells with tumors or other inhibitory immune mechanisms in / from tumors. In some cases, the more localized presence, for example, only in some tumors or in some lymph nodes can also be effective in the best way, for example, for targeting agents
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CTLA-4.
[010] An alternative approach to increase the anti-tumor immune response in cancer patients is to target (activate) immune co-stimulatory pathways, that is, as opposed to inhibiting immune co-inhibitory pathways. These pathways send activation signals to T cells and other immune cells, usually resulting from the interaction of the relevant ligands in the antigen presenting cells (APCs) and in the relevant receptors on the surface of T cells and other immune cells. These signals, depending on the ligand / receptor, can result in increased activation of T cells and / or APCs and / or NK cells and / or B cells, including particular subtypes, differentiation and increased proliferation of T cells and / or APCs and / or NK cells and / or B cells, including particular subtypes, or suppression of inhibitory T cell activity as regulatory T cells. Therefore, it is expected that the activation of these pathways will result in improved antitumor immune responses, but it can also be expected that the systemic activation of these pathways, that is, the activation of immune responses, in general, instead of specifically anti-tumor immune responses. or selectively, it would result in considerable off-target toxicity in non-tumor tissue, the degree of off-target toxicity depending on the immune co-stimulatory pathway being targeted. However, agents (mainly agonist antibodies or less frequently the soluble ligand of the receptor in question) that target immune co-stimulatory pathways, including agents that target GITR, 4-1-BB, 0X40, CD40 or ICOS, CD40 or ICOS, and are intended for systemic use (ie, intravenous delivery) have been proposed or are in clinical development.
[011] For many of these approaches that target the coibibory or coibibitory immune pathways to be successful, pre-existing immune responses to the tumors are necessary, that is, so that a pre-existing immune response can be potentiated or a block to a answer
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6/52 antitumor immune system can be relieved. The presence of an inflamed tumor microenvironment, which is indicative of this ongoing response, is also necessary. Pre-existing immune responses to neoantigens appear to be particularly important for the activity of blocking drugs and related to the immune co-inhibitory pathway. Only a few patients can have a continuous 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 that can induce an inflamed tumor microenvironment are attractive for use in combination with drugs that potentiate the blockade of the immune and coibitory pathways. This probably explains the promising combined antitumor effects of oncolytic and immune co-inhibitory pathway agents in mice and humans that have been observed so far.
[012] The above discussion demonstrates that there is still a lot of room for improvement of oncolytic agents and cancer therapies using oncolytic agents, antitumor immune responses and drugs that target immune co-stimulatory or co-stimulatory pathways.
Brief Description of the Invention [013] The present invention provides oncolytic virus that expresses a CTLA-4 inhibitor. The virus may also comprise other immunomodulatory agents. In particular, viruses can comprise GM-CSF and / or at least one molecule that targets an immune co-stimulatory pathway. The CTLA-4 inhibitor acts to block a co-inhibitory pathway, that is, it interferes with the interaction between CTLA-4 and B7. GM-CSF assists in the induction of an inflammatory tumor microenvironment and stimulates the proliferation and maturation of antigen presenting cells, including dendritic cells that assist in the induction of an antitumor immune response. These immune responses can be
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7/52 amplified through the activation of an immune co-stimulatory pathway or pathways that use a molecule that activates the immune co-stimulatory pathway or molecules also distributed by the oncolytic virus.
[014] Oncolytic viruses replicate within tumors, causing tumor cell lysis and the release of tumor antigens, combined with local inflammation and activation of innate immune responses, all of which are beneficial for the activation of an anti-tumor immune response and for the activity of inhibitors of CTLA-4 / B7 interaction.
[015] The distribution of molecules that inhibit CTLA4 / B7 interaction directly in a tumor that initiates an immune response, including where it is expected to travel to drainage lymph nodes, focus on immune enhancement by the inhibitor in the tumor and, therefore, antigens tumor present within it, reduce systemic toxicity and block the activation of regulatory T cells (Treg) that would otherwise inhibit the activation of T cells at the site of initiation of immune response. The use of an oncolytic virus to deliver molecules that target CTLA-4 and, optionally, molecules that target tumor immune co-stimulatory pathways focus on amplifying immune effects on antitumor immune responses, and reduces the amplification of immune responses to non-tumor antigens. Thus, immune cells in tumors and tumor draining lymph nodes are selectively affected by molecules expressed by the virus, rather than immune cells in general. This results in improved effectiveness of immune cell stimulation, and can also result in reduced off-target toxicity. It is also important to focus on the blocking effects of the combined systemic immune co-inhibitory pathway and activation of the immune co-stimulatory pathway in tumors, that is, so that amplified immune responses from which co-inhibitory blocks are released are anti-tumor immune responses, rather than responses to non-tumor antigens.
[016] The invention uses the fact that, when distributed by a
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8/52 oncolytic virus, the site of action of blocking CTLA-4 and, optionally, activation of the co-stimulatory pathway and the expression of GM-CSF is the tumor and / or tumor draining lymph nodes, but the results of this systemic activation antitumor (an amplified systemic antitumor immune response) are systemic. This affects target tumors in general, and not just tumors to which the oncolytic virus has distributed the immunomodulatory molecule or molecules. Oncolytic viruses of the invention, therefore, provide improved cancer treatment by generating enhanced immune responses focused on the tumor. The oncolytic virus of the invention also offers enhanced anti-tumor immune stimulating effects, so that the immune-mediated effects on tumors that are not destroyed by oncolysis, including micrometastatic diseases, are improved, resulting in the more effective destruction of these tumors, and anti-tumor vaccination of effective long-term approach to prevent future relapses and improve overall survival.
[017] Antitumor efficacy is enhanced when an oncolytic virus of the invention is used as a single agent and also when the virus is used in combination with other anticancer modalities, including chemotherapy, treatment with targeted agents, radiation and, in preferred embodiments, drugs blocking immunological control points (i.e., antagonists of an immune co-inhibitory pathway, for example, antibodies against PD1 or PD-L1) and / or agonists of an immune co-stimulatory pathway.
[018] Consequently, the present invention provides an oncolytic virus that encodes a CTLA-4 inhibitor. The CTLA-4 inhibitor is preferably an anti-CTLA-4 antibody or molecule similar to the antibody, or an antigen-binding fragment thereof.
[019] The virus may further comprise: (i) a gene encoding GM-CSF; and / or (ii) a molecule that activates the immune co-stimulatory pathway or a gene encoding a molecule that activates the immune co-stimulatory pathway. The virus
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9/52 can encode more than one molecule / gene that activates the immune co-stimulatory pathway.
[020] The molecule that activates the immune co-stimulatory pathway is preferably GITRL, 4-1-BBL, OX40L, ICOSL or CD40L or a modified version of any of these. Examples of modified versions include agonists of a co-stimulatory pathway, which are secreted instead of being bound to the membrane, and / or agonists modified so that protein multimers are formed.
[021] The virus can be a modified clinical isolate, such as a modified clinical isolate of a virus, in which the clinical isolate kills two or more tumor cell lines more quickly and / or at a lower dose in vitro than one or more clinical reference isolates of the same virus species.
[022] The virus is preferably a herpes simplex virus (HSV), like HSV1. HSV typically does not express functional ICP34.5 and / or functional ICP47 and / or expresses the US11 gene as an immediate initial gene.
[023] The invention also provides:
a pharmaceutical composition comprising a virus of the invention and a pharmaceutically acceptable carrier or diluent;
the virus of the invention for use in a method of treating the human or animal body by therapy;
the virus of the invention for use in a method for treating cancer, wherein the method optionally comprises administering an additional anti-cancer agent;
a manufacturing product comprising a virus of the invention, in a sterile vial, ampoule or syringe;
a method for treating cancer, which comprises administering a therapeutically effective amount of a virus, a pharmaceutical
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10/52 or a composition of the invention for a patient in need thereof, wherein the method optionally comprises administering an additional anti-cancer agent;
use of a virus of the invention in the manufacture of a medicament for use in a method for treating cancer, wherein the method optionally comprises administering an additional anti-cancer agent.
Brief Description of the Figures [024] Figure 1 represents the structures of the viruses used to construct exemplary viruses of the invention comprising human antiCTLA-4 or anti-mouse constructs, which are codon-optimized secreted scFv molecules linked to human IgG 1 Fc regions. or mouse. ScFvs contain light and heavy variable chains of 9D9 (the first mouse antibody initially used to validate CTLA-4; document WO 2007/123737 mouse version) or of ipilimumab. (WO 2014/066532; human version) linked by the 15-mer [G4Ô] 3 (GGGGSGGGGSGGGGS). Viruses are modified versions of the HSV1 strain RH018A (clinical strain 18). The ICP34.5 and ICP47 genes are inactivated in viruses. The US11 gene is placed under the control of the ICP47 early gene promoter by deletion of the ICP47 promoter. An expression cassette is inserted into the loci of the ICP34.5 gene. In virus 17, the expression cassette includes the human GM-CSF gene under the control of a CMV promoter and the GALV gene under the control of an RSV promoter. Virus 16 is the same as virus 17, except that human GM-CSF is included instead of mouse GM-CSF. Viruses 25 and 29 are the same as viruses 16 and 17, respectively, except that each additionally comprises a GFP gene under the control of an MMLV promoter in the expression cassette. Viruses 27 and 31 are the same as viruses 25 and 29, respectively, except that the GFP gene is replaced by mouse anti-CTLA4 and human anti-CTLA4, respectively.
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11/52 [025] Figure 2 represents the structures of the plasmids used to construct the exemplary viruses of the invention.
[026] Figure 3 shows the structure of human or anti-mouse anti-CTLA-4 constructs which are codonotimized secreted scFv molecules linked to human or mouse IgG1 Fc regions. ScFvs contain linked light and heavy ([G4S] s) variable chains of 9D9 (the first mouse antibody initially used to validate CTLA-4; US patent 2011044953 mouse version) or ipilimumab (US patent 20150283234; human version). The resulting structure of the CTLA-4 inhibitor is also shown.
[027] Figure 4 is a Western blot showing that mouse antiCTLA-4 is expressed from virus 27. The gel used was a reduced denatured PVDF membrane tris-glycine gel. Anti-CTLA-4 was detected with the use of a mouse anti-IgG1 antibody labeled with alkaline phosphatase. Lane 1: wide spectrum ladder; lane 2 virus 27 pure supernatant; lane 3 virus 27 supernatant diluted 1 in 2; lane 4 virus 27 supernatant diluted 1 in 4; lane 5 virus 27 supernatant diluted 1 in 8; lane 6 virus 27 supernatant diluted 1 in 16; lane 7 virus 27 supernatant diluted 1 in 32; lane 8 negative control virus (pure supernatant). The expected size of anti-CTLA-4 (reduced) is 57 kDa.
[028] Figure 5 shows superior tumor control and shrinkage in uninjected tumors of a virus that expresses anti-mCTLA4 (virus 27), compared to an identical virus that does not otherwise express CTLA-4 (virus 16) . The virus dose used was 5 x 10 ⁴ pfu (50 µl of 1 x 10 ⁶ pfu / ml in each case), given three times over a week. This virus dose level is subtherapeutic for tumors not injected for viruses 16, which allows the benefits of the distribution of the additional molecule encoded by virus 27 to be clearly seen.
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12/52 [029] Figure 6 shows superior tumor control and shrinkage in both injected and uninjected tumors, of a virus that expresses antimCTLA-4 (virus 27), compared to an identical virus that does not otherwise express CTLA-4 (virus 16). The dose of the virus used was 5 x 10 ⁴ pfu for one week in the right tumor, of a virus that expresses anti-mCTLA-4 (virus 27) compared to an identical virus that would not otherwise express CTLA4 (virus 16) . Each line represents a different mouse.
[030] Figure 7 shows the effect of the combined treatment of bilateral tumors of mouse A20 with the use of anti-PD1 and virus 27 that expresses mGM-CSF, GALVR and anti-mCTLA-4. The upper panel shows the effect of anti-PD1 alone on both injected (right) and non-injected (left) tumors. The middle panel shows the effect of virus 27 alone on both injected (right) and non-injected (left) tumors. The lower panel shows the superior tumor control of the tumor and the shrinkage obtained when anti-PD1 and virus 27 are both injected into the right tumor. The enhanced anti-tumor effect of the combined treatment is seen in both tumors, injected (right) and non-injected (left). Each line represents a different mouse.
[031] Figure 8 shows the superior tumor control and shrinkage effects obtained from virus 31 that expresses hGM-CSF, GALVR and human anti-CTLA4, compared to virus 17 that expresses only hGM-CSF and GALVR in MC38 tumors from knock-in mouse that express human CTLA-4. The anti-tumor effects of the virus 31 are observed when the virus is administered alone or in combination with anti-PD1. Superior tumor control and shrinkage in tumors injected with virus 31 that expresses human antiCTLA-4, compared to the identical virus that does not express human anti-CTLA4 (left panel). This effect is further enhanced when treatment with the virus is combined with treatment with anti-PD1. Superior tumor control and shrinkage are also seen in tumors not
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13/52 injected (right panel) when treatment with both viruses is combined with treatment with anti-PD1. This improvement is more pronounced for virus 31 that expresses anti-CTLA-4 than for virus 17, which does not. Each line represents a different mouse.
Brief Description of the Sequence Listing [032] SEQ ID NO: 1 is the light chain variable region amino acid sequence of the human CTLA-4 antibody used in the Examples.
[033] SEQ ID NOs: 2 is the complete light chain amino acid sequence comprising the light chain variable region amino acid sequence of the human CTLA-4 antibody used in the Examples.
[034] SEQ ID NO: 3 is the heavy chain variable region amino acid sequence of the human CTLA-4 antibody used in the Examples.
[035] SEQ ID NO: 4 is the heavy chain CH1 amino acid sequence of the human CTLA-4 antibody used in the Examples.
[036] SEQ ID NO: 5 is the CH2 / 3 heavy chain amino acid sequence of the human CTLA-4 antibody used in the Examples.
[037] SEQ ID NO: 6 is the complete heavy chain amino acid sequence of the human CTLA-4 antibody used in the Examples.
[038] SEQ ID NO: 7 is the amino acid sequence of the signal peptide present in the CTLA-4 antibodies of the Examples.
[039] SEQ ID NO: 8 is the amino acid sequence of the linker present between the light chain variable region of the heavy chain variable region in the CTLA-4 antibodies of the Examples.
[040] SEQ ID NO: 9 is the amino acid sequence of the human CTLA-4 scFv antibody of the Examples.
[041] SEQ ID NO: 10 is the nucleotide sequence of the human CTLA-4 scFv antibody of the Examples.
[042] SEQ ID NO: 11 is the region amino acid sequence
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14/52 murine CTLA-4 antibody light chain variable used in the Examples.
[043] SEQ ID NO: 12 is the heavy chain variable region amino acid sequence of the murine CTLA-4 antibody used in the Examples.
[044] SEQ ID NO: 13 is the complete heavy chain amino acid sequence of the murine CTLA-4 antibody used in the Examples.
[045] SEQ ID NO: 14 is the amino acid sequence of the murine CTLA-4 scFv antibody of the Examples.
[046] SEQ ID NO: 15 is the nucleotide sequence of the murine CTLA-4 scFv antibody of the Examples.
[047] SEQ ID NO: 16 is the nucleotide sequence of the murine CTLA-4 scFv antibody from the Examples with inserted restriction sites for cloning purposes located at the N and C terminations, which is present in the exemplary virus. The restriction sites are the first six and the last eight nucleotides in the sequence.
[048] SEQ ID NO: 17 is the nucleotide sequence of the human CTLA-4 scFv antibody of the Examples with inserted restriction sites for cloning purposes located at the N and C terminations, which is present in the exemplary virus. The restriction sites are the first six and the last eight nucleotides in the sequence.
[049] SEQ ID NO: 18 is the mouse GM-CSF nucleotide sequence.
[050] SEQ ID NO: 19 is the nucleotide sequence of a codon-optimized version of mouse GM-CSF.
[051] SEQ ID NO: 20 is the nucleotide sequence of human GM-CSF.
[052] SEQ ID NO: 21 is the nucleotide sequence of a codon-optimized version of human GM-CSF.
[053] SEQ ID NO: 22 is the amino acid sequence of GM-CSF
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15/52 of mouse.
[054] SEQ ID NO: 23 is the amino acid sequence of human GM-CSF.
[055] SEQ ID NO: 24 is the nucleotide sequence of GALV-R [056] SEQ ID NO: 25 is the nucleotide sequence of a codon-optimized version of GALV-R-.
[057] SEQ ID NO: 26 is the amino acid sequence of GALV-R-.
[058] SEQ ID NO: 27 is the nucleotide sequence, a codon-optimized version of a membrane-bound version of the human / mouse hybrid of CD40L.
[059] SEQ ID NO: 28 is the amino acid sequence of a human / mouse hybrid membrane-bound version of CD40L.
[060] SEQ ID NO: 29 is the nucleotide sequence of a codon-optimized version of a secreted multimeric version of human CD40L.
[061] SEQ ID NO: 30 is the amino acid sequence of a secreted multimeric version of human CD40L.
[062] SEQ ID NO: 31 is the nucleotide sequence of a codon-optimized version of a secreted multimeric version of mouse CD40L.
[063] SEQ ID NO: 32 is the amino acid sequence of a secreted multimeric version of mouse CD40L.
[064] SEQ ID NO: 33 is the nucleotide sequence of human wild-type CD40L.
[065] SEQ ID NO: 34 is the amino acid sequence of wild-type human CD40L.
[066] SEQ ID NO: 35 is the nucleotide sequence of CD40L from
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16/52 wild type mouse.
[067] SEQ ID NO: 36 is the amino acid sequence of wild type mouse CD40L.
[068] SEQ ID NO: 37 is the nucleotide sequence of the CMV promoter.
[069] SEQ ID NO: 38 is the nucleotide sequence of the RSV promoter.
[070] SEQ ID NO: 39 is the nucleotide sequence of polyA BGH.
[071] SEQ ID NO: 40 is the nucleotide sequence of the final polyA SV40.
[072] SEQ ID NO: 41 is the nucleotide sequence of rabbit polyA beta-globulin.
[073] SEQ ID NO: 42 is the nucleotide sequence of GFP.
[074] SEQ ID NO: 43 is the nucleotide sequence of MMLV retroviral LTR.
[075] SEQ ID NO: 44 is the nucleotide sequence of the EF1a promoter.
[076] SEQ ID NO: 45 is the nucleotide sequence of the SV40 promoter.
[077] SEQ ID NO: 46 is the nucleotide sequence of polyA HGH.
Detailed Description of the Invention
Oncolitic Virus [078] The virus of the invention is oncolitic. An oncolytic virus is a virus that infects and replicates itself in tumor cells, so that tumor cells are killed. Therefore, the virus of the invention is competent for replication. Preferably, the virus is selectively competent for replication in tumors.
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A virus is selectively competent to replicate in tumor tissue if it replicates more effectively in tumor tissue than in non-tumor tissue. The ability of a virus to replicate in different types of tissues can be determined using standard techniques.
[079] The virus of the invention can be any virus that has these properties, including a herpes virus, poxvirus, adenovirus, retrovirus, rabdovirus, paramixovirus or reovirus, or any species or strain within these larger groups. The viruses of the invention can be wild type (i.e., unchanged from parent virus species) or with gene interruptions or gene additions. Which of these is the case will depend on the type of virus to be used. Preferably, the virus is a species of herpes virus, more preferably an HSV strain, including HSV1 and HSV2 strains, and is most preferably an HSV1 strain. In particularly preferred embodiments, the virus of the invention is based on a clinical isolate of the virus species to be used. The clinical isolate may have been selected based on it, having particularly advantageous properties for the treatment of cancer.
[080] The virus can be a modified clinical isolate, in which the clinical isolate kills two or more tumor cell lines more quickly and / or at a lower dose in vitro than one or more reference clinical isolates of the same virus species . Typically, the clinical isolate kills two or more tumor cell lines within 48 hours, preferably within 24 hours, of infection in multiplicities of infection (MOI) less than or equal to 0.1. Preferably, the clinical isolate kills a wide variety of tumor cell lines, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or, for example, all of the following human tumor cell lines below: U87MG (glioma ), HT29 (colorectal), LNCaP (prostate), MDAMB-231 (breast), SK-MEL-28 (melanoma), Fadu (cell carcinoma)
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18/52 squamous), MCF7 (breast), A549 (lung), MIAPACA-2 (pancreas), CAPAN1 (pancreas), HT1080 (fibrosarcoma).
[081] In a preferred embodiment, the virus of the invention is a strain selected from:
• line RH018A that has accession number ECCAC 16121904;
• RH004A strain that has accession number ECCAC 16121902;
• RH031A strain that has ECCAC accession number 16121907;
• RH040B strain that has accession number ECCAC 16121908;
• RH015A strain that has accession number ECCAC 16121903;
• RH021A strain that has ECCAC accession number 16121905;
• RH023A strain that has accession number ECCAC 16121906; and • RH047A strain that has ECCAC accession number 16121909.
[082] More preferably, the virus of the invention is a strain selected from:
• line RH018A that has accession number ECCAC 16121904;
• RH004A strain that has accession number ECCAC 16121902;
• RH031A strain that has ECCAC accession number 16121907;
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19/52 • RH040B strain with ECCAC accession number 16121908; and • strain RH015A that has accession number ECCAC 16121903.
[083] Most preferably, the virus of the invention is from the RH018A strain that has accession number EACC 16121904. Any of the deposited strains can be modified as defined in the present application.
[084] An HSV of the invention is capable of selectively replicating itself in tumors, such as human tumors. Typically, an HSV replicates efficiently in target tumors but does not replicate efficiently in non-tumor tissue. This HSV can comprise one or more mutations in one or more viral genes that inhibit replication in normal tissue, but still allow replication in tumors. The mutation, for example, may be a mutation that prevents the expression of functional ICP34.5, ICP6 and / or thymidine kinase by HSV.
[085] In a preferred embodiment, the genes encoding ICP34.5 are mutated to confer selective oncolytic activity in HSV. Mutations of the genes encoding ICP34.5 that prevent the expression of functional ICP34.5 are described in Chou et al. (1990) Science 250: 1262-1266, Maclean et al. (1991) J. Gen. I will come. 72: 631-639 and Liu etal. (2003) Gene Therapy 10: 292-303, which are incorporated by reference into this application. The gene encoding ICP6 and / or gene encoding thymidine kinase can also be inactivated, as are other genes, as long as this inactivation does not prevent the virus from infecting or replicating in tumors.
[086] HSV may contain an additional mutation or mutations that improve HSV replication in tumors. The resulting improvement in viral replication in tumors not only results in the death of direct “oncolytic” tumor cells improved by the virus, but also improves the level of
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20/52 heterologous gene expression (that is, a gene inserted in the virus, in the case of the viruses of the genes of the invention that encode a CTLA-4 inhibitor, GM-CSF and / or a molecule (s) that activates (s) immune co-stimulatory pathway (s) and increases the amount of tumor antigen released as dead tumor cells, both of which may also enhance the immunogenic properties of therapy for the treatment of cancer. For example, in a preferred embodiment of the invention, deletion of the ICP47 encoding gene so that it puts the US11 gene under the control of the immediate initial promoter that normally controls the expression of the ICP47 encoding gene leading to improved replication in tumors (see Liu et al., 2003, which is incorporated into this application as a reference).
[087] Other mutations that place the US11 coding sequence, which is a late HSV gene, under the control of a promoter that is not dependent on viral replication can also be introduced into a virus of the invention. These mutations allow expression of US11 before HSV replication occurs and improve viral replication in tumors. In particular, these mutations improve the replication of HSV devoid of genes encoding functional ICP34.5.
[088] Consequently, in one embodiment, the HSV of the invention comprises a US11 gene functionally linked to a promoter, in which the activity of the promoter is not dependent on viral replication. The promoter can be an immediate initial promoter (IE) or a promoter without HSV that is active in mammals, preferably humans, tumor cells. The promoter can, for example, be a eukaryotic promoter, such as a promoter derived from the genome of a mammal, preferably a human. The promoter can be an omnipresent promoter (such as a β-actin or tubulin promoter) or a cell-specific promoter, such as tumor-specific promoter. The promoter can be a viral promoter, like the long terminal repeat of the leukemia virus
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21/52 murine Moloney (MMLV LTR) or the human or mouse cytomegalovirus (CMV) IE promoter. HSV immediate initial promoters (IE) are well known in the art. The HSV IE promoter may be the promoter that directs expression of ICPO, ICP4, PIC22, PIC27 or ICP47.
[089] The aforementioned genes, the functional inactivation that provides tumor selectivity to the virus, may become functionally inactive by any suitable method, for example, by deleting or replacing all or part of the gene and / or gene control sequence or by inserting one or more nucleic acids in or in place of the gene and / or the gene control sequence. For example, homologous recombination methods, which are standard in the art, can be used to generate the virus of the invention. Alternatively, approaches based on bacterial artificial chromosomes (BAC) can be used.
[090] As used in the present application, the term "gene" is intended to mean the sequence of nucleotides that encodes a protein, that is, the sequence encoding the gene. The various genes mentioned above can become non-functional by mutating the gene itself or the flanking control sequences of the gene, for example, the promoter sequence. Deletions can remove one or more portions of the gene, the entire gene or the entire gene and all or some of the control sequences. For example, only one nucleotide within the gene can be deleted, resulting in a change in the reading frame. However, a larger deletion can be made, for example, at least about 25%, more preferably at least about 50% of the total coding and / or non-coding sequence. In a preferred embodiment, the gene that is becoming functionally inactive is deleted. For example, the entire gene and optionally some of the flanking sequences can be removed from the virus. When two or more copies of the gene are present in the viral genome both copies of the gene become
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22/52 functionally inactive.
[091] A gene can be inactivated by replacing other sequences, for example, replacing all or part of the endogenous gene with a heterologous gene and optionally a promoter sequence. When no promoter sequence is replaced, the heterologous gene can be inserted so that it is controlled by the promoter of the gene that is becoming non-functional. In an HSV of the invention, it is preferred that genes encoding ICP34.5 become non-functional by inserting a heterologous gene or genes and a promoter sequence or sequences functionally linked to them, and optionally other regulatory elements such as sequences of polyadenylation, at each of the loci of the gene encoding ICP34.5.
[092] A virus of the invention is used to express a CTLA-4 inhibitor, and optionally GM-CSF and / or a molecule that activates the immune co-stimulatory pathway, in tumors. This is typically achieved by inserting a heterologous gene that encodes a CTLA-4 inhibitor, and optionally a heterologous gene that encodes GM-CSF and / or a heterologous gene that encodes the molecule that activates the immune co-stimulatory pathway into the genome of a virus selectively competent for replication where each gene is under the control of a promoter sequence. As the replication of this virus will occur selectively in the tumor tissue, the expression of the CTLA-4 inhibitor and, if present, the expression of GM-CSF and / or immune co-stimulatory activation protein by the virus, are also improved in the tumor tissue compared to non-tumor tissue in the body. Improved expression occurs where expression is greater in tumors compared to other tissues in the body. Proteins expressed by the oncolytic virus are also expected to also be present in tumor drainage lymph nodes infected by the oncolytic virus, including due to the expressed protein and virus traffic in or in tumor antigen presenting cells. Consequently, the invention provides benefits of
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23/52 expression of the CTLA-4 inhibitor and any coexpressed GM-CSF and / or molecule that selectively activates the immune co-stimulatory pathway in tumors and tumor drainage lymph nodes combined with the antitumor effect provided by replication of oncolytic virus.
[093] The virus of the invention comprises a CTLA-4 inhibitor. The CTLA-4 inhibitor is a molecule, typically a peptide or protein that binds to CTLA-4 and reduces or blocks signaling through CTLA-4. By reducing CTLA-4 signaling, the inhibitor reduces or removes the block of immune stimulatory pathways by CTLA-4.
[094] The CTLA-4 inhibitor is preferably an antibody or antigen-binding fragment thereof.
[095] The term "antibody" as quoted in the present application includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single strands thereof. An antibody refers to a glycoprotein that comprises at least two heavy chains (H) and two light chains (L) (kappa) interconnected by disulfide bond or antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated in the present application as VH) and a heavy chain constant region. Each light chain is comprised of a variable light chain region (abbreviated in the present application as VL) and a light chain constant region. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The VH and VL regions can still be subdivided into regions of hypervariability, called complementarity determining regions (CDR), interspersed with regions that are more conserved, called structure regions (FR). The antibody constant regions can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (for example, effector cells) and the first
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24/52 component (Clq) of the classic complement system.
[096] The antibody is typically a monoclonal antibody. The antibody can be a chimeric antibody. The antibody is preferably a humanized antibody and is more preferably a human antibody.
[097] The term "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that maintains the ability to specifically bind to CTLA-4. The antigen-binding fragment also maintains the ability to inhibit CTLA-4 and then reduce or remove the blocking of CTLA-4 from a stimulatory immune response. Examples of suitable fragments include an Fab fragment, an F (ab ') 2 fragment, an Fab' fragment, an Fd fragment, an Fv fragment, an dAb fragment and an isolated complementarity determining region (CDR). Single chain antibodies such as scFv and heavy chain antibodies such as VHH and camel antibodies are also intended to be encompassed within the term "antigen binding portion" of an antibody. In a preferred embodiment, the antibody is a scFv. Examples of scFv molecules are disclosed, for example, in WO 2007/123737 and WO 2014/066532, which are incorporated by reference into this application.
[098] Antibody coding sequences typically encode an antibody or antibody fragment that has an N-terminal signal sequence. The signal sequence can have the amino acid sequence shown in SEQ ID NO: 7. For example, this signal sequence is included in a scFv that has the amino acid sequence shown in SEQ ID NO: 9 and encoded by the nucleotide sequence shown in SEQ ID NO: 10, and in a scFv that has the amino acid sequence shown in SEQ ID NO: 14 and encoded by the nucleotide sequence shown in SEQ ID NO: 15.
[099] In the antibody or antibody fragment, the light chain and heavy chain sequences can be joined by an amino acid linker. O
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25/52 linker typically comprises from about 15 to about 25 amino acids, such as about 18 or 20 amino acids. Any suitable linker can be used, as linkers that comprise glycine and serine residues, for example, the amino acid sequence shown in SEQ ID NO: 8. For example, this linker is included in an scFv that has the amino acid sequence shown in SEQ ID NO: 9 and encoded by the nucleotide sequence shown in SEQ ID NO: 10, and in a scFv that has the amino acid sequence shown in SEQ ID NO: 14 and encoded by the nucleotide sequence shown in SEQ ID NO: 15. Both are fragments of preferred antibodies for use in the invention.
[100] Other antibody fragments that have similar structures are also preferred. Consequently, the virus of the invention can encode an antibody or fragment that comprises, or consists essentially 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 still encode a signal sequence at the N-terminus of the antibody.
[101] The antibodies or antibody fragments of the invention may preferably comprise an Fc region that is an IgG1, IgG2, IgG3 or IgG4 region, more preferably an IgG1 region. Preferably, the antibody is an scFv antibody in which the scFv is linked to the IgG heavy chain CH2 and CH3 domains.
[102] An antibody or CTLA-4 fragment comprises 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 can comprise the CH1 heavy chain domain that has the amino acid sequence shown in SEQ ID NO: 4 and / or
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26/52 the CH2 / CH3 domains shown in SEQ ID NO: 5. The antibody can comprise the light chain amino acid sequence shown in SEQ ID NO: 2. An antibody of the invention can optionally comprise a variant of one of these variable regions of heavy or light chain, or CDR sequences. For example, a variant can be a substitution, deletion or addition variant for any of the above amino acid sequences.
[103] A variant antibody can comprise 1, 2, 3, 4, 5, up to 10, up to 20, up to 30 or more amino acid substitutions and / or deletions of the specific sequences and fragments discussed above, while maintaining the activity of the antibodies described in this application. Variants of "deletion" may comprise the deletion, for example, of 1, 2, 3, 4 or 5 individual amino acids or of one or more groups of small amino acids, such as 2, 3, 4 or 5 amino acids. "Substitution" variants preferably involve replacing one or more amino acids with the same number of amino acids and producing conservative amino acid substitutions. For example, an amino acid can be replaced by an alternative amino acid that has similar properties, for example, another basic amino acid, another acidic amino acid, another neutral amino acid, another loaded amino acid, another hydrophilic amino acid, another hydrophobic amino acid, another polar amino acid aromatic or other aliphatic amino acid.
[104] The virus of the invention comprises one or more polynucleotide sequences encoding the CTLA-4 inhibitor. The polynucleotide sequence is under the control of a suitable promoter. The virus can comprise a first polynucleotide sequence that encodes an antibody heavy chain variable region and a second polynucleotide that encodes an antibody light chain variable region. The first polynucleotide can encode an entire heavy chain and / or the second
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27/52 polynucleotide can encode an entire light chain. The first and second polynucleotides may be under the control of a single promoter, optionally an IRES, or they may be under the control of two separate promoters. The separate promoters can be the same or different.
[105] The first polynucleotide can comprise, consists essentially of, or consists of the heavy chain variable region encoding the sequence shown in SEQ ID NO: 9 and / or the second polynucleotide can comprise, consists essentially of or consists of the variable chain region heavy encoding the sequence shown in SEQ ID NO: 10. The first polynucleotide can comprise, consists essentially of, or consists of the variable heavy chain region encoding the sequence shown in SEQ ID NO: 19 and / or the second polynucleotide can comprise, consists essentially of or consists of the heavy chain variable region encoding the sequence shown in SEQ ID NO: 20.
[106] A first and / or second polynucleotide sequence can be a variant of SEQ ID NO: 9,10,19 or 20. For example, a variant can be a substitution, deletion or addition variant for any of these sequences. amino acids. A variant polynucleotide can 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 of SEQ ID NO: 9,10 , 19 or 20.
[107] Suitable variants can be at least 70% homologous to a polynucleotide of any of the nucleic acid sequences disclosed in the present application, preferably at least 80 or 90%, and more preferably at least 95%, 97% or 99% homologous the same. Preferably homology and identity at these levels is present at least in relation to the polynucleotide coding regions. Methods for measuring homology are well known in the art and will be understood by those skilled in the art that, in the present context, homology is calculated based on
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28/52 on the identity of nucleic acids. Such homology can exist over a region of at least 15, preferably at least 30, for example, at least 40, 60, 100, 200 or more contiguous nucleotides. This homology can exist along the entire length of the unmodified polynucleotide sequence.
[108] Methods for measuring homology or identity of polynucleotides are known in the art. For example, the UWGCG package provides the BESTFIT program that can be used to calculate homology (for example, used in its standard configuration) (Devereux et al (1984) Nucleic Acids Research 12, pages 387-395).
[109] The PILEUP and BLAST algorithms can also be used to calculate homology or alignment sequences (typically in their standard configurations), for example, as described in Altschul S.F. (1993) J Mol Evol 36: 290-300; Altschul, S, F et al (1990) J Mol Biol 215: 403-10.
[110] The software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves the first identification of the high score sequence pair (HSPs) by identifying short words of length W in the query string that corresponds as much as it satisfies some positive value limit T scores when aligned with a word of the same length in a sequence of data. T is cited as the neighboring word score limit (Altschul et al, above). These first neighboring word hits act as seeds to start searching to find HSPs that contain them. The word hits are extended in both directions along each sequence as far as the cumulative alignment score can be increased. Extensions for word hits in each direction are interrupted when: the cumulative alignment score goes to zero or below, due to the accumulation of one or more alignments
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29/52 negative scoring residue; or the end of both strings is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program defaults to a word length (W) of 11, the BLOSUM62 punctuation matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50 , expectation (E) of 10, M = 5, N = 4, and a comparison of both tapes.
[111] The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see, for example, Karlin and Altschul (1993) Proc. Natl. Acad. Know. USA 90: 5873-5787. A measure of similarity provided by the BLAST algorithm is the least probability of sum (P (N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences occurs at random. For example, a sequence is considered similar to another sequence if the least probability of summing compared to the first sequence for the second sequence is less than about 1, preferably less than about 0.1, more preferably at least about 0, 01 and, with maximum preference, less than about 0.001.
[112] In one embodiment, a sequence variant may vary from the specific sequences given in the sequence listing due to the redundancy of the genetic code. The DNA code has 4 main nucleic acid residues (A, T, C and G) and uses them to "spell" three-letter codons that represent the amino acids in proteins encoded by an organism's genes. The linear sequence of codons across the DNA molecule is translated into the linear sequence of amino acids in the protein (s) encoded by these genes. The code is highly degenerate, with 61 codons that encode the 20 natural amino acids and 3 codons that represent “stop” signals. Thus, most amino acids are encoded by
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30/52 more than one codon, in fact several are coded by four or more different codons. A variant polynucleotide of the invention can therefore encode the same polypeptide sequence as another polynucleotide sequence of the invention, but it can have a different nucleic acid sequence due to the use of different codons to encode the same amino acids. Codons can be optimized in order to increase the expression levels of the proteins encoded in the target cells, in comparison if the unchanged sequence is used.
[113] The virus of the invention preferably comprises GMCSF. The sequence of the gene encoding GM-CSF can be codon-optimized, in order to increase the levels of expression of the respective proteins in the target cells, in comparison if the unchanged sequence is used.
[114] The virus of the invention preferably comprises one or more molecules that activate the immune co-stimulatory pathway and / or one or more genes that encode a molecule that activates the immune co-stimulatory pathway. Molecules that activate the immune co-stimulatory pathway include proteins and nucleic acid molecules (for example, aptamer sequences). Examples of molecules that activate the immune co-stimulatory pathway include CD40 ligand, GITR ligand, 4-1-BB ligand, 0X40 ligand, ICOS ligand, flt3 ligand, TL1A, CD30 ligand, CD70 and single chain antibodies that direct the respective receptors to these molecules (CD40, GITR, 4-1-BB, 0X40, ICOS, flt3, DR3, CD30, CD27).
[115] Activators of the immune co-stimulatory pathway include soluble, secreted, and / or membrane-bound mutant or wild-type ligands, and agonist antibodies including single chain antibodies. The viruses of the invention preferably encode one or more among CD40L, ICOSL, 4-1-BBL, GITRL or OX40L.
[116] The viruses of the invention can encode one or more molecules that activate the immune co-stimulatory pathway, preferably 1,2, 3 or
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31/52 molecules that activate the immune co-stimulatory pathway, more preferably 1 or 2 molecules that activate the immune co-stimulatory pathway.
[117] The sequence of the gene encoding the immune co-stimulating molecule can be codon-optimized to increase the expression levels of the respective protein (s) in the target cells, compared to the sequence unchanged is used.
[118] The virus of the invention may comprise one or more additional heterologous genes, in addition to a CTLA-4 and GM-CSF inhibitor and / or a molecule that activates the immune co-stimulatory pathway. In a preferred embodiment, the virus may further comprise a fusogenic protein such as GALVR-.
[119] The fusogenic protein can be any heterologous protein capable of fusing a cell infected with the virus of the invention to another cell. A fusogenic protein, preferably a wild-type or modified viral glycoprotein (that is, modified to increase its fusogenic properties), is a protein that is capable of inducing the cell to cell fusion (syncytial formation) of cells in which it is expressed. Examples of fusogenic glycoproteins include VSV-G, syncytine-1 (from human endogenous retrovirus-W (HERV-W)) or syncytine-2 (from HERVFRDE1), paramixovirus SV5 F, measles virus H, measles virus F , RSV-F, the retrovirus or lentivirus glycoprotein, such as gibbon monkey leukemia virus (GALV), murine leukemia virus (MLV), MasonPfizer monkey virus (MPMV) and equine infectious anemia virus (EIAV) with the transmembrane R peptide removed (R- versions). In a preferred embodiment, the fusogenic protein is GALV and has the R- peptide removed (GALV-R-).
[120] The virus of the invention can optionally comprise multiple copies of the gene encoding fusogenic protein, preferably 1 or 2 copies. The virus can comprise two or more different fusogenic proteins, including any of the fusogenic proteins listed above.
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32/52 [121] The fusogenic protein or proteins optionally expressed by a virus of the invention may be identical to a naturally occurring protein, or it may be a modified protein.
[122] The gene encoding the protein (fusogenic gene) may have a naturally occurring nucleic acid sequence or a modified sequence. The sequence of the fusogenic gene can, for example, be modified to increase the fusogenic properties of the encoded protein, or to provide codon optimization and, therefore, increase the expression efficiency of the encoded protein.
[123] | The invention also provides a virus, such as a poxvirus or an HSV, preferably HSV1, that expresses at least three heterologous genes, where each of the three heterologous genes is directed by a different promoter selected from the CMV promoter , RSV promoter, Ef 1 a promoter, SV40 promoter and a retroviral LTR promoter. The virus can, for example, express four heterologous genes, where each of the four heterologous genes is driven by a different promoter selected from the CMV promoter, RSV promoter, EF1a promoter and a retroviral LTR promoter. The retroviral LTR is preferably from MMLV. Heterologous genes can be terminated by polyadenylation sequences. The polyadenylation sequences can be the same or different. Preferably each heterologous gene is terminated by a different polyadenylation sequence, which is preferably selected from the polyadenylation sequences of BGH, SV40, HGH and RBG.The invention also provides a virus, such as a poxvirus or HSV, preferably HSV1, which expresses at least three heterologous genes, where each of the three heterologous genes is terminated by a different polyadenylation sequence selected from the polyadenylation sequences of BGH, SV40, HGH and RBG. The virus can, for example, express four heterologous genes terminated by each of the sequences
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33/52 polyadenylation of BGH, SV40, HGH and RBG, respectively.
[124] At least three heterologous genes can, for example, be selected from a CTLA-4 inhibitor, a gene encoding GM-CSF, a gene encoding a molecule that activates the immune co-stimulatory pathway and a fusogenic gene. Examples of the three heterologous genes are a CTLA-4 inhibitor, a gene that encodes GM-CSF and a gene that encodes a molecule that activates the immune co-stimulatory pathway; a CTLA-4 inhibitor, a gene encoding GM-CSF and a fusogenic gene; and a CTLA-4 inhibitor, a gene that encodes a molecule that activates the immune co-stimulatory pathway and a fusogenic gene. The four heterologous genes may, for example, be a CTLA-4 inhibitor, a gene that encodes GM-CSF, a gene that encodes a molecule that activates the immune co-stimulatory pathway and a fusogenic gene. The three or four heterologous genes can comprise, for example, two or more genes that encode molecules that activate the immune co-stimulatory pathway and / or two or more fusogenic genes.
[125] In one embodiment, the promoters that control the expression of the three heterologous genes are the CMV, RSV and MMLV promoters. For example, a preferred virus can comprise a GM-CSF gene under the control of a CMV promoter, a GALV gene under the control of an RSV promoter and a CTLA-4 inhibitor under the control of an MMLV promoter.
[126] In one embodiment, the terminating polyadenylation sequence of at least three heterologous genes are polyadenylation sequences of SV40, BGH and RBG and which controls the expression of the three heterologous genes are the CMV, RSV and MMLV promoters. For example, a preferred virus may comprise a GM-CSF gene terminated by a BGH polyadenylation sequence, a GALV gene terminated by an SV40 polyadenylation sequence and a CTLA-4 inhibitor terminated by an RGB polyadenylation sequence.
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34/52 [127] Any combination of the various promoters and polyadenylation can be used with any of the heterologous genes. For example, a preferred virus may comprise a GM-CSF gene under the control of a CMV promoter and terminated by a BGH polyadenylation sequence, a GALV gene under the control of an RSV promoter and terminated by an SV40 polyadenylation sequence , and a CTLA4 inhibitor under the control of an MMLV promoter terminated by an RGB polyadenylation sequence.
Virus Production [128] The viruses of the invention are constructed using methods well known in the art. For example, plasmids (for smaller viruses and viruses with competent RNA of multiple and single genomes) or BACs (for viruses with larger DNA including herpes viruses) that encode the viral genome to be packaged, including the genes that encode fusogenic molecules and immune stimuli under the proper regulatory control, can be constructed by standard molecular biology techniques and transfected into permissive cells from which recombinant viruses can be recovered.
[129] Alternatively, in a preferred embodiment, plasmids containing regions of DNA flanking the desired insertion site can be constructed and then cotransfected into permissive cells with viral genomic DNA, so that homologous recombination occurs between regions that flank the target insertion site on the plasmid and the same regions on the parental virus. Recombinant viruses can then be selected and purified by the loss or addition of a function inserted or deleted by the plasmid used for modification, for example, insertion or deletion of a marker gene such as GFP or lacZ of the parental virus at the intended insertion site. In a most preferred embodiment, the insertion site is
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35/52 the HSV ICP34.5 locus and, therefore, the plasmid used for manipulation contains HSV sequences that flank this insertion site, among which are an expression cassette encoding GM-CSF and the molecule that activates the pathway immune co-stimulatory. In this case, the parental virus may contain a cassette that encodes GFP in place of ICP34.5 and recombinant virus plaques are selected through the loss of GFP expression. In a most preferred embodiment, the HSV US11 gene is also expressed as an IE gene. This can be done by deleting the ICP47 coding region, or by other means.
[130] The CTLA-4 inhibitor, and optionally the GM-CSF coding sequences and the coding sequences of the molecule that activates the immune co-stimulatory pathway and / or the coding sequence for the additional protein, as a sequence encoding a fusogenic protein such as GALVR - are inserted into the viral genome under appropriate regulatory control. This may be under the regulatory control of natural promoters of the virus species used in the invention, depending on the species and insertion site or, preferably, under the control of heterologous promoters. Suitable heterologous promoters include mammalian promoters, such as the IEF2a promoter or the actin promoter. Most preferred are strong viral promoters such as the CME IE promoter, the RSV LTR, the MMLV LTR, other retroviral LTR promoters, or SV40-derived promoters. Preferably each exogenous gene (for example, which encodes GM-CSF and the molecule that activates the immune co-stimulatory pathway) will be under the control of the separate promoter, but can also be expressed from a single RNA transcript, for example, via insertion of internal ribosome entry sites (IRES) between protein coding sequences. The RNA derived from each promoter is typically terminated using a polyadenylation sequence (for example, mammalian sequences, such as growth hormone polyA sequence
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36/52 bovine or human (BGH), synthetic polyadenylation sequences, rabbit beta-globin polyadenylation sequence or viral sequences such as SV40 initial or final polyadenylation sequence).
[131] Each of the heterologous genes in the virus is typically under the control of a promoter. The promoters that control the expression of the heterologous genes can be the same or different. For example, antiCTLA-4, and one or more of GM-CSF, fusogenic gene and gene encoding molecule that activates the immune co-stimulatory pathway, may each be under the control of the CMV promoter, RSV promoter, promoter of EF1a, SV40 promoter or a retroviral LTR promoter. Alternatively, for example, antiCTLA-4 may be under the control of a retroviral LTR promoter such as the MMLV promoter, the GM-CSF gene may be under the control of the CMV promoter and / or the fusogenic gene, such as GALVR-, may be under the control of the RSV promoter.
Pharmaceutical Compositions [132] The invention provides a pharmaceutical composition comprising the virus and a pharmaceutically acceptable carrier or diluent. Suitable carriers and diluents include isotonic saline solutions, for example, phosphate buffered saline. The composition may also comprise other constituents such as sugars or proteins to improve properties such as product stability. Alternatively, a lyophilized formulation can be used, which is reconstituted in a pharmaceutically acceptable carrier or diluent prior to use.
[133] The choice of carrier, if necessary, is often a function of the composition's distribution route. Within this invention, the compositions can be formulated for any suitable route and means of administration. Pharmaceutically acceptable carriers or diluents are those used in compositions suitable for administration
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37/52 intratumoral, intravenous / intraarterial administration, administration to the brain or administration to a body cavity (eg, bladder, pleural cavity or by intraperitoneal administration). The composition can be administered in any suitable way, preferably as a liquid.
[134] The present invention also provides a manufacturing product comprising a virus of the invention in a sterile vial, ampoule or syringe.
Medical Uses / Methods for Treatment [135] The invention provides the virus of the invention for use in treating the human or animal body by therapy, particularly for use in a method to treat cancer. Cancer is typically in a mammal, preferably in a human. The virus kills the infected tumor cells by lysis and causing the infected tumor cells to fuse with each other. The virus of the invention also obtains a systemic anti-tumor immune response, enhanced through the expression of the CTLA-4 inhibitor, and optionally GM-CSF and the molecule that activates the immune co-stimulatory pathway, which also kill cancer cells.
[136] The invention also provides a method for treating cancer, the method comprising administering a therapeutically effective amount of the virus of the invention to an individual in need thereof.
[137] The invention additionally provides for the use of the virus of the invention in the manufacture of a drug to treat cancer.
[138] The virus of the invention is particularly useful in the treatment of any solid tumor, including any adenocarcinoma, carcinoma, melanoma or sarcoma. For example, the virus of the invention is useful in the treatment of cancers of the head and neck, prostate, breast, ovary, lung, liver, endometrium, bladder, gallbladder, pancreas, colon, kidney, gastric or cervical stomach / esophagus, mesothelioma, melanoma or other skin cancer, lymphoma,
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38/52 glioma or other cancer of the nervous system, or sarcomas, such as soft tissue sarcoma.
[139] The virus of the invention can be used to treat malignant tumors, including tumors metastasized from the original tumor. In this embodiment, the virus can be administered to the primary tumor or to one or more secondary tumors.
[140] The virus of the invention can be administered in combination with other therapeutic agents, including chemotherapy, targeted therapy, immunotherapy (including blocking immune control points, i.e., administration of one or more antagonists of an immune co-stimulatory pathway, and / or one or more agonists of an immune co-stimulatory pathway) and / or in combination with radiotherapy and / or in combination with any combination thereof. The therapeutic agent is preferably an anti-cancer agent.
[141] The virus of the invention can be administered in combination with a second virus, such as a second oncolytic virus.
[142] For example, the therapeutic agent may comprise an immunogen (including a recombinant or naturally occurring antigen, including that antigen or combination of antigens distributed as DNA or RNA into which he / they are encoded), to further stimulate an immune response , as a cellular or humoral immune response, to tumor cells, particularly tumor neoantigens. The therapeutic agent may be an agent designed to enhance or potentiate an immune response, such as a cytokine, an agent designed to inhibit an immune control point pathway or stimulate an immune potentiation pathway or an agent that inhibits the activity of regulatory T cells (Tregs) or myeloid-derived suppressor cells (MDSCs).
[143] The therapeutic agent may be an agent known for use in an existing therapeutic treatment of cancer. The therapeutic agent
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39/52 can be radiotherapy or a chemotherapeutic agent. The therapeutic agent can be selected from cyclophosphamide, as alkylating-like agents such as cisplatin or melphalan, plant alkaloids and terpenoids such as vincristine or paclitaxel (Taxol), antimetabolites such as 5-fluorouracil, topoisomerase type I or II inhibitors as camptothecin or doxorubicin , cytotoxic antibiotics like actinomycin, anthracyclines like epirubicin, glucocorticoids like triamcinolone, protein inhibitors, DNA and / or RNA synthesis like methotrexate and dacarbaxine, histone deacetylase inhibitors (HDAC), or any other chemotherapy agent.
[144] The therapeutic agent can be one, or a combination of: immunotherapeutic or immunomodulatory, such as TLR agonists; agents that downregulate regulatory T cells such as cyclophosphamide; or agents designed to block immune checkpoints or stimulate immune enhancement pathways, including, but not limited to, monoclonal antibodies, such as a CTLA-4 inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, a inhibitor of LAG-3, an inhibitor of TIM-3, an inhibitor of VISTA, an inhibitor of CSF1R, an inhibitor of IDO, an inhibitor of CEACAM1, an agonist of GITR, an agonist of 4-1-BB, an inhibitor of KIR, an SLAMF7 inhibitor, a 0X40 agonist, a CD40 agonist, an ICOS agonist or a CD47 inhibitor. In a preferred embodiment, the therapeutic agent is an inhibitor of CTLA-4 as an anti-CTLA-4 antibody, an inhibitor of PD1, as an anti-PD-1 antibody or an inhibitor of PD-L1 as an anti-PD antibody -L1. Such inhibitors, agonists and antibodies can be generated and tested by standard methods known in the art.
[145] Immunotherapeutic agents may also include bispecific antibodies, dendritic cells based on cell-based therapies, NK cells or genetically engineered T cells like CAR-T cells or T cells that express genetically engineered T cell receptors.
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40/52
Immunotherapeutic agents also include agents that target a specific genetic mutation that occurs in tumors, agents designed to induce immune responses to specific tumor antigens or combinations of tumor antigens, including neoantigens and / or agents designed to activate the STING / cGAS, TLR or another innate immune response and / or inflammatory pathway, including intratumor agents.
[146] For example, a virus of the invention can be used: in combination with dacarbazine, a BRAF inhibitor and / or blocking PD1 or PD-L1 to treat melanoma; in combination with taxol, doxorubicin, vinorelbine, cyclophosphamide and / or gemcitabine to treat breast cancer; in combination with 5-fluorouracil and optionally leucovorin, irinoteacano and / or oxaliplatin to treat colorectal cancer; in combination with taxol, carboplatin, vinorelbine and / or gemcitabine, blocking PD-1 or PD-L1 to treat lung cancer; in combination with cisplatin and / or radiation to treat head and neck cancer.
[147] The therapeutic agent may be an inhibitor of the idoleamine 2,3-dioxigenase (IDO) pathway. Examples of IDO inhibitors include epacadostate (INCB024360), 1-methyl-tryptophan, indoximode (1-methyl-Dtryptophan), GDC-0919 or F001287.
[148] The mechanism of action of IDO in suppressing anti-tumor immune responses can also suppress immune responses generated after therapy with oncolytic virus. IDO expression is induced by the activation of toll like receptors (TLR) and interferon-γ, both of which can result from infection by the oncolytic virus. An embodiment of the use of oncolytic virus therapy for cancer treatment includes the combination of an oncolytic virus, including a virus that expresses a CTLA-4 inhibitor, and optionally GMCSF and / or a molecule or molecules that activate the immune co-stimulatory pathway and / or one or more sequences coding for the additional protein, such as a
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41/52 sequence encoding a fusogenic protein such as GALVR-, with an inhibitor of the IDO pathway and optionally an additional antagonist of an immune co-inhibitory pathway and / or one or more agonists of an immune co-stimulatory pathway, including those that target PD-1 and / or PD-L1.
[149] When a therapeutic agent and / or radiation therapy are used in conjunction with a virus of the invention, the administration of the virus and the therapeutic agent and / or radiation therapy can be at the same time or separate. The composition of the invention can be administered before, together with, or after the therapeutic agent or radiotherapy. The method for treating cancer may comprise multiple administrations of the virus of the invention and / or the therapeutic agent and / or radiation therapy. In preferred embodiments, in the case of combination with blocking immune control points or other immune enhancing agents, the virus of the invention is administered once or several times before concomitant administration of blocking immune control points or other enhancing agent or agents immune, subsequently or concurrently with the administration of the blockade of immune control points or other immune enhancing agent or agents without prior administration of the virus of the invention.
[150] The virus of the invention can be administered to a subject by an appropriate route. Typically, a virus of the invention is administered by direct intratumor injection. Intratumoral injection includes direct injection into superficial, subcutaneous or nodal tumors, and image-guided injection (including CT, MRI or ultrasound) into deeper or more difficult to locate deposits, including visceral organs and elsewhere. The virus can be administered in a body cavity, for example, in the pleural cavity, bladder or by intraperitoneal administration. The virus can be injected into a blood vessel, preferably a blood vessel that supplies a tumor.
[151] Therapeutic agents that can be combined with a
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42/52 viruses of the invention can be administered to a human or animal subject in vivo using a variety of known routes and techniques. For example, the composition can be provided as a solution for injection, suspension or emulsion and administered parenterally, subcutaneously, orally, epidermally, intradermally, intramuscularly, interarterially, intraperitoneally, intravenously, using a conventional needle and syringe, or with a conventional syringe. the use of a liquid jet injection system. The composition can be administered topically to the skin or mucosal tissue, such as nasal, intratracheal, intestinal, sublingual, rectal or vaginal, or delivered as a finely divided spray suitable for respiratory or pulmonary administration. In preferred embodiments, the compositions are administered by intravenous infusion, orally or directly into a tumor.
[152] The virus and / or therapeutic agent can be administered to a subject in an amount that is compatible with the dosage composition that will be therapeutically effective. The administration of the virus of the invention is for a "therapeutic purpose". As used in the present application, the term "therapeutic" or "treatment" includes any one or more of the following as its objective: the prevention of any occurrence of metastasis or additional metastasis; the reduction or elimination of symptoms; the reduction or complete elimination of a tumor or cancer, an increase in the time to progression of the patient's cancer; an increase in the time of relapse after treatment; or an increase in survival time.
[153] Therapeutic treatment can be given to Stage I, II, III or IV cancers, Preferably Stage II, III or IV, more preferably Stage III or IV, pre- or post-surgical intervention (that is, after recurrence or incomplete removal of tumors after surgery), preferably before any surgical intervention (either by resection of primary or recurrent / metastatic disease), or after recurrence after surgery
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43/52 or after incomplete surgical removal of the disease, that is, while the residual tumor remains.
[154] Therapeutic treatment can be carried out after direct injection of the virus composition into the target tissue, which can be in the tumor, in a body cavity or in a blood vessel. As a guide, the amount of virus administered is in the case of HSV in the range from 10 ⁴ to 10 ¹⁰ pfu, preferably 10 ⁵ to 10 ⁹ pfu. In the case of HSV, a lower starting dose (for example, 10 ⁴ to 10 ⁷ pfu) can be given to patients for seroconversion in patients who are seronegative for HSV and to boost immunity in those who are seropositive, followed by a larger dose, then being given later (for example, 10 ⁶ 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 can be used for direct injection into tumors, or up to 50 ml for administration into a body cavity (which may be subject to further dilution) in an appropriate diluent prior to administration) or in the bloodstream. However, for some applications of oncolytic therapy, larger or smaller volumes may also be used, depending on the tumor and the route or location of administration.
[155] The routes of administration and doses described are intended for guidance only, as a person skilled in the art will be able to quickly determine the best route of administration and dosage. The dosage can be determined according to various parameters, especially 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 route of administration. Preferably, the virus is administered by direct injection into the tumor or into a body cavity. The virus can also be administered by injection into a blood vessel. The best route of administration will depend on the location and size of the tumor. Multiple doses may be necessary to achieve an immunological effect or
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44/52 clinical, which, if necessary, will typically be administered between 2 days to 12 weeks apart, preferably from 3 days to 3 weeks apart. Dose repetitions up to 5 years or more can be given, preferably for up to a month to two years depending on the response rate of the type of tumor to be treated and the response of a particular patient, and any combination therapy that may also be being given away.
[156] The following Examples illustrate the invention.
Example 1
Construction of a Virus of the Invention [157] The virus species used to exemplify the invention is HSV, specifically HSV1.
[158] The diagrams of the plasmids used are shown in Figure 2. The diagrams of the viruses are shown in Figure 1. All viruses were constructed using the RH018A strain of HSV1. The plasmids used for virus construction were generated by a combination of gene synthesis and subcloning, conducted by Genscript Inc.
[159] Viruses that express mouse anti-CTLA4 together with mouse GM-CSF and GALV were constructed by cotransfecting Plasmid 77 with Virus 16 DNA, in order to insert GFP into Virus 16 by selecting plates that express GFP to give Virus 25. GFP was inactivated from Virus 25 by co-transfecting DNA from Virus 25 with Plasmid 119. This resulted in Virus 27.
[160] Viruses expressing human anti-CTLA4 together with human GM-CSF and GALV were constructed by cotransfecting Plasmid 78 with Virus 17 DNA, in order to insert GFP into Virus 17 by selecting plates expressing GFP to give Virus 29 GFP was inactivated from Virus 29 by co-transfecting DNA from Virus 29 with Plasmid 122. This resulted in Virus 31.
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45/52 [161] Viruses that express mouse anti-CTLA-4 and co-stimulatory ligands together with mouse GM-CSF and GALV were constructed by cotransfection of a plasmid encoding GFP directed by an SV40 promoter between the GM-CSF of mouse and mouse anti-CTLA-4 coding sequences with Virus 27. GFP was then inactivated from the resulting virus with a plasmid encoding each of the individual mouse co-stimulatory ligands in place of GFP.
[162] Viruses expressing human anti-CTLA-4 and co-stimulatory ligands together with human GM-CSF and GALV were constructed by cotransfection of a plasmid encoding GFP directed by an SV40 promoter between human GM-CSF and anti coding sequences -CTLA-4 human with Virus 31. GFP was then inactivated from the resulting virus with a plasmid encoding each of the individual human co-stimulatory ligands in place of GFP.
[163] Figure 4 shows a Western blot showing expression of virus 27 anti-CTLA-4 mice.
Example 2 The Effect of the Combined Expression of GALV, GM-CSF and Anti-CTLA4 from an Oncolytic Virus [164] The utility of the invention is demonstrated as follows. A20 cells are administered on both sides of Balb / c mice and A 20 tumors are allowed to grow to approximately 0.5 cm in diameter.
[165] The following treatments are then given to groups of mice, on one flank of each mouse only (right tumor) 3 times a week for a week:
pL of vehicle (1 group);
pL of 10 ⁶ pfu / ml of HSV with mouse GM-CSF and inserted GALVR-only (Virus 16);
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46/52 μΙ_ of 10 ⁶ pfu / mL of HSV with GALVR-, mouse GM-CSF and the inserted mouse anti-CTLA-4 antibody (Virus 27);
[166] The effects on tumor growth are then seen for up to a month. The virus dose used was 5 x 10 ⁴ pfu (50 µL of 1 x 10 ⁶ pfu / ml in each case), given three times over a week. This virus dose level is subtherapeutic for tumors not injected for viruses 16, which allows the benefits of distributing the additional molecules encoded by virus 27 to be clearly seen. Figures 5 and 6 show superior tumor control and shrinkage in tumors not injected with the virus that expresses anti-CTLA-4 compared to virus 16, which does not express CTLA-4.
Example 3
The Effect of the Combined Expression of GALV, GM-CSF and Anti-CTLA4 From AN ONCOLYTIC VIRUS WITH ANTI-PD-1 [167] A20 cells are administered on both sides of Balb / c mice and tumors A 20 are allowed to grow up to approximately 0.5 cm in diameter.
[168] The following treatments are then given to groups of mice (10 per group), on one flank of each mouse only 3 times a week for a week:
vehicle pL;
Intraperitoneal mouse anti-PD1 (Bioxcell RMP-114 10 mg / kg every three days);
pL of 10 ⁷ pfu / ml of HSV with GALVR-, mouse GM-CSF and the inserted mouse anti-CTLA-4 antibody (Virus 27);
pL of 10 ⁷ pfu / mL, from HSV with GALVR-, mouse GM-CSF and the inserted mouse anti-CTLA-4 (Virus 27) together with intraperitoneal mouse anti-PD1 (10 mg / kg every three days ) (3
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47/52 groups).
[169] The effects on tumor growth are then seen for up to 80 days. Superior tumor control and shrinkage in both injected and uninjected tumors when treatment with the virus is combined treatment with anti-PD1. These data are shown in Figure 7.
Example 4
The Effect of Combined Expression of Human GALV, GM-CSF and Anti-CTLA4 from an Oncolytic Virus Alone and in Combination with Anti-PD-1 [170] MC38 cells are administered on both genetically engineered C57BL / 6 mice flanks by editing the gene to express human CTLA-4 instead of mouse. This makes the mice susceptible to human anti-CTLA-4 antibodies like ipilimumab. MC38 tumors are allowed to grow to approximately 0.5 cm in diameter.
[171] The following treatments are then given to groups of mice (10 per group), on one flank of each mouse only 3 times a week for two weeks:
vehicle pL;
pL of 10 ⁸ pfu / ml of Virus 17 (i.e., expressing hGMCSFe GALV);
pL of 10 ⁸ pfu / ml of Virus 31 (i.e., expressing hGMCSF, GALV and human anti-CTLA-4);
pL of 10 ⁸ pfu / ml of Virus 17 together with intraperitoneal mouse anti-PD1 (10 mg / kg every three days);
pL of 10 ⁸ pfu / ml of Virus 31 together with intraperitoneal mouse anti-PD1 (10 mg / kg every three days).
[172] The effects on tumor growth are then seen for up to 35 days. Superior tumor control was observed and
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48/52 shrinkage in tumors injected with the virus that expresses human anti-CTLA-4, which is further improved with combined treatment with anti-PD1. Superior tumor control and shrinkage in uninjected tumors are observed when treatment with any of the viruses is combined with anti-PD1 treatment. The improvement is more pronounced for the virus that expresses anti CTLA4. These data are shown in Figure 8.
Example 5
The Effect of the Combined Expression of GALV, GM-CSF and Anti-CTLA4 From AN ONCOLYTIC VIRUS WITH ANTI-PD-1 [173] A20 cells are administered in both flanks of Balb / c mice and tumors A 20 are allowed to grow up to approximately 0.5 cm in diameter.
[174] The following treatments are then given to groups of mice (10 per group), on one flank of each mouse only 3 times a week for two weeks:
pL of vehicle (1 group);
Intraperitoneal mouse anti-PD1 (Bioxcell RMP-114 10 mg / kg every three days);
pL of 10 ⁵ pfu / ml, 10 ⁶ pfu / ml or 10 ⁷ pfu / ml of HSV only with mouse GM-CSF and GALVR- inserted (3 groups);
pL of 10 ⁵ pfu / ml, 10 ⁶ pfu / ml or 10 ⁷ pfu / ml of HSV with GALVR-, mouse GM-CSF and mouse anti-CTLA-4 inserted together with intraperitoneal mouse anti-PD1 (10 mg / kg every three days) (3 groups);
pL of 10 ⁵ pfu / mL, 10 ⁶ pfu / mL or 10 ⁷ pfu / mL of HSV only with mouse GM-CSF and GALVR- inserted together with mouse anti-PD1 (10 mg / kg every three days) ( 3 groups);
pL of 10 ⁵ pfu / ml, 10 ⁶ pfu / ml or 10 ⁷ pfu / ml of HSV with
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49/52
GALVR-, mouse GM-CSF and mouse anti-CTLA-4 inserted together with mouse intraperitoneal anti-PD1 (10 mg / kg every three days) (3 groups).
[175] The effects on tumor growth are then seen for up to a month. Superior tumor control and shrinkage were observed in both injected and non-injected tumors with the virus that expresses anti-CTLA-4, which is further improved with combined treatment with anti-PD1, compared to other groups is observed, including through an improved dose-response curve.
Example 6
The Effect of Combined Expression of Human GALV, GM-CSF and Anti-CTLA4 from an Oncolytic Virus Alone and in Combination with Anti-PD-1 [176] MC38 cells are administered on both genetically engineered C57BL / 6 mice flanks by editing the gene to express human CTLA-4 instead of mouse. This makes the mice susceptible to human anti-CTLA-4 antibodies like ipilimumab. MC38 tumors are allowed to grow to approximately 0.5 cm in diameter.
[177] The following treatments are then given to groups of mice (10 per group), on one flank of each mouse only 3 times a week for two weeks:
pL of vehicle (1 group);
Intraperitoneal mouse anti-PD1 (Bioxcell RMP-114 10 mg / kg every three days);
pL of 10 ⁵ pfu / ml, 10 ⁶ pfu / ml or 10 ⁷ pfu / ml of HSV only with mouse GM-CSF and GALVR- inserted (3 groups);
pL of 10 ⁵ pfu / ml, 10 ⁶ pfu / ml or 10 ⁷ pfu / ml of HSV with GALVR-, mouse GM-CSF and mouse anti-CTLA-4 inserted
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50/52 together with intraperitoneal mouse anti-PD1 (10 mg / kg every three days) (3 groups);
pL of 10 ⁵ pfu / mL, 10 ⁶ pfu / mL or 10 ⁷ pfu / mL of HSV only with mouse GM-CSF and GALVR- inserted together with mouse anti-PD1 (10 mg / kg every three days) ( 3 groups);
μΙ_ of 10 ⁵ pfu / ml, 10 ⁶ pfu / ml or 10 ⁷ pfu / ml of HSV with GALVR-, mouse GM-CSF and mouse anti-CTLA-4 inserted together with intraperitoneal mouse anti-PD1 (10 mg / kg every three days) (3 groups).
[178] The effects on tumor growth are then seen for up to a month. Superior tumor control and shrinkage were observed in both injected and non-injected tumors with the virus that expresses anti-CTLA-4, which is further improved with combined treatment with anti-PD1, compared to other groups is observed, including through an improved dose-response curve.
Example 7
The GALV Combined Expression Effect. GM-CSF, Anti-CTLA4 and an Oncolytic Virus Immune Coestimulatory Pathway Activation Molecule [179] The experiment in Example 3 above is repeated, but the mice are dosed with the virus that additionally expresses well immune coestimulatory pathway ligands as they express GALV, mGM-CSF and antiCTLA4.
[180] More specifically, groups of mice received:
(1) Vehicle;
(2) Intraperitoneal mouse anti-PD1;
(3) HSV with mGM-CSF, GALVR- and anti-CTLA4 inserted as in Example 2;
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51/52 (4) HSV with inserted mouse mGM-CSF, GALVR-, anti-CTLA4 and CD4OL;
(5) HSV with mGM-CSF, GALVR-, anti-CTLA4 and 4-1 BBL of inserted mouse;
(6) HSV with mGM-CSF, GALVR-, anti-CTLA4 and inserted mouse GITRL;
(7) HSV with inserted mouse mGM-CSF, GALVR-, anti-CTLA4 and OX40L;
(8) HSV with mGM-CSF, GALVR-, anti-CTLA4 and inserted mouse ICOSL;
(9) HSV with mGM-CSF, GALVR- and anti-CTLA4 inserted as in Example 2, together with intraperitoneal anti-PD1;
(10) HSV with mGM-CSF, GALVR-, anti-CTLA4 and mouse CD40L inserted together with intraperitoneal anti-PD1;
(11) HSV with mGM-CSF, GALVR-, anti-CTLA4 and 4-1 mouse BBL inserted together with intraperitoneal anti-PD1;
(12) HSV with mGM-CSF, GALVR-, anti-CTLA4 and mouse GITRL inserted together with intraperitoneal anti-PD1;
(13) HSV with mGM-CSF, GALVR-, anti-CTLA4 and mouse OX40L inserted together with intraperitoneal anti-PD1; or (14) HSV with mGM-CSF, GALVR-, anti-CTLA4 and mouse ICOSL inserted together with intraperitoneal anti-PD1.
[181] Superior tumor control was observed with viruses that express immune co-stimulatory ligands.
Deposit Information [182] The following strains of HSV1 were deposited in the
ECACC, Culture Collections, Public Health England, Porton Down, Salisbury, SP4 OJG, United Kingdom on December 19, 2016 by Replimune Limited and
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52/52 the assigned access numbers were allocated:
[183] RH004A - Accession Number 16121902 [184] RH015A - Accession Number 16121903 [185] RH018A - Accession Number 16121904 [186] RH021A - Accession Number 16121905 [187] RH023A - Accession Number 16121906 [188] RH031A - Access Number 16121907 [189] RH040B - Access Number 16121908 [190] RH047A - Access Number 16121909.
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权利要求:
Claims (53)
[1]
Claims
1. ONCOLYTIC VIRUS, characterized by the fact that it encodes a CTLA-4 inhibitor.
[2]
2. VIRUS, according to claim 1, characterized in that the CTLA-4 inhibitor is an antibody or an antigen-binding fragment thereof.
[3]
3. VIRUS, according to claim 2, characterized in that the fragment comprises an scFv molecule.
[4]
4. VIRUS, according to claim 2, characterized in that the fragment is an scFv molecule linked to one or more regions of IgG1.
[5]
VIRUSES according to any one of claims 2 to
4, characterized in that the antibody or fragment comprises a variable region sequence of light chain linked to an IgG heavy chain.
[6]
6. VIRUSES according to any one of claims 2 to
5, characterized in that 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 NOs: 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.
[7]
7. VIRUS, according to claim 6, characterized in that 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.
[8]
8. VIRUS according to claim 7, characterized in that the antibody or fragment is encoded by (a) the nucleotide sequence of SEQ ID NO: 10; or (b) nucleotide sequence of SEQ ID NO: 15.
[9]
9. VIRUSES, according to any of the claims
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2/8 above, characterized by the fact that the virus still comprises a gene encoding GM-CSF.
[10]
10. VIRUS, according to any one of the preceding claims, characterized in that the virus still comprises a molecule that activates the immune co-stimulatory pathway or a gene encoding the molecule that activates the immune co-stimulatory pathway.
[11]
11. VIRUS, according to claim 10, characterized by the fact that the gene encoding the molecule that activates the immune co-stimulatory pathway encodes CD40 ligand (CD40L), ICOS ligand, GITR ligand, 4-1-BB ligand, 0X40, TL1A ligand, CD30 linker, CD27 or flt3 linker or a modified version of any of these.
[12]
12. VIRUS, according to claim 10 or 11, characterized in that the gene encoding the molecule that activates the immune co-stimulatory pathway encodes CD40 ligand, GITR ligand, 4-1-BB ligand, 0X40 ligand, ICOS ligand or a modified version of any of these.
[13]
13. VIRUS, according to any of the preceding claims, characterized by the fact that it still comprises a gene encoding fusogenic protein.
[14]
14. VIRUS, according to claim 13, characterized in that the fusogenic protein is selected from the group consisting of vesicular stomatitis virus (VSV) protein G, syncytine-1, syncytine-2, protein F of the simian virus 5 (SV5), H measles virus (MV) protein, MV protein F, respiratory syncytial virus protein (RSV) and a gibbon monkey leukemia virus (GALV) glycoprotein, murine leukemia virus (MLV) , Mason-Pfizer monkey virus (MPMV) or equine infectious anemia virus (EIAV) from which the R peptide was deleted.
[15]
15. VIRUS, according to claim 13 or 14, characterized by the fact that the fusogenic protein is the glycoprotein of
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3/8 gibbon monkey leukemia (GALV) and having the transmembrane peptide R mutated or removed (GALV-R-).
[16]
16. VIRUS, according to any of the preceding claims, characterized by the fact that it encodes more than one molecule that activates the immune co-stimulatory pathway.
[17]
17. VIRUS, according to any of the preceding claims, characterized by the fact that it is derived from a clinical isolate of a virus.
[18]
18. VIRUS, according to any of the preceding claims, characterized by the fact that it is a modified clinical isolate of a virus, in which the clinical isolate kills two or more tumor cell lines more quickly and / or at a lower dose in in vitro than one or more clinical reference isolates from the same virus species.
[19]
19. VIRUS, according to any of the preceding claims, characterized by the fact that it is selected from the group consisting of herpes, poxvirus, adenovirus, retrovirus, rabdovirus, paramixovirus and reovirus viruses.
[20]
20. VIRUS, according to any of the preceding claims, characterized by the fact that it is a herpes simplex virus (HSV).
[21]
21. VIRUS, according to claim 20, characterized by the fact that it is an HSV1.
[22]
22. VIRUS, according to claim 21, characterized by the fact that HSV:
(a) not expressing functional ICP34.5;
(b) not expressing functional ICP47; and / or (c) expressing the US11 gene as an immediate starting gene.
[23]
23. VIRUS according to any one of claims 20 to 22, characterized in that a gene encoding the anti-inhibitory protein
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4/8
CTLA-4 has been inserted into the ICP34.5 coding locus by insertion, partial deletion or complete deletion.
[24]
24. VIRUS according to claim 23, characterized in that the gene encoding the anti-CTLA-4 inhibitory protein is included in a cassette that also includes one or more immunity-stimulating genes such as GM-CSF and / or a molecule that activates the immune co-stimulatory pathway and / or a gene encoding fusogenic protein.
[25]
25. VIRUS according to any one of the preceding claims, characterized in that the coding sequence for the CTLA-4 inhibitor, the coding sequence for a molecule that activates the immune co-stimulatory pathway and / or the coding sequence for the fusogenic protein is codon- optimized in order to increase expression levels in target cells.
[26]
26. VIRUS, according to any of the previous claims, characterized by the fact that it expresses three heterologous genes, in which each of the three heterologous genes is directed by a different promoter selected from the CMV promoter, RSV promoter, promoter of SV40 (SEQ ID) and a retroviral LTR promoter.
[27]
27. VIRUS, according to claim 26, characterized by the fact that it expresses four heterologous genes directed by each of the CMV promoter, RSV promoter, SV40 promoter and a retroviral LTR promoter.
[28]
28. VIRUS, according to claim 26 or 27, characterized by the fact that the retroviral LTR is MMLV (SEQ ID).
[29]
29. VIRUS, according to any of the preceding claims, characterized by the fact that it expresses three heterologous genes, in which each of the three heterologous genes is terminated by a different polyadenylation sequence selected from the polyadenylation sequences
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5/8 of BGH, SV40, HGH and RBG.
[30]
30. VIRUS, according to claim 29, characterized by the fact that it expresses heterologous genes terminated by each of the polyadenylation sequences of BGH, SV40, HGH and RBG, respectively.
[31]
31. VIRUS according to any one of claims 26 to 30, characterized in that it is a poxvirus.
[32]
32. PHARMACEUTICAL COMPOSITION, characterized in that it comprises a virus, according to any one of claims 1 to 31, and a pharmaceutically acceptable carrier or diluent.
[33]
33. VIRUS according to any one of claims 1 to 31, characterized in that it is for use in a method of treating the human or animal body by therapy.
[34]
34. VIRUS according to any one of claims 1 to 31, characterized in that it is for use in a method for treating cancer.
[35]
35. VIRUS, for use according to claim 34, characterized in that the method comprises administering an additional anticancer agent.
[36]
36. VIRUS, for use according to claim 35, characterized in that the additional anticancer agent is selected from an immune co-stimulating pathway or immune co-stimulatory pathway, radiation and / or chemotherapy, an agent that directs a specific genetic mutation that occurs in tumors, an agent designed to induce an immune response to one or more tumor antigens or neoantigens, a cell product derived from T cells or NK cells, an agent designed to stimulate STING, cGAS, TLR or another immune response innate and / or inflammatory pathway, a second virus, optionally an oncolytic virus, and combinations thereof.
[37]
37. VIRUSES, for use according to claim 36,
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6/8 characterized by the fact that the targeting agent of an 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, IDO inhibitor, KIR inhibitor, SLAMF7 inhibitor, CEACAM1 inhibitor or CD47 inhibitor and / or the immune co-stimulatory pathway agent is a GITR agonist, a 4-agonist -1 -BB, a 0X40 agonist, a CD40 agonist or an ICOS agonist.
[38]
38. VIRUS, for use according to any one of claims 35 to 37, characterized in that the additional anti-cancer agent is an antibody.
[39]
39. VIRUS, for use according to any of claims 35 to 38, characterized in that it comprises administering an inhibitor of the indoleamine 2,3-dioxigenase (IDO) pathway and an additional antagonist of an immune co-inhibitory pathway or an agonist of an immune co-stimulatory pathway.
[40]
40. VIRUSES, for use according to any one of claims 34 to 39, characterized in that the virus and the additional anticancer agent (s) are administered separately.
[41]
41. VIRUSES, for use according to any one of claims 34 to 39, characterized in that the virus and the additional anticancer agent (s) are administered at the same time.
[42]
42. VIRUS, for use according to any one of claims 34 to 41, characterized in that the cancer is a solid tumor.
[43]
43. MANUFACTURING PRODUCT, characterized by the fact that it comprises a virus, according to any one of claims 1 to 31, in a sterile vial, ampoule or syringe.
[44]
44. METHOD FOR TREATING CANCER, characterized in that it comprises administering a therapeutically effective amount of the virus, according to any one of claims 1 to 31, or a composition
Petition 870190058870, of 06/25/2019, p. 129/145
Pharmaceutical 7/8 according to claim 32, for a patient in need thereof.
[45]
45. METHOD, according to claim 44, characterized by the fact that it still comprises administering an effective amount of an additional anticancer agent to a patient in need of it.
[46]
46. METHOD, according to claim 45, characterized in that the additional anticancer agent is selected from the group consisting of a targeting agent of an immune co-inhibitory pathway or immune co-stimulatory pathway, radiation and / or chemotherapy, an agent that targets a specific genetic mutation that occurs in tumors, an agent designed to induce an immune response to one or more tumor antigens or neoantigens, a cell product derived from T cells or NK cells, an agent designed to stimulate STING, cGAS, TLR or other innate immune response and / or inflammatory pathway, a second virus, optionally an oncolytic virus, and combinations thereof.
[47]
47. METHOD, according to claim 46, characterized in that the targeting agent of an immune co-inhibitory pathway is a PD-1 inhibitor, a PD-L1 inhibitor, a LAG-3 inhibitor, a TIM- 3, a VISTA inhibitor, a CSF1R inhibitor, an IDO inhibitor, a KIR inhibitor, an SLAMF7 inhibitor, a CEACAM1 inhibitor or a CD47 inhibitor and / or the targeting agent of an immune co-stimulatory pathway is an GITR agonist, 4-1-BB agonist, 0X40 agonist, CD40 agonist or ICOS agonist.
[48]
48. METHOD according to any one of claims 45 to 47, characterized in that the additional anti-cancer agent comprises an antibody.
[49]
49. METHOD according to any one of claims 45 to 48, characterized by the fact that the virus and the additional anticancer agent (s)
Petition 870190058870, of 06/25/2019, p. 130/145
8/8 be administered separately.
[50]
50. METHOD according to any of claims 45 to 48, characterized in that the virus and the additional anticancer agent (s) are administered at the same time.
[51]
51. METHOD, according to any of claims 44 to 50, characterized in that the cancer is a solid tumor.
[52]
52. USE of the virus according to any one of claims 1 to 31, characterized in that it is for the manufacture of a medicament for use in a method to treat cancer.
[53]
53. USE, according to claim 52, characterized in that the method comprises administering an additional anti-cancer agent.

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同族专利:

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公开号 | 公开日

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GB201700350D0|2017-02-22|

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WO2018127713A1|2018-07-12|

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MX2019008146A|2019-10-09|

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US20190343903A1|2019-11-14|

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IL267949D0|2019-09-26|

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JP2020503871A|2020-02-06|

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CA3049496A1|2018-07-12|

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CN110198724A|2019-09-03|

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AU2018205763A1|2019-06-20|

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KR20190104055A|2019-09-05|

---

EP3565568A1|2019-11-13|

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法律状态:
2021-10-13| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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GBGB1700350.0A|GB201700350D0|2017-01-09|2017-01-09|Altered virus|

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PCT/GB2018/050048|WO2018127713A1|2017-01-09|2018-01-09|Altered virus|

---